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Chemical studies of metabolites from Pacific Ocean marine sponges and the starfish Dermasterias imbricata Burgoyne, David L. 1992

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CHEMICAL STUDIES OF METABOLITES FROM PACIFIC OCEAN MARINESPONGES AND THE STARFISH DERMASTERIAS IMBRICATAbyDavid Lawrence BurgoyneB. Sc. (Hons), The University of British Columbia, 1987A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIES(Department of Chemistry)We accept this thesis as conformingto the required standard THE UNIVERSITY OF BRITISH COLUMBIASeptember 1992© David Lawrence Burgoyne, 1992In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature) Department of ^Che,/1/1 The University of British ColumbiaVancouver, CanadaDateDE-6 (2/88)AbstractA study of biologically active extracts of several Pacific Ocean marine spongeshas led to the isolation of eight new and nine previously known natural products. Thestructures of the compounds were determined by a combination of spectroscopic dataanalysis and chemical interconversions. In addition, the partial synthesis of imbricatine(3), a cytotoxic metabolite of the starfish Dermasterias imbricata, was accomplished.Crude extracts from the sponge Petrosia contignata collected in Papua NewGuinea showed potent cytotoxicity against the murine leukemia L1210 cell line. Theseextracts yielded contignasterol (24), a new highly oxygenated steroid that inhibitedhistamine release from rat peritoneal mast cells with an IC50 of 0.8 gM, and petrolactone(37), a new sesquiterpene with the drimane carbon skeleton. The structure ofcontignasterol, which contains the "unnatural" 1413 proton configuration and a cyclichemiacetal functionality in its side chain, was elucidated via spectroscopic studies of itstetraacetate and its reduction product pentaacetate. A known polybrominateddiphenylether 41 accounted for the cytotoxicity of the crude extracts.The sponge Acanthella sp. yielded ten sesquiterpenes, four of which were new.Compounds 80 -87, 89 and 90 contained isonitrile, isothiocyanate, formamide, carbamateand chloride functional groups. In addition, violacene (88), a halogenated monoterpenepreviously isolated from the red algae Plocamium violaceum, was isolated from thesponge extracts.The Northeastern Pacific sponge Neoesperiopsis digitata yielded neoesperlactone(96), an eighteen carbon fatty acid derived y-lactone. In addition, the major aromaticcomponent was found to be identical by tic and 1H NMR comparison to commerciallyobtained p-hydroxybenzaldehyde.The study of the Papua New Guinea sponge Pseudaxinella massa led to theidentification of a novel cyclic heptapeptide pseudaxinellin (103) which containediistandard protein amino acid residues with the L configuration. The study of two speciesof marine sponges, Ptilocaulis trachys and Amphimedon sp. , collected at Enewetaklagoon, led to the isolation and identification of the depsipeptide majusculamide C (104)which had previously been found in the blue green alga Lyngbya majuscula collected atEnewetak.The synthetic confirmation of the tetrahydroisoquinoline substructure of thestarfish metabolite imbricatine (3) was accomplished using a Pictet-Spengler reaction tocouple the starting materials 3-(3,5-dihydroxyphenyl) alanine methyl ester (143) andsodium 3-(4-benzyloxyphenyl) glycidate (141). A series of deprotections and protectionson the resulting racemic benzyltetrahydroisoquinoline led to a product 140 that wasidentical by 1H NMR and mass spectroscopic analysis to the protected Raney nickelreduction product of imbricatine (3).OHHeCH32437^ 41iiiCIBrCH(Cl^Cl88CH3HO^ .„‘CO2HOH3 v OH140AcO, .7...1182 R = NCS85 R = NC89 R = NHCO2CH390 R = NHCHOR^ l•IES80 R = Cl 8786 R = NCS96103er--NM CO2H/IN ,ivTable of ContentsAbstract^ iiTable of Contents^ vList of Figures viiiList of Schemes ^ xiiiList of Tables xvList of Abbreviations ^ xviiAcknowledgments xxMarine Natural Products Chemistry ^ 1Part I^Jsolation and Structure Elucidation of Metabolites from ThreePacific Ocean Marine Sponges Introduction to the Porifera^ 7^A.^Marine Natural Products from the Papua New Guinea SpongePetrosia contignataIntroduction ^ 9Results and Discussion ^  151. Isolation of metabolites from Petrosia contignata^ 152. Structure elucidation of metabolites from Petrosia contignata ^ 18^B.^Terpenoid Metabolites Isolated from the Northeastern PacificMarine Sponge Acanthella sp. Introduction ^ 66Results and Discussion ^ 711. Isolation of terpenoid metabolites from Acanthella sp^ 712. Structure elucidation of terpenoid metabolites fromAcanthella sp^ 73v3. Known compounds isolated from Acanthella sp^ 110Conclusions ^  113C.^Isolation and Structure Elucidation of a y-Lactone from theNortheastern Pacific Sponge Neoesperiopsis digitata. Introduction ^  117Results and Discussion ^ 1191. Isolation of metabolites from Neoesperiopsis digitata^ 1192. Structure elucidation of Neoesperlactone (96)^ 122Part II^Chemical Studies on Peptides from Three Species of South Pacific Ocean Marine SpongesIntroduction^ 127Results and Discussion^ 131A. The Isolation and Structure Elucidation of Pseudaxinellin (103),a Cyclic Heptapeptide from Pseudaxinella massa1. Taxonomy and Description of P. massa^ 1312. Isolation of Pseudaxinellin (103) 1333. Structure Elucidation of Pseudaxinellin (103)^ 134B. The Isolation of the Blue green Algal Metabolite MajusculamideC (104) from the Marine Sponges Ptilocaulis tract's andAmphimedon sp. 1. Isolation of Majusculamide C (104)^ 1482. Structural Information on Majusculamide C (104)^ 1493. Determination of the Stereochemistry of theMAP Residue^ 155Conclusions1. Origins of Peptides from Sponges^ 157viPart III^The Synthetic Confirmation of the TetrahydroisoquinolineSubstructure of ImbricatineIntroduction ^  166Results and Discussion^ 173Experimental^ 187References 216Appendix A 1H NMR Spectra of Known Compounds from Acanthella sp.^ 224Appendix B 1H NMR Spectra of Synthetic Products^ 232Appendix C Explanation of Tables^ 238viiList of FiguresFigure 1:^1H NMR spectrum of contignasterol (24) (500 MHz,DMSO-d6)^ 19Figure 2:^13C NMR spectrum of contignasterol (24) (125 MHz,DMSO-d6)^ 20Figure 3:^2D HMQC spectrum of contignasterol (24) (DMSO-d6)^ 21Figure 4:^Low resolution EI mass spectrum of contignasterol (24)^ 22Figure 5:^1H NMR spectrum of tetraacetate 25 (400 MHz, C6D6)^ 25Figure 6:^13C and APT NMR spectra of tetraacetate 25 (125 MHz, C6D6) ^26Figure 7:^2D COSY spectrum of tetraacetate 25 (400 MHz, C6D6)^ 27Figure 8:^2D HMQC spectrum of tetraacetate 25 (C6D6)^ 28Figure 9:^1H NMR spectrum of pentaacetate 26 (400 MHz, C6D6)^ 29Figure 10:^NOe results for rings A and B of tetraacetate 25^ 31Figure 11:^1H NMR spectrum of tetraacetate 25 (400 MHz,2:1 CC14/C6D6)^ 32Figure 12:^NOe results of tetraacetate 25 (400 MHz, 2:1 CC14/C6D6)^ 33Figure 13:^Relative stereochemistry of rings C and D of tetraacetate 25^ 34Figure 14:^Selected regions of the 2D HMBC spectrum oftetraacetate 25 (C6D6)^ 35Figure 15:^1H NMR spectrum of reduction product pentaacetate 28(400 MHz, C6D6)^ 36Figure 16:^COSY spectrum of reduction product pentaacetate 28(400 MHz, C6D6)^ 37Figure 17:^Relative stereochemistry of the tetrahydropyran ring oftetraacetate 25^ 40viiiFigure 18:^The effect of a C22 hydroxyl substituent on the 13C chemicalshifts of the C20 and C22 carbons in cholesteryl benzoates^ 40Figure 19:^The effect of two carbon branches at C24 on the 13C chemicalshifts of the C26 and C27^ 41Figure 20:^13C NMR spectrum of reduction product 29 (125 MHz,10:1 CDC13/DMSO-d6)^ 43Figure 21:^1H NMR spectrum of reduction product tetraacetate 30(400 MHz, C6D6)^ 44Figure 22:^13C NMR spectrum of reduction product tetraacetate 30(125 MHz, C6D6)^ 45Figure 23:^Inhibitory effect of contignasterol on histamine releasefrom rat mast cells^ 54Figure 24:^Spin system within rings B and C of petrolactone (37)^ 56Figure 25:^Selected HMBC correlations of petrolactone (37) 57Figure 26:^NOe results of petrolactone (37)^ 57Figure 27:^1H NMR spectrum of petrolactone (37) in CDC13(500 MHz)^ 60Figure 28:^1H NMR spectrum of petrolactone (37) in C6D6(400 MHz)^ 61Figure 29:^13C and APT NMR spectra of petrolactone (37)(75 MHz, CDC13)^ 62Figure 30:^Selected region of 2D HMBC spectrum of petrolactone(37) (C6D6)^ 63Figure 31:^1H NMR spectrum of acanthene A (80) (500 MHz,C6D6)^ 78Figure 32:^13C NMR spectrum of acanthene A (80) (100 MHz,C6D6)^ 79ixFigure 33:^Low resolution EI mass spectrum of acanthene A (80) ^80Figure 34:^Allylic coupling present in COSY spectrum of acanthene A(80)^ 82Figure 35:^HMBC correlations in acanthene A(80)^ 82Figure 36:^Selected region of HMBC spectrum of acanthene A (80)(C6D6)^ 83Figure 37:^NOe results for acanthene A (80)^ 84Figure 38:^COSY correlation assigned to W-coupling in acanthene A (80)^ 84Figure 39:^1H NMR spectrum of acanthene G (86) (400 MHz, CDC13)^ 87Figure 40:^13C and APT NMR spectra of acanthene G (86) (75 MHz,CDC13)^ 88Figure 41:^2D HMQC spectrum of acanthene G (86) (CDC13)^ 89Figure 42:^HMBC correlations in acanthene G (86)^ 90Figure 43:^NOe results for acanthene G (86) 91Figure 44:^Chemical shifts of protons associated with thiocyanates andisothiocyanates^ 92Figure 45:^NOe results of acanthene G (86) (CDC13)^ 93Figure 46:^1H NMR spectrum of acanthene H (87) (500 MHz, CDC13)^ 96Figure 47:^13C and APT NMR spectra of acanthene H (87) (75 MHz,CDC13)^ 97Figure 48:^HMBC correlations in acanthene H (87)^ 99Figure 49:^NOe results for acanthene H (87) 99Figure 50:^1H NMR spectrum of acanthene J (89) (500 MHz, CDC13)^ 103Figure 51:^13C and APT NMR spectra of acanthene J (89) (75 MHz,CDC13)^ 104Figure 52:^Selected region of COSY spectrum of acanthene J (89)(400 MHz, CDC13)^ 105Figure 53:^HMBC correlations for acanthene J (89)^  106Figure 54:^Selected region of HMBC spectrum of acanthene J (89)(CDC13)^ 107Figure 55:^NOe results for acanthene J (89)^  108Figure 56:^Fragment A of neoesperlactone (96)  121Figure 57:^Fragment B of neoesperlactone (96)^  121Figure 58:^HMBC results for neoesperlactone (96)  122Figure 59:^1H NMR spectrum of neoesperlactone (96) (500 MHz,CDC13)^ 124Figure 60:^13C and APT NMR spectra of neoesperlactone (96)(125 MHz, CDC13)^ 125Figure 61:^Selected region of HMBC spectrum of neoesperlactone (96) ^ 126Figure 62:^1H NMR spectrum of pseudaxinellin (103) (400 MHz,CDC13)^ 136Figure 63:^13C and APT spectra of pseudaxinellin (103) (125 MHz,CDC13)^ 137Figure 64:^NH region of HOHAHA spectrum of pseudaxinellin (103)(500 MHz, CDC13)^ 138Figure 65:^Connectivities in 103 based on HMBC results^ 143Figure 66:^Selected region of 2D HMBC spectrum of pseudaxinellin(103) (CDC13)^ 144Figure 67:^Connectivities in 103 based on NOe enhancements andROESY correlations^  145Figure 68:^NOe results for pseudaxinellin (103) (400 MHz, CDC13)^ 146Figure 69:^NOe and ROESY correlations for majusculamide C (104) in1:1 C6D6/CDCI3^ 150xiFigure 70:^1H NMR spectrum of majusculamide C (104) (500 MHz,1:1 CDC13/C6D6)^ 153Figure 71:^Selected region of ROESY spectrum of majusculamide C (104)(500 MHz, 1:1 CDC13/C6D6)^ 154Figure 72:^Perspective drawing of 106 156Figure 73:^1H NMR spectrum of cis tetraacetate 140 (500 MHz, CDC13) ^ 184Figure 74:^1H NMR of Raney nickel reduction product tetraacetate 140(400 MHz, CDC13)^ 185Figure 75:^1H NMR spectrum of compound 81 (400 MHz, CDC13)^ 225Figure 76:^1H NMR spectrum of compound 82 (400 MHz, CDC13)^ 226Figure 77:^1H NMR spectrum of compound 83 (400 MHz, CDC13)^ 227Figure 78:^1H NMR spectrum of compound 84 (400 MHz, CDC13)^ 228Figure 79:^1H NMR spectrum of compound 85 (400 MHz, CDC13)^ 229Figure 80:^1H NMR spectrum of compound 88 (400 MHz, CDC13)^ 230Figure 81:^1H NMR spectrum of compound 90 (400 MHz, CDC13)^ 231Figure 82:^1H NMR spectrum of alkylation product 144 (400 MHz,CDC13)^ 233Figure 83:^1H NMR spectrum of compound 150 (400 MHz, CD3OD)^ 234Figure 84:^1H NMR spectrum of compound 143 (400 MHz, CD3OD)^ 235Figure 85:^1H NMR spectrum of the mixture of compounds 153 and 154(400 MHz, CD3OD)^ 236Figure 86:^1H NMR spectrum of trans tetraacetate 162 (400 MHz, CDC13) ^ 237xiiList of SchemesScheme 1:Scheme 2:Scheme 3:Scheme 4:Scheme 5:Scheme 6:Scheme 7:Scheme 8:Scheme 9:Scheme 10:Scheme 11:Scheme 12:Scheme 13:Scheme 14:Scheme 15:Scheme 16:Scheme 17:Scheme 18:Scheme 19:The position of Petrosia contignata within the phylogeneticclassification of the Porifera according to Bergquist^ 10Isolation of metabolites from Petrosia contignata 17Epimerization and reduction with NaBH4^ 48The position of Acanthella sp. within the phylogeneticclassification of the Porifera according to Bergquist^ 67Isolation of metabolites from Acanthella sp. 72Attempted solvolysis of isothiocyanate 82^ 109Proposed biogenetic scheme for the functionalizedsesquiterpenes found in Acanthella sp.^  114The position of Neoesperiopsis digitata within the phylogeneticclassification of the Porifera according to Austin^ 118The position of Pseudaxinella massa within the phylogeneticclassification of the Porifera according to Bergquist^ 132Synthesis of the Marfey's derivatives of the MAP residues^ 155Retrosynthetic analysis of tetraacetate 140^ 171Retrosynthetic analysis of amino acid methyl ester 143^ 172Benzyl protection of methyl 3,5-dihydroxybenzoate 147^ 173Reduction and tosylation at benzylic position ^ 174Alkylation of diethylacetamidomalonate (145) withbenzyl tosylate 146^ 175Hydrolysis of alkylation product 144^ 175Preparation of compound 143 176Preparation of sodium p-benzyloxy glycidate (141)^ 177Pictet-Spengler reaction^  178Scheme 20:^Effect of methyl protection on Pictet-Spengler reaction^ 179Scheme 21:^Acetylation of Pictet-Spengler products^ 180Scheme 22:^Preparation of tetraacetates 140 and 162 180Scheme 23:^Preparation of model compounds for CD analysis^ 183xivList of TablesTable 1:^1H and 13C NMR data for contignasterol tetraacetate(25)recorded in C6D6^ 24Table 2:^1H NMR data for reduction product pentaacetate (28) recordedin C6D6 ^ 38Table 3:^1H and 13C NMR data for reduction product (29) recordedin 10:1 CDC13/DMSO-d6^ 46Table 4:^1H and 13C NMR data for reduction product tetraacetate(30)recorded in C6D6^ 47Table 5:^1H and 13C NMR data for contignasterol (24) recordedin DMSO-d6^ 50Table 6:^1H and 13C NMR data for petrolactone (37) recordedin C6D6^ 58Table 7:^1H and 13C NMR data for petrolactone (37) recordedin CDC13^ 59Table 8:^1H and 13C NMR data for acanthene A (80) recordedin CDC13^ 76Table 9:^1H and 13C NMR data for acanthene A (80) recordedin C6D6^ 77Table 10:^1H and 13C NMR data for acanthene G (86) recordedin CDC13^ 86Table 11:^1H and 13C NMR data for acanthene H (87) recordedin CDC13^ 95Table 12:^1H and 13C NMR data for acanthene J (89) recordedin CDC13^ 102xvTable 13:^1H nmr and 13C nmr data for lactone neoesperlactone (96)recorded in CDC13^ 123Table 14:^1H and 13C NMR data for pseudaxinellin (103) recordedin CDC13^ 135Table 15:^HOHAHA assignments for pseudaxinellin (103)^ 139Table 16:^1H NMR and 13C NMR data for majusculamide C (104)recorded in 1:1 C6D6/CDC13^ 151xviList of AbbreviationsAc^- acetylAc20^- acetic anhydrideAPT^- Attached Proton Testax^- axialbd^- broad doubletbm^- broad multipletbr^- broadbs^- broad singletbt^- broad tripletCD^-Circular DichroismCOSY^- COrrelation SpectroscopYd^- doubletDa^- Daltonsdbe^- double bond equivalentsAO^- difference in chemical shiftsdd^- doublet of doubletsAM^- difference in massDMF^- dimethyl formamideDMSO^- dimethyl sulphoxidedt^- doublet of tripletsED50^- Effective Dose resulting in 50% responseFDAA^- 1-Fluoro-2,4-Dinitropheny1-5-L-AlalninAmideHREIMS^- High Resolution Electron Impact Mass SpectrometryLREIMS^- Low Resolution Electron Impact Mass Spectrometryeq^- equatorialEt20^- diethyl etherEtOAc^- ethyl acetateFABMS^- Fast Atom Bombardment Mass SpectrometryFTIR^- Fourier Transform InfraRedGC^- Gas ChromatographyGC-MS^- Gas Chromatography-Mass Spectrometryh^- hourHETCOR^- HETeronuclear CORrelationHex^- hexaneHIV^- Human Immunodeficiency VirusHMBC^- 1H Detected Multiple Bond Heteronuclear Multiple Quantum coherenceHMQC^- 1 H Detected Heteronuclear Multiple Quantum coherenceHOHAHA - HOmonuclear HArtman-HAhn experimentHPLC^- High Performance Liquid ChromatographyHS V^- Herpes Simplex Virusi^- signal due to an impurityIC50^- Inhibitory Concentration resulting in 50% response.IgE^- Immunoglobulin EJ^- scalar coupling constantLD^- Lethal DoseLRCIMS^- Low Resolution Chemical Ionization Mass Spectrometrym^- multipletM+^- parent ionm.p.^- melting pointm/z^- mass to charge ratioMe^- methylMe0H^- methanolxviiiMIC^- Minimum Inhibitory Concentrationmin^- minutesmmu^- millimass unitsNMR^- Nuclear Magnetic ResonancenOe^- nuclear Overhauser effectppm^- parts per millionq^- quartetRf^- Ratio to frontrel. int.^- relative intensityROESY^- Rotating-frame Overhauser Enhancement SpectroscopYS^- signal due to solvents^- singletSCUBA^- Self-Contained Underwater Breathing Apparatussp.^- speciest^- triplettic^- thin layer chromatographyTMS^- tetramethylsilanew^- signal due to waterW1/2^- width at 1/2 heightxixAcknowledgmentsFirstly, I would like to express my appreciation to Dr. Raymond Andersen for hisconstant encouragement and guidance throughout the course of this work. It has been apleasure to work with and to learn from him.The assistance provided by the technical staffs of the Department of ChemistryNMR and mass spectrometry laboratories is thankfully acknowledged. I am also greatlyindebted to Mike LeBlanc, Eric Dumdei, Jana Pika and the rest of the members of myresearch group for their assistance in the collection of the organisms used in this work aswell as for their constant support and friendship. I would specifically like to thank Dr.David Williams and Dr. Charles Pathirana for being great sources of information andideas during my studies. Financial support provided by the University of BritishColumbia through MacMillan and F.J. Nicholson fellowships is gratefully acknowledged.Finally, I would like to thank my wife, Ann, for her unending patience,encouragement, and understanding during my years as a graduate student.xxNATURAL PRODUCTS CHEMISTRYNatural products chemistry is the root from which modem day organic chemistryhas grown. Throughout history marine natural products have been used as dyes,medicines and poisons. For example, the Phoenicians used natural extracts of theMediterranean gastropod molluscs Murex brandaris, Murex trunculus, and Purpurahaemastoma to produce the Royal Purple dye for fabric,' the Japanese used the seaweedChondria armata to rid infants of worms, 2 and Hawaiian natives used the marinecoelenterate Palythoa toxica to poison their spear tips for warfare. 3 Studies have shownthat Chondria armata contains domoic acid (WI the toxin responsible for shell fishpoisonings on the east coast of North America in 1987. 5 Palytoxin (2), the main toxiccomponent of P. toxica, is one of the most potent toxins known. It has a lethal dose tomice of < 0.45 .tg/kg. 3Natural products chemistry continues to be one of the important branches ofmodern day organic chemistry. The search for new compounds is driven by three mainforces: 1) the continued need for new clinically useful drugs, 2) the desire for greaterunderstanding of the ecological role of secondary metabolites, and 3) the pursuit ofknowledge of how nature synthesizes such complex structures. This thesis describes theisolation and structural elucidation of several novel secondary metabolites from marinesponges collected in the Pacific Ocean as well as the partial synthesis of the starfishmetabolite imbricatine (3).1OHOHOHOH.0 OH^OHHOHOH1^ 3OHOHOH^OH`OH HO'0H0 ^s'OHOH-OH„OHII OHOHOH`OH0^OH^HOHO,^__OHHO mWIL":":—"stiH^ OH2OHThe past three decades has witnessed the discovery of a large number of naturalproducts from marine organisms. This has been partly due to the greater accessibility ofthe marine environment made possible by the advent of SCUBA and manned2submersibles. Other factors that have greatly enhanced the productivity of marine naturalproducts chemistry are the continued developments in the areas of chromatography andspectroscopy. These developments have made possible the isolation and structuralelucidation of compounds that are available in very minor quantities and have forced a re-examination of many marine organisms in search of minor metabolites.The development of many effective clinical drugs has been based on naturalproducts research. The ability of pathogens to develop resistance to chemical treatmentsas well as the many extreme side effects of some drugs make the search for new clinicallyuseful compounds an unending process. It was evident from the earliest years of marinenatural products chemistry that the sea contained an enormous pool of unstudiedorganisms that contained compounds with potentially interesting biological activities. Inthe 1950's, Bergmann reported the nucleosides spongothymidine (4), spongosine (5), andspongouridine (6) from the marine sponge Cryptotethya crypta. The discovery of thesenucleosides led to the development of the clinically useful antiviral drugs ara-A (7) andara-C (8). Ara-A (7), which was later isolated from the Mediterranean gorgonianEunicella cavollini, has been in use since the late 1970's as a therapeutic agent againstHerpes encephalitis. These early findings clearly indicated that the marine environmentwas indeed an excellent source of leads for the development of new pharmaceuticals. 6HO OH4 R = CH36 R = H3HO HO ^OH^OH^7 8Examination of the role that marine natural products play in biological systemshas been very enlightening. It is widely believed that many if not all secondarymetabolites have specific roles within the host organism.? One of the suggested roles isas a chemical defense against predators. A good example is the observed lack ofpredation on nudibranchs and other shell-less molluscs. In 1963 Johannes showed thatmucus secretions from Phyllidia varicosa were toxic to fish and crustaceans,8 andScheuer et al. later identified the toxic agent as 9-isocyanopupukeanane (9). It was alsoshown that compound 9 was accumulated by the nudibranchs from their sponge diet. 9Since this example was reported, many isonitriles with fish antifeedant activities havebeen isolated from nudibranchs. 10NC9The isolation of any new natural product raises questions of its biogenetic origins.For example, the isolation of 9-isocyanopupukeanane (9) raised questions about the4biogenetic origin of the isocyano functionality. In contrast to terrestrial organisms,marine organisms do not appear to utilize amino acid precursors to produce the isonitrilefunctionality. In fact, Garson showed that cyanide is the direct precursor of the isonitrilecarbons in compound 10. 11 Subsequently, Scheuer et al. showed that cyanide was thesource of the isonitrile carbon and nitrogen in 9-isocyanopupukeanane (9) and relatedcompounds. 12,1310SummaryThe search for novel biologically active marine natural products continues to gainmomentum as new isolation, structural elucidation and bioassay techniques aredeveloped. The need for new clinical drugs, the desire to understand the intricacies ofnatural products biosynthesis, and the importance of increased knowledge of ecosystems,drive this field of research.The early work by Bergman and subsequent work by several other marine naturalproducts groups have demonstrated that among marine organisms sponges are very goodsources of secondary metabolites. In the following chapters I will describe the chemicalstudies of metabolites isolated from six Pacific Ocean sponges and the partial synthesis ofa starfish metabolite. Part I describes the isolation and structural elucidation of the majorconstituents of the Papua New Guinea sponge Petrosia contignata, several terpenoid5metabolites from the Northeastern Pacific sponge Acanthella sp., and a y-lactone fromanother Northeastern Pacific sponge Neoesperiopsis digitata. Part II describes theisolation and structural elucidation of the cyclic heptapeptide pseudaxinellin (103) fromthe Papua New Guinea sponge Pseudaxinella massa and the cyclic depsipeptidemajusculamide C (104) from Ptilocaulis trachys and Amphimedon sp., two species ofSouth Pacific sponges. Finally, part III describes the use of a Pictet-Spengler approachto provide synthetic confirmation of the tetrahydroisoquinoline substructure ofimbricatine, a novel alkaloid from the starfish Dermasterias imbricata.6Part I^The Isolation and Structure Elucidation of Metabolitesfrom Three Pacific Marine SpongesIntroduction to the PoriferaMembers of the phylum Porifera, commonly known as sponges, have beenstudied in detail by many marine natural products groups around the world. The researchto date has shown that sponges are very good sources of secondary metabolites. This hasbeen attributed to the fact that sponges are sessile and require secondary metabolites todiscourage predators and to prevent competing organisms from restricting their growth. 14The Porifera are filter feeding organisms that use a layer of flagellated cellsknown as choanocytes to pump water through the sponge body. 15 The organization ofsponges is quite simple with no organs, defined tissues, mouths or digestive cavities. 16Though sponge cells are virtually independent of one another, they act together to formpores, ostia, canals and chambers through which the water currents can travel." Spongesare split into three categories based on the degree of body wall folding. Asconoidsponges have very simple tube-like structures with no folding, syconoid sponges haveslight wall folding, and leuconoid sponges have the largest amount of folding leading tovery complex structures. Sponge skeletons are made up of siliceous or calcium carbonatespicules and protein fibers known as spongin fibers. 18The taxonomy of sponges is anything but clear. However, it is accepted that thereare four main classes: Calcarea, Hexactinellida, Demospongia and Sclerospongiae.Within these classes there are about 5,000 species. Sponges in the class Calcarea havecalcium carbonate spicules whereas the spicules found in all other classes are siliceous.In addition, the Calcarea are usually small and can be brightly coloured and are found7throughout the world in shallow coastal areas. All three structural types, asconoid,syconoid, and leuconoid are represented in this class. Sponges in the classHexactinellida, or glass sponges, are characterized by a syconoid structural type withhexaxon, or six-pointed, spicules. Glass sponges are generally pale and are mostly foundin deep Antarctic waters. The largest class, encompassing 95% of all sponge species, isthe Demospongiae. These sponges are often brightly coloured and are found at alldepths. All Demospongiae have a leuconoid structural type and the large spicules, incontrast to the glass sponges, are monoaxon or tetraxon in nature. The final class ofsponges is the Sclerospongiae. These sponges all have a leuconoid structure and areunique from other classes in that their skeleton consists of internal siliceous spicules andare covered by a calcium carbonate layer. The Sclerospongiae are found in many areasaround the world and they are always associated with coral reefs. 