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Novel nitrogenous metabolites from marine sponges Kong, Fangming 1995

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NOVEL NITROGENOUS METABOLITES FROM MARINE SPONGES by FANGMING KONG B Sc, Hangzhou University, China, 1982 M Sc, Hangzhou University, China, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF CHEMISTRY) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1995 © Fangming Kong 1995 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. (Signature) Department of C-W^^ry The University of British Columbia Vancouver, Canada Date W ^ ^ , tWf DE-6 (2/88) ABSTRACT u An investigation into the chemistry of four species of marine sponges has led to the isolation of twenty-two new natural products. The structures of these novel compounds were determined by spectroscopic analysis and chemical interconversion. The metabolites were tested in a variety of bioassays. Methanol extracts of the marine sponge Xestospongia ingens were found to be cytotoxic against murine leukemia P388. Fractionation of the crude extract led to the identification of two classes of biologically active alkaloids with novel heterocyclic skeletons. Ingenamine (27) is the first example of a new class of cytotoxic pentacyclic alkaloids that appears to arise biogenetically from an intramolecular [4+2] cycloaddition reaction. The discovery of the ingenamine type compounds strongly supported Baldwin and Whitehead's proposal for the biosynthesis of the manzamines, since the ingenamine skeleton corresponded to their proposed pentacyclic intermediate. Ingenamine showed in vitro cytotoxicity against murine leukemia P388 with an ED50 value of 1 |ig/mL. Madangamine A (30) represented the first example of another new class of pentacyclic alkaloids. It was proposed that the biosynthesis of madangamine involves the rearrangement of an ingenamine type precursor. Madangamine A also showed in vitro cytotoxicity against murine leukemia P388 (ED50 0.93 |Hg/mL), human lung A549 (ED50 14 |Lig/mL), brain U373 (ED50 5.1 (ig/mL), and breast MCF-7 (ED50 5.7 M-g/mL) cancer cell lines. The structures of ingenamine (27) and madangamine A (30) were elucidated by extensive analyses of spectroscopic data. The absolute configuration of ingenamine was determined by Mosher's ester methodology. Eight additional 'ingenamine' alkaloids (31 - 34, 36 - 39) and four additional 'madangamine' alkaloids (48 - 51) were also isolated from this sponge. Examination of the Norwegian sponge Polymastia boletiformis collected off the Korsnes Peninsula on Fanafjiord south of Bergen, Norway yielded six new metabolites, the steroid/amino acid conjugates, polymastiamides A-F (65, 68, 70, 72, 74, 76). The structure of polymastiamide A (65), isolated as the natural product, was elucidated by spectroscopic analysis as well as chemical degradation. The relative stereochemistry of the steroidal nucleus was obtained by i i i comparison with previously reported data. The remaining analogs, polymastiamides B-F (68, 70, 72, 74, 76), were hydrolyzed to remove the sulfate groups and then converted to methyl esters in order to exploit the convenience and efficiency of normal phase HPLC separation and purification techniques. Polymastiamide A (65) exhibited in vitro antimicrobial activity against various human and plant pathogens [MIC's in a 1/4-in. disk assay: Staphylococcus aureus (100jj,g/disk), Candida albicans (75 (j.g/disk) andpythium ultimum (25 fig/disk)]. The study of two Papua New Guinea sponges led to the isolation and identification of two new peptides. A novel cyclic heptapeptide pseudaxinellin (87) was isolated from Pseudaxinella massa. Pseudaxinellin (87) contained standard protein amino acid residues with the L configuration. A new cyclic depsipeptide, geodiamolide G (111), was obtained from the sponge Cymbastela sp., together with the known metabolites geodiamolides A-F (89 - 94). 65 IV HO 111 TABLE OF CONTENTS Abstract ii Table of Contents v List of Figures vi List of Schemes xv List of Tables xvi List of Abbreviations xviii Acknowledgment xxii Part I General Introduction 1 1.1. Sponges 1 1.2. Sponge Chemistry 3 Part II Ingenamines and Madangamines. Novel alkaloids from the PNG sponge Xestospongia ingens 9 2.1. Introduction 9 2.1.1. Taxonomy and Description of Xestospongia ingens 9 2.1.2. A Review of 3-Alkylpyridine Alkaloids Isolated from Marine Sponges 12 2.2. Results and Discussion 20 2.2.1. Isolation of Metabolites from Xestospongia ingens 20 2.2.2. Structure Elucidation of Ingenamine Alkaloids horn Xestospongia ingens 23 2.2.2.1. Ingenamine (27) 25 2.2.2.2. Ingamine A (31) 45 2.2.2.3. Ingamine B (32) 55 2.2.2.4. Ingenamine B (33) 65 2.2.2.5. Ingenamine C (34) and Ingenamine C Acetate (35) 72 2.2.2.6. Ingenamine D (36) 80 2.2.2.7. Ingenamine E (37) 85 VI 2.2.2.8. Ingenamine F (38) 92 2.2.2.9. Ingenamine G (39) and Ingenamine G Acetate (40) 97 2.2.2.10. Keramaphidin B (28) 105 2.2.2.11. Conclusions 109 2.2.3. The Absolute Configuration of Ingenamine Alkaloids Isolated from the Sponge Xestospongia Ingens 112 2.2.3.1. MTPA-Ingenamine (45) 113 2.2.3.2. MTPA-Ingamine A (46) 119 2.2.3.3. MTPA-Ingenamine E (47) 120 2.2.3.4. Conclusions 121 2.2.4. Structure Elucidation of Madangamine Alkaloids from the Sponge Xestospongia ingens 122 2.2.4.1. Madangamine A (30) 124 2.2.4.2. Madangamine B (48) 144 2.2.4.3. Madangamine C (49) 153 2.2.4.4. Conclusions 161 2.3. Experimental 162 Part III. Polymastiamides A-F. Novel Steroid/Amino Acid Conjugates. Isolated from the Norwegian Marine Sponge Polymastia boletiformis 174 3.1. Introduction 174 3.1.1. Taxonomy and Description of Polymastia boletiformis Classification of the sponge 174 3.1.2. A Brief Review of Marine Sponge Steroids Related to Polymastiamides 177 3.2. Results and Discussion 180 3.2.1. Isolation of Steroidal Metabolites from Polymastia boletiformis 180 3.2.2. Structure Elucidation of Polymastiamides A-F (65-76) and the Related Compounds 181 3.2.2.1. Polymastiamide A (65) 184 3.2.2.2. Polymastiamide B (68) 206 vii 3.2.2.3. Polymastiamide C (70) 211 3.2.2.4. Polymastiamide D (72) 216 3.2.2.5. Polymastiamide E (74) 221 3.2.2.6. Polymastiamide F (76) 224 3.2.2.7. Polymastiamide G (78) 231 3.2.2.8. Polymastiamides H (79), I (80), and J (81) 240 3.2.2.9. Polymastiamide K (82) 250 3.3. Conclusion 262 3.4. Experimental 264 Part IV. New Metabolites from Two Papua New Guinea Sponges 268 4.1. Introduction 268 4.1.1. A Review of Peptides Related to Pseudaxinellin and Geodiamolides 268 4.2. Results and Discussion 276 4.2.1. The Structure Elucidation of Pseudaxinellin (87). a Cyclic Heptapeptide from the Marine Sponge Pseudaxinella massa 276 4.2.1.1. Taxonomy and Description of Pseudaxinella massa 276 4.2.1.2. Isolation of Pseudaxinellin (87) from Pseudaxinella massa ••• 279 4.2.1.3. Structure Elucidation of Pseudaxinellin (87) 280 4.2.1.4. Conformational Analysis of Pseudaxinellin (87) 292 4.2.1.5. Conclusions 295 4.2.2. The Structure Elucidation of Geodiamolide G (111), a Depsipeptide from the Marine Sponge Cymbastela sp. 298 4.2.2.1. Taxonomy and Description of Cymbastela sp. 298 4.2.2.2. Isolation of Geodiamolide G (111) from Cymbastela sp. 299 4.2.2.3. Structure Elucidation of Geodiamolide G (111) 299 4.2.2.4. Conclusions 307 4.3. Experimental 308 LIST OF FIGURES vin Figure 1. Typical leuconoid plan of a sponge of the class Demospongaie 2 Figure 2. Anticancer leads with significant selective cytotoxic activity 6 Figure 3. Percentage of specimens per phyla with IC50 < 4 |ig/mL 6 Figure 4. Xestospongia ingens sponge 10 Figure 5. Low resolution positive FAB mass spectrum of ingenamine (27) 26 Figure 6. *H NMR spectrum of ingenamine (27) (500 MHz, MeOH-d4) 27 Figure 7. 13C and APT NMR spectra of ingenamine (27) (125 MHz, MeOH-d4) •• 28 Figure 8. 2D HMQC spectrum of ingenamine (27) in MeOH-d4 29 Figure 9. 2D COSY spectrum of ingenamine (27) in MeOH-d4 30 Figure 10. Selected regions of the 2D HMBC spectrum of ingenamine (27) inMeOH-af4 31 Figure 11. Substructure of fragment CsHioO and selected HMBC correlations in ingenamine (27) 33 Figure 12. The tricyclic core structure and four appendages, and selected HMBC correlations in ingenamine (27) 34 Figure 13. Selected NOE/ ROESY, COSYLR, and HMBC correlations in ingenamine (27) 36 Figure 14. Selected NOE results of ingenamine (27) in MeOH-d4 (400 MHz) 37 Figure 15. Substructures of the two side chains and selected HMBC correlations in ingenamine (27) 38 Figure 16. *H NMR spectrum of acid-free ingenamine (27) in MeOH-d4 (500 MHz) 40 Figure 17. 13C and APT NMR spectra of acid-free ingenamine (27) (125 MHz, MeOH-^4) 41 Figure 18. *H chemcal shift differences between the two forms of ingenamine (27) 43 Figure 19. 13C chemcal shift differences between the two forms of ingenamine (27) 44 Figure 20. Low resolution EI mass spectrum of ingamine A (31) 46 Figure 21. lH NMR spectrum of ingamine A (31) in CDCI3 (500 MHz) 47 Figure 22. 13C and APT NMR spectra of ingamine A (31) (125 MHz, CDCI3) 48 Figure 23. 2D HMQC spectrum of ingamine A (31) in CDCI3 49 IX Figure 24. 2D COSY spectrum of ingamine A (31) in CDCI3 50 Figure 25. Low resolution EI mass spectrum of ingamine B (32) 56 Figure 26. lH NMR spectrum of ingamine B (32) in CDCI3 (500 MHz) 57 Figure 27. 13C and APT NMR spectra of ingamine B (32) (125 MHz, CDCI3) 58 Figure 28. 2D HMQC spectrum of ingamine B (32) in CDCI3 59 Figure 29. 2D COSY spectrum of ingamine B (32) in CDCI3 60 Figure 30. *H NMR spectrum of ingenamine B (33) in MeOH-^4 (500 MHz) 66 Figure 31. 13C and APT NMR spectra of ingenamine B (33) in MeOH-J4 (125 MHz) 67 Figure 32. 2D COSY spectrum of ingenamine B (33) in MeOH-^4 68 Figure 33. 2D HMQC spectrum of ingenamine B (33) in MeOH-af4 69 Figure 34. Selected HMBC and COSYLR correlations in ingenamine B (33) 71 Figure 35. !H NMR spectrum of ingenamine C acetate (35) in MeOH-d4 (500 MHz) 73 Figure 36. APT NMR spectrum of ingenamine C acetate (35) in MeOH-^ (125 MHz) 74 Figure 37. 2D HMQC spectrum of ingenamine C acetate (35) in MeOH-^4 75 Figure 38. 2D COSY spectrum of ingenamine C acetate (35) in MeOH-d4 76 Figure 39. Selected COSY and HMBC correlations in ingenamine C acetate (35) •• 78 Figure 40. 2H NMR spectrum of ingenamine C (34) in MeOH-^4 (500 MHz) 79 Figure 41. JH NMR spectrum of ingenamine D (36) in MeOH-^4 (500 MHz) 81 Figure 42. 2D COSY spectrum of ingenamine D (36) in MeOH-^4 82 Figure 43. Selected HMBC and COSYLR correlations in ingenamine D (36) 84 Figure 44. JH NMR spectrum of ingenamine E (37) in MeOH-a?4 (500 MHz) 87 Figure 45. APT NMR spectrum of ingenamine E (37) in MeOH-c/4 (125 MHz) 88 Figure 46. 2D HMQC spectrum of ingenamine E (37) in MeOH-^4 89 Figure 47. 2D COSY spectrum of ingenamine E (37) in MeOH-d4 90 Figure 48. JH NMR spectrum of ingenamine F (38) in MeOH-^4 (400 MHz) 94 Figure 49. 13C and APT NMR spectra of ingenamine F (38) in MeOH-d4 (125 MHz) 95 Figure 50. !H NMR spectrum of ingenamine G (39) in MeOH-d4 (500 MHz) 98 Figure 51. APT NMR spectrum of ingenamine G (39) in MeOH-^4 (125 MHz) 99 Figure 52. 2D HMQC spectrum of ingenamine G (39) in MeOH-^4 100 Figure 53. 2D COSY spectrum of ingenamine G (39) in MeOH-^4 101 Figuer 54. Selected COSY and HMBC correlations in ingenamine G (39) 102 Figure 55. !H NMR spectrum of ingenamine G acetate (40) in MeOH-^4 (400 MHz) 104 Figure 56. *H NMR spectrum of keramaphidin B (28) in MeOH-d4 (500 MHz) 106 Figure 57. APT NMR spectrum of keramaphidin B (28) in MeOH-d4 (125 MHz) •• 107 Figure 58. *H NMR spectrum of (S)-MTPA-ingenamine (45a) in CD2CI2 (400 MHz) 114 Figure 59. *H NMR spectrum of (fl)-MTPA-ingenamine (45b) in CD2CI2 (500 MHz) 115 Figure 60. Chem3D-generated representations of (5)- and (^)-MTPA-ingenamines (45a and 45b) 116 Figure 61. The conformational representation of MTPA-ingenamine (45) with A5 (Hz) values recorded in CD2C12 (500 MHz) 117 Figure 62. The conformational representation of MTPA-ingamine A (46) with A8 (Hz) values recorded in CD2C12 (500 MHz) 119 Figure 63. The conformational representation of MTPA-ingenamine E (47) with A5 (Hz) values recorded in CD2C12 (500 MHz) 120 Figure 64. Low resolution EI mass spectrum of madangamine A (30) 125 Figure 65. !H NMR spectrum of madangamine A (30) in C6D6 (500 MHz) 126 Figure 66. 13C and APT NMR spectra of madangamine A (30) in C6D6 (125 MHz) 127 Figure 67. 2D HMQC spectrum of madangamine A (30) in C6D6 128 Figure 68. 2D COSY spectrum of madangamine A (30) in C6D6 129 Figure 69. Proton spin system of the central core and selected HMBC correlations in madangamine A (30) 131 Figure 70. 2D HMBC spectrum of madangamine A (30) in C6D6 132 Figure 71. 2D COSYLR spectrum of madangamine A (30) in C6D6 133 Figure 72. Selected COSYLR and HMBC correlations in madangamine A (30) 134 Figure 73. The central core structure and selected NOE, COSYLR, and HMBC correlations in madangamine A (30) 135 Figure 74. Selected NOE results of madangamine A (30) in C6D6 (400 MHz) 136 xi Figure 75. Relative stereochemistry of the tricyclic core and selected NOEs in madangamine A (30) 137 Figure 76. 2D HOHAHA spectrum of madangamine A (30) in C6D6 138 Figure 77. Structure of the side chain CI3-to-C20 and selected HOHAHA correlations in madangamine A (30) 139 Figure 78. Dreiding model of madangamine A (30) 143 Figure 79. !H NMR spectrum of madangamine B (48) in C6D6 (500 MHz) 145 Figure 80. 13C and APT NMR spectra of madangamine B (48) in C ^ (125 MHz) 146 Figure 81. 2D HMQC spectrum of madangamine B (48) in C6D6 147 Figure 82. 2D COSY spectrum of madangamine B (48) in C6D6 148 Figure 83. Selected HMBC and COSYLR correlations in madangamine B (48) 152 Figure 84. *H NMR spectrum of madangamine C (49) in C6D6 at 65 °C (500 MHz) 154 Figure 85. 13C and APT NMR spectra of madangamine C (49) in C6D6 at 65°C (125 MHz) 155 Figure 87. 2D HMQC spectrum of madangamine C (49) in C6D6 156 Figure 88. Selected NOE results of madangamine C (49) in C6D6 (400 MHz) 160 Figure 89. Polymastia boletiformis sponge 175 Figure 90. Low resolution FAB mass spectrum of polymastiamide A (65) 185 Figure 91. *H NMR spectrum of polymastiamide A (65) in DMSO-^6 (500 MHz) 186 Figure 92. 13C and APT NMR spectra of polymastiamide A (65) in DMSO-^6 (125 MHz) 187 Figure 93. Low resolution EI mass spectrum of polymastiamide A methyl ester 66 189 Figure 94. *H NMR spectrum of polymastiamide A methyl ester 66 in CDCI3 (500 MHz) 190 Figure 95. 13C NMR spectrum of polymastiamide A methyl ester 66 in CDCI3 (125 MHz) 191 Figure 96a. 2D HMQC spectrum of polymastiamide A methyl ester 66 in CDCI3 •••• 192 Figure 96b. Selected region of 2D HMQC spectrum of methyl ester 66 in CDCI3 •••• 193 Figure 97a. 2D COSY spectrum of polymastiamide A methyl ester 66 in CDCI3 194 Figure 97b. Selected region of 2D COSY spectrum of methyl ester 66 in CDCI3 195 Figure 98a. 2D HMBC spectrum of polymastiamide A methyl ester 66 in CDCI3 •••• 197 Xll Figure 98b. Selected region of 2D HMBC spectrum of methyl ester 66 in CDCI3 •••• 198 Figure 99. p-Methoxyphenylglycine methyl ester fragment and selected HMBC correlations in polymastiamide A methyl ester 66 200 Figure 100. Selected HMBC correlations for steroidal fragment in methyl ester 66 •• 202 Figure 101. Comparison of the 13C NMR assignments for 66 and 67 204 Figure 102. *H NMR spectrum of polymastiamide B methyl ester 69 in CDCI3 (400 MHz) 207 Figure 103. 13C NMR spectrum of polymastiamide B methyl ester 69 in CDCI3 (125 MHz) 208 Figure 104. Comparison of the 13C NMR assignments for 69 and 67 210 Figure 105. *H NMR spectrum of polymastiamide C methyl ester 71 in CDCI3 (500 MHz) 212 Figure 106. 13C and APT NMR spectra of polymastiamide C methyl ester 71 in CDCI3 (125 MHz) 213 Figure 107. Selected HMBC correlations in polymastiamide C methyl ester 71 215 Figure 108. *H NMR spectrum of polymastiamide D methyl ester 73 in CDCI3 (500 MHz) 218 Figure 109. 13C and APT NMR spectra of polymastiamide D methyl ester 73 in CDCI3 (125 MHz) 219 Figure 110. *H NMR spectrum of polymastiamide E methyl ester 75 in CDCI3 (500 MHz) 222 Figure 111. 2D COSY spectrum of polymastiamide E methyl ester 75 in CDCI3 223 Figure 112. *H NMR spectrum of polymastiamide F methyl ester 77 in CDCI3 (500 MHz) 225 Figure 113. 13C and APT NMR spectra of polymastiamide F methyl ester 77 in CDCI3 (125 MHz) 226 Figure 114. 2D COSY spectrum of polymastiamide F methyl ester 77 in CDCI3 227 Figure 115. 2D HMQC spectrum of polymastiamide F methyl ester 77 in CDCI3 228 Figure 116. Selected HMBC correlations in polymastiamide F methyl ester 77 230 Figure 117. *H NMR spectrum of polymastiamide G (78) in CDCI3 (500 MHz) 231 Figure 118. 13C and APT NMR spectra of polymastiamide G (78) in CDCI3 (125 MHz) 233 Figure 119. 2D HMQC spectrum of polymastiamide G (78) in CDCI3 234 Xll l Figure 120. 2D COSY spectrum of polymastiamide G (78) in CDCI3 235 Figure 121. 2D HMBC spectrum of polymastiamide G (78) in CDCI3 236 Figure 122. Comparison of the 13C NMR assignments for 78 and 61 237 Figure 123. !H NMR spectrum of polymastiamide H (79) in CDCI3 (500 MHz) 242 Figure 124. 13C and APT NMR spectra of polymastiamide H (79) in CDCI3 (125 MHz) 243 Figure 125. *H NMR spectrum of polymastiamide I (80) in CDCI3 (500 MHz) 245 Figure 126. 13C NMR spectrum of polymastiamide I (80) in CDCI3 (125 MHz) 246 Figure 127. *H NMR spectrum of polymastiamide J (81) in CDCI3 (500 MHz) 248 Figure 128. 13C and APT NMR spectra of polymastiamide J (81) in CDCI3 (125 MHz) 249 Figure 129. lH NMR spectrum of polymastiamide K (82) in CDCI3 (500 MHz) 252 Figure 130. 13C and APT NMR spectra of polymastiamide (82) in CDCI3 (125 MHz) 253 Figure 131. 2D HMQC spectrum of polymastiamide K (82) in CDCI3 254 Figure 132. 2D COSY spectrum of polymastiamide K (82) in CDCI3 255 Figure 133. 2D HMBC spectrum of polymastiamide K (82) in CDCI3 256 Figure 134. Selected HMBC correlations in polymastiamide K (82) 259 Figure 135. Pseudaxinella massa sponge 277 Figure 136. *H NMR spectrum of pseudaxinellin (87) recorded in CDCI3 (500 MHz) 280 Figure 137. 13C and APT NMR spectra of pseudaxinellin (87) (125 MHz, CDCI3) •• 281 Figure 138. NH region of HOHAHA spectrum of pseudaxinellin (87) in CDCI3 (500 MHz) 282 Figure 139. Selected regions of 2D HMBC spectrum of pseudaxinellin (87) (CDCI3) 289 Figure 140a. Connectivities in pseudaxinellin (87) based on HMBC results 288 Figure 140b. Connectivities in pseudaxinellin (87) based on HMBC results 290 Figure 141. Connectivities in pseudaxinellin (87) based on NOE and ROESY results 290 Figure 142. NOE results for pseudaxinellin (87) (400 MHz, CDCI3) 291 Figure 143. Conformational comparison of pseudaxinellin (87) with evolidine (110) 294 Figure 144. Carbonyl region of 13C NMR spectrum of pseudaxinellin (87) (125 MHz, DMSO-afe) 296 XIV Figure 145. lH NMR spectrum of geodiamolide G (111) in CDCI3 (500 MHz) 301 Figure 146. 2D HMQC spectrum of geodiamolide G (111) in CDCI3 302 Figure 147. 2D COSY spectrum of geodiamolide G (111) in CDCI3 303 Figure 148. 2D HMBC spectrum of geodiamolide G (111) in CDCI3 304 Figure 149. Selected HMBC correlations in geodiamolide G (111) 306 Figure Al. General pulse sequence for a one-dimensional NMR experiment 310 Figure A2. Pulse sequence for acquisition of a *H spectrum 311 Figure A3. Pulse sequence for acquisition of the NOE difference spectrum 311 Figure A4. Pulse sequence for acquisition of a 13C spectrum 312 Figure A5. Pulse sequence for the APT experiment 312 Figure A6. Pulse sequence for a two-dimensional NMR experiment 313 Figure A7. Pulse sequence for the COSY experiment 313 Figure A8. Pulse sequence for the COSYLR experiment 314 Figure A9. Pulse sequence for the HOHAHA experiment 315 Figure A10. Pulse sequence for the ROESY experiment 315 Figure Al l . Pulse sequence for the HMQC experiment 316 Figure A12. Pulse sequence for the HMBC experiment 317 LIST OF SCHEMES xv Scheme 1. The position of Xestospongia ingens within the phylogenetic classification of the Porifera according to van Soest 11 Scheme 2. to-3-alkyltetrahydropridinium 15, a plausible biogenetic precursor for petrosin (13) and araguspongine (14) 14 Scheme 3. Proposed biogenesis for saraine-1 (16) 15 Scheme 4. Biosynthetic proposal for manzamine B 17 Scheme 5. Isolation of alkaloids from Xestospongia ingens 22 Scheme 6. Proposed biogenesis of ingenamine (27) 39 Scheme 7. Fragmentation of ingamines A (31) and B (32) in EIMS 64 Scheme 8. Biogenetic relationship between ingamine A (31) and ingenamine E (37) 111 Scheme 9. Proposed biosynthesis of madangamine A (30) 141 Scheme 10. The position of Polymastia boletiformis within the phylogenetic classification of the Porifera according to W.C. Austin 176 Scheme 11. Proposed mechanism for the double bond migration 238 Scheme 12. Transformation of the compound 77 to 78 260 Scheme 13. Mechanism of C-ring aromatization proposed by Whaley et al 261 Scheme 14. Biosynthesis for steroid side chain expansion 263 Scheme 15. The position of Pseudaxinella massa within the phylogenetic classification of the Porifera according to Bergquest 278 LIST OF TABLES xvi Table 1. !H and 13C NMR data for ingenamine (27) recorded in MeOH-^4 30 Table 2. *H and 13C NMR data for non protonated ingenamine (27) recorded inMeOH-^4 42 Table 3. 2H and 13C NMR data for ingamine A (31) recorded in CDCI3 51 Table 4. XH and 13C NMR data for ingamine B (32) recorded in CDCI3 61 Table 5. 2H and 13C NMR data for ingenamine B (33) recorded in MeOH-d4 70 Table 6. *H and 13C NMR data for ingenamine C acetate (35) recorded in Table 7. Table 8. Table 9. Table 10. Table 11. Table 12. MeOH-^4 *H and 13C NMR data for ingenamine D (36) recorded in MeOH-^4 2H and 13C NMR data for ingenamine E (37) recorded in MeOH-^4 *H and 13C NMR data for ingenamine F (38) recorded in MeOH-^4 *H and 13C NMR data for ingenamine G (39) recorded in MeOH-a^ —• lH and 13C NMR data for keramaphidin B (28) recorded in MeOH-^4 •• JH NMR data for Mosher esters MTPA-ingenamine (45a/45b), MTPA-ingamine A (46a/46b), and MTPA-ingenamine E (47a/47b) 77 84 91 96 103 108 118 Table 13. lH and 13C NMR data for madangamine A (30) recorded in CeD6 130 Table 14. !H and 13C NMR data for madangamine B (48) recorded in C6D6 149 Table 15. !H and 13C NMR data for madangamine C (49) recorded in C6D6 157 Table 16. !H and 13C NMR data for polymastiamide A (65) in DMSO-^6 188 Table 17. *H and 13C NMR data for polymastiamide A methyl ester (66) in CDCI3 198 Table 18. *H and 13C NMR data for polymastiamide B methyl ester (69) in CDCI3 209 Table 19. *H and 13C NMR data for polymastiamide C methyl ester (71) in CDCI3 214 Table 20. JH and 13C NMR data for polymastiamide D methyl ester (73) in CDCI3 220 Table 21. lH and 13C NMR data for polymastiamide F methyl ester (77) in CDCI3 229 Table 22. !H and 13C NMR data for polymastiamide G (78) in CDCI3 239 Table 23. !H and 13C NMR data for polymastiamide H (79) in CDCI3 244 Table 24. lH and 13C NMR data for polymastiamide I (80) in CDCI3 247 xvii Table 25. !H and 13C NMR data for polymastiamide J (81) in CDCI3 250 Table 26. lH and 13C NMR data for polymastiamide K (82) in CDCI3 257 Table 27. !H and 13C NMR data for pseudaxinellin (87) recorded in CDCI3 284 Table 28. HOHAHA assignments for pseudaxinellin (87) recorded in CDCI3 285 Table 29. lH and 13C NMR data for pseudaxinellin (87) recorded in DMSO-^6 "" 297 Table 30. *H and 13C NMR data for geodiamolides G (111) and A (89) in CDCI3 305 LIST OF ABBREVIATIONS [a]D Ac AC2O APT aq. ax B.B.Dec. bs Bu C °C C6D6 CDCI3 cm-1 COSY d ID 2D Da 5 A5 dd ddd AM DMAP DMSO-^6 - specific rotation - acetyl - acetic anhydride - Attached Proton Test - aqueous - axial - Broad Band Decoupling - broad singlet - butyl - concentration (g/lOOmL) - degree Celsius - benzene-^ - chloroform-d - wave number - Correlation Spectroscopy - doublet - one-dimensional - two-dimensional - Daltons - chemical shift - difference in chemical shifts - doublet of doublets - doublet of doublets of doublets - difference in mass - 4-(dimethylamino)pyridine - dimethyl sulphoxide-rfg XIX dt - doublet of triplets ED50 - Effective dose resulting in 50% response eq - equatorial Et - ethyl Et20 - diethyl ether EtOAc - ethyl acetate FDAA - 5-fluoro-2,4-dinitrophenyl-L-alanineamide FTIR - Fourier Transform InfraRed g - gram GC - Gas Chromatography GC-MS - Gas Chromatography-Gas Chromatography h - hour Hex -hexane HRCIMS - High Resolution Chemical Ionization Mass Spectrometry HREIMS - High Resolution Electron Impact Mass Spectrometry HRFABMS - High Resolution Fast Atom Bombardment Mass Spectrometry HIV - Human Immunodeficiency Virus HMBC - Heteronuclear Multiple Bond Correlation HMQC - Heteronuclear Multiple Quantum Correlation HOHAHA - HOmonuclear HArtman-HAhn experiment HPLC - High Performance Liquid Chromatography Hz - hertz i - signal due to an impurity IC50 - Inhibitory Concentration resulting in 50% response / - scalar coupling constant X - wavelength L - liter L-1210 - murine leukemia cell line L-1210 XX LD LH-20 LRCIMS LREIMS LRFABMS m M+ m.p. m/z Me MeOH-^4 mg MHz MIC min. mL mmol mmu MTPA H nm NMR NOE org. P388 Ph ppm Pr - Lethal Dose - Sephadex LH-20 - Low Resolution Chemical Ionization Mass Spectrometry - Low Resolution Electron Impact Mass Spectrometry - Low Resolution Fast Atom Bombardment Gas Chromatography - multiplet - parent ion - melting point - mass to charge ratio - methyl - methanol-*^ - milligram - megahertz - Minimum Inhibitory Concentration - minutes - milliliter - millimole - millimass units - a-methoxy-a-(trifluoromethyl)phenylacetyl - micro (10"6) - nanometer - Nuclear Magnetic Resonance - nuclear Overhauser effect - organic - murine leukemia cell line P388 - phenyl - parts per million - propyl XXI prep. q R.f. r.m. rel. int ROESY S s SCUBA sp. t TFA TLC UV W - preparative - quartet - ratio to front - room temperature - relative intensity - Rotating-frame Overhauser Enhancement SpectroscopY - signal due to solvent - singlet - Self-Contained Underwater Breathing Apparatus - species - triplet - trifluroacetic acid - Thin Layer Chromatography - Ultra Violet - signal due to water xxii ACKNOWLEDGMENTS Firstly, I would like to express my gratitude to my research supervisor, Professor Raymond J. Andersen for his constant encouragement and detailed guidance throughout the course of my Ph.D. research. It has been a great pleasure to work with and learn from him. I would like to extend my thanks to Michael LeBlanc and my colleagues for assistance in the collection of sponge samples used in this work as well as for their cooperation and friendship. I am specifically indebted to Drs. David Williams, Shichang Miao, David Burgoyne, and Jana Pike for their invaluable helps and suggestions during my studies. The helpful proof-reading provided by my colleagues: Bruno Cinel, John Coleman, Jeffery Gerard, Edmund Graziani, Julie Kubanek, and Dr. Richard Lloyd in the preparation of this thesis is highly appreciated. The technical assistance provided by the staffs of the Chemistry Department NMR and mass spectrometry laboratories is thankfully acknowledged. Killam Predoctoral Fellowship, T. K. Lee Scholarship, and other financial support provided by the University of British Columbia are gratefully acknowledged. Part I General Introduction 1.1. Sponges Sponges, which constitute the phylum Porifera, are the oldest and most primitive metazoans. It has been estimated that there are over fifteen thousand species of sponges, the majority of which (ca. 99 %) are marine animals.1 They abound in all seas ranging from the shallow intertidal zone to the deep abyssal plain, wherever rocks, shells, or coral provide a suitable substratum. These multicellular animals lack any true tissues or organs and nervous system, and the cells display a considerable degree of independence. All members of the phylum are sessile and exhibit little detectable movement. As a result, their animal nature was not recognized until 1765.2 Sponges vary greatly in size, from the size of a grain of rice to massive structures. Some exceed a meter in height and diameter. Some sponges are radially symmetrical, but the majority are irregular and exhibit massive, erect, encrusting, or branching growth patterns. Most of the common species are brightly colored; green, yellow, orange, red, and purple sponges are frequently encountered. Sponges are divided into four classes according to their skeletal elements.3 The ones with calcium carbonate spicules belong to the class Calcarea. Sponges with hexagonal siliceous spicules belong to the class Hexactinellida. The Hexactinellida are mostly deep water sponges that are commonly known as glass sponges. More than 90 % of all sponge species belong to the class Demospongiae. All of the sponge species dealt with in this thesis belong to this large class. Demospongiae are leuconoid (Figure 1), and the majority are irregular, but all type of growth patterns are displayed. The skeletal composition of Demospongiae is variable. It may consist of siliceous spicules or spongin fibers or a combination of both. When both spongin fibers and spicules are present, the spicules are usually connected to or completely embedded in the spongin fibers. This class is represented in every aquatic environment including fresh water habitats. The fourth class of sponges, Sclerospongiae, has not only siliceous spicules and protein spongin fibers but also a solid basal skeleton of calcium carbonate. Sponges are filter-feeding animals and they obtain nutrition by pumping a large volume of water through their bodies. They feed on extremely fine particulate material such as organic detritus and planktonic organisms. The basic structure of a sponge, a collection of cells constructed around a system of canals and chambers (Figure l) ,2 is well designed for this purpose. Water enters at low pressure through dermal pores and flows into branching incurrent channels, which eventually open into flagellated chambers. Water leaves the chambers through excurrent channels, which become progressively larger as they are joined by other excurrent channels, and then outward to a large opening, the osculum. The water current supplies the animal with oxygen and food particles and takes away waste. Even sperm and eggs are moved in and out by the water currents. The rate of flow can be controlled by shifting the position of a central cell that the flagellated chambers of many leuconoid sponges contain. The volume of water pumped by a sponge is remarkable. A specimen of a fresh water species is capable of pumping up to ten times its body volume of water per hour.4 Osculum Excurrent canal Flagellated chamber Incurrent canal Figure 1. Typical leuconoid plan of a sponge of the class Demospongaie.2 Sponges reproduce both sexually and asexually.2 Asexual reproduction by the formation of buds that are liberated from the parent is not common. Most sponges are hermaphroditic, but cross fertilization is ensured by the nonsynchronous maturation of eggs and sperm in a single individual. Sperm leave one sponge and enter another in the currents flowing through the water canals. Eggs in the mesophyl are fertilized in situ. They may then be released by way of the water canals or brooded up to the larval stage. In most sponges the flagellated larva is a blastula, and reorganization equivalent to gastrulation occurs following settling. 1.2. Sponge Chemistry Although sponges are sedentary they have few predators. Many species produce toxic or distinctive odorous metabolites that may prevent other organisms from settling on their surfaces or may deter some potential grazing animals. In light of their apparent need for chemical defences, it is not surprising that sponges have proven to be an extremely rich source of novel bioactive metabolites. According to Ireland et al's survey, sponges have become the dominant source of newly reported marine natural products since 1986.5a Indeed, over 40 per cent of the total number (over 7,500) of known marine natural products have been isolated from sponges.5b'c Structural Novelty and Diversity Sponges contain the widest range of secondary metabolite types of any marine phylum.6 They synthesize a broad diversity of chemical structures that include terpenoids, polycyclic aromatics, peptides, steroids, alkaloids, etc. For instance, sponges have produced the most varied and biogenetically unprecedented array of sterols. Most of the 200 new monohydroxysterols found in marine organisms have been isolated from sponges.7 In addition to the monohydroxylated sterols, about 70 polyoxygenated sterols have been isolated from sponges in the last few years.8 It seems that every position in the sterol skeleton can potentially bear an oxygen functionality. An example is contignasterol (1), a highly oxygenated steroid with the 'unnatural' 14p-proton configuration and a cyclic hemiacetal functionality in its side chain. It was isolated from the Papua New Guinea sponge Petrosia contignata and it represented the first naturally occurring steroid with the 14(3-proton configuration (l).9 Because the metabolism of marine sponges is different from terrestrial organisms, sponges produce many unprecedented structural types. One example is manzamine A (2), a member of a very novel family of alkaloids isolated from the species of Haliclona, Xestospongia, Pellina, and P achy pellina}® It is a very unusual fi-carboline with a complex system of 5, 6, 8, and 13-membered rings. When it was discovered, the authors stated that "the provenance was biogenetically problematic". The biosynthetic origin of the unique ring system was proposed several years later.11 Medical Potential From the beginning, the field of marine natural products chemistry has focused on discovering bioactive metabolites. In the 1950's, Bergmann isolated the nucleosides spongothymidine (3), spongosine (4), and spongourine (5) from the Caribbean sponge Cryptotethya crypta}^ This discovery stimulated the synthesis of a whole series of ara-nucleosides such as ara-A (6) and ara-C (7), some of the first anti-viral agents. Ara-A (6), which was later isolated from the Mediterranean gorgonian Euniclla cavollini^, has been in use since the later 1970's as a therapeutic drug against Herpes encephalitis. HO. OH 3 R = CH3 5 R = H HO OH OH OCH, HO OH OH Since the development of effective antibiotics fifty years ago, cancer has emerged as a leading cause of death in industrialized countries. Research in the field of marine natural products has already provided some promising leads to new cancer chemotherapies. Recent statistical data from the National Cancer Institute shown in Figure 2 reveal an exciting trend for sponge researchers. There is a much higher percentage of leads emerging from screening marine animals than from screening terrestrial plants or microorganisms. Of the 6,500 marine animals screened to date, about 2 % show significant cytotoxic activity, whereas the testing of over 18,000 terrestrial plants and over 8,000 microorganisms has resulted in a hit rate of < 1 % in each case. When the data is analyzed by phylum, as shown in Figure 3, it is apparent that sponges are definitely rich sources for generating anticancer leads.14 Terrestrial Anlmals^B Terrestrial Plants H Marine Animals H Marine Plants H Microorganisms H 0 ^ ^ Anticancer Leaas witn signmcam selective ^ H 434 Cytotoxic Activity in NCI Preclinical ^ V Antitumour Drug Discovery Screen aaaafjM Absolute number screened in italics ^ ^ H 18293 • | H I | m i H | H H H m ^ H H | H | j ^ H | ^ f l ssto 1 7872 •^fls246 1 2 Percentage Figure 2. Anticancer leads with significant selective cytotoxic activity against a panel of 60 solid tumor cell lines. 1. • y f % cytolox 4-0 • -39 i Z < 15 i Z Z < 56 H 1 • 1 1 >-5 83 Absolute number screened 263 Wm 395 H H H H H i62 SS E | 40 io S9 n CIIO CNl CRU CYA F.CI1 MOL Phylum 1041 K B 100 H a. a. 179 O 2 Figure 3. Percentage of specimens per phyla with average in vitro IC50 < 4 |ig/mL. 7 Tools for Biology Research15 Many marine natural products exhibit highly selective receptor binding and enzyme inhibition activities. Potent cytotoxins from sponges may serve as molecular probes for active sites in enzymes and they may also be used to prepare affinity columns to isolate the enzymes. Information on active site interactions between toxins and enzymes will provide useful knowledge for rational drug design. Cytotoxins may also help identify important steps in cellular signal transduction. A number of sponge metabolites are already marketed as research tools for investigating signal transduction. For example, okadaic acid (8), a cytotoxic polyether isolated originally from the two sponges Halichondria okadai and Halichondria melanodocia,16 is: i) a potent inhibitor of protein phosphatases 1 and 2A, ii) a potent tumor promotor, and iii) it effects smooth muscle contraction. Manoalide (9), isolated from the sponge Luffariella variabilis, inhibits phospholipases A2 and C and has anti-inflammatory activity.17 i i Chemical Taxonomy 8 Traditionally the taxonomy of marine sponges has been based on their morphology. This can be problematic because the skeletal characters of sponges are frequently inconsistent. Many species of sponge grow in irregular shapes that encrust the substratum. Pigmentation, which can be highly variable, is not necessarily an aid in identification. Sponges which grow in direct light may develop deeper pigmentation as protection against ultraviolet radiation than those growing in the shade of rocks. The simplicity of these multicellular animals provides few morphological features for taxonomists to classify them. Marine natural products chemistry has helped to solve some of the problems faced by sponge taxonomists. The rich secondary metabolism exihibited by sponges can provide new information useful in classification. Bergmann played a pioneering role in this research field when he first attempted to classify marine invertebrates, particularly marine sponges, according to the sterols extracted from them.18a He pointed out the distinct differences between members of the Hymeniacidonidae, which predominantly contain saturated sterols, and Halichondridae, which contain unsaturated sterols. Bergquist has provided good evidence that species of the order Verongida predictably synthesize brominated amino acid derivatives18b. Recently, van Soest redefined the order Haplosclerida based on the chemical constituents of the sponges. He found that 3-alkylpiperidine derivatives were limited to marine sponges belonging to the order Haplosclerida.19 Part II Ingenamines and Madangamines. Novel alkaloids from the PNG sponge Xestospongia ingens 2.1. Introduction Specimens of Xestospongia ingens (Thiele, 1899) (Figure 4) were collected as part of a general collecting expedition to Papua New Guinea. The crude extracts of sample PNG-8-24-11-38, later identified as X. ingens,^ were found to be cytotoxic against murine leukemia P388 in preliminary bioassay screening. The isolation and structure elucidation of the secondary metabolites obtained from the X. ingens crude extracts are described in the following chapter. 2.1.1 Taxonomy and Description of Xestospongia ingens Sponges of the genus Xestospongia belong to the family Petrosiidae in the order Haplosclerida and class Demospongiae according to van Soest (Scheme l) .1 9 Most Xestospongia species have a similar morphology with large and thick spicules. However, Xestospongia species with small spicules are also known.20 There are approximately 30 species of Xestospongia found throughout out the world in both tropical and cold water habitats.21 The specimens of X. ingens examined in this study were collected on the tropical reefs near Sek Point off Madang, Papua New Guinea.22 The sponge was obtained by hand using SCUBA at depths of 15 - 20 meters of water in areas where there was little or no surge. The dark brown sponge had a relatively soft texture with fairly small spicules (Figure 4). It was identified by Dr. R. van Soest and a voucher sample has been deposited at the Zoologisch Museum, University of Amsterdam.20 10 WO o e >^ e a 3 6X1 11 Scheme 1. Phylogenetic classfication of Xestospongia ingens. 19 Subkingdom Metazoa (multi-cellular animals) Phylum Porifera (sponges) Class Calcarea Demospongiae Hexactinellida Sclerospongiae subclass Homoscleromorpha Ceractinomorpha Tetractinomorpha Order Halichondrida ^^Poecilosclerida Dendroceratida Haplosclerida Verongida Family Petrosiidae Genus Haliclona Xestospongia Petrosia Species X. ingens 12 2.1.2. A Review of 3-Alkylpiperidine Alkaloids Isolated from Marine Sponges Marine sponges have been an excellent source of novel nitrogenous secondary metabolites.6 Recently, a number of alkaloids containing a six-membered nitrogen heterocycle, which include monomers and oligomers of 3-alkylpyridine, macrocyclic dimers and more complex multicycles derived from the 3-alkylpiperidine or 3-alkyltetrahydropyridine structures, have been isolated from marine sponges. A recent review has adopted the name '3-alkylpiperidine' alkaloids for this group of biogenetically related metabolites.19 A brief review of this family of compounds is presented below. The monomeric structural unit of the '3-alkylpiperidine alkaloids' is readily apparent in a number of simple 3-alkylpyridines obtained from marine sponges. The alkyl component of the common structural motif is often a linear chain, either saturated or unsaturated, ranging in length from eight to sixteen carbons. In all known examples, the 3-alkyl component of the monomeric unit is attached to a primary or methyl amine,23-24 methoxy amine,25"28 oxime methyl ether,26"27 or imine N-oxide29 at the distal terminus. Niphatyne A (10), isolated from a Niphates sp. collected off Vitu Levu in the Fijian Islands, was the first reported example of a 3-alkylpyridine monomer from sponges.25 Niphatyne A had the alkyne and methoxy amine functionalities common to many of these compounds. It was found to be cytotoxic to murine leukemia P388 in vitro (ED50 0.5 fig/mL). H ^^^—^\^\x" soMe 10 The halitoxins (11), which are toxic to fish and mice, have been isolated from several sponges in the genus Amphimedon. They represented the first examples of 3-alkylpyridine 13 alkaloids reported from the sponges.30 The toxins were separated into molecular weight range fractions of 500 - 1,000, 1,000 - 25,000, and greater than 25,000 via membrane ultrafiltration, each of which showed the same spectroscopic and biological properties. Recently, a halitoxin was isolated from the sponge Callyspongia fibrosa by Faulkner's group.31 11 (n = 2, 3, 4, 5) The dimeric Ws-macrocycles belonging to the haliclamine and cyclostellettamine families were isolated by Fusetani's group in Japan.32'33 For example, the cytotoxic haliclamine A (12),33 obtained from a Haliclona sp., consists of two tetrahydropyridines linked through C9 and C12 alkyl chains. Haliclamine A (12) inhibited cell division of fertilized sea urchin eggs at concentrations of 5 |ig/mL. It also showed in vitro cytotoxicity against murine leukemia P388 (IC50 0.75 ng/mL). yJ 12 14 The petrosins,34 xestospongins,35 araguspongines,36 demethylxestospongin37 are members of a novel class of to-quinolizadine alkaloids. Petrosin (13), the first example of this structural type, was isolated from the sponge Petrosia seriata collected off Laing Island, Papua New Guinea. It was found to be toxic to fish (LD50 =10 mg/L). Araguspongine J (14), a bis-l-oxaquinolizadine alkaloid, was isolated form the sponge Xestospongia sp. collected in Okinawa, Japan.35 Both petrosin (13) and araguspongine J (14) were assumed to arise from the same precursor (15), a to-3-alkyltetrahydropyridinium macrocycle (Scheme 2).38 Intramolecular attack of the C-terminus of the enol on the iminium carbon of 15 generates 13 whereas O-terminus attack followed by reduction would lead to araguspongine J (14). Scheme 2. to-3-alkyltetrahydropyridinium 15, a plausible biogeneic precursor for petrosin (13) and araguspongine J (14). 38 O- attack OH 13 15 14 The sponge Reniera sarai collected in the Bay of Naples, Italy, yielded the saraines38 and isosaraines39. The work on the R. sarai alkaloids was initiated in the 1970's, however, the complexity of their lH NMR spectra coupled with the unavailability of suitable crystals delayed their structure elucidation. The partially solved structure of saraine-1 (16) was first reported383 in 1986 when 2D NMR experiments conducted on high field NMR spectrometers became routinely 15 available. It seems likely that biogenetic precursor to compound 16 is a to-3-alkylpiperidine macrocycle as shown. Scheme 3. Proposed biogenesis for saraine-1 (16) 38 16 HO ^> Halicyclamine A (17), a tetracycle with a transannular conjoint linkage between two piperidine rings, was isolated from a Haliclona sp. collected in Biak, Indonesia.40 It is apparent that the biogenetic precursor to this compound 17 is also a 6/.s-3-alkylpiperidine macrocycle. 17 Saraine A (18),41 originally named sarain A, was isolated from the same Bay of Naples Reniera sarai sample that was the source of the saraines 1-3 and isosaraines 1-3. The spectroscopic data obtained for saraine A (18) was very complicated and it contained a number of 16 pieces of conflicting information that prevented a structure elucidation. Fortunately, a diacetylated derivative 19 of saraine A gave crystals that were suitable for single crystal X-ray diffraction analysis. Based on the X-ray structure, the conflicting spectroscopic data (i.e. a strong IR band at 1660 cm"1 but no 13C NMR signal at the carbonyl region in the compound 18) could be rationalized by arguing that there was a strong 'proximity' effect42 between the tertiary amine at Nl and an aldehyde functionality at C2 (13C NMR: 5 98.0 in CDCI3/CD3CO2D) in saraine A (18).41b Manzamines A to H are a group of structurally unique alkaloids isolated from sponges of species of Haliclona, Xestospongia, Pellina, and Pachypellina}® The structure of manzamine B (20), a {3-carboline alkaloid, was determined by single crystal X-ray diffration analysis.10b At the time of discovery, the authors stated that the provenance of manzamine B (20) was biologically problematic.10b A few years later Baldwin and Whitehead put forth an elegant proposal suggesting that the manzamines also arise biogenetically from a bis-3-alkyldihydropyridine precursor (Scheme 2).11 Deoxygenation followed by removal of a tryptophan unit from 20 would lead to a tetracyclic intermediate 21. This intermediate 21 would form an iminium salt 22 by an intramolecular condensation between the aldehyde group and the secondary amine. Redox exchange between the two piperidine rings of 22 would give a pentacyclic intermediate 23, which is immediately revealed as the Diels-Alder adduct of &/.y-3-alkyldihydropyridine 24. This proposal not only explained the biogenetic origin of manzamine B (20), but also suggested the possible occurrence of two new classes of alkaloids, namely, the tetracyclic and pentacyclic intermediates (21 and 23). 