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Chemical Investigation of two Antarctic Invertebrates, Synoicum adareanum (Chordata: Ascidiaceae; Enterogona; Polyclinid...

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Title:
Chemical Investigation of two Antarctic Invertebrates, Synoicum adareanum (Chordata: Ascidiaceae; Enterogona; Polyclinidae) and Austrodoris kergulenensis (Molusca; Gastropoda; Nudibranchia; Dorididae)
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English
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Diyabalanage, Thushara Kelum Kaviraj
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
Antarctic tunicate
Palmerolide macrolide
Cytotoxicity
Nudibranch
Palmadorin
Dissertations, Academic -- Chemistry -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Synoicum adareanum is a colonial tunicate commonly found on the benthos around Palmer Station on Anvers Island, Antarctica. A comprehensive chemical investigation of the lipophilic extract of the frozen tunicates gave a new series of polyketide macrolides, palmerolides A-E and H. The structure elucidation of these compounds was accomplished by extensive NMR and mass spectral studies.The palmerolides are unusual 20-membered macrolides displaying functionality more commonly found in sponges or cyanobacteria. Palmerolide A, the major member of the group, shows significant and selective in vitro cytotoxicity against melanoma with three orders of greater sensitivity relative to other cell lines tested, in the National Cancer Institute (NCI) 60 human cancer cell-line panel. In addition, it displays potent cytostatic activity against several other cancer cell lines. Based on NCI's COMPARE analysis, palmerolide A was investigated as a V-ATPase inhibitor and shown to bind the V0 subunit with 2 nM inhibition.Austrodoris kerguelenensis is a common Antarctic nudibranch widely distributed in the High Antarctic and Sub Antarctic Zone. It is characterized by the presence of terpenoid glyceryl esters which are supposed to be involved in defense. Chemical investigations of several specimens of A. kerguelenensis collected near Palmer station Antarctica afforded hitherto undescribed series of clerodane diterpenoid glycerides. The structure elucidation of three major compounds of this series, palmadorin A, B and C was accomplished.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
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Includes bibliographical references.
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by Thushara Kelum Kaviraj Diyabalanage.
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Document formatted into pages; contains 188 pages.
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Includes vita.

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Chemical Investigation of two Antarctic Invertebrates, Synoicum adareanum (Chordata: Ascidiaceae; Enterogona; Polyclinidae) and Austrodoris kergulenensis (Molusca; Gastropoda; Nudibranchia; Dorididae)
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ABSTRACT: Synoicum adareanum is a colonial tunicate commonly found on the benthos around Palmer Station on Anvers Island, Antarctica. A comprehensive chemical investigation of the lipophilic extract of the frozen tunicates gave a new series of polyketide macrolides, palmerolides A-E and H. The structure elucidation of these compounds was accomplished by extensive NMR and mass spectral studies.The palmerolides are unusual 20-membered macrolides displaying functionality more commonly found in sponges or cyanobacteria. Palmerolide A, the major member of the group, shows significant and selective in vitro cytotoxicity against melanoma with three orders of greater sensitivity relative to other cell lines tested, in the National Cancer Institute (NCI) 60 human cancer cell-line panel. In addition, it displays potent cytostatic activity against several other cancer cell lines. Based on NCI's COMPARE analysis, palmerolide A was investigated as a V-ATPase inhibitor and shown to bind the V0 subunit with 2 nM inhibition.Austrodoris kerguelenensis is a common Antarctic nudibranch widely distributed in the High Antarctic and Sub Antarctic Zone. It is characterized by the presence of terpenoid glyceryl esters which are supposed to be involved in defense. Chemical investigations of several specimens of A. kerguelenensis collected near Palmer station Antarctica afforded hitherto undescribed series of clerodane diterpenoid glycerides. The structure elucidation of three major compounds of this series, palmadorin A, B and C was accomplished.
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Chemical Investigation of tw o Antarctic Invertebrates, Synoicum adareanum (Chordata: Ascidiaceae: Ente rogona:Polyclinidae) and Austrodoris kergulenensis (Molusca: Gastropoda:Nudi branchia: Dorididae) by Thushara Kelum Kaviraj Diyabalanage 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 J. Baker, Ph.D. Kirpal Bisht, Ph.D. Abdul Malik, Ph.D. Roman Manetsch, Ph.D. Date of Approval: April 11th 2006 Keywords: Antarctic tunicate, palmerolid e macrolide, cytotoxicity, nudibranch, palmadorin Copyright 2006 Thushara Diyabalanage

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DEDICATION This dissertation is dedicated to my family. To my parents, for their guidance, dedi cation and wisdom towards my education since my childhood. To my wife, Dilshani, for her understandi ng and endurance duri ng a difficult period in a foreign country. To my daughter Randima, for refreshing my mind with her childhood grace whenever I was tired. To my late Maternal Grand father Soma pala Ratnaweera, who wanted to see me become a medical doctor and help the mankind. To my late Paternal Grand father Diya balanage Podisinggno, who used to cure patients with snake bites using the herbs gr own in our back yard giving me the first lessons of biomolecules.

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AKNOWLEDGEMENT I wish to express my most sincere gratitude to my major advisor Dr Bill J Baker for his exemplary guidance in last five years. Hi s understanding, sheer dedi cation and patience have been as invaluable asset to me during this period of extensive research. I want to thank my committee members Dr Ki rpal Bisht, Dr Abdul Malik and Dr Roman Manetsch for their valuable comments and encouragement. I take this opportunity to extend my gratitude to Dr Sridevi Ankisett i and Dr Solomon Weldegirma, post docs at Baker lab. My sincere thanks are also due to all my past and present colleagues at Baker lab for their friendship, encouragements and words of wisdom. I wish to thank National Cancer Institute and Lee Moffit Cancer Research Institute for carrying out anticancer activity tests on Palmerolides. I would like to acknowledge US Antarctic Program and National Science Founda tion for providing financial assistance for my research.

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i TABLE OF CONTENTS LIST OF FIGURES vi LIST OF TABLES x LIST OF SCHEMES xi LIST OF ABBREVIATIONS xii ABSTRACT xiv CHAPTER 1. INTRODUCTION 1.1 Drugs from the Sea 1 1.2 Biodiversity of Antarctica 12 1.3 Research Objectives 19 1.4 Summary 20 CHAPTER 2. CHEMICAL INVESTIGATION OF THE ANTARCTIC TUNICATE SYNOICUM ADAREANUM 2.1 Introduction 21 2.1.1 Tunicates: a rich sour ce of bioactive natural products 21 2.1.2 Chemistry of the genus Synoicum 24 2.1.3 Research Objectives 26 2.2 Results and Discussion 27 2.2.1 Extraction and isolation of secondary metabolites 27 2.2.2 Characterization of palmerolide A 31 2.2.3 Stereochemical determination 41 2.2.3.1 Application of Mo sher’s method 41 2.2.3.1.1 R -MTPA monoester of palmerolide A ( 64 ) 42

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ii 2.2.3.1.2 R, R -MTPA diester of palmerolide A ( 66 ) 44 2.2.3.1.3 S -MTPA monoester of palmerolide A ( 65 ) 45 2.2.3.1.4 S, S -MTPA diester of palmerolide A ( 67 ) 47 2.2.3.1.5 Absolute stereochemistry assignment at C-7 of palmerolide A ( 58 ) 48 2.2.3.1.6 Absolute stereochemistry assignment at C-10 of palmerolide A ( 58 ) 49 2.2.3.2 Application of Murata’s method 50 2.2.4 Characterization of palmerolide C ( 59 ) 55 2.2.5 Characterization of palmerolide D ( 60 ) 63 2.2.6 Characterization of palmerolide E ( 61 ) 71 2.2.7 Characterization of palmerolide B ( 62 ) 78 2.2.8 Characterization of palmerolide H ( 63 ) 88 2.2.9 Bioactivity of palmerolides 96 2.2.9.1 In vitro cytotoxicity of palmerolide A ( 58 ) 96 2.2.9.2 In vivo cytotoxicity of palmerolide A ( 58 ) 97 2.2.9.3 In vitro cytotoxicity of palmerolide C ( 59 ) 98 2.2.9.4 In vitro cytotoxicity of palmerolide E ( 61 ) 99 2.2.9.5 Mechanism of action of palmerolides 99 2.2.9.6 V-ATPase 99 2.2.9.7 Known V-ATPase inhibitors 100 2.2.9.8 Palmerolides as V-ATPase inhibitors 103 2.3 Summary 103

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iii CHAPTER 3 CHEMICAL INVESTIGATION OF THE ANTARCTIC NUDIBRANCH AUSTRODORIS KERGUELENENSIS 3.1 Introduction 3.1.1 Nudibranchs 105 3.1.2 Chemistry of Austrodoris 111 3.1.3 Bioactivity 116 3.1.4 Research objectives 116 3.2 Results and Discussion 3.2.1 Extraction and isolation of secondary me tabolites 117 3.2.2 Characterization of palmadorin A ( 110 ) 118 3.2.3 Stereochemical determination of palmadorin A ( 110 ) 125 3.2.3.1 Absolute stereochemistry determination 128 3.2.4 Characterization of palmadorin B ( 111 ) 132 3.2.5 Stereochemistry determination of palmadorin B ( 111 ) 139 3.2.6 Characterization of palmadorin C ( 112 ) 141 3.2.7 Stereochemistry determination of palmadorin C ( 112 ) 147 3.2.7.1 Diacetyl derivative of palmadorin C ( 114 ) 148 3.2.7.2 RMTPA ester of palmadorin C diacetate ( 115 ) 152 3.2.7.3 S -MTPA ester of palmadorin C diacetate ( 116 ) 155 3.2.7.4 Application of Mosher’s method 160 3.3 Summary 161 CHAPTER 4 EXPERIMENTAL 162 4.1 General procedure 162

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iv 4.2 Isolation of secondary metabolites from Synoicum adareanum 163 4.2.1 Palmerolide A ( 58 ) 164 4.2.2 Preparation of MTPA esters of palmerolide A 165 4.2.2.1 Palmerolide A 7-( R )-MTPA ester ( 64 ) 165 4.2.2.2 Palmerolide A 7,10-( R,R )-MTPA diester ( 66 ) 166 4.2.2.3 Palmerolide A 7-( S )-MTPA ester ( 65 ) 167 4.2.2.4 Palmerolide A 7,10-( S,S )-MTPA diester ( 67 ) 167 4.2.3 Palmerolide C ( 59 ) 168 4.2.4 Palmerolide D ( 60 ) 169 4.2.5 Palmerolide E ( 61 ) 170 4.2.6 Palmerolide B ( 62 ) 171 4.2.7 Palmerolide H ( 63 ) 172 4.3 Isolation of secondary metabolites from Austrodoris kerguelenensis 173 4.3.1 Palmadorin A ( 110 ) 174 4.3.2 Ozonolysis of palmadorin A ( 110 ) 174 4.3.3 Ozonolyzed product of palmadorin A ( 113 ) 175 4.3.4 Palmadorin B ( 111 ) 175 4.3.5 Palmadorin C ( 112 ) 176 4.3.6 Acetylation of palmadorin C ( 112 ) 177 4.3.7 Palmadorin C diacetate ( 114 ) 177

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v 4.3.8 Preparation of R -MTPA esters of palmadorin C diacetate ( 114 ) 178 4.3.9 Palmadorin C diacetate R -MTPA ( 115 ) 178 4.3.10 Preparation of S -MTPA esters of palmadorin C diacetate ( 114 ) 179 4.3.11 Palmadorin C diacetate S -MTPA ( 116 ) 179 REFERENCES 180 APPENDICES 188 Appendix A Cytotoxicity profile of palmerolide A 188 ABOUT THE AUTHOR End Page

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vi LIST OF FIGURES Figure 1. Synoicum adareanum at Palmer Station Antarctica (photograph supplied by Bill J. Baker, University of South Florida) 26 Figure 2. LRFABMS positive mode spectrum of palmerolide A ( 58 ) 31 Figure 3. 13C NMR spectrum of palmerolide A ( 58 ) (125 MHz, DMSOd6) 32 Figure 4. DEPT 135 spectrum of palmerolide A ( 58 ) (125 MHz, DMSOd6 ) 33 Figure 5. 1H NMR spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) 34 Figure 6. gHMBC spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) 36 Figure 7. gHSQC spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) 37 Figure 8. Key gHMBC and gCOSY co rrelations of palmerolide A ( 58 ) 38 Figure 9. gCOSY spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) 39 Figure 10. Palmerolide A ( 58 ) planer structure 41 Figure 11. 1H NMR spectrum of palmerolide A R -MTPA monoester ( 64 ) (500 MHz, CD3OD) 43 Figure 12. Key gHMBC correlations demonstr ating the attachment of MTPA in compound 64. 44 Figure 13. 1H NMR spectrum palmerolide A R, R -MTPA diester ( 66 ) (500 MHz, CD3OD) 44 Figure 14. Key gHMBC correlations dem onstrating the attachment of MTPA moiety in compound 66 45 Figure 15. 1H NMR spectrum of palmerolide A SMTPA monoester ( 65 ) (500 MHz, CD3OD) 46

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vii Figure 16. Key gHMBC correlations demonstr ating the attachme nt of MTPA moiety in compound 65 46 Figure 17. 1H NMR spectrum of palmerolide A S, SMTPA diester ( 67 ) (500 MHz, CD3OD) 47 Figure 18. Key gHMBC correlations demons trating the attachment of MTPA moiety in compound 67 48 Figure 19. Chemial shift differences ( 1000) of palmerolide A MTPA Rand S -monoesters 49 Figure 20. MTPA model for config uration correlations 49 Figure 21. Chemical shift values ( 1000) of palmerolide A MTPA R, Rand S, Sdiesters 49 Figure 22. gHSQMBC spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) 50 Figure 23. 1D analysis of the gHSQMBC cross peak of the respective slice 51 Figure 24. Determination of the coupli ng constant by the subtraction of 1H NMR spectrum; 52 Figure 25. Coupling constant based configura tion analysis 52 Figure 26. ROESY spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) 53 Figure 27. Key ROESY correlations supporting the stereochemical determination of C-19 and C-20. 54 Figure 28. Full stereochemical assignment of palmerolide A ( 58 ) 54 Figure 29. Palmerolide C ( 59 ) HRESIMS spectrum 55

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viii Figure 30 13C NMR spectrum of palmerolide C ( 59 ) (125 MHz, DMSOd6) 56 Figure 31. DEPT 135 spectrum of palmerolide C ( 59 ) (125 MHz, DMSOd6) 56 Figure 32. gHMBC spectrum of palmerolide C ( 59 ) (500 MHz, DMSOd6) 57 Figure 33 gCOSY spectrum of palmerolide C ( 59 ) (500 MHz, DMSOd6) 58 Figure 34. Key gHMBC and gCOSY co rrelations of palmerolide C ( 59 ) 59 Figure 35. 1H NMR spectrum of compound 59 (500 MHz, DMSOd6) 59 Figure 36. gHMQC spectrum of palmerolide C ( 59 ) (500 MHz, DMSOd6) 60 Figure 37. HRESIMS spectrum of palmerolide D ( 60 ) 64 Figure 38 13C NMR spectrum of palmerolide D ( 60 ) (125 MHz, DMSOd6 ) 64 Figure 39. DEPT 135 spectrum of palmerolide D ( 60 ) (125 MHz, DMSOd6 ) 65 Figure 40. Key gHMBC and gCOSY co rrelations of Palmerolide D ( 60 ) 65 Figure 41 gHMBC spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6) 66 Figure 42. gCOSY spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6) 67 Figure 43. gHMQC spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6) 68 Figure 44. 1H NMR spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6) 69 Figure 45. Planer structure of palmerolide D ( 60 ) 71 Figure 46. Palmerolide E ( 61 ) HRESIMS spectrum 72 Figure 47. Key gHMBC and gCOSY co rrelations of palmerolide E ( 61 ) 73 Figure 48. 1H NMR spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6) 73 Figure 49 gHMQC spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6) 74 Figure 50. gHMBC spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6) 75

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ix Figure 51. gCOSY spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6) 77 Figure 52. 1H NMR spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) 78 Figure 53. 13 C NMR spectrum of palmerolide B ( 62 ) (125 MHz, CD3OD) 79 Figure 54. DEPT 135 spectrum of palmerolide B ( 62 ) (125 MHz, CD3OD) 79 Figure 55. gHSQC spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) 80 Figure 56. gHMBC spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) 81 Figure 57. Key gCOSY and gHMBC correlations of palmerolide B ( 62 ) 81 Figure 58. gCOSY spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) 83 Figure 59. HRESIMS spectrum of palmerolide B ( 62 ) 86 Figure 60. Fragmentation of palmerolide B ( 62 ) in LR ESIMS positive mode 87 Figure 61. 1H NMR spectrum of palmerolide H ( 63 ) (500 MHz, CD3OD) 88 Figure 62. 13C NMR spectrum of palmerolide H ( 63 ) (125 MHz, CD3OD) 89 Figure 63. gHSQC spectrum of palmerolide H( 63 )(500 MHz, CD3OD) 89 Figure 64. gHMBC spectrum of palmerolide H( 63 )(500 MHz, CD3OD) 90 Figure 65. gCOSY spectrum of palmerolide H ( 63 ) (500 MHz, CD3OD) 91 Figure 66. Key gHMBC and gCOSY co rrelations of palmerolide H ( 63 ) 91 Figure 67. HRESIMS spectrum of palmerolide H ( 63 ) 95 Figure 68. Austrodoris kerguelenensis at Bonaparte Point Antarctica (photograph supplied by Bill J. Baker, University of South Florida) 112 Figure 69. LRFABMS spectrum of palmadorin A ( 110 ) 119 Figure 70. 13C NMR spectrum of palmadorin A ( 110 ) (125 MHz, CDC13) 120 Figure 71. 1H NMR spectrum of palmadorin A ( 110 ) (500 MHz, CDC13) 121 Figure 72. gHSQC spectrum of palmadorin A ( 110 ) (500 MHz, CDCl3) 121

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x Figure 73. gHMBC spectrum of palmadorin A ( 110 ) (500 MHz, CDCl3) 122 Figure 74. Key gHMBC and gCOSY co rrelations of palmadorin A ( 110 ) 122 Figure 75. ROESY spectrum of palmadorin A ( 110 ) (500 MHz, CDCl3) 126 Figure 76. Selective 1D NOE experiments of palmadorin A (500 MHz, CDCl3) 127 Figure 77. Key ROESY correlations of palmadorin A ( 110) 128 Figure 78. Key gHMBC correlations of 113 130 Figure 79 Two possible trans decalin enantiomers of palmadorin A 130 Figure 80. CD spectrum of compound 113 showing a negative cotton effect 131 Figure 81. Absolute stereochemistry of palmadorin A ( 110 ) 131 Figure 82. LRFABMS spectrum of palmadorin B ( 111 ) 132 Figure 83 13C NMR spectrum of palmadorin B ( 111 ) (125 MHz, CDC13,) 133 Figure 84. gHMBC spectrum of palmadorin B ( 111 ) (500 MHz, CDCl3) 134 Figure 85. gHSQC spectrum of palmadorin B ( 111 ) (500 MHz, CDCl3) 134 Figure 86. gCOSY spectrum of palmadorin B ( 111 ) (500 MHz, CDCl3) 135 Figure 87. Key gHMBC and COSY co rrelations of palmadorin B ( 111 ) 135 Figure 88. 1H NMR spectrum of palmadorin B ( 111 ) (500 MHz, CDCl3) 136 Figure 89. ROESY spectrum of palmadorin B ( 109 ) (500 MHz, CDCl3) 140 Figure 90. Palmadorin B ( 111 ) ROESY correlations 140 Figure 91. Absolute stereochemistry of palmadorin B ( 111 ) 141 Figure 92. LRFABMS spectrum of palmadorin C ( 112 ) 141 Figure 93. 13C NMR spectrum of palmadorin C ( 112 ) (125 MHz, CDCl3) 142 Figure 94. gHSQC spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3) 143 Figure 95. 1H NMR spectrum of palmadorin C ( 112 ) (500 MHz, CDC13) 143

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xi Figure 96. gHMBC spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3) 144 Figure 97. gCOSY spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3) 145 Figure 98. Key HMBC and COSY correlations of palmadorin C ( 110 ) 145 Figure 99. Key ROESY correlations of palmadorin C ( 112 ) 148 Figure 100. 1H NMR spectrum of palm erolide C diacetate ( 114 ) (500MHz, CDCl3,) 149 Figure 101. 13C NMR spectrum of palmadorin C diacetate ( 114 ) (125 MHz, CDCl3) 150 Figure 102. Key HMBC correlations of palmadorin C diacetate ( 112 ) 151 Figure 103. LRESIMS spectrum of palmadorin C diacetate ( 112 ) 151 Figure 104. LRESIMS spectrum of palamadorin C diacetate R -MTPA ester ( 115 ) 152 Figure 105. 1H NMR spectrum of palmadorin C diacetate R -MTPA ester ( 115 ) (500 MHz, CDCl3) 154 Figure 106. gHSQC spectrum of palmadorin C diacetate R -MTPA ester ( 115 ) (500 MHz, CDCl3) 154 Figure 107. gHMBC spectrum of palmadorin C diacetate R -MTPA ester ( 115 ) (500 MHz, CDCl3) 155 Figure 108. LRESIMS of palmadorin C diacetate S MTPA ( 116 ) 156 Figure 109 13C NMR spectrum of palmadorin C diacetate SMTPA ester ( 116 ) (125 MHz, CDCl3) 157 Figure 110. 1H NMR spectrum of palmadorin C diacetate S -MTPA ester ( 116 ) (500 MHz, CDCl3) 158 Figure 111. gHSQC spectrum of palmadorin C diacetate S -MTPA ester ( 116 ) (500 MHz, CDCl3) 158 Figure 112. gHMBC spectrum of palmadorin C diacetate R -MTPA ester ( 114 ) (500 MHz, CDCl3) 159

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xii Figure 113. value assignments for palmadorin C diacetate MTPA esters 160 Figure 114 Absolute stereochemistry of palmadorin C ( 112 ) 160

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xiii LIST OF TABLES Table 1. Potential therapeutic comp ounds isolated from marine sources 3 Table 2. NMR data of palmerolide A ( 58 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 35 Table 3. NMR data of palmerolide C ( 59 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 61 Table 4. NMR data of palmerolide D ( 60 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 70 Table 5. NMR data of palmerolide E ( 61 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 76 Table 6. NMR data of palmerolide B ( 62 ) (1H, 500 MHz, 13C, 125 MHz, CD3OD) 83 Table 7. NMR data of palmerolide H ( 63 ) (1H, 500 MHz, 13C, 125 MHz, CD3OD) 93 Table 8 Comparison of cytotoxicity and V-ATPase activity 103 Table 9. NMR data of palmadorin A ( 108 ) (1H, 500 MHz, 13C, 125 MHz, CDCl3) 124 Table 10. NMR data of palmadorin B ( 109 ) (1H, 500 MHz, 13C, 125 MHz, CDCl3) 137 Table 11. NMR data of palmadorin C ( 110 ) (1H, 500 MHz, 13C, 125 MHz, CDCl3) 146

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xiv LIST OF SCHEMES Scheme 1. Extraction and purific ation of palmerolides 28 Scheme 2. Extraction and purific ation of palmadorins 118 Scheme 3. Ozonolysis of palmadorin A 129 Scheme 4. Acetylation of palmadorin C 149

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xv LIST OF ABBREVIATIONS Ac2O acetic anhydride [ ] specific rotation = 100 / lc CDCl3 deuterated chloroform CD3OD deuterated methanol C18 octadecyl bonded silica chemical shifts (NMR) DEPT distortionless enhan cement by polarization transfer DMAP dimethyl amino pyridine DMSOd6 deuterated dimethylsulfoxide EtOAc ethylacetate EtOH ethanol the molar extinction coefficient in UV spectroscopy gCOSY gradient correl ation spectroscopy (NMR) gHSQC gradient heteronuclear single quantum correlation (NMR) gHMQC gradient heteronuclear multiple quantum coherence (NMR) gHMBC gradient heteronuclear multiple bond connectivity (NMR) HRFABMS high resolution fast atom bombardment mass spectrometry HPLC high performance liquid chromatography IR infrared J coupling constant nJCH n-bond hydrogen to carbon correlation (n = 2,3 or 4) nJHH n-bond hydrogen to hydrogen correlation (n = 2,3 or 4)

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xvi LRESIMS low resolution electrospr ay ionization mass spectrometry LRFABMS low resolution fast atom bombardment mass spectrometry max the wavelength at which maximum absorption occurs MeCN acetonitrile MeOH methanol MTPA-Cl -methoxy-(trifluoromethyl)phe nylacetyl chloride m/z mass/charge ratio in mass spectrometry ROESY rotating-frame overhause r enhancement sp ectroscopy (NMR) TEA triethylamine UV ultraviolet

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xvii ABSTRACT Synoicum adareanum is a colonial tunicate co mmonly found on the benthos around Palmer Station on Anvers Island, Antarctica. A comprehensive chemical investigation of the lipophilic extract of the frozen tunicates gave a new series of polyketide macrolides, palmerolides A-E and H. The structur e elucidation of these compounds was accomplished by extensive NMR and mass spectral studies. The palmerolides are unusual 20-membered macrolides displaying functionality more commonly found in sponges or cyanobacteria. Palmerolide A, the major member of the group, shows significant and selective in vitro cytotoxicity against melanoma with three orders of greater sensitivity relative to other cell lines tested, in the National Cancer Institute (NCI) 60 human cancer cell-line panel. In addition, it displays potent cytostatic activity against several other cancer ce ll lines. Based on NCI’s COMPARE analysis, palmerolide A was investigated as a V-ATPase inhibitor and shown to bind the V0 subunit with 2 nM inhibition. Austrodoris kerguelenensis is a common Antarctic nudibranch widely distributed in the High Antarctic and Sub Antarctic Zone. It is characterized by the presence of terpenoid glyceryl esters which are supposed to be invol ved in defense. Chemical investigations of several specimens of A. kerguelenensis collected near Palmer st ation Antarctica afforded

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xviii hitherto undescribed series of clerodane dite rpenoid glycerides. The structure elucidation of three major compounds of this series, palmadorin A, B and C was accomplished.

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1 Chapter 1. INTRODUCTION 1.1 Drugs from the Sea The oceans compose more than 70% of the ear th’s surface and over 90% of its volume of its crust.1,2 The marine environment has been an ex ceptional reservoir of bioactive natural products.3 Due to the physical and chemical featur es of oceans, such as extreme variation of temperature, pressure and salinity, mari ne organisms have evolved biochemical and physiological mechanisms that give rise to bioactive compounds with unique structural features, often not found among terrestrial na tural products. These biomolecules play a pivotal role in reproduction, co mmunication and protection ag ainst predati on, infection and competition in the oceanic environment.4,5 Many marine organisms are soft-bodied or move slow whereas some of them have sedentary life styles. However, they ofte n do not have physical armament like hard external shells or spicules for their prot ection, making them extremely vulnerable to predation and competition.4,5 Such organisms need some form of defense. Many marine organisms have developed defenses employi ng bioactive natural pr oducts against their enemies.4,5 These compounds, belonging to the category of secondary metabolites, help them to either deter or have a competitive edge over the predators.4 Some of these marine organisms have evolved the ability to synthesize their own secondary metabolite defense chemicals via de novo biosynthesis. Other organisms have

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2 been found to derive their de fense chemicals by a symbiotic relationship or simply from their diet.4 Beyond chemical diversity, the marine e nvironment is known to hold enormous biological diversity as well. Evidence for th is coming from recent research on marine ecosystems like the deep sea floor and coral reef s. In fact, their biodi versity is known to be higher than that of tropical rain fore sts, a terrestrial ecosystem renown for its enormous biodiversity.2 For instance, according to recent estimates, out of the all species living on this planet only 10% are known and the unexplored majority live in the sea1,3 Meanwhile, out of the 34 fundamental phyla of life, 17 occur on land whereas 32 occur in the sea (with some overlap). The deep sea floor harbors millions of hither to unknown species including thousands of marine microorganisms.1, 2 It has been estimated that more than 10, 000 marine natural products have been isolated by 2000.6 Among them 25% are from algae, 33% fr om sponges, 24% from coelenterates and the remaining 24% from representatives of other invertebrate phyla such as ascidians, ophisthoibranch molluscs, echinoderms and bryozoans.2 Further analysis of this data highlighted that the search for drugs from th e sea progresses at a rate of 10% per year.2 During last few years several such marine natural products have successfully advanced into clinical trials (Table 1). However, none of th ese discoveries have reached a marketable stage.

