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Chemical investigation of three Antarctic marine sponges

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Title:
Chemical investigation of three Antarctic marine sponges
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Book
Language:
English
Creator:
Park, Young Chul, 1964-
Publisher:
University of South Florida
Place of Publication:
Tampa, Fla.
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Subjects

Subjects / Keywords:
erebusinone
Antarctic invertebrates
marine natural products
tryptophan catabolism
chemical defense
Dissertations, Academic -- Chemistry -- Doctoral -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Summary:
ABSTRACT: This thesis describes the chemical investigation of three marine sponges from Antarctica and the total syntheses of natural products erebusinone (12) and its derivative, erebusinonamine (52). Investigation of the yellow Antarctic marine sponge Isodictya setifera resulted in the isolation of two secondary metabolites, purine analog (32) and 3-hydroxykynurenine (24). Chemical investigation of Isodictya setifera led to the isolation of six secondary metabolites which included 5-methyl-2-deoxycytidine (25), uridine (28), 2-deoxycytidine (31), homarine (37), hydroxyquinoline (33), 3-hydroxykynurenine (24). The latter two compounds were found to be intermediates of tryptophan catabolism in crustaceans. From the Antarctic marine sponge Isodictya antractica ceramide analog (39) was isolated and its chemical structure was assigned by a combination of spectroscopic and chemical analyses. Stereochemistry was determined by modified Mosher's method. Erebusinone (12), a yellow pigment isolated from the Antarctic marine sponge Isodictya erinacea has been implicated in molt inhibition and mortality against the Antarctic crustacean amphipod, Orchomene plebs, possibly serving as a precursor of a xanthurenic acid analog. Thought to act as a 3-hydroxykynurenine 24 mimic, erebusinone (12) may be involved chemical defense.1 This appears to be the first example in the marine realm of an organism utilizing tryptophan catabolism to modulate molting as a defensive mechanism. To further investigate the bioactivity and ecological role of erebusinone (12), the synthesis of this pigment was carried out in an overall yield of 44% involving seven steps which were economical and convenient. Erebusinonamine (52) was also similarly synthesized in eight steps with an overall yield of 45%. Reference 1. Moon B. H.; Park Y. C.; McClintock J. B.; Baker B. J., Tetrahedron 2000, 56, 9057-9062.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2004.
Bibliography:
Includes bibliographical references.
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System requirements: World Wide Web browser and PDF reader.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Young Chul Park.
General Note:
Includes vita.
General Note:
Title from PDF of title page.
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Document formatted into pages; contains 217 pages.

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University of South Florida Library
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University of South Florida
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Resource Identifier:
aleph - 001469450
notis - AJR1204
usfldc doi - E14-SFE0000351
usfldc handle - e14.351
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SFS0025046:00001


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Chemical Investigation of Three Antarctic Marine Sponges by Young Chul Park 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 Robert Potter, Ph.D Edward Turos, Ph.D Abdul Malik, Ph.D Date of Approval: March 19, 2004 Keywords: marine natural products, tryptophan catabolism, chemical defense, Antarctic invertebrates, erebusinone Copyright 2004, Young Chul Park

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DEDICATION This dissertation is dedicated to my wife Jung Sook, who never fails to remind me how every day is most precious. I would also like to dedicate this work to my daughter, Yei Ryun and my son, Min Jun. I would like to dedicate this work to my dear parents, who gave me their love and patience. I would also like to dedicate this work to my father-in-law and my mother-in-law, for their patience and understanding on the value of education. Without their support, love, and patience, there is no way I could possibly have accomplished this.

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ACKNOWLEDGMENTS First and foremost, I would like to acknowledge my adviser, Dr. Bill J. Baker. This dissertation would not have been possible without his assistance and financial support. He has taught, inspired, and challenged me throughout this process. I would like to thank Jill Baker, for her concern towards my family. I would like to thanks Dr. James B. McClintock and Dr. Charles D. Amsler at the University of Alabama at Birmingham for the field and laboratory assistance. I would like to thank Dr. Steven Mullen at the University of Illinois at Urbana-Champaign, for the mass spectral data. Also I would like to thank Dr. Maya. P. Singh from Wyeth Pharmaceuticals and Dr. Fred Valeriote from Ford hospital for their bioactivity data. Finally, I would like to acknowledge my committee members, Dr. Robert Potter, Dr. Edward Turos and Dr. Abdul Malik, for their encouragement and guidance. Last but not the least I would like to thank Dr. Bakers students who have given me assistance in many ways.

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TABLE OF CONTENTS LIST OF FIGURES v LIST OF TABLES xvi LIST OF SCHEMES xviii LIST OF ABBREVIATIONS xvix ABSTRACT xxii CHAPTER 1. INTRODUCTION 1 1.1 A Brief History of Natural Products 1 1.2 Marine Natural Products Chemistry 1 1.3 Antarctic Marine Natural Products 8 1.4 Research Objectives 11 CHAPTER 2. CHEMICAL INVESTIGATION OF ANTARCTIC MARINE SPONGE ISODICTYA ERINACEA 12 2.1 Introduction 12 2.2 Extraction and Isolation of Secondary Metabolites 14 2.3 Characterization of Purine analog (23) 16 2.4 Characterization of 3-Hydroxykynurenine (24) 20 CHAPTER 3. CHEMICAL INVESTIGATION OF ANTARCTIC MARINE SPONGE ISODICTYA SETIFERA 25 3.1 Introduction 25 3.2 Extraction and Isolation of Secondary Metabolites 26 3.3 Characterization of 5-methyl-2-deoxycytidine (25) 28 i

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3.4 Characterization of Uridine (28) 33 3.5 Characterization of 2-Doxycytidine (31) 37 3.6 Characterization of 4, 8-Dihydroxyquinoline (33) 42 3.7 Characterization of Homarine (37) 46 CHAPTER 4. CHEMICAL INVESTIGATION OF ANTARCTIC MARINE SPONGE ISODICTYA ANTARCTICA 50 4.1 Extraction and Isolation of Secondary Metabolite 50 4.2 Characterization of Ceramide analog (39) 52 4.3 Determination of Stereochemistry 61 CHAPTER 5. TOTAL SYNTHESIS OF NATURAL PRODUCT EREBUSINONE AND EREBUSINONAMINE 70 5.1 Introduction 70 5.2 Synthesis of Erebusinone (12) 74 5.3 Results and Discussion 74 5.4 The synthesis of Erebusinonamine (52) 77 5.5 Results and Discussion 77 CHAPTER 6. BIOASSAY OF PURE COMPOUNDS 79 CHAPTER 7. DISCUSSION 81 ii

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CHAPTER 8. EXPERIMENTAL 84 8.1 General Procedure 84 8.2 Isolation of Secondary Metabolites from Isodicyta erinacea 86 8.2.1 Spectral data of Purine analog (23) 87 8.2.2 Spectral data of 3-Hydroxykynurenine (24) 88 8.3 Isolation of Secondary Metabolites from Isodictya setifera 89 8.3.1 Spectral data of 5-Methyl-2-deoxycytidine (25) 90 8.3.2 Spectral data of Uridine (28) 91 8.3.3 Spectral data of 2-Doxycytidine (31) 92 8.3.4 Spectral data of 4, 8-Dihydroxyquinoline (33) 93 8.3.5 Spectral data of Homarine (37) 94 8.4 Isolation of Secondary Metabolite from Isodictya antarctica 95 8.4.1 Spectral data of Ceramide analog (39) 96 8.4.2. Methanolysis of Ceramide analog (39) 97 8.4.3 Preparation of MTPA esters for Ceramide analog (39) 98 8.4.3.1 (S) MTPA Ester (43) 98 8.4.3.2 (R) MTPA Ester (44) 99 8.5. Synthesis of Erebusinone (12) 100 8.5.1 Preparation of Benzyl-3-(benzyloxy)-2-nitrobenzoate (46) 100 8.5.2 Preparation of 3-[3-(Benzyloxy)-2-nitrophenyl]-3oxopropanenitrile (47) 101 8.5.3 Preparation of 3-[3-(Benzyloxy)-2-nitrophenyl]-3-hydroxy propanenitrile (48) 102 iii

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8.5.4 Preparation of 3-Amino-1-[3-benzyloxy-2-nitrophenyl]propan-1-ol (49) 103 8.5.5 Preparation of N-{3-[3-(Benzyloxy)-2-nitrophenyl]-3-hydroxypropyl} acetamide (50) 104 8.5.6 Preparation of N-{3-[3-(Benzyloxy)-2-nitrophenyl]-3-oxopropyl} acetamide (51) 105 8.5.7. Preparation of Erebusinone, N-[3-(2-amino-3-hydroxyphenyl)3-oxopropyl] acetamide (12) 106 8.6 Synthesis of Erebusinonamine (52) 107 8.6.1 Preparation of tert-Butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropylcarbamate (53) 107 8.6.2. Preparation of tert-Butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropyl carbamate (54) 108 8.6.3. Preparation of 3-Amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) 109 8.6.4. Preparation of Erebusinonamine, 3-Amino-1-(2-amino-3hydroxyphenyl)propan-1-one hydrochloride (52) 110 REFERENCES 111 APPENDICES 119 ABOUT THE AUTHOR End Page iv

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LIST OF FIGURES Figure 1. Isodictya erinacea collected from at Erebus Bay, Ross Island, Antarctica 12 Figure 2. 1 H NMR spectrum of purine analog (23) (500 MHz, DMSO-d 6 ) 16 Figure 3. 13 NMR spectrum of purine analog (23) (125 MHz, DMSO-d 6 ) 17 Figure 4. gHMBC spectrum of purine analog (23) (500 MHz, DMSO-d 6 ) 18 Figure 5. Key gHMBC correlations of observed in purine analog (23) 18 Figure 6. 1 H NMR spectrum of 3-hydroxykynurenine (24) (250 MHz, MeOH-d 4 ) 21 Figure 7. 1 H NMR spectrum of synthetic erebusinone (12) (250 MHz, MeOH-d 4 ) 21 Figure 8. 13 C NMR spectrum of 3-hydroxykynurenine (24) (125 MHz, DMSO-d 6 ) 22 Figure 9. gHMBC spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ) 23 Figure 10. Key gHMBC correlations observed in 3-hydroxykynurenine (24) 23 Figure 11. Assigned NMR data of erebusinone and 3-hydroxykynurenine (MeOH-d 4 1 H NMR 250 MHz; 13 C NMR, 75, 125 MHz) 1 H chemical shifts 24 Figure 12. Isodictya setifera collected from Bahia Paraiso, Palmer Station, Antarctica 25 Figure 13. 1 H NMR spectrum of 5-Methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ) 28 Figure 14. 13 C NMR spectrum of 5-Methyl-2-deoxycytidine (25) (125 MHz, MeOH-d 4 ) 29 Figure 15. gCOSY correlations of 25 30 Figure 16. gHMBC spectrum of 5-Methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ) 31 Figure 17. Key gHMBC correlations observed in 5-methyl-2-deoxycytidine (25) 31 v

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Figure 18. Assigned 1 H NMR chemical shifts () of 5-methyl-2-deoxycytidine 25 (500 MHz, MeOH-d 4 ) from the current study data, 2-deoxycytidine 26 and thymidine 27 (D 2 O, 120 MHz) 32 Figure 19. 1 H NMR spectrum of uridine (28) (500 MHz, DMSO-d 6 ) 33 Figure 20. gCOSY correlations of uridine 28 34 Figure 21. 13 C NMR spectrum of uridine (28) (125 MHz, DMSO-d 6 ) 35 Figure 22. gHMBC spectrum of uridine (28) (500 MHz, DMSO-d 6 ) 35 Figure 23. Key gHMBC correlations observed in uridine (28) 36 Figure 24. Assigned 1 H NMR chemical shifts () of uridine 28 (500 MHz, DMSO-d 6 ) from the current study data, previous reported uridine 29 and uridine 30 (100 MHz, D 2 O + NaOD) 37 Figure 25. 1 H NMR spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ) 38 Figure 26. gCOSY correlations of 31 38 Figure 27. 13 C NMR spectrum of 2-deoxycytidine (31) (125 MHz, DMSO-d 6 ) 39 Figure 28. gHMBC spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ) 40 Figure 29. Key gHMBC correlations observed in 2-deoxycytidine (31) 40 Figure 30. Assigned 1 H NMR chemical shifts () of natural 2-deoxycytidine 31 (500 MHz, DMSO-d 6 ) from the current study data, previous reported 2-deoxycytidine 32 (MeOH-d 4 360 MHz) and authentic sample 26 (100 MHz, D 2 O + NaOD) 42 Figure 31. 1 H NMR spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ) 43 Figure 32. 13 C NMR spectrum of 4, 8-Dihydroxyquinoline (33) (125 MHz, DMSO-d 6 ) 43 vi

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Figure 33. gHMBC spectrum of 4, 8-Dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ) 44 Figure 34. Key gHMBC correlations observed in 4, 8-Dihydroxyquinoline (33) 45 Figure 35. Secondary metabolites of quinoline derivatives 45 Figure 36. 1 H NMR spectrum of homarine (37) (500 MHz, DMSO-d 6 ) 46 Figure 37. 13 C NMR spectrum of homarine (37) (125 MHz, DMSO-d 6 ) 47 Figure 38. gHMBC spectrum of homarine (37) (500 MHz, DMSO-d 6 ) 48 Figure 39. Key gHMBC correlations observed in homarine (37) 48 Figure 40. Assigned 1 H NMR chemical shifts () of natural homarine 37 (500 MHz, DMSO-d 6 ) from the current study data and authentic sample 38 (100 MHz, D 2 O) 49 Figure 41. Isodictya antarctica collected from Bahia Paraiso, Palmer Station, Antarctica 50 Figure 42. 1 H NMR spectrum of ceramide analog (39) (500 MHz, CDCl 3 ) 52 Figure 43. Key gCOSY (I) and TOCSY (II) correlations in partial structure (a) 53 Figure 44. 13 C NMR spectrum of ceramide analog (39) (125 MHz, CDCl 3 ) 54 Figure 45. DEPT-135 spectrum of ceramide analog (39) (125 MHz, CDCl 3 ) 55 Figure 46. DEPT-90 spectrum of ceramide analog (39) (125 MHz, CDCl 3 ) 55 Figure 47. Key gHMBC correlations observed in partial structure (a) of ceranalog (39) (125 MHz, CDCl 3 ) 56 Figure 48. gHMBC spectrum of ceramide analog (39) (125 MHz, CDCl 3 ) 56 Figure 49. 1 H NMR spectrum of methyl ester (40) (500 MHz, CDCl 3 ) 58 Figure 50. 13 C NMR spectrum of methyl ester (40) (125 MHz, CDCl 3 ) 59 Figure 51. 1 H NMR spectrum of acetylated aminodiol (42) (500 MHz, CDCl 3 ) 60 vii

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Figure 52. 13 C NMR spectrum of acetylated aminodiol (42) (125 MHz, CDCl 3 ) 60 Figure 53. The MTPA plane of the (R) MTPA and (S) MTPA esters of a secondary alcohol 62 Figure 54. Configurational correlation model for (R) MTPA and (S) MTPA esters 62 Figure 55. MTPA ester model to determine the absolute configurations of secondary alcohols ( = S R ) by the modified Moshers method assignment 63 Figure 56. 1 H NMR spectrum of (S) MTPA ester (43) (500 MHz, CDCl 3 ) 64 Figure 57. 1 H NMR spectrum of (R) MTPA ester (44) (500 MHz, CDCl 3 ) 64 Figure 58. values ( S R ) of the MTPA esters 43 and 44 in CDCl 3 65 Figure 59. Model to determine the absolute configuration of the ceramide analog (39) MTPA esters 66 Figure 60. Dihedral angles of H-3, H-4 proposed with C-3 (S) and C-2 (R) of ceramide analog (39) 67 Figure 61. Dihedral angles of H-3, H-4 proposed with C-3 (S) and C-2 (S) of ceramide analog (39) 67 Figure 62. Relative stereochemistry of ceramide analog (39) 68 Figure 63. Ceramide analog (39) 69 Figure 64. Occurrence of Mortality in O. plebs fed erebusinone in their diet 71 Figure 65. Occurrence of Molting in O. plebs fed erebusinone in their diet 71 Figure 66. Ecdysone metabolism 72 Figure 67. Tryptophan catabolism 73 Figure 68. Overlay of erebusinone (12) with 3-hydroxykynurenine (24) in a hypothetical receptor 73 viii

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Figure 69. UV spectrum of purine analog (23) in MeOH 120 Figure 70. IR spectrum of purine analog (23) 120 Figure 71. DEPT-135 spectrum of purine analog (23) (500 MHz, DMSO-d 6 ) 121 Figure 72. gCOSY spectrum of purine analog (23) (500 MHz, DMSO-d 6 ) 121 Figure 73. UV spectrum of 3-hydroxykynurenine (24) in MeOH 122 Figure 74. IR spectrum of 3-hydroxykynurenine (24) 122 Figure 75. gCOSY spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ) 123 Figure 76. DEPT-135 spectrum of 3-hydroxykynurenine (24) (125 MHz, DMSO-d 6 ) 123 Figure 77. gHSQC spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ) 124 Figure 78. gHMBC spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ) 124 Figure 79. UV spectrum of 5-methyl-2-deoxycytidine (25) in MeOH 125 Figure 80. IR spectrum of 5-methyl-2-deoxycytidine (25) 125 Figure 81. gCOSY spectrum of 5-methyl-2-deoxycitidine (25) (500 MHz, MeOH-d 4 ) 126 Figure 82. ROESY spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ) 127 Figure 83. DEPT-135 spectrum of 5-methyl-2-deoxycytidine (25) (125 MHz, MeOH-d 4 ) 127 Figure 84. gHSQC spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ) 128 Figure 85. gHMBC spectrum of 5-methyl-2-deoxycytidine (25) 129 Figure 86. UV spectrum of uridine (28) in MeOH 130 ix

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Figure 87. IR spectrum of uridine (28) 130 Figure 88. DEPT-135 spectrum of uridine (28) (125 MHz, DMSO-d 6 ) 131 Figure 89. ROESY spectrum of uridine (28) (500 MHz, DMSO-d 6 ) 131 Figure 90. gCOSY spectrum of uridine (28) (500 MHz, DMSO-d 6 ) 132 Figure 91. gHSQC spectrum of uridine (28) (500 MHz, DMSO-d 6 ) 133 Figure 92. gHMBC spectrum of uridine (28) (500 MHz, DMSO-d 6 ) 134 Figure 93. UV spectrum of 2-deoxycytidine (31) in MeOH 135 Figure 94. IR spectrum of 2-deoxycytidine (31) 135 Figure 95. DEPT-135 spectrum of 2-deoxycytidine (31) (125 MHz, DMSO-d 6 ) 136 Figure 96. ROESY spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ) 136 Figure 97. gCOSY spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ) 137 Figure 98. gHSQC spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ) 138 Figure 99. gHMBC spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ) 139 Figure 100. UV spectrum of 4, 8-dihydroxyquinoline (33) in MeOH 140 Figure 101. IR spectrum of 4, 8-dihydroxyquinoline (33) 140 Figure 102. DEPT-135 spectrum of 4, 8-dihydroxyquinoline (33) (125 MHz, DMSO-d 6 ) 141 Figure 103. ROSEY spectrum of 4, 8-Dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ) 141 Figure 104. gCOSY spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ) 142 Figure 105. gHSQC spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ) 143 x

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Figure 106. UV spectrum of homarine (37) in MeOH 144 Figure 107. IR spectrum of homarine (37) 144 Figure 108. DEPT-135 spectrum of homarine (37) (125 MHz, DMSO-d 6 ) 145 Figure 109. ROESY spectrum of homarine (37) (500 MHz, DMSO-d 6 ) 145 Figure 110. gCOSY spectrum of homarine (37) (500 MHz, DMSO-d 6 ) 146 Figure 111. gHSQC spectrum of homarine (37) (500 MHz, DMSO-d 6 ) 147 Figure 112. UV spectrum of ceramide analog (39) in CHCl 3 148 Figure 113. IR spectrum of ceramide analog (39) 148 Figure 114. gCOSY spectrum of ceramide analog (39) (500 MHz, CDCl 3 ) 149 Figure 115. TOCSY spectrum of ceramide analog (39) (500 MHz, CDCl 3 ) 150 Figure 116. DEPT 45 spectrum of ceramide analog (39) (125 MHz, CDCl 3 ) 151 Figure 117. gHSQC spectrum of ceramide analog (39) (500 MHz, CDCl 3 ) 152 Figure 118. gHMBC spectrum of ceramide analog (39) (500 MHz, CDCl 3 ) 153 Figure 119. NOESY spectrum of ceramide analog (39) (500 MHz, CDCl 3 ) 154 Figure 120. gCOSY spectrum of (S) MTPA ester (43) (500 MHz, CDCl 3 ) 155 Figure 121. gCOSY spectrum of (R) MTPA ester (44) (500 MHz, CDCl 3 ) 156 Figure 122. UV spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) in CHCl 3 157 Figrue 123. IR spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) 157 Figure 124. 1 H NMR spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) (250 MHz, CDCl 3 ) 158 Figure 125. 13 C NMR spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) (75 MHz, CDCl 3 ) 159 Figure 126. UV spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) xi

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in CHCl 3 160 Figure 127. IR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47)160 Figure 128. 1 H NMR spectrum 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) (250 MHz, MeOH-d 4 ) 161 Figure 129. 13 C NMR spectrum 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) (75 MHz, MeOH-d 4 ) 162 Figure 130. UV spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropanenitrile (48) in CHCl 3 163 Figure 131. IR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropanenitrile (48) 163 Figure 132. 1 H NMR spectrum of 3-[3-(benzyloxy)-2-nitroophenyl]-3hydroxypropanenitrile (48) (250 MHz, CDCl 3 ) 164 Figure 133. 13 C NMR spectrum of 3-[3-(benzyloxy)-2-nitroophenyl]-3hydroxypropanenitrile (48) (75 MHz, CDCl 3 ) 165 Figure 134. UV spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) in CHCl 3 166 Figure 135. IR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) 166 Figure 136. 1 H NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) (250 MHz, CDCl 3 ) 167 Figure 137. 13 C NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) (75 MHz, CDCl 3 ) 168 Figure 138. UV spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3xii

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hydroxypropyl}acetamide (50) in CHCl 3 169 Figure 139. IR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropyl}acetamide (50) 169 Figure 140. 1 H NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropyl}acetamide (50) (250 MHz, CDCl 3 ) 170 Figure 141. 13 C NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropyl}acetamide (50) (75 MHz, CDCl 3 ) 171 Figure 142. UV spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropyl}acetamide (51) 172 Figure 143. IR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropyl}acetamide (51) 172 Figure 144. 1 H NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropyl}acetamide (51) (250 MHz, CDCl 3 ) 173 Figure 145. 13 C NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropyl}acetamide (51) (75 MHz, CDCl 3 ) 174 Figure 146. UV spectrum of erebusinone (12) in MeOH 175 Figure 147. IR spectrum of erebusinone (12) 175 Figure 148. 1 H NMR spectrum of erebusinone (12) (250 MHz, MeOH-d 4 ) 176 Figure 149. 13 C NMR spectrum of erebusinone (12) (75 MHz, MeOH-d 4 ) 177 Figure 150. UV spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropylcarbamate (53) (250 MHz, CDCl 3 ) 178 Figure 151. IR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropylcarbamate (53) 178 xiii

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Figure 152. 1 H NMR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropylcarbamate (53) (250 MHz, CDCl 3 ) 179 Figure 153. 13 C NMR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3hydroxypropylcarbamate (53) (75 MHz, CDCl 3 ) 180 Figure 154. UV spectrum of tertbutyl-3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropylcarbamate (54) in CHCl 3 181 Figure 155. IR spectrum of tertbutyl-3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropylcarbamate (54) 181 Figure 156. 1 H NMR spectrum of tertbutyl-3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropylcarbamate (54) (250 MHz, CDCl 3 ) 182 Figure 157. 13 C NMR spectrum of tertbutyl-3-[3-(benzyloxy)-2-nitrophenyl]-3oxopropylcarbamate (54) (75 MHz, CDCl 3 ) 183 Figure 158. UV spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) 184 Figure 159. IR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) 184 Figure 160. 1 H NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) (250 MHz, MeOH-d 4 ) 185 Figure 161. 13 C NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) (75 MHz, MeOH-d 4 ) 186 Figure 162. UV spectrum of erebusinonamine (52) in MeOH 187 Figure 163. IR spectrum of erebusinonamine (52) 187 Figure 164. 1 H NMR spectrum of erebusinonamine (52) (250 MHz, MeOH-d 4 ) 188 xiv

