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Rearranged spongian diterpenoids from the nudibranch Chromodoris cavae that may serve as a chemical defense Dumdei, Eric J. 1988

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REARRANGED SPONGIAN DITERPENOIDS FROM THE NUDIBRANCH CHROMODORIS CAVAE THAT MAY SERVE AS A CHEMICAL DEFENSE. By ERIC J. DUMDEI B.A. St. Olaf College, 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Dept. of Chemistry) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1988 ©Eric J. Dumdei, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada DE-6 (2/88) Abstract Nudibranchs of the genus Chromodoris are known to selectively sequester sponge metabolites which have novel structures and often serve as deterrents to predation. Spectroscopic and X-ray diffraction studies on a biologically active metabolite from Chromodoris cavae have led to the discovery of a diterpenoid, chromodorolide A (100), with a new carbon skeleton. 19 - i i i -T A B L E OF CONTENTS Abstract i i List of Figures iv List of Schemes . v List of Tables vi Abbreviations vii Acknowledgements x I. Introduction 1 A . Chemical Defense in Nudibranchs ... 1 B. Spongian Diterpenes 9 II. Our Current Work 33 Part I«Chromodorolide A 34 Part TJ-Dendrillolide .62 Part m--Chromodorolide B 65 U l . Conclusion 76 IV. Experimental 77 V . Appendix - X-Ray Crystallography Data 84 - iv -LIST OF FIGURES 1. Typical Nudibranchs 2 2. Electron Impact mass spectrum of (100) 35 3. Chemical Ionization mass spectrum of (100) 36 4. * H nmr spectrum of chromodorolide A (100) in CDCI3 37 4a. Expanded upfield region of Fig. 4 38 4b. Expanded downfield region of Fig. 4 39 5 . 1 3 C nmr of chromodorolide A (100) in CDCI3 41 6 . 1 H nmr spectrum of (100) in C 6 D 6 .42 6a. Expanded upfield region of Fig. 6 43 6b. Expanded downfield region of Fig. 6 44 7 . 1 3 C nmr spectrum of (100) in C 6 D 6 45 8. COSY of (100) in C 6 D 6 47 8a. Expanded upper right quadrant of Fig. 8 48 9. PHDQCOSY of (100) in C 6 D 6 .49 10. Computer generated X-ray of (100) 51 11. ^ - ^ C H E T C O R of (100) in C 6 D 6 53 11a. Expanded lower right quadrant of Fig. 11 54 12. 2D-JRES plot of (100) in C 6 D 6 55 12a. Expansion of Fig. 12 56 13. Long range 1 H - 1 3 C H E T C O R of (100) in C 6 D 6 59 14. Infrared spectrum of (100), thin film 60 15. * H nmr spectrum of dendrillolide (101) in CDCI3 63 16. * H nmr spectrum of chromodorolide B (102) in CDCI3 66 16a. Expansion of upfield region of Fig. 16 67 16b. Expansion of downfield region of Fig. 16 < 68 17. Electron Impact mass spectrum of (102) 70 1 8 . 1 3 C nmr spectrum of (102) in CDCI3 ...71 19. COSY of (102) in CDCI3 72 19a. Expansion of upper right quadrant of Fig. 19 73 20. Infrared spectrum of (102), thin film 74 LIST OF SCHEMES Scheme I 11 Scheme II 27 Scheme III 28 vi LIST OF T A B L E S Table I 61 Table H 64 Table m 75 ABBREVIATIONS A c O H AcO A P T BB calcd. C a S 0 4 CDCI3 CHCI3 C H 2 C 1 2 C H 2 N 2 a acetic acid acetate Attached Proton Test BroadBand *H decoupled benzene-dg calculated calcium sulphate chloroform-d chloroform dichloromethane diazomethane Chemical Ionization COSY Correlated Spectroscopy COSYPHDQ PHase sensitive Double Quantum Correlated SpectroscopY CrO, ID 2D chromium (VI) trioxide one-dimensional two-dimensional 2 D J J-Resolved spectroscopy water-d2 1 EI electron impact Et2NH diethylamine Et20 diethyl ether fig. figure H E T C O R HETeronuclear CORrelation HCO2H formic acid hplc high performance liquid chromatography hrms high resolution mass spectrum (electron impact) ir infrared K2CO3 potassium carbonate L A H lithium aluminum hydride L i lithium M e O H methanol inp melting point range • •• ms mass spectrum (low resolution) NH3 ammonia * H nmr proton nuclear magnetic resonance l ^ C nmr carbon-13 nuclear magnetic resonance nOe nuclear Overhauser enhancement palladium on activated carbon phosphoryl chloride parts per million pyridine reference relative intensity solvent signal thin layer chromatography tetramethylsilane ultraviolet water signal X Acknowledgements I wish to sincerely thank my research supervisor, Dr. Raymond Andersen, for his encouragement and guidance during the course of this work. It has been my pleasure to work with him. Assistance given by E . Dilip DeSilva in collecting, extracting and transporting specimens of Chromodoris cavae from Sri Lanka is gratefully acknowledged. I wish to thank Sandra Millen, Zoology Dept., UBC, for identifying the species. Thanks also go to Mike LeBlanc for performing bioassays on B. subtilus and R. solani and to Dr. T. Allen, Dept. of Pharmacology, Univ. of Alberta, for perfonning cytotoxicity and antineoplastic assays. I wish also to express my deepest gratitude to Dr. Jon Clardy, M . Iqbal Choudhary, and the support staff of Cornell University's Baker Laboratory for determining the X-ray structure of Chromodorolide A . Finally, I'd like to express my appreciation for the assistance I received from the staff of U B C departmental nmr and mass spectrometry labs. -1-Introduction A . Chemical defense in nudibranchs. Shell-loss in marine opisthobranchs has been compensated for by the evolution of secondary defensive mechanisms f l l Nowhere is this more clearly demonstrated than in the completely shell-less nudibranchs (Fig. 1), whose secondary defensive mechanisms can be classified into three categories; behavioral (hiding), morphological and chemical.^ Morphological defenses include camouflage, spicule incorporation and partial autornization. Camouflaging can be achieved through cryptic colorization (homochromy) which enables the molluscs to resemble their food source; countershading which is thought to make their outline indistinct; or disruptive colorization which makes it hard to spot the organism as a whole. Some nudibranchs reinforce their mantles with spiny spicules from sponges, making them difficult to chew. Many nudibranchs employ autonomy; the ability to shed portions of their anatomy while under attack. Aeolid nudibranchs readily discard their cerata when harassed and some discodorids have been observed to shed their mantle edges P 1 . The most astounding morphological defense, however, can be observed in certain coelenterate-feeding aeolids which manage to salvage intact nematocysts from their diet and transport them to specialized areas at the tips of their cerata *4J. When disturbed the nudibranchs will release and fire these stinging cells. A few aeolids of this type are able to selectively retain only the most potent nematocysts of their [1] Faulkner D.J.; Marine Ecology Progress series 1983, 13, 295. [2] Karuso, P. "Chemical Ecology of the Nudibrachs", in Bloorganlc Marine Chemistry, P.J. Scheuer, Ed., Springer Verlag, Berlin, 1987: Vol. 1, pp. 31-60. [3] Ibid. [4] a) Grosvenor, G.H. J. Roy. Soc. London, 1903, 72, 462. b) Thompson, T.E. and Bennett, X. Science, 1969, 166, 1532. -2-TYPICAL CRYPTOBRANCH DORID NUDIBRANCH Dorsum Rhinophore Branchial Plume Foot' TYPICAL AEOLID NUDIBRANCH Rhinophores Ceratal Group Foot Corner Oral Tentacle Branchial Plume Rhinophores Extra-branchial Appendage Oral Tentacles Extra-rhinophoral Appendage \ Foot Corner TYPICAL PHANEROBRANCH DORID NUDIBRANCH Fig. 1 Typical Nudibranchs -3-coelenterate food, although how they distinguish among the various nematocysts or transport these alien cells intact through their bodies to their cerata tips is still a mystery. Perhaps the most interesting defense mechanism developed in nudibranchs is the selective aggregation of defensive metabolites from their food sources, using these in turn as their own chemical deterrents. Garstang observed this amazing ability in 1890 when he noted that many nudibranchs secreted acid as a defense, especially those observed to feed on acidic tunicates f 5 ]. Others subsequently noted that live nudibranchs would not be accepted as food by aquarium fishes and certain nudibranch species even killed fish that shared the aquarium. In 1960 Thompson found specialized skin glands in non-acidic nudibranchs containing bitter or tasteless fluids which he postulated contained compounds employed as chemical defenses by the shell-less molluscs [ 6 \ Since then, researchers have amassed considerable evidence supporting the role of unique secondary metabolites as chemical defenses in nudibranchs. Species from five families of nudibranchs contain organic compounds in their skin extracts which serve as either antifeedants or icthyotoxins [ 7 J. Such chemical defenses are particularly noticeable in dorids that feed on sponges. Sponges have long been recognized as having evolved elaborate chemical mechanisms to solve such problems as predation, fouling, and establishing a "home" within the marine environment. Screening and identifying any of these substances which might benefit humans has become a growing research concern [8 ]. [5] see note 2 above. [6] a) Thompson, T.E. J. Mar. Biol. Ass. U.K., 1960, 39, 115. b) Thompson, T.E. J. Mar. Biol. Ass. U.K., 1960, 39. 