17 Scheme 4. Baldwin and Whitehead's Biogenetic Proposal for Manzamine B (20) 11 CHO 20 ^> Tetracyclic Intermediate 21 <Jr Pentacyclic Intermediate ^ ^ ^ 23 Redox Exchange 22 [4 + 2] V ^ ^ 24 18 Shortly thereafter, the predicted existence of the tetracyclic and pentacyclic intermediates were confirmed by the discovery of the tetracyclic alkaloids, ircinals,43 and the pentacyclic alkaloids, ingenamines.45 Ircinal B (25),43 isolated from the Okinawan marine sponge of the genus Ircinia sp., had the overall skeleton and aldehyde functionality present in Baldwin and Whitehead's tetracyclic intermediate. The absolute stereochemistry of the compound 25 was shown by chemical correlation to be the same as that of manzamine B (20). Interestingly, the tetracyclic alkaloid ircinol B (26),44 the alcoholic form of ircinal (25), isolated from the same sponge Amphimedon sp., showed the opposite absolute configuration to that of ircinal (25). It is very rare that both enantiomeric forms have been simultaneously isolated from the same organism. The absolute configuration of ircinol (26) was based on the comparison of the specific rotation sign to that of the reduction product of ircinal B (25). CHO CH2OH OH OH X_/ 25 26 Ingenamine (27),45 isolated from the Papua New Guinea sponge Xestospongia ingens, was the first reported example of the pentacyclic alkaloids predicted by Baldwin and Whitehead's proposed biogenetic pathway to the manzamines. The structure of ingenamine (27) was elucidated by interpretation of the spectroscopic data, particularly, the NMR data. The absolute stereochemistry of 27 was determined employing Mosher's ester methodology.52 The configuration of ingenamine (27) was found to be antipodal to the configurations of manzamines A (2) and B (20). Ingenamine (27) showed in vitro cytotoxicity against murine leukemia P388 (ED50 = 1 p.g/mL). Subsequently, keramaphidin B (28) was isolated from an Amphimedon sp. 19 collected from the Kerma Islands, Okinawa, Japan.46 Its structure was solved by X-ray diffraction analysis. Keramaphidin B (28), which differs from ingenamine (27) only by the absence of the hydroxyl group, was reported to be a naturally occurring racemate. Subsequently, a revised structure of xestocyclamine A (29) was reported.4715 Xestocyclamine A was originally reported to have a skeleton derived from an intermolecular [4 + 2] cycloaddition reaction between two monomelic 3-alkyldihydropyridine macrocycles.47a The revised structure for xestocyclamine A (29) had an ingenamine type skeleton and it differed from ingenamine (27) only in the placement of the double bonds.47b OH 27 28 29 Madangamine A (30), the first example of a new type of pentacyclic alkaloid, was also isolated from the Papua New Guinea sponge Xestospongia ingensA^ The structure of 30 was determined by detailed analysis of ID and 2D lH and 13C NMR data. The biogenetic origin of 30 could be traced back to a £>/s-3-alkylhydropyridine macrocyclic precursor by analogy with the Baldwin and Whitehead's proposal for the biogenesis of the manzamines.48 2.2. Results and Discussion 2.2.1. Isolation of metabolites from Xestospongia ingens Specimens of Xestospongia ingens were collected by hand using SCUBA on reefs at depths of -15 to -20 m near Sek Point off Madang, Papua New Guinea in 1992. Freshly collected sponge was frozen on site and transported to Vancouver over dry ice. The sponge was identified by Dr. R van Soest. A voucher sample (ZMA 10701) has been deposited at the Zoologisch Museum, University of Amsterdam.20 The specimens of Xestospongia ingens were thawed and extracted exhaustively with methanol. The methanol extract was filtered and concentrated in vacuo to give a dark brown aqueous suspension which showed cytotoxic activity against murine leukemia LI210. The aqueous slurry was diluted with water and partitioned sequentially against hexanes and ethyl acetate. Purification of the hexanes extract was accomplished by repeated application of silica gel flash chromatography, preparative TLC, and normal phase HPLC (Scheme 3). Silica gel chromatography using a gradient elution (hexanes/ ethyl acetate 1:9 to 1:1) gave three fractions A, B, and C in sequence that were analyzed by TLC and *H NMR. Fractions B and C were found to be mainly ingamine B (32) and ingamine A (31), respectively. A second application of silica gel chromatography on fraction B (gradient elution: EtOAc:Hex:/-Pr2NH 50:50:1 to 100:0:1) yielded pure ingamine B (32). Normal phase HPLC of fraction A gave crude madangamine A (30) and madangamine C (49) (eluent: hexane/ ethyl acetate/ diisopropyl amine 98.4:1.5:0.1). Further independent recycling of these crude products under the same HPLC conditions yielded pure madangamine C (49), madangamine A (30), and madangamine B (48) in that order of elution, and a mixture of madangamines D (50) and E (51). The remaining fractions from the hexanes extract contained several fatty acid and steroid metabolites. 21 Separation and purification of the ethyl acetate-soluble extract was accomplished by repeated fractionation on Sephadex LH-20, silica gel flash chromatography, preparative TLC, and normal phase HPLC. Sephadex LH-20 size-exclusion chromatography (first in methanol and then ethyl acetate/ methanol/ water 40:10:4) afforded three major fractions (L, M, and N) that were analyzed by *H NMR and TLC. *H NMR spectra indicated that fraction L contained mainly ingamines A (31) and B (32) plus xestocyclamine B (41), fraction M contained a very complex mixture of ingenamine type compounds, and fraction N contained mainly ingenamine (27) and the trace metabolite ingenamine E (37). Recycling of fraction N as above (Sephadex LH-20, EtOAc/MeOH/H20 40:10:4) gave pure ingenamine (27) in a partially protonated form. Ingenamine F (38) was obtained from fraction M by Sephadex LH-20 chromatography (EtOAc/MeOH/H20 40:5:2) followed by preparative silica gel TLC (eluent: EtOAc/MeOH 75:25). Purification of fraction L on silica gel (eluent: EtOAc) and preparative normal phase TLC (eluent: EtOAc//-Pr2NH 92:8) gave pure ingamine A (31). Repeated purification of fractions M and N on normal-phase HPLC using EtOAc/hexane with a small amount of diisopropyl amine and/or methanol gave ingenamine B (33), keramaphidin B (28), 82A, ingenamine D (36), ingenamine E (37), and ingenamine (27). 82A was a mixture of two similar compounds. Repeated recrystallization of 82A from methanol yielded pure ingenamine G (39, colorless needles, m.p. 151-3 °C) and a mixture. Acetylation of this mixture in pyridine and acetic anhydride and followed by a normal phase HPLC separation (eluent: hexane/ ethyl acetate/ diisopropyl amine/ methanol 97:2.5:0.05:0.5) gave pure ingenamine C acetate (35) and ingenamine G acetate (40). Scheme 5. Isolation of ingenamine and madangamine alkaloids from Xestospongia ingens. Methanol extract 1. Concentrated 2. Diluted with water 3. Partitioned between aq. and org. solvents Hexanes fraction Ethyl acetate fraction Silica gel Hex/EtOAc(l:9tol:l) B N.P. HPLC C (Ingamine A) Silica gel column Ingamine B Madangamines A-E Silica gel column Prep. N.P. TLC Ingamine A Xestocyclamine B Sephadex LH-20 1. MeOH 2. EtOAc/MeOH/H20 (40 : 10 : 4) M N Sephadex Repeated LH-20 N.P. HPLC '^ , f Ingenamine Ingenamine Ingenamines B-E Ingenamine G Keramaphidin B Repeated N.P. HPLC 1. Sephadex LH-20 2. Prep. N.P. TLC Ingenamine F 23 2.2.2. Structure Elucidation of Ingenamine Alkaloids from Xestospongia ingens The structures of ingenamine (27), ingamines A (31) and B (32), ingenamines B (33), C (34), D (36), E (37), F (38), and G (39), keramaphidin B (28), and xestocyclamine B (41) were solved by extensive ID and 2D NMR, and mass spectrometric analyses. Proton-carbon attachments were determined by HMQC experiments and proton spin systems were based on COSY experiments. COSYLR, HOHAHA, and HMBC data also proved useful for the identification of all connectivities within the molecules. The assignments of the quaternary carbons and nitrogen atom interrupted systems were mainly dependent on HMBC results. The relative stereochemistries were determined using NOE and ROESY data, and in some cases long range COSY correlations attributed to W-couplings. The structure of ingenamine C (34) was deduced from its acetate derivative 35. The identification of the known compounds keramaphidin B (28) and xestocyclamine B (41) were confirmed by comparison of *H and 13C NMR and mass spectroscopic data with the reported values.46-4713 24 27 X = OH 28 X = H 31 X = OH 32 X = H 33 34 35 R = H R = CH 3 CO 36 39 R = H 40 R = CH 3 CO 37 X = OH 38 X = H Ingenamine (27)45 Ingenamine (27) was isolated as an optically active, white amorphous solid. The molecular formula of ingenamine (27) was determined from the parent ion in the positive FABHRMS (Figure 5). Compound 27 gave a very intense peak (M++ H) at m/z 397.32058 corresponding to a molecular formula of C26H40N2O (AM 3.30 ppm), requiring eight sites of unsaturation. Table 1 provides a summary of the NMR data acquired for 27. The *H NMR spectrum of 27 showed five olefinic protons and complex multiplets of aliphatic methylene and methine resonances between 8 0.8 and 3.5 ppm which were not well resolved, even at 500 MHz (Figure 6). All twenty-six carbons were observed in the 13C NMR spectrum (Figure 7), although two signals were quite broad. The APT experiment (Figure 7) revealed that there were two quaternary carbons, nine methine carbons, and fifteen methylene carbon atoms. The HMQC experiment (Figure 8) also demonstrated the presence of thirty-nine protons directly attached to the methine and methylene carbons. A broad band at 3398 cm"1 in the IR spectrum and NMR resonances at 8 66.1 (CHOH: C9) and 3.35 (CHOH) ppm were assigned to a secondary alcohol, which accounted for the one proton not attached to carbon. Since six deshielded carbon resonances could be assigned to three double bonds and no additional unsaturated functional groups were apparent from the 13C NMR data, it was apparent that the ingenamine (27) was pentacyclic. Isims 1267 Scan 1 (Av 7-13 Acq) 100%=99377 mv 20 Oct 93 13:11 LRP +LSIMS SL 5096 * Matrix : 3-Nitrobenzylalcohol 100 100 465 x20 500 550 600 650 700 750 800 Figure 5: Low resolution positive FAB mass spectrum of ingenamine (27) H to 27 cs ~ 1 0 \ ^ 00 ******** 3 26 17 25 18 4 mm****** fU*nt 27 *****&**m*****j**mi**Mm** 9 2 21 13 6 10 8 12 l*+**t***mm*mm*mm***mmmi**mm^ & iHHiiiJJUwfrmH 27 24 7 20 28 5 15/14 U mm ****** 23 16 19 22 «•» t^y'^rr1*'! r^ tininii't r »«r v mpm ffTTttn prrrr^i |IIMHIIIIMIIMIII|I ppa 140 120 100 00 40 20 Figure 7: ^C and APT NMR spectra of ingenamine (27) (125 MHz, MeOH-d 4) to 00 29 I- 25 I- 50 E- 75 H00 i I i i i i i i i i i i i i i i i i i i i | i i i i i i i i i i i i i i i i i i i | i i i i i i i i i i i i i pprn 4 2 -125 Eppm Figure 8: 2D HMQC spectrum of ingenamine (27) in MeOH-^ Table 1. NMR data for ingenamine (27) recorded in MeOH-a^ at 500 MHz (lH). C no. 2 3 4 5 6 6 ' 7 8 9 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 1 5 ' 16 1 6 ' 17 18 19 1 9 ' 20 2 0 ' 21 2 1 ' 22 2 2 ' 23 24 2 4 ' 25 26 27 2 7 ' 28 8 «C 63.8 143.0 125.8 34.8 53.8 45.2 51.6 66.1 52.5 50.8 55.1 26.9 27.4 23.8 133.3 130.2 21.2 41.6 56.7 20.2 26.04 26.26 132.6 133.7 26.3 37.2 2 8 ' | 8 XH 3.30, bs 6.01,d(6.4) 2.75, m 2.89,dd(9.4, 1.8) 1.76, dd(9.2, 2.4) 0.87, dd(10.5, 1.8) 3.35, td(l 1.2, 4.8) 3.00, dd(12.1,4.7) 2.92, t(12.2) 3.10,d(12.4) 2.22, bd(12.3) 3.02, td(12.7, 4.9) 2.24, m 1.53 1.31 1.62 1.48 2.41 1.62 5.70, td(10.2, 5.3) 5.65, td(10.4, 5.8) 2.40 1.82, m 1.93 1.71, m 3.49, m 2.88, dd(7.2,6.3) 1.92 1.54 1.53 2.31 2.07 5.32, bt(10.7) 5.45, bt(10.2) 2.37 2.15 2.41 2.36 a COSY Correlations H4 H2, H5, H8, H28, H28' H4, H6, H6', H8 H5, H6' H5, H6 H4, H5, H9 H8, H10, H10' H9, H10' H9, H10 H12' H12 H13',H14, H14' H13, H14, H14' H13, H13',H14\ H15, H15' H13, H13',H14, H15.H15' H14,H14',H15' H14, H14', H15, H16 H15',H16',H17 H16, H17 H16, H16',H18 H17, H19, H19' H18, H19', H20, H20' HI8, H19.H20, H20' H19, H19', H20' H19, H19',H20 H21',H22, H22' H21, H22, H22' H21,H21',H22' H21, H21',H22 H24, H24' H23, H24', H25 H23, H24, H25 H24, H24', H26 H25, H27, H27' H26, H27' H26, H27, H28 H4, H27', H28' b HMBC Correlations | c NOE H4, H6, H12', H13, H20', H28, H28' H2, H5, H28, H28' H2, H5, H6, H8, H28, H28' H4, H6, H6\ H8, H9 H2, H13, H13' H2, H5.H8.H12, H12\ H19, H20, H20' H2, H4, H6 H6\ H10, H12, H20 H8,H10,H10' H12 H2, H20, H21' H2, H6' H13 H13',H16 H15, H18 H16, H16',H19, H19' H16, H16',H19, H19',H20 H17, H18, H20, H20' H8, H12',H19, H19' H10, H10',H12' H21,H21' H25 H22, H25, H26 H24, H26, H27, H25, H27, H28, H28' H25, H26, H28, H28' H2, H4, H26, H27 H4, H28 H13, H13', H28, H28' H26, H28 H8 H4, 13' H6, H10', H20, H20' H4, H5,H12' H9 H2 H6' H8 H8 H25 H12 H23 H4, H25,H28 H2, H4, H26 a Correlated to proton resonance in 8 *H column; recorded at 400 MHz. b Correlated to carbon resonance in 8 13C column.c Resonance in 8 *H column was irradiated; recorded at 400 MHz. _LAM__L J J M ^ W O L & & 0 QS _ 3 6. 2 Figure 9: 4* P 5 G r 1 ' I ! ' ' ' I ' ' ' 5.a 4.3 3.0 PPM '" ' I 2.2 i . a 2D COSY spectrum of ingenamine (27) in MeOH-^4 'PM 27 </1 oo (N O r-~ 1 j ^ MO J-PPfl> ppm 10: Selected regions of the 2D HMBC spectrum of ingenamine (27) in MeOH-ck A C8H10O fragment, that included C2 to C6 and C8 to CIO and contained the secondary alcohol and a trisubstituted alkene functionalities, was routinely identified from the COSY, HMQC, and HMBC data (Table 1). COSY correlations were observed (Figure 9) between H4 (8 6.01) and H5 (8 2.75) and H2 (8 3.30, allylic coupling); between H5 (8 2.75) and H6 (8 2.89), H6' (8 1.76), and H8 (8 0.87); between H8 and H9 (8 3.35); and between H9 and both H10 (8 3.00) and H10' (8 2.92) for this contiguous spin system. HMBC correlations were observed between H4 (8 6.01) and C2 (8 63.8) and C5 (8 34.8); between H5 (8 2.75) and C3 (8 143.0) and C4 (8 125.8); between H6 (8 2.89) and C4 (8 125.8) and C5 (8 34.8); between H6' (8 1.76) and C8 (8 51.6); between H8 (8 0.87) and C5 (8 34.8); between H9 (8 3.35) and C5 (8 34.8); and between H10 (8 3.00) and C8 (8 51.6) (Figure 10). 1.76 Figure 11. Substructure of fragment CsHioO and selected HMBC correlations. The aliphatic quaternary carbon at 8 45.2 (C7), that was indicated by the APT experiment (Figure 7), could be positioned between C2 and C8 on this fragment by a series of HMBC correlations. Thus, H8 (8 0.87) and H2 (8 3.30) both showed two bond HMBC correlations to C7 (8 45.2) (Figure 10). In addition, H2 (8 3.30) was correlated to C8 (8 51.6) via a three bond coupling. A network of HMBC correlations identified C12 and C20 as the remaining two substituents flanking the quaternary carbon. A pair of geminal methylene protons at 8 3.10 (H12) and 2.22 (H12') that were attached to a carbon at 8 50.8 (C12) showed two bond HMBC correlations to the quaternary carbon (8 45.2 : C7). In addition, H12 (8 3.10) showed a three bond correlation to C8 (8 51.6) and HI 2' (8 2.22) showed a three bond correlation to C20 (8 41.6). The protons (8 1.93 and 1.71: H20, H20') attached to the methylene carbon at 8 41.6 (C20) in turn showed two bond HMBC correlations to the quaternary carbon (8 45.2: C7). Furthermore, H20 (8 1.93) showed a three bond correlation to C12 (5 50.8), H20' (8 1.71) showed a three bond correlation to C2 (8 63.8), and H8 (8 0.87) showed a three bond correlation to 8 41.6 (C20). H H H H - — ' Figure 12. The tricyclic core structure and four appendages with selected HMBC correlations. Since the single proton in 27 not attached to carbon could be assigned to an alcohol, it was apparent that there were no hydrogen atoms directly bonded to the two nitrogen atoms in ingenamine. COSY and HMBC data provided evidence that both nitrogens were present as tertiary amines and they were attached to the carbon atoms at C2/C6/C13 and C10/C12/C21, respectively. The COSY data (Table 1) suggested that the remaining substituents on C2, C6, CIO, and C12 had to be atoms without attached protons. The chemical shifts of these carbons (8 C2, 63.8; C6, 53.8; CIO, 52.5; C12, 50.8) were consistent with attachment to nitrogen. HMBC correlations between H2 (8 3.30) and C6 (8 53.8) and between H6 (8 2.89) and C2 (8 63.8) were consistent with a nitrogen bridge between C2 and C6. Further HMBC correlations between H6' (8 1.76) and a 35 methylene carbon at 8 55.1 (CI3), and between one of the protons (8 3.02: HI3) attached to the carbon at 8 55.1 and both C2 (8 63.8) and C6 (8 53.8) identified the methylene carbon (C13) as the third substituent of the tertiary amine. Similarly, HMBC correlations between H12 (8 3.10) and CIO (8 52.5) provided evidence for a nitrogen bridge between CIO and CI2, and correlations between a methylene carbon at 8 56.7 (C21) and H10 (8 3.00), H10' (8 2.92), and H12' (8 2.22) identified this methylene carbon (C21) as the third substituent of the second tertiary amine (Nil). The location of the two tertiary amines completed the structure of the tricyclic central core of 27 together with three methylene appendages at Nl, C7, and Ni l . The fourth methylene attachment to the core structure of 27 at C3 was simply identified by analysis of long range COSY, optimized for small proton-proton coupling constants, and HMBC data. Long range COSY correlations were observed between H4 (8 6.01) and both H28 (8 2.41) and H28' (8 2.36) resonances, which were due to allylic couplings. The presence of the allylic methylene (C28: 8 37.2) substituent at C3 was also confirmed by two bond HMBC correlations from the methylene protons at 8 2.41 (H28) and 2.36 ppm (H28') to the olefinic quaternary carbon C3 at 8 143.0, and by three bond HMBC correlations to both C2 at 8 63.8 and C4 at 8 125.8. The allylic methine proton H2 (8 3.30) and olefinic methine proton H4 (8 6.01) in turn showed three bond HMBC correlations to the appendage C28 (8 37.2). The relative stereostructure of ingenamine (27) was determined by difference NOE (Figure 11) and ROESY experiments. Difference NOEs were observed between H9 (8 3.35) and H4 (8 6.01), between H8 (8 0.87) and H6 (8 2.89), and between H8 and both geminal methylene protons H20 (8 1.93) and H20' (8 1.71). It was evident that the relative configurations at C2, C5, C7, and C8 had to be those as shown below. A long-range COSY correlation observed between the H4 (8 6.01) and H8 (8 0.87) resonances, that was attributed to W coupling, was consistent with the proposed relative configurations. Furthermore, the coupling constant of J = 11.2 Hz between H9 (8 3.35) and H8 (8 0.87) supported the trans relationship between H9 and H8 proposed for 27. The unusual upfield chemical shift of H6' at 8 1.76 ppm was mainly attributed to the anisotropic effect resulting from its location in the shielding region of the double bond A3-4. 36 NOE/ROESY COSYLR H M b L Figure 13. Selected NOE/ ROESY, COSYLR, and HMBC correlations. A ROESY correlation and a strong difference NOE (Figure 14) between H9 (8 3.35) and H12' (5 2.22) demonstrated that the C7 to C12 piperidine ring was in a boat conformation with H9 and H12' occupying flagpole orientations. The strong three bond HMBC correlations, that usually requires coplanar orientations between the coupled proton and carbon, were observed from HI2' (5 2.22) to C20 (5 41.6), and from H12 (5 3.10) to C8 (5 51.6) (Figure 10). This information confirmed that the H12' was in flagpole orientation while the H12 (6 3.10) in a pseudo equatorial orientation. A long range COSY correlation between H12' (8 2.22) and H20 (8 1.93), which was ascribed to W coupling, and an NOE correlation between H8 (8 0.87) and H10' (8 2.92) (Figure 14) were also consistent with the proposed conformation of this piperidine ring. The remaining portion of 27 had to account for eight aliphatic methylene and four olefinic methine carbons. Detailed analysis of the COSY, HMQC, and HMBC data (Table 1) showed these atoms comprised two linear six carbon chains (C14 to C19 and C22 to C27), each containing an alkene. Furthermore, the NMR data revealed that the two six carbon chains linked the C21 and C28, and the C13 and C20 methylene appendages, respectively, as shown in 27. Thus, COSY correlations observed between H13 (8 3.02) and H14/H14' (8 1.53/ 1.31); between H14/H14' H8 irradiated ~*_ H12' irradiated *^^*V-»vV*wfc, *|^fcK#»*A**A-^«/». *^>n—mi^ii'-V^i^i^^n^n^ Ws^y^ lA»y#-* ^t^h"1 ^^^V^^V^M"^^***^^^ JUUA. I—i—i—I—i—i—i—r—-J—i—i—i—r—^—i—i—i—r—|—i—i—r~i—|—I—i—i—i—|—i—i—•—i—|—•—I—i—i—|—•—'—'—r~~l ' ' ' > | ' ' ' • 1 • ' r 6.0 5.S 5.0 li.S 4.0 3.5 3.0 2.5 2.0 1-5 1.0 PPM Figure 14: Selected NOE results of ingenamine (27) in MeOH-f/4 (400 MHz) and H15 (5 1.62); between H15* (8 1.48) and H16 (5 2.41); between H16' and H17 (5 5.70); between H17 and H18 (5 5.65); between H18 and H19' (5 1.82); and between H19 (8 2.40) and H20 (8 1.93) completed the assignment of the first linkage from Nl to the aliphatic quaternary carbon C7. Three bond HMBC correlations from H20 (8 1.93) to the olefinic carbon C18 (8 130.2), from H18 (8 5.65) to C16 (8 23.8), and from H17 (8 5.70) to C19 (8 21.2) further confirmed the location of the A17-18 double bond 8 2.40 H H 1- 8 2 3.49 5.65 H 5.70H 1.53 HMBC 2.41 H5.45 Figure 15. Substructures of the two side chains and selected HMBC correlations. Similarly, interpretation of COSY, COSYLR, HMQC, and HMBC data identified the second chain bridging the C21 to C28 appendages. Comparison of the NMR data for ingenamine (27) with that reported for the ircinals4^ and the haliclamines33 confirmed the Z geometry for the A17-18 and A25-26 alkenes. The coupling constant of / = 10.5 Hz observed for olefinic protons H25 (8 5.32) and H26 (8 5.45) was also in agreement with Z double bond configuration.** * Z alkenes have allylic carbons at 8 < 27, E alkenes have allylic carbons at 8 > 30. ** Zprotons on alkene have coupling contant of/ = 10-12 Hz, E protons / = 14-16 Hz. Ingenamine (27) represents a new class of cytotoxic alkaloid (in vitro murine leukemia P388: ED50 = 1 |ig/mL). Its biosynthesis can be formally envisioned to arise from an intramolecular [4+2] cycloaddition reaction as shown in Scheme 6. The discovery of ingenamine (27) provides further support for Baldwin and Whitehead's proposal for the biosynthesis of the manzamines.11 Scheme 6. Proposed Biogenesis of Ingenamine (27). X = H or OH Upon further examination of the NMR data for ingenamine (27) we found that the ingenamine (27) isolated using Sephadex LH-20 chromatography existed in a partially protonated form. The following evidence supports this argument: 1). The *H NMR spectrum of ingenamine (27) isolated from Sephadex LH-20 column recorded in DMSO-^6 showed a small downfield peak at 8 10.4 ppm that was typical of an ammonium proton. The fact that this peak integrated to less than one proton (40 %) suggested that the molecule was only partially protonated. 2). The ingenamine (27) isolated using HPLC with 0.2 % diisopropyl amine in eluent displayed a quite different *H NMR spectrum (Figure 16). Qualitative titration of this non-protonated ingenamine with dilute triflouroacetic acid afforded the same lH NMR spectrum as that of partially protonated ingenamine (Figure 2). A few more drops of acid collapsed the proton NMR spectrum of 27 (peak broadening). 40 N «4 © ni ID OJ o m ID 's> o "•» X £ 8 i n 5 X o <o s c •c^ .-—-v r-CJ-4) C 6 «» c u 00 .s u <u * -a o ca «4-l o E 3 fa O a. W3 « s z X a> 3 SX1 *<M**toM4paMMttWIU|JM«*WlN*y«M#^^ *»«*<«M«*MM«*l#)*rtHMOT^ lJ*aMMMNtoniMMii*«M' MwJJ tthm**km tm < i •' 120 » i » 60 40 20 ppa 140 100 80 Figure 17: 13C and APT NMR spectra of acid-free ingenamine (27) (125 MHz, MeOH-^t) 42 Table 2. NMR data for non-protonated ingenamine (27) recorded in MeOH-ckj. at 500 MHz (lH). c # 2 3 4 5 6 6 ' 7 8 9 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 1 5 ' 16 1 6 ' 17 18 19 1 9 ' 20 2 0 ' 21 2 1 ' 22 2 2 ' 23 2 3 ' 24 2 4 ' 25 26 27 2 7 ' 28 2 8 ' 6 13C 65.2 143.8 124.0 35.2 54.3 46.9 53.1 69.3 54.7 51.5 55.2 27.1 27.7 23.80 132.4 131.4 21.6 42.9 56.9 22.1 25.67 26.0 132.6 133.1 26.5 37.9 5 XH 3.14,d(1.3) 5.85 d(6.5) 2.64, m 2.84, dd(9.2, 1.9) 1.71, dd(9.2, 2.9) 0.69 dddO.l, 2.1) 3.27, ddd(l 1.8, 10.1,4.8) 2.61, dd(12.0,4.8) 2.46, t(12.0) 2.26 d(10.8) 1.98,d(10.8) 2.97, td(12.6, 5.2) 2.21, m 1.48 1.27, m 1.58 1.50 2.41 1.54 5.63 5.63 2.34 1.73 1.82 1.71 3.04, ddd(14.1, 8.2, 6.1) 2.19 1.64 1.35 1.48 1.34 2.18 1.95 5.22, tt(10.8, 2.9) 5.36, m 2.31 2.08 2.35 2.28 aCOSY Correlation H4 H2, H5 H4, H6, H6\ H8 H5, H6' H5, H6 H5, H9 H8, H10, H10' H9.H10' H9.H10 H12' H12 H13',H14, H14' H13, H14 H13,H13',H14' H13, H14, H15 H14',H15' H15 H16',H17 H16, H17 H16, H16' H19, H19' H18, H19', H20, H20' H18, H19 H19, H20' H19, H20 H21',H22, H22' H21,H22, H22' H21,H21',H22*, H23w H21,H21',H22 H22w, H23', H24, H24' H23, H24, H24' H23, H23', H24', H25 H23, H23', H24 H24, H26 H25, H27 H26, H27' H27 H28' H28 bHMBC Correlation H4, H6, H13, H12',H20,H28, H28' H2, H4, H5, H28, H28' H2, H5, H6, H28, H28' H4, H6, H6' H2.H13, H13' H2, H5, H8, H12, H12', H20, H20' H2, H6, H10, H10' H12, H20' H8, H10, H10' H8, H12 H10, H10',H20', H21.H21' H6* H13 H13',H16' H15',H17, H18 H16, H19 H16, H19, H19' H17, H18, H20, H20' H8, H12, H12' H10, H10',H12',H22 H21 H21,H22 H22, H26 H26 H24, H25, H27 H25, H28, H28' H2, H4 Correlated to proton resonance in 5 *H column. *> Correlated to carbon resonance in 8 13C column, w = weak. 43 3). Comparison of *H NMR data of the non-protonated ingenamine (27) with that of the protonated form revealed that the chemical shift values of H10/H10', H12/H12', and H21/H21' were quite different for the two forms of ingenamine (27) while those of H2, H6/H6', and H13/H13' remained virtually the same. This result suggested that Nl 1 is more basic and thus the most easily protonated site in the molecule 27.49 0.03 H ^ ^ N j 0.05 11 H0.45 0.39 N- ~J^ H^ / U H 0 . 4 6 P ^ H 0.69 AS = 8pro - 5 n o n (ppm) values for the two forms of ingenamine (27). pro = protonated; non = non-protonated Figure 18. ! H chemical shift differences between the two forms of ingenamine (27). 4). The 13C NMR data (Figure 17) also provided evidence consistent with that from the lH NMR analysis. The chemical shifts of the carbons (C7, C9, and C22) B-positioned to N i l showed protonation effects,50 while the chemical shifts of C5 and C14 (B to Nl) were nearly identical for both forms of ingenamine (27). 11 AS -0.2 AS = 8pro - 8non (ppm) values for the two forms of ingenamine (27). pro = protonated; non = non-protonated Figure 19. 13C chemical shift differences between the two forms of ingenamine (27). 5). The very broad carbon signal of C22 at 8 20.2 ppm (Figure 2) could be partially attributed to the dynamic phenomenon of protonation and deprotonation at Nl 1. The 13C NMR of proton-free ingenamine displayed a relatively sharp carbon resonance for C22 (8 22.1, Figure 17) due to the removal of the labile proton. In addition, the sharpening of the carbon signals assigned to CIO and C21 was also in agreement with this assumption. Another possibility for the broadening of carbon signals around Ni l could arise from conformational equilibrium since the C7 to C12 piperidine ring is relatively flexible compared to the fixed piperidine ring (Nl to C6). This was ruled out as predominant contribution since we did not observe significant peak broadening in the 13C NMR spectrum of non-protonated ingenamine (27, Figure 17). Ingamine A (31)5 31 Ingamine A (31) was isolated as an optically active colorless glass that gave a parent ion in the HREIMS at mlz 448.3454 (Figure 20) corresponding to a molecular formula of C30H44N2O (AM +0.1 mmu). The !H NMR spectrum of ingamine A (31) in CDCI3 (Figure 21) exhibited complexity in both the aliphatic and olefinic regions. The l 3C NMR and APT spectra (Figure 22) of ingamine A (31), which showed resonances for all thirty carbon atoms (2 x C; 13 x CH; 15 x CH2, see Table 3), contained ten deshielded resonances that could be assigned to olefinic carbons. No additional unsaturated functional groups were apparent from the 13C NMR data, indicating that ingamine A (31) was also a pentacyclic compound. An IR band at 3307 cm-1 and NMR resonances at 8 68.9 (CHOH: C9) and 3.40 (CHOH) were assigned to a secondary alcohol, accounting for the one proton not attached to carbon. The ID and 2D ]H NMR and 13C NMR spectra of ingamine A (31) showed strong resemblances to the lH and 13C NMR spectra of ingenamine (27, see Table 1). In particular, the resonances corresponding to those assigned to the central tricyclic core (Nl to C12) and the linear alkyl bridge connecting Nl and C7 in the spectra of ingenamine (27) could be found in the NMR spectra of ingamine A (31), suggesting that the two molecules shared these structural features. Detailed analysis of the COSY, HMQC, HMBC, and NOE data obtained for ingamine A (31) confirmed the presence of the tricyclic core (Nl - CI2) and four attached methylene carbons at Nl, C3, C7, and Ni l also found in ingenamine (27). COSY correlations (Figure 24) were 142265.42 [TIC-3020864, 100X-96408] EI 100 90 80 70 60 50 40 30 20 10 0 448 319 338 407 417 295 , -|lly^Ulljij^ait UlliL-YiiI lllliJJllllliliYJl llil|u..|,iii|lll l|i, •[liii[au 1.^iiliJ|lil |.i.i|hll|., till IJll,^.|.llJ|illJ|l 275 300 325 350 375 (II 400 L 431 VT "~i—IT~I—T^T—r ~i—i 425 450 475 100 90 80 70 60 50 40 30 20 10 0 83 67 ,55 l-f 50 79 lH 108 75 100 II, il 1 Lb.ll 120 J.1 125 134 lb,' r 150 157 iljllll l|ll|UM|llll| ll.|bdjlf 188 242 £22 alijlll|ia )|Jlljk.|.lJ|llll|ln n.J 175 ' 200 225 250 Figure 20: Low resolution EI mass spectrum of ingamine A (31) ppa 6.0 9.6 3.0 2.5 2.0 1.5 1.0 B.O 4.8 4.0 3.8 Figure 21: *H NMR spectrum of ingamine A (31) (500 MHz, CDCI3) t^tm^mmmmmmtn "rtf *" 1""*" mmmtmmm^mmmmmmm^tmMmmmmtmmtmm MMMMMMIMMM 23 4 lH»WL*Uillil>'tli pNMfirfMMM* 1 •MMMMttoM* 9 2 - ,X.». 21 10 13/6 8 12 7 20 32 5 JUAJkw Li duniHUrf 60 T*1 40 20 DP* MO 120 100 BO Figure 22: 13c and APT NMR spectra of ingamine A (31) (125 MHz, CDCI3) 00 49 & — <• 20 40 60 rlOO 1-120 H140 i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i ppa 6 8 4 3 2 1 . :PP» Figure 23: 2D HMQC spectrum of ingamine A (31) in CDCI3 50 6 . 0 5 . 0 4 . 0 3 . 0 2 . 0 1.0 _ 1 . 0 _ 2 . _ 3 . 0 _ 4 . 0 5 . 0 _ 5 . 0 PPM Figure 24: 2D COSY spectrum of ingamine A (31) in CDCI3 51 C no. 2 3 4 5 6 6' 7 8 9 10 10' 12 12' 13 13' 14 14' 15 15" 16 16' 17 18 19 19' 20 20' 21 22 23 24 25 25' 26 27 28 28' 29 30 31 31' 32 32" Table. 3. NMR data for ingamine A (31) recorded in CDCI3 at 500 MHz ^H). 5 i 3 C 64.1 143.8 121.2 34.5 54.2 45.3 51.9 68.9 55.9 50.2 54.3 26.0 26.3 22.9 131.4 130.3 20.8 41.5 58.3 23.0 126.2 129.3 26.0 127.9 127.4 25.9 128.7 128.7 25.2 36.2 5 ! H 3.03 5.94,(1(6.1) 2.66 2.91 1.76 0.97, bd(8.9) 3.40, td(9.4, 4.2) 2.84 2.69 2.5,d(11.0) 2.07, d(l 1.0) 2.82 2.25 1.48 1.24 1.59 1.47 2.27 1.59 5.63 5.65 2.20 1.77 1.98 1.74 2.62 2.27 5.31 5.48 2.90 2.68 5.40 5.45 2.90 2.78 5.40 5.47 2.34 2.18 2.33 1.98 a C O S Y Correlation H4 H2, H5, H32, H32' H4, H6, H6\ H8 H6', H5 H6, H5 H5, H9 H8, H10, H10' H10', H9 H10.H9 H12' H12 H13\ H14, H14' H13, H14, H14' H14', H13.H13' H14, H13,H13',H15 H15', H14' H15 H16', H17 H16, H17 H16, H16' H19, H19' H19', H18, H20, H20' H19, H18, H20, H20' H20", H19, H19" H20, H19, H19' H22 H21, H23 H22, H24 H23, H25, H25' H25', H24, H26 H25, H24, H26 H25, H25' H28, H28' H28', H27, H29 H28, H27, H29 H28, H28' H31.H31' H31', H30 H31, H30, H32* H32', H4 H32, H4, H31" b H M B C Correlation H4, H6, H12', H13, H20, H32 H2, H5, H32, H32' H2, H5, H6, H6\ H8, H32, H32' H4, H6, H6\ H8, H9 H2, H8, H13, H13' H2, H5, H8, H12, H12', H20, H20' H2, H4, H5, H6, H6\ H12 H8 H8, H12, H21 H2, H8, H20\ H21 H2, H15, H6' H13 H13', H16.H16' H15, H15', H17, H18 H16.H16', H19 H16, H16', H19, H19' H17, H18, H20, H20' H8, HI2', H19, H19' H12', H22, H23 H23, H24 H22, H24, H25 H22, H23, H25 H23, H24 H25, H25', H28, H28' H25, H25', H28, H28' H26, H30 H28, H28',H31,H31' H28,H28',H31,H31',H32' H29, H32, H32' H2, H4, H31, H31' a R O E S Y H12',H13,H13', H31',H32,H32' H5, H6\ H9, H31, H31' H4, H9, H6\ H8 H6', H8, H14 H4, H5, H6, H13 H5, H6, H20, H20' H4, H5, H12' H10' H10 H12' H2, H9, H12 H2, H6',H13',H14' H2, H13, H14, H14' H6, H13', H14' H13,H13', H14, H15 H14' H17, H20' H16',H17 H16, H17 H15',H16, H16' H19, H19' H18, H19' H18, H19 H8, H20' H8, H15\ H20 H23 H22 H4 H2,H4 H2, H32' H2, H32 Correlated to proton resonance in 8 *H column; recorded at 400 MHz. ° Correlated to carbon resonance in 5 *-*C column. 52 observed between H4 (5 5.94) and H2 (5 3.03), H5 (5 2.66), H32 (8 2.33), and H32' (5 1.98); between H5 (8 2.66) and H6' (8 1.76), and H8 (8 0.97); between H8 (8 0.97) and H9 (8 3.40); and between H9 (8 3.40) and H10' (8 2.69). HMBC correlations were observed between H4 (8 5.94) and C2 (8 64.1), C5 (8 34.5), C8 (8 51.9), and C32 (8 36.2); between H5 (8 2.66) and C3 (8 143.8); between both H6 (8 2.91) and H6' (8 1.76) and C4 (8 121.2), C5 (8 34.5), and C8 (8 51.9); between H8 (8 0.97) and C5 (8 34.5), and C10 (8 55.9); and between H9 (8 3.40) and C5 (8 34.5). A network of HMBC correlations confirmed the placement of the aliphatic quaternary carbon (8 45.3: C7), indicated by the APT experiment, between C2 and C8. It also identified methylene carbons (CI2 and C20) as the other two substituents on the quaternary carbon. Thus, H8 (8 0.97) and H2 (8 3.03) both showed two bond HMBC correlations to 8 45.3 (C7), and H2 (8 3.03) was also correlated through three bonds to C8 (8 51.9). A pair of geminal methylene protons at 8 2.45 (H12) and 2.07 (H12'), that were correlated to a carbon at 8 50.2 in the HMQC spectrum, showed two bond HMBC correlations to the quaternary carbon (8 45.3: C7) and one of them (H12') showed a three bond correlation to a methylene carbon at 8 41.5 (C20). In addition, H12 (8 2.45) showed a three bond HMBC correlation to C8 (8 51.9) and H12' (8 2.07) showed a three bond correlations to C2 (8 64.1). The protons (8 1.98 and 1.74: H20, H20') attached to the methylene carbon at 8 41.5 (C20) showed two bond correlations to the quaternary carbon at 8 45.3 (C7). Furthermore, in the HMBC spectrum H20' (8 1.74) was correlated to C12 (8 50.2), H20 (8 1.98) was correlated to C2 (8 64.1), and H8 (8 0.97) was correlated to the methylene carbon (8 41.5: C20). The 13C NMR chemical shifts of C2, C6, C10, and C12 (Table 3) in ingamine A (31) were nearly identical to the chemical shifts of the corresponding carbons in ingenamine (27) (Table 1), which indicated that the two nitrogen atoms were also attached to C2 and C6, and C10 and C12, respectively, in 31. HMBC correlations between H2 (8 3.03) and C6 (8 54.2) and between H6 (8 2.91) and C2 (8 64.1) were consistent with the nitrogen bridge between C2 and C6. Additional correlations between H6' (8 1.76) and a methylene carbon at 8 54.3 (CI3), and between one of the methylene protons (8 2.82: HI3) and C2 (8 64.1)/ C6 (8 54.2) identified the 53 methylene carbon (C13) as the third substituent of the tertiary amine. Similarly, HMBC correlations between H12 (8 2.45) and CIO (8 55.9) provided evidence for the nitrogen bridge between CIO and C12, and correlations between a methylene carbon at 8 58.3 (C21) and H12' (8 2.07) identified this methylene carbon as the third substituent of the second tertiary amine. ROESY correlations were observed between H9 (8 3.40) and HI2" (8 2.07), between H9 and H4 (8 5.94), and between H8 (8 0.97) and H6 (8 2.91). This evidence confirmed that the central core of the ingamine A (31) had the same relative stereochemistry as in ingenamine (27) and that C7 to C12 piperidine ring still adopted a boat conformation. The remaining portion of ingamine A (31) was composed of eight aliphatic methylene and eight olefinic methine carbons. These sixteen carbons had to form a pair of linear bridges between the four appendages at Nl, C3, C7, and Ni l to complete the final two rings required by the unsaturation number. Detailed analysis of the COSY, HMQC, and HMBC data for ingamine A (31) clearly supported an eight carbon bridge spanning Nl and C7 that was identical to the N1/C7 bridge previously identified in ingenamine (27). HMQC and COSY correlations identified the chemical shifts of all of the pairs of geminal protons in this bridge (Table 3). COSY correlations were observed between H13 (8 2.82) and H14 (8 1.48)/ H14" (8 1.24); between H14' (8 1.24) and H15 (8 1.59); between H15' (8 1.47) and H16 (8 2.27, long range COSY); between H16 (8 2.27) and H17 (8 5.63); between H18 (8 5.65) and H19 (8 2.20); and between H19 (8 2.20) and H20 (8 1.98). The HMBC data were completely consistent with the above assignment (Table 3). Comparison of the 13C NMR chemical shifts of CI6 (8 22.9) and C19 (8 20.8) with the data reported for the ircinals43 and the haliclamines^ confirmed the Z geometry for the A17-1** alkene. With the nature of the Nl to C7 alkyl bridge established, it was apparent that the C3 to Ni l bridge had to be twelve carbons long and had to contain three olefins. The absence of an UV chromophore in ingamine A (31) indicated that there was no conjugation between the three double bonds. In addition, the ]H NMR spectrum contained two pairs of geminal methylene protons [H25 (8 2.90)/ H25' (8 2.68) and H28 (8 2.90)/ H28' (8 2.78)] that had chemical shifts typical of protons on doubly allylic carbons in polyunsaturated fatty acids. These H25/ H25' and H28/ H28' resonances were all correlated into the cluster of olefinic protons between 8 5.3 and 5.5 in the COSY spectrum (Figure 24). These two pieces of the evidence, along with the HMBC data (Table 3), were consistent with methylene interrupted triene substructure (C23 to C30). Furthermore, the COSY and HMBC data (Table 3) demonstrated that there were two methylene carbons between both Nl 1 and C3 and the respective ends (C23 and C30) of the triene substructure. Thus, COSY correlations were observed between H21 (5 2.62) and H22 (5 2.27), between H22 and H23 (5 5.31); and between H32' (6 1.98) and H31' (5 2.18), and between H31' and H30 (5 5.47). HMBC correlations were observed between H23 (5 5.31) and C21 (5 58.3), and between H32' (5 1.98) and C30 (5 128.7). Once again, the chemical shifts of the allylic carbons (C22: 5 23.0; C25: 5 26.0; C28: 8 25.9; C31: 8 25.2) indicated that the A23-24, A26 '27, and A29 '30 olefins had the Z geometry. Therefore, the complete structure of ingamine A (31) was as shown. Freshly isolated ingamine A (31) was an oil-like colorless glass. It (31) was not stable at normal conditions, slowly changed colors from yellowish to deep red-brown in NMR solvents, and finally decomposed to form a yellowish precipitate. This is likely attributable to the doubly allylic methylenes in the bridge Nl l-to-C3 and their susceptibility to air oxidation. Ingamine A (31) is a second member of the 'ingenamine' class of alkaloids.51 Its skeleton differs from that of ingenamine only by having a twelve-carbon alkyl bridge between C3 and Nl 1 instead of the eight-carbon alkyl bridge present in ingenamine (27). The putative biogenetic precursor to compound 31 would be an unsymmetrical Z?w-3-alkyldihydropyridine macrocycle with one eight-carbon and one-twelve carbon chain. Haliclamines A (12) and B, consisting of two tetrahydropyridines linked by C9 and CI2 alkyl chains, are examples of such an unsymmetrical macrocycle.33 Ingamine A (31) showed in vitro cytotoxicity against murine leukemia P388 with an ED50 of 1.5 |ig/mL. 55 Ingamine B (32)51 Ingamine B (32) was isolated as an optically active colorless glass. It gave a parent ion in the HREIMS at mlz 432.2504 (Figure 25) appropriate for a molecular formula of C30H44N2 (AM 0.0 mmu). The molecular formula obtained for ingamine B (32) differed from that of ingamine A (31) simply by the absence of an oxygen atom. The ID and 2D JH and 13C NMR data obtained for ingamine B (32) showed a close correspondence to the data obtained for ingamine A (31) (Table 4). The only significant differences in the data for the two compounds were the chemical shifts of the resonances assigned to C5, C8, C9, and CIO and their attached protons. Since ingamine A (31) contains an alcohol substituent at C9, and the molecular formula of ingamine B (32) contains no oxygen atoms, it was apparent that ingamine B (32) simply lacked the C9 hydroxyl group. The *H NMR spectrum (Figure 26) of ingamine B (32) confirmed the absence of methine proton resonance at 8 3.40 ppm present in ingamine A (31), a typical chemical shift for a secondary alcohol. The 13C and APT experiments (Figure 27) of ingamine B (32) contained one less methine carbon resonance at 8 68.9 ppm, typical for a carbon attached to oxygen, and one more methylene carbon resonance at 8 26.6 ppm when compared with that of ingamine A (31). A detailed analysis of the COSY, HMQC, HMBC, and ROESY data obtained for ingamine B (32) confirmed the structure of ingamine B (32). HMQC/ COSY correlations identified the chemical shifts of the carbon resonances assigned to C5, C8, C9, and CIO and their attached protons (Table 4). An examination of COSY and HMBC spectroscopic data established the connectivity. L42263.62 [TIC-1186816 , 100X-S96971 EI 100 90 80 70 60 50 40 30 20 10 0 432 335 391 I I2" I ll I h T..,|Wl|L^illL.yili|L^|llll m. r.i. Hllji,  {.•llfn. |.  ti.liji.. t .lillyl. (. il|ll, |li il|h^ T_rln<a,i )_i|l 275 300 325 350 375 400 425 -| i r~i—i—|—i—r—i—l—J-r 450 475 500 100 90 80 70 60 50 40 30 20 10 110 93 44 | MLJ ( 55 67 ia.Jl 50 1114-4 l.. TiJli 83 w 75 h 100 149 134 ll4lll|lllUl 206 244 188 n i|nll|llll |.,.i{JlllliL rnilll|lli.| .iillllllll., itl|llii t--^llL|iii n_^|lIll|L_^J|llL| ^ntl^-125 150 175 200 225 250 Figure 25: Low resolution EI mass spectrum of ingamine B (32) 0\ -i—i—•—•—.—i—•—• • •—r-—i— 4.S 3.0 2.5 2.0 l.S 1.0 ppa 5.5 5.0 4.0 3.5 Figure 26: J H NMR spectrum of ingamine B (32) in CDCI3 (500 MHz) *0* iwiPWiniip imm#mim**mm**mm •W^MWNMNMM^MMb«JLMMAM« MMMM fcWftWMN 17 18 24 29 30 26 27 23 JJUL 2 21 13 6 10 12 7 8 20 5 32 /U i •noi» „ II* rti lupin m If iitiiiiiilti ii|HiiiiiiiiiiMiiiii|in ppa 140 120 100 80 90 40 20 Figure 27: ^C and APT NMR spectra of ingamine B (32) (125 MHz, CDC13) 00 59 JUA .U\UMJ. S [ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I ] I I I I I I I I I I I I I I I I 8 4 3 2 1 ^ -rlOO r lM Figure 28: 2D HMQC spectrum of ingamine B (32) in CDCI3 60 * • * ! * _ 1.0 _ 3 .0 > 5.0 i — | — i — p — r — i — | i i i i | i i i i I 4.0 3 .0 2 .0 1.0 2. 0 _ 4 .0 5 .0 _ 6 .0 PPM Figure 29: 2D COSY spectrum of ingamine B (32) in CDCI3 Table 4. NMR data for Ingamine B (32) recorded in CDCI3 at 500 MHz (*H) #c 2 3 4 5 6 6' 7 8 9 9' 10 10' 12 12' 13 13' 14 14' 15 15' 16 16' 17 18 19 19' 20 20' 21 21' 22 23 24 25 25' 26 27 28 28' 29 30 31 31' 32 32' 8 ! 3 C 64.0 142.9 121.2 37.7 54.0 45.7 43.5 26.6 49.6 49.3 54.2 26.0 26.3 22.9 131.3 130.7 21.0 40.9 58.6 23.4 126.7 129.1 26.0 128.1 127.4 25.9 128.9 128.6 25.5 36.1 8 1H 2.98, bs 5.89, d(6.2) 2.25 2.88, dd(9.1, 1.9) 1.71, dd(9.1,2.6) 0.96, m 1.34 1.18, qd(13.3, 4.0) 2.95 2.59 2.4 , bd(10.2) 2.03, d(10.2) 2.83, td(12.7, 5.1) 2.23 1.46 1.23 1.55 1.47 2.29 1.55 5.62, td(10.3, 5.1) 5.68, td(10.3, 6.1) 2.23 1.76, m 1.90, td(l 1.9, 7.2) 1.64, q(11.9) 2.60, dt(12.4, 3.3) 2.51, td(12.4, 5.6) 2.26 5.32 5.48 2.91 2.69, dt(16.0, 6.0) 5.43 5.41 2.91 2.78, dt(16.0, 6) 5.40 5.47 2.34 2.18 2.32 aCOSY Correlation H4 H2, H5, H32, H32' H4, H6', H8 H6' H6, H5 H5, H9, H9' H9', H8, H10' H9, H8, H10, H10' H10', H9' H10, H9, H9' H12' H12 H13', H14, H14' H13, H14, H14' H14', H13, H13' H14, H13, H13',H15 H15', H14' H15, H16 H16', H15', H17 H16, H17 H16, H16', H18 H17.H19, H19' H19', H18, H20, H20' H19, H18, H20, H20' H20', H19, H19' H20, H19, H19' H21', H22 H21.H22 H21, H21', H23 H22, H24 H23, H25, H25' H25', H24, H26 H25, H24, H26 H25, H25' H28, H28' H28', H27, H29 H28, H27, H29 H28, H28 H31,H31' H31', H30 H31, H30, H32' H32', H4 1.99 |H32, H4, H31' b HMBC Correlation H4, H13, H20, H32, H32' H2, H31', H32, H32' H2, H6, H6', H8, H32, H32' H4, H6, H6', H8, H9' H2, H4, H8, H13, H13' H2, H5, H12', H19, H20' H2, H4, H5, H6, H6', H9', H10' H9*, H20' H20' H2, H6', H15 H13', H15, H15' H15, H15', H17, H18 H15', H16', H19 H16, H16', H19, H20' H17, H18, H20, H20' H8, H12', H19 H12', H22, H23 H23, H24 H24, H25 H23, H25, H25' H23, H24, H27 H25, H25' H28, H28 H26 H28, H28' H31,H31', H32' H29, H32, H32' H2, H4 a R O E S Y H13, H19', H31', H32, H32' H5, H6', H9', H31, H31' H4, H6', H7, H8, H9' H6', H8 H4, H5, H6 H5 H5, H6, H9', H20 H9' H4, H5, H8, H9, H12' H9' H2, H13', H14, H14' H13, H14, H14' H13, H13', H14' H13, H13', H14, H15 H15', H14', H17 H15, H17, H20' H16', H17 H16, H17 H15, H15', H16, H16" H19' H2, H19 H8, H20' H15', H20 H23 H22 H25, H25' H24, H26 H24, H26 H25, H25' H28, H28' H27, H29, H32 H27, H29 H28, H28' H31,H31' H4, H30 H2, H4, H30 H2, H28, H32' H2, H32 a Correlated to proton resonance in 8 *H column; recorded at 400 MHz. b Correlated to carbon resonance in 8 13C column. 62 Thus, COSY correlations were observed between H5 (5 2.25) and H8 (8 0.96), between H8 and H9 (8 1.34)/ H9' (8 1.18), and between H9' (8 1.18) and H10 (8 2.95)/ H10' (8 2.59). Two bond HMBC correlations were observed between H5 (8 2.25) and C8 (8 43.5), between C8 and H9' (8 1.18), and between H9' and CIO (8 49.6). Three bond HMBC correlations were observed between C5 (8 37.7) and H9' (8 1.18), and between C8 (8 43.5) and H10" (8 2.59). The 13C chemical shifts assigned to C5, C8, C9, and CIO of ingamine B (32), which differed from those of ingamine A (31), could be simply attributed to substituent effects. The absence of an oxygen atom at the C9 position dramatically changed its chemical shift from 68.9 ppm in ingamine A (2) to 26.6 ppm in ingamine B (32). The differences of carbon resonances (A8 = 831 - 832) of 8.4 ppm for C8 and 6.3 ppm for CIO between the two molecules were due to the P-effect of oxygen atom, while the negative y-gauche effect of heteroatom oxygen explained the downfield shift of 3.2 ppm for C5 in 32. 