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Terpenoid metabolites from three dorid nudibranchs and a new bromotryptamine derivative from the Northeast… Dumdei, Eric J. 1993

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We accept this thesis as conformingto the required standardTERPENOID METABOLITES FROM THREE DORID NUDIBRANCHSAND A NEW BROMOTRYPTAMINE DERIVATIVE FROM THE NORTHEASTPACIFIC SPONGE PLOCAMISSA IGZObyERIC JOHN DUMDEIB. A. St. Olaf College, Northfield, MN, 1986M. Sc. University of British Columbia, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Chemistry)THE UNIVERSITY OF BRITISH COLUMBIAJanuary 1993© Eric John Dumdei, 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.(SignatureDepartment of  C_The University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)AbstractInvestigation of the terpenoid metabolites present in the extractsof three dorid nudibranchs has led to the isolation of sixteen known andten new compounds. Structures were determined through extensivespectroscopic analysis. In addition, a new bromotryptamine derivedcompound was isolated from the Northeast Pacific spongePlocamissa igzo.Specimens of Chromodoris geminus collected off the coast of SriLanka were found to contain the known spongian diterpenoid 34 aswell as the new metabolites 61 and 62.A study investigating the geographical variation in themetabolites present in Cadlina luteomarginata along the BritishColumbian coast revealed the drimane sesquiterpenoid albicanylacetate (72) to be the most widely distributed metabolite. Specimensof the nudibranch collected at Rennell Sound contained thesesquiterpenoid metabolites 68, 69 and 78-80 which could be traced toa locally abundant prey sponge in the genus Acanthella. Interestingly,the algal metabolite violacene (81) was also present in both thesponge and nudibranch extracts. A single specimen of C.luteomarginata collected in the process of actively laying eggscontained the sesquiterpenoids 68, 69 and 78 as well as the diterpenoidmetabolite 82. The egg mass of this individual contained the drimanederivatives 83 and 84, acetylated analogs of 72. Specimens collectedoff Anthony Island contained the previously described compoundsmicrocionin-2 (70), marginatafuran (74) and 9,1 1-dihydrogracillin A (87)as well as the new metabolites acanthene K (8 5 ), 20-acetoxymarginatone (86) and the labdane diterpenoid lutenenolideii68 X=NC69 X = NCS78 X = NHCHO70R1^p A c::OAcR23412 1 = 0Ac R2 = H61 R i = H^R2 = OAc62 R i = OAc R2 = OAc(88). Samples of C. luteomarginato collected in AgamemnonChannel, Jervis Inlet, contained the previously isolated compoundsluteone (73) and diterpenoid 89 as well as the new metabolites 90 andcadlinaldehyde (91), a compound with a new carbon skeleton.Specimens of Archidoris pseudoargus collected off Norwaycontained the acyl glycerides 118 and 119, two compounds previouslyisolated from Archidoris spp. collected off the coast of British Columbia,as well as another as yet incompletely characterized glyceride.An investigation into the cytotoxic components of the extract ofthe Northeast Pacific sponge Plocamissa igzo resulted in the isolationof the new bromotryptamine derived compound igzamide (157).iiiR2 ',,.72R 1 =H^R2 =H83 R 1 = OAc R2 = OAc84R 1 =0AcR2 =HNH2Ac0„NHCHO I^ ' 8685087C I,,^R282 R i = OAc R2 = OH89 R i = 0Bu R2 = OH90 R I =H R2 = OAcCI^CI81CIBrCH2I"'79 X = NC80 X = NCSiv118R i =H^R2 =H^ 157119 R 1 = OAc R2 = HvTable of Contents.^ pg.Abstract^ iiTable of Contents^ vList of Figures ixList of Schemes^ xviiList of Tables xviiiList of Abbreviations^ xxAcknowledgments xxiiiI. Introduction to Modern Nuclear Magnetic Resonance Techniques^ 1A. Nuclear Spin States, Chemical Shift, Scalar and Dipolar Coupling^2B.Pulse Experiments^ 6i. One-dimensional Experiments^ 9a. 1 H Spectrum^ 9b. NOE Difference Spectra 9c. 13C Spectrum 10d. The Attached Proton Test (APT) Spectrum^ 11ii. Two-dimensional Experiments^ 12a. 1 H- 1 H COSY and Long Range COSY Spectra^ 13b. HMQC Spectra^ 14c. HMBC Spectra 15C. A Practical Example: The Structural Elucidation of a Compound^ 16II. Terpenoid Metabolites From Three Dorid Nudibranchs^ 31A. Introduction to the Nudibranchia^ 31i. Phyllidia varicose and an Isocyano Icthyotoxin^36viTable of Contents (cont.)^ pgii. Polygodial 37iii.Roboastra and Tambje Nudibranchs: Where DefensiveStrategy Fails^ 38B. The Skin Chemistry of Chromodoris geminus^ 40i. Introduction to the Chromodorids 40ii.Three Spongian Diterpenoids from the NudibranchChromodoris geminus (Rudman, 1987) Collected off theCoast of Sri Lanka^ 58a. Extraction and Purification^ 58b. Results and Discussion 60c. Conclusion^ 98C. Metabolites from the Pacific Dorid Nudibranch Cadlinaluteomarginata : Structures and Geographical Distribution^ 100i. Introduction to Cadlina luteomarginata^ 100ii.The Current Study^ 105a. Extraction and Purification^ 109b. Results and Discussion 1141) Metabolites from Cadlina luteomarginataCollected at Rennell Sound, B. C. ^ 1142) Metabolites from the Egg Mass of an ActivelyLaying Specimen of Cadlina luteomarginataCollected at Rennell Sound, B. C. ^ 1183) Metabolites from Cadlina luteomarginataCollected at Anthony Island, B. C. ^ 1384) Metabolites from Cadlina luteomarginataCollected at Agamemnon Channel,Jervis Inlet, B. C. ^ 175c. Conclusions^ 201D. Metabolites from the Nudibranch Archidorispseudoargus (Rapp, 1827)^ 203i. Introduction to the Archidorids 203viiTable of Contents (cont.)^ pgii. Acyl Glycerides Isolated from the Dorid NudibranchArchidoris pseudoargus (Rapp, 1827),Collected off the Coast of Norway^ 206a. Extraction and Purification 206b. Results and Discussion^ 208c. Conclusion^ 216E. Conclusion^ 217II. Igzamide, a New Bromotryptamine Derived Compound from theNortheast Pacific Sponge Plocamissa igzo^ 218A. Introduction to Poriferan Bromotryptamines 218B.A New Bromoindole Derivative from the Northeast Pacific SpongePlocamissa igzo (de Laubenfels, 1932)^ 224i. Extraction and Purification^ 225ii. Results and Discussion 226iii. Conclusion^ 233IV. Experimental^ 235A. General 235B.Metabolites from Chromodoris geminus^ 238C. Metabolites from Cadlino luleomorginata 241D. Metabolites from Archidoris psuedoargus^ 258E. Metabolites from Plocamissa igzo^ 260Appendix A. Spectra of the Previously Reported Metabolites Isolatedfrom the Three Dorid Nudibranchs^ 265viiiTable of Contents (cont.)^pgAppendix B. Spectra of the Previously Reported Metabolites Isolatedfrom Plocamissa igzo^ 285ixList of Figures^ PFigure 1. Perturbation of Transition (a->13) Energy Levels (vi, v2) byScalar Coupled Nuclei Results in the Removal ofDegeneracy^ 4Figure 2. Detail of the 400 MHz 1 H NMR Spectrum (CDCI3/Me2SO-do)of 4-methylcatechol (1) Showing Scalar Couplings (J)^5Figure 3. The Bulk Magnetization B is the Sum of the PrecessingNuclear Magnetic Dipoles^ 7Figure 4. Application of a Pulse Results in the Rotation of the BulkMagnetization B by the Flip Angle 0^ 8Figure 5. Pulse Sequence for Acquisition of a 1D 1 H spectrum^9Figure 6. Pulse Sequence for the Acquisition of NOE DifferenceSpectra^ 10Figure 7. Pulse Sequence for the Acquisition of a 13C Spectrum^ 11Figure 8. Pulse Sequence for the Acquisition of an APT Spectrum^ 12Figure 9. Prototype 2D Pulse Sequence^ 12Figure 10. Pulse Sequence used in the Acquisition of aCOSY Spectrum^ 13Figure 11. Pulse Sequence used for Acquisition of COSY-LR Spectra ^ 14Figure 12. Pulse Sequence for the Acquisition of an HMQCSpectrum^ 15Figure 13. Pulse Sequence for the Acquisition of an HMBCSpectrum 16Figure 14. 500 MHz 1 H NMR Spectrum (CDCI3/Me2SO-do) of4-methylcatechol (1)^ 17Figure 15. 125 MHz 13C NMR Spectrum (CDCI3/Me2SO-do) of4-methylcatechol (1)^ 18Figure 16. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3/Me2SO-do) of4-methylcatechol (1)^ 20Figure 17. 500 MHz HMQC Spectrum (CDCI3/Me2SO-do) of4-methylcatechol (1)^ 22xList of Figures (cont.)^ p gFigure 18. 500 MHz HMBC Spectrum (CDC13/Me2S0-c16) of4-methylcatechol (1) ^23Figure 19. HMBC Correlations from the Methyl 1 H Resonance^ 25Figure 20. HMBC Correlations from the Aromatic 1 H Resonances^ 26Figure 21. 400 MHz NOE Difference Spectra (CDC13/Me2S0-c16) of4-methylcatechol (1)^ 28Figure 22. NOE Summary for 1  27Figure 23. Typical Nudibranchs^  33Figure 24. Phylogenetic Classification of Opisthobranchs^34Figure 25. The Phylogenetic Classification of C. geminus 59Figure 26. 500 MHz 1 H NMR Spectrum (CDCI3) of120,15a,16a-triacetoxyspongian (34)^  62Figure 27. 125 MHz 13C NMR Spectrum (CDCI3) of120,15a,16a-triacetoxyspongian (34)^ 63Figure 28. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of12f3,15cc,16a-triacetoxyspongian (34)^ 65Figure 29. 500 MHz 1 H NMR Spectrum (C6D6) of1213,15a,16oc-triacetoxyspongian (34)^ 67Figure 30. 75 MHz 13C/APT NMR Spectra (C6D6) of1213,15a,16a-triacetoxyspongian (34)^ 68Figure 31. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) of143,15a,16a-triacetoxyspongian (34)^ 70Figure 32. C and D rings of 34^  69Figure 33. 500 MHz HMQC Spectrum (C6D6) of12f3,15a,16a-triacetoxyspongian (34)^ 72Figure 34. NOE Summary for 34^ 73xiList of Fig ures (cont.)^ PgFigure 35 . 500 MHz 1 H NMR Spectrum (CDCI3) ofOcc,15a,16a-triacetoxyspongian (61)^ 76Figure 36 . 125 MHz 130 NMR Spectrum (CDCI3) of6a,15a,16a-triacetoxyspongian (61)^ 78Figure 37 . 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of6a,15a,16a-triacetoxyspongian (61)^ 79Figure 38. Spin Systems for 61^ 77Figure 39 . 500 MHz 1 H NMR Spectrum (C6D6) of6a,15a,16a-triacetoxyspongian (61)^ 80Figure 40. 75 MHz 130 NMR Spectrum (C6D6) of6a,15a,16a-triacetoxyspongian (61)^ 82Figure 41. 500 MHz HMQC Spectrum (C6D6) of6a,15a,16a-triacetoxyspongian (61)^ 83Figure 42. 400 MHz 1 1-I- 1 H COSY Spectrum (C6D6) of6a,15a,16a-triacetoxyspongian (61)^ 84Figure 43. Spin Systems for 61^ 85Figure 44. NOE Summary for 61 86Figure 45. 500 MHz 1 H NMR Spectrum (CDCI3) of6a,12f3,15a,16a-tetracetoxyspongian (62)^89Figure 46. 125 MHz 13C NMR Spectrum (CDCI3) of6a,12f3,15a,16a-tetracetoxyspongian (62)^91Figure 47. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of6a,12(3,15a,16a-tetracetoxyspongian (62)^92Figure 48. B, C and D rings Spin Systems for 62^ 93Figure 49. 500 MHz 1 H NMR Spectrum (C6D6) of6a,1213,15a,16a-tetracetoxyspongian (62)^94XIIList of Figures (cont.)^ PgFigure 50. 75 MHz 13C/APT NMR Spectra (C6D6) of6a,120,15a,16a-tetracetoxyspongian (62)^ 95969798Figure 54. Phylogenetic Classification of C. luteomarginata^ 101102104111120122123124400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of^Albicanyl triacetate (83)^  125Figure 63. Ring A Spin System for 83 126Figure 64. NOE Summary for 83^ 127Figure 65. 400 MHz 1 H NMR Spectrum (C6D6) ofAlbicanyl diacetate (84)^ 131Figure 66. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) ofAlbicanyl diacetate (84)^ 132Figure 51. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) ofExa,1213,15a,16a-tetracetoxyspongian (62)^Figure 52. 500 MHz HMQC Spectrum (C6D6) of6a,120,15a,16a-tetracetoxyspongian (62)^Figure 53. NOE Summary for 62^collectedFigure 56. Metabolites from Cadlina luteomarginatathe coast of British Columbia^collected offFigure 57.Figure 58.Collection Areas for Cadlina luteomarginata^500 MHz 1 H NMR Spectrum (CDCI3) ofAlbicanyl triacetate (83)^Figure 59.Figure 60.Figure 61.Figure 62.500 MHz 1 H NMR Spectrum (CDCI3) ofAlbicanyl acetate (72)^125 MHz 13C NMR Spectrum (CDCI3) ofAlbicanyl acetate (72)^125 MHz 13C NMI? Spectrum (CDCI3) ofAlbicanyl triacetate (83)^Figure 55. Metabolites from Cadlina luteomarginatain Californian waters^xiiiPg130142144145146152154153155156158159160165166168169170List of Figures (cont.)Figure 67Figure 68 . 500 MHz 1 H NMR Spectrum (CDCI3) of Acanthene K (85) Figure 69 . 125 MHz 13C NMR Spectrum (CDCI3) of Acanthene K (85)Figure 70 . 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofAcanthene K (85)^. 500 MHz HMQC Spectrum (CDCI3) of Acanthene K (85)^. 500 MHz 1 H NMR Spectrum (CDCI3) of20-acetoxymarginatone (86)^. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of20-acetoxymarginatone (86)^COSY Spin Systems of 86^. 500 MHz 1 H NMR Spectrum (C6D6) of20-acetoxymarginatone (86)^. 75 MHz 13C/APT NMR Spectra (C6D6) of20-acetoxymarginatone (86)^. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) of20-acetoxymarginatone (86)^. 500 MHz HMQC Spectrum (C6D6) of20-acetoxymarginatone (86)^Figure 80. 500 MHz 1 H NMR Spectrum (CDCI3) of Lutenenolide (88)^Figure 81. 125 MHz 13C NMR Spectrum (CDCI3) of Lutenenolide (88)Figure 82. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofLutenenolide (88)^Figure 83. Spin Systems for 88Figure 84. 500 MHz HMQC Spectrum (CDCI3) of Lutenenolide (88)^. Spin Sysytems for 84Figure 71Figure 72Figure 73Figure 74.Figure 75Figure 76Figure 77Figure 78Figure 79. NOE Summary for 86xlvList of Figures (cont.)^ pgFigure 85. 400 MHz 1 H- 1 H COSY-LR Spectrum (CDCI3) ofLutenenolide (88)^ 171Figure 86. 500 MHz HMBC Spectrum (CDCI3) of Lutenenolide (88)^ 172Figure 87. 500 MHz 1 H NMR Spectrum (CDCI3) of17p-acetoxy-15,17-oxidospongian-16-one (90)^ 180Figure 88. 125 MHz 13C NMR Spectrum (CDCI3) of17 pracetoxy-15,17-oxidospongian-16-one (90)^ 181Figure 89. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of173-acetoxy-15,17-oxidospongian-16-one (90)^ 183Figure 90. 500 MHz HMQC Spectrum (CDCI3) of173-acetoxy-15,17-oxidospongian-16-one (90)^ 184Figure 91. 500 MHz 1 H NMR Spectrum (CDCI3) ofCadlinaldehyde (91)^ 188Figure 92. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofCadlinaldehyde (91)^ 189Figure 93. Spin Sysytems for Cadlinaldehyde (91)^ 190Figure 94. 75 MHz 13C NMR Spectrum (CDCI3) ofCadlinaldehyde (91)^ 192Figure 95. 500 MHz HMQC Spectrum (CDCI3) ofCadlinaldehyde (91)^ 193Figure 96. 500 MHz HMBC Spectrum (CDCI3) ofCadlinaldehyde (91)^ 194Figure 97. HMBC Correlations from the Methyl groups of 91^ 195Figure 98. Non-methyl HMBC Correlations of 91^ 197Figure 99. Metabolites Isolated from Archidoris odherni^204Figure 100. Metabolites Isolated from Archidoris montereyensis^205Figure 101. Phylogenetic Classification of A. pseudoargus^207Figure 102. Electron Impact Mass Spectrum of Compound 125^210List of Figures (cont.)^ p gFigure 103. 500 MHz 1 H NMR Spectrum (CDCI3) of Compound 125^211Figure 104. 125 MHz 13C NMR Spectrum (CDCI3) of Compound 125^213Figure 105.400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofCompound 125^ 214Figure 106. Simple Brominated Indole Metabolites from Sponges^219Figure 107. Brominated Aplysinopsin-type Metabolites^220Figure 108. Peptide-type Bromoindoles from Sponges 221Figure 109. Brominated bis(indole) Alkaloids from Sponges^223Figure 110 . 400 MHz 1 H NMR Spectrum (Me2SO-do) of lgzamide (157) 228Figure 111 . 75 MHz 13C NMR Spectrum (Me2SO-do) of Igzamide (157) 230Figure 112 . 400 MHz 1 H- 1 H COSY Spectrum (Me2SO-do) oflgzamide (157)^ 231Figure A01. 400 MHz 1 H NMR Spectrum (CDCI3) of 68^266Figure A02. 400 MHz 1 H NMR Spectrum (CDCI3) of 69 267Figure A03. 400 MHz 1 H NMR Spectrum (CDCI3) of 78^268Figure A04. 400 MHz 1 H NMR Spectrum (C6D6) of 79 269Figure A05. 400 MHz 1 H NMR Spectrum (CDCI3) of 80^270Figure A06. 400 MHz 1 H NMR Spectrum (CDCI3) of Violacene (81)^271Figure A07. 500 MHz 1 H NMR Spectrum (CDCI3) of 82^272Figure A08. 500 MHz 1 H NMR Spectrum (CDCI3) ofMarginatafuran (74)^ 273Figure A09. 125 MHz 13C NMR Spectrum (CDCI3) ofMarginatafuran (74)^ 274Figure A10. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofMarginatafuran (74)^ 275XVxviList of Figures (cont.)^ PgFigure Al 1 . 500 MHz HMQC Spectrum (CDCI3) ofMarginatafuran (74)^ 276Figure Al2 . 400 MHz 1 H NMR Spectrum (CDCI3) of9,11-dihydrogracillin A (87)^ 277Figure Al3 . 500 MHz 1 H NMR Spectrum (CDCI3) ofLuteone (73)^ 278Figure Al4 . 125 MHz 13C NMR Spectrum (CDCI3) ofLuteone (73)^ 279Figure Al5 . 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofLuteone (73)^ 280Figure A16. 500 MHz HMQC Spectrum (CDCI3) ofLuteone (73) 281Figure A17. 500 MHz 1 H NMR Spectrum (CDCI3) of 89^282Figure A18. 400 MHz 1 H NMR Spectrum (CDCI3) of 118 283Figure A19. 500 MHz 1 H NMR Spectrum (CDCI3) of 119^284Figure B01. 400 MHz 1 H NMR Spectrum (CDCI3) of3',5-diacetowthymidine (153)^ 286Figure B02. 400 MHz 1 H NMR Spectrum (CDCI3) of2',3',5-triacetoxyuridine (154)^ 287Figure B03. 400 MHz 1 H NMR Spectrum (CDCI3) of3',5'-diacetoxy-2'-deoxyuridine (155)^ 288Figure B04. 400 MHz 1H NMR Spectrum (CDCI3) of6-bromoindole-3-carboxaldehyde (156)^289xviiList of Schemes^ PgScheme I. Possible Formation Luteone (73) and Cadlinaldehyde(91) from a hypothetical sesterterpenoidprecursor^ 199List of Tables^ p gTable 1. Assignment of NMR Signals (CDC13/Me2S0-d6) for4-methycatechol (1)^ 30Table 2. Assignment of NMR Signals (C6D6) for120,15a,16a-triacetoxyspongian (34) ^ 74Table 3. Assignment of NMR Signals (Cope) for6a,15a,16a-triacetoxyspongion (61)^ 87Table 4. Assignment of NMR Signals (C6D6) for6a,120,15a,16a-tetracetoxyspongian (62)^99Table 5. Geographical Distrubution of Metabolites from Cadlinaluteomarginata Collected in British Columbian andAlaskan Coastal Waters^ 112Table 6. Assignment of NMR Signals (CDCI3) forAlbicanyl Triacetate (83)^ 128Table 7. Assignment of NMR Signals (CDCI3) forAlbicanyl Acetate (72) 129Table 8. Assignment of 1 H NMR Signals (C6D6) forAlbicanyl Diacetate (84)^ 134Table 9. Assignment of NMR Signals (CDCI3) for Acanthene K (85)^ 148Table 10. Assignment of NMR Signals (CDCI3) forMarginatafuran (74)^ 150Table 11. Assignment of NMR Signals (C6D6) for20-Acetoxymargincrtone (86)^ 161Table 12. Assignment of NMR Signals (CDCI3) for Lutenenolide (88) ^ 174Table 13. Assignment of NMR Signals (CDCI3) for Luteone (73)^ 177Table 14. Assignment of NMR Signals (CDCI3) forCompounds 82 and 90^ 185Table 15. Comparison of 1 H NMR Signals (CDCI3) forCompounds 90 and 110^ 186Table 16. Assignment of NMR Signals (CDCI3) forCadlinaldehyde (91)^ 198xixList of Tables (cont.)^ pgTable 17. Assignment of NMR Signals (Me2SO-do) forlgzamide (157) 234List of AbbreviationsPc^acetylAc20 acetic anhydrideAcOH^acetic acidamu atomic mass unitsAPT^Attached Proton Testbd broadened doubletBDT^6-bromo-8,9-didehydrotrypaminebs broadened singletCope)^benzene-d6CDCI3 chloroform-dCH2Cl2^dichioromethaneCHCI3 chloroformCOSY^COrrelation SpectroscopYCOSY-LR^Long Range COrrelation SpectroscopYd^doubletDCIMS^Desorption-Chemical Ionization Mass Spectrometrydd double doubletddd^double doublet of doubletsDFO Department of Fisheries and Oceans (Canada)Dn^delay timedt double tripletED50^Effective Dose which inhibits 50% cell proliferationEIMS Electron Impact Mass SpectrometryEt20^diethyl etherEtOAc ethyl acetateFABMS^Fast Atom Bombardment Mass Spectrometry)0(xxiFID^Free Induction DecayFT-NMR^Fourier Transform Pulse Nuclear Magnetic ResonanceFTIR Fourier Transform Infrared spectroscopyHMBC^1H detected Multiple Bond heteronuclear multiplequantum CoherenceHMQC^1H-detected Heteronuclear Multiple QuantumCoherenceHPLC^High Pressure Liquid ChromatographyHREIMS^High Resolution Electron Impact Mass SpectrometryHSV-1 Herpes Simplex Virus type IIR^Infrared spectroscopyi signal due to impurityJ^coupling constant in HertzJ Scalar CoupledL1210^murine leukemia cell line L1210LH-20 Sephadex LH-20M+^molecular ionm multipletm.p.^melting pointm /z mass to charge ratioMe^methylMe2S0-do^dimethylsulfoxide-d6Me0H methanolMIC^minimum inhibitory concentration to yield a zoneof inhibition 5 mm in diametermmu^0.001 atomic mass unitsNH3 ammoniaNMR^Nuclear Magnetic ResonanceNOE Nuclear Overhauser Enhancement (effect)P388^murine leukemia cell line P388PPM, ppm^chemical shift in parts per millionq^quartetrel. int.^relative intensityS nuclear spinSCUBA^Self Contained Underwater Breathing Apparatuss, S solvents^singletsp. speciesspp.^species (plural)t triplettic^thin layer chromatographyTMS tetramethylsilaneUV^Ultraviolet-Visible spectroscopyw signal due to watera^parallel spin statea below the plane of the ring(a)D^optical rotation at the sodium D line13^antiparallel spin stateR above the plane of the ringY^gyromagnetic ratioe8 difference in chemical shiftsAM^difference in mass8 chemical shift in parts per millionE^ extinction coefficient?.max^wavelength of absorbance maxima4) phase cyclingAcknowledgmentsI wish to express my sincere appreciation to Dr. RaymondAndersen for his guidance and encouragement throughout the courseof my studies. It has been a real pleasure to be a member of hisresearch team.I am greatly indebted to Michael LeBlanc, for ensuring thatthe collection expeditions were not only prosperous, but also a joyousexperience. My diving partners Jana Pika, Judy Needham and DavidBurgoyne are also gratefully acknowledged for their help andfriendship. I wish also to express my gratitude to Sandra Millen for her aidin collecting Archidoris pseudoargus as well as identifying thenudibranchs in the thesis, and Dr. Bill Austin for his identification ofPlocamissa igzo. Appreciation must also be expressed to Capt. JohnAndersen and the crew of the J. P. Tully for their aid in the collection ofCadlino luteomarginata. I also wish to thank Dr. E. Dillip de Silva forarranging the collection of Chromodoris geminus and the initialisolation of the terpenoids from this organism.Technical assistance provided by the staffs of theDepartment of Chemistry NMR and mass spectrometry laboratories isthankfully acknowledged with special thanks to Liane Darge for herassistance in running experiments on the Bruker AMX-500 NMRspectrometer.I. Introduction to Modem Nuclear Magnetic Resonance Techniques.The majority of work presented in this thesis involves theinterpretation of data obtained through the use of modern nuclearmagnetic resonance (NMR) techniques. In light of this, and in order tofacilitate the reading of the thesis, a brief introduction to NMR and theinformation provided by the experiments routinely used in the thesis,using the structural elucidation of 4-methylcatechol (1) as an example,seems appropriate.OHWith the production of highly stable superconductingmagnets in the 1970's, the uses of Fourier transform pulse NMR (FT-NMR) -expanded vastly. First used as an aid for structural studies in 1973, 1 FT-NMR has become the main technique used by chemists today todetermine or confirm the structure of organic molecules. A briefdiscussion of the general principles of nuclear magnetic resonanceand a few details about the experiments used in the thesis follows, butfor a more detailed examination of modern FT-NMR experiments the1 Gullo, V. P., Miura, I., Govindachari, T. R. and Nakanishi, K. J. Am. Chem. Soc., 1973,95 8749.1reader is referred to the review article by Kessler et. al. 2 and therelevant sections of either Derome's "Modern NMR Techniques forChemistry Research' 3 or Nakanishi's "One-dimensional and Two-dimensional NMR Spectra by Modern Pulse Techniques." 4A Nuclear Spin States, Chemical Shift, Scalar and Dipolar Coupling.One intrinsic property of all atomic nuclei is the nuclear spin(S), and while the theoretical basis for this property is beyond the graspof all but a few specialists, techniques such as NMR, which use thisproperty to obtain information about molecular structure, are in wideuse. A practical analogy for the basis of NMR is to imagine that theatomic nuclei are spinning on a nuclear axis and, since they arepositively charged particles, act in a way similar to an electric currentmoving through coiled loop; they induce a magnetic dipole along thisaxis. 5 When this magnetic dipole is placed within an externallyproduced magnetic field (I30), energy states arise according to theorientation between the two magnetic vectors. For the nucleiemployed most commonly in NMR by organic chemists ( 1 H and 13C),two such states exist, alignment parallel (a) and antiparallel (13) to theexternal field. Because of the angular momentum associated with thespinning charge, however, the nuclear magnetic dipole precessesabout the axis of the applied B0 field (by convention the z axis). The2 Kessler, H., Gehrke, M. and Griesinger, C. Angew. Chem. Int. Ed. Engl., 1988, 27, 490.3 Derome, A. E. Modem NMR Techniques for Chemistry Research, Pergamon Press;Oxford, 1987.4 Nakanishi, K., Ed. One-dimensional and Two-dimensional NMR Spectra by ModernPulse Techniques, University Science Books, Mill Valley, CA, 1990.5 Only nuclei with S *0 have a magnetic moment.2frequency of precession is called the Larmor frequency (coo) and isdependent on the external field strength and a factor called thegyromagnetic ratio (7, a constant for each nucleus) in the form coo =74 When the sample is irradiated with electromagnetic (EM) energyat this frequency (in the Radio frequencies (Rf) band of the EMspectrum) a condition of "resonance" is formed, allowing the nuclei toabsorb energy and exchange between the two nuclear spin states (a-> j3, 0 -> a) .If all the protons or carbon atoms within an organicmolecule experienced the external magnetic field (B0) as isolatednuclei, NMR would be little more than another way of determining theatomic composition of the compound. However, each of the nucleiwithin a molecule are surrounded by neighboring nuclei, electrons andsolvent molecules, each inducing their own currents and magneticfields, and so each nucleus experiences a slightly different magneticfield (B ell). These variations result in different Larmor frequencies (co ='YBeff) for atoms in different electronic environments and compose theproperty of NMR called chemical shift. Usually, chemical shifts arereported as a ratio (5) of the slight frequency differences caused byneighboring groups (co-coo) divided by the Larmor frequency (coo) of astandard reference compound (tetramethylsilane, TMS). This ratio istherefore independent of magnetic field strength.In addition, the spin state (a or 13) of one nucleus caninfluence the energy levels of transition (a -> 13) of neighboring nucleithrough the intervening electrons of a chemical bond. The result of suchinfluence is the perturbation of the normal transition energy levelsresulting in the removal of degeneracy (Figure 1). The number of non-3degenerate transitions produced (splitting) is dependent on thenumber of neighboring nuclei (within 2-4 bonds) and the'equivalence' (chemical shift and scalar couplings) of the nuclei;nuclei which are enantiotopic or at the same chemical shift do notshow scalar coupling to one another, while non equivalent nuclei do.For example, in the proton spectrum of 1 (Figure 2), the resonance forH6 (8 6.66) is split into a doublet by the neighboring H5 (8 6.44), while H5is split into a double doublet by neighboring atoms H6 and H3 (8 6.60).These perturbations which make up the property called scalarcoupling are an intrinsic property of the molecule and are thereforeindependent of the external magnetic field strength. When expressedas coupling constants (J) in Hertz, they can give the researcherinformation on the distance and geometry between coupled nuclei.4V I "aaV2^V1VI^ V213a 4--V2"af3f3a --4-1vi'al3aaV2 .Not Coupled Scalar CoupledFigure 1. Perturbation of Transition (a->13) Energy Levels (vi, v2) by ScalarCoupled Nuclei Results in the Removal of Degeneracy.One further property of the nuclear spin is employed inmodern NMR techniques, that of dipolar coupling. Dipolar couplingCH31J5-6nIIH6^.'-.J H3VI ,^ I^1^I^I^r r^I^I^1 I^I^1^I^,^ -1--6. 70 6.60 6.50PPMFigure 2. Detail of the 400 MHz 1 H NMR Spectrum (CDC13/Me2S0-d6) of 4-methylcatechol (1)Showing Scalar Couplings (J)arises out of a dipole-dipole mechanism, whereby the spin state ofone nucleus can affect the spin state of another nucleus through-space' rather than through the intervening electrons of a chemicalbond. When one nucleus' transition (a -> 13) is 'saturated" by irradiatingits resonant frequency and fluctuations in the orientation of thismagnetic dipole, due to molecular motion, have a component at theLarmor frequency of a nearby nucleus, the transferral of magnetizationcan occur through dipolar coupling. 