198A.^Secondary Metabolites from the Papua New Guinea Marine Sponge PetrosiaContignata IntroductionSpecimens of Petrosia contignata (Thiele, 1899) were collected as part of ageneral collecting expedition to Papua New Guinea. The extracts of sample PNG-5-25-5-137R, later identified as P. contignata,20 were among the most cytotoxic obtained fromthe collection trip. The isolation and structure elucidation of the major secondarymetabolites obtained from the P. contignata extracts is described in the followingchapter.1) Taxonomy and Description of Petrosia contignataSponges of the genus Petrosia are Demospongiae that belong to the familyNepheliospongiidae and the order Nepheliospongida according to Bergquist (Scheme1).21 Sponges of this genus and of this family, which includes the genera Xestospongiaand Strongylophora, are characterized by linear spicules with pointed or rounded endsthat give rise to a hard sponge surface. The approximately 20 species of Petrosia arefound throughout the world in both tropical and temperate waters. 22The specimens of P. contignata examined in this study were collected in tropicalwaters on the outer reef off Barracuda point near Madang on the north coast of PapuaNew Guinea. The sponge was obtained in 10 - 20 meters of water in areas where therewas little or no surge. 23 The grey/purple sponge had a stone-like surface with obviousoscula.9Scheme 1: The position of Petrosia contignata within the phylogenetic classificationof the Porifera according to Bergquist3 1Phylum: PoriferaClass:^Calcarea^Hexactinellida^Demospongiae^SclerospongiaeSubclass:^Homoscleromorpha^Ceratinomorpha^TetratinomorphaOrder: Halichondrida Poecilosclerida Haplosclerida DictyoceratidaDendroceratida Nepheliospongida VerongidaFamily:^Oceanapiidae NepheliospongiidaeGenus:^Xestospongia Petrosia Strongylophora Species: P. contignata102) A Review of Secondary Metabolites Previously Isolated from Petrosia SpeciesSponges of the genus Petrosia have been excellent sources of secondarymetabolites. The structural types that have been isolated include alkaloids, polyacetylenecompounds and a number of novel steroids.The petrosins, of which petrosin (11) was the first to be identified, are a novelclass of alkaloids containing a C16 macrocycle. 24 Compound 11 was isolated from thesponge P. seriata collected off Laing Island, Papua New Guinea and was found to betoxic to the fish Lebistes reticulatus (LD50 = 50 mg/L). The petrosins are quite unusualin that alkaloids with quinolizidine structures are more commonly found in plants than inanimals.11Another alkaloid, petrosamine (12), was isolated from a Petrosia sp. collected atCarrie Bow Cay, Belize. 25 The structure of 12 was solved by X-ray diffraction analysis.In the crystalline state, petrosamine exists as the diketone 12a whereas in solution itexists as the enol 12b.11OHN ,./'A a_  ^Cl.0^ 012a 12bA number of high molecular weight polyacetylenes have also been isolated fromsponges of the genus Petrosia.26 Included in this group is petrosynol (13), a compoundwith antifungal activity isolated from a Petrosia sp. collected in Hachijo-jima Island, IzuArchipelago. The tetraketo analog petrosynone (14) was isolated from the antimicrobialfractions in the same study. Compound 14 exhibited activity against Bacillus subtilis.OH^OHT.-OH^OH131412Petrosia spp. have yielded a number of steroids with cyclopropyl containing sidechains. One example is petrosterol (15) isolated from the marine sponge P .27,28ficiformis.^Originally the structure of petrosterol was assigned as 16 on the basis ofspectral data but was later revised to 15 by X-ray diffraction analysis. 29 The structure of15 differs from cholesterol only in the cyclopropyl containing side chain. A relatedsteroid 17 was also isolated from P. ficiformis.30 It was suggested that 17 might be theimmediate biosynthetic precursor of 23- and 24- methylcholesterols such as brassicasterol(18), and that in general biosynthetic C-alkylation might occur through a cyclopropylintermediate. 31 This suggestion was supported by the observation that acid-catalyzedring opening of 17 resulted in 18 as the major product. More recently,wienbersteroldisulfates A (19) and B (20) were isolated from P. wienbergi collected offAcklin Island, Bahamas, and identified on the basis of spectroscopic analysis. 32 Steroids19 and 20 also contain a cyclopropyl group in the side chain. Both are active in vitroagainst feline leukemia virus (FeLV) and 19 is active against HIV (in vitro). In the samestudy, a series of sterol disulfates 21-23 were isolated from P. weinbergi collected offAcklin Island and Long Island, Bahamas. 33 Compounds 21-23 have unusual ortho estercontaining side chains and all three exhibit antiviral activity.HO15 R =16 R =1321 R =Na*-03S0Na*-03S0‘‘23 R = .n/22 R .HO17 R =18 R =19 R 1 = H R2 = OH20 R 1 = OH R 2 = H14Results and Discussion1) Isolation of metabolites from Petrosia contignata .Specimens of P. contignata were collected by hand using SCUBA at Madang,Papua New Guinea and transported to Vancouver frozen over dry ice. The frozen spongespecimens (2.5 kg wet weight) were immersed in methanol (3L) and soaked at roomtemperature for 48 h. Concentration of the decanted methanol in vacuo gave an aqueoussuspension (1800 mL) which showed cytotoxic activity against murine leukemia L1210.The aqueous slurry was sequentially extracted with hexanes (4 x 500 mL) andchloroform (4 x 1L). The hexanes extract, which yielded a brown oil upon evaporationin vacuo, and the remaining aqueous fraction both showed cytotoxic activity (L1210:ED50 2 and 6 .tg/mL respectively). The chloroform extract, which gave a brown solid(2.1 g) after evaporation in vacuo, exhibited both antibacterial and antifungal activity.Purification of the hexanes extract was accomplished by repeated applications ofsilica gel chromatography, radial tic and normal phase HPLC. The silica gelchromatography (4:1 Hex/EtOAc) yielded fractions which were analyzed by 1 H NMRand tic and pooled to yield seven major fractions. 1 H NMR spectra indicated thepresence of the known tetrabromodiphenyl ether 41 in the second fraction (B) as well asthe new sesquiterpene, petrolactone (37), in the third fraction (C). A second applicationof silica gel chromatography on fraction B yielded pure compound 41 (Rf = 0.26 (4:1Hex/EtOAc)). Compound 41 was found to be the major cytotoxic component of thehexanes extract (L1210: ED50 < 1 p.g/mL). Purification of compound 37 wasaccomplished by sequential applications of radial silica gel tic (9:1 Hex/EtOAc) andnormal phase HPLC (9:1 Hex/EtOAc) to yield 6.2 mg of a white solid that was inactive15in the L1210 cytotoxicity assay. The remaining fractions from the hexanes extractcontained several steroid and fatty acid metabolites that were not pursued.Purification of the chloroform extract was accomplished by repeated bioassayguided fractionation using Sephadex LH-20, reversed phase flash chromatography andreversed phase HPLC. Sephadex LH-20 (4:1 Me0H/H20) yielded fractions that wereanalyzed by 1 H NMR and tic and pooled to yield five major fractions which showedvarying antibacterial and antifungal activity. The 1 H NMR spectrum of the fourthfraction (D) gave signals attributable to the highly oxygenated steroid, contignasterol(24). Tlc analysis of this fraction showed two major components (Rf = 0.14 and 0.20 onC18: 3:1 Me0H/H20 ) as well as several minor components. Further purification of thefourth fraction using reversed phase flash chromatography (4:1 Me0H/H20) gave anactive fraction which contained almost pure contignasterol (24). Final purification wasaccomplished using HPLC (3:1 Me01-1/1120) to yield pure contignasterol (24) ascolourless crystals (153 mg: mp 239-41 °C). HPLC purification of 24 gave two majorpeaks with retention times of 80 and 112 min. (Magnum-9 Partisil 10 ODS-3 column,flow rate 2.0 mL/min) in approximately a 2:1 ratio. Tlc analysis on each of the majorpeaks immediately after the HPLC run showed one major spot and one minor spotcorresponding to the two HPLC peaks. Tlc analysis of each of the major peaks 30minutes after the HPLC run showed a mixture of two major spots corresponding to theHPLC peaks. Reinjection of the individual fractions yielded HPLC traces that wereidentical to the original trace. This was the first indication that contignasterol existed insolution as two slowly epimerizing forms. Contignasterol (24) did not show antibacterialor antifungal activity and therefore the activity was attributed to the minor components offraction D.16Partitioned withchloroformOrganic Extract I I Aqueous layer I111,Normal phase HPLC9:1 Hex/EtOAcReversed phase flashPetrolactone (37)^4:1 Me0H/Hp6.2 mgIlySephadex LH-204:1 Me0H/H9Bromophenol 41Scheme 2: Isolation of metabolites from Petrosia contignata.I Sponge (wet weight 2.5 kg) I1)aqueous Me0H extract2) decanted and evaporated in vacuaI Aqueous suspension IPartitioned with hexanesI Aqueous layer IOrganic Extract ISilica gel4:1 Hex/EtOAcA^B^C^D-GFats Fats and steroidsSilica gel^Radial silica gel tic4:1 Hex/EtOAc 9:1 Hex/EtOAcReversed phase HPLC3:1 Me0H/HpContignasterol (24 )153 mg17OR29282425H3C2627CH3H F:OR'^OR24 R = Ri = H25 R = Ac R1 = H26 R = R 1 = Ac2) Structure elucidation of metabolites from Petrosia contignataContignasterol (24)Contignasterol (24) gave a parent ion in the HREIMS at m/z 508.3394 Da,corresponding to a molecular formula of C29H4807 (AM -0.6 mmu). The FTIR showed acarbonyl stretching frequency at 1719 cm -1 as well as an intense and broad OH stretchingband at 3381 cm -1 . The 1 H NMR spectrum of 24 (Fig. 1) showed a complex mixture ofcarbinol methine resonances between 8 3.2 and 5.2 ppm as well as many exchangeableprotons that were assignable to several hydroxyl groups in 24. A number of thedownfield proton resonances integrated for less than one proton. This informationcombined with the presence of 44 resolved resonances in the 13C NMR spectrum (Fig. 2)indicated that 24 existed in two slowly interconverting isomeric forms. Further evidencefor this was found in 13C NMR resonances at 8 95.6 and 90.4 ppm that had chemicalshifts appropriate for acetal carbons and which showed HMQC correlations (Fig. 3) into18to^ HCK. 7^6^5^4^PPM^3^2^I^0Fig. 1. 1H NMR spectrum of contignasterol (24) (500 MHz, DMSO-d6)OHMa^90^90^70^60^50^40^30^20Fig. 2. 13C NMR spectrum of contignasterol (24) (125 MHz, DMSO-d6)3^2^14pp. 590.4/5.160•95.6/4.50Os*0^•0oocCaAbV oo co %Br' r •Oo^0-100^;PR- 20- 40- 60_ BoFig. 3. 2D HMQC spectrum of contignasterol (24) (DMSO-d6)L35410.43 ITIC.2921104. 144/0.731241 E1!/Iet 17264417244111126II I1"^1I lh^I^ 11.'11'1 I .424^144114OS61141 4911   Iwilltillti444^544^524^511^561^SOS^611157164261319 448221 246^203 311^ 393111114111111111141111101111111^ 1 1 1 1 1 1 . 1 1^kt-rtiliertity,^elTr1111't et I t'l^t O't Tn224^244^261 284^314^324^314 361^344^111III ••^•1355OE 016964 —249520t'I^rill ^11t11 I tit 1411 1 11111'11114 60119 9 21335^47^1^1^1 591 1 1 . 1 11111/ 1 1 1 1 1 1^/ 4 1 1 1 11 I 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 11 1 1 1 1 1 1 1 1 1 1 1 1 1p181^I11 124^14%^164^104^211^221Fig. 4. Low resolution EI mass spectrum of contignasterol (24)1 H NMR resonances at 8 4.50 and 5.16 ppm, respectively, that each integrated to lessthan one proton.Acetylation of contignasterol (24) with acetic anhydride in pyridine gave amixture of polyacetates that were purified by HPLC (3:2 Hex/Et0Ac) to give thetetraacetate 25 as the major product. Several minor products included the pentaacetate 26( 1H NMR: see Fig. 9) and a tetraacetate which appeared to be the epimer of the majorproduct. The highest peak in the HREIMS of 25 was at m/z 616.3605 Da, appropriate fora fragment ion of C35H5209 (AM -0.6 mmu) resulting from loss of acetic acid (M+(C37H56011) - HOAc). The appearance of an OH stretch at 3477 cm -1 (br) in the FTIRand the isolation of 26 as a minor acetylation product indicated that 25 contained a freehydroxyl group. The presence of four acetate residues was evident from resonances inthe 1 H (8 1.61(s), 1.71(s), 1.82(s) and 1.88(s)) and 13C (8 20.4, 20.6, 20.7, 20.8, 169.1,169.3, 169.4, 172.7) NMR spectra (Figs. 5 and 6) which were appropriate for acetylresidues. The 13C NMR spectrum of 25 (Fig. 6) showed 37 resonances, demonstratingthat the complicating effects of hemiacetal epimerization had been eliminated byacetylation of 24.The presence of three methyl doublets (8 0.75, 0.77 and 0.77) and two methylsinglets (8 1.12 and 1.20) in the 1H NMR spectrum of 25 (Fig. 5) in conjunction with thenumber of carbons (HREIMS: C29H48 07) in the parent compound 24, provided the firstevidence that 24 was a steroidal metabolite. The five carbonyl functionalities (4 acetatecarbonyls and one saturated ketone ( 13C: 8 216.0)) and the four rings of the standardsteroid skeleton accounted for nine of the ten degrees of unsaturation in 25 required bythe molecular formula of C37H56011 (10 dbe). The four acetoxy groups, the hemiacetalfunctionality, the hydroxyl and the saturated ketone accounted for all of the elevenoxygen atoms in 25.A detailed analysis of the APT, COSY and HMQC spectra of 25 (Figs. 6-8, Table1) identified a series of resonances that could be assigned to the contiguous spin system23Table 1. 1 1-1 and 13C NMR data for contignasterol tetraacetate(25) recorded in C6D6 (see Appendix C).C# 1H(400 MHz) COSY nOesf 13C (125 MHz) HMBCg1 32.6131' 1.27 H2eQ,H2ax2., 2.02 H1',H2ax,H3 203a2,„ 1.60 H1 ',H2eq ,H33 5.24,bm H2ax,H2eq,H4 H2eq,H2ax,OH4,H4 71.6 OH44 3.87,bm H3,H5,0H4 H3,0H4,H5 66.6 H340H 3.05,bd H45 1.80 H4,H6 45.9 H3,H6,H196 5.40,dd(8.6,12.1) H5,H7 OH4,H19 73.8 H77 6.63,dd(8.6,10.4) H6,148 H5,H14 74.7 H68 1.95 H7,H9,H14 37.3 H79 1.26 H8 32.0 H1910 - 36.4 H1911 21.5a11'12ax 1.06 H12eq 36.3 H1812eq 1.33 H12ax,H1413 - 42.2 H16,H16',H1814 2.34,bs H8,H12eq H7,H8,H18,H22 51.7 H1815 - 216.0 H8,H16,H16'16 2.06,bd(19.7) H16',H17 40.516' 2.30,dd(19.7,9.9) H16,H1717 2.00 H16,H16',H20 43.0 H18,H2118 1.20,s 3H H14,H22 19.3c19 1.12,s 3H H2,0H4,H6,H8 14.5d20 1.68 H17,H21,H22 41.721 0.77 H2O 15.2d22 3.33,bt H20,H23ax,H23eq H14,H16,H18,H23,,,H2978.0 H21,H23ax23 ax 0.64,m(10.8,12.2) H22,H23eq,H24 32.8b23eq 1.41,bd(12.2) H22,H23ax,H2424 1.09 H23ax,H23eq,H25,H28eq 40.6 H26,H2725 1.23 H24,H26,H27 46.626 0.77e H25 19.8c27 0.75e H25 19.5c28ax 1.17 H28eq,H29 33Ab28eq 1.60 H24,H28ax,H2929 5.60,dd(2.2,9.5) H28 ax ,H28eq H22,H28eq 94.3 H28axOAc 1.61,s; 1.71,s;1.82,s; 1.88,s20.4; 20.6;20.7; 20.8;169.1; 169.3169.4; 172.7a,b,c,d,e May be interchanged.f Proton in Carbon # column irradiated.g Protons correlated to carbon resonances in 13 C NMR column.24CH3H3C=^.ts.)^ Hut OH OAcII..^ke..,i Inv r^ !my 1 ,1111111yr1vT,r1r r r vivII■li r r r1^Iv I TTII I I IITIIMIITVIIII1Tf f IFIFIIM^2 . 0^6 . 0^5 . 0^4 . 0 3 . 0^2 . 0^1 . 0^0 0PP MFig. 5. 1H NMR spectrum of tetraacetate 25 (400 MHz, C6D6)OAcCH3Ace;MI 22I3^260 ^140 ^160 ^140 ^IR) ^lOo ^81) ^60^40 ^20^15Fig. 6. 13C and APT NMR spectra of tetraacetate 25 (125 MHz, C6D6)CH3H3C6.0^5.0^4.0^3.0^2.0^1.0PPMFig. 7. 2D COSY spectrum of tetraacetate 25 (400 MHz, C6D6)CH 0*AcO`‘..^OAc11OH OAc• •ofthOPco bto•O ••O0oo••0I ^  I^  I^6 4 2Fig. 8. 2D HMQC spectrum of tetraacetate 25 (C6D6)pp.— 40OAoCH3— 80PP.— 60113C1w7.0 6.0^5.0^4.0^3.0^2.0^1.0^0.0PPMFig. 9. 1H NMR spectrum of pentaacetate 26 (400 MHz, C6D6)consisting of the H14 (5 2.34), H8 (1.95), H7(6.63), H6 (5.40), H5 (1.80), H4 (3.87), H3(5.24), H2ax (1.60) and H2eq (2.02) protons on a steroid nucleus. 1 H and 13C NMRchemical shifts placed the acetoxy substituents at C3 (5 71.6), C6 (73.8) and C7(74.7) andthe hydroxyl substituent at C4 (66.6). Further support for the positioning of the hydroxylsubstituent came from a COSY correlation (Fig. 7) between the OH proton at 5 3.05 ppmand H4 (5 3.87). Extensive HMBC correlations supported these assignments. A strongHMBC correlation between the Me19 proton resonance (5 1.12) and the C5 carbonresonance (5 45.9), and an overlapping network of correlations between the H3, OH4,H6 and H7 proton resonances and the C3 to C8 carbon resonances (see Table 1) were incomplete agreement with the proposed substitution pattern in the A and B rings.Difference nOe experiments established the stereochemistry of the substituents onthe A and B rings (Fig. 10). Irradiation of Me19 (5 1.12) induced nOe enhancements inthe resonances assigned to H2ax (6 1.60), H8 (1.95), OH4 (3.05) and H6 (5.40).Irradiation of H6 (5 5.40) induced enhancements in the resonances assigned to Me19(51.12) and OH4 (3.05). These results suggested that 25 had the standard steroidal ABring system configuration with a 1013-methyl and a 5a-proton in addition to 4P-hydroxyland 6a-acetoxy substituents. The magnitude of the scalar couplings observed in the H3and H7 protons showed that the remaining acetoxy groups at C3 and C7 were in the aand p configurations, respectively. The H3 resonance appeared as a broad singlet (W1/2= 8.3 Hz) with no large trans diaxial coupling constants and thus H3 had a p (equatorial)configuration. The H7/H8 coupling constant of 10.4 Hz demonstrated that the protonswere trans diaxial and therefore H7 had the a configuration. A ROESY correlationbetween H8 and Me 18 established their 1,3-diaxial nature and demonstrated that ring Cwas also in the standard steroidal conformation.30---^---,.,nOeFig. 10. NOe results for rings A and B of tetraacetate 25A series of nOe experiments led to the assignment of the 143 configuration. TheH14 proton resonance was observed as a broad singlet in the 1H NMR spectrum oftetraacetate 25 (Fig. 5) and the shape of the resonance was the first indication that H14had the p configuration (i.e. H14 did not appear to be in a trans diaxial relationship withH8 based on the small coupling constant). Irradiation of the H14 proton (5 2.34) in adifference nOe experiment resulted in enhancement of the Me18 protons (5 1.20).However, H14 was only partially resolved from one of the H16 methylene protons (52.30) and thus the nOe result could not be used as conclusive evidence of the 14P protonconfiguration. Titration of the NMR sample containing 25 in C6D6 with CC14 to a ratioof 2:1 CC14/C6D6, resulted in a shift upfield of H14 to 5 2.10 ppm where it wascompletely resolved ( 1 H NMR of 25 in 2:1 CC14/C6D6: see Fig. 11). An enhancement ofMe18 (5 1.10) was observed upon irradiation of H14 (5 2.10) in the subsequent nOeexperiment (Fig. 12). The back nOe from Me18 to H14 was also observed (Fig. 12). Theequivalent nOe was observed in the parent compound 24 between the H14 protons at 53.00 and 3.05 ppm and the Me18 protons at 5 1.13 ppm (DMSO-d6). Finally, a COSYcorrelation attributed to W-coupling between H14 and H1245 1.33) in 25 supported theH140 configuration (Fig. 13).31CH3OAcCH3HH3CAcO`‘‘..tr.)^ Hts.) OH^OAcHOAcS i11.0^3.0^2.11^1.0^ILOPPM1•'"1•""- I"' • I"^T"••1- " -•- "I'' 7 •1 '^1^I I^I^•"--1-""I'" I^I7 . 0^6.II 5.0Fig. 11. 114 NMR spectrum of tetraacetate 25 (400 MHz, 2:1 CCWC6D6)H14 irradiated44,44004.00.,,Arogire0004Mel 800)OAck -,--1 i^i^I^I^IPPM^7. II 6. 0 5. 0I^. I4.8 3.A^2.19i1. 0Fig. 12. NOe results of tetraacetate 25 (400 MHz, 2:1 Ca4/C6D6)1.20 (8 1.10; 2:1 CC1 4/C6D6)18013S 1.3314 2.34 8 2.10: 2:1 CC14/C6D6)5Fig. 13. Relative stereochemistry of rings C and D of tetraacetate 25The positioning of the saturated carbonyl functionality at the C15 position wasbased on chemical shift data and HMBC experiments on 24 and 25 as well as NMRanalysis of the reduction product pentaacetate (28). The chemical shifts of the H14resonances in both the parent compound 24 (DMSO-d6, 8 3.00 and 3.05) and thetetraacetate 25 (C6D6, 8 2.34) were relatively deshielded, the first indication that thecarbonyl was at C15. The C16 methylene protons at 8 2.06 and 2.30 ppm, which werethe beginning of a spin system that included the side chain, were also relativelydeshielded. In addition, HMBC correlations (Fig. 14) from the H16 (8 2.06) and H16' (82.30) to the carbonyl resonance (8 216) supported placement of the ketone at C15. Thepositioning of the ketone at C15 also provided an explanation for the extremelydeshielded chemical shift of the H7 methine proton in both 24 (DMSO-d6, 8 4.16) and 25(C6D6, 8 6.63). Drieding models showed that the H7 methine would be positioned nearthe C15 ketone functionality when the H14 proton was in the 3 position. Final supportfor the positioning of the ketone came from reduction of 24 with sodium borohydride andsubsequent acetylation in acetic anhydride and pyridine to yield, after purification byHPLC (1:1 Hex/EtOAc) the major product, pentaacetate 28 ( 1 H NMR, see Fig. 15). ACOSY correlation (Fig. 16, Table 2) between H14 (8 2.15) and the new methine at 8 5.25(H15) demonstrated that reduction took place at C15. Further support was found in the34Fig. 14. Selected regions of the 2D HMBC spectrum of tetraacetate 25 (C6D6)aH17/0Ac H29/OAc0PPM1^.6.0 55•^I^•6.5-168-172H6/OAc-174. PPM7 . 0^6.11^5.y^ .^ 2.0^1.0^0 0PPMFig. 15. 1H NMR spectrum of reduction product pentaacetate 28 (400 MHz, C6D6)S OAcAcO‘‘‘.CH3AcCeCH,5:6^ . ..^6.0•^•  ^• •  4.0 ' ' ' ' • ' • '3' '^,,2.0^1.0^PPMPPM .0Fig. 16. COSY spectrum of reduction product pentaacetate 28 (400 MHz, C6D6)upfield shifts of H14 (8 2.15) and H7 (8 5.07) in 28 relative to the respective protons intetraacetate 25 at 8 2.34 (H14) and 6.63 (H7).ORCH327 R = H28 R = AcTable 2. 1 H NMR data for reduction product pentaacetate (28) recorded in C6D6.C# 1 H(400 MHz) COSY Decouplinga nOesa7 5.07,dd H6,H88 1.99 H7,H14 H7,H1414 2.15,dd H8,H15 H1815 5.25,m H14,H16,H16' H14,H1616 2.49,m H15,H16',H1716' 1.26 H15,H16,H1717 1.80 H16,H16'18 1.07 H14,H22,H2919 1.04 H6a Proton resonance in carbon # column irradiated.After assignment of the substitution pattern and functionality in rings A-D in 25,several remaining structural features had to be accounted for in the side chain. Theseincluded three methyl doublets, the hemiacetal functionality, and the remaining acetate.38Analysis of the COSY spectrum of 25 (Fig. 7, Table 1) showed a spin system starting atthe C16 methylene protons H16/H16'(6 2.06 and 2.30) and continuing through H17(62.00) and on into H20(6 1.68). The H2O methine (6 1.68) was coupled into Me21 (60.77) as well as into a deshielded methine proton (H22: 6 3.33) which was in turn coupledinto the C23 methylene protons (H23/H23': 6 0.64 and 1.41). Further analysis of theCOSY spectrum (Fig. 7) showed that the remaining two methyl doublets at 6 0.75 and0.77 ppm were in the C26 and C27 positions giving rise to a standard steroid side chainbackbone with branches at C22 and C24. The H25 isopropyl methine (6 1.23) showedCOSY correlations into the Me26 and Me27 resonances (6 0.75 and 0.77) as well as intothe H24 methine (6 1.09). H24 (6 1.09) in turn showed COSY correlations into the C23methylene protons (6 0.64 and 1.41). Further analysis revealed a two carbon branch atC24 that consisted of a methylene group and the acetal functionality. The acetal protonH29 (6 5.60: HMQC correlation to 6 94.3) showed COSY correlations to the C28methylene protons H28 ax (1.17) and H28eq(1.60). A COSY correlation between H28eq (81.60) and H24(1.09) then connected C28 to C24. An HMBC correlation (Fig. 14) fromH29 (6 5.60) to an acetate carbonyl carbon (6 169) revealed that the fourth acetoxy groupin 25 was part of the acetal functionality. This accounted for all of the atoms in themolecular formula leaving one degree of unsaturation that was satisfied by attachment ofC29 to the C22 oxygen atom forming a six membered ring and completing the acetalfunctionality.The relative stereochemistry of the tetrahydropyran ring in the side chain of 25(Fig. 17) was determined using difference nOe and double resonance decouplingexperiments. A pair of nOe experiments (irradiated H29 (6 5.60) - nOe enhancement inH22 (3.33); irradiated H22 (6 3.33) - nOe enhancement in H29 (5.60)) along with a 9.5Hz trans diaxial scalar coupling between H29 (6 5.60) and H28 (6 1.17) showed that thering occupied a chair conformation with the C22 and C29 substituents in equatorialorientations. A coupling constant of 12.2 Hz between H23ax (6 0.64) and H24 (6 1.09),39HOAc^28H1.60CH32624H^CH31.09 27H8 3.33251.400 _______--...._ 290.64 H —assigned by a series of decoupling experiments, required these protons to be in a transdiaxial orientation and thus the isopropyl substituent had an equatorial orientation.1.17Fig. 17. Relative stereochemistry of the tetrahydropyran ring of tetraacetate 25An attempt was made to use empirical methods to assign the absolutestereochemistries at C22 and C24. In previous studies, 34-39 the absolutestereochemistries of side chains with substituents at C22 and C24 had been determined byempirical rules based on carbon chemical shift data. These studies showed that ahydroxyl group at C22 causes downfield shifts of 4.5 (22S) vs 6.8 (22R) ppm in C20 and9.4 (22S) vs 3.8 (22R) ppm in C23 as compared to the saturated side chain of cholesterylbenzoate (Fig. 18). 3435 A similar effect was observed with C22 amine substituents.R 13C shiftsR I C20^C23^H^35.8^23.9OH(22S)^40.3^33.3OH(22R) 42.6^27.5Fig. 18. The effect of a C22 hydroxyl substituent on the 13C chemical shifts of theC20 and C22 carbons in cholesteryl benzoates in a study done by Letourneux et al.3440For C24 substituents, the relative carbon chemical shifts of the C26 and C27methyls are characteristically different in 24R and 24S compounds. 36,37 In general, withsaturated side chains a chemical shift difference of AS > 1 ppm is observed for 24Rcompounds and of AS<0.5 ppm for 24S compounds (Fig. 19).27^13C shiftsR^I C26 C27 ASCH2OH(24R) 18.3 19.7 1.4CH2OH(24S) 18.9 19.3 0.4CO2Et(24R) 18.6 19.7 1.1CO2Et(24S) 19.2 19.3 0.1HO25Fig. 19. The effect of two carbon branches at C24 on the 13C chemical shifts of the C26and C27 carbons in a study done by Anastasia et al. 36Reduction of 24 with sodium borohydride under slightly harsher conditions thanused to produce 27 yielded the major product 29 after purification using reversed phaseHPLC (13:7 Me0H/H20). The 1H and 13C NMR assignments for 29 (Table 3) werebased on a detailed analysis of COSY, HMQC and HMBC experiments. Compound 29was further characterized by acetylation with acetic anhydride in pyridine to yieldtetraacetate 30 (Figs. 21-22 and Table 4) after purification using normal phase HPLC.Compound 29 contained a C22 hydroxyl substituent and a C24 ethyl alcohol substituentand was thought to be a good candidate for the empirical methods used by Letourneux etal. 34 and Anastasia et a1. 