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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 MARINEINVERTEBRATESbyJANA PIKAB. Sc., Concordia University, Montreal, P.Q., 1986A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Chemistry)We accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIAApril 1993©Jana Pika, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of „/CA-M1 i41A The University of British ColumbiaVancouver, CanadaDate 1 / / ,93DE-6 (2/88)AbstractAn investigation into the chemistry of four species of marine invertebrates which were found to producecytotoxic crude extracts (ED50 against the L1210 murine leukemia cell line 5 30 pg/mL) led to the isolation of ninenew and eleven previously known secondary metabolites. The structures of the novel compounds were elucidatedby a combination of spectroscopic analysis and chemical interconversion. The metabolites were tested against thein 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 ofthe novel 9,11-secosteroids glaciasterol A (100) and glaciasterol B (101). These compounds are the first examplesof secosteroids isolated from a sponge of the genus Aplysilla. Glaciasterol A (100) proved active in vitro against theMCF-7 human breast cancer cell line at an ED50 of 19 pg/mL. The ED50 for Glaciasterol A (100) against the drug-resistant MCF-7 Adr breast cancer cell line was 18 tig/mL. The structures of the glaciasterols were determined by aseries 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 ED50values of 1.8 pg/mL.A study of the metabolites of a gray-white sponge collected off the coast of Vancouver Island yielded thenew cytotoxic secosteroid blancasterol (102). Blancasterol (102) was found to be active against the MCF-7 anddrug-resistant MCF-7 Adr breast cancer cell lines with ED50 values of 3 g.ig/mL and 10.6 pg/mL, respectively. Thenew sesquiterpenes 0-methyl 9-oxofurodysinin lactone (72), 2-oxomicrocionin-2 lactone (73), 0-methyl2-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 wasuseful 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 againstL1210 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 newprenylated hydroquinone derivatives (160 and 161) as well as the known compounds 149, 150, 152, and 158.ll111Compound 152 was tested in vitro against the L1210 murine leukemia cell line and found to be cytotoxic with anED50 value of 3.9 pg/mL.HO HO100^ 1 01OR 72 73R =H74R = CH 3OH 0 OHHOOHOHOAc102 142HOHOHO H160^ 161Table of ContentsPageAbstractTable of Contents^ ivList of Tables viiList of Figures viiiList of Abbreviations^ xiiiList of Schemes xvAcknowledgments xviDedication^ xviiGeneral Introduction^1PART I. Isolation and Structure Elucidation of Metabolites from two North-Eastern Pacific Sponges5Introduction^5Classification of the Sponges^ 7Aplysilla glacialis and a Pleraplysilla Sp. Sponge^ 9Description of Aplysilla glacialis^ 9Description of the Pleraplysilla Sp. Sponge 10Taxonomy of Aplysilla glacialis and the Pleraplysilla Sp. Sponge^14Chemistry of Sponges of the Genera Aplysilla, Dysidea, and Pleraplysilla 17Secondary Metabolites of Aplysilla Species Sponges^ 17Natural Products of Sponges belonging to the Genus Dysidea^ 22The Chemistry of the Pleraplysilla Sponges^ 28Secosteroids and Related Steroids from Marine Invertebrates^30Introduction to Selected NMR Experiments^ 36A. Cytotoxic Metabolites of Aplysilla glacialis^56Results and Discussion^56Glaciasterol A (100)^ 57Glaciasterol B Diacetate (107)^ 765a,60-Dihydroxyglaciasterol A Diacetate (113) and 5a-HydroxyglacisterolA Triacetate (114)^ 93ivPageConclusions^9613. Natural Products of a Pleraplysilla Sp. Sponge^98Results and Discussion^98a) The 1990 and 1991 Pleraplysilla Sp. Sponge Collections^ 980-Methyl 9-Oxofurodysinin Lactone (72)^ 992-Oxomicrocionin-2 Lactone (73) 1090-Methyl 2-0xomicrocionin-2 Lactone (74) 118Known Metabolites of the Pleraplysilla Sp. Sponge (1990/1991Collections)^ 125b) The 1992 Pleraplysilla Sp. Sponge Collection^ 126Blancasterol (102) 127Known Metabolites of the Pleraplysilla Sp. Sponge (1992 Collection)^143Conclusions 145PART II. The Natural Products of Two Species of Colonial Tunicate from the Queen CharlotteIslands^152IntroductionA Th hemi LI • fan n^s •^/^e. from - • een h 1 eI 1 n^159i) Anthraquinones from Marine Invertebratesii) Results and DiscussionLangarin from an Undescribed Aplidium SpeciesLangarin (142)iii) ConclusionsB. Secondary Metabolites of Aplidium californicum from the Oueen Charlotte Islands159163163166178180i) Prenylated Quinones and Hydroquinones from Animals of the Subphylum^180UrochordataPageii) Results and Discussion^182Prenylated Quinone and Hydroquinone Derivatives from Aplidium californicum 182Calaplidol A (160)^ 185Calaplidol B (161) 194Known Metabolites from the Tunicate Aplidium californicum^205iii) Conclusions^206Experimental^208References^229viList of TablesPageTable 1^1H NMR and COSY Spectroscopic Data for 4-Methylcatechol (105)^ 45Table 2^Assignment of Protonated Carbons of 4-Methylcatechol (105) from the HMQCSpectrum^ 49Table 3^1H, 13C, and HMBC NMR Data for 4-Methylcatechol (105)^ 52Table 4^1H and 13C NMR Data for Glaciasterol A (100) (Recorded in CDC13)^58Table 5^1H and 13C NMR Data for Glaciasterol A Diacetate (106) (recorded in CDCI3)^67Table 6^1H and 13C NMR Data for Glaciasterol B Diacetate (107) (Recorded in CDC13)^78Table 7^1H and 13C NMR Data for 5a,60-Dihydroxyglaciasterol B Diacetate (111) (Recordedin CDC13)^ 83Table 8^1H NMR Data for 5a-Hydroxyglaciasterol B Triacetate (112)^ 90Table 9^1H NMR Data for 5a,613-Dihydroxyglaciasterol A Diacetate (1 1 4) and5a-Hydroxyglaciasterol A Triacetate (115)^ 94Table 10^Results of the Assay of Glaciasterol A (100) and Related Derivatives 107 and 112against In Vitro Murine Leukemia and Human Breast Cancer Cell Lines^97Table 11^1H and 13C NMR Data for 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded inCDC13)^ 100Table 12^1 H and 13C NMR Data for 2-Oxomicrocionin-2 Lactone (73) (Recorded in CDCI3)^110Table 13^1 H and 13C NMR Data for 0-Methyl 2-Oxomicrocionin-2 Lactone (74) (Recorded in^119CDC13)Table 14^1H and 13C NMR Data for Blancasterol (102) (Recorded in CDC13)^128Table 15^1 H NMR Shifts of Blancasterol (102) in CDC13 and in CDC13:C6D6 20:1 142Table 16^1 H and 13C NMR Data for Langarin (142) (Recorded in Me2SO)^ 167Table 17^11-1 and 13C NMR Data for Langarin Triacetate (143) (Recorded in Me2SO)^175Table 18^1H and 13C NMR Data for Calaplidol A (160) (Recorded in CDC13) 186Table 19^1 H and 13C NMR Data for Calaplidol B (161) (Recorded in CDC13)^195viiList of FiguresPageFigure 1.^Typical Body Plan of a Sponge of the Class Demospongaie^ 6Figure 2.^An Intertidal Specimen of Aplysilla glacialis^ 11Figure 3.^Aplysilla glacialis and Pleraplysilla Species Sponge Collection Sites^12Figure 4.^An Intertidal Specimen of a Pleraplysilla Species Sponge^ 13Figure 5.^Taxonomic Classification Schemes according to (A) Bergquist, (B) Boury-Esnault, and^16(C) Van SoestFigure 6.^Time Course for a One-Dimensional NMR Experiment^ 36Figure 7.^The Pulse Sequence for an NOEDS Experiment 37Figure 8.^1H NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at^39500 MHz)Figure 9.^NOE Difference Spectra of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO^40at 400 MHz)Figure 10. Result of NOE Difference Experiments on 4-Methylcatechol (105)^ 38Figure 11. Time Course for a Two-Dimensional NMR Experiment^ 41Figure 12. Fourier Transformation of the FID of a 2-D Experiment with Respect to a) t2 and b) ti^42and t2Figure 13. The Pulse Sequence for a Simple COSY Experiment^ 43Figure 14. COSY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at 400^44MHz)Figure 15. The Proton Connectivities of 4-Methyl Catechol (105) from the COSY Spectrum^45Figure 16. The Pulse Sequence for an HMQC Experiment^ 46Figure 17. 13C NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at^47125 MHz)Figure 18. HMQC Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 51.11 Me2SO at 500^48MHz)Figure 19. The Pulse Sequence for an HMBC experiment^ 50Figure 20. HMBC Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at 500^51MHz)Figure 21.^Selected HMBC Correlations for 4-Methylcatechol (105)^ 50Figure 22. The Pulse Sequence for a ROESY Experiment^ 53Figure 23. ROESY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 ml Me2SO at^55500 MHz)Figure 24. Results of a ROESY Experiment on 4-Methylcatechol (105)^ 54Figure 25.^1H NMR Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)^59viiiPageFigure 26. COSY Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)^60Figure 27. Double Resonance NMR Experiment on Glaciasterol A (100) (Recorded in CDC13 at^61400 MHz)Figure 28. 13C NMR Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 125 MHz)^62Figure 29. HMQC Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 500 MHz) 63Figure 30. 1 H NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400^68MHz)Figure 31. COSY Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400 MHz)^69Figure 32.^13C NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 100^70MHz)Figure 33. HMQC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 500 MHz)^71Figure 34. HMBC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 500 MHz)^72Figure 35. HMBC Correlations from H6, Me18, and Me19 of Glaciasterol A Diacetate (106)^74Figure 36. Results of Selected NOE Difference Experiments on Glaciasterol A Diacetate (106)^76Figure 37. 1H NMR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 400 MHz)^79Figure 38.^13C NMR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 100^80MHz)Figure 39. Selected NOE Difference Experiments on Glaciasterol B Diacetate (107) (Recorded in^81CDC13 at 400 MHz)Figure 40.^1 H NMR Spectrum of 5a,613-Dihydroxyglaciasterol B Diacetate (111) (Recorded in^84CDC13 at 500 MHz)Figure 41.^COSY Spectrum of 5a,613-Dihydroxyglaciasterol B Diacetate (111) (Recorded in^85CDC13 at 400 MHz)Figure 42.^13C NMR Spectrum of 5a,60-Dihydroxyglaciasterol B Diacetate (111) (Recorded in^86C6D6 at 125 MHz) with Expanded Regions a) Recorded in C6D6 and b) Recorded inCDC13Figure 43. a) HMQC Spectrum of 5a ,6(3-Dihydroxyglaciasterol B Diacetate (111) with^87b) Expanded Region (Recorded in CDC13 at 500 MHz)Figure 44.^1H NMR Spectra of 5a-Hydroxyglaciasterol B Triacetate (112) Recorded in a) CDC13^91and b) Pyridine-d5 at 400 MHzFigure 45^COSY Spectrum of 5a -Hydroxyglaciasterol B Triacetate (112) (Recorded in^92Pyridine-d5 at 400 MHz)Figure 46. The 1 H NMR Shifts of 111 in Pyridine-d5 Relative to CDC13^ 93Figure 47.^1H NMR Spectra of 5a-Hydroxyglaciasterol A Triacetate (114) Recorded in a) CDC13^95and b) Pyridine-d5 at 400 MHzixPageFigure 48. 1 H NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13^101at 500 MHz)Figure 49. 13C NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13^102at 125 MHz)Figure 50. COSY Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at^103400 MHz)Figure 51. HMQC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at^104500 MHz)Figure 52. HMBC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at^105500 MHz)Figure 53. HMBC Spectroscopic Correlations for 0-Methyl 9-Oxofurodysinin Lactone (72)^107Figure 54. Selected NOE Difference Experiments on 0-Methyl 9-0xofurodysinin Lactone (72)^108(Recorded in CDC13 at 400 MHz)Figure 55. 1H NMR Spectrum of 2-Oxomicrocionin-2 Lactone (73) (Recorded in CDC13 at 400^111MHz)Figure 56. COSY Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 400 MHz)^112Figure 57. HMQC Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 500^113MHz)Figure 58.^13C Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 125 MHz)^114Figure 59. HMBC Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 500^115MHz)Figure 60.^Selected HMBC Correlations Observed for 2-Oxomicrocionin-2 Lactone (73)^117Figure 61. Results of Selected NOE Difference Experiments on 2-Oxomicrocionin-2 Lactone (73)^118Figure 62. 1 H NMR Spectrum of 0-methyl 2-Oxomicrocionin-2 Lactone (74) (Recorded in^120CDC13 at 400 MHz)Figure 63. COSY Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in CDC13 at^121400 MHz)Figure 64.^13C NMR Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in^122CDC13 at 125 MHz)Figure 65. HMQC Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in CDC13^123at 500 MHz)Figure 66. HMBC Spectrum of 0-methyl 2-Oxomicrocionin-2 lactone (74) (Recorded in CDC13 at^124500 MHz)Figure 67. 1H NMR Spectrum of Blancasterol (102) (Recorded in CDC13 at 400 MHz)^129Figure 68. COSY Spectrum of Blancasterol (102) (Recorded in CDC13 at 400 MHz) 130Figure 69.^13C NMR Spectrum of Blancasterol (102) (Recorded in CDC13 at 125 MHz)^131xPageFigure 70. HMQC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz)^132Figure 71. HMBC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz) 133Figure 72. Selected HMBC Correlations for Blancasterol (102)^ 135Figure 73. Expanded Upfield Region of the APT Spectrum of Blancasterol (102) (Recorded in^138CDC13 at 125 MHz)Figure 74. 1H NMR Spectrum of Blancasterol (102) Recorded in CDC13:C6D6 20:1 at 400 MHz^139Figure 75. Double Resonance NMR Experiments on Blancasterol (102) (Recorded in^140CDC13:C6D6 20:1 at 400 MHz)Figure 76. ROESY Spectrum of Blancasterol (102) (Recorded in CDC13:C6D6 20:1 at 500 MHz)^141Figure 77. Selected ROESY Correlations for Blancasterol 102 (Recorded in CDC13:C6D6 20:1)^142Figure 78. Conformational Structure of Blancasterol (102) with a Cis-Fused A/B Ring System^143Figure 79.^The Classification of Tunicates of the Genus Aplidium^ 153Figure 80. The Body Plan of a Urochordate Larva^ 154Figure 81. The Body Plan of the Urochordate Aplidium californicum^ 156Figure 82.^The Aplidium Spp. Collection Site 164Figure 83.^A Freshly Collected Sample of the Undescribed Aplidium Species 165Figure 84. 1H NMR Spectrum of Langarin (142) (Recorded in Me2SO at 400 MHz)^168Figure 85. COSY Spectrum of Langarin (142) (Recorded in Me2S0 at 400 MHz) 169Figure 86. 13C NMR Spectrum of Langarin (142) (Recorded in Me2SO at 125 MHz)^170Figure 87. HMQC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz) 171Figure 88. HMBC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz)^173Figure 89. Selected HMBC Correlations for Langarin (142)^ 172Figure 90. 1H NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2SO at 400 MHz)^176Figure 91.^13C NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2S0 at 125 MHz)^177Figure 92. Freshly Collected Aplidium californicum^ 184Figure 93. 1H NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)^187Figure 94. 13C NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 100 MHz) 188Figure 95. COSY Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)^189Figure 96. APT Spectrum of Calaplidol A (160) (Recorded in CDC13 at 125 MHz) 190Figure 97. HMBC Spectrum of Calaplidol A (160) (Recorded in CDC13 at 500 MHz)^191Figure 98.^Selected HMBC Correlations for Calaplidol A (160)^ 193Figure 99. 1H NMR Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz)^196Figure 100. COSY Spectrum of Calaplidol B (161) (Recorded in CDC13 at 400 MHz) 197Figure 101. 13C NMR Spectrum of Calaplidol B (161) (Recorded in CDC13 at 100 MHz)^198Figure 102. HMQC Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz) 199Figure 103. HMBC Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz)^200xiPageFigure 104. Selected HMBC Spectroscopic Correlations for Calaplidol B (161)^ 201Figure 105. Selected 13C NMR Shifts of Calaplidol B (161), 2-Methylhydroquinone (163), and^2044-Methylcatechol (105)xiiList of AbbreviationsAc^— acetylAPT^— Attached Proton Testax^— axialbr^— broadCD^— Circular DichroismCOSY^— COrrelation SpectroscopYd^— doubletDa^— Daltonsdd^—doublet of doubletsAM^— difference in massdt^— doublet of tripletsED50^— Effective Dose resulting in 50% responseEIHRMS — Electron Impact High Resolution Mass SpectrometryEILRMS — Electron Impact Low Resolution Mass Spectrometryeq^— equatorialFABMS^— Fast Atom Bombardment Mass SpectrometryFTIR^— Fourier Transform InfraRedHETCOR — HETeronuclear CORrelationHIV^— Human Immunodeficiency VirusHMBC^— Heteronuclear Multiple Bond ConnectivityHMQC^— Heteronuclear Multiple Quantum Coherencei^— signal due to an impurityIC50^— Inhibitory Concentration resulting in 50% responseID50^— Inhibitory Dose resulting in 50% responseJ^— scalar coupling constantLRDCIMS — Low Resolution Desorption Chemical Ionization Mass Spectrometrym^— multipletxivM+^— molecular ionm/z^— mass to charge ratioMe^— methylMe2SO^— dimethyl sulfoxidemmu^— millimass unitsNOE^— Nuclear Overhauser Effectq^— quartetRf^- Ratio to frontROESY^— Rotating-frame Overhauser Enhancement SpectroscopyS^— signal due to solvents^— singletSCUBA^— Self-Contained Underwater Breathing Apparatussp.^— speciest^— tripletT/C^— Test versus ControlTLC^— Thin Layer Chromatographyw^— signal due to waterList of SchemesPageScheme 1 Proposed Biogenesis of Microcionin-2 (116) and Nakafuran-8 (41)^148Scheme 2^Proposed Biogenesis of Furodysin (35) and Furodysinin (36) 149Scheme 3^Proposed Biogenesis of Prenylated Quinones and Hydroquinones^ 207XVAcknowledgmentsI would like to thank my supervisor, Dr. Raymond J. Andersen, for the opportunity of studying in hisresearch group. Dr. Andersen's enthusiasm ensured that the group was always an exciting and dynamicenvironment 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 EtL'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 myappreciation for their help in collecting sample organisms, and for their contributions to a pleasant and intellectuallystimulating 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 toLiane 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 proofreadingthis thesis. I am grateful to Anna and to Kristinn Kristinsson for being wonderful friends. Thanks also to my oldfriends 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.xviFor Hanaxvii"Each of them in his own tempo and with his own voice discovered and reaffirmed with astonishment theknowledge that all things are one thing and that one thing is all things — plankton, a shimmering phosphorescence onthe sea and the spinning planets and an expanding universe, all bound together by the elastic string of time. It isadvisable to look from the tide pool to the stars and then back to the tide pool again."John Steinbeck and Edward F. RickettsThe Log from the Sea of Cortez, 1941GENERAL INTRODUCTIONRocky headlands swept by ocean waves characterize much of British Columbia's exposed coastline. Winterstorms can bring swells of such intensity that animals not well adapted to this environment may be torn from thesubstrate or abraded by the action of suspended particles. Despite the dangers this habitat poses, many invertebratessettle in the subtidal zones creating areas of high biomass and diversity. The rapid circulation of water provides arich diet of food particles for sessile organisms and the evolutionary advantage that wave action can disperse theirwater-borne larvae. 1 Organisms which grow in the inter- and subtidal zones on the exposed coast adapt to the highenergy environment in a number of ways. Many develop attachment devices such as the holdfasts characteristic oftunicates, others, such as sponges, may encrust the rock substrate, and still other organisms burrow into the rock tocreate shelters for themselves. All of the animals in such an environment must compete for the limited availableresources of food and space. While many species of arthropods, mollusks and echinoderms have shells which providea physical form of defense against predators and encroaching competitors, the method of defense of sessileinvertebrates 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 secondarymetabolites to render themselves poisonous or merely unpalatable. 2,3 Some of these toxic compounds may providevaluable leads to chemotherapeutic agents against such human diseases as cancer.Sponge taxonomists were among the first to investigate the nature of poriferan natural products, primarilyfor the purpose of classifying these animals. Bergmann studied the steroidal composition of the Porifera in the1940's to determine the chemotaxonomic significance of these metabolites. 4 His work led to the discovery ofsteroids unique to marine organisms. Bergmann's subsequent research on the chemistry of the sponge Tethya cryptaled to the isolation of the novel arabinosyl nucleosides spongouridine 1 3 and spongothymidine 2.50HN )1 •1(:). N -•00H^OH^ OH1 21HO HOCH3H3C CH3 CH3OH3OHWhile fish and algae have long been held in esteem as remedies in Chinese folk medicine, 6 western culturehas more commonly employed extracts of microbes and the higher plants for medicinal purposes. Bergmann'spioneering work sparked interest in the medicinal potential of marine invertebrate metabolites, particularly since asynthetic analog of compounds 1 and 2, cytosine arabinoside (Ara-C), has proven effective against adult leukemias. 3A decade later advances in SCUBA and submersible technologies improved access to the —200 000 species ofinvertebrates 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 fivethousand compounds reported between 1962 and 1991, an indication of the intense level of research activity in thisfield over the past three decades. 8 Many of these compounds have novel structures unique to aquatic organisms andexciting pharmacological activities.Roth et al. have proposed that some of the molecules of the human nervous and immune systems includinghormones and neural peptides resemble metabolites synthesized by parazoans and marine invertebrates because acommon evolutionary ancestor was capable of synthesizing related archaic molecules. 2,9 Certainly extracts ofmarine invertebrates can affect not only other invertebrates, 3 but some may show activity in bioassays which usehuman cancer as well as other pathological cell lines. Numerous compounds which display biological activity indiverse assays have been isolated. An example of a poriferan metabolite currently attracting a great deal of scientificattention is manoalide (3) which was first isolated from the tropical Pacific sponge Luffariella variabilis by Scheuerand de Silva in 1980. 10 Pharmacological investigations revealed it to have potent anti-inflammatory and analgesicproperties. 112Since the development of effective antibiotics fifty years ago cancer has emerged as a leading cause of deathin industrialized societies. 12 An ongoing challenge of modem medical therapeutics has been to identify compoundswhich inhibit cancer cell growth selectively without damaging the host organism. While chemotherapeutic agentswhich are effective against certain types of cancers, such as some forms of leukemia, have been developed, a widervariety of effective drugs is required. Successful chemotherapy requires drugs with improved selectivity, new modesof action as well as compounds which are effective against those cancers, solid tumors and drug-resistant cancer cellsfor example, which have proven refractory. 12Research in the field of marine natural products has already provided some promising leads to newchemotherapies. 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 laterpublished the structures of geodiamolides C (6), D (7), E (8), and F (9) and reported potent in vitro activities againstthe L1210 murine leukemia cell line for all of the geodiamolides with ED50 values ranging from 2.5 ng/mL forgeodiamolide C (6) to 39 ng/mL for geodiamolide D (7).143HO4 X=I,R=Me5 X =Br,R =Me6 X=C1,R=Me7 X=I,R=H8 X=Br,R=H9 X = Cl, R = HNovel and cytotoxic metabolites are by no means limited to organisms of the phylum Porifera. Bioassay-guided fractionation of extracts of the colonial tunicate Trididemnum solidum led to the isolation of the didemnins, afamily of cyclic peptides which exhibit potent cytotoxic, antiviral, and immunosuppressive activity. 15a DidemninB (10) is among the most cytotoxic of the didemnins with an ID50 value of 1.1 ng/mL against L1210 murineleukemia cells. Approximate immunosuppressive activity of the didemnins was cited as one thousand times that ofCH310cyclosporin. Didemnin B (10) was the first marine natural product to enter Phase I clinical trials as an anticanceragent and is currently in Phase II clinical trials at the National Cancer Institutes. 150^ 0^0^0II II II 11C —NH- CH- CHOHCH2 C - 0 — CHC CH(CH3) — CICH2CH(CH3)2^CH(CH3)2L-Lac-L-Pro-D-MeLeu NHCI^OCH3^CH2CH(CH3)2ICHO -C-C liN(CH3)-C^N COINII I-I0Another tunicate, Ecteinascidea turbinata, is reported to produce a "macromolecular" fraction ofapproximate molecular weight 10 000 g/mol which caused complete regression of Sarcoma solid tumors in two outof three mice tested and an average life extension of 92.5% was reported. 12It 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 newantitumor compounds. The isolation of novel, cytotoxic natural products combined with the production of syntheticderivatives and analogs is a promising strategy for the development of anticancer drugs. 12The objective of the research described in this thesis was to isolate cytotoxic compounds from marineinvertebrates collected off the North-Eastern Pacific coast. Two species of sponge, Aplysilla glacialis and aPleraplysilla sp., and two species of Aplidium, compound tunicates, were selected for study on the basis of thecytotoxicity of their extracts and because animals of these phyla are known to produce compounds which arebiologically active as well as structurally nove1. 124PART I. ISOLATION AND STRUCTURE ELUCIDATION OF METABOLITES FROM TWONORTH-EASTERN PACIFIC SPONGES INTRODUCTIONThe phylum Porifera is traditionally classified as belonging to the kingdom Metazoa although the spongeswhich constitute this phylum are intermediate between metazoans and colonial protozoans. 16 Poriferans areconsidered to be primitive organisms in that they have retained many ancestral characteristics, have no organs orclearly-defined tissues, lack a nervous system, and have little coordination between cells. Since the emergence of thephylum in Cambrian times, sponges have evolved successfully as evidenced by the greater than ten thousand speciesalive in the seas today. 17Sponges are essentially cooperating aggregations of cells. Water is pumped at low pressure into smallopenings, or ostia, in the surface of the sponge, through the inhalant pore to a single layer of choanocytes which areflagellated collar cells, and then outward via a larger opening, the osculum (Figure 1). 17,18 The movement of theflagella of the choanocytes is responsible for the unidirectional movement of water which passes through theorganism bringing in organic particles and microbes (ingested primarily by the choanocytes) and removing wasteproducts such as ammonia. Respiration takes place by diffusion of oxygen from the incoming flow of water acrosscell membranes. Sponges have very low metabolic rates but can pump high volumes of water. One fresh-waterspecies is capable of pumping up to seventy times its body volume of water per hour. 16 Sponges have flexibleforms and growth is heavily influenced by their environment. 17Poriferans are hermaphroditic and can reproduce sexually or asexually. Sexual reproduction may beviviparous or oviparous (a taxonomically significant variation). 19 In viviparous sponges, release of the larvae doesnot take place until the cells are in an advanced state of differentiation and the resultant larvae are free-swimming andhighly developed. If ciliated, the larvae may swim for three to forty-eight hours prior to subsiding to a creepingstate. Whether ciliated or not, the larvae creep for twenty to sixty hours showing sensitivity to the effects of gravityand light in choosing a substrate on which to settle. Once settled, the larvae metamorphose and within twenty-fourhours have established a functional pore system. 2° Two forms of asexual reproduction are known. The sponge mayform gemmules, clusters of totipotent cells protected by an outer layer of spicules and spongin, or fragments maybreak off the adult sponge which are capable of settling elsewhere and becoming separate individual sponges. 175osculum6inhalant poreostiachoanocytesFigure 1. Typical Body Plan of a Sponge of the Class Demospongaiel 7A sessile adult sponge has a single-cell outer epithelium known as the pinacoderm. The inner layer is themesohyl which contains mobile cells and skeletal elements. There is evidence that all of the cells of a poriferan aremobile and sponges have the ability to regenerate significant portions of their tissues. 17Although sponges are sedentary they have few predators presumably, in part, because many poriferans haveevolved arsenals of toxic or unpalatable secondary metabolites which are intolerable to other species. 2-3 Organismswhich are known to feed on sponges include asteroids and many species of opisthobranchs which have developed theability to sequester sponge secondary metabolites and employ them for their own protection. 3Sponges are classified according to their skeletal elements into the four classes Calcarea, the smallest of thesponges which possess skeletons composed of calcium carbonate, Hexactinellida, deep water sponges with siliceousspicules, Demospongiae, which will be described in greater detail, and Sclerospongiae, a class encompassing spongeswith siliceous spicules and an organic collagen skeleton within a thin layer of living tissue growing on a massivecalcium carbonate skeleton. 