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Novel secondary metabolites from selected cold water marine invertebrates Williams, David Ellis 1987

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NOVEL SECONDARY METABOLITES FROM SELECTED COLD WATER MARINE INVERTEBRATES By DAVID ELLIS WILLIAMS B.Sc.Hons, King's College, University of London, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Chemistry) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1987 ® David E l l i s Williams, 1987 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. The University of British Columbia 1956 Main Mall Vancouver, Canada Department DE-6(3/81) ABSTRACT. A study of the secondary metabolism of two nudibranchs and one soft coral has led to the i s o l a t i o n of eighteen new and two known secondary metabolites. The structures of a l l compounds were determined by a combination of the i n t e r p r e t a t i o n of s p e c t r a l data, chemical degradations and interconversions, and s i n g l e c r y s t a l x-ray d i f f r a c t i o n a n a l y s i s . The B r i t i s h Columbian dorid nudibranch Diaulula sandiegensis yielded two new s t e r o i d a l metabolites, d i a u l u s t e r o l s A (41) and B (42). The 25-(3-hydroxybutanoate) residue of d i a u l u s t e r o l A (41) and the 2a,3a-diol array of both 41 and 42 are not commonly encountered i n n a t u r a l l y occurring s t e r o i d s . Both metabolites exhibited considerable a n t i b a c t e r i a l and antifungal a c t i v i t y . Steroid 41 exhibited f i s h antifeedant a c t i v i t y . The r e l a t i v e concentration of 4.1 and 42 i n the skin extracts of D_. sandiegensis appears to be related to the animals' seasonal abundance. Extracts of the B r i t i s h Columbian so f t coral Gersemia  rubiformis yielded a s e r i e s of ten diterpenes possessing cembrane f170-175), pseudopterane (167-169) and gersolane (176) carbon skeletons. The structure of an eleventh diterpene remains unresolved. In addition, the structure of a degraded diterpene possessing a 13-membered ring (122) i s t e n t a t i v e l y proposed. G . rubiformis represents the f i r s t example of a s o f t c o r a l to - i i -y i e l d pseudopterane diterpenes. The organism i s the f i r s t to contain cembrane, pseudopterane and gersolane metabolites, a f a c t which has biogenetic implications. Two new sesquiterpenes were also i s o l a t e d . Tochuinyl acetate (165) and dihydrotochuinyl acetate (166) represent the f i r s t examples of cuparane sesquiterpenes to be i s o l a t e d from a s o f t c o r a l . A biogenesis i s proposed. Metabolite 166 exhibited f i s h antifeedant a c t i v i t y . Investigations of Gersemia rubiformis c o l l e c t e d i n Newfoundland waters revealed that the secondary metabolism d i f f e r e d from west coast specimens. The i s o l a t i o n of the new unstable sesquiterpene (+)-/?-cubebene-3-acetate (178) resulted. Skin extracts of the dendronotoid nudibranch Toquina  tetraquetra were examined in an attempt to c o r r e l a t e i t s feeding dependency and lack of predation to the presence of allomones. Metabolites 165. 166. 170. 179 and the new butanoate diterpene 180 could be traced to the coelenterates which make up the animal's d i e t . Tochuinyl acetate (165). dihydrotochuinyl acetate (166) and r u b i f o l i d e (170) were previously found i n extracts of Gersemia rubiformis. Ptilosarcenone (179) has been reported as one of the major metabolites of the sea pen Ptilosarcus 213 gurneyi . The exact o r i g i n of a s i x t h metabolite, pukalide ( £ 1 ) , remains unknown. I t i s proposed that Tochuina tetraquetra s e l e c t i v e l y sequesters d i e t a r y metabolites for defensive purposes. - i i i -Table OJL Contents. Page # Abstract i i L i s t of Figures v i L i s t of Schemes ix L i s t of Tables x Acknowledgements x i i Abbreviations x i i i I. Introduction 1 A. Overview 1 B. Natural Product Chemistry 1 C. Marine Natural Products 4 I I . Nudibranchs 10 A. introduction: Nudibranch Zoology 10 B. Nudibranch Defenses: Evolutionary and Ecolo g i c a l Influences 13 C. The Chemistry of Nudibranchs 19 I I I . Secondary Metabolites from the Dorid Nudibranch, Diaulula sandiegensis (Cooper, 1862) 23 A. Introduction 23 B. I s o l a t i o n and Structural E l u c i d a t i o n 25 C. B i o l o g i c a l A c t i v i t i e s of D i a u l u s t e r o l A (11) and D i a u l u s t e r o l B (12) 46 D. Further Observations and Discussion 47 - i v -IV. Soft Corals 52 A. Introduction: Alcyonacean Zoology 52 B. Symbiotic Associations Between Alcyonarians and Algae (Zooxanthellae): Implications to the O r i g i n of Isolated Metabolites 55 C. Why The Interest: The B i o l o g i c a l and Eco l o g i c a l Significance of Some Coelenterate Metabolites 58 D. Chemistry of Soft Corals and Alcyonarians In General . . . . . 61 E. Review of the Chemistry of the Family Nephtheidae 70 V. Secondary Metabolites from the Soft Coral, Gersemia rubiformis (Ehrenberg, 1834), and the Dendronotoid Nudibranch, Toouina tetraquetra ( P a l l a s , 1788) 78 A. Introduction 78 B. I s o l a t i o n of the Metabolites 83 C. S t r u c t u r a l E l u c i d a t i o n and Related Studies . . 87 (i) Sesquiterpenes 165 and 166 87 ( i i ) Pseudopterane diterpenoids, 167-169 . . . 105 ( i i i ) Cembrane diterpenoids, 170-175 125 (iv) New carbon s k e l e t a l metabolites, 176-177 . 175 (v) Sesquiterpene 123. 192 (vi) Pukalide (£3J 206 ( v i i ) B r i a r e i n diterpenoids, 179 and 180 . . . . 206 D. B i o l o g i c a l A c t i v i t i e s 210 E. Biosynthetic Postulates 212 F. Further Observations and Discussion 216 VI. Experimental 224 VII. Bibliography 258 - v -LAPt Of Figures. Figure Page # 1 Phylogenetic c l a s s i f i c a t i o n of two nudibranchs . 11 2 Typical cryptobranch dor id nudibranch . . . . . . 12 3 1 3 C NMR spectrum of d i a u l u s t e r o l A (H) 26 4 NMR spectrum of d i a u l u s t e r o l A (11) 27 13 5 C NMR spectrum of d i a u l u s t e r o l A t r i a c e t a t e (13J 29 6 *H NMR spectrum of d i a u l u s t e r o l A t r i a c e t a t e (12) 31 7 Contour p l o t of 2D-HETC0R NMR spectrum of d i a u l u s t e r o l A t r i a c e t a t e (12.) 33 8 Comparison of the coupling constants of 41 and 11 with models 15. and 49-52 38 9 *H NMR spectrum of d i a u l u s t e r o l B (12) 44 10 "^H NMR spectrum of d i a u l u s t e r o l B diacetate (52.) 46 11 Structure of a t y p i c a l alcyonarian 53 12 Phylogenetic c l a s s i f i c a t i o n of the s o f t c o r a l , Gersemia rubiformis 54 13 *H NMR spectrum of tochuinyl acetate (165) . . . 88 14 1 3 C NMR spectrum of tochuinyl acetate (165) . . . 89 15 Contour p l o t of 2D-HETCOR NMR spectrum of tochuinyl acetate (lfL5J 90 16 Gated decoupled JC NMR spectrum of tochuinyl acetate (1£5J 94 17 SINEPT NMR spectra of tochuinyl acetate (165) . . 96 - v i -18 H NMR spectrum of dihydrotochuinyl acetate (1£6J 101 19 C NMR spectrum of dihydrotochuinyl acetate ( 1 £ £ ) 102 20 1 3 C NMR spectrum of gersemolide (167) 107 21 DEPT NMR spectra of gersemolide (167) 108 22 *H NMR spectrum of gersemolide (167) 109 23 Computer generated perspective drawings of gersemolide (167) 113 24 1H NMR spectra of A 4 , 5 Z , A 7 , 1 7 - i s o g e r s e m o l i d e 119 25 1H NMR spectrum of A 4 ' 5 E , A 7 , 1 7 - i s o g e r s e m o l i d e (lfi&) 121 26 1 3 C NMR spectrum of A 4 , 5 E , A 7 , 1 7 - i s o g e r s e m o l i d e (169) 122 27 ^ NMR spectrum of r u b i f o l i d e (170) . . . . . . . 126 28 1 3 C NMR spectrum of r u b i f o l i d e (170) 127 29 400MHz -^H NMR spectrum of r u b i f o l i d e (170) . . . 136 30 400MHz lanthanide s h i f t e d 1H NMR spectra of r u b i f o l i d e (12H) 137 31 *H NMR spectrum of epilophodione (171) 146 32 1 3 C NMR spectrum of epilophodione (171) 147 33 ADEPT NMR spectra of epilophodione (171) . . . . 148 34 1H NMR spectrum of isoepilophodione A (122) . • . 151 35 1 3 C NMR spectrum of isoepilophodione A (172) . . 152 36 *H NMR spectrum of isoepilophodione B (173) . . . 155 37 Low temperature 1H NMR spectra of isoepilophodione B (122.) 157 38 NMR spectrum of isoepilophodione C (174) . . . 160 - v i i -39 C NMR spectrum of isoepilophodione C (174) . . 161 40 1H NMR spectrum of r u b i f o l (175) 165 41 1 3 C NMR spectrum of r u b i f o l (125.) 166 42 APT NMR spectrum of r u b i f o l (1751 . . . . . . . . 167 43 1H NMR spectrum of r u b i f o l (175) i n C gDg . . . . 171 44 "*"H NMR spectrum of unknown diterpene 172 45 Proposed structures for unknown diterpene . . . . 174 46 XH NMR spectrum of gersolide (176) 176 47 Computer generated perspective drawing of gersolide (176) 178 48 1H NMR spectrum of rubiformate (177) 182 49 1 3 C NMR spectrum of rubiformate (177) 183 50 1H NMR spectrum of rubiformate (177) i n C gD 6 . . 191 51 C NMR spectrum of ( + ) -y?-cubebene-3-acetate (1781 194 52 *H NMR spectrum of (+)-y?-cubebene-3-acetate (178) 196 53 1H NMR spectrum of the mixture of dihydro epimers 192 and 193 198 54 1H NMR spectrum of the mixture of dihydro epimers 192 and 193 i n C gDg 201 55 1H NMR spectrum of the butanoate 1M 207 56 Biogenetic speculation for (+)-/?-cubebene-3-acetate (12&) 216 - v i i i -ULs_t QL Schemes. Scheme Page # 1 Biogenetic proposal for tochuinyl acetate (165) and dyhydrotochuinyl acetate (166) . . . . 213 2 Biogenetic proposals for the cembrane, pseudopterane, gersolane and degraded 13-membered ring diterpenoids, 167-177 214 3 Biogenetic proposal for (+) -fj-cubebene-3-acetate (12SJ 217 - ix -L i s t ££ Tables. Table Page # 1 1 3 C NMR data comparison of 41 and 4 4 32 2 1H NMR data comparison of 41 and 45. 32 3 *H NMR data comparison of the s t e r o i d a l methyl resonances of A i r 45_, A3 and 42 35 13 4 C NMR data comparison of the s t e r o i d a l methyl resonances of 41f 4 £ and A3 36 5 P a r t i a l *H NMR assignments for d i a u l u s t e r o l A (11) , d i a u l u s t e r o l B (42), t r i a c e t a t e 41 and diacetate 5_3_ 40 6 P a r t i a l C NMR assignments for d i a u l u s t e r o l A (41) and t r i a c e t a t e 42 42 7 Results of i n v i t r o a n t i b i o t i c assays for the s t e r o i d a l metabolites, d i a u l u s t e r o l A (41) and d i a u l u s t e r o l B (42) 4 7 8 *H NMR data comparison of 165 and 181 91 9 1 3 C NMR data comparison of 165 and 1 £ 1 92 10 1H NMR assignments for tochuinyl acetate (165) and dihydrotochuinyl acetate (166) 99 11 1 3 C NMR assignments for tochuinyl acetate (1£5_) and dihydrotochuinyl acetate 166 100 12 1H NMR data comparison of 166. 183 and 184 . . . 104 13 1 3 C NMR data comparison of 166. 132. and 184 . . . 105 14 1 3 C NMR assignments for gersemolide (167) and 169. and s p e c t r a l comparison with 133. and 133 . • H I 15 1H NMR assignments and s p e c t r a l comparison for gersemolide ( 1 £ 2 ) , 133 and 1£9_ 116 - x -16 "^C NMR data comparison of 170 and 186 128 17 1 3 C NMR assignments for r u b i f o l i d e (HQ.) , epilophodione (171), isoepilophodiones A (172) and C (174) and r u b i f o l (175), and spe c t r a l comparison with JJL8. and 189 129 18 *H NMR assignments for r u b i f o l i d e (170), epilophodione f171), isoepilophodiones A (122), B (122.) and C (121) and r u b i f o l (125.), and spec t r a l comparison with 188 and 189 131 19 Coupling constant data for r u b i f o l i d e (170) . . . 141 20 Correlations observed i n a long range 2D-HETCOR NMR spectrum of r u b i f o l i d e (170) . . . 144 21 Subsequent NOE res u l t s observed for epilophodione (171) 149 22 NOE r e s u l t s observed for r u b i f o l (125.) 168 23 Spin-spin decoupling r e s u l t s for the eight-spin system found on C l l to C2 i n r u b i f o l (175) . . . 170 24 NOE re s u l t s observed for gersolide (176) . . . . 179 25 *H NMR assignments for gersolide (176) and rubiformate (122) 180 1 3 26 C NMR assignments for rubiformate (177) and spec t r a l comparison with epilophodione (171) . . 185 27 1 3 C NMR data comparison of 177 and 1 M 186 1 3 28 C NMR assignments for (+)-/?-cubebene-3-acetate (1781 195 29 NOE r e s u l t s observed for the dihydro epimers 1 £ 2 and 122 200 30 C NMR data comparison of the methyl resonances of 1 2 £ , U l r 123. and 1 £ £ 203 31 ''"H NMR assignments for ( + ) -/?-cubebene-3-acetate (1781 and the dihydro epimer 1E2 205 32 1H NMR assignments for butanoate 180 and spe c t r a l comparison with 179 208 - x i -Results of in v i t r o c y t o t o x i c i t y assays for r u b i f o l i d e f 170) and epilophodione (HI) Acknowledgements. I wish to express my appreciation to Professor Raymond J . Andersen for his patience, ceaseless encouragement and guidance throughout the course of t h i s work, and for his assistance during the preparation of the t h e s i s . It has been a pleasure and a learning experience to work with him. Assistance given by Mike LeBlanc for performing bioassays and c o l l e c t i n g specimens i s g r e a t f u l l y acknowleged. I thank the s t a f f of the Bamfield Marine Station and Drs. G. K. Eigendorf and S. 0. Chan and t h e i r s t a f f for e f f i c i e n t assistance throughout. I must express my deepest appreciation and apologies to the nudibranchs, Diaulula sandieaensis and Tpqvina tetraquetrfl, and the s o f t c o r a l , Gersemia rubiformis. I thank Mark T i s c h l e r for his assistance in drawing structures. F i n a l l y and not l e a s t , thanks to Peter Northcote and Peter C l i f f o r d for c o n t i n u a l l y bearing the cold waters of the P a c i f i c , and Judy Needham for u n f a i l i n g support. - x i i i -Abbreviations. A ADEPT APT c CaSO, C 6 D 6 CDC1. CH 2C1 2 DEPT e EIMS EtOAc EU(F0D) 3 FAB g GC GCMS 2D-HETCOR HPLC HRMS IR LRMS Angstrom units Automatic DEPT Attached proton test concentration, gram per lOOmL solvent Calcium sulphate Benzene-dg Chloroform-d^ Methylene chloride D i s t o r t i o n l e s s enhancement by p o l a r i s a t i o n transfer e l e c t r o n i c g l i t c h Electron impact mass spectrum or spectroscopy Ethyl acetate Europium-1,1,1,2,2,3,3-heptafluoro-7,7-dimethyl-4,6-octanedione Fast atom bombardment grease resonance Gas chromatography . Gas chromatographic mass spectrum Two dimensional heteronuclear c o r r e l a t i o n High performance l i q u i d chromatography High resol u t i o n mass spectrum Infra red Low resolu t i o n mass spectrum - xiv -M MeOH mins mp MS m/z 1H NMR 13 "LJC NMR NOE ODS r e l . , R f s SCUBA SINEPT TLC U UV w Wh/2 wt Parent Ion Methanol minutes melting point Mass spectrum or spectroscopy mass to charge r a t i o Proton nuclear magnetic resonance Carbon-13 nuclear magnetic resonance Nuclear Overhauser enhancement Octadecylsilane r e l a t i v e Rates of flow solvent resonance Self contained underwater breathing apparatus Selective i n s e n s i t i v e n u c l e i enhancement by p o l a r i s a t i o n transfer Thin layer chromatography unknown impurity resonance U l t r a v i o l e t water resonance width (Hz) of NMR resonance sign a l at half height weight - xv -Abbreviations for m u l t i p l i c i t i e s of NMR s i g n a l s : s = s i n g l e t d = doublet t = t r i p l e t ("apparent" t r i p l e t s are also referred as a t r i p l e t ) q = quartet dd = doublet of doublets, e tc.. b = broad s i g n a l m = multiplet Structures i n t h i s thesis are drawn, where appropriate, as depicted i n reference source. When r e l a t i v e stereochemistry ( a (down) and ft (up)) i s assigned to geminal protons the assignment i s made with respect to centres with a "known" r e l a t i v e stereochemistry i n the corresponding 2D s t r u c t u r a l drawing. - xvi -X. INTRODUCTION a., overview. The purpose of the research undertaken and described i n t h i s thesis was to i s o l a t e and elucidate the structures of previously unknown natural products from selected cold water marine invertebrates. The majority of i n t e r e s t i n g secondary metabolites* e x h i b i t 1 2 b i o l o g i c a l a c t i v i t i e s which are of pharmacological , e c o l o g i c a l or p h y s i o l o g i c a l i n t e r e s t . With t h i s i n mind, organisms for study were chosen on the basis of in. v i t r o screening for antifungal and a n t i b a c t e r i a l a c t i v i t i e s ( i t has been found that such a c t i v i t i e s can generally be correlated to wider b i o l o g i c a l a c t i v i t i e s ) and e c o l o g i c a l indicators such as a lack of predation, or an unfouled surface. These determinants may prove to be of l i t t l e importance i n terms of the roles that the metabolites a c t u a l l y play i n nature. However, they have proven to be an e f f e c t i v e guide to the discovery of i n t e r e s t i n g secondary 4 metabolism . £. Natural Product Chemistry. The main d r i v i n g force behind current natural product research has been the desire to discover new metabolites of po t e n t i a l use i n the pharmaceutical or a g r i c u l t u r a l i n d u s t r i e s . * natural products and secondary metabolites are i n t h i s thesis regarded as synomynous. 1 Encouragment for these e f f o r t s stems from the fact that many drugs in use today, such as morphine, atropine, p e n i c i l l i n and streptomycin, are natural products or semisynthetic derivatives thereof. Natural products chemistry (the chemistry of secondary metabolites) has t r a d i t i o n a l l y focused on t e r r e s t r i a l plants and microorganisms. H i s t o r i c a l l y only the major metabolites were characterised. Structures were solved by chemical degradations and interconversions and were confirmed by synthesis, since the 1960's, technological advances have enabled researchers to elucidate the structures of more and more complex metabolites present i n ever smaller amounts. The recent i s o l a t i o n and 5 s t r u c t u r a l e l u c i d a t i o n of the metabolites palytoxin (1) and brevetoxin B (2.)^ would have been unimaginable 20 years ago. As the process of structure e l u c i d a t i o n becomes more routine, there i s an increased e f f o r t by natural products chemists to understand why organisms produce such compounds. The h i s t o r i c a l d e f i n i t i o n of secondary metabolites as compounds that are not " e s s e n t i a l to the basic protoplasmic metabolism of the organism", or as "end products (waste or storage) of metabolism", i s becoming obsolete. I t i s now apparent that many secondary metabolites are involved i n the physiology of the producing organism. For example, b o n e l l i n (3J , is o l a t e d from the marine Echurian worm, Bo n e l l i a v i r i d i s . acts as a sex determinant and produces marked sexual dimorphism 3 3. Many other "secondary metabolites" are used to mediate e c o l o g i c a l interactions by acting as chemical communicants. Some allomones (interspecies chemical communicants) are used to ward o f f po t e n t i a l predators 2 3 C 0 2 H C 0 2 H 3 and pheromones (intraspecies chemical communicants) can be u t i l i s e d to warn members of the same species of a p o t e n t i a l OH "7 danger nearby ' . c. Marine Natural Products. Since i t s b i r t h i n the l a t e 1960*s, marine natural products research has undergone an explosive growth. The technological advances i n s t r u c t u r a l analysis and the advent of "cheap" safe SCUBA have greatly f a c i l i t a t e d the growth. New compounds are now discovered and reported at a rate that i s fa s t enough to r a p i d l y outdate any compilation of struc t u r e s . Research i n the f i e l d has spread geographically, and the active investigators have begun to explore i n earnest some f a s c i n a t i n g phenomena at the int e r f a c e between biology and chemistry. Most metabolites i s o l a t e d from marine organisms are exclusive to the marine environment, and many of the metabolites are r e s t r i c t e d to genera or even s i n g l e species. The p o t e n t i a l of the marine environment as a vast source of b i o a c t i v e compounds i s widely recognised. In the early 1970's, 1665 diverse marine species were screened for antitumor a c t i v i t y ^ and i t was found that nine percent contained compounds with a c t i v i t y compared with two or three percent of t e r r e s t r i a l organisms. Since there are more than a m i l l i o n species of marine invertebrates and more than 25000 known species of f i s h , such r e s u l t s are p a r t i c u l a r l y e x c i t i n g . Marine natural product chemists seeking to i s o l a t e i n t e r e s t i n g compounds have followed three basic search s t r a t e g i e s . One i s to screen f o r b i o l o g i c a l a c t i v i t y the extracts of large numbers of organisms c o l l e c t e d more or less randomly. A second strategy attempts to narrow the search by following promising l i n e s of evidence. For example, tumors are known to occur i n f i n f i s h and molluscs, but they are conspicuously absent from other marine animals such as sharks, sponges and tunicates. Extracts from many of the l a t t e r organisms have been examined^". The t h i r d search strategy c a p i t a l i z e s on clues about the presence of bi o a c t i v e metabolites by studying the ecology and behaviour of marine organisms. S e l e c t i o n pressures, i n densely populated habitats such as c o r a l r e e f s , have favoured the evolution of chemical defenses. Many organisms l i v i n g i n these habitats biosynthesise t o x i c metabolites or concentrate them from food sources i n order to deter predation, to keep competitors from approaching too c l o s e l y , and to keep themselves unfouled. To date, l i t e r a l l y thousands of compounds i s o l a t e d from marine invertebrates have shown b i o a c t i v i t i e s which give them p o t e n t i a l for pragmatic a p p l i c a t i o n , p a r t i c u l a r l y i n the areas of 5 human and veterinary medicine. The bryostatins i s o l a t e d by P e t i t g et a l . from the s e s s i l e plant l i k e bryozoan Bugula n e r i t i n a are strongly cytotoxic and anti-leukemic. An example of t h i s i n t e r e s t i n g family of metabolites i s provided by bryostatin 1 (A) • Extracts of the s o f t bodied s e s s i l e organisms Euplexaura f l a r a . a gorgonian, and a Haliclona species, a sponge, yielded g the a n t i - m f lammatory butenolides, 5_-fL , and the anti-leukemic a l k a l o i d manzamine A (j9_)*°, respectively. A unique metabolite imbricatine (1H) i s o l a t e d from the s t a r f i s h Dermasterias  i m b r i c a t a ^ i s capable of e l i c i t i n g the "swimming" response of the northeastern P a c i f i c anemone, Stomphia coccinea. Imbricatine (10) displays s i g n i f i c a n t a c t i v i t y i n the L1210 (ICgQ<l^g/mL) and P388 (T/C 139 at 0.5mg/Kg) antineoplastic assays. Unfortunately, many marine natural products that are a c t i v e in v i t r o are i n a c t i v e i n v i v o . In addition, many are so t o x i c that they k i l l the host organism. However, the marine environment i s gradually y i e l d i n g a number of compounds (or synthetic analogues), that have found, or show a p o t e n t i a l f o r , p r a c t i c a l a p p l i c a t i o n . Nereistoxin f11), which i s responsible for the i n s e c t i c i d a l a c t i v i t y of the marine polychaete worm Lumbricpnerejs heteropoda 1 2. led to the development of a 13 synthetic analogue padan (12) . which i s now i n common use i n Japan. The semisynthetic manipulation of spongouridine (Ara-U) 14 (12) i s o l a t e d from the sponge Cryptotethia crypta , and more 15 recently from the gorgonian E u n i c e l l a c a v o l i n i ^ , has l e d to 1-p-D-arabinofuranosylcytosine (Ara-C) ( 14 ) 1 6 an e f f e c t i v e antitumor drug that has been used f o r over a decade i n the treatment of leukemia. A number of other compounds are currently undergoing 17 18 c l i n i c a l t r i a l s An example i s provided by didemnin B (15.) i s o l a t e d from extracts of the tunicate Trididemnum l a 19 cyanophorum ' . Didemnin B (15.) i s a potent antitumor agent 20 (IC 5 00.00lMg/mL) and i n h i b i t o r of the Herpes simplex v i r u s , 7 CH3CHOHCO—•-N-CH-CO —^MeLeu—*Thr — ^ S t a — • H i p — ^ L e u — . „_ I ()-•—Me 2T y r-—Pro J 15 *" and i s undergoing c l i n i c a l t r i a l s as an anti-leukemic d r u g l b . F i n a l l y , a most s a t i s f y i n g aspect of marine natural products i s the high l e v e l of i n t e r e s t exhibited by s c i e n t i s t s outside the f i e l d . There i s a genuine desire to determine the functions of new compounds i n the marine environment. I n t e r d i s c i p l i n a r y research involving chemists, marine b i o l o g i s t s and marine ecol o g i s t s can only serve to strengthen a l l these f i e l d s . 