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Novel secondary metabolites isolated from selected marine invertebrates Morris, Sandra Anne 1990

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NOVEL SECONDARY METABOLITES ISOLATED FROM SELECTED MARINE INVERTEBRATES by SANDRA ANNE MORRIS B. Sc. (Hons) University of British Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA November 1990 © Sandra Anne Morris, 1990 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 C M g / V / J 7"3P^ The University of British Columbia Vancouver, Canada Date Nov- S~ , DE-6 (2/88) Abstract A study of the secondary metabolism of two northeastern Pacific sponges and two Sri Lankan nudibranchs has led to the isolation of thirteen new and one previously known natural products. The structures of all of the compounds were determined by a combination of spectroscopic data analysis and chemical interconversions. A study of the chemistry of the northeastern Pacific sponge Hexadella sp. has resulted in the isolation of six new brominated alkaloids. Two of these, hexadellins A (77) and B (78), are derived from dibromotyrosine. The structures of compounds 77 and 78 were determined via their acetylated derivatives 79 and 80. Four compounds possessing novel bis(indole) structures have also been isolated. Topsentin B2 (74) was isolated as a mixture of two slowly interconverting tautomers, 74a and 74b. Methylation of 74 resulted in the production of trimethyltopsentin B2 (75); the structure of 75 was determined spectroscopically. The structures of dragmacidons A (81), B (82), and C (83) were determined by a combination of spectral data interpretation and chemical interconversions. Dragmacidon C (83) was originally incorrectly assigned as 88; the correct structure was determined based upon synthesis of the model compound 94. Compounds 77 and 78 possess antimicrobial activities. Compounds 74 and 81 show considerable cytotoxic and antineoplastic activities. Five new triterpene glycosides have been isolated from the northeastern Pacific sponge Xestospongia vanilla. The structures of isoxestovanin A (125), xestovanin C (127), dehydroxestovanin A (129), epi-dehydroxestovanin A (131), and dehydroxestovanin C (132) were all determined by a combination of spectral data interpretation and chemical interconversions. These compounds all contain the deoxy sugars L-rhamnose and D-fucose. Isoxestovanin A (125) possesses a new carbon skeleton and xestovanin C (127) and dehydroxestovanin C (132) possess linear i i trisaccharide fragments which have not been previously encountered in triterpene glycosides isolated fromX. vanilla. The Sri Lankan nudibranch Chromodoris glenei has yielded the known compound 12-desacetoxyshahamin C (153) and the new metabolite shahamin K (155). Both compounds possess dendrillane diterpene skeletons. The compound chromodorolide B (156) was isolated from specimens of Chromodoris cavae. It is only the second known diterpene natural product possessing the chromodorane skeleton. i i i Table of Contents Page Abstract ii Table of Contents iv List of Figures viii List of Schemes xv List of Tables xvi List of Abbreviations xix Acknowledgements : xxii Marine Natural Products Chemistry 1 Part I Marine Natural Products Isolated from Two Species Within the Phylum Porifera (Sponges') 10 A. Novel Marine Natural Products Isolated from the Northeastern Pacific Sponge Hexadella sp. 12 Introduction 12 1. Taxonomy of and General Information About Hexadella sp 12 2. The Taxonomy of Verongiid Sponges 16 3. Natural Products from the Verongida 18 Results and Discussion 35 1. Isolation of the Metabolites from Hexadella sp. Collected at-100 m to-200 m 35 2. Novel Metabolites from Hexadella sp. Collected at -100 m to-200 m 35 Topsentin B2 (74) 35 iv Page 3. Isolation of the Metabolites from Hexadella sp. Collected at -40 m 46 4. Novel Metabolites from Hexadella sp. Collected at -40 m.... 47 Hexadellin A (77) and Hexadellin B (78) 47 Diacetylhexadellin A (79) 48 Diacetylhexadellin B (80) 55 Dragmacidon A (81) 58 Dragmacidon B (82) 65 Dragmacidon C (83) 69 Conclusions 84 B. Novel Marine Natural Products Isolated from the Northeastern Pacific Sponge Xestospongia vanilla (de Laubenfels. 1930). 92 Introduction 92 1. Taxonomy of and General Information About Xestospongia vanilla 92 2. The Chemistry of Petrosiid Sponges 96 3. Triterpene Glycosides from the Phylum Porifera (Sponges). 98 Results and Discussion 101 1. Isolation of the Metabolites from the Bamfield Collected Xestospongia vanilla 101 2. Novel Metabolites from the Bamfield Collected Xestospongia vanilla 102 Isoxestovanin A (125) 102 Xestovanin C (127) 127 Dehydroxestovanin A (129) 146 V Page 3. Isolation of the Metabolites from the Queen Charlotte Islands Collected Xestospongia vanilla, 164 4. Novel Metabolites from the Queen Charlotte Islands Collected Xestospongia vanilla 165 Epi-dehydroxestovanin A (131) 165 Dehydroxestovanin C (132) 177 5. Attempts at Determining the Absolute Stereochemistries of the Chiral Centres in Xestovanin A (110) 187 Conclusions 190 Part I I Marine Natural Products Isolated from Two Species of Nudibranch Belonging to the Genus Chromodoris 194 Introduction 194 1. General : 194 2. The Chemistry of Chromodorid Nudibranchs: Spongian Diterpenes 194 Results and Discussion 201 1. Isolation of the Metabolites from the Nudibranch Chromodoris glenei 201 2. Novel Metabolites Isolated from the Nudibranch Chromodoris glenei 201 12-Desacetoxyshahamin C (153) 201 Shahamin K (155) 208 3. Isolation of the Metabolites from the Nudibranch Chromodoris cavae 213 Vi Page 4. Novel Metabolites Isolated from the Nudibranch Chromodoris cavae 213 Chromodorolide B (156) 213 Conclusions 229 Experimental 233 References 286 v i i List of Figures Page Figure 1: The phylogenetic classification of Hexadella sp. (according to Austin^6) 13 Figure 2: Hexadella sp. collection sites 14 Figure 3: Freshly collected Hexadella sp 15 Figure 4: *H nmr spectrum of topsentin B2 (74) (acetone-d6; 300 MHz) 37 Figure 5: *H nmr spectrum of trimethyltopsentin B2 (75) (acetone-d6; 300 MHz) 38 Figure 6: nmr spectrum of trimethyltopsentin B2 (75) (acetone-d6; 75 MHz) 39 Figure 7: Selected nOe enhancements in trimethyltopsentin B2 (75).... 41 Figure 8: Selected nOe difference spectra of trimethyltopsentin B2 (75) (acetone-d6; 400 MHz) 42 Figure 9: Selected mass spectral fragment ions of trimethyltopsentin B2 (75) 44 Figure 10: Selected nOe enhancements in trimethyltopsentin B2 (75).... 45 Figure 11: SINEPT results for trimethyltopsentin B2 (75) 45 Figure 12: *H nmr spectrum of diacetylhexadellin A (79) (CDCI3; 400 MHz) 49 Figure 13: nmr and APT spectra of diacetylhexadellin A (79) (CDCI3; 75 MHz) 50 Figure 14: *H nmr spectrum of diacetylhexadellin B (80) (CDCI3; 400 MHz) 56 v i i i Page Figure 15: 1 3 C nmr spectrum of diacetylhexadellin B (80) (CDCI3; 75 MHz) 57 Figure 16: *H nmr spectrum of dragmacidon A (81) (acetone-d6; 400 MHz) 59 Figure 17: nmr spectrum of dragmacidon A (81) (acetone-d6; 75 MHz) 60 Figure 18: Selected nOe enhancements in dragmacidon A (81) 62 Figure 19: Selected nOe difference spectra of dragmacidon A (81) (acetone-d6; 400 MHz) 63 Figure 20: *H nmr spectrum of dragmacidon B (82) (acetone-d6; 400 MHz) 66 Figure 21: nmr spectrum of dragmacidon B (82) (acetone-d6; 75 MHz) 67 Figure 22: *H nmr spectrum of dragmacidon C (83) (acetone-d6; 400 MHz) 71 Figure 23: nmr spectrum of dragmacidon C (83) (acetone-d6; 75 MHz) 73 Figure 24: Selected nOe difference spectra of dragmacidon C (83) (acetone-d6; 400 MHz) 74 Figure 25: An expansion of the FLOCK spectrum recorded for model compound 94 (DMSO-d6) 80 Figure 26: The phylogenetic classification of Xestospongia vanilla (according to Austin^) 93 Figure 27: A sample of Xestospongia vanilla collected from Bamfield, B. C 94 ix Page Figure 28: Bamfield collection sites for Xestospongia vanilla 95 Figure 29: Queen Charlotte Islands collection site for Xestospongia vanilla 95 Figure 30: ^ C nmr and APT spectra of isoxestovanin A (125) (acetone-d6; 75 MHz) 105 Figure 31: HETCOR spectrum of isoxestovanin A (125) (acetone-d6; /(CH) = 140Hz) 106 Figure 32: ^H nmr spectrum of isoxestovanin A hexaacetate (126) (CDCl3;400MHz) 109 Figure 33: *H nmr spectrum of isoxestovanin A (125) (DMSO-d6; 400 MHz) 113 Figure 34: *H nmr spectrum of isoxestovanin A (125) (acetone-d6; 400 MHz) 116 Figure 35: *H COSY 60 spectrum of isoxestovanin A (125) (acetone-d6; 400 MHz) 117 Figure 36: Substructures A and B (chemical shifts are from ! H nmr spectrum recorded in DMS 0-d6). 119 Figure 37: ROESY spectrum of isoxestovanin A (125) (acetone-d6; 500 MHz) 120 Figure 38: Substructure C (chemical shifts are from *H nmr spectrum recorded in acetone-d6) 121 Figure 39: Aldol condensations of secoxestovanin A (111) (or its C10 epimer) resulting, in the formation of xestovanin A (110) and isoxestovanin A (125) 122 Figure 40: Selected nOes for compound 125 123 X Page Figure 41: Selected nOes for compound 125 125 Figure 42: Conformations of the bicyclic ring systems in compounds 125 and 110 125 Figure 43: Selected nOes for the disaccharide portion of 125 127 Figure 44: nmr and APT spectra of xestovanin C (127) (acetone-d6; 75 MHz) 132 Figure 45: HETCOR spectrum of xestovanin C (127) (acetone-d6; 7(CH) = 140Hz) 133 Figure 46: *H nmr spectrum of xestovanin C octaacetate (128) (CDCI3; 400 MHz) 136 Figure 47: *H nmr spectrum of xestovanin C (127) (DMSO-d6; 400 MHz) 139 Figure 48: *H nmr spectrum of xestovanin C (127) (acetone-d6; 400 MHz) ., 142 Figure 49: *H COSY 60 spectrum of xestovanin C (127) (acetone-d6; 400 MHz) 143 Figure 50: *H COSY 60 spectrum of xestovanin C (125) (DMSO-d6; 400 MHz) 144 Figure 51: Trisaccharide portion of 127 146 Figure 52: ^H nmr spectrum of dehydroxestovanin A hexaacetate (130) (benzene-d6; 400 MHz) 150 Figure 53: nmr and APT spectra of dehydroxestovanin A (129) (acetone-d6; 75 MHz) 153 Figure 54: *H nmr spectrum of dehydroxestovanin A (129) (DMSO-d6; 400 MHz) 156 xi Page Figure 55: *H nmr spectrum of dehydroxestovanin A (129) (acetone-d6; 400 MHz) 159 Figure 56: *H COSY 60 spectrum of dehydroxestovanin A (129) (acetone-d6; 400 MHz) 160 Figure 57: *H COSY 45 spectrum of the aliphatic *H nmr region of dehydroxestovanin A (129) (acetone-d6; 400 MHz) 161 Figure 58: HETCOR spectrum of dehydroxestovanin A (129) (acetone-d6; / (CH) = 140 Hz) 162 Figure 59: *H nmr spectrum of epi-dehydroxestovanin A (131) (DMSO-d6; 400 MHz) 169 Figure 60: *H nmr spectrum of epi-dehydroxestovanin A (131) (acetone-d6; 400 MHz) 172 Figure 61: nmr and APT spectra of epi-dehydroxestovanin A (131) (acetone-d6; 75 MHz) 175 Figure 62: Selected nuclear Overhauser enhancements observed in 129 and 131 176 Figure 63: *H nmr spectrum of dehydroxestovanin C (132) (DMSO-d6; 400 MHz) 180 Figure 64: *H nmr spectrum of dehydroxestovanin C (132) (acetone-d6; 400 MHz) 183 Figure 65: l^C nmr and APT spectra of dehydroxestovanin C (132) (acetone-d6; 75 MHz) 186 Figure 66: A specimen of Chromodoris glenei (top of picture) 195 Figure 67: A specimen of Chromodoris cavae. 196 X i i Page Figure 68: *H nmr spectrum of 12-desacetoxyshahamin C (153) (CDCI3; 400 MHz) 204 Figure 69: nmr and APT spectra of 12-desacetoxyshahamin C (153) (CDCI3; 75 MHz). 205 Figure 70: ! H nmr spectrum of shahamin K (155) (CDCI3; 400 MHz).. 206 Figure 71: 1 3 C nmr and APT spectra of shahamin K (155) (CDCI3; 75 MHz) 207 Figure 72: Selected nOes observed for shahamin K (155) 212 Figure 73: nmr spectrum of chromodorolide B (156) (CDCI3; 125 MHz) 215 Figure 74: *H nmr spectrum of chromodorolide B (156) (CDCI3; 400 MHz) 219 Figure 75: *H nmr spectrum of chromodorolide B (156) (benzene-d6; 400 MHz) 220 Figure 76: *H COSY spectrum of chromodorolide B (156) (CDCI3; 400 MHz) 221 Figure 77: HMQC spectrum of chromodorolide B (156) (CDCI3; / (CH) = 140 Hz) 222 Figure 78: Selected *H COSY correlations for the protons belonging to the hydrocarbon portion of 156 223 Figure 79: The chromodorane skeleton present in both 154 and 156 224 Figure 80: Selected nOe difference spectra of chromodorolide B (156) (benzene-d6; 400 MHz) 226 Figure 81: Selected nOe difference spectra of chromodorolide B (156) (benzene-d6; 400 MHz) 227 xi i i Page Figure 82: Selected nOe difference spectra of chromodorolide B (156) (CDCI3; 400 MHz) 228 Figure 83: Selected nOes observed in chromodorolide B (156) 229 x i v List of Schemes Page Scheme 1: Hypothetical biogenetic scheme for 7-bromocavernicolenone (38) proposed by^ Pietra and co-workers0^ 25 Scheme 2: Preparation of model compound 94 81 Scheme 3: Fragmentation pathway to give the base peak in the mass spectrum of dragmacidon C (83) 83 Scheme 4: Proposed biogenesis of the xestovanins 193 Scheme 5: Proposed biogenesis of 12-desacetoxyshahamin C (153) and shahamin K (155) 231 Scheme 6: Proposed biogenesis of chromodorolides A (154) and B (156) 232 xv List of Tables Page Table 1: 1 3 C nmr (75 MHz) and *H nmr (300 MHz) assignments for trimethyltopsentin B2 (75) 43 Table 2: *H nmr (400 MHz) assignments for diacetylhexadellins A (79) and B (80), diacetylaerothionin (84),73 tetraacetyl-fistalarin (85)71 and diacetylpsammaplysin-A (86)74 53 Table 3: * 3 C nmr (75 MHz) assignments for diacetylhexadellins A (79) and B (80), diacetylaerothionin (84),73 and diacetyl-psammaplysin-A (86)74 54 Table 4: 1 3 C nmr (75 MHz) and !H nmr (400 MHz) assignments for dragmacidon A (81) 64 Table 5: 1 3 C nmr (75 MHz) and *H nmr (400 MHz) assignments for dragmacidon B (82) 68 Table 6: *H nmr data for dragmacidon C (83) (400 MHz) and 4, 5-dihydro-6"-deoxybromotopsentin (89) (360 MHz);93 nOe data is given for compound 83 70 Table 7: 1 3 C nmr data for dragmacidon C (83) (75 MHz) and 4,5-dihydro-6"-deoxybromotopsentin (89) (95.5 MHz)93 72 Table 8: 1 3 C nmr (75 MHz) and one-bond HETCOR (optimized for J (CH) = 140 Hz) data for isoxestovanin A (125) 103 Table 9: *H nmr (400 MHz) data for isoxestovanin A hexaacetate (126) 107 Table 10: *H nmr (400 MHz) data for isoxestovanin A (125) (DMSO-d6) I l l XVi Page Table 11: i H nmr (400 MHz) data for isoxestovanin A (125) (acetone-d6) 114 Table 12: 1 3C nmr (75 MHz) data for xestovanin C (127) and xestovanin A (110). One-bond HETCOR correlations (/ (CH) = 140 Hz) are given for 127 130 Table 13: iH nmr (400 MHz) data for xestovanin C octaacetate (128) 134 Table 14: iH nmr (400 MHz) data for xestovanin C (127) and xestovanin A (110) (DMSO-d6). lH COSY data is given for 127 137 Table 15: i H nmr (400 MHz) data for xestovanin C (127) and xestovanin A (110) (DMSO-d6). *H COSY and nOe data are given for 127 140 Table 16: lH nmr (400 MHz) data for dehydroxestovanin A hexaacetate (130) 148 Table 17: 1 3 C nmr (75 MHz) and one-bond HETCOR (optimized for / (CH) = 140 Hz) data for dehydroisoxestovanin A (129) 151 Table 18: i H nmr (400 MHz) data for dehydroxestovanin A (129) (DMSO-d6) 154 Table 19: ! H nmr (400 MHz) data for dehydroxestovanin A (129) (acetone-d6) 157 Table 20: i H nmr (400 MHz) data for epi-dehydroxestovanin A (131) (DMSO-d6) 167 Table 21: i H nmr (400 MHz) data for epi-dehydroxestovanin A (131) (acetone-d6) 170 xvi i Page Table 22: 1 3 C nmr (75 MHz) and one-bond HETCOR (optimized for / (CH) = 140 Hz) data for epi-dehydroisoxestovanin A (131) 173 Table 23: *H nmr (400 MHz) data for dehydroxestovanin C (132) (DMSO-d6) 178 Table 24: *H nmr (400 MHz) data for dehydroxestovanin C (132) (acetone-d6) 181 Table 25: 1 3 C nmr (75 MHz) and one-bond HETCOR (optimized for J (CH) = 140 Hz) data for dehydroisoxestovanin C (132) 184 Table 26: *H nmr (400 MHz) data for 12-desacetoxyshahamin C (153) and shahamin K (155) 202 Table 27: 1 3 C nmr (75 MHz) data for 12-desacetoxyshahamin C (153) and shahamin K (155) 203 Table 28: *H COSY correlations and nOe enhancements for the protons in shahamin K (155) 210 Table 29: 1 3 C nmr data for chromodorolide A (154) (75 MHz) and chromodorolide B (156) (125 MHz). HMQC correlations (/ (CH) = 140 Hz) are given for 156 214 Table 30: lU nmr (400 MHz) data fro chromodorolides A (154) and B (156). i H COSY and nOe data are given for 156 217 xvi i i List of Abbreviations Ac acetyl APT Attached Proton Test BIRD Bilinear Rotation Decoupling br broad c concentration in g/100 mL C O L O C Correlation spectroscopy via LOng range Couplings COSY Correlation SpectroscopY (homonuclear) d doublet ID one-dimensional 2D two-dimensional DMSO dimethyl sulfoxide dq doublet of quartets E D 5 0 effective dose resulting in 50% response EIMS electron impact mass spectrum EtOAc ethyl acetate FABMS fast atom bombardment mass spectrum F L O C K a 13c-lH s n ^ 1 correlation pulse sequence incorporating three BIRD sequences FT fourier transform HETCOR HETeronuclear CORrelation H M Q C Heteronuclear Multiple-Quantum Coherence HPLC high performance liquid chromatography HREIMS high resolution electron impact mass spectrum i signal due to an impurity xix ir infrared / scalar coupling constant LR COSY long range Correlation SpectroscopY LRCIMS low resolution chemical ionization mass spectrum LREIMS low resolution electron impact mass spectrum m multiplet AM difference in mass M + parent ion Me methyl MeOH methanol MIC minimum inhibitory concentration min minutes mmu millimass units mp melting point mult multiplicity m/z charge to mass ratio nmr . nuclear magnetic resonance no number nOe nuclear Overhauser enhancement ppm parts per million q quartet quint quintet rel. int relative intensity ROESY Rotating-frame Overhauser Enhancement SpectroscopY s singlet xx SINEPT Selective Insensitive Nuclei Enhanced by Polarization Transfer t triplet T/C test compared to control T H F tetrahydrofuran tic thin layer chromatography TMS tetramethylsilane uv ultraviolet w signal due to water xxi Acknowledgements Firstly, I would like to extend my sincerest gratitude to my supervisor Dr. Raymond Andersen for his guidance, constant encouragement, insightful "suggestions", and considerable patience throughout the course of my studies. It has been a pleasure to work with him. I would like to thank the staffs of the chemistry department nmr and mass spectrometry facilities for their services during the years; thanks are also extended Mike LeBlanc and the members of my research group, both past and present, for assistance in collecting the samples and for their friendship. I am also greatly indebted to Dr. E. Dilip de Silva for both collecting the Sri Lankan nudibranchs and isolating the diterpene metabolites. The contributions made by Hexadella sp., Xestospongia vanilla, Chromodoris glenei, and Chromodoris cavae to this work are appreciated; I hope their deaths were not in vain. I am thankful for financial support provided by NSERC and UBC during my years at UBC. Support provided (in many forms) by my parents during these same years is also gratefully acknowledged. Finally, I would like to thank Richard Tillyer for always being there for me during the past four years. xxii M a r i n e N a t u r a l Products C h e m i s t r y Ever since the discipline of organic chemistry began, the chemistry of natural products from terrestrial sources such as plants and fungi has been studied extensively; in contrast, the chemistry of marine species has received relatively little attention. The reasons for this are simple. Firstly, from the earliest of times man has made use of terrestrial natural products, albeit as crude plant extracts, for many different purposes, such as for medicines, poisons, and perfumes. 1 In the light of this, it seems quite natural for chemists to have directed their efforts towards the isolation and characterization of the active principles in these traditionally used extracts. Secondly, and perhaps more importantly from a practical viewpoint, terrestrial species have always been easily accessible with respect to collection, whereas marine organisms have not. However, the last two decades have witnessed an explosive growth in the field of marine natural products chemistry, attested by the large number of reviews and books which have recently been written on the subject.2-8 This has been due in large part to the breakdown of the barrier between man and the oceans with the advent of SCUBA and the development of manned submersibles capable of collecting marine species at very great depths. Taking into account that the world's oceans, in terms of known species and habitats, are the richest resource of metabolic products (excluding insects),^  and that marine plants and animals often bear little or no relation to terrestrial species, the potential for marine natural products chemistry to yield secondary metabolites with unique and unusual carbon skeletons and structural features and, perhaps, with new biological activities is very great indeed. The major focus of marine natural products chemistry over the years has been to isolate and elucidate the structures of secondary metabolites which show promising biological activities, such as antiviral, antibacterial, antifungal, or antineoplastic activities. 1 Many groups of researchers have carried out comprehensive studies which note the incidence of biological activities in the various marine phyla.9-10 Recently, Munro and co-workers have completed a programme involving an extensive collection of marine organisms from many locations and differing habitats around the New Zealand coastline and from latitudes as far South as Antartica to as far North as Western Samoa in tropical waters. The aim of this program was targeted specifically towards the isolation of potential pharmaceuticals. Their findings from this undertaking suggest that within the various marine phyla, the incidence of activity, although high in many individual species from each phyla, was uniformly high in species of Porifera (sponges). H This may be one of the reasons that within marine natural products chemistry, the chemistry of sponges has continually been investigated and has yielded a rich assortment of novel secondary metabolites. In fact, between the years 1977 and 1985, of the 1009 new natural products isolated from marine invertebrates, almost one half were isolated from sponges. 12 in any case, screening programs such as the one undertaken by Munro and co-workers represent the most efficient means of isolating new, biologically active compounds from the marine environment. A number of medicinally useful products have already been developed as a result of leads found from the biological screening of marine organisms. In the 1950's, Bergmann and co-workers isolated three arabinosides from the marine sponge Cryptotethya cryptaX^ One of these, spongouridine (1), provided the basis for the development of the drug ara C (2), commonly used in cancer chemotherapy. A closely related compound, ara A (9-(i-arabinofuranosyladenine) (3), was recognized as an antiviral agent in 1964 and has been used therapeutically against the human viral infection Herpes encephalitis since the late 1970's.l0(*) This compound had been synthesized in 1960 following the synthesis of ara A and was subsequently isolated from the Mediterranean gorgonian Eunicella cavolini in 2 1984.14 Two different types of marine algae, Digenea simplex and Centrocerus clavulatum, have yielded (-)-a-kainic acid (4), 15,16 fae parent member of the kainoids, a unique group of pyrrolidine dicarboxylic acids which have attracted considerable interest primarily owing to their pronounced neuroexcitant properties,!^  although some members of the group have also been developed into antihelminthics for intestinal worms. 18 Finally, the compound tetrodotoxin (5), originally isolated from puffer fishes of the family Tetraodontidae, and derivatives of it have found use as analgesics and muscle relaxants in patients with terrninal cancer. 19 3 Although only a few marine secondary metabolites have to date been developed into clinically useful drugs, many more show potential for such development based upon preliminary biological screenings. The didemnins, a series of depsipeptides first isolated by Rinehart and co-workers in 1981,20 were responsible for the strongly cytotoxic and antiviral activities observed in crude extracts of the tunicate Trididemnum sp.. Two of the didemnins, didemnins A (6) and B (7), have shown quite promising antiviral activities in in vivo testing on mice inoculated with a number of human viral strains. 10(i) As well, didemnin B has been found active against a variety of human neoplastic cell types. Significant activity was found against ovarian, breast, and renal carcinomas as well as mesohelioma and sarcoma.^ l Currently, didemnin B is in phase II of human cancer clinical trials; it is the first marine metabolite to enter human cancer trials in its native form. Another series of compounds, the bryostatins, isolated from the bryozoan Bugula neritina by Pettit and co-workers,22 have all shown impressive activity in the in vivo P388 mouse leukemia screen, and bryostatin 1 (8) has been the subject of successful preclinical cancer testing at the National Cancer Institute (U. S. A.). 10(0 Studies done on the Californian colonial tunicate Aplidium californicum yielded the first examples of hemiterpenoid derivatives from the marine environment.23 Two of these compounds, prenylhydroquinone (9) and 6-hydroxy-2,2-dimethylchromene (10), have shown significant antineoplastic activity in tests against the P388 cell line; as well, both have been found to provide protection against various mutagens, including ultraviolet light. Finally, Pettit and co-workers isolated a series of compounds, the dolastatins, from the Indian Ocean sea hare Dolabella auricularia?^ One of these, dolastatin 1, has proven in preliminary testing to be one of the most active known antineoplastic agents from any source. Unfortunately, the structure of dolastatin 1 has not as yet been unequivocally assigned. The above examples represent only a few of the many marine natural products reported in the literature which show promising biological activities; these compounds have 4 progressed quite far in biological testing, but there are many more which have as yet only reached the first stages of screening and may one day prove to be just as active as, if not more active than, the above mentioned compounds. 3S,4R,5S-l.oStat Q 2S,4S,-Hlp In addition to determining the biological activities of metabolites isolated from marine organisms, marine natural products chemistry is necessarily concerned with determining the structures of the isolated metabolites. Traditionally, the structure elucidation of natural products has relied heavily upon chemical degradations and interconversions to substantiate structures derived by spectroscopic analyses. This usually meant that only the major metabolites in any given organism were able to have structures assigned to them, since fairly large quantities of material are needed to do chemical modifications on natural products (especially when their structures are uncertain). The use of X-ray crystallography has allowed the structures of many minor metabolites to be determined, since crystals of X-ray quality can be grown from relatively small amounts of material. However, many natural substances do not form crystals and many others, including many of the more bioactive ones, are isolated in such small quantities that crystallization is not feasible. The rapid growth of the field of nuclear magnetic resonance (nmr) during the last decade has witnessed the development of a remarkable array of both one and two dimensional experiments which have provided a new means of determining the structures of very complex metabolites which are produced in very minor amounts. Taking into account the highly increased levels of sensitivity attainable with today's modern nmr probes, these new experimental techniques have allowed the structure elucidation of complex compounds on sub-microgram quantities with quite high levels of confidence. A recent example of this is the determination of the structure of ciguatoxin (11) by Yasumoto and co-workers.25 The structure of compound 11, 0.35 mg in total isolated from 4000 kg of the moray eel Gymnothorax javanicus, was determined using a combination of spectroscopic techniques only. Other remarkable marine metabolite structures which have relied heavily upon spectroscopic techniques for their determination are those of palytoxin (12)26 and swinholide A (13).27 Although X-ray crystal structure determination and synthetic substantiation of structures still offer the best definitive proof 6 of proposed structures, the advanced spectroscopic techniques available to today's marine natural products chemist have made the structure determination of small amounts of complex, non-ciystalline substances much easier than was ever imagined possible. 11 7 With comprehensive, effective biological screening programs in place, and with structure elucidation becoming more and more "routine", marine natural products chemists have been able to focus upon new questions regarding the marine environment which they study. Two of the areas which are receiving a good deal of attention are the fields of chemical ecology and chemotaxonomy. In turning their attentions towards chemical ecology, marine natural products chemists are joining marine biologists in asking the question, "Why do marine organisms produce the elaborate secondary metabolites which are being isolated?". It has long been thought that secondary metabolites, although often of great use to man, were non-essential to the organism producing them;* however, recent studies in the marine environment have shown that many of these compounds do serve a variety of functions for their producing organisms. For example, certain marine molluscs of the order Nudibranchia which are are soft-bodied and physically unprotected have been found to sequester chemicals from their diet and use these as chemical defense agents 8 against their predators.28 Other marine metabolites have been found to act as stimulants of feeding behaviour in marine fish and crustaceans, chemical communication mediators, antifouling agents, and growth control factors.29 it is likely that as the chemistry of marine organisms is studied further, many of the metabolites which have already been or have yet to be isolated will also be found to play specific roles in their natural environment. Marine natural products chemistry has also helped to solve some of the problems faced by marine taxonomists. The taxonomy of marine organisms is not very easily definable, especially for organisms such as sponges which have few morphological characters to aid description. When it was demonstrated that organisms such as sponges were rich in novel secondary metabolites, many taxonomists recognized that the occurrence and relationships of these compounds could provide new information useful in classification. Bergquist has provided some very good examples of this by her redefinition of the classification of a number of orders within the class Demospongiae based upon the chemical constituents of the sponges within the various orders.3^ The continuing interaction of chemists and marine biologists and ecologists represents a trend which can only serve to strengthen both fields. The work which will be presented in this thesis describes the isolation and structure elucidation of thirteen new marine natural products. These metabolites have been isolated from two species of northeastern Pacific sponge and two species of Sri Lankan nudibranch. Some of these compounds were isolated as a result of there being significant levels of biological activity observed in the crude extracts of which they were a part; others were isolated in order to study further the chemistry of organisms previously worked on in this laboratory. Conclusions on how well these compounds fit into the already known chemistry of the organisms from which they were isolated will be made; the biological activities and possible biosynthetic origins of the compounds will also be presented. 9 Part I Marine Natural Products Isolated from Two Species Within the Phylum Porifera (Sponges) Members of the phylum Porifera, more commonly referred to as sponges, constitute what is believed to be the most primitive invertebrate group. These sessile organisms display so few of the characteristics normally attributable to animals that they were classified as plants up until the year 1825.31 Due to their general lack of mobility, sponges have faced a natural selective pressure to evolve defenses which thwart predation, encrustation, and encroachment by other organisms. Some of these defenses are outwardly visible; bright colouration and sharp spicules are two of the defenses which are possessed by certain species of sponge.32 However, some sponge species appear to have no outward defenses and yet they grow openly in areas surrounded by many possible predators and seem not to be harmed by any. Thus, it may be that sponges produce chemicals which act as defense agents against predation. When one considers the wide assortment of novel secondary metabolites that have been isolated from sponges in the last two decades,2-8,12 ^ is not unimaginable to think that many of these have been produced by the various sponges for specific purposes, such as for defense. Species of the order Nudibranchia, the shell-less marine molluscs, are known to feed on sponges, and it has been demonstrated that they selectively accumulate chemical defense agents from their diets. Indeed, many metabolites have been isolated from both the nudibranch and the sponge partner within predator-prey pairs/* Many of these shared metabolites have been found to possess either fish antifeedant or icthyotoxic activity. 28(f),(g),33 The presence of these compounds both in sponges and in the nudibranchs which feed on sponges lends support to the claim that sponges employ chemical defense as a means of detering predation. 1 0 The identification and classification of sponges is very difficult due to their primitive nature. The simple body plan and indeterminate growth pattern of sponges allow them to vary their sizes and shapes in response to various environmental factors, thus making identification based upon external morphology impossible. Therefore, taxonomists must rely on other factors, such as embryology, histology, skeletal characteristics, and biochemistry to classify sponges. Of the four factors mentioned, the nature of the skeleton is the one which is most crucial to the ultimate identification of a particular species.34 Four well-defined classes exist within the phylum Porifera. These classes are defined mainly by the nature of the sponge's inorganic skeletal material as well as the organization of the skeletal components. The class known as Hexactinellida (the glass sponges) is made up of sponges possessing skeletons formed entirely from siliceous spicules, often fused together. The skeletons of these sponges often account for over 90% of the organism's mass. The sponges comprising the class Calcarea have skeletons formed from crystalline calcium carbonate. These sponges are the smallest of all sponges and are found in the shallow waters of all of the world's oceans. The class Sclerospongiae is made up of sponges which have a massive calcareous skeleton encased in a soft tissue of collagen and siliceous spicules. There are no members of this class in the waters of the northeastern Pacific. The last class of sponge, the Demospongiae, is comprised of those sponges with skeletons formed from siliceous spicules and fibres of collagen. In some groups, the fibrous skeleton displaces the mineral skeleton completely. Ninety-five percent of all species of sponge belong to this class.34,35 The two species of sponge which were studied in this work, Hexadella sp. and Xestospongia vanilla, are members of the class Demospongiae. Both were collected in the cold temperate waters of the northeastern Pacific. The chemistry of each species will be dealt with separately. 1 1 A. Novel Marine Natural Products Isolated from the Northeastern Pacific Sponge Hexadella SD. Introduction 1. Taxonomy of and General Information about Hexadella sp. The sponge Hexadella sp. belongs to the order of sponges known as the Verongida (following the classification scheme of Austin), an order within the general class Demospongiae (see Figure 1).36,37 There has only been one species oi Hexadella identified in the waters of the northeastern Pacific and that is the species whose chemistry is described in this work. Two collections of Hexadella sp. were made for this study, both in the vicinity of Jervis Inlet, B. C. (see Figure 2). The first collection was made with the aid of the manned submersible PICES IV in an area of Jervis Inlet known as Dark Cove. The sponge specimens collected using the submersible were found growing at depths ranging from one hundred to two hundred meters below sea level. The second collection of Hexadella sp. was made with SCUBA in an area of Jervis Inlet referred to as Agamemnon Channel. The specimens collected at this location were found growing at depths of forty meters below sea level and greater. Hexadella sp. is always found growing on the skeletons of the dead hexactinellid glass sponges Aphrocallistes vastus and Chonelasma calyx, which are themselves found attached to the exposed faces of steep, rock walls at depths greater than forty meters below sea level. In its natural environment, Hexadella sp. is bright sulphur yellow in colour (see Figure 3). Upon death (for example, by immersion in methanol) or damage, the tissue undergoes a rapid colour change to dark purple, almost black. This colour change, due to 1 2 Kingdom: Metazoa (multi-cellular animals) Phylum: Porifera (sponges) Class: Hexactinellida Calcarea Demospongiae Sclerospongiae / Subclass: Homoscleromorpha Ceractinomorpha Tetractinomorpha / Order: Halichondria Haplosclerida Verongida Petrosiida Dictyoceratida Dendroceratida Poecilosclerida Family: Aplysinellidae Verongidae (Aplysinidae) Ianthellidae Genus: Aplysina (Verongia) Hexadella Verongula / Species: U n d e t e r m i n e d Figure 1 The phylogenetic classification of Hexadella sp. (according to Austin36) 13 14 Figure 3 Freshly collected Hexadella sp. the presence of the zoochrome 14 (a yellow compound which oxidizes readily in air to give the unstable blue quinone 15, which in turn decomposes to an insoluble black substance) in these sponges,3** is characteristic of the Verongida.3539 OH O O O 14 15 The initial incentive to begin studies on the chemistry of Hexadella sp. came when routine bioassays indicated significant in vitro cytotoxic (against the L1210 mouse leukemia cell line) and antimicrobial (against Staphylococcus aureus and Bacillus subtilis) activities in the crude methanolic extracts of sponge specimens collected from both locations. As well, to the best of our knowledge, there had been no previous investigations of the chemistry of any Hexadella species, and only a limited number of deep water sponge species had previously been examined4^ (although more have been examined since the initiation of our work on Hexadella sp.^l). Thus, Hexadella sp. appeared to be an ideal candidate for study. 