123. c) Thompson, T.E. Aust. J. Zool. 1969, 17, 755. [7] Gunthorpe, L. and Cameron, A.M. Mar. Biol., 1987, 94, 39. [8] Some current general references include: a) Thompson, J.E.; Walker, R.P.; Faulkner, J.D. Mar. Biol., 1985, 88, 11. b) McCaffrey, E . J . and Endean, R. Mar. Biol., 1985, 89, 1. c) Munro, M.H.G.; Luibrand, R.T.; Blunt, J .w. "The Search f or A n t i v i r a l and Anticancer compounds from Marine Organisms", i n Bioorganic Marine Chemistry, Vol. I, 1987, Springer-Verlag. -4-Nudibranchs in the family Chromodoridae are spongiverous, brightly colored and rarely, if ever, eaten by reef fishes making them prime candidates for studies of chemical defenses. Roughly twenty species of this family have been examined to date and all have been found to contain interesting metabolites that may be defensive allomones. The primitive chromodorid Cadlina luteomarginata has proven to be a rich source of interesting secondary metabolites P J . This common, fragrant nudibranch has been found to contain terpenes with isonitrile, isothiocyanate and furan functionalities. The isonitriles and associated isothiocyanates, such as 1 and 2 , from this nudibranch are icthyotoxic and inhibit feeding by goldfish and sculpin in lab assays. Furodysinin (3), idadione (4) and pallescensin-A (5) also serve as defensive allomones for C. luteomarginata, while the terpenoid albicanylacetate (6), isolated from specimens collected in British Columbian coastal waters, is the most potent antifeedant found in this nudibranch. Seasonal and regional variations indicate that C. luteomarginata obtains these metabolites from dietary sources. [9] a) Thompson, J.E.; Walker, R.P.; Wratten, S.J.; F a u l k n e r , D.J. Tetrahedron, 1982 , 38, 1 8 6 5 . b) H e l l o u , J . ; Andersen, R.J.; Thompson, J. E . Tetrahedron, 1 9 8 2 , 38, 1 8 7 5 . c) H e l l o u , J . and Andersen, R . J. Tetrahedron Lett., 1 9 8 1 , 22, 4 1 7 3 . Chromodoris elisabethina employs latmnculin-A (7), an icthyotoxin, for its defense V°\ as does C. lochi which obtains the metabolite from the sponge Spongia mycofijiensis[n\ The isomer, latrunculin-B (8), is the major defensive allomone for Glossodoris quadricolor and its prey, the red sponge Latrunculia magnified l l 2 l [10] Okuda, R . K . and Scheur, P . J . Experentia, 1985, 41, 1355. [11] Kakou, Y . ; Crews, P . ; Bakus, G . J . J . Nat . P r o d . , 1987, 50, 482. [12] Mebs, D. J. Chem. Ecol., 1985, 11, 713. Dysidea fragilis, a Pacific marine sponge, and two nudibranchs observed feeding on it, Chromodoris maridadilus and Hypselodoris godeffroyana, were all found to contain the furanosesquiterpenoids nakafuran-8 (9) and -9 (10) [ v i \ These two compounds proved to be effective antifeedants against common reef fishes. Other metabolites, found in D. fragilis but not common to all three organisms, failed to show any antifeedant activity suggesting that the nudibranchs selectively acquire only the effective defensive compounds in their diet Although only compounds 1-10 above are proven antifeedants, many other compounds of similar structure have been isolated from chromodorid nudibranchs. Potential defensive metabolites called scalarins, similar to 11, have been isolated from three species of Chromodoris; C. funerea, C. sedna and C youngbIeuthfi1A\ A variety of linear and cyclic furans such as marislin (12) and furodysinin (13) have been isolated from a number of chromodorid nudibranchs1151. Spiniferin-2 (14), a compound similar to albicanylacetate, has been isolated from Hawaiian specimens of C. albonotata I1*51. The rearranged diterpenoids norrisolide (15) and the macfarlandins (16-20), from C norrisi and C macfarlandi respectively1161, are degradation products of a class of sponge metabolites called "spongians" which may serve as defensive allomones in both phyla. [13] shulte, C ; Scheuer, P.J. and Mcconnell, O.J. Belv. chia. Acta, 1980, 63, 2159. [14] a) Kernan, M.R.; Baxrabee, E.B. and Faulkner, D.J. Comp. Biochem. Physiol., 1988, 89B, 275. b) Hochlovalci, J.E.; Faulkner, D.J.; Bass, L.S.; Clardy, J . J. Org. Chem., 1983, 48, 1738. c) Terem, B. and Scheuer, P.J. Tetrahedron, 19B€, 42, 4409. [15] a) Bochlovskl, J.E. and Faulkner, D.J. Tetrahedron Lett., 1981, 22, 271. b) Cimino, C ; De stefano, S.; De Rosa, s.; Sodano, G.; V i l l a n i , G. Bull. chem. soc. Belg., 1980, 89, 1069. c) Hochlowaki, J.E.; Walker, R.P.; Ireland, C ; Faulkner, D.J. J. Org. Chem., 1982, 47, 88. d) Shulte, G.R. and Scheuer, P.J. Tetrahedron, 1982, 38, 1857. [16] see notes [37], [39] and [42] -7-- 8 --9-B. Spongian Diterpenes. Among the most interesting of the secondary metabolites found in skin extracts from various Chromodoridae nudibranchs are those classified as containing or deriving from the novel hypothetical "spongian" diterpene skeleton (21). The large number of reports detailing the isolation and structures of related compounds published in recent years as well as discoveries in our laboratory have sparked our interest in this novel class of marine diterpenes. In light of the current interest in these compounds, a detailed review of previous work on their isolation and synthesis is warranted. 0 -10-The first diterpene of this novel class was reported by Cimino et. al [ 1 7 J who isolated it from the marine sponge Spongia officinalis. Because of its relation to isoagathic acid (23) the compound was named isoagatholactone (22). The mass spectral fragmentation pattern led to conclusion that the new compound is related to methyl isoagathate (23, R=Me). A detailed series of chemical reactions outlined in scheme I was used to confirm this hypothesis. Identical optical rotations and spectra were observed for isoagathic alcohol (25, scheme I) obtained from both grindellic acid (24, scheme I) and isoagatholactone ([a]D= -9®,-l(fl respectively) which also proved the absolute stereochemistry of the new terpenoid. [17] Cimino, G.; de Rosa, D.; de Stefano, S.; Minale, L. Tetrahedron, 1974, 30, 645. Scheme I -11-POCb , Py LAH (22) -12-Five years later Kazlaukas and coworkers reported the isolation and structure of a related spongian diterpene isolated from the sponge Apfysilla rosea118*. The compound, aplysillin (26, 12a,15a,16a-triacetoxy-spongian), had its structure confirmed by a single crystal X-ray experiment However, this work is not without controversy. A later effort to establish chemotaxonomic evidence for the classification of the so-called advanced sponges (those with reduced or absent spicules) has reclassified the sponge as a member of the genus DcuwinelldP^ and failed to detect any trace of aplysillin in fresh extracts of the sponge. A result which is consistent with chemotaxonomic evidence from closely related species. [18] Kazlauskas, R.; Murphy, P.T.; Wells, R.J. Tetrahedron Lett. 1979, 10, 903. [19] Karuso, P.; Bergquist, P.R.; cambie, R.C.; Buckleton, J i s . ; Clark G.R.; Rickard, C.E.F Aust. J. Chem., 1986, 39, 1643. -13-The unique skeleton shared by the spongians has been of some interest to synthetic chemists. Two South American groups have reported synthetic studies on the spongian diterpenes in the last seven years l 2 0 J . Although to date no stereospecific synthesis has been carried out on the spongian skeleton, enantiomeric isoagatholactone (28) as well as the parent hydrocarbon isocopalane (29) have been synthesized from methyl isocopalate (27) and racemic isoagatholactone has been synthesized from racemic labda-8(20),13-dien-15-oic acid (30). O [20] a) da Miranda, D.S.; Brendolan, G.; Imamura, P.M.; S i e r r a , M.G.; Marsaioli, A.J.; Ruveda, E.A. «7. Org. Chem., 1981, 46, 4851. b) Nakano, T. and Hernandez, M.I. Tetrahedron Lett., 1982, 14, 1423. c) Nakano, T. and Hernandez, M.I. J. Chem. Soc. Perkin. Trans. I., 1983, 135. d) Mischne, M.P.; s i e r r a , M.G.; Ruveda, E.A. J". Org. Chem., 1984, 49, 2035. -14-Following up earlier work on S. officinalis^, Cimino and coworkers examined a fresh specimen of the sponge and reported the isolation of four new metabolites 31-34pll In addition to isoagatholactone (22), S. officinalis was found to contain 15cr,16a-diacetoxyspongian (31), a compound similar to aplysillin (26) but lacking the C-12 acetoxy function, and three tricyclic diterpenes lacking the D ring system 32-34. These opened structures, enf-isocopal-12-en-15,16-dials, are considered to be possible precursors to the spongian system and are named from the base hydrocarbon isocopalane (29) previously synthesized from methyl isocopalate p o a j <PAc [21] cimino, G.; Marrone, R.; sodano, G. Tetrahedron Lett., 1982, 23, 4139. -15-Another group examinirig Spongia officinalis from the Canary Islands I 2 2 J isolated aplysillin (26) as well as four new spongian diterpenes closely related to isoagatholactone (22) indicating that this sponge is a rich source of compounds with the spongian carbon skeleton. Of the hydroxyspongia-12-en-16-ones (35-38) isolated, the 11/3-hydroxy- and 11/3-acetoxy- compounds are biologically active against broad spectrum bacteria and inhibit HeLa cell growth. [22] Gonzalez, A.G.; Estrada, D.M.; Martin, J.D.; Martin, V.S.; Perez, C ; Perez, R. Tetrahedron, 1984, 40, 4109. -16-Karuso and Taylor have also isolated spongian diterpenes closely related to isoagatholactone'23!. These two compounds, 7a-Acetoxyspongia-16-one (39) and 7a,17-diacetoxyspongia-16-one (40), were isolated from specimens of Apfysilla rosea collected in the Great Barrier Reef. (39) (40) The same year they reported the structure' of aplysillin, Kazlaukas et. o i [ 2 4 J reported the isolation of another variety of diterpenes related to spongian. The spongiols, 41-48, isolated from various species of Spongia sponges collected in the Great Barrier Reef, were the first diterpenoids related to isoagatholactone (22) to be reported. The term "spongian'' was used first in this paper and, following IUPAC guidelines, they proposed a sernisystematic nomenclature for naming diterpenes with the same carbon skeleton. Any functionality on the tetracyclic hydrocarbon is listed by the numbering scheme of the hypothetical spongian skeleton (21). Hence, isoagatholactone (22) is named under the new system as spongia-12-en-16-one and the compounds isolated by Kazlaukas and coworkers are named 3a, 19-dihydroxyspongia-13(16), 14-dien-2-one (41, spongiadiol); 3a,19-diacetoxyspongia-13(16),14-dien-2-one (42, spongiadiol diacetate); 3o,17,19-trihydroxyspongia-13(16),14-dien-2-one (43, spongiatriol); 3a,17,19-triacetoxyspongia-13(16),14-dien-2-[23] Karuso, P. and Taylor, W.C. Aust. J • Chem., 1986, 39, 1629. [24] Kaslaukas, R.; Murphy, P.T.; wells, R.J.; Noack, K.; Oberhansli, W.E.; Schttnholzer, P. Aust. J. Chem, 1979, 32, 867. -17-one (44, spongiatriol triacetate) as well as the 3/3 epimers of these four compounds 4^5-48, epispongiadiol etc.). While trivial names still persist in the literature, especially for highly functionalized derivatives, most authors follow the naming scheme outlined in this paper. Some years later