21 An excellent series of books edited by Scheuer and a 22 se r i e s of review a r t i c l e s by Faulkner provide timely, and exhaustive reviews of important aspects of marine natural 23 products research. Other au t h o r i t a t i v e reviews by Baker , 8 Christophersen , Faulkner et a l . , Fenical , Moore , 28 29 Scheuer and Shields et a l . , provide a d d i t i o n a l h i g h l i g h t s . 9 XI. NUDTBRANCHS. A . introduction: Nudibranch Zoology-Molluscs of the c l a s s Gastropoda, subclass Opisthobranchia (Figure 1.) are slow moving animals whose synonyms include sea slug and naked s n a i l . Of the 3000 described species of opisthobranchs 3 0' 3^" most are s h e l l - l e s s , though some have i n t e r n a l or g r e a t l y reduced s h e l l s . Nudibranchs comprise the l a r g e s t of seven orders i n the subclass Opisthobranchia, and are characterised by a complete absence of a s h e l l . Adult nudibranchs range i n s i z e from 3mm to 300mm and are found throughout the world. Over 130 species have 32 been described on the west coast of North America . Colouration ranges from c r y p t i c to very s t r i k i n g , conspicuous colours. The outer covering of a nudibranch i s termed the mantle or dorsum (Figure 2.). A pa i r of rhinophores located near the nudibranch*s head are used as a chemosensory apparatus. The branchial plume i s a r e s p i r a t o r y structure ( g i l l ) u s u a l l y located near the anus (some species of nudibranchs possess cerata which are finger l i k e or club-shaped structures arranged i n uniform 3 0 groups or c l u s t e r s along each side of the dorsum , these function as r e s p i r a t o r y or di g e s t i v e organs) and the foot anchors the animal and i s involved i n locomotion. When nudibranchs are disturbed, most species r e t r a c t t h e i r external structures leaving only the dorsum exposed. A few respond by swimming. The swimming response seems to be a transient means of escape, us u a l l y l a s t i n g for several seconds, swimming i s evoked only when the animals are 10 MOLLUSCA CLASS MONOPLACOPHORA POLYPLACOPHORA SCAPHOPODA GASTROPODA APLACOPHORA BIVALVIA CEPHALOPODA SUBCLASS PROSOBRANCHIA OPISTHOBRANCHIA ORDER PYRAMIDELLIDA PLATYHEDYLOIDA NOTASPIDEA THECOSOMATA SACOGLOSSA GYMNOMORPHA PULMONATA NUDIBRANCHIA ACHOCHLIDIOIDA CEPHALASPIDA ANASPIDA GYMNOSOMATA ENTOCHONCHIDA SUBORDER STTPFRFAMTI.Y AEOLIDACEA DENDRONOTOIDA ARMINOIDEA DORIDOIDA EUDORIDACEA SPECTER TRITONIIDAE TOCHUINA TETRAQUETRA DISCODORIDIDAE DISCODORIS (DIAULULA) SANDIEGENSIS Figure 1. Phylogenetic c l a s s i f i c a t i o n of two nudibranchs^ N.B. Organisms c l a s s i f i e d according to Austin . 11 alarmed and i t i s not used as an active form of locomotion under normal circumstances. A l l nudibranchs are predators on other invertebrates. Dorids (suborder Doridacea) predominantly eat sponges, bryozoans (ectoprocts) and tunicates, while members of the other three suborders (Dendronotacea, Arminacea and Aeolidacea) p r i m a r i l y feed on coelenterates (sea anemones, s o f t c o r a l s , e t c . . ) 3 0 . Nudibranchs are hermaphroditic. One advantage of hermaphrodism i s the increased p r o b a b i l i t y of f i n d i n g a mate, since every i n d i v i d u a l of the same species i s e l i g i b l e . L i t t l e i s known about how nudibranchs locate and recognise each other. They may secrete sex pheromones (a possible ro l e for some of the 33 secondary metabolites i s o l a t e d from nudibranchs) and the 3 4 rhinophores may act as the pheromone receptors . Nudibranch egg masses vary i n s i z e , shape and colour from species to species. Dorid egg masses are ribbon shaped, while aeolids and dendronotids lay egg " s t r i n g s " . Egg masses are very d i s t i n c t and many can be i d e n t i f i e d to species. Defenses: Evolutionary M Ecological Influences. Even though nudibranchs have exposed soft t i s s u e , l i m i t e d mobility, and are often b r i g h t l y coloured, they have few known p r e d a t o r s 3 5 . Only a very few selected members of four groups of animals, opisthobranchs, crustaceans, asteroids and f i s h e s , prey on n u d i b r a n c h s 3 4 ' 3 6 . This r e l a t i v e impunity to predation has led to speculation that nudibranchs u t i l i z e a v a r i e t y of sophisticated defensive s t r a t e g i e s . The primary l e v e l of defense employs colour which c r y p t i c a l l y mimics the preferred dietary substrate of a species. Some nudibranchs are known to concentrate pigments from t h e i r •57 d i e t as a means of achieving c r y p t i c colouration . The nudibranch AJLsLLSjg. sanouineae cooperi l i v e s e x c l u s i v e l y on the orange sponge Anthoarcuata graceae and apparently incorporates 38 3 5 carotenoid pigments from the sponge . However, Thompson demonstrated that c r y p t i c nudibranchs may s t i l l not be acceptable to f i s h when placed i n a s e t t i n g where colour offered no concealment. I t also appears that some well defended nudibranchs 37 b l a t e n t l y advertise themselves with conspicuous colouration . Thus, some nudibranchs apparently r e l y on a second l e v e l of defense. The second l e v e l of nudibranch defense may be some 3 6 combination of behavioral, morphological, or chemical e f f e c t s . The most basic behavioral defense mechanism i s the swimming response. On t h i s coast, accomplished swimmers are species of the genera T r i t o n i a . Melibe, Cumanotus. F l a b e l l i n o p s i s and 30 Dendronotus . Certain nudibranchs of the suborders 13 Dendronotacea, Aeolidacea and Arminacea possess large cerata which can be s a c r i f i c e d to predators at the s l i g h t e s t provocation, hence enabling captured nudibranchs to escape (the cerata are rap i d l y regenerated). Some dorid (suborder Doridacea) nudibranchs possess spicules acquired from t h e i r d i e t a r y sponges. The spicu l e s give the animal a r i g i d shape. A voracious nudibranch predator, the opisthobranch Navoanax inermis. does not eat spicule-containing dorids, while r e a d i l y consuming non-spiculose types . Spicules may provide some protection against predators, however, the r e l a t i v e importance of t h i s morphological defense i s believed to be rather l o w 3 4 . Certain a e o l i d nudibranchs s e l e c t i v e l y sequester and u t i l i z e i n t a c t nematocyst s t i n g i n g c e l l o r g a n e l l e s 3 5 ' 4 0 obtained from dietary coelenterates. The unfired nematocysts are stored i n a functional state i n s p e c i a l i s e d cynidocyst c e l l s located i n the ski n , t y p i c a l l y at the t i p s of the c e r a t a 3 4 . When an ae o l i d i s disturbed, clouds of st i n g i n g nematocysts are released causing some t r o p i c a l aeolids to be hazardous to man 4 0. It i s now widely accepted that opisthobranchs u t i l i z e some form of chemical defense at the secondary l e v e l , and i t i s the nature of these secretions that has led to an intense i n t e r e s t by marine natural product chemists i n recent years. Several dorids secrete sulphuric acid (pH=l) along t h e i r skin surface as a chemical d e t e r r e n t 4 * . Nudibranchs, p a r t i c u l a r l y dorids, possess abundant numbers of non-mucous secretory skin glands i n the 35 dorsum . The p o s i t i o n and function of the glands i s most 14 consistent with a defensive ro l e . Their placement explains the observation that most secondary metabolites from nudibranchs can be extracted by a r e l a t i v e l y short immersion of the i n t a c t animal i n a sui t a b l e solvent. These inferences are corroborated by studies that have found feeding i n h i b i t o r s i n the dorsum but not i n the gut, foot, or head parts of nudibranchs 4 2. I t has been found that a l l four nudibranch suborders secrete non-acidic 43 noxious substances , some of which taste b i t t e r , or b i t t e r s o u r 3 5 . Nudibranch secondary metabolites can have several o r i g i n s : (i) derived d i r e c t l y from a dietary source; ( i i ) derived i n d i r e c t l y by chemical modification of a metabolite obtained from a dietary source; ( i i ) or £e_ novo biosynthesis. Observations by 43c Johannes i n 1963 , suggested that the nudibranch P h y l l i d i a  varicosa secreted a to x i c mucus. This led to the i s o l a t i o n i n 1975 of a s t r u c t u r a l l y novel metabolite 9-isocyanopupukeanane (IfL) 4 4 which had f i s h antifeedant a c t i v i t y (one of the f i r s t examples of a marine metabolite with a known b i o l o g i c a l r o l e ) . The nudibranch obtained t h i s metabolite d i r e c t l y by concentrating 45 i t from a dietary Hymeniacidon species of sponge . In the nudibranch, Chromodoris marislae. a metabolite i s derived 46 i n d i r e c t l y by chemical modification . The major metabolite m a r i s l i n (12) i s related to the metabolite p l e r a p l y s i l l i n - 2 (12.), i s o l a t e d from the sponge P l e r a p l y s i l l a s p i n i f e r a 4 7 . by a simple rearrangement. Only a few species of nudibranchs appear to be capable of de  novo biosynthesis of defensive or other secondary metabolites. Dendrodoris limbata biosynthesises polygodial (12) 4 8, a 15 metabolite endowed with antifeedant properties. The incorporation 3 4 of [ C] mevalonic acid into the terpene portions of 2SL and 21 suggests that species of the genus Archidoris can also elaborate 49 terpenes . OH CHO 20 21 16 Since nudibranch metabolites are mainly of die t a r y o r i g i n 50 (the o r i g i n of a number remains unclear ) the skin chemistry of a species can d i f f e r g reatly from one c o l l e c t i n g l o c a t i o n to another. A number of clear examples are documented. Specimens of Cadlina luteomarginata from Vancouver, B r i t i s h Columbia contained 2a furodysin (22) i furodysinin (21) and microcionin-2 (24.) • Specimens collected i n the Queen Charlotte Islands, B r i t i s h 51 Columbia contained the furanoditerpene, mariginatafuran (25.) • Dihydropallescensin-2 (2fL) was found i n the skin extracts of specimens of £. luteomarginata c o l l e c t e d o f f of La J o l l a , 42c C a l i f o r n i a . I t was simultaneously shown that the majority of metabolites present i n the skin extracts of £.. luteomarginata were obtained from the sponges i n the nudibranch's d i e t , and that the observed v a r i a t i o n i n skin chemistry was a consequence of changing diet with changing c o l l e c t i o n s i t e . H H 22 23 24 25 26 17 The secondary metabolites i s o l a t e d from nudibranchs for which toxic or antifeedant properties have been established, f a l l A *5 A A very roughly into three groups: (i) isocyanosesquiterpenes ' ; 52 (i) furanoterpenes ; and ( i i i ) sesquiterpenes with a drimane 53 skeleton . The f i r s t two groups appear to be of dietary o r i g i n , while the drimane sesquiterpenes are biosynthesised d_e_ novo by the nudibranchs. It appears that compounds of these three groups may display antifeedant properties towards organisms other than f i s h 5 4 and that s i m i l a r defense strategies are operating among 55 opisthobranch molluscs and insects . The development of a "dietary-derived" chemical defense mechanism has probably had a profound influence on the evolutionary development of nudibranchs. It i s believed that the loss of the t y p i c a l molluscan s h e l l i s related to the gain of t h i s chemical defense. Circumstantial evidence supports the notion that the chemical defense was preadaptive to the loss of 37 the s h e l l , since a number of int e r e s t i n g secondary metabolites have been is o l a t e d from shelled Gastropod c o u s i n s 5 6 . Also the chemical defense may have played an important ro l e i n the adaptive radiation of nudibranchs through t h e i r dependency on 37 s p e c i a l i s t d i e t s . Further to t h i s , i t i s asserted that sponge-feeding nudibranchs tend to predominate i n t r o p i c a l faunas where t o x i c i t y among sponges as a general rule i s at i t s g r e a t e s t 3 7 1 , 5 7 ' 5 8 , while those that feed upon bryozoans and other 37 58 animals are more common i n temperate waters ' L i t t l e i s known about the r e l a t i v e importance of any s p e c i f i c nudibranch defense mechanism, but i t seems apparent that nudibranchs u t i l i z e a combination of st r a t e g i e s . It has not been 18 unequivocally proven, but circumstantial evidence and evolutionary hypothesis suggest that chemical defensive secretions (allomones) play an important r o l e . C. The Chemistry of. Nudibranchs. Many novel secondary metabolites have been is o l a t e d from gastropods, p a r t i c u l a r l y nudibranchs. The scope of t h i s work i s too extensive to be reviewed i n f u l l i n t h i s t h e s i s . Reviews of nudibranch chemistry up to 1982 were provided by Schulte 59 42c et a l . and Thompson et a l . . An update can be found i n 22 Faulkner's review papers . A d d i t i o n a l i n depth discussions are to be found i n the Ph.D., d i s s e r t a t i o n s of K. Gustafson 5 3 <^ and S. W. A y e r 6 0 . I t i s appropriate, however, that a b r i e f overview of the l i t e r a t u r e since Faulkner's l a s t review paper covering the published work up to and including J u l y 1985 be included i n t h i s t h e s i s . The following review covers the period from July 1985 up to the c i t a t i o n s reported i n CA Selects, issue 9, May 4 t n, 1987, and/or Chemical Abstracts, No. 20, May 1 8 t n , 1987. Although chemical studies of s h e l l e d molluscs are of increasing i n t e r e s t 6 1 , s h e l l - l e s s members of the subclass 32 Opisthobranchia, aplysiomorphs ( l a t e l y known as anaspids ) (sea 62 hares) and nudibranchs, continue to y i e l d i n t e r e s t i n g natural products. A p a r t i a l l y c l a s s i f i e d nudibranch belonging to the genus P h y l l i d i a from S r i Lanka, yielded the previously unreported 63 metabolite 3-isocyanotheonellin (22) • Extraction of the nudibranch, Chromadoris macfarlandi. has resulted i n the 19 i s o l a t i o n of the two aromatic norditerpenes macfarlandins A (28) and B ( 22 ) 6 4 * and the three diterpenes M , 21 and 2 2 6 5 . A l l of these compounds display a n t i m icrobial a c t i v i t i e s , and a l l appear to be derived from sponge metabolites i n the nudibranch's d i e t . S i m i l a r l y , extracts of £.. funera yielded the metabolites 22 and 34 f which appear to be oxidation products of compounds derived from the sponge Dysidea herbacea 6 6. Both metabolites cause food r e j e c t i o n i n the "spotted k e l p f i s h " Gibbonsia elegans. The nudibranch D_oxi£. verrucosu yielded the f i r s t n a t u r a l l y occurring analogue of methylthioadenosine 25., which possesses "diverse 67 regulatory a c t i v i t i e s " . 0 20 OAc Attention has also turned toward nudibranch eggmasses which are y i e l d i n g extraordinary macrolides. The b r i l l i a n t l y coloured nudibranch, Hexabranchus sanguineus, deposits i t s s t r i k i n g red eggmasses in underwater caves. Though exposed and vulnerable these eggs have only one predator, the aeolid nudibranch, Favorinus japonicus. This v i r t u a l immunity to predation led . 6 8 Scheuer et a l . to i n v e s t i g a t e the organic constituents of the eggs r e s u l t i n g i n the i s o l a t i o n of two extraordinary macrolides, ulapaulide A (3JL) and B (3_1) , which i n h i b i t L1210 leukemia c e l l p r o l i f e r a t i o n (IC 5 Q0.01-0.03 g/mL). Egg masses of an u n i d e n t i f i e d nudibranch c o l l e c t e d at Kabira Bay, located i n Japanese waters, yielded a s e r i e s of novel macrolides, for example kabiramide C (3JL) . This compound possesses antifungal a c t i v i t y and i n h i b i t s c e l l d i v i s i o n of f e r t i l i z e d s t a r f i s h eggs at low 21 XIX. SECONDARY METABOLITES FROM TJH£ DORID NUDIBRANCH, Diaulula sandieoensis (Cooper. 3 862). h. introduction. The i n t e r t i d a l and su b t i d a l regions of the P a c i f i c Coast of North America are inhabited by sizeable populations of dorid 32 nudibranchs . Many of the species have d i s t r i b u t i o n a l ranges that extend along the en t i r e c o a s t l i n e from Alaska to Mexico. PiauJ-ulfl sandjegepsis (recently Discodoris s a n d i e o e n s i s 3 2 ) . suborder Doridoida, family Discodorididae (Figure 1.), provides an example of a dorid which has a documented d i s t r i b u t i o n range that extends along the P a c i f i c Coast of North America from 32 southern Alaska to Mexico, and of f the coasts of Japan . Walker and Faulkner i s o l a t e d a se r i e s of nine chlorinated acetylenes related to 3_9_ from specimens of D_. sandieoensis c o l l e c t e d at Point Loma, C a l i f o r n i a 7 0 . Fuhrman et a l . 7 * i s o l a t e d isoguanosine (40) from the digestive glands of specimens c o l l e c t e d at Monterey Bay and the Channel Islands o f f Santa Barbara, C a l i f o r n i a . Consideration of the reported findings, the r e l a t i v e abundance of the nudibranchs and t h e i r apparent lack of predation led us to examine specimens of Diaulula sandieqensis c o l l e c t e d in Barkley Sound, B r i t i s h Columbia. The in v e s t i g a t i o n of the secondary metabolites from the skin extracts of D_. sandiegnesis was f i r s t i n i t i a t e d by S. W. Ayer in September 1980, and the studies reported herein were begun i n earnest i n A p r i l 1985. Diaulula sandieoensis was probably f i r s t i d e n t i f i e d by 3 2 Cooper i n 1862 . The animal t y p i c a l l y inhabits exposed rocky 23 channels at a depth of 1-5 meters, but can be found i n t e r t i d a l l y and to a depth of 30 meters. Body length can reach 8cm, but most f a l l into the range of 4-5cm. The dorsum has a velvety appearance produced by minute tubercles. The ground colour of t h i s dorid i s whitish-yellow to very pale brown broken by various numbers ( t y p i c a l l y 2-8) of brown to black markings shaped either as blotches or as d i s t i n c t i v e doughnut-shaped rings. Specimens having no spots have been reported. The rhinophores possess 20-30 lamellae and the s i x branchial plumes are white t r i p i n n a t e and are r e t r a c t a b l e . The animal supposedly feeds on i n t e r t i d a l and subtidal sponges of the genera Halichondria and H a l i c l o n a 3 0 ' 7 2 . Diaulula sandiegensis i s well camouflaged i n i t s natural environment, but with a l i t t l e e f f o r t and a sense of "tuning i n " on the objective they were r e a d i l y c o l l e c t e d by hand using SCUBA. D_. sandiegensis were most abundant on exposed rocky reefs, p a r t i c u l a r l y i n surge channels. The animal was c o l l e c t e d throughout the year at depths of 1 to 15 meters. 24 B_. i s o l a t i o n and Structural E l u c i d a t i o n . Freshly c o l l e c t e d animals (152 specimens) were immediately immersed i n methanol and allowed to extract at 2°C for two days. The extraction solvent was decanted from the nudibranchs, evaporated i n vacuo f and the residue was p a r t i t i o n e d between brine and "ethyl acetate. Fractionation of the ethyl acetate soluble material by f l a s h , Sephadex LH-20 and reverse-phase HPLC chromatographies produced pure samples of d i a u l u s t e r o l A (41) and d i a u l u s t e r o l B (42). 43 R' = Ac.R2=COCH2CH(OAc)Me 53 R^Ac. r2 = h D i a u l u s t e r o l A (41) was i s o l a t e d as a UV absorbing clear o i l that showed IR bands at 3380, 1722, 1664 and 1626cm"1, appropriate for hydroxyl, saturated carbonyl, unsaturated carbonyl and alkene f u n c t i o n a l i t i e s . The EIMS of 41 provided l i t t l e information since no parent ion, or recognisable fragment ions were observed. D i a u l u s t e r o l A (41) showed resonances for 13 t h i r t y one carbon atoms in i t s C NMR spectrum (Figure 3.) and i t s 1H NMR spectrum (Figure 4.) possessed a s e r i e s of f i v e methyl resonances at 0.65(s, 3H) , 0.96(d, J=6.8Hz, 3H), 1.17(s, 3H), 25 www I I I I I I I I I I I I I I I I I I I I M I I I I I I J I M I I I 60 40 20 PPM 0 27 1.21(d, J=7.0Hz, 1H) and 1.46(s f 6H)ppm. Other notable features i n the *H NMR spectrum of 41 were three very broad (W^y2=15-27Hz) one proton " s i n g l e t s " at 2.66, 2.87, 3.25ppm, three pos s i b l e carbinol methine proton resonances at 3.88(dt, J=10.7,4.9Hz, 1H), 4.15(m, 1H), 4.28(t, 4.9Hz, lH)ppm, and two o l e f i n i c proton resonances at 5.87(t, J=0.9Hz, 1H) and 6.54(d, J=4.9Hz, lH)ppm. Diau l u s t e r o l A (41) s t e a d i l y decomposed to give a complex mixture of very polar materials when i t was exposed to NMR solvents or other manipulations. Metabolite 41 possessed an IR 3 3 * band appropriate for hydroxyl and the C and ADEPT (optimised 73 for p o l a r i s a t i o n transfer through 140Hz coupling) NMR experiments indicated the presence of three secondary alcohol carbons resonating at 64.4(CH), 65.2(CH) and 66.7(CH)ppm. Confirmation f o r t h i s was provided by the three carbinol methine proton resonances and the three very broad one proton " s i n g l e t s " . Hence, 41 was expected to acetylate r e a d i l y . A cetylation of 41 with a c e t i c anhydride and pyridine at room temperature generated the t r i a c e t a t e 42. which proved to be a very stable substance. Triacetate 42. was shown by EIMS to have a molecular formula of C37H 5 40g (observed m/z 642.3779, requires 642.3768), which represents the addition of s i x carbon atoms to the t h i r t y 13 one that were observed i n the C NMR spectrum of 41. The t h i r t y seven resonances observed i n the C NMR spectrum of 42. (Figure 5.) and the three new methyl s i n g l e t resonances at 2.03, 2.06 and 2.10ppm i n addition to the observed downfield s h i f t s of the An experiment which allows for the f u l l proton count to be assigned to each carbon atom, for an example see Figure 33., page 148. For a d e t a i l e d discussion see reference 73. 