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Figure 165. 13 C NMR spectrum of erebusinonamine (52) (75 MHz, MeOH-d 4 ) 189 Figure 166. DEPT-135 spectrum of erebusinonamine (52) (125 MHz, MeOH-d 4 ) 190 xv

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LIST OF TABLES Table 1. NMR data of purine analog (23) (DMSO-d 6 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 19 Table 2. NMR data of 3-hydroxykynurenine (24) (DMSO-d 6 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 24 Table 3. NMR data of 5-methyl-2-deoxycytidine (25) (MeOH-d 4 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 32 Table 4. NMR data of uridine (28) (DMSO-d 6 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 36 Table 5. NMR data of 2-deoxycytidine (31) (DMSO-d 6 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 41 Table 6. NMR data of 4, 8-dihydroxyquinoline (33) (DMSO-d 6 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 45 Table 7. NMR data of homarine (37) (DMSO-d 6 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 49 Table 8. NMR data of partial structure (a) (CDCl 3 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 57 Table 9. NMR data of (S) MTPA ester (43) (CDCl 3 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 65 Table 10. NMR data of (R) MTPA ester (44) (CDCl 3 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 65 Table 11. NMR data of ceramide analog (39) (CDCl 3 ) ( 13 C, 125 MHz; 1 H NMR 500 MHz) 69 xvi

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Table 12. Antimicrobial activity of pure compounds (100 g/disk) using the disk Diffusion assay (Zone of Inhibition in mm) 79 xvii

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LIST OF SCHEMES Scheme 1. Isolation of purine analog 23 and 3-hydroxykynurenine 24. 15 Scheme 2. Proposed fragmentations of purine analog 23. 20 Scheme 3. Isolation of nucleosides, 3-hydroxykynurenine, 4, 8-dihydroxyquinoline, and homarine. 27 Scheme 4. Isolation of ceramide analog (39). 51 Scheme 5. Methanolysis and protection reactions of ceramide analog (39) 57 Scheme 6. MTPA reactions with ceramide analog (39) 63 Scheme 7. Synthetic route to erebusinone precursor 49 75 Scheme 8. Synthetic route from erebusinone precursor 49 to erebusinone (12) 76 Scheme 9. Synthetic route of erebusinonamine 52 from erebusinone precursor 49 78 xviii

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LISTS OF ABBREVIATIONS Ac 2 O acetic anhydride [] specific rotation = 100/lc BH 3 S(CH 3 ) 2 dimethylsulfide borane BnBr benzylbromide Boc 2 O di-tert-butyldicarbonate BuLi butyllithium BuOH butanol CaH 2 calcium hydride CDCl 3 deuterated chloroform CF 3 COOH trifluoroacetic acid CH 2 Cl 2 dichloromethane CH 3 CN acetonitrile CoCl 2 cobalt chloride C18 octadecyl bonded silica chemical shifts DEPT distortionless enhancement by polarization transfer DMSO-d 6 deuterated dimethylsulfoxide EtOAc ethylacetate EtOH ethanol the molar extinction coefficient in UV spectroscopy gCOSY gradient correlation spectroscopy xix

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gHSQC gradient heteronuclear single quantum correlation gHMBC gradient heteronuclear multiple bond connectivity HRFAMS high resolution fast atom bombardment mass spectrometry or spectrum HREIMS high resolution electron impact mass spectrometry or spectrum HRESIMS high resolution electrospray ionization mass spectrometry or spectrum HCl hydrochloric acid HPLC high performance liquid chromatography IR infrared J coupling constant n J CH n-bond hydrogen to carbon correlation (n = 2, 3 or 4) n J HH n-bond hydrogen to hydrogen correlation (n = 2, 3 or 4) K 2 CO 3 potassium carbonate KOH potassium hydroxide LiAlH 4 lithium aluminum hydride LREIMS low resolution electron impact mass spectrometry or spectrum LRESIMS low resolution electrospray ionization mass spectrometry or spectrum LRFAMS low resolution fast atom bombardment mass spectrometry or spectrum max the wavelength at which maximum absorption occurs MeOH methanol MeOH-d 4 deuterated methanol MTPA-Cl -methoxy-(trifluoromethyl)phenylacetyl chloride MgSO 4 magnesium sulfate m/z mass/charge for mass spectrometry xx

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NaHCO 3 sodium bicarbonate NaBH 4 sodium borohydride Na 2 SO 4 sodium sulfate Na 2 S 2 O 3 sodium thiosulfate NMR nuclear magnetic resonance Pd palladium ROESY rotating-frame overhauser enhancement spectroscopy S N 2 bimolecular nucleophilic substitution THF tetrahydrofuran TOCSY total correlation spectroscopy UV ultraviolet xxi

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Chemical Investigation of Three Antarctic Marine Sponges Young Chul Park ABSTRACT This thesis describes the chemical investigation of three marine sponges from Antarctica and the total syntheses of natural products erebusinone (12) and its derivative, erebusinonamine (52). Investigation of the yellow Antarctic marine sponge Isodictya setifera resulted in the isolation of two secondary metabolites, purine analog (32) and 3-hydroxykynurenine (24). Chemical investigation of Isodictya setifera led to the isolation of six secondary metabolites which included 5-methyl-2-deoxycytidine (25), uridine (28), 2-deoxycytidine (31), homarine (37), hydroxyquinoline (33), 3-hydroxykynurenine (24). The latter two compounds were found to be intermediates of tryptophan catabolism in crustaceans. From the Antarctic marine sponge Isodictya antractica ceramide analog (39) was isolated and its chemical structure was assigned by a combination of spectroscopic and chemical analyses. Stereochemistry was determined by modified Moshers method. Erebusinone (12), a yellow pigment isolated from the Antarctic marine sponge Isodictya erinacea has been implicated in molt inhibition and mortality against the Antarctic crustacean amphipod, Orchomene plebs, possibly serving as a precursor of a xanthurenic acid analog. xxii

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3-Hydroxykynurenic acid OH NH2 O Erebusinonamine OH NH2 O NH3Cl Erebusinone OH NH2 O NHAc NH2O OH (12)(52)(24) Thought to act as a 3-hydroxykynurenine 24 mimic, erebusinone (12) may be involved chemical defense. 1 This appears to be the first example in the marine realm of an organism utilizing tryptophan catabolism to modulate molting as a defensive mechanism. To further investigate the bioactivity and ecological role of erebusinone (12), the synthesis of this pigment was carried out in an overall yield of 44% involving seven steps which were economical and convenient. Erebusinonamine (52) was also similarly synthesized in eight steps with an overall yield of 45%. Reference 1. Moon B. H.; Park Y. C.; McClintock J. B.; Baker B. J., Tetrahedron 2000, 56, 9057-9062. xxiii

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Chapter 1. INTRODUCTION 1.1. A Brief History of Natural Products Natural products of terrestrial plants have been utilized for many purposes such as medicines, euphoria and hunting. The use of crude natural products to treat cancer has a long history, recorded at least as far back as the Ebers papyrus in 1550 B. C. 1 The origin of traditional Chinese medicine also dates back to around 2500 B. C. 2 With the origin of organic and biological chemistry in the 19 th century, chemists and biologists began the investigation of bioactive compounds in order to identify the individual active ingredients from the terrestrial plants, freshwater, and marine organisms. The study bioactive plant compounds such as morphine, strychnine, atropine, quinine and colchicine was begun in the early 1800s. However, the structures of these secondary metabolites were not elucidated until a century. 3 The invention of advanced instrumentation tools, such HPLC (high performance liquid chromatography) and NMR (nuclear magnetic resonance) spectrometers enabled the characterization of minute quantities of secondary metabolites such as maitotoxin and taxol. 4-6 1.2. Marine Natural Products Chemistry Marine organisms have been investigated by natural products chemists for the past 40 years. Many of the secondary metabolites produced are both complex and quite 1

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distinct from terrestrial natural products. Marine natural products chemistry has evolved from being the source of a handful of chemical curiosities 20 years ago to being one of the most productive areas of natural products research. 7-9 The oceans and seas have been the focus of considerable attention for their biological resources. Marine organisms have evolved under markedly distinctive chemical and physical conditions from terrestrial plants and animals. 10 Oceanic marine organisms are of scientific interest for two major reasons. First, they constitute a major share of the Earths biological resources. 11 Second, marine organisms often possess unique structures, metabolic pathways, reproductive systems, and sensory and chemical defense mechanisms, 12,13 because they have adapted to environmental conditions which have high biodiversity. Their range encompasses the cold polar Arctic and Antarctic seas at low temperature, the warm water tropics and the bright shallow waters to the great pressures of the deep ocean floor. Yet the potential of this domain as the basis for new biotechnology resources such as drug discovery remains largely underexplored. Recent improvements in underwater life-support systems, however, have facilitated the collection of organisms from previously unexplored regions of the oceans. Many bioactive chemicals from the marine environment have been isolated and characterized. 14-31 Same hold great promise for useful biotechnological applications, such as development of a wide range of pharmaceutical compounds, medical research materials, agricultural products and processes, novel energy sources and bioremediation techniques. 32,33 With knowledge of these factors, several marine invertebrates and microorganism groups have been noted for their potential for new drug discovery. 33,34 Many marine invertebrates such as algae, corals, sponges and ascidians (tunicates) use 2

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highly evolved chemical compounds for purposes such as reproduction, communication, and protection against predation, infection, and competition. 11 These bioactive chemical compounds may have antibiotic, anti-inflammatory, antiviral, cytotoxic, antitumor or antifungal properties. Secondary metabolites, which are not needed by the organism for basic or primary metabolic processes such as food digestion, energy storage, or metabolism, are believed to confer some evolutionary advantage because many marine invertebrates living in densely populated habitats are non-motile, have primitive immune systems, and are found symbiotic relationships with microorganisms. 35,36 Natural product chemists have been interested in sponge-microbe symbioses because of the possibility that the diverse, bioactive chemical structures found in sponges might be produced by microorganisms. More natural products have been reported from sponges 37 than from any other marine invertebrate phylum, and many of the most promising pharmaceuticals and agents for cell biological research were isolated from sponges. 37 Marine sponges belong to the phylum Porifera and are actually simple cell aggregates, which are usually referred to as the most undeveloped multicellular animals. 14-31 In contrast, ascidians belong to the phylum Chordata, which encompasses all vertebrate animals, including mammals. As much as 40% of the cell mass of sponges, ascidians and other marine macroorganisms could be composed of prokarytic organisms. 38,39 The first isolation of a secondary metabolite from a marine organism was Tyrian purple (6,6-dibromoindigotin) (1) from a marine mollusk. Identified in 1901, 7,40 3

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Tyrian purple (1) was also the first marine natural product to be used for commercial applications. NH HN O O Br Br Tyrian Purple (1) The marine environment became the focus of natural products drug discovery research because of its relatively unexplored biodiversity compared to terrestrial environments. The potential for marine natural products as pharmaceuticals was introduced by the pioneering work of Bergmann in 1950s which led to the discovery of Ara-A (2) 41 and Ara-C (3), 42 the only two marine derived pharmaceuticals that are clinically available today. The anticancer drug cytosine arabinoside (Ara-C) (3) is used to treat acute myelocytic leukemia. 43 The antiviral drug adenine arabonoside (Ara-A) (2) is used for the treatment of herpes virus infections. 44,45 NN NN NH2 O H OH H H H H HO O H OH H H H H HO NN NH2 O Adenine Arabinoside (Vidarbine)Ara-A (2)Cytosine Arabinoside (Cytarabine)Ara-C (3) 4

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Both are derived from nucleosides isolated from a shallow water marine sponge Cryptotethya crypta collected off the coast of Florida. The cytotoxic macrolide bryostantin (4), isolated from the bryozoan Bugula neritina, was found to have anticancer activity against murine P388 lymphocytic leukemia in vitro cell line. 46 It is now in clinical trials for the treatment of leukemias, lymphomas, melanoma and solid tumors. 47,48 O O MeOOC O OAc O COOMe O O OH OH OH OH Bryostatin (4) Another example of an important marine natural products include ecteinascidin 743 (5), a secondary metabolite displaying strong anticancer activity, which was first isolated from the mangrove tunicate, Ecteinascidia turbinata in 1990. 50, 51 5

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NNCH3 OO O O NH S OHCH3O HO CH3 OAc OCH3 HO CH3 H HHH Ecteinascidin 743 (5) Marine prostaglandins, first discovered in a gorgonian have since been isolated from other invertebrates and from red algae. 53 Punaglandins, 52 which are halogenated antitumor eicosanoids, were isolated from the octocoral Telesto riisei. These punaglandins (6, 7) are characterized by C-12 oxygen and unprecedented C-10 chlorine functions and inhibit L1210 leukemia cell proliferation with an IC 50 values in the range of 0.02 g/mL. 54, 55 O Cl OAc OAc O OMe HO Punaglandin 3 (6) O Cl OAc OAc O OMe HO Punaglandin 4 (7) Papuamine (8), an antifungal pentacyclic alkaloid from the marine sponge Haliclona sp., a thin, red, encrusting sponge, is formally derivable from a C 22 unbranched hydrocarbon and 1,3-diaminopropane and inhibits the growth of the fungus Trichophyton mentagrophytes. 56 6

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HN HN H H H H H H H H Papuamine (8) Spongia tubulifera found in the Florida Keys contains two types of toxins. The spongianolids, exemplified by spongianolide A-F (9,10) and kuropongin (11) are a series of cytotoxic sesterterpenes which display potent MCF-7 mammary tumor cell inhibition activity, inhibition of protein kinase C and antibiotic activity, displaying 5 mm zones of inhibition at 100 g/disk toward a host of antibiotic tester strains. 57 OR O O OH H H OH H H O O HO O RO H Spongianolides A B (9)Spongianolides C F (10) O O O O Kurospongin (11) Marine sponges are among the most prolific sources of diverse chemical compounds with therapeutic potential. Of the more than 5000 chemical compounds derived from marine organisms, more than 30 percent have been isolated from sponges. 49 7

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1.3. Antarctic Marine Natural Products The Antarctica marine benthos has diverse and abundant biological and ecological communities. For 20 million years the Antarctica benthos has had physically unique, isolated environments and ecology. These factors provide a basis to study the chemical and biological interactions such as competition and predation. Secondary metabolites from Antarctica have recently begun to be studied and they have been found to display unique biological activities such as cytotoxic, antimicrobial, antifungal, and antiviral properties. This may result from unique functional roles for Antarctic species. 58 On the benthos of McMurdo Sound, Antarctica, sponges are found to occupy 55% of the surface area. 59 The sea star Perknaster fuscus and the amphipod Orchomene plebs are predominant and voracious sponge predators. We reported that the bright yellow marine sponge Isodictya erinacea is a conspicuous member of the sponge community on the McMurdo Sound and suggested that despite a lack of physical protection such as spicules, I. erinacea is chemically defended against predator O. plebs. 60,61 Secondary metabolites, erebusinone (12), erinacine (13) were isolated from I. erinacea. Erebusinone (12) displayed molt inhibition leading to increased mortality in the predator amphipod, Orchomene plebs, serving as the first example in the marine realm of molt inhibition as a chemical defense mechanism. Erinacine (13) has shown 8

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cytotoxcity (LD 50 50 g/mL) against L5178Y mouse lymphoblastoid cells and displayed chemical deterrence toward the sea star Perknaster fuscus. 60,61,62 NN NHHN O NH HO2C OH NH2 O NH O Erinacean (13)Erebusinone (12) Sponges of the genus Latrunculia apicalis have been chemically studied. Ichthyotoxic and cytotoxic agents have been reported from the Red Sea Latrunculia magnifica 63 and South Pacific Latrunculia species. 64 Discohabdin C (14) and G (15), imino-quinone pigments having antibiotic and cytotoxic activity and influencing sea star (Perknaster fuscus) feeding behavior were isolated from the Antarctic sponge Latrunculia apicalis Ridley and Dendy from McMurdo Sound. 65 Variolin A (16) is unusual cytotoxic alkaloid, has been isolated from the bright red sponge Kirkpartrickia variolosa. 66 NH N HN O O +Br H Discorhabdin C (14)Br NH N HN O O +Br H Discorhabdin G (15)N N N NN -O NH2 OH Variolin A (16)++ 9

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Rhapsamine (17), a linear C28 polyene substituted by terminal 1,3-diaminoglycerol groups was isolated from the Antarctic sponge Leucetta leptorhapsis from the Ross sea. It was found to have potent cytotoxicity displaying LC 50 of 1.8 M in the KB nasopharyngeal cell line. 67 H2N HN OH HN OH H2N Rhapsamine (17) Secondary metabolites, membranolide (18), 9, 11-dihydrogracillin (19) and an isoquinoline pigment (20) were isolated from the bright yellow Antarctic sponge Dendrilla membranosa. The isoquinoline pigment, 4,5,8 trihydroxyquinoline-2-carboxylic acid displayed antibacterial activity against the marine bacteria Vibrio angullarum and Beneckea harveyii B-392. 68,69 CO2Me O O NH O OH OH COOH Membranolide (18)Isoquinoline pigment (20) O 9,11-Dihydrogracilin A (19)OAc OAc H 10

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1.4. Research Objectives Antarctic marine organisms have evolved differently under markedly distinctive chemical and physical conditions compared to terrestrial plants and animals. To study its chemical and ecological relationships of marine organisms in Antarctica, we have investigated three marine sponges. The benthos of McMurdo Sound is characterized by extensive sponge communities with spongevorous. Previously we reported that the secondary metabolite, erebusinone from Isodictya erinacea has been implicated in molt inhibition and mortality against an Antarctic crustacean, Orchomene plebs, perhaps serving as precursor of a xanthurenic acid analog. 61 With our previous metabolite profile, biological activities and ecological implications, we suggest that the Antarctic sponge, Isodictya setifera may have similar chemical defense mechanisms. To further investigate the chemical ecology of Antarctic sponges, we studied three Antarctic marine sponges, Isodictya erinacea, Isodictya setifera, and Isodictya antarctica. We also synthesized the natural product, erebusinone (12) and its derivative, erebusinonamine (52). 11

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Chapter 2. CHEMICAL INVESTIGATION OF ANTARCTIC MARINE SPONGE ISODICTYA ERINACEA 2.1 Introduction The marine sponge Isodictya erinacea (Topsent, 1916) (Family Esperiopsidae) is a conspicuous member of the Antarctic benthos which appears to be free of predation despite its lack of structural protection elements such as spicules (Figure 1). Figure 1. Isodictya erinacea collected from at Erebus Bay, Ross Island, Antarctica (Photograph supplied by Bill J. Baker, University of South Florida) We have previously reported that I. erinacea is chemically defended against the major Antarctic sponge predator, the sea star Perknaster fuscus and the isolation of 12

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secondary metabolites, erebusinone (12), erinacean (13), 1,9-dimethylguanine (21), 7-methyladenine (22). Erebusinone, a yellow pigment is a tryptophan catabolite and appears to protect the sponge from crustacean predation by inhibition of molting in predatory amphipods. In the course of our continuing search for secondary metabolites possessing ecological and biological significance, I. erinacea has been further studied. 60, 61,70 NN NHHN O NH HO2C OH NH2 O NH O Erinacean (13)Erebusinone (12)NN NN O H2N CH3 CH3 HNN NN NH2 CH3 1,9-Dimethylguanine (21)7-Methyladenine (22) 13

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2.2 Extraction and Isolation of Secondary Metabolites The chemical investigation of Isodictya erinacea collected from Erebus Bay on the western coast of Ross Island, Antarctica resulted in the isolation and structure elucidation of two compounds, purine (23) and 3-hydroxykynurenine (24) The wet sample of Isodictya erinacea was extracted (Scheme 1) with MeOH at 7 C. After concentration, the residue was subjected to Sephadex LH-20 column chromatography with MeOH/CH 2 Cl 2 (1:1) to provide five fractions. Fraction 4 was subjected to C18 column chromatography with a MeOH/CH 2 Cl 2 gradient system to give additional fractions. The first fraction 8 was chromatographed on silica gel to give 4 fractions. Fraction 11 (Scheme 1) was further chromatographed on silica gel yielding a purine analog (23) (68 mg, 0.0068% dry wt). Fraction 12 (104 mg) was chromatographed on silica gel to give 3-hydroxykynurenine (24) (13.5 mg, 0.00135% dry wt) (Scheme 1). NN NN CH3 CH3 HN O CH3 H (23)(24) OH NH2 O NH2O OH 14

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Isodictya erinacea1.0 Kg (frozen and wet)1. Extraction with MeOH, 2 L X 5 times at 7 oC 2. Filteration over C18 (8 cm X 2 cm)20 gFraction 1 0.21gFraction 2 6.34gFraction 3 5.71gFraction 5 0.73gFrction 6 510 mg LH-20 (5 cm X 60 cm), CH2Cl2/MeOH (1:1) C18 column (2.5 cm X 45 cm) 1. 100 % MeOH, 2.100% CH2Cl2Fraction 8 374 mgFraction 9 169mgsilica gel column (2.5 cm X 45 cm)H2O/MeOH/CHCl3 (0.4:3.0:6.6)Fraction 10 70 mgFraction 11 104 mgFraction 12 152 mgFraction 13 61 mgsilica gel (2.5 cm x 30 cm) H2O/MeOH/CH2Cl2 (0.4:3:6.6) Purine analog 23 (68 mg)0.0068 % yield3-Hydroxykynurenine 24 (13.5 mg) 0.00135% yieldsilica gel (2.5 cm X 30 cm)H2O/MeOH/CH2Cl2 (0.5:4:5.5)Fraction 4 1.1gFraction 7487 mg Scheme 1. Isolation of purine analog 23 and 3-hydroxykynurenine 24. 15

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2.3 Characterization of Purine analog (23) From fraction 11, purine analog 23 was obtained as a pale brown solid and assigned the molecular formula C 8 H 11 N 5 O, as deduced by HREIMS (observed m/z 193.0959, calculated 193.0964). The 1 H NMR spectrum (Figure 2) of purine analog (23) in DMSO-d 6 contained four signals at 3.36, 3.61, and 3.88 which suggested methyl protons bearing nitrogen and an aromatic methine at 8.17 with one exchangeable proton signal at 3.4. The 13 C NMR spectrum (Figure 3) showed three methyl groups at 33.4, 32.2, 29.3, one methine at 143.6 and four quaternary carbons at 152.5, 151.3, 148.2, 106.9. 8.172 3.887 3.614 3.435 3.382 3.367 8 7 6 5 4 3 2 1 PP M Figure 2. 1 H NMR spectrum of purine analog (23) (500 MHz, DMSO-d 6 ). 16

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Figure 3. 13 C NMR spectrum of purine analog (23) (125 MHz, DMSO-d 6 ). The IR spectrum of 23 showed one carbonyl stretching absorption band at 1715 cm -1 a C=N stretching absorption band at 1641 cm -1 and NH stretching absorption bands at 3303 cm -1 The basic chemical skeleton of this purine analog (23) was established by a gHMBC (gradient Heteronuclear Multiple Bond Connectivity) in DMSO-d 6 as shown in (Figure 4). Three methyl proton signals bearing nitrogen revealed connectivity (Figure 5) from N-1-CH 3 to C-2 (151.3) and C-6 (152.5); from N-3-CH 3 to C-2 (151.3) and C-4 (147.6); from N-7-CH 3 to C-5 (106.9) and C-8 (143.6) and one methine proton bearing carbon signal showed HMBC three-bond correlations with two quaternary carbons at C-5 (106.9) and C-8 (143.6). Table 1 provides a summary of NMR spectroscopic data. 17

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9 8 7 6 5 4 3 2 1 PPM 180 160 140 120 100 80 60 40 20 Figure 4. HMBC spectrum of purine analog (23) (500 MHz, DMSO-d 6 ). NN NN O CH3 HN CH3 123456789 CH3 H Figure 5. Key gHMBC correlations observed in purine analog (23). 18