2. The Taxonomy of Verongiid Sponges The recognition of the Verongida as a distinct order within the class Demospongiae (subclass Ceractinomorpha) is a relatively recent development.32»35,39 pri o r to 1978, the year that Bergquist grouped together the families Verongidae (Aplysinidae), Aplysinellidae, and Ianthellidae and placed them in a new order called the Verongida, these 16 families, along with those in the orders Dictyoceratida and Dendroceratida, had been placed together in a single order, the Keratosa, by many sponge taxonomists. The basis of the classification was that all of the genera included in this single order lacked mineral skeletons; they represented those sponges within the class Demospongiae which were composed of fibrous skeletons only. However, there were a large number of significant differences between the many families (and genera) included in the single order Keratosa, and many other sponge taxonomists disagreed that they should all be grouped together in this manner. Rather than establish a single order to contain all of the families (and genera) of sponges lacking mineral skeletons, a number of taxonomists instead divided the various families into two orders, the Dictyoceratida and the Dendroceratida, a division based largely upon the form of the fibrous skeleton possessed by all of the member sponges.32>35 Although this classification was more generally accepted than that which maintained the existence of a single order known as the Keratosa, it still did not, in the opinions of many sponge taxonomists, place the verongiid sponges in a phylogenetically acceptable position. Within the dual classification scheme, the verongiid sponges were placed together as a family in the order Dictyoceratida. There were, however, many factors which suggested that these sponges should be separated from typical dictyoceratid sponges. Biologically, the most significant factors supporting the separation were the distinctly different and complex histology of all verongiid sponges, the distinct pigmentation of most verongiids (in particular, the noticeable, rapid colour change from yellow to purple-black upon death or tissue damage), the distinct fibre construction of all verongiids, and their apparent oviparious reproduction as opposed to the viviparity seen in the Dictyoceratida.35 Although these biological factors constituted quite convincing evidence for the need to separate the verongiid sponges from the order Dictyoceratida, it was not until the extra 1 7 evidence provided by marine natural products chemists was presented and examined that Bergquist formally created a new order, the Verongida, which was distinct and separate from both the Dictyoceratida and the Dendroceratida.3^ 3. Natural Products from the Verongida During the period between 1965 and 1978, the field of marine natural products chemistry was experiencing a very rapid growth. A significant number of compounds isolated during this period came from the various genera of sponges which comprise the three families within the now recognized order Verongida (but were at the time considered to be a part of a single family within the Dictyoceratida). It became apparent that the types of compounds present in the verongiid sponges were very similar to one another, but were markedly different from the metabolites which were at the same time being discovered in other families of sponges belonging to the orders Dictyoceratida and Dendroceratida.3^ A number of very comprehensive studies were undertaken to compile the information obtained from marine natural products chemists and relate this to the more biological findings of sponge taxonomists in order to draw some definitive conclusions about the classification of the verongiid sponges with respect to the other dictyoceratids (ultimately resulting in the raising of the Verongida to the ordinal level).7,30(a),42-45 results of these studies showed that the chemistry of the verongiids is quite distinctive. It was found that these sponges are marked by a high quantity of sterols and a complete lack of terpenes (unlike the sponges in the orders Dictyoceratida and Dendroceratida, which are characterized by their unique terpene classes). In addition, it was also recognized that verongiid sponges all possess a unique class of brominated metabolite derived from tyrosine. In this introduction to the chemistry of the verongiids, no review of the sterol chemistry of these sponges will be presented (this subject is dealt with in a number of 18 reviews'» 30(a),43). A review of the characteristic bromotyrosine-derived metabolites will, however, now be presented.-The first bromotyrosine-derived metabolite obtained from a verongiid sponge was isolated as a result of the discovery of antibacterial activity in the methanolic extracts of the sponge Aplysina (Verongia) cauliformisA6A7 The compound which was responsible for the observed activity was assigned structure 16 based upon spectral analysis and synthesis. A subsequent investigation of the antibacterial methanolic extract of Aplysina (Verongia) fistularis by the same researchers revealed the presence of both the dienone 16 and the dimethoxyketal derivative of 16, compound 17.48 Compound 17, inactive in antibacterial testing, was deduced to be a true metabolite of the sponge and not an artifact produced during the extraction procedure based upon the failure to convert 16 into 17 by reaction with methanol under various conditions. O 16 17 Following the reports of the isolation of compounds 16 and 17 by Sharma et al., Andersen and Faulkner reported that ethanolic extracts of an unidentified species of Aplysina {Verongia) yielded the dienone 16 and the mixed ketal 18, which was shown to be a mixture of diasteriomers.4^ Isolation of this compound suggested that the ketal was not a natural product and led" the authors to propose that the dienone 16, the dimethoxyketal 17, and the mixed ketal 18 may all be derived from a single intermediate, 19 such as the arene oxide 19, by 1,4-addition of water, methanol, or ethanol during the extraction process. CH3CH2q Br^ rOCH3 .Br OCH3 Br Br CONH2 CONH2 18 19 The nitrile, aeroplysinin-1 (20a, 20b), has been isolated from species of both Aptysina (Verongia) and Pseudoceratina (family Aplysinellidae; originally misassigned in the literature as Ianthella ardis).^'^ This antibacterial metabolite was first isolated as the dextrorotatory isomer 20a by Fattorusso et al.; subsequendy, Fulmor and co-workers isolated this same compound as the laevorotatory isomer 20b. The absolute configurations of both isomers have been confirmed by X-ray studies.^ 3 The occurrence of both enantiomers in different genera of sponges within the same order is very unusual. Although aeroplysinin-1 was first reported as an antimicrobial agent, it has more recently been found to have in vivo antileukemic activity against certain mouse cell lines (only the (+)-enantiomer was used in the study). It has also been shown to be active against human mamma carcinoma cells and human colon carcinoma cells in vitro. Since aeroplysinin-1 has not as yet been found to be either a mutagen or a premutagen in test systems, it represents a very promising lead in the search for new anti-cancer agents.54 20 20a 20b Two more recently isolated compounds have been found to possess structures similar to that of aeroplysinin-1 (20a, 20b). These two dibromo nitriles, 21 and 22, were isolated from the sponge Aplysina (Verongia) laevis by Capon and MacLeod.^ 5 The compounds are reported to possess antimicrobial activity. O O The metabolite aeroplysinin-2 (23) has been isolated as both an optically active compound and a racemate from the sponges Aplysina (Verongia) aerophoba and Ianthella sp. respectively.56 In 1977, Chang and Weinheimer reported the amide 24 to be a metabolite of the sponge Psammaplysilla purpurea-^ This compound is very similar in structure to 25, a metabolite which had previously been isolated from Aplysina (Verongia) aurea by Rinehart and co-workers.^ 8 Compound 25 was shown to inhibit the growth of 21 the bacteria B. subtilis, E. coli, and P. atrovenetum. Structurally, 25 is quite unusual in that it is a skeletally rearranged dibromotyrosine-derived metabolite. A number of other metabolites which incorporate a single bromotyrosine moiety into their structures have also been reported. The sponge Aplysina (Verongia) cavernicola was found to contain the metabolites cavernicolin-1 (26) and cavernicolin-2 (27) in a 3:1 epimeric mixture.59 Both cavernicolins may be formally viewed as products of intramolecular cyclization of the dienone 16 with which they co-occur in A. cavernicola. Borders et al. have found the sponge Aplysina (Verongia) lacunosa to contain the metabolite 28.60 This compound was isolated in an attempt to find the metabolite(s) responsible for the antibacterial and antifungal activity observed in the crude extracts of the sponge; however, pure 28 did not show any significant activity. The dibromophenethylammonium salt 29, isolated from Aplysina (Verongia) fistularis,^ was studied in detail and found to produce pharmacological effects similar to epinephrine and acetylcholine. Aplysamine-1 (30), a tertiary amine salt similar in structure to 29, has recently been isolated from an unidentified Australian Aplysina (Verongia) species.62 No biological activity was noted for this compound. 22 Up until this point, the metabolites described have all contained two bromine atoms, suggesting that verongiid sponges produce secondary metabolites derived from 3, 5-dibromotyrosine. There have, however, been a number of metabolites isolated from the Verongida which have had one of the bromine atoms replaced by another atom or group, such as a hydrogen atom, a chlorine atom, or a hydroxyl group. These compounds (incorporating a single bromotyrosine moiety into their structures) have all been isolated by Pietra and co-workers from the sponge Aplysina (Verongia) cavernicola.6^-65 Two compounds similar to the dienone 16, (+)-3-bromoverongiaquinol (31) and (±)-3-bromo-5-chloroverongiaquinol (32), were shown to possess considerable antibacterial activity. 23 These compounds were isolated as racemates along with the formal cyclization products of 32, the C(7)-epimeric pair of 7P-bromo-5-chlorocavernicolin (33) and 7a-bromo-5-chlorocavernicolin (34), and the C(7)-epimeric pair of 5-bromo-7P-cholorcavernicolin (35) and 5-bromo-7a-chlorocavernicolin (36). Structures 31 and 32 were confirmed by synthesis of the racemic compounds. A similar compound to both 35 and 36, monobromocavernicolin (37), had previously been isolated and had been found to occur not as a racemate, but as a mixture which was 6% enriched in the (+)-enantiomer. This represents the first example of a marine natural product occurring naturally in a low enantiomerically pure state. The last of these compounds to be isolated, 7-bromocavernicolenone (38), was isolated as a racemate. Descriptively, the compound consists of a bicyclo [3.3.1] system, which has no precedent in previously known products of the Verongida. 33 34 35 36 24 The racemic nature of 38 led the authors to suggest a phenol oxidative biogenetic pathway for this compound (see Scheme 1). If 5-bromodopa (39) is taken to be the starting material from which 38 is formed, then oxidative spirocyclization of 39 to 40, followed by, in turn, hydrolysis of the y-lactone, conjugative addition of the amino group, and, finally, oxidative removal of the carboxyl group, would yield the desired product 38. This mechanism allows formation of a 1:1 diastereomeric mixture of intermediates (40) which may account for the racemic nature of 38. OH O 38 39 40 Scheme 1 Hypothetical biogenetic scheme for 7-bromocavernicolenone (38) proposed by Pietra and co-workers65 In addition to producing fairly simple secondary metabolites whose structures are derived from a single brominated tyrosine moiety, the verongiid sponges have been observed to produce a number of products resulting from the condensation of these 25 bromotyrosine units with either themselves or with other small molecules. The first of these compounds to be described were aerothionin (41) and homoaerothionin (42).66-68 These two compounds, isolated from the sponges Aplysina (Verongia) aerophoba and Aplysina {Verongia) thiona by Minale and co-workers, are interesting metabolites due to the presence of the unusual spirocyclohexadienyldihydroisoxazole functionality within their structures. The structures of both compounds were determined based upon spectral analysis and synthesis; the purification and structure elucidation of 42 was done on its diacetate. The structure and absolute configuration of aerothionin has since been deterrnined unequivocally by X-ray diffraction analysis.69 The conversion of a presumed monomelic precursor (such as 16 or 20a/20b) to these compounds likely involves an addition to the amide carbonyl group with concomitant productions of the spiroisoxazole ring. A quite recent publication has reported the isolation and structure elucidation of a dihydroxy derivative of aerothionin, compound 43, from the sponge Verongula rigidaJ® 26 The isomeric compounds fistularin-1 (44) and fistularin-2 (45), isolated from the sponge Aplysina (Verongia) fistularisPare similarly formed from two bromotyrosine moieties; however, only one of these groups has been transformed into a spirocyclohexadienyldihydroisoxazole structure. The two metabolites, fistularin-3 (46) and isofistularin-3 (47), isolated from Aplysina (Verongia) fistularis and Aplysina (Verongia) aerophoba respectively,71»7^ are composed of three dibromotyrosine units. The compounds 46 and 47 are known to be isomeric (epimeric), the difference between the two compounds lying in the stereochemistry of one (or more) of the chiral centres. As yet, it is not known which of the six chiral centres are involved. Two other compounds were reported to have been isolated from Aplysina (Verongia) aerophoba along with isofistularin-3.7^ These compounds, aerophobin-1 (48) and aerophobin-2 (49), incorporate an imidazole ring into their structures. 27 2 8 The sponge Psammaplysilla purpurea was found to contain two metabolites that were first misidentified73 and later accurately assigned structures 50 and 51 on the basis of X-ray diffraction analysis.7^ The two compounds, psammaplysin-A (50) and psammaplysin-B (51), exhibited in vitro antimicrobial activity. The biogenesis of these two compounds is thought to proceed through the oximino epoxide 52. Nakamura and co-workers have isolated four physiologically active dibromotyrosine metabolites from the sponge Psammaplysilla purea .75/76 The major compound, purealin (53), inhibited Na, K-ATPase and myosin Ca-ATPase, while it activated K,EDTA-ATPase.75 The three minor metabolites, lipopurealins-A, -B, and -C (54, 55, 56), were all found to inhibit Na, K-ATPase, lipopurealin-B being the most potent inhibitor.7^ 29 Two publications have reported the occurrence of bromotyrosine-derived metabolites containing disulfide moieties in unidentified verongiid sponges. Arabshahi and Schmitz reported the isolation and structure elucidation of two such compounds, 57 and 58, in 1987.^ 7 Compound 58 is stereoisomeric with compound 57 at one of the oxime centres. In the same year, Scheuer and co-workers isolated psammaplin-A (59), a compound with the same gross structure as both 57 and 58.78 This compound could possibly be identical to either 57 or 58; however, no definition of the stereochemistry at either of the oxime centres in 59 was made, and since both groups reported the nmr data for their compounds in different solvents, no conclusive comparison of these compounds can be made on the basis of the reported spectral data. Scheuer and co-workers also 30 reported the isolation of bisaprasin (60) in the same paper.7 8 This compound is a dimer of 59. Both 59 and 60 displayed antibacterial activity. 57 (E,E) 58 (E,Z) The compound aplysamine-2 (61) has been reported to be a metabolite of an unidentified Australian species of Aplysina (Verongia).^ This tertiary amine salt possesses structural features common to both aplysamine-1 (30) and compounds 57-60. 31 A unique series of dibromotyrosine-derived metabolites, the bastadins, have been isolated from the sponge Ianthella bastaP^&O The first of these compounds to be isolated were bastadins 1-7 (62-68). These metabolites were all found to originate from four bromotyrosine units. Bastadin-1 (62), bastadin-2 (63), and bastadin-3 (64) are derived from a single phenol oxidative coupling whilst the macrocyclic compounds bastadin-4 (65), bastadin-5 (66), bastadin-6 (67), and bastadin-7 (68) are formed from two phenol oxidative coupling reactions. Metabolites 63-68 showed potent in vitro antimicrobial activity against Gram positive organisms. A more recent study of the chemistry of the sponge Ianthella basta has revealed the presence of five new bastadins.81 These compounds, bastadins 8-13 (69-73), are structurally very similar to the original bastadins and have been found to have in vitro cytotoxic activity; this represents the first observation of cytotoxicity in this family of metabolites. 32 62 R = H 64 R = H 63 R = Br 66 R = H 65 R = Br 67 R = Br 68 R = H Ri u NOH 69 R = R 2 = Br,R 3 = H,R, = OH 70 R = H, Rj = OH, R 2 = R3 = Br 71 R = R 3 = Br,R 1 = R 2 = H 72 R = R 1 = R 2 = H, R 3=Br 73 R = R 3 = H, Rj = OH, R 2 = Br NOH 3 3 The occurrence of brominated alkaloids in marine organisms is not uncommon. However, when dealing with sponge metabolites, one can never be completely certain if the compounds isolated are actually true sponge metabolites or are produced instead by symbiotic microorganisms. Since sponges are filter feeding animals, it is conceivable that they could accumulate a high percentage of symbiots which could in turn be responsible for the production of the metabolites which are extracted from the sponge mass. In fact, it has been documented that some sponges consist of as much as 50% by weight of symbiotic bacteria and blue-green algae.5 The origins of the verongiid bromotyrosine metabolites have, therefore, been the subject of investigation. In one study, X-ray microanalysis was used to localize two brominated metabolites, aerothionin (41) and homoaerothionin (42), in the tissues of their producing sponge, Aplysina (Verongia) fistularis.%2 It was shown that these compounds are indeed produced in sponge cells and not by symbiotic microorganisms. Thus, it appears that these metabolites are true sponge-produced natural products. When we began our study of the chemistry of Hexadella sp., it was assumed that the majority of the metabolites which would be isolated would be related to the verongiid bromotyrosine metabolites discussed above. However, our investigation provided quite unexpected results, as will now be presented. 3 4 Results and Discussion 1. Isolation of the Metabolites from Hexadella sp. Collected at -100 to -200 m Hexadella sp. was collected at Dark Cove, Jervis Inlet, in November 1986 using the manned submersible PICES IV (-100 to -200 m). The freshly collected sponge (500 g, wet weight) was immersed in methanol immediately after collection. Concentration of the methanol extract under reduced pressure yielded 6 g of crude residue. A 400 mg portion of the crude residue was chromatographed on a Sephadex LH-20 column prepared and eluted with methanol/dichloromethane (7:3). Fractions containing a bright yellow, UV absorbing tic spot (Rf = 0.43 on silica; 4:1 EtOAc/hexane) were further purified via reversed phase HPLC (methanol/water; 3:1) to give 71 mg (1.8% of crude residue) of pure topsentin B2 (74). 2. Novel Metabolites from Hexadella sp. Collected at -100 to -200 m  Topsentin B2 (74) a H b H 74 Pure topsentin B2 (74) was isolated as a yellow/green solid. The LREIMS of this compound showed an intense molecular ion doublet (50:50) at m/z 420/422 daltons. High resolution measurements on this doublet provided a molecular formula of C2()Hl3BrN402 (AM, +0.2/-0.6 mmu) for the compound, a formula requiring sixteen sites of unsaturation. The high degree of unsaturation obtained from the mass spectral data, in addition to the intense absorbances in the uv spectrum (MeOH, X . m ax at 235 (sh), 260 (sh), 286 (e 17 000), 382 (e 15 500) nm) and the very weak C - H stretching vibrations in the ir spectrum, indicated that the compound possessed an aromatic skeleton. The absence of signals between 0 and 6.5 ppm in the *H nmr spectrum of 74 (see Figure 4) substantiated the aromatic nature of the compound. Although the behaviour of topsentin B2, both on tic and hplc, suggested that it was a pure compound, its *H nmr spectrum, recorded in acetone-dg, showed signals attributable to a mixture of two closely related compounds in a ratio of 2:1. Four of the peaks (amounting to eight protons) disappeared upon addition of D 2 O , indicating that there were four exchangeable protons in each of the two derivatives. Given the fact that the compound was aromatic in nature, possessed a number of exchangeable protons, and had a number of heteroatoms contained within its structure, it was thought that the two closely related compounds might be two slowly interconverting tautomers. It was necessary, then, to find a way to stop the tautomerization so that the structure of topsentin B2 could be defined. It was hoped that the two tautomeric forms of 74 could be trapped by derivatizing the compound. To this end, a 40 mg sample of topsentin B2 was methylated with dimethyl sulphate and potassium carbonate in refluxing acetone. 83 A tic analysis of the crude reaction mixture revealed the presence of two major components, neither of them starting material. Separation of the two components was carried out using reversed phase preparative tic (MeOH/CH3CN/H20; 82:10:8). The major compound was found to be trimethyltopsentin B2 (75); the minor compound was tetramethyltopsentin B2 (76). The structure determination was done on the major compound, trimethyltopsentin B2. 36 Figure 4 1 H nmr spectrum of topsentin B2 (74) (acetone-d6; 300 MHz) Figure 5 *H nmr spectrum of trimethyltopsentin B2 (75) (acetone-d6; 300 MHz) Pure 75 was isolated as a yellow/green oil. The LREIMS of this compound showed a molecular ion doublet at m/z 462/464 daltons. The high resolution mass measurements made on these peaks established the formula of 75 as C23Hi9BrN402 (AM, -1.8/+0.7 mmu), requiring sixteen degrees of unsaturation as in 74. Thus, compound 75 was a trimethyl derivative of topsentin B2. This was confirmed by the appearance of three methyl singlets at 8 3.91, 3.99, and 4.16 ppm in the *H nmr of compound 75 (see Figure 5 and Table 1), each integrating for three protons. The 13C nmr spectrum of trimethyltopsentin B2 (see Figure 6) showed resonances for twenty-three carbon atoms. An APT experiment^  established that there were three methyl carbons (again confirming that three methyl groups had been added to topsentin B2), nine methine carbons, and eleven quaternary carbons. A quaternary carbon signal at 8 178.4 ppm suggested the presence of an unsaturated carbonyl functionality; a band at 1706 cm'l in the ir spectrum of compound 75 supported this. The inclusion of a l-methyl-3-alkyl-6-methoxyindole fragment in 75 was evidenced by a series of nmr double resonance experiments, a homonuclear COSY experiment,^5 and nOe experiments^ ^ (see Figures 7 and 8). Two mass spectral fragments at m/z 188 (C11H10O2) and 174 (C10H8O2) daltons (see Figure 9) confirmed the existence of this indole moiety and also revealed that the C3 substituent was a carbonyl. The presence of a carbonyl at C3 of this indole moiety was also evidenced by the 40 substantial deshielding of the proton at position 2. Further decoupling and nOe experiments identified a second proton spin system (see Figures 7 and 8) which could be attributed to a 3-alkyl-6-bromoindole fragment by comparison with published *H nmr chemical shifts for molecules containing this substructure.^ ? The identification of two indole moieties and a carbonyl accounted for thirteen of the sixteen sites of unsaturation. The remaining atoms ( C 4 H 4 N 2 ) and unaccounted for spectral features in 75 which were required to account for these three sites of unsaturation (*H nmr, 8: 7.65 (s, IH) and 4.16 (s, 3 H ) ppm; 13c nmr, 8: 121.8 (d), 136.7 (s), 138.4 (s), and 36.5 (q) ppm) could be assigned to a 2,4-disubstituted-N-methyl-imidazole fragment. Figure 7 Selected nOe enhancements in trimethyltopsentin B2 (75) 41 H13 H22 H2 J L H23 irradiated H2 H13 H17 irradiated -Ttt" J L PPM 9.0 8.0 7.0 6.0 5.0 4.0 3.0 25-CH3 irradiated H8 J2L nrvrr 26-CH3 irradiated YQ. H8 J L H13 "ipvr 27-CH3 irradiated PPM 9.0 8.0 7.0 6.0 5.0 4.0 Figure 8 Selected nOe difference spectra of trimemyltopsentin B2 (75) (acetone-dy, 400 M H z ) 42 Table 1 liC nmr (75 MHz) and A H nmr (300 MHz) assignments for trimethyltopsentin B2 (75). Spectra were recorded in acetone-d6; *H nmr chemical shifts are reported in ppm using internal TMS as a reference. Position 1 3 C nmr shift (mult.)* *H nmr shift (mult; / in Hz) 2 140.5 (d) 9.07 (s) 3 115.1 (s) 4 122.4 (s) 5 123.6 (d) 8.40 (d; 8.7) 6 112.3 (d) 6.95 (dd; 8.7, 2.2) 7 157.9 (s) 8 94.2 (d) 7.09 (d; 2.4) 9 138.8 (s) 10 178.4 (s) 11 138.4 (s) 13 121.8 (d) 7.65 (s) 14 136.7 (s) 16 111.7 (s) 17 123.8 (d) 7.85 (s) 18 10.6 (br s) 19 143.8 (s) 20 115.2 (d) 7.68 (d; 2.4) 21 115.3 (s) 22 123.2 (d) 7.30 (dd; 8.7, 2.4) 23 122.6 (d) 8.12 (d; 8.7) 24 125.1 (s) 25 55.9 (q) 3.91 (s) 26 33.9 (q) 3.99 (s) 27 36.5 (q) 4.16 (s) *: multiplicities were determined via APT 75 43 188 daltons 174 daltons Figure 9 Selected mass spectral fragment ions of trimethyltopsentin B2 (75) Once all of the fragments comprising trimethyltopsentin B2 had been established, what remained was to assemble the fragments to form the complete molecule. A series of nOe experiments (see Figures 8 and 10) convincingly located the l-methyl-3-keto-6-methoxyindole, 6-bromoindole, and methyl substituents on the imidazole ring as shown in 75. Of particular importance were the observed nOe enhancements between the methyl singlet at 8 4.16 ppm (already established as being on one of the imidazole nitrogens) and the imidazole proton at 8 7.65 ppm; this placed the methyl substituent on the nitrogen next to the protonated imidazole carbon. The observation of an nOe enhancement between the imidazole proton (8 7.65 ppm; H13) and protons belonging to the 6-bromoindole fragment (8 7.85 and 8.12 ppm; H17 and H23) then placed this fragment as shown in 75. The structure of trimethyltopsentin B2 was further confirmed by the use of SINEPT experiments (optimized for a 7 Hz long range coupling).88 In particular, upon irradiation of H17, clear enhancements of carbons 14 and 13 were observed; when H2 was irradiated, no enhancements were observed into any of the imidazole carbons (see Figure 11). Finally, a HETCOR experiment^  (optimized for a 140 Hz one bond coupling) helped to confirm the assignment of the carbon resonances. 44 Figure 10 Selected nOe enhancements in trimethyltopsentin B2 (75) *: carbon showing enhancement { }: proton irradiated H 7 5 Figure 11 SINEPT results for rrimethyltopsentin B2 (75) 45 Having determined the structure of trimethyltopsentin B2, it was fairly routine to assign structures to both topsentin B2 (74) and tetramethyltopsentin B2 (76). The two tautomeric forms of 74 (74a and 74b) were deduced based upon the structures determined for the methylation products 75 and 76. 3. Isolation of the Metabolites from Hexadella sp. Collected at -40 m Hexadella sp. was collected using SCUBA (-40 m) in Agamemnon Channel, Jervis Inlet, B. C. in March 1987. The freshly collected sponge (800 g, wet weight) was immersed in methanol immediately following collection. Evaporation of the methanol under reduced pressure gave a residue that was suspended in water and sequentially extracted with hexane (2 x IL), chloroform (2 x IL), and ethyl acetate (2 x IL). The combined chloroform extracts were concentrated and chromatographed on Sephadex LH-20 (MeOH/CH2Cl2; 3:1) to give an antibacterial fraction containing a mixture (74 mg) of hexadellins A (77) and B (78). The compounds could not be separated in their native form; therefore, the mixture was acetylated with acetic anhydride and pyridine (0.5 mL of 46 each, room temperature, 24 hours) to give a mixture of 79 and 80. The acetylated derivatives were purified via gradient silica gel flash chromatography (EtOAc/hexane; 4:1 to EtOAc/MeOH; 4:1) and normal phase H P L C (EtOAc/MeOH; 95:5) to give pure diacetylhexadellin A (79) (30 mg) and pure diacetylhexadellin B (80) (19 mg). The combined ethyl acetate extracts were fractionated by Sephadex LH-20 chromatography (MeOH/CH2Cl2; 7:3). Early eluting fractions were further subjected to sequential application of silica gel flash (gradient: EtOAc/hexane; 7:3 to EtOAc/MeOH; 4:1), reversed phase flash (gradient: MeOH/H20; 7:3 to MeOH), and radial thin layer (silica gel: EtOAc/MeOH; 9:1) chromatographies to give pure samples of dragmacidons A (81) (6 mg) and B (82) (6 mg). The late eluting LH-20 fractions were further fractionated by radial thin layer chromatography (silica gel: EtOAc/hexane; 3:2) to give pure dragmacidon C (83) (3 mg). 4. Novel Metabolites from Hexadella sp. Collected at -40 m Hexadellin A (77) and Hexadellin B (78) The compounds hexadellin A (77) and hexadellin B (78) could not be separated in their native forms. Therefore, the structure determinations of the two compounds were done on their diacetyl derivatives, 79 and 80, which were readily separable. 47 79 Pure 79 was isolated as a pale yellow oil. The compound failed to show a molecular ion in either the electron impact or chemical ionization mass spectra. The highest mass ions in the LREIMS were a cluster at m/z 739/741/743/745/747 daltons. High resolution mass measurements provided a formula of C23H25Br4N3C«5 for the corresponding mass spectral fragment. However, a summation of the atoms present in a number of fragments that were readily identified from the *H nmr and 13C nmr spectra (see Figures 12 and 13 and Tables 2 and 3) provided a formula of C25H27Br4N3C»7 for the compound. This suggested that the highest observable mass in the mass spectrum corresponded not to the molecular ion, but instead to the molecular ion less C2H2O2 (58 daltons). This is consistent with what has previously been found for compounds similar to 48 6.0 5.0 4.0 3.0 2.0 Figure 12 *H nmr spectrum of diacetylhexadellin A (79) (CDC1 3 ; 400 M H z ) 50 79.90 The (facile) loss of C2H2O2 arises from a mass spectral fragmentation which involves opening of the spirocyclohexaaUenyldihydroisoxazole moiety and aromatization of the dibrominated six-membered ring.90 The ir spectrum of 79 showed stretching frequencies which suggested the presence of an acetate functionality (1752 cm~l) and an amide functionality (3338,1656 cm"l). The *H nmr resonances observed at 8 2.15 (s; -OCOCH3J, 3.09 (d; / = 18.0 Hz, H7), 3.46 (d; / = 18.0 Hz, H7), 3.76 (s; -OCH3J. 5.85 (s; HI), and 6.32 (s; H5) ppm in the spectrum of 79 could be assigned to a spirocyclohexadienyldihydroisoxazole fragment (A) by comparison with published chemical shifts for diacetylaerothionin (84)73 and tetraacetylfistularin-3 (85)71 ( s e e Table 2). A second set of resonances at 8 2.11 (tt; J = 6.2, 5.6 Hz; H12), 2.75 (t; / = 7.2 Hz; H18), 3.47 (df, / = 7.2, 5.2 Hz; H19), 3.71 (dt; / = 6.0, 5.2 Hz; Hll), 4.09 (t; / = 5.6 Hz; H13), 5.58 (br t; / = 6.0 Hz; H20), 7.17 (br t; J = 6.0 Hz; H10), and 7.35 (s; H16) ppm in the *H nmr spectrum of 79 could be assigned to fragment B by comparing them (see Table 2) with published values for the identical fragment found in diacetylpsammaplysin-A (86).74 Double resonance, COSY, and nOe experiments verified the structure of fragment B. A comparison of the nmr chemical shifts of the carbons in 79 with the values for the carbons in the relevant fragments in compounds 84 and 86 also provided evidence for the inclusion of these fragments (A and B) within the structure of diacetylhexadellin A (see Table 3). Finally, a methyl singlet at 8 1.98 ppm was assigned to an acetamide. Having identified all of the substructures of diacetylhexadellin A (79), it only remained to determine which of the terminal nitrogen atoms (N10 or N20) in fragment B was attached to the acetyl residue and which was attached to the carbonyl functionality in fragment A. Examination of the *H nmr chemical shifts of the NH protons in model compounds such as diacetylpsammaplysin-A (86) and tetraacetylfistularin-3 (85) (see Table 2) revealed that a proton on a nitrogen attached to fragment A would have a chemical shift in 51 the range of 8 6.60-7.30 ppm, while a proton on a nitrogen that was acetylated would have a chemical shift in the range of 8 5.50-6.10 ppm. Since H10 in compound 79 had a chemical shift of 8 7.17 ppm and H20 had a chemical shift of 8 5.58 ppm, it was possible to conclude that fragment A was attached to N10 and the acetyl group was attached to N20 of fragment B as shown in structure 79. Once the structure of diacetylhexadellin A had been assigned, the structure of the parent molecule, hexadellin A , could readily be assigned as having the structure 77. H B r H H 84 X = Y = A B 79 X = A , Y = -OCOCH3 OCOCH3 85 X = Y = A A C 5 2 Table 2 IH nmr (400 MHz) assignments for diacetylhexadellins A (79) and B (80), diacetylaerothionin (84)73 tetraacetylfistularin (85)?1 and diacetyl-psammaplysin-A (86)7^ All spectra were recorded in C D C I 3 ; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are given in Hz. Proton 79 80 84 85 86 1 5 10 11 12 13 16 18 19 20 OMe NHAc OAc 5.85 (s) 6.32 (s) 3.09 (d; 18.0) 3.46 (d; 18.0) 7.17 (brt; 6.0) 3.71 (dt; 6.0, 5.2) 2.11 (tt; 6.2, 5.6) 4.09 (t; 6.0) 7.35 (s) 2.75 (t; 7.2) 3.47 (dt; 7.2, 5.2) 5.58 (br t; 6.0) 3.76 (s) 1.98 (s) 2.15 (s) 5.84 (s) 6.32 (s) 3.08 (d; 18.0) 3.44 (d; 18.0) 6.10 (brt; 4.0)|6.68(brt; 6.0) 3.37 (m) 3.56 (dt; 6.0, 4.0) 2.07 (tt; 6.0, 5.2) 4.09 (t; 5.2) 7.39 (s) 2.81 (t; 7.6) 3.58 (dt; 7.6, 5.6) 6.65 (brt; 5.6) 3.78 (s) 2.01 (s) 2.15 (s) 5.84 (s) 6.32 (s) 3.07 (d; 18.1) 3.43 (d; 18.1) 1.62 (m) 3.77 (s) 2.13 (s) 5.86 (s) 6.33 (s) 3.08 (d; 18.0) 3.06 (d; 18.0) 3.45 (d; 18.0) 3.47 (d; 18.0) 7.07 (d; 6.5) 3.80 (m) 3.96 (m) 5.27 (quint; 4.0) 4.14 (dd; 10.0, 4.0) 4.21 (dd; 10.0, 4.0) 7.50 (s) 5.75 (dd; 7.0, 4.0) 3.57 (m) 3.70 (m) 6.82 (br t; 6.0) 3.77 (s) 3.78 (s) 1.98 (s) 2.14 (s) 7.07 (s) 3.04 (d; 16.0) 3.20 (d; 16.0) 6.34 (s) 7.10 (brt; 6.0) 3.66 (t; 6.3) 2.10 (tt; 6.3, 5.7) 4.08 (t; 5.7) 7.38 (s) 2.72 (t; 7.0) 3.46 (dt; 7.0, 5.5) 5.53 (br t; 7.0) 3.65 (s) 1.98 (s) 2.18 (s) 53 Table 3 l a C nmr (75 MHz) assignments for diacetylhexadellins A (79) and B (80), diacetylaerothionin (84) 73 a n (j diacetylpsammaplysin-A (86)74 All spectra were recorded in CDCI3. Chemical shifts are in ppm Carbon 79 (mult.)* 80 (mult.) 84 (mult.) 86 (mult.) 1 73.2 (d) 73.1 (d) 73.2 (d) 145.9 (d) 2 121.7 (s) 122.1 (s) 121.9 (s) 105.9 (s) 3 149.7 (s) 149.7 (s) 149.9 (s) 149.4 (s) 4 107.8 (s) 107.8 (s) 107.8 (s) 102.6 (s) 5 130.5 (d) 130.2 (d) 130.6 (d) 37.9 (t) 6 89.7 (s) 89.9 (s) 89.8 (s) 121.8 (s) 7 40.1 (t) 39.9 (t) 40.0 (t) 77.3 (d) 8 153.6 (s) 153.5 (s) 154.0 (s) 154.4 (s) 9 158.6 (s) 158.6 (s) 158.8 (s) 157.5 (s) 11 37.3 (t) 37.7 (t) 39.0 (t) 37.6 (t) 12 29.3 (t) 29.4 (t) 26.7 (t) 29.6 (t) 13 71.2 (t) 72.1 (t) 71.1 (t) 14 151.2 (s) 151.5 (s) 151.5 (s) 15 118.1 (s) 118.2 (s) 118.3 (s) 16 132.8 (d) 132.8 (d) 133.3 (d) 17 137.9 (s) 137.2 (s) 138.7 (s) 18 34.5 (t) 34.4 (t) 34.8 (t) 19 40.5 (t) 40.4 (t) 40.7 (t) OCH3 60.2 (q) 60.3 (q) 60.2 (q) 59.4 (q) OAc 169.3 (s) 169.4 (s) 169.6 (s) 168.4 (s) (CO) OAc 20.8 (q) 20.8 (q) 20.7 (q) 21.2 (q) (CH3) NAc 170.1 (s) 170.0 (s) 170.3 (s) (CO) NAc 23.4 (q) 23.6 (q) 23.3 (q) (CH3) *: multiplicities determined via APT 5 4 Diacetylhexadellin B (80) Pure diacetylhexadellin B (80) was isolated as a pale yellow oil. Compound 80, like compound 79, failed to show molecular ions in either the electron impact or chemical ionization mass spectra. Again, the highest mass ion observed was that corresponding to the loss of 58 daltons (C2H2O2) from the parent molecule. The formula (C25H27Br4N307) was, therefore, deduced from a summation of the atoms present in fragments that were clearly identified from nmr experiments. The structure elucidation of compound 80 was quite straightforward. The nmr data for this compound were nearly identical to those of 79 (see Figures 14 and 15 and Tables 2 and 3). By comparing the spectral data for 80 with the data for compounds 79, 84, 85, and 86, the two fragments A and B as well as an acetyl group could be easily identified. Once again, double resonance and COSY experiments confirmed the structures of the proposed fragments. The most noticeable difference between compounds 79 and 80 lay in the chemical shifts of the protons on N10 and N20. The chemical shift of H10 (8 6.10 ppm) and of H20 (8 6.65 ppm) in compound 80 indicated that fragment A was attached to N20 and that the acetyl group was attached to N10 as shown in structure 80 above. The structure of the parent metabolite, hexadellin B (78), was assigned based upon the determined structure of diacetylhexadellin B (80). 55 OI Figure 14 *H nmr spectrum of diacetylhexadellin B (80) (CDC13; 400 MHz) 57 Dragmacidon A (81) Pure dragmacidon A (81) was isolated as a pale yellow glass. The LREIMS of this compound showed a parent ion cluster at m/z 486/488/490 daltons (1:2:1). High resolution mass measurements on these peaks provided a formula of C2lH20Br2N4 (requiring thirteen sites of unsaturation) for dragmacidon A. Both the ^H and * 3 C nmr spectra of this compound contained resonances that could readily be assigned to two 3-alkyl-6-bromoindole fragments (see Figures 16 and 17 and Table 4) by comparison to the data obtained for trimethyltopsentin B2 (75). Confirmation of the presence of these two indole fragments was achieved by the use of double resonance, nOe, and COSY experiments. The two bromoindole fragments accounted for twelve of the thirteen sites of unsaturation. The atoms left unaccounted for after establishing the presence of these two moieties, C5H10N2, were thus responsible for only one of the sites of unsaturation. Since there was no evidence for any isolated double bonds in any of the spectral data of 81, it was assumed that the remaining fragment contained a single ring. A series of six aliphatic resonances (each integrating for one proton) in the ^H nmr spectrum of compound 81 were shown by double resonance experiments to belong to two separate 58 H • • | | " " I I ' ' I I • • I • - - i - • P " H . O IB.fl 9.0 8.0 7.8 6 B P | . H 5 ' B 3 ' " 2 Figure 16 !H nmr spectrum of dragmacidon A (81) (acetone-d6; 400 MHz) 60 three spin systems, each one consisting of a pair of geminal methylene protons and a neighbouring methine proton; the coupling constants of the protons in the two spin systems indicated that in each one, one of the methylene protons was axially oriented with respect to its neighbouring methine proton and the other was equatorially oriented with respect to the same proton (8 2.38 (dd; J = 11.0, 10.4 Hz; H6 a x ) , 3.18 (dd; / = 11.0, 2.6 Hz; H6 e q ) , and 4.43 (dd; / = 10.4, 2.6 Hz; H5 a x ) ppm; 8 3.07 (dd; / = 11.0, 3.0 Hz; H3 e q ) , 3.29 (dd; J = 11.0, 10.5 Hz; H3 a x ), and 3.41 (dd; J = 10.5, 3.0 Hz; H2 a x ) ppm). The remaining resonance in the *H nmr spectrum of 81 could be assigned to an N methyl group (8 2.09 (s) ppm). Identification of these two spin systems and the N methyl group accounted for all of the atoms in the unidentified remaining portion of the molecule except for one nitrogen and one hydrogen. Since the remaining hydrogen did not show a resonance in the ^H nmr spectrum of 81, it was assumed that this proton was exchangeable. Acetylation of 81 with acetic anhydride and pyridine gave the monoacetamide 87 (see experimental), revealing that the final proton required was an NH proton on a secondary amine. The aliphatic region of the 1 3C nmr spectrum of 81 contained a total of five resonances corresponding to the one N methyl (8 44.2 (q) ppm), two methine (8 63.3 (d) and 54.1 (d) ppm), and two methylene (8 64.2 (t) and 54.3 (t) ppm) carbons indicated by the *H nmr data. The *H nmr chemical shifts and coupling constants and the l^C nmr chemical shifts of the aliphatic resonances in dragmacidon A (81) indicated that 81 contained an N-methyl-2, 5-dialkylpiperazine moiety, the alkyl groups (in the case of 81, the 6-bromoindole groups) both having equatorial orientations.