8 2.59 37.7 The relative stereochemistry of ingamine B (32) appeared to be the same as ingamine A (31). ROESY correlations observed between H9' (8 1.18) and H12' (8 2.03), between H9' and H4 (8 5.89), and between H8 (8 0.96) and H6 (8 2.88) confirmed the relative stereochemistry of the central core structure of the ingamine B (32) and showed that the C7 to CI2 piperidine ring was still in a boat conformation. The mass spectrometric data (Scheme 7) also supported the proposed structures of ingamines A (31) and B (32). The LREIMS of ingamine B (32) gave two important fragment ions at m/z 244 (26 %) and 188 (17 %), corresponding to C17H26N and C13H18N, respectively. 63 These peaks are attributable to the parent ion carrying out a retro Diels-Alder reaction, which is quite common in the EI mass spectrometry experiment, followed by allylic-allylic bond cleavage between C19 and C20, and between C31 and C32 with a proton transfer (Scheme 7). The corresponding hydroxylated ingamine A (31) under the same EIMS condition yielded fragments at mlz 242 (39 %) and 188 (34 %). Analogously, the parent ion of ingamine A (31) in the EIMS lost water first, then underwent a retro Diels-Alder reaction, and finally the bonds between C19 and C20, and between C31 and C32 were cleaved to form fragment A (mlz 188) and fragment B (m/z 242), two mass units less than its counterpart in the spectrum of ingamine B (32). Similar to ingamine A (31), ingamine B (32) was also an unstable alkaloid. The fresh isolated colorless compound 32 gradually decomposed in air to yield a dark red solution and some yellowish precipitate. Ingamine B (32) showed in vitro cytotoxicity against murine leukemia P388 with an ED50 of 1.5 |ig/mL. 64 Scheme 7. Proposed Fragmentation of Ingamine B (32) and Ingamine A (31) in EIMS. 32 Retro [4 + 2] C 1 3 H 1 8 N + C 1 7 H 2 6 N mlz = 188 244 H , 0 31 Retro [4 + 2] C ^ H i o N + C i 7 H , 4 N 13"18 17"24 J mlz = 188 242 65 2.2.2.4. Ingenamine B (33)52 Ingenamine B (33) was obtained as a white powder. In the HREIMS spectrum it gave a parent ion at mlz 410.3299 corresponding to a molecular formula of C27H42N2O (AM 0.2 mmu), which differed from the molecular formula of ingenamine A (27) by one additional methylene unit. The structure of 33 was solved by analysis of a combination of ID and 2D lH and 13C NMR data (Table 5). Examination of the NMR data revealed that ingenamine B (33) had a hydroxylated tricyclic core (Nl to C12) and an eight carbon bridge (Nl to C7) that were identical to those previously identified in the non-protonated ingenamine (27, Table 2). For example, the *H NMR spectrum (Figure 30) recorded in MeOH-<i4 clearly showed two overlapping olefinic protons at a chemical shift of 5.64 ppm (H17 and HI8) that were very diagnostic for the ingenamine type compounds with an eight carbon chain bridged from Nl to C7 and the Z-double bond at the C17/C18 position. The 13C NMR and APT experiments (Figure 31) were also in complete agreement with the proposed structure 33, which showed all of the 27 resolved resonances, including one more methylene carbon resonance in the 8 22 to 28 ppm region compared with that of ingenamine (27). Subtraction of the atoms present in the already identified tricyclic core and the Nl to C7 bridge (C18H26N2O) from the molecular formula of ingenamine B (33) indicated that the C3 to Nl 1 bridge contained nine carbons with one alkene functionality (C9H16). Detailed analysis of COSY (Figure 32) and HMQC (Figure 33) data identified all of the carbons and their attached protons in the C3 to Nl 1 bridge (see Table 5). COSY correlations were w JulL . i i = /-PrNH 2 ppa 6.0 5.6 6.0 4.6 4.0 3.6 3.0 2.8 2.0 1.8 1.0 0.5 Figure 30: ] H NMR spectrum of ingenamine B (33) in MeOH-tf4 (500 MHz) ON ON ywa1  «»• III I>I • ••>!' .11 I* i « »u M<^MMl«*V««*M«»«nMii«^MHM>^^ •••JUAwW^'wtMMlJMMfpMMr i = /-Pr NH 2 I 1 1 • »' fTt p I I I I I I I I I I 'f'^1 T 1 >'T1'p » I « I » • • • » « r>'»^'«l I 1 |^'» I • '»'> 1' • ••'t'*l»'*'^ Tt"l I 1 | fl I I < ppa 140 120 100 80 60 40 20 Figure 31: 13C and APT NMR spectra of ingenamine B (33) in MeOH-</4 (125 MHz) ON -J 68 -i - A . 1U)IXJU'LJL pp« 1 • OF 1 o 0 l a * a o < J • < 8j Mfd • O • : • OO 0 : • 0 * . i 0 * m 0 rPe« Figure 32: 2D COSY spectrum of ingenamine B (33) in MeOH-^ 4 69 JJA JUiuU \_JL pp" * * • B 4 w < • 3 • « A ^ 9 o :ppa Figure 33: 2D HMQC spectrum of ingenamine B (33) in MeOH-^t Table 5. NMR data for ingenamine B (33) recorded in MeOH-^4 at 500 MHz ^H). #c 2 3 4 5 6 6 ' 7 8 9 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 1 5 ' 16 1 6 ' 17 18 19 1 9 ' 20 2 0 ' 2 1 2 1 ' 22 2 2 ' 23 2 3 ' 24 2 4 ' 2 5 2 5 ' 26 2 6 ' 27 28 29 2 9 ' 5 13c 65.25 144.8 121.2 35.03 55.01 47.75 52.61 69.54 54.72 50.19 55.27 27.11 27.40 23.89 132.5 131.3 21.59 42.59 57.26 24.16 28.41 27.81 28.60 26.26 132.4 125.6 34.55 8 lH 3.12, bs 5.90, d(6.3) 2.70 2.91, dd(9.1,1.8) 1.86, dd(9.1,2.6) 0.77 dd(9.9, 2.0) 3.32 ddd(l 1.9, 10.1,4.7) 2.68, dd(12.2,4.7) 2.57, t(12.2) 2.42, d(l 1.3) 2.25, d(l 1.3) 2.99, td(12.6, 5.0) 2.30, td(12.6, 4.2) 1.53 1.28 1.60 1.52 2.40 1.57 5.64 5.64 2.33 1.78 1.78 1.78 2.99 2.47, dt(14.1, 5.0) 1.50 1.43 1.39 1.35 1.40 1.40 1.53 1.32 2.33 1.79 5.54 , td(10.8, 4.4) 5.67, td(10.8, 4.4) 3.09,dd(18.1, 10.7) 2.72 a COSY Correlations H4 H2,H5 H4, H6, H6', H8 H5, H6' H5,H6 H5,H9 H8, H10, H10' H9, H10' H9, H10 H12' H12 H13',H14, H14' H13, H14 H13.H13', H14' H13, H14, H15 H14',H15' H15 H16\ H17 H16, H17 H16, H16' H19, H19' H18, H19' H18, H19 H21',H22, H22' H21, H22, H22' H21,H21',H22' H21, H21',H22 H25', H26, H26' H25, H26, H26' H25, H25', H26', H27 H25, H25', H26, H27 H26, H26', H28 H27, H29, H29' H28, H29' H28, H29 b HMBC Correlations H4, H6, H12', H13, H20 H2, H29, H29' H2, H6, H 6 \ H8, H29, H29' H4, H6, H6\ H8, H9 H2, H8, H13, H13' H2, H8, H12, H12', H20 H2, H6, H6NH10, H10', H20 H8, H10, H10' H12 H2, H10, H20, H21 H2, H6' H13 H13*,H16,H16' H17, H18 H16, H16' H16, H19, H20 H17, H18, H20 H8, H12, H12' H10, H10' H24, H26, H27 H27, H28 H26, H29, H29' H26, H29, H29' H2, H4, H27, H28 a Correlated to proton resonance in 8 ^H column. " Correlated to carbon resonance in 8 ! 3 C column. 1 71 observed between H21' (5 2.47) and both H22/ H22' (5 1.50, 1.43); between H25/ H25' (5 1.53, 1.32) and H26' (8 1.79); between H26/ H26' (5 2.33, 1.79) and H27 (8 5.54); between H27 and H28 (8 5.67); and between H28 and H29/ H29' (8 3.09, 2.72). Due to the severe overlap of the upfield proton resonances, the COSY correlations between the C22 and C23 methylene protons and between C24 and C25 protons were ambiguous. Fortunately, the well resolved proton H21' (8 2.47) showed a three bond HMBC correlation to the methylene carbon at 8 28.4 (C23), which clarified the connectivity of this chain. COSYLR data clearly confirmed the location of the alkene functionality in the bridge at the C27/C28 position. The olefinic proton H27 (8 5.54) showed long range COSY correlations to both doubly allylic protons H29 (8 3.09) and H29' (8 2.72) that in turn correlated to the central core olefinic proton H4 (8 5.90). Three bond HMBC correlations observed between H27 (8 5.54) and C29 (8 34.5); between both H29 (8 3.09) and H29* (8 2.72) and C4 (8 121.2), and in turn between H4 (8 5.90) and C29 (8 34.5) were consistent with the chain substructure. The 13C chemical shift of C26 (8 26.3) along with the observation of a 10.8 Hz coupling constant between H27 and H28 indicated that the olefin A27 '28 had the Z configuration. Figure 34. Selected HMBC and COSYLR correlations. 72 2.2.2.5. Ingenamine C (34) and Ingenamine C Acetate (35)52 11 34 R = H 35 R = CH 3 CO The isolation and purification of ingenamine C (34) was not straightforward. Repeated normal phase HPLC chromatography of the ingenamine type alkaloids gave only an inseparable mixture of ingenamine C (34) and ingenamine G (39). Acetylation of the mixture and followed by HPLC separation yielded ingenamine C acetate (35). A parent ion was observed at mis 452.3406 appropriate for a molecular formula of C29H44N2O2 (AM 0.3 mmu) in the EIHRMS of 35, indicating that the parent compound, ingenamine C (34), was isomeric with ingenamine B (33). The *H NMR spectrum of 35 (Figure 35) showed a downfield resonance at 8 4.57 that was attributed to H9, and an acetate methyl resonance at 2.00 ppm. An APT experiment (Figure 36) showed a methyl resonance at 19.8 ppm (C31) and an acetate carbonyl resonance at 5 172.3 (C30) that confirmed it (35) was an acetate derivative. Comparison of the NMR data (Table 6) collected for ingenamine C acetate (35) with that of the non-protonated ingenamine (27) (Table 2) revealed that ingenamine C (34), also had the hydroxylated tricyclic core (Nl to C12) and the eight carbon Nl to C7 bridge with Z double bond at A17-18. Analysis of the 13C, COSY, COSYLR, HMQC, and HMBC data obtained for ingenamine C acetate (35) indicated that the remaining part (C9H16) contained a linear nine carbon bridge (C3 to Ni l ) with a Z-double bond at A24-25. Examination of HMQC (Figure 37) and COSY (Figure 38) correlations identified all nine carbons and their attached protons (see Table 6). COSY correlations were observed between H21 (5 2.83) and both H22/ H22' (8 1.68/ 1.43); between both H22/ H22' and the allylic proton H23' (8 1.91); between both H23/ H23' (8 2.25/ 1.91) and the olefinic proton H24 (8 5.39); between H24 and H25 (8 5.42); between H25 and both H267 4 17/18 * 27 28 W X_A 35 V J -•—i— 4.0 Ac • 11...in, i ,t i „ , i.i.t i. .| ...» ..,.* 11 ^I.I  w. | i.f i 2.0 l.S 1.0 8.0 0.0 4.S 3.0 3.0 2.0 Figure 35: ] H NMR spectrum of ingenamine C acetate (35) in MeOH-^4 (500 MHz) • oc rS r # 74 rS i I O r -J3 -16 r8 6 1) -s -8 I </7 i g -S c =5 I— ^^  CJ <u < SO en « u 3 64 -8 75 35 _ J L J A _ JUL LLuAliAoL. - r 20 60 * •i i i r r - r i , VT ., , ,rT. •I •'?' I T 1 t 1 F I T T T T - I I I I ' riOO - r i M fPP« Figure 37: 2D HMQC spectrum of ingenamine C acetate (35) in MeOH-^ 4 76 JJLA- .1 1 lUuWJA, k-wA*. PP« e • 0 * a a i t • 0 > 4 0 0 3 • a a \ i $ • • 1 • ! J rPP* : 2D COSY spectrum of ingenamine C acetate (35) in MeOH-^ 4 Table 6. NMR data for ingenamine C acetate (35) recorded in MeOH-^4 at 500 MHz (lH). Carbon no. 2 3 4 5 6 6' 7 8 9 10 10' 12 12' 13 13' 14 14' 15 15' 16 16' 17 18 19 19' 20 20' 21 21 ' 22 22' 23 23' 24 25 26 26' 31 31 ' 32 32 33 33 ' 30 31 8 13C 65.80 145.0 121.1 35.38 54.39 48.03 50.07 73.1 52.41 51.36 55.04 26.99 27.57 23.87 132.5 131.2 21.69 43.35 57.43 27.07 28.39 130.7 131.2 27.50 28.82 25.29 34.75 172.3 19.75 8 !H 3.09, bs 5.91, dd(6.5, 1.5) 2.42 2.88, dd(9.3, 1.8) 1.79, dd(9.3, 2.7) 1.02, dd(9.7, 2.3) 4.57, ddddl.O, 9.7, 4.7) 2.70 dd(l 1.9,4.7) 2.49,1(11.5) 2.38, d(l 1.2) 2.26, d( 11.2) 2.93, td(12.4, 5.1) 2.24 1.48 1.27, m 1.58 1.48 2.40 1.56 5.63 5.63 2.35 1.74 1.83, m 1.72 2.83, ddd(13.8, 8.2, 3.9) 2.44 1.68, m 1.43, m 2.25 1.91, m 5.39, td(l 1.0, 4.8) 5.42, td(10.6, 5.5) 2.35 1.78 1.48 1.48 1.79 1.49 2.22 2.22 2.00, s a COSY Correlations H4 H2, H5, H29 H4, H6, H6', H8 H5, H6' H5.H6 H5, H9 H8, H10, H10' H9, H10" H9, H10 H12' H12 H13', H14, H14' H13, H14 H13, H13', H14' H13, H14, H15 H14', H15' H15 H16', H17 H16, H17 H16, H16' H19, H19' H18, H19', H20 H18, H19 HI9, H20' H20 H21', H22, H22' H21.H22, H22' H21,H21', H22', H23, H23' H21, H21", H22, H23' H22, H23', H24 H22, H22', H23, H24 H23, H23', H25 H24, H26, H26' H25, H26', H27 H25, H26 H26 H26 H28', H29 H28, H29 H4, H28, H28' H4, H28, H28' b HMBC Correlations H4, H6.H13, H20 H2, H5 H2, H8 H4, H6, H6', H8, H9 H2, H8, H13, H13' H2, H12, H12' H2, H6, H6', H9, H10, H10', H12 H8, H10, H10' H12, H21' H20' H2, H6' H13 H13', H16' H17, H18 H15',H16, H16', H19 H16,H16', H19, H19' H17, H18, H20, H20' H8, H12', H19 HIO, HIO', H12', H23' H23' H25 H24, H27 H28' H27 H2, H28' H9, H31 a Correlated to proton resonance in 8 ^H column, b Correlated to carbon resonance in 8 ^ C column. 78 H26' (8 2.35, 1.78); between H26 (5 2.35) and H27 (5 1.48); and between both H28/ H28' (5 1.79, 1.49) and H29 (8 2.22). The COSY correlation was not clear between H27 (8 1.48) and H28 (8 1.79) since the cross peak was buried under the strong geminal correlation between H28 (8 1.79) and H28' (8 1.49). HMBC correlations observed from H28' (8 1.49) to both C27 (8 28.8) and C29 (8 34.8), and in turn from H27 (8 1.48) to C28 (8 25.3) completed the connectivity of this bridge as illustrated below. Again, the chemical shifts of the allylic carbons (C23: 8 28.4, C26: 8 27.5) indicated that the A24>25 double bond had the Z geometry. Figure 39. Selected COSY and HMBC correlations. Once the structure of the ingenamine C (34) was deduced from its acetate derivative 35 NMR data, the natural product 34 was also isolated by extensive inspection of all the different fractions from the Sephadex LH-20 column and then purification of the crude sample on normal phase HPLC. All of the NMR experiments were repeated on the natural product 34 and the results (Figure 40: proton spectrum) were in complete agreement with the proposed structure. The HREIMS spectrum of ingenamine C (34), that gave a parent ion peak at mlz of 410.3294 appropriate for a molecular formula of C27H42N2O (AM - 0.3 mmu), further proved the structure of ingenamine C (34). COSY HMBC 79 V) 2.2.2.6. Ingenamine D (36)52 36 The minor metabolite ingenamine D (36) was obtained as a colorless glass that gave a parent ion in the HREIMS at mlz 422.3292 appropriate for a molecular formula of C28H42N2O (AM -0.2 mmu), differing from the molecular formula of ingenamine (27) by one additional vinyl group. The *H NMR spectrum (Figure 41) of 36 clearly showed all seven olefinic protons. The proton resonances at 8 5.95 (broad doublet, one proton) and 5.61 (two protons) were typical values for H4 and H17/ H18; and the magnitude of the chemical shift and scalar couplings at 8 3.41 was appropriate for the hydroxylated methine proton H9. These features of the proton spectrum indicated that ingenamine D (36) had a hydroxylated tricyclic core (Nl to CI2) and eight carbon bridge (Nl to C7) identical to those identified in ingenamine (27). Since only a small amount of ingenamine D (36) was isolated, its 13C NMR resonances were deduced from 2D data (HMQC and HMBC, see Table 7). Subtraction of the atoms present in the tricyclic core and Nl to C7 bridge (C18H26N2O) from the molecular formula of ingenamine D (36) revealed that the C3 to N i l bridge contained ten carbons and two alkene functionalities (C10H16). Detailed analysis of COSY and HMQC data identified all of the carbons and their attached protons in the C3 to Nl 1 bridge. It was apparent that the four olefinic methine protons were divided into two isolated double bonds (H24/H25 and H27/H28) since these two vinyl groups were not correlated into each other in the COSY spectrum (Figure 42). A pair of geminal methylene protons (H26 and H26') at chemical shifts of 3.06 and 2.53 ppm, typical of protons on doubly allylic carbons, showed aliphatic to vinyl COSY correlations to olefinic protons associated 81 X W> c c c c o <o Q. (/I Pi s Z 3 CD 82 Figure 42: 2D COSY spectrum of ingenamine D (36) in MeOH-^ 4 Table 7. NMR data for ingenamine D (36) recorded in MeOH-^4 at 500 MHz ( ! H ) . #c 2 3 4 5 6 6 ' 7 8 9 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 1 5 ' 16 1 6 ' 17 18 19 1 9 ' 20 2 0 ' 21 2 1 ' 22 2 2 ' 23 2 3 ' 24 25 26 2 6 ' 27 28 29 2 9 ' 30 5 13c 66.6 145.3 122.8 36.3 53.9 48.0 54.5 70.7 57.8 56.2 54.6 26.8 27.7 23.7 132.5 131.2 21.8 46.1 58.7 27.6 26.8 130.3 129.4 26.9 129.2 130.4 25.8 37.3 3 0 ' | 6 !H 3.06, bs 5.95, bd(6.5) 2.68, m 2.86 dd(9.3, 2.2) 1.80, dd(9.3, 2.6) 0.92, dd(8.7, 2.8) 3.41,ddd(14.0, 8.7, 5.0) 2.67 2.13 2.29,d(12.1) 2.21,d(12.1) 2.92, td(12.4, 5.1) 2.24 1.47 1.27, bd(l 1.8) 1.58 1.45 2.39 1.55 5.62 5.59 2.33 1.68 1.71 1.71 2.51 2.48 1.58 1.45 2.24 1.96 5.32 5.40 3.06 2.53 5.47 5.49 2.47 2.12 2.30 2.00 a COSY Correlations H4 H2, H5, H6, H30, H30* H4, H6, H6', H8 H4, H5, H6' H5, H6 H5, H9 H8.H10, H10' H9,H10' H9, H10 H12' H12 H13',H14,H14' H13, H14 H13,H13',H14' H13,H14, H15w H14'w, H15', H16 H15 H15, H16',H17 H16, H17 H16, H16\ H18 H17, H19, H19' H18, H19, H20 H18, H19, H20 H19,H19' H19, H19' H21',H22,H22' H21.H22, H22' H21,H21',H22\ H23, H23' H21,H21',H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H25 H24, H26, H26' H25, H26', H27 H25, H26, H27 H26, H26', H28 H27, H29, H29' H28, H29', H30, H30' H28, H29, H30, H30' H4, H29, H29', H30' H4, H29, H29', H30 b HMBC Correlations H4, HI3, H20, H30' H2, H5, H30 H2, H6, H8 H4, H6, H6' H2, H13, H13' H2, H8, H12, H20' H2, H5, H6, H6', H10 H5, H8 H12 H2 H2, H6, H6' H13,H13* H13' H16.H16' H20 H8, H12 H10, H12, H22, H23, H23' H23, H23' H25 H23 H23*, H26 H24 H26, H26' H2, H4 a Correlated to proton resonance in 8 *H column. ^ Correlated to carbon resonance in 5 13C column, w = weak. 1 84 with both double bonds [H25 (8 5.40) and H27 (8 5.47)]. The remaining vinyl to allylic correlations observed between H24 (8 5.32) and H23/ H23* (8 2.24, 1.96), and between H28 (8 5.49) and H29/ H29' (8 2.46, 2.12) expanded the fragment to seven carbons. The proton (8 2.51: H21) attached to carbon at chemical shift of 58.7 ppm (C21) showed COSY correlations to both the geminal methylene protons at 8 1.58 (H22) and 1.45 (H22'), that in turn correlated to the allylic protons H23/ H23' (8 2.24, 1.96). The final COSY correlations between H29/ H29' (8 2.46, 2.12) and H30/ H30' (8 2.30, 2.00) established the connectivity in this subunit. The long range COSY correlations observed between H24 (8 5.32) and H26' (8 2.53), between H27 (8 5.47) and H29' (8 2.12), and between H4 (8 5.95) and both H30/ H30' (8 2.30, 2.00) supported the above assignment. In addition, the HMBC correlations observed between H12 (8 2.29) and C21 (8 58.7), between C21 and H23/ H23" (8 2.24, 1.96), between H23/ H23' and C22 (8 5.34), and between C23 (8 26.8) and H25 (8 5.40) further confirmed the proposed structure. Figure 43. Selected HMBC and COSYLR correlations. 2.2.2.7. Ingenamine E (37)52 22 11 Ingenamine E (37) was isolated as an optically active, colorless glass. It (37) gave a parent ion in the HREIMS at mlz 448.3458 corresponding to a molecular formula of C30H44N2O (AM 0.5 mmu), indicating that it was isomeric with ingamine A (31). The sign of its optical rotation was negative, opposite to that of the other ingenamine type compounds so far discussed. Examination of the NMR data (Table 8) for 37 revealed that it contained a hydroxylated tricyclic core (Nl to C12) and eight carbon C3 to Ni l bridge with a Z olefin at A29>3() (C25 to C32 in ingenamine E; C21 to C28 in ingenamine). For example, the *H NMR spectrum (Figure 44) showed the following diagnostic proton resonances: H8 at the most upfield chemical shift (8 0.77), H4 at the most downfield chemical shift (8 5.89), and H9 at 3.27 ppm. The 13C NMR (Figure 45, APT) chemical shifts assigned to the C3 to Nl 1 bridge (Table 8) in ingenamine E (37) were nearly identical to the chemical shifts of the corresponding carbons in ingenamine (27) (Table 2). The COSY, HMQC, HMBC, and difference NOE data obtained for compound 37 were in complete agreement with this partial assignment. Subtraction of the atoms present in the tricyclic core and C3 to Ni l bridge (C18H26N2O) from the molecular formula of ingenamine E (37) indicated that the Nl to C7 bridge contained twelve carbons (C12H18). The 13C NMR data obtained for ingenamine E (37) showed that the Nl to C7 bridge contained six aliphatic methylene and six olefinic methine carbons, which required a linear chain with three alkene functionalities. Detailed analysis of the HMQC (Figure 46) and COSY (Figure 47) data identified all of the carbons and their attached protons in this bridge. The chemical shifts of two pairs of geminal methylene protons [H17 (8 2.88)/ H17' (5 2.78) and H20 (5 3.17)/ H20' (5 2.81)] were the typical resonances of protons on doubly allylic carbons. COSY correlations were observed between H17/ H17' (5 2.88/ 2.78) and the two olefinic protons H16 (5 5.52) and H18 (5 5.37), and between H20/ H20' (8 3.17/ 2.81) and another two olefinic protons H19 (8 5.34) and H21 (8 5.45). This evidence along with the absence of an UV chromophore in 37 indicated the presence of a methylene interrupted triene substructure (C15 to C22). In addition, the COSY and HMBC data demonstrated that there were two sets of two methylene carbons between both Nl 1 and C3 and the respective ends (CI5 and C22) of the triene substructure. Thus, COSY correlations were observed between H13/ H13' (8 2.49/ 2.22) and H14/ H14' (8 2.37/ 2.14), and between H14/ H14' and H15 (8 5.37) at one end of the triene substructure. COSY correlations were also observed between H22 (8 5.48) at the other end of the triene unit and H23/ H23' (8 2.34/ 1.82), and between H23 (8 2.34) and H24' (8 1.59). HMBC correlations were observed between H13/ H13' (8 2.49/ 2.22) and C14 (8 29.5), between C14 and H16 (8 5.52); and between H21 (8 5.45) and C23 (8 22.9), and between C23 and H24/ H24' (8 1.86/ 1.59). The 13C NMR shifts of the allylic carbons at C14, (8 29.5), C17 (8 27.2), C20 (8 26.8), and C23 (8 22.9) suggested that all three olefins at A15-16, A18>19, and A21-22 had the Z-geometry. The relative stereochemistry of ingenamine E (37) was found to be the same as ingenamine (27). Difference NOEs observed between H9 (8 3.27) and HI2' (8 2.02) confirmed that the C7 to C12 ring was still in boat conformation with H9 and HI2' occupying flagpole orientations. Difference NOEs observed between H9 (8 3.27) and H4 (8 5.89), between H8 (8 0.77) and H6 (8 2.99), between H8 and H10' (8 2.53), and between H8 and H24 (8 1.86) were consistent with the proposed relative configurations. The long range COSY correlations between H4 (8 5.89) and H8 (8 0.77) and between H12' (8 2.02) and H24 (8 1.86) could also be ascribed to W couplings. 87 M X X o w u c S S3 C <u an a o 8. Z X u 3 37 Mp#N*«tf*M»^ M**|*laM*WMM^^ 'W%x**»f ^j^yN^i^^iW^rt yfY^ **MUMW*#* **JwWWJvU* T 1 1 1 1 1—| 1 1 1 r 30 25 mk^MimmimmymmM 134 —r~ 132 130 128 i = /-Pr NH 2 11111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111 ppm 140 120 100 BO 60 40 20 Figure 45: APT NMR spectrum of ingenamine E (37) in MeOH-d4 (125 MHz) 00 00 89 37 PP» : 4 « * • < 3 4» « • C ~ a* <• o » I - £ -» ^ ! : : : : I i : i : : ! • j 20 100 120 Figure 46: 2D HMQC spectrum of ingenamine E (37) in MeOH-cU 90 37 ppa Figure 47: 2D COSY spectrum of ingenamine E (37) in MeOH-d4 Table 8. NMR data for ingenamine E (37) recorded in MeOH-^4 at 500 MHz (^H). #C | 6 13C 2 3 4 5 6 6 ' 7 8 9 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 16 17 1 7 ' 18 19 20 2 0 ' 2 1 22 23 2 3 ' 24 2 4 ' 25 2 5 ' 26 2 6 ' 27 2 7 ' 28 2 8 ' 29 30 3 1 3 1 ' 32 3 2 ' 63.40 143.46 124.46 35.16 55.59 45.76 52.53 69.13 54.15 51.21 59.41 29.46 129.74 129.44 27.22 128.57 129.02 26.75 129.14 131.09 22.89 40.41 56.78 21.81 27.40 25.94 132.61 133.18 26.47 38.22 5 *H 3.03,d(1.5) 5.89, bd(6.6) 2.62, m 2.99, dd(9.5, 1.9) 1.78, dd(9.5, 2.7) 0.77, dd(9.9, 1.9) 3.27, ddd( 11.9, 10.1, 4.7) 2.65, dd(12.2, 4.7) 2.53, t(12.0) 2.44, d(l 1.3) 2.02, d(l 1.3) 2.49, td(10.7, 4.4) 2.22, m 2.37 2.14 5.37 5.52 2.88, dt(15.3, 7.4) 2.78, m 5.37 5.34 3.17, td(15.8, 7.5) 2.81, m 5.45 5.48 2.34 1.82 1.86 1.59, bt(10.9) 3.09, dt(14.0, 7.1) 2.30 1.68, m 1.37 1.48, m 1.36 2.20 1.97, bd(14.4) 5.24, bt(10.6) 5.38 2.32 2.06, m 2.38 2.18 a COSY Correlations H4 H2, H5 H4, H6, H6', H8 H5, H6' H5, H6 H5, H9 H8, H10, H10' H9, H10' H9, H10 H12' HI2, H24(LR) H13',H14, H14' H13, H14, H14' H13, H13',H14',H15 H13, H13', H14, H15 H14,H14',H16 H15, H17, H17' H16, H7\H18 H16, H17, H18 H17, H17',H19 H18, H20, H20' H19, H20', H21 H19, H20, H21 H20, H20', H22 H21, H23, H23' H22, H23', H24, H24' H22, H23, H24, H24' H12'(LR), H23, H23\ H24' H23, H23', H24 H25', H26, H26' H25, H26, H26' H25, H25', H26', H27 H25, H25', H26 H26, H27', H28 H27, H28' H27, H28', H29 H27', H28, H29 H28, H28', H30 H29, H31.H31' H30, H31\ H32 H30, H31,H32' H31,H32' H31',H32 b HMBC Correlations H4, H6, H12',H13, H13', H24', H32, H32' H2, H5, H31, H32, H32' H2, H5, H6, H6', H8, H32, H32' H4, H6, H6', H8, H9 H2, H13, H13' H2, H5, H8, H12\ H24' H2, H4, H6, H6', H10, H10',H12 H8, H10, H10' H8, H12, H25' H2, H10, H24, H25, H25' H2, H6',H14' H13, H13',H15, H16 H13',H14, H14', H16 H15.H17, H17* H15, H16, H19 H17, H17' H20, H20' HI8, H22 H20, H20' H20, H23 H24, H24' H10, H10',H12',H26, H26' H25, H26 H26, H30 H31, H32, H32' H29, H32, H32' H2, H4.H30, H31 c N O E s H8 H6, H24 H4, H5, H12' H8, H24' a Correlated to proton resonance in 5 ^H column. " Correlated to carbon resonance in 8 13C column. c Resonance in 8 *H column was irradiated. LR = Long range COSY. 92 2.2.2.8. Ingenamine F (38)52 22 11 38 Ingenamine F (38) was isolated as an optically active, colorless glass. It gave a parent ion in the HREIMS at mlz 432.3497 appropriate for a molecular formula of C30H44N2 (AM -0.8 mmu). The molecular formula of 38 differed from that of ingenamine E (37) only by the absence of an oxygen atom, making it isomeric with ingamine B (32). Similar to ingenamine E (37), its optical rotation was also negative with the value of [a]o = -64.3°. The structure of ingenamine F (38) was initially elucidated by interpretation of ID and 2D NMR data acquired in deuterated chloroform.52 In order to compare its NMR data with that of other ingenamine type compounds, particularly with that of ingenamine E (37), all of the NMR experiments were repeated in MeOH-dd, (Table 9), and the result was in complete agreement with that in CDCI3. Comparison of the NMR data obtained for ingenamine F (38) in deuterated methanol with the data for ingenamine E (37) showed strong resemblance except for resonances assigned to C5, C8, C9, and CIO and their attached protons (Figures 48 and 49). It was apparent that the Nl to C7 and C3 to Nl 1 bridges were identical in both molecules, while the differences of chemical shifts assigned to C5, C8, C9, and CIO were simply due to the absence of a hydroxyl group in ingenamine F (38). Further comparison of the ingenamine F (38) NMR data with that obtained for ingamine B (32) and keramaphidin B (28) confirmed that ingenamine F (38) contained the same non-hydroxylated tricyclic core (Nl to C12) found in 32 and 28. For instance, the ^ C NMR chemical shifts of C5 (5 39.0), C8 (8 44.5), C9 (5 28.4), and CIO (8 48.2) in ingenamine F (38) were nearly indentical to the corresponding carbon resonances in keramaphidin B (28, 93 Table 11: C5, 5 38.8; C8, 44.1; C9, 26.8; CIO, 48.8) recorded in MeOH-d4. Similarly, difference NOEs observed between H9' (5 1.18) and H12' (5 2.05), and between H8 (5 0.96) and both H6 (8 3.00) and H24 (8 1.77) implied that ingenamine F (38) had the same ingenamine type stereochemistry. 94 X 8 X o u c ae c c 00 o p I o <u a . OS s z 90 3 95 _ 0 0 ^ <N _ o zn o 00 o o X o vO o 00 o o O t> 2 ,_ .« y * - \ 3C <**> U-<D c c <u so c Vl-i c p 1— u « a. v. OS 2 Z u 3 fa O •"3-Table 9. NMR data for ingenamine F (38) recorded in MeOH-^4 at 500 MHz (lH). Carbon n o . 2 3 4 5 6 6' 7 8 9 9 ' 10 10' 12 12 ' 13 13 ' 14 14 ' 15 16 17 17' 18 19 20 20 ' 21 22 23 2 3 ' 24 24 ' 25 25 ' 26 26 ' 27 27 ' 28 28 ' 29 30 31 3 1 ' 32 8 13c 63.7 142.9 124.5 39.0 55.9 45.1 44.5 28.4 48.2 50.9 59.1 29.5 129.8 129.4 27.2 128.5 129.2 26.7 128.8 131.4 23.1 40.0 57.1 21.7 27.6 26.1 132.7 133.2 26.6 38.4 3 2 ' | 8 *H 2.97,d(1.7) 5.86, d(6.5) 2.20 3.00, dd(9.3, 2.0) 1.71, dd(9.3, 2.7) 0.96, ddd(l 2.2, 5.8, 1.9) 1.36, m 1.18, qd(13.5,4.0) 2.74, td(13.4, 2.6) 2.63, dt(12.6, 3.5) 2.37, d(10.9) 2.05, d(10.9) 2.48, td(10.9, 4.4) 2.21 2.35 2.13, m 5.37 5.51 2.88, dt(14.5, 7.3) 2.78 5.35 5.36 3.17, dt(16.0, 8.0) 2.81 5.43 5.49 2.36 1.80 1.77 1.56, td(12.2, 3.6) 3.00 2.20 1.57, m 1.44, m 1.47, m 1.34, m 2.20 1.97, m 5.24, tt(10.7, 2.9) 5.36 2.32 2.07, m 2.36 2.17, m a COSY Correlations H4 H2, H5, H8LR, H32LR H4, H6, H6\ H8LR H5, H6' H5, H6 H4LR, H5LR, H9, H9' H8, H9',H10, H10' H8, H9, H10, H10' H9,H9',H10' H9, H9', H10 H12' HI2, H24LR H13',H14, H14' H13, H14, H14' H13, H13', H14',H15 H13,H13',H14,H15 H14, H14',H16 H15,H17, H17' H16, H17',H18 H16, H17, H18 H17, H17' H20, H20' H19, H20', H21 HI9, H20, H21 H20, H20', H22 H21,H23, H23' H22, H23', H24' H22, H23, H24 H12'LR,H23',H24' H23, H24 H25',H26, H26' H25, H26, H26' H25, H25', H26' H25, H25\ H26 H27', H28, H28' H27, H28LR, H28' H27, H27'LR, H28', H29 H27, H27', H28, H29 H28, H28', H30 H29, H31.H31' H30, H31' H30, H31,H32' H4LR, H32' H31',H32 b HMBC Correlations H4, H6, H24', H32, H32' H2, H5, H32, H32' H2, H6, H8, H32' H4, H6\ H8 H2, H5, H8, H13, H13' H2, H5, H8, H12', H24, H24' H2, H5, H6, H10', H24' H9',H12 H24, H25 H6',H14, H14" H13, H13',H16 H13, H14, H14' H17, H17' H15, H16, H18, H19 H17, H17' H20, H20' HI8, H22 H20, H20' H20, H23, H24' H21, H24, H24' H8.H12' H10, H10',H12',H26 H25 H26 H32, H32' H29, H32, H32' H2, H4, H31 c N O E s H5, H32 H5, H6, H9, H24 H5.H8 H12' H2 H9' H4 a Correlated to proton resonance in 8 XH column, b Correlated to carbon resonance in 8 ^ C column. c Resonance ino column was irradiated. LR = Long range COSY. 97 2.2.2.9. Ingenamine G (39) and Ingenamine G Acetate (40) 11 39 R = H 26 40 R = CH3C O Ingenamine G (39) was isolated as optically active colorless needles (m.p. 150 °C). The HREIMS of 39 gave a parent ion at mlz 424.3461 appropriate for a molecular formula of C28H44N2O (AM +0.8 mmu), which differed from the molecular formula of ingenamine (27) by two methylene units and was isomeric with xestocyclamine B (41).47b Xestocyclamine B (41) was isolated from the sponge Xestospongia sp. by Crews's group, which appeared in the literature after we have reported the structures of ingenamine (27),45 ingamines A (31) and B (32).51 However, the sponge Xestospongia ingens that we studied also contained compound 41. Comparison of the *H and 13C NMR data (Table 10) obtained for ingenamine G (39) with that of ingenamine (27, Table 2) showed that 39 also contained the Nl to CI2 tricyclic core and an eight carbon Nl to C20 bridge. Subtraction of the atoms present in the tricyclic core and Nl to C7 bridge (C18H26N2O) from the molecular formula of ingenamine G (39) indicated that the C3 to Ni l bridge (CioHis) contained ten carbons and one alkene functionality. Extensive analysis of ]H NMR (Figure 50), APT (Figure 51), and 2D NMR data identified eight aliphatic methylenes and two olefinic methines in this bridge. The appendage proton H30' (8 2.17) showed a COSY correlation to H29 (8 1.80) that in turn correlated to an allylic proton H28' (8 1.96). The routine vinyl and allylic COSY correlations (Figure 53) between H28/ H28' (8 2.36, 1.96) and H27 (8 5.34), which established the location of the double bond at A26-27 position, between H27 and H26 (8 5.26); and between H26 and H25/ H25' (8 2.25, 1.97) expanded the substructure to six carbons. Clear COSY correlations were observed between the allylic protons H25/ H25' and both H24 and H24' (8 1.57, 1.47), and between the well resolved 98 en X o o in 5 .5 O <u c £ c <u on c £ S o u Q. OS z o U 3 0\ ( ZHW ££l) ty'HCPW ui (6C) O auiuieuaSui jo uinjioads y^N xjy :iS aanSij oz I I I I I I I I I I 0* • In • 09 08 I i I i i i i i M | | f i t * I i i i i i i i i i « i I i 00T 031 on uidd i • • • 11 i i i i ii iftMW^fY*** 11** #** HwNMk >hflMi>WW#^l|MW)W^win|i^ 100 1 _L)UA_-PP« 6 « s -I 4 • * • • o — 0> • * «. 2 o -• • • o 1 « Eppn Figure 52: 2D HMQC spectrum of ingenamine G (39) in MeOH-dL* 101 _1LLK 3 ^n rr • 0 8 .' o m * a o " r r r p r r r r p a T T - t - i ' i m i"i •O s • 0 • 1 1 1 1 1 1 1 1 1 s k * 8 3° JZ*1 M 0 t i i i"T-'f—rT"r" 5ft Jf • e w n a • a • i i i i i i i i i a m Q r I I T I T i • P P « PP« 6 Figure 53: 2D COSY spectrum of ingenamine G (39) in MeOH-d^ proton H21 (5 2.79) and both H22 and H22' (5 1.51, 1.23). The last methylene unit had to be considered as the C23/ H23, H23' (8 25.7/ 1.26, 1.18) unit to complete the C3-to-Nll bridge, and the COSY correlation observed between H23' (5 1.18) and H24' (8 1.47) supported this assignment. HMBC correlations observed between H22' (8 1.23) and C23 (8 25.6), and in turn between C22 (8 21.0) and H23' (81.18) were consistent with the proposed connectivity. Figure 54. Selected COSY and HMBC correlations in ingenamine G (39). By comparison of the NMR data collected for ingenamine G (39) with the literature values of the isomer xestocyclamine B (41),47b it was found that both molecules exhibited similar 13C features except for the chemical shifts assigned to CIO (39: 41, 8 54.7: 59.2), C12 (8 49.5: 56.4), C20 (8 42.9: 46.1), and C22 (8 21.0: 26.6). When inspecting the 13C data of the whole family of ingenamine type compounds reported to date, we found that only ingenamine D (36, Table 7) had 13C NMR resonance values similar to that of xestocyclamine B (41) at CIO (36: 41, 8 57.8: 59.2), C12 (8 56.2: 56.4), C20 (8 46.1: 46.1), and C22 (8 27.6: 26.6). All the other 'ingenamine' compounds showed a close correspondence to ingenamine (27) at these positions. The reasons underlying the differences at the CIO, C12, C20, and C22 positions are not clear, however, it is very likely that the double bond at A27-28 in the ten carbon bridge (C3 to Ni l , compounds 36 and 41) might induce a conformational change. Ingenamine G acetate (40) was obtained as an optically active colorless glass. It gave a parent ion in HREIMS at m/z 466.3551 appropriate for a molecular formula of C30H46N2O2 (AM -0.8 mmu). A strong fragmention at m/z 407 (C28H43N2+, AM -0.9 mmu) in the LREIMS was consistent with the loss of an acetyl group (CH3CO). The *H NMR spectrum (Figure 55) of ingenamine G acetate (40) was nearly identical with that of the natural product 39. The most notable differences were that 40 had a methyl singlet at 8 2.02 ppm, typical for an acetyl group, and that the carbinol proton resonance (H9) showed a downfield shift from 8 3.30 in 39 to 8 4.53 in 40. All these differences indicated that the compound 40 was an acetate derivative. Table 10. NMR data for ingenamine G (39) recorded in MeOH-af4 at 500 MHz (!H). c# 2 3 4 5 6 6 ' 7 8 9 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 1 5 ' 16 1 6 ' 17 18 19 1 9 ' 20 2 0 ' 21 2 1 ' 22 2 2 ' 23 2 3 ' 24 2 4 ' 25 2 5 ' 26 27 28 2 8 ' 29 2 9 ' 30 3 0 ' 8 13c 65.63 144.3 122.2 35.13 54.70 47.29 53.34 69.43 54.70 49.49 55.2 27.12 27.48 23.80 132.4 131.4 21.57 42.93 58.35 20.98 25.67 27.70 25.84 130.8 131.5 27.26 27.18 36.2 8 lH 3.06, bs 5.91,(1(6.5) 2.69, m 2.86, dd(9.1,2.0) 1.77, dd(9.1,2.4) 0.74, dd(10.1,2.2) 3.30, td(l 1.2, 4.3) 2.66, dd(12.1,4.3) 2.51, t(12.1) 2.19d(10.4) 2.13,d(10.4) 2.95, td(12.6, 4.9) 2.22 1.49 1.27 1.59 1.51 2.38 1.56 5.63 5.63 2.31 1.73 1.86 1.71 2.79, ddd(13.7, 10.8, 5.5) 2.28 1.52 1.23 1.26 1.18 1.57 1.47 2.25 1.97 5.26 bt(10.5) 5.34 bt(10.2) 2.36 1.95 1.80 1.50 2.22 2.17 a COSY Correlations H4 H2, H5, H30, H30' H4, H6, H6', H8 H5, H6' H5, H6 H5, H9 H8, H10, H10' H9, H10' H9, H10 H12' H12 H13',H14, H14' H13, H14 H13, H13',H14' H13, H14, H15 H14' H16',H17 H16, H17 H16, H16' H19.H19' H18, H19\H20 H18,H19 H19, H20' H20 H21',H22, H22' H21,H22' H21,H22' H21,H21',H22 H23',H24 H23, H24' H23, H24', H25, H25' H23', H24, H25, H25' H24, H24', H25\ H26 H24, H24', H25, H26 H25, H25', H27 H26, H28, H28' H27, H28', H29' H27, H28, H29 H28', H29', H30' H29, H30 H4, H29', H30' H4, H29, H30 b HMBC Correlations H4, H6.H13, H20, H30' H2, H5, H30, H30' H2, H5, H6, H6 \ H30, H30' H4, H6, H 6 \ H8 H2, H8, H13, H13' H2, H5, H8, H12, H12',H20' H2, H6 H6', H10, H10', H12 H8, H10, H10' H12 H10,H20' H2, H6' H13.H13' H16.H16' H15',H17, H18 H16 H19, H19' H17, H18, H20, H20' H8, H12, H12',H19' H10, H10',H12' H23' H22' H27 H28 H26, H27, H30, H30' H30, H30' H2, H4, H28 Correlated to proton resonance in 8 *H column, b Correlated to carbon resonance in 8 13C column. 104 - 2: V) © - ) 2.2.2.10. Keramaphidin B (28)52 28 Keramaphidin B (28) isolated from sponge Xestospongia ingens was an optically active amorphous white solid. The HREIMS of 28 gave a parent ion at m/z 380.3191 correponding to the molecular formula C26H40N2 (AM -0.0 mmu), differing from ingenamine (27) simply by the absence of an oxygen atom. The structure was initially elucidated by extensive analysis of ID and 2D NMR data. Table 11 provides a summary of the NMR data recorded in deuterated methanol for compound 28. The proton NMR spectrum (Figure 56) and APT spectrum (Figure 57) of 28 showed a close resemblance to that of ingenamine (27, Figures 16 and 17). The most significant differences in the *H and 13C NMR data for the two compounds were the resonances assigned to C9 and its attached proton(s). Since keramaphidin B (28) contains no oxygen atoms, it was apparent that keramaphidin B (28) simply lacked the C9 alcohol functionality present in ingenamine (27). The constitution and relative configuration of 28 was as shown above. Shortly after we had the structure (28) in hand, Kobayashi's group reported the isolation of keramaphidin B (28) from an Amphimedon sp. collected off the Kerma Islands, Okinawa, Japan.46 Its structure was solved by single crystal X-ray diffraction analysis. Comparison of *H NMR, 13C NMR, and mass spectral data collected for 28 by our group with the literature values reported by Kobayashi's group confirmed the identity of the two compounds. Interestingly, the keramaphidin B (28) isolated from the sponge Amphimedon sp. was reported to be a naturally occurring racemate, while that obtained from Xestospongia ingens was optically active with [OC]D = +29.8°. 106 00 c/3 'm N O o 5 I X O u s 90 ca "2 IS & E 2 m M 3 J3 O a. S 2 X so <u u 3 CD E-a 107 CO — — — ^ - • ^ • • i ' E-8 u 3 ft* Table 11. NMR data for keramaphidin B (28) in MeOH-J4 at 500 MHz (^H). c# 2 3 4 5 6 6 ' 7 8 9 9 ' 10 1 0 ' 12 1 2 ' 13 1 3 ' 14 1 4 ' 15 1 5 ' 16 1 6 ' 17 18 19 1 9 ' 20 2 0 ' 2 1 2 1 ' 22 2 2 ' 2 3 2 3 ' 2 4 2 4 ' 2 5 26 27 2 7 ' 28 2 8 ' 8 13c 64.63 142.8 125.0 38.76 54.31 44.95 44.06 26.81 48.79 50.76 55.11 27.06 27.47 23.79 132.8 131.0 21.56 41.77 56.88 20.87 27.06 26.13 132.6 133.4 26.47 37.59 6 lH 3.18, bs 5.91,d(6.4) 2.30 2.89, dd(9.2, 1.9) 1.68 dd(9.2,2.6) 0.98, ddd(12.5, 5.5,2.1) 1.50 1.23 qd (14.0,4.1) 2.97, td(13.5, 2.6) 2.88 2.70, d(l 1.6) 2.16 d(l 1.6) 2.99, td(12.5, 5.2) 2.21, ddd(12.5, 5.2, 1.2) 1.53 1.27 1.61 1.50 2.41 1.56 5.65 5.65 2.38 1.76 1.75 1.75 3.24, dt(13.5, 7.5) 2.52, ddd(13.5, 7.5, 2.5) 1.73 1.49 1.52 1.44 2.26 2.02, bd(15.2) 5.28, tt(10.8, 2.8)) 5.41 2.35 2.11 2.38 2.31 a COSY Correlations H4 H2, H5 H4, H6, H6', H8 H5, H6' H5, H6 H5, H9, W H8, H9', H10, H10' H8, H9, H10, H10' H9, H9 \ H10' H9, H9', H10 H12' H12 H13\ H14.H14' H13.H14 H13, H13',H14\ H13, H14, H15 H14',H15' H15, H16 H15', H16',H17 H16, H17 H16.H16' H19,H19' H18.H19' H18.H19 H21',H22, H22' H21.H22, H22' H21,H21\H22' H21,H21',H22 H24, H24' H24' H23',H24',H25 H23, H23', H24, H25 H24, H24', H26 H25, H27, H27' H26, H27' H26, H27, H28 H27', H28* H28 b HMBC Correlations H4, H6, H12', H13, H28, H28' H2, H5, H28 H2, H5, H6, H6', H8, H28 H4, H6, H6 \ H8, H9 H2, H8, H13, H13' H2, H5, H8, H9, H12, H12', H19, H20 H2, H4, H6, H6', H10, HI2, H20 H8, H10 H9',H12,H21' H20, H21 H2, H6' H13.H15 H13', H16, H17 H15, H18 H16, H16', H19, H19' H16, H16', H19, H19' H17, H18, H20, H8, H12, H12', H19 H10, H10', H12',H22, H23 H21 H21, H22 H26 H26 H25, H27, H27' H25, H28, H28' H2, H4, H26 a Correlated to proton resonance in 8 ^H column. " Correlated to carbon resonance in 8 ^ C column. 2.2.2.11. Conclusions The 'ingenamine' type metabolites are a new class of cytotoxic alkaloids, of which ingenamine (27) was the first to be reported.45 The pentacyclic skeleton of the 'ingenamine' type alkaloids can be formally envisioned to arise from a biological intramolecular [4+2] cycloaddition reaction between two dehydropiperidine rings in a fr/s-3-alkylpiperidine macrocyclic precursor (Schemes 6 and 8). Interestingly, the occurrence of this new class of 'ingenamine' type alkaloids was predicted by Baldwin and Whitehead's proposal for the biosynthesis of the manzamines.11 The full connectivity and relative stereochemistries observed in the 'ingenamine' type alkaloids follow from the expected endo and regiochemical preferences of the Diels-Alder reaction. The suite of ingenamine alkaloids represented by ingenamine (27),45 ingamines A (31) and B (32),51 keramaphidin B (28),46>52 xestocyclamine A (29)4 7 and B (41),4 7 b and ingenamines B (33) to G (39 ) 5 2 illustrates some of the structural variations that are possible in this family. The only variation observed thus far in the central Nl to CI2 tricyclic core is the presence or absence of a hydroxyl functionality at C9. Both the Nl to C7 and the C3 to Nl 1 linear carbon bridges vary in length and position and degree of unsaturation. The greatest range of variation occurs in the C3 to Nl 1 bridge which has eight carbons and one alkene in ingenamine (27), keramaphidin B (28), xestocyclamine A (29), and ingenamines E (37) and F (38); nine carbons and one alkene in ingenamines B (33) and C (34); ten carbons and two alkenes in ingenamine D (36); ten carbons but one alkene in xestocyclamine B (41) and ingenamine G (39); and twelve carbons and three alkenes in ingamines A (31) and B (32). Only eight and twelve carbons have been identified in the Nl to C7 bridge. An interesting relationship exists between ingamine A (31) and ingenamine E (37), and between the corresponding non-hydroxylated analogs ingamine B (32) and ingenamine F (38). The Nl to C7 bridge in ingamines A (31) and B (32) is identical to the C3 to Nl 1 bridge in ingenamines E (37) and F (38), and the C3 to Ni l bridge in ingamines A (31) and B (32) is identical to the Nl to C7 bridge in ingenamines E (37) and F (38). Therefore, in principle, ingamines A (31) and B (32) and ingenamines E (37) and F (38) can all arise from a common 110 ftw-3-alkylpyridine macrocycle (43) precursor. Scheme 8 depicts the relationship of their biogenetic origins by analogy with the Baldwin and Whitehead proposal for manzamine biosynthesis.11 Macrocycles 44a and 44b are tautomers, which are derived from same precursor 43 by reduction. If a partially reduced ring A acts as the diene and a partially reduced ring B acts as the dieneophile (44a) in the biological [4+2] cyclization reaction, the skeleton of ingamines A (31) and B (32) is formed. Conversely, if a partially reduced ring A acts as the dieneophile and a partially reduced ring B acts as a diene (44b) in the condensation reaction, the skeleton of ingenamines E (37) and F (38) is formed. Interestingly, Mosher ester analysis (See next section) has shown that the absolute configuration of the tricyclic central core of ingamine A (31) and ingenamine E (37) is identical. I l l Scheme 8. The Skeleton Relationship between Alkaloids 31 and 37. Hi Ingamine A (31) f[0] Ingenamine E (37) Ingamine B (32) [4 + 2] & reduction Ingenamine F (38) [4 + 2] & reduction Tautomerization 44a 44b 112 2 .2 .3 . The Absolute Configuration of Ingenamine Alkaloids Isolated from the Sponge Xestospongia Ingens Ingenamine (27) isolated from Xestospongia ingen is a pentacyclic alkaloid, which represents the first reported example of the new 'ingenamine' family of cytotoxic sponge metabolites.45 Keramaphidin B (28),46 isolated from an Amphimedon sp., and xestocyclamine A (29) , 4 7 a ' b isolated from a Xestospongia sp., are the additional 'ingenamine' alkaloids. Interestingly, the existence of the ingenamine alkaloids was anticipated by Baldwin and Whitehead in their elegant proposal for the biogenetic origin of the of the manzamines (Scheme 4).1 1 Ingenamine (27), keramaphidin B (28), and xestocyclamine A (29) have carbon skeletons identical to the skeleton of the pentacyclic intermediate in Baldwin and Whitehead's proposal. Ircinal B (25),43 isolated from the same sponge Amphimedon sp. that yielded keramaphidin B (28), has the overall skeleton and aldehyde functionality identical to Baldwin and Whitehead's predicted tetracyclic intermediate (Scheme 4). The discovery of the the ingenamines and ircinals has provided strong support for Baldwin and Whitehead's proposed biogenetic pathway to the manzamines. If the 'ircinal' and 'ingenamine' alkaloids are produced by the same biosynthetic manifold as the manzamines, as suggested by Baldwin and Whitehead's proposal, it is reasonable to expect that all three families of alkaloids would have the same absolute configurations. Indeed, ircinals A and B (25) were shown by chemical correlation to have the same configuration as manzamines A (2) and B (20).43 However, two recent reports suggested that the situation might be more complex than anticipated. Keramaphidin B (28), whose structure was determined by x-ray diffraction analysis, was reported to be a naturally occuring racemate ,46 and ircinols A and B (26) were found to be antipodal to the corresponding ircinals and manzamines obtained from the same sample of sponge.44 In attempt to shed further light on the stereochemical features of this interesting biosynthetic manfold, we have utilized Mosher ester methodology to determine the absolute configurations of ingenamine (27), ingamine A (31), and ingenamine E (37). The details of this analysis are presented below. 113 Ingenamine (27), ingamine A (31), and ingenamine E (37) isolated from the crude extracts of X. ingens are optically active pentacyclic alkaloids. Compounds 27 and 31 had a positive sign for their optical rotations while 37 had a negative value. The extracts of X. ingens also yielded keramaphidin B (28) that unlike the racemic keramaphidin B (28) isolated from Amphimedon sp. is an optically active metabolite ([CC]D +29.8°, c 1.1; MeOH). The (R)- and (5)- Mosher's esters of ingenamine (27), ingamine A (31), and ingenamine E (37) were prepared according to literature procedures.53 The (/?)- and (S)- Mosher esters of cholesterol were prepared in parallel with each derivatization of the ingenamine alkaloids as a control for the chirality of the reagents. On the basis of lH, 13C, COSY, HMQC, HMBC, and difference NOE spectra of the Mosher esters 45a/45b (MTPA-ingenamine), 46a/46b (MTPA-ingamine A), and 47a/47b (MTPA-ingenamine E), the chemical shifts of each (/?)- and (S)-derivative were determined. The A5 values (AS = 5s - 5/? in Hz) for each proton were calculated and plotted on the conformational representations. 2.2.3.1. MTPA-ingenamine (45)52 The Mosher ester derivatives of (S)-MTPA-ingenamine (45a) and (/?)