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N-1-Me 3.36 (3.42)a 29.3 (27.8)a C2, C6C-2 151.3 (151.7)N-3-Me 3.61 (3.66) 32.2 (30.6) C2, C4C-4 147.6 (147.2)C-5 106.9 (106.8)C-6 152.5 (152.2)N-7-Me 3.88 (3.95) 33.4 (32.4) C5, C8C-8 143.6 (142.9)C-8-H(1H) 8.17 (8.14) C4, C5N-9 gHMBC position13C()1H() Table 1. NMR data of purine analog (23) (DMSO-d6) (13C, 125 MHz; 1H NMR 500 MHz)a Chemical shifts () measured in MeOH-d4 (13C, 75 MHz; 1H NMR 250 MHz) The UV spectrum of purine analog (23) showed at max 210 nm and 268 nm, which is similar to absorption properties of known compounds 1,3,7-trimethylguanine [UV (MeOH) max 213 nm and 288 nm] 71 and 1,3,7-trimethylisoguanine [UV (MeOH) max 214 nm and 288 nm]. 72 The mass spectrum of purine analog 23 and its 1-, 3-, and 7methyl derivatives have been analyzed in detail. 73 In the LREIMS spectrum of purine analog (23) (Scheme 2), fragments at m/z 178.1 [M-NH] + and m/z 164.1 [M-CHO] + were observed. The fragments of m/z 138.1 [M-HNCNCH 3 ] + due to the loss of methyl cyanamide at [M-55] + which then sequentially loses CO (m/z 109), HCN (m/z 82.1) as were reported in an N-1-methylated isoguanine purine 73 as the most characteristic mode of decomposition of this compound. Scheme 2 illustrates these fragmentations 19

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NN O HN CH3 CH3 NN CH3 H M-55, 56NHN C O N CH3 H NHN C O N CH3 H [137.1 CO]NN H N H3C CH3 m/z 193.1m/z 138.1 m/z 137.1m/z 109.1 HCN[109.1-17]CH3NC C NCH3 m/z 82.1 NN H N H3C CH3 Scheme 2. Proposed fragmentations of purine analog 23. 2.4 Characterization of 3-Hydroxykynurenine (24) 3-Hydroxykynurenine (24) was obtained as a bright yellow powder (C 10 H 12 N 2 O 3 ) and was soluble in DMSO and MeOH. The UV absorption in MeOH showed max 234, 273, 381 nm. The compound displayed a classic 1 H NMR (Figure 6) pattern for a 1, 2, 3-trisubstituted aromatic ring with signals at 7.26 (1H, dd, J = 1.2, 8.2 Hz), 6.79 (1H, dd, J = 1.3, 7.5 Hz), and 6.45 (1H, t, J = 8.2 Hz) in MeOH-d 4 The three aliphatic protons were observed including one methine at 3.98 (1H, dd, J = 2.7, 9.0 Hz), two methylene protons at 3.70 (1H, dd, J = 2.5, 18.6 Hz) and 3.47 (1H, dd, J = 9.5, 18.5 Hz). The 1 H NMR spectrum of aromatic proton chemical shifts of 3-hydroxykynurenine (24) was similar to that of erebusinone (12) (Figure 7) in MeOH-d 4 20

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7.274 7.241 6.805 6.778 6.484 6.452 6.421 4.894 3.991 3.963 3.953 3.668 3.659 3.526 3.489 3.324 3.285 8 7 6 5 4 PP M Figure 6. 1 H NMR spectrum of 3-hydroxykynurenine (24) (250 MHz, MeOH-d 4 ). 7.288 7.259 7.254 6.807 6.781 6.777 6.485 6.453 4.869 3.519 3.494 3.165 3.140 3.112 1.904 7 6 5 4 3 2 PP M Figure 7. 1 H NMR spectrum of synthetic erebusinone (12) (250 MHz, MeOH-d 4 ). 21

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The 13 C NMR spectrum (Figure 8) indicated the presence of ten carbons; six aromatic carbons at 145.5, 141.6, 121.6, 117.4, 117.1, 114.6, two carbonyl carbons at 170.3 and 200.0 and two aliphatic carbons at 50.5 and 41.1. The structure of 3-hydroxykynurenine (24) was deduced by analysis 2D NMR techniques [gCOSY (gradient COrrelation SpectroscopY), gHSQC (gradient Heteronuclear Single Quantum Correlation), and gHMBC)] (Figure 9, 10). Figure 8. 13 C NMR spectrum of 3-hydroxykynurenine (24) (125 MHz, DMSO-d 6 ). In the gHSQC experiment, mutual cross-peaks were observed between the aromatic protons at 7.19 (H-1), 6.85 (H-2), 6.40 (H-4) to carbons at 121.6, 117.4, and 114.6, respectively. Also, in the gHMBC spectrum (Figure 9) (Table 2), long range couplings ( 2 J CH / 3 J CH ) were observed between 7.12 (H-1) and C-12 ( 200.0), C-10 ( 22

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145.5), C-9 ( 141.6), C-8 ( 117.1), and C-2 ( 117.4) indicating the presence of the benzene ring (Figure 10). 8 7 6 5 4 3 2 1 PPM 200 150 100 50 PPM Figure 9. gHMBC spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ). OH NH2 O NH2O OH 8191024126511 Figure 10. Key gHMBC correlations observed in 3-hydroxykynurenine (24). 23

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Table 2. NMR data of 3-hydroxykynurenine (24) (DMSO-d6) (13C, 125 MHz; 1H, 500 MHz) 1 7.19 (1H, d, J = 8.2 Hz) 121.6 C12, C10, C9, C8, C22 6.85 (1H, d, J = 7.6 Hz) 117.4 C10, C9, C13 6.70 (2H, s) C10, C84 6.40 (1H, t, J = 8.0 Hz) 114.6 C10, C85 3.62 (1H, d, J = 7.8 Hz) 50.5 C12, C11, C6, C76 3.50 (1H, dd, J = 2.3, 18.1 Hz) 41.1 C12, C57 3.24 (1H, dd, J = 8.6, 17.5 Hz) 41.1 C12, C58 117.19 141.6 10 145.511 170.312 200.0 1H ()a13C()gHMBC a 1H NMR (250 MHz, MeOH-d4) 3.46 (1H, dd, J = 9.5, 18.5 Hz), 3.70 (1H, dd, J = 2.5, 18.7 Hz), 3.97 (dd, J = 2.5, 9.7 Hz), 6.45 (t, J = 8.2 Hz), 6.79 (d, J = 7.5 Hz), 7.26 (d, J = 8.2 Hz) The 3-hydroxykynurenine (24) displayed an optical rotation [] 25 D -47.6 (c = 0.17, MeOH) which is similar to that reported [] 25 D -45 [(c = 0.9, MeOH)] 74,75 suggesting absolute stereochemistry at position C-5 of our isolate to be the S configuration. The UV spectrum of compound 24 observed in max 234, 273, 381 nm in MeOH (pH = 7) was similar to previously reported absorption values, max 224, 266, 370 nm (0.005 M HCl) from literature. 74 3-Hydroxykynurenine OH NH2 O Erebusinone OH NH2 O NH NH2O OH (12)(24) 121.6114.6117.4145.5141.6117.1201.941.150.5170.3122.8146.4142.4118.0115.7118.9202.139.636.5173.522.7 O CH3 a'b'c'abc(7.26)*(6.79)(6.45)(7.28)(6.79)(6.45) Figure 11. Assigned NMR data of erebusinone 61 and 3-hydroxykynurenine (MeOH-d 4 1 H NMR 250 MHz; 13 C NMR, 75, 125 MHz) 1 H chemical shifts. 24

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Chapter 3. CHEMICAL INVESTIGATION OF ANTARCTIC MARINE SPONGE ISODICTYA SETIFERA 3.1 Introduction The Antarctic marine sponge Isodictya setifera Topsent (family Esperiopsidae) (Figure 12) has received attention due to its bioactive metabolites which were isolated from the sponge-associated with bacterial strain Pseudomonas aerugenosa, which showed strong antibacterial activity. 76 Although the chemistry of the I. setifera sponge-associated bacterium has been reported, 77,78 the secondary metabolites of the sponge have not been reported. Figure 12. Isodictya setifera collected from Bahia Paraiso, Palmer Station, Antartica (Photograph supplied by Bill J. Baker, University of South Florida). 25

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3.2 Extraction and Isolation of Secondary Metabolites In continuation of our study of Isodictya setifera, sponges were collected by SCUBA diving at the Bahia Paraiso shipwreck on the coast near Palmer station, Antarctica, and chemically studied, resulting in the isolation of six compounds, 5-methyl-2-deoxycytidine (25), uridine (27), 2-deoxycytidine (30), hydroxyquinoline (33), 3-hydroxykynurenine (24), and homarine (37). The freezedried sample of Isodictya setifera was exhaustively extracted with MeOH at room temperature (Scheme 2). After concentration, the crude extract was partitioned with hexane, MeOH, H 2 O and BuOH. The BuOH soluble material was subjected to Sephadex LH-20 column chromatography (5 cm X 60 cm) with MeOH to provide seven fractions. Fraction 12 was further subjected to normal phase column chromatography with MeOH/CH 2 Cl 2 and CH 3 CN (acetonitrile)/H 2 O and followed by reversed phase HPLC with a cyano-derivatized column using a gradient CH 3 CN/H 2 O to give pure compounds which are 5-methyl-2-deoxycytidine (25, 3.0 mg, 0.0007% dry wt), uridine (28, 2.0 mg, 0.0005% dry wt), 2-deoxycytidine (31, 1.5 mg, 0.0003% dry wt), hydroxyquinoline (33, 1.8 mg, 0.0004% dry wt), 3-hydroxykynurenine (24, 1.7 mg, 0.0004% dry wt), and homarine (37, 2.0 mg, 0.0005% dry wt). 26

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Isodictya setifera 435g(freeze dried)1. Extraction with MeOH (3 X 3 L)2. Hexane/MeOH/ H2O (4:5:1) CH2Cl2/MeOH/ H2O (4:5:1) BuOH/H2O (4:1) LH20, MeOH (2.5 X 50 cm)Fraction 6 85mgFraction 7 130 mgFraction 8 174 mgFraction 9 223 mgFraction10 190 mgFraction11 390 mgFraction 12 570 mgFraction 1 9.0 gFraction 2 2.3 gFraction 3 6.0 g Fraction 4 1.9 g(BuOH extract)Fraction 5 38g(H2O extract) (Hexane extract)(CH2Cl2/MeOH extract) 1. silica gel column (2.5 X 40 cm)2. HPLC with reversed cyano column CH3CN/H2O (from 99:1 to 80:20)5-Methyl-2'-deoxycitidine25, 3.0 mg (0.0007% yield) 2'-Deoxycytidine 311.5 mg (0.0003% yield) Uridine 28 2.0 mg (0.0005%)3-Hydroxykynurenine 24 1.7 mg (0.0004% yield) 4,8-Dihydroxyquinoline33, 1.8 mg (0.0004% yield) Homarine 372.0 mg ( 0.0005% yield) Scheme 3. Isolation of nucleosides, 3-hydroxykynurenine, 4, 8-dihydroxyquinoline, and homarine. 27

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3.3 Characterization of 5-Methyl-2-deoxycitidine (25) The 1 H NMR spectrum (Figure 13) of 5-methyl-2-deoxycitidine (25) showed typical chemical shifts of nucleosides, including the presence of one olefinic methine group ( 7.83), three aliphatic mithine groups ( 6.30. 4.42, 3.92), two aliphatic methylene groups ( 3.78, 2.25) and one methyl group ( 1.89). The 13 C NMR spectrum (Figure 14) showed ten carbon signals corresponding to one methyl ( 11.3), two methylenes ( 61.7, 40.0), four methines ( 136.7, 87.4, 85.1, 71.0), and three quaternary carbons ( 165.3, 151.2, 110.3) based on analysis by the gHSQC spectrum. 8 7 6 5 4 3 2 PP M Figure 13. 1 H NMR spectrum of 5-methyl-2-deoxycitidine (25) (500 MHz, MeOH-d 4 ). 28

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Figure 14. 13 C NMR spectrum of 5-methyl-2-deoxycitidine (25) (125 MHz, MeOH-d 4 ). First, the partial structure (a) of 5-methyl-2-deoxycitidine was established using a gCOSY (Table 3) (Figure 15) experiment. The olefinic methine at 7.83 (1H, s, H-1) showed allylic coupling with the methyl protons at 1.89 (3H, s, H 3 -7). A second partial structure (b) was evidenced by the aliphatic methines at 6.30 (1H, t, J = 6.8 Hz, H-2) coupled with neighbor methylene protons at 2.25 (2H, m, H-3) which were in turn correlated with methines at 4.42 (1H, m, H-4). The methine at 4.42 (H-4) showed 1, 3-correlations with methines at 3.92 (1H, q, J = 3.4, H-5) and 2.25 (2H, m, H-3). The methine at 3.92 (H-5) showed correlations with one methine at 4.42 (H-4) and one methylene at 3.81 (1H, dd, J = 3.2, 12.5 Hz, H a -6) and 3.74 (1H, dd, J = 3.5, 11.8 Hz, H b -6). The methylene protons at 3.81 (H a -6) and 3.74 (H b -6) 29

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showed correlation with one methine at 3.92 (H-5) and W-coupling with the methine at 4.42 (H-4), completing partial structure (b). N HO NH2 O HO 1532'3'4'5'6'6 CH3 NN O HO NH2 O HO 172'4'55'636'3'CH3 7 (b)(a) Figure 15. gCOSY correlations of 25. A methine proton at 7.83 (H-1) showed 2 J CH / 3 J CH correlations in the gHMBC spectrum (Figure 16) to C-2 ( 85.1), and C-7 ( 11.3) as well as with three quaternary carbons C-6 ( 110.4), C-3 ( 151.2), and C5 ( 165.3) supporting a pyrimidine moiety. The methine proton (H-2) at 6.30 showed 3 J CH correlation to C-1 ( 136.7) and a quaternary carbon at 151.20 (C-3). The methylene protons (H 2 -3) showed 2 J CH / 3 J CH correlations to C-2 ( 85.1), C-4 ( 71.1), and C-5 ( 87.6) supporting a sugar moiety. These data established the structure of 5-methyl-2-deoxycytidine (25) (Figure 17). 30

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9 8 7 6 5 4 3 2 1 0 PPM 200 150 100 50 0 Figure 16. gHMBC spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ). NN O HO NH2 O H O 172'4'55'636'3'CH3 Figure 17. Key gHMBC correlations observed in 5-methyl-2-deoxycitidine (25). 31

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Table 3 NMR data of 5-methyl-2'-deoxycytidine (25) (MeOH-d4) (13C, 125 MHz; 1H, 500 MHz) 1 7.83 (1H, s) 136.7 (CH) H7 C3, C5, C6, C7, C2'3 151.2 (C) 5 165.3 (C)6 110.4 (C) 7 1.89 (3H,s) 11.3 (CH3) H1 C5, C6, C1 2' 6.30 (1H, t, J = 6.8Hz) 85.1 (CH) H3' C3, C13' 2.25 (2H, m) 40.0 (CH2) H2', H4' C2', C4', C5'4' 4.42 (1H, m) 71.0 (CH) H3', H5' C2', C5'5' 3.92 (1H, q, J = 3.4 Hz) 87.6 (CH) H4', H6' C3'6' 3.81 (1H, dd, J = 3.2,12.5 Hz) 61.7 (CH2) H6' C4', C5' 3.74 (1H, dd, J = 3.5,11.8 Hz) 61.7 (CH2) H6' C4', C5' positiongCOSYgHMBC1H ()13C () The chemical shifts of 5-methyl-2-deoxycytidine (25) from the current study was compared with the literature and Aldrich Library of NMR spectra. 79,80 All assigned chemical shifts by 1 H NMR and gCOSY are similar those of published (Figure 18). O OH HO NN NH2 O CH3 (25)12'4'55'633'7.81.96.32.24.43.83.9O OH HO NNH O O CH3 (27)12'4'55'633'7.61.96.32.44.43.84.1O OH HO NN NH2 O (26)12'4'55'633'6'7.96.16.32.34.54.13.9 Figure 18. Assigned 1 H NMR chemical shifts () of 5-methyl-2-deoxycytidine 25 (500 MHz, DMSO-d 6 ) from the current study data, 2-deoxycytidine 26 80 and thymidine 27 80 (60 MHz, D 2 O). 32

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3.4 Characterization of Uridine (28) The 1 H NMR spectrum (Figure 19) of uridine (28) is similar to that of 5-methyl-2-deoxycytidine (25) except for the absence of the methyl group at 1.89. 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 PP M Figure 19. The 1 H NMR spectrum of uridine (28) (500 MHz, DMSO-d 6 ). The 1 H1 H gCOSY (Figure 20) of uridine (28) displayed 3 spin systems that include two olefinic methine groups ( 7.88, 5.64), four aliphatic methine groups ( 5.77, 4.01, 3.95, 3.83) and contiguous with one methylene group ( 3.61, 3.54). The aliphatic methines at 5.77 (H-2) coupled with its neighboring proton at 4.01 (H-3), which was in turn correlated with 3.95 and with one exchangeable proton. The methine at 3.95 (H-4) showed further correlation ( 3 J HH ) with 3.83 (H-5) and one exchangeable proton at 5.10 (H-4). The methine at 3.83 (H-5) showed additional correlation ( 3 J HH ) with the methylene protons (H-6) at 3.61 and 3.54 which showed further vicinal correlation ( 2 J HH ). 33

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HN HO O O HO 1532'3'4'5'6'6 HNN O HO O O HO 12'4'55'636'3' (b)(a)OH 3"4"OH 4"3" Figure 20. gCOSY correlation of 28. The 13 C NMR spectrum (Figure 21) displayed nine carbon signals. In the gHSQC experiment, mutual cross-peaks ( 1 J CH ) were observed between the aromatic protons at 7.88 (H-1) and 5.64 (H-6) to carbons at 141.4 and 102.4, respectively. Also, in the gHMBC spectrum (Figure 22), cross-peaks ( 2 J CH / 3 J CH ) were observed between 7.88 (H-1) and C-5 ( 163.8), C-3 ( 151.4), C-6 ( 102.4) and C-2 ( 88.3) (Figure 23) (Table 4). 34

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Figure 21. The 13 C NMR spectrum of uridine (28) (500 MHz, DMSO-d 6 ). 8 7 6 5 4 PPM 160 140 120 100 80 60 Figure 22. The gHMBC spectrum of uridine (28) (500 MHz, DMSO-d 6 ). 35

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HNN O HO O O HO 12'4'55'636'3' OH 3"4" Figure 23. Key gHMBC correlations observed in uridine (28). Table 4. NMR data of uridine (28) (DMSO-d6) (13C, 125 MHz; 1H, 500 MHz) 1 7.88 (1H, d, J = 7.9 Hz) 141.4 (CH) H6 C3, C5, C6, C2'3 151.4 (C)5 163.8 (C)6 5.64 (1H, d, J = 8.3 Hz) 102.4 (CH) H1 C12' 5.77 (1H, d, J = 5.8 Hz) 88.3 (CH) H3' C3, C1, C3'3' 4.01 (1H, q, J = 5.4 Hz) 74.2 (CH) H2', H4', H3" C2', C5'4' 3.95 (1H, q, J = 4.6 Hz) 70.5 (CH) H3', H5', H4" C2', C5'5' 3.83 (1H, q, J = 3.3 Hz) 85.5 (CH) H6', H4'6' 3.61 (m, 1H) 61.5 (CH) H5', H4" 3.54 (m, 1H) 61.5 (CH) H5', H4"3" 5.37 (1H, d, J = 5.9 Hz) H3' C2', C3', C4'4" 5.10 (1H, m) H4' C3', C4', C5' positiongCOSYgHMBC1H ()13C () The natural compound uridine (28) 70 was previously isolated from Isodictya erinacea and characterized structure by 2D NMR. In the current study, the isolated uridine 28 displayed two aromatic methines on the pyrimidine ring at 7.88 (H-1, J = 7.9 Hz) and 5.64 (H-6, J = 8.3 Hz), which were identical to chemical shifts (Figure 23) 36

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and coupling constant previously reported [ 7.86 (H-1, J = 8.1 Hz) 5.66 (H-6, J = 8.1 Hz)] Comparison of reported 1 H NMR data of uridine (Figure 24) showed the difference of chemical shifts attributed by solvent effect. O OH OH HO NNH O O (28)12'4'55'633'O OH OH HO NNH O O (30)12'4'55'633'7.885.645.774.013.953.833.613.54O OH OH HO NNH O O (29)12'4'55'633'7.865.665.874.124.123.963.753.657.95.96.03.94.24.24.2 Figure 24. Assigned 1 H NMR chemical shifts () of uridine (28) (500 MHz, DMSO-d 6 ) from the current study, previous reported uridine (29) 70 (360 MHz, MeOH-d 4 ) and uridine 30 81 (60 MHz, D 2 O+NaOD). 3.5 Characterization of 2-Deoxycytidine (31) 2-Deoxycytidine (31) was obtained as a pale brown solid. The 1 H NMR spectrum displayed (Figure 25) three spin systems including two olefinic methine groups at 7.84 (1H, d, J = 8.1 Hz, H-1), and 5.63 (1H, d, J = 8.1 Hz, H-6), as well as four aliphatic methine groups at 6.14 (1H, t, J = 6.2 Hz, H-2), 4.27 (1H, m, H-4), 3.77 (1H, m, H-5) coupled to two methylene groups at 3.55 (2H, m, H 2 -6), and 2.08 (2H, m, H 2 -3) (Figure 26). The aliphatic methines at 6.14 (H-2) coupled with 37

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neighboring methylene protons at 2.08 (H 2 -3) which were correlated with two methines at 4.27 (H-4) and 3.77 (H-5). 10 8 6 4 2 PP M Figure 25. 1 H NMR spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ). The methine at 4.27 (H-4) showed 1,3 correlation ( 3 J HH ) with one methine at 3.77 (H-5), one methylene at 2.08 (H-3) and with one exchangeable proton attached on the position at 5.23 (H-4). N HO NH2 O HO 1532'3'4'5'6'6 NN O HO NH2 O HO 12'4'55'636'3' (b)(a)4"4"6"6" Figure 26. gCOSY correlations of 31. 38

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The methine at 3.77 (H-5) showed correlations ( 3 J HH ) with one methine at 4.27 (H-4) and one methylene at 3.55 (H-6). The methylene protons at 3.55 (H 2 -6) also showed 1,3 correlation ( 3 J HH ) with one methine at 3.77 (H-5) and with one exchangeable proton at 5.00 (H 6). The 13 C NMR spectrum (Figure 27) showed nine carbons including one carbonyl carbon at 151.1. The positions of two quaternary carbons 151.1 (C-3) and 163.8 (C-5) were confirmed by correlation ( 2 J CH / 3 J CH ) with two methine protons at 7.84 (H-1) and 5.63 (H-6) by the gHMBC experiment as shown in Figure 28. The methylene protons (H-3) on furan ring also showed correlations ( 2 J CH / 3 J CH ) (Table 5) with three methine carbons at 84.8 (C-2), 71.1 (C-4), and 88.0 (C-5) (Figure 29). Figure 27. 13 C NMR spectrum of 2-deoxycytidine (31) (125 MHz, DMSO-d 6 ). 39

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8 7 6 5 4 3 2 PPM 180 160 140 120 100 80 60 40 Figure 28. gHMBC spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ). NN O HO NH2 O HO 12'4'55'636'3'4"6" Figure 29. Key gHMBC correlations observed in 2-deoxycytidine (31). 40