36 Unfortunately, attempts to assign the absolutestereochemistry at C22 and C24 of compound 29 were inconclusive. The chemical shiftof the C20 carbon in compound 29 was 5.9 ppm downfield from the C20 carbon incholesteryl benzoate. This result was between the 13-effects (on C20 as a result of the41C22 hydroxyl substituent) observed for the 22S(OH) and 22R(OH) compounds in themodel studies. The chemical shift of the C23 carbon in compound 29 was 9.3 ppmdownfield from the C23 carbon found in cholesteryl benzoate. This result was very closeto that observed for the 22S(OH) compounds in the model studies. The results for theC24 center were also inconclusive. The difference in chemical shifts between C26 andC27 in compound 29 was AS = 2.17 ppm. This result was more consistent with the 24Rconfiguration. However, it was much larger than observed for 24R compounds in themodel studies (AS = 1.4 ppm). 36 Although inconclusive, the results lean towards the 22Sand 24R configurations in 29, which correspond to 22S and 24S configurations in 25, andare consistent with the relative stereochemistry of the tetrahydropyran ring in 25. Thesuccess of an empirical method is based largely on its generality. In the empirical studieson C22 and C24 substituted steroids, all model compounds had either a C22 or a C24substituent but never both. Clearly, the functionalities and substitution pattern makecompound 29 a poor subject for the published empirical method resulting in ambiguousassignments.RO‘‘‘s29 R = H30 R = Ac42PPE^so^70^00^50^40^30^20^10Fig. 20. 13C NMR spectrum of reduction product 29 (10:1 CDC13/DMSO-d6)1 1111 111111111•1T4ITITT7,1W 1., IT T T I I I I T r 1^1^I I l I T I T T 1 Ill 11" I TA I I TT I TT T.1 ,7.0^6.0^5.0^4.0^3.0 2.0^1. 0^0.0PPMFig. 21. 1H NMR spectrum of reduction product tetraacetate 30 (C6D6)AcO'''..I,Stwi JivIFig. 22. 13C NMR spectrum of reduction product tetraacetate 30 (C6D6)Table 3. 1 H and 13C NMR data for reduction product (29) recorded in 10:1 CDC13/DMSO-d6.C# 1H(400 MHz) COSY 13C (125 MHz) HMBCd11'2 1.96 H2',H3 23.642 ' 1.58 H2,1133 3.91,bs H2,H2',H4 68.984 4.01,bs H2',H3,H5 68.245 1.37 H4,H6 46.04 HI96  3.64,m H5,H7 70.71 H77 3.30,bm H6,H8 79.50 H6,H88 1.91 H7,H9,H14 36.389 0.90 H8,H11,H11' 52.73b H1910 - 35.63 H1911 1.30 H9,H11' 31.76 111911' 1.49 H9,H11,H12,H12'12 1.90 H11,H11',H12' 40.51 H1812' 1.05 H11,H11',H1213 - 42.60 H15,H16,H1814 1.00 H8,H15 60.43 H1815 4.30 H14,H16,1416' 70.5516 1.47 H15,H16',H17 37.4416' 2.24 H15,H16,H1717 1.07 H16,H16',H20 52.69b H18,H2118 0.96,s 14.0519 1.08,s 14.8420 1.82 H17,1421 41.67 H2121 0.93,d H2O 12.1122 3.69,bm H23,H23' 71.98 H2123 1.67 H22,H23' 33.2423' 1.25 H22,H2324 1.62 36.54 H26,H2725 1.74 H26,H27 30.36 H26,H2726 0.82,d(7.0)a H25 19.69c H2727 0.90,d(7.0)a H25 17.52c H2628 1.67 H29,H29'28' 1.27 H29,H29'29 3.65 H28,H28' 60.2229' 3.65 H28,H28'OH 4.32,bs; 4.20,bs;4.08,bs; 3.75,bda•Nc May be interchanged.d Protons correlated to carbon resonances in 13C NMR column.46Table 4. 1 H and 13C NMR data for reduction product tetraacetate(30) recorded in C6D6.C# 1 H(400 MHz) COSY 13C (125 MHz) HMBCf1 1.18 HI ',H2,H2' 33.2 H191' 1.30 H1,H2,H2'2 2.09 H1,H1',H2',H3 21.92' 1.67 H1,H1',H2,H33 5.38,m H2',H2,H4 71.9 OH44 3.80,bs H3,H5,0H4 66.8 OH440H 3.44,d(3.5) H45 1.45 H4,H6 46.8 H196 5.11,dd(8.8,12.0) H5,H7 77.1 H5,H77 3.32,t(8.8) H6,H8 77.7 H6,H88 1.93 H7,H9,H14 37.8b H7,H149 0.64,bt(11.6) H8,H11,H11' 53.2 H8,H1910 - 36.6 H5,H1911 1.28 H9,H11',H12,H12' 20.411' 1.11 H9,H11,H12,H12'12 0.89 H11,H11',H12' 41.2 H1812' 1.80 H11,H11',H1213 - 43.6 H14,H15,H16',H1814 0.70,dd(5.4,10.8) H8,H15 61.0 H7,H8,H1815 4.20,m H14,H16,H16' 71.2 111616 1.94 H15,H16',H17 39.216' 2.39,dt(8.2,14.1) H15,H16,H1717 1.05 H16,H16',H20 53.7 1116'18 1.04,s 14.8c19 1.07,s 15.0c20 2.13 H17,H21,H22 40.021 1.04 H2O 13.5c22 5.32 1120,1123,1123' 74.8 H2123 1.4-1.6 H22 29.8d23' 1.4-1.6 H2224 1.40 37.8b H26,H2725 1.84 H26,H27 29.3d H26,H2726 0.88,d(6.8)a H25 20.2e 112727 0.81,d(6.8)a H25 17.5e H2628 1.66 H29,H29' 29.128' 1.32 H29,H29'29 4.20,m H28,H28',H29' 63.3 H28,H28'29' 4.07,dt(7.1,10.9) H28,H28',H29OAc 1.82,s,3H;1.75,s,3H;1.68,s,6H20.60; 20.63;20.9; 21.0;169.1; 170.1;170.3; 173.5a,b,c,d,e May be interchanged.f Protons correlated to carbon resonances in 13C NMR column.47OCH3During the stereochemical studies it was noticed that the H14 proton of 30 (H14:8 0.70) (Fig. 21, Table 4) had shifted upfield dramatically relative to that in 28 (H14: 52.15) (Fig. 15, Table 2). In addition, the coupling constants (H14: dd, J = 5.4,10.8 Hz)were significantly larger than observed for 25 (H 14: 5 2.34, broad singlet). A doubleresonance decoupling experiment on H15 of 30 showed the H14/H8 coupling constant tobe the larger of the two (H14/H8: J = 10.8 Hz). This information suggested that incompound 30 H14 and H8 had a trans diaxial relationship and that epimerization at theC14 center had occurred under the reduction conditions. The absence of an nOe betweenH14 and the Me18 protons in compound 30, which had been observed in the parentcompound 24 and in the derivatives 25 and 28, supported a trans CD ring junction.Previous work by Erman and Flautt (Scheme 3) showed that in the presence of NaBH4and hydroxylic solvents, epimerization at the a centre can occur prior to reduction. 40The example given was the reduction of the ketones shown in Scheme 3. In bothreactions, the sterically favored epimer was produced or maintained prior to reduction.OCH3Scheme 3. Epimerization and reduction with NaBH4 4048After the general constitution and relative stereochemistry of contignasterol (24)were established by spectroscopic analysis of the derivatives it was possible to assign themajority of the proton and carbon resonances in the parent compound based on COSY,HMQC and HMBC data (Table 5). Analysis of the COSY data allowed for theassignment of the spin systems in the ring structure as well as the side chain. In additionto the nOe observed between Me18(8 1.13) and H14(3.05/3.00), the W-coupling COSYcorrelation between H14(3.05/3.00) and H12 eq(1.29) was observed confirming the 0orientation of H14 in the parent compound. The doubling of proton and carbonresonances, caused by the presence of two epimers, was observed in the side chain andD-ring resonances. H14, H20, H22, H23,1123', H25, H28, H28' and H29 all had a cleardoubling of resonances. The HMQC experiment (Fig. 3) on compound 24 assigned anumber of these protons to be attached to individual carbon resonances.49Table 5. 1H and 13C NMR data for contignasterol (24) recorded in DMSO-d6.C# 1H(500 MHz) COSY 13C (125 MHz) HMBCd11'2 1.70 H2',H3 23.62' 1.30 H2,H33 3.62 H2',H2,H4 68.7 H44 3.88  H3,H5 67.85 1.15 H4,H6 44.96 H196 3.30 H5,H7 70.4 H77 4.16 H6,H8 73.98 1.45 H7,H14 38.29101112ax 1.08 H12ec, 36.9 H14,H1712", 1.29 H14,H12ax13 - 41 H14,H16',H17,H1814 3.05/3.00 H8,H12ec, 50.7/50.6 H1815 - 219.4/219.3 H8,H14,H16,H16'16 2.38 H16',H17 38.6b H1716' 2.09 H16,H1717 1.85 H16,H16' 384b H16,H16',H2118 1.13,s 18.919 0.93,s 14.920 1.85/1.90 H21,H22 45.9 H18,H2121 0.88 H2O 16.8/16.7 H2O22 3.78,bt/3.22,bt H20,H23,H23 ' 75.3 H2123 0.81/0.71 H22,H23'23' 1.55/1.50 H22,H232425 1.30/1.40 H26,H2726 0.82 H25 19.2-19.627 0.82 H25 19.2-19.628 1.12/0.85 H28 ',H2928' 1.50/1.60 H28,H2929 5.16/4.50 H28,H28' 90.4/95.6OW 5.95; 5.74; 4.53; 4.34; 4.16;4.04a 1 H NMR resonances disappeared upon addition of D20b May be interchanged.c HMQC correlation to H22(6 3.22)d Proton in Carbon # column irradiated.5031 R 1 = R2 = H32 R 1 = R2 = OHStructural Novelty of ContignasterolOne of the extraordinary features of steroids as a class of natural products is thatthey have, almost without exception, a particular set of configurations at the ring junctioncarbons. Occasionally, naturally occurring steroids are isolated that have an invertedcentre, most commonly at C5, giving rise to a cis rather than trans fused AB ringsystem. 41 Contignasterol (24) is one of a very small number of naturally occurringsteroids with the 1413 proton configuration. Xestobergsterols A (31) and B (32), tworelated steroids with the cis C/D ring junction (1401), were subsequently isolated fromthe marine sponge Xestospongia bergquistia.42 NOe studies on compound (31) by Shojiet al. demonstrated that the C ring had a boat conformation. In contrast, a ROESYexperiment on tetraacetate 25 showed correlations between Me18, H8 and H14demonstrating that ring C had the standard chair conformation. Of note is the fact thatthe xestobergsterols were isolated from a sponge, Xestospongia bergquistia, in the samefamily as Petrosia contignata (see Scheme 1, p10) and thus the 15 keto functionality andthe cis C/D ring junction configuration may be chemotaxonomically significant.The biogenetic origin of the 14P proton configuration is unclear. It could bederived through epimerization at C14 within the sponge from a compound that containedthe standard steroid proton configuration (14aH). Alternatively, it might arise from a51stereospecific ring opening of an epoxide intermediate. It is unlikely that contignasterol(24) is the product of epimerization during the isolation since the extraction and isolationtechniques employed involved mild conditions. The relative stabilities of the 14a and14(3 epimers of a number of semi-synthetic 15-keto steroids have been determined 43,44The 1413 epimer was found to be the most stable in all cases. For example, at equilibrium,14a-artebufogenin (33) and its 1413 epimer (34) are present in a ratio of 35:65 43 and the14a-steroid 35 and its 1413 epimer 36 have an equilibrium ratio of 13:87. 44 An attempt toequilibrate the contignasterol epimers with base (MeOH/20% NaOH, 40 °C, 18 h) resultedin a complex mixture of degradation products. Therefore, the relative stabilities of 14aand 1413 epimers of 24 were not determined.Ac033 14 alpha^ 35 14 alpha34 14 beta 36 14 betaAnother unusual feature of contignasterol (24) is the oxygenation pattern in thering system. Steroids with a 3P OH are common in the marine environment 45 andsteroids with dihydroxylation in ring A are also well documented.46 However, steroidswith the 3,4 hydroxylation pattern, especially with the hydroxyl at C3 in an a position asfound in contignasterol (24), are unusual. In addition, the six membered hemiacetal ringin the side chain is without precedent.52Biological Activity of Contignasterol.It is well known that histamine, released from tissue mast cells upon disruption ofthese cells, plays an active role in allergies including asthma. Histamine was firstidentified in normal tissues by Best et al. in 1927. Soon after, it was shown to beassociated with anaphylatic shock. The evidence that histamine played a role in allergyand anaphylaxis mounted and by the early 1930's scientists were searching for methodsof reducing histamine release. By the 1940's antihistamines were widely used and todaythey are used to treat various allergic disorders. 47The report that the xestobergsterols,42 which are structurally similar tocontignasterol (24), are potent inhibitors of histamine release from rat mast cells inducedby anti-IgE prompted us to test contignasterol (24) in the same assay. It was found thatcontignasterol also inhibited histamine release from rat mast cells induced by anti-IgE ina dose dependent manner (Fig. 23).48 The IC50 value of contignasterol (24) on histaminerelease in mast cells activated by anti-IgE was 0.8 ± 0.3 pM (n = 5). This effect is 330times more potent than the well known antiallergic drug disodium cromoglycate (IC50 =2621.1M). 42 Contignasterol (24) did not exhibit any cytotoxic, antifungal, or antibacterialactivities. The antiallergic activity and the lack of cytotoxicity suggest that contignasterol(24) and the xestobergsterols represent interesting leads to new antiallergy drugs.538020 -0100 -'1^•^• • ..^'1^'^.. • ••`..1^'.0001 .001^.01 1 10^100^1000Contignasterol (pM)Fig. 23. Inhibitory effect of contignasterol (24) on histamine release from rat mast cells.54Petrolactone(37)Petrolactone (37) was isolated as a white solid that gave a parent ion in theHREIMS at m/z 236.1768 Da, appropriate for a molecular formula of C15H2402 (AM-0.9 mmu). The FTIR showed a carbonyl stretching frequency at 1769 cm -1characteristic of y-lactones. The structure of 37 was solved by analysis of COSY,HMQC, HMBC and difference nOe experiments in two solvents, C6D6 and CDC13(Tables 6 and 7).The 1H NMR spectrum in CDC13 (Fig. 27) exhibited downfield resonances at 84.20 and 3.94 ppm which were appropriate for the y-methylene protons of a y-lactone.The 1H NMR spectrum in C6D6 (Fig. 28) supported this assignment as the protons wereshifted dramatically upfield to 8 3.62 and 3.30 ppm from the anisotropic effect of thesolvent on the protons that are in a close spatial relationship with the carbonyl. The 1HNMR spectra also exhibited three methyl singlet resonances.Due to the low concentration of sample, only 12 of the 15 carbons were visible inthe 13C NMR spectrum (CDC13, Fig. 29). The APT spectrum (Fig. 29) revealed sixresonances appropriate for 3 methine groups in addition to the 3 methyl groups.According to the molecular formula C15H2402, 12 protons remained, which wereassigned to the 6 APT signals appropriate for methylene groups. The three carbons notvisible in the 13C NMR spectrum (Fig. 29) included the carbonyl carbon and the two551.00H H^H H^.prrr1.15^2.01 0.41carbons bearing the tertiary methyl groups. The four degrees of unsaturation in themolecule were satisfied by the lactone carbonyl and a tricyclic structure.Analysis of COSY data established an isolated spin system that consisted of thelactone methylene protons (8 3.62 and 3.30), two consecutive methine protons followedby two sets of methylene protons, and finally a methine proton (Fig. 24, Table 6). Thisspin system incorporated all of the protons in the B and C rings. The other isolated spinsystem, which consisted of the six remaining methylene protons assigned to ring A, wasapparent from analysis of the COSY, HMQC and HMBC data (Table 6).8 3.30 3.62^1.65^1.35^0.77H^H H, ,, H^HFig. 24. Spin system within rings B and C of petrolactone (37).Proton-carbon connectivities were established by analysis of HMQC data. Theresults of an HMBC experiment (Figs. 25 and 30) completed the connectivities in themolecule. HMBC correlations from the C14 proton at 8 3.62 and the C8 methine protonat 8 1.65 to the carbonyl carbon at 8 176.5 completed the y-lactone ring. HMBCcorrelations from the methyl singlet at 8 0.40 ppm to the carbons at 8 38.5(C1),36.0(C10), 55.0(C5) and 55.5(C9) (the latter two were unresolved in the HMBC)established the connectivity of the isolated spin systems through C10. The two remainingmethyl singlets were shown to be geminal by HMBC correlations from the methylprotons at 8 0.70 and 0.65 ppm into the methyl carbons at 8 21.0(C11) and 33.5(C12)ppm respectively and into the carbons at 8 55.0(C5), 42.0(C3) and 32.0(C4) ppm. Thesecorrelations also connected the other termini of the spin systems, forming the drimanering skeleton.56CH8 3.300.40^H3.62H0.6513/.............„...■•■■........,s.s.,HMBCCH3 HiThi .----Thl3.300.40 8 0.65CH303,........^ H1.65Fig. 25. Selected HMBC correlations of petrolactone (37).The relative stereochemistry of 37 was assigned on the basis of observed nOes(Fig. 26, Table 6) and COSY correlations assigned to W-coupling. COSY correlationsassigned to W-coupling between the C15 methyl protons (8 0.40) and the Cl axial proton(0.60) and between the C11 methyl protons (0.65) and the C3 axial proton (0.95)demonstrated that both of these methyls were axial to ring A. Irradiation of Me15(8 0.40)in a difference nOe experiment led to the enhancement of the C11 methyl protons (60.65) as well as enhancements in H8 (1.65) and H14 (3.30). In addition, irradiations ofMel 1, H14 and H8 in three separate experiments all led to an enhancement in the Me15protons. These data established 1,3-diaxial relationships between Mel 1 (8 0.65) andMe15 (0.40) and between Me15 (0.40) and H8 (1.65) and also required the C14methylene group to be in a (3 orientation to ring B. Such a spatial relationship can only beachieved if both the A/B and B/C ring junctions are trans fused (Fig. 26).......,,--„,,........nOeFig. 26. NOe results of petrolactone (37).57Table 6. 1 H and 13C NMR data for Petrolactone (37) recorded in C 6.C# 1 1-1(500MHz) COSY nOes a 13C(125 MHz)b HMBCc1 0.87 H1',H2,H2' 38.5 H151" 0.60 H1,H2,H2',H152 1.18 HI,H1',H2',H3' 18.52' 1.32 HI ,HI ',H2,H3,H3'3 1.25 H2',H3' 42.0 HI1,H123' 0.95 H2,H2',H3,H114 - 32.0 H11,H125 0.41 H6,H6' 55.0 H11,H12,H156 0.77 H5,H6',H7,H7' 22.06 ' 1.35 H5,H6,H7,H7'7 2.01,m H6,H6',H7',H8 26.07' 1.00 H6,H6',H7,H88 1.65,m H7,H7',H9 H14,H15 39.0 H14'9 1.15 H8,H14,H14' 55.5 H14,H14',H1510 - 36.0 H1511 0.65,s H3' H15 21.0 H1212 0.70,s H6' 33.5 Nil13 - 176.5 H8,H14'14 3.30,dd H9,H14' H14',H15 67.0 H914' 3.62,dd H9,H14 H1415 0.40,s HI" H8,H14,HI1 13.5a Proton in Carbon # column irradiated.b Carbon chemical shifts were obtained from the HMQC spectrum.c Protons correlated to carbon resonances in 13C NMR column.3758Table 7. 1H and 13C NMR data for Petrolactone (37) recorded in CDC1 .C# 1H(500MHz) 13C(75MHz) HMBCa1 1.48 38.8 H151' 1.132 1.45 18.52' 1.603 1.48 42.0 H11,H123' 1.174 - 33.0 H11,H125 0.95 55.0 H11,H12,H156 1.30 25.96' 2.217 1.80 21.5T 1.308 2.27 39.1 H7,H14'9 1.87 56.0 H14,H14'10 - 36.0 H1511 0.85,s 21.4 F11212 0.89,s 33.6 H1113 - 178.0 H14'14 3.94,dd(8.4,10.4) 68.014' 4.20,dd(6.9,8.4)15 0.96,s 14.0a Protons correlated to carbon resonances in 13C NMR column.3759g^A^4.0^ 3.0^ppm^2.0^ 1.0^ 0.0Fig. 27. 1H NMR spectrum of petrolactone (37) in CDC13 (500 MHz).r/rv1 I^I3. 8^3. 6^3. Ai^I^I^'^-I I^•^I^I^ r^I^•I^I^I^I^T^I^13.2^3.0^2.8^2.6^2.4^2.2^2.0^1.8^1.6^1.4^I . 2^1.0^.8^.6 a^. 2^0. 0PPMFig. 28. 1H NMR spectrum of petrolactone (37) in C6D6 (400 MHz).I IIII I IU11 1 1111 1 111-11 tiiiitiiiirmitrilltrirrittiiiirtiIiirptirlimplIIIIIIrp wpm!90^80^70^60^50^40^30^20^10 PPM 0Fig. 29. 13C and APT NMR spectra of petrolactone (37) (75 MHz, CDC13).sps..........■••) L_J-20H12/C1 1-30H12/C4 HI 1/C4H12/C3^HI 1/C3CP C)HI 1/C12-40WO-50H12/C5^H 1 1/C5CO 0-PP.I^I^I^i0.7 0.6 0.5Fig. 30. Selected region of 2D HMBC spectrum of petrolactone (37) (C6D6).PP. 0.4Sesquiterpenes with the drimane carbon skeleton are well known from naturalsources. Euryfuran (38), the furan corresponding to petrolactone (37), was isolated fromthe marine sponge Euryspongia sp. collected at Casa Cove, La Jolla, CA . 4 9Dihydrocinnamolide (39), isolated from the plant Porella arboris-vitae, differs frompetrolactone (37) only in the relative stereochemistry at C8. 50 Compound 39 has alsobeen identified as one of the hydrogenation products of bemarivolide (40) isolated fromthe bark of Cinnamosma fragrans. 5138^ 394064BrOR OROMe OHBrBromophenol (41)The major cytotoxic component of P. contignata was assigned as the previouslyisolated bromophenol 41 on the basis of 1 H NMR and mass spectral data compared toliterature values.52 Compound 41 was originally isolated along with related compounds42-46 by Norton et al. from specimens of the sponge Dysidea herbacea collected fromthe Great Barrier Reef and a collection site off Fiji. In an independent study thebrominated phenolic compounds 47-52 were isolated from D. herbacea collected off theCaroline Islands in the Pacific Ocean. The presence of related compounds as indicatedby the 1 H NMR spectrum of some of the crude fractions of P. contignata was consistentwith the previous studies.41 R = H^43 R = H42 R = Me 44 R = MeOH^OH5'4647 3,4,5,2',4'-pentabromo48 4,5,6,2',4'-pentabromo49 3,5,2',4'-tetrabromo50 4,6,4'-tribromo51 3,4'-dibromo52 6,4'-dibromoOMe^OMe65B.^Terpenoid Metabolites Isolated from the Northeastern Pacific MarineSponge Acanthella sp. IntroductionThis chapter describes a study of the chemistry of a Northeastern Pacific sponge,Acanthella sp. Specimens of the sponge were acquired as part of a general collectionexpedition to the Queen Charlotte Islands, an area that had been relatively unstudied bymarine natural products chemists.1) Taxonomy and Physical Characteristics of Acanthella sp. The genus Acanthella is found in the family Axinellidae which is in the orderAxinellida (Scheme 4). 53 As is usual with sponges of the order Axinellida, Acanthellaspecies have rough surfaces with projecting spicules. 54 Species of Acanthella are foundthroughout the world in both tropical and temperate waters. The Acanthella studied inthis work, which apparently represents a new species according to Austin, 55 can be foundat various sites along the west coast of the Queen Charlotte Islands. The specimens ofAcanthella sp. used in this study were found in patches of up to 40 cm in diameter onrock walls at depths from 5 to 15 meters in semi-exposed areas in Rennell Sound, QueenCharlotte Islands. This orange coloured sponge had an irregular shape with a roughsurface and definite raised ridged oscula.66Scheme 4: The position ofAcanthella sp. within the phylogenetic classification of thePorifera according to BergquistPPhylum: PoriferaClass:^Calcarea^Hexactinellida^Demospongiae^SclerospongiaeSubclass:^Homoscleromorpha Tetratinomorpha CeratinomorphaOrder: Choristida Spirophorida Lithistida Axinel ida HadromeridaFamily:Genus: Axinella CeratopsionPtilocaulis PararaphoxyaAxinellidae1Acanthella Phakellia AulettaPseudaxinella BubarisHomaxinellaSpecies:^ Acanthella sp.671:.R56 R = NC57 R = NCS2) Secondary Metabolites Previously Isolated from Sponges of the Genus Acanthella. Previous chemical studies on sponges of the genus Acanthella have yielded anumber of functionalized terpenoids that appear to have chemotaxanomic significance. 56Isonitriles, which are very rare in nature, are produced by species of Acanthella as well asby sponges of the genus Axinella which is in the same family as Acanthella. Acanthellin-1 (53), isolated by Minale et al. from Acanthella acuta collected in the Bay of Naples,was the first isonitrile to be isolated from an Acanthella sponge.57 Compound 53, whichhas a 4-epi-eudesmane carbon skeleton, exhibits antimicrobial activity. Subsequently,Ciminello et al. identified the related compounds 54 and 55 from A. acuta collected inthe Bay of Naples58 and later work by Mayol et al. yielded the additional isonitrile-isothiocyanate pairs, 56-61.59 All of the above structures were solved by spectroscopicanalysis and chemical interconversions.=R54 R = NC55 R = NCS58 R = NC59 R = NCS68The largest group of compounds to be isolated from sponges of the genusAcanthella is a class of functionalized, biologically active diterpenoids, known as thekalihinols. The first of these compounds, kalihinol-A (62), was isolated by Chang et al.R 1^R2 R3 R4 R5 R1^R2 R3 R4 R5^R662 NC CH3 NC Cl H 63 OH CH3 NC H CH3^Cl66 NC CH3 NC H Cl 64 OH CH3 NC H =CH270 CH3 NCS NC H Cl 65 OH CH3 Cl H CH3 NC71 =CH2 NC H Cl 67 OH CH3 NC H CH3 NC72 CH3 NC NC H Cl 68 OH CH3 NC H CH3 NCS74 CH3 NCS NHCHO H Cl 69 10-NCS-F (67)75 CH3 NCS^NCS H Cl 73 CH3 NC^H^OH CH3 NCfrom a species of Acanthella collected in Apra Harbor, Guam. 60 Subsequentinvestigations by Chang et al. yielded kalihinols B(63), C(64), D(65), E(66), F(67), G(68)and H(69) from the same species collected off Guam as well as kalihinols X(70), Y(71)and Z(72) from an unidentified species of Acanthella collected off Fiji.61,62 Studies on aFijian collection of Acanthella carvenosa by Omar et al. yielded 62, 67 and 70 in additionto a new compound, isokalihinol F (73). 63 The same sponge, A. cavernosa, collected off69Thailand, yielded 70 and 71 as well as two new kalihinols, J (74) and I (75). 64 Finally,Fusetani et al. isolated kalihinene (76) and isokalihinol B (77) from A. klethra collectedoff Kuchinoerabu Island of the Satsuna Archipelago, Japan. 65 Biological activities werereported for each of the kalihinols with the exception of isokalihinol F (73). Theseinclude anthelmintic, cytotoxic and antibacterial activity.76^ 77In addition to the terpenoids, two guanidine containing metabolites have beenisolated from two different species of Acanthella. Compound 78, isolated from the RedSea sponge A. aurantiaca by Cimino et al., was solved by X-ray diffraction analysis. 66Compound 79, isolated from A. carteri collected off the coast of Madagascar, was alsosolved by X-ray diffraction analysis. 67078^ 7970Results and Discussion1) Isolation of terpenoid metabolites from Acanthella sp. Specimens of Acanthella sp. were collected by hand using SCUBA off ConeheadPoint in Rennell Sound. The frozen sponge (6.1 kg wet weight) was transported toVancouver, immersed in methanol (8L) and soaked at room temperature for 72 h. Thedecanted methanol was concentrated in vacuo to yield an aqueous suspension which waspartitioned against hexanes (2 X 1L), chloroform (2 X 1L), and ethyl acetate (2 X 1L).The 1 H NMR spectrum of the crude chloroform extract revealed the presence of theknown metabolite 90 which had previously been isolated in our laboratory. 68 The 1 HNMR spectrum of the crude hexanes extract revealed the presence of the isothiocyanate82 and the isonitrile 85 in the same series as 90. Initial purification of the hexanes extractwas performed using normal phase flash chromatography with a step gradient elutionfrom 100% hexanes to 100% ethyl acetate (in 25% increments). The fourth and fifthfractions were combined to yield four main fractions. The presence of several volatileterpenes was evident from the 1H NMR spectrum of the first fraction. Purification of thesecond fraction using normal phase HPLC (100% hexane) yielded pure acanthene A (80).Consecutive applications of normal phase HPLC (5% EtOAc/Hex and 100% Hex) on thethird fraction yielded in order of elution the known compounds 81, 82, 83, 84 and 85,novel metabolites acanthene G (86) and acanthene H (87), the algal metabolite violacene(88) and finally the novel carbamate 89. The fourth fraction contained the more polarformamide 90.71I Aqueous layer IPartitioned withchloroformiOrganicExtractimpure 90IAqueouslayerScheme 5: Isolation of metabolites from Acanthella sp.I Sponge (wet weight 6.1 kg)1)aqueous Me0H extract2) decanted and evaporated in vacuoI Aqueous suspension IPartitioned with hexanesOrganic Extract IFlash Silica gel Chrom.Step gradient Hex-Et0AcIlrB^C^DVolatile Formamide 90terpenes normal phase normal phaseHPLC HPLC100% Hex 19:1 Hex/EtOAcAcanthene80Anormal phaseHPLC100% HexCompounds 81-89722) Structure Elucidation of terpenoid metabolites from Acanthella sp. H -R80 R = Cl86 R = NCS 82 R = NCS85 R = NC89 R = NHCO2CH390 R = NHCHOThe structures of acanthenes A(80), G(86), H(87) and J(89) were solved byextensive 2D NMR and mass spectrometric analysis. Proton-carbon connectivities weredetermined by HMQC experiments and proton spin systems were determined by COSYexperiments. Final connectivities were based on HMBC results. Relativestereochemistries were determined using nOe data and supported by COSY correlationsattributed to W-coupling. Support for the relative stereochemistries was found in carbonchemical shift data. The structures of the previously identified compounds 81, 82, 83, 84,85, and 88 were assigned by comparison of 1H NMR, 13C NMR and mass spectral datawith literature values. Compound 90 was identified by comparison of 1 H NMR data tothat of an authentic sample isolated from the nudibranch Cadlina leuteomarginatacollected in Rennel Sound.697383 R = NCS84 R = NC88Cl74Acanthene A1580Acanthene A (80) was obtained as a colourless oil. The presence of an isotopecluster in the LREIMS (Fig. 33) at m/z 242 (rel. int. 6) and 240 (rel. int. 18) as well as afragment ion at m/z 204 (C15H24+, AM 0.8 mmu) assigned to the loss of HC1 indicatedthe presence of a chlorine atom in the parent ion. The HREIMS gave peaks at m/z242.1616 Da and m/z 240.1652 Da, corresponding to molecular formulae of C15H25 37C1(AM 0.1 mmu) and C15H25 35C1 (AM 0.8 mmu), which required three degrees ofunsaturation in the molecule.Initially, the one dimensional 1 H NMR, COSY and nOe experiments on 80 werecarried out in CDC13 (Table 8). However, it was found that recording the 1 H NMRspectrum in C6D6 (Fig. 31) gave much better dispersion in the upfield region andtherefore more definitive assignments. The 1H NMR spectrum (C6D6, Fig. 31) containedtwo broad singlets at 5 5.07 and 4.85 ppm integrating for one proton each, suggesting thepresence of an olefinic methylene. A triplet at 5 3.97 ppm integrating for one proton wasassigned to a secondary chloride functionality. An isopropyl moiety was evident from amultiplet at 5 2.64 ppm that was coupled to two methyl doublets (5 0.77 and 0.87). Atertiary methyl at 5 0.63 ppm was also present.75Table 8. 