^ ^ The establishment of the presence of this final moiety meant that all of the atoms in the molecule had been accounted for, thus, dragmacidon A could be assigned structure 81. Difference nOe experiments (see Figures 18 and 19) were consistent with the proposed structure and they clearly indicated that both indole residues resided in equatorial orientations. In particular, upon irradiation 61 of H5ax» enhancements were observed into H3ax» H6eq, H2', and H4'. When H2ax was irradiated, enhancements of H6ax, H3eq, H2", and H4" were observed. Irradiation of the N methyl group resulted in the enhancements of H6eq, H6ax» and H2ax. Finally, acetylation of 81 caused downfield shifts of the *H nmr resonances of H5, H3ax, and H3eq (see experimental), supporting the placement of the indole moieties on the N-methyl piperazine ring as depicted in 81. It is interesting to note that when dragmacidon A (81) was acetylated, the coupling constants for the aliphatic protons in the piperazine ring changed considerably (see experimental). This indicated that the addition of the acetamide functionality forced the ring to adopt a conformation different to that observed in dragmacidon A (81). Figure 18 Selected nOe enhancements in dragmacidon A (81) 62 H6, N-CH3 irradiated H2 H6, 'IX " " ' I I " ' 8.0 6.0 4.0 PPM H2' I " " 10. 0 T 2.0 H6 M irradiated H4" H6eq H2 JJ 10.0 8.0 H2" PPM 6. 0 4.0 H2 irradiated H4" 1 T 2.0 N-CH3 ra^ H6„ T 1 0 . 0 • " i • r " 8.0 6.0 H2* 4.0 2.0 H5 irradiated H3„ 1 1 10.0 1* and 1"-NH irradiated 1 1 ' " 8.0 H7* and H' 6.0 ™ PPM 7 H2' r i 4.0 2.0 1''"'•^•i ' i i • • - i ,1 r \ 10.0 8.0 6.0 PPM 4.0 2.0 Figure 19 Selected nOe difference spectra of dragmacidon A (81) (acetone-d6; 400 MHz) 63 Table 4 liC nmr (75 MHz) and A H nmr (400 MHz) assignments for dragmacidon A (81). Spectra were recorded in acetone-dft iH nmr chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are given in Hz. Position 1 3 C nmr shift (mult.)* iH nmr shift (mult) 2 63.3 (d) 3.41 (dd; 10.5, 3.0) 3ax 54.3 (t) 3.29 (dd; 11.0, 10.5) 3eq 3.07 (dd; 11.0, 3.0) 5 54.1 (d) 4.43 (dd; 10.4, 2.6) 6ax 64.2 (t) 2.38 (dd; 11.0, 10.4) 6eq r 3.18 (dd; 11.0, 2.6) 10.28 (br s) 2' 123.9 (d) 7.39 (d; 1.8) 3' 117.5 (s)a 3a' 124.9 (s)h 4' 122.1 (d)c 7.81 (d; 8.5) 5* 122.4 (d)c 7.17 (dd; 8.5, 1.2) 6' 115.1 (s)d 7' 115.0 (d) 7.61 (d; 1.2) 7a' 138.7 (s)e 1" 10.28 (br s) 2" 125.0 (d) 7.35 (d; 2.3) 3" 118.8 (s)a 3a" 126.6 (s)h 4" 122.6 (d)c 7.91 (d; 8.5) 5" 122.4 (d)c 7.18 (dd; 8.5, 1.4) 6" 115.2 (s)d 7" 115.0 (d) 7.61 (d; 1.4) 7a" 138.8 (s)e N C H 3 44.2 (q) 2.09 (s) *: multiplicities were deterrnined via APT a _ e : may be interchanged within a column * ^ \ J T % T H T 7a' N H 81 64 Dragmacidon B (82) Dragmacidon B (82) was isolated as a pale yellow powder. The HREIMS showed a parent ion cluster at m/z 500.0219/502.0196/504.0171 daltons, corresponding to a molecular formula of C22H22N4Br2 (AM, +0.7/+0.3/-0.2 mmu). Although the molecular formula indicated that the molecule contained twenty-two hydrogens and twenty-two carbons, the *H nmr spectrum of dragmacidon B (see Figure 20 and Table 5) showed resonances that integrated for a total of only eleven protons, and the l^C nmr spectrum (see Figure 21 and Table 5) contained only eleven resonances. This indicated that the molecule contained a twofold axis of symmetry and, therefore, each identified spin system was assumed to be contained in duplicate in 82. The aromatic region of the *H nmr spectrum of 82 (see Table 5) showed a set of resonances which could be readily assigned to a 3-alkyl-6-bromoindole residue by comparison with the data for dragmacidon A (81). Double resonance and nOe experiments confirmed the aromatic *H nmr assignments. Since 82 was known to possess two of each identified fragment, it was determined that the compound contained two symmetrically oriented 6-bromoindole moieties. The resonances in the aliphatic region of the ^H and A 3 C nmr spectra of 82 (see Table 5) were able to be 65 H i Br CD CO ^ 0 " V B r C H 3 82 w 1 • • I I 1 I ' 11.0 18.0 9.0 • • i i i i TTT 8.0 7.0 6.0 5.0 1.0 3.0 PPM 2.0 • I I 1.0 0.0 Figure 20 *H nmr spectrum of dragmacidon B (82) (acetone-d6; 400 MHz) 67 assigned to an N, N-dimethyl-2, 5-dialkylpiperazine ring by comparison with the data for dragmacidon A (81). It was apparent from the magnitude of the coupling constants observed for the H2 and H5 methine protons in 82 (dd; / = 10.6, 2.9 Hz) that the piperazine ring was in a chair conformation and that the two alkyl (indole) substituents were in equatorial orientations. Attachment of the 6-bromoindole rings to C2 and C5 of the piperazine ring gave the proposed symmetric structure 82 for dragmacidon B. T a b l e 5 1 3 C nmr (75 MHz) and *H nmr (400 MHz) assignments for dragmacidon B (82). Spectra were recorded in acetone-d6; *H nmr chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are given in Hz. Position 1 3 C nmr shift (mult.)* *H nmr shift (mult) 2 and 5 63.0 (d) 3.60 (dd; 10.6, 2.9) 3ax and 6ax 2.64 (dd; 11.0, 10.9) 3eq and 6eq 3 and 6 2.93 (dd; 11.0, 2.9) 64.2 (t) 1' and 1" 10.29 (br s) 2' and 2" 125.2 (d) 7.38 (d; 2.3) 3' and 3" 117.1 (s) 3a' and 3a" 126.7 (s) 4' and 4" 122.6 (d)a 7.92 (d; 8.5) 5' and 5" 122.8 (d)a 7.17 (dd; 8.5, 1.7) 6' and 6" 115.4 (s) 7" and 7" 115.2(d) 7.62 (d; 1.7) 7a' and 7a" 138.9 (s) 1- and 4-NCH3 43.8 (q) 2.09 (s) : multiplicities were determined via APT H N —\ I ^ ~ \ J L \ T CH " 82 68 Dragmacidon C (83) Dragmacidon C (83) was isolated as a pale yellow powder. This compound, the last of the bis(indole) compounds to be isolated, displayed a distinctive fluorescent yellow spot on dc when sprayed with vanillin stain reagent; 83 was the only one of the bis(indole) metabolites observed to stain in this manner, and it was as a result of this characteristic staining behaviour that small amounts of this compound were detected and subsequendy isolated. The LREIMS of dragmacidon C showed a parent ion cluster at m/z 498/500/502 daltons. High resolution measurements made on these peaks provided a formula of C2lHi6Br2N40, requiring fifteen sites of unsaturation, for dragmacidon C. The ir spectrum of compound 83 indicated the presence of a carbonyl moiety (Vmax: 1643 cm-*), thus accounting for the single oxygen atom contained in the molecular formula. The relatively low stretching frequency of the carbonyl indicated that it was either doubly a,/3-unsaturated or a,/3-unsaturated and next to a nitrogen.92 The *H and 1 3 C nmr spectra of 83 (see Figures 22 and 23 and Tables 6 and 7) indicated that there were two 3-alkyl-6-bromoindole fragments contained in this molecule, as there were in compounds 81 and 82. A series of double resonance, COSY, and nOe 69 Table 6 *H nmr data for dragmacidon C (83) (400 MHz) and 4, 5-dihydro-6"-deoxybromotopsentin (89) (360 MHz);93 nOe data is given for compound 83. Chemical shifts are reported in ppm and coupling constants are in Hz. Position 8 (mult.; J) (83)a nOe enhancements^ *5 8 (mult.; J) (89)c 3 4 5 6 r-NH V 3' 3a* 4* 5' 6' 7' 7a' 1"-NH 2" O i l (a) 4.27 (dd; 16.5, 5.3) (b) 4.41 (dd; 16.5, 5.2) 5.16 (ddd; 5.3, 5.2, <1) 10.34 (br s) 7.22 (dd; 2.5, <1) H4' H5(a), H2\ H4\ N-Me H2', H7' 8.47 (br s) (a) 3.60 (ddd; 12.1, 4.6, 4.1) (b) 3.45 (ddd; 12.1, 9.5, 2.1) 5.23 (dd; 9.5, 4.7) 11.13 (brs) 7.29 (br s) 7.69 (d; 8.5) 7.20 (dd; 8.5, 1.7) 7.67 (d; 8.5) 7.12 (dd; 8.5, 1.7) 7.63 (d; 1.7) 10.71 (brs) 8.62 (d; 2.7) H2", H7" 7.58 (d; 1.7) 11.52 (brs) 8.83 (br s) J 3a" 4" 5" 6" 7" 7a" N-Me 8.37 (d; 8.7) 7.20 (dd; 8.7, 1.8) 7.66 (d; 1.8) 3.05 (s) H6, H2\ H4' 8.37 (ddd; 8.0, 1.0, 0.7) 7.02 (ddd; 8.0, 8.0, 1.0) 7.13 (ddd; 8.0, 8.0, 1.0) 7.42 (ddd; 8.0, 1.0, 0.7) a : recorded in acetone-d6 and referenced to internal TMS D : protons enchanced upon irradiation of the proton in column 1 c : recorded in and referenced to DMSO-d6 Br H 83 70 J CH3 N H 8 3 — 1 — • — I — • — I — " — I — ' — I — 8.6 6. 4 8.2 8.0 7.8 PPH —I • 1 ' 1 ' r 7.6 7. 4 7.2 A J s I L — o w a j J w I I . • I I . • ' ' I ' ' 9.a B.B ' . I — r - 1 r— 6.1 S.I PPM 4.1 1.1 i—I— 7.1 Figure 22 *H nmr spectrum of dragmacidon C (83) (acetone-d6; 400 MHz) T a b l e 7 liC nmr data for dragmacidon C (83) (75 MHz) and 4, 5-dihydro-6 '-deoxybromotopsentin (89) (95.5 MHz);y^ chemical shifts are in ppm. Position 8 (mult.)f>* (83) 8 (mult.)g (89) 2 157.8 (s)a 160.5 (s) 3 158.0 (s)a 4 45.1 (t) 5 53.5 (t) 55.6 (d) 6 54.0 (d) 2' 124.4 (d)b 124.7 (d) 3' 123.7 (s) 116.2 (s) 3a' 125.3 (s)c 126.5 (s) 4' 121.2 (d)b 121.5 (d) 5' 123.3 (d)b 123.1 (d) 6' 116.2 (s) 116.0 (s) 7' 115.5 (d)d 132.0 (s)e 115.4(d) 7 a' 139.3 (s) 2" 133.8 (d) 132.9 (s) 3" 115.0 (s) 112.7 (s) 3a" 126.1 (s)c 127.6 (s) 4" 125.4 (d)*> 123.6 (d) 5" 125.5 (d)b 121.9 (d) 6" 115.8 (s) 123.8 (d) 7" 115.1 (d)<* 112.4 (d) 7a" 129.8 (s)e 138.1 (s) 8" 159.2 (s) N-Me 32.8 (q) r : recorded in acetone-d6 S: recorded in methanol-d4 a _ e : may be interchanged within column *: mult, determined via APT Br 7 2 7 3 H6 N-CH3 irradiated j£>. 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 PPM N-CH, H6 irradiated H4' . H2' I " T " 1 , ! 1 , , , , , , , , , , , , , , , , , , , , , n - 0 10-0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 PPM H2" 1"-NH irradiated H7" • • " • | " ' " M " 1 [ • • • . , , , , , , , , , , , | ^ . . . . . . . . p 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 PPM l'-NH irradiated H7 H2' JUL ' ' 1 1 • I " " ' " " 1 1 1 ' 1 ' ' ' " I I ' I ' 11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 PPM Figure 24 Selected nOe difference spectra of dragmacidon C (83) (acetone-dg; 400 MHz) 74 experiments (see Table 6 and Figure 24) substantiated the presence of the two indole groups. Once these two groups had been identified, thirteen of the fifteen sites of unsaturation were accounted for (including the carbonyl functionality). This left the remaining atoms, C5H6N2, accountable for two unsaturation sites. The aliphatic region of the iH nmr spectrum of 83 showed a spin system consisting of a methine proton (8 5.16 (ddd; / = 5.3, 5.2, <1 Hz) ppm) coupled to a pair of geminal methylene protons (8 4.27 (dd; / = 16.5, 5.3 Hz) and 4.41 (dd; / = 16.5, 5.2 Hz) ppm) (see Table 6). A singlet at 8 3.05 ppm integrating for three protons was attributed to an N methyl group. The aliphatic region of the l^C nmr spectrum of dragmacidon C showed resonances for an N methyl (8 32.8 (q) ppm), a methylene (8 53.5 (t) ppm), and a methine (8 54.0 (d) ppm) carbon (see Table 7), in support of the iH nmr data. The l^C nmr of 83 showed resonances for two sp -^hybridized carbons at 8 157.8 (s) and 158.0 (s) ppm. One of these resonances had to belong to the carbonyl carbon; since only one carbon atom and one nitrogen atom were now unaccounted for, the other deshielded carbon resonance had to be assigned to an irnine functionality. The final site of unsaturation in dragmacidon C had to be present as a ring. At this point, two possible structures (83 and 88) could be proposed for dragmacidon C which would fit the various spectral data. Br H H 83 88 7 5 During the period in which we were working on the structure elucidation of dragmacidon C (83), a number of other researchers began to find bis(indole) metabolites in a variety of sponges (see conclusions section). One of these new bis(indole) metabolites, 4, 5-dihydro-6"-deoxybromotopsentin (89), was isolated by Rinehart and co-workers from a sponge sample tentatively identified as Spongosorites sp..93 Since the very small amount of dragmacidon C which we had to work with prevented us from carrying out sophisticated nmr experiments on it, we initially assigned structure 88 to dragmacidon C (originally named topsentin C) based largely upon the similarity of its spectral data to that reported for 4, 5-dihydro-6"-deoxybromotopsentin (89).94 in particular, the chemical shifts of the resonances assigned to the carbonyl and imine carbons in 89 (methanol-d4; 8 159.1 and 160.5 ppm) were nearly identical to the chemical shifts of the resonances assigned to the corresponding carbons in dragmacidon C (acetone-d6; 8 157.8 and 158.0 ppm) and both molecules contained aliphatic spin systems consisting of a methine and a pair of adjacent gerriinal methylene protons (see Tables 6 and 7). As well, the mass spectra of both metabolites showed intense fragment ions corresponding to the loss of two protons, suggesting the facile formation of an aromatic ring. The authors who reported the structure of 4, 5-dihydro-6"-deoxybromotopsentin (89) based their structural determination heavily on data obtained from a COLOC experiment^ 5 optimized for an 8 Hz three bond carbon-hydrogen coupling. In particular, 76 they observed correlations between the hydrogens at both position 4 and position 5 with only one of the two sp -^hybridized carbons (8 160.5 ppm). The carbon showing the correlations was assigned to be the irnine carbon (C2) since it was assumed that both of the observed correlations were due to coupling through three bonds. The carbonyl was placed at position 8" based upon the observed low resolution electron impact mass spectral fragment ions of 89 and the deshielded *H nmr chemical shift of H2" (see Table 6). The structure assigned to 4, 5-dihydro-6"-deoxybromotopsentin (89) seemed reasonable since the two other metabolites isolated along with it, topsentin B2 (74) and topsentin A (90), had the same carbon skeleton93 Subsequent to our publication of the structure of dragmacidon C as 88,94 Braekman and co-workers published the structure of topsentin D (91), a metabolite of the Mediterranean sponge Topsentia genitrix.96 Topsentin D (91) was found to be air sensitive and it spontaneously converted to topsentin A (90) on standing. Very utile spectral data were reported for topsentin D (91); however, its transformation into topsentin A (90) provided rigourous proof of its structure because the structure of topsentin A (90) had been verified by synthesis.93, 97 Topsentin D (91) and our originally proposed structure 88 for dragmacidon C both contain similarly substituted dihydroimidazole 77 substructures and, therefore, they would be expected to have very similar susceptibilities to aerial oxidation. However, the observed instability of topsentin D (91) was not observed for dragmacidon C (83), which was found to be extremely stable. Since dragmacidon C showed no signs of spontaneous oxidation, we concluded that a reinvestigation of its originally proposed structure 88 was required. H 91 A re-examination of both our spectral data for dragmacidon C (83) and the published spectral data for 4, 5-dihydro-6"-deoxybromotopsentin (89)93 revealed a number of weaknesses in the structural assignments of these two metabolites, foremost among these being the unexplainedly low chemical shifts of the carbonyl carbons. The resonances assigned to the carbonyl carbons in dragmacidon C and 89 (8 158.0 and 160.5 ppm respectively) are significantly more shielded than would normally be expected for the 13C nmr resonance of a doubly a,/5-unsaturated carbonyl functionality. Usually, ketones which possess a,j3-unsaturation (due to either a double bond or an aryl group) on both sides show carbonyl resonances at approximately 8 170-180 ppm in their nmr spectra.98 For example, the resonance attributed to the carbonyl carbon in trimethyltopsentin B2 (75), a good model compound for the cross conjugated ketone functionality proposed to exist in 88 and 89, occurs at 8 178.4 ppm. The significant difference between the very similar carbonyl chemical shifts observed in dragmacidon C and 4, 5-dihydro-6"-deoxybromotopsentin (8 158.0 and 160.5 ppm respectively) and that 78 observed in trimethyltopsentin B2 (75) (8 178.4 ppm) is not easily rationalized if the structures originally assigned to the former two metabolites (88 and 89 respectively) are correct. Indeed, Rinehart and co-workers noted this fact when discussing the structure determination of compound 89.93 A second difficulty with the structural assignments of dragmacidon C and 4, 5-dihydro-6"-deoxybromotopsentin concerned the carbonyl stretching frequencies of these two compounds. The carbonyl stretching bands in the ir spectra of both topsentin B2 (74) (1702 cm - 1) and trimethyltopsentin B2 (75) (1706 cm_l) were observed at significantly higher frequencies than those in the ir spectra of the apparently related metabolites dragmacidon C (1643 cm-*) and 4, 5-dihydro-6"-deoxybromotopsentin (1665 cm'1). Taken together, the 13c nmr and ir evidence argued against the presence of a ketodihydroimidazole fragment and for the presence of a dihydropyrazinone fragment, which contains an unsaturated amide functionality, in both 89 and dragmacidon C, even though such a conclusion was at odds with the COLOC data. One simple explanation for the apparent discrepancy between between the COLOC data for 89 and the observed 1 3 c nmr chemical shifts and ir stretching frequencies would be that the COLOC correlation observed between the methylene protons 0H4) and the irnine carbon in 4, 5-dihydro-6"-deoxybromotopsentin (89) was actually due to coupling through four bonds. In order to test this possibility, we prepared the model compound 94 using standard procedures (see experimental and Scheme 2).99 Figure 25 shows an expansion of the FLOCK 100 spectrum (optimized for / (CH) = 8 Hz) recorded in DMSO-dg on the model compound 94 (the FLOCK experiment is a variation on the COLOC experiment which minimizes correlations due to one bond coupling but depends on the same coupling pathways to generate correlation peaks). A three bond correlation observed between the indole H2' proton (8 8.33 ppm) and the carbon resonance at 8 157.9 ppm identified that carbon resonance as belonging to the imine carbon (C3). The expected three 79 Figure 25 An expansion of the FLOCK spectrum recorded for model compound 94 (DMSO-d6) bond correlation between the H5 protons (8 3.78 ppm) and the imine carbon (C3; 8 157.9 ppm) was very strong; however, the three bond correlation between the H6 protons (8 3.29 ppm) and the carbonyl carbon (C2; 8 157.5 ppm) was extremely weak. The most interesting feature in the FLOCK spectrum of 94 was the relatively strong correlation observed between the H6 protons (8 3.29 ppm) and the imine carbon (C3; 8 157.9 ppm). This correlation corresponded to the correlation observed between the H4 methylene protons and the imine carbon in the COLOC data reported for 4, 5-dihydro-6"-deoxybromotopsentin (89).93 The assumption that the H4/imine carbon correlation in 89 was due to three bond coupling resulted in the proposal of a ketodihydroimidazole containing structure for 89. The FLOCK experiment conducted on the model compound 94 demonstrated that this assumption was not justified. O O H H 92 93 THF, A, N 2 molecular sieves N' i H 94 Scheme 2 Preparation of model compound 94 81 The spectral data for the model compound 94 provided strong evidence for the revision of the structure of dragmacidon C to 83 and also suggested that the structure proposed for 4, 5-dihydro-6"-deoxybromotopsentin (89.) might not be correct. The l^C nmr chemical shifts of the imine and carbonyl resonances in the model compound 94 (acetone-d6; 5 157.5 and 157.9 ppm; methanol-d4; 8 159.2 and 159.3 ppm) (see experimental) were nearly identical to the l^C nmr chemical shifts observed for the resonances assigned to these two carbons in dragmacidon C (83) (acetone-d6; 8 157.8 and 158.0 ppm) and in 4,5-dihydro-6"-deoxybromotopsentin (89) (methanol-d4; 8 159.1 and 160.5 ppm).93 j. n addition, the carbonyl stretching frequency observed for 94 (1668 cm-1) agrees well with that reported for 4, 5-dihydro-6"-deoxybromotopsentin (89) (1665 cm" 1)93 and also with that observed for dragmacidon C (83) (1643 cm~l) if one takes into account the 20 to 30 cm"l decrease in carbonyl stretching frequency expected when going from a secondary lactam, such as that in 94, to a tertiary lactam, such as that in 83. 1 0 1 Another piece of evidence supporting the revised structure 83 was that the base peak in the electron impact mass spectrum of dragmacidon C was a doublet (50:50) observed at m/z 237/239 daltons. This peak is easily obtained by a retro Diels-Alder fragmentation followed by addition of a proton to one of the resulting fragments, as shown in Scheme 3. The occurrence of a retro Diels-Alder mass spectral fragmentation in compounds possessing structures similar to 83 is common; thus, one would expect the peak at m/z 237/239 daltons to be"significantly large. However, the type of fragmentation which would lead to this fragment ion in 88 is not clear and, therefore, one would not expect it to be the base peak for this compound. Finally, the model compound 94 was observed to stain in the same manner as dragmacidon C (fluorescent yellow) when sprayed with vanillin stain reagent 82 o II c 237/239 daltons (HREIMS: m/z 239.0001 daltons; C 1 0H 1 0N 2Br; AM, -0.7 mmu) Scheme 3 Fragmentation pathway to give the base peak in the mass spectrum of dragmacidon C (83) Once it had been established that the spacer fragment in dragmacidon C (83) was a dihydropyrazinone ring, it remained to position the two indole moieties on this ring to complete the structural assignment. The downfield shift of the H2" proton demanded that the indole group possessing this proton be attached to the imine carbon. A small scalar coupling observed between H6 and H2' (see Table 6) and nOes between H5 (8 4.41 ppm) and H4' (8 7.69 ppm) and between H6 (8 5.16 ppm) and H4' and H2' (8 7.22 ppm) attached the second indole to C6 (see Table 6 and Figure 24). A strong nOe (10%) 83 between H6 and the N methyl protons located the methyl group at position 1 (see Table 6 and Figure 24). The evidence for the revision of the structure of dragmacidon C to 83 suggests that the structural assignment of 4, 5-dihydro-6"-deoxybromotopsentin (89) should also be reinvestigated. The spectral data reported for 4, 5-dihydro-6"-deoxybromotopsentin93 indicates that the structure of this compound should perhaps be revised to 95. H l 95 H Conclusions Previous to our work on Hexadella sp., the isolation of bis(indole) alkaloids similar in structure to topsentin B2 (74) and dragmacidons A (81), B (82), and C (83) from marine sponges was unprecedented. Not only were we surprised to find such novel compounds in Hexadella sp., we were also very surprised to find so few of the typical bromotyrosine-derived metabolites commonly encountered in sponges belonging to the order Verongida. When we first isolated topsentin B2 as the only significantly produced secondary metabolite from the submersible collected sample, we thought that perhaps the sponge had been incorrecdy identified. However, the deep water sponge was found growing on the dead remains of glass sponges, it underwent the colour change characteristic of the Verongida, and it was carefully examined both visually and 84 microscopically by an experienced sponge taxonomist!02 w n o assured us that it was a species of Hexadella. Upon the isolation of hexadellins A (77) and B (78) from the shallower collection of Hexadella sp. (which was examined by the same sponge taxonomist and found to be identical in all respects to the submersible collected sponge), we were confident that the sponge identification was correct. Therefore, although the isolation of compounds 77 and 78 fit in well with our original expectations, the isolation of the four bis(indole) alkaloids from a verongiid sponge was unexpected and difficult to reconcile within the framework of our knowledge about chemical taxonomy of the porifera.35 As mentioned previously in the section discussing the structure elucidation of dragmacidon C (83), a number of other researchers had also begun to find bis(indole) alkaloids in a variety of sponge species during the same period in which we were discovering these types of metabolites in Hexadella sp.. As in our work, the discovery of these metabolites by the other researchers was prompted by the observation of significant levels of biological activity in the crude extracts of the producing sponges. The first report of the isolation and structure elucidation of bis(indole) alkaloids from a marine sponge was by Braekman and co-workers. 103 They isolated the metabolites topsentin B2 (74), topsentin A (90), and topsentin BI (96) from the Mediterranean sponge Topsentia genitrix after it was found that extracts of this sponge were toxic to both fish and mice and were responsible for killing dissociated cells of the freshwater sponge Ephydatia fluviatilis before early aggregation. The compounds 74, 90, and 96 were found to be partially responsible for these activities, thus implicating them as being among the sponge's chemical defense agents. Shortly after this report was published, Rinehart and co-workers reported the discovery of topsentin B2 (74) and topsentin A (90) in methanolic extracts of four related Caribbean deep-sea sponges of the genus Spongosorites.93 These authors also reported the occurrence of 4, 5-dihydro-6"-deoxybromotopsentin B2 (89) (as 85 mentioned earlier) in one of the four sponge samples.93 Compounds 74, 89, and 90 were found to possess antiviral and antitumour properties. 74 Rl = OH, R2 = Br 90 Rl = H, R2 = H 96 Rl =OH,R2 = H (only one tautomeric \ form is depicted hereJ Three other papers were subsequently published which also reported the occurrence of bis(indole) alkaloids in marine sponges. Kashman and co-workers reported the isolation and structure elucidation of the cytotoxic compound dragmacidin (97) from the deep water marine sponge Dragmacidon sp..*04 This compound, like dragmacidons A (81) and B (82), contains an unoxidized piperazine ring. Following this report, Ireland and co-workers isolated the unusual antimicrobial pigment fascaplysin (98) from the Fijian sponge Fascaplysinopsis sp-.l^5 Finally, a recent publication by Braekman and co-workers reports the isolation of topsentin D (91), a dihydro derivative of topsentin A (90), from the Mediterranean sponge Topsentia genitrix.^ Since the first report of compound 74 was published by Braekman and co-workers shortly before our publication of this same compound, 106 we (and Rinehart and co-workers) followed the nomenclature scheme of these authors for this compound. Similarly, the naming of dragmacidons A (81), B (82) , and C (83) followed from the nomenclature used by Kashman and co-workers for dragmacidin (97). 86 H i H 91 As mentioned earlier, the occurrence of bis(indole) alkaloids in sponges belonging to the order Verongida is unexpected and not consistent with the chemical taxonomy of this order. In addition to this, the isolation of these types of metabolites from species of sponge belonging to many different orders raises questions as to the true origins of these compounds. A review of the papers reporting the isolations of the bis(indole) metabolites mentioned above shows that these metabolites are not restricted to a common order, as is most often the case for related groups of marine secondary metabolites. Instead, these compounds have not only been found in a number of different orders (Verongida,94,106 Halichondrida,93, 96, 103 Axinellida,104 and DictyoceratidalOS), they have also been encountered in two different subclasses of sponge (Ceractinomorpha93-94,96,103,105-106 and Tetractinomorphal04). if one considers further the origins of indole alkaloids in general in marine sponges, it becomes quite evident that there is no clear pattern as to which sponges produce these compounds. To illustrate this point, a brief (non-87 comprehensive) overview of the origins of marine indole alkaloids (other than the bis(indole) alkaloids described previously) will be presented Two orders within the subclass of sponges known as the Ceractinomorpha have yielded indole alkaloids. The verongiid sponge Aplysina (Verongia) spengelii has been found to contain aplysinopsin (99), an antineoplastic tryptophan derivative. 107 This same compound has also been isolated from the dictyoceratid sponges Thorecta aplysinopsis^* and Smenospongia sp..l09 Derivatives of aplysinopsin, as well as the tryptamine-derived metabolites 100 and 101, have been isolated from both Aplysina and Smenospongia species. 109 99 100 101 A number of orders within the subclass of sponges known as the Tetractinomorpha have yielded indole alkaloids. The sponge Axinella sp. (order Axinellida) contains three polyalkylated pentindoles, herbindoles A-C (102-104).! 10 These compounds are unusual in that they lack substitution at C3 and are, therefore, apparently not derived from tryptophan. Indole alkaloids have been encountered in three different genera within the order Choristida. Jaspamide (105), isolated from the sponge Jaspis sp.,111 is an unusual metabolite of mixed peptide/polyketide biosynthesis. Barretin (106) has been isolated from the marine sponge Geodia baretti.m The deep water sponge Dercitus sp. contains compound 107;113 a shallow water sample of Dercitus sp. has yielded aplysinopsin derivatives. 11^  The deep water sponge Pleroma menoui, a sponge belonging to the very 88 little studied order Lithistida, has been found to produce the simple indole derivatives 108 and 109.115 Finally, a series of indolic peptide alkaloids have been isolated from the burrowing sponge Cliona celata (order Hadromerida).87, 116-118 R 102 R = C H 3 103 R = C H 2 C H 3 104 R = s s ^ \ ^ 105 Although the above mentioned indole alkaloids are not the only ones to have been isolated from marine sponges, they do provide a good illustration of the point that indole containing metabolites are not formed selectively by sponges within a single order. This may indicate that these types of compounds are not true sponge metabolites, but are instead produced by symbiotic organisms associated with the sponges. In the case of Hexadella sp., this may account for the fact that the amounts of bis(indole) alkaloids isolated from the sponge changes considerably with change in depth. The shallow-water collection of sponge yielded typical verongiid metabolites in significantly greater amounts than the 8 9 bis(indole) metabolites; however, the deep water collection yielded only the bis(indole) alkaloid topsentin B2 (74) as the major constituent. Perhaps Hexadella sp. does not produce the typical bromotyrosine metabolites at very great depths but has more bis(indole) alkaloid-producing microorganisms living in or on it than does the shallower sponge (which yields very minor amounts of the indole alkaloids). In fact, if one examines the types of sponges found producing indole containing alkaloids, it is observed that many of these sponges are collected from quite deep waters. The idea that marine microorganisms are responsible for the production of metabolites isolated from sponges is not new, and studies have been done which prove that microorganisms, not their sponge hosts, produce the metabolites isolated from extraction of the sponge. 119-120 Considering the diversity of sponges found to contain bis(indole) alkaloids, it is not inconceivable that these may be produced by symbiotic organisms rather than the sponges themselves. As yet, no evidence has been provided for this theory; however, further work is planned to try and cultivate the bacteria associated with Hexadella sp. to see if these produce the observed bis(indole) alkaloids. As was mentioned earlier, the incentive to begin work on Hexadella sp. came when routine bioassays on the crude methanolic extracts of this sponge (both collections) showed significant levels of both in vitro cytotoxic (LI210 mouse leukemia cell line) and antimicrobial activities. The bis(indole) compounds (74,81,82, and 83) were found to be responsible (at least in part) for the observed activities. In our bioassays, pure topsentin B2 (74) showed significant in vitro cytotoxicity ( E D 5 0 : 5 p-g/ml) in the L1210 bioassay. In vivo testing showed that this compound was considerably antineoplastic (T/C: 135% in the P388 in vivo bioassay). Dragmacidon A (81) also displayed considerable levels of in vitro cytotoxicity ( E D 5 0 : 6.5 p.g/ml) and in vivo antineoplastic activity (T/C: 135%) in the same two bioassays. Topsentin B2 has recently been submitted for testing on solid breast tumour cultures; the results are still being awaited. 9 0 The two bromotyrosine metabolites (77 and 78) displayed antimicrobial activity against both Staphylococcus aureus and Bacillus subtilis. These compounds also showed in vitro cytotoxicity in the L1210 bioassay (ED50: <10 |ig/ml). Because compounds 77 and 78 could not be separated in their native forms, the biological testing was done on the isolated mixture of both. A more comprehensive biological screening programme of the bis(indole) alkaloids related in structure to topsentin B2 (74) has been carried out by Rinehart and co-workers.93 These authors have found that the majority of these compounds possess both cytotoxic and antineoplastic activities. As well, the compounds have been found active against a number of viral strains, including Herpes simples virus, type 1 and mouse hepatitis virus, strain A-59. A trend relating the cytotoxic activities of the compounds to substituents on the indole moieties has been found; addition of bromine atoms to the indole rings decreases the cytotoxicity of the metabolites, whereas addition of a hydroxyl group to the ring greatly enhances it. However, the absence of substituents on the indole rings results in a very marked reduction in cytotoxicity. This trend is not observed with respect to the antiviral activities of the metabolites. Finally, the biogenetic formation of the bis(indole) alkaloids mentioned above can be envisaged as arising from the condensation of two tryptophan or tryptarnine moieties. 91 B. Novel Marine Natural Products Isolated from the Northeastern Pacific Sponge Xestospongia vanilla (de Laubenfels. 1930). Introduction 1. Taxonomy of and General Information about Xestospongia vanilla Xestospongia vanilla is a species of sponge belonging to the family Petrosiidae within the order Petrosiida (see Figure 26). Like Hexadella sp., this sponge belongs to the general class of sponges known as the Demospongiiae. Xestospongia vanilla, an encrusting sponge, is found growing on vertical solid rock faces between the depths of 5 and 10 meters below sea level. The sponge is nearly always found in areas of high surge and is rarely observed to be either encrusted by or fed upon by other organisms. The sponge specimens used in this study were very hard textured, off-white coloured specimens (see Figure 27) which formed layers of between 1 and 10 cm on the rock substrate. Two collections of Xestospongia vanilla were made for this study. The first collection was made around a small group of islands near the Bamfield Marine Station on the west coast of Vancouver Island. In our collection areas at Bamfield, Xestospongia vanilla was never found to be a dominant part of the local sponge community; only two locations were found which provided a significant amount of the sponge (see Figure 28). The second collection of Xestospongia vanilla was made in a surge channel near Anthony Island in the Queen Charlotte Islands, B. C. (see Figure 29). Again, only small amounts of the sponge were able to be collected at this site. 92 Kingdom: Metazoa (multi-cellular animals) Phylum: Porifera (sponges) Class: Hexactinellida Calcarea Demospongiae Sclerospongiae / Subclass: Homoscleromorpha Ceractinomorpha Tetractinomorpha Order: Halichondria Haplosclerida Petrosiida Verongida Dictyoceratida Dendroceratida Family: Petrosiidae Poecilosclerida Genus: Strongylophora Xestospongia Petrosia / Species: muta vanilla caycedoi Figure 26 The phylogenetic classification of Xestospongia vanilla 36 (according to Austin ) 93 Figure 27 A sample of Xestospongia vanilla collected from Bamfield, B. C. Figure 28 Bamfield collection sites for Xestospongia vanilla Figure 29 Queen Charlotte Islands collection site for Xestospongia vanilla The crude organic extracts of Xestospongia vanilla collected from the two locations showed both antifungal and antimicrobial activities, thus providing the incentive to begin studying the chemistry of this sponge. 2. The Chemistry of Petrosiid Sponges The chemistry of sponges belonging to the order Petrosiida has recently been reviewed^ 1(a) therefore, no detailed review will be made here. In general, these sponges have been found to produce a wide variety of secondary metabolites which fall into four distinct groups: steroids, linear acetylenic compounds, alkaloids, and compounds of mixed polyketide and terpenoid biogenesis. The compounds which have received the most attention are the steroids, due to their biosynthetic novelty and their importance in the chemotaxonomy of the petrosiid sponges, and the alkaloids, due to their potent biological activities. The number of compounds of mixed polyketide and terpenoid biogenesis isolated from the petrosiids is relatively small compared to the number of compounds from the other three groups. In addition, except for those isolated in a very recent study of the chemistry of Xestospongia vani//a,121(a-e) there have been no purely terpenoid metabolites encountered in a petrosiid sponge. A recent study of the chemistry of Xestospongia vanilla made by Northcote and Andersen revealed the presence of unusual triterpenoid glycosides in this sponge. 121 (a-e) The compounds xestovanin A (110), secoxestovanin A (111), secodehydroxestovanin A (112), and xestovanin B (113) were all isolated from a Bamfield collection of Xestospongia vanilla. In addition, three degraded triterpenoid metabolites, xestenone (114), secoxestenone (115), and xestolide (116) were also isolated from the same sponge. All of these compounds can be envisaged as arising from a squalene precursor. 