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3 Table 1. Potential therapeutic compo unds isolated from marine sources (Adapted from reference 3) Condition Compound OrganismOrigin Cancer HIV Aplidine7 Bryostatin 18 Didemnin B9 Dolastanin 1010 Ecteinascidin 74311, 12 Halichondrin B13 Kahalaide F14 Mycaperoxide B15 Cyclodidemniserol trisulphate16 Lamellarin A 20 sulphate17 Tunicate Bryozoan Tunicate Sea hare Tunicate Sponge Mollusc Sponge Tunicate Tunicate Mediterranean Gulf of California Caribbean Indian Ocean Caribbean Okinawa Hawaii Thailand Palau Australia The history of marine derived drugs date s as far back as the 1950s when Bergmann et al. isolated several nucleoside anal ogues from the Caribbean sponge Tethya crypta .18 Two of these compounds, spongothymidine ( 1 ) and spongouridine ( 2 ) had a rare arabinose sugar rather than ribose, which is more common in nucleosides. Later, the discovery of their antiviral activity led the researchers to synthesize a wide range of analogues19, 20 such as Ara-A ( 3 ) and Ara-C ( 4 ), which have demonstrated signi ficantly improved activity. They

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4 represent the only marine derived drugs in current clinical usage.3 Bergmann’s initial discovery can be considered a significant breakthrough as it led to the introduction of nucleoside analogues in antiviral and antican cer therapy with profound success (eg. 3’azido-3’-deoxythymidine (AZT, Zidovudin) ( 5 ).21 HN N O O O OH HO HO HN N O O O OH HO HO N O OH HO HO N N N H2N Spongothymidine ( 1 ) Spongouridine ( 2 ) Ara-A ( 3 ) N N NH2 O OH HO HO O HN N O O N3 HO HO O Ara-C ( 4 ) 3’-azido-3’-deoxythymidine ( 5 ) Isolated form the bryozoan Bugula neritina bryostatin 18 ( 6 ) is one of the first drug candidates from the ocean to advance into clinical trials. Bryostatin 1, a complex polyketide which inhibits Prot ein Kinase C and thereby prev ents cancer is in Phase clinical trails.22 It has been found that bryostatin 1 is not effective in ca ncer treatment by itself, but seems to enhance the activity of other anticancer drugs such as taxol and

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5 cisplatin. Therefore, it may be used in combinat ion with taxol, in the treatment of breast, ovarian and lung cancers whic h usually respond to taxol.3 O O O O H OH O O O O OH O HO OH O O O O Bryostatin 1 ( 6 ) Discodermolide23 ( 7 ) is a polyketide isolated from the marine sponge Discodermia dissolute It was found to be a potent antitumor ag ent that inhibited the polymerization of microtubules. Structure-activity studies of discodermolide and its synthetic analogues have shown that it has a greater promise a nd versatility than taxol. Both, the natural product (+)-discodermolide and its syntheti c analogue (-)-discodermolide have been found to be active as i nhibitors of cell proliferation de spite their mechanism of action differing considerably.24 O OH O H HO OH OHOCONH2 (+)-Discodermolide ( 7 )

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6 Eleutherobin ( 8 )25 and sarcodictyin A ( 9 ) and B ( 10 )26 are a group of diterpenes isolated from different sources despite their distinct similarities in structure. Eleutherobin was isolated from Eleutherobia species of soft corral found in the Indian Ocean near Western Australia25 whereas sarcodictyins were isolated from the Mediterranean soft coral Sarcodictyon roseum.26 Both compounds showed microt ubule stabilization properties.24,27 O O O O N N O O O O OH O H OH O O O N N O OR Eleutherobin ( 8 ) Sarcodictyin A ( 9 ) R = Me Sarcodictyin B ( 10 ) R = Et Dolastatin 1010 ( 11 ), isolated from the sea hare Dolabella auricularia collected from the Indian ocean is a short polypeptide cont aining unique amino acids which showed microtubule stabilization properties.6 Later the same compound has been isolated from a nudibranch and a cyanobacterium. Dolastatine 10 is in Phase clinical trials as anticancer agent for use in the treatmen t of breast and liver cancer.24

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7 Dolastanin 10 ( 11 ) Ecteinascidins11,28 isolated from the colonial ascidian Ecteinascidia turbinata found in the Caribbean and Mediterranean, are novel cyto toxins that act via DNA intercalation. Ecteinascidin 743 ( 12 ) was selective against breast cancer and melanoma. Currently it is undergoing Phase clinical trials.3 N N NH O HO O O O O O HO H O O S H H H H OH Ecteinascidin 743 ( 12 ) Recent research on the anticancer activity of ecteinascidi n 743 suggestes its potential to prevent tumors from becoming drug resistant.29 It was discovered that it can prevent the formation of P-glycoprotein, a membrane protei n that transports toxins out of the cancer cell, thereby preventing such agents from destroying the tumor. Once chemotherapy is N H N O N O OMe O N O H N O N S

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8 administered this protein has the ability of preventing it reaching the intended target. In fact, it was found that when tumors are exposed to chemotherapeutic agents, they quickly boost the activity of the MDR1 gene, that is responsible for the formation of Pglycoprotein, which ultimately results in multidrug resistance.6 Therefore, ecteinascidin 743 has the potential to increase the suscep tibly of the tumor cells to chemotherapy by inhibiting the transcription of this gene,6 in addition to being a potent chemotherapeutic agent by itself.3 Kahalaide F ( 13 ), a cyclic depsipeptide isolated from the sea slug Elysia rufescens is another anticancer drug candi date in clinical trails.14 However, it is believed that actual organism which produces the compound is the green algae Bryopsis sp. on which it feeds. H2N N 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 Kahalalide F ( 13 )

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9 Kahalaide F has a unique pattern of cytotoxicity with selective in vitro and in vivo cytotoxicity against prostrate cancer. In pa rticular, it has been s hown to be selective against hormone-independent prostr ate tumor cells which are aggressive and hard to treat. Phase clinical trails of kahalaide F in pati ents with androgen independent prostrate cancer have already begun.3 Didemnin B ( 14 ), an unusual cyclic depsipeptide isol ated from the Caribbean tunicate Trididemnum solidom in the 1980s, generated great excitement due to its pronounced antitumor activity.7,16,30,31 Later, it was found to show antineoplastic, antiviral and immunosuppressive activities as well. Its m echanism of action was identified as due to the interference in protein synthesis. Despite it s toxicity being too high to be useful as an antiviral or immunosuppressive agent, it has been in Phase clinical trails as an anticancer agent and eventually promoted to Phase However, its further development as an anticancer drug was cancelled due to the hepataotoxic side effects.6 Dehydrodidemnin B ( 15 ), or aplidine, cyclic depsipeptide isolated from the Mediterranean tunicate Aplidium albicans showed less toxicity and more potency than didemnin B. It has shown a broad spectrum of anticancer activity and has been promoted to Phase clinical trails.

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10 N O O O NH NH N O N OH O O N O NH O O O O OH O O Didemnin B ( 14 ) N O O O NH NH N O N O O O N O NH O O O O OH O O Aplidine ( 15 )

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11 Manoalide32 ( 16 ), a sesquiterpene isolated from the Indo-Pacific sponge Luffariella variabilis is a potent analgesic and antiinflammatory agent. A low concentration of manoalide inhibited calcium channels. It has been undergone Phase I clinical trails for psoriasis. It is commercially avai lable as a standard probe for PLA2 inhibition.3, 33 O O O HO HO Manoalide ( 16 ) Pseudopterosin A ( 17 ) is a tricyclic diterpenoid glycos ide isolated from the Caribbean Sea whip (gorgonian) Pseudopterogorgia elisabethae (Gorgoniidae). It is a potent antiinflammatory and analgesi c agent. Further it was found th at it can inhibit eicosoniod biosynthesis by inhibition of both PLA2 and 5-lipoxygenase. It is thought that the cell type selectivity is due to the presence of the glycoside moiety.3, 33 H OH O O HO OH OH Pseudopterosin A ( 17 )

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12 1.2 Biodiversity of Antarctica Research on Antarctic ecosystems has revealed that marine biota ha ve thrived under ice covered seas for 20 million years, providing static environmental conditions for the evolution of broad biodiversity.34 Moreover, the continent has been isolated from its lower latitude neighbors even l onger and the circumpolar curre nt that encircles the land mass of Antarctica has prevented the Antarc tic species exchanging genetic information with Northern congenors. The factors such as physical stabilit y and isolation are important criteria for genetic divergence a nd have given rise to the high levels of endemism in Antarctica.34 Predation and competition are the dominant forces that determine the species composition and distribution of such an ecosy stem and eventually could facilitate the evolution of novel biogenetic pathways leadin g to bioactive secondary metabolites with structural diversity. In fact, being a very stable ecos ystem operating under very unique set of ecological conditions, the benthic co mmunity of Antarctic a has evolved many interspecies relationships where secondary metabolites perform an important role assisting defense against predators and competitors.35 A few decades back, it was conventional wisd om that competition and predation among marine species are most intense in tropical wa ters and as a result the chemical ecology of marine organisms dwelling in tropical waters received more attention in drug discovery programs. However, recent research on orga nisms from the Southern Ocean sea floor suggested that predation and competition similarly drive the chemical defense.4,36

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13 An expedition undertaken to study the bent hos of McMurdo Sound, Antarctica, in 1980 revealed that the sea floor under the ice is ri ch in marine life a nd is covered with an immense community of sponges, soft corals, molluscs, tunicates a nd echinoderms. Many of these organisms are immobile and theref ore cannot move to less densely populated regions if the area they occupy becomes overgrown with competitors, nor can they escape predators. Nevertheless, they manage to survive. Hence, it is apparent either these sessile organisms do not have predators or they have a defensive stategy.4 Further studies of Antarctic invertebrates showed that th ey do have many predators. These include swarms of the voracious Paramoera antractica a one centimeter-long crustacean resembling shrimp and dense populations of sea stars.4,35 In addition, it was evident that the benthic invertebrates are pressured by fouling diatoms, invertebrate larvae, algal spores and potentially infect ious water column microorganisms. These environmental features suggest the likelihood of the evolution of chemically mediated defensive strategies.35 Thus, the attention was focused on the chemistry of these bottom dwellers in search of secondary metabolite s that might provide chemical defense. Subsequently, chemical investigations have been performed on Antarctic organisms and numerous bioactive molecule s have been characterized.35 Sponges of McMurdo Sound Antarctica are subject to predation by sea stars, and it is considered to be a dominant ecological factor that might drive the production of defense chemicals.35 The bright yellow Antarctic sponge Isodictiya erinacea which lacks physical defense such as spicules and mucus, is one of several chemically defended sponges in the

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14 region. An investigation of its secondary meta bolites showed the pr esence of purine and nucleoside metabolites including previously unreported erinacean ( 18 ) which showed cytotoxicity.37 The tryptophane derivative erebusinone ( 19 ), which is a yellow pigment found in the sponge, showed molt inhibition in crustaceans, a possible strategy of chemical defense.38 N N N H H N NH O HO O OH NH2 O N H O Erinacean ( 18 ) Erebusinone ( 19 )39 The bright yellow Antarctic cactus sponge wa s observed as extremely slow growing, but never observed to be eaten by the spongivorous sea star Perknaster fuscus .34 It has neither apparent spicules nor mucus s uggesting a chemical defense. Chemical investigation of this sponge, Dendrilla membranosa yielded three new diterpenoids, membranolides B ( 20 ), C ( 21 ), D ( 22 ) and membranolide ( 23 ) which is reported to have antibiotic activity.39-42 O O O O COOH O R1 R2 O Membranolide B ( 20 ) Membranolide C ( 21 ) R1 = OMe R2 = H Membranolide D ( 22 ) R1 = H R2 = OMe

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15 O O O O Membranolide ( 23 ) Suberites sp. is a common McMurdo Sound sponge th at has a muted yellow coloration. Suberitenones A ( 24 ) and B ( 25 ) are two sesterterpenes that have been described from sponge collected at several sites around Antarc tica and are suspected to be involved in chemical defense of the sponge.43 Suberitenones have shown activ ity in both sea star tube foot retraction feeding dete rrent assay and an antibacterial assay using sympatric bacteria.44 The former assay was first develope d to study the feeding relationships between the sea star Perkanaster fuscus and the sponges in McMurdo Sound. The tube feet of the sea star are known to be chemosen sory, and when they ar e in contact with an unsuitable sponge extract, they illicit a re traction, indicative of feeding deterrence. Another sponge that elicits a significant sea star tube foot retraction is Latrunculia apicalis A subsequent fractionation of the spo nge produced discorhabdin alkaloids, a group of pigments first isolated from te mperate sponges, including discorhabdin G ( 26 ) and C ( 27 ), the latter of which was identif ied as a potent mammalian cytotoxin.35, 45

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16 H H H OH O O O H H H OH O O O OH Suberitenone A ( 24 ) Suberitenone B ( 25 ) +NH H N O O Br HN +NH H N O O Br HN Br Discorhabdin G ( 26 ) Discorhabdin C ( 27 ) Leucetta leptorhapsis, a calcareous sponge commonly known as the rubber sponge due to its appearance as being stretched, is a common member of the McMurdo Sound benthic community. Comprehensive study of th is organism yielded the cytotoxic agent rhapsamine ( 28 ).46 H N OH H2N H N HO NH2 Rhapsamine ( 28 )

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17 The pteropod Clione antarctica is a shell-less pelagic mollusc which blooms in each austral summer in McMurdo Sound. It has an intriguing relationship with the amphipod Hyperiella dilatata where the amphipod positions the mollusc on its dorsum and defends itself from predatory fish utilizing the defense chemicals of the mollusc.47 A bioassay guided fractionation of the mollusc afford ed the feeding deterrent pteroenone ( 29 ).48 O OH Pteroenone ( 29 ) It has been observed that am ong the Antarctic deep water oc tocorals, some are replete with calcareous spicules and ot hers are devoid of them. The octocorals that have spicules were found to yield chemical extracts palatable to Odontaster validus, a common predaceous sea star in Antarctica, whereas ocotcorals that did not have spicules produced extracts that deterred predation, suggesting th e presence of a chemi cal defense. Chemical investigation of one such horny corals, Ainigmaptilon antarctcus afforded two bioactive sesquiterpenoids, ainigmaptilone A ( 30 ) and B ( 31 ) based on the eudesmane carbon skeleton, which is fairly uncommon in corals.49 O OH O OH Ainigmaptilone A ( 30 ) Ainigmaptilone B ( 31 )

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18 Macroalgae are known to pay a role in stru cturing the near shor e Antarctic benthos. Delisea pulchra a marine red algae collected near Palmer Station, yielded three new dimeric halogenated furanones, pulchralides A ( 32 ), B ( 33 ), C ( 34 ) along with fimbrolide ( 35 ), acetoxyfimbrolide ( 36 ) and hydroxyfimbrolide ( 37 ). The latter two compounds have shown significant antibacterial properties.50 O O R2 Br R3 Br O O R R1 Br Br O Br R O Br Pulchraide A ( 32 ) R = R1= OAc, R2 = R3 = H Fimbrolide ( 35 ) R = H Pulchraide B ( 33 ) R = R1 = R2 = R3=H Acetoxyfimbrolide ( 36 ) R = OAc Pulchraide C ( 34 ) R= OAc, R1 = R2 = R3 = H Hydroxyfimbrolide ( 37 ) R = OH Plocamium cartilagineum a common red alga found in shallow-water Antarctic environments produces halogenated mo noterpenes including anverene ( 38 ), epiplocamene D ( 39 ) and pyranoid 40 .50 Anverene, which induces feeding deterrence in the amphipod Gondogeneia antarctica displayed modest but selective antibiotic activity whereas 40 showed selective antif ungal activity. Epiplocamene D exhibited a greater degree of feeding deterrence by the amphi pod than the other two compounds. However all three compounds had no effect on the sea star Odontaster validus .50

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19 Cl Br Cl Br Br Cl Cl Cl O C l Br Br OH Anverene ( 38 ) Epiplocamene D ( 39 ) 40 1.3 Research Objectives The benthos of Antarctica har bors many organisms that have not been investigated for their chemistry and bioactivity. The unique features of its ecosystem and dynamic relationship between the predators and prey c ould lead to the evolution of biosynthetic pathways leading to molecules with interesti ng biological activity. Ther efore, in terms of drug discovery a comprehensive chemical inve stigation of Antarctic species is very important. In addition, there are many parts of the An tarctic Peninsula that have never been explored. Considering the signific ant variations in chemistry of the species that have been studied from the regions of McMurdo Sound and Palmer Station35 a comprehensive investigation of the chemistry and the bioact ivity is of considerable interest from a chemical ecological stand point.

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20 1.4 Summary Due to the ecological features of their habitat, many mari ne organisms have adopted chemical defense strategies. As a result, the marine realm has been an exceptional reservoir of bioactive natura l products with a great stru ctural diversity. Numbers of secondary metabolites isolated from marine orga nisms have advanced in to clinical trials for drug development. The research on Antarcti c marine biota emphasizes its potential to give rise to novel biogenetic pathways lead ing to secondary meta bolites with attractive biological activities.

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21 Chapter 2. CHEMICAL INVESTGATION OF ANTARCTIC TUNICATE SYNOICUM ADAREANUM 2.1. INTRODUCTION 2.1.1 Tunicates: a rich source of bioactive natural products The tunicates, classified in the phylum Chordata under the subphylum Urochordata, represent one of the most evolved groups of animals that are known to produce secondary metabolites.51 They are commonly referred to as tunica tes, due to the sac like covering or tunic made of cellulose that covers their body.52 The subphylum Urochordata consists of three classes, Ascidiaceae, Larvacae and Thaliaceae. The members of class ascidiaceae are referred to as ascidians. They are also known as sea squirts, as many species expel water through a siphon when disturbed.52 The adult ascidians are exclusively marine, and share no resemblance with ot her Chordates. Their larvae are similar to amphibian tadpoles, they have notochords, pharyngeal slits and dorsal hollo w nerve chords, the features that make them classify as Chordates which are ultimately lost during the course of development.53 Adult ascidians are sessile filter feeders that have either colonial or solitary lifestyles. They prefer the regions where there is mini mal effect from the shock created by wave action, but have a considerable free flow of seawater. The asci dian morphology is diverse. A solitary tunicate can grow as long as 15 cm or as small as 1 cm, where as a colonial tunicates found encrusted in rock s, can be extremely thin and delicate.52

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22 The first interest on the chemistry of tunicates was ignited by the scientist’s curiosity to unfold the mysteries behind the color changes observed in the blood samples of tunicates as early as in 1847.54 Subsequent research led to the di scovery of a series of pigments called tunichromes ( 41 ) and vanadium sequestration.55, 56 The focus on other bioactive secondary metabolites from tunicates originated in the early 1970s when the first ascidian metabolite, geranyl hydroquinone ( 42 ), was isolated. It was found to be active against leukemia, Rous sarcoma and mammary carcinoma in animal tests.51 Since then, especially during the last two decades, tunica tes have emerged as a rich source of bioactive natural products bearing unique structural fe atures. Several such compounds have already advanced into clin ical stages of drug development. The majority of bioactive compounds describe d from ascidians are nitrogen containing metabolites and fall into the categories of peptides or amino acid derived alkaloids. However, they have given rise to a sm aller number of non-nitrogenous metabolites derived through diverse bioge netic pathways as well.51 O NH H2N OH HO HO H N O OH OH OH OH HO OH OH OH Tunichrome ( 41 ) Geranyl hydroquinone ( 42 )

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23 The tunicates have yielded a broad array of compounds with poten t cytotoxicity. The ecteinascidins ( 11 )11, 12 and didemnins9 ( 13 ) have already advanced into clinical trails as prospective anticancer drugs. Eudistomins, a group of -carboline alkaloids isolated from the colonial ascidian Eudistoma olivaceum are potent antiviral compounds.57-59 These compounds fall into five different general classes (Group 1: simple carbolines such as: eudistomin D ( 43 ), Group 2: pyrrolyl-carbolines such as eudistomin A ( 44 ), Group 3: pyrrolinyl-carbolines such as eudistomin G ( 45 ), Group 4: 2-phenylacetyl-carbolines such as eudistomin R ( 46 ) and Group 5: tetrahydro-carbolines such as eudistomin C ( 47 )). N H N Br HO Eudistomin D ( 43 ) N H N Br NH Eudistomin A ( 44 ) N H N Br O N H N Br N Eudistomin G ( 45 ) Eudistomin R ( 46 ) HO

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24 N H N HO Br S O H2N Eudistomin C ( 47 ) 2.12 Chemistry of the genus Synoicum Despite the fact that there is a growing interest in the chemistry of tunicates, out of many species of ascidians in the genus Synoicum only few have been subjected to chemical investigation. Studies on Synoicum prunum a colonial ascidian collected from North Stradbroke Island in Queensland, Australia, have yielded a series of tetraphenolic, bisspiroketals, prunolides A-C ( 48-50 ),60 which showed week cytotoxicity, along with rubrolide A ( 51 ).61 It is believed that prunolides ar ise from oxidative dimerization of a rubrolide precursor. O O Br OH Br Br HO Br O O O O O X RO X RO X X OR X OR X Y Y Prunolide A ( 48 ) R = H, X = Br, Y = Br Prunolide B ( 49 ) R = H, X = Br, Y = H Prunolide C ( 50 ) R = H, X = H, Y = H Rubrolide A ( 51 )

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25 The red colonial tunicate Synoicum blochmanni collected from Tarifa Island, Spain, afforded six new nitrogenous metabolites, rubrolides I N ( 51-56 ) along with four related known compounds. Rubrolides I, L, a nd M showed weak cytotoxicity.61 O O Br OH Br Br HO X O O OH Y HO X Br Rubrolide I ( 51 ) X = Cl Rubrolide K ( 53 ) X = Br, Y = Cl Rubrolide J ( 52 ) X = H Rubrolide M ( 55 ) X = Cl, Y = H Rubrolide L ( 54 ) X = Cl Rubrolide N ( 56 ) X = Br, Y = Cl Chemical investigation of the ascidian Synoicum macroglossum collected from the Indian Ocean near Tamilnadu, India, found the guanidino alkaloid, tiruchanduramine ( 57 ), which showed potent –glucocidase activity.62 N H N H O N H NH NH Tiruchanduramine ( 57 )

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26 2.1.3 Research Objectives Despite the enormous promise that the tunicate s have shown as a rich source of bioactive secondary metabolites, the chemistry of Anta rctic tunicates have not been completely explored. The unique ecological features found in Antarctica, and the dynamic interaction between these tunicates and other benthic orga nisms, could facilitate the development of biogenetic pathways leading to secondary metabolites with novel structural features. Therefore, it is of interest to study the chemistry of tu nicates found on the benthos of Antarctica. Figure 1. Synoicum adareanum at Palmer Station, Antarc tica (Photograph supplied by Bill J. Baker, University of South Florida)

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27 Synoicum adareanum is a colonial ascidian that is commonly found in the shallow water around Palmer Station, Antarctica. Its liphop hilic extract showed feeding deterrence towards the Antarctic sea star Odontaster validus indicating the presence of defense chemicals. Therefore, as part of our compre hensive chemical investigation of Antarctic tunicates, S. adareanum was studied to identify the sec ondary metabolites responsible for this activity. 2.2 RESULTS AND DISCUSSION 2.2.1 Extraction and isolation of secondary metabolites Synoicum adareanum tunicates were collected from the ocean near Anvers Island (64 46’ S, 64 03’W), Antarctica, in 2003. The freeze dried tunicates were extracted in CH2Cl2/MeOH for three days. After evaporating the solvent the resultant reddish-brown semi-solid was partitioned with EtOAc and water. The EtOAc layer was washed, dried and the solvent removed. The resultant EtOAc extract of the tunicat es was fractionated by flash chromatography to obtain 18 fractions (Scheme 1). Further separation of these factions with flash chromatography and subsequent purification by HPLC on C-18 afforded palmerolide A ( 58 ) (200 mg, 0.02% dry wt), palmerolide C ( 59 ) (4 mg, 0.0004% dry wt), palmerolide D ( 60 ) (2 mg, 0.0002% dry wt) and palmerolide E ( 61 ) (4 mg, 0.0004% dry wt) (Scheme 1). Fractions 8, 9 and 10, upon further purification by HPLC us ing 50% water in MeCN (isocrat ic elution, 2 mL per min) yielded palmerolide B ( 62 ) (2 mg, 0.0002% dry wt) and palmerolide H ( 63 ) (1 mg, 0.0001% dry wt).

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28Freeze dried Tunicates (520 g) 1. Extraction with CH2Cl2/MeOH 1:1 3 X 24 hrs 2. Evaporation of solvent 3. Partion with EtOAc/MeOH 4. EtOAc layer separated, washed and dried (anhydrous MgSO4) EtOAc Extract (3.1 g) Gradiant flash chrmatography over silica Hexane/ EtOAc / MeOH Fr 1, 2 ( 900 mg) Fr 3 (300 mg) Fr 4, 5 (310 mg) Fr 6, 7 (310 mg) Fr 8-10 (200 mg) Fr 11-18 (800 mg) Gradiant elution over silica gel 1-6% MeOH/CHCl3Gradiant elution over silica gel 1-20% MeOH/CHCl3Fr 40-48 Fr 54-60 Palmerolide A ( 58 ) (200 mg) Palmerolide C ( 59 ) (1.2 mg) Palmerolide B ( 62 ) (2 mg) PalmerolideH ( 63) (1 mg) HPLC ODS H2O : MeCN 4:6 Isochratic elution HPLC ODS H2O : MeCN 1:1 Isochratic elution Gradiant elution over silica gel 1-10% MeOH/CHCl3Fr-30-32 (20 mg) Fr 33-37 (9 mg) Fr 38-39 (9 mg) HPLC ODS H2O : MeCN 4:6 Isochratic elution HPLC ODS H2O : MeCN 4:6 Isochratic elution Palmerolide C ( 59 ) 3.5 mg Palmerolide E ( 61 ) 3.5 mg Palmerolide C ( 59 ) 3.5 mg Palmerolide D ( 60 ) 3.5 mg Palmerolide E ( 61 ) 3.5 mg Palmerolide D ( 60 ) 3.5 mg HPLC ODS H2O : MeCN 4:6 Isochratic elution Scheme 1. Extraction and purificatio n of the palmerolides

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29O O OH H N O HO O O NH2 7 10 11 19 20Palmerolide A ( 58 ) H N O O O OH OH O O NH2 1 2 3 4 6 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 27 22 23 24 1' 2' 3' 4' 5' 26 25Palmerolide C ( 59 ) O O OH H N O HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 27 26 25 1' 2' 3' 4' 5' 6' 7' 8' Palmerolide D ( 60 )

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30O O OH HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 27 26 25 O Palmerolide E ( 61 ) O O O H N O O S O O O NH2 HO OH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 27 26 25 1' 2' 3' 4' 5'Palmerolide B ( 62 ) O O O H N O O S O O O NH2 HO OH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 27 26 25 1' 2' 3' 4' 5' 7' 8' 6'Palmerolide H ( 63 )

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31 Compound 62 was first isolated from a 2001 collection of Synoicum adareanum. However it was found to be unstable in DMSO as it decomposed in the NMR tube while the 2D NMR data were being obtained. Theref ore, all the NMR experiments of fractions leading to palmerolides B and H were performed in CD3OD. 2.2.2 Characterization of Palmerolide A ( 58 ) Palmerolide A ( 58 ) was isolated as a white solid. The low resolution FABMS analysis displayed a prominent peak at m/z 585, which was assigned as [M + 1]+ (Figure 2). The HRFABMS provided a mol ecular formula of C33H48N2O7 (HRFAMBS m/z 585.3539, 0.1 mmu for [M + 1]+). Figure 2. LRFABMS spectrum of palmerolide A ( 58 ) The 13C NMR spectrum of compound 58 (Figure 3) displayed 33 carbon signals. These signals were further edited by DEPT 135 (F igure 4) and DEPT 90 experiments which

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32 established that palmerolide A is composed of five methyls, si x methylenes, sixteen methines and six quaternary carbon signals. The quaternary signals at 165.3, 156.6 and 163.1 were assigned as carbons at the oxidation state of a ca rboxylic acid, whereas the signals at 73.7, 72.5, 69.0 and 75.3 indicated hydroxymethines. Figure 3. 13C Spectrum of palmerolide A ( 58 ) (125 MHz, DMSOd6) 180 160 140 120 100 80 60 40 20 P C-1 C-1' OCONH2 C-3' C-3 C-8 C-22 C-14 C-17 C-21 C-9 C-15 C-24 C-2 C-2' C-23 C-19 C-7 C-11 C-10 C-18 C-6 C-20 C-4 C-13 C-12 C-4' C-5 C-5' C-26 C-25 C-27 135 130 125 120 115 PPM 40 35 30 25 20 15 PPM

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33 149.200 133.491 131.843 129.740 128.786 127.628 126.289 122.058 120.461 117.967 116.347 75.018 74.998 73.685 72.426 69.091 43.152 36.546 32.268 29.305 26.928 24.893 19.500 17.017 16.082 12.579 140 120 100 80 60 40 20 P P C-3 C-19 C-10 C-18 C-11 C-7 C-25 C-27 C-26 C-5` C-4` C-20 C-23 C-2` C-8 C-17 C-5 C-12/13 C-4 Figure 4. DEPT 135 Spectrum of palmerolide A ( 58 ) (125 MHz, DMSOd6) Analysis of the 1H NMR spectrum of palmerolide A ( 58 ) (Figure 5a), showed a series of proton signals in the region of 5-7 ppm (Figur e 5b), characteristic of olefins, confirming a high degree of unsaturation. The singlets (3H) at 1.61, 170, 1.83, 2.12 and the doublet 0.90 indicated five methyls (Figure 5c). A comp rehensive investigation of the structure of palmerolide A was undertaken using gHMQC and gHMBC techniques. The C-1 to C-24 carbon backbone of palmerolide A ( 58 ) could be unambiguously assigned based on 1H-13C assignments (Table 2) from the gHMBC spectrum (Figure 6), to establish a 20 member macrocycle. The ester carbonyl 166.1 (C-1) correlated with the H-2 and H-3 olefins, the latter of wh ich must be disposed trans ( J = 15.2 Hz).