1 H and 13C NMR data for acanthene A (80)recorded in CDC1 .C# 1 H(400 MHz) COSY nOes1 not observed2 1.61 H2',H3,H3'2' 1.40 H2,H33 2.35,m H2,H2 ',H3 ' H14'3' 1.99,m H2,H3,H14' H34 - -5 2.17,d H6,H14,H14' H76 3.95,t H5,H7 H12,H14,H157 1.60 H6,H118 not observed9 not observed10 - -11 2.48,m H7,H12,H13 H12,H1312 0.84,d H11 H6,H1113 , 0.95,d H11 H1114 4.65,s H5,H14' H6,H14'14' 5.01,s H3',H5,H14 H1415 0.73,s H61513128076Table 9. 1 H and 13C NMR data for acanthene A (80)recorded in C6D6.C# 1H(400 MHz) COSY nOes 13C(125 MHz) HMBC1 1.20 H2',H3 42.2 H151'  1.08 H2,H152 1.43 H1',H2',H3' 24.32' 1.52 H1,H2,H3,H3 'a3 2.24,m HI ,H2',H3" 38.2 H14,H14'3' 1.87,dt(6.1,12.8) H2,H2'a,H3,H14'4 - - 145.9a5 _2.11,d(10.9) H6,H14,H14' 58.9 H14a,H14',H156 3.97,t(10.9) H5,H7 H12,H14,H15 62.07 1.59 H6,H8,H8' 51.6 H12,H138 1.10 H7,H8' 19.78' 1.29 H7,H89 40.2b H1510 - - 38.9a•b H1511 2.64,m H12,H13 27.5 H12,H1312 0.77,d(7.0) H11 21.3 H1313 0.87,d(7.0) H11 15.3 H1214 4.85,s H5,H14' 109.014' 5.07,s H3',H5,H1415 0.63,s HI' H6 17.1a weakb may be interchanged1513128077 iiCIICI)/PPM^7^6^5^4^3^2^1Fig. 31. 1H NMR spectrum of acanthene A (80) (500 MHz, C6D6).I130^120^110^100^90^80^70PPM^60^50^40^30^20^10Fig. 32. 13C NMR spectrum of acanthene A (80) (100 MHz, C6D6).115161131Ill4156 671A!71  1^111 [1t1`11/ri l le 11,1 ^1 1*1 1, IlvTlit^1 1 1 vt, 1 1 1 11 ^ i °41C-^ I ^ 1 1 1 ^1611^211 221 2411 261 211 3111122621161611,69616766666463621II611691111176616641/3621II6137136.• MC.1692764, 161/4■697641 (11971191491 1 I 1 . 1 I I 1 „A l l I I I„ 1 1 1 1  k OW kAk tll I I k iiIIII 1 1 01 1 1 1 1I lardt 1 1 1 1 II PP! iirri,1411^66^1111^116^121^III^161Fig. 33. Low resolution EI mass spectrum of acanthene A (80).The 13C NMR spectrum recorded in either C6D6 (Fig. 32) or CDC13 contained 13observable resonances attributable to compound 80. The carbon resonance at 8 109.0ppm and the corresponding proton resonances at 8 5.07 and 4.85 ppm, assigned to anolefinic methylene, were the only indications of unsaturated functionality. Theremaining two sites of unsaturation required by the molecular formula (C15H25C1 dbe 3)were attributed to a bicyclic structure.A detailed analysis of the COSY, HMQC, and HMBC data recorded in C6D6(Table 9) confirmed the presence of an olefinic methylene and identified two otherisolated 1 H NMR spin systems. The first spin system contained resonances at 81.20(H1), 1.08(H1'), 1.43(H2), 1.52(H2'), 2.24(H3), and 1.87(H3') ppm assigned to threecontiguous methylene groups. The H3'(8 1.87) proton showed allylic coupling to theH14'(5.07) olefinic methylene proton (Fig. 34). The second spin system contained theresonance at 8 2.11 ppm assigned to the ring junction proton H5 which showed a COSYcorrelation to the proton at 8 3.97 (H6). The chemical shifts of H6 and C6 ( 1 H: 8 3.97,13C: 8 62.0) indicated that the chlorine atom was attached to the C6 carbon. The ringjunction methine proton H5(2.11) also showed allylic coupling to the olefinic methyleneprotons (Fig. 34: COSY correlations between 8 5.07(H14') and 2.11(H5) and between 84.85(H15) and 2.11(H5)). The assignments of proton resonances H7(6 1.59), H8(1.10),H8'(1.29), H11(2.64), H12(0.77) and H13(0.87) in the second spin system were based onthe results of a COSY experiment carried out in C6D6 (Table 9) and the H7/H11 couplingwas supported by the data from a COSY experiment carried out in CDC13 (Table 8). InC6D6 no correlation was observed between the isopropyl methine proton at 8 2.64(H11)and the methine proton at 6 1.59(H7). However, the correlation was observed in theCOSY spectrum recorded in CDC13 likely due to an increased concentration of thesample. The protons of the remaining methylene group at C9 were not resolved due tothe complexity of the upfield region of the 1H NMR spectrum (Fig. 31).81Allylic couplingCOSY correlationFig. 34. Allylic coupling present in COSY spectrum of Acanthene A (80)Data from an HMBC experiment (Figs. 35 and 36) assigned the positions of theremaining methyl group Me15 (8 0.63), the quaternary carbon C10 and the remainingmethylene carbon C9. The data included HMBC correlations from the Me15 protons tothe carbon resonances at 8 40.2 and 38.9 ppm (Fig. 35) assigning them to the C9 and C10positions. These correlations in conjunction with HMBC correlations from Me15 (80.63) to Cl (8 42.2) and C5 (58.9) (Fig. 35) attached Cl, C5, and C9 to C10 and resultedin two fused six-membered rings. These assignments completed the constitution ofacanthene A.5 0.63----"-.. kHMBC correlationFig. 35. HMBC correlations in acanthene A (80)Finally, the relative stereochemistry of the molecule was established through aseries of nOe experiments and coupling constants. A trans fused ring system was evidentfrom the coupling constant of the H6 proton (8 3.97, t, J = 10.9 Hz) in combination with82I I I I0.7^0.6pm 0.9^0.9Lowasull•■■■•••■g# ‘j'^a^Fig. 36. Selected region of HMBC spectrum of acanthene A (80) (C6D6).cco==xt•...03==os••■cl:C=12,^cie=s2-30■Tallts•H1 5/C 10ns==ft-H 15/C9 42 :1'•H1 5/C 1-401=901.^40III=N*H 1 5/C5 c= ,....../^----..,....,.nOeFig. 37. NOe results for acanthene A (80)an nOe enhancement in Me15 upon irradiation of H6 (Fig. 37). These data revealed atrans diaxial relationship between 115 and H6 and between H6 and H7 and also a 1,3-diaxial relationship between Me15 and H6. Such a spatial relationship can only beachieved in a trans fused system. In addition, irradiation of H6 induced an nOeenhancement in H14 and irradiation of H14 in turn induced an nOe enhancement in H6(Fig. 37). These data required C4 to be in a 13 orientation to ring B which in conjunctionwith the Me15/1-16 1,3-diaxial relationship supported a trans fused ring system. The transdiaxial relationship between H6 and H7 placed the isopropyl in a p orientation. This wassupported by an nOe enhancement in H6 upon irradiation of the Me12 resonance (Fig.37). Finally, a COSY correlation assigned to W-coupling between Me15 (8 0.63) andH1' (1.08) supported the positioning of Me15 axial to ring A (Fig. 38).HFig. 38. COSY correlation assigned to W-coupling in acanthene A (80)84Acanthene G15131286Acanthene G (86) was obtained as an optically active white solid Gab - 34° (c0.18 CHC13)). The HREIMS gave a parent ion at m/z 263.1714 Da corresponding to themolecular formula C16H25NS (AM 0.1 mmu), which requires 5 degrees of unsaturation.A fragment ion at m/z 204 (Ci5H24+ AM -0.3) resulting from loss of HNCS indicated thepresence of an isothiocyanate or thiocyanate functionality. The presence of a broad andintense IR stretch at 2080-2180 cm -1 (with two absorption maxima at 2103 and 2165cm -1 ) suggested an isothiocyanate rather than a thiocyanate functionality. Thiocyanatestypically show sharp stretching bands of medium intensity at 2150 cm - 1 .67The 1H NMR spectrum of 86 (Fig. 39) was in most respects very similar to the 1HNMR spectrum of 80 (in CDC13, Table 8). The most notable difference was the chemicalshift of the resonance assigned to the H6 methine which appeared at 5 3.95 ppm in 80 andat 8 3.57 ppm in 86. The spectrum of 86 (Fig. 39) also contained two singlets at 55.02(1H) and 4.61(1H) ppm indicating the presence of an olefinic methylene. Theisopropyl moiety was identifiable from the presence of a multiplet at 5 2.17(1H) ppmcoupled to two upfield methyl doublets at 8 0.87 (H12) and 0.98 (H13) ppm. These datasuggested that acanthenes A (80) and G (86) were identical except for the functionalgroups attached to C6.85Table 10. 1 H and 13C NMR data for acanthene G (86) recorded in CDC13.C# 1 H(500MHz) COSY nOes 13C(75MHz) HMBC1 1.42 H2,H2',H3 42.0 H15l' 1.30  H2,H2'2 1.54 H1,H1',H3,H3' 23.9 H3'2' 1.60 H1,H1',H3,H3'3 2.36,m H1,H2,H2',H3' H3 ',H14 ' 37.5 H1",H14,H14'3' 1.99,dt(5.8,13.0) H2,H2',H3,H14' H34 - - 146.0 H3',H55 2.06,d(10.8) H6,H14,H14' 56.3 H6,H9,H14,H14',H156  3.57,t(10.8) H5,H7 H12,H14,H15 56.5 H57 1.55 H6 49.8 H6,H9,H12,H138 1.54 18.5 H118' 1.25. 9 1.50 39.8 H159' 1.2310 - - 37.6 H1511 2.17,m H12,H13 H12,H13 27.7 H12,H1312 0.87,d(7.0) H11 H6,H11 16.0 H11,H1313 0.98,d(7.0) H11 H11 21.0 H11,H1214 4.61,s H5,H14' H6,H14' 108.0 H3',H514' 5.02,s H3',H5,H14 H3,H1415 0.71,s H1 'a, H9'a H6 17.0 HI 'a,H5,H9 'a16 - - not observeda H1' and H9' are unresolved in the COSY and HMBC1513128686fbyHi NCS7.e^6.e^5. II^4.1^3. II^2.1^I II^.IIPPMFig. 39. IH NMR spectrum of acanthene G (86) (400 MHz, CDC13).- "I - r r^t ---r-r--/^r r^ -^r r • .f - -I' 7 7'140 120 100 80'vim,^■Irwir I I60^40^20 PP"Fig. 40. 13C and APT NMR spectra of acanthene G (86) (75 MHz, CDC13). Fig. 41.41. 2D HMQC spectrum of acanthene G (86) (CDC13)8 0.71HMBC correlationsA detailed analysis of the COSY, HMQC and HMBC data (Table 10) collected on86 confirmed the relationship between acanthene A and G. For example, the two spinssystems connected through allylic coupling to the olefinic methylene found in 80 werealso present in 86. The lack of dispersion in the upfield region in the 1 H NMR spectrumof 86 (Fig. 39) made the COSY spectrum difficult to interpret. Therefore, HMQC (Fig.41) and HMBC experiments were used to make most proton assignments. The results aresummarized in Figures 41 and 42 and Table 10.Fig. 42. HMBC correlations in Acanthene G (86)The relative stereochemistry of acanthene G (86) was also established by analysisof difference nOe (Figs. 43 and 45, Table 10), coupling constant, and COSY data. Apair of difference nOe experiments (irradiation of Me 15(8 0.71) led to an nOeenhancement in H6(3.57) and irradiation of H6(5 3.57) led to an nOe enhancement inMe15(0.71)) established the 1,3-diaxial relationship between the C15 methyl and the H6methine (Figs. 43 and 45). As in acanthene A, the Me15/H6 1,3-diaxial relationship inconjunction with the large coupling constant of H6 (10.8 Hz) showed that the rings weretrans fused. A strong nOe from the olefinic proton at 8 4.61(H14) to the proton at 53.57(H6), which required C4 to be in a p orientation to ring B, was consistent with a transfused ring system (Fig. 43). The H6/H7 coupling constant (10.8 Hz) and an nOeenhancement in the H6 methine upon irradiation of the C12 methyl doublet (5 0.87)90showed that the isopropyl group was also in the 13 position (Fig. 43). Finally, the methylsinglet at 8 0.71 ppm showed a broad COSY correlation to the resonances between 8 1.20and 1.33 (8 1.23 (H9ax ) and 1.30 (H l ax )). These correlations were assigned to W-coupling which supported the placement of Me15 in an axial orientation to both rings.nOeFig. 43. NOe results for acanthene G (86)Further support for an isothiocyanate rather than a thiocyanate functionality camefrom the chemical shift of the H6 proton. Literature examples revealed that a protonsassociated with secondary isothiocyanates typically have chemical shifts between 5 3.46and 4.09 ppin,58,59,70,71 whereas those associated with thiocyanates have chemical shiftsranging from 5 2.99 to 3.43 ppm (Fig. 44). 72,73 One anomaly was observed where thechemical shift of an a methine of an isothiocyanate had a chemical shift at 5 3.32 ppm.However, this could possibly be due to an anisotropic shielding effect from a double bondin the molecule. The a methine (H6) in acanthene G (86) at 5 3.57 ppm was consistentwith an isothiocyanate functionality. Unfortunately, not enough material was available toperform chemical degradations which could have provided additional support for theisothiocyanate functionality.91SCNH3.654.091/111-13.43SCNFig. 44. Chemical shifts of protons associated with thiocyanates and isothiocyanates.92H15 irradiated H6H12 irradiated 1-16H6 irradiated^H14^H12^H1511-^ .......44^H14' n^H14 irradiated 1 H6^---------..-4----A.-",.........4,^H14' irradiated H14I-114'^F114I^ ,^111-1`^1^IV'5.0^ 4.0^ 3.0 2.0 1.0Fig. 45. NOe results of acanthene G (86) (CDC13).Acanthene H15131287Acanthene H (87) was obtained as a colourless oil that showed a broad andintense isothiocyanate band at 2073 cm -1 in the FTIR. The HREIMS of 87 showed aparent ion at m/z 263.1715 Da, appropriate for a molecular formula of C16H25NS (AM0.8 mmu) and a fragment ion at m/z 204.1881 (C15H24+ AM 0.4 mmu) resulting fromloss of HNCS. The 1H NMR spectrum of 87 (Fig. 46) contained a broad singlet at 5 4.73ppm integrating for two protons as well as three methyl singlets. The 13C NMR and APTspectra (Fig. 47) contained 15 resonances, 10 of which were appropriate for methyleneand quaternary carbons and 5 of which were appropriate for methyl and methine carbons.The presence of three methyls in the 1 H NMR spectrum (Fig. 46) required that twomethines be present in the molecule. Therefore, seven methylenes and four quaternarycarbons were required to account for the remaining fourteen protons and eleven carbonsin the molecular formula C16H25NS.The chemical shifts of the methyl groups provided a good indication of thepositioning of the functionalities in the molecule. The downfield shift of the methylsinglet at 5 1.32 ppm indicated that it was attached to the carbon bearing theisothiocyanate functionality. An HMBC correlation from the Me14 protons to the carbonC4 (5 65.3) supported the assignment. The methyl singlet at 5 1.77 ppm had anappropriate chemical shift for an olefinic methyl. The methyl resonances at 5 1.32 and94Table 11. 1 H and 13C NMR data for acanthene H (87)recorded in  CDC13.C# 1 H(500MHz) COSY nOes 13C(75MHz) HMBC1 1.42 H1',H2',H3 40.3 H151' 1.12,dt(4.7,12.6) H1,H2',H152 1.30 H3 26.8a2' 1.56 H1,H3,H3'3 2.02 H1,H2,H2',H3' 42.0 H143' 1.75 H2',H34 - - 65.3 H5,H145 1.52 H6,H6' 53.1 H14,H156 1.28 H5,H6' 27.66' 1.79 H5,H6,H77 1.99 H6' H12,H12' 46.0 H12,H12',H138 1.58 H9,H9' 18.9a8'9 1.43 H8,H9' 44.7 H159' 1.22 H8,H9,H1510 - - 34.6 H1511 - - 149.9 H1312 4.73,s H13 H13 108.6 H1312' 4.73,s H13 H1313 1.77,s H12,H12' H7,H12,H12' 21.1 H12,H12'14 1.32,s H3,H15 22.015 0.90,s H1 ',H9' H14 19.016 not observeda may be interchanged1513128795MrINCASaPPM^i i 5^ 4 3^ 2IFig. 46. 1H NMR spectrum of acanthene H (87) (500 MHz, CDC13).i•0 140^1 01 1 0^80^60^40^20 PPM^0Fig. 47. 13C and APT NMR spectra of acanthene H (87) (75 MHz, CDC13).1.77 ppm were similar to those found in the known compounds 82 (Me14: 5 1.42 ppm)and 91 (Me13: 8 1.77), respectively. 10,70 HMBC correlations from the Me13 protons (61.77) to the olefinic carbons at 8 108.6 and 149.9 ppm (Fig. 48) as well as allyliccoupling observed in the COSY spectrum between the Me13 protons (8 1.77) and theolefinic protons at 6 4.73 ppm confirmed the presence of an olefinic methyl. The APT(Fig. 47) and HMQC experiments revealed that C12 (6 108.6) was a methylene carbonbearing the two protons at 6 4.73. The absence of unsaturated functionalities, other thanthe olefinic and isothiocyanate groups, required a bicyclic structure to make up the 5degrees of unsaturation in the molecule.82^ 91A detailed analysis of the COSY, HMQC and HMBC spectra (Table 11) revealedtwo independent spin systems joined at one end by the functionalized quaternary carbonat 8 65.3 ppm and at the other end by a quaternary ring junction carbon bearing an axialmethyl group. HMBC correlations from the protons at 8 4.73 and the methyl at 6 1.77 toC7(6 46.0) established the connectivity of the propenyl system (Fig. 48). The ringstructure became clear from HMBC correlations from the methyl singlet at 8 0.90(Me15) to carbons Cl (6 40.3), C5 (53.1) and C9 (44.7) (Fig. 48). HMBC correlationsfrom the Me14 (6 1.32) protons to C3 (42.0), C4 (65.3) and C5 (53.1) established thefinal connectivity in ring A (Fig. 48).981.77CH346.0 4H ^HSCN1-1^ 1.99 H.,^12 CZ 4.73^■,■._/------.HMBC correlationFig. 48. HMBC correlations in acanthene H (87)As with acanthenes A and G, the relative stereochemistry was established throughanalysis of nOe experiments (Table 11) and COSY data. Irradiation of the Me15 protonsinduced enhancement in the methyl singlet at 8 1.32 ppm (Me14) and established a 1,3diaxial relationship between Me14 and Me15 (Fig. 49). COSY correlations assigned toW-coupling from the 6 0.90 methyl singlet (Me15) to the axial protons 1-11'(6 1.12) andH9'(1.22) required Me15 to be axial to both rings and showed that the ring system wastrans fused. The relative stereochemistry of the unsaturated isopropyl group was not asclear. The 1H NMR spectrum of 87 at 500 MHz (Fig. 46) showed H7 to be a partiallyresolved multiplet at 6 1.99 ppm. The multiplet appeared to be a triplet of triplets withone large coupling and one small coupling. This data was consistent with an axial protonwhich has two trans diaxial and two axial-equatorial relationships with the four adjacentprotons and suggested that H7 had the a orientation.Fig. 49. NOe results for acanthene H (87)nOe99Subsequently, Konig et al. reported the isolation of acanthene H (87) fromAcanthella klethra collected on the Great Barrier Reef, Australia. 74 The structure wasdetermined by analysis of spectral data and the structure of the C7 isomer 92, which wasisolated along with 87, was confirmed by X-ray analysis. The 1 H and 13C NMR data ofacanthene H (87) obtained in our study was identical to that reported for the compoundisolated from A. klethra.87^ 92100Acanthene J1589Acanthene J (89) was the last compound to be eluted from the HPLC duringpurification of crude fraction C. Compound 89 was isolated as an optically active whitesolid gab + 24° (c 0.64 CHC13)) that gave a parent ion in the HREIMS at m/z 279.2193Da, appropriate for a molecular formula of C17H29NO2 (AM -0.5 mmu). A strongfragment ion at m/z 204 (C15H24+, AM - 0.4 mmu) in the LREIMS was consistent withthe loss of a functional group and a proton consisting of C2H5NO2. The FTIR spectrumcontained a strong carbonyl stretching frequency at 1732 cm -1 which is typical of acarbamate functionality.The 1 H NMR spectrum of 89 (Fig. 50) contained several resolved resonancesincluding two broad singlets at 8 4.60 and 3.59 ppm integrating for one and three protonsrespectively. The chemical shifts as well as the coupling constants of two upfieldmethine resonances at 8 0.43 and 0.65 ppm were very similar to the cyclopropyl protonsfound in compound 82 and 85. The presence of four tertiary methyls at 8 0.92, 0.94, 1.04and 1.35 ppm in 89 supported a structure similar to 82 and 85.A detailed analysis of the COSY spectrum (Fig 52, Table 12) revealed twoindependent spin systems similar to those found in 80, 86, and 87. Three contiguous101C# 1 H(500MHz) COSY noes 13C(75MHz) HMBC1 1.33 H1 ',H2,H2',H3 39.5 H151' 1.01 HI ,H2,H2'2 1.57 H1,H1',H3,H3' 19.22' 1.57 H1,H1',H33 2.26,bd(12.5) HI ,H2,H2',H3' H2,H2',H3',H14 38.5 H143 - 1.72 H2,H2',H34 56.1 1-16,H145 1.20 H6 46.1 H9,H14,H156 0.43,dd(6.8,9.1) H5,H7 20.3 H5,H8,H12,H137 0.65,t(9.1) H6,H8' 19.5 H8,H8 ',H9,H9 '8 1.52 H8 ',H9,H9 ' 15.48' 1.80,m H7,H8,H9,H9'9 1.13,dd(8.3,13) H8,H8',H9' 41.9 H7,H8,H8',H159' 0.79,dt(7.5,13) H8,H8',H9,H1510 32.7 H8,H1511 17.7 H8,H8',H12,H1312 1.04,s H6,H7 29.0 H6,H7,H1313 0.94,s 15.5 H12,H6a,H7a14 1.35,s H3,H6,H15 19.915 0.92,s H9' 19.316 153 H1717 3.59,s(3H) 51.2NH 4.60,bs H5,H6,H 14a weak15140=(^12OCH389E .., ENH171^1,,,r_ 13Table 12. 1 H and 13C NMR data for acanthene J (89)recorded in CDC13.1024.5^4.0^3.3^3.0^2.5^2.0^1.5^1.0^0.5^0.0Fig. 50. 1H NMR spectrum of acanthene J (89) (500 MHz, CDC13).1100^80^60^40^20 PPMIII I I IIT11111111-111111111111 -111111111111I111111110Fig. 51. 13C and APT NMR spectra of acanthene J (89) (75 MHz, CDC13).2.4^2.2^2.0^1.8^1.6^1.4^1.2^1.0^0.8^0.6^0.4^PPMPPMFig. 52. Selected region of COSY spectrum of acanthene J (89) (400 MHz, CDC13).methylene groups contained the resonances assigned to H1(8 1.33), H1'(1.01), H2(1.57),H2'(1.57), H3(2.26), and H3'(1.72) by the COSY experiment (Fig. 52). The COSYexperiment also showed that the second spin system contained three contiguous methineprotons followed by two sets of methylene protons assigned to H5(1.20), H6(0.43),H7(0.65), H8(1.52), H8'(1.80), H9(1.13) and H9'(0.79) (Fig. 52). Support for thegeminal dimethyl system at C11 was found in HMBC correlations (Fig. 54) from theMe12 and Me13 protons (8 1.04 and 0.94) into carbon C11(8 17.7). In addition, protonsH6(0.43) and H7(0.65) both gave HMBC correlations into C12 confirming the presenceof the cyclopropyl residue (Fig. 53). The methyl at 8 0.92 ppm was placed at the ringjunction position based on HMBC correlations from the Me15 protons to C1(39.5),C5(46.1), C9(41.9) and C10(32.7). The quaternary carbon bearing the Me14 and thefunctional group was placed in the same position as in 87 based on HMBC correlationsfrom the C14 methyl protons to C3(38.5), C4(56.1) and C5(46.1).,.----^---..,,H JO . I 0.65^HMBC correlations38.5^46.1HNH (31^H^15.5H3H3C0Fig. 53. HMBC correlations for Acanthene J (89)The relative stereochemistry was again based on a series of nOe experiments (Fig.55, Table 12). Irradiation of the methyl singlet at 5 1.35 ppm (Me14) induced an nOeenhancement in the Me15 protons thereby establishing the Me14/Me15 1,3-diaxialrelationship (Fig. 55). Independent irradiations of the C14 methyl and the NH proton106PPE.^.PPE• i^•1.5• I^•1.0-10-204PCPCMCP40.^CD^<11-^4toco^4C1 4.Ovt) CID-30.-40C CD 410-50ft*-60INDFig. 54. Selected region of HMBC spectrum of acanthene J (89) (CDC13).1 '35H3C '^CH31.04H3C0H 1.20both induced an nOe enhancement in H6 which in conjunction with the Me14/Me15 1,3diaxial relationship established a trans fused ring system (Fig. 55). The couplingconstants observed in the H6 resonance (J = 6.8, 9.1 Hz) suggested a trans diaxialrelationship between H5 and H6. This stereochemistry was consistent with that of theisothiocyanate 82 and the isonitrile 85. The Me12 group (8 1.04) was then assigned tothe 13 position based on nOe enhancements in H6 and H7 upon irradiation of the C12methyl protons (Fig. 55).8 0.92CH32.26 H^ 0.65^nOeFig. 55. NOe results for Acanthene J (89)Acanthene J (89) is likely an isolation artifact due to extraction of the sponge withmethanol. If this is the case, the precursor would have to contain a functional group withthe ability to react with Me0H under aqueous conditions at room temperature to produce89. One possible precursor is the major metabolite 82 which might react with methanoland water to yield 89. An attempt was made to convert 82 to 89 by stirring in aqueousmethanol for 3 days (MeOH/H2O/acetic acid, pH 4, 40°) but 82 remained unreacted(Scheme 6). Another possible precursor is an isocyanate. There is literature precedentfor isocyanate functionalities being isolated along with isonitriles and isothiocyanates ofthe same carbon skeleton. 75 Unfortunately, attempts to isolate the isocyanate throughextraction with CH2C12 were unsuccessful.10882 89E^••., :z ,IN HCO1CH11''""'"W-^-...Scheme 6. Attempted solvolysis of isothiocyanate 821093) Known compounds isolated from Acanthella sp.Several previously identified compounds were isolated along with acanthenes A,G, H and J. A total of seven known compounds were identified in Acanthella sp. bycomparison of 1 H NMR (see appendix A, Figs. 75-81), 13C NMR, IR and mass spectraldata to literature values.The first known compound to be isolated from the sponge was the formamide 90which had been previously isolated in our lab from specimens of the nudibranch Cadlinaluteomarginata collected very near the sponge collection site. 68 In fact, it was laterdiscovered that the nudibranch also contained compounds 82 and 85 which had beenisolated from the sponge. It has been well documented that nudibranchs selectivelysequester defense chemicals from their diets. Thompson et al. initially identifiedcompounds 82 and 85 from Cadlina luteomarginata collected at Scripps Canyon, LaJolla, California and showed that these compounds exhibited antifeedant activity. 10 Fromanalysis of the gut content of the nudibranchs, Thompson et al. concluded that thenudibranchs had concentrated the selected metabolites from their diet.82 R = NCS85 R = NC90 R = NHCHOIn addition to the above series, there were four other known compounds including83 and 84 that were found to be metabolites of Acanthella sp. collected at Rennel Sound.Both compounds 83 and 84 had been previously isolated by Ciminello et al. from arelated marine sponge Axinella cannabina (see Scheme 4, p67) collected off the coast ofTaranto near Porto Cesareo, Italy. 76 No biological activity was reported. Compound 83110was identified by comparison to literature values of 1 H, 13C NMR and massspectroscopic data and compound 84 was identified solely from the comparison of 1HNMR data.83 R = NCS84 R = NCAnother sesquiterpene, axisothiocyanate-3 (81), with the very interestingspiroaxane carbon skeleton was also isolated. Compound 81, along with 94 and 95, hadbeen previously isolated by Blasio et al. from Axinella cannabina again collected off thecoast of Taranto. 7181 R = NCS93 R = NC94 R = NHCHOFinally, violacene (88), a halogenated monoterpene was isolated from Acanthellasp. Violacene is a known metabolite of the red algae Plocamium violaceum. 