96 112 113 97 116 3. Triterpenoid Glycosides from the Phylum Porifera (Sponges') Although many triterpenoid glycosides, most notably saponins, have been isolated from various classes of echinoderms (especially starfish and holothurians),^  the occurrence of these metabolites in marine sponges is not common. Prior to the isolation of compounds 110-113, there had only been eight other triterpenoid glycosides encountered in the phylum Porifera. In 1988, Schmitz and co-workers isolated a series of five triterpenoid glycosides with unusual carotenoid-like carbon skeletons, pouosides A-E (117-121),122 from the sponge Asteropus sp.. From the same sponge, these authors also reported the isolation of the cytotoxic saponin sarainoside A l (122).123 This same compound, and two closely related derivatives of it, have also been isolated by Kitagawa and co-workers. * 24 98 117 = OAc, R 2 = Ac, R3 = H, R 4 = H 118 Rx = OAc, R 2 = H, R3 = H, R 4 = H 119 Ri = H, R 2 = Ac, R 3 = H, R 4 = H 120 Ri = OAc, R 2 = Ac, R3 = Ac, R4 = H 121 Rx = OAc, R 2 = Ac, R3 = H, R 4 = Ac OH Subsequent to the publications of structures 110-113 and 117-122, there have appeared two reports of steroidal glycosides from marine sponges. In 1989, Kashman and co-workers isolated eryloside A (123), an antitumour and antifungal steroidal glycoside, from the sponge Erylus LendenfeldiX^ The structure of pachastrelloside A (124) has 99 very recently been reported; this compound, isolated from the sponge Pachastrella sp.,1^ 6 inhibits cell division, but not nuclear division, of starfish eggs. OH 124 The work done on Xestospongia vanilla in this study provides a continuation of work done previously in this laboratory.!21 (a-e) When the current work was initiated, it was thought that extracts of this sponge, especially extracts from the specimens collected in the Queen Charlotte Islands, would yield additional novel triterpenoid glycosides, perhaps with new carbon skeletons. As well, it was hoped that the absolute configurations of the chiral centres in xestovanin A (110) could be solved, either by crystallization of a derivative (or derivatives) of 110 or by chemical degradation. 100 Results a n d Discussion 1. Isolation of the Metabolites from the Bamfield Collected Xestospongia vanilla Xestospongia vanilla was collected in the Sandford surge channel at Bamfield, B. C. (May 1989) at depths ranging from five to fifteen feet below sea level. The freshly collected sponge (1000 g, dry weight) was immersed in methanol immediately after collection. After having soaked for three days, the methanol was decanted from the sponge and concentrated under reduced pressure. The aqueous suspension that remained was made up to 500 mL with distilled water and extracted with hexane (3 x 500 mL) followed by dichloromethane (3 x 500 mL). The combined dichloromethane extracts were concentrated under reduced pressure to yield a brown gummy residue (3.5 g). A portion of this (500 mg) was chromatographed on a Sephadex LH-20 column prepared and eluted with methanol/dichloromethane (9:1). The early eluting fractions contained complex mixtures of compounds which stained heavily (brown) with vanillin; these fractions were combined and further purified by open column reverse phase chromatography (gradient: acetone/H20; 2:3 to acetone/H20; 7:3). 127 Final purification of the compounds was achieved using reverse phase HPLC. Pure samples of isoxestovanin A (125) (11 mg), xestovanin C (127) (10 mg), xestovanin A (110) (50 mg), and xestovanin B (113) (12 mg) were obtained using a solvent system consisting of acetone/H20 (55:45). A pure sample of dehydroxestovanin A (129) (12 mg) was obtained using a solvent system consisting of methanol/H20 (3:1). 101 2. Novel Metabolites from the Barnfield Collected Xestospongia vanilla Isoxestovanin A (125) 125 A very intense parent ion was observed at m/z 787 daltons (C42H68O12 (764) + Na(23)) in the FABMS of isoxestovanin A (125). This mass ion was also observed as the parent ion in the FABMS of xestovanin A (110), 121(d) indicating that the two compounds likely possessed the same molecular formulae. The highest observed mass ion in the HREIMS of 125 was at m/z 436.3336 daltons (M + - disaccharide - H 2 O ) , corresponding to an elemental composition of C 3 0 H 4 4 O 2 (AM, -0.5 mmu). An intense peak in the FABMS of 125 at m/z 311 daltons corresponded to the disaccharide portion ( C 1 2 H 2 3 O 9 ) of the molecule (plus one extra proton). A total of 42 carbons were observed in the * 3 C nmr spectrum of isoxestovanin A (125) and an APT experiment revealed 61 protons attached to carbon atoms (see Table 8 and Figure 30). A one bond HETCOR experiment (optimized for J (CH) = 140 Hz) (see Table 8 and Figure 31) assigned the protonated carbon resonances. Isoxestovanin A (125) gave the hexaacetate 126 (*H nmr ( C D C I 3 ) 8 1.92 (s, 3 H ) , 1 .93 (s, 3 H ) , 1 .94 (s, 3 H ) , 1.98 (s, 3 H ) , 1 . 9 9 (s, 3 H ) , and 2.06 (s, 3 H ) ppm) (see Table 9 and Figure 32) when reacted with acetic anhydride in pyridine. 102 Table 8 1 3 Cnmr (75 MHz) and one-bond HETCOR (optimized for / (CH) = 140 Hz) data for isoxestovanin A (125). Spectra were recorded in acetone-d6. Position Chemical shift (ppm) mult.* HETCOR correlation 1 18.0 q 1.60 (brs) 2 133.1a s 3 121.2 d 5.01 (brt) 4 35.2 t 2.31 (m), 2.24 (m) 5 82.9 d 4.21 (dd) 6 135.7 s 7 128.0 d 5.76 (br d) 8 53.2 d 3.92 (d) 9 210.5 s 10 46.9 d 2.17 (dq) 11 55.5 d 1.81 (m) 12 33.7 t 13 21.6 t 14 37.1 t 15 52.6 s i 16 83.6 s 17 32.3 t 18 122.5 d 5.41 (dd) 19 138.4 s 20 77.9 d 3.94 (d) 21 35.2 t 2.21 (m) 22 122.2 d 5.16 (brt) 23 133.0a s 24 26.0 q 1.66 (br s) 25 25.8 q 1.60 (brs) 26 12.2 q 1.57 (br s) 27 13.5 q 0.95 (d) 28 21.6 q 1.09 (s) 29 11.1 q 1.63 (brs) 30 18.0 q 1.59 (brs) 103 Table 8 (continued) Position Chemical shift (ppm) mult.* HETCOR correlation 1' 99.4 d 4.33 (d) V 72.0 d 3.52 (dd) 3* 76.2 d 3.59 (dd) 4' . 78.3 d 3.79 (m) 5' 70.8 d 3.80 (m) 6* 17.8 q 1.28 (d) 1" 102.5 d 5.27 (d) 2" 72.0 d 4.01 (m) 3" 72.5 d 3.71 (brd) 4" 73.7 d 3.40 (t) 5" 69.7 d 3.80 (m) 6" 18.1 <1 1.22 (d) *: multiplicities were determined via APT a : may be interchanged within a column 24 125 104 105 F2 (PPM) 10- — 20-30-40 H 110 H 120 -130 -J 1 , — 1 1 1 1 1 1 r 1— 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 F I (PPM) Figure 31 HETCOR spectrum of isoxestovanin A (125) (acetone-d6; / (CH) = 140 Hz) Table 9 *H nmr (400 MHz) data for isoxestovanin A hexaacetate (126). Spectra were recorded in CDCI3; chemical shifts are reported in ppm using internal TMS as a reference. Position 8 (mult.; / (Hz); number of H) 1 1.52(brs;3H) 3 4.80 (brt;7.1; IH) 4a 2.17 (m; IH) 4b 2.40 (ddd; 13.4, 7.1, 5.4; IH) 5 4.20 (br dd; 9.7, 5.4; IH) 7 5.58 (br d; 10.0; IH) 8 3.65 (d; 10.1; IH) 10 2.06 (brdq; 11.0, 6.5; IH) 11 1.73 (m; IH) 12a 1.44 (m; IH) 12b 1.95 (m; IH) 13a 1.57 (m; IH) 13b 1.59 (m; IH) 14a 1.63 (m; IH) 14b 1.96 (m; IH) 17a 2.11 (m; IH) 17b 2.37 (br dd; 13.4, 7.6; IH) 18 5.43 (m; IH) 20 5.00 (br t; 6.5; IH) 21a 2.18 (m; IH) 21b 2.29 (m; IH) 22 4.91 (br t; 7.0; IH) 24 1.61 (br s; 3H) 25 1.48 (br s; 3H) 26 1.39 (brs; 3H) 27 0.97 (d; 6.5; 3H) 28 0.98 (s; 3H) 29 1.56 (br s; 3H) 30 1.54 (br s; 3H) 107 Table 9 (continued) Position 8 (mult; / (Hz), number of H) r 4.44 (d; 8.1; IH) 2* 5.13 (dd; 10.3, 8.1; IH) 3' 4.95 (dd; 10.3, 2.8; IH) 4' 3.76 (br d; 2.8; IH) 5' 3.84 (br q; 6.4; IH) 6' 1.28 (d; 6.4; 3H) 1" 4.77 (d; 1.6; IH) 2" 5.36 (m; IH) 3" 5.34 (m; IH) 4" 5.01 (t; 9.6; IH) 5" 4.06 (dq; 9.6, 6.2; IH) 6" 1.12 (d; 6.2; 3H) OAc 1.92 (s; 3H) OAc 1.93 (s; 3H) OAc 1.94 (s; 3H) OAc 1.98 (s; 3H) OAc 1.99 (s; 3H) OAc 2.06 (s; 3H) 24 126 108 An OH stretching vibration (3400 cm"*) in the ir spectrum of hexaacetate 126 and the presence of six hydroxyl proton resonances showing vicinal coupling (DMSO-d6; 8 4.46 (d; / = 5.5 Hz), 4.48 (d; / = 4.6 Hz), 4.54 (d; J = 4.3 Hz), 4.60 (d; / = 5.7 Hz), 4.73 (d; / = 4.4 Hz), and 4.90 (d; / = 4.4 Hz) ppm) and one hydroxyl proton resonance lacking vicinal coupling (8 4.42 (s) ppm) in the *H nmr spectrum of 125 (see Table 10 and Figure 33) indicated that a tertiary alcohol in 125 had not been acetylated. The acetylation reaction, in addition to the *H nmr data for 125, established that the seven protons not attached to carbon atoms in isoxestovanin A (125) were part of alcohol functionalities. A summation of the numbers of carbon atoms and protons evidenced in the 13 C nmr data (see Table 8 and Figure 30) and the *H nmr data (see Tables 10 and 11 and Figures 33 and 34) of 125 supported the molecular formula of C42H68O12 deduced from the FABMS. The molecular formula C42H68O12 required nine sites of unsaturation in isoxestovanin A (125). Five of these could be readily identified from the * 3 C nmr spectrum of 125. A downfield singlet (8 210.5 (s) ppm) revealed a saturated ketone (ir stretch 1710 cm-1), and eight olefinic resonances (8 121.2 (d), 122.2 (d), 122.5 (d), 128.0 (d), 133.0 (s), 133.1 (s), 135.7 (s), and 138.4 (s) ppm) revealed four carbon-carbon double bonds, each trisubstituted (there were only four olefinic protons in the *H nmr spectrum of 125 (acetone-d6; 8 5.01 (br t), 5.16 (br t), 5.41 (br t), and 5.76 (br d) ppm) and none coupled into any of the others). The lack of evidence for additional unsaturated functional groups indicated that isoxestovanin A (125) was tetracyclic. The observation of ketal methine carbon resonances at 8 99.4 (d) and 102.5 (d) ppm in the l^C nmr spectrum of isoxestovanin A (125) suggested the presence of two monosaccharide residues in the molecule, as in xestovanin A (110). This was substantiated by the large number of oxygenated methine carbon atoms (8 69-77 ppm) and methine protons (acetone-d6; 8 3.3-4.1 ppm) in the l^C nmr and *H nmr spectra of 125 respectively. A AH COSY 60 nmr experiment, carried out on 125 in DMSO-d6 so that 110 Table 10 *H nmr (400 MHz) data for isoxestovanin A (125). Spectra were recorded in DMSO-dft chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mulL; /; number of H) COS Y correlations 1 1.55 (br s; 3H) H3 3 4.86 (br t; 6.5; IH) HI, H4a, H4b, H25 4a 2.14 (m; IH) H3, H4b, H5 4b 2.29 (ddd; 13.6, 6.5, 5.7; IH) H3, H4a, H5 5 4.08 (br dd; 9.5, 5.7; IH) H4a, H4b 7 5.58 (br d; 10.6; IH) H8, H26 8 3.79 (d; 10.6; IH) H7 10 2.10 (brdq; 10.9, 6.3; IH) Hl l , H27 11 1.69 (m; IH) H10, H12a, H12B 12a 1.47 (m; IH) Hl l , H12B, H13a 12B 1.81 (m; IH) Hl l , H12a, H13a 13a 1.52 (m; IH) H12a, H12B, H13a, H14B 13b 1.60 (m; IH) H13a 14B 1.42 (m; IH) HI4a, HI3a 14a 2.04 (m; IH) H14B 16-OH 4.42 (s; IH) 17a 1.92 (m; IH) H17b, H18 17b 2.42 (br dd; 14.2, 10.2; IH) H17a, H18 18 5.12 (brd; 10.2; IH) H17a, H17b, H29 20 3.72 (br dt; 7.0, 4.6; IH) H21, 20-OH 20-OH 4.48 (d; 4.6; IH) H20 21 2.09 (m; 2H) H20, H22 22 5.05 (br t; 6.6; IH) H21.H24, H30 24 1.61 (br s; 3H) H22 25 1.52 (br s; 3H) H3 26 1.49 (br s; 3H) H7 27 0.88 (d; 6.3; 3H) H10 28 0.96 (s; 3H) 29 1.50 (br s; 3H) H18 30 1.53 (br s; 3H) H22 111 Table 10 (continued) Position 8 (mult; J; number of H) COSY correlations r 4.10 (d; 7.7; IH) H2* V 3.22 (m; IH) HI', H3', 2'-OH 2'-OH 4.73 (d; 4.4; IH) H2' 3' 3.30 (m; IH) H2\ H4', 3'-OH 3'-OH 4.90 (d; 4.4; IH) H3' 4' 3.60 (m; IH) H3', H5' 5* 3.32 (m; IH) H4', H6' 6' 1.15 (d; 6.4; 3H) H5' 1" 5.08 (d; 1.8; IH) H2" 2" 3.77 (m; IH) HI", H3", 2"-OH 2"-OH 4.54 (d; 4.3; IH) H2" 3" 3.48 (ddd; 9.5, 5.5, 3.3; IH) H2", H4", 3"-OH 3"-OH 4.46 (d; 5.5; IH) H3" 4" 3.19 (ddd; 9.5, 9.3, 5.7; IH) H3", H5", 4"-OH 4"-OH 4.60 (d; 5.7; IH) H4" 5" 3.57 (m; IH) H4", H6" 6" 1.12 (d; 6.2; 3H) H5" 2 4 125 112 w Table 11 A H nmr (400 MHz) data for isoxestovanin A (125). Spectra were recorded in acetone-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult.; / ; number of H) COSY correlations nOe enhancements3 1 1.60 (br s; 3H) H3, H4a*. H4b* 3 5.01 (brt; 7.3; IH) HI, H4a, H4b, H25 4a 2.24 (m; IH) HI*, H3, H4b, H5, H25* 4b 2.31 (ddd; 13.7, 7.3, 5.8; HI*, H3, H4a, H5, IH) H25* 5 4.21 (dd; 8.5, 5.8; IH) H4a, H4b, H7* HI', H7, H3, H4a, H4b 7 5.76 (br d; 10.2; IH) H5*, H8, H26 H5 b , 16-OHb, H29 b 8 3.92 (d; 10.2; IH) H7 H10C 10 2.17 (dq; 11.1,6.3; IH) H l l , H27 H8 b , H27 b 11 1.81 (m; IH) H10, H12a, H12B H27, H28, 16-OH 12a 1.58 (m; IH) H l l , H12B, H13a, H13b 126 1.95 (m; IH) H l l , H12a, H13a, H l lc A € rliF H13b 13a 1.60 (m; IH) H12a, H12B, H13b, H14a, H14B 13b 1.62 (m; IH) H12a, H12B, H13a, H14a, H14B 14B 1.65 (m; IH) H13a, H13b, H14a 14a 2.18 (m; IH) H13a, H13b, H14B, H10 c, H20 c H28* 16-OH 3.49 (s; IH) 17a 2.20 (dd; 13.9, 8.8; IH) H17b, H18, H29* 17b 2.59 (dd; 13.9, 8.8; IH) H17a, H18, H29* H17ab, H29 b 18 5.41 (br t; 8.8; IH) H17a, H17b, H29 H8, H20 20 3.94 (br dd; 7.4, 4.8; IH) H21 (a, b) H18c 21(a, b) 2.21 (m; IH) H20, H22, H30* 22 5.16 (br t; 7.0; IH) H21, H24, H30 H21 b, H24b 24 1.66 (br s; 3H) H22 25 1.60 (br s; 3H) HI*, H3 26 1.57 (br s; 3H) H7 27 0.95 (d; 6.3; 3H) H10 H10, H12a,Hll 28 1.09 (s; 3H) H14a* H l l , 16-OH, H14B 29 1.63 (br s; 3H) H17a*, H17b*, H18 30 1.59 (br s; 3H) H21*, H22 114 Table 11 (continued) Position 8 (mult.; /; number of H) COSY correlations nOe enhancementsa r 4.33 (d; 7.5; IH) H2' H5, H5', H3' V 3.52 (dd; 9.6, 7.5; IH) HI', H3' 3' 3.59 (dd; 9.6, 2.7; IH) H2\ H4' Hl'C 4' 3.79 (m; IH) H3\ H5' H2"c 5* 3.80 (m; IH) H4', H6' Hl' c , H6'c 6' 1.28 (d; 6.4; 3H) H5' H5*c 1" 5.27 (d; 1.6; IH) H2" H2", H4' 2" 4.01 (m; IH) HI", H3" H4*c, H3"c 3" 3.71 (dd; 9.0, 3.2; IH) H2", H4" H2"c 4" 3.40 (t; 9.0; IH) H3", H5" 5" 3.80 (m; IH) H4", H6" H6"c 6" 1.22 (d; 6.2; 3H) H5" H5"c *: correlations observed in long range COSY a : the values in this column represent those protons whose resonances were enhanced upon irradiation of the protons in column 1 (substantiated by a 2D-ROESY experiment) °: these ID nOe results were obtained in a 10:1 solution of acetone-do" and benzene-dtf c: these nOe results are from the 2D ROES Y experiment only 24 125 115 o 125 Figure 35 lU COSY 60 spectrum of isoxestovanin A (125) (acetone-d6; 400 MHz) scalar couplings to the alcohol protons could be observed, confirmed the existence of the sugar residues (see Table 10), and it demonstrated that both monosaccharides were 6-deoxyhexoses, as in xestovanin A (110). Subtracting the 12 carbons of the two hexose residues from the total of 42 carbons present in isoxestovanin A (125) indicated a bicyclic C30 aglycone. The *H nmr spectrum of 125 contained resonances that could be assigned to four aliphatic and six olefinic methyls (see Tables 10 and 11). A H COSY correlations established that two of the aliphatic methyl residues were contained in the two 6-deoxyhexose fragments. The remaining eight methyl residues, which had to be assigned to the aglycone portion, were the number required by a triterpenoid skeleton. It was apparent, therefore, that isoxestovanin A (125), like xestovanin A (110) and the other members of the xestovanin series of metabolites (111-113), was a triterpenoid glycoside. The HREIMS spectrum of 125, which showed an intense fragment ion at m/z 436.3336 daltons (C30H44O2; AM, -0.5 mmu) corresponding to the loss of two sugar residues plus one water molecule, and the presence of peaks corresponding to C30 aglycone fragment ions in the LREIMS (m/z 418 (436 - H2O) daltons) and FABMS (m/z 436 and 418 daltons) of 125 supported this. Two ten carbon fragments (substructures A and B) (see Figure 36) belonging to the aglycone could be identified from a series of double resonance, *H COSY (see Tables 10 and 11) (both normal and long range), and nOe nmr experiments (see Table 11). Scalar coupling observed between H20 (DMSO-d6; 8 3.72 (br dt; / = 7.6, 4.6 Hz) ppm) and the neighbouring alcohol proton (8 4.48 (d; / = 4.6 Hz) ppm) and the lack of such a coupling into H5 (8 4.08 (br dd; / = 9.5, 5.7 Hz) ppm) established the presence of alcohol and ether functionalities at C20 and C5 respectively. This was supported by the acetylation of 125, which caused the H20 *H nmr resonance, but not the H5 *H nmr resonance, to shift downfield. Strong nOe enhancements (acetone-d6) between H7 and H5 (14%) and between H20 and H18 (12%) provided connectivities between the two independent scalar-118 coupled spin systems in each ten-carbon fragment and also established the E configuration for both the C 6 - C 7 and C18-C19 olefins. The nOes were observed both in I D difference nOe spectra and in a 2D R O E S Y 1 2 8 spectrum (see Figure 37) of 125 (acetone-d6). A s we l l , an a l ly l ic scalar coupl ing between H 7 and H 5 was observed in the * H C O S Y (acetone-d6; opt imized for long range couplings) spectrum of 125 (see Table 11), providing additional evidence for the constitution of substructure A . Substructures A and B are identical with two of the substructures present in xestovanin A (110).121(d) 81.52 52.14 82.29 81-49 53.79 2 5 C H 3 H H 2 6 ^ 3 H V 2 \ / \ / 54.86 84.08 H OR H 85.58 81.50 8 1 " 82.09 52.09 n v  3oCH 3 H H , 0 C H 3 H H 81.92 82.42 H H OH H 85.05 5 3 7 2 84.48 55.12 B Figure 36 Substructures A and B (chemical shifts are from 1 H nmr spectrum recorded in D M S O - d 6 ) The remaining portion o f the aglycone had to contain ten carbon atoms, had to incorporate ketone (ir 1710 c m - 1; 13c nmr 8 210.5 ppm), tertiary alcohol (13c nmr 119 120 8 83.6 (s) ppm; *H nmr (DMSO-d6) 8 4.42 (s, IH) ppm), secondary methyl ( 1 3C nmr 8 13.5 (q) ppm; ! H nmr (DMS0-d6) 8 0.88 (d; / = 6.3 Hz; 3H) ppm), and tertiary methyl (!3c nmr 8 21.6 (q) ppm; *H nmr (DMSO-d6) 8 0.96 (s; 3H) ppm) functionalities and had to be bicyclic. In addition, the bicyclic portion had to contain substructure C (see Figure 38), identified by careful examination of both *H COSY 45 (0.5-3 ppm) and AH COSY (optimized for long range couplings) spectra of 125 (see Tables 10 and 11). Since it was assumed that isoxestovanin A (125) had an unrearranged triterpenoid carbon skeleton and that substructure C was part of a bicyclic system similar to that present in xestovanin A (110), the structure 125 was proposed for isoxestovanin A. This structure could be envisaged as arising from an aldol condensation (alternative to the one which results in production of xestovanin A (110)) of the previously reported metabolite secoxestovanin A (111) (see Figure 39).121(d) The proposal of structure 125 for isoxestovanin A raises questions as to the correctness of the relative stereochemistry assigned to CIO in 111. This stereochemical assignment was originally made based upon the relative stereochemistry of C10 in xestovanin A; however, the relative stereochemistry at C10 in isoxestovanin A (125) differs with that observed for the same centre in 110 (as will shortly be proven). Therefore, it is not clear whether the compound isolated and reported!21(a,d) w a s actually secoxestovanin A (111) or its C10 epimer. 82.17 80.95 81.58 81.95 81.65 82.18 81.81 81.62 81.60 81.09 C Figure 38 Substructure C (chemical shifts are from *H nmr spectrum recorded in acetone-dg) 121 122 A number of pieces of spectral evidence, in addition to the *H COSY data which identified substructure C, supported the aglycone structure shown in 125. The ketone stretching frequency of 1710 cm_l was consistent with the inclusion of a cyclohexanone moiety in the molecule. Irradiation of the secondary methyl protons at position 27 (acetone-d6; 8 0.95 ppm) of 125 in a SINEPT experiment optimized for a carbon-hydrogen coupling constant of 8 Hz gave strong polarization transfer through three-bond coupling to C9 (8 210.5 ppm); in the same experiment, irradiation of the tertiary methyl protons at position 28 (acetone-d6; 8 1.09 ppm) gave strong polarization transfer through three-bond coupling to C16 (8 83.6 ppm). These results are consistent with the proposed structure 125. A series of nOe difference experiments done in acetone-d6 helped to establish the relative stereochemistry of the bicyclic ring system. Irradiation of the tertiary methyl protons (8 1.09 ppm; H28) induced nuclear Overhauser enhancements into H l l (8 1.81 ppm), H14JJ (8 1.65 ppm), and the tertiary alcohol proton at C16 (8 3.49 ppm). Irradiation of H l l (8 1.81 ppm) caused enhancements of H27 (8 0.95 ppm), H28 (8 1.09 ppm), and the C16 hydroxyl proton (8 3.49 ppm) (see Figure 40). These ID-difference nOe results were substantiated by a 2D ROES Y experiment (see Figure 37). Figure 40 Selected nOes for compound 125 123 In order to determine unequivocally that H10 and H8 were both on the a face of the bicyclic ring system in isoxestovanin A (125), it was necessary to do an nOe experiment to see if an enhancement could be observed into one of these proton resonances upon irradiation of the other. In either acetone-d6 or DMSO-d6, both the H8 and H10 resonances were too close to other resonances to be selectively irradiated. Acetylation of 125 caused the resonance for H8 to become well dispersed from other resonances; however, the resonance for H10 was buried under a number of proton resonances and it could not be determined with certainty that it was the one enhanced upon irradiation of H8. Addition of a small amount of benzene-d6 to an acetone-d6 solution of 125 (final solution composition: 10:1 acetone-d6/benzene-d6) resulted in a downfield shift of the allylic protons which had been overlaying the resonance for H10 (causing the H10 resonance to be completely dispersed from all other resonances). Irradiation of H10 caused a fairly strong nOe into H8 (8%) and also into H27 (see Figure 41). This result was substantiated by a 2D ROESY experiment done in acetone-d6 (see Figure 37). The stereochemistry of the bicyclic ring system was further confirmed by measuring the coupling constant between H10 and H l l (10:1 acetone-d6/benzene-d6). Irradiation of the H27 protons in a double resonance experiment caused the H10 resonance to collapse into a doublet with coupling constant / = 11.1 Hz, thus establishing that H10 and H l l were trans diaxially oriented with respect to one another. The coupling constant between these same protons in xestovanin A (110) was found to be much smaller (/ = 5.9 Hz) due to the different relative stereochemistry of the substituents on the bicyclic ring system (see Figure 42). 124 H Figure 41 Selected nOes for compound 125 10 /(H10,H11) = 11.1 Hz /(H10,Hll) = 5.9Hz Bicyclic ring system of 125 Bicyclic ring system of 110 Figure 42 Conformations of the bicyclic ring systems in compounds 125 and 110 Having determined the structure of the aglycone portion of 125, it remained to determine the structures of the two 6-deoxyhexoses present in isoxestovanin A (125). The iH and 1 3 C nmr chemical shifts of the protons and carbons associated with the two sugars (see Tables 8,10, and 11) suggested that they might be the same as those found in xestovanin A (110) (L-rhamnose and D-fucose). 121(d) j n order to prove this, the water soluble material formed from the treatment of isoxestovanin A (125) with potassium hydroxide was hydrolyzed with aqueous trifluoroacetic acid. The resulting monosaccharides were treated with (+)-2-octanol in trifluoroacetic acid followed by acetic anhydride and pyridine. Analysis of the resulting acetylated (+)-2-octyl glycosides by capillary GC (compared to standards prepared in the same manner (see experimental) with 1 2 5 sugars of known chirality) led to the identification of L-rhamnose and D-fucose,1-^ " as expected. The AH nmr resonances for all of the fucose and rhamnose protons in 125 could be assigned by careful examination of the *H COSY data (see Tables 10 and 11). The iH COSY data obtained in DMSO-d6 was of particular use since it allowed the couplings between each of the hydroxyl protons and its adjacent methine to be observed. The fucose H4' methine and the two anomeric protons, HI1 and HI", failed to show vicinal couplings to adjacent hydroxyl protons indicating the presence of ether linkages at C4\ Cl', and Cl". This was supported by the acetylation of 125, which resulted the downfield shift of only the *H nmr resonances for H2", H3', H2", H3", and H4". The magnitude of the vicinal coupling constant between the fucose anomeric proton (HI1) and H2' (J = 1.1 Hz; DMSO-d6) suggested that these two protons were trans diaxially oriented, thus requiring the fucose to have a [3-anomeric configuration. This was substantiated by the magnitude of the one bond carbon-hydrogen coupling constant between HI' and Cl' (/ (CH) = 156.8 Hz).130 Irradiation of the fucose anomeric proton (acetone-d6) induced nOes into H3' and H5' as well as into H5, the ether methine proton of fragment A of the aglycone (see Table 11). Therefore, the fucose had to be attached to the aglycone through a P-glycosidic linkage to C5. An nOe observed between H4' and the rhamnose anomeric proton (HI") demonstrated that the rhamnose moiety was linked to the fucose via a 1,4-glycosidic linkage (see Figure 43). The small vicinal coupling constant between HI" and H2" (7 = 1.8 Hz; DMSO-d6) did not allow the determination of the glycosidic linkage of the rhamnose. The one-bond carbon-hydrogen coupling constant between the rhamnose anomeric carbon and its attached proton was, however, found to be 171.0 Hz, proving that the rhamnose had the a-anomeric configuration. 130 126 <r H Figure 43 Selected nOes for the disaccharide portion of 125 The arguments presented above established that the structure of isoxestovanin A was that depicted by 125. In addition, the absolute configurations of the chiral centres in the disaccharide portion of the molecule are as depicted. The chiral centres in the bicyclic portion of the aglycone are shown with the correct stereochemistries relative to one another. The absolute configurations of these centres have not been determined; therefore, the set of configurations shown for these centres is arbitrary. No conclusions could be made about the configurations at C5 and C20. Xestovanin C (127) 127 The electron impact mass spectrum of xestovanin C (127) failed to show a parent ion; the highest observable mass ion in the HREIMS was at m/z 436.3342 daltons (AM, 0.0 mmu), corresponding to an elemental composition of C30H44O2 (M+ - trisaccharide -H2O). The FABMS of 127 showed an intense pseudomolecular ion at m/z 933 daltons (C48H78Oi6(910) + Na(23)), the same as that observed for xestovanin B (113).121(a) As well, the FABMS of 127 exhibited an intense mass ion at m/z 455 daltons, corresponding to a trisaccharide fragment (C18H32O13). The ir spectrum of xestovanin C showed a very intense OH stretching vibration (3423 cm"l) and a carbonyl stretching frequency (1708 cm_l) which was correct for a cyclohexanone carbonyl functionality. A total of 48 carbons were observed in the l^C nmr spectrum of 127 and an APT experiment established that there were 11 methyl, 6 methylene, 24 methine, and 7 quaternary carbons (see Table 12 and Figure 44). The protonated carbons were assigned based upon a careful analysis of HETCOR (optimized for J (CH) = 140 Hz) data obtained for xestovanin C (127) (see Table 12 and Figure 45). Identification of the various types of carbons established that 69 of the 78 protons were bound to carbon atoms. The remaining protons had to be associated with oxygen atoms. Acetylation of xestovanin C (127) resulted in the incorporation of eight acetate groups (*H nmr (CDCI3) 8 1.99 (s, 3H), 2.01 (s, 3H), 2.03 (s, 3H), 2.05 (s, 3H), 2.06 (s, 3H), 2.10 (s, 3H), 2.14 (s, 3H), and 2.17 (s, 3H) ppm) (see Table 13 and Figure 46). An OH stretching vibration (3385 cm'1) i n me ir spectrum of xestovanin C octaacetate 128 and the presence of eight hydroxyl resonances showing vicinal coupling (8 4.42 (d; J = 5.8 Hz), 4.55 (d; / = 4.2 Hz), 4.62 (d; / = 5.6 Hz), 4.64 (d; / = 4.1 Hz), 4.68 (d; J = 6.8 Hz), 4.76 (d; / = 4.5 Hz), 4.90 (d; / = 4.3 Hz), 5.03 (d; J = 4.0 Hz) ppm) and one hydroxyl proton resonance lacking vicinal coupling (8 4.37 (s) ppm) in the DMSO-d6 *H nmr spectrum of xestovanin C (127) (see Table 14 and Figure 47) indicated that a tertiary alcohol had not been acetylated. The acetylation reaction, in addition to the iH nmr data for 127, substantiated 1 2 8 the fact that the nine protons not attached to carbon atoms were part of alcohol functionalities. The molecular formula of xestovanin C (127), C48H78O16, required ten unsaturation sites. A comparison of the *H nmr data (see Tables 14 and 15 and Figures 47 and 48) and the * 3 C n m r data (see Table 12 and Figure 44) obtained for xestovanin C (127) with the *!! nmr and 1 3 C nmr data of xestovanin A (110) (see Tables 12, 14, and 15) showed that the aglycone portion of 127 was identical with that present in 110. A careful analysis of the *H COSY data (see Tables 14 and 15 and Figures 49 and 50) collected for 127 supported this. A w-coupling was observed between the C9 hydroxyl proton and H8 in the COSY spectrum (DMSO-d6; optimized for long range couplings) of xestovanin C (127) . This correlation was observed in the COSY spectrum of xestovanin A (110) and was used to argue that the tertiary hydroxyl group was on the a face of the bicyclic aglycone ring system. 121(d) The structure of the aglycone was further substantiated by nOe data obtained for 127 in acetone-d6 (see Table 15) and by a series of decoupling experiments done on both 127 and 128. The triterpenoid aglycone portion of 127 accounted for seven of the nine sites of unsaturation, leaving only three to be accounted for. An analysis of the *H nmr and 1 3 C nmr data for xestovanin C (127) suggested that there were three 6-deoxyhexose moieties (representing the last three unsaturation sites in 127) present in the molecule. This was in agreement with the mass spectral data, which supported the presence of sixteen oxygen atoms in the molecule, only four of which were associated with the aglycone. Spectral evidence suggested that the three sugar residues had to be joined together to form a trisaccharide. In particular, only one of the two possible positions for a glycosidic linkage in xestovanin C (C5 and C20) was observed to possess an ether functionality. Acetylation caused the resonance for H20, and not that for H5, to shift downfield. As well, 129 Table 12 YiC nmr (75 MHz) data for xestovanin C (127) and xestovanin A (110). One-bond HETCOR correlations (/(CH) = 140 Hz) are given for 127. Spectra were recorded in acetone-d6. Chemical shifts are in ppm. Position 8 (mult)* (127) HETCOR correlation 8 (mult.)* (110) 1 26.0a (q) 1.65 (br s) 2 133.1° (s) 3 121.2 (d) 5.04 (br t) 4 34.9C (t) 5 83.1 (d) 4.20 (dd) 6 135.4 (s) 7 127.0 (d) 5.43 (br dd) 8 37.8 (t) 2.21 (m) 9 83.4 (s) 10 35.0 (d) 2.34 (br dq) 11 56.3 (d) 2.04 (m) 12 28.0 (t) 13 22.4 (t) 14 35.4 (t) 2.56 (m) 15 55.9 (s) 16 213.0 (s) 17 52.0 (d) 3.75 (d) 18 122.2 (d) 5.61 (br d) 19 141.1 (s) 20 78.4 (d) 4.06 (br dd) 21 32.6c (t) 22 122.1 (d) 5.09 (br t) 23 132.7b (S) 24 25.9a (q) 1.65 (br s) 25 18.0 (q) 1.60 (brs) 26 11.0 (q) 1.62 (br s) 27 13.7 (q) 1.08 (d) 28 26.0 (q) 1.38 (s) 29 11.6 (q) 1.53 (br s) 30 18.0 (q) 1.60 (brs) r 99.8 (d) 4.19 (d) 2' 71.8 (d) 3.52 (m) 3' 79.7<* (d) 3.57 (m) 4' 78.6 (d) 3.81 (brd) 5' 76. id (d) 3.60 (m) 6* 17.8 (q) 1.29 (d) 26.0a (q) 133.1° (s) 121.1 (d) 34.9C (t) 83.0 (d) 135.4 (s) 127.0 (d) 37.7 (t) 83.3 (s) 34.9 (d) 56.2 (d) 27.9 (t) 22.3 (t) 35.7 (t) 55.8 (s) 213.3 (s) 52.0 (d) 122.1 (d) 141.1 (s) 78.3 (d) 32.5C (t) 122.1 (d) 132.7° (s) 25.9a (q) 18.0d(q) 11.0 (q) 13.7 (q) 25.9a (q) 11.5 (q) 18.ld(q) 99.7 (d) 71.9 (d) 76.0 (d) 78.1 (d) 71.2 (d) 17.8 (q) 130 Table 12 (continued) Position 8 (mult)* (127) HETCOR correlation 8 (mult.)* (110) 1" 102.5 (d) 5.25 (d) 102.5 (d) 2" 72.5 (d) 4.01 (br s) 71.7 (d) 3" 73.0 (d) 3.88 (m) 72.4 (d) 4" 72.4 (d) 3.57 (m) 73.5 (d) 5" 68.3 (d) 3.80 (m) 69.7 (d) 6" 18.7 (q) 1.25 (d) 18.1 (q) 1"' 102.2 (d) 5.29 (d) 2"' 72.0 (d) 3.95 (br s) 3 „ , 71.2d (d) 3.61 (m) 4"' 73.7 (d) 3.41 (br t) 5*" 69.8 (d) 3.69 (m) 6"* 18.0 (q) 1.21 (d) *: multiplicities were determined via APT a-d; may be interchanged within a column 127 110 131 132 F2 (PPM) 10-f-FI (PPM) Figure 45 HETCOR spectrum of xestovanin C (127) (acetone-d6; J (CH) = 140 Hz) Table 13 *H nmr (400 MHz) data for xestovanin C octaacetate (128). Spectra were recorded in CDCI3; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult; /; number of H) COSY correlations 1 1.64 (br s; 3H) H3 3 4.90 (br t; 7.9; IH) HI, H4a, H4b, H25 4a 2.22 (m; IH) H3, H4b, H5 4b 2.43 (m; IH) H3, H4a, H5 5 4.10 (dd; 9.3, 5.6; IH) H4a, H4b 7 5.26 (br d; 9.4; IH) H8a, H8b, H26 8a 2.15 (m; IH) H7, H8b 8b 2.28 (m; IH) H7, H8a 10 2.27 (dq; 7.0, 5.9; IH) Hll, H27 11 1.99 (m; IH) H10, H12a, H12b 12a 1.70 (m; IH) Hll,H12b, H13b 12b 1.81(m; IH) Hll,H12a, H13a, H13b 13a 1.42 (m; IH) H12b, H13b, H14a, HI 4b 13b 1.50 (m; IH) H12a, H12b, H13a, H14a, H14b 14a 1.20 (m; IH) H13a, H13b, H14b 14b 2.63 (ddd; 13.0, 8.4, 5.5; IH) H13a, H13b, H14a 17 3.60 (d; 9.6; IH) H18 18 5.65 (br d; 9.8; IH) H17, H29 20 5.17 (brt; 6.9; IH) H21a, H21b 21a 2.30 (m; IH) H20, H21b, H22 21b 2.42 (m; IH) H20, H21a, H22 22 5.01 (br t; 7.3; IH) H21a, H21b, H24, H30 24 1.67 (br s; 3H) H22 25 1.60 (br s; 3H) H3 26 1.50 (br s; 3H) H7 27 1.08 (d; 7.0; 3H) H10 28 1.35 (s; 3H) 29 1.52 (br s; 3H) H18 30 1.61 (br s; 3H) H22 134 Table 13 (continued) Position 8 (mult; /; number of H) COSY correlations r 4.30 (d; 7.9; IH) H21 2* 5.20 (dd; 10.4, 7.9; IH) HI', H3' 3' 4.93 (dd; 10.4, 3.1; IH) H2\ H4' 4' 3.81 (dd; 3.0, <1; IH) H3\ H5' 5' 3.55 (dq; 6.4, <1; IH) H4\ H6* 6' 1.34 (d; 6.4; 3H) H5' 1" 4.82 (d; 2.0; IH) H2" 2" 5.42 (dd; 3.2, 2.0; IH) HI", H3" 3" 5.30 (dd; 9.4, 3.2; IH) H2", H4" 4" 3.70 (t; 9.4; IH) H3", H5" 5" 4.00 (dq; 9.5, 6.4; IH) H4", H6" 6" 1.33 (d; 6.4; 3H) H5" 1"' 5.00 (d; 1.8; IH) H2"' 2'" 5.16 (dd; 3.3, 2.0; IH) HI"', H3"' 3"' 5.26 (dd; 9.8, 3.3; IH) H2"\ H4"' 4'" 5.07 (t; 9.8; IH) H3"', H5"' 5"' 3.98 (dq; 9.8, 6.3; IH) H4"', H6"' 6"' 1.21 (d; 6.3; 3H) H5"' OAc 1.99 (s, 3H) OAc 2.01 (s, 3H) OAc 2.03 (s, 3H) OAc 2.05 (s, 3H) OAc 2.06 (s, 3H) OAc 2.10 (s, 3H) OAc 2.14 (s, 3H) OAc 2.17 (s, 3H) 128 135 Table 14 *H nmr (400 MHz) data for xestovanin C (127) and xestovanin A (110). IH COSY data is given for 127. Spectra were recorded in DMSO-dft chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8(mult.;/; no.of H) (127) COSY correlations 8(mult;/) (110)* 1 1.59 (br s; 3H) H3 1.60 (brs) 3 4.93 (br t; 6.9; IH) HI, H4a, H4b, H25 4.93 (t; 7.0) 4a 2.23 (m; IH) H3, H4b, H5 2.20 (m) 4b 2.29 (m; IH) H3, H4a, H5 2.25 (m) 5 4.03 (dd; 8.7, 6.0; IH) H4a, H4b 4.04 (dd; 8.7, 5.9) 7 5.20 (br d; 7.4; IH) H8a, H8b, H26 5.22 (brd; 8.1) 8a 2.01 (m; IH) H7, H8b 1.97 (m) 8b 2.25 (m; IH) H7, H8a 2.24 (m) 9-OH 4.37 (s; IH) H17 a 4.40 (s) 10 2.11 (dq; 6.9, 5.6; IH) H27 2.07 (dq; 6.2, 5.5) 11 1.86 (m; IH) H10, H12a, H12b 1.86 (m) 12a 1.55 (m; IH) H l l , H12b, H13a, H13b 12b 1.60 (m; IH) H l l , H12a, H13a, H13b 13a 1.34 (m; IH) H12a, H12b, H13b, 1.32 (m) H14a, H14b 13b 1.40 (m; IH) H12a, H12b, H13a, H14a, H14b 14a 1.14 (m; IH) H13a, H13b, H14b 1.10 (m) 14b 2.43 (ddd; 13.0, 7.5, 5.4; H13a, H13b, H14a 2.43 (ddd; 13.0, IH) 8.0, 5.5) 17 3.57 (d; 9.9; IH) 9-OH a, HI 8 3.59 (d; 9.9) 18 5.45 (br d; 9.9; IH) H17 5.46 (br d; 9.9) 20 3.88 (br dt; -7.4, 4.1; IH) 20-OH, H21a, H21b 3.90 (dt; 7.5, 4.1) 20-OH 4.64 (d; 4.1; IH) H20 4.67 (d; 4.1) 21a 2.08 (m; IH) H20, H21b, H22 2.05 (m) 21b 2.18 (m; IH) H20, H21a, H22 2.17 (m) 22 4.96 (br t; 9.2; IH) H21a, H21b, H24, 4.96 (t) H30 24 1.57 (br s, 3H) H22 1.58 (br s) 25 1.54 (br s, 3H) H3 1.55 (brs) 26 1.52 (br s, 3H) H7 1.53 (brs) 27 0.92 (d; 6.8; IH) H10 0.95 (d; 6.2) 28 1.26 (s, 3H) 1.30 (s) 29 1.40 (br s; 3H) H18 1.41 (br s) 30 1.52 (br s; 3H) H22 1.53 (brs) 137 T a b l e 14 (continued) Position 5 (mult.;/; no. of H) (127) COSY correlations 8 (mult;/) (110)* r 3.94 (d; 7.3; IH) H2' 3.97 (d; 7.3) 2' 3.25 (m; IH) HI", 2'-OH, H3' 3.28 (ddd; 9.6, 7.3, 4.3) 2'-OH 4.90 (d; 4.3; IH) H2' 4.92 (d; 4.3) 3' 3.30 (m; IH) H2', 3'-OH, H4' 3.31 (ddd; 9.6, 4.0, 1.8) 3'-OH 5.03 (d; 4.0; IH) H3' 5.03 (d; 4.0) 4' 3.60 (br s; IH) H3', H5' 3.61 (brd; 1.8) 5' 3.39 (m; IH) H4\ H6' 3.39 (br q; 6.3) 6' 1.13 (d; 6.1; 3H) H5' 1.15 (d; 6.3) 1" 5.06 (br s; IH) H2" 5.10 (d; 2.0) 2" 3.75 (m; IH) HI", 2"-OH, H3" 3.76 (ddd; 4.2, 3.1, 2.0) 2"-OH 4.76 (d; 4.5; IH)) H2" 4.57 (d; 4.2) 3" 3.61 (m; IH) H2", 3"-OH, H4" 3.46 (ddd; 9.3, 5.7, 3.1) 3"-OH 4.68 (d; 6.8; IH) H3" 4.48 (d; 5.7) 4" 3.39 (m; IH) H3", H5" 3.18 (dt; 9.3, 5.8) 5" 3.64 (m; IH) H4", H6" 3.56 (dq; 9.2, 6.2) 6" 1.14 (d; 6.1; 3H) H5" 1.10 (d; 6.2) 1,M 5.06 (br s; IH) H2 , M 2"' 3.71 (m; IH) HI"', 2'"-OH, 3"' 2"'-OH 4.55 (d; 4.2; IH) H2 , M 3*" 3.37 (m; IH) H2"', 3"'-OH, H4,M 3"'-OH 4.42 (d; 5.8; IH) H3"* 4"' 3.19 (ddd; 9.4, 9.2, 5.6; H3"', H5"' IH) 4 , , ,-OH 4.62 (d; 5.6; IH) H4 , M 5"' 3.49 (dq; 9.2, 6.2; IH) H4"', H6"' 6"' 1.11 (d; 6.2; 3H) H5"' *: from reference 121(d) a : correlations observed in long range COSY 138 Table 15 A H nmr (400 MHz) data for xestovanin C (127) and xestovanin A (110). ! H COSY and nOe data are given for 127. Spectra were recorded in acetone-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult.; J) (127) COSY correlation nOe* 8 (mult; 7) (110)a 1 1.65 (br s) H3 1.65 (br s) 3 5.04 (br t; 6.7) HI, H4a, H4b, H25 5.10 (t; 7.2) 4a 2.26 (m) H3, H5 2.28 (m) 4b 2.31 (m) H3, H5 2.32 (m) 5 4.20 (dd; 8.3, 6.5) H4a, H4b H3, H7 4.20 (dd; 8.3, 6.4) 7 5.43 (br d; 9.8) H8a, H8b, H26 5.44 (br d; 10.0) 8a 2.21 (m) H7, H8b 2.22 (m) 8b 2.41 (dd; 15.0, 9.6) H7, H8a 2.43 (dd; 15.1, 9.5) 10 2.34 (dq; 6.9, 5.8) Hl l , H27 2.36 (m) 11 2.04 (m) H10, H12a, H12b 2.06 (m) 12a 1.64 (m) Hl l , H12b, 1.62 (m) HI 3a, HI 3b 12b 1.97 (m) Hl l , H12a, 2.01 (m) H13a, H13b 13a 1.35 (m) HI2a, HI2b, 1.39 (m) H13b, H14a, H14b 13b 1.44 (m) H12a, H12b, 1.47 (m) H13a, H14a, HI 4b 14a 1.18 (m) H13a, H13b, 1.17 (m) H14b 14b 2.56 (ddd; 13.2, H13a, H13b, 2.58 (ddd; 12.9, 8.3, 5.2) H14a 8.1, 5.1) 17 3.75 (d; 9.8) H18 3.75 (d; 9.9) 18 5.61 (br d; 9.7) H17, H29 5.63 (d; 9.9) 20 4.06 (br t; 7.5) H21a, H21b 4.02 (t; 7.3) 21a 2.18 (m) H20, H21b, 2.21 (m) H22 21b 2.28 (m) H20, H21a, 2.26 (m) H22 22 5.09 (br t; 7.2) H21a, H21b, 5.05 (t; 6.9) H24, H30 24 1.65 (brs) H22 1.66 (br s) 25 1.60 (br s) H3 1.60 (br s) 26 1.62 (br s) H7, H8ab, H8b*> 1.63 (br s) 27 1.08 (d; 6.9) H10 H10 1.08 (d; 6.9) 28 1.38 (s) H8, H l l 1.38 (s) 29 1.53 (br s) H18 1.54 (br s) 30 1.60 (br s) H22 1.61 (br s) 140 Tab le 15 (continued) Posit ion 8 (mult.; J) (127) C O S Y correlation nOe* 8 (mul t ; J) (110)a r 4.19 (d; 7.2) H 2 * 4.17 (d; 7.3) 2 ' 3.52 (m) H I ' , H 3 ' 3.56 (m) 3 ' 3.57 (m) H 2 \ H 4 ' 3.58 (m) 4 ' 3.81 (b rd ; 2.1) H 3 ' , H 5 ' 3.82 (br d; 3.0) 5 ' 3.60 (m) H 4 \ H 6 ' 3.61 (m) 6 ' 1.29 (d; 6.3) H 5 ' 1.29 (d; 6.4) 1" 5.25 (d; 1.2) H 2 " 5.27 (d; 1.2) 2 " 4.01 (br s) H I " , H 3 " 4.02 (br s) 3 " 3.88 (m) H 2 " , H 4 " 3.71 (dd) 4 " 3.57 (m) H 3 " , H 5 " 3.41 (t; 9.1) 5 " 3.80 (m) H 4 " , H 6 " 3.80 (m) 6" 1.25 (d; 6.2) H 5 " 1.21 (d; 6.2) 1" ' 5.29 (d; 1.1) H 2 ' " H 2 m , H 4 " 2 " . 