6 This transferred magnetizationresults in an increase or decrease in the intensity of the NMRresonances of the receiving nuclei. This change in intensity is termedthe nuclear Overhauser effect. Since the nuclear Overhauser effectresults from a "through space" mechanism, it can give the chemistinformation on the spatial relationships between atoms in the moleculewhich are not scalar coupled (i.e. conformation and relativeconfiguration).B. Pulse Experiments.Older, continuous wave NMR techniques involvedinstruments which scanned through the Rf band (u -> u') and measuredthe absorbed radiation at the resonance conditions (u = co) to obtaina spectrum. When multiple scans needed to be performed andaveraged (for dilute samples or nuclei, such as 13C, with a low naturalabundance) this method was very time consuming. Modern pulseexperiments have the advantage of utilizing the bulk magnetization of6 Nuclei which are not spatially close may still exhibit dipolar coupling, providedrelaxation does not take place through other mechanisms.6the sample and thus affecting all spin state transitions at once. Thisenables multiple scans to be performed in a fraction of the timepreviously required.Since alignment parallel to B0 (a) is lower in energy thanthe antiparallel alignment ((3), the population in the a state is greaterthan the p state and a bulk magnetization (B) for the sample existsalong the B0 field (Figure 3). Pulses are short term oscillating magneticfields in the form Bicoscoot where coo is the carrier frequency (i.e. theLarmor frequency of the nucleus monitored) t is the duration and B1 isthe applied field strength (determined by the power level of thetransmitter coil). Because the nuclear magnetic dipoles areprecessing about the axis of the external field (z axis), a very complex7INFigure 3. The Bulk Magnetization B is the Sum of the Precessing NuclearMagnetic Dipolesmotion results in the magnetization vectors following a pulse. Thismotion can be simplified by considering a "rotating frame" coordinatesystem wherein the coordinate system rotates about the z axis at theLarmor frequency (coo) of the nuclear transition. When this coordinatesystem is used, the magnetization appears to rotate about Bi alone.Pulses are usually described as rotations of a certain angle(Figure 4). Thus, a 90°x pulse rotates the bulk magnetization vector 90°clockwise about the x axis resulting in its orientation along the (+) y axis(My). After the application of a pulse, the vector My will rotate about thez axis at a precession frequency (co) which corresponds to its resonantfrequency. It should be noted that, in the rotating frame of reference,the detector is viewed as rotating with the coordinate system atfrequency coo, and hence experiences the precessing magneticvectors in the form co - coo. As the magnetic vectors precess about the zaxis, they induce a measurable electric current in the detector coil.which is recorded in the form of a Free Induction Decay (FID). The ADis then subjected to a computer algorithm, which performs a FourierTransform, resulting in a frequency spectrum.Figure 4. Application of a Pulse Results in the Rotation of the BulkMagnetization by the Flip Angle 8.While it is beyond the intent of this introduction to describe indetail how the various pulse sequences used in the acquisition of thespectra for the thesis affect the magnetization vectors to result in thevarious spectra, the sequences as well as factors which influenced thechoice of the various delay times are briefly discussed below.8i. One dimensional Experimentsa. 1 H SpectrumThe most fundamental pulse experiment, used to acquirea proton spectrum, is simply a pulse followed by the acquisition of anFID (Figure 5). Usually a short delay follows the pulse to allow thetransmitter coil (also the receiver coil) to return to equilibrium. The initialdelay (Di) is termed the relaxation delay and should be of sufficientduration to allow the bulk magnetization vector to reestablish alongthe axis of the Bo field. 8°I FIDD1Figure 5. Pulse Sequence for Acquisition of a 1D 1 H spectrum.?b. NOE Difference SpectraIn essence, the pulse sequence for the acquisition of NOEdifference spectra is merely the sequential acquisition of 1 H spectrawith the added factor of gated decoupling (Figure 6). In gateddecoupling, a second transmitter coil is used to irradiate a specificresonant frequency prior to the application of a pulse (saturation and7 Reference 4. pg. 29IRR 1st ResonanceDec. Chan.8'Obs. Chan.^D I^ IIRR 2nd Resonance8°D 1FIDbuildup of NOE). The pulse is then applied and the decoupler is shut offduring the acquisition of the FID. This is repeated sequentially, irradiatingeach resonant frequency being studied and also an 'off resonance'frequency. Subtraction of the FID for the 'off resonance' spectrum fromthe other FIDs gives the NOE difference spectra.10Figure 6. Pulse Sequence for the Acquisition of NOE Difference Spectra 8c. 13C SpectrumThe standard method of obtaining a 13C spectrum is toirradiate the entire proton region (broad band decoupling) andapply the basic pulse-FID sequence to the carbon nuclei (Figure 7).This has the advantage of eliminating all the heteronuclear (H-C)coupling, resulting in the carbon signals appearing as singlets in thespectrum. It also increases the intensity of protonated carbons due toheteronuclear NOE enhancement.8 Ibid. pg. 168°1113C Observe ID1 FID1H BB DecouplingFigure 7. Pulse Sequence for the Acquisition of a 13C spectrum 9d. The Attached Proton Test (APT) SpectrumThe APT pulse sequence employs gated 1 H broadbanddecoupling to achieve a modulation of the 13 C transversemagnetization due to spin spin (scalar) coupling with protons (Figure 8).Because the decoupler is switched on during the acquisition of the FID,all signals in the spectrum are singlets. When D2 in the pulse sequencebelow is set to 1 /JcH (7 ms), however, the phase of the signals in the 13Cspectrum will be dependent on the number of protons attached to thecarbons, with an even number of attached protons yielding positivesignals and an odd number of attached protons resulting in negativepeaks. The final delays (D3, 1 ms) and pulse are compensation factorsto increase the data acquisition rate and allow flip angles (8) of lessthan 90° to be used.9 Ibid. pg. 32128 9^180°^180°13 C Observe I ID 24- D3 ID3 FIDD1 D 21H BB DecouplingFigure 8. Pulse Sequence for the Acquisition of an APT Spectrum.'°ii. Two-dimensional ExperimentsThe prototype of two-dimensional experiments involvesthree discreet stages (Figure 9): 1) a Preparation time, in which the bulkmagnetization realigns on the z axis through spin lattice relaxation, orNOE enhancement is built up under the influence of the decoupler, 2)Evolution and Mixing periods following the first pulse, in which themagnetization vectors "evolve" under the influence of additionalpulses or other factors and the duration of which is incrementallyincreased (T1), 3) the acquisition of an FID (T2).B.TiPreparation I Evolution& Mixing DetectionFigure 9. Prototype 2D Pulse Sequence.Fourier transformation of the FIDs gives a series of spectra10 Part, S. I. and Shoolery. J. N. J. Magn. Res., 1982, 46, 535.which are modulated under the influence of the second (T1) timevariable. A second Fourier transformation over Ti "perpendicular" to thefirst transformation results in a spectrum as a function of two frequencies.a. 1 H- 1 H COSY and Long Range COSY SpectraHomonuclear c_ofrelation spectroscopy. is the two-dimensional NMR technique most frequently used by organic chemists.The pulse sequence used for the acquisition of a COSY spectrum(Figure 10) is simply two pulses separated by an incremented delay (3gs increments). The second pulse modulates the FIDs in such a waythat after the second Fourier transformation, off diagonal peakscorrelate spins which share a scalar coupling (J). An additional featureof two-dimensional experiments is "phase cycling" (0) of one or moreof the pulses and the detector to compensate for inexactly calibratedpulses. In phase cycling, the phase of the pulses and detector arevaried under regular repeating patterns. 90°^4)60°*1^1IT1D IFigure 10. Pulse Sequence used in the Acquisition of a COSYSpectrum"11 Aue, W. P., Bartholdi, E. and Ernst, R. R. J. Chem. Phys., 1976, 64, 2229.13A variation on the above pulse sequence, which increasesthe intensity of off-diagonal correlations resulting from smaller scalarcoupling constants is used to determine long range (allylic, homoallylicand w) couplings (Figure 11). The additional delay (D2, .08 s) enhancesthe modulating effect of small scalar couplings on the FID. 90°1^602D1^10Ti D2 ^D 2Figure 11. Pulse Sequence used for Acquisition of COSY-LR Spectra. 12b. HMQC SpectraThe 1 H-detected heteronuclear multiple quantumcoherence (HMQC) pulse sequence is designed for the observationof the 1 H- 13C connectivity with direct coupling (Figure 12). Becauseearlier methods of detecting the 1 H- 13C connectivity acquired the FIDin the 13C domain, the HMQC experiment (and the analogous, longrange, HMBC technique) is often referred to as an "inverse detected"experiment. Inverse detection has the advantage of utilizing thegreater sensitivity of the 1 H nucleus, dramatically reducing the amountof sample and/or time required to obtain the connectivity data. Thesecond Fourier transformation results in a two-dimensional plot with 1 Hfrequencies along the x (F2) axis, 13C frequencies along the y (F1) axis,and correlations corresponding to direct connectivity (C-H).12 BOX, A. and Freeman, R. J. Magn. Res., 1981, 44, 542.14I15180*^900^900D i D2 I D2 D3 D2 T_L Ti. D4 D21 .1 1 °2^910;31 x13C Dec.^___ _2^2^BB Dec.Figure 12. Pulse Sequence for the Acquisition of an HMQC SpectrumnTypical delay times used for the acquisition of HMQCspectra in the thesis were: Di 2 s (relaxation delay); D2 3.5 ms (1/2J CH);D3 0.7 s (optimized to eliminate signals due to 1 H bonded to 12C); D43p,s (compensation factor). The normal incrementalization factor of Tiwas 3µs.c. HMBC SpectraThe 1 H-detected multiple bond heteronuclear multiplequantum Qoherence pulse sequence (Figure 13), gives long range (2-4 bond) 1 H- 13C connectivity. After the Fourier transformations aspectrum with 1 H frequencies along the x axis, 13C frequencies alongthe y axis and correlations resulting from long range H-C couplingresults. One bond correlations are screened out instrumentally by usinga low pass J filter.13 Bax, A. and Subramanian, S. J. Magn. Res., 1986, 67, 565.16 90°):^1800x^ I ^I-/H Observe FID90*^90°13C Dec.I Oi 1 02D 1 D2 ID3 I T1-L.900 °Ti 1 3 T22^2Figure 13. Pulse Sequence for the Acquisition of an HMBC Spectrumi 4The initial delays (Di and D2) are the same as those in theHMQC sequence (2 s and 3.5 ms, respectively), while D3 is optimizedfor long range CH J values (D3 = 1/2J CHAR). Correlations to sp 2hybridized carbons were maximized with a D3 value of 60 ms whilecorrelations into sp3 hybridized carbons were strongest when D3 was90-110 ms. The normal Ti incrementalization factor of this experimentwas 2µs.C. A Practical Example: The Structural Elucidation of a Compound.Once a molecular formula has been determined,examination of the one dimensional 1 H and 13C NMR spectra (Figures14 and 15 respectively) is the first stage in the process of structuralelucidation. Information on the functional groups present in themolecule (by diagnostic chemical shifts and coupling patterns) canbe readily garnered from these basic experiments. 15 In the 1 H NMR14 Bax, A. and Summers, M. F. J. Am. Chem. Soc., 1986, 108, 2093.15 Many texts exist which tabulate the relationships between a nucleus' chemical shiftand the electronic environment (functional groups) near the atom. An excellentintroductory level test is: Silverstein, R. M., Bassler, G. C. and Morrill, T. C.Spectromectric Identification of Organic Compounds., 4th edition, John Wiley &Sons, New York, 1981.14 3 2 0OHH 61^ OH4CH313 HH2H1MeFigure 14. 500 MHz 1 H NMR Spectrum (CDCI3/Me2SO-do) of 4-methylcatechol (1)Figure 15. 125 MHz 13C NMR Spectrum (CDC13/Me2S0-do) of 4-methylcatechol (1)00spectrum (CDC13/Me2S0-d6, Figure 14) of 4-methylcatechol (1),resonances appropriate for methyl (8 2.20) and substituted phenolgroups (5 7.38, bs, 1.5H; 5 6.74, d, J = 8 Hz; 5 6.68, d, J = 2 Hz; 5 6.52, dd, J =2, 8 Hz) can be distinguished. The 13C spectrum (CDCI3/Me2SO-d6,Figure 15) contains all seven carbon resonances. Once again,resonances at chemical shifts appropriate for methyl (5 20.5) andsubstituted phenol (5 144.0 (0-0), 141.9 (0-0), 129.6 (C), 120.4 (CH), 116.1(CH) and 115.1 (CH)) groups can be identified. 16The next stage involves identifying the proton spin systemspresent in portions of the molecule. For most of the compounds in thethesis, this entailed the use of the two dimensional 1 H- 1 H COSYexperiment. Figure 16 is a COSY spectrum of 4-methylcatechol (1). Offdiagonal correlations can be seen between the doublet resonancesat 5 6.74 or 6.68 ppm and the double doublet resonance at 5 6.52. Alsoapparent are off diagonal correlations of low intensity between themethyl resonance at 5 2.20 and the resonances at 5 6.68 and 6.52 ppm.These low intensity correlations arise from allylic coupling, and point outone of the strengths of the COSY technique: coupling which is notreadily apparent in the 1 H NMR spectrum, due to line broadening, willgive clear correlations in the two-dimensional technique. When theone-dimensional spectrum is complicated due to overlappingresonances, a COSY spectrum is often the only way to easily discernspin systems. Interpretation of the COSY in Figure 16 gives the following16 Carbons with protons attached can be discerned by the increased intensitycaused by an (H-C) heteronuclear NOE197. 0 E.0 . 0 4.0PPM3.0 2. 0 1 . 01IIItC •I1I1 iIIL1;$:.H3,H15.415H3-Me• H5-Me40IiIIFigure 16.400 MHz 1 H- 1 H COSY Spectrum (CDCI3/Me2SO-d6) of4-methylcatechol (1)MeH5H2HI20OHH 6 1 OHCH313 H41. 02. 0. 0._^5.0_ E. 07.0_ s.0PPMH86.748 6.52 v .4_18 6.688 2.20COSY correlationsspin system.The one-dimensional 13C NMR spectrum (Figure 15)contained four resonances (8 20.5, 115.1, 116.1 and 120.4)corresponding to carbons with attached protons. Which of the protonresonances in Figure 14 correspond to these protons? Assignmentsbetween carbon resonances and corresponding proton resonancesare accomplished through the use of an HMQC experiment. TheHMQC spectrum of 1 (CDC13/Me2S0-d6, Figure 17) contains four peakswhich allow the co-assignment of the proton and carbon resonances.For example: the proton with a resonance at 8 6.52 can be assignedas being attached to the carbon whose resonance is at 120.4 ppm.Assignments for all the 1 H and 130 resonances can be found in Table 1.Connectivity between isolated proton spin systems andthe assignment of quaternary carbon resonances is accomplishedthrough the use of an HMBC spectrum. The HMBC spectrum of 4-methylcatechol (CDCI3/Me2SO-do, Figure 18) provides the remaininginformation required to complete the assignment of all the resonancesin both 1 H and 13C NMR spectra of 1. Correlations can be observed21MeOHH 6 1 OH3 H4CH3H3H1 H6H2 H5- 50-100-150PPEFT V 1 I IFFTTI 111-1 Tl^TVITI■fl I VII■UrTTTTVIITMTTTTT11-17111FTTTITI22PPE^6^4^2Figure 17. 500 MHz HMQC Spectrum (CDCI3/Me2SO-do) of4-methylcotechol (1)H5H6H2 H3H1OH6^1 OHCH314 3 HMe6^4^2Figure 18. 500 MHz HMBC Spectrum (CDCI3/Me2SO-do) of4-methylcatechol (1)23• • • •t• 50••-100ti se•Ir•-150Pimfrom the proton methyl resonance (8 2.20) to the carbon resonancesat 5 116.1, 120.4 and 129.6 ppm (Figure 19). The correlations into theprotonated carbons (8 116.1, 120.4) support the spin system identified inthe COSY spectrum and the third correlation allows us to assign aquaternary carbon resonance (C4). Correlations from the aromaticproton resonances (Figure 20) allow the assignment of the remainingtwo quaternary carbon resonances (see Table 1). Since an HMBCspectrum contains correlations from proton resonances to resonancescorresponding to carbons which are anywhere from two to four bondsdistant, assignments based on data obtained by this technique mustbe checked for internal consistency before being accepted.A final experimental sequence yielding information on thespatial arrangement of the molecular structure (conformation andstereochemistry) is nuclear Overhauser enhancement (NOE)difference spectroscopy. 17 The increased intensity of one nucleusbrought about through dipolar coupling to nearby (saturated) nucleican be measured either through a comparison of integration valuesor, more accurately, by subtraction of a reference spectrum whereinno transition is irradiated (usually referred to as an off-resonancespectrum). When the subtraction method is used, the saturated signalappears as a negative peak, while resonances for the receivingnuclei are usually positive for small molecules in a nonviscous solvent.Resonances pertaining to nuclei which are not dipolarly coupled donot appear in the difference spectrum.17 Sanders, J. K. M. and Mersh, J. D. Prog. NucL Mogn. Reson., 1982, 15, 353.24OHFigure 19. HMBC Correlations from the Methyl 1 H Resonance2526^iu,^H3-Me c=1. .111111P H5-Me40- 60- 80-100..^... ' e==. H5-C3cmc• H3-05H6-C1 ...... cira H3-C1^4==. H&c 1H6-C2 caw, • H3-C2I^I T^f6.8 6.620-1406.45 6.74 C H3Figure 20. HMBC Correlations from the Aromatic 1 H ResonancesAn example of the spectra resulting from theseexperiments can be found in Figure 21. Here, the reference (off-resonance) spectrum is the bottom-most spectrum while thedifference spectra resulting from the various irradiations are presentedabove. As an example, with 4-methylcatechol, irradiation of the methylproton resonance (8 2.20) gives positive peaks in the differencespectrum for the resonances corresponding to H3 (8 6.68) and H5 (86.52). In the thesis, such a result is described as observing anenhancement of the resonances corresponding to H3 and H5, and asummary of all the NOE difference experiments done is presented inthe manner of Figure 22 below and tabulated as in Table 1.OH27^-.-Observed nOeFigure 22. NOE Summary for 1Two problems exist with the NOE difference technique,examples of which can be seen in Figure 21. Because the gateddecoupler has a relatively wide band width, only signals which are welldispersed will give accurate results. The upper most spectrum in Figure21 is the difference spectrum resulting from the irradiation of the H3resonance (8 6.68). However, because the resonance corresponding7. 0 6. 0 5. 0 4 .0PPMz a 2.a 1.028Figure 21. 400 MHz nOe Difference Spectra (CDC13/Me2S0-45) of4-methylcatechol (1)to H6 is at a frequency (8 6.74) which is within the bandwidth of thedecoupler, both resonances are irradiated and any observedenhancements (Me, H1 or H2, H5) cannot be ascribed as resulting fromdipolar coupling to H3. Because of this only well dispersed (46,8 > 0.2ppm) are amenable to this technique. The second source of possibleerror lies in the slight effects the very act of irradiating a sample has onthe chemical shifts and line widths of resonances. Applying energy tothe sample through the decoupler raises the temperature of thesample slightly, which in turn affects the chemical shifts of resonances.While this effect is not usually very large, for exchangeable protons orsharp intense signals such as methyls, it can be enough to result inpeaks which have a significant phase error after the subtraction routine.For example, in Figure 21, irradiation of the H4 proton resonance (8 6.52)results in a dispersion peak corresponding to the phenolic protonsresonance (8 7.38) and dispersive character to the methyl resonance(8 2.20). Such peaks may represent actual proximity data, but cannotbe used as structural evidence and must be discounted.29Table 1. Assignment of NMR Signals (CDC13/Me2S0-c16) for4-methylcatechol (1)C# 5 13C 8 1H COSY* HMBC** NOE***1 141.9 7.38 (OH) H3, H5,H62 144.0 7.38 (OH) H3, H63 116.1 6.68 H5, Me H5, Me4 129.6 H6, Me5 120.4 6.52 H3, H6, Me H3, Me6 115.1 6.74 H5Me 20.5 2.20 H3, H5 H3, H5 H3, H5OHCH3130* Correlations to proton in column 3.** Correlations to the carbon in column 2.*** Proton in column 3 irradiated.II. Terpenoid Metabolites From Three Dorid NudibranchsA. Introduction to the Nudibranchia"The opisthobranch gastropoda are to the Mollusca whatthe orchids are to the angiosperms or the butterflies to the arthropods.Although their approximately 3000 living species cannot rival theprosobranch gastropods (snails) in terms purely of numbers, they showmore variety in their behavior, diets, defensive adaptations, and in theflamboyance of their epidermal color patterns and the extravagance oftheir somatic ornamentation. In short, they are among the mostinteresting and visually exciting invertebrate animals." 18So begins T. E. Thompson's two volume monograph on thebiology of British opisthobranchs. While his detailed study deals with allthe opisthobranchia, the statement is also valid for the largest subclass,nudibranchia.The nudibranchia are a large and phylogeneticallyconfusing order. Thompson proposes that they may be polyphyletic(arising from several ancestral lines through convergent evolution) inorigin and therefore the true relationships among the thousands ofspecies may never be fully sorted out. 19Habits and diets of the nudibranchs vary widely. They seemto have adapted to fill most carnivorous niches in the oceans' ecology.Some of these adaptations can be extreme. The planktonic Glaucusatlanticus lives circumtropically in the upper surface waters, staying18 Thompson T. E. The Biology of Opisthobronch Molluscs. The Ray Society, London,1976: page 3.19 Ibid.31afloat with gulps of air into a specialized stomach, feeding on Physallia(Portuguese-man-o-war) and able to retain and transport the mostpowerful and painful nematocysts from its food source, intact to itscerata. 20 Melibe leonida, a dendronotid nudibranch of the EasternPacific, catches zooplankton through the use of an enlarged and highlyspecialized oral veil. Other nudibranchs feed on a variety of ascidians,cnidarians, bryozoans, molluscs and sponges.Nudibranchs are grouped into four general subordersbased on body plan and organ distribution. Figure 22 shows sometypical nudibranchs. Aeolid nudibranchs, such as the above mentionedGlaucus sp. have highly developed cerata which contain intactnematocysts isolated from their cnidarian prey. Dendronotidnudibranchs have developed ceras which contain tributaries of thedigestive gland. Dorids, the largest suborder, lack the above processesand usually have a spiculose mantle and a gill plume circlet surroundingthe postero-dorsal anal opening. Arminacean nudibranchs have anonspiculose mantle and no gill plume circlet. The phylogeneticclassification of the Nudibranchia is outlined in Figure 23.Loss of the shell/operculum system of passive defense inopisthobranch molluscs has necessitated the adoption of otherdefensive strategies. These are usually classified into three categories;behavioral, morphological and chemical. 21 Behavioral adaptationsinclude hiding and escape activities, such as swimming. Morphologicaldefenses include the use of nematocysts, spiculose mantles, cryptic20 Thompson, T. E. and Bennett, I. Science, 1969, 166, 1532.21 Karuso, P. "Chemical Ecology of the Nudibranchs ", in Bioorganic Marine Chemistry,P. J. Scheuer, Ed., Springer Verlag, Berlin, 1987: Vol. 1,.page 33.3233A typical Aeolid Nudibranch^A typical Dendronotid NudibranchA typical Dorid Nudibranch A typical Arminacean NudibranchFigure 23. Typical Nudibranchs(From Thompson)Aplaysiamorpha^PleurobranchomorphaAcochlidlacea^Bullomorpha^Pyramidellomorpha^ThecosomataSacoglassa^GymnosomataORDERPHYLUM^ MolluscaCLASS^ GastropodaSUBCLASS^ OpisthobranchiaNudibranchiaSUBORDER^Aeolidacea^Arminacea^Doridacea^DendronotaceaFigure 24. Phylogenetic Classification of Opisthobranchs(from Thompson)coloration and autonomy of certain body parts. Chemical defenses arebased on the secretion, from specialized dorsal glands, of distasteful ortoxic compounds obtained from their diets or synthesized de novo .Often a species of opisthobranch will employ more thanone defensive strategy. The completely mimetic sacoglossan Oxynoeolivacea, when molested, secretes a white fluid containing theicthyotoxic metabolite 2 and automizes its tail while the rest of theanimal makes its escape. 22 With one sacoglossan employing so manydefensive mechanisms (cryptis, autonomy, swimming escape andchemical secretion) it is difficult to point to a particular defensemechanism for any opisthobranch. One thing is clear, however, of the24 species of opisthobranchs tested by Thompson,23 all were rejected asfood by fish in aquarium studies, even those which seem employ classicbehavioral defensive strategies.35CHO2With the exception of the Aeolids, which contain potentnematocysts as a primary means of defense, Thompson's results, as wellas those of Gunthorpe and Cameron on 21 species of Australiandorids,24 would seem to implicate an important role for chemicalsecretions in deterring predation. Many chemical studies on22 Cimino, G. and Sodano, G. Chem. Scr. 1989, 29, 391.23 Thompson, T. E. J. mar. biol. Ass. U. K. 1960, 39, 123.24 Gunthorpe, L. and Cameron, A. M. Mor. Biol., 1987, 94, 39.nudibranchs have been conducted to discover the structures, originsand roles of these allomones. 25 Below are results from three of the mostinteresting of these studies.i. PhvIlidi• varicosa and an isocvano icthvotoxin. 26The first reported isolation of a defensive allomone from anudibranch was that of 9-isocyanopupukeane (3) isolated from themucus secretions of Phyllidia varicosa.26 It had been demonstratedearlier that Phyllidia spp. killed other organisms when the nudibranchswere placed in an occupied aquarium. The animal's mucus secretionalone had a similar effect, on both crustaceans and fish. Scheuer'sgroup in Hawaii isolated the active agent and determined its structure,through X-ray diffraction analysis, as 9-isocyanopupukeane (3). They alsotraced the compound to the sponge Hymeniacidon sp. on whichPhyllidia varicosa feeds, demonstrating that nudibranchs are able tosequester toxic substances from their diets and utilize them for their owndefensive strategy.25 For examples see: a) Williams, D. E. and Andersen, R. J. Can. J. Chem. 1987. 65,2244. b) Gustafson, k. and Andersen, R. J. Tetrahedron, 1985, 41, 1101. c) Lindquist, N.and Fenical, W. Experientia, 1991, 47, 504. d) de Petrocellis, L., Di Marzo, V., Arco, B.,Gavagnin, M., Minei, R. and Cimino, G. Comp. Biochem. Physiol. , 1991, 100C, 603.26 Shulte, G. R. and Scheuer, P. J. Tetrahedron, 1982, 38, 1857. and references therein.36ii. PolygodialvOne of the best studied nudibranch metabolites, polygodial(4), has been found in several species of Dendrodoris . 27a Masked formsof polygodial (the drimane esters (5), and olepupuane (6)) , have alsobeen found in Dendrodoris and Doriopsilla species.27Lb374^5 R i = RCO, R2 = H6 R i = Ac, R2 = Ac0Polygodial was first isolated as the compound responsiblefor the "hot" taste in several species of plants. 22.27 Biosynthetic studies onDendrodoris limbata have demonstrated the ability of the dorid tosynthesize these compounds de novo,27c indicating similarities inmolluscan and terrestrial plant biochemical pathways.The portion of the molecule responsible for the diversebiological effects has been shown to be the dialdehyde functionality,which reacts with amines. 27d Although compounds 4 and 5 aresynthesized by Dendrodoris limbata, polygodial (4) is toxic when injectedinto the mollusc's hepatopancreas, and is strictly localized along theborder of the dorid's mantle. The drimane esters (5), however, werelocalized in the hermaphroditic gland and egg masses and are27 a) Okuda, R. K., Scheuer, P. J., Hochlowski, J. E., Walker, R. P. and Faulkner, D. J. J.Org. Chem., 1983, 48, 1866, and references therein. b) Cimino, G., de Stefano, S., deRosa, S., Sodano, G. and Villani, G. Bull. Soc. Chim. Belg., 1980, 89, 1069. c) Cimino,G., de Rosa, S., de Stefano, S. and Sodano, G. Experientio, 1985, 41, 1335. d) Avila,C., Cimino, G., Crispino, A. and Spinella, A. Experientio, 1991, 47, 306.speculated to possess a different biological role, perhaps indevelopment. 27diii. Roboastra and Tambie nudibranchs -where defensive strategy failst28The ability to tolerate the noxious chemicals in their preyand utilize these substances as defensive metabolites works well formany species of nudibranchs. But what if the animal's chief predatoralso has this ability?Nembrothid nudibranchs in the genus Tambje, sequestertambjamines (7- 10) from the bryozoan Sessibugula translucens, on whichthey feed, and release them when harassed. 28 The predatorynudibranch Roboastra tigris, which feeds on the smaller nembrothid/OMeI....