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New cytotoxic natural products from North-Eastern Pacific marine invertebrates Pika, Jana 1993

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NEW CYTOTOXIC NATURAL PRODUCTS FROM NORTH-EASTERN PACIFIC MARINE INVERTEBRATES  by JANA PIKA  B. Sc., Concordia University, Montreal, P.Q., 1986  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  THE UNIVERSITY OF BRITISH COLUMBIA April 1993 ©Jana Pika, 1993  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  „/CA-M1  i41A  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  1 / / ,93  ll  Abstract  An investigation into the chemistry of four species of marine invertebrates which were found to produce cytotoxic crude extracts (ED50 against the L1210 murine leukemia cell line 5 30 pg/mL) led to the isolation of nine new and eleven previously known secondary metabolites. The structures of the novel compounds were elucidated by a combination of spectroscopic analysis and chemical interconversion. The metabolites were tested against the in vitro L1210 murine leukemia, the drug-sensitive MCF-7, or the drug-resistant MCF-7 Adr breast cancer cell lines. Investigation of the cytotoxic extracts of the marine sponge Aplysilla glacialis led to the identification of the novel 9,11-secosteroids glaciasterol A (100) and glaciasterol B (101). These compounds are the first examples of secosteroids isolated from a sponge of the genus Aplysilla. Glaciasterol A (100) proved active in vitro against the MCF-7 human breast cancer cell line at an ED50 of 19 pg/mL. The ED50 for Glaciasterol A (100) against the drugresistant MCF-7 Adr breast cancer cell line was 18 tig/mL. The structures of the glaciasterols were determined by a series of chemical interconversions in addition to extensive analysis of spectroscopic data. Glaciasterol B diacetate (107) was one of the derivatives synthesized and it proved to be the most active in the cytotoxicity assays. Glaciasterol B diacetate was active against both the MCF-7 and the drug-resistant MCF-7 Adr cell lines with ED50 values of 1.8 pg/mL. A study of the metabolites of a gray-white sponge collected off the coast of Vancouver Island yielded the new cytotoxic secosteroid blancasterol (102). Blancasterol (102) was found to be active against the MCF-7 and drug-resistant MCF-7 Adr breast cancer cell lines with ED50 values of 3 g.ig/mL and 10.6 pg/mL, respectively. The new sesquiterpenes 0-methyl 9-oxofurodysinin lactone (72), 2-oxomicrocionin-2 lactone (73), 0-methyl 2-oxomicrocionin-2 lactone (74) as well as seven previously reported sesquiterpenes (35, 36, 56, 57, 41, 43, and 44) were also isolated from the sponge extract. An understanding of the secondary metabolism of the invertebrate was useful in identifying the sponge as a Pleraplysilla species, the first found off the west coast of North America. Langarin (142) proved to be the cytotoxic component of the extract of an undescribed species of Aplidium, a compound tunicate found in the waters off the Queen Charlotte Islands. Langarin (142) was active in vitro against L1210 murine leukemia cells with an ED50 of 0.44g/mL. Examination of extracts of the colonial tunicate Aplidium californicum resulted in the isolation of two new prenylated hydroquinone derivatives (160 and 161) as well as the known compounds 149, 150, 152, and 158.  111 Compound 152 was tested in vitro against the L1210 murine leukemia cell line and found to be cytotoxic with an ED50 value of 3.9 pg/mL.  HO  HO 100  ^  1 01  OR  72  73R =H 74R = CH 3  OH 0 OH  OH HO  OH OAc  102  142  HO  HO  HO H  160  ^  161  iv Table of Contents  Page Abstract Table of Contents^  iv  List of Tables^  vii  List of Figures^  viii  List of Abbreviations^  xiii  List of Schemes^  xv  Acknowledgments^  xvi  Dedication^  xvii  General Introduction ^1  PART I. Isolation and Structure Elucidation of Metabolites from two North-Eastern Pacific Sponges 5 Introduction ^5  Classification of the Sponges^ Aplysilla glacialis and a Pleraplysilla Sp. Sponge^  7  9  Description of Aplysilla glacialis^  9  Description of the Pleraplysilla Sp. Sponge^  10  Taxonomy of Aplysilla glacialis and the Pleraplysilla Sp. Sponge^14 Chemistry of Sponges of the Genera Aplysilla, Dysidea, and Pleraplysilla^17 Secondary Metabolites of Aplysilla Species Sponges^  17  Natural Products of Sponges belonging to the Genus Dysidea^ 22 The Chemistry of the Pleraplysilla Sponges^ 28 Secosteroids and Related Steroids from Marine Invertebrates ^30 Introduction to Selected NMR Experiments^  36  A. Cytotoxic Metabolites of Aplysilla glacialis ^56 Results and Discussion^56  Glaciasterol A (100)^  57  Glaciasterol B Diacetate (107)^  76  5a,60-Dihydroxyglaciasterol A Diacetate (113) and 5a-Hydroxyglacisterol A Triacetate (114)^  93  Page Conclusions ^96  13. Natural Products of a Pleraplysilla Sp. Sponge^98 Results and Discussion^98  a) The 1990 and 1991 Pleraplysilla Sp. Sponge Collections^ 98 0-Methyl 9-Oxofurodysinin Lactone (72)^  99  2-Oxomicrocionin-2 Lactone (73)^  109  0-Methyl 2-0xomicrocionin-2 Lactone (74)^ Known Metabolites of the Pleraplysilla Sp. Sponge (1990/1991  118  Collections)^ b) The 1992 Pleraplysilla Sp. Sponge Collection^  125 126  Blancasterol (102)^ 127 Known Metabolites of the Pleraplysilla Sp. Sponge (1992 Collection)^143 Conclusions  145  PART II. The Natural Products of Two Species of Colonial Tunicate from the Queen Charlotte Islands^152 Introduction^152  A Th hemi  LI •  fan n^  s •^/^e.  from  - • een h 1 eI 1  n  ^  159  i) Anthraquinones from Marine Invertebrates  159  ii) Results and Discussion  163  Langarin from an Undescribed Aplidium Species Langarin (142) iii) Conclusions  B. Secondary Metabolites of Aplidium californicum from the Oueen Charlotte Islands  163 166 178 180  i) Prenylated Quinones and Hydroquinones from Animals of the Subphylum^180 Urochordata  vi Page ii) Results and Discussion ^182  Prenylated Quinone and Hydroquinone Derivatives from Aplidium californicum 182 Calaplidol A (160)^  185  Calaplidol B (161)^ 194 Known Metabolites from the Tunicate Aplidium californicum^205 iii) Conclusions ^206 Experimental ^208 References ^229  vii List of Tables  Page Table 1^1H NMR and COSY Spectroscopic Data for 4-Methylcatechol (105)^ 45 Table 2^Assignment of Protonated Carbons of 4-Methylcatechol (105) from the HMQC Spectrum^ Table 3^1H, 13 C, and HMBC NMR Data for 4-Methylcatechol (105)^  49 52  Table 4^1H and 13 C NMR Data for Glaciasterol A (100) (Recorded in CDC13)^58 Table 5^1H and 13 C NMR Data for Glaciasterol A Diacetate (106) (recorded in CDCI3)^67 Table 6^1H and 13 C NMR Data for Glaciasterol B Diacetate (107) (Recorded in CDC13)^78 Table 7^1H and 13 C NMR Data for 5a,60-Dihydroxyglaciasterol B Diacetate (111) (Recorded in CDC13)^ Table 8^1H NMR Data for 5a-Hydroxyglaciasterol B Triacetate (112)^  83 90  Table 9^1H NMR Data for 5a,613-Dihydroxyglaciasterol A Diacetate (1 1 4) and 5a-Hydroxyglaciasterol A Triacetate (115)^  94  Table 10^Results of the Assay of Glaciasterol A (100) and Related Derivatives 107 and 112 against In Vitro Murine Leukemia and Human Breast Cancer Cell Lines^97 Table 11^1H and 13 C NMR Data for 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13)^  100  Table 12^1 H and 13 C NMR Data for 2-Oxomicrocionin-2 Lactone (73) (Recorded in CDCI3) ^110 Table 13^1 H and 13 C NMR Data for 0-Methyl 2-Oxomicrocionin-2 Lactone (74) (Recorded in^119 CDC13) Table 14^1 H and 13 C NMR Data for Blancasterol (102) (Recorded in CDC13)^128 Table 15^1 H NMR Shifts of Blancasterol (102) in CDC13 and in CDC13:C6D6 20:1^142 Table 16^1 H and 13 C NMR Data for Langarin (142) (Recorded in Me2SO)^ 167 Table 17^1 1-1 and 13 C NMR Data for Langarin Triacetate (143) (Recorded in Me2SO)^175 Table 18^1 H and 13 C NMR Data for Calaplidol A (160) (Recorded in CDC13)^186 Table 19^1 H and 13 C NMR Data for Calaplidol B (161) (Recorded in CDC13)^195  viii List of Figures  Page Figure 1.^Typical Body Plan of a Sponge of the Class Demospongaie ^ Figure 2.^An Intertidal Specimen of Aplysilla glacialis^  6 11  Figure 3.^Aplysilla glacialis and Pleraplysilla Species Sponge Collection Sites^12 Figure 4.^An Intertidal Specimen of a Pleraplysilla Species Sponge^ 13 Figure 5.^Taxonomic Classification Schemes according to (A) Bergquist, (B) Boury-Esnault, and ^16 (C) Van Soest Figure 6.^Time Course for a One-Dimensional NMR Experiment ^  36  Figure 7.^The Pulse Sequence for an NOEDS Experiment ^ 37 Figure 8.^1H NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at ^39 500 MHz) Figure 9.^NOE Difference Spectra of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO^40 at 400 MHz) Figure 10. Result of NOE Difference Experiments on 4-Methylcatechol (105) ^ 38 Figure 11. Time Course for a Two-Dimensional NMR Experiment ^ 41 Figure 12. Fourier Transformation of the FID of a 2-D Experiment with Respect to a) t2 and b) ti ^42 and t2 Figure 13. The Pulse Sequence for a Simple COSY Experiment^ 43 Figure 14. COSY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at 400^44 MHz) Figure 15. The Proton Connectivities of 4-Methyl Catechol (105) from the COSY Spectrum^45 Figure 16. The Pulse Sequence for an HMQC Experiment ^ 46 Figure 17. 13 C NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at ^47 125 MHz) Figure 18. HMQC Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 51.11 Me2SO at 500 ^48 MHz) Figure 19. The Pulse Sequence for an HMBC experiment^  50  Figure 20. HMBC Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at 500 ^51 MHz) Figure 21.^Selected HMBC Correlations for 4-Methylcatechol (105)^ Figure 22. The Pulse Sequence for a ROESY Experiment ^  50 53  Figure 23. ROESY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at ^55 500 MHz) Figure 24. Results of a ROESY Experiment on 4-Methylcatechol (105)^ 54 Figure 25.^1 H NMR Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 400 MHz) ^59  ix Page Figure 26. COSY Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)^60 Figure 27. Double Resonance NMR Experiment on Glaciasterol A (100) (Recorded in CDC13 at^61 400 MHz) Figure 28. 13 C NMR Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 125 MHz) ^62 Figure 29. HMQC Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 500 MHz)^63 Figure 30. 1 H NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400^68 MHz) Figure 31. COSY Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400 MHz)^69 Figure 32.^13 C NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 100 ^70 MHz) Figure 33. HMQC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 500 MHz)^71 Figure 34. HMBC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 500 MHz)^72 Figure 35. HMBC Correlations from H6, Me18, and Me19 of Glaciasterol A Diacetate (106)^74 Figure 36. Results of Selected NOE Difference Experiments on Glaciasterol A Diacetate (106)^76 Figure 37. 1 H NMR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 400 MHz)^79 Figure 38.^13 C NMR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 100^80 MHz) Figure 39. Selected NOE Difference Experiments on Glaciasterol B Diacetate (107) (Recorded in^81 CDC13 at 400 MHz) Figure 40.^1 H NMR Spectrum of 5a,613-Dihydroxyglaciasterol B Diacetate (111) (Recorded in^84 CDC13 at 500 MHz) Figure 41.^COSY Spectrum of 5a,613-Dihydroxyglaciasterol B Diacetate (111) (Recorded in ^85 CDC13 at 400 MHz) Figure 42.^13 C NMR Spectrum of 5a,60-Dihydroxyglaciasterol B Diacetate (111) (Recorded in^86 C6D6 at 125 MHz) with Expanded Regions a) Recorded in C6D6 and b) Recorded in CDC13 Figure 43. a) HMQC Spectrum of 5a ,6(3-Dihydroxyglaciasterol B Diacetate (111) with^87 b) Expanded Region (Recorded in CDC13 at 500 MHz) Figure 44.^1 H NMR Spectra of 5a-Hydroxyglaciasterol B Triacetate (112) Recorded in a) CDC13 ^91 and b) Pyridine-d5 at 400 MHz Figure 45^COSY Spectrum of 5a -Hydroxyglaciasterol B Triacetate (112) (Recorded in ^92 Pyridine-d5 at 400 MHz) Figure 46. The 1 H NMR Shifts of 111 in Pyridine-d5 Relative to CDC13^ 93 Figure 47.^1 H NMR Spectra of 5a-Hydroxyglaciasterol A Triacetate (114) Recorded in a) CDC13^95 and b) Pyridine-d5 at 400 MHz  x Page Figure 48. 1 H NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 ^101 at 500 MHz) Figure 49. 13 C NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 ^102 at 125 MHz) Figure 50. COSY Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at ^103 400 MHz) Figure 51. HMQC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at ^104 500 MHz) Figure 52. HMBC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at ^105 500 MHz) Figure 53. HMBC Spectroscopic Correlations for 0-Methyl 9-Oxofurodysinin Lactone (72)^107 Figure 54. Selected NOE Difference Experiments on 0-Methyl 9-0xofurodysinin Lactone (72) ^108 (Recorded in CDC13 at 400 MHz) Figure 55. 1 H NMR Spectrum of 2-Oxomicrocionin-2 Lactone (73) (Recorded in CDC13 at 400 ^111 MHz) Figure 56. COSY Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 400 MHz) ^112 Figure 57. HMQC Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 500 ^113 MHz) Figure 58.^13 C Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 125 MHz)^114 Figure 59. HMBC Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 500^115 MHz) Figure 60.^Selected HMBC Correlations Observed for 2-Oxomicrocionin-2 Lactone (73) ^117 Figure 61. Results of Selected NOE Difference Experiments on 2-Oxomicrocionin-2 Lactone (73) ^118 Figure 62. 1 H NMR Spectrum of 0-methyl 2-Oxomicrocionin-2 Lactone (74) (Recorded in^120 CDC13 at 400 MHz) Figure 63. COSY Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in CDC13 at ^121 400 MHz) Figure 64.^13 C NMR Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in^122 CDC13 at 125 MHz) Figure 65. HMQC Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in CDC13 ^123 at 500 MHz) Figure 66. HMBC Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in CDC13 at^124 500 MHz) Figure 67. 1 H NMR Spectrum of Blancasterol (102) (Recorded in CDC13 at 400 MHz)^129 Figure 68. COSY Spectrum of Blancasterol (102) (Recorded in CDC13 at 400 MHz)^130 Figure 69.^13 C NMR Spectrum of Blancasterol (102) (Recorded in CDC13 at 125 MHz) ^131  xi Page Figure 70. HMQC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz) ^132 Figure 71. HMBC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz) ^133 Figure 72. Selected HMBC Correlations for Blancasterol (102) ^  135  Figure 73. Expanded Upfield Region of the APT Spectrum of Blancasterol (102) (Recorded in ^138 CDC13 at 125 MHz) Figure 74. 1 H NMR Spectrum of Blancasterol (102) Recorded in CDC13:C6D6 20:1 at 400 MHz ^139 Figure 75. Double Resonance NMR Experiments on Blancasterol (102) (Recorded in ^140 CDC13:C6D6 20:1 at 400 MHz) Figure 76. ROESY Spectrum of Blancasterol (102) (Recorded in CDC13:C6D6 20:1 at 500 MHz)^141 Figure 77. Selected ROESY Correlations for Blancasterol 102 (Recorded in CDC13:C6D6 20:1) ^142 Figure 78. Conformational Structure of Blancasterol (102) with a Cis-Fused A/B Ring System ^143 Figure 79.^The Classification of Tunicates of the Genus Aplidium^ 153 Figure 80. The Body Plan of a Urochordate Larva ^ Figure 81. The Body Plan of the Urochordate Aplidium californicum^  154 156  Figure 82.^The Aplidium Spp. Collection Site^ 164 Figure 83.^A Freshly Collected Sample of the Undescribed Aplidium Species^ 165 Figure 84. 1 H NMR Spectrum of Langarin (142) (Recorded in Me2SO at 400 MHz) ^168 Figure 85. COSY Spectrum of Langarin (142) (Recorded in Me2S0 at 400 MHz)^169 Figure 86. 13 C NMR Spectrum of Langarin (142) (Recorded in Me2SO at 125 MHz)^170 Figure 87. HMQC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz) ^171 Figure 88. HMBC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz)^173 Figure 89. Selected HMBC Correlations for Langarin (142)^  172  Figure 90. 1 H NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2SO at 400 MHz)^176 Figure 91.^13 C NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2S0 at 125 MHz)^177 Figure 92. Freshly Collected Aplidium californicum^  184  Figure 93. 1 H NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)^187 Figure 94. 13 C NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 100 MHz) ^188 Figure 95. COSY Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz) ^189 Figure 96. APT Spectrum of Calaplidol A (160) (Recorded in CDC13 at 125 MHz) ^190 Figure 97. HMBC Spectrum of Calaplidol A (160) (Recorded in CDC13 at 500 MHz) ^191 Figure 98.^Selected HMBC Correlations for Calaplidol A (160) ^ 193 Figure 99. 1 H NMR Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz)^196 Figure 100. COSY Spectrum of Calaplidol B (161) (Recorded in CDC13 at 400 MHz) ^197 Figure 101. 13 C NMR Spectrum of Calaplidol B (161) (Recorded in CDC13 at 100 MHz) ^198 Figure 102. HMQC Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz) ^199 Figure 103. HMBC Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz)^200  xii  Page Figure 104. Selected HMBC Spectroscopic Correlations for Calaplidol B (161) ^  201  Figure 105. Selected 13 C NMR Shifts of Calaplidol B (161), 2-Methylhydroquinone (163), and^204 4-Methylcatechol (105)  List of Abbreviations  Ac^— acetyl APT^— Attached Proton Test ax^— axial br^— broad CD^— Circular Dichroism COSY^— COrrelation SpectroscopY d^— doublet Da^— Daltons dd^—doublet of doublets AM^— difference in mass dt^— doublet of triplets ED50^— Effective Dose resulting in 50% response EIHRMS — Electron Impact High Resolution Mass Spectrometry EILRMS — Electron Impact Low Resolution Mass Spectrometry eq^— equatorial FABMS^— Fast Atom Bombardment Mass Spectrometry FTIR^— Fourier Transform InfraRed HETCOR — HETeronuclear CORrelation HIV^— Human Immunodeficiency Virus HMBC^— Heteronuclear Multiple Bond Connectivity HMQC^— Heteronuclear Multiple Quantum Coherence i^— signal due to an impurity IC50^— Inhibitory Concentration resulting in 50% response ID50^— Inhibitory Dose resulting in 50% response J^— scalar coupling constant LRDCIMS — Low Resolution Desorption Chemical Ionization Mass Spectrometry m^— multiplet  xiv M+^— molecular ion m/z^— mass to charge ratio Me^— methyl Me2SO^— dimethyl sulfoxide mmu^— millimass units NOE^— Nuclear Overhauser Effect q^— quartet Rf^- Ratio to front  ROESY^— Rotating-frame Overhauser Enhancement Spectroscopy S^— signal due to solvent s^— singlet SCUBA^— Self-Contained Underwater Breathing Apparatus sp.^— species t^— triplet T/C^— Test versus Control TLC^— Thin Layer Chromatography w^— signal due to water  XV  List of Schemes  Page Scheme 1 Proposed Biogenesis of Microcionin-2 (116) and Nakafuran-8 (41)^ 148 Scheme 2^Proposed Biogenesis of Furodysin (35) and Furodysinin (36)^  149  Scheme 3^Proposed Biogenesis of Prenylated Quinones and Hydroquinones^ 207  xvi  Acknowledgments  I would like to thank my supervisor, Dr. Raymond J. Andersen, for the opportunity of studying in his research group. Dr. Andersen's enthusiasm ensured that the group was always an exciting and dynamic environment for scientific endeavor. I consider it a tremendous privilege to have had Dr. Andersen's instruction, guidance, and support in the work I have done for my doctoral degree. I would like to acknowledge the financial support of Les Fonds Pour La Formation De Chercheurs Et L'Aide A La Recherche and UBC. I would like to thank Mike LeBlanc for assistance in collecting specimens for study and for his friendship. Eric Dumdei, Shi-Chang Miao, Dave Burgoyne, Dave Williams, Barb Shaw, and Judy Needham have my appreciation for their help in collecting sample organisms, and for their contributions to a pleasant and intellectually stimulating research group. I would like also to express my appreciation to Captain John Anderson, master of the C. S. S. John P. Tully, and her crew. Thanks are extended to the chemistry department NMR facility, in particular to Liane Darge whose assistance frequently proved invaluable, and to the mass spectrometry service. Finally, I would like to thank Anna Dora Gudmundsdottir and Jacques Y. Roberge for help in proofreading this thesis. I am grateful to Anna and to Kristinn Kristinsson for being wonderful friends. Thanks also to my old friends Beth and Dave. I would like to thank my parents and my sister, Hana, for their encouragement and support. A heartfelt thank you to Jacques for love, warmth, and emotional sustenance.  xvii  For Hana  "Each of them in his own tempo and with his own voice discovered and reaffirmed with astonishment the knowledge that all things are one thing and that one thing is all things — plankton, a shimmering phosphorescence on the sea and the spinning planets and an expanding universe, all bound together by the elastic string of time. It is advisable to look from the tide pool to the stars and then back to the tide pool again." John Steinbeck and Edward F. Ricketts The Log from the Sea of Cortez, 1941  ^  1 GENERAL INTRODUCTION  Rocky headlands swept by ocean waves characterize much of British Columbia's exposed coastline. Winter storms can bring swells of such intensity that animals not well adapted to this environment may be torn from the substrate or abraded by the action of suspended particles. Despite the dangers this habitat poses, many invertebrates settle in the subtidal zones creating areas of high biomass and diversity. The rapid circulation of water provides a rich diet of food particles for sessile organisms and the evolutionary advantage that wave action can disperse their water-borne larvae. 1 Organisms which grow in the inter- and subtidal zones on the exposed coast adapt to the high energy environment in a number of ways. Many develop attachment devices such as the holdfasts characteristic of tunicates, others, such as sponges, may encrust the rock substrate, and still other organisms burrow into the rock to create shelters for themselves. All of the animals in such an environment must compete for the limited available resources of food and space. While many species of arthropods, mollusks and echinoderms have shells which provide a physical form of defense against predators and encroaching competitors, the method of defense of sessile invertebrates lacking shells or claws is less obvious. It has been suggested that many invertebrates such as porifera (particularly those lacking prominent spicules), coelenterates, bryozoans, and urochordates employ secondary metabolites to render themselves poisonous or merely unpalatable. 2,3 Some of these toxic compounds may provide valuable leads to chemotherapeutic agents against such human diseases as cancer. Sponge taxonomists were among the first to investigate the nature of poriferan natural products, primarily for the purpose of classifying these animals. Bergmann studied the steroidal composition of the Porifera in the 1940's to determine the chemotaxonomic significance of these metabolites. 4 His work led to the discovery of steroids unique to marine organisms. Bergmann's subsequent research on the chemistry of the sponge Tethya crypta led to the isolation of the novel arabinosyl nucleosides spongouridine 1 3 and spongothymidine 2. 5 0 CH3  HN )1 •1 HO  (:).  N -• 0 0H  HO  ^ OH ^ 1  OH 2  2 While fish and algae have long been held in esteem as remedies in Chinese folk medicine, 6 western culture has more commonly employed extracts of microbes and the higher plants for medicinal purposes. Bergmann's pioneering work sparked interest in the medicinal potential of marine invertebrate metabolites, particularly since a synthetic analog of compounds 1 and 2, cytosine arabinoside (Ara-C), has proven effective against adult leukemias.  3  A decade later advances in SCUBA and submersible technologies improved access to the —200 000 species of invertebrates found in the aquatic environment and made available a vast resource of new natural products?  Faulkner's comprehensive reviews of natural products isolated from marine invertebrates list over five thousand compounds reported between 1962 and 1991, an indication of the intense level of research activity in this field over the past three decades. 8 Many of these compounds have novel structures unique to aquatic organisms and exciting pharmacological activities.  Roth et al. have proposed that some of the molecules of the human nervous and immune systems including hormones and neural peptides resemble metabolites synthesized by parazoans and marine invertebrates because a common evolutionary ancestor was capable of synthesizing related archaic molecules. 2,9 Certainly extracts of marine invertebrates can affect not only other invertebrates, 3 but some may show activity in bioassays which use human cancer as well as other pathological cell lines. Numerous compounds which display biological activity in diverse assays have been isolated. An example of a poriferan metabolite currently attracting a great deal of scientific attention is manoalide (3) which was first isolated from the tropical Pacific sponge Luffariella variabilis by Scheuer and de Silva in 1980. 10 Pharmacological investigations revealed it to have potent anti-inflammatory and analgesic properties. 11  H3 C CH3  CH3  OH  OH 3  3 Since the development of effective antibiotics fifty years ago cancer has emerged as a leading cause of death in industrialized societies. 12 An ongoing challenge of modem medical therapeutics has been to identify compounds which inhibit cancer cell growth selectively without damaging the host organism. While chemotherapeutic agents which are effective against certain types of cancers, such as some forms of leukemia, have been developed, a wider variety of effective drugs is required. Successful chemotherapy requires drugs with improved selectivity, new modes of action as well as compounds which are effective against those cancers, solid tumors and drug-resistant cancer cells for example, which have proven refractory. 12  Research in the field of marine natural products has already provided some promising leads to new chemotherapies. The geodiamolides A—F (4-9) constitute a group of sponge metabolites with potent cytotoxicities. Geodiamolides A (4) and B (5) were originally reported by Chan and coworkers. 13 Andersen and de Silva later published the structures of geodiamolides C (6), D (7), E (8), and F (9) and reported potent in vitro activities against the L1210 murine leukemia cell line for all of the geodiamolides with ED50 values ranging from 2.5 ng/mL for geodiamolide C (6) to 39 ng/mL for geodiamolide D (7).14  HO  4 X=I,R=Me 5 X =Br,R =Me 6 X=C1,R=Me 7 X=I,R=H 8 X=Br,R=H 9 X = Cl, R = H  Novel and cytotoxic metabolites are by no means limited to organisms of the phylum Porifera. Bioassayguided fractionation of extracts of the colonial tunicate Trididemnum solidum led to the isolation of the didemnins, a family of cyclic peptides which exhibit potent cytotoxic, antiviral, and immunosuppressive activity. 15 a Didemnin B (10) is among the most cytotoxic of the didemnins with an ID50 value of 1.1 ng/mL against L1210 murine leukemia cells. Approximate immunosuppressive activity of the didemnins was cited as one thousand times that of  4 cyclosporin. Didemnin B (10) was the first marine natural product to enter Phase I clinical trials as an anticancer agent and is currently in Phase II clinical trials at the National Cancer Institutes. 15 0^ 0^0^0 II^ II^II^11 C —NH- CH- CHOHCH2 C - 0 — CHC CH(CH 3) — C I CH2CH(CH 3 )2^CH(CH3)2 L-Lac-L-Pro-D-MeLeu NH OCH3^CH2CH(CH3)2 C I^ I CHO -C-C liN(CH3)-C ^N COIN II  0  CH3  I-I  10  Another tunicate, Ecteinascidea turbinata, is reported to produce a "macromolecular" fraction of approximate molecular weight 10 000 g/mol which caused complete regression of Sarcoma solid tumors in two out of three mice tested and an average life extension of 92.5% was reported. 12  It is clear from the common clinical use of cytosine arabinoside (Ara-C), the current trials of clidemnin B (10) and the potent cytotoxic activities reported for such compounds as the geodiamolides A to F (4-9) and the "macromolecular" fraction of E. turbinata that marine invertebrate natural products represent a rich source of new antitumor compounds. The isolation of novel, cytotoxic natural products combined with the production of synthetic derivatives and analogs is a promising strategy for the development of anticancer drugs. 12  The objective of the research described in this thesis was to isolate cytotoxic compounds from marine invertebrates collected off the North-Eastern Pacific coast. Two species of sponge, Aplysilla glacialis and a Pleraplysilla sp., and two species of Aplidium, compound tunicates, were selected for study on the basis of the cytotoxicity of their extracts and because animals of these phyla are known to produce compounds which are biologically active as well as structurally nove1. 12  5 PART I. ISOLATION AND STRUCTURE ELUCIDATION OF METABOLITES FROM TWO NORTH-EASTERN PACIFIC SPONGES  INTRODUCTION  The phylum Porifera is traditionally classified as belonging to the kingdom Metazoa although the sponges which constitute this phylum are intermediate between metazoans and colonial protozoans. 16 Poriferans are considered to be primitive organisms in that they have retained many ancestral characteristics, have no organs or clearly-defined tissues, lack a nervous system, and have little coordination between cells. Since the emergence of the phylum in Cambrian times, sponges have evolved successfully as evidenced by the greater than ten thousand species alive in the seas today. 17 Sponges are essentially cooperating aggregations of cells. Water is pumped at low pressure into small openings, or ostia, in the surface of the sponge, through the inhalant pore to a single layer of choanocytes which are flagellated collar cells, and then outward via a larger opening, the osculum (Figure 1).  17,18  The movement of the  flagella of the choanocytes is responsible for the unidirectional movement of water which passes through the organism bringing in organic particles and microbes (ingested primarily by the choanocytes) and removing waste products such as ammonia. Respiration takes place by diffusion of oxygen from the incoming flow of water across cell membranes. Sponges have very low metabolic rates but can pump high volumes of water. One fresh-water species is capable of pumping up to seventy times its body volume of water per hour. forms and growth is heavily influenced by their environment.  16  Sponges have flexible  17  Poriferans are hermaphroditic and can reproduce sexually or asexually. Sexual reproduction may be viviparous or oviparous (a taxonomically significant variation). 19 In viviparous sponges, release of the larvae does not take place until the cells are in an advanced state of differentiation and the resultant larvae are free-swimming and highly developed. If ciliated, the larvae may swim for three to forty-eight hours prior to subsiding to a creeping state. Whether ciliated or not, the larvae creep for twenty to sixty hours showing sensitivity to the effects of gravity and light in choosing a substrate on which to settle. Once settled, the larvae metamorphose and within twenty-four hours have established a functional pore system. 2° Two forms of asexual reproduction are known. The sponge may form gemmules, clusters of totipotent cells protected by an outer layer of spicules and spongin, or fragments may break off the adult sponge which are capable of settling elsewhere and becoming separate individual sponges. 17  6  osculum  inhalant pore  choanocytes  ostia  Figure 1. Typical Body Plan of a Sponge of the Class Demospongaiel 7  A sessile adult sponge has a single-cell outer epithelium known as the pinacoderm. The inner layer is the mesohyl which contains mobile cells and skeletal elements. There is evidence that all of the cells of a poriferan are mobile and sponges have the ability to regenerate significant portions of their tissues. 17 Although sponges are sedentary they have few predators presumably, in part, because many poriferans have evolved arsenals of toxic or unpalatable secondary metabolites which are intolerable to other species. 2-3 Organisms which are known to feed on sponges include asteroids and many species of opisthobranchs which have developed the ability to sequester sponge secondary metabolites and employ them for their own protection. 3 Sponges are classified according to their skeletal elements into the four classes Calcarea, the smallest of the sponges which possess skeletons composed of calcium carbonate, Hexactinellida, deep water sponges with siliceous  7 spicules, Demospongiae, which will be described in greater detail, and Sclerospongiae, a class encompassing sponges with siliceous spicules and an organic collagen skeleton within a thin layer of living tissue growing on a massive calcium carbonate skeleton. 16,17 This thesis is concerned with two species of sponge belonging to the class Demospongiae. The Demospongiae are characterized by skeletons comprised of either or both siliceous spicules and an organic skeleton which may be dispersed throughout the mesohyl or secreted as fibres of a protein known as 16,17^ 16,18 spongin.^This class is the most common comprising 95% of recent sponges. ^It is represented in every  aquatic environment including fresh-water habitats and is found throughout the world's oceans from the intertidal shallows to the deepest abysses. 21  CLASSIFICATION OF THE SPONGES  Classification of the Porifera at the ordinal, generic, and class levels can be problematic because morphology is frequently inconsistent and the simplicity of the organism provides few features on which to base a classification system. 22 Many species of sponge respond to their environments by encrusting the substratum and growing in irregular, unpredictable symmetries. Some species react to the direction and force of water currents by growing oscula which are oriented to minimize mixing of inhalant and exhalant water currents. 23 Pigmentation is not necessarily an aid in identification as color morphs within species are known. Sponges which grow in direct light may develop deeper pigmentation as protection against ultraviolet radiation than animals which grow in shade or on the undersides of rocks. 23  Traditionally classification is based on the skeletal characteristics of the sponge. The chemical nature of spicules is used to determine class. Further identification considers the relative sizes of the spicules, their shapes, and the degree of incorporation of sand or debris. The structure, organization, form, and extent of the overall skeleton are also considered. Additional morphological factors include any external characteristics of the sponge, its consistency, and surface features. 24  8  In 1956 Levi proposed division of the Demospongiae, on the basis of reproductive biology, into the orders Tetractinomorpha (the oviparous sponges) and Ceractinomorpha (viviparous sponges which release larvae). 25 Since Levi's initial attempt to classify sponges according to characters other than skeletal features, the advent of electron microscopy has led to the study of the histology of differentiated sponge cells and the resultant information has been used for taxonomic classification. 