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Terpenoids from the marine sponge Aplysilla glacialis and the nudibranch Cadlina luteomarginata Tischler, Mark 1989-02-16

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TERPENOIDS FROM THE MARINE SPONGE APLYSILLA GLACIAUS AND THE NUDIB RANCH CADLINA L UTEOMAR GIN A TA by MARK TISCHLER M.Sc. University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept this thesis as confonning to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1989 ©Mark Tischler, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of r^/V^&M/J 77?^ The University of British Columbia Vancouver, Canada Date 77>?/i/ 3V f DE-6 (2/88) n Abstract A chemical study of the pink encrusting sponge Aptysilla glacialis collected in Barkley Sound, B.C., has led to the isolation and structure elucidation of terpenes which are believed to be derived biogenetically from the hypothetical "spongian" precursor. In addition, the first example of a diterpene from a sponge containing a "marginatane" skeleton has been found. Cadlinolide A (75) was isolated and its structure elucidated by a combination of spectroscopic interpretation, chemical degradation, and confirmed by a single crystal x-ray diffraction analysis. The structure of a related metabolite, cadlinolide B (761. was also isolated and elucidated by spectroscopic interpretation and conversion to the known metabolite tetrahydroaplysulphurin-1 (72). The stracture of a nor-diterpene, aplysilloUde A (1011 was determined by spectroscopic interpretation and chemical interconversion along with its dehydrated analogue, aplysillolide B (102). Glaciolide (110). a degraded and highly rearranged diterpene was solved by extensive NMR analysis of both the parent compound and its chemically interconverted derivatives. Glaciolide (110) represents only the second known example of a metabolite containing a "glaciane" skeleton. Marginatone (112) is the first example of a diterpene containing a "marginatane" skeleton from a sponge. The "marginatane" skeleton was first encountered in a metabolite, majginatafuran (111), isolated from the nudibranch Cadlina luteomarginata which is generally found in the same location as Aptysilla glacialis. The structure of cadlinolide C (J__L)» containing both methyl ester and y lactone moieties, was elucidated by spectroscopic interpretation. This compound is believed to be an isolation artifact Examination of the chemical constituents of the nudibranch Cadlina luteomarginata found feeding on the sponge Aptysilla glacialis yielded a mixture of terpenes mcluding m cadlinolide A (75). glaciolide (110) and tetrahydroaplysulphurin-1 (72). Compound 72 was previously isolated from a New Zealand sponge. A review of "spongian" and "marginatane" derived metabolites from sponges and nudibranchs as well as a review of Cadlina luteomarginata terpenoids is presented. IV Table of Contents Abstract II Table of Contents V List of Tables VH List of Figures LX List of Schemes XTV List of Abbreviations XAcknowledgements XVII A. Introduction To The Sponges 1 0 biologyii) Marine Natural Products Chemistry 3 -Spongian and Marginatane Derived Diterpenes 4 -Spongian Skeleton. 4 -Norisane Skeleton 16 -Macfarlandin Skeleton 18 -Aplysulphurane Skeleton 21 -Denririllane Skeleton 23 -Degraded Spongian Skeleton 28 -Chromodorane Skeleton 31 V -Gladane Skeleton 32 -Marginatane Skeleton. 33 -Biogenetic Proposals 33 B. TERPENOID METABOLITES FROM THE SPONGE APLYSELLA GLACIALIS MEREJKOWSKI1878 43 1. Introduction 42. Isolation and Structure Elucidation 46 3 A. CadlinoUdeA(2_) 46 3 B. Cadlinolide B W 60 3 C. Aplysillolide A (lfll) 71 3D. Aplysillolide B (J_t_) 88 3E. Glaciolide 94 3F. Marginatone (JL12) 126 3G. Cacfflnotide C 03J_) 138 C-I. INTRODUCTION TO THE NUDIBRANCHS 148 -METABOLITES OF CADLINA LUTEOMARG1NATA.... 151 C-II. SPONGIAN METABOLITES FROM THE NUDIBRANCH CADLINA LUTEOMARGINATA MACFARLAND 1966 156 L Introduction 156 2. Isolation and Structure Elucidation 157 3. Tetrahydroaplysulphurin-l (22) 158 Conclusion 166 D. EXPERIMENTAL 170 E. List of References 182 vn List of Tables Page Table 1: 75MHz 13c NMR/APT Data for CadlmoUde A (25) in CDCI3 48 Table 2: 4(X)MHz !H NMR Data for <_adlinoUde A CZ_D in CDCI3 50 Table 3: 75MHz 13C NMR/APT Data for CadlinoUde B Q£) in CDCI3 62 Table 4: 400MHz lH NMR Data for CadlinoUde B (2© in CDCI3 63 Table 5: 75MHz 13C NMR Data for AplysiUoUde A (____) and GracilinA(23JinCDa3 73 Table 6: 400MHz JH NMR Data for AplysilloUde A (Jj_l) in CDCI3 75 Table 7: 400MHz ^ NMR Data for Triacetate 125 in CDCI3 83 Table 8: 400MHz lH NMR Data for AplysilloUde B Q02) in CDa3 90 Table 9: 75MHz 13c NMR/APT Data for GlacioUde (HQ) in CDCI3 96 Table 10: 400MHz lH NMR Data for GlacioUde (lift) in CDCI3 97 Table 11: 400MHz lH NMR Data for GlacioUde (110) in QD6 100 Table 12: 400MHz lH NMR Data for Diol 127 in CDCI3 107 Table 13: 400MHz lH NMR Data for Diacetate 128 in CDCI3 112 Table 14: 400MHz lH NMR Data for RUO4 Product 129 in CDCI3 117 Table 15: 400MHzNMR Data for RUO4 Product 130 in CDCI3..... 122 Table 16: 400MHz !H NMR Data for Marginatone (112) in CDCI3.... 128 Table 17: 400MHz lR NMR Data for Marginatone (ill) in 130 Table 18: 75MHz 13C NMR/APT Data for Marginatone (JU_) in CDCI3 133 vm Table 19: 75MHz 13c NMR/APT Data for Odlinolide C (131) in CDCI3 140 Table 20: 400MHz NMR Data for Gidlmokde C (121) in CDCI3 142 Table 21: 400MHz lH NMR Data for Tctrahyd^plysulphurin-1 (22) inCDCki 161 Table 22: 75MHz 13C NMR Data for Tetrahytoaplysulphurin-1 (22) in CDQ3 163 List of Figures Page Figure 1: Phylogenic Qassification of the Sponge Aptysilla glacialis (Merejkowski 1878) According to Austin (1989) 44 Figure 2: 75MHz 13c NMR/APT Spectra for CadlinoUde A (25) in CDCI3.... 47 Figure 3: 400MHz lH NMR Spectrum of CadlinoUde A (25) in CDCI3 49 Figure 4: 400MHz COSY Spectrum of CadlinoUde A (25) in CDCI3 52 Figure 5: Isolated Spin Systems from COSY Spectra of (ZadlinoUde A (25)... 53 Figure 6: NOe Enhancements Observed for (L^adlinoUde A CIS) 54 Figure 7: 300MHz lH NMR of Diacetate 123 in CDCI3. 56 Figure 8: 400MHz COSY Spectrum of Diacetate 123 in CDCI3 57 Figure 9: Isolated Spin Systems for Diacetate 123 58 Figure 10: 13C NMR Chemical Slurts for Ring A 59 Figure 11: Computer CteneratedORTEP Drawing of CadlinoUde A (25)... 59 Figure 12: 75MHz 13C NMR/APT Spectra for CadlinoUde B Q£) in CDCI3.... 61 Figure 13: 400MHz lH NMR Spectrum of Cadhnolide B (2£) in CDCI3 64 Figure 14: 400MHz COSY Spectrum of (jadlinolide B (7j6J in CDQ3 66 Figurel5: Assignment of Spin Systems for C!adlinohde B CI6) from COSY Spectra. 67 X Figure 16: Nee Enhancements Observed for Cadlinolide B (7j_> 67 Figure 17: 100MHz lH NMR Spectrum of authentic Tetrahy(iroaplysulphuiin-l (22) in CDCI3 68 Figure 18: 75MHz 13C NMR/APT Spectra for Aplysillolide A (Jill) in CDQ3 72 Figurel9a: 400MHz !H NMR Spectrum of AplysilloUde A (lfll) in CDCI3.... 74 Figurel9b: Offset, Irradiation at 8l.66ppnx 74 Figure 20: 400MHz COSY Spectrum of AplysUUoUde A (10_D in CDCI3 77 Figure 21: Spin Systems from COSY/ Double resonance Spectra for Substructure C 78 Figure 22: SINEPT Results for AplysiUoUde A Ofll) 79 Figure 23: NOe Enhancements for AplysiUoUde A (1Q1) 80 Figure 24: 400MHz lH NMR Spectrum of Triacetate 125 in CDCI3 82 Figure 25: 400MHz COSY Spectrum of Triacetate 125 in CDCI3 84 Figure 26: Spin Systems for Triacetate 125 86 Figure 27: Summary of NOe Enhancements for Triacetate 125 87 Figure 28: 400MHz lH NMR Spectrum of AplysiUoUde B Q_2) in CDCI3 89 Figure 29: 400MHz COSY Spectrum of AplysiUoUde B &Q2) in CDCI3 91 Figure 30: NOe Enhancements Observed for AplysiUoUde B (1Q2) 93 Figure 31: 75MHz 13c NMR/APT Spectra for GlacioUde OIQ) in CDCI3 95 XI Figure 32: 400MHz iH NMR Spectrum of Glaciolide (HQ) in C6D6 98 Figure 33: 400MHz lH NMR Spectram of Glaciolide (JIQ) in CDCI3 99 Figure 34: 400MHzOOSY Spectrum of Glariohte (110J in CDCI3 101 Figure 35: 400MHz COSY Spectrum of GlacioUde (lift) in C6De 102 Figure 36: Isolated Spin Systems from COSY Data for Glaciolide (HQ) 103 Figure 37: 400MHz Long Range COSY Spectrum of Glaciolide (lift) inCDCl3 104 Figure 38: Noe Enhancements Observed for Glaciolide (HQ) 105 Figure 39: 400MHz JH NMR Spectrum of Diol 127 in CDCI3 106 Figure 40: 4(X)MHz(X)SYSrjectrumofDioll27inCDa3 109 Figure 41: 400MHz *H NMR Spectrum of Diacetate 128 in Cf£>6 HI Figure 42: 400MHz COSY Spectrum of Diacetate 128 in (_6D6 113 Figure 43: 400MHz lH NMR Spectrum of RUO4 Product 129 in CDCI3 116 Figure 44: 400MHzCOSY Spectrum of RUO4 Product 129 inCDCl3 118 Figure 45: FT-IR Spectrum of Product 129 0 Figure 46: 400MHz *H NMR Spectrum of RUO4 Product 130 in CDCI3 121 Figure 47: 400MHzCOSY Spectrum of RUO4 ftxxluct 130 in CDCI3 123 Figure 48: FT-IR Spectrum of Product 130 125 xn Figure 49: 400MHz *H NMR Spectrum of Marginatone (112) in CDCI3 127 Figure 50: 400MHz lH NMR Spectrum of Marginatone (112) in C6D6 129 Figure 51: 75MHz 13C NMR/APT Spectra for Marginatone (112) in 0)03... 132 Figure 52: 400MHzCOSY Spectrum of Marginatone (112) in 0X33 134 Figure 53: 400MHz CDS Y Spectrum of Marginatone (112) in C6E>6 135 Figure 54: NOe Enhancements Observed for Marginatone (112) 136 Figure 55: 400MHz Long Range COSY Spectrum of Marginatone (112) in CDCI3 137 Figure 56: 75MHz 13c NMR/APT Spectra for CadlinoUde C (Ul) in CD CI 3. 139 Figure 57: 400MHz lH NMR Spectrum of CadlinoUde C (121) in CDCI3 141 Figure 58: 400MHz COSY Spectrum of CadlinoUde C (121) U1CDCI3 144 Figure 59: Isolated Spin Systems in CadUnoUde C (121) 145 Figure 60: Phylogenic Classification of Nudibranchs (Classification acccoding to Behrens) 149 Figure 61: Typical Dorid Nudibranch 150 Figure 62: 400MHz !H NMR Spectrum of Tetrahydroaplysidphurin-1 (22) in CDa3 16Figure 63: 15MHz 13C NMR/APT Spectra for Tetrahydroaplysulphurin-1 (22) inCDa3 162 Figure 64: 400MHz COSY Spectrum for Tetrahydroaplysulphurin-1 (22) inCDCk 164 Figure 65: NOe results for Tetrahydroaplysulphurin-1 (Z2) Figure 66: 400MHz lH NMR Spectrum of Compound D 132 and 76 List of Schemes Page Scheme 1: Biogenetic Proposals far Spongian and Marginatane Skeletons 38 Scheme 2: Spongian metabolites Prom Geranylgeraniol 39 Scheme 3: Biogenetic Proposals for Degraded and Rearranged Spongian Metabolites 40 Scheme 4: Biogenetic Proposals for Rearranged Spongian Deri 41 Scheme 5: Biogenesis of the Glaciane Skeleton via an Epoxide 41 Scheme 6: Biogenetic Proposal for the Gracillane Skeleton via an Epoxide 42 Scheme 7: Isolation Scheme for Diterpenes from Aptysilla glacialis 45 Scheme 8: IJAIH4 Reduction of CiadlinoUde A Q5J 54 Scheme 9: Acetylation of CkdtinoUde B Q6J 70 Scheme 10: Mclarfferty Rearrangement of AplysiUoUde A (101) 81 Scheme 11: Reduction and Acetylation of AplysiUoUde A (J__l) 81 Scheme 12: McLafferty Rearrangement of AplysiUoUde B (_Q2) 92 Scheme 13: Chemical Interconversion of GlacioUde (llfl) : 114 Scheme 14: Conversion of QdTinoUde A QS) to C^ad_noUde C (J_3J_).... 146 Scheme 15: Methanolysis of CadlinoUde A (75) 167 List of Abbreviations XV APT = Attached Proton Test br = broad CDC-3 = Chloroform-di (CD3)2CO = acetone-d6 COSY = Homonuclear correlation d = doublet DQMS = Desorption Chemical Ionization Mass Spectrometry ED50 = Concentration that ellicits a 50% response in Cells EIHRMS = Electron Impact High Resolution Mass Spectrum Ell-RMS = Electron Impact Low Resolution Mass Spectrum EtOAc = Ethyl Acetate Et20 = Diethyl ether HETCOR = Heteronuclear Correlation HPLC = High Performance Liquid Chromatography HPLC-MS = High Performance Liquid Chromatography Mass Spectrum IC50 = Concentration that inhibits 50% of the cell growth IR = mfrared J = Scalar coupling constant LD50 = Dose that inhibits growth of 50% of cells M+ = Parent ion XVI MeOH = Methanol m = proton resonance with unresolvable couplings MIC = Minimum Inhibitory Concentration mult = multiplicity mp. = melting point m/z = mass to charge ratio nOe = nuclear Overhauser effect ppm = parts per million PS = in vitro lymphocytic teukemia q = quartet rel. int. = relative intensity s = singlet S INEPT = Selective Insensitive Nuclei Enhanced by Polarization Transfer T/C = Test compared to Control TLC = Thin Layer Chromatography lH NMR = Proton nuclear magnetic resonance 13C NMR = Carbon-13 nuclear magnetic resonance Acknowledgements xvn I would like to express my appreciation to Professor Raymond Andersen for his encouragement and guidance throughout the course of this work, and for his assistance during the preparation of this thesis. Also, I wish to thank the members of our group, especially Mr. Mike LeBlanc, who have assisted me in the collection of the organisms studied. I thank Dr. Guenter Eigendorf of the B.C. Regional Mass Spectrometry Facility for his training and friendship as well as Dr. S. Orson Chan and his staff for their assistance with my NMR studies. Finally, I wish to extend a very special thanks to my parents for their patience, constant encouragement and support throughout the course of my studies. A. Introduction To The Sponges l 0 Biology Sponges (phylum Porifera) are the most primitive multicellular animals. Their way of life is so unlike that of other animals that up to 1825 they were classified as plants.1 All members of this phylum are sessile and exhibit very little movement. Due to the porous nature of their body, particles suspended in water near a living sponge enter the many small encurrent pores, or ostia, and emerge by way of a complex system of passageways and cavities from the large excurrent pores, or oscula. As water passes through these channels aided by choanocytes, which are cells on the outermost part of the sponge possessing a flagellum which propels water through the passageways, the body is nourished and aerated.2 Sponges vary gready in size and shape depending on the nature of the substratum, available space, and the velocity and type of water current. Thus, taxonomic confusion often results because specimens of the same species growing in different environments can have quite different appearances. Although some sponges are radially symmetrical, the majority are irregular and exhibit massive, erect, encrusting, or branching growth patterns. The significance of the often observed bright colouration of sponges is uncertain, however, protection from solar radiation and predation have been suggested.3 It is only in obtaining food and other materials from the environment that sponges have capitalized on their multicellular organization. Sponges feed chiefly on bacteria, dinoflagellates and other plankton in addition to absorbing oxygen, silica and calcium salts from incoming streams of water.4 The constant influx of water through the sponge provides conditions for respiratory exchange since no special respiratory organs are present. Sponges are quite sensitive to oxygen availability and they appear to possess some form of oxygen debt system, closing down the oscula during oxygen shortage. As a result, when metabolism is carried out during this shortage, complex organic end products are formed and accumulated which are later oxidized when oxygen becomes available.5 Even though sponges lack special sensory organs and the ability to escape, they are far from helpless. Fishes, for example, tend to avoid sponges perhaps due to chemical defences or the presence of sharp bristles which can penetrate soft tissue. However, sponges do have predators, particularly molluscs which have the ability to selectively sequester defensive allomones from their sponge diet.6 The approximately 10,000 known species of marine sponges can be placed into four main classes based on the nature of their skeleton. Class Calcarea, contains all sponges which have calcium carbonate spicules (known as calcareous sponges). Sponge spicules vary in size and shape and often serve as useful characters in identifying sponge species. Spicules are normally labelled by the number of axes or rays they possess by adding the appropriate numerical prefix to the ending -axons (when referring to the number of axes) or -actine (when referring to the number of rays or points). The spicules of the Calcarea are monaxons or three or four pronged types. The colours encountered in this sponge class vary from greyish white to brilliant yellow, red, or lavender. Species in this class are the smallest of all sponges, normally less than 10cm in height, and generally can be found in the shallow waters of all the oceans in the world.7 Class Hexactinellida have spicules which are always of the triaxon or six pointed type. Some of the spicules are occasionally fused to form a lattice like skeleton built of long siliceous fibers, hence they are commonly called "glass sponges". This class elaborates the most symmetrical sponges, which have cup, vase, or urnlike shapes averaging 10 to 30cm in height. Hexactinellidae are mainly deep water sponges, commonly found at depths of 400 to 950m, mainly in the tropical waters of the West Indies and the Eastern Pacific.7 Class Demospongiae contains the greatest number of sponge species, nearly 95 percent of all those known, including most of the North American sponges. They are distributed from shallow water to great depth. Different species are characterized by various bright colours due to pigment granules in their cells. Their skeletons vary, consisting of siliceous spicules or spongian fibers or a combination of both. The spicule containing species differ from those in Class Hexactinellidae in that their spicules are larger monaxons or tetraxons rather than triaxons.7 Finally, Class Sclerospongiae sponges account for a small number of species that are found mainly in tunnels associated with coral reefs in various parts of the world. These sponges differ from other classes in that they have an internal skeleton of siliceous spicules and spongin fibers and an outer encasement of calcium carbonate.7 ii) Marine Natural Products Chemistry Chemists and biochemists have been particularly interested in the wide diversity of compounds isolated from sponge species in the class Demospongiae. These metabolites often possess unique chemical structures as well as significant biological activity. Review articles outhning the various classes of compounds reported, including alkaloids, steroids and terpenes, have been prepared by Scheuer8 and Faulkner.9 Of these classes, terpenes are the most abundant non-steroidal secondary metabolites which have been isolated from sponges. Of particular interest over the past 15 years, has been the isolation of an interesting class of terpenoid metabolites derived