16,17 This thesis is concerned with two species of sponge belonging to the classDemospongiae. The Demospongiae are characterized by skeletons comprised of either or both siliceous spicules andan organic skeleton which may be dispersed throughout the mesohyl or secreted as fibres of a protein known as16,17^ 16,18spongin.^This class is the most common comprising 95% of recent sponges.^It is represented in everyaquatic environment including fresh-water habitats and is found throughout the world's oceans from the intertidalshallows to the deepest abysses.21CLASSIFICATION OF THE SPONGESClassification of the Porifera at the ordinal, generic, and class levels can be problematic becausemorphology is frequently inconsistent and the simplicity of the organism provides few features on which to base aclassification system.22 Many species of sponge respond to their environments by encrusting the substratum andgrowing in irregular, unpredictable symmetries. Some species react to the direction and force of water currents bygrowing oscula which are oriented to minimize mixing of inhalant and exhalant water currents. 23 Pigmentation isnot necessarily an aid in identification as color morphs within species are known. Sponges which grow in directlight may develop deeper pigmentation as protection against ultraviolet radiation than animals which grow in shadeor on the undersides of rocks.23Traditionally classification is based on the skeletal characteristics of the sponge. The chemical nature ofspicules 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 overallskeleton are also considered. Additional morphological factors include any external characteristics of the sponge, itsconsistency, and surface features.247In 1956 Levi proposed division of the Demospongiae, on the basis of reproductive biology, into the ordersTetractinomorpha (the oviparous sponges) and Ceractinomorpha (viviparous sponges which release larvae).25 SinceLevi's initial attempt to classify sponges according to characters other than skeletal features, the advent of electronmicroscopy has led to the study of the histology of differentiated sponge cells and the resultant information has beenused for taxonomic classification. 26In a number of species, the secondary metabolites synthesized by the sponge may be of utility inidentifying the animal. Bergmann pioneered this area of research when he first attempted to classify marineinvertebrates according to the steroids produced by the animals.4 Bergquist and other workers have shown that somesecondary metabolites do have chemotaxonomic significance. 27 Species of the order Verongida, for example,predictably synthesize brominated amino acid derivatives. 27This thesis describes studies of the secondary metabolites of two species of Demospongiae which belong tothe order Keratosa (or Horny sponges), poriferans which lack spicules entirely but which secrete skeletons of sponginfibres. Faulkner has suggested that, in the absence of spicules which might serve to physically deter predators, theKeratosa sponges have developed diverse suites of terpenoids and other natural products for the purpose of chemicaldefense.8d Numerous researchers have isolated a variety of chemically novel compounds from this group ofsponges. 28 Many have proved active as antibiotics, antifeedants and antifouling agents. 28a-c Since the absence ofspicules makes classification of this order particularly difficult, studies of their natural products may be a valuable aidin grouping these organisms. Bergquist warns that several factors must be considered for chemotaxonomy to be aneffective and reliable tool. Obviously the secondary metabolites must be carefully characterized. The spongetaxonomy must be reliable and many zoologists emphasize the importance of storing voucher samples forverification by future workers. Another factor which must be taken into account is the presence of extraneousorganisms which grow on the surface, in the pores and in the mesohyl of many sponge species. Microscopic speciesare difficult to remove and may contribute to the observed chemistry. 22a8APLYSILLA GLACIALIS AND A PLERAPLYSILLA SP. SPONGEAplysilla glacialis is an encrusting pink sponge found in exposed habitats off the west coast of BritishColumbia. In 1991 Andersen et al. reported the isolation of six novel diterpenes from extracts of A. glacialis.28dWe 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 identifiedas a new species of Aplysilla. Our interest in the chemistry of the white sponge was prompted by the possibilitythat it might contain cytotoxic metabolites similar to those produced by Aplysilla glacialis. It was proposed that aninvestigation of the natural products of the white sponge would also determine whether it was a color morph ofAplysilla glacialis or if not, might aid in establishing it as a new Aplysilla species. Our study provided evidenceagainst the classification of the gray-white sponge in the genus Aplysilla and suggested that the genus Dysideawould be a more appropriate classification. Reexamination of the specimen in light of its secondary metabolismindicated that it is a member of the Pleraplysilla, intermediate between the Aplysilla and the Dysidea, and a genuspreviously unknown on the west coast of North America. 24 Aplysilla glacialis and the Pleraplysilla speciessponges will be described. The taxonomy of these sponges will be discussed and the secondary metabolites ofsponges of the genera Aplysilla, Dysidea, and Pleraplysilla briefly reviewed.DESCRIPTION OF APLYSILLA GLACIALISAustin describes Aplysilla glacialis as an encrusting, non-branching sponge, typically rose to rose-red butsometimes varying to tan or ivory. 29 The organism is soft and slippery with a surface characterized by conuleswhich project 1-2 mm in height. Austin indicates that the cones are irregular and may have spongin fibresprojecting from the tips. Oscula of —0.6 mm diameter are visible flush with the sponge surface. The skeleton isformed of unbranched or sparsely branched fibres which start at a basal plate and end at the pinacoderm or projectfrom the conules. The sponge does not incorporate foreign material such as sand or detritus into its skeletalnetwork. Individual specimens are several square decimeters in area and reach 3-6 mm in thickness intertidally and atleast 12 mm subtidally. Some individuals can grow to cover several square meters. Austin states that A. glacialis9has 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 ashaded environment such as the underside of a raised rock, overhanging cliff, or cave. 29The specimens used for this study (Figure 2) were collected subtidally by SCUBA off the Deer Group ofIslands near the Bamfield Marine Station and off the north shore of Sydney Inlet (Figure 3). The organisms grow at2-5 m depth where they encrust overhanging rockwalls at the mouths of surge channels. They grow to a squaremeter in area and 2 cm in thickness. All of the specimens used were of a very bright pink color and the surfacemorphology was identical to that described by Austin. Aplysilla glacialis has been observed growing at many ofour exposed dive sites off the coast of British Columbia but it is not abundant. This sponge was never overgrownby other encrusting organisms and the surface appeared free of algal growth. The nudibranch Cadlinaluteomarginata has been observed to feed on Aplysilla glacialis 30DESCRIPTION OF THE PLERAPLYSILLA SP. SPONGEBergquist describes Pleraplysilla poriferans as thin, encrusting sponges with simple fibres which maybranch once or twice but which are of constant thickness from the basal plate to the surface. 24 An importantdistinguishing characteristic is that the sponge incorporates sand and debris into the core of its spongin fibres. 24In 1990 specimens of what appeared to be the same Pleraplysilla species were collected intertidally atBotanical 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 theundersides of boulders at sea level in exposed areas. The Botanical Beach specimen (Figure 4) is —20 cm in diameterand 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 thePleraplysilla species nor was any algal fouling observed. This species of sponge seems to be rare as we have notobserved it on collecting trips to other exposed coastal areas of Vancouver Island.1011Figure 2.^An Intertidal Specimen of Aplysilla glacialis50°11129°W 128°WI 127°W^126°W1 124°W1 123°oN-1......\...) 125°W1BRITISHCOLUMBIASydney Inlet Collection SiteBamfield Collection Site^Botanical Beach Collection sitePACIFIC OCEAN47°N129°W 1^1128°W^127°W 126°W 125ovv, 47°N123°W50°N49°N48°N47°N50°N49°N47°N12Figure 3.^Aplysilla glacialis and Pleraplysilla Species Sponge Collection SitesFigure 4.^An Intertidal Specimen of a Pleraplysilla Species Sponge13TAXONOMY OF APLYSILLA GLACIAL IS AND THE PLERAPLYSILLA SP. SPONGEAplysilla glacialis and the Pleraplysilla sponge species belong to the class Demospongiae and to thesubclass Ceractinomorpha, the sponges which reproduce sexually by releasing fully developed larvae. 19 Both areconsidered to be "horny" sponges in that these species lack spicules but have skeletons of secreted spongin fibres. 24The lack of spicules greatly reduces the characters on which taxonomy can be based 24 and consequently there is littleagreement among sponge taxonomists about the classification of these organisms. Based on skeletal characteristicsalone, both the Aplysilla and Pleraplysilla sponges were traditionally placed in the order Dendroceratida which ischaracterized by fragile, dendritic skeletons of pure spongin fibres which branch from a basal point withoutanastomosing.24 Recently Bergquist has proposed that the secondary metabolites of Pleraplysilla species spongesindicate a closer relationship to the Dysidea sponges of the order Dictyoceratida than to the Dendroceratida. 22b TheDictyoceratids are also "horny" sponges characterized by anastomosing networks of tough spongin fibres whichcontain detritus.31 Consequently Bergquist moves the Pleraplysilla to the Dictyoceratida but leaves the Aplysillaspecies sponges in the order Dendroceratida (Figure 5A). 22bBoury-Esnault et al. have studied the histology of the Demospongiae by electron microscopy.2 6Comparison of the choanocytes, the choanocyte chambers, the mesohyl, and apopinocytes (cells lining the exhalantpores) led them to conclude as did Bergquist and coworkers that Aplysilla and Dysidea are closely related. UnlikeBergquist, however, Boury-Esnault et al. propose that Aplysilla, Pleraplysilla, and Dysidea be grouped together inthe order Dendroceratida (Figure 5B). 26Van Soest questions the validity of the classification system introduced by Levi and applied, withmodifications, by Bergquist. 32 Van Soest suggests that species be grouped only on the basis of shared charactersinherited from a common ancestor. He feels that logic argues against dividing the "horny" sponges into distinctorders and classifies them in a single order, the Keratosa. Van Soest proposes that the appropriate status for theDendroceratida and Dictyoceratida is subordinal. On the basis of common characters, Van Soest proposes classifyingthe Dendroceratida and the Dysideidae together as both lack spicules, have sand in their skeletal fibres, are rich interpenes and have choanocyte chambers greater than 30 jim in diameter (Figure 5C). 32b14It 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 establishingporiferan classification systems and in dictating which classification system will eventually gain wide acceptance anduse.15KINGDOM^ Metazoa^ Metazoa^ Metazoa/'^ 1 1PHYLUM^ Porifera Porifera^ Porifera/^ /CLASS^ Demospongiae^ Demospongiae^ DemospongiaeI^ !ISUBCLASS^Ceractinomorpha^ CeractinomorphaI ORDER^Dendroceratida Minchin^Dictyoceratida Minchin^Dendroceratida Minchin^ KeratosaSUBORDER^ DendroceratidaDysideidaeFAMILY Aplysillidae Vosmaer^Dysideidae Gray^Aplysillidae Vosmaer^Dysideidae GrayI^ '1GENUS^Aplysilla Schulze^Pleraplysi la Topsent Aplysilla Schulze^Dysidea Johnston'/Dysidea JohnstonSPECIES^A. glacialis^ A. glacialisA^ B^ CFigure 5. Taxonomic Classification Schemes according to (A) Bergquist,22b (B) Boury-Esnault,26 and (C) Van Soest.32b5CHEMISTRY OF SPONGES OF THE GENERA APLYSILLA, DYSIDEA, AND PLERAPLYSILLAA study of the chemistry of Aplysilla glacialis resulted in the isolation of two novel cytotoxic9,11-secosteroids. Investigation of the natural products of a Pleraplysilla species sponge resulted in the isolation ofseven known and three new sesquiterpene derivatives in addition to a third novel, cytotoxic 9,11-secosteroid. Thissection of the thesis will briefly review the known chemistry of sponges of the genera Aplysilla, Dysidea, andPleraplysilla in order to place the chemistry of A. glacialis and the Pleraplysilla sponge in a chemotaxonomiccontext. The known secosteroid chemistry of marine invertebrates will also be reviewed.SECONDARY METABOLITES OF APLYSILLA SP. SPONGESThe natural products chemistry of Aplysilla sp. sponges is characterized by diterpene derivatives of thehypothetical "spongian" skeleton 11. Of forty compounds reported to date from Aplysilla sponges, twenty-sevenwere diterpenoids. The remaining thirteen compounds included eleven steroids, one purine and one amino acidderivative. Microscope studies of the mesohyls of Dendroceratid sponges prompted Bergquist and Wells to reportthat these poriferans "have negligible matrix microorganism populations". 22 It is likely that Aplysilla spongessynthesize their spongian-derived diterpenoids endogenously.18^1911Bergquist and coworkers have proposed that the mechanism followed in the spongian skeleton derivatizationand degradation by Dendroceratid sponges may be a reliable marker for taxonomic classification at the specificleve1.22b 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 of1718Aplysilla sponge chemistry will select relevant examples but will not attempt to present all of the secondarymetabolites reported from this genus.Molinski and Faulkner reported the isolation of three diterpenes from an undescribed pink species ofAplysilla from Australia.34 Of these metabolites, ambliofuran had been previously reported and one of the othercompounds 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 classfrom 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 Chelonaplysillaviolacea,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 knownfrom 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 (13-16). Polyrhaphin C (15) was found to be a fish antifeedant at concentrations of 100 pg/mL and exhibitedantibacterial activity against Staphylococcus aureus at 100 pg/disk and Bacillus subtilis at 10 tg/disk.35126a,7a,1713-Trihydroxy-150,17-oxidospongian-16-one7-butyrateSpongian skeleton13^ 14Polyrhaphin A Polyrhaphin BDendrillane skeleton^Dendrillane skeleton0OAc1 5Polyrhaphin CMacfarlandin skeleton16Polyrhaphin DIsospongian skeleton(= marginatane skeleton)19Aplysilla glacialis collected off Vancouver Island, British Columbia yielded six novel diterpenoidmetabolites 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 diterpenesrepresented the glaciane, aplysulphurane, gracilane,33 and marginatane28d carbon skeletons.17^ 18Glaciolide Cadlinolide AGlaciane skeleton^Aplysulphurane skeletonOH19^ 20Cadlinolide B Aplysillolide AAplysulphurane skeleton^Gracilane skeleton202 1^ 22Aplysillolide B MarginatoneGracilane skeleton Marginatane skeletonIn 1990 Poiner and Taylor reported that a study of the natural products of the orange Aplysilla tango fromAustralian waters had yielded the known diterpene gracilin A, four novel diterpenes, and a steroid fraction. The newditerpenes were aplytandiene-1 (23), aplytandiene-2 (24), aplytangene-1 (25), and aplytangene-2 (26).37 Theaplytandienes possessed the gracilane skeleton while the aplytangenes were nor-diterpenes. A sterol fractioncontaining a mixture of ergosterols exemplified by compound 27 was also isolated. The ergosterols shared acommon nucleus and varied only in the side chain constitution. A fraction of endo-peroxy sterols (28) withcommon nuclei and diverse side chains was also isolated, but Poiner et al. suggested that these were artifacts ofergosterol reaction with singlet oxygen. 3723^ 24Aplytandiene-1 Aplytandiene-2Gracilane skeleton Gracilane skeletonCH3 CH25^ 2 6Aplytangene-1 Aplytangene-2Nor-diterpene Nor-diterpeneHO HO212 7 Recently Faulkner and Bobzin reported the isolation of four diterpenes, two endoperoxy sterols, an aminoacid derivative and 1-methyladenine from Aplysilla glacialis collected at Crooked Island in the Bahamas. 38 None ofthese compounds was novel. Manotil was known from terrestrial sources and the endoperoxy sterols are commonlyisolated from marine organisms. Of the diterpenoids, spongia-16-one, had been reported from a Dendroceratidsponge, atisane-30,16a-diol had been isolated from the sponge Tedania ignis and spongia-15a,16a-diacetate wasalso a metabolite of Spongia officinalis.39 Faulkner and Bobzin investigated the antimicrobial, antifeedant, andantifouling activities of these compounds. They reported that manotil and cholesterol endoperoxide were active indeterring fish predation. The antifouling assays proved inconclusive but 1-methyladenine was identified as theantibacterial component of the sponge, showing activity against an Acinetobacter species, a Flavobacterium, andtwo Vibrio strains.38 The chemistry of the Bahamian Aplysilla glacialis differed markedly from that of the BritishColumbian Aplysilla glacialis although the presence of spongian derivatives in both sponges suggested taxonomicrelatedness.This review has illustrated that spongian-derived diterpenes are characteristic of sponges of the genusAplysilla. Bergquist's argument that carbon skeletons of metabolites can be used for classification at the specieslevel is not supported.22b In the case of each of the Aplysilla sponges studied, metabolites with a diverse array ofcarbon skeletons were isolated. Furthermore, compounds which had previously been reported in the literature wereby no means exclusive to the genus Aplysilla but represented many Dendroceratid and, in one case, Dictyoceratidsponges.The ecological role the Aplysilla sponges' diterpene metabolites play is not yet fully understood. Faulknerhas suggested that non-siliceous sponges such as these elaborate intricate arrays of metabolites to serve as a chemicaldefense against predation. 8d No conclusive studies support this hypothesis as far as the Aplysilla sponges areconcerned. The antimicrobial activity of some of the reported metabolites, polyrhaphin C and 1-methyladenine forexample, may indicate a defense against microbial fouling. 38 Another role has been suggested for antibacterialcompounds. Bergquist proposed that the sponge may secrete antimicrobial agents via its pinacoderm to preventbacteria from settling as they are drawn into the sponge inhalant pores towards the choanocytes where feeding takesplace.3NATURAL PRODUCTS OF SPONGES BELONGING TO THE GENUS DYSIDEASponges of the genus Dysidea produce a diverse assortment of natural products which have beenextensively studied. Faulkner has reviewed reports of one hundred and twenty-seven metabolites from Dysideaspecies sponges.8 Of these compounds, —50% were sesquiterpenes or sesquiterpene derivatives. Terpenes are widelyaccepted as the "true" metabolites of Dysidea sponges.8c The majority of terpenoidal compounds reported to date arederivatives of the furan sesquiterpene 29.29Sesquiterpene furans and sesquiterpene quinones have been found from Dysidea sponges collected around theworld.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 ofterpene syntheses. Electron micrograph studies have shown Dysidea sponges to possess high concentrations ofmatrix cyanophytes.22 It has been suggested that the polychlorinated amino acid derivatives which have beenisolated from the Dysidea and in particular from Dysidea herbacea, are metabolites of the sponges' bacterialpopulations 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 againstleukemia cells in vivo and inhibit HIV virus replication in vitro. 42 A number of related natural products have beenisolated and derivatives have been synthesized for biological testing. 4222OH30^ 3133^34HO 0HH36 R = H38 R = AcSIn 1975 Minale et al. reported ten sesquiterpene furan derivatives from the sponge Dysidea pallescens.43Of particular note are the pallescensins 1-3 (32-34, respectively). Minale discussed the possibility that 34 was anoxidation artifact of compound 33 but suggested that the absence of the corresponding hydroxybutenolide ofmetabolite 32 indicated that 34 was produced by the sponge. 43 aWells 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 productsshowed that the a-substituted-y-hydroxy-a,13-butenolide of 15-acetylthioxy-furodysinin (38) had a strong affinity andspecificity for human leukotriene B4 (LTB4) receptors. This suggested a possible defense role as this compoundwould initiate an pro-inflammatory response in humans and possibly also in predators. 452335 R = H37 R = AcS24The first terpene isolated from D. herbacea, a sponge which had yielded a number of chlorinated amino acidderivatives, was spirodysin (39) reported by Wells et a1.46 Wells suggested that spirodysin may be a precursor offurodysin (35) and furodysinin (36).Upial (40) is a sesquiterpene rearranged to form a bicyclo(3.3.1)nonane aldehyde lactone which was reportedfrom the Hawaiian sponge Dysidea fragilis by Scheuer and coworkers. 47 Also isolated from D. fragilis by Scheueret al. were two sesquiterpene furans; nakafuran-8 (41) and nakafuran-9 (42). 28b Scheuer noted the isolation of thehydroxybutenolide of nakafuran-8 (43) and the corresponding methyl ketal 44 but suggested that these compoundsmay be artifacts. Nakafuran-8 and nakafuran-9 were effective fish anti-feedants but upial (40) proved inactive. 28b4142^ 43 R = H4 4 R = CH3Wells and coworkers have reported the isolation of furodysin derivatives 45 and 46, furodysinin derivatives47-50, the drimane furan 51, and the linear furan 52 from a collection of Dysidea herbacea 40H45 R=f3OH46 R=f30AcH47 R=POH48 R=I30Ac49 R=aOH0. 5154H3502552Faulkner 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 ofpallescensin A.43e 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 produceambliolide leading Faulkner to conclude that oxidation to the butenolide was likely a process of the sponge althoughhe noted that formation of the methoxy group might be due to exposure of the compound to methanol."ORH56 R = H57 R = CH326In 1984 Cardellina and Grode reported the isolation of the sesquiterpene lactone of furodysinin (56) from thesponge Dysidea etheria.49 In order to elucidate the structure they attempted to form compound 56 by oxidation offurodysinin (36). A light—induced oxidation in methanol yielded only 57, the methyl ketal of 56. Eventually thesesquiterpene lactone was formed with meta—chloroperbenzoic acid followed by Jones' reagent. 49Cardellina and Bamekow have recently reported the structures of the oxidized nakafuran derivatives 58-60from the sponge Dysidea etheria.50 Since no nakafuran-8 (41) was isolated from any of a number of spongecollections, it was suggested that the oxidized nakafurans (58-60) were unlikely to be artifacts of an autooxidationreaction.R58 R = OAc, R' = H59 R = OH, R' = H60 R, R' = 0Terpene metabolites other than sesquiterpenes have been isolated from Dysidea species sponges. The firstmonoterpene reported from a marine sponge was adriadysiolide (61) isolated from a north Adriatic Dysideaspecies.51OI^„PO6127Dysidea 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 of62.53HOOH62As previously mentioned, Faulkner has reported the isolation of pallescensolide (53), a sesquiterpenoid, andambliol (54), and ambliolide (55), related diterpenes from the same sponge.48 Faulkner noted that this is unusualamong sponges but cites precedents among Dysidea species.48 Recently Kashman and coworkers have publishedthe structures of thirteen novel rearranged spongian-derived diterpenes from a Red Sea Dysidea species.54 These newcompounds are exemplified by shahamin E (63)54a and norrlandin (64). 54b Norrlandin was found to be cytotoxic(IC50 1.2 Ltg/mL).54bH OAc63 64Non-terpene natural products of Dysidea sponges include a diverse array of chlorinated amino acidderivatives which may co-occur with sesquiterpene metabolites.° Compound 65 55 is an example of thesemetabolites. It has been suggested that these natural products are synthesized by the sponges' symbiotic bacteria 40HO -Cl3C70CC1365A variety of novel steroids have been isolated from Dysidea species sponges. These will be discussed insome 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) andpleraplysillin (67) from Pleraplysilla spinifera. 56 The same researchers later published the structure ofpleraplysillin-2 (68), an ester formed of a hemiterpene alcohol and a sesquiterpene furan acid, isolated from polarextracts of the same collection of P. spinifera.5728 6668In a later collection of what appeared to be P. spinifera, Minale and coworkers failed to detect the threepreviously isolated metabolites 66-68. 58 They isolated compound 69, longifolin, known from a terrestrial plantand 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 thatthis was a distinct species of Pleraplysilla sponge, distinguishable only by its chemistry.58a Minale et al. presentedtwo alternatives each for the structures of the spiniferans, and it was not until a later publication that the structureswere confirmed to be 70 for spiniferan-1 and 71 for spiniferan-2. 58b29This thesis presents a study of the chemistry of a Pleraplysilla sp. sponge. The sponge yielded the knowncompounds furodysinin lactone (56),49 nakafuran-8 lactone (43),28b their respective 0—methyl ketals (57 and 44),in addition to furodysinin (36)," furodysin (35),44 and nakafuran-8 (41). 28b The new sesquiterpenes isolatedfrom the sponge were 0—methyl 9—oxofurodysinin lactone (72), 2-oxomicrocionin-2 lactone (73), and 0—methyl2-oxomicrocionin-2 lactone (74).72^ 73 R = H74 R = MeThe chemistry of sponges of the genus Pleraplysilla has not been extensively studied. Those naturalproducts which have been isolated suggest that Pleraplysilla sponges characteristically produce sesquiterpene furanmetabolites. This is in keeping with Bergquist's observation that the chemistry of the Pleraplysilla sponges "fitsbetter with an identification as Dysidea" than as Aplysilla sponges.22 Bergquist further remarked concerning thetaxonomy of the Pleraplysilla, that they are "strictly intermediate between the two groups" Aplysilla andDysidea.22bSECOSTEROIDS AND RELATED STEROIDS FROM MARINE INVERTEBRATES Steroidal natural products which incorporate many unique chemical features have been isolated from marineinvertebrates. Among these features are highly oxygenated skeletons, epoxides, polyenone functionalities, andunusual side chain structures such as the rare cyclopropene ring. 59 Secosteroids are uncommon among steroidalmetabolites of marine invertebrates but a number of interesting compounds, some biologically active, have beenreported.The secoderivative (75) of gorgosterol was the first secosteroid reported from a marine invertebrate. 60Compound 75 was isolated from the gorgonian Pseudopterogorgia americana. The structure was solved by X—raycrystallography of the 3—p—iodobenzoate,11—acetate derivative. The side chain of secogorgosterol incorporated anunusual cyclopropane functionality. 60HO75Two C—ring secocholestane derivatives, 76 and 77, possessing a common nucleus identical to that of seco-gorgosterol and differing only in side chain structure were isolated from the soft coral Sinularia species.61 Wells etal. reported the structures of compounds 76 and 77 without determining relative stereochemistry. Djerassi andcoworkers 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 the3080R'0 „HHO31structures of the closely related secosteroids 78-80. 62 The authors suggested that the presence in the animal ofsecosteroids 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 ofcompounds 75-80.62The first secosteroid reported from a poriferan was herbasterol (81) isolated from an Australian specimen ofDysidea herbacea. 63 Herbasterol (81) was a highly oxidized 9,11-secosteroid with an unusual 19-hydroxylatedmethyl 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.OH81Sica 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—90) differing from hipposterol only in the structures of the side chains were later isolated from H. communis by thesame researchers. 6532HOOH82 R =84 R =83 R =^ir.85 R = ,MM86 R=88 R =90 R =Ciminiello and coworkers reported the structures of the highly degraded incisterols (91-94) from theMediterranean sponge, Dictyonella incisa (family: Hymeniacidonidae). 