28 carbinol methine resonances to 5.11(dt, J=13.8f4.8Hz, 1H) , 5.24(m, 1H) and 5.60(t, J=5.4Hz, lH)ppm and the loss of the three very broad " s i n g l e t s " i n the *H NMR spectrum of 42 (Figure 6.) confirmed the formation of a t r i a c e t a t e . Subtraction of the atoms present i n the three a c e t y l residues from the molecular formula of 42 resulted i n a molecular formula C3i H48°s ^ o r d i a u l u s t e r o l A (41)t which corresponds to a very low i n t e n s i t y M + peak ( r e l . , i n t e n s i t y <0.2) at a m/z of 516 daltons i n the FAB MS of 41. Three of the oxygen atoms i n t h i s formula could be accounted for by the three secondary alcohols which underwent a c e t y l a t i o n . The IR bands at 1722 and 1664cm - 1 13 and the C NMR resonances at 172.4(C) and 188.7(C)ppm indicated that the remaining oxygen atoms were possibly present as ester and cross-conjugated ketone f u n t i o n a l i t i e s . Compare for example, the p a r t i a l 1 3 C NMR resonances of the cross conjugated keto "7 A 1 s t e r o i d 44 and the H NMR resonances of the ecdysone-like 75 metabolite, acetylpinnasterol 45. » with those of 41 (Tables 1. and 2.). The molecular formula C 3 1H 4 gOg requires 8 units of unsaturation. Two of these could be accounted for by the two carbonyls. The *H NMR spectrum of d i a u l u s t e r o l A 41 possessed two o l e f i n i c proton resonances at 5.87 and 6.54ppm. The short range 2D-HETC0R NMR spectrum 7 6 of t r i a c e t a t e 42 (see Figure 7.) by analogy correlated the o l e f i n i c proton resonances with the carbon resonances at 123.6(CH) and 128.1(CH)ppm, respectively, and the resonances at 146.2(C) and 166.9(C)ppm were assigned to the remaining carbons of two t r i s u b s t i t u t e d o l e f i n s . Comparison of 13 74 the C NMR chemical s h i f t values with those for metabolite 44 30 44 45 R=Ac 57 R=H 13 Table 1. C NMR data comparison of A l and 44. Chemical s h i f t , ppm. Chemical s h i f t , ppm. Carbon # 6 7 8 A l 188.7 123.6 166.9 Carbon # 3 4 5 A A a 186.3 123.9 168.9 reference 74, 25.20MHz, CDCl-j. Table 2. H NMR data comparison of A l and 45. Chemical s h i f t , ppm. Carbon # A l 4 6.54(d, J=4.9Hz, 1H) 7 5.87(t, J=0.9HZ, 1H) 19 1.17(s, 3H) AST 6.82(d, J=2.0Hz, 1H) 6.03(t, J=2.0Hz, 1H) 1.14(s, 3H) reference 75, 400MHz, C 5D 5N. 32 (Table 1.) provided a d d i t i o n a l credence to the assignments. The remaining four units of unsaturation could be accounted for by four ring s . The presence of four rings, the methyl resonances i n the *H NMR spectrum at 0.65, 0.96, 1.17 and 1.46ppm, and the existence 13 of methyl carbons i n the C NMR spectrum of AX at 12.5(CH 3), 18.7(CH 3), 22.3(CH 3), 26.1(CH 3) and 26.2(CH3)ppm, implied that d i a u l u s t e r o l A (11) consisted of a s t e r o i d a l nucleus. Supporting 1 13 t h i s hypothesis was the close agreement of the H and C NMR methyl chemical s h i f t s of Al and the s t e r o i d a l metabolites 4 5 7 5 , AL, 4 2 7 7 and 1 £ 7 8 (Tables 3. and 4.). A se r i e s of decoupling experiments resulted i n the elaboration of the ester containing fragment. I r r a d i a t i o n of the carbinol methine resonance at 4.15ppm i n the *H NMR spectrum of 41 collapsed the methyl doublet at 1.21ppm to a s i n g l e t , and s i m p l i f i e d the two resonances at 2.34(dd, J=16.4,9.0Hz, 1H) and 2.24(dd, J=16.4,3.8Hz, lH)ppm to an AB quartet (J=16.4Hz). This i s o l a t e d s i x proton spin system was assigned to a 3-hydroxybutanoate moiety. Subtraction of the four carbons of t h i s fragment from the molecular formula of Al leaves the twenty seven carbons of a basic s t e r o i d skeleton, supporting the hypothesis of a s t e r o i d a l structure for 41. The short range 2D-HETCOR NMR spectrum of the t r i a c e t a t e 42 correlated the 1H NMR resonance at 1.46(s, 6H)ppm i n H with the 1 3 C NMR resonances at 26.1(CH 3) and 26.2(CH3)ppm (C26 and C27). The values are a l l somewhat deshielded from t y p i c a l s t e r o i d a l chemical s h i f t s at the C26 and C27 po s i t i o n s (see Table 3. and 4.). By inference, the presence of a 1 3 C NMR resonance at 83.6(C)ppm indicated that the 34 OS0 3Na OAc Table 3. H NMR data comparison of the s t e r o i d a l methyl resonances of A l , AJLr A£ and Al Chemical s h i f t , ppm. Carbon # A l A5. a AL A 2 b 18 10.65s 1.05s 10.67s 0.78s 19 21.17s 21.14s 1.00s 1.02s 21 30.96d 1.51s 30.92d 1.28d 26 41.46s 0.97d z 0.87d 41.42s 27 41.46s 0.98d z 0.87d 41.42s areference 75, 400MHz, CgDgN. b r e f e r e n c e 77, 500MHz, CgDgN. zassignments within a column may be interchanged. Compare the values of the chemical s h i f t s with the same superscripts 1, 2, 3, or 4. 35 Chemical s h i f t , ppm. Carbon # 4 1 M 4 £ a 18 112.5 112.0 15.5 19 222.3 19.3 224.0 21 318.7 318.6 13.3 26 426.1 22.5 429.7 Z 27 426.2 22.7 429.4 Z reference 78, p. 238, C 5D 5N, spectrometer frequency not reported. Assignments within a column may be interchanged. Compare the values of the chemical s h i f t s with the same superscripts 1, 2, 3, or 4. butanoate fragment was attached at C25 of the s t e r o i d sidechain. The existence of a butanoate moiety was confirmed by the observation of a fragment ion at a m/z of 496 daltons i n the MS of the t r i a c e t a t e 41r which corresponds to the loss of 3-acetoxybutanoic acid v i a a McLafferty rearrangement. A second series of *H NMR decoupling experiments allowed the placement of much of the remaining f u n c t i o n a l i t y on the s t e r o i d nucleus. I r r a d i a t i o n of the methine proton at 3.88ppm i n the spectrum of 41 collapsed the methine t r i p l e t at 4.28ppm to a doublet (J=4.9Hz) and s i m p l i f i e d a p a i r of resonances at 1.75-1.85ppm to an apparent AB quartet. I r r a d i a t i o n of the methine resonance at 4.28ppm s i m p l i f i e d the doublet of t r i p l e t s at 3.88ppm to a doublet of doublets (J=10.7,4.9Hz) and collapsed the o l e f i n i c doublet at 6.54(J=4.9Hz)ppm to a s i n g l e t , while i r r a d i a t i o n at 6.54ppm s i m p l i f i e d the methine resonance at 4.28ppm to a doublet (J=4.9Hz). The only way to si t u a t e t h i s f i v e proton spin system on a s t e r o i d nucleus was to assign the reson-ance at 1.75-1.85ppm to a pai r of geminal protons at CI, the resonance at 3.88 and 4.28ppm to carbinol methine protons at C2 and C3, respectively, and the resonance at 6.54ppm to an o l e f i n i c proton at C4. The demonstration of a NOE between the methyl protons at 1.23ppm (C19) and a methine proton at 5.10ppm (H2) i n the t r i a c e t a t e 41 required that H2 be /?. An additional NOE between the methine proton at 5.10ppm (H2) and i t s v i c i n a l neigh-bour, which appears at 5.60ppm (H3) i n the *H NMR spectrum of 41, showed that H3 was also p. Comparison of the observed coupling constants for t h i s spin system i n 41 and 41 to the corresponding 75 7 9 8 0 8 1 coupling constants i n the model compounds 45. and 45_-52 ' confirmed the configurational assignment (see Figure 8.). 37 H ° ^ 2 ^ v l / f A c O ^ ^ s f ^ j A c O v ^ J / i H0JkA; A c o - ' - ^ i H O - ' ^ A J 41 J 1 2 = 1 0 7 H z 43 J l > 2=13-8Hz 45 J 1 ( 2 = 10 5.3 5Hz J 2 ' 3 = 4 9 H z J 2 3 = 4-8Hz J 2 3=7 0Hz J 3 , V * 9 H z J 3 # 4 = 5-*Hz J 3 ; = 20Hz 6=3 90.dt 49 J , 2 - -100,4-5Hz 50 J 1 2 M 2 , A 5 H z J 2 3 = 8 0Hz J 2 3 = A 5Hz 51 R = H, J 2 3 = 1 0 H z 52 8 1J 3 i < = 5Hz R = A c , J 2 3 = 9Hz J 3 > A =<1Hz Figure 8. Comparison of the coupling constants of 41, 42. and the models 45. and 49-52. Since the nature of the A ring in 41 and 42. had been established, the remaining f u n c t i o n a l i t y indicated by the 38 s p e c t r a l data and required by the molecular formula, the cross conjugated ketone and the associated t r i s u b s t i t u t e d o l e f i n , had to be placed with the ketone f u n c t i o n a l i t y at C6 and the o l e f i n 1 13 at C7-C8. The r e s u l t i n g H and C NMR assignments for d i a u l u s t e r o l A (11) and t r i a c e t a t e 12 ( f a c i l i t a t e d by the short range 2D-HETCOR NMR spectrum of 12) are given i n Tables 5. and 6., respectively, and are i n agreement with the proposed s t r u c t u r e . Consistent with the s t r u c t u r a l assignment i s the s p l i t t i n g of the H7 o l e f i n i c proton resonance (5.87ppm) i n H into a t r i p l e t (J=0.9Hz) v i a a l l y l i c coupling to H9 and H14, which resonate at 2.08(ddm, J=12.0,7.2Hz, 1H) and 2.41(m, lH)ppm. The a l l y l i c coupling was v e r i f i e d by i r r a d i a t i n g the o l e f i n i c proton at 5.87ppm (H7) which resulted i n a sharpening of both the H9 and H14 resonances. The observation of intense fragment ions at m/z Of 582(M + - HOAc), 522(M + - 2xHOAc), 376(M + - (2xHOAc + 3-acetoxybutanoic a c i d ) ) , 361(M + - (2xHOAc + 3-acetoxybutanoic a c i d + CH 3)), 265(M + - (sidechain + 2xHOAc)) and 496(M + - (3-acetoxybutanoic acid)) daltons i n the MS of ! 2r provides further confirmation of the proposed s t r u c t u r e . The r e l a t i v e stereochemistry f o r ring A was established by NMR as discussed i n the preceeding paragraphs. The observed chemical s h i f t s for the C18 (^ 0.65ppm, 1 3 C 12.5ppm) and C21 ( 1H 13 0.96ppm, C 18.7ppm) methyl groups i n H were v i r t u a l l y i d e n t i c a l to those for c h o l e s t e r o l (Ifi.) (C18 0.67, 12.0ppm: C21 0.92, 18.6ppm), suggesting that the r e l a t i v e configurations at C13, C14, C17 and C20 were i d e n t i c a l i n the two molecules. The 39 Table 5. P a r t i a l NMR assignments for di a u l u s t e r o l A (11) , d i a u l u s t e r o l B (12) t t r i a c e t a t e 12 and diacetate 5_3_. Chemical s h i f t , ppm. Carbon # 11 12 1 1.75-1.85 2 3.88(dt, J=10.7f4.9Hz, IH) 3.88(m, IH) 3 4.28(t, J=4.9Hz, IH) 4.27(t, J=5. 2Hz, IH) 4 6.54(d f J=4.9Hz, IH) 6.54(d, J=5. 2Hz, IH) 7 5.87(t, J=0.9Hz, IH) 5.87(bs r IH) 9 2.08(ddm, J=12.0 f7.2Hz f 1H) Z 2.07(m, 1H) Z 14 2.41(m/ 1H) 2' 1 2.42(m, 1H) Z 18 0.65(s, 3H) 0.65(s, 3H) 19 1.17(s, 3H) 1.17(s, 3H) 21 0.96(d f J=6.8Hz, 3H) 0.97(d, J=7. 2Hz, 3H) 26 1.46(s) 1.23(s) 27 1.46(s) 1.23(s) 29 2.34(dd, J=16.4,9.0Hz, 2.42(dd, J=16.4,3.8Hz, IH) IH) / / 30 4.15(m, IH) / 31 1.21(d, J=7.0Hz, 3H) / -OH 2.66(bs, W, / 9=27Hz, IH) 2.87(bs, W?^=27Hz, IH) 3.25(bs, W^/2 = 1 5 H z' 1 R ) / / / cont'd, absolute stereochemistry at C20 in cholesterol ( M ) i s 20R. I t 82 has been demonstrated by Vanderah and Djerassi that i n 20S 40 Table 5. continued. Chemical s h i f t , ppm. Carbon # 4JL 1 ? 2 5.10(dt, J=13.8,4.8Hz, 1H) 3 5.60(m, 1H) 4 6.43(d, J=5.4Hz, 1H) 7 5.89(bs, 1H) 9 ? 14 ? 18 0.65(s, 3H) 19 1.23(s, 3H) 21 0.96(d, J=7.2Hz, 3H) 26 1.42(s) 27 1.42(s) 29 2.42(dd, J=16.2,6.3Hz, 1H) 2.57(dd, J=16.2,7.7HZ, 1H) 30 5.23(m, 1H) 31 1.29(d, J=7.6Hz, 3H) -OC(0)CH^ 2.03(s, 3H), 2.06(s, 3H), J 2.10(s, 3H) 52. ? 5.11(dt, J=13.4,3.9Hz, 1H) 5.60(m, 1H) 6.43(d, J=6.9Hz, 1H) 5.89(bs, 1H) ? Z 2.42(tm, J=9.5Hz, 1H) Z 0.65(s, 3H) 1.24(s) 0.98(d, J=7.lHz, 3H) 1.24(s) 1.24(s) / / / / 2.04(s, 3H), 2.08(S,3H) Assignments within a column may be interchanged. A i t s underneath doublet of doublets at 2.42ppm. "? nunable to assign. s t e r o i d s , for comparable molecules, the C21 methyl group i s s h i f t e d approximately O.lppm u p f i e l d i n the *H NMR spectrum. This 41 Table 6. P a r t i a l C NMR assignments for d i a u l u s t e r o l A (H) and t r i a c e t a t e 43. Chemical s h i f t , ppm. Carbon # l l 3 12 2 65.2(CH) Z 67.5(CH) Z 3 64.4(CH) 64.7(CH) 4 128.KCH) 124.2(CH) 5 146.2(C) 147.6(C) 6 188.7(C) 187.6(C) 7 123.6(CH) 123.7(CH) 8 166.9(C) 166.5(C) 18 12.5(CH3) 12.5(CH 3) 19 22.3(CH 3) y 20.4(CH 3) 21 18.7(CH3) 18.6(CH 3) 25 83.6(C) 83.1(C) 26 26.1(CH 3) X 26.0(CH 3) X 27 26.2(CH 3) X 25.9(CH 3) X 28 172.4(C) 170.1(C) W 29 ? 47.7(CH 2) 30 66.7(CH) Z 67.6(CH) Z 31 21.1{CH 3) y 19.8(CH 3) -oc C O - / 169.4 W, 170 / 170.1 W. "?"unable to assign C29 by comparison with 12. "Assignments within a column may be interchanged. 42 further supports the hypothesis that the r e l a t i v e configuration at C20 i n A l i s i d e n t i c a l to that i n cholesterol (AfL) • I r r a d i a t i o n of the C18 methyl protons in A3, at 0.65ppm f a i l e d to induce an observable NOE into the C14 proton and i r r a d i a t i o n of the C19 methyl protons at 1.23ppm likewise f a i l e d to induce an NOE into the C9 proton. This negative evidence, while not proving the stereochemical assignment, i s at le a s t consistent with that shown. I t i s postulated from biosynthetic reasoning that d i a u l u s t e r o l A (Al) possesses the absolute stereochemistry t y p i c a l l y associated with n a t u r a l l y occurring s t e r o i d s . The r e l a t i v e stereochemistry at C30, the carbinol c h i r a l carbon i n the butanoate moiety, remains undetermined. Attempts to make the 2,3-acetonide of A l / with intention of subsequent a p p l i c a t i o n of 83 the Horeau method to determine the absolute configuration at C30, f a i l e d . D i a u l u s t e r o l B (A2) was also i s o l a t e d as a clear o i l that was shown by EIMS to have a molecular formula of C27 H42°4 ( o b s e r v e d m/z 430.3055, requires 430.3083), requiring 7 units of unsaturation. The 1H NMR spectrum of A2 (Figure 9.) was i d e n t i c a l to that of A l i n a l l respects except for the absence of the resonances due to the 3-hydroxybutanoate residue. S i m i l a r l y , the IR was e s s e n t i a l l y i d e n t i c a l apart from the absence of an ester absorption band (1722cm"1 i n 141). The observation of intense fragment ions at m/z of 412(M + - H 20), 397(M + - (H 20 + CH 3)), 379(M + - (2xH 20 + CH 3)), 301(M + -(sidechain)) and 283(M + - (K 20 + sidechain)) daltons i n the MS of A2r further confirmed that d i a u l u s t e r o l B should be assigned structure A2. Treatment of the t r i o l A2 with a c e t i c anhydride and pyridine at room 43 temperature generated the diacetate 52. as expected. The H NMR spectrum (Figure 10.) was p e r f e c t l y consistent with the proposed structure 52., as was the IR and mass spectrum. P a r t i a l *H NMR assignments for 42 and 52. are included in Table 5. Dia u l u s t e r o l B (42) was i s o l a t e d from rapidly processed extracts of Diaulula sandieqensis and in a l l the i s o l a t i o n s undertaken the r a t i o of 41 to 42 was v i r t u a l l y i d e n t i c a l (7 1/2:1), thereby eliminating the p o s s i b i l i t y that 42 i s an a r t i f a c t formed by hydrolysis of d i a u l u s t e r o l A (41). £. B i o l o g i c a l A c t i v i t i e s of. D i a u l u s t e r o l A. (41) a M Diaulusterol £ (42). D i a u l u s t e r o l A (41) and d i a u l u s t e r o l B (42) were present i n r e l a t i v e l y high concentrations (0.16mg (0.06% dry wt.) and 0.02mg (0.007% dry wt.) per animal, r e s p e c t i v e l y ) . It seems reasonable, therefore, to expect that these two s t e r o i d a l metabolites might have a b i o l o g i c a l r o l e . The metabolites 41 and 42 e x h i b i t considerable a n t i m i c r o b i a l a c t i v i t y as demonstrated by the r e s u l t s of the i n v i t r o a n t i b a c t e r i a l and antifungal assays, Table 7. A f i s h antifeedant bioassay was undertaken using commercial g o l d f i s h tCarassius auratus) as the t e s t species. The r e s u l t s showed that 41 i n h i b i t s feeding at a concentration of 0.20mg/pellet (0.0074rag/mg of food p e l l e t ) , and there was a marked preference for the control at concentrations of l e s s than 0.05mg/pellet (0.0019mg/mg of food p e l l e t ) . Metabolite 42 showed no a c t i v i t y up to the maximum concentration tested, 0.20mg/pellet. Since the average concentration of metabolite 41 45 Table 7. Results of j j i v i t r o a n t i b i o t i c assays for the s t e r o i d a l metabolites, d i a u l u s t e r o l A (41) and d i a u l u s t e r o l B (42). (Minimum i n h i b i t o r y concentration reported i n /ig/disc.) Micro-organism. Compound ScA BS PythD RhyzS d i a u l u s t e r o l A (41) 1270 63 1270 63 d i a u l u s t e r o l B (42) 48 10 387 48 B a c t e r i a : Fungi: ScA: Staphylococcus aureus. PythU: Pvthium ultiftium.. BS: B a c i l l u s s u b t i l i s . RhyzS: Rhizoctonia s o l a n i . per animal was 0.16mg the experimental i n h i b i t o r y concentration i s quite comparable. The comparability i s even more apparent when you consider that loses w i l l have been incurred during the i s o l a t i o n and that the metabolites were not released d i r e c t l y 84 into the mouth of an i n q u i s i t i v e predator . D_. Further Observations and Discussion. The 25-(3-hydroxybutanoate) residue and the 2a,3c»-diol array of the d i a u l u s t e r o l s A (41) and B (42) are not commonly encountered i n n a t u r a l l y occuring s t e r o i d s . Three of the small number of steroids containing the 2a, 3e*-diol moieties include compound 54., which was recently i s o l a t e d from the marine hydroid F.udendrium qlomeratum 8 5. the potent anticancer reagent 2a-8 6 hydroxyhippuristanol (5_5J from the gorgonian Xs_is_ hippuris and 47 azedarachol (5JL) i s o l a t e d from the root bark of Melia 87 azedarach , which also possesses an unsaturated sec-butanoate moiety at the C20 p o s i t i o n of the pregnane skeleton. D i a u l u s t e r o l A (11) and d i a u l u s t e r o l B (12) are related to the two phytosterols, a cetylpinnosterol (15.) and pinnosterol (.52), which were i s o l a t e d from the red alga Laurencia p i n n a t a 7 5 . A l l four steroids share s t r u c t u r a l features with the ecdysones, for example ot-ecdysone (12.). Metabolites 15. and 5_2 showed b i o l o g i c a l a c t i v i t y as insect moulting hormones, however, metabolites H and 12 exhibited no such a c t i v i t y . The r e l a t i v e concentrations of d i a u l u s t e r o l s A (H) and B (12)f i n the skin extracts of Diaulula sandiegnesis. v a r i e s greatly throughout the year. Minimum concentrations, sometimes so low that i t was impossible to i s o l a t e the metabolites (they were 48 apparently not present at a l l on occasions), were observed from February to A p r i l . During the summer the concentrations gradually increased reaching a maximum in September/October. During the f a l l the concentrations decreased to the minimum values observed in l a t e winter. During the winter months the nudibranchs generally hid themselves deeply i n cracks and f i s s u r e s and became very d i f f i c u l t to c o l l e c t . Presumably t h i s provides protection from the battering that exposed rocky reefs experience from winter storms. This behavior must also provide protection from predation at a time when the chemical defense reserves are at a minimum. The retreat to p h y s i c a l l y inaccessible habitats appears to coincide with the depletion i n the nudibranch*s chemical arsenal and not with the approach of winter storms which s t a r t i n November (the animals can be found i n reasonable numbers from May through to early January). The seasonal v a r i a t i o n i n the r e l a t i v e concentrations of d i a u l u s t e r o l s A (11) and B (12) i s probably the re s u l t of one of two scenarios: i ) The nudibranchs take s h e l t e r from the winter storms and stop feeding, and thus the chemical arsenal becomes depleted; Or i i ) they take shelter as a r e s u l t of a spent chemical defense? Further study i s required to d i s t i n g u i s h which of the two cause and e f f e c t s operates. In addition to Diaulula sandiegensis. A l d i s a sanguinea cooperi provides another example of a dorid nudibranch that sequesters a s t e r o i d a l f i s h antifeedant, 3-oxo-chol-4-ene-24-oic acid (JLBJ . Further s t e r o i d a l metabolites have been i s o l a t e d from nudibranchs for which no defensive role i s ascribed. For example, the highly oxygenated steroid 5_9_ from Hervie peregrina. F l a b e l l i n a a f f i n i s and r.oryphella l i n e a t a 8 9 . and the s t e r o i d a l 49 peroxide 5a,8a-epidioxysteroid (££.) i s o l a t e d from a species of 90 Adalaria . The use of steroids for chemical defense i s not l i m i t e d to nudibranchs. Throughout nature steroids e x h i b i t a wide v a r i e t y of defensive b i o l o g i c a l a c t i v i t i e s , depending on o f t -91 times subtle molecular changes . In conclusion, these r e s u l t s , i n combination with the 7 0 71 previous findings of Walker et a l . and Fuhrman et a l . , have shown that Diaulula sandiegensis represents another example of a dorid nudibranch possessing d i f f e r e n t skin chemistry at d i f f e r e n t c o l l e c t i n g s i t e s (Fuhrman et a l . 7 1 only investigated extracts i s o l a t e d from d i g e s t i v e glands). It seems reasonable to assume that the steroids found i n the B r i t i s h Columbian specimens, and 50 the chlorinated acetylenes found i n the C a l i f o r n i a n specimens are being produced by the d i f f e r e n t dietary organisms found at the two locations. In f a c t , i t has been demonstrated that the chlorinated acetylenes are derived d i r e c t l y from a dietary 92 sponge . A dietary source for the s t e r o i d a l metabolites has not yet been found. 51 IY. SOFT CORALS. A.. Introduction: Alcyonacean Zoology. Soft corals are members of the phylum Coelenterata (or Cnidaria), and except for the hydras and a few other freshwater hydrozoans, a l l coelenterates are marine. Coelenterates are rather p r i m i t i v e animals that e x h i b i t two d i f f e r e n t body forms, the medusa which i s adapted to a pelagic existence, and the polyp which i s adapted to a s e s s i l e benthic existence. The phylum i s composed of approximately 9000 l i v i n g species, and has a r i c h f o s s i l record. The coelenterates show a number of d i s t i n g u i s h i n g 93—97 features . They a l l lack organs or d i f f e r e n t i a t e d muscle c e l l s , many species are b r i l l i a n t l y coloured, a l l have a r a d i a l symmetry and the gastrovascular c a v i t y (Figure 11.) opens to the outside at just one end to form the mouth. There are three classes of coelenterates: The Hydrozoa (hydroids and hydras); the Scyphozoa (mainly j e l l y f i s h ) ; and the Anthozoa (sea-anemones and most corals) (Figure 12.). The c l a s s Anthozoa i s the l a r g e s t of the three coelenterate classes and consists of over 6000 species. In t o t a l , 123 species of anthozoans 3 2 are known from Central C a l i f o r n i a to Southern Alaska . The c l a s s Anthozoa can be divided into the two subclasses Zoantharia (sea anemones, stony corals) and Alcyonaria (soft c o r a l s , sea pens, sea pansies, sea fans, whip c o r a l s , pipe c o r a l s ) . The 98 Alcyonarians are very abundant in t r o p i c a l reef environments . The order Alcyonacea (soft corals) dominates almost e x c l u s i v e l y 52 I tube Figure 11. Structure of a t y p i c a l alcyonarian . i n Indo P a c i f i c reefs, while Western A t l a n t i c reefs are populated p r i m a r i l y by species of the order Gorgonacea. In the order Alcyonacea (soft corals) the i n t e r n a l skeleton supports the colony and consists of separate calcareous spicules integrated i nto the t i s s u e (the coenenchyme). Hence, these 53 PHYLUM CNIDARIA (or COELENTERATA) CLASS. HYDROZOA (hydroids, hydra) ANTHOZOA (sea anemones, corals) SCYPHOZOA (large j e l l y f i s h ) SUBCLASS ALCYONARIA ORDER GORGONACEA (horny corals, sea whips and sea fans) STOLONIFERA (organ pipe corals) PROTOALCYONARIA ZOANTHARIA (sea anemones, stony corals) HELIOPORACEA PENNATULACEA (sea pens or sea pansies) GASTRAXONACEA ALCYONACEA EAUILi ALCYONIIDAE ASTEROSPICULARIIDAE NIDALIIDAE XENIIDAE PARALCYONIIDAE CEffllS SPECIES NEPHTHEIDAE GERSEMIA RUBIFORMIS Figure 12. Phylogenetic c l a s s i f i c a t i o n of the s o f t c o r a l , Gersemia rubiformis. N.B. Organisms c l a s s i f i e d according to Austin . 