26  In a number of species, the secondary metabolites synthesized by the sponge may be of utility in identifying the animal. Bergmann pioneered this area of research when he first attempted to classify marine invertebrates according to the steroids produced by the animals. 4 Bergquist and other workers have shown that some secondary metabolites do have chemotaxonomic significance. 27 Species of the order Verongida, for example, predictably synthesize brominated amino acid derivatives. 27  This thesis describes studies of the secondary metabolites of two species of Demospongiae which belong to the order Keratosa (or Horny sponges), poriferans which lack spicules entirely but which secrete skeletons of spongin fibres. Faulkner has suggested that, in the absence of spicules which might serve to physically deter predators, the Keratosa sponges have developed diverse suites of terpenoids and other natural products for the purpose of chemical defense. 8 d Numerous researchers have isolated a variety of chemically novel compounds from this group of sponges. 28 Many have proved active as antibiotics, antifeedants and antifouling agents. 28 a-c Since the absence of spicules makes classification of this order particularly difficult, studies of their natural products may be a valuable aid in grouping these organisms. Bergquist warns that several factors must be considered for chemotaxonomy to be an effective and reliable tool. Obviously the secondary metabolites must be carefully characterized. The sponge taxonomy must be reliable and many zoologists emphasize the importance of storing voucher samples for verification by future workers. Another factor which must be taken into account is the presence of extraneous organisms which grow on the surface, in the pores and in the mesohyl of many sponge species. Microscopic species are difficult to remove and may contribute to the observed chemistry. 22 a  9 APLYSILLA GLACIALIS AND A PLERAPLYSILLA SP. SPONGE  Aplysilla glacialis is an encrusting pink sponge found in exposed habitats off the west coast of British  Columbia. In 1991 Andersen et al. reported the isolation of six novel diterpenes from extracts of A. glacialis.28d We proposed to study the more polar extracts of Aplysilla glacialis in order to isolate novel cytotoxic compounds. When this study was underway we received a small collection of a gay-white sponge which was initially identified as a new species of Aplysilla. Our interest in the chemistry of the white sponge was prompted by the possibility that it might contain cytotoxic metabolites similar to those produced by Aplysilla glacialis. It was proposed that an investigation of the natural products of the white sponge would also determine whether it was a color morph of Aplysilla glacialis or if not, might aid in establishing it as a new Aplysilla species. Our study provided evidence  against the classification of the gray-white sponge in the genus Aplysilla and suggested that the genus Dysidea would be a more appropriate classification. Reexamination of the specimen in light of its secondary metabolism indicated that it is a member of the Pleraplysilla, intermediate between the Aplysilla and the Dysidea, and a genus previously unknown on the west coast of North America. 24 Aplysilla glacialis and the Pleraplysilla species sponges will be described. The taxonomy of these sponges will be discussed and the secondary metabolites of sponges of the genera Aplysilla, Dysidea, and Pleraplysilla briefly reviewed.  DESCRIPTION OF APLYSILLA GLACIALIS  Austin describes Aplysilla glacialis as an encrusting, non-branching sponge, typically rose to rose-red but sometimes varying to tan or ivory. 29 The organism is soft and slippery with a surface characterized by conules which project 1-2 mm in height. Austin indicates that the cones are irregular and may have spongin fibres projecting from the tips. Oscula of —0.6 mm diameter are visible flush with the sponge surface. The skeleton is formed of unbranched or sparsely branched fibres which start at a basal plate and end at the pinacoderm or project from the conules. The sponge does not incorporate foreign material such as sand or detritus into its skeletal network. Individual specimens are several square decimeters in area and reach 3-6 mm in thickness intertidally and at least 12 mm subtidally. Some individuals can grow to cover several square meters. Austin states that A. glacialis  10 has been observed to grow as far north as Kuiu Island, Alaska and as far south as Hermosa Beach, California. A. glacialis is found in exposed coastal areas with high energy wave circulation. This sponge generally prefers a  shaded environment such as the underside of a raised rock, overhanging cliff, or cave. 29  The specimens used for this study (Figure 2) were collected subtidally by SCUBA off the Deer Group of Islands near the Bamfield Marine Station and off the north shore of Sydney Inlet (Figure 3). The organisms grow at 2-5 m depth where they encrust overhanging rockwalls at the mouths of surge channels. They grow to a square meter in area and 2 cm in thickness. All of the specimens used were of a very bright pink color and the surface morphology was identical to that described by Austin. Aplysilla glacialis has been observed growing at many of our exposed dive sites off the coast of British Columbia but it is not abundant. This sponge was never overgrown by other encrusting organisms and the surface appeared free of algal growth. The nudibranch Cadlina luteomarginata has been observed to feed on Aplysilla glacialis 30  DESCRIPTION OF THE PLERAPLYSILLA SP. SPONGE  Bergquist describes Pleraplysilla poriferans as thin, encrusting sponges with simple fibres which may branch once or twice but which are of constant thickness from the basal plate to the surface.  24  An important  distinguishing characteristic is that the sponge incorporates sand and debris into the core of its spongin fibres. 24  In 1990 specimens of what appeared to be the same Pleraplysilla species were collected intertidally at Botanical Beach and at Checleset Sound. Both sites are off the west coast of Vancouver Island, British Columbia (Figure 3). In subsequent years, only the Botanical Beach collecting site was revisited. The sponge grows on the undersides of boulders at sea level in exposed areas. The Botanical Beach specimen (Figure 4) is —20 cm in diameter and 3-10 mm thick. It is gray-tan in color with many prominent conules rising 1-2 mm above the sponge surface. Spongin fibres are visible protruding from some conules. Oscula flush with the pinacoderm can be observed. Although the Botanical Beach boulder was home to at least three species of sponges there was no overgrowth of the Pleraplysilla species nor was any algal fouling observed. This species of sponge seems to be rare as we have not  observed it on collecting trips to other exposed coastal areas of Vancouver Island.  11  Figure 2.^An Intertidal Specimen of Aplysilla glacialis  12  50° 11129° W  128°W I  -  127°W^126 °W 1  1......\...)  125°W 1  124°W 1  123°o N  BRITISH COLUMBIA  50° N  50 ° N  °  49 N  °  49 N  Sydney Inlet Collection Site  Bamfield Collection Site ^  °  48 N  Botanical Beach Collection site PACIFIC OCEAN  °  °  47 N  47 N  °  47 N 129 W  °  1^1 128 W^127°W  °  126°W  125 ovv,  °  47 N 123 W  Figure 3.^Aplysilla glacialis and Pleraplysilla Species Sponge Collection Sites  °  13  Figure 4.^An Intertidal Specimen of a Pleraplysilla Species Sponge  14  TAXONOMY OF APLYSILLA GLACIAL IS AND THE PLERAPLYSILLA SP. SPONGE  Aplysilla glacialis and the Pleraplysilla sponge species belong to the class Demospongiae and to the  subclass Ceractinomorpha, the sponges which reproduce sexually by releasing fully developed larvae. 19 Both are considered to be "horny" sponges in that these species lack spicules but have skeletons of secreted spongin fibres. 24 The lack of spicules greatly reduces the characters on which taxonomy can be based 24 and consequently there is little agreement among sponge taxonomists about the classification of these organisms. Based on skeletal characteristics alone, both the Aplysilla and Pleraplysilla sponges were traditionally placed in the order Dendroceratida which is characterized by fragile, dendritic skeletons of pure spongin fibres which branch from a basal point without anastomosing. 24 Recently Bergquist has proposed that the secondary metabolites of Pleraplysilla species sponges indicate a closer relationship to the Dysidea sponges of the order Dictyoceratida than to the Dendroceratida. 22b The Dictyoceratids are also "horny" sponges characterized by anastomosing networks of tough spongin fibres which contain detritus. 31 Consequently Bergquist moves the Pleraplysilla to the Dictyoceratida but leaves the Aplysilla species sponges in the order Dendroceratida (Figure 5A). 22b Boury-Esnault et al. have studied the histology of the Demospongiae by electron microscopy.2  6  Comparison of the choanocytes, the choanocyte chambers, the mesohyl, and apopinocytes (cells lining the exhalant pores) led them to conclude as did Bergquist and coworkers that Aplysilla and Dysidea are closely related. Unlike Bergquist, however, Boury-Esnault et al. propose that Aplysilla, Pleraplysilla, and Dysidea be grouped together in the order Dendroceratida (Figure 5B). 26 Van Soest questions the validity of the classification system introduced by Levi and applied, with modifications, by Bergquist. 32 Van Soest suggests that species be grouped only on the basis of shared characters inherited from a common ancestor. He feels that logic argues against dividing the "horny" sponges into distinct orders and classifies them in a single order, the Keratosa. Van Soest proposes that the appropriate status for the Dendroceratida and Dictyoceratida is subordinal. On the basis of common characters, Van Soest proposes classifying the Dendroceratida and the Dysideidae together as both lack spicules, have sand in their skeletal fibres, are rich in terpenes and have choanocyte chambers greater than 30 jim in diameter (Figure 5C). 32b  15 It is beyond the scope of this thesis to critically evaluate the classification schemes proposed by Bergquist, Boury-Esnault, and Van Soest. It is likely that chemotaxonomy will continue to play a role in establishing poriferan classification systems and in dictating which classification system will eventually gain wide acceptance and use.  KINGDOM^  Metazoa^  Metazoa^  Metazoa  PHYLUM^  /'^ Porifera^  Porifera^  Porifera  CLASS^  1^1  /^ Demospongiae^  Demospongiae^  I^ SUBCLASS^Ceractinomorpha^  !I Ceractinomorpha  I  ORDER^Dendroceratida Minchin^Dictyoceratida Minchin^Dendroceratida Minchin^  SUBORDER  / Demospongiae  ^  Keratosa  Dendroceratida Dysideidae  FAMILY Aplysillidae Vosmaer^Dysideidae Gray^Aplysillidae Vosmaer^Dysideidae Gray  '1 I^ GENUS^Aplysilla Schulze^Pleraplysi la Topsent Aplysilla Schulze^Dysidea Johnston ^ Dysidea Johnston '/ ^ SPECIES^A. glacialis  A  ^  A. glacialis  B^  C  Figure 5. Taxonomic Classification Schemes according to (A) Bergquist,22b (B) Boury-Esnault, 26 and (C) Van Soest. 32b  5  17 CHEMISTRY OF SPONGES OF THE GENERA APLYSILLA, DYSIDEA, AND PLERAPLYSILLA  A study of the chemistry of Aplysilla glacialis resulted in the isolation of two novel cytotoxic 9,11-secosteroids. Investigation of the natural products of a Pleraplysilla species sponge resulted in the isolation of seven known and three new sesquiterpene derivatives in addition to a third novel, cytotoxic 9,11-secosteroid. This section of the thesis will briefly review the known chemistry of sponges of the genera Aplysilla, Dysidea, and Pleraplysilla in order to place the chemistry of A. glacialis and the Pleraplysilla sponge in a chemotaxonomic  context. The known secosteroid chemistry of marine invertebrates will also be reviewed. SECONDARY METABOLITES OF APLYSILLA SP. SPONGES  The natural products chemistry of Aplysilla sp. sponges is characterized by diterpene derivatives of the hypothetical "spongian" skeleton 11. Of forty compounds reported to date from Aplysilla sponges, twenty-seven were diterpenoids. The remaining thirteen compounds included eleven steroids, one purine and one amino acid derivative. Microscope studies of the mesohyls of Dendroceratid sponges prompted Bergquist and Wells to report that these poriferans "have negligible matrix microorganism populations". 22 It is likely that Aplysilla sponges synthesize their spongian-derived diterpenoids endogenously.  18  ^  19  11  Bergquist and coworkers have proposed that the mechanism followed in the spongian skeleton derivatization and degradation by Dendroceratid sponges may be a reliable marker for taxonomic classification at the specific leve1. 22b This hypothesis will be considered with respect to the reported chemistry of the Aplysilla sponges. Tischler recently prepared an exhaustive review of spongian derived diterpenes 33 therefore the following review of  18 Aplysilla sponge chemistry will select relevant examples but will not attempt to present all of the secondary  metabolites reported from this genus. Molinski and Faulkner reported the isolation of three diterpenes from an undescribed pink species of Aplysilla from Australia. 34 Of these metabolites, ambliofuran had been previously reported and one of the other  compounds appeared to be an artifact. The one novel natural product was 6a , 7 a , 17 - tri h y dr ox y 150,17-oxidospongian-16-one-7-butyrate 12, a diterpene with a spongian skeleton elaborated by oxidation. Faulkner and Bobzin reported the isolation of five known and four new diterpenes of the spongian class from the purple sponge Aplysilla polyrhaphis collected in the Gulf of California. 35 Of these compounds, macfarlandin E and aplyviolene had previously been isolated from the Dendroceratid sponge Chelonaplysilla violacea, 35 norrisolide was known from a Dendrilla sponge, 35 shahamin C had been reported from a Dysidea sp.  sponge (order: Dictyoceratida) and (5R * ,7S * ,8S * ,9S * ,10R * ,13S * ,14S * )-16-oxospongian-7-y1 acetate was known from a Darwinella sp. sponge. The known compounds represented three types of carbon skeletons; the dendrillane, norrisane and spongian. The four novel diterpenes isolated from A. polyrhaphis were the polyrhaphins A—D (1316). Polyrhaphin C (15) was found to be a fish antifeedant at concentrations of 100 pg/mL and exhibited antibacterial activity against Staphylococcus aureus at 100 pg/disk and Bacillus subtilis at 10 tg/disk. 35  12 6a,7a,1713-Trihydroxy-150,17-oxidospongian-16-one 7-butyrate Spongian skeleton  ^ 13 14 ^ Polyrhaphin A Polyrhaphin B Dendrillane skeleton ^Dendrillane skeleton  19  OAc  0  15  16  Polyrhaphin C Macfarlandin skeleton  Polyrhaphin D Isospongian skeleton (= marginatane skeleton)  Aplysilla glacialis collected off Vancouver Island, British Columbia yielded six novel diterpenoid  metabolites 28d,36 The natural products glaciolide (17), cadlinolide A (18), cadlinolide B (19), aplysillolide A (20), aplysillolide B (21), and marginatone (22) were reported by Tischler and Andersen. These six diterpenes represented the glaciane, aplysulphurane, gracilane, 33 and marginatane 28 d carbon skeletons.  ^ 17 18 ^ Glaciolide Cadlinolide A ^ Glaciane skeleton Aplysulphurane skeleton OH  ^ 19 20 ^ Cadlinolide B Aplysillolide A ^ Aplysulphurane skeleton Gracilane skeleton  20  ^ 22 21 ^ Marginatone Aplysillolide B ^ Marginatane skeleton Gracilane skeleton In 1990 Poiner and Taylor reported that a study of the natural products of the orange Aplysilla tango from Australian waters had yielded the known diterpene gracilin A, four novel diterpenes, and a steroid fraction. The new diterpenes were aplytandiene-1 (23), aplytandiene-2 (24), aplytangene-1 (25), and aplytangene-2 (26). 37 The aplytandienes possessed the gracilane skeleton while the aplytangenes were nor-diterpenes. A sterol fraction containing a mixture of ergosterols exemplified by compound 27 was also isolated. The ergosterols shared a common nucleus and varied only in the side chain constitution. A fraction of endo-peroxy sterols (28) with common nuclei and diverse side chains was also isolated, but Poiner et al. suggested that these were artifacts of ergosterol reaction with singlet oxygen. 37  ^ 23 24 ^ Aplytandiene-1 Aplytandiene-2 ^ Gracilane skeleton Gracilane skeleton  CH3  CH  ^ 25 ^ Aplytangene-1 ^ Nor-diterpene  26 Aplytangene-2 Nor-diterpene  21  HO  HO 27  Recently Faulkner and Bobzin reported the isolation of four diterpenes, two endoperoxy sterols, an amino acid derivative and 1-methyladenine from Aplysilla glacialis collected at Crooked Island in the Bahamas. 38 None of these compounds was novel. Manotil was known from terrestrial sources and the endoperoxy sterols are commonly isolated from marine organisms. Of the diterpenoids, spongia-16-one, had been reported from a Dendroceratid sponge, atisane-30,16a-diol had been isolated from the sponge Tedania ignis and spongia-15a,16a-diacetate was also a metabolite of Spongia officinalis. 39 Faulkner and Bobzin investigated the antimicrobial, antifeedant, and antifouling activities of these compounds. They reported that manotil and cholesterol endoperoxide were active in deterring fish predation. The antifouling assays proved inconclusive but 1-methyladenine was identified as the antibacterial component of the sponge, showing activity against an Acinetobacter species, a Flavobacterium, and two Vibrio strains. 38 The chemistry of the Bahamian Aplysilla glacialis differed markedly from that of the British Columbian Aplysilla glacialis although the presence of spongian derivatives in both sponges suggested taxonomic relatedness. This review has illustrated that spongian-derived diterpenes are characteristic of sponges of the genus Aplysilla. Bergquist's argument that carbon skeletons of metabolites can be used for classification at the species  level is not supported. 22 b In the case of each of the Aplysilla sponges studied, metabolites with a diverse array of carbon skeletons were isolated. Furthermore, compounds which had previously been reported in the literature were by no means exclusive to the genus Aplysilla but represented many Dendroceratid and, in one case, Dictyoceratid sponges. The ecological role the Aplysilla sponges' diterpene metabolites play is not yet fully understood. Faulkner has suggested that non-siliceous sponges such as these elaborate intricate arrays of metabolites to serve as a chemical defense against predation. 8 d No conclusive studies support this hypothesis as far as the Aplysilla sponges are concerned. The antimicrobial activity of some of the reported metabolites, polyrhaphin C and 1-methyladenine for example, may indicate a defense against microbial fouling. 38 Another role has been suggested for antibacterial  22 compounds. Bergquist proposed that the sponge may secrete antimicrobial agents via its pinacoderm to prevent bacteria from settling as they are drawn into the sponge inhalant pores towards the choanocytes where feeding takes place. 3  NATURAL PRODUCTS OF SPONGES BELONGING TO THE GENUS DYSIDEA  Sponges of the genus Dysidea produce a diverse assortment of natural products which have been extensively studied. Faulkner has reviewed reports of one hundred and twenty-seven metabolites from Dysidea species sponges. 8 Of these compounds, —50% were sesquiterpenes or sesquiterpene derivatives. Terpenes are widely accepted as the "true" metabolites of Dysidea sponges. 8 c The majority of terpenoidal compounds reported to date are derivatives of the furan sesquiterpene 29.  29  Sesquiterpene furans and sesquiterpene quinones have been found from Dysidea sponges collected around the world. 22b The secondary metabolites are not limited to sesquiterpenes and the isolations of a novel monoterpene, diterpenes and sesterterpenes seem to suggest that sponges of the genus Dysidea are capable of a wide variety of terpene syntheses. Electron micrograph studies have shown Dysidea sponges to possess high concentrations of matrix cyanophytes. 22 It has been suggested that the polychlorinated amino acid derivatives which have been isolated from the Dysidea and in particular from Dysidea herbacea, are metabolites of the sponges' bacterial populations 22,40 A review of examples of metabolites from Dysidea sponges relevant to this thesis follows.  The first sesquiterpene quinones reported from a Dysidea species sponge were avarol (30) and avarone (31) from Dysidea avara. 41 These compounds continue to attract interest because they exhibit cytotoxic activity against leukemia cells in vivo and inhibit HIV virus replication in vitro. 42 A number of related natural products have been isolated and derivatives have been synthesized for biological testing. 42  23  OH  30^  31  In 1975 Minale et al. reported ten sesquiterpene furan derivatives from the sponge Dysidea pallescens. 43 Of particular note are the pallescensins 1-3 (32-34, respectively). Minale discussed the possibility that 34 was an oxidation artifact of compound 33 but suggested that the absence of the corresponding hydroxybutenolide of metabolite 32 indicated that 34 was produced by the sponge. 43 a HO 0 H  33^34  Wells and coworkers published the structures of the four new sesquiterpenes furodysin (35), furodysinin (36), and their respective thioacetates 37 and 38. 44 Studies on compounds 35-38 and several oxidation products showed that the a-substituted-y-hydroxy-a,13-butenolide of 15-acetylthioxy-furodysinin (38) had a strong affinity and specificity for human leukotriene B4 (LTB4) receptors. This suggested a possible defense role as this compound would initiate an pro-inflammatory response in humans and possibly also in predators.  45  H 35 R = H 37 R = AcS  36 R = H 38 R = AcS  24 The first terpene isolated from D. herbacea, a sponge which had yielded a number of chlorinated amino acid derivatives, was spirodysin (39) reported by Wells et a1. 46 Wells suggested that spirodysin may be a precursor of furodysin (35) and furodysinin (36).  Upial (40) is a sesquiterpene rearranged to form a bicyclo(3.3.1)nonane aldehyde lactone which was reported from the Hawaiian sponge Dysidea fragilis by Scheuer and coworkers. 47 Also isolated from D. fragilis by Scheuer et al. were two sesquiterpene furans; nakafuran-8 (41) and nakafuran-9 (42).  28 b Scheuer noted the isolation of the  hydroxybutenolide of nakafuran-8 (43) and the corresponding methyl ketal 44 but suggested that these compounds may be artifacts. Nakafuran-8 and nakafuran-9 were effective fish anti-feedants but upial (40) proved inactive.  28 b  41  42  ^  43 R = H 4 4 R = CH3  Wells and coworkers have reported the isolation of furodysin derivatives 45 and 46, furodysinin derivatives 47-50, the drimane furan 51, and the linear furan 52 from a collection of Dysidea herbacea 40  25  H  H 45 R=f3OH 46 R=f30Ac  47 R=POH 48 R=I30Ac 49 R=aOH 0  . 51  50  52 Faulkner reported the isolation of pallescensolide (53) from the Californian sponge Dysidea amblia." The author addressed the question of whether the butenolide 53 might be an artifact as a result of the oxidation of pallescensin A.43 e A similar relationship had been observed between the diterpenes ambliol A (54) and ambliolide (55) both previously isolated from D. amblia. Singlet oxygen oxidation of ambliol A in methanol did not produce ambliolide leading Faulkner to conclude that oxidation to the butenolide was likely a process of the sponge although he noted that formation of the methoxy group might be due to exposure of the compound to methanol."  54  H3  26 In 1984 Cardellina and Grode reported the isolation of the sesquiterpene lactone of furodysinin (56) from the sponge Dysidea etheria. 49 In order to elucidate the structure they attempted to form compound 56 by oxidation of furodysinin (36). A light—induced oxidation in methanol yielded only 57, the methyl ketal of 56. Eventually the sesquiterpene lactone was formed with meta—chloroperbenzoic acid followed by Jones' reagent. 49 OR  H  56 R = H 57 R = CH3 Cardellina and Bamekow have recently reported the structures of the oxidized nakafuran derivatives 58-60 from the sponge Dysidea etheria. 50 Since no nakafuran-8 (41) was isolated from any of a number of sponge collections, it was suggested that the oxidized nakafurans (58-60) were unlikely to be artifacts of an autooxidation reaction.  R  58 R = OAc, R' = H 59 R = OH, R' = H 60 R, R' = 0 Terpene metabolites other than sesquiterpenes have been isolated from Dysidea species sponges. The first monoterpene reported from a marine sponge was adriadysiolide (61) isolated from a north Adriatic Dysidea species. 51 O  I^„P O  61  27 Dysidea pallescens from the Bay of Naples yielded a scalarane sesterterpene hydroxyhydroquinone, disidein  (62). 52 The absolute stereochemistry was assigned by X—ray crystallographic analysis of a brominated derivative of 62. 53 HO OH  62  As previously mentioned, Faulkner has reported the isolation of pallescensolide (53), a sesquiterpenoid, and ambliol (54), and ambliolide (55), related diterpenes from the same sponge. 48 Faulkner noted that this is unusual among sponges but cites precedents among Dysidea species. 48 Recently Kashman and coworkers have published the structures of thirteen novel rearranged spongian-derived diterpenes from a Red Sea Dysidea species. 54 These new compounds are exemplified by shahamin E (63) 54 a and norrlandin (64). 54b Norrlandin was found to be cytotoxic (IC50 1.2 Ltg/mL). 54 b H OAc  63  64  Non-terpene natural products of Dysidea sponges include a diverse array of chlorinated amino acid derivatives which may co-occur with sesquiterpene metabolites.° Compound 65 55 is an example of these metabolites. It has been suggested that these natural products are synthesized by the sponges' symbiotic bacteria 40  28 HO -  Cl3C  7  CC13  65 A variety of novel steroids have been isolated from Dysidea species sponges. These will be discussed in some detail in a later section.  THE CHEMISTRY OF THE PLERAPLYSILLA SPONGES  In 1972 Minale and coworkers reported the isolation of the sesquiterpene furans dehydrodendrolasin (66) and pleraplysillin (67) from Pleraplysilla spinifera. 56 The same researchers later published the structure of pleraplysillin-2 (68), an ester formed of a hemiterpene alcohol and a sesquiterpene furan acid, isolated from polar extracts of the same collection of P. spinifera.57  66  0 68  In a later collection of what appeared to be P. spinifera, Minale and coworkers failed to detect the three previously isolated metabolites 66-68. 58 They isolated compound 69, longifolin, known from a terrestrial plant and the novel sesquiterpenes spiniferin-1 (70) and spiniferin-2 (71). Their failure to find dehydrodendralosin (66) and the pleraplysillins (67 and 68) in the later collection of Pleraplysilla prompted Minale et al. to conclude that this was a distinct species of Pleraplysilla sponge, distinguishable only by its chemistry. 58 a Minale et al. presented two alternatives each for the structures of the spiniferans, and it was not until a later publication that the structures were confirmed to be 70 for spiniferan-1 and 71 for spiniferan-2. 58 b  29  This thesis presents a study of the chemistry of a Pleraplysilla sp. sponge. The sponge yielded the known compounds furodysinin lactone (56), 49 nakafuran-8 lactone (43), 28 b their respective 0—methyl ketals (57 and 44), in addition to furodysinin (36)," furodysin (35), 44 and nakafuran-8 (41). 28 b The new sesquiterpenes isolated from the sponge were 0—methyl 9—oxofurodysinin lactone (72), 2-oxomicrocionin-2 lactone (73), and 0—methyl 2-oxomicrocionin-2 lactone (74).  72  ^  73 R = H 74 R = Me  The chemistry of sponges of the genus Pleraplysilla has not been extensively studied. Those natural products which have been isolated suggest that Pleraplysilla sponges characteristically produce sesquiterpene furan metabolites. This is in keeping with Bergquist's observation that the chemistry of the Pleraplysilla sponges "fits better with an identification as Dysidea" than as Aplysilla sponges. 22 Bergquist further remarked concerning the taxonomy of the Pleraplysilla, that they are "strictly intermediate between the two groups" Aplysilla and Dysidea. 22 b  30  SECOSTEROIDS AND RELATED STEROIDS FROM MARINE INVERTEBRATES  Steroidal natural products which incorporate many unique chemical features have been isolated from marine invertebrates. Among these features are highly oxygenated skeletons, epoxides, polyenone functionalities, and unusual side chain structures such as the rare cyclopropene ring. 59 Secosteroids are uncommon among steroidal metabolites of marine invertebrates but a number of interesting compounds, some biologically active, have been reported.  The secoderivative (75) of gorgosterol was the first secosteroid reported from a marine invertebrate.  60  Compound 75 was isolated from the gorgonian Pseudopterogorgia americana. The structure was solved by X—ray crystallography of the 3—p—iodobenzoate,11—acetate derivative. The side chain of secogorgosterol incorporated an unusual cyclopropane functionality. 60  HO  75  Two C—ring secocholestane derivatives, 76 and 77, possessing a common nucleus identical to that of secogorgosterol and differing only in side chain structure were isolated from the soft coral Sinularia species. 61 Wells et al. reported the structures of compounds 76 and 77 without determining relative stereochemistry. Djerassi and coworkers further investigated secosteroids of the same soft coral species. 62 They re-isolated compounds 75-77, defined the relative stereochemistry of metabolites 76 and 77 by use of circular dichroism spectra and reported the  31 structures of the closely related secosteroids 78-80. 62 The authors suggested that the presence in the animal of secosteroids with a variety of side chains was the result of the soft corals' cleaving of the C-rings of dietary steroids. Support for this argument was provided by the isolation of steroids with standard nuclei possessing the side chains of compounds 75-80. 62  R'0  HO  „H  80  The first secosteroid reported from a poriferan was herbasterol (81) isolated from an Australian specimen of Dysidea herbacea. 63 Herbasterol (81) was a highly oxidized 9,11-secosteroid with an unusual 19-hydroxylated  methyl and an A/B cis ring junction. Faulkner and coworkers reported that compound 81 was toxic to goldfish (Carassius auratus) at 101.1.g/mL. 63 Mild antimicrobial activity was also recorded.  OH  81  Sica and coworkers have reported the isolation of the first naturally occuring B-ring secosteroid, hipposterol (82), from the sponge Hippospongia communis. 64 The structure was confirmed by synthesis. Eight steroids (83—  32 90) differing from hipposterol only in the structures of the side chains were later isolated from H. communis by the  same researchers. 65  HO OH 82 R =  83 R =^ir.  84 R =  85 R = ,MM  86 R=  88 R =  90 R =  Ciminiello and coworkers reported the structures of the highly degraded incisterols (91-94) from the Mediterranean sponge, Dictyonella incisa (family: Hymeniacidonidae). 59 In this case, the entire A-ring and Me19 functionality have been cleaved away from the steroid nucleus.  33  1  /  T  '''''' .•■•■■„,..../..• /' 92 R-=  The New Caledonian sponge Jereicopsis graphidiophora yielded the novel 8,14-secosteroids, jereisterols A (95) and B (96). 66 In addition to the rare cleavage point, these compounds have an unusual 3-methoxy functionality. Steroids with the conventional 3-hydroxy group were absent from extracts of the sponge.  96 Recently a new C-ring secosteroid was reported from the sponge  Spongia officinalis. 67  The structure of  secosterol 97 was confirmed by synthesis. Further examination of extracts of S. officinalis by the same researchers yielded the closely related 9,11-secosteroids 98 and 99. 68  34  HO  HO ,  OH  98 R=  97  99 R=  This thesis will dicuss the recent isolation and structure elucidation of three cytotoxic secosteroids from the sponges Aplysilla glacialis and a Pleraplysilla sp. The marine sponge Aplysilla glacialis was found to produce glaciasterols A (100) and B (101). 69 A study of a Pleraplysilla sp. sponge resulted in the isolation of a single secosteroid, blancasterol (102).  100 R=  1 01  ovvv. ..//■./ R .MM  102 It is interesting to note that sponges of the genera Dysidea, Hippospongia, and Spongia belong to the single order Dictyoceratida. The steroidal metabolites, including secosteroids, produced by these sponges tend to be highly oxidized. Wells and coworkers have suggested that the biosynthesis of the secosteroidal skeleton proceeds via  35 oxidative cleavage of the 9,11 or, in the case of the hipposterols (82-90), the 5,6-7c bond of an exogenously introduced steroid. 61 A number of steroids have been isolated from Dysidea species sponges which are considered attractive precursors or intermediates for the synthesis of secosteroids via oxidative cleavage. These are exemplified by steroid 103 which was isolated from the Bermudian sponge Dysidea etheria. 7 ° This polyhydroxylated steroid may result from the oxidation of 5,6— and 9,11—double bonds. Oxidative cleavage of such an intermediate would result in secosteroids similar to those which have been isolated from the Dictyoceratid sponges. 61 This review and the results presented in this thesis suggest that, while not limited to the Dictyoceratid sponges, highly oxidized secosteroids are characteristic of sponges of this order.  103 The biological roles of highly oxidized steroids in marine invertebrates is not well understood. Cardellina and coworkers have suggested that these compounds are too polar to play an important function in maintaining the cell membrane. 71 They point out a similarity between functionalized steroids from poriferans and plant defense hormones such as cyasterone (104). Cyasterone is related to the growth hormones of the plants' insect predators and causes uncontrolled growth and deformation in those predators. 2 Cardellina et al. speculate that highly oxidized poriferan steroids may stimulate a similar effect in sponge predators such as crustaceans or dorid nudibranchs.  HO HO 104  71  36  INTRODUCTION TO SELECTED NMR EXPERIMENTS  Analyses of the results of various NMR experiments were used to elucidate the structures of the natural products described in this thesis. Since NMR is a complex and rapidly developing field, the focus of this section will be to give a simple (non-quantum mechanical) explanation of an NOE difference experiment and of each of the two-dimensional NMR experiments used to elucidate the structures of compounds discussed in the Results and Discussion section. This section will also demonstrate the application of each NMR technique to a simple model compound and show how the data is reported. Nearly half a century has passed since the first report of a nuclear magnetic resonance (NMR) signal which was at that time termed a "nuclear induction". 