PAGE 55

34 Figure 5. 1H NMR spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) Three methylene carbons ( 32.6, 24.8 and 38.3) were obser ved by both gCOSY (Figures 8, 9) and gHMBC between the C-2/C3 olefin and a hydroxymethine at 3.82 (H-7). A trans -disubstituted olefin ( J = 15 Hz) could be positioned between that hydroxymethine and another at 4.14 (H-10). While H-10 showed no HM BC correlations, H-8, -9 and -11 all correlated to C-10. H-11 was found correla ted not only with C-10 and C-12/13 (C-12 and -13 were coincident in the 13C NMR spectrum) but also w ith an ester-type carbonyl (OC OX) which displayed no further conn ectivity using th ese techniques. 10 8 6 4 2 0 P NH H-27 H-25 H-26 H-4` H-5` H-20 H-24 H-3 H-23 H-15 H-19 H-21 H-2` H-10 H-11 H-7 H-18 7.0 6.5 6.0 5.5 5.0 4.5 4.0 PPM 2.5 2.0 1.5 1.0 PPM(a) (b) (c)

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35 Table 2. NMR data of palmerolide A ( 58 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6)a 13C 1H (ppm, mult, J (Hz)) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 OCONH2 24-NH 10-OH 7-OH 165.3 120.3 149.2 32.6 24.8 24.8 38.0 38.0 72.5 133.6 128.9 69.0 72.3 29.5 29.5 29.5 132.0 126.1 125.7 131.5 43.6 43.6 73.7 36.9 129.7 132.5 116.4 121.8 12.6 16.9 16.2 163.1 117.9 151.7 27.1 19.2 156.6 5.77 (1H, d, 15.3) 6.71 (1H, ddd, 5.0, 9.9, 15.2) 2.11 (2H, m) 1.30 (1H, m) 1.05 (1H, m) 1.50 (1H, ddd, 4.5, 8.2, 11.2) 1.30 (1H, m) 3.82 (1H, ddd, 4.4, 7.4, 7.6) 5.54 (1H, dd, 7.7, 15.0) 5.48 (1H, dd, 2.9, 15.6) 4.14 (1H, br s) 4.48 (1H, dd, 5.0, 10.5) 1.59 (1H, m) 0.98 (1H, m) 1.96 (2H, m) 5.41 (1H, ddd, 4.7, 10.1, 14.6) 6.04 (1H, dd, 11.1, 14.6) 5.59 (1H, d, 11.4) 2.17 (1H, dd, 1.3, 13.2) 2.00 (1H, dd, 11.2, 13.2) 4.84 (1H, ddd, 1.3, 7.4, 11.2) 2.68 (1H, qdd, 6.5, 7.4, 9.6) 5.13 (1H, d, 9.6) 5.85 (1H, d, 14.2) 6.85 (1H, dd, 10.1, 14.2) 1.70 (3H, s) 0.90 (3H, d, 6.5) 1.61 (3H, s) 5.69 (1H, br s, 1.0) 1.83 (3H, s) 2.12 (3H, s) 6.49 (2H, br) 9.84 (1H, d, 10.1) 5.20 (1H, d, 4.9) 4.72 (1H, d, 3.9) 1, 4 1, 2, 4, 5 2, 3, 5, 6 7 6 5, 7, 8 5, 7, 8 5, 9 6, 7, 9, 10 7, 8, 10 9, 10, 12/13, OC ONH2 12/13 11, 12/13 12/13, 14, 15 12/13, 16 12/13, 16, 17 14, 15, 18, 25 8, 9, 11, 12/13 16, 17, 19, 25 16, 17, 19, 20, 25 1, 17, 18, 20, 21, 26 18, 19, 21, 22, 26 19, 20, 23, 26, 27 21, 22, 24, 27 22, 23, 1` 16, 17, 18 19, 20, 21 21, 22, 23 1 3 4 5 1 2 3 5 1 2 3 4 23, 24, 1 9, 10, 11 6, 7, 8 aProton carbon assignments based on gHSQC spectrum (Figure 7)

PAGE 57

36 10 8 6 4 2 PPM Direct Dimension 160 140 120 100 80 60 40 20 0 PPM Indirect Dimension 1 Figure 6. gHMBC spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6)

PAGE 58

37 140 120 100 80 60 40 20 PPM Indirect Dimension 1 7 6 5 4 3 2 1 0 PPM Direct Dimension Figure 7. gHSQC spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) In the gHMBC spectrum, H-13 was coupled back in to the olefinic region to C’s -14 and 15. The C-14/15 trans -olefin ( J = 14.6 Hz) was shown to be c onjugated to a trisubstituted olefin in positions C-16 and C-17 by gHM BC correlations of H-14, -15, -18 and -19 as well as H3-25. The C-16/C-17 olefin must be trans based on the correlations of the ROESY spectrum for H3-25 and H-15. The methylene group at C-18 ( 43.9) intervenes between the C-16/C-17 olefin and an oxygen bearing methine (C-19, 75.9), based on gHMBC correlations of H-16 and H3-25 to C-18 as well as H-19 and H-20 correlations to C-18. The macrocycle was completed by obs ervation of correlation between H-19 and the C-1 ester carbonyl.

PAGE 59

38 The features of the C20 macrocycle were established by extensive analysis of 2D NMR data. In addition to the four trans olefins described above, one methyl group and three oxygen atoms were pendant on the macrocycle Hydroxymethine protons at H-7 and H10 were conclusively assigned based on the observation of coupling of hydroxyl protons in both the gHMBC and gCOSY spectra; in the gHMBC spectrum, the hydroxyl protons correlated to the respective and -carbons, while the gHMBC correlations were observed between the hydroxyl protons and their respective hydroxylmethines. The third oxygen bearing carbon (C-11) as described earli er correlates with an ester-type carbonyl (OC OX). O O OH H N O HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1' 2' 3' 4' 5' gCOSY gHMBC Figure 8. Key gHMBC and gCOSY correlations of palmerolide A

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39 10 8 6 4 2 PPM Indirect Dimension 1 10 8 6 4 2 0 PPM Direct Dimension Figure 9. gCOSY spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) Also pendant on the macrocycle was the C-19 side chain. gHMBC and gCOSY correlations of the multiplet at 2.68 (H-20), which was clear ly coupled (gCOSY) to a methyl group (C-26, 0.90), could be extended to a c onjugated diene system based on gHMBC correlations of H-19 and H-20 to olefin C-21 ( 129.7). Both the C-21 / C-22 and C-23 / C-24 olefins were determined to be trans based, in the former case of by ROESY correlation between H3-27 and H-19, and in the latter case, J =14.2 Hz. Connectivity of the C-23/ C-24 olefin could be established based on gHMBC correlations of H-23 to C-21, -22, -24 and -27. C-24 marked the terminus of the continuous carbon

PAGE 61

40 chain and could be shown to bear an –NH group due to gHMBC correlations of a amide proton at 9.84 to carbons C-23, C-24 and an amide carbonyl, C-1 ( 163.1). The presence of amide group was confirmed by an 15N gHSQC experiment. The isopentenoyl substructure (C-1 to C-5 ) was unusual in displaying 4J CH coupling in the gHMBC spectrum between the amide carbonyl (C-1 ) and both vinyl methyl groups (C-4 and C-5 ). Only one vinyl methyl can be placed within the 3J CH reach of the typical HMBC experiment optimized for 8 Hz. Presence of another alternativ e structure, the 2methyl-2-butenoyl isomer, wh erein one vinyl met hyl resides three bonds away from the carbonyl and the second resides four bonds di stant, is consistent with these HMBC correlations. However, it was rules out on ch emical shift grounds and by the observations of gHMBC correlations between both vinyl methyls. The substructure was secured as the isopentenoyl group by observation of very small coupling constants ( J = 1.0 Hz) of the vinyl proton (H-2 ) to both vinyl methyl groups (C-4 and C-5 ), excluding a vicinal relationship between the vinyl proton and th e vinyl methyl required by the 2-methyl-2 butenoyl isomer. The isopentenoyl structure could be connected to the vinyl proton at 6.82 (C-24) based on gHMBC cross peak between amide carbonyl (C-1 ) and H-24. The above described connectivity established the full planer structure of palmerolide A (Figure 10) with the exception of a single ope n valence, the ester-t ype carbonyl attached to the macrolide at C-11. Remaining to be accounted from the known molecular formula was –NH2. That the C-11 functional group was a carbamate is supported by the precedence of that functional gr oup on other polyketides, mo st notably the anticancer agent discodermolide ( 7 ).23

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41O O OH H N O HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 26 26 25 1' 2' 3' 4' 5' Figure 10. Plainer structure of palmerolide A ( 58 ). 2.2.3 Stereochemical determination Stereochemical assignment of the five stereo centers of palmerolide A was established by application of the modified Mosher’s63 and Murata64 methods. Further support for stereochemistry assignment was obtaine d by ROESY and selective 1D NOESY experiments. 2.2.3.1 Application of Mosher’s method Mosher’s method has been a very successf ul method to determine the absolute stereochemistry of chiral centers associated with secondary alcohols.64 Out of the five stereocenters in palmerolide A ( 58 ), C-7 and C-10 both have secondary alcohols making them suitable candidates to employ Mosher’s method. Palmerolide A ( 58 ) was treated with both R and S -MTPACl separately and the reaction was monitored by TLC. It was observed that in each case two products were formed. In each case, the formation of the first product, which was the major product, was observed

PAGE 63

42 after one hour and the formation of the s econd product was noticed after 16 hours. This can be explained as due to the substituti on of one secondary hydroxyl in palmerolide A giving rise to R and S monoesters ( 64 and 65 ) initially, and then subsequen tly substitution of the other secondary hydroxyl giving rise to R and S diesters ( 66 and 67 ). The products 64 and 65 were less polar than 66 and 67 in agreement with above explanation. Each product was isolated by chromatogr aphy on silica gel and purified by HPLC. A comprehensive structure elucidation was undert aken of each of these products using 2D NMR and mass spectroscopy. 2.2.3.1.1 R -MTPA monoester of palmerolide A ( 64 ) Analysis of the more polar product 64 by ESIMS showed a mass peak at m/z = 823.1 representing [M + Na]+. The HRESIMS experiment performed on this ion showed a molecular weight of 823.3776 amu consis tent with molecular formulae of O O OR1 H N O R2O O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 27 26 25 1' 2' 3' 4' 5' 64 R1 = R -MTPA, R2 = H 65 R1 = SMTPA, R2 = H 66 R1 = R -MTPA, R2 = R -MTPA 67 R1 = S -MTPA, R2 = S -MTPA

PAGE 64

43 C43H55N2O9F3Na suggesting the incorporation of one MTPA group. This explains the more polar nature and the fa ster rate of formation of 64 as compared to the disubstituted product 66 The 1H NMR spectrum (Figure 11) indica ted significant chemical shift changes around C-7 (Figure 12). The multiplets at 7.51 and 7.43 represented the MTPA phenyl group whereas the singlet at 3.63 indicated the OCH3 group. The multiplet 5.47 assigned for H-7, show a significant deshie lding accounting for an esterification at C-7. The observation of a gHMB C correlation between H-7 and 165.9 assigned as the carbonyl carbon of the MTPA gr oup, confirmed the attachment of MTPA at C-7, giving rise to palmerolide A R -MTPA monoester ( 64 ). 7.515 7.500 7.437 7.427 6.957 6.812 6.098 5.793 5.769 5.730 5.464 5.182 4.667 4.590 4.328 3.535 2.756 2.180 1.905 1.804 1.677 0.983 0.969 7 6 5 4 3 2 1 PP M Figure 11. 1H NMR spectrum palmerolide A R -MTPA monoester ( 64 ) (500 MHz, CD3OD)

PAGE 65

44 O OH H H O H H H H H H H O MeO Ph CF3 NH2 O 1.40 1.82 1.30 31.9 1.64 165.85 5.45 4.34 4.68 158.64 32.16 5.76 5.97 70.16 134.89 128.32 76.25 H H 1.63 30.3 78.13 Figure 12. Key gHMBC correlations demonstrati ng the attachment of the MTPA moiety in compound ( 64 ) 2.2.3.1.2 R, R -MTPA diester of palmerolide A ( 66 ) The less polar product ( 66 ) from the reaction of palmerolide A ( 58 ) with MTPA-Cl showed a m/z = 1039.1 in the ESIMS, c onsistent with [M + Na]+. HRESIMS indicated a molecular weight of 1039.4145 amu, consis tent with molecular formula of C53H62N2O11F6Na and indicative of incorporat ion of two MTPA moieties. The 1H NMR spectrum (Figure 13) indicated significant ch emical shift changes around C-7 and C-10 (Figure 14). 7 .4 6 7 7 .4 5 5 7 .3 5 6 7 .3 4 2 6 .9 6 3 6 .7 7 4 6 .1 2 2 5 .9 5 0 5 .7 6 1 5 .7 3 2 5 .6 5 4 5 .5 4 8 5 .4 3 4 5 .1 7 6 4 .9 3 9 4 .5 9 2 3 .5 6 9 3 .4 7 9 2 .7 7 8 2 .1 8 4 1 .9 1 1 1 .8 0 5 1 .6 7 4 0 .9 7 1 7 6 5 4 3 2 1 PP M Figure 13. 1H NMR spectrum of palmerolide A R R -MTPA diester ( 66 ) (500 MHz, CD3OD)

PAGE 66

45 The multiplets at 7.46 and 7.35 could be assigned as the MTPA phenyl group whereas the two singlets at 3.47 (3H) and 3.56 (3H) were due to the two OCH3 groups of two MTPA moieties. H-7 and H-10 both displaye d significant deshieldi ng demonstrating the formation of ester bonds. A spectroscopic i nvestigation using gHMBC COSY and HSQC experiments around C-7 and C-10 confirmed the attachment of two MTPA molecules at C-7 and C-10 to give rise to palmerolide A R R -MTPA diester ( 66 ). O O H H O H H H H H H H O MeO Ph CF3 CF3 MeO Ph O NH2 O 1.12 1.64 1.29 29.5 1.49 165.38 5.53 5.55 4.83 158.30 75.63 5.56 5.73 131.80 128.00 165.61 75.80 72.06 33.2 H H 1.42 1.10 31.2 Figure 14. Key gHMBC correlations demons trating the attachment of MTPA in compound ( 66 ) 2.2.3.1.3 S -MTPA monoester of palmerolide A ( 65 ) The HRESIMS of 65 showed a molecular weight of 823.3747 amu consistent with molecular formula of C43H55N2O9F3Na, affirming the substitu tion of one MTPA moiety. In addition, it explained the more polar natu re and the faster rate of formation of 65 as compared to the disubstituted product 67 The 1H NMR spectrum (Figure 15) indicated significant chemical shift changes around C-7 (Figure 16). The multiplets at 7.41, 7.44, 7.50 and 7.60 can account for the aromatic proton s of the MTPA moieties and the singlet

PAGE 67

46 at 3.55 was due to the OCH3 group of MTPA moiety. H-7 showed a significant deshielding demonstrating the formation of an ester linkage. In addition it displayed gHMBC correlation with 165.8 assigned for the ester carbonyl of MTPA moiety, further accounting for the attachme nt of a MTPA molecule at C-7. 7.60 4 7.501 7.449 7.416 7.406 6.968 6.846 6.108 5.962 5.930 5.815 5.737 5.685 5.459 5.185 4.655 4.482 4.320 3.553 2.771 2.19 4 1.919 1.816 1.688 0.992 7 6 5 4 3 2 1 PP M Figure 15. 1H NMR spectrum of palmerolide A SMTPA monoester ( 65 ) (500 MHz, CD3OD) O OH H H O H H H H H H H O MeO Ph CF3 NH2 O 1.46 1.82 1.30 24.5 1.71 165.5 5.45 4.32 4.65 158.64 34.34 5.67 5.92 70.16 134.50 128.32 76.02 H H 1.15 1.63 30.3 Figure 16. Key gHMBC correlations demonstrati ng the attachment of the MTPA moiety in compound 65

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47 2.2.3.1.4 S, S MTPA diester of palmerolide A ( 67 ) The less polar product from esterification of palmerolide A ( 58 ), 67, showed a prominent mass peak at m/z = 1039.1 in the ESIMS i ndicative of [M + Na]+. HRESIMS indicated a molecular weight of 1039.4151 amu that was consistent with molecular formula of C53H62N2O11F3Na, indicative of incorporation of two MTPA moieties. The 1H NMR spectrums (Figure 17) indicated significant chemical shift changes around C-7 and C-10 (Figure 18). The multiplets at 7.61, 7.50, 7.43 and 7.39 represented the aromatic protons of the MTPA moieties wh ereas the two singlets (3H) at 3.41, 3.57 indicated the two OCH3 groups of two MTPA groups. H-7 and H-10 both displayed significant deshielding due to the formation of ester bonds that attach two MTPA groups at C-7 and C-10 in order to form palmerolide A S, S -MTPA diester ( 67 ). 7.610 7.502 7.435 7.395 6.962 6.811 6.093 5.955 5.791 5.729 5.675 5.614 5.307 5.179 3.573 3.418 2.779 2.189 1.913 1.808 1.672 0.990 0.977 7 6 5 4 3 2 1 PP M Figure 17. 1H NMR spectrum of palmerolide A S, SMTPA diester ( 67 ) (500 MHz, CD3OD)

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48 O O H H O H H H H H H H O MeO Ph CF3 CF3 MeO Ph O NH2 O 1.40 1.68 1.27 24.5 1.69 165.38 5.54 5.62 4.71 157.55 75.02 5.53 5.69 131.80 128.00 165.61 75.02 72.06 33.2 H H 1.40 1.24 Figure 18. Key gHMBC correlations demonstr ating the attachment of the MTPA in compound ( 67 ) 2.2.3.1.5 Absolute stereochemistry assi gnment at C-7 of palmerolide A ( 58 ) According to Mosher’s method,63 the chemical shifts of all the neighboring protons in the either side of the desired chiral center of both R and S -MTPA monoesters of palmerolide A ( 58 ) were assigned as shown in previous secti ons. In the next step, for each proton, the value of the chemical shifts were calcula ted employing following formula: S – R = values were placed on the model designe d as described in Mosher’s method (Figure 19) so that all positive chemical shift differences are assigned to the right hand side of the MTPA plane, whereas all negative chemical shift difference are assigned to the left hand side. H H H H H H OH OCONH2 H MTPAO H H -10 -20 -50 -90 0.00 +70 +60 Figure 19. Chemial shift differences ( 1000) of palmerolide A MTPA Rand S monoesters

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49 Once a model is built satisfying all requi rements described in Mosher’s method,63 the model (Figure 20) should have the absolute st ereochemistry of the desired chiral center involving a secondary hydroxyl group. Therefor e, in the case of palmerolide A the absolute stereochemistry at C-7 was assigned as R OMe Ph CF3 O O ! H ! ! HAHBHCHZHYHX (OMe) (Ph) RMTPA SMTPA MTPA Plane !C OMTPA H >O
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50 2.2.3.2 Application of Murata’s method64 Matura’s method is based on configurati on analysis using he teronuclear coupling constants. It has been successfully applied to the stereochemical elucidation of number of natural products.64 The gHSQMBC experiment based on this principle has proven as a very successful method to determin e stereochemistry of macrolides.65 Hence, a gHSQMBC experiment was acquired using a co ncentrated sample of palmerolide A ( 58 ) (Figure 22). Heteronuclear c oupling constants were obtaine d by analyzing the coupled proton and carbon signals in the correspondin g 1D spectrum (Figure 23). Based on the magnitude of the coupling constants the st ereochemical relationships between the respective atoms are determined. 10 8 6 4 2 PPM Direct Dimension 180 160 140 120 100 80 60 40 20 0 PPM Indirect Dimension 1 Figure 22. gHSQMBC spectrum of plamerolide A ( 58 ) (500 MHz, DMSOd6)

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51 In order to determine the heteroneuclear coupling constants, for each gHSQMBC cross peak, the 1D gHSQMBC slice was extracted. The heteroneuclear coupling constant was derived by the subtraction of the 1H NMR spectrum from itself, offset by the equivalent magnitude of the coupling constant, to pr oduce a difference spectrum which matched the gHSQMBC slice. 2JC-12/ H-10 Figure 23. 1D analysis of the gH SQMBC cross peak of the respective slice Figure 24. Determination of the coupli ng constant by the subtraction of 1H NMR spectrum; Offset = 2JC-12 /H-10

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52 Configuration analysis of fragment C-10 /C -11 showed a gauche relationship between H10 and H-11, based on the small 3JH-10/ H-11 observed between the vi cinal protons and the large 3JCH for both the H-10/C-12 a nd H-11/C-9 relationships. Further support for the conformation was found in 2JC-11/H-10 and 2JC-10/H-11 both of which are large and negative defining the absolute stereochemistry of C-11 as R .64 Similarly, configurational analysis of C-19/C-20 system suggested an anti relationship for the respective protons, based on the large 3JH-19/H-20, small 3JC-21/H-19 and 3JC-18/H-20 as well as large 2JC-19/H-20. The relative position of C-18 wa s secured by an observation of ROESY correlations (Figures 25, 26) between H2-18 as well as H-20 and H3-26 while no ROESY correlation was observed between H2-18 and H-21 requiring the relative configuration 19 R 20 S C9 O H10 H11 O C12 H H H20 C26 C21 H19 O C18 H H H ROESY correlations 3JC-12/H-10 = 6.9 Hz Large 3JC-21/H-19 = 3.4 Hz Small 3JC-9/H-11 = 5.2 Hz Large 3JC-18/H-20 = 2.9 Hz Small 2JC-11/H-10 = -6.2 Hz Large 3JC-26/H-19 = 3.9 Hz Small 2JC-10/H-11 = -4.8 Hz Large 2JC-19/H-20 = -5.9 Hz large Figure 25. Coupling c onstant based configuration analysis64

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53 10 8 6 4 2 PPM Direct Dimensio n 10 8 6 4 2 0 PPM Indirect Dimension 1 Figure 26. ROESY spectrum of palmerolide A ( 58 ) (500 MHz, DMSOd6) The four olefins in the macrocycle constrai n the flexibility often found in macrolides, facilitating stereochemical analysis by NO E studies. Further analysis of the ROESY spectrum (Figure 26) revealed th e macrolide to adopt two largely planer sides of a tear drop shaped cycle, one side consisting of C-1 through C-6, the other C-11 through C-19, with C-7 through C-10 providing a curvilin ear connection. In particular, H-9, H3-27, H15 and H2-13 are sequentially correlated in the ROESY Spectrum, as are H3-26, H2-18, H-16, H-14 and H-12, defining the periphery of the top and bottom face of the western

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54 hemisphere (Figure 27). H-11 correlates onl y to the top series of protons, a result consistent only with C-19 and C-11 both adopting the R configuration. The absolute stereochemistry of the C-19/C-20 frag ment is therefore assigned as 19 R 20 S (Figure 28). Figure 27. Key ROESY correlati ons supporting the stereochem istry determination of C19 and C-20. O O OH H N O HO O O NH2 7 10 11 19 20 Figure 28. Full stereochemical assignment of palmerolide A ( 58 ) The presence of type 1 pol yketides like palmerolide A ( 58 ), is not a common feature among tunicates. Palmerolide A, bearing unu sual 20-membered macrolide, display functionality more commonly found in sponge s or cyanobacteria than the secondary metabolites generally found in tunicates. For instance, the vinyl amide is a feature very commonly associated with cyanophyte de rived macrolides such as tolytoxin.

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55 2.2.4. Characterization of palmerolide C ( 59 ) Palmerolide C ( 59 ), was isolated as a yellow solid and showed a molecular ion at m / z 585.3 in the low resolution LRESIMS. The HRESIMS (Figure 28) showed that it was consistent with the molecular formula of C33H49N2O7, identical to that of palmerolide A ( 58 ). Figure 29. Palmerolide C ( 59 ) HRESIMS spectrum Analysis of the 13C NMR spectrum (Figure 29) with DEPT 135 (Figure 30) suggested that palmerolide C ( 59 ) resemble palmerolide A further by having five methyls, six methylenes, sixteen methines and six quatern ary carbon signals. In fact, out of the six methines, four were assigned to hydroxy methines as found in palmerolide A ( 58 ).

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56 Figure 30. 13C NMR spectrum of palmerolide C ( 59 ) (125 MHz, DMSOd6) 149.825 129.082 127.402 123.052 122.199 118.978 74.563 72.982 31.840 28.753 27.948 20.527 18.118 16.812 13.585 140 120 100 80 60 40 20 PP M C-25 C-26 C-27 C-9 C-8 C-10 C-19 C-3` C-2` C-4` C-5` Figure 31. DEPT 135 spectrum of palmerolide C ( 59 ) (125 MHz, DMSOd6) 160 140 120 100 80 60 40 20 P C-1 C-1' OCONH2 C-3' C-3 C-18 C-20 C-25 C-27 C-26 C-5' C-4' C-11 C-12/13 C-4 C-5 C-2' C-2 C-23 C-24 C-15 C-16 C-21 C-6 C-7 C-14 C-17 C-22 C-8 C-10 C-9 C-19 135 130 125 120 PPM 50 45 40 35 30 25 20 15 PPM(a) (b) (c)

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57 Interpretation of HMBC (Figure 32) and COSY (Figure 33) spectrum indicated that palmerolide C ( 59 ) is also composed of a 20 member macrocyclic ring with a side chain (Figure 34). The structure of the side chain was the same as that of palmerolide A ( 58) however, there were significant rearrangements of the structural features in C-3 through C-13 in the macrocyclic ring. 10 8 6 4 2 PPM Direct Dimension 180 160 140 120 100 80 60 40 20 0 PPM Indirect Dimension 1 Figure 32. gHMBC spectrum of palmerolide C ( 59 ) (500 MHz, DMSOd6)

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58 10 8 6 4 2 0 PPM Indirect Dimension 1 10 8 6 4 2 0 PPM Direct Dimension Figure 33. gCOSY spectrum of palmerolide C ( 59 ) (500 MHz, DMSOd6) The doublet 5.73 (1H, J = 15.5 Hz) assigned to H-2 (Figure 35) showed HMBC coupling to ester carbonyl (C-1) 166.9 and methylene at 31.7 (C-4). The multiplet at 6.77 (H-3) correlated to C-1 and a methine at 149.8 (C-2). All these observations were consistent with a trans olefin conjugated to an ester carbonyl as found in palmerolide A. H-3 showed further HMBC correlation to tw o methylenes, C-4 and C-5 (Table 3), indicating the extension of the carbon backbone by two met hylenes after the double bond. The COSY correlations of H-5 and H-6 a nd HMBC correlation of H-6 to C-5 and -7 confirmed this observation.

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59 H N O O O OH OH O O NH2 1 2 3 4 6 5 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 25 22 23 24 1' 2' 3' 4' 5' 26 27 gCOSY gHMBC Figure 34. Key gHMBC and gCOSY correlations of palmerolide C ( 59 ) 10 8 6 4 2 0 PP M NH2 H-26 H-5` H-4` H-27 H-25 H-24 H-3 H-15 H-23 H-16 H-21 H-19 H-20 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 PPM 7.0 6.5 6.0 5.5 5.0 PPM9-OH 8-OH H-18b H-18a H-14 H-6/7 CONH 2Figure 35. 1H NMR spectrum of compound ( 59 ) (500 MHz, DMSOd6)

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60 7 6 5 4 3 2 1 PPM Direct Dimension 140 120 100 80 60 40 20 0 PPM Indirect Dimension 1 Figure 36. gHMQC spectrum of palmerolide C ( 59 ) (500 MHz, DMSOd6) Based on the gCOSY and gHMBC correlati ons of H-8, H-9 and H-10, the three hydroxymethines C-8, C-9 and C-10 were posit ioned between the C-7 and C-11. Further H-10 was correlated to an ester-type carbonyl (OCOX). COSY correla tions of H-11a, and H-11b with H2-12 and that of H2-12 with H-13a and H-13b established the connectivity between methylenes C-11, C-12 and C-13. The latter two showed overlapping carbon signals in the carbon spectrum.

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61 Table 3. NMR data of palmerolide C ( 59 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 13C 1H (ppm, mult, J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 OCONH2 24-NH 8-OH 9-OH 166.9 122.0 149.8 31.7 31.7 32.2 32.2 131.8 131.1 72.8 75.6 74.2 28.7 28.7 30.1 30.1 30.1 132.3 127.2 128.8 132.5 44.1 44.1 74.7 37.4 130.5 133.3 117.2 122.8 13.3 17.8 16.5 164.0 118.8 152.5 27.4 20.3 5.73 (1H, d, 15.5) 6.77 (1H, ddd, 7.4, 7.5,15.4) 1.30 (1H, m) 2.13 (1H, m) 1.89 (1H, m) 1.98 (1H, m) 5.54 (1H, m) 5.58 (1H, m) 3.96 (1H, m) 3.56 (1H, m) 4.56 (1H, ddd, 3, 7.5, 10.5) 1.30 (1H, m) 1.49 (1H, m) 1.95 (1H, m) 1.90 (1H, m) 1.99 (1H, m) 5.46 (1H, ddd, 5, 10, 15) 6.08 (1H, dd, 12, 15) 5.63 (1H, d, 11) 2.07 (1H, m) 2.18 (1H, m) 4.85 (1H, ddd, 2.8, 7.8, 10.5) 2.70 (1H, qdd, 6.7, 6.7, 9.8) 5.15 (1H, d, 9.5) 5.85 (1H, d, 14.5) 6.85 (1H, dd, 9, 14.6) 1.69 (3H, s) 0.90 (3H, d, 7) 1.59 (3H, s) 5.68 (1H, s) 1.82 (3H, s) 2.11 (3H, s) 6.37 (2H, br) 9.85 (1H, d, 10) 4.62 (1H, d, 5) 4.72 (1H, d, 4.5) 1, 4 1, 2, 4, 5 2, 3. 5, 6 2, 6 6, 7 3, 7 5, 7 6, 8 6, 7, 9 10 11, OC ONH2 10, 12 10, 12 10 15 15 13, 15,16 13, 14, 16, 17 14, 15, 25 16, 17, 19, 25 16, 17, 19, 20, 25 1, 17, 18, 20, 21, 26 16, 19, 21, 22, 26 19, 20, 23, 26, 27 21, 22, 24, 27 22, 23, 1` 16, 17, 18 19, 20, 21 21, 22, 23 1 3 4 5 1 2 3 5 1 2 3 4 23, 24, 1 7, 8, 9 8, 9, 10

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62 The remaining portion of the carbon backbone of the macrocycle was identical to that of palmerolide A ( 58 ). Connectivity of double bond C-14/ C-15 to C-13 and was established by COSY correlation between H-13b and H-14 and the coupling observed in the HMBC between H-14 and H-15 with C13. A large coupling constant ( J = 15 Hz) indicated that H-14 and H-15 have a trans relationship. This olefin was conjugated to a trisubstituted double bond based on HMBC correlation of H16 with carbons C-14, C-15 and C-25. H325 showed HMBC correlations with C-16, C-17 and C-18, consistent with this assignment. H-19 demonstrated HMBC correla tions with C-1, C-17 and C-18 completing the 20 memebered macrocycle. The side chain of palmerolide C ( 59 ) showed features similar to that of palmerolide A ( 58 ). The doublet at 0.90 (H3-26) showed coupling in th e COSY spectrum with H-20. In addition, this methyl group displaye d gHMBC correlation to C-19, C-20 and C-21, confirming, that it is located between C19 and C-21 and attached to C-20. Meanwhile, olefinic methine H-21 was correlated to another olefinic methine C-23 and C-27 in the gHMBC spectrum. However, evidence from the gHSQC spectrum showed that the latter carbon belongs to the vinylic methyl H3-27. Based on the coupling constant ( J = 14 Hz) the relationship between olefin ic protons H-23 and H-24 was trans These correlations are consistent with a trisubstituted double bond conjugated to a trans olefin at C-23/C-24. The doublet 9.85 representing N-H, which was coupled to H-24 as established by gCOSY, displayed HMBC correlations to C-23, C-24 and C-1 The latter assigned to a carbonyl. The gHMBC relationshi p of broad singlet H-2 with C-1’ and the unusual 4JCH

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63 HMBC coupling of two vinyl methyl groups C-4 and C-5 as observed in palmerolide A suggested an isopentenoyl connected to the amide described earlier. These data established the full planer structure of palmerolide C ( 59 ) with the exception of a single open valence, the ester carbonyl at tached to the macrolide at C-10. Remaining to be accounted from the establis hed molecular formula was an -NH2, suggesting the presence of a carbamate group as it was observed in the case of palmerolide A ( 58 ). 2.2.5 Characterization of palmerolide D ( 60 ) Palmerolide D ( 60 ) which was separated as a ye llow solid, showed a [M + 1]+ peak at m/z 625.6 in the low resonance ESIMS spectrum. Analysis by HREISMS (Figure 37) indicated a molecular formula of C36H53N2O7 (625.3864, calc 625.3853). The 13C NMR spectrum of ( 60 ) (Figure 38), taken with the DEPT 135 spectrum (Figure 39) showed the presence of fi ve methyls, eights methylenes sixteen methines and seven quaternary carbon signals. 2D NMR investigation (Figure 40) of palmerolide D ( 60 ) indicated that it has a macrocyclic ring similar to that of palmerolide A ( 58 ). The quaternary carbon at 166.1 was assigned as an ester ca rbonyl (C-1) and was correlated to olefinic protons H-2 and H-3 by gHMBC (Figure 41). These two protons showed a coupling constant of 15.8 Hz indicating a trans double bond.