3.95 (br s) H I " ' , H 3 ' " 3 " ' 3.61 (m) H 2 " ' , H 4 " ' 4 - 3.41 (br t; 8.5) H3" * , H 5 , M H 6 " ' 5 " ' 3.69 (m) H 4 " ' , H 6 " ' 6 " ' 1.21 (d; 6.0) H 5 " ' * : protons enhanced upon irradiation of the proton in the first column a : from reference 121(d) D : correlations observed in long range C O S Y 6" 111 1 1 0 141 Figure 48 ! H nmr spectrum of xestovanin C (127) (acetone-d6; 400 MHz) Figure 49 ! H COSY 60 spectrum of xestovanin C (127) (acetone-d6; 400 MHz) P P M Figure 50 *H COSY 60 spectrum of xestovanin C (127) (DMSO-d6; 400 MHz) H20 (8 3.88 (br dt; / = ~7.4, 4.1 Hz) ppm) clearly showed scalar coupling to a hydroxyl resonance (8 4.64 (d; / = 4.1 Hz) ppm) in DMSO-d6. Thus, only C5 possessed an ether functionality. Capillary GC analysis of the (+)-2-octyl glycosides formed from the monosaccharides present in xestovanin C (127) established the presence of L-rhamnose and D-fucose in the molecule (see experimental). The anomeric proton and carbon resonances indicated that the trisaccharide was composed of one fucose (*H nmr (acetone-d6) 8 4.19 (d; / = 7.2 Hz) ppm; 13c nmr (acetone-d6) 8 99.8 (d) ppm) and two rhamnose (*H nmr (acetone-d6) 8 5.25 (d; J = 1.2 Hz) and 5.29 (d; / = 1.1 Hz) ppm; 13C nmr (acetone-d6) 8 102.2 (d) and 102.5 (d) ppm) moieties. Irradiation of the fucose anomeric proton of xestovanin C octaacetate 128 (HI', 8 4.30 (d; / = 7.9 Hz) ppm) (this proton was not well enough dispersed in 127 to be selectively irradiated) induced nuclear Overhauser enhancements in the *H nmr resonances belonging to H5 (8 4.10 ppm), H3' (8 4.93 ppm), and H5' (8 3.55 ppm). This established that D-fucose was attached to the aglycone portion of 127 at C5. Analysis of the *H COSY data obtained for xestovanin C (127) in DMSO-d6 showed that one rhamnose was linked to fucose at C4' and that the other rhamnose was linked to the first rhamnose moiety at C4" (see Figure 51). This was supported by the acetylation of 127, which resulted in downfield shifts of the *H nmr resonances of H2\ H3\ H2", H3", H2'", H3"\ and H4 ,M. The resonances for H4' and H4" were not shifted, indicating that ether functionalities were present at C4' and C4". The large coupling constant observed between HI' and H2' (7.2 Hz; acetone-d6) and the value obtained for the coupling constant between HI' and C l ' (156.3 Hz) established a (5-glycosidic linkage for the fucose residue. The two rhamnose residues were found to have ot-glycosidic linkages based upon the observed coupling constants between HI" and C l " (170.0 Hz) and between HI'" and Cl'" (171.0 Hz).130 145 Dehydroxestovanin A (129) A very intense parent ion was observed at m/z 785 daltons ( C 4 2 H 6 6 0 1 2(762) + Na(23)), corresponding to a parent ion of two mass units less than the parent ions for both xestovanin A (110) and isoxestovanin A (125). The highest observed mass ion in the HREIMS of dehydroxestovanin A (129) corresponded to the aglycone portion of 129 less H 2 O (m/z 434.3191 daltons; AM, + 0 . 6 mmu). The molecular formula obtained for dehydroxestovanin A (129) ( C 4 2 H 6 6 O 1 2 ) required ten sites of unsaturation. Since the elemental composition of the aglycone portion of dehydroxestovanin A (129) 146 (C30H42O2) was found to be two hydrogens less than the elemental compositions of the aglycone portions of xestovanin A (110) and isoxestovanin A (125), the extra site of unsaturation in 129 had to be associated with this part of the molecule. The ir spectrum of dehydroxestovanin A (129) showed a cyclohexanone carbonyl stretching vibration (1701 cm -l) and a very strong OH stretching vibration (3406 cm"*). Acetylation of 129 with acetic anhydride and pyridine resulted in the formation of the hexaacetate 130 (see Table 16 and Figure 52). The ir spectrum of dehydroxestovanin A hexaacetate 130 showed a weak OH stretching vibration (3497 cm"l) indicating that an unacetylated hydroxyl functionality remained in 130. All 42 carbons were observed in the 1 3 C nmr spectrum of dehydroxestovanin A (129) and an APT experiment showed that 59 of the 66 protons present in 129 were attached to carbon atoms (see Table 17 and Figure 53). Analysis of the *H nmr data obtained for dehydroxestovanin A (129) in DMSO-d6 (see Table 18 and Figure 54) indicated that six of the protons not associated with carbon atoms belonged to secondary alcohol functionalities since they were vicinally coupled into their neighbouring methine protons (5 4.47 (d; / = 5.8 Hz), 4.55 (d; / = 4.2 Hz), 4.63 (d; J = 5.9 Hz), 4.64 (d; / = 4.6 Hz), 4.81 (d; J = 4.7 Hz), and 4.97 (d; / = 4.3 Hz) ppm); the other proton not bound to a carbon atom was associated with a tertiary alcohol functionality (8 4.64 (s) ppm) which failed to acetylate. It was noticed that the l^C nmr spectrum of dehydroxestovanin A (129) had one less methyl and one less aliphatic methine carbon resonance than the spectra for both xestovanin A (110) and isoxestovanin A (125) (see Tables 8, 12, and 17). As well, the characteristic *H nmr resonances for the methyl doublet corresponding to H27 and the methine proton it couples to, H10, in compounds 110,125, and 127 (see Tables 10, 11, 14, and 15) were noticeably absent from the *H nmr spectra of 129 (see Tables 18 and 19 and Figures 54 and 55). Instead, two new sets of signals corresponding to an olefinic 1 4 7 Table 16 *H nmr (400 MHz) data for dehydroxestovanin A hexaacetate (130). Spectra were recorded in benzene-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult.; / ; number of H) COSY correlations nOe enhancementsa 1 1.74 (br s; 3H) H3, H4a*, H4b* 3 5.17 (brt; 7.2; IH) HI, H4a, H4b, H25 4a 2.40 (m; IH) HI*, H3, H4b, H5 4b 2.58 (dd; 14.9, 7.2; IH) HI*, H3, H4a, H5 5 4.45 (dd; 8.9, 5.9; IH) H4a, H4b 7 5.71 (brt; 6.8; IH) H8a, H8b, H26 8(a, b) 2.53 (m; 2H) H7, H26* 11 2.54 (m; IH) H12a, H12b 12a 1.88 (m; IH) H l l , H12b, H13a, H13b 12b 2.16 (m; IH) H l l , H12a, H13a, H13b 13a 1.62 (m; IH) H12a, H12b, H13b, H14a, H14b 13b 1.76 (m; IH) H12a, H12b, H13a, H14a, H14b 14a 1.50 (m; IH) H13a, H13b, H14b 14b 2.12 (m; IH) H13a, H13b, H14a 17 3.86 (d; 9.4; IH) H18 H28, H29 18 5.78 (d; 9.4; IH) H17, H29 20 5.33 (br t; 6.9; IH) H21 (a, b) 21(a, b) 2.52 (m; 2H) H20, H22 22 5.18 (brt; 7.1; IH) H21(a, b), H24, H30 24 1.75 (br s; 3H) H22, H21(a, b)* 25 1.55 (br s; 3H) H3 26 1.74 (br s; 3H) H7 21E 5.23 (br s; IH) H27Z 27Z 5.60 (br s; IH) H27£ 28 1.28 (s; 3H) H l l , H17 29 1.92 (d; 1.3; 3H) H18 30 1.59 (br s; 3H) H22 148 Table 16 (continued) Position 8 (mult.; /; number of H) COSY correlations nOe enhancementsa 1' 4.75 (d; 8.0; IH) H2' H5, H5', H3' V 5.80 (m; IH) HI', H3' 3' 5.30 (dd; 10.3, 3.1; IH) H2', H4' 4' 3.58 (br d; 3.1; IH) H3\ H5' 5' 3.38 (br q; 6.6; IH) H4', H6' 6' 1.34 (d; 6.4; 3H) H5* 1" 5.02 (d; 2.1; IH) H2" 2" 5.88 (dd; 3.2, 2.1; IH) HI", H3" 3" 5.81 (dd; 9.7, 3.3; IH) H2", H4" 4" 5.59 (t; 9.7; IH) H3", H5" 5" 4.29 (dq; 9.7, 6.3; IH) H4", H6" 6" 1.29 (d; 6.3; 3H) H5" H4", H5" OAc 1.74 (s; 3H) OAc 1.75 (s; 6H) OAc 1.79 (s; 3H) OAc 1.98 (s; 3H) OAc 2.20 (s; 3H) *: correlations observed in long range COSY a : the values in this column represent those protons whose resonances were enhanced upon irradiation of the protons in column 1 OAc O 28 130 1 4 9 o Figure 52 *H nmr spectrum of dehydroxestovanin A hexaacetate (130) (benzene-d6; 400 MHz) Table 17 liC nmr (75 MHz) and one-bond HETCOR (optimized for / (CH) = 140 Hz) data for dehydroxestovanin A (129). Spectra were recorded in acetone-d6-Position Chemical shift (ppm) mult.* HETCOR correlation 1 26.0 q 1.64 (br s) 2 132.9 s 3 121.2 d 5.00 (br t) 4 34.8a t 2.22 (m), 2.27 (m) 5 83.1 d 4.07 (dd) 6 136.1 s 7 126.1 d 5.42 (br t) 8 38.8 t 2.33 (m), 2.46 (m) 9 79.4 s 10 150.4 s 11 52.5 d 2.67 (m) 12 29.0 t 13 22.9 t 1.64 (m) 14 36.4 t 2.03 (m) 15 56.2 s 16 212.7 s 17 57.6 d 3.67 (d) 18 120.3 d 5.60 (br d) 19 142.7 s 20 77.5 d 4.01 (m) 21 32.7* t 2.21 (brt) 22 122.1 d 5.12 (brt) 23 132.9 s 24 26.0 q 1.65 (brs) 25 18.0 q 1.57 (br s) 26 11.1 q 1.54 (br s) 27 111.0 t 5.17(brt), 5.33 28 (brt) 25.9 q 1.21 (s) 29 12.7 q 1.61 (brs) 30 18.0 q 1.57 (br s) 1 5 1 Table 17 (continued) Position Chemical shift (ppm) mult.* HETCOR correlation 1* 99.9 d 4.13 (d) V 71.9 d 3.49 (dd) 3' 76.0 d 3.57 (dd) 4' 78.1 d 3.78 (dd) 5' 70.9 d 3.60 (dq) 6' 17.7 q 1.22 (d) 1" 102.5 d 5.24 (d) 2" 71.9 d 4.00 (br d) 3" 72.5 d 3.69 (dd) 4" 73.6 d 3.39 (dd) 5" 69.7 d 3.79 (dq) 6" 18.0 q 1.19 (d) : multiplicities were determined via APT a : may be interchanged within a column 129 152 153 Table 18 *H nmr (400 MHz) data for dehydroxestovanin A (129). Spectra were recorded in DMSO-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult; /; number of H) COSY correlations 1 1.60 (br s; 3H) H3 3 4.90 (br t; 7.0; IH) HI, H4a, H4b, H25 4a 2.18 (m; IH) H3, H4b, H5 4b 2.21 (m; IH) H3, H4a, H5 5 3.95 (dd; 8.8, 5.7; IH) H4a, H4b 7 5.26 (br t; 5.9; IH) H8a, H8b, H26 8a 2.20 (m; IH) H7, H8b, H26* 8b 2.34 (dd; 14.5, 5.6; IH) H7, H8a, H26* 9-OH 4.64 (s; IH) 11 2.57 (brt; 6.1; IH) H12a, H12b, H27£, H27Z* 12a 1.89 (m; IH) Hll, H12b, H13a*, H13b* 12b 2.01 (m; IH) Hll , H12a, H13a, H13b 13a 1.53 (m; IH) H12a*, H12b, H13b, H14a, H14B 13b 1.59 (m; IH) H12a*. H12b, H13a, H14a, H146 146 1.24 (m; IH) H13a, H13b, H14a 14a 1.93 (m; IH) H13a, H13b, H14B, H28* 17 3.56 (d; 8.8; IH) H18 18 5.44 (br d; 8.8; IH) H17, H29 20 3.84 (brdt; 6.6, 4.1; IH) H21(a, b), 20-OH 20-OH 4.64 (d; 4.6; IH) H20 21(a, b) 2.09 (m; 2H) H20, H22, H24*, H30* 22 5.02 (br t; 5.7; IH) H21(a, b), H24, H30 24 1.60 (br s; 3H) H22 25 1.54 (br s; 3H) H3 26 1.49 (br s; 3H) H7, H8a, H8b HE 5.04 (br s; IH) Hll , H27Z 27Z 5.17 (brs; IH) Hll*, H27£ 28 1.12 (s; 3H) 29 1.48 (br s; 3H) H17*, H18 30 1.53 (br s; 3H) H22 154 Table 18 (continued) Position 5 (mult; / ; number of H) COSY correlations r 3.92 (d; 7.7; IH) H2' V 3.21 (ddd; 9.6, 7.7, 4.7; IH) HI', H3', 2'-OH 2'-OH 4.81 (d; 4.7, IH) H2' 3' 3.33 (m; IH) H2', H4', 3*-OH 3'-OH 4.97 (d; 4.3; IH) H3" 4* 3.56 (m; IH) H3\ H5' 5' 3.44 (br q; 6.2; IH) H4', H6' 6' 1.12 (d; 6.2; 3H) H5' 1" 5.07 (d; 1.4; IH) H2" 2" 3.75 (m; IH) HI", H3", 2"-OH 2"-OH 4.55 (d; 4.2; IH) H2" 3" 3.45 (m; IH) H2", H4", 3"-OH 3"-OH 4.47 (d; 5.8; IH) H3" 4" 3.17 (dt; 9.5, 5.9; IH) H3", H5", 4"-OH 4"-OH 4.63 (d; 5.9; IH) H4" 5" 3.58 (m; IH) H4", H6" 6" 1.09 (d; 6.2; 3H) H5" *: correlations observed in a long range COSY 129 1 5 5 156 Table 19 A H nmr (400 MHz) data for dehydroxestovanin A (129). Spectra were recorded in acetone-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult; J; number of H) COSY correlations nOe enhancementsa 1 1.64 (br s; 3H) H3 3 5.00 (brt; 7.1; IH) HI, H4a, H4b, H25 4a 2.22 (m; IH) HI*, H3, H4b, H5 4b 2.27 (m; IH) HI*, H3, H4a, H5 5 4.07 (dd; 8.6, 6.1; IH) H4a, H4b 7 5.42 (br t; 7.2; IH) H8a, H8b, H26 H5, H17 8a 2.33 (dd; 14.3, 7.2; IH) H7, H8b, H26* 8b 2.46 (dd; 14.3, 7.1; IH) H7, H8a, H26* 11 2.67 (br t; 5.8; IH) H12a, H12b, H27E, H27Z H17, H27E, H28 12a 1.99 (m; IH) H l l , H12b, H13a, H13b 12b 2.14 (m; IH) H l l , H12a, H13b 13(a, b) 1.64 (m; 2H) H12a, H12b, H14a, H14B H14B 1.33 (ddd; 12.9, 7.9, 5.3; IH) H13a, H13b, H14a H14a 2.03 (m; IH) H13b, H14B 17 3.67 (d; 9.3; IH) H18 18 5.60 (br d; 9.3; IH) H17, H29 H17, H20 20 4.01 (m; IH) H21 (a, b) 21(a, b) 2.21 (br t; 6.7; 2H)) H20, H22 22 5.12 (brt; 7.1; IH) H21(a, b), H24, H30 24 1.65 (br s; 3H) H22 25 1.57 (br s; 3H) H3 26 1.54 (br s; 3H) H7, H8a*, H8b* TIE 5.17 (brt; 1.2; IH) H l l , H27Z H l l , H27Z 27Z 5.33 (brt; 1.3; IH) H11,H27£ H27E 28 1.21 (s; 3H) H17 29 1.61 (br s; 3H) H18 30 1.57 (br s; 3H) H22 157 Table 19 (continued) Position 8 (mult.; / ; number of H) COSY correlations nOe enhancementsa r 4.13 (d; 7.6; TH) H2* V 3.49 (br dd; 9.8, 7.6; IH) HI', H3' 3' 3.57 (dd; 9.8, 3.0; IH) H2\ H4' 4' 3.78 (m; IH) H3', H5' 5' 3.60 (dq; 6.4, 1.2; IH) H4\ H6' 6' 1.22 (d; 6.4; 3H) H5' H5' 1" 5.24 (d; 1.5; IH) H2" H5" 2" 4.00 (br s; IH) HI", H3" 3" 3.69 (m; IH) H2", H4" 4" 3.39 (brt; 9.1; IH) H3", H5" 5" 3.79 (m; IH) H4", H6" 6" 1.19 (d; 6.2; 3H) H5" H5" *: correlations observed in long range COSY a : the values in this column represent those protons whose resonances were enhanced upon irradiation of the protons in column 1 OH O 28 129 158 Figure 55 ! H nmr spectrum of dehydroxestovanin A (129) (acetone-d6; 400 MHz) Figure 56 *H COSY 60 spectrum of dehydroxestovanin A (129) (acetone-d6; 400 MHz) Figure 58 HETCOR spectrum of dehydroxestovanin A (129) (acetone-d6; J (CH) = 140 Hz) methylene functionality appeared in both the *H and A 3 C nmr spectra of dehydroxestovanin A (129) (*H nmr (DMS0-d6) 5 5.04 (br s) and 5.17 (br s) ppm; *H nmr (acetone-d6) 8 5.17 (br t) and 5.33 (br t) ppm; l^C nmr (acetone-d6) 8 111.0 (t) and 150.4 (s) ppm) (see Tables 16, 18, and 19). Analysis of the lH COSY spectra of 129 showed that the two new olefinic protons coupled to one another and also coupled into H l l (see Tables 18 and 19). This indicated that dehydroxestovanin A (129) possessed an exocyclic methylene group at CIO of the aglycone bicyclic ring system. An nOe observed between H27£ (8 5.17 ppm) and H l l (8 2.67 (br t; J = 5.8 Hz) ppm) supported this. Comparison of the various spectral data obtained for compounds 110,127,128,129, and 130 showed that, except for the replacement of the methyl substituent at C10 with a double bond, the aglycone portion of dehydroxestovanin A (129) was identical to the aglycone portions of xestovanin A (110) and xestovanin C (127). The relative stereochemistries of the substituents on the bicyclic portion of the aglycone were determined by nOe experiments done on both dehydroxestovanin A (129) and its hexaacetate 130 (see Tables 17 and 19). In particular, irradiation of H l l (acetone-d6; 8 2.67 (br t; J = 5.8 Hz) ppm) in compound 129 induced enhancements into H28 (8 1.21 (s) ppm), H17 (8 3.67 (d; J = 9.3 Hz) ppm), and H27£ (8 5.17 (br t; / = 1.2 Hz) ppm); irradiation of H28 caused an enhancement of H17. An observed nOe between H7 and H17 placed the alkyl substituent at C9 on the same face of the bicyclic ring system as H17 (the (3 face). This meant that the tertiary alcohol group was on the a face, as in compounds 110 and 127. Capillary GC analysis of the (+)-2-octyl glycosides formed from the monosaccharides present in dehydroxestovanin A (129) established the presence of L-rhamnose and D-fucose in the molecule (see experimental). An nOe observed between HI' and H5 in compound 130 (benzene-d6) supported evidence obtained from the acetylation of 129 and the *H nmr data of 129 that the disaccharide moiety was linked to 163 the aglycone at C5, as in all of the previously isolated xestovanins. The resonances assigned to the fucose and rhamnose fragments of 110 and 125 were virtually duplicated in the spectra of 129 indicating that the two molecules contained identical disaccharide substructures. The glycosidic linkage of the rhamnose moiety was assumed to be the same in 129 as it was found to be in 110 and 125 (i.e. a). The structural assignment of compound 129 is supported by the structure of the previously isolated Xestospongia vanilla metabolite secodehydroxestovanin A (112).121(a) Dehydroxestovanin A (129) can arise by either an aldol condensation of 112 or by dehydrogenation of xestovanin A (110). 112 3. Isolation of the Metabolites from the Queen Charlotte Islands Collected Xestospongia vanilla Xestospongia vanilla was collected in a surge channel near Anthony Island in the Queen Charlotte Islands, B. C. (April 1989). The freshly collected sponge (400 g, dry weight) was frozen immediately after collection. Extraction of the metabolites was HO CH 3 OH o CH 3 164 accomplished by soaking the frozen sponge in methanol for three days, after which time the methanol was decanted from the sponge and concentrated under reduced pressure. The aqueous suspension that remained was made up to 300 mL with distilled water and extracted with hexane (3 x 300 mL) followed by dichloromethane (3 x 300 mL). The dichloromethane extracts were combined and concentrated under reduced pressure to yield a brown gummy residue (1.5 g). A portion of this (500 mg) was chromatographed on a Sephadex LH-20 column prepared and eluted with methanol/dichloromethane (9:1). The early eluting fractions contained complex mixtures of compounds which stained heavily (brown) with vanillin; these fractions were combined and further purified by open column reverse phase chromatography (gradient: acetone/H20; 2:3 to acetone/H20; 7:3). 127 Final purification of the compounds was achieved using reverse phase HPLC. Pure samples of dehydroxestovanin A (129) (15 mg) and epi-dehydroxestovanin A (131) (14 mg) were obtained using a solvent system consisting of methanol/H20 (3:1). Pure samples of secodehydroxestovanin A (112) (20 mg) and dehydroxestovanin C (132) (10 mg) were obtained using a solvent system of acetone/H20 (55:45). 4. Novel Metabolites from the Queen Charlotte Islands Collected Xestospongia vanilla Epi-dehvdroxestovanin A (131) 165 Epi-dehydroxestovanin A (131) failed to show a parent ion in its electron impact mass spectra. The highest mass ion observed in the HREIMS of 131, m/z 434.3189 daltons, corresponded to the fragment ion of an aglycone with the same elemental composition as the aglycone in dehydroxestovanin A (129) (C30H42O2; AM, +0.5 mmu). The compound showed a very intense parent ion in its FABMS at m/z 785 daltons (C42H660i2(762) + Na(23)). This mass ion was also observed as the parent ion in the FABMS of dehydroxestovanin A (129). The ir spectrum of epi-dehydroxestovanin A (131) contained stretching frequencies attributable to hydroxyl functionalities (3429 cm_l, very intense) and a carbonyl group (1703 cm_l). Examination of the nmr data of 131 (see Tables 20 and 21 and Figures 59 and 60) confirmed the presence of seven hydroxyl groups, six of them secondary and one tertiary. A resonance at 8 212.6 ppm (s) in the l^C nmr spectrum of 131 (see Table 22 and Figure 61) substantiated the presence of a carbonyl functionality. A comparison of the lHand nmr data for epi-dehydroxestovanin A (131) and dehydroxestovanin A (129) revealed that the two compounds were almost identical in structure. The l^C nmr resonances of the carbons in 129 were virtually duplicated by the resonances for the carbon atoms in 131. As well, analysis of the various iH COSY spectra obtained for epi-dehydroxestovanin A (131) showed that the aglycone portions of compounds 131 and 129 possessed the same scalar coupled spin systems. Although the spectral data for dehydroxestovanin A (129) and epi-dehydroxestovanin A (131) were very similar, there were a few noticeable iH nmr spectral features which differentiated the two compounds. In particular, H l l and H28 were slightly more shielded in epi-dehydroxestovanin A (131) (acetone-d6; 8 2.54 (br t; / = 6.3 Hz) and 1.13 (s) ppm respectively) than they were in dehydroxestovanin A (129) (acetone-d6; 8 2.67 (br t; / = 5.8 Hz) and 1.21 (s) ppm respectively). A series of nOe difference experiments done in 166 Table 20 *H nmr (400 MHz) data for epi-dehydroxestovanin A (131). Spectra were recorded in DMSO-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult; /; number of H) COSY correlations 1 1.63 (br s; 3H) H3, H4a*, H4b* 3 4.89 (brt; 7.1; IH) HI, H4a, H4b, H25 4a 2.16 (m; IH) HI*, H3, H4b, H5, H25* 4b 2.22 (m; IH) HI*, H3, H4a, H5, H25* 5 3.93 (m; IH) H4a, H4b 7 5.32 (br t; 6.6; IH) H8a, H8b, H26 8(a, b) 2.22 (m; 2H) H7, H26* 9-OH 4.57 (s; IH) 11 2.45 (br t; 6.8; IH) H12a, H12b, H27£* 12a 1.90 (m; IH) H l l , H12b, H13a, H13b 12b 2.03 (m; IH) H l l , H12a, H13a, H13b 13a 1.50 (m; IH) H12a, H12b, H13b, H14a, H14B 13b 1.61 (m; IH) H12a, H12b, H13a, H14a, H14B T4B 1.37 (ddd; 12.2, 7.5, 4.5; IH) H13a, H13b, H14a 14a 2.04 (m; IH) H13a, H13b, H14B 17 3.38 (d; 9.5; IH) H18 18 5.27 (br d; 9.5; IH) H17, H29 20 3.81 (brdt; 6.2, 4.0; IH) H21(a, b), 20-OH 20-OH 4.56 (d; 4.0; IH) H20 21(a, b) 2.09 (m; 2H) H20, H22, H24*, H30* 22 5.01 (br t; 7.0; IH) H21(a, b), H24, H30 24 1.59 (br s; 3H) H21(a, b)*, H22 25 1.54 (br s; 3H) H3, H4a*, H4b* 26 1.46 (br s; 3H) H7, H8a*, H8b* TIE 5.07 (br s; IH) Hl l* , H27Z 27Z 5.14 (br s; IH) H27E 28 1.05 (s; 3H) 29 1.52 (br s; 3H) H17*, H18 30 1.52 (br s; 3H) H21(a, b)*, H22 167 Table 20 (continued) Position 8 (mulL; / ; number of H) COSY correlations r 3.94 (d; 7.6; IH) H2' 2* 3.23 (ddd; 9.6, 7.6, 4.6; IH) HI', H3\ 2'-OH 2'-OH 4.76 (d; 4.7; IH) H2' 3' 3.35 (m; IH) H2', H4', 3'-OH 3'-OH 4.94 (d; 4.2; IH) H3' 4' 3.55 (br s; IH) H3\ H5' 5' 3.43 (br q; 6.2; IH) H4', H6' 6' 1.09 (d; 6.2; 3H) H5' 1" 5.07 (br s; IH) H2" 2" 3.75 (m; IH) HI", H3", 2"-OH 2"-OH 4.53 (d; 4.2; IH) H2" 3" 3.45 (m; IH) H2", H4", 3"-OH 3"-OH 4.46 (d; 5.7; IH) H3" 4" 3.17 (ddd; 9.5, 9.3, 5.7; IH) H3", H5", 4"-OH 4"-OH 4.61 (d; 5.7; IH) H4" 5" 3.57 (m; IH) H4", H6" 6" 1.09 (d; 6.2; 3H) H5" *: correlations observed in a long range COSY 131 168 - , , , 1 1 r 1 1 i 1 1 1 1 • 1 1 1 1 1 ' ' 1 • ' ' 1 1 ' 1 ' 1 1 1 ' 1 ' I ^ ~ 5 0 4 . 5 4 . 0 3 . 5 3 . 0 2 . 5 2 . 0 1 . 5 PPM Figure 59 *H nmr spectrum of epi-dehydroxestovanin A (131) ( D M S O - d 6 ; 400 M H z ) Table 21 *H nmr (400 MHz) data for epi-dehydroxestovanin A (131). Spectra were recorded in acetone-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult.; / ; number of H) COSY correlations nOe enhancement^  1 1.65 (br s; 3H) H3 3 4.98 (brt; 7.1; IH) HI, H4a, H4b, H25 4(a, b) 2.27 (m; 2H) H3, H5 5 4.06 (m; IH) H4a, H4b 7 5.43 (br t; 6.5; IH) H8a, H8b, H26 H5, H8a, H8b 8a 2.32 (dd; 14.9, 7.0; IH) H7, H8b 8b 2.36 (dd; 15.0, 6.6; IH) H7, H8a 11 2.54 (br t; 6.3; IH) H12a, H12b H12a, H12b, H27£, H28 12a 1.99 (m; IH) H l l , H12b, H13a, H13b 12b 2.16 (m; IH) H l l , H12a, H13a 13a 1.62 (m; IH) H12a, H12b, H13b, H14a, H14B 13b 1.73 (m; IH) H12a, H13a, H14a, H14B H148 1.42 (ddd; 12.5, 7.8, 4.3; IH) H13a, H13b, H14a H14a 2.03 (m; IH) H13a, H13b, H14B 17 3.58 (d; 9.5; IH) H18 18 5.52 (br d; 9.5; IH) H17, H29 H20 20 4.05 (m; IH) H21 (a, b) 21 (a, b) 2.26 (m; 2H)) H20, H22 22 5.15 (brt; 7.3; IH) H21(a, b), H24, H30 24 1.65 (br s; 3H) H22 25 1.58 (br s; 3H) H3 26 1.54 (br s; 3H) H7 TIE 5.15 (br s; IH) H27Z 27Z 5.33 (br s; IH) H27£ 28 1.13 (s; 3H) H l l , H14B, H28 29 1.64 (br s; 3H) H18 30 1.58 (brs; 3H) H22 170 Table 21 (continued) Position 8 (mult.; /; number of H) COSY correlations nOe enhancements* r 4.12 (d; 7.6; IH) H2' V 3.50 (dd; 10.0, 7.6; IH) HI', H3' V 3.58 (m; IH) H2\ H4' 4' 3.76 (dd; 2.7, 1.0; IH) H3", H5" 5* 3.60 (dq; 6.4, 1.0; IH) H4', H6' 6' 1.22 (d; 6.4; 3H) H5' 1" 5.24 (d; 1.4; IH) H2" 2" 4.00 (br s; IH) HI", H3" 3" 3.69 (m; IH) H2", H4" 4" 3.39 (br t; 9.3; IH) H3", H5" 5" 3.78 (m; IH) H4", H6" 6" 1.19 (d; 6.2; 3H) H5" *: correlations observed in long range COSY a : the values in this column represent those protons whose resonances were enhanced upon irradiation of the protons in column 1 OH O 2 8 131 171 ' ' « ! | ' <.'• l.'a ' l . ' l 3 !4 i'.2 S.'l I.'B ' I.'S i l * ' 2.'l K B ' I.'« PPM Figure 60 *H nmr spectrum of epi-dehydroxestovanin A (131) (acetone-d6; 400 MHz) Table 22 ™C nmr (75 MHz) and one-bond H E T C O R (optimized for / (CH) = 140 Hz) data for epi-dehydroxestovanin A (131). Spectra were recorded in acetone-d6-Position Chemical shift (ppm) mult.* H E T C O R correlation 1 26.0 q 1.65 (br s) 2 133.0 s 3 121.3 d 4.98 (br t) 4 34.7 a t 2.27 (m) 5 83.1 d 4.06 (m) 6 136.5 s 7 125.3 d 5.43 (br t) 8 38.7 t 2.32 (m), 2.36 (m) 9 79.4 s 10 150.3 s 11 52.8 d 2.54 (br t) 12 32.6 t 13 23.4 t 14 34.7 a t 15 55.5 s 16 212.6 s 17 59.0 d 3.58 (d) 18 119.7 d 5.52 (d) 19 143.9 s 20 77.5 d 4.05 (m) 21 32.9 a t 2.26 (m) 22 121.9 d 5.15 (brt) 23 133.0 s 24 26.0 q 1.65 (brs) 25 18.1 q 1.58 (br s) 26 11.1 q 1.54 (br s) 27 110.3 t 5.15(brs), 5.33 28 (brs) 26.6 q 1.13 (s) 29 13.0 q 1.64 (br s) 30 18.1 q 1.58 (br s) 173 Table 22 (continued) Position Chemical shift (ppm) mult.* HETCOR correlation r 110.1 d 4.12 (d) 2 ' 71 .0 d 3.50 (dd) 3 * 7 5 . 9 d 3.58 (m) 4' 7 8 . 2 d 3.76 (dd) 5 ' 7 0 . 8 d 3.60 (dq) 6 ' 17.7 q 1.22 (d) 1" 102.5 d 5.24 (d) 2" 71.0 d 4 . 0 0 (br s) 3 " 7 2 . 5 d 3.69 (m) 4 " 7 3 . 6 d 3.39 (br t) 5 " 69.7 d 3.78 (dq) 6" 18.0 q 1.19 (d) *: multiplicities were determined via APT a : may be interchanged within a column 131 174 175 acetone-d.6 revealed that epi-dehydroxestovanin A (131) was epimeric with dehydroxestovanin A (129) at C17 (see Figure 62). Irradiation of H l l induced enhancements in H27E (5 5.15 (br s) ppm), H8a and H8b (5 2.32 (dd; / = 14.9, 7.0 Hz) and 2.36 (dd; J = 15.0, 6.6 Hz) ppm), H12a and H12b (8 1.99 (m) and 2.16 (m) ppm), and H28 (8 1.13 (s) ppm). Irradiation of H28 induced enhancements in H14{3 (8 1.42 (ddd; J = 12.5, 7.8, 4.3 Hz) ppm), HI 1 (8 2.54 (br t; J = 6.3 Hz) ppm), and H18 (8 5.52 (br d; / = 9.5 Hz) ppm). The observation of an nOe between H28 and HI8 was not expected, since all of the other compounds in the xestovanin series which have bicyclic aglycones showed an enhancement of H17 upon irradiation of H28. In order to see an enhancement between H28 and HI8, the alkyl substituent at C17 had to be on the P face of the bicyclic substructure. In keeping with what has previously been found for compounds in the xestovanin family, the tertiary OH group at C9 was assigned to be on the a face of the bicyclic ring system. Figure 62 Selected nuclear Overhauser enhancements observed in 129 and 131 Capillary GC analysis of the (+)-2-octyl glycosides formed from the monosaccharides present in epi-dehydroxestovanin A (131) established that the two R 129 131 176 sugars present in the molecule were L-rhamnose and D-fucose (see experimental).1^ The resonances assigned to the fucose and rhamnose fragments of dehydroxestovanin A (129) were virtually duplicated in the spectra of 131 indicating that the two molecules contained identical disaccharide substructures. The glycosidic linkage of the rhamnose moiety was assumed to be the same in epi-dehydroxestovanin A (131) as it was found to be in xestovanin A (110), isoxestovanin A (125), and dehydroxestovanin A (129) (i.e. a). Dehydroxestovanin C (132) A very intense parent ion was observed at m/z 931 daltons (C48H76Oi6(908) + Na(23)) in the FABMS of dehydroxestovanin C (132). This ion was two mass units less than the parent ion observed for xestovanin C (127). The HREIMS of compound 132 possessed a mass ion at m/z 434.3191 daltons, corresponding to an elemental composition of C30H42O2 (AM, +0.6 mmu). This mass ion represented the aglycone portion of dehydroxestovanin C (132); the elemental formula calculated for the aglycone of 132 was identical with that obtained for the aglycone of dehydroxestovanin A (129). OH O 132 1 7 7 Table 23 *H nmr (400 MHz) data for dehydroxestovanin C (132). Spectra were recorded in DMSO-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult; / ; number of H) COSY correlations 1 1.61 (br s; 3H) H3, H4a*, H4b* 3 4.90 (br t; 6.6; IH) HI , H4a, H4b, H25 4a 2.16 (m; IH) HI*, H3, H4b, H5 4b 2.19 (m; IH) HI* , H3, H4a, H5 5 3.95 (dd; 8.7, 6.1; IH) H4a, H4b 7 5.26 (br t; 6.8; IH) H8a, H8b, H26 8a 2.20 (m; IH) H7, H8b, H26* 8b 2.34 (dd; 15.0, 6.8; IH) H7, H8a, H26* 9-OH 4.64 (s; IH) 11 2.62 (m; IH) H12a, H12b 12a 1.86 (m; IH) H l l , H12b, H13a, H13b 12b 2.01 (m; IH) H l l , H12a, H13a, HI 3b 13(a, b) 1.57 (m; 2H) H12a, H12b, H14a, H14B 14B 1.24 (m; IH) H13a, H13b, H14a 14a 1.94 (m; IH) H13a, H13b, H14B 17 3.56 (d; 8.9; IH) H18 18 5.44 (d; 8.8; IH) HI7, H29 20 3.84 (dt; 7.1, 4.9; IH) H21(a, b), 20-OH 20-OH 4.63 (d; 5.0; IH) H20 21(a, b) 2.09 (m; 2H) H20, H22, H24*, H30* 22 5.02 (m; IH) H21(a, b), H24, H30 24 1.60(brs;3H) H21(a, b)*, H22 25 1.54 (br s; 3H) H3 26 1.47 (br s; 3H) H7, H8a*, H8b* 21E 5.04 (br s; IH) H27Z 27Z 5.17 (br s; IH) miE 28 1.10 (s; 3H) 29 1.48 (br s; 3H) H18 30 1.53 (br s; 3H) H21(a, b)*, H22 178 T a b l e 23 Position (continued) 8 (mult; / ; number of H) COSY correlations r 3.92 (d; 7.6; IH) H2' 2' 3.22 (m; IH) HI', H3', 2'-OH 2'-OH 4.80 (d; 4.7; IH) H2' 3' 3.30 (m; IH) H2', H4', 3'-OH 3'-OH 4.99 (d; 4.3; IH) H3' 4' 3.57 (m; IH) H3', H5' 5' 3.39 (m; IH) H4', H6' 6' 1.09 (d; 6.1; 3H) H5' 1" 5.06 (br s; IH) H2" 2" 3.74 (br s; IH) HI", H3", 2"-OH 2"-OH 4.76 (d; 4.5; IH) H2" 3" 3.60 (m; IH) H2", H4", 3"-OH 3"-OH 4.69 (d; 6.8; IH) H3" 4" 3.39 (t; 9.3; IH) H3", H5" 5" 3.62 (m; IH) H4", H6" 6" 1.13 (d; 6.1; 3H) H5" 1"' 5.06 (br s; IH) H2'" 2'" 3.71 (br s; IH) HI"', H3'", 2"'-OH 2"'-OH 4.56 (d; 4.3; IH) H2"' 3"' 3.38 (m; IH) H2'", H4"', 3"*-OH 3'"-OH 4.43 (d; 5.8; IH) H3'" 4'" 3.18 (m; IH) H3'", H5'", 4"'-OH 4"'-OH 4.62 (d; 5.9; IH) H4'" 5'" 3.49 (dq; 9.3, 6.2; IH) H4'", H6"* 6"' 1.10 (d; 6.2; 3H) H5"' *: correlations observed in a long range COSY 132 179 Table 24 lH nmr (400 MHz) data for dehydroxestovanin C (132). Spectra were recorded in acetone-d6; chemical shifts are reported in ppm using internal TMS as a reference. Coupling constants are in Hz. Position 8 (mult; / ; number of H) COSY correlations 1 1.65 (br s; 3H) H3 3 5.00 (br t; 6.8; IH) HI, H4a, H4b, H25 4a 2.21 (m; IH) HI*, H3, H4b, H5 4b 2.27 (m; IH) HI*, H3, H4a, H5 5 4.07 (dd; 8.7, 6.1; IH) H4a, H4b 7 5.44 (br t; 6.0; IH) H8a, H8b, H26 8a 2.35 (dd; 15.1, 6.6; IH) H7, H8b, H26* 8b 2.46 (dd; 15.1, 6.7; IH) H7, H8a, H26* 11 2.67 (br dd; 8.0, 3.9; IH) H12a, H12b, H27E 12a 1.99 (m; IH) H l l , H12b, H13a, HI 3b 12b 2.14 (m; IH) Hll ,H12a, H13a, H13b 13(a, b) 1.65 (m; 2H) H12a, H12b, H14a, H146 146 1.33 (m; IH) H13a, H13b, H14a 14a 2.03 (m; IH) H13a, H13b, H146 17 3.67 (d; 9.3; IH) HI 8 18 5.58 (d; 9.3; IH) HI7, H29 20 4.01 (m; IH) H21(a, b) 21(a, b) 2.21 (m; 2H) H20, H22 22 5.12 (brt; 7.0; IH) H21(a, b), H24, H30 24 1.65 (br s; 3H) H22 25 1.58 (br s; 3H) H3 26 1.56 (brs; 3H) H7, H8a*, H8b* 21E 5.17 (brs; IH) H27Z, H l l 27Z 5.33 (br s; IH) H27£ 28 1.21 (s; 3H) 29 1.63 (d; 1.3; 3H) H18 30 1.58 (br s; 3H) H22 1 8 1 Table 24 (continued) Position 8 (mult; / ; number of H) COSY correlations r 4.13 (d; 7.6; IH) H2' 2' 3.51 (brt; 7.8; IH) HI', H3' 3' 3.58 (m; IH) H2\ H4' 4' 3.75 (br d; 2.0; IH) H3', H5' 5' 3.60 (m; IH) H4\ H6* 6' 1.23 (d; 6.4; 3H) H5' 1" 5.21 (d; 1.4; IH) H2" 2" 4.00 (br s; IH) HI", H3" 3" 3.71 (brd; 10.2; IH) H2", H4" 4" 3.56 (m; IH) H3", H5" 5" 3.83 (m; IH) H4", H6" 6" 1.23 (d; 6.2; 3H) H5" 1"' 5.25 (d; 1.4; IH) H2 , M 2"' 3.93 (br s; IH) HI"', H3"' 3"' 3.61 (br d; 9.5; IH) H2"', H 4 m 4"' 3.39 (br t; 9.4; IH) H3"', H5"' 5"' 3.69 (m; IH) H4"', H6"' 6"' 1.20 (d; 6.0; 3H) H5"' *: correlations observed in a long range COSY 132 182 00 CO —, , , 1 r—. 1 • | . . . i 1 • • ' ' 1 ' ' ' ' 1 ' ' ' ' 1 1 1 ' ' I ' ' r 4 5 4.0 3.5 3.0 2.5 2.0 1.5 PPM Figure 64 *H nmr spectrum of dehydroxestovanin C (132) (acetone-d6; 400 MHz) Table 25 1 3 C nmr (75 MHz) and one-bond HETCOR (optimized for / (CH) =140 Hz) data for dehydroxestovanin C (132). Spectra were recorded in acetone-d6-Position Chemical shift (ppm) mult.* HETCOR correlation 1 26.0 q 1.65 (br s) 2 132.9 s 3 121.3 d 5.00 (br t) 4 34.8a t 2.21 (m), 2.27 (m) 5 83.1 d 4.07 (dd) 6 136.1 s 7 126.1 d 5.44 (br t) 8 38.9 t 9 79.4 s 10 150.5 s 11 52.5 d 2.67 (br dd) 12 29.0 t 13 22.9 t 1.65 (m) 14 36.4 t 2.03 (m) 15 56.2 s 16 212.7 s 17 57.6 d 3.67 (d) 18 120.4 d 5.58 (d) 19 142.7 s 20 77.5 d 4.01 (m) 21 32.7a t 22 122.1 d 5.12 (brt) 23 132.9 s 24 26.0 q 1.65 (brs) 25 18.0b q 1.58 (br s) 26 11.1 q 1.56 (br s) 27 111.0 t 28 25.9 q 1.21 (s) 29 12.7 q 1.63 (d) 30 18.lb q 1.58 (br s) 184 Table 25 (continued) Position Chemical shift (ppm) mult.* HETCOR correlation r 99.9 d 4.13 (d) 2' 71.9 d 3.51 (brt) 3* 79.6 d 3.58 (m) 4" 78.6 d 3.75 (br d) 5' 75.9 d 3.60 (m) 6* 17.7 q 1.23 (d) 1" 102.4 d 5.21 (d) 2" 72.5 d 4.00 (br s) 3" 73.0 d 3.71 (brd) 4" 72.4 d 3.56 (m) 5" 68.2 d 3.83 (m) 6" 18.7 q 1.23 (d) 1"' 102.2 d 5.25 (d) 2'" 72.0 d 3.93 (br s) 3'" 70.8 d 3.61 (br d) 4"' 73.6 d 3.39 (br t) 5"' 69.7 d 3.69 (m) 6"' 18.0 q 1.20 (d) *: multiplicities were determined via APT ': may be interchanged within a column 185 1 8 6 The structure of dehydroxestovanin C (132) was readily deduced by comparing the spectral data obtained for this compound with the spectral data for both dehydroxestovanin A (129) and xestovanin C (127). Analysis of the *H nmr data (see Tables 23 and 24 and Figures 63 and 64) and the nmr data (see Table 25 and Figure 65) obtained for 132 showed that the aglycone portion of this metabolite was identical with the aglycone of dehydroxestovanin A (129). Capillary GC analysis of the (+)-2-octyl glycosides formed from the monosaccharides present in dehydroxestovanin C (132) determined that the only sugars present in 132 were D-fucose and L-rhamnose (see experimental). 129 The resonances assigned to the fucose and rhamnose fragments of xestovanin C (127) were virtually duplicated in the spectra of dehydroxestovanin C (132) indicating that the two molecules contained identical trisaccharide substructures. The structural assignment of dehydroxestovanin C (132) was supported by extensive ! H COSY and HETCOR (optimized for / (CH) = 140 Hz) analyses (see Tables 23-25). 5. Attempts at Determining the Absolute Stereochemistries of the Chiral Centres in  Xestovanin A (110) The first attempt to determine the absolute stereochemistries of the chiral centres in xestovanin A (110) was to make a crystalline derivative of the entire molecule. To this end, two derivatives of xestovanin A (110), xestovanin A hexaacetate 133 and xestovanin A hexaparabromobenzoate 134, were synthesized (see experimental). Pure xestovanin A hexaacetate 133 was isolated as an oil and showed no signs of crystallization even after repeated HPLC purification (both normal and reversed phase). Pure 134 was also isolated as an oil; however, repeated attempts at crystallization did result in this compound forming an amorphous powder. Unfortunately, no signs of crystallization were observed. The failure of both 133 and 134 to show any signs of crystallization led us to believe that the 187 determination of the absolute configuration of 110 might not be best accomplished by crystallization of a derivative of the native metabolite. Treatment of xestovanin A (110) with potassium hydroxide in MeOH (80°C) resulted in elimination of the disaccharide to form compound 135. It was thought that either 135 itself or a derivative of it would form suitable X-ray quality crystals. However, the pure compound was continually isolated as an oil after repeated HPLC purifications and no signs of crystallization were observed in any solvent system tested. Acetyl and parabromobenzoyl derivatives of 135 were found to be unstable and were, therefore, very difficult to purify for crystallization purposes; these derivatives readily underwent double bond isomerization, dehydration, and elimination when purified on silica or when left in nmr solvents for short periods of time. Perhaps if the secondary alcohol in 135 were derivatized with a less efficient leaving group, the resulting derivative would be stable and crystallization might be achieved. 135 The most promising results towards solving the absolute stereochemistries of some of the chiral centres in xestovanin A (110) came from the ozonolysis of both xestovanin A hexaacetate 133 and xestovanin A hexaparabromobenzoate 134. Ozonolysis of 133 resulted in the production of two major products, 136 and 137. Compound 136 has 188 been observed to form microcrystalline needles from diethyl ether, cyclohexane, and mixtures of diethyl ether and petroleum ether. If a suitable crystal of 136 can be obtained, then the absolute configuration of the C5 centre in xestovanin A (110) can be determined. Unfortunately, none of the crystals have as yet been large enough for X-ray crystallographic analysis. The second product obtained from the ozonolysis of xestovanin A hexaacetate, 137, has not shown signs of crystallization in its native form; however, if the hemi-ketal functionality in 137 can be suitably derivatized, then crystallization may be possible and the absolute configuration of the bicyclic portion of the aglycone of xestovanin A (110) could then be determined. Another possibility exists for the determination of the absolute stereochemistry of the bicyclic ring system present in 110 through use of compound 137. This compound possesses an enone functionality as well as a hydroxyl group (present in the hemi-ketal functionality) which can be derivatized with a para-substituted benzoate moiety. If the hydroxyl group can be derivatized with a para-substituted benzoate group possessing a ^ max at a wavelength close to that of the enone, then the absolute configurations of the chiral centres in 137 (and, thus, of those in the bicyclic portion of 110) might be determined using the CD exciton chirality method. 131 Ozonolysis of xestovanin A hexaparabromobenzoate 134 resulted in the production of two isolatable compounds, 138 and 139. Compound 138, like 136, has shown signs of crystallization but as yet has not produced suitable X-ray quality crystals. The fairly unstable compound 139 has been isolated only as an oil and does not show much promise for crystallization. 189 139 Conclusions The five new secondary metabolites isolated from the two collections of Xestospongia vanilla add to the number of triterpenoid glycosides already isolated from this species of sponge. The new compounds all share similar structural features with those isolated previously. In particular, all of the compounds contain only the deoxy sugars D-fucose and L-rhamnose and all have unrearranged triterpenoid carbon skeletons. Isoxestovanin A (125) possesses a new triterpenoid carbon skeleton and xestovanin C (127) and dehydroxestovanin C (132) contain linear trisaccharide substructures which had not been previously encountered in the xestovanin series of compounds. The unique structures of the xestovanins and their abundance in extracts from both the Bamfield and the Queen Charlotte Islands collected Xestospongia vanilla suggest that 190 they are biosynthesized by the sponge. The likely precursor to all of these compounds is squalene. Allylic oxidation of squalene at four equivalent positions followed by attachment of the sugar moieties (either di- or trisaccharide) and cyclization (joining C l 1 and C15) to form a five membered ring would result in the production of secodehydroxestovanin A (112) or the equivalent trisaccharide containing compound (as yet, a trisaccharide version of 112 has not been found in extracts of X. vanilla). The aglycone present in 112 has the correct oxidation level and carbon skeleton expected for the first product of such a cyclization of a squalene-type precursor. An aldol condensation of 112 or its possible trisaccharide equivalent would then result in the production of the metabolites dehydroxestovanin A (129), epi-dehydroxestovanin A (131), and dehydroxestovanin C (132). If the product of the initial cyclization of the squalene precursor undergoes reduction of the C10-C27 double bond, then a compound possessing the aglycone present in secoxestovanin A (111) would be formed. Aldol condensations of triterpenoid glycosides possessing this aglycone would enable the formation of metabolites such as xestovanin A (110), xestovanin B (113), xestovanin C (127), isoxestovanin A (125). The above biogenetic proposals for the xestovanins are illustrated in Scheme 4. No evidence has as yet been found to determine the order of the biosynthetic transformations which result in the production of the xestovanins. For example, it may be that the sugars are added prior to or following the original cyclization to form the monocyclic triterpenoid glycosides. Isolation of compounds 111 and 112 suggests that glycosidation occurs prior to the aldol condensations in the metabolites possessing disaccharide moieties. Since no monocyclic triterpenoid glycosides possessing trisaccharide fragments have as yet been isolated, it is not known whether the third sugar is added prior to or following the aldol condensation. Although it is clear that the triterpenoid glycosides isolated form the Bamfield and the Queen Charlotte Islands collected samples of X. vanilla are derived from the same 191 biogenetic pathway, there is a very noticeable difference between the metabolites from the two sources. The major metabolite in all of the extracts looked at to date from the Bamfield collected sponge is xestovanin A (110). No trace of this compound or any compound possessing a reduced C10-C27 double bond was observed in X. vanilla extracts from the Queen Charlotte Islands. Perhaps the Queen Charlotte Islands sponge lacks the enzyme responsible for the reduction of this bond. The observation of a difference in the chemistry of X. vanilla due to a relatively minor change in its geographical location is very interesting. To our knowledge, very little work has been done on how the chemistry of a particular species of sponge changes with geographical location. Our findings suggest that this may be an area of marine natural products chemistry which warrants more attention. At present, the absolute stereochemistries of the chiral centres in the aglycone of xestovanin A (110) remain undetermined. Work is still in progress in this area and it is hoped that some of the methods mentioned previously may lead eventually to the determination of one or more of the aglycone chiral centre absolute configurations. None of the work to date has held any promise for the determination of the absolute stereochemistry of the C20 centre. The determination of this centre's configuration may be best accomplished by degradation of a derivative of xestovanin B (113) rather than xestovanin A (110). Xestovanin B possesses a glycosidic linkage at C20 whereas 110 has only a hydroxyl group at this position. Derivatization of 113 followed by ozonolysis would produce a compound similar in structure to compounds 136 and 138, both of which have shown signs of crystallization. Not all of the xestovanins displayed biological activities. Both xestovanin A (110) and dehydroxestovanin A (129) were found to have significant levels of antifungal activity (see experimental). Epi-dehydroxestovanin A (131) displayed both antibacterial and antifungal activities. Dehydroxestovanin C (132) was found to be active against both 192 bacteria and the yeast Candida albicans. in all of our biological screens. The other xestovanins were found to be inactive squalene oxidation glycosidation R = di- or trisaccharide reduction OH O 111 (or CIO epimer) R = disaccharide 1 1 0 (R = disaccharide) 1 2 7 (R = trisaccharide) t 1 2 5 (R = disaccharide) OH O 112 R = disaccharide 129 (R = disaccharide) aldol condensation 1 3 2 (R = trisaccharide) 1 3 1 (R = disaccharide) Scheme 4 Proposed biogenesis of the xestovanins 193 P a r t II M a r i n e N a t u r a l Products Isolated from T w o Species of N u d i b r a n c h Belonging to the Genus Chromodoris Introduction 1. General The order Nudibranchia comprises the largest of seven orders within the subclass Opisthobranchia (class Gastopoda; Phylum Mollusca). Nudibranchs, often referred to as sea slugs, are slow-moving, shell-less marine molluscs. They are often very brighdy coloured and sometimes possess quite striking patterns on their body surfaces. Outwardly, nudibranchs appear to be very vulnerable to predation. However, they are not generally observed to be fed upon by other marine organisms due a number of factors, among these being their employment of secondary metabolites, acquired from the sponges in their diets, as chemical defense allomones.28,132(a-c) As a part of our laboratory's on-going studies of the chemistry of nudibranch skin extracts, we have recendy examined the chemistry of a number of Chromodoris species collected in the coastal waters of Sri Lanka. 