54 animals consist t y p i c a l l y of a rubbery mass with a massive mushroom, or a variously lobate growth form. Alcyonacean colonies can be reproductively hermaphroditic, dioceous (one colony male, another female), or asexual. However, in general the Alcyonaceans are asexual and the singl e polyps that are produced and released by sexual reproduction, attaches and by asexual budding becomes the parent of a l l other members of the colony. Alcyonaceans feed by catching prey i n the tentacles surrounding the or a l c a v i t y . The presence of a mouth and a diges t i v e cavity permits the use of a wide range of food s i z e s , which includes zooplankton and f i n e p a r t i c u l a t e matter. Upon capture, prey i s paralyzed by stinging structures c a l l e d nematocysts and carried to the mouth. Nematocysts are a unique structure of a l l coelenterates. They are contained i n sp e c i a l i s e d c e l l s known as cnidoblasts which are located throughout the epidermis, p a r t i c u l a r l y i n the ten t a c l e s . £. Symbiotic Associations Between Alcyonarians and Algae f zooxantheiiae): implications For Tiie. Origin oL Isolated Metabolites. Many coelenterates, p a r t i c u l a r l y those l i v i n g i n shallow t r o p i c a l seas (eg., stony and s o f t c o r a l s , gorgonians and sea 99 anemones), contain symbiotic algae known as zooxanthellae . The algae are c l a s s i f i e d as d i n o f l a g e l l a t e s 1 0 0 and were assigned to the s i n g l e species, Zooxanthella m i c r o a d r i a t i c a 1 0 1 . This s i n g l e 102 species designation i s now i n question . I t has been proposed 55 from studies of coelenterate zooxanthellae associations that the photosynthetic algae provide substantive l e v e l s of n u t r i t i o n for 103 the host . The host, i n turn, provides e s s e n t i a l elements fo r autotrophic metabolism with the zooxanthellae augmenting the re c y c l i n g of nutrients ("waste products") 9 3 , 1 0 3 C f < ^ ' 1 0 4 . I t would appear that the very existence of c o r a l reefs rests on the photosynthetic a c t i v i t y of the u n i c e l l u l a r algae and t h e i r 1 05 association with coelenterates . In order to maintain the symbiotic a s s o c i a t i o n , most coelenterates reside i n the upper photic zone and c o r a l reefs are r e s t r i c t e d to shallow, c l e a r waters. Soft corals (order Alcyonacea), i n v a r i a b l y t r o p i c a l species containing a l g a l symbionts, have been a r i c h source of i n t e r e s t i n g terpenoid natural products (other Alcyonarians have 22 been a r i c h source of prostanoids as well as terpen.es) . The very existence of the symbiotic association obscures the o r i g i n of the substances i s o l a t e d from zooxanthellae bearing-c o e l e n t e r a t e s 1 0 6 . I t has been established that the s t e r o l composition of such animals r e f l e c t s both a dietary accumulation and a contribution from t h e i r symbiotic zooxanthellae. Zooxanthellae-bearing coelenterates show the presence of several unique, highly alkylated s t e r o l s such as the cyclopropane containing s t e r o l s gorgosterol ( £ 1 ) 1 0 7 and 23-demethylgorgosterol 108 ( £ 2 ) . However, the p o s i t i o n and degree of a l k y l a t i o n appear dependent on whether the algae l i v e symbiotically or a l o n e 1 0 2 a _ ( ^ . Direct evidence i n the l i t e r a t u r e indicates that both gorgonians and s o f t corals are capable of independent terpene synthesis. 56 61 62 rC0 2Me 63 64 Pukalide (£2.) was i s o l a t e d from Lophoaoraia r i g i d a 1 0 9 ' 1 1 0 and L_. alba. 1 1 1. P a c i f i a o r o i a pulchra e^ilis. yielded furanodiene ( £ 1 ) 1 1 2 . These species are t r o p i c a l gorgonians which lack a l g a l symbionts. Other examples are k n o w n 1 0 9 ' 1 1 1 ' 1 1 3 . Pukalide (£2.) and furnodiene (£A) have also been i s o l a t e d from the s o f t c o r a l s , S i n u l a r i a a b r u p t a 1 1 4 . and an E f f l a t o u n a r i a s p e c i e s 1 1 5 , r e s p e c t i v e l y , which possess symbionts. Further to t h i s , measurements of the 1 3 C / 1 2 C r a t i o s of i n d i v i d u a l metabolites has determined that terpenes can only have been produced by the host c o r a l or g o r g o n i a n 1 0 2 ^ , and these conclusions have been confirmed by a se r i e s of incorporation studies with l a b e l l e d p r e c u r s o r s 1 1 6 . 57 £. Why The Interest: The. B i o l o g i c a l and. Ecological S i g n i f i c a n c e OL Some Coelenterate Metabolites. The members of the class Scyphozoa ( j e l l y f i s h , etc.) which u t i l i z e nematocysts to deter predators have, to date, provided no int e r e s t i n g natural products. S i m i l a r l y , the p h y s i c a l l y protected hard corals have yielded l i t t l e of i n t e r e s t to the marine natural products chemist. The majority of i n t e r e s t i n g coelenterate metabolites have been i s o l a t e d from s o f t corals and gorgonians, 117 with fewer examples from sea pens (order Pennatulacea) , 118 zoanthids (sea anemones, etc..) and species of the order 119 Sto l o n i f e r a . The tissues of many of these exposed c o r a l reef invertebrates are t o x i c or show antifeedant properties to 120 121 f i s h ' . Soft corals and gorgonians i n p a r t i c u l a r show very 120 122 l i t t l e evidence of predation . Only a few gastropods feed on 123 these animals. An example of a compound that i s t o x i c to f i s h i s provided by c r a s s o l i d e (65). is o l a t e d from the s o f t c o r a l , LPbpphyturo c r a s s u m 1 2 4 . OAc 65 In addition to predatory defensives i t appears that s o f t coral metabolites can be released into the water column 125 surrounding a colony to i n h i b i t the growth of competitors . 58 67 126 F l e x i b i l i d e (££), from S i n u l a r i a f l e x i b i l i s . and the diterpene 127 67. from Lobophytum pauciflorum-1"*' . can k i l l or retard the growth 128 of the hard c o r a l Acropora formosa . S e s s i l e reef coelenterates need to ensure that they are not invaded by microorganisms, larvae and/or algae. A chemical defense strategy i s once again apparently employed to handle the p o t e n t i a l problem. A c t i v i t y against marine u n i c e l l u l a r algae i s 123 widespread . Further to t h i s , a sea pen (order Pennatulacea), a S t y l a t u l a species, y i e l d e d s t y l a t u l i d e (£8J which i s t o x i c to 129 copepod larvae . The growth of diatoms i s i n h i b i t e d by the muricins 1-4 (69-72). These saponins appear to contribute to reduced f o u l i n g of the gorgonian Muricea f r u c t i c o s a by epiphytes and e p i z o a 1 3 0 . 59 Me 69 R 1 = R 2 = A c 70 R 1 = C 0 P r n , R 2 = A c 71 R 1 = A c , R 2 =C0Pr n 72 R ' = R 2 = C 0 P r n C9H19 NH(CH2)3N(CH2)4NMe2 73 Terpenoids from s o f t corals e x h i b i t other diverse biodynamic 131 132 properties. P h y s i o l o g i c a l , ichthyotoxic and cor a l metabolites. The i n vivo antimicrobial a c t i v i t y of the spermidine d e r i v a t i v e 22, i s o l a t e d from a S i n u l a r i a species, i s of n o t e 1 3 4 . Alcyonarians i n general appear to be capable of £e_ novo terpene synthesis. Many species of t h i s subclass possess no s k e l e t a l p r o t e c t i o n , and hence t h e i r s u r v i v a l appears dependent on a l t e r n a t i v e s t r a t e g i e s . These st r a t e g i e s are based c e r t a i n l y , i n part, on the production of noxious and toxic chemicals. The wide d i s t r i b u t i o n of b i o l o g i c a l l y and e c o l o g i c a l l y active metabolites associated with these animals make them a prime target for marine natural products chemists. antineoplastic 133 a c t i v i t i e s have a l l been a t t r i b u t e d to s o f t 60 EL. chemistry pJL s o f t corals and Alcyonarians In. General. Soft corals have been a r i c h source of novel terpenoid metabolites. A f u l l review of the l i t e r a t u r e f a r exceeds the scope of t h i s t h e s i s , and i s quite unnecessary. An excellent 9 8 review by Tursch et a l . covers the l i t e r a t u r e on coelenterates up to 1978, and diterpenes, i n p a r t i c u l a r cembranoid metabolites, which are widely d i s t r i b u t e d i n s o f t c o r a l s , were reviewed by F e n i c a l 1 3 5 i n 1978, and Weinheimer et a l . 1 3 6 i n 1979. The l i t e r a t u r e i s further reviewed and updated i n Faulkner's review 22 papers . However, i t i s appropiate that a b r i e f overview of the l i t e r a t u r e since Faulkner's l a s t review paper covering work published up to and including J u l y 1985, excluding s t e r o i d a l 137 metabolites , be included i n t h i s t h e s i s . The following review covers the period from J u l y 1985 up to the c i t a t i o n s reported i n CA Selects, issue 9, 1987, and/or Chemical Abstracts, No. 20, May 1 8 t h , 1987. Many sesquiterpenes have been i s o l a t e d from s o f t c o r a l s . The majority of sesquiterpenes i s o l a t e d from marine coelenterates e x i s t as the o p t i c a l antipode of the form found, when known, i n t e r r e s t r i a l or marine p l a n t s . A number of sesquiterpenes have been reported from s o f t c o rals over the past two years. Two new 138 139 capnellane sesquiterpenes, 74 and 75 , were i s o l a t e d from the Chinese s o f t c o r a l , Capnella imbricata. Extracts of the A u s t r a l i a n s o f t c o r a l , Lemnalia c e r v i c o r n i s yielded eleven calamenane sesquiterpenes, including compound 7JL and two non-140 aromatic minor metabolites 22 and 78 . Studies on several A u s t r a l i a n Xenia species resulted i n the i s o l a t i o n of the new 61 sesquiterpenes 79-81 . The Mediterranean alcyonacean, Alcyonium c o r a l l c i d e s . yielded the two metabolites (+)-corlloidin-A (82) and (-)-coralloidin-B (JL2.) , where £2 i s the f i r s t n a t u r a l l y 142 occurring 5,6-dehydroeudesmane 74 75 62 H -ChUOAc 82 83 Diterpenes are the most frequently encountered metabolites of s o f t c o r a l s , and of these cembranoids are probably the most common. D e n t i c u l a t o l i d e ( M ) i s an ichthyotoxic peroxide-containing cembranolide from the soft c o r a l , Lobophytum 143 denticulatum . Studies of Australian s o f t corals of the genus E f f l a t o u n a r i a revealed the presence of two isomeric 7,8-144 epoxycembra-3,ll,15-trien-16,2-olides, £5_ and 86 C o r a l l o i d o l i d e s A ( £ 2 ) and B ( M ) r the f i r s t and rather unusual cembranoids from a Mediterranean organism, were i s o l a t e d from the 145 so f t coral Acyonium c o r a l l o i d e s . Extraction of the s o f t c o r a l , S i n u l a r i a mayi. resulted i n the i s o l a t i o n of a neocembranoid metabolite, 13-hydroxyneocembrene ( & 9 J 1 4 6 . A P a c i f i c s o f t coral of the genus S i n u l a r i a . collected i n Palau, Western Caroline Islands, gave £Q. and three other new norcembranoid m e t a b o l i t e s 1 4 7 . Ten new diterpenoids, including minabein-10 (£1) , 148 possessing the known b r i a r e i n skeleton have been i s o l a t e d from a species of Minabea col l e c t e d i n the Eastern Caroline 149 Islands . This i s the f i r s t report of the occurrence of t h i s class of compounds in s o f t corals. Briareins are t y p i c a l l y 22 associated with gorgonians and sea pens (order Pennatulacea) 63 C o l l et a l . have looked at a se r i e s of so f t corals of the genus Lobophvtum and noted the co-occurrence of d i f f e r e n t diterpene skeletons in the same colony. The two diterpenes 22. and 93 which might be considered as a prenylated sesquiterpene cubebol and a prenylated germacrene, respectively, were found to co-occur with known cembranoid diterpenes i n two seperate species of Lobophytum. The new b i c y c l i c diterpene 2A was isolated from another species of Lobophytum along with a known cembranoid. 87 88 64 90 91 94 Species of the family Xeniidae had u n t i l recently yielded only xenicane and xeniaphyllane diterpenes such as xenicin f 95) 150 from Xenia elongata and xeniaphyllenol (2A) from m a c r a s p i c u l a t a 1 5 1 . Recently, a species of C e s p i t u l a r i a of the family Xeniidae yielded 4,5-deoxyneodolabelline (JLD / which can be related to the dolabellane skeleton by migration of a methyl from CI to C l l 1 5 2 . The two c y c l i z e d cembranes 2JL and 22. were also i s o l a t e d . A xenia species from Okinawan waters yielded two 65 p a i r s of diasteriomeric diterpenes. One pair consisted of the perhydroazulene d e r i v a t i v e s hydratoxeniolone (ULQ.) and hydratoisoxeniolone (101). The putative biogenetic precursor 153 germacrexeniolone (1£2) was also noted. 96 97 95 99 100 101 66 103 HO 104 A number of s t e r o i d a l metabolites have been i s o l a t e d from 137 s o f t c o rals , but only two unique metabolites w i l l be mentioned here. A Capnella species contained the norpregane C 2 0 154 d e r i v a t i v e s , 103 and 104 . Naturally occuring pregnane-derived s t e r o i d s , p a r t i c u l a r l y from the marine environment, are rare. 105 106 F i n a l l y , two very unusual metabolites, methyl sartortuoate f 1 0 5 ) 1 5 5 and methyl isosartortuoate ( 1 0 6 ) 1 5 6 f have been i s o l a t e d from the s o f t c o r a l Sarcophvton tartuosum c o l l e c t e d in the South China Sea. They both represent unprecedented t e t r a c y c l i c tetraterpenoids with unknown biogenetic o r i g i n s . However, since diterpenes of the cembrene class are commonly found i n s o f t 67 c o r a l s , a p l a u s i b l e biogenesis would involve generation of the cyclohexene ring and hence the required carbon skeleton by a Diels-Alder coupling of two cembrenes. Other orders of the subclass Alcyonaria, p a r t i c u l a r l y gorgonians, also continue to y i e l d interesting metabolites. The anti-inflammatory diterpenoid k a l l o l i d e A (107) possessing the rare pseudopterane skeleton, was isolated from Pseudopterogorgia 157 k a l l o s . A new hydroquinone glycoside, moritoside (108) f which i n h i b i t s the c e l l d i v i s i o n of f e r t i l i z e d s t a r f i s h eggs, was 158 i s o l a t e d from a gorgonian of the genus Euplexaura . A marine sea whip, Pseudopterogorgia elisabethae. yielded pseudopterosin C (109), which represents a new class of anti-inflammatory and 159 analgesic diterpene pentosides . Species of the order S t o l o n i f e r a continue to y i e l d p r o s t a n o i d s 1 6 0 , such as the antitumor agents bromovulone (110) and iodovulone (111) i s o l a t e d from C l a v u l a r i a v i r i d i s 1 6 1 . Tubipora musica yielded tubipofuran (112) r which i s ichthyotoxic toward a k i l l i f i s h , O r i z i a s  l a t i p e s . C l a v u l a r i a k o e l l i k e r i yielded f i v e kericembrenolides related to metabolite 113. which a l l i n h i b i t e d the growth of B-16 3 63 melanoma c e l l s . The s t o l o n i f e r , Sarcodictyon roseum, contained 164 sarcodictyenone (114) , which represents the f i r s t example of a c h i r a l , o p t i c a l l y a c t i v e prenyl d e r i v a t i v e of a reduced benzoquinone. F i n a l l y , the sea pansy (order Pennatulacea), R e n i l l a reniformis contained the metabolite 115 which i n h i b i t s the settlement of larvae of the barnacle Balanus a m p h i t r i t e 1 6 5 68 69 R. Review Of. the. Chemistry Ol. the. Family Nephtheidae. The chemistry of the s o f t c o r a l , Gersemia rubiformis P a species of the family Nephtheidae, i s to be discussed i n t h i s t h e s i s . Hence, a review of the chemistry of t h i s family i s i n order. The family Nephtheidea comprises many genera of which Lemnflliflf Paralemnalia, Capnella. Litophvton and Nephthea have received considerable attention from organic chemists. In the f i r s t three genera, only sesquiterpenes have been reported while the l a t t e r two genera have afforded mainly cembranoid 22 98 diterpenes ' . The sesquiterpenes observed include the simple t e r r e s t r i a l sesquiterpene germacrene-C (116) is o l a t e d from Nephthea c h a b r o l i i together with the guaiane-derived alcohol H I 1 6 6 . Two new metabolites, eudesma-4,7(ll)-dien-8B-ol (1181 and eudesma-4,7(11)-diene-8-one (119) f were isol a t e d from an 45a u n i d e n t i f i e d species of Nephthea . Numerous sesquiterpenes bearing the capnellane carbon skeleton, but d i f f e r i n g i n the l e v e l of oxygenation and unsaturation, have been i s o l a t e d from 22 98 so f t corals of the genus Capnella ' . Examples include metabolites H 1 3 8 , 2 5 1 3 9 , 12£ and 1 2 1 1 6 7 which were a l l i s o l a t e d from Capnella imbricata. 116 117 118 70 119 ! H OH 120 121 Soft corals of the genera Lemnalia and Paralemnalia have been characterised by the exclusive production of sesquiterpenes. The majority of sesquiterpenes i s o l a t e d from a number of d i f f e r e n t species have been based on a nardosinane skeleton. An example i s provided by lemnacarnol (122) i s o l a t e d from L . c a r n o s a 1 6 8 . L . africana has yielded a wide v a r i e t y of sesquiterpenes which includes the germacrene alcohols 123 and 1 2 4 1 6 9 , the racemic d i o l , 125_1 6 6, a f r i c a n o l (126J 1 7 0 (which was also noted i n extracts of L . n i t i d a 1 7 1 ) , a 17 2 norsesquiterpene 127 and the two sesquiterpenes 128 and 172 129 . Extracts of h.. africana have also yielded the two unusual compounds the monoacetate 130 and the diacetate 131. that appear to be derived from the nardosinane skeleton (lemnalane skeleton) by ring expansion. These two sesquiterpenes were found 173 to co-occur with the eremophilane d e r i v a t i v e 132 71 122 R=OH 123 124 138 R=H ^OCHO 125 126 127 72 Lemnacarnol (122) was found to co-occur with 7-epi-lemnalactone ( i l l ) 1 7 1 . Lemnalol (134) was i s o l a t e d from L. 174 tenuis and L.. c e r v i c o r n i s yielded metabolites 7JL, 22 and 140 78 . The discovery of two ar i s t o l a n e d e r i v a t i v e s 135 and 136 i n L. humesi could indicate that the nardosinane der i v a t i v e s are derived from precursors having a cyclopropane r i n g 1 7 5 . 2-Deoxy-12-oxolemnacarnol (137) was found to co-occur with 2-deoxylemnacarnol (US.) i n Paralemnalia 171 thyroides , the presence of 138 was also noted i n Lemnalia af rieana and L.. i a e x i s . 1 7 1 . Extracts of Paralemnalia d i a i t i f o r m i s resulted i n the i s o l a t i o n of both 133 and 176 lemnalactone (139) . A specimen of p_. thyroides from the Townsville area was the source of two sesquiterpenes, 140 and 177 141. together with 2-deoxylemncarnol (138) . Specimens of E« thyroides from Palau contained, along with 2-deoxylemnacarnol (138), the two new norsesquiterpenes 142 178 and 143 . I t i s i n t e r e s t i n g to note that the carbon skeleton of lemnacarnol, lemnalactone and t h e i r d e r i v a t i v e s i s antipodal to that of known nardosinane sesquiterpenes i s o l a t e d 98 from t e r r e s t r i a l sources OH Y 133 134 135 73 136 137 139 142 143 Diterpenes are the most frequently encountered Nephtheidae metabolites and again they are dominated by cembranoid derived compounds. For example, Schmitz and co-workers described nephthenol f144) and epoxynephthenol acetate (145), as 179 metabolites of a Nephthea species from Enewetrak . Metabolite 144 was l a t e r found i n extracts from Litophyton flrbpreum and L. 74 180 v i r i d i s . 2-Hydroxynephthenol (146) was also noted from L. 181 v i r i d i s . A large s e r i e s of "simple" cembranoids (144 and 147-182 166 155) have been i s o l a t e d from H. brassica . H. c h a b r o l i i and 183 184 unknown species of Nephthea J'- L O ,* t Further s t r u c t u r a l l y novel diterpenes include the two xeniaphyllanes 156 and 157. which were 185 i s o l a t e d from H. c h a b r o l i i 144 145 146 147 166,182,183 148 166,182,184 75 153 182 156 R="V 157 R=X' Steroidal related metabolites i s o l a t e d from the family, Nephtheidea, include the two pregnane d e r i v a t i v e s , 158 and 159. which together with the two s t e r o i d a l metabolites, 160 and 161. were obtained from the cold water species, Gersemia 186 rubiformis . An u n i d e n t i f i e d species, reportedly a Nephthea species, contained the d i o l 162 187 158 159 76 0 OH 160 R = H 161 R=OAc HO 162 Very few aromatic compounds have been i s o l a t e d from s o f t c o r a l s . However, the f a m i l i a r 5-methyl-2-tetraprenyl-l,4-benzoquinone and the corresponding benzopyran, 164. were 1 88 i s o l a t e d from a species of the genus Nephthea . where they may play a ro l e as symbiont a t t r a c t a n t s . The chemistry of ju s t a few t r o p i c a l and one cold water species of the family Nephtheidea have been studied. However, numerous secondary metabolites have been uncovered possessing diverse s t r u c t u r a l forms. This family by i t s e l f provides a prime target for marine natural products chemists. 163 164 77 Y. SECONDARY METABOLITES ££0J1 THE. SOFT CQBAL., Gersemia rubjfprmjs (Ehrenbera. 1834) , AHE THE. DKNnRONOTOTD NUDIBRANCHi Toauina tetraauetra ( P a l l a s . 1788). A.. Introduction. ( i ) . Gersemia rubiformis (Ehrenbera. 18JL4) . Soft corals and gorgonians, two groups of coelenterates that belong to the orders Alcyonacea and Gorgonacea re s p e c t i v e l y , are o p very abundant i n t r o p i c a l reef environments . Indo P a c i f i c reefs tend to be dominated almost e x c l u s i v e l y by s o f t c o r a l s , while western A t l a n t i c reefs are populated p r i m a r i l y by gorgonians. The abundance and ease of c o l l e c t i o n of these invertebrates has f a c i l i t a t e d an extensive i n v e s t i g a t i o n of t h e i r secondary 22 98 135 136 metabolism Sesquiterpenes and diterpenes, having a wide v a r i e t y of both well known and rare carbon skeletons, are the most common s o f t c o r a l and gorgonian metabolites. In contrast to t r o p i c a l waters the cold temperate waters of B r i t i s h Columbia harbour only one reported species of i n t e r t i d a l 32 and s u b t i d a l s o f t c o r a l , Gersemia rubiformis . G_. rubiformis. a so f t coral of the family Nephtheidae (Figure 12.), has a documented d i s t r i b u t i o n range in the P a c i f i c that extends from Japan north to the Bering Sea and down along the west coast of North America from Alaska to Northern C a l i f o r n i a (north of Point Arena). £. rubiformis i s also reported from the northern 32 A t l a n t i c . The species was probably f i r s t i d e n t i f i e d by 3 2 Ehrenberg i n 1834 and i s known l o c a l l y as the "sea strawberry". 78 The animal can obtain a height of up to 15cm and consists of a branching mass with polyps having eight tentacles. Colour varies from pale orange to red. When the tentacles are expanded, the t i p s of the polyps and the whole colony takes on a whitish hue. £. rubiformis i s found attached to rocks at the very lowest tides and s u b t i d a l l y to a depth of 15 meters in current swept 72 189 surge channels and other exposed areas ' 1 86 Kingston et a l . previously reported the i s o l a t i o n of the rare C 2^ pregnane steroids I 5 8 1 8 6 a and 159 1 8 6 t 3, and the two unusual C 2 g steroids 12/j-hydroxy-24-norcholesta-l,4,22-triene-3-one (JL£Q.) and i t s acetate 1 6 1 1 8 6 c , d from specimens of Gersemia  rubiformis c o l l e c t e d o f f the Newfoundland c o a s t l i n e . U n t i l now, t h i s work probably represented the f i r s t and only study on a cold temperate or cold water species of s o f t c o r a l . No i n v i t r o antimicrobial or other b i o l o g i c a l a c t i v i t i e s were reported for any of these s t e r o i d a l metabolites. 