-MTPA-ingenamine (45b), prepared according the literature procedures53 (See experimental), were colorless viscous glasses. The lack of NMR and chromatography evidence for more than one diastereomer of each Mosher ester indicated that ingenamine (27) obtained from X. ingens was an optically pure natural product. The derivatives (45a) and (45b) gave parent ions in the HREIMS at m/z 612.3543 and 612.3544, respectively, corresponding to a molecular formula of C36H47N2O3F3 (AM both 0.5 mmu). The proton spectra (Figures 58 and 59) of both 45a and 45b showed a close resemblance to that of the original compound ingenamine (27) except for the aromatic and methoxyl proton resonances from the MTPA group, and a downfield shift of the carbinol proton H9 (8 4.82 and 4.85). Detailed analysis of the esters 45a and 45b using COSY, HMQC, HMBC, and NOE experiments led to complete assignment of the *H NMR spectra (Table 12). J 41a X = (S)-MTPA AVL_A J I -]—i—i—i—i—]—i—i—i—i—i—i—i—i—i—i—i—i—i—r- ]—i—i—i—r- ]—i—i—i—i—|—i—i—i—r—j—i—i—i—r?)—i—i—i i-| i—i—i—i—|—i—i—n—p 7.5 7.0 G.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM Figure 58: 'H NMR spectrum of (5)-MTPA-ingenamine (45a) in CD2CI2 (400 MHz) 115 - z co N X o o in O cs 8 JO <u a c c g s o 6 5 o u o. c/5 z U 3 OX) (S)- MTPA-ingenamines (45a) (tf)-MTPA-ingenamines (45b) Figure 60. Chem3D-generated representations of (S)- and (/?)-MTPA-ingenamines (45a and 45b) based on the MM2 program. All the hydrogens have been omitted for clarity. C atoms are represented as grey circles with medium-size dots, N atoms as white circles with fine dots, O atoms as white circles with big dots, and F atoms as small circles with medium-size dots. 117 Coupling constant analysis and difference NOE results demonstrated that the C7 to C12 piperidine ring in the Mosher's ester 45 was in a boat conformation with H9 and HI2' occupying flagpole positions as in the parent compound 27. Examination of Dreiding models (Figure 60) indicated that there are no steric impediments to the MTPA group adopting the 'ideal conformation', i.e. having the trifluoromethyl, ester carbonyl, and carbinol methine proton coplanar in derivatives 45a and 45b. As can be seen below (Figure 61), when the MTPA plane contains the X (= MTPA) and H groups at C9 as required by the 'ideal conformation', the A5 values for derivative 45 are all positive on one side of the plane and negative on the other side. Following the empirical rule for analyzing the data as put forth by Ohtani et al.,53a ^ j s apparent that ingenamine (27) has the absolute configuration (2R, 55, 75, SR, 95) as shown for the MTPA derivative 45 in Figure 61. Figure 61. A5 = 5<j -5^ (Hz) values for the Mosher ester of ingenamine recorded in CD2C12 at 500 MHz. 118 Table 12. *H NMR data of Mosher esters 45a/45b (MTPA-ingenamine), 46a/46b (MTPA-ingamine A), and 47a/47b (MTPA-ingenamine E) recorded in CD2CI2 (500 MHz). c# 2 4 5 6 6' 8 9 10 10' 12 12' 13 13" 14 14' 15 15' 16 16' 17 18 19 19' 20 20' 21 21' 22 22' 23 24 25 25' 26 27 28 28' 29 30 31 31" 32 32' ArH ArH ArH ArH ArH CH3 MTPA-ingenamine (45) A 8 -0.04 -0.22 -0.11 -0.07 -0.10 -0.03 +0.07 +0.16 +0.03 -0.04 -0.03 -0.07 -0.04 -0.03 -0.04 -0.04 -0.05 -0.05 +0.13 +0.05 +0.05 +0.04 -0.03 5 *H (5) 3.09 5.80 2.20 2.73 1.63 1.07 4.82 2.91 2.40 2.30 2.21 2.86 2.20 1.43 1.24 1.53 1.36 2.31 1.54 5.64 5.63 2.27 1.61 1.63 1.63 2.77 2.23 1.54 1.37 1.52 1.33 2.06 2.01 5.42 5.47 2.24 2.20 2.37 2.19 7.51 7.43 7.42 7.43 7.51 3.52 5 lE (R) 3.11 5.84 2.42 2.84 1.70 1.17 4.85 2.84 2.24 2.29 2.18 2.88 2.24 1.44 1.27 1.60 1.40 2.34 1.58 5.64 5.62 2.29 1.65 1.68 1.68 2.64 2.18 1.49 1.33 1.50 1.33 2.04 2.01 5.41 5.48 2.22 2.22 2.36 2.22 MTPA-Ingamine (46) AS -0.05 -0.24 -0.09 -0.08 -0.08 -0.02 +0.07 +0.19 -0.04 -0.04 -0.03 -0.05 +0.05 +0.05 +0.04 -0.04 8 ^ ( 5 ) 3.09 5.88 2.21 2.79 1.68 1.09 4.77 2.85 2.74 2.38 2.20 2.86 2.24 1.46 1.26 1.58 1.43 2.30 1.57 5.63 5.62 2.23 1.65 1.85 1.65 2.58 2.54 2.28 2.26 5.35 5.50 2.93 2.77 5.45 5.45 3.01 2.78 5.45 5.49 2.39 2.20 2.33 2.05 8 *H (/?) 3.11 5.93 2.45 2.88 1.76 1.17 4.79 2.78 2.55 2.36 2.21 2.88 2.23 1.47 1.26 1.59 1.47 2.32 1.58 5.64 5.63 2.27 1.68 1.87 1.70 2.53 2.49 2.24 2.24 5.35 5.50 2.912 2.78 5.44 5.44 3.03 2.78 5.44 5.49 2.40 2.24 2.34 2.05 7.50 7.43 7.43 7.43 7.50 3.53 MTPA-ingenamine E (47) C# 2 4 5 6 6' 8 9 10 10' 12 12' 13 13' 14 14' 15 16 17 17' 18 19 20 20' 21 22 23 23' 24 24' 25 25' 26 26' 27 27' 28 28' 29 30 31 31' 32 32' ArH ArH ArH ArH ArH CH3 A 8 -0.20 -0.09 -0.09 -0.10 -0.01 +0.07 +0.14 -0.03 -0.04 -0.09 -0.08 +0.10 +0.06 +0.04 +0.05 8 *H (5) 2.98 5.85 2.18 2.90 1.68 1.13 4.81 2.91 2.46 2.30 2.23 2.45 2.20 2.29 2.10 5.36 5.51 2.84 2.79 5.37 5.37 3.15 2.77 5.43 5.38 2.26 1.72 1.73 1.44 2.78 2.26 1.54 1.38 1.50 1.32 2.07 2.01 5.40 5.44 2.22 2.22 2.34 2.11 7.50 7.43 7.43 7.43 7.50 3.53 8 *H (/?) 3.00 5.85 2.38 2.99 1.77 1.23 4.82 2.84 2.32 2.31 2.23 2.47 2.22 2.29 2.12 5.37 5.52 2.83 2.79 5.36 5.38 3.16 2.78 5.42 5.40 2.29 1.76 1.82 1.52 2.68 2.20 1.50 1.33 1.50 1.32 2.05 2.01 5.42 5.45 2.21 2.21 2.36 2.12 7.49 7.43 7.43 7.43 7.49 3.52 119 2.2.3.2. MTPA-Ingamine A (46)52 The Mosher ester derivatives (S)-MTPA-ingamine A (46a) and (#)-MTPA-ingamine A (46b) were colorless viscous glasses, which gave parent ions in the HREIMS at mlz 664.3850 and 664.3852, respectively, appropriate for a molecular formula of C40H51N2O3F3 (AM -0.2 and +0.1 mmu). Similar to the ingenamine case, analysis of the Mosher's esters revealed that the original compound ingamine A (31) isolated from X. ingens was optically pure. Detailed analysis of the esters 46a and 46b using COSY, HMQC, HMBC, and NOE experiments led to complete assignment of the *H NMR spectra (Table 12). The A5 values (AS = £>s - &R in Hz) for each proton were calculated and plotted on the conformational representation. As can be seen in Figure 62, the A5 values for derivative 46 are all positive on one side of the plane and negative on the other side. Therefore, according to the empirical rule,5-53 ingamine A (31) has the absolute configuration (2R, 55, 75, &R, 95) like ingenamine (27). 25 H -120 X .1 0 Figure 62. AS = 85 -Sy? (Hz) values for the Mosher ester of ingamine A recorded in CD2C12 at 500 MHz. 2.2.3.3. MTPA-Ingenamine E (47)52 Ingenamines E (37) and F (38) showed the negative optical rotations that are opposite in sign to all of the other 'ingenamine' alkaloids obtained from the same specimens of Xestospongia ingens. Mosher's method was used to determine whether the molecule 37 had the same or opposite absolute configuration as ingenamine (27). The Mosher's ester derivatives (S)-MTPA-ingenamine E (47a) and (ft)-MTPA-ingenamine E (47b) were prepared according the literature procedures.53 The HREIMS spectra of 47a and 47b showed parent ions at m/z 664.38501 and 664.38435, respectively, appropriate for the molecular formula C40H51N2O3F3 (AM -0.2 and -0.8 mmu). Analysis of ID and 2D NMR data collected for the Mosher's ester derivatives 47a and 47b led to complete assignment of the *H NMR spectra (Table 12). The result showed that the natural product ingenamine E (37) was an enantiomerically pure compound that had the same absolute configuration as that of ingenamine (27) and ingamine A (31). As can be seen in Figure 63, when AS values (AS = 85 - 87? in Hz) for each proton were plotted on the conformational representation, the A8 values for derivative 47 are all positive on one side of the plane and negative on the other side. Thus, ingenamine E (37) had the absolute configuration (2R, 5S, IS, SR, 9S) as shown below. Figure 63. A8 = 85 -8^ (Hz) values for the Mosher ester of ingenamine E recorded in CD2C12 at 500 MHz. 2.2.3.4. Conclusions Since the alkaloids ingenamine (27), ingamine A (31), and ingenamine E (37) isolated from X. ingens share the same absolute configuration (2R, 55, 75, 8R, 95) as described above, it is apparent that the negative sign of the optical rotaion of 37 arises from the different arrangement of the two bridges in the molecule. Therefore, some relationship might exist between the magnitude of the optical rotations and the length of the side chains. Ingenamine (27) had two symmtrical eight-carbon bridges spanning Nl-to-C7 and C3-to-Nl 1, respectively, and it showed a positive [OC]D of +62 degrees. When the length of C3-to-Nl 1 bridge increases to twelve carbons, the molecule [i.e. ingamine A (31)] showed a larger positive value for its specific rotation ([OC]D = +131.0°). Conversely, if the length of Nl-to-C7 bridge increases to twelve carbons, as is the case for ingenamine E (37), the magnitude of specific rotation decreased to give a negative value ([OC]D = -23.80). The absolute configurations of ingenamine (27), ingamine A (31), and ingenamine E (37) isolated from X. ingens described above are antipodal to the absolute configurations reported for manzamines A (2) and B (20), and ircinals A and B (25). However, their configurations are identical to the absolute configurations recently reported for the ircinols A and B (26). Interestingly, the keramaphidin B (28) isolated by Kobayashi et al. from the same Amphimedon sp. that yielded ircinals A and B (25), manzamines A, B, G, and H, and ircinols A and B (26) was reported to be a naturally occurring racemate, while keramaphidin B (28) isolated from X. ingens was found to be optically active. The structural relationship between the ingenamine, ircinol, ircinal, and manzamine alkaloids provides compelling evidence for the Baldwin and Whitehead biogenetic proposal that they arise from an achiral &/.y-3-alkyldihydropyridine macrocycle as shown in Scheme 4. It is clear, however, that there are two antipodal series of alkaloids formed from this pathway. One of the enantiomers of keramaphidin B (28) isolated from Amphimedon sp., the ircinals A and B (25), and the manzamines A (2) and B (20) all belong to one configurational series. The other enantiomer of keramaphidin B (28), ircinols A and B (26), ingenamine (27) and ingamine A (31) belong to the other configurational series. According to the Baldwin and Whitehead proposal, the chirality of these alkaloids is established by the biological equivalent of an intramolecular [4 + 2] cycloaddition reaction of an achiral bis-3-alkyldihydropyridine macrocycle (24) (Scheme 4).11 Therefore, it appears that there are enantiomeric enzymes capable of catalyzing this intramolecular condensation. 2.2.4. Structure Elucidation of Madangamine Alkaloids from Xestospongia ingens The Papua New Guinea marine sponge Xestospongia Ingens produced another new class of alkaloids, the madangamines. The structures of madangamines A (30), B (48), C (49), D (50), and E (51) were elucidated by extensive analysis of spectroscopic data, particularly, NMR data. Proton-carbon attachments were determined by HMQC experiments and proton spin systems were based on COSY experiments. COSYLR, HOHAHA, and HMBC data also proved useful for identification of the spin systems. The final connectivities of the quaternary carbons and nitrogen atom interrupted systems were mainly dependent on HMBC results. The relative stereochemistries were determined using NOE and ROESY data, and in some cases long range COSY correlations attributed to VK-couplings. The absolute stereochemistry of the madangamines is still not known, but it might be deduced by correlation to the putative precursor ingenamines. A detailed discussion of the structure elucidation of madangamines A (30), B (48), and C (49) is presented below, while the description of madangamines D (50) and E (51) will be reported elsewhere. 123 30 48 " h i m " 49 50 51 2.2.4.1. Madangamine A (30)48 29 30 Madangamine A (30) was isolated as an optically active colorless glass. An intense parent ion (base peak) at mlz 432.3503 in the HREIMS (Figure 64) of 30 was appropriate for a molecular formula of C30H44N2, which required 10 sites of unsaturation. Table 13 provides a summary of the NMR data acquired for compound 30. The *H NMR spectrum of 30 showed severely overlapped olefinic proton resonances and complex multiplets of poorly resolved aliphatic methylene and methine resonances (Figure 65). The 13C and APT NMR spectra showed signals for all 30 carbons: two quaternary carbons, twelve methine carbons, and sixteen methylene carbon atoms, among which ten were sp2 hybridized carbons (Figure 66). Since ten deshielded carbon resonances could be assigned to five double bonds and no additional unsaturated functional groups were apparent from the 13C NMR data, it was obvious that the madangamine A (30) was pentacyclic. The HMQC spectrum (Figure 67) confirmed that all 44 hydrogen atoms were attached to carbons. A detailed analysis of the COSY, HMQC, HMBC, and NOE data collected for madangamine A (30) identified the constitution and relative configurations of the tricyclic core (Nl to C12) and four additional carbons attached at Nl, C3, N7, and C9. The lH.-lU COSY experiment (Figure 68) routinely established the proton spin system for the central core. Thus, significant COSY and long-range COSY correlations were observed between H2 (8 3.69) and H l l a x (5 2.40)/ H l l e q (8 1.27); between H l W H l l e q and H12 (8 1.14); between H12 and H5 L42401.75 CTIC-14300160, 100X-592864J EI 100 90 80 70 60 50 40 30 20 10 0 432 283 391 297 320 337 I i I I351  I365 i|[lll r ,,lill|L, „|llih, r„i|ilil[. ^ilili.,,, |.tltfi..,, iiiii,,. , il| i—i—i—r 325 350 "T. "'-i i "I"'i T"'!"1 '. r'Tvr'i fy(,-*>1 f\\0 1—I—I—I—I—I—I—I—I—I—f~ 450 476 500 275 300 375 400 425 100 _ 90 80 70 60 50 40 30 20 10 79 91 41 55 iL 67 lu+ f-r 108 iiu 117 |Li^^ h n 240 111 II 254 50 75 100 125 150 175 200 225 250 Figure 64: Low resolution EI mass spectrum of madangamine A (30) 126 m ^ £ r f " - S -N DC o o Q c © C c « s o 4) U 3 en 127 CN CN J CN 00 ON r » 2 CN cn Os CN :8 r -a- m CN O cn CN 00 rS :S as •cv • § PS IS CN CO X CN o o c s c3 c •o E o s o U a. a* Z r-(X < e u cn s s 41 3 CD 128 29 30 21 l ^ 5 Hi ' 6 4 12 u \ H ^ / 18 A 20 17 30 20 = <o i 9 o o °o - r 40 <<s Oe " ^ > -r 60 80 • • rlOO <^^ i-120 i l l ! — r - l — 1 1 I 1—1—I—1—1—1—1—1—1—p-1—1—1—1—r—i—1—1—1—I—1—1—1—r-i—r—1—1—1—|—1—1—1—1—I—1—I 1 1 | 1 1 ppa 5 4 3 2 1 _ : p p « Figure 67: 2D HMQC spectrum of madangamine A (30) in C6D6 129 29 30 N-J3. 21 L-~\ i 4 30 ppm Figure 68: 2D COSY spectrum of madangamine A (30) in C6Dg Table 13. NMR data of madangamine A (30) recorded in C6D6.at 500 MHz (^H). c# 2 3 4ax 4eq 5 6eq 6ax 8eq 8 ax 9 lOax lOeq l l a x l leq 12 13 13' 14 14' 15 16 16' 17 18 19 19' 20 21 21 ' 22 22' 23 24 25 25' 26 27 28 28' 29 30 31 31 ' 32 32' 8 i 3 C 51.8 139.3 38.3 37.1 61.6 59.4 37.0 52.4 32.4 39.1 55.6 23.2 25.4 25.8 129 129 26.8 121.8 57.7 25.5 129.2 128 26.6 127.5 128.3 26.7 125.9 132.8 22.7 36.1 5 *H 3.69 bs 3.07 t(16.5) 2.21 dd(16.5, 7.7) 1.70 m 2.31 bd(10.8) 2.11 dd(10.8, 3.1) 2.72 d(11.2) 1.38 d(11.2) 3.10 d(12.0) 2.45 d(12.0) 2.40 dt(12.4, 3.4) 1.27 dt(12.4, 2.6) 1.14 m 2.81 ddd(13.5, 11.9, 5.8) 2.63 bt(13.5) 1.81 1.42 m 1.19 2.24 1.80 5.38 5.43 td(10.7, 4.1) 3.34 dt(13.3, 11.3) 2.32 dd(13.3, 3.0) 5.16 dt(11.5, 2.8) 2.47 td(l l , 5.9) 2.14 ddd(ll, 5.5, 3.3) 2.40 1.94 5.36 5.36 3.07 2.60 bd(16.9) 5.35 5.35 3.12 2.56 bd(16.5) 5.31 5.55 m 2.25 1.93 2.50 ddd(13, 10.5, 3.0) 1.00 bdd(13, 7.2) *COSY HII , Hir H4e, H5, H20 H4a,H5 H4a, H4e, HI2 H6a, H8e H6e H8a, H6e H8e HlOe HlOa HIT, H2, H12 H l l , H2, H12 H5, Hl l , H l l ' H13', H14, H14' H13, H14, H14' H14', H13, H13', H15 H14, H13, H13', H15 H14, H14', H16, H16' H16', H15, H17 H16, H15, H17 H16, H16' H19, H19' H19', H18, H20 H19, H18, H20 H4a, H19, H19' H21', H22, H22' H21, H22' H22', H21 H22, H21, H21' H22, H22' H25, H25' H25', H24, H26 H25, H24, H26 H25, H25' H28, H28' H28\ H27, H29 H28, H27, H29 H28, H28', H30 H29, H31, H31' H31', H30, H32, H32' H31, H30, H32, H32' H32', H31, H31' H32, H31, H31' *COSYLR H4e, HlOe, H19 H20 H2 H6, H6 H5, H8e H5 H6e H32' H2, H12 HlOe H18 H19 H16' H2, H17 H4a H30 H30 H31, H31' H28, H28' H29 H29 HlOa tHMBC H4e, HlOe, Hl l , H13, H20 H4e, H19 H2, H6a, H6e, HI2, H20 H4e, H6e, Hl l , Hll ' , H12 H8a, H8e, H21 H6a, H6e, HlOe, H21, H32, H32' H5, H8a, H8e, HlOe, HIT, H31', H32, H32' H2, H8a, H12, H13', H32' H2, H12 H2, H4e, H5, H6a, H6e, H8a. H8e, HlOe, Hl l , Hll ' , H32' HlOe, H14, H15 H16' H14, H16 H14', H18 H16', H19 H19 H18 H4e, H19 H6e, H8a, H8e, H22, H22' H21, H21', H24 H25 H25 H27 H25, H28 H25, H28 H26, H29, H30 H28, H31, H31' H28, H31, H31', H32, H32' H29, H30, H32, H32' H8a, H8e, HlOa, HlOe, H31' *NOE H13', H19 H20 H l l ' H8a, H12 HlOe H6a, H12 H5 H8a, H32' HlOe H2 H2 H17 H19 H20 H2 H4e, H18 H31 H31* H30 H32, H8a, H12 Correlated to proton resonance in 8 *H column. '''Correlated to carbon resonance in 8 ^C column. $ Resonance in 8lH column was irradiated; recorded at 400 MHz. a = axial, e = equatorial, LR = Long range COSY. 131 (5 1.70); between H5 and H4ax (5 3.07)/ H4eq (6 2.21), and between H5 and H6ax (5 2.11, LR)/ H6eq (5 2.31, LR). The HMBC data also supported this assignment (Figure 70). Two bond HMBC correlations were observed between C2 (5 51.8) and H l l a x (5 2.40); between C3 (8 139.3) and H4e q (5 2.21); between C5 (8 37.1) and both H4eq (8 2.21) and H6e q (8 2.31); between Cl l (8 32.4) and both H2 (8 3.69) and H12 (8 1.14); and between C12 (8 39.1) and H l l a x (8 2.40). Three bond HMBC correlations were observed between H2 (8 3.69) and both C4 (8 38.3) and C12 (8 39.1); between H l l a x (8 2.40) and C5 (8 37.1); between H6eq (8 2.31) and C12 (8 39.1); and between H6ax (8 2.11) and C4 (8 38.3). 2.40 H 8 1.14H Jb^Jfc* 3.69 H1.27 2.11 H-2.31 H 1.70 H H3.07 2.21 H H HMBC Figure 69. Proton spin system of the central core and selected HMBC correlations in madangamine A (30). A long-range COSY correlation observed between H6eq (8 2.31) and H8eq (8 2.72) was attributed to W-coupling. Another W-coupling was observed between the axial proton H10ax (8 3.10) and one appendage proton H32' (8 1.00) in the COSYLR experiment (Figure 71). The aliphatic quaternary carbon at 8 37.0 (C9), that was indicated by the APT experiment (Figure 66), could be positioned between C8, C10, C12, and C32 based on COSYLR (Figure 72) and more importantly on a series of HMBC correlations (Figure 72). Thus, HS^ (8 2.72), H10eq (8 2.45), and H32' (8 1.00) all showed two bond HMBC correlations to C9 (8 37.0). In addition, both H8a x (8 1.38)/ H8eq (8 2.72) were correlated into C10 (8 52.4), C12 (8 39.1), and C32 (8 36.1) via three bond couplings. H10ax (8 3.10)/ H10eq (8 2.45) showed three bond correlations to C32 (8 36.1), and H10eq was also correlated into C8 (8 59.4) and C12 (8 39.1). In turn, H32* (8 1.00) showed three bond HMBC correlations to C8 (8 59.4), C10 (8 52.4), and C12 (8 39.1). 29 30 27, 132 015 17 , ... 4 30 _jJ{^M^hAAjM^ 0o Q Q fi& D eft)" ' i ppm 3 . 5 o o -ta 9 'i Oo 0 0 Cft — q—O—S— Do MQ"c3 k B O o O o B . • o 3.0 0 «*5S ° i ^ °o i "i r-3.0 2.5 10 So fro C) o a 0 Q 0 aruji 2.0 0 —1 1 — 1 1 ! 1 . 5 1.0 •20 •30 •40 •50 60 ppm Figure 70: 2D HMBC spectrum of madangamine A (30) in C6D6 133 29 30 vis *%3-2 1 l ^ . . , . ."" , L 5=5S / 18 4 30 ppm ppm Figure 71: 2D COSYLR spectrum of madangamine A (30) in C6D6 1.00 H ^ / H 2.50 Y7 J 6 j ^ 5 '< 3 ^ H '-M /^~\ „. ' fc=^ COSYLR HMBC Figure 72. Selected COSYLR (W coupling) and HMBC correlations in madangamine A (30). The chemical shifts of the C2 (8 51.8), CIO (5 52.4), and C13 (8 55.6) resonances indicated that these three carbons were attached to nitrogen. The three bond HMBC correlations (Figures 70 and 73) observed between C2 (8 51.8) and H10eq (8 2.45)/ H13 (8 2.81); between C10 (8 52.4) and H2 (8 3.69)/ H13' (8 2.63); and between the appendage C13 (8 55.6) and H10eq (8 2.45) demonstrated that all the three carbons (C2, C10, C13) were attached to the same tertiary amine nitrogen atom. Irradiation of H2 (8 3.69) induced an NOE in HI3 (8 2.81), supporting the attachment of CI3 to Nl. A similar chemical shift and HMBC correlation analysis showed that C6 (8 61.6), C8 (8 59.4), and C21 (8 57.7) were attached to the second tertiary amine nitrogen N7 (Figures 70 and 73). The three bond HMBC correlations between H6ax (8 2.11)/ H6eq (8 2.31) and C8 (8 59.4), and in turn between H8ax (8 1.38)/ H8e q (8 2.72) and C6 (8 61.6) provided evidence for the nitrogen bridge between C6 and C8. The correlations between a methylene carbon at 8 57.7 (C21) and H6eq (8 2.31)/ H8ax (8 1.38)/ H8eq (8 2.72) identified this methylene carbon as the third substituent of the second tertiary amine. The broadening of the carbon resonances of C2, C10, and CI3 suggested that there might be a slow inversion of the nitrogen lone pair at Nl. Figure 73. The central core structure and selected NOE, COSYLR and HMBC correlations in madangamine A (30). A well resolved olefinic proton resonance at 5.16 ppm (H20) showed a strong long range COSY correlation into an allylic proton resonance at 5 3.07 (H4ax). The observation of this coupling indicated that H4ax was situated almost perpendicular to the double bond plane. HMBC correlations observed between H20 (5 5.16) and C2 (5 51.8)/ C4 (5 38.3), and in turn between H4 e q (8 2.21) and C20 (8 121.8) confirmed that the olefinic carbon C20 was attached to the tricyclic core at C3. Difference NOE experiments (Figure 74) along with the long range COSY information established the relative stereochemistry of madangamine A (30). The long range COSY correlation ascribed to ^-coupling between the axial proton H10ax (8 3.10) and one appendage proton H32' (8 1.00) indicated that the piperidine ring containing Nl was in a chair conformation and that the appendage carbon C32 was axial (Figure 72). An NOE observed between H32' (8 1.00) and H12 (8 1.14) revealed that the second piperidine ring was as-fused to the first one. Irradiation of H12 (8 1.14) induced NOEs in resonances assigned to H6ax (5 2.11), H8ax (8 1.38), and H5 (8 1.70); and irradiation of the H6ax (8 2.11) induced enhancements in H5, H12, and H8ax (Figure 74). These results indicated that the second piperidine ring containing N7 was also in a chair conformation. The exjunction of the two piperidine rings further required that C3 be attached in the axial orientation at C2 and that C4 be attached in axial orientation at C5, resulting in a slightly flattened chair conformation for the six membered carbocyclic ring. 136 H32' irradiated H8ax irradiated V^|^^sA> / \ rj^^ H19 irradiated H2 irradiated H2 H6, eq H6 ax iJ« H8 ax H5 H12 A u« H32' 3.5 3.0 — I — i — i — i — i — | — i — i — i — i — | — i — i — i — i — | — r ~ 2.5 2 .0 1.5 1.0 PPM Figure 74: Selected NOE results of madangamine A (30) in C ^ (400 MHz) Figure 75. Relative stereochemistry of the tricyclic core and selected NOEs in madangamine A (30). The remaining portion of 30, consisting of eight aliphatic methylene and eight olefinic methine carbons, had to form a pair of linear bridges between the attachment carbons CI 3, C20, C21, and C32 to complete the final two rings required by the unsaturation number. Detailed analysis of the COSY, HOHAHA, HMQC, and HMBC data supported an eight-carbon chain spanning Nl and C3 as shown. COSY correlations were observed between H20 (5 5.16) and H19 (5 3.34)/ H19' (8 2.32); between H19/ H19' and H18 (5 5.43); between H18 and H17 (8 5.38); between H17 and H16 (8 2.24)/ H161 (8 1.80); between H16/ H16' and H15 (8 1.19, both); between H15 and H14 (8 1.81)/ H14'(8 1.42); and between H14/ H14' and H13 (8 2.81)/ H13'(8 2.63). The HOHAHA (Figure 76) and HMBC data supported the connectivity indicated by the COSY data. For example, HI3 (8 2.81) showed HOHAHA correlations to HI3' (8 2.63), H14/H14' (8 1.81/1.42), H15/H15' (8 1.19), H16/H16' (8 2.24/1.80), and H17 (8 5.38); H19 (8 3.34) showed HOHAHA correlations to H20 (8 5.16), H19' (8 2.32), H18 (8 5.43), H17 (8 5.38), H16' (8 1.80), and H15/H15' (8 1.19). HMBC correlations were observed between C13 (8 55.6) and H14 (8 1.81)/ H15 (8 1.19); between C14 (8 23.2) and H13 (8 2.81)/ H16' (8 1.80); between C15 (8 25.4) and H14 (8 1.81)/ H16' (8 1.80); between C16 (8 25.8) and H18 (8 5.43); between C17 (8 129.0) and H16' (8 1.80); between C18 (8 129.0) and H19 (8 3.34); and between C20 (8 121.8) and H19 (8 3.34). 138 29 30 30 ppm PPa Figure 76: 2D HOHAHA speorum of madangamine A (30) in eg* 139 Figure 77. Structure of the side chain C13-to-C20 and selected HOHAHA correlations in madangamine A (30). Difference NOEs observed between H2 (8 3.69) and H19 (8 3.34) established the Z configuration for the A3-20 olefin (Figures 74 and 75). The 13C NMR resonances of C19 at 26.8 ppm and C16 at 25.8 ppm were consistent with the Z geometry for the A17 '18 olefin (Z alkenes have allylic carbons at 8 < 27; E alkenes have allylic carbons at 8 > 30).33»43 Two very broad 13C NMR signals assigned to C14 (8 23.2) and C16 (8 25.8) respectively suggested a slow interconversion of low-energy conformers in this part of the molecule. The remaining N7-to-C9 bridge had to account for six aliphatic methylene carbons and six olefinic methine carbons. The absence of an UV chromophore in madangamine A (30) indicated the lack of the conjugation between the three alkenes. Two pairs of geminal methylene protons [H25 (8 3.07)/ H25' (8 2.60) and H28 (8 3.12)/ H28' (8 2.56)], that had chemical shifts typical of protons on doubly allylic carbons, were all correlated into the very congested olefinic proton region around 8 5.35 in the COSY spectrum, suggesting a methylene interrupted triene substructure (C23 to C30). In addition, detailed analysis of the combined COSY and HMQC data identified that there were two sets of two aliphatic methylene carbons between both N7 and C9 and the respective ends of the triene substructure (C23 to C30). For example, both geminal protons H21/H21' (8 2.47/2.14) showed the COSY correlations to H22/H22" (8 2.40/1.93) that were in turn coupled to the olefinic proton H24 (8 5.36). A well resolved olefinic proton at a chemical shift of 5.55 ppm (H30) showed COSY correlations to H31/H31' (6 2.25/1.93) and the latter were further correlated to the appendage protons H32/H32'(5 2.50/1.00). The HOHAHA and HMBC data were in complete agreement with the indicated constitution of the N7-to-C9 bridge. For example, the well resolved olefinic proton H30 (8 5.55) showed HOHAHA correlations to the H28/H28' (5 3.12/2.56), H29 (5 5.31), H31/H31' (5 2.25/1.93), and H32/H32' (8 2.50/1.00). Again, the 13C chemical shifts of the allylic carbons (C22: 8 25.5; C25: 8 26.6; C28: 8 26.7; C31: 8 22.7) indicated that the A23-24, A26-27, and A29-30 olefins had the Z geometry. Therefore, the complete structure of madangamine A (30) is as shown. Madangamine A (30) represents the first example of a new class of pentacyclic alkaloids. The unusual central tricyclic core structure with the chair-chair-chair conformation is, to our knowledge, without precedent. The biosynthesis of madangamine A (30) could be envisioned as arising via the rearrangement of an ingenamine-type intermediate originating from a bis-3-alkylhydropyridine macrocycle. Scheme 9 outlines the proposed biogenesis for 30 by analogy with Baldwin and Whitehead's proposal for the biogenesis of the manzamines.11-4^ An intramolecular [4 + 2] cycloaddition reaction of a partially reduced to-3-alkylpyridine macrocycle generates the ingenamine type intermediate that is equivalent to ingenamine F (38). Compound 38 would undergo allylic type chemical interconversion and reduction to give intermediate 52. Fragmentation54 of 52 yields a ring opened intermediate, iminium salt 53. Redox exchange between the two nitrogen atoms followed by cyclization immediately reveals the madangamine type skeleton. The madangamines are the only examples to date of 3-alkylpiperidine alkaloids with rearranged carbon skeletons.19 141 Scheme 9. Proposed Biosynthesis of Madangamine A (30). [4 + 2] >-38 I 53 J Redox exchange 52 Cyclization >-& - H+ / -^""•„ Madangamine A (30) 54 The absolute configuration of madangamine A (30) is still unknown. All the attempts to obtain a suitable crystal for X-ray diffraction analysis have failed. However, based on the above proposed biosynthetic pathway, it is apparent that the absolute configuration of madangamine A (30) could be related to that of ingenamine F (38). Since ingenamine F (38) has the same carbon skeleton as ingenamine E (37), and also the same sign of optical rotation, it is very likely that both ingenamine E (37) and ingenamine F (38) share the same absolute chemistry. Thus, ingenamine F (38) would have the absolute configuration (2R, 55, IS, 8R). Therefore, madangamine A (30), by correlation with its biosynthetic precursor ingenamine F (38), would have the absolute configuration (25, 55, 9R, 12R) as shown in Dreiding model (Figure 78). Although madangamine A (30) has the same molecular formula as that of ingenamine F (38), its polarity is dramatically different from that of 38. The madangamine alkaloids are extremely non-polar. They were hexane soluble while the ingenamine type compounds stayed in the aqueous layer when the crude extract of Xestospongia ingens was partitioned between water and hexanes. The significant difference of the polarity between these two type compounds can be attributed to their different core structures. In madangamine A (30), for example, one of the two nitrogen atoms, namely N7, is quite rigid. Because of steric hindrance, the inversion of nitrogen atom N7 cannot occur as the cavity of the central core is too small to fit the alkyl group C21. The lone pair on the nitrogen N7 is in such an orientation that it is situated in the cage of the tricyclic core and shielded by the surrounding hydrocarbon atoms. This would result in decreasing the basicity for the N7 amine, and consequently the lower polarity of the molecule. For the ingenamine alkaloids, both lone pairs on Nl and Nl 1 are available for protonation and hydrogen bonding. Therefore, the ingenamine type compounds exhibit greater polarity. Madangamine A (30) showed in vitro cytotoxicity against murine leukemia P388 (ED50 0.93 Jig/mL) and human lung A549 (ED50 14 ng/mL), brain U373 (ED50 5.1 \ig/mL), and breast MCF-7 (ED50 5.7 jig/mL) cancer cell lines. Figure 78. Dreiding model of madangamine A (30) generated by Chem3D based on the MM2 program. C atoms are represented as grey circles with medium-size dots, N atoms as white circles with fine dots, and H atoms as small white circles. M 2.2.4.2. Madangamine B (48) 29 30 Madangamine B (48) was isolated as an optically active colorless glass. The compound 48 gave an intense parent ion in the HREIMS of at mlz 432.3498 appropriate for a molecular formula of C30H44N2 (AM -0.6 mmu). This indicated that madangamine B (48) was an isomer of madangamine A (30) with 10 sites of unsaturation as well. The structure of 48 was elucidated by analysis of the COSY, COSYLR, HMQC, HMBC, NOE, and HOHAHA data. Table 14 lists the NMR data acquired in deuterated benzene for compound 48. The *H NMR spectrum of 48 (Figure 79) showed a close correspondence to the *H NMR spectrum of the madangamine A (30), particularly the proton resonances assigned to the central core and the linear alkyl bridge connecting Nl and C3. The significant difference was the disappearance of two well resolved signals at chemical shifts of 8 1.00 ppm (H32') and 5.55 ppm (H30) present in 30, and the appearance of a broad doublet of doublet resonance at 8 1.62 (H32') and an overlapped olefinic proton at 8 5.20 (H27) in compound 48. As expected, the 1 3C and APT NMR spectra (Figure 80) of madangamine B (48) were nearly identical to those of madangamine A (30) except for the resonances assigned to the C28-to-C32 fragment (See Tables 13 and 14). The 2D NMR and NOE data were completely consistent with the proposed structure of madangamine B (48) as shown. The HMQC spectrum (Figure 81) showed all methylene and methine C-H correlations, indicating that all 44 hydrogen atoms were attached to carbons. Detailed analysis of the COSY, COSYLR, and HMBC data obtained for madangamine B (48) confirmed 29 30 N-^I3 21 /^ , ,3 ' 4 48 —i • 1 r-"" ' _• •—'—'—•—i—•——>—•—r— 5.0 4.5 4.0 •'• • • 1—-r • r-3.5 11  l • • i 1 r-3.0 2.5 "" • 1 • • i • 1 r-2.0 1.5 -»—•—|— 1.0 Figure 79: J H NMR spectrum of madangamine B (48) in C6D6 (500 MHz) 146 J S - z o en ON CO f CS ^ v NO TON 1 -*sA ^  \ « ^ r •* 9 -s •s CO -eu • a. a. § hS NO Q so « c 6 « (50 c c3 T3 S3 E I * * o 2 o Q, 2 T3 C u rn O so <u 3 i 147 29 30 2 1 / ^ . . H " " 1 ^ / is 4 48 T T- r r i PpB -=V T-I—f—i—i i l—i—r O * ^o%| •I I I I 1 I I 40 60 -100 -120 i i i—i i i r :PP" Figure 81: 2D HMQC spectrum of madangamine B (48) in C<5D6 29 30 21 Q15 1 7 5 '""I 6 4 48 ppa Figure 82: 2D COSY spectrum of madangamine B (48) in C6D6 Table 14. NMR data of madangamine B (48) recorded in C6D6. C# 2 3 4ax 4eq 5 6eq 6ax 8eq 8ax 9 lOax lOeq llax lleq 12 13 13' 14 14' 15 15' 16 16' 17 18 19 19' 20 21 21' 22 22' 23 24 25 25' 26 27 28 28' 29 29' 30 31 32 32' 8 1 3 C 51.76 139.2 38.34 37.07 61.12 59.26 37.10 52.96 32.38 38.38 56.16 23.8 25.40 26.0 128.9 129.1 26.83 122.0 57.68 24.67 129.5 128.0 26.34 129.6 129.2 28.89 31.74 133.1 128.5 40.03 8 *H 3.71, t(3.1) 3.11, m 2.24, dd(16.9, 8.4) 1.72, m 2.32, bd(10.8) 2.11, dd(10.8, 3.1) 2.48, dd(l 1.4, 1.6) 1.38,d(11.4) 3.14,d(12.2) 2.47, d(12.2) 2.37, dt(12.4, 3.5) 1.28, dt(12.4, 2.8) 1.22, m 2.89, ddd(13.8, 11.8,5.6) 2.69, td(12.5, 2.1) 1.88, m 1.46, m 1.20 1.20 2.27 1.82, m 5.40 5.45 3.34, dt(13.3, 11.3) 2.33 5.20, m 2.53, ddd(12.0, 10.4, 6.8) 2.17, ddd(9.7, 6.4, 3.2) 2.37, m 1.96, m 5.41 5.44 3.00 2.54, bd(l 7.0) 5.38 5.20, m 2.14 1.96 2.11 1.95 5.43 5.48 3.00 dd(12.3, 6.8) 1.62, ddd(12.3, 7.4, 1) *COSY H l l a , H l l e H4e, H5, H20 H4a, H5 H4a, H4e, H6e(LR), H6a(LR), H12 H6a, H5(LR), H8e H6e, H5(LR) H8a, H6e H8e HlOe HlOa Hlle , H2, H12 Hlla, H2, H12 H5, Hlla, Hlle H13', H14, H14' H13, H14, H14' H14', H13, H13', H15 H14, H13, H13', H15 H14, H14', H16, H16' H16', H15, H17 H16, H15, H17 H16, H16' H19, H19' H19', H18, H20 H19, H18, H20 H4a, H19, H19' H21', H22, H22' H21, H22, H22' H22', H21, H21', H23 H22, H21,H21*, H23 H22, H22' H25, H25' H25', H24, H26 H25, H24, H26, H27 H25, H25', H27 H25', H26, H28, H28' H28', H27 H28, H27 H29', H30 H29, H30 H29, H29' H32, H32' H32', H31 H32, H31 tHMBC H4e,H10e, H13, H20 H4e,H19 H2, H6a, H6e, H20 H4e,H6e, Hlla, Hl le H8a, H8e, H21 H6a, H6e, HlOa, H21, H32, H32' H8e, Hl le , H32.H32' H2, H8a, H8e, H32' H2 H2, H4e, H6a, H6e, H8e, HlOe H14', H15 H14, H14' H15, H16' H19 H17 H2, H4e, H19 H6e, H22 H21.H24 H21", H22, H24 H22, H25 H23 H25, H25', H27 H28, H28' H26, H29' H31 H29,H29',H31, H32.H32' H30, H32, H32' HlOa, H30, H31 *NOE Hlle H6a, H21' H5 H2,H10e H2, HlOe H2, HlOa H22 H8a, H12 Correlated to proton resonance in 8 *H column. 'Correlated to carbon resonance in 8 ^ C column. -^Resonance in 8 *H column was irradiated; recorded at 400 MHz. a = axial, e = equatorial, LR = Long Range COSY. the presence of the tricyclic core (Nl - C12) and four additional carbons attached at Nl, C3, N7, and C9 that were also found in madangamine A (30). The COSY spectrum (Figure 82) showed a spin system starting at the methine proton H2 (8 3.71) and continuing through both methylene protons H l l a x / H l l e q (8 2.37/1.28) and on into H12 (8 1.22). H12 was correlated to another methine proton H5 (8 1.72), and the latter was further coupled into a pair of rather deshielded methylene protons H4ax/H4eq (8 3.11/2.24) in the COSY spectrum. Similar to madangamine A (30), only COSYLR correlations were observed between the methine proton H5 (8 1.72) and the methylene protons H6ax/H6eq (8 2.11/2.32) in molecule 48. A long range COSY correlation observed between H4ax (8 3.11) and the vinyl proton H20 (8 5.20) identified the attachment of carbon C20. Another long range COSY correlation (W-coupling) observed between H10ax (8 3.14) and one of the methylene protons H32' (8 1.62) established connectivity between C9 and C32, and suggested the Nl piperidine ring was in a chair conformation. A network of HMBC correlations located the aliphatic quaternary carbon (8 37.1) at the C9 position. For example, three bond HMBC correlations were observed between C8 (8 59.3) and both H10eq (8 3.14) and H32 (8 3.00); between C10 (8 53.0) and both H8ax (8 1.38) and H32' (8 1.62); between C12 (8 38.4) and both H8e q (8 2.48) and H10eq (8 2.47); and between C32 (8 40.0) and H10ax (8 3.14). Similar to madangamine A (30), the chemical shifts of the C2 (8 51.8), C10 (8 53.0), and CI3 (8 56.2) resonances in madangamine B (48) indicated that these three carbons were attached to nitrogen. HMBC correlations between H2 (8 3.71) and C10 (8 53.0), and in turn between H10eq (8 2.47) and C2 (8 51.8) were consistent with the nitrogen bridge between C2 and C10. Additional correlations between H13 (8 2.89) and C2 (8 51.8) identified the methylene carbon (C13: 8 56.2) as the third substituent of the tertiary amine. A similar chemical shift and HMBC correlation analysis revealed that C6 (8 61.1), C8 (8 59.3), and C21 (8 57.7) were attached to the second tertiary amine nitrogen N7. Thus, the mutual HMBC correlations were observed between C6 (8 61.1) and H8ax (8 1.38)/ H8eq (8 2.48)/ H21 (8 2.53); between C8 (8 59.3) and H6eq (8 2.32)/H21 (8 2.53); and between C21 (8 57.7) and H6eq (8 2.32). Again, a series of NOE correlations together with the long range COSY information established the relative stereochemistry of madangamine B (48). The long range COSY correlation ascribed to ^-coupling between axial proton H10ax (8 3.14) and one appendage proton H32' (5 1.62) indicated that the appendage carbon C32 was in an axial orientation, and the first piperidine ring containing Nl was in a chair conformation. An NOE observed between H32 (8 3.00) and H l l a x (8 2.37) was consistent with the assignment as these two protons had a 1,3-diaxial relationship. NOE enhancement observed between H32' (8 1.62) and H12 (8 1.22) demonstrated that the two piperidine rings were c/s-fused to each other. ROESY and NOE correlations observed between H6ax (8 2.11) and H8ax (8 1.38) and H12 (8 1.22) revealed that the second piperidine ring containing N7 was also in a chair conformation. Irradiation of H5 (8 1.72) induced an NOE in Hlleq (8 1.28); and irradiation of the HI le q in turn induced enhancement in H5. These NOE results suggested the third six-membered ring was also in chair conformation as H5 and HI 1^ had the 1,3-diaxial relationship in the carbocyclic ring (C2-to-C5-to-C12). The remaining portion of 48, eight aliphatic methylene and eight olefinic methine carbons, had to form two linear bridges between the appendage carbons CI3, C20, C21, and C32 to complete the final two rings required by the unsaturation number. The 13C NMR chemical shifts (Table 14) assigned to the Nl-to-C7 bridge in madangamine B (48) were nearly identical to that of the corresponding carbons in madangamine A (30) (Table 13), proving the presence of side chain Nl-to-C3 with a cis-olefin at A17>18. Subtraction of the atoms present in the already identified tricyclic core and Nl-to-C7 bridge (C18H26N2) from the molecular formula of madangamine B (48) indicated that the N7-to-C9 bridge contained twelve carbons with three alkene functionalities (C12H18). Detailed analysis of the COSY, COSYLR, HOHAHA, HMQC, and HMBC data supported a twelve-carbon chain spanning N7 and C9. COSY correlations were observed from H21/H21' (8 2.53/2.17) into H22/H22' (8 2.37/1.96) that further coupled into a vinyl proton at 8 5.41 (H23). Both methylene protons H25/H25', that had typical doubly allylic resonances at chemical shifts of 3.00 and 2.54 ppm, were correlated into resonances in the severely overlapped olefinic proton region at 8 5.44 (H24) and 8 5.38 (H26), respectively. The H26 resonance (8 5.38) showed 152 further coupling with a relatively shielded olefinic proton resonance at 5 5.20 (H27), and the latter was correlated into the allylic protons H28/H28' (8 2.14/1.96). A COSYLR correlation observed between H27 and a doubly allylic proton H25 (5 3.00) confirmed the assigned connectivity. Further analysis of the COSY spectrum revealed the remaining two sets of vinyl to allylic correlations between H32/H32' (8 3.00/1.62) and H31 (8 5.48); and between H30 (8 5.43) and H29/H29' (8 2.11/1.95). Three bond HMBC correlations observed between both diagnostic protons H32/H32' (8 3.00/1.62) and the most deshielded olefinic methine carbon C30 (8 133.1); and the two bond HMBC correlations observed between C30 and H29/H29' (8 2.11/1.95) were in complete agreement with the proposed structure as shown below. Again, the chemical shifts of the allylic carbons at C22 (8 24.7), C25 (8 26.3), and C28 (8 28.9) were consistent with the Z configuration for the alkenes at A23-24 and A26'27. The chemical shift of C29 (8 31.7) indicated that the A30>31 double bond had the E geometry. HMBC H H H H H H H V H ^ COSYLR Figure 83. Selected HMBC and COSYLR correlations in madangamine B (48). Madangamine B (48) is the first metabolite isolated from the Papua New Guinea sponge Xestospongia ingens having an E double bond configuration. It is unlikely that the E olefin at A3 0 '3 1 in compound 48 is the product of isomerization of madangamine A (30) during the isolation since the extraction and separation techniques employed very mild conditions. Furthermore, it is well known that a molecule with conjugated alkenes is thermodynamically more stable than its isomer with isolated double bonds. If the isomerization did occur during the isolation, the double bond at A29>30 in madangamine A (30) would be expected to migrate to A28 '29 to yield a conjugated system instead of forming madangamine B (48) with an isolated E olefin at A30-31. 2.2.4.3. Madangamine C (49) Madangamine C (49) was obtained as an optically active colorless glass ([OCJD = +140.8° (c 0.09, EtOAc)). The HREIMS of 49 gave an intense parent ion at mlz 408.3507 corresponding to a molecular formula of C28H44N2 (AM 0.3 mmu), which required eight degrees of unsaturation in the molecule. Initially, the ID and 2D NMR data for 49 were carried out at room temperature. However, it was found that the 13C and APT NMR spectra recorded at room temperature only showed sixteen strong signals, fewer than expected twenty-six proton-attached carbon peaks and two quaternary carbon signals. Recording the spectrum at a higher temperature not only sharpened the 13C signals in the ID spectrum, it also improved the 2D spectra. The structure of madangamine C (49) was determined by extensive analysis of ID and 2D NMR data. The *H NMR spectrum (Figure 84) of 49 was similar in most respects to the lH NMR spectrum (Figure 65) of madangamine A (30). The most notable difference was the fewer numbers of olefinic proton resonances and more crowding in the upfield region for compound 49. The 13C NMR and APT spectra (Figure 85) of madangamine C (49) acquired at 65 °C contained all twenty-eight carbons although some signals were still broad. Since six deshielded carbon signals could be assigned to the three olefins, it was apparent that madangamine C (49) was also pentacyclic, as required by its unsaturation number. Comparison of the 13C NMR data obtained for madangamine C (49) with that of madangamine A (30) indicated that compound 49 also contained the tricyclic core and a linear eight carbon chain (Nl to C3). 