133,134 The work presented here describes the chemistry of two of the Sri Lankan chromodorid nudibranchs, Chromodoris glenei (see Figure 66) and Chromodoris cavae (see Figure 67). 2. The Chemistry of Chromodorid Nudibranchs: Spongian Diterpenes The chemistry of nudibranchs belonging to the genus Chromodoris is characterized by the presence of terpenoid compounds. Among the most interesting of the terpenoid 1 9 4 Figure 66 A specimen of Chromodoris glenei (top of picture) Figure 67 A specimen of Chromodoris cavae metabolites that have been isolated from skin extracts of chromodorid nudibranchs are those whose structures are thought to be derived from the hypothetical "spongian" diterpene skeleton 140. These metabolites are not true nudibranch metabolites but are derived from the sponges upon which the nudibranchs feed; hence, spongian diterpenes have also been isolated from many species of sponge. A comprehensive review of these compounds has recently been made 135 a n ( j ? therefore, no such review will be presented here. An overview of the previously isolated spongian diterpenes which are related to the compounds described in this work will, however, be presented. 140 A number of spongian diterpenes possessing the perhydroazulene-containing "dendrillane" skeleton have been isolated from both nudibranchs belonging to the genus Chromodoris and a variety of sponge species. The first report of the isolation of dendrillane diterpenoids was made by Sullivan and Faulkner. These authors reported the compounds dendrillolide A (141), dendrillolide B, and dendrillolide C (142) to be constituents of the deep water sponge Dendrilla sp..!36 Subsequent to this publication, Hambley et al. reported the isolation of aplyviolene (143) and aplyviolacene (144) from the sponge Chelonaplysilla violacea?^ The structure of aplyviolene (143), confirmed by X-ray diffraction analysis, was identical to the structure originally proposed for dendrillolide A (141) by Sullivan and Faulkner. Because of this, a reinvestigation of the 197 structural assignment of dendrillolide A (141) was made. 138 The revised structure of 141 is identical to that previously assigned to dendrillolide B, the correct structure of which has not yet been determined. The compound macfarlandin E (144), isolated from the nudibranch Chromodoris macfarlandi, was reported concurrently with the structure determinations of aplyviolene and aplyviolacene.139 This compound has been assigned a structure identical with that proposed for aplyviolacene (144). The authors of the latter paper chose to rename the compound based upon a lack of reported spectral evidence for 144 in the original report 143 144 Six rearranged diterpene metabolites possessing the dendrillane skeleton were isolated from extracts of the Red Sea sponge Dysidea sp. by Kashman and co-198 workers. 140 The structures of shahamins A-E (145-149) were elucidated from spectral data; the structure of the sixth compound, macfarlandin E (144), was determined by comparison with published spectral data. Aplysilla polyrhaphis, collected in the Gulf of California, contained two dendrillane derivatives, polyrhaphins A (150) and B (151). 141 Specimens of the nudibranch Chromodoris norrisi, found at the same site as the Aplysilla polyrhaphis sample, were also found to contain compound 150. Finally, Bobzin and Faulkner recently reported the isolation of 12-desacetoxypolyrhaphin A (152) and 12-desacetoxyshahamin C (153) from the Palauan sponge Dendrilla sp..l38 148 149 1 9 9 152 153 154 The chemistry of the nudibranch Chromodoris cavae has been studied previously by Andersen and co-workers. 133 A novel diterpenoid compound, chromodorolide A (154), was isolated from this nudibranch and its structure was determined by single crystal X-ray diffraction analysis. Chromodorolide A (154) possesses a new rearranged spongian skeleton which has been named the "chromodorane" skeleton. The compound displayed both cytotoxic and antimicrobial activities. 200 Results and Discussion 1. Isolation of the Metabolites from the Nudibranch Chromodoris glenei A collection of twelve animals was made in December 1987 in the coastal waters near Mt. Lavenia, Sri Lanka. Freshly collected animals were preserved in a 1:1 rnixture of dichloromethane and methanol which was kept at -4°C until workup. The organic extract was decanted off and concentrated under reduced pressure to give 200 mg of an orange residue which was fractionated by silica flash chromatography (gradient: CH2CI2 to CH2Cl2/MeOH; 100:1). A mixture of 12-desacetoxyshahamin C (153) and shahamin K (155) was isolated in the more polar fractions. Pure samples of 12-desacetoxyshahamin C (153) (7.5 mg) and shahamin K (155) (3 mg) were obtained from the combined polar fractions by sequential application of Sephadex LH-20 (hexane/CH2Cl2/MeOH; 2:1:1) and silica gel flash (CH2Cl2/MeOH; 100:1) chromatographies. 2. Novel Metabolites Isolated from the Nudibranch Chromodoris glenei  12-Desacetoxvshahamin C (153) OAc 153 201 Table 26 * H nmr (400 M H z ) data for 12-desacetoxyshahamin C (153) and shahamin K (155). Spectra were recorded in CDCI3; chemical shifts are given in ppm using internal T M S as a reference. Coupling constants are in H z . Posit ion 8 (mult.; / ; number of H) (153)* 8 (mult.; J; number of H) (155)* l a 1.80 (m, IH) 1.95 (dd; 12.7, 12.6; IH) l b 2.36 (br dd; IH) 2.39 (dd; 12.7, 5.1; IH) 2a 1.39 (m; IH) 1.40 (m; IH) 2b 1.80 (m; IH) 1.78 (m; IH) 3a 1.27 (brdt; IH) 1.30 (ddd; 14.0, ~4, 3.7; IH) 3b 1.62 (dt; IH) 1.57 (ddd; 14 .0 , -12 , 3.7; IH) 5 1.92 (ddd; 10.6, 8.9, 8.7; IH) 1.82 (m, IH) 6 1.72 (m; 2H) (a, b) 1.80 (m; IH) (B) 2.17 (m; IH) (a) 7 1.51 (m; lH ) (a ) 4.98 (br dd; 9.0, 5.3; IH) (a) 1.56 (m; IH) (b) 9 2.74 (d; 8.7; IH) 2.80 (br d; 7.7; IH) 12 2.55 (m; 2H) (a, b) 2.55 (d; 3.8; IH) (a) 2.56 (d; 6.5; IH) (b) 13 2.48 (m; IH) 2.49 (m; IH) 14 1.77 (m; IH) 1.80 (m; IH) 15B 4.21 (dd; 11.8, 10.0; IH) 4.20 (dd; 12.0, 9.8; IH) 15a 4.32 (dd; 11.8,6.1; IH) 4.28 (dd; 12.0, 6.0; IH) 16 3.85 (dd; 11.2, 7.8; IH) (a) 3.89 (dd; 11.2,7.5; IH) (a) 4.19 (dd; 11.2, 4.2; IH) (b) 4.21 (dd; 11.4,4.4; IH) (b) 17 0.93 (s; 3H) 0.93 (s; 3H) 18 0.95 (s; 3H) 0.95 (s; 3H) 19 1.00 (s; 3H) 1.01 (s; 3H) 20Z 4.63 (b rd ; 2.1; IH) 4.69 (br d; 1.7; IH) 20E 4.86 (brd ; 2.1; IH) 4.94 (br d; 1.7; IH) OAc 2.08 (s; 3H) 2.06 (s; 3H) OAc 2.07 (s; 3H) * : assignments based upon * H C O S Y and double resonance experiments 2 0 2 Table 27 liC nmr (75 MHz) data for 12-desacetoxyshahamin C (153) and shahamin K (155). Spectra were recorded in CDCI3; chemical shifts are in ppm. Position 8 (mult.)* (153) 8 (mult.)* (155) 1 37.0 (t) 36.7 (t) 2 28.8 (t) 28.8 (t) 3 37.6 (t)a 37.8 (t) 4 48.5 (s) 49.7 (s) 5 54.4 (d) 48.7 (d) 6 25.9 (t) 32.5 (t) 7 37.7 (t) 78.5 (d) 8 36.2 (s) 36.0 (s) 9 54.8 (d) 54.2 (d) 10 153.7 (s) 153.0 (s) 11 170.8 (s) 170.8 (s) 12 32.1 (t) 32.2 (t) 13 32.0 (d) 31.5 (d) 14 44.1 (d) 44.6 (d) 15 68.2 (t) 67.9 (t) 16 67.5 (t) 67.0 (t) 17 21.3 (q) 21.2 (q) 18 34.4 (q) 34.4 (q) 19 25.6 (q) 25.3 (q) 20 115.0 (t) 116.3 (t) OAc 20.7 (q) 20.8 (q) 172.9 (s) 172.6 (s) OAc 16.1 (q) 170.7 (s) *: multiplicites determined by APT; protonated carbons were assigned based upon one bond HETCOR data 203 OCOCH 3 Figure 68 ! H nmr spectrum of 12-desacetoxyshahamin C (153) (CDC13; 400 MHz) 205 OCOCH3 Figure 70 *H nmr spectrum of shahamin K (155) (CDC13; 400 MHz) 207 12-Desacetoxyshahamin C (153) showed a parent ion in the HREIMS at m/z 362.2541 daltons, appropriate for a molecular formula of C22H34O4 (AM, +0.6 mmu). The molecular formula indicated that there were six sites of unsaturation in 153, two of which could be accounted for by carbonyl functionalities (ir: 1748 cm* 1; 13C nmr (CDCI3) 8 170.8 (s) and 172.9 (s) ppm) and another by an exocyclic double bond (13c nmr (CDCI3) 8 115.0 (t) and 153.7 (s) ppm; iH nmr (CDCI3) 8 4.63 (d; J = 2.1 Hz) and 4.86 (d; J = 2.1 Hz) ppm). The remaining three sites of unsaturation could be accounted for by assigning a tricyclic structure to 153. A major fragment ion in the HREIMS of 153 (m/z 191.1813 daltons; C14H23; AM, +1.3 mmu) suggested the presence of a perhydroazulene fragment, indicating that the compound might be related to the dendrillane spongian diterpenes. Compound 153 was then readily identified as 12-desacetoxyshahamin C (153) based upon a comparison of the iH and 13c nmr data which we obtained for this compound (see Tables 26 and 27 and Figures 68 and 69) with literature values. 138 Shahamin K (155) 155 Shahamin K (155) was obtained as a clear oil that showed a parent ion in the HREIMS at m/z 420.2517 daltons, corresponding to a molecular formula of C24H36O6 208 (AM, -0.5 mmu). The molecular formula obtained for 155, requiring seven sites of unsaturation, differed from that of 12-desacetoxyshahamin C (153) by the addition of C2H2O2. The *H and ^ C nmr data for shahamin K (155) were very similar to those for 153 (see Tables 26 and 27), indicating that the two metabolites were closely related. The presence of two acetoxy functionalities in 155 was evidenced in both the *H and nmr spectra (see Figures 70 and 71) of shahamin K (155) (*H nmr (CDCI3) 8 2.06 (s, 3H) and 2.07 (s, 3H) ppm; l^C nmr (CDCI3) 8 170.7 (s) and 172.6 (s) ppm). A downfield methine proton (8 4.98 ppm) not present in the *H nmr spectrum of 153 (see Table 26) indicated that the extra atoms in shahamin K (155) could be assigned to a secondary acetate group. Thus, it was apparent that shahamin K (155) was simply an acetoxy derivative of 12-desacetoxyshahamin C (153). The *H and nmr resonances assigned to the 8 lactone fragment of shahamin K (155) were virtually identical to those previously assigned to the same fragment in 12-desacetoxyshahamin C (153) (see Tables 26 and 27), indicating that this portion of the two molecules was identical. The presence of the 8 lactone fragment accounted for three of the seven sites of unsaturation -in 155 (including one of the acetate groups). The remaining four sites of unsaturation could be attributed to an acetylated version of the perhydroazulene moiety contained in 153. Two methyl singlets which were shown to be w-coupled to one another by a COSY experiment optimized for long range scalar couplings (iH nmr 8 0.95 (3H) and 1.01 (3H) ppm) were assigned as the C4 geminal methyl pair (see Table 28). nmr resonances at 8 116.3 (t) and 153.0 (s) ppm and pair of scalar coupled *H nmr signals at 8 4.69 (d; / = 1.7 Hz) and 4.94 (d; J = 1.7 Hz) ppm were assigned to the exocyclic double bond at C10 of the perhydroazulene system. In a long range COSY experiment, the olefinic resonance at 8 4.69 (H20Z) ppm showed a correlation to a methine resonance at 8 2.80 0or d; J = 7.7 Hz; H9) ppm and in a normal 209 Table 28 *H COSY correlations and nOe enhancements for the protons in shahamin K (155). Spectra were recorded in CDCI3 and were referenced to internal TMS. Position i H COSY correlations nOe enhancementsa la Hlb, H2a, H2b*, H20Z, H20£* lb Hla, H2a, H2b, H20£* 2a Hla, Hlb, H2b, H3a, H3b 2b Hla*, Hlb, H2a, H3a, H3b 3a H2a, H2b, H3b 3b H2a, H2b, H3a, H19 5 H6a, H7*, H9 6a H5, H6B, H7, H9* H5 and H6B, H7, H18 6B H6a, H7 7a H6a, H6B, H9* H6a, H15a, HI 6b (H5 and/or H6B and/or H14) 9 H5, H6a*, H7*, H20Z* H5 or 14, H13, H19, H20Z 12a H13 12b H13 13 H12 (a, b), H14*, H16a, H16b H7, HI6a, HI6b, H20Z 14 H13*, H15a, H15B 15a H14, H15B 15B H14, H15a 16a H13, H16b H16b, H13 16b H13, H16a 17 18 H19* H5 and H6B, H6a b 19 H3b, H18* H5 and H6B, H9 20Z Hla, H9*, H20£ 20E Hla*, Hlb*, H20Z OAc OAc a : proton resonances enhanced when the proton in column 1 was irradiated D : recorded in benzene-d6; see experimental for partial *H nmr assignments for 155 in benzene-d6-*: observed in the long range *H COSY spectrum OCOCH3 210 COSY experiment, this same proton showed a correlation to one of the HI methylene resonances (5 1.95 (br dd; / = 12.7, 12.6 Hz) ppm); the olefinic resonance at 8 4.94 (H20E) ppm showed correlations to both HI methylene resonances (8 1.95 and 2.39 ppm) (see Table 28). The H9 methine proton of 155 showed additional correlations into a resonance at 8 1.82 (m; H5) ppm (normal COSY), into a methylene proton at 8 2.17 (m; H6oc) ppm (long range COSY), and into the downfield methine proton at 8 4.98 (br dd; / = 9.0, 5.3 Hz; H7a) ppm (long range COSY) (see Table 28). The observation of correlations between H9 and both H6ct and H7a placed these protons on the same face of the five-membered ring (to satisfy the geometry requirements necessary to observe w couplings between these protons). A correlation between H5 (8 1.82 ppm) and the resonance at 8 2.17 ppm was assigned to a vicinal coupling between H5 and H6oc. H6a was further correlated into its geminal partner H6{3 (8 1.80 ppm) and into the downfield acetoxy methine resonance at 8 4.98 ppm (H7a), which itself showed a correlation into H6(3. A series of double resonance experiments involving selective irradiation of H7a (8 4.98 ppm), H6a (8 2.17 ppm), and simultaneous irradiation of H5 (8 1.82 ppm) and H6(3 (8 1.80 ppm) confirmed the proton assignments for the five-membered ring portion of the perhydroazulene system. In particular, the irradiation of H6ct (8 2.17 ppm) converted the H7ct resonance (8 4.98 ppm) into a doublet with a scalar coupling constant of 9 Hz. This observation is consistent with the placement of the acetate group at C7. Difference nOe experiments demonstrated that the downfield methine proton (8 4.98 ppm, H7a) was cis to both H9 and the 8 lactone substituent at C8 in shahamin K (155) (see Table 28). Thus, irradiation of H7a (8 4.98 ppm) resulted in the enhancements of H15a (8 4.28 ppm), H16 (8 4.21 ppm), H6a (8 2.17 ppm), and the complex multiplet at 8 1.8 ppm (H5, H14, and H6|3); irradiation of H9 (8 2.80 ppm) caused enhancements in HI3 (8 2.49 ppm), H19 (8 1.01 ppm), H20Z (8 4.69 ppm), and the complex multiplet at 8 1.8 ppm (H5 or H14). Unfortunately, the coincidental chemical 21 1 shifts of H5, H6fi, and H14 prevented the determination of the relative stereochemistry between H9 and H5 by nOe experiments. Attempts at dispersing these signals by the use of different nmr solvents and combinations of nmr solvents failed. However, the magnitude of the scalar coupling constant between H9 and H5 in shahamin K (155) (7.7 Hz) was very similar to that observed in 12-desacetoxyshahamin C (153) (8.7 Hz). As well, the *H and nmr resonances assigned to these two centres were similar in both molecules (see Tables 26 and 27), indicating that H9 and H5 were cis to one another in 155 as they were in 153. A series of nOe experiments confirmed the placement of the acetoxy moiety at C7 (see Figure 72). Irradiation of HI 8 induced an enhancement in both H6oc and the complex multiplet belonging to H5 and H6P; irradiation of H19 induced enhancements in H9 and the complex multiplet belonging to H5 and H6P. Similarly, irradiation of H6a induced enhancements in H7, HI 8, and the complex multiplet belonging to H5 and H6P and irradiation of H9 resulted in enhancement of H19 (as mentioned previously) (see Table 28). An enhancement of the downfield acetoxy methine proton was not observed upon irradiation of either H18 or HI9. Finally, the *H and 13C nmr data for the 8 lactone portion of 155 were virtually identical to those for 153 (see Tables 26 and 27), indicating that both molecules have the same relative configurations about this ring as shown. Figure 72 Selected nOes observed for shahamin K (155) 212 3 . Isolation of the Metabolites from the Nudibranch Chromodoris cavae Fifty-four specimens of Chromodoris cavae (69 g, wet weight) were collected from the waters near Jaffna, Sri Lanka in January 1990. The whole animals were extracted with C H 2 C I 2 and the extract thus obtained was concentrated under reduced pressure. The resulting oil was chromatographed on silica (hexane/EtOAc; 5:2) to afford 6 mg of pure chromodorolide A (154) and a fraction containing a mixture of chromodorolides A (154) and B (156). Further purification of the latter fraction by normal phase H P L C (hexane/EtOAc; 2:1) gave a further 1 mg of pure chromodorolide A (154) and 2 mg of pure chromodorolide B (156). 4. Novel Metabolites Isolated from the Nudibranch Chromodoris cavae  Chromodorolide B (156) O 156 Chromodorolide B (156) was isolated as an optically active colourless oil that gave a parent ion in the H R E I M S at m/z 492.2361 daltons, appropriate for a molecular formula of C 2 6 H 3 6 O 9 ( A M , +0.2 mmu). Resonances at 8 169.1, 169.2, 170.0, and 170.2 ppm in the 13 Q n m r spectrum of chromodorolide B (156) (see Table 29 and Figure 7 3 ) indicated that there were four ester functionalites in the molecule. Resonances at 8 2.04 (s, 213 Table 29 1 3 C nmr data for chromodorolide A (154) (75 MHz) and chromodorolide B (156) (125 MHz). HMQC correlations (J (CH) = 140 Hz) are given for 156. Spectra were recorded in CDCI3; chemical shifts are in ppm. Position S (mult.)a (154) S (mult.)a (156) HMQC correlations 1 41.1 (t) 40.9 (t) 1.03 (dt), 1.38 (m) 2 20.8 (t) 21.1 (t) 1.55 (m) 3 40.4 (t) 39.1 (t) 0.95 (m), 1.51 (m) 4 33.1 (s) 33.1 (s) 5 57.8(d) 57.0 (s) 1.09 (dd) 6 19.8 (t) 19.9 (t) 1.40 (m), 1.56 (m) 7 25.6 (t) 25.2 (t) 1.48 (m), 1.58 (m) 8 44.6 (d) 48.0 (d) 2.57 (ddd) 9 51.6 (d) 50.3 (d) 1.69 (brdd) 10 42.5 (s) 43.9 (s) 169.1 (s)* 11 172.4 (s)* 12 79.9 (s) 81.4 (s) 13 52.0 (d) 50.4 (d) 3.79 (dd) 14 46.3 (d) 45.6 (d) 2.93 (br t) 15 104.2 (d) 97.8 (d) 6.50 (br s) 16 95.5 (d) 103.4 (d) 6.08 (d) 17 78.8 (d) 73.9 (d) 5.30 (d) 18 33.4 (q) 33.4 (q) 0.79 (s) 19 20.8 (q) 21.0 (q) 0.83 (s) 20 13.6 (q) 13.7 (q) 0.84 (s) OAc 20.8 (q) 168.8 (s)* 20.8 (q) 169.2 (s)* OAc 20.8 (q) 20.8 (q) 170.0 (s)* 170.0 (s)* OAc 20.9 (q) 170.2 (s)* *: may be interchanged within a column : multiplicities determined via APT 214 215 3H), 2.11 (s, 3H), and 2.19 (s, 3H) ppm in the C D C I 3 * H nmr spectrum of 156 (see Table 30 and Figure 74) could be assigned to the methyl protons of three acetate groups. A very intense band in the ir spectrum of chromodorolide B (156) at 1751 cm~l was assigned to the stretching vibrations of the acetate carbonyls; a second carbonyl stretching band at 1812 cm"l indicated the presence of a y lactone moiety in chromodorolide B (156), accounting for the fourth ester functionality. The lack of spectral evidence for additional unsaturated functionalities in 156 indicated that chromodorolide B had to be pentacyclic to satisfy the unsaturation number (nine) required by its molecular formula. A comparison of the * H and l ^ C nmr data for chromodorolide B (156) with that for chromodorolide A (154) showed that the two metabolites had closely related structures. The C12H21 bicyclic hydrocarbon portion of chromodorolide A (154) could be identified in chromodorolide B (156) by a detailed analysis of the data obtained from * H C O S Y and H M Q C 1 4 2 (optimized for J (CH) = 140 Hz) experiments (see Tables 29 and 30 and Figures 76 and 77). Analysis of the spectra generated from these experiments allowed the complete assignment of the proton resonances belonging to this system. i H C O S Y correlations due to long range w couplings between H20 (8 (CDCI3) 0.84 ppm) and H l a x (5 (CDCI3) 1.03 ppm), and between H18 (8 (CDCI3) 0.79 ppm) and H 3 a x (8 (CDCI3) 0.95 ppm), H5ax (8 (CDCI3) 1.09 ppm), and H19 (8 (CDCI3) 0.83 ppm) (see Table 30) provided crucial connectivities through the quaternary centres at C10 and C4 (see Figure 78). The * H C O S Y assignments of the geminal methylene pairs of protons were substantiated by the data generated from the H M Q C experiment, which showed all of the methylene carbons correlating into their two attached methylene protons (see Table 29 and Figure 77). The bicyclic hydrocarbon portion of chromodorolide B (156) was shown to be linked to the rest of the molecule through C 9 by the observation of a * H C O S Y correlation between H9 and a proton not belonging to the hydrocarbon portion (8 (CDCI3) 2.57 ppm; H8). 216 Table 30 A H nmr (400 MHz) data for chromodorolides A (154) and B (156). A H C O S Y and nOe data are given for 156. Listed C O S Y correlations apply to both CDCI3 and benzene-d6 spectral data. Chemical shifts are given in ppm and are referenced to internal T M S . Position 8 (mult.; J; #H) (154)* 8 (mult.; / ; #H) (156) a 8 (mult; / ; #H) (156) b i H C O S Y nOe* correlations enhancements lax 1.03 (dt; 13.5, 4.2; IH) 1.03 (dt; 12.3, 3.6; IH) 0.90 (m; IH) H l e q , H2, H20 leq 1.40 (m; IH) 1.38 (m; IH) 1.43 (m; IH) H l a x , H2 2(ax. eq) 1.50-1.52 (m; 2H) 1.55 (m; 2H) 1.46-1.50 (m; 2H) H l a x . H l e q , H3ax» H3eq 3ax 1.08 (m; IH) 0.95 (m; IH) 1.00 (m; IH) H2 , H3eq, H18 3eq 1.69 (m; IH) 1.51 (m; IH) 1.38 (m; IH) H2, H 3 a x 5 1.11 (m; IH) 1.09 (dd; 13.1, 6.6; IH) 0.68 (dd; 11.0, 7.0; IH) H6a, H6b, H18 6a 1.31 (m; IH) 1.40 (m; IH) 1.14 (m; IH) H5 , H6b, H7a, H7b 6b 1.56 (m; IH) 1.56 (m; IH) 1.37 (m; IH) H5 , H6a, H7a, H7b 7a 1.15 (m; IH) 1.48 (m; IH) 1.42 (m; IH) H6a, H6b, H7b, H9 7b 1.76 (m; IH) 1.58 (m; IH) 1.44 (m; IH) H6a, H6b, H7a, H9 8 2.39 (ddd; 12.0, 6.6, 3.0; IH) 2.57 (ddd; 12.1, 11.6, 7.9; IH) 2.51 (ddd; 12.2, 11.0, 7.5; IH) H9 , H14, H17 H13 a > b , H20 a > b 9 1.55 (m; IH) 1.69 (brdd; 12.0, 9.9; IH) 1.16 (m; IH) H7a, H7b, H 8 13 3.00 (m; IH) 3.79 (dd; 8.9, 6.1; IH) 3.50 (dd; 8.8, 6.0; IH) H14, H16 H 1 4 a « b H 1 6 a » b 217 Table 30 (continued) Position 8 (mult.; 7; #H) (154)a 8 (mult.; /; #H) (156)a 8 (mult; /; #H) (156)b !H COSY correlations nOe* enhancements 14 3.00 (m; IH) 2.93 (br t; 8.2; IH) 2.62 (br dd; 8.2, 7.5; IH) H8, H13, H15 H8a, HI3a H15a 15 5.73 (brt; 2.1; IH) 6.50 (br s; IH) 6.63 (br s; IH) H14 H9 b, H14b, H17b 16 6.30 (s; IH) 6.08 (d; 6.1; IH) 5.80 (d; 6.0; IH) H13 H13b 17 4.77 (d; 2.8; IH) 5.30 (d; 11.6; IH) 5.70 (d; 12.2; IH) H8 H15b 18 0.79 (s; 3H) 0.79 (s; 3H) 0.81 (s; 3H) H3ax» H5, H19 19 0.80 (s; 3H) 0.83 (s; 3H) 0.84 (s; 3H) H18 20 0.82 (s; 3H) 0.84 (s; 3H) 0.63 (s; 3H) Hlax H8b, H18b OAc 2.02 (s; 3H) 2.04 (s; 3H) 1.63 (s; 3H) OAc 2.06 (s; 3H) 2.11 (s; 3H) 1.66 (s; 3H) OAc 2.19 (s; 3H) 1.84 (s; 3H) *: these represent the proton resonances enhanced upon irradiation of the proton in column one a : recorded in CDCI3 b : recorded in benzene-d6 218 Figure 75 ! H nmr spectrum of chromodorolide B (156) (benzene-d6; 400 MHz) Figure 76 *H COSY spectrum of chromodorolide B (156) (CDC13; 400 MHz) Figure 77 HMQC spectrum of chromodorolide B (156) (CDC13; J (CH) = 140 Hz) Figure 78 Selected H COSY correlations for the protons belonging to the hydrocarbon portion of 156 The remaining fragment of chromodorolide B (156) had to have the elemental composition C14H15O9, had to be tricyclic, and had to contain the acetate and y lactone functionalities identified from the ir and l^C nmr data. The presence of two ketal functionalities in this fragment was evidenced by the resonances at 5 97.8 and 103.4 ppm in the l^C nmr spectrum of chromodorolide B (156); these two resonances showed correlations in the HMQC spectrum of 156 to *H nmr resonances (CDCI3) at 8 6.50 and 6.08 ppm respectively. Two additional deshielded nmr resonances at 8 73.9 (d) and 81.4 (s) ppm were assigned to sp3-hybridized carbon atoms singly bonded to one oxygen atom apiece. *H COSY correlations connected the ketal methine resonance at 8 6.50 (CDCI3; br s; H15) ppm through two intervening methine resonances at 8 2.93 (CDCI3; br t; J = 8.2 Hz; H14) and 3.79 (CDCI3; dd; / = 8.9, 6.1 Hz; H13) ppm to the second ketal methine resonance at 8 6.08 (CDCI3; d; / = 6.1 Hz; H16) ppm. The methine resonance at 8 2.93 ppm (H14) correlated into an additional methine resonance at 8 2.57 (CDCI3; ddd; / = 12.1, 11.6, 7.9 Hz; H8) ppm which was in turn correlated into a 223 deshielded methine resonance at 5 5.30 (CDCI3; d; / = 11.5 Hz; H17) ppm and also into the resonance assigned to H9 of the bicyclic hydrocarbon fragment (8 1.69 (CDCI3; br dd; / = 12.0, 9.9 Hz) ppm) (see Table 30). The six proton spin system described above for the tricyclic fragment of chromodorolide B (156) corresponded exactly to the six proton spin system (H8, H13, H14, H15, H16, and H17) present in the tricyclic portion of chromodorolide A (154) (see Table 30). In addition, the nmr resonances assigned to the tricyclic fragments of chromodorolides A (154) and B (156), with the exception of the resonances assigned to the acetate groups, showed a direct one to one correspondence (that is, the l^c chemical shifts observed for carbons 8 and 12-17 in 154 were very similar to those for the same carbon atoms in 156) (see Table 29). Therefore, it was concluded that chromodorolide B (156) possessed a chromodorane skeleton with a bisketal-oxolane ring fused 3, 4 to a cyclopentane ring functionalized similarly to that present in chromodorolide A (154) (encompassing C8 and C12-C17; see Figure 79). Figure 79 The chromodorane skeleton present in both 154 and 156 The most apparent spectral differences between chromodorolide B (156) and chromodorolide A (154) were the ir stretching frequencies of the lactone carbonyls 224 (1812 cm"l in 156 (appropriate for a y lactone) and 1770 cm~l (due to a 8 lactone) in 154) and the systematic difference in the relative chemical shifts of both the proton and carbon resonances assigned to C15 and C16. *H COSY and HETCOR (optimized for / (CH) = 140 Hz) experiments assigned the most deshielded proton resonance (CDCI3; 8 6.67 ppm) and the most shielded carbon resonance (8 95.9 ppm) in chromodorolide A (154) to the ketal carbon that was bonded to the acetate (C16); these same experiments assigned the most shielded ketal proton resonance (CDCI3; 8 5.59 ppm) and the most deshielded carbon resonance (8 104.2 ppm) in chromodorolide A (154) to the ketal carbon bonded to the C l l 8 lactone carbonyl functionality (C15). By analogy, the relative chemical shifts of the proton and carbon resonances assigned to the C15 and C16 ketal functionalities in chromodorolide B (156) (see Tables 29 and 30) indicated that the C15 ketal carbon was bonded to an acetate (CDCI3; *H nmr 8 6.50 ppm; 13c nmr 8 97.8 ppm) and that the C16 ketal carbon was bonded to the C l l lactone carbonyl functionality ( C D C I 3 ; iH nmr 8 6.08 ppm; 13C nmr 8 103.4 ppm). The resulting tricyclic structure contained a y lactone moiety, consistent with the observed stretching band at 1812 cm~l in the ir spectrum of chromodorolide B (156). Placement of the two remaining acetate functionalities at C17 (^C nmr 8 73.9 (d) ppm) and C12 (13c nmr 8 81.4 (s) ppm) in 156, positions occupied in chromodorolide A (154) by acetate and tertiary hydroxyl functionalities respectively, was consistent with the *H and 13c nmr data obtained for chromodorolide B (156) (see Tables 29 and 30). Difference nOe experiments were in complete agreement with the proposed structure 156 for chromodorolide B and they defined the relative stereochemistry of the tricyclic fragment as shown in structure 156 (see Table 30 and Figures 80-83). The relative stereochemistries at C8 and C9 are assumed to be the same in chromodorolide B (156) as they were found to be in chromodorolide A (154). 133 225 i JUL 1 1 1 1 i 1 1 1 1 i 1 1 1 1 i 1 ' 1 1 i ' ' 1 ' i 1 ' ' ' i • ' ' • i ' i i i i i • • 1 1 i i i 1 1 i I I i | i i i 1 1 i . i i 6.5 6.0 5.S 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1 5 1 0 PPM H17 irradiated H15 HI3 irradiated H16 H15 irradiated H17 H14 Figure 80 Selected nOe difference spectra of chromodorolide B (156) (benzene-d6; 400 MHz) 226 i Ji I JL_JI ~r—i—i—|—i—t—i—i—|—i—l—i l | i i l l | l i i i | i i i i — | l l — i i | i — i — i i | i i—i i | i i — i i | i i—i i | i i i i | i i i l 6.5 6.0 S.5 5.0 a.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 PPM H14 irradiated H15 ' I S I ' K ] J I H13 1 H8 irradiated H13 H20 H16 irradiated H13 ' I ' ' • ' I I T l I I l l l l I i l l l I i l l i I l l l l I i i i i I i i i i [ i i i i i i i i i i i i i i i i i i 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.S 2.0 1.5 1.0 P P H Figure 81 Selected nOe difference spectra of chromodorolide B (156) (benzene-d6; 400 MHz) 227 228 H Figure 83 Selected nOes observed in chromodorolide B (156) C o n c l u s i o n s The isolation of 12-desacetoxyshahamin C (153), shahamin K (155), and chromodorolide B (156) adds to the number of spongian-derived diterpenoid metabolites which have already been found in species of nudibranch belonging to the genus Chromodoris. The origin of these metabolites in the nudibranchs is almost certainly dietary, since 153 has also been found to be a metabolite of the sponge Dendrilla sp.138 and a number of compounds very similar in structure to both 153 and 155 have 229 previously been isolated from a variety of sponge species.136-141 T 0 date, no diterpenoid metabolites possessing the chromodorane skeleton have been found in sponges; however, it is likely that both chromodorolides A (154) and B (156) are obtained from a sponge in the diet of Chromodoris cavae. As yet, the identity of the sponge(s) from which Chromodoris glenei and Chromodoris cavae sequester metabolites 153-156 remains unknown. i The dendrillane skeleton possessed by 12-desacetoxyshahamin C (153) and shahamin K (155) can be envisaged as arising from a spongian precursor by the transformations shown in Scheme 5.136,140 Scheme 6 shows a proposed biogenetic pathway to chromodorolides A (154) and B (156) which illustrates that the two metabolites represent alternate modes of cyclization of the Cl 1 carboxyl group with the bisketal-oxolane ring. Metabolites 153,155, and 156 have not as yet been screened for biological activities. Chromodorolide A (154) displayed significant cytotoxic and antineoplastic activities (L1210 in vitro mouse leukemia cell line bioassay: ED50 20 u.g/mL; P388 in vivo mouse leukemia cell line bioassay: T/C 125% at 4 mg/ Kg) as well as antibacterial and antifungal activities (Bacillus subtilis: MIC 60 ng/disc; Rhizoctonia solani: MIC 60 u,g/disc). It is anticipated that chromodorolide B (156) will possess similar levels of biological activity. 230 oxidative cleavage of C9-C11 11 155 Scheme 5 Proposed biogenesis of 12-desacetoxyshahamin C (153) and shahamin K (155) 2 3 1 154 Scheme 6 Proposed biogenesis of chromodorolides A (154) and B (156) 232 Experimental A. General The i H nmr spectra were recorded on either a Bruker AMX-500, a Bruker WH-400, or a Varian XL-300 nmr spectrometer. Tetramethylsilane (8 = 0 ppm) was used as an internal standard for the *H nmr spectra. The nmr spectra were recorded either at 75 MHz on a Varian XL-300 nmr spectrometer or at 125 MHz on a Bruker AMX-500 nmr spectrometer. The solvents used in the recording of the nmr spectra were used as the internal standards unless otherwise indicated. The reference positions used are: acetone-d6 (8 = 29.8 ppm, CH3 multiplet), benzene-d6 (8 = 128.0 ppm), chloroform-d (8 = 77.0 ppm), and dimethylsulfoxide-d6 (8 = 39.5 ppm). All COSY, nOe difference, and double resonance nmr experiments were carried out using a Bruker WH-400 nmr spectrometer. The APT spectra were recorded either on a Varian XL-300 or a Bruker AMX-500 nmr spectrometer. All carbon-detected HETCOR, FLOCK, and SINEPT experiments were performed on a Varian XL-300 nmr spectrometer. The ROESY and inverse-detected HETCOR (HMQC) spectra were recorded on a Bruker AMX-500 nmr spectrometer. Low resolution and high resolution electron impact mass spectra were recorded on Kratos AEI MS-59 and AEI MS-50 mass spectrometers respectively. Low resolution chemical ionisation mass spectra were recorded on a Delsi-Nermag R-10-10 quadrupole mass spectrometer using either methane or ammonia as the reagent gasses. Low resolution fast atom bombardment mass spectra were recorded on a Kratos AEI MS-59/DS55SM mass spectrometer employing xenon as the bombarding gas. Infrared spectra were recorded on either a Perkin-Elmer 1600 Fourier Transform (FT) or 1710 FT spectrometer (internal calibration). Ultraviolet-visible spectra were recorded using a Bausch and Lomb Spectronic-2000 spectrophotometer. Optical rotations were measured using an Optical 233 Activity Ltd. AA-1000 polarimeter (10 cm path length; 1 mL cell; sodium source). Uncorrected melting points were determined using a Fisher-Johns melting point apparatus. Gas-liquid chromatography was carried out using a Hewlett-Packard 5890 capillary gas chromatograph employing a 25 m by 0.21 mm fused silica column coated with cross-linked SE-54 and equipped with a flame-ionisation detector. High performance liquid chromatography was carried out on a Perkin-Elmer Series 2 instrument using either a Perkin-Elmer LC-55 spectrophotometer or a Perkin-Elmer LC-25 refractive index detector for peak detection. Whatman Magnum-9 Partisil 10 ODS-3 reversed phase and Whatman Magnum-9 Partisil 10 normal phase columns were used for preparative HPLC. Whatman Partisil 5 ODS-3 reversed phase and Whatman Partisil PXS 5/25 normal phase columns were used for analytical HPLC. All solvents used for HPLC were BDH Omnisolve grade; the water used in HPLC solvent systems was glass distilled. Normal phase thin layer chromatography (tic) analyses were done on commercial aluminum backed silica gel plates (E. Merck, Type 5554). Normal phase flash chromatography was carried out on Merck silica gel (230-400 mesh); radial chromatography was performed using plates coated with a dried layer of an aqueous slurry of Merck silica gel 60 PF-254 and CaS04-1/2 H2O on a Harrison Research chromatotron (model 7924) equipped with an FMI lab pump (model R PG-150). Reversed phase dc analyses were done on glass backed Whatman KC18F dc plates (either 20 x 20 cm or 1 x 3 inches). Preparative reversed phase tic was done on glass backed 20 x 20 cm Whatman KC18F dc plates (200 um thickness). Reversed phase open column chromatography was carried out using reversed phase silica prepared according to Kuhler et al.H7 Visualisation of compounds (for analytical tic, preparative tic, and radial dc analyses) was accomplished with ultraviolet light, iodine, or staining with either vanillin (0.5 g of vanillin in 100 mL of H2S04/ethanol, 4:1) or ninhydrin (0.2 g of ninhydrin in 100 mL of ethanol) solutions. Sephadex LH-20 resin was used for molecular exclusion chromatography 234 unless otherwise stated. All solvents used for chromatography were BDH Omnisolve grade. The acetic anhydride used in acetylation reactions was stirred over calcium carbide for several days and then distilled (138*-140°C). The pyridine used in acetylation reactions was allowed to reflux over barium oxide for 12 hours and then distilled (114°-116'C). All solvents used for reactions were BDH Omnisolve grade. All antibacterial and antifungal bioassays were performed by Mike LeBlanc (UBC). All cytotoxicity bioassays, both in vitro (using the L1210 mouse leukemia cell line) and in vivo (using the P388 mouse leukemia cell line), were carried out under the direction of Dr. Terry Allen at the University of Alberta. B . Metabolites From the Northeastern Pacific Sponge Hexadella sp. 1. Hexadella sp. collected at -100 to -200 m Hexadella sp. was collected at Dark Cove, Jervis Inlet, in November 1986 using the manned submersible PICES IV (-100 to -200 m). The freshly collected sponge (500 g, wet weight) was immersed in methanol immediately after collection. Concentration of the methanol extract under reduced pressure yielded 6 g of crude residue. A 400 mg portion of the crude residue was chromatographed on a Sephadex LH-20 column prepared and eluted with methanol/dichloromethane (7:3). Fractions containing 74 were further purified via reversed phase HPLC (methanol/water, 3:1) to give 71 mg (1.8% of crude residue) of pure topsentin B2 (74). 