59 In this case, the entire A-ring and Me19functionality have been cleaved away from the steroid nucleus./'''''' 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-methoxyfunctionality. Steroids with the conventional 3-hydroxy group were absent from extracts of the sponge.96Recently a new C-ring secosteroid was reported from the sponge Spongia officinalis. 67 The structure ofsecosterol 97 was confirmed by synthesis. Further examination of extracts of S. officinalis by the same researchersyielded the closely related 9,11-secosteroids 98 and 99. 683334HOOH98 R=,99 R=9 7HO100 R=ovvv.101 R ..//■./.MM102This thesis will dicuss the recent isolation and structure elucidation of three cytotoxic secosteroids from thesponges Aplysilla glacialis and a Pleraplysilla sp. The marine sponge Aplysilla glacialis was found to produceglaciasterols A (100) and B (101). 69 A study of a Pleraplysilla sp. sponge resulted in the isolation of a singlesecosteroid, blancasterol (102).It is interesting to note that sponges of the genera Dysidea, Hippospongia, and Spongia belong to thesingle order Dictyoceratida. The steroidal metabolites, including secosteroids, produced by these sponges tend to behighly oxidized. Wells and coworkers have suggested that the biosynthesis of the secosteroidal skeleton proceeds viaoxidative cleavage of the 9,11 or, in the case of the hipposterols (82-90), the 5,6-7c bond of an exogenouslyintroduced steroid.61 A number of steroids have been isolated from Dysidea species sponges which are consideredattractive precursors or intermediates for the synthesis of secosteroids via oxidative cleavage. These are exemplifiedby steroid 103 which was isolated from the Bermudian sponge Dysidea etheria.7° This polyhydroxylated steroidmay result from the oxidation of 5,6— and 9,11—double bonds. Oxidative cleavage of such an intermediate wouldresult in secosteroids similar to those which have been isolated from the Dictyoceratid sponges.61 This review andthe results presented in this thesis suggest that, while not limited to the Dictyoceratid sponges, highly oxidizedsecosteroids are characteristic of sponges of this order.103The biological roles of highly oxidized steroids in marine invertebrates is not well understood. Cardellinaand coworkers have suggested that these compounds are too polar to play an important function in maintaining thecell membrane.71 They point out a similarity between functionalized steroids from poriferans and plant defensehormones such as cyasterone (104). Cyasterone is related to the growth hormones of the plants' insect predators andcauses uncontrolled growth and deformation in those predators. 2 Cardellina et al. speculate that highly oxidizedporiferan steroids may stimulate a similar effect in sponge predators such as crustaceans or dorid nudibranchs. 7135HOHO104INTRODUCTION TO SELECTED NMR EXPERIMENTSAnalyses of the results of various NMR experiments were used to elucidate the structures of the naturalproducts described in this thesis. Since NMR is a complex and rapidly developing field, the focus of this sectionwill be to give a simple (non-quantum mechanical) explanation of an NOE difference experiment and of each of thetwo-dimensional NMR experiments used to elucidate the structures of compounds discussed in the Results andDiscussion section. This section will also demonstrate the application of each NMR technique to a simple modelcompound and show how the data is reported.Nearly half a century has passed since the first report of a nuclear magnetic resonance (NMR) signal whichwas at that time termed a "nuclear induction". 72 In the ensuing fifty years, NMR has developed from an interestingphysical phenomenon to an analytical tool widely used in a variety of disciplines. NMR spectroscopy is currentlyone of the most powerful techniques available for the elucidation of natural product structures. With thedevelopment 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 detailedexplanations of the theory of NMR spectroscopy and of one-dimensional experiments. 73A typical time course for a one dimensional NMR experiment is shown in Figure 6. The preparationperiod allows the magnetization of the sample to reach thermal equilibrium and ends with the application of the firstradiofrequency pulse. During the evolution time (a constant time Di) additional pulses may be applied, thedecoupler channel may be gated to remove scalar through-bond coupling interactions, or through-space dipolarcouplings may take place. The resultant signal is acquired during the detection period 73cPULSE PREPARATION^EVOLUTIONDiDETECTIONt236Figure 6. Time Course for a One-Dimensional NMR Experiment.THE 1 H HOMONUCLEAR NOE DIFFERENCE EXPERIMENTThe NOEDS (Nuclear Qverhauser Effect Difference .pectrum) experiment was used in this thesis todetermine structure and assign relative stereochemistry to the natural products which were isolated. When a proton atchemical shift 5A is saturated by application of a selective radiofrequency pulse, a change in the intensity of signal5X, known as a Nuclear Overhauser Effect (NOE), can occur. The appearance of a NOE is a consequence of throughspace dipolar coupling between nuclei A and X. Subtraction of a spectrum irradiated off-resonance (i.e. with noNOE) 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, andsignals at 5X, the chemical shift of the NOE altered resonance. NOEs may be positive or negative and the intensityof the signal is inversely proportional to the sixth power of the through-space distance between the protons 5A and5X.73cFigure 7 shows the pulse sequence of an NOEDS experiment. 73c The NOEDS experiments discussed inthis thesis were recorded on a 400 MHz spectrometer. Typically the decoupler power used for the selectivecontinuous irradiation was attenuated at 50 dB and a 6 s preparation delay allowed the system to come to completethermal equilibrium between experiments.90037tHtHDECOUPLERSELECTIVE CONTINUOUSIRRADIATIONFigure 7. The Pulse Sequence for an NOEDS Experiment. 73cFigure 8 is the 1H NMR spectrum of 4-methylcatechol (105). This commercially-available compound waschosen to illustrate the applications of the NOEDS and 2-D NMR experiments because of its simple structure andbecause 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 atthe 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 signalsand the NOE difference spectrum 9(a) shows enhancement of the signals at 6.52 (H5) and 6.68 (H3). The data is nottabulated but is presented in the form of an illustration, as shown in Figure 10. In Figure 10, the tail of the arroworiginates 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 spectrum9(b) that the NOEDS experiment is limited when the region to be irradiated is congested. The results are not reliableas all three protons in the aromatic region (6.5-6.8) show a negative signal in spectrum 9(b). This indicates that theirradiating pulse was not sufficiently selective to differentiate the aromatic protons.OH10538Figure 10. Result of NOE Difference Experiment on 4-Methylcatechol (105).Mel OHOHMO^7.71^^7.M^7..00^•..70^I. ^1.ssOH^H6 H3 115ppm^i^e^ii^4^3^i^iFigure 8.^111 NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 AlMe2S0 at 500 MHz)b^115a^ Mell's"444#44140woMe.,•••,•fteNseve740OHOH7.0^6 . 0^5 . 0^4. 0^3. 0^2.0^1 . 0PPMFigure 9. NOE Difference Spectra of 4-Methylcatechol (105) (Recorded in CEC13 + 5 p.1 Me2S0 at 400MHz)TWO DIMENSIONAL NMR EXPERIMENTSTwo dimensional NMR pulse sequences share a common time course, illustrated by Figure 11. 74 As inthe 1-D experiment (Figure 6), the preparation period allows the sample to come to thermal equilibrium and endswith the application of the first radiofrequency pulse. The evolution time, ti , is incremented by a constant valueit over the course of the 2-D experiment which results in modulation of the signals in the ti domain. The mixingperiod 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 transformationprovides 1-D spectra of the observed nucleus along the F2 axis as shown in Figure 12(a). By varying the delays andpulses introduced into the pulse sequence during the evolution period ti , the spectra along the F2 axis will bemagnitude or phase modulated, encoding information relating to some chosen parameter, for instance, scalar couplingto homo- or heteronuclei or dipolar coupling to another nucleus in the vicinity. A second Fourier transformation ofthe modulated signals along the F2 axis generates frequency domain spectra in the Fl direction which contain crosssignals in the 2-D spectrum at (SA, SX) where SA and SX are the chemical shifts of two nuclei which are coupled toone another (Figure 12(b)).7241PULSEPREPARATION^EVOLUTIONtiMIXING DETECTIONt2Figure 11. Time Course for a Two-Dimensional NMR Experiment.4.4 4.2 4. ^IS 3.11 pPPMIbl0^042tt(a)• • 09 ° it/o • /FIDFigure 12. Fourier Transformation of the FID of a 2-D Experiment with Respect to a) t2 and b) ti and t2.THE COSY EXPERIMENTA simple homonuclear COSY (COrrelated Spectroscopy) experiment was employed to obtain informationabout the proton connectivities of the natural products studied. The COSY experiment is a 2-D technique whichprovides comparable information to a series of 1-D homonuclear decoupling experiments. The advantage thistechnique has over its 1-D equivalent is that all scalar couplings can be mapped in a short time span whereas in acomplex 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 NMRspectra while 1-D homonuclear decoupling relies on selective irradiation of a chosen proton which may beimpossible if the resonance is not well resolved. 75Figure 13 shows the pulse sequence for a simple COSY experiment. 73b The first 90° pulse inducesmagnetization in the sample proton spins. These then precess at their Larmour frequency, Do , during the evolutionperiod ti. The second 60° pulse transfers the coherence (or polarization) of the protons which have evolved in ti toprotons to which they are scalar coupled. This mechanism has been explained by quantum mechanics and also byvector product formalism. It is not trivial, however, and a detailed explanation is outside the scope of this thesis.43The proton spectra which result from Fourier transformation of FIDs (Free Induction Decay) acquired during thedetection period are consequently modulated by scalar coupling. A second Fourier transform produces off-diagonalcorrelations between scalar-coupled protons. Where vi = v2 (i.e. correlation of a proton to itself) the signal appearson the diagonal.76Figure 13. The Pulse Sequence for a Simple COSY Experiment. 73bFigure 14(a) shows the entire COSY spectrum of 4-methylcatechol (105) while Figure 14(b) is anexpansion of the aromatic region. Figure 14(b) shows correlations from the doublet at 8 6.74 (H6) into a doublet ofdoublets at 6.52 (H5) which is further correlated to a doublet at 6.68 (H3). From the chemical shifts, it was evidentthat these were protons on an aromatic ring. The coupling constants were determined from the 1-D 1 H NMRspectrum. The coupling constant of the doublet at 8 6.74 (8 Hz) was consistent with coupling to a proton at theortho position. From the COSY spectrum, we knew this was the doublet of doublets at 8 6.52 (H5). The resonanceat 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 protonresonance at 8 6.68 (H3) and 6.52 (H5) to a singlet at 2.20 (Me7) which integrated for three protons. These weakcorrelations are typical of long-range coupling and they indicated that a methyl substituent separated the aromaticprotons at 8 6.68 (H3) and 6.52 (H5). The broad singlet at 8 7.35 was attributed to the exchangeable phenolprotons. From the information provided by the COSY spectrum it was possible to construct a fragment with allproton connectivities defined, such as that shown in Figure 15. The data is summarized in Table 1. The firstcolumn in Table 1 lists the carbon number. The second column contains the chemical shifts of protons attached tothe carbons listed in column 1. The proton resonances which show COSY correlations to the resonances listed inthe second column are found in column 3.(alFigure 14. COSY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 511.1 Me2SO at 400 MHz)H6113 H5MelOHOH1. I V i,^• I I 'VI I TV w I 1 l7.07 0^6 0^5 0^4.0^3 0^2 0^i 0PPM116 113 H5OH^1 t.^H5/1160• •H5/Me7113/Me70\6.74H2.207CH36.68^6.52H HFigure 15. The Proton Connectivities of 4-Methylcatechol (105) from the COSY Spectrum.Table 1. 1H NMR and COSY Spectroscopic Data for 4-Methylcatechol (105).Carbon no. 51H NMR (ppm) (recorded in CDC13 +5 pL Me2S0 at 400 MHz)COSY (recorded in CDC13 + 5 RI,Me2SO at 400 MHz)1 — —2 — —3 6.68, d, J = 2 Hz H5, Me74 — —5 6.52, dd, J = 2, 8 Hz H3, H6, Me76 6.74, d, J = 8 Hz H57 2.20, s H3, H5-OH 7.35, br s —THE HMOC EXPERIMENTFor 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 illustratesthe pulse sequence used for the HMQC experiment.77 Again the mechanism of the experiment involves quantummechanics and is beyond the scope of this thesis. Typical values of the delays 0 and T used for this experiment were3.5 x 10-3 s and 0.7 s, respectively. After Fourier transformation of the FID with respect to t2, the resultant 2-Dspectrum has proton spectra, accumulated during the acquisition period t2, along the F2 axis. The protonmagnetization is modulated by scalar one-bond coupling to 13C during the evolution period, ti . Consequently,following a second Fourier transformation with respect to ti, the 2-D spectrum contains signals which correspond tocorrelations from the proton chemical shifts (F2 axis) to the chemical shifts of attached carbons (F1 axis).454690°,^180°.^90°_.^90°,^180%t H13C DETEC'ITONA^1/2t1^1/2t1^AT180° 90°^90°^90°xDECOUPLE-41111—BIRD —3■0- iFigure 16. The Pulse Sequence for an HMQC Experiment. 77Visually 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 detectedwhereas the HETCOR experiment detects the 13C RD. The greater abundance of 1 H versus 13C and its highersensitivity in the NMR experiment means that the HMQC experiment is significantly more sensitive than theHETCOR experiment. 74Figure 17 shows the 13C NMR spectrum of 4-methylcatechol (105). In order to assign the protonatedcarbons, an HMQC spectrum was employed (Figure 18). Figure 18(a) shows the entire HMQC spectrum. Anintense 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). Inthe expanded HMQC spectrum it could be seen that the H6 resonance at 5 6.74 was correlated to the carbonresonance at 5 115.1, the H3 doublet at 6.68 showed a correlation to a carbon signal at 116.1, and a correlation wasalso evident from the H5 resonance at 6.52 to a carbon resonance at 120.4. The data is reported in tabulated form asshown in Table 2.7OHOHSS*40 tooFigure 17.^13C NMR Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 g1Me2S0 at 125 MHz)OH[al7MelOHH6 1-13 H5—IRO1P" 6.9^6.7PPI6.5ce)UFigure 18.^HMQC Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 pi Me2SO at 500 MHz)Table 2. Assignment of Protonated Carbons of 4-Methylcatechol (105) from the HMQC Spectrum.Carbon no. 81H NMR (ppm) (recorded in CDC13 +5 ilL Me2S0 at 400 MHz)813C NMR signals assigned fromHMQC correlations (ppm) (recorded inCDC13 + 51.1L Me2SO at 125 MHz)1 — —2 — —3 6.68, d, J = 2 Hz 116.14 — —5 6.52, dd, J = 2, 8 Hz 120.46 6.74,d,J= 8 Hz 115.17 2.20, s 20.5-OH 7.35, br sTHE HMBC EXPERIMENTThe HMBC (Ideteronuclear Multiple Bond Connectivity) experiment is a variation of the HMQCexperiment which has a filter for one-bond correlations and has been optimized for two and three bond 1 H- 13Ccorrelations (theoretically, other nuclei may be substituted for 13C).78 This experiment is equivalent in terms of theinformation it provides to the 13C-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. TheHMBC is a very powerful experiment for establishing connectivity because the correlations can span heteroatomsand quaternary carbons. 79 By use of HMBC, proton spin systems which are separated by heteroatoms or quaternarycarbons may be assembled to give the molecular structure. The chemical shifts of quaternary carbons may also beassigned with greater confidence.The HMBC pulse sequence is illustrated in Figure 19. 78 The BIRD pulse sequence used for the HMQCexperiment is omitted here. The first 90° proton pulse is applied and time Ai later, a 900 13C pulse follows. Thissequence acts as a low-pass J filter so that the final spectrum does not show one-bond correlations.78 For HMBCspectra 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 results49A l^A2^1 /2 t,1 H50from the Fourier transformation has proton chemical shifts along the F2 axis and 13C chemical shifts along the F1axis. Correlations are from protons to carbons two and three bonds distant.900x^ 1800„DETECTION1 /2t,90 ° 90° 90°x13C A2..■■■••■■.Figure 19. Pulse Sequence for an HMBC experiment. 78Figure 20 shows the HMBC spectrum for the model compound 105. Assignment of the protonatedcarbons was possible from the HMQC spectrum but the quaternary carbons remained unassigned. Figure 21 showssome selected HMBC correlations which facilitated assignment of the quaternary carbons.2.20Figure 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 fromthe aromatic protons, the methyl singlet at 8 2.20 has a noisy line of correlations running along the F1 axis. Notall of the signals which make up the line correlate to carbon signals. This is known as ti noise. It generallyC6C3C5120C4 —130 cl ^C2 -PM-100•• •0f.I .• .^ .^ J,...4^.2 MU^2.2• •se 6• . IC6-50^C3C5C4 —CQ)44D^i^7OHOHMelH6 H3 H5Alm_________tts 0^tIlD 46')0^0 120e•^-130it :^-1406.8^6.4lal Ibl^ Ic)Figure 20. HMBC Spectrum of 4 -Methylcatechol (105) (Recorded in CDC13 + 5 µ1 Me2SO at 500 MHz)52occurs along the Fl axis and is associated with intense peaks in the F2 spectrum. It is caused primarily by technicalfactors such as fluctuations in pulses, temperature, and magnetic field. 74 Figure 20(b) shows an expansion of thecorrelations 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 finalcarbon, 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 thearomatic region of the HMBC spectrum. The proton resonances at 5 6.68 and 6.74 showed correlations into bothphenolic carbon signals at 8 141.9 and 144.0. This indicated that the phenolic carbons are side by side and that theprotons which resonate at 8 6.68 and 6.74 are each attached to a carbon adjacent to a phenolic carbon. The protonresonance at 8 6.52 (H5) showed a correlation into the carbon resonance at 6 141.9 but not the signal at 144.0. Thisallowed assignment of the carbon resonance at 8141.9 as Cl since a three bond HMBC correlation from H5 to Cl islikely 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) aresummarized in Table 3. The data contained in columns 1 and 2 is the same as in Table 1. The 13C chemical shiftsof the carbons in the first column are contained in column 3. The fourth column lists carbon resonances whichshow HMBC correlations to the proton resonance in the 5 1H column.Table 3. 1 H, 13C, and HMBC NMR Data for 4-Methylcatechol (105).Carbon no. 81H NMR (ppm) (recordedin CDC13 + 5 ill- Me2S 0at 400 MHz)613C NMR (ppm)(recorded in CDC13 + 5 'ILMe2SO at 100 MHz)HMBC Correlationsa (500MHz)1 - 141.9 -2 - 144.0 -3 6.68, d, J = 2 Hz 116.1 CI, C2, C5, C74 129.6 -5 6.52, dd, J = 2, 8 Hz 120.4 Cl, C3, C76 6.74, d, J = 8 Hz 115.1 Cl, C2, C47 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.THE ROESY EXPERIMENTThe ROESY experiment (_Rotating Frame averhauser Enhancement .pectroscopy, originally named theCAMELSPIN experiment) provides information comparable to NOE difference experiments. 80 It is closely relatedto the NOESY experiment but is the technique of choice for molecules with molecular weights of less than1000 g/mol. Figure 22 illustrates the pulse sequence used to record a ROESY spectrum. 80 A 900YU pulse is appliedand the system evolves as a function of scalar coupling during the evolution period ti . Subsequently aradiofrequency field, known as the spin-lock pulse, is applied for a mixing time 'rm . A mixing time of 0.225 s wasused for ROESY experiments discussed in this thesis. During the mixing time, the magnetization which evolved asa result of scalar coupling is "locked" and cross-relaxation, i.e. dipolar relaxation from one proton to a proton in thevicinity, takes place. The proton spectra detected during the acquisition period are therefore modulated by dipolarrelaxation. 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. 80,741H^90°Figure 22. The Pulse Sequence for a ROESY Experiment. 80The ROESY experiment has advantages over NOE difference experiments that are similar to the advantagesof COSY over 1-D decoupling experiments. Again, much more information can be acquired in a shorter period oftime and the ROESY experiment can be used to observe correlations in congested areas where selective irradiation53would be difficult. A limitation of this experiment is that signals close to the diagonal may be difficult todistinguish.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 observedfrom the methyl resonance at S 2.20 (Me7) to the aromatic proton resonances at 6.52 and 6.68. These correlationswere in good agreement with the results of the NOE difference experiments (Figures 9 and 10). The aromatic regionof Figure 23 is noisy and provides no information as the aromatic proton resonances are not sufficiently wellresolved. 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 experimentresults).OH105Figure 24. Results of a ROESY Experiment on 4-Methylcatechol (105).54MelH6 H3 H541 111U.■..755OHOHFigure 23. ROESY Spectrum of 4-Methylcatechol (105) (Recorded in CDC13 + 5 tilMe2S0 at 500 MHz)A. CYTOTOXIC METABOLITES OF APLYSILLA GLACIALISRESULTS AND DISCUSSIONAndersen and Tischler studied the secondary metabolites of the marine sponge Aplysilla glacialis andreported the isolation of six new diterpenes. 28d36 The research described in this thesis was prompted by routinescreening of A. glacialis silica gel column fractions for biological activity. A polar fraction with 1 H NMR signalswhich did not correspond to those of any of the previously isolated diterpenes proved to be cytotoxic. Furtherseparation of the polar fraction led to the isolation of glaciasterols A (100) and B (101), members of a new familyof cytotoxic 9,11-secosteroids.Specimens of A. glacialis were collected in Barkley Sound and Sydney Inlet, British Columbia. Thefreshly-collected sponge was extracted sequentially with methanol and methanol/dichloromethane (1:1). The extractswere combined, concentrated, and partitioned between ethyl acetate and brine. Chromatography of the ethyl acetateextract yielded glaciasterol A (100) and an inseparable mixture of related steroids. A 1 H NMR spectrum of this crudemixture contained no strong singlets at —6 2 indicating that the parent glaciasterols did not contain acetatefunctionalities. Acetylation of the steroidal mixture made it possible to purify one of the major products, thediacetate 107 of glaciasterol B (101). The structures of glaciasterols A (100) and B (101) were deduced by analysisof the spectroscopic data of glaciasterol A, the diacetates 106 and 107 of glaciasterols A and B, respectively, andfrom the results of chemical interconversions. 100 R =^, R'. H 106 R= , R .= Ac56101 R =^, R'. H 107 R= I^I , R'= AcHO^21 22181219 02517 23^26HO57Glaciasterol A (100)Glaciasterol A was isolated as an optically active, amorphous, white powder. The EIHRMS spectrumshowed 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 NMRspectrum (Figure 25) of glaciasterol A (100) indicated that the molecule contained five methyl groups. The methylsignals included; two sets of doublets, one at 8 0.94 which integrated for six protons and one at 1.02 whichcorresponded 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 correspondto 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 sidechain a and the singlets were due to Me18 and Me19 of the steroid nucleus. The connectivity of the side chain a wasdetermined from the COSY spectrum (Figure 26). A correlation was observed from the Me25 and Me26 doublet intoa methine at 8 2.22. This methine (H24) showed a correlation into an olefinic proton at 8 5.29 (H23) which wascoupled 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). Adouble resonance experiment in which H2O (8 2.13) and H24 (2.22) were simultaneously irradiated (possible becausethe signals are not well resolved in the 1 H NMR spectrum in CDC13) resulted in simplification of the multiplet at5.28 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 ofseveral of the side chain carbon resonances. The Me25 and Me26 doublet showed correlations into carbon signals at8 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.5.272.13 H1.02^HCH3580.94CH3132.136.0^CH30.94H 2.225.29aTable 4. 1H and 13C NMR Data for Glaciasterol A (100) (Recorded in CDC13)Carbon no. 81H (ppm) (400 MHz) COSY (400 MHz) 813C (ppm) (125MHz)813C for 97 (ppm)(100 mHz)c,671 1.8 H1', H2, H2' 26.6a 32.81' 2.1 HI, H2, H2' - -2 1.68 HI, H1', H2', H3 30Aa 31.72' 2.13 H1, H1', H2, H3 - -3 3.99, m H2, H2', H4, H4' 68.4 69.84 1.59 H3, H4' 37.4 34.34' 2.19 H3, H4 - -5 - - 63.5 49.86 3.39, d, J = 4.6 Hz H7 53.5 68.87 6.80, dd, J = 4.6, 0.9 Hz H6 139.3 151.08 - - 134.2 133.09 201.8 205.110 - - 45.6 45.311 3.69, m H11', H12, H12' 59.1 204.011' 3.81, m H11, H12, H12' -12 1.15 H11, H11', H12' 40.4 51.012' 1.63 H11, H11', H12 -13 46.1 46.414 3.39, ddd, J = 11.3, 8.1,0.9 HzH15, H15' 43.9 43.515 1.58 H14, H15' 26.9a 26.715' 1.66 H14, H15 - -16 - - 29.8a 26.417 49.6 52.018 0.68, s - 17.8 -19 1.24, s 21.5 -20 2.13 H21, H22 38.5 -21 1.02, d, J = 6.8 Hz H2O 21.3 -22 5.27, dd, J = 15.3, 7.6 Hz H20, H23 136.0 -23 5.29, dd, J = 15.3, 6.1 Hz H22, H24 132.2 -24 2.22, m H23, H25, H26 31.0 -25 0.94, d, J = 6.7 Hz H24 22.6b -26 0.94, d, J = 6.7 Hz H24 22.7ba,b May be interchanged. C Spectrum of secosteroid 97 was recorded in pyridine-d5.HO 11 21181219 022^2523^26HOOH11'H3^H11Me19Me26Me25Me21H7H14H23^ H6H22H12'^H1'^H4HI-1244'111220'^HH215wH12 ,I6. 5^6. 0^5 . 5^5.0^4.5^4.0^3.5^3 . 0^2.5PPMFigure 25.^Ill NMR Spectrum of Glaciasterol A (100) (Recorded in CDCI3 at 400 MHz)I^' " I2.0 1.5^1.0Me18HOFigure 26.^COSY Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)60H3/H4 gab 12H3/H4'...., H14/H15-g- H14/H15'H11/H121_H11/H 12'AoH11'/H12^'V H11 '/H12'- eaH3/H2' H3/1-12•3.5- 4.5_^s. 0H22/H23^ H22/H20H24/H23 5 .564).H14/H7•,.. 7. 01^• • I " " 1" "^• .'• 1••••1••••, •• ••■■■••T • ••^•^• • I • • • • 1 • • • • ppm7.0^6.5 6.^5.5^5.0^6. 5^4. FIT 3.5^3.0^2.5^2.0^1.5^1.1^.5"ftCSH20/Me21,1111H12/H12'Me25/H24Me26/H243.0sl.1-^ 5.5 5.0Ir^ V-^V^V^ V-7.0 6.0^5.0^4.0^3.0^2.0 1.0^O.()Figure 27. Double Resonance NMR Experiment on Glaciasterol A (100) (Recorded in CDC13 at 400 MHz)o.- •^•^ 1.--L^ slab 11 NI I" I MI Ai . 1 1AULLA^41141464..1.^*LAFigure 28.^13C NMR Spectrum of Glaciasterol A (100) (Recorded in CDC13 at 125 MHz)HOH11' H14H3 H11 7 6H7 1-1H22PPP 6^5^4^3•4 0•PI• 0^0Qa 0102 • c21C 19C25 C26C18•• ••0—40'I^ IPPMPOP•El r••C61- 50C14. C11C3-100•• •C23C22-C7pp.a4, 4Figure 29.^HMQC Spectrum of Glaciasterol A (100) (Recorded in CDCI3 at 500 MHz)A peak in the mass spectrum at m/z 301.1814 Da (5% intensity, C19H2503) which corresponded to themolecular ion with loss of one equivalent of water and the side chain (C7H13) supported the assigned constitution aof the side chain.All twenty-six carbons were visible in the 13C NMR spectrum of glaciasterol A (100) (Figure 28). Aresonance at 5 201.8 indicated that the molecule contained an a,13-unsaturated ketone which was corroborated by thepresence of a carbonyl stretch at 1681 cm -1 in the IR spectrum. 