-^.X^NH NHR7X=H Y=H R=H8X=Br Y=H R=H9X=H Y=H R=Bu10X=H Y=Br R=Bunudibranchs, is able to detect trace amounts of tambjamines in theslime trail of Tambje spp., and use them to track its preferred prey. Afterdevouring the smaller nudibranch, R. tigris sequesters the tambjaminesfor its own defense. However, when confronted with highconcentrations of compounds 7- 10 which have been excreted by T.abdere, R. tigris will break off its attack, demonstrating the defensive28 Carte, B. and Faulkner, D. J. J. Org. Chem., 1983, 48, 2314.38/Yproperties of compounds 7-10. While the tambjamines seem to provideprotection for Tambje species from most predators, anothernudibranch, R. tigris, has developed the ability to broach this defensivestrategy.The following sections of this thesis detail our investigationsinto the chemical constituents found in three species of nudibranchscollected off the coasts of Sri Lanka, British Columbia and Norway.39B. The Skin Chemistry of Chromodoris geminusi. Introduction to the ChromodoridsNudibranchs of the genus Chromodoris (Suborder: Doridacae,Superfamily: Eudoridoidea, Family: Chromodorididae) are brightlycolored (non-cryptic) gastropods found throughout the tropical andsemitropical oceans of the world. Such flamboyantly coloredinvertebrates should be easily recognized prey species for marinecarnivores such as crabs and fish, but are rarely, if ever, observed beingeaten. In the absence of obvious biological deterrents to predation(escape behavior, nematocysts or cryptic coloration) it has beensuggested that the nudibranchs are employing a defensive strategybased upon a chemical arsenal. Many investigations into the skinchemistry of members of this genus have been carried out in order todetermine the plausibility of this hypothesis. 29 - 3229 a) Schulte, G. , Scheuer, P. J. and McConnell, 0. J. Hely. Chim. Acta. 1980, 63, 2159.b) Hochlowski, J. E. and Faulkner, D. J. Tetrahedron Left. 1981, 22, 271. c) Schulte, G.and Scheuer, P. J. Tetrahedron, 1982, 38, 1857. d) Carte, B. , Keman, M. R. , Barrabee,E. B. , Faulkner, D. J. , Matsumoto, G. K. and Clardy, J. J. Org. Chem. 1986, 51, 3528. e)Kakou, Y., Crews, P. and Bakus, G. J. Nat. Prod., 1987, 50, 482.30 a)Hochlowski, J. E. , Faulkner, D. J. , Matsumoto, G. K. and Clardy, J. J. Org. Chem.1983, 48, 1142. b) Bobzin, S. C. and Faulkner, D. J. J. Org. Chem. 1989, 54, 3902.c)Molinski, T. F. and Faulkner, D. J. J. Org. Chem. 1986, 51, 2601. d) Molinski, T. F. ,Faulkner, D. J. , Cun-heng, H. , Van Duyne, G. D. and Clardy, J. J. Org. Chem. 1986,51, 4564. e) Cimino, G. , Crispino, A. , Govagnin, M. and Sodano, G. J. Nat. Prod.1990, 53, 102. f) Gavagnin, M., Vardaro, R. R., Avila, C., Cimino, G. and Ortea, J. J.Nat. Prod., 1992, 55, 368.g) Dumdei, E. J. , de Silva, E. D. , Andersen, R. J. ,Choudhary, M. I. and Clardy, J. J. Am. Chem. Soc. , 1989, 111, 2712. h) Morris , S. A. ,de Silva, E. D. and Andersen, R. J. Can. J. Chem, 1991, 69, 768. i) de Silva, E. D. ,Morris, S. A. , Miao, S. , Dumdei, E. and Andersen, R. J. J. Nat. Prod. 1991, 54, 993.31 a) Hochlowski, J. E. , Faulkner, D. J. , Bass, L. S. and Clardy, J. J. Org. Chem., 1983, 48,1738. b) Terem, B. and Scheuer, P. J. Tetrahedron, 1986, 42, 4409. c) Keman, M. K. ,Barrabee, E. B. and Faulkner D. J. Comp. Biochem. Physiot 1988, 898, 275.32 a) Corley, D. G. , Herb, R. , Moore, R. E. , Scheuer, P. J. and Paul, V. J. J. Org. Chem.1988, 53, 3644. b) Okuda, R. K. and Scheuer, P. J. Experientio, 1985, 41, 1355.40The wide variety of chemicals which have been isolated fromvarious species of chromodorids are summarized in the following pages.Many of these metabolites can be linked directly to a sponge sourceupon which the animal feeds, while all others show similarities to knownsponge metabolites. Considering the wide variety of structural classesand the link to poriferan metabolites, it seems likely that chromodoridnudibranchs have a general mechanism for isolating potentialallomones from their prey sponges. Differences in composition of thesemetabolites between species or between geographic sites couldrepresent either an association between a particular food source andthe individual species, or a different array of potential food spongesavailable at the site.There are at present four classes of metabolites isolated fromvarious chromodorid nudibranchs. Sesquiterpenes 11-23; 29 diterpenesand modified diterpenes 24-45; 30 sesterterpenes and methylatedsesterterpenes 46-57; 31 and the macrolides laulimalide (58),isolaulimalide (59) and latrunculin A (60) . 29e ,32SesquiterpenesThe antifeedant tricyclic furans nakafuran 8 (11) and nakafuran 9(12), isolated from C. maridadilus, were traced to the mollusc's poriferanprey, Dysidea frogilis. They were also present in a closely relatednudibranch, Hypselodoris godeffroyana, which was collected while alsofeeding on the sponge. 29aMarislin (13a), a sesquiterpenoid from C. marislae, is closelyrelated to pleraplysillin-2 (13b), a metabolite from the sponge41R=^a\b013a, 13b14a, MbOW"CHO 11 12Chromodoris marldadilus 29.^ Chromodoris albonotato 290Chromodoris marlslae 29bPleraplysilla spinifera. 29b The nudibranch was also found to contain thelinear ketone metabolites 14a and 14b.Chromodoris albonotato contained pu'ulenal (15) 29c, a maskedform of the known icthyotoxin polygodial (4) found in Dendrodorisspp.. 27Chromodoris funerea was found to contain furodysin (19),furodysinin (20), and related metabolites. 29d Furodysin and furodysininare known metabolites of the sponge Dysidea herbacea, while most ofthe other compounds present in C. funerea at this locale can beexplained as singlet oxygen oxidation products of furodysin andfurodysinin. It is of interest to note that identical isolation procedures forboth the nudibranch and its prey sponge resulted in the isolation of theoxidation products only in the nudibranch extracts. This result has beenexplained by the suggestion that the nudibranch is responsible for thechemical modification of dietary furanosesquiterpenoids.Dendrolasin (23), was isolated from both the nudibranch C. lochiand its prey sponge Spongia mycofijiensis. 29eDiterpenesDiterpenoid metabolites are by far the largest class of compoundsisolated from chromodorids. They are usually formally related to ahypothetical "spongian" precursor and their structures are oftenexplained as cationic or oxidative rearrangements of such a precursor. 33Many sponges in the orders Dictyoceratida and Dedroceratida are33 For an excellent review of all the carbon skeletons formally derived from a spongianprecursor as well as the proposed biogenetic pathways see: Tischler, M. PhD thesisUniversity of British Columbia, 1990.4320 21OR^ OMeOOH16 R = Me17 R=HsH 1816-22Chromodoris funerea 29dChromodoris loch! 29ekknown to produce spongian and rearranged spongian metabolites andprobably serve as the dietary sources for these compounds inchromodorids.M45010SpongianChromodoris norrisi from the Gulf of California was found tocontain the metabolites norrisolide (24), macfarlandin E (25), shahaminC (26) and polyrhaphin A (27) all of which were also isolated from thesponge Aplysilla polyrhaphis.300Another Californian chromodorid, C. macfarlandi, collected atScripps Canyon in California, contained the diterpenoid macfarlandinsA-E (25, 28-31). 30c4Macfarlandin A (28), luteorosin (32) and the 12-epi-aplysillincompounds (33-34) were isolated from C. luteorosea collected in thebay of Taranto ,30e while 28, 32, polyraphin C (35), norrisolide (24) andcheonaplysillin C (36) were isolated from specimens collected in theCantabrian Sea, North Spain. 30f Such differences in composition ofallomones within this species may reflect differences in dietary spongepopulations at the two sites.34 For examples see: a) Kcalauskas, R., Murphy, P. T., Wells, R. J. and Daly, J. J.Tetrohedron Lett., 1979, 903, b) Karuso, P., Sketton, B. W., Taylor, W. C. and White, A. H.Aust. J. Chem., 1984, 37, 1081. c) Carmely, S., Cojocaru, M., Loya, Y. and Kashman,Y. J. Org. Chem., 1988, 53, 4801.Ac0 OAcI 0.„.26AcOChromodoris norrisi 30a,e31 25OAc28 = H,^R2 = OAc^3029 R i = OAc, R2 = H0A„0 c^AcOHChromodoris macfarlandi mbxAcO 32Chromodoris luteorosea 3".'The chromodorolides (37 & 38), isolated from Chromodoris cavae,are the only reported compounds with the interesting chromodoraneskeleton. The structure of chromodorolide A (37) was determinedthrough X-ray diffraction analysis, 30g while chromodorolide B (38) is thelactone variant of 37. 30hSeveral chromodorid nudibranchs collected in Sri Lankan coastalwaters contained a rich array of diterpenoid metabolites. 30 iChromodoris gleneii contained the metabolites dendrillolide A (39), 12-desacetoxyshahamin C (40) and shahamin K (41). Chromodorisannulata contained shahamin F (45), while aplyroseol 2 (42) and thespongian lactones 43 and 44 were found in C. inopinata. These sevenmetabolites as well as the chromodorolides, all from nudibranchscollected in Sri Lanka are presumed to derive from dietary sources,although no extensive studies on poriferans from this location have beendone.SesterterpenesThree species of chromodorids have been found to containmetabolites in the relatively rare sesterterpenoid class. 31 Chromodorissedna contains C-20 methylated scalarins (compounds 46-50), 31 a whileChromodoris youngbleuthi contains the scalaradials (51-53). 31b Sca la rinsare known to be metabolites of Cacospongia sponges,35 though Teremand Scheuer also report them from Spongia oceania. 3 lb35 for examples see: a) Cafieri, F., de Napoli, L., Fafforuso, E., Santacroce, C. and Sica,D. Gozz. Chim. Ital. 1977, 107, 71. b) Yasucla, F. and Tada, H. Experientio, 1981, 37,110. and references therin.490Chromodoris cavae 30d.it40 R = H41 R = OAcChromodoris glenell 30h84542 43 R = H44 R = OAcChromodoris Inopinata 30,Chromodoris annulata 3°hChromodoris sedna 31a OH CHOCHO CHOCHOCHO53Chromodoris youngbleuthi "bChromodoris funerea, collected within an isolated marine lake ofPalau, was found to contain the sesterterpenes 54-57 31c which differedsignificantly from the sesquiterpene metabolites isolated in the previousstudy. 29d The marine lakes of Palau offer researchers a "naturallaboratory" as they contain ecologys differing from that in thesurrounding ocean, with which the lakes have limited exchange. Theisolation of sesquiterpenoids from one population of C. funerea andsesterterpenoids from another suggests that the nudibranch is able todistinguish allomones from other dietary metabolites and is notdependent on a specific sponge to provide these defensive chemicals.MacrolidesThe final and perhaps most interesting class of compoundsisolated from chromodorid nudibranchs are the three cytotoxicmacrolides 58-60 isolated from Chromodoris lochi and its preyspecies. 29e,32a Latrunculin A (60) has also been isolated from C.elizabethina 32b and several sponges, 36 while the laulimalides have beenisolated from a sponge in the genus Hyatella. 32a While these sponges areundoubtedly the source of the metabolites, it is surprising that anudibranch predator has developed a resistance to such potenttoxins. 3736 see reference 12e as well as Kashman, Y., Groweiss, A. and Shmueli, U. TetrahedronLeff., 1980, 21, 3629.37 The related nudibranch, Glossodoris quadricolor, also has been shown to possessresistance to this class of metabolites and sequesters latrunculin B from Latrunculomagnifica. : Mebs, D. J. Chem. Ecol. 1985, 11, 713.54X55 X = 1 or 3 oxo57OHChromodoris funerea 3I0g/ /00HOHOOHO%rc4 .758OHCD,HN^ S° 6058-60 Chromodoris loch! 29e' 32160 Chromodorls ellsabethina 32bgThe hypothesis that these varied chemicals are sequestered bythe chromodorids for defensive reasons is strengthened by results ofantipredation or toxicity assays on many of these metabolites. Amongthe sesquiterpenoids, the nakafurans (11-12), have been shown to actas antifeedants as have furodysinin (20) and related metabolites 16-18.The diterpenoid polyrhaphin C (26) has antifeedant properties vs theGulf of California rainbow wrasse, Thalossoma lucasanum , while all ofthe metabolites from C. luteorosea at both locations are icthyotoxic.The scalaradials (51-53) have the same functionality as polygodial (4),and may act in a similar fashion, and the three macrolides 58-60, foundin extracts of C. lochi, are very cytotoxic.5734ii. Three Spongian Diterpenoids from the Nudibranch Chromodorisgeminus (Rudman, 1987) Collected off the Coast of Sri Lanka.As part of an ongoing study into metabolites from Sri Lankanchromodorids30g.h.i we have isolated three diterpenoids (34, 61 and 62)from the nudibranch Chromodoris geminus (Rudman, 1987). 38a. Extraction and Purification 39A collection of twelve C. geminus was made in the coastalwaters of Sri Lanka in January 1988. Live animals were immediatelyimmersed in Me0H/CH2C12 (1/1) and stored in a freezer. At the time of38 See Figure 25 for the phylogenetic taxonomy of C. geminus.39 The isolation and purification of compounds 34, 61 and 62 was accomplished by Dr.E. Dilip de Silva, a visiting professor working in our laboratory.58SUBORDER^ DoridaceaSUPERFAMILY^Anadorldoldea^Eudoridoidea^Porodorldoldea^GnathodoridoideaHexabranchldae^Aldisidae^DorIdldae^Discodorldidae^KentrodoridideaFAMILY^CadlInIdae^Homolodorldidae^Baptodorldidae^AsteronotidaeRostangldae^Actlnocyclidae^Chromodorididae^Platydorididae^ArchidorididaeGENUS^Casella^Hypselodoris^Chromodoris^Ceratosoma^MiamiraSPECIES C. geminusFigure 25. The phylogenetic classification of C. geminus(from Thompson)workup, the organic solvents were decanted and evaporated in vacuoto give an aqueous residue that was partitioned between H2O andCH2Cl2. Fractionation of the organic soluble materials through repeatedsilica gel flash chromatographies (hexane/ EtOAc gradients) andnormal phase HPLC (hexane/ EtOAc: 1/1) yielded 23 mg of 12f3,15a,16a-triacetoxyspongian (34), 10 mg of 6a,15a,16a-triacetoxyspongian (61)and 34 mg of 6a,12f3,15a,16a-tetracetoxyspongian (62) in order ofelution.b. Results and Discussion1213,15a,16a-triacetoxyspongian (34):OAcHOAc...,:1213,15a,1 ba-triacetoxyspongian (34) was isolated as anoptically active colorless glass (23 mg; 1.9 mg/animal, (a)D +8.4, CHCI3)which failed to give a molecular ion peak in either high resolutionelectron impact mass spectrometry (HREIMS) or desorption chemicalionization mass spectrometry (DCIMS). The highest mass observed ineither method was at m/z 404.2568 amu, corresponding to a molecularformula C24H3605 (A -0.5 mmu). Examination of the 1 H (Figure 26, three60acetate singlet resonances; 8 2.04, 2.03, 1.95) and 13C NMR spectra(Figure 27, twenty-six signals; three acetate carbonyl resonances: 8169.9, 169.8, 169.7) for 34, enabled this peak to be interpreted ascorresponding to the loss of one equivalent of acetic acid from themolecular ion. The molecular formula thus established (C26H4007) hasan unsaturation index of seven. Three of these sites of unsaturation couldbe accounted for as acetate carbonyls, and the lack of any signals inthe 13C NMR spectra at chemical shifts appropriate for olefinic carbons(8 105-140) indicated 34 was a tetracyclic diterpenoid.Closer examination of the 1 H NMR spectra of 34 in CDCI3(Figure 26), indicated a triacetoxy diterpenoid with a strongresemblance to the spongian derived diterpenes isolated in ourlaboratory from other chromodorids.30g.h.i The characteristic features of:two acetal 1 H NMR resonances at 8 6.34 (d, J = 7.6 Hz, H16) and 8 6.04 (s,H15); a deshielded methine resonance at a chemical shift (8 5.05, m,H12) appropriate for attachment to an acetoxy substituent; anothermethine signal at 8 3.07 (q, J = 7.6 Hz, H13); three singlet resonancesappropriate for acetate methyls (8 2.04, 2.03 and 1.95), and four upfieldmethyl singlet resonances (8 0.97, 0.84, 0.83 and 0.78) could easily bedistinguished. In particular, the four upfield methyl resonances indicatedthat 34 likely possessed an unrearranged spongian skeleton.The 13C NMR spectrum (CDCI3, Figure 27) displayed twenty-six signals appropriate for a triacetoxy diterpenoid. Resonancesattributable to the carbons of three acetates (8 169.9, 169.8, 169.7, 21.4,21.1 and 20.8), two acetal carbons (8 99.2 and 98.4) and a carbinolmethine carbon (8 71.1) could easily be assigned. As well, resonancesappropriate for four other methine (8 59.5, 56.6, 55.0 and 41.9), six61Figure 26. 500 MHz 1 H NMR Spectrum (CDCI3) of 120,1 5a, 1 6a-triacetoxyspongian (34)Figure 27. 125 MHz 13C NMR Spectrum (CDCI3) of 1213,15a,16a-triacetoxyspongion (34)8 1.58 1.78H 80.80 12, H8196AOAc OAcH 8 3.07^H 86.348 5.05 Hmethylene (8 42.2, 41.9, 40.0, 23.4, 18.4 and 18.0), four methyl (8 33.3,21.3, 17.1 and 16.6) and three quaternary carbons (8 37.4, 34.7 and 33.2)could be distinguished.1 H- 1 H COSY (Figure 28) correlations could be observedbetween the methine resonance at 8 3.07 (H 13) and three othermethine proton resonances at 8 6.34 (H16), 5.05 (H 12) and 1.96 ppm (d,J = 7.5 Hz, H14). The acetoxy methine (8 5.05, H12) was further coupledinto two protons at 8 1.78 (m, H11 a) and 8 1.5 (H1 10) both of whichshowed coupling into an unresolved methine at 8 0.80 (H9), completingthe spin system for the partial structure A below.The above partial structure strongly resembled the C and Dring systems of aplysillin (63), a metabolite isolated from the spongeAplysilla rosea, and solved by X-ray diffraction analysis. 34a In fact, 63possessed exactly the same features characteristic of 34 (e. g. four rings,three acetates, two acetals and four methyls attached to quaternarycarbons). The differences between the spectroscopic data reported for63 and the data obtained for 34 implied a different stereochemical643.04. 05. 0E . 07. 0P PM H1565H16r^, & H110-H121- 8'lar*. iierIra go1:16 4,,, -viidre 4 jH9-1-H9+— H a- H13-H14 CO 1f. ' •^'0^11136 s• 0 80 I H12-H13 1 a0• c• H13-H166 0^5 0^4 0^3 0^2 0^1 0PPMFigure 28. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of1213,15a,16a-triacetoxyspongian (34)relationship between the chiral centers, however, and suggested that34 was the C12 epimer of aplysillin.63It had been noticed in our earlier work that resonances forthe a axial protons of trans fused substituted decalins shiftedsignificantly upfield in deuterated benzene. Therefore, we redisolvedcompound 34 in C6D6 in an attempt to help delineate the remainingspin systems on the A and B rings as well as the stereochemistry of 34.The 1 H NMR spectrum (C6D6, Figure 29) again shows twoacetal resonances at 8 6.73 (d, J = 7.5 Hz, H16) and 8 6.26 (s, H15); anacetoxy methine resonance at 8 5.08 (m, H12); the H13 methineresonance at 8 3.21 (q, J = 7.5 Hz, H13); three acetate methyl singletresonances (8 1.71, 1.70 and 1.66), and four upfield singlet resonancescorresponding to methyl groups (8 0.82, 0.72, 0.71 and 0.57). Theresonance for the H9 methine becomes well resolved at 8 0.32 (bd, J =12 Hz), and another resonance at 8 0.5 integrating for two protons (Hla,H5) emerges from the hydrocarbon envelope.In the 13C NMR spectrum recorded in C6D6 (Figure 30)twenty-six resonances are clearly resolved. An APT experiment allowedthe resonances to be assigned as corresponding to the carbons of three66Mel?Me19Me18Me20H16H15H13H12HlaH5H14Figure 29. 500 MHz 1 H NMR Spectrum (C6D6) of 1213,15a,16a-triacetoxyspongian (34)OAcH ?'160,15.OAcOAc2019^"18 34tPASI***C3C1 3C C18C9C14C12^C5C15 C16volowovii4401.0010441^III^1^1^T^IIIIIIIIIII111111111IIIIIIIWTIIIIIIIIIIIIIITIIIIIIIIIIIIIIIIIti180 160 140 120^100^ppm^80^60^40^20Figure 30. 75 MHz 13C/APT NMR Spectra (Copts) of 1 213,15a,16a-triacetoxyspongian (34)85.08H8 1.258 1.60 HOAc OAc==H83.21 : H 8 6.730H 80.32^H81.62 OAcH8 6.26acetates (8 169.3, 169.1, 169.0, 20.9, 20.7, 20.4), two acetals (8 99.6, 98.7),five methines (8 71.1, 59.6, 56.4, 54.5, 42.3), six methylenes (8 42.2, 42.1,39.9, 23.6, 18.2, 18.0) four methyls (8 33.5, 21.5, 16.9, 16.6) and threequaternary carbons (8 37.4, 34.7, 33.3).Once again, 1 H- 1 H COSY (C6D6, Figure 31) correlationscould be observed between the resonance at 8 3.21 (H13) and thethree methine resonances at 8 6.73 (H 16), 5.08 (H 12) and 1.62 ppm (d, J= 7.6 Hz, H14). H12 (8 5.08) was further coupled into two protons withresonances at 8 1.60 (dd, J = 6.5, 13 Hz, H11 a) and 8 1.25 (H1113) whichwere coupled into the H9 proton resonance at 8 0.32, yielding the samepartial structure A. Further comparison of the spectroscopic data for 34to the data reported for 63 indicated that partial structure A could beplaced on the C and D rings of a spongian skeleton (Figure 32). Inaddition, the coupling pattern of the two acetal protons of 34 (H 15, s;H16, d, J = 7.5 Hz) was similar to that reported for aplysillin and gave astrong indication that the stereochemistry at C15 and C16 was the samein the two compounds.69Figure 32. C and D Rings of 34H97.0^6.0^5.0^4.0 PPM 3.0^2.0 1.0^0.0• , aagigaal kL^. 1 , ^/?:,,i,^ 'it°H13-H14,.,)e0 H9-HIo0H11(3-H12Hlla-H12r---0 s 0 0a a H12-H13 0 eO.• H13-H16Figure 31. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) of12I3,15a,16a-triocetoxyspongian (34)70- 0.0- 2.0- 3.0- 4.0- 5.06.0- 7.0PPMThe spin systems on the A and B rings were establishedthrough careful examination of the COSY spectra in conjunction with aheteronuclear multiple quantum coherence (HMQC) spectrum (Figure33). Starting from the upfield (8 0.5) a axial protons (Hla, H5), contiguousspin systems could be followed in the COSY spectra which alsomatched, in the HMQC spectrum, the pattern of 130 chemical shiftsfound on substituted (trans) decalin systems (Table 2).Protons not showing correlations in the COSY spectrum of 34(methyls and H15) as well as the stereochemistry at the C12, C15 andC16 positions were assigned through the use of a series of nuclearOverhauser enhancement (NOE) difference experiments (Figure 34).Irradiation of the H 12 resonance (8 5.08) resulted in the enhancement ofresonances at 8 3.21 (H13), 1.60 (H11a), 1.62 (H14) and 0.32 ppm (H9).Irradiation at 8 0.32 (H9) enhanced resonances corresponding to Hlla,H12 and H14. Irradiation at 8 3.21 (H13) gave enhancement of thesignals corresponding to H12 and H16 (8 6.73). Also observed were NOEsfrom the H16 proton resonance to resonances corresponding to H13and Me17 (8 0.72) and from the H15 resonance (8 6.26) to the Me17signal. The enhancements observed between H9, H12 and H14indicated a diaxial relationship between the three methines andtherefore the acetate at C12 could be assigned in the f3 orientation.Likewise, observed NOEs from both acetal resonances (8 6.24 and 6.69)to the methyl at 8 0.72 indicated that the acetates at C15 and C16 werein the a configuration. Methyl 18 was assigned by its distinctive carbonchemical shill (8 33.5: indicative of an equatorial position) while methyls71H7ti Pc H113 H2a^i Me18 H9 H5H14^Me17 Me20 Hla1436pp. H13^3Hop Me19728IYr el lfrI•ilt11 14 ' +"!I/CllC21C.611?i,ci401• ♦.^.C.1tIs, 3°Clcit4oC3C'01 4, c:«•04^44.^.i^...^•. C5C1460PPIC20C73Ac1Figure 33. 500 MHz HMQC Spectrum (C6D6) of12f3,15a,16a-triacetoxyspongian (34)Me19 and Me20 were assigned by comparison of their 1 H and 13Cchemical shifts with those reported for other metabolites in this class. 30iFigure 34. NOE Summary for 34Subsequent to the structural elucidation of 34 from C.geminus, but prior to publication, another laboratory reported itsisolation from the Mediterranean nudibranch C. luteorosea. 30e73Table 2. Assignment of NMR Signals (C6D6) for120,15a,16a-triacetoxyspongian (34)C# 8 13C 8 1H COSY* NOE**1a 39.9 0.54 H113, H213113 1.45 H la, H2a2a 18.0 1.4 H113, H211, H3a213 1.6 H la, H2a. H3133a 42.2 1.05 H213, H313313 1.3 H2a, H3a4 33.3a5 54.5 0.50 H6a, H6136a 18.2 1.35 H5, H613, H7a, H7I3613 1.00 H5, H6a, H7a, H7137a 42.1 0.8 H6a, HO, H7137f 1.65 H6a, HO, H7a8 37.4a9 56.4 0.32 H11a,H1111 Hlla, H12, H1410 34.7alla 23.6 1.60 H9, H110,H121 113 1.25 H9, H1 la, H1212 71.1 5.08 H11a,H1113,H13 H9, H1 la, H13,H1413 42.3 3.21 H12, H14, H16 H12, H1614 59.6 1.62 H1315 99.6 6.26 Me1716 98.7 6.73 H13 H13, Me 1717 21.5 0.7218 33.5 0.8219 16.9 0.7120 16.6 0.57Ac 20.9, 20.7, 20.4169.3, 169.1,169.01.71, 1.70,1.66* Correlations to proton in column 3.** Proton In column 3 Irradiated.a May be Interchanged.746a,15a,16a-triacetoxyspongian (61):Compound 61 was isolated as an optically active, colorlessglass (10 mg, 0.8 mg/animal; (a)D +3.0, CHCI3) which also failed to givea molecular ion peak in either the HREIMS or DCIMS spectra. The highestmass detected, at m/z 404.2537 amu, corresponded to a molecularformula of C24H3605 (A -2.5 mmu). As with 34, through examination ofthe 1 H (Figure 35, three acetate methyls: 8 2.08, 2.03, 2.02) and 13C NMRspectra (Figure 36, twenty six resonances; three acetate carbonyls: S170.5, 170.1, 169.8) of 61, this peak could be interpreted as the loss ofone equivalent of acetic acid from the molecular ion. Compound 61therefore has the same molecular formula (C261-14007) and unsaturationindex as 34.The 1 H NMR spectrum (CDCI3, Figure 35) again displayedtwo acetal methine resonances (5 6.06, d, J = 7.3 Hz, H16 and 8 6.05, s,H15), a downfield methine resonance at a chemical shift appropriatefor an acetoxy substituent (8 5.21, dt, J = 3.5, 11.0 Hz, F16) and anothermethine resonance at 8 2.57 (q, J = 7.4 Hz, H 13). Three acetate methylresonances (8 2.08, 2.03 and 2.02) and four upfield methyl singletresonances (8 1.08, 1.00, 0.94 and 0.85), were also apparent, indicating75Figure 35. 500 MHz 1 H NMR Spectrum (CDCI3) of 6a,15a,16a-triacetoxyspongian (61)AcO H8 52181.3 H^HS 2.12s s5Lc, HS 1.92a very close structural similarity to compound 34 and suggesting that 61was a positional isomer of 1213,15a,16a-triacetoxyspongian (34). Otherresonances at 8 2.12 (dd, J = 3.4, 12.4 Hz H7f3), 8 1.92 (d, J = 7.9 Hz, H14)and 8 0.78 (m) could be clearly distinguished from the hydrocarbonenvelope.The 13C NMR spectrum of 61 (CDCI3, Figure 36) displayedtwenty-six carbon resonances corresponding to three acetates (8 170.5,170.1, 169.8, 22.0, 21.4, 21.3), two acetal methine (8 101.7, 99.6), anacetoxy methine (8 69.8), four methyl (8 35.7, 22.1, 18.0, 17.2) andthirteen other carbons.1 H- 1 H COSY (CDCI3, Figure 37) correlations could beobserved between the H13 1 H resonance (8 2.57) and three otherproton resonances at 8 6.06 (H16), 1.92 (H 14) and 1.6 ppm (H 12).Correlations from the acetoxy methine 1 H resonance at 8 5.21 (H6) toresonances at 8 2.12 (H713) and 1.3 ppm could also be observed to givethe two spin systems outlined in Figure 38 below.77OAcH82.57^H 8 6.06Figure 38. Spin Systems for 611.Redisolving 61 in C6D6 once again helped to furtherdelineate the spin systems. The 1 H NMR spectrum (Figure 39) shows,OAc17:1'20^11^13 014.15OAcOAc61Figure 36. 125 MHz 13C NMR Spectrum (CDCI3) of 6a,15a,16a-triacetoxyspongian (61)0.00.51 .0152.02.53.0- 334.04.55.0556.0PPM79 61-1--—1• CM%.• "... '0• .7 ,• .H12-H13 9^'^-H13-H14 a^vj^ala.‘...L•L$,S H6-H7r3 s A^k,•••" •• c•O, I- . ..•0••0 H13-H16.^.6.0^5.5^5 0^45^40^33^3.0^2.5^2.0^13^10^0.5^0.0PPMFigure 37. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of6cc,15a,16cc-triacetoxyspongian (61)Figure 39. 500 MHz 1 H NMR Spectrum (C6D6) of 6a ,1 5a,16a-triacetoxyspongian (61)increased dispersion in the downfield region: 8 6.34, d, J = 7.4 Hz, H16; 56.28, s, H15; 5 5.38, dt, J = 3.6, 11.2 Hz, F16; 8 2.59, q, J = 7.2 Hz, H13; 8 2.29,dd, J = 3.6, 12.2 Hz, H713. As well, a axial methine resonances appearupheld out of the hydrocarbon envelope: 8 0.35, bd, J = 10.3 Hz, H9; 80.55, m, Hla.The 13C NMR spectrum (C6D6, Figure 40) displays increaseddispersion which aided in assigning HMQC (Figure 41) correlations. Table3 lists the assignment of all NMR signals of 61 in C6D6.COSY (C6D6, Figure 42) correlations from the acetoxymethine resonance at 8 5.38 (dt, J = 3.6, 11.2 Hz, H6) to three otherproton resonances (a methine 1 H resonance at 5 1.12 (d, J = 11 Hz, H5)and a pair of geminal methylene resonances at 5 1.24 (H7a) and 8 2.29(dd, J = 3.6, 12.2, H7f3)) completed an isolated spin system (substructureA, Figure 43). Given the similarity of the acetal resonances and otherspectroscopic features (four methyl singlet resonances; a methineresonance (H 13) which was split into a quartet; a single carbinol methine1 H resonance) to those of 34, it could be concluded that 61 alsopossessed an unrearranged spongian skeleton. In order toaccommodate the spin system of partial structure A on the spongianskeleton the acetate must be placed in either the C6 or C 11 positions.The C ring placement could be dismissed due to theobservation of coupling from the H16 acetal resonance (8 6.34) to theresonance for the H13 proton at 5 2.59, which showed further couplinginto two proton resonances at 5 1.72 (H 14) and 8 1.3 (H120) (substructureB, Figure 43). The H1213 resonance was further coupled into resonancescorresponding to H12a (8 1.7) and H1 la (8 1.1) both of which werecoupled into a resonance at 8 0.75 (H 1113) which coupled into the H9 1 H81SpAc• 161314.15OAc2061ItivIIII^II 111,11111g^!miller,'^111111111111180^160^140^120^100^PPM^80^•^60 40^20Figure 40. 75 MHz 13C NMR Spectrum (C6D6) of 6a ,1 5a,16cc-triacetoxyspongian (61)1r •iillra:4fi020* 6^....1<a>*.--_-_-_,4g (zits• A• PPruts^25• 1 I •201 II I 0 1^•^a^71 5 I110II 1^•051^7 7Figure 41. 