77 Thoughother halogenated monoterpenes have been isolated from other marine organisms andlinked to red algal metabolism, 78 to our knowledge 88 has not previously been isolatedfrom a marine sponge. It was initially thought that alga may have been growing on thesponge but an examination of the sponge did not reveal any obvious algal growth. It maybe that the sponge itself is accumulating this compound through its filter feedingmechanism. Even more remarkable was the fact that the nudibranch Cadlina111luteomarginata collected near the sponge collection site also contained violacene (88).This is a very good example of how chemicals are carried through the food chain andsequestered by feeding organisms as suggested by Thompson et al. 10Cl88In addition to the compounds listed above, several other terpenoid metaboliteswere present in the sponge as indicated from the 1 H NMR spectrum of the crude mixture.Several volatile sesquiterpenes in the non-polar fractions were indeed too volatile topermit collection of sufficient data for structural elucidation. Based on previous studies itis probable that the formamide and isonitrile counterparts of acanthenes G (86) and H(87) are also present. A triplet at 6 3.39 ppm and the presence of an olefinic methylenelikely belonging to the same compound were apparent in the 1 H NMR spectrum of apartially purified fraction. This was a clear indication that another compound belongingto the same series as acanthenes A (80) and G (86) was present in the sponge.Unfortunately, the compound was inseparable from the major isonitrile 85.112ConclusionsAcanthenes A (80), G (86) and H (87) represent further examples offunctionalized sesquiterpenes with the eudesmane carbon skeleton. Acanthene A (80)appears to represent the first example of a chloro-sesquiterpene to be isolated in the sameseries as the isothiocyanates and isonitriles. Acanthene G (86) is very similar to thepreviously isolated isothiocyanate 95 from the Mediterranean sponge Axinella cannabina, differing only in the stereochemistry at C7. 70 In addition, this is the only example ofcompounds 81-85 and 90 having been isolated from a sponge of the genus Acanthella.The algal metabolite, violacene (88), was isolated from a sponge for the first time.The biogenesis of the carbon skeletons of the Acanthenes presumably takes placeby standard isoprenoid pathways through farnesyl pyrophosphate. A series of wellprecedented ring closures and hydride shifts would give rise to the immediate precursors.In contrast, the biogenesis of the functional groups has very little precedent and istherefore of considerable interest. All naturally occurring isonitriles from terrestrialsources show strong resemblances to amino acids whereas none of the marine isonitrilesdo. 12,13 Garson et al. showed that the marine isonitrile terpenoids arise through theuptake of inorganic cyanide likely through the quenching of a carbocation. 79 In addition,it has been shown by Scheuer et al. that the isonitrile functionality is the immediate1131NC-NCOPPXScheme 7. Proposed biogenetic scheme for the functionalized sesquiterpenes found inAcanthella sp.114. RNCS--.--RNCRNHCOHScheme 7. Continued.115precursor of isothiocyanate and formamide functionalities that occur with theisonitriles.80 This suggests that the isonitrile precursors of acanthenes G (86) and H (87)are also produced by and are present in Acanthella sp. collected at Rennel Sound. As foracanthene A (80), one can envision a chloride ion quenching the carbocation precursor.In conclusion, our study of the secondary metabolites of the Northeastern Pacificmarine sponge Acanthella sp. has led to the isolation and structure elucidation of fournovel functionalized sesquiterpenes, acanthenes A (80), G (86), H (87) and J (89), as wellas seven known compounds 81-85, 88 and 90 which had not previously been isolatedfrom Acanthella sp.116C. •^II^• 1 •^LI^•Pacific Sponge Neoesperiopsis digitataIntroductionSpecimens of Neoesperiopsis digitata (Miklucho-Maclay, 1870) 81 were collectedas part of a general collection expedition along the coast of British Columbia. The crudeextracts of N. digitata showed antibacterial and antifungal activity. This project wasprompted by the fact that the chemistry of very few sponges in the genus Neoesperiopsishas been studied.Taxonomy and Description of Neoesperiopsis digitataNeoesperiopsis digitata is a Demospongiae that according to Austin belongs tothe order Poecilosclerida and to the family Esperiopsidae. 81 Poecilosclerida is thelargest and most structurally diverse order within the Demospongiae. All Poeciloscleridaskeletons are composed of a mixture of spicules and spongian fibres. 82The specimens of Neoesperiopsis digitata examined in the present study werecollected off Bull Island in Rivers Inlet on the coast of British Columbia. The spongespecimens were found at depths of 10-15 meters in areas of moderate to heavy surge. N.digitata was observed in patches of up to 20 cm (diameter) on rock bottoms or walls.The sponge had a soft texture and was tan in colour.117HalichondridaOrder:^ Nepheliospongida HaploscleridaDictyoceratidaScheme 8: The position ofNeoesperiopsis digitata within the phylogenetic classificationof the Porifera according to Austin 81.Phylum: PoriferaClass:^Calcarea^Hexactinellida^Demospongiae^SclerospongiaeSubclass:^Homoscleromorpha^Ceratinomorp ha TetratinomorphaDendroceratida^ VerongidaPoeciloscleridaFamily: EsperiopsidaeGenus: Esperiopsis Neoesperiopsis MonanchoraSpecies: N. digitata118Results and Discussion1) Isolation of metabolites from Neoesperiopsis digitataSpecimens of Neoesperiopsis digitata (850 g wet weight) were collected by handusing SCUBA off Bull Island, Rivers Inlet, B.C., and transported to Vancouver frozen. Asmall sample (25 g) of the sponge was ground in a blender and extracted with Me0H andthis extract was used for biological testing. The extract showed antibacterial andantifungal activity. The remaining portion of the sponge (825 g) was immersed inmethanol (3L) and soaked at room temperature for 72 h. Concentration of the decantedMe0H in vacuo gave an aqueous suspension (1L) that was sequentially extracted withhexanes (2 X 1L), chloroform (2 x 1L) and ethyl acetate (2 x 500 mL). Evaporation ofthe combined hexanes extracts yielded a dark oil (2.14 g) that was inactive. Evaporationof the chloroform extract yielded a dark oil that exhibited antibacterial and antifungalactivity. The ethyl acetate and aqueous layers after evaporation in vacuo did not displayantimicrobial activity.Purification of the hexanes extract using Sephadex LH-20 (4:1 Me0H/CH2C12)yielded a complex mixture of fats and steroids that were not pursued. Initial purificationof the chloroform extract was accomplished using Sephadex LH-20 (4:1 Me0H/CH2C12).1 H NMR analysis indicated the presence of neoesperlactone (96) in the early elutingfractions and aromatic resonances in the later eluting fractions. Flash silicachromatography (1:1 Hex/EtOAc) led to the isolation of the major aromatic compoundwhich was identical by 1 H NMR and tic to a commercial sample of p-hydroxybenzaldehyde 83 which was shown to possess both antibacterial and antifungalactivity. Final purification of 96 was accomplished using successive applications of flashsilica gel chromatography (step gradient 3:1 Hex/EtOAc - 100% EtOAc), preparative tic(2:3 Hex/EtOAc), and normal phase HPLC (9:11 Hex/EtOAc) to yield 4.2 mg of acolourless oil.11911^12^14^16^18CH310^13^15^172) Structure elucidation of Neoesperlactone (961OH96Neoesperlactone (96) was obtained as an optically active ([0.]D -620 (c 0.08))colourless oil that gave a parent ion in the HREIMS at m/z 296.2354 Da (AM 0.3 mmu)appropriate for a molecular formula of CigH3203. The FTIR (film) showed hydroxyl andcarbonyl stretching bands at 3414 and 1769 cm -1 respectively. The latter indicated thepresence of a y-lactone. The 1 H NMR spectrum (Fig. 59) showed a complex multiplet at8 5.36 ppm integrating to two protons as well as methine protons at 8 4.53 and 3.65 ppm.The upfield region consisted of several resolved multiplets between 8 1.70 and 2.60 ppm,a methylene envelope between 8 1.28 and 1.32 ppm and a methyl triplet at 8 0.89 ppm.The 13 C/APT NMR spectra (Fig. 60) showed resonances attributable to 17 of the 18carbons in 96, appropriate for 4 methines, 12 methylenes and 1 methyl.The connectivities were determined by extensive COSY, HMQC and HMBCanalysis (Table 13). The COSY data revealed a fragment (Fig. 56) containing H2/H2'(62.53), H3(2.33), H3'(1.87), H4(4.53), H5(1.89), H5'(1.73), H6/H6'(1.60), H7(3.65) andH8/H8'(1.49). The upfield region of the COSY spectrum was too congested to elaboratethe spin system further.1201.32H2.05^2.01H H H H2.05^ 2.011.89^1.73 3.65H^H HO H0S 2.53H 1.60H4.53H1.601.49H H1.492.53 HH^1.872.33Fig. 56. Fragment A of neoesperlactone (96)The carbonyl, that was apparent from the FTIR spectrum, was attached to C2 onthe basis of the chemical shifts of H2 and H2' as well as HMBC correlations (Figs. 58and 61) from H2/H2'(5 2.53) to a carbon resonance at 8 177 ppm. Connecting thecarbonyl carbon to the oxygen attached to C4 completed the y-lactone which accountedfor 2 of the 3 degrees of unsaturation required by the molecular formula of Ci8H3203(dbe 3).The COSY spectrum was also able to identify the two methylenes on either sideof the cis olefinic bond. This part of the spin system (Fig. 57) contained H9(5 1.50),H9'(1.40), H10/H10'(2.05), H11/H12(5.36), H13/H13'(2.01) and H14/H14'(1.32). Thedetermination that the double bond was cis was based on the carbon chemical shifts of theFig. 57. Fragment B of neoesperlactone (96)C10 (27.3) and C13 (27.0) carbons which are more typical of allylic carbons of cis (525-30 ppm) olefins than of trans (6 35-40 ppm) olefins in otherwise saturated aliphatic121systems.84 The COSY data also showed that the methyl triplet was coupled into a pair ofmethylene protons at 5 1.30 ppm.Attachment of C8 to C9 was accomplished by HMBC correlations from H9 andH9' to C7 and from H8 and H8' to C9 (Figs. 58 and 61). The chemical shifts of bothH7( 1 H: 8 3.65) and C7( 13C: 5 71.2) indicated that the hydroxyl group was at C7. Onlytwo methylene groups remained and they were placed between C14 and the terminalethyl group.CH3,,.,....,,....,..................,.,,,,..,,.....,,kHMBCFig. 58. HMBC results for neoesperlactone (96).12211^12^14^16^1810^13^15^17CH3Table 13. 1 H nmr and 13C nmr data for neoesperlactone (96) recorded in CDC13.C# 1H(400 MHz) COSY 13C(125 MHz) HMBC1 177 H2,H2',H32 2.53,m H3,H3' 28.82' 2.53,m H3,H3'3 2.33,m H2,H2',H3,H4 28.03' 1.87 H2,H2',H3,H44 4.53,m H3,H3',H5,H5' 80.7 H2,H2',H3',H5,H6,H6'5 1.89 H4,H6,H6' 31.65' 1.73,m H4,H6,H6'6 1.60 H5,H5',H7 32.76' 1.60 H5,H5',H77 3.65,m H6,H6',H8,H8' 71.2 H5,H6,H6',H9,H9'8 1.49 H7 37.2 H6,H6',H9,H10,H10'8' 1.49 H79 1.50 H9',H10,H10 - 25.7 H8,H8',H10,H10'9' 1.40 H9,H10,H10'10 2.05 H9,H9',H11 27.3 a10' 2.05 H9,H9',H1111 5.36,m H10,H10' 130.6b H10,H10',H13,H13'12 5.36,m H13,H13' 129.1 b H10,H10',H13,H13'13 2.01 H12,H14,H14' 27.0a13' 2.01 H12,H14,H14'14 1.32 H13,H13' 29.0c H13,H13'14' 1.32 H13,H13'15 1.28-1.32 29.7c H13,H13'15' 1.28-1.3216 1.28 31.8d H1816' 1.2817 1.30 H18 22.6d H1817' 1.30 H1818 0.89,0 = 6.7 Hz) H17,H17' 14.1a,b,c,d may be interchangede Protons correlated to carbon in carbon # column.OH96123PP. 7 6 5 4 s 2 1^0Fig 59. 1H NMR spectrum of neoesperlactone (96) (500 MHz, CDC13).Fig. 60. 13C and APT NMR spectra of neoesperlactone (96) (125 MHz, CDC13).41•11,■11:10 "I". "Xt.^.•=1 Or1.0.^.^1^.pp.^2.5.^..^1^• ^I^.2.0 1.5—asid:Zio■Za•^ ... ■...50-100150■IC:21.^QOM"PP.OHFig. 61. Selected region of HMBC spectrum of neoesperlactone (96).Part II Chemical Studies on Peptides from Three Species of TropicalPacific Ocean Marine SpongesIntroductionMarine sponges have been an excellent source of secondary metabolites.Recently, an increasing number of the reported sponge metabolites are peptides or arebiogenetically derived from peptides. Many of these peptides have highly modifiedstructures containing unusual amino acid residues and they often possess potentbiological activities. A number of the sponge peptides resemble blue-green algalmetabolites and it has been speculated for some time that these metabolites are theproducts of microorganisms and not of sponges. A small number of peptides containingstandard protein amino acid residues all with the natural L configurations have also beenisolated from sponges. These more normal peptides do not in general have as potentbiological activities as those with unusual residues. In addition, there is no clear linkbetween these "standard residue" peptides and blue-green algal metabolites.The following sections describe the isolation and structural elucidation of twodistinctly different peptides, each falling into one of the categories discussed above, fromthree species of Tropical Pacific marine sponges. The first section describes the isolationand structure elucidation of pseudaxinellin (103), a cyclic heptapeptide with standardamino acid residues. Pseudaxinellin (103) was isolated from the Papua New Guineasponge Pseudaxinella massa. It is the first example of a cyclic heptapeptide to beisolated from a marine source.* , 85 The second section describes the identification of the* The marine peptide Dolastatin-3 (cyclo(L-Pro-L-Leu-L-Val-(gly)Thz-(gly)Thz), which contains fiveamide bonds and two thiazole residues, may also be considered a cyclic heptapeptide (see ref. 85).127algal metabolite majusculamide C (104) from the sponges Amphimedon sp. andPtilocaulis trachys collected at Enewetak Atoll in the Marshall Islands. MajusculamideC (104) contains several unusual amino acid residues and possesses potent biologicalactivity.1) A review of metabolites previously isolated from sponges in the genera Pseudaxinella,Amphimedon and Ptilocualisi) PseudaxinellaIn contrast to many other marine sponges, few chemical studies have beenreported on the genera Pseudaxinella, Ptilocaulis or Amphimedon. The only chemicalstudy found in the literature on sponges of the genus Pseudaxinella reported the isolationof several steroidal metabolites. Pseudaxinella lunaecharta collected off the Senegalesecoast yielded two novel steroids with normal steroid nuclei but with modified sidechains. 86 Compounds 97 and 98 possess the rare feature of having two double bonds inthe side chains. At least thirteen other steroids were identified from P. lunaecharta bycomparison of GC and HPLC relative retention times and by GC-MS data of knownstructures. 97 R =HO 98 R =128ii) Ptilocaulis and AmphimedonPrevious work carried out on sponges of the genera Ptilocaulis and Amphimedonled to the isolation of some very biologically active metabolites. In fact, the crude extractof Ptilocaulis spiculifer, collected at Burn Cay, Honduras, was listed among the mostactive extracts obtained from a Caribbean collection expedition in 1978. 87 The extractswere active against gram-positive and gram-negative bacteria, yeasts, and fungi. Themajor antibacterial compounds identified from this sponge were the guanidine containingcompounds, ptilocaulin (99) and isoptilocaulin (100). Partitioning of the methanol-toluene extract of the sponge with 1 N sodium nitrate and subsequent organic extractionand purification led to the isolation of the nitrates of 99 and 100. Ptilocaulin (99), whichwas the most active of the two, had an IC50 of 0.39 1.1g/mL against L1210 murineleukemia cells as well as antimicrobial minimum inhibitory concentrations varying from3.9-62.5 1..tg/mL. Isoptilocaulin (100) had an IC50 of 1.4 i.tg/mL (L1210 leukemia cells)and antimicrobial minimum inhibitory concentrations of 25-100 gg/mL.99^100Another study on a Caribbean collection of Ptilocaulis spiculifer yieldedptilomycalin A (101). 88 This alkaloid also possesses a guanidine moiety and a widevariety of biological activity including cytotoxicity against P388 (IC50 = 0.1 1.1g/mL),antifungal activity against Candida albicans (MIC = 0.8 1.1g/mL) and antiviral activity129(against HSV at 0.2 ilg/mL). The structure of the bis(trifluoroacetyl) derivative wasdetermined using mass spectrometry and extensive 1D and 2D NMR spectroscopy.101The Pacific sponge, Amphimedon sp., collected at Guam, yielded an aromaticalkaloid, amphimedine (102).89 The structure of this cytotoxic alkaloid was solved usinglong-range proton-carbon couplings as well as natural abundance carbon-carboncouplings.0102130Result and DiscussionA.^The Structure Elucidation of Pseudaxinellin. a Cylic Heptapeptide from theSouth Pacific Marine Sponge Pseudaxinella massa This section describes the structure elucidation of a cyclic heptapeptide isolatedfrom the marine sponge Pseudaxinella massa.90 Specimens of P. massa were collectedas part of a general collection expedition to Papua New Guinea. The isolation and initialscreening of pseudaxinellin (103) was carried out in our laboratory by Fangming Kong.1) Taxonomy and Description of Pseudaxinella massaPseudaxinella belongs to the family Axinellidae and the order Axinellida. 91Sponges of this order contain a stiff axial area which is independent of a softer extra-axialarea.92 Both massive sponges like Pseudaxinella and encrusting sponges are foundwithin this order. All genera of Axinellida have rough surfaces due to projectingspicules. Pseudaxinella obtained for this study was collected off Loloata Island on thesouth coast of Papua New Guinea near Port Moresby. The specimens were collected in10 - 25 meters of water in areas exposed to little or no surge.131Scheme 9: The position of Pseudaxinella massa within the phylogenetic classificationof the Porifera according to Bergquist. 91Phylum:^ PoriferaClass:^Calcarea^Hexactinellida^Demospongiae^Sclerospongiae Subclass:^Homoscleromorpha Tetratinomorpha CeratinomorphaOrder: Choristida SpirophoridaLithistida Axinel ida^HadromeridaFamily: AxinellidaeGenus: Axinella CeratopsionPseudaxinella Phakellia^AulettaPtilocaulis Pararaphoxya Acanthella^BubarisHomaxinellaSpecies: P. massa1322) Isolation of Pseudaxinellin from Pseudaxinella massa.93Pseudaxinella massa was collected by hand using SCUBA, frozen and transportedto Vancouver over dry ice. Frozen specimens were homogenized in methanol andextracted at room temperature for 48 h. The mixture was filtered and the filtrateevaporated in vacuo to yield a residue that was suspended in water and sequentiallyextracted with hexanes, dichloromethane and ethyl acetate. 1 H NMR analysis of thedichloromethane extract indicated the presence of a moderately complex peptide.Purification of the dichloromethane soluble portion using Sephadex LH-20 (eluent:hexanes/CHC13/Me0H 10:10:1), followed by silica flash chromatography (EtOAc toMe0H, step gradient) and finally by silica preparative thin layer chromatography(Et0Ac/Me0H 4:1) yielded pseudaxinellin (103) as an optically active ([oc]D -100.1, c0.34, CHC13) clear glass.133034 3029 -N032 260 240 NH273) Structure Elucidation of Pseudaxinellin (103)38^3736HN 355H^2016232117^013^CONH218103The structure of pseudaxinellin (103) was determined by a detailed analysis ofone and two dimensional NMR spectra and mass spectrometric analysis. The structuresof the individual residues were determined by analysis of COSY, HOHAHA, HMQC andHMBC experiments. The sequence of attachment of these residues was determined by acombination of ROESY, HMBC and one dimensional difference nOe experiments.The HREIMS of 103 gave a parent ion at m/z 752.4218 Da appropriate for amolecular formula of C381-156N808 (AM -0.3 mmu), which requires fifteen degrees ofunsaturation in the molecule. The 1 H and 13C NMR spectra of 103 (Figs. 62 and 63)contained resonances that were characteristic of peptides. The 1 H NMR spectrum (Fig62) contained several protons that were attached to heteroatoms as well as five aromaticprotons. Seven methine resonances between 5 4.0 and 4.9 ppm, that could be assigned toa-protons of amino acids, and several methyl multiplets were also present. TheHOHAHA data (Table 15, Fig. 64) facilitated the assignment of the resonances22134Table 14. 1H and 13C NMR data for Pseudaxinellin (103) recorded in CDC13.C# 1H(400 MHz) COSY noes' 13C(125 MHz) HMBCbPhe-1 - 172.13 NH(7.32)2 4.77, m H3,H3',NH(7.55) H5,H9 55.4 H33 2.94 H2,H3' 37.73' 3.23 H2,H34 - 137.5 H6,H8,H3,H3'5,9 7.17 128.9 H3,H3',H76,8 7.24 128.3 H8,H67 7.18 126.6 H5,H9NH 7.55, d(9.5 ) H2Prol 10 - 170.31 H11,NH(7.55)11 4.08 H12,H12' 63.512 2.28,m H11,H12',H13,H13' 29.612' 1.36,m H11,H12,H1313 1.80 H12,H12',H14' 25.813' 1.92 H12,H14,H14'14 3.58, bt(7.3) H13',H14' H16 48.014' 3.48 H13,H13',H14NAsn 15 - 172.6 H17'16 4.61, bm H17,H17',NH(8.01) - 50.2 H17'17^, 2.94 H16,H17 36.2  NH2(5.43)17' 3.20 H16,H1718 - 169.0 H17'NH2 5.43, bs; 6.66, bs 6.66,5.43NH 8.01, d(5.3) H16Vall 19 - 172.26 H20,NH(8.01)20 4.05 H21,H25,NH(8.22) 62.121 2.36, m H20,1-122,H23 29.522 1.04 H21 18.823 1.04 H21 18.7NH 8.22, d(8.0) H2OPro2 24 - 171.5 H25,NH(8.22)25 4.53, d(6.9) H20,H26' H30 61.226 2.57, m H26',H27,H27' 31.2 H2526'  1.90 H25,H2627 1.93 H26,H27' 21.8 H2527' 1.70 H26,H2728 3.70, m H27,H27',H28' 46.0 H25,H2628' 3.48 H27,H27',H28NVal2 29 - 171.8 H3030 4.16, dd(4.4,7.3) H31,NH(6.80) H25 58.531 1.95 H30,H32,H33 30.132 1.04 H31 19.833 0.95 H31 18.5NH 6.80, d(4.4) H30 1135Va13 34 - 171.18 H3635 4.21, t(9.4) H36,NH(7.32) NH(6.80) 57.336 2.01 H35,H37,H38 29.1 H3537 0.95 H36 19.938 0.95 H36 19.3_ NH 7.32, d(9.4) H35a Proton in C# column irradiated. b Protons correlated to carbons in C# column.135I • • •^• • • •^• • • -^•8. 8^7. 6. 8^5.e^4.8^3.e^2. 0PPMFig. 62. 1H NMR spectrum of pseudaxinellin (103) (400 MHz, CDC13).^- Trig wrru^Tv, wyrwly1.e 0.0Fig. 63. 13C and AN' spectra of pseudaxinellin (103) (125 MHz, CDC13)..80 I oa 0 0o o 41.00^0PININoI ^  I ^  I'9 2 14Fig. 64. NH region of HOHAHA spectrum of pseudaxinellin (103) (500 MHz, CDC13).-7.5within all of the seven amino acid residues. The 13C NMR spectrum (Fig. 63) containedeight resolved carbonyl resonances between 5 169 and 173 ppm and four aromatic carbonresonances.Table 15. HOHAHA assignments for seudaxinellin (103)Residue HOHAHA assigned spin systemsPhe 5 7.55, 4.77, 3.23, 2.94 ppmPro 1 8 4.08, 3.58, 2.28 ppmA sn 1) 5 8.01, 4.61, 3.20, 2.94 ppm 2) 5 6.66, 5.43 ppmVal i 6 8.22, 4.05, 2.36, 1.04 ppmProt 5 4.53, 3.48, 2.57, 1.90 ppmVal2 8 6.80, 4.16, 1.95, 1.04 ppmVa13 5 7.32, 4.21, 2.01, 0.95 ppmAnalysis of the HMQC and APT spectra demonstrated the presence of a mono-substituted aromatic ring. The aromatic proton resonances, which integrated to fiveprotons, were attached to three of the four 13C aromatic resonances that were shown to bemethine carbons by the APT experiment (Fig. 63). This data was consistent with asymmetrically substituted benzene ring where C3 and C5 as well as C2 and C6 arechemically equivalent. The phenylalanine residue was confirmed by HMBC correlationsfrom the methylene protons at 6 2.94 and 3.23 ppm to the aromatic carbon at 6 137.5ppm. The methylene protons showed COSY correlations into the methine at 5 4.77 ppmwhich in turn gave a COSY correlation to the NH proton at 6 7.55 ppm. The carbonyl ofthe phenylalanine residue (6 172.13 ppm) was the only one of the eight carbonyls that didnot show HMBC correlations.1397.24 HH4.772.947.18 H8 7.55H_/.N7.17H "5-"../...........■.................‘HMBCPheThe HOHAHA spectrum indicated that the group of methyl multiplets between 80.9 and 1.1 ppm belonged to a series of three valine residues. The first system, Vali,contained resonances at 8 8.22, 4.05, 2.36 and 1.04 ppm (Table 15, Fig. 64). Analysis ofthe COSY spectrum revealed that the NH proton (8 8.22) was coupled into the a-methine at 8 4.05 ppm and that the isopropyl methine, Vali-PH (8 2.36), was coupled intoVali-aH (8 4.05) and also into the unresolved methyls at 8 1.04 ppm. The Vali-CO (8172.26) assignment was based on an HMBC correlation to the carbonyl from Vali-aH (64.05).ValiThe second valine spin system contained resonances at 8 6.80, 4.16, 1.95 and 1.04ppm according to the HOHAHA data (Table 15). As with Vali, the COSY experimentcould uniquely assign the resonances to NH(6 6.80), aH(4.16), 1 H(1.95) and the methylprotons (1.04). Further analysis of the COSY revealed that the isopropyl proton (6 1.95)140was coupled into a second methyl at 8 0.95 ppm that was not visible in the HOHAHAexperiment. The Va12-CO (8 171.8) was assigned on the basis of an HMBC correlationto Va12-aH(8 4.16).The final valine spin system, Va13, consisted of resonances at 8 7.32, 4.21, 2.01and 0.95 ppm (Table 15, Fig. 64). Again, analysis of the COSY spectrum assigned theresonances to NH (8 7.32), aH (4.21), 13H (2.01) and the unresolved methyls (0.95). AnHMBC correlation from the Va13-13H (2.01) to the Va13-CO (8 171.18) assigned thecarbonyl carbon .8 7.32H,N0 13C: 171.18H3C0.95H2.01^CH30.95Va13COSY and HOHAHA analysis identified two proline spin systems, eachcontaining an a-methine connected to three contiguous methylene carbons. The chemicalshifts of the terminal methylene protons in each system were appropriate for attachmentto heteroatoms. The cyclic structures accounted for two of the fifteen degrees ofH 4.211418 3.58H03.48 HH1.80ProsH 4.082 .28H1.36H1.928 3.70H03.4PH „„1.70^n1.93H4.53H 2.57H1.90unsaturation in the molecule. HMBC correlations from Prol-aH (8 4.08) and Pro2-aH (84.53) to carbon resonances at 8 170.31 and 171.5 ppm assigned the proline carbonyls(Fig. 66).Pro2The remaining a-methine proton at 8 4.61 ppm was part of a spin systemcontaining resonances at 8 8.01, 4.61, 3.20 and 2.94 ppm. Analysis of the COSYspectrum showed correlations from 8 4.61 ppm to the NH proton (8 8.01) and a pair ofmethylene protons(8 3.20 and 2.94). The two remaining carbonyls in the moleculeshowed HMBC correlations from the methylene proton at 8 3.20 ppm suggesting that thespin system was terminated in the side chain by a carbonyl functionality. Only the NH2protons at 8 5.43 and 6.66 ppm, which were coupled only into each other, remainedunassigned. An HMBC correlation from the proton at 8 5.43 ppm to the methylenecarbon (8 36.2) attached the NH2 group to the carbonyl in the side chain generating anasparagine residue (Asn).,....------...HMBC142The functionality present in the seven residues accounted for fourteen of therequired fifteen degrees of unsaturation. This, along with the failure of 103 to react withninhydrin, was consistent with a cyclic structure.The sequence of the amino acid residues was determined from analysis of HMBC,ROESY and difference nOe data. Two fragments composed of three residues each wereestablished by HMBC correlations from NH protons to the carbonyl carbon of theadjacent residue (Figs. 65 and 66). HMBC correlations from Phe-NH (8 7.55) to Proi-CO (8 170.31) and from Va13-NH (8 7.32) to Phe-CO (8 172.13) established the partialsequence Proi-Phe-Va13. HMBC correlations from Vali-NH (8 8.22) to Prot-CO (8171.5) and from Asn-NH (8 8.01) to Vali-CO (8 172.26) established the second threeresidue sequence, Prot-Vale-Asn.O(—INI f HMBCProl-Phe-Va13 HMBCNI-12Prot-Val 1 -AsnFig. 