13C NMR resonances at 6 139.3, 136.0, 134.2 and132.2 ppm indicated that glaciasterol A had two double bonds. The HMQC spectrum established that one doublebond 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 the13C NMR resonance at 5 201.8 (C9). The molecular formula C26H4004 required seven sites of unsaturation. Thecarbonyl and two double bonds left four sites of unsaturation unaccounted for, thus it was concluded that themolecule must be tetracyclic. Acetylation of glaciasterol A gave a single product, 106, which was determined to bea diacetate by analysis of 1 H NMR (Figure 30) and mass spectrometric data. This indicated that the nativecompound, glaciasterol A (100), had two hydroxyl functionalities. One oxygen remained unassigned. Since it didnot belong to a hydroxyl function, and the IR contained only one carbonyl stretch, it was concluded that one of therings must be a cyclic ether. This was confirmed by the 13C 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 oxygenfunctionalities. From the formation of the diacetate 106, it was clear that two of these carbon resonances must beattached to hydroxyls in the parent compound 100. The IR spectrum of glaciasterol A diacetate (106) contained nohydroxyl stretching bands at 3300-3500 cm -1 indicating that glaciasterol A (100) did not contain more than twohydroxyls. Consequently, the two remaining carbons between 5 50 and 60 ppm were both necessarily attached to theremaining oxygen, forming an ether linkage. As a normal steroid skeleton does not contain a cyclic ether moiety, 37this provided the first suggestion that the compound might be a secosteroid, with only three of the standardcarbocyclic steroid rings.6465The COSY spectrum of 100 (Figure 26) indicated the presence of a number of spin systems in addition tothe 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 thecarbon and its attached protons indicated the methylene was attached to a hydroxyl function. Acetylation of 100yielded the diacetate 106 with a corresponding downfield shift of the HI1 and HI1' resonances from 6 3.69 and 3.81to 4.16 and 4.10, respectively. This result confirmed that C11 bore a hydroxyl terminus. The COSY spectrum of100 showed correlations from 5 3.69 and 3.81 (H11, H11') to a pair of geminal methylene proton resonances at1.15 and 1.63 (H12, H12') indicating that the molecule contained the hydroxyethyl fragment b. A lack of furthercorrelations suggested that fragment b was an isolated spin system.1.63^1.15H^HHO^59.1H^H3.81^3.69bGood agreement between the 1 H and 13C 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 indicatedthat glaciasterol A (100) might also be a 9,11-secosteroid.81COSY (Figure 31), HMQC (Figure 33) and HMBC (Figure 34) data for glaciasterol A diacetate (106) wereused to establish the structure of glaciasterol A (100). Glaciasterol A diacetate (106) was a white solid which gavea 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 inthe spectrum of 100 was no longer apparent but that it had been replaced by an acetate carbonyl stretch at 1737 cm -1which was observed in addition to a carbonyl stretch at 1685 cm -1 . The latter frequency was attributed to thea,(3-unsaturated ketone also present in the parent compound 100. The UV spectrum of diacetate 106 had a Amax at254 nm which was in good agreement with the UV spectrum of model compound 108 (, max at 242 nm)82 once anincrement of 10 nm for the additional a-alkyl substituent had been added.OH108Table 5 summarizes the NMR data for glaciasterol A diacetate (106). The significant changes in the1 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 106.66Table 5. 1H and 13C NMR Data for Glaciasterol A Diacetate (106) (recorded in CDC13)Carbon no. 8 1H (ppm) (400 MHz) COSY (400 MHz) 613C (ppm) (125MHz)HMBCa (500 MHz)1 1.77 H1', H2, H2' 27.6 —1' 2.10 H1, H2, H2' —2 1.70 H1, H1', H2', H3 26.6 —2' 2.17 H1, H1', H2, H3 — 3 4.99, m H2, H2', H4, H4' 70.71.65—4eu H3, H4' 34.0 C34'ax 2.26, t, J = 12.5 Hz H3, H4 — —5 — 63.0 —6 3.37, d, J = 4.5 Hz H7 53.4 C7, C87 6.76, d, J = 4.5 Hz H6 138.6 C5, C6, C9, C148 — — 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 3.23, dd, J = 10.7, 7.5 Hz H15, H15' 43.8 C7,C8,C13,C15,C1815 1.57 H14, H15' 27.4 —15' 1.71 H14, H15 — —16 — 25.1 —17 1.74 H2O 50.4 —18 0.72, s — 17.6 C12,C13,C14,C1719 1.22, s 21.1 C1,C5,C9,C1020 2.17 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 24 2.21 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 22.72.01, s; 2.04, s—0Ac 21.2; 21.2; 170.1;171.0a The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 H column.6719 0 Me196 . 5IT^1-1-1-11 T 1 T T 11^I^r 16 . 0 2.0^1 . 5^1 . 05 . 5^5.0^4 . 5 4 . 0^3.5^3.0^2.5PPMOCOCII3OCOCE3^Ac0 11 21^22^18^23^2612AcOOwH23H22H7}16^ H24H4'1420^H12'H2' H1'H11' H11^H14Me26Me25Figure 30.^1 H NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400 MHz)t —^1 . 04. 0_^5 . 0pt6 . 0_^6.0H3/H2,H3/HT H20/Me21esMe25/H24Me26/H24_^2. 0H14/H15'eraH14/H15H11/1112: H11/H12H11/H11'^ H11'/1112' H11'/H12Ira^VIH3/H4' H3/H4ectH24/H23_^3. 0H6/H7- •^ • • •^1^I^PPMMe19Me18Me26Me25Ne25. 0^4. 0^3. 0 2. 0 I. 0PPM69 Figure 31.^COSY Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 400 MHz)AcOI200I ,140 1201^I^1^I80 60 40 20Figure 32. 13C NMR Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 100 MHz)r100PPM1160180H7 HO• C14•- C6— 80▪ C11Me26Me19 Me25Me21 Me18H3^Hll'Hll^H14414:•%• to.• 4100820C21C19C 25 C26:C1— 40giO- C3—80•Ac0 11 21181219 02226H23H22AcO- C23-C22LC7ppm• Oks •71— 100• ••—120•-11111-1111-1.71-17-1-TIT T/ 11111-T1111 -1rivrtfri-Timi5^4^9^2^1^O^•^ppa^6Figure 33.^HMQC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDC13 at 500 MHz)Me19 Me18Ac0 11 21181219 o22Ac0ppe^5.9^ 5.I^8 ^ 5.7• Me19/C5e C511••00%^ ■^ili,• Me19/C1:51..s.'Me19/C101[1:MO -^Me18/COO z Mel 8111I C1417 Me18/C1345*•4="1"-...Me18/C12-100•• ODAID-150• •2.0•1.5C9"VPMe19/C9ppmClC12C14- C17C13Figure 34.^HMBC Spectrum of Glaciasterol A Diacetate (106) (Recorded in CDCI3 at 500 MHz)C^6.761122(13.0"'0^H3.37()200.0 '777.412124^1 141.2sr, 53.4 138.6H73A resonance in the 13C NMR spectrum (Figure 32) at 8 200.0 in addition to the IR band at 1685 cm -1indicated that 106 contained an a,13-unsaturated ketone. From the HMQC data it was established that the carbonresonances at 8 136.2 and 131.9 formed the side chain double bond and this permitted the assignment of the tworemaining olefinic carbon resonances at 141.2 and 138.6 to the double bond a to the ketone. From the HMQCspectrum (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 forvicinal coupling which established that the protonated olefinic carbon (C7, 8 141.2) must be 0 to the ketone. TheHMQC spectrum indicated that the proton at 8 3.37 (H6) was bound to a carbon at 8 53.4, presumably one of twocarbons attached by an ether linkage. This data resulted in the elucidation of fragment c .cExamination of the basic steroid skeleton showed that no cleavage point other than the 9,11-carbon bondwould allow construction of a molecule containing an isolated acetoxyethyl fragment and an a,f3-unsaturated ketonewhere the double bond is trisubstituted and bears a proton at the p position. The cleavage of the 9,11 carbon bondindicated 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 theremaining unassigned hydroxyl from its shift to 4.99 upon acetylation of 100 to the diacetate 106. The COSYspectrum of glaciasterol A diacetate (106) (Figure 31) showed correlations from the multiplet at 8 4.99 (H3) to twopairs of geminal methylene protons at 1.70 and 2.17 (H2, H2') and at 1.65 and 2.26 (H4, H4'). The methyleneprotons 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.H H2.26 1.65dMe19 was assigned from a correlation in the HMBC spectrum of 106 from the methyl singlet at 6 1.22 tothe carbonyl resonance at 6 200.0 (C9) (Figure 34, Figure 35). Another HMBC correlation was observed from theMe19 resonance (6 1.22) to a 13C resonance at 6 27.6 (C1) which had been assigned to the terminal methylenefurthest from the acetoxy functionality of fragment d from the HMQC data. The HMBC correlation from the Me19resonance into this carbon permitted its assignment as Cl which indicated that the acetoxy functionality was situatedat the standard C3 position. An HMBC correlation from the Me19 proton resonance (6 1.22) into a 13C resonanceat 5 63.0 (C5) provided evidence that glaciasterol A diacetate (106) was a 5,6 epoxide. HMBC correlations from theH7 resonance (6 6.76) to the carbon signals at 6 63.0 (C5) and 53.4 (C6) further substantiated the location of a5,6-epoxide moiety adjacent to the (4-unsaturated ketone.74AcOFigure 35. HMBC Correlations from H6, Me18, and Me19 of Glaciasterol A Diacetate (106).75The final clearly resolved downfield resonance in the 1 H NMR of 106 (Figure 30) was a doublet ofdoublets at 8 3.23. This was tentatively assigned as H14 of the D ring. The H14 multiplet showed COSYcorrelations to a pair of methylene protons at 6 1.57 and 1.71 ppm (H15, H15'). Further correlations were obscuredby the methylene envelope. An HMBC correlation from the H7 resonance at 6 6.76 to a 13C resonance at 8 43.9(C14, HMQC correlation to 3.23) established the C8/C14 linkage. The carbon resonances for the D rings of 100and 106 were assigned from the HMQC spectra (Figures 29 and 33, respectively) and by comparison with the valuesfor a closely related secosteroid (97) 67 (see Table 4).HOH OH97The Me18 proton resonance (8 0.72) had an HMBC correlation to a 13C resonance at 6 36.7 (C12) whichconclusively situated the acetoxyethyl fragment. A correlation was also observed from the Me18 resonance to the13C resonance at 8 43.8 (C14) which further supported the assignment of H14. Finally, correlations from the1 H NMR resonance at 8 0.72 (Me18) to 13C resonances at 8 46.1 and 50.4 permitted the assignment of these carbonshifts as C13 and Cu,7 respectively.Figure 36 shows the results of an NOEDS experiment on 106 in which the Me19 signal at 6 1.22 wasirradiated. NOE enhancement was observed in the proton resonances at 6 1.70 (H2ax ) and 2.26 (H4'ax). Theobserved NOEs from Me19 to H2ax and H4ax , in conjunction with the trans diaxial coupling of H4ax with H383(6 2.26, t, J = 12.5 Hz), indicated that the A ring was in a chair conformation and the C3 acetoxy functionality hadthe R configuration. Irradiation of the H3 methine (8 4.99) resulted in NOE enhancements of the proton signals at2.17 (H2'eq) and 1.65 (H4eq).je,..----1.70 /..---.., 1.22H CH32.26HHH 1.65j. ---- 4.99•••^•■■=O.6'Ac0^21^22^24^2623^2516^2715AcOFigure 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 topermit determination of the relative stereochemistry of chiral centres with the exception of C3. The more ubiquitousglaciasterol B diacetate (107) and several chemical derivatives were studied to obtain additional information about thestereochemistry of the glaciasterols.Glaciasterol B Diacetate (107)Glaciasterol B diacetate (107) was isolated from a mixture of acetylated secosteroids and recrystallized fromaqueous methanol to yield white needles. The melting point of the crystals was found to be 55-57°C. GlaciasterolB diacetate (107) gave a molecular ion in the EIHRMS at m/z 516.3451 Da, appropriate for a molecular formula ofC31 H4806 (AM 0.0 mmu). The IR spectrum of 107 was similar to that of 106 with carbonyl stretches at1735 cm -1 and 1684 cm -1 . The UV spectrum recorded in methanol had a Xmax at 258 nm.7677From 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. Themolecular formula C31H4806 indicated that glaciasterol B diacetate (107) had one more methylene and one lessdegree of unsaturation than glaciasterol A diacetate (106). The 1H NMR spectrum of 107 (Figure 37) lacked themultiplet at 6 5.28 (H22 and H23) which was apparent in the 1 H NMR spectrum of 106 and which resulted fromthe olefmic protons in the side chain of 106. The chemical shifts of the methyl doublets in the 1 H NMR spectrumof 107 (Figure 37) at 8 0.97 (3H) and 0.86 (6H) agreed with those expected for a cholesterol side chain. Analysis ofthe 13C NMR (Figure 38), HMQC, and COSY data for 107 permitted the assignment of the side chain carbonresonances. Comparison of the side chain 13C NMR shifts of 107 with those of secosteroid 97 indicated that bothcompounds possessed the same saturated C8H17 side chain (see Table 6). 68 A peak in the EIHRMS of 107 atm/z 283.1729 Da (22.9% intensity) corresponding to the molecular ion with loss of two equivalents of acetic acidand the C8H17 side chain corroborated the assignment of the side chain structure. The resonances in the 1 H and13C NMR spectra of 107 were assigned by use of the COSY and HMQC data and by comparison with the valuesfor glaciasterol A diacetate (106) (Table 6).HO97Figure 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 H213resonance, and at 2.24, assigned as the H413 signal. The NOEDS results indicated that, as in the case of glaciasterolA diacetate (106), the A ring of glaciasterol B diacetate (107) was in a chair conformation and the 3-acetoxysubstituent had the 13 configuration as shown in Figure 39. Irradiation of the H14 resonance at 8 3.23 induced NOEenhancements in the H11 multiplet at 4.10 and the H15a resonance at 1.68. From the NOE data it was concludedthat H14 and Me18 existed in the trans relationship common to most steroids. C17 and C20 were assumed to havethe standard steroidal configuration but the stereochemistry was not verified by experiment.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) (75MHz)813C NMR shiftsfor 97 (ppm) (100mHz)68,c1 1.77 H1', H2, H2' 27.6a -1' 2.10 H1, H2, H2' -2 1.72 HI, H1', H2', H3 26.6a -2' 2.17 HI, H1', H2, H3 - -3 4.99, m H2, H2', H4, H4' 70.6 -4eq 1.64 H3, H4' 33.9 -4'ax 2.24, dd, J = 12.5, 12.6 Hz H3, H4 - -5 - - 63.0 -6 3.36, d, J = 4.5 Hz H7 53.4 -7 6.75, d, J = 4.5 Hz H6 138.6 -8 - 141.3 -9 200.0 -10 45.3 -11 4.10 H11', H12, H12' 61.1 -11' 4.19 H11, H12, 1112' -12 1.23 H11, H11', H12' 36.6 -12' 1.69 H11, H11', H12 - -13 46.0 -14 3.23, dd, J = 10.8, 7.5 Hz H15, H15' 43.5 -15 1.58 H14, H15' 25.9a -15' 1.68 H14, H15 - -16 - - 27Aa -17 1.83 1120 49.8 -18 0.73, s - 17.2 -19 1.23, s - 21.1b -20 1.44 H17, H21 34.8 35.321 0.97, d, J = 6.7 Hz H2O 18.9 19.422 - - 35.4 35.723 - - 24.5 24.524 - 39.4 39.625 1.54 H26, H27 28.0 28.226 0.86, d, J = 6.6 Hz 1125 22.6 22.727 0.86, d, J = 6.6 Hz 1125 22.8 22.9OAc 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-d5.782) 22 24^2618•,27SH7H3OCOCH3OCOCE3Me19Me18Me27Me26Me21w1-16Hll'Hll^1 H14I •^ '7.5^7.0^6.5^6.0^5.5^5.0 ,, ^ -^"-^14.5^4.0^3.5^3:0^2.5 2.0 1.5^t.0^0.5^00ppmFigure 37.^1 111s1MR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 400 MHz)ITIOITIIIIIII^IIII^200^16011 1 11^11111 1 111111^1 1111 1111 1 11 11 11 1 110^140^1 0^100IIIIIIiiiIIII^Itiii r 'tilt 1 lilt,'60^40^20ppmFigure 38.^13C NMR Spectrum of Glaciasterol B Diacetate (107) (Recorded in CDC13 at 100 MHz)n4.191.231.72 H ^CH3H2.24AcOAcOHH.683.23Figure 39. Selected NOE Difference Experiments on Glaciasterol B Diacetate (107) (Recorded in CDC13 at400 MHz).While NOE difference experiments were successful in determining some aspects of the stereochemistry ofthe 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 inTHE proceeded via SN2 attack at the less hindered secondary, rather than the more sterically hindered tertiary, carbonto yield the trans diol 110 (see Equation 1).84 Treatment of glaciasterol B diacetate (107) with aqueous perchloricacid under similar conditions was also found to result in ring-opening of the epoxide via SN2 attack to yield thecorresponding trans diol (Equation 2).81HC104 aq . / THE1 0 9 Equation 1. Ring-Opening of a 4a,5a-Epoxide with 60% Perchloric Acid in TI-1F to yield the 40,5a-Dio1. 84In order to ascertain that ring-opening of glaciasterol B diacetate (107) had indeed proceeded via nucleophilicattack at the less hindered C6, the reaction was carried out using 69% perchloric acid in —1:1 H2 61 0/H2 180 andAcOAc20 / pyridineOHR111 R = -1:1 160H / 180H107112 R' = -1:1 16OAc / 18OAc•WOAcO^- .^ AcO69% HC104 / -4:1 H2 160:H2180 /THF82THF. The signal for the carbon which undergoes nucleophilic attack by the labelled water should exhibit 160/180isotope-induced splitting in the 13C NMR spectrum of product 111. The diol 111 was eventually acetylated toyield the triacetylated ring-opened glaciasterol B derivative 112 (see Equation 2).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 amolecular formula of C31HSO 1607 (AM 0.3 mmu) and at m/z 536.3596 Da which corresponded to a molecularformula of C31 H SO 1606 180 (Am -0.3 mmu). This result indicated that glaciasterol B diacetate 107 hadincorporated one equivalent of -1:1 160/ 180-labelled water. The fragmentation pattern observed for the massspectrum was consistent with this conclusion (see experimental section). The IR spectrum showed an 0-H stretchat 3432 cm-1 as well as carbonyl stretches at 1739 and 1677 cm -1 . The derivative 111 was completely characterizedby NMR spectroscopy and the results are summarized in Table 7. The 1 H NMR resonances (Figure 40) wereassigned from the COSY spectrum (Figure 41). The major changes from the spectrum of the parent compound weredownfield shifts of the equatorial H4 resonance from 8 1.64 in the epoxide 107 to 1.85 in the diol 111 and of theH6 resonance from 3.36 in the parent compound 107 to 4.03 in the derivative 111. The resonance attributed to H7in 107 (8 6.75) was observed to shift upfield to 6.42 in product 111.Table 7. 1H and 13C NMR Data for 5a,613-Dihydroxyglaciasterol B Diacetate (111) (Recorded in CDC13)Carbon no. S1H (ppm) (400 MHz) COSY (400 MHz) 813C (ppm) (125 MHz)1 - 26.41' - - -2 1.65 H2', H3 26.12' 2.04 H2, H3 -3 5.11, m H2, H2', H4, H4' 70.44ea 1.85, dd, J = 12.8, 3.2 H3, H4' 34.74'ax 2.21, dd, J = 12.8, 11.9 H3, H4 -5 - - 76.56 4.03, d, J = 5 Hz H7 72.28, 72.30a7 6.42, d, J = 5 Hz H6 138.08 - - 137.39 - 202.310 - - 46.111 4.18, m H11', H12, H12' 61.411' 4.18, m H11, H12, H12' -12 1.30 H11, H11', H12' 36.912' 1.68 H11, H11', H12 -13 - 48.014 3.29, dd, J = 11.0, 8.6 Hz H15, H15' 42.715 1.60 H14, H15' 27.115' 1.70 H14, H15 -16 - - 27.417 - 50.318 0.76, s - 17.119 1.38, s - 21.620 1.46 H21 35.421 0.99, d, J = 6.7 Hz H2O 19.022 - 35.523 - 24.524 39.525 1.50 H26, H27 27.926 0.87, d, J = 6.6 Hz H25 22.5b27 0.87, d, J = 6.6 Hz H25 22.8bOAc 2.00, s; 2.04, s 21.1; 21.4; 170.1; 170.3aSplit into doublet by 160/180 isotope shift. b May be interchanged.83SAc0OH OHOCOCL130c0d113 wMe1921 22 2418 is'AcO 11 121926271 2^Figure 40.^1 H NMR Spectrum of 5a,6P-Dihydroxyglaciasterol B Diacetate (111) (Recorded in CDC13 at 500MHz)PP• 7 410,411%.Me27Me26Me 18Me285OCOCH3OCOCH3AcO OHOH21 22 24AcO 11 12262719H7^ H11'Me19H3^H11H6 H14Mee18VMe26Me21-4 siti eV ', ..el) it)* 40 Ilk* •7. I^6^S. I^4.^3^2 0^t o^PPMPPM1 02.03.04 a6 07 0Figure 41.^COSY Spectrum of 5a,613-Dihydroxyglwiasterol B Diacetate (111) (Recorded in CDC13 at 400MHz)AcOISO^140Figure 42.^13C NMR Spectrum of 5agl-Dihydroxyglaciasterol B Diacetate (111) (Recorded in C6D6 at 125MHz) with Expanded Regions a) Recorded in C6D6 and b) Recorded in cDa32) 22 24^2618 11'AcO 112719H3 AcO^OH^H11^a'H11H6OH•141111!*• .I•es °NI" •C14Ida^ 50•^NM*^ C11•is^.410 •C3C6100• •^• •II^WTI ^pl)al^6 4^2la]^ blFigure 43.^a) HMQC Spectrum of 5a,60-Dihydroxyglaciasterol B Diacetate (111) with b) Expanded Region(Recorded in CDC13 at 500 MHz).^acetic anhydridelOOOHOHpyridineThe 13C NMR resonances seen in Figure 42 were assigned from the HMQC spectrum and by comparisonwith the assignments for the parent compound 107. A correlation was observed in the HMQC spectrum from theH6 resonance at 5 4.03 to a 13C resonance at 6 72.30 (Figure 43). The quaternary carbon resonance at 6 76.5 ppmwas assigned to C5. The 13C NMR spectrum clearly showed an 160/180 isotope-induced splitting of the signal at6 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 13C NMR resonance at 6 76.5 ppm (C5) was a sharp singlet as were all of the other resonances inthe 13C NMR spectrum (Figure 42).Having established that the acid-catalyzed ring opening of the glaciasterol 5,6-epoxide proceeded via attack atcarbon 6, it was necessary to determine whether the product, 111, was the 5a,613-diol or the 513,6a-diol. Earlierstudies of the model compound 11082,84 had demonstrated that reaction of 110 with acetic anhydride resulted inacetylation of only the secondary hydroxyl while the tertiary alcohol functionality remained underivatized(Equation 3).88110Equation 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 majorproduct, the expected triacetate 112 (Equation 4). The glaciasterol B derivative 112 gave a molecular ion atm/z 576.3670 (C33HSO 1608, AM +0.8 mmu). The IR spectrum showed stretching frequencies at 3448, 1739 and1639 cm" 1 .OHR111 R = —1:1 160H / 180HAcOAcO •^0=0 69% HC104 / —1:1 H2 160:H2 180 /THFAc0^= .10789Ac20 / pyridine AcOOHR '112 R' = —1:1 16OAc / 18OAcEquation 4. Ring-Opening of Glaciasterol B Diacetate (107) to form 111 followed by Acetylation to yield 112It has been shown by Demarco and coworkers 85 that protons which are situated in the vicinity of ahydroxyl are shifted downfield in pyridine-d5 relative to deuterochloroform. Demarco et al. speculated that pyridinecomplexes with the hydroxyl via hydrogen-bonding. Protons located nearby are deshielded by the anisotropy of thepyridine aromatic ring. The 1 H NMR data for 5a-hydroxyglaciasterol B triacetate (112) recorded in CDC13 and inpyridine-d5 is summarized in Table 8 and shown in Figure 44. The proton resonances were assigned from COSYspectra (Figure 45) and the correlations are tabulated in Table 8. It can be seen from Figure 46 that H3 (8 5.10 inCDC13; 5.62 in pyridine-d5) was shifted downfield by 00.52 ppm relative to a downfield shift of only 00.19 ppmfor Me19 (1.36 in CDC13; 1.55 in pyridine-d5). Since an NOE difference experiment on the parent compoundglaciasterol B diacetate (107) had shown that H3 had the a configuration (Figure 39), the much greater downfieldshift of a proton on the a face (H3) compared to protons on the p face (Me19) of the steroid indicated that the3a-methine had a closer proximity to the 5-hydroxyl than did the n-oriented Me19 moiety. This evidence establishedthat the 5-hydroxyl functionality had the a configuration. The results indicated that the ring-opened product 111was the 5a,6(3-dihydroxyl derivative of glaciasterol B diacetate and that the 5,6-epoxide moiety of the parentcompound, glaciasterol B diacetate (107), had the a configuration.Table 8. 1H NMR Data for 5a-Hydroxyglaciasterol B Triacetate (112).Carbon no. 81H NMR recorded inCDC13 (ppm) (400 MHz)81H NMR recorded inpyridine-d5 (ppm) (400MHz)COSY recorded in pyridine-d5 (ppm) (400 MHz)1 1.73 1.93 Hi', H2, H2'1' 1.76 2.60, dd, J = 17.7, 12.8 Hz H1, 1-12, H2'2 2.05 1.73 H1, H1',112'2' 2.13 2.06 H1, H1', H23 5.10, m 5.62, m H2, H2', H4, H4'4 1.83 1.14 H3, H4'4' 1.95 2.25, br d H3, H45 — — —6 5.19, d, J = 5 Hz 5.85, d, J= 5 Hz H77 6.36, d, J = 5 Hz 6.62, d, J= 5 Hz H68 — — —9 — — —10 —11 4.14, m 4.47, m H11', H12, H12'11' 4.18, m 4.47, m H11, H12, H12'12 1.25 1.47 H11, H11', H12'12' 1.66 1.83 H11, H11',111213 —14 3.30, dd, J = 11.3, 8.9 Hz 3.53, m H15, H15'15 1.47 H14, 1115'15' 1.58 H14, H1516 —17 — — —18 0.70, s 0.80, s —19 1.36, s 1.55, s 20 1.45 1.35 H2121 0.98, d, J = 6.8 Hz 1.03, d, J= 6.6 Hz H2O22 — — —23 — —24 — — —25 1.53 1.50, m 1126, 112726 0.84, d, J = 6.7 Hz 0.88, d, J= 6.6 Hz H2527 0.84, d, J = 6.7 Hz 0.88, d, J= 6.6 Hz 1125OAc^_ 2.01, s; 2.04, s; 2.13, s 1.98, s; 2.02, s; 2.14, s —90AcOOH OAc6.0^5.0 4.0 3.0 2.0 1.0AcO 111921 22 24 2618 .'11227lalH3ppm6.0^5.0^4.0^3.0^2.0^1.0ppmFigure 44. 1 11 NMR Spectra of 5a-Hydroxyglaciasterol B Triacetate (112) Recorded in a) CDC13 and b)Pyridine-d5 at 400 MHz910 dgaH11/H12' H11/H1221 22 24 2618 '''AcO 11 1219OH OAc27AcO•6ss 2"PMs : s 1PPII 3-3H14/H15'aoH14/H15•H3/H2'a^ 0^•o^ H3/114'^113/}12OH6/H7•5-692Figure 45^COSY Spectrum of 5a-Hydroxyglaciasterol B Triacetate (112) (Recorded in Pyridine-d5 at 400MHz)AcO00.19 ppmCH3OAc 0AcOHA0.52 ppm18^2317161512AcO 1119AcOFigure 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 (11412521^22^2426OHOR113 R = H114 R = AcThe similarity of the 1 H and 13C NMR data for glaciasterol A diacetate (106, Table 5) and glaciasterol Bdiacetate (107, Table 6) provided evidence for a common nucleus for these compounds. The configuration of the5,6-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 withsubstitution of distilled water of standard isotopic composition for the —1:1 160/180 aliquot. Only a small portion93of the resulting diol was isolated and characterized by 1 H NMR and mass spectrometry. Derivative 113 gave amolecular 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 theanalogous 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. 8 1 H NMR of Diacetate 113recorded in CDC13 (ppm)(400 MHz)8 1 H NMR of Triacetate 114recorded in CDC13 (ppm) (400MHz)8 1 H NMR of Triacetate 114recorded in pyridine-d5 (ppm)(400 MHz)1 — — —2 — 1.83 —2' — 1.96 —3 5.12, m 5.09, m 5.67, m4 — 1.60 —4' — 2.03 —5 — — —6 4.04,d 5.19, d, J = 5 Hz 5.91,d7 6.40, d 6.34, d, J = 5 Hz 6.66, d8 — — —9 — — —10 — — —11 4.16, m 4.17, m 4.50, m11' 4.16, m 4.17, m 4.50, m12 — 1.30 —12' — 1.68 —13 — — —14 3.28,m 3.29, dd, J = 10A, 8 Hz 3.54,m15 — 1.57 —15' — 1.70 —16 —17 — — —18 0.75, s 0.68, s 0.81, s19 1.37, s 1.35, s 1.52, s20 2.19 —21 1.05,d 1.03, d, J = 6.8 Hz 1.09,d22 5.28, m 5.28, m 5.33, m23 5.28, m 5.28, m 5.33, m24 — 2.21 —25 0.95, d 0.95, d, J = 6.7 Hz 0.96, d26 0.95, d 0.95, d, J = 6.7 Hz 0.96, dOAc 2.02, s; 2.05, s 2.01, s; 2.04, s; 2.13, s 2.00, s; 2.05, s; 2.15, s94(alwMe19Ac0H3" • . I "5.5^5.0^4.5^4.0^3.5^3.0^2.5^2.0^1.5PPM 1.06. 5 6.0I blMe19195Figure 47.^1 H NMR Spectra of 5a-Hydroxyglaciasterol A Triacetate (114) Recorded in a) CDC13 and b)Pyridine-d5 at 400 MHz965a-Hydroxyglaciasterol A triacetate (114) was formed by reacting intermediate 113 with acetic anhydridein pyridine. The product 114 gave a molecular ion in the EIHRMS at m/z 560.3340 Da, appropriate for a molecularformula of C32H4808 (AM -0.9 mmu). The IR spectrum of 114 showed a band at 3402 cm -1 , attributed to thehydroxyl, and carbonyl stretching frequencies at 1738 and 1688 cm -1 . The compound was fully characterized byNMR spectroscopy. Table 9 summarizes 1 H NMR shifts of 114 in CDC13, confirmed by the COSY data, and inpyridine-d5, determined by comparison of the spectrum with that of the corresponding glaciasterol B derivative 112in pyridine-d5 (Figure 47). The H3a resonance was shifted t10.58 ppm downfield (5 5.09 in CDC13; 5.67 inpyridine-d5), significantly further than the Me19 (1.35 in CDC13; 1.52 in pyridine-d5) which was shifted downfieldby A0.17 ppm in pyridine-d5 relative to CDC13. The results in Table 9 established that glaciasterol A (100) wasalso a 5a,6a-epoxy secosteroid.CONCLUSIONSGlaciasterols A (100 ) and B (101 ) are the first secosteroids reported to be cytotoxic and the firstexamples of this class of compound identified from a sponge in the genus Aplysilla. These compounds add to thenumber of secosteroids which have been isolated from marine invertebrates. To date these are the only secosteroidsreported 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-bondof a highly oxidized intermediate such as 103, a polyhydroxylated steroid which has been reported from the spongeDysidea etheria.7°The cytotoxicity of a crude fraction which contained the glaciasterols had first attracted our interest tocompounds 100 and 101. Pure glaciasterol A (100), glaciasterol B diacetate (107), and 5a—hydroxyglaciasterol Btriacetate (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. Itis particularly interesting that glaciasterol A (100) and the acetylated compounds 107 and 112 were equally activeagainst both the MCF-7 and the multidrug-resistant MCF-7 Adr cell lines. The multidrug resistance of the MCF-7Adr cell line has been attributed to increased levels of a plasma membrane protein which appears to actively transportcytotoxic drugs out of the ce11. 86 Since steroids pass through plasma membranes by simple diffusion 87 it ispossible compounds 100, 107, and 112 are retained in the drug-resistant cells. Acetylation of the glaciasterolswould be expected to produce a more lipophilic derivative which would presumably be even better suited for transportacross cell membranes. The fact that glaciasterol B diacetate (107) was approximately ten times as active againstthe drug-sensitive MCF-7 and the drug-resistant MCF-7 Adr breast cancer cell lines as the unacetylated nativesecosteroid 100 is consistent with this hypothesis.Table 10. Results of the Assay of Glaciasterol A (100) and Related Derivatives 107 and 112 against In VitroMurine Leukemia and Human Breast Cancer Cell Lines.COMPOUND ED50 against L1210Cell Line (gg/mL)ID50 against MCF-7Cell Line (p.gimL)ID50 against MCF-7 Adr DrugResistant Cell Line (pg/mL)Glaciasterol A (100) 2.1 19 18Glaciasterol B Diacetate (107) 2.5 1.8 1.85a-HydroxyglaciasterolB Triacetate (112 )5.66 2 4.597B NATURAL PRODUCTS OF A PLERAPLYSILLA SP. SPONGERESULTS AND DISCUSSIONa) 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 smallsample of a gray-white sponge which had been identified by Dr. W. C. Austin as a new species of Aplysilla on thebasis of morphology alone. 29 The white sponge appeared to be an excellent candidate for the study of naturalproduct chemistry on the basis that it might contain cytotoxic metabolites similar to those produced by Aplysillaglacialis. It was proposed that a study of the metabolites of the sponge would also determine whether it was a colorvariant of Aplysilla glacialis or an entirely new Aplysilla species. The sponge was later reidentified as anundescribed Pleraplysilla species by Dr. R. W. M. van Soest on the basis of chemistry as well as morphology. 88Small collections of the sponge were made at Checleset Bay and Botanical Beach in British Columbia in 1990. Thefollowing year, the sponge patch at Botanical Beach was harvested again. These initial collections were treated in asimilar 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 werepooled and concentrated to yield a brown oil (67.8 mg). Separation of the sponge metabolites was accomplished bysequential application of size-exclusion gel (Sephadex LH-20 eluted with ethyl acetate/methanol/water; 20:5:2) andsilica gel (eluted with a polarity gradient of diethyl ether/dichloromethane/ethyl acetate) chromatographies. Finalpurification was accomplished by normal phase HPLC separation. Eluted in order of polarity were the knownsesquiterpenes 0-methyl furodysinin lactone (57) 89 and 0-methyl nakafuran-8 lactone (44). 