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Table 5. NMR data of 2'-deoxycytidine (31) (DMSO-d6) (13C, 125 MHz; 1H, 500 MHz) NH2 11.27 (s, 1H)1 7.84 (1H, d, J = 8.1 Hz) 141.2 (CH) H6 C12, C113 151.1 (C)5 163.8 (C)6 5.63 (1H, d, J = 8.1 Hz) 102.4 (CH) H1 C12, C22' 6.14 (1H, t, J = 6.1Hz) 84.8 (CH) H3' C11, C23' 2.08 (m,2H) 40.2 (CH2) H2', H4' C8, C3, C74' 4.27 (m, 1H) 71.1 (CH) H5', H3',H4"5' 3.77 (m, 1H) 88.0 (CH) H4', H6' 6' 3.55 (m, 2H) 61.9 (CH2) H5', H6"4" 5.23 (br,s, 1H) H4' 6" 5.00 (br,s, 1H) H6' positiongCOSYgHMBC1H ()13C () 2-Deoxycytidine (31) was isolated previously from the marine sponge Isodictya erinacea and characterized structure by 2D NMR. 70 The 1 H NMR data were compared with the current compound (31), previous compound (32) and authentic 2-deoxycytine (26). In the current study (31), two aromatic methines attached to the pyrimidine ring of 2-deoxycytidine are found at 7.88 (H-1, J = 8.1 Hz) and 5.64 (H-6, J = 8.1 Hz), which is identical to chemical shifts and coupling constant from the authentic sample (Figure 30), compound (31) [ 7.86 (H-1, J = 8.1 Hz) 5.66 (H-6, J = 8.1 Hz)]. 71,75 The difference in chemical shifts is attributed to solvent effect. 41

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O OH HO NN NH2 O (31)12'4'55'633'6'O OH HO NN NH2 O (32)12'4'55'633'6'O OH HO NN NH2 O (26)12'4'55'633'6'7.845.636.142.084.273.773.557.965.856.214.323.893.712.197.96.16.32.34.54.13.9 Figure 30. Assigned 1 H NMR chemical shifts () of natural 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ), previously isolated 2-deoxycytidine (32) 70 (360 MHz, MeOH-d 4 ) and authentic sample (26) 80 (60 MHz, D 2 O+NaOD). 3.6 Characterization of 4, 8-Dihydroxyquinoline (33) 4, 8-Dihydroxyquinoline (33) was obtained as an amorphous powder. The structure elucidation, complete proton and carbon assignment were achieved by DEPT, gCOSY, gHSQC, and gHMBC techniques. The 1 H NMR spectrum (Figure 31) of 4,8-dihydroxyquinoline showed chemical shifts of a classic pattern for a 1,2,3-trisubstituted aromatic ring at 7.49 (1H, dd, J = 8.0, 1.0, 1.5 Hz), 7.09 (1H, t, J = 7.8 Hz), 7.04 (1H, dd, J = 7.7 1.5 Hz) and pyridine moiety at 7.72 ( 1H, d, J = 7.2 Hz), 6.0 (1H, d, J = 7.2 Hz). The 13 C NMR spectrum (Figure 32) of 4, 8-dihydroxyquinoline (33) displayed nine aromatic carbons assigned with gHSQC indicating that five methine cabons ( 139.4, 123.6, 115.4, 114.8, 109.2) and four quaternary carbons ( 177.8, 147.8, 131.1, 127.4). 42

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7.730 7.716 7.504 7.503 7.489 7.486 7.110 7.095 7.080 7.050 7.046 7.034 7.031 6.004 5.990 2.500 8 7 6 5 4 3 2 1 PP M Figure 31. 1 H NMR spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ). Figure 32. 13 C NMR spectrum of 4, 8-dihydroxyquinoline (33) (125 MHz, DMSO-d 6 ). 43

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In the gHMBC spectrum (Figure 33), correlations ( 2 J CH / 3 J CH ) between proton and carbon were observed that the aromatic proton at H-1 ( 7.72) to C-9 ( 177.8), C-7 ( 131.1), and C-5 ( 109.2) and H-5 ( 6.00) to C-9 ( 177.8), C-6 ( 127.4), and C-1 ( 139.4). A benzene moiety was evident in the gHMBC experiment (Figure 34) based on correlations ( 2 J CH / 3 J CH / 4 J CH ) of H-2 ( 7.50) to C-8 ( 147.8), C-7 ( 131.1), C-3 ( 123.6), and C-4 ( 114.8) while H-3 ( 7.09) correlated with C-8 ( 147.8), C-7 ( 131.1), C-6 ( 127.4), C-4 ( 114.8) and C-2 ( 115.4). 8.0 7.5 7.0 6.5 6.0 PPM 190 180 170 160 150 140 130 120 110 100 Figure 33. gHMBC spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ). 44

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N OH OH 519762348 Figure 34. Key gHMBC correlations observed in 4, 8-dihydroxyquinoline (33). Table 6. NMR data of 4, 8-dihydroxyquinoline (33) (DMSO-d6) (13C, 125 MHz; 1H, 500 MHz) 1 7.72 (1H, d, J = 7.2 Hz) 139.4 (CH) H5 C9, C7, C6, C52 7.50 (1H, dd, J = 1.4, 7.9 Hz) 115.4 (CH) H3, H4 C9, C8, C7, C3, C43 7.09 (1H, t, J = 7.7 Hz) 123.6 (CH) H2, H4 C8, C7, C6, C4, C24 7.04 (1H, dd, J = 1.5, 7.7 Hz) 114.8 (CH) H3, H2 C8, C7, C6, C25 6.00 (1H, d, J = 7.3 Hz) 109.2 (CH) H1 C9, C6, C7, C1 6 127.4 (C) 7 131.1 (C) 8 147.8 (C) 9 177.8 (C) gCOSYgHMBC1H ()13C () N OH OH 519762348 N OH OH N OH OH OH COOH OH (33)(36)(35) N OH OH COOH (34) Figure 35. Secondary metabolites of quinoline derivatives. 45

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Quinoline derivatives including quinoline (34), 4,5,8-trihydroxyquinoline-2-carboxylic acid (35) are known secondary metabolites isolated from sponges Dendrilla membranosa and the genus Verogia 69 and are suggested to be intermediates of tryptophan catabolism (Figure 35). The chemical shifts of isolated 4,8-dihydroxyquinoline (33) were compared relative to known quinoline derivatives 34, 35 69 and xanthurenic acid 36 from the Aldrich Library of NMR spectra. 82 The quaternary C-9 at 177.8 in 4, 8-dihydroxyquinoline (33) showed a similar chemical shift with quaternary C-9 at 183.6 in trihydroxyquinoline carboxylic acid (36). 69 3.7 Characterization of Homarine (37) Homarine (N-methyl picolinic acid) (37) was isolated as pale brown solid. The 1 H NMR spectrum of homarine (Figure 36) showed four methine groups at 8.72 (d, J = 6.0 Hz), 8.42 (t, J = 7.9 Hz), 7.90 (dd, J = 1.3, 7.8 Hz), 7.85 (t, J = 6.9 Hz) and one methyl group at 4.28 (s). 8 6 4 2 PP M Figure 36. 1 H NMR spectrum of homarine (37) (500 MHz, DMSO-d 6 ). 46

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In the 13 C NMR spectrum (Figure 37), the five carbons of the pyridine ring resonated at 156.7, 145.7, 145.0, 126.9, 125.5, the quaternary carbon of carboxylic acid resonated at 161.8 and the carbon of methyl bearing nitrogen was seen at 46.5. Figure 37. 13 C NMR spectrum of homarine (37) (125 MHz, DMSO-d 6 ). In the gHMBC spectrum (Figure 38), correlations (Table 7) ( 2 J CH / 3 J CH ) from the aromatic proton at H-1 ( 8.72) to C-2 ( 145.7), C-6 ( 156.7), C-4 ( 125.5), C-5 ( 46.5), and H-2 ( 8.42) to C-1 ( 145.0), C-6 ( 156.7). H-3 ( 7.90) correlated to C-4 ( 125.5), C-6 ( 156.7), and C-7 ( 161.8) and H-4 ( 7.85) correlated with C-1 ( 145.0), and C-3 ( 126.0), H 3 -5 ( 4.26) to C-1 ( 145.0), and C-6 ( 156.7). Thus, the structure of homarine was deduced by gCOSY and gHMBC (Figure 39). 47

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9 8 7 6 5 4 3 PPM 180 160 140 120 100 80 60 40 2 0 Figure 38. The gHMBC spectrum of homarine (37) (500 MHz, DMSO-d 6 ). N CH3 6321 O O 475 Figure 39. Key gHMBC correlations observed in homarine (37). 48

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Table 7. NMR data of homarine (37) (DMSO-d6) (13C, 125 MHz; 1H, 500 MHz) 1 8.72 (1H, d, J = 6.0 Hz) 145.0 (CH) H2, H4 C2, C6, C4, C5 2 8.42 (1H, t, J = 7.9 Hz) 145.7 (CH) H3, H4 C1, C6 3 7.90 (1H, dd, J = 1.3, 7.8 Hz) 126.0 (CH) H2 C4, C6, C7 4 7.85 (1H, t, J = 6.9 Hz) 125.5 (CH) H1, H2, H3 C1, C3 5 4.28 (3H, s) 46.5 (CH3) C1, C6 6 156.7 (C) 7 161.8 (C) positiongCOSYgHMBC1H ()13C () Homarine was isolated from Dendrilla membranosa and showed feeding deterrence to the Antarctic seastar Odonataster validus 83 and suggested chemical interactions between sponge and sea stars. 83 The feeding deterrence of homarine also was reported Antarctic mollusk Marseniopsts mollis against seastars. 84 The chemical shifts of homarine 37 were compared with authentic 1 H NMR spectrum data (Figure 40) from the Aldrich Library of NMR spectra 85 and suggest homarine. The difference in chemical shifts is attributed to solvent effect. N CH3 6321 O O 475 N CH3 6321 O O 475 8.728.427.907.864.288.98.68.38.14.5(37)(38) Figure 40. Assigned 1 H NMR chemical shifts () of natural homarine (37) (500 MHz, DMSO-d 6 ), and authentic sample (38) 85 (60 MHz, D 2 O). 49

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Chapter 4. CHEMICAL INVESTIGATION OF ANTARCTIC MARINE SPONGE ISODICTYA ANTARCTICA 4.1 Extraction and Isolation of Secondary Metabolite The sponge Isodictya antarctica (Figure 41) was collected by SCUBA diving on the shipwreck Bahia Paraiso near Palmer station, Antarctica. The freeze-dried sample of Isodictya antarctica was first extracted with methanol at room temperature (Scheme 4). Figure 41. Isodictya antarctica collected from Bahia Paraiso, Palmer Station, Antartica (Photograph supplied by Bill J. Baker, University of South Florida). 50

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Isodictya antarctica 141g (freeze dried)1. Extraction with MeOH (3 X 1 L) 2. Partition with CH2Cl2/MeOH/ H2O (500 ml/100ml/100 ml)3. Partition with BuOH/H2O (400 ml/100 ml) 1. LH20 (2.5 cm X 50 cm), MeOH/CH2Cl2 (50:50)2. silica gel column chromatography Hexane/EtOAc (from 80:20 to 0:100)fraction 1EtOAc:hexane(25:75)1.8 gfraction 2EtOAc:hexane(50:50)560 mgfraction 3EtOAc:hexane(75:25)325 mgfraction 4100 % EtOAc136 mgfraction 5EtOAc:MeOH(90:10)420 mgCH2Cl2extract 5.7gBuOHextract 1.7g H2O extract 1.8gsilica gel column (2.5 cm X 45 cm) with gradient elutionfraction 6 EtOAc:MeOH(70:30)627 mgCeramide analog 39, 104 mg (0.07% yield) Scheme 4. Isolation of ceramide analog (39). After concentration, the crude extract was partitioned with CH 2 Cl 2 MeOH, and BuOH. The CH 2 Cl 2 soluble material was initially chromatographed on silica gel with gradient elution (from EtOAc/hexane to EtOAc/MeOH) to afford six fractions. After combining fraction 4 and fraction 5, the resulting fraction was subjected to Sephadex LH-20 column chromatography and then further purified by silica gel column chromatography to afford a ceramide analog (39) (104 mg, 0.07% dry wt). 51

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4.2 Characterization of Ceramide analog (39) Ceramide analog (39) was obtained as a colorless amorphous solid. The HRESIMS showed the [M + H] + ion peak at m/z 664.6599 (calculated 664.6607) corresponding to the molecular formula C 43 H 85 NO 3. The IR spectrum showed absorptions at 3300 cm -1 for a hydroxyl group, 1642 cm -1 for a carbonyl. The 1 H NMR spectrum (Figure 42) displayed a large number of methylene groups at 1.28. The connectivities of the aliphatic moiety were determined by gCOSY, TOCSY and the 1 J CH connectivities were assigned by gHSQC. 7 6 5 4 3 2 1 PP M Figure 42. 1 H NMR spectrum of ceramide analog (39) (500 MHz, CDCl 3 ). 52

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A substructure is represented by the nonequivalent methylene proton (H-1) signals at 3.97 (dd, J = 3.8, 11.0 Hz) and 3.71 (dd, J= 3.3, 11.3 Hz), two olefinic proton signals at 5.80 (H-5) (dt, J = 7.4, 15.5 Hz) and 5.54 (H-4) (dd, J = 6.6, 15.6 Hz) and two methine protons at 4.34 (H-3) (t, J = 5.0 Hz) and 3.91 (m, H-2). The large coupling constants (J = 15.5, 15.6 Hz) of two methine protons at 5.80 and 5.54 revealed the (E) configuration of the double bond. The gCOSY and TOCSY (TOtal Correlation SpectroscopY) experiments (Figure 43) confirmed the coupling of an amide proton ( 6.24) with the methine proton at 3.91 (1H, m, H-2), as well as with the methylene proton (H 2 -1) signals at 3.97 (1H, dd, J = 3.8, 11.0), 3.71 (1H, dd, J = 3.3, 11.3) and methine signal at 4.34 (1H, t, J = 5.0, H-3). The methylene proton at 2.02 (2H, m, H-6) also showed the coupling with methine signal at 5.54 (H-4) and 4.34 (H-3). Thus the 1 H, 13 C NMR (Figure 44), gCOSY, TOCSY, gHSQC, and gHMBC experiments led to confirm the partial structure (a) of ceramide analog (39). HNOHO 62'3'2345 1 OH HNOHO 62'3'2345 1 OH (I)(II) Figure 43. Key gCOSY (I) and TOCSY (II) correlations in partial structure (a). 53

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Figure 44. 13 C NMR spectrum of ceramide analog (39) (125 MHz, CDCl 3 ). The DEPT (Distortionless Enhancement by Polarization Transfer)-135 (Figure 45) and 90 (Figure 46) data showed five methine carbon groups at 134.2, 129.1, 74.2, 54.9, 28.2 and three methyl carbons at 22.9, 22.8 indicating terminal isopropyl group and 14.3. The partial gHMBC data (Table 8) revealed correlations (Figure 47, 48) from amide proton ( 6.24) to the methine carbons at 74.2 (C-3), 55.0 (C-2) and the methylene carbon at 62.4 (C-1) along with quaternary carbonyl carbon at 174.5 (C-1). 54

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Figure 45. DEPT-135 spectrum of ceramide analog (39) (125 MHz, CDCl 3 ). Figure 46. DEPT-90 spectrum of ceramide analog (39) (125 MHz, CDCl 3 ). 55

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HNOHO 62'3'2345 1 OH Figure 47. Key gHMBC correlations observed in partial structure (a) of ceramide analog (39) (125 MHz, CDCl 3 ). 7 6 5 4 3 2 1 PPM 200 150 100 50 Figure 48. gHMBC spectrum of ceramide analog (39) (500 MHz, CDCl 3 ). 56

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1 3.97 (1H, dd, 3.8, 11.0) 62.4 (CH2) H2 H2, H3, NH C2, C3 3.71 (1H, dd, 3.3, 11.3) H2 H2, H3, NH2 3.91 (1H, m) 54.9 (CH) H3, NH H1, H4, H5 C4, C1'3 4.34 (1H, t, 5.0) 74.2 (CH) H2, H4 H1, H2, H4, C1, C2, C4, H5, H6, NH C54 5.54 (1H, dd, 6.6, 15.6) 129.1 (CH) H3, H5 H3, H5, H6 C2, C3, C6 H7, NH5 5.80 (1H, dt, 7.4, 15.5) 134.2 (CH) H4, H6 H3, H4, H6 C3, C6 H76 2.02 (2H, m) 32.6 (CH2) H5, H7 H3, H4, H5 C4, C5, CH21' 174.5 (C) 2' 2.24 (2H, t, 7.5) 37.0 (CH2) H3' H3', CH2 C1', C3', CH23' 1.60 (2H, m) 26.0 (CH2) H2' H2', CH2 C1', C2', CH2 NH 6.24 (1H, d, 7.7) H2 H1, H2, H3 C1, C2, C3, H4, H5, H6 C1' 1H (J, z)13C ()gCOSYgHMBC TOCSY Table 8. NMR data of partial structure (a) (CDCl3) (13C, 125 MHz; 1H, 500 MHz) To determine the number of methylene carbons of ceramide analog (39), the natural product ceramide analog was subjected to methanolysis (Scheme 5) with 1.25 M HCl MeOH for 20 hr under mild reflux and then partitioned into petroleum ether and MeOH layers. NHOOH HO nm HCl, MeOHreflux20 hrNH2OOH HO nmOCH3 NH2OH HO m Ac2OpyridineovernightNHAcOAc AcO m(39)(40)(41)(41)(42) Scheme 5. Methanolysis and protection reactions of ceramide analog (39). 57

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The MeOH layer was treated with acetic anhydride to give the protected compound (42). The 1 H NMR spectrum (Figure 49) of compound (40) in the petroleum ether layer displayed typical fatty ester moiety with methoxy proton at 3.59 and provided single terminal methyl proton at 0.81 (3H, t, J = 6.7 Hz). The 13 C NMR spectrum (Figure 50) showed quaternary carbonyl carbon at 174.6 indicating an ester unit. The molecular formula of compound 40 was determined by HREIMS as C 25 H 50 O 2 (observed m/z 382.3804, calculated 382.3811) indicating a C24 n-nervonic acid moiety. 3.594 2.245 2.229 2.215 1.560 1.545 1.530 1.214 1.183 0.823 0.809 0.796 3.5 3.0 2.5 2.0 1.5 1.0 0.5 PP M Figure 49. 1 H NMR spectrum of methyl ester (40) (500 MHz, CDCl 3 ). 58

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Figure 50. 13 C NMR spectrum of methylester 40 (125 MHz, CDCl 3 ). The 1 H NMR spectrum (Figure 51) of protected acetylated aminodiol 42 showed three acetoxy methyl groups at 2.10, 2.09, 2.01 along with isopropyl unit at 0.87 (6H, d, J = 6.3 Hz) and the 13 C NMR spectrum (Figure 52) displayed two esters and one amide carbonyls at 171.2, 170.2, and 169.8. The HRESIMS data of compound 42 displayed an ion peak at m/z 440.3372 [M + H] + (calculated m/z 440.3376) corresponding to the molecular formula C 25 H 45 NO 5 59

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7.290 2.099 2.092 2.011 1.608 1.603 1.282 0.895 0.883 7 6 5 4 3 2 1 PP M 5.836 5.819 5.805 5.682 5.662 5.442 5.426 5.411 5.397 5.318 5.304 5.291 4.472 4.465 4.461 4.454 4.446 4.347 4.336 4.325 4.312 4.086 4.078 4.063 4.055 6.0 5.5 5.0 4.5 4.0 PPM Figure 51. 1 H NMR spectrum of acetylated aminodiol (42) (500 MHz, CDCl 3 ). Figure 52. 13 C NMR spectrum of acetylated aminodiol (42) (125 MHz, CDCl 3 ). 60

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4.3 Determination of Stereochemistry The modified Moshers method was used for the determination of absolute stereochemistry of ceramide analog (39) at C-3 using a MTPA-Cl [-methoxy--(trifluoromethyl)phenylacetyl chloride]. 86 The most important factor of modified Moshers method is the difference in steric bulkiness of the substitutions of the two and -carbons. The steric repulsion between the phenyl group of MTPA moiety and the -substitutions is essential to bring about the of the CF 3 ( 19 F NMR) or OMe ( 1 H NMR) of the (R) and (S) MTPA esters which is then used to determine the absolute stereochemistry. However, some erroneous predictions of absolute stereochemistry have reported using the original Moshers method, 87 due to the or -substituents of certain compounds being placed far away from the MTPA moiety or other substituents (-, -, positions) having a greater steric interaction with the MTPA groups. 86, 87 The modified Moshers method uses the same principles as the initial Moshers method, however these assigned values must meet certain conditions before the absolute stereochemistry can be confidently assigned. 86,88 Thus, the modified method is generally more reliable than initial Moshers method. However, there were some reports incorrect absolute stereochemistry assignments using this technique on secondary alcohols with the hydroxyl group located in crowded environments. 86 If a molecule has a less steric crowding or hindrance around hydroxyl group, the preferred conformation of the MTPA ester should be obtained, and the modified Moshers method will give the correct absolute configuration. This preferred orientation consists of the carbinyl proton, ester carbonyl and trifluoromethyl groups of the MTPA ester all 61

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lie in the same plane (Figure 53). In this orientation, protons (H A,B,C ) of an (R) MTPA ester should have an upfield 1 H NMR chemical shift compared to the protons (H X,Y,Z ) of (S) MTPA ester due to the anisotropic effect of the MTPA phenyl rings (Figure 54). This shielding effect of the phenyl group has assumed to alter the chemical shifts of proton from the ring. 86,89 With the modified Moshers method, after the determination of the values ( = S R ) for the particular compound, a molecular model is constructed and then confirmed that all the assigned protons with positive and negative values are actually found on the right and left sides of the MTPA plane, respectively (Figure 54). C MeO CF3 O O C H HAHBHCHZHYHX''' MTPA plane(R) MTPA ester(Ph)(OMe) (S) MTPA ester Figure 53. The MTPA plane of the (R) MTPA and (S) MTPA esters of a secondary alcohol. (R) MTPA esterCF3Ph MeO O HA' (S) MTPA esterCF3OMe Ph O HA' HZ(') in (R)-MTPA ester >HZ (') in (S) MTPA esterHA() in (R)-MTPA ester
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The absolute values of the = S R for each proton should be proportional to the distance from the MTPA moiety due to the diamagnetic effect of the benzene ring of the MTPA to the particular proton (Figure 55). When these conditions are satisfied, the MTPA model indicates the correct absolute stereochemistry. OMTPA H HCHBHAHZHYHX Figure 55. MTPA ester model to determine the absolute configurations of secondary alcohols ( = S R ) by the modified Moshers method assignment. After construction of a simple molecular model, the ceramide analog (39) was subjected to esterification with (R) MTPA-Cl and (S) MTPA-Cl in the presence of pyridine (Scheme 6). NHO 62'3'2345 HO 1 NHO 62'3'2345 RO 1 (S) or (R) MTPA-Clpyridine, rtovernight R = (S) MTPA (43)R = (R) MTPA (44)OH OR Scheme 6. MTPA reactions with ceramide analog (39). The MTPA esters were chromatographed by normal phase silica gel with EtOAc/hexane (5:95). The absolute stereochemistry of ceramide analog (39) was confirmed using 1 H NMR spectra (Figure 56 and 57) and gCOSY analysis (Table 9 and 63

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10). All proton signals for both (S) MTPA ester (43) and (R) MTPA ester (44) were assigned and 1 H NMR chemical shift differences ( = S R ) (Figure 58) were determined for neighboring protons. 8 7 6 5 4 3 2 1 PP M Figure 56. 1 H NMR spectrum of (S) MTPA ester (43) (500 MHz, CDCl 3 ). 7 6 5 4 3 2 1 PP M Figure 57. 1 H NMR spectrum of (R) MTPA ester (44) (500 MHz, CDCl 3 ). 64

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1 4.39 (1H, dd, J = 5.2, 11.6 Hz) H2 4.34 (1H, dd, J = 4.0, 11.8 Hz)2 4.55 (1H, m) H1, H3, NH3 5.39 (1H, t, J = 7.8 Hz) H2, H44 5.29 (1H, m) H2, H3, H5 5 5.81 (1H, dt, J = 7.9, 13.4 Hz) H4, H66 2.01 (2H, m) H4, H5 2' 1.99 (2H, t, J = 8.0 Hz) H3' 3' 1.49 (2H, m) H2'NH 5.27 (1H, d) H2 1H()gCOSYTable 9. NMR data of (S)-MTPA ester (43) (CDCl3) (13C, 125 MHz; 1H NMR 500 MHz) 1 4.27 (1H, dd, J = 3.6, 11.5 Hz) H2 4.20 (1H, dd, J = 5.5, 11.3 Hz) 2 4.49 (1H, m) H1, H3, NH 3 5.42 (1H, t, J = 8.5 Hz) H2, H4 4 5.39 (1H, m) H2, H3, H5 5 5.90 (1H, dt, J = 5.5, 14.6 Hz) H4, H6 6 2.02 (2H, m) H4 ,H5 2' 2.02 (2H, m) H3' 3' 1.51 (2H, m) H2'NH 5.23 (1H, d) H2 1H()gCOSYTable 10. NMR data of (R)-MTPA ester (44) (CDCl3)(13C, 125 MHz; 1H NMR 500 MHz) HNORO 62'3'2345 1 OR + 0.06-0.03+ 0.12,+ 0.140.100.090.01+ 0.03 H -0.030.02 R = (S)-MTPA (43)R = (R)-MTPA (44) Figure 58. values ( S R ) of the MTPA esters 43 and 44 in CDCl 3 65