27 28 21 /-i .M..""3, 49 ppa 5.0 4.S 4.0 3.5 3.0 2.5 2.0 1.5 1.0 Figure 84: l H NMR spectrum of madangamine C (49) in C6De at 65 °C (500 MHz) 155 •s 00 I I | o ro __/o\ _^^^ " -00 I ~^ \ ^ ^ *"* * 1 , — ""J ~ • • — 1 <0 \ \ \ ©\ IS » X in U o >o *-» 45 C E 60 C C3 -o C3 E <*_ o is o Q. C/3 C * £ 2 fc T3 C u CO oc U 3 &X) 156 27 28 1 l^i .,,. L i ^ / is 5 "i|i 4 49 Ik LkJJijM^ 40 - r 60 80 -100 T—I— - I — I — 1 — I I I I - i—i—I—i—I—r i i i i i—r--rl20 :pp» Figure 87: 2D HMQC spectrum of madangamine C (49) in C ^ Table 15. NMR data of madangamine C (49) recorded in C6D6 at 500 MHz (*H) at 65 °C. c# 2 3 4a 4e 5 6e 6a 8e 8a 9 10a lOe 11a l ie 12 13 13" 14 14' 15 15' 16 16' 17 18 19 19' 20 21 21" 22 22' 23 23' 24 24' 25 25' 26 26' 27 28 29 29' 30 30" 8 1 3 C 51.6 139.2 38.6 37.4 62.6 63.3 38.2 53.8 32.1 39.9 56.2 24.5 25.5 26.4 129.1 129.0 26.8 122.0 55.6 30.1 24.9 26.0 28.0 25.0 129.1 133.7 23.3 36.2 8 lH 3.70, t(3.2) 3.11, ddt(16.4, 11.7,3.2) 2.23 1.71 m 2.32, bd(10.7) 2.15, dd(l0.7, 3.4) 2.73, dd(l 1.8, 1.8) 1.52, d(l 1.8) 3.40, dd(l 1.9, 1.6) 2.67, d(l 1.9) 2.34, dt(12.5, 3.4) 1.26, dt(12.5, 2.9) 1.20, m 2.82, ddd(13.8, 11.7, 5.4) 2.59, td(13.8, 4.5) 1.82 1.43 1.22 1.12, m 2.24 1.84 5.39 5.43 3.33, dt(13.4, 11.1) 2.31 5.19, dt(11.7, 3.3) 2.31 2.18 1.68, m 1.43, m 1.22 1.15, m 1.62, m 1.27, m 1.49, m 1.39, m 2.20 2.06, m 5.16, m 5.44 2.25 2.09, m 2.70 ddd(l2, 3.7, 2.0) 1 0.89 ddt(12, 9.2, 1.9) *COSY Hlla, Hl le H4e, H5, H20 H4a, H5 H4a, H4e, H6e(LR), H6a(LR), H12 H6a, H5(LR) H6e, H5(LR) H8a, H6e H8e HlOe, H30'(LR) HlOa Hlle , H2, H12 Hlla, H2, H12 H5, Hlla, Hl l e H13', H14, H14' H13, H14, H14' H14', H13, H13', H15 H14, H13, H13', H15' H14, H16' H14',H16 H16', H15', H17 H16, H15, H17 H16, H16' H19, H19' H19', H18, H20 H19, H18, H20 H4a(LR), H19, H19' H21', H22, H22' H21, H22, H22' H22', H21, H21' H22, H21, H21', H23' H24 H22' H24', H23, H25 H24, H23, H23' H25 H25', H24, H24', H26, H26' H25', H26, H26 H26', H25, H25' H27 H26, H25, H25" H27 H26, H26' H28 H27, H29, H29' H29', H28, H30' H29, H28, H30 H30\ H29' H30, H10a(LR),H29 t H M B C H4e, HlOe, H13, H13', H20 H4a, H4e, H19 H2, H6a, H6e, H20 H4e H8a, H8e, H21 H6a, H6e, HlOa, H21, H30 H8e, HlOe, Hlle, H29',H30' H2, H8a, H8e, HI3', H30 H2 H2, H4e, H6e, H8e, HlOe HlOe, H14, H14', H15, H15' H13, H13', H15, H15', H16 H14, H14' H14, H14', H15', H18 H15, H15', H16' H19 H17 H2, H4e, H19 H6e, H8a H28 H24 H29, H29', H30, H30' H27, H30, H30' H8a, HlOa, HlOe, H29 *NOE 10a Hl le , H12 H8a 4a H12 H2, H16 H2 H4 Hlla |H8a ,H12 | Correlated to proton resonance in 8 ^H column. ^Correlated to carbon resonance in 8 13C column. + Resonance in 8 *H column was irradiated; recorded at 400 MHz at room temperature. a = axial, e = equatorial, LR = Long Range COSY. Detailed analysis of the COSY, COSYLR, and HMBC data obtained for madangamine C (49) confirmed the presence of the tricyclic core (Nl - CI2) and four attached carbons CI3, C20, C21, and C30 at Nl, C3, N7, and C9, respectively. The HMQC spectrum (Figure 83) showed all carbon-proton correlations, indicating that all 44 hydrogen atoms were attached to carbons. The COSY spectrum routinely identified the spin system for the central core. The most deshielded aliphatic methine proton H2 (8 3.70) showed COSY correlations through both methylene protons Hllax/Hlleq (5 2.34/1.26) into H12 (8 1.20). The H12 resonance was correlated to another methine proton H5 (8 1.71), that was further coupled into a pair of methylene protons H4ax/H4eq (8 3.11/2.23). Only COSYLR correlations were observed between the methine proton H5 (8 1.71) and methylene protons H6ax/H6eq (8 2.15/2.32) in 49. A long range COSY correlation observed between H4ax (8 3.11) and the vinyl proton H20 (8 5.19) identified the olefinic methine carbon C20 (8 122.0) via HMQC coupling. The most upfield proton resonance assigned to H30' (8 0.89) showed COSYLR correlation into a proton at 8 3.40 (H10ax), which indicated the Nl piperidine ring was still in a chair conformation. The doublet of doublet splitting of H10ax with coupling constants of 11.9 and 1.6 Hertz were appropriate for geminal and W couplings, respectively. A series of HMBC correlations led to the assignment of aliphatic quaternary carbon 8 38.2 at the C9 position. The chemical shifts of C2 (8 51.6), C6 (8 62.6), C8 (8 63.3), C10 (8 53.8), C13 (8 56.2), and C21 (8 55.6) were typical for the carbons attached to nitrogen. HMBC correlations observed between C2 (8 51.6) and H10eq (8 2.67)/H13 (8 2.83); and between C10 (8 53.8) and H2 (8 3.70) identified that C2, C10, and C13 were attached to the first tertiary amine nitrogen atom Nl. A similar analysis established connectivity of the second tertiary amine N7. Once again, the assignment of the relative stereochemistry of madangamine C (49) was based on a series of NOE correlations. For example, NOE enhancements observed between H30' (8 0.89) and H12 (8 1.20) and between H12 and H5 (8 1.71) demonstrated that the second piperidine ring containing N7 was cw-fused to the first one, and that the third carbocyclic ring (C2-to-C5-to-C12) was m-fused to the second piperidine ring. NOE correlations observed between H30 (8 2.70) and H l l a x (6 2.34); between H6ax (8 2.15) and H8ax (8 1.52), and H12 (8 1.20); and between H5 (8 1.71) and HI leq (5 1.26) confirmed that all three six-membered rings were in chair conformations. Since the proton resonances assigned to H4ax (5 3.11) and H10ax (8 3.40) were well dispersed in madangamine C (49), strong transannular NOEs (Figure 87) were observed between these two protons. Irradiation of H4ax induced 9% NOE enhancement in HlOaxi and in turn irradiation of HlOax induced 11% enhancement in H4ax. The remaining portion of 49 had to form two linear bridges between the appendage carbons C13, C20, C21, and C32 to complete the final two rings. The 13C NMR chemical shifts of C13 (8 56.2), C14 (8 24.5), C15 (8 25.5), C16 (8 26.4), C17 (8 129.1), C18 (8 129.0), C19 (8 26.8), and C20 (8 122.0) in madangamine C (49, Table 15) were nearly identical to those of the corresponding carbons in madangamine A (30) (Table 13: CI3/8 55.6, C14/23.2, C15/25.4, C16/25.8, C17/129.0, C18/129.0, C19/26.8, C20/121.8), proving the presence of Nl-to-C3 side chain with a Z-olefin at A17>18. Subtraction of the atoms present in the already identified tricyclic core and Nl-to-C7 bridge (C18H26N2) from the molecular formula of madangamine C (49) indicated that the N7-to-C9 bridge contained ten carbons with one alkene functionality (CioHis). Detailed analysis of the COSY, COSYLR, HOHAHA, HMQC, and HMBC data supported a ten-carbon chain spanning N7 and C9 as shown. The most upfield resonance at 0.89 ppm (H30') showed a COSY correlation to one of the methylene protons attached to C29 (H29, 8 2.25), which was further coupled to the olefinic proton H28 (8 5.44). H28 was correlated to another olefinic proton H27 (8 5.16) which was in turn coupled into the allylic protons H26/H26' (8 2.20/2.06). The very diagnostic proton H30' (8 0.89) showed a two bond HMBC correlation into the most shielded methylene carbon C29 (8 23.3) and a three bond coupling into the most deshielded methine carbon C28 (8 133.7), confirming the location of the vinyl group at A27-28. COSYLR correlations observed between H26 (8 2.20) and H28 (8 5.44); and between H27 (8 5.16) and H29' (8 2.09) were consistent with the above assignments. Similarly, the chemical shifts of the allylic carbons of C26 (8 25.0) and C29 (8 23.3) indicated that the alkene at A27 '28 had the Z-geometry. Some of the NMR resonances assigned to carbon atoms in this bridge were very broad when the 13C NMR spectrum of 49 was acquired at room temperature. This is probably due to the slow conformational exchange of the atoms in this bridge. 27 28 N-J2 21 L~*\ i 5 "i|i 4 4 9 H4 ax irradiated H10ax irradiated -r—1 1 1 1 1 1 1 1—i r——i 1—i 1 1 1 1 1 1 1 1—|' i r -1 1 1 1 1 r—J 1 1 1 1 1 r—i 1 1 1 1—i P—i 1 1 1 i I" 3.5 3.0 2.5 2.0 1.5 1.0 PPM 5.0 1.5 4.0 Figure 88: Selected NOE results of madangamine C (49) in C6D6 (400 MHz) 8 2.2.4.4. Conclusions The 'madangamines' type metabolites represent another new class of cytotoxic alkaloids. The basic skeleton of the madangamine metabolites contains a tricyclic core and two linear bridges. The pentacyclic skeleton of the madangamines can be envisaged to arise from the rearrangement of an ingenamine type precursor (Scheme 9). The madangamines are the only examples to date of 3-alkylpiperidine alkaloids with rearranged skeletons.19 Madangamines A (30), B (48), C (49), and D (50)* and E (51)* all share the same tricyclic core and Nl-to-C3 linear eight carbon bridge. The only variation observed in this group of alkaloids occurs in the N7-to-C9 bridge that varies in length and position and degree of unsaturation. Both madangamines A (30) and B (48) have a linear chain with twelve carbons and three unconjugated alkenes spanning N7 and C9, but B (48) has one E geometry double bond. Madangamine C (49) has a ten-carbon with one alkene functionality bridge between N7 and C9. Madangamine D (50) and E (51) contain saturated ten- and eleven-carbon bridges (N7 to C9), respectively. The tricyclic core of the madangamines possesses a diamond-lattice structure. All the three six-membered rings are in chair conformations. The two axial protons H4ax and H10ax in madangamine molecules should repulse each other as these two protons occupy the methylene carbon position of an adamantane type skeleton. Strong transannular NOEs observed between H4ax and HIO^ in madangamine C (49) provide the support for this argument. The magnitude (8 >3) of chemical shifts assigned to H4ax and H10ax is the further evidence that steric repulsion may exist between these two protons. In turn, because of this repulsion the carbons bearing these two protons demonstrate upfield shifts in the 13C NMR spectrum. The sp2 hybridized carbon situated at the C3 in the central core forces the carbocyclic ring to be in a slightly flattened chair conformation, which results in a bit more space in the central cage of the core, easing the severe steric interaction between H4ax and HIO^. The structural elucidation of madangamines D (50) and E (51) was accomplished by Edmund I. Graziani. Detailed discussion will be reported elsewhere. 2.3. Experimental 1. General Details Optical rotations were measured with a JASCO J-700 spectropolarimeter and the [OC]D values are given in 10"1 degcrr^g"1. UV, IR spectra were taken on Bausch-Lomb Spectronic-2000 spectrophotometer and Perkin-Elmer 1600 FT spectrometer, respectively. Low resolution and high resolution EI mass spectra were recorded on Kratos MS50/DS55SM mass spectrometer. The low resolution and high resolution FAB mass spectra were recorded on a Kratos Concept II HQ mass spectrometer. Gel permeation chromatography was performed using Sephadex LH-20 resin. Normal phase column chromatography was carried out either on Merck silica gel G60 (230-400 mesh) or Sigma silica gel (size: 10-40 |i). HPLC was performed on Waters instruments (996, 486, 440) with a normal phase silica gel column packed with 8MP10(i using either RI (Perkin Elmer LC-25) or UV as a detector. TLC was conducted on precoated Kieselgel 60 F254 (Art 5554; Merck) and the spots were detected by UV and/or heating after spraying with vanillin reagent. *H and 13C NMR spectra were recorded on Bruker spectrometers (WH-400 and AMX-500) at r.t., unless otherwise indicated. The chemical shifts are reported in ppm downfield from the tetramethylsilane resonance with the solvent residual peaks as the references (lH: DMSO-cfe 2.49 ppm, CD3OD 3.30 ppm, CD2C12 5.32 ppm, CDCI3 7.24 ppm, C6D6 7.15 ppm; 13C: DMSO-^6 39.5ppm, CD3OD 49.0 ppm, CD2CI2 53.8 ppm, CDCI3 77.0 ppm, C6D6 128.0 ppm). The coupling constants (J) are given in Hz. The criterion of compound purity used in this thesis is based on both lH and 13C NMR and HPLC purity. 2. Materials Specimens of Xestospongia ingens were collected by hand using SCUBA on reefs at depths of -15 to -20 m near Sek Point off Madang, Papua New Guinea in 1992. Freshly collected sponge were frozen on site and transported to Vancouver over dry ice. The sponge was identified by Dr. R van Soest. A voucher sample (ZMA 10701) has been deposited at the Zoologisch Museum, University of Amsterdam. 3. Extraction and Isolation of Ingenamines and Madangamines The sponge (200 g, wet) of Xestospongia ingens was thawed and extracted with methanol (500 mL x 3, 24 hr. between extractions). The methanol extract was filtered and concentrated in vacuo to give a dark brown aqueous suspension which was then diluted up to 300 mL and partitioned sequentially agaist hexanes (400mL x 3) and ethyl acetate (400mL x 3). The hexanes-soluble fraction (770 mg) was subjected to silica gel flash chromatography using a gradient elution of hexanes/ethyl acetate ( 1:9 to 1:1) to give three fractions A (120 mg), B (290 mg), and C (200 mg) in sequence. Fractions B and C were found mainly to be ingamine B and ingamine A respectively. Fraction B was further subjected to silica gel column (gradient elution: EtOAc:Hex;j-Pr2NH 50:50:1 to 100:0:1) to give pure ingamine B (32 mg from 50 mg crude). A normal phase HPLC chromatography of fraction A, mainly madangamine A, using an eluent of hexane:ethyl acetate:diisopropyl amine 98.4:1.5:0.1 gave crude madangamine C (20 mg) and madangamine A (60 mg). Further recycling independently of these crude products under the same HPLC conditions yielded madangamines E and D, pure madangamine C (11 mg), madangamine A (45 mg), and the minor component madangamine B (5.5 mg) in their eluting out sequence. The ethyl acetate-soluble fraction (650 mg) was chromatographed on sephadex LH-20 column using methanol first, and then ethyl acetate:methanol:water 40:10:4 as eluent to afford three fractions: fraction L (190 mg), mainly ingamines A and B plus xestocyclamine B; fraction M (440 mg), a very complex mixture of ingenamine type compounds; and fraction N (65 mg), mainly ingenamine and minor component ingenamine E. Recycling of fraction N via same column (Sephadex LH-20, EtOAc/MeOH/H20 40:10:4) gave pure ingenamine which was in a protonated form (20 mg from 200 g wet wt. of sponge). Ingrenamine F (4 mg) was obtained from fraction M (100 mg) by Sephadex LH-20 column (EtOAc/MeOH/H20 40:5:2) and then preparative silica gel TLC (eluent: EtOAc/MeOH 75:25). Fraction L was further subjected to a silica gel column (eluent: EtOAc) and followed by a preparative silica gel TLC (eluent: EtOAc//-Pr2NH 92:8) to give ingamine A (40 mg). Repeated fractionation of fractions M and N on normal-phase HPLC using an eluent of ethyl acetate/ hexane modified by small amount of diisopropyl amine and/or methanol to give ingenamine B (25 mg), keramaphidin B (17 mg), 82A (30 mg), ingenamine D (1 mg), ingenamine E (7 mg), and unprotonated ingenamine (25 mg). 82A was a mixture of two similar compounds and was subjected to repeated recrystallization from methanol to afford a pure ingenamine G (5.7 mg, colorless needle (acetonitile:methanol 4:1), m.p. 151-3 °C) and a mixture of the remaining two. Acetylation of this mixture (23 mg) in 1 mL of pyridine and 1 mL of acetic anhydride at room temperature with stirring overnight, followed by a normal phase HPLC separation (eluent: hexane / ethyl acetate / diisopropyl amine / methanol 97.5:2.5:0.05:0.05) gave pure ingenamine C acetate (7 mg) and ingenamine G acetate (6 mg), respectively. Ingenamine (27) White amorphous solid; [OC]D = +62° (c 0.14; MeOH); HRFABMS: [M + H+] mlz 397.32058 (C26H40N2O, AM 3.30 ppm); HREIMS (M+), mil 396.3137 (C26H4oN20, AM -0.3 mmu); LREIMS, mlz [formula (HREIMS), relative intensity % ] : 396 (M+ C26H40N2O, 28), 379 (C26H39N2, 13), 365 (C25H37N2, 14), 281 (C19H25N2, 29), 267 (Ci8H23N2) 32), 217 (Ci4H2iN2,22), 188 (Ci3Hi8N, 36), 93 (C6H7N, 100); *H NMR (MeOH-rf4, 500 MHz) and !3C NMR (MeOH-^4, 125 MHz), and 2D NMR data are listed in Tables 1 and 2. Ingamine A (31) Colorless glass; [oc]D = +131° (c 0.8; MeOH); IR vmax (neat film): 3307, 3010, 2943, 2924, 1660, 755, 723 cm"1; HREIMS (M+), m/z 448.3454 (C30H44N2O, AM -0.1 mmu); LREIMS, mlz [formula (HREIMS), relative intensity %] : 448 (M+ C30H44N2O, 100), 431 (C30H43N2, 22), 319 (C22H27N2, 27), 242 (C17H24N 39), 188 (Ci 3Hi 8N, 34), 108 (C7H10N, 92), 93 (C6H7N, 99); *H NMR (CDCI3, 500 MHz) and 13C NMR (CDCI3, 125 MHz), and 2D NMR data are listed in Table 3. Ingamine B (32) Colorless glass; [<X]D= +108° (c 0.5; MeOH); IR vmax (neat film): 3010, 2924, 2852, 1655, 755, 723 cm"1; HREIMS (M+), mlz 432.3504 (C30H44N2, AM 0.0 mmu); LREIMS, mlz [formula (HREIMS), relative intensity % ] : 432 (M+, C30H44N2, 100), 244 (Ci7H2 6N 26), 206 (Ci4H24N, 77), 188 (C13Hi8N, 17), 149 (C10H15N, 36), 110 (C7H12N, 81); *H NMR (CDCI3, 500 MHz) and 13C NMR (CDCI3, 125 MHz), and 2D NMR data are listed in Table 4. Ingenamine B (33) White powder or colorless glass; [CC]D = +22.4° (c 0.25, MeOH); HREIMS (M+), mlz 410.3299 (C27H42N2O, AM 0.2 mmu); LREIMS mlz (formula, relative intensity %), 410 (C27H42N2O 100), 393 (C27H41N2 30), 392 (C27H40N2 27), 391 (C27H39N2 37), 379 (C26H39N2 56), 367 (C25H37NO 19), 295 (C20H27N2 61), 281 (C19H25N2 93), 231 (C15H23N2 30), 202 (C14H20N 34), 190 (Ci3H20N 24), 189 (C13H19N 19), 188 (Ci3H18N 20), 146 (C10H12N 30), 132 (C9H10N 33), 120 (C8Hi0N 17), 106 (C7H8N22), 93 (C6H7N43),79 (C6H7 19), 67 (C5H7 27), 55 (C3H5N 26); *H NMR (MeOH-d4, 500 MHz): 8 0.77 (dd,7=9.9, 2.0 Hz, 1H), 1.28 (1H), 1.32 (1H), 1.35 (1H), 1.39 (1H), 1.40 (2H), 1.43 (1H), 1.50 (1H), 1.52 (1H), 1.53 (1H), 1.53 (1H), 1.57 (1H), 1.60 (1H), 1.78 (1H), 1.78 (2H), 1.79 (1H), 1.86 (dd,/=9.1, 2.6 Hz, 1H), 2.25 (d,/=11.3 Hz, 1H), 2.30 (td,/=12.6, 4.2 Hz, 1H), 2.33 (1H), 2.33 (1H), 2.40 (1H), 2.42 (d,/=11.3 Hz, 1H), 2.47 (dt,/=14.1, 5.0 Hz, 1H), 2.57 (t,/=12.2 Hz, 1H), 2.68 (dd,/=12.2, 4.7 Hz, 1H), 2.70 (1H), 2.72 (1H), 2.91 (dd,7=9.1, 1.8 Hz, 1H), 2.99 (td,/=12.6, 5.0 Hz, 1H), 2.99 (1H), 3.09 (dd,/=18.1, 10.7 Hz, 1H), 3.12 (bs, 1H), 3.32 (ddd,7=11.9, 10.1, 4.7 Hz, 1H), 5.54 (td,/=10.8, 4.4 Hz, 1H), 5.64 (1H), 5.64 (1H), 5.67 (td,/=10.8, 4.4 Hz, 1H), 5.90 (d,/=6.3 Hz, lH)ppm; 13C NMR (MeOH-J4, 125 MHz): 5 21.59, 23.89, 24.16, 26.26, 27.11, 27.40, 27.81, 28.41, 28.60, 34.55, 35.03, 42.59, 47.75, 50.19, 52.61, 54.72, 55.01, 55.27, 57.26, 65.25, 69.54, 121.2, 125.6, 131.3, 132.4, 132.5, 144.8ppm; 2D NMR data are listed in Table 5. Ingenamine C (34) Colorless glass; HREIMS (M+), mlz 410.3294 (C27H42N2O, AM - 0.3 mmu); LREIMS mlz (formula, relative intensity %), 410 (C27H42N2O, 100), 393 (C27H41N2, 32), 379 (C26H39N2, 13), 297 (C20H29N2, 36), 295 (C20H27N2, 44), 281 (C19H25N2, 73), 202 (C14H20N, 30), 190 (C13H20N, 31), 188 (C13H18N, 30), 174 (Ci2Hi6N, 23), 162 (CnHi 6N 21), 148 (C10H14N 23), 134 (C9Hi2N,45), 120 (C8Hi0N48), 107 (C7H9N 58), 106 (C7H8N 50), 93 (C6H7N, 96), 79 (C6H7, 42), 67 (C5H7, 60), 55 (C4H7, 55); lH NMR (MeOH-d4, 500 MHz), see Figure 40. Ingenamine C Acetate (35) Colorless glass; [oc]D = +41.6 (c 0.09, MeOH); HREIMS M+ mlz 452.3406 (C29H44N2O2, AM 0.3 mmu); LREIMS mlz (formula, relative intensity %), 452 (C29H44N2O2 49), 409 (C27H41N2O 10), 393 (C27H41N2 100), 379 (C26H39N2 96), 297 (C20H29N2 36), 295 (C20H25N2 29), 283 (C19H27N2 26), 281 (C19H25N2 34), 190 (Ci3H20N 22), 189 (C13H19N 38); 188 (Ci 3Hi 8N29), 174 (Ci 2Hi 6N24), 162 (CnHi 6 N 22), 148 (C10H14N 30), 134 (C9Hi2N37), 120 (C8Hi0N42), 107 (C7H9N 47), 106 (C7H8N46), 93 (C6H7N 75), 79 (C6H7 40), 67 (C5H7 53), 55 (C4H7 33); *H NMR (MeOH-rf4, 500 MHz): 8 1.02 (dd, 7=9.7, 2.3 Hz, IH), 1.27 (m, IH), 1.43 (m, IH), 1.48 (IH), 1.48 (IH), 1.48 (2H), 1.49 (IH), 1.56 (IH), 1.58 (IH), 1.68 (m, IH), 1.72 (IH), 1.74 (IH), 1.78 (IH), 1.79 (dd, 7=9.3, 2.7 Hz, IH), 1.79 (IH), 1.83 (m, IH), 1.91 (m, IH), 2.00 (s, 3H), 2.22 (2H), 2.24 (IH), 2.25 (IH), 2.26 (d, 7=11.2 Hz, IH), 2.35 (IH), 2.35 (IH), 2.38 (d, 7=11.2 Hz, IH), 2.40 (IH), 2.42 (IH), 2.44 (IH), 2.49 (t, 7=11.5 Hz, IH), 2.70 (dd, 7=11.9, 4.7 Hz, IH), 2.83 (ddd, 7=13.8, 8.2, 3.9 Hz, IH), 2.88 (dd, 7=9.3, 1.8 Hz, IH), 2.93 (td, 7=12.4, 5.1 Hz, IH), 3.09 (bs, IH), 4.57 (ddd, 7=11.0, 9.7, 4.7 Hz, IH), 5.39 (td, 7=11.0, 4.8 Hz, IH), 5.42 (td, 7=10.6, 5.5 Hz, IH), 5.63 (IH), 5.63 (IH), 5.91 (dd, 7=6.5, 1.5 Hz, IH) ppm; 13C NMR (MeOH-^4, 125 MHz) : 5 19.75, 21.69, 23.87, 25.29, 26.99, 27.07, 27.50, 27.57, 28.39, 28.82, 34.75, 35.38, 43.35, 48.03, 50.07, 51.36, 52.41, 54.39, 55.04, 57.43, 65.80, 73.10, 121.1, 130.7, 131.2, 131.2, 132.5, 145.0, 172.3 ppm; 2D NMR data are listed in Table 6. Ingenamine D (36) Colorless glass; HREIMS (M+), mlz 422.3292 (C28H42N2O, AM -0.5 mmu); LREIMS mlz (formula, relative intensity %), 422 (C28H42N2O 47), 405 (C28H41N2 15), 391 (C27H39N2 40), 321 (C22H29N2 11), 307 (C21H27N2 17), 293 (C20H25N2 30), 216 (C15H22N 27), 214 (C15H20N 29), 188 (Ci3Hi8N 31), 162 (C n H 1 6 N 20), 148 (C10H14N 19), 134 (C9H12N33), 120 (C8HioN26), 107 (C7H9N 29), 93 (C6H7N 100), 79 (C6H7 34), 67 (C5H7 43), 55 (C4H7 33); JH NMR (MeOH-d4, 500 MHz): 5 0.92 (dd,7=8.7, 2.8 Hz, IH), 1.27 (bd,7=11.8 Hz, IH), 1.45 (IH), 1.45 (IH), 1.47 (IH), 1.55 (IH), 1.58 (IH), 1.58 (IH), 1.68 (IH), 1.71 (2H), 1.80 (dd,7=9.3, 2.6 Hz, IH), 1.96 (IH), 2.00 (IH), 2.12 (IH), 2.13 (IH), 2.21 (dd,7=12.1 Hz, IH), 2.24 (IH), 2.24 (IH), 2.29 (d,7=12.1 Hz, IH), 2.30 (dd,7=9.9, 2.0 Hz, IH), 2.33 (IH), 2.39 (IH), 2.47 (IH), 2.48 (IH), 2.51 (IH), 2.53 (IH), 2.67 (IH), 2.68 (m, IH), 2.86 (dd,7=9.3, 2.2 Hz, IH), 2.92 (td,7=12.4, 5.1 Hz, IH), 3.06 (IH), 3.06 (bs, IH), 3.41 (ddd,7=14.0, 8.7, 5.0 Hz, IH), 5.32 (IH), 5.40 (IH), 5.47 (IH), 5.49 (IH), 5.59 (IH), 5.62 (IH), 5.95 (bd,7=6.5 Hz, IH) ppm; 13C NMR (MeOH-J4, 125 MHz): 8 21.8, 23.7, 25.8, 26.8, 26.8, 26.9, 27.6, 27.7, 36.3, 37.3, 46.1, 48.0, 53.9, 54.5, 54.6, 56.2, 57.8, 58.7, 66.6, 70.7, 122.8, 129.2, 129.4, 130.3, 130.4, 131.2, 132.5, 145.3 ppm; 2D NMR data are listed in Table 7. Ingenamine E (37) Colorless glass film; [oc]D = -23.8 (c 0.062, MeOH); HREIMS (M+), mlz 448.3458 (C30H44N2O, AM 0.5 mmu); LREIMS mlz (formula, relative intensity %), 448 (C30H44N2O 100), 431 (C30H43N2 35), 417 (C29H41N2 34), 405 (C28H41N2 23), 335 (C23H31N2 26), 281 (C19H25N2 58), 267 (Ci8H23N2 51), 242 (C17H24N 26), 217 (Ci4H2iN2 31), 188 (C13H18N 51), 148 (C10H14N 18), 146 (C10H12N 23), 134 (C9Hi2N25), 120 (CgHioN 19), 107 (C7H9N 45), 93 (C6H7N 81), 79 (C6H7 39), 67 (C5H7 36), 55 (C4H7 27); 2H NMR (MeOH-^4, 500 MHz): 8 0.77 (dd, 7=9.9, 1.9 Hz, IH), 1.36 (IH), 1.37 (IH), 1.48 (m, IH), 1.59 (bt, 7=10.9 Hz, IH), 1.68 (m, IH), 1.78 (dd, 7=9.5, 2.7 Hz, IH), 1.82 (IH), 1.86 (IH), 1.97 (bd, 7=14.4 Hz, IH), 2.02 (d, 7=11.3 Hz, IH), 2.06 (m, IH), 2.14 (IH), 2.18 (IH), 2.20 (IH), 2.22 (m, IH), 2.30 (IH), 2.32 (IH), 2.34 (IH), 2.37 (IH), 2.38 (IH), 2.44 (d, 7=11.3 Hz, IH), 2.49 (td, 7=10.7, 4.4 Hz, IH), 2.53 (t, 7=12.0 Hz, IH), 2.62 (m, IH), 2.65 (dd, 7=12.2, 4.7 Hz, IH), 2.78 (m, IH), 2.81 (m, IH), 2.88 (dt, 7=15.3, 7.4 Hz, IH), 2.99 (dd, 7=9.5, 1.9 Hz, IH), 3.03 (d, 7=1.5 Hz, IH), 3.09 (dt, 7=14.0, 7.1 Hz, IH), 3.17 (td, 7=15.8, 7.5 Hz, IH), 3.27 (ddd, 7=11.9, 10.1, 4.7 Hz, IH), 5.24 (bt, 7=10.6 Hz, IH), 5.34 (IH), 5.37 (IH), 5.37 (IH), 5.38 (IH), 5.45 (IH), 5.48 (IH), 5.52 (IH), 5.89 (d, 7=6.6 Hz, IH), ppm; 13C NMR (MeOH-^4, 125 MHz): 8 21.81, 22.89, 25.94, 26.47, 26.75, 27.22, 27.40, 29.46, 35.16, 38.22, 40.41, 45.76, 51.21, 52.53, 54.15, 55.59, 56.78, 59.41, 63.40, 69.13, 124.46, 128.57, 129.02, 129.14, 129.44, 129.74, 131.09, 132.61, 133.18, 143.46 ppm; 2D NMR data are listed in Table 8. Ingenamine F (38) Colorless glass; HREIMS (M+), mlz 432.34965 (C30H44N2, AM -0.8 mmu); LREIMS mlz (formula, relative intensity %), 432 (C30H44N2 92), 417 (C29H41N2 13), 391 (C27H39N2 20), 351 (C24H35N2 12), 337 (C23H33N2 27), 311 (C21H31N2 13), 297 (C20H29N2 21), 283 (C19H27N2 100), 217 (Ci4H2iN2 31), 192 (C13H22N 28), 190 (C13H20N 17), 188 (C13H18N 18), 174 (Ci2Hi6N 10), 160 (C11H14N 11), 110 (C7Hi2N53), 107 (C7H9N 35), 93 (C6H7N 35), 79 (C6H7 34), 67 (C5H7 36), 55 (C4H7 27); lH NMR (McOH-d4, 500 MHz) and 13C NMR (MeOH-^j, 125 MHz), and 2D data are listed in Table 9. Ingenamine G (39) Colorless needles (acetonitrile:methanol 4:1), m.p. 151-3 °C; [CC]D = +60.1 (c 0.057, MeOH); HREIMS (M+), mlz 424.3461 (C28H44N2O, AM 0.8 mmu); LREIMS mlz (formula, relative intensity %), 424 (C28H44N2O 78), 407 (C28H43N2 20), 406 (C28H42N2 20), 405 (C28H41N2 30), 393 (C27H41N2 71), 311 (C21H31N2 30), 309 (C21H29N2 56), 295 (C20H27N2 100), 281 (C19H25N2 17), 245 (C16H25N2 43), 231 (C15H23N2 16), 216 (C15H22N 34), 202 (C14H20N 30), 190 (C13H20N 40), 188 (Ci 3 H ] 8 N33) , 174 (Ci2H1 6N37), 148 (C10H14N 31), 134 (C9Hi2N40), 120 (C8Hi0N46), 106 (C7H8N 99), 94 (C6H8N 94), 93 (C6H7N 89), 79 (C6H7 44), 67 (C5H7 60), 55 (C4H7 38); *H NMR (MeOH-rf4, 500 MHz) and " C NMR (MeOH-^4, 125 MHz), and 2D NMR data are listed in Table 10. Ingenamine G Acetate (40) Colorless glass; HREIMS (M+), mlz 466.3551 (C30H46N2O2, AM -0.8 mmu); LREIMS mlz (formula, relative intensity %), 466 (C30H46N2O2 35), 423 (C28H43N2O 7), 407 (C28H43N2 58), 393 (C27H41N2 100), 311 (C2iH31N2 24), 309 (C21H29N2 20), 297 (C20H29N2 100), 295 (C20H27N2 100), 245 (Ci6H25N2 17), 231 (C15H23N2 17), 216 (C15H22N 24), 202 (Ci4H20N 25), 188 (Ci3Hi8N 17), 174 (Ci2Hi6N 18), 148 (CioHi4N31), 134 (C9H12N28), 120 (C8Hi0N29), 106 (C7H8N47), 94 (C6H8N50), 93 (C6H7N 50), 79 (C6H7 32), 67 (C5H7 43), 55 (C4H7 30); ]H NMR (MeOH-J4, 400 MHz), see Figure 55. Keramaphidin B (28) Amorphous white solid; [OC]D +29.8O (C 1.1; MeOH); HREIMS, C26H40N2 (M+) mlz: 380.319 (AM -0.0 mmu); LREIMS mlz (formula (HR), relative intensity); 380 (C26H40N2, 76), 283 (Q9H27N2, 100), 217 (C14H21N2, 25), 206 (C14H24N 27), 192 (C13H22N, 62), 190 (C13H20N, 33), 188 (C13H18N, 45), 148 (C10H14N, 27), 134 (C9H12N1 29), 110 (C7H12N 77), 93 (C6H7N 38); *H NMR (MeOH-</4, 500 MHz), 5: 0.98 (ddd, J = 12.5, 5.5, 2.1 Hz, H8), 1.23 (qd, J = 14.0, 4.1 Hz, H9'), 1.27 (m, H14'), 1.44 (m, H23'), 1.49 (m, H22'), 1.50 (m, H9), 1.50, (m, H15'), 1.52 (m, H23), 1.53 (m, H14), 1.56 (m, H16'), 1.61 (m, H15), 1.68 (dd, J = 9.2, 2.6 Hz, H6'), 1.73 (m, H22), 1.75 (2H, m, H20), 1.76 (m, H19'), 2.02 (bd, J = 15.2 Hz, H241), 2.11 (m, H27'), 2.16 (d, J = 11.6 Hz), 2.21 (ddd, J = 12.5, 5.2, 1.2 Hz, HI3'), 2.26 (m, H24), 2.30 (m, H5), 2.31 (m, H28'), 2.35 (m, H27), 2.38 (m, H19), 2.38 (m, H28), 2.41 (m, H16), 2.52 (ddd, J = 13.5, 7.5, 2.5 Hz H21'), 2.70 (d, J = 11.6 Hz, H12), 2.88 (m, H10'), 2.89 (dd, J = 9.2, 1.9 Hz, H6), 2.97 (td, J = 13.5, 2.6 Hz, H10), 2.99 (td, J = 12.5, 5.2 Hz, H13), 3.18 (bs, H2), 3.24 (dt, J = 13.5, 7.5Hz, H21), 5.28(tt, J = 10.8, 2.8 Hz, H25), 5.41 (m, H26), 5.65 (m, H17), 5.65 (m, H18), 5.91 (d, J = 6.4 Hz, H4) ppm; 13C NMR (MeOH-d4, 125 MHz), 5: 20.87 (C22), 21.56 (C19), 23.79 (C16), 26.13 (C24), 26.47 (C27), 26.81 (C9), 27.06 (C14), 27.06 (C23), 27.47 (C15), 37.59 (C28), 38.76 (C5), 41.77 (C20), 44.06 (C8), 44.95 (C7), 48.79 (CIO), 50.76 (C12), 54.31 (C6), 55.11 (C13), 56.88 (C21), 64.63 (C2), 125.0 (C4), 131.0 (C18), 132.6 (C25), 132.8 (C17), 133.4 (C26), 142.8 (C3) ppm; 2D NMR data listed in Table 11. Preparation and Purification of Mosher Ester Derivitives of Ingenamine, Ingamine A, and Ingenamine E General procedures: To a solution of 3.5mg of ingenamine and 6 mg of 4-(dimethylamino)pryridine (DMAP) in 1 mL of methylene chloride was added 8 |uL of (R)-(-)-a-methoxy-a-(trifluoromethyl)phenylacetyl chloride (MTPA-C1) prepared according to the literature. After the mixture was stirred at room temperature for about 4 hours, 2 drops of triethyl amine was sequentially added. The reaction mixture was allowed to stir overnight at same temperature and the solution became yellowish gradually. After the evaporation of the solvent under the reduced pressure, the residue obtained was washed by water to give relatively pure (S)-Moshor ester (5 mg). Further purification by normal phase HPLC (eluent: 90:10:0.1 hexane/ethyl acetate/diisopropyl amine) yieled the pure (S)-MTPA ester (3.5 mg). Using (S)-MTPA-Cl gave the (/?)-Mosher ester. The (S)- and (7?)-Mosher esters of ingamine A and ingenamine E were prepared as described above and purified by normal phase HPLC (eluent: 95:5:0.1 haxane/ethyl acetate/ diisopropyl amine, and 92:8:0.1 haxane/ethyl acetate/diisopropyl amine, respectively). 170 (S)-MTPA-Ingenamine (45a) Colorless viscous glass ; HREIMS, C36H47N2O3F3 (M+) mlz: 612.3543 (AM 0.5 mmu); LREIMS mlz [formula (HREIMS), relative intensity]: 612 (C36H47N2O3F3, 13), 396 (C26H40N2O, 10), 379 (C26H39N2, 59), 365 (C25H37N2) 100), 283 (C19H27N2, 12), 269 (C18H25N2, 15), 190 (Ci3H2oN, 18), 189 (C9H8OF3, C13Hi9N, 41), 188 (C13H18N, 16), 134 (C9H12N, 18), 93 (C6H7N, 33); *H NMR (CD2CI2, 500 MHz) are listed in Table 12. (fl)-MTPA-Ingenamine (45b) Colorless viscous glass; HREIMS, C36H47N2O3F3 (M+) mlz: 612.3544 (AM 0.5 mmu); LREIMS mlz [formula (HREIMS), relative intensity]: 612 (C36H47N2O3F3, 24), 396 (C26H40N2O, 10), 379 (C26H39N2, 71), 365 (C25H37N2, 110), 283 (C19H27N2, 15), 269 (C18H25N2, 16), 190 (C13H20N, 14), 189 (C9H8OF3, C13H19N, 50), 188 (C13H18N, 18), 134 (C9H12N, 19), 93 (C6H7N, 34); JH NMR (CD2CI2, 500 MHz) listed in Table 12. (S)-MTPA-Ingamine A (46a) Colorless viscous glass; HREIMS, C40H51N2O3F3 (M+) mlz: 664.3850 (AM -0.2 mmu); LREIMS mlz (formula, relative intensity): 664 (C40H51N2O3F3, 0.7), 417 (C29H41N2, 11), 190 (C13H20N, 18), 189 (C9H8OF3, C13H19N, 100); lH NMR (CD2C12) 500 MHz) listed in Table 12. (#)-MTPA-Ingamine A (46b) Colorless glass film; HREIMS, C40H51N2O3F3 (M+) mlz: 664.3852 (AM 0.1 mmu); LREIMS mlz (formula, relative intensity): 664 (C40H51N2O3F3, 44), 431 (C30H43N2, 95), 417 (C29H41N2, 100), 321 (C22H29N2, 14), 283 (Q9H27N2, 12), 269 (C18H25N2, 11), 190 (C13H20N, 17), 189 (C9H8OF3, Ci3H19N, 60), 148 (C10H14N, 28), 134 (C9H12N, 27), 93 (C6H7N, 77); lH NMR (CD2C12, 500 MHz) listed in Table 12. (S)-MTPA-Ingenamine E (47a) Colorless glass film; HREIMS, C40H51N2O3F3 (M+) mlz: 664.3850 (AM -0.2 mmu); LREIMS mlz (formula, relative intensity): 664 (C40H51N2O3F3, 44), 417 (C29H41N2, 11), 190 (C13H21N, 18), 189 (C9H8OF3, C13H19N, 100); JH NMR (CD2C12, 500 MHz) 5 (AS in Hz, proton number) 2.98 (-10, H2), 5.85 (0, H4), 2.18 (-100, H5), 2.90 (-45, H6), 1.68 (-45, H6'), 1.13 (-50, H8), 4.81 (0, H9), 2.91 (+35, H10), 2.46 (+70, H10'), 2.31 (0, H12), 2.23 (0, H12'), 2.45 (-10, H13), 2.20 (-10, H13'), 2.29 (0, H14), 171 2.10 (-10, H14'), 5.37 (0, H15), 5.51 (0, H16), 2.84 (0, H17), 2.79 (0, H17'), 5.37 (0, H18), 5.38 (0, H19), 3.15 (0, H20), 2.78 (0, H20'), 5.43 (0, H21), 5.38 (-10, H22), 2.26 (-15, H23), 1.72 (-20, H23'), 1-73 (-45, H24), 1.44 (-40, H24'), 2.78 (+50, H25), 2.26 (+30, H25'), 1.54 (+20, H26), 1.38 (+25, H26'), 1.50 (0, H27), 1.32 (0, H27'), 2.06 (0, H28), 2.01 (0, H28'), 5.41 (0, H29), 5.44 (0, H30), 2.22 (0, H31), 2.22 (0, H31'), 2.34 (-10, H32), 2.12 (0, H32'), 7.50 (ArH), 7.43 (ArH), 7.43 (ArH), 7.43 (ArH), 7.50 (ArH), 3.53 (MeO). (fl)-MTPA-Ingenamine E (47b) White glass film;7t HREIMS, C40H51N2O3F3 (M+) mlz: 664.3852 (AM 0.1 mmu); LREIMS mlz (formula, relative intensity): 664 (C40H51N2O3F3, 0.7), 431 (C30H43N2, 95), 417 (C29H41N2, 100), 321 (C22H29N2) 14), 283 (Q9H27N2, 12), 269 (C18H25N2, 11), 190 (C13H21N, 17), 189 (C9H8OF3, C13H19N, 60), 148 (Q0H14N, 28), 134 (C9H12N, 27), 93 (C6H7N, 77); !H NMR (CD2C12, 500 MHz) 5 3.00 (H2), 5.85 (H4), 2.38 (H5), 2.99 (H6), 1.76 (H6'), 1.23 (H8), 4.81 (H9 ), 2.84 (H10), 2.32 (H10'), 2.31 (H12), 2.23 (H12'), 2.47 (H13), 2.22 (H13'), 2.29 (H14), 2.12 (H14'), 5.37 (H15), 5.51 (H16), 2.84 (H17), 2.79 (H17'), 5.37 (H18), 5.38 (H19), 3.15 (H20), 2.78 (H20'), 5.43 (H21), 5.40 (H22), 2.29 (H23), 1.76 (H23'), 1.82 (H24), 1.52 (H24'), 2.68 (H25), 2.20 (H25'), 1.50 (H26), 1.33 (H26'), 1.50 (H27), 1.32 (H27'), 2.06 (H28), 2.01 (H28'), 5.41 (H29), 5.44 (H30), 2.22 (H31), 2.22 (H3T), 2.36 (H32), 2.12 (H32'), 7.49 (ArH), 7.43 (ArH), 7.43 (ArH), 7.43 (ArH), 7.49 (ArH), 3.52 (MeO). Madangamine A (30) Colorless glass; [ a ] D = +319° (c 1.0, EtOAc); HREIMS (M+), mlz 432.3503 (C30H44N2, AM -0.1 mmu); LREIMS mlz (formula, relative intensity %), 432 (C30H44N2 100), 391 (C27H39N2 27), 337 (C23H33N2 13), 297 (C20H29N2 15), 283 (C19H27N25I), 254 (Ci8H24N35),240 (Ci7H22N 25), 108 (C7Hi0N27), 107 (C8Hn 21), 91 (C7H7 51), 79 (C6H7 47), 67 (C5H7 38); IR (film), v m a x: 3006, 2927, 2912, 2874, 2793, 2759, 1684, 1654, 1488, 1460, 1441, 1374, 1351, 1262, 1128, 1091, 924, 917, 724, 689, 668; lH NMR (C6D6 , 500 MHz): 5 1.00 (bdd,/=13, 7.2 Hz, IH), 1.14 (bs, IH), 1.19 (2H), 1.27eq (dt,/=12.4, 2.6 Hz, IH), 1.38ax (d,/=11.2 Hz, IH), 1.42 (m, IH), 1.70 (m, IH), 1.80 (IH), 1.81 (IH), 1.93 (IH), 1.93 (IH), 2.1 lax (dd,./=10.8, 3.1 Hz, IH), 2.14 (ddd,7=ll, 5.5, 3.3 Hz, IH), 2.21eq (dd,/=16.5, 7.7 Hz, IH), 2.24 (IH), 2.25 (IH), 2.31eq (bd,/=10.8 Hz, IH), 2.32 (dd,/=13.3, 3.0 Hz, IH), 2.40ax (dt,/=12.4, 3.4 Hz, IH), 2.40 (IH), 2.45eq (d,/=12.0 Hz, IH), 2.47 (tdj=ll, 5.9 Hz, IH), 2.50 (ddd,/=13, 10.5, 3.0 Hz, IH), 2.56 (bd,/=16.5 Hz, IH), 2.60 (bd,/=16.9 Hz, IH), 2.63 (bt,/=13.5 Hz, IH), 2.72eq (d,/=11.2 Hz, IH), 2.81 (ddd,/=13.5, 11.9, 5.8 Hz, IH), 3.07ax (t,/=16.5 Hz, IH), 3.07 (IH), 3.10ax (d,/=12.0 Hz, IH), 3.12 (IH), 3.34 (dt,/=13.3, 11.3 Hz, IH), 3.69 (bs, IH), 5.16 (dt,/=11.5, 2.8 Hz, IH), 5.31 (m, IH), 5.35 (IH), 5.35 (IH), 5.36 (IH), 5.36 (IH), 5.38 (IH), 5.43 (td,/=10.7, 4.1 Hz, IH), 5.55 (m, IH) ppm; 13C NMR (C6D6> 125 MHz): 5 22.7, 23.2, 25.4, 25.5, 25.8, 26.6, 26.7, 26.8, 32.4, 36.1, 37.0, 37.1, 38.3, 39.1, 51.8, 52.4, 55.6, 57.7, 59.3, 61.6, 121.8, 125.9, 127.5, 128.0, 128.3, 129.0, 129.0, 129.2, 132.8, 139.3 ppm; 2D NMR data are listed in Table 13. Madangamine B (48) Colorless glass; [oc]D = +150.7° (c 0.067, EtOAc); HREIMS (M+), miz 432.3498 (C30H44N2, AM -0.6 mmu); LREIMS miz (formula, relative intensity %), 432 (C30H44N2, 100), 378 (C26H38N2 17), 337 (C23H33N2 16), 283 (C19H27N2 33), 269 (C18H25N2 19), 254 (C18H24N 16), 242 (C17H24N 22), 91 (C7H7 27), 79 (C6H7 25), 67 (C5H7 20); *H NMR (C6D6, 500 MHz): 5 1.20 (m, 2H), 1.22 (m, IH), 1.28 (dt,/=12.4, 2.8 Hz, IH), 1.38 (d,/=11.4 Hz, IH), 1.46 (m, IH), 1.62 (ddd,/=12.3, 7.4, 1 Hz, IH), 1.72 (m, IH), 1.82 (m, IH), 1.88 (m, IH), 1.95 (IH), 1.96 (m, IH), 1.96 (IH), 2.11 (dd,/=10.8, 3.1 Hz, IH), 2.11 (IH), 2.14 (IH), 2.17 (ddd,/=9.7, 6.4, 3.2 Hz, IH), 2.24 (dd,7=16.9, 8.4 Hz, IH), 2.27 (IH), 2.32 (bd,/=10.8 Hz, IH), 2.33 (IH), 2.37 (dt,/=12.4, 3.5 Hz, IH), 2.37 (m, IH), 2.47 (d,/=12.2 Hz, IH), 2.48 (dd,/=11.4, 1.6 Hz, IH), 2.53 (ddd,/=12.0, 10.4, 6.8 Hz, IH), 2.54 (bd,/=17.0 Hz, IH), 2.69 (td,/=12.5, 2.1 Hz, IH), 2.89 (ddd,/=13.8, 11.8, 5.6 Hz, IH), 3.00 (dd,/=12.3, 6.8 Hz, IH), 3.00 (IH), 3.11 (m, IH), 3.14 (d,/=12.2 Hz, IH), 3.34 (dt,/=13.3, 11.3 Hz, IH), 3.71 (t,/=3.1 Hz, IH), 5.20 (m, IH), 5.20 (m, IH), 5.40 (IH), 5.38 (IH), 5.41 (IH), 5.43 (IH), 5.44 (IH), 5.45 (IH), 5.48 (IH), ppm; 13C NMR (C6D6, 125 MHz): 5 23.8, 24.67, 25.40, 26.0, 26.34, 26.83, 28.89, 31.74, 32.38, 37.07, 37.10, 38.34, 38.38, 40.03, 51.76, 52.96, 56.16, 57.68, 59.26, 61.12, 122.0, 128.0, 128.5, 128.9, 129.1, 129.2, 129.5, 129.6, 133.1, 139.2 ppm. 2D NMR data are listed in Table 14. Madangamine C (49) Colorless glass; [ a ] D = +140.8° (c 0.09, EtOAc); HREIMS (M+), miz 408.3507 (C28H44N2, AM 0.3 mmu); LREIMS miz (formula, relative intensity %), 408 (C28H44N2, 100), 296 (C21H30N 18), 282 (C2oH28N 13), 230 (Ci6H24N33), 216 (Ci5H22N 24); *H NMR (C6D6, 500 MHz): 8 0.89 (ddt,/=12, 9.2, 1.9 Hz, IH), 1.12 (m, IH), 1.15 (m, IH), 1.20 (m, IH), 1.22 (IH), 1.22 (IH), 1.26 (dt,/=12.5, 2.9 Hz, IH), 1.27 (m, IH), 1.39 (m, IH), 1.43 (m, IH), 1.43 (IH), 1.49 (m, IH), 1.52 (d,/=11.8 Hz, IH), 1.62 (m, IH), 1.68 (m, IH), 1.71 (m, IH), 1.82 (IH), 1.84 (IH), 2.06 (m, IH), 2.09 (m, IH), 2.15 (dd,/=10.7, 3.4 Hz, IH), 2.18 (IH), 2.20 (IH), 2.23 (IH), 2.24 (IH), 2.25 (IH), 2.31 (IH), 2.31 (IH), 2.32 (bd,/=10.7 Hz, IH), 2.34 (dt,/=12.5, 3.4 Hz, IH), 2.59 (td,./=13.8, 4.5 Hz, IH), 2.67 (d,/=11.9 Hz, IH), 2.71 (ddd,/=12, 3.7, 2.0 Hz, IH), 2.73 (dd,/=11.8, 1.8 Hz, IH), 2.82 (ddd,/=13.8, 11.7, 5.4 Hz, IH), 3.11 (ddt,/=16.4, 11.7, 3.2 Hz, IH), 3.33 (dt,/=13.4, 11.1 Hz, IH), 3.40 (dd,/=11.9, 1.6 Hz, IH), 3.70 (t,/=3.2 Hz, IH), 5.16 (m, IH), 5.19 (dt,/=11.7, 3.3 Hz, IH), 5.39 (IH), 5.43 (IH), 5.44 (IH) ppm; 13C NMR (C6D6, 125 MHz): 6 23.32, 24.54, 24.91, 25.00, 25.48, 26.00, 26.39, 26.84, 27.99, 30.12, 32.07, 36.23, 37.35, 38.16, 38.58, 39.92, 51.59, 53.75, 55.61, 56.15, 62.59, 63.28, 122.0, 129.0, 129.1, 129.1, 133.7, 139.2 ppm; 2D NMR data are listed in Table 15. Part III. Polymastiamides A-F. Novel Steroid/Amino Acid Conjugates. Isolated from the Norwegian Marine Sponge Polymastia boletiformis 3.1. Introduction This chapter describes the isolation, structure elucidation, and biological activities of polymastiamides A-F (65, 68, 70, 72, 74, 76), the steroid and amino acid conjugates, from the Norwegian sponge Polymastia boletiformis (Lamarck, 1815). Specimens of the sponge (Figure 89) were collected in Norway in 1992 as part of an ongoing investigation of the secondary metabolites of cold water marine invertebrates. The crude extracts of sample NOR-92-41 exhibited antimicrobial activity in preliminary bioassay screening. 3.1.1. Taxonomy and Description of Polymastia boletiformis Sponges of the genus Polymastia are Demospongiae belonging to the family Polymastiidae within the order Tetractinomorpha according to W.C. Austin (Scheme 10).55a The specimens used for this study were collected by hand using SCUBA at depths of 20 - 25 meters of water near Bergen, Norway.55b The organisms had a firm textured body with raised oscula on the surface. They were bright orange in color and round in shape, and up to 12 cm in diameter and thickness. The sponge was identified by Dr. R. van Soest and a voucher sample has been deposited at the Zoological Museum, University of Amsterdam (ZMA POR 10170). **»?»»»>.-«*W •^gJWJSB^ -•** "•"•.iiif&W^ Figure 89. Polymastia boletiformis sponge. Scheme 10. Phylogenetic classfication of Polymastia boletiformis. 55a Phylum Porifera (sponges) Class Calcarea Demospongiae Hexactinellida Sclera spongiae subclass Homoscleromorpha Tetractinomorpha Ceractinomorpha Order Choristida Spirophorida , Lithisida Axinellida Hardromerida Family Genus Polymastiidae Sphaerotylus Polymastia Weberella Species P. boletiformis 3.1.2. A Brief Review of Marine Sponge Steroids Related to the Polymastiamides Sponges have proven to be the extremely rich source of novel sterols. Most of the 200 new monohydroxysterols found in marine organisms have been isolated from sponges.7 In addition, a number of polyoxygenated steroids, of which most are biologically active and structurally unique, have been reported in the last few years.8 Halistanol sulfate (55) isolated from Halichrondria moorei56* is the first example of the growing list of sulfated polyhydroxysteroids from sponges. Containing a rm-butyl moiety at the end of side chain and the 2p\3oc,6a-trisulfate, it was reported to be active against the HIV virus. Steroid 56, isolated from the sponge Toxadocia zumi,56b has the cholesterol skeleton with a sulfate at C-3 and the 19-methyl group oxidized to a carboxyl group. Compound 55 together with two other analogs showed antimicrobial, antifeedant, and cytotoxic activities that might be in part responsible for the lack of fouling organisms on Toxadocia zumi.56b s • Na +"03S O ^ ^ V L ^ Y ^ - ^ Na+"03S O ^ ^ ^ ^ T OS03"Na + 55 A novel group of sterol sulfates were discovered as antiviral substances in the sponge Petrosia weinbergi,57 collected off Acklin Island, Bahamas. An example is the orthoester disulfate B (57) that has an unprecedented cyclopropyl and ortho ester-containing side chain. The structure of 57 was determined on the basis of spectroscopic analysis. Contignasterol (1) is a highly oxygenated steroid with a cyclic hemiacetal functionality in its side chain. It was isolated from the Papua New Guinea sponge Petrosia contignata and it represented the first naturally occurring steroid with the 14pVproton configuration.9 NaT 0 3 S O HOOC 56 Recently, several secosterols have been isolated from sponges. Blancsterol (58), a polyhydroxylated 9,11-secosteroid isolated from the Northeastern Pacific marine sponge Pleraplysilla sp.,58 showed in vitro cytotoxicity against LI210 murine leukemia, drug sensitive MCF-7 human breast cancer, and drug resistant MCF-7 Adr human breast cancer cell lines. Jereisterol B (59), a unique 8,14-secosteroid with 3P-methoxy group, was isolated from the Pacific sponge Jereicopsis graphidiophora.59 Steroids with a A8-14 double bond are not frequently encountered. An example is A8-14-3B,7oc-dihydroxysterol 60, obtained from the Mediterranean Pellina semitubulosa.60 Sterols with a D-ring unsaturation are extremely rare. A rare A14 '15 unsaturated sterol, the 3B,16a-diol 61, was recently isolated from the sponge Topsentia aurantiaca.61 The only other examples are found in the starfish Echinaster sepositus^ and from cultured marine dinoflagellates.6^ HO' 60 61 The polymastiamides, which will be discussed in detail in this chapter, have been isolated from the Norwegian sponge Polymastia boletiformis. The interesting side chain modification in polymastiamides, involving an amide bond linking the steroid part to a non-protein oc-amino acid, is to the best of our knowledge the first example of this type in nature.64 Carolisterols A-C (62-64), a group of starfish steroids, were isolated from the starfish Styracaster caroli collected off the New Caledonia coast after our discovering of the polymastiamides.65 The amide linkage connecting the steroid side chain and D-cysteinolic acid in carolisterols A-C (62-64) is the only example in the literature related to the polymastiamides. OH HO S03 'Na + 62. Carolisterol A R=OH; RJ= -*OH 63. Carolisterol B R=OH; R*= = 0 64. Carolisterol C R=H; R ' = " " O H 180 3.2. Results and Discussion 3.2.1. Isolation of steroidal metabolites from Polymastia boletiformis Specimens of Polymastia boletiformis were collected by hand using SCUBA off Korsnes Peninsula, Norway, in July 1992. Freshly collected sponge was frozen on site, transported to Vancouver over dry ice, and stored frozen. A portion of the frozen sponge was chopped, immersed in MeOH, and soaked at room temperature for 2 days. Concentration of the decanted methanol extract in vacuo gave a red suspension that was partitioned between an aqueous solution (1:1 H20/MeOH) and hexanes. The hexane-soluble fraction, appearing to be mainly fatty acids and normal steroids, was not further pursued. The aqueous layer was concentrated, lyophilized, and then suspended in methanol. The methanol filtrate was concentrated in vacuo and chromatographed on Sephadex LH-20 (eluent: MeOH) to give several fractions. lH NMR spectra indicated that the major fraction contained a complex mixture of steroidal compounds containing aromatic resonances. The mixture was chromatographed by reversed-phase silica column (gradient: H20/MeOH = 100:0 to 50:50), and subsequently Sephadex LH-20 column (eluent: EtOAc/MeOH/H20 = 40:10:4) chromatographies to yield crude polymastiamide A (65) and a complex mixture of closely related steroids. Repeated Sephadex LH-20 chromatography (eluent: EtOAc/MeOH/H20 = 40:10:4) of the crude sample successfully led to the isolation of the pure natural product polymastiamide A (65), but failed to purify other closely related metabolites. A minor fraction containing a number of nucleosides as indicated by the *H NMR was not further investigated. Once the pure compound polymastiamide A (65) was in hand, it was recognized that the difficulty in separating other related polymastiamides could be attributed to the very polar sulfate and carboxylic acid groups, which dominated the polarity of these molecules. In order to exploit the convenience and efficiency of normal phase HPLC separation and purification techniques, all the other fractions containing polymastiamide analogs were combined and methylated in MeOH/HCl, and purified by normal phase HPLC separation to give pure desulfated polymastiamide methyl ester derivatives 66, 69, 71, 73, 75, and 77. Polymastiamides B-F (68, 70, 72, 74, 76) were isolated only as their methyl ester counterparts 69, 71, 73, 75, 77. In addition, the methylation reaction mixture also yielded the rearranged products 7 8 - 8 1 and a C-ring benzenoid sterol 82. 3.2.2. Structure Elucidation of Polymastiamides A-F (65-76) and the Related Compounds Both the natural product polymastiamide A (65) and its methyl ester derivative (66) were obtained and their structures were solved by extensive analyses of 2D NMR and mass spectrometric data. The carbon-carbon connectivities were mainly based on HMBC results and COSY correlations. Relative stereochemistries were determined using NOE and ROESY data, and finally by comparison with previously reported data. The absolute configuration of the amino acid residue was determined using Marfey's methodology. The structures of the remaining analogs polymastiamides B-F (68, 70, 72, 74, 76) were deduced from their desulphated methyl ester derivatives 69, 71, 73, 75, 77. In addition, the structures of the rearranged products 78, 79, 80, 8 1 , which were thought to be derived from the natural metabolites 65, 68, 70, 76, respectively, were obtained by spectroscopic methods and by comparison with the literature data. A rare C-ring benzenoid steroid was also isolated from the reaction mixture and its lH and 13C NMR assignments were characterized by an interpretation of ID and 2D NMR data collected for 82. Compound 82 appeared to be a synthetic artifact since its structural features were very similar to the previously reported synthetic products. 65 R = H Rx = S 0 3 ' R" = CH3 X = OCH3 66 R = CH3 R1 = H R2 = CH 3 X = OCH3 68 R = H R1 = S0 3 " R2 = H X = OCH3 69 R = CH3 R1 = H R2 = H X = OCH3 74 R = H R1 = S0 3 " R2 = CH3 X = H 75 R = CH3 R1 = H R2 = CH3 X = H 70 R = H R1 = S0 3 " R2 = CH 3 X = OCH3 71 R = CH3 R1 = H R2 = CH3 X = OCH3 72 R = H R1 = SO, R = H 73 R = CH3 R = H R = H X = OCH3 X = OCH3 76 R = H R1 = S0 3 " R2 = CH 3 X = H 77 R = CHi R1 = H R^ = CH* X = H 183 31 39 Q C 0 2 C H 3 30 33 HO r 4 Y 5 78 R" = C H 3 X = O C H 3 Y = 28 26 79 Rz = H X = OCH3 80 R z = C H 3 X = OCH3 81 R^ =CflU X = H 26 24 Y = V ^ 25 26 24 25 26 HO 37 ^ T "*OCH3 36 38 3 29 8 2 3.2.2.1. Polymastiamide A (65)64 29 65 R = H R1 = S 0 3 ' 66 R = CH3 R1 = H Polymastiamide A (65) was isolated as an optically active amorphous solid. It gave a parent ion at mlz 684.35480 in the negative ion HRFABMS (Figure 90) appropriate for a molecular formula of C38H54NO8S (AM -3.24 ppm). An intense IR band at 1249 cm-1 suggested that the sulfur atom was present as a sulfate functionality. A broad OH/NH stretching band that extended from 3640 to 2400 cm-1 with a maximum at 3519 cm-1 and carbonyl stretching bands at 1730 and 1656 cm-1 were tentatively assigned to carboxylic acid and amide functional groups. The confirmation of the presence of the sulfate and carboxylic functionalities came from the reaction of polymastiamide A (65) with MeOH/HCl to give the methyl ester (66). Compound 66 gave a parent ion in the HREIMS (Figure 93) at mlz 619.4235 appropriate for a molecular formula of C39H57NO5 (AM -0.2 mmu), indicating that the transformation of 65 to 66 involved the loss of the one sulfur and three oxygen atoms and the gain of one carbon and three hydrogen atoms. The water-soluble fraction from the methanolysis reaction mixture gave a white precipitate with BaCl2, suggesting the presence of sulfate ion. The intense band at 1249 cm-1 in the IR spectrum of 65, which was attributed to the sulfate functionality, was absent in the IR spectrum of the derivative 66. The loss of a carboxylic acid OH stretching band in the IR spectrum of compound 66 was consistent with the conversion of the carboxylic acid in polymastiamide A (65) to a methyl ester 66. Isimsn0104 Scan 1 (Av 1-5 Acq) 100%=11438 mv 31 May 93 16:48 LRF -LS1MS SL 4910 * Matrix : 3-Nitrobenzylalcohol 153 97 80 k__Ji. 122 * 137 I II I 168 199 233 182 I I 216 ,„.. .il |Jl,,.,..,,.rl., f||.i..lii ... ,..ll () .,.,,,, ,)[.. 306 255 289 325 369 |,.JU ,mi,li,.,.....nlHii...i...iJll 1, .....i.!...),. ,, V I I I | I I I I | I I'""""l I" 250 300 393 •['•'"•'[•"'"I 400 100 150 200 350 421 Iu-449 475 1 1 450 fuT~T 505 rui 527 560 583  6i 0 684 .^JILI—^—..J ,.i.i...i.| ,JI li. 640 + 500 550 600 7 650 706 i... f r i r 700 746 I 750 Figure 90: Low resolution FAB mass spectrum of polymastiamide A (65) 186 6 Q .2 * • » ca E _>, o Q . O S a. 5/3 at 2 X 3 £0 '03so 4^5 29 65 m*m*m*mmmmbmiim**mmmmm M»*«*4kM«MNINWMiewVI*^ IT *4MMrt* mmmm NMW mmmmm mmmmmMM*mrmmma***mimm>mi* 0W ft* ftMff «* inn ri-r^-i « r ••• i i ft if iTrrmiTiTfTi n ri nni'iinrin fi rt ririTii'riTiTrn r^ ifr-ri Ti'tTrrrrTH i «'^| niii'iri r» run HI T|1'*I ITTTTITI T-I w i iffryTfrrt !•»••< n r« T-I TIT* |* t^ »T^ H »t I 140 120 100 80 60 40 20 ppn 160 Figure 92: 13C and APT NMR spectra of polymastiamide A (65) in DMSO-fife (125 MHz) c# leq lax 2eq 2ax 3ax 4ax 5ax 6eq 6ax 7eq 7ax 8 9ax 10 lleq llax 12eq 12ax 13 14 15 15" 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 26' 27 28 29 NH 30 31 COOH 32 33 34 35 36 37 [38 Table 16. lH and 13C NMR data of polymastiamide A (65) recorded in DMSO-^6-13C 35.8 28.1 80.1 37.1 50.7 24.7 29.2 125.8 48.7 36.8 19.5 36.9 42.1 141.4 25.3 26.5 56.3 18.0 13.6 33.9 18.9 32.5 31.3 34.9 149.5 115.8 168.5 19.6 15.5 55.6 172.2 129.3 129.1 113.6 158.8 113.6 129.1 55.1 *H 1.60 1.03 2.10 1.28 3.53 1.21 0.89 1.64 0.87 2.32 1.63 1.60 1.55 1.40 1.85 1.02 2.16 2.08 1.69 1.25 0.97 0.76, s 0.63, s 1.34 0.83, d(6.5) 1.32 0.96 1.46 1.13 2.54 5.61, bs 5.21, bs 0.98, d(6.8) 0.87, d(6.2) 8.48, d(7.5) 5.35, d(7.6) 11.0 7.31,d(8.7) 6.88, d(8.7) 6.88, d(8.7) 7.31,d(8.7) 3.72, s * C O S Y HI a, H2a, H2e Hle,H2a,H2e H2a, H3a, Hla, Hie H2e, H3a, Hla, Hie H2a, H2e, H4a H29, H3a, H5a H4a, H6e H6a, H5a, H7e H6e, H7a, H7e H7a, H6a, H6e H7e, H6a H l l a . H l l e Hlla,H9a,H12a,H12e Hlle,H9a,H12a,H12e H12a,Hl la ,Hl le H12e,Hl la ,Hl le H15', H16, H16' H15, H16, H16" H16', H15, H15',H17 H16, H15, H15', H17 H16, H16', H20 H21, H17, H22' H20 H22', H23, H23', H22, H20, H23, H23' H23', H22, H22',H24 H23, H22, H22*. H24 H28, H23, H23' H26' H26 H24 H4 H30 NH H34 H33 H37 H36 t H M B C H19 H29, Hie H29 H19, H29, H7e, H9a H7a,H7e,H 15',H6e,H9a,H 1 le H19, H12e, H7e H19,H6e,H9a,Hlla H18,H9,Hlla H18,Hlle,H12e H18,H12e,H15,H15',H9a,H16 H18, H21, H16' H12a H9a, Hie H21 H21 H28 H28, H26, H26' H28, H24, H26, H26' H24 NH, H26, H26', H24 H24, H23' H4 NH, H33, H37 H30 H30, H33,H34,H36,H37 H30, H34, H37 H36 H33, H34,H36,H37,H38 H34 H30, H33, H36 * R O E S Y Hla, H2a, H2e, H19 Hie, H2e, H3a, H9a H2a, Hla, Hie, H3a H2e,Hle,H19 Hla, H2e, H5a, H29 H19, H29 H3a, H6e, H7a, H9a H5a, H6a, H7e, H29 H6e, H7a, H7e, H19 H6a, H6e, H15, H15' H7e, H6a, H5a Hla,H5a,H12a HI la, H12a, H12e Hl le , H12e, H18, H19 Hlla , Hl le , H21.H18 H12e,H9a,Hlle H7e, H16, H16', H18 H7e, H15, H16, H16' H15', H16', H17, H15 H15, H15', H16, H18 H16, H20 Hlla,H12e,H15,H16',H20 Hle,H2a,H4a,Hlla,H6a H17, H18, H21 H12e, H20, H22, H23' H21, H23', H24, H28 H22, H24 H21, H24, H26', H28 H21.H22, H24, H28 H22, H26', H28, H22' H26', NH H23, H24, H26, H28 H22, H23, H23', H26' H3a, H4a, H6e H26, H30, H33, H37 H33, H37, NH H30, H34, NH H33, H38 H37, H38 H30, H36, NH H34, H36 Correlated to proton resonance in 8 column. COSY was recorded at 400 MHz. ' Correlated to carbon resonance in o column, a = axial, e = equatorial. 