235 Topsentin B2 (74) Topsentin B2 was isolated as a yellow/green solid (2:1 mixture of tautomers); mp: 260-262°C; [cc]D: 0° (c = 0.37, MeOH); uv (MeOH), X m a x : 286 nm (e 17 000), 382 nm (e 15 500) with shoulders at 235 nm and 260 nm, changing upon addition of NaOH to m^ax: 230 nm (e 10 000), 302 nm (e 4000) and 370 nm (e 3200); ir (film), vmax- 3300, 1702, 1524, 1158, 868 cm"1; lH nmr (acetone-d6, 300 MHz), major tautomer, 8: 6.87 (dd, / = 8.7, 2.2 Hz, IH, H6), 7.02 (d, / = 2.2 Hz, IH, H8), 7.27 (dd, / = 8.6, 1.5 Hz, IH, H22), 7.68 (d, / = 1.5 Hz, IH, H20), 7.72 (d, / = 2.1 Hz, IH, H13), 7.86 (d, / = 2.5 Hz, IH, H17), 8.09 (s, IH, -OH), 8.22 (d, J = 8.6 Hz, IH, H23), 8.32 (d, / = 8.7 Hz, IH, H5), 9.43 (s, IH, H2), 10.59 (br s, IH, 18-NH), 10.90 (br s, IH, 15-NH), and 12.20 (br s, IH, 1-NH) ppm; minor tautomer, 8: 6.84 (dd, / = 8.7, 2.2 Hz, IH, H6), 6.99 (d, J = 2.2 Hz, IH, H8), 7.33 (dd, / = 8.6, 1.5 Hz, IH, H22), 7.61 (d, / = 1.5 Hz, IH, H20), 7.73 (d, / = 1.5 Hz, IH, HI3), 7.87 (d, / = 2.6 Hz, IH, H17), 7.92 (d, / = 8.6 Hz, IH, H23), 8.09 (s, IH, -OH), 8.29 (d, J = 8.7 Hz, IH, H5), 9.22 (s, IH, H2), 10.80 (br s, IH, 18-NH), 10.90 (br s, IH, 12-NH), 12.20 (br s, IH, 1-NH) ppm; LRCIMS (NH3; positive ion detection), pseudomolecular ion m/z: 421/423 (M+(C20Hl3BrN4O2) + 1); LREIMS, m/z (formula, relative intensity): 420/422 (M+, 23), 287/289 (Ci2H7BrN30, 5), 261/263 (CnH8BrN3, 4), 160 (C9H6NO2, 44), 133 (C8H8NO, 100); HREIMS, C20Hl3BrN4O2 (M+) m/z: 420.0222/422.0203 (AM, +0.2 236 mmu/-0.6 mmu). ED50: 5 jig/mL (in vitro L1210 bioassay). T/C: 135% (in vivo P388 bioassay). Methylation of Topsentin B2: 40 mg of 74 (0.09 mmol) were allowed to reflux overnight under a nitrogen atmosphere with 35 |iL (0.4 mmol) of dimethyl sulphate and 66 mg (0.5 mmol) of potassium carbonate in 15 mL of freshly distilled acetone.83 Removal of excess reagents and solvent under reduced pressure gave an oily residue that was purified via reversed phase preparatory dc (eluant: MeOH/CH3CN/H20; 82:10:8) to yield 11 mg of pure trimethyltopsentin B2 (75) (Rf = 0.52) and 1 mg of pure tetramethyltopsentin B2 (76) (Rf = 0.44). Trimethyltopsentin B2 (75) Trimethyltopsentin B2 was isolated as a yellow/green oil; ir (film), vmax^ 3134, 1706, 1593, 1520, 1250, 1072, 926, 836 cm"1; *H nmr (acetone-d6, 300 MHz), 8: 3.91 (s, 3H, H25), 3.99 (s, 3H, H26), 4.16 (s, 3H, H27), 6.95 (dd, / = 8.7, 2.2 Hz, IH, H6), 7.09 (d, / = 2.4 Hz, IH, H8), 7.30 (dd, J = 8.7, 2.4 Hz, IH, H22), 7.65 (s, IH, H13), 7.68 (d, / = 2.4 Hz, IH, H20), 7.85 (s, IH, H17), 8.12 (d, / = 8.7 Hz, IH, H23), 8.40 (d, / = 8.7 Hz, IH, H5), 9.07 (s, IH, H2), 10.60 (br s, IH, 18-NE) ppm; 1 3 C nmr (acetone-d6, 75 MHz), 8: 33.9 (q, C26), 36.5 (q, C27), 55.9 (q, C25), 94.2 (d, C8), 237 111.7 (s, C16), 112.3 (d, C6), 115.1 (s, C3), 115.2 (d, C20), 115.3 (s, C21), 121.8 (d, C13), 122.4 (s, C4), 122.6 (d, C23), 123.2 (d, C22), 123.6 (d, C5), 123.8 (d, C17), 125.1 (s, C24), 136.7 (s, C14), 138.4 (s, Cll), 138.8 (s, C9), 140.5 (d, C2), 143.8 (s, C19), 157.9 (s, C7), 178.4 (s, CIO) ppm; LREIMS, m/z (formula, relative intensity): 462/464 (M+, 96), 273/275 (Ci2Hi()BrN3, 1), 188 (C11H10NO2, 100), 174 ( C 1 0 H 8 N O 2 , 7), 145 (C9H7NO, 20); HREIMS, C23Hi9BrN402 (M+) m/z: 462.0690/464.0670 (AM, -1.8 mmu/+0.7 mmu). Tetramethyltopsentin B2 (76) Tetramethyltopsentin B2 was isolated as a yellow/green oil; ir (film), Vmax: 1709, 1595, 1522, 1076, 843 cirri; 1H nmr (acetone-d6, 400 MHz), 5: 3.90 (s, 3H, H25), 3.96 (s, 3H, H28), 3.99 (s, 3H, H26), 4.04 (s, 3H, H27), 6.93 (dd, / = 8.6, 2.4 Hz, IH, H6), 7.08 (d, / = 2.4 Hz, IH, H8), 7.26 (s, IH, H17), 7.31 (dd, / = 8.7, 2.5 Hz, IH, H22), 7.59 (d, / = 8.7 Hz, IH, H23), 7.63 (s, IH, H13), 7.77 (d, / = 2.5 Hz, IH, H20), 8.39 (d, / = 8.6 Hz, IH, H5), 8.90 (s, IH, H2) ppm; HREIMS, C24H2lBrN402 (M+) m/z: 476.0828/478.0813 (AM, +0.2 mmu/-1.3 mmu). 28 238 2. Hexadella sp. collected at -40 m Hexadella sp. was collected by S C U B A (-40 m) in Agamemnon Channel, Jervis Inlet, B. C . in March 1987. The freshly collected sponge (800 g, wet weight) was immersed in methanol immediately following collection. Evaporation of the methanol under reduced pressure gave a residue that was suspended in water and sequentially extracted with hexane (2 x IL), chloroform (2 x IL), and ethyl acetate (2 x IL). The combined chloroform extracts were concentrated and chromatographed on Sephadex LH-20 (MeOH/CH2Cl2; 3:1) to give an antibacterial fraction containing a mixture (74 mg) of hexadellins A (77) and B (78) (ED50 (mixture): <10 | ig/mL (in vitro L1210 bioassay)). The compounds could not be separated in their native form; therefore, the mixture was acetylated with acetic anhydride and pyridine (0.5 m L of each, room temperature, 24 hours) to give a mixture of 79 and 80. The acetylated derivatives were purified via gradient silica gel flash chromatography (EtOAc/hexane; 4:1 to EtOAc/MeOH; 4:1) and normal phase H P L C (EtOAc/MeOH; 95:5) to give pure diacetylhexadellin A (79) (30 mg) and pure diacetylhexadellin B (80) (19 mg). The combined ethyl acetate extracts were fractionated by Sephadex LH-20 chromatography (MeOH/CH2Cl2; 7:3). Early eluting fractions were further subjected to sequential application of silica gel flash (gradient: EtOAc/hexane; 7:3 to EtOAc/MeOH; 4:1), reverse phase flash (gradient: MeOH/H20; 7:3 to MeOH), and radial thin layer (silica gel: EtOAc/MeOH; 9:1) chromatographies to give pure samples of dragmacidons A (81) (6 mg) and B (82) (6 mg). The late eluting LH-20 fractions were further fractionated by radial thin layer chromatography (silica gel: EtOAc/hexane; 3:2) to give pure dragmacidon C (83) (3 mg). 239 Diacetylhexadellin A (79) Diacetylhexadellin A was isolated as a pale yellow oil; ir (film), v m a x : 3338,2928,1752, 1656, 1544, 1215, 737 cm-1; 1 H n m r (CDCI3, 400 MHz), 8: 1.98 (s, 3H, -NHCOCH3J, 2.11 (tt, / = 6.2, 5.6 Hz, 2H, H12), 2.15 (s, 3H, -OCOCHV). 2.75 (t, / = 7.2 Hz, 2H, H18), 3.09 (d, J = 18.0 Hz, IH, H7), 3.46 (d, / = 18.0 Hz, IH, H7), 3.47 (dt, / = 7.2, 5.2 Hz, 2H, H19), 3.71 (dt, J = 6.0, 5.2 Hz, 2H, H l l ) , 3.76 (s, 3H, -OCH3J, 4.09 (t, J = 6.0 Hz, 2H, H13), 5.58 (br t, / = 6.0 Hz, IH, H20), 5.85 (s, IH, HI), 6.32 (s, IH, H5), 7.17 (br t, J = 6.0 Hz, IH, H10), 7.35 (s, 2H, H16) ppm; 13c nmr (CDCI3,75 MHz), 8: 20.8 (q, -OCOCH^), 23.4 (q, -NCOCH3J), 29.3 (t, C12), 34.5 (t, C18), 37.3 (t, C l l ) , 40.1 (t, C7), 40.5 (t, C19), 60.2 (q, -OCHg). 71.2 (t, C13), 73.2 (d, C l ) , 89.7 (s, C6), 107.8 (s, C4), 118.1 (s, C15), 121.7 (s, C2), 130.5 (d, C5), 132.8 (d, C16), 137.9 (s, C17), 149.7 (s, C3), 151.2 (s, C14), 153.6 (s, C8), 158.6 (s, C9), 169.3 (s, -OCOCH3), 170.1 (s, -NCOCH3) ppm; LREIMS, m/z (relative intensity): 739^741/743/745/747 (M+ - C2H2O2, 0.5), 418/420/422 (0.5:1:0.5), 405/407/409 (1:2:1), 359/361/363 (12:24:12), 303/305/307 (18:36:18), 276/278/280 (50:100:50); HREIMS, C23H25 7 9Br38lBrN305 (M+ - C2H2O2) m/z: 740.8505 (AM, -0.1 mmu). 240 Diacetylhexadellin B (80) Diacetylhexadellin B was isolated as a pale yellow oil; ir (film), Vmax: 3305, 2936,1758, 1657, 1543, 1216, 772, 737 cm"1; *H nmr ( C D C I 3 , 400 MHz), 8: 2.01 (s, 3H, - N H C O C H 3 J ) , 2.07 (tt, J = 6.0, 5.2 Hz, 2H, HI 8), 2.15 (s, 3H, - O C O C H 3 ) , 2.81 (t, / = 7.6 Hz, 2H, H12), 3.08 (d, J = 18.0 Hz, IH, H7), 3.44 (d, / = 18.0 Hz, IH, H7), 3.56 (dt, J = 6.0, 4.0 Hz, 2H, H19), 3.58 (dt, / = 7.6, 5.6 Hz, 2H, Hl l ) , 3.78 (s, 3H, - O C H 3 ) , 4.09 (t, J = 5.2 Hz, 2H, H17), 5.84 (s, IH, HI), 6.10 (br t, / = 4.0 Hz, IH, H20), 6.32 (s, IH, H5), 6.65 (br t, J = 5.6 Hz, IH, H10), 7.39 (s, 2H, H14) ppm; 13c nmr ( C D C I 3 , 7 5 MHz), 8: 20.8 (q, - O C O C H 3 J , 23.6 (q, -NCOCH3J), 2 9 A (*> c l g ) ' 34.4 (t, C12), 37.7 (t, C19), 39.9 (t, C 7 ) , 40.4 (t, Cl l ) , 60.3 (q, -OCH3J, 72.1 (t, C17), 73.1 (d, Cl), 89.9 (s, C6), 107.8 (s, C4), 118.2 (s, C15), 122.1 (s, C2), 130.2 (d, C5), 132.8 (d, C14), 137.2 (s, C13), 149.7 (s, C3), 151.5 (s, C16), 153.5 (s, C8), 158.6 (s, C9), 169.4 (s, -0£OCH3), 170.0 (s, -N£OCH3) ppm; LREIMS, m/z (relative intensity): 755/757/759/761/763 (<0.5), 739/741/743/745/747 (<0.5), 638/640/642/644/646 (<0.5), 303/305/307 (50:100:50), 276/278/280 (7:14:7); HREIMS, C23H2579Br28lBr2N305 (M+ - C 2 H 2 O 2 ) m/z: 742.8490 (AM, +0.4 mmu). 241 Dragmacidon A (81) H H Dragmacidon A was isolated as a pale yellow glass (8 x 10"4% of the wet weight of the sponge); [cc]D: + 122.5° (c = 0.04, MeOH); ir (film), vmax: 3413, 3284, 2947, 2837, 2790, 1615, 1546 cm'1; iH nmr (acetone-d6, 400 MHz), 8: 2.09 (s, 3H, I-NCH3J), 2.38 (dd, J = 11.0, 10.4 Hz, IH, H6ax), 3.07 (dd, / = 11.0, 3.0 Hz, IH, H3eq), 3.18 (dd, / = 11.0, 2.6 Hz, IH, H6eq), 3.29 (dd, J = 11.0, 10.5 Hz, IH, H3ax), 3.41 (dd, / = 10.5, 3.0 Hz, IH, H2ax), 4.43 (dd, / = 10.4, 2.6 Hz, IH, H5ax), 7.17 (dd, J = 8.5, 1.2 Hz, IH, H5'), 7.18 (dd, / = 8.5, 1.4 Hz, IH, H5"), 7.35 (d, J = 2.3 Hz, IH, H2"), 7.39 (d, / = 1.8 Hz, IH, H2*), 7.61 (d, / = 1.2 Hz, IH, H7'), 7.61 (d, / = 1.4 Hz, IH, H7"), 7.81 (d, / = 8.5 Hz, IH, H41), 7.91 (d, J = 8.5 Hz, IH, H4"), 10.28 (br s, 2H, l'-NH and 1"-NH) ppm; 13c nmr (acetone-d6, 75 MHz), 8: 44.2 (q, 1-NCH3_), 54.1 (d, C5), 54.3 ( t , C3), 63.3 (d, C2), 64.2 (t, C6), 115.0 (d, C7"), 115.0 (d, C7"), 115.1 (s, C6')a, 115.2 (s, C6")a, 117.5 (s, C3')b, 118.8 (s, C3")b, 122.1 (d, C4')c, 122.4 (d, C5")C, 122.4 (d, C5')c, 122.6 (d, C4")c, 123.9 (d, C2'), 124.9 (s, C3a')d, 125.0 (d, C2"), 126.6 (s, C3a")d, 138.7 (s, C7a')e, 138.8 (s, C7a")ep m (»-e: m a y be interchanged); LRCIMS (NH3; positive ion detection), pseudomolecular ion m/z: 487/489/491 (M+(C2lH20Br2N4) + 1); LREIMS, m/z (formula, relative intensity): 486/488/490 (C2lH20Br2N4, 1:2:1), 291/293 (Cl3Hi4BrN3, 11:11), 251/253 (CnHi3BrN2, 242 80:80), 221/223 ( C i f j H g B r N , 60:60), 208/210 ( C o H 7 B r N , 12:12), 195/197 (CsH6BrN, 100:100); H R E I M S , C 2 l H 2 0 B r 2 N 4 (M+) m/z: 486.0042/488.0027/490.0027 ( A M , -1.4 mmu/-0.9 mmu/+0.8 mmu). ED50: 6.5 | ig /mL (in vitro L1210 bioassay). T / C : 135% (in vivo P388 bioassay). A c e t y l d r a g m a c i d o n A (87) A 2 mg sample of dragmacidon A (81) was stirred overnight in a 1:1 mixture of acetic anhydride and pyridine (5 m L of each). The reagents were removed under reduced pressure and the o i l thus obtained was purified by radial thin layer chromatography to give pure acetyldragmacidon A (87). Acetyldragmacidon A was isolated as a pale yel low o i l ; i H nmr (CDCI3, 300 MHz), 8: 1.88 (s, 3H, I-NCOCH3), 2.15 (s, 3H, 4-NCH3), 2.98 (dd, J = 12.3, 1.8 Hz, IH, H3p), 3.06 (dd, / = 12.3, 4.6 Hz, IH, H3a), 3.64 (br m, IH, H6a), 3.82 (dd, J = 13.1, 3.7 Hz, IH, H6p), 4.33 (br s, IH, H5p), 6.21 (br s, IH, H2a), 7.20 (d, / = 2.6 Hz, IH, H2'), 7.22 (dd, J = 8.6, 1.7 Hz, IH, H5'), 7.26 (dd, / = 8.6, 1.7 Hz, IH, H5"), 7.49 (d, / = 8.5 Hz, IH, H4"), 7.52 (d, / = 1.4 Hz, IH, H7'), 7.59 (d, / = 1.6 Hz, IH, H7"), 7.67 (br s, IH, H2"), 7.75 (d, / = 8.5 Hz, IH, H4'), 8.20 (br s, IH, l'-NH), 8.57 (br s, IH, 1"-NH) ppm; LREIMS, C23H22Br2N40 (M+) m/z: 528/530/532. H 1 r N 7a» 7" H 243 Dragmacidon B (82) Dragmacidon B was isolated as a pale yellow powder (8 x 10"4% of the wet weight of the sponge); [tx]D: + 65.0° (c = 0.02, MeOH); ir (film), v m a x : 3257, 2947, 2800, 1615, 1546 cm"1; J H nmr (acetone-d6, 400 MHz), 8: 2.09 (s, 6H, 1- and 4-NCH.3), 2.64 (dd, / = 11.0, 10.9 Hz, 2H, H 3 a x and H6 a x), 2.93 (dd, / = 11.0, 2.9 Hz, 2H, H 3 e q and H6 e q), 3.60 (dd, / = 10.6, 2.9 Hz, 2H, H2 a x and H5 a x), 7.17 (dd, J = 8.5, 1.7 Hz, 2H, H5' and H5"), 7.38 (d, / = 2.3 Hz, 2H, H2' and H2"), 7.62 (d, / = 1.7 Hz, 2H, H7" and H7"), 7.92 (d, / = 8.5 Hz, 2H, H4" and H4"), 10.29 (br s, 2H, l'-NH and 1"-NH) ppm; !3c nmr (acetone-d6, 75 MHz), 8: 43.8 (q, 1- and 4-NCJJ3), 63.0 (d, C2 and C5), 64.2 (t, C3 and C6), 115.2 (d, C7' and Cl"), 115.4 (s, C6' and C6"), 117.1 (s, C3" and C3"), 122.6 (d, C4' and C4")a, 122.8 (d, C5' and C5")a, 125.2 (d, C2* and CT), 126.7 (s, C3a' and C3a"), 138.9 (s, C7a' and C7a") ppm (a: may be interchanged); LRCIMS (NH3; positive ion detection), psuedomolecular ion m/z: 501/503/505 (M+(C22H22Br2N4) + 1); LREIMS, m/z (formula, relative intensity): 500/502/504 (C22H22Br2N4, 12:21:12), 422/424 (C22H23BrN4, 5:5), 250/252 (CnHnBrN2, 7:7), 237/239 (CioH8BrN2, 100:100), 221/223 (CioHgBrN, 60:60), 195/197 (CsHsBrN, 6:6); HREIMS, C22H22Br2N4 (M+) m/z: 500.0219/502.0196/504.0171 (AM, +0.7 mmu/+0.3 mmu/-0.2 mmu). 244 Dragmacidon C (83) Dragmacidon C was isolated as a pale yel low powder (4 x 10"2% of the wet weight of the sponge); [ a ] D : + 60.0° (c = 0.02, M e O H ) ; ir (f i lm), v m a x : 3413, 2919, 2849, 1643, 1590 c m - 1 ; 1 H nmr (acetone-d6, 400 M H z ) , 8: 3.05 (s, 3H, 1-NCH.3_), 4.27 (dd, / = 16.5, 5.3 H z , I H , H5fJ), 4.41 (dd, J = 16.5, 5.2 H z , I H , H5cc), 5.16 (ddd, / = 5.3, 5.2, <1 H z , I H , H6p), 7.20 (dd, J = 8.5, 1.7 H z , I H , H5'), 7.20 (dd, / = 8.7, 1.8 H z , I H , H5"), 7.22 (dd, J = 2.5, <1 H z , I H , H2'), 7.63 (d, / = 1.7 H z , I H , H7'), 7.66 (d, / = 1.8 H z , I H , H7"), 7.69 (d, / = 8.5 H z , I H , H4'), 8.37 (d, J = 8.7 H z , I H , H4"), 8.62 (d, / = 2.7 H z , I H , H2"), 10.34 (br s, l ' - N H ) , 10.71 (br s, 1" -NH) ppm; 13c nmr (acetone-d6, 75 M H z ) , 8: 32.8 (q, -NCH3), 53.5 (t, C5) , 54.0 (d, C6), 115.0 (s, C3"), 115.1 (d, C 7 " ) a , 115.5 (d, C 7 ' ) a , 115.8 (s, C6")b, 116.2 (s, C6')b, 121.2 (d), 123.3 (d), 123.7 (s, C3'), 124.4 (d), 125.3 (s, C 3 a ' ) c , 125.4 (d), 125.5 (d), 126.1 (s, C3a")c , 129.8 (s, C 7 a " ) d , 132.0 (s, C 7 a ' ) d , 133.8 (d, C2"), 157.8 (s, C2)e, 158.0 (s, C3)e p p m ( a _ e : may be interchanged); L R E I M S , m/z (formula, relative intensity): 498/500/502 ( C 2 l H i 6 B r 2 N 4 0 , 38:76:38), 496/498/500 ( C 2 l H i 4 B r 2 N 4 0 , 4:8:4) , 237/239 ( C l Q H 9 B r N 2 , 100:100), 221/223 ( C 9 H 5 B r N O , 30:30) , 208/210 ( C 9 H 6 B r N , 245 15:15), 195/197 ( C g H s B r N , 20:20); H R E I M S , C 2 l H i 6 B r 2 N 4 0 (M+) m/z: 497.9672/499.9674/501.9644 (AM, -2.0 mmu/+0.2 mmu/-0.8 mmu). 3-IndoIeglyoxylic ac id methyl ester (93) T o a stirred solution of commercially available 3-indoleglyoxylic acid (92) (500 mg, 2.64 mmol) in 50 m L of methanol were added 5 drops of concentrated sulphuric acid. The bright yellow solution was allowed to reflux under a nitrogen atmosphere for two hours, after which time the solution colour had changed to bright pink. The solvent was removed under reduced pressure and the resulting solid was purified by silica flash chromatography (EtOAc/hexane; 3:2) to yield 515 mg of pure 93 (96% yield). Compound 93 crystallized from T H F as pink needles; mp: decomposes at 213-216°C; ir (KBr), v m a x : 3200,1730, 1617, 1489, 1421, 1262,1229,1118,752 cm-1; 1H n m r (DMSO-d6, 500 MHz) , 8: 3.88 (s, 3H, H10), 7.26 (dt, / = 7.2, 1.4 Hz , IH, H5)*, 7.29 (dt, / = 7.2, 1.5 H z , IH , H6)*, 7.54 (dd, J = 7.1, 1.4 H z , IH, H7), 8.16 (dd, / = 7.2, 1.5 H z , IH, H4), 8.44 (d, / = 3.3 H z , IH , H2), 12.4 (s, IH , 1-NH) ppm (*: may be interchanged); 1 3 C nmr (DMSO-d6 , 125 M H z ) , 8: 52.6 (q), 112.4 (s), 112.8 (d), 121.2 (d), 122.9 (d), 123.9 (d), 125.5 (s), 136.7 (s), 138.6 (d), 164.0 (s), 178.7 (s) ppm; L R E I M S , m/z (relative intensity): 203 (14), 144 (100), 116 (31), 89 (27), 84 (24); H R E I M S , C11H9O (M+) m/z: 203.0586 ( A M , +0.4 mmu). O H 2 4 6 l,2,5,6-Tetrahydro-3-indole-2-pyrazinone (94) Into a 100 mL three-necked flask fitted with a nitrogen inlet, a pressure-equalized addition funnel, and a condenser was added a solution of freshly distilled ethylenediamine (162 uX, 2.41 mmol) in 20 mL of THF. The solution was brought to reflux over flame-dried, ground 4 A molecular sieves. The addition funnel was charged with a solution of 3-indoleglyoxylic acid methyl ester (93) (490 mg, 2.41 mmol) in 30 mL of hot THF. This was added to the refluxing solution over a period of twenty minutes. After the addition was complete, the reaction mixture was allowed to reflux overnight The reaction mixture was then cooled to room temperature and the excess solvent was removed under reduced pressure. The crude yellow oil which remained was purified by silica flash chromatography (EtOAc/hexane; 4:1) to yield 474 mg of pure 94 (92% yield). The pure material was isolated as an amorphous yellow solid; mp: 153-157°C; ir (film), v m a x -3298, 1668, 1582, 1530, 1434, 1342, 1247, 1166, 1125, 1015, 843, 751 cm-1; 1 H nmr (DMSO-d6, 500 MHz), 5: 3.29 (m, 2H, H6), 3.78 (t, J = 6.2 Hz, 2H, H5), 7.08 (dt, / = 7.5, 1.0 Hz, IH, H5')*, 7.14 (dt, / = 7.3, 1.2 Hz, IH, H6')*, 7.41 (d, / = 8.0 Hz, IH, H7*), 8.33 (d, / = 2.7 Hz, IH, H2'), 8.36 (d, / = 7.9 Hz, IH, H4'), 8.39 (br s, IH, 1-NH), 11.46 (br s, IH, l'-NH) ppm (*: may be interchanged); i H nmr (acetone-d6,400 MHz), 8: 3.46 (m, 2H, H6), 3.88 (t, / = 6.1 Hz, 2H, H5), 7.10 (dt, / = 7.1, 1.2 Hz, IH, H5')*, 7.15 (dt, / = 7.1, 1.4 Hz, IH, H6')*, 7.38 (br s, IH, 1-NH), 7.44 (dm, / = 247 7.7 Hz, IH, H7'), 8.49 (br s, IH, H21), 8.51 (dm, / = 7.7 Hz, IH, H4'), 10.53 (br s, IH, l'-NH) ppm (*: may be interchanged); 1 3 C nmr (DMSO-d6,75 MHz), 5: 38.0 (t), 47.4 (t), 111.1 (s), 111.6 (d), 120.4 (d), 122.1 (d), 122.5 (d), 125.9 (s), 131.7 (d), 136.1 (s), 157.5 (s, C2), 157.9 (s, C3) ppm; 13c nmr (acetone-d6, 125 MHz), 8: 39.6 (t), 48.6 (t), 112.2 (d), 113.1 (s), 121.4 (d), 123.1 (d), 124.0 (d), 127.5 (s), 132.7 (d), 137.5 (s), 158.7 (s), 159.0 (s) ppm; 13c nmr (methanol-d4, 125 MHz), 5: 39.2 (t), 46.9 (t), 111.2 (s), 113.4 (d), 123.1 (d), 123.1 (d), 124.6 (d), 126.7 (s), 137.1 (d), 138.2 (s), 159.2 (s), 159.3 (s) ppm; LREIMS, m/z (relative intensity): 213 (25), 184 (14), 156 (35), 142 (19), 115 (13), 58 (25); HREIMS, C12H11N3O (M+) m/z: 231.0902 (AM, 0.0 mmu). C . Metabolites from the Northeastern Pacific Sponge Xestosnoneia vanilla (de Laubenfels. 1930) 1. Xestospongia vanilla collected from Bamfield. B. C. Xestospongia vanilla was collected in the Sandford surge channel at Bamfield, B. C. (May 1989) at depths ranging from five to fifteen feet below sea level. The freshly collected sponge (1000 g, dry weight) was immersed in methanol immediately after collection. After having soaked for three days, the methanol was decanted from the sponge and concentrated under reduced pressure. The aqueous suspension that remained was made up to 500 mL with distilled water and extracted with hexane (3 x 500 mL) followed by dichloromethane (3 x 500 mL). The combined dichloromethane extracts were concentrated under reduced pressure to yield a brown gummy residue (3.5 g). A portion of this (500 mg) was chromatographed on a Sephadex LH-20 column prepared and eluted with methanol/dichloromethane (9:1). The early eluting fractions contained complex 248 mixtures of compounds which stained heavily with vanillin; these fractions were combined and further purified by open column reverse phase chromatography (gradient: acetone/H20; 2:3 to acetone/H20; 7:3). ^ 7 Final purification of the compounds was achieved using reverse phase HPLC. Pure samples of isoxestovanin A (125) (11 mg), xestovanin C (127) (10 mg), xestovanin A (110) (50 mg)a, and xestovanin B (113) (12 mg) were obtained using a solvent system consisting of acetone/H20 (55:45). A pure sample of dehydroxestovanin A (129) (12 mg) was obtained using a solvent system consisting of methanol/H20 (3:1) (a: larger quantities of xestovanin A which were needed for the work on the determination of its absolute stereochemistry were obtained in the same manner). Isoxestovanin A (125) 2 4 The pure compound was isolated as a colourless glass; [cc]D: - 4.4° (c = 0.16, MeOH); ir (film), v m a x : 3406, 1710, 1512, 1448, 1070, 1030 cm"1; lH nmr (acetone-d6, 400 MHz), 8: 0.95 (d, / = 6.3 Hz, 3H, H27), 1.09 (s, 3H, H28), 1.22 (d, / = 6.2 Hz, 3H, 249 H6"), 1.28 (d, / = 6.4 Hz, 3H, H6'), 1.57 (br s, 3H, H26), 1.58 (m, IH, H12a), 1.59 (br s, 3H, H30), 1.60 (br s, 3H, HI), 1.60 (m, IH, H13), 1.60 (br s, 3H, H25), 1.62 (m, IH, H13), 1.63 (br s, 3H, H29), 1.65 (m, IH, H14p), 1.66 (br s, 3H, H24), 1.81 (m, IH, H l l ) , 1.95 (m, IH, H12p), 2.17 (dq, J = 11.1, 6.3 Hz, IH, H10), 2.18 (m, IH, H14a), 2.20 (dd, / = 13.9, 8.8 Hz, IH, H17), 2.21 (m, 2H, H21), 2.24 (m, IH, H4), 2.31 (ddd, J = 13.7, 7.3, 5.8 Hz, IH, H4), 2.59 (dd, / = 13.9, 8.8 Hz, IH, H17), 3.40 (t, / = 9.0 Hz, IH, H4"), 3.49 (br s, IH, 16-OH), 3.52 (dd, / = 9.6, 7.5 Hz, IH, H2'), 3.59 (dd, / = 9.6, 2.7 Hz, IH, H3'), 3.71 (dd, / = 9.0, 3.2 Hz, IH, H3"), 3.79 (m, IH, H4'), 3.80 (m, 2H, H5' and H5"), 3.92 (d, J = 10.2 Hz, IH, H8), 3.94 (br dd, j = TA, 4.8 Hz, IH, H20), 4.01 (dd, / = 3.2, 1.6 Hz, IH, H2"), 4.21 (dd, / =8.5, 5.8 Hz, IH, H5), 4.33 (d, / = 7.5 Hz, IH, HI'), 5.01 (br t, / = 7.1 Hz, IH, H3), 5.16 (br t, J = 7.0 Hz, IH, H22), 5.27 (d, J = 1.6 Hz, IH, HI"), 5.41 (br t, / = 8.8 Hz, IH, H18), 5.76 (br d, / = 10.2 Hz, IH, H7) ppm; lH nmr (DMSO-d6, 400 MHz), 8: 0.88 (d, / = 6.3 Hz, 3H, H27), 0.96 (s, 3H, H28), 1.12 (d, / = 6.2 Hz, 3H, H6"), 1.15 (d, J = 6.4 Hz, 3H, H6'), 1.42 (m, IH, H14p), 1.47 (m, IH, H12a), 1.49 (br s, 3H, H26), 1.50 (br s, 3H, H29), 1.52 (br s, 3H, H25), 1.52 (m, IH, HI 3), 1.53 (br s, 3H, H30), 1.55 (br s, 3H, HI), 1.60 (m, IH, H13), 1.61 (br s, 3H, H24), 1.69 (m, IH, Hl l ) , 1.81 (m, IH, H12P), 1.92 (m, IH, H17), 2.04 (m, IH, H14a), 2.09 (m, 2H, H21), 2.10 (dq, J = 10.9, 6.3 Hz, IH, H10), 2.14 (m, IH, H4), 2.29 (ddd, / = 13.6, 6.5, 5.7 Hz, IH, H4), 2.42 (br dd, / = 14.2, 10.2 Hz, IH, H17), 3.19 (ddd, / = 9.5, 9.3, 5.7 Hz, IH, H4"), 3.22 (m, IH, H2'), 3.30 (m, IH, H3"), 3.32 (m, IH, H5'), 3.48 (ddd, / = 9.5, 5.5, 3.3 Hz, IH, H3"), 3.57 (m, IH, H5"), 3.60 (m, IH, H4'), 3.72 (br dd, / = 7.0, 4.6 Hz, IH, H20), 3.77 (m, IH, H2"), 3.79 (d, / = 10.6 Hz, IH, H8), 4.08 (br dd, / = 9.5, 5.7 Hz, IH, H5), 4.10 (d, / = 7.7 Hz, IH, HI'), 4.42 (s, IH, 16-OH), 4.46 (d, / = 5.5 Hz, IH, 3"-OH), 4.48 (d, / = 4.6 Hz, IH, 20-OH), 4.54 (d, J = 4.3 Hz, IH, 2"-0H), 4.60 (d, / = 5.7 Hz, IH, 4"-0H), 4.73 (d, / = 4.4 Hz, IH, 2'-0H), 4.86 (br t, / = 6.5 Hz, IH, 250 H3), 4.90 (d, / = 4.4 Hz, IH, 3'-OH), 5.05 (br t, / = 6.6 Hz, IH, H22), 5.08 (d, 7 = 1.8 Hz, IH, HI"), 5.12 (br d, / = 10.2 Hz, IH, H7), 5.58 (br d, / = 10.6 Hz, IH, H7) ppm; 13c nmr (acetone-d6,75 MHz), 8: 11.1 (q, C29), 12.2 (q, C26), 13.5 (q, C27), 17.8 (q, C6'), 18.0 (q, C25), 18.0 (q, C30), 18.1 (q, C6"), 21.6 (q, C28), 21.6 (t, C13), 25.8 (q, Cl), 26.0 (q, C24), 32.3 (t, C17), 33.7 (t, C12), 35.2 (t, C4), 35.2 (t, C21), 37.1 (t, C14), 46.9 (d, CIO), 52.6 (s, C15), 53.2 (d, C8), 55.5 (d, Cll), 69.7 (d, C5"), 70.8 (d, C5'), 72.0 (d, C2'), 72.0 (d, C2"), 72.5 (d, C3"), 73.7 (d, C4"), 76.2 (d, C3'), 77.9 (d, C20), 78.3 (d, C4'), 82.9 (d, C5), 83.6 (s, C16), 99.4 (d, Cl'), 102.5 (d, Cl"), 121.2 (d, C3), 122.2 (d, C22), 122.5 (d, C18), 128.0 (d, C7), 133.0 (s, C23)a, 133.1 (s, C2)a, 135.7 (s, C6), 138.4 (s, C19), 210.5 (s, C9) ppm (a: may be interchanged); LRFABMS, pseudomolecular ion m/z: 787 (M+(C42H680l2) + 23 (Na)), 437 ( C 3 0 H 4 5 O 2 ) , 418 ( C 3 0 H 4 2 O ) , 367 ( C 2 5 H 3 5 O 2 ) , 311 ( C 1 2 H 2 3 O 9 ) ; LREIMS, m/z (formula, relative intensity): 436 ( C 3 0 H 4 4 O 2 , 29), 418 ( C 3 0 H 4 2 O , 4), 367 ( C 2 5 H 3 5 O 2 , 88), 349 ( C 2 5 H 3 3 O , 10), 163 ( C 1 1 H 1 5 O , 40); HREIMS, C 3 0 H 4 4 O 2 (M+ - C 1 2 H 2 2 O 9 - H20) m/z: 436.3336 (AM, -0.5 mmu). Isoxestovanin A showed neither antibacterial nor antifungal activity (150 jig/disk). Isoxestovanin A hexaacetate (126) 25 27 AcO-CH 3 6" 30 24 2 5 1 A 4 m g sample of isoxestovanin A was stirred overnight in a 1:1 mixture of acetic anhydride and pyridine (1 m L of each). The reagents were removed under reduced pressure to yield the hexaacetate of isoxestovanin A , which was isolated as a pale yellow glass; i r (f i lm), v m a x : 3400,2990, 2935,1752, 1532,1370,1224, 1190, 1040 cm-1; 1 H nmr (CDCI3, 400 M H z ) , 8: 0.97 (d, / = 6.5 H z , 3 H , H27) , 0.98 (s, 3 H , H28) , 1.12 (d, / = 6.2 H z , 3 H , H6" ) , 1.28 (d, / = 6.4 H z , 3 H , H6 ' ) , 1.39 (br s, 3 H , H26) , 1.44 (m, I H , H12cc), 1.48 (br s, 3 H , H25), 1.52 (br s, 3 H , H i ) , 1.54 (br s, 3 H , H30) , 1.56 (br s, 3 H , H29) , 1.57 (m, 1H, H13) , 1.59 (m, I H , H13) , 1.61 (br s, 3 H , H24) , 1.63 (m, I H , H 1 4 p ) , 1.73 (m, I H , H l l ) , 1.92 (s, 3 H , -COCH_3_), 1.93 (s, 3 H , -COCH3J), 1.94 (s, 3 H , -COCH3J), 1.95 (m, I H , H120) , 1.96 (m, 1 H , H 1 4 a ) , 1.98 (s, 3 H , -COCH_3_), 1.99 (s, 3 H , - C O C H V ) . 2.05 (s, 3 H , -COCH3J), 2.06 (br dq, J = 11.0, 6.5 H z , 1H , H10) , 2.11 (m, I H , H17) , 2.17 (m, I H , H4) , 2.18 (m, 1H, H21) , 2.29 (m, I H , H21) , 2.37 (br dd , / = 13.4, 7.6 H z , 1H, H17) , 2.40 (ddd, / = 13.4, 7.1, 5.4 H z , I H , H4) , 3.65 (d, 7 = 10.1 H z , I H , H8) , 3.76 (br d,J = 2.8 H z , I H , H4 ' ) , 3.84 (br q, / = 6.4 H z , I H , H5 ' ) , 4.06 (dq, J = 9.6, 6.2 H z , I H , H5" ) , 4.20 (br dd, / = 9.7, 5.4 H z , I H , H5) , 4.44 (d, J = 8.1 H z , I H , H I ' ) , 4.77 (d, / = 1.6 H z , I H , H I " ) , 4.80 (br t, J = 7.1 H z , I H , H3) , 4.91 (br t, / = 7.0 H z , I H , H22) , 4.95 (dd, J = 10.3, 2.8 H z , I H , H3') , 5.00 (br t, / = 6.5 H z , I H , H20) , 5.01 (br t, J = 9.6 H z , I H , H4 " ) , 5.13 (dd, / = 10.3, 8.1 H z , I H , H2 ' ) , 5.34 (m, I H , H3" ) , 5.36 (m, I H , H2 " ) , 5.43 (m, I H , H I 8 ) , 5.58 (br d , / = 10.0 H z , I H , H7) ; L R F A B M S , psuedomolecular ion m/z: 1039 (M+(C54H8oOi8) + 23 (Na)). D e t e r m i n a t i o n of the Absolute C o n f i g u r a t i o n s of the Sugars: 129 A 5 mg sample of isoxestovanin A dissolved in 1 m L of M e O H was mixed with 1 m L of 0.5 M K O H ( a q ) and heated to 90° C for 2 hours with stirring. The resulting aqueous suspension was extracted with CH2CI2 to remove the organic soluble hydrolysis products. The 252 aqueous soluble material was taken to dryness by lyophilization. Aqueous trifluoroacetic acid (3 M, 5 mL) was added to the dried residue and the stirred solution was heated to 100°C for 2 hours. Removal of the water and trifluoroacetic acid under reduced pressure gave a clear gum that was transfered, along with 250 |iL of (+)-2-octanol and 1 drop of trifluoroacetic acid, to a vial. The vial was sealed and the solution was heated, with stirring, in an oil bath at 130°C for 12 hours The solution was concentrated to dryness under reduced pressure at a temperature of 55°C. The resulting product was then dissolved in a 1:1 mixture of acetic anhydride and pyridine (0.5 mL of each) and was kept at 100°C for 30 minutes. Excess reagents were removed under reduced pressure to yield a mixture of peracetylated (+)-2-octylglycosides. Standards were prepared similarly with D-and L-fucose and L-rhamnose. A sample of D-rhamnose was not available; therefore, L-rhamnose was derivatized in the manner described above with (-)-2-octanol to provide a standard which would mimic (+)-2-octyl-D-rhamnoside. The product mixture and standards were dissolved in CH2CI2 and analysed by capillary GC (HP SE54 column; temperature programme: 120°C for 2 minutes followed by an increase in temperature of 20°C per minute to a final temperature of 250°C). The retention times of the (+)-2-octylglycosides obtained from the degradation of isoxestovanin A were compared with those obtained for the standards to determine the absolute configurations of the sugars. The observed retention times for the standards were as follows: peracetylated (+)-2-octyl-D-fucoside (5.24 min, 5.33 min, 5.40 min); peracetylated (+)-2-octyl-L-fucoside (5.31 min, 5.45 min, 5.63 min); peracetylated (+)-2-octyl-L-rhamnoside (5.00 min, 5.07 min, 5.21 min, 5.36 min); peracetylated (-)-2-octyl-L-rhamnoside (5.14 min, 5.28 min, 5.52 min). The observed retention times for the peracetylated (+)-2-octyl-isoxestovanin A sugars were as follows: 5.02 min, 5.08 min, 5.23 min, 5.34 min, and 5.40 min. These retention times closely matched those expected for a mixture of derivatives formed from D-fucose and L-rhamnose. As well, the absence of peaks with 253 retention times greater than or equal to 5.45 min indicates that L-fucose and D-rhamnose are not present.. These results are consistent with those obtained previously for compounds in the xestovanin series.l21(a,d) Xestovanin C (127) The pure compound was isolated as a colourless glass; [oc]D: - 25.0* (c = 0.12, MeOH); ir (film), Vmax: 3423, 2933, 1708, 1366, 1225, 1045, 838, 801, 766 cm"1; lK nmr (acetone-d6, 400 MHz), 8: 1.08 (d, J = 6.9 Hz, 3H, H27), 1.18 (m, IH, H14), 1.21 (d, / = 6.0 Hz, 3H, H6"'), 1.25 (d, / = 6.2 Hz, 3H, H6"), 1.29 (d, / = 6.3 Hz, 3H, H6'), 1.35 (m, IH, H13), 1.38 (s, 3H, H28), 1.44 (m, IH, H13), 1.53 (br s, 3H, H29), 1.60 (br s, 6H, H25 and H30), 1.62 (br s, 3H, H26), 1.64 (m, IH, H12), 1.65 (br s, 6H, HI and H24), 1.97 (m, IH, H12), 2.04 (m, IH, Hll), 2.18 (m, IH, H21), 2.21 (m, IH, H8), 2.26 (m, IH, H4), 2.28 (m, IH, H21), 2.31 (m, IH, H4), 3.34 (dq, / = 6.9, 5.8 Hz, IH, H10), 2.41 (dd, / = 15.0, 9.6 Hz, IH, H8), 2.56 (ddd, / = 13.2, 8.3, 5.2 Hz, IH, H14), 3.41 (br t, J = 8.5 Hz, IH, H4"'), 3.52 (m, IH, H2'), 3.57 (m, 2H, H3' and 254 H4" ) , 3.60 (m, I H , H5 ' ) , 3.61 (m, I H , H 3 , M ) , 3.69 (m, I H , H5" ' ) , 3.75 (d, J = 9.8 H z , I H , H17) , 3.80 (m, I H , H5 " ) , 3.81 (br d, / = 2.1 H z , I H , H4 ' ) , 3.88 (m, I H , H3" ) , 3.95 (br s, I H , H 2 m ) , 4.01 (br s, I H , H2 " ) , 4.06 (br t, J = 7.5 H z , I H , H20) , 4.19 (d, J = 7.2 H z , I H , H I ' ) , 4.20 (dd, J = 8.3, 6.5 H z , I H , H5 ) , 5.04 (br t, / = 6.7 H z , I H , H3) , 5.09 (br t, / = 7.2 H z , I H , H22) , 5.25 (d, / = 1.2 H z , I H , H I " ) , 5.29 (d, J = 1.1 H z , I H , H I " ' ) , 5.43 (br d, / = 9.8 H z , I H , H7 ) , 5.61 (br d, / = 9.7 H z , I H , H18) ppm; i H nmr ( D M S O - d 6 , 400 MHz) , *8 : 0.92 (d, J = 6.8 H z , 3 H , H27) , 1.11 (d, J = 6.2 H z , 3 H , H6" ' ) , 1.13 (d, / = 6.1 H z , 3 H , H6' ) , 1.14 (d, / = 6.1 H z , 3 H , H6" ) , 1.14 (m, I H , H14) , 1.26 (s, 3 H , H28) , 1.34 (m, I H , H13) , 1.40 (m, I H , H13) , 1.40 (br s, 3 H , H29), 1.52 (br s, 6 H , H26 and H30) , 1.54 (br s, 3 H , H25) , 1.55 (m, I H , H12) , 1.57 (br s, 3 H , H24) , 1.59 (br s, 3 H , H I ) , 1.60 (m, I H , H12) , 1.86 (m, I H , H l l ) , 2.01 (m, I H , H8) , 2.08 (m, I H , H21) , 2.11 (dq, / = 6.9, 5.6 H z , I H , H10) , 2.18 (m, I H , H21), 2.23 (m, I H , H4 ) , 2.25 (m, I H , H8) , 2.29 (m, I H , H4 ) , 2.43 (ddd, / = 13.0, 7.5, 5.4 H z , I H , H14) , 3.19 (ddd, / = 9.4, 9.2, 5.6 H z , I H , H4" ' ) , 3.25 (m, I H , H2' ) , 3.30 (m, I H , H3"), 3.37 (m, I H , H3" ' ) , 3.39 (m, I H , H5 ' ) , 3.39 (m, I H , H 4 " ) , 3.49 (dq, J = 9.2, 6.2 H z , I H , H5" ' ) , 3.57 (d, / = 9.9 H z , I H , H17) , 3.60 (br s, I H , H4 ' ) , 3.61 (m, I H , H3" ) , 3.64 (m, I H , H5" ) , 3.71 (m, I H , H2" ' ) , 3.75 (m, I H , H2 " ) , 3.88 (br dt, J = 7.4, 4.1 H z , I H , H20) , 3.94 (d, J = 7.3 H z , I H , H I ' ) , 4.03 (dd, J = 8.7, 6.0 H z , I H , H5) , 4.37 (s, I H , 9 - O H ) , 4.42 (d, / = 5.6 H z , I H , 3 " ' - 0 H ) , 4.55 (d, / = 4.2 H z , I H , 2 ' " - 0 H ) , 4.62 (d, / = 5.6 H z , I H , 4 " ' -OH) , 4.64 (d, / = 4.1 H z , I H , 20-OH) , 4.68 (d, / = 6.8 H z , I H , 3 " - O H ) , 4.76 (d, / = 4.5 H z , I H , 2 " - O H ) , 4.90 (d, / = 4.3 H z , I H , 2 ' -OH) , 4.93 (br t, / = 6.9 H z , I H , H3) , 4.96 (br t, J = 9.2 H z , I H , H22) , 5.03 (d, / = 4.0 H z , I H , 3*-0H), 5.06 (br s, 2 H , H I " and H I " ' ) , 5.20 (br d , / = 7.4 H z , I H , H7) , 5.45 (br d , / = 9.9 H z , I H , H18) ppm; 13c nmr (acetone-d6, 75 M H z ) , 5: 11.0 (q, C26) , 11.6 (q, C29) , 13.7 (q, C27) , 17.8 (q, C6 ' ) , 18.0 (q, C25) , 18.0 (q, C30) , 18.0 (q, C6 " ' ) , 18.7 (q, C 6 " ) , 22.4 (t, C13) , 25.9 (q, C 2 4 ) * , 26.0 (q, C l ) * , 26.0 (q, C28) , 2 5 5 28.0 (t, C12), 32.6 (t, C21)°, 34.9 (t, C4)b, 35.0 (d, CIO), 35.4 (t, C14), 37.8 (t, C8), 52.0 (d, C17), 55.9 (s, C15), 56.3 (d, Cll), 68.3 (d, C5"), 69.8 (d, C5"'), 71.2 (d, C3"')c, 71.8 (d, C2'), 72.0 (d, C2"'), 72.4 (d, C4"), 72.5 (d, C2"), 73.0 (d, C3"), 73.7 (d, C4"'), 76.1 (d, C5')c, 78.4 (d, C20), 78.6 (d, C4'), 79.7 (d, C3*)c, 83.1 (d, C5), 83.4 (s, C9), 99.8 (d, Cl'), 102.2 (d, Cl"'), 102.5 (d, Cl"), 121.2 (d, C3), 122.1 (d, C22), 122.2 (d, C18), 127.0 (d, C7), 132.7 (s, C23)d, 133.1 (s, C2)d 135.4 (s, C6), 141.1 (s, C19), 213.0 (s, C16) ppm (*-<*: m a y be interchanged); LRFABMS, pseudomolecular ion m/z: 933 (M+(C48H780i6) + 23 (Na)), 455 (C18H32O13 - H), 436 (C30H44O2); LREIMS, m/z (formula, relative intensity): 436 (C30H44O2,34), 418 (C30H42O, 7), 385 (C25H37O3, 9), 367 (C25H35O2, 33), 349 (C25H33O, 18), 301 (C20H29O2, 14), 229 (C16H21O, 14), 203 (C14H19O, 26), 163 (C11H15O, 15), 147 (C11H15, 14), 139 (C9H15O, 10); HREIMS, C30H44O2 (M+ - C18H32O13 - H20) m/z: 436.3342 (AM, 0.0 mmu). Xestovanin C showed neither antibacterial nor antifungal activity (148 jig/disk). Xestovanin C octaacetate (128) 256 A 5 mg sample of xestovanin C was acetylated in the manner described for the acetylation of isoxestovanin A. The pure compound was isolated as a pale yellow glass; ir (film), Vmax: 3385, 3000, 2929, 1751, 1716, 1371, 1242, 1227, 1072, 1041 cm"l; iH nmr (CDCI3, 400 MHz), 8: 1.08 (d, / = 7.0 Hz, 3H, H27), 1.20 (m, IH, H14), 1.21 (d, / = 6.3 Hz, 3H, H6'"), 1.33 (d, / = 6.4 Hz, 3H, H6"), 1.34 (d, / = 6.4 Hz, 3H, H6'), 1.35 (s, 3H, H28), 1.42 (m, IH, H13), 1.50 (m, IH, H13), 1.50 (br s, 3H, H26), 1.52 (br s, 3H, H29), 1.60 (br s, 3H, H25), 1.61 (br s, 3H, H30), 1.64 (br s, 3H, HI), 1.67 (br s, 3H, H24), 1.70 (m, IH, H12), 1.81 (m, IH, H12), 1.99 (m, IH, Hll), 1.99 (s, 3H, -OCOCJi3_), 2.01 (s, 3H, -OCOCHg;). 2.03 (s, 3H, -OCOCHg). 2.05 (s, 3H, - O C O C H 3 J , 2.06 (s, 3H, -OCOCH3_), 2.10 (s, 3H, -OCOCHg). 2.14 (s, 3H, -OCOCH3J, 2.15 (m, IH, H8), 2.17 (s, 3H, -OCOCHg), 2.22 (m, IH, H4), 2.27 (dq, J = 7.0, 5.9 Hz, IH, H10), 2.28 (m, IH, H8), 2.30 (m, IH, H21), 2.42 (m, IH, H21), 2.43 (m, IH, H4), 3.55 (dq, / = 6.4, <1 Hz, IH, H5'), 3.60 (d, J = 9.6 Hz, IH, H17), 3.70 (t, J = 9.4 Hz, IH, H4"), 3.81 (dd, / = 3.0, <1 Hz, IH, H4'), 3.98 (dq, / = 9.8, 6.3 Hz, IH, H5m), 4.00 (dq, / = 9.5, 6.4 Hz, IH, H5"), 4.20 (dd, / = 9.3, 5.6 Hz, IH, H5), 4.30 (d, J = 7.9 Hz, IH, HI"), 4.82 (d, J = 2.0 Hz, IH, HI"), 4.90 (br t, / = 7.9 Hz, IH, H3), 4.93 (dd, / = 10.4, 3.1 Hz, IH, H3"), 5.00 (d, J = 1.8 Hz, IH, HI"'), 5.01 (br t, / = 7.3 Hz, IH, H22), 5.07 (t, J = 9.8 Hz, IH, H4"'), 5.16 (dd, / = 3.3, 2.0 Hz, 12H, H2"'), 5.17 (br t, J = 6.9 Hz, IH, H20), 5.20 (dd, / = 10.4, 7.9 Hz, IH, H2'), 5.26 (br d, / = 9.4 Hz, IH, H7), 5.26 (dd, J = 9.8, 3.3 Hz, IH, H3"'), 5.30 (dd, / = 9.4, 3.2 Hz, IH, H3"), 5.42 (dd, J = 3.2, 2.0 Hz, IH, H2"), 5.65 (br d, / = 9.8 Hz, IH, H18) ppm; LRFABMS, pseudomolecular ion m/z: 1269 (M+(C64H94C»24) + 23 (Na)). Determination of the absolute configurations of the sugars: The determination of the absolute configurations of the sugars forming the trisaccharide portion of xestovanin 257 C was carried out in the same manner as for isoxestovanin AA 29 A 3 mg sample of xestovanin C was used. The retention times found for the peracetylated (+)-2-octylglycosides of 127 were: 5.00 min, 5.05 min, 5.20 min, 5.32 min, and 5.38 min . A comparison of these values with those obtained for the standards shows that the sugar portion of xestovanin C is comprised of D-fucose and L-rhamnose. X e s t o v a n i n A (110) OH O 28 The data obtained for this compound was the same as that found previously!21(a, d) Additional data is the following: [ct]D: - 42.0° (c = 0.30, MeOH); *3c nmr (acetone-d6, 75 MHz), 8: 11.0 (q, C26), 11.5 (q, C29), 13.7 (q, C27), 17.8 (q, C6'), 18.0 (q, C25), 18.1 (q, C30 and C6"), 22.3 (t, C13), 25.9 (q, C24)a, 25.9 (q, C28)a, 26.0 (q, Cl)a, 27.9 (t, C12), 32.5 (t, C21)b, 34.7 (t, C14), 34.9 (t, C4)b 34.9 (d, C10), 37.7 (t, C8), 52.0 (d, C17), 55.8 (s, C15), 56.2 (d, Cll), 69.7 (d, C5"), 71.2 (d, C5'), 71.7 (d, C2"), 71.9 (d, C2'), 72.4 (d, C3"), 73.5 (d, C4"), 76.0 (d, C3'), 78.1 (d, 4'), 78.3 (d, C20), 258 83.0 (d, C5), 83.3 (s, C9), 99.7 (d, Cl'), 102.5 (d, Cl"), 121.1 (d, C3), 122.1 (d, C18), 122.1 d, C22), 127.0 (d, C7), 132.7 (s, C23)c, 133.1 (s, C2)C, 135.4 (s, C6), 141.1 (s, C19), 213.3 (s, C16) ppm (a~c: may be interchanged). The compound showed no antibacterial activity but showed considerable antifungal activity against the fungus Pythium ultimum (264 ug/disk). Xestovanin B (113) The data obtained for this compound was the same as that found previously 121(a). Additional data is the following: [a]D: - 45.5° (c = 0.29, MeOH); l^ C nmr (acetone-d6, 75 MHz), 8: 11.2 (q, C26)a, 11.3 (q, C29)a, 13.7 (q, C27), 17.8 (q, C6')b, 17.9 (q, C6")b, 18.0 (q, C6m)b 18.0 (q, C25)b 18.2 (q, C30)b, 22.4 (t, C13), 25.9 (q, C28), 2 5 9 26.0 (q, Cl), 26.0 (q, C24), 28 (t, C12), 32.7 (t, C21)C, 32.9 (t, C4)C, 34.8 (d, CIO), 35.5 (t, C14), 37.9 (t, C8), 52.4 (d, C17), 55.8 (s, C15), 56.1 (d, Cll), 69.1 (d, C5m), 69.7 (d, C5"), 71.2 (d, C51), 71.8 (d, C2")d, 71.9 (d, C2')d, 72.2 (d, C2"')d 72.5 (d, C3")d, 72.6 (d, C3"')d, 73.6 (d, C4")e, 73.9 (d, C4"*)e, 76.1 (d, C31), 78.0 (d, C4*), 81.4 (d, C20)f, 82.9 (d, C5)f, 83.0 (s, C9), 96.9 (d, Cl*"), 99.9 (d, Cl'), 102.5 (d, Cl"), 121.1 (d, C3)S, 121.7 (d, C22)g, 126.2 (d, C18)h, 126.4 (d, C7)h, 133.1 (s, C2)i, 133.6 (s, C23)i, 135.9 (s, C6)J, 136.8 (s, C19)J, 212.5 (s, C16) ppm (a-j: may be interchanged). The compound showed neither antibacterial nor antifungal activity (172 u,g/disk). D e h y d r o x e s t o v a n i n A (129) OH O 28 The pure compound was isolated as a pale yellow glass; [a]D: - 45.0° (c = 0.78, MeOH); ir (fdm), vmax: 3406, 2979, 2938, 2896 (sh), 1701, 1660 (sh), 1464, 1381, 1253, 1129, 1072, 1046, 1031, 981 cm"l; *H nmr (acetone-d6, 400 MHz), 8: 1.19 (d, / = 6.2 Hz, 260 3H, H6"), 1.21 (s, 3H, H28), 1.22 (d, / = 6.4 Hz, 3H, H6'), 1.33 (ddd, / = 12.9, 7.9, 5.3 Hz, IH, H14P), 1.54 (br s, 3H, H26), 1.57 (br s, 6H, H25 and H30), 1.61 (br s, 3H, H29), 1.64 (br s, 3H, HI), 1.64 (m, 2H, HI3), 1.65 (br s, 3H, H24), 1.99 (m, IH, H12), 2.03 (m, IH, H14a), 2.14 (m, IH, H12), 2.21 (m, 2H, H21), 2.22 (m, IH, H4), 2.27 (m, IH, H4), 2.33 (dd, J = 14.3, 7.2 Hz, IH, H8), 2.46 (dd, / = 14.3, 7.1, IH, H8), 2.67 (br t, / = 5.8 Hz, IH, Hll), 3.39 (br t, / = 9.1 Hz, IH, H4"), 3.49 (br dd, J = 9.8, 7.6 Hz, IH, H2'), 3.57 (dd, J = 9.8, 3.0 Hz, IH, H3"), 3.60 (dq, / = 6.4, 1.2 Hz, IH, H5'), 3.67 (d, / = 9.3 Hz, IH, H17), 3.69 (m, IH, H3"), 3.78 (m, IH, H4'), 3.79 (m, IH, H5"), 4.00 (br s, IH, H2"), 4.01 (m, IH, H20), 4.07 (dd, / = 8.6, 6.1 Hz, IH, H5), 4.13 (d, J = 7.6 Hz, IH, HI'), 5.00 (br t, J = 7.1 Hz, IH, H3), 5.12 (br t, / = 7.1 Hz, IH, H22), 5.17 (br t, / = 1.2 Hz, IH, H27E), 5.24 (d, J = 1.5 Hz, IH, HI"), 5.33 (br t, / = 1.3 Hz, IH, H27Z), 5.42 (br t, / = 7.2 Hz, IH, H7), 5.60 (br d, / = 9.3 Hz, IH, H18) ppm; iH nmr (DMSO-d6, 400 MHz), 5: 1.09 (d, / = 6.2 Hz, 3H, H6"), 1.12 d, / = 6.2 Hz, 3H, H6'), 1.12 (s, 3H, H28), 1.24 (m, IH, H14p), 1.48 (br s, 3H, H29), 1.49 (br s, 3H, H26), 1.53 (m, IH, H13), 1.53 (br s, 3H, H30), 1.54 (br s, 3H, H25), 1.59 (m, IH, H13), 1.60 (br s, 6H, HI and H24), 1.89 (m, IH, H12), 1.93 (m, IH, H14ct), 2.01 (m, IH, H12), 2.09 (m, 2H, H21), 2.18 (m, IH, H4), 2.20 (m, IH, H8), 2.21 (m, IH, H4), 2.34 (dd, J = 14.5, 5.6 Hz, IH, H8), 3.57 (br t, / = 6.1 Hz, IH, Hll), 3.17 (dt, J = 9.5, 5.9 Hz, IH, H4"), 3.21 (ddd, / = 9.6, 7.7, 4.7 Hz, IH, H2'), 3.33 (m, IH, H3'), 3.44 (br q, J = 6.2 Hz, IH, H5'), 3.45 (m, IH, H3"), 3.56 (m, IH, H4'), 3.56 (d, / = 8.8 Hz, IH, H17), 3.58 (m, IH, H5"), 3.75 (m, IH, H2"), 3.84 (br dt, J = 6.6, 4.1 Hz, IH, H20), 3.92 (d, J = 7.7 Hz, IH, HI'), 3.95 (dd, J = 8.8, 5.7 Hz, IH, H5), 4.47 (d, / = 5.8 Hz, IH, 3"-OH), 4.55 (d, / = 4.2 Hz, IH, 2"-OH), 4.63 (d, / = 5.9 Hz, IH, 4"-OH), 4.64 (s, IH, 9-OH), 4.64 (d, / = 4.6 Hz, IH, 20-OH), 4.81 (d, J = 4.7 Hz, IH, 2'-OH), 4.90 (br t, / = 7.0 Hz, IH, H3), 4.97 (d, / = 4.3 Hz, IH, 3'-OH), 5.02 (br t, / = 5.7 Hz, IH, H22), 5.04 (br s, IH, H27£), 5.07 (d, / = 1.4 Hz, 261 I H , H I " ) , 5.17 (br s, I H , H27Z) , 5.26 (br t, J = 5.9 H z , I H , H7) , 5.44 (br d, J = 8.8 H z , I H , H I 8 ) ppm; 13c nmr (acetone-d6, 75 MHz) ,8: 11.1 (q, C26) , 12.7 (q, C29) , 17.7 (q, C6'), 18.0 (q, C25) , 18.0 (q, C6"), 18.0 (q, C30) , 22.9 (t, C13) , 25.9 (q, C28) , 26.0 (q, C l ) , 26.0 (q, C24) , 29.0 (t, C12)*, 32.7 (t, C21)*, 34.8 (t, C4 )a, 36.4 (t, C14) , 38.8 (t, C8), 52.5 (d, C l l ) , 56.2 (s, C15), 57.6 (d, C17) , 69.7 (d, C5"), 70.9 (d, C5'), 71.9 (d, C2'), 71.9 (d, C2"), 72.5 (d, C3"), 73.6 (d, C4"), 76.0 (d, C3'), 77.5 (d, C20), 78.1 (d, C4'), 79.4 (s, C9), 83.1 (d, C5), 99.9 (d, C l ' ) , 102.5 (d, C l " ) , 111.0 (t, C27) , 120.3 (d, C18) , 121.2 (d, C3), 122.1 (d, C22), 126.1 (d, C7), 132.9 (s, C2), 132.9 (s, C23) , 136.1 (s, C6), 142.7 (s, C19) , 150.4 (s, CIO), 212.7 (s, C16) ppm; (*: may be interchanged) (*: there is uncertainty in the position of this peak due to the overlapping solvent mult iplet); L R F A B M S , pseudomolecular ion m/z: 785 ( M + ( C 4 2 H 6 6 0 l 2 ) + 23(Na)), 435 (M+ - C 1 2 H 2 1 O 9 - H 2 O ) , 309 (C12H21O9); L R E I M S , m/z (formula, relative intensity): 434 (C30H42O2, 41), 416 (C30H40O, 14), 365 (C25H33O2, 69), 349 ( C 2 5 H 3 3 O , 10), 265 (C19H21O, 13); H R E I M S , C 3 0 H 4 2 O 2 (M+ - C 1 2 H 2 2 O 9 - H 2 O ) m/z: 434.3191 ( A M , +0.6 mmu). Dehydroxestovanin A displayed no antibacterial activity but displayed moderate antifungal activity against the fungi Pythium ultimum and Rhizoctonia solani (144 |ig/disk). 