from a hypothetical "spongian" (1) precursor. The following section is a review of all the "spongian" and related "marginatane" derived metabolites which have been isolated from sponges and from nudibranchs which are known to obtain these compounds from sponges in their diet.lu Spongian and Marginatane Derived Diterpenes Spongian Skeleton Until the mid 1970's, very few examples of sponge diterpenoids were known. In fact, only two different skeletal types had been discovered. Three metabolites from a Halichondria species were reported as the isonitrile, isothiocyanate and formamide analogues of geranyllinalool, 2-4. Their structures were solved by a combination of spectroscopic analysis and chemical interconversion.11 R 1 R=N--C 1 R= NHCHO £ R= N=C=S The second group of diterpenes were based on the isoagathic acid skeleton (5J, which was first obtained by Ruzicka and Hosking in 1930 upon acid treatment of agathic acid (£).12 Surprisingly, diterpenes possessing the isoagthic acid skeleton were not known from nature prior to the isolation of isoagatholactone (Z) by Cimino et al. from the Mediterranean marine sponge Spongia officinalis.13 Previous investigations of 5. officinalis (order Dictyoceratida) had yielded a series of linear C21 and C25 (order Dictyoceratida) had yielded a series of linear C21 and C25 furanoterpenes.14 Of interest is the fact that samples of the sponge containing isoagatholactone (2) were devoid of the linear furanoterpenes, while samples of sponge containing the linear furanoterpenes did not contain any of the diterpene lactone 7. Since both samples were identified as Spongia officinalis, which on comparative analysis showed only slight morphological differences, it was concluded that the two samples represented different subspecies. Subsequent work by Kazlauskas et al. on the extracts of several Spongia species collected on the Australian Great Barrier Reef led to the isolation and structure elucidation of eight new diterpenes which were initially given the trivial names spongiadiol (8J, spongiadiol diacetate (9J, spongiatriol QOJ, spongiatriol triacetate (JJD, epispongiadiol (12). epispongiadiol diacetate (13J, epispongiatriol (i£), and epispongiatriol triacetate (lfj.15 As a result of the 1976 IUPAC recommendations on the naming of natural products, these compounds were renamed as derivatives of the hypothetical compound "spongian".15 Spongian Skeleton (sponge) [nudibranch] "Spongian" S. R=H Spongiadiol 2 R=Ac Spongiadiol diacetate (Spongia sp.15) [Glossodoris atromarginata20] spongi-12-en-16-one Isoagatholactone (Spongia officinalis ,-,13.22 J_ R=H Spongiatriol 11 R=Ac Spongiatriol triacetate (Spongia sp.15) (Spongia arabica ) [Glossodoris atromarginata20] OR 12 R=H Epispongiadiol 13 R=Ac Epispongiadiol diacetate (Spongia sp.15) (Spongia arabica ) In 1979, Kazlauskas et al. reported the isolation of a novel diterpene triacetate, aplysillin (16). from the sponge Aplysilla rosea (order Dendroceratid) collected in New Zealand.16 The relative stereochemistry of this compound was deterrnined by a single crystal x-ray diffraction analysis. Four new metabolites, including a related compound, lSa.^a-diacetoxyspongian (17). along with three tricyclic diterpenes enr-isocopal-12-en-15,16-dial (18J, 14-iso-enr-isocopal-12-en-15,16-dial (12) and 15-acetoxy-enr-isocopal-12-en-16-al (2jOJ were reported in 1982 by Cimino et al. from a collection of Spongia officinalis.17 The structures of these compounds were solved by a combination of spectroscopic analysis and chemical interconversions. It is interesting to note that Cimino offers the hypothesis that the tricyclic metabolites could be precursors to the metabolites possessing the "spongian" type skeletons. Also, of particular interest in this set of compounds is dialdehyde 18 since sesquiterpene and diterpene dialdehydes having two aldehydes in a similar structural arrangement have been shown to exhibit a number of interesting biological properties mcluding a very hot peppery taste to humans.18 It has been suggested the biological activity is related to the ability of these compounds to interact with NH2 groups of the taste receptors.19 Compound 18 has, however, been shown to be tasteless, indicating that the overall molecular structure together with the functionality are relevant for the biological activity. An examination by de Silva et al. of the extracts of the dorid nudibranch, Glossodoris (previously Casella) atromarginata from Sri Lanka, revealed the presence of spongiadiol diacetate (9_), and spongiatriol triacetate (11) found previously from an Australian Spongia species as well as four new compounds.20 Two of the compounds, 21 and 22, are minor structural variants of the furanoditerpenes reported by Kazlauskas et a/.15 while two others, 23 and 24 contain a more highly oxidized A-ring. These compounds were believed to originate from a Spongia species since nudibranchs have been shown to have the ability to selectively sequester defensive allomones from their prey.21 Spongian Skeleton During the course of studies on the chemical constituents of Spongia officinalis collected in the Canary Islands in 1984, Gonzalez et al. observed that crude methanol extracts exhibited antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Bacillus sphaericus in a disk assay.22 The extract also inhibited the growth of HeLa cells with ID50 of l-5^ig/mL. Further extraction and purification yielded four new diterpenes, 25 to 28, which were closely related to isoagatholactone (2). differing only in the added oxidation of the caibocyclic skeleton at the C7 or Cn position. The extract also contained isoagatholactone (2) and aplysUlin (16).13»16 Bioassays conducted on the pure metabolites showed that only 27 and 28 were inactive. In a continuing search for biologically active compounds, Schmitz et al. reported the isolation of three "spongian" diterpene lactones, 29 to 31, from the Caribbean sponge Igernella notabilis.^ The structure of lactone 30 was solved by single crystal x-ray diffraction analysis, while the structures of the other compounds were solved by a subsequent spectroscopic comparison. Lactones 29 to 31 have a different oxygenation pattern than all the spongians isolated by Kazlauskas;15 lacking the oxidation in the A-ring while displaying an alternative oxidation at C15 and C17. Schmitz suggests that the lactone/tetrahydrofuran rings in 29 to 31 seem conveniently arranged to serve as a complexing moiety for cations which could give rise to the biological activity observed for these compounds. Lactone 30 has been found to be mildly cytotoxic with an ED50 of 6.5 |!g/mL against the PS cell line.24 Subsequent to the completion of this work, Karuso et al. reported isolation of compound 32, similar to 29 but with the functional groups at C16 and C17 interchanged and undefined stereochemisty.25 The identification of sponges that are closely related to other species can be extremely difficult. Karuso et al. in 1986 reported that an encrusting sponge previously referred to as Aplysilla rosea 16 should be renamed Darwinella sp. and that the sponge Spongian Skeleton R2 21 Ri=H, R2=R3=OAc 22 R,=R3=H, R2=OAc [Glossodoris atromarginata20] (Spongia arabica32) 25. R=OH llp-Hydroxyspongi-12-en-16-one 2£ R=OAc lip-Acetoxyspongi-12-en-16-one (Spongia officinalis22) OR2 22 Rn=H,R2=Ac 24 R,=R2=H [Glossodoris atromarginata2^] 27 R1=OH,R2=H 7P,lip-dihydroxy-spongi-12-en-16-one 2S R^H, R2=OH 7P,lla-dihydroxy-spongi-12-en-16-one (Spongia officinalis22) Spongian Skeleton 5 OH OR o 22 R= C-CH2CH2CH3 7a,17P-dihydroxy-15,17-oxidospongian-16-one 7 butyrate O _Q R= "-CH3 7a,17P-dihydroxy-15,17-oxidospongian-16-one 7 acetate 21 R=H 7a,17P-dihydroxy-15,17-oxidospongian-16-one (Igernella notibilis ) previously named Aptysilla sulphurea, should be renamed Darwinella oxeata.26 In a study undertaken in order to observe any possible geographical variations in terpene content, extraction of the sponge Dendrilla rosea, which is morphologically similar to Darwinella sp., yielded a mixture of aplyroseols-1 (__), -2 CM), -3 (_5J, -5 (__), -6 C_2), -7 (__), each of which was identified by comparison with authentic samples.26-27 In addition, four new compounds designated as dendrillols 1-4 (39) to (42) were isolated and their structures elucidated by spectroscopic and x-ray diffraction analyses.26 It is interesting to note that aplyroseol-1 QD and aplyroseol-2 (34) were identical to compounds 29 and 30 previously reported from the Caribbean sponge Igernella notdbilis?^ Molinski et al. working on an Australian Aptysilla species, reported the isolation of two new diterpene lactones 43 and 44, which are similar to lactone 29 previously reported by Schmitz et at?* from Igernella notabilis, differing only in the oxygenation at Q;.2** Nine "spongian" type diterpenes were reported by Ksebati et al. in 1987, from the nudibranch Ceratosoma brevicaudatum collected in South Australia.29 The structures of compounds 45 to 52, and the previously reported metabolite, 39, were elucidated by detailed spectroscopic analyses and comparison to published results.26-27 In fact, the authors cited errors made by Karuso et al.26 in the assignment of the lH and 13C data of lactone 39. Metabolites 45 to 52 differed from all the other compounds in this series by the absence of the IR absorption due to the y lactone and its replacement by IR and 13C data con-esponcling to the C13 methyl ester (1740cm-1, 8174.4 (s), 51.9 (q)). A regioisomer of spongiadiol (8J and epispongiadiol (12), previously reported by Kazlauskas et al.,15 was isolated by Khomoto et al. from a deepwater Caribbean sponge, Spongia sp., whose crude extracts exhibited activity against Herpes simplex virus type 1 (HSV-1), and P388 murine leukemia cells.30 Extraction of this sponge, followed by chromatographic separation, yielded three active compounds including spongiadiol (JD, epispongiadiol (12) and a structurally related compound, isospongiadiol (5_3J. Assignment Spongian Skeleton (Darwinella sp.26) (Aplysilla sp.28) [Igernella notibilis23] 22 R1=H, R2= OCO(CH2)2CH3 Aplyroseol-1 24 R,=H,R2=OAc Aplyroseol-2 25 R1=OH,R2=OCO(CH2)2CH3 AplyroseoI-3 2$ R1= OCO(CH2)2CH3 R2=OH AplyroseoI-5 2Z R^OCOfCH^CHaR^OAc Aplyroseol-6 22 Ri=H,R2=H Dendrillol-1 4fi R,=OAc, R2=OAc Dendrillol-2 2S R,=H, R2=CH2OAc AplyroseoI-7 41 R=H Dendrillol-3 42 R=OH Dendrillol-4 Spongian Skeleton 45 R=OAc 4j£ R=OC(0)Pr 42 R=H [C. brevicaudatum29] 42 Rt=C(0)Pr, R2=H, 17p 5Q Rj=Ac, R2=H, 17P 51 R,=Ac, R2=Ac, 17p 52 Rt=C(0)Pr, R2=Ac, 17P [C. brevicaudatum ] Spongian Skeleton AcO X:OOH 51 Isospongiadiol (Spongia sp.30) 54 (Hyatella intestinalis31) SS Spongialactone A (Spongia arabica32) [Chromodoris norrisi ] of the ring A oxidation pattern as well as the absolute configuration was facilitated by comparison of *H NMR spectra and optical rotations of the reduction products of compounds 8,12, 53. In vitro assays against P388 cells yielded IC50 values of 0.5, 8, and 5 ug/mL for compounds 8,12,53, respectively. Against HSV-1, the IC50 values for 8,12, 53 were 0.25, 12.5 and 2 u^g/mL, respectively. The sponge Hyatella intestinalis, collected off the coast of Northern Australia, yielded the known compounds 12 and 13 as well as the structurally similar compound 54.31 A novel metabolite, spongialactone A (551. was isolated as a minor constituent of the lipophilic extract of the Red Sea sponge, Spongia arabica?2 The structure of compound 55, which represents the first "spongian" with a ring-A lactone, was based on spectroscopic analyses and chemical interconversion. Recently, Bobzin et al. have reported the isolation of compound 56, a dihydro-analogue of isoagatholactone (2), from the sponge Aptysilla polyrhaphis and the nudibranch Chromodoris norrisi collected in the same locale.33 Dumdei et al. have reported the isolation of three new metabolites, compounds 57, 58 and 59 from the nudibranch Chromodoris geminus, collected in Sri Lanka.34 Norrisane Skeleton Norrisolide (60). the first of the rearranged "spongian" diterpenes, was isolated by Hochlowski et al. in 1983 from a dorid nudibranch, Chromodoris norrisi, collected at San Carlos Bay, Sonora, Mexico.35 This metabolite, whose structure was ultimately solved by a single crystal x-ray diffraction analysis, was later found as a very niinor constituent of the sponges Aptysilla polyrhaphis and Dendrilla sp. collected at Palau, Western Caroline Norrisane Skeleton Norrisolide [Chromodoris norrisi ] (Aptysilla polyrhaphis ) (Dendrilla sp. 36) Islands. However, neither of these sponges or any related sponge was found in the Gulf of California.33'36 Since Dendrilla sp. is taxonomically related to Aplysilla rosea from which Kazlauskas et al. obtained aplysillin (16).16 it was proposed that norrisolide (60) represented the first example of a "norrisane" skeleton, derived from the rearrangment of a "spongian" skeleton (Scheme 4). Macfarlandin Skeleton An examination of the nudibranch Chromodoris macfarlandi, collected at Scripps Canyon, La Jolla, yielded a mixture of diterpenes including macfarlandins C (61) and D (62).37 The structure of macfarlandin C (61) was solved by single crystal x-ray methods while macfarlandin D (62) was solved by a subsequent comparison of spectral data. Carmely et al. also found a specimen of the sponge Dysidea sp. to contain shahamin F (63) and shahamin G (64). A second Dysidea sp. collected in the same habitat yielded shahamin F (63). shahamin H (65). shahamin I (66) and shahamin J (67).38 Recendy, Bobzin and Faulkner reported the isolation and structure elucidation of polyrhaphin C (68) from the Gulf of California sponge, Aplysilla polyrhaphis?^ as well as dendrillolides D (69) and E (70). from the Palauan sponge, Dendrilla sp..36 These diterpenes are all believed to be derived biosynthetically from a "spongian" precursor (Scheme 4). Macfarlandin Skeleton £2 R,=R2=H, Shamamin F M R]=H,R2=OH Shahamin G £5 Rj=OH,R2=H Shahamin H {Dysidea sp.38) OAc Polyrhaphin C (Aplysilla polyrhaphis ) Macfarlandin Skeleton AcQ _2 Dendrillolide D (Dendrilla sp.36) IQ Dendrillolide E (Dendrilla sp.36) Aplysulphurane Skeleton 21 In 1984, Karuso et al. reported the isolation of an aromatic diterpene, aplysulphurin (21), from the bright yellow sponge Aplysilla sulphurea (renamed Darwinella oxeata), collected at depths of up to 30m in the waters of the Eastern Australian seaboard.39 The structure of this compound, thought to originate from a "spongian" type precursor (Scheme 3), was deduced from a combination of spectroscopic, chemical, and x-ray crystallographic evidence. This metabolite was the first terpenoid with an "aplysulphurane" skeleton. Examination of a Darwinella sp. (previously Aplysilla rosea) afforded aplysulphurin (21) as well as a new minor metabolite, tetrahydroaplysulphurin-1 (22), whose structure was later confirmed by single crystal x-ray diffraction analysis.26'66 In a study established in order to observe any geographical variation in terpene content, Darwinella oxeata (previously Aplysilla sulphurea), collected from various locations around New Zealand, was shown to contain the major component, aplysulphurin (71). in addition to the minor metabolites, tetrahydroaplysulphurins-1 (72). -2 (73). and -3 (74). 26 The work described in this thesis describes a chemical study of the sponge Aplysilla glacialis, collected at Barkley Sound, B.C. A. glacialis extracts have yielded a mixture of diterpenes including cadlinolide A (2_), and cadlinolide B (76). which are structurally similar to tetrahydroaplysulphurins-1 to -3 (22-2D- In addition, minor amounts of cadlinolide A (2_) and tetrahydroaplysulphurin-1 (22) were isolated from the dorid nudibranch Cadlina luteomarginata found feeding on Aplysilla glacialis. Since no trace of tetrahydroaplysulphurin-1 (22) was found in the extracts of Aplysilla glacialis, it was suggested that the nudibranch might be acetylating cadlinolide B (2_0 in vivo.40 Aplysulphurin (Aplysilla sulphurea*9) (Darwinella oxeata26) (Darwinella sp.26) 24 Tetrahydroaplysulphurin-3 (Darwinella oxeata26) Aplysulphurane Skeleton 75 7_ Cadlinolide A Cadlinolide B (Aplysilla glacialis40) (Aplysilla glacialis40) [Cadlina luteomarginata40] Dendrillane Skeleton Sullivan et al. examined the deep purple sponge Dendrilla sp. collected in a marine lake on an island in Iwayama Bay, Western Caroline Islands, and isolated a number of diterpenes with the "dendrillane" skeleton including the three compounds, dendrillolide A (21), dendrillolide B (2&) and dendrillolide C (22).41 The "dendrillane" skeleton, possessing a perhydroazulene portion, is thought to be derived from a "spongian" precursor (Scheme 4). Hambley et al. subsequendy reported that the major diterpene constituents of the sponge Chelonaplysilla violacea (family Aplysillidae, order Dendroceratida), collected off the coast of Eastern Australia, were aplyviolene (80) and aplyviolacene (SI).42 The structures of these compounds, which differ only in the oxidation level of C12. were proposed from spectroscopic analysis and confirmed by single crystal x-ray diffraction analysis of aplyviolene (80). A small discrepancy is noted in the naming of macfarlandin E (81). reported simultaneously by Molinski et al. from the nudibranch, Chromodoris macfarlandin It would appear that the two compounds aplyviolacene (81) and macfarlandin E (81) are identical, however, the authors of the latter paper chose to rename the structure because of the lack of reported evidence by the previous authors for the assigned structure of aplyviolacene (8JJ. Also, as a result of the structural assignment of aplyviolene (80) via x-ray diffraction analysis yielding the same structure originally proposed by Sullivan et al. for dendrillolide A (22), it was clear the structures of dendrillolides A (77) and B (78) had to be reassigned.41 Carmely et al. reported the isolation of ten new rearranged spongian diterpenes from two Dysidea sponge species.38 The structures of these compounds were elucidated from Dendrillane Skeleton 22 2fi 22 Dendrillolide A Dendrillolide B/A Dendrillolide C (Dendrilla sp.41) (Dendrilla sp.41'36) (Dendrilla sp 41) Dendrillane Skeleton 22 M 22 Shahamin B Shahamin C Shahamin D (Dysidea sp.38) (D^asp.38) (D^V/easp.38) 33 (Aplysilla polyrhaphis ) 33 [Chromodoris norrisi ] examination of the spectral data and comparison to known diterpenes. Extraction of Dysidea sp. collected near Shaab Mahamud in the Red Sea at a depth of 15m gave a mixture of six rearranged metabolites possessing the "dendrillane" skeleton; namely, shahamin A (82), shahamin B (S3J, shahamin C (84). shahamin D (85J, shahamin E (86) and the known metabolite macfarlandin E (81) (aplyviolacene). Shahamin A (82) possesses a dihydrofuran moiety, shahamin B (Si) has a tetrahydrofuran moiety, while shahamins C-E (84-86) encompass a trisubstituted 5-lactone functionality linked to the perhydroazulene system. Aplysilla polyrhaphis, collected in the Gulf of California, contained two "dendrillane" derivatives, polyrhaphins A (82) and B (SS).33 Polyrhaphin A (S2) was also isolated from the nudibranch Chromodoris norrisi collected at the same site. Investigation of Chromodoris gleniei collected in the Indian Ocean has yielded two related metabolites, compounds 89 and 90.43 The structures of these compounds, also possessing a perhydroazulene portion as well as a disubstituted 6 lactone functionality, were solved by spectroscopic analysis and comparison to the known metabolites shahamins A (82) to E(M). Bobzin et al. have recendy corrected the structure for dendrillolide A (78) on the basis of interpretation of new spectral data, particularly the two-dimensional heteronuclear NMR shift correlation experiments (HETCOR).36 The revised structure of dendrillolide A (78) is identical to the structure previously assigned to dendrillolide B (2S). Dendrillohde B was not examined and its structure remains undeterrnined. Bobzin et al. also reported the structures of the related diterpenes dendrillohde C (91). 