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An MTPA molecular model was constructed with the protons on the right and + protons on the left side of the MTPA plane (Figure 59). After checking that the values were qualitatively proportional to the distance from the MTPA moiety, the substituents about the chiral centre indicated priority by the Cahn-Ingold-Prelog rules. 90 The results, shown in Figure 59, established that the absolute stereochemistry of C-3 was 3S configuration. -+ OMTPA H 423 OMTPA H S 1234 + Figure 59. Model to determine the absolute configuration of the cearamide analog (39) MTPA esters. The relative stereochemistry of C-2 was determined with Chem3D software from Cambridge Soft Corporation. The carbon-2 was assumed to have R configuration and carbon-3 to have S configuration. The dihedral angles (Figure 60) of H-2 (C-2), H-3 (C-3), and H-4 (C-4) were obtained from Chem3D with minimized energy condition by computational calculations. The possible proton-proton dihedral angles showed 173 between H-3 and H-2, 163 between H-3 and H-4. The relative dihedral angles suggested that H-3 was located in anti configuration between H-4 and H-2 and the possible coupling constant ( 1,3 J HH ) should be triplet multiplicity. The relative dihedral angles showed 91 indicating gauch form between H-2 and H-1a, 147 indicating anti form between H-2 and H-1b suggesting that H-2 is multiplet. 66

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R CHHOHCH2OH NH H 173 o CH2163 o H OH H HC 3243 OHHHCH2OH H1b2 1aHN CH 91 o147 oHNOHO 62'3'2345 1 OH S Figure 60. Dihedral angles of H-3, H-4 proposed with 3 (S) and 2 (R) of ceramide analog (39). When the carbon-2 was assumed to have S configuration, the possible proton-proton dihedral angles (Figure 61) calculated by Chem3D showed 63 between H-3 and H-2, 166 between H-3 and H-4. HNOHO 62'3'2345 1 OH SS CH3HOHCH2OH H HN 63 o CH2166 o H 3243 H HC OH OHHHNH CH H 1b 1a263 o173 o Figure 61. Dihedral angles of H-3, H-4 proposed with 3 (S) and 2 (S) in ceramide analog (39). The dihedral angle showing 63 suggested a gauche arrangement between H2 and H3 and the dihedral angle showing 166 suggested an anti arrangement between H3 and H4. Therefore, H3 when assumed as 3S and 2S configurations has to display two different multiplicities which are doublet and doublet, however the actual multiplicity of 67

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C-3 in ceramide analog (9) is triplet ( 1,3 J HH = 4.9 Hz) between H-3 and H-6. The relative dihedral angles also showed 63 indicating gauch form between H-2 and H-1a, 173 anti form between H-2 and H-1b suggesting that H-2 is multiplet. Thus C-3 (S) and C-2 (R) are favorable configurations than C-3 (S) and C-2 (S) in ceramide analog (9) by Chem3D. The relative configuration (Figure 62) of C-6 is suggested to be R and C-3 (S) indicates triplet multiplicity of H-3. HNOHO OH RS Figure 62. Relative stereochemistry of ceramide analog (39). The Chem 3D models with C-3 in S configurations and C-2 in R configuration showed almost the same dihedral angle between H-2 and H-4 which are 173 and 163. This indicates that H-3 is not a double-doublet, but a triplet. However, theoretically the coupling constant ( 1,3 J HH ) of H-3 between two protons, H-2 and H-4 with dihedral angles, 163 and 173 should be above 1,3 J HH = 5 Hz. The Moshers method and the Chem3D models are showing that C-3 is in the S configuration and C-2 is in R-configuration. Also, the acetylated aminodiol 42 showed that H-4 is a triplet with coupling constant with 1,3 J HH = 6.6 Hz. The dihedral angle between H-2 and H-3 in this product is also the same as that in ceramide analog (39). Except for the coupling constants in ceramide, all the other models are showing as C-3 (S) and C-2 (R) rather than C-3 (S) and C-2 (S). Hence, the relative configuration of C-2 is suggested to be R configuration from the results of Chem3D and C-3 (S) indicates triplet multiplicity of 68

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H-3 from the Moshers method. From the mass spectrometric data of ceramide analog (39), methanolysis reaction, and Moshers method, the chemical structure of ceramide analog (39) was suggested in Figure 63 with Table 11. HN OH OH O 1'2'24'23' 2012345 106171819 Figure 63. Ceramide analog (39). 1 3.97 (1H, dd, 3.8, 11.0) 62.4 (CH2) H2 H2, H3, NH C2, C3 3.71 (1H, dd, 3.3, 11.3) H2 H2, H3, NH2 3.91 (1H, m) 54.9 (CH) H3, NH H1, H4, H5 C4, C1'3 4.34 (1H, t, 5.0) 74.2 (CH) H2, H4 H1, H2, H4, C1, C2, C4, H5, H6, NH C54 5.54 (1H, dd, 6.6, 15.6) 129.1 (CH) H3, H5 H3, H5, H6 C2, C3, C6 H7, NH5 5.80 (1H, dt, 7.4, 15.5) 134.2 (CH) H4, H6 H3, H4, H6, H7 C3, C66 2.02 (2H, m) 32.6 (CH2) H5, H7 H3, H4, H5, H7 C4, C5, CH27 1.40 (2H, m) 27.9 (CH2) H6, H8 H3, H4, H5, H6 C6, CH2 8-15 1.28 (16H, m)16 1.17 (2H, m) 39.8 (CH2) H17 H17, H18, C17, C18, H19 C19, CH2 17 1.54 (1H, heptet, 6.7) 28.2 (CH) H16, H18 H16, H18, H19 C16, C18, H19 C19 18 0.88 (3H, d, 6.8) 22.9 (CH3) H16, H17 H16, H17 C16, C1719 0.88 (3H, d, 6.8) 22.8 (CH3) H16, H17 H16, H17 C16, C171' 174.5 (C) 2' 2.24 (2H, t, 7.5) 37.0 (CH2) H3' H3', CH2 C1', C3', CH23' 1.60 (2H, m) 26.0 (CH2) H2' H2', CH2 C1', C2', CH24'23' 1.28 (34H, m) 24' 0.90 (3H, t, 7.0) 14.3 (CH3) H23' H23', CH2 NH 6.24 (1H, d, 7.7) H2 H1, H2, H3, H4 C1, C2, C3, H5, H6 C1' 1H (J, z)13C ()gCOSYgHMBC TOCSY Table 11. NMR data of ceramide analog (39) (CDCl3) (13C, 125 MHz; 1H, 500 MHz) 69

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Chapter 5. TOTAL SYNTHESIS OF THE NATURAL PRODUCT EREBUSINONE AND EREBUSINONAMINE 5.1 Introduction. The Antarctic benthos is characterized by a dominant sponge community. Sponge predators include sea stars, nudibranchs and amphipods, though some sponge species appear to lack predation. Our own chemical ecological investigations of Antarctic invertebrates led to us to investigate the sponge Isodictya erinacea (Topsent, 1916) (Family Esperiopsidae), collected in McMurdo Sound, Antarctica, because I. erinacea appeared to be free of predation despite its lack of structural protection elements such as spicules. 59 I. erinacea was found to elaborate a host of secondary metabolites bearing cytotoxicity and ecological activities. 60, 61 We have documented molt-inhibition activity of sponge extracts toward the sympatric amphipod Orchomene plebs, a common predator of McMurdo Sound sponges. In laboratory assays carried out at McMurdo Station, amphipods were fed diets composed of either nutritionally rich control agar pellets or the same pellets to which an ecologically relevant (i.e., on a volume/volume basis relative to that found in the sponge) sponge extract had been added; the sponge extracts were substantially enriched with erebusinone (12), a yellow pigment we had previously isolated from the sponge. 61 We monitored food consumption, molt events and mortality. Feeding was vigorous in both groups of amphipods as control and experimental (erebusinone70

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enriched) diets. Over the 4 week experiment, the consumption and mortality of amphipods (Figure 64) were significantly higher in erebusinone-enriched diet and molt events significantly reduced; leading us to suggest that erebusinone interferes with molting in O. Plebs leading to premature mortality (Figure 65). 05101520253005101520253035Time (days)Mortality (% ) Sponge Extract Control Figure 64. Occurrence of mortality in O. plebs fed erebusinone in their diet. Figure 65. Occurrence of molting in O. plebs fed erebusinone in their diet. 0246810121405101520253035Time (days)Molt (%) Control S p on g e Extrac t 71

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3-Hydroxykynurenic acid OH NH2 O Erebusinone OH NH2 O NHAc NH2O OH (12)(24) Molt inhibition as a chemical defense mechanism has not previously been documented in marine systems. We believe that erebusinone (12) is acting as a 3-hydroxykynurenine (24) mimic. 3-Hydroxykynurenine is a tryptophan catabolite intermediate in the biosynthesis of xanthurenic acid, a quinoline alkaloid inhibitor of the cytochrome P450 oxidase responsible for oxidizing ecdysone (Figure 66) to 20-hydroxyecdysone, the latter of which is the molt regulator of crustaceans (Figure 67, 68). 74,91 To further investigate the bioactivity and ecological role of erebusinone, we have carried out the synthesis of the yellow pigment, erebusinone. HOHOOOHOHOH EcdysoneHOHOOOHOHOHHO 20-HydroxyecdysoneMolting Hormone in CrustaceansXanthurenic Acid x Figure 66. Ecdysone metabolism. 72

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NH COOH NH2 O NH2 COOH NH2 OH N OH OH COOH N OH OH Xanthurenic AcidCrustacean Molt InhibitorTryptophan Figure 67. Tryptophan Catabolism. Receptor siteE+E+NH2ONH2HOOOHO NHOONH2OH NH2ONH2OHCO2H Erebusinone (1) 3-Hydroxy kynurenine (8) Figure 68. Overlay of erebusinone (12) with 3-hydroxykynurenine (24) in a hypothetical receptor. 73

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5.2 Synthesis of Erebusinone (12) The yellow pigment from the Antarctic sponge Isodictya erinacea, erebusinone has been implicated in molt inhibition and mortality against Antarctic crustacean amphipods, perhaps serving as a precursor of a xanthurenic acid analog. Our interest in erebusinone bioactivity led us to develop the synthesis, which we have achieved in an overall yield of 44% involving seven steps. The synthetic pathway also features an economical and efficient preparation of erebusinone. 5.3 Results and Discussion We developed a convenient and efficient total synthetic strategy of erebusinone (12). As shown in Scheme 7, we initially pursued the synthesis of the 3-benzyloxy-2-nitrobenzylbenzoate (46) using 3-hydroxy-2-nitro benzoic acid (45) as a starting material. Benzyl bromide treatment with solid K 2 CO 3 92 in dry DMSO at ambient temperature gave the dibenzyl ester (46) in 95% yield. To elaborate the side chain, we envisioned that the extension of aliphatic carbon chains could be achieved by lithium acetonitrile generated in situ by the treatment of acetonitrile and n-butyllithium. 93 The treatment of lithium acetonitrile with dibenzyl ester 46 through direct S N 2 substitution provided a 90% yield of the resulting ketontrile 47. To prepare amino alcohol 49, we had to establish the reduction conditions for the conversion of the nitrile to amine. At this stage, our initial attempts of sequential 74

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reduction of ketone and nitrile proved to be more difficult than we had expected. Numerous attempts to perform reduction of 47 and 48 with LiAlH 4 NaBH 4 with CoCl 2 94,95 and hydrogenation failed. 96 However we finally achieved a successful reduction to afford amino alcohol 49 in a two step procedure by NaBH 4 and dimethylsulfide borane (BH 3 SMe 2 ). 97 The reduction of compound 48 with 2.0 equiv of aqueous sodium borohydride (NaBH 4 ) afforded the corresponding intermediate 48 in 95 % yield, which was further treated with 2.4 equiv of BH 3 SMe 2 in refluxing dry THF, providing the desired hydroxyl amine 49 in 76% overall yield via two-step reduction sequence involving ketone reduction of 47 and nitrile reduction of 48. OBn NO2 O OBn OH NO2 O OH K2CO3/BnBrDMSO95% OBn NO2 O CN BuLi/CH3CNTHF/78 oC90%NaBH4/H2OMeOH95 % OBn NO2 OH CN THF83% BH3S(CH3)2 OBn NO2 OH NH2 (45)(46)(47)(48)(49) Scheme 7. Synthetic route to erebusinone precursor 49 The selective protection reaction (Scheme 8) of the intermediate 49 was carried out by the selective protection of the primary amine 98 with 1.2 equiv of acetic 75

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anhydride in the presence of triethylamine in dry dichloromethane at ambient temperature providing 82% yield of the protected acetylamide 50. Oxidation of secondary alcohol 50 by Dess-Martin oxidation 99 led to the formation of the desired the ketone 51 in 90 % yield. OBn NO2 OH NHAc OBn NO2 OH NH2 Ac2OtriethylamineCH2Cl282% OBn NO2 O NHAc Dess MartinoxidationCH2Cl290%Erebusinone (12)H2, Pd/C, 1 atmEtOH93% OH NH2 O NHAc (49)(50)(51) Scheme 8. Synthetic route from erebusinone precursor 49 to erebusinone (12). To complete the synthesis of erebusinone 12, we needed to remove the protected benzyl group and reduce the nitro group. For the purpose of this study, we considered that the intermediate 51 had to be converted into erebusinone 12 in a one-pot reaction. Exposure of 51 in absolute ethanol to Pd/C (10%) 96 at ambient temperature under a hydrogen atmosphere (1 atm) worked well with mild conditions leading to erebusinone 12 in 93% yield (Scheme 8). In summary, we have developed a highly convenient synthetic pathway of erebusinone (12) providing an overall 44% yield. To further evaluate the biological activities of erebusinone (12) and its analogs, the explored synthetic method should 76

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hold promise for the preparation of various structural types of erebusinone congeners with potential molt inhibition activity. We have envisioned analogs of erebusinone with different substitution patterns on the aromatic ring, with differing side chain lengths, and with additional oxidation levels. 5.4 Synthesis of Erebusinonamine (52) The utility of our synthesis of erebusinone (12), besides being brief and high-yielding, is the opportunity to afford its analogue synthesis. Our ongoing research focuses on the preparation of analogues and the evaluation of the potential role of these compounds as molt inhibitors. Analogues with different substitution patterns on the aromatic ring, with differing side chain lengths, and with additional oxidation levels are envisioned. The synthesis of erebusinononamine (52), the deacetylated terminal amine was the first erebusinone analog synthesis attempted. OH NH2 O NHAc OH NH2 O NH3Cl Erebusinone (12)Erebusinonamine (52) 5.5 Results and Discussion 77

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In our previous study, the synthesis of erebusinone (12) was achieved and the synthesis of erebusinonamine (52) was pursued from an intermediate of erebusinone, hydroxylamine (49). Erebusinonamine (52) was synthesized following the reaction sequence depicted in Scheme 8. Hydroxylamine 49 was reacted with di-tert-butyl dicarbonate (Boc 2 O) 100 to afford the tert-BOC carbamate 53 in 100% yield and then followed by Dess-Martin oxidation to give compound 54 in 80% yield. The deprotection of t-BOC group 101 was achieved in the presence 55 with HCl (1.25 M) to afford precursor of erebusinonamine in 100% yield. Finally, erebusinonamine (52) was prepared by reaction of 55 with Pd/C 96 under 3.5 atm in 90% yield. OBn NO2 OH NH OBn NO2 OH NH2 t-BOC2OtriethylamineMeOH100% O OC(CH3)3 OBn NO2 O NH 1.25 M HClMeOH100% O OC(CH3)3 Dess MartinoxidationCH2Cl280% OBn NO2 O NH3Cl Erebusinonamine (52)H2, Pd on C, 3.5 atmMeOH90% OH NH2 O NH3Cl (49)(53)(54)(55) Scheme 9. Synthetic route of erebusinonamine (52) from erebusinone precursor 49. 78

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Chapter 6. BIOASSAY OF PURE COMPOUNDS Bioassays for the pure compounds were done at Wyeth Pharmaceuticals and also at Marine Natural Products Lab at University of South Florida. Antimicrobial activities were carried out with the isolated compounds purine analog (23), ceramide analog (39), synthetic compounds, erebusinone (12) and erebusinonamine (52) using standard disk diffusion method against two gram-positive bacteria, Staphylococcus aureus (Sa, strain 375) and Staphylococcus meth resistant (Sa, strain 310), against one gram-negative bacteria Escherichia faecium van resistant (Ef, strain 379), and against a fungus Candida albicans (Ca, strain 54). 102 Assay plates were prepared by pouring 125 mL volume of agar medium (tempered at 50C) inoculated with an overnight broth culture of the test organisms. Sample concentrations of 100 g in 10 L aliquots were spotted onto agar surface and the plates were incubated at 37C for 24-48 h. The zones of growth inhibition were measured from the edge of the disk to the edge of the clear inhibition zone in mm, respectively. Control disks were treated with solvent alone (MeOH or CHCl 3 ). Synthetic erebusinone (12)Erebusinonamine (52)Sa375Sa310Ef379Ca54 000088610 Sa375: Staphylococcus aureus, Sa310: Staphylococcus meth resistant, Ef379: Escherichia faecium van resistant, Ca54: Candida albicansCeramide analog (39)7769Purine analog (23)0000Table 12 Antimicrobial activity of pure compounds (100 g/disk) using the disk diffusion assay (Zone of Inhibition in mm) 79

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The purine analog (23) did not show considerable antimicrobial activity. Ceramide analog (39) showed modest antimicrobial activity against Staphylococcus aureus (Sa, strain 375), Staphylococcus meth resistant (Sa, strain 310), Escherichia faecium van resistant (Ef, strain 379) and Candida albicans (Ca, strain 54). Erebusinonamine (52) showed relatively better activity against Candida albicans (Ca, strain 54) than Staphylococcus aureus (Sa, strain 375), Staphylococcus meth resistant (Sa, strain 310) and Escherichia faecium van resistant (Ef, strain 379). Erebusinone (12) did not show considerable antimicrobial activity. Erebusinone (12) is presently being tested for insecticidal activity. 80

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Chapter 7. DISCUSSION Isodictya erinacea has been found to produce unusual molt inhibitor precursor analog erebusinone (12), purine derivatives (13, 21, 22, 23) and nucleosides metabolites and exhibits an unusual chemical defense role against a major sponge predator, the sea star Perkanster fuscus, as well as the amphipod Orchomene plebs. 60,61 Our chemical and ecological investigations of the Antarctic invertebrates led us study the marine sponges, Isodictya erinacea, Isodictya setifera and Isodictya antarctica. Isodictya erinacea was found to produce an unusual N-methylated purine analog 23 and 3-hydroxykynurenine (24), the precursor of molting inhibitor xanthurenic acid. N-methylated purine base analogues previously reported including from various sponges, 1,9-dimethyl-6-imino-8-oxopurine from Hymeniacidon sanguinea, 103,104 1,3,7-trimethylguanine from Latrunculia brevis, 71 herbipoline (7,9-dimethylguanidium salt) and 1-methyl adenine from Geodia gigas, 105 longamide (3,7-dimethylisoguanine) from Agelas lingissima, 106 1,3-dimethylisoguanine from Amphimedon viridis, 107 mucronatine (2-methoxy-3-methylisoguanine) from Styphnus mucronatus. 108 The purine 1,3,7-trimethylisoguanine was also isolated from the Ascidian Pseudodistoma cereum. 72 Chemical investigation of Isodictya setifera led to the isolation of known secondary metabolites, homarine (37), 5-methyl-2-deoxycytidine (25), uridine (28), 2-deoxycytidine (31), 4, 8-dihydroxyquinoline (33), 3-hydroxykynurenine (24), of 81

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which the latter two are known biosynthetic intermediates of tryptophan catabolism in crustaceans. The first discovery of purine and nucleosides were isolated from the marine sponge Cryptothethya crypta by Bergmann and co-workers in the 1950s. 41,42,109,110 The modified unusual halogenated nucleosides also isolated from sponges and algae. 111-115 Therefore, marine sponges are well known as source of nucleosides and purine derivatives. Among the potential ecological roles of the sponges ion the Antarctic benthos, previously we reported that the secondary metabolite, erebusinone (12) from Isodictya erinacea has been implicated in molt inhibition and mortality against an Antarctic crustacean, Orchomene plebs, perhaps serving as a precursor of xanthurenic acid analog. We documented significant molt inhibition activity against common sponge predator crustacean amphipods. 60,61 The secondary metabolites, 4,8-dihydroxyquinoline (33), 3-hydroxykynurenine (24), from the Antarctic sponge I. setifera, are known to participate in the regulatory pathway of crustacean molting via tryptophan catabolism. 60,61 This appears to be the first example in marine realm that sponges produce tryptophan catabolism intermediates to that interfere molting in O. plebs for chemical defense. According to our previous metabolite profile, biological activities and ecological implications, we believe species of the genus Isodictya is distinctive in producing biosynthetically unique and various secondary metabolites and suggest that I. setifera and I. erinacea may play a role in chemical defense. 82

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Chemical investigation of I. antarctica was found to produce ceramide analog which showed antimicrobial activity against Staphylococcus aureus, Staphylococcus meth resistant, Escherichia faecium van resistant and Candida albicans. A variety of ceramide derivatives have been reported from marine sponges. 116-118 Relative stereochemistry of ceramide analog (39) was assigned by Moshers method and Chem3D. 86 We have developed mild, simple, and efficient synthetic methods for erebusinone (12) and erebusinonamine (52) in excellent yields from 3-hydroxy-2-nitro benzoic acid (45) and we also optimized each synthetic conditions of erebusinone (12) and erebusinonamine (52) from 1 to 20 gram. The bioassays of synthetic natural product, erebusinone (12) did not show antimicrobial activity but, erebusinonamine (52) showed moderate antimicrobial activity against Staphylococcus aureus, Staphylococcus meth resistant, Escherichia faecium van resistant and Candida albicans. The ecological and biological assays of these compounds as molt inhibitors are currently under investigation. 83

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Chapter 8. EXPERIMENTAL 8.1 General Procedure. Flash column chromatography was performed on EM Science normal phase Silica gel 60 (200 400 mesh). Thin layer chromatography (tlc) was carried out using Whatman normal phase Silica gel 60 Partisil K6F, reversed phase Silica gel 60 Partisil KC18F, and CNF 254 s plates 0.25 mm thickness. There were visualized by spraying with 5% phosphomolybdic acid in EtOH and heating and 2 % ninhydrin in BuOH/acetic acid (95:5). HPLC (high performance liquid chromatography) was performed on Waters 510 equipped with a Waters 490E programmable multiwavelength UV detector at 254 nm and Shimadzu LC-8A equipped with a multisolvent delivery system connected to a Shimadzu SPD-10A UV-VIS tunable absorbance detector and/or an Alltech ELSD 2000 using a YMC-Pack ODS-AQ C-18 analytical column, a Waters prepLC (25 mm X 30 cm) C-18 column for reversed phase, or Waters Sphereclone (250 X 10 mm) for normal phase. Infrared (IR) spectra were obtained with Nicolet Avatar 320FT-IR in solid state. Ultraviolet-Visible (UV) experiments were measured on a Hewlett-Packard 8452A diode array UV/Vis spectrometer. High resolution mass spectra ESI-MS (negative or positive mode), EI-MS (negative or positive mode) and CI-MS were obtained on Micromass 70-VSE spectrometer at the University of Illinois. Optical rotation was determined on a Rudolph Research Analytical AUTOPOL IV with a sodium lamp (589 nm) and 0.5 dm cell. 1 H NMR 84