28b The next set ofsilica column fractions contained the new sesquiterpenes 0-methyl 9-oxofurodysinin lactone (72) and 0-methyl2-oxomicrocionin-2 lactone (74). The previously reported compounds furodysinin lactone (56) 49 and nakafuran-8lactone (43)28b were the major components of the next fractions. The final fraction contained the novel metabolite2-oxomicrocionin-2 lactone (73) as its major component. All of the compounds isolated were hydroxybutenolidesor their corresponding methyl ketals. The possibility that these compounds were artifacts of autooxidation andreaction with the methanol extracting solvent was considered but examination of the 1 H NMR spectrum of the crudeethyl acetate sponge extract revealed no signals at —6 7.1 and —6.1, the expected chemical shifts of the protons of an98a,13-disubstituted furan. 28b The chromatographic separation also provided no evidence for the presence of furans inthe Pleraplysilla sp. sponge.99OR56 R . H^ 7257 R = CH343 R = H^ 73 R = H44 R = CH3 74 R = CH30-methyl 9-Oxofurodysinin Lactone (72)14 150-Methyl 9-oxofurodysinin lactone (72) was isolated as a colorless glass which gave a molecular ion in theEIHRMS at m/z 276.1371 Da, appropriate for a molecular formula of C16H2004 (AM 0.9 mmu). The molecularformula indicated that 72 had seven sites of unsaturation. Two carbonyl stretching bands were observed in the IRspectrum. The absorption band at 1767 cm -1 was at the appropriate frequency for a 8-lactone, 2813,89 while thesecond band at 1665 cm -1 indicated the molecule contained an a,(3-unsaturated ketone. A signal at 8 198.1 ppm inthe 13C 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 at6 3.20 (OCH3), which integrated for three protons, established the presence of a methyl ether (Figure 48). The10013C 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 13C shift of 8 106.8 ppm, was the onlyother carbon single-bonded to an oxygen atom. The chemical shift of C4 (8 106.8) was appropriate for a ketalfunctionality. Since three oxygens had previously been assigned to the unsaturated ketone and the butenolide, theseresults suggested that the molecule contained the methoxybutenolide moiety a.0SS5^168.8006.8^OCH3aTable 11. 1H and 13C 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)HMBCb1 168.8 —2 5.92, s — 118.7 Cl, C43 — — 171.0 —4 106.8 —5 1.64, dd, J = 13.5, 13.6 Hz H5', H6 38.6 —5' 2.47, m 115, H6 —6 3.09, m H5, H5', H7 31.0 —7 6.74, br d, J = 6.3 Hz H6, H13 146.5 C98 135.8 —9 198.1 10 2.05, dd, J = 14.7, 16.8 Hz H10', H11 35.0 C910' 2.47, m H10, H11 —11 2.25, dt, J = 4.2, 4.2, 14.7 Hz H10, H10' 47.1 —12 — 37.6 —13 1.77, s H7 15.6 C8, C914 1.40, s — 25.0a C3, C11, C1215 1.22, s — 24.7a C3, C11, C12OCH3 3.20, s 50.6 C4a May be interchanged.b The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 Hcolumn.1^,^,^,^1^,^,^I^1^,^,^,^1^rvi^,7 . 0^6 . 5^6. 0^5.5^5.0^4.5^4.0^3. 5^3.0^2.5^2.0^1.5^1.0PPM^Figure 48.^1H NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 400 MHz) 0Me15Me 13 Me14OCH3131^II^ 1,1^,,,t^,Itt^1^64^u/ibl1110 140 100Figure 49.^13C NMR Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 125MHz)Figure 50. COSY Spectrum of 0-Methyl 9-Ozofurodysinin Lactone (72) (Recorded in CDC13 at 400 MHz). 51. s 1.82.5 2.4I^3.5•P63. 14.575^71^65^III^5.5^51IJo.'.."\.it.•,_.-6H11/H1O'S6H11/Hle 's, -..•A.. - - aH5/H5'8 I: te H6/H5'.- OD116/1-15•f• _^.etS•H7/116to •H7/Me013el.^. .^.^. - . . . .^.^. .^.^.^. . .6.3S.81038.8. s1.1t. 52.12.53. 15Mel3Me14104If 0'C13C14C15C6C11OC.H 3.1 soBO100I.140C76 5 4 3 2 1Figure 51.^HMQC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 500 MHz)Figure 52, HMBC Spectrum of 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13 at 500 MHz)105Me14Me13 Me154 2P011^6• •.• • •• •..• •Me14/C15044 Me15/C14• .^• • • •^•• Me14/C1 UPOMe15/C12• • o. 4,• Me14/C11 Q qMe15/C114r 5 •I. . •• •• •.^•.^:I H2/C4 OCH3/C4 • • • • 4 •^.•• .•Me13/C8 4• Me13/C74 • • •4 H2/C1 . Me14/C341••Me15/C3• • • • •.^.• • H7/C9 H107C9 Me 13/C9. H10/C9 • —^•C8170ClC3C9C14 C15C12C100C410613C NMR signals at 8 118.7, 135.8, 146.5, and 171.0 ppm confirmed that 72 had two double bonds eachconjugated 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 forone proton. The signal at 8 6.74 (117) also showed a correlation to a broad methyl singlet at 1.77 (Me13). Thedeshielded chemical shift of the methyl resonance at 8 1.77 (Me13) in conjunction with the observed COSYcorrelation indicated that the methyl was allylic to the proton at 6.74 (H7). The spin system was further extendedby a correlation from the multiplet at 8 3.09 (H6) to a pair of geminal methylene proton resonances at 1.64 (H5) and2.47 (H5'). These results allowed the assembly of fragment b.6.74^1.64, 2.47H^H HbA 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'). Thechemical shift of the methylene pair (H10, H10') was deshielded, suggesting that it might be adjacent to the carbonat 8 198.1 (C9) or the olefinic carbon at 171.0 (C3). The 1 H NMR spectrum also contained two aliphatic methylsinglets 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 carbonresonances at 8 106.8 (C4) and 168.8 (C1) confirmed the constitution of the methoxybutenolide ring and thechemical 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.6.74H H H1.77 H OCH31.4H( '`2.47 2.05 1.22107The connectivity of the A ring was also clarified by the HMBC data (Figure 52). Correlations from boththe H7 resonance (8 6.74) and the Me13 resonance (1.77) into the carbonyl signal at 8 198.1 located spin system bnext to the ketone at C9. The methylene resonances at 8 2.47 and 2.05 (H10, H10') were also correlated to thecarbonyl resonance at 6 198.1 (C9) which placed the methylene (H10, H10') on the other side of the unsaturatedketone. As mentioned earlier, these proton resonances (H10, H10') were further coupled to a methine resonance at8 2.25 (Hi 1) in the COSY spectrum. The HMQC spectrum indicated the methine at 8 2.25 (H11) was attached to acarbon at 8 47.1 (Figure 51). The methyl singlets at 8 1.22 (Me15) and 1.40 (Me14) showed HMBC correlations tothe C11 carbon resonance at 8 47.1 which established that the methoxybutenolide fragment a and the A ring wereconnected via a quaternary carbon with two methyl substituents.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 assignmentof 72 as the structure of the metabolite. The only feature inconsistent with the proposed structure was the absenceof a COSY correlation between the ring junction hydrogens [i.e. from H6 (8 3.09) to H11 (2.25)]. NOE differenceexperiments, illustrated in Figure 54, clarified the stereochemistry of 72.1086.74\■.____w2.25^3.09H1.77CH3^)1.40/1.22CH3/35 36H^ HFigure 54. Selected NOE Difference Experiments on 0-Methyl 9-Oxofurodysinin Lactone (72) (Recorded in CDC13at 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. Itshould be noted that NOEs were also observed from H6 to H11 and vice versa but these were weak. A doubleresonance experiment in which the multiplet at 8 3.09 (H6) was irradiated did result in slight simplification of themultiplet 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 relativeto 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 methylresonance at 1.22 (Me15) and vice versa (Figure 54). This located the methoxy butenolide with the ketal functionadjacent to C5 and with C3 attached to C12. The final structure suggested that the biosynthesis of compound 72proceeded via an intermediate which more closely resembled furodysinin (36) than furodysin (35).2-Oxomicrocionin-2 Lactone (73)2-Oxomicrocionin-2 lactone (73) was isolated as a colorless oil. A molecular ion was evident in theEIHRMS at m/z 264.1317 Da, appropriate for a molecular formula of C15H2004 (AM -4.5 mmu). The IRspectrum indicated that the molecule contained an 41-unsaturated ketone with a stretching frequency of 1650 cm -1and a 6-lactone which absorbed at 1758 cm -1 . A band at 3317 cm -1 in the IR spectrum suggested that the moleculecontained a hydroxyl group.It was clear from the COSY spectrum that compound 73 was not a disubstituted butenolide with a carbonskeleton similar to furodysinin (Table 12). The 1 H NMR spectrum, shown in Figure 55, contained no isolatedolefinic proton but rather a deshielded multiplet at 6 6.87 (H10) which displayed allylic coupling in the COSYspectrum (Figure 56) to another downfield resonance at 6.11 (H12). The proton resonance at 8 6.87 (H10) showedfurther allylic coupling to a pair of geminal methylene proton resonances at 2.03 (H8) and 2.33 (H8'). Thedownfield proton resonance at 8 6.11 (H12) showed a COSY correlation to an isolated resonance at 3.50 (OH). TheHMQC spectrum (Figure 57) showed that the proton signal at 6 6.11 (H12) was correlated to a carbon resonance at8 96.4, an appropriate chemical shift for a ketal. No HMQC correlation was observed for the 1 H NMR resonance at6 3.50 (OH) indicating that it was bound to an oxygen, rather than a carbon atom. From these results it wasdetermined that 73 contained the mono-substituted hydroxybutenolide fragment c.109H H2.33^2.03Table 12. 1H and 13C NMR Data for 2-Oxomicrocionin-2 Lactone (73) (Recorded in CDC13)Carbon no. 81H (ppm) (400 MHz) COSY (400 MHz) 813C (ppm)(125 MHz)HMBCa1 5.91, s Me13 128.8 C3, C132 — — 198.8 —3 2.29, m H3', H4 41.8 C23' 2.37, m H3, H4 — —4 2.33, m H3, 1-13', Me14 33.8 —5 — — 42.1 —6 — 167.3 —7 1.81, m H7', H8, H8' 33.9 C87' 1.81, m H7, H8, 118' — —8 2.03, m 1410, H7, H7', H8' 20.5 C78' 2.33, m H10, H7, 117', H8 — —9 — 142 —10 6.87, d, J = 1.0 Hz H8, H8', H12 143.1 C11, C1211 — 170.9 —12 6.11, br s —OH 96.4 —13 1.98, s H1 20.2 Cl, C5, C614 1.00, d, J = 6.1 Hz H4 15.4 C3, C4, C515 1.06, s 19.4 C4, C5, C6, C7OH 3.50, br s H12 — —a The carbon resonances in the HMBC column are correlated to proton resonances in the 8 1 H column.1106^5^4^3^2^1PPmFigure 55.^1 H NMR Spectrum of 2-0xomicrocionin -2 Lactone (73) (Recorded in CDC13 at 400 MHz)6.5 6.1 5.5^^4.0^3.5.^3.1^2.5^2.0^1.5^1 . 1PPM112Figure 56. COSY Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 400 MHz)Me15... C IC IIINO '1Ow . 5if C740C30000C12120Cl140doPP—100IlitVIII.aI$Ial I II4I 1 ^NE 5 5 -4 3 2 11133 C15Figure 57.^HMQC Spectrum of 2-Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 500 MHz)Figure 58.^13C Spectrum of 2 -Oxomicrocionin-2 lactone (73) (Recorded in CDC13 at 125 MHz)Me15HI.H1/C13d* HI/C3ClC3I0Cl.SOO150CI500PP10am 1110/C12ow• H10/C11 ..^.^.^• .^.^.^. .^.^.^. .^.^.^.^5 0 5 5 Le^1.5^Figure 59.^HMBC Spectrum of 2-0xomicrocionin-2 lactone (73) (Recorded in CDC13 at 500 MHz)HIO,2C7 C4CSasClC6C200•0........•■•^Me13/C5..ab......•v.. a..Me15/C7 tMe15/C4 V111-1Mei5/c5 dime.1.Me14/C4Me 14/C5ImoMe13/C1 •14;;•.,®..r••Me13/C6 49mum. H3/C2 _.-• •—01Me15/C6'if.H3'/C2^...116The IR band at 1758 cm -1 was attributed to the lactone carbonyl. This value was in close agreement withthe value recorded for the equivalent functionality in manoalide (3) (1765 cm -1 ). 103Three 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 signalat 5 1.81 was attributed to H7 and 117'. Fragment d, an isolated spin system evident from the COSY data, containedan 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 aCOSY 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').2H.29S-53.^2.37^H1.00CH31.98CH3sS^(1217S2.33d eFrom the molecular formula C15H2004 it was determined that the molecule contained six sites ofunsaturation. The unsaturated ketone and the hydroxybutenolide moiety together accounted for five of thoseunsaturations. The final site of unsaturation was attributed to a second ring. The allylic coupling of H10 to the H8methylene 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) and2.37 (H3') allowed the location of this moiety a to the ketone carbonyl. These assignments were corroborated bythe 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 methylene5 .9111 .%■.../)115.9170.90H6.87117pair at 8 2.29 (H3) and 2.37 (H3') to a carbon at 8 41.8 ppm, consistent with the expected shift for a methylenecarbon adjacent to the ketone of a cyclohexanone.90The structure 73 was assembled on the basis of the fragments pieced together from the HMBCspectroscopic data. Figure 59 shows the HMBC spectrum of 73 and Figure 60 illustrates selected HMBCcorrelations.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 at8 1.98 (Me13), the methyl doublet at 1.00 (Me14), and the methyl singlet at 1.06 (Me15) into a single quaternarycarbon resonance at 8 42.1 (C5). In conjunction with the COSY data, this result provided evidence for thesix-membered ring. A correlation from the Me15 singlet (8 1.06) to a carbon resonance at 8 33.9 (C7) linked thecyclohexenone ring to the hydroxybutenolide by a fragment consisting of two contiguous methylene carbons. Thelocation 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 observedfrom the HI resonance (8 5.91) to the C3 resonance (8 41.8). HMBC correlations also substantiated the assignmentof the hydroxybutenolide moiety as illustrated in Figure 60. The HMBC correlation confirmed the chemical shifts ofthe carbonyl carbons, since these were somewhat obscured by noise in the 13C spectrum (Figure 58) and could notbe assigned with confidence.118Figure 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 methylshould 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 themultiplet 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 providedconclusive 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 amolecular 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)119suggested that the compounds were closely related. The only significant difference in the 1H NMR spectra was theabsence of the hydroxyl proton at 6 3.50 in the spectrum of 74 (Figure 62) and its replacement by a singlet at 3.58which integrated for three protons (see Table 13). The infrared spectrum lacked the hydroxyl stretch at —3300 cm -1but retained the two carbonyl bands at 1764 cm -1 (butenolide) and 1663 cm-1 (a,(3-unsaturated ketone). The COSYspectrum (Figure 63) indicated that the 1112 proton resonance was no longer coupled to a hydroxy proton resonanceand that it had shifted upfield from 5 6.11 in compound 73 to 5.73 in 74. The data suggested that metabolite 74was the methyl ether of 2-oxomicrocionin-2 lactone 73. The 13C NMR (Figure 64), HMQC (Figure 65), andHMBC (Figure 66) data are summarized in Table 13. The data corroborated the assignment of the structure as 74.Table 13. 1H and 13C NMR Data for 0-Methyl 2-Oxomicrocionin-2 Lactone (74) (Recorded in CDC13)Carbon no. 61H (ppm) (400 MHz) COSY (400 MHz) 613C (ppm)(125 MHz)HMBCa1 5.90, br s Me13 128.8 C3, C132 — 198 —3 2.26, m H3', H4 41.9 —3' 2.35, m H3, H4 — C24 2.30, m H3, H3', Me14 33.6 —5 — — 42.1 6 — — 167 —7 1.77, m H7', H8, H8' 33.9 —7' 1.83, m H7, H8, H8' — —8 2.03, m H10, H7, H7', H8' 20.2 —8' 2.31, m H7, H7', H8 — —9 — 129 —10 6.79, br s H8, 1112 142.0 C11, C1211 — — — —12 5.73, br s H10 102.513 1.97, br s HI 20.5 Cl, C5, C614 0.99, d, J = 4.2 Hz 114 15.4 C3, C515 1.06, s — 19.4 C4, C5, C6OCH3 3.58, s 57.2 C12a The carbon resonances in the HMBC column are correlated to proton resonances in the 6 1 H column.SwHIOHI H12 S2^1^,ppmFigure 62.^11-1 NMR Spectrum of 0-methyl 2 -Oxomicrocionin-2 Lactone (74) (Recorded in CDCI3 at 400MHz)0T.4.1^3.5^3 . 1PPM•H10/H12013^1015 11012OCH3146.5^6.1^5.S^S.11^4.5121Figure 63.^COSY Spectrum of O-methyl2-oxomicrocionin-2 lactone (74) (Recorded in CDC13 at 400 MHz)OCH3pp soe^ieo^g^the^No^so N o NFigure 64.^13C NMR Spectrum of 0-methyl 2-ozomicrocionin-2 lactone (74) (Recorded in CDC13 at 125MHz)^ k'0— 50HMQC Spectrum of 0-methyl 2-oxomicrocionin-2 lactone (74) (Recorded in CDC13 at 500 MHz)Figure 65.-C14—20 Cl3C15NI•••IPMI—40WIID•■NM: OC_H 3—60PPr00I4I ^ I ^ I'3 2 1PP.H10^HI H12i—100C120i  ^iPP.^6^ 5C lC10PPII•H10/C12 • • el0• OCE_3/C12 A5• 9^I•^C13Me15/C4Melf4• •^• C40 0 C3 CS?mMaeii54/c/C55 so•• •Mel /C5•••• •Me13/C1 I• ••Me13/C6H3/C2Me15/C6ea C631Hl/C13••• Hl/C3a•••C12C l124H10^H1Figure 66. HMBC Spectrum of 0-methyl 2-ozomicrocionin-2 lactone (74) (Recorded in CDC13 at 500 MHz)OCkL3^ Me15Me13Me14125Known Metabolites of the Pleraplysilla Sp. Sponge (1990/1991 Collections)The 1990 and 1991 collections of the Pleraplysilla species of sponge were processed individually. Bothcontained the same known compounds, furodysinin lactone (56) 49 , nakafuran-8 lactone (43), 28b and their respectivemethoxybutenolide derivatives 57 89 and 4428b in sufficiently high concentrations that structure elucidation wasfeasible. In addition, the 1990 collection contained at least five metabolites which were isolated and identified assesquiterpenes but which were present in too small concentrations to permit complete structure elucidation. Thesecompounds did not appear to be present in the 1991 collection of the Pleraplysilla sponge.OR56 R = H57 R = CH3 43 R = H44 R = CH3The structures of furodysinin lactone (56) and its corresponding methoxybutenolide (57) were determinedby comparison of infrared, 1 H NMR and 13 C NMR and mass spectroscopic data to literature data (seeexperimental) 49,89The structure elucidations of nakafuran-8 lactone (43) and its corresponding methyl ether 44 requiredcomplete characterization to distinguish the compounds from butenolides of nakafuran-9 (42).28b The HMBCspectrum clearly indicated that the compounds isolated were derivatives of nakafuran-8 (41). The structures wereconfirmed by comparison of 1 H NMR, 13C NMR, infrared, and mass spectra with published values (seeexperimental) 28b4 1^ 42126b) The 1992 Pleraplysilla Sp. Sponge CollectionAlthough the crude methanolic extracts of the Pleraplysilla sp. sponge collected in 1990 and 1991contained only hydroxybutenolides and their corresponding methyl ether derivatives, the possibility that thesecompounds might be autooxidation artifacts could not be ignored. Consequently a final collection of spongeharvested in 1992 was immediately frozen, freeze-dried, and extracted with ethyl acetate. The ethyl acetate extract wasseparated into its component metabolites by silica gel column chromatography eluted with a polarity gradient ofhexane to dichloromethane to ethyl acetate. Final purification was achieved by normal phase HPLC. The only threesesquiterpenoid compounds isolated from the ethyl acetate extracts of the 1992 collection were furodysinin (36)44and nakafuran-8 (41)28b which was contaminated by a small concentration of a sesquiterpene tentatively identified asfurodysin (35)44 from the 1 H NMR spectrum. All three structures had been previously reported.28b ,44Sesquiterpenes 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 the1 H NMR spectrum, the major compound was purified by normal phase HPLC. By use of extensive NMRspectroscopic 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 indicatedthat metabolite 102 was present in those extracts as well but had escaped notice.41^ 35HO_,E OHOAc10236 Blancasterol (102)127HOBlancasterol was isolated as an amorphous white solid which had an M+H peak in the FABHRMS atm/z 551.36006 Da corresponding to the molecular formula C31145108 (M+1, AM 3.01 mmu). The 1 H NMRspectrum (Figure 67, Table 14) contained signals diagnostic of a steroid side chain. Specifically, there was a doubletat 6 1.00 which integrated for three protons (Me21) and a second doublet at 0.88 (Me26, Me27) which integrated forsix protons. Interestingly, the molecule possessed only one aliphatic methyl singlet, at 5 0.74 (Me18). Analysis ofthe NMR data contained in Table 14 indicated that 102 possessed the same fully saturated side chain as glaciasterolB diacetate (107). The methyl doublet at 5 0.88 (Me26, Me27) showed COSY correlations (Figure 68) to a signalat 1.52 (H25) which was further correlated into geminal methylene resonances at 1.73 (H24) and 1.83 (H24'). TheH24 and H24' resonances were correlated into one another but further correlations were obscured by the methyleneenvelope. The Me21 (8 1.00) resonance showed a COSY correlation into a resonance at 1.45 (H20) and furthercorrelations into a methylene pair at 1.83 (H22) and 1.67 (H22') were discernible. 13C NMR resonances (Figure 69)were assigned to the side chain by analysis of the HMQC (Figure 70) and HMBC spectra (Figure 71) and bycomparison with the appropriate values for glaciasterol B diacetate (107) (see Table 6).Table 14. 1 H and 13C NMR Data for Blancasterol (102) (Recorded in CDC13)Carbon no. 511-1 (ppm) (400 MHz) COSY (400 MHz) 613C (ppm) (125MHz)HMBCb (500MHz)1 2.26, dt, J = 14.8 Hz H1', H2, H2' 19.7 —1' 1.88, ddd, J = 14.8, 13.2,3.3 HzH1, H2, 112' — —2 2.09, m H1, H1', H2', H3 26.0a —2' 1.62, m H1, H1', H2, H33 4.01, m H2, H2', H4 69.9 —4 4.98, d, J = 9.5 Hz H3 78.8 C3, OAc5 — — 76.7 —6 2.56, dd, J = 19.8, 2.4 Hz H6', H7 34.5 C86' 2.35, dd, J = 19.8, 5.8 Hz H6, H7 — —7 6.44, dd, J = 5.8, 2.4 Hz H6, H6' 1382 C5, C98 — 137.3 —9 — 199.7 —10 — 56.3 —11 4.22, ddd, J = 15.8, 10.6,5.3 HzH11', H12, H12' 62.0 —11' 4.05, m H11, 1112, H12' — —12 1.62, m H11, H11', H12' 37.1 —12' 1.40, m 1111, H11', H12 — —13 — — 45.8 —14 3.28, t, J = 11.6 Hz H15, 1115' 42.7 C8, C9, C13,C16, C1815 1.73, m H14, H15' 24.4 —15' 1.46, m H14, H15 —16 — — 28.0a —17 — — 50.8 18 0.74, s — 16.7 C11, C12, C13,C14, C1719 4.05, d, J= 11.2 Hz H19' 61.5 —19' 3.91, d, J = 11.2 Hz H19 — Cl, C920 1.45, m H21, H22 34.8 C17, C20, C2221 1.00, d, J = 6.7 Hz H2O 19.7 —22 1.83, m H20, H22' 35.5 —22' 1.67, m H20, H22 — —23 — 29.7 —24 1.83, m H25, H24' 39.5 —24' 1.73, m H24, H25 — —25 1.52, m H24, H24', H26, H27 28.5 —26 0.88, d, J = 6.6 Hz H25 22.8 C24, C25, C2727 0.88, d, J = 6.6 Hz H25 22.5 C24, C25, C26OAc 2.00, s; 2.20, s — 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 Hcolumn.128HOOCOCff3°COQ:13ppmFigure 67.^I H NMR Spectrum of Blancasterol (102) (Recorded in CDCI3 at 400 MHz).51.83 5^3.6 2 5 2 11^1.5• IPPM75^711^65^66^SS^50^4521 22 24Ac0 11 12HO 27HOMe21/H20_;,,—,19., ' 4, /z0%a 1 ii_Me26/H25TilMe27/H25iea 4H3/H2' itIIIIH3/H2- ..',^2)^dis H2/H2''41 CO CIHUH 1'^H2'/H1.e •P ''^'H6/H6'1iH144415 H14/H15'I^• TH19/H19' . IH11'/H12 !Hll'/H12'. .It llA' H11/H11'11^ftaH11/H12 ®ItH1 1/H12'...--40 el.H4/H3. ..0H7/H6a 0H7/H6' •..•.• ._..-Figure 68. COSY Spectrum of Blancasterol (102) (Recorded in CDC13 at 400 MHz)8.8PPM130OCOCH3^Me27OCOCH3 Me26Mel 8H6' t^Me21OHOAcH19H11'H3H7 H4 H11 H19' H14 H6.s1. 57.87.5 SHO 1 OHOAc200 ISO 140 100Figure 69.^13C NMR Spectrum of Blancasterol (102) (Recorded in CDC13 at 125 MHz)Me27Me26Me18OCOCIj3OCOCkI3HO132OHOAcH19H11'H7^H4^H1 1 H3H19' H1411111^s^4 : 4 : c21,:C26C6401 11.010^IIA t.4 4 1WOI^.I^1IPtI00C 1 4..-cis.- C3.- C4BoAl.i4i.r- 100-'.120•.-C7. I I •O • PPIIen. a^5^4. 3 2^1C19C21C1C27Figure 70. HMQC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz)-OH H7OAcHO1-14 H 19' H14Me27Me26Me21 Me18I.■ -..— _ _._.1H 1 9 7C 1 • •orgo4, H7/C5.•ob H4/C3•...,..•• _I. aH14/CSO..-40- H7/C9. CP H4i000CH3H19'/C9.— •• • f• ata• .2^t0^•-• Me26/C27Me26/C24Me21/C20 40Me21/C22 .Me21/C17 wafir`Vr-^eGt ^•^rMe18/^41^C12i• a•• •....^..• •w•^••a.^.^.IP^••■•• :21..•al.• • . •.•.•• OPi+41 .^t•.•Cl10050C3C5C8150C90P6C26 C27C24c2°c12soC17100160pp.6.0^5.5^6.0^4.0^4.0^3.5^006^2.5^1.0^1.5^1.0Figure 71.^HMBC Spectrum of Blancasterol (102) (Recorded in CDC13 at 500 MHz)134The molecular formula C31HSO08 indicated that steroid 102 had seven sites of unsaturation. Threecarbonyl 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 13C NMR spectrum, accounting for four sites of unsaturation. Three unsaturationsremained 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 carbonylstretches at 1734 and 1678 cm-1 . The IR band at 1734 cm -1 was attributed to an ester carbonyl. Methyl singlets at8 2.00 and 2.20 in the 1 H NMR (Figure 67), shifts diagnostic of acetate methyls, were consistent with theassignment of the IR absorbance at 1734 cm -1 to ester stretches. The IR stretch at 1678 cm -1 was typical of ana,13-unsaturated ketone. This assignment was corroborated by the 13C NMR spectrum (Figure 69) which containeda carbon resonance at 5 198.7 (C9). The molecular formula indicated that blancasterol (102) contained eight oxygenatoms. Two acetates and a carbonyl accounted for five oxygen atoms leaving three to be assigned. Five carbons wereat chemical shifts appropriate for single bond attachment to oxygen atoms, specifically, the carbon resonances at5 69.9 (C3), 78.8 (C4), 76.7 (C5), 62.0 (C11) and 61.5 (C19). Two of these carbons must bear the acetatefunctionalities but three carbons remained unassigned suggesting that metabolite 102 contained three hydroxylgroups.Table 14 contains a summary of the NMR data recorded for 102. One spin system was particularly notablein 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 at1.62 (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 thesame proton resonances in glaciasterol A (100) (H11, 3.69; H11', 3.81) but were in better agreement with theobserved shifts for glaciasterol A diactate (106) (H11, 4.10; H11', 4.16) (Tables 4 and 5). This led to theconclusion 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) wascorrelated to an olefinic carbon resonance at 5 138.2. The other olefinic carbon resonance (5 137.3 (C8)) had noHMQC 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 methyleneresonances at 2.56 (H6) and 2.35 (H6'). The coupling constant 416' ,H7 = 5.8 Hz was a typical value for vicinalcoupling but was too large to attribute to allylic coupling. 73a This result indicated that the olefinic proton at 5 6.44CH0^3% C^I ^1.00^0 H3C0.74135must be to the ketone. The functionalized ethyl fragment and the a,(3-unsaturated ketone could be accomodated bya 9,11-secosteroid.Figure 72 illustrates selected correlations observed in the HMBC spectrum (Figure 71). The protonresonance at 5 6.44 (H7) had HMBC correlations to carbon resonances at 5 199.7 (C9) and 76.7 (C5). The chemicalshift of the C5 resonance indicated that the carbon had a hydroxyl or acetyl substituent. No further COSYcorrelations were observed from the C6 methylene protons and the carbon resonance at 5 76.7 had no correlations inthe HMQC spectrum consequently it was concluded that the C5 ring junction was tetrasubstituted.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 otherresonances in the same vicinity (H11', 4.05; H19, 4.05). Correlations from the signal at 5 4.01 (H3) were observedinto a pair of methylene proton resonances at 2.09 (H2) and 1.62 (H2') which in turn were coupled into one otherpair of methylene resonances at 2.26 (H1) and 1.88 (H1'). This spin system, illustrated as fragment f, could only beaccomodated by the A ring of the steroid.1361.88 2.26H HfAs was mentioned earlier, only one aliphatic methyl singlet (5 0.74 (Me18)) was visible in the 1 H NMRspectrum. 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 theprotons and the attached carbon led to the proposal that either Me19 or Me18 had been oxidized to ahydroxymethylene. 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 theMe18, had been oxidized in blancasterol from a correlation in the HMBC spectrum (Figure 71) from the protonresonance at 8 3.91 (H19') to the C9 carbonyl resonance at 5 199.7 ppm. A second HMBC correlation was observedfrom the H19' resonance (8 3.91) into a carbon resonance at 5 19.7 (C1). The carbon resonance (5 19.7 ppm) wasassigned as Cl from HMQC (Figure 70) and APT data (Figure 73). This result permitted the orientation of spinsystem f with methylenes at Cl and C2 and the hydroxy or acetoxy substituents at C3 and C4. An HMBCcorrelation 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 C11carbons. Consequently it was concluded that C3, C5, and C19 had hydroxyl substituents.137A triplet at 5 3.28 in the 1 H NMR spectrum was at the expected chemical shift for H14. COSYcorrelations 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 assignedfrom the HMQC spectrum (Figure 70) and these were in close agreement with the values observed for the D ringcarbons of the glaciasterols (glaciasterol A (100); C14, 43.9; C15, 26.9). An HMBC correlation from the 1-114resonance (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 theHMQC spectrum. This result substantiated the location of the acetoxyethyl fragment. Further HMBC correlationsfrom the Me18 resonance (5 0.74) into carbon signals at 8 45.8 and 50.8 permitted assignment of these resonancesto C13 and C17, respectively (Figure 72).As mentioned earlier, HMBC correlations from the side chain methyls aided in the assignment of the sidechain carbon resonances. Figure 72 shows that correlations were observed from the Me21 resonance (5 1.00) tocarbon 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 toassign 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. Titrationof 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 inFigure 74. A series of double resonance experiments on 102 in CDC13: C6D6 20:1 permitted assignment of thenewly resolved signals (Figure 75). Table 15 lists the assignments of selected protons of blancasterol in CDC13 andin CDC13:C6D6 20:1.HOOHOAcas^s^as^34^as^ 24^a^20^a^toFigure 73.^Expanded Upfield Region of the APT Spectrum of Blancasterol (102) (Recorded in CDC13 at 125MHz)HOOHOAc2; 22 24^2618 ss•AcO 11HO127SSH3H19H11' H19'H11H4H7H14H600C113OCOCjj3Me27Me26Me18Me21M•H6fiv, r ,^rr--r^, r^I^,^, r-r-r"^ 1"11'' 1 17.5^7.0^6.5^6.0^5.5^5.0^4.5^4.0^3.5^3.0^2.5^2.0^1.5^1.0^.5PPM^Figure 74.^1 H NMR Spectrum of Blancasterol (102) Recorded in CDC13:C6D6 20:1 at 400 MHzH19H11' H3H19'H11 H14H4HO1405.9^4.9^4.5^3:7^4.5^4:5^4.4^4:3^- 4.2^IA^as^3:•^1:-11^3:7^3:5^3:5^3:4^3.1^3.2PPMFigure 75.