72 In the ensuing fifty years, NMR has developed from an interesting physical phenomenon to an analytical tool widely used in a variety of disciplines. NMR spectroscopy is currently one of the most powerful techniques available for the elucidation of natural product structures. With the development of increasingly sensitive instrumentation, NMR can be used to determine the structures of large, complex compounds with even very small quantities of material. A number of excellent references provide detailed explanations of the theory of NMR spectroscopy and of one-dimensional experiments.  73  A typical time course for a one dimensional NMR experiment is shown in Figure 6. The preparation period allows the magnetization of the sample to reach thermal equilibrium and ends with the application of the first radiofrequency pulse. During the evolution time (a constant time Di) additional pulses may be applied, the decoupler channel may be gated to remove scalar through-bond coupling interactions, or through-space dipolar couplings may take place. The resultant signal is acquired during the detection period 73c PULSE PREPARATION^EVOLUTION  Di  Figure 6. Time Course for a One-Dimensional NMR Experiment.  DETECTION  t2  37  THE 1 H HOMONUCLEAR NOE DIFFERENCE EXPERIMENT  The NOEDS (Nuclear Qverhauser Effect Difference .pectrum) experiment was used in this thesis to determine structure and assign relative stereochemistry to the natural products which were isolated. When a proton at chemical shift 5A is saturated by application of a selective radiofrequency pulse, a change in the intensity of signal 5X, known as a Nuclear Overhauser Effect (NOE), can occur. The appearance of a NOE is a consequence of through space dipolar coupling between nuclei A and X. Subtraction of a spectrum irradiated off-resonance (i.e. with no NOE) from the spectrum containing NOE enhanced signals results in one-dimensional difference spectra (NOEDS). The NOE difference spectra contain a strong negative signal at 5A, the chemical shift of the irradiated proton, and signals at 5X, the chemical shift of the NOE altered resonance. NOEs may be positive or negative and the intensity of the signal is inversely proportional to the sixth power of the through-space distance between the protons 5A and 5X. 73 c  Figure 7 shows the pulse sequence of an NOEDS experiment. 73 c The NOEDS experiments discussed in this thesis were recorded on a 400 MHz spectrometer. Typically the decoupler power used for the selective continuous irradiation was attenuated at 50 dB and a 6 s preparation delay allowed the system to come to complete thermal equilibrium between experiments. 90  t  H  tH DECOUPLER  SELECTIVE CONTINUOUS IRRADIATION  Figure 7. The Pulse Sequence for an NOEDS Experiment. 73 c  0  38  Figure 8 is the 1 H NMR spectrum of 4-methylcatechol (105). This commercially-available compound was chosen to illustrate the applications of the NOEDS and 2-D NMR experiments because of its simple structure and because it was used as a model compound for comparison with natural product 161, described later in the thesis. NOE difference spectra of 4-methylcatechol (105) are shown in Figure 9. The off-resonance spectrum is shown at the bottom of the figure. Spectra 9(a) and 9(b) correspond to irradiations of the resonances at 8 2.20 and 6.52, respectively. The first signal irradiated, the methyl singlet at 8 2.20, was well resolved from the remaining signals and the NOE difference spectrum 9(a) shows enhancement of the signals at 6.52 (H5) and 6.68 (H3). The data is not tabulated but is presented in the form of an illustration, as shown in Figure 10. In Figure 10, the tail of the arrow originates at the irradiated proton and the head of the arrow points to the proton which shows NOE enhancement. Spectrum 9(b) shows irradiation of the signal at 8 6.52. It can be seen from the resultant NOE difference spectrum 9(b) that the NOEDS experiment is limited when the region to be irradiated is congested. The results are not reliable as all three protons in the aromatic region (6.5-6.8) show a negative signal in spectrum 9(b). This indicates that the irradiating pulse was not sufficiently selective to differentiate the aromatic protons.  OH 105  Figure 10. Result of NOE Difference Experiment on 4-Methylcatechol (105).  OH  Mel  ^H6 H3 115  OH OH  MO^7.71^7.as^7.M^7..00^•..70^I.  ^1.ss  ppm^i^e^ii^4^3^i^i  Figure 8.^111 NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 AlMe2S0 at 500 MHz)  40 7  OH OH b  ^115  l's"444#44140woMe.,•••,•fteNseve a  ^  Mel  7.0^6 . 0^5 . 0^4. 0^3. 0^2.0^1 . 0 PPM  Figure 9.  NOE Difference Spectra of 4-Methylcatechol (105) (Recorded in CEC13 + 5 p.1 Me2S0 at 400 MHz)  41  TWO DIMENSIONAL NMR EXPERIMENTS  Two dimensional NMR pulse sequences share a common time course, illustrated by Figure 11.  74  As in  the 1-D experiment (Figure 6), the preparation period allows the sample to come to thermal equilibrium and ends with the application of the first radiofrequency pulse. The evolution time, ti , is incremented by a constant value it over the course of the 2-D experiment which results in modulation of the signals in the ti domain. The mixing period consists of pulses and fixed delays and the signal is observed during the detection period as a function of t2. During the t2 acquisition period, the chemical shifts of the observed nuclei are recorded. Fourier transformation provides 1-D spectra of the observed nucleus along the F2 axis as shown in Figure 12(a). By varying the delays and pulses introduced into the pulse sequence during the evolution period ti , the spectra along the F2 axis will be magnitude or phase modulated, encoding information relating to some chosen parameter, for instance, scalar coupling to homo- or heteronuclei or dipolar coupling to another nucleus in the vicinity. A second Fourier transformation of the modulated signals along the F2 axis generates frequency domain spectra in the Fl direction which contain cross signals in the 2-D spectrum at (SA, SX) where SA and SX are the chemical shifts of two nuclei which are coupled to one another (Figure 12(b)). 72  PULSE PREPARATION^EVOLUTION  ti  Figure 11. Time Course for a Two-Dimensional NMR Experiment.  MIXING  DETECTION  t2  42  tt  • • 09 o  ° it/  •/  0^0 4.4 4.2 4. ^IS 3.11 p PPM  (a)  Ibl  FID Figure 12. Fourier Transformation of the FID of a 2-D Experiment with Respect to a) t2 and b) ti and t2.  THE COSY EXPERIMENT  A simple homonuclear COSY (COrrelated Spectroscopy) experiment was employed to obtain information about the proton connectivities of the natural products studied. The COSY experiment is a 2-D technique which provides comparable information to a series of 1-D homonuclear decoupling experiments. The advantage this technique has over its 1-D equivalent is that all scalar couplings can be mapped in a short time span whereas in a complex molecule the 1-D technique may require numerous irradiations to determine all couplings of interest. Furthermore, COSY spectra show all connectivity networks, even for molecules with congested 1-D 1 H NMR spectra while 1-D homonuclear decoupling relies on selective irradiation of a chosen proton which may be impossible if the resonance is not well resolved. 75 Figure 13 shows the pulse sequence for a simple COSY experiment. 73 b The first 90° pulse induces magnetization in the sample proton spins. These then precess at their Larmour frequency, D o , during the evolution period ti. The second 60° pulse transfers the coherence (or polarization) of the protons which have evolved in ti to protons to which they are scalar coupled. This mechanism has been explained by quantum mechanics and also by vector product formalism. It is not trivial, however, and a detailed explanation is outside the scope of this thesis.  43 The proton spectra which result from Fourier transformation of FIDs (Free Induction Decay) acquired during the detection period are consequently modulated by scalar coupling. A second Fourier transform produces off-diagonal correlations between scalar-coupled protons. Where vi = v2 (i.e. correlation of a proton to itself) the signal appears on the diagonal.76  Figure 13. The Pulse Sequence for a Simple COSY Experiment. 73 b  Figure 14(a) shows the entire COSY spectrum of 4-methylcatechol (105) while Figure 14(b) is an expansion of the aromatic region. Figure 14(b) shows correlations from the doublet at 8 6.74 (H6) into a doublet of doublets at 6.52 (H5) which is further correlated to a doublet at 6.68 (H3). From the chemical shifts, it was evident that these were protons on an aromatic ring. The coupling constants were determined from the 1-D 1 H NMR spectrum. The coupling constant of the doublet at 8 6.74 (8 Hz) was consistent with coupling to a proton at the ortho position. From the COSY spectrum, we knew this was the doublet of doublets at 8 6.52 (H5). The resonance at 6 6.52 (H5) was coupled into a doublet at 6.68 (H3) with a splitting of 2 Hz, consistent with meta substitution. The COSY spectrum displaying the entire spectral width (Figure 14(a)) also showed weak coupling from the proton resonance at 8 6.68 (H3) and 6.52 (H5) to a singlet at 2.20 (Me7) which integrated for three protons. These weak correlations are typical of long-range coupling and they indicated that a methyl substituent separated the aromatic protons at 8 6.68 (H3) and 6.52 (H5). The broad singlet at 8 7.35 was attributed to the exchangeable phenol protons. From the information provided by the COSY spectrum it was possible to construct a fragment with all proton connectivities defined, such as that shown in Figure 15. The data is summarized in Table 1. The first column in Table 1 lists the carbon number. The second column contains the chemical shifts of protons attached to the carbons listed in column 1. The proton resonances which show COSY correlations to the resonances listed in the second column are found in column 3.  H6113 H5  Mel 116 113 H5 OH  OH OH  ^1 t.^  1.0  2.0  ••  3.0  H5/116  4.0  0  5.0  6.0  H5/Me7 113/Me7  7.0  0  PPM  7 0^6 0^5 0^4.0^3 0^2 0^i 0 PPM  8.0  I  V i,^• I  I  'VI  I  7.0  (al Figure 14. COSY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 511.1 Me2SO at 400 MHz)  TV w  I 1 l  45 2.20 CH3  7  6.68^6.52  H^H  6.74 H  \  Figure 15. The Proton Connectivities of 4-Methylcatechol (105) from the COSY Spectrum. Table 1. 1 H NMR and COSY Spectroscopic Data for 4-Methylcatechol (105). Carbon no.  51H NMR (ppm) (recorded in CDC13 +  COSY (recorded in CDC13 + 5 RI,  5 pL Me2S0 at 400 MHz)  Me2SO at 400 MHz)  1  —  2  —  —  3  6.68, d, J = 2 Hz  H5, Me7  4  —  —  5  6.52, dd, J = 2, 8 Hz  H3, H6, Me7  6  6.74, d, J = 8 Hz  H5  7  2.20, s  H3, H5  -OH  7.35, br s  —  —  THE HMOC EXPERIMENT  For the compounds reported in this thesis, the HMQC (ifeteronuclear Multiple Quantum Coherence) experiment was used to correlate proton chemical shifts to the shifts of their attached carbons. Figure 16 illustrates the pulse sequence used for the HMQC experiment. 77 Again the mechanism of the experiment involves quantum mechanics and is beyond the scope of this thesis. Typical values of the delays 0 and T used for this experiment were 3.5 x 10 -3 s and 0.7 s, respectively. After Fourier transformation of the FID with respect to t2, the resultant 2-D spectrum has proton spectra, accumulated during the acquisition period t2, along the F2 axis. The proton magnetization is modulated by scalar one-bond coupling to 13 C during the evolution period, ti . Consequently, following a second Fourier transformation with respect to ti, the 2-D spectrum contains signals which correspond to correlations from the proton chemical shifts (F2 axis) to the chemical shifts of attached carbons (F1 axis).  46  90°,^180 ° .^90°_.^90°,^180%  DETEC'ITON t  H  T  180 °  13  A  ^  1/2t1  ^  1/2t1^A  90 °^90°^90°x  C  DECOUPLE -41111—BIRD —3■0- i  Figure 16. The Pulse Sequence for an HMQC Experiment. 77  Visually the HMQC spectrum resembles and provides the same information as a HETCOR experiment (HETeronuclear CORrelation experiment). The advantage of an HMQC, however, is that the 1 H FID is detected whereas the HETCOR experiment detects the 13 C RD. The greater abundance of 1 H versus 13 C and its higher sensitivity in the NMR experiment means that the HMQC experiment is significantly more sensitive than the HETCOR experiment. 74  Figure 17 shows the 13 C NMR spectrum of 4-methylcatechol (105). In order to assign the protonated carbons, an HMQC spectrum was employed (Figure 18). Figure 18(a) shows the entire HMQC spectrum. An intense correlation was evident from the upfield methyl singlet at 5 2.20 (Me7) to the carbon resonance at 5 20.5 (C7). The aromatic region was somewhat congested and so an expansion was required, shown by Figure 18(b). In the expanded HMQC spectrum it could be seen that the H6 resonance at 5 6.74 was correlated to the carbon resonance at 5 115.1, the H3 doublet at 6.68 showed a correlation to a carbon signal at 116.1, and a correlation was also evident from the H5 resonance at 6.52 to a carbon resonance at 120.4. The data is reported in tabulated form as shown in Table 2.  7  OH OH  S  S  *40  too  Figure 17.^13C NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 g1Me2S0 at 125 MHz)  7 Mel OH OH H6 1-13  H5  ce)  U  —IRO  P"  [al  Figure 18. ^HMQC Spectrum of 4-  PPI  1  6.9^6.7  Methylcatechol (105) (Recorded in CDC13 + 5 pi Me2SO at 500 MHz)  6.5  49  Table 2. Assignment of Protonated Carbons of 4-Methylcatechol (105) from the HMQC Spectrum. 81H NMR (ppm) (recorded in CDC13 +  813C NMR signals assigned from  5 ilL Me2S0 at 400 MHz)  HMQC correlations (ppm) (recorded in CDC13 + 51.1L Me2SO at 125 MHz)  1  —  —  2  —  —  3  6.68, d, J = 2 Hz  116.1  4  —  5  6.52, dd, J = 2, 8 Hz  120.4  6  6.74,d,J= 8 Hz  115.1  7  2.20, s  20.5  -OH  7.35, br s  Carbon no.  —  THE HMBC EXPERIMENT  The HMBC (Ideteronuclear Multiple Bond Connectivity) experiment is a variation of the HMQC experiment which has a filter for one-bond correlations and has been optimized for two and three bond 1 H- 13 C correlations (theoretically, other nuclei may be substituted for 13 C). 78 This experiment is equivalent in terms of the information it provides to the 13 C-detected COLOC (COrrelation to LOng Range coupling) technique but, again, the HMBC is a proton-detected experiment and consequently is considerably more sensitive than COLOC. The HMBC is a very powerful experiment for establishing connectivity because the correlations can span heteroatoms and quaternary carbons. 79 By use of HMBC, proton spin systems which are separated by heteroatoms or quaternary carbons may be assembled to give the molecular structure. The chemical shifts of quaternary carbons may also be assigned with greater confidence. The HMBC pulse sequence is illustrated in Figure 19. 78 The BIRD pulse sequence used for the HMQC experiment is omitted here. The first 90° proton pulse is applied and time Ai later, a 90 0 13 C pulse follows. This sequence acts as a low-pass J filter so that the final spectrum does not show one-bond correlations. 78 For HMBC spectra discussed in this thesis, Al was set to 3.5 x 10 -3 s and 02 was 5 x 10 -2 s. The 2-D spectrum which results  50 from the Fourier transformation has proton chemical shifts along the F2 axis and 13 C chemical shifts along the F1 axis. Correlations are from protons to carbons two and three bonds distant. 900x^  1H  DETECTION A l^A2^1 /2 t,  90 °  13  C  1800„  90°  1 /2t,  90° x  A2  ..■■■••■■.  Figure 19. Pulse Sequence for an HMBC experiment. 78 Figure 20 shows the HMBC spectrum for the model compound 105. Assignment of the protonated carbons was possible from the HMQC spectrum but the quaternary carbons remained unassigned. Figure 21 shows some selected HMBC correlations which facilitated assignment of the quaternary carbons. 2.20  Figure 21. Selected HMBC Correlations for 4 -Methylcatechol (105).  Figure 20(a) shows the entire HMBC spectrum. At the level of intensity required to see correlations from the aromatic protons, the methyl singlet at 8 2.20 has a noisy line of correlations running along the F1 axis. Not all of the signals which make up the line correlate to carbon signals. This is known as ti noise. It generally  7  OH OH  Mel  H6 H3 H5 Alm_________  ••  •• •0  • .^ .  CQ)  C5  44D  C4 —  .^ . ^ J,... .  se 6  4^  lal  tts 0  ^i^ MU^2.2  120  C4 —  130 cl ^ C2  2  PM  ^  0  -  ^  Figure 20.  C6 C3 C5  -100  f  .  I•  I  C6 -50^C3  ^  0  120  e•^-130  i:  ^  t  6.8^6.4 Ibl^  HMBC Spectrum of 4 Methylcatechol (105) (Recorded in CDC13 + 5 µ1 Me2SO at 500 MHz) -  tIlD 46' )  Ic)  -140  52 occurs along the Fl axis and is associated with intense peaks in the F2 spectrum. It is caused primarily by technical factors such as fluctuations in pulses, temperature, and magnetic field. 74 Figure 20(b) shows an expansion of the correlations to the Mel (see also Figure 21). The intensity level has been lowered to eliminate most of the noise. The methyl singlet at 6 2.20 showed correlations into carbon resonances at 8 116.1 (C3), 120.4 (C5), and 129.6 (C4). The first two carbons were known to be C3 and C5, respectively, from the HMQC spectrum. The final carbon, at 6 129.6, was thus assigned to C4 by process of elimination. Correlations were also observed from the H3 (8 6.68) and H5 (6.52) resonances into the methyl resonance at 5 20.5 (C7). Figure 20(c) is an expansion of the aromatic region of the HMBC spectrum. The proton resonances at 5 6.68 and 6.74 showed correlations into both phenolic carbon signals at 8 141.9 and 144.0. This indicated that the phenolic carbons are side by side and that the protons which resonate at 8 6.68 and 6.74 are each attached to a carbon adjacent to a phenolic carbon. The proton resonance at 8 6.52 (H5) showed a correlation into the carbon resonance at 6 141.9 but not the signal at 144.0. This allowed assignment of the carbon resonance at 8141.9 as Cl since a three bond HMBC correlation from H5 to Cl is likely but a four bond correlation is not. The remaining phenolic carbon, at 5 144.0 can therefore be assigned as C2. The two and three bond correlations which appear in the HMBC spectrum of 4-methylcatechol (105) (Figure 20) are summarized in Table 3. The data contained in columns 1 and 2 is the same as in Table 1. The 13 C chemical shifts of the carbons in the first column are contained in column 3. The fourth column lists carbon resonances which show HMBC correlations to the proton resonance in the 5 1 H column.  Table 3. 1 H, 13 C, and HMBC NMR Data for 4-Methylcatechol (105). Carbon no.  81H NMR (ppm) (recorded 613C NMR (ppm) HMBC Correlationsa (500 in CDC13 + 5 ill- Me2S 0 (recorded in CDC13 + 5 'IL MHz) at 400 MHz)  Me2SO at 100 MHz)  1  -  141.9  2  -  144.0  -  3  6.68, d, J = 2 Hz  116.1  CI, C2, C5, C7  4  129.6  -  -  5  6.52, dd, J = 2, 8 Hz  120.4  Cl, C3, C7  6  6.74, d, J = 8 Hz  115.1  Cl, C2, C4  7  2.20, s  20.5  C3, C4, C5  -OH  7.35  -  -  a The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 H column.  53  THE ROESY EXPERIMENT  Rotating Frame averhauser Enhancement .pectroscopy, originally named the The ROESY experiment (_ CAMELSPIN experiment) provides information comparable to NOE difference experiments.  80  It is closely related  to the NOESY experiment but is the technique of choice for molecules with molecular weights of less than 1000 g/mol. Figure 22 illustrates the pulse sequence used to record a ROESY spectrum.  80  A 90 YU 0 pulse is applied  and the system evolves as a function of scalar coupling during the evolution period ti . Subsequently a radiofrequency field, known as the spin-lock pulse, is applied for a mixing time 'r m . A mixing time of 0.225 s was used for ROESY experiments discussed in this thesis. During the mixing time, the magnetization which evolved as a result of scalar coupling is "locked" and cross-relaxation, i.e. dipolar relaxation from one proton to a proton in the vicinity, takes place. The proton spectra detected during the acquisition period are therefore modulated by dipolar relaxation. The resultant 2-D spectrum resembles a COSY spectrum but an off-diagonal correlation at (SX, SA) corresponds to a through space nuclear Overhauser effect between the protons with chemical shifts SX and SA. Where SA = SX, the signal will appear on the diagonal but it will be antiphase relative to the cross peaks. 80,74  1 ^ H  90°  Figure 22. The Pulse Sequence for a ROESY Experiment. 80  The ROESY experiment has advantages over NOE difference experiments that are similar to the advantages of COSY over 1-D decoupling experiments. Again, much more information can be acquired in a shorter period of time and the ROESY experiment can be used to observe correlations in congested areas where selective irradiation  54 would be difficult. A limitation of this experiment is that signals close to the diagonal may be difficult to distinguish.  Figure 23 is the ROESY spectrum of 4-methylcatechol (105). Only positive signals have been plotted, therefore, the diagonal, which is antiphase, does not appear on the spectrum. In Figure 23 correlations were observed from the methyl resonance at S 2.20 (Me7) to the aromatic proton resonances at 6.52 and 6.68. These correlations were in good agreement with the results of the NOE difference experiments (Figures 9 and 10). The aromatic region of Figure 23 is noisy and provides no information as the aromatic proton resonances are not sufficiently well resolved. As a result correlations are too close to the antiphase diagonal and can not be distinguished reliably. Figure 24 presents the data and these results are the same as those illustrated in Figure 10 (NOEDS experiment results).  OH 105  Figure 24. Results of a ROESY Experiment on 4-Methylcatechol (105).  55 7  OH OH H6 H3 H5  Mel  41 111U.■..  Figure 23. ROESY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 tilMe2S0 at 500 MHz)  56 A. CYTOTOXIC METABOLITES OF APLYSILLA GLACIALIS RESULTS AND DISCUSSION  Andersen and Tischler studied the secondary metabolites of the marine sponge Aplysilla glacialis and reported the isolation of six new diterpenes. 28 d36 The research described in this thesis was prompted by routine screening of A. glacialis silica gel column fractions for biological activity. A polar fraction with 1 H NMR signals which did not correspond to those of any of the previously isolated diterpenes proved to be cytotoxic. Further separation of the polar fraction led to the isolation of glaciasterols A (100) and B (101), members of a new family of cytotoxic 9,11-secosteroids. Specimens of A. glacialis were collected in Barkley Sound and Sydney Inlet, British Columbia. The freshly-collected sponge was extracted sequentially with methanol and methanol/dichloromethane (1:1). The extracts were combined, concentrated, and partitioned between ethyl acetate and brine. Chromatography of the ethyl acetate extract yielded glaciasterol A (100) and an inseparable mixture of related steroids. A 1 H NMR spectrum of this crude mixture contained no strong singlets at —6 2 indicating that the parent glaciasterols did not contain acetate functionalities. Acetylation of the steroidal mixture made it possible to purify one of the major products, the diacetate 107 of glaciasterol B (101). The structures of glaciasterols A (100) and B (101) were deduced by analysis of the spectroscopic data of glaciasterol A, the diacetates 106 and 107 of glaciasterols A and B, respectively, and from the results of chemical interconversions.  100 R =^, R'. H 106 R=  , R .= Ac  101 R =^, R'. H 107 R= I^I , R'= Ac  57 Glaciasterol A (100)  25 HO^21 22 18 ^26 17 23 12 19 0  HO  Glaciasterol A was isolated as an optically active, amorphous, white powder. The EIHRMS spectrum showed a molecular ion at  m/z 416.2936  Da corresponding to a molecular formula of C26H4004 (AM +0.9 mmu).  Table 4 provides a summary of the NMR data acquired for compound 100. The upfield region of the 1 H NMR spectrum (Figure 25) of glaciasterol A (100) indicated that the molecule contained five methyl groups. The methyl signals included; two sets of doublets, one at 8 0.94 which integrated for six protons and one at 1.02 which corresponded to three protons, and two methyl singlets at 8 0.68 and 1.24 which integrated for three protons each. The observed pattern of methyl resonances was typical of a steroidal compound where the methyl doublets correspond to Me21, Me26, and Me27 of the side chain and the singlets to Me18 and Me19. In the case of glaciasterol A (100), the methyl doublets corresponded to Me21, Me25, and Me26 of the side chain a and the singlets were due to Me18 and Me19 of the steroid nucleus. The connectivity of the side chain a was determined from the COSY spectrum (Figure 26). A correlation was observed from the Me25 and Me26 doublet into a methine at 8 2.22. This methine (H24) showed a correlation into an olefinic proton at 8 5.29 (H23) which was coupled in turn to a proton at 5.27 (H22). A double resonance experiment indicated that the proton at 8 5.27 (H22) was further coupled to a signal at 2.13 (H20) (Figure 27), which gave a COSY correlation to 1.02 (Me21). A double resonance experiment in which H2O (8 2.13) and H24 (2.22) were simultaneously irradiated (possible because the signals are not well resolved in the 1 H NMR spectrum in CDC13) resulted in simplification of the multiplet at 5.28 to a pair of doublets corresponding to 1122 and H23 (Figure 27). The H22/H23 coupling constant was 15.3 Hz, appropriate for the E stereochemistry at the side chain double bond. 81 HMQC correlations permitted assignment of several of the side chain carbon resonances. The Me25 and Me26 doublet showed correlations into carbon signals at 8 22.6 and 22.7 ppm and the Me21 doublet was correlated to 21.3 ppm. The olefinic hydrogen resonances at 8 5.27 (H22) and 5.29 (1423) were correlated to carbon resonances at 8 136.0 and 132.2 ppm respectively.  58 0.94 CH3  5.27 2.13 H 1.02^H CH 3  132. 136.0^CH3  H 2.22 5.29  0.94  a  Table 4. 1 H and 13 C NMR Data for Glaciasterol A (100) (Recorded in CDC13) Carbon no.  81H (ppm) (400 MHz)  COSY (400 MHz)  1 1' 2 2' 3 4 4' 5 6 7 8 9 10  1.8 2.1 1.68 2.13 3.99, m 1.59 2.19 3.39, d, J = 4.6 Hz 6.80, dd, J = 4.6, 0.9 Hz -  H1', H2, H2' HI, H2, H2' HI, H1', H2', H3 H1, H1', H2, H3 H2, H2', H4, H4' H3, H4' H3, H4 H7 H6 -  3.69, m 3.81, m 1.15 1.63  H11', H12, H12' H11, H12, H12' H11, H11', H12' H11, H11', H12  3.39, ddd, J = 11.3, 8.1, 0.9 Hz 1.58 1.66 -  H15, H15'  11  11' 12 12' 13 14  813C (ppm) (125 MHz) 26.6a 30Aa 68.4 37.4 63.5 53.5 139.3 134.2 201.8 45.6 59.1 40.4 46.1 43.9  H14, H15' 15 26.9a H14, H15 15' 16 29.8a 49.6 17 17.8 18 0.68, s 21.5 1.24, s 19 H21, H22 2.13 38.5 20 H2O 21.3 21 1.02, d, J = 6.8 Hz H20, H23 136.0 5.27, dd, J = 15.3, 7.6 Hz 22 H22, H24 132.2 5.29, dd, J = 15.3, 6.1 Hz 23 H23, H25, H26 24 2.22, m 31.0 H24 0.94, d, J = 6.7 Hz 25 22.6b H24 0.94, d, J = 6.7 Hz 26 22.7b a,b May be interchanged. C Spectrum of secosteroid 97 was recorded in pyridine-d5.  8 13 C for 97 (ppm) (100 mHz)c,67 32.8 31.7 69.8 34.3 49.8 68.8 151.0 133.0 205.1 45.3 204.0 51.0 46.4 43.5 26.7 26.4 52.0 -  -  -  -  ^  HO 11 21 18 12 19 0  22  ^25  Me26 Me25  23^26  Me19 w  Me18  HO O  Me21  H14 H6  H23^ H22  H12' H1'^H4 HI-1 244'1111220'^HH215  H7  H12  H11' H3^H11  6. 5  ^ I  6. 0^5 . 5^5.0^4.5^4.0^3.5^3 . 0^2.5 PPM Figure 25.^Ill NMR Spectrum of Glaciasterol A (100) (Recorded in CDCI3 at 400 MHz)  ^  I^' " I  2.0  ,  1.5^1.0  60  HO  s "ft  l.1  CS H20/Me21,  -^4.5  H3/H4 gab 12  1111 H12/H12'  2.0  Me25/H24 Me26/H24  H3/H4'  2.5  3.0  ...., H14/H15 -g- H14/H15' Ao 11/H 12' H11/H121_H H11'/H12^'V H11 '/H12' - ea H3/H2' H3/1-12  •  3.5  - 4.5  _^s. 0  H22/H20  H22/H23^  H24/H23  5  .5  6  •  4).  ,..  H14/H7 • • I • • • • 1 • • .'• 1••••1•••• , •• ••■■■••T • ••^•^ 1^• • I " " 1" "^ 3.5^3.0^2.5^2.0^1.5^1.1^.5 7.0^6.5^6.^5.5^5.0^6. 5^4.  FIT  Figure 26.^COSY Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)  • ••  ppm  7.  0  HO  5.0  5.5  Ir^  V-^V^V^  V-  7.0^6.0^5.0^4.0^3.0^2.0  Figure 27.  1.0^O.()  Double Resonance NMR Experiment on Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)  o.  - •^•^  1.--L^  slab  41141464..1.^*LA 1 11 NI I" I MI Ai .11AULLA^  Figure 28.^13 C NMR Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 125 MHz)  HO H7  1-1H222  H11' H14 H3 H11 7 6 •  • •El  r  C6 50 C14 . C11  •  1-  •  • 0^0  C3  Qa  •• -  • •  •  0  4  0102  •PI  •  C18 c21C 19 C25 C26  •  100  0 —40  4, PPP  a 4  C23 C22 -C7  pp.  6^5^4^3  'I^  PPM  I  Figure 29.^HMQC Spectrum of Glaciasterol A (100) (Recorded in CDCI3 at 500 MHz)  POP  64  A peak in the mass spectrum at m/z 301.1814 Da (5% intensity, C19H2503) which corresponded to the molecular ion with loss of one equivalent of water and the side chain (C7H13) supported the assigned constitution a of the side chain.  All twenty-six carbons were visible in the 13 C NMR spectrum of glaciasterol A (100) (Figure 28). A resonance at 5 201.8 indicated that the molecule contained an a,13-unsaturated ketone which was corroborated by the presence of a carbonyl stretch at 1681 cm -1 in the IR spectrum. 13 C NMR resonances at 6 139.3, 136.0, 134.2 and 132.2 ppm indicated that glaciasterol A had two double bonds. The HMQC spectrum established that one double bond was disubstituted (that belonging to the side chain as previously shown) and the other was trisubstituted (Figure 29). By process of elimination, the trisubstituted double bond must be a to the ketone responsible for the 13 C NMR resonance at 5 201.8 (C9). The molecular formula C26H4004 required seven sites of unsaturation. The  carbonyl and two double bonds left four sites of unsaturation unaccounted for, thus it was concluded that the molecule must be tetracyclic. Acetylation of glaciasterol A gave a single product, 106, which was determined to be a diacetate by analysis of 1 H NMR (Figure 30) and mass spectrometric data. This indicated that the native compound, glaciasterol A (100), had two hydroxyl functionalities. One oxygen remained unassigned. Since it did not belong to a hydroxyl function, and the IR contained only one carbonyl stretch, it was concluded that one of the rings must be a cyclic ether. This was confirmed by the 13 C NMR spectrum which contained signals at 5 53.5, 59.1, 63.5, and 68.4 (Figure 28), chemical shifts which are typical of carbons attached via single-bonds to oxygen functionalities. From the formation of the diacetate 106, it was clear that two of these carbon resonances must be attached to hydroxyls in the parent compound 100. The IR spectrum of glaciasterol A diacetate (106) contained no hydroxyl stretching bands at 3300-3500 cm -1 indicating that glaciasterol A (100) did not contain more than two hydroxyls. Consequently, the two remaining carbons between 5 50 and 60 ppm were both necessarily attached to the remaining oxygen, forming an ether linkage. As a normal steroid skeleton does not contain a cyclic ether moiety, 37 this provided the first suggestion that the compound might be a secosteroid, with only three of the standard carbocyclic steroid rings.  65 The COSY spectrum of 100 (Figure 26) indicated the presence of a number of spin systems in addition to the side chain. A pair of broad multiplets at 5 3.81 and 3.69 were correlated into one another. The HMQC spectrum (Figure 29) showed that both protons were correlated to a single carbon at 5 59.1 (C11). The chemical shifts of the carbon and its attached protons indicated the methylene was attached to a hydroxyl function. Acetylation of 100 yielded the diacetate 106 with a corresponding downfield shift of the HI1 and HI1' resonances from 6 3.69 and 3.81 to 4.16 and 4.10, respectively. This result confirmed that C11 bore a hydroxyl terminus. The COSY spectrum of 100 showed correlations from 5 3.69 and 3.81 (H11, H11') to a pair of geminal methylene proton resonances at  1.15 and 1.63 (H12, H12') indicating that the molecule contained the hydroxyethyl fragment b. A lack of further correlations suggested that fragment b was an isolated spin system.  1.63^1.15 H^H HO^  59.1  H^H 3.81^3.69 b  Good agreement between the 1 H and 13 C chemical shifts assigned to fragment b of glaciasterol A (100) and the hydroxyethyl fragment of herbasterol (81), a 9,11-secosteroid reported by Capon and Faulkner, 63 indicated that glaciasterol A (100) might also be a 9,11-secosteroid.  81  66  COSY (Figure 31), HMQC (Figure 33) and HMBC (Figure 34) data for glaciasterol A diacetate (106) were used to establish the structure of glaciasterol A (100). Glaciasterol A diacetate (106) was a white solid which gave a molecular ion in the mass spectrum at m/z 500.3139 Da, appropriate for a molecular formula of C30H4406 (AM +0.1 mmu). As mentioned earlier, the IR spectrum of 106 showed that the 0—H stretch observed at 3359 cm -1 in the spectrum of 100 was no longer apparent but that it had been replaced by an acetate carbonyl stretch at 1737 cm -1 which was observed in addition to a carbonyl stretch at 1685 cm -1 . The latter frequency was attributed to the a,(3-unsaturated ketone also present in the parent compound 100. The UV spectrum of diacetate 106 had a A max at 254 nm which was in good agreement with the UV spectrum of model compound 108 (, max at 242 nm) 82 once an increment of 10 nm for the additional a-alkyl substituent had been added.  OH 108  Table 5 summarizes the NMR data for glaciasterol A diacetate (106). The significant changes in the 1 H NMR spectrum (Figure 30) of 106 relative to that of 100 were downfield shifts of the H3, H11 and H11'  resonances and the appearance of two acetate methyl resonances at 8 2.01 and 2.04 in the spectrum of 106.  67 Table 5. 1 H and 13 C NMR Data for Glaciasterol A Diacetate (106) (recorded in CDC13)  Carbon no.  8 1 H (ppm) (400 MHz)  COSY (400 MHz)  613C (ppm) (125 HMBCa (500 MHz) MHz) 1 1.77 H1', H2, H2' 27.6 — 1' 2.10 H1, H2, H2' — 2 H1, H1', H2', H3 1.70 26.6 — 2.17 H1, H1', H2, H3 2' — — H2, H2', H4, H4' 3 4.99, m 70.7 — 1.65 H3, H4' 34.0 C3 4eu 4' ax 2.26, t, J = 12.5 Hz H3, H4 — — — 5 63.0 3.37, d, J = 4.5 Hz 6 H7 53.4 C7, C8 7 6.76, d, J = 4.5 Hz H6 138.6 C5, C6, C9, C14 — — 8 141.2 — 9 — 200.0 10 — 45.3 11 4.10, m H11', H12, H12' 61.1 11' 4.16, m H11, H12, H12' — — 12 1.26 H11, H11', H12' — 36.7 12' 1.64 H11, H11', H12 — — 13 — 46.1 14 H15, H15' 3.23, dd, J = 10.7, 7.5 Hz 43.8 C7,C8,C13,C15,C18 15 1.57 H14, H15' 27.4 — H14, H15 15' 1.71 — — 16 — 25.1 — 17 1.74 H2O 50.4 — 18 0.72, s — 17.6 C12,C13,C14,C17 1.22, s 19 21.1 C1,C5,C9,C10 2.17 20 H21, H22 38.2 21 1.03, d, J = 6.8 Hz H2O 21.5 — 22 5.27, dd, J = 15.3, 6.2 Hz H21, 1123 — 136.2 23 5.28, dd, J = 15.3, 6.2 Hz 1122, H24 131.9 — 2.21 24 H23, H25, H26 31.0 — 25 0.95, d, J = 6.8 Hz H24 22.6 — 26 0.95, d, J = 6.8 Hz H24 22.7 — 0Ac 2.01, s; 2.04, s 21.2; 21.2; 170.1; 171.0 a The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 H column. —  —  —  —  —  —  ^  Me26 Me25  OCOCII3 O COCE3 ^Ac0  22 21^ 18^ 23^26 12  11  Me19  19 0  AcO  O  w  H23 H22  }16^ H24 H4'1420^H12' H2' H1'  H7  H11' H11^H14  1^I^r 1  6.5  6.0  IT^1-1-1-11  T  1  T  T  1  5 . 