another Australian group reexamined the same sponge Kazlaukas had worked on and classified it as belonging to a new genus, Rhopaloeides t251. R odorablie, as the species is now named, was found consistently to contain the four 3a epimers 41-44, though samples genetically identical to each other produced varying amounts of the spongiols depending on environmental factors. No evidence was found for the natural presence of any 3/3-spongiol and it was suggested that these compounds may have resulted by silica catalyzed epimerization in Kazlaukas' purification. R Ri R 2 R3 (41) OH H OH H (42) OAc H OAc H (43) OH H OH OH (44) OAc H OAc OAc (45) H OH OH H (46) H OAc OAc H (47) H OH OH OH (48) H OAc OAc OAc [25] Thompson, J.E.; Murphy, P.T.; Bergquist, P.R.; Evans, E.A. Biochem. System. Ecol. 1987, 15, 595. -18-The marine sponge Spongia officinalis continued to provide diterpenes related to isoagatholactone in subsequent research efforts. A Belgian group working on specimens of this sponge collected in the waters near Papua-New Guinea isolated three new spongians 49-51 in 19801261. These three compounds all contain the furan moiety of the spongiadiols but lack any oxidation of the A ring system. In fact, spongia-13(16),14-dien-19-oic acid (49) is remarkably similar to the isoagathic acid and may represent an early stage in biosynthesis. [26] Cape l i e , N.; Braekman, J . C ; Daloze, D.; Tursch, B. Bull. Soc. Chlm. Belg., 1 9 8 0 , 89, 3 9 9 . .19-Six spongian diterpenes 42,44,52-55 were isolated from a dorid nudibranch, Casella atromarginata, by de Silva and Scheuer in 1982 p 7 ] . Two of these were the previously reported peracetates of spongiadiol and spongjatriol (42 and 44); two were the 3a hydroxyl derivatives of the same structures 52-53; and two were enolized diones 54-55. These last two, 2^1749-trihyo!roxyspongia-13(16),14-dien-3-one (54) and its 17,19-diacetoxy ester are related closely enough to poriferan spongian diterpenes that the authors suggest that C atromarginata has oxidized the A ring of a dietary precursor. (54) Rj = R 2 = H (55) Rj = H R 2 = A c [27] da S i l v a , E.D. and Scheuer, P.J. Beterocyclea, 1982, 17, 167. -20-Wbile reports of isolation and structural elucidation of spongian-related terpenoids have been fairly frequent, details concerning biological activity of these interesting compounds have been relatively rare. One such report came from the SeaPharm Project Laboratories { 2 8 1 . A deep water Caribbean species of Spongia was found to contain previously reported spongiadiol and epispongiadiol (41 and 45) as well as the new compound isospongiadiol (56, 2a,19-dihydroxyspongia-13(16),14-dien-3-one). Cytotoxicity assays against P388 cells of 41, 45 and 56 yielded IC^ values of 0.5,8,and 5 pg/ml, respectively. Antiviral activity against Herpes Simplex Virus I gave the values of (L25, 12.5, and 2 pg/ml, respectively. Comparison with standards indicates that while only mildly cytotoxic, spongiadiol and isospongiadiol are fairly effective as antiviral agents in in vitro assays. While technically not a spongian diterpene, marginatafuran (57), isolated from the nudibranch Cadlina luteomarginata by Gustafson and Andersen [ 2 9 \ has a remarkably similar skeleton. The nudibranch was collected in the waters off the Queen Charlotte Islands of British Columbia, a region thought to have remained ice-free during the last glaciation period. Perhaps the resulting evolutionary isolation from the Indo-Pacific marine basin has allowed unique development in the marine invertebrates near the islands. The amazingly slight difference in structure between marginatafuran and diterpenoids from other parts of the world certainly may be evidence for such a claim. [28] Kohmoto, S . ; M c C o n n e l l , O . J . ; Wright , A . J . ; C r o s s , S. Chem. Lett., 1987, 1687. [29] Gus ta f son , K . and Andersen , R . J . Tetrahedron Lett., 1985, 26, 2521. -21-A Caribbean sponge, Igemella notabilis, has also proven to contain spongian terpenoids [ 3 0 ) . The 7a,17)3-dihydroxy-15,17-oxidospongian-16-ones isolated from this sponge 58-60 have lactone-ketal functionalities and an interesting cyclization pattern from Me-17. Whether this functionality serves any biological function may be pondered but no evidence has been offered in either case. O (58) R = COPr (59) R = Ac (60) R = H Several reports of compounds related to those from Igemella notabilis came out the following year P1!. Molinski and Faulkner reported the isolation of 6a,7a47p-trihydroxy-15/3,17-oxidospongian-16-one 7-buryrate (61) from an Australian Apfysilla species. Karuso and Taylor reported the isolation of the above compounds (58, 59, and 61) from Apfysilla rosea as well as three related compounds which he called the aplyroseols (62-64). The aplyroseols and four related compounds 65-68, were also isolated from the sponge Dendrilla rosea. [30] Schmitz, F.J.; Chang, J.S.; Hossain, M.B.; van der Helm, D. J. Org. Chem., 1985, 50, 2862. [31] a) Molinski, T.F. and Faulkner, D.J. J. Org. Chem., 1986, 51, 1144. b) see note [23]. c) see note [19]. -22-O Ri R2 (61) OCOPr OH (62) OCOPr OAc (63) OH OCOPr (64) OAc OCOPr (65) H H (66) OAc OAc Eight compounds 69-76 related to the aplyroseols 61-66 were reported isolated from an Australian nudibranch classified as Ceratosoma brewccmdatumP®. The wide variety in oxidation and cyclization patterns of these nine compounds poses some interesting questions about the role of functionality in the spongians. (67) R = H - (68) R = OH [32] Ksebati, M.B. and Schmitz, F . J . J. org. chem., 1 9 8 7 , 52, 3766. -23-O (70) Ac H (71) Ac Ac (72) COPr Ac (73) R = OAc (74) R = OCOPr (75) R = H -24-An interesting degraded spongian diterpene was isolated from eastern Australian samples of Apfysilla sulphweal33\ Aplysulphurin (77), is related to the spongian skeleton through a single methyl shift (C17) and an opening of ring B. Karuso and coworkers later reexamined the same sponge'341, now reclassified as DarwineUa oxeata, and again found aplysulphurin as well as the tetrahydroaplysulphurins 1 (78), 2 (79) and 3 (80). OAc [33] Karuso, P.; Skeleton, B.W.; Taylor, H.C.; White, A.B. Aust. J. Chem., 1984, 37, 1081. [34] see note [19] above. - 25 -The sponge Spongionella gracilis provided a compound related to the aplysulphurins but completely lacking the C17 methyl1351. Gracilin A (81), a norditerpene which is similar to 15a,16a-diacetoxyspongian (31) could possibly be derived from a precursor similar to this undegraded terpenoid isolated earlier by Cimino et al from Spongia officinalis. A dinorditerpene, gracilin B (82), found in conjunction with gracilin A in 5. gracilis^ has a highly unusual carbon skeleton that appears to derive from a spongian-type diterpene, but if so rearrangement involves a more complex mechanism than the simple cationic shifts and oxidations which account for other rearrangements. OAc OAc [35] Mayol, L.; F i c c i a l l i , v.; Slca, D. Tetrahedron £ett., 1985, 26, 1357. [36] Mayol, L.; P i c c i a l l i , v.; Slca, D. Tetrahedron L e t t . , 1985, 26, 1253. -26-From the nudibranch Chromodoris macfarlandi two terpenoids similar to aplysulphurin have been isolated p 7 J . Macfarlandins A and B (16 and 17) are epimeric aromatic norditerpenes, familial to aplysulphurin (77) but without the C-17 methyl and the 6 lactone, and appear to represent more highly oxidized relatives of gracilin A (81). O (16) Rj = H R 2 = OAc (17) R x = OAc R 2 = H Molinski and Faulknef reported the isolation of two more terpenoids in this family, 9,11-dihydrogracilin A (83) and membranolide (84), found in the Antarctic sponge Dendrilla membranosaP8*. Since D. membranosa grows slowly, contains no spicules and has never been observed to be eaten, it is likely that some form of chemical defense exists. These two compounds are likely candidates. OAc [37] M o l i n s k i , T.F. and F a u l k n e r , D.J. J. Org. Chem., 1986, 51, 2601 [38] M o l i n s k i , T.F. and F a u l k n e r , D.J. J. Org. Chem., 1987, 52, 296. -27-Faulkner's research group at Scripps Institution of Oceanography was the first to report the elucidation of a "rearranged spongian diterpene" containing a novel carbon skeleton related to a spongian precursor. Norrisolide (15), which they isolated from the nudibranch Chromodoris norrisP 9^, has also been found as a trace component in extracts of the sponge DendriUa sp. collected at Palau, though this sponge is not native to the Gulf of California where C norrisi was collected. A possible biogenesis of norrisolide was proposed starting from a spongian skeleton and following the series of bond transformations outlined in scheme n. AcO [39] Hochlowski, J.E.; Faulkner, D.J.; Matsumoto G.K.; clardy, j . J". Org. Chem., 1983, 48, 1141. -28-Besides norrisolide, Dendrilla sp. contains three other diterpenes which can be envisioned as being derived through rearrangement of the spongian skeleton. The dendrillolides A, B and C (85-87), isolated from Palauan marine lake specimens of the sponge by Sullivan and Faulkner1401, can be thought of as arising from spongian diterpenes via the mechanism proposed in scheme DX [40] Sullivan, B and Faulkner, D.J. J. Org. chem., 1984, 49, 3204. -29-Rearranged spongians continued to be found in a variety of organisms. The marine sponge Chelonapfysilla violacea has been found to contain the two terpenoids aplyviolene (88) and aplyviolacene (89)[41l Aplyviolene has the same structure proposed by Sullivan and Faulkner as dendrillolide A (50), but differences in spectral data have brought into question the actual structure of dendrillolide A. Aplyviolene was solved by an X-ray diffraction study. H (88) (89)R1 = OAc R 2 = H (20)R 1 = H R 2 = OAc From the nudibranch Chromodoris macfarlandi three more rearranged spongian terpenoids have been isolated'421. Macfarlandins C (18) and D (19) are lactone cyclization isomers of each other, bearing a carbon skeleton that is only slightly modified through shift of Me-20 