^Double Resonance NMR Experiments on Blancasterol (102) (Recorded in CDC13:C6D6 20:1 at400 MHz)HOaH4f3H14• iI H7/H6a• •0H14/H11H6f3/H190 •00H4f3/H19'•H413/H6f3OHOAcH7H3Hii H19 H19'H11HO141246or • 40 •MEM■1.•0.11011111^11-11 lllllllllllllll llll4 21111^I^il-I f I6Figure 76.^ROESY Spectrum of Blancasterol (102) (Recorded in CDC13:C6D6 20:1 at 500 MHz)142Table 15. 1H NMR Shifts of Blancasterol (102) in CDC13 and in CDC13:C6D6 20:1.Carbon no. 81H NMR recorded in CDC13 (ppm) 8 111 NMR recorded in CDC13:C6D620:1 (ppm)3 4.01 3.954 4.98 4.936 2.56 2.466' 2.35 2.247 6.44 6.3411 4.22 4.2211' 4.05 4.0514 3.28 3.2719 4.05 3.9819' 3.91 3.83A ROESY spectrum of blancasterol was recorded in CDC13:C6D6 20:1 (Figure 76) and the correlationsprovided information concerning the relative stereochemistry of 102. Figure 77 illustrates a number of informativeROESY 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 H4ax (8 4.93, d, J = 9.5 Hz) and H3, in conjunction with theROESY correlations established that the A ring of blancasterol (102) was in a chair conformation and that H4 (4.93)143and H6 (2.46) were 13 to the A/B ring system. From the results of the ROESY experiment it was clear that the Aand B rings of 102 must be trans fused. Figure 78 shows the alternative structure with the A and B ring junctioncis fused. The ROESY correlation from H19' into H6ax would probably be observed, however no correlation wouldbe 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 toMe18 found in most steroids.60,67 The side chain was assumed to have the normal 13 configuration but this was notverified 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 thePleraplysilla species sponge. The structures of furodysinin (36), 44 nakafuran-8 (41),28b and furodysin (35)44were determined by comparison of their 1 H NMR spectra with published results (see experimental). COSY spectraconfirmed the connectivities.14435^ 3641An NMR sample of nakafuran-8 in CDC13 (41) was left in the refrigerator overnight. The following daynone of the starting material remained but there appeared to be a product with a characteristic aldehyde resonance at6 9.61 in the 1 H NMR spectrum. The structure of the degradation product was determined to be 116, the y-ketoenal of nakafuran-8 (41), which Scheuer and coworkers had reported to be a product of meta-chloroperbenzoic acidoxidation of 41. 28b Scheuer et al. found that oxidation of the intermediate 116 under Jones' conditions gave thehydroxybutenolide 56 (Equation 5).MCPBA CHO 41^ 116Jones'oxidation56Equation 5. Oxidation of Nakafuran-8 (41) to the Hydroxybutenolide 56. 2813In our case, it is likely that the combination of acidity in the CDC13, oxygen, and a small amount ofatmospheric moisture was sufficient to catalyze the conversion of 41 to 116. The reaction of 41 to form 116145indicates oxidation of 41 to the corresponding the y-keto enal is a facile reaction and suggests this to be a likelyroute to the hydroxybutenolide 56.CONCLUSIONSA study of a gray-white sponge, initially identified as an Aplysilla sp. on the basis of morphology alone,29resulted 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 secosteroidblancasterol (102). The isolation of blancasterol (102) adds to the number of secosteroids reported from marinesponges. The biogenesis of the 9,11-secosteroid is expected to proceed in a similar manner as in the case ofglaciasterols 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 becytotoxic. Pure blancasterol (102) was submitted for biological testing against the in vitro L1210 murine leukemiaand human breast cancer solid tumor cell lines. Blancasterol (102) was active against the L1210 cell line at anED50 of 8.8 lig/mL. The compound proved active against the drug-sensitive MCF-7 breast cancer cell line at anID50 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 gray-white sponge. These metabolites are furodysin (35),44 furodysinin (36),44 furodysinin lactone (56), 49 0-methylfurodysinin lactone (57),49 nakafuran-8 (41),28b nakafuran-8 lactone (43),28b and 0-methyl nakafuran-8 lactone(44).28b The presence of sesquiterpene chemistry in the white sponge indicated that the sponge may have beenmisidentified. As discussed previously, the terpenoid metabolites of sponges of the genus Aplysilla are invariablyditerpenes. 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 theinconsistency of the metabolite chemistry with the original classification and consequently reidentified the sponge asa species of the genus Pleraplysilla, biologically a genus intermediate between Aplysilla and Dysidea.88 NoPleraplysilla sponges have previously been identified from the west coast of North America and it is possible thatthis is a new species of the genus.88OR7 2 73 R = H74 R = CH33 64 1 43 R = H44 R = CH335HO0-Methyl 9-oxofurodysinin lactone (72) is an oxidized analog of 0-methyl furodysinin lactone (57). 49Compounds 47 and 49, which have been reported from a collection of Dysidea herbacea,4° can be envisaged to bepossible biosynthetic intermediates in the formation of 72 from a precursor such as furodysinin (36).1463 6^ 47 R=H,W=OH49 R = OH, R' = H14772 57Faulkner and coworkers investigated the stability of furodysin (35) and furodysinin (36) in methano1.91 Asample of Dysidea species sponge which had been soaked in methanol for two years was found to containfurodysinin (36) but none of the oxidized furodysinin lactone 56. Faulkner et al. proposed that the methyl ketalmoiety of 57 was an artifact of the reaction of furodysinin lactone (56) with the methanol solvent but thatChromodoris funerea, the animal from which 0-methyl furodysinin lactone (57) had been isolated, was certainlyproducing 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 resultsof Faulkner and coworkers, it can be postulated that 0-methyl 9-oxofurodysinin lactone (72) is very likely theproduct 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^ 57Equation 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 ofmicrocionin-2 (117), a sesquiterpene metabolite of the sponge Microciona toxystila.92 While it is improbable thatthe C9 carbonyl is an artifact, the methyl ketal of 74 is likely to result from a reaction of 73 with the methanolused for extraction (Equation 6). Further experimental data is required to determine whether the hydroxybutenolidemoiety of 73 is an artifact of autooxidation upon workup.117OHcyclizationOPPH+ inducedcvclization c+(.,/..H shift\ \0-H+117=CH3 shift[0]H+ / cyclization148ME41Scheme 1. Proposed Biogenesis of Microcionin-2 (117) and Nakafuran-8 (41).2813Scheuer has proposed that the biogenesis of the nakafuran-8 (41) and the microcionin-2 (117) carbonskeletons follow the same mechanistic pathway (Scheme l).28b The biogenesis proceeds via conversion of farnesylpyrophosphate to the furan intermediate followed by cyclization to form the six-membered ring, a proton shift, a149methyl shift, and elimination to yield sesquiterpenes possessing the microcionin-2 (117) carbon skeleton. Scheuersuggested 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 twohydroxybutenolide derivatives of microcionin-2 (73 and 74). It can be seen from Scheme 1 that elimination ofhydrogen from microcionin-2 (117) followed by addition of a proton and cyclization leads to formation of thenakafuran-8 carbon skeleton. 28bThe biogenesis of furodysin (35), furodysinin (36), and the related derivatives 56, 57, and 72 can beenvisaged as shown in Scheme 2. Conversion of one end of a unit of farnesyl pyrophosphate leads to the terminalfuran followed by cyclization to an intermediate such as spirodysin (39), a metabolite which has been isolated fromthe sponge Dysidea herbacea." Rearrangement of spirodysin (39) could conceivably lead to both furodysin (35)and furodysinin (36).46OPP[0]3935^ 36Scheme 2. The Proposed Biogenesis of Furodysin (35) and Furodysinin (36)150It is puzzling that each year that the Pleraplysilla sponge was collected the sponge extracts yielded adecreasing diversity of metabolites. The 1990 and 1991 collections contained the same major metabolites withrepresentative derivatives of the microcionin-2 (117), furodysinin (35), and nakafuran-8 (41) carbon skeletons. Asmentioned earlier, the 1990 collection contained at least five sesquiterpenes, present in minor amounts, which wereabsent from the 1991 sponge collection. The following year, the sponge collection contained only threesesquiterpene derivatives and there was no trace of compounds with the microcionin-2 (117) carbon skeleton. Whileit is possible that the hydroxybutenolide moieties of the sesquiterpenes isolated from the 1990 and 1991 collectionswere 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 the9-oxo and 2-oxo functionalities found in compounds 72, 73, and 74 isolated from collections of previous years. Itwas evident that some factor, possibly the ecological pressure applied by collecting the sponge, was influencing thenature of the secondary metabolites produced by the organism. The sponge was collected at the same time each yearso seasonal variation was not responsible for the change in metabolite composition. Physically the sponge did notchange in appearance, however, the rock on which it grows is home to at least three other species of sponge andthese 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 timeframe of our collections. Little is known about the kinetics of sponge secondary metabolism and this idea mightmerit further investigation.The second possibility is that the sponge, under pressure from competing organisms and suffering reducedcompetitive effectiveness due to the annual collections is in some way producing those compounds which are mosteffective against predators and competing animals. It has been reported that nakafuran-8 (41) and furodysinin (36)are effective fish antifeedants.91,28b This explanation is inconsistent, however, with a report that the oxidizedproduct of furodysinin (36), 0-methyl furodysinin lactone (57), is in fact a significantly more active fish antifeedantthan the proposed precursor furodysinin (36) (furodysinin [36] was effective as a fish antifeedant at 50 µg/mg foodpellet, while 0-methyl furodysinin lactone [57] required 10 tg/mg food pellet. 91 ). To our knowledge this is the151first report of an instance where collection of a sponge appeared to produce an effect on the chemistry of the sampleorganism. It would be interesting to further investigate the effect of harvesting sponges on the composition of thesponge metabolites.PART II. THE NATURAL PRODUCTS OF TWO SPECIES OF COLONIAL TUNICATE FROM THE OUEENCHARLOTTE ISLANDSINTRODUCTIONPhylogenetic schemes depicting the subkingdom Metazoa generally place the phylum Porifera at thebottom, among the least organized of organisms. The phylum Chordata, in contrast, is considered to contain themost organized and highly evolved species of animals. 93 The phylum Chordata is comprised of the subphylaVertebrata, 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 ofcytotoxic secondary metabolites of two tunicate species, members of the subphylum Urochordata. This section willdiscuss the biology of these animals.While the solitary tunicates are generally not difficult to identify, compound ascidians which are colonies oftunicates, may resemble sponges in both their sedentary lifestyles and general appearance. An uninformed observerwould 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 withthe larval state. The urochordates produce free swimming larvae, several mm in size, which resemble the tadpoles offrogs (see Figure 80) and have many morphological features in common with the other phyla of the Urochordata. 94A particularly remarkable characteristic of the larva is the notochord, a fibrous sheath which constitutes a primitivespinal column94 and provides the body with firmness and flexibility. 93 The larvae have a number of adaptations tooptimize selection of a safe stratum on which to settle and develop into adults. These adaptations include musculartails which allow swimming, and an anterior end which is equipped with a multicellular light receptor (ocellus) aswell as a single-celled statocyst to differentiate up from down.95152153Subkingdom^ MetazoaPhylum^ ChordataSubphylumClassOrderFamilyGenus^Vertebrata^Urochordata^Cephalochordata'11AscidiacealirAspiraculata^Aplousobranchia^Stolidobranchia^PhlebobranchiaSynoicidae1(AplidiumFigure 79. The Classification of Tunicates of the Genus AplidiumFigure 80. The Body Plan of a Urochordate Larva96The 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 theorders Aspiraculata, the deep-sea ascidians, the Phlebobranchia and Stolidobranchia, which include primarily solitarytunicates and the suborder Aplousobranchia, which contains most compound tunicates. 95 . 96 The orderAplousobranchia can be further divided into a number of families including the Synoicidae which includes the genusAplidium.94 The cytotoxic natural products of two species of this genus, Aplidium californicum and an undescribedspecies of Aplidium, are discussed in this thesis.The adult tunicate bears little resemblance to its larvae. After swimming several hours to two days, thelarvae attaches itself by the three fixation papillae at its anterior end to a suitable substratum. 93 .94 It appears that asite where adult tunicates of the same species grow is preferred. 95 Metamorphosis involves resorption of thenotochord and tail. The dorsal nerve cord degenerates into a simple ganglion and the digestive tract and organs rotate180° to the free end of the body. The mouth area, unused until now because the larvae never feed, expands and thepharynx, the central body cavity, enlarges and develops numerous slits until it resembles a sieve. The siphons beginto pump water through the body cavities initiating adult life activities such as feeding, respiration, reproduction and154155excretion.93 The ascidian may reproduce in one of two ways; sexually or asexually. Species of the genus Aplidiumare compound or colonial tunicates which bud asexually by means of lengthwise constriction to produce colonies ofthe anima1.95 These colonies may share a common protective outer layer and some blood vessels. Colonies of acompound tunicate may grow to a meter in diameter even though each individual tunicate, or zooid, may be as smallas 3 mm. All Aplidium tunicates are hermaphrodites which contain a single ovary and testis. In species of thisgenus, the ovum is fertilized in situ and develops into a larva in one of the parent's body cavities. 96Figure 81 illustrates the body plan of a single zooid of the compound tunicate Aplidium californicum, oneof the species studied." The other Aplidium species has a similar body plan. The ascidian body is covered with anepidermally-secreted layer referred to as a tunic composed of cellulose and protein. The tunic provides protection andfixes the tunicate to its substratum. Within the tunic is the true external body-wall of the ascidian, a membrane-likesac called the mantle.93 As can be seen from Figure 81, zooids of the genus Aplidium are segmented into threesections; the anterior segment is the thorax which contains the two siphons and the branchial sac, the abdomenencloses the digestive organs and the post-abdomen contains the reproductive organs and the heart." Tunicates havea buccal and an atrial siphon. The buccal siphon is the incurrent opening permitting water to enter the body. Thebase of the opening is surrounded by tentacles which act to exclude large particles. The incurrent siphon opens intoa large body cavity, the pharynx, which occupies most of the volume of the animal's body. The pharynx isperforated with many slits, known as stigmata, so that it resembles a net. The stigmata are covered with cilia whichsweep water into the pharynx. The incoming current passes through the stigmata where any planktonic foodparticles are trapped, into the atrium (a second body cavity) and out through the atrial siphon. A vertical slit in thepharyngeal wall is the site of production of a mucus which moves in a continuous sheet across the pharyngeal wallcollecting food particles. The food-mucus sheet is rolled into a rope which is delivered to the esophageal opening onthe pharyngeal floor. Food travels through a narrow esophagus, into a sphinctered stomach and through a longintestine.93 The entire digestive tract is ciliated. Excrement is discharged through the anus which is located underthe atrial siphon to permit flushing of waste with the excurrent water flow. In the case of colonial tunicates such asAplidium species, the atrial siphons of a number of zooids may open into a common cloacal cavity prior to flushingaway."156Figure 81. Body Plan of the Urochordate Aplidium caWornicum94Urochordates have open circulatory systems including a poorly defined heart which is comprised of a fold inthe tissue of the pericardial region. The heart pumps blood through a system of vessels and periodically reverses theflow of pumping. The heart's primary function is to saturate the vessels of the pharynx with blood. Sinceorganisms belonging to the subphylum Urochordata are sessile, they have very low rates of metabolism andtypically extract only 10% of available oxygen from incoming seawater. 93Excretion is the function of multipurpose cells known as amoebocytes which absorb uric acid, and travel tothe walls of the digestive system where they are stored for the duration of the animal's life. Ammonia whichconstitutes 90% of the nitrogenous waste of the organism diffuses out through the body walls. 93Amoebocytes are known in some species to concentrate heavy metals such as vanadium, niobium,chromium and iron at levels 105-106 times higher than the concentration in seawater. This phenomenon is subjectto intensive study but is not yet well understood. 93 .95 .96The cerebral ganglion, a vestige of the larval nerve chord, is located in the thorax between the mouth andthe atrial siphon. A number of single nerves branch from it but the nervous system is decentralized, as evidenced by120 R =121 R =0122 R= )1s.HNR157the fact that removal of the cerebral ganglion has little effect on the organism. If threatened, the animal can expelwater from its body cavities and close its siphons, hence one of its names, the seasquirt. 93Taxonomy of the Ascidiacea is difficult because, like the Porifera, these animals are soft-bodied with fewfeatures to distinguish them." Classification is based mainly on the structure of the pharyngeal wall, particularlythe stigmata and the location of the gonads.96 The fossil record of the Urochordata is minimal as soft-bodiedanimals do not fossilize well." It has been suggested, however, that ascidians alive today evolved from a muchgreater specific diversity in past geological periods. 96 On the basis of the high level of organization of theurochordate larva, it has been proposed that tunicates are products of retrograde evolution, stemming from morehighly organized ancestors."The urochordates are known to have predators, including humans, asteroids, gastropods, and some fishes. 95Nevertheless, in spite of their sedentary ways of life and lack of physical means of defense, many tunicates are notfed upon by other marine animals. There is circumstantial evidence which suggests that some species of tunicatesynthesize natural products which provide a chemical defense system against predators. For example, shermilamineB (118) and the kuanoniamines A—D (119-122) were isolated from both an unidentified purple compound tunicateas well as a prosobranch mollusk, Chelynotus semperi. The prosobranchs, snails characterized by reduced shells, areknown to be predators of invertebrates toxic to other species. It has been suggested that these mollusks sequester thebioactive secondary metabolites of their prey and use them for their own chemical defense.97HNAc118^ 119158Natural products chemists have only recently started to show significant interest in the secondarymetabolites of urochordates. Until 1986 approximately fifty natural products had been reported from tunicates. 7Since 1987 however more than one hundred and eighty structures of urochordate metabolites have been published. 8e-iTunicate chemistry is remarkable in that it is dominated by amino acid metabolism. Approximately 90% of thereported natural products are nitrogenous.? The sudden surge of excitement surrounding tunicate metabolites hasbeen prompted by the high levels of bioactivity of urochordate extracts and interesting physiological effects of someof the reported compounds. 811-1 One example is didemnin B (10), a natural product which exhibits cytotoxicity andimmunosuppressive activity. 150^ 0^0I IC —NH—CH— CHOHCH2 C -0 —CHC CH(CH3)CH2CH(CH3)2^CH(CH3)2L-Lac-L-Pro-D-MeLeu NHOCH3^CH2CH(CH3)2N CCII^O^0CH310The interesting chemical and pharmacological activities of metabolites of the Urochordata are likely tocontinue to attract the attention of chemists in the future, yet the secondary metabolites of these animals are unlikelyto be of great utility in classification. This is due primarily to the fact that the tunics and cloacal cavities of theascidians make excellent habitats for high concentrations of symbiotic bacteria. These organs are rich in nutrientsand carbon dioxide and provide physical protection from predators. Consequently many species of tunicate, includingspecies of the genus Aplidium, have been found to have large colonies of symbiotic bacteria living in the tests andcloacal aperatures.98 It has been suggested that some compounds isolated from collections of urochordates are in factproducts 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. MooreCHO —C - C FIN(CH3) -CIOCNOH OCH3CH3H3CH3CCH3OH159and Corley suggest that this compound, which is of mixed biosynthesis but is primarily polyketide-derived, is likelyto originate as a secondary metabolite of the tunicates' algal symbionts. 99This thesis presents the isolation and structure determination of an anthracene quinone which is thecytotoxic component of an undescribed Aplidium species from the Queen Charlotte Islands. The isolation andstructure elucidation of a family of related prenylated hydroquinones from a collection of Aplidium californicum willalso be discussed. The following section will discuss relevant examples of anthracene quinones from marineinvertebrates.A. THE CHEMISTRY OF AN UNDESCRIBED APLIDIUM SP. FROM THE QUEEN CHARLO 1 lE ISLANDS i) ANTHRAQUINONES FROM MARINE INVERTEBRATESIt is of particular relevance that marine bacteria may produce compounds related to metabolites isolated fromascidians since, as previously mentioned, bacteria frequently inhabit the tunics of ascidians. It is possible thatcompounds isolated from tunicates are, in fact, produced by symbiotic microorganisms. 98,99 A marinemicroorganism, Chaina species, was reported to produce antibiotic SS-228Y (124). The structure was initiallymisassigned 1 °° but synthesis proved the compound to be 124. 101 SS-228Y is active against gram-positivebacteria. 1 °°OH 0 OH160OH 01 2 4The 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 bacteriaStaphylococcus aureus and Bacillus subtilis. 102 Xestoquinone proved to have potent cardiotonic activity. 103 Anumber of closely related reduction products were also reported from an Adocia species marine sponge. 1040^0125 R,R' = H126  R, R' = 0Coelenterates which have been reported to produce polyaromatic quinones include a stony coral and a speciesof hydroid. Sanduja et al. reported the isolation of three related polyketide-derived metabolites, 127 to 129, fromthe Pacific stony coral Tubastraea micranth. 105 The authors speculated that these compounds might deter feeding ofthe coral predator the Crown-of-Thorn seastar Acanthaster planci. 1050^R4127 ^128 R 1 , R6 = OCH3, R2 , R3 = OCH20,R4 = H, R5 = OH129  R 1 = H, R2 = Br, R3 , R5 , R6 = H,R4 = CO2H132 1331300H3C161Twenty-one related anthracenone derivatives have been reported from the British Columbian hydroidGarveia annulata. 106 These are exemplified by garveatin A (130)9106a garvin A quinone (131), 106b annulin A(132),106c garvalone A (133), and garvalone B (134). 106d The G. annulata metabolites are characterized byantimicrobial activity. Garvin A quinone, for instance, was active against Staphylococcus aureus and Rhizoctoniasolani with ED50 values of 3 tg/mL for both assays. 1 °610134The crinoids are flowerlike animals commonly known as sealilies forming a class of the phylumEchinodermata. Sutherland and coworkers have studied the pigments of these organisms extensively and report thatthey produce a colorful array of anthraquinone-derived compounds. 107 Among these Sutherland et al. listrhodocomatulin 6-monomethyl ether (135) and rhodocomatulin 6,8-dimethyl ether (136) from the crinoid speciesComatula cratera and Comatula pectinata. 107a The crinoid Ptilometra australis yielded rhodoptilometrin (137),OH 0 OHOH 0 OH OH 0 OHHOHO0137OH 0138CH3OOH 0 OH OH 0 OHO COCH3140 X = H141 X = OH0139HO162.107cisorhodoptilometrin (138), and ptilometric acid (139)^The Caribbean crinoid Comactinia meridionalismeridionalis gave the polyaromatic quinones 140 and 141. 107d Sutherland and coworkers proposed that crinoidssynthesize these compounds endogenously since the anthraquinones do not appear to be randomly dispersed amongthe organisms. Rather, the distribution of the anthraquinoid metabolites seems to be species specific.lificThis 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 hasbeen reported to date from the urochoniates.ii) RESULTS AND DISCUSSIONLANGARIN FROM AN UNDESCRIBED APLIDIUM SP.In May of 1991, a previously undescribed species of colonial tunicate of the genus Aplidium was collectedat the southern tip of Langara Island in the Queen Charlotte Islands (Figure 82). The tunicate grows on exposed rockwalls 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 indiameter. A voucher sample (100 g) and a small collection (310 g) were harvested. A methanolic extract of thevoucher 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. Thetunicate was ground with methanol, filtered and reextracted with methanol. Again the methanolic extract wasfiltered, the extracts were combined and concentrated to an aqueous slurry. The slurry was extracted sequentially withhexanes and ethyl acetate, however, screening by TLC and 1 H NMR revealed both organic extracts to have the samemajor product so the hexane and ethyl acetate extracts were pooled. Purification of the major active compound wasaccomplished by sequential application of size exclusion chromatography (Sephadex LH-20 eluted with ethylacetate/methanol/water. 20:5:2) and normal phase silica radial TLC eluted with ethyl acetate and hexane. The majorcompound, 142, was recrystallized from methanol to yield bright orange needles (mp 225-228 °C).163164Figure 82.^The Aplidium Spp. Collection SiteFigure 83.^A Freshly Collected Sample of the Undescribed Aplidium Species165Langarin (142)166OHLangarin was isolated as bright yellow—orange needles which turned deep red upon the addition of 0.1 Maqueous sodium hydroxide. 107a A molecular ion was observed at m/z 270.0527 Da in the EIHRMS correspondingto a molecular formula of C15111005 (AM -0.1 mmu). The molecular formula indicated that langarin had elevensites of unsaturation and must be polyaromatic. The IR spectrum contained two carbonyl stretching bands at 1623and 1675 cm -1 . The dark yellow color of 142 and the carbonyl bands in its IR spectrum indicated that thecompound might be a quinone. This proposal was substantiated by the UV spectrum (recorded in methanol) whichhad three absorption bands at 429 nm, 286 nm, and 254 nm characteristic of a para-quinone. 108 The IR band at1623 cm -1 was at an appropriate frequency for a chelated quinone carbony1. 109 A resonance in the 13C NMRspectrum at 8 192.0 ppm provided further evidence for the chelated quinone (see Table 16). The IR band at1675 cm -1 was at the appropriate frequency for a non-chelated quinone carbonyl and a corresponding carbonylresonance was observed at 8 181.8 ppm in the 13C NMR spectrum.Compound 142 was completely characterized by NMR spectroscopy and the data is summarized inTable 16. The 1H 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. Fiveprotons were observed in the aromatic region from 8 7.9 to 7.1 ppm. A triplet which integrated for one proton had achemical shift of 8 5.59 ppm. Again no proton-carbon correlation was observed in the HMQC spectrum suggestingthat 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 COSYcorrelation from a doublet at 8 7.36 (H7) into a doublet of doublets at 7.80 (H6) which was further correlated into adoublet at 8 7.70 (H5) established that 142 contained an isolated spin system consisting of three contiguousaromatic 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 signal7.36a bH7.80H7.70H7.68H H4.61OH5.59H^'244167at 4.61 (H11, H11'). This data indicated the presence of a pair of aromatic protons meta to one another and ortho tothe hydroxymethylene substituent which separated them (fragment b).Table 16. 