12-desacetoxyshahamin A (22) and 12-desacetoxy shahamin C (82)36 Dendrillane Skeleton OAc OAc 12-Desacetoxyshahamin A (Dendrilla sp.36) 28 Degraded Spongian Skeletons An unique nOT-diterpene metabolite was isolated in 1985 by Mayol et al. from the Mediterranean sponge Spongionella gracilis.*4 The structure of gracilin A (93), a nor-diteipene diacetate, was solved by a combination of spectroscopic analysis and chemical interconversion. Subsequent studies carried out on the extracts of S. gracilis afforded the related nor-diterpenes gracilin E (94), gracilin F (95), compound 96, and three bis-nor-diterpenes, gracilins B-D (104-106) and spongiolactone (107) 45>46 It has been suggested that the skeleton of nor-diterpenes 93 to 95 could be derived from a common "spongian" derivative (Scheme 2,4), while the skeleton of the bis-nor-diterpenes, 104 to 106, although reminiscent of that of the other metabolites, cannot simply be related to a "spongian" precursor and are open to biosynthetic speculation. It is interesting to note that both nor- and bis-nor diterpenes are very rare from marine sources.47 Two new aromatic nor-diterpenes were isolated in 1986 by Molinski et al. from the dorid nudibranch Chromodoris macfarlandi.48 Twenty two specimens collected in Scripps Canyon, La Jolla, yielded macfarlandin A (97) and macfarlandin B (98s). closely related to the previously reported aplysulphurin (71). Macfarlandin A (2Z) inhibited the growth of B. subtilis at lOu-g/disc while macfarlandin B (98) was active against!?, subtilis and S. aureus at lOug/disc, using the standard disc-assay procedure. Although a sponge source for these compounds has not been found, the authors propose that the nudibranchs are selectively sequestering these metabolites from a Dendroceratid sponge for defensive purposes. Examination of the Benthic community at McMurdo Sound, Antarctica by Dayton et al., revealed that the sponge Dendrilla membranosa was extremely slow growing and was never observed to be eaten. Dayton concluded that D. membranosa, which lacks apparent physical protection from spicules or mucus, must be chemically defended.49 Molinski et al. Degraded Diterpenes H "OR* 23 R*=OAc RJ=Ac Gracilin A 24 R*=H R2=Ac Gracilin E 25 R*=H R2=H Gracilin F 24 R*=0 R2=Ac (Spongionella gracilis44'45'46) H OAC 2ZRi=H,R2=OAc Macfarlandin A 28 R,=OAc, R2=H Macfarlandin B (Chromodoris macfarlandt**) 22 9,11-dihydrogracilin A (Dendrilla membranosa50) 100 Membranolide (Dendrilla membranosa5®) 101 Aplysillolide A (Aplysilla glacialis4®) m. Aplysillolide B (Aplysilla glacialis40) 103 Spongionellin (Spongionella gracilis45) Bis-Nor-Diterpenes 47^ (SpongionelJa gracilis ) KM R=Ac Gracilin B lOi R=Propionyl Gracilin C 106 Gracilin D OCOCH2CH(CH3)2 107 Spongiolactone o set out to survey the chemistry of this sponge in order to establish a chemical explanation for these observations.50 Extraction of D. membranosa yielded two degraded "spongian" metabolites, 9,11-dihydrogracilin A (22) and membranolide (100'). Compound 99 appears to incorporate one less double bond as compared to gracilin A (22), while compound 100 afforded signals in the 1H and 13C NMR spectra rerniniscent to those observed in the aromatic metabolites aplysulphurin (21), macfarlandins A (22), and B (SS)- Both 99 and 100 inhibited the growth of B. subtilis at lOOjig per disk and 100 was also mildly active against 5. aureus. Antifeedant studies on the isolated compounds could not be carried out using the major Antarctic spongivores, the sea stars Perknaster fuscus antarticus and Acodontaster conspicuus, however, the authors offer that increasing circumstantial evidence suggests these "spongian" type diterpenes are distasteful to all but specialized predator nudibranch s.49 The work in this thesis describes the isolation and structure elucidation of two nor-diterpenes, aplysillolides A (101) and B (1021 which both uniquely possess a carbonyl functionality at Cn.40 The structure of spongionellin (103). possessing a novel carbocyclic skeleton, was deduced by detailed spectroscopic analyses and chemical interconversion.45 Degraded diterpenes such as compounds 93 to 99 and 101 to 102 can all be said to possess a "gracilane " skeleton, which could be formed from a "spongian" precursor (Scheme 3,6). Chromodorane Skeleton A novel rearranged diterpene, chromodorolide A (1081. was isolated recently by Dumdei et al. from Indian Ocean Nudibranch, Chromodoris cavae.51 Chromodorolide A (1081 encompassess a new rearranged "spongian" diterpene skeleton which has been named the "chromodorane" skeleton (Scheme 4). This new skeleton could be derived by the formation of a bond between C17 and C12 subsequent to the degradation and rearrangement steps that generate the "norrisane" skeleton (Scheme 4), This compound appears to provide further evidence for Chromodorid nudibranchs acquiring diterpenes from dietary sponges.52 The structure of chromodorolide A (108). which possesses a unique heterocyclic portion, was ultimately solved by a single crystal x-ray diffraction analysis. Chromodorolide A OM) displayed both cytotoxic (L1210 ED50 2tyg/mL; P388 T/C 125% 4ug/kg) and antimicrobial activity (B. subtilis MIC 60ug/disc; R. solani MIC 60p:g/disc). Glaciane Skeleton Mayol et al. observed the unique ability of the Mediterranean sponge Spongionella gracilis to elaborate a large variety of degraded diterpenes including the gracilins.45'46 Recently, Mayol et al. reported the isolation and structure elucidation of compound 109, a degraded and rearranged diterpene 45 The structure of 109 was solved by a combination of spectroscopic interpretation and chemical interconversion. A related metabolite, glaciolide (ULtt), has since been isolated from the pink encrusting sponge, Aplysilla glacialis, collected in Barkley Sound, B.C., and also characterized by spectroscopic analysis and chemical interconversion.53 Glaciolide (110) was also isolated as a niinor component of the extract of the nudibranch, Cadlina luteomarginata, found feeding on A. glacialis.40 The unique skeleton of 109 and 110 was named the "glaciane" skeleton and it could be envisaged as being derived from a "spongian" precursor (Scheme 3,5). Marginatane Skeleton 33 Marginatafuran (ill), a furanoditerpene with a new carbon skeleton, was isolated in 1985 by Gustafson et al. from the dorid nudibranch Cadlina luteomarginata, collected in the Queen Charlotte Islands.54 The structure of this metabolite was solved by single crystal x-ray diffraction methods. The new carbon skeleton was subsequently named the "marginatane" skeleton (Scheme 1). A recent collection of Aplysilla glacialis made in Barkley Sound, B.C., has yielded a similar metabolite, marginatone (112). also possessing a "marginatane" skeleton with a ketone functionality at C12.40 This was the first example of a compound possessing a "marginatane" skeleton from a sponge and offers evidence for the true origin of marginatafuran (111), which was believed to be selectively sequestered by the nudibranch from a sponge prey. Dumdei et al. have since isolated marginatafuran (111) as well as a similar metabolite, 113, from a Queen Charlotte Island collection of C. luteomarginata.^ Bobzin et al. have reported the isolation of a similar compound, polyrhaphin-D (114). from the sponge Aplysilla polyrhaphis, collected in the Gulf of California. The authors described this compound as the first example of a diterpene containing an "isospongian" skeleton, which appears to be identical to the "marginatane" skeleton.33 Biogenetic Proposals The proposed biogenetic origin of this wide array of terpenes and norditerpenes starts with a hypothetical tetracyclic "spongian" precursor. It is possible to construct a Chromodorane Skeleton Glaciane Skeleton O. IS 19 ms. Chromodorolide A (Chromodoris cavae51) Marginatane Skeleton 111 Marginatafuran [Cadlina luteomarginata54] ms. (Spongionella gracilis45) JJ_ R=CH3 Marginatone (Aplysilla glacialis40) HJ R=CH2OAc [Cadlina luteomarginata*4] HQ Glaciolide (Aplysilla glacialis40-53) [Cadlina luteomarginata40,53] m polyrhaphin D (Aplysilla polyrhaphis33) simple model describing the origin of the "spongian" intermediate from a linear terpenoid precursor. Several of the sponges containing "spongian" metabolites, for example Spongia species,15 also elaborate linear furano-terpenes. Of particular interest is the metabolite ambliofuran (115) isolated from the marine sponge, Dysidea amblia, by Walker and Faulkner in 1981.54 This metabolite is believed to be the precursor of four compounds, ambliol-A (H6J, ambliol-B (112), dehydroambliol-A (Ufi), and ambliolide (H9_) found in this same sponge. A recent examination of the Palauan sponge, Dendrilla sp., has yielded dehydroambliol-A (118). l-bromo-8-ketoambliol-A acetate (120) as well as a mixture of "spongian" derivatives.36 If ambliofuran (115) serves as a starting point in the biosynthesis of di- and tri-cyclic terpenes, perhaps this can be extended to the formation of tetracyclic compounds. One can envisage the proton initiated cyclization of ambhofuran (115) to afford products containing either the "spongian" or "marginatane" skeletons (Scheme 1). The wide variety of metabolites possessing the "spongian" skeleton can be formed by subsequent biological interconversions involving enzyme catalyzed oxidations and reductions. The alternate cyclization product, having the "marginatane" skeleton, can similarly be converted to its derivatives. Alternatively, Fenical has pointed out that the well known stereospecific cyclization of all-franj-geranylgeraniol (121) using a known terrestrial route (Scheme 2) can also lead to the "spongian" skeleton.8 Suggestions as to the biogenetic origin of the rearranged and degraded metabolites have been put forth by the various investigators following accepted biogenetic principles. Carmely et al. have suggested that compounds having the "macfarlandin" and "dendrillane" skeletons are rearranged oxidative cleavage products of the "spongian" precursor as shown in Scheme 3.38 Similar oxidative cleavage and rearrangement reactions can also give rise to the "aplysulphurane", "glaciane", or "norrisane", and "chromodorane" skeletons (Scheme 36 3,4). Mayol et al. have proposed mechanisms involving epoxide intermediates for the biognesis of the "glaciane" and "gracilane" skeletons (Scheme 5,6).45 While it is generally assumed that the pathways employed in the biosynthesis of marine natural products are identical to the well documented mechanisms established in metabolites isolated from terrestrial sources, experimental evidence which would allow for the confirmation of this assumption is still lacking. What is known is that there are certain obvious differences, for instance, the frequent occurrence of halogen and isocyanide functionalities in marine terpenoids and the frequent occurance of optical antipodes of terrestrial skeletons.56 While the reasons for such differences are not clear, some of the variables such as individuality of the producer organism, evolutionary significance as well as the marine environment itself give rise to a whole new set of biosynthetic conditions compared to those found on land. Scheme 1: Biogenetic Proposals For Spongian and Marginatane Skeletons Scheme 2: Spongian Metabolites From Geranylgeraniol "Spongian" Scheme 3: Biogenetic Proposals For Degraded and Rearranged Spongian Metabolites oxidative cleavage CH3 migration Scheme 4: Biogenetic Proposals for Rearranged Spongian Derivatives Scheme 5: Biogenesis of the Glaciane Skeleton via an Epoxide "glaciane" 42 Scheme 6: Biogenetic Proposal for the Gracillane Skeleton via a 6,7-Epoxide B. TERPENOID METABOLITES FROM THE SPONGE APLYSILLA GLACIALIS MEREJKOWSKI 1878 1. Introduction Aplysilla glacialis (Merejkowski 1878) (Family Aplysillidae, Order Dendroceratida, Class Demospongiae) (Figure 1) is a pink encrusting sponge commonly found in exposed surge channels on the Pacific coast of North America from Alaska to California. Specimens of this species have also been identified in the North Adantic as well as the waters of Australia and South America.57 The dorid nudibranch Cadlina luteomarginata, which is commonly found along the west coast of British Columbia, was found feeding on A. glacialis collected at Sanford Island and the Queen Charlotte Islands. Our chemical studies on Aplysilla glacialis were initially prompted by an interest in establishing the source of skin metabolites previously isolated during chemical investigations of Cadlina luteomarginata collected off the coasts of British Columbia and California.58 Secondly, there have been numerous examples of interesting metabolites isolated from encrusting sponges collected in surge channels.59 A third reason for interest in A. glacialis was the intensely sweet smelling methanol extracts of the sponge which indicated the presence of terpenoid metabolites. Although A. glacialis turned out to lack the metabolites isolated thus far from C. luteomarginata (see section C), a preliminary investigation of the methanol extracts of the sponge using Thin Layer Chromatography (TLC) and Nuclear Magnetic Resonance (NMR) spectroscopy indicated the presence of a series of interesting new "spongian" and "marginatane" derived metabolites. Figure 1: Phylogenic Classification of the Sponge Aplysilla glacialis (Merejkowski 1878) According To Austin (1989)57 Metazoa (multi-cellular animals) Porifera I Hexactinellida Demospongia Calcarea (sponges) Homoscleromorpha Ceractinomorpha Tetractinormorpha Haplosclerida Halichondrida Dendroceratida Poecilosclerida r Halisdrcidea Aplysillidae Dictyodendrillidae Aplysilla glacialis polyraphis KINGDOM PHYLUM CLASS SUBCLASS ORDER FAMILY GENUS SPECIES Scheme 7: Isolation Scheme For Terpenes from Aplysilla glacialis Whole sponge in MeOH aq. MeOH decanted Evaporation in vacuo Partition Between Ethyl Acetate/Water Aqueous: Red Solid on Lyophilization Organic Extract Flash Chromatography 8 major fractions screened by NMR Terpenes 2. Isolation and Structure Elucidation Aplysilla glacialis was collected by hand using SCUBA (0 - 3m depth) and immediately immersed in methanol. After soaking in methanol at room temperature for one to three days, the methanol layer was decanted, vacuum filtered and evaporated in vacuo to yield an aqueous methanolic suspension. This suspension was partitioned between brine and ethyl acetate, and the organic layer was dried over anhydrous Na2S04. The sponge was soaked in methanol for one additional day, before being ground in a Waring blender. The suspension of ground sponge in methanol was vacuum filtered, and the filtrate was evaporated in vacuo, partitioned between brine and ethyl acetate and the organic layer was dried over anhydrous Na2SC«4. The combined organic layers were vacuum filtered and evaporated in vacuo affording a dark green crude oil which was fractionated by silica gel flash chromatography60 to give a complex mixture of fats, pigments, steroids and terpenoids as detected by analytical TLC analysis. Further separation and purification guided by lH NMR analysis yielded a series of pure terpenoid metabolites, namely, cadlinolide A (7_5J, cadlinolide B (2_), aplysillolide A (IM), aplysillolide B (JJ__), glaciolide (1101. marginatone (112) and cadlinolide C (J22) (Scheme 7). 3A. Cadlinolide A (75) Cadlinolide A (7__), obtained as colourless needles from hexane (mp 126-127 °C), had a molecular formula of C20H28O4 (EIHRMS found 332.1982, calc'd 332.1983) that 47 Table 1: 15MHz NMR Data For CarJlinohde A Q_) i° CDQ3 Carbon 6 ppm mult8 1 39.19 t 2 19.94 t 3 39.90 t 4 31.31 s 5 50.15 t Me6 16.68 q 7 38.90b d 8 118.85 8 9 147.29 5 10 39.81 8 11 20.57d t 12 23.25d t 13 35.07b d 14 38.20b d 15 99.43 d 16 169.89 s 17 173.26 s Mel8 28.14c q Me 19 31.38c q Me20 31.89c q 8 Assignments based on APT and JH-13C cxjrrelation experiments " 'fc-d Interchangable Table 2: 400MHz *H NMR Data for CadlinoUde A (71) in CDCI3 Proton 6 ppm COSY Correlations nOesa H5 1.72 H5' 1.78 Me6 1.48,d, J=7.4 H7 H7,H14,H15(weak) H7 4.28,q, J=7.4 Me6 H5,Me6,Me20 Hll 2.35,bd, J=17.9 H11',H12,H12' HIT 2.19,m H11,H12,H12',H14 H12 2.06,m H11,H11',H12,H13 H12' 1.69,m H11,H11',H12,H13 H13 3.12,dt, J=7.9,4.6 H12,H12',H14 H14.H15 H14 3.48,m H11',H13,H15 H13,H15,Me6 H15 6.16,d, J=5.3 H14 H13.H14 Mel 8 0.77,s* Mel9 Mel9 0.92,s* Mel8 Me20 1.13,s H7 a Resonance in Proton column irradiated * Interchangable 2 3 required 7 degrees of unsaturation. Resonances for all 20 carbon atoms were well resolved in the 13C NMR spectrum of cadlinolide A (75) and an APT experiment61 (Figure 2) indicated that all 28 hydrogen atoms were attached to carbon (4xCH3, 6xCH2, 4xCH, 6xC) (Table 1). Infrared bands at 1789 and 1760 cm-1 indicated the presence of two ester functionalities, which was further supported by the resonances in the 13C NMR at 8169.89 (s) and 173.26 (s) ppm, accounting for the 4 oxygens in the molecule. The frequency of one of the ester carbonyl stretching vibrations (1789 cm"1) suggested the presence of a y lactone in cadlinolide A (75). Further exarnination of the 13C NMR spectrum revealed a deshielded resonance at 8 99.43 (d) ppm indicating the presence of a ketal functionality. Since cadlinolide A (75) contained only 4 oxygen atoms, the alkoxy oxygens of the two esters had to be attached to the ketal carbon. Also apparent from the 13C NMR of cadlinolide A (75) was a tetrasubstituted double bond with resonances at 8 118.85 (s) and 147.29 (s) ppm, which accounted for the final unsaturated functionality in the molecule. Therefore, four rings had to be incorporated into the structure of cadlinolide A (75.) in order to account for the reniaining sites of unsaturation required by the molecular formula. The NMR spectrum of cadlinolide A (75). which was well dispersed and extremely informative (Figure 3), contained a deshielded resonance at 8 6.16 (d, J=5.3Hz, IH) which was found to be coupled to the ketal carbon at 99.43 (d) ppm in a HETCOR62 experiment optimized for one bond 13C-!H coupling. 