5^5.0^4 . 5  4 . 0^3.5^3.0^2.5 PPM  2.0^1 . 5^1 . 0  Figure 30.^1 H NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400 MHz)  69 Me19 Me18 Me26 Me25  Ne2  —^1 . 0  t  H3/H2  _^2. 0  H20/Me21  ,H3/HT  es  Me25/H24 Me26/H24  _^3. 0  H14/H15'  era  H14/H15  4. 0  H11/1112: H11/H12 H11/H11'^  H11'/1112' H11'/H12  _^5 . 0  Ira^VI  H3/H4' H3/H4  p  ect  H24/H23  _^6.0 H6/H7  t  6.0  5. 0^4. 0^3. 0^2. 0^I. 0 PPM - •^  • • •^1^  I^  PPM  Figure 31.^COSY Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400 MHz)  AcO  I  200  1  I  r  ,  1  ^  ^  I  ^ ^ 1 I  ^  180  160  Figure 32.  13 C NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 100 MHz)  140  120  PPM  100  80  60  40  ^  20  71 22  Ac0 11 21 18 12  26 Me26 Me19 Me25 Me21 Me18  19 0  AcO H23 H22 H7  HO H3^Hll'Hll^H14  08 2 0C21C19 C 25 C26 :C1  414:  •  %  •  •  to.  410  Oks  •  •  •  — 40  C14 gi  O  - C6  •  — 80 ▪ C11  •  - C3  —80  —  1 00  • •• —120 •  ^O ^ppa  ^  ^  6  •  - C23 -C22 LC7 -11111-1111-1.71-17-1-TIT  T/  11111- T1111 -1rivrtfri Timi  5^4^9^2^1  ppm  Figure 33.^HMQC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 500 MHz)  Ac0 11 21 18 12  Me19  22  Me18  19 o  Ac0  11•• 00%^  ■^  ili,  45*• 4="1"-... Me18/C12 MO COO -^Me18/ z Mel 8111I C14 Me19/C101[1:  •  Me19/C1:51..s.'  17  •  Me19/C5e  • •  Me18/C13  Cl  C12 C14 - C17  C13 C5  -100  OD  AID  -150  ppe^5.9^ ^8  I  5. ^  5.7  ppm  •  2.0  •  •  Me19/C9 1.5  Figure 34.^HMBC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDCI3 at 500 MHz)  C9  "VP  73 A resonance in the 13 C NMR spectrum (Figure 32) at 8 200.0 in addition to the IR band at 1685 cm -1 indicated that 106 contained an a,13-unsaturated ketone. From the HMQC data it was established that the carbon resonances at 8 136.2 and 131.9 formed the side chain double bond and this permitted the assignment of the two remaining olefinic carbon resonances at 141.2 and 138.6 to the double bond a to the ketone. From the HMQC spectrum (Figure 33) it could be seen that the more shielded olefinic carbon (8 138.6) had one attached proton (86.76 , H7) while the downfield carbon (8 141.2) was fully substituted. The olefinic proton resonance at 8 6.76 (H7) had a COSY correlation to a doublet at 3.37 (H6). The H6,H7 coupling constant of 4.5 Hz was appropriate for vicinal coupling which established that the protonated olefinic carbon (C7, 8 141.2) must be 0 to the ketone. The HMQC spectrum indicated that the proton at 8 3.37 (H6) was bound to a carbon at 8 53.4, presumably one of two carbons attached by an ether linkage. This data resulted in the elucidation of fragment c .  ()  200.0 '777.4 12124^  1  141.2  53.4 138.6  sr,  H C^6.76  112 (13.0"'0^H 2  3.37  c Examination of the basic steroid skeleton showed that no cleavage point other than the 9,11-carbon bond would allow construction of a molecule containing an isolated acetoxyethyl fragment and an a,f3-unsaturated ketone where the double bond is trisubstituted and bears a proton at the  p position. The cleavage of the 9,11 carbon bond  indicated that the position of the carbonyl functionality was likely to be C9. The multiplet at 8 3.99 in the 1 H NMR spectrum of 100 was identified as the methine adjacent to the remaining unassigned hydroxyl from its shift to 4.99 upon acetylation of 100 to the diacetate 106. The COSY spectrum of glaciasterol A diacetate (106) (Figure 31) showed correlations from the multiplet at 8 4.99 (H3) to two pairs of geminal methylene protons at 1.70 and 2.17 (H2, H2') and at 1.65 and 2.26 (H4, H4'). The methylene protons at 6 1.70 and 2.17 (H2, H2') were further correlated to another geminal methylene pair at 1.77 and 2.10 ppm (HI, H1'). Consequently it was possible to assemble fragment d which could only form the A ring of 106.  74  HH  2.26 1.65  d  Me19 was assigned from a correlation in the HMBC spectrum of 106 from the methyl singlet at 6 1.22 to the carbonyl resonance at 6 200.0 (C9) (Figure 34, Figure 35). Another HMBC correlation was observed from the Me19 resonance (6 1.22) to a 13 C resonance at 6 27.6 (C1) which had been assigned to the terminal methylene furthest from the acetoxy functionality of fragment d from the HMQC data. The HMBC correlation from the Me19 resonance into this carbon permitted its assignment as Cl which indicated that the acetoxy functionality was situated at the standard C3 position. An HMBC correlation from the Me19 proton resonance (6 1.22) into a 13 C resonance at 5 63.0 (C5) provided evidence that glaciasterol A diacetate (106) was a 5,6 epoxide. HMBC correlations from the H7 resonance (6 6.76) to the carbon signals at 6 63.0 (C5) and 53.4 (C6) further substantiated the location of a 5,6-epoxide moiety adjacent to the (4-unsaturated ketone.  AcO  Figure 35. HMBC Correlations from H6, Me18, and Me19 of Glaciasterol A Diacetate (106).  75 The final clearly resolved downfield resonance in the 1 H NMR of 106 (Figure 30) was a doublet of doublets at 8 3.23. This was tentatively assigned as H14 of the D ring. The H14 multiplet showed COSY correlations to a pair of methylene protons at 6 1.57 and 1.71 ppm (H15, H15'). Further correlations were obscured by the methylene envelope. An HMBC correlation from the H7 resonance at 6 6.76 to a 13 C resonance at 8 43.9 (C14, HMQC correlation to 3.23) established the C8/C14 linkage. The carbon resonances for the D rings of 100 and 106 were assigned from the HMQC spectra (Figures 29 and 33, respectively) and by comparison with the values for a closely related secosteroid (97) 67 (see Table 4).  HO  H  OH 97  The Me18 proton resonance (8 0.72) had an HMBC correlation to a 13 C resonance at 6 36.7 (C12) which conclusively situated the acetoxyethyl fragment. A correlation was also observed from the Me18 resonance to the 13 C resonance at 8 43.8 (C14) which further supported the assignment of H14. Finally, correlations from the 1 H NMR resonance at 8 0.72 (Me18) to 13 C resonances at 8 46.1 and 50.4 permitted the assignment of these carbon  7 respectively. shifts as C13 and Cu,  Figure 36 shows the results of an NOEDS experiment on 106 in which the Me19 signal at 6 1.22 was irradiated. NOE enhancement was observed in the proton resonances at 6 1.70 (H2 ax ) and 2.26 (H4' ax ). The observed NOEs from Me19 to H2 ax and H4 ax , in conjunction with the trans diaxial coupling of H4 ax with H3 83 (6 2.26, t, J = 12.5 Hz), indicated that the A ring was in a chair conformation and the C3 acetoxy functionality had the R configuration. Irradiation of the H3 methine (8 4.99) resulted in NOE enhancements of the proton signals at 2.17 (H2' eq) and 1.65 (H4 eq).  76 ,..----  je  1.70 /..---.., 1.22 H CH3 2.26 H  H H 1.65  j. ---- 4.99  =  •••^•■■ O.  6'  Figure 36. Results of Selected NOE Difference Experiments on Glaciasterol A Diacetate (106). The amounts of the glaciasterol A (100) and its diacetate (106) which were isolated were too small to permit determination of the relative stereochemistry of chiral centres with the exception of C3. The more ubiquitous glaciasterol B diacetate (107) and several chemical derivatives were studied to obtain additional information about the stereochemistry of the glaciasterols. Glaciasterol B Diacetate (107)  Ac0^21^22^24^26 23^25 16^27 15  AcO  Glaciasterol B diacetate (107) was isolated from a mixture of acetylated secosteroids and recrystallized from aqueous methanol to yield white needles. The melting point of the crystals was found to be 55-57°C. Glaciasterol B diacetate (107) gave a molecular ion in the EIHRMS at m/z 516.3451 Da, appropriate for a molecular formula of C31 H4806 (AM 0.0 mmu). The IR spectrum of 107 was similar to that of 106 with carbonyl stretches at 1735 cm -1 and 1684 cm -1 . The UV spectrum recorded in methanol had a Xmax at 258 nm.  77 From inspection of the NMR data summarized in Table 6 it was evident that glaciasterol B diacetate (107) had the same nucleus as glaciasterol A diacetate (106) and differed only in the constitution of the side chain. The molecular formula C31H4806 indicated that glaciasterol B diacetate (107) had one more methylene and one less degree of unsaturation than glaciasterol A diacetate (106). The 1 H NMR spectrum of 107 (Figure 37) lacked the multiplet at 6 5.28 (H22 and H23) which was apparent in the 1 H NMR spectrum of 106 and which resulted from the olefmic protons in the side chain of 106. The chemical shifts of the methyl doublets in the 1 H NMR spectrum of 107 (Figure 37) at 8 0.97 (3H) and 0.86 (6H) agreed with those expected for a cholesterol side chain. Analysis of the 13 C NMR (Figure 38), HMQC, and COSY data for 107 permitted the assignment of the side chain carbon resonances. Comparison of the side chain 13 C NMR shifts of 107 with those of secosteroid 97 indicated that both compounds possessed the same saturated C8H17 side chain (see Table 6). 68 A peak in the EIHRMS of 107 at m/z 283.1729 Da (22.9% intensity) corresponding to the molecular ion with loss of two equivalents of acetic acid  and the C8H17 side chain corroborated the assignment of the side chain structure. The resonances in the 1 H and 13 C NMR spectra of 107 were assigned by use of the COSY and HMQC data and by comparison with the values  for glaciasterol A diacetate (106) (Table 6).  HO 97 Figure 39 shows the results of NOE difference experiments carried out on glaciasterol B diacetate (107). Irradiation of the Me19 resonance at 8 1.23 ppm caused enhancement in multiplets at 1.72, attributed to the H213 resonance, and at 2.24, assigned as the H413 signal. The NOEDS results indicated that, as in the case of glaciasterol A diacetate (106), the A ring of glaciasterol B diacetate (107) was in a chair conformation and the 3-acetoxy substituent had the 13 configuration as shown in Figure 39. Irradiation of the H14 resonance at 8 3.23 induced NOE enhancements in the H11 multiplet at 4.10 and the H15a resonance at 1.68. From the NOE data it was concluded that H14 and Me18 existed in the trans relationship common to most steroids. C17 and C20 were assumed to have the standard steroidal configuration but the stereochemistry was not verified by experiment.  78  Table 6. 1 H and 13C NMR Data for Glaciasterol B Diacetate (107) (Recorded in CDC13) Carbon no.  51H (ppm) (400 MHz)  COSY (400 MHz)  813C (ppm) (75 MHz)  813C NMR shifts for 97 (ppm) (100 mH z)68,c  1 1' 2 2' 3 4eq 4' ax 5 6 7 8 9 10 11 11' 12 12' 13 14 15 15' 16 17 18 19 20 21 22 23 24 25 26 27 OAc  1.77 2.10 1.72 2.17 4.99, m 1.64 2.24, dd, J = 12.5, 12.6 Hz 3.36, d, J = 4.5 Hz 6.75, d, J = 4.5 Hz -  H1', H2, H2' H1, H2, H2' HI, H1', H2', H3 HI, H1', H2, H3 H2, H2', H4, H4' H3, H4' H3, H4 H7 H6  27.6a  -  26.6a 70.6 33.9  -  -  -  4.10 4.19 1.23 1.69  H11', H12, H12' H11, H12, 1112' H11, H11', H12' H11, H11', H12  3.23, dd, J = 10.8, 7.5 Hz 1.58 1.68 1.83 0.73, s 1.23, s 1.44 0.97, d, J = 6.7 Hz -  H15, H15' H14, H15' H14, H15 1120 H17, H21 H2O H26, H27 1125 1125 -  -  63.0 53.4 138.6 141.3 200.0 45.3 61.1  -  -  -  36.6 46.0 43.5 25.9a  -  -  -  -  -  27Aa 49.8 17.2 21.1b 34.8 18.9 35.4 24.5 39.4 1.54 28.0 22.6 0.86, d, J = 6.6 Hz 0.86, d, J = 6.6 Hz 22.8 2.00, s; 2.04, s 21.1b, 21.3b, 170.1, 170.1 a,b May be interchanged .c 13C NMR spectrum of 97 was recorded in pyridine-d5.  -  -  -  -  35.3 19.4 35.7 24.5 39.6 28.2 22.7 22.9 -  2) 22 24^26 18• ,  27  OCOCH3 OCOCE3  Me19 Me18 Me27 Me26  S  w  H7  1-16 H3  I •^  Me21  '  7.5^7.0^6.5^6.0^5.5^5.0  Hll'Hll^1 H14  -^" -^1 4.5^4.0^3.5^3:0^2.5^2.0^1.5^t.0^0.5^00 ,,  ^  ppm  Figure 37.^1 111s1MR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 400 MHz)  ITIOITIIIIIII^IIII^  200^160  11 1 11^11111 1 111111^1 1111 1111 1 11 11 11 1 11  0^140^1 0^100  IIIIIIiiiIIII^Itiii r 'tilt 1 lilt,' 60^40^20  ppm  Figure 38.^13 C NMR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 100 MHz)  n  81  AcO 1.23 ^CH3  1.72 H  4.19  H  H2.24 3.23  AcO  H .68  Figure 39. Selected NOE Difference Experiments on Glaciasterol B Diacetate (107) (Recorded in CDC13 at 400 MHz).  While NOE difference experiments were successful in determining some aspects of the stereochemistry of the A and D rings of the glaciasterols, it remained unclear whether the 5,6-epoxide moiety was a or 13 to the B ring. It had previously been demonstrated that reaction of a related compound, 109, with 60% aqueous perchloric acid in  THE proceeded via SN2 attack at the less hindered secondary, rather than the more sterically hindered tertiary, carbon to yield the trans diol 110 (see Equation 1). 84 Treatment of glaciasterol B diacetate (107) with aqueous perchloric acid under similar conditions was also found to result in ring-opening of the epoxide via SN2 attack to yield the corresponding trans diol (Equation 2).  HC104 aq . / THE  109  Equation 1. Ring-Opening of a 4a,5a-Epoxide with 60% Perchloric Acid in TI-1F to yield the 40,5a-Dio1. 84 In order to ascertain that ring-opening of glaciasterol B diacetate (107) had indeed proceeded via nucleophilic attack at the less hindered C6, the reaction was carried out using 69% perchloric acid in —1:1 H2160/H 2 180 and  82 THF. The signal for the carbon which undergoes nucleophilic attack by the labelled water should exhibit 160/180 isotope-induced splitting in the 13 C NMR spectrum of product 111. The diol 111 was eventually acetylated to yield the triacetylated ring-opened glaciasterol B derivative 112 (see Equation 2).  •  16  69% HC104 / -4:1 H2 0: 18 H2 0 /THF  WO AcO^ - .^ 107  AcO 111 R = -1:1  OH 16  R  0H  18  / 0H  Ac20 / pyridine AcO 112 R' = -1:1 16OAc / 18 OAc Equation 2. Ring-Opening of Glaciasterol B Diacetate (107).  The ring-opened derivative 111 gave molecular ions in the EIHRMS at m/z 534.356 Da appropriate for a molecular formula of C31HSO 16 07 (AM 0.3 mmu) and at m/z 536.3596 Da which corresponded to a molecular formula of C31 H SO 160 6 180 (Am -0.3 mmu). This result indicated that glaciasterol B diacetate 107 had incorporated one equivalent of -1:1 160/ 18 0-labelled water. The fragmentation pattern observed for the mass spectrum was consistent with this conclusion (see experimental section). The IR spectrum showed an 0-H stretch at 3432 cm -1 as well as carbonyl stretches at 1739 and 1677 cm -1 . The derivative 111 was completely characterized by NMR spectroscopy and the results are summarized in Table 7. The 1 H NMR resonances (Figure 40) were assigned from the COSY spectrum (Figure 41). The major changes from the spectrum of the parent compound were downfield shifts of the equatorial H4 resonance from 8 1.64 in the epoxide 107 to 1.85 in the diol 111 and of the H6 resonance from 3.36 in the parent compound 107 to 4.03 in the derivative 111. The resonance attributed to H7 in 107 (8 6.75) was observed to shift upfield to 6.42 in product 111.  83 Table 7. 1 H and 13 C NMR Data for 5a,613-Dihydroxyglaciasterol B Diacetate (111) (Recorded in CDC13) S1H (ppm) (400 MHz) COSY (400 MHz) Carbon no. 1 1' 2 1.65 H2', H3 H2, H3 2' 2.04 H2, H2', H4, H4' 5.11, m 3 4ea 1.85, dd, J = 12.8, 3.2 H3, H4' 4' ax 2.21, dd, J = 12.8, 11.9 H3, H4 5 H7 4.03, d, J = 5 Hz 6 7 6.42, d, J = 5 Hz H6 8 9 10 4.18, m H11', H12, H12' 11 11' 4.18, m H11, H12, H12' 12 1.30 H11, H11', H12' 12' 1.68 H11, H11', H12 13 14 3.29, dd, J = 11.0, 8.6 Hz H15, H15' H14, H15' 15 1.60 H14, H15 15' 1.70 16 17 18 0.76, s 1.38, s 19 1.46 H21 20 0.99, d, J = 6.7 Hz H2O 21 22 23 24 1.50 H26, H27 25 0.87, d, J = 6.6 Hz 26 H25 0.87, d, J = 6.6 Hz 27 H25 OAc 2.00, s; 2.04, s aSplit into doublet by 160/ 18 0 isotope shift. b May be interchanged.  813C (ppm) (125 MHz) 26.4 26.1 70.4 34.7 76.5 72.28, 72.30a 138.0 137.3 202.3 46.1 61.4 36.9 48.0 42.7 27.1 27.4 50.3 17.1 21.6 35.4 19.0 35.5 24.5 39.5 27.9 22.5b 22.8b 21.1; 21.4; 170.1; 170.3  OCOCL13 0 c0 d113 w 26  21 22 24 18 is  S  Me27 Me26  '  AcO 11 12  Me19  27  19  Me 18 Ac0  OH  OH  Me2  10,411%. PP•  7  4  1^2  Figure 40.^1 H NMR Spectrum of 5a,6P-Dihydroxyglaciasterol B Diacetate (111) (Recorded in CDC13 at 500 MHz)  AcO 11 12  85  26  21 22 24  27  19  AcO  OH  OCOCH3 OCOCH3  OH Me19 Me V Me26  H7  e18  Me21  ^  ^H11' H11H6 H14 H3  1 0  t .  -4  siti  eV  '  it-  ,.  3.0  el) *  2.0  it)  4  4  a  60  0  Ilk •  * ^ ^ ^ ^ ^ ^ 7. I 6 S. I 4. 3 t 2 0 PPM  o  70  ^PPM  Figure 41.^COSY Spectrum of 5a,613-Dihydroxyglwiasterol B Diacetate (111) (Recorded in CDC13 at 400 MHz)  AcO  ISO  ^  140  Figure 42.^13 C NMR Spectrum of 5agl-Dihydroxyglaciasterol B Diacetate (111) (Recorded in C6D6 at 125 MHz) with Expanded Regions a) Recorded in C6D6 and b) Recorded in  cDa3  la]^  2) 22 24^26 18 '  bl  11  AcO 11  27  19  H3 AcO^  •  a 'H11H6 OH^H11^ OH  141111!* • .I•es °NI"  •  C14 Ida^  •  ^NM*^  •  is^.410  •  50  C11 C3 C6  100  •  II^WTI  •^• •  ^  pl)al^6^4^2 Figure 43.^a) HMQC Spectrum of 5a,60-Dihydroxyglaciasterol B Diacetate (111) with b) Expanded Region (Recorded in CDC13 at 500 MHz)  88  The 13 C NMR resonances seen in Figure 42 were assigned from the HMQC spectrum and by comparison with the assignments for the parent compound 107. A correlation was observed in the HMQC spectrum from the H6 resonance at 5 4.03 to a 13 C resonance at 6 72.30 (Figure 43). The quaternary carbon resonance at 6 76.5 ppm was assigned to C5. The 13 C NMR spectrum clearly showed an 160/ 18 0 isotope-induced splitting of the signal at 6 72.28/72.30 (C6) of 2.9 Hz indicating that the ring-opening had taken place via nucleophilic attack at C6 (Figure 42). The 13 C NMR resonance at 6 76.5 ppm (C5) was a sharp singlet as were all of the other resonances in the 13 C NMR spectrum (Figure 42).  Having established that the acid-catalyzed ring opening of the glaciasterol 5,6-epoxide proceeded via attack at carbon 6, it was necessary to determine whether the product, 111, was the 5a,613-diol or the 513,6a-diol. Earlier studies of the model compound 110 82,84 had demonstrated that reaction of 110 with acetic anhydride resulted in acetylation of only the secondary hydroxyl while the tertiary alcohol functionality remained underivatized (Equation 3).  .^  lOO OH OH  acetic anhydride pyridine  110  Equation 3. The Acetylation Reaction of Model Compound 110 to produce the Corresponding Monoacetate.  Consequently, the ring-opened diacetate 111 was treated with acetic anhydride in pyridine to yield one major product, the expected triacetate 112 (Equation 4). The glaciasterol B derivative 112 gave a molecular ion at m/z 576.3670 (C33HSO 16 08, AM +0.8 mmu). The IR spectrum showed stretching frequencies at 3448, 1739 and 1639 cm" 1 .  ^ 89  • 0=0 AcO  16  69% HC104 / —1:1 H2 0: 18 H2 0 /THF  Ac0^= .  AcO  OH 16  R  18  111 R = —1:1 0H / 0H  107  Ac20 / pyridine AcO  OH  R'  16  18  112 R' = —1:1 OAc / OAc Equation 4. Ring-Opening of Glaciasterol B Diacetate (107) to form 111 followed by Acetylation to yield 112  It has been shown by Demarco and coworkers 85 that protons which are situated in the vicinity of a hydroxyl are shifted downfield in pyridine-d5 relative to deuterochloroform. Demarco et al. speculated that pyridine complexes with the hydroxyl via hydrogen-bonding. Protons located nearby are deshielded by the anisotropy of the pyridine aromatic ring. The 1 H NMR data for 5a-hydroxyglaciasterol B triacetate (112) recorded in CDC13 and in pyridine-d5 is summarized in Table 8 and shown in Figure 44. The proton resonances were assigned from COSY spectra (Figure 45) and the correlations are tabulated in Table 8. It can be seen from Figure 46 that H3 (8 5.10 in CDC13; 5.62 in pyridine-d5) was shifted downfield by 00.52 ppm relative to a downfield shift of only 00.19 ppm for Me19 (1.36 in CDC13; 1.55 in pyridine-d5). Since an NOE difference experiment on the parent compound glaciasterol B diacetate (107) had shown that H3 had the a configuration (Figure 39), the much greater downfield shift of a proton on the a face (H3) compared to protons on the p face (Me19) of the steroid indicated that the 3a-methine had a closer proximity to the 5-hydroxyl than did the n-oriented Me19 moiety. This evidence established that the 5-hydroxyl functionality had the a configuration. The results indicated that the ring-opened product 111 was the 5a,6(3-dihydroxyl derivative of glaciasterol B diacetate and that the 5,6-epoxide moiety of the parent compound, glaciasterol B diacetate (107), had the a configuration.  90 Table 8. 1 H NMR Data for 5a-Hydroxyglaciasterol B Triacetate (112). Carbon no.  81H NMR recorded in CDC13 (ppm) (400 MHz)  1 1.73 1' 1.76 2 2.05 2' 2.13 3 5.10, m 4 1.83 4' 1.95 — 5 6 5.19, d, J = 5 Hz 7 6.36, d, J = 5 Hz 8 — 9 10 4.14, m 11 4.18, m 11' 12 1.25 12' 1.66 13 3.30, dd, J = 11.3, 8.9 Hz 14 15 15' 16 17 — 18 0.70, s 19 1.36, s 1.45 20 21 0.98, d, J = 6.8 Hz 22 — — 23 24 1.53 25 0.84, d, J = 6.7 Hz 26 0.84, d, J = 6.7 Hz 27 OAc^_ 2.01, s; 2.04, s; 2.13, s —  —  81H NMR recorded in pyridine-d5 (ppm) (400 MHz) 1.93 2.60, dd, J = 17.7, 12.8 Hz 1.73 2.06 5.62, m 1.14 2.25, br d — 5.85, d, J= 5 Hz 6.62, d, J= 5 Hz  COSY recorded in pyridined5 (ppm) (400 MHz) Hi', H2, H2' H1, 1-12, H2' H1, H1',112' H1, H1', H2 H2, H2', H4, H4' H3, H4' H3, H4 — H7 H6  —  —  —  —  — H11', H12, H12' H11, H12, H12' H11, H11', H12' H11, H11',1112 — H15, H15' H14, 1115' H14, H15 —  4.47, m 4.47, m 1.47 1.83 3.53, m 1.47 1.58 — 0.80, s 1.55, s 1.35 1.03, d, J= 6.6 Hz —  —  —  —  H21 H2O —  — —  —  1.50, m 0.88, d, J= 6.6 Hz 0.88, d, J= 6.6 Hz 1.98, s; 2.02, s; 2.14, s  1126, 1127 H25 1125 —  91  lal  AcO 11  12  21 22 24 26 18 .'1 27  19  AcO  OH  OAc  H3  6.0^5.0  4.0  3.0  2.0  1.0  ppm  6.0  ^  5.0  ^  4.0  ^  3.0  ^  2.0  ^  1.0  ppm Figure 44. 1 11 NMR Spectra of 5a-Hydroxyglaciasterol B Triacetate (112) Recorded in a) CDC13 and b) Pyridine-d5 at 400 MHz  92  AcO 11  21 22 24 26 18 '''  12  27  19  AcO  OH  OAc  •  6  2  ss  -3 H14/H15'  ao  H14/H15  0  dga  H11/H12' H11/H12 5  •  H3/H2'  a^  0^•  H3/114'^113/}12  o^  O  -6  H6/H7  •  s:s  Figure 45  ^  PPII  3  1  "PM  COSY Spectrum of 5a-Hydroxyglaciasterol B Triacetate (112) (Recorded in Pyridine-d5 at 400 MHz)  93  AcO 00.19 ppm CH3 OAc 0 AcO H  A0.52 ppm  Figure 46. The 1 H NMR Shifts of 112 in Pyridine-d5 Relative to CDC13.  5a.613-Dihydroxyglaciasterol A Diacetate (113) and 5a-Hydroxyglaciasterol A Triacetate (1141 25 21^22^24 18^23  AcO  12 11  26  17  19  16 15  AcO OH OR 113 R = H 114 R = Ac The similarity of the 1 H and 13 C NMR data for glaciasterol A diacetate (106, Table 5) and glaciasterol B diacetate (107, Table 6) provided evidence for a common nucleus for these compounds. The configuration of the 5,6-epoxide of glaciasterol A was nevertheless confirmed by reaction with perchloric acid. Glaciasterol A diacetate (106) was subjected to perchloric acid under the same reaction conditions as shown in Equation 2 but with substitution of distilled water of standard isotopic composition for the —1:1 16 0/ 18 0 aliquot. Only a small portion  94 of the resulting diol was isolated and characterized by 1 H NMR and mass spectrometry. Derivative 113 gave a molecular ion in the EIHRMS at m/z 518.3242 which was appropriate for the molecular formula C30H4607 (AM -0.1 mmu). The crude 1 H NMR spectrum of 113 was analyzed by comparison with the spectrum of the analogous glaciasterol B derivative (111) and the data for 113 is contained in Table 9. Table 9. 1 H NMR Data for 5a,60-Dihydroxyglaciasterol A Diacetate (113) and 5a-Hydroxyglaciasterol A Triacetate (114). Carbon no.  1 2 2' 3 4 4' 5 6 7 8 9 10 11 11' 12 12' 13 14 15 15' 16 17 18 19 20 21 22 23 24 25 26 OAc  8 1 H NMR of Diacetate 113 recorded in CDC13 (ppm) (400 MHz) — — — 5.12, m — — — 4.04,d 6.40, d — — — 4.16, m 4.16, m — — — 3.28,m — — — 0.75, s 1.37, s 1.05,d 5.28, m 5.28, m — 0.95, d 0.95, d 2.02, s; 2.05, s  8 1 H NMR of Triacetate 114 8 1 H NMR of Triacetate 114 recorded in CDC13 (ppm) (400 recorded in pyridine-d5 (ppm) MHz) (400 MHz) — — 1.83 — 1.96 — 5.09, m 5.67, m 1.60 — 2.03 — 5.19, d, J = 5 Hz 5.91,d 6.34, d, J = 5 Hz 6.66, d — — — — — — 4.17, m 4.50, m 4.17, m 4.50, m 1.30 — 1.68 — — 3.29, dd, J = 10A, 8 Hz 3.54,m 1.57 — 1.70 — — 0.68, s 0.81, s 1.35, s 1.52, s 2.19 — 1.03, d, J = 6.8 Hz 1.09,d 5.28, m 5.33, m 5.28, m 5.33, m 2.21 — 0.95, d, J = 6.7 Hz 0.96, d 0.95, d, J = 6.7 Hz 0.96, d 2.01, s; 2.04, s; 2.13, s 2.00, s; 2.05, s; 2.15, s —  —  —  —  —  95 w  (al  Me19  Ac0  H3  6. 5  6.0  " • . I "  ^ 5.5^5.0^4.5^4.0^3.5^ ^ 3.0^2.5^2.0 1.5 PPM  1.0  I bl Me19  1  Figure 47.^1 H NMR Spectra of 5a-Hydroxyglaciasterol A Triacetate (114) Recorded in a) CDC13 and b) Pyridine-d5 at 400 MHz  96 5a-Hydroxyglaciasterol A triacetate (114) was formed by reacting intermediate 113 with acetic anhydride in pyridine. The product 114 gave a molecular ion in the EIHRMS at m/z 560.3340 Da, appropriate for a molecular formula of C32H4808 (AM -0.9 mmu). The IR spectrum of 114 showed a band at 3402 cm -1 , attributed to the hydroxyl, and carbonyl stretching frequencies at 1738 and 1688 cm -1 . The compound was fully characterized by NMR spectroscopy. Table 9 summarizes 1 H NMR shifts of 114 in CDC13, confirmed by the COSY data, and in pyridine-d5, determined by comparison of the spectrum with that of the corresponding glaciasterol B derivative 112 in pyridine-d5 (Figure 47). The H3a resonance was shifted t10.58 ppm downfield (5 5.09 in CDC13; 5.67 in pyridine-d5), significantly further than the Me19 (1.35 in CDC13; 1.52 in pyridine-d5) which was shifted downfield by A0.17 ppm in pyridine-d5 relative to CDC13. The results in Table 9 established that glaciasterol A (100) was also a 5a,6a-epoxy secosteroid.  CONCLUSIONS  Glaciasterols A (100 ) and B (101 ) are the first secosteroids reported to be cytotoxic and the first examples of this class of compound identified from a sponge in the genus Aplysilla. These compounds add to the number of secosteroids which have been isolated from marine invertebrates. To date these are the only secosteroids reported which possess an epoxide moiety adjacent to an a,(3-unsaturated ketone.  100 R=^  ,  101 R= JNAIV.  The biosynthesis of metabolites 100 and 101 most likely proceeds via oxidative cleavage of the 9,11-bond of a highly oxidized intermediate such as 103, a polyhydroxylated steroid which has been reported from the sponge Dysidea etheria. 7 °  97  The cytotoxicity of a crude fraction which contained the glaciasterols had first attracted our interest to compounds 100 and 101. Pure glaciasterol A (100), glaciasterol B diacetate (107), and 5a—hydroxyglaciasterol B triacetate (112) were tested against the in vitro L1210 murine leukemia, MCF-7 human solid tumor breast cancer, and MCF-7 Adr multidrug-resistant breast cancer cell lines. 86 The results of these assays are listed in Table 10. It is particularly interesting that glaciasterol A (100) and the acetylated compounds 107 and 112 were equally active against both the MCF-7 and the multidrug-resistant MCF-7 Adr cell lines. The multidrug resistance of the MCF-7 Adr cell line has been attributed to increased levels of a plasma membrane protein which appears to actively transport cytotoxic drugs out of the ce11. 86 Since steroids pass through plasma membranes by simple diffusion 87 it is possible compounds 100, 107, and 112 are retained in the drug-resistant cells. Acetylation of the glaciasterols would be expected to produce a more lipophilic derivative which would presumably be even better suited for transport across cell membranes. The fact that glaciasterol B diacetate (107) was approximately ten times as active against the drug-sensitive MCF-7 and the drug-resistant MCF-7 Adr breast cancer cell lines as the unacetylated native secosteroid 100 is consistent with this hypothesis.  Table 10. Results of the Assay of Glaciasterol A (100) and Related Derivatives 107 and 112 against In Vitro Murine Leukemia and Human Breast Cancer Cell Lines. ED50 against L1210  ID50 against MCF-7  ID50 against MCF-7 Adr Drug  Cell Line (gg/mL)  Cell Line (p.gimL)  Resistant Cell Line (pg/mL)  Glaciasterol A (100)  2.1  19  18  Glaciasterol B Diacetate (107)  2.5  1.8  1.8  5a-Hydroxyglaciasterol  5.66  2  4.5  COMPOUND  B Triacetate (112 )  98 B NATURAL PRODUCTS OF A PLERAPLYSILLA SP. SPONGE  RESULTS AND DISCUSSION  a) The 1990 and 1991 Pleraplysilla Sp. Sponge Collections  This study of the chemistry of a Pleraplysilla sp. sponge was initiated in 1990 upon receiving a small sample of a gray-white sponge which had been identified by Dr. W. C. Austin as a new species of Aplysilla on the basis of morphology alone. 29 The white sponge appeared to be an excellent candidate for the study of natural product chemistry on the basis that it might contain cytotoxic metabolites similar to those produced by Aplysilla glacialis. It was proposed that a study of the metabolites of the sponge would also determine whether it was a color  variant of Aplysilla glacialis or an entirely new Aplysilla species. The sponge was later reidentified as an undescribed Pleraplysilla species by Dr. R. W. M. van Soest on the basis of chemistry as well as morphology. 88 Small collections of the sponge were made at Checleset Bay and Botanical Beach in British Columbia in 1990. The following year, the sponge patch at Botanical Beach was harvested again. These initial collections were treated in a similar manner. Sponge tissue from the 1991 collection was soaked in methanol at +7°C for three to five weeks. The extract was filtered, concentrated, and partitioned between ethyl acetate and brine. The ethyl acetate extracts were pooled and concentrated to yield a brown oil (67.8 mg). Separation of the sponge metabolites was accomplished by sequential application of size-exclusion gel (Sephadex LH-20 eluted with ethyl acetate/methanol/water; 20:5:2) and silica gel (eluted with a polarity gradient of diethyl ether/dichloromethane/ethyl acetate) chromatographies. Final purification was accomplished by normal phase HPLC separation. Eluted in order of polarity were the known sesquiterpenes 0-methyl furodysinin lactone (57) 89 and 0-methyl nakafuran-8 lactone (44). 28 b The next set of silica column fractions contained the new sesquiterpenes 0-methyl 9-oxofurodysinin lactone (72) and 0-methyl 2-oxomicrocionin-2 lactone (74). The previously reported compounds furodysinin lactone (56) 49 and nakafuran-8 lactone (43) 28 b were the major components of the next fractions. The final fraction contained the novel metabolite 2-oxomicrocionin-2 lactone (73) as its major component. All of the compounds isolated were hydroxybutenolides or their corresponding methyl ketals. The possibility that these compounds were artifacts of autooxidation and reaction with the methanol extracting solvent was considered but examination of the 1 H NMR spectrum of the crude ethyl acetate sponge extract revealed no signals at —6 7.1 and —6.1, the expected chemical shifts of the protons of an  99 a,13-disubstituted furan. 28 b The chromatographic separation also provided no evidence for the presence of furans in the Pleraplysilla sp. sponge. OR  ^ 72 56 R . H 57 R = CH3  ^ 43 R = H ^ 73 R = H 44 R = CH3 74 R = CH3  0-methyl 9-Oxofurodysinin Lactone (72)  14 15  0-Methyl 9-oxofurodysinin lactone (72) was isolated as a colorless glass which gave a molecular ion in the EIHRMS at m/z 276.1371 Da, appropriate for a molecular formula of C16H2004 (AM 0.9 mmu). The molecular formula indicated that 72 had seven sites of unsaturation. Two carbonyl stretching bands were observed in the IR spectrum. The absorption band at 1767 cm -1 was at the appropriate frequency for a 8-lactone, 2813,89 while the second band at 1665 cm -1 indicated the molecule contained an a,(3-unsaturated ketone. A signal at 8 198.1 ppm in the 13 C NMR spectrum (Table 11) confirmed the presence of an unsaturated ketone while a resonance at 168.8 ppm, in conjunction with the IR stretch at 1767 cm -1 was consistent with a butenolide moiety. A 1 H NMR singlet at 6 3.20 (OCH3), which integrated for three protons, established the presence of a methyl ether (Figure 48). The  100 13 C NMR spectrum, shown in Figure 49, contained the corresponding signal for the methyl carbon at 8 50.6 ppm  (HMQC correlation). From Figure 49 it was evident that C4, which had a 13 C shift of 8 106.8 ppm, was the only other carbon single-bonded to an oxygen atom. The chemical shift of C4 (8 106.8) was appropriate for a ketal functionality. Since three oxygens had previously been assigned to the unsaturated ketone and the butenolide, these results suggested that the molecule contained the methoxybutenolide moiety a. 0 SS5^  168.8 0 06.8 ^ OCH 3  a  Table 11. 1 H and 13 C NMR Data for 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13) Carbon no.  61H (ppm) (400 MHz)  COSY (400 MHz)  613C (ppm) (125 MHz) 168.8 118.7 171.0 106.8 38.6  HMBCb  1 — — 2 5.92, s Cl, C4 — — 3 — 4 — 1.64, dd, J = 13.5, 13.6 Hz H5', H6 5 2.47, m 115, H6 — 5' H5, H5', H7 31.0 — 6 3.09, m 6.74, br d, J = 6.3 Hz H6, H13 146.5 7 C9 135.8 8 198.1 9 H10', H11 2.05, dd, J = 14.7, 16.8 Hz 35.0 10 C9 H10, H11 — 2.47, m 10' H10, H10' — 11 2.25, dt, J = 4.2, 4.2, 14.7 Hz 47.1 37.6 — 12 — H7 13 1.77, s 15.6 C8, C9 14 1.40, s — 25.0a C3, C11, C12 1.22, s 24.7a C3, C11, C12 — 15 OCH3 3.20, s 50.6 C4 a May be interchanged.b The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 H column. —  —  —  OCH3  Me15 Me 13 Me14  13  1^,^,^,^1^,^,^I^1^,^,^,^1^ 7 . 0^6 . 5^6. 0^5.5^5.0^4.5^4.0^ 3. 5^3.0^2.5^2.0^1.5^1.0rvi^, PPM ^Figure 48.^1 H NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 400 MHz)  0  1^II^  1,1^,,,t^  1110  ,Itt^1^64^u/ibl 140  100  Figure 49.^13C NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 125 MHz)  103  Jo.  I  8.8  '.."\. .  it  . •  1.1  -  .  ,_  6  6  t. 5  H11/Hle H11/H1O'S .. •A.. a H5/H5' '  s,  -  -  8  I f  •  : te  2.1  2.5  -  .H6/H5'  3. 8  OD 116/1-15  3.5  _^.  •  s  4.1  4.5  et  5.8  S  5.5  •  6.  0  6.5  H7/Me 13 •  H7/116 to  7.1  7.5  el  .^.  75^71^65^III^5.5^51  4.5  .^.^. - . . .  4I^3.5 • P6  .^.^.  3. 1  .^.^.^.  2.5  2.  1. s  1.8  .  5  .  S.8  .  6.3  Figure 50. COSY Spectrum of 0-Methyl 9-Ozofurodysinin Lactone (72) (Recorded in CDC13 at 400 MHz)  104  Me 15 Mel3Me14  OCEL3  C13 C14C15 C6  .1 If 0  C11  OC.H 3  '  so  BO  100  I. 140  C7 6  5  4  3  2  1  Figure 51.^HMQC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 500 MHz)  105  Me14 Me13 Me15  •• .  •  4r  . •  •.. • • • Me14/C15044 Me15/C14 .^• • • •^• Me14/C1 UPOMe15/C12 • o. 4, Me14/C11 Q qMe15/C11 •  • • •  •  •  5•  I  C14 C15 C12 C  . •• •  • I H2/C4  .^• .^: • • • 4 •^.  •  OCH3/C4  •  •  100  C4  .  •  • •  4  17 0  Cl C3  ••• .^. H107C9  .  C8  • • Me14/C341 Me15/C3  •  • • H7/C9  4  Me13/C74 • • •  .  