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and 13 C NMR spectra were recorded at 250 MHz and 75 MHz, respectively on a Bruker AMX-250 or 500 MHz and 125 MHz, respectively on a Varian INOVA-500. 1 H and 13 C NMR chemical shifts are listed relative to CDCl 3 ( 7.27), MeOH-d 4 ( 3.31), or DMSO-d 6 ( 2.50) and ( 77.00), ( 49.00), or ( 39.50) respectively. The 13 C resonance multiplicities were determined by DEPT experiments. 1 H1 H correlations were determined by using gCOSY and TOCSY experiments optimized coupling constant (J HH of 7 Hz). One bond connectivities ( 1 J CH ) of 1 H13 C were determined via the 2-D proton-detected gHSQC experiment. The interpulse delays were optimized for average 1 J CH of 120 MHz. Twoor three-bond heteronuclear multiple-bonds ( 3 J CH / 2 J CH ) were recorded via the 2D proton detected gHMBC experiment optimized for a long range coupling constant (J CH of 7 Hz). Unless otherwise specified, materials were purchased from commercial suppliers and used without further purification. Low-temperature baths of C and 40 C were obtained with an immersion cooler bath using acetone and CH 3 CN with dry ice (CO 2 ). THF was distilled from sodium and benzophenone immediately before use. CH 2 Cl 2 and CH 3 CN were distilled from CaH 2 under argon and triethylamine from KOH. Moisture-sensitive reactions were conducted in oven or flame-dried glassware under and argon atmosphere. Unless otherwise noted, all organic layers were dried with over either anhydrous Na 2 SO 4 or MgSO 4 and concentration of all solutions was accomplished by a rotary evaporator at water aspirator pressures. Melting points were uncorrected. 85

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8.2 Isolation of Secondary Metabolites from Isodictya erinacea A specimen of Isodictya erinacea was collected by using SCUBA diving (-20 30 m) at Erebus Bay on the western coast of Ross Island, Antarctica in summer 1999. The freshly collected marine sponge was frozen in liquid nitrogen immediately to prevent degradation of the pigment. A voucher specimen has been deposited in a 70 C freezer at the Department of Chemistry, University of South Florida. The body of I. erinacea is yellow or light brown both inside and outside, and the surface is globular and spiny. The marine sponge (1.0 kg, wet) which was kept frozen until use was exhaustively extracted with MeOH (2L X 5 times) at 7 C. After concentration, the extract was re-dissolved with MeOH to remove salt. The residual fraction (20g) was subjected to Sephadex LH-20 column chromatography (5 cm X 60 cm) with CH 2 Cl 2 /MeOH (1:1) to provide 5 fractions. Fraction 4 (1.1g) was further subjected to C18 column chromatography (2.5 X 45 cm) with a MeOH/CH 2 Cl 2 (100:0 0:100) gradient system to give four fractions. Fraction 8 (374 mg) was chromatographed on silica gel column (2.5 cm X 45 cm) with H 2 O/MeOH/CHCl 3 (0.4:3.0:6.6) to provide 4 fractions. Purine analog (23) (0.0068 %, 68 mg) was obtained from fraction 11 (104 mg) on silica gel column (2.5 cm X 30 cm) with H 2 O/MeOH/CH 2 Cl 2 (0.4:30:66). Fraction 12 was chromatographed on silica gel (2.5 cm X 30 cm) eluting with H 2 O/MeOH/CH 2 Cl 2 (0.5:4:5.5) to yield 3-hydroxykynurene (24), fraction 20 (0.00135%, 13.5 mg) (Scheme 1). 86

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8.2.1 Spectral data of Purine analog (23) NN NN O CH3 HN CH3 123456789CH3 H 1,3,7-Trimethylisoguanine: a pale brown crystal; mp decomposed at 230 C ; IR v max 3303, 3241, 3089,1715, 1641, 1567, 1026 cm -1 ; UV (MeOH) max (log ) 210 (2.02), 268 (0.75) nm; 1 H NMR (DMSO-d 6, 500 MHz): 8.17 (s, 1H, H-8), 3.88 (s, 3H, N-7-CH 3 ), 3.61 (s, 3H, N-3-CH 3 ), 3.36 (s, 3H, N-1-CH 3 ); 13 C NMR (DMSO-d 6 125 MHz) 152.5 (s, C-6), 151.3 (s, C-2), 147.6 (s, C-4), 143.6 (d, C-8), 106.9 (s, C-5), 33.4 (q, N-7-CH 3 ), 32.2 (q, N-3-CH 3 ), 29.3 (q, N-1-CH 3 ); HREIMS m/z 193.0959 (calcd for C 8 H 11 N 5 O 1 193.0964), LREIMS m/z [M + ] 193.1 (100 %), [M-NH] + 178.1, [M-CHO] + 164.1 (20 %), [M-HNCNCH 3 ] + 138.1 (12 %), [M-CO] + 109.0 (30 %), [M-HCN] + 82.1 (24 %). 87

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8.2.2 Spectral data of 3-Hydroxykynurenine (24) OH NH2 O NH2O OH 8191024126511 3-Hydroxykynurenic acid; bright yellow solid; [] 25 D -47.6 (c = 0.17, MeOH); IR v max 3339, 2981,1685, 1611, 1533, 1275 cm -1 ; UV (MeOH) max (log ) 234 (1.06), 273 (0.36), 381 (0.21) nm; 1 H NMR (DMSO-d 6 500 MHz) 7.19 (1H, d, J = 8.2 Hz, H-1), 6.85 (1H, d, J = 7.6 Hz, H-2), 6.40 (1H, t, J = 8.0 Hz, H-4), 3.62 (1H, d, J = 7.8 Hz, H-5), 3.50 (1H, dd, J = 2.3, 18.1 Hz, H-6), 3.24 (1H, dd, J = 8.6, 17.5 Hz); 13 C NMR (DMSO-d 6 125 MHz) 200.0 (s, C-12), 170.3 (s, C-11), 145.5 (s, C-10), 141.6 (s, C-9), 121.6 (d, C-1), 117.4 (d, C-2), 117.1 (s, C-8), 114.6 (d, C-4), 50.5 (d, C-5), 41.1 (t, C-6). 88

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8.3 Isolation of Secondary Metabolites from Isodictya setifera The marine sponge Isodictya setifera was collected by using SCUBA from Bahia Paraiso on the coast near Palmer station, Antartica at depths of 40-50 m on 28 Jan 2003. The marine sponge, Isodictya setifera (435g, freeze dried) was first extracted with methanol (3L X 3 times) at room temperature. After concentration, the crude extract was partitioned with hexane/MeOH/H 2 O (400 mL/500 mL/100 mL). The hexane layer was separated and concentrated to give a crude extract (9.0 g) and aqueous layer was partitioned with CH 2 Cl 2 /MeOH/H 2 O (400 mL/500 mL/100 mL) and the organic layer was separated and concentrated to give CH 2 Cl 2 /MeOH extract (8.3 g). The aqueous layer was extracted with BuOH which gave a BuOH extract (1.9 g) and the aqueous extract was concentrated to provide the H 2 O extract (38g). BuOH extract (1.9g) was subjected to Sephadex LH-20 column (5 cm X 60 cm) with MeOH to provide seven fractions. Fraction 12 (570 mg) was further subjected to normal phase silica gel column (2.5 X 45 cm) with a MeOH/CH 2 Cl 2 (0:100 30:70) and CH 3 CN/H 2 O (100:0 80:20) and followed by HPLC with reversed cyano column using AcCN/H 2 O (99:1 80:20) to give pure compounds which are 3-hydroxykynurenine (24) (1.7 mg, 0.0004% yield), 5-methyl-2-deoxycytidine (25) (3 mg, 0.0007% yield), uridine (28) (2.0 mg, 0.0005% yield), 2-deoxycytidine (31) (1.5 mg, 0.0003% yield), 4,8-dihydroxyquinoline (33) (1.8 mg, 0.0004% yield), homarine (37) (2.0 mg, 0.0005% yield) (Scheme 3). 89

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8.3.1. Spectral data of 5-Methyl-2-deoxycytidine (25) 12'4'55'636'3'O H O H H H H H HO NN NH2 O CH3 5-Methyl-2deoxycytidine; white solid: [] 25 D +5.3 (c = 0.15, MeOH); IR v max 2918, 1688, 1469, 1236, 1090 cm -1 ; UV (MeOH) max (log ) 209 (0.88), 268 (0.83) nm; 1 H NMR (MeOH-d 4 500 MHz) 7.83 (1H, s, H-1), 6.30 (1H, t, J = 6.8 Hz, H-2), 4.42 (1H, m, H-4), 3.92 (1H, q, J = 3.4, H-5), 3.81 (1H, dd, J = 3.2, 12.5 Hz, H a -6), 3.74 (1H, dd, J = 3.5, 11.8 Hz, H b -6), 2.25 (2H, m, H-3), 1.89 (3H, s, H-7); 13 C NMR (MeOH-d 4 125 MHz) 165.3 (s, C-5), 151.2 (s, C-3), 136.7 (d, C-1), 110.4 (s, C-6), 87.6 (d, C-5), 85.1 (d, C-2), 71.0 (d, C-4), 61.7 (t, C-6), 40.0 (t, C-3), 11.3 (q, C-7). 90

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8.3.2. Spectral data of Uridine (28) O OH OH H H H H HO NN NH2 12'4'55'636'3'3"4"O 5-Methyl-2deoxycytidine; white solid: [] 25 D 0 (c = 0.1, MeOH); IR v max 2984, 2902, 1687, 1235, 1207, 1089 cm -1 ; UV (MeOH) max (log ) 209 (1.01), 265 (0.56) nm; 1 H NMR (DMSO-d 6 500 MHz) 7.88 (1H, d, J = 7.9 Hz, H-1), 5.77 (1H, d, J = 5.8 Hz, H-2), 5.64 (1H, d, J = 8.3 Hz, H-6), 5.37 (1H, d, J = 5.9 Hz, H-3), 5.10 (1H, m, H-4), 4.01 (1H, q, J = 5.4 Hz, H-3), 3.95 (1H, q, J = 4.6 Hz, H-4), 3.83 (1H, q, J = 3.3 Hz, H-5), 3.61 (1H, m, H a -6), 3.54 (1H, m, H b -6); 13 C NMR (DMSO-d 6 500 MHz): 163.8 (s, C-5), 151.4 (s, C-3), 141.4 (d, C-1), 102.4 (d, C-6), 88.3 (d, C-2), 85.5 (d, C-5), 74.2 (d, C-3), 70.5 (d, C-4), 61.5 (t, C-6). 91

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8.3.3. Spectral data of 2-Deoxycytidine (31) 12'4'55'636'3'O H OH H H H H HO NN NH2 O 4"6" 2-Deoxycytidine; white solid: [] 25 D +7.1 (c = 0.07, MeOH); IR v max 2977, 2917, 1685, 1235, 1089 cm -1 ; UV (MeOH) max (log ) 209 (0.75), 265 (0.65) nm; 1 H NMR (DMSO-d 6 500 MHz) 11.27 (2H, br s, NH 2 ), 7.84 (1H, d, J = 8.1 Hz, H-1), 6.14 (1H, t, J = 6.2 Hz, H-2), 5.63 (1H, d, J = 8.1 Hz, H-6), 5.23 (1H, br s, OH-4), 5.00 (1H, br s, OH-6), 4.27 (1H, m, H-4), 3.77 (1H, m, H-5), 3.55 (2H, m, H-6), 2.08 (2H, m, H-3); 13 C NMR (DMSO-d 6 125 MHz) 163.8 (s, C-5), 151.1 (s, C-3), 141.2 (d, C-1), 102.4 (d, C-6), 88.0 (d, C-5), 84.8 (d, C-2), 71.1 (d, C-4), 61.9 (t, C-6), 40.2 (t, C-3). 92

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8.3.4. Spectral data of 4, 8-Dihydroxyquinoline (33) N OH OH 519762348 4,8-Dihydroxyquinoline; white solid: IR v max 2976, 1574, 1525, 1285, 1235, 1089 cm -1 ; UV (MeOH) max (log ) 227 (1.2), 263 (0.3), 325 (0.4) nm; 1 H NMR (DMSO-d 6 500 MHz) 7.72 (1H, d, J = 7.2 Hz, H-1), 7.50 (1H, dd, J = 1.4, 7.9 Hz, H-2), 7.09 (1H, t, J = 7.7 Hz, H-3), 7.04 (1H, dd, J = 1.5, 7.7 Hz, H-4), 6.00 (1H, d, J = 7.3 Hz, H-5); 13 C NMR (DMSO-d 6 125 MHz) 177.8 (s, C-9), 147.8 (s, C-8), 139.4 (d, C-1), 131.1 (s, C-7), 127.4 (s, C-6), 123.6 (d, C-3), 115.4 (d, C-2), 114.8 (d, C-4), 109.2 (d, C-5). 93

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8.3.5. Spectral data of Homarine (37) N CH3 6321 O O 475 Homarine; white solid: IR v max 2976, 1652, 1610, 1373, 1235, 1089 cm -1 ; UV (MeOH) max (log ) 206 (0.8), 280 (0.6); 1 H NMR (DMSO-d 6 500 MHz) 8.72 (1H, d, J = 6.0 Hz, H-1), 8.42 (1H, t, J = 7.9 Hz, H-2), 7.90 (1H, dd, J = 1.3, 7.8 Hz, H-3), 7.85 (1H, t, J = 6.9 Hz, H-4), 4.28 (3H, s, N-CH 3 ); 13 C NMR (DMSO-d 6 125 MHz) 161.8 (s, C-7), 156.7 (s, C-6), 145.7 (d, C-2), 145.0 (d, C-1), 126.0 (d, C-3), 125.5 (d, C-4), 46.5 (q, C-5). 94

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8.4 Isolation of Secondary Metabolite from Isodictya antarctica The marine sponge Isodictya antarctica was collected by using SCUBA on the Bahia Paraiso on the coast near Palmer station, Antartica 2001. A voucher specimen was freeze dried in the laboratory of marine natural products laboratory in the department chemistry at University of South Florida. The marine sponge, Isodictya antarctica (141g, freeze dried) was first extracted with methanol (1L X 3 times) at room temperature. After concentration, the crude extract (10g) was partitioned with CH 2 Cl 2 /MeOH/H 2 O (500 mL/100 mL/ 100 mL). The organic layer was separated and concentrated to give CH 2 Cl 2 extract (5.7 g). An aqueous layer was partitioned with BuOH/H 2 O (400 mL/100 mL). The BuOH layer was separated and concentrated to give BuOH extract (1.7 g). An aqueous layer was concentrated to provide H 2 O extract (1.8 g). The CH 2 Cl 2 extract (5.7 g) was subjected to normal phase silica gel (2.5 X 45 cm) with EtOAc/hexane (25:75 100:0) and EtOAc/MeOH (90:10 70:30) to give six fractions. Fraction 4 and fraction 5 (556 mg) was combined and subjected over Sephadex LH-20 column (5 cm X 60 cm) with MeOH/CH 2 Cl 2 (1:1) followed by silica gel with EtOAc/hexane (20:80 100:0) to give ceramide analog (39) (104 mg, 0.07% yield) (Scheme 4). 95

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8.4.1 Spectral data of Ceramide analog (39) HN OH OH O 1'2'24'23' 2012345 106171819 Ceramide analog; white solid: [] 25 D -7.3 (c = 0.165, CHCl 3 ); mp 73 C; IR v max 3300, 2916, 2849, 1642, 1615, 1547, 1466 cm -1 ; UV (CHCl 3 ) max (log ) 243 (0.3) nm; 1 H NMR (CDCl 3 500 MHz) 6.24 (1H, d, J = 7.7 Hz, NH), 5.80 (1H, dt, J = 7.4, 15.5 Hz, H-5), 5.54 (1H, dt, J = 6.6, 15.6 Hz, H-4), 4.34 (1H, t, J = 5.0 Hz, H-3), 3.97 (1H, dd, J = 3.8, 11.0 Hz, H-1 a ), 3.71 (1H, dd, J = 3.3, 11.3 Hz, H-1 b ), 3.91 (1H, m, H-2), 2.24 (2H, t, J = 7.5 Hz, H-2), 2.02 (2H, m, H-6), 1.60 (2H, m, H-3), 1.54 (1H, heptet, J = 6.7 Hz, H-17), 1.40 (2H, m, H-7), 1.28 (16H, m, H (8-15)), 1.28 (34H, m, H (423)), 1.17 (2H, m, H-16), 0.90 (3H, t, J = 7.0 Hz, H-24), 0.88 (3H, d, J = 6.8 Hz, H-18), 0.88 (3H, d, J = 6.8 Hz, H-19); 13 C NMR (CDCl 3 125 MHz) 174.5 (s, C-1), 134.2 (d, C-5), 129.19 (d, C-4), 74.2 (d, C-3), 62.4 (t, C-1), 54.9 (d, C-2), 39.8 (t, C-16), 37.0 (t, C-2), 32.6 (t, C-6), 29.9 (t, C8-15, C4-23), 28.2 (d, C-17), 27.9 (t, C-7), 26.0 (t, C-3), 22.9 (q, C-18), 22.8 (q, C-19), 14.3 (q, C-24); LRFABMS m/z 664.5 [M + H] + HRESIMS as C 43 H 86 NO 3 (observed m/z 664.6599 [M + H] + calculated for 664.6607). 96

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8.4.2 Methanolysis of Ceramide analog (39) NHOOH HO nm HCl,MeOHreflux20 hrNH2OOH HO nmOCH3 NH2OH HO m Ac2OpyridineovernightNHAcOAc AcO m(39)(40)(41)(41)(42) Ceramide analog (39) (5 mg) was heated with 1.25 M HCl in MeOH (3 mL) at 70 C for 20 h in 5 mL flask. The reaction mixture was extracted with petroleum ether, the extract concentrated in vacuo to give fatty acid methyl ester 40 (1.7 mg): 1 H NMR (CDCl 3 500 MHz) 3.60 (3H, s, OCH 3 ), 2.22 (2H, t, J = 6.5 Hz), 1.55 (2H, m), 1.18 (br s), 0.81 (3H, t, J = 6.7 Hz); 13 C NMR (CDCl 3 125 MHz) 174.5 (s), 51.7 (q, OCH 3 ), 34.4 (t), 32.2 (t), 29.9 (t), 29.8 (t), 29.7 (t), 29.6 (t), 29.5 (t), 29.4 (t), 25.2 (t), 22.9 (t), 14.31 (q, CH 3 ); LRFABMS m/z 383.3 [M + H] + LREIMS m/z 382.5 [M] + HREIMS as C 25 H 50 O 2 (observed m/z 382.3804 [M] + calculated 382.3811). The MeOH layer was concentrated in vacuo to give a long chain base aminodiol (41) and then reacted with acetic anhydride in the presence of pyridine (1mL) overnight at room temperature. The reaction mixture was concentrated in vacuo to remove pyridine and diluted with CH 2 Cl 2 (5 mL) with H 2 O (1 mL). The organic layer washed with saturated NaHCO 3 solution, H 2 O, brine solution and then dried with anhydrous MgSO 4 97

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The filtrate was concentrated in vacuo to give protected compound 42 and subjected to silica gel column chromatography using 40% EtOAc in hexane to give protected compound 42 (0.8 mg): 1 H NMR (CDCl 3 500 MHz) 5.80 (1H, dt, J = 7.2, 14.8 Hz), 5.65 (1H, J = 9.4 Hz, NH,), 5.40 (1H, dd, J = 7.6, 15.8 Hz), 5.28 (1H, t, J = 6.1 Hz), 3.44 (1H, m), 4.31 (1H, dd, J = 6.2, 11.8 Hz), 4.05 (1H, dd, J = 3.8, 11.6 Hz), 2.08 (3H, s), 2.07 (3H, s), 2.04 (m), 1.99 (3H, s), 1.58 (br s), 1.52 (1H, m), 1.36 (2H, m), 1.26 (br s), 1.15 (2H, m), 0.87 (6H, d, 6.3 Hz); 13 C NMR (CDCl 3 125 MHz) 171.2 (s), 170.2 (s), 169.8 (s), 137.7 (d, CH), 124.4 (d, CH), 74.1 (d, CH), 62.8 (t), 50.9 (d, CH), 39.3 (t), 32.5 (t), 29.9 (t), 29.8 (t), 29.6 (t), 29.4 (t), 29.1 (t), 28.2 (d, CH), 23.6 (q, acetoxy CH 3 ), 22.9 (q, isopropyl CH 3 ), 22.9 (q, isopropyl CH 3 ), 21.3 (q, acetoxy CH 3 ), 21.1 (q, acetoxy CH 3 ); LRFABMS m/z 440.3 [M + H] + HRESIMS as C 25 H 46 NO 5 (observed m/z 440.3372 [M + H] + calculated 440.3376). 8.4.3 Preparation of MTPA esters for Ceramide analog (39) NHO 62'3'2345 HO 1 NHO 62'3'2345 RO 1 (S) or (R) MTPA-Clpyridine, rtovernight R = (S) MTPA (43)R = (R) MTPA (44)OH OR 8.4.3.1 (S) MTPA Ester (43) To a solution of ceramide analog (39) (1.5 mg) in pyridine (500 L) was added (R)-MTPA-Cl (30 L) at room temperature for 16 h and concentrated in vacuo. The residue was diluted with EtOAc (4mL)/H 2 O (1 mL) and then washed with saturated 98

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NaHCO 3 solution, H 2 O, brine solution and then dried with anhydrous MgSO 4 The filtrate was concentrated in vacuo and chromatographed on silica gel by EtOAc/hexane (5:95) to give (S) MTPA ester (43) (0.7 mg) as colorless oil: [] 25 D +60.0 (c = 0.015, CHCl 3 ); 1 H NMR (CDCl 3 500 MHz) 7.45 (10H, 2 MTPAs aromatic protons), 5.81 (1H, dt, J = 7.9, 13.4 Hz), 5.39 (1H, t, J = 7.8 Hz), 5.29 (1H, m), 5.27 (1H, d, J = 8.9 Hz, NH), 4.55 (1H, m), 4.39 (1H, dd, J = 5.2, 11.6 Hz), 4.34 (1H, dd, J = 4.0, 11.8 Hz), 3.53 (3H, s), 3.48 (3H, s), 1.99 (m), 1.55 (br s), 1.49 (m), 1.26 (br s), 0.89 (3H, t, J = 6.8 Hz), 0.87 (6H, d, J = 6.6 Hz). 8.4.3.2 (R) MTPA Ester (44) Ceramide analog (39) (1.5 mg) was reacted with (S) MTPA-Cl (30 L) as described above and chromatographed to give (R) MTPA ester (44) (0.3 mg) as colorless oil: [] 25 D -29.0 (c = 0.0165, CHCl 3 ); 1 H NMR (CDCl 3 500 MHz) 7.46 (10H, 2 MTPAs aromatic protons), 5.90 (1H, dt, J = 5.5, 14.6 Hz), 5.42 (1H, t, J = 8.5 Hz), 5.39 (1H, m), 5.23 (1H, d, J = 9.4 Hz, NH), 4.49 (1H, m), 4.27 (1H, dd, J = 3.6, 11.5 Hz), 4.20 (1H, dd, J = 5.6, 11.3 Hz), 3.53 (3H, s), 3.50 (3H, s), 2.02 (m), 1.56 (br s), 1.49 (m), 1.26 (br s), 1.15 (2H, m), 0.89 (3H, t, J = 6.8 Hz), 0.87 (6H, d, J = 6.6 Hz). 99

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8.5 Synthesis of Erebusinone (12) 8.5.1 Preparation of Benzyl-3-(benzyloxy)-2-nitrobenzoate (46) OBn NO2 O OBn OH NO2 O OH K2CO3/BnBrDMSO95% (45)(46) To DMSO (100 ml) was added solid K 2 CO 3 (9.05 g, 65.5 mmol, 6 eq.). After stirring for 5 min, 3-hydroxy-2-nitrobenzoic acid (45) (2.0 g, 10.9 mmol, 1 eq.) was added all at once followed by benzylbromide (7.47g, 43.7 mmol, 4 eq.). The reaction mixture was stirred at room temperature for 3 h and then poured into ice water (300 mL). After cooling, the solution was adjusted to pH 3-4 with HCl (10%) and extracted with CH 2 Cl 2 (3 X 100 mL). The organic layer washed with brine (2 X 50 mL), dried with MgSO 4 and concentrated. The residue was chromatographed on silica gel by EtOAc/hexane (5:95) to give 46 3.76g (95% yield) as a white solid: mp 82-83 C; IR v max 3081, 2931, 1714, 1541, 1454, 1282 cm -1 ; UV (CHCl 3 ) max (log ) 243 (0.7), 303 (0.44) nm; 1 H NMR (CDCl 3 250 MHz) 7.61 (1H, d, J = 7.2 Hz), 7.38 (11H, m), 7.24 (1H, d, J = 8.7 Hz), 5.34 (2H, s), 5.19 (2H, s); 13 C NMR (CDCl 3 75 MHz) 162.7 (s), 149.8 (s), 141.2 (s),134.9 (s), 134.7 (s), 130.5 (d), 128.7 (d), 128.6 (d), 128.3 (d), 126.9 (d), 123.6 (s), 122.5 (d), 118.6 (d), 71.2 (t), 67.9 (t); HRESIMS [M + Na] + m/z observed 386.0998 (calculated for C 21 H 17 NO 5 Na 386.1005). 100