2D-COSY63 (Figure 4) and double resonance experiments carried out on cadlinolide A (75) identified a seven proton spin system that started with the ketal proton resonance (8 6.16, HI5) and continued uninterrupted through two contiguous methines (83.48, H14; 3.12, H13 ), before terminating in a pair of adjacent methylenes (82.06, H12) and (81.69, H12'), (82.35, Hll) and (82.19, Hll*) (Figure 5) (Table 2). The chemical shifts of Hll (82.35) and HIT (82.19) implied that they were allylic and a weak COSY correlation 52 Figure 4: 400MHz COSY Spectrum of (^dlinolide A (25J in CDCI3 53 observed between HIT and H14 (83.48) was attributed to homoallylic coupling. Therefore, Cn and C14 had to be connected by the tetrasubstituted double bond in the molecule. Figure5: Isolated Spin Systems from COSY Spectra of Cadlinolide A ( __) A second spin system, consisting of a single deshielded proton at 8 4.28 (q, J=7.4Hz, IH) attached to a carbon bearing a deshielded methyl group at 1.48 (d, J=7.4Hz, 3H) ppm was readily identified from the COSY spectrum (Figure 4) (Table 2). The deshielded chemical shift of the methine proton (84.28) in this spin system implied that the carbon atom to which it was attached had to be adjacent to both the tetrasubstituted double bond and one of the ester carbonyls. Combining all the above structural evidence led to the indicated constitution of the tricyclic bis-lactone fragment of cadlinolide A (75V Assignment of the cis relationship between the three contiguous methines, H15, H14, H13 as well as Me6 in this fragment was detemrined by nOe enhancement experiments (Figure 6) (Table 2). The weak nOe observed between Me6 and H15 protons indicated that the 8 lactone is in a boat-like conformation with Me6 and H15 being flagpole substituents. 54 Figure 6: NOe Enhancements Observed for Cadlinolide A (_5J o tricyclic bis-lactone The stucture of the bis-lactone fragment comprising both y and 8 lactone moieties was confirmed by the conversion of cadlinolide A (751 to the diacetate 123 by reduction with LiAlH4 followed by acetylation with acetic anhydride and pyridine (Scheme 8). Four new deshielded resonances were present in the *H NMR spectrum (Figure 7) of diacetate 123 which could be assigned to two sets of geminal methylene protons attached to carbon atoms singly bonded to oxygen. One spin system, identified through correlations obtained from a COSY spectrum (Figure 8), consisted of a pair of geminal methylene protons resonating at 8 3.62 (dd, J=l 1.3,4.9Hz, H17) and 3.69 (dd, J=11.3,4.9Hz, H17') coupled to a methine resonance at 3.29 (m, H7) that was in turn coupled to a methyl doublet at 1.17 (d, J=6.7Hz, Me6) ppm (Figure 9). The observation of this spin system in diacetate 123 confirmed the placement of the methy Vmethine spin system identified in cadlinolide A Q5J adjacent to the 8-lactone carbonyl. A second spin system identified from the COSY spectrum (Figure 8) of diacetate 123 linked the second pair of methylene proton resonances at 8 3.81 (dd, J=ll.l,7.6Hz, H16) and 4.16 (dd, J=ll.l,6.1Hz, H16') through two methine protons at 2.14 (m, H13) and 2.64 (m, H14) to a ketal proton at 5.66(d, J=9.0Hz, HI5) ppm in agreement with the expected course of the LiAlH* reduction of cadlinolide A (751. 56 a a r~ o e f- s B 2 * 5 — m CO a •in Figure 9: Isolated Spin Systems for Diacetate 123 58 3.814.16 2.14. H H 1.17 3.29 H H 3.63 3.69 The tricyclic bis-lactone fragment of cadlinolide A (751 showed a great resemblance to two spongian derived metabolites previously reported, namely aplysulphurin (16) and tetrahydroaplysulphurin-1 (72)- The remaining pieces of 75 (3xCH3, 4xCH2, 2xC) were also consistent with the ring A functionality found in 16 and 72 (Table 7,2). This was further confirmed by comparison of the 13C NMR chemical shifts for carbons 1 to 5 in substructure A with identical systems seen in gracilin A (93) and 9,11 dmydrogracilin A (99) (Figure 10).50 However, it was not possible to unambiguously establish the A Figure 10: 13C NMR Chemical Shifts for Ring A Figure 11: Computer Generated ORTEP Drawing of Cadlinolide A Q5J Ol cm 60 interrelationship of these compounds by spectroscopic means and it was also impossible to establish the relative stereochemistry between the remaining chiral center in cadlinolide A (75) at CIO and the chiral centers at C7, C13, C14 and C15 in the tricyclic bis-lactone fragment. Therefore, cadlinolide A (75) was subjected to a single crystal x-ray diffraction analysis.64 A computer generated ORTEP drawing of cadlinolide A (7_D is shown in Figure 11, demonstrating the structure assigned to cadlinolide A (_5J-3B. Cadlinolide B (7j_) Cadlinolide B (76). isolated as a colourless oil, had a molecular formula of C20H30O4 (EEHRMS found 334.2152, calc'd 334.2144) differing from that of cadlinolide A (21) by the addition of two protons. Examination of the 13C NMR/APT (Table 3), 1H NMR (Table 4), and IR data obtained for cadlinolide B (76) revealed that it was a derivative of cadlinolide A (75) with the CI6 y lactone functionality reduced to a lactol. Figure 12: 75MHz ,3C NMPv/APT Spectra for Cadlinolide B W in CDCI3 Table 3: 75MHz 13C NMR/APT Data for CadlinoUde B Q6J in CDCI3 Carbon 8 ppm multipUcity3 CI 39.36 t C2 20.71 t C3 39.10 t C4 31.33 s C5 50.99 t C8 146.28b s C9 122.96b s C15 102.62c d C16 101.81c d C17 171.66 s a Assigned from APT b_c Shifts interchangable 1A Table 4: 400MHz *H NMR Data for Cadlinolide B Q£) in CDQ3 Proton 6 ppm COSY Correlation nOes» Me6 1.41,d\ J=7.4 Hz H7 H7 4.20,q, J=7.4 Hz Mc6 Mc6,Me20 Hll 2.36.m Hir.Hn.Hir Hll' 2.04,m H11312312' H12 1.92,m H11,H11',H12*,H13 H12' 1.20,m H11,H11',H12,H13 H13 2.40,m H12,H12',H14,H16 H14 3.23,m H13.H15 H15,H13,Me6 H15 6.054, J=6.2 Hz H14 H14 H16 5.394 J=3.9 Hz H13 HI3 (weak) Mel 8 0.77,s* Mel9 0.92,s* Me20 1.13,s * Resonance in Proton column irradiated * Interchangable 7JL 64 Thus, the 13C NMR spectrum of 76 (Figure 12) displayed only one ester carbonyl resonance at 8 171.66 (s) and two ketal carbon resonances at 101.81 (d) and 102.62 (d) ppm, while the IR spectrum of 76 exhibited only a single carbonyl stretching band at 1730 cm-1 and a strong OH stretching band at 3369 cm"1. The existence of an equilibrium mixture of epimers at C16 (5:1, A:B) was apparent from the presence of minor shadow peaks of many of the resonances in the *H NMR spectrum of 76 (Figure 13). The two deshielded resonances at 8 6.05 (d, J=6.2Hz, H15) and 5.39 (d, J=3.9Hz, H16) ppm were assigned to ketal protons in the major epimer. Using the methine at 8 6.05 (H16) as a starting point, correlations in the COSY spectrum (Figure 14) of 76 (Figure 15) provided a means by which connectivity through to the second ketal at 8 5.39 (HI5) could be achieved via two intervening methine resonances at 3.23 (t, J=7.4Hz, HI4) and 2.40 (m, HI3) ppm. From the H13 (82.40) methine resonance, correlations also exist in the COSY spectrum of cadlinolide B (76) to a vicinal methylene with protons resonating at 81.92 (m, H12), 1.20 (m, H12') which are further coupled to a second methylene system with resonances at 2.36 (m, Hll) and 2.04 (m, HIT) ppm (Figure 15). The deshielded character of the H14 and Hll, HIT resonances (83.23, 2.36, 2.04 ppm respectively) as seen before in cadlinolide A (7__) can be attributed to their allylic nature, further verifying the positioning of the tetrasubstituted double bond between C8, C9. CadlinoUde B (7_D, like CadlinoUde A (J5J, possessed the characteristic deshielded methyl doublet in the *H NMR spectrum at 8 1.41 (d, J=7.4Hz) assigned to Me6 coupled to a deshielded methine at 4.20 (q, J=7.4Hz) assigned to H7 (Figure 15). The couphng between the resonances was observed by COSY correlations (Figure 14) as well as double resonance experiments. The resonances assigned to the methyl groups in ring A were found in the !H NMR spectrum of cadlinohde B (J6J at 8 0.77 (s, 3H), 0.91 (s, 3H) and 1.13 (s, 3H) ppm. Their chemical shifts were in close agreement with the shifts found for the corresponding methyl groups in cadlinotide A (75). Figure 15: Assignment of Spin Systems for Cadlinolide B (76) From COSY Spectra 4.20 O Figure 16: NOe Enhancements Observed For Cadlinolide B(76) 26 NOe experiments (Figure 16) carried out on cadlinolide B (7JD (Table 4) allowed assignment of the relative configurations at centers at C7, C13, C14 and C15. The relative stereochemistry of C7 was established by the observation of enhancement of the Me6, H15 and H13 resonances on irradiation of the H14 methine. The independent irradiation of the H15 and H13 methines gave enhancement of the H14 methine resonance. This established that Me6, H13, H14, and H15 were all cis with respect to each other as in cadlinolide A (75) with the 8 lactone in a boat conformation. NOe experiments failed to give the definitive proof for the configurations at CI6 in the major and minor epimers. The observation of small vicinal H13-H16 coupling constants of 5.4 and 3.9Hz for the major and minor epimers in cadlinolide B (7Ji) precluded the use of this information to make an assignment of configuration at C16. Cadlinolide B (16) was treated with acetic anhydride and pyridine (Scheme 9) to form a single monoacetate 124, which was constitutionally identical to the known metabolite tetrahydroaplysulphurin-1 CJ2).26 A comparison of corrected lH NMR spectral data for tetrahydroaplysulphurin-1 (22), supplied by Cambie (Figure J7),65 with that of monoacetate 124 revealed that the two compounds were identical. NOe experiments and the magnitude of the vicinal H13-H16 coupling constant for monoacetate 124 again failed to provide unambiguous proof of the relative configuration at C16. The original assignment of the relative configuration at C16 of tetrahydroaplysulphurin-1 (72) made by Karuso et al.26 based on the observed H13-H16 vicinal coupling constant of 3Hz also remained in question until it was later confirmed by a single crystal x-ray diffraction analysis carried out by Buckleton et al..66 3C. Aplysillolide A (101) Aplysillolide A (101). isolated as a clear colourless oil, gave a pseudo-molecular ion at m/z 307 (M++1) in the DCIMS and an ion at m/z 288.2088 (M+-H20) (calc'd 288.2089) in the EIHRMS appropriate for a molecular formula of CioH3o03( requiring 5 degrees of unsaturation. All nineteen carbons could be accounted for in the 13C NMR spectrum of aplysillolide A (101). while an APT experiment revealed the presence of only 29 protons attached to carbon atoms (4xCH3, 6xCH2,5xCH, 4xC) (Figure 18) (Table 5). An OH stretching band in the IR spectrum at 3420 cm*1 revealed that the remaining proton atom was part of a hydroxyl functionality. Resonances at 8125.28 (d) and 132.07(s) in the 13C NMR spectrum could be assigned to a trisubstituted olefin (A) and a resonance at 212.64 (s) ppm was assigned to a saturated carbonyl (IR band at 1701 cm"1) (B). Since only two units of unsaturation in aplysillolide A (101) could be attributed to olefinic and ketone functionalities, it was apparent that the molecule must be tricyclic. The remaining two oxygens in aplysillolide A (101) were located by the observation of 13C NMR spectral resonances at 8 102.60 (d) and 71.24 (t) ppm assigned to a ketal carbon, and a methylene carbon singly bonded to an oxygen atom, respectively. Since there were only three oxygen atoms present in the molecule, the hydroxyl oxygen and the oxygen attached to the methylene carbon had to be attached to the ketal carbon to O A B 72 Table 5: 75MHz 13C NMR Data for Aplysillolide A (lfll) and Gracilin A (22) IM 22 Carbon 8 ppm 8 ppm mult8 1 37.41 36.2 t 2 18.94 19.2 t 3 38.90 39.0 t 4 31.08 31.1 s 5 49.07 50.3 t Me6 14.69 15.9 q 7 125.28 130.1 d 8 132.07 133.9 s 11 212.64 - s 15 71.24 - t 16 102.60 - d Mel8 27.85 27.5 q Mel9 35.37 36.0 q Me20 23.86 24.0 8 Assignments based on APT results Figure 19a: 400MHz 'H NMR Spectrum of Aplysillolide A (IQD in CDCI3 19b: Offset, Irradiation at 6l.66ppm Table 6: 400 MHz JH NMR Data for Aplysillolide A (101) in CDCI3 Proton 5 ppm COSY Correlation nOesa Me6 1.65,dd, J=6.8,2.4 Hz H7.H14 H9,H7 H7 5.80,dd, J=2.3,6.8 Hz Me6 Me6,H15 H9 3.11.S H12 2.18,dd, J=l 1.5,16.6 Hz H12',H13 H12',H13,H16, H16' H12' 2.36,dd, J=5.5,16.6 Hz H12.H13 H12.H13 H13 2.88,m H12,H12',H14,H16 ,H16' H12,H12',H16 H14 3.04,rri Me6,H13,H15 H15 5.63,d, J=2.3Hz H14 H7,H14 H16 4.23,dd, J=6.4,8.7 Hz H13.H16' H13.H16* H16' 3.54,dd, J=3.9,8.7 Hz H13.H16 H12,H12',H15, H16 Me 18 0.88.S* Mel9 0.97,s* Me20 1.13,s H13.H14 a Resonance in Proton column irradiated 101 form a hemiketal functionality. Further proof of this moiety came from the *H NMR spectrum of 101 (Figure 19a) (Table 6) which showed a methine resonance at 8 5.63 (d, J=2.3Hz, H15) corresponding to the proton on the hemiketal carbon in addition to a pair of geminal proton resonances at 4.23 (dd, J=8.7,6.4Hz, HI6) and 3.54 (dd, J=8.8,3.9Hz, H16') ppm assigned to the protons on a methylene carbon singly bonded to the hemiketal oxygen atom. 2.88 3.05 H H H H H OH 3.54 5.63 4.23 Correlations in the COSY spectrum (Figure 20) of aplysillolide A (101) provided a linkage of the ketal methine resonance (8 5.63, H15) through two intervening methine protons resonating at 8 3.05 (dd, J=Hz, H14) and 2.88 (m, HI3) to the methylene proton resonances at 4.23 (H16) and 3.54 (H16') ppm, indicating that the hemiketal functionality was part of a y lactol system (Figure 21). Vicinal coupling between an olefinic methyl resonance at 8 1.66 (dd, J=2.4, 6.7Hz, Me6) and an olefinic proton resonance at 5.80 (ddd, J=2.3,6.8,13.8Hz, H7) ppm indicated the methyl and olefinic proton were geminal substituents on the trisubstituted double bond in aplysillolide A (101). This vicinal coupling was observed in both the COSY spectrum (Figure 20) as well as through double resonance experiments where irradiation of the olefinic proton resonance at 8 5.80 (H7) collapsed the methyl signal at 1.66 to a sharp doublet (J=2.4Hz), while irradiation of the olefinic methyl (Me6) collapsed the olefinic resonance at 5.80 to a doublet (J=2.3Hz) (Figure 19b). Homoallylic coupling observed in the COSY spectrum 77 of aplysillolide A (101) between the olefinic methyl protons (8 1.66,Me6) and the methine proton resonating at 8 3.05 ppm (H14) indicated that the fully substituted carbon of the trisubstituted olefinic system had to be attached to the methine carbon (C14). Figure2 h Spin Systems From COSY / Double Resonance Spectra for Substructure C COSY correlations also showed that the methine proton resonating at 8 2.88 (m, HI 3) was further coupled into a pair of geminal methylene protons resonating at 2.18 (dd, J=5.1,11.5Hz, H12) and 2.36 (dd, J=5.5,16.6Hz, H12') ppm (Figure 21). The lack of further coupling into protons H12 and H12', in addition to their downfield chemical shifts, prompted the placement of the saturated ketone functionality at Cll. This was further supported by the presence of an allylic singlet resonating at 8 3.11 (s) ppm, assigned to H9, to give substructure C. SINEPT67 experiments carried out on aplysillolide A (101) (Figure 22) also supported the positioning of the ketone functionality. Irradiation of the methine signal at 8 3.11 (H9) gave strong polarization transfer into the carbonyl carbon 1.66 5.80 5.63 C resonance (8 212.64 (s), 2 bond) and into the olefinic carbon signals (8 125.28 (d), 132.07 (s), 3 and 2 bond respectively). Irradiation of the other methine at 8 2.88 (HI3) also gave polarization transfer into the carbonyl carbon, while only the irradiation of the deshielded equatorial proton on the adjacent methylene system (8 2.36, H121), and not its upfield axial partner (8 2.18, H12), yielded an enhancement of the carbonyl carbon (2 bond) presumably due to the magnitude of the ^C-lH coupling constant selected in the experiment (7 Hz). An intense peak in the EILRMS mass spectrum of aplysillolide A (101) at m/z 182 (EIHRMS for C10H14O3 calc'd 182.0943, found 182.0938) due to a McLafferty rearrangement68 (Scheme 10) also supported the ketone placement. Figure22: SINEPT Results for Aplysillolide A (10D This substructure thus far identified in aplysillolide A (101) closely resembled the ring C and D functionality previously reported for the metabolites 9,1 l-dihycfrogracilin A (99) and gracilin A (93). The remaining functionality indicated in the spectral data of aplysillolide A (101) could also be accommodated by the tricyclic framework present in 93 and 99. The chemical shifts (Table 5) of resonances in the 13C NMR of aplysillolide A 80 (101) assigned to the carbon atoms of ring A (8 37.41 (CI), 18.94 (C2), 38.90 (C3), 31.08 (C4), 49.07 (C5), 40.62 (C10), 27.35 (Me)) and *H NMR signals (Table 6) due to methyl protons (8 0.88, 0.97, and 1.13), were in excellent agreement with the carbon resonances (8 36.2 (CI), 19.2 (C2), 39.0 (C3), 31.1 (C4), 50.3 (C5), 39.0 (C10), 27.5 (Mel8), 36.0 (Mel9), 24.0 (Me20)) and methyl proton assignments (8 0.89, 0.96, 1.03) ppm, reported for 9,11-dihydrogracilin A (99).50 The relative stereochemistries at centers C13, C14 and C15 as well as the geometry of the A7*8 double bond as shown were determined by nOe experiments (Table 6) (Figure 23), however, it was not possible to determine the relative configurations at C9 and C10 by spectroscopic means on the parent compound. The difficulty encountered in the Figure 23: NOe Enhancements Observed for Aplysillolide A (101) assignment of the relative configuration at C9 by nOe was believed to arise from the geometric constraints present in ring C as a result of two sp2 centers flattening the ring. As 101 Scheme 10: McLafferty Rearrangement of Aplysillolide A (101) m/z 164.0837 (found) 164.0837 (calc'd) Scheme 11: Reduction and Acetylation of Aplysillolide A (101). 