1H and 13C NMR Data for Langarin (142) (Recorded in Me2SO)Carbon no. 81H (ppm) (400 MHz) COSY (400 MHz) 813C (ppm) (125MHz)HMBCb1 — — 162.0 —2 7.28, br s H4, H11, 1111' 121.0 Cl, C4, C9a, C113 — — 154.1 -4 7.68, br s 112, H11, H11' 117.5 C2, C9a, C10,C114a 133.5a —5 7.70, d, J = 7.9 Hz 116, H7 119.7 C7, C8a, C106 7.80, dd, J = 8.3, 7.9 Hz H5, H7 137.7 C8, ClOa7 7.36, d, J = 8.3 Hz H5, 116 124.7 C5, C8, C8a8 — 161.7 —8a — — 116.2 -9 — — 192.0 —9a — — 114.7 -10 — — 181.8 -l0a — — 133.-la11, 11' 4.61, d, J = 5.8 Hz H2, H4, H11, H11',-OH62.4 C2, C3, C4CH2-01I 5.59, t, J = 5.8 Hz H11, H11' —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 11column.OHH11'0 H11H4H51-16^H2OHOHH7' ' ' ' '^ I^'^" I " '^'^I^I^' '^I^'12.0^11.0^10.0^9.0^8.0^2.0^6.0^5.0^4.0^3.0PPM1 1-1 NMR Spectrum of Langarin (142) (Recorded in Me2SO at 400 MHz)Figure 84.H61 H2H710H4H5 1OH.IJ,.....-10OHH11'H11OH OH1^i^I "^ ' " '  ' ' " " " ' " '  "pp m6.I 5^6.0^5.I 5^5.I 0^4.5PPMFigure 85. COSY Spectrum of Langarin (142) (Recorded in Me2SO at 400 MHz)1698.0 7.5 7.0190^180^170^160^150 120^110140^130OHFigure 86.^13C NMR Spectrum of Langarin (142) (Recorded in Me2SO at 125 MHz)171OH 0 OHOHOH4H5^H2H6 H7 —so-C11—SO—100•dI 0 C50.-C4-C6—140pplH11'011^H11pp.^ 7^ B^ 5Figure 87. HMQC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz)-150C3CSH11/C3co. H2/C1H7/C8 •H4/C1 I^•OH2/C1 IH5/C8aH4/C9aO H7/C8a ti2/c9a2* go.23 H2/C4H4/C20,.,^0H5/C70 a-or17/C:*H6/C10a0co^coH4/C10H5/C10C 1 I▪ 50—75—100••^••HI 1/C4 1'11111/C2 411,c5c.9C811 C4C2—125—175'C10172OH 0 OHOH0H6 H4^H2H5^H7 H11PP•^8 7^6^ 5Figure 88 . HMBC Spectrum of Langarin (142) (Recorded in Me2SO at 500 MHz)173The five oxygen atoms in the molecular formula of 142 were assigned to two quinone carbonyls and threehydroxyl functionalities. The quinone carbonyls accounted for two sites of unsaturation, thus the remaining nineunsaturations must be double bonds or rings. The 13C NMR spectrum (Figure 86) contained twelve aromaticcarbon resonances (including two with chemical shifts of 5 161.7 (C8) and 162.0 (C1) which were assigned toaromatic carbons bearing hydroxyl groups) indicating that langarin had six double bonds and consequently must betricyclic. Protonated carbons in the 13C NMR spectrum were assigned from correlations in the HMQC spectrumshown 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 thoseHMBC correlations which proved particularly helpful in determining the structure of 142. The aromatic protonresonances 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 wereconsequently 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 twohydroxyl groups on C8 and Cl.174Further analysis of the HMBC spectrum (Figure 88) permitted assignment of all of the quaternary carbonswith the exception of C9 and C4a. The chemical shift of the chelated quinone carbonyl (C9) could not be confirmedfrom correlations in the HMBC spectrum because it was further than three bonds away from the nearest proton. AnHMBC correlation from a proton signal at 5 7.36 (H7) to a carbon resonance at 5 116.2 permitted the carbon to beassigned as C8a. Likewise, a correlation from the H2 resonance (8 7.28) into a carbon signal at 5 114.7 resulted inthe assignment of the carbon as C9a. HMBC correlations from the H7 (5 7.36) and H2 (7.28) resonances to carbonresonances at 5 161.7 and 162.0 permitted the assignment of these resonances to C8 and Cl, respectively. AnHMBC correlation from the H6 resonance (5 7.80) into a carbon resonance at 6 133.7 ppm allowed assignment ofthe carbon as ClOa. No HMBC correlations were observed into the C4a signal so the shift was assigned bycomparison with the chemical shift of ClOa. A correlation from the signal at 5 4.61 (H11, H11') into a carbonresonance 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 asample of langarin with acetic anhydride in pyridine overnight under anhydrous conditions resulted in a singleproduct. The excess reagents were removed in vacuo to yield langarin triacetate (143), a compound which gave amolecular 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 threecarbonyl stretches, a band at 1675 cm -1 , appropriate for a non-chelated quinone carbonyl, an absorbance at1770 cm -1 corresponding to the phenol acetate stretch, and a band at 1744 cm -1 resulting from the C11acetoxymethylene acetate stretch. Table 17 summarizes the 1 H (Figure 90) and 13C NMR (Figure 91) data recordedfor the triacetate 143. The shifts were assigned by comparison with the spectroscopic data of the parentcompound 142.OAc0143Table 17. 1H and 13C NMR Data for Langarin Triacetate (143) (Recorded in Me2SO)Carbon no. 51H (ppm) (400 MHz) 513C (ppm) (125 MHz)1 — 149.7a2 7.61, d, J = 1.7 Hz —3 —4 8.09, d, J = 1.7 Hz —4a — —5 8.13, dd, J = 8.0, 1.2 Hz —6 7.93, t, J = 8.0 Hz —7 7.62, dd, J = 8.0, 1.2 Hz —8 149.5a8a — —9 180.3b9a — —10 181.4b10a — —11, 11' 5.26, s 63.9CH2-OAS 2.13, s 21.1, 170.1Ar—OA 2.38, s; 2.39, s 20.6, 20.8, 169.0, 168.9a,b May be interchanged.As expected, the hydroxyl protons at 5 11.96, 11.90, and 5.59 in the 1 H NMR spectrum of 142 were nolonger 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 13C NMR spectrum (Figure 91) wasthe upfield shift of one quinone carbonyl (C9) from 5 192.0 in the parent quinone 142 to 180.3 in the triacetate143. The other carbonyl quinone resonance (C10) did not change position significantly, but had a chemical shift of5 181.8 in langarin (142) and 181.4 in langarin triacetate (143). This result is consistent with the assignment oflangarin as 142 since acetylation disrupts the hydrogen bonding of the Cl and C8 hydroxyl groups with the C9carbonyl 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 cytotoxiccomponent of an undescribed Queen Charlotte Islands Aplidium species, was assigned as the anthraquinone 142.175OAc0H11'H11H6 H2H4 H7H5OCOCkL3OCOCJI3 OCOCH,3 I^' ,9. 0^8.0^7.0^6.0^5. 0^4.0^3.0PPM12. 0^1 1. 0^10.0Figure 90.^1 H NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2SO at 400 MHz)us^MO^IN^100^II0^soFigure 91.^13C NMR Spectrum of Langarin Triacetate (143) (Recorded in Me2SO at 125 MHz)178iii) CONCLUSIONSA study of an undescribed species of Aplidium from Langara Island resulted in the isolation and structuralelucidation of the new compound langarin (142). This is the first reported anthraquinone from an animal of thesubphylum Urochordata. As previously mentioned, the related compound 128 has been isolated from the stonycoral 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 includingrhubarb root. 110 The leaf exudates of the Aloe plant are rich in a variety of related natural products including thebarbaloin isomers 145, 10-C-rhamnosyl aloe-emodin anthrone (146), and 11-0-rhamnosyl aloe emodin (147)isolated from the leaves of Aloe rebaiensis. 111OH 0 OH^OH 0 OHOH0^0142 128OH 0 OH^OH 0^OHOH^0^R144 145 R = glucose146 R = rhamnoseOH 0 OH0-rhamnose0147It can be postulated that the biogenesis of langarin (142) results from folding, cyclization, andaromatization of a single octaketide unit as shown in Equation 7. 112 Reduction of the carbonyl at C6,decarboxylation000^1W;CO2H0 0 08 CH3CO2H11t[0]^[0]OH 0 OH OH0142Equation 7. The Proposed Biogenesis of Langarin (142).179decarboxylation, and oxidation of C10 and C11 yields an anthraquinone skeleton with the appropriate oxidationpattern.As mentioned earlier, the impetus for this investigation was the cytotoxicity of the methanolic extract ofthe 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 cytotoxicityof langarin (142) was of particular interest since the compound proved completely inactive in bacterial and fungalassays indicating a surprising level of specificity in the L1210 cytotoxicity assay. The results of an in vivo testagainst leukemia in mice are still awaited.180B. SECONDARY METABOLITES OF APL/Q/J/M_CA/IFORN/CUM FROM THE OUEEN CHARLOTTEISLANDS PRENYLATED OUINONES AND HYDROOUINONES FROM ANIMALS OF THE SUBPHYLUMUROCHORDATAThis thesis presents the isolation of five known and two new prenylated quinones and hydroquinones fromthe tunicate Aplidium californicum. Consequently, the following section will review reports of prenylated quinonesfrom animals of the subphylum Urochordata. All organisms produce prenylated quinones which are believed to playa crucial role in the electron transport of living systems. 113 Simple prenylated quinones as well as intricatelyelaborated derivatives are found in marine organisms of every phylum. 8Fenical first reported the isolation of the geranylhydroquinone 148 from an Aplidium species. 114 Howardand Clarkson reported the structures of prenylhydroquinone (149), 6-hydroxy-2,2-dimethylchromene (150), andprenylquinone (151) from Aplidium californicum collected near San Francisco, Califomia. 115 Prenylhydroquinone149 proved active in vivo against P388 murine lymphocytic leukemia with a maximum T/C of 138% at3.12 mg/kg. Addition of compounds 149 and 150 to Salmonella typhimurium which had been treated with thecarcinogens benzo(a)pyrene and aflatoxin B1 resulted in a considerable decrease in mutations observed inS. typhimurium. Similarly, 149 decreased the mutagenic effects of ultraviolet radiation. 115OH148HOHO149^ 150^151Targett and Keeran reported the chromenol 152 from Aplidium constellatum. 116a This compound hadbeen previously isolated from the tropical American tree Cordia alliodora. 116bHO1811 5 2Pietra and coworkers described the isolation of the verapliquinones A to D (153-156, respectively) from anAplidium species collected off the Atlantic coast of France. 117 Verapliquinones B (154) and D (156) are the firstcompounds with a neryl-type linear monoterpene side chain, where the A 2 ' ,3 '-olefin has the Z rather than the morecommon E configuration. 117CH3O CH3O153^ 154CH3O CH3O155 156 OHThe colonial tunicate Amaroucium (synonymous with Aplidium) multiplicatum was found to contain theknown quinone derivatives 148 and 152 and the three novel compounds, chromenol 157, and the hydroquinonederivatives 158 and 159. 118 All of the isolated compounds were found to inhibit lipid peroxide formation and15-lipoxygenase, with ID50 values ranging from 0.3-1.0 pg/mL for the former assay and 0.2-3.0 lig/mL for thelatter. Formation of lipid peroxides has been implicated in development of arteriosclerosis in humans. 118H3COHO182OH^OHOH1 5 7^ 158 C-1'—C2' = single bond159 C1'—C2' = E double bondAlthough the majority of compounds reported from animals belonging to the Urochordata are nitrogenous, anumber of mono- and diprenylated quinone derivatives have been reported. Some of these compounds have provenactive in a variety of pharmacological bioassays and it is notable, albeit not surprising, that all of these activities areassociated with the antioxidant properties of the prenylated hydroquinones.ii) RESULTS AND DISCUSSIONPRENYLATED OUINONE AND HYDROOUINONE DERIVATIVES FROM APLIDIUM CALIFORNICUMA small collection of Aplidium californicum was made off Langara Island, one of British Columbia'sQueen Charlotte Islands in May of 1991 (Figure 82). Abbott and Newberry state that A. californicum is commonto semiprotected coastal areas ranging from southern California (La Paz) to Vancouver Island. 95 It is found at depthsof 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 smoothto irregular.95The colony of A. californicum collected off Langara Island (Figure 92) was pale yellow in color and soft intexture. The colony was 1 cm thick and 20 cm across with an irregular but smooth surface. The tunicate wascommon at the collecting site and grew on exposed rock walls at an average depth of 5 m. The collection included avoucher sample (100 g) which was extracted with methanol and a small sample (350 g) which was frozen for laterstudy. 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 cellline) and prompted study of the natural products of the Aplidium californicum sample.183One 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 TLCand 1 H NMR indicated that these extracts had much the same chemical composition. In vitro testing against theL1210 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 but the 1 H NMR spectrumof this extract looked less interesting. The hexanes, chloroform, and ethyl acetate extracts were combined andchromatographed 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 fromhexane 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 mostpolar fractions eluted by radial TLC gave the known simple prenylated hydroquinones 149 and 150. The less polarfractions yielded the previously reported hydroquinone derivatives 152 and 158 and the two new prenylatedhydroquinones calaplidol A (160) and calaplidol B (161). All of the prenylated quinones and hydroquinones wereunstable and decomposed rapidly, in some cases limiting the data which could be collected in order to elucidate thestructures.HOHO149^150 HOHO152^158HOHOHO H160^ 161Figure 92.^Freshly Collected Aplidium californicum184Calaplidol A (1601185HOCalaplidol A (160) was isolated as a colorless glass which gave an M+1 peak in the LRDCIMS atm/z 261 Da (2.40 % relative intensity), appropriate for a molecular formula of C161-12003. The EIHRMS did notshow a molecular ion but had an intense peak (9.06 % relative intensity) at m/z 242.1303 Da corresponding to themolecular ion with loss of one equivalent of water (C161-11802 [AM -0.3 mmu]). The molecular formula indicatedthat 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) whichindicated that calaplidol A had a trisubstituted benzene ring. The coupling patterns suggested 1,2,4-substitution (seeTable 18).The connectivity of the side chain was determined by analysis of the spectroscopic data which issummarized in Table 18. The 13C NMR spectrum is shown by Figure 94. The carbon resonances were assignedfrom the HMQC and APT (Figure 96) spectra. Two methyl singlets at 5 1.49 (Me9') and 1.70 (Me8') in the1 H NMR spectrum both had weak COSY correlations (Figure 95) consistent with allylic coupling into a singleproton resonance at 5.20 (H6'). The chemical shift of this proton resonance confirmed that it was olefinic as did acorrelation 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'). TheHMQC spectrum showed a correlation from the proton resonance at 5 4.48 into a carbon signal at 5 82.0 ppm (C4'),186a chemical shift appropriate for a carbon bearing an oxygen functionality. The above data was used to elucidatefragment a.aTable 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 — — — —2 — 147 —3 6.64, ma 117.1 —4 — — 151 —5 6.63, ma H6 115.7 6 6.82, d, J = 9.1 Hz H5 122.2 —1' 6.36, d, J = 11.7 Hz H2' 127.4 —2' 6.19, d, J = 11.7 Hz H1', H10' 131.1 C2, C103' — — — —4' 4.48, m H5'a, H5'b 82.0 —5'a 2.36, m H5'b, H6' 30.8 —5'b 2.50, m H5'a, H6' — —6' 5.20, m H5'a, H5'b, H8', H9' 120.0 —7' — — 134 —8' 1.70, sa H6', H9' 25.8b C6', C7', C9'9' 1.49, sa H6', H8' 16.5b C6', C7', C8'10'a 5.09, br s H10'b, H2' 115.9 C2', C4'10'b 5.16, br s H10'a — C2', C4'a,b May be interchanged. c The carbon resonances in the HMBC column are correlated to proton resonances in the8 1 H column.HOHO H7^ , - 5^3Figure 93.^1 11 NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)^ ppmFigure 94,^13C NMR Spectrum of Calaplidol A (160) (Recorded in CDC13 at 125 MHz)HO7 5 7 a 6 5 6 3 5.5 5 8 4 5 4.3 3 5 3 8 2 5 2 8 t5 1.3PPMPPMa aHO H1 1^.•I■i^:i IMe9'/H6'. ,Me8'/H6' •s, ,,H5/H510,^•I^:I. H5'a/H4'H5'b/H4'H2'/H10'. A,i1 H10'a/H1O'b H6'/H5'a°s 4 1ii11 H6'/H5'b !i H1'/H2'.H6/H5LIAS. .iATP..! .Figure 95.^COSY Spectrum of Calaplidol A (160) (Recorded in CDC13 at 400 MHz)1898.3.5131. a4. HI T T^ r^ u^r^1140^1i0^100 1;0^SO 40 20^IPPMFigure 96. APT Spectrum of Calaplidol A (160) (Recorded in CDC13 at 100 MHz)gSFigure 97 . HMBC Spectrum of Calaplido! A (160) (Recorded in CDC13 at 500 MHz)%S.HO5HO HH1O'b FIllr aH 10'b/C4'••H10',/C4'o^•co•PPM ie 1.0^0.5t.0^1.5C4'•H1O'b/C2'H I O' a/C2'•, • .^ •^ ,—, •7.0^8.5^5.0^0.5^0.0—10017 PP—100—lesMe8'/C9'• •^Me9'/C8'Me8'/C6'^Me9'/C6'Me8'/C7' a .ro Me9'/C7'•C9'C8'100C6'CTH6.27152131.02' H122.6 1'5.59HOHO192The COSY spectrum (Figure 95) showed a correlation from a deshielded proton resonance at 8 6.36 (111') toanother downfield proton resonance at 6.19 (H2'). The chemical shifts of these protons in conjunction withcorrelations in the HMQC spectrum from the proton signals at 8 6.36 and 6.19 into carbon resonances at 8 127.4and 131.1, respectively, indicated that 160 contained a disubstituted double bond. The coupling constant of 11.7 Hzfor the resonances at 8 6.36 (H1') and 6.19 Hz (H2') was in better agreement with the coupling constant for H1' andH2' 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 13C 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 than159 and the A 1 ' ,2 ' bond of metabolite 160 was consequently assigned the Z configuration.HO H^H HO H6.36 6.19160,7,..........,01{ 6H.10I^125.3134.7 1'H^HO6.97159A weak COSY correlation (Figure 95) was observed from the proton signal at 8 6.19 (H2') to a broadsinglet 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.1.70( CH3‘k 134.0,".w ...._.CH3120.0^1.49",..._...."HO 131. 82.06.19H\5.9H^H5.09^5.16bDouble bonds in the side chain accounted for three sites of unsaturation leaving four unsaturations for thearomatic 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 thevicinity of 1675 cm-1 , the expected frequency for a quinone carbony1. 109 A strong band which could be seen at3369 cm -1 in the IR spectrum was attributed to hydroxyl stretching. The phenols of the hydroquinone ringaccounted for two of the oxygen atoms in the molecular formula leaving one unassigned. The final oxygen wasassigned 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' andpermitted the latter carbon resonance to be assigned to C7'.HO H193Figure 98. Selected HMBC Correlations for Calaplidol A (160).194The H10'a and H10'b proton resonances (8 5.09, 5.16) had HMBC correlations to carbon resonances at8 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 sidechain with A 1 ' ,2 ' and A3 ' ,1I3 'unsaturations and a hydroxyl substituent at C4'. Once the side chain had beenassembled, the hydroquinone could only be attached to C1'. The structure of calaplidol A was tentatively assigned as160.Calaplidol B (161)HO8 'CH3Calaplidol B (161) was isolated as an unstable colorless glass. The EIHRMS had a peak corresponding tothe molecular ion at m/z 352.1669 Da appropriate for a formula of C22H2404 (AM -0.6 mmu). From themolecular formula it was evident that 161 had eleven sites of unsaturation. The IR spectrum of 161 had a stronghydroxyl stretch at 3361 cm -1 . No carbonyl stretch was evident in the IR spectrum of calaplidol B indicating thatthe compound was not a quinone.The structure of 161 was proposed from analysis of the spectroscopic data which is summarized inTable 19. The aromatic region of the 1 H NMR spectrum (Figure 99) was congested with signals which integratedfor a total of eight protons of chemical shift 8 6.2 to 7.0 ppm. Another olefinic proton was apparent at 8 5.10. Apair of olefinic methyls were evident at 8 1.79 and a final methyl singlet had a chemical shift of 1.47 ppm.Table 19. 1H and 13C NMR Data for Calaplidol B (161) (Recorded in CDC13)Carbon no. 81H (ppm) (400 MHz) COSY (400 MHz) 813C (ppm)(100 MHz)HMBCd (500 MHz)1 — — 147c —2 — — 125 —3 6.86, br s H5 113.5 Cl, C54 148c —5 6.62, m H3, H6 115.4 Cl6 6.76, d, J = 8.6 Hz H5 118.0 C2, C41' 6.84, br d, J = 16.2 Hz H2' 121.6 C3'2' 6.33, d, J = 16.2 Hz H1' 137.3 C23' — — 76.4 —4'a 1.78, dd H4'b, H5' 39.2 4'b 1.93, dd H4'a, H5' — —5' 3.73, m H4'a, H4'b, H6' 32.0 —6' 5.10, m H5', H8', H9' 127.1 C7', C8', C9'7' — — 134 —8' 1.79, br s H6' 25.8b C6', C7', C9'9' 1.79, br s H6' 17.6b C6', C7', C8'10' 1.47, s — 23.6 C2', C3', C4'1" — — 147a —2" — 125 —3" 6.57, dd, J = 3.0, 0.8 Hz H5", H6' 114.9 Cl", C5"4" — 149a —5" 6.62, m H3", H6" 115.0 Cl"6" 6.68, d, J = 8.6 Hz H5" 116.9 C2", C4"Ar-OH 4.55, s — — —Ar-OH 4.35, s — — _Ar-OH 4.28, sa,b,c May be interchanged.d The carbon resonances in the HMBC column are correlated to proton resonances in the8 1 H column.1956 5 4 3 2 i 0PPRFigure 99 1 11 NMR Spectrum of Calaplidol B (1611 (Recorded in CDC13 at 500 MHz)gFigure 100 COSY Spectrum of Calaplidol B (161) (Recorded in CDC13 at 400 MHz) 04 0PPM6 0 5 07 0HOMe9'Me8'••..^... . •..,.1.4.%.4...,^4,-^6•,e.i:.IC.^.....4...9. H5'/H4'b1btv r .H5'/H4'a--o-Me8'/H6'Me9'/H6'. • -‘,.•C.0-H6'/H5'0 •a'.'^•H3"/H5" -_H3;-3"/H5'-.. , .H2'/Hl'- ...,^. N.^ .g.^•-^--1970. 0PM• i40•80r POI2010 01 40 1 2 0• •60SHOFigure 101, 13C NMR Spectrum of Calaplidol B (161) (Recorded in CDCI3 at 100 MHz)00C9'• 4 cg , C10'C5'0 0^C4'0^ C6'• ppm C2'■..-. . IPP,^6^4^250I  I•il1001^1^.^I6.0 6.0 6.4ppmFigure 102 HMQC Spectrum of Calaplidol B (161) (Recorded in CDC13 at 500 MHz)Figure 103 HMBC Spectrum of Calaplidol B (161) (Recorded in CDCI3 at 500 MHz)PIM1^I^1^ 12.0^1.5C9'Me8'/C9' •Me9'/C8' C8'F PP.HO5"H1'^H5"H3 H6 H6" H5 H3"- 7..OHH2'Me10'/C4' C4'— 50dd. Me107C3' C3'—100Me8'/C6' •Me9'/C6 '^ Me8'/C7'Me9'/C7' Me107C2'C6'CTC2'1.781.93^5.10H H3 .73 HH 1.79CH31.74CFI125H127.1^1'79CH3134CHH 1.746.57\1496.76147 OHH6.866.846.33H 1.4739.2137.3H8HO 14H6.68^115.0- H201The spin systems of the side chain were elucidated from the COSY spectrum (Figure 100). The olefinicmethyl 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 at8 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 (Figure103) from the proton resonance at 8 1.79 (Me8' and Me9') to carbon resonances at 8 127.1 and 134 established theallylic relationship of H6' to Me8' and Me9' and permitted the assignment of the carbon resonance at 8 134 to C7'(Figure 104).Figure 104. Selected HMBC Spectroscopic Correlations for Calaplidol B (161).202In the COSY spectrum (Figure 100) a correlation was observed from a doublet at 5 6.84 (H1') to a signal at6.33 (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 wereobserved 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 aquaternary 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 shiftof 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 thesewere 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 of3.0 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 oneanother. The smaller coupling constant of 0.8 Hz had the appropriate magnitude for allylic coupling. A weakCOSY correlation was in fact evident from the signal at 5 6.57 (H3") to the multiplet at 3.73 (H5'). This wasinterpreted 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). Theseresonances both had coupling constants of 8.6 Hz indicating that each is an aromatic proton ortho to one of the twoprotons which overlap to form the multiplet at 5 6.62 (H5, H5"). From the COSY spectrum it could not bedetermined 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") showedfurther coupling into a broad singlet at 6.86 (H3). The downfield shift of the A-ring proton at 5 6.86 (H3) wasattributed to deshielding resulting from extended conjugation into the A 1 ' ,2 'double bond. These data were interpretedto mean that ring A, too, is a 1,2,4-trisubstituted benzene ring.203As shown in Figure 104, HMBC correlations from the aromatic protons of rings A and B permitted theassignment 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 phenoliccarbons meant that the signals can not be confidently differentiated, but it also substantiated the conclusion that bothrings were similarly substituted. Three singlets in the 1 H NMR spectrum at 8 4.55, 4.35, and 4.28 were notcorrelated to carbons in the HMQC spectrum and were consequently assigned to the hydroxyl protons. The chemicalshifts of the hydroxyl protons were in good agreement with the data for the related compound 162 (8 4.55 inCDC13). 1194.55OH162The substitution pattern of the B ring of 161 was confirmed by comparison of the 13C NMR spectrumwith 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 forcalaplidol A (160), the B ring seemed to contain a multiplet at 8 6.62 with fine coupling of —0.8 Hz. Thiscoupling constant was appropriate for allylic coupling and suggested that ring B might be a 1,2,4-substitutedcatechol rather than a hydroquinone, where H6" also had allylic coupling to H5'. Allylic coupling from the protonsof ring A to H1' was unlikely because the proton resonance for Hr (8 6.84) was a sharp doublet (coupled to H2',-11-11' ,H2' = 16.2 Hz). Figure 101 shows the 13C NMR spectrum of calaplidol B (161). The carbon shifts of 161were assigned from the HMQC (Figure 102) and HMBC spectra (Figure 103). The HMBC data was of no assistancein determining the substitution pattern of ring B so the carbon shifts of calaplidol B (161) were compared to thecarbon shifts of the model compounds 4-methylcatechol (105) (see Table 3) and 2-methylhydroquinone (163)(Figure 105).204HO115.6125^OH^125.2 OH113.1^ 120.4117.1^115.1115.0OH1 6 1^ 163^ 105Figure 105. Selected 13C 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 of2-methylhydroquinone indicated that ring B was more likely to be a 2-substituted 1,4-hydroquinone than a4-substituted 1,2-catechol.Two hydroquinone rings in addition to the two double bonds in the side chain accounted for ten of theeleven 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 chainat C5'. An HMBC correlation from the proton resonance at 8 6.33 (H2') to a carbon signal at 8 125 situated theA 1 ' ,2 '-double bond adjacent to ring A (Figure 103, Figure 104). One site of unsaturation remained unassigned. Thespectroscopic data contained no evidence for additional unsaturated functional groups and consequently the finalunsaturation was attributed to a third ring. The chemical shift of C3' (8 76.4) indicated that the final ring consistedof a cyclization where C3' was attached to the hydroxyl of one of the hydroquinone rings to form a chromenol. TheE stereochemistry of the A 1 ' ,2 '-double bond obviated the possibilty of a chromenol moiety which incorporated the Aring. The only structure which can accomodate all the functionalities of calaplidol B and three rings was determinedto be 161 where C3' was attached to the Cl" oxygen to form a chromenol fragment incorporating ring B. Thestructure was tentatively assigned to be 161 but further analysis is required to confirm the structure and to elucidatethe stereochemistry of the C3' and C5' centres.HOHOHO150OHHO158149152HOHO205161Known Metabolites from the Tunicate Aplidium californicumIn addition to the new compounds calaplidol A (160), and calaplidol B (161), our collection of Aplidiumcalifornicum yielded four known compounds. The structures of the simple prenylated hydroquinone 149 and thechromenol 150 were determined from the mass spectra and by comparison of the 1 H NMR with published data (seeexperimental). 115The structures of the hydroquinone 158 118 and the chromenol 152 116b were elucidated from spectroscopicdata including mass spectra, 1 H NMR, 13C NMR, COSY, HMQC, and HMBC spectra (see experimental). Thestructure of 158 was confirmed by comparison of the chemical shifts of the 1 H NMR and 13C NMR spectra withreported results. 118 The structure of 152 was in turn substantiated by comparing the 1 H NMR spectrum withpublished values. 116b206iii) CONCLUSIONSExamination of extracts of the colonial tunicate Aplidium californium led to the isolation of the two newmetabolites 160 and 161. The known compounds 149, 150, 152, and 158 were also isolated. All of theisolated metabolites are closely related and originate from a hemiterpene or monoterpene substituted quinone moiety.HO HO149^ 150HOHO 158HO HOHO H160^ 161It can be postulated that the biogenesis is comparable to that of ubiquinone (164) and follows the shikimic acidpathway. As shown in Scheme 3 the first step is the formation of p-hydroxybenzoic acid. This is accomplished bythe loss of pyruvic acid from chorismic acid in bacteria or by degradation of phenyl alanine in higher plants andmammals. 112 The prenyl side chain is subsequently introduced ortho to the hydroxyl function. Decarboxylationoccurs followed by introduction of a second hydroxy para to the first to yield the hydroquinone which may beoxidized to form the corresponding quinone.H3CO^CH3H3CO164 R = prenyl sidechain207CO2H^ CO2HPhenylalanineorChorismic Acid OH^ OHp-hydroxybenzoicacidOHOH OHScheme 3. The Proposed Biogenesis of Prenylated Quinones and HydroquinonesIt  is likely that calaplidol A (160) results from oxidation of the side chain of a monoterpene-substitutedhydroquinone followed by elimination to form the 1 3 ' ,10 ' double bond. Calaplidol B (161) may be the product of acondensation reaction involving two hemiterpene-substituted hydroquinone units or of the substitution of amonoterpene 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 promisingcytotoxic activity of the methanolic extract of a voucher sample. Compound 152 was tested in vitro against theL1210 murine leukemia cell line and found to be cytotoxic with an ED50 value of 3.9 As mentionedearlier, Howard and Clarkson have reported that the prenylated hydroquinone 149 is active against P388 murineleukemia 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 testthese compounds for biological activity and also to determine the stereochemistry of C4' in 160 and C3' and CS' in161. NOE difference or ROESY experiments should be conducted to confirm the structure of calaplidol B (161).208GENERAL EXPERIMENTALProton NMR spectra were recorded on Bruker WH-400 and AMX-500 spectrometers. The spectra werereferenced to tetramethylsilane (8 0.00 ppm) or residual solvent peaks as secondary references (Me2SO-d6 82.49 ppm, pyridine-d5 8 7.19 ppm, C6D6 8 7.15 ppm, CDC13 8 7.26 ppm). 