4 H2/C1  P011^6  Me13/C8  H10/C9  Me 13/C9 • —^•  C9  2  Figure 52, HMBC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 500 MHz)  106  13 C NMR signals at 8 118.7, 135.8, 146.5, and 171.0 ppm confirmed that 72 had two double bonds each  conjugated to a carbonyl group. The remaining unsaturations were attributed to three rings. The COSY spectrum (Figure 50) indicated that a broad doublet at 8 6.74 (H7) was coupled to a multiplet at 3.09 (H6) which integrated for one proton. The signal at 8 6.74 (117) also showed a correlation to a broad methyl singlet at 1.77 (Me13). The deshielded chemical shift of the methyl resonance at 8 1.77 (Me13) in conjunction with the observed COSY correlation indicated that the methyl was allylic to the proton at 6.74 (H7). The spin system was further extended by a correlation from the multiplet at 8 3.09 (H6) to a pair of geminal methylene proton resonances at 1.64 (H5) and 2.47 (H5'). These results allowed the assembly of fragment b. ^ 6.74 1.64, 2.47 H^H H  b A second spin system could be elucidated from the COSY spectrum (Figure 50). A multiplet at 8 2.25 (H11) showed correlations into a pair of geminal methylene proton resonances at 2.05 (H10) and 2.47 (H10'). The chemical shift of the methylene pair (H10, H10') was deshielded, suggesting that it might be adjacent to the carbon at 8 198.1 (C9) or the olefinic carbon at 171.0 (C3). The 1 H NMR spectrum also contained two aliphatic methyl singlets at 8 1.22 (Me15) and 1.40 (Me14) and a deshielded olefinic singlet at 5.92 ppm (H2). The proton at 8 5.92 (H2) was tentatively assigned a to the carbonyl of the methoxybutenolide ring on account of its chemical shift. HMQC data permitted assignment of the chemical shifts of protonated carbons (see Table 11, Figure 51). Correlations in the HMBC spectrum (Figure 52) were useful in elucidating the structure of 72 (Table 11). Selected HMBC correlations are illustrated in Figure 53. Correlations from the 1-12 singlet at 8 5.92 into carbon resonances at 8 106.8 (C4) and 168.8 (C1) confirmed the constitution of the methoxybutenolide ring and the chemical shift of the Cl carbonyl. HMBC correlations from the methyl singlets at 8 1.22 (Me15) and 1.40 (Me14) to carbon signals at 8 171.0 ppm (C3) and 37.6 ppm (C12) located both methyls on one quaternary carbon (C12, 37.6) adjacent to the methoxy butenolide.  107  The connectivity of the A ring was also clarified by the HMBC data (Figure 52). Correlations from both the H7 resonance (8 6.74) and the Me13 resonance (1.77) into the carbonyl signal at 8 198.1 located spin system b next to the ketone at C9. The methylene resonances at 8 2.47 and 2.05 (H10, H10') were also correlated to the carbonyl resonance at 6 198.1 (C9) which placed the methylene (H10, H10') on the other side of the unsaturated ketone. As mentioned earlier, these proton resonances (H10, H10') were further coupled to a methine resonance at 8 2.25 (Hi 1) in the COSY spectrum. The HMQC spectrum indicated the methine at 8 2.25 (H11) was attached to a  carbon at 8 47.1 (Figure 51). The methyl singlets at 8 1.22 (Me15) and 1.40 (Me14) showed HMBC correlations to the C11 carbon resonance at 8 47.1 which established that the methoxybutenolide fragment a and the A ring were connected via a quaternary carbon with two methyl substituents.  6.74 H H H 1.77 H OCH3  H(` '  2.47 2.05 1.22  1.4  Figure 53. HMBC Spectroscopic Correlations for 0-Methyl 9-Oxofurodysinin Lactone (72).  Analysis of the spectroscopic data and comparison with the data for furodysinin (56) 49 allowed assignment of 72 as the structure of the metabolite. The only feature inconsistent with the proposed structure was the absence of a COSY correlation between the ring junction hydrogens [i.e. from H6 (8 3.09) to H11 (2.25)]. NOE difference experiments, illustrated in Figure 54, clarified the stereochemistry of 72.  108 6.74 \■.____w 3.09 2.25^ H 1.77 ^)1.40 CH3 / 1.22 CH3 /  Figure 54. Selected NOE Difference Experiments on 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 400 MHz).  As can be seen from Figure 54 an NOE enhancement was observed in signals at 8 2.25 (H11) and 3.09 (H6) upon irradiation of the methyl singlet at 1.40 (Me14). This result indicated that the A and B rings are cis fused. It should be noted that NOEs were also observed from H6 to H11 and vice versa but these were weak. A double resonance experiment in which the multiplet at 8 3.09 (H6) was irradiated did result in slight simplification of the multiplet at 2.25 (H11) indicating that some coupling did occur. Irradiation of the methyl singlet at 6 1.40 (Me14) also produced enhancement of the methoxy singlet at 3.20 permitting tentative assignment of the methoxy 13 relative to the plane of the molecule (Figure 54). A weak NOE enhancement which was observed from the methoxy singlet (8 3.20) to the Me14 (1.40) resonance substantiated the assignment of the methoxy stereochemistry. Upon irradiation of the proton singlet at 8 5.92 (H2) NOE enhancement was observed in the methyl resonance at 1.22 (Me15) and vice versa (Figure 54). This located the methoxy butenolide with the ketal function adjacent to C5 and with C3 attached to C12. The final structure suggested that the biosynthesis of compound 72 proceeded via an intermediate which more closely resembled furodysinin (36) than furodysin (35). H^ H  35  36  109  2-Oxomicrocionin-2 Lactone (73)  2-Oxomicrocionin-2 lactone (73) was isolated as a colorless oil. A molecular ion was evident in the EIHRMS at m/z 264.1317 Da, appropriate for a molecular formula of C15H2004 (AM -4.5 mmu). The IR spectrum indicated that the molecule contained an 41-unsaturated ketone with a stretching frequency of 1650 cm -1 and a 6-lactone which absorbed at 1758 cm -1 . A band at 3317 cm -1 in the IR spectrum suggested that the molecule contained a hydroxyl group. It was clear from the COSY spectrum that compound 73 was not a disubstituted butenolide with a carbon skeleton similar to furodysinin (Table 12). The 1 H NMR spectrum, shown in Figure 55, contained no isolated olefinic proton but rather a deshielded multiplet at 6 6.87 (H10) which displayed allylic coupling in the COSY spectrum (Figure 56) to another downfield resonance at 6.11 (H12). The proton resonance at 8 6.87 (H10) showed further allylic coupling to a pair of geminal methylene proton resonances at 2.03 (H8) and 2.33 (H8'). The downfield proton resonance at 8 6.11 (H12) showed a COSY correlation to an isolated resonance at 3.50 (OH). The HMQC spectrum (Figure 57) showed that the proton signal at 6 6.11 (H12) was correlated to a carbon resonance at 8 96.4, an appropriate chemical shift for a ketal. No HMQC correlation was observed for the 1 H NMR resonance at 6 3.50 (OH) indicating that it was bound to an oxygen, rather than a carbon atom. From these results it was determined that 73 contained the mono-substituted hydroxybutenolide fragment c.  H H  2.33^2.03  110  Table 12. 1H and 13C NMR Data for 2-Oxomicrocionin-2 Lactone (73) (Recorded in CDC13)  813C (ppm) HMBCa (125 MHz) 128.8 C3, C13 Me13 1 5.91, s 198.8 — — 2 41.8 C2 H3', H4 2.29, m 3 H3, H4 — — 2.37, m 3' — H3, 1-13', Me14 33.8 2.33, m 4 5 42.1 — — — — 167.3 6 33.9 H7', H8, H8' C8 1.81, m 7 — H7, H8, 118' — 1.81, m 7' 1410, H7, H7', H8' 20.5 C7 2.03, m 8 — H10, H7, 117', H8 2.33, m 8' 142 — — 9 143.1 C11, C12 H8, H8', H12 6.87, d, J = 1.0 Hz 10 — 170.9 — 11 —OH 96.4 6.11, br s 12 20.2 H1 Cl, C5, C6 1.98, s 13 15.4 H4 C3, C4, C5 1.00, d, J = 6.1 Hz 14 19.4 C4, C5, C6, C7 1.06, s 15 — — H12 3.50, br s OH H column. 1 a The carbon resonances in the HMBC column are correlated to proton resonances in the 8 Carbon no.  81H (ppm) (400 MHz)  COSY (400 MHz)  —  —  —  —  6  ^ ^ ^ 5 ^ ^ 4 3 2 1  PPm  Figure 55.^1 H NMR Spectrum of 2  -  0xomicrocionin 2 Lactone (73) (Recorded in CDC13 at 400 MHz) -  112  6.5  6.1  7.0 5.5^5.0  4.5^4.0^3.5.^3.1^2.5^2.0^1.5^1 . 1 PPM  Figure 56. COSY Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 400 MHz)  113  Me15  ..  II 1 NO  '  Ow  CI C I 3 C15 5  if  .  40  C3  VIII. I  C7  a  lit  00  I  00  I  $ C12  al I  —100  I I  120 Cl  4  NE  5  I 5  1 4  -^  3  2  1  140 do PP  Figure 57.^HMQC Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 500 MHz)  Figure 58.  ^  13 C Spectrum of 2 -Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 125 MHz)  Me15  HI  HIO,  v.  Cl  Imo H1/C13  C3 I0  d* HI/C3  .  ..  ...  ...... .. •■•^Me13/C5 ab  .  ..  a  .1.  Me15/C7 t Me15/C4 V111-1 Me14/C4 Mei5/c5 dime Me 14/C5  ...  •  C7 C4  CS as  10 am 1110/C12  Cl. 2 SOO  , ow  a.  .  r  ®  •  ..  •  Me13/C1 •14;;•  Cl  150  01  ga• H10/C11  .  CI  Me13/C6 49  -• •  .  .^.^.^•  ^5  ^Figure  .^.^.^.  0  ^ .^.^.^.  55  500 PP ^ .^.^.^.  mum. H3/C2  H3'/C2^...  _ Le  C6  Me15/C6'if  . —  ^  00• 1.5  59.^HMBC Spectrum of 2-0xomicrocionin-2 lactone (73) (Recorded in CDC13 at 500 MHz)  0  C2  116 The IR band at 1758 cm -1 was attributed to the lactone carbonyl. This value was in close agreement with the value recorded for the equivalent functionality in manoalide (3) (1765 cm -1 ). 10  3 Three more spin systems were evident from the COSY spectrum (Figure 56). The methylene pair at 5 2.03 (H8) and 2.33 (H8') showed COSY correlations to a multiplet at 1.81 which integrated for two protons. The signal at 5 1.81 was attributed to H7 and 117'. Fragment d, an isolated spin system evident from the COSY data, contained an olefinic proton resonance at 5 5.91 (H1) which showed allylic coupling to a broad methyl singlet at 1.98 (Me13). The final spin system is illustrated by fragment e and consisted of a methyl doublet at 5 1.00 (Me14) which gave a COSY correlation to a multiplet at 2.33 (H4). Further correlations were observed from the resonance at 5 2.33 (H4) to a pair of geminal methylene proton resonances at 2.29 (H3) and 2.37 (H3'). 2 .29 H 5 .9111 ■.../)11 S-53.^2.37 ^H 1.00 CH 3 CH3sS ^(1217S 1.98 2.33 d^ e .%  From the molecular formula C15H2004 it was determined that the molecule contained six sites of unsaturation. The unsaturated ketone and the hydroxybutenolide moiety together accounted for five of those unsaturations. The final site of unsaturation was attributed to a second ring. The allylic coupling of H10 to the H8 methylene pair allowed linking of the hydroxybutenolide fragment to the C8 methylene. The chemical shift of HI (5 5.91) led to its placement a to the ketone carbonyl. Likewise, the shifts of the methylene pair at 5 2.29 (H3) and 2.37 (H3') allowed the location of this moiety a to the ketone carbonyl. These assignments were corroborated by the HMQC spectrum (Figure 57) which had a correlation from the proton at 5 5.91 (H1) to a carbon at 8 128.8, appropriate for an olefinic carbon a to a ketone." Another HMQC correlation was observed from the methylene  117 pair at 8 2.29 (H3) and 2.37 (H3') to a carbon at 8 41.8 ppm, consistent with the expected shift for a methylene carbon adjacent to the ketone of a cyclohexanone. 90 The structure 73 was assembled on the basis of the fragments pieced together from the HMBC spectroscopic data. Figure 59 shows the HMBC spectrum of 73 and Figure 60 illustrates selected HMBC correlations.  5.91 70.9 H 6.87  0  Figure 60. Selected HMBC Correlations Observed for 2-Oxomicrocionin-2 Lactone (73). As can be seen from Figure 60, HMBC correlations were observed from the allylic methyl resonance at 8 1.98 (Me13), the methyl doublet at 1.00 (Me14), and the methyl singlet at 1.06 (Me15) into a single quaternary carbon resonance at 8 42.1 (C5). In conjunction with the COSY data, this result provided evidence for the six-membered ring. A correlation from the Me15 singlet (8 1.06) to a carbon resonance at 8 33.9 (C7) linked the cyclohexenone ring to the hydroxybutenolide by a fragment consisting of two contiguous methylene carbons. The location of the C2 ketone was confirmed by HMBC correlations from methylene proton resonances at 8 2.29 (H3) and 2.37 (H3') to a carbon resonance at 8 198.8 (C2). Further confirmation was provided by a correlation observed from the HI resonance (8 5.91) to the C3 resonance (8 41.8). HMBC correlations also substantiated the assignment of the hydroxybutenolide moiety as illustrated in Figure 60. The HMBC correlation confirmed the chemical shifts of the carbonyl carbons, since these were somewhat obscured by noise in the 13 C spectrum (Figure 58) and could not be assigned with confidence.  118  Figure 61. Results of Selected NOE Difference Experiments on 2-Oxomicrocionin-2 Lactone (73). The relative stereochemistry of 73 was determined by NOE difference experiments as shown in Figure 61. Upon irradiation of the methyl singlet at 8 1.06 (Me15) NOE enhancements were observed in signals at 1.98 (Me13), 2.33 (H4), and 1.81 (H7) . The enhancement at 6 1.98 (Me13) was predictable since the olefinic methyl should lie in the plane of the cyclohexane ring which was expected to be flattened by the unsaturated ketone moiety. The NOE observed in the H7 methylene resonance (8 1.81) was also not surprising but the enhancement of the multiplet at 2.33 (H4) suggested that this proton was on the same side of the cyclohexane ring as the methyl at 1.06 (Me15). Irradiating the Me14 doublet (8 1.00) produced an NOE in the H7 resonance at 1.81 which provided conclusive evidence for locating Me14 (1.00) on the same side of the six-membered ring as the side chain. Me15 (8 1.06) was consequently assigned to the same side of the cyclohexane ring as H4 (2.33). 0-Methyl 2-Oxomicrocionin-2 Lactone (74)  0-Methyl 2-oxomicrocionin-2 lactone (74) was isolated as a translucent white glass which gave a molecular ion in the EIHRMS at m/z 278.1508 Da, appropriate for the molecular formula C16H2204 (AM -1.0 mmu). The similarity of the 1 H NMR spectrum (Figure 62) to that of 2-oxomicrocionin-2 lactone (73)  119 suggested that the compounds were closely related. The only significant difference in the 1 H NMR spectra was the absence of the hydroxyl proton at 6 3.50 in the spectrum of 74 (Figure 62) and its replacement by a singlet at 3.58 which integrated for three protons (see Table 13). The infrared spectrum lacked the hydroxyl stretch at —3300 cm -1 but retained the two carbonyl bands at 1764 cm -1 (butenolide) and 1663 cm -1 (a,(3-unsaturated ketone). The COSY spectrum (Figure 63) indicated that the 1112 proton resonance was no longer coupled to a hydroxy proton resonance and that it had shifted upfield from 5 6.11 in compound 73 to 5.73 in 74. The data suggested that metabolite 74 was the methyl ether of 2-oxomicrocionin-2 lactone 73. The 13 C NMR (Figure 64), HMQC (Figure 65), and HMBC (Figure 66) data are summarized in Table 13. The data corroborated the assignment of the structure as 74.  Table 13. 1 H and 13 C NMR Data for 0-Methyl 2-Oxomicrocionin-2 Lactone (74) (Recorded in CDC13)  Carbon no.  61H (ppm) (400 MHz)  COSY (400 MHz)  1 2 3 3' 4 5 6 7 7' 8 8' 9 10  5.90, br s  Me13 — H3', H4 H3, H4 H3, H3', Me14 — — H7', H8, H8' H7, H8, H8' H10, H7, H7', H8' H7, H7', H8  11  2.26, m 2.35, m 2.30, m — — 1.77, m 1.83, m 2.03, m 2.31, m — 6.79, br s —  613C (ppm ) HMBCa (125 MHz) 128.8 C3, C13 198 — 41.9 — C2 — 33.6 — 42.1 167 — 33.9 — — — 20.2 — — — 129 142.0 C11, C12 —  —  H8, 1112 —  —  —  12 5.73, br s H10 102.5 HI 13 1.97, br s 20.5 Cl, C5, C6 114 15.4 14 0.99, d, J = 4.2 Hz C3, C5 15 — 19.4 1.06, s C4, C5, C6 OCH3 C12 3.58, s 57.2 a The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 H column.  S w  HIO HI H12  S  2  Figure 62.^  ^^  11-1 NMR Spectrum of 0-methyl 2 -Oxomicrocionin-2 Lactone (74) (Recorded in CDCI3 at 400 MHz)  1  ,  ppm  121  0  13^10 15^11 0 12 OCH3 14  •  0  H10/H12 6.5  ^  6.1^5.S  ^  T.  S.11^4.5  3.1  4.1^3.5^ PPM  Figure 63.^COSY Spectrum of O-methyl2-oxomicrocionin-2 lactone (74) (Recorded in CDC13 at 400 MHz)  OCH 3  pp soe^ieo^g^the^No^so  Figure 64.  N  o  N  ^13C NMR Spectrum of 0-methyl 2-ozomicrocionin-2 lactone MHz)  (74) (Recorded in CDC13 at 125 ^  k'  H10  ^  HI H12 i  0  I I  — 50  100  —  C12  00  Cl 0  C10  i ^i PP.^6^ 5 Figure 65.  r  PP  4 PP.  I  -C14 —20 Cl3C15 NI  ••• IP  MI  —40 WI  ID  •■ NM  I ^ I ^ I' 3^2^1  HMQC Spectrum of 0-methyl 2-oxomicrocionin-2 lactone (74) (Recorded in CDC13 at 500 MHz)  : OC_H 3 —60 PP  124  OCkL3  ^  Me15  Me13 Me14 H10^H1  1 Hl/C13  •  •  • 9^I •^C13  Me15/C4  •  •  • • Hl/C3 a  Melf4 • •^• C4 00 C3 CS /C55 ?m Meaeii54/c  •  • •  •  Mel /C5  so  •  •  •  0 A • OCE_3/C12  H10/C12  • • el  •  C12  • • Me13/C1 I  Cl  • •  Me15/C6 Me13/C6  ea  C6  H3/C2  5  3  Figure 66. HMBC Spectrum of 0-methyl 2-ozomicrocionin-2 lactone (74) (Recorded in CDC13 at 500 MHz)  125 Known Metabolites of the Pleraplysilla Sp. Sponge (1990/1991 Collections) The 1990 and 1991 collections of the Pleraplysilla species of sponge were processed individually. Both contained the same known compounds, furodysinin lactone (56) 49 , nakafuran-8 lactone (43), 28 b and their respective methoxybutenolide derivatives 57 89 and 44 28 b in sufficiently high concentrations that structure elucidation was feasible. In addition, the 1990 collection contained at least five metabolites which were isolated and identified as sesquiterpenes but which were present in too small concentrations to permit complete structure elucidation. These compounds did not appear to be present in the 1991 collection of the Pleraplysilla sponge.  OR  56 R = H 57 R = CH3  43 R = H 44 R = CH3  The structures of furodysinin lactone (56) and its corresponding methoxybutenolide (57) were determined by comparison of infrared, 1 H NMR and 13 C NMR and mass spectroscopic data to literature data (see experimental) 49,89 The structure elucidations of nakafuran-8 lactone (43) and its corresponding methyl ether 44 required complete characterization to distinguish the compounds from butenolides of nakafuran-9 (42).28b The HMBC spectrum clearly indicated that the compounds isolated were derivatives of nakafuran-8 (41). The structures were confirmed by comparison of 1 H NMR, 13 C NMR, infrared, and mass spectra with published values (see experimental) 28b  41  ^  42  126 b) The 1992 Pleraplysilla Sp. Sponge Collection  Although the crude methanolic extracts of the Pleraplysilla sp. sponge collected in 1990 and 1991 contained only hydroxybutenolides and their corresponding methyl ether derivatives, the possibility that these compounds might be autooxidation artifacts could not be ignored. Consequently a final collection of sponge harvested in 1992 was immediately frozen, freeze-dried, and extracted with ethyl acetate. The ethyl acetate extract was separated into its component metabolites by silica gel column chromatography eluted with a polarity gradient of hexane to dichloromethane to ethyl acetate. Final purification was achieved by normal phase HPLC. The only three sesquiterpenoid compounds isolated from the ethyl acetate extracts of the 1992 collection were furodysinin (36) 44 and nakafuran-8 (41) 28 b which was contaminated by a small concentration of a sesquiterpene tentatively identified as furodysin (35) 44 from the 1 H NMR spectrum. All three structures had been previously reported. 28 b ,44 Sesquiterpenes possessing the microcionin-2 carbon skeleton (see structures 73 and 74) were notably absent. As the most polar silica gel fraction (eluted with 100% ethyl acetate) had interesting signals in the 1 H NMR spectrum, the major compound was purified by normal phase HPLC.  By use of extensive NMR  spectroscopic analysis the structure of blancasterol (102), a novel, cytotoxic secosteroid, was elucidated. Reexamination of the 1 H NMR spectra of extracts of the earlier collections of the Pleraplysilla sponge indicated that metabolite 102 was present in those extracts as well but had escaped notice.  41  ^  HO 36  35  _,E OH OAc  102  127  Blancasterol (102)  HO  Blancasterol was isolated as an amorphous white solid which had an M+H peak in the FABHRMS at m/z 551.36006 Da corresponding to the molecular formula C31145108 (M+1, AM 3.01 mmu). The 1 H NMR  spectrum (Figure 67, Table 14) contained signals diagnostic of a steroid side chain. Specifically, there was a doublet at 6 1.00 which integrated for three protons (Me21) and a second doublet at 0.88 (Me26, Me27) which integrated for six protons. Interestingly, the molecule possessed only one aliphatic methyl singlet, at 5 0.74 (Me18). Analysis of the NMR data contained in Table 14 indicated that 102 possessed the same fully saturated side chain as glaciasterol B diacetate (107). The methyl doublet at 5 0.88 (Me26, Me27) showed COSY correlations (Figure 68) to a signal at 1.52 (H25) which was further correlated into geminal methylene resonances at 1.73 (H24) and 1.83 (H24'). The H24 and H24' resonances were correlated into one another but further correlations were obscured by the methylene envelope. The Me21 (8 1.00) resonance showed a COSY correlation into a resonance at 1.45 (H20) and further correlations into a methylene pair at 1.83 (H22) and 1.67 (H22') were discernible. 13 C NMR resonances (Figure 69) were assigned to the side chain by analysis of the HMQC (Figure 70) and HMBC spectra (Figure 71) and by comparison with the appropriate values for glaciasterol B diacetate (107) (see Table 6).  128  Table 14. 1 H and 13C NMR Data for Blancasterol (102) (Recorded in CDC13) Carbon no.  511 1 (ppm) (400 MHz)  COSY (400 MHz)  1 1'  2.26, dt, J = 14.8 Hz 1.88, ddd, J = 14.8, 13.2, 3.3 Hz 2.09, m 1.62, m 4.01, m 4.98, d, J = 9.5 Hz — 2.56, dd, J = 19.8, 2.4 Hz 2.35, dd, J = 19.8, 5.8 Hz 6.44, dd, J = 5.8, 2.4 Hz — —  H1', H2, H2' H1, H2, 112'  2 2' 3 4 5 6 6' 7 8 9 10 11  -  H1, H1', H2', H3 H1, H1', H2, H3 H2, H2', H4 H3 — H6', H7 H6, H7 H6, H6'  613C (ppm) (125 MHz) 19.7 —  HMBCb (500 MHz) — —  26.0a  — — C3, OAc — C8 — C5, C9  11' 12 12' 13 14  4.22, ddd, J = 15.8, 10.6, 5.3 Hz 4.05, m 1.62, m 1.40, m — 3.28, t, J = 11.6 Hz  — H11', H12, H12'  69.9 78.8 76.7 34.5 — 1382 137.3 199.7 56.3 62.0  H11, 1112, H12' H11, H11', H12' 1111, H11', H12 — H15, 1115'  — 37.1 — 45.8 42.7  15 15' 16 17 18  1.73, m 1.46, m — — 0.74, s  H14, H15' H14, H15 — — —  24.4  19 19' 20 21 22 22' 23 24 24' 25 26 27 OAc  4.05, d, J= 11.2 Hz 3.91, d, J = 11.2 Hz 1.45, m 1.00, d, J = 6.7 Hz 1.83, m 1.67, m — 1.83, m 1.73, m 1.52, m 0.88, d, J = 6.6 Hz 0.88, d, J = 6.6 Hz 2.00, s; 2.20, s  H19' H19 H21, H22 H2O H20, H22' H20, H22  28.0a 50.8 16.7  —  — —  — — — — — C8, C9, C13, C16, C18 — — —  —  C11, C12, C13, C14, C17 — Cl, C9 C17, C20, C22 — — —  61.5 — 34.8 19.7 35.5 — 29.7 39.5 H25, H24' — H24, H25 — — H24, H24', H26, H27 28.5 — H25 22.8 C24, C25, C27 H25 22.5 C24, C25, C26 20.9, 21.1, 171.0, — — 171.8 a May be interchanged. b The carbon resonances in the HMBC column are correlated to proton resonances in the 8 1 H column. —  HO  OCOC ff3 °C OQ :13  Figure 67.  ^  ppm I H NMR Spectrum of Blancasterol  (102) (Recorded in CDCI3 at 400 MHz)  130 21 22 24 Ac0 11 12 HO  HO  OH OAc  27  OCOCH3^Me27 H19 OCOCH3^Me26 H11' Mel 8 H3 H6' t^Me21 H7 H4 H11 H19' H14 H6  .s 1.8  Me21/H20 _ %a  1  ea 4  it  H3/H2' II H3/H2  II  ii_Me26/H25  ;,,—,19  ., ' 4, /z0 i - ..',^2)^dis H2/H2' '41 CO CI H2'/H1 HUH 1'^  e  1.5  TilMe27/H25  2.6  ''^ •PH6/H6' 1  2.5  .  '  T  i  I^• I H11'/H12 !Hll'/H12' 11^ft a I®t H11/H12 H1 1/H12'  H19/H19' .  It  All  ' H11/H11'  . . --40  3.0  H144415 H14/H15'  3.5  .  ... 4.5  el H4/H3  5.6  .  . .  .  5.5  6.8  0 .  H7/H6 H7/H6' a0  •  •.  S. 5  . 7.8  •  . _..-  75^711^65^66^SS^50^45  • I PPM  3 5^3.6  25  2 11^1.5  1.8  .5  Figure 68. COSY Spectrum of Blancasterol (102) (Recorded in CDC13 at 400 MHz)  PPM  7.5  8.8  S  HO  1 OH  OAc  200  ISO  140  100  Figure 69.^13C NMR Spectrum of Blancasterol (102) (Recorded in CDC13 at 125 MHz)  132  HO  OH OAc  Me27 Me26 Me18  OCOCIj3 OCOCkI3  H19 H11' H7^H4^H3 H19' H14 H1 1  s^4 4 11. 11111^ A t. t :  1  0  10^II  0  4 4 1WO  : c21, C21C1 :C26 C27 C6  1 I^.I^ IP I  40 C1  4  .  0  Al.  -cis C19  i  . - C3  .  i  - C4Bo  4  .  r  - 100 .  I  I  •  '.  120  O en.  • .  -C7  •  a^5^4.  PPII  3  2^1  Figure 70. HMQC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz)  ^  Me27 Me26 Me21 Me18  I  1-14 HO  OH H7 OAc  H 19'  —__ _  ■-  H14  1 H 1 9 7C 1 •  •  Cl  ...re-  •  . ^eGt or .  ..  4,  . ob H4/C3  .  H7/C5  go  .  •  •  .,  .a  •  • .  •  _  CP  •  -  •  Me21/C17 wafir`Vr • a  -  •■•  •  • :21  150  al.  c2 °c12 soC17  ••  100  •  .  . •  •  •  i  1  .  •  •  OP  160  .^t  +4  H4i000CH3  C26 C27 C24  ....^. .  .. • C8  r  Me26/C27  Me26/C24 ^•^ Me21/C20 40 iMe18/ Me21/C22 . 41^C12  IP^•  .  .  •  w•^•• a .^.^.  .  H14/CSO .  50  100  I  0^  2^t  C3 C5  .  .  •. • f• ata  • .•  -40- H7/C9 6.0  •  H19'/C9 — ^  5.5  ^  6.0  ^  4.0  ^  4.0  ^  3.5  C9  0P 6 ^  pp. 006  ^  2.5  ^  1.0  ^  1.5  Figure 71.^HMBC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz)  ^  1.0  134 The molecular formula C31HSO08 indicated that steroid 102 had seven sites of unsaturation. Three carbonyl carbon resonances (5 199.7, 171.8, and 171.0 ppm) and two olefinic carbons (138.2 (CH) and 137.3 ppm (C)) were visible in the 13 C NMR spectrum, accounting for four sites of unsaturation. Three unsaturations remained indicating that the molecule was tricyclic and was most likely a secosteroid. The infrared spectrum of blancasterol (102) contained a hydroxyl stretch at 3458 cm -1 as well as carbonyl stretches at 1734 and 1678 cm -1 . The IR band at 1734 cm -1 was attributed to an ester carbonyl. Methyl singlets at 8 2.00 and 2.20 in the 1 H NMR (Figure 67), shifts diagnostic of acetate methyls, were consistent with the assignment of the IR absorbance at 1734 cm -1 to ester stretches. The IR stretch at 1678 cm -1 was typical of an a,13-unsaturated ketone. This assignment was corroborated by the 13 C NMR spectrum (Figure 69) which contained a carbon resonance at 5 198.7 (C9). The molecular formula indicated that blancasterol (102) contained eight oxygen atoms. Two acetates and a carbonyl accounted for five oxygen atoms leaving three to be assigned. Five carbons were at chemical shifts appropriate for single bond attachment to oxygen atoms, specifically, the carbon resonances at 5 69.9 (C3), 78.8 (C4), 76.7 (C5), 62.0 (C11) and 61.5 (C19). Two of these carbons must bear the acetate functionalities but three carbons remained unassigned suggesting that metabolite 102 contained three hydroxyl groups. Table 14 contains a summary of the NMR data recorded for 102. One spin system was particularly notable in the COSY spectrum (Figure 68). A pair of methylene proton resonances at 5 4.22 (H11) and 4.05 (HIP), deshielded by an undetermined substituent, showed correlations to another pair of methylene proton resonances at 1.62 (H12) and 1.40 (H12'). No further correlations were discernible indicating that the spin system was isolated. The 1 H NMR shifts of the H11 (8 4.22) and H11' (4.05) resonances were considerably more deshielded than the same proton resonances in glaciasterol A (100) (H11, 3.69; H11', 3.81) but were in better agreement with the observed shifts for glaciasterol A diactate (106) (H11, 4.10; H11', 4.16) (Tables 4 and 5). This led to the conclusion that the native blancasterol (102) had an acetoxy terminus at the C11 methylene. Examination of the HMQC spectrum of 102 (Figure 70) indicated a proton resonance at 5 6.44 (H7) was correlated to an olefinic carbon resonance at 5 138.2. The other olefinic carbon resonance (5 137.3 (C8)) had no HMQC correlation to a proton resonance which suggested that the only double bond in 102 was trisubstituted. Correlations were observed in the COSY spectrum from the resonance at 5 6.44 (H7) to a pair of geminal methylene resonances at 2.56 (H6) and 2.35 (H6'). The coupling constant 416' ,H7 = 5.8 Hz was a typical value for vicinal coupling but was too large to attribute to allylic coupling. 73 a This result indicated that the olefinic proton at 5 6.44  ^  135 must be to the ketone. The functionalized ethyl fragment and the a,(3-unsaturated ketone could be accomodated by a 9,11-secosteroid. Figure 72 illustrates selected correlations observed in the HMBC spectrum (Figure 71). The proton resonance at 5 6.44 (H7) had HMBC correlations to carbon resonances at 5 199.7 (C9) and 76.7 (C5). The chemical shift of the C5 resonance indicated that the carbon had a hydroxyl or acetyl substituent. No further COSY correlations were observed from the C6 methylene protons and the carbon resonance at 5 76.7 had no correlations in the HMQC spectrum consequently it was concluded that the C5 ring junction was tetrasubstituted. CH3 0 ^ %  ^I  C  ^1.00 0^H3C 0.74  Figure 72. Selected HMBC Correlations for Blancasterol (102).  One other notable spin system in the COSY spectrum (Figure 68) consisted of a doublet at 5 4.98 (H4) which was correlated to a signal at 4.01 (H3). The multiplicity of the latter signal was obscured by two other resonances in the same vicinity (H11', 4.05; H19, 4.05). Correlations from the signal at 5 4.01 (H3) were observed into a pair of methylene proton resonances at 2.09 (H2) and 1.62 (H2') which in turn were coupled into one other pair of methylene resonances at 2.26 (H1) and 1.88 (H1'). This spin system, illustrated as fragment f, could only be accomodated by the A ring of the steroid.  136 1.88 2.26 HH  f As was mentioned earlier, only one aliphatic methyl singlet (5 0.74 (Me18)) was visible in the 1 H NMR spectrum. The COSY spectrum (Figure 68) contained an isolated spin system consisting of a doublet at 5 4.05 (H19) which was correlated to a doublet at 3.91 (I-119'). Both of the proton resonances at 8 4.05 (H19) and 3.91 (H19') had HMQC correlations to a single carbon resonance at 8 61.5 (Figure 70). The chemical shifts of the protons and the attached carbon led to the proposal that either Me19 or Me18 had been oxidized to a hydroxymethylene. Such functional groups, while not common among marine natural products, have been reported. A representative example is the polyhydroxylated steroid 103. 71 It was determined that the Me19, rather than the Me18, had been oxidized in blancasterol from a correlation in the HMBC spectrum (Figure 71) from the proton resonance at 8 3.91 (H19') to the C9 carbonyl resonance at 5 199.7 ppm. A second HMBC correlation was observed from the H19' resonance (8 3.91) into a carbon resonance at 5 19.7 (C1). The carbon resonance (5 19.7 ppm) was assigned as Cl from HMQC (Figure 70) and APT data (Figure 73). This result permitted the orientation of spin system f with methylenes at Cl and C2 and the hydroxy or acetoxy substituents at C3 and C4. An HMBC correlation was observed from the proton resonance at 8 4.98 (H4) to an acetate carbonyl resonance at 5 171.0 ppm. This correlation along with the chemical shifts of the Hll and 1111' protons located the acetates at the C4 and C11 carbons. Consequently it was concluded that C3, C5, and C19 had hydroxyl substituents.  137  A triplet at 5 3.28 in the 1 H NMR spectrum was at the expected chemical shift for H14. COSY correlations were observed from the resonance at 5 3.28 (1114) into proton resonances at 1.73 (H15) and 1.46 (H15') which were attributed to a geminal methylene pair. The carbon shifts for C 14 (5 42.7) and C15 (24.4) were assigned from the HMQC spectrum (Figure 70) and these were in close agreement with the values observed for the D ring carbons of the glaciasterols (glaciasterol A (100); C14, 43.9; C15, 26.9). An HMBC correlation from the 1-114 resonance (6 3.28) to the olefinic carbon at 5 137.3 (C8) confirmed the C8/C14 linkage.  An HMBC correlation was observed from the Me18 singlet (5 0.74) to the C14 carbon resonance (5 42.7). This correlation was consistent with the assignment of the singlet at 8 0.74 to Mel 8. The methyl singlet at 5 0.74 (Me18) also had an HMBC correlation to a carbon resonance at 5 37.1 which had been assigned to C12 from the HMQC spectrum. This result substantiated the location of the acetoxyethyl fragment. Further HMBC correlations from the Me18 resonance (5 0.74) into carbon signals at 8 45.8 and 50.8 permitted assignment of these resonances to C13 and C17, respectively (Figure 72).  As mentioned earlier, HMBC correlations from the side chain methyls aided in the assignment of the side chain carbon resonances. Figure 72 shows that correlations were observed from the Me21 resonance (5 1.00) to carbon resonances at 6 34.8, 35.5, and 50.8 ppm. In conjunction with the COSY data (Figure 68), the HMQC data (Figure 70), and by comparison with the side chain of glaciasterol B diacetate (107) (see Table 6), it was possible to assign these resonances as C20, C22, and C17, respectively. HMBC correlations from the methyl doublet at 8 0.88 (C26 and C27) into carbon signals at 5 28.5 and 39.5 allowed the assignment of these carbons to C25 and C24, respectively.  The structure of blancasterol was determined to be the trihydroxy 9,11-secosteroid diacetate 102. Titration of a deuterochloroform NMR sample of 102 with deuterobenzene dispersed the complex, crowded region at —5 4.10 ppm in the CDC13 1 H NMR spectrum. The 1 H NMR spectrum in CDC13: C6D6 20:1 is shown in Figure 74. A series of double resonance experiments on 102 in CDC13: C6D6 20:1 permitted assignment of the newly resolved signals (Figure 75). Table 15 lists the assignments of selected protons of blancasterol in CDC13 and in CDC13:C6D6 20:1.  HO  OH  OAc  as^s^as^34^as  ^  24^a^20^a^to  Figure 73.^Expanded Upfield Region of the APT Spectrum of Blancasterol (102) (Recorded in CDC13 at 125 MHz)  S  S  AcO 11 HO  HO  1  00C113 OCOCjj3  2; 22 24^26 18 ss• 27  Me27 Me26  OH  OAc  Me18  Me21  H4 H7  M•  H3 H19 H11' H19' H11  H14  H6  H6  fiv  , r ,^rr--r^,^r^I^,^ , r-r-r"^ 1 7.5^7.0^6.5^6.0^5.5^5.0^4.5^4.0^3.5 ^3.0^2.5^2.0^1.5^1.0^.5 PPM  ^Figure  1"11''  1  74.^1 H NMR Spectrum of Blancasterol (102) Recorded in CDC13:C6D6 20:1 at 400 MHz  140  HO  H4 H19 H11  H11'  H3  H19'  H14  5.9^4.9^4.5^3:7^4.5^4:5^4.4^4:3^- 4.2^IA^as^3:•^1:-11^3:7^3:5^3:5^3:4^3.1^3.2 PPM  Figure 75.  ^Double Resonance NMR Experiments on Blancasterol (102) (Recorded in CDC13:C6D6 20:1 at 400 MHz)  141  HO  OH OAc H7  H4f3  H3  Hii H19 H19'  H11  •  H14  HO  2  i  I H7/H6a  •  •  •  0  a  0  4  H6f3/H19  H14/H11  00 H4f3/H19'  •  H413/H6f3  6 •  or  40 • MEM  ■  1.• 0.  1111^I^il-I  6  f  ^  11-11 lllllllllllllll I 11011111^  4  llll  2  Figure 76.^ROESY Spectrum of Blancasterol (102) (Recorded in CDC13:C6D6 20:1 at 500 MHz)  142 Table 15. 1 H NMR Shifts of Blancasterol (102) in CDC13 and in CDC13:C6D6 20:1.  Carbon no.  81H NMR recorded in CDC13 (ppm) 8 1 11 NMR recorded in CDC13:C6D6 20:1 (ppm)  3  4.01  3.95  4  4.98  4.93  6  2.56  2.46  6'  2.35  2.24  7  6.44  6.34  11  4.22  4.22  11'  4.05  4.05  14  3.28  3.27  19  4.05  3.98  19'  3.91  3.83  A ROESY spectrum of blancasterol was recorded in CDC13:C6D6 20:1 (Figure 76) and the correlations provided information concerning the relative stereochemistry of 102. Figure 77 illustrates a number of informative ROESY correlations.  Figure 77. Selected ROESY Correlations for Blancasterol 102 (Recorded in CDC13:C6D6 20:1).  ROESY correlations were observed from the H4 resonance at 8 4.93 into proton resonances at 2.46 (H6(3) and 3.83 (H19'). The trans diaxial coupling between H4 ax (8 4.93, d, J = 9.5 Hz) and H3, in conjunction with the ROESY correlations established that the A ring of blancasterol (102) was in a chair conformation and that H4 (4.93)  143 and H6 (2.46) were 13 to the A/B ring system. From the results of the ROESY experiment it was clear that the A and B rings of 102 must be trans fused. Figure 78 shows the alternative structure with the A and B ring junction cis  fused. The ROESY correlation from H19' into H6 ax would probably be observed, however no correlation would  be expected from the 1119' resonance to the H4 doublet.  Figure 78. Conformational Structure of Blancasterol (102) with a Cis-Fused A/B Ring System.  