126 82 Table 7: 400MHz *H NMR Data for Triacetate 125 in CDCI3 Proton 5 ppm COSY Correlation nOea H7 5.32,q, J=6.7Hz Me6 H14.H15 H9 3.1'l,m Hll Me6,Hll,Me20 Hll 5.15,m H9,H12,H12' H9 H12 2.17,m H11,H12',H13 H12' 1.42,m H11,H12,H13 H14 H13 2.36,m H12,H12',H14, H14,Me20 H16,H16' H14 2.98,m H13.H15.H15' H13,H12*,Me20 H15 4.21,dd, H15',H14 J=l 1.2,7.9Hz H15' 4.30,dd, H14.H15 J=11.2,7.7Hz H16 3.89,dd, H13.H16' J=11.2,6.8Hz H16' 4.00,dd, H13.H16' J=l 1.2,7.1Hz Me6 1.65,dd, H7 H7,H9,Me20 J=1.2,6.8Hz Mel 8 0.86,s* Mel9 0.98,s* Me20 1.15,s Me6,H13,H14 OAc 2.02,s 2.06,s 2.09,s a Resonance in Proton column irradiated * Interchangable Figure 25: 400MHz COSY Spectrum of Triacetate 125 in CDC13 84 a result, aplysillolide A (1011 was reduced with LiAlHU and immediately acetylated with acetic anhydride and pyridine to furnish triacetate 125 as well as minor amounts of its epimer, triacetate 126, which was chromatographically inseparable from 125 (Scheme 11). In carrying out this interconversion, it was hoped that ring C would adopt a more chair like conformation allowing for more predictable nOe results and coupling constant values. *H NMR resonances attributable to the major epimer, triacetate 125, were well dispersed (Figure 24) facilitating the assignments of the various spin systems from the COSY spectra (Figure 25). Triacetylation was confirmed by the presence of three methyl singlets (8 2.02, 2.06, 2.09), two sets of geminal methylene protons attached to carbons singly bonded to oxygen atoms 8 3.89 (dd, J=l 1.2,6.8Hz, H16), 4.00 (dd, J=11.2,7.1Hz, H16'), 4.21 (dd, J=11.2,7.9Hz, H15), and 4.30 (dd, J=11.2,7.7Hz, H15') and one deshielded methine at 5.15 (m, Hll) ppm attached to a carbon singly bonded to an oxygen atom (Figure 26). Formation of an acetoxy methine center at Cll converted the allylic singlet originally at 8 3.11 (H9) in aplysillolide A (1011 into an allylic doublet resonating at 2.7 l(d, J=6.3Hz, H9) ppm in the major epimer, triacetate 125 (Figure 24). The difficulty involved in the assignment of the stereochemistry of C9 based on the observed coupling constants was immediately apparent. Since a coupling constant of 6.3Hz for a cyclohexane system could indicate either aa, ae or ee coupling to a vicinal proton, or more likely a non chair conformation, it was impossible to assign the relative configuration of this center with confidence based on coupling constants. Figure 26: Isolated Spin Systems for Triacetate 125 2.36 165 5.32 Using H9. as a starting point in the COSY spectrum of triacetate 125 (Figure 25), it was possible to assign all of the proton resonances around ring C (Table 7). With the assignment of the proton resonances in ring C secure, nOe experiments were carried out in order to confirm the relative stereochemistry at centers C9, Cll, C13 and C14 (Table 7). One of the key experiments performed was the irradiation of the methyl signal at 5 1.15 (s, Me20) which gave enhancements of signals at 2.71 (H9), and 2.98 (H14) ppm. In addition, irradiation of the H14 methine (52.98) afforded an enhancement of the resonances at 8 1.15 (Me20), 2.36 (H13), 1.31 (H12') and the acetoxy methylene system, 4.21 (HI5) and 4.30 (H15') ppm (Figure 27) mdicating H9 was equitorial with the A ring system axial. However, a strong nOe of Hll (8 5.15) was also observed on irradiation of Me20 which was only possible if H9 were axial and the A ring system were equatorial. Figure 2 7: S ummary of NOe Enhancements For Triacetate 125 This ambiguous nOe result made any conclusion regarding the relative stereochemistry of C9 as identical or contrary to that found in other "spongian" metabolites including gracilin A (93) and 9,11 dihydrogracilin A (99)SQ (see p. 29) impossible, however, the CI3 and C14 relative stereochemistries appeared to be identical based on the data. 3D. Aplysillolide B (102) Aplysillolide B (102), isolated as a colourless oil, had a molecular formula of C19H28O2 (EIHRMS found 288.2043, calc'd 288.2038) differing from the molecular formula of aplysillolide A (101) simply by loss of H2O. Examination of the IR spectrum indicated the presence of a saturated ketone functionality with a band at 1700cm"1 similar to that found with 101. The lH NMR spectrum of aplysillolide B (102) (Figure 28) displayed the typical methyl doublet at 8 1.65 (d, J=7.2Hz, Me6) as well as the deshielded olefinic quartet at 5.75 (q, J=7.2Hz, H7) seen in 101 for the trisubstituted double bond as well as another deshielded olefinic methine proton doublet at 6.33 (d, J=2.4Hz, H15) ppm (Table 8). The remaining units of unsaturation could be attributed to the presence of three rings in the molecule. Examination of the COSY spectrum (Figure 29) showed the presence of a five proton spin system starting with a pair of geniinal methylene protons resonating at 8 3.92 (dd, J=9.1, 10.9Hz, H16') and 4.60 (t, J=9.3Hz, H16) attached to a carbon singly bonded to an oxygen. These methylene protons (H16.H16') were coupled to an allylic methine at 3.27 (m, HI3) which is further coupled to a second set of deshielded gerninal methylene JL„ i i i <Vl _m t* >i ' AJ u f"-» S.S S.H I.S «.B 1.', J n ri-M Figure 2tf: 400MHz 'H NMR Spectrum of Aplysillolide B (1Q2J in CDCI3 Table 8: 400 MHz *H NMR Data For Aplysillolide B (1Q2J in CDCI3 90 Proton 5 ppm COSY Correlation nOesa Me6 1.65,d, J=7.2Hz H7 H9.H7 H7 5.75,q, J=7.2Hz Me6 Me6^15 H9 3.11,s Me6,Me20 H12 2.73,dd, J=13.3,6.3Hz H12',H13 H12',H13 H12' 2.49,dd, J=13.6,l 1.9Hz H12.H13 H12,H13(weak) H13 3.27,m H12,H12',H14, H16.H16' H12.H16 H15 6.33,d. J=2.4Hz H13 H16 4.60,t, J=9.3Hz H16',H13 H13.H16' H16' 3.92,dd, J=9.1,10.9Hz H16.H13 H16.H12' Mel 8 0.88,s* Mel9 0.96,s* Me20 1.12.S H12',H9,H15 (weak) a Resonance in Proton column irradiated * Interchangable 91 protons resonating at 2.49 (dd, J=13.6,11.9Hz) and 2.73 (dd, J=13.3, 6.3Hz) ppm assigned to H12 and H12' (Table 8). Also present was a deshielded methine singlet at 5 3.03 (s, H9) ppm reminiscent of the singlet at 3.11 (H9) ppm in aplysillolide A (1112). The remaining downfield signal at 8 6.33 ppm was attributed to an olefinic proton (H15) of a dihydrofuran moiety. It showed allylic coupling (2.4Hz) to the H13 methine resonance. lH NMR resonances due to the three methyl groups of ring A at 8 0.88, 0.96 and 1.12 ppm (Table 8) were also present in the spectrum of 102. It was quite clear that compound 102 was simply the dehydration product of aplysillolide A (101). This was apparent from the presence of short wave UV activity expected for a diene system which was not seen with aplysillolide A (102). The EILRMS of aplysillolide B (1112) yielded an intense peak at m/z 164 corresponding to a fragment with molecular formula C10H12O2 (EDHRMS found 164.0829, calc'd 164.0837) due to cleavage via a McLafferty rearrangement68 (Scheme 12) in agreement with the structural assignment. Scheme 12: McLafferty Rearrangement of Aplysillolide B (102) m/z 288.2043 (found) 288.2038 (calc'd) m/z 164.0829 (found) 164.0837 (calc'd) m/z 124.1253(found) 124.1252 (calc'd) The assigned geometry of the A7-8 double bond in aplysillolide B (102) as E was based on nOe experiments (Table 8). Enhancements of the H15 olefinic methine and Me6 olefinic methyl doublet resonances were observed on irradiation of the H7 olefinic quartet. There was also an enhancement of the H7 proton resonance on irradiation of the H15 olefinic resonance (Figure 30). Irradiation of the H9 singlet gave a strong enhancement of the Me6 methyl doublet as well as the Me20 singlet confirming the stereochemical assignment of the trisubstituted double bond. Figure 30: NOe Enhancements Observed For Aplysillolide B (102) An nOe experiment involving irradiation of the downfield component of the carbinol methylene system (5 4.60.H16) afforded an enhancement of the H13 methine proton, while irradiation of the upfield component (3.92, HI6') gave an enhancement of its geminal partner and the axial component (2.49, H12') of the methylene system adjacent to the ketone functionality (Figure 30). Assignment of the relative stereochemistry at C9 was established by the observed nOe enhancements of signals assigned to HI5, H9 and H12 on irradiation of Me20 indicating a similar relative configuration assigned to the "spongian" metabolite 9,11 dihydrogracilin A (99) (Table 8). This leads one to suggest that the relative stereochemistry at C9 in 101 is identical considering the chemical shifts of H9 are identical (8 3.11). 3G. Glaciolide (110) Glaciolide (110) was isolated as clear colourless needles from hexane (mp 102-103 °C) and determined to have a molecular formula of C19H30O2 (EIHRMS found 290.2246, calc'd 290.2248) which required five units of unsaturation. The 13C NMR spectrum of 110 showed resolved resonances for all nineteen carbon atoms and an APT experiment (Figure 31) (Table 9) demonstrated that all 30 protons were attached to carbon atoms (Cx5; CHx3; CH2X6; CH3X5). A band at 1776 cm"1 in the IR spectrum and a 13C NMR resonance at 8 178.9 (s) ppm suggested the presence of a y lactone functionality, accounting for the two oxygen atoms in the molecule. In addition, a tetrasubstituted double bond was indicated by the two olefinic carbon resonances observed in the 13C NMR spectrum of 110 at 8 145.2 (s) and 128.5 (s) ppm. The remaining three sites of unsaturation required by the molecular formula had to be due to rings. The *H NMR spectrum of glaciolide (1101. recorded in either CDCI3 (Figure 32) or benzene-d^ (Figure 33), was sufficiendy dispersed to allow assignment of the spin systems in the major fragments. A pair of deshielded methylene protons at 8 4.15 (dd, J=9.8,5.3Hz, H13) and 4.26 (d, J=9.8Hz, H13') ppm, which had to be attached to the y carbon of the lactone (8 68.0 (t)), established a starting point from which one spin system, H13,HI3' to H7, in glaciolide (110) could be assigned via the 2D COSY data (Table 10,11, Figure 34J5). Double resonance experiments confirmed the assignments 95 Table 9: 15MRz NMR Data for Glaciolide (110) and 109 in CDCI3 W 169 Carbon 5 ppma 8 ppma mult8 1 $4.34 34.4 t 2 22.66 22.6 t 3 46.74 46.8 t 4 34.34b 34.3b 8 5 128.54C 128.3C 8 6 145.18C 145.6C 8 7 46.67 46.9 d 8 41.38b 41.4b 8 9 48.678 56.9 d 10 37.798 39.8 d 11 21.51 21.9 t 12 23.8 23.7 t 13 68.0 - t 14 178.89 - s Mel 5 29.65<U 29.7d q Mel6 29.1 id,e 29.1d q Mel7 18.52 18.6e q Mel 8 27.86f.e 29.2* q Mel9 18.15f 20.4 q a Assignments based on APT or !H-13C correlation data b-g Assignments can be interchanged within each column 110 JJL2 Table 10: 400MHz *H NMR Data for Glaciolide (IM) in CDCI3 Proton 5 ppm COSY Correlations nOes8 HI 2.2-2.3,m H2J47,Mel7 H2 1.56,m HI Hl,Mel5,Mel6 H3 1.56,m H7 2.48,dd, J=12.0,2.3Hz H2.H12JH2' H93H.Mcl5, Mel6,Mel9 H9 2.23,dd, J=7.8,5.3Hz H10,H13,H13' H10 2.62,bt, J=7.8Hz H9,H11,H12 H9,H13'311 Hll 1.77,m H10,H11' Hll' 2.28,m H11,H10,H12' H12 1.79,m H11',H7,H12' H12* 1.26,m H7,H10,H11',H12 H13 4.26,d, J=9.8Hz H9.H13' Mel8,Mel9, H13' H13' 4.15,dd, J=9.8,5.3Hz H9.H13 H10.H9.H13 Mel5 1.17,8 Mel6 1.23,8 H7 Mel7 1.49,t, J=1.3Hz HI Mel8JH Mel8ax 0.93,s Mel9eq 0.91,8 H9 8 Resonance in Proton column Irradiated 98 99 100 Table 11: 400MHz lH NMR Data For GlacioUde flMV) in C6D6 Proton 5 ppm COSY Correlation nOesa HI 2.0-2.2,m H2,Mel7 H2 1.48,m HI H3 1.55,m H7 2.21,dd, J=2.3,12.4Hz Hl.Hl^JH^, MelS.Meie.Hl^, Mel9eq H10,H13,13' Me 19,Mel7 H9 1.42,m H10^1el9eaJHlax H10 1.42,m H9JHlaxJHlea HI 1^312^312^ H9, HI lax 2.33,dd, J=5.1,14.0Hz H9 Hlleq 1.93,t, J=7.9Hz Hllax,H12ltt,H12eq H12ax H12ax 1.12,m Hllax3Heq312eq37 HI leq,H12eq, Mel8ax H12eq 1.70,m H7,Hllax,Hlleq,H12ax H7,H12ax H13 3.74,d, J=9.7Hz H9.H13' Mel8ax,H13' H13' 3.45,dd, J=5.3,9.6Hz H9.H13 H13 Mel5 l.ll.s* H7 Mel 6 1.17.S* H7 Mel7 1.33,t, J=l.lHz HI, H7^el8ax Mel8ax 0.62.S Mel9«i Hl^.MenjHn, Mel9eq H7J19,Mel8ax Mel9eq 0.82.S Mel8ax 8 Resonance in Proton column irradiated * Interchangable LLQ 101 102 103 shown for this spin system, however, failure to detect appreciable vicinal coupling between HI3 (5 4.26) and H9 (8 2.62) could be attributed to an unfavourable dihedral angle (ca. 90°) between this pair of protons (Figure 36). The coupling constants observed for H7 Figure36: Isolated Spin Systems From COSY Data For Glaciolide ( HQ (8 ppm) 1.26 4.15 2.2-2.31.56 2.28 2.62 1.77 (J=2.3,12.0 Hz) suggested that it was a methine proton having an axial orientation in a cyclohexane ring occupying a chair conformation. Long range COSY correlations between a methyl resonance at 80.93 (Me 18) and the H7 (82.48) and H9 (82.23) resonances were attributed to W coupling, which indicated that a quaternary carbon bearing an axial methyl had to be situated between the C7 and C9 carbons of this fragment (Figure 37). 18 19 104 105 The placement of a second methyl group at C8, forrning a geminal dimethyl system was based on observed nOes between H7 and Mel9 (5 0.91), between H13 (6 4.26) and Mel8 and Mel9, and between Mel9 and H9 (5 2.23) (Figure 38) (Table 10). Figure3 & NOe Enhancements Observed for Glaciolide ( HQ 110 ^ 110 Assignment of the protons around the cyclohexane ring fused through C9 and CIO to the y lactone moiety was aided by nOe experiments (Table 10) (Figure 38). Irradiation of the H7 proton gave enhancement of signals HIT and H9 as a result of 1,3 diaxial interactions. The observed nOe between H7 and Mel9 (8 0.91) also showed Mel9 must be equatorial and Mel8 (8 0.93) must be axial. Since H9 was shown to be axial, the observed nOe enhancement of H9, HIT and H13 on irradiation of H10 (8 2.62) proved H10 was cis to H9 and, therefore, equatorial while HI 1* was axial. The final assignment to be made involved the protons at C12 0H12, H12'). The assignment of H12 as equatorial 106 Table 12: 400MHz *H NMR Data For Diol 127 in CDCI3 Proton 6 ppm COSY Correlation HI 2.2-2.3,m H2,Mel7 H2 1.53,m HI H3 1.53,m H7 2.54,dd, J=2.6,l 1.9Hz H12ax<H12eq H9 2.22,m H13J113\H10 H10 1.66,m Hllax.Hlleq.H14 H10J411eq312eqfll2ax HI lax 1.66,m HI leq 1.90,m H10JlllaxJH2eqJ112ax H7JH2eqJHleq\Hllax H12ax 1.24,m H12eq 1.75,m H13 3.93,dd, J=6.1,10.1Hz H13',H9 H13' 3.63,dd, J=6.9,9.8Hz H13.H9 H14 3.83,m H10 Mel5 1.21.S* Mel6 1.16,s* Mel7 1.48,t, J=l.lHz HI Mel8ax 0.94,s Mel9eo 0.92,s a Resonance in Proton column irradiated * Interchangable 121 108 (81.26) was based on the observed long range W coupling in the COSY spectrum (Figure 37) between H12 and H10, which also gave further proof that H10 was equatorial. The cyclohexane ring fused to the y lactone provided the major fragment of glaciolide (110). 2.48 2.23 Support for the structure of this major fragment was furnished by the transformation of glaciolide (110) to diol 127 via L1AIH4 reduction (Scheme 13). Examination of the ]H COSY spectrum (Figure 40) of diol 127 (CDCI3) gave evidence for the presence of two hydroxymethyl functionalities with resonances (Figure 39) at 8 3.63 (dd, J=6.9,9.8Hz, H13), 3.94 (dd, J=6.1,10.1Hz, H13') and 3.84 (m, H14.H14') which were linked through two methine resonances at 2.24 (m, H9) and 1.68 (m, H10) ppm as confirmed by double resonance and nOe experiments (Table 12). The spin system linking methine H10 through to methine H7 was confirmed by correlations in the COSY spectrum, double resonance and nOe experiments (Table 12). The methine resonance at H10 was coupled to a pair of geminal methylene protons at 8 1.90 (m, HI 1) and 1.75 (m, HIT) which were in turn coupled to another pair of geminal methylene protons resonating at 1.62 (m, H12) and 1.25 (m, H12') ppm. The H12' proton was shown to be further coupled to the methine proton at 2.54 (dd, J=2.6,11.9Hz, H7) ppm completing the assignment of the eleven proton spin system from C13 to C7. As noted previously, the 109 methine proton at C7 could be assigned to the axial position on the basis of observed coupling constants (J=2.6,11.9Hz). The polar nature of Diol 127 gave rise to poor solubility which often led to partial or total precipitation in normal NMR solvents resulting in broadened signals. Diol 127 also had a tendency to decompose during routine handling. Therefore, diol 127 was H 2.54 acetylated yielding diacetate 128 (Scheme 13), which gave much sharper signals in the *H NMR (Figure 41) (Table 13). The COSY spectrum of diacetate 128 (benzene-d$) (Figure 42) contained evidence for the presence of two acetoxymethyl groups with resonances at 8 4.32 (dd, J=4.9,11.3Hz, H13), 4.19 (dd, J=2.0,11.3Hz, H13') and 4.24 (bm, H14.H14'), as well as methyl singlets due to the acetates at 1.70,1.76 ppm. The two acetoxymethyl groups were linked through two methine carbons with JH NMR resonances at 6 2.35 (m, H10) and 1.76 (m, H9) as would be expected from the reduction of the y lactone in glaciolide (UOJ. The H10 methine signal was further connected through two geminal methylene protons resonating at 1.89 (m, HI 1) and 1.75 (m, HI 1'), to the next set of methylene protons, 1.75 (H12), 1.22 (H12') which were linked to the axial methine proton at 2.48 (dd, J=2.6,12.3Hz, H7) ppm. This completed the assignment of the spin Ill Table 13: 400MHz *H NMR Data For Diacetate 128 in 112 Proton 8 ppm COSY Correlation nOesa HI 2.1-2.2,m H2,Mel7 H2 1.49,m HI H3 1.49,m H7 2.48,dd, J=2.6,12.3Hz H12ax>H12eq H9 1.77,m H13.H13' H10 2.35,m H14 Hllax 1.47,m HI leq,H12ax»H12eq HI leq 1.90,m HI lax»H12ax»H12eq H12ax 1.22,m H7Jllleq,Hllax,Hl2eq Hllax H12eq 1.75,m H7JH2ax,Hllax,Hlleq H9,H13* H13 4.32,dd, J=4.9,11.3Hz H13' H13' 4.20,dd, J=1.7,l 1.3Hz H9.H13 H13 H14 4.24,m H10 Mel5 1.13.S H7 Mel 6 1.17.S H7 Mel7 1.38,s HI H7.H1, Mel8 Mel8ax 0.70,s H13.H13', H14,Mel7, Mel5,Mel6 Mel9eq 0.88,s H7.H9.H14, Mel6Mel7, Mel 8 OAc 1.70.S OAc 1.76,s a Resonance In Proton column irradiated 113 Scheme 13: Chemical interconversion of Glaciolide (1101 115 system from C14 to C13 and on to C7. The gerninal relationship between Mel9 (5 0.88) and Mel 8 (5 0.70) was confirmed via the nOe enhancement induced on the Mel8 singlet on irradiation of Me 19 as well as by the observed long range coupling (W coupling) between the two methyl groups in the COSY spectrum (Figure 41). The remaining portion of glaciolide (110) (C9H15) had to contain a tetrasubstituted double bond to account for signals observed in the 13C NMR at 8 145.18 (s), 128.54 (s). In addition, an olefinic methyl group (8 1.49 (bs)), two aliphatic methyls (1.17 (s), 1.23 (s) ppm), a quaternary carbon, and three methylenes had to be present in one ring. The positioning of the tetrasubstituted double bond was established once the