and cleavage of the C9-C11 bond of a spongian precursor. Macfarlandin E (20) on the other hand bears the carbon skeleton of the dendrillolides and appears to be an epimer of aplyviolacene (89). [41] Hambley, T.W.; Poiner, A.; Taylor, W.C Tetrahedron Lett., 1986, 28, 3281. [42] Molinski, T.F.; Faulkner, D.J.; Cun-heng, H.; Van Duyne, G.D.; Clardy, J . J. org. Chem., 1986, 51, 4564. - 3 0 -O The latest report of spongian type diterpenoids reports the structure of ten compounds related to the macfarlandinsl431. Five compounds (90-94, shahamins A-£ ) related to macfarlandin E have been isolated from one Dysidea species of sponge and five others (95-99, shahamins F-J), related to macfarlandins C and D, but containing a unique C5-C10 double bond, have been isolated from another. Shahamins A-E display a wide variety of cyclization in the heterocyclic portion of the molecule and again calls into question the biological role the various cyclization isomers may play. Shahamins F-J are unique in that these five compounds contain a tetrasubstituted double bond across the decalin system. Such compounds are rare in marine systems and this once again indicates the spongians are a novel and fascinating class of compounds. [43] Carmely, s.; Cojocaru, M.; Loya, Y.; Kashman, Y. J. Org. Chem., 1 9 8 8 , 53, 4801. -32 -O (99) R1 = OH R 2 = H -sa-i l . Our Current Work The opportunity arose to study the organic skin extract of a previously uninvestigated chromodorid nudibranch, Chromodoris cavae, collected in the Indian Ocean. In light of the interesting metabolites isolated to date from molluscs in this genus, we were very interested in investigating the metabolites a new species collected in a different geographical region. A series of silica flash and thin-layer chromatography procedures led to the purification of three diterpenoid metabolites 100-102 from the crude skin extract of specimens of Chromodoris cavae. The metabolite isolated in greatest quantity, chromodorolide A (100) proved to be a rearranged spongian diacetate with a new carbon skeleton. The two minor metabolites each proved to be rearranged spongian monoacetates, one of these, 101, being the previously isolated dendrillolide A. - 3 4 -Discussion Part I—Chromodorolide A Electron impact mass spectrometry (Fig. 2) failed to show a parent ion for the compound. A peak at 390.2043 amu corresponding to a formula of C22**30^6 (calcd. 390.2044) was the highest mass observed, due to the facile loss of acetic acid from the molecular ion. Losses of a methyl (375.1807 C 2 1 H 2 7 0 6 , calcd. 375.1807), C 0 2 (346.2144 C21H3QO4, calcd. 346.2144) and another acetic acid molecule (330.1825 C^QH^C^, calcd. 330.1831) from the M-60 mass at 390 gave further indication of a parent formula of C ^ F ^ O g . A CI mass spectrum (Fig. 3) did show this parent ion as an M+1 peak at 451 amu while the largest peak observed in the CI was at 468 indicating incorporation of the ionizing agent NH3. * H nmr spectroscopy of chromodorolide A in CDCI3 (Fig. 4) revealed many interesting features. Two acetate methyl resonances were present at 6 2.12 and 2.08 ppm. Three aliphatic methyl resonances at 6 0.88, 0.86, and 0.85 ppm, an exchangeable proton signal at 6 3.31 ppm, and two ketal protons resonating at 6 6.36(s) and 5.79 (dd, J=2.9,1.8 Hz) ppm gave some indication that the molecule might be a rearranged spongian diterpenoid. One dimensional decoupling experiments helped to work out the spin system for the heterocyclic portion of the molecule (see structure A below). Irradiation of the multiplet at 6 5.79 ppm simplified the signals at 3.06 ppm. Irradiation of the doublet at 6 4.83 ppm simplified the 2.45 ppm resonance to a doublet of doublets. Irradiation at 6 2.45 ppm simplified 3.06 ppm, collapsed 4.83 ppm into a singlet and increased the intensity of an upfield signal near 1.3 ppm. When complications arose due to two coincidental multiplet signals at 6 3.06 ppm, careful examination of the decoupled spectra suggested that these two protons were coupled to each other giving the spin system outlined below. IflV it tt It it I f AW it Zt I f t 3C2 2 M 275 3 f f 328 3Bf 37B M f ST — i — | — i — r 37S " i — | — i — i — i — i — j — i — i — i — i — j — i — r «ff 428 49* I f * It • f 7t «f tt At it tt It t 13 II I 10 C9 Ii [Jl ll,l,„ . , . . . 75 1 I f f lf» It.l.lil u 123 153 139 LJilL [•..i|ilH p 12S tit I7i Figure 2. Electron Impact mass spectrum of (100) • I. , ,. I" •|",,»'" I1 ' '| " I1 !• 2 f f 225 2Sf in C M O M O O l O U O t * I C O C H I L JJ 5 , 5 5 - ° 4 5 4 - ° P ^ A"O 2.5 i.S 1.5 7 J " ~ Flgure4 400 MHz % nmr spectrum of Chromodorilide A (100) in CDCh 0.5 0.0 EJDSLN14.500 DATE 22-10-88 SF ST 01 SI TD SV HZ/PT PV RO no RG NS TE 408.080 0.0 6290.000 32788 32768 4000.000 .244 16.5 0.0 4.096 1 48 298 FV 5000 02 8800.000 OP 50L PO LB GB CX CT FI F2 HZ/CM PPM/CH . IBB 8.8 35.88 8.8 6.9BBP -.IBBP 88.BB1 .288 SR 4412.B7 CtlRONOOOROLIOC « ICOCLSI Figure 4a 400 MHz % nmr CDCI3 upfield region EJOSLN14.500 DATE 22-10-88 SF 400.000 ST 0.0 01 6290.000 SI 32768 TD 32768 SV 4000.000 HZ/PT .244 PV 16.5 RO 0.0 PO 4.096 RC 1 NS 48 TE 298 FV 5000 02 8800.000 OP 50L PO LB .100 CB 0.0 CX 35.00 CT 0.0 FI 6.900P F2 -.100P HZ/CN 80.001 PPM/CM .200 SR 4412.07 CNflOllOOOROLIOC • I C O U l l JL 5 . 5 5 . 0 4 . 5 4.0 3 . 5 3 . 0 Figure 4b. 400 MHz % nmr CDCI, downfield region EJDSLN14.500 DATE 22-10-88 SF 400.000 ST 0.0 01 6290.000 SI 32768 TO 32768 SW 4000.000 HZ/PT .244 PV 16.5 RO 0.0 AO 4.096 RG 1 NS 48 TE 298 FW 5000 02 8800.000 OP 50L PO LB .100 GB 0.0 CX 35.00 CT 0.0 Fl 6.900P F2 -.100P HZ/CM 80.001 PPM/CH .200 SR 4412.07 vo -40-nmr spectroscopy in CDCI3 (Fig. 5) revealed twenty-two of the twenty-four signals expected for a diterpenoid diacetate. Three carbonyl signals at 6 172, 170 and 169 ppm accounted for the diacetate and indicated the possible presence of a lactone in the molecule. Two ketal methines at 6 104 and 95 5 ppm as well as highly deshielded quaternary and methine resonances at 80 and 79 ppm respectively accounted for the remaining oxygen containing sites. Only three methyl resonances were observed clearly, at 6 33.4,20.8 and 13.6 ppm, however, the peak at 20.8 is the most intense in the entire spectrum. The relatively low intensity of this peak in the APT spectrum1461 indicates that the signal may be due to more than one carbon. In order to clear up some of the ambiguities created by coincidental signals in the C D C I 3 nmr spectra of chromodorolide A (100), C^Dg was tried as a solvent. Some fairly dramatic shifts were observed in both the *H and nmr spectra (Figs. 6 and 7, respectively) as a result [46] Patt, S.L.; Schoolery, J.N. J. Mag. Reaon. 1982, 46, 5 3 5 . C H R O M O D O R O L I D E A P T Figure 5. 75.4 M H z 1 3 C nmr spectrum and A P T of (100) in C D C 1 3 CftROHOOO*OUOE a A— A— JL_JL^ 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 EJDSLN14.505 DATE 12-11-88 SF 400.000 ST 0.0 01 6290.000 SI 32768 TD 32768 sw 4000.000 HZ/PT .244 PV 12.0 RD 0.0 no 4.096 RC 1 NS 48 TE 298 FW 5000 02 5935.996 DP 71 PO LB 0.0 GB 0.0 CX 35. 00 CT 0.0 FI 6.900P F2 -.100P HZ/CM 80.001 PPM/CM .200 SR 4395.22 1.0 0.5 0.0 Figure 6. 400 MHz *H nmr spectrum of (100) in C 6 D 6 E J D S L N 1 4 . 5 0 5 OflTE 1 2 - 1 1 - 8 8 SF ST 01 SI TD SV HZ/PT PV RD 00 RG NS TE 4 0 0 . 0 0 0 0. 0 6 2 9 0 . 0 0 0 32768 32768 4 0 0 0 . 0 0 0 . 2 4 4 1 2 . 0 0 . 0 4. 096 1 48 298 FV 5000 . 02 5 9 3 5 . 9 9 6 DP 7L PO 0. 0 0. 0 3 5 . 0 0 0 . 0 6 . 9 0 0 P - . 1 0 0 P 8 0 . 0 0 1 . 2 0 0 4 3 9 5 . 2 2 Li r — T 1 • .1 • • • | • i • | • • • | I'""' 1 ' 1 ' I ' ' ' I 1 ' I ' ' '• I ' 1 ' 1 ' ' ' I ' 1 -R—' 1 • • I • I ' • ' I ' • I • I • • i 6.80 6.63 6.10 6.20 6.00 5.60 5.60 5.40 S.20 5.00 4.80 4.60' 4.10 4.20 4.00 3.S0 3.60 3.40 3.20 3.03 2.32 2.60 PPM Figure 6b. Expanded downfield region of fig. 6 C r i R O M O D O R O i . I D A P T E A i . , 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 1 1 1 1 1 1 1 I i ' 160 140 120 T1" IOC I I I I I I I I 80 ' I • ' ' i | 60 T r 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 C B B D E C 0 U P L E 0 40 20 PPM | • ) I I | I I I I | I I I I | I I II | II I I | I I I I | I II I | I I I I | I I I I | I I I 1 | • I I i i I I I i | i i t i | i i i i | ' ' i i l ' i i i | i i i i | ' 1 1 ' | U\ ISO 16C 140 120 100 80 60 40 20 PPM 0 Figure 7. 75.4 MHz 1 3 C nmr spectrum and APT of (100) in C 6 D 6 - 4 6 -The proton ketal singlet at 6 6.36 ppm was shifted downfield to 6.67 ppm in deuterobenzene. The apparent triplet at 5 5.79 in CDCI3 clearly became a doublet of doublets at 5.59 ppm in deuterobenzene and the doublet at 4.83 ppm moved downfield to 5.03 ppm. The exchangeable proton shifted downfield slightly and the coincidental signals fonning a multiplet at 6 3.06 in CDCI3 resolved into a doublet of doublets at 2.96 ppm and a double doublet of doublets at 2.76 ppm in CgDg. The acetates moved upfield to 6 1.80 and 1.60 ppm and the aliphatic methyls shifted to 0.80, 0.77 and 0.45 ppm. Two highly shielded multiplets at 6 0.87 and 0.57 ppm which each integrated to a single proton were also now clearly resolved. The 1JC nmr spectrum recorded in CgDg again showed three carbonyl signals, two ketal methines, as well as down field quaternary and me thine carbons. Five methines, two quaternary and five methylene resonances accounted for all but the five methyls. Four of the methyls were easily observed at 6 33.4,20.44,20.36 and 13.4 ppm. The remaining methyl resonance appears to be nearly coincident with the methylene at 6 20.9 ppm. The intensity of the peak in the BB decoupled spectrum, as well as a small negative peak in the APT spectrum, indicates that this is indeed the case. The molecular formula of C ^ r T ^ O g , with three sites of unsaturation accounted for by the carbonyls, indicated that the molecule was pentacyclic. Although ID decoupling, COSY' 4 7 ! (Fig. 8) and COSYPHDQ' 4 8 ! (Fig. 9) experiments helped to work out the spin system in structure A , this system could not be made to fit any previously reported pentacyclic spongian diterpenoid carbon skeleton. A n aliphatic [4,3,0] bicyclic system as in norrisolide could be deduced from the five methylene resonances in the nmr spectrum as well as the mass spectral fragment at m/z 165 corresponding to the ionic form of structure B. Therefore the heterocyclic portion of the molecule must be a tricyclic system. Since no such [47] Bax, A "Two-Dimensional Nuclear Magnetic Resonance i n L i q u i d s " , R e i d e l , Boston, 1982. Chapter 2. [48] Sanders, J . K . M . and Hunter, B . K . "Modern NMR Spectroscopy, A Guide f o r Chemists", Oxford U n i v . Press , New York, 1987. Chapter 4. EJDHCST.SHX RU PROC: cosr.mi ORTE S-II-8B SI2 1024 Sll 512 SV2 2283.IIS SVI 1141.5S3 NDI 1 KOV2 S tfOMI S SSB2 I SSBI | HC2 H PL1N HOW: 'I 3.227P .3ISP ONO COLUNN: 'J 3.294P .3ISP 01 I. • • • m i 16.SI 01 .1111131 RO l . l l . l Of 273.11 NS 16 OS 2 NC | IN .1114361 CHROHOOOROliOC A IH-IH COST Figure 8a. Expanded upper right quadrant of fig. 8 EJOMCST.SNX AU PROC: CQST.flU DATE 5 - 1 1 - 8 8 S I 2 ID 2 4 S M S I 2 SJJ2 2 2 8 3 . 