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8.5.2 Preparation of 3-[3-(Benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) (47)(46) OBn NO2 O OBn OBn NO2 O CN BuLi/CH3CNTHF/78 oC90 % Anhydrous neat acetonitrile (1.06 g, 25.8 mmol, 2.5 eq) was added to a solution of n-butyllithium (1.99 g, 31.0 mmol, 3 eq) in THF (100 mL) at C. After a white suspension formed, the solution of 46 (3.76g, 10.3 mmol, 1 eq) in THF (5 mL) was added into the reaction mixture. The light brown reaction mixture was warmed to 45 C and then stirred for 2.5 h and the aqueous HCl (10%) was added until pH 4. The reaction mixture was concentrated in vacuo. The residue was diluted with dichloromethane (300 mL) and then washed with water (2 X 50 mL) and brine (50 mL) and dried with MgSO 4 and the solvent removed in vacuo. Flash chromatography eluting with EtOAc/hexane (from10:90 to 20:80) to afford 2.77 g (90% yield) of 47 as a brown solid: mp 160-161 C; IR v max 3081, 2931, 2220, 1708, 1544, 1449, 1285 cm -1 ; UV (CHCl 3 ) max (log ) 210 (1.21), 249 (0.30), 313 (0.13) nm; 1 H NMR (CDCl 3 250 MHz) 7.54 (1H, t, J = 8.3 Hz), 7.37 (6H, m), 7.26 (1H, d, J = 6.8 Hz), 5.26 (2H, s), 4.00 (2H, s); 13 C NMR (CDCl 3 75 MHz) 187.8 (s), 149.5 (s), 138.6 (s), 135.5 (d), 131.7 (s), 128.6 (d), 128.3 (d), 127.5 (d), 126.9 (d), 122.3 (d), 120.8 (d), 115.1 (s), 70.7 (t), 31.3 (t); HREIMS [M] + m/z observed 296.0799 (calculated for C 16 H 12 N 2 O 4 296.0797). 101

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8.5.3 Preparation of 3-[3-(Benzyloxy)-2-nitrophenyl]-3-hydroxypropanenitrile (48) (47)(48) OBn NO2 O CN NaBH4/H2OMeOH95 % OBn NO2 OH CN To a solution of 46 (300 mg, 1.0 mmol) in MeOH (10 mL) was added slowly a solution of NaBH 4 (77 mg, 2.0 mmol), which was prepared with the solution (12 mL, 4:1, methanol and H 2 O, pH 9) at 5 C. The reaction mixture was warmed to room temperature and stirred for 3 h. The HCl (10%) solution was added to the reaction mixture until getting pH 4 and then concentrated in vacuo and diluted with EtOAc (100 mL). The organic layer was washed with brine and dried with MgSO 4 and The residue was chromatographed on silica gel by EtOAc/hexane (10:90) to give 0.29g (95% yield) of 48 as a light brown solid: [] 25 D 0 (c = 0.193, CHCl 3 ); mp 72-73 C; IR v max 3389, 2928, 2264, 1630, 1455, 1275 cm -1 ; UV (CHCl 3 ) max (log ) 244 (0.50) nm; 1 H NMR (CDCl 3 250 MHz) 7.47 (1H, t, J = 8.1 Hz), 7.36 (7H, m), 7.08 (1H, d, J = 8.1 Hz), 5.15 (2H, s), 5.08 (1H, br s), 2.38 (2H, m); 13 C NMR (CDCl 3 75 MHz) 149.6 (s), 139.4 (s), 135.1 (s), 134.3 (s), 131.6 (d), 128.5 (d), 128.2 (d), 126.9 (d), 118.6 (d), 116.7 (d), 114.1 (s), 71.0 (t), 65.1 (t), 27.2 (d); HRESIMS [M + Na] + m/z observed 321.0841 (calculated for C 16 H 14 N 2 O 4 Na 321.0852). 102

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8.5.4 Preparation of 3-Amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) (49)(48) OBn NO2 OH CN THF83 % BH3S(CH3)2 OBn NO2 OH NH2 To a solution of 48 (288 mg, 0.97 mmol) in THF (5 mL) was added BH 3 SMe 2 (183 mg, 2.41 mmol) in THF (10 mL) at 0 C. The reaction mixture was refluxed for 3h and then cooled to room temperature. MeOH (2 mL) followed by 6 N HCl (2 mL) was added to reaction mixture and then refluxed again for additional 0.5h and cooled to room temperature and concentrated under reduced pressure. The residue was partitioned between EtOAc (50 mL) and 1N HCl solution (30 mL). The phases were separated and then aqueous layer was basified with 5 N NaOH and extracted with EtOAc (3 X 50 mL). The combined organic phases were dried with K 2 CO 3 and concentrated under reduced pressure. The residue was chromatographed on silica gel by MeOH/CH 2 Cl 2 (10:90) to give 292 mg (81% yield) of 49 as a pale brown solid: [] 25 D 0 (c = 0.210, CHCl 3 ); mp 122-123 C; IR v max 3320, 3104, 2929,1608, 1525, 1455, 1273 cm -1 ; UV (CHCl 3 ) max (log ) 245 (0.5) nm; 1 H NMR (CDCl 3 250 MHz) 7.23 (m, 7H), 6.85 (1H, d, J = 8.2 Hz), 5.06 (2H, s), 4.80 (1H, d, J = 8.8 Hz), 3.03 (2H, m), 1.78 (2H, m); 13 C NMR (CDCl 3 75 MHz) 149.2 (s), 139.8 (s), 137.9 (s), 135.5 (s), 130.8 (d), 128.5 (d), 128.1 (d),126.9 (d), 119.1 (d), 112.5 (d), 70.8 (t), 70.5 (t), 39.9 (t), 38.5 (d); HREIMS [M] + m/z observed 302.1261 (calculated for C 16 H 18 N 2 O 4 302.1267). 103

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8.5.5. Preparation of N-{3-[3-(Benzyloxy)-2-nitrophenyl]-3-hydroxypropyl} acetamide (50) (49) OBn NO2 OH NH2 Ac2OtriethylamineMeOH82 % (50) OBn NO2 OH NHAc To a stirred solution of 49 (136 mg, 0.45 mmol) and triethylamine (68 mg, 0.68 mmol, 1.5 eq) in CH 2 Cl 2 (5 mL) was added Ac 2 O (55 mg, 0.54 mmol, 1.2 eq) at 0 C. The reaction mixture was stirred at room temperature for overnight and then poured to dilute saturated NaHCO 3 solution (5 mL) and the separated organic layer washed with brine and then dried with MgSO 4 and concentrated. The residue was chromatographed on silica gel by MeOH/CH 2 Cl 2 (1:99) to afford 127 mg (82% yield) of 50 as a pale brown solid: [] 25 D 0 (c = 0.193, CHCl 3 ); mp 102-103 C; IR v max 3425, 3264, 3097, 1525, 1449 cm -1 ; UV (CHCl 3 ) max (log ) 243 (0.36) nm; 1 H NMR (CDCl 3 250 MHz) 7.30 (6H, m), 7.18 (1H, d, J = 8.2 Hz), 6.93 (1H, d, J = 7.9 Hz), 5.93 (1H, br s), 5.14 (2H, s), 4.68 (1H, dd, J = 2.3, 9.2 Hz), 4.21 (1H, br s), 3.75 (1H, m), 3.10 (1H, m), 1.99 (3H, s), 1.82 (2H, m); 13 C NMR (CDCl 3, 75 MHz) 171.8 (s), 149.3 (s), 139.9 (s), 137.4 (s), 135.5 (s), 131.2 (d), 128.6 (d), 128.3 (d), 127.0 (d), 118.8 (d), 112.8 (d), 71.0 (t), 66.5 (t), 38.8 (t), 36.4 (t), 23.0 (q); HRESIMS [M + H] + m/z observed 345.1445 (calculated for C 18 H 21 N 2 O 5 345.1450), [M + Na] + m/z observed 367.1268 (calculated for C 18 H 20 N 2 O 5 Na 367.1270). 104

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8.5.6 Preparation of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropyl}acetamide (51) (51)(50) OBn NO2 OH NHAc OBn NO2 O NHAc Dess MartinoxidationCH2Cl290% To a mixture of Dess-Martin periodinane (360 mg, 0.85 mmol, 3 eq) and pyridine (100 mg, 1.5 eq of Dess-Martin periodinane) in dichloromethane (3 mL) was added a solution of 50 (97 mg, 0.28 mmol) in dichloromethane (1.5 mL) at 0 C and then stirred at room temperature for 2 h. The reaction mixture was diluted with saturated NaHCO 3 solution (6 mL) at 5 C and the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 The combined organic extracts were washed with saturated Na 2 S 2 O 3 (10 mL) and then water (2 X 10 mL) and brine and dried with MgSO 4 and concentrated. The residue was chromatographed on silica gel by EtOAc/hexane (80:20) to give 88 mg (92% yield) of 51 as a white solid: mp 73-74 C; IR v max 3320, 3104, 2929, 1608, 1525, 1455, 1273 cm -1 ; UV (CHCl 3 ) max (log ) 248 (1.1), 312 (0.47) nm; 1 H NMR (CDCl 3 250 MHz) 7.43 (1H, t, J = 8.1 Hz), 7.23 (m, 6H), 7.22 (1H, d, J = 8.1 Hz), 5.17 (2H, s), 3.54 (2H, q, J = 5.6 Hz), 3.14 (2H, t, J = 5.7 Hz), 1.90 (3H,s); 13 C NMR (CDCl 3 75 MHz) 197.7 (s), 170.4 (s), 150.3 (s), 139.3 (s), 134.8 (s), 131.2 (s), 130.7 (d), 128.7 (s), 128.4 (s), 126.9 (s), 120.6 (s), 118.6 (s), 71.3 (t), 39.7 (t), 34.1 (t), 23.1 (q); HREIMS [M] + m/z observed 342.1222 (calculated for C 18 H 18 N 2 O 5 342.1216). 105

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8.5.7. Preparation of Erebusinone, N-[3-(2-amino-3-hydroxyphenyl)-3-oxopropyl] acetamide (12) (51) OBn NO2 O NHAc Erebusinone (12)H2, Pd/C, 1 atmEtOH93% OH NH2 O NHAc A mixture of 51 (30 mg, 0.09 mmol) and 10 % Pd/C (15 mg) in dried ethanol (15 mL) was shaken with hydrogen atmosphere (1 atm) for 3 h. The reaction mixture was filtered to remove the catalyst and then concentrated. The residue was chromatographed on silica gel by MeOH/CH 2 Cl 2 (2:98) to give 18.2 mg (93% yield) of erebusinone (12) as a bright yellow solid: mp 150-151 C; IR v max 3488, 3362, 2925, 1619, 1568, 1458, 1221 cm -1 ; UV (MeOH) max (log ) 231 (3.2), 272 (1.5), 375 (1.1) nm; 1 H NMR (MeOH-d 4 250 MHz) 7.28 (1H, dd, J = 8.3, 1.0 Hz), 6.79 (1H, dd J = 7.6, 1.2 Hz), 6.45 (1H, t, J = 7.9 Hz), 3.40 (2H, t, J = 6.5 Hz), 3.12 (2H, t, J = 6.5 Hz), 1.9 (3H, s); 13 C NMR (MeOH-d 4 75 MHz) 201.2 (s), 173.5 (s), 146.4 (s), 142.4 (s), 122.8 (s), 118.9 (d), 118.0 (d), 115.8 (d), 39.6 (t), 36.5 (t), 22.7 (q); HREIMS [M] + m/z observed 222.1007 (calculated for C 11 H 14 N 2 O 3 222.1004). 106

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8.6 Synthesis of Erebusinonamine (52) 8.6.1 Preparation of tert-Butyl 3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropylcarbamate (53) (49) OBn NO2 OH NH OBn NO2 OH NH2 t-BOC2OtriethylamineMeOH100% O OC(CH3)3 (53) To a stirred solution of 42 (2.0 g, 66.15 mmol) and triethylamine (0.67 g, 66.20 mmol, 1 eq) in MeOH (100 mL) was added di-tert-butyldicarbonate (2.17 g, 99.20 mmol, 1.5 eq) in MeOH (20 mL) at 0 C. The reaction mixture was stirred at room temperature for 3 h and then concentrated. The crude mixture was diluted with CH 2 Cl 2 (50 mL) and washed with saturated NaHCO 3 solution (2 X 15 mL) and the separated organic layer washed with brine and then dried with MgSO 4 and concentrated to give 2.66 g (100% yield) of 45 as a pale yellow oil which was directly used in the next step without purification: [] 25 D 0 (c = 0.140, CHCl 3 ); IR v max 3416, 2979, 1686, 1609, 1532, 1368, 1278, 1069 cm -1 ; UV (MeOH) max (log ) 224 (3.5) nm; 1 H NMR (CDCl 3 250 MHz) 7.30 (m, 6H), 7.20 (1H, d, J = 8.0 Hz), 6.93 (1H, d, J = 8.4 Hz), 5.13 (2H, s), 4.88 (1H, br s), 4.72 (1H, d, J = 8.8 Hz), 3.53 (1H, m), 3.07 (1H, m), 1.79 (2H, m), 1.42 (9H, s); 13 C NMR (CDCl 3 75 MHz) 157.0 (s), 148.9 (s), 139.6 (s), 136.9 (s), 135.1 (s), 107

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130.7 (d), 128.2 (d), 127.7 (d), 126.6 (s), 118.4 (s), 112.4 (s), 79.6 (s), 70.6 (t), 65.8 (d), 39.0 (t), 36.6 (t), 27.8 (q). 8.6.2. Preparation of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropylcarbamate (54) (54)(53) OBn NO2 OH NH O OC(CH3)3 OBn NO2 O NH O OC(CH3)3 Dess MartinoxidationCH2Cl280 % To a mixture of Dess-Martin periodinane (5.27g, 124 mmol, 2 eq.) and pyridine (1.97g, 248 mmol, 2.0 eq of Dess-Martin periodinane) in CH 2 Cl 2 (60 mL) was added a solution of 53 (97 mg, 0.28 mmol) in CH 2 Cl 2 (30 mL) at 0C and then stirred at room temperature for 2h. The reaction mixture was diluted with saturated NaHCO 3 solution (60 mL) at 5 C and the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 The combined organic extracts were washed with saturated Na 2 S 2 O 3 (60 mL) and then H 2 O (2 X 30 mL) and brine and dried with MgSO 4 and concentrated. The residue was chromatographed on silica gel by EtOAc/hexane (80:20) to give 1.98 g (80%) of 54 as a white solid: mp 66-67 C; IR v max 3355, 2977, 2928, 1681, 1568, 1514, 1221 cm -1 ; UV (CHCl 3 ) max (log ) 247 (1.0), 313 (0.4) nm; 1 H NMR (CDCl 3 250 MHz) 7.44 (1H, t, J = 8.1 Hz), 7.23 (6H, m), 7.16 (1H, d, J = 7.7 Hz), 5.17 (2H, s), 3.45 (2H, q, J = 5.8 Hz), 3.10 (2H, t, J = 5.5 Hz), 1.39 (9H, s); 13 C NMR (CDCl 3 75 108

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MHz) 196.9 (s), 155.5 (s), 149.9 (s), 138.9 (s), 134.5 (s), 130.7 (s), 130.3 (d), 128.3 (d), 128.0 (d), 126.5 (d), 120.3 (d), 118.2 (d), 78.9 (s), 70.8 (t), 39.7 (t), 34.8 (t), 27.9 (q); HRESIMS [M + Na] + m/z observed 423.1535 (calculated for C 21 H 24 N 2 O 6 Na 423.1532), [M + K] + m/z observed 439.1271 (calculated for C 21 H 24 N 2 O 6 K 439.1271). 8.6.3 Preparation of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) (54)(55) OBn NO2 O NH 1.25 M HClMeOH100% O OC(CH3)3 OBn NO2 O NH3Cl To a mixture of t-BOC protected compound 54 (0.2g, 0.5 mmol) in MeOH (2 mL) was added HCl solution (7 mL, 1.25 M in MeOH) at 0 C and then stirred at room temperature for 24 h. The reaction mixture was concentrated to give 168 mg (100% yield) of 55 as a white solid which was directly used without any purification for the next step: mp 169-170 C; IR v max 2957, 2856, 1693, 1539, 1284 cm -1 ; UV (MeOH) max (log ) 213 (1.1), 250 (0.2), 313 (0.1) nm; 1 H NMR (MeOH-d 4 250 MHz) 7.59 (3H, m), 7.35 (5H, m), 5.22 (2H, s), 3.44 (2H, m), 3.27 (2H, m); 13 C NMR (MeOH-d 4 75 MHz) 196.8 (s), 151.5 (s), 140.6 (s), 136.9 (s), 136.9 (s), 132.6 (d), 130.5 (d), 129.6 (d), 129.3 (d), 128.4 (d), 122.5 (d), 120.9 (d), 72.3 (t), 37.9 (t), 35.5 (t); HRESIMS [M Cl] + m/z observed 301.1187 (calculated for C 16 H 17 N 2 O 4 301.1188). 109

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8.6.4 Preparation of erebusinonamine, 3-amino-1-[2-amino-3-hydroxyphenyl]propan-1-one hydrochloride (52) (55) OBn NO2 O NH3Cl Erebusinonamine (52)H2, Pd on C, 3.5 atmMeOH90% OH NH2 O NH3Cl A mixture of precursor of erebusinonamine (55) (66 mg, 0.2 mmol) and 10 % Pd/C (120 mg) in MeOH (50 mL) was shaken with hydrogen atmosphere (3.5 atm) for overnight. The reaction mixture was filtered to remove the catalyst and then concentrated. The residue was chromatographed on silica gel by eluting with MeOH/CH 2 Cl 2 /CF 3 COOH (20:80:0.1) to give 42.5 mg (89.5% yield) of erebusinonamine (52) as a brown wet solid: IR v max 3200, 2925, 1671, 1184 cm -1 ; UV (MeOH) max (log ) 235 (0.6), 273 (0.2), 381 (0.1) nm; 1 H NMR (MeOH-d 4 250 MHz) 7.18 (1H, d, J = 8.8 Hz), 6.77 (1H, d, J = 7.6 Hz), 6.50 (t, 1H, J = 8.2 Hz), 3.23 (4H, m); 13 C NMR (MeOH-d 4 75 MHz) 200.04 (s), 146.9 (s),122.0 (d), 119.3 (s), 118.4 (s),117.4 (d), 114.2 (d), 35.6 (t), 35.1.(t); HRESIMS [M Cl] + m/z observed 181.0969 (calculated for C 9 H 13 N 2 O 2 181.0977). 110

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REFERENCES 1. Hartwell, J.L., Lloydia, 1967, 30, 379-436. 2. Ody, P. The Complete Medicinal Herbal,1 st Ed., Darling Kindersley Limited, 1993. 3. Barton, D.; Nakanishi, K.; Meth-Cohn, O. Ed., Comprehensive Natural Products Chemistry,1 st Ed., Pergman Press, 1999,Vol. 1. 4. Murata, M.; Naoki, H.; Iwashita, T.; Matsunaga, S; Sasaki, M.; Yokoyama, A.; Yasumoto, T. J. Am. Chem. Soc. 1993, 115, 2060-2062. 5. Nonomura, T.; Sasaki, M.; Matsumori, N.; Murata, M.; Tachibana, K.; Yasumoto, T. Angew. Chem. Int. Ed. Engl. 1996, 35, 1675-1678. 6. Wani, M.C.; Taylor, H.L.; Wall, M.E.; McPhail, A.T. J. Am. Chem. Soc. 1971, 93, 2325-2327 7. Scheuer, P.J. In Chemistry of Marine Natural Products; 1 st Ed.; Academic Press, 1973. 8. Scheuer, P.J. In Marine Natural Products: Chemical and Biological Perspectives; Academic Press, New York, 1978-1983, Vol. I-V. 9. Fenical, W.H. Science 1982, 215, 923-928. 10. Scheuer, P.J. In Biomedical Importance of Marine Organisms; Fauntin, D., Ed.; Memories of the California Academy of Sciences, San Francisco, 1988, Vol. 13, 37-40. 11. Paul, V.J. In Biomedical Importance of Marine Organisms; Fauntin, D., Ed.; Memories of the California Academy of Sciences, San Francisco, 1988, Vol. 13, 23-27. 111

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50. Rinehart, K.L.; Holt, T.G.; Fregeau, N.L.; Stroh, J.G.; Keifer, P.A.; Sun, F.; Li, L.H.; Martin, D.G. J. Org. Chem. 1990, 55, 4512-4515. 51. Wright, A.E.; Forleo, D.A.; Gunawardana, F.P.; Gunasekera, S.P.; Koehn, F.E.; McConnell, O.J. J. Org. Chem. 1990, 55, 4508-4512. 52. Weinheimer, A. J.; Spraggins, R.L. Tetrahedron Lett. 1969, 5785-5188. 53. Okuda, R. K.; Klein, D.; Kinnel, R.B.; Li, M.; Scheuer, P.J. Pure Appl. Chem. 1982, 54, 1907-1914 and reference therein. 54. Baker, B.J.; Okuda, R.K.; Yu, P. T.K.; Scheuer, P.J. J. Am. Chem. Soc. 1985, 107, 2976-2977. 55. Suzuki, M.; Morita, Y.; Yanagisawa, A.; Noyori, R.; Baker, B.J.; Scheuer, P.J J. Am. Chem. Soc. 1986, 108, 5021-5022. 56. Baker, B.J.; Scheuer, P.J.; Shoolery, J.M. J. Am. Chem. Soc., 1988, 110, 965-966. 57. He, H.; Kulanthaivel, P.; Baker, B.J. Tetrahedron Lett. 1994, 35, 7189-7192. 58. Amsler, C.D.; Iken, K.B.; McClintock, J.B.; Baker, B.J. In Marine Chemical Ecology, McClintock, J.B.; Baker, B.J., Eds., CRC Press, Boca Raton, FL, 2001, 267-300. 59. Dayton, P.K.; Robilliaird, G.A.; Paine, R.T.; Dayton, L.B. Ecol. Monog. 1974, 44, 105-128. 60. Moon B.H.; Baker, B.J.; McClintock, J.B. J. Nat. Prod. 1998, 61, 116-118. 61. Moon B.H.; Park, Y.C.; Baker, B.J.; McClintock, J.B. Tetrahedron 2000, 9057-9062. 62. Pearse, V.; Pearse, J.; Bushsbaum, M.; Buschsbaum, R. In Living Invertebrates, The Blackwood Press, Pacific Grove, CA, 1987, 71-90. 114