41-00 80 60 40 20 0 00 80 60 40 20 0 00 80 60 40 20 0 162-C L41009.75 mC = 504848, 100X = 4701H EI <""5>-;>-601 586 "]—r~ r~i—r—| r~i r~i—]—i—i—i—i—p-r 450 475 500 525 , 542 560 olli 550 I , I In I. 1 IL rX. i—i—i—i—r~i—r 575 600 619 |633 ML ~m—r i—1—i—r 650 625 285 ij.^—JAj1 .i.,|.. 1.|,i.lj?MY.n |..li|u J-250 275 300 1—f^i—r~^—i '"i "i—r 325 I ' ' ' ' I 350 375 I" I ''I"'"I" 179 400 406 ,422 f^l—r' l,ll7 |' -r 425 T 1—r 450 121 1 i T T 55 169 81 -l,J,Jlll|. • ,J-xk 95 ll..,. ., lIllll^-X. ,136 ,151 194 50 75 100 li|,..l |l,l![lll,r.,li|llll,l l.l|,l,l.[|l,[,^,l,ll|,i.i i 125 150 175 i i | ..i • .i ll 111 I , U I • I 11 I r U-, I . I • I • I i I |-u pY-Yu,,.,,,.!.!,^-,^. 200 225 Figure 93: Low resolution EI mass spectrum of polymastiamide A methyl ester 66 28 31 39 O C0 2CH3 ~30 33 HO NH •r—r^"f "T • i' i i i 7 29 26 26* 30 I 66 38 39 12 18 19 28 29 21 2 1MJM^M I UL -T—* 1 1 1-5 T •.».. y ... . r .1^11. ,i  1i. ?. i.y - ,.. 1 i-n ———y— T •• > •' r "i • ppa Figure 94: ] H NMR spectrum of polymastiamide A methyl ester 66 in CDCI3 (500 MHz) O 191 n C l i S o ~0 g co O n i < TT m \ _ /oT" CO X ,~ o in / ^ c y \ vo _ / ~ e ' -e n en so so cN u Q O \© u 4> **^  "O in a u ex 5/5 OS 2 U u 3 192 HO 4 29 66 J_L Li * • jUUCJtaA^W^L ^ 9 ° : 25 * 'i 111111111111111111111111M111111111 [ i M111111111 i i 11111111111111 ppa 6 4 2 - 50 78 -100 125 ppn Figure 96a: 2D HMQC spectrum of polymastiamide A methyl ester 66 in CDCI3 193 28 31 39 C 0 2 C H 3 30 33 HO 29 66 ppa Figure 96b: Selected region of 2D HMQC spectrum of methyl ester 66 in CDCI3 194 HO 0 A 27 N ' 1 l H 31 39 C 0 2 C H 3 = 30 33 If) 36 ,34 L35 0CH 3 38 3 ty L 1.0 L 2.0 ^ a a. »" P P P P 1' 1 1 1 1 1 1 1 I 1 r T 1 1 1 1 I 1 1 7-0 e'0 5"0 frj 3.0 2.0 l-B 3.0 L 4.0 U 5.0 6. 0 L 7.0 PM Figure 97a: 2D COSY spectrum of polymastiamide A methyl ester 66 in CDCI3 31 39 C02CH3 i.30 33 OCH-38 % h i . a 3-0 2 .5 Figure 97b: Selected PPM 1. 5 1. 0 M - 5 h 2 . 0 h 2 . 5 M . 0 'PPM region of 2D COSY spectrum of methyl ester 66 in CDC13 196 HO 0 27^N" i 1 H 31 39 C 0 2 C H 3 J^30 33 22^ || 36 34 .35 OCH3 38 3 9 0 0 • 66 JLJLi 9 o JUUCJtaA****^ .•'.• / » • .6.8 i- 50 0 .V rlOO -150 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ' ppn 6 4 2 :PP« Figure 98a: 2D HMBC spectrum of polymastiamide A methyl ester 66 in CDCI3 197 HO H C HMBC Correlation 17 40 60 80 -100 -120 14 25 ppa 1.0 0.9 0.8 0.7 -140 :pp» Figure 98b: Selected region of 2D HMBC spectrum of methyl ester 66 in CDCI3 Table 17. *H and 1 3 C NMR data of polymastiamide A methyl ester 66 recorded in CDCI3. #c le la 2e 2a 3a 4a 5a 6e 6a 7e 7a 8 9a 10 l i e 11a 12e 12a 13 14 15 15' 16 16' 17 18 19 20 21 22 22' 23 23 ' 24 25 26 26' 27 28 29 NH 30 31 32 33 34 35 36 37 38 Ti~ "" 36.3 31.2 76.5 39.7 50.7 24.9 29.7 126.0 49.4 37.5 20.0 37.4 42.6 142.1 25.7 27.0 56.6 18.1 13.9 34.5 19.1 33.1 31.8 35.8 150.9 115.6 168.9 19.8 15.2 55.8 171.7 128.7 128.5 114.4 159.8 114.4 128.5 55.3 39 152.7 lH 1.68 1.12 1.81, dq (13, 4) 1.42 3.11,td (10.8,4.4) 1.26 0.84 1.69 0.98 2.38, m 1.65 1.61 1.58 1.43 1.88, dt (12.2, 4) 1.06 2.16 2.16 1.73 1.27 1.06 0.78, s 0.69, s 1.37 0.86, d (6.6) 1.36 1.01 1.52 1.17 2.57, dt (6.8) 5.57, bs 5.22, bs 1.05, d (6.8) 0.97, d (6.3) 6.65, d (7.1) 5.55, d (7.1) 7.27, d (8.7) 6.85, d (8.7) 6.85, d (8.7) 7.27, d (8.7) 3.77, s *COSY Hla,H2a, H2e Hie, H2e Hla, Hie, H2a, H3a Hie, H2e, H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6a, H6e H5a, H6e, H7a, H7e H5a, H6e, H7a, H7e H6a, H6e, H7a H6a, H6e, H7e Hlla , H l l e H9, Hl la , H12a,H12e H9, Hl le , H12a,H12e Hlla , Hl le , H12a Hlla, Hlle , H12e H15', H16, H16' H15, H16, H16' H15, H15', H16', H17 H15, H15', H16, H17 H16, H16', H20 H17, H21, H22' H20 H22', H23, H23' H20, H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H28 H26' H26 H4a H30 NH H34 H33 H37 H36 3.72, s 1 tHMBC H2e, H19 Hie Hie, H4a, H2a, H29 H29 Hie, H6e,H7e, H19, H29, H4 H6e, H7a,H7e, Hlle , H15 H7e, H12e, H19 Hie, H6e, H9a, H19 H9a, H12e H9a, HI8 Hlle , H12e, H15, H16, H18 H7a, H12e, H15, H15', H18 H16' H15, H15' H16', H18, H20, H21 H9a H16', H21 H20, H22 H20, H21, H23, H24 H20, H22, H24, H28 H23, H26, H26', H28 H24, H26, H26', H28 H24 H24, H26, H26', NH H23, H23', H24 H33, H37, NH H30, H39 H30, H33, H34, H36, H37 H30, H34, H37 H36 H33, H34. H36, H37, H38 H34 H30, H33, H36 * ROESY H19 H3a, H5a, H9a H19 Hie, H5a, H29 HI9, H29 H3a, H9a, Hla H29 H19 H15, H15' Hla, H5a H18, H19 H18, H21 H7e, H17, H18 H7e, H17, H18 H21 H15, H15' Hl la , H20, H15, H12e, H22 Hie, H2a, H4a, H6a, Hl la H18 H12e, H16', H22, H23.H23' H18, H24, H28 H21, H26', H28 H21, H28 H22, H26' NH H23, H24, H28 H22, H26' H3a, H6e H26, H33, H37 H33, H37 H30, NH H38 H38 H30, NH H34, H36 *NOE H19 Hla, H29 H19 H6e H29 H15, H15' H18, H19 H18, H21 H7e H7e Hl la , H12e H2a,Hlla H12e H22, NH NH H26, H28 H26' H6e H26 H33, H37 H30, NH H38 H38 H30, NH H34, H36 Correlated to proton resonance in 8 *H column. COSY was recorded at 400 MHz. t Correlated to carbon resonance in 6 13C column. * Resonance in 8 *H column was irradiated; recorded at 400 MHz. a = axial, e = equatorial. The *H NMR spectrum (Figure 91) of the natural product 65 showed a series of methyl resonances in the most upfield region, diagnostic of steroids. A very broad peak at 5 11.0 was typical for a carboxylic acid proton. The NH proton was a rather broad doublet at 5 8.48, which correlated into an amino acid methine proton H30 (8 5.35) in the COSY experiment. A pair of scalar coupled doublets integrating for two protons each in the aromatic region (8 7.31 and 6.88) was appropriate for a para-substituted benzene ring. Two broad singlets at 8 5.61 and 5.21 ppm that correlated to same carbon (C26, 8 115.8) in the HMQC spectrum were assigned to olefinic methylene protons (H26 and H26'). A sharp singlet integrating for three protons at 8 3.72 suggested the methyl group was directly bonded to an oxygen atom. A proton resonance at 3.53 ppm was typical for a carbinol methine proton (H3ax). The *H NMR spectrum (Figure 94) of polymastiamide A methyl ester (66) showed a close correspondence to that of natural product 65 (Figure 91). The significant difference was the disappearance of the carboxylic acid proton resonance at 8 11.0 present in 65 and the appearance of a new methoxy proton singlet at 8 3.72 in 66. The upfield shift of the NH proton from 8.48 ppm in 65 to 6.65 ppm in methyl ester derivative 66 was due to the solvent effect of DMSO-o?6 which, being a good H-bond acceptor, can form a hydrogen bond with the NH proton. The fact that the carbinol proton (H3ax) resonance was shifted to 3.11 ppm in the ester derivative 66 from 3.53 ppm in 65 was in good agreement with the desulfation. The !3C and APT spectra also provided evidence for the transformation of 65 to 66. The ester derivative 66 of polymastiamide A (Figure 95) showed one more carbon signal at 8 52.7, an appropriate chemical shift for a methoxy carbon, than its precursor 65. Due to the deshielding effect of the sulfate group, the carbinol carbon (C3) resonance in the natural product 65 was at 80.1 ppm (Figure 92), whilst its desulfated derivative 66 appeared at 76.5 ppm. A series of ID and 2D NMR experiments, which led to the proposed structure for polymastiamide A (65), were initially carried out on the derivative 66 in order to exploit the convenience of collecting data in CDCI3. Subsequently, all of the experiments were repeated on a solution of the natural product 65 in DMSO-flf6- The NMR data for both the natural metabolite polymastiamide A (65) and its methyl ester 66 are listed in Tables 16 and 17, respectively. 200 The 13C NMR spectrum (Figure 95) of methyl ester 66 contained only 37 resolved resonances, which suggested that some element of symmetry was present in the molecule. Detailed analysis of the COSY, HMQC, and HMBC data collected for 66 revealed that a. p-methoxyphenylglycine methyl ester fragment accounted for the symmetry. A pair of scalar coupled doublets, each integrating for two protons, in the *H NMR at 8 6.85 (H34/H36) and 5 7.27 (H33/H37) could be assigned to apora-disubstituted benzene ring. A deshielded methyl resonance H38 (8 3.77) showed a three bond HMBC correlation to a deshielded aromatic carbon at 8 159.8 (C35), which was in turn coupled to H33/H37 (8 7.27) via three bonds. This pair of HMBC correlations established the presence of a methoxy substituent on the aromatic ring. HMBC 6.85 Figure 99. p-Methoxyphenylglycine methyl ester fragment and selected HMBC correlations in polymastiamide A methyl ester 66. A deshielded broad doublet at 8 6.65 was assigned to an NH proton because it did not show a correlation to any carbon in the HMQC spectrum (Figure 96). A COSY correlation (Figure 97) was observed between this NH proton (8 6.65) and a methine proton H30 (8 5.55). The methine proton H30 (8 5.55) showed a HMBC correlation (Figure 98) to a carbonyl carbon at 8 171.7 (C31), which was also coupled into the methyl ester proton resonance at 8 3.72 (H39). Further HMBC correlations demonstrated that the methine carbon (C30) was the second substituent on the benzene ring. Thus, HMBC correlations were observed between the methine proton H30 (8 5.55) and the aromatic carbon at 8 128.5 (C33/C37); and in turn between H33/H37 (8 7.27) and C30 (8 55.8). Intense fragment ions at mlz 179 (base peak, C10H11O3) and 194 (90 %, C10H12NO3) in the HREIMS of the derivative 66 (Figure 93) confirmed the presence of thep-methoxyphenylglycine methyl ester fragment. Subtraction of thep-methoxyphenylglycine methyl ester fragment (C10H12NO3) from the molecular formula of 66 (C39H57NO5) revealed that the remaining portion of the molecule had to account for an elemental composition of C29H45O2, which required seven sites of unsaturation. Since five sp2 carbon resonances at 8 168.9 (s, C27), 126.0 (s, C8), 142.1 (s, C14), 150.9 (s, C25), and 115.6 (t, C27) in the 13C and APT spectra of 66 could be assigned to an amide carbonyl and two olefins, and no additional unsaturated functional groups were detected in the 13C and APT NMR data, it was apparent that this second portion of the molecule was tetracyclic. The presence of a series of diagnostic methyl resonances between 8 0.69 to 0.96 in the *H NMR spectrum indicated that this remaining fragment was steroidal in nature. An extensive analysis of the HMBC, HMQC, and COSY data obtained for derivative 66 confirmed a steroidal substructure. A series of strong HMBC correlations from methyl protons (Mel8, Mel9, Me21, Me28, and Me29) to the neighboring carbons led to the assignment of carbons CI, C3, C4, C5, C9, CIO, C l l , C12, C13, C14, C17, C20, C22, C23, C24, and C25 (Figure 98) and also to the assignment of the methyl carbons C18, C19, C21, C28, and C29 by HMQC correlations. The most downfield aliphatic carbon resonance at 8 76.5, a typical chemical shift for a carbon attached to an oxygen, was assigned to the carbinol methine carbon C3. This carbinol carbon C3 (8 76.5) was correlated to a methyl doublet at 8 0.97 (Me29) in the HMBC spectrum. Three bond HMBC correlations were observed from both the methyl doublet (Me29, 8 0.97) and the most upfield methyl singlet (Mel9, 8 0.69) into a carbon resonance at 8 50.7, which positively identified this carbon as C5. An additional HMBC correlation was observed from the methyl doublet (Me29, 8 0.97) to the methine carbon C4 (8 39.7). All of these data indicated that the methyl Me29 was attached at C4. Further HMBC correlations were observed from the methyl singlet (Mel9, 8 0.69) to a methylene carbon CI (8 36.3), a quaternary carbon C10 (8 37.5), and a methine carbon C9 (8 49.4). By one bond proton-carbon correlations (HMQC), 13C resonances at 8 15.2 and 13.9 were assigned to the methyl doublet carbon (C29) and methyl singlet carbon 202 (C19), respectively; and the methine and methylene proton resonances were assigned on the basis of the readily identified methine and methylene carbons (CI, C4, C5, and C9). Figure 100. Selected HMBC correlations of the steroidal fragment in methyl ester 66. Similarly, a methine carbon resonance at 5 56.6 was correlated to the second methyl singlet at 8 0.78 (Mel8) and a methyl doublet at 8 0.86 (Me21) in the HMBC spectrum, suggesting that the carbon resonance at 8 56.6 was C17. The methyl singlet (Mel8, 8 0.78) showed additional HMBC correlations to the methylene carbon C12 (8 37.4), the aliphatic quaternary carbon C13 (8 42.6), and the fully substituted olefinic carbon C14 (8 142.1). HMBC correlations were also observed between the methyl doublet (Me21, 8 0.86) and the methine carbon C20 (8 34.5) and the methylene carbon C22 (8 33.1). The final methyl doublet at 8 1.05 (Me28) showed three cross-peaks at carbon resonances 8 31.8, 35.8, and 150.9, corresponding to the methylene carbon C23, the methine carbon C24, and the sp2 quaternary carbon C25, in the HMBC spectrum. This information indicated that the methyl doublet (Me28, 8 1.05) had to be attached at the methine carbon C24 (8 35.8). The carbon resonances assigned to Mel9 (8 13.9), Me21 (8 19.1), and Me28 (8 19.8), and the proton resonances attached to C10, C17, C20, C22, C23, and C24 were identified by HMQC correlations. 203 The remaining connectivity and the location of the functional groups in the steroidal substructure were established by analyzing COSY, APT, and HMBC data. Since the Mel8/C14 HMBC correlation already located the olefinic carbon at C14, and 13C APT data showed that the three remaining olefinic carbons in 66 included two quaternary carbons and one methylene carbon, it was apparent that the second olefinic carbon in the steroid nucleus had to be the quaternary carbon located at C8 (5 126.0). A deshielded methine resonance at 5 3.11 in the *H NMR spectrum (Figure 77) was assigned to the carbinol proton H3 since it was coupled to the oxygenated carbon resonance C3 (5 76.5) in the HMQC spectrum. In the COSY spectrum of 66, the carbinol proton H3a x (8 3.11) showed correlations to a pair of geminal methylene proton resonances at 8 1.81 and 1.41 (H2eq/H2ax), and the latter were further correlated to Hleq (8 1.68) and H l a x (8 1.12). Further analysis of the COSY data established the whole spin system beginning from the Hleq/Hlax through H5ax (8 0.84) to H6eq/H6ax (8 1.69/ 0.81) and the allylic methylene protons H7eq/H7ax (5 2.38/ 1.65). Analogously, spin systems H9ax to H12eq/H12ax and H15/H15' through H17 to the side chain protons H24 and Me28 were identified on the basis of COSY data (Table 17). Comparison of the 13C NMR assignments for the steroid nucleus of 66, obtained from HMBC, HMQC, and COSY data, with the literature values for the steroid 6766 confirmed the proposed steroidal substructure and also provided evidence for the relative configurations at C5, C9, CIO, C13, C17, and C20 as shown. The magnitude of the scalar coupling constants of H3ax (td, / = 10.8 and 4.4 Hz) indicated that the carbinol proton H3 was axial and further required that H43X occupy the axial orientation with the methyl group Me29 equatorial. A series of difference NOEs and ROESY correlations observed between Mel9 and H2ax, H4ax, H6ax, and H l l ^ ; and between Mel8 and Hl l a x , and H20 was completely consistent with the standard steroidal ring system. 204 Figure 101. Comparison of 13C NMR assignments for 66 with literature values for 67. Further analysis of the HMBC, HMQC, and COSY data for 66 revealed the nature of the link between the steroid and p-methoxyphenylglycine fragments. A pair of broad proton singlets at 8 5.57 (H26) and 5.22 (H26'), which were both correlated to a carbon resonance at 8 115.6 in the HMQC spectrum, were assigned to an olefinic methylene functionality. HMBC correlations were observed from both of the olefinic methylene protons (H26/H26') to a quaternary olefinic carbon resonance at 8 150.9 (C25) and to a carbonyl resonance at 8 168.9 (C27). The magnitude of the chemical shifts assigned to C25 (8 150.9) and C27 (8 168.9) suggested that there may exist an a/J-unsaturated carbonyl system. An additional HMBC correlation between the NH proton resonance at 8 6.65 and the carbonyl resonance C27 (8 168.9) revealed that the p-methoxyphenylglycine fragment was linked to the a,/?-unsaturated carbonyl via an amide bond. The acrylamide substructure defined above could be most readily accommodated by the C25, C26, and C27 carbons of a standard steroidal side chain skeleton. HMBC correlations from the olefinic methylene protons H26/H26' (8 5.57/ 5.22) to the methine carbon at 8 35.8 (C24) further confirmed the indicated side chain connectivity in 66. Irradiation of olefinic methylene proton H26 (8 5.57) induced an NOE enhancement in the NH proton; and irradiation of the other olefinic methylene proton H26' (8 5.22) induced an NOE enhancement in Me28. This information indicated that H26 (8 5.57) was cis to amide functionality and that H26' (8 5.22) was cis to Me28. Reaction of the methyl ester 66 with aqueous HBr followed by derivatization with Marfey's reagent67 and HPLC analysis showed that p-methoxyphenylglycine had been formed in the hydrolysis reaction. Comparison of the HPLC retention times of the Marfey's reagent derivative of the p-methoxyphenylglycine liberated from 66 by hydrolysis with authentic standards revealed that the amino acid residue in 66 had the L-configuration. The initial conversion of polymastiamide A (65) to the derivative 66 involved hydrolysis of a sulfate functionality and formation of a methyl ester. With the structure of 66 established, it was apparent that the sulfate group had to be attached to the C3 hydroxyl group and that the p-methoxyphenylglycine residue contained a free carboxylic acid in the natural product 65. The NMR and MS data for polymastiamide A (65) (Tables 16) were completely consistent with these conclusions. Polymastiamide A (65) exhibited in vitro antimicrobial activity against various human and plant pathogens [MIC's in a 1/4-in. disk assay: Staphylococcus aureus (100 (ig/disk), Candida albicans (75 jig/disk) and Pythium ultimum (25 |j,g/disk)]. 3.2.2.2. Polymastiamide B (68) 206 68 R = H R1 = S0 3 " 69 R = CH3 R1 = H Polymastiamide B (68) was obtained as its methyl ester derivative 69. A parent ion was observed in the HREIMS of 69 at rnlz 605.40806 appropriate for a molecular formula of C38H55NO5 (AM +0.0 mmu), differing from that of polymastiamide A methyl ester (66) only by one carbon and two hydrogen atoms. The *H NMR spectrum (Figure 102) of methyl ester 69 displayed a close resemblance to that of methyl ester 66, particularly in the downfield region. For example, a pair of doublets at 5 7.27 (H33/H37) and 6.85 (H34/36) that were assigned to the aromatic ring, the NH proton doublet (8 6.65), a methine proton resonance (H30, 8 5.55), and two methoxy singlets at 8 3.77 and 3.72 in 69 had exactly the same chemical shifts and coupling constants as that of 66, implying that compound 69 also contained a p-hydroxyphenylglycine methyl ester fragment. The most significant difference in the *H NMR spectrum was that 69 only showed four methyl resonances in the upfield region, missing the methyl doublet at 8 0.97 present in 66. Another noticeable difference was the carbinol proton resonance assigned to H3, which showed a downfield shift from 8 3.11 in 66 to 8 3.60 in 69. This evidence suggested that compound 69 differed from 66 simply by the absence of the Me29 methyl group at the C4 position. The 13C NMR and APT spectra (Figure 103) of the methyl ester 69 showed four upfield methyl carbon resonances at 8 18.2, 12.8, 19.1, and 19.7 assigned to Mel8, Mel9, Me21, and HO 31 39 C0 2CH3 = 30 33 \*\ H 37kx 36 T JU5 OCH3 38 69 LL. J ' u_ -i—i —i—i—i—i—i—i—i—i—i—i—i—i—|—i—i—i—r—i—r~i—i—i—|—i—i—i—r—i—i—i—i—i—|—i—i—i—i—r~ 6 5 4 3 -,—-, 1 , ,—, ,—, j—j ,—, p^-, ,—f ,—|—r 2 I Figure 102: *H NMR spectrum of polymastiamide B methyl ester 69 in CDCI3 (400 MHz) 3 28 31 39 O C0 2CH3 ~30 33 HO IJJUM^^ m mmm U mm mm ri'iiMitniiiMnn>Mi> rni i rwrt ni'iniMnnniniii rnffri imitttfiMiiiH|i ffwi iitirti umi | trti i nrw> iiinMiiiHiiimiiiiinnniiiinniiiinntiii r»* n iftTi u PP» 160 140 120 100 80 60 40 20 Figure 103: 13C NMR spectrum of polymastiamide B methyl ester 69 in CDCI3 (125 MHz) to o 00 Table 18. *H and 13C NMR data of polymastiamide B methyl ester (69) recorded in CDCI3. c# le la 2e 2a 3a 4e 4a 5a 6e 6a 7e 7a 8 9a 10 l i e 11a 12e 12a 13 14 15 15' 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 26' 27 28 NH 30 31 32 33 34 35 36 37 38 39 5 1 3 C 36.5 31.6 71.3 38.3 44.3 28.9 29.6 126.3 49.3 36.8 19.9 37.2 42.7 142.7 25.8 27.0 56.6 18.2 12.8 34.5 19.1 33.1 31.8 35.8 150.9 115.8 169.0 19.7 55.8 171.7 128.7 128.5 114.4 159.8 114.4 128.5 55.3 52.7 8 *H 1.67, dt (13.8, 3.4) 1.08 1.80 1.33 3.60, tt (10.0, 5.0) 1.61 1.25 1.24 1.33 1.19 2.34, m 1.74 1.61 1.58 1.45 1.88, dt (12.7, 3.6) 1.08 2.17, m 2.14, m 1.73 1.27 1.06 0.78, s 0.66, s 1.37 0.86, d (6.6) 1.36 1.01 1.50 1.18 2.57, dt (6.8) 5.57, bs 5.22, bs 1.05, d (6.8) 6.65, d (7.2) 5.55, d (7.2) 7.27, d (8.8) 6.85, d (8.8) 6.85, d (8.8) 7.27, d (8.8) 3.77, s 3.72, s COSY HI a, H2a, H2e Hie, H2a, H2e Hie, Hla, H2a, H3a Hla,Hle, H2e, H3a H2a, H2e, H4a, H4e H3a, H4a H3a, H4e H6a, H6e H5a, H6a, H7a, H7e H5a, H6e, H7a, H7e H6a, H6e, H7a H6a, H6e, H7e HI la, HI le H9a,Hlla, H12a, H12e H9a,Hlle, H12a,H12e Hlla ,Hlle ,H12a Hlla ,Hlle ,H12e H15', H16, H16' H15, H16, H16' H15, H15', H16', H17 H15, H15', H16, H17 H16, H16', H20 H17, H21,H22' H20 H22', H23, H23' H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H28 H26' H26 H24 H30 NH H34 H33 H37 H36 HMBC H19 H19 H6e,H7a, H7e, H9a,Hlle, H15, H15' H19 H19 H18 H12e,H18 H7a, H7e, H9a, H12e, H15, H15', H18 H18, H21 H21 H21 H28 H23, H23', H26', H28 H24, H28 H24 H24, H26, H26', NH H33, H37 H30, H39 H30, H34, H36 H30, H34, H37 H36 H33, H34, H36, H37, H38 H34 H30, H33, H36 NOE H15, H15' NH H26', NH H26, H28 H24, H26, H30 H33, H37, NH 210 Me28, respectively, differing from that of methyl ester 66 (Mel8, 8 18.1; Mel9, 13.9; Me21, 19.1; Me28, 19.8; Me29, 15.2) by the absence of the Me29 resonance. The upfield shift of the carbinol carbon C3 at 5 71.3 in 69, instead of 8 76.5 in 66, was in good agreement with the lack of a (3-positioned methyl group Me29 (|3-effect). Detailed analysis of COSY, HMQC, HMBC, and NOE data (Table 18) demonstrated that the structure of 69 was as shown. Final comparison of the 13C NMR assignments for the steroid nucleus of 69 with the standard steroid 6766 further confirmed the proposed structure and relative stereochemistry of the steroid nucleus for the methyl ester 69. Figure 104. Comparison of 13C NMR assignments for 69 with literature values for 67. 211 3.2.2.3. Polymastiamide C (70) 29 70 R = H R1 = S0 3 " 71 R = CH3 R1 = H Polymastiamide C (70) was isolated as its methyl ester 71 , a colorless solid. The HREIMS of 71 showed a parent ion at mlz 605.40820 appropriate for a molecular formula of C38H55NO5 (AM +0.2 mmu), indicating that it differed from that of methyl ester 66 only by one carbon and two hydrogen atoms and that it was isomeric with methyl ester 69. The *H NMR spectrum (Figure 105) of methyl ester 71 showed some resemblace to that of methyl ester 66. The resonances assigned to the steroidal nucleus and the p-hydroxyphenylglycine methyl ester fragment in 71 could also be found in 66. The most notable difference was that a broad triplet at 5 6.38 (H24, / = 6.7 Hz) in the *H NMR spectrum of 71 replaced two broad singlets assigned to the olefinic methylene protons at 8 5.57 (H26) and 5.22 (H26') in 66. Another significant difference was that compound 71 showed a broad methyl singlet at 8 1.83 (Me26), which had a chemical shift appropriate for an allylic methyl group. A further difference was that 71 only showed four upfield methyl resonances all below 1 ppm, while methyl ester 66 contained five methyl resonances in the most upfield region, one of which had a chemical shift of 1.05 ppm was assigned to Me28. This evidence suggested that the 71 differed from that of 66 only in the steroidal side chain. The 13C NMR and APT spectra of methyl ester 71 reinforced the evidence for the *H NMR spectrum. All the carbon peaks in the spectrum (Figure 106) of 71 could be found in that of 66 except for the resonances assigned to the steroidal side chain. Detailed analysis of COSY, 21 31 39 C0 2CH3 r 30 33 HO 24 Ui I 26 MA/WVAAI 7 6 5 4 ppa 3 2 1 Figure 105: 'H NMR spectrum of polymastiamide C methyl ester 71 in CDCI3 (500 MHz) 21 31 39 O C0 2CH3 24 || 5 3() 33 OCH< 38 * 29 71 Jmmmmmmmm mm mmmm IMWimNMMMMMM Jl mmm *m mm mmmm tmmtm mm Ml MM IMP Mm mmmm ppa 160 140 120 100 80 60 40 20 Figure 106: 13C and APT NMR spectra of polymastiamide C methyl ester 71 inCDC^ (125 MHz) Table 19. *H and 13C NMR data of polymastiamide C methyl ester (71) recorded in CDCI3. c# le la 2e 2a 3a 4a 5a 6e 6a 7 7* 8 9a 10 l i e 11a 12e 12a 13 14 15 15' 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 27 29 NH 30 31 32 33 34 35 36 37 38 39 1 3 C 36.3 31.2 76.5 39.7 50.6 24.8 29.6 126.2 49.4 37.5 19.9 37.3 42.7 141.9 25.7 27.0 56.6 18.2 13.9 34.3 18.9 34.6 25.1 137.9 129.8 12.5 168.5 15.2 56.0 171.9 128.8 128.6 114.4 159.7 114.4 128.6 55.3 52.70 l H 1.68, dt(13.4, 3.4) 1.12 1.81 1.42 3.10, tdC10.5, 4.8) 1.26 0.86 1.69 0.98 2.38, m 1.65 1.60 1.58 1.44 1.90, dt(12.5, 3.5) 1.08, td(13.2, 3.5) 2.20 2.18 1.79 1.35 1.12 0.81, s 0.69, s 1.49 0.94, d(6.6) 1.51 1.20 2.17, m 2.01, m 6.38, t(6.7) 1.83, bs 0.96, d(6.3) 6.59, d(6.8) 5.53, d(6.8) 7.28, d(8.7) 6.86, d(8.7) 6.86, d(8.7) 7.28, d(8.7) 3.77, s 3.71, s * C O S Y Hla, H2a, H2e Hle,H2a,H2e,H19a Hla, Hie, H2a, H3a Hla, Hle,H2e,H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6a, H6e H5a, H6a, H7 H5a, H6e, H7, H7' H6a, H6e, H7' H6a, H7 Hi la Hlla,H12a,H12e H9a,Hlle,H12a,H12e Hl l a ,Hl l e , H12a Hlla, Hlle, H12e H15', H16, H16' H15, H16, H16' H15, H15', H16', H17 H15, H15', H16, H17 H16, H16*, H20 Hla H17, H21, H22' H20 H22', H23, H23' H20, H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23*, H26 H24 H4 H30 NH H34 H33 H37 H36 t H M B C H2e, H19 Hla, Hie Hie, H4a, H2a, H29 H29 H6e, H7, Hi9, H29, H7 H6eH7, Hl le , H15, H15' H7, H12e, H19 Hie, H6e, H7, H9a, H19 H9a, H12e Hlla, H18 Hlle,H12e,H15',H16,H17, H18 H7, H7', H12e, H15, H15', H18 H16' H15, H15', H17, H20 H15, H16', H18, H20, H21 H12e,H17 Hla, H5a, H9a H16', H17, H21,H23,H23' HI7, H22, H22' H17, H21, H23, H23', H24 H20, H22, H24 H23, H23', H26 H23, H23', H26 H24 H24, H26, H30, NH H2e, H3a, H5a NH H30, H39, NH H30, H34, H36 H30, H37 H36 H33, H34, H36, H37, H38 H34 H30, H33 * R O E S Y H3a, H5a, H9a H19 Hla, H5a, H29 H19 Hla, H3a, H9a H29 H6a, H15, H15' Hla, H5a H18, H19 H18, H21 H7 H7 H20 Hlla , H12e,H20 H2a,H4a,H6a,Hlla H16, H18 H12e, H23, H23' H24 H21, H26 H21 H22, H26, NH H23, NH H3a, H6e H24, H26 H33, H37 H38 H38 H30 H34, H36 Correlated to proton resonance in 8 *H column. COSY was recorded at 400 MHz. t Correlated to carbon resonance in o 13C column, a = axial, e = equatorial 215 HMQC, HMBC, and APT data obtained for 71 established the side chain substructure. The COSY spectrum showed a long range coupling between the broad methyl singlet (8 1.83, Me26) and a broad triplet at 8 6.38 (H24) that was correlated in the HMQC spectrum to an olefinic methine carbon at 5 137.9 (C24). The olefinic proton H24 (8 6.38) showed scalar coupling to a pair of methylene protons at 8 2.17 and 2.01 (H23/H23'), typical chemical shifts for allylic protons. These allylic protons were further correlated to another pair of methylene protons at 8 1.51 and 1.20 (H22/H22'). These methylene protons showed HMQC correlations to a carbon at 8 34.6 (C22) that was strongly coupled to a methyl doublet at 8 0.94 (Me21, J = 6.6) in the HMBC spectrum of 71. Further HMBC correlations observed from the allylic methyl protons Me26 (8 1.83) to an olefinic methine carbon at 8 137.9 (C24), a fully substituted olefinic carbon at 8 129.8 (C25), and a carbonyl carbon at 8 168.5 (C27) established the NMR assignments for the side chain substructure and revealed the existence of the a,/?-unsaturated carbonyl system. An HMBC correlation between the NH proton resonance at 8 6.59 and the carbonyl resonance C27 (8 168.5) further confirmed that the p-methoxyphenylglycine fragment was linked to the a,/J-unsaturated carbonyl via an amide bond. The chemical shift of the 13C NMR resonance assigned to the olefinic methyl Me26 (8 12.5)* and the lack of NOE or ROESY correlations between Me26 and the olefinic proton H24 (8 6.38) indicated that the alkene in the side chain at A24-25 had E-configuration. Figure 107. Selected HMBC correlations in methyl ester 71. * chemical shift of methyl group on double bond cis to methylene is usually <20 ppm, cis to proton >20 ppm.5^b 3.2.2.4. Polymastiamide D (72) 216 72 R = H R1 = S0 3 " 73 R = CH3 R1 = H Polymastiamide D (72) was obtained as its methyl ester derivative 73, a colorless solid. It gave a parent ion at mlz 591.39203 in the HREIMS corresponding to a molecular formula of C37H53NO5 (AM -0.4 mmu), which differed from that of methyl ester 66 by two carbons and four hydrogen atoms. The lH NMR spectrum (Figure 108) of methyl ester 73 showed all the typical resonances assigned to thep-hydroxyphenylglycine methyl ester fragment. A pair of scalar coupled doublets at 5 7.28 (H33/H37) and 6.86 (H34/36), each integrating for two protons, were very diagnostic for apara-disubstituted benzene ring with one electron donating substituent. A broad NH proton doublet (8 6.58) and a methine proton doublet (H30, 8 5.55), which were correlated to one another in the COSY spectrum, had typical chemical shift values for an amide proton and an a-methine portion. Two sharp singlets at 8 3.77 (Me38) and 3.72 (Me39) in 73 were appropriate for two methoxy groups present in the p-hydroxyphenylglycine methyl ester fragment. Other characteristic lH NMR signals were an olefinic triplet at 8 6.38 (H24) and a broad methyl singlet at 8 1.83 (Me26) that were found in the methyl ester 71. This result suggested that methyl ester 73 contained a side chain that was already identified in methyl ester 71. A significant feature in the *H NMR spectrum was that compound 73 only contained three upfield methyl resonances, of which two singlets at 8 0.81 and 0.66 were very diagnostic for two angular methyl groups Mel8 and Mel9 in the steroidal nucleus, and the doublet at 8 0.94 was appropriate for the Me21 methyl group. This indicated that compound 73 simply lacked the two methyl groups assigned to Me28 and Me29 present in methyl ester 66. The 13C NMR and APT spectra (Figure 109) of methyl ester 73 showed 35 resolved resonances, two less than that of methyl ester 66. Detailed analysis of COSY, HMQC, and HMBC data obtained for 73 led to the assignments of all protons and carbons for polymastiamide D derivative 73. Comparison of the 13C NMR assignments for the methyl ester 73 with that of methyl esters 66, 69, and 71 confirmed the proposed structure for 73 and revealed that methyl ester 73 was a hybrid of methyl esters 69 and 71, i.e. a combination of the steroidal nucleus present in 69 and the side chain present in 71. ROESY correlations observed between H3ax and both Hl a x and H5ax; between H9ax and H l ^ , H5ax, and H12ax; between Mel9 and H2ax, H4ax, H6ax, and HI lax; and between Mel8 and HI lax, and H20 further confirmed that molecule 73 had a standard steroid nucleus. 31 39 O C0 2CH3 30 33 HO •*•*. iAj. ppi 7 6 5 4 3 2 1 Figure 108: *H NMR spectrum of polymastiamide D methyl ester 73 in CDCI3 (500 MHz) to •—* 00 HO 0 TOT 1 H 31 39 C0 2CH3 J^O 33 32] 37 r 36 I)34 h OCH3 38 73 iiimiHiitumtyimtiriiiiiiiTi w^n rtiiTiiiTiiiiiniH't» wv • n • •'•*•• i*ri irqTri'TTTtmti 11 m »| 111 n in 111 n 11 n 11| 111 >i nil in 111 m 1 |i 111 if 11 yii 1I'I^TT'ITT^ nr'iiiniiii PPB 160 140 120 100 BO 60 40 SO Figure 109: ] 3C and APT NMR spectra of polymastiamide D methyl ester 73 in CDCI3 (125 MHz) to Table 20. *H and 13C NMR data of polymastiamide D methyl ester (73) recorded in CDCI3. c# leq lax 2eq 2ax 3ax 4eq 4ax 5ax 6eq 6ax 7eq 7ax 8 9ax 10 lleq llax 12eq 12ax 13 14 15 15' 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 27 NH 30 31 32 33 34 35 36 37 38 3 9 .. 1 3 C 36.5 31.6 71.3 38.3 44.3 28.9 29.6 126.6 49.3 36.8 20.0 37.3 42.8 142.4 25.8 27.0 56.7 18.3 12.8 34.3 18.9 34.7 25.1 137.8 129.8 12.5 168.5 56.0 171.9 128.9 128.6 114.4 159.7 114.4 128.6 55.3 52.70 lH 1.68, dt 1.09 1.80 1.35 3.60,td(10.8, 4.4) 1.61 1.24 1.24 1.33 1.20 2.34, m 1.74 1.61 1.59 1.46 1.91,dt(12.2,4) 1.09 2.20 2.20 1.80 1.33 1.12 0.81, s 0.66, s 1.49 0.94, d(6.6) 1.52 1.20 2.17 2.01, m 6.38, bt(6.7) 1.83, bs 6.58, d(6.8) 5.54, d(6.8) 7.28, d(8.7) 6.86, d(8.7) 6.86, d(8.7) 7.28, d(8.7) 3.77, s 3.72, s * C O S Y HI a, H2a, H2e Hie, H2a, H2e Hla,Hle,H2a, H3a Hla,H2a,H3a H2a, H2e, H4a, H4e H3a, H4a, H5a H3a, H4e, H5a H4a, H4e, H6a, H6e H5a, H6a, H7a, H7e H5a, H6e, H7a, H7e H6a, H6e, H7a H6a, H6e, H7e Hlla,H12a,H12e Hlle,H12a, H12e Hlla, Hlle,H12a Hlla ,Hl le ,H12e H15', H16, H16' H15, H16, H16' H15, H15', H16', H17 H15, H15', H16, H17 H16, H16', H20 H17, H21, H22' H20 H22', H23, H23' H20, H22, H23, H23' H22, H22", H23', H24 H22, H22', H23, H24 H23, H23', H26 H24 H30 NH H34 H33 H37 H36 t HMBC H19 H4a, H6a, H19 H5a H7e,H19 H4e,H19 H18 H12a,H17, H18 H12e, H15, H15', H18 H18, H21 H12a H21 H21, H24 H24 H23', H26 H26 H24 H24, H26, NH NH, H33, H37 H30, H39 H30, H34, H36 H30, H37 H36 H33,H34,H36,H37,H38 H34 H30, H33 , . *ROESY H3a, H5a, H9a H3a H3a,H19 Hla, H5a H19 H3a, H9a H19 H15 H5a H18, H19 H21 H7e H7e H18, H21 Hlla, H16', H17, H20 H2a,H4a,H6a,Hlla, H16, H18 H12e, H23,H23' H24 H24 H21 H21 H22, H22', NH NH H24, H26 H33, H37 H30 H38 H38 H30 H34, H36 Correlated to proton resonance in 8 lH column. COSY was recorded at 400 MHz. t Correlated to carbon resonance in 0 13C column, a = axial, e = equatorial. 3.2.2.5. Polymastiamide E (74) 29 74 R = H R1 = S0 3 " 75 R = CH3 R1 = H Polymastiamide E (74) was obtained as its methyl ester 75, a colorless solid. The HREIMS spectrum of compound 75 gave a parent ion at mlz 589.41223 appropriate for a molecular formula of C38H55NO4 (AM -0.9 mmu), differing from that of methyl ester 66 only by a CH2O unit. The lH NMR spectrum (Figure 110) of methyl ester 75 was almost identical to that of polymastiamide A methyl ester 66. For example, five upfield methyl resonances at 5 0.69 (s), 0.78 (s), 0.86 (d), 0.97 (d), and 1.06 (d) assigned to Mel9, Mel8, Me21, Me29, and Me28 in methyl ester 75 could also be found in that of methyl ester 66 (Mel8, 8 0.78, s; Mel9, 0.69, s; Me21, 0.86, d; Me28, 1.05, d; Me29, 0.97, d). The only notable difference in the !H NMR was that a pair of doublets at 8 7.28 (H33/H37) and 6.86 (H34/36), and a sharp methoxy singlet at 8 3.77 (Me38) assigned to p-methoxyphenyl group in 66 were replaced by a cluster of aromatic protons at 8 7.35, typical chemical shifts for a phenyl group. This evidence suggested that methyl ester 75 simply contained steroidal and phenylglycine methyl ester fragments. The COSY data (Figure 111) collected for 75 were consistent with the proposed structure as shown. Since only a very limited amount of compound 75 was obtained, the 13C NMR data of 75 were inconclusive. 28 29 75 31 39 C0 2CH3 = 30 33 W _A X. •mm * »' -m i w^»^*v- >^»«iir i^ •»»*•«»> _JLU~„A iuU*<J*r™' HI -i—r~T—!~T—i~i—i—r—i—i—i—i—i—r—i—\~ i—r~ vr—t -r—i"r—i—]—i—i—i—r—i—r—i—r—i—p--i—i—r~t-• -T--j —f~i— T—T—,—,—!—1~-1 -|-Figure 110: *H NMR spectrum of polymastiamide E methyl ester 75 in CDCI3 (500 MHz) 223 31 39 Q C02CH3 Jbo 33 27 > T HO 4 29 75 I jL>, ...A-. JUJ^J^ ^ Figure 111: 2D COSY spectrum of polymastiamide E methyl ester 75 in CDCI3 224 3.2.2.6. Polymastiamide F (76) 29 76 R = H R1 = S0 3 " 77 R = CH3 R1 = H Polymastiamide F (76) was isolated as its methyl ester 77, a colorless solid. The ester 77 gave a parent ion in the HREIMS at mlz 575.39667 appropriate for a molecular formula of C37H53NO4 (AM -0.8 mmu), differing from that of polymastiamide C methyl ester 71 simply by a CH2O unit. The JH NMR spectrum (Figure 112) of methyl ester 77 was nearly identical to that of methyl ester 71 except that a cluster of resonances, integrating for five protons, around 8 7.35 in 77 replaced a pair of doublets at 5 7.28 (H33/H37) and 6.86 (H34/36) and a sharp methoxy singlet at 8 3.77 (Me38) assigned to p-methoxyphenyl group in 71. This information suggested that methyl ester 77 differed from methyl ester 71 only by the lack of a methoxy group on the aromatic ring at the C35 position. The 13C NMR and APT data obtained for methyl ester 77 were in complete agreement with the proposed constitution for 77. All the 13C NMR signals in the spectrum (Figure 113) of methyl ester 77 could also be found in that of methyl ester 71 except for those resonances assigned to the aromatic ring. Detailed analysis of COSY, HMQC, HMBC, and APT data collected for 77 confirmed the presence of a phenylglycine methyl ester fragment in 77. A broad doublet at 8 6.65 (NH) showed a COSY correlation to a methine proton H30 (8 5.61). In the HMBC spectrum this methine proton H30 (8 5.61) was correlated to an aromatic quaternary carbon at 8 136.8 (C32), to aromatic methine carbons at 8 127.3 (C33/C37), and to a carbonyl carbon at 8 171.7 (C31). The carbonyl carbon (C31) was further correlated to a deshielded methyl proton resonance at 8 3.72 (H39). HO 29 JUL A-77 •«*A. .. JLAAJI PPH 7 6 5 4 3 2 1 Figure 112: *H NMR spectrum of polymastiamide F methyl ester 77 in CDCI3 (500 MHz) to to 31 39 O C0 2CH3 21 22 24 || =™ 33 mmm*qmmmmmm mm *mmmmm ± mmm** WW ^ifrrtH +** mm wm mm n*t I'rw-rrrmi n rr»i t 11 irrtt • • i m mi HI HIMIIIIIHTMIII rri pmini 160 140 120 100 80 • • i ••' 60 "i i PP« Figure 113: 13C and APT NMR spectra of polymastiamide F methyl ester 77 in CDCI3 (125 MHz) ON 227 31 39 O C0 2 CH 3 30 33 HO 29 JL JUL 77 A . A JUJ^KM, n / p p & I I I I—i—i—i—i—i—i—i—i—i—p—i—i—i—i—i—r Q oa * i i i i i r i i i _ 1 . 0 _ 2 . 0 _ 3 . 0 _ 4 . 0 5. 0 _ 6. 0 7. 0 7 . 0 6 . 0 5 . 0 4 . 0 3 . 0 2 . 0 1 .0 PPM PPM Figure 114: 2D COSY spectrum of polymastiamide F methyl ester 77 in CDCI3 21 31 39 Q C02CH3 ~30 33 HO Figure 115: 2D HMQC spectrum of polymastiamide F methyl ester 77 in CDCI3 Table 21. *H and 13C NMR data of polymastiamide F methyl ester (77) recorded in CDC^. c# leq lax 2eq 2ax 3ax 4ax 5ax 6eq 6ax 7 T 8 9ax 10 lleq llax 12eq 12ax 13 14 15 15' 16 16' 17 18 19 20 21 22 22" 23 23' 24 25 26 27 29 NH 30 31 32 33 34 35 36 37 39 8 i 3c 36.3 31.2 76.5 39.7 50.7 24.9 29.7 126.2 49.4 37.5 19.9 37.3 42.7 141.9 25.7 27.0 56.6 18.2 13.9 34.3 18.9 34.7 25.1 137.9 129.8 12.5 168.5 15.2 56.6 171.7 136.8 127.3 129.2 128.5 129.2 127.3 52.8 8*H 1.68 1.12 1.81 1.42 3.10, td(10.5, 4.7) 1.26 0.85 1.69 0.98 2.39, m 1.66 1.61 1.58 1.44 1.90, dt(12.5, 3.5) 1.09 2.22 2.18 1.80 1.35 1.13 0.81, s 0.69, s 1.50 0.94, d (6.6) 1.52 1.20 2.17 2.02, m 6.40, t (6.7) 1.84, bs 0.97, d (6.3) 6.65, d (7.0) 5.61, d (7.0) 7.35 7.36 7.32 7.36 7.35 3.72, s * C O S Y Hla, H2a, H2e Hie, H2a, H2e Hla, Hie, H2a, H3a Hla,Hle,H2e, H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6a, H6e H5a, H6a, H7, H7' H5a, H6e, H7, H7* H6a, H6e, H7' H6a, H6e, H7 H l l a , H l l e H9a,Hlla, H12a,H12e H9a,Hlle, H12a,H12e Hlla ,Hlle ,H12a Hlla ,Hlle ,H12e H15', H16, H16' H15, H16, H16' H15, H15', H16', H17 H15, H15', H16, H17 H16, H16', H20 H17, H21.H22' H20 H22", H23, H23' H20, H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H26 H24 H4a H30 NH t H M B C H2a, H2e, H9a, H19 Hla, Hie Hie, H2a, H29 H29 Hie, H4a, H7, H19.H29 H6eH7,H9a,H15 H7, H12e, H19 Hie, H6e, H9a, H19 H9a H18 Hlle , H12e, H15', H16, H17, H18 H7', H12e, H15, H15' H16', H17 H15, H15', H17, H20 H16', H18, H20, H21 H12a Hla H16', H17, H21 H20, H21,H23, H23', H24 H24 H23, H23', H26 H23, H23', H26 H24 H24, H26, NH NH, H33, H37 H30, H39 H30, H34, H36 H30, H37 H36 H33, H37 H34 H30, H33 Correlated to proton resonance in 5 *H column, recorded at 400 MHz. < Correlated to carbon resonance in 5 13C column, a = axial, e = equatorial 230 Additional HMBC correlations were observed between the aromatic protons at 5 7.36 (H33/H37) and a-methine carbon at 8 56.6 (C30). These HMBC correlations revealed that there was a phenyl ring attached to the a-methine carbon at 5 56.6 (C30). Further analysis of HMBC data demonstrated that the phenylglycine methyl ester fragment was linked to a steroidal side chain via an amide bond. Particularly, HMBC correlations were observed between the amide carbonyl carbon C27 (5 168.5) and the NH proton (8 6.65), the allylic methyl protons Me26 (8 1.84), and the olefinic proton H24 (8 6.40). \^S\JS HMBC Correlation 29 Figure 116. Selected HMBC correlations in polymastiamide F methyl ester 77. The structure of steroidal portion in 77 was routinely determined by analysis of HMBC, HMQC, APT, and COSY data. Strong HMBC correlations observed between the methyl proton resonances (Mel8, Mel9, Me21, Me26, and Me29) and the surrounding carbons identified all the carbon resonances except those belonging to C2, C6, C7, C l l , C15, and C16. An interpretation of COSY data for 77 revealed three spin systems present in the steroidal portion, i.e. Hlax/Hleq through H5 to H7/H7', H9ax to H12ax/H12eq, and HI5/15' through H17 and H20 to Me26, and established the remaining connectivity in 77. The magnitude of the scalar coupling constants of H3 a x (td, / = 10.5 and 4.7 Hz) indicated that the carbinol proton H3ax and the coupled proton H4ax were both axial, while the methyl group Me29 was equatorial. 3.2.2.7. Polymastiamide G (78) 78 Polymastiamide G (78) was obtained as colorless solid that gave a parent ion at mlz 619.42404 in the HREIMS appropriate for a molecular formula of C39H57NO5 (AM +0.4 mmu), indicating that it was isomeric with polymastiamide A methyl ester 66. The *H NMR spectrum (Figure 117) of polymastiamide G (78) showed a close correspondence to that of methyl ester 66, particularly, the resonances assigned to the /?-methoxyphenylglycine methyl ester fragment and the steroid side chain. The most significant difference was that compound 78 showed an extra deshielded proton resonance at 8 5.08 (HI5) that was correlated into an olefinic methine carbon resonance at 5 116.9 (C16) in the HMQC spectrum (Figure 119). Another noticeable difference was that the two upfield methyl proton singlets, which were assigned to Me 18 and Me 19, respectively, shifted to 5 0.84 and 0.82 in 78. The 13C and APT spectra (Figure 118) of polymastiamide G (78) showed 37 resolved signals, two less than the molecular formula indicated. This was attributed to the presence of ap-disubstituted benzene ring in the molecule 78. Comparison of the 13C NMR data for compound 78 with that of methyl ester 66 revealed that 78 also contained ap-methoxyphenylglycine methyl ester fragment and a steroidal side chain identical to those in methyl ester 66. The significant differences between these two molecules were the resonances assigned to the C-ring and D-ring carbons of the steroid nucleus. A detailed analysis of the COSY, HMQC, and APT data obtained for 78 identified all the proton resonances on the steroid nucleus that comprised a contiguous spin system, starting from ppm HO 29 I 27 N' H 78 uJdvU 31 39 C0 2CH3 ^30 33 32^ 37^5/ 36 il34 lb OCH3 38 3 ^_^AhJv^ AJW. 7 6 5 4 3 2 1 Figure 117: *H NMR spectrum of polymastiamide G (78) in CDCI3 (500 MHz) 28 31 39 Q C0 2CH3 ~30 33 HO OCH3 36 38 mmUriitmikJmJm* 29 78 L V+mm* «**•*******«** mmimim*m(H**mmf tmmmm J, I 11, J IHIMI H»ii* ***• mmmmmj)*mmmm JULMU *** pTI'lTHtfTT pp« 160 140 120 100 80 60 40 Figure 118: ^Cand APT NMR spectra of polymastiamide G (78) in CDCI3 (125 MHz) 20 to HO i I H 31 39 C 0 2 C H 3 = 30 33 '£f^ 3 7 ^ / 36 s34 lbs OCH 3 38 3 29 78 A 1A 1 II T -i ° A K j^Jvm^ •. L i t ..!_-. 1 M 11 t ; i i * i i i 11 i i i i | i t i i i i i i i ppa 7 6 S i i i i i i | i f i i i i i i I j i i i i i i i i i I i i i i i i i i « | i •• i i i i i i i j i i i i r 40 SO 30 rlOO 120 PP» Figure 119: 2D HMQC spectrum of polymastiamide G (78) in CDCI3 31 39 C 0 2 C H 3 Al0 JL ^f 3 7 ^ 36 A 3 4 lbs OCH 3 38 3 235 HO £• £• £• & & 7 . 0 6 .0 5 . 0 4 . 3 3 .G 2 . 0 1.0 FPU _ 1.0 _ 2 . 2 _ 3 . 0 4 . 0 3 . b _ 6. 0 _ 7. 0 PPM Figure 120: 2D COSY spectrum of polymastiamide G (78) in CDCI3 HO 29 78 1 1A 1 ll 1 A A J A J W I I I I I I I I 0 o t w • I I 9 so -100 • ; -150 i i i i i i [ i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i i i i i i i | i i i pp» 7 6 5 4 3 2 1 :PP» Figure 121: 2D HMBC spectrum of polymastiamideG (78) in CDCI3 237 Hl a x /Hl eq to H12ax/H12eq by vicinal scalar couplings and continuing from H8ax through the olefinic proton H15 by a long range COSY correlation on into H17 (Figure 120). Extensive HMBC correlations supported these assignments. A strong HMBC correlation between the Me 18 proton resonance (5 0.84) and the C14 carbon resonance (8 155.5); and an overlapping network of correlations between the olefinic proton H15 (8 5.08) and CI 3 (8 47.0), C16 (8 35.5), and C17 (8 58.4) and between the allylic proton H16 (8 2.18) and C13 (8 47.0), C14 (8 155.5), C15 (8 116.9), and C17 (8 58.4) were completely consistent with the proposed D-ring constitution. The relative configuration at C8 could be deduced from the vicinal ^ ^ H coupling constants. The splitting pattern and 3 / w c value of H9ax (8 0.63, td, / = 11.6, 2.8) unequivocally defined the axial orientation of H9ax (8 0.63), which further required that there exist two vicinal axial protons (H8ax, 8 1.93 and HI lax , $ 1-28) and one vicinal equatorial proton (Hlleq, 8 1.55) coupled with H9ax. Finally, comparison of the 13C NMR assignments for the steroidal nucleus of 78 with the literature values for steroid 3P,16oc-diols (61)61 confirmed the structure of 78. Therefore, it was clear that compound 78 differed from methyl ester 66 only in the placement of the double bond on the steroid nucleus. 