195 III " " « 3 NOB 1 VOV2 S VOW I S SSB2 0 SSBI | HC2 N PLIM ROW: BHO COLUMN?"' Jl S.294P « .Slfip OJ l . l l l l l l l £1 , 6 - 5 1 OB . I B I I I 3 I 2 . . . » • ' • ••• 2| "3.81 NS 16 OS 2 NC 1 IN .1114381 CHR0M0O0ROLI3E fl OOUBLE QUANTUM COST 2.2 —""I 2.1 l.B — r -1.6 • i * ' •• i 1.* 1.2 PPN -r——r~ .61 EJDUPDQ.SMX AU PROG: COSTPHDQ.AU DATE 18-9-88 SI2 1024 SI1 1024 SW2 800.000 SW1 400.000 ND0 2 MDV2 S VDV1 S SSB2 0 SSB1 0 MC2 W PLIM ROW: F l 2.261P F2 .265P AND COLUMN: F l 2.26 Figure 9. PHDQCOSy of (100, aliphatic resonances) in C 6 D 6 - 5 0 -structure had been reported to date and nmr experiments could not unequivocally determine the pattern of cyclization, an X-ray diffraction experiment was performed, by Jon Clardy and coworkers at Cornell University, on a chromodorolide A crystal grown from hot methanol1491 The computer-generated drawing of the X-ray crystal structure of chromodorolide A (100) (Fig. 10) indicated that the molecule generally followed the norrisane (scheme JJ) skeleton but a new ring was formed through an unprecedented C-17-C-12 carbon-carbon bond. We suggest the new carbon skeleton resulting from this bond be named "chromodorane" (structure C). [49] Crystals of chromodorolide A belonged to the space group P212121 with a=8.653(2), b=9.662(3), c»30.743(9) A and one molecule of composition C24H34°8 C H3O H forming the asymmetric u n i t . Additional data can be found i n the appendix. -51-C(31) C(18) Figure 10. Computer generated X-ray structure of (100) -52-With the structure of chromodorolide A (100) t50' in hand, assignment of most of the * H and nmr resonances was fairly straightforward. It is interesting to note that the downfield ketal is a singlet due to a dihedral angle of nearly 90 degrees between H-16 and H-13. A l l other resonances in the down field * H nmr spectrum (Fig. 6b) could be assigned to appropriate protons by coupling patterns and chemical shifts (see Table I). In order to assign the carbons and decipher the couplings observed in the PHDQCOSY plot of the aliphatic system, a single bond heteronuclear correlation (HETCOR) experiment1511 was performed (Fig. 11). Since the * H nmr spectrum for the heterocyclic portion of the molecule had already been assigned, assignment of this portion of the was easily accomplished by examination of the H E T C O R contour plot (Table I). Assignment of the aliphatic portion was more difficult owing to the fact that two isolated spin systems with many overlapping multiplets were involved. In an attempt to sort out the chemical shifts of all the aliphatic protons, a 2-D J-resolved1521 experiment was performed (Fig. 12). By careful correlation between the HETCOR, PHDQCOSY and 2D J contour plots it could be seen that the H-8 methine (6 2.16 ppm) was coupled into a methine at 6 1.26 ppm (H-9). This methine was further coupled into one methylene proton at 6 1.47 ppm (H-7a). This resonance was coupled into its upfield geminal partner at 6 1.03 ppm (H-7/3) as well as another methylene proton at 1.10 ppm (H-6a). Coupling can be seen from H-6a into its geminal partner at 6 1.28 ppm (H-6/3) and an upfield methine at 0.67 ppm (H-5). Correlations from 6 1.28 ppm (H-6/3) to 0.67 (H-5) and 1.03 (H-7/3) ppm complete the spin system. Assignment of the remaining six protons was more difficult since they form a completely isolated spin system. It was possible to get a handle on the system [50] The abso lute s tereochemistry shown i n (100) as w e l l as the numbering system used i s based on the assumption t h a t the chromodorane ske le ton i s generated from a spongian p r e c u r s o r . [51] Bax, chapter 2. [52] Bax, Chapter 3. F2 (PPM) 110 -100 -90 80 H 70 60 -50 -40 30 -20 -10 J L J L PPfA 6 —T" 4 9-Figure 11. ^ - ^ C HETCOR of (100) in C 6 D 6 VARIAN XL-300 13C OBSERVE EXP9 PULSE SEQUENCE: HETCOR DATE 04-10-88 SOLVENT C6D6 FILE HETCOR ACQUISITION TN 13.750 SW • 12500.0 AT 0. 082 NP 2048 PW 18. 0 P1 0 D1 1. 000 02 0 TO -400 NT 128 CT 0 PW90 18. 0 -iW2 700. 0 Nl 32 BS 64 SS 0 IL Y JN Y DP Y HS NN J1XH 80. 0 JNXH 8. 0 PRESAT N HMULT Y GAIN 0 DEC. Sc VT DN 1. 750 DO -800.0 DM NNY DMM CCS DMF 8000 DHP N DLP 0 PP 81. 9 PROCESSING RE 0. 005 FN 2048 AF 0. 020 MATH F FN2 128 RE2 0. 003 AF2 0. 01 1 DISPLAY SP 672. 0 W 12500.0 VS 180 SP2 55. 6 WI 700. 0 SC 0 WC 200 IS 500 RFL 8981. 0 RFP 9654. 9 TH 9 SC2 290 WC2 200 INS 1. 000 RFL2 -51.6 F2 (PPM) VARIAN XL-300 13C OBSERVE EXP9 PULSE SEQUENCE: HETCOR DATE 23-09-88 SOLVENT C606 FILE HETCOR ACQUISITION DEC. & VT TN 13.750 DN 1. 750 sw 8703.2 DO -100.0 AT 0. 118 DM NNY NP 2048 DMM CCS PW 18.0 DMF 6700 PI 0 DHP N Dl 0: 400 DLP 0 D2 0 PP 81. 9 TO -3000 -. • NT 512 PROCESSING CT 512 RE 0 007 PW90 18. 0 FN 2048 SW2 2100.0 AF 0. 029 NI 64 MATH F BS 32 FN2 256 SS 0 RE2 0. 002 IL Y AF2 0 008 IN Y DP Y DISPLAY - ; HS NN SP 8. S J1XH 140. 0 WP 5019 5 JNXH 0 VS 3028 . PRESAT N SP2 -16.5 HMULT N W»2 1256 7 SC 0 wc .. 200 IS 500 RFL 9681.5 RFP . 96926 TH 9 SC2 290 WC2 200 INS 1.000 RFL2 2161.9 RFP2 2144.6 AI DC AV Figure 11a. Expanded lower right quadrant of fig. 11 Figure 12. 2D-JRES plot of (100) in C 6 D 6 SLNU2DJ.SHX RU PROG: JRES.qu ORTE 3-10-B6 SI2 1024 SI1 256 SW2 1039.501 SW1 32.468 NQ0 2 W0W2 s WDW1 S SSB2 0 SSB1 0 HC2 H PLIM ROW: 2.49SP JNO C0LUHN?98P l\ -081P F 2 -.B81P 01 l.L g§ .0000038 RO 0 . 0 p« 0.0 NS ^ OS 2 NE 128 IN .8B770BB L -10 U 16 AU PROC: JRES.AU DATE 3-10-88 SI2 S l l SV2 SW| NOO 1124 256 1839.501 J2.«68 2 V0V2 S VOVI S SSB2 e SSB1 0 HC2 H PL IN ROW: 2.495P »N0 C0LUMN?98P 1\ .B8IP F 2 -.B8IP 01 PI 00 P2 RO PH OE NS OS NE IN I.I8BBBBB 25. BB .1008830 SB. BB B. 0 _ 8.8 601.BB 32 2 128 .I077BBB NERTI - 5 7 -through the use of nOe difference spectroscopy.153' Irradiation of the methine at 6 5.59 ppm (H-15) resulted in a 6% positive enhancement of the multiplet at 0.56 ppm (H-la), and a 62% enhancement of H-14. Irradiation of the upfield multiplet at 6 0.56 ppm (H-la) resulted in a 12.5% enhancement of the ketal proton at 5.59 ppm (H-15) as well as a 27.5% enhancement to a proton signal at 1.05 ppm (H-l/3). Irradiation of the methine at 6 2.75 ppm (H-14) also displayed a 3.9% positive nOe to the same 1.05 ppm (H-l/3) proton signal. Such nOes can be explained by examining a molecular model of chromodorolide A (see structure D, below). If, as the X-ray structure shows, ring A exists in a chair conformation with both Me-20 and Me-19 axial, then H-15 can be brought close enough to H-la and H-l/3 to account for the observed enhancements. The H-14 methine on the other hand is only close enough to the H-l/3 proton to exhibit a significant nOe. O 0.80 H 0.67 [53] Sanders and Hunter, Chapter 6. -58-Examination of the COSYPHDQ contour plot (Fig. 9) showed the H - l a proton at 6 0.56 ppm coupling into its geminal partner at 1.05 (H-l/3) as well as another methylene signal at 1.18 ppm (H-2/3). This latter proton signal showed coupling into protons at 6 1.05 (H-l/3), 1.3 (H-2a or H-3/3) and 0.87 ppm (H-3a). Examination of the H E T C O R plot indicated that a 6 1.3 ppm proton was geminal to the proton at 1.18 ppm (H-2a and H-2/3 respectively). However, the proton at 6 0.87 ppm (H-3a) also has a geminal partner at 1.3 ppm (H-3/3). H-3a couples into one of the protons at 6 1.3 ppm as well as the H-2/3 signal. Examination of the PHDQCOSY and 2D-J contour plots did not permit the exact determination of the chemical shifts or coupling patterns of the two equatorial protons at 5 13 ppm (H-2a and H-3/3). A l l that remained to be assigned were the five methyl peaks and the quaternary carbon signals. Irradiation of the methyl at 6 0.45 ppm (Me-20) resulted in a 6.5% positive nOe to the methine at 2.16 ppm (H-8) as well as a 7.5% enhancement of the methyl at 0.77 ppm (Me-19). Me-20 is thus assigned as the upfield resonance while 6 0.77 must be Me-19. The * H nmr methyl resonance at 6 0.80 ppm (Me-18) has a highly deshielded methyl carbon (33.4 ppm C-18) which is characteristic of Me-18 in other spongian diterpenoids, thus supporting the other aliphatic methyl assignments. Acetate methyls were assigned to their respective carbonyls by a long range heteronuclear correlation experiment (Fig. 13) optimized for a 2 or 3 bond coupling constant of 8 Hz. A clear correlation can be seen between the methyl * H resonance at 1.80 and the carbonyl at 169.5 ppm as well as acetate methyl signal at 1.60 ppm to the carbonyl at 168.0 ppm. Tentative assignment of the acetates to their respective methinyl protons (H-16, H-17) was based on the observation of a weak nOe (3.8%) from the methyl resonance at 1.60 ppm to the methine proton at 6.67 ppm, however this assignment may be erroneous, since no other nOe's were observed to either acetate. Aliphatic quaternary carbons are assigned by comparison to other spongians. Complete assignments of both *H and ^ C spectra are listed in Table I. L O N G - R A N G E H E T C O R C O N T O U R 60 • 40 -20 -• „ — i 1 I i 1 1 i i 1 1 r i' 1 » u 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0 4 0. 