allylic protons in the molecule were identified. One allylic proton was H7 (8 2.48) as indicated by its chemical shift while the two other protons with allylic chemical shifts were the methylene protons resonating at 8 2.2-2.3 (m, Hl^H'). A double resonance experiment involving irradiation of a complex four proton multiplet at 8 1.56 (H2,H2'; H3.H3') ppm converted the allylic proton resonances at 8 2.2-2.3 into a pair of broad doublets typical of an AB spin system. These data were logically accommodated by two possible substructures, A and B. However, the COSY spectrum of glaciolide (110). which showed strong long range 4.24 OAc H 2.48 116 117 Table 14: 400MHz JH NMR Data for R11O4 Product 129 in CDCI3 Proton 5 ppm COSY Correlation nOesa HI 1.67,m HlfH8 H2ax 1.75,m H132eq33 H2eq 1.60,m H132ax,H3 H3 2.33,dd, J=3.0,11.0Hz H2aXrH2eq H5 2.22,m H6.H6' H6 4.36,dd, J=9.7,1.7Hz H5.H6' H6',Mellax, Mel2«j H6' 4.17,dd, J=5.8,9.7Hz H5.H6 H6.H8 H8 2.61,dt, J=2.8,7.4Hz H1,H5 H5 Me 10 2.18,s H2ax,Mellax, Mel2eq Mel lax 0.93,s H12eq H2ax,H6,Mel0, Mel2cq Mel2eq 1.05,s HI lax H3,H5,H6,Mel0, Mel lax 8 Resonance in Proton column irradiated 129 Figure 44: 400MHz COSY Spectrum of RUO4 Product 129 in CDCI3 118 119 A B correlations attributable to homoallylic coupling between the ally he protons (HI HY) and both the Mel7 protons and H7 methine, (Figure 35,37) was only compatible with substructure A. Therefore, the olefinic methyl (Mel7) and C7 of the major fragment had to be geminal substituents on one of the olefinic carbons. Since there was evidence for only one pair of allylic methylene protons, the second substituent on the other olefinic carbon had to be the quaternary carbon bearing the two aliphatic methyls. Linking the allylic methylene to the quaternary carbon with the two remaining methylene carbons led to the constitution shown for glaciolide (110). LUQ 122 Table 15: 400MHz *H NMR Data For RuC-4 Product 130 in CDCI3 COSY Proton 5 ppm Correlation nOes8 Hlax 1.45,m H1 eq Ji2ax J12eq,H8 Si"1 1.79,m Hlax»H2ax32eq»H8 H2ax 1.72,m H2eq 1.58,m H^T 2.42,dd, J=10.3,3.8Hz H2axJi2eq Hlax»H2eq35, MelOJvlel2eq H5 1.75,m H6.H6* H6 4.12,m H5.H6' H6' 4.37,dd, J=11.7,4.5Hz H5.H6 H7 4.14,m H8 H8 2.22,m H1 ax »H 1 eq,H5 ,H7 MelO 2.17.S Mel ^ 0.97,s H2ax,H6,H6', l.lO.s Mel0^1el2eq Mel2eq H3,H5,H6',Mel0, Mel lax OAc 2'.04,s OAc 2.04,s 8 Resonance in Proton column irradiated 130 Figure 47: 400MHz COSY Spectrum of RUO4 Product 130 in CDCI3 B 4&> 0 a 124 The stereochemistry about the tetrasubstituted double bond was shown to be Z by the observation of intense nOes between H7 and the Mel5 and Mel6 protons, as well as between the Me 17 protons and the allylic protons at CI (Table 10) (Figure 38). The final proof for the structure of glaciolide (110) came from its reaction with R.UO469 to give the degradation product 129 in excellent yield (Scheme 13). Compound 129 had a molecular formula of C12H18O3 (EIHRMS calc'd 210.1256, found 210.1254) requiring four degrees of unsaturation. The methyl ketone functionality expected from the cleavage of glaciolide (1101 was represented in the NMR (Figure 43,44) (Table 14) as a methyl singlet appearing at 8 2.18 (s) ppm as well as in the IR spectrum by a band at 129 IM 1688 cnr1 (Figure 45)..The y lactone was represented by a band at 1762 cm-1 in the IR spectrum and only one set of geminal dimethyl protons (8 0.93,1.05 ppm) was present in the lH NMR spectrum. The remaining two units of unsaturation were due to the cyclohexane and lactone rings. This same reaction was carried out on diacetate 128 yielding compound 130 in good yield (Scheme 13). The NMR (Figure 46,47) (Table 15), IR (Figure 48) and mass spectral data for 128 gave further evidence for the assigned structure of glaciolide (HOI.55 3500 3000 2500 2000 1500 1000 Figure 48: FT-IR Spectrum of Product 130 126 3H. Marginatone (112) Marginatone (112). obtained as a white solid, gave a parent ion in the EIHRMS at m/z 300.2093 Daltons corresponding to a molecular formula of C20H28O2 (calc'd 300.2090) requiring seven units of unsaturation. The *H NMR spectra of marginatone (112). run in either CDCI3 (Figure 49) (Table 16) or C6D6 (Figure 50) (Table 17), were well dispersed and extremely informative. A pair of deshielded doublets at 8 6.59 (d, J=2Hz, H15) and 7.26 (d, J=2Hz, H16) in the !H NMR spectrum of 112 (CDCI3) and resonances in the 13C NMR (CDCI3) (Figure 51) at 106.18 (d, C15), 118.18 (s, C13), 142.25 (d, C16) and 161.73 (s, C14) ppm (Table 18) were assigned to a disubstituted furan ring. The observation of nOes between the two deshielded proton resonances in conjunction with their relative chemical shifts and scalar coupling of 2Hz (Table 16) demonstrated that the two furan protons were a (8 7.26) and |3 (8 6.59) substituents on adjacent carbons and the furan must, therefore, be 2,3-disubstituted. An IR band at 1680 cm-1 and a 13C NMR resonance at 8 195.19 (s) ppm were assigned to an a,(5 unsaturated Table 16: 400 MHz !H NMR Data for Marginatone (1121 in CDCI3 Proton 5 ppm COSY Correlation nOes8 H7 1.63,m H7' H7' 2.28,dt, J=12.8,3.1Hz H7 Mel9 H9 1.90,dd. J=3.8,12.9Hz Hll,Hll,,Me20 Hll 2.54,dd, J=3.8,17.2Hz H9.HH' Hll' 2.46,dd, J=12.9,17.2Hz H9.HH Mel9,Me20 H15 6.59,d, J=2.0Hz H16 H16 H16 7.26,d, J=2.0Hz H15 Mel7 0.86,s Mel 8 0.88,s Mel9 1.29.S Me20 Me20 0.99.S Mel7,Mel9 8 Resonance in Proton column irradiated 112 129 Table 17: 400MHz !H NMR Data For Marginatone (U2) in C&D6 Proton 6 ppm COSY Correlation nOes8 H6 1.38,m* H7 H6' 1.19,m* H7 H7 1.35,m H7' H7' 2.06,dt, J=12.2,3.1Hz H7 Mel9 H9 1.44,dd, J=3.1,13.5Hz HlltHll',Me20 Hll 2.45,dd, J=3.1,16.9Hz H9,HH' Hll' 2.21,dd, J=13.6,16.9Hz H9.H11 Mel9,Me20 H15 6.63 ,d, J= 1.9Hz H16 H16 H16 6.78,d, J=1.9Hz H15 Me 17 0.62,s Mel8 0.70,s Mel9 0.95,s Me20 Me20 0.75.S Mel7,Mel9 a Resonance in Proton column irradiated 131 ketone functionality in marginatone (112). With no 13C NMR evidence for olefinic functionalities other than the furan ring, it was clear that the a,P unsaturated ketone moiety had to be conjugated into the furan ring. Subtraction of the four sites of unsaturation accounted for by the furan and ketone carbonyl from the seven sites required by the molecular formula revealed that the molecule contained three rings in addition to the furan. The incorporation of the 2,3-disubstituted furan ring and the four tertiary methyl residues apparent in the !H NMR (CDCI3) (8 0.86, s; 0.88, s; 0.99, s; 1.29, s) into a tetracyclic diterpenoid metabolite could readily be accomplished by assuming that marginatone (112) had the "marginatane" carbon skeleton first encountered in the metabolite marginatafuran (HI).54 O 17 18 ill 112 Support for the placement of the ketone functionality at C12 as shown was found in the lH NMR spectra (Figure 49£0) where a pair of deshielded doublet of doublets reminiscent of geminal methylene protons on a carbon adjacent to a carbonyl were identified. Double resonance and COSY experiments (Figure 52,53) on marginatone (112) identified a three proton spin system incorporating these deshielded protons resonating at 8 2.46 (dd, 132 Table 18: 75MHz 13C NMR Data For Marginatone (112) (CDCI3) Carbon 8 ppm mult8 12 195.19 s 13 118.18 s 14 161.73 s 15 106.18 d 16 142.25 d a Assigned from APT experiment 134 135 Figure 53: 400MHz COSY Spectrum of Marginatone (112) in CePe O • i • ' i I i •'— i i i 1 7.0 6.0.5.0 4.0 3.0 2.0 1.0 PPM 136 J=12.9,17.2Hz, Hll') and 2.54 (dd, J=3.8, 17.2Hz, Hll) and a methine at 1.90 (dd, J=3.8, 12.9Hz, H9) ppm. The relative stereochemistry at centers C5, CIO, C9, and C8 was established by the use of nOe (Figure 54) (Table 16) and long range COSY experiments (Figure 55). Irradiation of a methyl singlet at 8 1.29 ppm induced enhancements in the HIT (axial) proton resonance at 8 2.46 as well as in a second resonance at 2.28 (dt, J=12.8,3.1Hz) assigned to H7 (equatorial). Therefore, the methyl singlet resonance could be assigned to the axial Mel9 protons (Figure 54) (Table 16). An nOe from HIT (axial) to a second methyl singlet at 8 0.99 ppm identified this resonance as belonging to the axial Me20 protons. A correlation observed in the long range COSY spectrum of marginatone (112). attributed to W coupling between resonances at 8 0.99 (Me20) and 1.90 (H9) ppm, provided support for their assignment (Figure 55). Irradiation of the Me20 singlet (81.29) resonance gave an nOe enhancement of a methyl resonance at 8 0.86, assigned to Me 17 (axial). Therefore, the remaining methyl singlet at 8 0.88 ppm was Figure 5 4' NOe enhancements Observed for Marginatone ( 112 O nOe 112 137 138 assigned to Mel8 (equatorial). The observed nOes from HIT (axial) to Mel9 (axial) and Me20 (axial), the diaxial coupling constant of 12.9Hz observed between HIT and H9, observed W coupling between H9 and Me20 as well as the nOe between Me20 and Mel7, estabhshed the presence of a trans-wxi-trans fused tricyclic ring system. 31. Cadlinolide C (131) Cadlinolide C (131). isolated as a colourless oil, gave a parent ion in the EIHRMS at m/z 364.2246 (calc'd 364.2250) Daltons corresponding to a molecular formula of C21H32O5 requiring six units of unsaturation. Well resolved resonances for all 21 carbon atoms were apparent in the 13C NMR spectrum of cadlinolide C (131) while an APT experiment indicated 31 hydrogens were attached to carbon (5xCH3, 6XCH2, 4xCH, 6xC) {Figure 56) (Tablel9). The remaining hydrogen atom, unaccounted for in the APT experiment, was assigned to an hydroxyl functionality based on the presence in the IR of a band at 3389 cm-1 (-OH stretch) as well as an intense peak in the EILRMS at m/z 346 corresponding to the loss of H2O (EIHRMS found for C21H30O4 346.2144, calc'd 346.2144). The 13C NMR (8 175.34 (s) and 179.23 (s)) in conjunction with IR bands at 1777 and 1737 cm-1 indicated the presence of two ester functionalities accounting for the four remaining oxygen atoms in cadlinolide C (131). The frequency of one of the ester carbonyl stretching vibrations (1777 cm-1) suggested the presence of a y lactone. The frequency of the other ester carbonyl (1737 cm-1) in addition to 139 Table 19: 75MHz 13C NMR Data For Cadlinolide C Oil) in CDCI3 Carbon S ppm mult8 1 $8.99 t 2 19.77 t 3 39.88 t 4 31.58 s 5 50.63 t 6 16.52 q 7 41.29b d 8 127.38d s 9 147.02d s 10 41.95 s 11 26.74c t 12 26.69* t 13 41.52b d 14 45.75b d 15 104.08 d 16 175.34 s 17 179.23 s Mel 8 27.50 q Mel9 30.59 q Me20 32.86 q Me21 52.17 q 8 Assignment made by APT experiments b-d Interchangable 121 Table 20: 400MHz *H NMR Data for Cadlinolide C (121) in CDCI3 Proton 5 ppm COSY Correlation nOe* Me6 1.21,d. J=6.9Hz H7 7 4.30,q, J=7.0Hz Me6 H11313315,Me6 11 2.33,m H11\H12,H12' H11',H12,H12', Me20 11' 1.48,m H11,H12,H12' 12 2.09.m H11,H11',H12,H13 H7.H12' 12' 1.30,m H11,H11\H12,H13 13 2.99,m H12,H12',H14 Me637,H15 14 2.99,m H13.H15 Me6,H7,H15 15 5.4l,d, J=3.5Hz H14 H7.H13 Mel8 0.86,s* Me 19 0.88,s* Me20 1.07.S H7.H13.H14, OMe 3.71.S a Resonance in Proton column irradiated * Interchangable 121 143 O [5 bH y lactone the observation of a sharp methyl singlet in the lH NMR at 5 3.71 and a methyl resonance at 52.17 (q) in the 13C NMR, indicated the presence of a methyl ester functionality. A deshielded ketal methine in the JH NMR resonating at 8 5.41 (d, J=3.5Hz) plus a deshielded ketal carbon resonance at 104.08 (d) ppm, rerniniscent of the hemi-ketal moiety found in cadlinolide B (1311. suggested the hydroxyl functionality must be attached to the carbon attached to the alkoxy oxygen of the y lactone. The remaining unsaturated functionality in cadlinolide C (1311 that could be identified from the 13C NMR data was a tetrasubstituted double bond with resonances at 8 127.38 (s) and 147.02 (s) ppm. Three remaining degrees of unsaturation had to belong to three rings in order to satisfy the sites of unsaturation required by the molecular formula. The !H NMR spectrum of cadlinolide C (1311 (Figure 57) (Table 20) was well enough dispersed to facilitate the assignment of the key spin systems in the molecule using COSY spectra (Figure 58). Starting with the most deshielded resonance, assigned to a ketal methine proton (8 5.41,d, H15), a correlation was observed to a deshielded two proton multiplet at 8 2.99 (m, H13.H14) consisting of two overlapping methine resonances which are either allylic or adjacent to a carbonyl group. Further coupling was observed into Figure 58: 400MHz COSY Spectrum of Cadlinolide C (121) in CDCI3 145 Figure 5 9. Isolated Spin System in Cadlinolide C (131) a pair of geminal methylene protons resonating at 8 2.09 (m, H12) and 1.30 (m, H12') which were in turn coupled into a second pair of allylic geminal methylene protons at 2.33 (m, Hll) and 1.48 (m, HIT) ppm completing a seven proton spin system (Figure 57). A second spin system immediately identifiable from the *H NMR and COSY spectra consisted of a deshielded methine quartet at 8 4.30 (J=7.0Hz, H7) and a downfield methyl doublet resonating at 1.21 (d, J=6.9Hz, Me6) ppm, resembling the systems found in cadlinolides A (751 and B (761 (Figure 59). The deshielded character of these two 146 resonances indicates that, as before, the methyl and its corresponding methine are located between a double bond and a carbonyl. Based on this data, two possible substructures A and B, incorporating a y lactone, a hemi-ketal and a methyl ester with an adjacent allylic methine/methyl system were put forth. An nOe experiment demonstrating an nOe enhancement between the ketal methine resonance (8 5.41) and the allylic methine quartet (8 4.30) (Table 20) suggested that substructure A contained the correct regiochemistry for cadlinolide C (131). *H and 13C NMR data indicated the remaining portion of the molecule was identical to the ring A system of cadlinolides A (75). B (76). and tetrahydroaplysulphurin-1 (7_2J (Table 19,20). It would appear that cadlinolide C (131) is an isolation artifact formed via the nucleophilic attack by the extraction solvent methanol on cadlinolide A (75) at the C17 position (Scheme 14) forming the methyl ester and ketal functionalities, while Scheme 14: Conversion of Cadlinolide A (75) to Cadlinolide C (131) H O H O MeOH. OH O 71 MeOH 121 147 maintaining the y lactone moiety. Should this be the case, assignment of the relative stereochemistry at C14 and C13 could be based on the assignments made for cadlinolide A (751 with the two ring junction protons cis to each other. Since the chemical shifts of the two methine protons H13 and H14 are so similar, this cis arrangement was impossible to verify via nOe experiments (Table 20). C-I. INTRODUCTION TO THE NUDIBRANCHS 148 Nudibranchs (Phylum Mollusca, class Gastropoda, subclass Opisthobranchia) have been the subject of much interest by natural products chemists in recent years. The large phylum Mollusca, estimated to contain about 75,000 living species and 35,000 fossil species can be subdivided into seven classes. Members of the class Gastropoda (Figure 60) have been examined in greatest detail by chemists.70 Nudibranchs have been named "sea slug" or "naked snail" because of their slow movement and greatly reduced or totally absent shell. Nudibranchs have very few known predators despite an apparent lack of physical protection and often brightly coloured soft outer tissue.71 Faulkner72 and Thompson,73 in separate studies, concluded that nudibranchs had preadaptively developed biological and chemical defences enabling the animals to dispense with the shell. This conclusion was based on fish antifeedant studies carried out on partially shelled nudibranchs which were rejected as food by fish. Further investigation has led to the observation that nudibranchs are able to employ defence mechanisms in a hierarchical fashion.73 The primary form of defence is to avoid detection by adopting a reclusive habit and cryptic colouration.74 One of the most interesting defensive adaptations is the ability of the nudibranch to attain the colouration on their outermost tissue through the ingestion and accumulation of pigments and carotenoids from organisms, such as sponges, upon which they feed.73 Alternatively, many nudibranchs, notably chromodorids and polycerids, are not cryptic and often possess strikingly bright colouration making no effort to conceal themselves. These animals, thought to possess aposomatic or "warning colouration", are often found to be toxic to fish and crustaceans.75 Further examples of primary defence mechanisms noted in the literature MOLLUSCA PHYLUM GASTROPODA OPJSTHOBRANCHIA CLASS SUBCLASS 1 BULLOMORPHA APLYSIAMORPHA PLEUOBRANCHOMORPHA PTEROPODA ORDER SACOGLASSA NUDIBRANCHIA PYRAMIDELLA AEOLIDACEA ARMINACEA DENDRONOTACEA DORWACEA SUBORDER Figure 60: Phylogenic Classification of Nudubranchs 79 (Classification according to Behrens ) *—» 150 include swimming responses,76 changes in colouration77 and the presence of spiney spicules78 on the outer mantle (dorsum) (Figure 6119). Figure 61: Typical Dorid Nudibranch The final line of nudibranch defence involves chemicals secreted by the animals in times of distress. It had been noted since the late 1800's that nudibranchs were rejected as food by aquarium fish.80 It was first noted by Garstang in 1890 and later by Thompson that dorids secrete acid when aggravated.81-80 This form of defence has since been recognized in many dorids and is believed to originate from an acidic tunicate diet.82 Thompson has also noted that non-acidic dorids contained fluids in their glands that were bitter tasting, suggesting their possible use as defensive allomones.80 In all early studies, very little attention was paid to the chemistry of the these allomones. It was not until the 1960's before Yamamura and Hirata first investigated the secretions of an opisthobranch, reporting the isolation of brorninated terpenoids from the sea hare Aplysia kurodai.