65. Connectivities in 103 based on HMBC results.143H17'/C18(H11/C10-170H25/C24-171-172VH30/C29II^V^I^•1.07173:ppm3.5H20/C19PPG 9.0^7.6^7.6^7.4Fig. 66. Selected regions of 2D HMBC spectrum of pseudaxinellin (103) (CDC13).0Difference nOes (Figs. 67 and 68) and ROESY correlations between Prol-8H (83.58) and Asn-aH (8 4.61) and between Va13-aH (8 4.21) and Va12-NH (8 6.80)completed the linear sequence, Prot-Va11-Asn-Pros -Phe-Va13-Va12. A ROESYcorrelation and a strong nOe (8.5%) between Pro2-aH (8 4.53) and the Va12-aH (8 4.16)resonances established the Va12-Pro2 connectivity and demonstrated that the Va12-CO/Pro2-N amide bond had the cis geometry. A long range COSY correlation fromVal i-aH (8 4.05) to Pro2-aH (8 4.53) supported the Prot-Vale linkage.Val3 Va120Phe ProtnOe/ROESYVal iPro t0CONH2AsnFig. 67. Connectivities in 103 based on NOe enhancements and ROESY correlations.Finally, hydrolysis of 103 with 6N HCI, followed by derivatization with Marfey'sreagent" and HPLC analysis confirmed the presence of Phe, Pro, Asn and Val andshowed that all of the amino acids had the L(S) configuration.*The determination of the absolute stereochemistries of the amino acid residues was accomplished byFangming Kong.H145Fig. 68. NOe results for pseudaxinellin (103) (400 MHz, CDC13).8.0 3.06.0 5.0I • •• • 1PPM. • , • .4.0H35Vale NH113011251116Va12 NH irradiatedH35 irradiated1125 irradiated1130 irradiatedH14 irradiatedHN0^NH'N.,z.N1CH3104 R = CH3105 R =HB.^Identification of the Algal Metabolite Majusculamide C from the MarineSponges Ptilocaulis trachys and Amphimedon sp. In addition to sponges, blue-green algae have also been excellent sources of novelpeptides. Examples include the cyclic depsipeptides majusculamide C (104) and 57-normajusculamide C (105) isolated from the blue-green alga, Lyngbya majuscula. 95 Asdiscussed earlier, it is believed that a large number of sponge peptides are products ofmicroorganisms. The following discussion describes the identification of majusculamideC (104) from the marine sponges Ptilocualis trachys and Amphimedon sp. This workwas accomplished as part of a joint project between Dr. R. J. Andersen (Depts. ofChemistry and Oceanography, U.B.C.) and Dr. T. M. Allen (Dept. of Pharmacology,Univ. of Alberta). The extraction and isolation of majusculamide C (104) wasaccomplished by Ziba R. Fathi-Afshar (Univ. of Alberta). The structure elucidation wasthen carried out by our group at U.B.C.1471) Isolation of majusculamide C (104). 96i) Isolation from Ptilocaulis trachysPtilocaulis trachys was collected by hand using SCUBA at Enewetak Atoll in 15meters of water, frozen, and transported to Edmonton. The frozen sponge (450 g wetweight) was homogenized in 95% ethanol (800 mL) and filtered. The residue was againextracted with ethanol (1600 mL) and the combined extracts were evaporated to drynessin vacuo. The resulting residue (22 g) was suspended in methanol (200 mL) and filteredto yield an inactive (against P388) solid (9.71 g) and an active methanol filtrate whichwas evaporated to dryness in vacuo. The active residue was taken up in methanol/water(9:1) and partitioned with hexane yielding an inactive hexane layer and an active aqueousmethanol layer. The active layer, after evaporation to dryness, was subjected to repeatedapplications of normal phase flash chromatography (CHC13), while tracking the activity.Further purification of the active fractions was accomplished using Sephadex LH-20(ethanol) and finally silica open column chromatography to yield majusculamide C (104)(1 mg).ii) Isolation from Amphimedon sp.Frozen specimens of Amphimedon sp. (1480 g wet weight) were extracted with95% ethanol, filtered and the filtrate evaporated to dryness in vacuo to yield the cruderesidue (68.3 g). The crude extract was taken up in methanol/water (9:1) and partitionedwith hexane. The active aqueous methanol layer was adjusted to 3:1 methanol-water andextracted with CC14 to yield, after evaporation to dryness, an active mixture (0.88 g).Purification of the mixture using Sephadex LH-20 (ethanol), normal phase flashchromatography (2% Me0H in EtOAc) and successive applications of silica opencolumn chromatography (1% Me0H in CHC13 and EtOAc/MeOH) yieldedmajusculamide C (104) (0.5 mg).148310 CH316 1,22 021 n3223^33^37C H3N 34^353825O^40117^ 81911HN433610HN 1202830CH3O 27 262) Structural information on majusculamide C (104) based on extensive one and twodimensional NMR analysis. 0^NH41 053H 52 4350 N41. 45 46^4751CH349^4858 5505657104The original structure elucidation of majusculamide C (104) carried out by Mooreet al. 95 was accomplished by NMR analysis, chemical degradation and extensive FABMS analysis. In the original study, 5 g of pure majusculamide C (104) was isolated fromone collection of the blue-green algae Lyngbya majuscula from the lagoon of Enewetak.In the present study, we were provided with 0.5 mg of 104 and therefore destructive•techniques such as FABMS and chemical degradations were avoided. Consequently, theidentification of majusculamide C (104), including the determination of the constitutionof the individual residues as well as the sequence of these residues, had to beaccomplished exclusively by NMR (Table 16). This led to a very common problem whendealing with moderately complex peptides in that several of the protons in the a-methineregion were not resolved. However, the proton spectrum in a 1:1 mixture of CDC13 andC6D6 displayed a-methine resonances that were completely resolved (Fig. 70).149Subsequent ROESY and one dimensional nOe experiments provided unambiguoussequence information (Figs. 69 and 71). An example of this was the MeIle a-methine at8 4.93 ppm (H43) which was unresolved from the Ala residue in CDC13. In 1:1CDC13/C6D6 this resonance, now at 8 4.90 (H43), was completely resolved and gave aclear nOe and ROESY correlation to the Gly2 NH proton (H41: 8 7.32). In addition, theVal and Ile N-methyls, which were unresolved in CDC13, were completely resolved in1:1 C6D6/CDCI3 and gave ROESY correlations into the Glyi (H40: 8 4.54) andGly2(H51: 5 4.24) protons respectively. In the original study, the MeVal-Gly-Melle-Glysequence was determined by analysis of the FABMS spectrum of the saponificationproduct of majusculamide C (104). The analysis of the data obtained in 1:1 CDC13/C6D6on 0.5 mg of majusculamide C (104) provides direct NMR evidence for this sequence anddemonstrates the power of modem NMR techniques.Fig. 69. NOe and ROESY correlations for majusculamide C (104) in 1:1 C6D6/CDC13.1500 C H 30.f.„, t,j,,,00TABLE 16: 1 H NMR and 13C NMR data for majusculamide C (104) in 1:1 C6D6/CDC13C# 1H(500MHz) COSY ROESY 13C(125 MHz)a HMBCb1 - 172.5 H52 2.53,m H5 H3 42.7 H53 4.46(bm) H6,NH H2,H6 51.0 H5,H75 0.95,d(7.0) H2 9.86 1.25 H3,H7 H3 26.5 H76'7 0.75 }16 11.0NH 7.03 H3 H9 -8 -9 4.31,m H11,NH H11,NH(7.03) 48.5 H1111 0.98,d(6.5) H9 H9 15.8NH 7.74,d(8.5) H9 H17 -12 - 172.0 H17,H1813 - 55.2 H17,H1814 - 210.0 H17,H1815 4.86 H19,NH H19 52.0 H1917 1.38,s NH(7.74) 22.2 H1818 1.27,s 21.8 H1719 1.08,d(7.0) H15 H15 19.5NH 7.26(bm) H15 H21 -20 - 168.2 H2121 5.16,t(7.5) H23,H23' H23,H33,NH(7.26) 61.223 3.16,dd(7.5,13.8) H21 H2123' 2.69,dd(7.5,13.8) H2124 - 129.0 H26,H2825,29 7.01_4(8.5), H26,H28 131.0 H25,H2926,28 6.61,d(8.5) H25,H29 114.027 - 159.0 H25,H29,OMeOMe 3.37(s) 55.0NMe 2.90(s) 29.4a Carbon chemical shifts obtained from HMQC and HMBC spectra.b Protons correlated to carbons in Carbon # column.151HN420-.••••„.'OIN1 22'CH3^0.1\^00 HvN 5(:NICH3TABLE 16 continued: 1 1-INNIR and 13C NMR data for ma usculamide C (104) in 1:1 C^CDC1C# 1 H(500MHz) COSY ROESY 13C(125 MHz)a HMBCb32 - 170.3 NMe(2.90)33 4.76,d(10.5) H35 H21,H35,H36,H37 58.5 H36,H37,NMe(2.63)35 2.11 H33,H36,H37 H33 27.5 H36,H3736 0.31,d(6.5) H35 H33 19.2 H3737 0.57,d(6.5) H35 H33 19.0 H36NMe 2.63(s) H40 29.139 - 169.2 H40,NMe(2.64)40 4.54,dd(8.5,17.3) H40',NH H40',NMe(2.63) 41.040' 3.29,d(17.3) H40 H40,NH(7.54) „NH 7.54(bd) 1440 H40',H4342 -43 4.90,d(11.5) H45 H48,NH(7.54) 61.3 H4845 2.01 H43,H48 33.0 H48,H4746 1.35 H46',H47 25.0 H48,H4746' 1.03 H46,H47 .47 0.78 H46,H46'48 0.98,d(6.5) H45 H43 15.8NMe 2.96(s) H51 30.550 - 170.0 NMe(2.96)51 4.24,dd(7.0,16.0) H51 ',NH NME(2.96) 41.051' 3.35 H51,NH NH(7.32) ,,NH 7.32(bt) H51,H51' H51' -53 -54 5.21,d(3.5) H55 H55,H58 78.8 H5855 2.03 H54,H58 H54 32.5 (H57 or H58)56 1.45 H56',H57 23.0 (H57 or H58)56' 1.15 H56,H5757 0.81 H56,H56'58 0.80 H55 H54a Carbon chemical shifts were obtained from the HMQC and HMBC spectra.b Protons correlated to carbon in Carbon # column.152Fig. 70. 1H NMR spectrum of majusculamide C (104) (500 MHz, 1:1 CDC13/C6D6)2i^6^5^4^iSSFig. 71. Selected region of ROESY spectrum of majusculamide C (104) (500 MHz, 1:1 CDC13/C6D6).1)NH4Ac, NaBH 3CNMe0H, r.t., 67 h.N../2)Et3N/H20 work up NH2 0^/.OH=_: HH N2 ^N0106 2S,3R107 2R,3SNO2108 2S,3S109 2R,3R3)^Determination of the stereochemistry of the MAP residue. As part of the present study on majusculamide C (104) the absolutestereochemistry of the 2-methyl-3-amino pentanoic acid residue (MAP) wasdetermined.* ,97 This was accomplished by the synthesis of four possible diastereomers ofthe MAP residue followed by derivatization using Marfey's reagent 94 to yieldcompounds 106-109 (Scheme 10). These derivatives and the Marfey's derivatizedhydrolysis product of majusculamide C (104) were then compared by HPLC analysis. X-ray diffraction analysis (Fig. 72) of the synthetic product 106 (which was known to havethe S stereochemistry at C13) that corresponded to the derivatized hydrolysis product ofmajusculamide C (104) identified the stereochemistry at C2 to be S and at C3 to be R.**,■Thr10 ../ NaOEt06N HC1108 °C, 16 hFDAA, acetone, NaHCO340 °C, 1 hOHNH2 0Scheme 10. Synthesis of the Marfey's derivatives of the MAP residues* The work involving the synthesis of the MAP residue and the Marfey's derivative and the subsequentdetermination of the absolute stereochemistry of this residue was accomplished by Dr. D. E. Williams inour laboratory.** For experimental details on the synthesis of compounds 106-109, the HPLC analysis of the Marfey'sderivatives and the X-ray diffraction analysis see reference 97.155H13^H14H19H21^ C9C13Fig. 72. Perspective drawing of 106.156ConclusionsOrigins of Peptides from Marine SpongesThe Porifera have been an excellent source of unusual peptides whose structuralnovelty and bioactivity is of great interest. Of almost equal interest are the possibleorigins of these compounds. Are they actually produced by the sponges or are theyproduced by microorganisms? More and more evidence is being gathered to support thelatter, including the isolation of the algal metabolite majusculamide C (104) from twospecies of marine sponges in the present study. However, it is unlikely that this is auniversal rule. There is no evidence to suggest that pseudaxinellin (103) is produced by amicroorganism. This poses the question; are there structural features that one can use todistinguish between those peptides produced by microbes and those produced bysponges?Marine sponge peptides can be divided into two categories based on structure: 1)those that contain highly modified and unusual residues, and 2) those that containstandard protein amino acid residues. The unusual structural features that exist inpeptides of category 1 include a high incidence of N-methylated amino acids, n-aminoacids and residues with the a D-configuration. These peptides often possess potentbiological activities. In contrast, the amino acids in peptides of category 2 are all aamino acids with the standard L-configuration and they rarely possess significantbiological activity.The literature indicates that sponge peptides in category 1 most often resemblealgal metabolites and therefore may be produced by blue-green algae. One of the clearestexamples of this is the isolation of the cyclic pentapeptide motuporin (110) from thesponge Theonella swinhoei collected off Motupore Island, Papua New Guinea. 98157I--;,.,..,,,, ^MeO^0,.... ,, NHH/^NRHNs,,..,,,,CO2R ...,„.0 NMeCO2R 0110 R = CH(CH3)2111 R = (CH2)3NHC(=NH)NHMotuporin is very closely related to the algal metabolites nodularin (111) andmicrocystin-LR (112). Compound 110 differs from nodularin only in the replacement ofan arginine residue with a valine residue. All three peptides contain the 13-amino acidAdda, D-glutamic acid and D-erythro 13-methyl aspartic acid. As with nodularin andmicrocystin-LR, motuporin possesses potent biological activity. Motuporin (110) inhibitsprotein phosphatase-1 (IC50 < 1 nmolar) and displays considerable in vitro cytotoxicityagainst several cancer cell lines.112Another peptide that has been isolated from a sponge of the genusTheonella is thedodecapeptide theonellamide F (113).99 Compound 113 also contains several unusual158residues including 13-alanine, 3-hydroxyasparagine, 2-amino-4-hydroxyadipic acid, L-p-bromophenylalanine, 3-amino-4-hydroxy-6-methyl-8-(9p-bromophenyl)-5,7-octadienoicacid (Aboa) and an unprecedented histidinoalanine bridge. Theonellamide F exhibitsantifungal activity against Candida spp., Trichophyton spp., and Aspergillus spp., as wellas cytotoxicity against L1210 and P388 leukemia cells (IC50 3.2 and 2.7 gg/mLrespectively). As with the other peptides with unusual residues, compound 113 hasremarkable structural similarities with blue-green algae metabolites. The Aboa residue isbiogenetically related to the 13-Aminophenyldecanoic acids found in the peptides fromblue-green algae.113Collections of the Okinawan sponge Theonella sp. have also yielded two relatedbiologically active peptides, keramamide A (114) and konbamide (115).100,101159N^Br/z.,.....0 0H^I-INClCompounds 114 and 115 are cyclic hexapeptides that contain unusual halogenatedtryptophan residues as well as a ureido bond. The authors suggest that the presence ofseveral unusual amino acid residues in keramamide A (114) and konbamide (115)indicates that they might be produced by microorganisms.OH114160OH115A series of biologically active tetradecapeptides were isolated from the marinesponge Discodermia kiiensis. 102 Discodermins A-D (116-119) contain several D-aminoacid residues, an MeGln residue and at least one tert-Leu residue. The structures of thediscodermins vary at the fourth and fifth residues from the N-terminus having differentcombinations of valine and tert-leucine. All of the discodermins exhibit significantantimicrobial activity and A and C inhibit the development of starfish embryos at 5tg/mL. The discodermins can also be linked to microorganism metabolism. Previously,tert-Leu residues had only been found in the bottromycins isolated from actinomycetes.In addition, the sponge was shown to contain symbiotic bacteria and blue-green alga.HCO -D-Ala-L-Phe -D-Pro-X -D-Trp-L -Arg-D-Cys(03H) -L-yr-L-MeGln-D-Leu -L-Asn -L-Thr-S7116 X = D-t-Leu-L-t-Leu^118 X = D-t-Leu-L-Val117 X = D-Val-L-t-Leu^119 X = D-Val-L-Val161More recently, another biologically active peptide, polydiscamide A (120), wasisolated from a Discodermia Sp . 1 °3 Polydiscamide A (120) also contains a tert-Leuresidue as well as the new residue 3-methylisoleucine. Compound 120 exhibits in vitroactivity against the cultured human lung cancer A549 cell line (IC50 0.7 1.4/mL). Againthe presence of the tert-Leu residue indicates that polydiscamide A (120) might be theproduct of a microorganism.120In contrast to the peptides with unusual residues, pseudaxinellin represents agrowing class of sponge peptides that contain standard L a-amino acid residues. Amongthese are fenestins A (121) and B (122) isolated from the sponge Leucophloeus fenestratacollected off Fiji.t 04 The fenestins were the first examples of medium ring cyclicpolypeptides. Both compounds 121 and 122 contain two proline residues which seem tooccur frequently in peptides of this type. A known diketopiperazine, cyclo-(L-Pro-L-Val)162(123), also containing a proline residue was isolated along with the fenestins. All of theamino acid residues in each of the three peptides had the L-configuration. The authorssuggest that the fenestins might be products of microorganisms due to the fact that theywere obtained from only one of two Fiji collections that contained similar amorphanesesquiterpenes. However, unlike the peptides with unusual residues there is no clearstructural resemblance between the fenestins and known blue-green algae peptides. Likepseudaxinellin (103), the fenestins were not found to have biological activity in a limitednumber of assays.121^ 122In addition to cyclo-(L-Pro-L-Val) (123) two other diketopiperazines, cyclo-(L-Pro-L-Ala) (124) and cyclo-(L-Pro-L-Leu) (125), were isolated from the sponge Tedanisignis collected near Summerland Key, Florida Keys. 105 These structures all containedproline and all residues had the L-configuration.1630RNOHN0123 R = CH(CH3)2124 R = CH3125 R = CH2CH(CH3)2Finally, a cyclic octapeptide hymenistatin 1 (126) was isolated from the WesternPacific Ocean sponge Hymeniacidon sp. collected off the Western Caroline Islands in thePalau Archipelago. 106 Hymenistatin . 1 contained three proline residues and all of theresidues had the L-configuration. Hymenistatin 1 is the only peptide in this category todisplay biological activity, exhibiting cytotoxic activity against the P388 leukemia cellline (ED50 3.5 .tg/mL).126The identification of the two distinctly different peptides, pseudaxinellin (103)and majusculamide C (104) from species of marine sponges, may be indicative of the164origins of peptides from marine sponges. The isolation of the Lyngbya majusculametabolite majusculamide C (104) from the marine sponges P. trachys and Amphimedonsp. demonstrates that at least some peptides isolated from sponges are products of blue-green algae. It is clear that peptides that contain unusual residues, the unnaturalD-configuration and 13-amino acid residues bear a remarkable resemblance to blue-greenalgal peptides. Alternatively, there is no clear correlation between the heptapeptidepseudaxinellin (103), a representative of the general group of marine sponge peptidescontaining only residues having the natural L-configuration and frequently containingprolines, and blue-green algal metabolites.1650HO --- 11...\-- 0HO^OCH30.„NOHOHOHHO127 R = H128 R = OHPart III The Synthetic Confirmation of the TetrahydroisoquinolineSubstructure of Imbricatine Introduction1) Introduction to the StelleroideaThe common seastars belong to the class Stelleroidea (Subclasses Asteroidea andOphiuroidea) within the phylum Ecinodermata. There are 1600 known species ofseastars of which all are marine. Many species of seastars are located in the NortheasternPacific and some seventy species are restricted to the Vancouver Island area. 107Natural products research on the seastars has yielded an enormous number ofsaponins. Many triterpenoid and steroid glycosides have been isolated from terrestrialplants and used as chemotaxonomic markers. 108 In the marine environment theechinoderms produce the majority of these compounds. Some examples are the manysteroidal glycosides isolated from the seastar Crossaster popposus which includecompounds 127 and 128. 109166Though the majority of natural products from Asteroidea have been steroid andtriterpenoid glycosides, there have been some very interesting examples of alkaloids andunusual amino acids isolated. Imbricatine (3) from Dermasterias imbricata was the firstexample of a benzyltetrahydroisoquinoline to be isolated from a marine source. 110 Otherrelated compounds include the ovathiols (129-131) and the disulphides 132 and 133 fromenchinoderm eggs. 111,112SHO3NR 1R2NCH3-^CO2HNSH129 R 1 = R2 = H130 R 1 = CH3, R2 = H131 R 1 = R2 = CH3CO2Hs_s,_ NCH3N)R2N CO2H132 R = H133 R = CH3CH167More recently , fuscusine (134), an alkaloid very closely related to imbricatine,was isolated from the seastar Perknaster fuscus antarcticus. Fuscusine (134) is the onlyother tetrahydroisoquinoline alkaloid to be isolated from a starfish. 1132) Introduction to the Current Study. The study of marine organisms has led to the discovery of some very uniquepredator/prey relationships. One of these is the interaction between the sea anemoneStomphia coccinea and its predator, the starfish Dermasterias imbricata. S. coccinearesponds to contact from D. imbricata first by releasing its basal disc from the substrateand then "swimming" through the water by flexing its elongated column, effectivelyevading its predator. 114 Chemical studies on D. imbricata by Ayer et al., using ananenome response guided isolation, led to the purification of a single component as thecausative agent for the anenome response. 115 Later, Pathirana and Andersen reported thegeneral constitution and partial stereochemistry of the active metabolite, imbricatine (3).The structure of 3 was determined by a combination of spectroscopic and chemicalanalysis. 110 It was found that imbricatine (3) contained the unprecedented C6/C8hydroxylation pattern as well as the unusual C3 carboxyl functionality. Imbricatine (3) isthe first benzyltetrahydroisoquinoline alkaloid to be isolated from a non plant source.1683) Objectives. The hydroxylation pattern in the tetrahydroisoquinoline portion as well as thepositioning of the carboxyl group raised some very interesting questions about thebiosynthesis of imbricatine. Therefore, it was decided that a synthesis of thetetrahydroisoquinoline portion of imbricatine was required to verify the structure as wellas to develop some of the more obvious biosynthetic precursors for future studies.There is considerable evidence to support the hypothesis that, in mammaliantissue, dopamine (135) condenses with 3,4-dihydroxyphenylacetaldehyde to yield the 6,7-dihydroxytetrahydroisoquinoline 136. 116 Likewise, it is probable that the previouslyunknown amino acid 137 and a tyrosine derivative are the immediate precursors of thetetrahydroisoquinoline portion of imbricatine. It is also reasonable to assume that, in thenatural system, the thiohistidine is added to the intact amino acid 137 or possibly to theintact tetrahydroisoquinoline 139.HOHOHO HOOR2OR2135^ 136 R 1 = H R2 = H138 R 1 = CO2Me R 2 = Me169OR2OH137^ 139 R 1 = H R2 =H140 R 1 = Me R2 = Ac4) Synthetic PlanAs part of the structure elucidation of imbricatine (3), Pathirana and Andersenemployed a Raney nickel reduction to cleave the thiohistidine bond and liberate the freetetrahydroisoquinoline alkaloid 139. 110 Compound 139 was derivatized to form theprotected methyl ester tetraacetate 140. At the outset of the present study, it was decidedthat compound 140 was an appropriate target molecule for synthetic verification of theconstitution of the tetrahydroisoquinoline portion of imbricatine (3). In addition, thedeprotected tetrahydroisoquinoline 139 and the new amino acid 137, were thought to bepotential intermediates in the biosynthesis of imbricatine and therefore good synthetictargets.Retrosynthetic analysis of 140 revealed that the most obvious precursors of thetetrahydroisoquinoline were a tyrosine derivative and a meta disubstituted phenylalaninederivative (Schemes 11 and 12). These precursors could be coupled in a Pictet-Spenglercondensation similar to that used previously to produce the tetrahydroisoquinoline138. 117 The tyrosine moiety could be introduced using the sodium glycidate 141 whichcan be produced in a three step procedure from the readily available para hydroxy170OH143+ORROCO2Na140 R = AcHO141 R = C6H5CH2142RObenzaldehyde (142). 118 The phenylalanine derivative 143 could be produced byalkylation of diethyl acetamidomalonate (145) with the appropriate benzyl derivativefollowed by hydrolysis and decarboxylation. The benzyl derivative could be preparedfrom the protected 3,5-dihydroxy benzoate by reduction and tosylation at the benzylicposition.Scheme 11. Retrosynthetic analysis of tetraacetate 140.171OTsOHOR147 ^146 R = C6H 5 CH2CO2EtH .1.__ CO2EtNHCOCH3145HO CO2MeRO+HORO OH1 4 3OR144 R = C6H 5 CH 2Scheme 12. Retrosynthetic analysis of amino acid methyl ester 143.The following discussion describes the synthesis of the racemic methyl esterprotected amino acid 143 as well as the synthesis of the protected tetrahydroisoquinolinealkaloid 140. Compound 140 was found, by tic, 1H NMR (Fig. 73) and mass spectralcomparison, to be identical to the protected Raney nickel reduction product of imbricatineproduced by Pathirana and Andersen.110172Results and DiscussionAll of the reactions attempted were based on literature procedures on relatedcompounds. In all cases, the literature reaction conditions were used in initial attemptsand in some cases subsequent variations were made to increase yields.HO OHK2CO3 /C6H5CH2Br^111.-acetone, reflux, 1 hRO OR147^148 R = C6H5CH2Scheme 13. Benzyl protection of methyl 3,5-dihydroxybenzoate 147Initially, methyl protecting groups were chosen for the hydroxyl functionalities ofmethyl 3,5-dihydroxybenzoate 119 (147 in NaOH and dimethylsulphate, reflux, 2 h). 1 20However, attempts at deprotection after further manipulations resulted in complexmixtures of degradation products. This was also true for the methoxy methyl (MOM)protected derivative (Chloromethyl methyl ether, K2CO3, 147, CH3CN). 121 In bothcases, the addition of concentrated acid for deprotection resulted in resinous tars. Thebenzyl protected derivative of 147 was prepared by stirring 147, K2CO3 and benzylbromide in anhydrous acetone under reflux. 122 The benzyl protecting groups were foundto be stable to all of the reaction conditions used in subsequent steps and were easilyremoved under catalytic hydrogenation conditions. Compound 148 was isolated ascolourless crystals (m.p. 66.0-67.5 °C) along with the monoprotected compound whichwas recycled. Ester 148 gave a parent ion in the HREIMS at m/z 348.1364 (AM 0.2mmu) appropriate for the molecular formula of C22H20O4. The presence of 13 aromaticprotons and 4 methylene protons (8 4.97) in the 1 H NMR and the absence of a phenolicstretching band in the FTIR identified compound 148 as the major product.173ROLiAIH4, Et20reflux, 2.5 hOR^RONaHasCIC6H6 , r.t., 9 hOR^RO148 R = C6H5CH2^149 R = C6H5CH2 146 R = C6H5CH2Scheme 14. Reduction and tosylation at benzylic position.Reduction of the methyl benzoate 148 with LiA1H4 in diethyl ether under refluxquantitatively produced 3,5-dibenzyloxy benzyl alcohol (149). 123 Alcohol 149 existed aswhite crystals (m.p. 78.5-79 °C) that gave a parent ion in the HREIMS at m/z 320.1420Da corresponding to a molecular formula of C211-12003. The 1 H NMR containedresonances appropriate for a benzylic methylene (8 4.50, s, 2H) and an OH proton (82.29, bs, 1H). An OH stretching frequency at 3356 cm -1 in the FTIR confirmed thepresence of the hydroxyl group.The benzyl alcohol was converted to the benzyl tosylate according to theprocedure used by Williams et al . 