Figure 76 shows that the H14 resonance at 5 3.27 had a ROESY correlation into the H11 and H11' methylene signals at 4.05 and 4.22. This indicated that the H14 proton has the  trans  configuration with respect to  Me18 found in most steroids. 60,67 The side chain was assumed to have the normal 13 configuration but this was not verified experimentally.  Known Metabolites of the Pleraplysilla Sp. Sponge (1992 Collection)  Three known sesquiterpenes were isolated from the ethyl acetate extract of the 1992 collection of the Pleraplysilla  species sponge. The structures of furodysinin (36), 44 nakafuran-8 (41), 28 b and furodysin (35)44  were determined by comparison of their 1 H NMR spectra with published results (see experimental). COSY spectra confirmed the connectivities.  144  35  ^  36  41  An NMR sample of nakafuran-8 in CDC13 (41) was left in the refrigerator overnight. The following day none of the starting material remained but there appeared to be a product with a characteristic aldehyde resonance at 6 9.61 in the 1 H NMR spectrum. The structure of the degradation product was determined to be 116, the y-keto enal of nakafuran-8 (41), which Scheuer and coworkers had reported to be a product of meta-chloroperbenzoic acid oxidation of 41. 28 b Scheuer et al. found that oxidation of the intermediate 116 under Jones' conditions gave the hydroxybutenolide 56 (Equation 5).  MCPBA  41  ^  CHO 116  Jones' oxidation  56  Equation 5. Oxidation of Nakafuran-8 (41) to the Hydroxybutenolide 56.  2813  In our case, it is likely that the combination of acidity in the CDC13, oxygen, and a small amount of atmospheric moisture was sufficient to catalyze the conversion of 41 to 116. The reaction of 41 to form 116  145 indicates oxidation of 41 to the corresponding the y-keto enal is a facile reaction and suggests this to be a likely route to the hydroxybutenolide 56.  CONCLUSIONS  A study of a gray-white sponge, initially identified as an Aplysilla sp. on the basis of morphology alone, 29 resulted in the isolation of four new compounds; the sesquiterpenes 0-methyl 9-oxofurodysinin lactone (72), 2-oxomicrocionin-2 lactone (73), 0-methyl 2-oxomicrocionin-2 lactone (74), and the cytotoxic secosteroid blancasterol (102). The isolation of blancasterol (102) adds to the number of secosteroids reported from marine sponges. The biogenesis of the 9,11-secosteroid is expected to proceed in a similar manner as in the case of glaciasterols A and B (100 and 101) via oxidative cleavage of the 9,11-carbon bond. The structural similarity of blancasterol (102) to glaciasterol A (100) indicated that it might also be cytotoxic. Pure blancasterol (102) was submitted for biological testing against the in vitro L1210 murine leukemia and human breast cancer solid tumor cell lines. Blancasterol (102) was active against the L1210 cell line at an ED50 of 8.8 lig/mL. The compound proved active against the drug-sensitive MCF-7 breast cancer cell line at an ID50 of 3 gg/mL. The ID50 against the drug-resistant MCF-7 Adr breast cancer cell line was 10.6 gg/mL. In addition to the new compounds, seven known sesquiterpenes were also isolated from extracts of the graywhite sponge. These metabolites are furodysin (35), 44 furodysinin (36), 44 furodysinin lactone (56), 49 0-methyl furodysinin lactone (57), 49 nakafuran-8 (41), 28 b nakafuran-8 lactone (43), 28 b and 0-methyl nakafuran-8 lactone (44).28b  The presence of sesquiterpene chemistry in the white sponge indicated that the sponge may have been  misidentified. As discussed previously, the terpenoid metabolites of sponges of the genus Aplysilla are invariably diterpenes. The sesquiterpene chemistry was more appropriate for the closely related Dysidea sp. sponges. In fact, compounds 35, 36, 56, 57, 41, 43, and 44 have been reported from sponges of the genus Dysidea. Dr. R. W. M. van Soest of the University of Amsterdam reexamined a sample of the sponge in consideration of the inconsistency of the metabolite chemistry with the original classification and consequently reidentified the sponge as a species of the genus Pleraplysilla, biologically a genus intermediate between Aplysilla and Dysidea. 88 No Pleraplysilla sponges have previously been identified from the west coast of North America and it is possible that  this is a new species of the genus. 88  146  OR  72  73 R = H 74 R = CH3  HO 35  36  41  43 R = H  44 R = CH3  0-Methyl 9-oxofurodysinin lactone (72) is an oxidized analog of 0-methyl furodysinin lactone (57). 49 Compounds 47 and 49, which have been reported from a collection of Dysidea herbacea,4 ° can be envisaged to be possible biosynthetic intermediates in the formation of 72 from a precursor such as furodysinin (36).  36  ^  47 R=H,W=OH 49 R = OH, R' = H  147  72  57  Faulkner and coworkers investigated the stability of furodysin (35) and furodysinin (36) in methano1. 91 A sample of Dysidea species sponge which had been soaked in methanol for two years was found to contain furodysinin (36) but none of the oxidized furodysinin lactone 56. Faulkner et al. proposed that the methyl ketal moiety of 57 was an artifact of the reaction of furodysinin lactone (56) with the methanol solvent but that Chromodoris funerea, the animal from which 0-methyl furodysinin lactone (57) had been isolated, was certainly  producing some oxidized derivative of furodysinin (36), if not the hydroxybutenolide (56) then perhaps a precursor, such as an endoperoxide, which decomposes to form 56 under the isolation conditions. 91 Extrapolating the results of Faulkner and coworkers, it can be postulated that 0-methyl 9-oxofurodysinin lactone (72) is very likely the product of a reaction of 9-oxofurodysinin lactone with the extracting solvent, methanol. It seems unlikely, however, that 72 is an artifact resulting from oxidation of furodysinin (36) during the isolation procedure. methanol  56  ^  57  Equation 6. Reaction of Furodysinin Lactone (56) with Methanol to form 57. 2-Oxomicrocionin-2 lactone (73) and 0-methyl 2-oxomicrocionin-2 lactone (74) are oxidized derivatives of microcionin-2 (117), a sesquiterpene metabolite of the sponge Microciona toxystila. 92 While it is improbable that the C9 carbonyl is an artifact, the methyl ketal of 74 is likely to result from a reaction of 73 with the methanol used for extraction (Equation 6). Further experimental data is required to determine whether the hydroxybutenolide moiety of 73 is an artifact of autooxidation upon workup.  117  148  OPP [0] H+ induced cvclization  =  c+(.,/.. CH3 shift  H shift \ \ 0 -H+  117  cyclization OH  H + / cyclization  ME  41 Scheme 1. Proposed Biogenesis of Microcionin-2 (117) and Nakafuran-8 (41).2813  Scheuer has proposed that the biogenesis of the nakafuran-8 (41) and the microcionin-2 (117) carbon skeletons follow the same mechanistic pathway (Scheme  l).28b  The biogenesis proceeds via conversion of farnesyl  pyrophosphate to the furan intermediate followed by cyclization to form the six-membered ring, a proton shift, a  149 methyl shift, and elimination to yield sesquiterpenes possessing the microcionin-2 (117) carbon skeleton. Scheuer suggested that oxidation of microcionin-2 to form the hydroxybutenolide is intermediate in the biosynthesis of upial (40), a natural product known from Dysidea fragilis. 47 This is of interest in view of our isolation of two hydroxybutenolide derivatives of microcionin-2 (73 and 74). It can be seen from Scheme 1 that elimination of hydrogen from microcionin-2 (117) followed by addition of a proton and cyclization leads to formation of the nakafuran-8 carbon skeleton. 28 b The biogenesis of furodysin (35), furodysinin (36), and the related derivatives 56, 57, and 72 can be envisaged as shown in Scheme 2. Conversion of one end of a unit of farnesyl pyrophosphate leads to the terminal furan followed by cyclization to an intermediate such as spirodysin (39), a metabolite which has been isolated from the sponge Dysidea herbacea." Rearrangement of spirodysin (39) could conceivably lead to both furodysin (35) and furodysinin (36). 46  OPP [0]  39  35  ^  Scheme 2. The Proposed Biogenesis of Furodysin (35) and Furodysinin (36)  36  150 It is puzzling that each year that the Pleraplysilla sponge was collected the sponge extracts yielded a decreasing diversity of metabolites. The 1990 and 1991 collections contained the same major metabolites with representative derivatives of the microcionin-2 (117), furodysinin (35), and nakafuran-8 (41) carbon skeletons. As mentioned earlier, the 1990 collection contained at least five sesquiterpenes, present in minor amounts, which were absent from the 1991 sponge collection. The following year, the sponge collection contained only three sesquiterpene derivatives and there was no trace of compounds with the microcionin-2 (117) carbon skeleton. While it is possible that the hydroxybutenolide moieties of the sesquiterpenes isolated from the 1990 and 1991 collections were artifacts of methanol extraction, it seems unlikely that the microcionin-2 carbon skeleton is an artifact. Furthermore, the compounds isolated in 1992, furodysin (35), furodysinin (36), and nakafuran-8 (41), lacked the 9-oxo and 2-oxo functionalities found in compounds 72, 73, and 74 isolated from collections of previous years. It was evident that some factor, possibly the ecological pressure applied by collecting the sponge, was influencing the nature of the secondary metabolites produced by the organism. The sponge was collected at the same time each year so seasonal variation was not responsible for the change in metabolite composition. Physically the sponge did not change in appearance, however, the rock on which it grows is home to at least three other species of sponge and these were clearly attempting to encroach on the areas cleared by collecting the sponge.  Two explanations of the change in the composition of metabolites in the sponge extract come to mind. First, it is possible that the sponge did not have time to elaborate its full array of secondary metabolites in the time frame of our collections. Little is known about the kinetics of sponge secondary metabolism and this idea might merit further investigation.  The second possibility is that the sponge, under pressure from competing organisms and suffering reduced competitive effectiveness due to the annual collections is in some way producing those compounds which are most effective against predators and competing animals. It has been reported that nakafuran-8 (41) and furodysinin (36) are effective fish antifeedants. 91,28 b This explanation is inconsistent, however, with a report that the oxidized product of furodysinin (36), 0-methyl furodysinin lactone (57), is in fact a significantly more active fish antifeedant  than the proposed precursor furodysinin (36) (furodysinin [36] was effective as a fish antifeedant at 50 µg/mg food pellet, while 0-methyl furodysinin lactone [57] required 10 tg/mg food pellet. 91 ). To our knowledge this is the  151 first report of an instance where collection of a sponge appeared to produce an effect on the chemistry of the sample organism. It would be interesting to further investigate the effect of harvesting sponges on the composition of the sponge metabolites.  152  PART II. THE NATURAL PRODUCTS OF TWO SPECIES OF COLONIAL TUNICATE FROM THE OUEEN CHARLOTTE ISLANDS  INTRODUCTION  Phylogenetic schemes depicting the subkingdom Metazoa generally place the phylum Porifera at the bottom, among the least organized of organisms. The phylum Chordata, in contrast, is considered to contain the most organized and highly evolved species of animals. 93 The phylum Chordata is comprised of the subphyla Vertebrata, which contains the vertebrate animals including humans, the Cephalochordata, and the Urochordata (Figure 79). The two latter subphyla are exclusively marine. The work presented in this thesis includes studies of cytotoxic secondary metabolites of two tunicate species, members of the subphylum Urochordata. This section will discuss the biology of these animals. While the solitary tunicates are generally not difficult to identify, compound ascidians which are colonies of tunicates, may resemble sponges in both their sedentary lifestyles and general appearance. An uninformed observer would find it difficult to differentiate tunicates from sponges, yet phylogenetically the distance between them is vast. The reason the urochordates are classified among the higher animals has less to do with the adult organism than with the larval state. The urochordates produce free swimming larvae, several mm in size, which resemble the tadpoles of frogs (see Figure 80) and have many morphological features in common with the other phyla of the Urochordata. 94 A particularly remarkable characteristic of the larva is the notochord, a fibrous sheath which constitutes a primitive spinal column 94 and provides the body with firmness and flexibility. 93 The larvae have a number of adaptations to optimize selection of a safe stratum on which to settle and develop into adults. These adaptations include muscular tails which allow swimming, and an anterior end which is equipped with a multicellular light receptor (ocellus) as well as a single-celled statocyst to differentiate up from down. 95  ^  153  Subkingdom  Phylum  ^ Metazoa  ^  Subphylum  Chordata  Vertebrata^Urochordata^Cephalochordata  '11 Class  Order  Family  Ascidiacea  lir Aspiraculata^Aplousobranchia^Stolidobranchia^Phlebobranchia  Synoicidae  1( Genus  Aplidium  Figure 79. The Classification of Tunicates of the Genus Aplidium  154  Figure 80. The Body Plan of a Urochordate Larva 96  The subpylum Urochordata is subdivided into three classes, including the Ascidiacea which contains —90% of the —2000 species of urochordates. As can be seen from Figure 79, the Ascidiacea is in turn comprised of the orders Aspiraculata, the deep-sea ascidians, the Phlebobranchia and Stolidobranchia, which include primarily solitary tunicates and the suborder Aplousobranchia, which contains most compound tunicates.  95 . 96  The order  Aplousobranchia can be further divided into a number of families including the Synoicidae which includes the genus Aplidium. 94 The cytotoxic natural products of two species of this genus, Aplidium californicum and an undescribed  species of Aplidium, are discussed in this thesis.  The adult tunicate bears little resemblance to its larvae. After swimming several hours to two days, the larvae attaches itself by the three fixation papillae at its anterior end to a suitable substratum. 93 . 94 It appears that a site where adult tunicates of the same species grow is preferred. 95 Metamorphosis involves resorption of the notochord and tail. The dorsal nerve cord degenerates into a simple ganglion and the digestive tract and organs rotate 180° to the free end of the body. The mouth area, unused until now because the larvae never feed, expands and the pharynx, the central body cavity, enlarges and develops numerous slits until it resembles a sieve. The siphons begin to pump water through the body cavities initiating adult life activities such as feeding, respiration, reproduction and  155 excretion. 93 The ascidian may reproduce in one of two ways; sexually or asexually. Species of the genus Aplidium are compound or colonial tunicates which bud asexually by means of lengthwise constriction to produce colonies of the anima1. 95 These colonies may share a common protective outer layer and some blood vessels. Colonies of a compound tunicate may grow to a meter in diameter even though each individual tunicate, or zooid, may be as small as 3 mm. All Aplidium tunicates are hermaphrodites which contain a single ovary and testis. In species of this genus, the ovum is fertilized in situ and develops into a larva in one of the parent's body cavities.  96  Figure 81 illustrates the body plan of a single zooid of the compound tunicate Aplidium californicum, one of the species studied." The other Aplidium species has a similar body plan. The ascidian body is covered with an epidermally-secreted layer referred to as a tunic composed of cellulose and protein. The tunic provides protection and fixes the tunicate to its substratum. Within the tunic is the true external body-wall of the ascidian, a membrane-like sac called the mantle. 93 As can be seen from Figure 81, zooids of the genus Aplidium are segmented into three sections; the anterior segment is the thorax which contains the two siphons and the branchial sac, the abdomen encloses the digestive organs and the post-abdomen contains the reproductive organs and the heart." Tunicates have a buccal and an atrial siphon. The buccal siphon is the incurrent opening permitting water to enter the body. The base of the opening is surrounded by tentacles which act to exclude large particles. The incurrent siphon opens into a large body cavity, the pharynx, which occupies most of the volume of the animal's body. The pharynx is perforated with many slits, known as stigmata, so that it resembles a net. The stigmata are covered with cilia which sweep water into the pharynx. The incoming current passes through the stigmata where any planktonic food particles are trapped, into the atrium (a second body cavity) and out through the atrial siphon. A vertical slit in the pharyngeal wall is the site of production of a mucus which moves in a continuous sheet across the pharyngeal wall collecting food particles. The food-mucus sheet is rolled into a rope which is delivered to the esophageal opening on the pharyngeal floor. Food travels through a narrow esophagus, into a sphinctered stomach and through a long intestine. 93 The entire digestive tract is ciliated. Excrement is discharged through the anus which is located under the atrial siphon to permit flushing of waste with the excurrent water flow. In the case of colonial tunicates such as Aplidium species, the atrial siphons of a number of zooids may open into a common cloacal cavity prior to flushing  away."  156  Figure 81. Body Plan of the Urochordate Aplidium caWornicum 94 Urochordates have open circulatory systems including a poorly defined heart which is comprised of a fold in the tissue of the pericardial region. The heart pumps blood through a system of vessels and periodically reverses the flow of pumping. The heart's primary function is to saturate the vessels of the pharynx with blood. Since organisms belonging to the subphylum Urochordata are sessile, they have very low rates of metabolism and typically extract only 10% of available oxygen from incoming seawater. 93 Excretion is the function of multipurpose cells known as amoebocytes which absorb uric acid, and travel to the walls of the digestive system where they are stored for the duration of the animal's life. Ammonia which constitutes 90% of the nitrogenous waste of the organism diffuses out through the body walls.  93  Amoebocytes are known in some species to concentrate heavy metals such as vanadium, niobium, chromium and iron at levels 10 5 -106 times higher than the concentration in seawater. This phenomenon is subject to intensive study but is not yet well understood. 93 . 95 . 96 The cerebral ganglion, a vestige of the larval nerve chord, is located in the thorax between the mouth and the atrial siphon. A number of single nerves branch from it but the nervous system is decentralized, as evidenced by  157 the fact that removal of the cerebral ganglion has little effect on the organism. If threatened, the animal can expel water from its body cavities and close its siphons, hence one of its names, the seasquirt. 93 Taxonomy of the Ascidiacea is difficult because, like the Porifera, these animals are soft-bodied with few features to distinguish them." Classification is based mainly on the structure of the pharyngeal wall, particularly the stigmata and the location of the gonads. 96 The fossil record of the Urochordata is minimal as soft-bodied animals do not fossilize well." It has been suggested, however, that ascidians alive today evolved from a much greater specific diversity in past geological periods. 96 On the basis of the high level of organization of the urochordate larva, it has been proposed that tunicates are products of retrograde evolution, stemming from more highly organized ancestors." The urochordates are known to have predators, including humans, asteroids, gastropods, and some fishes. 95 Nevertheless, in spite of their sedentary ways of life and lack of physical means of defense, many tunicates are not fed upon by other marine animals. There is circumstantial evidence which suggests that some species of tunicate synthesize natural products which provide a chemical defense system against predators. For example, shermilamine B (118)  and the kuanoniamines A—D (119-122) were isolated from both an unidentified purple compound tunicate  as well as a prosobranch mollusk, Chelynotus semperi. The prosobranchs, snails characterized by reduced shells, are known to be predators of invertebrates toxic to other species. It has been suggested that these mollusks sequester the  bioactive secondary metabolites of their prey and use them for their own chemical defense. 97  HNAc ^ 118  119  120 R =  121 R = 0  HNR  122 R= )1s.  158  Natural products chemists have only recently started to show significant interest in the secondary metabolites of urochordates. Until 1986 approximately fifty natural products had been reported from tunicates. 7 Since 1987 however more than one hundred and eighty structures of urochordate metabolites have been published. 8e - i Tunicate chemistry is remarkable in that it is dominated by amino acid metabolism. Approximately 90% of the reported natural products are nitrogenous.? The sudden surge of excitement surrounding tunicate metabolites has been prompted by the high levels of bioactivity of urochordate extracts and interesting physiological effects of some of the reported compounds. 811-1 One example is didemnin B (10), a natural product which exhibits cytotoxicity and immunosuppressive activity. 15 0^ 0^0 II C —NH—CH— CHOHCH 2 C -0 —CHC CH(CH3)  O C  CH2C H(CH3)2^CH(CH3)2  L-Lac-L-Pro-D-MeLeu NH OCH3^CH2CH(CH3)2 CHO —C - C FIN(CH3 ) -C N CCII^ N I O^0 CH3 10  The interesting chemical and pharmacological activities of metabolites of the Urochordata are likely to continue to attract the attention of chemists in the future, yet the secondary metabolites of these animals are unlikely to be of great utility in classification. This is due primarily to the fact that the tunics and cloacal cavities of the ascidians make excellent habitats for high concentrations of symbiotic bacteria. These organs are rich in nutrients and carbon dioxide and provide physical protection from predators. Consequently many species of tunicate, including species of the genus Aplidium, have been found to have large colonies of symbiotic bacteria living in the tests and cloacal aperatures. 98 It has been suggested that some compounds isolated from collections of urochordates are in fact products of the metabolisms of the tunicates' symbiotic bacterial populations. This is exemplified by patellazole B (123) isolated from the marine tunicate Lissoclinum patella which is known to host colonies of bacteria. Moore  159 and Corley suggest that this compound, which is of mixed biosynthesis but is primarily polyketide-derived, is likely to originate as a secondary metabolite of the tunicates' algal symbionts. 99 OH OCH3 CH3  H3C  H3 C  OH  CH3  This thesis presents the isolation and structure determination of an anthracene quinone which is the cytotoxic component of an undescribed Aplidium species from the Queen Charlotte Islands. The isolation and structure elucidation of a family of related prenylated hydroquinones from a collection of Aplidium californicum will also be discussed. The following section will discuss relevant examples of anthracene quinones from marine invertebrates.  A. THE CHEMISTRY OF AN UNDESCRIBED APLIDIUM SP. FROM THE QUEEN CHARLO 1 lE ISLANDS  i) ANTHRAQUINONES FROM MARINE INVERTEBRATES  It is of particular relevance that marine bacteria may produce compounds related to metabolites isolated from ascidians since, as previously mentioned, bacteria frequently inhabit the tunics of ascidians. It is possible that compounds isolated from tunicates are, in fact, produced by symbiotic microorganisms.  98,99  A marine  microorganism, Chaina species, was reported to produce antibiotic SS-228Y (124). The structure was initially misassigned 1 °° but synthesis proved the compound to be 124. bacteria. 1 °°  101  SS-228Y is active against gram-positive  160  OH 0 124 The sponges Xestospongia exigua and Xestospongia sapra (phylum: Porifera) yielded helenaquinone (125) 102 and xestoquinone (126) 103 , respectively. Helenaquinone (125) was active in vitro against the bacteria Staphylococcus aureus and Bacillus subtilis. 102 Xestoquinone proved to have potent cardiotonic activity. 103 A  number of closely related reduction products were also reported from an Adocia species marine sponge. 104  0^0 125 R,R' = H 126 R, R' = 0 Coelenterates which have been reported to produce polyaromatic quinones include a stony coral and a species of hydroid. Sanduja et al. reported the isolation of three related polyketide-derived metabolites, 127 to 129, from the Pacific stony coral Tubastraea micranth. 105 The authors speculated that these compounds might deter feeding of the coral predator the Crown-of-Thorn seastar Acanthaster planci. 105 OH 0  OH  0^R4 1  6  2  3  12 7 ^128 R , R = OCH3, R , R = OCH20, 4 5 R = H, R = OH 2 129 R 1 = H, R = Br, R 3 , R 5 , R 6 = H, 4 R = CO2H  161 Twenty-one related anthracenone derivatives have been reported from the British Columbian hydroid Garveia annulata. 106 These are exemplified by garveatin A (130)9106a garvin A quinone (131), 106 b annulin A  (132),106c garvalone A (133), and garvalone B (134). 106 d The G. annulata metabolites are characterized by antimicrobial activity. Garvin A quinone, for instance, was active against Staphylococcus aureus and Rhizoctonia solani with ED50 values of 3 tg/mL for both assays. 1 ° 610  130  H3C  0 132  133  134  The crinoids are flowerlike animals commonly known as sealilies forming a class of the phylum Echinodermata. Sutherland and coworkers have studied the pigments of these organisms extensively and report that they produce a colorful array of anthraquinone-derived compounds. 107 Among these Sutherland et al. list rhodocomatulin 6-monomethyl ether (135) and rhodocomatulin 6,8-dimethyl ether (136) from the crinoid species Comatula cratera and Comatula pectinata. 107 a The crinoid Ptilometra australis yielded rhodoptilometrin (137),  162 .107c Caribbean crinoid Comactinia meridionalis isorhodoptilometrin (138), and ptilometric acid (139)^The  meridionalis gave the polyaromatic quinones 140 and 141. 107 d Sutherland and coworkers proposed that crinoids  synthesize these compounds endogenously since the anthraquinones do not appear to be randomly dispersed among the organisms. Rather, the distribution of the anthraquinoid metabolites seems to be species specific.lific OH 0  OH  CH3 O  OH 0  OH  OH 0  OH  HO  HO OH  0 137 OH 0  OH  0 138 OH 0  OH  HO 0 139  O COCH3 140 X = H 141 X = OH  This section has presented a summary of anthraquinoid metabolites isolated from marine invertebrates. While these polyaromatic quinones are widely dispersed among phyla, as mentioned earlier no anthraquinone has been reported to date from the urochoniates.  163  ii) RESULTS AND DISCUSSION  LANGARIN FROM AN UNDESCRIBED APLIDIUM SP.  In May of 1991, a previously undescribed species of colonial tunicate of the genus Aplidium was collected at the southern tip of Langara Island in the Queen Charlotte Islands (Figure 82). The tunicate grows on exposed rock walls at a depth of 5-10 m and is composed of small, globular colonies, orange in color and soft in texture (Figure 83). The colonies grew abundantly at the collecting site to an average thickness of 3 cm and to —15 cm in diameter. A voucher sample (100 g) and a small collection (310 g) were harvested. A methanolic extract of the voucher sample proved to be cytotoxic against the in vitro L1210 murine leukemia cell line (ED50 = 141.1.g/mL), prompting study of the secondary metabolites of the undescribed Aplidium sp.  A small specimen (310 g) of Aplidium sp. tunicate was stored frozen for a period of three months. The tunicate was ground with methanol, filtered and reextracted with methanol. Again the methanolic extract was filtered, the extracts were combined and concentrated to an aqueous slurry. The slurry was extracted sequentially with hexanes and ethyl acetate, however, screening by TLC and 1 H NMR revealed both organic extracts to have the same major product so the hexane and ethyl acetate extracts were pooled. Purification of the major active compound was accomplished by sequential application of size exclusion chromatography (Sephadex LH-20 eluted with ethyl acetate/methanol/water. 20:5:2) and normal phase silica radial TLC eluted with ethyl acetate and hexane. The major compound, 142, was recrystallized from methanol to yield bright orange needles (mp 225-228 °C).  164  Figure 82.^The Aplidium Spp. Collection Site  165  Figure 83.^A Freshly Collected Sample of the Undescribed Aplidium Species  166 Langarin (142)  OH  Langarin was isolated as bright yellow—orange needles which turned deep red upon the addition of 0.1 M aqueous sodium hydroxide. 107 a A molecular ion was observed at m/z 270.0527 Da in the EIHRMS corresponding to a molecular formula of C15111005 (AM -0.1 mmu). The molecular formula indicated that langarin had eleven sites of unsaturation and must be polyaromatic. The IR spectrum contained two carbonyl stretching bands at 1623 and 1675 cm -1 . The dark yellow color of 142 and the carbonyl bands in its IR spectrum indicated that the compound might be a quinone. This proposal was substantiated by the UV spectrum (recorded in methanol) which had three absorption bands at 429 nm, 286 nm, and 254 nm characteristic of a para-quinone. 108 The IR band at 1623 cm -1 was at an appropriate frequency for a chelated quinone carbony1. 109 A resonance in the 13 C NMR spectrum at 8 192.0 ppm provided further evidence for the chelated quinone (see Table 16). The IR band at 1675 cm -1 was at the appropriate frequency for a non-chelated quinone carbonyl and a corresponding carbonyl resonance was observed at 8 181.8 ppm in the 13 C NMR spectrum. Compound 142 was completely characterized by NMR spectroscopy and the data is summarized in Table 16. The 1 H NMR spectrum, shown in Figure 84, contained two deshielded singlets at 6 11.90 and 11.96. These singlets showed no HMQC correlations to carbon resonances and so were assigned as phenolic protons. Five protons were observed in the aromatic region from 8 7.9 to 7.1 ppm. A triplet which integrated for one proton had a chemical shift of 8 5.59 ppm. Again no proton-carbon correlation was observed in the HMQC spectrum suggesting that this was an aliphatic hydroxyl. Finally a broad doublet at 8 4.61 (H11, H11') integrated for two protons. The COSY spectrum (Figure 85) indicated that langarin had two isolated spin systems. A COSY correlation from a doublet at 8 7.36 (H7) into a doublet of doublets at 7.80 (H6) which was further correlated into a doublet at 8 7.70 (H5) established that 142 contained an isolated spin system consisting of three contiguous aromatic protons (fragment a). The second system consisted of two broad singlets at 6 7.28 (H2) and 7.68 (H4) which exhibited strong correlations into one another and less intense correlations into the hydroxymethylene signal  167 at 4.61 (H11, H11'). This data indicated the presence of a pair of aromatic protons meta to one another and ortho to the hydroxymethylene substituent which separated them (fragment b).  7.36 H^'244 OH 5.59  H 7.80 H 7.70  H 7.68  a  b  HH 4.61  Table 16. 1 H and 13 C NMR Data for Langarin (142) (Recorded in Me2SO) Carbon no.  81H (ppm) (400 MHz)  COSY (400 MHz)  1 2 3 4  — 7.28, br s — 7.68, br s  — H4, H11, 1111' — 112, H11, H11'  4a 5 6 7 8 8a 9 9a 10 l0a 11, 11'  7.70, d, J = 7.9 Hz 7.80, dd, J = 8.3, 7.9 Hz 7.36, d, J = 8.3 Hz — — — — — — 4.61, d, J = 5.8 Hz  116, H7 H5, H7 H5, 116  813C (ppm) (125 MHz) 162.0 121.0 154.1 117.5  HMBCb — Cl, C4, C9a, C11  -  133.5a 119.7 137.7 124.7 161.7 116.2 192.0 114.7 181.8 133.-la 62.4  C2, C9a, C10, C11 — C7, C8a, C10 C8, ClOa C5, C8, C8a —  — — — — — — H2, H4, H11, H11', C2, C3, C4 -OH CH2-01I H11, H11' — 5.59, t, J = 5.8 Hz Ar—Off 11.90, br s; 11.96, br s — — a May be interchanged. b The carbon resonances in the HMBC column are correlated to proton resonances in the 5 1 11 column. -  -  -  OH H11' H11  0  H4 H5 1-16^H2 H7  OH OH  '' ^  I^'^" I " '^'^  12.0^11.0^10.0^9.0^8.0^2.0^6.0^ I^I^' '^I^' 5.0^4.0^3.0 PPM ' ' '  Figure 84.  1 1-1 NMR Spectrum of Langarin (142) (Recorded in Me2SO at 400 MHz)  169 0  OH  H11' H11  OH  OH 0  H4 H5 1 H6 1  H2  H  71  OH. ,.....-1  I  J  1^i^I "^I  8.0^7.5^7.0  ' " '  ' ' "I " " 'I " '  "pp m  6. 5^6.0^5. 5^5. 0^4.5 PPM  Figure 85. COSY Spectrum of Langarin (142) (Recorded in Me2SO at 400 MHz)  OH  190^180^170^160^150  Figure 86.  ^  140^130  13C NMR Spectrum of Langarin (142) (Recorded in Me2SO at 125 MHz)  120^110  171  OH 0 OH  OH  O H4 H5^H2 H6^H7  011  ^H11' H11  —so -C11  —SO  —100  •  dI  0 C5  -C4 -C6  0. pp.  ^  —140 ppl  7^ B^ 5  Figure 87. HMQC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz)  172  OH 0 OH  OH 0  H6 H4^H2 H5^H7  H11  ▪ 50 H4/C1 I^• O  ••^••  H2/C1 I  C1I  —75  —100 H5/C8a H4/C9aO H7/C8a ti2/c9a 2* go.23 H2/C4 H4/C20,.,^0 H5/C70 a-or 17 /C:*  HI 1/C4 1'1 1111/C2 411 ,  .911 C4c5 cC8 C2  —125  H6/C10a0 co^co  H11/C3 co. H2/C1 H7/C8 • H4/C10 H5/C10  PP•^8  ^ ^ ^ 7 6 5  Figure 88 . HMBC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz)  -150 C3 CS —175 'C10  173 The five oxygen atoms in the molecular formula of 142 were assigned to two quinone carbonyls and three hydroxyl functionalities. The quinone carbonyls accounted for two sites of unsaturation, thus the remaining nine unsaturations must be double bonds or rings. The 13 C NMR spectrum (Figure 86) contained twelve aromatic carbon resonances (including two with chemical shifts of 5 161.7 (C8) and 162.0 (C1) which were assigned to aromatic carbons bearing hydroxyl groups) indicating that langarin had six double bonds and consequently must be tricyclic. Protonated carbons in the 13 C NMR spectrum were assigned from correlations in the HMQC spectrum shown in Figure 87.  Figure 89. Selected HMBC Correlations for Langarin (142).  