78 61 Figure 122. Comparison of the 13C NMR assignments for 78 and 61. Since the preliminary *H NMR screening of the sponge crude extracts and the fractions eluted from Sephadex LH-20 chromatography did not contain an olefinic proton resonance that could be assigned to an olefinic proton H15 on a steroid nucleus, polymastiamide G (78) appeared 238 to be an artifact derived from the natural product polymastiamide A (65). It was very likely that the double bond at A8-14 in the natural product 65 migrated during the methylation reaction with MeOH/HCl to yield compound 78. Further evidence came from the reaction of pure methyl ester 66 with MeOH/HCl under methylation conditions (12 hr., 40 °C), which gave a mixture of the isomerized porduct 78 and the remaining methyl ester 66. The presence of other rearranged products, polymastiamides H (79), I (80), and J (81) derived respectively from polymastiamides B (68), C (70), and F (77), supported this argument (see section 3.2.2.8.). Scheme 11 outlines the possible mechanism for isomerization of the A8-14 double bond in the steroid nucleus. Scheme 11. Proposed mechanism for the double bond migration. H+ HO 78 Table 22. *H and 13C NMR data of polymastiamide G (78) recorded in CDC13. c# leq lax 2eq 2ax 3ax 4ax 5ax 6eq 6ax 7eq 7ax 8ax 9ax 10 lleq llax 12eq 12ax 13 14 15 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 26' 27 28 29 NH 30 31 32 33 34 35 36 37 38 39 5 i 3 C 36.8 31.0 76.5 39.1 50.5 23.9 30.2 34.4 53.8 36.2 21.7 42.4 47.0 155.5 116.9 35.5 58.4 16.8 13.1 33.9 18.9 33.1 31.9 35.7 150.9 115.6 168.9 19.8 15.2 55.8 171.7 128.7 128.4 114.4 159.7 114.4 128.4 55.3 52.70 5 1 H 1.77 0.97, td(13.4, 4.3) 1.79 1.47 3.06, ddd(l 1.1,9.9,4.3) 1.28 0.68, td(l 1.3, 2.9) 1.71, dq(12.5, 3.3) 1.07 1.92, dq(13.8, 3.3) 1.14 1.93 0.63, td(l 1.6, 2.8) 1.55 1.28 1.94, dt(12.6, 3.1) 1.16 5.08, bs 2.18, ddd(15.8, 9.6, 2.2) 1.79 1.43 0.84, s 0.82, s 1.50 0.835, d(6.4) 1.34, m 0.96 1.53 1.18 2.58, dt(6.6) 5.57, bs 5.23, bs 1.05, d(6.9) 0.95, d(6.3) 6.67, d(7.1) 5.54, d(7.1) 7.27, d(8.8) 6.85, d(8.8) 6.85, d(8.8) 7.27, d(8.8) 3.77, s 3.72, s * COSY Hla Hle,H2a H2a, H3a Hla, H2e, H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6a, H6e, H19 H5a, H6a, H7a, H7e H5a, H6e, H7a, H7e H6a, H6e, H7a H6a, H6e, H7e, H8a H7a, H9a, H15 H8a,Hl la ,Hl le H9a,Hlla, H12a, H12e H9a, Hlle , H12a,H12e Hlla ,Hlle ,H12a HI la, Hlle,H12e H8a, H16,H16' H15, H16', H17 H15, H16, H17 H16, H16' H5a H21, H22, H22' H20 H20, H22', H23, H23' H20, H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H28 H24 H4a H30 NH H34 H33 H37 H36 t H M B C H19 Hie, H2a, H4a, H29 H29 Hie, H4a, H7e, H19, H29 H5a, H7a, H7e H6e H6e, Hl le H7e, H8a, Hlla,H12e, H19 H19 H9a,H12a,H12e Hlla, Hlle,H17,H18 H l l e , H12e, H15, H16, H17, H18 H8a, H16, H16', H18 H16, H16' H15, H17 H15.H16, H18, H22 Hla,H5a,H9a H17, H21 H17 H17, H21 H28 H23, H26, H26', H28 H23, H23', H24, H28 H24 H24, H26, H26', NH H23, H24 H3a H33, H37 H30, H39 H30, H34, H36 H30, H35, H37 H36 H33, H34, H36, H37, H38 H34 H30, H33, H35 * N O E H3a Hla, H12a 7e H15 Correlated to proton resonance in 8 *H column, t Correlated to carbon resonance in 8 13C column. * Resonance in 8 *H column was irradiated; recorded at 400 MHz. a = axial, e = equatorial. 3.2.2.8. Polymastiamides H (79), I (80), and J (81) 240 31 39 O C02CH3 4- 6 7 9 R = H X = OCH3 Y = R 80 R = CH3 X = OCH3 Y = 26 81 R =CH3 X = H Y = 26 Polymastiamides H (79), I (80), and J (81) were obtained as colorless solids. Polymastiamide H (79) gave a parent ion in the HREIMS at mlz 605.40936 appropriate for a molecular formula of C38H55NO5 (AM +1.3 mmu); polymastiamide I (80) gave a parent ion in the HREIMS at mlz 605.40839 appropriate for a molecular formula of C38H55NO5 (AM +0.4 mmu); and polymastiamide J (81) gave a parent ion in the HREIMS at mlz 575.39762 appropriate for a molecular formula of C37H53NO4 (AM +0.2 mmu), respectively. All three compounds showed a broad deshielded proton resonance at 5.1 ppm that could be assigned to the olefinic proton H15 as discussed above (Section 3.2.2.7.). Upon further inspection of the JH NMR spectra for these three compounds, it was found that polymastiamides H (79), I (80), and J (81) were the rearranged products of polymastiamides B (68), C (70), and F (76), respectively. The most diagnostic signals were the following: Two broad singlets at 8 5.57 and 5.32, and a methyl doublet at 8 1.05 (Me28) in the JH NMR spectrum (Figure 123) of polymastiamide H (79) indicated that it contained an olefinic methylene (C26) and a methyl group (Me28) at C24 in the 128 24 . 24 25 steroidal side chain. The chemical shift and 3 /v / c value of the carbinol proton H3 (8 3.57) suggested that 79 contained no Me29 at the C4 position. Both polymastiamides I (80) and J (81) showed an olefinic triplet at 8 6.4 (H24) and carbinol proton at 8 3.06 (H3), indicating the absence of a methyl group (Me28) at the C24 position and the presence of Me29 at the C4 position in the steroidal portion (Figures 125 and 127). The difference between 80 and 81 was the resonances assigned to protons on the aromatic ring. It was obvious that polymastiamide I (80) contained ap-methoxyphenyl group while polymastiamide J (81) had a phenyl substituent. Detailed analysis of ID and 2D NMR data (See Tables 23, 24, and 25) collected for compounds 79, 80, and 81; and comparison with that of the readily identified compounds 69, 71, 77, and 78 established and confirmed the structures of 79, 80, and 81 as shown. HO 79 UL ^ * H JhA r r—* 'i i •'•'i | -• i "i i ^ * ^ i •< | i • t "i PI f •• i j r i • "t 't"i •'•'T—t f"T '^-^ rBil f r- T fi ppi 7 6 5 4 3 if i, i, yi t fi.n, ,.,i, t- , ^, 1Mr..T.-^ ., ». , •,,,, i T |... T.„T..i^.,.n |n, f- | Figure 123: ] H NMR spectrum of polymastiamide H (79) in CDCI3 (500 MHz) to to Mmtmmmm mmmmmm m*mmmmmm**wmmm J\i0MmJMmm m ^Mmmm • i > 11 111111111»111111111111111 iiniiiiii PPM iiiiiiiiiiriii|iiiiiiiiiiiiiiiiiii|iiiiiiiiiiiiiiiiiii|iiiiiiiiiiiiiliiiii|iiiiiiiiiiiiiiiiiii|iiiiiiiiiiiiliiiiii|iiiiiiiiliiiiil 80 F 60 40 T 20 TTTTTTTTTTTTTTTTTTT 160 140 120 100 Figure 124: *3c and APT NMR spectra ofpolymastiamideH (79) in CDC13 (125 MHz) to •p. Table 23. lH and 1 3C NMR data of polymastiamide H (79) recorded in CDC13. c# le la 2e 2a 3a 4e 4a 5a 6e 6a 7e 7a 8a 9a 10 l ie 11a 12e 12a 13 14 15 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 26' 27 28 29 NH 30 31 32 33 34 35 36 37 38 39 8 13c 37.1 31.5 71.3 38.4 44.4 28.5 30.1 35.1 53.7 35.7 21.9 42.4 47.3 155.4 117.0 35.5 58.6 16.8 12.0 34.0 19.0 33.3 32.1 35.9 151.1 115.6 168.9 19.8 55.9 171.7 128.9 128.5 114.5 159.8 114.5 128.5 55.3 8 1 H 1.75 0.95, td(13.8, 4.3) 1.78 1.54 3.57, ttC10.8, 4.7) 1.57 1.27 1.07, td(11.4, 2.9) 1.32 1.32 1.87 1.15 1.97 0.64, td(12.0, 2.8) 1.53 1.29 1.94, dt(12.8, 3.2) 1.18 5.08, bs 2.17, ddd(15.4, 7.5, 1.9) 1.77 1.44 0.87, s 0.82, s 1.51 0.835, d(6.4) 1.35 0.96 1.53 1.19 2.58, dt(6.7) 5.57, bs 5.23, bs 1.05,d(6.9) 6.67,d(7.1) 5.54,d(7.1) 7.27, d(8.7) 6.85, d(8.7) 6.85, d(8.7) 7.27, d(8.7) 3.77, s *COSY Hla Hie, H2a H2a, H3a Hla,H2e,H3a H2a, H2e, H4a H3a, H5a H4a, H6a, H6e,H19 H5a, H6a, H7a, H7e H5a, H6e, H7a, H7e H6a, H6e, H7a H6a, H6e, H7e, H8a H7a,H9a,H15 H8a,Hl la ,Hl le H9a,Hlla, H12a,H12e H9a,Hlle, H12a,H12e Hlla ,Hlle ,H12a Hlla ,Hl le ,H12e H8a, H16, H16' H15, H16', H17 H16, H16, H17 H16.H16', H20 H5a H21,H22, H22' H20 H20, H22', H23, H23' H20, H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H28 H24 H30 NH H34 H33 H37 H36 52.6 3.72, s tHMBC H2a, H19 H4e Hle,H4a,H4e Hie, H4a, H7e,H19 H6 Hlle H7e, H8a, H12e,H19 H9a, H19 H12a H17, H18 H15, H16, H17, H18 H12e, H16, H18 H16, H16' H15 H15, H16, H18, H20, H21 H12a,H17 H17, H21, H23 H21,H23 H24, H28 H26, H26', H28 H23, H23', H26, H26', H24, H28 H24 H24, H26, H26', NH H23', H24 H33, H37 H30, H39, NH H30, H34, H36 H30, H37 H36 H33, H34, H36, H37, H38 H34 H30, H33 Correlated to proton resonance in 8 *H column, t Correlated to carbon resonance in 8 ^ C column. a = axial, e = equatorial. HO 31 39 0 C0 2CH3 I =30 J3 27  ^32^ H 3 7k/ 36 n34 h 0CH3 38 3 80 Lui JI k j w i_JL J vW u. —I—I—I I ""f ' PPIB | I * I I T" » fM, l I 1 1 T ' I "f "t T111* t "1" j "T » T—'» > '•* t"-f » i.-y..'^' -t — t ' 1 ••»•• 1 t"> "T "1 '• ?' I 'I I T 'I • T' 'I •t"l,| 1 ' 1 " t T" T •t T ' ' T ' f "T"r 'I » "I » I'" 7 6 5 4 3 2 1 Figure 125: *H NMR spectrum of polymastiamide I (80) in CDCI3 (500 MHz) to 246 L i :S § •s =5 CO CTv CM S3 8 si 0 Q U c o so s ea s a 3 (~ U a, OS Z u I „ o. . a. Table 24. *H and 13C NMR data of polymastiamide I (80) recorded in CDC13. c# leq lax 2eq 2ax 3ax 4ax 5ax 6eq 6ax 7eq 7ax 8ax 9ax 10 lleq llax 12eq 12ax 13 14 15 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 27 28 29 NH 30 31 32 33 34 35 36 37 38 5 13c 36.8 31.0 76.5 39.1 50.5 23.9 30.1 34.4 53.8 36.2 21.7 42.4 47.1 155.6 116.8 35.5 58.5 16.8 13.1 33.8 18.8 34.8 25.1 137.8 129.8 12.5 168.5 15.2 56.0 171.9 128.8 128.6 114.4 159.7 114.4 128.6 55.3 39 | 52.70 5 !H 1.79 0.97, td(13.8, 4.3) 1.79 1.47 3.06, td(10.5, 4.7) 1.29 0.69, td(l 1.4, 2.9) 1.72, dq(12.7, 3.0) 1.08, qd(12.8, 3.1) 1.94 1.15 1.97 0.64, td(12.0, 2.8) 1.57 1.30 1.97, dt(12.7, 3.2) 1.20 5.12, bs 2.25, ddd(15.3, 7.7, 2.9) 1.87 1.50 0.87, s 0.82, s 1.62 0.92, d(6.6) 1.51 1.17 2.18, m 2.03, m 6.38, t(6.7) 1.84, bs 0.95, d(6.3) 6.59, d(6.8) 5.54, d(6.8) 7.29, d(8.6) 6.86, d(8.6) 6.86, d(8.6) 7.29, d(8.6) 3.77, s 3.71, s *COSY Hla Hle,H2a H2a, H3a Hla, H2e, H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6a,H6e, H19 H5a, H6a, H7a, H7e H5a, H6e, H7a, H7e H6a, H6e, H7a H6a, H6e, H7e, H8a H7a, H9a, HI5 H8a, HI la, HI le H9a, Hlla,H12a, H12e H9a, Hlle , H12a, H12e Hlla, Hlle,H12a Hlla, Hlle, H12e H8a, H16, H16' H15, H16', H17 H16, H16, H17 H16, H16', H20 H5a H17, H21 H20 H22\ H23, H23' H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H26 H24 H4 H30 NH H34 H33 H37 H36 tHMBC H19 Hie, H29 H5a, H29 Hie, H7e, H19, H29 Hlle H7e, H8a, H12e,H19 H2e, H9a, H19 H12a H17, H18 H15, H16, H17, H18 H8a, H12e, H16, H18 H16, H16" H15, H17 H15, H18, H21 H12a,H17 H17, H21,H23, H23' H21,H23', H24 H24 H23, H23', H26 H23, H23', H26 H24 H24, H26, H30, NH NH, H33, H37 H30, H39 H30, H34, H36 H30, H37 H36 H33, H34, H36, H37, H38 H34 H30, H33 Correlated to proton resonance in 8 ^H column, t Correlated to carbon resonance in 8 13C column, a = axial, e = equatorial. 248 * * < •i A M CO o Q 00. •o S •a a e *o O s 3 fa U 4> D , V] S z 4) 3 e a. • a. HO m m^mhmmmmmmmmmwmm U | H 31 COj ^30 32 17 39 >CH3 33 r^s ^S, T4 ^35 36 1 mmimmmmmmmmmmmf H mm mm* mm mmmm i*mm* wmtm+rfJ* mmmmt mm m mm mmm* wmmmmmm pp» 1ST 140 120 100 80 60 40 rrf" 20 Figure 128: 13C and APT NMR spectra of polymastiamide J (81) in CDCI3 (125 MHz) Table 25. *H and 13C NMR data of polymastiamide J (81) recorded in CDC13. c# leg lax 2eq 2ax 3ax 4ax 5ax 6eq 6ax 7eq 7ax 8ax 9ax 10 lleq llax 12eq 12ax 13 14 15 16 16" 17 18 19 20 21 22 22' 23 23' 24 25 26 27 29 NH 30 31 32 33 34 35 36 37 38 1 39 8 13c 36.8 30.9 76.5 39.1 50.5 23.9 30.1 34.4 53.8 36.2 21.7 42.4 47.1 155.6 116.8 35.5 58.5 16.8 13.1 33.8 18.8 34.8 25.1 137.9 129.8 12.5 168.5 15.2 56.6 171.7 136.7 127.3 129.0 128.5 129.0 127.3 52.8 8 1H 1.79 0.98, td(13.6, 4.2) 1.78 1.46 3.06, ddd(10.8,10.1,4.6) 1.30 0.68, td(l 1.5, 2.6) 1.71, dq(12.6, 2.9) 1.07, qd(12.6, 2.7) 1.93 1.12 1.97 0.64, td(l 1.2, 2.6) 1.58 1.28 1.97, dt(12.5, 3.2) 1.21 5.12, bs 2.25, ddd(15.2, 7.4, 2.3) 1.89 1.51 0.88, s 0.82, s 1.61 0.92, d(6.6) 1.50 1.15 2.18, m 2.05, m 6.40, t(6.7) 1.85, bs 0.95, d(6.3) 6.65, d(7.0) 5.61,d(7.0) 7.37 7.35 7.33 7.35 7.37 3.72, s *COSY Hla,H2a Hie, H2a, H2e HI a, H2a, H3a Hla,Hle,H2e?H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6a, H6e H5a, H6a, H7a7 H7e H5a, H6e, H7e H6a, H6e, H7a H6e, H7e, H8a H7a, H9a, H15 H8a, H l l a , H l l e H9a,Hlla,H12a,H12e H9a,Hlle,H12e Hlla ,Hlle ,H12a Hlle, H12e H8a,H16 H15, H16', H17 H16, H17 H16, H16', H20 H17, H21,H22 H20 H22', H23, H23' H20, H22, H23, H23' H22, H22', H23', H24 H22, H22', H23, H24 H23, H23', H26 H24 H4 H30 NH t H M B C H5a, H9a, H19 H29 H29 Hie, H7e, H19, H29 H5a, H7a, H8a H6e,H9a,Hlle H7e,H8a,H12e,H19 H5a, H9a, H19 H9a,H12a Hlle , H17,H18 Hl le , H15, H16.H17, H18 H8a, H15, H16, H16', H18 H16, H16' H15, H17 H15, H18, H21 H12a,H17 H9a H17, H21, H22 H17 H21,H23', H24 H24 H23, H23', H26 H23, H23', H26 H24 H24, H26, H30, NH H3 NH, H33, H37 H30, H39 H30, H34, H36 H30, H35, H37 H36 H33, H37 H34 H30, H33, H35 Correlated to proton resonance in 8 XH column, t Correlated to carbon resonance in 8 13C column, a = axial, e = equatorial. 3.2.2.9. Polymastiamide K (82) 251 29 82 Polymastiamide K (82) was obtained as a colorless solid. It gave a parent ion in the HREIMS at mlz 615.39208 appropriate for a molecular formula of C39H53NO5 (AM -0.3 mmu), differing from that of polymasitamide A methyl ester 66 only by the lack of four hydrogen atoms. The *H NMR spectrum (Figure 129) of 82 exhibited a close similarity in the downfield region (5 > 3.5 ppm) to that of 66, except that 82 had one more aromatic proton resonance at 8 6.85 (HI 1). This suggested that compound 82 contained a p-methoxyphenylglycine methyl ester fragment and a side chain identical to 66. Comparison of the 13C NMR data collected for 82 with that of 66 supported this postulate. All of the carbon resonances assigned to the /?-methoxyphenylglycine methyl ester fragment and steroidal side chain of 82 could be found in the counterpart 66 except for slight differences at C20 and C21 (82/66: C20, 8 36.0/34.5; C21, 8 14.3/19.1). Analysis of the COSY spectrum of 82 (Figure 132, Table 26) identified a series of resonances that belonged to the side chain spin system. The most shielded methyl doublet at 8 0.49 (Me21, / = 6.6 Hz) was correlated to the H20 methine proton (8 1.87) which was in turn weakly coupled to the C22 methylene protons (H22/H22': 8 1.38 and 1.23). The H24 proton (8 2.65) showed COSY correlations to another methyl doublet Me28 (8 1.08) as well as to H23' (8 1.33). A COSY correlation observed between H22' (8 1.23) and H23 (8 1.56) completed the whole spin system in the steroid side chain. Strong HMBC correlations observed from the methyl 11 JJLJX 31 39 C0 2CH3 ^30 33^ 32^ 37^X 36 N34 Jbs OCH3 38 3 HO 4r I 1 1— w "T- •"••! 'r 'f PP» Figure 129: *H NMR spectrum of polymastiamide K (82) in CDCI3 (500 MHz) 253 S o GO :S • 8 o Q c so CO S3 s a o C3 -8 i= a. CO •a c S3 o u 3 254 HO 4 = 31 39 C 0 2 C H 3 = 30 33 ^f^ 3 7 ^ / 36 s34 n OCH3 38 3 -4__JJL 1 ^JIL-XJJJ • » • 0 • 0 0 » * - 50 -100 r-> n » i > 111 i n - ; 111 »i 111111 n i n i 1 1 ; f 111 f i 111 11 ; i i • 111 n i'r r i r ' » ' ' " ' 111111 111 ppei rr*- p »-n 2 PfJ» Figure 131: 2D HMQC spectrum of polymastiamide K (82) in CDCI3 255 18 HO 22 £20 23 16 27 N" I 26 H 31 39 C0 2 CH 3 ^30 33 32] 37 r 36 j,34 11.35 OCH3 38 3 29 82 ^AJJL,... Li WJW^-AJAA^ - 1 . 0 - 2 . 0 3.0 - a. 0 - 5 . 0 - 6 . 0 - 7 . 0 PPM Figure 132: 2D COSY spectrum of polymastiamide K (82) in CDCI3 256 HO 29 31 39 O C02CH3 OCH. 36 3 8 • 82 I t • \ JL^M-_XJIAJ I » 6 9 I t * » r 5 0 riOO \ E-150 111111111[ 1111111111111ri 111 n 11111111111111111111111111111111111' :PP« ppa Figure 133: 2D HMBC spectrum of polyrnastiamide K (82) in CDCI3 Table 26. *H and 13C NMR data of polymastiamide K (82) recorded in CDC13. c# leg lax 2eq 2ax 3ax 4ax 5ax 6eq 6ax 7 7' 8 9 10 11 12 13 14 15 15' 16 16' 17 18 19 20 21 22 22' 23 23' 24 25 26 26' 27 28 29 NH 30 31 32 33 34 35 36 37 38 39 8 13 C 36.5 31.4 76.4 39.5 46.5 21.0 31.4 128.5 145.7 36.5 124.5 130.4 141.9 142.8 27.0 24.5 48.2 19.2 23.0 36.0 14.3 33.7 33.8 35.8 151.0 115.6 168.9 19.4 15.2 56.0 171.7 128.7 128.5 114.4 159.8 114.4 128.5 55.3 52.7 5 *H 2.26, dt(13.5, 3.5) 1.48 1.96 1.67, qd 3.16, td(10.5, 4.3) 1.41 1.20, td 1.98 1.48 2.64 2.57 6.85, bs 2.67 2.54 1.88 1.88 3.22, dt 2.19, bs 1.11, s 1.87 0.49, d (6.6) 1.38 1.23 1.56 1.33 2.65 5.57, bs 5.25, bs 1.08, d(7) 1.05,d(6.3) 6.59, d (6.8) 5.54, d (6.8) 7.28, d (8.7) 6.84, d (8.7) 6.84, d (8.7) 7.28, d (8.7) 3.75, s 3.72, s * C O S Y Hla,H2a,H2e Hle,H2a,H2e Hla,Hle, H2a,H3a HI a, Hie, H2e, H3a H2a, H2e, H4a H3a, H5a, H29 H4a, H6e H5a, H6a, H7, H7' H6e, H7, H7' H6a, H6e, H7' H6a, H6e, H7 H19 H15', H16 H15,H16 H15, H15', H17 H16, H20 Hll H17, H21, H22, H22' H20 H20, H22' H20, H22, H23 H22', H23", H24 H23. H24 H23, H23', H28 H24 H4 H30 NH H34 H33 H37 H36 t H M B C H19 H29 H7, H19, H29 Hl l , H15.H15' H11.H19 H6, H11.H19 H18 H18 H18 H7' H21 Hll H21 H21 H24, H28 H26, H26' H24, H28 H26, H26', NH H33, H37 H39 H30, H33, H34, H36, H37 H34, H37 H37 H33, H34, H36, H37, H38 H34 H33, H36 *NOE Hle,H18 Hl l Correlated to proton resonance in 8 lH column. COSY was recorded at 400 MHz. t Correlated to carbon resonance in 0 column. + Resonance in S *H column was irradiated; recorded at 400 MHz. a = axial, e = equatorial. 258 proton resonance at 8 0.49 (Me21) to a methine carbon at 8 36.0 (C20) and a methylene carbon at 8 33.7 (C22); and from another methyl proton resonance at 8 1.08 (Me28) to a methylene carbon at 8 33.8 (C23), a methine carbon at 8 35.8 (C24), and a fully substituted sp2 carbon at 8 151.0 (C25) confirmed the existence of a steroid side chain in 82. Once the side chain and the p-methoxyphenylglycine methyl ester fragment were established, the remaining structure of 82 had to account for a composition of C20H27O that required 7 sites of un saturation. Since there existed sixteen sp2 carbons in the 13C NMR spectrum of 82, ten of which were assigned to the steroid side chain and p-methoxyphenylglycine methyl ester fragment, the steroid nucleus had to contain six sp2 carbons that comprised three double bonds. The chemical shift and scalar couplings of the carbinol proton H3 (8 3.16, td J = 10.5, 4.3), which was correlated in the HMQC spectrum (Figure 131) to a carbon resonance at 8 76.4 appropriate for an oxygen bonded carbon, suggested that H3 had an axial orientaion and was shielded by the equatorial methyl group at C4 (Me29, 8 1.05) as discussed previously for 66. Detailed analysis of the COSY spectrum (Figure 132) confirmed the existence of Me29 (8 1.05), which showed scalar coupling to a methine proton at 8 1.41 (H4). H4 (8 1.41) showed additional COSY correlations to the carbinol methine proton H3 (8 3.16) as well as to H5 (8 1.20). Further COSY correlations were observed between H5 (8 1.20) and H6eq (8 1.98) and between H6eq/H6ax (8 1.98/ 1.48) and H7/H7" (8 2.64/ 2.57). The carbinol methine proton H3 (8 3.16) was also coupled to both methylene protons H2eq/H2ax (8 1.96/ 1.67) and the latter exhibited further correlations to protons Hleq/Hlax (8 2.26/ 1.48), completing the spin system in the A/B rings. Analysis of the HMBC data supported the above assignment for the A/B rings and also provided evidence that C9 was an sp2 hybridized carbon. Strong HMBC correlations were observed from the methyl doublet at 8 1.05 (Me29) into a carbinol carbon (C3: 8 76.5), a methine carbon (C4; 8 39.5), and another methine carbon (C5: 8 46.5). The C5 methine carbon (8 46.5) was also correlated to a methyl singlet resonance at 8 1.11 (Mel9) via three bonds in the HMBC spectrum, suggesting the methyl singlet (Mel9) was attached to C10. The methyl singlet (Mel9, 8 1.11) exhibited additional HMBC correlations to CI and C10 (both 8 36.5), and to an sp2 hybridized quaternary carbon resonance at 8 145.7 that must be assigned to C9. 259 A broad singlet integrating to three protons at 8 2.19 (Mel8) was assigned to a benzylic or allylic methyl group based on its chemical shift. In the HMBC spectrum (Figure 133), this methyl proton resonance showed correlations into a me thine carbon at 8 124.5 (Cll) and into two non-protonated carbon resonances at 8 130.4 (C12) and 141.9 (C13), respectively. This information implied that the methyl group (Mel8, 8 2.19) had to be attached to one of these two non-protonated carbons. A three bond HMBC correlation* observed between the aromatic proton at 8 6.85 (Hll) , which was correlated to the already identified carbon Cl l (8 124.5) in the HMQC spectrum, and a carbon resonance at 8 141.9 (CI3) ruled out the attachment of Me 18 at 8 141.9 (C13). An additional three bond HMBC correlation observed between the aromatic proton HI 1 (8 6.85) and the aliphatic quaternary carbon CIO (8 36.5) located the aromatic proton at Cl l . The aromatic proton Hl l (8 6.85) showed further HMBC correlations to a non-protonated carbon resonance at 8 128.5 (C8) and the benzylic methyl M18 carbon (8 19.2) as shown below. The remaining olefinic quaternary carbon at 8 142.8 had to be assigned to C14 to form an aromatic C-ring. The base peak in both the LREIMS and HREIMS of 82 at mlz 283.20643 was appropriate for the steroid nucleus ion of Q20H27O (AM +0.3 mmu) resulting from loss of the side chain by an easy benzylic cleavage. VA HO H C HMBC Correlation Figure 134. Selected HMBC correlations in polymastiamide K (82). * Protons on a benzene ring usually show three bond HMBC correlations, but no or very weak two bond HMBC correlations. The HMBC experiment is optimized for coupling constant of 6-8 Hz, while the two bond coupling constant between an aromatic or olefinic proton and an sp^ carbon is 0-3 Hz.68 260 The substructure of the D-ring was routinely identified by analysis of the COSY data of 82. A deshielded methine proton resonance at 8 3.22 (H17), which was coupled to a carbon at 8 48.2 (C17) in the HMQC spectrum, showed scalar couplings with H16/H16' (both 8 1.88). The latter were further correlated to H15/H15' (8 2.67/ 2.54). Finally, the stereochemistry of C17 was established by comparison of *H NMR chemical shifts assigned to H17 and Me21 in 82 with the literature values.69 Steroids with aromatic C-rings are very rare. There appear to be no naturally occurring examples reported to date. All C-ring benzenoid steroids in the literature were prepared by the aromatization of suitable sterols. For example, the treatment of 3(3-acetoxy-5a-cholesta-8,14-dien-7(3-ol 83 with hydrochloric acid in refluxing ethanol gave the aromatic sterol 84 in good yield (scheme 12). The mechanism of C-ring aromatization proposed by Whalley et a/.70 involved protonation, methyl group (Mel9) 1,2-shift from C13 to C12, and deprotonation as depicted in scheme 13. The side chain configuration was inverted in the aromatization promoted by hydrochloric acid.69 Scheme 12. Transformation of the compound 83 to 84. C«H 8 n 17 HCI / EtOH >-Reflux HO p 8 H 1 7 83 84 Scheme 13. Mechanism of C-ring aromatization proposed by Whaley et al. C17 inversion H R Since HCl/MeOH was utilized in derivatization of the polymastiamide mixture, the polymastiamide K (82) obtained in our investigation was very likely a rearranged product. The real metabolite that leads to polymastiamide K (82) is not known at present. However, it is certain that the polymastiamide A (65) could not be the precursor of polymastiamide K (82) as the two compounds have different oxidation states. The evidence that the reaction of pure methyl ester 66 with MeOH/HCl under the methylation conditions (12 hr., 40 °C) did not give any trace of polymastiamide K (82) is consistent with the above hypothesis. 3.3. Conclusions 262 Steroids isolated from marine sponges frequently contain substantially modified side chains.7 The most commonly encountered modifications involve extensive alkylation and/or dealkylation.71 Recently, a small group of biologically active steroids containing more complex side chain functional groups have been reported from sponges.8 The interesting side chain modification in the polymastiamide series (65, 68, 70, 72, 74, 76), which involves linkage to a nonprotein amino acid via an amide bond, represents a new type of marine natural product. The discovery of polymastiamide steroids, among which polymastiamide A (65) was the first to be identified and reported,72 enriches the diversity of steroid modifications. Structurally, the polymastiamides are conjugates of steroids and amino acids. The amino acid part varies only by the presence or absence of the methoxy substituent on the phenyl ring, i.e. the p-methoxyphenylglycine in polymastiamides A (65), B (68), C (70), and D (72) or simply phenylglycine in E (74) and F (76). As for the steroid fragment, it is apparent that the skeletons of the steroidal part of the polymastiamides are quite normal. The variations in the steroid fragment depend on whether the methyl substituents at C4 and/or C24 positions are present. If both methyl groups exist at these two positions, then we have polymastiamides A (65) and E (74). Polymastiamides B (68), C (70), and F (76) only contain one methyl group at either C28 or C4, and B (68) and C (70) are isomeric with each other. Polymastiamide D (72) has no methyl group at either the C24 or C4 position, and therefore it has the cholesterol carbon skeleton. It is not uncommon in marine sterols for a methyl group to be present at C4 and a methyl or ethyl group at the C24 position. The methyl group at C4 is believed to be directly derived from a squalene precursor.73 However, the biogenetic origin of carbon atom at C24 is not readily apparent. Biosynthetic studies of marine sponges have demonstrated that the side chain extension at C24 involves SAM (S-adenosylmethionine) dependent enzymatic methylation, and that in the conversion of desmosterol (85) to 24-methylenecholesterol (86) there is a hydride shift (3H) from C24 to C25 (Scheme 15).74 Scheme 14. Biosynthesis for steroidal side chain extension. 263 W W W 85 SAM H-shift 86 In conclusion, our chemical investigation of the Norwegian sponge Polymastia boletiformis has led to the isolation and identification of the novel sponge metabolites, polymastiamides A (65), B (68), C (70), D (72), E (74), and F (76), as well as the rearranged by-products, polymastiamides G (78), H (79), I (80), J (81), and K (82). Experimental 264 General See the experimental section of chapter II. Collection of sponge Polymastia boletiformis and isolation and derivatization of polymastiamides Specimens of Polymastia boletiformis were collected by hand using SCUBA at depths of 20-25 m on vertical rock faces off Korsnes Peninsula on Fanafjiord south of Bergen, Norway, in July 1992. Freshly collected sponge (2 kg wet weight) was frozen on site, transported to Vancouver over dry ice, and stored in freezer. A portion of the frozen sponge (152 g) was cut into small pieces, immersed in MeOH, and soaked at room temperature for 2 days. The methanol extract was concentrated in vacuo to give a red suspension that was partitioned between an aqueous solution (300 mL) of 1:1 H20/MeOH and hexanes (200 mL x 2). The aqueous layer was concentrated and lyophilized to yield a brown solid (12.2 g, mostly salt). The solid was suspended in MeOH, and the suspension then filtered. The filtrate was concentrated in vacuo and chromatographed on Sephadex LH-20 (eluent: MeOH) to give a fraction containing steroidal compounds (378 mg). This fraction was desalted by reversed-phase silica column chromatography (eluent: first water, then H20/MeOH 50:50) to afford 193 mg of steroid mixture. Subsequent chromatography on Sephadex LH-20 (eluent: EtOAc/MeOH/H20 = 40:10:4) yielded almost pure polymastiamide A (65). Final purification was achieved by repeated Sephadex LH-20 chromatography (eluent: EtOAc/MeOH/H20 = 40:10:4) to give pure polymastiamide A (65) (34 mg). Polymastiamide A (65) off-white amorphous solid; [OC]21D = +67.4° (MeOH, c = 1.1); UV Xmax 224 nm (e 8840), 272 nm (8 1520), and 278 nm (£ 1270); IR (KBr) 3519, 2945, 1730, 1656, 1613, 1514, 1465, 1380, 1249, 980, 827, and 630 cnr1; *H NMR see Table 16; 13C NMR see Table 16; negative ion HRFABMS mlz 684.35480 (C38H54NO8S AM -3.24 ppm). 265 Methanolysis of Polymastiamide A (65): Polymastiamide A (65) (12 mg) was dissolved in 3 mL of MeOH containing five drops of concentrated HC1, and the reaction mixture was stirred overnight at room temperature. The reaction mixture was partitioned between EtOAc and H2O. Evaporation of EtOAc gave the methyl ester 66 (9 mg) that was further purified on normal phase HPLC (eluent: 3:1 hexane/EtOAc) (yield 4.5 mg). Polymastiamide A Methyl Ester (66): amorphous white solid; [OC]21D = +66.6° (MeOH, c = 0.35); UV ^max 226 nm (E 10620), 272 nm (£ 1590), and 278 nm (£ 1420); IR (KBr) 3316,2933, 2872, 1741, 1651, 1615, and 1507 cm"1; lH NMR see Table 17; 13C NMR see Table 17; LREIMS m/z 619 (19.3), 601 (15.6), 586 (10.5), 285 (7.6), 194 (90), and 179 (100); HREIMS (M+) m/z 619.4235 (C39H57NO5 AM -0.2 mmu). Determination of the Absolute Configuration of the /?-Methoxyphenylglycine Residue: Methyl ester 66 (3 mg) was dissolved in 4 mL of 5 N HBr and refluxed overnight with stirring. The cooled reaction mixture was evaporated to dryness, and traces of HBr were removed from the residue by repeated evaporation from 10 mL aliquots of H2O. The residual hydrolysate was treated with excess Marfey's reagent,67 5-fluoro-2,4-dinitrophenyl-L-alanineamide (FDAA), for 1 hour at 40 °C. The FDAA derivative was analyzed by reversed-phase HPLC. A linear gradient of (A) 9:1 triethylammonium phosphate (50 mM, pH 3.0)/MeCN and (B) MeCN with 0 % at the start to 40 % over 40 minutes (flow rate 0.7 mL/min.) was used to separate the FDAA derivative which was detected by UV absorbance at 340 nm. The peaks in the chromatograph were identified by comparing their retention times and photodiode array UV spectra with those of pure amino acid standards and by coinjection. The hydrolysate showed peaks at 54.2 (major) and 57.1 (minor). The amino acid standards gave the following retention times in minutes: 57.9 for D-p-hydroxyphenylglycine, 55.1 for L-p-hydroxyphenylglycine injected as a racemic mixture; 57.7 for £>-/?-hydroxyphenylglycine injected as a pure enantiomer. In all cases a peak at 42 minute was observed, which was attributed to the excess FDAA. Coinjection of the hydrolysate sample with the authentic amino acid derivatives confirmed that the p-hydroxyphenylglycine residue in polymastiamide A (65) had theL-configuration. Methyl Esters 69, 71, 73, 75, 77; Polymastiamides G, H, I, J (78, 79, 80, 81); and Polymastiamide K (82): All fractions containing polymastiamide analogs were combined and treated with 2 N hydrochloric acid in MeOH-H20 (1:1) solution at 50 °C for 4 hours. The reaction mixture was evaporated in vacuo and partitioned between ethyl acetate and water. The ethyl acetate layer was concentrated and chromatographed on normal phase silica gel column (eluent: 30:70 EtOAc/Hexane) to give crude products. Repeated fractionation of the crude products on normal phase HPLC (eluent: 1. EtOAc/Hexane = 25:75 to 50:50; 2. EtOAc/CH2Cl2 = 8:92) led to the separation and purification of methyl esters 75, 77, polymastiamide J (81), methyl ester 66, polymastiamide G (78), polymastiamide K (82), methyl ester 71, polymastiamide I (80), methyl ester 69, polymastiamide H (79), and methyl ester 73 in eluting out sequence. Polymastiamide B Methyl Ester (69): colorless solid; lU NMR see Table 18; 13C NMR see Table 18; LREIMS mlz (relative intensity): 605 (2.9), 587 (2.4), 546 (1.6), 194 (98), 179 (100); HREIMS (M+) mlz 605.40806 (C38H55NO5 AM +0.0 mmu). Polymastiamide C Methyl Ester (71): colorless solid; !H NMR see Table 19; 13C NMR see Table 19; LREIMS mlz (relative intensity): 605 (14.9), 587 (5.8), 572 (3.6), 546 (3.2), 194 (100), 179 (74); HREIMS (M+) mlz 605.40820 (C38H55NO5 AM +0.2 mmu). Polymastiamide D Methyl Ester (73): colorless solid; !H NMR see Table 20; 13C NMR see Table 20; LREIMS mlz (relative intensity): 591 (12.8), 573 (3.4), 559 (2.7), 532 (2.9), 297 (2.3), 194 (100), 179 (74.8); HREIMS (M+) mlz 591.39203 (C37H53NO5 AM -0.4 mmu). Polymastiamide E Methyl Ester (75): colorless solid; JH NMR (CDCI3, 400 MHz) 8: Hl e q , 1.68, Hl a x , 1.13; H2eq, 1.81 (dq, 7=13, 4 Hz), Hl^ 1.42; H3, 3.11 (td, 7=10.8, 4.5 Hz); H4, 1.26; H5, 0.84; H6eq, 1.69, H6ax, 0.97; H7cq, 2.38, m, H7ax, 1.64; H9, 1.61; Hl l e q , 1.58, H l l a x , 1.43; H12eq, 1.88 (dt, 7=12.2, 4 Hz), H12ax, 1.06; H15, 2.16 (2H); H16, 1.72, H16', 1.27; H17, 1.06; H18, 0.78 (s, 3H); H19, 0.69 (s, 3H); H20, 1.37; H21, 0.85 (d, 7=6.7 Hz); H22, 1.36, H22', 1.01; H23, 1.50, H23', 1.16; H24, 2.57 (dt, 7=6.9, 6.9 Hz); H26, 5.58 (bs), H26', 5.23 (bs); H28, 1.05 (d, 7=6.8, 3H); H29, 0.97 (d, 7=6.4, 3H); NH, 6.72 (d, 7=7.1 Hz); H30, 5.61 (d, J=7.1 Hz); H33/H37, 7.34; H34/H36, 7.35; H35, 7.31; H39, 3.73 (s, 3H); LREIMS mlz 589 (18.5), 571 (28), 556 (36), 285 (21), 283 (29), 166 (100), and 149 (27); HREIMS (M+) mlz 589.41223 (C38H55NO4 AM -0.9 mmu). Polymastiamide F Methyl Ester (77): colorless solid; *H NMR see Table 21; 13C NMR see Table 21; LREIMS mlz (relative intensity): 575 (93), 557 (54), 542 (52), 516 (14), 410 (21), 392 (22), 382 (34), 367 (23), 283 (81), 260 (55), 247 (41), 221 (68), 166 (78), 164 (48), 149 (44); HREIMS (M+) mlz 575.39667 (C37H53NO4 AM -0.8 mmu). Polymastiamide G (78): colorless solid; *H NMR see Table 22; 13C NMR see Table 22; LREIMS mlz (relative intensity): 619 (9.4), 601 (1.9), 587 (1.4), 560 (2.0), 285 (16), 194 (100), 179 (65); HREIMS (M+) mlz 619.42404 (C39H57NO5 AM +0.4 mmu). Polymastiamide H (79): colorless solid; ]H NMR see Table 23; 13C NMR see Table 23; LREIMS mlz (relative intensity): 605 (7.7), 587 (1.7), 573 (1.7), 546 (3.0), 426 (2.2), 410 (2.4), 382 (2.4), 299 (7.7), 271 (16), 255 (8.1), 194 (100), 179 (59); HREIMS (M+) mlz 605.40936 (C38H55NO5 AM +1.3 mmu). Polymastiamide I (80): colorless solid; *H NMR see Table 24; 13C NMR see Table 24; LREIMS mlz (relative intensity): 605 (14), 587 (1.7), 573 (0.9), 546 (2.5), 410 (10.3), 383 (2.9), 355 (7.2), 329 (5.3), 313 (12), 302 (27.6), 285 (19.4), 251 (11.7), 194 (93), 179 (100); HREIMS (M+) mlz 605.40839 (C38H55NO5 AM +0.4 mmu). Polymastiamide J (81): colorless solid; *H NMR see Table 25; 13C NMR see Table 25; LREIMS mlz (relative intensity): 575 (2.6), 557 (0.9), 542 (1.7), 516 (3.1), 499 (1.3), 410 (27.2), 383 (9.8), 367 (7.7), 329 (8.4), 313 (25), 285 (46), 260 (46), 221 (29), 163 (37), 149 (27); HREIMS (M+) mlz 575.39762 (C37H53NO4 AM +0.2 mmu). Polymastiamide K (82): colorless solid; ]H NMR see Table 26; 13C NMR see Table 26; LREIMS mlz (relative intensity): 615 (8.6), 597 (0.8), 582 (1.0), 556 (0.8), 283 (100), 194 (22), 179 (32), 143 (15.2); HREIMS (M+) mlz 615.39208 (C39H53NO5 AM -0.3 mmu). 268 Part IV New Metabolites from Two Papua New Guinea Sponges 4.1. Introduction This chapter describes the results of two individual projects: 1) the isolation and structure elucidation of pseudaxinellin (87), a cyclic heptapeptide isolated from the sponge Pseudaxinella massa; and 2) the identification of geodiamolide G (111), a cyclodepsipeptide from the sponge Cymbastela sp. The projects were accomplished in cooperation with Dr. David L. Burgoyne and Dr. E. Dilip de Silva within our research group. 4.1.1. A review of peptides related to pseudaxinellin (87) and geodiamolide G f i l l ) Marine sponges continue to be a very rich source of secondary metabolites. Recently a number of cyclic peptides and cyclic depsipeptides have been isolated from marine sponges.75 Many of these peptides have highly modified structures including a high incidence of N-methylated amino acids, (3-amino acids, and residues with the ^ -configuration. These peptides often possess potent biological activities. More and more evidence is being gathered to support the speculation that these metabolites with 'unnatural' amino acid residues are the products of symbionts and not of sponges. A small number of peptides containing standard protein amino acid residues all with the natural L-configurations have also been isolated from sponges, but these peptides usually do not have as potent biological activities as those with unusual residues. The two distinctly different peptides, pseudaxinellin (87) and geodiamolide G (111), isolated from two species of South Pacific marine sponges represent examples of each of two the categories discussed above. Jaspamide (88), a four-residue cyclic depsipeptide, was isolated independently from the South Pacific sponges of the genus Jaspis (order: Choristida) by two research groups.76 The structures of the amino acid residues, and the amino acid sequence were determined by an 269 interpretation of the spectroscopic data. An analysis of the x-ray diffraction data of the acetate derivative firmly established the structure of jaspamide including the relative stereochemistry. The absolute stereochemistry was secured from the configuration of the alanine residue based on chiral HPLC analysis of the acid hydrolyzate. Jaspamide contains a 12-carbon polypropionate unit and two rare amino acids, (3-tyrosine and 2-bromoabrine with the 'unnatural' D-configuration. Conformational analysis, molecular mechanics and molecular dynamics calculations have demonstrated that jaspamide adopts two major conformations.77 Recently, Crews et al. reported that specimens of Auletta c.f. constricta (order: Choristida) collected in Papua New Guinea also contained jaspamide.78 Jaspamide exhibited potent insecticidal activity against Heliothis virescens and antimicrobial activity against Candida albicans as well as in vitro cytotoxicity against larynx epithelial carcinoma at 0.32 |i.g/mL and human embryonic lung cancer cells at 0.01 fj.g/mL. OH Shortly after the structure of jaspamide was reported, two closely related cyclic depsipeptides, geodiamolides A (89) and B (90), were isolated from the Caribbean sponge Geodia sp.(order: Choristida).79 The structures of the fragments in geodiamolides A and B were determined by spectroscopic analysis, and their stereostructures were determined by x-ray 270 crystallography.79 Similar to jaspamide (88), geodiamolides A and B (89 and 90) contain a tripeptide unit with one ^ -configuration residue and a twelve-carbon polypropionate unit linked to form an 18-membered ring. Geodiamolides A (89) and B (90), together with the new metabolites C (91) to F (94), were also isolated from a Papua New Guinean sponge identified as a Pseudoaxinyssa sp., belonging to the order Axinellida that is taxonomically distant from Geodia.%Ga Geodiamolides A (89), B (90), and C (91) are differed only in the halogen atoms present in the tyrosine residues, while geodiamolides D (92), E (93), and F (94) contain a glycine residue instead of the alanine residue found in geodiamolides A, B, and C, respectively. Recently, jaspamide (88) and geodiamolide TA (95) were reported to be isolated from the sponge Hemiastrella minor (order: Hadromerida) collected in South Africa.81 Geodiamolide TA (95) differs from geodiamolide D (92) only in the replacement of an alanine residue with a valine residue. Isolation of the same family compounds from taxonomically remote species suggests that they might be produced by symbiotic microorganisms.82 The geodiamolides showed in vitro cytotoxicity against L1210 murine leukemia cells: geodiamolide A (ED50 0.0032 |ig/mL), B (ED50 0.0026 |ig/mL), C (ED50 0.0025 (ig/mL), D (ED50 0.039 Hg/mL), E (ED50 0.014 |ig/mL), and F (ED50 0.006 (ig/mL). 89 X = I, R = R* = Me 90 X = Br, R = R' = Me 91 X = CI, R = R' = Me 92 X = I, R = H, R' = Me 93 X = Br, R = H, R' = Me 94 X = CI, R = H, R' = Me 95 X = I, R = Me, R' = i-Pr There are numerous examples that support the speculation that many of the cyclic peptides found in sponges have their origin in symbiotic blue-green algae. One of the clear examples of this is the isolation of the cyclic pentapeptide motuporin (96) from the sponge Theonella swinhoei collected off Motupore Island, Papua New Guinea.833 Motuporin is closely related to the HO 271 nodularins (97, 98)83b>c and microcystin-LR (99)84 found in fresh-water blue-green algae. The structures of all three peptides are characterized by the presence of three unusual amino acids, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (Adda), D-glutamic acid, and £)-erythro p-methyl aspartic acid (Masp). Motuporin (96) possesses potent inhibitory activity against protein phosphatase-1 (IC50 < 1 nM) and exhibits considerable in vitro cytotoxicity against several cancer cell lines. C 0 2 H 96 R = CH(CH3)2 R' = CH3 97 R = (CH 2 ) 3 NHC(=NH)NH 2 R' = CH3 98 R = (CH 2 ) 3 NHC(=NH)NH 2 R ' = H O C 0 2 H 99 In contrast to the peptides with unusual residues, certain peptides with standard L-a-amino acid residues have also been obtained from the marine sponges. Among these are the fenestins A 272 (100) and B (101) isolated from the sponge Leucophloeus fenestrata collected off Fiji.85 The fenestins are the first examples of medium ring size cyclic peptides. Both compounds contain two prolines and valine/leucine residues. All of the amino acid residues in these two peptides have the L-configuration. The fenestins were found to have no biological activity in a limited number of assays. 100 101 Recently, a growing number of cyclic hepta and octapeptides have been isolated from marine sponges. All these peptides are proline rich with standard amino acid residues. Phakellistatin 1 (102) was isolated from two Indo-Pacific sponges, Phakellia costate and Stylotella aurantium.^ 102 OH 273 The structure was determined by analyses of NMR and MS data, and involved the use of a chiral GC method for absolute configuration determination. The assignment was finally confirmed by a single crystal x-ray diffraction analysis. Phakellistatin 1 is a proline rich cyclopeptide with L-configurations for all the amino acid residues . The X-ray structure of phakellistatin 1 suggests the amide bonds at CO(Phe)/N(Pro) and CO(Ile)/N(Pro) have the cis geometries. A series of cycloheptapeptides, hymenamides A-E containing two or three proline residues, have been isolated from the Okinawan marine sponge Hymeniacidon sp..87 Hymenamides C (103), D (104), and E (105) share the same backbone structure in solution with one cis amide bond at the Pro2 position and three transannular hydrogen bonds incorporating a {3-bulge motif, indicating that the proline residues play an important role in the backbone conformation. The absolute stereochemistry of each amino acid was found to be L by a combination of chiral HPLC and chiral GC analyses of the acid hydrolysate. 103 Prol-Phe-Gly-Pro4-Glu-Leu-Trp 104 Prol-Tyr-Asp-Pro4-Leu-Ala-IIe 105 Prol-Thr-Thr-Pro4-Tyr-Phe-Phe More recently, two additional cyclic heptapeptides, axinastatins 2 (106) and 3 (107), were isolated from a West Caroline Island marine sponge Axinella sp..88 Structures of axinastatins 2 and 3 were elucidated by HRFABMS and tandem MS/MS techniques and 2D NMR spectral analysis. The absolute stereochemistry was established by a combination of hydrolysis, derivatization, and chiral GC method, and found to have L-configurations. Both axinastatins 2 and 3 contain two proline residues having the same sequence as that of hymenamides C-E (103-105), with valine and/or leucine-isoleucine residues in high rate. Axinastatin 3 differs from axinastatin 2 only by replacement of valine with isoleucine. 274 O V T S „ \ ^ O ^ N H 0 0 106 R = H 107 R = CH3 Hymenistatin 1 (108) is a cyclic octapeptide isolated from the western Pacific Ocean sponge Hymeniacidon sp. collected off the Western Caroline Islands in Palau Archipelago.89 The amino acid composition of hymenistatin 1 was deduced from an interpretation of the NMR data, while the chirality of the constituent amino acids was determined by chiral GC analysis. The sequence of the amino acid residues was determined by NOE experiments and confirmed by FABMS/MS. In the FABMS/MS spectrum the protonation of each proline nitrogen gave three different series of ions. Hymenistatin 1 exhibited in vitro cytotoxic activity against the P388 murine leukemia cell line (ED50 3.5 |j.g/mL). OH 108 R1 = Me R2 = Et 109 R1 = H R2 = i-Pr 275 The collection of the Okinawan sponge Hymeniacidon sp. also yielded the cyclic octapeptides, hymenamides G, H, J, and K,90 in addition to cycloheptapeptides, hymenamides A-E. The structures of hymenamides G, H, J, and K were elucidated on the basis of 2D NMR data and Edman degradation experiments of their partial hydrolysis products. The absolute stereochemistry of each amino acid was determined to be L by HPLC analyses of FDAA derivatives (Marfey's method) of the acid hydrolysates. Hymenamide G (109) defers from hymenistatin 1 (108) by replacement of the isoleucine in 108 with a leucine residue in 109. It is clear now that sponge peptides are in general divided into two categories. Category one contains peptides having amino acids with the unnatural D-configuration and p-amino acid residues. Peptides in this category usually possess significant biological activities. These peptides are conceivably associated with symbiotic microorganisms, particularly blue-green algae (cyanobacteria). Alternatively, the category two sponge peptides contain only protein amino acids having the natural L-configuration and they frequently contain proline residues. There is no clear correlation between these peptides and blue-green algal metabolites. 4.2. Results and Discussion 276 4 . 2 . 1 . The Structure Elucidation of Pseudaxinellin. a Cyclic Heptapeptide from the Marine Sponge Pseudaxinella massa This section describes the structure elucidation of a cyclic heptapeptide isolated from the marine sponge Pseudaxinella massa.95a Specimens of P. massa were collected as part of a general collection expedition off the islands of Papua New Guinea. 