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64 Figure 37. HRESIMS spectrum of palmerolide D ( 60 ) Figure 38. 13C NMR spectrum of palmerolide D ( 60 ) (125 MHz, DMSOd6) 180 160 140 120 100 80 60 40 20 P C-1 C-1' OCONH2 C-3' C-3 C-5' C-8 C-14 C-17 C-21 C-16 C-15 C-6 C-24 C-2 C-2' C-23 C-6' C-27 C-25 C-26 C-7' C-8' C-12 C-4 C-20 C-6 C-18 C-4' C-9 C-15 135 130 125 120 115 PPM 45 40 35 30 25 20 15 PPM(a) (b) (c)

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65 134.216 129.585 128.397 122.728 121.214 120.292 112.552 79.782 75.810 73.173 69.907 40.872 38.386 33.020 30.089 30.083 24.772 22.582 17.746 16.872 16.848 13.318 10.141 140 120 100 80 60 40 20 PP M C-25 C-4` C-10 C-7 C-11 C-19 C-4 C-5 C-26 C-27 C-7` C-8` C-6` C-8 C-16 C-2` Figure 39. DEPT 135 spectrum of palmerolide D ( 60 ) (125 MHz, DMSOd6) O O OH H N O HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1' 2' 3' 4' 5' 6' 7' 8' gCOSY gHMBC Figure 40. Key gHMBC and gCOSY co rrelations of palmerolide D ( 60 )

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66 10 8 6 4 2 PPM Direct Dimension 180 160 140 120 100 80 60 40 20 PPM Indirect Dimension 1 Figure 41. gHMBC spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6) Three methylenes were positioned between C-3 and hydroxymethine C-7 based on gHMBC correlation of H-4 with C-5, H-5 with C-3 and that of H-5 and H-7 with C-6. In addition, H-7 displayed HMBC resonance w ith C-8 and C-9, assigned as olefinic methines (Figure 43) indicating the pres ence of a double bond attached to C-7. The location of two hydroxymethines C-10 and C-11 adjacent to each other was established

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67 by the COSY correlations (Fi gure 42) of H-10 and H-11 and further confirmed by HMBC correlations observed for H-11 with C-9 a nd C-10. In addition, H-11 showed HMBC correlations to the quaternary carbon at 156.4 which was assigned as an ester-type carbonyl carbon. 10 8 6 4 2 0 PPM Indirect Dimension 1 10 8 6 4 2 0 PPM Direct Dimension Figure 42. gCOSY spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6)

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68 7 6 5 4 3 2 1 PPM Direct Dimension 140 120 100 80 60 40 20 0 PPM Indirect Dimension 1 Figure 43. gHMQC spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6) The two methylenes C-12 and C-13, appeared overlapped with each other as a one carbon signal in the 13C NMR spectrum. Their linkage with hydroxymethine C-11 and olefinic methine C-14 was established by gHMBC co rrelations with H-10, H-11 and H-14. Based on the HMBC spectrum it was evident that the trans ( J = 14.6 Hz) double bond (C-14 and C-15) is conjugated to a trisubstituted double bond (C-16 and -17). The methylene C18 was positioned between the hydroxymethin e C-19 and quaternary carbon C-17 based on gHMBC correlation of H-19, observed with C-18 and C-17. The 20 member macrocylce was completed by the observati on of HMBC cross peak identified for H-19 and C-1.

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69 The doublet at 0.89 in the 1H NMR spectrum (Figure 44) was assigned as a methyl (H326) and showed COSY coupling to H-20. In addition, this methyl group displayed gHMBC correlation to C-19, C-20 and C-21 c onfirming that it is located between C-19 and C-21 attached to C-20. Meanwhile, H-21 representing an olef inic methine was correlated to another olefinic methin e C-23 and C-25 by HMBC. However, HSQC evidence showed that the latter carbon belong to the vinylic methyl H3-27. Based on the coupling constant ( J = 14.6 Hz) the realtionship between olefinic protons H-23 and H-24 was identified as trans These correlations are consistent with a trisubstituted double bond conjugated with a trans olefin at C-23/C-24. The doublet 9.85 representing N-H, which was coupled to H-24 by gCOSY, disp layed gHMBC resonanc e with C-23, C-24 and C-1`. 10 8 6 4 2 0 PP M NH2 C-24 C-3 H-15 H-6` H-21 H-2 H-23 H-9 H-26 H-25/H-7` H-25 H-8` H-20 7.0 6.5 6.0 5.5 5.0 PPM 2.5 2.0 1.5 1.0 PPM Figure 44. 1H NMR spectrum of palmerolide D ( 60 ) (500 MHz, DMSOd6)

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70 Table 4. NMR data of palmerolide D ( 60 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 13C 1H (ppm, mult, J (Hz)) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 6 7 8 OCONH2 24-NH 7-OH 10-OH 166.1 121.2 150.0 33.0 33.0 30.1 38.4 38.4 73.2 134.2 129.6 69.9 75.8 30.0 30.0 133.6 127.0 128.4 132.2 43.9 43.9 74.5 37.3 130.7 133.3 117.5 122.7 16.8 17.7 13.3 163.5 120.3 153.2 40.8 143.6 112.6 22.7 24.8 157.4 5.76 (1H, d 15.8) 6.71 (1H, ddd 4, 11.5, 15.7) 2.11 (1H, m) 2.15 (1H, m) 5, 1.98 (1H, m) 1.30 (1H, m) 1.48 (1H, m) 3.82 (1H, m) 5.53 (1H, m) 5.49 (1H, m) 4.15 (1H, m) 4.48 (1H, m) 1.94 (2H, m) 1.94 (2H, m) 5.41 (1H, ddd 10, 14.9) 6.04 (1H, dd, 11.6, 14) 5.59 (1H, d, 12) 2.16 (1H, m) 2.00 (1H, m) 4.84 (1H, m) 2.68 (1H, m) 5.14 (1H, d, 9.7) 5.86 (1H, d, 14.6) 6.85 (1H, dd, 10.4, 15 ) 1.60 (3H, s) 0.89 (3H, d, 6,7) 1.70 (3H, s) 5.81 (1H, s) 3.34 (2H, s) 4.72 (2H, d, ) 1.61 (3H, s) 1.76 (3H, s) 6.45 (2H, br) 9.94 (1H, d, 10.3) 4.53 (1H, m) 5.19 (1H, m) 1, 3, 4 1, 2, 4 2, 3, 5 2, 3 3, 4 3, 6 6, 8, 9 7, 9, 10 7, 8, 11, 12 8. 9. 12 9, 10, 13 OC ONH2 13, 15, 16 14, 16 14, 15, 25 15, 16, 18, 25 16, 19, 25 16. 19 17, 18, 20, 26 18, 19, 21, 26 19, 20, 26, 27 21, 23, 27 21, 24, 27 22, 23, 1` 15, 16, 18 19, 20, 21 21, 23 1 4 7 2 3 5 7 4 8 4 5 6 2 3 4

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71 From C-1 onwards the side chain showed furt her modification by the elongation of the C-1 C-5 moiety in palmerolide A ( 58 ). The quaternary carbon at 153.2 (C-3 ) and methine at 120.3 assigned for C-2 showed distinct gHMBC correlation with the proton signal at 3.34 a methylene, by the analysis of gHMQC and DEPT 135 spectra. This peak further displays gHMBC correlation to quaternary carbons at 143.6 (C-5 ) and 112.6 (C-6 ), further defining an extended side chain. The carbon signal at 112.6 (C-6 ) was found to be an exomethylene based on its HMQC correlation to the proton signal at 4.72, which integrates for two hydrogens. A ttachment of the vinylic methyl at 1.61 to C-5 was based on gHMBC correlations with C-4 C-5 and C-6 The above discussed correlations established the full planer structure of palmerolide D (Figure 45) with the exception of a single open valence, the ester type carbonyl attached to the macrolide at C-11. As it was previously observed in respect of palmerolides A and C, to be accounted from molecular fo rmula obtained by HREIMS, was an –NH2, suggesting the presence of a carbamate group. Figure 45. Planer structure of palmerolide D ( 60 ) O O OH H N O HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1' 2' 3' 4' 5' 6' 7' 8'

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72 2.2.6 Characterization of palmerolide E ( 61 ) Palmerolide E ( 61 ), isolated as a pale yellow solid, displayed a [M + Na]+ peak at m/z 512 in the LRESIMS spectrum. Analysis by HRESIMS (Figure 46) afforded the molecular formula of C27H39NO7Na ( m/z 512.2634, calc 512.2624). Figure 46. Palmerolide E ( 61 ) HRESIMS spectrum 2D NMR study (Figure 47) of palmerolide E ( 61 ) indicated that it has a 20 member macrocyclic ring similar to that of palmerolide A ( 58 ). However, the side chain attached to C-19 was shorter with only one ol efinic group. The sharp singlet at 9.41 in the 1H NMR spectrum (Figure 48) disp layed gHMQC correlation (Fi gure 49) with a quaternary

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73 carbon resonating at 196.3 suggesting the presence of an aldehyde. The aldehyde was further correlated to olefins C-21 and C-22 in the gHMBC spectrum (Figure 50), confirming the presence of an aldehyde carbonyl conjugated to the C-21/C-22 double bond at the terminus of the side chain. O O OH H HO O O NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 27 26 25 gCOSY gHMBC O Figure 47. Key gHMBC and gCOSY co rrelations of palmerolide E ( 61 ) 9 8 7 6 5 4 3 2 1 PP M H-23 H-3 H-21 H-15 H-2 H-11 H-10 H-19 H-9 H-25 H-24 H-26 H-20 H-7 H-4a 7.0 6.5 6.0 5.5 5.0 4.5 PPM 3.0 2.5 2.0 1.5 1.0 PPM Figure 48. 1H NMR spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6)

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74 10 9 8 7 6 5 4 3 2 1 PPM Direct Dimension 200 150 100 50 0 PPM Indirect Dimension 1 Figure 49. gHMQC spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6) The quaternary carbon at 166.1 was assigned as an es ter carbonyl (C-1), which correlated to olefinic protons H-2 and H-3 in the gHMBC. These two protons showed a coupling constant of 15.7 Hz indicating a trans double bond. Three methylenes were positioned between C-3 and hydroxymethine C7, based on gHMBC correlations of H-2 with C-4, H-5 with C-6 and H-8 with C-6. Th is was further supported by the observation of gCOSY correlations (Figure 51) between H-4a and H-5b, H-5a and H-6b and H-7 with H-6a and H-6b. In addition, H-7 displayed a HMBC correlation to olefinic methines C-8 and C-9 (Table 5). Location of two hydroxym ethines, C-10 and C-11 adjacent to each

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75 other was established by the COSY corre lations of H-9, H-10 and H-11 and further confirmed by HMBC correlation observed for H-11 with C-9 and C-10. In addition, H-11 showed HMBC correlation to the quaternary carbon at 156.4 which was assigned as the carbonyl carbon of the carbamate group. 10 8 6 4 2 0 PPM Direct Dimensio n 200 150 100 50 0 PPM Indirect Dimension 1 Figure 50. gHMBC spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6)

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76 Table 5. NMR data of palmerolide E ( 61 ) (1H, 500 MHz, 13C, 125 MHz, DMSOd6) 13C 1H (ppm, mult, J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 OCONH 2 166.1 121.2 150.8 33.4 33.4 25.9 25.9 38.5 38.5 73.3 134.3 129.6 70.1 76.1 29.8 29.8 132.9 127.3 128.7 133.0 43.9 43.9 73.0 38.1 155.8 139.6 196.3 17.0 16.5 9.9 5.78 (1H, d, 15.7) 6.74 (1H, ddd 4.3, 11.5, 15.7 ) 2.11 (1H, m) 2.14 (1H, m) 1.30 (1H, m) 1.05 (1H, m) 1.49 (1H, m) 1.29 (1H, m) 3.81 (1H, m) 5.53 (1H, dd, 1.4, 8) 5.49 (1H, d, 2.9) 4.12 (1H, m) 4.47 (1H, ddd, 1.5, 5.1,10.7) 1.05 (1H, m) 1.95 (1H, m) 5.42 (1H, m) 6.05 (1H, dd, 10.8, 14.8) 5.61 (1H, d, 10.6) 2.09 (1H, m) 2.16 (1H, m) 5.02 (1H, ddd, 2.1, 7.5, 10.) 2.64 (1H, qdd, 6.8, 7.1, 9.3) 6.55 (1H, dd, 1.5, 10.2) 9.41 (1H, s) 1.63 (3H, s) 1.01 (3H, d 6.8) 1.67 (3H, d 1.2) 6.48 (2H, br) 1, 3, 4 1, 2, 2, 3, 2, 3, 6 8, 9 6, 9, 10 8, 10 8, 9, 11, 12/13 9, 10, 12/13, C ONH2 10, 13 12, 14, 15 12/13, 16, 13, 14, 16 14, 15, 18, 24 15, 16, 18, 24 16, 17, 19, 24 16, 17, 19, 24 1, 17, 20, 23, 25, 19, 21, 22, 25 19, 23, 25, 26 21, 22, 26 16, 17, 18 19, 20, 21 21, 22, 23

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77 10 8 6 4 2 0 PPM Indirect Dimension 1 10 8 6 4 2 0 PPM Direct Dimension Fig Figure 51. gCOSY spectrum of palmerolide E ( 61 ) (500 MHz, DMSOd6) The two methylenes, C-12 and C-13, appeared overlapped with each other as a one carbon signal in the 13C NMR spectrum. Their linkage with hydroxymethine C-11 and olefinic methine C-14 was established by HMBC correlati ons of H-10, H-11 and H-14. Based on the HMBC relations of H-14, H-15 and H3-25, it was evident that the trans ( J = 14.8 Hz) double bond (C-14 and C-15) is conj ugated to a trisubstituted double bond (C16 and 17). Methylene C-18 was positione d between the hydroxymethine C-19 and quaternary carbon (C-17) based on gHMBC re lationships of H-19, observed with C-18 and C-17. The 20 member macrocycle was co mpleted by the observation of the HMBC cross peak identified for H-19 and C-1.

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78 2.2.7 Characterization of palmerolide B ( 62 ) Palmerolide B ( 62 ), was isolated as a colorless, sticky solid. Its 1H NMR spectrum (Figure 52) showed signals charac teristic of palmerolides despite that it was isolated form a more polar fraction than the fractions th at contained other palmerolides. Further, palmerolide B showed a distinctly low Rf value when compared with other palmerolides in thin layer chromatography in norma l phase, affirming its higher polarity. 7 6 5 4 3 2 1 PP M H-24 H-3 H-15 H-23 H-8 H-7 H-11 H-19 H-21 H-14 H-9 H-10 H-26 H-5` H-4` H-25 H-27 H-20 7.0 6.5 6.0 5.5 5.0 4.5 PPM Figure 52. 1H NMR spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) The 13C NMR spectrum (Figure 53) showed that compound 62 had 33 carbon as observed in palmerolide A ( 58 ). Further analysis of DEPT 135 (Figure 54) and gHSQC (Figure 55) spectra suggested that it is composed of fi ve methyls, six methylenes, sixteen methines and six quaternary carbon signals. Out of th e six methines, four were indicative of hydroxymethines. All these observati ons were also consistent w ith the structural features of palmerolide A.

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79 Figure 53. 13C NMR spectrum of palmerolide B ( 62 ) (125 MHz, CD3OD) 133.436 133.425 133.333 133.316 132.227 131.048 129.767 128.039 122.750 122.291 119.578 118.867 81.620 77.439 76.155 72.116 45.187 38.709 36.608 33.958 33.946 31.755 30.887 30.851 27.673 26.108 20.354 17.688 16.653 13.205 140 120 100 80 60 40 20 PP M C-3 C-8 C-19 C-7 C-11 C-18 C-20 C-25 C-27 C-26 C-5` C-4`C-13C-2' C-23 C-15 C-16 C-9 Figure 54. DEPT 135 spectrum of palmerolide B ( 62 ) (125 MHz, CD3OD) 180 160 140 120 100 80 60 40 20 P C-1 C-1' OCONH2 C-3' C-3 C-22 C-9 C-17 C-21 C-10 C-16 C-15C-24 C-2 C-2' C-23 C-11 C-7 C-19 C-8 C-18 C-6 C-4 C-12 C-20 C-13 C-4' C-5 C-5' C-26 C-27 C-25 136 134 132 130 128 126 124 122 120 PPM 45 40 35 30 25 20 15 PPM(a) (b) (c)

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80 180 160 140 120 100 80 60 40 20 PPM Indirect Dimension 1 7 6 5 4 3 2 1 PPM Direct Dimension Figure 55. gHSQC spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) The interpretation of gHMBC (Figure 56) and gCOSY (Figures 57, 58) data of palmerolide B ( 62 ) revealed that it has a 20 member macrocyclic ring with a significant rearrangement of the structural features in C-3 through C-12. Nevert heless, structure of the side chain was identified as identical to that of palmerolide A ( 58 ). A quaternary carbon 168.2 which was assigned as an este r carbonyl (C-1), showed gHMBC correlation to H-3, which in turn displayed HMBC correlation to two adjacent methylenes, C-4 and C-5. The linkage between the nest two methylenes, C-5 and C-6, was established by observation of COSY correlations betw een them. These spectral evidence was consistent with an ester carbonyl with a conjugated trans olefin ( J = 15.3 Hz) attached to these methylenes.

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81 7 6 5 4 3 2 PPM Direct Dimension 160 140 120 100 80 60 40 20 PPM Indirect Dimension 1 Figure 56. gHMBC spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) O O O H N O O S O O O NH2 HO O1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 27 26 25 1' 2' 3' 4' 5' gCOSY gHMBC Figure 57. Key gCOSY and gHMBC correlations of palmerolide B ( 62 )

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82 7 6 5 4 3 2 1 PPM Indirect Dimension 1 8 7 6 5 4 3 2 1 PPM Direct Dimension Figure 58. gCOSY spectrum of palmerolide B ( 62 ) (500 MHz, CD3OD) The hydroxymethine 77.4 can be positioned at C-7, between the methylene C-5 and another hydroxymethine 72.1 (C-8) based on gHMBC correlations of H-7 with C-6 and the COSY correlations of H-7 with H-6a and H-8. The presence of another olefin at C9/C-10 attached to the latter hydroxymethi ne was supported by gHMBC correlations of H-8 with C-9 and that of H-9 with C-8. This assignment was consistent with the observation of COSY correaltions between H7 and H-8 and H-8 and H-9. In addition, H7 displayed further HMBC correla tions with the quaternary carbon 159.8 characteristic of the carbonyl attached to carbamate group present in all palmerolides.

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83 Table 6. NMR data of palmerolide B ( 62 ) (1H, 500 MHz, 13C, 125 MHz, CD3OD) 13C 1H (ppm, mult, J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 OCONH2 168.2 122.3 150.7 34.0 34.0 26.1 26.1 31.8 31.8 77.4 72.1 133.4 131.0 81.6 36.6 36.6 30.9 30.9 133.3 128.0 129.8 132.8 45.2 45.2 76.6 38.7 132.2 134.5 119.6 122.7 16.7 17.7 13.2 166.7 118.9 154.9 27.7 20.4 159.8 5.70 (1H, d, 15) 6.74 (1H, ddd, 4.0, 11, 15) 2.14 (2H, m) 2.10 (1H, m) 1.34 (1H, m) 1.14 (1H, m) 1.08 (1H, m) 1.55 (1H, m) 4.57 (1H, m) 4.20 (1H, m) 5.72 (1H, m) 5.64 (1H, m) 4.64 (1H, m) 1.53 (1H, m) 1.79 (2H, m) 1.21 (1H, m) 1.96 (1H, m) 5.38 (1H, ddd, 14.5, 10.5, 4) 6.01 (1H, dd, 10.5, 14.5) 5.57 (1H, d, 10.9) 1.99 (1H, m) 2.15 (1H 4.82 (1H, m) 2.68(1H, qdd, 5, 10, 10) 5.08 (1H, d, 10) 5.85 (1H, d, 15) 6.86 (1H, d, 14.5) 1.58 (3H, s) 0.88 (3H, d, 6.5) 1.71 (3H, s) 5.64 (1H, br s) 1.81 (3H, s) 2.09 (3H, s) 1, 4 1, 4, 5 6, 8, OC ONH2 9 8, 10 8, 11 11 11, 15 14, 17 15, 18 20 1, 20 19, 20, 22, 25 21, 22, 24, 25 22, 23, 1 16, 17, 18 19, 20, 21 21, 22, 23 1 3 4 5 1 2 3 5 1 2 3 4

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84 Another hydroxymethine (C-11) could be plac ed between the former olefin and another methylene (C-12) based on HMBC correlati ons of H-10, H-12a and H-12b with C-11. The connectivity between the methylenes C-12 and C-13 wa s confirmed by gCOSY data. The latter methine protons displaye d COSY coupling with proton signal 5.38 indicating the presence of two methylenes between hydroxymethine C-11 and olefinic methine C14. The stereochemistry of this olefin was determined as trans based on the coupling constant between H-14 and H-15. Consider ing the gHMBC correlations of olefinic protons H-15 to C-14 and the quaternary ca rbon C-17 and that of vinylic methyl at 1.58 (H3-25) to C-18, C-17 and C-15 a trisubstitute d double bond was positioned at C-17/C-16 conjugated to the former olefin. The hydroxymethine at 76.1 was assigned to C-19, based on HMBC correlations between H-18a and H-18b and COSY correlations of the latter protons to 4.82, representing H-19. The 20 macrolide was completed by the observations of gHMBC correlation between H-19 and C-1. The side chain of palmerolide B showed featur es similar to that of palmerolide A. The doublet at 0.88 in the 1H NMR spectrum assigned for the methyl (H3-26) showed COSY correlation to H-20. In addition this methyl group displayed gHMBC correlation to C-19, C-20 and C-21 confirming that its lo cated between C-19 and C-21, and attached to C-20. Meanwhile, H-21, an olefinic methine, was correlated to olefinic methines C-23 and C-25 in the gHMBC spectrum. However, gHSQC evidence showed that the latter carbon belong to the vinylic methyl H3-27. Based on the coupling constant ( J = 15 Hz),

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85 the stereochemistry of olefinic protons H-23 and H-24 was identified as trans These correlations are consistent with a trisubstituted doubl e bond conjugated with a trans olefin at C-23/C-24. H-24 displayed gH MBC correlation to C-23, C-24 and C-1 assigned for an ester carbonyl. The HMBC co rrelation of broad singlet H-2 with C-1 and the unusual 4J HMBC coupling of two vi nyl methyl groups C-4 and C-5 as observed in palmerolide A suggested an isopentenoyl c onnected to C-24 via an ester linkage. When the compound 62 was analyzed by LRESIMS it displayed a major peak at m/z = 663.2 indicative of [M H]+. However, in LRESIMS it afforded a major peak at m/z = 567.4. This was explained as due to [M + H H2SO4]+, suggesting the presence of a sulphate group attached to one of the seconda ry hydroxyl groups of palmerolide B. These observations were further confirmed by performing HRESIMS experiments on both above peaks using respective modes of ioni zation. The HREIMS negative mode (Figure 59) gave a molecular mass of 663.2957 consistent with a formula of C33H48N2O10S. In addition this data indicates the presence of two nitrogens confirming the presence of a carbamate attached to C-7 and an amide between C-24 and C-1 as was observed in other palmerolides. The site of attachment of the sulphate gr oup was assigned as C-11 by the observation of relatively high proton and carbon chemical sh ifts for H-11 and C-11 as compared to corresponding chemicals shifts in other palmer olides. In addition, generation of a stable mass peak at m/z = 567.3 in ESIMS can be explained as due to forma tion of a double bond between C-11 and C-12, after the loss of a sulphuric acid (Figure 60).

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86 Figure 59. HRESIMS spectrum of palmerolide B ( 62 )

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87O O O H N O O S O O O NH2 HO OH O O O H N O O NH2 HO -(H2SO4) m/z = 567.3 Figure 60. Fragmentation of palmerolide B ( 62 ) in ESIMS (positive mode)

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88 2.2.8 Characterization of palmerolide H ( 63 ) Further purification of the HPLC fr actions containing palmerolide B ( 62 ) afforded another compound with 1H NMR signals (Figure 61) indicative of palmerolides, which was identified as palmerolide H ( 63 ), after a comprehensive stru cture elucidation with 2D NMR spectroscopy and mass spectrometry. 7 6 5 4 3 2 1 PP M H-24 H-3 H-20 H-15 H-23 H-2` H-14 H-21 H-19 H-26 H-25 H-7` H-27 H-8` H-4` 7.0 6.5 6.0 5.5 5.0 4.5 PPM 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 PPM Figure 61. 1H NMR spectrum of palmerolide H ( 63 ) (500 MHz, CD3OD) The 13C NMR spectrum (125 MHz) (Figure 62) s howed that palmerolide H has 36 carbon signals. Further analysis of gHSQC spectra (500 MHz) (Figure 63) accounted for five methyls, eight methylenes, sixteen methines and six quaternary carbon signals. Out of these methines, four repr esented hydroxymethines.

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89 1 80 160 140 120 100 80 60 40 20 PP M C-1 C-1` OCONH2 C-5` C-11 C-6` C-8 C-19 C-7 C-4` C-18 C-27 C-25 C-26 C-7` C-8` C-2 C-2` Figure 62. 13C NMR spectrum of palmerolide H ( 63 ) (125 MHz, CD3OD) 160 140 120 100 80 60 40 20 PPM Indirect Dimension 1 7 6 5 4 3 2 1 PPM Direct Dimension Figure 63. gHSQC spectrum of palmerolide H ( 63 ) (500 MHz, CD3OD)

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90 Extensive study of the gHMBC (Figure 64) and gCOSY spectra (Figure 65) of palmerolide H ( 63 ) indicated that it has a 20 member m acrocyclic ring similar to that of palmerolide B, in planer structur e (Figure 66). The quaternary carbon at 168.2 could be assigned to an ester carbonyl (C-1) base d on analogy with other palmerolides, and showed HMBC correlation to H-3, which in tu rn displayed gHMBC correlation with two adjacent methylenes, C-4 and C-5. The conne ctivity between two methylenes, C-5 and C6, was established by gCOSY correlations. These spectral data were consistent with an ester carbonyl bearing a conjugated trans olefin ( J = 15.5 Hz) followed by C-4 and C-5. 7 6 5 4 3 2 1 PPM Direct Dimension 200 150 100 50 0 PPM Indirect Dimension 1 Figure 64. gHMBC Spectrum of palmerolide H ( 63 ) (500 MHz,CD3OD)

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91 7 6 5 4 3 2 1 PPM Indirect Dimension 1 7 6 5 4 3 2 1 PPM Direct Dimension Figure 65. gCOSY spectrum of palmerolide H ( 63 ) (500 MHz, CD3OD). O O O H N O O S O O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 27 26 25 1' 2' 3' 4' 5' 6' 7' 8' gCOSY gHMBC OO NH2 HO Figure 66. Key gHMBC and gCOSY co rrelations of palmerolide H ( 63 ).

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92 The hydroxymethine at 77.5 can be positioned between the methine at C-6 and another hydroxymethine 72.2 (C-8) based on the gHMBC corre lation of H-8 to C-6 and C-7. This assignment was confirmed by respectiv e gCOSY correlations. H-7 was correlated to an olefinic methine 133.4 (C-9) by HMBC suggesting the presence of an olefin at C9/C-10, which was further supported by gCOS Y correlations between H-8 and H-9. H-7 displayed further gHMBC correlation with the quaternary carbon at 159.9 indicative of carbonyl that was characteristic of the carbamate group present in other palmerolides. Another hydroxymethine (C-11) could be plac ed between the former olefin and another methylene 35.5 (C-12) based on gHMBC correla tion of H-10, H-12a and H-12b with C-11. The connectivity between the met hylenes C-12 and C-13 was confirmed by gCOSY data. The latter methine protons disp layed COSY correlation with a proton signal at 5.38 indicating the presence of two methyls between hydroxymethine C-11 and olefinic methine C-14. The stereochemistry of this olefin was determined to be trans based on the coupling constant between H-14 and H-15. Considering the gHMBC correlation of olefinic proton H-15 with C-14 and the quaternary car bon C-17 and that of vinylic methyl at 1.58 (H3-25) to C-18, C-17 and C-15, a trisubstituted double bond was positioned at C-17/C-16, conjugated to the former olefin. The hydroxymethine 76.2 was assigned to C-19 based on HMBC correlations between H-18a and H-18b and COSY correlations of the latter pr otons with and that of 4.82, H-19. The 20 membered macrolide was completed by the observati on of gHMBC correlation between H-19 and C-1 (Table 7).