The HMBC spectrum (Figure 88) aided in elucidating the structure of langarin. Figure 89 illustrates those HMBC correlations which proved particularly helpful in determining the structure of 142. The aromatic proton resonances at 5 7.68 (H5) and 7.70 (H4) were both correlated into the carbonyl resonance at 5 181.8 ppm (C 10). From this result it was possible to locate spin systems a and b f3 to C10. The hydroxyl functionalities were consequently situated on C8 and Cl. The large difference in the chemical shifts of the two quinone carbonyls (C10, 5 181.8; C9, 192.0) was attributed to deshielding of C9 by hydrogen bonding of the C9 carbonyl with the two hydroxyl groups on C8 and Cl.  174 Further analysis of the HMBC spectrum (Figure 88) permitted assignment of all of the quaternary carbons with the exception of C9 and C4a. The chemical shift of the chelated quinone carbonyl (C9) could not be confirmed from correlations in the HMBC spectrum because it was further than three bonds away from the nearest proton. An HMBC correlation from a proton signal at 5 7.36 (H7) to a carbon resonance at 5 116.2 permitted the carbon to be assigned as C8a. Likewise, a correlation from the H2 resonance (8 7.28) into a carbon signal at 5 114.7 resulted in the assignment of the carbon as C9a. HMBC correlations from the H7 (5 7.36) and H2 (7.28) resonances to carbon resonances at 5 161.7 and 162.0 permitted the assignment of these resonances to C8 and Cl, respectively. An HMBC correlation from the H6 resonance (5 7.80) into a carbon resonance at 6 133.7 ppm allowed assignment of the carbon as ClOa. No HMBC correlations were observed into the C4a signal so the shift was assigned by comparison with the chemical shift of ClOa. A correlation from the signal at 5 4.61 (H11, H11') into a carbon resonance at 6154.1 resulted in the assignment of C3.  The structure of langarin (142) was confirmed by acetylating a portion of the sample. Reaction of a sample of langarin with acetic anhydride in pyridine overnight under anhydrous conditions resulted in a single product. The excess reagents were removed in vacuo to yield langarin triacetate (143), a compound which gave a molecular ion at m/z 396 Da (4.5 % relative intensity) in EILRMS, appropriate for a molecular ion of C2114608. The IR spectrum no longer had the chelated quinone carbonyl stretch at 1623 cm -1 . The spectrum showed three carbonyl stretches, a band at 1675 cm -1 , appropriate for a non-chelated quinone carbonyl, an absorbance at 1770 cm -1 corresponding to the phenol acetate stretch, and a band at 1744 cm -1 resulting from the C11 acetoxymethylene acetate stretch. Table 17 summarizes the 1 H (Figure 90) and 13 C NMR (Figure 91) data recorded for the triacetate 143. The shifts were assigned by comparison with the spectroscopic data of the parent compound 142.  OAc 0 143  175  Table 17. 1 H and 13 C NMR Data for Langarin Triacetate (143) (Recorded in Me2SO)  Carbon no. 1 2 3 4 4a 5 6 7 8 8a 9 9a  10 10a 11, 11' CH2-OAS Ar—OA a,b May be interchanged.  51H (ppm) (400 MHz) — 7.61, d, J = 1.7 Hz 8.09, d, J = 1.7 Hz — 8.13, dd, J = 8.0, 1.2 Hz 7.93, t, J = 8.0 Hz 7.62, dd, J = 8.0, 1.2 Hz —  513C (ppm) (125 MHz) 149.7a — — — — — — — 149.5a — 180.3b  —  —  — 5.26, s 2.13, s 2.38, s; 2.39, s  181.4b — 63.9 21.1, 170.1 20.6, 20.8, 169.0, 168.9  As expected, the hydroxyl protons at 5 11.96, 11.90, and 5.59 in the 1 H NMR spectrum of 142 were no longer apparent in the 1 H NMR spectrum of 143 (Figure 90) but had been replaced by three methyl singlets at 2.13, 2.38, and 2.39, appropriate shifts for acetate methyls. A notable change in the 13 C NMR spectrum (Figure 91) was the upfield shift of one quinone carbonyl (C9) from 5 192.0 in the parent quinone 142 to 180.3 in the triacetate 143. The other carbonyl quinone resonance (C10) did not change position significantly, but had a chemical shift of 5 181.8 in langarin (142) and 181.4 in langarin triacetate (143). This result is consistent with the assignment of langarin as 142 since acetylation disrupts the hydrogen bonding of the Cl and C8 hydroxyl groups with the C9 carbonyl resulting in an upfield shift of the C9 resonance in acetylation product 143. By analysis of spectroscopic data and by chemical interconversion, the structure of langarin, the cytotoxic component of an undescribed Queen Charlotte Islands Aplidium species, was assigned as the anthraquinone 142.  OCOCkL3 OCOCJI3 OCOCH,3  OAc 0  H11' H11  H6 H2 H4 H7 H5  12. 0^1 1. 0^10.0  , I^'^ 9. 0^8.0^7.0^6.0^5. 0^4.0^3.0 PPM  Figure 90.^1 H NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2SO at 400 MHz)  us  ^  Figure 91.  ^  MO^IN^100^II0  ^  so  13 C NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2SO at 125 MHz)  ^ ^  178 iii) CONCLUSIONS  A study of an undescribed species of Aplidium from Langara Island resulted in the isolation and structural elucidation of the new compound langarin (142). This is the first reported anthraquinone from an animal of the subphylum Urochordata. As previously mentioned, the related compound 128 has been isolated from the stony coral Tubastraea micranth, and a number of related compounds have been also reported from terrestrial plants. These include the plant metabolite emodin (144) which has been isolated from a number of sources including rhubarb root. 110 The leaf exudates of the Aloe plant are rich in a variety of related natural products including the barbaloin isomers 145, 10-C-rhamnosyl aloe-emodin anthrone (146), and 11-0-rhamnosyl aloe emodin (147) isolated from the leaves of Aloe rebaiensis. 111 OH 0 OH  ^  OH 0 OH  OH ^ 0 ^ 142 OH 0 OH  ^  0  128 OH 0  ^  OH  OH ^ 0 ^ 144  R 145 R = glucose 146 R = rhamnose  OH 0 OH  0-rhamnose 0 147  It can be postulated that the biogenesis of langarin (142) results from folding, cyclization, and aromatization of a single octaketide unit as shown in Equation 7. 112 Reduction of the carbonyl at C6,  179 decarboxylation, and oxidation of C10 and C11 yields an anthraquinone skeleton with the appropriate oxidation pattern.  decarboxylation 000^1 W;CO2 H 0 0 0  8 CH3CO2H  11  [0]  ^  t  [0]  OH 0 OH  OH 0 142 Equation 7. The Proposed Biogenesis of Langarin (142).  As mentioned earlier, the impetus for this investigation was the cytotoxicity of the methanolic extract of the Aplidium species. Langarin (142) proved to be the compound responsible for this activity. Pure langarin (142) was found to be active in vitro against murine L1210 leukemia cells with an ED50 of 0.4 ttg/mL. The cytotoxicity of langarin (142) was of particular interest since the compound proved completely inactive in bacterial and fungal assays indicating a surprising level of specificity in the L1210 cytotoxicity assay. The results of an in vivo test against leukemia in mice are still awaited.  180 B. SECONDARY METABOLITES OF APL/Q/J/M_CA/IFORN/CUM FROM THE OUEEN CHARLOTTE ISLANDS PRENYLATED OUINONES AND HYDROOUINONES FROM ANIMALS OF THE SUBPHYLUM UROCHORDATA  This thesis presents the isolation of five known and two new prenylated quinones and hydroquinones from the tunicate Aplidium californicum. Consequently, the following section will review reports of prenylated quinones from animals of the subphylum Urochordata. All organisms produce prenylated quinones which are believed to play a crucial role in the electron transport of living systems. 113 Simple prenylated quinones as well as intricately elaborated derivatives are found in marine organisms of every phylum. 8 Fenical first reported the isolation of the geranylhydroquinone 148 from an Aplidium species. 114 Howard and Clarkson reported the structures of prenylhydroquinone (149), 6-hydroxy-2,2-dimethylchromene (150), and prenylquinone (151) from Aplidium californicum collected near San Francisco, Califomia. 115 Prenylhydroquinone 149 proved active in vivo against P388 murine lymphocytic leukemia with a maximum T/C of 138% at 3.12 mg/kg. Addition of compounds 149 and 150 to Salmonella typhimurium which had been treated with the carcinogens benzo(a)pyrene and aflatoxin B1 resulted in a considerable decrease in mutations observed in S. typhimurium. Similarly, 149 decreased the mutagenic effects of ultraviolet radiation. 115  OH  HO  148  HO 149  ^  150  ^  151  Targett and Keeran reported the chromenol 152 from Aplidium constellatum. 116 a This compound had been previously isolated from the tropical American tree Cordia alliodora. 116b  181  HO 152  Pietra and coworkers described the isolation of the verapliquinones A to D (153-156, respectively) from an Aplidium species collected off the Atlantic coast of France. 117 Verapliquinones B (154) and D (156) are the first  compounds with a neryl-type linear monoterpene side chain, where the A 2 ' ,3 '-olefin has the Z rather than the more common E configuration. 117  CH3O  CH 3 O  153  CH 3 O  ^  154  CH 3 O  155  156 OH  The colonial tunicate Amaroucium (synonymous with Aplidium) multiplicatum was found to contain the known quinone derivatives 148 and 152 and the three novel compounds, chromenol 157, and the hydroquinone derivatives 158 and 159. 118 All of the isolated compounds were found to inhibit lipid peroxide formation and 15-lipoxygenase, with ID50 values ranging from 0.3-1.0 pg/mL for the former assay and 0.2-3.0 lig/mL for the latter. Formation of lipid peroxides has been implicated in development of arteriosclerosis in humans.  118  182 OH^OH  H3 CO  HO 157  ^  OH  158 C-1'—C2' = single bond 159 C1'—C2' = E double bond  Although the majority of compounds reported from animals belonging to the Urochordata are nitrogenous, a number of mono- and diprenylated quinone derivatives have been reported. Some of these compounds have proven active in a variety of pharmacological bioassays and it is notable, albeit not surprising, that all of these activities are associated with the antioxidant properties of the prenylated hydroquinones.  ii) RESULTS AND DISCUSSION  PRENYLATED OUINONE AND HYDROOUINONE DERIVATIVES FROM APLIDIUM CALIFORNICUM  A small collection of Aplidium californicum was made off Langara Island, one of British Columbia's Queen Charlotte Islands in May of 1991 (Figure 82). Abbott and Newberry state that A. californicum is common to semiprotected coastal areas ranging from southern California (La Paz) to Vancouver Island. 95 It is found at depths of 0-30 m but has been seen growing as deep as 85 m. Colonies are generally —15 cm in diameter and —1 cm thick. The colors may vary from gray to yellow, white, transparent, or reddish-brown. The surface is described as smooth to irregular.95 The colony of A. californicum collected off Langara Island (Figure 92) was pale yellow in color and soft in texture. The colony was 1 cm thick and 20 cm across with an irregular but smooth surface. The tunicate was common at the collecting site and grew on exposed rock walls at an average depth of 5 m. The collection included a voucher sample (100 g) which was extracted with methanol and a small sample (350 g) which was frozen for later study. The methanolic extract of the voucher sample exhibited mild activity against bacterial and fungal cultures. The cytotoxicity of the extract was promising (ED50 = 6 pg/mL against the in vitro L1210 murine leukemia cell line) and prompted study of the natural products of the Aplidium californicum sample.  183 One half of the Aplidium californicum collection was freeze-dried and extracted sequentially with hexanes, chloroform, ethyl acetate, and methanol. Screening of the hexanes, chloroform, and ethyl acetate extracts by TLC and 1 H NMR indicated that these extracts had much the same chemical composition. In vitro testing against the L1210 cell line indicated that the ED50 values were 11 lig/mL, 5 i_tg/mL, and 3 gg,/mL for the hexanes, chloroform, and ethyl acetate extracts, respectively. The ED50 of the methanol extract was 6 j.tg/mL but the 1 H NMR spectrum of this extract looked less interesting. The hexanes, chloroform, and ethyl acetate extracts were combined and chromatographed by sequential application to a size exclusion gel column (Sephadex LH-20 eluted with ethyl acetate/ methanol/water 20:5:2) and normal phase silica radial TLC (eluted with a polarity gradient of solvents ranging from hexane to ethyl acetate). Final purification was accomplished by use of normal phase HPLC. The lipophilic extracts of the yellow Aplidium yielded four known and two new compounds. The most polar fractions eluted by radial TLC gave the known simple prenylated hydroquinones 149 and 150. The less polar fractions yielded the previously reported hydroquinone derivatives 152 and 158 and the two new prenylated hydroquinones calaplidol A (160) and calaplidol B (161). All of the prenylated quinones and hydroquinones were unstable and decomposed rapidly, in some cases limiting the data which could be collected in order to elucidate the structures.  HO 149  ^  HO 150  HO  HO 152  ^  158  HO HO HO H  160  ^  161  184  Figure 92.^Freshly Collected Aplidium californicum  185  Calaplidol A (1601  HO  Calaplidol A (160) was isolated as a colorless glass which gave an M+1 peak in the LRDCIMS at m/z 261 Da (2.40 % relative intensity), appropriate for a molecular formula of C161-12003. The EIHRMS did not  show a molecular ion but had an intense peak (9.06 % relative intensity) at m/z 242.1303 Da corresponding to the molecular ion with loss of one equivalent of water (C161-11802 [AM -0.3 mmu]). The molecular formula indicated that the molecule had seven sites of unsaturation.  The 1 H NMR spectrum (Figure 93) contained resonances at 5 6.63 (H5), 6.64 (H3) and 6.82 (H6) which indicated that calaplidol A had a trisubstituted benzene ring. The coupling patterns suggested 1,2,4-substitution (see Table 18).  The connectivity of the side chain was determined by analysis of the spectroscopic data which is summarized in Table 18. The 13 C NMR spectrum is shown by Figure 94. The carbon resonances were assigned from the HMQC and APT (Figure 96) spectra. Two methyl singlets at 5 1.49 (Me9') and 1.70 (Me8') in the 1 H NMR spectrum both had weak COSY correlations (Figure 95) consistent with allylic coupling into a single  proton resonance at 5.20 (H6'). The chemical shift of this proton resonance confirmed that it was olefinic as did a correlation in the HMQC spectrum to a carbon resonance at 5 120.0 ppm. The olefinic proton resonance at 5 5.20 (H6') showed further COSY correlations into a pair of geminal methylene resonances at 2.36 (H5' a) and 2.50 (H5'b). The pair of methylene resonances (H5' a , H5'b) were further coupled into a single proton signal at 8 4.48 (H4'). The HMQC spectrum showed a correlation from the proton resonance at 5 4.48 into a carbon signal at 5 82.0 ppm (C4'),  186 a chemical shift appropriate for a carbon bearing an oxygen functionality. The above data was used to elucidate fragment a.  a  Table 18. 1 H and 13C NMR Data for Calaplidol A (160) (Recorded in CDC13) Carbon no.  81H (ppm) (400 MHz)  COSY (400 MHz)  813C (ppm) (100 MHz)  HMBCc (500 MHz)  1 — 147 2 — 117.1 — 3 6.64, ma — 151 4 — 115.7 H6 — 5 6.63, ma 122.2 — 6.82, d, J = 9.1 Hz H5 6 H2' 127.4 — 1' 6.36, d, J = 11.7 Hz H1', H10' 131.1 6.19, d, J = 11.7 Hz C2, C10 2' 3' — 4.48, m 4' 82.0 — H5'a, H5'b H5'b, H6' 30.8 5' a 2.36, m 5'b 2.50, m H5'a, H6' — — 120.0 — 5.20, m H5'a, H5'b, H8', H9' 6' — 134 — — 7' H6', H9' C6', C7', C9' 8' 1.70, sa 25.8b H6', H8' C6', C7', C8' 9' 1.49, sa 16.5b H10'b, H2' 115.9 5.09, br s C2', C4' 10'a C2', C4' 5.16, br s H10'a 10'b — a,b May be interchanged. c The carbon resonances in the HMBC column are correlated to proton resonances in the 8 1 H column. —  —  —  —  —  —  —  —  —  HO HO H  7  ^  , - 5  Figure 93.  ^  ^3  1 11 NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)  ^  ppm  Figure 94,^13 C NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 125 MHz)  189  HO HO H  1 .  1^.•  8.3  I  i^ : i^ I  .5  ■  ,  13  Me9'/H6'  s ,, Me8'/H6' •  1.5  ,  2.0 2.5  H5/H510 ,^ • I^ : I H5'b/H4' H5'a/H4'  .  4.  i,  H2'/H10' H10'a/H1O'b  i i H6/H5LIAS ATP  1  i  1  s °4  H6'/H5'a H6'/H5'b  5.5  .  . i !  7 5  5.8  1 !  6.3  H1'/H2'  .  . . .  7 a 6 5 6 3 5.5 5 8 4 5 4.3 3 5 3 8 2 5 2 8 t5 1.3  PPM  a  4.5  .A 1  3.5  a a PPM  Figure 95.^COSY Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)  6.5  7.a 7.5  S  HO HO H  I T T^ r^ u^r^ 140^1i0^100^1;0^SO^40^20^ 1 PPM  Figure 96. APT Spectrum of Calaplidol A (160) (Recorded in CDC13 at 100 MHz)  I  g  5 HO HO H H1O'b FIllr a  C9' C8'  Me8'/C9' •  o^•co  •^  Me9'/C8'  H 10'b/C4' •• C4' H10',/C4' —100  •  100  H1O'b/C2' H I O' a/C2' •  Me8'/C6'^Me9'/C6'  C6'  Me8'/C7' a .ro Me9'/C7'  CT  —les  •  —100 , • .^ •^ ,—, • 7.0^8.5^5.0^0.5^0.0  17 PP PPM  ie  t.0^1.5  Figure 97 . HMBC Spectrum of Calaplido! A (160) (Recorded in CDC13 at 500 MHz)  1.0^0.5  •  %S.  192  The COSY spectrum (Figure 95) showed a correlation from a deshielded proton resonance at 8 6.36 (111') to another downfield proton resonance at 6.19 (H2'). The chemical shifts of these protons in conjunction with correlations in the HMQC spectrum from the proton signals at 8 6.36 and 6.19 into carbon resonances at 8 127.4 and 131.1, respectively, indicated that 160 contained a disubstituted double bond. The coupling constant of 11.7 Hz for the resonances at 8 6.36 (H1') and 6.19 Hz (H2') was in better agreement with the coupling constant for H1' and H2' of the chromenol 152 (J = 9.8 Hz) which had the Z configuration than with the coupling constant of 159 (JH1',H2' = 18 Hz) 117 which had the E configuration. The 13 C NMR resonances for C1' (8 127.4) and C2'  (131.1) of 160 were compared with the corresponding chemical shifts for 152 (C1', 122.6; C2', 131.0) and 159 (C1', 134.7; C2', 125.3). 117 Again, the chemical shifts for 160 were in better agreement with those of 152 than 159 and the A 1 ' ,2 ' bond of metabolite 160 was consequently assigned the Z configuration.  HO H^H HO H ^ 6.36 6.19 160  ,7,..........,01{ 6 H.10 I^125.3  HO  122.6 1'  H  6.27 152  131.0 2' H 5.59  HO  134.7 1' H^HO 6.97 159  A weak COSY correlation (Figure 95) was observed from the proton signal at 8 6.19 (H2') to a broad singlet at 5.09 (H10' a). The latter signal showed a COSY correlation into another broad singlet at 8 5.16 (H10'b). The HMQC spectrum (see also APT spectrum (Figure 96)) indicated that the protons at 8 5.09 (HIO' a ) and 5.16 (H1O'b) were attached to the same olefinic carbon at 8 115.9 ppm. The data permitted the assembly of fragment b.  193 6.19 H  \ 5.9 H^H 5.09^5.16 b  Double bonds in the side chain accounted for three sites of unsaturation leaving four unsaturations for the aromatic ring. This suggested that 160 was a hydroquinone as a quinone would require five sites of unsaturation. The IR spectrum confirmed that calaplidol A was a hydroquinone since no carbonyl stretch was observed in the vicinity of 1675 cm -1 , the expected frequency for a quinone carbony1. 109 A strong band which could be seen at 3369 cm -1 in the IR spectrum was attributed to hydroxyl stretching. The phenols of the hydroquinone ring accounted for two of the oxygen atoms in the molecular formula leaving one unassigned. The final oxygen was assigned to a hydroxyl functionality at C4'.  The structure was assembled from the individual fragments a and b by use of the HMBC data (Figure 97). Selected HMBC correlations are presented in Figure 98. HMBC correlations from the Me8' (5 1.70) and Me9' (1.49) resonances to the carbon signals at 5 120.0 and 134.0 confirmed the allylic relationship of Me8' and Me9' and permitted the latter carbon resonance to be assigned to C7'.  1.70 ( CH 3 ‘k  HO  82.0  131.  HO H  Figure 98. Selected HMBC Correlations for Calaplidol A (160).  134.0  ,".w ...._.CH 3 120.0^1.49 ",..._...."  194 The H10'a and H10'b proton resonances (8 5.09, 5.16) had HMBC correlations to carbon resonances at 8 131.1 (C2') and 82.0 (C4'). The latter resonance had been assigned to C4', the hydroxy terminus of fragment a, from the HMQC data. The HMBC data established that fragments a and b were linked to form a monoterpene side chain with A 1 ' ,2 ' and A 3 ' ,1I3 'unsaturations and a hydroxyl substituent at C4'. Once the side chain had been assembled, the hydroquinone could only be attached to C1'. The structure of calaplidol A was tentatively assigned as 160.  Calaplidol B (161)  8 CH 3 '  HO  Calaplidol B (161) was isolated as an unstable colorless glass. The EIHRMS had a peak corresponding to the molecular ion at m/z 352.1669 Da appropriate for a formula of C22H2404 (AM -0.6 mmu). From the molecular formula it was evident that 161 had eleven sites of unsaturation. The IR spectrum of 161 had a strong hydroxyl stretch at 3361 cm -1 . No carbonyl stretch was evident in the IR spectrum of calaplidol B indicating that the compound was not a quinone.  The structure of 161 was proposed from analysis of the spectroscopic data which is summarized in Table 19. The aromatic region of the 1 H NMR spectrum (Figure 99) was congested with signals which integrated for a total of eight protons of chemical shift 8 6.2 to 7.0 ppm. Another olefinic proton was apparent at 8 5.10. A pair of olefinic methyls were evident at 8 1.79 and a final methyl singlet had a chemical shift of 1.47 ppm.  195  Table 19. 1 H and 13 C NMR Data for Calaplidol B (161) (Recorded in CDC13)  Carbon no.  81H (ppm) (400 MHz)  COSY (400 MHz)  1 2 3 4 5 6 1' 2' 3' 4'a 4'b 5' 6' 7' 8' 9' 10' 1" 2" 3" 4" 5" 6" Ar-OH Ar-OH Ar-OH  — — 6.86, br s  — — H5  6.62, m 6.76, d, J = 8.6 Hz 6.84, br d, J = 16.2 Hz 6.33, d, J = 16.2 Hz — 1.78, dd 1.93, dd 3.73, m 5.10, m — 1.79, br s 1.79, br s 1.47, s — — 6.57, dd, J = 3.0, 0.8 Hz — 6.62, m 6.68, d, J = 8.6 Hz 4.55, s 4.35, s 4.28, s  H3, H6 H5 H2' H1' — H4'b, H5' H4'a, H5' H4'a, H4'b, H6' H5', H8', H9' — H6' H6' — — H5", H6' H3", H6" H5" — —  813C (ppm) (100 MHz) 147c 125 113.5 148c 115.4 118.0 121.6 137.3 76.4 39.2 — 32.0 127.1 134 25.8b 17.6b 23.6 147a 125 114.9 149a 115.0 116.9 — —  HMBCd (500 MHz) — — Cl, C5 —  Cl C2, C4 C3' C2 —  —  — —  C7', C8', C9' — C6', C7', C9' C6', C7', C8' C2', C3', C4' — —  Cl", C5" — Cl" C2", C4" —  _  a,b,c May be interchanged.d The carbon resonances in the HMBC column are correlated to proton resonances in the 8 1 H column.  PP R  6  5  4  3  2  i  0  Figure 99 1 11 NMR Spectrum of Calaplidol B (1611 (Recorded in CDC13 at 500 MHz)  g  197  HO  Me9' Me8'  •  . .^...  •  •  . 1.4  ..  ,.  %  0.0 •  .  . .  ..,^4, -^6  4  1.0  ,  2.0 e.  .^.  i:.  IC  .  . ....4..  3.0  H5'/H4'b btv  9  1  r  .  .•  _  ..  6.0  -  ;-3  H3"/H5"  , .  -  H3 "/H5' H2'/Hl' -  70  .  . • -  •a  0  '^•  .  60  5 0  7.0  N. N^ .g.^•  ..,^.  40 PPM  5.0  Me9'/H6'  H6'/H5'  .  ‘,  --o-Me8'/H6' '  0-  C  4.0  H5'/H4' a  -^--  30  2.0  1.0  0.0  PM  8. 0  Figure 100 COSY Spectrum of Calaplidol B (161) (Recorded in CDC13 at 400 MHz)  S  HO  1 40  120  10 0  • r POI  80  •  60  •  •  i 40  Figure 101, 13C NMR Spectrum of Calaplidol B (161) (Recorded in CDCI3 at 100 MHz)  20  • 4  C9' cg C10' C5' ,  0 0^C4'  •  50  il  100 0  0  0^  •  ■..-. . I I^I PP ^6^ 4^ 2 ,  C6'  ppm C2' ppm  1^1^.^I 6.0^6.0^6.4  Figure 102 HMQC Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz)  HO  OH 5" H1'^H5" H3 H6 H6" H5 H3"  H2' C9'  Me8'/C9' • Me9'/C8'  C8' Me10'/C4'  C4' —  50  C3'  dd. Me107C3'  —100  Me8'/C6' • Me9'/C6  C6'  Me8'/C7' Me9'/C7'  ' ^  1^  - 7..  PIM  Me107C2'  I^1^  2.0^1.5  Figure 103 HMBC Spectrum of Calaplidol B (161) (Recorded in CDCI3 at 500 MHz)  CT  C2'  1  F PP.  201 The spin systems of the side chain were elucidated from the COSY spectrum (Figure 100). The olefinic methyl signal at 8 1.79 (Me8', Me9') was weakly correlated to a single olefinic proton resonance at 5.10 (H6') (HMQC correlation to 8 127.1, Figure 102). This resonance in turn had a COSY correlation to a multiplet at 8 3.73 (H5') which integrated for one proton and was further correlated to geminal methylene resonances at 1.78 (H4' a) and 1.93 (H4'b). This spin system is shown by fragment c. Correlations in the HMBC spectrum (Figure 103) from the proton resonance at 8 1.79 (Me8' and Me9') to carbon resonances at 8 127.1 and 134 established the allylic relationship of H6' to Me8' and Me9' and permitted the assignment of the carbon resonance at 8 134 to C7' (Figure 104). 1.781.93^5.10 H H3 . 73 H H  1.79 CH3  CFI 1.74  6.76 -H  H  147 OH  6.33 H 1.47  H 127.1^1'79 CH 3  39.2  HO  14 8  137.3 H 6.86  134  CH  125 6.84  H 1.74  6.57\ 149  H 6.68^115.0  Figure 104. Selected HMBC Spectroscopic Correlations for Calaplidol B (161).  202 In the COSY spectrum (Figure 100) a correlation was observed from a doublet at 5 6.84 (H1') to a signal at 6.33 (H2'). This constituted an isolated double bond. The coupling constant of 16.2 Hz for the resonances at 5 6.84 (H1') and 6.33 (H2') established that the Al "2 '-bond had the E configuration.  The constitution of the isoprene-derived side chain was elucidated from HMBC correlations which were observed from the methyl singlet at 5 1.47 (Me10') to carbon resonances at 5 137.3 (C2'), 76.4 (C3'), and 39.2 (C4') (Figure 103, Figure 104). This data established that fragment c and the A 1 ' ,2 '-double bond were linked via a quaternary carbon at 5 76.4 (C3') (HMQC spectrum, Figure 102). From the HMBC correlation of the Me10' resonance (6 1.47) to the C3' signal (8 76.4) it was determined that Me10' was attached to C3'. The chemical shift of C3' (576.4) was diagnostic of a carbon with an attached oxygen functionality.  Six protons in the aromatic region remained unassigned. From the coupling constants it appeared that these were protons belonging to two tri-substituted benzene rings. The most upfield aromatic proton signal, at 5 6.57 (H3") was split into a doublet of doublets with coupling constants of 3.0 Hz and 0.8 Hz. The coupling constant of 3.0 Hz corresponded to meta coupling to another proton on the aromatic ring. A COSY correlation (Figure 100) from the signal at 5 6.57 (H3") to a multiplet at 6.62 (H5") confirmed that the protons are meta relative to one another. The smaller coupling constant of 0.8 Hz had the appropriate magnitude for allylic coupling. A weak COSY correlation was in fact evident from the signal at 5 6.57 (H3") to the multiplet at 3.73 (H5'). This was interpreted to mean that C5' is the point of attachment for a benzene ring. The aromatic multiplet at 5 6.62 (H5, H5") integrated for two protons and was further coupled to two resonances at 6.68 (H6") and 6.76 (H6). These resonances both had coupling constants of 8.6 Hz indicating that each is an aromatic proton ortho to one of the two protons which overlap to form the multiplet at 5 6.62 (H5, H5"). From the COSY spectrum it could not be determined which of the signals at 8 6.68 (H6") and 6.76 (H6) belonged to ring A and which to ring B. It appeared, however, that ring B was a 1,2,4-trisubstituted aromatic ring. The aromatic multiplet at 5 6.62 (H5, H5") showed further coupling into a broad singlet at 6.86 (H3). The downfield shift of the A-ring proton at 5 6.86 (H3) was attributed to deshielding resulting from extended conjugation into the A 1 ' ,2 'double bond. These data were interpreted to mean that ring A, too, is a 1,2,4-trisubstituted benzene ring.  203 As shown in Figure 104, HMBC correlations from the aromatic protons of rings A and B permitted the assignment of the carbon resonances at 8 125 (C2 and C2") in addition to the phenolic carbon signals at 147 (C1), 148 (C4), 147 (Cl"), and 149 (C4"). From the COSY and HMBC data it was determined that calaplidol B (161) contained two substituted hydroquinone rings. The similarity in the shifts of C2 and C2" and of the phenolic carbons meant that the signals can not be confidently differentiated, but it also substantiated the conclusion that both rings were similarly substituted. Three singlets in the 1 H NMR spectrum at 8 4.55, 4.35, and 4.28 were not correlated to carbons in the HMQC spectrum and were consequently assigned to the hydroxyl protons. The chemical shifts of the hydroxyl protons were in good agreement with the data for the related compound 162 (8 4.55 in CDC13). 119 4.55 OH  162 The substitution pattern of the B ring of 161 was confirmed by comparison of the 13 C NMR spectrum with the corresponding chemical shifts for model compounds 4-methylcatechol (105) and 2-methylhydroquinone (163). While the chemical shifts and coupling pattern of ring A of 161 appeared to be consistent with the data for  calaplidol A (160), the B ring seemed to contain a multiplet at 8 6.62 with fine coupling of —0.8 Hz. This coupling constant was appropriate for allylic coupling and suggested that ring B might be a 1,2,4-substituted catechol rather than a hydroquinone, where H6" also had allylic coupling to H5'. Allylic coupling from the protons of ring A to H1' was unlikely because the proton resonance for Hr (8 6.84) was a sharp doublet (coupled to H2', -1 1-11' ,H2' = 16.2 Hz). Figure 101 shows the 13 C NMR spectrum of calaplidol B (161). The carbon shifts of 161 were assigned from the HMQC (Figure 102) and HMBC spectra (Figure 103). The HMBC data was of no assistance in determining the substitution pattern of ring B so the carbon shifts of calaplidol B (161) were compared to the carbon shifts of the model compounds 4-methylcatechol (105) (see Table 3) and 2-methylhydroquinone (163) (Figure 105).  204  125^OH^125.2  OH  113.1^  115.0  161  HO  ^  120.4  117.1^115.1 115.6  163  ^  OH 105  Figure 105. Selected 13 C NMR Shifts of Calaplidol B (161), 2-Methylhydroquinone (163), and 4-Methylcatechol (105).  It can be seen from Figure 105 that the close agreement of the carbon shifts of ring B with those of 2-methylhydroquinone indicated that ring B was more likely to be a 2-substituted 1,4-hydroquinone than a 4-substituted 1,2-catechol.  Two hydroquinone rings in addition to the two double bonds in the side chain accounted for ten of the eleven sites of unsaturation of 161. As previously mentioned, a COSY correlation from the H3" resonance (8 6.57) to the H5' multiplet (3.73) provided evidence that the B ring was attached to the isoprene-derived side chain at C5'. An HMBC correlation from the proton resonance at 8 6.33 (H2') to a carbon signal at 8 125 situated the A 1 ' ,2 '-double bond adjacent to ring A (Figure 103, Figure 104). One site of unsaturation remained unassigned. The spectroscopic data contained no evidence for additional unsaturated functional groups and consequently the final unsaturation was attributed to a third ring. The chemical shift of C3' (8 76.4) indicated that the final ring consisted of a cyclization where C3' was attached to the hydroxyl of one of the hydroquinone rings to form a chromenol. The E stereochemistry of the A 1 ' ,2 '-double bond obviated the possibilty of a chromenol moiety which incorporated the A ring. The only structure which can accomodate all the functionalities of calaplidol B and three rings was determined to be 161 where C3' was attached to the Cl" oxygen to form a chromenol fragment incorporating ring B. The structure was tentatively assigned to be 161 but further analysis is required to confirm the structure and to elucidate the stereochemistry of the C3' and C5' centres.  205  HO  161  Known Metabolites from the Tunicate Aplidium californicum  In addition to the new compounds calaplidol A (160), and calaplidol B (161), our collection of Aplidium californicum yielded four known compounds. The structures of the simple prenylated hydroquinone 149 and the  chromenol 150 were determined from the mass spectra and by comparison of the 1 H NMR with published data (see experimental). 115  The structures of the hydroquinone 158 118 and the chromenol 152 116b were elucidated from spectroscopic data including mass spectra, 1 H NMR, 13 C NMR, COSY, HMQC, and HMBC spectra (see experimental). The structure of 158 was confirmed by comparison of the chemical shifts of the 1 H NMR and 13 C NMR spectra with reported results. 118 The structure of 152 was in turn substantiated by comparing the 1 H NMR spectrum with published values. 116b  HO  HO 150  149  OH HO  HO  HO 152  158  206 iii) CONCLUSIONS  Examination of extracts of the colonial tunicate Aplidium californium led to the isolation of the two new metabolites 160 and 161. The known compounds 149, 150, 152, and 158 were also isolated. All of the isolated metabolites are closely related and originate from a hemiterpene or monoterpene substituted quinone moiety.  HO 149  ^  HO 150  HO  HO  158  HO  HO HO H  160  ^  161  It can be postulated that the biogenesis is comparable to that of ubiquinone (164) and follows the shikimic acid pathway. As shown in Scheme 3 the first step is the formation of p-hydroxybenzoic acid. This is accomplished by the loss of pyruvic acid from chorismic acid in bacteria or by degradation of phenyl alanine in higher plants and mammals. 112 The prenyl side chain is subsequently introduced ortho to the hydroxyl function. Decarboxylation occurs followed by introduction of a second hydroxy para to the first to yield the hydroquinone which may be oxidized to form the corresponding quinone. H 3CO^CH3  H3 CO  164 R = prenyl sidechain  207 CO2H^  CO2H  Phenylalanine or Chorismic Acid OH  ^  OH  p-hydroxybenzoic acid OH  OH  OH  Scheme 3. The Proposed Biogenesis of Prenylated Quinones and Hydroquinones It is likely that calaplidol A (160) results from oxidation of the side chain of a monoterpene-substituted hydroquinone followed by elimination to form the 1 3 ' ,10 ' double bond. Calaplidol B (161) may be the product of a condensation reaction involving two hemiterpene-substituted hydroquinone units or of the substitution of a monoterpene chain by two aromatic rings followed by cyclization to form the chromenol moiety. Our initial interest in the Langara Island collection of Aplidium californicum stemmed from the promising cytotoxic activity of the methanolic extract of a voucher sample. Compound 152 was tested in vitro against the L1210 murine leukemia cell line and found to be cytotoxic with an ED50 value of 3.9 j.tg/mL. As mentioned earlier, Howard and Clarkson have reported that the prenylated hydroquinone 149 is active against P388 murine leukemia in vivo. 115 It would be interesting to test the cytotoxicities of calaplidol A (160) and calaplidol B (161) but the assays were precluded in this instance by the instability of these compounds. It would be worthwhile to reisolate calaplidol A (160) and, particularly, calaplidol B (161) in order to test these compounds for biological activity and also to determine the stereochemistry of C4' in 160 and C3' and CS' in 161. NOE difference or ROESY experiments should be conducted to confirm the structure of calaplidol B (161).  208 GENERAL EXPERIMENTAL  Proton NMR spectra were recorded on Bruker WH-400 and AMX-500 spectrometers. The spectra were referenced to tetramethylsilane (8 0.00 ppm) or residual solvent peaks as secondary references (Me2SO-d6 8 2.49 ppm, pyridine-d5 8 7.19 ppm, C6D6 8 7.15 ppm, CDC13 8 7.26 ppm). 13 C and APT NMR spectra were recorded on Varian XL-300, Bruker AM-400 and AMX-500 spectrometers. 