13C and APT NMR spectra wererecorded on Varian XL-300, Bruker AM-400 and AMX-500 spectrometers. 13C spectra were referenced to residualsolvent peaks (Me2SO-d6 8 39.5 ppm, CDC13 8 77.0 ppm). COSY, 73b NOE difference, 73c and double resonanceexperiments were performed on a Bruker WH-400 spectrometer. ROESY, 80 HMQC,77 and HMBC78 experimentswere conducted on a Bruker AMX-500 spectrometer.Low and high resolution FAB mass spectra were recorded on a Kratos Concept II HQ spectrometer. Lowand high resolution electron impact mass spectra were performed on a Kratos MS-50 spectrometer. DCI massspectra 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-IRspectrophotometer. UV spectra were recorded by use of Bausch and Lomb Spectronic 2000 and Perkin-ElmerLambda 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-backedKieselgel 60 F254 and Whatman MKC18F reversed phase TLC plates, respectively. TLC plates were visualized byUV or by use of H2SO4 and vanillin sprays. Further detail concerning spray preparation is provided by Stah1. 120Size exclusion chromatography used Sigma Sephadex LH-20-100 gel (bead size 25-100 11). Normal phase flashchromatography 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 ResearchChromatotron model 7924). For the purification of glaciasterol A (100) AgNO3 (5.6 g) was added to the silica gelslurry prior to pouring the chromatotron plate. 122Samples were prepared for HPLC purification by application to Waters Sep-Pak silica and C18 cartridgesprepared according to package instructions. HPLC separations used one of two systems. The first is comprised of aWaters 501 HPLC pump equipped with a Waters model 440 absorbance detector and a Perkin-Elmer LC-25RI detector. The other system consists of a Perkin-Elmer series 2 liquid chromatograph and LC-55spectrophotometer coupled to a Waters 410 differential refractometer. Reversed phase separations required Alltech209Associates Econosil C18 5 p. analytical and Whatman Partisil 10 ODS-3 magnum columns. Normal phase HPLCseparations 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 furtherpurification. 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 assaysfor effectiveness against the mouse L1210 (in vitro) leukemia cell lines were supervised by Dr. Theresa M. Allen atthe 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 ofAnatomy, UBC.Glaciasterols A and B from the sponge Aplysilla glacialisCollection and Isolation DataIsolation of Glaciasterol A (100)Aplysilla glacialis 123 was collected by SCUBA at -5m in Sydney Inlet, Vancouver Island and off SanfordIsland 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 washomogenized 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 betweenbrine (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 astep gradient of solvents beginning with hexane through ethyl acetate to methanol. Twenty-nine fractions werecollected, 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 furtherpurified by application to a normal phase SepPak eluted with ethyl acetate / benzene (3:7). The glaciasterol fraction210was enriched in glaciasterol A (100) by radial TLC chromatography using a normal phase silica plate saturated withAgNO3 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 containedglaciasterol A (100) in the highest concentration. The fourth fraction was prepared for HPLC by application to areversed phase Sep-Pak cartridge eluted with water/methanol (3:7, 10 mL). HPLC purification by use of a reversedphase 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;^13C 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, 201.8ppm; EILRMS, m/z (formula, relative intensity): 400 (C26H4003, 8), 382 (C26113802, 5), 285 (C19l-12502, 10),97 (C71113, 69); EIHRMS m/z: 416.2936 (C26H4004, AM +0.9 mmu); ED50: 2.1 p.g/mL (L1210 in vitromurine leukemia cell line); 1D50: 19 µg/mL (MCF-7 in vitro human breast cancer cell line) and 18 pg/mL (MCF-7Adr multidrug resistant in vitro human breast cancer cell line).211Acetylation of Glaciasterol A (100): Glaciasterol A (100) (1 mg) was stirred overnight under a nitrogenatmosphere with pyridine and acetic anhydride (0.5 mL each). Excess reagent was removed in vacuo to yield pureglaciasterol A diacetate (106).Glaciasterol A Diacetate (106) AcOGlaciasterol A diacetate (106) was isolated as a white solid; UV max (CH2C12) 254 nm (e 2850); IR 1737, 1685cm -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; 13C 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 with5% NaHSO3(aq.) (20 mL). The aqueous layer was extracted sequentially with dichloromethane (3 x 20 mL). Theorganic extracts were combined and washed with brine (2 x 20 mL). The dichloromethane layer was dried overNa2SO4, filtered, and evaporated in vacuo to yield 5a,613-dihydroxyglaciasterol A diacetate (113). The diol was212acetylated as previously described and purified by reversed phase HPLC (water/methanol 1:4) to yield5a-hydroxyglaciasterol A triacetate (114).5a.60-Dihydroxvglaciasterol A Diacetate (113)AcO5a,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)AcO5a-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,2131H), 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 inmethanol (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 togive a brown oil (82.8 g). A portion of the crude extract (31 g) was partitioned between brine (500 mL) and ethylacetate (4 x 250 mL). The ethyl acetate extract was dried over Na2SO4, filtered and evaporated in vacuo to yield adark 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 givefourteen fractions which were screened by 1 H NMR. The ninth fraction contained a signal at 8 6.8 which isdiagnostic 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/dichloromethane1:99), by radial TLC (methanol / dichloromethane 1:99) and by reversed phase HPLC (water/methanol 3:17).Glaciasterol B Diacetate (107)AcO214Glaciasterol 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; 13C 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 breastcancer cell line).Acid-catalyzed Ring Opening of the 5,6-Epoxide of Glaciasterol B Diacetate (107) with H218j Glaciasterol Bdiacetate (107) (4 mg) was stirred for four hours with THE (0.25 mL), H2O (100 41..), H2 180 (97.5 atom% 180,1251AL) and perchloric acid (251.1L). The reaction workup was identical to that previously described and yielded 180labeled 5a,613-dihydroxyglaciasterol B diacetate (111). The diol 111 was reacted with pyridine and acetic anhydrideas previously described. The acetylation reaction yielded 5a-hydroxyglaciasterol B triacetate (112).5a.60-Dihydroxyglaciasterol B Diacetate (111)AcO5a,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), 1.38215(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; 13C 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 1607, AM +0.3 mmu),536.3596 (C311150 1606 180 - ,I AM -0.3 mmu).5a-Hydroxvglaciasterol B Triacetate (1121AcO5a-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 1608, AM +0.8 mmu); ED50: 5.66 ptg/mL (L1210 in vitro murine leukemiacell line); ID50: 2 ptg/mL (MCF-7 in vitro human breast cancer cell line) and 4.5 .tg/mL (MCF-7 Adr multidrugresistant in vitro human breast cancer cell line).216Sesquiterpenes and Blancasterol from the Sponge Pleraplysilla sp.Collection and Isolation DataA small patch of a Pleraplysilla sp. sponge124 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 theintertidal zone at Botanical Beach, British Columbia. The boulder was replaced in its original position with care toallow 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 1991collections. 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) andethyl acetate (4 x 25 mL). The ethyl acetate extracts were pooled, dried over Na2SO4, and evaporated to yield abrown 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 NMRspectra of these fractions indicated that fourth, fifth and sixth fractions were rich in terpenoidal metabolites and thesewere pooled prior to further separation.Fractions 4-6 (44 mg) were applied to a TLC silica flash column9 and eluted with a polarity step gradientof solvents ranging from diethyl ether/dichloromethane (1:19) to ethyl acetate (100%). Twenty-eight fractions werecollected, analyzed by TLC and pooled to yield ten fractions. The second fraction contained a mixture of 0-methylfurodysinin lactone (57) (1 mg) and 0-methyl nakafuran-8 lactone (44) (0.2 mg). These were separated and purifiedby normal phase HPLC eluted with hexane/dichloromethane (3:7). The fourth column fraction contained 0-methyl9-oxofurodysinin lactone (72) (0.5 mg). 0-methyl 2-oxomicrocionin-2 lactone (74) was the major component ofthe fifth fraction. The sixth fraction contained furodysinin lactone (56) (2.0 mg). HPLC purification of the seventhfraction 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 normalphase 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 1764cm -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; EIHRMSm/z: 262.1573 (C16H2203, AM +0.4 mmu).0-Methyl Nakafuran-8 Lactone (4410-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)218ppm; 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, 198.1ppm; 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)OCH30-methyl 2-oxomicrocionin-2 lactone (74) was isolated as a translucent white glass; IR 1764, 1663 cm -1 ; 1 1-1NMR (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),2192.03 (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 (561OHFurodysinin 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, 125MHz) 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, 21.0,22024.4, 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)OH2-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, 198.8ppm 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 eighteenhours 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. Thesponge was extracted again, overnight, filtered, and the extracts were pooled and concentrated in vacuo to yield a darkgreen oil (68.7 mg). 1H NMR spectra and TLC showed no indication of the sesquiterpene lactones isolated from the1990 and 1991 collections.A portion of the ethyl acetate extract (25 mg) was applied to a TLC silica flash column and eluted with apolarity 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 firstfraction contained terpenoidal metabolites. This was further purified by normal phase HPLC eluted with hexane toyield 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.222Furodysin (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 (411CHOThe 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)HOOHOAcBlancasterol (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 = 5.8,2232.4 Hz, 1H) ppm; 13C 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 cellline); ID50: (MCF-7 in vitro human breast cancer cell line) and 10.6 1.1g/mL (MCF-7 Adr multidrugresistant in vitro human breast cancer cell line).Langarin from an Aplidium speciesCollection and Isolation DataA 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. Thecrude methanolic extract was found to be active against the in vitro L1210 murine leukemia cell line with an ED50of 14 µg/mL. The remainder of the orange colonial tunicate was stored frozen for three months, then ground in ablender and extracted with methanol (500 mL). The extract was filtered and the organism was re-extracted withmethanol (500 mL) overnight. The second extract was filtered and pooled with the first. The combined extracts wereconcentrated 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 thesame major component (yellow spot on TLC, Rf=0.33 in methanol/dichloromethane 3:99) thus the extracts werepooled. 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.1H NMR indicated that the second fraction (42 mg) contained aromatic compounds and bioassay indicated that thisfraction 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).Langarin (142)OH 0 OH224OHLangarin (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 (brs, 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 vitromurine leukemia cell line).Acetylation of Langarin (142): Langarin (142) was stirred overnight with acetic anhydride and pyridine (0.5 mLeach) under an atmosphere of nitrogen. Excess reagent was removed in vacuo to yield langarin triacetate (143).Langarin Triacetate (143)OAc 0 OAcOAcLangarin 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)225S 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, relativeintesity): 396 (C20-11608, 5), 354 (C01 -4407, 35), 312 (C17141206, 32), 270 (C15H1005, 73).Metabolites from Aplidium californicumCollection and Isolation DataAplidium californicum (350 g), a pale yellow colonial tunicate, was collected by SCUBA at -5m offLangara Island in the Queen Charlotte Islands, B.C. A voucher sample (100 g) was reserved for screening and theremaining organisms were stored frozen for three months. A crude methanolic extract of the voucher sample provedmildly active against Bacillis subtilis and Rhizoctonia solani. The extract was active against the in vitro L1210murine leukemia cell line at an ED50 of 6 One half of the remaining, frozen animals (160 g) were freeze-dried to give a dry weight of 9.9 g. The dry animals were extracted sequentially with hexanes, chloroform, ethylacetate, and methanol (2 x 500 mL each). In each case the tunicates were soaked in the solvent, broken up with aspatula, sonicated, and then filtered. The animals were reimmersed in solvent and soaked overnight prior tofiltration. Extracts of like solvents were pooled. By 1 H NMR and TLC, the hexane, dichloromethane and ethylacetate 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.1H NMR spectra of the fractions indicated that the four initial fractions eluted contained fats and steroids. The latterfractions 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 hydroquinones149 and 150.Column fractions five through seven were also pooled and further purified by radial TLC eluted with apolarity gradient of solvents ranging from ethyl acetate/hexane (1:19) to ethyl acetate (100%). The final purificationwas accomplished by use of normal phase HPLC eluted with methanol / dichloromethane (1:99). Extraction andpurification of the entire A. californicum collection by the isolation procedure described above yielded calapliquinoneA (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).Calaplidol A (160)OH HHOHO2261 4 91H 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).HO1501 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).HO HCalaplidol 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 = 11.7Hz, 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, relativeintensity): 242 (C16141802, 9), 199 (C13H1102, 7), 173 (Ci iH902, 24); DCIMS m/z (NH3, M+H): 261(C16H2003, 2.4% relative intensity).Calaplidol B (161)227HOCalaplidol 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; 13C 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). HO152Chromenol 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; 13C NMR (CDC13, 100 MHz) 8 17.6, 22.7, 25.6, 26.0,22840.9, 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).HO158Hydroquinone 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 = 2.7Hz, 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).REFERENCES 1 a) Carefoot, T. Pacific Seashores; J.J.Douglas: Vancouver, B.C., 1977, and b) Carefoot, T., University ofBritish 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. Res. 1949, 8, 137-176.5 Bergmann, W.; Feeney, R. J. J. Amer. Chem. Soc. 1950, 72, 2809-2810.6 Scheuer, P. J. Biomedical Importance of Marine Organisms; Fautin, D. G., Ed.; California Academy ofSciences: San Francisco, C.A., 1988, 37-40.7 Ireland, C. M.; Roll, D. M.; Molinski, T. F.; McKee, T. C.; Zabriskie, T. M.; Swersey, J. C. BiomedicalImportance of Marine Organisms; Fautin, D. G., Ed.; California Academy of Sciences: San Francisco, C.A.,1988, 41-57.8 a) Faulkner, D. J. Tetrahedron, 1977, 33, 1421-1443, b) Faulkner, D. J. Nat. Products Rep. 1984, 1(3), 251-280, c) Faulkner, D. J. Nat. Products Rep. 1984, 1(6), 552-598, d) Faulkner, D. J. Nat. Products Rep.1986, 3(1), 1-33, e) Faulkner, D. J. Nat. Products Rep. 1987, 4, 539-576, 0 Faulkner, D. J. Nat. ProductsRep. 1988, 5, 613-663, g) Faulkner, D. J. Nat. Products Rep. 1990, 7, 269-309, h) Faulkner, D. J. Nat.Products Rep. 1991, 8, 97-147, and i) Faulkner, D. J. Nat. Products Rep. 1992, 5, 323-364.9 Roth, J.; LeRoith, D.; Collier, E. S.; Watkinson, A.; Lesniak, M. A. Ann. N.Y. Acad. Sci. 1986, 463, 1-11.10 De Silva, E. D.; Scheuer, P. J. Tetrahedron Lett. 1980, 21, 1611-1614.11 a) Jacobs, R. S.; Culver, P.; Landon, R.; O'Brien, T.; White, S. Tetrahedron, 1985, 41(6), 981-984,b) Mann, J. Nature, 1992, 358, 540, and c) Mayer, A. M. S. ; Jacobs, R. S. Biomedical Importance ofMarine Organisms; Fautin, D. G., Ed.; California Academy of Sciences: San Francisco, C.A., 1988, 133-142.12 a) Greenspan, E. M. Clinical Interpretation and Practice of Cancer Chemotherapy; Greenspan, E. M., Ed.;Raven: New York, 1982, pp. 1-16, and b) Munro, M. H. G.; Luibrand, R. T.; Blunt, J. W. BioorganicMarine Chemistry; Scheuer, P. J., Ed.; Springer-Verlag: Berlin, 1987, pp. 93-176.13 Chan, W. R.; Tinto, W. F.; Manchand, P. S.; Todaro, L. J. J. Org . Chem. 1987, 52, 3091-3093.14 De Silva, E. D.; Andersen, R. J.; Allen, T. M. Tetrahedron Lett. 1990, 31(4), 489-492.15 a) Rinehart, K. L.; Gloer, J. B.; Hugh es, R. G. Jr.; Renis, H. E.; McGovern, J. P.; Swynenberg, E. B.;Stringfellow, D. A.; Kuentzel, S. L.; Li, L. H. Science, 1981, 212, 933-935 and b) Rinehart, K. L.; Sakai,R.; Holt, T. G.; Fregeau, N. L.; Perun, T. J. Jr.; Seigler, D. S.; Wilson, G. R.; Shield, L. S. Pure andAppl. Chem. 1990, 62(7), 1277-1280.16 Barth, R. H.; Broshears, R. E. The Invertebrate World; CBS College: Philadelphia, PA, 1982, pp. 71-85.22923017 Barnes, R. S. K.; Calow, P.; Olive, P. J. W.; Golding, D. W. The Invertebrates, A New Synthesis;Blackwell Scientific: Oxford, 1988, pp.49-53.18 Bergquist, P. R. The Origins and Relationships of Lower Invertebrates; Conway Morris, S.; George, J. D.;Gibson, R.; Platt, H. H., Ed.; Clarendon: Oxford, 1985, 28, 14-27.19 Reference 3, p.100.20 Reference 3, p.105.21 Reference 3, p.10.22 a) Bergquist, P. R.; Wells, R. J. Marine Natural Products, Chemical and Biological Perspectives, Vol. V;Scheuer, P. J., Ed.; Academic Press: New York, N.Y., 1983, 1-50 and b) Bergquist, P. R.; Karuso, P.;Cambie, R. C.; New Perspectives in Sponge Biology; Rutzler, K., Ed.; Smithsonian: Washington, D.C.,1991, 72-78.23 Reference 3, p.143.24 Bergquist, P. R. N.Z. J. Zool. 1980, 7, 443-503.25 Bergquist, P. R. N.Z. J. Zool. 1980, 7, 1-6.26 Boury-Esnault, N.; De Vos, L.; Donadey, C.; Vacelet, J. New Perspectives in Sponge Biology; Rutzler, K.,Ed.; Smithsonian: Washington, D.C., 1991, 237-244.27 Reference 3, p.214.28 a) Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lett. 1974, 38, 3401-3404, b) Schulte, G.; Scheuer, P.J.; McConnell, 0. J. Hely. Chim. Acta, 1980, 63(8), 2159-2167, c) Bobzin, S. C.; Faulkner, D. J. J.Chem. Ecol. 1992, /8(3), 309-331, and d) Tischler, M.; Andersen, R.J .; Choudhary, M. I.; Clardy, J. J.Org . Chem. 1991, 56, 42-47.29 Austin, W.C., Khoyotan Marine Laboratories, personal communication, 1990.30 Dumdei, E.J. Ph.D. Thesis, UBC, Vancouver, B.C., 1992.31 Simpson, T. L. The Cell Biology of Sponges; Springer-Verlag: New York, N.Y., 1984.32 a) Van Soest, R. W. M. New perspectives in Sponge Biology; Rutzler, K., Ed.; Smithsonian: Washington,D.C., 1991, 344-348 and b) Van Soest, R. W. M. Fossil and Recent Sponges; Reitner, J.; Keupp, H., Ed.;Springer-Verlag: Berlin, 1991, 54-71.33 Tischler, M. Ph.D. Thesis, UBC, Vancouver, B.C., 1989.34 Molinski, T. F.; Faulkner, D. J. J. Org. Chem. 1986, 51, 1144-1146.35 Bobzin, S. C.; Faulkner, D. J. J. Org. Chem. 1989, 54, 3902-3907.36 Tischler, M.; Andersen, R. J. Tetrahedron Lett. 1989, 30, 5717-5720.37 Poiner, A.; Taylor, W. C. Aust. J. Chem. 1990, 43, 1713-1727.23138 Bobzin, S. C.; Faulkner, D. J. J. Chem. Ecol. 1992,18(3), 309-332.39 Reference 38 and references cited therein.40 Dunlop, R. W.; Kazlauskas, R.; March, G.; Murphy, P. T.; Wells, R. J. Aust. J. Chem. 1982, 35, 103.41 Minale, L.; Riccio, R.; Sodano, G. Tetrahedron Lea. 1974, 38, 3401-3404.42 Cozzolino, R.; De Giulio, A.; De Rosa, S.; Strazzullo, G.; Gasic, M. J.; Sladic, D.; Zlatovic, M. J. Nat.Prod. 1990, 53(3), 699-702. and references cited therein.43 a) Cimino, G.; De Stefano, S.; Guerriero, A.; Minale, L. Tetrahedron Lea. 1975, / 7, 1417-1420, b)Cimino, G.; De Stefano, S.; Guerriero, A.; Minale, L. Tetrahedron Lett. 1975, /7, 1421-1424, and c)Cimino, G.; De Stefano, S.; Guerriero, A.; Minale, L. Tetrahedron Lea. 1975, / 7, 1425-1428.44 Kazlauskas, R.; Murphy, P. T.; Wells, R. J. Tetrahedron Lett. 1978, 49, 4951 -4954.45 Mong, S.; Votta, B.; Sarau, H. M.; Foley, J. J.; Schmidt, D.; Carte, B. K.; Poehland, B.; Westley, J.Prostaglandins, 1990, 39(1), 89-97.46 Kazlauskas, R.; Murphy, P. T.; Wells, R. J. Tetrahedron Lett. 1978, 49, 4949-4950.47 Schulte, G.; Scheuer, P. J.; McConnell, 0. J. J. Org. Chem. 1980, 45(3), 552-554.48 Walker, R. P.; Rosser, R. M.; Faulkner, D. J. J. Org. Chem. 1984, 49, 5160-5163.49 Grode, S. H.; Cardellina, J. H., II J. Nat. Prod. 1984, 47(1), 76-83.50 Cardellina, J. H., II; Barnekow, D. E. J. Org. Chem. 1988, 53, 882-884.51 Mancini, I.; Guella, G.; Guerriero, A.; Boldrin, A.; Pietra, F. Helv. Chim. Acta, 1987, 70, 2011-2018.52 Cimino, G.; De Luca, P.; De Stephano, S.; Minale, L. Tetrahedron, 1975, 31, 271-275.53 Cimino, G.; De Rosa, S.; De Stephano, S.; Puliti, R.; Strazzullo, G.; Mattia, C.A.; Mazzarella, L.Tetrahedron, 1987, 43, 4777-4784.54 a) Carmely, S.; Cojucaru, M.; Loya, Y.; Kashman, Y. J. Org . Chem. 1988, 53, 4801-4807, and b) Rudi, A.;Kashman, Y. Tetrahedron, 1990, 46(11), 4019-4022.55 Carmely, S.; Gebreyesus, T.; Kashman, Y.; Skelton, B. W.; White, A. H.; Yosief, T. Aust. J. Chem. 1990,43, 1881-1888.56 Cimino, G.; De Stephan, S.; Minale, L.; Trivellone, E. Tetrahedron, 1972, 28, 4761-7.57 Cimino, G.; De Stephano, S.; Minale, L. Experientia, 1974, 30, 846.58 a) Cimino, G.; De Stephano, S.; Minale, L.; Trivellone, E. Tetrahedron Lett. 1975, 43, 3727-30, and b)Cimino, G.; De Stephano, S.; Minale, L.; Trivellone, E. Experientia, 1978, 34, 1424-7.59 Ciminiello, P.; Fattorusso, E.; Magno, S.; Mangoni, A.; Pansini, M. J. Am. Chem. Soc. 1990, 112, 3505-3509.23260 Enwall, E. L.; Van Der Helm, D.; Nan Hsu, I.; Pattabhiraman, T.; Schmitz, F. J.; Spraggins, R. L.;Weinheimer, A. J. J.C.S. Chem. Comm. 1972,215-216.61 Kazlauskas, R.; Murphy, P. T.; Ravi, B. N.; Sanders, R. L.; Wells, R. J. Aust. J. Chem. 1982,35, 69-75.62 Bonini, C.; Cooper, C. B.; Kazlauskas, R.; Wells, R. B.; Djerassi, C. J. Org . Chem. 1983, 48, 2108-2111.63 Capon, R. J.; Faulkner, D. J. J. Org . Chem. 1985,50,4771-4773.64 Madaio, A.; Piccialli, V.; Sica, D. Tetrahedron Lett. 1988, 29(46), 5999-6000.65 Madaio, A.; Notaro, G.; Piccialli, V.; Sica, D. J. Nat. Prod. 1990,53(3), 565-572.66 D'Auria, M. V.; Paloma, L. G.; Minale, L.; Riccio, R.; Debitus, C. Tetrahedron Lett. 1991, 32(19),2149-2152.67 Migliuolo, A.; Piccialli, V.; Sica, D. Tetrahedron, 1991, 47(37), 7937-7950.68 Migliuolo, A.; Piccialli, V.; Sica, D. Steroids, 1992,57,344-347.69 Pika, J.; Tischler, M.; Andersen, R. J. Can. J. Chem. 1992, 70, 1506.70 West, R. R.; Cardellina, J. H. II J. Org. Chem. 1988,53, 2782-2787.71 West, R. R.; Cardellina, J. H. II J. Org. Chem. 1989,54, 3234-3236.72 Martin, G. E.; Zektzer, A. S. Two-Dimensional NMR Methods for Establishing Molecular Connectivity— AChemist's Guide to Experiment Selection, Performance, and Interpretation; VCH: New York, N.Y., 1988,p.l.73 a) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric Identification of Organic Compounds,Fourth Ed.; John Wiley and Sons: New York, N.Y., 1981, pp.181-304, b) Derome, A. E. Modern NMRTechniques for Chemistry Research; Pergamon: New York, N.Y., 1987, and c) Sadler, I.H. Nat. Prod. Rep.1988, 101-127.74 Kessler, H.; Gehrke, M.; Griesinger, C. Angew. Chem. Int. Ed. Engl. 1988,27,490-536.75 Reference 72, pp.59-60.76 Reference 73b), pp.188-190.77 Bax, A.; Subramanian, S. J. Magn. Res. 1986, 67, 565-569.78 Bax, A.; Summers, M. F. J. Amer. Chem. Soc. 1986, /08, 2093-2094.79 Martin, G. E.; Crouch, R. C. J. Nat. Prod. 1991,54(/), 1-70.80 Bax, A.; Davis, D. G. J. Magn. Res. 1985, 63, 207-213.81 Stonard, R. J.; Petrovich, J. C.; Andersen, R. J. Steroids„ 1980, 36, 81-86.23382 Ishiguro, M.; Kajikawa, A.; Haruyama, Y.; Ogura, Y.; Okabayashi, M.; Morisaki, M.; Ikekawa, N., J.Chem. Soc. Perkin Trans. 1,1975, 2295-2302.83 Reference 73a), p.209.84 Ishiguro, M.; Saito, H.; Hirano, Y.; Ikekawa, N. J. Chem. Soc. Perkin Trans. 1 1980, 2503-2506.85 Demarco, P. V.; Farkas, E.; Doddrell, D.; Mylari, B. L.; Wenkert, E. J. Amer. Chem. Soc. 1968, 90 (20),5480-5486.86 s ting' , J .; Andersen, R. J.; Emerman, J. T. Cancer Chemother Pharmacol 1992, 30, 401-406.87 Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. D. Molecular Biology of the Cell, 3rdEd.; Garland: New York, N.Y., 1983, p.730.88 Van Soest, R. W. M., University of Amsterdam, The Netherlands, personal communication, 1992.89 Carte, B.; Keman, M. R.; Barrabee, E. B.; Faulkner, D. J. J. Org. Chem. 1986, 51, 3528-3532.90 Reference 73a), 270.91 Carte, B.; Kernan, M. R.; Barrabee, E. B.; Faulkner, D. J.; Matsumoto, G. K.; Clardy, J. J. Org. Chem.1986, 51, 3528-3532.92 Cimino, G.; De Stephano, S.; Guerriero, A.; Minale, L. Tetrahedron Lett. 1975, 43, 3723-3726.93 Reference 16, pp 558-567.94 Van Name, W. G. Bulletin of the American Museum of Natural History, 1945, 84, 12.95 Abbott, D. P.; Newberry, A. T. Intertidal Invertebrates of California; Morris, R. H., Abbott, D. P.;Haderlie, E. C., Ed.; Stanford University: California, 1990, 177-184.96 Reference 17, pp. 208-211.97 Carroll, A. R.; Scheuer, P. J. J. Org . Chem. 1990, 55, 4426 4131.98 Parry, D. L.; Kott, P. Bull. Mar. Sci. 1988, 42 (1), 149-153.99 Corley, D. G.; Moore, R. E. J. Am. Chem. Soc. 1988, 110, 7920-7922.100 Kitahara, T.; Naganawa, H.; Okazaki, T.; Okami, Y.; Umezawa, H. J. Antibiot. 1975, 28, 280-285.101 Tamura, —.i ; Fukata, F.; Sasho, M.; Tsugoshi, T.; Kita, Y. J. Org . Chem. 1985, 50, 2273-2277.102 Roll, D. M.; Scheuer, P. J. J. Am. Chem. Soc. 1983, 105, 6177-6178.103 Nakamura, H.; Kobayashi, J.; Kobayashi, M.; Ohizumi, Y.; Hirata, Y. Chem. Lett. 1985, 713-716.104 Schm itz, F. J.; Bloor, S. J. J. Org . Chem. 1988, 53, 3922-3925.105 Sanduja, R.; Alam, M.; Wellington, G. M. J. Chem. Research, 1986, 450-451.234106 a) Fahy, E.; Andersen, R. J.; Cun-Heng, H; Clardy, J. J. Org. Chem. 1985, 50, 1149, b) Fahy, E.;Andersen, R. J.; Van Duyne, G. D.; Clardy, J. J. Org. Chem., 1986,51, 57-61, c) Fahy, E.; Andersen, R.J.; Xu, C.; Clardy, J. J. Org . Chem., 1986,51, 5145-5148, d) Fahy, E.; Andersen, R. J. Can. J. Chem.1987, 65, 376-383, and e) Fahy, E. Ph. D. Thesis, UBC, Vancouver, B.C., 1986.107 a) Sutherland, M. D.; Wells, J. W. Aust. J. Chem. 1967, 20, 515-534, b) Powell, V. H.; Sutherland, M. D.;Wells, J. W. Aust. .1. Chem. 1967, 20, 534-540, c) Powell, V. H.; Sutherland, M. D. Aust. J. Chem. 1967,20, 541-543, and d) Rideout, J. A.; Sutherland, M. D. Aust. J. Chem. 1985, 38, 793-808.108 Pasto, D. J.; Johnson, C. R. Organic Structure Determination; Prentice-Hall: NJ., 1969, p.98.109 Kitahara, T.; Naganawa, H.; Okazaki, T.; Okami, Y.; Umezawa, H. J. Antibiot. 1975, 280-285.110 The Merck Index, An Encyclopedia of Chemicals and Drugs, 9th Ed.; Merck: N.J., 1976, p.3509.111 a) Conner, J. M.; Gray, A. I.; Reynolds, T.; Waterman, P. G. Phytochem. 1987, 26(11), 2995-2997, andb) Conner, J.M.; Gray, A.I.; Reynolds, T.; Waterman, P.G. Phytochem. 1989, 28(12), 3551-3553.112 Torssell, K.G.B. Natural Product Chemistry; A Mechanistic and Biosynthetic Approach to SecondaryMetabolism; John Wiley and Sons: New York, N.Y., 1983, p.126.113 Britton, G. Nat. Prod. Rep. 1991, p.243.114 Fenical, W. Food-Drugs Sea, Proc. 4, 1976, 388.115 Howard, B. M.; Clarkson, K. Tetrahedron Lett., 1979, 46, 4449-4452.116 a) Targett, N. M.; Keeran , W. S. J. Nat. Prod. 1984, 47, 556-57, and b) Manners, G. D.; Jurd, L. J.C.S.Perkin 1 1977, 405-410.117 Guella, G.; Mancini, I.; Pietra, F. Hely. Chim. Acta, 1987, 70, 621-626.118 Sato, A.; Shindo, T.; Kasanuki, N.; Hasegawa, K. J. Nat. Prods. 1989,52(5), 975-981.119 Fenical, W.; McConnell, 0. Experientia, 1975, 1004-1005.120 Stahl, E. Thin -Layer Chromatography, A Laboratory Handbook, translated by M.R.F. Ashworth; Springer-Verlag: Berlin; 1969.121 Taber, D. F. J.Org.Chem. 1982,47, 1351-1352.122 Kokke, W. C. M. C.; Pak, C. S.; Fenical, W.; Djerassi, C. Helv.Chim.Acta, 1979, 62 (Fasc. 4), 1310-1318.123 A sample of Aplysilla glacialis was deposited in the Zoological Museum of Amsterdam (registration numberZMA POR. 10,083).124 A sample of the Pleraplysilla sp. sponge was deposited in the Zoological Museum of Amsterdam (registrationnumber ZMA POR. 10,084).


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