500 MHz HMQC Spectrum (C6D6) of6cc,15a,16a-triacetoxyspongian (61)838461r 0.0▪ 2.0- 3.04.05.06.07.0PPM1.0-1-^H5416a HO-H7aHO-H713H12-H13a aa a8o OH13-H1•6•7.0^6.0^5.0^4.0^3.0PPMIr --r2.0^1.0^0.0Figure 42. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) of6a,15a,16a-triacetoxyspongian (61)8 1.1 H  8 1.24 H H8 2.295AcO^H8 5.38Aresonance at 80.35 to complete the spin system in substructure B below.Therefore, the third acetate was at C6.8551.7 8 1.38 2.59OAc-^H 86.3450.75 1681.1 1-1'0•' 110139 14^AS^1..^IIWAAP(A8 0.35 H81.72BFigure 43. Spin Systems for 61The relative stereochemistry of 61 was again elucidatedthrough a series of NOE difference experiments (Figure 44). Irradiation ofthe resonance at 8 5.38 (HO) led to enhancement of the protonresonance at 8 2.29 (H7(3) as well as three upfield methyl singletresonances at 8 0.93 (Me17), 0.92 (Me19) and 0.52 ppm (Me20). Adiaxial relationship between H6 and the three methyl groups canaccount for the observed enhancements, while the small couplingconstant between H6 and H713 (3.6 Hz) indicates an axial-equatorialrelationship between the two vicinal protons. The acetate functionalityat C6 is therefore in the a orientation. An equatorial acetate can alsoexplain the downfield shift of resonances of the equatorial groups inclose proximity: H713 (8 2.29) and Me 18 (8 1.16). Irradiation of theresonance at 8 2.29 (H713) gave NOE enhancements in the H6resonance (8 5.38), a triplet at 81.24 (J = 11 Hz, H7a) and the singlet at 86.28 (H 15). The observed NOE from H713 to H15 as well as the lack ofcoupling between H15 and H14 confirmed that the C15 acetate wasa. 40 The acetate at C16 was assigned as a by comparison to thespectroscopic data of compounds 34 and 63.Figure 44. NOE Summary for 61864° A model of compound 61 accomodating a 90° bond angle between H15 and H14brings H 15 into close proximity to H7f3 and puts H 16 at approximately 120° to H 13.Table 3. Assignment of NMR signals (C6D6) forOa, 15a,16cc-triacetoxyspongian (61)C# 6 13C 8 1 H COSY* NOE**1 a 40.4 0.5518 1.452a 17.7 1.128 1.33a 44.2 1.20313 1.44 34.0a5 59.5 1.1 H66 70.0 5.38 H5, H7a, H713 H713, Me17,Mel 9,Me207a 50.2 1.24 H6, H7878 2.29 H6H7a H6, H7a, H158 40.1a9 56.4 0.35 Hllb10 36.3alla 23.3 1.1 H118, H12a, H12811 13 0.75 H9,H11a,H12a12a 19.0 1.7 H1 la, H1113, H1213128 1.3 H1 la, H12a, H1313 39.9 2.59 H128, H14, H16 H12a,H14, H1614 59.8 1.72 H1315 100.6 6.3416 102.4 6.28 H1317 22.3 0.9318 37.0 1.1619 18.4 0.9220 18.1 0.52OAc 22.0,21.4, 21.2,170.3,169.9, 169.41.69, 1.66,1.66 ,* Correlations to proton in column 3** Proton in column 3 irradiateda May be interchanged87OAc6a,12b,15a,16a-tetracetoxyspongian (62):OAcHpAc88Compound 62 was isolated as an optically active, colorlessglass (34 mg, 2.8 mg/animal; (a)D +40.0, CHCI3) which also failed to givea parent ion in either HREIMS or DCIMS spectra. The largest observedmass in the HREIMS spectrum was at m/z 402.2405 amu (C24H3405, A -0.1mmu), which in conjunction with the 1 H (Figure 45, four acetate methylresonances: 8 2.05, 2.03, 2.02, 1.95) and 13C NMR spectra (Figure 46,twenty-eight carbon resonances, four acetate carbonyls: 8 169.6, 169.5,169.5, 169.4) of 62 could be interpreted as the loss of two equivalents ofacetic acid from the molecular ion. A peak of low intensity at m/z 540(M+ + NH4), detected in the soft ionization technique (DCIMS),supported this interpretation of the HREIMS data.The 1 H NMR spectrum of 62 (CDCI3, Figure 45) containedelements of both compounds 34 and 61: two acetal proton resonancesat 8 6.24 (d, J = 7.4 Hz, H16) and 5.86 (s, H15); two acetoxy methineresonances at 8 5.08 (dt, J = 3.4, 11.1 Hz, Ho) and 8 4.84 (m, H12);resonances for H13 (8 2.84, q, J = 7.5 Hz) and H713 (8 1.99, dd, J = 3.4, 12.4Hz); four acetate methyl resonances (8 1.88, 1.87, 1.87 and 1.79) andfour upfield methyl singlet resonances (8 0.99, 0.94, 0.86 and 0.78).i^i^i^3^2^1^0PPM 7Figure 45. 500 MHz 1 H NMR Spectrum (CDCI3) of 6a,1 2 13,1 5a,16a-tetracetoxyspongian (62)The 130 NMR spectra (CDCI3, Figure 46) displayed twenty-eight carbon resonances for a tetracetoxyspongian diterpenoid. Thecharacteristic features of: two acetal carbon resonances (8 98.9, 98.2);two carbinol methine resonances (8 70.6, 69.4); four acetate substituents(8 1696, 169.5, 169.5, 169.4, 21.8, 21.2, 21.0, 20.7) and four methylresonances (8 35.9, 21.9, 18.0, 17.9) could be clearly identified. Alsopresent were three quaternary (8 39.4, 35.5, 33.2) four methine (8 59.2,58.7, 54.3, 41.8) and five methylene (8 48.5, 43.0, 39.8, 23.4, 18.8) carbonresonances.1 H- 1 H COSY (CDCI3, Figure 47) correlations were observedthat established, through comparison to the data obtained forcompounds 34 and 61, the B, C and D ring systems of 62 (Figure 48). TheH6 methine resonance at 5 5.20 was coupled into a methine resonanceat 5 1.18 (H5) as well as signals corresponding to a geminal methylenepair at 8 2.14 (H7(3) and 1.24 ppm (H7a). The H12 methine resonance at 85.06 was coupled into the resonances of another pair of geminalmethylene protons at 8 1.82(H11a) and 8 1.48 (H110). Both of theseproton resonances showed further coupling into the resonance of anunresolved methine at 8 0.85 (H9). The H12 resonance also had acorrelation corresponding to coupling to the H13 resonance (8 3.08).Finally, the H 13 resonance was further coupled into methine resonancesat 8 6.34 (H 16) and 2.01 ppm (H 14) to complete the spin systems (Figure48).90pAc0.15-0Ac20Shike.^ ri Lb. Mika 1.41Atil AAA Au150 125 100Figure 46. 125 MHz 13C NMR Spectrum (CDCI3) of 6a, 1213,15a,16a-tetracetoxyspongion (62)- 0.0031.0132.02.53.03.54.0435.0536.063..______ —di'.^.'--- .-[jl1. oil lbH5-H6H6417a•fp-§twa? •:-.,..,.;^n, .1:'••0H11/3-H12Hlla-H12 1..^.si.7:,^„ ,.......431;,-, ••sH6-H7I3 010 H13-H14 -fr' • • 0 1 9Q CO a a.^. .p 8 H12-H1 lec,AI'0 •ecl H13-H1PPM•6.5^6 0^5 5^5.0^4 5^40^3.5^3.0^23^20^13^10^03^0.0PPMFigure 47. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of6a,1213,15a,1ba-tetracetoxyspongian (62)925AcO H8 5.2071381.18 H^7a81.24 H^ H82.149;01 /14Hs 0.85 ^H8 2.018 1.488 1.82 11a938 5 .06 12 OAc^OAc16H8 6.34110^13Figure 48. B, C and D Ring Spin Systems for 62Redisolving 62 in C6D6 resulted in 1 H (Figure 49), 13C (Figure50), 1 H- 1 H COSY (Figure 51), and HMQC (Figure 52) spectra in which theelements of both compounds 34 and 61 could be distinguished (Table4). Compound 62 therefore possesses acetates in both the C6 and C12positions on the spongian skeleton.Confirmation of the stereochemistry at the acetatepositions was again accomplished through NOE difference experiments(Figure 53). Irradiation of the H6 resonance (8 5.31) led to theenhancement of three methyl resonances (8 0.62, 0.87, 0.88). Onceagain, the enhancements can be explained if the proton (H6) is in adiaxial relationship (0 orientation) with the methyl groups. The C6acetate is therefore in the a orientation. Irradiation of the H 15 resonance(8 6.15) induced signal enhancement in resonances corresponding toH7f3 (8 2.21) and Me17 (8 0.87), again confirming the a orientation of theacetate. Irradiation of the resonance at 8 5.01 (H12) yieldedenhancement of the signal for H9 (8 0.28), consistent with a 0 acetate,and irradiation of the H16 resonance (8 6.60) gave an NOE into thesignal for Mel 7 (8 0.87), confirming the a orientation of the final acetate.Z9 ov0 et---9l.$Hr-IOvd0V0(0) uoleuodsAxoleocui.e039039 l'ClZ 039 Jo (9090) wrupedS dIAIN HI zHIA1 009 '6t, enlald°^! ^°^° ^!^... 9^9^odd7.6"4 nS1 1 1 I 1 I^I I r 1 1 t 1 I 1^1 1 1 1 I I I 1 1 1 t 1 I I 1 1 I 1 1 1 1 1 I I 1 I 1 1 1100^PPM^80^ 60^40^201 1 1 1 1 1 I I 1 I 1 T I t I I 11 I 1^1 I 1 -11 1 T 1180^160^140 120Figure 50. 75 MHz 13C/APT NMR Spectra (C6D6) of 6a,12 13,15a, 16a-tetracetoxyspongian (62)dr 90rii H11 ft-H12• Hlla-H121•aH13-H14• .0ttH6-H7 f3 • o eae • e•• H12-H13•• •it H5-H6kl6-147a0o• H13-H145o7.0^6.0Figure 51. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) of6a,1213,15a,16a-tetracetoxyspongian (62)2.0PPM-r^•^-T1.0 0.03.04.05.06.07.04.0^3.0PPM5.02.00.0fFI1 .096OAcpAcH11613 014.15OAc" 18 OAc 62•••. .4ortir."7,9 ., -■.----...- CO........-----• .4C+,1P1-4.:•.- -id&<11411V•al oi' mon -Op.^2.0^1.5^1.0^05971020304050pp.Figure 52. 500 MHz HMQC Spectrum (C6D6) of6a ,1 213,15a,16a-tetracetoxyspongian (62)1 , H^OAcobserved NOEOAcCH3Figure 53. NOE Summary for 62c. ConclusionThe three spongian diterpenoids described above add tothe growing number of compounds isolated from chromodoridnudibranchs. The reported icthyotoxicity of compound 34 30e ,f addscredence to the idea that terpenoids of this type are sequestered bythe molluscs for use as chemical defensive agents.98Table 4. Assignment of NMR signals (C6D6) for6a,1213,15a,16a-tetracetoxyspongian(62)C# 8 13C 8 1H COSY* NOE**10 39.8 0.51f3 1.32a 18.4 1.428 1.253a 43.6 1.3313 1.154 33.4a5 58.7 1.02 H66 69.4 5.31 Me17, Me 19,Me207a 48.9 1.15 H5, H7a, H7ii78 2.21 H6, H7I38 39.4a H6, H7a9 53.9 0.2810 35.6a H11I3lla 23.6 1.51 H1113, H12118 1.13 H9, H1 la, H1212 70.7 5.01 H11a,H118,H13 H913 42.1 3.15 H12, H14, H1614 59.4 1.65 H1315 99.7 6.15 H713, Me1716 98.6 6.60 H13 Me1717 22.1 0.8718 36.4 1.1419 17.8 0.8820 17.8 0.62Ac 21.4, 20.8, 20.7,20.4169.3, 169.2,169.0, 168.91.71, 1.67,1.66, 1.65OAc OAcH 11620^11^13 014.15H^-OAc*--,18 OAc 62* Correlations to proton in column 3** Proton in column 3 irradiateda May be interchanged99C. Metabolites from the Dorid Nudibranch Cadlina luteomarginata,Collected in the Northeastern Pacific Ocean: Structures andGeographical Distribution.i. Introduction to Cadlina luteomarginataThe dorid nudibranch, Cadlina luteomarginata,(Opisthobranchia, Nudibranchia, Cadlinidae41 ) is a noncrypticnudibranch known to feed on a wide variety of sponges. 42 Commonalong the rocky headlands of the North American Pacific coast, thismollusc shows remarkable variation in its dorsal metabolites. One studyin California42a and several in British Columbia42b-e have demonstratedthe ability of C. luteomarginata to sequester terpenoids with a range ofcarbon numbers and functionalities from its various dietary sponges.Californian C. luteomarginata specimens were found tocontain a variety of metabolites (Figure 55) including furodysinin (20).dendrolasin (23), pleraplysillin-1 (64), pallescensin-A (65),dihydropallescensin-2 (66), idiadione (67), the isonitrile 68 and theisothiocyanate 69 as well as other isocyano and isothiocyanatocompounds for which structures were not determined.42a A markedseasonal variation in the metabolites was also found for nudibranchscollected at the same dive site at different times of the year.Compounds 20, 23, 64-5 and 67 were found in January collections whilecompounds 64, 66 and 68-9 predominated in July, perhaps reflecting41 See Figure 54 for the phylogenetic classification of Cadlina luteomarginata.42 a)Thompson, J. E., Walker, R. P., Wratten, S. J. and Faulkner, D. J. Tetrahedron,1982,38, 1865. b) Hellou. J. Andersen, R. J., Rafii, S., Arnold, E. and Clardy, J.Tetrahedron Left., 1981, 22, 4173. c) Hellou, J. Andersen, R. J. and Thompson, J. E.Tetrahedron, 1982, 38, 1875. d) Gustafson, K., Andersen, R. J., Cun-Heng, H. andClardy, J. Tetrahedron Lett., 1985, 26, 2521.e) Tischler, M. and Andersen, R. J.Tetrahedron Lett, 1989, 30, 5717.100ORDER^ NudibranchiaSUBORDER^Aeolldacea^Arminacea^Doridacea^DendronotaceaSUPERFAMILY^AhadorldoIdea^Eudoridoidea^Porodoridoidea^GnathodoridoldeaHexabranchidae^Archidorididae^Discodorididae^KentrodoridideaFAMILY^Chromodoridldoe^Homoiodorididae^Dorldidae^Aldisidae^AsteronotidaeRostangidae^Actinocyclidae^Cadlinidae^Baptodorididae^PlatydorididaeGENUS^ CadlinaSPECIES^ C. luteomarginataFigure 54. Phylogenetic Classification of C. luteomarginata(from Thompson)065X68 X = NC69 X = NCS10223 2067Figure 55. Metabolites from Cadlina luteomarginotaCollected in Californian Waters 42'either a seasonal grazing preference of the nudibranchs or a seasonaldifference in metabolite production within the prey sponges.Several of the compounds exhibited some icthyotoxiceffects against goldfish (Carassius auratus) at a concentration of 100u.g/ml_ and antifeedant effects (rejection of food pellets when thecompound was added) against either goldfish (compounds 68, 69 and20) or woolly sculpin (Clinocottus analis, compounds 20, 65 and 67).Specimens of Cadlino luteomarginata collected off thecoast of British Columbia were found to contain the metabolitesfurodysin (19), furodysinin (20), microcionin-2 (70), albicanol (71) and itsacetate 7 2,42b the degraded sesterterpenoid, luteone (73),42emarginatafuran (74) 42d and the spongian derived diterpenoidscadlinolide B (75), tetrahydroaplysulphurin (76) and glaciolide (77)42e(Figure 56).Furodysin (19) and furodysinin (20) are metabolitescommonly found in sponges belonging to the genus Dysidea,43 whilemicrocionin-2 has been reported as a metabolite of sponges comprisingthe genus Microciona.44 The diterpenoids 75-77 were isolated fromspecimens of the opisthobranch that were collected while activelyfeeding on the sponge Aplysilla glacialis, which was also shown to bethe dietary source of these metabolites. 42e Marginatafuran (74), ametabolite related to the spongians, although with a different C-D ringclosure, was isolated from a few specimens collected along the coast of43 For examples see reference 27a and a) Grode, S. H. and Cardellina, J. H. II J. Nat.Prod., 1984, 47, 76. b) Capon, R. J. and MacLeod, J. K. J. Nat. Prod., 1987, 50, 1136.44 a) Cimino, G., De Stefano, S., Guerriero, A. and Minale, L. Tetrahedron Lett., 1975,3723. b) Cimino, G., De Rosa, S., De Stefano, S. and Sodano, G. Pure AppL Chem.,1986, 58, 375 and references therin.103075 R i = R2 = 076 12 1 = OAc, R2 = H 7710419^20Figure 56. Metabolites from Cadlina luteomarginatacollected off the coast of British Columbiathe Queen Charlotte Islands. 42d Luteone (73) is a unique degraded C23terpenoid with a new carbon skeleton. Its structure was solved by X-raydiffraction analysis of a dinitrophenylhydrazone derivative .42bConsidering the noted variation in the skin chemistry ofCadlino luteomarginata with geographic location, and the mollusc'sability to sequester a wide variety of metabolites from dietary sponges, itwas felt it would be instructive to take a look at the variation inmetabolites found in the nudibranch along the entire coast of BritishColumbia. This chapter details the results of our study which involved tencollection sites along the coast of B. C. and two in Alaska.ii. The Current Study.An opportunity to participate in a joint oceanographiccruise aboard the Department of Fisheries and Oceans vessel J. P. Tully ,in May 1990, enabled us to collect specimens of Cadlinaluteomarginata, at many previously inaccessible sites along the coast ofBritish Columbia. Several new and many previously describedmetabolites were isolated at the various collection sites (Table 5). Themost common metabolite encountered was albicanyl acetate (72),which was often the only non-steroidal mevalonate derived metabolitepresent in the extracts. Three collection areas in particular (RennellSound, Anthony Island and Agamemnon Channel) proved to be rich interpenoid metabolites and yielded several new compounds.The Rennell Sound collection was found to containmetabolites 68, 69 and 78-80 which could be traced to a locally105abundant Acanthella species of sponge, as well as the algalmetabolite violacene (81), which interestingly enough was also found inthe sponge. The spongian derived compound 82 was isolated from asingle specimen of C. luteomarginata collected while actively layingeggs. The egg mass of this individual contained the new metabolitesalbicanyl triacetate (83) and albicanyl diacetate (84), modified versionsof 72.Specimens collected off Anthony Island (Ninstints) had themost chemical diversity; containing the sesquiterpenoid compounds 68-69, 70, 72, 78 and 85, the marginatanes 74 and 86, 9,1 1-dihydrogracillinA (87) and the new diterpenoid lutenenolide (88).Molluscs collected at Agamemnon Channel in Jervis Inlet,were found to contain the known compounds albicanyl acetate (72)and luteone (7 3), the oxidospongian compounds 8 9-90 andcadlinaldehyde (91), a C21 compound with a new terpenoid carbonskeleton.10672^68X=NC69 X = NCS78 X = NHCHO81CI10779X=NC80 X = NCS."---„^AcO OH82...,--...j^R85 X = NHCHOO7074CI^CICIBrCH2, "%**1088789 R 1 = 08u, R2 = OH90 R2 = H^R2 = OAC91a. Extraction and PurificationAll specimens of Cadlina luteomarginata were handled inthe following manner.Samples were collected during our scientific cruise aboardthe DFO vessel J. P. Tully during the month of May 1990 with thefollowing exceptions: Barkley Sound (June 1990); Jervis Inlet (October1990 and August 1992); Hetta Inlet and Moira Sound, Alaska (May 1991);Howe Sound (various times 1990-1992). Specimens were collected ateach dive site where the nudibranchs were present. 45 Samples werekept separate if the choice of dive site was different from the generalcollection for the area (deep dives, highly polluted waters), if thenudibranch was displaying markedly different behavior (mating, layingeggs) or found actively feeding. Where possible a sample of the spongefood source for those nudibranchs collected while feeding was alsoobtained and extracted for chemical comparisons.Freshly collected specimens were immediately immersed inmethanol and labeled with dive site and collection date for later work-up in the laboratory. During workup, the methanolic extract wasdecanted and the animals were immersed in a fresh portion ofmethanol for 24 hours. This second extract was combined with the firstmethanolic extract and reduced in vacua to yield an aqueoussuspension. This suspension was brought up to approximately 100 mLwith distilled water and partitioned with 100 mL of 1/1 CH2Cl2/MeOHfollowed by two successive 50 mL portions of CH2Cl2. The organicextract was rotary evaporated to dryness, taken up in 1-2 mL CH2Cl245 See the map in Figure 57 for the locations of the various dive sites.109and applied to a silica flash column (20 X 1.5 cm). A step gradientelution pattern was then followed consisting of 100 mL portions of; 40%hexanes in CH2Cl2, 20% hexanes in CH2Cl2, 100% CH2Cl2, 20% Et20 inCH2Cl2, 40% Et20 in CH2Cl2, and 40% Me0H in CH2Cl2. All fractionswere rotary evaporated to dryness and screened on the WH-400 NMRspectrometer for interesting resonances. The two most polar fractionsinvariably consisted of fats, peroxysteroids and phthalate esters from theplastic Ziploc® bags used in collection of individual samples. The lesspolar fractions varied considerably in content and complexity with divesite (see Table 5), and contained the terpenoids described in thischapter as well as cholesterol and other steroids which were not purifiedas such work was beyond the scope of this study.Individual compounds were purified through successive useof radial tic and normal phase HPLC. Choice of eluant was dictated bythe size and functionality of the compounds. Sesquiterpenoid isonitrileswere eluted with pure hexane, other sesquiterpenes eluted either withhexane or hexane/ethyl acetate solvent mixtures. Diterpenoids andsesquiterpene formamides were chromatographed using eitherhexane/ethyl acetate (3/1) or dichloromethane/diethyl ether (88/12).Final purification and elucidation of the structures forcompounds 79-81 was accomplished in our laboratory by David L.Burgoyne after recognition that the compounds were identical tocompounds he was isolating from the sponge Acanthella sp. collectedat the same dive sites as the nudibranchs.1105 50435o W^132°W^29°Wvtio u-',C25,266-8o23126oW o20 5/°Nna rlottes la nds9-1213-1BRITISHCOL UM BIA16-2153oN51 °N-i 4 9°NPACIFICOCEANUSA530N51 0N49°N47°N 47°N4 50 ^I^ IN 4 5oNo135 W 132°W 129°W^126°W^123°W^120°W1 1 1Numbers correspond to the Dive Sites listed in Table 5.Figure 57. Collection Areas for Cadlina luteomarginataTable 5. Geographical Distribution of Metabolites fromCadlina luteomarginata Collected in British Columbianand Alaskan Coastal WatersDive site # animals Chemistry1.Pendrell Sound  29 72, steroids2. East Redonda Island 50 72, steroids3. Rivers Inlet, Bull Island 2 72,drimane type C154. Kitimat An-n, Emilia Is. 4 unidentified C15 acetate5. Kitimat Arm 3 steroids6. Langara Island A 10 72, steroids7.Langara Island B 18 unidentified furans8.Langara Island C 8 steroids9.Rennell Sound A 13 68, 69, 78, other isonitrilesand compounds fromAcanthella sp.10. Rennell^Sound^--Laying eggs1 68, 69, 78, 82, otherisonitriles andcompounds fromAcanthella sp.11. Rennell^Sound^--Egg Mass83, 8412.Rennell Sound B 3 72, steroids13.Tasu Narrows — 110 Ft 1 steroids14.Tasu Narrows — 30 Ft 1 72, steroids15.Tasu Sound 13 72, steroids16.Anthony Island A 4 72, steroids17.Anthony Island A --feeding1 72, steroids18.Anthony Island B 1 68, 69, 78, 85112Table 5. cont.19.Anthony Island C--feeding1 72, steroids20. Anthony Island C --feeding on Aplysilla sp14 74, 86-8721.Anthony Island C 116 70, 72, 74, 78, 86, 8822.Quatsino Sound 5 72, steroids23.Barkley Sound 10 72, steroids24. Barkley^Sound^--Sanford Island.18 7525.Moira Sound, Alaska 33 72, steroids26.Heffa Inlet, Alaska 33 72, steroids27.Howe Sound, KelvinGrove16 72, steroids28. Howe^Sound,Passage Island22 72, steroids29. Jervis Inlet 30,55 72, 73, 89-9111379X=NC80 X = NCS68 X = NC69 X = NCS78 X = NHCHOCIBrCH2 ,""CICI^CI81b. Results and Discussion1) Compounds from Cadlina luteomarginata Collected at RennellSound British Columbia.Nudibranch specimens that made up the generalcollection at Rennell Sound B. C. contained the previously describedmetabolites 68-69, 7842a 79-80,46 81,47 and 82.48 Structures were solved bycomparison of the spectroscopic data with published values (seeExperimental and Appendix A).46 Ciminiello, P., Fattorusso, E., Magno, s. and Mayol, L. Can. J. Chem. 1987, 65, 518.47 a) Mynderse, J. S. and Faulkner, D. J. J. Am. Chem. Soc., 1974, 96, 6771. b) Mynderse,J. S., Faulkner, D. J., Finer, J. and Clardy, J. Tetrahedron Lett., 1975, 16, 2175. c) VanEngen, D., Clardy, J., Kho-Wiseman, E., Crews, P., Higgs, M. D. and Faulkner, D. J.Tetrahedron Lett., 1978, 19, 2948 Schmitz, F. J., Chang, J. S., Hossain, M. B. and van der Helm, D. J. Org. Chem., 1985,50, 2862.114Although no new compounds were isolated from thegeneral collection at Rennell Sound, the above known compounds 78-82 have not previously been reported as metabolites from Cadlinaluteomarginata, and the compounds from this site raise interestingpoints about the chemical ecology of the nudibranch.Compounds 68 and 69 had previously been isolated fromspecimens of C. luteomarginata collected in Southern California andtraced to an Axinella species of sponge.42a In our collection fromRennell Sound, metabolites 68-69 and 78-80 could be traced to thesponge Acanthella sp. which was very abundant in the area.Compounds 68-9 have previously been shown to be effectiveantifeedants. Our finding that the nudibranch sequesters the samemetabolites from different sponges adds credence to the hypothesis 49that opisthobranch molluscs have developed an ability to detectallomones present in their diet and treat them differently than nutritivecomponents.One finding of particular interest is the presence ofviolacene (81) in both sponge and nudibranch extracts from the RennellSound collection site. Non-aromatic, polyhalogenated metabolites arerelatively common among red algal species 50 and are often found tocomprise the majority of metabolites in herbivorous sea hare extracts 5149 Faulkner, D. J. and Ghiselin, M. T. Mar. Ecol. Prog. Ser., 1983, 13, 295.50 for examples see references in note 29 and: a) McCombs, J. D., Blunt, J. W.,Chambers, M. V., Munro, M. H. G., and Robinson, W. T. Tetrahedron, 1988, 44, 1489.b) Kennedy, D. J., Selby, I. A. and Thomson, R. H. Phytochem., 1988, 27, 1761. c) Coll,J. C. and Wright, A. D. Aust. J. Chem., 1989, 42, 1685. d) Watanabe, K., Miyakado, M.,Ohno, N., Okado, A., Yanagi, K. and Moriguchi, K. Phytochem., 1989, 28, 77.51 a) Inouye, Y., Uchida, H. Kusumi, T. and Kakisaw, H. J. Chem. Soc. Chem. Comm.1987, 346. b) Kusumi, T., Uchida, U., Inouye, Y., Ishitsuka, M., Yamamoto, H. andKakisawa, H. J. Org. Chem., 1987, 52, 4597. c) Sakai, R., Higa, T., Jefford, C. W. andBemardinelli, G. Helv. Chim. Acta, 1986, 69, 91.115I^IOH o ,BrHH DI Br92H„,93but are relatively rare in sponges 52 and never before found innudibranchs. The nudibranch is undoubtedly obtaining the metabolitefrom the sponge Acanthella, but the sponge's source is more uncertain.It seems unlikely that Acanthella is synthesizing violacene asit does not fit into structural series with the other terpenoid metabolites(isonitriles, isothiocyanates and formamides) found in the sponge. Themost likely explanation, therefore, is that the sponge is obtaining thecompound as a product from an external source such as a symbiotic,fouling, or dietary organism.A recently reported analogous case was published bygroup operating at the University di Napoli, working on extracts of thesponge Mycale rotilis. 52 They found, in the sponge extract, threemetabolites (92-94) which are of a class typically found in red algalspecies of the genus Laurencia. 50 Mycale is often found growingamongst macrophytes, but none of the algal species collected with ornear the sponge were Laurencia spp., nor did the epiphytic algaecontain compounds52 a) Giordano, F., Mayol, L., Notaro, G., Piccialli, V. and Sica, D. J. Chem. Soc., Chem.Commun., 1990. 1559. b) Notaro. G., Piccialli, V., Sica, D., Mayol, L. and Giordano, F. J.Nat. Prod., 1992, 55, 626.116^HO^Br^-...^I HBri,„,/^ 0\-0.:^H$H i.....194BrH'Br11792-94 when their extracts were examined. The authors have suggestedthat perhaps the sponge could have completely overgrown aLaurencia macrophyte, but the relative growth rates of the twoorganisms makes this unlikely. A more plausible source for the presenceof compounds 92-94 in Mycale, and compound 81 in Acanthella, is thatthe sponges have obtained the metabolites either through ingestion ofdetritus from decaying seaweeds, or by concentrating trace amounts ofthe metabolites found in the large volumes of water they circulatethrough their tissues each day. It is interesting to note that bothviolacene and compounds 92-94 are polyhalogenated metabolites,and in this regard similar to industrial insecticides such as dieldrin andaldrin which are resistant to metabolic catabolism and persist asresidues in the environment for many years. Considering this, perhapsthe presence of violacene in extracts of Cadlina luteomarginata isanalogous to the traces of DDT which were found in species of raptorsafter its widespread use as an agricultural insecticide during the 1960's.The spongian derived compound 82 was isolated from asingle specimen collected while in the process of laying eggs. It was firstisolated from the Caribbean sponge Igemella notabilis 48 and itsstructure was solved through X-ray diffraction analysis. Subsequently ithas been shown to also be present in Australian Aplysillidae sponges. 53Compounds 68, 69 and 78 were also present in this individual.2). Metabolites Isolated from the Egg Mass of an Actively LayingSpecimen of Cadlina luteomarginata Collected at Rennell Sound.One specimen of C. luteomarginata collected at RennellSound B.C. was in the process of laying eggs. The egg mass from thisindividual was extracted and worked up according to the procedureoutlined above. The fraction eluting off the silica flash column with 20%hexanes/CH2Cl2 had 1 H NMR signals that were characteristic ofalbicanyl acetate (72), but upon further purification could be assignedto two new compounds, albicanyl triacetate (83) and albicanyldiacetate (84).AcOR1 157^1214^'1372R 1 = R2 = H8312i = R2 = OAc84R 1 = OAc, R2 = H53 a) Karuso P. and Taylor, W. C. Aust. J. Chem., 1986, 39. 1629. b) Karuso, P., Bergquist,P. R., Cambie, R. C., Buckleton, J. S., Clark, G. R. and Rickard, C. E. F. Aust. J. Chem.,1986, 39, 1643.11811Albicanyl Triacetate (83).Albicanyl triacetate (83, 1.5 mg) was isolated as a colorlessoil which failed to show a molecular ion peak in the HREIMS spectrum. Apeak at m/z 320.1985 amu, corresponding to a molecular formula ofCi9H2804 (D M = -1.3 mmu) was the highest mass observed. Throughexamination of the 1 H (Figure 58, three acetate resonances at 8 2.16,1.96 and 1.94) and 13C NMR spectra (Figure 60, two carbinol methines at8 73.2 and 68.4 as well as a carbinol methylene at 8 60.8), this peakcould be interpreted as corresponding to the loss of one equivalent ofacetic acid from the molecular ion.In addition to the three acetate methyl singlet resonances(8 2.16, 1.96, 1.94), the 1 H NMR spectrum (CDCI3, Figure 58) displayedresonances which could be attributed to: two acetoxy methine protons(8 5.25, bs, H1; 8 5.18, dm, J = 10.6 Hz, H2), a geminal pair of acetoxymethylene protons (8 4.18, dd, J = 3.4, 11.4 Hz, H1 I a; 8 4.08, dd, J = 9.9,11.4 Hz, H 11 b), the protons of an exocyclic methylene (8 4.95, bs, H12a; 84.65, bs, H12b), three allylic protons (8 2.48, dd, J = 3.4, 9.9 Hz, H9; 8 2.40,m, H7a; 8 2.00, m, H713) as well as three tertiary methyls (8 1.02, 0.93, 0.92).119H7iiH9AcAAc c83wMel 5Me14Mel 3H12bH12aH1^HllaHl lbH2wITIT.P.T.T.0Figure 58. 