190 Our routine b i o a c t i v i t y t e s t i n g program revealed that extracts of Gersemia rubiformis. c o l l e c t e d off the B r i t i s h Columbian c o a s t l i n e , exhibited potent in v i t r o a n t i m i c r o b i a l a c t i v i t y . The organism competes extremely well i n a very crowded environment and i s very clean and unfouled. G_. rubiformis has, as far we know, just two predators, the dendronotoid nudibranch, 122a Toquina tetraquetra i n the P a c i f i c and the prosobranch gastropod, Calliostoma occidentale (Mighels and Adams, 1842) i n the A t l a n t i c 1 2 2 1 3 . Considering just how b l a t e n t l y G_. rubiformis' advertises i t s e l f and that X. tetraquetra grazes on t h i s s o f t coral with such apparent culinary delight (we have observed up to a dozen on a s i n g l e wall (approximate area 3 x 7 meters) covered 79 with G. rubiformis, each rasping a large groove into the colony) i t i s extremely s u r p r i s i n g that no other organism, not even another nudibranch, i s observed to feed on £. rubiformis on the west coast. Prompted by these observations, we undertook an investigation of the secondary metabolites of £. rubiformis c o l l e c t e d off the east and west coasts of Canada. The investigation was i n i t i a t e d i n August 1985. Gersemia rubiformis was c o l l e c t e d on several exposed rocky reefs located within the Gordon Group of Islands, Port Hardy, B r i t i s h Columbia, and at Admiral's Cove, Cape Broyle and Bay B u l l s , Newfoundland (south of St. Johns). Once a colony of £. rubiformis had been located i t was re a d i l y c o l l e c t e d by hand using SCUBA. The B r i t i s h Columbian animals were c o l l e c t e d on four occasions in the spring, l a t e summer and early f a l l . The Newfoundland animals were c o l l e c t e d on a number of occasions between October 1986 and January 1987. Although found i n an assortment of habitats, G_. rubiformis was most abundant on exposed c l i f f faces at a depth of 10-15 meters and also i n exposed surge channels at depths of 1-3 meters. ( i i ) . Toauina tetraquetra (Pallas, 1788). The cold temperate waters of North America are inhabited by more than one hundred and t h i r t y species of subtidal and 32 i n t e r t i d a l nudibranchs . The skin extracts of nudibranchs have 22 been found to contain i n t e r e s t i n g secondary metabolites . The majority of the compounds i s o l a t e d to date have been sesquiterpenoids or diterpenoids. It i s found that many of the 80 1 Q T metabolites are of a dietary o r i g i n . A v a r i a t i o n i n skin chemistry from one c o l l e c t i n g s i t e to the next, r e f l e c t i n g a change in the nudibranch's d i e t , provides strong evidence for a dietary o r i g i n 2 3 ' 4 2 0 ' 1 9 1 . It has been demonstrated that nudibranchs can sequester secondary metabolites, sometimes 2a 42c 19 2 s e l e c t i v e l y , from dietary sponges ' , bryozoans , s o f t c o r a l s 4 5 3 and h y d r o i d s 1 9 3 . Toguina tetraquetra. suborder Dendronotoida, family T r i t o n i i d a e (Figure 1.) feeds primarily on two coelenterates, the cold water s o f t c o r a l , Gersemia rubiformis (order 122a 72 189 Alcyonacea) ' ' and the sea pen, PtUPSarcus aurneyi 1 pq (Gray, 1860) (order Pennatulacea) . Tochuina tetraquetra i s found i n the north P a c i f i c from central Japan north to the Bering Sea and down along the west coast of North America from Alaska to Southern C a l i f o r n i a (south 32 of Point Conception to the Mexican border) . The animal 3 2 was probably f i r s t i d e n t i f i e d by Pallas i n 1788 , and i s known l o c a l l y as the "orange peel nudibranch". This s t r i k i n g dendronotoid i s found at depths from 3-100 meters i n rocky areas when feeding on Gersemia rubiformis and f l a t sandy-mud bottoms when feeding on the sea pen Ptilosarcus aurneyi. I . tetraquetra i s one of the worlds largest nudibranchs, reaching 30cm i n length. The body i s a deep orange-yellow covered with tubercles tipped with white and the foot i s salmon pink to yellow. A white band marks the margin of the dorsum and branchial plumes form an ir r e g u l a r series of low, white t u f t s along the undulating body margins. A point of i n t e r e s t i s that these molluscs were eaten by Aleutian indians raw or cooked i n the K u r i l Islands, U.S.S.R., 81 where i t i s known as "Tochni". I t may also have been consumed by 3 0 189 North American west coast indians i n times of hardship Specimens of Toquina tetraquetra were co l l e c t e d throughout the year by hand using SCUBA at depths of 3-15 meters from two l o c a t i o n s . The f i r s t from several exposed rocky surge channels located within the Gordon Group of Islands, Port Hardy, B r i t i s h Columbia, where they were found feeding on or s i t t i n g nearby the s o f t c o r a l , Gersemia rubiformis. The second c o l l e c t i o n s were made on sandy bottoms located within the Deer Group of Islands, Bamfield, B r i t i s h Columbia, where the sea pen Ptilosarcus aurneyi i s commonly found. The i n v e s t i g a t i o n of the skin extracts of Toquina tetraquetra was prompted by a s e r i e s of observations. F i r s t l y , the nudibranch grazes with apparent "delight" on the s o f t c o r a l , Gersemia rubiformis. for which no other predator i s known on the west coast of North America. Observations indicate that t h i s nudibranch i s so s p e c i a l i s e d that the animal would preferably starve to death in the absence of £. rubiformis even when offered a v a r i e t y of other coelenterates (the sea pen, P t i l o s a r c u s 122a gurneyi was not offered) . Hence, the soft c o r a l may possess a chemical defense which t h i s one s p e c i a l i s e d nudibranch has learned to ignore and quite probably modify for i t s own defensive purposes. In addition, I . tetraquetra has no known natural predators. Also the animal's s i z e , r e l a t i v e abundance and " t o t a l l y excessive" mucus covering makes the i s o l a t i o n of secondary metabolites a r e l a t i v e l y undemanding task. Encouraged by these observations an i n v e s t i g a t i o n of the skin extracts of I . 82 tetraquetra was i n i t i a t e d i n November 1986. £. I s o l a t i o n Q_f Th_e_ Metabolites. ( i ) . Gersemia rubiformis (Ehrenbera. 1834). (a). B r i t i s h Columbian specimens: Freshly c o l l e c t e d specimens were immediately immersed i n methanol, homogenised and allowed to extract at room temperature. The extraction solvent was f i l t e r e d o f f , concentrated i n vacuo and p a r t i t i o n e d between brine and organics. Fractionation of the organic soluble material through a complex combination of f l a s h , Sephadex LH-20, preparative TLC, r a d i a l TLC, reverse and normal phase HPLC chromatographies produced pure samples of two new 194 sesquiterpenes 165 and 166 , and eleven new diterpenes, possessing pseudopterane f 1 6 7 - 1 6 9 ) l 9 0 d . cembrane f170-1 7 5 ) 1 9 0 d ' 1 9 4 and the new gersolane ( 1 7 6 ) 1 9 5 carbon skeletons (the structure of the eleventh remains unresolved). In addition, a degraded diterpene (177) was i s o l a t e d . 2 165 166 83 84 175 176 177 (b). Newfoundland specimens: Freshly c o l l e c t e d specimens were immediately frozen and stored at -5°C. After d e f r o s t i n g , the animals were homogenised i n methanol and allowed to extract at room temperature. The extraction solvent was f i l t e r e d o f f , concentrated i n vacuo and the residue was p a r t i t i o n e d between brine and ethyl acetate. Fractionation of the organic extract by a combination of f l a s h , r a d i a l TLC, normal and reverse phase HPLC chromatographies produced a pure sample of sesquiterpene 178. The presence of the s t e r o i d a l metabolites 15B-161 1 8 6 was noted by 300MHz XH NMR analysis on crude f r a c t i o n s . These metabolites were never f u l l y p u r i f i e d . 15 178 85 ( i i ) . Toauina tetraquetra f P a l l a s , 3788). Freshly c o l l e c t e d animals were immediately immersed i n methanol and allowed to extract at room temperature. The methanol was decanted, concentrated i n vacuo and p a r t i t i o n e d between brine and ethyl acetate. Fractionation of the organic soluble material by a combination of preparative TLC and reverse phase HPLC chromatographies yielded two unrelated sets of pure secondary metabolites. (a) . Port Hardy animals: From Port Hardy animals pure samples of the two 194 sesquiterpenes 165 and 166 and two cembranes, the diterpene 1 7 Q190d,194 a n ( 3 t j i e previously reported diterpene r , 109, 111, 114,194 L i - j 63 i were obtained. (b) . Bamfield animals: From Bamfield animals pure samples of two diterpenes 148 possessing the previously reported b r i a r e i n carbon skeleton , the known metabolite 179 and the previously unreported butanoate 194 analogue 180 , were obtained. 86 C Structural Elucidation ajid. Related Studies. ( i ) . sesquiterpenes 165 and 166; Tochuinyl acetate (165) was obtained as a clear o i l that gave a parent ion at a m/z of 260.1777 daltons i n the EIMS appropriate for a molecular formula of ci7 H24°2 ( r e c3 u^ r e^ m / z * s 260.1777 daltons) which required 6 units of unsaturation. A carbonyl band at 1734cm"1 i n the IR, i n combination with a methyl s i n g l e t resonance at 1.94ppm i n the "^H NMR spectrum (Figure 13.) 13 and a carbonyl resonance at 171.2ppm i n the C NMR spectrum (Figure 14.), i d e n t i f i e d an acetate residue. The carbinol 7 6 methylene (a short range 2D-HETCOR NMR experiment gave a proton 13 count, see Figure 15.) resonance at 70.6ppm i n the C NMR spectrum of 1£5_ suggested that the acetate was an ester of a primary a l c o h o l . The a d d i t i o n a l s i n g l e t s at 1.13, 1.33 and 2.30ppm i n the NMR spectrum suggested that the remaining f i f t e e n carbon atoms of tochuinyl acetate (165) constituted a sesquiterpene fragment. One degree of unsaturation was accounted for by the acetate carbonyl. Four more were r e a d i l y assigned to the 4-methylphenyl moiety indicated by the NMR methyl resonance at 2.30(s, 3H)ppm and the two aromatic resonances at 7.08(d, J=8.1Hz, 2H) and 7.22(d, J=8.lHz, 2H)ppm. These resonances were correlated i n the 13 short range 2D-HETCOR NMR spectrum (Figure 15.) with the C NMR resonances at 20.8(CH 3), 128.6(2xCH) and 126.7(2xCH)ppm, resp e c t i v e l y . The resonances at 135.3(C) and 142.9(C)ppm were assigned to the remaining two aromatic carbons. 87 o co-Details: 187mg 5mm tube 7hours 42-1mins Figure 15. Contour p l o t of 2D-HETC0R NMR spectrum of tochuinyl acetate (1£5_). I I I I | t ( 1 I ] I I I I | I I ! I | 1 I I I | I I I I | I I I I j 1 i i I | i I ( I | I I I I | I I 1 I j I ! I I | I I I I j I I I I | I i I PPM F1 (PPM) Comparison of the p a r t i a l H and C NMR data of the model metabolite 1 8 1 1 9 6 with those for 165 (see Tables 8. and 9.), supports the assignment. The 4-methylphenyl moiety was further confirmed by a MS fragment ion at an m/z of 91 daltons (C^H7) which i s i n d i c a t i v e of a tropylium ion derived from an a l k y l substituted 197 benzene ring 181 Table 8. 1H NMR data comparison of 165 and 181• Chemical s h i f t , ppm. Carbon # 1£5_ l£l a 1,5 7.22(d, J=8.1Hz, 2H) 7.26(d, J=8.3Hz, 2H) Z 2,4 7.08(d, J=8.1HZ, 2H) 7.20(d, J=8.3Hz, 2H) Z 12 2.30(s, 3H) 2.33(s, 3H) 13 1.33(s, 3H) 1.42(s, 3H) reference 196, 360MHz, solvent not reported. Assignments within a column may be interchanged. The short range 2D-HETC0R NMR spectrum correlated the NMR resonances at 3.36(d, J = l l . l H z , IH) and 3.59(d, J = l l . l H z , lH)ppm with the 1 3 C NMR carbinol methylene resonance at 70.6ppm. The 91 Table 9. 13 C NMR data comparison of 165 and 181. Chemical s h i f t , ppm. Carbon # 165 12 13 3 6 7 8 1,5 2,4 126.7 128.6 135.3 142.9 49.8 37.6 20.8 25.0 144.44 40.46 20.97 29.71 reference 196, 90MHz, solvent not given. No chemical s h i f t s reported for "?" (C3 and C7). re s u l t s of two decoupling experiments were consistent with the c o r r e l a t i o n . I r r a d i a t i o n of the signa l at 3.36ppm collapsed the doublet at 3.59ppm to a s i n g l e t and i r r a d i a t i o n at 3.59ppm s i m i l a r l y collapsed the doublet at 3.36ppm to a s i n g l e t . Hence, the resonances at 3.36 and 3.59ppm were assigned to the geminal carbinol protons of a t e r t i a r y acetoxymethyl group. Since the molecular formula of tochuinyl acetate f165) required s ix units of unsaturation and only f i v e could be accounted for by the s t r u c t u r a l fragments so far ascribed (acetate and 4-methylphenyl moieties), i t was assumed that metabolite 165 possessed an additional carbocyclic r i n g . The short range 2D-HETCOR NMR spectrum indicated that the structure had to further accomodate: two additional quaternary t e t r a - a l k y l substituted carbons (47.4(C) and 49.8(C)ppm); three a d d i t i o n a l 92 methylene carbons (20.2(CH 2) f 34.8(CH2) and 37.6(CH2)ppm); and two a d d i t i o n a l methyl carbons (19.SfCH^) and 25.0(CHj)ppm). The three methylene carbons were correlated to the NMR multiplets resonating at 1.58(m, IE), 1.75-1.88(m, 4H) and 2.48(m, lH)ppm. A serie s of decoupling experiments established that these three sets of mu l t i p l e t s were highly spin-coupled. I r r a d i a t i o n of the multiplet at 2.48ppm sharpened the multiplets at 1.58 and 1.75-1.88ppm. S i m i l a r l y i r r a d i a t i o n at 1.58ppm sharpened the multiplets at 1.75-1.88 and 2.48ppm. Two b i o g e n e t i c a l l y sound sesquiterpenes, 165 and 182. accomodated a l l the s t r u c t u r a l fragments thus f a r ascribed to 1 13 tochuinyl acetate. Comparison of the p a r t i a l H and C NMR chemical s h i f t assignments for 1 8 1 1 9 6 with 165 (Tables 8. and 9.), suggested that the correct gross structure was represented by 165. However, more d e f i n i t i v e proof was sought. A gated decoupled 1 3 C NMR spectrum (Figure 16.) (which allows long range couplings to be observed) distinguished between the two structures, 165 and 182. In Figure 16., the carbinol carbon at 70.6ppm appears as a t r i p l e t (large one bond C-H coupling) of quartets (small three bond C-H coupling to the methyl protons resonating at either 1.33 or 1.13ppm). The methyl resonating at 182 93 s 13 J . 15 OAc 165 IB 71 IS 1« » • 1 I ' 1 1 ' -'- 80 60 40 20 PPM 0 160 1 40 1 20 100 Figure 16. Gated decoupled 1 3 C NMR spectrum of tochuinyl acetate (liLS.) . 25.0ppm (correlated to the methyl s i n g l e t at 1.33ppm in H NMR spectrum) appears as a quartet (large one bond coupling) of t r i p l e t s (small three bond coupling). The results were consistent with the gross structure 165 and not 182. Further consistencies were noted. The C12 resonance at 20.8ppm appeared as a quartet of t r i p l e t s , the C17 methyl at 20.9ppra appeared as a straig h t quartet. With the ad d i t i o n a l knowledge that aromatic two (and four) bond C-H coupling i s small r e l a t i v e to three bond ( J C H = 1 -3 198 2Hz, J C H=7-12Hz) the experiment also allowed for the chemical s h i f t assignment of the two sets of aromatic methine carbons. The CI and C5 resonance at 126.7ppm appeared as a doublet (one bond coupling) of doublets (three bond coupling to H5 and Hi res p e c t i v e l y , as they are not magnetically equivalent), and the C2 and C4 resonance at 128.6ppm appeared as a doublet (one bond coupling) of quintets (three bond coupling to C12 methyl protons and the H4 and H2 protons, r e s p e c t i v e l y ) . Structure 165. based on a cuparane sesquiterpene skeleton, e f f e c t i v e l y accounted for a l l the spectral features of tochuinyl 199 acetate. Two SINEPT experiments, optimised for p o l a r i s a t i o n transfer through 7Hz coupling, connected the s t r u c t u r a l fragments of 165 together and i n so doing unambiguously confirmed the proposed s t r u c t u r a l c o n s t i t u t i o n . I r r a d i a t i o n of the methyl resonance at 1.13ppm (Mel5) showed p o l a r i s a t i o n transfer to carbon resonances at 34.8(C10), 47.4(C7 or C l l ) , 49.8(C7 or C l l ) and 70.6(C14)ppm (Figure 17a.). Thus, the f i v e membered ring and the acetate moiety were connected through C l l . I r r a d i a t i o n of the methyl resonance at 1.33ppm (Mel3) showed transfer to carbon 95 Details for each: 187mg llhours 21 2mins 12^3 OAc 165 (a). Irradiated at M 3 p p m (Me15) (b). Irradiated at 133ppm (Me13) | i i i i I i i i i | i i i i I 1 1 1 1 I i i i i ]• i i i 200 I i i i i | i i i i | i i i r j i i r r j • i \ r i | i t 1 " i | r"i • i r -| i • i • 180 160 140 120 i I i i i i | i i i i | i i i i [ i i i i | i i i i | i i i i | i i i 100 80 60 40 20 PPM Figure 17. SINEPT NMR spectra of tochuinyl acetate (UL5.) resonances at 37.6(C8), 47.4(C7 or C l l ) , 49.8(C7 or C l l ) and 142.9(C6)ppm (Figure 17b.). Thus, the f i v e membered ring and the 4-methylphenyl moiety were connected through the C7-C6 bond. The experiment also allowed for further chemical s h i f t assignments. The 1 3 C NMR resonances at 19.5, 25.0, 142.9ppm and hence 135.3ppm could be assigned to the C15, C13, C6 and C3 carbons, respectively, and the resonances at 34.8, 37.6 and hence 20.2ppm to the CIO, C8 and C9 carbons, respectively. A series of dif f e r e n c e NOE experiments established that the r e l a t i v e stereochemistry about the cyclopentane ring was as shown in 165. I r r a d i a t i o n of the methyl resonance at 1.13ppm (Mel5) induced NOE's into the methyl resonance at 1.33ppm (Mel3), the geminal carbinol protons at 3.36 and 3.59ppm (H14 protons) and into the aromatic protons at 7.22ppm (Hi and H5). I r r a d i a t i o n of the methyl resonance at 1.33ppm (Mel3) induced NOE's into the methyl resonance at 1.13ppm (Mel5) and the aromatic resonance at 7.22ppm (HI and H5). The reverse i r r a d i a t i o n s resulted i n the expected NOE's. Inducement of an NOE into the aromatic protons at 7.08ppm (H2 and H4), on i r r a d i a t i o n of the methyl resonating at 2.30ppm (Mel2), provided a further consistency. One small ambiguity needs to be explained. Decoupling experiments indicated that the H8b proton, resonating at 2.48ppm in the *H NMR spectrum of 165. was coupled to the HlOa proton resonating at 1.58ppm. W-coupling can readily account f o r t h i s observation. Molecular models indicate that the conformation required for W-coupling i s quite strained. Since an NOE was observed between the H8b (2.48ppm) and the aromatic HI and H5 protons (7.22ppm), the H8b proton has to possess an ct-97 configuration and hence, the HlOa must also be a to allow for the planar configuration required for W-coupling. The deshielded chemical s h i f t of the H8b proton (2.58ppm) i s consistent with an a-configuration, for i t would be positioned within the deshielding region established by the ring current of the aromatic r i n g . S i m i l a r l y , the Mel3 protons, resonating at 1.33ppm, would to some extent be situated within the same deshielding region. These protons show a much stronger NOE with the aromatic protons at 7.22ppm (HI and H5) than do the methyl protons at 1.13ppm (Mel5), which are situated outside the deshielding region. F u l l ^H and 13 C NMR assignments for 165 are given i n Tables 10. and 11., respectively. Dihydrotochuinyl acetate (166) was also i s o l a t e d as a colourless o i l . I t gave a parent ion at a m/z of 262.1926 daltons in the EIMS appropriate f o r a molecular formula of ci7 H26°2 (required m/z i s 262.1934 daltons) that required 5 units of unsaturation. The s i m i l a r i t y of the MS of 165 and 1 £ £ and a f a c i l e loss of two mass units from the parent ion of 166 suggested that i t was the dihydro derivative of The *H and 1 3 C NMR spectra of 166 (Figures 18. and 19.) were f u l l y consistent with the proposal. Resonances assigned to the 4-methylphenyl fragment i n the H and -LJC NMR spectra of 165 (Tables 10. and 11.), were replaced in the NMR spectra of dihydrotochuinyl acetate f166) with H^ resonances at 1.65(s, 3H), 2.61(m, 2H), 2.70(m, 2H), 5.39(m, IH) and 5.53(bs, lH)ppm, and 1 3 C resonances at 28.4(CH 2), 31.9(CH 2), 119.0(CH), 119.KCH), 130.5(C) and 138.5(C)ppm, which could be assigned to a l - a l k y l - 4 -98 Table 10. H NMR assignments for tochuinyl acetate (165) and dihydrotochuinyl acetate (166). Chemical s h i f t , ppm. Carbon # 1£5_ 1 M 1 7.22(d, J=8.1Hz, IH) 2.70(m, 2 H ) z l 2 7.08(d, J=8.lHz, IH) 5.39(m, l H ) y l 4 7.08(d, J=8.1Hz, IH) 2.61(m, 2 H ) z 2 5 7.22(d, J=8.1Hz, IH) 5.53(bs, l H ) y 2 8a 1.75-1. 88(m) 1.65-1.80(m)x 8b 2.48(m, IH) 2.20(m, IH) 9 1.75-1. 88(m) 1.42-1.55(m)x 1.65-1.80(m)x 10a 1.58(m, IH) 1.42-1.55(m) 10b 1.75-1. 88(m) 1.65-1.80(m)x 12 2.30(s, 3H) 1.65(s, 3H) 13 1.33(s, 3H) 1.07(s, 3H) W 14 3.36(d, J= l l . l H z , IH) 3.74(d, J=11.0Hz, IH) 3.59(d, J= l l . l H z , IH) 3.81(d, J=11.0Hz, IH) 15 1.13(s, 3H) 1.04(s, 3H) W 17 1.94(s, 3H) 2.02(s, 3H) Assignments within a column may be interchanged. Were appropriate interchange as complete pairs according to the second superscript 1 or 2. 99 Table 11. C NMR assignments for tochuinyl acetate (165) and dihydrotochuinyl acetate (166). Chemical s h i f t , ppm. Carbon # 165 166 1 126.7(CH) 28.4 Z 2 128.6(CH) 119.0 y 3 135.3(C) 130.5 X 4 128.6(CH) 31.9 2 5 126.7(CH) 119.l y 6 142.9(C) 138.5 X 7 49.8(C) 2 50.3 W 8 37.6(CH 2) 37.0 V 9 20.2(CH 2) 20.1 10 34.8(CH 2) 35.3 V 11 47.4(C) 2 46.9 W 12 20.8(CH 3) 22.7 U 13 25.0(CH 3) 22.8 U 14 70.6(CH 2) 70.1 15 19.5(CH 3) 19.5 16 171.2(C) 171.4 17 20.9(CH 3) 21.0 assignments within a column may be interchanged. 100 101 o to r OAc 166 P I i i I I | I I I I | I I I i | i I I i | i I I I [ i I i i | I I I i | I l M | M M | l l l I | I I I I | I I I I | I I i I | I I I i | n i i | i m i | i i i i | 160 140 120 100 80 60 40 20 PPM 0 Figure 19. 1 3 C NMR spectrum of dihydrotochuinyl acetate (!££). methyl-1,4-cyclohexadiene residue. Apart from these differences the two sets of spectra were v i r t u a l l y i d e n t i c a l . Comparison of the p a r t i a l -^H and 1 3 C NMR data of 1£6_ with the two models 1£3_ 1 9 6 and 1 8 4 2 0 0 (Tables 12. and 13.) provided further confirmation and allowed for a number of chemical s h i f t assignments. Assignment of the NMR resonances a t t r i b u t e d to the l - a l k y l - 4 - m e t h y l - l f 4 -cyclohexadiene moiety was p a r t i a l l y achieved by a s e r i e s of decoupling experiments. I r r a d i a t i o n of the multiplet at 2.61ppm markedly sharpened the o l e f i n i c resonance at 5.53ppm and weakly sharpened the m u l t i p l e t at 5.39ppm. I r r a d i a t i o n at 2.70ppm markedly sharpened the resonance at 5.39ppm and weakly sharpened the broad s i n g l e t at 5.53ppm. The proposed assignments (see Table 10.) are based on the knowledge that v i c i n y l coupling i s l a r g e r 197 than a l l y l i c coupling i n such spin systems. However, as indicated, the appropriate interchanges are also p o s s i b l e . Consistent with the proposed structure for dihydrotochuinyl acetate f166) were the NMR resonances (Table 10. and 11.) and an IR band (1731cm - 1) appropriate for the acetate f u n c t i o n a l i t y . Decoupling the three sets of multiplets at 1.42-1.55, 1.65-1.80 and 2.20ppm established that they were a l l once again highly spin-coupled. F i n a l l y , dihydrotochuinyl acetate (1£&) was q u a n t i t a t i v e l y converted to tochuinyl acetate (1£5_) with 1 13 palladium on charcoal i n refluxing ethanol. Proposed H and C NMR assignments for 166 are included i n Tables 10. and 11., respectively. 