262 Dehydroxestovanin A hexaacetate (130) H r ^ O A c -OAc 25 o 24 13 A 5 mg sample of dehydroxestovanin A was acetylated in the manner described for the acetylation of isoxestovanin A . The pure compound was isolated as a pale yel low glass; ir (f i lm), v m a x : 3497, 2974, 2933, 2872, 1749, 1369, 1241 (sh), 1226, 1174, 1128, 1072, 1041 cm-1 ; ! H nmr ( D M S O - d 6 , 400 M H z ) , 8: 1.10 (d, / = 6.3 H z , 3 H , H6" ) , 1.13 (s, 3 H , H28) , 1.17 (d, / = 6.3 H z , 3 H , H6"), 1.23 (m, I H , H14p ) , 1.38 (br s, 3 H , H26) , 1.53 (m, I H , H13) , 1.54 (br s, 3 H , H25) , 1.55 (br s, 3 H , H29) , 1.56 (br s, 3 H , H30) , 1.58 (m, I H , H13) , 1.61 (br s, 3 H , H I ) , 1.62 (br s, 3 H , H24) , 1.87 (m, I H , H12) , 1.91 (m, I H , H14ct) , 1.93 (s, 3 H , - O C O C H 3 ) , 1.94 (s, 3 H , - O C O C H 3 ) , 1.95 (s, 3 H , - O C O C H 3 _ ) , 1.97 (s, 3 H , - O C O C H 3 ) , 2.04 (s, 3 H , - O C O C H 3 ) , 2.04 (m, I H , H12) , 2.10 (s, 3 H , - O C O C H 3 ) , 2.10 (m, I H , H4) , 2.20 (m, 2 H , H 4 and H8) , 2.24 (m, I H , H21) , 2.25 (m, I H , H21) , 2.33 (m, I H , H8) , 2.47 (m, I H , H l l ) , 3.55 (d, / = 9.0 H z , I H , H17) , 3.70 (br q, / = 6.3 H z , I H , H5 ' ) , 3.87 (br d, J = 3.5 H z , I H , H4 ' ) , 3.92 (dd, / = 9.2, 5.9 H z , I H , H5) , 3.98 (dq, / = 9.6, 6.3 H z , I H , H5 " ) , 4.33 (d, / = 7.9 H z , I H , H I ' ) , 4.88 (m, I H , H3) , 4.88 (m, I H , H 4 " ) , 4.89 (d, J = 1.7 H z , I H , H I " ) , 4.89 (m, 263 I H , H2 ' ) , 4.98 (br d, / = 8.6 H z , I H , H3') , 4.98 (br t, / = 7.0 H z , I H , H22) , 5.01 (dd, / = 9.6, 6.5 H z , I H , H20) , 5.10 (br s, I H , H27£) , 5.17 (dd, J = 10.0, 3.5 H z , I H , H 3 " ) , 5.21 (br s, I H , H27Z) , 5.29 (dd, / = 3.5, 1.8 H z , 1H, H2" ) , 5.33 (br t, / = 5.6 H z , I H , H7) , 5.54 (d, / = 9.0 H z , I H , H18) ppm; i H nmr (benzene-d6, 400 M H z ) , 8: 1.28 (s, 3 H , H28) , 1.29 (d, / = 6.3 H z , 3 H , H6" ) , 1.34 (d, / = 6.4 H z , 3 H , H6' ) , 1.50 (m, I H , H14) , 1.55 (br s, 3 H , H25) , 1.59 (br s, 3 H , H30) , 1.62 (m, I H , H13) , 1.74 (br s, 3 H , H26) , 1.74 (br s, 3 H , H I ) , 1.74 (s, 3 H , - O C O C H ^ ) . 1.75 (br s, 3 H , H24) , 1.75 (s, 3 H , - O C O C H 3 _ ) , 1.75 (s, 3 H , -OCOCJ i3_ ) , 1.76 (m, I H , H13) , 1.79 (s, 3 H , -OCOCH3J), 1.88 (m, I H , H12) , 1.92 (d, / = 1.3 H z , 3 H , H29) , 1.98 (s, 3 H , -OCOCm). 2.12 (m, I H , H14) , 2.16 (m, I H , H12), 2.20 (s, 3 H , - O C O C T U ) . 2.40 (m, I H , H 4 ) , 2.52 (m, 2 H , H21) , 2.53 (m, 2 H , H8) , 2.54 (m, I H , H l l ) , 2.58 (dd, J = 14.9, 7.2 H z , I H , H4 ) , 3.38 (br q, / = 6.6 H z , I H , H5') , 3.58 (br d, J = 3.1 H z , I H , H4 ' ) , 3.86 (d, / = 9.4 H z , I H , H17) , 4.29 (dq, J = 9.7, 6.3 H z , I H , H5" ) , 4.45 (dd, J = 8.9, 5.9 H z , I H , H5 ) , 4.75 (d, / = 8.0 H z , I H , HI") , 5.02 (d, / = 2.1 H z , I H , H I " ) , 5.18 (br t, / = 7.1 H z , I H , H22) , 5.23 (br s, I H , H 2 7 E ) , 5.30 (dd, / = 10.3, 3.1 H z , I H , H3 ' ) , 5.33 (br t, / = 6.9 H z , I H , H20) , 5.59 (t, / = 9.7 H z , I H , H4" ) , 5.60 (br s, I H , H 2 7 Z ) , 5.71 (br t, / = 6.8 H z , I H , H7) , 5.78 (d, J = 9.4 H z , I H , H18) , 5.80 (m, I H , H2"), 5.81 (dd, / = 9.7, 3.3 H z , I H , H3" ) , 5.88 (dd, / = 3.2, 2.1 H z , I H , H2" ) ppm; L R F A B M S , pseudomolecular ion m/z: 1037 ( M + ( C 5 4 H 7 8 0 i 8 ) + 23(Na)). Determination of the absolute configurations of the sugars: The determination o f the absolute configurations o f the sugars forming the disaccharide portion of dehydroxestovanin A was carried out in the same manner as for isoxestovanin A . 129 A 5 mg sample o f dehydroxestovanin A was used. The retention times found for the peracetylated (+)-2-octylglycosides of 129 were: 5.00 min, 5.07 min, 5.21 min, 5.33 min, and 5.41 min. A comparison of these values with those obtained for the standards 264 shows that the sugar portion of dehydroxestovanin A is comprised of D-fucose and L-rhamnose. 2. Xestospongia vanilla collected in the Queen Charlotte Islands Xestospongia vanilla was collected in a surge channel near Anthony Island in the Queen Charlotte Islands, B. C. (April 1989). The freshly collected sponge (400 g, dry weight) was frozen immediately after collection and immersed in methanol after being transported to our laboratory; after three days of soaking, the methanol was decanted from the sponge and concentrated under reduced pressure. The aqueous suspension that remained was made up to 300 mL with distilled water and extracted with hexane (3 x 300 mL) followed by dichloromethane (3 x 300 mL). The dichloromethane extracts were combined and concentrated under reduced pressure to yield a brown gummy residue (1.5 g). A portion of this (500 mg) was chromatographed on a Sephadex LH-20 column prepared and eluted with methanol/dichloromethane (9:1). Again, the early eluting fractions contained complex mixtures of compounds which stained heavily (brown) with vanillin; these fractions were combined and further purified by open column reverse phase chromatography (gradient: acetone/H20; 2:3 to acetone/H20; 7:3). ^ 7 Final purification of the compounds was achieved using reverse phase HPLC. Pure samples of dehydroxestovanin A (129) (15 mg) and epi-dehydroxestovanin A (131) (14 mg) were obtained using a solvent system consisting of methanol/H20 (3:1). Pure samples of secodehydroxestovanin A (112) (20 mg) and dehydroxestovanin C (132) (10 mg) were obtained using a solvent system of acetone/H20 (55:45). 265 Epi-dehydroxestovanin A (131) The pure compound was isolated as a pale yel low glass; [cc]D: - 3.8° (c = 3.8, M e O H ) ; i r (f i lm), Vmax: 3493, 2919, 1703, 1509, 1376, 1229, 1070, 991, 909, 838, 808 c m ' l ; i H nmr (acetone-d6, 400 M H z ) , 8: 1.13 (s,< 3 H , H28) , 1.19 (d, J = 6.2 H z , 3 H , H6" ) , 1.22 (d, / = 6.4 H z , 3 H , H6 ' ) , 1.42 (ddd, / = 12.5, 7.8, 4.3 H z , I H , H14) , 1.54 (br s, 3 H , H26) , 1.58 (br s, 6 H , H25 and H30) , 1.62 (m, I H , H13) , 1.64 (br s, 3 H , H29) , 1.65 (br s, 6 H , H I and H24) , 1.73 (m, I H , H13) , 1.99 (m, I H , H12), 2.03 (m, I H , H14) , 2.26 (m, 2 H , H21) , 2.27 (m, 2 H , H4) , 2.32 (dd, / = 14.9, 7.0 H z , I H , H8) , 2.36 (dd, / = 15.0, 6.6 H z , I H , H8) , 2.54 (br t, / = 6.5 H z , I H , H l l ) , 3.39 (br t, J = 9.3 H z , I H , H 4 " ) , 3.50 (dd, J = 10.0, 7.6 H z , I H , H2' ) , 3.58 (d, J = 9.5 H z , I H , H17) , 3.58 (m, I H , H3*), 3.60 (dq, / = 6.4, 1.0 H z , I H , H5' ) , 3.69 (m, I H , H3" ) , 3.76 (dd, / = 2.7 H z , 1.0 H z , I H , H4"), 3.78 (m, I H , H5 " ) , 4.00 (br s, I H , H2 " ) , 4.05 (m, I H , H20) , 4.06 (m, I H , H5) , 4.12 (d, J = 7.6 H z , I H , H I ' ) , 4.98 (br t, / = 7.1 H z , I H , H3) , 5.15 (br t, / = 7.3 H z , I H , H22) , 5.15 (br s, I H , H27£) , 5.24 (d, J = 1.4 H z , I H , H I " ) , 5.33 266 (br s, IH, H27Z), 5.43 (br t, / = 6.5 Hz, IH, H7), 5.52 (br d, J = 9.5 Hz, IH, HI8) ppm; ! H nmr (DMS0-d6, 400 MHz),8: 1.05 (s, 3H, H28), 1.09 (d, / = 6.2 Hz, 6H, H6* and H6"), 1.37 (ddd, / = 12.2, 7.5, 4.5 Hz, IH, H14p), 1.46 (br s, 3H, H26), 1.50 (m, IH, H13), 1.52 (br s, 6H, H29 and H30), 1.54 (br s, 3H, H25), 1.59 (br s, 3H, H24), 1.61 (m, IH, H13), 1.63 (br s, 3H, HI), 1.90 (m, IH, H12), 2.03 (m, IH, H12), 2.04 (m, IH, H14cc), 2.09 (m, 2H, H21), 2.16 (m, IH, H4), 2.22 (m, 3H, H4 and H8), 2.45 (br t, J = 6.8 Hz, IH, Hl l ) , 3.17 (ddd, J = 9.5, 9.3, 5.7 Hz, IH, H4"), 2.32 (ddd, / = 9.6, 7.6, 4.6 Hz, IH, H2'), 3.35 (m, IH, H3'), 3.38 (d, / = 9.5 Hz, IH, H17), 3.43 (br q, / = 6.2 Hz, IH, H5'), 3.45 (m, IH, H3"), 3.55 (br s, IH, H4'), 3.57 (m, IH, H5"), 3.75 (m, IH, H2"), 3.81 (br dt, J = 6.2, 4.0 Hz, IH, H20), 3.93 (m, IH, H5), 3.94 (d, J = 7.6 Hz, IH, HI'), 4.46 (d, J = 5.7 Hz, IH, 3"-OH), 4.53 (d, / = 4.2 Hz, IH, 2"-OH), 4.56 (d, / = 4.0 Hz, IH, 20-OH), 4.57 (s, IH, 9-OH), 4.61 (d, J = 5.7 Hz, IH, 4"-OH), 4.76 (d, / = 4.7 Hz, IH, 2'-OH), 4.89 (br t, J = 7.1 Hz, IH, H3), 4.94 (d, / = 4.2 Hz, IH, 3-OH), 5.01 (br t, J = 7.0 Hz, IH, H22), 5.07 (br s, 2H, HI" and H27E), 5.14 (br s, IH, H27Z), 5.27 (br d, / = 9.5 Hz, IH, H18), 5.32 (br t, / = 6.6 Hz, IH, H7) ppm; 13c nmr (acetone-d6, 75 MHz),8: 11.1 (q, C26), 13.0 (q, C29), 17.7 (q, C6'), 18.0 (q, C6"), 18.1 (q, C25), 18.1 (q, C30), 23.4 (t, C13), 26.0 (q, Cl), 26.0 (q, C24), 26.6 (q, C28), 32.6 (t, C12), 32.9 (t, C21)a, 34.7 (t, C4)a, 3 4 j a (t, C14), 38.7 (t, C8), 52.8 (d, Cl l ) , 55.5 (s, C15), 59.0 (d, C17), 69.7 (d, C5"), 70.8 (d, C5'), 71.0 (d, C2'), 71.0 (d, C2"), 72.5 (d, C3"), 73.6 (d, C4"), 75.9 (d, C3'), 77.5 (d, C20), 78.2 (d, C4'), 79.4 (s, C9), 83.1 (d, C5), 100.1 (d, Cl'), 102.5 (d, Cl"), 110.3 (t, C27), 119.7 (d, C18), 121.3 (d, C3), 121.9 (d, C22), 125.3 (d, C7), 133.0 (s, C2), 133.0 (s, C23), 136.5 (s, C6), 143.9 (s, C19), 150.3 (s, CIO), 212.6 (s, C16) ppm (a: may be interchanged); LRFABMS, pseudomolecular ion m/z: 785 (M+(C42H660l2) + 23(Na)), 435 (M+ - C 1 2 H 2 1 O 9 - H 2 O ) , 309 ( C 1 2 H 2 1 O 9 ) ; LREIMS, m/z (formula, relative intensity): 434 (C30H42O2, 10), 365 (C25H33O2, 24), 349 (C25H33O, 4); HREIMS, 2 6 7 C 3 0 H 4 2 O 2 ( M + - C12H22O9 - H 2 O ) m/z: 434.3189 (AM, +0.5 mmu). The compound showed considerable antibacterial activity against both Staphylococcus aureus and Bacillus subtilis and also showed considerable antifungal activity against Pythium ultimum (625 H.g/disk). Determination of the absolute configurations of the sugars: The determination of the absolute configurations of the sugars forming the disaccharide portion of epi-dehydroxestovanin A was carried out in the same manner as for isoxestovanin A . 129 A 4 mg sample of epi-dehydroxestovanin A was used. The retention times found for the peracetylated (+)-2-octylglycosides of 131 were: 5.00 min, 5.06 min, 5.21 min, 5.33 min, and 5.39 min. A comparison of these values with those obtained for the standards shows that the sugar portion of epi-dehydroxestovanin A is comprised of D-fucose and L-rhamnose. Dehydroxestovanin C (132) OH O 28 268 The pure compound was isolated as a colourless glass; [ a ] D : - 24.6° (c = 0.08, M e O H ) ; i r ( f i lm), v m a x : 3385, 3036, 2933, 2329, 1698, 1538, 1376, 1192, 1044, 856 c m " l ; * H nmr (acetone-d6, 400 M H z ) , 1.20 (d, / = 6.0 H z , 3 H , H 6 m ) , 1.21 (s, 3 H , H28) , 1.23 (d, / = 6.4 H z , 3 H , H6' ) , 1.23 (/ = 6.2 H z , 3 H , H 6 " ) , 1.33 (m, I H , H14J5), 1.56 (br s, 3 H , H26) , 1.58 (br s, 6 H , H25 and H30) , 1.63 (br s, 3 H , H29) , 1.65 (br s, 6 H , H I and H24 ) , 1.65 (m, 2 H , H13) , 1.99 (m, I H , H12) , 2.03 (m, I H , H 1 4 a ) , 2.14 (m, I H , H12) , 2.21 (m, 3 H , H 4 and H21) , 2.27 (m, I H , H4) , 2.35 (dd, / = 15.1, 6.6 H z , I H , H8) , 2.46 (dd, / = 15.1, 6.7 H z , I H , H8) , 2.67 (br dd, / = 8.0, 3.9 H z , I H , H l l ) , 3.39 (br t, J = 9.4 H z , I H , H4" ' ) , 3.51 (br t, / = 7.8 H z , I H , H2"), 3.56 (m, I H , H4 " ) , 3.58 (m, I H , H3 ' ) , 3.60 (m, I H , H5 ' ) , 3.61 (br d , / = 9.5 H z , I H , H3 ' " ) , 3.67 (d, / = 9.3 H z , I H , H17) , 3.69 (m, I H , H 5 m ) , 3.71 (br d, J = 10.2 H z , I H , H3 " ) , 3.75 (br d, / = 2.0 H z , I H , H4 ' ) , 3.83 (m, I H , H5" ) , 3.93 (br s, I H , H 2 , M ) , 4.00 (br s, I H , H 2 " ) , 4.01 (m, I H , H20) , 4.07 (dd, / = 8.7, 6.1 H z , I H , H5) , 4.13 (d, / = 7.6 H z , I H , H I ' ) , 5.00 (br t,J = 6.8 H z , I H , H3 ) , 5.12 (br t, / = 7.0 H z , I H , H22) , 5.17 (br s, I H , H 2 7 £ ) , 5.21 (d, / = 1.4 H z , I H , H I " ) , 5.25 (d, / = 1.4 H z , I H , H I " ' ) , 5.33 (br s, I H , H27Z ) , 5.44 (br t, / = 6.0 H z , I H , H7) , 5.58(d, / = 9.3 H z , I H , H18) ppm; i H nmr ( D M S O - d 6 , 400 M H z ) , 5: 1.09 (d, / = 6.1 H z , 3 H , H6' ) , 1.10 (d, / = 6.2 H z , 3 H , H6" ' ) , 1.10 (s, 3 H , H28) , 1.13 (d, J = 6.1 H z , 3 H , H 6 " ) , 1.24 (m, I H , H14(3), 1.47 (br s, 3 H , H26) , 1.48 (br s, 3 H , H29) , 1.53 (br s, 3 H , H30) , 1.54 (br s, 3 H , H25) , 1.57 (m, 2 H , H I 3 ) , 1.60 (br s, 3 H , H24) , 1.61 (br s, 3 H , H I ) , 1.86 (m, I H , H12) , 1.94 (m, I H , H 1 4 a ) , 2.01 (m, I H , H12) , 2.09 (m, 2 H , H21) , 2.16 (m, I H , H4) , 2.19 (m, I H , H4) , 2.20 (m, I H , H8) , 2.34 (dd, / = 15.0, 6.8 H z , I H , H8) , 2.62 (m, I H , H l l ) , 3.18 (m, I H , H4" ' ) , 3.22 (m, I H , H2 ' ) , 3.30 (m, I H , H3 ' ) , 3.38 (m, I H , H3" ' ) , 3.39 (m, 2 H , H 5 ' and H 4 " ) , 3.49 (dq, J = 9.3, 6.2 H z , I H , H5" ' ) , 3.56 (d, / = 8.9 H z , I H , H17) , 3.57 (m, I H , H4 ' ) , 3.60 (m, I H , H3 " ) , 3.62 (m, I H , H 5 " ) , 3.71 (br s, I H , H 2 , M ) , 3.74 (br s, 2 6 9 I H , H 2 " ) , 3.84 (dt, J = 7.1, 4.9 H z , I H , H20) , 3.92 (d, J = 7.6 H z , I H , H I ' ) , 3.95 (dd, / = 8.7, 6.1 H z , I H , H5) , 4.43 (d, / = 5.8 H z , I H , 3 ' " -0H) , 4.56 (d, / = 4.3 H z , I H , 2 M , - O H ) , 4.62 (d, J = 5.9 H z , I H , 4 " ' - 0 H ) , 4.63 (s, I H , 9 -OH) , 4.63 (d, / = 5.0 H z , I H , 2 0 - O H ) , 4.69 (d, J = 6.8 H z , I H , 3 " - O H ) , 4.76 (d, J = 4.5 H z , I H , 2 " -OH) , 4.80 (d, / = 4.7 H z , I H , 2 ' -OH) , 4.90 (br t, / = 6.6 H z , I H , H3) , 4.99 (d, / = 4.3 H z , I H , 3 ' - O H ) , 5.02 (m, I H , H22) , 5.04 (br s, I H , H 2 7 E ) , 5.06 (br s, 2 H , H I " and H I " ' ) , 5.17 (br s, I H , H27Z) , 5.26 (br t, / = 6.8 H z , I H , H7) , 5.44 (d, J = 8.8 H z , I H , H18) ppm; 13c nmr (acetone-d6, 75 M H z ) , 11.1 (q, C26) , 12.7 (q, C29), 17.7 (q, C6' ) , 18.0 (q, C 2 5 ) a , 18.0 (q, C 6 , M ) , 18.1 (q, C30)a, 18.7 (q, C6" ) , 22.9 (t, C13), 25.9 (q, C28) , 26.0 (q, C l ) , 26.0 (q, C24) , 29.0 (t, C12) , 32.7 (t, C21)b, 34.9 (t, C4)b, 36.4 (t, C14) , 38.9 (t, C8 ) , 52.5 (d, C l l ) , 56.2 (s, C15) , 57.6 (d, C17) , 68.2 (d, C5" ) , 69.7 (d, C5" ' ) , 70.8 (d, C 3 , M ) , 71.9 (d, C2 ' ) , 72.0 (d, C 2 , n ) , 72.4 (d, C 4 " ) , 72.5 (d, C 2 " ) , 73.0 (d, C 3 " ) , 73.6 (d, C4 ' " ) , 75.9 (d, C5 ' ) , 77.5 (d, C20) , 78.6 (d, C4' ) , 79.4 (s, C9) , 79.6 (d, C3 ' ) , 83.1 (d, C5 ) , 99.9 (d, C l 1 ) , 102.2 (d, C l ' " ) , 102.4 (d, C l " ) , 111.0 (t, C27) , 120.4 (d, C18) , 121.3 (d, C3) , 122.1 (d, C22) , 126.1 (d, C7) , 132.9 (s, C2) , 132.9 (s, C23) , 136.1 (s, C6 ) , 142.7 (s, C19) , 150.5 (s, CIO) , 212.7 (s, C16) ppm (a-b : m a y be interchanged); L R F A B M S , pseudomolecular ion m/z: 931 ( M + ( C 4 8 H 7 6 0 i 6 ) + 23(Na)), 453 (C18H29O13), 435 (M+ - C18H31O13 - H 2 0 ) , 309 (C12H21O9); L R E I M S , m/z ( formula, relat ive intensity): 434 ( C 3 0 H 4 2 O 2 , 11), 383 ( C 2 5 H 3 5 O 3 , 16), 365 (C25H33O2,41); H R E I M S , C30H42O2 (M+ - C12H22O9 - H2O) m/z: 434.3191 ( A M , +0.6 mmu) . The compound d isp layed m i l d antibacterial activity against both Staphylococcus aureus and Bacillus subtilis and also showed mild activity against the yeast Candida albicans (130 p,g/disk). D e t e r m i n a t i o n of the absolute configurations of the sugars: The determination of the absolute configurations o f the sugars forming the trisaccharide portion of 270 dehydroxestovanin C was carried out in the same manner as for isoxestovanin A . 129 A 5 mg sample o f dehydroxestovanin C was used. The retention times found for the peracetylated (+)-2-octylglycosides of 132 were: 5.00 min, 5.06 min , 5.20 min, 5.32 min , and 5.40 min. A comparison of these values with those obtained for the standards shows that the sugar portion of dehydroxestovanin C is comprised of D-fucose and L -rhamnose. 3 . Derivatives of xestovanin A X e s t o v a n i n A hexaacetate (133) A 150 mg sample of xestovanin A (110) was acetylated in the manner described for the acetylation of isoxestovanin A . The data obtained for this compound was the same as that found previously. 121 (a, d) 271 Ozonolysis of xestovanin A hexaacetate: A 75 mg (7.4 x 10" 5 mol) sample of xestovanin A hexaacetate (133) was dissolved in 10 m L of a mixture of dichloromethane and methanol (3:2). The pale ye l low solution was cooled to -78°C and a continuous stream of ozone gas was bubbled into it unti l it maintained a persistent, faint blue colour. Once the colour change had been observed, the stream of ozone gas was replaced with oxygen gas; the oxygen was bubbled into the solution until the blue colour completely disappeared. Dimethy l sulphide (0.5 m L ) was added and the solution was warmed to room temperature. 143 The excess solvents and reagents were removed under reduced pressure. The yel low o i l thus obtained was purif ied by flash si l ica chromatography (gradient elution: ethyl acetate/hexane; 1:4 to 4:1). The 4-ketopentanal substituted at the 3 position with the peracetylated disaccharide, acetyl X V A ozonolysis product 1 (136), was purified by normal phase H P L C (ethyl acetate/hexane; 3:2) to yield 28 mg (4.7 x 10"^ mol, 64%) o f pure material. The tr icycl ic product, acetyl X V A ozonolysis product 2 (137), was also purified by normal phase H P L C (ethyl acetate/hexane; 2:3) to yield 9.8 mg (3.9 x 10"5 mol , 53%) of pure material. Acetyl X V A ozonolysis product 1 (136) A c O 6Zj 5 o 272 The pure compound was isolated as white microcrystall ine needles from diethyl ether; [ a ] D : - 9.5° (c = 0.19, CH2CI2); i r (KBr) , v m a x : 2985, 2944, 1749, 1431, 1369, 1246 (sh), 1226, 1169, 1128, 1072, 1041, 913, 733 c m " l ; i H nmr (benzene-d6, 400 M H z ) , 8: 1.11 (d, / = 6.4 H z , 3 H , H6' ) , 1,27 (d, J = 6.2 H z , 3 H , H6" ) , 1.65 (s, 3 H , -OCOCHsJ, 1.66 (s, 3 H , -OCOCH3). 1.68 (s, 3 H , -OCOCH3). 1.75 (s, 3 H , H 5 ) * , 2.00 (s, 3 H , -OCOCJi3_)*, 2.11 (s, 3 H , -OCOCH3_) * , 2.51 (ddd, / = 17.4, 5.2, 1.3 H z , I H , H2) , 2.57 (ddd, J = 17.4, 4.9, 1.0 H z , I H , H2) , 2.93 (br q, / = 6.6 H z , I H , H5 ' ) , 3.37 (br d, / = 2.6 H z , I H , H4' ) , 4.23 (dq, / = 9.7, 6.4 H z , I H , H5 " ) , 4.31 (d, / = 8.0 H z , I H , H I ' ) , 4.40 (dd, / = 6.5, 4.9 H z , I H , H3) , 4.93 (d, / = 1.9 H z , I H , H I " ) , 5.01 (dd, / = 10.4, 3.1 H z , I H , H3' ) , 5.55 (t, / = 9.7 H z , I H , H4 " ) , 5.61 (dd, / = 10.4, 8.0 H z , I H , H2 ' ) , 5.74 (dd, / = 9.7, 3.2 H z , I H , H3" ) , 5.76 (dd, / = 3.3, 2.0 H z , I H , H 2 " ) , 9.32 (br s, I H , H I ) ppm; L R C I M S (NH3; positive ion detection), pseudomolecular ion m/z: 636 ( M + ( C 2 7 H 3 8 0 i 6 ) + NH4+) ; L R C I M S , m/z (formula, relative intensity): 503 (C22H31O13, 2); H R E I M S , C22H31O13 m/z: 503.1762 ( A M , -0.2 mmu). A c e t y l X V A Ozono lys i s P r o d u c t 2 (137) The pure compound was isolated as a colourless o i l ; [cc]D: - 21.7* (c = 0.06, CH2CI2); i r ( f i lm), v m a x : 3419, 2958, 2871, 1664, 1448, 1415, 1396, 1227, 1142, 1070, 1035, 972, 937 c m " 1 ; i H nmr (benzene-d6, 400 M H z ) , 8: 0.64 (d, / = 7.3 H z , 3 H , H14) , 1.04 273 (s, 3 H , H13 ) , 1.25-1.37 (m, 4 H , H 6 and H7) , 1.44 (m, I H , H5) , 1.47 (m, I H , H 8 ) , 1.91 (ddd, / ' = 18.4, 3.1, 1.3 H z , I H , H l l ) , 2.26 (m, I H , H9) , 2.34 (br dd, / = 18.4, 8.8 H z , I H , H l l ) , 2.81 (ddd, / = 18.7, 8.8, 2.7 H z , I H , H5) , 2.91 (d, / = 3.6 H z , I H , H15) , 5.09 (dd, J = 8.9, 3.2 H z , I H , H12) , 6.11 (br d, J = 3.5 H z , I H , H I ) ppm; L R E I M S , m/z (formula, relative intensity): 252 ( C 1 4 H 2 0 O 4 , <1), 234 ( C 1 4 H 1 8 O 3 , 10), 216 ( C 1 4 H 1 6 O 2 , 34), 204 ( C 1 3 H 1 6 O 2 , 16), 201 ( C 1 3 H 1 3 O 2 , 40), 173 ( C 1 1 H 9 O 2 , 23), 163 ( C 1 1 H 1 5 O , 25), 141 ( C 1 1 H 9 , 37); H R E I M S , C 1 4 H 2 0 O 4 (M+) m/z: 252.1355 ( A M , -0.7 mmu). X e s t o v a n i n A hexapa rab romobenzoa te (134) B r B z O 6Z O B r B z O 28 A 40 mg (5.1 x 10"5 mol) sample of xestovanin A was suspended in 5 m L of benzene under a nitrogen atmosphere. To this was added a solution of 332 mg (1.53 mmol) of parabromobenzoyl chloride (freshly prepared by reacting parabromobenzoic acid (307 mg, 1.53 mmol) with oxaly l chloride (394 \iL, 4.58 mmol) in benzene; the reaction mixture 274 was allowed to reflux for two hours under an inert atmosphere after which time the excess solvent and reagents were removed under reduced pressure, leaving the freshly prepared acid chloride) in 5 m L of benzene. The reaction mixture was allowed to reflux overnight under an inert atmosphere. After the solution had cooled, it was extracted twice with water. The organic layer was removed and the solvent was evaporated off under reduced pressure. The crude yel low o i l thus obtained was purif ied by reversed phase preparative tic (acetone/H20; 17:3) to y ie ld 52 mg (2.79 x 10-5 mol , 92%) of pure 134. The pure compound was isolated as a colourless o i l ; [cc]D: + 131.6° (c = 0.32, CH2CI2); i r (f i lm), Vmax: 3518, 2964, 2923, 2851, 1728, 1590, 1482, 1400, 1267, 1262, 1174, 1113, 1097, 1067, 1010, 908, 841, 749, 733 cm-1; 1 H nmr (benzene-d6, 400 M H z ) , 8: 1.06 (d, / = 6.9 H z , 3 H , H27) , 1.20 (m, I H , H14) , 1.29 (s, 3 H , H28) , 1.38 (d, J = 6.2 H z , 3 H , H 6 " ) , 1.44 (br s, 3 H , H25) , 1.45 (m, I H , H13) , 1.49 (d, / = 6.3 H z , 3 H , H6' ) , 1.54 (br s, 3 H , H26) , 1.60 (br s, 3 H , H30) , 1.63 (br s, 3 H , H I ) , 1.66 (br s, 3 H , H29) , 1.67 (br s, 3 H , H24) , 1.69 (m, I H , H12) , 1.77 (m, I H , H13) , 1.88 (m, I H , H l l ) , 2.11 (m, I H , H12) , 2.18 (br t, J = 7.5 H z , 2 H , H8) , 2.29 (dq, J = 7.0, 6.3 H z , I H , H10) , 2.32 (m, I H , H4 ) , 2.52 (m, I H , H4) , 2.57 (m, I H , H21) , 2.69 (m, I H , H21) , 2.99 (ddd, / = 12.8, 8.4, 4.7 H z , I H , H14) , 3.60 (br q, / = 6.2 H z , I H , H5 ' ) , 3.70 (d, / = 9.6 H z , I H , H17) , 3.96 (br d , / = 2.8 H z , I H , H4 ' ) , 4.47 (dd, J = 8.9, 5.7 H z , I H , H5) , 4.62 (dq, / = 9.9, 6.2 H z , I H , H5" ) , 4.92 (d, / = 7.9 H z , I H , H I ' ) , 5.03 (br t, J = 6.9 H z , I H , H3) , 5.22 (d, / = 1.5 H z , I H , H I " ) , 5.34 (br t, / = 7.2 H z , I H , H22) , 5.49 (br t, / = 6.7 H z , I H , H7) , 5.64 (t, J = 7.8 H z , I H , H20) , 5.65 (dd, J = 10.5, 3.3 H z , I H , H3 ' ) , 6.01 (t, / = 10.0 H z , I H , H4 " ) , 6.06 (d, / = 9.6 H z , I H , H18) , 6.25 (dd, J = 10.0, 3.3 H z , I H , H3 " ) , 6.30 (dd, / = 3.2, 1.6 H z , I H , H2 " ) , 6.37 (dd, / = 10.5, 7.9 H z , I H , H2 ' ) , 6.95 (d, / = 8.6 H z , 2 H , H B ) , 7.08 (d, / = 8.6 H z , 2 H , H B ) , 7.13 (d, / = 8.6 H z , 2 H , H B ) , 7.17 (d, / = 8.7 H z , 2 H , H B ) , 7.18 (d, / = 8.7 H z , 2 H , H B ) , 7.22 (d, / = 8.6 H z , 2 H , H B ) , 7.66 (d, / = 8.6 H z , 4 H , H A ) , 7.72 (d, / = 8.6 H z , 2 H , H A ) , 7.85 2 7 5 (d, / = 8.6 H z , 2 H , H A ) , 7.89 (d, / = 8.5 H z , 2 H , H A ) , 8.00 (d, / = 8.6 H z , 2 H , H A ) ppm; L R F A B M S , largest peak measured m/z: 1678 (multiplet) ( M + ( C 8 4 H 8 6 B r 6 0 i 8 ) - B r B z ( C 7 H 4 B r O ) ) ; L R C I M S , largest mass measured m/z: 1494 (multiplet) (M+ - 2BrBz ) . Ozonolysis of xestovanin A hexaparabromobenzoate: A 30 mg (1.6 x 10" 5 mol) sample of xestovanin A hexaparabromobenzoate (134) was subjected to ozonolysis in the manner described for xestovanin A hexaacetate. The yellow oi l thus obtained was purified by reversed phase preparative tic (acetone/H20; 3:1), yielding the two major compounds which had been detected by tic, benzoyl X V A ozonolysis product 1 (138) (15 mg, 71%) and benzoyl X V A ozonolysis product 2 (139) (2.5 mg, 49%). Benzoyl X V A Ozonolysis Product 1 (138) The pure compound was isolated as an amorphous white solid; ir (KBr ) Vmax: 2954, 2923, 2851, 1729, 1590, 1395, 1272, 1174, 1097, 1010, 846, 748 cm-1; 1 H nmr (benzene-d6, 400 M H z ) , 8: 1.28 (d, J = 6.4 H z , 3 H , H6 ' ) , 1.39 (d, J = 6.2 H z , 3 H , H 6 " ) , 1.86 (s, 3 H , H5) , 2.52 (dd, J = 17.6, 6.8 H z , I H , H2) , 2.58 (dd, J = 17.6, 5.0 276 H z , I H , H2 ) , 3.22 (br q, / = 6.6 H z , I H , H5') , 3.77 (br d , / = 2.5 H z , I H , H4 ' ) , 4.49 (dd, / = 6.6, 4.8 H z , I H , H3) , 4.57 (d, / = 7.8 H z , I H , HI*), 4.60 (dq, / = 9.8, 6.3 H z , I H , H5 " ) , 5.13 (d, / = 1.5 H z , I H , H I " ) , 5.47 (dd, / = 10.5, 3.0 H z , I H , H3') , 6.02 (t, / = 9.9 H z , I H , I H , H4" ) , 6.21 (dd, / = 10.5, 8.0 H z , I H , H2*), 6.24 (dd, J = 9.9, 3.4 H z , I H , H3 " ) , 6.29 (dd, / = 3.3, 1.5 H z , I H , H2" ) , 6.93 (d, / = 8.6 H z , 2 H , H B ) , 7.07 (d, J = 8.8 H z , 2 H , H B ) , 7.09 (d, J = 8.8 H z , 2 H , H B ) , 7.12 (d, / = 8.6 H z , 2 H , H B ) , 7.14 (d, / = 8.6 H z , 2 H , H B ) , 7.64 (d, / = 8.6 H z , 2 H , H A ) , 7.65 (d, / = 8.7 H z , 2 H , H A ) , 7.71 (d, / = 8.6 H z , 2 H , H A ) , 7.76 (d, / = 8.6 H z , 2 H , H A ) , 7.98 (d, / = 8.6 H z , 2 H , H A ) , 9.32 (s, I H , H I ) ppm; L R F A B M S , largest mass measured m/z : 1201/1203/1205/1207/1209/1211 (benzoylated disaccharide; M + ( C 5 2 H 4 3 B r 5 0 i 6 ) -C5H9O3). Benzoyl X V A ozonolysis product 2 (139) The pure compound was isolated as a colourless o i l ; i r (f i lm), v m a x ' 3385, 2958, 2917, 2844, 1703, 1667, 1615, 1458, 1375, 1125, 1021 c m " l ; i H nmr (benzene-d6, 400 M H z ) , 8: 0.75 (d, / = 7.1 H z , I H , H19) , 1.13 (m, I H , H9) , 1.14 (s, 3 H , H18) , 1.32 (br s, 3 H , H17) , 1.35 (m, I H , H10) , 1.54 (m, I H , H l l ) , 1.67 (m, I H , H12) , 1.72 (m, I H , H10) , 1.86 (dd, / = 17.6, 1.0 H z , I H , H15) , 1.92 (m, I H , H l l ) , 1.98 (dd, / = 17.6, 1.4 H z , I H , H15) , 2.17 (dq, / = 7.1, 5.8 H z , I H , H13) , 2.93 (ddd, / = 13.0, 8.3, 277 5.1 H z , I H , H9 ) , 3.82 (d, / = 10.2 H z , I H , H6) , 6.01 (dd, / = 15.7, 7.6 H z , I H , H2) , 6.14 (br d, / = 10.4 H z , I H , H5) , 6.64 (d, J = 15.7 H z , I H , H3) , 9.04 (br s, I H , H16), 9.46 (d, / = 7.6 H z , I H , H I ) ppm; L R E I M S , m/z (formula, relative intensity): 318 (C19H26O4, 9), 300 (C19H24O3, 10), 274 (C17H22O3, 24), 231 (C15H19O2, 100), 209 (C12H17O3, 27), 109 (C7H9O, 30); H R E I M S , C19H26O4 (M+) m/z: 318.1827 ( A M , -0.4 mmu). Base e l im ina t i on p roduc t of xes tovan in A (135) 25 26 T o a 25 mg sample of xestovanin A dissolved in 5 m L of M e O H were added 5 drops of 0.5 N KOH(aq ) . The solution was kept at 80°C with stirring for two hours. After this time, the solution was cooled and extracted with CH2CI2 ( 3 x 5 mL) . The organic extracts were combined and the solvent was removed under reduced pressure, yielding a yellow o i l . A tic analysis of the crude product showed one major vani l l in staining, U V active compound. This compound was purif ied by normal phase flash chromatography (ethyl acetate/hexane; 45:55) fol lowed by normal phase H P L C (ethyl acetate/hexane; 2:3). The pure compound was isolated as a colourless o i l ; [ a ] D : - 15.5° (c = 0.11, CH2CI2); ir ( f i lm), v m a x : 3436, 2964, 2872, 1712, 1692, 1646, 1559, 1451, 1374, 1159, 1040, 913, 728 c m ' 1 ; A H nmr (benzene-d6; 400 M H z ) , 8: 0.98 (d, / = 7.0 H z , 3 H , H27), 1.21 278 (s, 3 H , H28) , 1.22 (m, I H , H14) , 1.48 (m, I H , H13) , 1.58 (br s, 3 H , H29) , 1.68 (br s, 6 H , H25 and H30) , 1.69 (br s, 6 H , H I and H24) , 1.69 (m, I H , H12) , 1.75 (br s, 3 H , H26) , 1.81 (m, I H , H13) , 1.84 (m, I H , H l l ) , 2.04 (dq, / = 7.0, 5.6 H z , I H , H10) , 2.15 (m, I H , H12) , 2.18 (m, I H , H21) , 2.41 (dt, J = 15.1, 6.9 H z , I H , H21) , 2.86 (br t, / = 7.1 H z , 2 H , H4) , 3.06 (ddd, / = 12.6, 8.5, 5.2 H z , I H , H14) , 3.58 (d, / = 9.7 H z , I H , H17) , 4.06 (br t, / = 6.7 H z , I H , H20) , 5.22 (m, I H , H3) , 5.23 (m, I H , H22) , 5.32 (d, / = 15.7 H z , I H , H8) , 5.52 (m, I H , H5) , 5.78 (d, / = 9.6 H z , I H , H18) , 6.08 (d, / = 15.7 H z , I H , H7 ) ppm; L R E I M S , m/z (formula, relative intensity): 454 (C30H46O3, <D, 436 (C30H44O2, 2), 418 (C30H42O, 2), 367 (C24H31O3, 5), 301 (C19H25O3,10); H R E I M S , C30H46O3 (M+) m/z: 454.3453 ( A M , +0.6 mmu). D . Metabo l i tes F r o m the S r i L a n k a n N u d i b r a n c h Chromodoris glenei., ( K e l a a r t . 1858) A collection of twelve animals was made in December 1987 in the coastal waters near M t . Laven ia , Sr i Lanka. The fresh animals were preserved in a 1:1 mixture of dichloromethane/methanol which was kept at -4*C until workup. The organic extract was decanted off and concentrated under reduced pressure to give 200 mg of an orange residue wh ich was fractionated by s i l i ca f lash chromatography (gradient: CH2CI2 to C H 2 C l 2 / M e O H ; 100:1). A mixture of 12-desacetoxyshahamin C (153) and shahamin K (155) was isolated in the more polar fractions. A partial separation o f these two compounds was achieved by chromatographing the mixture on Sephadex L H - 2 0 (hexane /CH2C l2 /MeOH; 2:1:1). The final purif ication of each of the compounds was accomplished using si l ica flash chromatography. A sample of 7.5 mg (3.5% of the crude organic extract) of pure 12-desacetoxyshahamin C (153) was obtained by elution with 279 C H 2 C l 2 / M e O H (100:1) and a sample of 3 mg (1.5% of the crude organic extract) of pure shahamin K (155) was obtained after elution with C H 2 C l 2 / M e O H (75:1). 12-Desacetoxyshahamin C (153) O C O C H 3 The pure compound was isolated as a colourless o i l ; [ct] D: + 72.0° (c = 0.49, CH2CI2); i r (f i lm), v m a x : 2953, 2938, 2866, 1748, 1385, 1365, 1233, 1135, 1109, 1039 cm-1; 1 H nmr (CDCI3, 400 M H z ) , 5: 0.93 (s, 3 H , H17) . 0.95 (s, 3 H , H18) , 1.00 (s, 3 H , H19), 1.27 (br dt, I H , H3) , 1.39 (m, I H , H2) , 1.51 (m, I H , H7 ) , 1.56 (m, I H , H7) , 1.62 (dt, I H , H3) , 1.72 (m, I H , H6) , 1.72 (m, I H , H6) , 1.77 (m, I H , H14) , 1.80 (m, I H , H I ) , 1.80 (m, I H , H 2 ) , 1.92 (ddd, J = 10.6, 8.9, 8.7 H z , I H , H 5 ) , 2.08 (s, 3 H , -OCOCH3J, 2.36 (br dd, I H , H I ) , 2.48 (m, I H , H13) , 2.55 (d, / = 5.2 H z , I H , H12), 2.55 (d, / = 5.1 H z , I H , H12) , 2.74 (d, / = 8.7 H z , I H , H9) , 3.85 (dd, / = 11.2, 7.8 H z , I H , H16) , 4.19 (dd, / = 11.2, 4.2 H z , I H , H16) , 4.21 (dd, / = 11.8, 10.0 H z , I H , H 1 5 p ) , 4.32 (dd, / = 11.8, 6.1 H z , I H , H15oc), 4.63 (br d, / = 2.1 H z , I H , H20Z) , 4.86 (d, / = 2.1Hz, I H , H20£) ppm; 13c nmr (CDCI3), 8: 20.7 (q, -OCOCH3J, 21.3 (q, C17) , 25.6 (q, C19) , 25.9 (t, C6 ) , 28.8 (t, C2), 32.0 (d, C13) , 32.1 (t, C12) , 34.4 (q, C18) , 36.2 (s, C8 ) , 37.0 (t, C l ) , 37.6 (t, C3 ) , 37.7 (t, C7 ) , 44.1 (d, C14) , 48.5 (s, C4) , 54.4 (d, C5 ) , 54.8 (d, C9 ) , 67.5 (t, C16) , 68.2 (t, C15) , 115.0 (t, C20) , 153.7 (s, 280 CIO), 170.8 (s, C l l ) , 172.9 (s, -OCOCH3) ppm; L R C I M S (NH3; positive ion detection), psuedomolecular ion m/z: 380 ( M + ( C 2 2 H 2 2 B i 2 N 4 ) + NH4+), 363 ( M + ( C 2 2 H 2 2 B r 2 N 4 ) + H + ) ; L R E I M S , m/z (formula, relative intensity): 362 (C22H34O4, 8), 347 (C21H32O4, 3), 302 (C20H30O2, 8) , 287 (C19H27O2, 9), 191 (C14H23, 24), 177 (C13H21, 2), 173 (C13H17, 11), 166 (C10H14O2, 15), 164 (C10H12O2, 12), 161 (C12H17, 13), 159 (C12H15, 16), 137 (C10H17, 100), 136 ( C i 0 H i 6 , 54), 135 (C10H15, 45), 121 (C9H13, 67); H R E I M S , C22H34O4 (M+) m/z: 362.2451 ( A M , -0.6 mmu). S h a h a m i n K (155) OCOCH3 The pure compound was isolated as a colourless o i l ; [oc]D: + 84.0° (c = 0.10, CH2CI2); i r (f i lm), v m a x : 2952, 2931, 2867, 1745(sh), 1740, 1366, 1243, 1135, 1110, 615 c m " l ; ! H nmr (CDCI3, 400 M H z ) , 8: 0.93 (s, 3H, H17) , 0.95 (s, 3H, H18) , 1.01 (s, 3H, H19) , 1.30 (ddd, / = 14.0, ~4, 3.7 H z , I H , H3), 1.40 (m, I H , H2), 1.57 (ddd, / = 14.0, -12, 3.7 H z , I H , H3), 1.78 (m, I H , H2), 1.80 (m, I H , H6p), 1.80 (m, I H , H14) , 1.82 (m, I H , H5ce), 1.95 (br dd, / = 12.7, 12.6 H z , I H , H I ) , 2.06 (s, 3H, - O C O C H 3 ) , 2.07 (s, 3H, -OCOCH3.), 2.18 (m, I H , H6a), 2.39 (br dd, / = 12.7, 5.1 H z , I H , H I ) , 2.49 (m, I H , H13) , 2.55 (d, J = 3.8 H z , I H , H12), 2.56 (d, J = 6.5 H z , 281 I H , H12) , 2.80 (br d , / = 7.7 H z , I H , H 9 a ) , 3.89 (dd, / = 11.2, 7.5 H z , I H , H16) , 4.20 (dd, / = 12.0, 9.8 H z , I H , H150) , 4.21 (dd, J = 11.4, 4.4 H z , I H , H16) , 4.28 (dd, / = 12.0, 6.0 H z , I H , H 1 5 a ) , 4.69 (br d, / = 1.7 H z , I H , H 2 0 Z ) , 4.94 (br d, / = 1.7 H z , I H , H 2 0 £ ) , 4.98 (br dd , J = 9.0, 5.3 H z , I H , H 7 a ) ppm; i H nmr (partial assignment; benzene-d6, 400 M H z ) , 8: 0.74 (s, 3 H , H17) , 0.87 (s, 3 H , H18) , 0.98 (s, 3 H , H19) , 1.62 (m, 3 H , H 5 , H6p, H14 ) , 1.69 (s, 3 H , - O C O C H 3 _ ) , 1.74 (s, 3 H , -OCOCH3J, 2.02 (br t, I H , H6a), 3.75 (dd, I H , H15) , 3.83 (dd, I H , H16) , 3.96 (dd, I H , H15) , 4.09 (dd, I H , H16) , 4.67 (br d, I H , H20Z) , 4.83 (br d , I H , H 2 0 E ) , 4.91 (br dd, I H , H 7 a ) ppm; l ^ C nmr (CDCI3), 8: 16.1 (q, - O C O C H ^ ) , 20.8 (q, -OCOCH3J, 21.2 (q, C17) , 25.3 (q, C19) , 28.8 (t, C2 ) , 31.5 (d, C13) , 32.2 (t, C12) , 32.5 (t, C6) , 34.4 (q, C18) , 36.0 (s, C8) , 36.7 (t, C l ) , 37.8 (t, C3 ) , 44.6 (d, C14) , 48.7 (d, C5) , 49.7 (s, C4) , 54.2 (d, C9) , 67.0 (t, C16) , 67.9 (t, C15) , 78.5 (d, C7 ) , 116.3 (t, C20), 153.0 (s, CIO) , 170.7 (s, -OCOCH3), 170.8 (s, C l l ) , 172.6 (s, -OCOCH3) ppm; L R C I M S (NH3; positive ion detection), psuedomolecular ion m/z: 438 ( M + ( C 2 4 H 3 6 0 6 ) + NH4+); L R E D v l S , m/z (formula, relative intensity): 420 (C24H36O6, 18), 378 (C22H34O5, 2), 360 (C22H32O4, 8), 300 (C20H28O2, 8), 191 (C14H23, 2), 177 (C11H13O2, 3), 171 (C13H15, 6), 166 (C10H14O2, 3), 164 (C10H12O2, 5), 161 (C12H17, 7), 159 (C12H15, 9), 158 (C12H14, 3), 150 (C11H18, 23), 137 (C10H17, 31), 136 ( C i 0 H i 6 , 88), 121 (C9H13, 39); H R E I M S , C24H36O6 (M+) m/z: 420.2517 ( A M , +0.5 mmu). E. Metabolites From the Sri Lankan Nudibranch Chromodoris cavae (Eliot. 1904) Fifty-four specimens of Chromodoris cavae (69 g, wet weight) were collected from the waters near Jaffna, Sr i Lanka in January 1990. The whole animals were extracted with CH2CI2 and the extract thus obtained was concentrated under reduced pressure. The 282 resulting o i l was chromatographed on si l ica (hexane/EtOAc; 5:2) to afford 6 mg of pure chromodorolide A (154) and a fraction containing a mixture of chromodorolides A and B (156). Further purification of the latter fraction by normal phase H P L C (hexane/EtOAc; 2:1) gave a further 1 mg of pure chromodorolide A and 2 mg of pure chromodorolide B . C h r o m o d o r o l i d e A (154) O The data obtained for this compound was the same as that found previously.* 33 Addit ional data is the fol lowing: [ a ] D : - 74.5° (c = 0.11, CH2CI2); * H nmr (CDCI3,400 M H z ) , 8: 0.79 (s, 3H, H18) , 0.80 (s, 3H, H19) , 0.82 (s, 3H, H20), 1.03 (br dt, / = 13.5, 4.2 H z , I H , H l a ) , 1.08 (m, I H , H3a), 1.11 (m, I H , H5), 1.15 (m, I H , H7), 1.31 (m, I H , H6), 1.40 (br d , J = 13.3 H z , I H , Hl|3), 1.50-1.52 (m, 2H, H2), 1.55 (m, I H , H9), 1.56 (m, I H , H6), 1.69 (br d, J = 11.8 H z , I H , H3p), 1.76 (m, I H , H7), 2.02 (s, 3H, -OCOCH3_), 2.06 (s, 3H, -OCOCH.3J), 2.39 (ddd, / = 12.0, 6.6, 3.0 H z , I H , H8), 3.00 (m, 2H, H13 and H14) , 3.22 (s, I H , 12-OH), 4.77 (d, / = 2.8 H z , I H , H17) , 5.73 (br t, / = 2.1 H z , I H , H15) , 6.30 (s, I H , H16) ppm; ^ C nmr (CDCI3, 75 M H z ) , 8: 13.6 (q, C20)a, 19.8 (t, C6), 20.8 (t, C2), 20.8 (q, C19)a, 20.8 (q, -OCOCH3J), 20.8 (q, -OCOCH3_), 25.6 (t, C7), 33.1 (s, C4)b, 33.4 (q, C18)a 40.4 (t, C3), 41.1 (t, C l ) , 42.5 (s, C10)b, 44.6 (d, C8), 46.3 (d, C14) , 51.6 (d, C9), 52.0 (d, C13) , 57.8 (d, C5), 283 78.8 (d, C17) , 79.9 (s, C12) , 95.5 (d, C16) , 104.2 (d, C15) , 168.8 (s, - O C O C H 3)C, 170.0 (s, - O C O C H 3 ) c , 172.4 (s, - O C O C H 3 ) c ppm may be interchanged). C h r o m o d o r o l i d e B (156) 19 The pure compound was isolated as a colourless o i l ; [ a ] D : - 95.0° (c = 0.10, CH2CI2); ir ( f i lm) Vmax: 2917, 2846, 1812, 1751, 1368, 1214, 1096, 999, 964 c m " l ; i H nmr (benzene-d6; 400 M H z ) , 8: 0.63 (s, 3 H , H20) , 0.68 (dd, / = 11.0, 7.0 H z , I H , H5) , 0.81 (s, 3 H , H18) , 0.84 (s, 3 H , H19) , 0.90 (m, I H , H l a ) , 1.00 (m, I H , H3a), 1.14 (m, I H , H6) , 1.16 (m, I H , H9 ) , 1.37 (m, I H , H 6 ) , 1.38 (m, I H , 3p), 1.42 (m, I H , H7 ) , 1.43 (m, I H , H i p ) , 1.44 (m, I H , H7) , 1.46 (m, I H , H2) , 1.50 (m, I H , H2) , 1.63 (s, 3 H , -OCOCH3), 1.66 (s, 3 H , -OCOCH3), 1.84 (s, 3 H , -OCOCH3), 2.51 (ddd, / = 12.2, 11.0, 7.0 H z , I H , H8) , 2.62 (t, J = 8.7, 7.5 H z , I H , H14) , 3.50 (dd, / = 8.8, 6.0 H z , I H , H13) , 5.70 (d, / = 12.2 H z , I H , H17) , 5.80 (d, J = 6.0 H z , I H , H16) , 6.63 (br s, I H , H15) ppm; i H nmr (CDCI3, 400 M H z ) , 8: 0.79 (s, 3 H , H18) , 0.83 (s, 3 H , H19) , 0.84 (s, 3 H , H20) , 0.95 (br dd , / = 10.4, 3.5 H z , I H , H3a), 1.03 (br dt, / = 12.3, 3.6 H z , I H , H l a ) , 1.09 (br dd, / = 13.1, 6.6 H z , I H , H 5 ) , 1.38 (m, I H , H i p ) , 1.40 (m, I H , H6 ) , 1.48 (m, I H , H7) , 1.51 (m, I H , H3p), 1.55 (m, 2 H , H2) , 1.56 (m, I H , H6) , 1.58 (m, I H , H7 ) , 1.69 (br dd, / = 12.0, 9.9 H z , I H , H 9 ) , 2.04 (s, 3 H , 284 - 0 C 0 C H 3 _ ) , 2.11 (s, 3 H , -0C0CJJ3_) , 2.19 (s, 3 H , -0C0CH_3_), 2.57 (ddd, J = 12.1, 11.6, 7.9 H z , I H , H8) , 2.93 (br t, J = 8.2 H z , I H , H14) , 3.79 (dd, / = 8.9, 6.1 H z , I H , H13) , 5.30 (d, J = 11.5 H z , I H , H17) , 6.08 (d, / = 6.1 H z , I H , H16), 6.50 (br s, I H , H15) ppm; 13c nmr (CDCI3, 125 M H z ) , 8: 13.7 (q, C20), 19.9 (t, C6 ) , 20.8 (q, -OCOCH3J, 20.8 (q, -OCOCH3_) , 20.9 (q, -OCOCH3J, 21.0 (q, C 1 9 ) a , 21.1 (t, C2), 25.2 (t, C7 ) , 33.1 (s, C 4 ) b , 33.4 (q, C 1 8 ) a , 39.1 (t, C3 ) , 40.9 (t, C l ) , 43.9 (s, C 1 0 ) b , 45.6 (d, C14) , 48.0 (d, C8) , 50.3 (d, C9)c, 50.4 (d, C13)c , 57.0 (d, C5) , 73.9 (d, C17) , 81.4 (s, C12) , 97.8 (d, C15) , 103.4 (d, C16) , 169.1 (s, C l l ) d , 169.2 (s, -OC_OCH3)d , 170.0 (s, - O C O C H 3 ) d 170.2 (s, - O C O C H 3 ) d ppm (a-d : m a y be interchanged); 1 3 C nmr (benzene-d6), 8: 13.5, 20.2, 20.4, 20.6, 21.0, 21.1, 25.5, 33.1, 33.3, 39.1, 41.2, 43.7, 45.8, 48.2, 49.7, 50.4, 53.3, 56.0, 74.5, 81.8, 97.9, 103.4 ppm (no carbonyls discernible in benzene-d6); L R E I M S , m/z (formula, relative intensity): 492 (C26H3609. <1), 477 (C25H33O9,4), 432 (C24H32O7, 11), 417 (C23H29O7, 23), 285 ( C i 2 H i 3 0 8 , 4), 165 (C12H21, 5), 163 (C12H19, 10), 138 ( C i o H i g , 38), 123 (C9H-5, 100), 122 ( C 9 H 1 4 , 15), 109 (C9H13, 24), 95 (C7H11, 36), 82 (C 6 Hi() , 25), 69 (C5H9, 30); HPxEIMS, C26H36O9 (M+) m/z: 492.2361 ( A M , +0.2 mmu). 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