500 MHz 1 H NMR Spectrum (CDCI3) of Albicanyl triacetate (83)_..8The relationship to albicanyl acetate could be seen clearly bycomparison to the 1 H NMR spectrum of 72 (Figure 59).The 130 NMR spectrum of 83 (CDCI3, Figure 60) containedsixteen carbon resonances, accounting for all but the quaternarycarbons of the molecule. Once again the relationship to 72 could beclearly seen. Resonances attributable to: three tertiary methyl carbons(8 14.7, C15; 8 23.4, C14; 8 33.4, C13), the carbons of an exocyclicmethylene olefin (8 145.4, C8, S 109.6, C12), an acetoxy methylenecarbon (8 60.8, C11), two methine carbons (5 48.4, C9; 8 48.5, C5) andthree methylene carbons (8 22.7, C6; 8 37.4, C7; 8 40.0, C3) could bedistinguished with chemical shifts which closely matched similarresonances in the 130 spectrum of 72 (CDCI3, Figure 61). Resonances atchemical shifts appropriate for two additional carbinol methine carbonsat 8 73.2 (Cl) and 8 68.4 (C2) as well as the presence of two upfieldmethyl carbon resonances at 8 20.8 and 8 20.5 confirmed that 83 was adiacetoxy derivative of 72.Groups of correlations in the 1 H- 1 H COSY spectrum of 83(CDCI3, Figure 62) established the spin systems of the molecule (seeTable 6). Coupling could be observed between the methine resonanceat 8 5.18 (H2) and the resonances of a geminal methylene pair at 8 1.80(H3a) and 8 1.42 (H30). Irradiation of the methine resonance at 8 5.18during a double resonance experiment sharpened the methine signal at8 5.25 (H1), while the reverse experiment reduced the methineresonance at 8 5.18 to a doublet of doublets, indicating a very smallcoupling between the two acetoxy methine protons and suggesting acis relationship between them (Figure 63). The remaining spin systems of83 were similar in all respects to the spin systems on the B ring of 72121- r^17.5^7.0^6.5^6:0^5.5 5.0 1.5^4.0^34PPM 3.5^3.0 2:5^2.0 1.5^1.0Figure 59. 400 MHz 1 H NMR Spectrum (CDCI3) of Albicanyl acetate (72)C14C6CSC9C11C7C3 C1383C12CI C2120^100 80 60^40 20Figure 60. 125 MHz 13C NMR Spectrum (CDCI3) of Albicanyl triacetate (83)ISO^140^120^100Figure 61. 125 MHz 13C NMR Spectrum (CDCI3) of Albicanyl acetate (72)- 1383Hi20 H12bH1^Hl loHllbMel 5Me14Me13H7f3H9 J- 1 .0- 13_ 2.0- 23- 3.0- 334.04.55.01255.0^43^4.0^3.0^2.5^2.0^13^1.0PPMFigure 62.400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofAlbicanyl triacetate (83)(Table 7). The spin system in Figure 63 thus had to be on the A ring of themolecule, requiring placement of the acetates either at Cl and C2, orC2 and C3.5.18^OAc126 decoupling correlationCOSY correlationsH51.80^H51.42^8 5.25 OAcFigure 63. Ring A Spin System for 83Evidence for the placement at positions Cl and C2, as wellas the relative stereochemistry of the substituents, was obtained throughthe use of a series of NOE difference experiments (Figure 64), carried outon a C6D6 solution of 83 for improved dispersion. Irradiation of themethine resonance at 8 5.52 (bs, H1) resulted in enhancement of thesignals at 8 5.34 (m, H2), 4.45 (dd, H1 la) and 0.78 ppm (s, 3H, Me 15).Irradiation of the other acetoxy methine resonance (8 5.34, H2)enhanced proton signals at 8 5.52 (H1), 0.71 (s, 3H, Me14) and Me15.Enhancement of the acetoxy methylene proton at 8 4.45 can only beexplained if the methine proton at C 1 is equatorial (1p), placing theacetate in the la (axial) position. Placement of the other acetate in the2a (equatorial) orientation can then account for the coupling patternwithin ring A and the remaining observed NOEs from the H2 methineresonance to the resonances of Me14 and Me 15. Irradiation of the Me15resonance gave NOE enhancement into resonances corresponding to2^ 15H -0- CH3 ------- 4"-- Hllb.......•^--,111/4 .^.,HAC ----^H1 -- ----- -b.-', - 14^--------1Hop,^,Ac0,-, 1,313,OAcH3C13^OAc^ observed NOEH1, H2 and Me14 as well as a resonance at 8 3.94 (t, H11b), whileirradiation of the Me14 resonance resulted in enhancement ofresonances corresponding to Me15, Me13 (8 0.83) and H2. Alsoobserved were NOE enhancements of resonances at 8 1.1 (m, HO), and8 1.4 (m, H3I3) when the Me14 signal was irradiated. All these results areconsistent with a acetates at Cl and C2.Figure 64. NOE Summary for 83Albicanyl diacetate (84).84Compound 84 was isolated as a colorless oil (0.3 mg) whichgave a highest mass peak in the HREIMS spectrum at m/z 322.2140 amu,127Table 6. Assignment of NMR Signals (CDCI3) for Albicanyl Triacetate (83).C# 813C 8 1H COSY* COSY LR NOE**1 73.2 5.25 H2, H1 la, Me152 68.4 5.18 H3a, H3fl H1, Me153a 40.0 1.80 H2, H303f3 1.42 H2, H3a H545 48.5 1.59 H6a, H68 H386a 22.7 1.75 H5, H68, H7a, H71368 1.33 H5, H6a, H7a, H787a 37.4 2.00 H6a, H68, H70 H12a, H12bIii 2.40 H6a, H6f3, H7a8 145.49 48.4 2.48 Hlla,H1lb H12a, H12b1011 a 60.8 4.18 H9, H1 lb1 lb 4.08 H9, H1 la12a 109.6 4.95 H12b H7a, H912b 4.65 H12a H7a, H913 33.4 1.0214 23.4 0.93 H2, H68, H38,Me13, Me1515 14.7 0.92 H1, H2, Hl lbMeCOCH3CO 21.0,20.9,20.52.16,1.96,1.94128AcO,„.83* Correlations to proton in column 3** Proton in column 3 irradiatedTable 7. Assignment of NMR Signals (CDCI3) for Albicanyl Acetate (72).C# 8130 81H COSY* HMBC**hx 39.0 1.25 H18, H2a, H21318 1.70 Hla, H2a, H2132a 19.2 1.55 Hla, H18, H28, H3a, H31328 1.45 Hla, H18, H2a, H3a, H3133a 41.9 1.19 H2a, H28, H313 Me13, Me1438 1.39 H2a, H213, H3a4 33.5 Me13, Me 145 55.1 1.11 H6a, H613 H6a, Hop, Me 13.Me14, Me156a 23.9 1.70 H5, H613, H7a, H713613 1.32 H5, H6a, H7a, H7137a 37.8 2.01 H6a, H6f3, H78, H12a, H12b H12a, H12b713 2.39 H6a, H6f3, H7a, H12a8 146.8 Hlla,H11b,H12a, H12b9 54.7 2.02 H1 la, Hllb, H12b H713, H12a, H12b10 38.9 Me15, Hlalla 61.6 4.32 H9,H1lb1 lb 4.17 H9, Hl la12a 107.1 4.83 H7a, H713, H12b12b 4.50 H78, H9, H12a13 33.6 0.8614 21.7 0.79 Me1315 15.1 0.74MeCO 171.4 CH3COCH3CO 21.1 1.99* Correlations to proton in column 3** Correlations to carbon in column 2129H82.68corresponding to a molecular ion with a formula of C 1 013004(calculated mass of 322.2144), which indicated that 84 was amonoacetoxy derivative of 72.Several features were observed in the 1 H NMR spectrum of84 (C6D6, Figure 65) which were also common to the spectra of 72 and83. A broad singlet resonance at 8 4.95 integrating for two protons (H1,H12a), another broad singlet resonance at 8 4.81 (H12b), resonances fora geminal acetoxy methylene at 8 4.49 (dd, J = 4.0, 11.8 Hz, Hlla) and S4.15 (t, J = 11.6 Hz, Hllb), two multiplet resonances at 8 2.68 (H9) and2.30 ppm (H7f3), two acetate methyl singlet resonances (8 2.06, 1.75)and three upfield methyl singlet resonances (8 0.88, 0.75 and 0.64).Unfortunately the small amount of material available (0.3mg) did not permit us to obtain a 13C NMR spectrum.Examination of the 1 H- 1 H COSY spectrum (Figure 66) againled to the elucidation of the spin systems for the drimane derivative(Figure 67). Correlations could be observed between resonances which1308 1.88 HH mH 4H H OAc81.6 ---8 1.04 84.95OAc54.95^'""-•\ 54.81^H --:—°"- HH H^84.49 HS 4.15COSY correlationsFigure 67. Spin Systems for 84indicated the acetoxy methylene proton resonances at 8 4.49 and 4.15coupled into one another and into a methine resonance at 8 2.68 (H9)7.5^7.0•-6.5^6.0^5.5^5.0^4.5^4.0 ^3.5^3.0^2.5^ 2.0^1.5PPM1.0 013^0 •Figure 65.400 MHz 1 H NMR Spectrum (C6D6) of Albicanyl diacetate (84)132Figure 66. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) ofAlbicanyl diacetate (84)as well, the exocyclic methylene proton resonances at 8 4.95 and 8 4.81were coupled into each other. The resonance for H1 (8 4.95) showedcoupling into a resonance at 8 1.88 (H2/H2') which was further coupledinto two proton resonances at 8 1.6 (H313) and 8 1.04 ppm (H3a). Anotherspin system containing five protons and accounting for the remainder ofthe molecule (C5-C7) could also be clearly distinguished (Table 8).Comparison to the data for albicanyl triacetate led to the conclusionthat 84 was the Cl monoacetoxy derivative of 72.Our finding of acetoxy derivatives of albicanyl acetate (72)in the egg mass of Cadlina luteomarginata is similar to the report fromCimino's group in Italy that Dendrodoris limbata produced esterifiedmasked versions of polygodial and deposited them in thehermaphrodite gland and egg mass. 27b Such similarities, as well as thefact that 72 was the most common metabolite encountered along theentire British Columbian coast suggested that Cadlina luteomarginatamight be capable of synthesizing this allomone de novo, andsecondarily obtains other allomones from its diet. Biosynthetic studiesusing radiolabled acetate and mevalonate as well as 13C2 labledacetate were conducted to test this hypothesis (see Experimentalsection for the conditions of the experiments).It was found that after purifying the albicanyl acetate (72)isolated from the biosynthetic test specimens several times there was noevidence for significant incorporation of the injected precursors.The lack of any significant incorporation from the injectedprecursors into albicanyl acetate can be interpreted in several ways: 1)the seven day course of the experiment was not long enough (i.e. 72 is133Table 8. Assignment of 1 H NMR Signals (C6D6) forAlbicanyl Diacetate (84).C# 8 1 H COSY*1 4.95 H22 1.88 H1, H3a, H3b3a 1.6 H2, H3b30 1.04 H2, H3a45 1.15 H6a, H6b6a 1.82 H5, H6b, H7a, H7bop 1.6 H5, H6a, H7a, H7b7a 1.95 H6a, H6b, H7b7f3 2.30 H6a, H6b, H7a89 2.68 Hl la, Hl lb1011a 4.49 H9,H1lb11b 4.15 H9,H1la12a 4.95 H12b12b 4.81 H12a13 0.8814 0.6415 0.75CH3CO 2.06, 1.75134* Correlations to proton in column 2synthesized at a very slow rate and retained for long periods within thedorsal glands of the nudibranch); 2) neither acetate normevalonolactone are effective precursors for 72; 3) there is anundiscovered dietary source of 72 which grows along the entire BritishColumbian coastal range of the nudibranch, and for which the molluschas a strong grazing association.In light of the range of metabolites found within the extractsof Cadlina luteomarginata, and the reported lack of 72 in extracts fromSouthern California 42a the third explanation seems the most plausible.Our finding of 83 and 84 in the egg mass of C. luteomarginata istherefore more akin to the reports of Matsunaga et. al. 54 and Roesenerand Sheuer55 who found compounds (kabiramides A-E (95-99),dihydrohalichondramides (100-1), and ulapualides A and B (102-3).respectively) in nudibranch egg masses, which were related tohalichondramide (104) isolated from a Halichondria species of spongeby Kernan and Faulkner. 56 Given the structural similarity of the molluscanand poriferan metabolites, it is most likely that compounds 95-103 aredietary in origin and sequestered by the nudibranchs (perhaps aftermodification) into the egg masses.54 a) Matsunaga. S., Fusetani, N., Hashimoto, K., Koseki, K. and Noma. M. J. Am. Chem.Soc., 1986, 108, 847. b) Matsunaga, S., Fusetani, N., Hashimoto, K., Koseki, K., Noma,M., Noguchi, H. and Sankawa, U. J. Org. Chem., 1989. 54, 1360.55 Roesener, J. A. and Scheuer, P. J. J. Am. Chem. Soc., 1986, 108, 846.56 Keman, M. R. and Faulkner, D. J. Tetrahedron Lett., 1987, 28, 2809. Keman andFaulkner also note that kabiramide C was isolated from this sponge, but provide nospectroscopic data.135CH3^MeO 0^OMe 0O100 R = H101 R = Me0Ri R2 R395 CONH2 H Me% CONH2 OH Me97 CONH2 H H98 H H Me99 COMe H MeOH OMeOMeOMeOH102 R = 0 H103R= =(o0OMe136A further biosynthetic experiment utilizing radiolabledacetate in an attempt to determine whether Cadlino luteomarginata isresponsible for the modification of 72 into the acetoxy derivatives 83and 84 should be attempted when the nudibranchs enter the egglaying stage of their biological cycle.Dorid nudibranchs, and other opisthobranchs, seem to fallinto two general categories; those which exhibit variation in thecomposition of their metabolites with geographical location, and thosewhose extract compositions are constant throughout their geographicalrange. Faulkner and coworkers57 have recently put forward a hypothesisthat such differences can be used to distinguish between allomoneswhich are of dietary origin and those possibly synthesized de novo bythe molluscs, offering targets for biosynthetic studies.Since most extracts of Cadlino luteomarginata collectedin British Columbian waters contained albicanyl acetate (72), and it hasnot been reported as a metabolite from any other marine source,57 Faulkner, D. J., Molinski, T. F., Andersen, R. J.. Dumdei, E. J. and de Silva, E. D. Comp.Biochem. PhysioL, 1990, 97C, 233.137biosynthetic studies had to be conducted to examine the possibility thenudibranch was capable of synthesizing this allomone. Theseexperiments failed to show any incorporation of the injected precursorsinto the drimane metabolite and suggest that there is an unidentifiedporiferan source for the compound. Since 72 was most prevalent innudibranch specimens collected in areas of lower biological diversity(within coastal inlets such as Howe Sound and Hetta Inlet), is should bepossible to identify the sponge responsible for its production.3). Metabolites Isolated from Cadlina luteomarginata Collected atAnthony Island, British Columbia.Metabolites isolated from the Anthony Island collections ofCadlina luteomarginata could be classified in three types and roughlycorresponded to the location at which the opisthobranchs werecollected.Nudibranchs collected at the most seaward dive sitecontained predominantly sesquiterpenes like those found in the RennellSound collection and they could be linked to feeding on Acanthellawhich was also prevalent at this locale. Compounds 68, 69 and 78 wereonce again isolated along with the new sesquiterpenoid formamideacanthene K (85) which is related to metabolites isolated from theRennell Sound collection of Acanthella sp. by David L. Burgoyne in ourlaboratory.13868X=NC^85 X = NHCHO69 X = NCS78 X = NHCHOSpecimens collected while actively feeding on the spongeAplysilla glaciallis at the more Easterly dive site were found to containditerpenoids possessing either the marginatane or spongian carbonskeletons. Marginatafuran (74) was isolated previously from C.luteomarginata collected in the Queen Charlotte Islands42d and cannow be linked to the sponge Aplysilla glacial's. Other diterpenoids fromthis collection included 20-acetoxymarginatone (86), a compoundrelated to marginatone (105) which had been isolated from A. glacial'scollected in Barkley Sound, 58 and 9,11-dihydrogracillin A (87), acompound previously isolated from the Antarctic marine spongeDendrilla membranosa. 5958 Taschler, M., Andersen, R. J., Choudhary, M. I. and Clardy, J. J. Org. Chem., 1991, 56,42.59 Molinski, T. F. and Faulkner, D. J. J. Org. Chem., 1987, 52, 296.139HOOC14074^ 86 R = Ac0pAc 105 R=H87Specimens in the general collection from this dive sitecontained many of the above metabolites as well as microcionin-2 (70)and the new labdane diterpenoid lutenenolide (88).7013114^ 215Acanthene K (85).In addition to the above mentioned compounds 68, 69 and78, the extract from C. luteomarginata specimens collected at the firstAnthony Island dive site also contained the new sesquiterpenoidformamide acanthene K (85), a metabolite related to acanthene G(106), isolated from Rennell Sound collections of the sponge Acanthellasp..85 X = NHCHO106 X = NCSAcanthene K (85) was isolated as an amorphous white solid(3 mg, 1 individual) which gave a molecular ion peak at m/z 249.2098amu in the HREIMS spectrum, corresponding to a molecular formula ofC16H27NO (A M = 0.6 mmu). A mass of m/z 250 amu (M + H+) was also .the highest mass and most intense peak observed in the DCIMSspectrum of 85.The 1 H NMR spectrum (CDCI3, Figure 68) contained featuresthat clearly indicated the relationship of 85 to acanthene G (106). Twosinglet resonances at 8 4.84 (H14b) and 8 4.64 (H14a) appropriate for anexocyclic methylene; a methine resonance at 8 4.05 (q, J = 10.6 Hz, H6);a resonance integrating for a single proton at 8 2.31 (bd, J = 12.7 Hz,H3a); a resonance which integrated for six protons at 8 0.91 (d, J = 6.9141131 2)11485 X = NHCHO.........rr•rwirup•r•••••• ............y..............r^ ••••■•••••1.11.41•••••TI,••••••••• •••••••• .1P...1"i5^5^4 3^2^1........pp. 91 5Figure 68. 500 MHz 1 H NMR Spectrum (CDCI3) of Acanthene K (85)Hz, Me12, Me13), and a methyl singlet resonance at 8 0.77 (Me15). Theformamide functionality could be discerned from the signals at 8 8.31 (s)and 8 4.5 (br, exch.).The 130 NMR spectrum of 85 (CDCI3, Figure 69) displayed 15carbon resonances corresponding to: three methyls (8 21.6, 17.3, 16.1),five methylenes (8 42.2, 40.4, 38.3, 24.2, 18.4), four methines (8 57.0, 50.5,46.1, 26.5), an exocyclic methylene (8 145.7, 108.0) and a formyl group(8 160.8). These resonances accounted for all but the 010 carbon of themolecule.1 H- 1 H COSY correlations (CDCI3, Figure 70) could beobserved between the six proton doublet resonance at 8 0.91 (Me12,Me13) and the resonance of an unresolved methine at 8 1.90 (H11),constituting an isopropyl group. Another spin system was defined byobservation of coupling between the apparent quartet at 8 4.05 (F16)and two methines at 8 1.75 (d, J =10.9 Hz, H5) and 8 1.12 (m, H7).Interpretation of the remaining spin systems was accomplished with thehelp of a Heteronuclear Multiple Quantum Coherence (HMQC)spectrum (Figure 71), and is summarized in Table 9.The relative stereochemistry between the chiral carbons C5-C7 of 85 has been assigned as the same as acanthene G (106), isolatedfrom the nudibranch's prey sponge, 60 on the basis of observed couplingconstants (10.6-10.9 Hz, signifying axial-axial relationships). The trans ringfusion was assigned by a comparison of the 13C NMR chemical shifts ofthe C15 methyl of 85 to those reported for the C15 methyl of 106 andthe cis eudesmane compound 107, isolated by Ciminello et. al. from6° Burgoyne, David L., PhD thesis, University of British Columbia, 1992143ppm NO^140^120^100^BO^5U^49Figure 69. 125 MHz 13C NMR Spectrum (CDCI3) of Acanthene K (85)13114^285 X = NHCHO150.0-^1.0- 2.0• 3.0- 4.0- 5.06.0• 7.0- 8.01H11-Mel2JVIel3i108..•,• ,11-15-H6 el^.:- .':•`,.t...Al^;-•.^8-1-1A-^s, AlV^41•_...—.._1458.0 7.0 6.0 5.0 4.0PPM3.0^2.0 1 .0 0.0Figure 70. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofAcanthene K (85)151460500050P01385 X = NHCHO.•..4" IP.1.4,••44 • .•12PPPS 6 4^2Figure 71. 500 MHz HMQC Spectrum (CDCI3) of Acanthene K (85)the sponge Axinella cannabina collected off the coast of Italy 61 (5 28.0(cis 107)vs 5 17.1 (trans 106)vs 5 17.3 (85) ).-15The isolation of acanthene K from Cadlina luteomarginataonce again demonstrates that the nudibranch is an excellent source ofmetabolites from its prey sponges. Although 85 was not isolated fromAcanthella, it is unlikely that it is a hydrolysis product of acanthene G,caused either by the isolation procedure or the nudibranch's digestiveprocesses, since compound 69 is also present in the extract. Rather, it isa demonstration of how opisthobranchs are able to sequester andconcentrate interesting metabolites which may be in concentrationstoo small to detect in their source organisms.14761 Ciminello, P., Magno, S., Mayo!, L. and Piccialli, V. J. Not. Prod.,1987 , 50, 21711^1315Table 9. Assignment of NMR Signals (CDCI3) for Acanthene K (85).C# 8130 81H COSY*la 42.2 1.2 H1f3, H210 1.5 Hla, H22 24.2 1.6 (2H) H1 a, H113, H3a, H3f33a 38.3 2.31 H2, H313313 1.89 H2, H3a4 145.75 57.0 1.75 H66 46.1 4.05 H5, H77 50.5 1.12 H6, H8a, H8138a 18.4 1.52 H7, H8f3, H9a, H913813 1.37 H7, H8a, H9a, H9139a 40.4 1.25 H8a, H8f3, H9fi913 1.55 H8a, H813, H9a1011 26.5 1.90 Me12, Me1312 16.1 0.91 H1113 21.6 0.91 H1114a 108.0 4.64 H14b14b 4.84 H14a15 17.3 0.77NHCHO  160.8 4.5, 8.1314^k^1285 X = NHCHO148* Correlations to proton in column 3Marginatafuran (74)Previously isolated from C. luteomarginata collected in theQueen Charlotte Islands, marginatafuran (72) was the first reportedexample of a diterpenoid with the C8-C16 C ring closure. 42d Thestructure was solved by X-ray diffraction analysis.In the current study, 74 has been reisolated from specimens,found feeding on the sponge Aplysilla glacialis which were collectedoff Anthony Island.Although the structure was solved by comparison to thepublished data, the reported NMR data were incomplete and thereforethe usual experiments ( 1 H and 13C NMR, COSY, HMQC) were run on 74in order to flesh out the available information and provide a basis forcomparison with other marginatane diterpenoids. The results aresummarized in Table 10 and the spectra (Figures A08-A11) can be foundin Appendix A.149Table 10. Assignment of NMR Signals (CDCI3) for Marginatafuran (74)C# 5 13C 8 1 H COSY*la 38.6 0.67 H113, H2a, H213113 2.52 Hla, H2a, H2132a 20.5 1.4 Hla, H113, H213, H3a, H313213 1.4 Hla, H113, H2a, H3a, H3133a 42.5 1.08 H2a, H213, H313313 1.35 H2a, H213, H3a4 33.85 56.4 0.84 H6a, H6136a 19.0 1.56 H5, H613, H7a, H713613 2.3 H5, H6a, H7a, H7137a 37.3 1.4 H6a, Haft, H713713 2.3 H6a, H6f3, H7a8 48.59 55.9 1.42 Hlla,H111310 37.41 la 20.9 1.70 H9, H1113, H12a, H12131113 2.05 H9, H1 la, H12a, H121312a 23.3 2.22 Hlla,H1113,H1213Hlla,H11f3,H12a1213 2.313 113.914 110.2 6.00 H1515 140.5 7.09 H1416 159.517 23.0 1.0318 33.8 0.8519 20.0 1.3120 182.2-„19^"18 74150* Correlations to the proton in column 3.20-Acetoxymarginatone (86)The most abundant metabolite present in this collection ofC. luteomarginata, was the diterpenoid acetate, 20-acetoxymarginat-12-one (86).,,,,'1 819^8620-acetoxymarginatone (86) was isolated as a colorless oil(26 mg, 1.8 mg/animal) which displayed a molecular ion at m/z358.2144 amu in the HREIMS spectrum, corresponding to a molecularformula of C22H3004 (A M = 0.0 mmu).The 1 H NMR spectrum (CDCI3, Figure 72) containedresonances characteristic of furan (8 7.24, bs, H15; 8 6.56, bs, H14) andacetoxy methylene (8 4.65, d, J = 12.2 Hz, H20a; 8 4.25 d, J = 12.2 Hz,H20b; 8 2.03 s, 3H) functionalities. As well, three upfield methyl singletresonances (8, 1.32, 0.87, 0.84), resonances for a geminal methylene pairat 8 2.82 (dd, J = 13.6, 17.0 Hz, 1111a) and 8 2.70 (dd, J = 2.4, 17.0 Hz,Hllb) and a broad doublet resonance at 8 2.33 (J = 13.0 Hz, H7a) couldbe distinguished from the hydrocarbon envelope.1511415H15 H14Me19Me17 Me18H2°21 H20bHllbH1laH7aFigure 72. 500 MHz 1 H NMR Spectrum (CDCI3) of 20-acetoxymorginatone (86)1 H- 1 H COSY (CDCI3, Figure 73) correlations could beobserved which defined three isolated spin systems composed of: thetwo furan proton resonances; the two acetoxy methylene protonresonances and the proton resonances at 8 2.82, 2.70 and 1.99 ppm(H9) (Figure 74).153H8 425/H86.5611 5^ 141^I  1_1"8 7.24/82.82 H1 H8 2.70H8 4.658 1.99Figure 74. COSY Spin Systems of 86Redisolving 86 in C6D6 aided the interpretation of the 1 HNMR spectrum (Figure 75). The two H11 methylene resonancescollapsed into a two proton multiplet at 8 2.75 (H11/H11'). The furanproton resonances shifted to 8 6.84 (d, J = 1.8 Hz, H15) and 8 6.65 (d, J =1.8 Hz, H14) while the acetoxy methylene proton resonances shiftedslightly to 8 4.67 (d, J = 12.2 Hz, H20a) and 8 4.05 (d, J = 12.2 Hz, H20b). Allfour methyl resonances shifted upfield (8 1.58, Ac; 8 1.10, Me 17; 8 0.71,Me18; 8 0.67, Me19) and several other resonances became resolvedfrom the hydrocarbon envelope (8 2.08, bd, J = 12.5 Hz, H7a; 8 1.79, bd, J= 12.6 Hz, H1(3; 8 0.94, dt, J = 4.4, 14.3 Hz, H3a; 8 0.62, bd, J = 12.2 Hz, H5; 80.41, bt, J = 12.1 Hz, H1 a).The 13C NMR and APT spectra of 86 (C6D6, Figure 76)showed all 22 carbon resonances. Carbon resonances corresponding tofuran (8 170.0, 142.3, 118.9, 106.7) and acetoxy methylene (8 174.4, 64.2,21.6) functionalities could easily be distinguished, as well as theFigure 73.400 MHz 1 H- 1 HCOSY Spectrum (CDCI3) of20-ocetoxymorginotone (86)0.01 .02.03.0- 4.0- 5.0- 6.07.0PPM• ,5.0 4.•^3PPM .0•^•^-^,^•7.0 6.0 2.0^1.0^0.014 Mel 8Me1915H20a H20bH15 H14154AcH1 laH1 lbH7aMel 7S 0 • OgefbH200-H20b/ 1•• H154114a.• ••Mel 7Mel 8Me19HH15 H14HllaHllb8 *I;er-■•••••■•■■rorp-7 15^4^3^2^I^0PON 7Figure 75. 500 MHz 1 H NMR Spectrum (C6D6) of 20-acetoxymarginatone (86)gC20C15C14 C5 C9Cla C7c3 c8 C18C11 ClC2Ac COC17C19• • 1^1 •••• 1• • • • I • • • 11$0 160^140^1^100^SO^60^40Flip* 76. 75 MHz 13C/APT NMR Spectra (C6D6) of 20-acetoxyrnorginotone (86)20 PPM .0resonances of an a,13 unsaturated ketone carbon at 8 192.3 and threemethyl carbons at 8 33.5, 20.4 and 19.2 ppm.1 H- 1 H COSY (C6D6, Figure 77) correlations could beobserved that delineated the spin systems of 86. The H14 resonance (86.65) was coupled to the H15 resonance (8 6.64). The resonance forproton H20a (8 4.67) displayed coupling into the resonance of itsgeminal proton H20b (8 4.05) as well as w coupling into the H9resonance (8 1.5). The H20b resonance, however, showed w couplinginto the resonance at 8 0.41 (Hl a), indicating that rotation around theC10-C20 bond is sterically hindered. The geminal methylene H11 protons(8 2.70) also gave a strong correlation into the H9 resonance.The HMQC spectrum of 86 (C6D6, Figure 78) allowed theassignment of protons to the carbons to which they were attached andgreatly aided the interpretation of the A and B ring spin systems of 86 inthe COSY spectrum. The complete assignment of NMR chemical shiftscan be found in Table 11.While the relative stereochemistry is without ambiguitiesonce trans ring fusions are established, nuclear Overhauser differenceexperiments did aid in assigning some of the signals, and revealed a fewinteresting features. Irradiation of the H11/H11' resonance (8 2.70) led tothe enhancement of signals corresponding to H113 (8 1.79), H9 (8 1.5),Me17 (8 1.10) and, interestingly, the acetate methyl at 8 1.58. Irradiationof the H20a proton resonance (8 4.67) led to enhancement of signalscorresponding to H20b (8 4.05), Me19 (8 0.67) and H2O (8 1.5), whileirradiating the resonance of H20b gave enhancement at resonancescorresponding to H20a, Me17 and H60 (8 1.16). These results indicatethat the C20 acetate has a specific orientation with respect to the rest1571415I^•^I1580.0031.01.52.02.53.0334.04.55.0536.06.57.0PPM7.0^6.5 6.0^53^5.0^4.5 4.0^33^3.0^2.5^2.0^13^1.0^0.5^0.0PPMFigure 77. 400 MHz 1 H- 1 H COSY Spectrum (C6D6) of20-ocetoxymorginotone (86)1I• 44+ 440.. 4• ••4.i-4.100C14C15C6 C2. Ac C17 C19Cl C7 C18C11C350CC5 9C201415159PPII^H14 6H15H20a^4H20bH11 H7132 Fua H9 Me19Ac^H5Me18Me 17I-12a H2f3H3ct 143t3 HlaHPFigure 78. 500 MHz HMQC Spectrum (C6D6) of20-ocetoxymarginatone (86)of the molecule (Figure 79) and offer an explanation for the w couplingobserved from the H2O protons into the methine protons at C5 and C9.Figure 79. NOE Summary for 869,11-Dihydrogracillin A (87):A small amount (-1 mg) of compound 87 was also isolatedfrom the Cadlino luteomarginata collected while feeding on Aplysillaglacialis at this dive site. Comparison of the 1 H NMR spectra and EIMSresults (see Figure Al2 in Appendix A and Experimental section) topublished data59 confirmed the compound was 9,11-dihydrogracillin A.pAc160Table 11. Assignment of NMR Signals (C6D6) for20-Acetoxymarginatone (86)C# 8 13C 6 1H COSY* NOE**1 a —^34.2 0.41 H10, H2a, H213, H20b113 1.79 Hla, H2a, H2132a 17.9 1.27 H1 a, H 113, H213, H3a, H313213 1.5 Hla, H113, H2a, H3a, H3133a 41.5 0.94 H2a, H213, H31338 1.25 H2a, H213, H3s4 32.85 56.3 0.62 H6a, HOP6a 18.3 1.35 H5, H613, H7a, H713613 1.16 H5, H6a, H7a, H7137a 36.0 2.08 H6a, H613, H71370 1.24 Hem, H613, H7a8 37.49 55.1 1.5 H11, H20010 40.611 38.4 2.75, (m, 2H) H9 H10, H9, Me17,Ac12 193.613 118.914 106.7 6.65 H1515 142.3 6.84 H1416 170.017 19.2 1.1018 33.5 0.7119 20.4 0.6720a 64.2 4.67 H9, H20b H20b, Me 19, H2(320b 4.05 Hla,H20a H20a, Me 17, H6pAc 21.6, 174.4 1.58* Correlations to the proton in column 3** Proton in column 3 irradiated161The isolation of the above three compounds fromnudibranchs found feeding on Aplysilla glacialis raises a few points.Aplysilla sponges are known to produce compounds related to 74, 86and 87,62 although these compounds themselves have not been foundin sponges of that genus. It is also of interest to note that themarginatanes 74 and 8 6 are both oxidized at C20, even thoughcompound 105, isolated from Aplysilla glacialis collected in BarkleySound, is not. The small amount of A. glacialis collected at AnthonyIsland did not allow for a chemical comparison with the nudibranchmetabolites. It would also be worthwhile to investigate the spongemetabolites from this dive site in order to check for geographic variationwithin the sponge.Precedent for a geographical variation between BarkleySound and Anthony Island poriferan populations was found recently byother investigators in our laboratory. 