103 183 184 Table 12. H NMR data comparison of 166. 183 and 184. Chemical s h i f t , ppm. Carbon # 166 184 b 1 2.70(m, 2H) Z 2.57(bs) 2.50(m) 2 5.39(m, l H ) y 5.40(bs, 1H) Z 5.34(m) 4 2.61(m, 2H) Z 2.57(bs) 2.50(m) 5 5.53(bs, 1H) Y 5.56(bs, 1H) Z 5.34(m) 8b 2.20(m, 1H) ? 2.19(bq, 1H) 12 1.65(s, 3H) 1.64(s, 3H) 1.63(bs, 3H) 13 1.07(s, 3H) 1.15(s, 3H) 0.98(s, 3H) reference 196, 360MHz, solvent not given. b r e f e r e n c e 200, 100MHz, CC1.. No chemical s h i f t reported for B ? n (H8b). 4 z yassignments within a column may be interchanged. 104 Table 13. C NMR data comparison of 166. 1£2 and ISA.. Chemical s h i f t , ppm. Carbon # 166 l£l a 184 b 1 28.4 Z 26.45 28.6 2 119.0 y 119.19 117.8 3 130.5 X 130.92 130.9 4 31.9 Z 32.22 31.8 5 1 1 9 . l v 119.51 118.8 6 138.5 X 138.47 139.2 7 50.3 52.13 49.5 8 37.0 36.61 ? 11 46.9 159.08 48.8( 12 22.7 W 22.85 22.8 13 22. 8 W 26.71 25.8 d) a r e f e r e n c e 196, 90MHz, solvent not given. ^reference 200, 25MHz, CDC1,. Resonances were not assigned. Not possible to d i s t i n g u i s h between 27.4(t) or 32.8(t)ppm (C8/C9) for n <p n z Assignments within a column may be interchanged. ( i i ) . Pseudopterane diterpenoids. 167~169: Gersemolide (167). obtained as colourless needles, was shown by EIMS to possess a molecular formula of C20 H24°4 ( ° b s e r v e d m/z 328.1686, requires 328.1675) having 9 units of unsaturation. The IR bands at 1699, 1648 and 1762cm"1 and resonances at 208.3, 105 197.6 and 173.7ppm i n the 1 3 C NMR spectrum of 167 (Figure 20.) could be assigned to the carbonyls of two ketones and one ester f u n c t i o n a l i t y , r e s p e c t i v e l y , thereby accounting for a l l four 201 202 oxygen atoms. Eight o l e f i n i c carbon resonances i n the DEPT ' (optimised for p o l a r i s a t i o n transfer through 140Hz coupling) (Figure 21.) and 1 3 C NMR spectra at 112.3(CH 2), 116.1(CH 2), 123.8(CH), 134.2(C), 138.1(C), 146.4(C), 150.0(CH) and 156.2(C)ppm indicated four carbon-carbon double bonds. Subtracting the seven s i t e s of unsaturation required by the carbonyl and o l e f i n i c f u n c t i o n a l i t i e s from the t o t a l of nine i n the molecule, indicated that gersemolide f167) was b i c y c l i c . Proton resonances at 7.12(bs, 1H) and 5.38(m, lH)ppm i n the 1H NMR spectrum (Figure 22.), and 1 3 C NMR resonances at 173.7(C), 150.0(CH), 134.2(C) and 79.0(CH)ppm could be assigned to an a,r-disubstituted a, ^-unsaturated y-lactone by comparison with the reported s p e c t r a l data for the model compounds pseudopterolide (1£5J 2 0 3, k a l l o l i d e s A-C ( l f i l , 1 £ £ and 1£2) 1 5 7 and lophodione f 1 8 8 ) w h i c h a l l contain such a fragment (see Table 14.). The lactone accounts for one of the required rings i n gersemolide f167). The assignment of the two proton resonances at 5.38 and 7.12ppm to the H8 and H9 lactone protons, respectively, was consistent with the r e s u l t s of a decoupling experiment. The H8 resonance (5.38ppm) was markedly sharpened on i r r a d i a t i o n of the o l e f i n i c H9 resonance at 7.12ppm. Two isopropenyl groups were i d e n t i f i e d from the 1 3 C NMR resonances at 112.3(CH2) and 116.1(CH2)ppm and the 1H NMR resonances at 1.64(bs, 3H), 1.73(bs, 3H), 4.76(m, 2H), 5.20(bs, 1H) and 5.47(bs, lH)ppm. Again, 106 107 Figure 21. DEPT NMR spectra of gersemolide (167) Details for each: 3-5mg 3hours 67mins i i i i i i i i i i i i i i i i i i i i i i i i i i ' i i i i ' i 1 1 1 1 i 60 40 20 PPM 0 H--0 0 185 186 HQ 0 187 188 compare the p a r t i a l C NMR resonances of the respective carbons for metabolites 1 8 6 1 5 7 and 1 8 8 1 1 1 with those of 1£I i n Table 14. A decoupling experiment indicated that the two proton m u l t i p l e t at 4.76ppm was coupled to the broad methyl s i n g l e t at 1.64ppm, since the methyl was markedly sharpened on i r r a d i a t i o n at 4.76ppm. Also, the methyl doublet resonating at 1.83(J=1.2Hz)ppm collapsed to a s i n g l e t on i r r a d i a t i o n of the o l e f i n i c quartet (J=1.2Hz) at 6.33ppm. Presumably the methyl resonance at 1.83ppm and the o l e f i n i c resonance at 6.33ppm constituted part of a t r i s u b s t i t u t e d double bond. The two o l e f i n i c protons resonating at 5.20 and 5.47ppm were by inference, therefore, associated with the a l l y l i c methyl protons, resonating at 1.73ppm, of the second isopropenyl group. 110 Table 14. C NMR assignments for gersemolide (167) and 169. and spectral comparison with 186 and 188. Chemical s h i f t , ppm. Chemical s h i f t , ppm. Carbon # 1£2 1 £ £ 186 a Carbon # l B 8 b 1 41.2(CH) 43.5 Z 42.1 1 41.3 2 44.9(CH2) 44.3 2 / 2 45.7 3 208.3(C) 2 203.3 y / 3 205.4 4 156.2(C) 150.0 X / 4 144.8 2 5 123.8(CH) 130.8 X / 5 133.4 6 197.6(C) 2 193.8 y / 6 190.7 7 62.4(CH) 145.3 X 48.8 / 8 79.0(CH) 77.6 81.2 10 80.1 9 150.0(CH) 14 8 . l x 147.1 11 148.4 10 134.2(C) Y 1 3 5 . l x 136.8 12 134.1 11 22.0(CH2) 23.1 V 23.1 13 25.7 12 31.4(CH2) 28.4 34.5 14 30.3 13 146.4(C) Y 147.3 X 148.2 2 15 145.5 Z 14 112.3(CH 2) X 112.7 110.7 y 16 115.9 15 19.8(CH 3) W 21. 9 V 19.5 X 17 17.0 16 21.9(CH 3) W 23.8 V / 18 21.5 17 138.1(C) y 131.4 X 142.7 Z / 18 116.1(CH 2) X 14.2 V 114.5 y / 19 23.1(CH 3) W 18.4 V 21.8 X / 20 173.7(C) 172.4 175.6 20 173.1 cont'd. I l l Table 14. continued. areference 157, 50MHz, CDC13, "/" means no advantage i n comparing, ^reference 111, 20MHz, CDCl^, "/" means no advantage i n comparing, z~ vassignments within a column may be interchanged. The preceeding arguments suggested that gersemolide (167) possessed a pseudopterane carbon skeleton with the 12-membered ring accounting for the remaining s i t e of unsaturation. At t h i s stage of the project only a small quantity of gersemolide (167) had been is o l a t e d (<4.0mg) and the spec t r a l information obtained thus far did not allow for an unambiguous s t r u c t u r a l proof, or for the determination of r e l a t i v e stereochemistry. With some e f f o r t reasonable c r y s t a l s of gersemolide (167) were obtained by slow evaporation from 1:1 d i e t h y l ether/methanol at 0°C. The complete structure of gersemolide (167) was secured v i a s i n g l e c r y s t a l x-ray d i f f r a c t i o n a n a l y s i s , performed by G. D. Van Duyne and J . Clardy of Cornell U n i v e r s i t y . A computer generated perspective drawing of the f i n a l x-ray model of gersemolide (167) i s represented i n Figure 23a. I t should be noted that the x-ray experiment did not define the absolute configuration, so the enantiomer shown i s a r b i t r a r y . While the structure in Figure 23a., was completely compatible with the spectral data, Van Duyne and Clardy indicated that there * were some troubling aspects i n the molecular geometry . The C10-C11-C12-C1 region of the x-ray model was unacceptable. The C l l -C12 distance was 1.417A, much too short for a sp -sp bond, and 112 C10-C11-C12-C1 C10-C11-C12-C1 TORSION OF -37* TORSION OF +43* CM. (c). Figure 23. Computer generated perspective drawings of gersemolide f167). 113 the C10-C11-C12-C1 t o r s i o n a l angle was -8°, an e s s e n t i a l l y eclipsed conformation. This type of s i t u a t i o n i s not unprecedented and i s usually explained by assuming a disordered structure. There were presumably at l e a s t two low energy conformations which occur i n the c r y s t a l , and the r e s u l t i n g x-ray structure was t h e i r superposition. Van Duyne and Clardy c a r e f u l l y inspected various electron density syntheses, t h i s did not reveal discrete peaks fo r the disordered mates, but did reveal that the electron d e n s i t i e s of CIO and C l l were rather broad. Since the x-ray analysis did not resolve the problem, they used molecular mechanics to f i n d the suspected minima. Two l o c a l minima on the three dimensional surface were found by varying the C10-C11-C12-C l t o r s i o n a l angle. The f i r s t had a t o r s i o n a l angle of -37° (Figure 23b.) and the second, +43° (Figure 2 3 c ) . The region around 0° represented a broad maximum. The calculated heats of formation for the minima d i f f e r e d by only 0.5kcal/mole, and hence, the x-ray geometry (Figure 23a.) was, as expected, the superposition of the two minima. Neither conformation allows for both the C3 and C6 ketones to be conjugated with the C4-C5 t r i s u b s t i t u t e d o l e f i n . Figures 23b. and c , indicate that the C6 ketone i s almost co-planar with the C4-C5 bond, which i s i n 1 3 agreement with the C NMR resonances at 208.3 (non-conjugated) and 197.6ppm (somewhat conjugated) for C3 and C6, r e s p e c t i v e l y . A l l x-ray analysis and associated work kindly performed and interpreted by G. D. Van Duyne and J . Clardy, without whom I would be at a complete loss here. Hence, only interpreted r e s u l t s are reported. For supplementary material concerning t h i s analysis see reference 190d. 114 Comparison of the *H NMR chemical s h i f t s of 167 with those of the model compounds l f i ^ - l M 1 1 1 ' 1 5 7 ' 2 0 3 and the diterpenes 3 68-177 (to be discussed in the following pages), combined with a s e r i e s of decoupling experiments allowed for a complete assignment of *H NMR resonances. I r r a d i a t i o n of the m u l t i p l e t at 2.26ppm (HI) s i m p l i f i e d the resonances at 2.38(dd, J=15.1,9.5Hzf H2a) and 2.53(dd, J=15.1,4.5Hz, H2b)ppm to two doublets (J=15.lHz). The HI resonance was also observed to couple with the rather shielded two proton multiplet resonating at 1.28ppm. One or both of the H12 protons (or equivalent protons (H14) i n cembrane metabolites) generally l i e i n the u p f i e l d region from 0.4 to 1 . 3 p p m i n ' 1 5 7 , 1 9 0 d ' 2 0 3 . This presumably results from anisotropic s h i e l d i n g by either the nearby isopropenyl moiety or the lactone carbonyl, or by a combination of the two e f f e c t s . Molecular models r e a d i l y demonstrate that either e f f e c t i s f e a s i b l e . These arguments are consistent with the decoupling re s u l t s and hence the H12 protons were assigned to the resonance at 1.28ppm. The H12 protons were subsequently shown to couple to a multiplet resonating at 2.01ppm (Hlla) and one i n the region 2.32-2.45ppm (Hllb) l y i n g underneath the H2 doublet of doublets at 2.38ppm. I r r a d i a t i o n at 2.0lppm (Hlla) markedly affected the geminal partner resonating i n the region 2.32-2.45ppm ( H l l b ) . These assignments were confirmed by the observation that i r r a d i a t i o n of the lactone o l e f i n i c resonance at 7.12ppm (H9) weakly sharpened the region 2.32-2.45ppm, which i s consistent with a l l y l i c coupling between the H9 and the Hllb protons. Proposed *H NMR assignments for 167 are included i n Table 15. 1 3 Proposed C NMR assignments, based on comparison to model 115 Table 15. H NMR assignments and spectral comparison for gersemolide f167). 168 and 169. Chemical s h i f t , ppm. Carbon 1 2a b 5 7 8 9 11a b 12 14a b 15 16 18 19 1£1 2.26(m, 1H) 2.38(dd, J=15.1,9.5Hz, 1H) 2.53(dd f J=15.1,4.5Hz, 1H) 6.33(q, J=1.2Hz, 1H) 3.29(d, J=0.6Hz, 1H) 5.38(m, 1H) 7.12(bs f 1H) 2.01(m, 1H) 2.32-2.45(m, 1H) 1 1.28(m, 2H) 5.20(bs, 1H) 5.47(bs f 1H) 1.73(bs, 3H) z l z l y i 1.83(d, J=1.2Hz, 3H) 4.76(mf 2 H ) z 2 1.64(bs, 3H) y2 1LS. 1.95-2.06(m ,1H) Z 2.31-2.49(m)z 2.72(dd, J=13.6,3.8Hzr 1H) 6.11(q, 1.6Hz, 1H) / 5.93(bs f 1H) 7.10(bs f 1H) 2.31-2.49(m)Z 1.18(mr 1H) 1.26(mf 1H) 4.94(m, 1H) 5.07(bs f 1H) 1.73(bs, 3H) 2.14(d, J=1.6Hz, 3H) 1.85(s r 3H) 1.93(s, 3H) cont'd. compounds, p a r t i c u l a r l y k a l l o l i d e B (186) , lophodione (JJL2.) and the diterpenes 168-177. to be discussed i n the following pages, are included i n Table 14. A 4' 5Z, 2 ' 1 7-Isogersemolide (168). a very minor metabolite 116 Table 15. continued. Chemical s h i f t , ppm. Carbon # 169 1 2.33(m, IH) 2a 2.49-2.57(m) b 2.71(dd, J=14.0,5.7Hz, IH) 5 6.21(q, J=1.2Hzr IH) 7 / 8 5.92(d, J=0.6Hz, IH) 9 7.02(t r J=0.6Hz, IH) 11a 2.17(tdm, J=12.2,1.5Hz, IH) b 2.49-2.57(m) 12 1.26-1.41(m, 2H) 14a 4.76(bs, IH) b 4.86(q, J=0.9Hz, IH) 15 1.72(bs, 3H) 16 2.08(d, J=1.2Hz, 3H) 18 2.01(s, 3H) 19 2.06(s f 3H) ' yassignments within a column may be interchanged. Were appropriate interchange as complete groups according to the second superscript 1 or 2. 1 l i e s underneath the H2 resonance at 2.38ppm. (<1.5mg) obtained as clear c r y s t a l l i n e needles, was shown by EIMS to possess a molecular formula of C2o H24°4 ( o b s e r v e d m/z 328.1684, requires 328.1675), i d e n t i c a l to that of gersemolide f167) . A number of fragments were t e n t a t i v e l y i d e n t i f i e d by the 117 combination of the r e s u l t s of a serie s of NOE experiments and spe c t r a l comparison of the *H NMR resonances of 168 (Figure 24.) and gersemolide (167) (see Table 15.). Induction of an NOE into the o l e f i n i c proton at 6.11(q, J=1.6Hz, lH)ppm upon i r r a d i a t i o n of the methyl doublet (J=1.6Hz) at 2.14ppmf i d e n t i f i e d a methyl substituted ene-dione, i d e n t i c a l with the corresponding substructure of gersemolide (167). I r r a d i a t i o n of the methyl resonance at 1.73(bs, 3H)ppm induced NOE's into the o l e f i n i c resonance at 4.94(m, lH)ppm and part of the mu l t i p l e t region centred at 2.44ppm. Comparison with gersemolide (167) allowed for the tentative i d e n t i f i c a t i o n of an isopropenyl residue. Two methyl s i n g l e t s resonating at 1.85 and 1.93ppm and the two u p f i e l d one proton m u l t i p l e t s at 1.18 and 1.26ppmf t y p i c a l of H12 protons situated i n a pseudopterane skeleton, were also observed. The pseudopterane structure 168 e f f e c t i v e l y accomodates a l l the i d e n t i f i e d s t r u c t u r a l fragments of A 4' 5Z,A 7' 1 7-isogersemolide. A s e r i e s of NOE experiments v e r i f i e d that 168 represented the correct s t r u c t u r e . Simultaneous i r r a d i a t i o n of the Mel9 resonance (1.93ppm) and the HI mu l t i p l e t (1.95-2.06ppm) induced NOE's into the doubly a l l y l i c lactone carbinol resonance at 5.93(bs f lH)ppm (H8, from Mel9), the H9 o l e f i n i c resonance at 7.10ppm (from Mel9), the methyl resonance at 1.85ppm (Mel8, from Mel9), the H12 mul t i p l e t at 1.18ppm (from Hi) and the Hl4b resonance at 5.07(bs, lH)ppm (from HI). I r r a d i a t i o n of the methyl (Mel8) resonance at 1.85ppm induced a small NOE into the o l e f i n i c proton at 6.12ppm (H5). Thus, the Mel8 and Mel9 methyls had to be located i n the v i c i n i t y of both the methyl substituted 118 ene-dione and lactone moieties, i . e . , at the C17 p o s i t i o n . I r r a d i a t i o n at 2.14ppm (Mel6) not only induced an NOE into the H5 proton (6.11ppm), but also into the resonances at 2.72ppm (H2b) and 5.07ppm (H14b). This set of experiments demonstrated the expected c o n n e c t i v i t i e s about the 12-membered r i n g . F i n a l l y , gersemolide (167) was p a r t i a l l y isomerised to 168 i n the presence of formic a c i d . The gentle ref l u x i n g of reactants yielded a 30:3:20 mixture of 167. 168 and one other u n i d e n t i f i e d compound 4 5 (presumably a A ' E isomer of gersemolide), respectively, as evidenced by 300MHz *H NMR a n a l y s i s . Proposed ^H NMR assignments for A 4 ' 5 Z , A 7 , 1 7 - i s o g e r s e m o l i d e (168) are included i n Table 15. A 4 , 5 E , A 7 , 1 7 - l s o g e r s e m o l i d e (169). obtained as colourless needles, was shown by EIMS to possess a molecular formula of C20 H24°4 ( ° b s e r v e d m / z r 328.1669, requires 328.1675), i d e n t i c a l to that of both gersemolide (167) and A 4 ' 5 Z , A 7 , 1 7 - i s o g e r s e m o l i d e (168). A number of fragments could r e a d i l y be i d e n t i f i e d from the XH and 1 3 C NMR spectra of A 4 , 5 E , A 7' 1 7-isogersemolide (1£9_) (Figures 25. and 26.). These included: a methyl substituted ene-dione ( 1 3C NMR: 203.3, 150.0, 130.8, 193.8 and 23.8ppm; XH NMR: 2.08(d, J=1.2Hz, 3H) and 6.21(q, J=1.2Hz, lH)ppm); o , y -13 disubstituted os,^-unsaturated r-lactone ( C NMR: 77.6, 148.1, 135.1 and 172.4ppm; XH NMR: 7.02(t, J=0.6Hz, 1H) and 5.92(d, J=0.6Hz, lH)ppm); and isopropenyl ( 1 3C NMR: 147.3, 112.7 and 21.9ppm; XH NMR: 1.72(bs, 3H), 4.76(bs, 1H) and 4.86(q, J=0.9Hz, lH)ppm) residues. The NMR resonances for each of these residues were v i r t u a l l y i d e n t i c a l with those assigned to the corresponding substructures i n e i t h e r gersemolide (1£1) and/or A 4 ' 5 Z , A 7 ' 1 7 -isogersemolide (168) (see Tables 14. and 15.). A marked 120 121 122 s i m i l a r i t y i n the H NMR spectra of 169 and 16J1, p a r t i c u l a r l y i n the methyl s i n g l e t s resonating at 2.01 and 2.06ppm, was noted. These methyl resonances were comparable to those assigned to the Mel8 and Mel9 protons of 168. which resonated at 1.85 and 1.93ppm, r e s p e c t i v e l y . The above arguments, hence suggested that 168 and 169 had the same c o n s t i t u t i o n but d i f f e r e d i n some stereochemical sense. The geometrical isomer of A 4 f ^ Z , A 7 , l 7 - i s o g e r s e m o l i d e (16J1) possessing the pseudopteranoid structure 169 e f f e c t i v e l y accomodates a l l the i d e n t i f i e d s t r u c t u r a l fragments. A s e r i e s of NOE and spin-spin decoupling experiments v e r i f i e d that 169 represented the correct structure. I r r a d i a t i o n of the methine t r i p l e t (J=0.6Hz) at 7.02ppm (H9) collapsed the doublet (J=0.6Hz) at 5.92ppm (H8) to a s i n g l e t and sharpened the methylene H l l resonance at 2.17(tdm, J=12.2,1.5Hz, lH)ppm. I r r a d i a t i o n of the H8 carbinol doublet at 5.92ppm s i m p l i f i e d the H9 t r i p l e t at 7.02ppm to a doublet (J=0.6Hz). Hence, H8 through to H l l were connected v i a s c a l a r coupling. I r r a d i a t i o n of the o l e f i n i c H5 resonance at 6.21(q, J=1.2Hz, lH)ppm collapsed the methyl doublet (J=1.2Hz) at 2.08ppm (Mel6) to a s i n g l e t and i r r a d i a t i o n of both the H14 protons, resonating at 4.76 and 4.86ppm, sharpened the methyl s i n g l e t at 1.72ppm (Mel5). Also an NOE was observed between Mel5 and the Hl4b resonance at 4.86ppm. The stereochemical arrangement about the C4-C5 double bond was proposed on the basis of s p e c t r a l evidence. Unlike A 4 f 5Z ,^ 7'" 1 7-isogersemolide (168) no NOE could be demonstrated between the H5 and Mel6 protons, and although t h i s does not prove the stereochemical assignment i t was at l e a s t consistent with 123 proposed structure. Secondly, the H5, Mel6 proton coupling constant i n 168 was 1.6Hz while in 169 i t was 1.2Hz. This i s consistent with the general observation that a l l y l i c c i s coupling 197 i s larger than a l l y l i c trans coupling . To confirm the proposed structure A 4 , 5 E , A 7'"^-isogersemolide (169) was treated with a c a t a l y t i c amount of iodine i n benzene. *H NMR analysis showed that a 1:3 mixture of 168 and 169. res p e c t i v e l y , had res u l t e d . To allow for a complete assignment of the NMR resonances exhibited by 169. a second seri e s of decoupling experiments were performed. I r r a d i a t i o n of the H l l a methylene resonance at 2.17ppm markedly sharpened the multiplet regions resonating between 2.49-2.57 (consisting of H2a and Hllb) and 1.26-1.41ppm (Hl2a and Hl2b). I r r a d i a t i o n of the Hl2a, Hl2b two proton m u l t i p l e t at 1.26-1.41ppm sharpened the HI multiplet at 2.33ppm, s i m p l i f i e d the H l l a at 2.17(tdm, J=12.2,1.5Hz)ppm to a doublet of m u l t i p l e t s (J=12.2Hz) and sharpened the Hllb m u l t i p l e t at 2.49-2.57(m)ppm. Ir r a d i a t i o n of the Hi mul t i p l e t at 2.33ppm sharpened the Hl2a, H12b two proton m u l t i p l e t (1.26-1.41ppm) and the mu l t i p l e t region 2.49-2.57ppm (H2a and H l l b ) , and reduced the H2b doublet of doublets (J=14.0,5.7Hz) at 2.71ppm to a doublet (J=14.0Hz). F i n a l l y , i r r a d i a t i o n of the H2b resonance at 2.71ppm affected the geminal H2a resonating i n the multiplet region 2.49-2.57ppm and s i m p l i f i e d the HI mu l t i p l e t at 2.33ppm to an apparent broad t r i p l e t ( J - 8 . 2 H Z ) . Hence, a l l the proton resonances were assigned (see Table 15.). 124 ( i i i ) . Cembrane diterpenoids. 120.-125. Rubifolide f170). obtained as a white c r y s t a l l i n e s o l i d , was shown by EIMS to possess a molecular formula of C 2 o H24°3 (observed m/z 312.1275, requires 312.1276), requiring 9 units of unsaturation. A number of fragments could r e a d i l y be i d e n t i f i e d from the H and X J C NMR spectra (Figures 27, and 28.). The f i r s t 13 fragment, a 2,5-dialkyl-3-methylfuran, was indicated by the C NMR resonances at 9.5(q) 149.4(s), 117.l(s), 113.8(d) and 149.9(s)ppm (carbon m u l t i p l i c i t i e s deduced from a f u l l y coupled 13 C NMR spectrum) which were almost i d e n t i c a l i n chemical s h i f t with the resonances assigned to the corresponding fragment i n k a l l o l i d e B ( 1 £ £ ) 1 5 7 (Table 16.). In the model compound 3 8 6 1 5 7 the /3-proton and the methyl substituent were assigned *H NMR resonances at 5.85(s, IH) and 1.96(s, 3H)ppm, respect i v e l y . I r r a d i a t i o n of the o l e f i n i c resonance at 5.99(bs, lH)ppm sharpened the methyl s i n g l e t at 1.92(bs, 3H)ppm. Thus these resonances were assigned to the equivalent protons i n the methylfuran fragment of 170. The fragment accounted for three units of unsaturation. Carbon resonances at 78.7(d), 152.1(d), 132.8(s) and 174.5(s)ppm, and a NMR resonance at 6.86(bs, lH)ppm, i d e n t i f i e d the second fragment as an «i?'-disubstituted a,f3-unsaturated y-lactone i d e n t i c a l with the corresponding 13 substructure of gersemolide f167). The appropriate C NMR comparisons are given i n Tables 14. and 17. The *H NMR resonance at 6.86ppm and the IR band at 1753cm - 1 were, as expected, t y p i c a l of such a s y s t e m 1 1 1 ' 1 ^ 7 ' 1 9 7 ' 2 0 3 . The lactone moiety accounted for 125 126 Table 16. C NMR data comparison of 170 and 186. Chemical S h i f t , ppm. Chemical s h i f t , ppm. Carbon # Carbon # 3 86a 3 4 5 6 18 149.4 117.1 113.8 149.9 9.5 3 4 5 6 16 149.6 Z 116.4 112.4 150.0 Z 9.7 reference 157, 50MHz, CDC1 3. Assignments within a column may be interchanged. a further 3 units of unsaturation. The t h i r d s t r u c t u r a l fragment, an isoproprenyl residue, was re a d i l y apparent from the 1 3 C NMR resonances at 145.4(s), 112.9(t) and 19.2(q), and ''"H NMR resonances, t y p i c a l of such a s y s t e m 1 1 1 ' 1 5 7 ' 1 9 7 ' 2 0 3 , at 4.88(bs, 1H), 4.90(sharp m, 1H) and 1.74(bs, 3H)ppm (see Tables 14. and 17., 15. and 18., f o r spect r a l comparisons). The fragment was confirmed by two observations. F i r s t , on i r r a d i a t i n g the 1H NMR resonance at 4.90ppm the methyl s i g n a l at 1.74ppm sharpened and second, an NOE was demonstrated between t h i s o l e f i n i c proton and the methyl. F i n a l l y , a fourth s t r u c t u r a l fragment, a t r i s u b s t i t u t e d 13 o l e f i n bearing a methyl, was evidenced by the C NMR resonances at 117.4(d), 127.0(s) and 25.7(q)ppm, and 1H NMR resonances at 6.07(bs, 1H) and 1.98(bs, 3H)ppm. The preceeding arguments indicated that r u b i f o l i d e f170) 128 Table 17. C NMR assignments for r u b i f o l i d e (170) , epilophodione (171) , isoepilophodiones A (122) and C (121) and r u b i f o l (125.) , and s p e c t r a l comparison with 188 and 189. Chemical s h i f t , ppm. Carbon # 120. 