13 C spectra were referenced to residual solvent peaks (Me2SO-d6 8 39.5 ppm, CDC13 8 77.0 ppm). COSY, 73 b NOE difference, 73 c and double resonance experiments were performed on a Bruker WH-400 spectrometer. ROESY, 80 HMQC,77 and HMBC 78 experiments were conducted on a Bruker AMX-500 spectrometer. Low and high resolution FAB mass spectra were recorded on a Kratos Concept II HQ spectrometer. Low and high resolution electron impact mass spectra were performed on a Kratos MS-50 spectrometer. DCI mass spectra were recorded by use of a Delsi-Nermag R10-10C instrument. Infra-red spectra of sample films on sodium chloride plates were recorded on a Perkin-Elmer 1600 FT-IR spectrophotometer. UV spectra were recorded by use of Bausch and Lomb Spectronic 2000 and Perkin-Elmer Lambda 4B spectrometers (1 cm quartz cells). CD spectra were obtained by use of a Jasco J-710 spectropolarimeter (1 mm quartz cells). Uncorrected melting points were measured on a Fisher-Johns melting point apparatus. Normal and reversed phase thin layer chromatography was done using Merck type 5554 aluminium-backed Kieselgel 60 F254 and Whatman MKC18F reversed phase TLC plates, respectively. TLC plates were visualized by UV or by use of H2SO4 and vanillin sprays. Further detail concerning spray preparation is provided by Stah1. 120 Size exclusion chromatography used Sigma Sephadex LH-20-100 gel (bead size 25-100 11). Normal phase flash chromatography employed either Merck silica gel 060 (230-400 mesh) or Sigma type H TLC grade silica gel (10— 401.t, no binder). 121 Merck silica gel 60 PF-254 with CaSO4.1/2 H2O was used for radial TLC (Harrison Research Chromatotron model 7924). For the purification of glaciasterol A (100) AgNO3 (5.6 g) was added to the silica gel slurry prior to pouring the chromatotron plate. 122 Samples were prepared for HPLC purification by application to Waters Sep-Pak silica and C18 cartridges prepared according to package instructions. HPLC separations used one of two systems. The first is comprised of a Waters 501 HPLC pump equipped with a Waters model 440 absorbance detector and a Perkin-Elmer LC-25 RI detector. The other system consists of a Perkin-Elmer series 2 liquid chromatograph and LC-55 spectrophotometer coupled to a Waters 410 differential refractometer. Reversed phase separations required Alltech  209 Associates Econosil C18 5 p. analytical and Whatman Partisil 10 ODS-3 magnum columns. Normal phase HPLC separations were accomplished by use of Alltech silica 5p. analytical and Whatman Partisil 10 magnum columns. All HPLC solvents were BDH Omnisolve grade. All reagents were commercial grade and were used without further purification. An exception is pyridine which was distilled from and stored over BaO. Homogenization of invertebrates was achieved by use of an Osterizer 1 L capacity blender. In vitro antibacterial and antifungal assays were carried out by Mike Leblanc (UBC.). Cytotoxicity assays for effectiveness against the mouse L1210 (in vitro) leukemia cell lines were supervised by Dr. Theresa M. Allen at the University of Alberta (Department of Pharmacology). The breast cancer MCF-7 and MCF-7 Adr cell line assays (in vitro) were carried out by John Stingl under the direction of Dr. Joanne T. Emerman of the Department of Anatomy, UBC.  Glaciasterols A and B from the sponge Aplysilla glacialis Collection and Isolation Data  Isolation of Glaciasterol A (100)  Aplysilla glacialis 123 was collected by SCUBA at -5m in Sydney Inlet, Vancouver Island and off Sanford  Island in Barkley Sound, British Columbia. The following procedure was typically used to isolate glaciasterol A (100). Sponge freshly collected in Sydney Inlet (160 g, wet weight) was soaked in methanol (1 L) and stored at +7°C for four months. The methanolic extract was decanted, filtered and concentrated. The sponge was homogenized and soaked for 16 hours in methanol/dichloromethane (1:1). Again the organic extract was decanted, filtered, and concentrated in vacuo to give an aqueous slurry. The crude extracts were pooled and partitioned between brine (500 mL) and ethyl acetate (3 x 250 mL). The combined ethyl acetate extracts were dried over Na2SO4, filtered, and concentrated in vacuo to yield a pungent brown oil (1.67 g). The ethyl acetate extract (1.67 g) was applied to a normal phase silica gel flash column and eluted with a step gradient of solvents beginning with hexane through ethyl acetate to methanol. Twenty-nine fractions were collected, analyzed by TLC and pooled to give eight fractions. The seventh fraction (148 mg, eluted in methanol/ ethyl acetate 1:3) had 1 H NMR signals diagnostic of a mixture of glaciasterols. The glaciasterols were further purified by application to a normal phase SepPak eluted with ethyl acetate / benzene (3:7). The glaciasterol fraction  210 was enriched in glaciasterol A (100) by radial TLC chromatography using a normal phase silica plate saturated with AgNO3 10 and eluted with ethyl acetate/benzene (4:6). One hundred and seventy one fractions were collected, analyzed by TLC, and pooled to yield thirteen fractions. 1 H NMR showed that the fourth fraction contained glaciasterol A (100) in the highest concentration. The fourth fraction was prepared for HPLC by application to a reversed phase Sep-Pak cartridge eluted with water/methanol (3:7, 10 mL). HPLC purification by use of a reversed phase analytical column eluted with water/methanol (3:7) yielded pure glaciasterol A (100) (1 mg).  Glaciasterol A (100)  Glaciasterol A (100) was isolated as an amorphous, white solid; IR 3359, 2953, 1681 cm -1 ; CD (methanol) ( 0 )204 -24000, ( 8 )263 - 22000, (9)336 9900; 1 H NMR (CDC13, 400 MHz) 8 0.68 (s, 3H), 0.94 (d, J = 6.7 Hz, 6H), 1.02 (d, J = 6.8 Hz, 3H), 1.15 (1H), 1.24 (s, 3H), 1.58 (1H), 1.59 (1H), 1.63 (1H), 1.66 (1H), 1.68 (1H), 1.8 (1H), 2.1 (1H), 2.13 (2H), 2.19 (m, 1H), 2.22 (m, 1H), 3.39 (d, J = 4.6 Hz, 1H), 3.39 (ddd, J = 11.3, 8.1, 0.9 Hz), 3.69 (m, 1H), 3.81 (m, 1H), 3.99 (m, 1H), 5.27 (dd, J = 15.3, 7.6 Hz, 1H), 5.29 (dd, J = 15.3, 6.1 Hz, 1H), 6.80 (dd, J = 4.6, 0.9 Hz, 1H) ppm;^13 C NMR (CDC13, 125 MHz) 8 17.8, 21.3, 21.5, 22.6, 22.7, 26.6, 26.9, 29.8, 30.4, 31.0, 37.4, 38.5, 40.4, 43.9, 45.6, 46.1, 49.6, 53.5, 59.1, 63.5, 68.4, 132.2, 134.2, 136.0, 139.3, 201.8 ppm; EILRMS, m/z (formula, relative intensity): 400 (C26H4003, 8), 382 (C26 113802, 5), 285 (C19l-1 2502, 10), 97 (C71113, 69); EIHRMS m/z: 416.2936 (C26H4004, AM +0.9 mmu); ED50: 2.1 p.g/mL (L1210 in vitro murine leukemia cell line); 1D50: 19 µg/mL (MCF-7 in vitro human breast cancer cell line) and 18 pg/mL (MCF-7 Adr multidrug resistant in vitro human breast cancer cell line).  211 Acetylation of Glaciasterol A (100): Glaciasterol A (100) (1 mg) was stirred overnight under a nitrogen  atmosphere with pyridine and acetic anhydride (0.5 mL each). Excess reagent was removed in vacuo to yield pure glaciasterol A diacetate (106). Glaciasterol A Diacetate (106)  AcO  Glaciasterol A diacetate (106) was isolated as a white solid; UV max (CH2C12) 254 nm (e 2850); IR 1737, 1685  cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 0.72 (s, 3H), 0.95 (d, J = 6.8 Hz, 6H), 1.03 (d, J = 6.8 Hz, 3H), 1.22 (s, 3H), 1.26 (1H), 1.57 (1H), 1.64 (1H), 1.65 (1H), 1.70 (1H), 1.71 (1H), 1.74 (1H), 1.77 (1H), 2.01 (s, 3H), 2.04 (s, 3H), 2.10 (1H), 2.17 (1H), 2.17 (1H), 2.21 (1H), 2.26 (t, J = 12.5 Hz, 1H), 3.23 (dd, J = 10.7, 7.5, 1H), 3.37 (d, J = 4.5 Hz, 1H), 4.10 (m, 1H), 4.16 (m, 1H), 4.99 (m, 1H), 5.27 (dd, J = 15.3, 6.2 Hz, 1H), 5.28 (dd, J = 15.3,  6.2 Hz, 1H), 6.67 (d, J = 4.5 Hz, 1H) ppm; 13 C NMR (CDC13, 125 MHz) 8 17.6, 21.1, 21.2, 21.2, 21.5, 22.6, 22.7, 25.1, 26.6, 27.4, 27.6, 31.0, 34.0, 36.7, 38.2, 43.8, 45.3, 46.1, 50.4, 53.4, 61.1, 63.0, 70.7, 131.9, 136.2, 138.6, 141.2, 170.0, 170.1, 200.0 ppm; EILRMS, m/z (relative intensity): 500 (2), 482 (1), 440 (3), 424 (7), 364 (7), 97 (56); EIHRMS m/z: 500.3139 (C301 -14406, AM +0.1 mmu).  Acid-Catalyzed Ring-Opening of the 5.6-Epoxide of Glaciasterol A Diacetate (106): Glaciasterol A diacetate (106)  was stirred for two hours with THE (0.5 mL) and perchloric acid (20 p.L, 69-72% aqueous solution). Dichloromethane (20 mL) was added to the reaction and excess acid was neutralized by washing once with 5% NaHSO3(aq.) (20 mL). The aqueous layer was extracted sequentially with dichloromethane (3 x 20 mL). The organic extracts were combined and washed with brine (2 x 20 mL). The dichloromethane layer was dried over Na2SO4, filtered, and evaporated in vacuo to yield 5a,613-dihydroxyglaciasterol A diacetate (113). The diol was  212 acetylated as previously described and purified by reversed phase HPLC (water/methanol 1:4) to yield 5a-hydroxyglaciasterol A triacetate (114).  5a.60-Dihydroxvglaciasterol A Diacetate (113)  AcO  5a,60-Dihydroxyglaciasterol A diacetate (113) was isolated as a white solid; 1 H NMR (CDC13, 400 MHz) 6 0.75 (s, 3H), 0.95 (d, 6H), 1.05 (d, 3H), 1.37 (s, 3H), 2.02 (s, 3H), 2.05 (s, 3H), 3.28 (m, 1H), 4.04 (d, 1H), 4.16 (m, 2H), 5.12 (m, 1H), 5.28 (m, 2H), 6.40 (d, 1H) ppm; EILRMS, m/z (relative intensity): 500 (5), 440 (6), 343 (6), 97 (79); EIHRMS m/z: 518.3242 (C30H4607, AM -0.1 mmu).  5a-Hydroxyglaciasterol A Triacetate (114)  AcO  5a-Hydroxyglaciasterol A triacetate (114) was isolated as a white solid; IR 3402, 2920, 1738, 1688 cm -1 ; 1 H NMR (CDC13, 400 MHz) 5 0.68 (s, 3H), 0.95 (d, J = 6.7 Hz, 6H), 1.03 (d, J = 6.8 Hz, 3H), 1.30 (1H), 1.35  (s, 3H), 1.57 (1H), 1.60 (1H), 1.68 (1H), 1.70 (1H), 1.83 (1H), 1.96 (1H), 2.01 (s, 3H), 2.03 (1H), 2.04 (s, 3H), 2.13 (s, 3H), 2.19 (1H), 2.21 (1H), 3.29 (dd, J = 10.4, 8 Hz, 1H), 4.17 (m, 2H), 5.09 (m, 1H), 5.19 (d, J = 5 Hz,  213 1H), 528 (m, 2H), 6.34 (d, J = 5 Hz, 1H) ppm; 1 H NMR (Pyridine-d5, 400 MHz) 8 0.81 (s, 3H), 0.96 (d, 6H), 1.09 (d, 3H), 1.52 (s, 3H), 2.00 (s, 3H), 2.05 (s, 3H), 2.15 (s, 3H), 3.54 (m, 1H), 4.50 (m, 2H), 5.33 (m, 2H), 5.67 (m, 1H), 5.91 (d, 1H), 6.66 (d, 1H); EILRMS, m/z (relative intensity): 500 (5), 440 (4), 97 (65); EIHRMS m/z: 560.3340 (C32H4808, AM -0.9 mmu).  Isolation of Glaciasterol B Diacetate (107)  Aplysilla glacialis (5 kg, wet weight) was collected off Sanford Island, homogenized, and soaked in  methanol (6 L) for 48 hours. The sponge was filtered and re-extracted with methanol/dichloromethane (1:1) overnight. The second sponge extract was decanted, the organic extracts were pooled and concentrated in vacuo to give a brown oil (82.8 g). A portion of the crude extract (31 g) was partitioned between brine (500 mL) and ethyl acetate (4 x 250 mL). The ethyl acetate extract was dried over Na2SO4, filtered and evaporated in vacuo to yield a dark oil (2.8 g). The extract was applied to a silica gel flash column and eluted with a polarity step gradient (hexanes /ethyl acetate/methanol). Two hundred and twenty fractions were collected, analyzed by TLC and pooled to give fourteen fractions which were screened by 1 H NMR. The ninth fraction contained a signal at 8 6.8 which is diagnostic of the proton (3 to the a,13-unsaturated ketone common to the glaciasterols. Consequently a portion (125 mg) of the ninth fraction was acetylated as previously described in this section. Glaciasterol B diacetate (107) was purified from the acetylated portion by sequential separation on a silica flash column (methanol/dichloromethane 1:99), by radial TLC (methanol / dichloromethane 1:99) and by reversed phase HPLC (water/methanol 3:17).  Glaciasterol B Diacetate (107)  AcO  214 Glaciasterol B diacetate (107) was recrystallized from aqueous methanol to yield white needles; mp 55-57°C; UV max (CH2C12) 257 nm (e 4400); IR 2956, 1735, 1684 cm -1 ; CD (methanol) ( 8 )261 -20000, (8)336 8900; 1 H NMR (CDC13, 400 MHz) 8 0.73 (s, 3H), 0.86 (d, J = 6.6 Hz, 6H), 0.97 (d, J = 6.7 Hz, 3H), 1.23 (1H), 1.23  (s, 3H), 1.44 (1H), 1.54 (1H), 1.58 (111), 1.64 (1H), 1.68 (1H), 1.69 (1H), 1.72 (1H), 1.77 (1H), 1.83 (1H), 2.00 (s, 3H), 2.04 (s, 3H), 2.10 (1H), 2.17 (1H), 2.24 (dd, J = 12.6, 12.5 Hz, 1H), 3.23 (dd, J = 10.8, 7.5 Hz, 1H), 3.36 (d, J = 4.5 Hz), 4.10 (1H), 4.19 (1H), 4.99 (m, 1H), 6.75 (d, J = 4.5 Hz, 1H) ppm; 13 C NMR (CDC13, 75 MHz) 8D(1)17.2, 18.9, 21.1, 21.1, 21.3, 22.6, 22.8, 24.5, 25.9, 26.6, 27.4, 27.6, 28.0, 33.9, 34.8, 35.4, 36.6, 39.4, 43.5, 45.3, 46.0, 49.8, 53.4, 61.1, 63.0, 70.6, 138.6, 141.3, 170.1, 170.1, 200.0 ppm;^EILRMS, m/z (formula, relative intensity): 456 (C29H4404, 39), 396 (C27H4002, 26), 283 (C19H2302, 23); EIHRMS m/z: 516.3451 (C31H4806, AM 0.0 mmu); ED50: 2.5 1.ig/mL (L1210 in vitro murine leukemia cell line); ID50: 1.8 tg/mL (MCF-7 in vitro human breast cancer cell line) and 1.8 µg/mL (MCF-7 Adr multidrug resistant in vitro human breast cancer cell line). Acid-catalyzed Ring Opening of the 5,6-Epoxide of Glaciasterol B Diacetate (107) with H218j Glaciasterol B  diacetate (107) (4 mg) was stirred for four hours with THE (0.25 mL), H2O (100 41..), H2 18 0 (97.5 atom% 18 0, 1251AL) and perchloric acid (251.1L). The reaction workup was identical to that previously described and yielded 180 labeled 5a,613-dihydroxyglaciasterol B diacetate (111). The diol 111 was reacted with pyridine and acetic anhydride as previously described. The acetylation reaction yielded 5a-hydroxyglaciasterol B triacetate (112).  5a.60-Dihydroxyglaciasterol B Diacetate (111)  AcO  5a,613-Dihydroxyglaciasterol B diacetate (111) was isolated as a white solid; IR 3432, 2951, 1739, 1677 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 0.76 (s, 3H), 0.87 (d, J = 6.6 Hz, 6H), 0.99 (d, J = 6.7 Hz, 3H), 1.30 (1H), 1.38  215 (s, 3H), 1.46 (1H), 1.50 (1H), 1.60 (1H), 1.65 (1H), 1.68 (1H), 1.70 (1H), 1.85 (dd, J = 12.8, 3.2 Hz, 1H), 2.00 (s, 3H), 2.04 (s, 3H), 2.04 (1H), 2.21 (dd, J = 12.8, 11.9 Hz, 1H), 3.29 (dd, J = 11.0, 8.6 Hz, 1H), 4.03 (d, J = 5 Hz, 1H), 4.18 (m, 2H), 5.11 (m, 111), 6.42 (d, J = 5 Hz, 1H) ppm; 13 C NMR (CDC13, 125 MHz) 8 17.1, 21.1, 21.4, 21.6, 22.5, 22.8, 24.5, 26.1, 26.4, 27.1, 27.4, 27.9, 34.7, 35.4, 35.5, 36.9, 39.5, 42.7, 46.1, 48.0, 50.3, 61.4, 70.4, 72.28, 72.30, 76.5, 137.3, 138.0, 170.1, 170.3, 202.3 ppm; EILRMS, m/z (relative intensity): 474 (12), 476 (13), 456 (14), 458 (12), 343 (6), 345 (5); EIHRMS m/z: 534.3560 (C3 0450 16 07, AM +0.3 mmu), 536.3596 (C311150 1606 18 0 -I , AM -0.3 mmu).  5a-Hydroxvglaciasterol B Triacetate (1121  AcO  5a-Hydroxyglaciasterol B triacetate (112) was isolated as a white solid; IR 3448, 2958, 1739, 1639 cm -1 ; CD (methanol) (8)215 10000, (8)243 -19000; 1 H NMR (CDC13, 400 MHz) 8 0.70 (s, 3H), 0.84 (d, J = 6.7 Hz, 6H), 0.98 (d, J = 6.8 Hz, 3H), 1.25 (1H), 1.36 (s, 3H), 1.45 (1H), 1.53 (1H), 1.66 (1H), 1.73 (1H), 1.76 (1H), 1.83 (1H), 1.95 (1H), 2.01 (s, 3H), 2.04 (s, 3H), 2.05 (1H), 2.13 (s, 3H), 2.13 (1H), 3.30 (dd, J = 11.3, 8.9 Hz, 1H), 4.14 (m, 1H), 4.18 (m, 1H), 5.10 (m, 1H), 5.19 (d, J = 5 Hz, 1H), 6.36 (d, J = 5 Hz, 1H) ppm; 1 H NMR (Pyridine-d5, 400 MHz) 8 0.80 (s, 3H), 0.88 (d, J = 6.6 Hz, 6H), 1.03 (d, J = 6.6 Hz, 3H), 1.14 (1H), 1.35 (1H), 1.47 (2H), 1.50 (m, 1H) 1.55 (s, 3H), 1.58 (1H), 1.73 (1H), 1.83 (1H), 1.93 (1H), 1.98 (s, 3H), 2.02 (s, 3H), 2.06 (1H), 2.14 (s, 3H), 2.25 (br d, 1H), 2.60 (dd, J = 17.7, 12.8 Hz, 1H), 3.53 (m, 1H), 4.47 (m, 2H), 5.62 (m, 1H), 5.85 (d, J = 5 Hz, 1H), 6.62 (d, J = 5 Hz, 1H) ppm; EILRMS, m/z (relative intensity): 456 (4), 343 (2), 283 (7); EIHRMS m/z: 576.3670 (C33H52 16 08, AM +0.8 mmu); ED50: 5.66 ptg/mL (L1210 in vitro murine leukemia cell line); ID50: 2 ptg/mL (MCF-7 in vitro human breast cancer cell line) and 4.5 .tg/mL (MCF-7 Adr multidrug resistant in vitro human breast cancer cell line).  216 Sesquiterpenes and Blancasterol from the Sponge Pleraplysilla sp. Collection and Isolation Data  A small patch of a Pleraplysilla sp. sponge 124 was harvested annually for three consecutive years. Approximately 2/3 of the sponge colony was scraped from the underside of a boulder which lies in a tide pool in the intertidal zone at Botanical Beach, British Columbia. The boulder was replaced in its original position with care to allow the sponge to regrow. Isolation Procedure (1990 and 1991)  The following is typical of the procedure used to isolate sponge metabolites from the 1990 and 1991 collections. Freshly collected sponge (20 g) was soaked in methanol (30 mL) and stored at +7°C for five weeks. The extract was filtered and concentrated in vacuo. The resultant dark oil was partitioned between brine (50 mL) and ethyl acetate (4 x 25 mL). The ethyl acetate extracts were pooled, dried over Na2SO4, and evaporated to yield a brown oil (67.8 mg). The ethyl acetate extract was chromatographed on an LH-20 column eluted with ethyl acetate/methanol/ water (20:5:2). Sixty-two fractions were collected, analyzed by TLC and pooled to yield six fractions. 1 1-1 NMR spectra of these fractions indicated that fourth, fifth and sixth fractions were rich in terpenoidal metabolites and these were pooled prior to further separation.  Fractions 4-6 (44 mg) were applied to a TLC silica flash column 9 and eluted with a polarity step gradient of solvents ranging from diethyl ether/dichloromethane (1:19) to ethyl acetate (100%). Twenty-eight fractions were collected, analyzed by TLC and pooled to yield ten fractions. The second fraction contained a mixture of 0-methyl furodysinin lactone (57) (1 mg) and 0-methyl nakafuran-8 lactone (44) (0.2 mg). These were separated and purified by normal phase HPLC eluted with hexane/dichloromethane (3:7). The fourth column fraction contained 0-methyl 9-oxofurodysinin lactone (72) (0.5 mg). 0-methyl 2-oxomicrocionin-2 lactone (74) was the major component of the fifth fraction. The sixth fraction contained furodysinin lactone (56) (2.0 mg). HPLC purification of the seventh fraction yielded nakafuran-8 lactone (43) (1.1 mg) and the final fraction contained 2-oxomicrocionin-2 lactone (73)  217 (0.8 mg) as its major component. Purification of sesquiterpene lactones 72 and 73 was accomplished by normal phase HPLC eluted with diethyl ether/clichloromethane (1:9).  0-Methyl Furodysinin Lactone (52)  0-Methyl furodysinin lactone (57) was isolated as a colorless glass; UV max (hexane) 220 nm (e 2837); IR 1764 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 1.23 (s, 3H), 1.36 (s, 3H), 1.52 (m, 1H), 1.62 (s, 31I), 1.66 (m, 1H), 2.34 (dd, J = 13.5, 3.4 Hz, 1H), 2.76 (m, 1H), 3.17 (s, 3H), 5.36 (dd, J = 3.8 Hz, 1H), 5.81 (s, 1H) ppm; EIHRMS m/z: 262.1573 (C16H2203, AM +0.4 mmu).  0-Methyl Nakafuran-8 Lactone (441  0-Methyl nakafuran-8 lactone (44) was isolated as a colorless glass: UV max (hexane) 198 nm (c= 6216); IR: 1764 cm -1 ; CD (methanol) (8)220 -1700, ( 19)256 9800; 1 H NMR (CDC13, 400 MHz) 8 0.85 (d, J = 6.8 Hz, 3H), 1.06 (s, 3H), 1.21 (m, 111), 1.54 (m, 1H), 1.66 (m, 1H), 1.67 (s, 31.1), 1.73 (m, 1H), 1.82 (m, 1H), 2.18 (ddd, J = 25.1, 12.5, 5.2 Hz, 1H), 2.44 (m, 1H), 2.90 (m, 1H), 3.15 (s, 3H), 5.85 (d, J = 7.4 Hz, 1H), 5.89 (s, 1H)  218 ppm; 13C NMR (CDC13, 125 MHz) 8 20.9, 21.2, 24.2, 25.7, 31.1, 37.9, 38.2, 40.2, 43.7, 50.6, 112, 120.0, 123.7, 139.1, 170.3 ppm; EIHRMS m/z: 262.1565 (C16H2203, AM -0.4).  0-Methyl 9-Oxofurodysinin Lactone (72)  0-Methyl 9-oxofurodysinin lactone (72) was isolated as a colorless glass; UV max (hexane) 220 nm (e 2724 ); IR 1767, 1665 cm-1 ; CD (hexane) (8)223 -31000; 1 H NMR (CDC13, 400 MHz) 8 1.22 (s, 3H), 1.40 (s, 3H), 1.64 (dd, J = 13.6, 13.5 Hz, 1H), 1.77 (s, 3H), 2.05 (dd, J = 16.8, 14.7 Hz, 1H), 2.25 (dt, J = 14.7, 4.2, 4.2 Hz, 1H), 2.47 (m, 2H), 3.09 (m, 1H), 3.20 (s, 3H), 5.92 (s, 1H), 6.74 (br d, J = 6.3 Hz, 1H) ppm; 13C NMR (CDC13, 125 MHz) 8 15.6, 24.7, 25.0, 31.0, 35.0, 37.6, 38.6, 47.1, 50.6, 106.8, 118.7, 135.8, 146.5, 168.8, 171.0, 198.1 ppm; EILRMS, m/z (formula, relative intensity): 244 (C15H1603, 3), 216 (C14H1602, 1), 150 (C10H140, 17); EIHRMS m/z: 276.1371 (C16H2004, +0.9 mmu).  0-Methyl 2-Oxomicrocionin-2 Lactone (74)  OCH 3  0-methyl 2-oxomicrocionin-2 lactone (74) was isolated as a translucent white glass; IR 1764, 1663 cm -1 ; 1 1-1 NMR (CDC13, 400 MHz) 8 0.99 (d, J = 4.2 Hz, 3H), 1.06 (s, 3H), 1.77 (m, 1H), 1.83 (m, 1H), 1.97 (br s, 3H),  219 2.03 (m, 1H), 2.26 (m, 1H), 2.30 (m, 1H), 2.31 (m, 1H), 2.35 (m, 1H), 3.58 (s, 3H), 5.73 (br s, 1H), 5.90 (br s, 1H), 6.79 (br s, 1H) ppm; 13C NMR (CDC13, 125 MHz) 8 15.4, 19.4, 20.2, 20.5, 33.6, 33.9, 41.9, 42.1, 57.2, 102.5, 128.8, 129, 142.0, 167, 198 ppm; EILRMS, m/z (formula, relative intensity): 246 (C15111803, 2), 234 (C15H2202, 1), 138 (C9H140, 10), 109 (C6HSO2, 1); EIHRMS m/z: 278.1508 (C16H2204, AM -1.0 mmu).  Furodysinin Lactone (561  OH  Furodysinin lactone (56) was isolated as a white solid; IR 1739 cm -1 ; CD (hexane) (8)208 -1900; 1 11 NMR (CDC13, 400 MHz) 8 1.24 (s, 3H), 1.41 (s, 3H), 1.61, 1.63 (s, 3H), 1.69 (m, 1H), 1.96 (m, 2H), 2.30 (dd, J = 13.9, 3.8 Hz, 1H), 2.82 (m, 1H), 5.37 (br d, J = 4.3 Hz, 1H), 5.69 (s, 1H) ppm; 13C NMR (CDC13, 125 MHz) 6 18.6, 23.1, 25.3, 26.8, 30.3, 30.9, 38.5, 40.9, 47.2, 115.2, 123.4, 193.6; EIHRMS m/z: 248.1408 (C15H2003, AM -0.4 mmu).  Nakafuran-8 Lactone (43)  Nakafuran-8 lactone (43) was isolated as a crystalline white powder (2:1 mixture of epimers ); UV max (hexane) 220 nm (c 2272); IR 3354, 1739 cm* 1 H NMR (CDC13, 400 MHz) 8 0.85 (d, J = 6.7 Hz, 3H), 1.07 (s, 3H), 1.23, 1.62, 1.65 (m, 1H), 1.69 (s, 3H), 1.72 (m, 1H), 1.85 (m, 1H), 2.34 (ddd, J = 12.9, 12.7, 4.8, 1H), 2.49 (m, 11.1), 2.88 (m, 1H), 2.91, 5.78 (s, 1H), 5.87 (d, J = 6.3 Hz, 1H) ppm; 13C NMR (CDC13, 125 MHz) 8 20.9, 21.0,  220 24.4, 25.6, 31.3, 37.8, 38.9, 40.3, 44.1, 109.8, 118.3, 123.2, 140.4, 172.3 ppm; EIHRMS m/z: 248.1410 (C15H2003, AM -0.2 mmu).  2-Oxomicrocionin-2 Lactone (73)  OH  2-Oxomicrocionin-2 Lactone (73) was isolated as a colorless oil; IR 3317, 1758, 1650 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 1.00 (d, J = 6.1 Hz, 3H), 1.06 (s, 3H), 1.81 (m, 2H), 1.98 (s, 3H), 2.03 (m, 1H), 2.29 (m, 1H), 2.33 (m, 2H), 2.37 (m, 1H), 3.50 (br s, OH), 5.91 (s, 1H), 6.11 (br s, 1H), 6.87 (d, J = 1.0 Hz, 1H) ppm; 13 C NMR (CDC13, 125 MHz) 8 15.4, 19.4, 20.2, 20.5, 33.8, 33.9, 41.8, 42.1, 96.4, 128.8, 142, 143.1, 167.3, 170.9, 198.8 ppm 219 (C14H1902, 2), 138 (C911140, 11); EIHRMS m/z: 264.1317 (C15H2004, AM -4.5 mmu).  Isolation Procedure (1992)  Pleraplysilla sp. (20 g) was collected at low tide and frozen immediately over CO2(solid)• After eighteen  hours transit time, the sponge was subjected to freeze-drying for thirty-six hours to yield the dry animal (1.79 g). The freeze-dried sponge was extracted with ethyl acetate (50 mL), crushed with a spatula, sonicated, and filtered. The sponge was extracted again, overnight, filtered, and the extracts were pooled and concentrated in vacuo to yield a dark green oil (68.7 mg). 1 H NMR spectra and TLC showed no indication of the sesquiterpene lactones isolated from the 1990 and 1991 collections. A portion of the ethyl acetate extract (25 mg) was applied to a TLC silica flash column and eluted with a polarity step gradient of solvents (hexane to dichloromethane to ethyl acetate). Fifty-five fractions were collected, analyzed by TLC, and pooled to yield fourteen major fractions. Screening by 1 H NMR indicated that only the first fraction contained terpenoidal metabolites. This was further purified by normal phase HPLC eluted with hexane to yield furodysinin (36) and nakafuran-8 (41) which was contaminated with a small amount of furodysin (35)  221 (nakafuran-8: furodysin 3:1). Upon standing, nakafuran-8 (41) decomposed to the corresponding y-keto enal 116. The fourteenth TLC silica fraction contained 1 H NMR signals diagnostic of blancasterol (102) (0.95 mg). Purification of 102 was accomplished by normal phase HPLC eluted with ethyl acetate/hexane (4:1).  Furodysinin (36)  Furodysinin (36) was isolated as a volatile, crystalline white solid; UV max (hexane) 221 nm (e 1377); 1 H NMR (CDC13, 400 MHz) 6 1.20 (s, 3H), 1.21 (s, 3H), 1.27 (m, 1H), 1.55 (m, 1H), 1.66 (s, 3H), 1.75 (m, 2H), 2.08 (m, 1H), 2.30 (m, 1H), 2.69 (m, 1H), 2.74 (m, 1H), 5.62 (br d, J = 3.8 Hz, 1H), 6.23 (d, J = 1.9 Hz, 1H), 7.21 (d, J = 1.3 Hz, 1H) ppm.  Nakafuran-8 (41)  Nakafuran-8 (41) was isolated as a volatile, colorless glass; 1 H NMR (CDC13, 400 MHz) 6 0.89 (d, J = 7.1 Hz, 3H), 1.06 (s, 3H), 1.29 (m, 1H), 1.70 (s, 3H), 1.75 (m, 1H), 1.82 (m, 2H), 1.86 (m, 1H), 2.26 (m, 1H), 2.44 (m, 1H), 3.45 (m, 1H), 5.96 (br d, J = 7.7 Hz, 1H), 6.07 (d, J = 1.5 Hz, 1H), 7.13 (d, J = 1.7 Hz, 1H) ppm.  222  Furodysin (35)  Furodysin (35) was a minor contaminant of nakafuran-8 (41); 1 H NMR (CDC13, 400 MHz) 81.25 (s, 3H), 1.26 (s, 3H), 1.66 (s, 3H), 5.60 (m, 1H), 6.10 (d, J = 1.9 Hz, 1H), 7.22 (d, J = 1.9 Hz, 1H) ppm.  y-Keto Enal (116) from Nakafuran-8 (411  CHO  The y-keto enal (116) from nakafuran-8 was a colorless glass; 1 H NMR (CDC13, 400 MHz) 6 0.90, 1.05, 1.70 (s, 3H), 1.70, 1.75, 2.25 (m, 1H), 2.49 (m, 1H), 3.33 (m, 1H), 5.55 (br d, 1H), 5.96 (d, 1H), 9.61 (d, 1H) ppm.  Blancasterol (102)  HO  OH  OAc  Blancasterol (102) was isolated as an amorphous white solid; UV max (methanol) 288 nm (e 1048), 240 nm (e 5102); IR 3476, 2955, 1711, 1681 cm -1 ; CD (methanol) ( 8 )235 15600, (8)266 -670, (8)330 556; 1 H NMR (CDC13, 400 MHz) 6 0.74 (s, 3H), 0.88 (d, J = 6.6 Hz, 61-1), 1.00 (d, J = 6.7 Hz, 3H), 1.40 (m, 1H), 1.45 (m, 1H), 1.46 (m, 1H), 1.52 (m, 1H), 1.62 (m, 2H), 1.67 (m, 1H), 1.73 (m, 2H), 1.83 (m, 2H), 1.88 (ddd, J = 14.8, 13.2, 3.3 Hz, 1H), 2.09 (m, 1H), 2.00 (s, 31.1), 2.20 (s, 3H), 2.26 (dt, J = 14.8 Hz, 1H), 2.35 (dd, J = 19.8, 5.8 Hz, 1H), 2.56 (dd, J = 19.8, 2.4 Hz, 1H), 3.28 (t, J = 11.6 Hz, 1H), 3.91 (d, J = 11.2 Hz, 1H), 4.01 (m, 1H), 4.05 (d, J = 11.2 Hz, 111), 4.05 (m, 1H), 4.22 (ddd, J = 15.8, 10.6, 5.3 Hz, 1H), 4.98 (d, J = 9.5 Hz, 1H), 6.44 (dd, J = 5.8,  223 2.4 Hz, 1H) ppm; 13 C NMR (CDC13, 125 MHz) 8 16.7, 19.7, 19.7, 20.9, 21.1, 22.5, 22.8, 24.4, 26.0, 28.0, 28.5, 29.7, 34.5, 34.8, 35.5, 37.1, 39.5, 42.7, 45.8, 50.8, 56.3, 61.5, 62.0, 69.9, 76.7, 78.8, 137.3, 138.2, 171.0, 171.8, 199.7 ppm; EILRMS, m/z (relative intensity):^532 (1), 490 (3), 430 (2), 412 (2), 269 (21); FABHRMS (M+H) m/z: 551.36006 (C31145108, AM 30.1 ppm); ED50: 8.8 p.g/mL (L1210 in vitro murine leukemia cell line); ID50: 31..tg/mL (MCF-7 in vitro human breast cancer cell line) and 10.6 1.1g/mL (MCF-7 Adr multidrug resistant in vitro human breast cancer cell line).  Langarin from an Aplidium species Collection and Isolation Data  A previously undescribed Aplidium species (310 g) was collected by SCUBA at -5m off Langara Island, one of British Columbia's Queen Charlotte Islands. A voucher sample (100 g) was extracted with methanol. The crude methanolic extract was found to be active against the in vitro L1210 murine leukemia cell line with an ED50 of 14 µg/mL. The remainder of the orange colonial tunicate was stored frozen for three months, then ground in a blender and extracted with methanol (500 mL). The extract was filtered and the organism was re-extracted with methanol (500 mL) overnight. The second extract was filtered and pooled with the first. The combined extracts were concentrated in vacuo to an aqueous slurry. The slurry was partitioned between water (500 mL) and, first, hexanes (3 x 250 mL) and then ethyl acetate (3 x 250 mL). TLC analysis showed the two organic extracts to contain the same major component (yellow spot on TLC, Rf=0.33 in methanol/dichloromethane 3:99) thus the extracts were pooled. Evaporation of the organic extract in vacuo yielded a dark red oil (464 mg).  The organic extract was applied to an LH-20 size exclusion column eluted with ethyl acetate/methanol/ water (20:5:2). Twenty-eight fractions were collected, screened by TLC, and pooled to give three major fractions. 1 H NMR indicated that the second fraction (42 mg) contained aromatic compounds and bioassay indicated that this  fraction contained the active compound(s). Fraction two was further purified by radial TLC eluted with ethyl acetate /hexane (1:4) to yield langarin (13.7 mg) (142).  224 Langarin (142)  OH 0 OH  OH  Langarin (142) was recrystallized from methanol to give bright orange needles; mp 225-228°C; UV max (methanol) 429 nm (e 8608), 286 nm (e 8120), 254 nm (e 16724), 225 nm (e 31020); IR 1675, 1627 cm -1 ; 1 H NMR (Me2SO-d6, 400 MHz) 8 4.61 (d, J = 5.8 Hz, 2H), 5.59 (t, .1 = 5.8 Hz, OH), 7.28 (br s, 1H), 7.36 (d, J = 8.3 Hz, 1H), 7.68 (br s, 1H), 7.70 (d, J = 7.9 Hz, 1H), 7.80 (dd, J = 8.3, 7.9 Hz, 1H), 11.90 (br s, OH), 11.96 (br s, OH) ppm; 13 C NMR (Me2SO-d6, 125 MHz) 8 62.4, 114.7, 116.2, 117.5, 119.7, 121.0, 124.7, 133.5, 133.7, 137.7, 154.1, 161.7, 162.0, 181.8, 192.0 ppm; EILRMS, m/z (formula, relative intensity): 272 (C15111205, 2), 271 (C15H1105, 17), 254 (C15111004, 6), 252 (C151-1804, 6), 243 (C14111104, 2), 242 (C14H1004, 22), 241 (C14H904, 93); EIHRMS m/z: 270.0527 (C15111005, AM -0.1 mmu); ED50: 0.4 mg/mL (L1210 in vitro murine leukemia cell line).  Acetylation of Langarin (142): Langarin (142) was stirred overnight with acetic anhydride and pyridine (0.5 mL  each) under an atmosphere of nitrogen. Excess reagent was removed in vacuo to yield langarin triacetate (143).  Langarin Triacetate (143)  OAc 0 OAc  OAc  Langarin triacetate (143) was a yellow solid; IR 1675, 1744, 1770 cm* 1 H NMR (Me2SO-d6, 400 MHz) 8 2.13 (s, 3H), 2.38 (s, 3H), 2.39 (s, 3H), 5.26 (s, 2H), 7.61 (d, J = 1.7 Hz, 1H), 7.62 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 8.09 (d, J = 1.7 Hz, 1H), 8.13 (dd, J = 8.0, 1.2 Hz, 1H) ppm; 13C NMR (Me2SO-d6, 125 MHz)  225 S 20.6, 20.8, 21.2, 63.9, 149.5, 149.7, 168.9, 169.0, 170.1, 180.3, 181.4 ppm; EILRMS m/z (formula, relative intesity): 396 (C20-11608, 5), 354 (C01 -4407, 35), 312 (C17141206, 32), 270 (C15H1005, 73).  Metabolites from Aplidium californicum Collection and Isolation Data  Aplidium californicum (350 g), a pale yellow colonial tunicate, was collected by SCUBA at -5m off  Langara Island in the Queen Charlotte Islands, B.C. A voucher sample (100 g) was reserved for screening and the remaining organisms were stored frozen for three months. A crude methanolic extract of the voucher sample proved mildly active against Bacillis subtilis and Rhizoctonia solani. The extract was active against the in vitro L1210 murine leukemia cell line at an ED50 of 6 g.tg/mL. One half of the remaining, frozen animals (160 g) were freezedried to give a dry weight of 9.9 g. The dry animals were extracted sequentially with hexanes, chloroform, ethyl acetate, and methanol (2 x 500 mL each). In each case the tunicates were soaked in the solvent, broken up with a spatula, sonicated, and then filtered. The animals were reimmersed in solvent and soaked overnight prior to filtration. Extracts of like solvents were pooled. By 1 H NMR and TLC, the hexane, dichloromethane and ethyl acetate extracts appeared to contain related compounds thus these extracts were pooled (304 mg). A portion (100 mg) of the organic extract was applied to an LH-20 size exclusion column eluted with ethyl acetate/methanol/water (20:5:2). Sixty-seven fractions were collected, analyzed by TLC and pooled to yield eleven major fractions. 1 H NMR spectra of the fractions indicated that the four initial fractions eluted contained fats and steroids. The latter  fractions contained terpenoidal metabolites. Radial TLC eluted with a polarity gradient from ethyl acetate/hexane (3:17) to ethyl acetate (100%) of combined fractions nine through eleven yielded known prenylated hydroquinones 149 and 150. Column fractions five through seven were also pooled and further purified by radial TLC eluted with a polarity gradient of solvents ranging from ethyl acetate/hexane (1:19) to ethyl acetate (100%). The final purification was accomplished by use of normal phase HPLC eluted with methanol / dichloromethane (1:99). Extraction and purification of the entire A. californicum collection by the isolation procedure described above yielded calapliquinone A (160), calaplidol A (160) (1.0 mg), and calaplidol B (161), in addition to the known compounds 152 (1.3 mg) and 158 (1.6 mg).  226  HO 149 1 H NMR (CDC13, 400 MHZ) 8 1.76 (d, 6H), 3.30 (d, 2H), 4.32 (s, 1H), 4.65 (s, 1H), 5.29 (m, 11-1), 6.57 (dd, 1H),  6.62 (d, 1H), 6.67 (d, 111) ppm; EILRMS m/z (relative intensity): 178 (25.6).  HO 150  1 H NMR (C6D6, 500 MHz) 6 0.42 (s, 611), 3.58 (br s, 1H), 5.27 (d, 1H), 6.01 (d, 1H), 6.21 (d, 1H), 6.26 (dd, 1H),  6.75 (d, 1H) ppm; EILRMS m/z (relative intensity): 176 (38.6).  Calaplidol A (160)  OH H HO HO H  Calaplidol A (160) was isolated as a colorless glass; UV max (methanol) 321 nm (e 3399), 291 nm (e 9388), 281 (e 10298); IR 3380, 2920 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 1.49 (s, 3H), 1.70 (s, 3H), 2.36 (m, 1H), 2.50 (m, 1H), 4.48 (m, 1H), 5.09 (br s, 1H), 5.16 (br s, 1H), 5.20 (m, 1H), 6.19 (d, J = 11.7 Hz, 1H), 6.36 (d, J = 11.7 Hz, 1H), 6.63 (m, 1H), 6.64 (m, 1H), 6.82 (d, J = 9.1 Hz, 1H) ppm; 13 C NMR (CDC13, 100 MHz) 8 16.5, 25.8, 30.8, 82.0, 115.7, 115.9, 117.1, 120.0, 122.2, 127.4, 131.1, 134, 147, 151 ppm; EILRMS m/z (formula, relative intensity): 242 (C16141802, 9), 199 (C13H1102, 7), 173 (Ci iH902, 24); DCIMS m/z (NH3, M+H): 261 (C16H2003, 2.4% relative intensity).  227  Calaplidol B (161)  HO  Calaplidol B (161) was an unstable, colorless glass; IR 3361, 2924 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 1.47 (s, 3H), 1.78 (dd, 1H), 1.79 (br s, 6H), 1.93 (dd, 1H), 3.73 (m, 1H), 4.28 (s, Ar—OH), 4.35 (s, Ar—OH), 4.55 (s, Ar-01a), 5.10 (m, 1H), 6.33 (d, J = 16.2 Hz, 1H), 6.57 (dd, J = 3.0, 0.8 Hz, 1H), 6.62 (m, 2H), 6.68 (d, J = 8.6 Hz, 1H), 6.76 (d, J = 8.6 Hz, 1H), 6.84 (d, J = 16.2 Hz, 1H), 6.86 (br s, 1H) ppm; 13 C NMR (CDC13, 100 MHz) 8 18.0, 23.6, 25.8, 32.0, 39.2, 76.0, 113.5, 114.9, 115.0, 115.4, 116.9, 118.0, 121.6, 125, 126, 127.1, 134, 138, 146, 148, 149.5 ppm; EILRMS m/z (formula, relative intensity): 366.1459 (C22H2205, 0.3), 352.1669 (C22H2404, 11), 309 (C19111704, 3), 176 (C11141202, 9), 161 (C10H902, 100); EIHRMS m/z: 366.1459 (C22H2205, AM -0.8), 352.1669 (C22H24O4, AM -0.6 mmu).  HO 152  Chromenol 152 was isolated as a white solid; UV max (methanol) 328 nm (c 3829 ), 249 nm (c 13822), 223 nm (e 18591); IR 3380, 2920 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 1.36 (s, 3H), 1.57 (s, 3H), 1.63 (m, 1H), 1.66 (s, 3H), 2.11 (m, 1H), 5.09 (m, 1H), 5.59 (d, J = 9.8 Hz, 1H), 6.27 (d, J = 9.8 Hz, 1H), 6.64 (d, J = 8.6 Hz, 1H), 6.57 (dd, J = 8.6, 2.9 Hz), 6.47 (d, J = 2.9 Hz, 1H) ppm; 13 C NMR (CDC13, 100 MHz) 8 17.6, 22.7, 25.6, 26.0,  228 40.9, 78.1, 112.8, 115.4, 116.7, 122.6, 124.1, 131.0, 131.7, 148.7, 149.2 ppm; EIHRMS m/z: 244.1467 (C16H2002, AM 0.4 mmu); ED50: 3.9 gg/mL (L1210 in vitro murine leukemia cell line).  HO  158  Hydroquinone 158 was isolated as a white solid; UV max (methanol) 297 nm (e 2742); IR 3388, 2927 cm -1 ; 1 H NMR (CDC13, 400 MHz) 8 1.24 (s, 3H), 1.63 (s, 3H), 1.69 (s, 3H), 1.57 (m, 1H), 1.61 (m, 1H), 1.65, 1.80, 2.10 (m, 1H), 2.65 (ddd, J = 7.7, 7.3, 1.8 Hz, 1H), 5.13 (m, 1H), 6.57 (dd, J = 6.8, 3.1 Hz, 1H), 6.59 (d, J = 2.7 Hz, 1H), 6.70 (d, J = 8.3 Hz, 111) ppm; 13C NMR (CDC13, 100 MHz) 8 17.7, 22.9, 26.6, 26.6, 28.0, 41.4, 41.8, 73.8, 113.8, 116.6, 116.9, 124.0, 130.2, 132.4, 148.0, 148.9 ppm; EIHRMS m/z: 264.1723 (C16H24O3, AM -0.2 mmu).  229  REFERENCES  1 a)  Carefoot, T. Pacific Seashores; J.J.Douglas: Vancouver, B.C., 1977, and b) Carefoot, T., University of British Columbia, personal communication, 1992.  2  Williams, D. H.; Stone, M. J.; Hauck, P. R.; Rahman, S. K. J. Nat. Prod. 1989, 52(6), 1189-1208.  3  Bergquist, P. R. Sponges; University of California: Berkeley and Los Angeles, C.A., 1978, p.204.  4  Bergmann, W. J. Mar. 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