2$f|A Figure 13. Long range ^ -^C HETCOR of (100) in C 6 D 6 EXP9 PULSE SEQUENCE: HETCOR DATE 04-10-88 SOLVENT C6D6 F I L E LRHTCR ACQUISITION DEC. & VT TN 13.750 DN 1. 750 SW 12500.0 DO - 8 0 0 . 0 AT 0. 082 DM NNY NP 2048 DMM CCS PW 18. 0 DMF 8000 PI 0 DHP N 01 1. 000 DLP 0 D2 0 PP 81. 9 TO -400 NT 128 PROCESSING CT 0 RE 0. 005 PW90 18. 0 FN 2048 SW2 7 0 0 . 0 AF 0. 020 NI 32 MATH F BS 64 FN2 128 SS 0 RE2 0. 003 IL Y AF2 0. 011 IN Y DP Y DISPLAY HS NN SP 806. 5 J1XH 80. 0 VP 6 1 0 9 . 5 JNXH 8 . 0 VS 175 PRESAT N SP2 111.1 HMULT Y WP2 255. 6 SC 0 WC 200 IS 500 RFL 8981. 0 RFP 9654. 9 TH 9 SC2 290 WC2 200 INS 1. 000 22.65 4000 3500 3000 88/11/10 10:54 8lnl4: 16 scans, 4.0cm-i chromodorolide A 2500 2000 1500 1000 car* 500 OS © Figure 14. Infrared spectrum of (100). thin film -61-TABLEI Complete Assignment of *H and ^ C nmr Spectra ofChromodorolide A (100). CARBON NUMBER ^(CgDg) 1 3 C ( C 6 D 6 ) la 0.57 1/3 1.05 40.0 2a 13 2/3 1.18 20.1* 3a 0.87 3/3 13 41.2 4 33.0 5 0.72 56.8 6a 1.10 6/3 128 21.0* 7a 1.45 7/3 1.03 26.7 8 2.17 44.5 9 126 512 10 * 423 11 172.6 12 3.65 (O-H) 80.4 13 2.96 52.7 14 2.76 465 15 5.59 1042 16 6.67 95.9 17 5.05 792 18 0.80 33.4 19 0.77 20.9 20 0.45 13.4 1 6 -MEC0 2 168.0 16- H 3 C - C 0 2 1.60 20.4 17- M E C 0 2 169.5 17-H 3C-C0 2 1.80 20.4 assignments may be exchanged - 6 2 -Part n—DendrilloUde (101). One of the monoacetates isolated has been identified as the previously reported dendrillolide A. Only 6.1 mg of this terpenoid was isolated and it degraded in a subsequent purification step. However, comparison of the *H nmr spectrum (Fig. 15) with that of a monoacetate isolated in good quantity by E . Dilip de Silva from Chromodoris gleniei proved that the two metabolites were identical. Dr de Silva has identified the compound as dendrillolide A based on a comparison of nmr chemical shifts. Table II lists the nmr shift assignments of the monoacetate compared to reported values for dendrillolide A. Dr de Silva is currently trying to establish the stereochemistry of the heterocyclic portion of the molecule in order to resolve the conflicting evidence on the structure of dendrillolide A. The structure reported previously, 85, has been shown to be incorrect through comparison to spectral data of aplyviolene (88). Such discrepancies are thought to have arisen by differences in the stereochemistry at position 15. OAc -64-Table II Comparison of ^ C nmr Chemical shifts (CgD^) for Dendrillolide A isolated from C. gleniei to reported values. Carbon # Found Reported1541 1 28.7 28.7 2 38.1 38.7 3 37.7 37.7 4 46.7 46.7 5 54.9 54.9 6 27.1 27.1 7 37.7 37.7 8 36.0 36.0 9 55.7 55.7 10 153.8 153.7 11 175.1 175.2 12 28.8 28.8 13 41.9 41.9 14 54.6 54.5 15 105.0 105.0 16 97.1 97.1 17 24.1 24.1 ... 18 25.7 25.8 19 34.4 34.5 20 114.3 113.3 OAc 20.7 20.7 169.0 169.1 [54] see note [40] above. -65-Part m—Chromodorolide B (102) The second monoacetate (chromodorolide B, 102) seems to be related to chromodorolide A (100). The % nmr of (102) (Fig. 16) is similar to that of (100), (Fig. 4), but has some very interesting differences. Three aliphatic methyls at 6 0.70, 0.77 and 0.80 ppm and a single acetate resonance at 2.13 ppm are present in the CDCI3 *H nmr spectrum of (102). Interestingly, however, a methyl ether resonance at 6 333 ppm is present instead of a second acetate. The heterocyclic portion of the chromodorolide B also displays a different coupling pattern than that observed in (100). One-dimensional decoupling and *H-*H COSY (Fig. 19) experiments have worked out the spin system outlined in partial structure E below. The downfield doublet at 6 6.04 ppm (J= 7 Hz) is coupled into a doublet of doublets coincidental with the methyl ether at 333 ppm (dd, J=7,10 Hz). The proton at 6 333 ppm is further coupled into a resonance at 2.85 ppm (ddd, J=53,7,10 Hz) which is coupled into the two protons resonating at 5.07 ppm (d, J=5.5 Hz) and 235 (ddd, J=7,9,ll Hz). The methine proton at 6 235 ppm also couples into the ketal doublet at 5.48 (J=9 Hz) and into a multiplet at 1.80 ppm. CH40N000R01I01 II JJ J L Jl k h — i — '6.5 • " I T -'6.6 EJOSLN04.500 DATE 23-10-88 400.B08 0.0 6290.000 32768 32768 4000.000 .244 16.5 0.0 4.096 1 32 298 FW 5008 02 8888.880 OP SOL PO .300 0.0 35.00 0.0 6.900P -.100P 80.001 .200 4413.01 • i • ~5."b 4.5 4.0 3.5 3.0 2.5 Fig. 16 *H nmr spectrum of chromodorolide B (102) in CDCI3 PPH Fig. 16a Expansion of upfield region of fig. 16 t ' I ' • • | • • • | ' • • | • I • | • I ' | ' I • | . I . - | - ' 1 • | • I • | • . • • . , . • • , . . r ~ . • • • • • • I • . . . I 6.88 b.60 6.41 6.21 B.H 5.81 5.6B 5.48 5.20 S.88 4.88 4.bit 4.40 4>» 4.00 3.80 3.61 J.«B 3.2B 3.11 2.8B 2.M ON 00 PPM Fig. 16b Expansion of downfield region of fig. 16 -69-The strongest evidence supporting a relationship between (100) and (102) is the mass spectral fragmentation patterns of the two compounds (Figs. 2 and 17 respectively). Chromodorolide B has an almost identical fragmentation pattern in the low mass (<200 amu) region of the spectra. The peak at m/z 163 seems to indicate that the chromodorolides both contain the [4,3,0] bicyclic system of partial structure B. Comparison of the 1 3 C nmr spectra of (100) and (102) (Figs. 5 and 18 respectively) also implies a familial relationship between the two compounds. Two ketal methines at 6 106.7 and 104.1 ppm in the chromodorolide B spectrum as well as another oxymethine at 77.7 ppm are very reminiscent of chromodorolide A . Five aliphatic methines and five methylenes indicate a skeleton very similar to (100) in chromodorolide B. A l l five methyl resonances can be observed. A downfield methyl at 6 33.4 ppm is indicative of Me-18 in spongian diterpenoids. Two resonances at 6 21.3 and 20.7 ppm account for the acetate and Me-19 while the peak at 13.3 is likely Me-20. The fifth methyl is a methyl ether resonance 6 54.7 ppm. Because of small sample size and its relative impurity, quaternary carbons and carbonyls could not be observed. Table III lists a comparison of ^ C nmr resonances for chromodorolide A (100) and B (102). Although undeniably similar to (100), the spin system of chromodorolide B outlined in partial structure E cannot be reconciled with the chromodorane skeleton. Work is currently underway to elucidate the structure of chromodorolide B which should prove to be another highly unusual rearranged spongian diterpenoid. 100 90 •0 70 60 60 40 30 20 10 0 35* 375 - ••|ll..,--.-|tf-|0».|l- 1..t.|..tl|. j,,,)!,.., , ,|, |||| 276 300 326 360 362 407 376 litliil^ lllljlili, i I,., 111, I I , 400 426 I | 1" I I I | ' I I I I | 460 478 600 100 90 80 70 60 50 40 30 20 10 '1 43 86 T 5 0 ml—hi iLllpl • i 95 75 109 123 ,138 463 100 203 175 2 0 0 260 j.llip.llj.i.i^ . 1^ O Fig. 17 EI mass spectrum of chromodorolide B (102) Fig. 18 1 3 C nmr spectrum of (102) in CDCI3 - 1.00 _ 1 . 50 . 2.00 - 2.50 . 3.00 . 3.50 . 4.00 - 4.50 . 5.00 . 5.50 , 6.00 PPM EJDSLN04.SMX flU PROC: C03Y.AU DATE 23-10-88 SI2 1024 SI1 512 SW2 2283.105 SW 1 1141.553 NO0 1 WDW2 S WDW1 S SSB2 0 SSB1 0 MC2 M PLIM ROW: F 1 6.228P F2 . 531P AND COLUMN. F l 6.228P F2 . 531P Dl 1.0000000 PI 16. 50 D0 .0000030 P2 16. 50 RD 0.0 ,PW 0.0 DE 273.80 NS 80 DS 2 NE 256 IN .0004380 EJDSLN04.SMX flU PROG: COSY.flU DATE 23-10-88 SI2 1024 SI 1 512 SW2 2283. 105 SW 1 1141. 553 ND0 1 WDW2 S WDW1 S SSB2 0 SSB1 0 MC2 M PLIM ROW. F l 3. 006P F2 531P AND COLUMN F l 3. 006P F2 • 531P Dl 1 . 0000000 Pt 16 . 50 D0 . 0000030 P2 16 . 50 RD 0.0 PW ' 0 . 0 DE 273 . 80 NS 80 DS 2 NE 256 IN .0004380 88/09/13 16:01 SCAN: 64 scans, 4.0CB-1 sln04 monoacetate #2 Fig. 20 Infrared spectrum of (102), thin film. - 7 5 -TABLE III Partial Comparison of 1 3 C nmr resonances for Chromodorolides A (100) and B (102). )N NUMBER (100) (102) 1 40.4 40.2 2 19.8 19.9 3 41.1 41.3 5 57.8 57.4 6 20.8 21.0 7 26.6 25.2 8 44.6 47.2 9 51.6 50.0 13 52.1 55.4 14 46.3 48.4 15 104.3 106.7 16 95.6 104.1 . 17 78.8 77.7 18 33.4 33.4 19 20.8 20.7 (21.3) 20 13.6 13.3 -76-CONCLUSION Three rearranged spongian metabolites 100-102 have been isolated from the Indian Ocean nudibranch Chromodoris cavae. At least one of these, chromodorolide A , (100), displays a wide range of biological activity and may serve as a chemical antifeedant. Work is currently underway to elucidate the structures of the other two minor metabolites, and plans to examine their possible role in inhibiting fish predation are being made. -77-Experimental The * H and nmr spectra were recorded on Bruker WH-400 and Varian XL-300 spectrometers. Most spectra were run using either CDCI3 or CgDg as an internal standard, though the peaks were referenced to internal tetramethylsilane (6=0 ppm) in order to correct for solvent variance. Low-resolution electron impact mass spectra were recorded on an A.E.I. MS-902 spectrometer and high resolution mass spectra were recorded on an A.E.I. MS-50 spectrometer. Chemical ionization mass spectra were recorded on a Nermag R 10-10 C spectrometer employing NH3 as the ionizing agent. Infrared spectra were recorded on a Perkin-Elmer series 1600 FTTR. Optical rotations were measured on a Perkin-Elmer model 141 polarimeter using a 1.000 dm microcell, and a Fisher-Johns apparatus was used to determine melting points which are uncorrected. A Perkin-Elmer Series 2 instrument was used for hplc wherein a Perkin-Elmer LC-55 uv detector linked to a Hewlett-Packard 18850A recorder was employed for peak detection. A Whatman Magnum-9 Partisil 10 column was used for preparative hplc. A l l hplc, chromatotron and crystallization solvents were B D H Omni-solv grade or Fisher hplc grade. A l l other chromatography solvents were reagent grade. Merck Silica Gel 230-400 mesh was used for flash chromatography employing N 2 as the propulsive gas. Preparative tic was carried out with Merck Silica Gel 60 PF-254 on glass plates. A Harrison Research model 7924 Chromatotron was used with Merck Silica Gel 60 PF-254 / C a S O ^ V ^ O for radial tic. -78-Collection Data Ninety specimens of Chromodoris cavae [ 4 4 ] were collected from the waters near Jaffna on the northern coast of Sri Lanka during the months of January to March 1988. Extraction and Purification The fresh animals were preserved in dichloromethane/methanol (1:1) and stored in the freezer. The organic layer was separated from thawed samples, the aqueous layer was concentrated at reduced pressure and extracted with more methylene chloride. The combined organic layers were evaporated at reduced pressure to yield 2.74 g of crude extract as a red-orange oil. One half of the residue was dissolved in dichloromethane (10 mL) and fractionated by silica flash chromatography using a step gradient elution of increasing amounts of diethyl ether or methanol in dichloromethane. The less polar fractions (0-10% diethyl ether / methylene chloride) were found to contain mainly long chain hydrocarbons and steroids, while those eluting at high polarity (60% diethyl ether and 10-50% methanol in dichloromethane) contained mainly triacyl glycerides and fatty acids. The fractions eluting between twenty and thirty percent diethyl ether in dichloromethane were shown by tic and nmr to be of the greatest interest. These fractions were further purified by preparative scale silica thin layer chromatography (prep tic) using a solvent system of 12% diethyl ether in methylene chloride. The major component, (100), (Rf 0.42) was shown to be pure by tic and nmr. Minor components failed to purify completely on a series of silica high [44] identified by Sandra Millen, Zoology Dept. UBC A voucher sample i s deposited at UBC. -79-performance chromatography (hplc) which followed. The very weak chromophores of the minor components made it difficult to interpret the ultraviolet detector traces. Better response was often seen for impurities than the compounds of interest. Further purification was achieved using the chromatotron with a solvent system of 15% Et20/CH2Cl2. Analysis of the eluting fractions by nmr spectroscopy indicated a high degree of purity for both a diacetate (chromodorolide A , 100) and two monoacetates. Chromodorolide A was easily separated from trace colored impurities by crystalization from hot methanol to give 31 mg of colorless needles (melting point 133-134°C), [a]D -0.4 (c 3, CDCI3). Further purification of the monoacetates was not judged necessary. Evaporation under reduced pressure yielded 6.2 mg of dendrillolide A (101) and 4 mg of a previously unreported diterpene (chromodorolide B, 102). Chromodorolide A (100) is biologically active, inhibiting growth of Bacillus subtilis and Rhizoctonia solani at a rninimum inhibitory concentration of 60 ^ g/disc. Cytotoxic activity against L1210 gave an E D ^ value of 20^g/ml, and antineoplastic activity in the P388 mouse leukemia assay resulted in a T / C value of 125% at 4mg/kg with no toxicity observed at this level.1 4 5 1 Chromodorolide A (100):Electron Impact Mass Spectrometry m/z (rel. int.): Low Resolution: 390 (1), 375 (1), 362 (3), 334 (5), 319 (6), 274 (8) 164 (27), 163 (31), 123 (58), 109 (36), 95 (53), 81 (55), 69 (57), 55 (30), 43 (100); High Resolution: 390.2043 ( C 2 2 H 3 0 O 6 , calcd. 390.2042), 375.1807 ( C ^ H ^ O g calcd. 375.1807), 334.2152 ( C 2 0 H 3 0 ° 4 c a l c d 334.2144), 330.1825 ( C 2 o H 2 6 0 4 calcd. 330.1831), 274.1939 [45] EDgQ refers to an "effective dose" causing 50% inhibition of cell growth. L1210 is a human lymphocytic leukemia c e l l line. T/C is a ratio of survivors in test/control groups of leukemiatic mice. For particulars on the assay procedures employed contact Dr. T. Allen, Dept. of Pharmacology, Univ. of Alberta, Edmonton, Alta, Canada. -80-(CjgH2g02 calcd. 274.1933). Chemical Ionization Mass Spectroscopy m/z: 469, 468,451, 408, 392, 391, 362, 332, 319, 287, 267,224, 218, 177, 163, 138,123, 109, 95, 81. Fourier Transform Infrared Spectroscopy, thin film, cm"*: 3852, 3464, 2948, 2869, 2846, 1770, 1654, 1220. XH nmr in CDCI3 (400 MHz, ppm from TMS):6 6.36 (s,lH), 5.79 (dd,lH, J= 1.9,1.7 Hz), 4.83 (d,lH, J=2.9 Hz), 3.31 (s,lH,exch.), 3.06 (m,2H), 2.45 (ddd,lH, J=2.9,5.6,12.2 Hz), 2.12 (s,3H), 2.08 (s,3H), 1.82 (m,lH), 1.34 (m,lH), 1.07 (m,lH), 0.88 (s,3H), 0.86 (s,3H,), 0.85 (s,3H). 1 3 C nmr in CDCI3 (75.4 MHz, ppm from TMS):6 172.3 (C), 170.0 (C), 168.9 (C), 104.3 (CH), 95.55 (CH), 79.9 (C), 78.8 (CH), 57.8 (CH), 53.4 (C), 52.1 (CH), 51.6 (CH), 46.3 (CH), 44.6 (CH), 42.5 (C), 41.1 (CH 2 ) , 40.4 (CH 2 ) , 33.4 (CH 3 ) , 26.6 (CH 2 ) , 20.8 ( C H 2 , C H 3 ) , 19.8 (CHj), 13.6 (CH 3 ) . *H nmr in C 6 D 6 (400 MHz, ppm from TMS):6 6.67 (s,lH), 5.59 (dd,lH,J=3.5,1.0 Hz), 5.03 (d,lH,J=2.6Hz), 3.61 (s,lH,exc), 2.96 (dd,lHJ=5.4,1.0Hz), 2.76 (ddd,lHJ=3.5,6.8,5.5 Hz), 2.17 (ddd,lHrT=6.8,2.9,12.0Hz), 1.80 (s,3H), 1.60 (s,3H), 1.45(m,lH), 0.87(m,lH), 0.80 (s,3H), 0.77 (s,3H), 0.74 (dd,lH,J=5.3,11.6 Hz), 0.57 (m,lH), 0.45 (s,3H). 1 3 C nmr in C 6 D 6 (75.4 MHz, ppm from TMS):6 172.6 (C),' 169.4 (C), 168.0 (C), 104.2 (CH), 95.9 (CH), 80.4 (C), 79.2 (CH), 57.3 (CH), 52.7 (CH), 51.7 (CH), 46.5 (CH),45.1 (CH), 42.3 (C), 41.2 (CH 2 ) , 40.0 (CH 2 ) , 33.4 (CH3), 33.0 (C), 26.7 (CH 2 ) , 20.9 ( C H 2 , C H 3 ) , 20.44 (CH 3 ) , 20.36 (CH3), 20.1 (CH 2 ) , 13.4 (CH3). Dendrillolide (101). Electron impact mass spectrometry, low resolution, m/z (rel. int.); 374 (1), 316 (5), 301 (6), 255 (5), 237 (5), 166 (25), 137 (51), 121 (28), 93 (28), 69 (53), 55 (33), 43 (100). Fourier transform infrared spectrometry, thin film, cm"*; 2950, 1799, 1752, 1451, 1366, 1225, 986. *H nmr (400 MHz, ppm from TMS in CDC1 3):6 6.49 (d,lH, J=7.5Hz), 6.10 (d,lH, J=4.4Hz), 4.87 (d, lH, J=2.2Hz), 4.60 (d,lH, J=2.2Hz), 3.20 (m,lH), 2.74 (m,2xlH), 2.56 (dd,lH, J=9.4,17Hz), 2.16 (s,3H), 1.01 (s,3H), 0.98 (s,3H), 0.97 (s,3H). 1 3 C nmr (75.4 MHz, ppm from TMS in CDC1 3):6 175.48 (C), 169.63 (C), 153.25 (C), 114.41 (CH 2 ) , 104.86 (CH), 96.95 (CH), 55.89 (CH), 54.44 (CH), 54.34 (CH), 46.61 (C), 41.85 (CH), 37.95 (CH 2 ) , 37.84 (CH 2 ) , 37.41 (CH 2 ) , 35.88 (C), 34.23 (CH3), 28.92 (CH 2 ) , 28.28 (CH 2 ) , 26.75 -81-( C H ^ , 25.56 (CH 3 ) , 23.97 (CH 3 ) , 21.07 (CH 3 ) . See Table H for comparison to reported * 3 C nmr values of dendrillolide A . Chromodorolide B (102): Electron impact mass spectrometry, low resolution, m/z (rel. int.); 407 (2), 391 (2), 390 (3), 375 (2), 362 (2), 286 (14), 250 (26), 235 (22), 203 (45), 163 (45), 123 (67), 109 (35), 95 (44), 85 (59), 71 (100). Fourier transform infrared spectrometry, thin film, cm - 1 ; 2924,1787,1727,1691,1462, 1451,1258, 974. % nmr spectroscopy (CDCI3, 400 MHz, ppm from TMS); 6 6.04 (d,lH, J=7 Hz), 5.48 (d, lH, J=9 Hz), 5.07 (d,lH, J=5.5 Hz), 3.33 (dd,lH, J=7,10 Hz, s,3H), 2.85 (ddd,lH, J=5.5,7,10 Hz), 2.35 (ddd,lH, J=7,9,ll Hz), 2.13 (s,3H), 0.80 (s,3H), 0.77 (s,3H), 0.70 (s,3H). 1 3 C nmr spectroscopy (CDC13, 75.4 MHz, ppm from TMS); 6 106.7 (CH), 104.1 (CH), 77.7 (CH), 57.5 (CH), 55.4 (CH), 54.7 (CH), 50.0 (CH), 48.4 (CH), 47.2 (CH), 43.0 (C), 41.3 (CH 2 ) , 40.2 (CH2), 33.4 (CH 3 ) , 33.1 (C), 29.8 (CH2), 25,2 (CH 2 ) , 21.3 (CH 3 ) , 21,0 (CH 2 ) , 20.8 (CH 3 ) , 19.9 (CH 2 ) , 13.2 (CH 3 ) . -J -1-5 A H and lJC nmr experiments: Parameters used in the individual experiments are listed to the left of the spectra. Abbreviations used are as follows. SF, standard proton frequency; O l , deuterium lock reference; SI, size (in bits) of FTD acquired; SW, sweep width in Hz; PW, pulse length in /jsec; R D , relaxation delay; A Q (or AT), acquisition time in sec; NS (or NT), number of scans; NE, number of experiments. 2D nmr Experiments: A l l parameters used in the acquisition and processing of two-dimensional nmr experiments (i.e. FID size, sweepwidths, offsets, etc.) can be found in the parameter lists contained with the contour plots within the discussion. Variations on the parameters' abbreviations above are as follows: PI P2, pulse lengths in psec; SI1 SI2, relative FTD sizes in two dimensions; ND0, number of phase switches; D l , relaxation delay. General guidelines and pulse sequences are listed below. -82-1 H - 1 H COSY: All COSY experiments were performed on a Bruker WH-400 nmr instrument using the following pulse sequence: D l - PI - DO - P2 - FID where D l is a relaxation delay of 1.0 second, PI is a 90 degree pulse, P2 is either a 60 or 90 degree pulse, and DO is a small delay of 3 usee to allow the shift and coupling patterns to evolve. Generally, 256 IK FIDs completed the matrix which allowed zero-filling in the F l domain but not in F2. COSYPHDQ: A PHase sensitive Double Quantum COSY experiment was performed on a Bruker WH-400 nmr spectrometer employing the following pulse sequence; D l - PI - DO - PI - D3 - PI - FTD where D l is a relaxation delay of 1.2 seconds, PI is a 90 degree pulse, DO is a short (3 psec) delay for evolution and D3 is a fixed delay (3 usee) for phase switching. Phase correction on the 2D matrix was accomplished by obtaining phasing parameters from a ID spectrum acquired using a 90 degree pulse and a relaxation delay of 12 seconds. A 512K matrix was employed for this experiment. Homonuclear J-Resolved 2-D nmr: 2D-J spectroscopy was performed on the Bruker WH-400 nmr spectrometer using the Hahn spin-echo pulse sequence below; D l PI - DO - P2 - DO - FTD where D l is a relaxation delay of 1.0 seconds, DO is an evolution delay of 3 /jsec, and PI, P2 are 90 and 180 degree pulses respectively. Sweep width in the F l domain was set equal to or larger than one half the width of the largest multiplet, chosen such that the ratio I2D (Hz/pt/Hz/pt) is a multiple of two, in order to allow symmetrization and tilting of the transformed 128K 2D matrix. 