4.2.1.1. Taxonomy and Description of Pseudaxinella massa Pseudaxinella belongs to the family Axinellidae and the order Axinellida.95b Sponges of this order contain a stiff axial area. The ectosome is thick with sparsely scattered spicules. Both massive and encrusting sponges are found within this order and Pseudaxinella has a massive structure. All species of Axinellida have rough surfaces due to projecting spicules.950 Pseudaxinella obtained for this study was collected off Loloata Island on the south coast of Papua New Guinea near Port Moresby. The specimens were collected in 10-25 meters of water in areas exposed to little or no surge.95d Figure135.Pseudaxinellamassasponge.L’3-J-a278 Scheme 15. Phylogenetic classfication of Pseudcainella massa according to Bergquist 95b Phylum Porifera (sponges) Class Calcarea Demospongiae Hexactinellida Sclerospongiae subclass Homoscleromorpha Tetractinomorpha Ceractinomorpha Order Choristida Spirophorida , r Lithisida Hadromerida Axinellida Family Axinellidae Genus Axinella Pseudaxinella Ceratopsion PhakeIlia Auletta I Acanthella Ptilocaulis Pararaphoxya Homxinella / Bubairs Species P. massa 279 4.2.1.2. Isolation of pseudaxinellin (87) from Pseudoaxinella massa Specimens of Pseudoaxinella massa were collected by hand using SCUBA, frozen on site, and transported to Vancouver over dry ice. Frozen specimens were homogenized and extracted in methanol at room temperature for two days. The mixture was filtered and the filtrate evaporated in vacuo to yield a residue that was suspended in water and sequentially extracted with hexanes, dichloromethane, and ethyl acetate. A routine TLC examination and lH NMR analysis indicated the presence of a moderately complex peptide. Purification of the methylene chloride soluble fraction using Sephadex LH-20 (eluent: hexanes/CHCl3/MeOH 10:10:1) followed by silica gel flash chromatography (eluent: EtOAc/MeOH, 10:1.5) and finally by silica gel preparative thin layer chromatography (eluent: EtOAc/MeOH 4:1) yielded pseudaxinellin (87) as an optically active ([CC]D -100.1°, c 0.34, CHC13) colorless glass. 4.2.1.3. Structure Elucidation of Pseudoaxinellin (87) 280 Yah Val-Phe HN 35 >o ° 3 2 ^ N 3 Pro2 Pro lor ° NH 1 13 14 V « „ u 22 87 CONH 2 18 Asn The structure of 87 was elucidated by a detailed analysis of one and two dimensional NMR spectra and mass spectroscopic analysis. The structures of the individual residues were determined by analysis of COSY, HOHAHA, HMQC, and HMBC experiments. The sequence of these residues was determined by a combination of ROESY, HMBC, and one dimensional difference NOE experiments. The EIHRMS of 87 gave a parent ion at mlz 752.4218 appropriate for a molecular formula of C38H56N8O8 (AM -0.3 mmu) which required fifteen degrees of unsaturation in the molecule. The *H and 13C NMR spectra of 87 contained resonances that were characteristic of peptides. The *H NMR spectrum (Figure 105) contained several protons that were attached to heteroatoms as well as five aromatic protons. Seven methine resonances between 8 4.0 and 4.9 ppm, that could be assigned to cc-protons of amino acid residues, and several methyl multiplets were also present in the *H NMR spectrum. The HOHAHA data (Table 28, Figure 107) established the majority of the resonances within all of the nine isolated spin systems. The 13C NMR spectrum (Figure 106) contained eight resolved carbonyl resonances between 8 169 and 173 ppm and four aromatic carbon resonances. »™t~»—'I « I I »" PP" 8 > 8 LJUL Val. Val. 27 Pro 2 Pro 13 14 X. " 22 23 Val, CONH2 18 * Asn 87 37 38 32 33 22 23 Figure 136: l H NMR spectrum of pseudaxinellin (87) recorded in CDCI3 (500 MHz) 00 Vah 38 37 Val2 Proi .<WrY», J \—L v? o I CONHo Asn 18 t,vw»"vyfw » 9 tf» • llll|IIIMIIIIIMIM?ff«|f1 160 140 W*99*9 9V9,9*WV*mwmwwm9*VW9<*vV)jm>* V9 9 99.W »»•»«••'>•»»» | l»IIHH»Wmi lt»f W«»»»T»» 120 100 66 IT^WKAX1 * u J*JW. www v-vVAvwe^c^ 4© Sf »o Figure 137: 13Cand APT NMR spectra of pseudaxinellin (87) (125 MHz, CDCI3) to 00 Val3 Val2 38 37 H | P r 0 * Phe H N^35Sf N v ^29^N- " 5 3 I H z [25 >27 p - ° 3 2 ^ V 2. 8 Pro, 13 CONH2 2 2 V a , l 18 Asn 87 __AiLJtoA__>AA^jL_^^ V Vali < i i i i l i i i i 4 I i i i i i i i i i I i i i i i i i i i i I I i -7.0 -7.5 •8.0 PP" gure 138: NH region of HOHAHA spectrum of pseudaxinellin (87) in CDCI3 284 Phe Prol Asp Vail Pro2 Val2 Val3 Table 27. lHand 1 3 CNM C# 1 2 3 3 ' 4 5,9 6,8 7 NH 10 11 12 12' 13 13' 14 14' N 15 16 17 17' 18 NH2 NH 19 20 21 22 23 NH 24 25 26 26' 27 27' 28 28' N 29 30 31 32 33 NH 34 35 36 37 38 NH ^ ( 4 0 0 MHz) -4.77, m 2.94 3.08 -7.13 7.24 7.18 7.52, d(J=9.5 Hz) -4.08 2.28, m 1.36, m 1.78 1.92 3.58, bt(J=7.3 Hz) 3.48 -4.61, bm 2.94 3.20 -5.43, bs; 6.66, bs 8.01, d(J=5.3 Hz) -4.05 2.36, m 1.04 1.04 8.22, d(J=8.0 Hz) -4.53, d(J=6.9 Hz) 2.57, m 1.90, m 1.93, m 1.70, m 3.70, m 3.48 -4.16, dd(J=4.7, 7.3 Hz) 1.95 1.04 0.95 6.80, d(J=5.2 Hz) -4.21, t(J=9.4 Hz) 2.01 0.95 0.95 7.32, d(J=9.4 Hz) R. data for pseudaxinellin (87) recorded in CDCI3. aCOSY H3, H3', NH(7.55) H2, H3' H2, H3 H2 H12, H12' Hl l , H12', H13, H13' H l l , H12, H13 H12, H12', H14' H12, H14, H14' H13', H14' H13, H13', H14 H17, H17', NH(8.01) H16, H17' H16, H17 6.66, 5.43 H16 H21, NH(8.22) H20, H22, H23 H21 H21 H20 H26' H26', H27, H27' H25, H26 H26, H27' H26.H27 H27, H27', H28' H27, H27', H28 H31, NH(6.80) H30, H32, H33 H31 H31 H30 H36, NH(7.32) H35, H37, H38 H36 H36 H35 b NOE H5, H9 H16 -H30 H25 H35 NH(6.80) 1 3C(125 MHz) j 172.1 55.4 37.7 137.5 128.9 128.3 126.6 170.3 63.5 29.6 25.8 48.0 172.6 50.2 36.2 169.0 172.3 62.1 29.5 18.8 18.7 171.5 61.2 31.2 21.8 46.0 171.8 58.5 30.1 19.8 18.5 171.2 57.3 29.1 19.9 19.3 CHMBC NH(7.32) H3 H3, H3', H6, H8 H3, H3\ H7 H8.H6 H5, H9 Hl l , NH(7.55) H17' H17' NH2(5.43) H17' H20, NH(8.01) H25, NH(8.22) H25 H25 H25, H26 H30 H30 H36 H35 a Protons correlated to proton resonance in *H column. b Proton in C# column irradiated. c Protons correlated to carbons in C# column. 285 Table 28. HOHAHA assignments for pseudaxinellin (87) recorded in CDCJ3. Residue Phe Proi Asn Vali Pro2 Val2 Val3 HOHAHA assigned spin systems 8 7.55, 4.77, 3.23, 2.94 ppm 8 4.08, 3.58, 2.28 ppm 1) 6 8.01, 4.61, 3.20, 2.94 ppm; 2) 5 6.66, 5.43 ppm 6 8.22, 4.05, 2.36, 1.04 ppm 8 4.53, 3.48, 2.57, 1.90 ppm 5 6.80,4.16, 1.95, 1.04 ppm 8 7.32, 4.21, 2.01, 0.95 ppm Analysis of the HMQC and APT spectra demonstrated the presence of a mono-substituted aromatic ring. The APT revealed four 13C aromatic resonances that had to account for five methine carbons and one quaternary carbon. This data is consistent with a symmetrical aromatic system where C5 and C9 as well as C6 and C8 are chemically equivalent. The presence of a phenyl group at the P-position of an alanine residue was confirmed by HMBC correlations from the methylene protons at 8 2.94 and 3.23 ppm to the aromatic carbon at 8 137.5 (C4) and 128.9 (C5/C9) ppm. The methylene protons showed COSY correlations into the methine at 8 4.77 ppm which in turn gave a COSY correlation to the NH proton at 8 7.55 ppm. 7.24 H H 7.18 The HOHAHA spectrum (Figure 107) indicated that the group of methyl multiplets between 8 0.9 and 1.1 ppm belonged to a series of three valine residues. The first system, Vali, contained resonances at 8 8.22, 4.05, 2.36, and 1.04 ppm. Analysis of the COSY spectrum 8 7.55 HMBC Phe 286 revealed that the NH proton (8 8.22) was coupled into the cc-methine at 8 4.05 ppm, and that the isopropyl methine, Vali-pH (8 2.36), was coupled into Vali-aH (8 4.05) and also into the unresolved methyls at 8 1.04 ppm. The Vali-CO (8 172.3) assignment was based on an HMBC correlation to the carbonyl from Vali-aH (8 4.05). l.04H3C C: 172.3 As with Vali, the COSY experiment could uniquely assign the resonances of NH (8 6.80), aH (4.16), PH (1.95), and the methyl protons (1.04) to the second valine spin system. The HOHAHA spectrum (Figure 107) of 87 showed the spin system that contained resonances at 8 6.80 (NH), 4.16 (Val2-ocH), 1.95 (Val2-pH), and 1.04 and 0.95 (two methyls) ppm. The Val2-CO (8 171.8) was assigned based on an HMBC correlation to Val2-aH (8 4.16). C: 171.8 H 4.16 0.95 H3C-L 9 5 H C H3i.Q4 Val-The final valine spin system, Val3, consisted of resonances at 8 7.32, 4.21, 2.01, and 0.95 ppm. Again, analysis of the COSY spectrum as well as the HOHAHA spectrum assigned the resonances to NH (8 7.32), aH (4.21), pH (2.01) and the unresolved methyls (0.95). An HMBC correlation from the Val3-aH (4.21) to the Val3-CO (8 171.2) assigned the carbonyl carbon. 13C: 171.2 0.95 H 3 C H 4.21 2 .01 H C H 3 0 . 9 5 Vah COSY and HOHAHA analysis identified two proline spin systems, each containing an oc-methine connected to three contiguous methylene carbons. The chemical shifts of the terminal methylene protons in each system were appropriate for attachment to heteroatoms. The cyclic structures accounted for two of the fifteen degrees of unsaturation in the molecule. The assignment of the proline carbonyls at 8 170.3 and 171.5 ppm were based on HMBC correlations from Proi-ceH (5 4.08) and Pro2-aH (5 4.53), respectively. '4.08 1.92 H H L 3 6 1.80 Pro 1 The remaining oc-methine proton at 8 4.61 ppm was part of a spin system containing resonances at 8 8.01, 4.61, 3.20, and 2.94 ppm (Figure 107). Analysis of the COSY spectrum showed correlations from 8 4.61 ppm to the NH proton (8 8.01) and a pair of methylene protons (8 3.20 and 2.94). The two remaining carbonyls in the molecule showed HMBC correlations from the methylene proton at 8 3.20 ppm suggesting that the spin system was terminated in the side chain by a carbonyl functionality. Only the NH2 protons at 8 5.43 and 6.66 ppm, which were only coupled into each other, remained unassigned. An HMBC correlation from the proton at 8 288 5.43 ppm to the methylene carbon (5 36.2) attached the NH2 group to the carbonyl in the side chain and completed the asparagine residue (Asn). 8 8.01 6.66 H HMBC Asn The functionality present in the seven residues accounted for fourteen of the required fifteen degrees of unsaturation. It was apparent that pseudaxinellin (87) was a cyclic peptide. The failure of 87 to react with ninhydrin was consistent with a cyclic structure. The sequence of the amino acid residues was determined by a combination of HMBC, ROESY, and one dimensional NOE experiments. Two fragments composed of three residues each were established based on HMBC correlations from NH proton to the carbonyl carbon of the adjacent residue (Figure 139). HMBC correlations from Phe-NH (5 7.52) to Proi-CO (8 170.3) and from Val3-NH (8 7.32) to Phe-CO (8 172.1) established the partial sequence Proi-Phe-Val3. HMBC Pro rPhe-Val3 Figure 140a. Connectivities in pseudaxinellin (87) based on HMBC results. 683 290 HMBC correlations from Vali-NH (5 8.22) to Pr02-CO (5 171.5) and from Asn-NH (8 8.01) to Vali-CO (5 172.3) established the second three residue sequence, Prc>2-Vali-Asn. HMBC 3.20 \ ~ y |169-0 NH2 Pro2-Vali-Asn Figure 140b. Connectivities in pseudaxinellin (87) based on HMBC results. Difference NOE (Figures 141 and 142) and ROESY correlations between the Proi-8H (5 3.58) and the Asn-aH (5 4.61), and between the Val3-ccH (5 4.21) and the Val2-NH (5 6.80) completed the linear sequence, Pro2-Vali-Asn-Proi-Phe-Val3-Val2. Val , -N Val-Pro, NOE/ROESY Val i : CONH2 Asn Figure 141. Connectivities in pseudaxinellin (87) based on NOE and ROESY results. A ROESY correlation and a strong NOE (8.5%) between Pro2-aH (6 4.53) and the Vah-aH (5 4.16) resonances established the Val2-Pro2 connectivity, and therefore completed the macrocyclic Val2 NH inadialcd H3S irradiated 11 Val2NH H2S irradiated H30 irradiated y^^^^« * — ^^^y <**'++— *•• -P» "*»»fc«A/ A^». Wi»*W*>M H25 f^^d*"! *i*~fc fiim« — -\_f *m *+» S»^«Vr^ ^•'•r*V ^f^^^lMrfhtMw^^^^^^^ H14 irradiated HI6 JL_ H**-V-**-*> A-—*r ^W* • WJ¥W#*'*M /LA. XL__AiiLj4_^M_>VLjL_A p-r-i 8.0 r-|—i 7.0 6.0 PPM 5.0 r-|-i 4.0 3.0 Figure 142: NOE results for pseudaxinellin (87) (400 MHz, CDC1 3) to 292 ring. This was the only NOE correlation observed between cc-protons of adjacent amino-acid residues in 87, which implied that the CO(Val2)/N(Pro2) amide bond had the cis geometry. A long range COSY correlation from Vali-aH (8 4.05) to Pro2-aH (5 4.53) supported the Pro2-Vali linkage. The absolute configurations of the amino acids were determined using Marfey's method. Hydrolysis of 87 with 6N HC1 yielded the corresponding amino acids (note: Asn was converted to Asp under these conditions). The acid hydrolysate was treated with Marfey's reagent, 5-fluoro-2,4-dinitrophenyl-L-alanine amide (FDAA)67 and the resulting FDAA derivatives were analyzed by reverse phase HPLC. Each peak in the chromatographic trace was identified by comparing the retention time with that of the FDAA derivative of the authentic amino acid and by coinjection. The Marfey's reagent derivatives of the amino acids liberated from 87 showed peaks at 34.64, 40.63, 47.92, 54.51 minutes. The amino acid standards gave the following retention times in minutes: 31.51 for L, 32.47 for D-Asn; 34.52 for L, 36.02 for D-Asp; 40.28 for L, 42.83 for D-Pro; 47.62 for L, 53.42 for D-Val; 54.15 for L-, 58.40 for D-Phe. In all cases a peak at 45 minute was observed which was attributed to excess FDAA. Therefore, it was confirmed that pseudaxinellin (87) contained Asn, Pro, Val, and Phe and that all of the amino acids had the L-configuration. 4.2.1.4. Conformational Analysis of Pseudaxinellin (87) There are growing numbers of naturally occurring cyclic heptapeptides containing standard protein a-amino acids reported in the literature. Examples in this group are the plant peptide evolidine (HO)91 and recently reported sponge peptides, hymenamides A-E (103-105)87 and axinastatins 2 (106) and 3 (107).88 Pseudaxinellin was the first cyclic heptapeptide isolated from a marine source. Perhaps due to their limited occurrence in nature, cyclic heptapeptides have not been subjected to extensive conformational analysis. A recent NMR, molecular mechanics, and x-ray diffraction examination of evolidine (110) [cyclo(Ser-Phe-Leu-Pro-Val-Asn-Leu)], a cyclic heptapeptide first isolated in 1950s from the plant Evodia Xanthoxy hides, showed that it contained a cis amide bond at proline (Leu-CO/Pro-N) and a two-turn p-bulge backbone 293 conformational motif.92 A question that arises from the study of evolidine is whether its backbone conformation is unique or if it represents a preferred conformation for a broad range of cyclic heptapeptides. In this regard, pseudaxinellin (87) represents a useful analog of evolidine (110) for comparison purposes. It is interesting to note that the partial sequence Val-Pro-Val-Asn in 87 is almost identical to the partial sequence Leu-Pro-Val-Asn in evolidine (110). Figure 143 shows a representation of evolidine (110) illustrating the location of the (3-bulge present in the preferred conformation. Also shown is a representation of pseudaxinellin folded according to the evolidine template which suggests that pseudaxinellin might have a {3-bulge involving transannular hydrogen bonding between the valine/(Val3) carbonyl oxygen and both valine/(Vali) and asparagine amide NH protons. The NMR data collected for 87 strongly support this backbone conformation. As discussed above, pseudaxinellin also contains a cis amide bond at proline (Val2-CO/Pro2-N) based on the NOE effect observed between the a-protons of these two adjacent amino acid residues. The carbon chemical shift at 8 21.8 for yC-Pro2 further supports the amide bond to be cis.* Difference NOEs and/or ROESY correlations between Proi-8H (8 3.58) and Asn-ocH (8 4.61), and between NH and the a-proton of the preceding amino acid at every other amide bond confirm that all other amide bonds are in the frans-conformation. Analysis of NH proton resonances in two different solvents (CDCI3 and DMSO-d6) reveals that the NH signals of Asn, Vali, and Val3 are almost identical (See Tables 27 and 29), indicating these NH protons are involved in intramolecular H-bonding. On the contrary, the chemical shifts of other amide protons seem to be significantly dependent of the solvent used. In DMSO solution, the NH protons of Val2, Phe, and Asn's Y-CONH2 are largely shifted to low field in comparison with the corresponding proton resonances recorded in CDCI3. This information illustrates these protons to be in an external orientation where they can form hydrogen bonds with the solvent DMSO, a good H-bond acceptor. * The carbon chemical shifts of •yC-Pro of cyclic peptides with cis X-Proamide bonds appeared at 8 20 ~ 22, while trans X-Proamide bonds appeared at 8 ~ 25.9° 294 HO. P bulge cis amide Evolidine (110) Asn NHc Yah Val2 Proi Pseudaxinellin (87) Figure 143. Conformational comparison of pseudaxinellin (87) with evolidine (110). 295 With the evidence that the NH groups of Asn, Vali, and Val3 are involved in intramolecular H-bonds, the question now is, which CO group acts as acceptor. It is well known that the carbonyl carbons usually exhibit downfield 13C NMR shifts when they are involved in a H-bond, although the carbonyl group of an amide is less sensitive to such effect.93 Upon inspection of the 13C NMR spectrum (Figure 144) recorded in DMSO-cfc for 87, it is apparent that among eight carbonyl carbons of pseudaxinellin (87), the two most deshielded resonances at 5 172.5 and 171.5 ppm can be assigned to the amide carbonyl carbons of Asn and Val3, respectively. The chemical shifts of all other six carbonyls are less than 170.7 ppm (Figure 144, Table 29). Both of the two carbonyl groups (Asn and Val3) are expected to be involved in intramolecular hydrogen bonding as depicted in Figure 143. Thus, the backbone structure of pseudaxinellin (87) in solution includes two (3-turns, one of type-I at Proi-Phe and one of type-VI(a) at Pro2-Val2, and three transannular hydrogen bonds between Val3-NH and Asn-CO, and between Val3-CO and both Vali-NH and Asn-NH, incorporating a p-bulge motif. The incorporation of the p-bulge motif in evolidine (110) and in pseudaxinellin (87) perhaps represents a template for cyclic heptapeptide backbones containing a proline residue. 4.2.1.5. Conclusions Our chemical study of the Papua New Guinea sponge Pseudaxinella massa led to the isolation of a cycloheptapeptide pseudaxinellin (87). The structure elucidation of pseudaxinellin (87) was accomplished using a variety of one and two dimensional NMR techniques. An analysis of the NMR data recorded in two different deuterated solvents indicated that the solution backbone conformation of pseudaxinellin (87) contained one cis peptide bond and three transannular hydrogen bonds, comprising a classical p-bulge. It should be mentioned that the same compound 87 was also isolated concurrently from the Palauan sponge Axinella sp. by Pettit's group, and named axinastatin 1 (87) .9 4 a Very recently, pseudaxinellin (87) was reported to be synthesized,94b and the synthetic product was found to be identical to the natural product 87 in every respect. 296 P u 13 > > Table 29. lH and 13C NMR data for pseudaxinellin (87) recorded in DMSO. Phe Prol Asp Vail Pro2 Val2 Val3 C# 1 2 3 3 ' 4 5,9 6,8 7 NH 10 11 12 12" 13 13' 14 14' N 15 16 17 17' 18 NH2 NH 19 20 21 22 23 NH 24 25 26 26' 27 27' 28 28 ' N 29 30 31 32 33 NH 34 35 36 37 38 NH ^ ( 4 0 0 MHz) | aCOSY -4.28, m 2.93 3.08 -7.13, d(J=7.1 Hz) 7.25, t(J=7.5 Hz) 7.18, t(J=7.3Hz) 8.11, d(J=9.6 Hz) -3.93, t(J=8.9 Hz) 2.02, m 0.99 1.73, m 1.73, m 3.76, bt(J=7.3 Hz) 3.35 -4.61, bq(J=5.0 Hz) 2.95 3.00 -7.23, bs; 7.97, bs 8.02, d(J=5.7 Hz) -3.85, t(J=8.4 Hz) 2.18, bq(J=7.5 Hz) 0.85 0.91, d(J=6.7 Hz) 8.35, d(J=8.8 Hz) -4.65, d(J=7.0 Hz) 2.42, m 1.73, m 1.85, m 1.50, m 3.54, m 3.17 -4.07,dd(J=5.1,7.7 Hz) 1.95m 0.88 0.83 8.67, d(J=5.2 Hz) -4.26, t(J=8.9 Hz) 1.95, m 0.96, d(J=6.6 Hz) 0.86 H3,H3',NH(8.11) H2.H3' H2,H3 H6.H8 H5,H7,H9 H6.H8 H2 H12,H12' H11,H12',H13,H13' H11,H12,H13,H13' H12',H14,H14' H12',H14,H14' H13,H13',H14' H13,H13',H14 H17,H17',NH(8.02) H16.H17' H16,H17 7.97, 7.23 H16 H21,NH(8.35) H20,H22,H23 H21 H21 H20 H26' H26',H27' H25.H26 H27',H28,H28' H26,H27,H28,H28' H27,H27',H28' H27,H27',H28 H31,NH(8.67) H30,H32,H33 H31 H31 H30 H36,NH(7.29) H35,H37,H38 H36 H36 7.29, d(J=9.2 Hz) | H35 bNOE H5,H9 H16 -H20 H29 H27' H24,NH(6.80) H35 1 3C(125 MHz) 170.6 55.1 37.2 138.4 128.9 128.1 126.3 170.2 62.4 29.2 25.2 47.7 172.5 49.5 35.1 169.7 170.3 61.7 29.6 18.4 19.8 170.2 60.7 29.7 21.3 45.4 170.7 57.8 29.2 19.2 18.9 171.5 57.0 30.3 18.8 18.5 H35 1 CHMBC NH(7.29),H2 H3 H5.H9 H6,H8,H3 H3,H3',H7,H5,9 H8,H6 H5,H9 H11,NH(8.11) H14.H11 H14 H16 NH2(7.23) H20,NH(8.02) H21 H20 H20.H21 H25,NH(8.35) H25 H25 H25 H30 H30 H30 H35,NH(8.67) H36 H35 H35.H36 Protons correlated to proton resonance in !H column. b Proton in C# column irradiated. c Protons correlated to carbons in C# column. 4.2.2. The Structure Elucidation of Geodiamolide G (111), a Depsipeptide from the Marine Sponge Cymbastela sp. This section describes the structure elucidation of a cyclic depsipeptide geodiamolide G (111) isolated from the marine sponge Cymbastela sp.. Our original small collection (50 g dry wt.) of the sponge identified as a Pseudaxinyssa sp. did not provide the quantities of geodiamolides required for in vivo evaluation in animal models relevant to the observed in vitro cytotoxicities against human cancer cell lines.80 In order to obtain sufficient amounts of the geodiamolides for in vivo testing, the source sponge was recollected from the original dive sites at Motupore and Madang in Papua New Guinea. Taxonomic identification of a voucher sample of the recollected sponge verified that it was identical to the original sample, however, in the period between collections the classification of the sponge had been changed from Pseudaxinyssa sp. to Cymbastela sp. (probably an undescribed species). Bioassay-guided fractionation of extracts from the recollected Cymbastela sp. led to the isolation of the known metabolites, geodiamolides A (89) to F (94), and the novel metabolite geodiamolide G (111). The isolation and initial screening was carried out in our laboratory by Dr. E. Dilip de Silva. 4.2.2.1. Taxonomy and Description of Cymbastela sp. Sponges of the genus Cymbastela belong to the class Demospongiae. According to Bergquist's system95 and Fusetani's paper,75 Cymbastela sp. {Pseudaxinyssa sp.) belongs to the family Axinellidae and the order Axinellida, although there exists disagreement about using Axinellida as a polyphyletic ordinal taxon.96 The specimens obtained for this study were collected on outer reefs in 1 0 - 2 5 meters of water near Port Moresby, Papua New Guinea. This Cymbastela sp. is a tan yellow stalked sponge, grows upright with finger-like projections. Its overall size is up to 20 cm high and 1 cm thick. 299 4.2.2.2. Isolation of Geodiamolide G (111) from the Sponge Cymbastela sp. Specimens of Cymbastela sp. were collected by hand using SCUBA on nearshore reefs near Port Moresby, Papua New Guinea. Freshly collected sponge was frozen on site and transported to Vancouver over dry ice. The thawed sponge was extracted exhaustively with a solution of CH2CI2/ MeOH (1:1). The organic extract was filtered and concentrated in vacuo to give an aqueous suspension. To the aqueous suspension, methanol was added to yield a 9:1 MeOH:H20 solution, which was extracted with hexanes. The water/methanol layer was diluted with water to a 1:1 MeOH:H20 solution, and then extracted with CHCI3. The chloroform soluble portion was concentrated, and then fractionated by sequential application of Sephadex LH-20 (eluent: MeOH), silica-gel drip column (step gradient: from 3:2 to 1:2, hexane/EtOAc), and reversed phase HPLC (eluent: MeOH/H20 60:40) chromatographies to give pure samples of geodiamolide G (111) as well as the known metabolites geodiamolides A to F (89-94). 4.2.2.3. Structure Elucidation of Geodiamolide G (111) 111 The structure of geodiamolide G (111) was determined using spectroscopic methods, particularly, by extensive analysis of 2D NMR data. The structures of the individual residues were determined by analysis of COSY, HMQC, and HMBC experiments. The quaternary carbon 300 resonances were identified on the basis of the HMBC correlations. The stereochemistry of 111 was established by comparison of its 13C NMR resonances with that of the literature values for geodiamolide A (89).79 Geodiamolide G (111) was obtained as a clear glass that gave a parent ion in the HREIMS at mlz 655.1760 appropriate for a molecular formula of C28H38N3O7I (AM +0.6 mmu). The molecular formula of 111 differed from that of geodiamolide A (89) simply by the gain of one oxygen atom and the loss of two hydrogen atoms. Examination of the *H NMR (Figure 145) of 111 revealed that it showed a correspondence with that of geodiamolide A (89). The most significant difference was that 111 lacked the resonances that could be assigned to the olefinic methine proton H5 (8 4.91) and the olefinic methyl protons H23 (5 1.48) found in geodiamolide A (89). However, the *H NMR of 111 contained an extra pair of resonances at 8 5.78 (bs: H23') and 5.90 (bs: H23) that were both correlated to the same olefinic methylene carbon resonance at 8 127.8 in the HMQC spectrum (Figure 146). Detailed analysis of the *H NMR, COSY, HMQC, and HMBC spectra of 111 showed that it contained a tripeptide fragment identical to that present in geodiamolide A (89) (Table 30), but differed in the polyketide fragment. A series of COSY and HMBC correlations established the substructure of this twelve carbon polypropionate unit. Analysis of the COSY spectrum (Figure 47) of 111 identified the two isolated spin systems present in this unit. A methyl doublet at 8 1.15 (H22) showed scalar coupling to a methine proton H2 (8 2.46) and the latter was further correlated to a pair of methylene protons H3/H3' (8 2.55/ 2.14). Another spin system in this unit started at methyl doublet at 8 1.09 (H24) that was coupled to the methine proton H6 (8 2.95). Additional COSY correlations were observed between H6 (8 2.95) and H7' (8 1.61) and between H7 (8 1.82) and H8 (8 5.11). The deshielded methine proton H8 (8 5.11) was finally coupled to the methyl doublet at 8 1.28 (H25). The HMBC spectrum (Figure 148) of 111 contained strong correlations from both the olefinic methylene proton resonances at 8 5.78 (H23') and 5.90 (H23) to a ketone carbonyl resonance at 8 205.1 (C5) and to an aliphatic methylene carbon resonance at 8 37.7 (C3). A series of additional strong HMBC correlations between the methyl proton resonances and the adjacent 301 -CU N m U 8 c 2 — X en (W)HN (Ol)HN U <o ."2 "3 £ '•3 o U O s 5 o <u ex c/5 2 u 3 to C/3 302 20 A-M 28" H 22 111 - 50 -100 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppn ppa Figure 146: 2D HMQC spectrum of geodiamoUde G (111) in CDCI3 303 ( 23 ^. H0'191T 27" *f °- N f * 0 28 A-M H 22 111 JLUJL-UJLL^ J U ^ J L > ^ V ppn ppa Figure 147: 2D COSY spectrum of geodiamolide G (111) in CDCI3 304 25 - '21 /N>^° „ V**. 20 27 13 14 28' 111 N 1 I H 4 O 2 / ^ 3 2~2 jLLdLjuJLji J U >Lou>\ ppa Figure 148: 2D HMBC spectrum of geodiamolide G (111) in CDCI3 Table 30. NMR data for geodiamolides G (111) and A (89) recorded in CDCI3 (500 MHz). c# 1 2 3 3' 4 5 6 7 7 8 9 10 NH(10) 11 12 13 14 NH(14) 15 15' 16 17 18 19 20 21 22 23 23' 24 25 26 27 Geodiamolide G (111) 5 13c 5 i H 174.5 41.0 37.7 143.6 205.1 36.2 38.8 69.7 170.6 49.1 168.9 57.1 174.4 45.1 33.2 130.3 138.2 85.2 154.5 115.6 130.6 17.9 127.8 17.8 20.8 18.3 30.7 28 19.2 2.46 m 2.55 dd(12.3, 3.7) 2.14 t(l 1.9) 2.95 1.82 ddd(14.6, 9.5, 2.8) 1.61 ddd(14.6, 10.9, 2.8) 5.11 m 4.51 quin(7.3) 6.35 d(7.3) 5.06 t(8.2) 4.72 qu(6.9) 6.19d(7.0) 3.12 dd(14.6, 7.9) 2.90 dd(14.6, 8.9) 7.45 d(l.4) 6.88 d(8.2) 7.04dd(8.2, 1.4) 1.15d(6.4) 5.90 bs 5.78 bs 1.09d(7.1) 1.28d(6.3) 1.32 d(7.2) 2.97 s 1.04 d(6.9) a C O S Y H3, H3', H22 H2, H3' H2, H3 H7', H24 H7', H8 H6, H7 H7, H25 H26, NH(10) H10 H15, H15 H28, NH(14) H14 H12, H15' H12, H15 H21 H21 HI7, H20 H2 H6 H8 H10 H14 b H M B C H3, H3\ H14, H22, NH(14) H3', H22 H22, H23, H23' H3, H23' H3', H23, H23', H24 H24 H24, H25 H25 H10, H26 H26 H12, H15' H15, H15', H27 H12, H14, H27, H28,NH(14) H28 H12, H17, H21 H15, H15', H20 H15, H15', H21 H17, H20 H17, H21 H17 H3' H10 H12 a Protons correlated to proton resonance in 'H column. b Protons correlated to carbons in C# column. Geodiamolide A (89) 5 1 3 C 175.3 42.3 43.3 130.1 131.7 28.9 43.6 71.0 170.8 49.0 168.8 56.7 174.5 45.8 32.4 130.1 138.4 85.2 154.3 115.0 130.4 18.6 17.6 20.3 20.6 18.2 30.6 6 !H 2.32 m 2.11 dd(13.7,11.2) 2.02 dd(13.9, 3.7) 4.91 bd(8.8) 2.15 m 1.59 ddd(13.7, 7.9, 5.6) 1.34 m 4.86 m 4.45 quin(7.4) 6.55 d(7.8) 5.18 dd(9.1,7.3) 4.71 qu(6.8) 6.49 d(6.6) 3.12 dd(14.8, 7.2) 2.90 dda4.8,9.0) 7.46d(2.1) 6.84 d(8.3) 7.02 dd(8.3, 2.1) 1.13 d(6.8) 1.48d(1.0) 0.84 d(6.6) 1.21 d(6.2) 1.33d(7.1) 2.95 s 18.7 1.01 d(6.8) 306 carbons located the ketone at C5 and the olefin at A4-23 in 111 as shown. For example, the HMBC correlation between the methyl proton resonance at 5 1.09 (H24) and a most deshielded carbon at 5 205.1, appropriate for a conjugated ketone carbon, identified this carbon as C5. The HMBC correlations between the methylene carbon resonance (8 37.7: C3) and a methyl proton resonance at 8 1.15 (H22), and between the methyl proton resonance at 8 1.15 (H22) and a carbonyl carbon resonance at 8 174.5 (CI) further confirmed the location of the enone functionality in 111. Difference NOEs observed between H23 (8 5.90) and H6 (8 2.95), and between H23' (8 5.78) and H3' (8 2.14) demonstrated that H23 was cis and H23' was trans to the C5 ketone. The configuration at C2, C6, and C8 in geodiamolide G (111) were assumed to be identical to the configurations at the same centers in geodiamolide A (89). Figure 149. Selected HMBC correlations for polypropionate unit in 111. The assignment of the remaining carbonyl carbon resonances in geodiamolide G (111) was accomplished unambiguously by a series of strong and well-resolved long range proton-carbon correlations in the HMBC spectrum. A carbon resonance at 8 174.4 was assigned to C13 as it was coupled to both N-Me (H27) and Me28 methyl protons. The HMBC correlation observed between the methyl protons (H26: 8 1.32) and a carbon resonance at 8 170.6 assigned this resonance to the ester carbonyl carbon C9. The most upfield carbonyl carbon resonance remaining at 8 168.9 must be assigned to the tyrosine residue at CI 1. Indeed, the HMBC correlations between this carbon (CI 1: 8 168.9) and the Tyr oc-methine proton H12 (8 5.06) and one of the P-methylene protons (H15': 8 2.90) provided evidence for this assumption. It is worth noting that the original structure of geodiamolide A (89) was established by an X-ray crystallography. The JH and 13C NMR assignments of geodiamolide A (89) were based on the chemical shifts, and DEPT and proton decoupling experiments. The authors claimed that the carbon-13 assignments were tentative. We recollected the ID and 2D NMR data for geodiamolide A (89) and found the original assignments for the carbonyl carbons of 89 were incorrect. The reassigned *H and 13C NMR data based on our experiments for geodiamolide A (89) are listed in Table 30. Geodiamolide G (111) represents the first member of the jaspamide/geodiamolide family with a modification in the polyketide fragment. Comparison of the cytotoxicities of jaspamide (88) and the previously reported geodiamolides A to F (89-94) and TA (95) shows that significant variation in the three amino acid residues causes only minor changes in the levels of cytotoxicity exhibited by the compounds. By contrast, geodiamolide G (111) with the modified polyketide fragment (in vitro human glioblastoma/astrocytoma U373, ED50 = 7.7 fig/mL; in vitro human ovarian carcinoma HEY, ED50 = 8.6 |ig/mL) is significantly less cytotoxic than the analog geodiamolide A (89) (average in vitro ED50 against a panel of more than ten human solid tumor cell lines = 0.2 |ig/mL) 4.2.2.4. Conclusions In conclusion, the structure elucidation of the novel marine cyclic depsipeptide geodiamolide G (111) was accomplished using a variety of one and two dimensional NMR techniques. Geodiamolide G (111) is a new member of the jaspamide/geodiamolide family and it is the first example containing a modified polypropionate unit. The *H and 13C NMR data of geodiamolide A (89) have been recollected and the original assignments revised. Experimental 308 General See the experimental section of chapter II. Pseudaxinellin (87) from Pseudaxinella massa Collection and Isolation data: (See section 4.2.1.2.) Pseudaxinellin (87): clear glass; [oc]21D = -100.1° (CHCI3, c = 0.34); *H NMR in CDCI3 see Table 27; *H NMR in DMSO-af6 see Table 29; 13C NMR in CDCI3 see Table 27; 13C NMR in DMSO-^6 see Table 29; LREIMS mlz (relative intensity): 752 (8.9), 734 (0.9), 710 (0.9), 683 (4.2), 640 (7.4), 612 (2.0), 584 (1.2), 541 (5.6), 527 (1.8), 513 (1.1), 443 (5.2), 70 (100); HREIMS (M+) mlz 752.4218 (C38H56N808 AM -0.3 mmu). Determination of the Absolute Configurations of Amino Acid Residues: Pseudaxinellin (87, 1 mg) in 6N HCl (0.5 mL) was heated at 108 °C in a sealed Pyrex tube overnight to yield the corresponding amino acids (note: Asn was converted to Asp under these conditions). The cooled reaction mixture was evaporated to dryness and the traces of HCl was removed from the residual acid hydrolysate by repeated evaporation from frozen water (10 mL). The amino acid mixture (acid hydrolysate) was treated with Marfey's reagent 5-fluoro-2,4-dinitrophenyl-L-alanine amide (FDAA, 8 (imol) in acetone solution (250 |iL) followed by IN NaHCC<3 (50 jiL). The mixture was heated for 1 hr at 40 °C. After cooling to room temperature, 2N HCL (25 |i.L) was added, and the resulting solution of the FDAA derivatives was filtered through a 4.5fj, filter and analyzed by reverse phase HPLC. A linear gradient of (A) 9:1 triethylammonium phosphate (50 mM, pH 3.0)/MeCN and (B) MeCN with 0 % at the start to 40 % (B) over 40 minutes (flow rate 0.7 mL/min.) was used to separate the FDAA derivatives which were detected by UV absorbance at 340 nm. The Marfey's reagent derivatives of the amino acids liberated from 87 showed peaks at 34.64, 40.63, 47.92, 54.51 minutes. The amino acid 309 standards gave the following retention times in minutes: 31.51 for L, 32.47 for D-Asn; 34.52 for L, 36.02 for D-Asp; 40.28 for L, 42.83 for D-Pro; 47.62 for L, 53.42 for D-Val; 54.15 for L-, 58.40 for D-Phe. In all cases a peak at 45 minute was observed which was attributed to excess FDAA. Therefore, it was confirmed that pseudaxinellin contained Asn, Pro, Val, and Phe, and that all of the amino acids had the L-configuration. Geodiamolide G (111) from Cymbastela sp. Collection and Isolation data: Specimens of Cymbastela sp. were collected by hand using SCUBA on nearshore reefs near Port Moresby, Papua New Guinea. Freshly collected sponge was frozen on site and transported to Vancouver over dry ice. The thawed sponge (260 g dry wt.) was extracted exhaustively with a solution of CH2CI2/ MeOH (1:1). The organic extract was filtered and the filtrate evaporated in vacuo to give an aqueous suspension. The methanol was added to yield a 9:1 MeOH:H20 solution (1 L), which was extracted with hexanes (4 x 250 mL). The hexane extracts were combined and concentrated in vacuo to give an orange oil. The water/methanol layer was diluted with water to a 1:1 MeOH:H20 solution, and then extracted with CHCI3 (4 x 250 mL). The combined CHCI3 layers were concentrated in vacuo to yield an orange oil (3.5 g), which was repeatedly chromatographed on Sephadex LH-20 column (eluent: MeOH) and silica-gel drip column (step gradient: from 3:2 to 1:2, hexane/EtOAc) to yield crude geodiamolide G (111) as well as the known metabolites geodiamolide A (89) to F (94). Finally, a reversed-phase HPLC purification (eluent: MeOH/H20 60:40) yielded pure geodiamolide G (111) as a colorless glass (1 mg). Geodiamolide G (111): colorless glass; IR (neat) 3313, 2977, 2933, 1732, 1675, 1635, 1505, 1455, 1417, 1377, 1285, 1217, 1102, 1083, 1052, 952, 827, 754 cm"1; *H NMR in CDCI3 see Table 30; 13C NMR in CDCI3 see Table 16; LREIMS m/z (relative intensity): 655 (41), 624 (38), 597 (13), 581 (8.7), 549 (12), 535 (7.2), 422 (9.6), 276 (58), 264 (60), 236 (48), 230 (18), 193 (87), 127 (62); HREIMS (M+) m/z: 655.1760 (C28H38N3O7I; AM 0.6 mmu). 310 Appendix. Nuclear Magnetic Resonance Techniques Nuclear magnetic resonance (NMR) techniques have been used to elucidate the structures of the marine natural products in this thesis. A simple description of the *H and 13C, APT, NOE, COSY, COSYLR, HOHAHA, ROESY, HMQC, and HMBC experiments will be presented in this appendix. For more detailed explanation of the theory of one-dimensional and two-dimensional NMR, please refer to the NMR books and review articles.97 1. One-dimensional NMR experiments Shown below (Figure 1A) is a typical pulse sequence for one-dimensional experiment. The preparation time di allows the sample to reach thermal equilibrium. A short pulse (radiofrequency) pi is applied to the nuclei to tip the equilibrium magnetization through an angle given by: 0 = y B i p i Commonly, the time (pi, - 5-20 |is) is chosen so that 6 is 90°, called 7t/2 pulses. During the evolution time ti, additional pulses may be applied. The resultant FID is acquired during the detection period t2- The decoupler channel may be gated to remove scalar coupling interactions. ^ Pulse 0° preparation Evolution Detection FID Figure Al . General pulse sequence for a one-dimensional NMR experiment.98 a. *H Spectrum The most common pulse experiment used to acquire a proton spectrum is simply a pulse followed by the acquisition of an FID (Figure A2). Usually a short delay follows the pulse to allow the transmitter coil to return to equilibrium. The relaxation delay (di, usually 1-2 seconds) should be of sufficient duration to allow the bulk magnetization to reestablish along the BQ field. e° 311 Figure A2. Pulse sequence for acquisition of a *H spectrum. b. NOE Difference Spectra In essence, the pulse sequence for the NOE difference spectrum is merely the sequential acquisition of lH spectra with gated decoupling (Figure A3). In gated decoupling, a second transmitter coil is used to irradiate a specific resonant frequency to build up NOEs. This is a slow process and normally takes 5-6 seconds. The decoupler is then shut off during the application of a pulse and the acquisition of the FID. Obs. Chan. Dec. Chan. Decoupler on Figure A3. Pulse sequence for acquisition of the NOE difference spectrum.100 c. 13C Spectrum The pulse sequence for obtaining a 13C spectrum is to irradiate the entire proton region (broad band decoupling) and apply the standard pulse-FID sequence to the carbon channel simultaneously (Figure A4). This method eliminates all the proton-carbon couplings, resulting in the carbon signals appearing as singlets in the spectrum. It also increases the intensity of protonated carbons due to heteronuclear NOE enhancement. 13 C Observe H BB Dec. Decoupler on Figure A4. Pulse sequence for acquisition of a l 3C spectrum.101 d. The Attached Proton Test (APT) Spectrum The APT experiment employs gated *H broad band decoupling to achieve a modulation of the 13C transverse magnetization due to scalar coupling with protons (Figure A5). Because the decoupler of proton channel is switched on during the acquisition of the FID, all signals in the spectrum are singlets. When the evolution time is set to 1//CH (d2, 7 ms), however, the phases of the 13C magnetization will depend on the number of the attached protons. The carbon with an even number of attached protons will give positive signals, while an odd number of attached protons yields negative peaks. The final spin-echo sequence (d3, 1 ms) is compensation factors to increase the data acquisition rate and allow flip angle (9) not equal to exactly 90° to be used. e° 13 C Observe 180 d2 do + dq l H BB Dec. Decoupler on Decoupler on Figure A5. Pulse sequence for the APT experiment.10^ 2. Two-dimensional NMR experiments The pulse sequence for a two-dimensional (2D) NMR experiment is illustrated in Figure A6. It involves three discreet stages "preparation-evolution-detection" as in the ID experiment. 313 The evolution time ti in 2D experiment is incrementally increased, during which the magnetization vectors "evolve" under the influence of additional pulses or other factors. Fourier transformation of the FIDs gives a series of spectra which are modulated under the influence of the second time (ti) variable. A second Fourier transformation over ti results in a spectrum as a function of two frequencies. 0° Preparation Evolution & Mixing Detection dj t! + x Figure A6. Pulse sequence for a two-dimensional NMR experiment. a. lH-lH COSY and Long Range COSY Spectra Homonuclear correlation spectroscopy, is the most frequently used 2D NMR technique. The pulse sequence used for the acquisition of a COSY spectrum (Figure A7) is simply two pulses separated by an incremented delay (3 JLLS). The second pulse modulates the FIDs in such a way that after the second Fourier transformation, off diagonal peaks correlate spins as a result of scalar coupling (/). The magnitude of the second pulse can be 20° to 90°. The 90° pulse maximizes the cross peaks, while 45° pulse minimizes the diagonal signals. The COSY-45 spectrum also provides information on the sign of the spin-spin coupling constant. The cross peak due to geminal coupling is raised toward the right side, while that due to vicinal coupling is tilted upward the left side. An additional feature of two-dimensional experiments is "phase cycling" ((()) to compensate for inexactly calibrated pulses. In phase cycling, the phase of the pulses and detector are varied under regular repeating patterns. 90° <!>! ti e_>2 Figure A7. Pulse sequence for the COSY experiment.103 A variation on the above pulse sequence, which optimizes the intensity of cross peaks resulting from smaller scalar coupling constants, is used to determine long range couplings (Figure A8). The additional delay (d2, ~0.1 s) suppresses the modulating effect of large spin-spin couplings. It should be noted that when the decay (FID) is rapid, signal to noise is poor. 90° eh 90° <j>2 di tj + d2 Figure A8. Pulse sequence for the COSYLR experiment.104 b. HOHAHA experiment The HOHAHA experiment (Homonuclear Hartmann-Hahn spectroscopy) can be thought of as an alternative version of COSY. However, the mechanism of coherence transfer in HOHAHA is fundamentally different from that in COSY. This technique relies on the principle of cross polarization, which can be obtained using a single coherent radiofrequency field. If the effective radiofrequency fields experienced by two nuclei are identical, the Hartmann-Hahn condition is established, resulting in oscillatory exchange of spin-locked magnetization between the two nuclei. The pulse sequence for this experiment is illustrated in Figure A9. The experiment is carried out in the phase-sensitive mode. An advantage of cross polarization coherent transfer is the achievement of relay and multiple-relay peaks in the spectrum. The relay distance is dependent on the mixing (spin locking) time. Usually, the spin-lock duration is constructed with a short (2.5 ms) spin-lock field and MLEV-17 decoupling sequences to achieve efficient spin-locking of resonances. This technique is especially useful for determining networks of mutually coupled protons. Therefore, HOHAHA spectra are frequently used for determination of the amino acid constituents of peptides. 90°<|> SLx MLEV-17 SLx • i i i tl i Figure A9. Pulse sequence for the HOHAHA experiment.105 c. ROESY experiment The ROESY experiment (Rotating Frame Overhauser Enhancement Spectroscopy) provides information comparable to difference NOE experiments. Figure A10 illustrates the pulse sequence used to record a ROESY spectrum. Essentially, this pulse program is identical to that used for HOHAHA. However, the r.f. power used to achieve spin locking is significantly smaller in ROESY. In addition, it is usual to place the transmitter carrier at the low-field end of the spectrum. The combined low power and transmitter offset disables the Hartmann-Hahn condition, and therefore prevents the appearance of HOHAHA crosspeaks in the ROESY spectrum. In addition, the ROESY crosspeaks appear with opposite phase to HOHAHA crosspeaks. A 90° pulse is applied and the bulk magnetization evolves as a function of the evolution time (ti). A second pulse, known as the spin-lock pulse, is then applied for a mixing time Tm (-0.225 s), during which the magnetization is "locked" and the cross-relaxation, namely dipolar relaxation, takes place. Therefore, the FID detected during the acquisition time t2 is modulated by dipolar relaxation. The resultant 2D spectrum resembles a COSY spectrum but the cross peaks represent through space coupled protons. 90° <J) spin lock x i i i l i Figure A10. Pulse sequence for the ROESY experiment.106 316 d. HMQC experiment The ^-detected heteronuclear multiple quantum coherence (HMQC) experiment is used to determine one bond 2H-13C connectivity. An HMQC spectrum provides the same information as that of the 13C-detected heteronuclear correlation (HETCOR) experiment. The advantage of the HMQC experiment is that the FID is acquired in the sensitive *H magnetization, and hence less sample and/or time are required to obtain the data in the HMQC experiment. The pulse sequence for the HMQC experiment is illustrated in Figure Al l . The Bird pulse is applied at the beginning to suppress signals from protons not coupled to 13C nuclei. Typical values used for the acquisition of HMQC spectra in the thesis were: di was 1.5-2.0 s (relaxation delay); d2 3.5 ms (1 /2 /CH); ^3 0.7 s (optimized to eliminate lH signals bonded to 12C); (I4 3 JIS (compensation factor); ti 3 |is (normal incremented factor). Fourier transformation with respect to t2 results in a series of *H spectra modulated by one bond !H-13C scalar coupling. A second Fourier transformation with respect to ti results in a two-dimensional plot with *H frequencies along the f2 axis, 13C frequencies along the fi axis. The cross peaks in a HMQC spectrum represent direct ^ - ^ C connectivity. 1 H Observe 13 CDec. 90°x 180°x 90°_x 90°x 180°x 180 &2 d 2 Bird pulse 90°,,, 90° 90 B.B.Dec. d3 d2 t2/2 tj/2 d4 d2 t2 Figure A l l . Pulse sequence for the HMQC experiment. 317 e. HMBC experiment The ^-detected heteronuclear multiple bond correlation (HMBC) is one of the most powerful techniques for structural elucidation of complicated natural products. This experiment is similar to the HMQC experiment but has been optimized for long range, usually two and three bond, iH-^C connectivity. After the Fourier transformations a 2D spectrum with *H frequencies along the f2 axis, 13C frequencies along the f[ axis results. The two and three bond ^H-^C correlations are observed as cross peaks at (fi, f2) The pulse sequence for the HMBC experiment is shown in Figure A12. One bond correlations are screen out instrumentally by using a low pass / filter, consisting of a 90° proton pulse, a delay d2, followed by a 90° carbon pulse. The initial delays (di and d2) are the same as those in the HMQC experiment, while d3 is optimized for long range C-H J values (d3 = 1/2/cH-LR)- Typical value for the d3 used for the HMBC experiments in this thesis was 60 ms. 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