63 The sponge Xestospongia vanillawas found to contain a series of triterpenoid and degraded triterpenoidglycosides at both sites, of which xestovanin A (1 0 8) anddehydroxestovanin A (109) are examples. The Anthony Islandcollections, however, were found to contain only the 10,27-dehydroversions of the compounds (e. g. 109), while both forms were present inthe Barkley Sound collections. Considering this, perhaps the presence of74 and 86 in the extracts of C. luteomarginata is a sensitive indicator ofa similar variation in the chemistry of A. glacialis. It would also be of62 For examples see references 13b, 37 and Poiner, A. and Taylor, W. C. Aust. J. Chem.,1990, 43, 171363 Morris, S. A., Northcote, P. T. and Andersen, R. J. Con. J. Chem., 1991, 69, 1352.162interest to see if the sponge contains compound 8 7 which hasheretofore only been isolated from an Antarctic species. 59163OR'108OR'109Microcionin -2 (70):70The most prevalent metabolite (28 mg, 0.25 mg/animal) inthe general collection of Cadlina luteomarginata, collected at AnthonyIsland, was microcionin-2 (70). Compound 70 had previously beenisolated from sponges in the genus Microcionia 44 and from collectionsof C. luteomarginata from Southern British Columbia. Andersen andHellou have shown compound 70 to be an effective antifeedant. 42bLutenenolide (88):Lutenenolide (88) was isolated as colorless oil (5.2 mg, .05mg/ animal) which failed to give rise to a molecular ion peak in theHREIMS spectrum. The highest mass observed was at m/z 300.2091 amu,corresponding to a molecular formula of C20E -I2802 M = 0.2 mmu).Through examination of the 1 H NMR spectra (Figure 80, an acetateresonance at 5 1.99), this peak could be interpreted as corresponding tothe loss of one equivalent of acetic acid from the molecular ion. Themolecular formula thus established (C22H3204) has an unsaturationindex of seven.The 1 H NMR spectrum (CDCI3, Figure 80) displayedresonances with coupling patterns and chemical shifts characteristic ofexocyclic methylene (5 5.12, bs, H17a; 5 5.00, bs, H17b) and acetoxymethine (5 5.59, m, H11; 1.99, s, 3H) moieties. Three upfield methyl singletresonances (5 0.83, Me18; 0.81, Me20; 0.79, Me19), a methine 1 Hresonance at 5 7.19 (s, H14) and a two proton multiplet resonance at S4.74 ppm (H15/H15") could also be easily distinguished.The 13C NMR spectrum (CDCI3, Figure 81) containedeighteen carbon resonances accounting for all but the carbonyl and164 sFigure 80. 500 MHz 1 H NMR Spectrum (CDCI3) of Lutenenolide (88)40^20116, 1.1^ Licii^Limi.1^IP08 140^120 100^80Figure 81. 125 MHz 13C NMR Spectrum (CDCI3) of Lutenenolide (88)quaternary carbon signals. Resonances at 8 110.4 (C17) and 144.4 (C8)ppm confirmed the exocyclic methylene, while a methine carbonresonance at 8 145.8 (C14) along with an IR absorption at 1752 cm-1suggested a butenolide functionality. Resonances appropriate formethine and methylene carbons attached to oxygen atoms (8 70.2,Cll; 8 70.2, C15) were present, as well as additional resonances for twomethine (8 59.6, C9; 8 56.2, C5), six methylene (8 41.9, C3; 8 39.2, C7; 839.0, Cl; 8 30.5, C12; 8 24.4, C6 and 8 19.1 C2) and four methyl carbons(8 33.7, C18; 8 21.7, 019; 8 21.5, Ac and 8 15.8, C20). Five of the sevensites of unsaturation could be accounted for by the acetate, butenolideand exocyclic methylene moieties. The presence of two additional ringscould therefore be deduced.The 1 H- 1 H COSY spectrum (CDCI3, Figure 82) was able tooutline several spin systems for the molecule. For one spin system,coupling could be observed between the methine resonance at 8 7.19(H 14) and the multiplet resonance integrating for two protons at 8 4.74ppm (H 15) which showed further weak (homoallylic) coupling into aresonance at 8 2.61 (ddd, J = 1.9, 7.9, 15.6 Hz, H12b). The H12bresonance was further coupled into the resonance at 8 2.83 (ddd, J =1.6, 5.7, 15.6 Hz, H12a). Both of these resonances (H12a, H12b) possessedfurther coupling into the acetoxy methine proton resonance at 8 5.59(H11) (Spin system A, Figure 83). A second spin system was evidenced bycoupling from a resonance at 8 2.35 (dm, J = 12.2Hz, H7a) to three otherproton resonances at 8 1.96 (H7B), 1.69 (H6a) and 1.34 ppm (H60). TheH6 proton resonances were further coupled into a resonance at 8 0.99ppm (dd, J= 2.9, 12.6 Hz, H5) (Spin system B, Figure 83). This spin systemwas very similar to the spin system on the B ring of albicanyl acetate1670.01.0- 2.0168) 8H 88go •0H11-H12b 3^1 •^H12a-H12bI' 4 H11-H12ai 1^!^ 1^11^i3.04.0- 5.0•6.07.0• H14-H156.015 0^4.0^3.0PPM 2.0^1.0^0.0H12b-H15Figure 82. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofLutenenolide (88)AcO 1214H7.19A85.59 H(72). A closer examination of the 13C NMR data in conjunction with theCOSY and an HMQC spectrum (Figure 84) revealed many similarities inthe spectral data for 88 and 72 which suggested a similar AB ring systemfor the two molecules (See Tables 7 and 12).169H 85.1217 8 5.00HH 8 1.96H82.83 H 82.611-H H84.7468 0.99 H^H H8 2 •358 1.69BFigure 83. Spin Systems for 88The Long Range COSY spectrum of 88 (CDCI3, Figure 85).designed to maximize the correlations resulting from weak couplings,picked out the allylic and homoallyic couplings within the moleculewhich allowed the two spin systems in Figure 83 above to be joined, andcomplete the structure of lutenenolide. Correlations were nowobservable from the H15/H15' resonance (8 4.74) to both H12resonances (8 2.83, 2.61) and from the H11 resonance (8 5.59) into aresonance at 8 1.82 (bs, H9). The H9 resonance was also coupled intothe resonance for H17a (8 5.12, bs), while the H17b resonance (8 5.00)showed coupling to the resonance at 8 1.96 (H713).Additional, supporting evidence for the proposed structurewas found in the HMBC spectrum of 88 (Figure 86). Correlations could beobserved from the 1 H methyl resonance at 8 0.81 (Me 20) into theresonances of the Cl (8 39.0) and C9 (8 59.6) carbons. The Me19 1 H0a* ••il41.• Idr a+6•4• 14-• Now .. • .^.fi•f IPI1 ^PPM'.161714-1-111111-14444^...................44141-1-ITUTTITITTUTr21705000Figure 84. 500 MHz HMQC Spectrum (CDCI3) of Lutenenolide (88)-118^88171. .H9-H11. • H9-H17a^e/ ^/Ib^I.^le lb^04^, 4,' ,,r ofh e00 a Hlho-Ha H12a-H55,, r^•"^c1.•• •H7a-H17b ----- -^-1• is• a .40^30PPMFigure 85. 400 MHz 1 H- 1 H COSY-LR Spectrum (CDCI3) ofLutenenolide (88)7.0 6.0 5.0 20^1 00 .01.02.03.0- 4.0- 5.06.0- 7.0PPM00r-7 0••* •^• i^a^•^• 4^•H15-C13 •^H12a-C13 •rPP.172Figure 86. 500 MHz HMBC Spectrum (CDCI3) of Lutenenolide (88)resonance (8 0.79) gave correlations into carbon resonancescorresponding to C3 (8 41.9), C5 (8 56.2) and Me 18 (8 33.7). Correlationsinto the carbon resonances of C3 and C5 were also observed from theMe18 1 H resonance (8 0.83). The Me18 1 H resonance also gave acorrelation into the Me19 carbon resonance at 8 21.7. Heteronuclearmultiple bond correlations could also be observed from the H17a andH17b resonances (8 5.12, 5.00) into C9 and C7 (8 39.2) carbonresonances, confirming the allylic couplings observed in the long rangeCOSY. Correlations from proton resonances at 8 2.83 (H12a) and 4.74(H15's) into a carbon signal at 130 ppm accounted for one of themissing carbon resonances (C13). The acetate carbonyl (8 171) couldalso be assigned from the HMBC spectrum. See Table 12 for thecomplete assignment of NMR data.An attempt to determine the relative stereochemistry at theC11 acetate through the use of NOE difference experiments yieldedmixed results. The free rotation about all the bonds in the C9-C 13 portionof the molecule makes interpretation of the observed enhancementsproblematic. Irradiation at the H11 resonance (8 5.59) enhanced signalscorresponding to H9 (8 1.82), H12a (8 2.83), H1(3 (8 1.75) and H14 (8 7.19).These NOEs are equally explainable from either stereochemical isomerdepending on the conformational placement of the Cl 1-C16 sidechain. Observed NOEs between the H5 resonance (8 0.99) and H9confirmed the relative stereochemistry at these centers.A review of the literature failed to reveal any previousreports for the presence of labdane diterpenoids in opisthobranch173Table 12. Assignment of NMR Signals (CDCI3) for Lutenenolide (88)C# s 13C 8 1 H COSY* COSY LR* HMBC** NOE***la 39.0 1.07 H1f3, H2 H9, Me20113 1.75 Hla,H22 19.1 1.5 (2H) Hla, H113, H3a, H3133a 41.9 1.14 H2, H3f3 Me18, Me1938 1.37 H2, H3a5 56.2 0.99 H613 H6a Me18 H96a 24.4 1.69 H6f3, H7a, H713 H560 1.34 H5, H6a, H7a, H7137a 39.2 2.35 H6a, H6f3, H7137fi 1.96 H6a, H613, H7a H17b H9, H17a,H17b8 144.8 H99 59.6 1.82 H 17a H11,H17a H17a,H17b,Me20H511 70.2 5.59 H12a, H12b H9 H12a H1B, H9,H14, H1212a 30.5 2.83 H11,H12b H14, H1512b 2.61 H11, H12a H14, H15 H9, H12a13 (130) H12a, H1514 145.8 7.19 H15 H12a, H12b H15 H1515 70.2 4.74(2H) H14, H12b H12a, H12b H14 H1417a 110.4 5.12 H9 H70 H17b17b 5.00 H70 H70, H17018 33.7 0.83 Me1919 21.7 0.79 Me1820 15.8 0.81 H9OAc 21.5(171)1.99 OAc* Correlations to the proton in column 3** Correlations to the carbon in column 2*** Proton in column 3 irradiated174Hpi OR289 R1 = °COP!" R2 = H^90121=H^R2 = AC^110R1 =H R2 = H73extracts. Lutenenolide (88) therefore widens the scope of metabolitesfound in marine molluscs.4). Metabolites from Cadlina luteomarginata Collected at AgamemnonChannel, Jervis Inlet, B. C.Specimens of C. luteomarginata collected at AgamemnonChannel, Jervis Inlet, B. C. contained two previously reported and twonew terpenoid metabolites. Luteone (73) was previously isolated fromBritish Columbian specimens of C. luteomarginata collected in Howeand Barkley Sounds. 42. The oxidospongian 89 is related to compound 82,described above, and was isolated from the same Caribbean sponge. 48Compound 90, another oxidospongian, has not been previouslyreported, though the desacetoxy version (110) has been found in thenudibranch Ceratosoma brevicaudatum, collected off Australia. 64Cadlinaldehyde (91) is a unique C21 diterpenoid metabolite with a newcarbon skeleton.17564 Ksebati, M. B. and Schmitz, F. J. J. Org. Chem., 1987, 52, 3766.Luteone (73):19Previously isolated from specimens of the nudibranchcollected in Howe and Barkley Sounds, luteone's structure was solved byX-ray diffraction analysis of its 2,4-dinitrophenyl hydrazone derivative. 42cThe reported NMR data, however, were incomplete on the nativecompound. As with marginatafuran (74), spectra 0 H and 13C NMR,HMQC, HMBC: Figures A13 - A16 in Appendix A) were run for this knowncompound to flesh out and complete the spectroscopic assignmentsand to facilitate data comparison with related compounds. The resultsare summarized in Table 13 below.176Table 13. Assignment of NMR Signals (CDCI3) for Luteone (73)C# 8 13C 8 1 H COSY* HMBC**la 34.4 0.6 H1B, H2a, H2B, H23113 2.52 H la, H2a, H282a 19.4 1.35 Hla, H1f3, H213, H3a, H313 H la213 1.15 Hla, H113, H2a, H3a, H3133a 41.7 1.3 H2a, H213, H313 Me21, Me223[3 1.1 H2a, H213, H3a4 33.6 Me21, Me225 55.1 1.2 H6 Me21, Me226 18.4 1.7 (2H) H5, H7a, H7137a 39.5 1.99 H6, H713 Me207f3 1.3 H6, H7a8 39.89 60.2 1.33 Hlla,H1113 Me2010 53.8 H231 la 23.5 1.78 H9, H1113, H12a1113 0.95 H9, H1 la, H12a, H121312a 37.8 2.3 Hlla,H1113,H1213 H19a, H19b1213 1.8 H1 la, H1113, H12a, H19a13 147.114 55.2 1.55 H19a, H19b H19a, H19b, Me2015a 17.6 1.85 H15b, H16a, H16b15b 1.45 H15a, H16a, H16b16a 42.5 2.34 H15a, H15b, H16b16b 2.58 H15a, H15b, H16a17 210.5 Me1818 30.1 2.0219a 106.9 4.81 H14, H1213,H19b19b 4.43 H14,H19a20 16.3 0.5721 31.9 0.91 H313, Me2222 20.7 0.75 Me2123 206.3 10.119* Correlations to the proton in column 3** Correlations to the carbon in column 2177Oxidospongians (89-90):89 R i = OBU, R2 = OH9012 1 =H^R2 = OAcTwo compounds related to the spongian diterpenoids werealso isolated from specimens in this collection. Compound 89 (7a,178-dihydroxy- 15,17-oxidospongian-16-one 7-butyrate) has been reportedas a metabolite of the Caribbean sponge Ingemella nobilis 48 and is verysimilar to compound 82 isolated in the Rennell sound collection, differingonly in the ester at C7. The structure was solved by comparison withreported data (see Experimental). The 1 H NMR spectrum for 89 can befound in Appendix A (Figure A 17).A second compound, 90, eluted off the HPLC slightly laterthan compound 89. Spectroscopic evidence and comparison to knownmetabolites led to the conclusion that the compound was 17f3-acetoxy-15,17-oxidospongian-16-one.178Compound 90 (1713-acetoxy-15,17-oxidospongian-16-one):Compound 90 was isolated as a colorless oil (1.7 mg, 0.03mg/animal) which gave a molecular ion peak in the HREIMS spectrumat m/z 376.2249 amu, corresponding to a molecular formula ofC22H3205 M = 0.0 mmu). The base peak in the DCIMS spectrum of 90was at m/z 393 amu, which could be assigned as an M+ + NH3 peak.Examination of the 1 H NMR spectrum of 90 (CDCI3, Figure87) indicated a close similarity to the other oxidospongian compounds(82 and 89) isolated in collections of Cadlina luteomarginata. Adownfield singlet resonance at 8 6.29 (H 17), a doublet resonance at 86.10 (J = 6.0 Hz, H15), three proton resonances at 8 2.76 (dd J = 6.9, 11.2Hz, H13), 2.65 (dd, J = 6.0, 11.2 Hz, H14), and 2.45 ppm (dm J = 12.5 Hz,H1213), an acetate methyl resonance (8 2.04, s) and three methyl singletresonances (8 0.86, 0.81, 0.72) were all well resolved.The 13C NMR spectrum of 90 (CDCI3, Figure 88) displayedseventeen resonances corresponding to: four methyls (8 33.4, 21.5, 21.3,15.6); seven methylenes (8 41.8, 41.7, 39.0, 23.7, 19.9, 18.7, 16.6) and sixmethines (8 104.2, 100.6, 56.8, 55.3, 49.2, 37.5) which accounted for allbut the quaternary carbons in the molecule. Of particular note was the179Figure 87. 500 MHz 1 H NMR Spectrum (CDCI3) of 1711-acetoxy-15,17-oxidospongian-16-one (90)890 R2 = H R2 = OACiLa/ Is ALA latA100Figure 88. 125 MHz 13C NMR Spectrum (CDCI3) of 1713-acetoxy-15,17-oxidospongian-16-one (90)presence of two acetal carbon resonances at 8 104.2 and 100.6 ppm,and the lack of any other carbinol methine resonance.Analysis of the 1 H- 1 H COSY spectrum (CDCI3, Figure 89)coupled with an HMQC spectrum (Figure 90) elucidated the spinsystems in the C and D rings. The acetal doublet resonance at 8 6.10(H 15) was coupled into the methine resonance at 8 2.65 (H 14). The H14resonance was further coupled into the methine resonance at 8 2.76(H 13) which was also coupled into the resonances of a geminalmethylene pair at 8 2.45 (H12(3) and 1.6 ppm (H12a). Further couplingfrom the H 12 proton resonances into resonances at 8 1.94 (H 11 0) and1.19 ppm (H11 a) and from these resonances into a methine resonanceat 8 1.3 (H9) completed the spin system below.1820H86.10Comparison with the data obtained for compound 82gable 14) indicated that the acetate was attached to the acetal atC17 and that the C7 position was unoxidized. Further comparison withthe data published for the desacetoxy compound (110 see Table 15)confirmed our assignments and the structure.Figure 89.400 MHz 1 H- 1 H COSY Spectrum (CDCI3) of178-acetoxy-15,17-oxidospongian-16-one (90)2.0 10^00BA/H12a-H13 el^#'8 VW/&^.i4 '0 /-*. IS0 en pat0 H14-H15 H13-H14 xSPaI•pto■P7 0^6 0^5.0^4 0 —^3.0PPM- 0.01.02.03.04.05.0- 6.0- 7.0PPM18390 R2 = H^R2 = OAC1.0IP•,1 ,It .•i4 •es•. 1.II ie 5^4 318440so100PPR: 20Figure 90. 500 MHz HMQC Spectrum (CDCI3) of1713-acetoxy-15,17-oxidospongian-16-one (90)Table 14. Assignment of NMR Signals (CDCI3) for Compounds 82 and 90170-acetoxy-15,17-oxidospongian-16-one (90) compound 82C# 8 13c 8 1H COSY* 813C 8 1 H1a 39.0 0.9 38.9 0.91(s 1.69 1.692a 18.7 1.5 18.7 1.6213 1.42 1.53a 41.8 1.1 41.8 1.13(3 1.4 1.44 32.85 55.3 0.9 48.5 1.286a 19.9 1.6 24.6 1.66f3 1.57 1.847a 41.7 1.13 73.0713 1.9 4.738 31.99 56.8 1.3 Hlla,H118 49.5 1.4810 38.01 la 16.6 1.19 H9, H118, H12a, H128 16.1 1.51113 1.94 H9, H1 la, H12a, H1213 1.9512a 23.7 1.6 H1 la, H1113, H128, H13 23.3 1.512t3 2.45 H1 la, Hi 1p, H12a 2.3613 37.5 2.76 H12a, H14 37.6 2.7414 49.2 2.65 H13,H15 42.2 2.8515 104.2 6.10 H14 104.3  6.0116 17717 100.6 6.29 103.6 5.4418 33.4 0.86 33.0 0.7519 21.3 0.80 21.2 0.7920 15.6 0.72 15.3 0.87Ester 21.5 2.04 21.4,170 2.12(OAc)2.66 (OH)82 R i = OAc, R2 = OH90 R2 = H R2 = OAC* Correlations to the proton in column 3185Table 15. Comparison of 1 H NMR Signals (CDCI3) forCompounds 90 and 11090 110C# 81H 81H6a 1.6 1.62 (m)61i 1.57 1.34 (dq, 3,12.4)7a 1.13 (m) 1.08 (dq, 4,12.4)7f1 1.9 1.87 (dt, 2.9, 12.4)9 1.3 (br d, 12) 1.46 (br d, 12.5)lla 1.19 (m) 1.64 (m)11 ii 1.94 (dm, 13.5) 1.93 (dq, 4, 12.5)'Mr. 1.6 1.60 (m)1 211 2.45 (dm, 12.5) 2.40 (br dt, 2, 12.5)13 2.76 (br dd, 6.9, 12.2) 2.72 (br dd, 8.3, 12)14 2.65 (dd, 6.0, 12.2) 2.58 (dd, 6.2, 12)15 6.10 (d, 6.0) 6.08 (d, 6.2)17 6.29 (s) 5.50 (br s)18 0.86 0.82 (s)19 0.80 0.86 (s)20 0.72 0.91 (s)OR 2.0490 R = OAc110 R = OH186Cadlinaldehyde (91):21Cadlinaldehyde (91) was isolated as an amorphous whitesolid (12 mg, 0.2 mg/animal) which was subsequently recrystallized frommethanol (colorless needles, mp = 65°C). The highest mass observed inthe HREIMS spectrum of 91 was at m/z 303.2308 amu, corresponding toa molecular formula of C20H3102 (A M = -1.6 mmu).The 1 H NMR spectrum of cadlinaldehyde (CDCI3, Figure 91)exhibited features that were reminiscent of luteone (73 see Figure A13).An aldehydic proton resonance at 5 10.1 ppm (s, H20) as well as a pairof resonances for geminal methylene protons at 5 2.52 (bd, J = 12 Hz,H113) and 0.7 ppm (m, Hla) gave evidence for close structural similarityto luteone. However, the exocyclic methylene and methyl ketonefunctionalities, present in 71, are absent in cadlinaldehyde. Instead,resonances for a geminal methylene pair at 5 3.83 (apparent q, J = 8.3Hz, H16a) and 3.92 ppm (dt, J = 2, 8.4 Hz, H16b), and a fourth upfieldmethyl resonance at 5 1.01 (Me21) are present.Interpretation of the 1 H- 1 H COSY spectrum (CDCI3, Figure92) proved to be difficult owing to the lack of dispersion in the upfield (1-2 ppm) region. However, some couplings could be clearly distinguished.187Figure 91. 500 MHz 1 H NMR Spectrum (CDCI3) of Codlinaldehyde (91)• 0.51 .0• 1.5- 2.0▪ 2.5- 3.03.5- 4.0- 4.5PPMMe18Me21 1  M919IMe17189 H16 A__""--r"-r4.5^4.0^3.5^3.0^2.5^2.0^1.5^1.0^0.5PPMFigure 92.400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofCadlinaldehyde (91)81.3H H 81.4 H81.7 H31.65H 812H^ H82.52H\ / 8 0.7 83.83 H83.92 8 1.4The resonances at 8 3.83 and 8 3.92 (H16a, H161)) were coupled into twoproton resonances at 8 1.7 (H15b) and 1.65 ppm (H15a) which werefurther coupled into a proton resonance at 8 1.4 (H14). The protonresonance at 8 2.52 (H1 r) was coupled into the resonance of its geminalpartner at 8 0.7 (Hla) and into two proton resonances at 8 1.3 (H2a) and1.4 ppm (H2(3). Both H2 proton resonances also displayed coupling intoanother resonance at 8 1.2 (H3a) (Figure 93).Figure 93. Spin Systems for Cadlinaldehyde (91)At first, given the EIMS evidence, it was believed 91 was asimple diterpenoid. However no C20 skeleton could accommodate thenumber of methyl singlets (five, including the aldehyde) and thecoupling patterns observed. The small amount of 91 isolated (<3 mg)prevented us from obtaining a complete 13C NMR spectrum andseverely hindered the interpretation of the heteronuclear coherencespectra. Therefore, a second collection of Cadlina luteomarginata atAgamemnon Channel was undertaken, during August 1992, in order toobtain more of the compound for further spectroscopic analysis. The 1 HNMR and tic analysis of 91 isolated from this newer collection (12 mg)were identical with the compound isolated previously. With this largeramount of material, crystallization from methanol yielded 91 of greaterpurity for further spectroscopic analysis.190Although El mass spectrometric techniques yieldedidentical results for both collections, the crystalline material resulted in abase peak of m/z 319 in the DCIMS spectrum with a highest mass at m/z336. Examination of the 130 NMR spectrum (Figure 94, twenty-oneresonances) allowed the interpretation of these peaks as the molecularion plus a proton (M + H+) and the molecular ion following the additionof the ionizing agent (M + NH4).-The 130/APT NMR spectra (Figure 94) of 91 now clearlydisplayed twenty-one resonances. Resonances corresponding to analdehydic carbon (8 206.4), three methine carbons (8 61.4, 59.5 and 55.6ppm), nine methylene carbons (8 64.9, 41.7, 40.4, 39.3, 34.4, 23.0, 19.9,19.4 and 17.5 ppm), four methyl carbons (8 32.0, 20.8, 20.8 and 16.6ppm) and four quaternary carbons (8 79.0, 53.4, 36.5 and 33.7 ppm)could be discerned.An HMQC spectrum (Figure 95) now enabled thecorrelation of the carbon resonances to the resonances of attachedprotons and facilitated the interpretation of the COSY spectrum for theremaining spin systems (Table 16).The HMBC spectrum of 91 (Figure 96) was by far the mostinformative of all the spectroscopic techniques employed. Correlationscould be observed which confirmed the D ring connectivity, assignedthe quaternary carbons and led to the final structure.HMBC correlations from the four methyl groups (Figure 97)were very helpful in establishing connectivities within the molecule. Themost downfield methyl 1 H resonance (8 1.01, Me21) gave clearcorrelations into the carbon resonances corresponding to C12 (8 39.3),C13 (8 79.5) and C14 (8 59.5). The Me17 1 H resonance (8 0.73) gave191C13 C10C19C21C20C16 C14C9C5C4C8C7 Cl^C11C3 C12 C18^C2C15 C6C171 40 r^I^11101 I 1 rr 1"i TT FT TT" FrrBO^60^40^11111111112^0 20PPMFigure 94. 75 MHz 13C NMR Spectrum (CDCI3) of Cadlinaldehyde (91). 0Co ..3/40• 4 a•4140 4:91/4i4••iI• .s1iiC17 C11co C220 C19 C21C15C18ClC12 C74° C3C5-GO C14C9C1621193NW H16 3 H1f3 2 H15 H14 H9^Me19H12a^H5^Me18 HlaH118 H7a H128 Me21 Me17H1 la H30 H3aHO I-12aFigure 95. 500 MHz HMQC Spectrum (CDCI3) ofCadlinaldehyde (91)• a•6 .•A11/0• H2O-C10• H16b-C13• 61• 1I.•^•iH5,H9-1C20 • :PPS^6^6^4^2Figure 96. 500 MHz HMBC Spectrum (CDCI3) ofCadlinaldehyde (91)1945010015019516HFigure 97. HMBC Correlations from the Methyl groups of 91correlations into the C7 (8 40.4), C8 (8 36.5), C9 (8 61.4) and C 14 carbonresonances. The Me18 1 H resonance (8 0.94) correlated into the 13Cresonances of C3 (8 41.7), C4 (8 33.7), C5 (8 55.6) and C19 (8 20.8), whileMe19 1 H resonance (8 0.80) yielded correlations into the C3, C4, C5 andC18 (8 32.0) 13C resonances.Several other correlations could be observed which alsoaided in the structural elucidation and assignment of NMR signals for 91(Figure 98, Table 16). the resonance for H18 (8 2.52) was found tocorrelate into resonances for C3 (8 41.7) and C5 (8 33.7), while both H6proton resonances (8 1.35, 1.7) also correlated into the 05 resonance.The Hlla proton resonance (8 1.7) gave correlations into both the C9 (861.4) and C12 (8 39.3) carbon signals, while the H12a resonance (8 1.85)was found to correlate into the C11 (8 17.5) and C 13 (8 79.5) carbonsignals. The 1 H resonances of H128 (8 1.35), H14 (8 1.4), H15a (8 1.65) andH16b (8 3.93) also all gave correlations into the C13 carbon resonance.In addition, the H14 resonance correlated into the carbon resonance at20.8 (C21) and the H15a resonance correlated into 8 64.9 (C 16). EitherH5 (8 1.27) or H9 (8 1.25) gave a correlation into the C20 aldehyde .carbon (8 206.4), and the H2O resonance (8 10.1) correlated into 010 (853.4).196HMBC Correlation/^..-^21 MeFigure 98. Non-methyl HMBC Correlations of 91The relative stereochemistry was confirmed through the useof a series of NOE difference experiments. Irradiation of the aldehydicproton resonance (8 10.1) gave enhancement of signals correspondingto Me19 (8 0.80) and Me17 (8 0.73). Irradiation of the Me17 resonancegave enhancement of the Me21 resonance (8 1.01) and the aldehydeproton resonance establishing the diaxial relationship among methylsC17, C19 C21 and the aldehyde at C20. The trans CD ring junction hasbeen proposed based on the argument that cadlinaldehyde (91) isbiogenetically related to luteone (73), perhaps by deriving from thesame sesterterpenoid precursor (Scheme I).197198Table 16. Assignment of signals (CDCI3) for Cadlinaldehyde (91)C# 8 13c 8 1 H COSY* HMBC** NOE***l ot 34.4 0.7 HI 0, H2a, H2O (H20)10 2.52 Hla, H2a, H2O H1 a,H1 la2a ,^19.4 1.3 HI a, H10, H213, H3a1.4 H la. H10, H2a, H3a3a 41.7 1.2 H2a, H30 Me18, Me19, H1030 1.4 H3cc4 33.7 Me18, Me195 55.6 1.27 H6a, H60 Me18, Me19, H10, H6a,H606a 19.9 1.35 H5, H60, H701.7 H5, H6a, H707a 40.4 1.7 H71378 1.10 H6a, H60, H7a8 36.59 61.4 1.25 Hlla,H110 Me17, H1 la, H12a10 53.4 H20, H5 or H9lla 17.5 1.7 H9, H11f3,1113 1.9 H9, H1 la, H121312a 39.3 1.85 H120 Me21, H1 la120 1.35 H110, H12a -13 79.5 Me21, H1 2a. H120, H14,H15b, H16a14 59.5 1.4 H15a Me17, Me21, H12a15a 23.0 1.65 H14,H15b, H16a, H16b H14, H16a* 15b 1.7 H15a, H16a, H16b16a 64.9 3.83 H15a, H15b, H16b H15a16b 3.92 H15a, H15b, H16a17 16.6 0.73 H20, Me2118 32.0 0.94 Me1919 20.8 0.80 Me1820 206.4 10.1 (H10) H5 or H9 Me17, Me19, H6f321 20.8 1.01^_ H14 Me1721* Correlations to the proton in column 3** Correlations to the carbon in column 2*** Proton in column 3 irradiated.HOC,HOC73:4kScheme I. Possible Formation of Luteone (73) andCadlinaldehyde (91)from a hypothetical sesterterpenoid precursor199Cadlinaldehyde (91) is the first fully cyclized C21 terpenoidto be found in the marine environment, although linear C2 1furanoterpenes, such as furospongin-2 (111) and ircinin-3 (112), havebeen isolated from a variety of Dictyoceratid sponges. 65 It has beensuggested that these structures arise biogenetically from thedegradation of related sesterterpenoids, such as ircinin-1 (113) whichare often also found in the same extract.111200COOH112OHThe presence of both luteone (73) and cadlinaldehyde (91)in the extracts of the Agamemnon Channel collection of C.luteomarginato, likely reflects a similar pattern within one of its preysponges. It would be of interest to locate the sponge responsible for the65 Minale, L. In 'Marine Natural Products, Chemical and Biological Perspectives',Scheuer, P. J., Ed.; Academic Press: New York, 1978; Vol. I, Chapter 4, and referencestherein.production of these compounds in order to investigate the terpenoidmetabolites and possibly discover an undegraded precursor of 73 and91.c. ConclusionsThe ability of the dorid nudibranch, Cadlinaluteomarginata, to sequester terpenoids from its varied poriferan dietmeans that•this mollusc will continue to be a source of interesting anddiverse chemistry. Several of the compounds isolated have been the firstrepresentations of new carbon skeletons (73, 74, 77 and 91), while othercompounds either have not been found in opisthobranchs before (88)or are new representatives in their class (86, 90). Many of these structurespose interesting synthetic problems. Albicanyl acetate (72), for example,has been a target of several synthetic routes. 66In addition to being a source of unique metabolites, thenudibranch is a natural indicator of the ecological diversity of thePacific Coast rocky headlands. Areas which foster higher diversityamong poriferan species (high current or wind-wave surge) arereflected in the diversity of metabolites found in C. luteomarginata. Thethree sites detailed in this section of the thesis were all areas with a highecological diversity, while areas with less diversity were reflected in the66 a) Armstrong, R. J., Harris, F. L. and Weiler, L. Con. J. Chem., 1986, 64, 1002. b)Shishido, K., Tokunaga, Y., Omachi, N., Hiroya. K. and Fukumoto, K. J. Chem. Soc.Perkin Trans. 1, 1990, 2481.201isolation of only the single metabolite, albicanyl acetate (72), fromcollected nudibranch specimens. 67With a large geographical range and varied poriferan diet,this nudibranch will continue to provide the natural products chemistwith challenges.20267 Specimens from several collection sites (Kitimat Arm, Langara Island, Anthony Islandand Barkley Sound) contained terpenoid metabolites which could not be properlyidentified and should be reinvestigated.D. Metabolites from the Nudibranch Archidoris pseudoargus(Rapp, 1827)i. Introduction to the Archidorids.Nudibranchs in the genus Archidoris (Suborder: Doridacae,Superfamily: Eudoridoidea, Family: Archidoridae) are largecryptobranch dorids found along diverse coastal regions. Welladapted to the rigours of intertidal existence, these nudibranchs are thespecies most commonly encountered in tide pools.Investigations into the chemical constituents from twospecies, A. odheneri and A. montereyensis collected in the coastalwaters of British Columbia 68 have elucidated a defensive strategy forthese molluscs based on derivatives of glycerol.Archidoris odhneri was found to contain the farnesic acidglycerides 114-116 as the major metabolites, 68a with the positionalisomer 117, the cyclized terpenoic glycerides 118-121 and the glycerolether 122 as minor constituents68c.d (Figure 99). Biosynthetic studies usingradiolabled mevalonic acid have demonstrated the ability of A.odhneri to synthesize at least one of these metabolites (114) andantifeedant studies using the tide pool sculpin (Oligocottus maculosis)have shown that compounds 120 and 122 are effective deferents topredation. 