^ These compounds were later isolated from the red alga Laurencia sp. upon which they feed.84 This result created the impetus for chemists to further investigate the defensive allomones secreted by nudibranchs. Subsequent work in the field of nudibranch chemistry has provided numerous examples of repugnant compounds isolated from skin extracts. These compounds are thought to be selectively sequestered from dietary sources and stored in non-mucous glands on the dorsum where they can be secreted for immediate effect when the animals are 151 perturbed by potential predators.85 The dietary origin of many of these "noxious" metabolites is reflected in the large variety of compounds isolated from the nudibranch Cadlina luteomarginata collected at different sites. METABOLITES OF CADLINA LUTEOMARGINATA The chemistry of the dorid nudibranch Cadlina luteomarginata has been investigated from collections made on the west coast of North America ranging from as far south as Punta Eugenia, Baja California, to as far north as the Queen Charlotte Islands, British Columbia. Of particular interest to marine natural products chemists has been the wide variety of metabolites isolated from C. luteomarginata reflecting the cosmopolitan nature of its diet. Samples of Cadlina luteomarginata were collected at Scripps Canyon, La Jolla, California during January, July and October 1977 and at Point Loma, San Diego, California during October 1978 and July to September 1980.86 Examination of the January 1977 collection of 25 animals yielded dendrolasin (1331. pallescensin-A (134). pleraplysillin-1 (135). furodysinin (136) and idiadione (122). The July 1977 collection of about 100 specimens yielded isonitrile (138). as the major metabolite as well as the corresponding isothiocyanate (139). isonitrile (140). pallescensin-A (134) and dihydropallescensin-2 (141) a derivative of pallescensin-2 (JL&2J-86 The October 1977 collection from Scripps Canyon was used for gut content analysis exclusively, while those collected from Point Loma in 1978 yielded an unknown isonitrile, as well as isonitrile (140).86 Samples of C. luteomarginata collected at Point Loma in the summer of 1980 yielded isonitrile (140). two unknown isonitriles as well as their corresponding isothiocyanates, dendrolasin (133). pallescensin-A (134). pleraplysillin-1 ("1351. furodysinin (136). idiadione (137). isothiocyanate (139). and dihydropallescensin-2 (141).86 Through a careful investigation of the gut contents of C. luteomarginata, Thompson et al. were able to deduce the origin for each of the metabolites 133-142 which were all previously known from various sponges.86 Ul The methanol extracts of Cadlina luteomarginata collected in Howe Sound and Barkley Sound, British Columbia have afforded a variety of terpenoids which were not found in the California extracts. Hellou and Andersen87 reported the isolation of albicanol acetate (143) as well as minor amounts of albicanol (144). Sesquiterpenes 143 and 144 contain a drimane skeleton like compounds isolated from the Dysidea sponge species.88 Luteone (145). an odoriferous compound possessing a novel degraded terpenoid skeleton, as well as three furanosesquiterpenoids, furodysinin (1361. furodysin (146) and microcionin-2 (147). were also reported.89 Compounds 136,146 and 147 were already known from sponge sources and were identified by a comparison with published data.90*91 The structure of luteone (145J, believed to be a degraded sesterterpene, was solved by 123. R= NC iM R= NC 139 R= NCS ill 112 single crystal x-ray diffraction analysis of its 2,4-dinirrophenylhydrazone derivative. The origins of albicanol acetate (143). albicanol (144) and luteone (145) are unknown. However, since these compounds were only found in collections made in British Columbia, a dietary source such as a sponge is highly likely. Marginatafuran (111), a furanoditerpene with a new carbon skeleton, was isolated by Gustafson et al. in 1985 from a collection of C. luteomarginata made in the Queen Charlotte Islands.54 The structure of this compound, which contained the new 154 "marginatane" skeleton, was solved by single crystal x-ray diffraction analysis. Recently, a similar diterpene, compound 113, was isolated from C. luteomarginata collected in the Queen Charlotte Islands.34 The discovery of marginatone (JJ2) from the sponge Aplysilla glacialis, an observed prey of C. luteomarginata indicates that these compounds have a dietary origin 40 Three other diterpenes, cadlinolide A (75). glaciolide (110) and tetrahydroaplysulpurin-1 (72). were also isolated from C. luteomarginata specimens found40 143 R=Ac 14J. 144 R=H 146 112 155 feeding on the A. glacialis. Of these three compounds, only compound 72, previously reported by Karuso from a sponge collected in New Zealand,27 was not found in the extracts of A. glacialis. With the isolation of cadlinolide B (76) in minor amounts from A. glacialis, it has been suggested that the nudibranch could be selectively sequestering compound 76 and converting it to compound 72 by in vivo acetylation. ill 112 156 "C-II. SPONGIAN METABOLITES FROM THE NUDIBRANCH CADLINA LUTEOMARGINATA (MACFARLAND 1966) 1. Introduction Cadlina luteomarginata (MacFarland 1966) (Class Gastropoda, Subclass Opisthobranchia, Order Nudibranchia, Suborder Doridacea, Family Cadlinidae), is commonly found on the Pacific coast of North America ranging from Auke Bay, Alaska, to Point Eugenia, Mexico.92 In the field, C. luteomarginata is characterized by a translucent white dorsum which is edged by a yellow hne. Numerous samples of Cadlina have been collected from sites off the coast of British Columbia, especially, Howe Sound, Sanford Island and the Queen Charlotte Islands. Our chemical studies on the nudibranch Cadlina luteomarginata were prompted by an interest in the variety of terpenoid metabolites which have been isolated from this species which survives in a competitive environment despite its bright coloration and apparent lack of physical defence. One theory is that the nudibranch, which is known to feed on a variety of sponges, might be sequestering sponge metabolites which it can use and sometimes alter slightly for defensive purposes.93 The current collection of C. luteomarginata was made while the nudibranch was feeding on the sponge Aplysilla glacialis, which is known to contain a wide variety of "spongian" derived metabolites.40 It was believed that this clear case of a host predator relationship would yield conclusive evidence with respect to the origin of some of the metabolites isolated from C. luteomarginata. 157 2. Isolation and Structure Elucidation Cadlina luteomarginata was collected by hand using SCUBA in an exposed surge channel on Sanford Island, Barkley Sound, B.C., at depths of 0 to -3 m and immediately immersed in methanol. After soaking in methanol for up to three days at room temperature, the methanol layer was decanted, vacuum filtered and evaporated in vacuo to yield an aqueous methanolic suspension. This suspension was partitioned between brine and ethyl acetate, and the organic layer was dried over anhydrous Na2S04. This procedure was repeated four times at one hour intervals. The combined organic layers were then vacuum filtered and evaporated in vacuo affording a sweet smelling viscous yellow oil which was fractionated by flash chromatography to give a mixture of fats, pigments, steroids and terpenoids as detected by analytical TLC analysis. Further separation and purification guided by *H NMR analysis yielded a mixture of terpenoid metabolites including cadlinolide A (75). glaciolide (1101 previously isolated from Aplysilla glacialis, 40>53 as well as tetrahydroaplysulphurin-1 (22)26 15 1LQ 12 158 3. Tetrahydroaplysulphurin-1 (22) Tetrahydroaplysulphurin-1 (72). isolated as a clear colourless oil, gave an intense ion at m/z 394 (M++NH4+) and at m/z 334 (M++NH4-HOAc) in the DCIMS appropriate for a di terpenoid acetate with a molecular formula C22H32O5, requiring seven units of unsaturation. This molecular formula was confirmed from the EDHRMS which gave a weak ion at m/z 376.2243 (C22H32O5) (calc'd 376.2250). Initial examination of the !H NMR of compound 72 (Figure 62) suggested it was simply the acetate of cadlinolide B (76). The presence of an acetoxy functionality was indicated by a peak in t he EILRMS at m/z 316 (M+-HOAc) (EDHRMS calc'd 316.2039, found 316.2040 for C20H2XO3) and confirmed by the presence of a three proton singlet in the *H NMR at 8 2.04 and signals in the 13C NMR/APT (Figure 62,63) at 21.19 (q) and 169.87 (s) ppm (Tables 2122). A second carbonyl resonance in the 13C NMR spectrum at 8 170.94 (s) ppm as well as an IR absorption at 1750 cnr1 and a strong peak in the EILRMS due to loss of CO2, from the M+-HOAc fragment, at m/z 272, suggested the presence of a 5 lactone. Two olefinic singlets in the 13C NMR at 8 121.25 and 146.48 ppm, resembled the resonances assigned to the tetrasubstituted olefinic systems found in 75 and 76. The presence of methine signals in the !H NMR at 8 6.00 (d, J=6.2Hz) and 6.18 (d, J=2.4Hz) corresponding to ketal protons and in the 13C NMR at 100.57 (d) and 102.71 (d) ppm, attributable to ketal carbons, allowed for the assignment of all the oxygen atoms in the molecule. It was clear from the functionality deterrnined from the spectral data thusfar, including the acetoxy, 8 lactone and tetrasubstituted double bond moieties that the molecule must be tetracyclic in order to satisfy the degrees of unsaturation prescribed by the molecular formula. O tricyclic portion Ring A The presence of the familiar tricyclic and ring A portions shown was established by examination of the !H NMR, COSY, and nOe data (Figure 62,64,65) (Table 21). Acetylation of cadlinolide B (761 gave a product which was spectroscopically identical to acetate 72 isolated from C. luteomarginata. A search through the literature revealed the metabolite, tetrahydroaplysulphurin-1 (721. isolated from a New Zealand sponge,26 was constitutionally identical to the acetylated derivative of 76, however, on comparison of 160 Table 21: 400 MHz !H NMR Data for Tetrahydroaplysulphurin-1 Q2) in CDCI3 Proton 6 ppm COSY Correlation nOesa Me6 1.42,d, J=7.4Hz H7 H7 4.2 l,q, J<=7.4Hz Me6 Me6,Me20 Hll 2.36,m H11\H12,H12' Hll' 2.09,m Hmil2JH2'314 H12 1.90,m H11,H11\H12',H13 H12' 1.28,m H11,H11',H12,H13 H13 2.52,m H12,H12',H14,H16 H14 3:22,m H11',H13,H15 H13,H15,Me6 H15 6.00,d, J=6.2Hz H14 H14 H16 6.18,d, J=2.4Hz H13 H13 (weak) Mel 8 0.78.S* Mel9 0.91,s* Me20 1.13,s OAc 2.08,s a Resonance in Proton column irradiated * Interchangable 12 162 Table 22: 75MHz 13c NMR Data For Tctrahydxoaplysulphurin-1 (22) (CDCI3) Carbon 8 ppm mult8 1 39.03 t 2 20.73 t 3 39.51 t 4 31.58 s 5 50.88 t Me6 14.74 q 7 42.06d d 8 121.71 s 9 146.48 8 10 39.73 8 11 23.99b t 12 25.03^ t 13 40.63d d 14 38.05d d 15 100.57e d 16 102.71* d 17 170.94' 8 Mel 8 28.288 q Mel9 31.078 q Me20 32.52 q OAc 21.19 q 169.87* 8 8 Assignment based on APT exreriment b-8 Assignments interchangeable 22 164 Figure 65: NOe Enhancements Observed For Tetrahydroaplysulphurin-1 (72) 165 NMR data collected versus the reported data, certain discrepancies were found. Of particular concern were the differences in the chemical shifts quoted for nearly all the *H NMR resonances (Table 21), suggesting that the two metabolites were in fact not identical, whereas, the 13C NMR data was nearly identical. Consultation with the original authors proved the discrepancy was due to an error on their part in reporting of the *H NMR chemical shifts. From an original *H NMR spectrum furnished by Professor Cambie65 (Figure 17), it was evident that the two metabolites were identical. Their assignment of the relative stereochemistry at CI6, which was first proposed based on the vicinal coupling constant of 3Hz for H16, was later confirmed by a single crystal x-ray diffraction analysis.66 Based on the observed coupling constants observed previously for the two epimers of cadlinolide B (76). the unambiguous spectroscopic assignment of the relative stereochemistry at CI6 would be virtually impossible. Conclusion Cadlinolides A (25J and B (761 are further examples of "spongian" derived diterpenes possessing the "aplysulphurane" skeleton first reported by Cambie et al.26 Biogentically, it is easy to see that metabolite 76 can be simply formed via selective reduction of the y lactone in 75. Cadlinolide C (1311 is believed to be an isolation artifact formed by attack by methanol on the 5 lactone carbonyl forming the methyl ester and hemiketal functionalities. In addition to cadlinolide C (1311. some evidence existed for an alternate isolation artifact, compound D 132, which could be formed via attack of methanol at the ketal centre forming the methyl ether and carboxylic acid as shown in Scheme 14. Attempts to separate the trace amounts of compound 132 from cadlinolide B (761 on silica gel resulted in the rapid conversion of the entire mixture to cadlinolide B (761 (Scheme 15). Figure 60 displays the JH NMR spectrum of the mixture of compound D 132 and cadlinolide B (761 before purification, showing the presence of the resonances due to three methyl singlets, a methyl doublet, a methyl ether singlet, an allylic methine, a downfield methine quartet and a deshielded ketal methine required for compound D 132. 167 What is more intriguing is the isolation of the acetylated metabolite, tetrahydroaplysulphurin-1 (72) as the major component in the extract of the dorid nudibranch Cadlina luteomarginata. Careful examination of the extracts of several collections of the sponge, Aplysilla glacialis, has failed to reveal the presence of 72. A possible explanation for this is that the nudibranch is selectively sequestering cadlinolide B (76) and converting it to acetate 72 in vivo. Thus far, attempts to inject purified samples of cadlinolide B (76) into the gut of live C. luteomarginata have not confirmed this hypothesis. Scheme 15: Methanolysis of CadlinoUde A (75) AplysilloUdes A (101) and B (102) which are degraded diterpenes possessing the "gracilane" skeleton are notable for the presence of the ketone functionaUty at Cll, the center that becomes oxidized to the carboxyUc acid functionaUty during the formation of several rearranged spongian derived metabolites including macfarlandin A (97).48 dendriUoUde A Q&),36 norrisolide (6J1)35 and chromodoroUde A (108)52. A further point of interest is the alternate stereochemistry observed at the C9 position in comparison to all Figure 66: 400MHz lH NMR Spectrum of Compound D 132 and 76 ~ oo the known "spongian" derived metabolites, presumably due to an isomerization of this acidic center to form the least sterically hindered configuration. The isolation of marginatone (1121. possessing a "marginatane" skeleton, from the sponge Aplysilla glacialis addresses the problem of the origin of the related metabolites marginatafuran (111').54 and compound 113, isolated from the dorid nudibranch Cadlina luteomarginata. Numerous examples exist in the literature describing the isolation of identical compounds from both sponges and nudibranchs collected in the same location. Since "marginatane" diterpenoids have appeared only where Aplysilla species are known to exist, it would appear the sponge Aplysilla glacialis is a possible dietary source for related metabolites marginatafuran (111), and compound 113 isolated from Cadlina luteomarginata. 170 D. EXPERIMENTAL The *H and 13C NMR spectra were recorded on either the Bruker WH-400 or the Varian XL-300 spectrometers. Tetramethylsilane (8=0) was employed as the internal standard for JH NMR spectra and CDCI3 (8=77.Oppm) or Benzene-d6 (8=128.0ppm) were used both as internal standards as well as solvents for 13C NMR spectra unless otherwise indicated. Low resolution and high resolution electron impact mass spectra were recorded on the Kratos MS-59 and MS-50 spectrometers respectively. Low resolution chemical ionisation mass spectra were recorded on the Delsi-Nermag R-10-10 quadrupole mass spectrometer either using methane or ammonia as the reagent gasses. Infrared spectra were recorded on a Perkin- Elmer 1600 FT spectrometer. Optical rotations were measured on the Perkin- Elmer model 141 polarimeter using a 10cm cell, while uncorrected melting points were determined on a Fisher-Johns melting point apparatus. HPLC was carried out on either a Perkin-Elmer Series 2 instrument equipped with a Perkin-Elmer LC-55 UV and refractive index detector or a Waters model 501 system equipped with a Waters 440 dual wavelength detector for peak detection. The HPLC columns used were either the Whatman Magnum-9 ODS-3 reverse phase or Whatman Magnum-9 Partisil 10 normal phase preparative columns. The solvents used for HPLC were BDH Omnisolve grade and the water was glass-distilled. All other solvents used were at least reagent grade unless otherwise indicated. Silica gel types used were Merck silica gel 60 PF-254 for preparative TLC, Merck silical gel 60 230-400 mesh for flash chromatography and Merck silica gel 60 PF-254 with CaS04-l/2H20 for radial TLC. All Rf values were calculated on analytical TLC plates using Macherey-Nagel Sil G/UV 254 precoated sheets 0.25mm thick. 171 All 2D-COSY Spectra were run on the Bruker WH-400 spectrometer using the following general parameters: SI=1K; SI=TD=1024; NE=256; TD 1=256; SI1=SI/2=512; SWl=SW/2; Dl= 1.2 s; PW=0; RD=0; Pl=9.0ms; P2 (60°)=6.0ms; Df=3ms; NS= variable; D2 (optional for long range COSY experiments)=0.08s. All nOe difference data were accumulated on the Bruker WH-400 spectrometer using the following general parameters: SI=16K; PW=9.0ms; RD=0; Dl=6.0s; DS=2; LB=0.3; NE=variable; NS=8. APLYSILLA GLACIALIS (Merejkowski 1878) Collection Data Aplysilla glacialis was collected during all seasons in exposed surge channels of Sanford Island, Barkley Sound, B.C. at depths of 0 to -3 metres. Immediately after collection, the sponge was immersed in methanol and stored at room temperature for up to three days. If the sponge was not worked up immediately, it was stored a low temperatures (4-(-5) °C) until used (typically within 2 weeks). Extraction and Chromatographic Separation During the course of this study on the extracts of the marine sponge Aplysilla glacialis, a number of collections were made yielding lirtle or no observed variation in metabolites. Therefore, the following represents a typical procedure. After storage at room temperature for 2 days, the aqueous methanolic layer was decanted and stored while the sponge, approximately 1600g (dry weight after extraction) was again soaked in methanol (4L) for 1 hour before the two aqueous methanolic portions were vacuum filtered and concentrated in vacuo to about 300ml before being partitioned between brine (200ml) and ethyl acetate (4 x 250 ml). The combined dark green ethyl acetate layers were dried over anhydrous Na2SC>4. Filtration, followed by evaporation, in vacuo, gave 12.4g (0.78%) of a dark green crude oil. Flash chromatography (40 mm diameter column, 15cm silica gel, step gradient 100% hexanes to 100% ethyl acetate) yielded fractions containing fats, pigments as well as intensely charring spots on TLC (1:1 hexanes:ethyl acetate) exhibiting deep red to bright pink spots using vanillin-H2S04 spray reagent corresponding to terpenoids. Purification of these components are described below. CADLINA LUTEOMARGINATA (MacFarland 1966) Collection Data Cadlina luteomarginata was collected using SCUBA in an exposed surge channel on Sanford Island, Barkley Sound, B.C., at depths of 0 to -3 metres feeding on Aplysilla glacialis. Immediately after collection, 27 whole animals were immersed in methanol and stored at room temperature for up to 2 days before being stored at lower temperature (about 2 °C) for 7 days before workup. Extraction and Chromatographic Separation Cadlina luteomarginata ( 150g dry weight after extraction) was stored at reduced temperature for 7 days before the aqueous methanolic layer was decanted and stored at room temperature while the nudibranchs were further soaked with methanol (50ml) and decanted 3 times at 1 hour intervals. The combined skin extracts were then vacuum filtered, concentrated in vacuo, and partitioned between brine (50ml) and ethyl acetate (4x75ml). The organic soluble extracts were combined, dried over anhydrous Na2SC>4, filtered and evaporated in vacuo, to yield a sweet smelling yellow oil 2.5g (1.7%). Flash chromatography ( 20mm column, 15cm silical gel, step gradient 100% hexanes to 100% ethyl acetate) followed by further purification yielded cadlinolide A (75). tetrahydroaplysulphurin (72). and glaciolide (110). Aplvsilla glacialis Compounds: A) Cadlinolide A (751 was purified by repeated flash chromatography (20mm column, 15cm silica gel, step gradient 100% hexanes to 1:1 hexanes/ethyl acetate) to yield clear colourless needles, 94.3mg (.006% of dry weight sponge) recrystallized from hexane at 2 °C. mp 126-127 °C; Compound 75: IR (film) vmax 2948, 2874, 1789, 1760, 1147, 984, 756 cm-1; EILRMS m/z (relative intensity) 332 (M+, 3), 317(1), 304(2), 303(2), 289(2), 259(4), 243(4), 231(4), 223(4), 203(4), 195(5), 191(4), 189(4), 177(7), 175(4), 166(5), 163(5), 147(10), 145(8), 135(9). 