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63. Kashman, Y.; Groweiss, A.; Lidor, R.; Blasberger, D.; Carmely, S. Tetrahedron 1985, 41, 1905-1914. 64. Copp, B.R.; Fulton, K.F.; Perry, N.B.; Blunt, J.W.; Munro, M.H.G. J. Org. Chem. 1994, 59, 8233 -8238. 65. Yang, A.; Baker, B.J.; Grimwade, J.E.; Leonard, A.C.; McClintock, J.B. J. Nat. Prod. 1995, 58, 1596-1599. 66. Trimurtulu, G.; Faulkner, D.J.; Perry, N.B.; Ettouati, L.; Litaudon, M.; Blunt, J.W.; Munro, M.H.G.; Jameson, G.B. Tetrahedron, 1994, 50, 3993-4000. 67. Jayatilake, G.S.; Baker, B.J.; McClintock, J.B. Tetrahedron Lett. 1997, 38, 7507-7510. 68. Molinski, T.F.; Faulkner, D.J. J. Org. Chem. 1987, 52, 296-298. 69. Molinski, T.F.; Faulkner, D.J. Tetrahedron Lett. 1988, 29, 2137-2138. 70. Moon B.H. Ph.D. Dissertation., Florida Institute of Technology, Melbourne, FL 1997. 71. Perry, N.B.; Blunt, J.W.; Munro, M.H.G. J. Nat. Prod. 1987, 50, 307-308. 72. Copp, B.R.; Wassvik, C.M.; Lambert, G.; Page, M.J. J. Nat. Prod. 2000, 63, 1168-1169. 73. Rice, J.M.; Dudek, G.O. J. Am. Chem. Soc. 1967, 89, 2719-2725. 74. Naya, Y.; Kishida, K.; Sugiyama, M.; Murata, M.; Miki, W.; Ohinishi, M.; Nakanish, K. Experientia 1988, 44, 50-52. 75. Buckingham, J. In Dictionary of Organic Compounds, 5 th Ed., Chapman and Hall, 1982, Vol. 3, 3091. 76. Mclintock, J.B.; Baker, B.J.; Slattery, M.; Hamann, M.; Kopitzke, R.; Heine, J. J. 115

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107. Chehade, C.C.; Dias, R.L.A.; Berlinck, R.G.S.; Gerreira, A.G.; Costa, L.V.; Rangel M.; Malpezzi, E.L.A.; de Freitas, J.C.; Hajdu, E. J. Nat. Prod. 1997, 60, 729-731. 108. Bourguet-Kondracki, M.-L.; Martin, M.-T.; Vacelet, J.; Guyot, M. Tetrahedron Lett. 2001, 42, 7257-7259. 109. Bergmann, W.; Burke, D.C. J. Org. Chem. 1956, 21, 225-228. 110. Bergmann, W.; Feeney, R.J. J. Am. Chem. Soc. 1950, 72, 2809-2810. 111. Searle, P.A.; Molinski, T.F. J. Org. Chem. 1995, 60, 4296-4298. 112. Isono, K. J. Antibiot. 1988, 41, 1711-1739. 113. Isono, K. Pharmacol. Ther. 1991, 52, 269-286. 114. Isono, K.; Uramoto, M.; Kusakabe, H.; Miyato, N.; Koyama, T.; Ubukata, M.; Sethi, S.; McCloskey, J.A. J. Antibiot. 1984, 37, 670-672. 115. Takahasi, E.; Beppu, T. J. Antibiot. 1982, 32, 930-947. 116. Hattori, T.; Adachi, K.; Shizuri, Y. J. Nat. Prod. 1998, 61, 823-826. 117. Chakrabarty, M.; Batabyal, A.; Barua, A.K. J. Nat. Prod. 1994, 57, 393-395. 118. Shin J.; Seo, Y. J. Nat. Prod. 1995, 58, 948-953. 118

PAGE 145

APPENDICES 119

PAGE 146

268, 0.745438099210, 2.025354385-0.500.511.522.5190230270310350390430470510550590 Figure 69. UV spectrum of purine analog (23) in MeOH. Figure 70. IR spectrum of purine analog (23). 120

PAGE 147

Figure 71. DEPT-135 spectrum of purine analog (23) (125 MHz, DMSO-d 6 ). 9 8 7 6 5 4 3 2 PPM 9 8 7 6 5 4 3 2 1 Figure 72. gCOSY spectrum of purine analog (23) (500 MHz, DMSO-d 6 ). 121

PAGE 148

381, 0.214275837273, 0.359592915234, 1.068024635-0.200.20.40.60.811.2190230270310350390430470510550590 Figure 73. UV spectrum of 3-hydroxykynurenine (24) in MeOH. Figure 74. IR spectrum of 3-hydroxykynurenine (24). 122

PAGE 149

8 7 6 5 4 3 2 PPM 10 9 8 7 6 5 4 3 2 1 Figure 75. gCOSY spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ). Figure 76. DEPT-135 spectrum of 3-hydroxkynurenine (24) (125 MHz, DMSO-d 6 ). 123

PAGE 150

140 120 100 80 60 40 20 0 PPM 12 10 8 6 4 2 0 Figure 77. gHSQC spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ). 7 6 5 4 3 PPM 220 200 180 160 140 120 100 80 60 40 Figure 78. gHMBC spectrum of 3-hydroxykynurenine (24) (500 MHz, DMSO-d 6 ). 124

PAGE 151

268, 0.827114105208.9, 0.88-0.100.10.20.30.40.50.60.70.80.91190230270310350390430470510550590 Figure 79. UV spectrum of 5-methyl-2-deoxycytidine (25) in MeOH. Figure 80. IR spectrum of 5-methyl-2-deoxycytidine (25). 125

PAGE 152

Figure 81. gCOSY spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ). 9 8 7 6 5 4 3 2 1 0 PPM 9 8 7 6 5 4 3 2 1 0 126

PAGE 153

9 8 7 6 5 4 3 2 1 PPM 9 8 7 6 5 4 3 2 1 Figure 82. ROESY spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ). Figure 83. DEPT-135 spectrum of 5-methyl-2-deoxycytidine (25) (125 MHz, MeOH-d 4 ). 127

PAGE 154

Figure 84. gHSQC spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ). 160 140 120 100 80 60 40 20 PPM 8 7 6 5 4 3 2 128

PAGE 155

8 7 6 5 4 3 2 PPM 180 160 140 120 100 80 60 40 20 0 Figure 85. gHMBC spectrum of 5-methyl-2-deoxycytidine (25) (500 MHz, MeOH-d 4 ). 129

PAGE 156

265, 0.557020664209, 1.012714863-0.200.20.40.60.811.2190230270310350390430470510550590 Figure 86. UV spectrum of uridine (28) in MeOH. Figure 87. IR spectrum of uridine (28). 130

PAGE 157

Figure 88. DEPT-135 spectrum of uridine (28) (125 MHz, DMSO-d 6 ). 12 10 8 6 4 2 PPM 12 10 8 6 4 2 Figure 89. ROESY spectrum of uridine (28) (500 MHz, DMSO-d 6 ). 131

PAGE 158

9 8 7 6 5 4 3 2 1 PPM 9 8 7 6 5 4 3 2 Figure 90. gCOSY spectrum of uridine (28) (500 MHz, DMSO-d 6 ). 132

PAGE 159

160 140 120 100 80 60 PPM 9 8 7 6 5 4 3 Figure 91. gHSQC spectrum of uridine (28) (500 MHz, DMSO-d 6 ). 133

PAGE 160

8 7 6 5 4 PPM 180 160 140 120 100 80 60 Figure 92. gHMBC spectrum of uridine (28) (500 MHz, DMSO-d 6 ). 134

PAGE 161

265, 0.652936935209, 0.749296665-0.100.10.20.30.40.50.60.70.8190230270310350390430470510550590 Figure 93. UV spectrum of 2-deoxycytidine (31) in MeOH. Figure 94. IR spectrum of 2-deoxycytidine (31). 135

PAGE 162

Figure 95. DEPT-135 spectrum of 2-deoxycytidine (31) (125 MHz, DMSO-d 6 ). 9 8 7 6 5 4 3 2 1 PPM 8 7 6 5 4 3 2 1 Figure 96. ROESY spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ). 136

PAGE 163

9 8 7 6 5 4 3 2 1 PPM 9 8 7 6 5 4 3 2 1 Figure 97. gCOSY spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ). 137

PAGE 164

180 160 140 120 100 80 60 40 20 PPM 9 8 7 6 5 4 3 2 1 Figure 98. gHSQC spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ). 138

PAGE 165

8 7 6 5 4 3 2 1 PPM 200 180 160 140 120 100 80 60 40 Figure 99. gHMBC spectrum of 2-deoxycytidine (31) (500 MHz, DMSO-d 6 ). 139

PAGE 166

200, 0.581551552263, 0.284370422325, 0.404864311227, 1.230090618-0.200.20.40.60.811.21.4190230270310350390430470510550590 Figure 100. UV spectrum of 4, 8-dihydroxyquinoline (33) in MeOH. Figure 101. IR spectrum of 4, 8-dihydroxyquinoline (33). 140

PAGE 167

Figure 102. DEPT-135 spectrum of 4, 8-dihydroxyquinoline (33) (125 MHz, DMSO-d 6 ). 8 7 6 5 4 3 2 1 PPM 8 7 6 5 4 3 2 1 Figure 103. ROESY spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ). 141

PAGE 168

8.0 7.5 7.0 6.5 6.0 PPM 8.0 7.5 7.0 6.5 6.0 5.5 Figure 104. gCOSY spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ). 142

PAGE 169

180 170 160 150 140 130 120 110 100 PPM 8.5 8.0 7.5 7.0 6.5 6.0 5.5 Figure 105. gHSQC spectrum of 4, 8-dihydroxyquinoline (33) (500 MHz, DMSO-d 6 ). 143

PAGE 170

280, 0.634979725206, 0.844995975-0.100.10.20.30.40.50.60.70.80.9190230270310350390430470510550590 Figure 106. UV spectrum of homarine (37) in MeOH. Figure 107. IR spectrum of homarine (37). 144

PAGE 171

Figure 108. DEPT-135 spectrum of homarine (37) (125 MHz, DMSO-d 6 ). 8 6 4 2 PPM 10 8 6 4 2 0 Figure 109. ROESY spectrum of homarine (37) (500 MHz, DMSO-d 6 ). 145

PAGE 172

9 8 7 6 5 4 3 PPM 9 8 7 6 5 4 3 Figure 110. gCOSY spectrum of homarine (37) (500 MHz, DMSO-d 6 ). 146

PAGE 173

8 0 160 140 120 100 80 60 40 PPM 9 8 7 6 5 4 Figure 111. gHSQC spectrum of homarine (37) (500 MHz, DMSO-d 6 ). 147

PAGE 174

243, 0.2969517710.10.150.20.250.30.35200240280320360400440480520560600 Figure 112. UV spectrum of ceramide analog (39) in CHCl 3 Figure 113. IR spectrum of ceramide analog (39). 148

PAGE 175

Figure 114. gCOSY spectrum of ceramide analog (39) (500 MHz, CDCl 3 ). 7 6 5 4 3 2 1 PPM 8 7 6 5 4 3 2 1 149

PAGE 176

Figure 115. TOCSY spectrum of ceramide (39) (500 MHz, CDCl 3 ). 150 7 6 5 4 3 2 1 0 PPM 7 6 5 4 3 2 1

PAGE 177

120 100 80 60 40 20 PP M Figure 116. DEPT-45 spectrum of ceramide analog (39) (125 MHz, CDCl 3 ). 151

PAGE 178

Figure 117. gHSQC spectrum of ceramide analog (39) (500 MHz, CDCl 3 ). 180 160 140 120 100 80 60 40 20 PPM 8 7 6 5 4 3 2 1 152

PAGE 179

7 6 5 4 3 2 1 PPM 180 160 140 120 100 80 60 40 20 Figure 118. gHMBC spectrum of ceramide analog (39) (500 MHz, CDCl 3 ). 153

PAGE 180

Figure 119. NOESY spectrum of ceramide analog (39) (500 MHz, CDCl 3 ). 8 7 6 5 4 3 2 1 PPM 7 6 5 4 3 2 1 154

PAGE 181

Figure 120. gCOSY spectrum of (S)-MTPA ester (43) (500 MHz, CDCl 3 ). 155 8 7 6 5 4 3 2 1 PPM 8 7 6 5 4 3 2 1 0

PAGE 182

Figure 121. gCOSY spectrum of (R)-MTPA ester (44) (500 MHz, CDCl 3 ). 156 8 7 6 5 4 3 2 1 0 PPM 8 7 6 5 4 3 2 1 0

PAGE 183

303, 0.445133209243, 0.797145367-0.100.10.20.30.40.50.60.70.80.9190230270310350390430470510550590 Figure 122. UV spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) in CHCl 3. Figure 123. IR spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46). 157

PAGE 184

7.628 7.601 7.597 7.454 7.429 7.421 7.416 7.409 7.404 7.400 7.388 7.382 7.368 7.357 7.349 7.275 7.271 5.340 5.201 8 7 6 5 4 3 2 1 PP M Figure 124. 1 H NMR spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) (250 MHz, CDCl 3 ). 158

PAGE 185

162.690 149.812 141.317 134.954 134.720 130.535 128.679 128.559 128.332 126.947 123.553 122.484 118.644 71.235 67.885 160 140 120 100 80 60 PP M Figure 125. 13 C NMR spectrum of benzyl-3-(benzyloxy)-2-nitrobenzoate (46) (75 MHz, CDCl 3 ). 159

PAGE 186

249, 0.304718494313, 0.129708767210, 1.210551739-0.200.20.40.60.811.21.4190230270310350390430470510550590 Figure 126. UV spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) in CHCl 3. Figure 127. IR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47). 160

PAGE 187

7.545 7.513 7.374 7.359 7.323 7.283 7.258 5.245 3.986 8 7 6 5 4 3 PP M Figure 128. 1 H NMR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) (250 MHz, CDCl 3 ). 161

PAGE 188

187.753 149.513 135.512 131.711 128.597 128.288 127.501 122.336 120.770 70.663 31.234 200 150 100 50 0 PP M Figure 129. 13 C NMR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropanenitrile (47) (250 MHz, CDCl 3 ). 162

PAGE 189

244, 0.49794054-0.100.10.20.30.40.50.6190290390490590 Figure 130. UV spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropanenitrile (48) in CHCl 3. Figure 131. IR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropanenitrile (48). 163

PAGE 190

7.473 7.439 7.374 7.323 7.292 7.096 7.063 5.189 5.030 2.908 2.888 2.859 2.831 1.263 8 7 6 5 4 3 2 1 0PP M Figure 132. 1 H NMR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropanenitrile (48) (250 MHz, CDCl 3 ). 164

PAGE 191

149.629 139.429 135.128 134.340 131.687 128.595 128.243 126.983 118.607 116.759 114.190 71.004 65.111 27.232 140 120 100 80 60 40 PP M Figure 133. 13 C NMR spectrum of 3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropanenitrile (48) (75 MHz, CDCl 3 ). 165

PAGE 192

245, 0.506969929-0.100.10.20.30.40.50.6190230270310350390430470510550590 Figure 134. UV spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) in CHCl 3. Figure 135. IR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49). 166

PAGE 193

7.386 7.354 7.340 7.322 7.306 7.284 7.262 7.239 6.936 6.905 5.139 5.003 4.992 4.967 4.955 3.457 3.106 2.923 2.460 1.850 1.840 1.830 1.724 1.231 8 7 6 5 4 3 2 1 0PP M Figure 136. 1 H NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) (250 MHz, CDCl 3 ). 167

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149.204 139.866 137.953 135.548 130.827 128.544 128.080 126.953 119.095 112.509 70.876 70.514 39.938 38.496 140 120 100 80 60 40 PP M Figure 137. 13 C NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-ol (49) (75 MHz, CDCl 3 ). 168

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243, 0.360056877-0.0500.050.10.150.20.250.30.350.4190290390490590 Figure 138. UV spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropyl}acetamide (50) in CHCl 3. Figure 139. IR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropyl}acetamide (50). 169

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7.397 7.363 7.357 7.340 7.314 7.289 7.239 7.213 6.954 6.922 5.934 5.276 5.142 4.682 4.644 4.224 4.213 3.118 3.098 1.992 1.845 1.821 1.803 1.783 1.764 1.655 1.230 7 6 5 4 3 2 1 PP M Figure 140. 1 H NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropyl}acetamide (50) (250 MHz, CDCl 3 ). 170

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171.857 149.332 139.873 137.422 135.481 131.229 128.633 128.213 127.012 118.789 112.792 70.998 66.510 38.817 36.385 23.020 160 140 120 100 80 60 40 20 PP M Figure 141. 13 C NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropyl}acetamide (50) (75 MHz, CDCl 3 ). 171

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312, 0.470425606246, 1.156972885-0.200.20.40.60.811.21.4190240290340390440490540590 Figure 142. UV spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-oxoypropyl}acetamide (51) in CHCl 3. Figure 143. IR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropyl}acetamide (51). 172

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7.436 7.403 7.321 7.304 7.248 7.240 7.215 5.169 3.569 3.546 3.434 3.157 3.135 3.113 1.901 8 7 6 5 4 3 2 1 PP M Figure 144. 1 H NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-oxoypropyl}acetamide (51) (250 MHz, CDCl 3 ). 173

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197.674 170.422 150.284 139.243 134.828 131.183 130.717 128.710 128.393 126.945 120.565 118.630 71.241 39.645 34.072 23.102 2 00 150 100 50 PP M Figure 145. 13 C NMR spectrum of N-{3-[3-(benzyloxy)-2-nitrophenyl]-3-oxoypropyl}acetamide (51) (75 MHz, CDCl 3 ). 174

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375, 1.08142519272, 1.557867527231, 3.252184391-0.500.511.522.533.54190230270310350390430470510550590 Figure 146. UV spectrum of erebusinone (12) in MeOH. Figure 147. IR spectrum of erebusinone (12). 175

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7.288 7.259 7.254 6.807 6.781 6.777 6.485 6.453 4.869 3.519 3.494 3.165 3.140 3.112 1.904 7 6 5 4 3 2 1 PP M Figure 148. 1 H NMR spectrum of erebusinone (12) (250 MHz, MeOH-d 4 ). 176

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202.192 173.501 146.418 142.427 122.811 118.933 117.984 115.740 39.627 36.513 22.662 200 150 100 50 0 PP M Figure 149. 13 C NMR spectrum of erebusinone (12) (75 MHz, MeOH-d 4 ). 177

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-0.500.511.522.533.544.5200250300350400450500 Figure 150. UV spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropylcarbamate (53). Figure 151. IR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropylcarbamate (53). 178

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7.383 7.351 7.335 7.318 7.306 7.282 7.268 7.261 7.239 7.223 7.191 6.947 6.913 5.134 4.880 4.738 4.710 4.107 4.077 4.063 4.051 4.038 3.548 3.513 3.118 3.099 3.079 3.060 3.041 2.017 1.818 1.799 1.781 1.764 1.425 1.261 8 7 6 5 4 3 2 PP M Figure 152. 1 H NMR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropylcarbamate (53) (250 MHz, CDCl 3 ). 179

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156.987 148.930 139.648 136.969 135.126 130.726 128.233 127.787 126.591 118.371 112.412 79.555 70.596 65.879 38.965 36.582 27.861 160 140 120 100 80 60 40 20PP M Figure 153. 13 C NMR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-hydroxypropylcarbamate (53) (75 MHz, CDCl 3 ). 180

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313, 0.425293922247, 1.029672146-0.200.20.40.60.811.2190230270310350390430470510550590 Figure 154. UV spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropylcarbamate (54) in CHCl 3. Figure 155. IR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropylcarbamate (54). 181

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7.439 7.407 7.329 7.244 5.175 3.467 3.445 3.130 3.108 1.394 8 7 6 5 4 3 2 1 PP M Figure 156. 1 H NMR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropylcarbamate (54) (250 MHz, CDCl 3 ). 182

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196.986 155.515 149.908 138.950 134.525 130.753 130.342 128.335 127.990 126.581 120.281 118.227 78.941 70.862 39.746 34.790 27.928 200 150 100 50 PP M Figure 157. 13 C NMR spectrum of tert-butyl-3-[3-(benzyloxy)-2-nitrophenyl]-3-oxopropylcarbamate (54) (75 MHz, CDCl 3 ). 183

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313, 0.096655846250, 0.240504265213, 1.110908985-0.200.20.40.60.811.2190230270310350390430470510550590 Figure 158. UV spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) in MeOH. Figure 159. IR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55). 184

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7.614 7.592 7.570 7.348 7.327 7.303 7.294 5.230 4.845 3.444 3.420 3.291 3.283 3.278 3.270 8 7 6 5 4 3 2 1 PP M Figure 160. 1 H NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) (250 MHz, MeOH-d 4 ). 185

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196.821 151.537 140.574 136.922 132.672 130.561 129.635 129.346 128.416 122.503 120.913 72.322 37.962 35.528 200 150 100 50 0 PP M Figure 161. 13 C NMR spectrum of 3-amino-1-[3-(benzyloxy)-2-nitrophenyl]propan-1-one hydrochloride (55) (75 MHz, MeOH-d 4 ). 186

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381, 0.114315033273, 0.243011475235, 0.56055975-0.100.10.20.30.40.50.6190230270310350390430470510550590 Figure 162. UV spectrum of erebusinonamine (52) in MeOH. Figure 163. IR spectrum of erebusinonamine (52). 187

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7.204 7.174 7.172 6.790 6.788 6.760 6.757 6.531 6.500 6.469 5.038 3.305 3.274 3.253 3.199 3.179 8 7 6 5 4 3 2 1 0PP M Figure 164. 1 H NMR spectrum of erebusinonamine (52) (250 MHz, MeOH-d 4 ). 188

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200.047 146.955 122.037 119.388 118.458 117.619 117.498 36.543 35.870 200 150 100 50 0 PP M Figure 165. 13 C NMR spectrum of erebusinonamine (52) (75 MHz, MeOH-d 4 ). 189

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121.133 117.042 114.762 114.726 35.594 35.184 120 100 80 60 40 PP M Figure 166. DEPT-135 spectrum of erebusinonamine (52) (125 MHz, MeOH-d 4 ). 190

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ABOUT THE AUTHOR Young Chul Park received a M.S. with honors in chemistry from Dankook University, Seoul, Korea in the year 1990. He then worked in Korea for 8 years on natural products chemistry and also on tota l synthesis of natural products at Esung pharmaceutical company. He joined the Department of Chemistry at Florida Institute of Technology in 1999 for his Ph.D program under the supervision of Dr. Bill J. Baker. His research is based on th e isolation of secondary metabolites from Antarctic marine organisms and total synt hesis of natural products. He relocated along with his supervisor Dr. Bill J. Baker to Department of Chemistry, University of South Florida in 2001 to continue his Ph.D. program He has coauthored two publications in Tetrahedron and J Nat Prod. He also gave seve ral presentations at international meetings of the American Ch emical Society and the American Society of Pharmacognosy in 2003.


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Chemical investigation of three Antarctic marine sponges
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[Tampa, Fla.] :
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Thesis (Ph.D.)--University of South Florida, 2004.
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ABSTRACT: This thesis describes the chemical investigation of three marine sponges from Antarctica and the total syntheses of natural products erebusinone (12) and its derivative, erebusinonamine (52). Investigation of the yellow Antarctic marine sponge Isodictya setifera resulted in the isolation of two secondary metabolites, purine analog (32) and 3-hydroxykynurenine (24). Chemical investigation of Isodictya setifera led to the isolation of six secondary metabolites which included 5-methyl-2-deoxycytidine (25), uridine (28), 2-deoxycytidine (31), homarine (37), hydroxyquinoline (33), 3-hydroxykynurenine (24). The latter two compounds were found to be intermediates of tryptophan catabolism in crustaceans. From the Antarctic marine sponge Isodictya antractica ceramide analog (39) was isolated and its chemical structure was assigned by a combination of spectroscopic and chemical analyses. Stereochemistry was determined by modified Mosher's method. Erebusinone (12), a yellow pigment isolated from the Antarctic marine sponge Isodictya erinacea has been implicated in molt inhibition and mortality against the Antarctic crustacean amphipod, Orchomene plebs, possibly serving as a precursor of a xanthurenic acid analog. Thought to act as a 3-hydroxykynurenine 24 mimic, erebusinone (12) may be involved chemical defense.1 This appears to be the first example in the marine realm of an organism utilizing tryptophan catabolism to modulate molting as a defensive mechanism. To further investigate the bioactivity and ecological role of erebusinone (12), the synthesis of this pigment was carried out in an overall yield of 44% involving seven steps which were economical and convenient. Erebusinonamine (52) was also similarly synthesized in eight steps with an overall yield of 45%. Reference 1. Moon B. H.; Park Y. C.; McClintock J. B.; Baker B. J., Tetrahedron 2000, 56, 9057-9062.
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erebusinone.
Antarctic invertebrates.
marine natural products.
tryptophan catabolism.
chemical defense.
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