68c68 a) Andersen, R. J. and Sum, F. W. Tetrahedron Left., 1980, 21. 797. b) Gustafson, K.,Andersen, R. J., Chen, M. H. M., Clardy, J. and Hochlowski, J. E. Tetrahedron Lett., 1984,25, 11. c) Gustafson, K. and Andersen, R. J. Tetrahedron, 1985, 41, 1101. d) Gustafson,K. PhD. Thesis. 1984, University of British Columbia.203114 R 1 = R2 = H115 =Ac R2 = H116R 1 =H R2=AcOR 1OR2OROR20118 R i = R2 = Fl119 R1=AcR 2 =H120 R = HHO^C 16H33OH122121HO204Figure 99. Metabolites Isolated from Archidoris odherni 68&'•ORS0 R2011812 1 =R2 =H119R i =Ac R2 =H123R D =H R 2 =AcOHOOR120R=H124R= Ac 121Archidoris montereyensis contained compounds 118-120,and 123 as the major metabolites, with compounds 121-122 and 124present as minor constituents (Figure 100). Biosynthetic studies on A.montereyensis resulted in significant incorporation of 14C mevalonicacid into compounds 118 and 120, indicating the compounds aresynthesized de novo. 68b -dHO^IC 6H33CYOH122Figure 100. Metabolites Isolated from Archidoris montereyensis mad20511 Acyl Glycerides Isolated from the Dorid Nudibranch Archidorispsuedoargus (Rapp, 1827), Collected off the Coast of Norway.A recent collecting expedition off the coast of Norway hasallowed us to investigate the chemical constituents of the nudibranchArchidoris pseudoargus (Rapp, 1827) 69 . The skin extract from twospecimens of A. pseudoargus was found to contain the diterpeneglyceride 118 as the major (19 mg) metabolite with compound 119and an as yet incompletely determined glyceride 125 as minorconstituents.206OR ]OR20118 P, =R2 =H119R 1 =Ac R2 =Ha. Extraction and Purification.Two specimens of A. pseudoargus were collected off thecoast of Norway, near Bergen, in August 1992. The animals wereimmersed in methanol and stored for later workup in Vancouver. At thetime of workup, the methanolic extract was decanted and a secondportion of methanol was added to the specimens. This secondmethanolic extract was combined with the first extract and reduced invacuo to yield an aqueous suspension which was subsequently69 See Figure 101 for phylogenetic relationshipsORDER^ NudibranchiaSUBORDER^Aeolldacea^Arminacea^Doridacea^DendronotaceaSUPERFAMILY^Anadorldoldea^Eudoridoidea^Porodoridoldea^GnathodoridoideaHexabranchidae^Cadlinidae^Discodorididae^KentrodoridideaFAMILY^Chromodorldldae^Homoiodorididae^Dorididae^Aldisidae^AsteronotidaeRostongldae^Actinocyclidae^Archidorididae^Baptodorididae^PlatydorididaeGENUS^ ArchidorisSPECIES^ A. pseudoargusFigure 101. Phylogenetic classification of A. pseudoargus(from Thompson)118 P 1 =R2 =H119P 1 =Ac R2 =Hextracted with two 100 ml portions of chloroform. The organic phasewas again reduced in vacuo to yield 60 mg of a slightly yellow oil.Subsequent normal phase silica flash (1.5 X 20 cm, EtOAc)and HPLC (hexane/EtOAc: 1/1) chromatographies yielded in order ofelution: 1.2 mg of 119, 19 mg of 118 and 2.7 mg 125.b. Results and Discussion.Compounds 118 and 119:Previously isolated from two species of Archidorisnudibranchs collected in British Columbia, compounds 118 and 119were solved by comparison of their spectra to published data. 68 The1 H NMR spectra (Figures A18 and A19) can be found in Appendix A.208125 R = ?1'13OHO^OOR1512Compound 125:Compound 125 was isolated as a white amorphous solid(2.7 mg) which gave molecular cluster ions in the EIMS spectrum (Figure102). The largest cluster was centered at m/z 1036 amu. No distinct ionsabove an m/z of 357 amu could be detected in the FABMS spectrum,though peaks of low intensity could be observed at each mass unitfrom m/z 379 to 785. In each of several attempts at obtaining a DCIMSspectrum no peaks were ever observed and the small amount ofsample remaining prevented further attempts at obtaining a molecularformula for the compound.A very large resonance at 8 1.26 in the 1 H NMR spectrum of125 (CDCI3, Figure 103) was at first believed to be due to fatty acidimpurities, but analysis by both normal and reverse phase tic failed toreveal any resolution into components. Coupled with the fact that therelative intensity of the NMR signals remained constant through severalHPLC purifications, under a variety of solvent ratios, it could be surmisedthat compound 125 possessed a long hydrocarbon side chain. A seriesof complex multiplet resonances at 8 4.3-4.1 (3H), 8 3.94 (1H), 8 3.85 (1H)and 8 3.75-3.4 (7H) indicated the presence of at least one glycerol209100526409442n'r-urth4504920500778^8041---8000 --- x102641000 5550165^)111^Ath^b1. ill^111111135 149^11 .1100^150218^2411111111116111111111111111111111110116111161111200^250282Ili1„1111111h^365300^339^d^388111111111111i11101101111111 Ithillroilyall1111111111111111111111111tbahlololit300^350^400109x20564^618^675^757r 1 r . 1 "1""1"T"f"...rtalar^- -1 ---r""A"1"550^600^650 700^750x20 ------100 —1036863^902r- ri"1"")1r1"1-1^Ii...^ r I -mill -4^6,1 -it^ilitIri850^900^950^1000^1050^1100^1150Figure 102. Electron Impact Mass Spectrum of Compound 125Figure 103. 500 MHz 1 H NMR Spectrum (CDCI3) of Compound 125moiety and several additional heteroatoms. Other resonancescorresponding to allylic methine (8 2.95, bs) and methyl (8 1.61, bs, 3H)protons as well as three methyl singlet resonances (8 0.97, 0.91, 0.88) anda methyl triplet resonance (8 0.88, J = 6.5 Hz) could be discerned.The 13C NMR spectrum of 125 (CDCI3, Figure 104) was alsohighly complex. Resonances at chemical shifts appropriate for olefiniccarbons (8 124.6, 114.2, 113.6) and carbons attached to hetero atoms (872.6, 71.9, 70.4, 70.3, 65.2, 64.3, 62.2 and 61.9) could be observed. A veryintense group of resonances centered at 8 29.7, corresponding toseveral methylene carbons, provided collaborative evidence for along aliphatic side chain.Correlations in the 1 H- 1 H COSY spectrum (CDCI3, Figure 105)could be observed between the allylic methine resonance (8 2.95)and the allylic methyl resonance at 8 1.61, while both of these signalsgave a weak correlation into the olefinic methine at 8 5.56. Couplingcould also be observed between the methine resonance at 8 3.94and three other proton resonances at 8 4.23, 4.15 and 3.6 ppm.The two spin systems outlined in the COSY spectrum as wellas the three upfiled methyl singlet resonances in the 1 H NMR spectrumindicated that 125 was related to compound 120. Further comparison ofthe 1 H and 13C NMR spectra obtained for 125 to the reportedSpectroscopic data for 120 indicated that compound 125 had thesame drimanoyl glygeride as 120 with an additional side chain etherlinked through the 3' oxygen atom.While the structure of the hydrocarbon side chain could notbe completely determined in the absence of a molecular formula,several features could be determined through the examination of the212PI*^150^125^100^75^50^25Figure 104. 125 MHz 13C NMR Spectrum (CDCI3) of Compound 1251.5 1.0^0.53.5^3.0^2.5^2.0PPMalp( 4..i•- CAI^(I - •C :i/1i ii:,^. •"4to-, AlfLd,^ iii,_.-r11IIfr.)t, 40.'•6'Lik,„1Figure 105. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofCompound 1252144.5 4.0Os1.01.52.02.53.04.04.5PPMspectroscopic data. The presence of one double bond could bededuced from the broad multiplet resonance in the 1 H NMR spectrumat 8 5.05 (2H) as well as the resonances at 8 114.2 and 113.6 in the 13CNMR spectrum. Four carbon resonances at 8 72.6, 71.9, 70.3 and 64.3ppm as well as COSY correlations from the methine resonance at 8 3.85into several proton resonances between 8 3.5-3.75 and from aresonance at 8 3.45 into a resonance at 8 1.6, suggested the possibilityof a further glygerol ether linkage. Finally the pesence only one methyltriplet at 8 0.88 gave an indication that the hydrocarbon chain is linear toyield the working structure below.O ((CH2)n   CH2)mOH R?Considering the reported presence of the glycerol ether122 in extracts from two Archidoris species, the above structure isplausible, however, the triplet at 8 2.35 (J = 7.5 Hz, 2H) in the 1 H NMRspectral remains unexplained. Attempts at crystallization have thus faronly yielded very small crystals of too poor a quality for analysis by X-ray difraction methods. Further attempts at crystalization are in progress.Although the diterpene glyceride metabolites 118 and 119have no reported antifeedant activity, they closely resemble theverrucosins (126 -7), two icthyotoxic metabolites isolated from thenudibranch Doris verrucosa. 70 The verrucosins are potent protien kinase7° Cimino, G., Gavagnin, M., Sodano, G., Puliti, R., Matti°, C. A. and Mozzarella, LTetrahedron, 1988, 44, 2301.215OR2126 R i =H R2 =Ac127 R 1 = AC R2 = HC activators, and it has been speculated that the high toxicity of thesecompounds is a result of the phosphoralation of the Ca2+ channelproteins. 25d Studies are underway in a collaborating laboratory to see ifthe compounds isolated from Archidoris pseudoorgus have a similarpotentiating effect of protien kinases.c. ConclusionThe isolation of terpenoic glycosides from a third species ofArchidoris nudibranch is indication that these compounds are ageneral feature at the genera level. The reported antifeedant activityof several of these compounds as well as their similarity to theicthyotoxic verrucosins suggests that molluscs in this genus haveevolved the strategy of synthesizing their own defensive metabolites.216E. ConclusionSixteen known and ten new terpenoid metabolites havebeen found in the extracts from three species of nudibranchs. Two ofthese species ( Chromodoris geminus and Cadlino luteomarginata)contained metabolites which resembled terpenoids found in spongesand are therefore probably sequestered from a dietary source. Thethird nudibranch species (Archidoris pseudoorgus ) was found tocontain metabolites which were either identical to the metabolitesisolated from other members of this genus or closely related, whichsuggests that the de novo biosynthesis of these compounds is afeature of all nudibranchs in this genera.217218III. Igzamide, a New Bromotryptamine Derived Compound from theNortheast Pacific Sponge Plocamissa igzo.A. Introduction to Poriferan BromotryptaminesA number of bioactive metabolites that are formallyderivatives of tryptophan or tryptamine have been reported in theliterature. 71-74The first of these metabolites, simple dibrominatedtryptamines (128-9), were isolated by Van Lear et. al. from the Carribeansponge Polyfibrospongia maynardii. 71 a Compounds 128 and 129inhibited the growth of several strains of bacteria in vitro, but did notprevent the growth of these infectious agents in vivo. Subsequently Djuraet. al. reported the isolation of compounds 130 and131 from the relatedsponges Polyfibrospongia (reclassified as Smenospongia) echina andS. aurea, collected in the Gulf of Mexico. 71 b Rhineharrs group has alsoreported the isolation of compounds 131 and the related 6-bromoindole132 from independent collections of S. aurea. 71 c Other simplebromoindole derivatives isolated from marine sponges include71 a) Van Lear, G. E., Morton, G. 0. and Fulmor, W. Tetrahedron Lett., 1973, 299. b) Djura.P., Stierle, D. B., Sullivan, B.. Faulkner, J. D., Arnold, E. and Clardy, J. J. Org. Chem., 1980,1435. c) Tymiak, A. A., Rhinehart, K. L. Jr. and Bakus, G. J. Tetrahedron, 1985, 41, 1039. d)Kobayashi, J., Cheng, J-f., Yamamura, S. Sasaki, T. and Ohtumi, Y. Heterocycles, 1990,31, 2205. e) Faulkner, D. J. Nat. Prod. Repts., 1991, 8, 97, and references within.72 Djura, P. and Faulkner, D. J. J. Org. Chem., 1980, 45, 735.73 a) Andersen, R. J. and Stonard, R. J. Can. J. Chem., 1979, 57, 2325. b) Stonard. R. J. PhD.thesis, University of British Columbia. 1981. c) Lidgren G., Bohlin, L. and Bergman, J.Tetrahedron Lett., 1986, 27, 3283. d) Bobzin, S. C. and Faulkner, D. J. J. Org. Chem., 1991,56, 4403.74 a) Bartik, K., Braekman, J-C., Daloze, D., Stoller, C., Huysecom, J., Vaddedyer, G. andOttinger, R. Con. J. Chem., 1987, 65, 2118. b) Kohmoto. S., Kashman, Y., McConnell, 0. J.,Rinehart, K. L. Jr., Wright, A. and Koehn, F. J. Org. Chem., 1988, 53, 3116. c) Tsujii, S.,Rinehart, K. L., Gunasekera, S. P., Kashman, Y., Cross, S. S., Lui, M. S., Pomponi, S. A.and Dim, M. C. J. Org. Chem., 1988, 53. 5446. d) Morris, S. A. and Andersen, R. J. Can. J.Chem., 1989, 67, 677. e) Morris, S. A. and Andersen, R. J. Tetrahedron, 1990, 46, 715.compound 133,  isolated from an Okinawan Penares specieeld andcompounds 134-5 isolated from the deep water Indopacific spongePleroma menoui 71 e (Figure 106).219^/5".■^1^I^INR1R2N^ Br^NH 133X Y R i^R2128 Br Br H^H129 Br Br H Me130 Br Br Me Me131 Br H Me Me132 H Br Me MeNH134 R= OEt135 R = CH2OHFigure 106. Simple Brominated Indole Metabolites from Sponges"Among the more interesting bromoindole metabolitesreported from sponges are the aplysinopsins (136-139, Figure 107), foundin several species and possessing diverse potent biological effects.Djura and Faulkner72 reported the first brominated aplysinopsin 136 fromcollections of a Dercitus species. Subsequently, both they and Rhinehartreported the isolation of related compounds (1 3 7-1 3 9) fromSmenospongia aurea. 71b ,c Rhinehart's suggestion that such compounds(and bromoindoles in general) can be used as chemotaxonomicmarkers for the genus, seems unlikely given the more recent report ofGuella et. al. who found aplysinopsin-type compounds in a Swedish softcoral,75 and the bromoindole-derived eudistomins (e. g. 140) isolatedfrom a variety of tunicates. 76 Aplysinopsins have aroused considerable76 Guella, G., Mancini, I., Zibrowius, H. and Pietra, F. He/v. Chim. Acta, 1988, 71, 773.76 for examples see: a) Blunt, J. W., Lake, R. J. and Munro, H. G. Tetrahedron Leff., 1987,28, 1825. b) Kinzer, K. F. and Cardellina, J. H. II Tetrahedron Lett., 1987, 28, 925.COOHN./;\__,I^I^.-- N H2Br^N^Os-NHH138220140interest due to their diverse biological effects ranging from preventingneurotransmission to cancer specific cytotoxicity.H..4.„_.____.„,-....„,,^N,..^I^IBr ''....%--..'-'%..- N " Ci:>--- NH^R136 Z = NH, R = Me137Z=0, R=HFigure 107. Brominated Aplysinopsin-type Metabolites. 71b'c.72Another group of bromoindoles (Figure 108) from sponges isexemplified by clionamide (141), the major metabolite of the Pacificsponge Cliona celata, and implicated in the sponge's ability to burrowinto organic calcium carbonate. 73a Such metabolites are typical ofporiferan alkaloids in that they derive from amino acid starter units, in thiscase 6-bromotryptophan and dopa. Several other peptidalbromoindole compounds have also been isolated from C. celatamore recently. 73b The celenamides (142-4) are unique aromatic221OHHOHO142 X = OH, R = CH(CH 3)2143 X = OH, R = CH2CH(CH3)2144 X = H, R = CH2CH(CH3)2OMeFigure 108. Peptide-type Bromoindoles from Sponges"alkaloids similar to the tunichrome blood pigments found in severalascidians. 77 Other bromoindole metabolites in this class include thediketopiperazine metabolite 145, isolated from Geodia baretti,73c andcompound 146 isolated from Chelonaplysilla sp. collected in Palau. 73dThe final class of bromoindole metabolites isolated frommarine sponges are the bis-indole topsentins and dragmacidins (Figure109). 74 Bartik et. al. isolated topsentin B2 (147) from the sponge Topsentiagenitrix collected near Banyuls (France). 74a This compound, along withthe debromo and debromo-dehydroxy analogs was found to beslightly toxic to the fish Lebistes reticulatis, as well as inhibiting theaggregation of dissociated cells of the freshwater sponge Ephydatiafluviatilis, and may therefore be an important defensive metabolite for T.genitrix. Susbsequently, Tsujii et. al. isolated both compound 147 and thedihydro anlalog 148 from Carribean deep-sea sponges in the genusSpongosorites, 74c and Morris and Andersen have isolated 147 fromdeep-sea specimens of Hexadella sp. 74d and 149 from specimenscollected at shallower depths. 74e In addition to the above mentionedbioactivities, the topsentins have been shown to have inhibitory effectson the proliferation of both murine leukemia (P388) and the Herpessimplex virus (HSV- 1 ). 74cDragmicidin (150), a compound similar to the topsentins butwith a piperazine, rather than imidazole, ring bridging the indoles, wasisolated from the sponge Dragmacidon sp. collected at -148 m off theBahamas Islands. 74b Two other dragmicidins (151-2) were isolated fromshallow (-40 m) collections of Hexadella sp. by Morris and Andersen. 74e77 Bruening, R. C., Ottz, E. M.. Furukawa, J. and Nakanishi, K. J. Nat. Prod., 1986, 49, 193, andreferences within.222Compounds 150 and 152 are cytotoxic to a variety of cancer cell lines.H147 R = OH R' = Br148 R=H R'=Br RN=H149 R = Br R' = Br R" = MeH223MeNNR150R=H X=OH Y=6r151 R=H X=Y=H152 R=Me X=Y=HFigure 109. Brominated bis(indole) Alkaloids from Sponges. 74HO 153 HO 1550).s.,^NH1■,,,0 HOH2C^N ,,..s...0 HOH2CH3CHOH2CNH0ONH2156 HHO OH154224B. A New Bromoindole Derivative from the Northeast Pacific SpongePlocamissa igzo (de Laubenfels, 1932)The Northeast Pacific sponge Plocamissa igzo d eLaubenfels, 1932 (Demospongiae, Poecilosclerida, Plocamidae) is anunusual looking poriferan with a smooth, pastel, pinkish orangeappearance underwater. Preliminary extracts exhibited bothanitbacterial (vs Staohylococcus aureus and Bacillus subtilus ) andcytotoxic (L1210) activities.A bioassay guided fractionation of the methanolic extractsof P. izgo resulted in the isolation of three nucleosides (153-5),responsible in part for the cytoxicity, 6-bromoindole-carboxoldehyde(156), and the weakly cytotoxic, new brominated indole derivativeigzamide (157)i. Extraction and PurificationSamples of Plocamissa igzo were collected by hand usingSCUBA (-5 m) off Anthony Island B.C. during April 1989, May 1990 and May1991. Freshly collected specimens were frozen for later workup in thelaboratory. Freeze dried specimens (400 g) were extracted first withCH2Cl2 (700 mL) to remove fatty acid and carotenoid componentsfollowed by sequential extraction with EtOAc (700 mL) and Me0H (700mL). The more polar extracts were subjected to a bioassy guided (L1210cytotoxicity) fractionation procedure involving repeated gel exclusion(LH-20) and a variety of reverse phase (C18 silica, Me0H/H20 solventmixtures) and normal phase (silica, EtOAc/MeOH solvent mixtures)chromatographies. Most of the cytotoxicity could be traced to a fractionwhich yielded two components (156 and 157) and a more polar fractionwhich failed to resolve into components under normal chromatographicconditions. Following acetylation with Ac20/Pyr, the mixture was resolvedinto the three nucleosides (153-5), isolated as their per 0-acetylatedproducts.225IL Results and DiscussionNucleosides (153-155):Isolated as their per-O-acetylated derivatives, compounds153-155 were solved by analysis of their spectroscopic data (seeExperimental and Appendix B) and confirmed by comparison withauthentic commercial samples of thymidine (153), uridine (154) and 2'-deoxyuridine (155). Commercial grade compounds were submitted tothe standard cytotoxicity assay, resulting in ED50's of 0.5-5 µg/ml, andaccounting for much of the cytotoxicity exhibited in the sponge extract.6-Bromoindole-3-carboxaldehyde (156):226Compound 156 was isolated as a white powder (6 mg, UVXmax 216 nm, £ = 9600) which was one of the major components of thecytotoxic fraction from P. igzo. The structure was solved by comparisonof the spectroscopic data to published reports. 78 (See Experimental andAppendix B)Igzamide (157):227012013NH2157lgzamide was isolated as a yellow solid (14 mg UV Xmaxt 294nm, e = 4900; Xmax2 228 nm, e = 12800) which yielded a molecular iondoublet at 308.9937/306.9967 amu in the HREIMS spectrum. Thecorresponding molecular formula of Ci2Hi0N302 81 Br/Ci2H1oN302 79Br (AM = 0.0/1.0 mmu) has an unsaturation index of nine, indicating a highdegree of aromaticrty.Examination of the 1 H NMR spectrum (dmso-do, Figure 110)of igzamide, indicated the compound was a 6-bromoindole derivative.A broad singlet resonance at 8 11.52 exchangeable with D20 (H1), anaromatic singlet resonance at 8 7.53 (H2), doublet resonances at 8 7.55 (J= 7.4 Hz, H4) and 8 7.61 (J = 1.8 Hz, H7) and a double doublet resonanceat 8 7.19 (J = 7.4, 1.8 Hz, H5) closely matched data reported for other 6-bromoindoles substituted at C3. 73,74 A cis olefin conjugated into another78 Wratten, S. J., Wolfe, M. S., Andersen, R. J. and Faulkner, D. J. Antimicrob. AgentsChemother, 1977, 11, 411.NH1013NyNH20PPM 12.5°^12.01^11‘.51^11.0^ I10.5^10. ^9.5^9.0^8.5^8.0^7.5^7.0^6:5 .^10^ 5 .5Figure 110. 400 MHz 1 H NMR Spectrum (Me2SO-do) of Igzamide (157)nitrogen aton (8 6.15, d, J = 9.1 Hz, H8; 8 6.70, dd, J = 7.6, 9.1 Hz, H9; 8 9.48, d,J = 7.6 Hz, H10) and broad singlets at 8 8.07 and 8.36 ppm (H13's,exchangeable) were also present in the 1 H spectrum.The 130 NMR spectrum (Me2SO-do, Figure 111) displayed alltwelve carbons. Signals appropriate for bromoindole (8 109.7 (0), 114.3(CH), 114.7 (C), 120.3 (CH), 122.3 (CH), 124.4 (CH), 130.8 (C), 136.7(0) ) andenamine (8 105.3 (CH), 118.0 (CH) ) functionalities could be observed, aswell as two additional carbons at chemical shifts appropriate for amides(8 157.2, 161.4) which could only fit into the structure as an oxalamidefunctionality.1 H- 1 H COSY correlations (Me2SO-do, Figure 112) could beobserved between the broad doublet resonance at 8 9.48 (H 10) andthe H9 resonance at d 6.70 which was further coupled into the resonanceat 8 6.15 (H8). Other correlations between the resonance at d 7.19 (H5)and the two resonances at d 7.55 (H4) and 8 7.61 (H7) supported thesubstitution pattern of the indole subunit. A correlation between the twobroad singlet resonances at 8 8.07 and 8 8.36 could also be observed.Comparison of the spectroscopic data with that reported for the BDTresidue of the halocyclamines 79 confirmed the 6-bromoindole-3-enamine portion of the molecule.Nuclear Overhauser enhancement difference experimentsconfirmed the substitution pattern on the indole ring, as well as the Z olefingeometry (Figure 112). Irradiation of the resonance at 8 11.52 (H1),resulted in enhancement of signals at 8 7.53 (H2) and 7.61 ppm (H7), whileirradiation of the doublet resonance at 8 6.15 (H8) enhancedresonances at 8 6.70 (H9), 7.55 (H4) and 7.53 ppm (H2). Irradiation of either79 crzumi, K., Yokosawa, H. and Ishii, S-i. Biochemistry, 1990, 29, 159.2290Figure 111. 75 MHz 13C NMR Spectrum (Me2SO-do) of Igzamide (157)H8-H9 alp 030 H9-H100O O93^9.0^83^8:0 PPM 73^7.0^63^6.0Figure 112. 400 MHz 1 H- 1 H COSY Spectrum (ME2SO-d6) ofIgzomide (157)2336.0.. 63- 7.0▪ 7.58.0839.093PPM0goH5-H7 H44-15a0of the downfield amide protons (d 8.07, 8.36, H13a/H13b) led only to theenhancement of the other amide proton, indicating a primary amidewell distanced from the rest of the molecule.Figure 113. NOE Summary for lgzamide (157).The oxalamide functionality of the molecule was confirmedby comparison to the synthetic model compound 158. The 13C NMRchemical shifts of the carbonyl resonances in 158 (8 162.3, 158.9) closelymatched those in igzamide (8 161.4, 157.2), and the EIMS fragmentationpattern was quite similar (m/z 264/262 and 237/235 vs m/z 119 and 94).0lgzamide (157) exhibited very weak cytotoxicity (L1210: ED501914/m1) and mild antimicrobial activity vs Staphylococcus aureus andBadIlis subtilis (MIC 1001.1.g/disk).232it Conclusionlgzamide (157) is a new member of the family of 6-bromotryptophan and 6-bromtryptamine derived metabolites isolatedfrom marine sources. A review of the literature failed to offer any otherexamples of naturally occuring oxalyl diamides in the marineenvironment.233Table 17. Assignment of NMR Signals (Me2SO-do) for lgzamide (157)C# 8 13C 8 1 H (m) COSY* J (Hz) nOe**1 11.52(bs) H2, H72 124.4 7.53(s)3 114.73a 130.84 120.3 7.55(d) H5 7A5 122.3 7.19(dd) H4, H7 7.4, 1.86 136.77 109.7 7.61(d) H5 1.87a 114.38 105.3 6.15(d) H9 9.1 H9,H2,H49 118.0 6.70(dd) H8, H10 9.1, 7.610 9.48(d) H9 7.611 161.4a12 157.213a 8.36(bs) 13b13b 8.07(bs) 13a234* Correlations to the proton in column 3** Proton in column 3 irradiated.a may be interchanged.IV. ExperimentalA. GeneralThe 1 H nmr spectra were recorded on either a Bruker AMX-500 or a Bruker WH-400 spectrometer. Chemical shifts are reported usingthe internal standard tetramethylsilane (TMS, 8 0.00 ppm) as areference. The 13C nmr spectra were recorded on either a Varian XL-300 (75 MHz) or a Bruker AMX-500 (125 MHz) spectrometer. Chemicalshifts are reported with respect to TMS using the deuterated solventpeak as a secondary reference (Me2SO-do: 39.5 ppm; CDCI3: 77.0PPM C6D6: 128.0 ppm). COSY, nOe difference and double resonanceexperiments were performed on the Bruker WH-400 spectrometer. AllMultiple Quantum Coherence experiments were conducted on theBruker AMX-500 spectrometer.Low resolution and high resolution El mass spectra wererecorded on Kratos AEI MS-59 and AEI MS-50 mass spectrometers,respectively. Desorption chemical ionization mass spectra wererecorded on a Delsi-Nermag R-10-10 quadrupole mass spectrometer.Infrared spectra were recorded on a Perkin-Elmer 1600Fourier Transform spectrometer. All FTIR spectra were acquired as filmson sodium chloride plates. The uncorrected melting point ofcadlinaldehyde (91) was obtained with a Fischer-Johns melting pointapparatus. Optical rotations of compounds 34, 61 and 62 weremeasured using a Jasco J-710 spectropolarimeter.Normal phase flash chromatographies were performedusing Merck silica gel G60 (230-400 mesh). Normal and reverse phasethin layer chromatographies were performed using Merck Type 5554235aluminum backed Kieselgel 60 F254 silica gel plates and WhatmanMKC18F tic plates respectively. Visualization of the compounds on thetic plates was accomplished either by UV (254 nm) or by the use ofH2SO4 and Vanillin spray reagents. Further information on thepreparation of the spray reagents is provided by Stah1.80 Size exclusionchromatography was accomplished using Sigma Sephadex LH-20resin (bead size 25-100 11). Radial thin layer chromatography wasperformed on a Harrison research Model 7924 chromatotron (on platesmade of Merck silica gel 60 PF-254 with CaSO4.1/2 H2O as a binder)using an FMI Model R PG-150 lab pump. High performance liquidchromatography was accomplished one of two systems. The first iscomprised of a Perkin-Elmer Series 2 liquid chromatograph attached toeither a Waters 410 differential refractometer or a Perkin-Elmer LC-55spectrophotometer. The second system employed a Waters 501 HPLCpump attached to either a Waters Associates Model 440 absorbancedetector or a Perkin-Elmer LC-25 refractive index detector. Normalphase HPLC was performed using an Alltech 5 micron column, whilereverse phase HPLC was performed using an Alltech 5 micron C18column.Solvents for extractions and column chromatographieswere either Fisher or BDH reagent grade. Solvents for radial thin layerand high performance liquid chromatographies were either BDHOmnisolve grade or Fisher HPLC grade. All reagents were commercialgrade and were used without further purification. An exception was80 Stahl.E. (translated by M. R. F. Ashworth. - Thin-Layer Chromotography, ALaboratory Handbook', Springer-Verlag. Berlin. 1969.236pyridine which was distilled over BaO and stored over Linde type 4Amolecular sieves.Antimicrobial assays were performed by Michael LeBlanc(Department of Oceanography, U. B. C.). Cytotoxicity bioassays (invitro L1210 and P388 murine leukemia cell lines) were performed underthe supervision of Dr. Theresa M. Allen (Department of Pharmacology,U. of A.).237B. Metabolites from Chromodoris geminusCollection and Isolation Procedure. See Extraction and Purificationsection in text.1213,15cc,16a-triacetoxyspongian (34):34Compound 34 (23 mg; 1.9 mg/animal) was isolated as acolorless glass.