133(11), 125(12), 122(9), 121(13), 119(14), 110(13), 109(25), 105(20), 95(23), 93(15), 91(26), 85(30), 83(45), 81(19), 79(17), 77(15), 69(65), 67(19), 57(17), 55(48); iH NMR (400MHz, CDC13) 8 0.77(s, 3H), 0.92(s, 3H), 1.13(s, 3H), 1.48(d, J=7.4Hz, 3H), 1.69(m, IH), 1.72(m, IH), 1.78(m, IH), 2.06(m, IH), 2.19(m, IH), 2.35(bd, J=17.9Hz, IH), 3.12(dt, J=7.9,4.3Hz, IH), 3.48(m, IH), 4.28(q, J=7.4Hz, IH), 6.16(d, J=5.3Hz, lH)ppm; 13C NMR (75MHz, CDCI3) 8 16.68(d), 19.94(f), 20.57(f), 23.25(t), 28.14(s), 31.31(f), 31.38(d), 31.89(d), 35.07(d), 38.20(t), 38.90(d), 39.19(f), 39.90(d), 50.15(t), 99.43(d), 118.85(s), 147.29(s), 169.89(s), 173.26(s)ppm; EDHRMS m/z calc'd for C20H20O4 332.1982, found 332.1983. B) Cadlinolide B (J6J was purified by radial preparative TLC (1mm thick silica plate, step gradient 1:1 ethyl acetate/hexanes to 100% ethyl acetate) to yield 5.4mg (0.0003% of dry weight sponge) as a clear colourless oil; Compound 76: DR (film) vmax 3369, 2931, 1730, 1457, 1028, 606 cm*1; EELRMS m/z (relative intensity) 334(M+,1), 316(15), 301(4), 262(4), 206(35), 178(27), 177(33), 175(10), 163(12), 149(35), 147(16), 137(10), 135(14), 133(15), 125(28), 124(14), 121(16), 109(52), 95(26), 91(22), 83(31), 81(30), 69(100), 67(30); *H NMR (400MHz, CDC13) 8 0.77(s, 3H), 0.92(s, 3H), 1.13(s, 3H), 1.20(m, IH), 1.41(d, J=7.4Hz, 3H), 1.92(m, IH), 2.04(m, IH), 2.36(m, IH), 2.40(m, IH), 3.23(m, IH), 4.20(q, J=7.4Hz, IH), 5.39(d, J=3.9Hz, IH), 6.05(d, J=6.2Hz, lH)ppm; 13c NMR (75MHz, CDCI3) 8 14.53, 20.71, 24.20, 25.62, 28.06, 29.71, 31.33, 31.57, 32.65, 39.11, 39.36, 39.55, 40.81, 43.81, 50.99, 101.81, 102.62, 122.96, 146.28, 171.66ppm; EIHRMS calc'd for C20H30O4 334.2144, found 334.2152. C) Aplysillolide A (1011 was purified by flash chromatography (10mm column, 15cm silica, step gradient 100% hexanes to 100% ethyl acetate) followed by repeated radial preparative TLC (1mm silica plate, step gradient 100% hexanes to 1:1 hexane/ethyl acetate) to yield 24.3mg (.002% of dry sponge weight) of a clear colourless glass; Compound 101: IR (film) vmax 3421, 1701 cm"1; EILRMS m/z (relative intensity) 288(M+-H20, 9), 182(89), 164(72), 136(81), 121(47), 107(28), 91(37), 83(43), 69(100), 55(69); *H NMR (400MHz, CDCI3) 8 0.88(s, 3H), 0.97(s, 3H), 1.13(s, 3H), 1.65(dd, J=2.4,6.8Hz, 3H), 2.18(dd, J=11.5, 16.6Hz, IH), 2.36(dd, J=5.5, 16.6Hz, IH), 2.88(m, IH), 3.04(m, IH), 3.11(s, IH), 3.54(dd, J=3.9. 8.7Hz, IH), 4.23(dd, J=8.7,6.4Hz, IH), 5.63(d, J=2.3Hz, IH), 5.80(dd, J=2.3,6.8Hz, lH)ppm; NMR (75MHz, CDCI3) 8 14.69(q), 18.94(t), 23.86(q), 27.35(q), 31.08(s), 35.37(q), 35.86(d), 37.41(t), 38.88(t), 40.62(s), 42.74(t), 49.07(t), 50.41(d), 62.47(d), 71.24(t), 102.60(d), 125.28(d), 132.07(s), 212.64(s)ppm; EIHRMS calc'd for C19H28O2 (M+-H20) 288.2090, found 288.2088. D) Aplysillolide B (1021 was purified by radial preparative TLC (1mm silica plate, step gradient 100% hexanes to 4:1 hexanes/ethyl acetate followed by radial preparative TLC (1mm silica plate, step gradient 100% hexanes to 100% diethyl ether) to give 15.6mg (.002% dry sponge weight) as colorless oil; Compound 102: IR (film) vmax 2928, 2868, 1700, 1462, 1338, 1365, 1231, 1097, 911cm-1; EILRMS m/z (relative intensity) 288 (M+,2), 164(100), 134(16), 121(9), 69(47); lH NMR (400MHz, CDC13) 8 0.88(s, 3H), 0.96(s, 3H), 1.12(s, 3H), 1.65(d, J=7.2Hz, 3H), 2.49(dd, J=13.6,l 1.9Hz, IH), 2.73(dd, J=13.3,6.3Hz, IH), 3.03(s, IH), 3.11(s, IH), 3.27(m, IH), 3.92(dd, J=9.1,10.9Hz, IH), 4.60(t, J=9.3Hz, IH), 5.75(q, 7.2Hz, IH), 6.33(d, J=2.4Hz, IH) ppm; EIHRMS m/z calc'd for C19H28O2 288.2089, found 288.2084. E) Glaciolide (1101 was purified by flash chromatography (20mm column, 15cm silica, step gradient 100% hexanes to 1:1 hexanes/ethyl acetate) followed by radial preparative TLC (1mm silica plate, step gradient 100% hexanes to 4:1 hexanes/ethyl acetate) to yield 32.6mg (.001% of dry weight sponge) as needles recrystallised from hexane/chloroform (9:1); Compound 110: mp 102-103 °C; IR (film) vmax 2947, 2921, 2867, 2853, 1776, 1682 cm"1; EILRMS m/z (relative intensity) 290 (M+, 21), 275(8), 247(3), 163(100), 162(17), 159(3), 150(8), 149(9), 147(33), 135(29), 133(10), 127(15), 123(29), 122(18), 121(39), 119(15), 109(21), 108(13), 107(50), 106(24), 105(19), 95(33), 91(27), 85(28), 77(17), 69(54), 55(42); *H NMR (400MHz, CDCI3) 8 0.91 (s, 3H), 0.93(s, 3H), 1.17(s, 3H), 1.23(s, 3H), 2.62(bt, J=7.8Hz, IH), 2.23(dd, J=7.8,5.3Hz, IH), 2.48(dd, J=12.0, 2.3Hz, IH), 4.15(dd, J=9.8,5.3Hz, IH), 4.26(d, J=9.8Hz, IH) ppm; *H NMR (400MHz, C6D6) d 0.62(s, 3H), 0.82(s, 3H), 1.10(s, 3H), 1.17(s, 3H), 1.33(t, J=), 3.44(dd, J=5.3,9.7Hz, IH), 3.74(d, J=9.7Hz, IH) ppm; 13C NMR (75MHz, CDCI3) 8 18.15(q), 18.52(q), 21.51(t), 22.66(t), 23.80(t), 27.9(q), 29.11(q), 29.65(q), 34.34(t), 35.23(s), 37.79(d), 41.38(s), 46.67(d), 46.74(t), 48.67(d), 67.94(t), 128.54(s), 145.18(s), 178.89(s) ppm; EIHRMS m/z calc'd for C19H30O2 290.2246, found 290.2248. F) Marginatone (112) was purified by radial preparative TLC (1mm silica plate, step gradient 100% hexanes to 4:1 hexanes/ethyl acetate) to yield 9.5mg (.001% of sponge dry weight) of a white solid; Compound 112: IR (film) Vmax 2925, 2866, 1680, 1440, 1387, 1262, 1046, 719, 644, 617 cm*1; EILRMS m/z (relative intensity) 300 (M+, 38), 285(26), 258(19), 243(9), 203(13), 201(12), 189(14), 187(11), 176(23), 175(14), 164(38), 163(83), 162(40), 161(84), 150(36), 149(82), 148(45), 147(100), 137(73), 136(14), 135(47), 133(19), 127(27), 121(29), 119(26), 109(72), 108(16), 107(19), 95(58), 93(24), 91(59), 83(17), 81(60), 79(36), 77(44), 69(77), 69(39), 65(17), 44(36); *H NMR (400MHz, CDC13) 8 0.86(s, 3H), 0.88(s, 3H), 0.99(s, 3H), 1.29(s, 3H), 1.63(m, IH), 2.28(dt, J=3.1,12.8Hz, IH), 1.90(dd, J=3.8,12.9Hz, IH), 2.46(dd, J=12.9,17.2Hz, IH), 2.54(dd, J=3.8,17.2Hz, lH),6.59(d, J=1.9Hz, IH), 7.26(d, J=2.0Hz, IH) ppm; *H NMR (400MHz, C6D6) 6 0.62(s, 3H), 0.70(s, 3H), 0.75(s, 3H), 0.95( s, 3H), 1.44(dd, J=3.1,13.5Hz, IH), 2.06(dt, J=3.1,6.8,12.2Hz, IH), 2.21(dd, J=13.6,16.9Hz, IH), 2.45(dd, J=3.1,16.9Hz, IH), 6.63(d, J=1.9Hz, IH), 6.78(d, J=1.9Hz, IH) ppm; 13c NMR (75MHz, CDCI3) 6 16.06(q), 17.94(f), 18.26(f), 20.52(q), 21.30(q), 33.24(q), 35.32(f), 35.3l(t), 37.42(s), 39.32(t), 41.83(t), 56.03(d), 56.48(d), 106.18(d), 118.18(s), 142.25(d), 161.73(s), 195.19(s) ppm; EDHRMS m/z calc'd for C20H28O2 300.2090, found 300.2093. G) Cadlinolide C (131) was purified by normal phase preparative HPLC (20:80 ethyl acetate/hexane, using a 15cm Whatman Partisil-10 analytical column, 0.8mL/min., refractive index detection, retention time 3.25min.) to yield 131 (13.2mg) as a clear colourless oil: Compound 131; DR (film) vmax 3381, 2948, 1737, 1451, 1208, 958, 754 cm-1; EILRMS m/z (relative intensity) 364(M+,1), 346(1), 332(4), 290(95), 203(35), 180(48), 119(47), 105(48), 88(79), 69(100), 55(70); *H NMR (400MHz, CDCI3) 8 0.86(s, 3H), 0.88(s, 3H), 1.07(s, 3H), 1.21(d, J=6.9Hz, 3H), 1.30(m, IH), 1.48(m, IH), 2.09(m, IH), 2.33(m, IH), 2.99(m, 2H), 3.71(s, 3H), 4.30(q, J=7.0Hz, IH), 5.4 l(d, J=3.5Hz, IH) ppm; 13c NMR (75MHz, CDCI3) 5 16.52(q), 19.77(t), 26.74(t), 27.50(t), 30.60(q), 31.58(s), 32.85(q), 32.86(q), 38.99(t), 39.88(t), 41.29(d), 41.52(d), 41.95(s), 45.75(d), 50.63(t), 52.17(q), 104.08(d), 127.38(s), 147.02(s), 175.34(s), 179.23(s) ppm; EIHRMS calc'd for C21H32O5 364.2250, found 364.2246. Cadlina luteomarginata Compounds H) Tetrahydroaplysulphurin-1 (72) was purified by flash chromatography (10mm column, 15cm silica, step gradient 100% hexanes to 1:1 hexanes/ethyl acetate) followed by radial preparative TLC (1mm thick silica plate, step gradient 100% hexanes to 1:1 hexanes/ethyl acetate) to yield 12.8mg (.009% of dry weight nudibranch) of a clear colourless oil. Compound 72: IR (film) vmax 2944, 1750, 1458, 1372, 1230, 995, 557 cm-1: MS (DCI-, NH3) m/z (relative intensity) 394(M++NH4+, 79), 334(100), 317(71), 288(16), 272(30), 225(7), 180(18), 163(45), 147(24), 109(12), 69(10); *H NMR (400MHz, CDCI3) 8 0.78(s, 3H), 0.91(s, 3H), 1.13(s, 3H), 1.28(m, IH), 1.42(d, J=7.4Hz, 3H), 1.90(m, IH), 2.08(s, 3H), 2.09(m, IH), 2.36(m, IH), 3.23(m, IH), 4.21(q, J=7.4Hz, IH), 6.00(d, J=6.2Hz, IH), 6.18(d, J=2.4Hz, IH) ppm; «c NMR (75MHz, CDCI3) 8 14.74(q), 20.73(t), 21.19(q), 23.99(t), 25.03(t), 28.28(q), 31.07(q), 31.58(s), 32.52(q), 38.05(d), 39.03(t), 39.51(t), 39.73(s), 40.63(d), 42.06(d), 50.88(t), 100.57(d), 102.71(d), 121.25(s), 146.48(s), 169.87(s), 170.94(s)ppm; HRMS calc'd for C22H32O5 376.2250, found 376.2248. Synthetic Derivatives: I) Reduction/Acetylation of Cadlinolide A (75) to give Compound 123: Cadlinolide A (75) (5mg, .015mmole) dissolved in diethyl ether (2mL) was added dropwise to a solution of lithium aluminum hydride (lOmg) in diethyl ether (3mL) and allowed to stirr at room temperature under an atmosphere of nitrogen. After .5h, ethyl acetate was added dropwise and allowed to stirr for .25h, .IN hydrochloric acid was added dropwise before the reaction mixture was poured onto water (lOmL) and extracted with chloroform (4x15mL). The combined organic extracts were dried over anhydrous sodium sulphate, filtered and evaporated in vacuo to yield a white solid (4.8mg) which was immediately acetylated by treatment with 1:1 Ac20/pyridine (3mL). The reaction was allowed to stirr ovenight at room temperature and then evaporated under vacuum. The residue (4.5mg) was shown to contain a single product by TLC and *H NMR analyses: oil; Compound 123: IR(film) vmax 2949, 2870, 1743, 1612, 1463, 1369, 1240, 1101, 1036,1009,923, 755, 603, 552 cm-1; EILRMS m/z (relative intensity) 346 (M+-C2H4C»2, 26), 331 (10), 286 (44), 271 (29), 201 (13), 176 (41), 161 (30), 105 (31), 91 (34), 69 (75), 43 (100) ; lH NMR (400MHz, CDC13) 6 0.85(s, 3H), 0.91(s, 3H), 1.12(s, 3H), 1.17(d, J=6.7Hz, 3H), 2.04(s, 3H), 2.09(s, 3H), 2.65(m, IH), 3.29(q, J=5.8Hz, IH), 3.62(dd, J=11.3,4.9Hz, IH), 3.69(dd, J=l 1.3,4.9Hz, IH), 3.81(dd, J=ll.l,7.6Hz, IH), 4.16(dd, J=ll.l,6.1Hz, IH), 5.66(d, J=9.0Hz, IH) ppm; HRMS calc'd for C22H34O3 (M+-C2H402) 346.2508, found 346.2511. J) Reduction and Acetylation of Aplysillolide A(101): Aplysillolide A (101) (14.5mg, .047mmol) was dissolved in dry diethyl ether (lmL) and added to a suspension of lithium aluminum hydride (15mg) in dry diethyl ether (2mL) at room temperature. After 0.5 h, the reaction was quenched by the addition of ethyl acetate (3mL) and 0.5N hydrochloric acid (2mL). The solution was extracted with ethyl acetate (4xl0mL) and dried over anhydrous sodium sulfate. Filtration and evaporation of solvent yielded a white solid (13.2mg) which was immediately dissolved in pyridine (lmL) and acetylated with acetic anhydride (2mL). After 14 h, excess pyridine and acetic anydride was evaporated in vacuo to yield triacetate 125 (10.5mg, .025mmol, 53%) as a clear colourless oil: Compound 125; IR (film) vmax 2947, 1741, 1444, 1369, 1235, 1034, 976, 605 cm-1; MS (DCI+, NH3) m/z (relative intensity) 454 (M++NH4+, 100); !H NMR (400MHz, CDCI3) 8 0.86(s, 3H), 0.98(s, 3H), 1.15(s, 3H), 1.42(m, IH), 1.65(dd, J=1.2,6.8Hz, 3H), 2.02(s, 3H), 2.05(s, 3H), 2.07(s, 3H), 2.17(m, IH), 2.36(dd, J=11.9,5.9Hz, IH), 2.71(d, J=6.3Hz, IH), 2.98(m, IH), 3.89(dd, 11.2,6.8Hz, IH), 4.00(dd, J=l 1.2,7.1Hz, IH), 4.21(dd, J=11.2,7.9Hz, IH), 4.30(dd, J=11.2,7.7Hz, IH), 5.15(m, IH), 5.32(q, J=6.7Hz, IH) ppm; EDHRMS calc'd for C23H36O4 (M+- CH3CO2H) 376.2613, found 376.2605. K) Reduction of Glaciolide (110) With Lithium Aluminum Hydride To Give Compound 127: A solution of glaciolide (1101 (6.8mg, 0.0234mmol) in dry diethyl ether (3mL) was added to a solution of lithium aluminum hydride (_15mg) in dry diethyl ether (5mL), and the mixture was allowed to stirr for .5h at room temperature. The excess reagent was destroyed by addition on ethyl acetate (lmL) followed by a dropwise addition of .IN hydrochloric acid. The reaction mixture was then poured over water (lOmL) and extracted with chloroform (4xl5mL). The combined organic layers were dried over anhydrous sodium sulphate and evaporated in vacuo to give a white solid which was purified by flash chromatography (5mm column, 15cm silica, step gradient 1:4 to 1:1 ethyl acetate/hexanes) to yield a white solid (4.9mg, 71%); Compound 127: DR(film) vmax 3354, 2945, 2362, 1771, 1455, 1022, 653, 542 cm-»; EILRMS m/z (relative intensity) 294(M+, 8), 276(10), 263(9), 233(16), 191(21), 189(19), 175(17), 167(18), 163(28), 162(13), 150(26), 149(44), 147(31), 135(33), 129(61), 123(84), 121(63), 109(63), 95(74), 81(62), 69(100), 55(80); lH NMR (400MHz, CDCI3) 8 0.92(s, 3H), 0.94(s, 3H), 1.16(s, 3H), 1.21(s, 3H), 1.24(m, IH), 1.48(t, J=l.lHz, 3H), 1.53(m, 2H), 1.66(m, 2H), 1.75(m, IH), 1.90(m, IH), 2.22(m, IH), 2.54(dd, J=2.6,11.9Hz, IH), 3.63(dd, J=6.9,9.8Hz, IH), 3.83(m, 2H), 3.93(dd, J=6.1,10.1Hz, IH) ppm; ^C NMR (75MHz, CDCI3) 8 18.61(q), 21.09(q), 22.65(f), 24.45(f), 29.04(q), 29.19(f), 29.41(q), 29.5l(q), 34.42(f), 37.14(s), 37.96(d), 41.40(s), 46.86(t), 50.80(d), 53.30(d), 60.89(t), 180 63.47(t), 129.23(s), 145.18(s) ppm; EDHRMS m/z calc'd for C19H34O2 294.2559, found 294.2564. L) Acetylation of Diol 127, To give Diacetate 128: Diol 127 (8.3mg, .028mmol) was treated with 2:1 Ac20/pyridine (2mL). The reaction mixture was allowed to stirr overnight at room temperature before being evaporated under vacuum. The resulting residue was purified by radial TLC (1mm thick silica plate, step gradient 100% hexanes to 1:1 hexanes/ethyl acetate) to yield a white solid (8.5mg, 80%) which was a single compound by TLC and lH NMR analyses; Compound 128: IR(film) vmax 2964, 1739, 1574, 1240,1032 cnr1; EILRMS m/z (relative intensity) 378(M+, 48), 318(100), 303(19), 268(2), 258(24), 243(30), 215(43), 189(36), 188(26), 187(23), 176(22), 175(47), 173(18), 164(10), 163(50), 162(60), 161(53), 159(20), 150(17), 149(29), 147(35), 135(42), 121(67), 107(50), 95(41), 81(33), 69(28): *H NMR (400MHz, C6D6) 8 0.70(s, 3H), .88(s, 3H), 1.13(s, 3H), 1.17(s, 3H), 1.22(m, IH), 1.38(brs, 3H), 1.47(m, IH), 1.49(m, 2H), 1.70(s, 3H), 1.76(s, 3H), 1.77(m, IH), 2.16(m, 2H), 2.35(m, IH), 2.48(dd, J=2.6,12.3Hz, IH), 4.20(dd, J=1.7,11.3Hz, IH), 4.24(m, 2H), 4.32(dd, J=4.9,11.3Hz, IH) ppm; EDHRMS m/z calc'd for C23H3804 378.2769, found 378.2765. M) Reaction Of Glaciolide (1101 With Ruthenium Tetroxide To Give Compound 129: Ruthenium tetroxide reagent was formed by treatment of ruthenium dioxide (.04g) in CCI4 (5mL) stirred at 0 °C in an erlenmeyer flask with sodium metaperiodate (0.32g) dissolved in water (5mL). The black oxide dissolved in about lh and the yellow CCI4 layer was separated, filtered and added to a stirring CCI4 (5mL) solution of glaciolide (110') (6.5mg, 0.0224mmol) at room temperature. The reaction mixture immediately turned black on addition of ruthenium tetroxide and was allowed to stirr for 2h before adding MeOH (lmL). Filtration of the reaction mixture through glass wool, evaporation in vacuo, followed by purification using preparative TLC on silica (1:1 hexanes/ethyl acetate) furnished compound 129 (4.5mg, 0.0214mmol, 95%) as a clear colourless oil; Compound 129: IR(film) vmax 2992, 2968, 2905, 2888, 1762, 1688, 1485, 1376, 1356, 1194, 1141, 950 cm"1; EILRMS m/z (relative intensity) 210(M+ 20), 195(12), 168(27), 167(12), 153(14), 126(18), 125(10), 123(13), 121(20), 111(14), 109(41), 107(18), 95(14), 93(18), 86(17), 83(30), 67(26), 55(22), 43(100); *H NMR (400MHz, CDC13) 6 0.93(s, 3H), 1.05(s, 3H), 1.60(m, IH), 1.67(m, 2H), 1.75(m, IH), 2.18(s, 3H), 2.22(m, IH), 2.33(dd, J=3.0,11.0Hz, IH), 2.61(dt, J=7.4,2.8Hz, IH), 4.17(dd, J=5.8,9.7Hz, IH), 4.36(dd, J=9.7,1.7Hz, IH) ppm; EIHRMS m/z calc'd for C12H18O3 210.1256, found 210.1254. N) Reaction Of Diacetate 128 With Ruthenium Tetroxide, To give Compound 130: Treatment of diacetate 128 (7.5mg, 0.0198mmol) dissolved in CCI4 (3mL) overnight with ruthenium tetroxide (5mL) (as described above) yielded compound 13 (5.8mg, 0.0161mmol, 81%) as a clear colourless oil; Compound 130: IR(film) vmax 2940, 2357, 1738, 1713, 1651, 1557, 1506, 1456, 1395, 1369, 1238, 1033, 653 cm-l; EILRMS m/z (relative intensity) 238(M+-AcOH, 7), 223(1), 195(4), 178(15), 163(8), 135(38), 120(21), 121(15), 107(15), 95(12), 93(28), 82(11); JH NMR (400MHz, CDCI3) 8 0.97(s, 3H), 1.10(s, 3H), 1.45(m, IH), 1.58(m, IH), 1.72(m, IH), 1.75(m, IH), 1.79(m, IH), 2.04(s, 3H), 2.17(s, 3H), 2.22(m, IH), 2.42(dd, J=10.3,3.8Hz, IH), 4.12(m, IH), 4.37(dd, J=11.7,4.5Hz, IH) ppm; EIHRMS m/z calc'd for C14H22O3 (M+- CH3CO2H) 238.1571, found 238.1570. 182 E. List of References I. Barnes, R.D. "Invertebrate Zoology", W.B.Saunders, Toronto, 1974, p.76. 2 . Bergquist, P.R. 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