121 122 121 1 43.3(d) 41.8(CH) 41.5 2 35.7 2 2 31.2(t) 45.5(CH2) 42.8 Z 41.7 Z 3 149.4(s) 205.5(C) 202.6 202.0 4 117.l(s) 143.9(C) 2 149.8 y 154.3 y 5 113.8(d) 133.5(CH) 132.3 128.3 6 149.9(s) 192.1(C) 194.6 196.9 7 117.4(d) 127.KCH) 130.2 48.3 2 8 127.0(s) 151.8(C) 140.0 y 143.7 y 9 39.5(t) 43.6(CH2) 38.9 2 41.6 Z 10 78.7(d) 78.4(CH) 78.1 80.0 11 152.1(d) 148.1(CB) 147.8 148.0 12 132.8(s) 136.2(C) 134.1 132.9 13 20.0(t) 21.0(CH2) 22.1 X 21.2 X 14 30.5(t) 33.5(CH 2) 29.0 27.6 15 145.4(s) 145.4(C) 2 144.9 y 145.8 y 16 112.9(t) 115.6(CH2) 113.2 113.0 17 19.2(q) 17.2(CH3) 14.3 18.1 18 9.5(q) 21.1(CH 3) y 18.3 X 27.3 X 19 25.7(g) 22.3(CH 3) y 23.4 X 125.3 20 174.5(s) 173.6(C) 172.2 173.9 cont'd. 129 Table 17. continued. Chemical s h i f t , ppm. Carbon # 125. 188 3 189 a 1 40.7(CH) 41.3 40.5 2 39.3(CH 2) Z 45.7 44.8 3 217.3(C) y 205.4 208.3 4 155.2(C) X 144.8 Z 154.1 5 126.3(CH) 133.4 126.5 6 202.9(C) Y 190.7 189.0 7 50.2(CH 2) Z 125.9 128.0 8 80.7(C) 156.3 146.4 9 37.6(CH 2) Z 43.5 37.3 10 78.3(CH) 80.1 77.9 11 147.2(CH) 148.4 148.6 12 132.3(C) 134.1 133.1 13 21.3(CH 2) 25.7 22.2 14 26.6(CH 2) 30.3 31.2 15 145.2(C) X 145.5 Z 146.4 16 112.9(CH2) 115.9 114.0 17 18.2(CH 3) 17.0 19.1 18 26.7(CH 3) W 21.5 22.2 19 24.9(CH 3) W 22.6 26.9 20 173.6 173.1 173.1 a r e f e r e n c e 111, 20MHz, CDC1 3. Hydrogen attachments 3were determined using a s i n g l e frequency o f f resonance C NMR spectrum. z _ wassignments within a column may be interchanged. 130 Table 18. H NMR assignments for r u b i f o l i d e (170) . epilophodione (121), isoepilophodiones A (122), B (173) and C (174) and r u b i f o l (175), and s p e c t r a l comparison with 188 and 189. Chemical s h i f t , ppm. Carbon # 1 2a b 5 7 9a b 10 11 13a b 14a b 16a b 17 18 19 120. 121 2.36(td, J=12.6f4.2Hz, 1H) 2.20-2.47(m) 2.55(m, 2H) 5.99(bs, 1H) 6.07(bs, 1H) 2.68(dd, J=11.4,4.2Hz, 1H) 3.22(t, J=11.4Hz, 1H) 4.95(dm, J=11.4Hz, 1H) 6.86(bs, 1H) 2.08(dm, J=14.0Hz, 1H) 2.43(td, J=14.0,2.7Hz, 1H) 1.18(tq, J=14.0,2.7Hz, 1H) 1.64(m, 1H) 4.88(bs, 1H) 4.90(m, 1H) 1.74(bs, 3H) 1.92(bs, 3H) 1.98(br, 3H) 2.51(m, 2H) 6.38(q, J=1.6Hz, 1H) 6.12(bs, 1H) 2.67(dd, J=13.3,4.5Hz, 1H) 2.82(dd, J=13.3,4.8Hz, 1H) 5.27(m, 1H) 7.15(bd, J=0.9Hz, 1H) 2.20-2.47(m) 1.38(m, 1H) 1.86(m, 1H) 4.92(m, 2H) 1.61(bs, 3H) 1.91(d, J=1.6Hz, 3H) 2.19(d, J=1.2Hz, 3H) cont'd. possessed f i v e carbon-carbon double bonds, a lactone carbonyl and the two rings, the furan and lactone. Thus, 170 had to possess one further c a r b o c y c l i c ring to account for the t o t a l of nine 131 Table 18. continued. Chemical s h i f t , ppm. Carbon # 112 173' 1 2.33-2.45(m)z 2.20-2.62(b) 2 2a 2.50(m, 2H) Z 2.20-2.62(b) 2 b 2.68-2.92(b) 5 6.68(q, J=1.2Hz, 1H) 6.21(bm, 1H) 7 6.07(bs, 1H) 6.25(bs, 1H) 9a 2.98(ddm, J=13.5 ,5.1Hz, 1H) 2.68-2.92(b) b 3.04(ddm, J=13.5 ,4.5Hz, 1H) 4.03(b, 1H) 10 5.23(bm, 1H) 5.27(b, 1H) 11 7.19(bs, 1H) 7.16(b, 1H) 13a b 2.33-2.45(m)z 2.2g-2.62(b) z 14a b 2.06(m, 1H) Z ? 1.41(b, 1H) 16a 4.54(bs, 1H) 4.64(b, 1H) b 4.76(q, J=0.6Hz, 1H) 4.83(bm, 1H) 17 1.66(bs, 3H) 1.70(bs, 3H) 18 2.16(d, J=1.2Hz, 3H) 1.88(bs, 3H) 19 1.99(bs, 3H) 2.06(ra, 3H) cont'd. units of unsaturation. Further to t h i s , the ring had to accomodate the remaining unassigned a l i p h a t i c methine and four a l i p h a t i c methylene carbons ( 1 3C NMR: 43.3(d), 20.0(t), 30.5(t), 31.2(t) and 39.5(t)ppm). The cembranoid structure 170 e f f e c t i v e l y accomodates a l l the i d e n t i f i e d s t r u c t u r a l fragments of r u b i f o l i d e . 132 Table 18. continued. Chemical s h i f t , ppm. Carbon # 174 175 1 1.85-2.06(m)z 3.34(bd, J=10.7Hz, IH) 2a 2.20(m, 2H) Z 2.09-2.19(m) b 2.21-2.37(m) u p f i e l d part 5 6.29(q, J=1.2Hz, IH) 6.31(bs, IH) 7a 3.08(d, J=15.0Hz, IH) 2.47(d, J=17.3Hz, IH) b 3.73(d, J=15.0Hz, IH) 3.37(d, J=17.3Hz, IH) 9a 2.14(m, IH) 2.59(dd, J=10.8,3.9Hz, IH) b 3.05(dd, J=ll.l,1.8Hz, IH) 3.91(bt, J=10.8Hz, IH) 10 5.06(bm, IH) 4.83(m, IH) 11 6.79(d, J=1.8Hz, IH) 6.64(d, J=0.6Hz, IH) 13a 1.85-2.06(m)z 2.09-2.19(m) b 2.21-2.37(m) downfield part 14a 1.70-1.82(m, 2H) Z 1.67-1.77(m, IH) b 1.82(bt, J=10.7Hz, IH) 16a 4.69(bs, IH) 4.82(bs, IH) b 4.81(m, IH) 4.84(bs, IH) 17 1.61(bs, 3H) 1.70(bs, 3H) 18 1.95(d, J=1.2Hz, 3H) 2.06(bs, 3H) 19 5.90(d, J=0.9Hz, IH) 1.20(s, 3H) 6.08(s, IH) cont'd. NOE and spin-spin decoupling experiments v e r i f i e d that 170 represented the correct s t r u c t u r e . I r r a d i a t i o n of the methyl resonance at 1.98ppm (Mel9) i n the 1H NMR spectrum of r u b i f o l i d e 1110) induced p o s i t i v e NOE's into an o l e f i n i c proton at 6.07ppm (H7), a methylene proton at 2.68ppm (H9a), the H10 carb i n o l 133 Table 18. continued. Chemical s h i f t , ppm. Carbon # l££b 189 b 1 / / 2 2.40(m, 1H) / 2.64(bd, J=14.0Hz, 1H) 5 6.42(bs, 1H) 6.15(bs, 1H) 7 6.12(bs, 1H) 6.26(bs, 1H) 9a 2.61(bd, J=13.3Hz, 1H) 2.41(m, 1H) b 3.04(dd, J=13.3,4.6Hz, 1H) 2.67(m, 1H) 10 5.31(m, 1H) 4.99(m, 1H) 11 6.97(bs, 1H) 7.00(bs, 1H) 13a / / b / / 14a / / b / / 16a 4.72(bs, 1H) 4.70(bs, 1H) b 4.97(bs, 1H) 4.90(bs, 1H) 17 1.60(bs, 3H) 1.68(bs, 3H) 18 1.84(bs, 3H) 1.89(bs, 3H) 19 2.19(bs, 3H) 2.04(bs, 3H) a w i t h i n t h i s column "b" means very broad signals with no f i n e structure. breference 111, 220 and 360MHz, CDC1,. No chemical s h i f t s reported for "/" (HI, H13a and b, and H14a and b). zassignments within a column may be interchanged. "?"could not assign from data a v a i l a b l e . 134 proton at 4.95ppm, and induced a weak negative NOE into a methylene proton at 3.22ppm (H9b). The lactone methine proton at 4.95ppm (H10) showed v i c i n a l coupling to the methylene protons H9a and H9b (2.68 and 3.22ppm, respectively) and to the o l e f i n i c H l l (6.86ppm) and i t showed homoallylic coupling to the H13a methylene proton at 2.08ppm. I r r a d i a t i o n of the furan methyl resonance at 1.92ppm (Mel8) induced NOE's into an o l e f i n i c proton at 5.99ppm (H5) and into the H2 methylene protons at 2.55ppm. S i m i l a r l y , i r r a d i a t i o n of the isopropenyl methyl resonance at 1.74ppm (Mel7) also induced an NOE into the H2 protons (2.55ppm), and with t h i s the Mel7 and Mel8 were connected through space. Hence, connectivity was established from the isopropenyl group to H5 and from H7 to H13a. The chemical s h i f t assignments, thus f a r , are summarised i n Table 18. A second set of decoupling experiments extended the connectivity to include a l l protons from H7 to HI. The r e s u l t s are summarised i n Table 18. However, due to the overlap of resonance signals i n the region from 2.32 to 2.60ppm the chemical s h i f t assignments, coupling constants and co n n e c t i v i t i e s for Hi, H2a, H2b and Hl3b were confused. A lanthanide s h i f t experiment with decoupling resolved the problem and allowed a l l protons, but the H2 methylene protons, to be viewed as f i r s t order i n the 400MHz •'"H NMR spectrum (Figure 29.). The re s u l t s of the addition of Eu(FOD) 3 are shown i n Figures 30a-c. It i s seen that the region from 2.32 to 2.46ppm, i n Figure 29., can be viewed as two p a i r s of t r i p l e t of doublets resonating at 2.36(td> J=12.6,4.2Hz, IH) and 2.43(td, J=14.0,2.7Hz, lH)ppm, and the resonance at 135 136 Figure 30. 400MHz lanthanide shifted XH NMR spectra of r u b i f o l i d e (170). 2.55ppm as a second order two proton m u l t i p l e t . A f i n a l s e r i e s of decoupling experiments allowed for the unambiguous assignment of resonances and extended the scalar coupling connectivity to include the H2 protons. I r r a d i a t i o n of the broad t r i p l e t , i n Figure 30c, resonating at 3.93ppm (H13b) s i m p l i f i e d the broad t r i p l e t at 1.69ppm (H14a) to a doublet, affected the resonance l y i n g underneath the two methyl resonances between 2.07 to 2.13ppm (Hl4b) and dramatically sharpened the H13a resonance presumably centered at 2.76ppm. I r r a d i a t i o n of the Hi m u l t i p l e t (3.32ppm) affected the H14b resonance (2.07-2.13ppm), markedly sharpened the H2a, H2b multiplet at 2.78ppm and weakly sharpened 3+ the H14a resonance (1.69ppm). Plots of Eu volume (ul) against observed chemical s h i f t resulted i n a se r i e s of s t r a i g h t l i n e p l o t s . The plots that corresponded to the resonances at 2.76ppm (Hl3a), 3.32ppm (Hi), 3.93ppm (Hl3b) and 2.78ppm (H2a, H2b), i n Figure 30c, crossed the chemical s h i f t axis at 2.075, 2.340, 2.390 and 2.545ppm, res p e c t i v e l y . Although each p l o t was based on only three data points the r e s u l t s allowed for the assignments given i n Table 18. A summary of the coupling constant data for 170 i s given i n Table 19. The spin-spin decoupling experiments demonstrated that H14a and Hi zero coupled (or had a very small coupling i n the s h i f t e d spectra). A dihedral angle of 90° (required for zero coupling) was r e a d i l y obtainable i n molecular models for either of the H14 protons i n a manner that allowed for a l l the s t r u c t u r a l r e s t r a i n t s imposed by the NOE r e s u l t s . The model and NOE r e s u l t s demonstrated that the resonances at 3.22(t, J=11.4Hz, H9b) and 140 Table 19. Coupling constant data for r u b i f o l i d e (170). Observed Proton # m u l t i p l i c i t y Jx,y Magnitude, Hz HI td J l , 2 a 12.6 J l , 2 b 4.2 J l r 1 4 a 0.0 Jl,14b 12.6 H2 m J l , 2 4.2 and 12.6 J2,2 ? H5 bs J5,18 sharpened H7 bs J7,9b sharpened J7,19 0.8 H9a dd J9a,9b 11.4 J9a,10 4.2 H9b t J7,9b sharpened J9a f9b 11.4 J9b,10 11.4 H10 dm J9a /10 4.2 J9b,10 11.4 J 1 0 f H 1.4 J10 r13a 2.0 H l l bs J 1 0 , l l 1.4 J l l , 1 3 a 1.0 H13a dm J10,13a 2.0 J l l , 1 3 a 1.0 cont'd. 141 Table 19. continued. Observed Proton # m u l t i p l i c i t y J v Magnitude, Hz J13a,13b 14.0 J13a,14a 2.7 J13a,14b 2.7 H13b td J13a,13b 14.0 J13b,14a 14.0 J13b,14b 2.7 H14a td J13a,14a 2.7 J13b,14a 14.0 J14a,14b 14.0 J l , 1 4 a 0.0 H14b m J13a,14b 2.7 J13b,14b 2.7 J14a,14b 14.0 Jl,14b 12.6 -means not observed and/or unable to deduce. sharpened-on i r r a d i a t i n g e i t h e r of the appropriate protons the other proton was sharpened, but an actual J value could not be assigned. The J's are probably, therefore <0.5Hz. 2.68(dd, J=11.4,4.2Hz, H9a)ppm were to be assigned to the H9/? and H9a protons, r e s p e c t i v e l y . The H10 and H9a (H9a) would e f f e c t i v e l y display an a x i a l - e q u a t o r i a l array (with an approximate dihedral angle of 50°), with the observed coupling constant of 4.2Hz, and the H10 and H9/3 (H9b) protons an a x i a l -142 a x i a l array (with an approximate dihedral angle of 175°) with coupling of 11.4Hz. The magnitude of the two coupling constants 197 are consistent with the proposed configuration . 13 Since there was some ambiguity over the C NMR assignments for the C3 and C6 carbons (at 149.4(s) or 149.9(s)ppm), and as connectivity had only been established from H7 through to H5f a long range 2D-HETC0R NMR s p e c t r u m 7 6 ' 2 0 4 of 170 was obtained. As 7 6 with the short range experiment (see pages 33. and 90.) co n n e c t i v i t i e s between two d i f f e r e n t n u c l e i are established. However, by u t i l i s i n g the appropriate delay times the 2 3 experiment can be optimised to detect long range J C H and J C H couplings while suppressing the much larger ^JQ^ coupling. The experiment was optimised for p o l a r i s a t i o n transfer through 8Hz coupling, since a long range coupling of approximately 8Hz had been observed into the carbon resonating at 149.9ppm i n the f u l l y 1 3 coupled C NMR spectrum of 170. Both the H5 and H7 protons transferred magnetisation to the 1 3 C NMR resonance at 149.9(s)ppm i d e n t i f y i n g i t as C6, while only H5 transferred magnetisation to 149.4(s)ppm i d e n t i f y i n g i t as C3. Hence, the experiment allowed for the assignment of C3 and C6, and completed the connectivity about the 14-membered ring of 170. Further c o n n e c t i v i t i e s were apparent, these were f u l l y consistent with structure 170. and 13 allowed for the unambiguous assignment of C NMR resonances. The c o r r e l a t i o n r e s u l t s are summarised i n Table 20., and the subsequent chemical s h i f t assignments are summarised in Table 17. The occurrence of an NOE between the C19 methyl protons (1.98ppm) and the H7 o l e f i n i c proton (6.07ppm) requires that the C7-C8 o l e f i n i c bond of r u b i f o l i d e (170) has a Z configuration. 143 Table 20. Correlations observed i n a long range 2D-HETCOR NMR spectrum of r u b i f o l i d e (170). Proton Coupling Carbon coupled to H2af H2b 2-bond C3 H5 3-bond C3 2-bond C6 1-bond C5 H7 3-bond C9 2-bond C6 1-bond C7 H l l 3-bond C20 1-bond C l l Mel7 3-bond C16 2-bond C15 Mel8 3-bond C5 3-bond C3 2-bond C4 1-bond C18 Mel9 3-bond C9 3-bond C7 2-bond C8 1-bond C19 F i n a l l y , i t was assumed that the r e l a t i v e configurations at the CI and CIO carbons i n r u b i f o l i d e (170) were the same as those assigned to the corresponding carbons i n gersemolide (167). The enantiomer shown i s a r b i t r a r y . Epilophodione f171), obtained as colourless needles, was shown by EIMS to possess a molecular formula of C20 H24°4 (observed m/z 328.1680, requires 328.1675) which required nine 144 1 13 units of unsaturation. The H and C NMR spectra (Figures 31. 73 and 32.), in combination with the ADEPT spectrum (Figure 33), r e a d i l y i d e n t i f i e d methyl substituted ene-dione, a,r-disubstituted a,/?-unsaturated r - l a c t o n e , isopropenyl and t r i s u b s t i t u t e d o l e f i n residues, i d e n t i c a l with the corresponding substructures i n gersemolide (167) and r u b i f o l i d e (170). The NMR resonances are compared in Tables 14, 15, 17 and 18. The 1H and 1 3 C NMR data (Tables 18. and 17.) observed for epilophodione (171) showed a s t r i k i n g resemblence to the data reported by Bandurraga et a l . f o r lophodione (188,) 1 1 J " r suggesting that the two metabolites had the same constitution and d i f f e r e d only i n some stereochemical sense. The structure 171 was proposed for epilophodione and was confirmed by a series of NOE experiments. I r r a d i a t i o n of the Mel8 methyl protons (1.91ppm) i n 171 induced an NOE into the H5 o l e f i n i c proton (6.38ppra) in d i c a t i n g that the C4-C5 o l e f i n i c bond in epilophodione (171) had the Z configuration i n common with the corresponding bond i n lophodione ( 1 8 8 ) 1 1 1 . No NOE could be demonstrated between the Mel9 protons (2.19ppm) and the H7 o l e f i n i c proton (6.12ppm) i n epilophodione (171), a r e s u l t that was consistent with the C7-C8 o l e f i n i c bond having the E configuration found i n lophodione (188) 1 1 ]". The r e s u l t s of subsequent NOE i r r a d i a t i o n s were consistent with the proposed structure and are summarised in Table 21. The gross c o n s t i t u t i o n was thus confirmed, and proposed 1H and 1 3 C NMR assignments are given i n Table 18. and 17., r e s p e c t i v e l y . Since the o l e f i n i c geometries in epilophodione (171) were i d e n t i c a l to those of lophodione ( l f l S . ) 1 1 1 / the stereochemical 145 147 Details for each: 16-0mg 2hours 20 Omins C H 3 P e a k s s 00 CH2 P e a k s CH P e a k s C H , C H 2,CH3 P e a k s *i>iUi)Mi»«Mi|i|4iWMi'M»NiWi»W<fr>^ I I I I j I I I I I I I i I I I I I I I I I I I J I 1 1 1 1 t 1 1 1 J I I I 1 I I I I I J I I I I j I I I I I I I 1 I I I I I I J I I I 1 I T 200 180 160 140 120 100 80 I I | I I I T ~ r ~ r n 1 1 | 1 1 1 1 60 40 i i 1 | 1 1 1 1 1 1 1 20 PPM I Figure 33. ADEPT NMR spectra of epilophodione (111). Table 21. Subsequent NOE r e s u l t s observed for epilophodione (111). Proton i r r a d i a t e d Protons with observed NOE H7 H5 H10 H9a H9b H l l Mel7 H16(a and/or b) H2a r H2b Mel8 H16(a or b) H2a and/or H2b Mel9 H l l H10 H9a difference i n the two structures had to involve the r e l a t i v e configurations at the CI and CIO c h i r a l centers. The reported r e l a t i v e configurations, derived from an x-ray c r y s t a l l o g r a p h i c a n a l y s i s , for the CI and CIO c h i r a l centers i n lophodione are * * S ,S . I f the two c h i r a l centers i n epilophodione (121) have the same configurations as the corresponding c h i r a l centers i n gersemolide (167), the r e l a t i v e configurations at CI and CIO must be R ,S , respectively, making 171 epimeric with 188. This i s perhaps supported by the observation that the two metabolites rotate the plane of polarised l i g h t i n opposite d i r e c t i o n s ; i n epilophodione (171) the o p t i c a l rotation was p o s i t i v e , for lophodione (188) i t was negative. The absolute configuration of lophodione ( 1 8 8 ) 1 1 1 was not reported and the absolute 149 configuration of epilophodione (121) has not been determined, so i t i s not possible to say which of the two c h i r a l centers has the opposite configuration i n the two metabolites. Isoepilophodione A (122) i i s o l a t e d as a pale yellow o i l , was shown by EIMS to possess a molecular formula of C 2 o H 2 4 ° 4 (observed m/z 328.1686, requires 328.1675), i d e n t i c a l to that of 1 13 epilophodione (171). Examination of the IR, DV and the H and X"X NMR spectra (Figures 34. and 35.) of isoepilophodione A (172) i d e n t i f i e d a number of s t r u c t u r a l fragments; a f r - d i s u b s t i t u t e d a» ^-unsaturated r-lactone, methyl substituted ene-dione, t r i s u b s t i t u t e d o l e f i n and isopropenyl moieties, i d e n t i c a l with the corresponding substructures of epilophodione (171) ( a l l b e i t with very s l i g h t l y d i f f e r e n t chemical s h i f t s , see Tables 17. and 18.). The strong c o r r e l a t i o n of a l l s p e c t r a l data with 171 suggested that the two compounds could be geometrical isomers of one another at either or both of the t r i s u b s t i t u t e d o l e f i n s . This type of s i t u a t i o n has a precedent. The C4-C5 double bond i n the pseudopterolides, 167-169r can possess either the Z or E configuration, and lophodione (188) was known to co-exist with the C4-C5 E and C7-C8 Z geometric isomer, isolophodione ( 1 8 9 ) 1 1 1 . 0 189 150 The AH and •LJC NMR data for 189 are included i n Tables 17. and 18. Hence, isomerization of epilophodione (121) to isoepilophodione A (172), and v i s a - v e r s a f was accomplished using iodine i n benzene, providing confirmation of the proposal. However, the geometry of the two t r i s u b s t i t u t e d o l e f i n s i n 172 was s t i l l in question: the C4-C5 Z and C7-C8 E o l e f i n s i n epilophodione (171) could p o t e n t i a l l y isomerize to either the corresponding E,E, Z,Z, or E,Z o l e f i n s . I r r a d i a t i o n of the methyl protons resonating at 1.99ppm induced NOE's into the o l e f i n i c proton resonating at 6.07ppm and the lactone carbinol methine proton at 5.23ppm (H10), i d e n t i f y i n g them as the Mel9 and H7 protons, respectively. The NOE experiment demonstrated that the C7-C8 carbon-carbon double bond possessed a Z configuration. Consistent with t h i s assignment was the observation that i r r a d i a t i o n of H7 (6.07ppm) only induced an NOE into Mel9 (1.99ppm). I r r a d i a t i o n of the methyl protons resonating at 2.16ppm only induced a small p o s i t i v e NOE into the H16a proton resonating at 4.54ppm, i d e n t i f y i n g t h i s as the Mel8. Assignment of the E configuration to the C4-C5 double bond of 172 was consistent with t h i s NOE r e s u l t . Construction of a molecular model f o r 172. possessing the s t r u c t u r a l r e s t r a i n t s imposed by the above NOE r e s u l t s , r e a d i l y demonstrated that the H5 o l e f i n i c proton (6.68ppm) pointed into the i n t e r i o r of the r i n g . For a number of conformations the H2, H9 and H13 (or H14, but not both H13 and H14 at the same time) protons, s i m i l a r l y , a l l pointed into the i n t e r i o r , so that one or both protons i n each of the methylene p a i r s were situated close to the H5 proton. I r r a d i a t i o n of H5 (6.68ppm) induced NOE's into the H9a doublet of doublets resonating at 2.98ppm and into a 153 number of protons resonating i n the two multiplet regions, 2.33-2.45ppm and the one centred at 2.50ppm (H2a, H2b). The preceeding 1 13 arguments a l l support the proposed structure, 372. and C NMR assignments are included i n Tables 18. and 17. Interestingly for both'isoepilophodione A (172) and A 4 , 5 E , 7 17 A ' -isogersemolide (1£9_) the chemical s h i f t of the C3 carbon was more shielded (more conjugated) compared to epilophodione (171) and gersemolide (167) by 2.9 and 5.0ppm, re s p e c t i v e l y . The C6 ketone resonance of 172 was more deshielded (less conjugated), compared to 121, by 2.5ppm. I t appears that p l a n a r i t y between the C6 ketone and C4-C5 double bond i s p a r t i a l l y l o s t while p l a n a r i t y between the C3 ketone and C4-C5 double bond i s p a r t i a l l y gained i n going from a Z to E configuration. Isoepilophodione B (173). obtained as a pale yellow o i l , was shown by EIMS to possess a molecular formular of C 2o H24°4 (observed m/z 328.1670, requires 328.1675), i d e n t i c a l to that of epilophodione (171) and isoepilophodione A (172). The *H NMR spectrum of 173 (Figure 36.) consisted of very broad s i g n a l s so that very l i t t l e s t r u c t u r a l information could be gained. Fortunately, however, isomerization of both epilophodione (171) and isoepilophodione A (172) had also generated isoepilophodione B (122.). Isomer i z a t i o n of 173 to 171 and 172f as expected, was r e a d i l y accomplished using iodine i n benzene. Thus, 173 had to ex i s t as one of the two remaining geometrical isomers of epilophodione (171). i e . , with C4-C5 and C7-C8 carbon-carbon double bonds i n either the E,E or Z,Z configurations. I r r a d i a t i o n of the methyl protons resonating at 1.88ppm induced an NOE in t o 154 the o l e f i n i c proton at 6.21ppm. S i m i l a r l y , i r r a d i a t i o n of the methyl protons resonating at 2.06ppm induced an NOE into the proton at 6.25ppm. Hence, both double bonds had to possess the Z configuration and structure 173 was proposed for isoepilophodione B. Isoepilophodione B (173) must exi s t i n at l e a s t two slowly interconverting low energy conformations i n s o l u t i o n , and hence, those protons that s i t i n d i f f e r e n t chemical environments due to the exchange are excessively broad. I n i t i a l attempts to resolve the problem involved high temperature *H NMR experiments. At 50°C the problem was s t i l l not removed and resulted i n the p a r t i a l decomposition of 173. A series of low temperature NMR experiments resulted i n the freezing out of two conformations ( i n a r a t i o of 4:1) (see Figure 37., -40°C appeared to be the optimum temperature, going to lower temperatures had no apparent e f f e c t ) . Spectral comparison of the low temperature ^H NMR data of 173 to the room temperature data given i n Table 18., for 170. 171. 