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93 Table 7. NMR data of palmerolide H ( 63 ) (1H, 500 MHz, 13C, 125 MHz, CD3OD) 13C 1H (ppm, mult, J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 1 2 3 4 5 6 7 8 OC ONH2 168.2 122.3 150.7 33.9 33.9 26.1 26.1 31.8 31.8 77.5 72.2 132.3 131.1 81.7 36.6 36.6 30.8 30.8 133.4 128.1 129.7 133.0 45.2 45.2 76.2 38.7 132.3 134.5 119.7 122.8 13.2 17.7 16.6 166.0 120.5 155.7 42.1 144.6 112.6 22.4 24.8 159.9 5.70 (1H, d, 15.5) 6.75 (1H, ddd, 4, 11.5, 15.7) 2.10 (1H, m) 2.14 (1H, m) 1.18 (1H, m) 1.32 (1H, m) 1.10 (1H, m) 1.56 (1H, m) 4.58 (1H, m) 4.20 (1H, m) 5.74 (1H, m) 5.63 (1H, m) 4.66 (1H, m) 1.55 (1H, m) 1.80 (1H, m) 1.21 (1H, m) 1.96 (1H, m) 5.38 (1H, m) 6.02 (1H, dd, 10, 14.5) 5.57 (1H, d, 11.5) 2.00 (1H, m) 2.15 (1H, m) 4.84 (1H, m) 2.68 (1H, m) 5.09 (1H, d, 10) 5.87(1H, d, 14.6) 6.87 (1H, d, 14.5) 1.59 (3H, s) 0.89 (3H, d, 6, 7) 1.72 (3H, s) 5.76 (1H, s) 3.44 (2H, s) 4.69 (2H, s) 1.61 (3H, s) 1.76 (3H, s) 1, 3, 4 1, 2, 4 2, 3, 5 2, 3 3, 4 3, 6 6, 8, 9 7, 9, 10 7, 8, 11, 12 8. 9. 12 9, 10, 13 OC ONH2 13, 15, 16 14, 16 14, 15, 25 15, 16, 18, 25 16, 19, 25 16. 19 17, 18, 20, 26 18, 19, 21, 26 19, 20, 26, 27 21, 23, 27 21, 24, 27 22, 23, 1’ 15, 16, 18 22, 24.25 22, 23, C-1` 16, 18 19, 20, 21 22, 23 1 4 8 2 3 5 7 4 8 4 5 6 2 3 4

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94 The side chain of palmerolide H ( 63 ) showed features similar to that of palmerolide D ( 60 ), The doublet at 0.89 in the proton NMR spectrum assigned for the methyl (H3-26), showed COSY correlation to H-20. In addi tion, this methyl group displayed gHMBC correlations with C-19, C-20 and C-21 confirmi ng that it is positioned between C-19 and C-21, and attached to C-20. Mean while, H-21, an olefinic methine, was correlated to another olefinic methine (C-23) and C-25 by observation of a cross peak in the gHMBC spectrum. However, gHSQC data showed that the latter carbon bel ong to the vinylic methyl H3-27. Considering the coupling constant ( J = 14.5 Hz), the orientation of olefinic protons H-23 and H-24 was assigned as trans These correlations are consistent with a trisubstituted double bond conjugated with a trans olefin at C-23/C-24. The doublet at 6.87 (H-24) displayed gHMBC correla tion to the quaternary carbon at 166.0 (C-1 ). The singlet at 5.83 (H-2 ) was correlated to C-1 the methyl at 23.8 (C8 ) and a methylene at 42.1 (C-4 ) in the HMBC spectrum. The sharp singlet observed at 3.54, (H-4 ) in turn showed HMBC correlation with C-3 and C-8 constituting a trisubstituted double bond between the a bove carbonyl and latter methylene. The singlet at 3.54 showed further gHMBC correlations with the vinylic methyl C-7 the quaternary carbon at 144.6 (C-5 ) and an exomethylene carbon at 112.6 (C-6 ), demonstrating the further elongation of the side chain, giving rise to another disubstituted double bond which defines the terminus in the form of an exomethylene at C-6

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95 In the LRESIMS spectrum, the compound ( 63 ) displayed a major peak at m/z = 703.3 indicative of [M H]+. However, the LRESIMS positive mode spectrum demonstrated a major peak at m/z = 607.3, which was explained as due to [M + H H2SO4]+ suggesting the presence of a sulphate group attached to one of th e secondary hydroxyl groups of palmerolide H ( 63 ). These observations were furthe r confirmed by performing HRESIMS analysis (Figure 67). Figure 67. HRESIMS spectrum of palmerolide H ( 63 )

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96 The HRESIMS spectrum gave a molecular ma ss of 703.3258 consistent with a formula of C36H52N2O10S (Figure 67). In addition, this mass sp ectral data indicated the presence of two nitrogens affirming the presence of a carbamate attached to C-7 and an amide between C-24 and C-1 as was observed in other palmerolides. 2.2.9 Bioactivity of palmerolides Unique structural features of palmerolide A ( 58 ), and the attractive biological activity reported from macrolides with similar appendages, warranted an investigation of its biological activity. Therefore, palmerolide A was tested for anticancer properties at the National Cancer Institute.66 The very promising antica ncer activity displayed by palmerolide A prompted the bioactivit y investigation of plamerolide C ( 59 ) and D ( 60 ) as well. 2.2.9.1 In vitro cytotoxicity of palmerolide A (58) Palmerolide A displayed reproducible in vitro cytotoxicity towards several melanoma cell lines (Appendix A), UACC-62 (LC50 0.018 M), MI14 (LC50 0.076 M), SK-MEL-5 (LC50 6.8 M) and LOX IMVI (LC50 9.8 M) in the NCI 60 human cancer cell line panel. Besides melanoma, it showed cytot oxicity against one co lon cancer cell line, (HCC-2998, LC50 6.5 M) and one renal cancer cell line (RFX 393, LC50 6.5 M). One of the main features of its activity profile was that it was largely devoid of other cytotoxicity (LC50>10 M) making it a promising anticancer drug candidate.

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97 Apart from that, palmerolide A ( 58 ) showed potent cytostatic activity against, leukemia (RPMI-8226), colon cancer (HT-116), me lanoma cell lines (LOXIMVI, SKMEL-5, UACC-62), ovarian cancer (OVCAR-3) a nd breast cancel (MDA-MB-231A/ATCC). 2.2.9.2 In vivo cytotoxicity of palmerolide A ( 58 ) Palmerolide A was subjected to the NCI hollow fiber assay,67 a standard bioassay used by National Cancer Institute to determine in vivo cytotoxicity of an ticancer agents. The hollow fiber assay has established as a ve ry efficient bioassay that can provide quantitative information of drug efficacy with minimum expenditures of time and materials. Therefore, it is curren tly being utilized as the initial in vivo test for agents found to have reproducible activity in the initial in vitro anticancer drug screen. The hollow fiber assay involves standard pane l of 12 tumor cell lines. The cancer cell suspensions are flushed into pol yvinylidene fluoride hollow fibers that are heat-sealed at 2 cm intervals. The samples generated from these seals are placed into tissue culture medium and incubated prior to implantation. Each mouse receives three intraperitoneal (Ip) implants (1 of each tumor line) and 3 s ubcutaneous (Sc) implants (1 of each tumor line). On the day of implantation, sample s of each tumor cell line preparation are quantitated for viable cell mass so that the time zero cell mass is known. Mice are treated with experimental agents starting on da y 3 or 4 following fiber implantation and continuing daily for 4 days. Each agent is admi nistered by intraperitone al (Ip) injection at 2 dose levels. The fibers are collected from the mice on the day following the fourth

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98 compound treatment and the viable cell mass in determined again to analyze the efficacy of the drug.67 The percent net growth for each cell line in each treatment group is calculated and compared to the percent net growth in the c ontrols. A 50% or greater reduction in percent net growth in the treated samples compared to the control samples is considered a positive result. Each positive result is given a score of 2 and all of the scores are totaled for a given compound. The maxi mum possible score for an agent is 96 (12 cell lines X 2 sites X 2 dose levels X 2 [score]). A compound is advanced to the xenograft assay, if it has a combined Ip + Sc score of 20 or greater, a Sc score of 8 or greater, or produces cell kill of any cell line at either dose level evaluated.67 Palmerolide A showed a score of 12 for intr aperitoneal implants and a score of 16 of subcutaneous implants giving a total score of 28 which was reproduc ible, indicating its potent in vivo cytotoxicity. Therefore it was advanced to th e xenograft assay. 2.2.9.3 In vitro Cytotoxicity of palmerolide C ( 59 ) Palmerolide C showed in vitro cytotoxicity, and cytostatic activity against leukemia (RPMI-8226, LC50 7.33 M), colon cancer (HCC-2998, LC50 7.10 M and HCT-15, LC50 9.87 M), CNS cancer (SF-295, LC50 4.02 M), melanoma (M-14, LC50 1.69 M), ovarian cancer (OVCAR-3, LC50 7.89 M) and beast cancer (MDM-MB-435, LC50 7.75 M).

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99 2.2.9.4 In vitro cytotoxicity of palmerolide E ( 61 ) Palmerolide E showed in vitro cytotoxicity agai nst non small cell l ung cancer cell line (HOP-62, LC50 9.58 M), colon cancer (HCC-2998, LC50 7.60 M and HCT-116 LC50 7.16 M), CNS cancer (SF-295, LC50 7.35 M) and melanoma cell line (M-14, LC50 0.56 M). It also showed cytostat ic activity against melanoma. 2.2.9.5 Mechanism of action of palmerolides By comparison with the activity profiles of the other known anticancer agents, the mode of action of a novel compound can often be predicted.68 It was found that the activity profiles of palmerolides are similar to a seri es of compounds isolated from several other organisms that have been identified as vacuolar ATPase (V-ATPase) inhibitors. 2.2.9.6 V-ATPase The vacuolar ATPase comprises a class of en zymes that is widely distributed throughout eukaryotes. These enzymes occur in many tissu es of multicellular organisms. The major function of V-ATPase is to pump protons from one side of a membrane to the other in order to regulate the pH levels. Hence v acuolar-ATPase performs this function in membranes of vacuoles.69 It has been found that V-AT Pases are involved in the onset of diseases such as osteoporosis, diabetes, pancreat itis and melanoma. In bones, resorption and remodeling is conducted by osteocasts, specialized cells which possess a V-ATPase on their plasma membrane. Release of V-ATPase acidifies the bone surface thus dissolving the bone

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100 matrix and activating osteoclast secreted acid hydrolases. Defects in osteoclast V-ATPase function have been found to be related to th e defects of this enzyme. Involvement of VATPase has been proposed in metastatic invasion through degradation of the extra cellular matrix by tumor cells as well. Besides, it has been implicat ed in the development of melanoma as it was found that organellar pH contributes to the lack of pigmentation in tyrosinase–positive amelanotic melanoma cells Therefore, V-ATPase inhibitors have emerged as a very attractive target for drug development against osteoporosis and melanoma.69 2.2.9.7 Known V-ATPase inhibitors Salicylihalamides A ( 64 ) and B ( 65 ), two macrolides isolated from the marine sponge Haliciona sp. showed very selective cytotoxicity against melanoma in the NCI 60 cell line screen.70 However, their activity profile did not match that of any standard clinical agent although a correlation wa s observed with the patter ns of two known V-ATPase inhibitors, concanamycin ( 66 )71 and bafilomycin A ( 67 ),72 which were considered unsuitable as anticancer drugs due to high cytotoxicity. Lobatamide A ( 68 ),73,74 belongs to a group of structura lly related macrocyclic enamids, isolated from the ascidian Aplidium lobatom and showed a cell growth inhibition profile similar that of salicylihalamides. Later, apicularin A ( 69 ),75,76 oximidine I ( 70 ) and oximidine II ( 71 ),77 which resemble the structure of salicylihalamides more closely, were isolated from marine microbes and also demonstrated similar activity.69

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101 O OH O OH H H N O Salicylihalamide A ( 64 ) O OH O OH H N H O Salicylihalamide B ( 65 ) Concanamycin ( 66 ) O O OH OH OH O O O Bafilomycin A ( 67 ) O O O HO H O HO HO O O HO O OH O NH2

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102 O O O OH O H N O N O H O O O OH N H O OH O Lobatamide A ( 68 ) Apicularin A ( 69 ) O OH O O H N N O O H OH O OH O O H N N O H OH Oximidine I ( 70 ) Oximidine II ( 71 ) Based on the strong correlations observed be tween the patterns of cytotoxicity of salicylihalamide A ( 64 ) and bafilomycin derivatives, the V-ATPase inhibition activity of salicylihalamides and lobatamides have been tested and they have shown excellent inhibitory activity.69 These observations have simulate d interest in these compounds and the functional role of the enamide group, a feat ure common to most of these compounds in V-ATPase inhibition.68 However, low yields of above compounds from their natural sources have limited the progr ess of further research.

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103 2.2.9.8 Palmerolides as V-ATPase inhibitor. Palmerolide A ( 58 ) showed inhibition of V-ATPase at 2 nM concentration (Table 8 ). Further, it was found out that it binds with V-ATPase enzyme at nearly 1:1 molar ratio. Palmerolide C, which has a different ring stru cture from palmerolide A, displayed lesser activity against V-ATPase at a concentra tion of 150 nM. Both palmerolides A and C possess an enamide as a part of its side chai n and above results are consistent with other macrolides having similar features.68,69 Nevertheless, palmerolide E which does not have an enamide in its structure has only week V-ATPase activity (10 M) despite submicromolar cytotoxicity against melanoma. These observations suggest the presence of mechanism of action independent of the enamide. Table 8.. Comparison of cytotoxicity and V-ATPase activity Palmerolide A Palmerolide C Palmerolide E V-ATPase activity 2 nM 150 nM 10 M Activity against Melanoma 18 nM UACC-62 1.7 M MI-14 560 nM MI-14 2.3 Summary Antarctic tunicate Synoicum adareanum elaborates a new series of polyketide macrolides, palmerolides. They are composed of a 20 member macrocycle and a side chain. Characteristic features of th e palmerolides include a carb amate group and two secondary hydroxyls attached to the macrolide ring. Ho wever, within the series, they show variations in the arrangement of the functional group attached to the ring and side chain.

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104 The structural novelty of palmerolides prompt ed their bioactivity i nvestigation in NCI 60 cancer cell line panel. The principal member of the series, palmerolide A, showed very potent and selective in vitro cytotoxicity against several melanoma at nanomolar concentrations. In addition, it showed potent cy tostatic activity agains t a number of other cell lines. The in vivo cytotoxicity of palmerolide A was determined by the hollow fiber assay. Palmerolides have been investigated as V-ATPase inhibitors.

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105 Chapter 3 CHEMICAL INVESTIGATION OF THE ANTARCTIC NUDIBRANCH AUSTRODORIS KERGUELENENSIS 3.1 Introduction 3.1.1 Nudibranchs Nudibranchs are a group of marine molluscs th at do not have extern al shells. They are soft bodied, very often brightly colored and do not have external armaments. The name nudibranchs literaly means “naked gills”, as th eir gills are prominently displayed in the dorsal side in many species. In gene ral, the nudibranchs are carnivores.5 They feed on variety of other animals, sponges, anemones, tunicates, corals, bryozoans, barnacles and sometimes other nudibranchs.5, 78 Their high visibility, low mobility and lack of physical defense make the nudibranchs extremely vulnerable to predators. Neverthe less, reports of predation are virtually nonexistent as they appear to employ chemical defense.79 Most nudibranch s obtain their defensive chemicals from their diet, whereas there is a small group of nudibranchs that can synthesize them via de novo biosynthesis.5 The secondary metabolite composition of the former category of nudibranchs most often portrays their choice of food. In addition, th eir chemistry varies according to the location.78 Many such sequestered chemicals obtai ned from other species are toxic and often stored in specialized s pherical dorsal glands called man tle dermal formations, a part of the animal that is most su sceptible to predation. This furt her facilitates the usage of a bioactive chemical as a defense wea pon with minimal toxic side effects.5, 78

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106 It is understood that nudibran chs can selectively concentrat e certain allochemicals from their diet or modify the chemistry of the ingested compounds.80 For instance, the Mediterranean nudibranch Hypselodoris orsini has shown the remarkable ability to convert dietary scalaradial ( 72 ) into deoxoscalarin ( 73 ) which is concentrated in viscera, and to 6-keto-deoxoscalarin ( 74 ), located in mantle dermal formations.5, 81 H H CHO CHO OAc H H H OAc H X O HO Scalaradial ( 72 ) Deoxoscalarin ( 73 ) X = H2 6-keto-deoxoscalarin ( 74 ) X = O It has been reported that nudibranch Glossodoris pallida sequesters scalaradial ( 72 ), a sesterterpene identified as a sponge meta bolite, and converts it into deoxoscalarin ( 73 ). However, it was also noted that it does not sequest er scalarin ( 75 ), another sesterterpene which is the major sponge metabolite.80 On the other hand, th e concentration of deoxyscalarin was found to be significan tly higher than th e corresponding sponge metabolites. Deoxoscalarin is found in the reproductive system, eggs, and mantle border of G. pallida Although removal of the nudibranch ma ntle increase susceptibility to predation by reef fish, the specific loca tion of the diet-deriv ed compounds was not significant. It was conclude d that the localization of co mpounds in the mantle tissue, may prevent autotoxicity. However, the egg masses of G. pallida were found to be low

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107 in the major sesterterpenoid secondary metabolites and are reported to be eaten by a variety of fish.5, 80 H H OAc H O HO O Scalarin ( 75 ) The nudibranchs have also adapted to feed on soft corals, ascidians, bryozoans, or on other molluscs.5, 78 The nudibranch Ovula ovum for example, was reported to sequester the toxic terpene sarcophytoxide ( 76 ) from soft coral Sarcophyton sp. and transform it into the less toxic derivative 77. 82 O O O Sarcophytoxide ( 76 ) 77 A distinct feature of nudibranchs that have the ability to biosynthesize their own defense chemicals was their preference to an inverteb rate diet lacking secondary metabolites. In addition, their chemistry is independent of the location that they are collected.

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108 Feeding experiments performed using 14Clabeled precursors illustrated the ability of the some nudibranchs to undertake de novo biosynthesis.5 Injection of 14C labeled mevalonate into three speci es of dorid nudibranchs ( Dendrodoris limbata, D. grandiflora, and D. arborescens ) demonstrated incorporation of label into three drimane sesquiterponoids, polygodial ( 78 ), 6-acetoxyolepupuane ( 79) and the sesquiterpene ester 80 Incorporation of 13C-labelled glucose confirmed these terpenes were synthesized de novo via the classical mevalonic acid pathway.5, 83 H O O H O OAc OAc AcO H O OAc OAc O O R Poligodial ( 78 ) 6-acetoxyolepupuane ( 79 ) 80 In biosynthetic studies involvi ng the Canadian nudibranchs Archidoris montereyensis and A. odhneri a low rate of incorporation of meval onic acid into the sesquiterpenoids and diterpenoid acid gly ceride metabolites 81, 82 and 83 was reported.5, 83 O OH OH O H H O OH OH O 81 82

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109 O O HO HO 83 Use of stable isotopically labeled precursor s has led to the convincing experimental evidence for de novo biosynthesis in nudibranchs. It was found that effective incorporation levels of respect ive isotopes can be achieved by simulation of the particular ecological roles of the metabolites. Theref ore, handling of the nudibranchs prior to injection leads to release of defensive substances in their mucus and activates the biosynthetic pathways so that the chemical defenses can be unleashed.5 Implementation of this strategy demonstrated signif icantly high incorporations of [1, 2-13C2] acetate into the aldehydes nanaimoal ( 84 ), acanthodoral ( 85 ), and isoacanthodoral ( 86 ) in Acanthodoris nanaimoensis .84 O H O H O Nanaimol ( 84 ) Acanthodoral ( 85 ) Isoacanthodoral ( 86 ) Subsequent 13C studies by the Faulkner and Andersen groups established the mevalonate origin of the terpene porti on of glyceride metabolites ( 81) ( 83) and tanyolide B ( 87 ) in various nudibranchs. Similarly de novo biosynthesis of the sesquiterpene metabolites albicanylacetate ( 88 ), cadlinaldehyde ( 89 ), and luteone ( 90 ), isolated from Cadlina

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110 luteomarginate was established. It was further unders tood that the biosynthesis of above metabolites can be regulated according to the need.84,85 O O OH OH O O H O H H H O H H H O O Tanyolide B ( 87 ) Albicanyl acetate ( 88 ) Cadlinaldehide ( 89 ) Luteone ( 90 ) The cadlinaldehyde and luteane skeletons were shown to be formed by degradation of a sesterterpenoid precursor. The acetyl resi due of albicanylacetate was labeled and its incorporation into the terpene portion wa s monitored. However, it was only evident during the egg-laying period illustrating that the biosynthe sis is regulated.85 It has been found that some nudibranchs utiliz e both biosynthetic stra tegies and are able to carry out de novo biosynthesis as well as seque stering metabolites. The North American nudibranch Cadlina luteomarginata is characterized by the presence number of different sesquiterponoid and diterpenoid cons tituents representing 37 carbon skeletons. The origin of most of these have been trac ed to local sponges. However, the nudibranch

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111 consistently yielded albicanyl acetate ( 88 ), a reasonably potent fish deterrent metabolite, from all collection sites. Meanwhile, 1 ,2 -albicanyl acetate ( 91 ) was found in egg masses but never found in skin or whole body ex tracts suggesting that it was produced by the nudibranch. Thus, the nudibranch produces major defense compounds de novo in order to ensure reproductive success, but makes use of local sponge toxins for additional protection.86,87 Similarly, Dendrodoris grandiflora was found to produce drimane sesquiterpenes via de novo biosynthesis for egg masses, even though is known to store some sponge metabolites. 88 O OAc AcO O 1 2 -albicanyl acetate ( 91 ) It has been debated as to whether the nudibr anchs lost their shell, a major defensive structure, due to the availability of elaborate and efficient defense strategies in the form of chemical defense. On the other hand, the ability to use the toxic substances biosynthesized in other organi sms has reduced the metabolic cost to make them on their own.78 3.1.2 Chemistry of Austrodoris Nudibranchs belonging to the family Do ridae are known to employ ichthyotoxic acyl glyceryl esters of diterpenoids or sesquite rpenoids for their prot ection against predators.5 They are only present in the mantle tissues of the animals sugges ting their function in

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112 defending the shell-less molluscs. These gl ycerols were first found in the British Columbian nudibranchs Archidoris monotereyensis and Archidoris odhneri .89-91 Subsequently, they have been isolated from the Mediterranean nudibranch Doris verrucosa, The Antarctic nudibranch Austrodoris kerguelenensis and two Archidoris species A. tuberculata and A. carvi which were collected from Northern Spain and Argentina respectively.83,92,93 Austrodris kerguelenensis, a common Antarctic nudibranch widely distributed in the high Antarctic and Subantarctic Zone has been subj ected to extensive studies for the presence of defensive chemicals. Studies on different collections of A. kerguelenensis have led to a series of ent -labdane, halimane and isocopalane d iterpenoid glycerols along with some nor -sesquiterpenoids.91 Figure 68. Austrodoris kerguelenensis at Bonaparte Point, Antarctica (Photograph supplied by Bill J. Baker, University of South Florida)

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113 Chemical investigation of the mantle tissues of a specimen of Austrodoris kerguelenensis collected from McMurdo Sound Antarctica, ga ve five diterpenoid glyceryl esters 92-96 belonging to labdane family of diterpenoids.79 It has been proposed that the oxidative cleavage of the B ring of 92 and 93 between C-8 and C-9 would have given rise to diketones 94 and 95 .79 O O OR1 OR2 H O O O OR1 OR2 O H 92 R1= OAc, R2 = H 94 R1 = OAc, R2 = H 93 R1 = H, R2 = OAc 95 R1 = H, R2 = OAc O O OH OH O H ( 96 ) Austrodorin ( 97 ), a diterpenoid with an unusual halimane skeleton, was reported with its diaceylated derivative from a sample colle cted from Tethys Bay, of Antarctica.93 It may have arisen by a rearrangement of analogous compound having a labd ane skeleton. Later, more compounds with this skeleton 98, and 99 were isolated along with labdanes 92 and 93.90,94 After synthetic studies to determine the absolute stereochemistry, the absolute

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114 configuration of C-2 of all these glycerides was established as S .95 Consequently, the stereochemistry of C-2 of 92 and 93 was assigned as in 100 and 101 .91 Further, reinvestigation of the structures of compounds 99 and 101 led to their revision to 102 and 103 .91 O O OR1 OR2 O O OR1 OR2 Austrodorin ( 97 ) R1 = H, R2 =H ( 100) R1 = H, R2 = OAc 98 R1 = H, R2 = OAc ( 101) R1 = OAc, R2 = H 99 R1 = OAc, R2=H O O OH OH O O OH OH ( 107 ) ( 108 ) Another specimen of Austrodoris kerguelenensis collected at South Shetland Island Antarctica, yielded two isocopalane diterpenoids austrodorin A ( 104 ) and B ( 105 ).90

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115 O O OH OH O O O O O O OH OH O O O O Austrodorin A ( 104 ) Austrodorin B ( 105 ) The skin extracts of A. kerguelenensis nudibranch collected from Terra Nova Bay, Antarctica, gave two norsesquiterpenoids austrodoral ( 106 ) and its oxidized product austrodoric acid ( 107 ).96 In addition, it afforded cler odane diterpenoi d acyl glyceride ( 108 ) which had the same clerodane dite rpenoid residue as archiodrin ( 109 ) isolated from Archidoris tuberculata.91 H O H O OH Austrodoral ( 106 ) Austrodoric acid ( 107 ) O O OH OH O O OH O O 108 Archidorin ( 109 )

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116 The observation of the close resemblan ce of the diterpenoi d glycerides of Austrodris kerguelenensis to that isolated from Archidoris montereyensis, Archidoris odhnery and D. verrucosa which are established to be products of de novo biosynthesis, led to the suggestion that they also may be biosynthesized de novo .83, 97 3.1.3 Bioactivity The biological role of these di terpenoid esters only present in the mantle tissue is linked to the protection of the shell-less molluscs. They have shown toxic ity against fresh water fish98 and antifeedant activity against marine fish99 in different bioassays. The 1,2diacylglycerols have demons trated more activity than corresponding 1,3-diacylglycerols. Further, 1,2-diaclyglycerols have exhibited pot ent activity in activation of protein kinase C and in the regenerative test s with fresh water hydrazoan, Hydra vulgaris .100 3.1.4 Research Objectives Different collections of Austrodris kerguelenensis have shown differences in chemical composition based on the location of the sample collection.79,93 Significant variations of chemistry have been already observed in McMurdo Sound and Palmer Station, in Antarctica.35 Therefore, it was of interest to unde rtake a chemical investigation on the specimens of A. kerguelenensis collected from Palmer Station.

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117 3.2 Results and Discussion 3.2.1 Extraction and isolation of secondary metabolites Austodoris kerguelenensis nudibranchs were collected from the ocean near Palmer Station, Antarctica. The CHCl3 extract of the nudibranchs were fractionated to 12 factions by flash chromatography on silica (S cheme 2). Further purification of fractions 7, 9 and 10 by HPLC on silica gel and C-18 yielded palmadorin A ( 110 ) (24 mg, 0.006% dry wt), palmadorin B ( 111 ) (7 mg, 0.002% dry wt) and palmadorin C ( 112 ) (7 mg 0.002% dry wt). O O OH OH 4 5 8 9 10 14 15 16 17 18 19 2' 20 O O OH O 4 5 8 9 10 14 15 16 17 18 19 2' 20 O 1' 3' palmadorin A ( 110 ) palmadorin B ( 111 ) O O OH OH 4 5 8 9 10 14 15 16 17 18 19 2' 20 OH 7H palmadorin C ( 112 )

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118 Scheme 2. Extraction and pur ification of palmadorins 3.2.2 Characterization of palmadorin A ( 110 ) Palmadorin A ( 110 ), isolated as a viscous oil, showed a [M + 1] + peak at m/z = 379.3 (Figure 69) in the LRFABMS spectrum. The HRFABMS analysis provided a molecular weight of 379.2842 amu consistent with the molecular formula of C23H38O4. Fr 7 (120 mg) Fr 8 (160 mg) Fr 9 (268 mg) Fr 10 (112 mg) Fr 11 & 12 (38 mg) Fr 6 (350 mg) Fr 5 (280 mg) AK/1S/7/4 (20 mg) Palmadorin B ( 109 ) (7 mg) HPLC C-18 20% H2O/ MeCN AK/1S/9/8 (20 mg) HPLC C-18 20% H2O/MeCN HPLC Silica 60% EtOAc/hexane AK/1S/10/7 (20 mg) Mixture of Steroides Palmadorin C ( 110 ) (24 mg) HPLC C-18 25% H2O/MeCN Palmadorin A 108 ) (24 mg) HPLC C-18 25% H2O/MeCN Austrodoris kerguelenensis freeze dried (2.5g ) Fractionation on Silica EtOAc / hexane increasing polarity gradiant Fr 1 & 2 (410 mg) Fr 3 &4 (327 mg) Mixture or sterols and fatty acids Exracted with CHCl3 (24hrs, 3X) CHCl3 Extract AK/1S/9/6 ( 55 mg) AK/1S/9/7 (60 mg) HPLC Silica 40% EtOAc/ hexane

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119 Figure 69. LRFABMS spectrum of palmadorin A ( 110 ) The 13C NMR spectrum of palmadorin A ( 110 ) showed 22 carbon signals (Figure 70). The signals at 163.9, 160.7, 114.6 and 102.8 were assigned as olefins whereas the 167.0 was assigned as an ester carbonyl. The carbon resonances 74.6 and 62.9 could be assigned as oxygen bearing carbons. The latter signal exhibited unusually high intensity indicative of coincident signals.