124 This method was chosen mainly due to the ease ofworkup which simply involved centrifugation of the mixture followed by evaporation ofthe supernatant to dryness. Compound 149 and sodium hydride were stirred in benzeneand p-toluenesulfonyl chloride was slowly added to the mixture. The solution was stirredat room temperature for 9 h. After workup and purification by flash chromatographycompound 146 was obtained as a white solid which upon standing turned a yellow colour.Due to its instability, the product was either used immediately in the next reaction orstored under nitrogen in the dark at 4 °C. In addition to the 1 H NMR signals attributableto the dibenzyloxy benzyl residue, the spectrum of compound 146 contained a pair ofortho coupled aromatic resonances and a methyl singlet at 8 2.39 ppm attributable to thetosylate group. The FTIR of 146 did not contain an OH stretching band.174CO2EtH \/...-- 0O2EtNHCOCH3OTsNaH, RO^OR146C6H6/DMF, r.t., 6 hROOROR144OR150CO2EtCO2Et 1) 20% NaOH,dioxane, reflux, 3 hNHCOCH3 2) acidify with AcOHreflux, 1 hRO RO145^144Scheme 15. Alkylation of diethyl acetamidomalonate (145) with benzyl tosylate 146.The use of diethyl acetamidomalonate to introduce a glycine residue is a standardliterature method which has been used to produce amino acids such as phenylalanine,serine, leucine, ornithine and tryptophan. 125 The enolate anion of 145 was producedusing NaH (145, NaH in 1:1 C6H6/DMF). 124 The subsequent addition of the benzyltosylate 146 and stirring at room temperature for 6 h gave a mixture which was purifiedby flash chromatography to yield compound 144 as white crystals (m.p. 138.5-140.0 °C).Diester 144 gave a parent ion in the mass spectrum at m/z 519.2254 Da (AM -0.3 mmu)appropriate for a molecular formula of C30H33N07. The 1 H NMR (Appendix B, Fig. 82)showed resonances that could be uniquely assigned to the protons in 144. The ethyl estermoieties were observed as a multiplet at 8 4.23 ppm and a triplet at 1.25 ppm and theamide group was apparent from the NH proton at 8 6.56 ppm and the methyl at 8 1.95ppm. The three meta substituted aromatic protons were observed as two singlets at 8 6.51(1H) and 6.26 (2H) ppm.Scheme 16. Hydrolysis of alkylation product 144.175143Hydrolysis and decarboxylation was accomplished by refluxing a solution of 144in dioxane and 20% NaOH for 3 h followed by acidification with acetic acid andrefluxing for an additional 1 h. Acetic acid was used in the decarboxylation step ratherthan HC1, which was used by Haudegond et al., 125 because the latter afforded a complexmixture of products. After workup and evaporation in vacuo, compound 150 was isolatedin excellent yield (93%). The presence of an a-methine resonance (8 4.51, bs) and (3-methylene protons at 8 3.15 and 2.90 ppm in the 1H NMR of the crude mixture (appendixB, Fig. 83) showed that the reaction went virtually to completion.CO2HNHCOCH31) NH2NH2, 100°C, 24 h2) Me0H/HCI, reflux, 1 h3) H2, Pt/C, r.t., 10 hRO^OR^ HO150 R = C6H5CH2Scheme 17. Preparation of compound 143.The next three steps were accomplished without purification and with minimalworkup between each step in order to increase yields. Removal of the N-acetyl groupwas accomplished by heating a solution of compound 150 in hydrazine hydrate for 24 h.This procedure has been used effectively for removing N-benzoyl groups frompolybenzoylnucleosides as well as for regenerating amino functionalities fromacetamidodeoxy sugars. 126 The excess hydrazine was removed in vacuo and the mixtureesterified in Me0H/FIC1 under reflux. Phenolic deprotection was then accomplishedunder catalytic hydrogenation conditions. Purification of the resulting mixture usingreversed phase flash chromatography (1:1 Me0H/H20) afforded the methyl esterprotected amino acid 143. The HREIMS of 143 gave a parent ion of m/z 211.0851 Da,176appropriate for the molecular formula of C10H1304N (AM 0.7 mmu). The 1 H NMRspectrum (Appendix B, Fig. 84) showed resonances appropriate for a methyl ester (53.83), an a-methine (5 4.26) and a benzylic methylene (5 3.08, 2H), as well as metasubstituted aromatic protons (5 6.25(1H) and 6.22(2H)). The FTIR showed a carbonylstretching frequency at 1743 cm -1 . H^K2co3 ,c6H5cH2BrHOacetone, reflux, 1 hRO142 151 R = C6H5CH2C1CH2CO2Me,Na0Me/Me0H,0°C, 5.5 hr.t., 3 hCO2NaNaOMe/MeOHCO2MeROC6H6, 10°C, 3 hRO141 R = C6H5CH2 152 R = C6H5CH2Scheme 18. Preparation of sodium p -benzyloxy glycidate (141).The other starting material for the Pictet-Spengler condensation, sodium-3-(p-benzyloxyphenyl) glycidate (141), was prepared from p-hydroxy benzaldehyde (142) 83 ina three step procedure. Reaction of p-benzyloxy benzaldehyde (151) withmethylchloroacetate in sodium methoxide in methanol yielded methyl-3-(p-benzyloxyphenyl) glycidate (152). The next step involved preparation of the sodium gycidate 141by addition of sodium methoxide to a solution of 152 in benzene. 118 A slight excess ofwater was added (1.2 equiv.) and the resultant precipitate was filtered to yield 141 as awhite solid that was used directly in the Pictet-Spengler condensation withoutpurification.177HO+ROCO2NaOH143^ 141 R = C6H 5CH2Me0H/H20,HOAc,35°C, 17 hOR153 R = C6H5 CH2^154 R = C6H5CH 2Scheme 19. Pictet -Spengler reaction.Initially, the Pictet-Spengler reaction was attempted using the methoxyl protectedderivative of amino acid 143. 127 This resulted in a mixture of products, none of whichcould be identified as the desired tetrahydroisoquinoline. In previous studies on Pictet-Spengler reactions it was shown that protection of the phenolic group para to the site ofcondensation on the ring prevented reaction from occurring (Scheme 20). 128 Clearly thephenolic group at C6 participates in the reaction by making the C9 position morenucleophilic. Therefore, the Pictet-Spengler reaction was accomplished using thedeprotected amino acid methyl ester 143. A water suspension of 141 was added to asolution of 143 in methanol. The mixture was acidified with acetic acid and stirred at35°C for 17 h under N2. Work up and purification using open column silica gelchromatography yielded an inseparable mixture of diastereomeric178R 10CH3CHOR 10R20 R20CH3R1 R2 % YieldH H 79H CH3 89CH3 H 0CH3 CH3 0Scheme 20. Effect of methyl protection on Pictet-Spengler reaction. 128tetrahydroisoquinolines. The HREIMS of the mixture gave a parent ion at m/z 419.1728Da appropriate for a molecular formula of C25H25N05 (AM -0.4 mmu). The 1 H NMRspectrum (Appendix B, Fig. 85) of the mixture demonstrated signals representative of thetwo racemic diastereomers 153 and 154. The aromatic doublets from the tyrosine moietywere observed at 8 7.37 and 7.06 ppm in the major racemate and at 8 7.17 and 6.97 ppmin the minor racemate. The aromatic protons assigned to H5 and H7 were observed asbroad singlets at 8 6.22 and 6.13 ppm in the major racemate and at 8 6.26 and 6.06 ppmin the minor racemate. The ratio of the two diastereomers in the mixture, calculated bythe integration of like resonances in the 1 H NMR spectrum (Appendix B, Fig. 85), wasapproximately 6:1. Several attempts to purify the mixture using normal phase preparativetic (3:7 CH2C12/EtOAc), normal phase HPLC (1:4 CH2C12/EtOAc), and reversed phaseHPLC(4:1 Me0H/H20) resulted in either isolation of the same mixture or complete lossof the minor diastereomer.179OR153 cis R = C6H5CH2154 trans R = C6H5CH2Ac20, pyridiner.t., 19 hOHNAcOAcOROAcAc0^ CO2Me1) H2, Pt/C, Me0H, r.t., 3 h2) Ac20, pyridine, r.t., 15 hNAcOAcOR157 cis R = C6H5CH2158 trans R = C6H5CH2140 cis162 transOAcHO^ CO2Me^Ac0^ CO2Me157 cis R = C6H5CH2158 trans R = C6H5CH2Scheme 21. Acetylation of Pictet -Spengler products.Acetylation of the tetrahydroisoquinoline mixture in acetic anhydride and pyridineyielded after purification by preparative tic (1:1 Hex/EtOAc) the triacetates of the majorand the minor diastereomers, 158 and 157 respectively. The 1 H NMR spectrum ofcompound 158 contained signals representative of two distinct conformers inapproximately a 3:2 ratio. The 1 H NMR spectrum of compound 157 indicated that it alsoexisted as two distinct conformers with one being predominant (>90%).Scheme 22. Preparation of tetraacetates 140 and 162.180Debenzylation of the cis racemate 157 under hydrogenation conditions (H2 Pt/C,Me0H, 3 h) yielded after work up compound 159. Acetylation of the resulting phenolicproduct after purification gave the cis benzyl tetrahydroisoquinoline derivative 140 as theracemate which was identical by 1 H NMR comparison to the tetraacetate prepared byPathirana et al. from imbricatine. 129 The HREIMS of 140 gave a parent ion at m/z497.1691 (AM 0.5 mmu) appropriate for the molecular formula of C26H27N09,accounting for all of the atoms in 140. The 1H NMR (Fig. 73) contained resonancesappropriate for two meta aromatic protons (8 6.94 and 6.88) as well as for a symmetricaltyrosine residue (8 7.29 and 7.04). The four acetate methyls (6 2.290, 2.287, 2.20 and1.78) and one methyl ester (8 3.85) had the identical chemical shifts to those in the 1HNMR spectrum of the tetracetate produced from the Raney nickel reduction product ofimbricatine (Fig. 74). The H1 and H3 methine protons appeared as doublets of doublets(8 5.06(H3) and 4.47(H1)) coupled to the upfield benzylic methylenes. The 1H NMR ofthe synthetic racemate 140 (Fig. 73) contained what was first believed to be a inseparableminor impurity from the synthesis. However, it was noticed that the 1 H NMR of theprotected Raney nickel reduction product (Fig. 74) 129 also contained these resonances.This suggests that the cis tetrahydroisquinoline tetraacetate also exists in two conformerswith one conformer being greatly favored and further supports the conclusion that thetetraacetates prepared from the Pictet-Spengler condensation and the Raney nickelreduction of imbricatine have the same constitution and relative stereochemistry.Debenzylation followed by acetylation of the trans compound 158 resulted in a product162 ( 1 H NMR: Appendix B, Fig. 86) that was different from 140.As part of the structure elucidation of imbricatine 130 nOe difference experimentsdemonstrated that the tetrahydroisoquinoline substructure had the cis relativestereochemistry. An nOe was observed between H1 and H3 in the methyl ester protectedRaney nickel reduction product indicating that these two protons had a 1,3-diaxialrelationship. Thus, in the Pictet-Spengler reaction the H 1/H3 trans product was greatly181favored. During the synthesis of the naturally occurring alkaloid S-(+)-laudanosine byKonda et al., 117 L-Dopa methyl ester hydrochloride (163) was condensed with sodium 3-(3,4-dimethoxyphenyl) glycidate under Pictet-Spengler conditions to yield the cistetrahydroisoquinoline 138 as the major product (yields: cis 30.8%, trans 9.9%). Theseresults indicated that the ratio of products in the present study was directly related to thesteric effect of the C8 hydroxyl during the condensation reaction which was not present inthe synthesis of S-(+)-laudanosine.HOHOHOHO163 A poor yield was obtained in the Pictet-Spengler condensation reaction where theyield of the diastereomeric mixture was only 36 % and therefore the yield of the cisproduct was 6 %. Nevertheless, the goal of structure verification was accomplished.Subsequently, Miao et al. demonstrated that imbricatine had the 1R, 3R, and 7'Sabsolute stereochemistry. 130 Determination of the configuration of thetetrahydroisoquinoline was accomplished by comparison of the Raney nickel reductionproduct CD spectra with that of the model compounds 165 and 166 prepared fromL-Dopa methyl ester (163) and 164 (Scheme 22). It was found that the cis modelcompound 165 had the opposite Cotton effect of the Raney nickel reduction productmethyl ester of imbricatine (3). Since model compound 165 had the 1S,3S absoluteconfiguration it was concluded that the Raney nickel reduction product methyl ester andits tetraacetate 140 had the 1R,3R configuration.182+HOHO MeOCO2Na163 164165 1661) H2O, HOAc, 35*C, 36 h2) Ac2O, pyridine, r.t., 24 hCO2Me^HO A^CO2MeHOHO HOOMe OMeScheme 23. Preparation of model compounds for CD analysis by Miao et al. 130183S wAcO A^„.0O2MeNAcOAcOAcFig. 73. 1H NMR spectrum of cis tetraacetate 140 (500 MHz, CDC13).9 8 7 6^5 4^3 2 1 0Fig. 74. 1H NMR of Raney nickel reduction product tetraacetate 140 (400 MHz, CDC13).ConclusionIn conclusion, the verification of the constitution of the tetrahydroisoquinolineportion of imbricatine was accomplished by synthesis of the protected derivative of theRaney nickel reduction product of imbricatine 140 as the racemate. In addition,compounds 140 and 143 were developed as the methyl ester and acetyl protectedderivatives of the probable biosynthetic precursors. Deprotection of these compoundscould afford the biosynthetic precursors for future studies.186ExperimentalA.^General The 1 H NMR spectra were recorded on either a Bruker AMX-500 or a BrukerWH-400 NMR spectrometer. Chemical shifts are reported with respect to the internalstandard TMS (8 0.00 ppm) or the residual solvent peak (CDC13: 7.26 ppm; C6D6: 7.15ppm). The 13C NMR and APT 131 spectra were recorded on either a Varian XL-300 (75MHz), a Bruker AM-400(100MHz) or a Bruker AMX-500 (125 MHz) NMRspectrometer. All carbon chemical shifts are reported with respect to TMS (8 0.00 ppm)using the solvent peak as a secondary reference (DMSO-d6: 39.5 ppm; CDC13: 77.0ppm; C6D6: 128.0 ppm). COSY 132 , nOe difference 133 and double resonance 134experiments were conducted on either Bruker WH-400 or Bruker AMX-500 NMRspectrometers. All ROESY, 135 HOHAHA, 136 inverse-detected HETCOR (HMQC) 137and long range HETCOR (HMBC) 138 experiments were recorded on a Bruker AMX-500NMR spectrometer.Low resolution and high resolution EI mass spectra were recorded on Kratos AEIMS-59 and AEI MS-50 mass spectrometers, respectively. Chemical ionization massspectra were recorded on a Delsi-Nermag R-10-10 quadrupole mass spectrometer usingeither methane or ammonia as the ionization gas. The low resolution and high resolutionFAB mass spectra of majusculamide C (104) were recorded on a Kratos Concept HQspectrometer.Infrared spectra were recorded on a Perkin-Elmer 1600 Fourier Transformspectrometer. All FTIR spectra were acquired as films on sodium chloride plates.Melting points were obtained with a Fisher-Johns melting point apparatus. Opticalrotations were measured using a Jasco J-710 spectrophotometer.187Normal phase flash chromatography was accomplished on Merck silica gel G60(230 - 400 mesh). Reversed phase flash chromatography was accomplished on reversedphase silica prepared from silica according to Kuhler et a1. 139 Normal phase andreversed phase thin layer chromatography was accomplished using Merck Type 5554silica gel plates and Whatman MKC18F tic plates respectively. Size exclusionchromatography was accomplished using Sephadex LH-20 resin. Radial chromatographywas performed using a Model 7924 chromatotron (Harrison research), an FMI Model RPG-150 lab pump and plates coated with a dried layer of an aqueous slurry of Mercksilica gel 60 PF-254 and CaSO4.1/2H20. Normal phase preparative chromatography wasaccomplished using Merck Type 5554 silica plates. Reversed phase preparativechromatography was performed using Whatman KC18F tic plates. High performanceliquid chromatography was done on a Perkin-Elmer Series 2 liquid chromatographattached to a Perkin-Elmer LC-25 refractive index detector. Preparative HPLC wasaccomplished using Whatman Magnum-9 Partisal (normal phase) and WhatmanMagnum-9 Partisal 10 ODS-3 (reversed phase) columns. Analytical HPLC wasaccomplished using Alltech 5 micron silica(normal phase) and 10 micron C18 (reversedphase) columns. All solvents used for HPLC were BDH Omnisolve grade. All reagentswere commercial grade and unless otherwise stated were used directly without furtherpurification. An exception was pyridine which was distilled over BaO and stored incontact with Linde type 4A molecular sieves.Antifungal and antibacterial bioassays were performed by Mike LeBlanc (Dept. ofOceanography, UBC). Cytotoxicity bioassays (in vitro L1210 murine leukemia cell lineand in vivo P388 murine leukemia cell line) were performed in Alberta by those under thesupervision of Theresa M. Allen (Dept. of Pharmacology, University of Alberta).188B.^Metabolites from the Papua New Guinea Sponge Petrosia contignataCollection and Isolation dataSpecimens of P. contignata (2.5 kg wet weight) were collected by hand usingSCUBA at Madang Papua New Guinea and transported to Vancouver frozen over dry ice.The frozen sponge specimens were immersed in methanol (3 L) and soaked at roomtemperature for 48 h. Concentration of the decanted methanol in vacuo gave an aqueoussuspension (1800 mL) that was sequentially extracted with hexanes (4 X 500 mL) andchloroform (4 X 1 L).Evaporation of the combined hexanes extract in vacuo gave a brown oil that wassubjected to silica gel chromatography (4:1 Hex/EtOAc). A total of eighty fractions werecollected and analysed by tic and pooled to yield seven major fractions. 1 H NMR of thefractions indicated the presence of the known tetrabromodiphenyl ether 41 in fraction Band the new sesquiterpene 37 in fraction C. A second application of silica gelchromatography (4:1 Hex/EtOAc) on fraction B yielded 41. Radial silica gel tic (9:1Hex/EtOAc) on fraction C followed by normal phase HPLC (9:1 Hex/EtOAc) yielded 37(6.2 mg).Evaporation of the combined chloroform extracts in vacuo gave a brown solid(2.1 g). The crude chloroform extract was subjected to Sephadex LH-20 (4:1Me0H/H20) which yielded fractions that were analysed by 1 H NMR and tic and pooledto yield five major fractions. The 1 H NMR spectrum of the fourth fraction showedsignals attributable to contignasterol (24). Final purification was achieved by sequentialapplications of reversed phase flash chromatography (4:1 Me0H/H20) and reversedphase HPLC (3:1 Me011/1120) to give contignasterol (24) as colorless crystals (153 mg).189Contignasterol (24):OHHO"CH324Compound 24: obtained as colourless needles from Me0H/H20 (10:1) mp 239-41 °C; Rf = 0.14 and 0.20 (reversed phase tic: 3:1 Me0H/H20); [alp +45 (MeOH, c0.4); FTIR (film) 3381, 1719 cm -1 ; 1 H NMR (500 MHz, DMSO-d6) 8 6.21(bs),5.95(bs), 5.74(bs), 5.16(bs), 4.53(bm), 4.50(bm), 4.34(bs), 4.16(bm), 4.04(bs), 3.88(bs),3.78(bt), 3.62(bs), 3.22(bt), 3.05(bs), 3.00(bs), 2.38(m), 2.09(bd,J=20.0 Hz), 1.13(s),0.93(s), ppm; 13C NMR (125 MHz, DMSO-d6) 8 219.4, 219.3, 95.6, 90.4, 75.3, 73.9,73.8, 70.4, 70.2, 68.7, 68.1, 67.8, 50.7, 50.6, 46.3, 45.9, 45.04, 44.96, 41.3, 41.2, 40.1,38.9, 38.6, 38.4, 38.2, 36.9, 35.8, 35.5, 34.7, 34.0, 32.6, 32.2, 32.0, 31.9, 23.6, 20.2, 19.6,19.3, 19.2, 18.95, 18.9, 16.8, 16.7, 14.9 ppm; HREIMS M± 508.3394 (C29H4807 AM-0.6 mmu); LREIMS m/z(relative intensity): 508(0.8), 493(9.2), 490(16.4), 472(78.5),457(14.0), 454(29.6), 447(34.8), 426(13.6), 408(37.3), 393(12.2), 319(38.7), 301(14.7),283(19.9), 264(45.4), 246(21.0), 221(23.6), 203(34.4), 155(23.3), 119(37.1), 109(43.4),95(59.1), 93(51.3), 55(100.0), 43(55.5).190CH3RC 0.P25 R = Ac R1 = H26 R = R1 = AcContignasterol tetraacetate (25):Contignasterol (24) (18.0 mg, 0.031 mmol) was stirred in pyridine (2 mL) andacetic anhydride (2 mL) at room temperature for 18 h. The reagents were removed invacuo, and the resulting gum was purified using normal-phase HPLC (3:2 Hex/EtOAc) toyield the tetraacetate (25) (5.8 mg) and the pentaacetate (26) (1.0 mg). Compound 25:colorless oil; fah) +63 (CH2C12, c 0.34); FTIR (film) 3477, 1748, 1736 cm -1 ; 1H NMR(400 MHz, C6D6) 8 0.64(m, J = 10.8, 12.2 Hz, H23), 1.12(s, Me19), 1.20(s, Me18),1.61(s, OAc), 1.71(s, OAc), 1.82(s, OAc), 1.88(s, OAc), 2.06(bd, J = 19.7 Hz, H16),2.30(dd, J = 19.7, 9.9 Hz, H16'), 2.34(bs, H14), 3.05(bd, OH4), 3.33(bt, H22), 3.87(bm,H4), 5.24(bm, H3), 5.40(dd, J = 8.6, 12.1 Hz, H6), 5.60(dd, J = 2.2, 9.5 Hz, H29),6.63(dd, J = 8.6, 10.4 Hz, H7) ppm; 13C NMR (125 MHz, C6D6) 8 14.5, 15.2, 19.3, 19.5,19.8, 20.4, 20.5, 20.6, 20.7, 20.8, 21.5, 32.6, 32.6, 32.8, 33.4, 36.3, 36.4, 37.3, 40.5, 40.6,41.7, 42.2, 43.0, 45.9, 46.6, 51.7, 66.6, 71.6, 73.8, 74.7, 78.0, 94.3, 169.1, 169.3, 169.4,172.7, 216.0 ppm; HREIMS (M+ - HOAc) m/z 616.3605 (C35H5209 AM -0.6 mmu);191Contignasterol reduction product (27):ORCH3CH3RO'‘H E.OH OR27 R = H28 R =AcLREIMS m/z(relative intensity): 616(0.5), 556(1.0), 513(7.2), 496(3.2), 436(5.3),421(1.9), 361(3.8), 123(100.0), 60(59.8), 43(68.5).Contignasterol pentaacetate (26): obtained as a colourless oil; 1 H NMR (400MHz, C6D6) 8 0.64(m), 0.75(d, 3H), 0.76(d, 3H), 0.77(d, 3H), 0.94(s, 3H), 1.24(s, 3H),1.54(s, 3H), 1.80(s, 3H), 1.86(s, 3H), 1.89(s, 3H), 1.95(s, 3H), 2.31(dd, J = 10.3, 20.0Hz), 2.39(bs), 3.32(bt), 5.10(m), 5.45(dd, J = 9.0, 12.0 Hz), 5.47(bs), 5.60(dd, J = 2.2, 9.0Hz), 6.54(dd, J = 9.0, 10.6 Hz) ppm.NaBH4 (2 lmg) was added to a solution of contignasterol (24) (12.5mg) inisopropyl alcohol (10m1). The reaction mixture was stirred at room temperature for 1 hand quenched with H20(10m1). The resulting suspension was extracted with EtOAc(2 x10m1) and the organic layer was washed with 1N HC1(10m1) and H20(10m1). Theorganic layer was dried over magnesium sulphate. The mixture was purified usingreversed phase HPLC (3:1 Me0H/H20) to yield the reduction product 27 (7.6mg,61%) asa white solid.192Reduction product pentaacetate (28): Reduction product 27 (7.6mg) wasstirred in pyridine (1m1) and acetic anhydride (1m1) at room temperature for 17 h. Thereagents were removed in vacuo and the resulting gum was purified on normal phaseHPLC (1:1 Hex/Et0Ac) to yield the pentaacetate 28. Compound 28: colourless oil; 1HNMR(400 MHz, C6D6) 8 0.74(d, J=6.8 Hz, H27), 0.76(d, J=6.8 Hz, H26), 1.04(s,Me19), 1.07(s, Me18), 1.48(m, H23'), 1.59(s, OAc), 1.72(s, OAc), 1.76(s, OAc), 1.80(m,H17), 1.82(s, OAc), 1.91(m, H20), 1.99(m, H8), 2.00(m, H2), 2.08(s, OAc), 2.15(dd,J=3.6,7.8 Hz, H14), 2.49(m, H16), 2.63(d, OH4), 3.54(dd, J=5.9,9.4 Hz, H22), 3.82(bm,H4), 5.07(dd, J=8.9,11.2 Hz, H7), 5.18(bm, H3), 5.25(m, H15), 5.32(dd, J=8.9,12.2 Hz,H6), 5.75(dd, J=2.2,9.7 Hz, H29) ppm; HREIMS (M+ - HOAc) 660.3871 (C371 -156010AM -0.2 mmu); LREIMS m/z(relative intensity): 660, 642, 615, 600(1.2), 540(2.3),497(2.8), 481(3.8), 462(1.0), 437(2.1), 420(6.3), 123(100.0).Contignasterol reduction product 29:29 R = H30 R = AcNaBH4 (40 mg) was added to a stirring mixture of contignasterol (24) (25.3 mg,0.05 mmol) in isopropyl alcohol (10 ml). The solution was refluxed for 0.5 h, cooled toroom temperature and quenched with H2O (10 mL). The resulting mixture was extracted193with ethyl acetate (3 x 15 mL) and the combined extracts were dried over Na2SO4.Purification was accomplished using reversed phase HPLC (13:7 Me0H/H20) to yieldthe reduction product 29 (14.3 mg) as a white solid; FTIR (film) 3384 cm -1 ; 1 H NMR(500 MHz, 10:1 CDC13/DMSO) 8 0.82(d, J = 7.0 Hz, H26), 0.90(d, J = 7.0 Hz, H27),0.93(d, J = 7.0 Hz, H21), 0.96(s, H18), 1.08(s, H19), 2.24(m, H16'), 3.30(bm, H7),3.75(bd, OH), 3.91(bs, H3), 4.01(bs, H4), 4.08(bs, OH), 4.20(bs, OH), 4.32(bs, OH) ppm;13C NMR (125 MHz, 10:1 CDC13/DMSO) 8 12.11, 14.05, 14.84, 17.52, 19.69, 19.72,23.64, 29.70, 30.36, 31.76, 33.24, 35.63, 36.38, 36.54, 37.44, 40.51, 41.67, 42.60, 46.04,52.69, 52.73, 60.22, 60.43, 68.24, 68.98, 70.55, 70.71, 71.98, 79.50 ppm.Reduction product tetraacetate 30: Reduction product 29 (8.3 mg) was stirredin pyridine (2 mL) and acetic anhydride (2 mL) at room temperature for 14 h. Themixture was evaporated to dryness in vacuo and passed through a normal phase sep pak(EtOAc eluent). Purification was accomplished using normal-phase HPLC (7:13Hex/EtOAc) to yield the tetraacetate 30: colourless oil; FTIR (film) 3454, 2941, 1736cm-1 ; 1H NMR (500 MHz, C6D6) 8 0.64(bt, J = 11.6 Hz, H9), 0.70(dd, J = 5.4, 10.8 Hz,H14), 0.81(d, J = 6.8 Hz, H27), 0.88(d, J = 6.8 Hz, H26), 1.04(s, H18), 1.07(s, H19),1.68(s, 6H, 2 X OAc), 1.75(s, OAc), 1.82(s, OAc), 2.39(dt, J = 8.2, 14.1 Hz, H16'),3.32(t, J = 8.8 Hz, H7), 3.44(d, J = 3.5 Hz, OH), 3.80(bs, H4), 4.07(dt, J = 7.1, 10.9 Hz,H29'), 4.20(m, H29), 5.11(dd, J = 8.8, 12.0 Hz, H6), 5.32(m, H22), 5.38(m, H3) ppm;13C NMR (125 MHz, C6D6) 8 13.5, 14.8, 15.0, 17.5, 20.2, 20.4, 20.60, 20.63, 20.9, 21.0,21.9, 29.1, 29.3, 29.8, 33.2, 36.6, 37.81, 37.83, 39.2, 40.0, 41.2, 43.6, 46.8, 53.2, 53.7,61.0, 63.3, 66.8, 71.2, 71.9, 74.8, 77.1, 77.7, 169.1, 170.1, 170.3, 173.5 ppm; HREIMS(M+ - H2O) 662.4350 (C37}158010 AM -0.1 mmu); LREIMS m/z(relative intensity):680(0.4), 662(0.9), 620(4.3), 602(3.1), 542.5(20.9), 524(9.0), 482(31.6), 464(14.6),421(24.6), 361(9.7), 344(6.9), 267(14.8), 229(0.9), 109(36.6).194Petrolactone (37):37Compound 37: white solid; FTIR (film) 1769 cm -1 ; 1 H NMR (500 MHz, C6D6) 80.40(s, 3H, H15), 0.65(s, 3H, H11), 0.70(s, 3H, H12), 1.65(m, H8), 2.01(m, H7), 3.30(dd,H14), 3.62(dd, H14') ppm; 1H NMR (400 MHz, CDC13) 8 0.85(s, 3H, H11), 0.89(s, 3H,H12), 0.96(s, 3H, H15), 1.80(m, H7), 1.87(m, H9), 2.21(m, H6'), 2.27(m, H8), 3.94(dd, J= 8.4, 10.4, H14), 4.20(dd, J = 6.9, 8.4, H14') ppm; 13C NMR (75 MHz, CDC13) 8 14.0,18.5, 21.4, 21.5, 25.9, 33.6, 38.8, 39.1, 42.0, 55.0, 56.0, 68.0 ppm; HREIMS M+236.1768 (C15H2402 A M -0.9 mmu); LREIMS m/z(relative intensity): 236(2.5),221(29.5), 203(1.7), 193(2.2), 175(9.0), 149(13.8), 123(24.3), 121(15.6), 109(23.7),107(26.3), 105(79.0), 95(34.4), 93(29.1), 91(29.5), 81(43.0), 79(32.9), 69(50.4), 67(41.6),57(30.4), 55(69.2), 43(61.3), 41(100.0).Diphenylether (41):OH^OMeCompound 41: obtained as a colourless oil; 1 H NMR (400 MHz, CDC13) 87.45(d, J = 2.2 Hz), 7.38(d, J = 2.2 Hz), 7.18(d, J = 2.2 Hz), 6.79(d, J = 2.2 Hz), 4.03(s);1951H NMR (500 MHz, CD3OD) 8 7.38(d), 7.35(d), 7.13(d), 6.50(d), 3.96(s); HREIMS M+533.7120 (C131180379Br81Br3 AM -3.0 mmu), 531.7176 (C13H803 79Br281 Br2 AM 0.6mmu), 529.7217 (C13H8O379Br381 Br AM 2.7 mmu); LREIMS m/z(relative intensity):536(14.4), 534(62.4), 532(100.0), 530(62.1), 528(16.0), 440(16.8), 438(48.9), 436(52.4),434(17.9), 374(19.6), 372(41.8), 370(20.4).196C.