(a)D +8.4 (CHCI3); DCIMS m/z (rel. int.): 362 (18), 344 (57),302 (66), 285 (100), 191(17); HREIMS: m/z 404.2568 (404.2573 calculated forC24H3605); EIMS m/z (rel. int.): 404 (1.2), 345 (29.9), 344 (100), 302 (55.6), 285(22.8), 274 (11.5), 265 (11.8), 191 (24.1), 137 (17.2), 123 (23.3), 109 (19.4), 95(24.3), 81 (23.1), 69 (29.6), 55 (19.9), 43 (83.1), 41 (16.9); 13C nmr (8, CDCI3):169.9, 169.8, 169.7, 99.2, 98.4, 71.1, 59.5, 56.6, 55.0, 42.2, 41.9, 41.9, 40.0, 37.4, 34.7,33.3, 33.2, 23.4, 21.4, 21.3, 21.1, 20.8, 18.4, 18.0, 17.1, 16.6; 1 H nmr (8, CDCI3):6.34 (d, J = 7.5 Hz); 6.04 (s); 5.05 (m); 3.07 (q, J = 7.5 Hz); 2.04 (s, 3H); 2.03 (s,3H); 1.95 (s, 3H); 1.78 (dd, J = 12.8, 6.0 Hz); 1.67 (bd, J = 12.0 Hz); 1.11 (m);0.97 (s, 3H); 0.84 (s, 3H); 0.83 (s, 3H); 0.78 (s, 3H); 13C nmr (8, C6D6): seeTable 2; 1 H nmr (8, C6D6): see Table 2; FTIR (film): 2938, 1743, 1368, 1241,1039, 939, 788 cm-1 .2386a,15a,16a-trlacetoxyspongian (61):Compound 61 (10 mg; 0.8 mg/animal) was isolated as acolorless glass. (a)D +3.0 (CHCI3); DCIMS m/z (rel. int.): 402 (16.3), 344(46.2), 343 (67.1), 342 (28.1), 285 (44.9), 284 (54.8), 284 (100); HREIMS: m/z404.2537 (404.2573 calculated for C24H3605); EIMS m/z (rel. int.): 404 (0.1),344 (35.2), 302 (4.7), 285 (12.3), 269 (100), 187 (12.5), 119 (12.4), 109 (10.3).95(11.4), 69 (16.4), 55 (10.5), 43 (46.9), 41 (10.1); 13C nmr (5, CDCI3): 170.5,170.1, 169.8, 101.7, 99.6, 69.8, 58.9, 56.1, 49.3, 43.3, 39.9, 39.6, 39.2, 36.1, 35.7, 33.3,23.4, 22.7, 22.1, 22.0, 21.4, 21.3, 18.2, 18.0, 17.7, 17.2; 1 H nmr (8, CDCI3): 6.06 (d,J = 7.3 Hz); 6.05 (s); 5.21 (dt, J = 3.5, 11.0 Hz); 2.57 (q, J = 7.4 Hz); 2.12 (dd, J =3.4, 12.4 Hz); 2.08 (s, 3H); 2.03 (s, 3H); 2.02 (s, 3H); 1.92 (d, J = 7.9 Hz); 1.86(bd, J = 14.5 Hz); 1.08 (s, 3H); 1.00 (s, 3H); 0.94 (s, 3H); 0.85 (s, 3H); 0.78 (m);13C nmr C6D6): see Table 3; 1 H nmr (5, Cope.): see Table 3; FTIR (film):2931, 1734, 1366, 1244, 1028, 953, 788 cm-1 .2396a,120,15cc,16a-tetracetoxyspongian (62):Compound 62 was isolated as a colorless glass (34 mg; 2.8mg/animal). (a)D +40.0 (CHCI3); DCIMS m/z (rel. int.): 540 (6), 420 (71), 402(27), 360 (12), 343 (64), 300 (13), 283 (100), 272 (9), 267 (19); HREIMS: m/z403.2405 (402.2406 calculated for C24H3405); EIMS m/z (rel. int.): 402 (15.7),360 (6.5), 342 (12.0), 300 (12.9), 285 (22.3), 267 (12.8), 188 (16.3), 173 (12.1), 119(14.2), 109 (12.8), 95 (12.9), 81 (15.2), 69 (23.5), 55 (14.3), 43 (100), 41 (11.7); 13Cnmr (8, CDCI3): 169.6, 169.5, 169.5, 169.4, 98.9, 98.2, 70.6, 69.4, 59.2, 58.7, 54.3,48.5, 43.0, 41.8, 39.8, 39.4, 35.9, 35.5, 33.2, 23.4, 21.9, 21.8, 21.2, 21.0, 20.7, 18.8,18.0, 17.9; 1 H nmr Co. CDCI3): 6.34 (d, J = 7.5 Hz), 6.02 (s), 5.20 (dt, J = 3.5, 11.0Hz), 5.06 (m), 3.08 q, J = &.5 Hz), 2.14 (dd, J = 3.5, 12.5), 2.05 (s, 3H), 2.03 (s,3H), 2.02 (s, 3H), 1.95 (s, 3H), 1.82 (dd, J = 5.5, 12.5 Hz), 1.69 (bd, J = 12.5 Hz),1.20 (d, J = 11.5 Hz), 1.10 (s, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.84 (s, 3H); 13C nmr(8, C6D6): see Table 4; 1 H nmr (5, C6D6): see Table 4; FTIR (cm-1 ): 2931,1738, 1368, 1241, 1036,914, 732 cm -1 .240C. Metabolites from Cadlina luteomarginata.Collection and Isolation Procedure. See Extraction and Purificationsection in text.Biosynthetic Studies of Albicanyl Acetate:14C Incorporation Study: Twenty-eight specimens of C. luteomarginata were collectedby hand using SCUBA (-15 m) in Howe Sound B. C. The animals weretransported back to the laboratory and each was injected with 1 p.Ci of1- 14C acetic acid sodium salt (Sigma, 48.9 mCi/mmol) in an artificialseawater medium (0.01 mL). The animals were then maintained in anaerated 40L seawater aquarium for a period of one week. The watertemperature was kept at ca. 15 °C by cooling the aquarium withrunning water. Following the incubation period, the animals were flashfrozen with dry ice and dissected to separate the mantle andreproductive organs from the foot and other internal organs. The threefractions provided by this dissection were extracted separately withmethanol (40 mL) for 24 hrs. Evaporation of the methanolic extractsfollowed by screening by tic (silica gel, hexane) and 1 H nmr indicatedthat albicanyl acetate (72) was present only in the mantle of thenudibranchs and provided no indication for the presence ofcompounds 83 or 84 in any fraction. Purification by silica gel flashchromatography (hexane) and repeated normal phase HPLC(hexane, 1/19 EtOAc/hexane, 1/99 Me0H/pentane) resulted in theisolation of 3.3 mg of 72. The radioactivity was measured on a Beckman241liquid scintillation counter at each stage in the purification. Theradioactivity of the final purified compound was found to be onlyslightly higher than background. The incorporation was thereforedeemed to small to be significant.3H-Incorporation Study: Twenty-five specimens of C. luteomarginata were collected byhand using SCUBA (-15 m) in Howe Sound B. C. and each animal wasinjected immediately with 2 p.Ci of mevalonolactone-5-3H (Sigma, 20Ci/mmol) in an artificial seawater medium (0.02 mL). The animals werethen transported back to the laboratory. The same procedure outlinedabove was followed, resulting in the isolation of 2.7 mg of 72. Theradioactivity was counted on a liquid scintillation counter and wasfound to be statistically equal to the background.13C Incorporation Study: Thirty specimens of C. luteomarginata were collected by handusing SCUBA (-15 m) in Howe Sound B. C. and each was injectedimmediately with 0.2 mg of acetic- 13C2 acid sodium salt in .02 mLartificial seawater. The animals were maintained in a running seawateraquarium for one week. Following the incubation period, the wholeanimals were extracted with methanol. Purification according to theabove procedure yielded 3.0 mg of 72 which gave no indication ofsignificant incorporation of the injected precursor in the 13C nmrspectrum.242Isonfirie 68:243ANC 1\65Compound 68 was isolated as a colorless oil: HREIMS; m/z204.1869 (204.1878 calculated for C16H25N); EIMS m/z (rel. int.); 204 (43.3),189 (47.0), 161 (59.9), 147 (19.6), 133 (30.7), 119 (26.4), 105 (46.0), 93 (43.6), 81(100); 13C nmr (8, CDCI3): 48.6, 41.7, 41.6, 38.9, 28.9, 22.2, 20.6, 19.3, 19.0, 18.6,15.5, 15.3; 1 H nmr (8, CDCI3): 2.06 (m), 1.47 (bs, 3H), 1.09 (s, 3H). 0.99 (s. 3H),0.88 (s, 3H), 0.69 (dd, J = 8.5, 9.0 Hz), 0.58 (dd, J = 6.5, 9.0 Hz); FTIR (film); 2126cm-1 .Isothlocyanate 69:Compound 69 was isolated as a colorless oil. HREIMS; m/z263.1699 (263.1708 calculated for Ci6H25NS); EIMS m/z (rel. int.): 263 (9.1),204 (32.1), 189 (33.0), 161 (42.8), 149 (15.5), 133 (22.8), 123 (38.7), 105 (48.8), 93(61.1), 81 (100), 67 (30.4), 55 (41.1), 40 (54.4); DCIMS (NH3) m/z: 264, 205, 123,81; 13C nmr (8, CDCI3): 49.2, 41.6, 41.5, kO, 29.5, 22.0, 20.5, 19.1, 19.0, 15.7,15.5; 1 H nmr (8, CDCI3): 1.95 (dm, J = 12.6Hz); 1.43 (s, 3H); 1.34 (dm, J = 13.4Hz); 1.25 (d, J = 6.4Hz); 1.12 (s, 3H); 0.99 (s, 3H); 0.86 (s, 3H); 0.78 (m); 0.64 (t, J= 8.8Hz); 0.54 (dd, J=9.3, 6.5 Hz); FTIR (film); 2085 cm -1 .Microcionin 2 (70):Microcionin 2 was isolated as a colorless oil (28 mg; 0.2 mg/animal). HREIMS; m/z 218.1674 (218.1671 calculated for C15H220); ELMSm/z (rel. int.): 219 (0.1), 218 (23.1), 203 (40.5), 147 (15.4), 123 (100), 109 (66.5),95 (72.5), 81 (97.1), 67 (40.6), 55 (32.9), 41 (56.6); 13C nmr (8, C6D6): 142.7,140.0, 138.4, 125.8, 123.4, 111.1, 42.3, 38.0, 36.7, 28.0, 26.8, 24.4, 21.4, 20.1, 16.6; 1 Hnmr (8, CDCI3): 7.23 (bs); 7.16 (bs); 6.18 (bs); 5.40 (m); 1.66 (d, J = 1.8 Hz,3H); 1.10 (s, 3H); 1.00 (d, J = 7 Hz, 3H).244Albicanyl acetate (72):Compound 72 was isolated as a colorless oil (numerouscollections; 0.1-1 mg/animal). EIMS m/z (rel. int.): 264 (0.1), 249 (0.3), 204(31.6), 189 (19.6), 137 (100), 123 (47.3), 107 (56.7), 93 (84.1), 81 (91.6), 69 (93.4),55 (54.7), 43 (74.8); 13C nmr (8, CDCI3): 171.4, 146.8, 107.1, 61.6, 55.1, 54.7, 41.9,39.0, 38.9, 37.8, 33.6, 33.5, 23.9, 21.7, 21.1, 19.2, 15.1; 1 H nmr (8, CDCI3): 4.83(bs); 4.50 (bs); 4.32 (dd, J = 3.7, 11.3 Hz); 4.17 (dd, J = 9.3, 11.1 Hz); 2.39 (dm,J = 12.9 Hz); 1.99 (s, 3H); 1.70 (bd, J = 12.5 Hz); 0.86 (s, 3H); 0.79 (s, 3H); 0.74(s, 3H).Luteone (73):Luteone was isolated as a white solid (5 mg 0.1mg/animal). HREIMS: m/z 344.2717 (344.2715 calculated for C23H3602):EIMS m/z (rel. int.): 344 (13.8), 326 (21.6), 315 (16.7), 297 (11.9), 287 (12.2), 274245(11.4), 268 (25.5), 202 (38.5), 189 (37.7), 177 (35.7), 159 (29.2), 149 (25.1), 133(32.9), 119 (43.5), 107 (42.5), 95 (55.0), 81 (55.1), 69 (41.5), 55 (49.3), 43 (100);13C nmr (d, CDC13): 210.5, 206.3, 147.1, 106.9, 60.2, 55.2, 55.1, 53.8, 42.5, 41.7,39.8, 39.5, 37.8, 34.4, 33.6, 31.9, 30.1, 23.5, 20.7, 19.4, 18.4, 17.6, 16.3; 1 H nmr (d,CDCI3): 10.1 (s), 4.81 (bs), 4.43 (bs), 2.58 (m), 2.52 (bd, J = 13.7 Hz), 2.34 (m),2.3 (m), 2.02 (s, 3H),1.99 (ctt, J = 10.3, 3.3 Hz), 0.91 (s, 3H), 0.75 (s, 3H), 0.57 (s,3H); FT1R (film): 2932, 1714 cm -1 .Marginatafuran (74):74Marginatafuran was isolated as a white solid (20 mg; 1.4mg/animal). HREIMS: m/z 316.2031 (316.2038 calculated for C20H2803);EIMS m/z (rel. int.): 316 (40.5), 301 (91.5), 270 (12.6), 255 (100), 147 (15.4), 131(13.2), 109 (73.6), 91 (13.0), 69 (10.3), 55 (11.8), 41 (14.6); 13C nmr (8,CDQ3):182.2, 159.5, 140.5, 113.9, 110.2, 56.4, 55.9, 48.5, 42.5, 38.6, 37.4, 37.3,33.8,33.8, 23.3, 23.0, 20.9, 20.5, 20.0, 19.0; 1 H nmr (5 CDCI3): 7.09 (d, J = 1.8 Hz), 6.00(d, J= 1.8 Hz), 2.52 (bd, J = 12.2 Hz), 2.22 (m), 2.05, (dd, J = 13.3, 5.6 Hz), 1.56(bd, 13.8 Hz), 1.31 (s, 3H), 1.03 (s, 3H), 0.85 (s, 3H); FT1R: 2965, 2923, 2861, 1698,1455, 1238, 788 cm -1 .246Tetrahydroaplysulphurin-1 (75):75Compound 75 was isolated as a colorless oil (4.0 mg, 0.2mg/animal). HREIMS; m/z 376.2248 (376.2250 calculated for C22H3205);13C nmr (8, CDCI3): 170.9, 169.9, 146.5, 121.3, 102.7, 100.6, 50.9, 42.1, 40.6, 39.7,39.5, 39.0, 38.0, 32.5, 31.6, 31.1, 28.3, 25.0, 24.0, 21.2, 20.7, 14.7; 1 H nmr (8,CDCI3): 6.18 (d, J = 2.4 Hz); 6.00 (d, J = 6.2 Hz); 4.21 (q, J = 7.4 Hz); 3.23 (m);2.36 (m), 2.09 (m); 2.08 (s, 3H); 1.90 (m); 1.42 (d, J = 7.4 Hz, 3H); 1.28 (m); 1.13(s, 3H); 0.91 (s, 3H); 0.78 (s, 3H); FTIR (film): 2944, 1750, 1458, 1372, 1230, 995,557 cm-1 .247Formarnicle 78:248H^'''',,,=NHCHO b78Compound 78 was isolated as a white solid (8 mg; 0.6mg/animal). HREIMS; m/z 249.2090 (249.2093 calculated for C161127N0);EIMS m/z (rel. int.): 249 (3.3), 234 (1.1), 204 (100), 189 (80.5), 161 (66.6), 147(14.2), 133 (30.4), 122 (31.0), 121 (25.6), 107 (42.1), 93 (45.1), 91 (43.9), 87 (44.5),81 (50.3), 67 (31.2), 55 (36.9); 1 H nmr (8, CDCI3): doubling of some signalsobserved due to differing conformational positions of the amide, 8.23(d, J = 12.3 Hz) & 8.01 (s); 5.82 & 5.12 (bs); 2.43 (bd, J = 8.3 Hz) & 1.96 (bd, J= 8.8 Hz); 1.74 (m); 1.42 & 1.38 (s, 3H); 1.03 (s, 3H); 0.94 & 0.90 (s, 3H); 0.91 (s,3H); 0.66 (t, J = 8.7 Hz); 0.44 & 0.38 (dd, J = 9.3, 6.9 Hz); FTIR (film): 3184, 3047,3008, 2979, 2925, 2856, 1689, 1459, 1320 cm -1 .!son!Inle 79:79Compound 79 was obtained as a colorless oil. 1 H nmr (8,C6D6): 5.36 (d, J = 3.1 Hz), 2.40 (m), 1.88 (m), 1.72 (m), 1.12 (d, J = 7.5 Hz,3H), 1.03 (s, 9H).Isothlocyanate 80:80Compound 80 was obtained as a colorless oil. EIMS m/z(rel. int.); 263 (2.0), 205 (1.4), 189 (1.8), 163 (26.9), 119 (3.1), 93 (26.1), 81 (100);13C nmr (8, CDCI3): 151.8, 119.1, 64.3, 44.7, 40.8, 39.0, 38.9, 34.5, 33.3, 27.1, 22.2,20.3, 18.9, 17.5; 1 H nmr (8, CDCI3): 5.35 (d, J = 3.0 Hz), 2.52 (m), 2.19 (m), 1.40(s, 3H), 1.38 (s, 3H), 1.16 (d, J = 7.6 Hz), 1.10 (s, 3H); FTIR (film); 2082 cm-1 .Violacene (81):249CIBrCHit""CICI^CI81Violacene (81) was obtained as a colorless oil. HREIMS;m/z 355.8903 (355.8906 calculated for CioH13 35C1337C1 81 Br, 355.8896calculated for C101-11335Cl237C1279Br); EIMS M/z (rel. int.): 358 (3.2). 356 (5.1),354 (6.3), 352 (2.6), 319 (6.1), 283 (10.6), 273 (9.8), 271 (20.2), 269 (13.9), 247(14.6), 245 (14.5), 239 (11.9), 237 (16.3), 235 (13.6), 205 (10.0), 204 (14.2). 203(20.4), 201 (31.2), 189 (14.3), 167 (17.7), 165 (2932), 163 (14.5), 161 (23.6), 131(16.1), 129 (23.6), 127 (25.6), 125 (31.6), 115 (29.6), 105 (30.2), 91 (69.0), 84(78.6), 77 (54.7), 49 (100); 13C nmr (8, CDCI3): 135.4, 119.1, 64.1, 59.1, 48.8, 38.6,38.2, 27.4; 1 H nmr (8, CDCI3): 6.56 (d, J = 13.6 Hz), 6.08 (d, J = 13.6Hz), 4.34(dd, J = 4.7, 12.5 Hz), 3.96 (d, J = 10.6 Hz), 3.70 (dd, J = 4.0, 12.5 Hz), 3.55 (d,J = 10.6 Hz), 2.64 (q, J = 13.1 Hz), 2.46 (ctt, J = 4.0, 13.1 Hz), 2.37 (d, J = 15.3Hz), 2.20 (d, J = 15.3 Hz), 1.30 (s, 3H).7a,170-dihydroxy-15,17-oxidospongIan-16-one 7 acetate (82):Compound 82 was obtained as a white solid (3.8 mg; 1animal). HREIMS: m/z 332.1979 (332.1987 calculated for C20H2804); EIMSm/z (rel. int.): 332 (0.7), 286 (16.1), 258 (16.8), 137 (20.8), 124 (31.3), 109 (57.3),95 (20.6), 91 (30.1), 81 (31.0), 69 (39.0), 55 (29.3); 13C nmr (8, CDCI3): 177.2,170.0, 104.3, 103.6, 73.0, 49.5, 48.5, 42.2, 41.8, 38.9, 38.0, 37.6, 33.4, 32.8, 29.7, 24.6,23.3, 21.4, 21.2, 18.7, 16.1, 15.3; 1 H nmr (8, CDCI3): 6.01 (d, J = 6.0 Hz), 5.44(bs), 4.73 (m), 2.85 (dd, J = 11.5, 6.0 Hz), 2.73 (dd, J = 11.6, 7.6 Hz), 2.35 (dm,J = 14.1 Hz), 2.12 (s, 3H), 1.95 (dq, J = 12.6, 4.0 Hz), 1.84 (dm, J = 14.6 Hz), 0.91250(s, 3H), 0.79 (s, 3H), 0.76 (s, 3H); RIR (film); 3419, 2923, 2850, 1780, 1743, 1463,1366, 1240 cm-1 .Albicanyl triacetate (83):251AcOAcOAc0„„.. 083Albicanyl triacetate (83) was isolated as a colorless oil (1.5mg). HREIMS; m/z 320.1985 (320.1988 calculated for C191 -12804 M-HOAc);EIMS m/z (rel. int.): 321 (4.3), 260 (1.4), 218 (6.6), 200 (29.9), 185 (11.9), 121(9.9), 107 (10.7), 93 (17.8), 86 (17.4), 81 (19.0), 69 (20.7), 55 (28.3), 43 (100), 41(37.3); 13C nmr (8, CDCI3):145.4, 109.4, 73.3, 68.4, 60.7, 48.2, 48.0, 39.5, 37.0,33.4, 23.2, 22.8, 21.0, 20.8, 20.5, 16.7; 1 H nmr (8, CDCI3): 5.25 (bs); 5.18 (m);4.95 (bs); 4.65 (bs); 4.18 (dd, J = 3.4, 11.4 Hz); 4.08 (dd, J = 9.9, 11.4 Hz); 2.48(m); 2.40 (m); 2.16 (s, 3H); 1.96 (s, 3H); 1.94 (s, 3H); 1.80 (t, J = 11.8 Hz); 1.59(dd, J = 3.6, 11.3 Hz); 1.42 (dd, J = 3.5, 11.9 Hz); 1.02 (s, 3H); 0.93 (s, 3H); 0.92(s, 3H); FTIR (film): 22937, 1736, 1702, 1458, 1364, 1248, 1039 cm -1 .Albicanyl diacetate (84):84Compound 84 was isolated as a colorless oil (0.3 mg).HREIMS; m/z 322.2140 (322.2144 calculated for C19H30404); EIMS m/z (rel.int.): 322 (0.2), 262 (6.3), 203 (18.2), 202 (99.7), 187 (53.7), 159 (24.7), 152 (15.1),43 (100); 1 H nmr (8, C6D6): 4.95 (bs, 2H), 4.81 (bs), 4.49 (dd, J = 11.8, 4.0 Hz),3.94 (t, J = 11.6 Hz), 2.68 (m), 2.30 (m), 2.06 (s, 3H), 1.75 (s, 3H), 0.88 (s, 3H),0.75 (s, 3H), 0.64 (s, 3H).Acanthene K (85):Acanthene K (85) was isolated as a white amorphous solid(3 mg; 1 animal). HREIMS; m/z 249.2098 (249.2092 calculated forC161-127N0); EIMS m/z (rel. int.): 249 (7.4), 234 (9.7), 204 (84.3), 189 (17.8), 161(100), 133 (33.5), 119 (30.8), 105 (47.5), 91 (47.7), 81 (39.8), 67 (29.4), 55 (33.5),41 (26.7); DCIMS m/z (rel. int.) 250 (100); 13C nmr (8, CDCI3): 160.8, 107.9,25257.0, 50.5, 46.0, 42.2, 40.4, 38.3, 26.5, 24.2, 21.6, 18.4, 17.3, 16.9; 1 H nmr (8,CDCI3): 8.13 (s); 4.84 (bs); 4.64 (bs); 4.05 (q, J = 10.6 Hz); 2.31 (bd, J = 12.7);1.74 (d, J = 10.9 Hz); 0.91 (d, J = 6H); 0.77 (s, 3H); FTIR (film); 3271, 3058, 2955,2932, 2846, 1659, 1548, 1382, 901, 765 cm -1 .20-acetoxymorginatone (86):86Compound 86 was isolated as a colorless oil (26 mg; 1.8mg/animal). HREIMS; m/z 358.2144 (358.2144 calculated for C22H3004);EIMS m/z (rel. int.): 359 (13.8), 358 (56.5), 343 (15.5), 283(12.4), 187 (26.8), 174 (93.7), 161 (100), 147 (92.1), 135(74.4), 91 (52.8), 81 (38.6), 69 (42.5), 55 (36.8), 43 (86.1); 13C nmr (d, C6D6):192.3, 174.4, 169.5, 142.0, 118.8, 107.0, 64.2, 57.4, 55.9, 42.0, 41.0, 38.2, 37.7, 36.6,34.8, 34.2, 33.4, 22.2, 21.1, 19.8, 18.5, 18.3; 1 H nmr (d, CDCI3): 7.24 (bs); 6.57(bs); 4.66 (d, J = 12.2Hz); 4.26 (d, J = 12.2 Hz); 2.83 (dd, J = 13.6, 17.0 Hz); 2.71(dd, J = 2.4, 17.0 Hz); 2.34 (bd, J = 13 Hz); 2.04 (s, 3H); 1.76 (bd, J = 15 Hz);1.66 (m); 1.57 (m); 1.46 (m); 1.33 (s, 3H); 1.16 (m); 1.12 (m); 0.88 (s, 3H); 0.85(s. 3H); 1 H nmr (8, C6D6): See Table 11; FTIR: 2937, 2870, 1738, 1681, 1455,1441, 1234, 1047, 788, 762 cm-1 .253(20.9), 243 (42.7), 227(50.6), 123 (28.4), 1099,11-dihydrograciffin A (87):87HREIMS; m/z 332.2352 (332.2352 calculated for C21H3203 M+-AcOH); EIMS m/z (rel. int.): 332 (0.2), 273 (6.1), 272 (1.8), 148 (100), 125 (17.1),120 (17.9), 109 (16.9), 91 (20.1), 83 (24.2), 69 (91.7), 55 (25.2), 43 (54.8), 41(35.6); 1 H nmr (8, CDCI3): 6.45 (d, J = 5.4 Hz); 5.97 (s); 5.65^J = 6.9 Hz);3.14 (dd, J = 7.4, 5.5 Hz); 2.42 (ddd, J = 9.0, 7.7, 7.5 Hz); 2.35 (dd, J = 7.9, 7.7Hz); 2.10 (s, 3H); 2.08 (s, 3H); 1.85 (n); 1.65 (d, J = 6.9 Hz, 3H); 1.03 (s, 3H);0.96 (s, 3H); 0.90 (s, 3H).lutenenolide (88):Lutenenolide (88) was isolated as a colorless oil.(5.0 mg, .05mg/animal). HREIMS: m/z 300.2091 (300.2089 calculated for C20E12802.254M+-AcOH); EIMS m/z (rel. int.): 300 (24.6), 285 (14.8), 221 (23.1), 203 (28.4),189 (19.8), 147 (16.5), 137 (61.3), 123 (50.2), 119 (50.4), 109 (73.0), 95 (71.5), 91(53.6), 81 (83.7), 69 (92.1), 55 (64.5), 43 (100); DCIMS (NH3) m/z: 378, 302; 13Cnmr (d, CDCI3): 145.8, 144.8, 110.4, 70.2, 70.2, 59.6, 56.2, 41.9, 39.2, 39.0, 33.7,33.6, 30.5, 24.4, 21.7, 21.5, 19.1, 15.8; 1 H nmr (d, CDCI3): 7.19 (s), 5.59 (m), 5.12(bs), 5.00 (bs), 4.74 (bs, 2H), 2.83 (ddd, J = 1.6, 5.7, 15.6 Hz), 2.61 (ddd, J =1.9, 7.9, 15.6 Hz), 2.35 (dm, J = 12.2 Hz), 1.99 (s, 3H), 1.82 (bs), 0.99 (dd, J =2.9, 12.6 Hz), 0.83 (s, 3H), 0.81 (s, 3H), 0.79 (s, 3H); MR (film); 2928, 1752, 1735,1654, 1235, 1061 cm -1 .7a,17P-dihydroxy-15,17-oxidosponglan-16-one 7 butyrate (89):OCompound 89 was isolated as a colorless oil (2.6 mg, .05mg/animal). HREIMS: m/z 402.2414 (402.2406 calculated for C24H3405 M+-H20); EIMS m/z (rel int.): 402 (0.4), 332 (4.0), 314 (10.0), 286 (100), 271 (19.4),258 (81.1), 229 (29.2), 162 (32.7), 137 (33.2), 123 (41.4), 109 (60.5), 91 (29.8), 81(36.6), 71 (47.2), 69 (42.2), 55 (40.1), 43 (81.5); 1 H nmr (8, CDCI3): 6.04 (d, J =6.0 Hz), 5.47 (bs), 4.77 (m), 2.86 (dd, J = 6.0, 11.4 Hz), 2.74 (bdd, J = 7.1, 11.0Hz), 2.61 (d, J = 1.9 Hz), 2.37 (t, J = 7.4 Hz), 1.00 (t, J = 7.5 Hz, 3H), 0.93 (s, 3H),2550.81 (s, 3H), 0.77 (s, 3H); FTIR (film): 3420, 2956, 2869, 1782, 1733, 1071, 976cm -1 .1711-acetoxy-15,17-oxidospongion-16-one (90):SOCompound^90 was isolated as a colorless oil (1.7 mg,0.03mg/animal). HREIMS: m/z 376.2249 amu (376.2249 calculated forC22H3205); EIMS m/z (rel. int.): 376 (0.4), 316 (100), 301 (38.5), 288 (67.9), 273(22.8), 260 (48.2), 177 (21.7), 150 (59.2), 137 (41.0), 123 (46.2), 109 (53.4). 95(58.2), 91 (55.4), 81 (68.1), 69 (74.8), 55 (64.5), 43 (99.1); 13C nmr (8, CDCI3):104.2, 103.6, 56.8, 55.3, 49.2,41.8,41.7,39.0,37.5, 33.4, 23.7,21.5, 21.3, 19.9, 18.7,16.6, 15.6; 1 H nmr (8, CDCI3): 6.29 (s), 6.10 (d, J = 6.0 Hz), 2.76 (dd, J = 6.9,11.2 Hz), 2.65 (dd, J = 6.0, 11.2 Hz), 2.45 (dm, J = 12.5 Hz), 2.04 (s, 3H), 0.86 (s,3H), 0.81 (s, 3H), 0.72 (s, 3H); F'1112 (film): 2952, 2930, 1784, 1755, 1221, 1029, 941cm -1 .256Cadlinaldehyde (91):91Cadlinaldehyde (91) was isolated as a white amorphoussolid which was subsequently recrystallized from methanol (12 mg, 0.2mg/animal; colorless needles, mp = 65 °C). DCIMS m/z (rel. int.): 336(11.5), 319 (100), 303 (27.7); HREIMS: m/z 303.2308 amu (303.2324calculated for C20H3102, M+ - 15); EIMS m/z (rel. int.): 304 (26.4), 303 (100),285 (9.1), 275 (8.3), 191 (7.0), 149 (9.9), 137 (24.9), 121 (13.4), 109 (23.0), 97(77.6), 81 (30.8), 69 (34.6), 55 (51.0), 43 (82.4), 41 (64.7); 13C nmr (8, CDCI3):206.4, 79.0, 64.9, 61.4, 59.5, 55.6, 53.4, 41.7, 40.4, 39.3, 36.5, 34.4, 33.7, 32.0, 23.0,20.8, 20.8, 19.9, 19.4, 17.5, 16.6; 1 H nmr (8, CDCI3): 10.1 (s), 3.92 (dt, J = 2.0, 8.4Hz), 3.83 (q, J = 8.3 Hz), 2.52 (bd J = 12.2 Hz), 1.01 (s, 3H), 0.94 (s, 3H), 0.80 (s,3H), 0.73 (s, 3H), 0.7 (m); FTIR (film): 2959, 2928, 1704, 1456, 1376, 1001.257D. Metabolites from Archidoris pseudoargus.Collection and Isolation Procedure. See Extraction and Purificationsection in text.Compound 118:Compound 118 was isolated as an amorphous white solid(19.0 mg, 9.5 mg/animal). DCIMS m/z (rel. int.): 396 (45.3), 379 (100), 287(19.6); EIMS m/z (rel. int.) 378 (2.5), 304 (4.7), 286 (61.5), 258 (14.8), 192 (91.2),177 (65.8), 147 (28.7), 137 (38.0), 123 (79.7), 121 (69.9), 105 (74.8), 95 (100), 81(68.2), 69 (98.7), 55 (44.2); 13C nmr (d, CDCI3): 173.5, 128.4, 124.4, 70.3, 65.1,63.5, 62.5, 56.4, 54.2, 41.8, 39.8, 37.4, 36.6, 33.4, 33.1, 29.7, 22.6, 21.2, 18.6, 18.4,15.7, 15.6; 1 H nmr (d, CDCI3): 5.53 (bs), 4.22 (dd, J = 11.5, 14.7 Hz), 4.16 (dd, J= 6.0, 11.5 Hz), 3.95 (m), 3.71 (dd J = 3.9, 11.5 Hz), 3.62 (dd J = 5.8, 11.5), 2.96(bs), 2.32 (bs), 1.96 (bm), 1.61 (bs, 3H), 0.95 (s, 3H), 0.91 (s, 3H), 0.86 (s, 3H),0.82 (s, 3H); FTIR (film): 3435, 3358, 2924, 2846, 1723, 1169 cm- 1 .258Compound 119:OR S0119R1 =Ac R2 =HCompound 119 was isolated as a colorless oil (0.5 mg, 0.25mg/animal). DCIMS m/z (rel. int.): 438 (100), 421 (25.4); 1 H nmr (8, CDCI3):5.51 (m), 5.05 (m), 4.32 (dd, J = 4.5, 12 Hz), 4.25 (dd, J = 5.0, 12 Hz), 3.74 (d, J= 4.5 Hz, 2H), 2.93 (bs), 2.08 (s, 3H), 1.58 (bs, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.85(s, 3H), 0.80 (s, 3H). FTIR (film): 3446, 2927, 2854, 1733, 1653, 1234, 1157 cm -1 .Compound 125:OHO OOR125R= ?Compound 125 was isolated as a white amorphous solid(3.5 mg, 1.8 mg/animal). EIMS m/z (rel. int.): 1036 (1.1), 902 (0.2), 863 (0.2),(1.5), 365 (1.9), 282 (3.5), 264 (5.3), 218 (2.6),(40.7), 83 (53.9), 69 (72.5), 55 (100), 43 (92.9),41 (90.8); 13C nmr (8, CDCI3): 124.6, 114.2, 113.6, 72.6, 71.9, 70.4, 70.3, 65.2, 64.3,63.3, 62.2, 61.9, 49.4, 42.0, 40.4, 34.2, 33.3, 31.9, multiple 13C resonancesbetween 29 and 30 ppm, 29.1, 26.1, 24.9, 23.7, 22.7, 21.9, 21.4, 18.7, 14.9, 14.1;1 H nmr (8, CDCI3): 5.56 (bs), 5.35 (bm, 2H), 4.23 (dd, J = 4.4, 11.6 Hz), 4.15(m), 3.94 (m), 3.85 (m), 3.75-3.44 (unresolved muttiplets, 7H), 2.96 (bs), 2.35259804 (0.l), 757 (0.4), 618 (0.5), 526191 (6.2), 135 (18.3), 109 (39.8), 95(t, J = 7.8 Hz), 1.61 (bs, 3H), 1.26 (multiple 1 H resonances), 0.97 (s, 3H), 0.91(s, 3H), 0.88 (s, 3H), 0.88 (t, J = 6.5 Hz, 3H); FT1R (film): 3391, 2926, 2854, 1738,1456, 1110, 787, 764 cm -1 .E. Metabolites from Plocamissa Igzo.Collection and Isolation Procedure. Plocamisso igzo was collected byhand using SCUBA ( -2m, Anthony Island B.C.) and kept frozen until timeof work up. Workups entailed freeze-drying the sponge (500g),extracting with 700mL CH2Cl2 , 700mL EtOAc and 700mL Me0Hsequentially. The EtOAc extract was evaporated to yield a viscous,brown, foul-smelling oil (1.7 g). This residue was passed through asephadex LH-20 column (1 X 40cm) using Me0H as the elluent. Thefractions with highest cytotoxic activity (L1210) were further partiallypurified using a second LH-20 column (0.5 X 170cm) and a 40:10:4EtOAc:MeOH:H20 solvent mixture. The cytotoxic components eluted intwo main fractions. The most cytoxic could not be resolved intocomponents by chromatographic methods and was acetylated bydissolving in an excess of pyridine/acetic anhydride (1 / 1) and stirring atroom temperature overnight. Subsequent purification by normal phaseHPLC (EtOAc) yielded; 6.0 mg 3',5'-diacetoxythymidine (153), 6.8 mg2',3',5'-triacetoxyuridine (154) and 2.4 mg 3',5'-diacetoxy-2'-deoxyuridine(155) in order of elution. Final purification of the second cytotoxic fractionon RP-HPLC (35% water in methanol) yielded; 6.0 mg 6-bromoindole-3-carboxyaldehyde (156) and 14 mg igzarnide (157) in order of ellution.26031,5'-diacetoxythymidine (153):AcO 153Compound 153 was isolated as a colorless oil (6.0 mg). 1 Hnmr (d, CDCI3): 6.24 (dd, J = 5.7, 8.4 Hz), 5.14 (m), 4.27 (m, 2H), 4.17 (m),2.38 (dm, J = 14.3 Hz), 2.12 (m), 2.03 (s, 3H), 1.95 (s, 3H), 1.84 (s, 3H).2',3',5Arlacetoxyuridine (154):0NHIIAcOH2C nN''LO1AcO OAc154Compound 154 was isolated as a colorless oil (6.8 mg). 1 Hnmr (d, CDCI3): 8.62 (d, J = 4.3 Hz), 7.40 (d, J = 8.1 Hz), 6.02 (d, J = 4.7 Hz),5.79 (d, J = 6.1 Hz), 5.34 (m), 4.34 (m, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.08 (s,3H).2613',5'-diacetoxy-2'-deoxyuddine (155):0NHAcOH2C N 0AcO 155Compound 155 was isolated as a colorless oil (2.4 mg). 1 Hnmr (d, CDCI3): 7.49 (d, J = 8.2 Hz), 6.27 (dd J = 5.7, 8.3 Hz), 5.78 (d, J = 8.2Hz), 5.20 (m), 4.34 (m, 2H), 4.27 (m), 2.53 (dm, J = 14.3 Hz), 2.14 (m), 2.11 (s,3H), 2.10 (s, 3H).6-bromoindole-3-carboxaldehyde (156):Compound 156 was isolated as a white amorphous solid(6.0 mg, UV; ,ax 216 nm e 9600); EIMS m/z (rel. int.): 225 (96.1) 224 (27.7),223 (100), 222 (90.0), 196 (25.3), 194 (25.1), 143 (24.3), 115 (47.4); 1 H nmr (d,Me2SO-do): 10.04 (s), 8.25 (s), 8.16 (d, J = 7.5 Hz), 7.77 (d, J = 1.8 Hz), 7.40(dd, J = 1.8, 7.5 Hz).262Igzamide (157):263NH2lgzamide (157) was isolated as a yellow soilid (14 mg, UV;Xrnaxl 294 nm, e = 4900; Xmax2 228 nm, E = 12800); HREIMS: m/z 308.9944/306.9961 amu (308.9937/306.9957 calculated for Ci2H1ON302Br); EIMSm/z (rel. int.): 309 (4.4), 308 (1.1), 307 (4.9), 264 (4.6), 262 (3.7), 149 (13.7), 129(10.5), 97 (12.6), 87 (10.0), 85 (19.5), 84 (28.0), 83 (23.5), 82 (16.5), 81 (16.1), 43(100).13C nmr (8, Me2S0-d6): 161.4, 157.2, 136.7, 130.8, 124.4, 122.3, 120.3,118.0, 114.7, 114.3, 109.7, 105.3; 1 H nmr (5, Me2S0-d6): 11.52 (bs), 9.48 (d, J =7.6 Hz), 8.36 (bs), 8.07 (bs), 7.61 (d, J = 1.8 Hz), 7.55 (d, J = 7.4 Hz), 7.53 (s),7.19 (dd, J = 1.8, 7.4 Hz), 6.70 (dd, J = 7.6, 9.1 Hz), 6.15 (d, J = 9.1 Hz); FTIR(film); 3300, 1678, 1613, 1536 cm -1 . Biological activity L1210 (ED50) 19.0p.g/mL (Et0H), B.s. (MIC) >100 p.g, S.a.a. (MIC) >100 p.gAniline oxalamide (158):0Excess oxalyl chloride was added with rapid stirring to 50mg of aniline in dry THF. After 1 hr, ammonia gas was bubbled throughthe reaction mixture. Insoluble oxalyldiamide was removed by gravityfiltration and the aniline oxalamide was purified on silica (EtOAc, 35 mg158 after purification). HREIMS: 164.0591 amu (164.0585 calculated forC8H8N202); EIMS m/z (rel. int.): 164 (54.2), 121 (11.8), 120 (59.7), 119 (25.7),93 (58.5), 92 (35.0), 77, (100), 66 (31.2), 65 (30.2), 64 (11.1), 51 (33), 44 (60); 13Cnmr (8, Me2S0-d6); 162.3, 158.9, 137.8, 128.7, 124.3, 120.3; FTIR (film): 3395,3300, 1659, 1651, 1598, 1537, 1445, 1401,751 cm -1 .264Appendix A. Spectra of the Previously Reported MetabolitesIsolated from the Three Dorid Nudibranchs26522 2.0 1.8 1.6 1.4 12^1.0PPM0.8 0.6 0.4 02^0.0Figure A01.400 MHz 1 H NMR Spectrum (CDCI3) of 682.0^1.8 ^1 .6^1 .4^1 2^1.0PPM0.4^02 0.00.8 0.6Figure A02. 400 MHz 1 H NMR Spectrum (CDCI3) of 69^ vX78 X = NHCHOSII^.,..^.^3.0^4---71.0 ----"-----7- 67.0^.0^---. ^.0PPM3.0^2.0Figure A03. 400 MHz 1 H NMR Spectrum (CDCI3) of 781.0 0.0g2.0 1.0^0.07.03.04.05.06.0PPMFigure A04. 400 MHz 1 H NMR Spectrum (C6D6) of 7980 X = NCS1-.--r-7.0^6.0^5.0^4.0^3.0^2.0^1.0PPMFigure A05. 400 MHz 1 H NMR Spectrum (CDCI3) of 800.0CICIBrCH20 "CI CI817.0^6.0^5.0^4.0^3.0^2.0PPMFigure A06. 400 MHz 1 H NMR Spectrum (CDCI3) ofViolacene (81)1.0S I^w1elliagnalk^FW.W.I.TTWW.r.V.TO^ ■r.f I me■Tr.I.N.^■t•M••••••TT7^T.V.r e^e^4^3 2^i^0PPMFigure A07.500 MHz 1 H NMR Spectrum (CDCI3) of 82•••••■■■TP.P.IPPPMFigure A08. 500 MHz 1 H NMR Spectrum (CDCI3) of Marginatofuran (74)■ Vie,^► II VIVI!!!► VIII ► TWIT^ I 1 ►SIN ► ' ► "vie' ► ► J  ► "A" ► I  ►^► ► " ► VIV I 111 ► 1 1 ►NI PPM^Id►411► ►^1 IFigure A09. 75 MHz 13C/APT nmr Spectra (C6D6) ofMargInatafuran (74)Figure A10. 400 MHz 1 H- 1 H COSY Spectrum (CDCI3) ofMarginatafuran (74)275I••••.I8•••• . ^•4• • .. • • .... • • ^04 i  .••• •..^• 9 •• • WO•• • ••1.• •A••u• 4• •^.• • • •••• ••••..,• ••• ••••••••••••••■•••••••••• • ••■•• •••■••••■•••••••••^•111/••••■••••.. mom ea ea .••••••■•••••••■•■•■•,....SI^-^I^- 15.0^4.5^4.0^3.5^3.0 2.5^2.0^1.5^1.0^0.5PPMFigure Al2. 400 MHz 1 H NMR Spectrum (CDCI3) of 9,11-dihydrogrocillin A (87)7.5^7.0Os)6.5^6.0^5.5S.Figure A13.500 MHz 1 H NMR Spectrum (CDCI3) of Luteone (73)OS 100Figure A14. 125 MHz 13C NMR Spectrum (CDCI3) of Luteone (73)OA0.60.81D121.4I . 61.82.0222A2.62.8PPM. - .2.8 24 2:4 -22 2.0^1 B 1.6^1.4 1 12^1 1.0 0.8 0,6^OAPPMFigure A15. 400 MI-lz 1 H- 1 H COSY Spectrum (CDCL3) of ofLuteone (73)280___._ 0.• 1.9.46..^.Abe,it . ,o^•awl50ele •4111*100......1502DC. .. POI001 a^5^4^2Figure M6. 500 MHz HMQC Spectrum (CDCI3) ofLuteone (73)281a^a s^2^1^o4Figure A17. 500 MHz 1 H NMR Spectrum (CDCI3) of 89H0R1 R289 R i = OBu, R2 = OH•1.7.5 4.0 PPM,^-^- •7.0^6.5^6.0^5.5^5.0^4.5 3.0^2.5^2.0^1.5^1.0^0.5 ^0.0OR2Figure A18. 400 MHz 1 H NMR Spectrum (CDCI3) of 118I Figure A19. 500 MHz 1 H NMR Spectrum (CDCI3) of 119iAppendix B. Spectra of the Previously Reported MetabolitesIsolated from Plocomisso igzo2850AcO 153 ^k^r^-r7.6 6.0 5. 0 4P.9PM1^ -3.0 2.0^t.Figure 801. 400 MHz 1 H NMR Spectrum (CDCI3) of 3',5'-dlacetoxythymidine (153)QNpDDNHAc0H2C N 00AcO OAc154a . e^7.d^6.e^5. I^4. 0^3. 0^2.0^L . 0FPMN.)CO^Figure B02. 400 MHz 1 H NMR Spectrum (CDCI3) of 2',3',5-triacetowuridine (154)^ .10AcO,s[^ 7. 0^6.0^5.0^4.0^3.0^2.0^t.0?PHFigure B03. 400 MHz 1 H NMR Spectrum (CDCI3) of 3',5-diacetoxy-2'-deoxyuridine (155) DD••••■••••■•••••■•••••■s-••••.T-..--•p•-•.•Nip-•■••■••-••■•••■r8.0^7.5^1.09.0 PPM8:5••••••••■■=m4,••••••ipor•••••••••9.5103 10.0Figure B04. 400 MHz 1H NMR Spectrum (CDCI3) of 6-bromolndole-3-carboxaldehyde (156)

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