1 H - 1 3 C HETCOR: Both single bond and long range HETCOR experiments were performed on the Varian XL-300 nmr spectrometer with the following pulse sequence; -83-! H D l - 90° x - t l - A X - 90° y - ^ 2 " ^ - B B 1 3 C 180° x 90° x F E D where D l is a set relaxation delay and all other delays (tl, and t\j) are set by choice of single and multiple bond coupling constants. The single bond experiment was performed using a D l of 0.4 sec and a coupling constant of 140.0 Hz. The long range H E T C O R was run using a D l of 1.0 sec, a single bond J value of 80.0 H z and a multiple bond J value of 8.0 Hz. - 8 4 -Appendix Material in this appendix includes interatomic distances and angles determined in the X-ray crystallography experiment performed at Cornell University. The numbering system employed is one arbitrarily assigned by the computer and bears no relation to the spongian numbering system employed throughout the text of this thesis. A copy of the X-ray structure numbered as per the data listed in this appendix precedes the tables of interatomic distances and angles. C(25> -86-Table 1. Atomic coordinates (xlO ) and equivalent isotropic 2 3 displacement parameters (A xlO ) X y z U(eq) C(l) 4325(9) 6654(10) 593(3) 62(3) C(2) 3340(12) 5612(12) 334(3) 78(4) C(3) 4134(11) 5108(11) -89(3) 78(4) C(4) 5749(10) 4455(10) -17(3) 56(3) C(5) 6635(10) 5486(9) 262(2) 50(3) C(6) 8331(10) 5139(9) 392(3) 60(3) C(7) .8724(10) 6241(9) 737(3) 56(3) C(8) 7205(10) 7551(9) 1306(3) 51(3) C(9) 7198(10) 7025(9) 834(2) 50(3) C(10) 5918(10) 6008(9) 692(3) ' 49(3) C ( l l ) 7620(12) 10775(11) 1300(3) 66(4) C(12) 7933(10) 9741(10) 1663(3) 57(3) C(13) 6435(10) 9169(9) 1867(3) 56(3) C(U) 5795(10) 8366(10) 1475(3) . 54(3) C(15) 5209(11) 9619(10) 1225(3) 64(3) C(16) 5227(10) 10236(12) 1956(3) 73(4) C(17) 8608(10) 8461(9) 1423(3) " 50(3) C(18) 6536(13) 4343(11) -466(3) 88(4) C(19) 5624(13) 2981(9) 167(3) 82(4) C(20) 5721(11) 4840(9) 1047(3) 67(3) 0(21) 9541(7) 7662(6) 1720(2) 56(2) C(22) 11134(11) 7657(10) 1658(3) 59(4) C(23) 11866(12) 6640(9) 1963(3) 77(4) 0(24) 11745(8) 8359(8) 1399(2) 80(3) 0(25) 8871(7) 10330(7) 1987(2) 74(2) 0(26) 6450(7) 10399(6) 1023(2) 64(2) 0(27) 8350(9) 11778(7) 1227(2) 83(3) 0(28) 4540(7) 10522(7) 1540(2) 73(3) 0(29) 4008(7) 9577(8) 2217(2) 83(3) C(30) 3590(13) 10179(13) 2586(3) 83(5) C(31) 2208(11) 9485(13) 2775(3) 102(5) 0(32) 4259(11) 11133(11) 2729(3) 153(5) C(33) 1746(17) 2883(13) 1652(6) 200(11) 0(34). 1565(9) 1576(9) 1738(3) 126(4) * Equivalent isotropic U defined as one t h i r d of the trace of the orthogonalized U.. tensor -87-Table 2. INTERATOMIC DISTANCES (A) C(l)-C(2) 1.542 (14) C(l)-C(10) 1. 544 (12) C(2)-C(3) 1.548 (13) t(3)-C(4) 1. 549 (13) C(4)-C(5) 1.522 (12) C(4)-C(18) 1. 543 (12) C(4)-C(19) 1.536 (13) C(5)-C(6) 1. 557 (12) C(5)-C(10) 1.545 (11) C(6)-C(7) 1. 540 (12) C(7)-C(9) 1.552 (12) C(8)-C(9) 1. 538 ( I D C(8)-C(14) 1.543 (12) C(8)-C(17) 1. 541 (12) C(9)-C(10) 1.544 (12) C(10)-C(20) 1. 580 (12) C(ll)-C(12) 1.523 (13) C(ll)-0(26) 1. 373 (12) C(ll)-0(27) 1.178 (12) C(12)-C(13) 1. 542 (12) C(12)-C(17) 1.555 (12) C(12)"-0(25) 1. 403 (10) C(13)-C(14) 1.538 (12) C(13)-C(16) 1. 493 (14) C(14)-C(15) 1.520 (13) C(15)-0(26) 1. 453 (11) C(15)-0(28) 1.425 (11) C(16)-0(28) 1. 437 (11) C(16)-0(29) 1.470 (12) C(17)-0(21) 1. 442 (10) 0(21)-C(22) 1.391 (11) C(22)-C(23) 1.498 (13) C(22)-0(24) 1.172 (12) .0(29).-C(30) 1. 325 (13) C(30)-C(31) 1.489 (15) C(30)-O(32) 1. 175 (16) C(33)-0(34) 1.299 (15) -88-Table 3. INTERATOMIC ANGLES (°) C(2)-C(l)-C(10) 109. 3 (8) C(2)-C(3)-C(4) 114. 2 7) C(3)-C(4)-C(18) 107. 4 (7) C(3)-C(4)-C(19) 111. 5 [8) C(18)-C(4)-C(19) 107. 2< (8) C(4)-C(5)-C(10) 119. 5l [7) C(5)-C(6)-C(7) 103. 7( [7) C(9)-C(8)-C(14) 118. 9< [7) C(14)-C(8)-C(17) 104.71 [7) C(7)-C(9)-C(10) 104. 2< [7) C(l)-C(10)-C(5) 108. 8< [6) C(5)-C(10)-C(9) 99. 3< :6> C(5)-C(10)-C(20) 113. 8( [7) C(12)-C(ll)-0(26) 114. 4< [8) 0(26)-C(ll)-0(27) 119. 7( :9) C(ll)-C(12)-C(17) 103. 91 [7) C(ll)-C(12)-0(25) 110. 9< [8) C(17)-C(12)-0(25) 116. 2( 7) C(12)-C(13)-C(16) 114. 5( :8> C(8)-C(14)-C(13) 103. 7< 7) C(13)-C(14)-C(15) 96. 5< 7) C(14)-C(15)-0(28) 106. 3( 7) C(13)-C(16)-0(28) 105. K 7) 0(28)-C(16)-0(29) 105. 8( 7) C(8)-C(17)-0(21) 106. 4< 7) C(17)-0(21)-C(22) 118. 1< :6) 0(21)-C(22)-0(24) 122. 5( :9) C(ll)-0(26)-C(15) 114. 6( 7) C(16)-0(29)-C(30) 118. 3< :8> O(29)-C(30)-O(32) 122. 1( : i i ) C(l)-C(2)-C(3) 113. 2 (8) C(3)-C(4)-C'5) 105. 5 (7) C(5)-C(4)-C(18) 109. 1 [7) C(5)-C(4)-C(19) 115. 8 [7) C(4)-C(5)-C(6) 118. 6i 7) C(6)-C(5)-C(10) 103. 2< [6) C(6)-C(7)-C(9) 106. 3i 7) C(9)-C(8)-C(17) 114. 3l 7) C(7)-C(9)-C(8) 109. 81 7) C(8)-C(9)-C(10) 118. 7( 7) C(l)-C(10)-C(9) 116. Oi 7) C(l)-C(10)-C(20) 109. 21 7) C(9)-C(10)-C(20) 109. 6| :6) C(12)-C(ll)-0(27) 125. 71 :9) C(ll)-C(12)-C(13) 112. 6< 7) C(13)-C(12)-C(17) 102. 9i 7) C(13)-C(12)-0(25) 110. 1( :6> C(12)-C(13)-C(14) 99. 5< :6) C(14)-C(13)-C(16) 103. 8( 7) C(8)-C(14)-C(15) 120. 1< 7) C(14)-C(15)-0(26) 112. 5( 7) 0(26)-C(15)-0(28) 105. 9( 7) C(13)-C(16)-0(29) 107. 7< :8) C(8)-C(17)-C(12) 105. 6( 7) C(12)-C(17)-0(21) 109. 6< :6) 0(21)-C(22)-C(23) 109. 6< :8) C(23)-C(22)-0(24) 127. 9< :9) C(15)-0(28)-C(16) 108. 5< 7) 0(29)-C(30)-C(31) 110. 9< : i o ) C(31)-C(30)-0(32) 127. o< : i o ) -89-Table 4. Anisotropic displacement parameters (A xlO ) UU U22 U33 U23 U13 U12 C(l) 53(6) 73(6) 59(6) -5(6) -6(5) 10(6 C(2) 73(7) 82(7) 79(7) 0(7) -11(6) -2(7 C(3) 79(7) 81(7) 73(6) -5(6) -18(6) -13(7 C(4) 62(6) 58(6) 49(5) -5(5) -13(5) 0(5 C(5) 62(6) 46(5) 43(5) -1(A) 5(5) -1(5 C(6) 63(6) 60(6) 57(5) -7(5) 0(5) 6(5 C(7) 55(6) 59(6) 53(5) -10(5) -1(5) 5(5 C(8) 65(6) 41(5) 47(5) -1(4) 2(5) 7(5 C(9) 62(6) 48(5) 39(4) -1(4) 5(4) -8(5 C(10) 56(5) 49(5) 42(5) 4(4) -2(4) -2(5 C(U) 74(7) 60(7) 63(6) -10(6) 5(6) 2(6 C(12) 60(6) 64(6) 47(5) -11(5) -5(5) -9(5 C(13) 60(6) 65(6) 44(5) -3(5) 9(5) -3(6 C(14) 53(5) 57(6) 52(5) -7(5) 10(5) -3(5 C(15) 66(6) 70(6) 57(5) -14(5) -2(5) 7(6 C(16) 59(6) 92(8) 69(7) -35(7) 20(6) -7(6 C(17) 51(5) 59(6) 40(5) 3(5) -1(5) -1(5 C(18) 102(8) 101(8) 60(6) -27C6) -14(6) 0(8 C(19) 105(9) 59(6) 82(7) " -11(5) -18(7) -4(7 C(20) 89(7) 59(6) 54(5) 7(5) -6(5) -10(6 0(21) 55(4) 62(4) 50(3) 7(3) 0(3) -1(3 C(22) 58(7) 55(6) 64(6) -5(5) -3(6) -9(6 C(23) 72(7) 69(6) 90(7) 6(6) -17(7) 10(6 0(24) 57(4) 100(6) 83(5) 26(5) 3(4) -5(4 0(25) 65(4) 98(5) 61(4) -16(4) 1(3) -16(4 0(26) 72(4) 62(4) 59(4) 7(3) 2(4) 4(4 0(27) 87(5) 56(4) 105(6) 9(4) 2(5) -11(4 0(28) 69(4) 79(5) 72(4) -21(4) 8(4) 14(4 0(29) 78(5) 97(5) 75(4) -42(4) 28(4) -14(5 C(30) 82(8) 93(8) 74(7) -35(7) 11(7) -4(8 C(31) 84(8) 145(11) 79(7) -41(8) 31(7) -21(9 0(32) 152(9) 192(10) 116(7) -102(7) 58(6) -77(8 C(33) 94(11) 80(10) 425(31) -68(15) 46(17) -17(9 0(34) 65(5) 95(6) 219(10) -14(7) 25(6) -20(5 The anisotropic displacement exponent takes the form: - 2 » r2( h2a *2U . i + ' • • • + 2hka*b*TT ) Tab le 5. H-Atom c o o r d i n a t e s (x lO ) and i s o t r o p i c 2 3 d i sp l a cemen t parameters (A x lO ) X y z U H(1A) 3804 6850 862 80 H U B ) 4441 7506 436 80 H(2A) 2374 6037 258 80 H(2B) 3114 4817 510 80 H(3B) 4242 5888 -280 80 H(3C) 3479 4447 -231 80 H(5B) 6718 6304 86 80 H(6A) 9011 5219 147 80 H(6B) 8410 4219 508 80 H(7A) 9505 6862 631 80 H(7B) 9104 5797 995 80 H(8A) 7270 6747 1489 "80 H(9B) 7163 7815 645 80 H(13A) 6625 8573 2111 80 H(14B) 4965 7764 1560 80 H(15B) 4451 9343 1014 80 H(16B) 5638 11053 2091 80 H(17B) 9170 8726 1167 80 H(18B) 7542 3935 -437 80 H(18C) 5913 3781 -655 80 H(18D) 6633 5254 -587 80 H(19B) 6636 2593 205 80 H(19C) 5107 3020 443 80 H(19D) 5040 2413 -30 80 H(20C) 4929 4201 960 80 H(20D) 6680 4354 1082 80 H(20E) 5437 5265 1318 80 H(23A) 12963 6675 1919 80 H(23B) 11632 6871 2259 80 H(23C) 11497 5724 1901 80 H(25B) 9675 • 10679 1872 80 H(31D) 1951 9951 3041 80 H(31E) 1349 9538 2578 80 H(31F) 2438 8532 2835 80 H(33A) 937 3416 1785 80 H(33B) 1704 2998 1342 80 H(33C) 2730 3194 1758 80 H(34A) 1009 793 1849 80 rOKSION ANGLES FOR CHROHODOROLIOE CIO Cl C2 C3 -55.7 (0.0) C2 Cl CIO C20 -71.9 (0.0) C? C3 C4 CIS -166.4 (0.0) C3 C4 CS CIO 52.9 (0.0) CIS C4 CS C6 56.5(0.0) Clt) CS C6 C? -34.6 (0.0) C4 C5 CIO C2C 65.5 (0.0) C6 CS CIO C20 -68.8 [0.0) C6 C7 C9 CIO 22.0 (0.0) CU C8 CS C7 SS.4 [0.0) C9 C8 CI4 CIS -53.0 [0.0) C9 C8 CU C12 133.1 [0.0) CU C8 CI7 021 117.8 [0.0) c; CS CIO C20 76.91 r0.0) C8 C9 CIO C20 -45.6 [0.0) 026 c n C12 025 -166.21 [o.o) 02? c n Cl? 025 18.11 [0.0) c n C12 C13 CU 65.SI [0.0) Cl 7 Cl? CI3 CI6 -155.41 [CO) Cll C12 C17 C8 -89.9( 0.0) C13 C12 CI7 021 -86.71 [0.0) C12 C13 CH C8 46.71 k0.C) C16 Ct3 CU CIS 4t.9< [0.0) CU C13 C16 028 -30.01 ;o.o) C8 C14 CIS 028 -150.31 ;o.c) CH CIS 026 c n -52.8< 0.0) 026 CIS 028 C16 -95.61 '0.0) C13 CIS 029 C30 127,1< 0.0) Cl? C17 021 C22 -109.8< 0.0) C16 023 C30 C31 172.3< O.C) C2 Cl CIO 52.8 (0.0) Cl C2 C3 c< S6.1 (o.o; C2 C3 C4 CIS 76.5 (0.0) C18 C4 C5 C6 -64.5 f n n\ y. vy CIS C4 C5 fs * ft -70.S CO) C4 CS CIO Cl -56.5 [0.0) C6 C5 CIO Cl 16S.3 [0.0) CS C6 C7 CS 7.6 [0.0) c u C8 C9 C7 17S.S '0.0) C17 C8 CS C10 175.1 '0.0) CI7 C8 CU CI3 -23.9 '0.0) CS C8 CU 021 -110.5 [CO) C7 C9 CIO Cl -158.8 [0.0) C8 C9 CIO C1 78.J| [0.0) 026 Cll CJ2 CI3 -42.4, '0.0) 027 C11 C12 Cl 3 143.0, CO) Cl? Cll 026 C15 32.S( '0.0) c n C12 C13 C16 -44.2( ft ft \ St. v> 025 C12 C13 CU -163.8, 0.0) C11 C12 CU 021 15S.8( ft r, \ v. v; 025 C12 CJ7 C8 148.0< 0.0) C12 C13 CU C15 -76.5( v .u) CJ2 CI3 CI6 028 17.4( n nl v. v) CU C13 C16 029 82.4| 0.0) C13 CU CI5 026 75.1( CC) 028 C15 026 C11 62.S( fl ft\ CI3 CI6 028 CIS 3.3i 0.0) 028 C16 023 C30 -123.9< 0.0) C17 021 C22 C23 -173.2< 0.0) C16 023 C30 032 -7.5< 0.0) 91-f*ft w Cl r* i n CS 163.7(0.0) C2 C3 C4 C5 -50.1(0.0) C3 C4 C5 C5 -US.7(0.0} /*1 ft »y 1 O C4 CS C1C 168.1(0.0} C4 C5 C6 C7 -163.3(0.0) C4 C5 C1C CS -178.2(0.0) C6 C5 CIO CS 47.6(0.0) C6 C7 CS C8 150.2(0.0) cu C8 CS CIO -60.4(0.0) CS C8 CU C13 -1SS.0(0.0) CI7 C8 CU C15 76.1(0.0) cu C8 CU C12 1.3(0.0) C7 CS CIO C5 -42.5(0.0) C8 CS n viu C5 -155.0(0.0) 026 Cll C12 CU 68.2(0.0) 027 CU C12 CU -106.4(0.0) 027 Cll 026 C55 -152.2(0.0) Cl? C12 C13 CH -45.3(0.0) 025 C12 C53 C16 80.1(0.0) C13 CU CU C8 2?.6(0.0) C12 M l v-l 1 021 33.7(0.0) C16 C13 CH C8 185.0(0.0) CI2 C!3 CIS 028 -170.1(0.0) C8 CH CIS 026 -34.8(0.0) CI3 CU C15 028 -40.3(0.0) CH C15 028 C16 24.3(0.0) 023 C15 028 CIS -109.9(0.0) C8 CU 021 C22 136.5(0.0) r-»t Kf 1 t 021 C22 024 6.2(0.0) 

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