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120 Figure 70. 13C NMR spectrum of palmadorin A ( 110 ) (125 MHz, CDC13) The 1H NMR spectrum of compound 110 (Figure 71) showed a distinct sharp doublet at 4.48 (H-18) that integrated for two hydr ogens. H-18 exhibited gHSQC correlation (Figure 72) with carbon signal at 102.8, suggesting the presence of an exomethylene. These exomethylene protons displayed strong HMBC (Figures 73, 74) correlations to the quaternary carbon resonances at 160.7 (C-4), 40.2 (C-5) and the methylene carbon signal at 33.2 (C-3). The proton signal 2.09 (H-3a) displayed HMBC correlations with carbons 160.4 (C-4), 40.0 (C-5) and 102.5 (C-18) In addition, H-3a shows HMBC correlation with one carbon resonance at 21.9 (C-1). On the other hand, the proton signal at 2.27 (H-3b) displays HM BC correlations with carbons 160.4 (C-4), 102.5 (C-18) and 28.9 (C-2). 180 160 140 120 100 80 60 40 20 P C-15 C-13 C-4 C-14 C-18 C-2' C-1'/ 3' C-10 C-5 C-9 C-6 C-8 C-11 C-12 C-3 C-2C-7 C-1C-19 C-16 C-17 C-20 40 35 30 25 20 15 PPM(a) (b)

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121 5.685 5.683 4.913 4.488 4.485 3.830 3.821 2.137 2.134 1.477 1.459 1.438 1.434 1.421 1.415 1.020 0.791 0.779 0.722 5 4 3 2 1 PP M H-14 H-2` H-18 H-1`/ H-3` H-16 H-19 H-17 H-20 H-3b Figure 71. 1H NMR spectrum of palmadorin A ( 110 ) (500 MHz, CDC13 ) 100 80 60 40 20 PPM Indirect Dimension 1 5 4 3 2 1 PPM Direct Dimension Figure 72. gHSQC spectrum of palmadorin A ( 110 ) (500 MHz, CDCl3)

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122 6 5 4 3 2 1 PPM Direct Dimension 200 150 100 50 0 PPM Indirect Dimension 1 Figure 73. gHMBC spectrum of palmadorin A ( 110 ) (500 MHz, CDCl3) O O OH OH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1' 2' 3' gCOSY gHMBC Figure 74. Key gHMBC and gCOS Y correlations of palmadorin A ( 110 )

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123 The singlet at 1.02 (H-19) integrated for th ree protons and exhibited HMBC correlations to quaternary carbons 1 60.4 (C-4), C-5 (40.0), methine at 48.9 (C-10) and a methylene at 37.4 (C-6). The proton signal at 1.03 (H-10), in turn shows HMBC correlations to 28.6 (C-2), 40.0 (C-5 ) and 20.8 (C-19), confir ming the structural assignments for the A ring of palmadorin A. The 3H singlet at 0.71 shows HMBC correlation to 48.7 (C-10), the quaternary carbon at 39.5 (C-9) and a methine at 36.9 (C-8). Meanwhile, the doublet at 0.79, indicative of another methyl, exhibits HMBC correlations to the same carbons suggesting that they are located on adjacent carbons. In addition, it shows further HMBC correlations with the methine at 27.6 (C-7). Both prot ons attached to C-7, 1.44 (H-7a) and 1.47 (H-7b) exhibit HMBC connectivity to C-8 and C-5 constituting the B ring of palmadorin A ( 110 ) (Table 8). The broad singlet at 5.68 indicative of an olefin displayed HMBC correlation to the quaternary carbon 167.0 (C-15), assigned as an ester carbonyl, as well as methylene 34.8 and methyl at 19.7 (C-16). The proton resonance of the latter methyl signal was observed as a doublet at 2.14 in turn displayed HMBC correlations to the former carbonyl, another quaternary carbon signal at 163.9 and the above methylene. These assignments are consistent with a trisubstituted double bond attached to the ester carbonyl and of a methylene group adjacent to the qua ternary carbon (C-13). Both protons attached to this methine show distinct HMBC correlations with 163.7 (C-13), 114.4 (C-14), 19.5 (C-16) and 36.1 (C-11). The proton signals at 1.33 and 1.43 (H-11a and H-11b) show

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124 HMBC connectivity to carbons 36.6 (C-8), 39.3(C-9), 48 .7 (C-10), 34.6 (C-12), 163.7 (C-13) and 18.1 (C-20) defining the side chain of the B ring at C-9. Table 9. NMR data for palmadorin A ( 108 ) (1H, 500 MHz, 13C, 125 MHz, CDC13) 13 C 1H (ppm, mult. J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1` 2` 3` 21.6 28.6 33.0 160.4 40.0 37.2 27.4 36.7 39.3 48.7 36.1 34.6 163.7 114.3 166.7 19.5 16.0 102.5 20.8 18.0 62.7 74.4 62.7 1.43 (1H, m) 1.48 (1H, m) 1.23 (1H, m) 1.87 (1H, m) 2.09 (1H, m) 2.27 (1H, m) 1.50 (1H, m) 1.58 (1H, m) 1.44 (1H, m) 1.47 (1H, m) 1.39 (1H, m) 1.03 (1H, m) 1.33 (1H, m) 1.44 (1H, m) 1.85 (1H, m) 1.96 (1H, m) 5.68 (brs s)) 2.14 (3H, s) 0.79 (3H, d, 6) 4.48 (2H, d, 1.5) 1.02 (3H, s) 0.71 (3H, s, 6.0) 3.83 (2H, d, 4.5) 4.91 (1H, m) 3.83 (2H, d, 4.5) 1, 3 1, 4, 5, 18 2, 4, 18 5, 8 5, 7, 10, 19 5, 8 5, 8 2, 5, 19, 20 8, 9, 10, 12, 13, 20 8, 9, 10, 12, 13, 20 11, 13, 14, 16 11, 13, 14, 16 12, 15, 16 12, 13, 14 7, 8, 9 3, 4, 5 4, 5, 6, 10 8, 9, 10, 11 2 3 1 3 15 1 2

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125 The C-15 ester carbonyl at 166.7 displays HMBC correlation to the multiplet at 4.91 (H-2 ). H-2 in turn displayed COSY correlation to the doublet at 3.83 (H-1 / H-3 ) suggesting their presence on neighboring carbon s that have downfield shifts due to an oxygen substituent. Meanwhile the latter signal, 3.83 (H-1 / H-3 ) integrated for four protons and the carbon attached to it showed significantly incr eased signal in tensity in the 13C NMR spectrum suggesting they are hydroxy methylene groups with identical carbon ( 62.7) and proton ( 3.83) chemical shifts. Therefor e, a glyceride moiety must be attached to the es ter carbonyl at C-2 of the glycerol. The diterpenoid acid involved in the formati on of palmadorin A belongs to the category of clerodane diterpenoids. This carbon skelet on has been reported ea rlier from tropical plants belonging to the family Annonaceae.101,102 3.2.3 Stereochemical determination of palmadorin A ( 110 ) The decalin of palmadorin A ( 110 ) can exist in either cis or trans conformations. In order to determine the correct conf ormation, the ROESY spectrum (Figure 75) of palmadorin A was investigated. In addition, the specific proton signals be longing to the appendages that are most likely to reflect the stereochemistry were irradiated selectively employing a 1D NOESY experiments (Figure 76) to further confirm those observations.

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126 6 5 4 3 2 1 PPM Direct Dimensio n 6 5 4 3 2 1 PPM Indirect Dimension 1 Figure 75 ROESY spectrum of palmadorin A ( 110 ) (500 MHz, CDC13)

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127 Selective Irradiation of H-19 2.0 1.5 1.0 0.5 0.0 PP M H-20 H-19 Selective Irradiation of H-20 1.5 1.0 0.5 0.0 PP M H-19 H-17 H-20 Figure 76. Selective 1D NOE experiments of palmadorin A ( 110 ) (500 MHz, CDCl3)

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128 The protons of the C-19 met hyl group of palmadorin A ( 110 ) show distinct ROESY correlation to H-3b, H-7b and H11b defining the top face of the A ring of palmadorin A (Figure 75). The axial orientation of H3-20 on the top face of the decalin was confirmed by the observation of strong NOE between H3-20 and H-19, when both peaks were irradiated separately (Figures 76, 77) H-2b and H-10 show ROESY cross peaks demonstrating that they belong on the bo ttom face of the A ring. The exomethylene protons at C-18 show strong co rrelations with H-3a and H-6b affirming that the former was equatorial and the latter was axial. H H H H CH3 H H CH3 CH3 H CH2 H H 5 4 19 18 10119 8 2 3 17 20 6 R Figure 77. Key ROESY correlations of palmadorin A ( 110 ) H3-20 shows further cross peaks with H-11b, H-12b and H3-17, confirming the axial orientation of C-20. These assign ments were consistent with the trans decalin conformation for palmadorin A as depicted in Figure 77. 3.2.3.1 Absolute stereochemistry determination Circular dichroism spectroscopy is a very convenient method to determine absolute stereochemistry of organic compounds. In the case of compound 110 this technique can be implemented to determine the absolute stereochemistry of C-5 by converting the A ring that has an exomethylene at C-4 into a cyclohexanone which co uld give rise to n* transitions creating a CD spectrum i ndicative of its stereochemistry.

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129 Hence, palmadorin A ( 110 ) was subjected to ozonolysis under oxidative conditions and the product was separated and pu rified by silica gel chromat ography. Interpretation of the 2D NMR data of the product establis hed its identity as the diketone 111 (Scheme 3). O O OH OH 18 O O 4 4 13 14 13 1. O3 / CH2Cl2 30 mins (-80 C) 2. DMS14 15 16 17 Palmadorin A ( 110 ) Diketone 113 Scheme 3. Ozonolysis of palmadorin A ( 110 ) The product showed two quaternary carbon signals at 216.3 (C-4) and 209.1 (C-13) indicating the presence of two ketones. The former carbon resonance showed distinct HMBC connectivity (Figure 79) with proton signals 2.58 (H-3a) and 1.15 (H3-15) resulting in its assignment as C-4. The latter quaternary carbon was correlated to proton signals 2.16 (H3-14) and 2.34 (H-12b) providing eviden ce for the presence of another ketone at C-13. Based on the key HMBC correlations of the methyls H3-15, H3-17 and methine proton H-10 th e structure of the pr oduct was confirmed as 113 This demonstrated the cleavage of bot h double bonds in palmadorin A ( 110 ) during the ozonolysis to give rise to two ketones.

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130 O O 4 5 10 13 14 15 16 17 Figure 78. Key gHMBC correlations of 113 The ROESY correlations discussed earlier confirmed the trans decalin conformation of the A and B rings of palmerolide A. Nevertheless, this trans decalin conformation can exist in two possible enantio meric forms (Figure 78). Iden tification of the correct enantiomer was accomplished using circular dichroism spectroscopy. Cotton effect value 96 = +0.07 Figure 79. Two possible trans decalin enatiomers for palmadorin A ozonolysis product According to the principles of CD spectroscop y when the octant rule is applied to the above two enantiomers, 113a should give a positive cotton effect where as 113b should indicate a negative cotton effect.103 Therefore, the cotton effect observed for the diketone O O 113a 113b O

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131 113 can be used to distinguish the particular enatiomeric form of palmadorin A. When the CD spectrum of diketone 113 was recorded (Figure 80), it displayed a negative cotton effect confirming that palmadorin A exist as enantiomeric form 113b Hence the absolute stereochemistry of palmadorin A was established as 5 S 8 S 9 R and 10 S ( 110 ). 260280300320340360 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Palamdorin A diketoneMolar ElepticityWavelength (nm) Figure 80. CD spectrum of compound 113 showing a negative cotton effect O O OH OH 4 5 8 9 10 14 15 16 17 18 19 2' 20 Figure 81. Absolute stereochemistry of Palmadorin A ( 108 )

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132 3.2.4 Characterization of palmadorin B ( 111 ) Palmadorin B ( 111 ) was isolated as colorless, viscous oil and showed an intense FABMS peak at m/z = 379.4 (Figure 82), indicative of [M (CH3CO)]+. It further displayed another prominent peak at m/z = 421.4, indicative of [M H]+. Analysis of the HRFABMS data, gave an accurate mass of 421.2974, consistent with the molecular formula C25H41O5. Figure 82. LRFABMS spectrum of palmadorin B ( 111 )

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133 The 13C NMR spectrum (Figure 83) of palmador in exhibited 25 carbon signals supporting the HRFBMS data. The carbon signals at 171.2 and 166.3 were assigned as ester carbonyls whereas the signals at 163.6, 160.6, 114.6 and 102.8 appear ed to be olefinic carbons. The three carbon signals at 71.6, 62.7 and 61.9 were char acteristic of carbons bearing oxygen. Figure 83. 13C NMR spectrum of palmadorin B ( 111 ) (125 MHz, CDC13) A complete structure elucidation of palm adorin B was accomplished by an extensive analysis of its HMBC (Figure 84) and HSQC (Figure 85) data. The COSY spectrum (Figure 86, 87) was used to get supporting information wherever it was appropriate. 180 160 140 120 100 80 60 40 20 P C-15 OCOCH3 C-13 C-4 C-14 C-18 C-2' C-1' C-1' C-10 C-5 C-9 C-6 C-8 C-11 C-12 C-3 C-2 C-7 C-19 C-1 C-16 C-17 C-20 COOCH3 40 35 30 25 20 PPM(a) (b)

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134 6 5 4 3 2 1 PPM Direct Dimension 200 150 100 50 0 PPM Indirect Dimension 1 Figure 84. gHMBC spectrum of palmadorin B ( 111 ) (500 MHz, CDC13) 160 140 120 100 80 60 40 PPM Indirect Dimension 1 6 5 4 3 2 1 PPM Direct Dimension Figure 85. gHSQC spectrum of palmadorin B ( 111 ) (500 MHz, CDC13)

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135 6 5 4 3 2 1 PPM Indirect Dimension 1 6 5 4 3 2 1 PPM Direct Dimension Figure 86. gCOSY spectrum of palmadorin B ( 111 ) (500 MHz, CDC13) O O OH O 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1' 2' 3' gCOSY gHMBC O Figure 87. Key HMBC and COSY correlations of palmadorin B ( 111 )

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136 The sharp doublet at 4.48 in the 1H NMR spectrum (Figure 88) of the compound ( 111 ) integrated for two hydrogens and showed HS QC correlations with a single carbon at 102.8 demonstrating that it was an exomethyl ene. These exomethylene protons showed distinct HMBC correlations to carbons 34.8 (C-3), 160.6 (C-4) and 40.2 (C-5), respectively. The methylene proton H-3a ( 2.08) exhibited HMBC correlation to 29.0 (C-2), 40.4 (C-5) and 10 2.9 (C-18) whereas H-3b ( 2.26) is related to only 29.4 (C-2) and 102.9 (C-18) (Table 9). Figure 88. 1H NMR spectrum of palmadorin B ( 111 ) (500 MHz, CDC13) 5.642 5.063 4.481 4.479 4.268 4.259 3.726 3.718 2.139 2.137 2.119 2.073 2.070 2.052 1.474 1.452 1.432 1.424 1.412 1.398 1.017 0.967 0.789 0.776 0.720 0.706 6 5 4 3 2 1 PP M H-14 H-2` H-18 H-3` H-1` H-16 OCOCH3 H-20 H-17H-19H-3b H-2b H-6a H-2a H-1b

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137 Table 10. NMR data of palmadorin B ( 111 ) (1H, 500 MHz, 13C, 125 MHz, CDC13) 13C 1H (ppm, mult. J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 3 COOC H3 C OOCH3 21.8 29.0 34.4 160.7 40.4 37.7 27.8 37.0 39.7 49.1 36.4 34.9 163.9 114.7 166.8 19.8 16.4 102.9 22.3 18.5 62.9 72.0 62.2 21.2 171.2 1.42 (m) 1.46 (m) 1.21 (m) 1.86 (m) 2.09 (m) 2.27 (m) 1.47 (m) 1.57 (m) 1.44 (m) 1.47 (m) 1.39 (m) 1.03 (m) 1.33 (m) 1.44 (m) 1.85 (m) 1.96 (m) 5.68 (br s) 2.14 (3H, s) 0.78 (3H, d, 6.5) 4.48 (2H, d, 1) 1.02 (3H, s) 0.72 (3H, s) 4.26 (2H, m ) 5.06 (1H, m) 3.72 (2H, d, 4) 2.05 (3H, s) 3 2, 5, 18 2, 18 8, 19 5, 7, 8, 10, 19 5, 6, 7, 8 9, 10, 12 8, 9, 10, 12, 20 11, 16 11, 16 12, 15, 16 12, 13, 14 7, 8, 9 3, 4, 5 4, 5, 6, 10 8, 9, 10, 11 2 3 C OOCH3 15, 1 1 2 C OOCH3

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138 The 3H singlet at 1.02 (C-19), exhibited HMBC correlation to the quaternary carbons 160.6 (C-4), 40.4 (C-5), 37.7 (C -6) and 49.1 (C-10), establis hing a major portion of the A ring (Figure 87). The rest of the six me mebered ring was characterized by the COSY correlations between H-2a, H-2b and H-1a, H-1b and H-10. The sharp 3H singlet at 0.72 (H3-20) displayed HMBC co rrelations to carbons 37.0 (C-8), 39.7 (C-9), 49.1(C-10), 36.4 (C11). Meanwhile, the 3H doublet at 0.78 (H3-17) exhibited HMBC cross peaks with carbons 27.8 (C-7), 37.0 (C-8), and 39.7 (C-9). Unambiguous assignment of the rest of the B ring was accomplished on the basis of HMBC correlation of H-6b ( 1.57) with carbons 40.4 (C-5), 37.0 (C-8) and 49.1 (C10) and that of H-6a ( 1.47) with 37.0 (C-8). The broad singlet observed at 5.64 (H-14) showed HMBC correlation to the ester carbonyl 166.3 (C-15) and the methylene 34.9 (C-12). On the other hand, the 3H doublet at 2.12, indicative of a vinyl methyl, displayed HMBC correlation with 34.9, a quaternary carbon at 163.6 and a methine at 114.6. All these observation were consistent with a trisubstituted double bond conjugated to an ester carbonyl. The connectivity between this side chain into th e A and B rings could be established by the HMBC correlations of 1.33 (H-11a) and 1.44 (H-11b) with 39.7 (C-9), 49.1 (C-10) and 34.9 (C-12). The ester carbonyl at 166.3 (C-15) exhibited HMBC corre lation to the proton signal at 5.06, observed as a multiplet. The doublets at 3.73 and 4.26 correlated in the COSY

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139 spectrum with the proton at 5.06 (H-2 ), establishing the presence of the glyceride moiety. The proton resonances at 4.26 (H-1 ) not only shows HMBC coupling with carbon shifts 72.0 (C-2 ) and 62.2 (C-3 ), but also with th e ester carbonyl at 171.2. The proton at 4.26 (H-1 ) also correlates to the methyl singlet 2.05 which suggest the presence of an acetyl group. All these obser vations are consistent with a glyceride moiety attached to a carbonyl via an ester linkage at C-2 which is acetylated at C-1 3.2.5 Stereochemistry determination of palmadorin B ( 111 ) The stereochemical elucidation of palmadorin B ( 111 ) was undertaken by analysis of its ROESY spectrum. Selective one dimensional NOE experiments were also employed to confirm these assignments. The carbon and proton spectral data of the A and B rings of palmadorin A and B showed significant similarities suggesti ng that they have similar ster eochemistry. As observed in palmadorin A, methyl group C-19 showed RO ESY correlations (Fi gure 89) to H-3b, H7a indicating that they define the top face of the A ring (Figure 90). In addition, the H3-19 methyl shows a strong ROESY correlation with H3-20 demonstrating th at they both are on the top face of the decalin wi th an axial orientation. Furt her, the exomethylene protons at C-18 displayed ROSEY cross peaks with H-3a and H-6a, in agreement with this observation.

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140 8 7 6 5 4 3 2 1 PPM Direct Dimension 8 7 6 5 4 3 2 1 PPM Indirect Dimension 1 Figure 89. ROESY spectrum of palmadorin B ( 111 ) (500 MHz, CDC13) H H H H CH3 H H CH3 CH3 5 4 19 18 10 11 9 8 2 3 17 20 6 H H H H 71 Figure 90. Palmadorin B ( 111 ) ROESY correlations Therefore, the stereochemistry of compound 109 can be illustrated as displayed in Figure 91. Hence, by analogy with the stereochemical features of palmadorin A, the absolute stereochemistry of palmadorin B is assigned as 5 S 8 S 9 R and 10 S

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141 O O OH O 4 5 8 9 10 14 15 16 17 18 19 2' 20 O 1' 3' Figure 91. Absolute stereochemistry of palmadorin B ( 111 ) 3.2.6 Characterization of palmadorin C ( 112 ) Palmadorin C ( 112 ) was isolated as colorless oil. The LRFABMS (Figure 92) provided a m/z = 377.3 indicative of [M H2O]+. HRFABMS on this mass peak suggested molecular formulae of C23H38O4. Figure 92. LRFABMS spectrum of palmadorin C ( 112 )

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142 Analysis of the 13C NMR spectrum (Figure 93) of palmadorin C ( 112 ) showed 23 signals. The carbon resonance at 166.9 was assigned as an ester car bonyl where as the signals at 163.5, 163.5, 145.1, 120.0 and 114.8 were indicative of olefins. Meanwhile, the signals 74.6, 73.8, 63.0 and 62.9 indicated f our carbons bearing oxygen. Figure 93. 13C NMR spectrum of palmadorin C ( 112 ) (125 MHz, CDCl3) According to HSQC spectrum (Figure 94) the 1H NMR peak at 5.13 (Figure 95) correlated to the carbon resonance at 120.0. The carbon signals at 145.1 (C-4) and 37.8 (C-5) did not show HSQC correlation sugge sting that they were quaternary. The 3H signal at 1.60 correlated in the HMBC (Figure 96) spectrum with all above three carbons indicating a trisubstituted double bond. 150 100 50 0 P C-15 C-13 C-4 C-3 C-14 C-2' C-7 C-1'/ C-3' C-10 C-6 C-8 C-5 C-11 C-9 C-12 C-2 C-19 C-20 C-16 C-18 C-1 C-17 40 35 30 25 20 15 PPM(a) (b)

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143 160 140 120 100 80 60 40 20 PPM Indirect Dimension 1 6 5 4 3 2 1 PPM Direct Dimension Figure 94. gHSQC spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3) 5.704 5.131 4.918 4.908 4.019 4.013 3.830 3.821 2.177 2.146 2.099 1.942 1.931 1.919 1.906 1.601 1.574 1.402 1.394 1.388 1.270 1.040 1.026 6 5 4 3 2 1 PP M H-14 H-3 H-2` H-7 H-1`/ H-3` H-16 H-18 H-17 H-19 H-20 Figure 95. 1H NMR spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3)

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144 6 5 4 3 2 1 PPM Direct Dimension 160 140 120 100 80 60 40 20 0 PPM Indirect Dimension 1 Figure 96. gHMBC spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3) The 1H NMR signals at 1.27, depicting a si nglet and integrating for three protons, displayed HMBC correlation to carbons C-4 ( 145.2), C-5 ( 37.80), C-6 ( 43.14) and C-10 ( 38.5). The connectivity from C-3 ( 120.0) to C-10 ( 46.9) via two methylenes C-1 ( 18.2) and C-2 ( 27.0) was established by COSY correlations (Figures 97, 98) giving rise to the A ring of palmadorin C. The 3H doublet at 1.01 displayed HMBC correlation to 73.9 (C-7) and 39.5 (C-8). A 3H singlet at 0.99 correlated in the HMBC with 39.5 (C-8), 38.5 (C-9), 46.9 (C-10) and 37.6 (C-11) (Table 10). In addition, the 1H NMR signal at 1.37 (H-10) showed HMBC correlations to 18.2 (C-1), 145.2 (C-4), 37.8 (C-5 ), 43.1 (C-6), 22.0 (C-19) and

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145 20.3 (C-20). Meanwhile, H-6b ( 2.10) demonstrated HMBC cross peaks to 22.0 (C19), 73.9 (C-7) and 39.5 (C-8), establishing the B ring. 6 5 4 3 2 PPM Indirect Dimension 1 6 5 4 3 2 1 PPM Direct Dimension Figure 97. gCOSY spectrum of palmadorin C ( 112 ) (500 MHz, CDCl3) O O OH OH 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1' 2' 3' gCOSY gHMBC OH Figure 98. Key HMBC and COSY correlations of palmadorin C ( 112 )

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146 Table 11. NMR data of palmadorin C ( 112 ) (1H, 500 MHz, 13C, 125 MHz, CDC13) 13 C 1H (ppm, mult. J (Hz) gHMBC 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 3 18.2 27.0 120.0 145.2 37.8 43.1 73.9 39.5 38.5 46.9 37.6 35.1 163.4 114.8 167.0 19.7 12.7 18.3 22.0 20.3 63.1 74.8 63.1 1.54 (1H, m) 1.58 (1H, m) 1.98 (1H, m) 2.08 (1H, m) 5.13 (1H, m) 1.39 (1H, m) 2.10 (1H, m) 4.02 (1H, m) 1.51 (1H, m) 1.37 (1H, m) 1.39 (1H, m) 1.51 (1H, m) 1.93 (1H, m) 1.98 (1H, m) 5.70 (1H, s) 2.16 (3H, s) 1.01 (3H, d, 7.5) 1.60 (3H, s) 1.27 (3H, s) 0.99 (1H, s) 3.82 (2H, d, 4.5) 4.91 (1H, m) 3.82 (2H, d, 4.5) 4, 5, 10, 19, 5, 7, 8, 9, 10, 19 17 1, 4, 5, 6, 9, 19, 20 5, 10, 19 8 16 12, 15, 16 12, 13, 14 7, 8 3, 4, 5 4, 5, 6, 10 8, 9, 10, 11 The singlet at 5.70 (H-14) indicated distinct HMBC correlations to 35.10 (C-12), 167.0 (C-15) and 19.7 (C-16). Due to its relatively low field chemical shift, the methyl at 2.16 (H3-16) could be assigned as a vinyl methyl. It show ed further HMBC correlation to the methine C-12 ( 35.1), quaternary C-13 ( 163.4) and C-14 ( 114.9). These assignments are consistent with a trisub stituted double bond conjugated to an ester

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147 carbonyl. The connectivity of C-12 ( 35.10) and C-9 ( 38.5) via C-11 ( 37.6) was established by the observed CO SY correlations of H-11a ( 1.39), H-11b ( 1.51) with H12a ( 1.93), H-12b ( 1.98). HMBC correlation was observed between the multiplet at 4.92 (H-2 ), attached to a hydroxymethine 74.8 and the ester carbonyl at 167.0. In addition, the multiplet at 4.92 (H-2 ) shows COSY correlation to the 4H doublet at 3.82 (H-1 /H-3 ) which showed HSQC correlations to two hydroxymethines C-1 and C-3 ( 63.1). Based on these data palmadorin C ( 112 ) was characterized as a di terpenoid glyceride. It shares similar pattern of glyceride formation with palmadorin A ( 110 ) and B ( 111 ) with the ester linkage established at C-2 of the glycerol. This is relatively uncommon among diterpenoid glycerides. 3.2.7 Stereochemistry determination of palmadorin C ( 112 ) The relative stereochemistry of palmador in C was studied by NOE spectroscopy. The olefinic proton H-3 displayed strong ROESY correlati ons to H-2b and th e vinylic methyl at 1.60 (H3-18). A cross peak for the methyls at H3-19 and H3-20 indicated that they both are on the same face of the decalin syst em. Similarly H-7 shows its spatial proximity with H-8 and H-6 by means of ROSEY correlati ons and H-6a in turn correlated to H-10. H-6a, H-7 and H-8 are therefore all oriented toward the opposite face of the decalin compared to H3-19 and H3-20 (Figure 99).

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148 H3C H H CH3 H H OH H H CH3 H3C R H 1 2 3 45 6 7 8 9 10 18 19 20 17 Figure 99. Key ROESY correla tions of palmadorin C ( 112 ) Mosher’s method was chosen to determine th e stereochemistry of C-7 taking advantage of the secondary hydroxyl group. However, pr ior to that the prim ary hydroxyl groups in the glyceride moiety needed to be protected. 3.2.7. 1 Diacetyl derivative of palmadorin C ( 114 ) Palmadorin C ( 112 ) was allowed to react with acetic anhydride overnight and the resultant product was separated and purified by chromatography on silica. Two equivalents of acetic anhydride was used to ma ke sure the acetylation takes place only on desired primary hydroxyl groups. The purified product was identified as palmadorin C diacetate ( 114 ) by NMR spectroscopy (Figure 100, 101) and mass spectroscopy.

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149 O O OH OH 3 4 5 8 9 13 14 15 16 17 18 19 20 1' 2' 3' O O OAc OAc 3 4 5 8 9 13 14 15 16 17 18 19 20 1' 2' 3' Ac2O (2eq) DMAP TEA CH2Cl225 C, 12hr OH OH 112 114 Scheme 4. Acetylation of palmadorin C ( 112 ) 5.650 5.272 5.253 5.136 4.276 4.268 4.262 4.244 4.202 4.191 4.178 4.167 2.131 2.131 2.056 2.023 1.654 1.604 1.276 1.209 1.024 1.010 1.000 6 5 4 3 2 1 PP M Figure 100. 1H NMR spectrum of palmadorin C diacetate ( 114 ) (500 MHz, CDCl3) The 1H NMR spectrum of the diacet ate showed a singlet at 2.06 indicative of a methyl group of an acetyl affirming that acetylation has taken place. In a ddition, instead of the doublet at 3.82 representing the H-1 a, H-1 b, H-3 a and H-3 b, a multiplet was observed at 4.23. The HSQC correlations confirmed their attached to C-1 and C-3 A

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150 HMBC correlation observed between the carbon at 170.8 representing the carbonyl of the acetyl group and the multiplet at 4.23 provided further confirmation for acetylation (Figure 102). The hydroxymethine proton at C7 did not show a noticeable difference in its chemical shift affirming that it was not esterified. Fu rther it did not show HMBC correlation to carbonyls. The other proton and carbon signals showed HMBC and COSY correlations in agreement with palmadorin C. 170.791 165.744 163.287 145.166 120.010 114.720 73.917 68.228 62.700 46.859 43.179 39.505 35.094 31.812 29.928 26.948 22.882 22.022 22.013 12.712 160 140 120 100 80 60 40 20 PP M Figure 101. 13C NMR spectrum of palmadorin C diacetate ( 114 ) (125 MHz, CDCl3)

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151 O O O O 3 4 5 8 9 13 14 15 16 17 18 19 20 1' 2' 3' O O OH Figure 102. Key HMBC correlations of palmadorin C diacetate ( 114 ) Analysis of the compound 114 by FABMS (Figure 103) displayed a prominent peak at m/z = 477.2 which was attributed to [M H]+. This indicates a molecular weight of 478, consistent with the addition of two acetyl substituents. Figure 103. LRESIMS spectrum of palmadorin C diacetate ( 114 )

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152 3.2.7.2 R -MTPA ester of palmadorin C diacetate ( 115 ) The diacetate of palmadorin C ( 114 ) was treated with R -MTPACl for 48 hours and the product was separated and purified by chroma tography on silica gel. The purified R MTPA ester 115 was analyzed by NMR and mass sp ectroscopic methods in order to confirm the formation of MTPA derivative. LRESIMS (Figure 104) of compound 115 produced a peak at m/z = 717.1 attributed to [M + Na]+. A HRFABMS produced a molecular mass of 717.3213 consistent with molecular formulae of C37H49F3O9 (calc. 717.3219) Figure 104. LRESIMS spectrum of palmadorin C diacetate R -MTPA ester ( 115 )

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153 O O O O 3 4 5 8 9 13 14 15 16 17 18 19 20 1' 2' 3' O O OR ( 115 ) R= R -MTPA ( 116 ) R = S -MTPA The 1H NMR spectrum (Figure 105) confirmed the attachment of MTPA moiety by displaying two multiplets at 7.35 and 7.52, indicating the aromatic protons of the phenyl ring and the 3H singlet at 3.58 representing the methoxy group. The HSQC spectrum (Figure 106) showed that the prot on that gives rise to the signal at 5.35 is attached to C-7. Attachment of the MTPA moie ty to C-7 by means of an ester group was further illustrated by the significant decrease of the chemical shif t of the neighboring protons. The sharp singlet at 0.53 was identified as the methyl group at C-19 by HMBC (Figure 107) correlations to the quaternary 144.5 (C-4), methine 46.4 (C-10) and methylene 40.4 (C-6). The 3H doublet at 0.92 (H3-17) and the 3H singlet 0.80 (H3-20), both show HMBC correlations to the carbons at 38.4 and 38.9, assigned to C-9 and C-8 respectively. This assignment was furthe r confirmed by the HMBC cross peaks observed between the singlet 0.80 and the carbon 46.4 (C-10). H-7 exhibi ted COSY correlation with proton signals 1.92, 1.37 and 1.69 indicating that th ey represent H-6a, H-6b and H-8. The 3H singlet at 1.43 was identified as the methyl at C-18 based on its HMBC correlations to 120.6 (C-3), 145.2 (C-4) and 37.3 (C-5).