^Metabolites from the Northeastern Pacific Sponge Acanthella sp. Collection and Isolation dataSpecimens of Acanthella sp. (6.1 kg wet weight) were collected by hand usingSCUBA in Rennel Sound, Queen Charlotte Islands, B.C. and transported to Vancouverfrozen. The sponge was immersed in methanol (8L) and soaked at room temperature for72 h. Concentration of the decanted methanol in vacuo gave an aqueous suspension (1L)that was sequentially extracted with hexanes (2 x 1L), chloroform (2 x 1L), and ethylacetate (2 x 1L). Evaporation of the combined hexanes extracts gave a brown oil (1.91g). Initial purification of the hexane extract was accomplished using normal phase flashchromatography using a step gradient elution from 100% hexanes to 100% ethyl acetatein 25% increments. The fourth and fifth fractions were combined to yield four mainfractions. The first fraction contained several as yet to be identified volatile turpenes.The second fraction was subjected to normal phase HPLC (100% Hex) yieldingacanthene A (80). The third fraction contained several compounds that were purifiedusing successive applications of normal phase HPLC (5% EtOAc/Hex and 100% Hex) toyield, in order of elution, compounds 81-89. The fourth fraction contained the more polarformamide 90.197Compound 80:Compound 80: obtained as a colorless oil; 1 H NMR (400 MHz, C6D6) 5 0.63(s,H15), 0.77(d, J = 7.0 Hz, H12), 0.87(d, J = 7.0 Hz, H13), 1.87(dt, J = 6.1, 12.8Hz, H3'),2.11(d, J = 10.9 Hz, H5), 2.24(m, H3), 2.64(m, HI1), 3.97(t, J = 10.9 Hz, H6), 4.85(s,H14), 5.07(s, H14') ppm; 13C NMR (125 MHz, C6D6) 8 15.3, 17.1, 19.7, 21.3, 24.3,27.5, 38.2, 38.9, 40.2, 42.2, 51.6, 58.9, 62.0, 109.0 ppm; HREIMS M+ 242.1616(C15H25 37C1 AM 0.1 mmu) and 240.1652 (Ci5H25 35 C1 AM 0.8 mmu); LREIMSm/z(relative intensity) 242(6.0), 240(18.1), 227(8.6), 225(27.9), 205(9.6), 204(14.1),197(51.9), 191(37.0), 189(33.9), 169(23.1), 161(45.8), 149(27.2), 135(43.2), 133(39.7),121(30.1), 119(35.1), 105(62.3), 91(86.6), 81(100.0), 67(60.2), 55(58.7), 41(96.6).Compound 81:Compound 81: obtained as a colorless oil; FTIR (film) 2120 cm -1 ; 1 H NMR(400 MHz, CDC13) 8 0.77(d, J = 7.0 Hz), 0.92(d, J = 7.0 Hz), 0.94(d, J = 7.0 Hz),1.11(m), 1.75(s), 3.65(s), 5.11(s) ppm; 13C NMR (75 MHz, CDC13) 8 16.2, 17.1, 20.4,21.0, 25.6, 30.1, 31.5, 35.1, 36.0, 45.7, 59.0, 67.5, 124.0, 129.2, 145.0 ppm.198I, ,,,Compound 82:Compound 82: obtained as a colorless oil; FTIR (film) 2085 cm -1 ; 1 H NMR(400 MHz, CDC13) 6 0.56(dd, J= 6.5, 9.0 Hz), 0.66(dd,J= 8.5, 9.0 Hz), 0.88(s, 3H),1.01(s, 3H), 1.13(s, 3H), 1.26(d, J = 6.4 Hz), 1.35(m), 1.43(s, 3H), 1.97(m, H3) ppm; 13CNMR (75 MHz, CDC13) 8 15.5, 15.7, 19.0, 19.1, 20.5, 22.0, 29.5, 39, 41.5, 41.6, 49.2ppm; LRCIMS (NH3) m/z: 264(M+1), 205, 123, 81.Compound 83:83 R = NCS84 R = NCCompound 83: obtained as a colorless oil; FTIR (film) 2082 cm -1 ; 1 H NMR(400 MHz, CDC13) 6 1.10(s, 3H), 1.16(d, J= 7.6 Hz), 1.38(s, 3H), 1.40(s, 3H,), 2.19(m),2.52(m), 5.35(d, J = 3.0 Hz) ppm; 13C NMR (75 MHz, CDC13) 8 17.5, 18.9, 20.3, 22.2,27.1, 33.3, 34.5, 38.9, 39.0, 40.8, 44.7, 64.3, 119.1, 151.8 ppm; LREIMS m/z(relativeintensity): 263(2.0), 205(1.4), 204(1.4), 189(1.8), 163(26.9), 123(3.1), 121(3.0),119(3.1), 93(26.1), 91(11.7), 81(100.0).199Compound 84: obtained as a colourless oil; 1 1-1 NMR (400 MHz, C6D6) 5 1.03(s, 9H),1.12(d, 3H), 1.72(m), 1.88(bm), 2.40(m), 5.36(d) ppm.Compound 85:Compound 85: obtained as a colorless oil; FTIR (film) 2126 cm -1 ; 1H NMR(400MHz, CDC13) 8 0.58(dd), 0.69(dd), 0.88(s, 3H), 0.99(s, 3H), 1.09(s, 3H), 1.47(bs, 3H),2.06(m) ppm; 13C NMR(75 MHz, CDC13) 8 15.3, 15.5, 18.6, 19.0, 19.3, 20.6, 22.2, 28.9,38.9, 41.6, 41.7, 48.6 ppm; HREIMS M+ 204.1869 (C16F 125N - HCN, AM -0.9 mmu);LREIMS m/z(relative intensity): 204(43.3), 189(47.0), 161(59.9), 147(19.6), 133(30.7),119(26.4), 107(40.4), 105(46.0), 93(43.6), 91(36.4), 81(100.0); LRCIMS m/z: 232(M +1), 205.Compound 86:Compound 86: obtained as a white solid; [a]D - 34° (c 0.18 CHC13); FTIR (film)2080-2160 (br) cm -1 ; 1 H NMR(500MHz, CDC13) 8 0.71(s, 3H, H15), 0.87(d, J = 7.0 Hz,200H12), 0.98(d, J = 7.0 Hz, H13), 1.99(dt, J = 5.8 ,13.0 Hz, H3'), 2.06(d, J = 10.8 Hz, H5),2.17(m, H11), 2.36(m, H3), 3.57(t, J = 10.8 Hz, H6), 4.61(s, H14), 5.02(s, H14') ppm;13C NMR (75 MHz, CDC13) 8 16.0, 17.0, 18.5, 21.0, 23.9, 27.7, 37.5, 37.6, 39.8, 42.0,49.8, 56.3, 56.5, 108.0, 146.0 ppm; HREIMS M+ m/z 263.1714 (C16H25NS AM 0.6mmu); LREIMS m/z(relative intensity): 263(27.3), 248(19.1), 221(73.9), 205(22.8),204(5.9), 161(16.0), 149(50.2), 135(34.5), 109(100.0), 95(83.6), 81(56.7), 69(42.4),67(33.7),.55(44.7), 41(50.5).Compound 87:Compound 87: obtained as a colorless oil; FTIR(film) 2073 cm -1 ; 1HNMR(500MHz, CDC13) 8 0.90(s,3H,H15), 1.12(dt,J=4.7,12.6,H1'), 1.32(s,3H,H14),1.77(s,3H,H13), 4.73(s,2H,H12,H12') ppm; 13C NMR (75 MHz, CDC13) 8 18.9, 19.0,21.1, 22.0, 26.8, 27.6, 34.6, 40.3, 42.0, 44.7, 46.0, 53.1, 65.3, 108.6, 149.9 ppm;HREIMS M+ 263.1715 (C16H25NS AM 0.8 mmu); LREIMS m/z(relative intensity):263(3.0), 248(0.9), 205(100.0), 204(21.1), 189(25.5), 149(48.3), 135(41.9), 123(64.8),109(81.0), 95(92.3), 81(88.2).201ClBrCHe" .Cl ClCompound 88:ClCompound 88: obtained as a colorless oil; 1H NMR(400 MHz, CDC13) 8 1.30(s,3H), 2.20(d, J = 15.3 Hz), 2.37(d, J = 15.3 Hz), 2.46(dt, J = 4.0, 13.1 Hz), 2.64(q, J = 13.1Hz), 3.55(d, J = 10.6 Hz), 3.70(dd, J = 4.0, 12.5 Hz), 3.96(d, J = 10.6 Hz), 4.34(dd, J =4.7, 12.5 Hz), 6.08(d, J = 13.6 Hz), 6.56(d,J = 13.6 Hz) ppm; 13C NMR(75 MHz,CDC13) 6 27.4, 38.2, 38.6, 48.8, 59.1, 64.1, 119.1, 135.4 ppm; HREIMS M+ m/z355.8903 (C101113 35 C13 37081 Br AM -0.3 mmu and C101-113 35 C1237 C1279Br AM 0.7mmu), 353.8938 (C10H13 35C1481Br AM 0.2 mmu and C101113 35C13 37C179Br AM 1.2mmu) and 351.8961 (C101113C14Br AM 0.6 mmu); LREIMS m/z(relative intensity):358(3.2), 356(5.1), 354(6.3), 352(2.6),^319(6.1), 283(10.6), 273(9.8), 271(20.2),269(13.9), 247(14.6), 245(14.5), 239(11.9), 237(16.3), 235(13.6), 205(10.0), 204(14.2),203(20.4), 201(31.2), 189(14.3), 167(17.7), 165(29.2), 163(14.5), 161(23.6), 131(16.1),129(23.6), 127(25.6), 125(31.6), 115(29.6), 105(30.2), 91(69.0), 84(78.6), 77(54.7),49(100.0).Compound 89:202NHCHO i...""Compound 89: obtained as a white solid; FTIR(film) 1732 cm -1 ; [o]D + 24° (c0.64 CHC13); 1H NMR (500MHz, CDC13) 8 0.43(dd, J = 6.8, 9.1 Hz, H6), 0.65(t, J = 9.1Hz, H7), 0.79(dt, J = 7.5, 13.0 Hz, H9'), 0.92(s, 3H, H15), 0.94(s, 3H, H13), 1.04(s, 3H,H12), 1.13(dd,J = 8.3, 13.0 Hz, H9), 1.35(s, 3H, H14), 2.26(bd, J = 12.5 Hz, H3), 3.59(s,3H, H17), 4.60(bs, NH) ppm; 13C NMR(75 MHz, CDC13) 8 15.4, 15.5, 17.7, 19.2, 19.3,19.5, 19.9, 20.3, 29.0, 32.7, 38.5, 39.5, 41.9, 46.1, 51.2, 56.1 ppm; HREIMS M± m/z279.2193(Ci7H29N 02 AM -0.5 mmu); LREIMS m/z(relative intensity): 279(16.2),205(40.2), 204(100.0), 190(19.2), 189(74.1), 161(71.1), 122(66.0).Compound 90:Compound 90: 1 H NMR (400 MHz, CDC13) 8 0.39(m), 0.43(m), 0.65(t), 0.89(s),0.90(s), 1.00(s), 1.35(s), 1.40(s), 2.43(bd), 5.11(bs), 5.82(bs), 8.01(s), 8.24(d) ppm.203D.^Neoesperlactone (96) from the Marine Sponge Neoesperiopsis digitataCollection and Isolation dataSpecimens of Neoesperiopsis digitata (0.850 kg wet weight) were collected byhand using SCUBA at Rivers Inlet B. C. and transported to Vancouver frozen. A sampleof the sponge (25 g) was ground in a blender and extracted with Me0H and this extractwas used for biological testing. The extract showed antibacterial and antifungal activity.The remaining portion of the sponge (825 g) was immersed in Me0H (3L) and soaked atroom temperature for 72 h. Concentration of the decanted Me0H in vacuo gave anaqueous suspension (1L) that was sequentially extracted with hexanes (2 X 1L),chloroform (2 X 1L) and ethyl acetate (2 X 500 mL). Evaporation of the combinedhexanes extracts yielded a dark oil (2.14 g). Purification of the hexanes extract usingSephadex LH-20 (4:1 Me0H/CH2C12) yielded a complex mixture of fatty acids andsteroids. Evaporation of the combine chloroform extracts yielded a dark oil (2.45 g).Initial purification of the chloroform extract was accomplished using Sephadex LH-20(4:1 Me0H/CH2C12). 1 H NMR of the resulting fractions indicated the presence of 96 inthe early eluting fractions and several aromatic compounds in the late eluting fractions.Purification of the fraction that contained the major aromatic compound using flash silcagel chromatography (1:1 Hex/EtOAc) yielded p-hydroxybenzaldehyde which wasidentical by tic and 1 H NMR to a commercial sample (Aldrich cat. # 14,408-8). Finalpurification of 96 was accomplished using flash silica gel chromatography (step gradient,3:1 Hex/EtOAc - EtOAc) followed by preparative tic (2:3 Hex/EtOAc) and normal phaseHPLC (9:11 Hex/EtOAc).204CH3OHNeoesperlactone(96):Compound 96: obtained as a colourless oil; 4.2 mg; Rf = 0.24 (2:3 Hex/EtOAc); [a]c•-620 (c 0.08); FTIR (film) 3414 (br), 1769 cm-1 ; 1H NMR (400 MHz, CDC13) 6 0.89(t,J = 6.7 Hz, H18), 1.73(m, H5'), 2.33(m, H3), 2.53(m, H2, H2'), 3.65(m, H7), 4.53(m,H4), 5.36(m, H11, H12) ppm; 13C NMR (125 MHz, CDC13) 6 14.1, 22.6, 25.7, 27.0,27.3, 28.0, 28.8, 29.0, 29.7, 31.6, 31.8, 32.7, 37.2, 71.2, 80.7, 129.1, 130.6 ppm;HREIMS (M+) 296.2354 (C18113203 AM 0.3 mmu); LREIMS Ink 296(7.2), 278(6.0),253(7.2), 225(12.2), 169(78.3), 156(34.8), 151(36.4), 138(66.0), 125(35.5), 123(46.6),110(37.0), 109(36.5), 96(72.0), 81(100.0), 67(96.0), 55(92.9), 41(82.2).205E.^pseudaxinellin (103) A Cyclic Heptapeptide from Pseudaxinella massaPseudaxinellin (103):Pseudaxinellin (103): obtained as a clear glass; [oc]D -100.1 ° (c 0.34, CHC13);1 H NMR (500 MHz, CDC13) 8 1.36(m, H12'), 2.28(m, H12), 2.36(m, H21), 2.57(m,H26), 3.58(bt, J = 7.3 Hz, H14), 3.70(m, H28), 4.16(dd, J = 4.4, 7.3 Hz, H30), 4.21(t, J =9.4 Hz, H35), 4.53(d, J = 6.9 Hz, H25), 4.61(bm, H16), 4.77(m, H2), 5.43(bs, 1H,NH2Asn), 6.66(bs, 1H, NH2Asn), 6.80(d, J = 4.4 Hz, NHVa12), 7.32(d, J = 9.4 Hz,NHVa13), 7.54(d, J = 9.5 Hz, NHPhe), 8.01(d, J = 5.3 Hz, NHAsn), 8.22(d, J = 8.0 Hz,NHVal 1) ppm; 13C NMR (125 MHz, CDC13) 6 18.5, 18.7, 18.8, 19.3,25.8, 29.1, 29.5, 29.6, 30.1, 31.2, 36.2, 37.7, 46.0, 48.0, 50.2, 55.4, 57.3,63.5, 126.6, 128.3, 128.9, 137.5, 169.0, 170.31, 171.18, 171.5, 171.8,19.8, 19.9, 21.8,58.5, 61.2, 62.1,172.13, 172.26,172.6 ppm; HREIMS (Mt) 752.4218 (C381 -156N808 AM -0.3 mmu); LREIMSm/z(relative intensity): 752(8.9), 734(0.9), 710(0.9), 683(4.2), 640(7.4), 612(2.0),584(1.2), 541(5.6), 527(1.8), 513(1.1), 443(5.2), 70(100.0).206F.^Majusculamide C (104) from Ptilocualis trachys and Amphimedon sp yMajusculamide C (104):Compound 104: isolated as a white solid96; the absolute stereochemistry of the2-methyl-3-amino pentanoic acid residue was determined to be 2S,3R 97; 1H NMR (500MHz, 1:1 C6D6/CDC13 (referenced to C6D6 residual solvent peak at 8 7.15 ppm)) 80.31(d, J = 6.5 Hz, H36), 0.57(d, J = 6.5 Hz, H37), 0.95(d, J = 7.0 Hz, H5), 0.98(d, J =6.5 Hz, H11), 0.98(d, J = 6.5 Hz, H48), 1.08(d, J = 7.0 Hz, H19), 1.27(s, H18), 1.38(s,H17), 2.01(m, H45), 2.03(m, H55), 2.11(m, H35), 2.53(m, H2), 2.63(s, NMe), 2.69(dd, J= 7.5, 13.8 Hz, H23'), 2.90(s, NMe), 2.96(s, NMe), 3.16(dd, J = 7.5, 13.8 Hz, H23),3.29(d, J = 17.3 Hz, H40'), 3.35(m, H51'), 3.37(s, OMe), 4.24(dd, J = 7.0, 16.0 Hz, H51),4.31(m, H9), 4.46(bm, H3), 4.54(dd, J = 8.5, 17.3 Hz, H40), 4.76(d, J = 10.5 Hz, H33),4.86(m, H15), 4.90(d, J = 11.5 Hz, H43), 5.16(t, J = 7.5 Hz,H21), 5.21(d, J = 3.5 Hz,H54), 6.61(d, J = 8.5 Hz, H26, H28), 7.01(d, J = 8.5 Hz, H25, H29), 7.03(NH), 7.26(bm,NH), 7.32(bt, NH), 7.54(bd, NH), 7.74(d, J = 8.5 Hz, NH); HRFABMS (M+ + H)985.5975 (C501181N8012 AM 0.2 mmu); LRFABMS ink 985.4, 753.4, 719.4, 688.3,551.4, 523.3, 441.2, 413.2, 391.2, 367.2, 338.3, 313.2, 285.2, 267.2.207G.^synthesis of the Tetrahydroisoqjinoline Portion of ImbricatinePreparation of methyl 3,5-dibenzyloxy benzoate (148):Methyl 3,5-dihydroxy benzoate (147: 5.00 g, 29.7 mmol) and K2CO3 (20.0 g)were stirred in 100 mL of anhydrous acetone for 0.5 h at room temperature. Benzylbromide (7.50 mL) was added slowly over a period of 5 minutes and the mixture wasrefluxed for 1 h. Filtration and evaporation to dryness in vacuo yielded a crude mixture(13.24 g). Purification using flash chromatography (400 mL 9:1 Hex/EtOAc, 400 mL 4:1Hex/EtOAc, 400 mL 7:3 Hex/EtOAc) yielded methyl 3,5-dibenzyloxy benzoate (148:9.48 g, 27.2 mmol) for a 91.5 % yield after recovery of the starting material and theintermediate monobenzyloxy product. Compound 148: 140 m.p. = 66.0-67.5 °C; FTIR(film) 3033, 2950, 1724, 1595, 1498, 1444 cm -1 ; 1H NMR (400 MHz, CDC13) 5 3.82(s,3H), 4.97(s, 4H), 6.76(t, J = 2.3 Hz, 1H), 7.28(d, J = 2.3 Hz, 2H), 7.32(m, 10H) ppm;HREIMS (Mt) 348.1364 (C22H2004 AM 0.2 mmu); LREIMS m/z(relative intensity):348(29.3), 317(11.8), 225(3.3), 197(2.8), 181(33.8), 91(100.0), 65(34.0).Preparation of 3,5-dibenzyloxy benzyl alcohol (149):LiA1H4 (0.0108 g) was added slowly to a stirring solution of methyl 3,5-dibenzyloxy benzoate (148: 0.0995 g, 0.2855 mmol) in diethyl ether (10 mL). Themixture was refluxed for 2.5 h and quenched with water (10 mL). NaOH (10 mL, 20%)was added and the mixture was extracted with diethyl ether (3 X 15 mL). The combinedorganic extracts were washed with water (10 mL) and dried over magnesium sulphate.Evaporation to dryness in vacuo gave 3,5-dibenzyloxy benzyl alcohol (0.087 g, 0.272mmol, 95%). Compound 149: 140 m.p. = 78.5-79.0 °C; FTIR (film) 3500-3000(br),1596, 1498, 1453 cm-1 ; 1 H NMR (400 MHz, CDC13) 8 2.29(bs, 1H), 4.50(s, 2H), 4.94(s,2084H), 6.50(bs, 1H), 6.56(bs, 2H), 7.31(m, 10H) ppm; HREIMS (M+) 320.1420(C21H2003 AM 0.8 mmu); LREIMS (M+) 320(11.4), 181(13.6), 91(100.0), 65(21.4).Preparation of 3,5-dibenzyloxy benzyl tosylate (146):Sodium hydride (0.0189 g: 80% oil dispersion) was added to a stirring solution of3,5-dibenzyloxy benzyl alcohol (149: 0.2013 g, 0.6283 mmol) in benzene (10 mL).p-Toluenesulfonyl chloride (0.1199 g, previously washed with 20% NaOH andrecrystalized from Et20) was added slowly over 10 minutes and the mixture was stirredat room temperature for 9 h. The resulting solution was centrifuged and the supernatantwithdrawn. Evaporation to dryness in vacuo yielded a mixture of the product 3,5-dibenzyloxy benzyl tosylate (146) and starting material. Purification using normal phaseflash chromatography (7:3 Hex/EtOAc) yielded 146 (0.1082 g, 0.2279 mmol) in 73 %yield after recovery of starting material. Compound 146: FTIR (film) 3033, 2925, 1598,1497, 1452, 1360, 1175 cm -1 ; 1 H NMR (400 MHz, CDC13) 8 2.39(s, 3H), 4.94(s, 4H),4.96(s, 2H), 6.46(d, J = 2.2 Hz, 2H), 6.54(t, J = 2.2 Hz, 1H), 7.28(d, J = 8.2 Hz, 2H),7.34(m, 10H), 7.76(d, J = 8.2 Hz, 2H) ppm.Preparation of diester 144:Diethyl acetamidomalonate (145: 0.133 g, 0.612 mmol) and sodium hydride(0.018 g: 80% oil dispersion) were stirred in benzene (5 mL) and DMF (5 mL) for 15minutes. 3,5-dibenzyloxy benzyl tosylate (146: 0.068 g, 0.144 mmol) was added slowlyand the mixture was stirred at room temperature for 6 h. Evaporation in vacuo followedby normal phase flash chromatography (2:3 Hex/EtOAc) gave 144 (0.069 g, 0.132 mmol,92%). Compound 144: m.p. = 138.5-140.0 °C; FTIR (film) 3255, 1745, 1597 cm-1 ; 1 HNMR (400 MHz, CDC13) 8 1.25(t, J = 7.1 Hz, 6H), 1.95(s, 3H), 3.58(s, 2H), 4.23(m,2094H), 4.98(s, 4H), 6.26(d, J = 2.2 Hz, 2H), 6.51(t, J = 2.2 Hz, 1H), 6.56(s, 1H), 7.34(10H)ppm; HREIMS (M+) 519.2254 (C30H33N07 AM -0.3 mmu); LREIMS m/z(relativeintensity): 519(0.8), 181(4.0), 115(7.6), 91(100.0).Hydrolysis of alkylation product 144:Alkylation product 144 (0.055 g, 0.106 mmol) was dissolved in dioxane (10 mL).NaOH (2 mL, 20%) was added and the mixture was stirred under reflux for 3 h. Aftercooling, glacial acetic acid was added and the acidic mixture was refluxed for anadditional 1 h. After cooling, the mixture was extracted with ethyl acetate (3 X 10 mL).The combined organic extracts were washed with water (10 mL) and dried overmagnesium sulphate. Evaporation in vacuo gave the crude product 150 (0.041 g, 0.099mmol, 93%) which was used directly in the next step. Compound 150: FTIR (film) 3398(br), 1650, 1594 cm -1 ; 1 H NMR (400 MHz, CD3OD) 8 1.88(s, 3H), 2.90(dd, J = 7.5,13.6 Hz, 1H), 3.15(dd, J = 4.1, 13.6 Hz, 1H), 4.51(bs, 1H), 5.02(s, 4H), 6.45(bs, 1H),6.53(bs, 2H), 7.35(10H) ppm; LRCIMS (CH4) m/z 420(M++ H (100.0)), 403(61.6),379(36.8), 360(17.3), 343(7.0), 331(8.0), 315(11.5), 181(17.8), 91(40.3).Preparation of methyl 3-(3,5-dihydroxyphenyl) alanine (143):Hydrolysis product 150 (0.028 g) was stirred in hydrazine hydrate (2 mL) at 100°C for 24 h. The mixture was evaporated to dryness in vacuo, dissolved in a saturatedMe0H/HC1 solution (10 mL) and refluxed for 1 h. The mixture was evaporated todryness and dissolved in Me0H (10 mL). Stirring under H2 in the presence of Pt/C atroom temperature for 10 h gave 143 after reversed phase flash chromatography (1:1Mc0H/H20). Compound 143: FTIR (film) 3193(br), 1743, 1603, 1509, 1454 cm -1 ; 1 HNMR (400 MHz, CD3OD) 8 3.08(m, 2H), 3.83(s, 3H), 4.26(t, J = 6.6 Hz, 1H), 6.22(d, J =2101.8 Hz, 2H), 6.25(t, J = 1.8 Hz, 1H) ppm; HREIMS (M+) 211.0851 (CI0H13N04 AM0.7 mmu); LREIMS m/z(relative intensity): 211(16.0), 153(26.5), 152(65.7), 125(42.6),124(70.4), 123(25.9), 112(26.5), 107(19.9), 88(100.0).Preparation of p-benzyloxy benzaldehyde (151):p-Hydroxy benzaldehyde (142: 5.00 g, 41.0 mmol) and K2CO3 (10 g) were stirredin acetone (100 mL) for 0.5 h. Benzyl bromide (5 mL) was added slowly and the mixturewas then refluxed for 1 h. The reaction was filtered and evaporated to dryness in vacuoand recrystalized from hexane to give 151 (8.34 g, 40.0 mmol, 97%). Compound 151: 141m.p. = 65.5-68.0 °C; FTIR (film) 1688, 1600, 1508, 1454 cm -1 ; 1H NMR (400 MHz,CDC13) 8 5.07(s, 2H), 7.02(d, J = 8.9 Hz, 2H), 7.34(m, 5H), 7.77(d, J = 8.9 Hz, 2H),9.82(s, 1H) ppm; HREIMS (Mt) 212.0834 (C14111202 AM -0.3 mmu); LREIMSm/z(relative intensity): 212(25.4), 92(39.1), 91(100.0), 90(14.4), 89(22.7), 86(7.8),84(10.9), 77(10.9), 65(40.0).Preparation of methyl 3-(p-benzyloxyphenyl) glycidate (152):p-Benzyloxy benzaldehyde (151: 3.00 g, 0.014 mol) and methylchloroacetate(1.53 g, 0.014 mmol) were added alternately over 2.5 h to an ice cold solution of freshlyprepared NaOCH3/CH3OH. The solution was then stirred for 3 h at 0 0C followed by 3h at room temperature and poured onto ice water. After filtering, the white precipitate152 remained (3.87 g, 0.0136 mol, 97%). Compound 152: 141 m.p. = 97.0-101.0 °C;FTIR (film) 1727, 1604, 1512, 1245 cm -1 ; 1H NMR (400 MHz, CDC13) 8 3.49(d, J = 1.7Hz, 1H), 3.80(s, 3H), 4.04(d, J = 1.7 Hz, 1H), 5.05(s, 2H), 6.95(d, J = 6.8 Hz, 2H),7.19(d, J = 6.8 Hz, 2H), 7.36(m, 5H) ppm; HREIMS (M+) 284.1041 (C17H1604 AM -0.7211mmu); LREIMS m/z(relative intensity): 284(9.6), 227(9.6), 211(3.8), 197(3.0),92(22.7), 91(100.0), 77(6.0), 65(21.8).Preparation of sodium 3-(p-benzyloxyphenyl) glycidate (141):Me0H (1.2 mL) was added dropwise to sodium metal (0.083 g) at 5 °C. Themixture was added slowly to a solution of 152 (1.00 g, 3.52 mmol) in benzene (5 mL)and was stirred at 10 °C for 3 h. H2O (1.2 equiv.) was added and the reaction mixturewas filtered. The precipitate 141 141 was washed with ether and used directly in the nextreaction.Preparation of the tetrahydroisoquinolines 153 and 154:Compound 141 (0.154 g, 0.528 mmol) in water (10 mL) was added to a solutionof 143 (0.112 g, 0.528 mmol) in Me0H (15 mL). Acetic acid (2 mL) was added and theresulting acidic mixture was stirred at 35 °C for 17 h under N2. The solvent wasevaporated in vacuo and the crude material was dissolved in ethyl acetate. The organiclayer was washed with 1N HCl followed by H2O then dried over sodium sulphate.Purification of the crude mixture on silica (1:1 Hex/Et0Ac) yielded an inseparablemixture of the tetrahydroisoquinolines 153 and 154 (0.079 g, 0.188 mmol, 36%).Compounds 153 and 154: FTIR (film) 3161(br), 1730, 1603, 1509, 1455 cm -1 ; 1 H NMR(400 MHz, CD3OD) 5 2.78(m), 2.85(m), 2.97(m), 3.29(m), 3.44(m), 3.74(s), 4.01(d, J =11.3 Hz), 4.31(d, J = 10.0 Hz), 4.46(bs), 5.03(s), 5.06(s), 6.06(s), 6.13(s), 6.22(s), 6.26(s),6.88(d, J= 8.1 Hz), 6.97(d, J = 8.1 Hz), 7.06(d, J = 8.1 Hz), 7.17(d, J = 8.1 Hz), 7.37(m)ppm; HREIMS (M+) 419.1728 (C25H25N05 AM -0.4 mmu); LREIMS m/z(relativeintensity): 419(0.2), 360(0.9), 324(1.3), 236(5.0), 222(69.2), 176(4.9), 162(100.0),91(76.8).212Acetylation of Compounds 153 and 154:The mixture of compounds 153 and 154 (0.0303 g, 0.0722 mmol) was stirred inpyridine (2 mL) and acetic anhydride (2 mL) for 19 h. Evaporation in vacuo yielded thecrude mixture (0.0478 g) which was purified using preparative tic (1:1 Hex/EtOAc) togive 157 (0.0035 g, 0.0064 mmol) and 158 (0.0228 g, 0.0418 mmol). 157: FTIR (film)1769, 1652, 1616 cm-1 ; 1 H NMR (400 MHz, CDC13) 8 1.71(s, 3H), 2.20(s, 3H), 2.29(s,3H), 2.81(dd, J = 5.3, 13.7 Hz, 1H), 3.25(m, 3H), 3.85(s, 3H), 4.46(t, J = 9.6 Hz, 1H),5.03(dd, J = 5.3, 8.0 Hz, 1H), 5.06(s, 2H), 6.88(d, J = 2.1 Hz, 1H), 6.93(d, J = 2.1 Hz,1H), 6.92(d, J = 8.7 Hz, 2H), 7.22(d, J = 8.7 Hz, 2H), 7.40(m, 5H) ppm; LREIMS (M+)545.2043 (C311-131N08 AM -0.7 mmu); LREIMS m/z(relative intensity): 545(<0.1),514(0.4), 486(1.5), 444(1.8), 348(94.6), 306(98.4), 264(95.3), 246(14.1), 222(100.0),204(23.0), 162(98.7), 91(64.4). 158: FTIR (film) 1770, 1754(sh), 1651, 1614 cm -1 ; 1 HNMR (400 MHz, CDC13) 8 1.99(s), 2.10(m), 2.12(s), 2.16(s), 2.22(s), 2.25(s), 2.26(s),2.86(m), 2.93(m), 2,95(m), 3.02(dd, J = 3.5, 16.3 Hz), 3.24(dd, J = 6.7, 13.2 Hz), 3.49(s),3.57(s), 4.54(dd, J = 2.4, 5.4 Hz), 4.83(dd, J = 3.5, 6.4 Hz), 5.01(s), 5.04(s), 5.18(t, J =6.3 Hz), 5.64(dd, J = 2.8, 6.7 Hz), 6.66(d, J = 8.6 Hz), 6.67(d, J = 2.2 Hz), 6.75(d, J = 8.6Hz), 6.79(d, J = 2.2 Hz), 6.86(s), 6.89(d, J = 2.2 Hz), 6.93(d, J = 2.2 Hz), 7.33(m) ppm;HREIMS (M+) 545.2043 (C311 -131N08 AM -0.7 mmu); LREIMS m/z(relative intensity):545(<0.1), 514(0.1), 486(1.4), 444(1.5), 348(94.6), 306(98.6), 264(90.4), 246(10.0),222(99.5), 204(17.5), 162(100.0), 91(55.2).Debenzylation of compound 157:Compound 157 (0.0052 g, 0.0095 mmol) was stirred in Me0H (2 mL) under H2in the presence of Pt/C catalyst for 3 h. The mixture was filtered and evaporated todryness in vacuo. Purification was accomplished using preparative tic (9:1 ethylacetate/hexane) to yield 159 (0.0027 g, 0.0059 mmol, 62%). Compound 159: 1 H NMR213(400 MHz, CDC13) 8 2.00(s), 2.13(s), 2.19(s), 2.26(s), 2.27(s), 2.90(m), 3.04(dd, J =3.4,16.3 Hz), 3.22(dd, J = 6.8, 13.2 Hz), 3.49(s), 3.57(s), 4.56(dd, J = 2.3, 5.4 Hz),4.85(dd, J = 3.4, 6.5 Hz), 5.18(t, J = 6.3 Hz), 5.64(dd, J = 2.9, 6.8 Hz), 6.60(s), 6.69(d, J =2.0 Hz), 6.72(d, J = 8.5 Hz), 6.80(d, J = 2.1 Hz), 683(d, J = 8.5 Hz), 6.89(d, J = 2.0 Hz),6.93(d, J = 2.1 Hz) ppm.Acetylation of compound 159:Compound 159 (0.0027 g, 0.0059 mmol) was stirred in pyridine (1 mL) and aceticanhydride (1 mL) at room temperature for 15 h. Evaporation to dryness in vacuo andpurification using preparative tic (9:1 EtOAc/Hex) followed by normal phase HPLC (8:2EtOAc/Hex) yielded 140 (0.0017 g, 0.0034 mmol). Compound 140: FTIR (film) 1760,1648, 1507, 1438 cm -1 ; 1 H NMR (400 MHz, CDC13) 8 1.78(s), 2.20(s), 2.287(s),2.290(s), 2.88(dd, J = 6.0, 13.5 Hz), 3.26(m), 3.32(dd, J = 7.6, 13.5 Hz), 3.85(s), 4.47(dd,J = 8.6, 10.6 Hz), 5.06(dd, J = 6.0, 7.6 Hz), 6.88(d, J = 2.1 Hz), 6.94(d, J = 2.1 Hz),7.04(d, J = 8.5 Hz), 7.29(d, J = 8.5 Hz) ppm; HREIMS (Mt) 497.1691 (C261127N09 AM0.5 mmu); LREIMS m/z(relative intensity): 497(<0.1), 466(0.3), 438(0.3), 396(2.3),348(37.8), 306(48.3), 264(46.7), 246(4.6), 222(79.0), 204(9.4), 162(100.0), 107(40.9).Debenzylation of compound 158: Compound 158 (0.0039 g, 0.0071 mmol) was stirredin Me0H (2 mL) under H2 in the presence of Pt/C catalyst for 3 h. The mixture wasfiltered and evaporated to dryness in vacuo. Purification was accomplished usingpreparative tic (9:1 EtOAc/Hex) to yield 160. Compound 160: 1 H NMR (400 MHz,CDC13) 8 1.70(s), 2.25(s), 2.29(s), 2.80(dd, J = 5.0, 13.6 Hz), 3.24(m), 3.84(s), 4.47(dd, J= 8.6, 11.0 Hz), 5.03(dd, J = 5.0, 8.2 Hz), 6.79(d, J = 8.5 Hz), 6.89(d, J = 2.1 Hz), 6.94 (d,J = 2.1 Hz), 7.20(d, J = 8.5 Hz) ppm.214Acetylation of compound 160: Compound 160 (0.0052 g, 0.0114 mmol) was stirred inpyridine (1 mL) and acetic anhydride (1 mL) at room temperature for 15 h. Evaporationto dryness in vacuo followed by purification using preparative tic (9:1 EtOAc/Hex)yielded 162 (0.0048 g, 0.0096 mmol, 85%). Compound 162: FTIR (film) 1761, 1651,1505, 1437 cm -1 ; 1 H NMR (400 MHz, CDC13) 8 2.06(s), 2.13(s), 2.15(s), 2.22(s),2.25(s), 2.26(s), 2.27(s), 2.28(s), 2.95(m), 2.97(m), 3.08(dd, J = 3.5, 16.3 Hz), 3.21(dd, J= 7.2, 13.0 Hz), 3.49(s), 3.57(s), 4.59(dd, J = 2.5, 5.3 Hz), 4.86(dd, J = 3.5, 6.3 Hz),5.21(t, J = 6.2 Hz), 5.65(dd, J = 2.8, 7.2 Hz), 6.71(d, J = 2.1 Hz), 6.79(d, J = 8.6 Hz),6.82(d, J = 2.6 Hz), 6.86(d, J = 8.6 Hz), 6.88(d, J = 2.1 Hz), 6.94(d, J = 2.6 Hz), 6.99(m)ppm; HREIMS (M+ - OCH3) 466.1505 (C25H24N08 AM 0.3 mmu); LREIMSm/z(relative intensity): 466(0.1), 438(0.6), 396(1.6), 348(36.3), 306(44.5), 264(43.0),246(4.5), 222(74.4), 204(9.7), 162(100.0), 107(40.7).215References1. P. E. McGovern and R. H. Michel, Analytical Chemistry, 1985, 57, 1515A.2. Y. Hashimoto, Marine Toxins and Other Bioactive Marine Metabolites, JapanScientific Societies Press, Tokyo, 1979, 229.3. R. E. Moore, in Progress in the Chemistry of Organic Natural Producta, W.Herz, H. Grisebach, G. W. 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C. de Lorenzo, and G. Mann,Tetrahedron, 1972, 28, 5999.142. Fig. 81 is the 1 H NMR spectrum of formamide 90 isolated from Cadlinaluteomarginata by E. J. Dumdei.223Appendix A. 1 H NMR Spectra of Known Compounds from Acanthelaa sp.2240.01.08.0^7.0^6.0^5.0 PPM^4.0^3.0^2.0Fig. 75. 1H NMR spectrum of compound 81 (400 MHz, CDC13).►1.4►2. 8-r1. 6►1 . 211. 81I . 8PPM►^'^►^ I^-^ ►.8 .6 . 4 .2^8.8Fig. 76. 1H NMR spectrum of compound 82 (400 MHz, CDC13).S1""I'•••1`•••1`• - `•1•"•1 • "^1^ 1^'••1v-'-v.-1.•-•-•1•'•^1-7.11^6.^5.11 4. 0 3.0 2.0^1.0PPMFig. 77. 1H NMR spectrum of compound 83 (400 MHz, CDC13).0. 03.0 2.0 1.0 0.07.0^6.0^5.0^ATPPMFig. 78. 1H NMR spectrum of compound 84 (400 MHz, C6D6). )41t 12.2^ 1.8^1.6^114^1.2^1.8^.8^.6^.4^.2^0.0PPMFig. 79. 1H NMR spectrum of compound 85 (400 MHz, CDC13).7.0,-^'6.0 -^- - -5:0- 4.0^PPM 3.0-- 2.6,1.0 0.0Fig. 80. 1H NMR spectrum of compound 88 (400 MHz, CDC13).1.0 0.0810^t^7.0^6.0^5.0^ppm 4.0^3.0^2.0Fig. 81. 1H NMR spectrum of compound 90 (400 MHz, CDC13). 142SJNHCHOAppendix B. 1 H NMR Spectra of Synthetic Products2321CO2EtCO2EtNHCOCH3ROOR7.0^6.0^5.0^4.0^PPM^3.0^2.0^1.0^0.0Fig. 82. 1H NMR spectrum of alkylation product 144 (400 MHz, CDC13).3.0 2.0 1.0 0.07.0^6.0^5.0^4.0 PPMFig. 83. 1H NMR spectrum of compound 150 (400 MHz, CD3OD).HO ■^4.0^3.0^2:0PPMFig. 84. 1H NMR spectrum of compound 143 (400 MHz, CD3OD).7.0 6.0■5.0■0.0Fig. 85. 1 H NMR spectrum of the mixture of compounds153 and 154(400 MHz, CD3OD).A J,7.0HOR = C61-15C142S5 .0 ^0PPM '3.0- ---,- ^2.0 1.0^0.0,8.01^^ ,.^.^.^ .8.0    ^7.0 6.0^5.0 4.0^3.0^2.0PPMFig. 86. 1H NMR spectrum of trans tetraacetate 162 (400 MHz, CDC13).1.0.0.0Appendix C. Explanation of Tables.The tables presented in the text are a summary of the data accumulated on thecompound listed at the top of each table. The first column is a list of the carbon numbers(C #) of the particular compound. The 1H NMR column lists the proton resonancesassigned to be attached to the carbon in the C # column. The COSY column indicates allproton resonances that showed COSY correlations to the proton resonances in the 1HNMR column. The nOe column lists the proton resonances that showed enhancementswhen the proton resonance in the 1 H NMR column was irradiated. The 13C NMRcolumn lists the carbon resonances that were assigned to the carbons in the C # column.The HMBC column lists the proton resonances that showed 2 or 3 bond correlations tothe carbons in the C # column. The ROESY column lists the proton resonances thatshowed correlations into the proton resonance in the 1 H NMR column in the ROESYexperiment.238

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