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154 7.530 7.364 7.355 5.628 5.353 5.263 5.073 4.239 4.181 3.596 2.129 2.051 2.048 1.537 1.429 0.946 0.917 0.806 0.803 0.532 8 7 6 5 4 3 2 1 PP M H-14 H-7 H-2` OCH3 H-3 H-18 H-20 H-17 H-19 Figure 105. 1H NMR spectrum of palmadorin C diacetate R -MTPA ester ( 115 ) (500 MHz, CDCl3) 200 150 100 50 PPM Indirect Dimension 1 8 7 6 5 4 3 2 1 PPM Direct Dimension Figure 106. gHSQC spectrum of palmadorin C diacetate R -MTPA ester ( 115 ) (500 MHz, CDCl3)

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155 8 7 6 5 4 3 2 PPM Direct Dimension 200 150 100 50 0 PPM Indirect Dimension 1 Figure 107. gHMBC spectrum of palmadorin C diacetate R -MTPA ester ( 115 ) (500 MHz, CDCl3) The rest of the carbon and proton resonances we re not affected by the attachments of the MTPA moiety. 3.2.7.3 S -MTPA ester of palmadorin C diacetate ( 116 ) The diacetate of palmadorin C ( 114 ) was treated with S -MTPACl for 48 hours and the product was separated and pur ified by chromatography on silica gel. The purified S MTPA ester ( 116 ) was analysed by NMR and mass spectroscopic methods in order to confirm the formation of the MTPA derivative.

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156 LRESIMS of compound 116 (Figure 108) displayed m/z = 717.1 attributed to [M + Na]+. HRFABMS analysis produced a molecular ma ss of 717.322 consistent with molecular formula of C37H49F3O9 (Calc. 717.3219). Figure 108. LRESIMS of palmadorin C diacetate S -MTPA ( 116 ) The 13C NMR spectrum (Figure 109) of the pr oduct confirmed the formation of the MTPA derivative by demonstrating additional carbon signals at 127.36, 128.47 and 129.65 indicative of the aromatic ring and 166.95 representing the carbonyl involved in the ester group.

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157 170.778 165.653 162.695 144.054 129.729 128.577 127.983 127.587 120.586 114.864 78.013 68.274 62.670 46.406 40.442 39.109 38.184 37.180 37.056 34.885 29.926 26.754 21.161 20.969 19.621 19.149 18.145 17.927 11.996 160 140 120 100 80 60 40 20 PP M C-7 C-2` C-1`/ C-3` Figure 109. 13C NMR spectrum of palm adorin C diacetate S -MTPA ester ( 116 ) (125 MHz, CDCl3) The 1H NMR spectrum of 116 (Figure 110) provided evidence for the attachment of MTPA moiety by displaying two multiplets at 7.53and 7.37 indicating the aromatic protons belong to the phenyl ring and the 3H singlet at 3.49 representing the methoxy group. The HSQC correlations (Figure 111) showed that the proton that gives rise to the signal at 5.44 is attached to C-7. Attachment of the MTPA moiety to C-7 by means of an ester group was further illustrated by the si gnificant decrease of th e chemical shift of the neighboring protons. The sharp singlet at 0.93 (3H) displayed HMBC correlations (Figure 112) to the quaternary carbon 143.6 (C-4), the methine 46.6 (C-10) and methylene 40.1 (C-6). The doublet at 0.75 (3H) and the singlet 0.63 (3H) both show HM BC correlations to carbons at 38.4 (C-9) and 39.3 (C-8) indicating that th ey represent the methyls at C-17

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158 and C-20 respectively. The latter singlet further displayed HMBC correlations with 46.6 (C-10) and 37.2 (C-11) further supportin g this assignment. Th e singlet (3H) at 0.93 was assigned as C-19 consider ing its HMBC cross peaks with 143.6 (C-4), 37.4 (C-5) and methine 40.06 (C-6). 7.446 7.028 5.612 5.443 5.251 5.132 4.261 4.168 3.508 2.116 2.050 1.535 1.232 0.937 0.746 0.625 7 6 5 4 3 2 1 PP M H-7 H-14 H-2` H-3 OCH3 H-20 H-17 H-19 H-18 Figure 110. 1H NMR spectrum of palmadorin C diacetate S -MTPA ester ( 116 ) (500 MHz, CDCl3) 180 160 140 120 100 80 60 40 20 PPM Indirect Dimension 1 8 7 6 5 4 3 2 1 0 PPM Direct Dimension Figure 111. gHSQC spectrum of palmadorin C diacetate S -MTPA ester ( 116 ) (500 MHz, CDCl3)

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159 9 8 7 6 5 4 3 2 1 PPM Direct Dimension 200 150 100 50 0 PPM Indirect Dimension 1 Figure 112. gHMBC spectrum of palmadorin C diacetate S -MTPA ester ( 116 ) (500 MHz, CDCl3) Based on COSY correlations exhibited by H-7, the proton signals 1.48, 2.06 and 1.65 were identified as H-6a, H-6b and H-8 respec tively. The vinyl methyl C-18 appeared at 1.54 was assigned based on the HMBC relationships with 120.7 (C-3), 144.0 (C-4) and 37.4 (C-5). The rest of the carbon and proton shif ts were consistent with that observed for the starting material.

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160 3.2.7.4 Application of Mosher’s method Once all the signals are assigned for the respec tive protons in a model according to the Mosher’s method (Figure 113), the stereo chemistry of C-7 was found to be R. Considering the ROESY correlations observed, the final structure of palmadorin C was assigned as 5 R 7 R 8 S 9 R and 10 R ( Figure 114). H3C CH3 H H OMTPA H H CH3 H3C +0.11 +0.40 +0.11 +0.40 +0.09 -0.17 -0.18 -0.043 4 5 6 7 8 9 10 18 19 17 20 Figure 113. value assignments for palmadorin C diacetate MTPA esters O O OH OH 4 5 8 9 10 14 15 16 17 18 19 2' 20 OH 7H Figure 114. Absolute stereochemistry of palmadorin C ( 112 )

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161 3.3 Summary Nudibranches, being shell-less molluscs, ar e known to employ defensive chemicals in order to survive against predators. A comp rehensive chemical investigation of the Antarctic nudibranch Austrodoris kerguelenensis afforded a series of new clerodane diterpenoid glyceride esters named palmadorins, which may be involved in its chemical defense. The structure elucidation of palmerodorin A-C was accomplished by spectroscopic methods and semi-synthetic derivatizations.

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162 Chapter 4 EXPERIMENTAL 4.1 General Procedure Optical rotations were measured on an Autopol IV automatic polarimeter using Na lamp at 25 C. Infrared spectra were obtained with Nicolete Avatar 320FT-IR as films. Ultraviolet-Visible e xperiments were measured on a He wlett-Packard 8452A diode array UV-Vis spectrometer. 1H and 13C, gHMQC, gHSQC, gHMBC and 1H-1H COSY NMR spectra were obtained on a Varian Inova 500 instrument, operating at 500 MHz for 1H and 125 MHz for 13C, using residual protonated solvent as 1H internal standard or 13C absorption lines of solvents for 13C internal standard. 1H chemical shifts were recorded relative to 7.24 (CDCl3), 4.78 (CD3OD) and 2.50 (DMSOd6) whereas the 13C shifts are referanced to 77.2 (CDCl3), 49.2 (CD3OD) and 39.5 (DMSO-d6). Low resonance mass spectra were recorded on a Aligent Teconologies LC/MSD VL electrospray ionization mass spectrometer. High resonance mass spectra were obtained on an Aligent Teconologies LC/MSD TOF el ectrospray ionization mass spectrometer. CD spectra were obtained with Aviv Inst ruments model 215 CD spectrometer. HPLC was performed on with a Shimadzu LC-8A mu ltisolvent delivery system connected to a Shimadzu SPD-10A UV-VIS tunable absorb ance detector and using YMC-Pack ODSAQ C-18 column. EM Science silica gel 60 of 230-400 mesh was used in flash column chromatography. TLC was carried out on What man K6F silica gel 60A TLC plates with 0.25 mm thickness. They were visualized by spraying with 5% phosphomolybdic acid in EtOH and heating.

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163 4.2 Isolation of Secondary metabolites from Synoicum adareanum Synoicum adareanum was collected at a depth of 80-100 feet at number of locations near Palmerstation in Antarctica by SCUBA diving, and the animals were frozen immediately after the collection. The samples were identif ied by Dr. Linda Cole, at the Smithsonian Institute, Washington, D.C. After freeze drying, 180 g of animals was sequentially extracted 3X with 1:1 CH2Cl2/MeOH for one day each. Upon evaporat ion of the solvent under reduced pressure, a reddish brown highly viscous oil was obtained. It was partitioned with EtOAc/H2O and the EtOAc layer was separate d, washed and dried with anhydrous Na2SO4. The removal of solvent un der reduced pressure gave a reddish brown semisolid (2.2 g). Fractionation of the EtOAc extract was perf ormed by step gradient flash chromatography on silica using 100 mL each of hexane, 2% 5%, 7%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 80% and 100% EtOAc in hexane fo llowed by 5%, 10%, 15%, 20% MeOH in EtOAc. The factions 3, 5 and 6 were furthe r separated by gradient elusion of 1%, 2%, 5%, 7% and 10% MeOH in CHCl3. Subsequent purification of eluate by HPLC using 40% water in MeCN (isocratic eluti on, 2 mL per min) afforded compounds 58 59 60 and 61 as white solids. The fract ions 8, 9 and 10 upon further purification with HPLC using 50% water in MeCN (isocratic el ution, 2 mL per min) yielded compounds 62 and 63 Since 62 and 63 found to be unstable in DMSO, the NMR investigations were performed in CD3OD.

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164 4.2.1 Palmerolide A ( 58 ) White solid; [ ]24D –1.6 ( c 0.5, MeOH); IR (thin film) 3360 (br), 2925, 2856, 1696, 1633, 1517, 1392, 1275, 1190 and 1080. UV (MeOH) max ( ): 224 (2670), 242 (2800), 296 (1775); 1H NMR (500 MHz, DMSOd6 ) (multiplicity, J (Hz), assignment): 9.84 (1H, d, 10.1, 24-NH ) 6.85 (1H, dd, 10.1, 14.2, H-24) 6.71 (1H, ddd, 5.0, 9.9, 15.2, H-3) 6.49 (2H, br OCONH 2 ) 6.04 (1H, dd 11.1, 14.6, H-15) 5.85 (1H, d 14.2, H-23) 5.77 (1H, d 15.3, H-2) 5.69 (1H, br s, 1.0, H-2 ) 5.59 (1H, d, 11.4, H-16) 5.54 (1H, dd, 7.7, 15.0, H-8) 5.48 (1H, dd, 2.9, 15.6, H-9) ) 5.41 (1H, ddd, 4.7, 10.1, 14.6, H-14) 5.20 (1H, d, 4.9, 10OH ) 5.13 (1H, d, 9.6, H-21) 4.84 (1H, ddd, 1.3, 7.4, 11.2, H-19) 4.72 (1H, d, 3.9, 7-OH ) 4.48 (1H, dd, 5.0, 10.5, H-11) 4.14 (1H, br s, H-10) 3.82 (1H, ddd, 4.4, 7.4, 7.6, H-7) 2.68 (1H, qdd, 6.5, 7.4, 9.6, H-20) 2.17 (1H, dd, 1.3, 13.2, H-18) 2.12 (3H, s, H-5 ) 2.11 (2H, m, H-4) 2.00 (1H, dd, 11.2, 13.2, H-18) 1.96 (2H, m, H-13) 1.83 (3H, s, H3-4 ) 1.70 (3H, s, H3-27) 1.61 (3H, s, H3-25) 1.59 (1H, m, H-12) 1.50 (1H, ddd, 4.5, 8.2, 11.2, H-6) 1.30 (1H, m H-6) 1.30 (1H, m H-5) 1.05 (1H, m, H-5) 0.98 (1H, m, H-12) 0.90 (3H, d, 6.5, H3-26) 13C NMR (125 MHz, DMSOd6) (assignment): 165.3 (C-1), 163.1 (C-1 ), 156.6 (OC ONH2), 151.7 (C-3 ), 149.2 (C-3), 133.6 (C-8), 132.5 (C-22), 132.0 (C-14), 131.5 (C-17), 129.7 (C-21), 128.9 (C-9), 126.1 (C -15), 125.7 (C-16), 121.8 (C-24), 120.3 (C-2), 117.9 (C-2 ), 116.4 (C-23), 73.7 (C-19), 72.5 (C-7), 72.3 (C-11), 69.00 (C-10), 43.6 (C-18), 38.1 (C-6), 36.9 (C-20), 32.6 (C-4), 29.5 (C-13), 29.5 (12), 27.1 (C-4 ), 24.8 (C-5), 19.2 (C-5 ), 16.9 (C-26), 16.2 (C-25), 12.6 (C-27) ; FABMS m/z (%) 737.5 (5, [M+ + dtt]+ ), 585.5 (10 [M + H] +), 501.4 (200), 309.1 (13), 275.1 (5), 195.1 (15), 155.1 (45): HRFABMS m/z 585.3539 (C33H49N2O7 requires 585.3540).

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165 4.2.2 Preparation of MTPA esters of palmerolide A Palmerolide A ( 58 ) (5 mg, 0.0086 mmol) was dissolved in CH2Cl2 and treated with Ror Smethoxytrifluorophenylacetyl chloride (MTPAC l) (10 eq) in the presence of Hunig’s base and DMAP. The reaction was stirred for 48 hrs. Upon concentr ation of the reaction mixture the two main products were purif ied by chromatography on silica gel (2% MeOH/CH2Cl2) followed by HPLC (silica gel 2% MeOH:CH2Cl2). 4.2.2.1 Palmerolide A 7-[( R )-MTPA] Ester ( 64 ) Colorless solid; 1H NMR (500 MHz, CD3OD) (multiplicity, J (Hz), assignment): 7.92 (s, NH), 7.52 – 7.45 (5H, m, Ph), 6.97 ( d, 14.4, H-24), 6.83 (ddd, 5.2, 10.1, 15.5, H-3), 6.12 (dd, 11.1, 14.2, H-15), 5.96 (d, 14.4, H-23) 5.99 (dd, 3.5, 15.5, H-9), 5.80 (d,15.5, H-2), 5.78 (ddd, 1.8, 8.6, 15.5, H-8), 5.74 (br t, 1.2, H-2 ), 5.67 (d, 11.1, H-16), 5.48 (m, H-7), 5.47 (m, H-14), 5.19 (dd, 0.7, 9.9, H21), 4.95 (m, H-19), 4.68 (ddd, 2.0, 5.3, 10.8, H-11), 4.34 (ddd, 1.9, 3.4, 5.2, H-10), 3.55 (s, OCH3), 2.77 (ddq, 6.6, 8.0, 9.9, H-20), 2.26 (br d,12.5, H-18a), 2.20 (d, 1.2, H3-5 ), 2.18 (m, H-4a), 2.10 (dd, 11.5, 12.8, H-18b), 2.07 (m, H-4b), 2.06 (m, H2-13), 1.92 (d, 1.2, H3-4), 1.82 (d, 0.7, H3-25), 1.69 (s, H3-27), 1.68 (m, H-6a), 1.65 (m, H-12a), 1.62 (m, H-6b), 1.38 (m, H-5a), 1.24 (m, H-5b), 1.19 (m, H-12b), 0.99 (d, 6.7, H3-26). 13C NMR (125 MHz, CD3OD) (assignment): 166.8 (C-1), 165.1 (C-1 ), 158.3 (OCONH2), 153.7 (C-3 ), 149.1 (C-3), 135.0 (C-9), 133.0 (C22), 132.0 (C-14), 131.8 (C-17), 130.8 (C-21), 129.7 (Ph), 128.4 (Ph) 128.4 (C-16) 127.5 (Ph), 127.5 (C-8), 126.9 (C-15), 121.5 (C -24), 121.2 (C-2), 118.2 (C-23), 117.6 (C-2 ), 78.2 (C-7), 76.1 (C-11), 74.9 (C-19), 70.4 (C-10), 54.8 (OCH3), 43.7 (C-18), 37.3 (C-20),

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166 33.9 (C-6), 32.2 (C-4), 30.1 (C -12), 29.6 (C-13), 26.2 (C-4 ), 24.4 (C-5) 19.0 (C-5 ), 16.2 (C-26), 15.0 (C-27), 11.8 (C-25); LRESIMS m/z 823.1 [M + Na]+; HRESIMS m/z 823.3779 (C43H55N2O9F3Na requires 823.3757). 4.2.2.2 Palmerolide A 7, 10–[( R, R )-MTPA] diester ( 66 ). 1H NMR (500 MHz, CD3OD) (multiplicity, J (Hz), assignment): 7.91 (s, NH), 7.367.47 (10H, m, Ph), 6.97 (d, 14.4, H-24) 6.78 (ddd, 5.6, 9.1, 15.3, H-3), 6.13 (dd, 10.7, 5.1, 11-15), 5.96 (d, 14.4, H-23), 5. 76 (d, 15.5, H-2), 5.74 (br t, 1.1, H-2`), 5.73 (dd, 5.8, 15, 11-9), 5.66 (d, 10.6, H-16), 5.57 (m, H-8) 5.55 (m, H-10), 5.53 (m, H-7), 5.44 (dd, 4.6, 10.0, H-14), 5.19 (d, 9.5, H-21), 4.95 (m, H-19), 4.84 (m, H-11), 3.58 (s, OCH3), 3.49 (s, OCH3), 2.78 (ddq, 7.0, 7.1, 10.5, H-20), 2.26 (b r d, 13.0, H-18a), 2.19 (d, 1.1, H5 ), 2.16 (m, H-4a), 2.11 ( dd, 11.5, 13.1, H-18b), 2.05 (m, H13), 2.03 (m, H-4b), 1.92 (d, 1.1, H-4 ), 1.81 (d, 1.0, H-25), 1.68 (s, H-27), 1. 65 (m, H-6a), 1.49 (m, H-6b), 1.43 (m, H-12a), 1.29 (m, H-5a), 1.12 (m, H5b), 1.10 (m, H-12b), 0.99 (d, 7.0, H-26). 13C NMR (125 MHz, CD3OD) (assignment): 166.7 (C-1), 165.1 (C-1 ), 153.9 (C-3 ), 148.6 (C-3), 133.3 (C-22), 132.0 (C-17), 131.8 (C-8), 131.0 (C -14), 130.9 (C-21), 130.0 (Ph), 128.7 (Ph), 128.3 (C-16), 128.0 (C-9), 127.5 (C -15), 127.3 (Ph), 121.7 (C-24), 121.6 (C-2), 118.3 (C-23), 117.7 (C-2`), 75.8 (C-10), 75 .6 (C-7), 75.1 (C-19), 72.6(C-11), 55.4 (OCH3), 54.8 (OCH3) 43.4 (C-18), 37.2 (C-20), 33.2 (C-6 ), 31.8 (C-4), 31.2 (C-12), 29.5 (C-5), 28.0 (C-13), 26.1 (C-4 ), 19.0 (C-5 ), 16.4 (C-26), 14.8 (C -27), 11.5 (C-25). LR ESIMS m/z 1039.1 [M + Na]+. HRFABMS m/z 1039.4145 (C53H62N2O11F6 Na requires 1039.4155).

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167 4.2.2.3 Palmerolide A 7-[( S )-MTPA ester] ( 65 ) 1H NMR (500 MHz, CD3OD) (multiplicity, J (Hz), assignment): 7.92 (s, NH), 7.50 – 7.45 (5H, m, Ph), 6.97 (d, 14.4, H-24), 6.85 (ddd, 5.1, 10.0, 15.5, H-3), 6.11 (dd, 11.2, 15.0, H-15), 5.96 (d, 14.7, H-23), 5.92 (dd, 3.4, 15.6, H-9), 5.82 (d,15.2, H-2), 5.74 (br t, 1.2, H-2’), 5.68 (ddd, 1.8, 8.6, 15.2, H-8), 5.67 (d,15.0, H-16), 5.46 (m, H-7), 5.45 (dd, 4.9, 8.8, H-14), 5.19 (d, 9.8, H-21),4.95 (m, H19), 4.32 (ddd, 1.9, 3.2, 5.3, H-10), 4.66 (ddd, 2.0, 5.2, 10.8, H-11), 3.55 (s, OCH3), 2.78 (ddq, 6.8, 7.5, 9.8, H-20), 2.26 (br d, 12.7, H-18a), 2.24 (m, H-4a), 2.19 (d, 0.8, H-5’), 2.16 (m, H-4b), 2.09 (dd, 11.5, 12.4, H18b), 2.07 (m, H2-13), 1.92 (d, 0.8, H-4’), 1.83 (m, H-6a), 1.82 (s, H3-25), 1.72 (m, H6b), 1.69 (s, H3-27), 1.64 (m, H-12a), 1.46 (m, H-5a ), 1.31 (m, H-5b), 1.17 (m, H-12b), 0.99 (d, 6.9, H3-26). 13C NMR (125 MHz, CD3OD) (assignment): 167.0 (C-1), 165.2 (C1 ), 158.3 (OCONH2), 153.8(C-3 ), 149.2 (C-3), 134.8 (C-9), 133.2 (C-22), 132.0 (C-14), 131.7(C-17), 130.8 (C-21), 129.6 (Ph), 128.5 (C16), 128.4 (Ph), 127.4 (Ph), 127.3 (C-8), 126.9 (C-15), 121.6 (C-24), 121.3 (C -2), 118.3 (C-23), 117.6 (C-2 ), 78.5 (C-7), 76.2 (C11), 74.8 (C-19), 70.5 (C-10), 54.8 (OCH3) 43.8 (C-18), 37.3 (C-20), 34.2 (C-6) 32.3 (C4), 30.3 (C-12), 29.7(C-13), 26.3 (C-4 ), 24.6 (C-5), 19.1 (C-5), 16.4 (C-26), 15.2 (C-27), 11.9 (C-25). LRESIMS m/z 823.1 [M + Na]+, HRFABMS m/z 823.3747 (C43H55N2O11F3Na requires 823.3757). 4.2.2.4 Palmerolide A 7, 10-[( S, S )-MTPA] diester ( 67 ) 1H NMR (500 MHz, CD3OD) (multiplicity, J (Hz), assignment): 7.92 (s, NH), 7.387.50 (m, Ph), 6.97 (d, 14.4, H-24), 6.82 (ddd, 6.1, 8.3, 15.5, H-3), 6.10 (dd,11.1, 14.6, H15), 5.96 (d, 14.5, H-23), 5.80 (d, 15.5, H-2), 5.74 (br t, 1.1, H-2 ), 5.70 (m, H-9), 5.64 (d,

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168 11.1, H-16), 5.63 (m, H-10), 5.54 (m, H-7), 5.54 (m, H-8), 5.31 (dt, 4.2, 14.6, H-14), 5.19 (d, 10.0, H-21), 4.96 (m, H-19), 4.71 (dd, 6.5, 12.1, H-19), 3.58 (s, OCH3), 3.43 (s, OCH3), 2.79 (ddq, 6.8, 7.5, 10.0, H-20), 2.26 (br d, 13.4, H-18a), 2.22 (m, H-4a), 2.19 (d, 1.0, H-5 ), 2.178 (m, H-4b), 2.11 (dd, 11.5, 11.9, H-18b), 1.99 (m, H-13b), 1.92 (d, 1.0, H-4’), 1.90 (m, H-13a), 1.82 (d,1.0, H-25), 1.78 (m, H-6a), 1.69 (m, H-6b), 1.68 (s, H27), 1.43 (m, H-12a), 1.41 (m, H-5a), 1.27 (m, H-5b), 1.24 (m, H-12b), 0.99 (d, 6.7, H26); 13C NMR (125 MHz, CD3OD) (assignment): 166.9 (C-1), 165.2 (C-1 ), 153.6 (C3 ), 149.1 (C-3), 133.5 (C-22), 132.1 (C-9), 132.0 (C-17), 131.1 (C-14), 130.8 (C-21), 129.8 (C-8). 129.6 (Ph), 128.4 (Ph), 128.3 (C-1 6), 127.4 (Ph), 127.3 (C -15) 121.6 (C-24), 121.5 (C-2), 118.3 (C-23), 117.6 (C-2 ), 76.1 (C-7), 75.0 (C-10), 75.0 (C-19), 72.7 (C11), 55.2 (OCH3), 49.3 (OCH3), 42.6 (C-18), 37.3 (C-20), 34.2 (C-6), 32.1 (C-4), 30.8 (C-12), 26.3 (C-4 ), 26.1 (C-13), 24.6 (C-5), 19.2 (C-5 ),16.0 (C-26), 15.3 (C-27), 11.9 (C-25). LRESIMS m/z 1039.1 [M + Na]+, HRFABMS m/z 1039.4151 (C53H62N2O11F6Na requires 1039.4155). 4.2.3 Palmerolide C ( 59 ) White solid; [ ]25 D -27.1 ( c 0.1, MeOH); IR (thin film) cm-1: 3364 (br), 2933, 1697, 1637, 1446, 1387, 1274, 1182, 1018, 978; UV (MeOH) max ( ): 216 (1002), 248 (635); 1H NMR (500 MHz, DMSOd6) (multiplicity, J (Hz), assignment): 9.85 (1H, d, 10, 24NH ), 6.85 (1H, dd, 9, 14.6, H-24), 6.77 (1H, ddd, 7.4, 7.5, 15.4, H-3), 6.37 (2H, br, CONH 2 ), 6.08 (1H, dd, 12, 15, H-15), 5.85 (1H, d, 14.5, H-23), 5.73 (1H, d, 15.5, H-2), 5.68 (1H, s, H-2 ), 5.63 (1H, d, 11, H-16), 5.58 (1H, m, H-7), 5.54 (1H, m, H-6), 5.46 (1H, ddd, 5, 10, 15, H-14), 5.15 (1H, d, 9.5, H-21), 4.85 (1H, ddd, 2.8, 7.8, 10.5, H-19),

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169 4.72 (1H, d, 4.5, 9-OH ), 4.62 (1H, d, 5, 8-OH ), 4.56 (1H, ddd, 3, 7.5, 10.5, H-10), 3.96 (1H, m, H-8), 3.56 (1H, m, H-9), 2.70 (1 H, qdd, 6.7, 6.7, 9.8, H-20), 2.07 (1H, m), 2.18 (1H, m, H-18), 2.13 (1H, m, H-4), 2.11 (3H, s, H3-5 ), 2.07 (1H, m, H-18), 1.99 (1H, m, H-13), 1.98 (1H, m, H-5), 1.95 (1 H, m, H-12), 1.90 (1H, m, H-13), 1.89 (1H, m, H-5), 1.82 (3H, s, H3-4 ), 1.69 (3H, s, H3-27), 1.59 (3H, s, H3-25), 1.49 (1H, m, H-11), 1.30 (1H, m, H-11), 1.30 (1H, m, H-4), 0.90 (3H, d, 7, H3-26); 13C NMR (125 MHz, DMSOd6) (assignment): 166 .9 (C-1), 164.0 (C-1 ), 152.5 (C-3 ), 149.8 (C-3), 133.3 (C-22), 132.5 (C-17), 132.3 (C-14), 131.8 (C-6), 131.1 (C-7 ), 130.5 (C-21), 128.8 (C-16), 127.2 (C-15), 122.8 (C-24), 122.0(C-2), 118.9 (C-2’), 117.2 (C-23), 75.6 (C-9), 74.7 (C-19), 74.2 (C-10), 72.8 (C-8), 44.1(C-18), 37.4 (C20), 33.3. 32.16 (C-5), 31.7 (C-4), 30.1 (C14), 30.1 (13), 28.7 (C-12), 28.7 (C-11), 27.4 (C-4 ), 20.3 (C-5 ), 17.7 (C-26), 16.5 (C25), 13.3 (C-27); ESIMS m/z (%) 608.3 (37, [M + H + Na]+), 607.3 (38, [M + Na]+), 591.3 (40), 585.3 (15, [M + H]+), 567.3 (15), 503.3 (10), 485.3 (30), 424.2 (25), 389.2 (46), 321 (20). HRESIMS m/z 585.3534 (C33H49N2O7 requires 585.3540). 4.2.4 Palmerolide D ( 60 ) Colorless solid; [ ]25 D +67 ( c 0.5, MeOH); IR (thin film) cm-1 3327, 2939, 2829, 2061, 1716, 1558. 1455, 1261, 1025, 975; max ( ): 216 (1742), 248 (528); 1H NMR (500 MHz, DMSOd6) (multiplicity, J (Hz), assignment): 9.94 (1H, d 10.3, 24-NH ), 6.85 (1H, dd, 10.4, 15, H-24 ), 6.71(1H, ddd, 4, 11.5, 15.7, H-3), 6.45 (2H, br, OC ONH2), 6.04 (1H, dd, 11.6, 14, H-15), 5.86 (1H, d, 14.6, H-23), 5.81 (1H, s, H-2 ), 5.76 (1H, d, 15.8, H-2), 5.59 (1H, d, 12, H-16), 5.53 (1H, m, H-8) 5.49 (1H, m, H-9), 5.41 (1H, ddd, 5, 10, 14.9, H-14), 5.19 (1H, m, 10-OH ), 5.14 (1H, d, 9.7, H-21), 4.84 (1 H, m, H-19), 4.72 (2H,

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170 d, H-6), 4.53 (1H, m, 7-OH ), 4.48 (1H, m, H-11), 4.15 (1H, m, H-10), 3.82 (1H, m, H-7), 2.68 (1H, m, H-20), 2.16 (1H, m, H-18b) 2.15 (1H, m, H-4b), 2.11 (1H, m, H-4a), 2.00 (1H, m, H-18a), 1.98 (1H, m, H-5), 1.94 (2H, m, H-13), 1.94 (2H, m, 12), 1.76 (3H, s, H3-8 ) 1.70 (3H, s, H3-27) 1.61 (3H, s, H3-7 ) 1.60(3H, s, H3-25), 1.48 (1H, m, H-6b) 1.30 (1H, m, H-6a) 0.89 (3H, d, 6.7, H3-26); 13C NMR (125 MHz, DMSOd6) (assignment): 166.1 (C-1), 163.5 (C-1 ), 157.4 (C ONH2), 153.2 (C-3 ), 150.0 (C-3), 143.6 (C-5 ), 134.2 (C-8), 133.6 (C-14), 133.3 (C-22) 132.2 (C-17), 130.7 (C-21), 129.6 (C-9), 128.4 (C-16), 127.0 (C-15) 122.7 (C-24), 121.2 (C-2), 120.3 (C-2 ), 117.5 (C-23), 112.6 (C-6’), 75.8 (C-11), 74.5 (C-19), 73.2 (C-7 ), 69.9 (C-10), 43.9 (C-18), 40.8 (C4 ), 38.4 (C-6), 37.3 (C-20), 30.1 (C-5), 33.0 (C-4), 30.0 (C-13) 30.0 (C-12), 24.8 (C-8 ), 22.7 (C-7 ), 17.7 (C-26), 16.8 (C-25), 13.3 (C-27). ESIMS m/z (%) 625.6 (45, [M + H]+), 615 (35), 585.3 (30), 520.4 (10), 432.3 (20) 349.4 (50), 305.2 (52); HR ESMS m/z 625.3864 (C36H53N2O7 requires 625.3853) 4.2.5 Palmerolide E ( 61 ) Colorless solid; [ ]25 D +17 ( c 0.1, MeOH); IR (thin film) cm-1 3635, 2940, 2830, 1715, 1637, 1540, 1387, 1276, 1194, 1079, 938 ; UV (MeOH) max ( ): 216 (1295), 248 (645); 1H NMR (500 MHz, DMSOd6 ) (multiplicity, J (Hz), assignment): 9.41 (1H, s, H-23), 6.74 (1H, ddd, 4.3, 11.5, 15.7, H-3), 6.55 (1H, dd, 1.5, 10.2, H-21), 6.48 (2H, br, CONH 2 ), 6.05 (1H, dd, 10.8, 14.8, H-15), 5.78 (1H, d, 15.7, H-2), 5.61 (1H, d, 10.6, H16), 5.53 (1H, dd, 1.4, 8, H-8), 5.49 (1H, d, 2.9, H-9), 5.02 (1H, ddd, 2.1, 7.5, 10. H-19), 4.47 (1H, ddd, 1.5, 5.1, 10.7, H-11), 4.12 (1H, m, H-10), 3.81 (1H, m, H-7), 2.94 (1H, qdd, 6.8, 7.1, 9.3, H-20), 2.16 (1H, m, H-18b), 2.14 (1H, m, H-4b), 2.11 (1H, m, H-4a),

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171 2.09 (1H, m, H-18a), 1.95 (1H, m, H-13), 1.67 (3H, s, H3-26), 1.63 (3H, s, H3-24), 1.49 (1H, m, H-6b), 1.30 (1H, m, H-5b), 1.29 (1H, m, H-6a), 1.05 (1H, m, H-5a), 1.01 (3H, d, 6.8, H3-25); 13C NMR (125 MHz, DMSOd6) (assignment): 196.3 (C-23), 166.1 (C-1), 155.8 (C-21), 150.8 (C-3), 139.6 (C-22), 134.4 (C -8), 133.0 (C-17), 132.9 (C-14), 129.6 (C-9), 128.7 (C-16), 127.3 (C-15), 121.2 (C-2), 76.1 (C-11), 73.3 (C-7), 73.0 (C-19), 70.1 (C-10), 43.9 (C-18), 38.5 (C-6), 38.1 (C-20), 33.4 (C-4), 29.8 (C-12), 25.9 (C-5), 17.0 (C-24), 16.5 (C-25), 9.9 (C-26); ESIMS m/z (%) 601.5 (40), 512.3 (30, [M + Na]+), 502.3 (36), 490.3 (80), 472.4 (90), 437.2 (25), 393.3 (50), 349.2 (60), 305.2 (65), 273.1 (80), 195.0 (64), 153 (90); HRESMS m/z 512.2634 (C27H39NO7Na requires 512.2624) 4.2.6 Palmerolide B ( 62 ) White solid; [ ]25 D +1.6 ( c 0.1, MeOH); IR (thin film) cm-1: 3514, 3433 (br), 1648, 1633, 1510, 1392, 1275, 1190; UV (MeOH) max ( ): 216 (1756), 240(603); 1H NMR (500 MHz, DMSOd6) (multiplicity, J (Hz), assignment): 6.8 6 (1H, d, 14.5, H-24), 6.74 (1H, ddd, 5.0, 9.9, 15.2, H-3), 6.01 (1H, dd, 10.5, 14.5, H-15), 5.85 (1H, d, 15, H-23), 5.72 (1H, m, H-9), 5.70 (1H, d, 13, H-2), 5.64 (1H, m, H-10), 5.64 (1H, br s, H-2 ), 5.57 (1H, d, 10.9, H-16), 5.38 (1H, m, H-14), 5.08 (1H, d, 10, H-21), 4.82 (1H, m, H-19), 4.64 (1H, m, H-11), 4.57 (1H, m, H-7), 4.20 (1H, m, H-8), 2.68 (1H, qdd, 6.5, 7.4, 9.6, H-20), 2.15 (1H, m, H-18b), 2.14 (2H, m, H-4b), 2.10 (1H, m, H-4a), 2.09 (3H, s, H3-5 ), 1.99 (1H, m, H-18a), 1.96 (1H, m, H-13b), 1.81 (3H, s, H3-4 ), 1.79 (1H, m, H-12b), 1.71 (3H, s, H3-25), 1.58 (3H, s, H3-27), 1.55 (1H, m, H-6b), 1.53 (1H, m, H-12a), 1.34 (1H, m, H-5b), 1.21 (1H, m, H-13a), 1.14 (1H, m, H-5a), 1.08 (1H, m, H-6a), 0.88 (3H, d, 6.5, H3-26); 13C NMR (125 MHz, DMSOd6) (assignment): 16 8.2 (C-1) 166.7 (C-1 ), 159.8

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172 (C ONH2), 154.9 (C-3 ), 150.7 (C-3), 134.5 (C-22), 133.4 (C-9), 133.3 (C-14), 132.8 (C7), 132.2 (C-21), 131.0 (C-10), 129.8 (C-16) 128.0 (C-15), 122.7 (C-24), 122.3 (C-2), 119.6 (C-23), 118.9 (C-2 ), 81.6 (C-11), 77.4 (C-7), 76.2 (C -19), 72.1 (C-8), 45.2 (C-18), 38.7 (C-20), 36.6 (C-12), 34.0 (C-4), 31.8 (C-6), 30.9 (C-13), 27.7 (C-4 ), 26.1 (C-5), 20.4 (C-5 ), 17.7 (C-26), 16.6 (C-27), 13.2 (C-25); LR ESIMS (-) m/ z 663.3[M H]+, LRESIMS (+) 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) 4.2.7 Palmerolide H ( 63 ) Colorless solid; [ ]25 D -27 ( c 0.1, MeOH); IR (thin film) cm-1: 3515, 3400 (br), 2925, 2856, 1653, 1633, 1517, 1208, 1040; UV (MeOH) max ( ): 217 (1232), 248 (712); 1H NMR (500 MHz, DMSOd6 ) (multiplicity, J (Hz), assignment): 6.87 (1H, d, 14.5, H24), 6.75 (1H, ddd, 4, 11.5, 15.7, H-3), 6.02 (1 H, dd, 10, 14.5, H-15) 5.87 (1H, d, 14.6, H-24), 5.76 (1H, s, H-2 ), 5.74 (1H, m, H-9) 5.70 (1H, d, 14, H-2), 5.63 (1H, m, H-10), 5.57 (1H, d, 11.5, H-16), 5.38 (1H, m, H-14), 5.09 (1H, d, 10, H-21), 4.84 (1H, m, H-19), 4.69 (2H, s, H-6 ), 4.66(1H, m, H-11), 4.58 (1H, m, H7), 4.20 (1H, m, H-8), 3.44 (2H, s, H-4 ), 2.68 (1H, m, H-20), 2.15 (1H, m, H-18b), 2.14 (1H, m, H-4b), 2.11 (1H, m, H-4a), 2.00 (1H, m, H-18a), 1.96 (1H, m, H-13b) 1.80 (1H, m, H-12b), 1.76 (3H, s, H3-8 ), 1.72 (3H, s, H3-25) 1.61 (3H, s, H3-7 ) 1.59 (3H, S, H3-27), 1.56 (1H, m, H-b), 1.55 (1H, m, H12a), 1.32 (1H, m, H-5a), 1.21 (1H, m, H-13a), 1.18 (1H, m, H-5a), 1.10 (1H, m, H-6a) 0.89 (3H, d, 6.7, H3-26); 13C NMR (125 MHz, DMSOd6) (assignment): 168.2 (C-1), 166.0 (C-1 ), 159.9 (CONH2), 155.7 (C-3 ), 150.7 (C-3), 144.6 (C-5 ), 134.5 (C-22),

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173 133.4 (C-14), 132.3 (C-21), 132.3 (C-9), 131.1 (C -10), 129.7 (C-16), 128.1 (C-15), 122.8 (C-24), 122.3 (C-2), 120.5 (C-2 ), 119.7 (C-23), 112.6 (C-6 ) 81.7 (C-11), 77.5 (C-7), 76.2 (C-19), 72.2 (C-8), 45.2 (C-18), 42.1(C-4 ), 38.7 (C-19), 36.6 (C-12), 33.9 (C-4), 31.8 (C-6), 30.8 (C-13), 26.1 (C-5), 24.8 (C-8 ), 22.4 (C-7 ), 17.7 (C-26), 16.6 (C-27); LRESIMS (-) m/ z 703.3 [M H]+, LRESIMS (+) m/z 607.3 [M + H -H2SO4]+, HRESIMS 703.3258 (C36H52N2O10S requires 703.3270 ) 4.3 Isolation of Secondary metabolites from Austrodoris kerguelenensis Austrodoris kerguelenensis nudibranches were collec ted by SCUBA around Palmer Station Antarctica. They were frozen imme diately after the collection. Upon freeze drying, the nudibranchs were subs equently extracted with CHCl3 three times for 24 hours, sequentially. After evaporat ing the solvent under reduced pressure, a gummy, reddish brown solid 2.5 g was obtained. The CHCl3 extract was further fractionated into 12 fractions by elution of increasing polarity gradient of EtOAc in n -hexane on silica gel. Investigation of each fraction by 1H NMR spectroscopy indicated that fractions 1-4 contained a mixture of ster ols and fatty acids. Fractions 5 and 6 displayed peaks characteristic of steroids. Fractions 711 showed signals indi cative of terpenoid glycerides. Further purificatio n of factions 7, 9 and 10 by HPLC, first on silica gel (EtOAc/ n -hexane, 2 mL/min) and then on C-18 (MeCN / H2O, 2 mL, min) afforded palmadorin A ( 110 ) (24 mg), palmadorin B ( 111 ) (7 mg) and palamadorin C ( 112 ) (7 mg).

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174 4.3.1 Palmadorin A (110) Colorless oil; [ ]25 D +18 ( c 0.05, MeOH); IR (thin film) cm-1: 3400 (br), 2969, 2864, 1712, 1640, 1488, 1382, 1281, 1147, 1100, 1040 and 975. UV (MeOH) max ( ): 215 (1001), 248 (499), 266 (484); 1H NMR (500 MHz, CDCl3) (multiplicity, J (Hz), assignment): 5.68 (br s, H-14), 4.91 (1H, m, H-2 ), 4.48 (2H, d, 1.5, H-18), 3.83 (4H, d, 4.8, H-1 and H-3 ), 2.27 (1H, m, H-3b), 2.14 (3H, S), 2.09 (1H, m, H-3a), 1.96 (1H, m, H-12b), 1.87 (1H, m, H-2), 1.85 (1H, m), 1.58 (1H, m, H-6b), 1.50 (1H, m, H-6a), 1.48 (1H, m), 1.47 (1H, m, H-7b), 1.44 (1H, m, H7a), 1.44 (1H, m, H-11b), 1.43 (1H, m, H-1) 1.39 (1H, m, C-8), 1.33 (1H, m, H-11a), 1.23 (1H, m, H-2) 1.03 (1H, m, H-10), 1.02 (3H, s, H3-19), 0.79 (3H, d, 6, H3-17), 0.71 (3H, s, H3-20), 13C NMR(125 MHz, CDCl3) (assignment): 166.7 (C-15), 163.7 (C-13), 160.4 (C-4), 114.3 (C-14) 102.5 (C-18), 74.4 (C-2 ), 62.7 (C-1 ), 62.7 (C-3 ), 48.7 (C-10), 40.0 (C-5), 39.3 (C-9), 37.2 (C-6), 36.7 (C-8), 36.1 (C-11), 34.6 (C-12), 33.0 (C-3), 28.6 (C-2), 27.4 (C-7), 21.6 (C-1), 20.8 (C-19), 19.5 (C-16), 16.00 (C-17), 18.04 (C-20), LRFABMS m/z ( % ) 379.3 (52 [M]+), 361.2 (10); HRFABMS m/z 379.284200 (C23H39O4 requires 379.2844835). 4.3.2 Ozonolysis of Palmadorin A ( 110 ) Compound 110 (5mg, 0.0132 mmol) was dissolved in 2mL of CH2Cl2 and allowed to react with O3 for 25 mins. The temperature of the reaction vessel was controlled at -80C. After 25 minitues the O3 supply was disconnected and DMS (5 drops) was added and allowed to react for 1.5 hours while allowing the reaction mixture to warm to room temperature. The solvent was evaporated under reduced pressure to obtain a crude product 8 mg as a colorless solid. This cr ude product was purified by chromatography

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175 over silica using 30% EtOAc/hexan e yeilding the ozonolysis product 113 as a white amorphous solid 1mg (0.038 mmol, 28.65%). 4.3.3 Ozonolyzed product of Palmadorin A ( 113 ) Colorless solid; [ ]25 D +0.6 ( c 0.05, MeOH); 1H NMR (500 MHz, CDCl3) (multiplicity, J (Hz), assignment): 2.58 (1 H, m, H-3b), 2.34 (1H, m, H12b), 2.23 (1H, m, H-3a), 2.16 (3H, s, H3-14), 2.13 (1H, m, H-12a), 1.72 (1H, m, H-11b), 1.60(1H, m, H-6b), 1.56(1H, m, H-6a), 1.53 (1H, m, H-7b), 1.46 (1H, m, H-2b), 1.44 (1H, m, H-11a), 1.28 (1H, m, H7a), 1.24 (1H, m, H-2a), 1.20 (1 H, m, H-10), 1.15 (3H, s, H3-17), 0.82 (3H, d, H3-17), 0.73 (3H, s, H3-18); 13C NMR (125 MHz, CDCl3) (assignment): 216.3 (C-4), 209.0 (C13), 50.0 (C-10), 40.6 (C-9) 39.4 (C-5), 37.7 (C-12), 37.6 (C-3), 36.6 (C-8), 33.1 (C-6), 30.8 (C-11), 30. 6 (C-14), 26.5 (C-2), 26.5 (C -7), 18.8 (C-17), 17.00 (C-18), 15.5 (C-15). EIMS m/z ( % ) 175 (22), 122 (10), 95 (20) 43 (100), 41 (45), 28 (30). 4.3.4 Palmadorin B ( 111 ) Colorless oil; [ ]25 D +24( c 0.05, MeOH); IR (thin film) cm-1 3360 (br), 2933, 2359, 1718, 1642, 1448, 1383, 1219, 1145, 110, 909. UV (MeOH) max ( ): 214 (1063), 248 (537), 264 (524); 1H NMR (500MHz, CDCl3) (multiplicity, J (Hz), assignment): 5.68 (br s, H-14), 4.91 (1H, m, H-2 ) 4.48 (2H, d, 1, H-18) 4.26 (2H, m, H-1 ) 3.72 (2H, d, H3’) 2.27 (1H, m, H-3b), 2.14 (3H, s, H3-16), 2.09 (1H, m, H-3a), 2.05 (3H, S, COOCH 3 ), 1.86 (1H, m, H-2b), 1.85 (1H, m, H-12a), 1.57 (1H, m, H-6b), 1.47 (1H, m, H-6a), 1.47 (1H, m, H-7b), 1.46 (1H, m, H-1b), 1.44 (1H, m, H-7a), 1.42 (1H, m, H-1a), 1.39 (1H, m, H-8), 1.33 (1H, m, H-11a), 1.21 (1H, m, H2a), 1.03 (1H, m, H-10), 1.02 (3H, s, H3-19), 0.78 (3H, d, 6.5, H3-17), 0.72 (3H, s, H-20) 13C NMR (125 MHz, CDCl3) (assignment);

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176 171.2 (C OOCH3), 166.8 (C-15), 163.9 (C-13), 160.7 (C-4), 114.7 (C-14), 102.9 (C-18), 72.0 (C-2 ), 62.9 (C-1 ), 62.2 (C-3 ), 49.1 (C-10), 40.4 (C-5), 39.7 (C-9), 37.7 (C-6), 37.0 (C-8), 36.4 (C-11), 34.9 (C -12), 34.4 (C-3), 29.0 (C-2), 27.8 (C-7), 22.3 (C-19), 21.8 (C-1), 21.2 (COOC H3), 19.8 (C-16), 18.5 (C-20), 16.4 (C-17); LRFABMS m/z ( % ) 421.4 (36 [M+1]+), 379.4(8), 321.3(8), 287.3 (90); HRFABMS m/z 421.2964 (C25H41O5 requires 421.2954). 4.3.5. Palmadorin C ( 112 ) Colorless oil; [ ]25 D +8( c 0.05, MeOH); IR (thin film) cm-1: 3389 (br), 2840, 1648, 1408, 1228, 1219, 1109, 1015, 894. UV (MeOH) max ( ): 216 (1002), 248 (635); 1H NMR (500 MHz, CDCl3) (multiplicity, J (Hz), assignment): 5.70 (1H, s, H-14), 5.13 (1H, m, H-3), 4.91 (1H, m, H-2 ), 4.02 (1H, m, H-7), 3.82 (4H, d, 4.5, H-1 and H-3 ) 2.16 (3H s, H316) 2.10 (1H, m, H-6b), 2.08 (1H, m, H-2b) 1.98 (1H, m, H-2a), 1.98 (1H, m, H-12b), 1.93 (1H, m, H-12a), 1.60 (3H, s, H-18), 1.58 (1 H, m, H-1b), 1.54 (1H, m, H-1a), 1.51 (1H, m, H-11b), 1.51 (1H, m, H-8a), 1.39 (1H, m, H-11a), 1.39 (1H, m, H-6a), 1.37 (1H, m, H-10), 1.27 (3H, s, H3-19), 1.01 (3H, d, 7.5, H-17), 0.99 (1H, s, H3-20); 13C NMR(125 MHz, CDCl3) (assignment): 166.9 (C OOCH3), 163.4 (C-13), 145.2 (C-4), 120.0 (C-3), 114.8 (C-14), 74.8 (C -2’), 73.9 (C-7), 63.1 (C-1 and C-3 ), 46.86 (C-10), 43.1 (C-6), 39.5 (C-8), 38.5 (C-9), 37.8 (C-5), 37.57 (C-11), 35.1 (C-12), 27.0 (C-2), 22.0 (C-19), 20.3 (C-20), 19.7 (C -16), 18.3 (C-18), 18.2 (C-1 ), 12.7 (C-17): FABMS (+) m/z ( % ) 377.4 (15, [M + H H2O]+), 309.1(14), 195.1 (15), 153.1 (45), 135 (45). ESIMS (+) m/z ( % ) 377.4 (15 [M + HH2O]+, HRFABMS m/z 378.1503 (C23H36O4 requires 378.1467).

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177 4.3.6 Acetylation of Palmadorin C (112) Palmadorin C (5mg, 0.0127 mm ol) was dissolved in CH2Cl2 (1 mL) and treated with Ac2O (30 L, 0.025 mmol) in the presen ce of DMAP (3 mg), Et3N (30 L) for 24 hrs at 25C. The reaction was quenched by the ad dition of few drops of MeOH. Upon evaporation of the solvent by reduced pr essure the crude product was separated by chromatography over silica gel (EtOAc /hexane gradient elution) to obtain palmadorin C diacetate ( 114 ) (6mg, 0.0125 mmol, 98%). 4.3.7 Palamadorin C diacetate ( 114 ) Colorless solid; [ ]25 D +12( c 0.05, MeOH); IR (thin film) cm-1: 3389 (br), 2834, 1648, 1408, 1228, 1210, 1040, 1109, 1015, 894. 1H NMR(500 MHz, CDCl3) (multiplicity, J (Hz), assignment): 5.65 (1H, s, H-14), 5.26 (1H, m. H-2 ), 4.23 (4H, m, H-1 and 3 ), 4.03 (1H. m, H-7), 2.15 (3H, s, H3-16), 2.08 (1H, m, H-6b), 2.06 (3H, s, COOCH 3 ), 2.05(1H, m H-2b), 1.98 (1H, m, H-2b), 1.93 (1H, m, H-12b), 1.90 (1H, m, H-12a), 1.60 (3H, s, H3-18), 1.52 (1H, m, H-8), 1.48 (1H, m, H-11a ), 1.38 (1H, m, H-6a), 1.38 (1H, m, H-10), 1.37 (1H, m, H-11), 1.27 (3H, s, H3-19), 1.02 (3H, d, 7, H3-17), 1.00 (3H, s, H3-20).13C NMR (125 MHz, CDCl3) (assignment); 170.8 (C OOCH3), 165.7 (C-15), 163.3 (C-13), 145.2 (C-4), 120.0 (C-3), 114.7 (C-14), 73.9 (C-7), 68.2 (C-2 ), 62.7 (C-1 & C-3 ), 54.8 (OCH3), 46.9 (C-10), 43.2 (C-6), 39. 5 (C-8), 38.0 (C-9), 37.8 (C -5), 37.5 (C-11), 35.1 (C12), 26.9 (C-2), 21.8 (C-19), 20.2 (C-20), 19.7 (C-16), 12.7 (C-17); LRESIMS (-) m/z (%) 477.2 (78, [M 1]+), 478.2 (20, [M]+).

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178 4.3.8 Preparation of R -MTPA esters of Palmadorin C diacetate (114) Palamadorin A diacetate (3 mg, 0.0063 mmol) was dissolved in CH2Cl2 (500 L) and allowed to reacted with R -MTPACl (50 mg, 0.07 mmol) in the presence of Hunig’s base (100 L) and DMAP (1mg) for 48hrs Conversion of the starting material to the products was monitored by TLC. Evaporation of the so lvents gave a crude product of 20 mg. Further purification of this mixture by ch romatography on slica gel (EtOAc/hexane gradient elution) gave compound ( 115 ) (1.5 mg, 0.0022 mmol, 34%). 4.3.9 Palmadorin C diacetate R -MTPA ( 115 ) Colorless solid; [ ]25 D +10 ( c 0.05, MeOH); IR (thin film) cm-1 3401 (br), 2825, 1662, 1510, 1340, 1235, 1032, 1006, 860. 1H NMR (500 MHz, CDCl3) (multiplicity, J (Hz), assignment): 7.53 (2H, m, Ph), 7.35 (3H, m Ph ), 5.62 (1H, s, H-14), 5.25 (1H, m. H-2`), 4.22 (4H, m, H-1` & 3`), 5.35 (1H. m, H-7), 3.59 (3H, s, OCH 3) 2.13(3H, s, H3-16), 2.05 (3H, s, COOCH 3 ), 2.05 (1H, m H-2b), 1.98 (1H, m, H-2b), 1.97 (1H, m, H-6b), 1.90 (1H, m, H-12b), 1.88 (1H, m, H-12a), 1.43 (3H, s, H3-18), 1.52 (1H, m, H-8), 1.48 (1H, m, H-11a), 1.37 (1H, m, H-6a), 1.37 (1H, m, H-11), 1.31 (1H, m, H-10), 0.92 (3H, d, H317), 0.80 (3H, s, H3-20) 0.53 (3H, s, H3-19). 13C NMR (125 MHz, CDCl3) (assignment); 170.79(C OOCH3), 166.9 (C OOMTP), 165.7 (C-15), 162.7 (C -13), 145.2 (C-4), 129.7 (Ph) 128.5 (Ph), 127.36 (Ph), 120.6 (C-3), 114.9 (C -14), 78.0 (C-7), 68.3 (C-2’), 62.7 (C1`& C-3`), 55.8 (OCH3) 46.4 (C-10), 40.4 (C-6), 39.1 (C-8 ), 38.2 (C-9), 37.3 (C-5), 37.1 (C-11), 34.9 (C-12), 26.8 (C -2), 21.8 (C-19), 20.2 (C20), 19.6 (C-16), 12.00 (C-17). HRESIMS m/z 717.3220 (C37H49O9F3 requires 717.3219).

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179 4.3.10 Preparation of S -MTPA esters of Palmadorin C diacetate (114) Palamadorin A diacetate ( 114 ) (1mg, 0.0021 mmol) was dissolved in CH2Cl2 (300 L) and allowed to reacted with S -MTPACl (50 mg, 0.07 mmol) in the presence of Hunig’s base (100 L) and DMAP (1 mg) for 48 hrs. Convers ion of the starting material to the products was monitored by TLC. Evaporation of the solvents gave a crude product of 20 mg. Further purification of this mixture by chromatography on sli ca gel (EtOAc/hexane gradient elution) gave compound ( 116 ) (1 mg, 0.0014 mmol, 69%) 4.3.11 Palmadorin C diacetate S -MTPA ( 116 ) Colorless solid; [ ]25 D -5( c 0.02, MeOH); IR (thin film) cm-1 3342 (br), 2905, 1658, 1408, 1340, 1235, 1048, 1109, 1020, 870. 1H NMR (500 MHz, CDCl3) (multiplicity, J (Hz), assignment); 7.59 (1H, m, Ph), 7.53 (2H, m, Ph), 7.37 (2H, m, Ph) 5.61 (1H, s, H-14), 5.24(1H, m. H-2 ), 4.22 (4H, m, H-1 & 3 ), 5.44 (1H. m, H-7), 3.59 (3H, s, OCH 3) 2.13 (3H, s, H3-16), 2.07 (1H, m, H-2b), 2.05 (3H, s, COOCH 3 ), 2.05 (1H, m H-2b), 1.98 (1H, m, H-2b), 2.06 (1H, m, H-6b), 1.90 (1H, m, H-2a), 1.90 (1H, m, H-12b), 1.86 (1H, m, H-12a), 1.65 (1H, m, H-8), 1.54 (3H, s, H3-18), 1.48 (1H, m, H-6a), 1.46 (1H, m, H11b), 1.35 (1H, m, H-10), 1.26 (1H, m, H-11a), 0.75 (3H, d, H3-17), 0.63 (3H, s, H3-20) 0.94 (3H, s, H3-19). 13CNMR (125 MHz, CDCl3) (assignment): 170.8 (C OOCH3), 167.0 (C OOMTP), 165.7 (C-15), 163.3 (C-13), 144.1 (C-4), 129.7 (Ph), 128.6 (Ph), 128.0 (Ph), 127.6 ( Ph), 127.6 ( Ph), 120.0 (C-3), 114.7 (C14), 73.9 (C-7), 68.2 (C-2’), 62.7 (C-1’& C-3’), 46.6 (C-10), 43.2 (C-6), 39.5 (C-8), 38.1 (C-9), 37.5 (C-11), 37.4 (C-5), 35.1 (C12), 26.9 (C-2), 20.8 (C-19), 20.2 (C-20) 19.7 (C-16), 12.7 (C-17). HRESIMS m/z 717.3220 (C37H49O9 F3 requires 717.3219).

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188 APPENDICES Appendix A Cytotoxicity profile of palmerolide A

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About the Author Thushara Diyabalanage gradua ted with a BSc degree in Biology from the University of Peradeniya Sri Lanka in 1992. In 1996, he completed a Masters Degree in Organic chemistry at the same university after some extensive research on Sri Lankan medicinal plants. Having spent next three years in indus try, where he was employed as a research and development chemist, he came to USA in 2000 to pursue doctoral studies. His research with Dr Bill J Baker in marine natural products chemistry at the University of South Florida has been very successful, as it led to the discovery of a series of new anticancer agents that show great promise as drug leads. In recognition of this, he has been awarded the ASP student research award 2005, annually given to the most outstanding student research performed by a gr aduate student, by the American Society of Pharmacognosy. He has presented his resear ch in several nationa l and international conferences.