122* 188 and 189 indicated that at -40°C the H10 lactone carbinol proton resonated at 5.42ppm (see Figure 37b.). With t h i s a s e r i e s of spin-spin decoupling experiments at -40°C allowed for proton NMR assignments. I r r a d i a t i o n of the broad doublet, of the major conformer, resonating at 4.07ppm (J=11.2Hz, H9b) s i m p l i f i e d the broad doublet of doublets at 2.79(J=11.2,8.0Hz, H9a)ppm to a doublet (J=8.0Hz) and sharpened the broad o l e f i n i c s i n g l e t at 6.30ppm (H7). I r r a d i a t i o n of the doublet of doublets at 2.79ppm (H9a) collapsed the methylene doublet at 4.07ppm (H9b) to a broad s i n g l e t and sharpened the methine proton at 5.42ppm (H10) to a broad s i n g l e t . These assignments were correlated to the room temperature H NMR assignments given i n Table 18. H5 could be assigned to 6.21ppm since H7 was assigned the resonance at 6.25ppm, and hence the Mel8 protons were assigned to the resonance at 1.88ppm and Mel9 to 2.06ppm. F i n a l l y , at -40°C i r r a d i a t i o n of the H9b broad t r i p l e t (3.68ppm), of the minor conformer, sharpened H9a (resonating underneath the H9a broad doublet of doublets (2.79ppm) of the major conformer). Previously i r r a d i a t i o n at 2.79ppm had also s i m p l i f i e d the broad t r i p l e t at 3.68ppm (H9b) to a broad doublet and collapsed the H10 doublet of mu l t i p l e t s (5.01ppm) to a broad s i n g l e t . Thus, the above p a r t i a l 1H NMR assignments for the minor conformer were also made. Isoepilophodione C (174) f i s o l a t e d as a clear o i l , was shown by EIMS to possess a molecular formula of C 2 o H 2 4 ° 4 ( ° D s e r v e d m / z 328.1671, requires 328.1675) which required nine units of 1 1 3 unsaturation. The H and C NMR spectra (Figures 38. and 39.) and IR spectra r e a d i l y i d e n t i f i e d methyl substituted ene-dione, a»r-disubstituted a,^-unsaturated r-lactone and isopropenyl residues, i d e n t i c a l with the corresponding substructures i n gersemolide (167). r u b i f o l i d e (170) and epilophodione (171) (see 13 Tables 14, 15, 17 and 18). Further to these fragments, the C NMR resonances at 125.3 and 143.7ppm and the 1H NMR resonances at 5.90(d, J=0.9Hz, IH) and 6.08(s, lH)ppm t e n t a t i v e l y i d e n t i f i e d an exocyclic methylene moiety. This fragment was consistent with the observation that on i r r a d i a t i o n of the o l e f i n i c proton resonating at 5.90ppm an NOE was induced into the proton at 6.08ppm. Isoepilophodione A (172) p a r t i a l l y isomerized to isoepilophodione C (174) on treatment with formic acid under reflux conditions, suggesting that the two metabolites were 159 related by a simple double bond migration. The cembranoid structure 174 e f f e c t i v e l y accomodated a l l the i d e n t i f i e d s t r u c t u r a l fragments of isoepilophodione C. A serie s of NOE, 199 spin-spin decoupling and SINEPT NMR experiments v e r i f i e d that 174 represented the correct structure. I r r a d i a t i o n of the Mel8 protons resonating at 1.95ppm induced NOE's into the H5 proton at 6.29ppm and the H16a at 4.69ppm, and an across ring NOE was also observed into the o l e f i n i c lactone proton (Hll) (6.79ppm). I r r a d i a t i o n of the methyl resonance at 1.61ppm (Mel7) induced an NOE into the H16b proton at 4.81(m, lH)ppm, and under decoupling conditions the same i r r a d i a t i o n sharpened the H16b resonance. Thus, assignment of the Z configuration to the C4-C5 double bond was confirmed and the Mel8 protons and the isopropenyl residue were connected through space and could be assigned to t h e i r "usual" p o s i t i o n s at C4 and CI, respectively. I r r a d i a t i o n of the H7b broad doublet (J=15.0Hz) resonating at 3.73ppm collapsed the H7a doublet (J=15.0Hz) at 3.08ppm to a s i n g l e t and sharpened the Hl9a doublet at 5.90ppm to a s i n g l e t . As expected, i r r a d i a t i o n at 3.08ppm (H7a) collapsed H7b (3.73ppm) to a s i n g l e t . I r r a d i a t i o n of H7b (3.73ppm) under NOE conditions induced NOE's into the H7a doublet (3.08ppm) and H5 (6.29ppm). Simultaneous i r r a d i a t i o n of H7a (3.08ppm) and H9b (3.05ppm) induced NOE's into H7b (3.73ppm), Hl9b (6.08ppm) and the H9a multiplet at 2.14ppm and small NOE's were observed into H5 (6.29ppm) and H19a (5.90ppm). I r r a d i a t i o n of the exocyclic methylene H19a proton at 5.90ppm not only induced an NOE into Hl9b, but also H7a, and i r r a d i a t i o n of H19b induced an NOE into the H9b proton resonating at 3.05ppm. This s e r i e s of experiments connected the H5 to the H9 protons through 162 s p a t i a l and scalar coupling c o n n e c t i v i t i e s of the two H19 protons with the H7 and H9 protons. F i n a l l y , i r r a d i a t i o n of the lactone H10 proton resonating at 5.06ppm sharpened both the H9a multiplet centered at 2.14ppm and the lactone o l e f i n i c proton (Hll) at 6.79ppm, and i r r a d i a t i o n at 2.14ppm (H9a) reduced H9b (3.05ppm) to a broad s i n g l e t . Hence, the isopropenyl moiety was connected s p a t i a l l y to the Mel8 protons which were subsequently connected around the ring to the lactone H l l proton through both dipolar and s c a l a r coupling c o r r e l a t i o n s . A molecular model demonstrated that the H7a, H7b, H9a and H9b protons were to be assigned to the Elft, H7a, H9a and H9/3 protons, r e s p e c t i v e l y . 199 Three SINEPT experiments, optimised for p o l a r i s a t i o n t r a n s f e r through 7Hz coupling, connected the exocyclic methylene and ene-dione fragments, and allowed for 1 3 C NMR assignments to be made. F i r s t , i r r a d i a t i o n of the methylene proton at 3.73ppm (H7b) showed p o l a r i s t i o n transfer to the carbon resonances at 128.3(C5), 143.7(C8) and 196.9(C6)ppm. Second, i r r a d i a t i o n of the ex o c y c l i c methylene proton resonating at 5.90ppm (H19a) showed p o l a r i s t i o n transfer to carbon resonances at 48.3(C7 or C9, more l i k e l y C7) and 143.7(C8)ppm. Thus, the s t r u c t u r a l c o n s t i t u t i o n was further supported. The t h i r d experiment involved i r r a d i a t i o n of the H10 lactone methine proton (5.06ppm) and i t confirmed the assignments made for C12 and C20, since transfer of p o l a r i s a t i o n was observed into the carbon resonances at 132.9(C12) and 1 13 173.9(C20)ppm. Proposed H and C NMR assignments are included in Tables 18. and 17. Rubifol f175). i s o l a t e d as colourless needles, was shown by 163 EIMS to possess a molecular formula of C20 H26°5 ( ° D S e r v e ( ^ m / z 346.1785, requires 346.1781) which required 8 units of unsaturation. The IR, 1H, 1 3 C and A P T 2 0 5 NMR spectra (for l a t t e r three, see Figures 40., 41., and 42.) r e a d i l y i d e n t i f i e d methyl substituted ene-dione, a,y-disubstituted a, ^-unsaturated r-lactone and isopropenyl residues, i d e n t i c a l with the corresponding substructures i n gersemolide (167), r u b i f o l i d e (170) and epilophodione (171) (see Tables 14., 15., 17. and 18.). — 1 13 The IR band at 3509cm and a C NMR resonance at 80.7(C)ppm i d e n t i f i e d a fourth fragment, a t e r t i a r y a l cohol. The presence of the alcohol was confirmed by the l o s s of water from the parent ion (m/z 346 daltons) i n the MS to give a fragment ion at an m/z 197 of 328 daltons . Hence, seven degrees of unsaturation and a l l f i v e oxygens were accounted f o r . The preceeding arguments indicated that r u b i f o l (175) had to possess a second ring to account for the t o t a l of eight units of unsaturation. The cembranoid structure 175 e f f e c t i v e l y accomodated a l l the i d e n t i f i e d s t r u c t u r a l fragments. An extensive s e r i e s of NOE and spin-spin decoupling experiments v e r i f i e d that 175 represented the correct s t r u c t u r e . The NOE resul t s are summarized i n Table 22. Decoupling experiments, in combination with the NOE r e s u l t s , demonstrated that the *H NMR resonances at 2.47 and 3.37ppm were to be assigned to a geminal p a i r , the H7a and H7b protons, r e s p e c t i v e l y , and s i m i l a r l y the C9 methylene protons were assigned the resonances at 2.59 and 3.91ppm. The H9a, H9b protons also showed v i c i n a l coupling to the lactone methine proton at 4.83ppm (H10). The decoupling and NOE r e s u l t s , discussed thus f a r , were consistent with the proposed structure 164 s C ' s , C H 2 ' s up, C H ' s , C H 3 ' s down J>|»>i||l0l^ wL»llNi>>l^ l>l*^ W>*W^ i(l Details: 76mg lOhours 50-1mins 1 1 1 1 1 1 i 1 1 1 i | 1 1 i 11 i 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i 1 1 i i 1 1 1 11 i i i 11 1 i 11 1 1 i 1 1 1 1 i 1 1 1 1 1 i 11 i i i | i i i i 11 i 1 1 | i i i i i i 1 1 i | i i i 11 1 1 1 1 i 1 1 1 1 1 ' 1 1 1 i 220 200 180 160 140 120 100 80 60 40 20 PPM 0 Figure 42. APT NMR spectrum of r u b i f o l f175). Table 22. NOE r e s u l t s observed for r u b i f o l (175). Proton i r r a d i a t e d HI H2a and H13a a H7a H7b H9a H9b H l l Protons with observed NOE H2b H14b Mel9 H9a H l l Hl4a H7b Mel9 H7a H5 Mel9 H9b H10 H9a H l l H2a, H2b H9b Mel7 and H14a a H16b H2a H2b Hl3b Mel8 H5 H16a, H16b Mel9 HI H7a f H7b re s u l t of a simultaneous i r r a d i a t i o n . and connected through from the isopropenyl moiety to the o l e f i n i c lactone proton ( H l l ) . Assuming that the CI and CIO carbons i n 168 r u b i f o l (175) possess the same r e l a t i v e configuration as the appropriate carbons i n gersemolide f167), a molecular model possessing the c o n f i g u r a t i o n a l r e s t r a i n t s imposed by the NOE r e s u l t s demonstrated that the r e l a t i v e configuration at C8 had to be S . A second s e r i e s of spin-spin decoupling experiments i n combination with the NOE r e s u l t s and a molecular model c l e a r l y demonstrated the connectivity i n the eight-spin system found on C l l to C2 and thus completed the connectivity about the 14-membered r i n g . The r e s u l t s of the decoupling experiments are summarised i n Table 23., and the subsequent *H NMR assignments 13 are included i n Table 18. Proposed C NMR assignments are included i n Table 17. F i n a l l y , to remove ambiguities associated with the assignment of the lactone methine proton (H10) to the resonance at 4.83(m, lH)ppm ( l y i n g underneath the Hl6a (4.82ppm) and H16b (4.84ppm) resonances) a *H NMR spectrum of r u b i f o l (175) was obtained i n deuterated benzene (C gDg). The chemical s h i f t assignment and the m u l t i p l e t coupling pattern had been based on the observation that i r r a d i a t i o n of the H9a resonance at 2.59ppm induced an NOE into the resonance at 4.83ppm (H10). The *H NMR spectrum run i n CgDg i s shown i n Figure 43. As expected most resonances are s h i f t e d . I r r a d i a t i o n of the H10 mu l t i p l e t resonance (4.18ppm) reduced the H9b t r i p l e t (J=10.8Hz) at 3.78ppm to a doublet (J=10.8Hz) and sharpened the lactone H l l resonance at 6.40ppm. Thus, the carbinol lactone proton (H10) decoupled as expected and the previously discussed decoupling and NOE r e s u l t s i n CDClj were consistent with the proposed assignment. 169 Table 23. Spin-spin decoupling results for the eight-spin system found on C l l to C2 i n r u b i f o l (US.) Proton Coupling Proton M u l t i p l i c i t y p r i o r R e s u l t 3 i r r a d i a t e d coupled to to i r r a d i a t i o n H2a v i c i n a l HI bd sh H2b v i c i n a l HI bd sh H l l a l l y l i c H13a m sh Hl3a a l l y l i c H l l d sh v i c i n a l Hl4a m sh v i c i n a l Hl4b bt sh H13b v i c i n a l Hl4a m sh v i c i n a l Hl4b bt dm H14b v i c i n a l HI bd bs v i c i n a l Hl3a m sh v i c i n a l Hl3b m sh m u l t i p l i c i t y a f t e r i r r a d i a t i o n , sh-means sharpened. A seventh metabolite*, possibly a cembranoid, was i s o l a t e d only once. I t was obtained as a clear o i l that was shown by EIMS to possess a molecular formular of C21 H28°5 ( ° b s e r v e d m/z 360.1949, requires 360.1937), which required eight units of unsaturation. The *H NMR spectrum (Figure 44.) consisted of rather broad peaks, suggesting that the metabolite existed i n at l e a s t two low energy conformations i n s o l u t i o n . A *H NMR methyl resonance at 3.18(s, 3H)ppm suggested the existence of a methyl ether. The low i n t e n s i t y mass sp e c t r a l fragment at a m/z of 345 daltons, corresponding to the loss of a methyl, supported the * Referred to as the/an unknown diterpene in t h i s t h e s i s . 170 notion. Due to the lack of material (2.2mg) and the conformational 13 problem, i t was possible to obtain only a p a r t i a l C NMR spectrum of the unknown diterpene. However, the *H and the 13 a v a i l a b l e C NMR data did allow for the i d e n t i f i c a t i o n of the following s t r u c t u r a l fragments: an a.,j^-disubstituted a,f1-unsaturated r-lactone ( 1 3C NMR: 80.9, 132.0, 148,6 and 174.1ppm; 1H NMR: 5.17(very bs, IH) and 6.98(bs, lH)ppm); a isopropenyl moiety ( 1 3C NMR: 17.8, 112.8 and 143.9ppm; 1H NMR: 1.62(bs, 3H), 4.58(bs, IH) and 4.79(bs, lH)ppm); and a t r i s u b s t i t u t e d o l e f i n ( 1 3C NMR: 126.1ppm; XH NMR: 1.97(d, J=1.5Hz, 3H) and 6.19(bs, lH)ppm). Compare these chemical s h i f t s with those given i n Tables 17. and 18. The presence of isopropenyl and t r i s u b s t i t u t e d o l e f i n moieties was confirmed by two NOE experiments. I r r a d i a t i o n of the methyl resonance at 1.62ppm induced an NOE into the isopropenyl o l e f i n i c proton at 4.79(bs, lH)ppm, and i r r a d i a t i o n of the methyl resonance at 1.97ppm induced NOE's i n t o the o l e f i n i c proton at 6.19ppm and into the second o l e f i n i c isopropenyl proton resonating at 4.58(bs, lH)ppm. The broad "bump" on the baseline centered at 3.83(IH)ppm i n the NMR spectrum of the unknown diterpene indicated that an 13 alcohol residue may have been present. Also a C NMR resonance at 50.6ppm may have corresponded to the methyl ether carbon, but t h i s resonance would also be appropriate for the carbon at C7 i n Figure 45 (a). Not only was the metabolite i s o l a t e d in small quantity i t was also rather unstable and IR, UV and low or high temperature *H NMR spectra were not obtained p r i o r to decomposition. Hence, further evidence for the presence of the 173 (a). (b). (c). Figure 44. Proposed structures for the unknown diterpene. alcohol etc., was not obtained. Three possible structures for the metabolite are proposed i n Figure 45. Extensive NOE and spin-spin decoupling experiments suggested that a l l three were reasonable candidates. The NOE's previously discussed allowed the isopropenyl and t r i s u b s t i t u t e d o l e f i n moieties to be placed i n the "usual" p o s i t i o n s , at CI and C4-C5, respectively, with the C4-C5 double bond possessing a Z configuration. NOE's were observed between the methyl ether protons at 3.18ppm and the methyl resonance at 1.43(s, 3H)ppm. The doublet at 3.15(J=11.7Hz, lH)ppm, as evidenced by both NOE and decoupling r e s u l t s , was the geminal or v i c i n a l neighbour of the resonance at 1.80(td, J=11.7, 0.3Hz, lH)ppm. I r r a d i a t i o n of the o l e f i n i c proton at 6.19ppm induced NOE's into the i s o l a t e d methylene AB quartet resonating at 2.84(d, J=17.9Hz, 1H) and 3.00(d, J=17.9Hz, lH)ppm. The three proposed structures f i t t h i s data accordingly. However, each structure exhibits discrepencies. For example, in Figure 45 (a)., the arguments for the presence of a secondary alcohol at C3 are weak and for the structures (b). 174 and ( c ) . , there was no apparent coupling to, or protons that could be r e a d i l y associated with the C9 p o s i t i o n . I t was thought that the unknown diterpene might be an a r t i f a c t of i s o l a t i o n . However, attempts to generate methyl ethers of the cembranoids 171-174. through treatment with methanol i n the presence of a c a t a l y t i c amount of formic a c i d , have f a i l e d to generate t h i s compound. Obviously the s t r u c t u r a l s o l u t i o n of t h i s unknown diterpene needs further work. (iv) . Hew. CarbOP Skeletal Metabolites. 17_£ and 122: Gersolide (176). i s o l a t e d as a very minor metabolite (1.5mg) and obtained as colourless needles, was shown by EIMS to possess a molecular formula of C 2 o H 2 4 ° 4 ( ° D s e r v e d m / z 328.1674, requires 328.1675) which required nine units of unsaturation. A number of fragments were i d e n t i f i e d from the IR and *H NMR spectra (Figure 46.). The f i r s t fragment, a a i ^ - d i s u b s t i t u t e d a»fl-unsaturated Y-lactone, was indicated by the IR band at 1758cm"1 and the "^H NMR resonances at 5.14(d, J=0.6Hz, 1H) and 6.76(bs, lH)ppm. 1H NMR resonances at 2.06(d, J=0.6Hz, 3H) and 5.96(q, J=0.6Hz, lH)ppm, and the IR bands at 1683, 1679 and 1609cm - 1 i d e n t i f i e d a second fragment as a methyl substituted ene-dione. The observation of an NOE between the o l e f i n i c proton and the methyl resonance provided further support for the proposed fragment. F i n a l l y , the t h i r d s t r u c t u r a l fragment, a isopropenyl group, was evidenced by the 1H NMR resonances at 1.72(d, J=0.6Hz, 3H) , 4.97(bs, 1H) and 5.22(bs, lH)ppm. On i r r a d i a t i n g the methyl resonance at 1.72ppm an NOE was observed into the o l e f i n i c 175 isopropenyl resonance at 4.97ppm. The three s t r u c t u r a l fragments thus far proposed were i d e n t i c a l with the corresponding substructures i n gersemolide (167), r u b i f o l i d e f170) and epilophodione (171). The s p e c t r a l data reported i s t y p i c a l of such s y s t e m s 1 1 1 , 1 5 7 ' 1 9 7 , 2 ° 3 (see Tables 15. and 18.). The three i d e n t i f i e d s t r u c t u r a l fragments ascribed to gersolide (176) accounted for a l l the oxygen atoms and seven units of unsaturation. Thus, the two degrees of unsaturation not accounted for had to be c a r b o c y c l i c rings. A 1H NMR resonance at 1.30(s, 3H)ppm required that a methyl substituent be situated at one of the ring junctions. No known diterpenoid carbon skeleton could account for the above features and further s p e c t r a l data was lacking since only a small quantity of gersolide (176) had been is o l a t e d (1.5mg). C r y s t a l s of gersolide f176) were obtained by slow evaporation from methanol at 0°C and the complete structure of gersolide (176) was secured v i a s i n g l e c r y s t a l x-ray d i f f r a c t i o n a n a l y s i s , k i n d l y performed by L. Parkanyi and J . Clardy of Cornell U n i v e r s i t y . A computer generated perspective drawing of the f i n a l x-ray model of gersolide (176) i s represented in Figure 47. Gersolide (176) represents a diterpenoid with a new rearranged carbon skeleton possessing a 13-membered carbocyclic ring . I t should be noted that the x-ray experiment did not define the absolute configuration, so the enantiomer drawn i s a r b i t r a r y . The a,ft-unsaturated ^-lactone fragment was planar within experimental e r r o r . The C4-C5 double bond was not co-planar with ei t h e r the C6 *We propose the name "gersolane" for the new diterpeniod carbon skeleton of g e r s o l i d e (176). 177 Figure 47. Computer generated perspective drawing of gersolide (1Z£>. or the C3 carbonyl groups. These two s t r u c t u r a l observations are consistent with those observed for gersemolide (167) and are also 1 13 consistent with the H and JC NMR chemical s h i f t assignments made for the diterpenes, 167-175. An intensive s e r i e s of NOE and spin-spin decoupling experiments with reference to a molecular model allowed for the assignment of the majority of the NMR resonances, and provided confirmation of the st r u c t u r e , 176. The NOE r e s u l t s are summarized i n Table 24. The H9a cyclopropyl proton (0.98(dd, J=8.3, 5.8Hz, lH)ppm) showed geminal coupling to H9b (1.83(dd, 178 Table 24. NOE r e s u l t s observed for gersolide f176). Proton i r r a d i a t e d Protons with observed NOE H9a H7 H9b H l l H9b H9a Mel7 H2b Hl6a Mel8 H2b H5 H16b Mel9 H5 H9b H10 J=8.3, 5.8Hzf lH)ppm) and v i c i n a l coupling to H7 (1.43(t f J=5.8Hz, H7)ppm), and i r r a d i a t i o n of the H2b doublet (J=12.3Hz) at 2.81ppm s i m p l i f i e d the H2a t r i p l e t at 2.18ppm to a broad doublet (J=12.3Hz). The subsequent "'"H NMR assignments are given i n Table 25. F i n a l l y , i t could be r e a d i l y demonstrated, with a molecular model, that the methylene H9a and H9b protons (resonating at 0.98 and 1.83ppm, respectively) had to be assigned to the H9a and H9/3 protons, r e s p e c t i v e l y . Rubiformate (177). i s o l a t e d as a minor metabolite (<4.0mg) and obtained as a clear o i l , was shown by EIMS to possess a molecular formula of C20 H24°5 ( ° b s e r v e d m/z 344.1623, requires 344.1624) which required nine units of unsaturation. A number of 179 Table 25. H NMR assignments for gersolide f176) and rubiformate (122). Carbon 1 2a b 5 7 9a b 10 11 13a b 14a b 16a b 17 18 19 Chemical s h i f t , ppm. \ 176 Carbon 2.31(tdm, J=12.3,5.2Hz, 1H) 2.18(t, J=12.3Hz, 1H) 2.81(bd, J=12.3Hz, 1H) 5.96(g, J=0.6Hz, 1H) 1.43(t, J=5.8Hz, 1H) 0.98(dd, J=8.3,5.8Hz, 1H) 1.83(dd, J=8.3,5.8Hz, 1H) 5.14(d, J=0.6Hz, 1H) 6.76(bs, 1H) 2.33-2.48(m, 2H) Z 1.25-1.36(m, 1H) Z 1.98-2.13(m, 1H) Z 4.97(bs, 1H) 5.22(bs, 1H) 1.72(d, J=0.6Hz, 3H) 2.06(d, J=0.6Hz, 1H) 1.30(s, 3H) 2 1 2 8a b 9 10 12a 12b 13a b 15a b 16 17 18 20 Chemical s h i f t , ppm. # 122 2.68(m, 1H) 2.70-2.78(m, 2H) 7.03(s, 1H) / 2.98(ddm, J=6.6,2.7Hz, 1H) 3.04(ddm, J=6.6,2.7Hz, 1H) 5.29(m, 1H) 7.11(m, 1H) 1.63(m, 1H) 2.20(m, 1H) 2.55-2.65(m, 2H) 4.69(bs, 1H) 4.78(q, J=0.9Hz, 1H) 1.67(bs, 3H) 2.01(s, 3H) 2.23("d n, 3H) 9.46(s, 1H) 'assignments within a column may be interchanged. 180 1 13 fragments could be i d e n t i f i e d from the H and C NMR (Figures 48. and 49.) and other s p e c t r a l evidence. The f i r s t fragment, a O i ^ - d i s u b s t i t u t e d ot,^-unsaturated r-lactone, was i d e n t i f i e d from 1 3 C NMR resonances at 76.8, 134.2, 147.7 and 173.1ppm and XH NMR resonances at 5.29(m, IH) and 7.11(m, lH)ppm. An IR band at 1758cm"1, t y p i c a l of such a s y s t e m 1 1 1 , 1 5 7 , 1 9 7 f 2 0 3 , and the observation that i r r a d i a t i n g either proton resonance sharpened the other, were consistent with the proposal. Carbon resonances at 18.4, 113.3 and 145.0ppm and NMR resonances at 1.67(bs, IH) , 4.69(bs, IH) and 4.78(q, J=0.9Hz, lH)ppm i d e n t i f i e d a second fragment, an isopropenyl residue. Confirmation was provided by decoupling and NOE experiments. I r r a d i a t i o n at 1.67ppm collapsed the downfield quartet at 4.78ppm to a s i n g l e t and sharpened the u p f i e l d resonance at 4.69ppm. The same i r r a d i a t i o n under NOE conditions induced an NOE into the resonance at 4.78ppm. IR absorptions at 1676 and 1606cm"1 i d e n t i f i e d a t h i r d s t r u c t u r a l fragment as a a,^-unsaturated ketone with the 206 p o s s i b i l i t y of further conjugation . The proposal was supported by the presence of a carbonyl resonance at 204.5ppm and o l e f i n i c resonances at 125.5, 157.8, 119.2 and 150.7ppm i n the 1 3 C NMR spectrum of rubiformate (177). Evidence for an enone with extended conjugation was provided by the DV absorption at 297nm (s 16780). I r r a d i a t i o n of the methyl resonance at 2.01(s, 3H)ppm sharpened the o l e f i n i c s i n g l e t at 7.03ppm. The same i r r a d i a t i o n under NOE conditions induced an NOE into t h i s o l e f i n i c s i n g l e t and into part of the m u l t i p l e t region resonating between 2.70-2.78ppm; the induced part appeared as a t r i p l e t (7.3Hz) centered 181 at 2.73ppm. On i r r a d i a t i n g the isopropenyl methyl resonance at 1.67ppm an NOE was also observed into the t r i p l e t at 2.73ppm. This i s t y p i c a l for metabolites 167-175 and i s the r e s u l t of both an isopropenyl and a methyl substituted ene-dione l y i n g in the v i c i n i t y of the methylene H2 protons. Hence, the enone was to be treated as a methyl d i a l k y l s u b s t i t u t e d enone. The three s t r u c t u r a l fragments thus far i d e n t i f i e d were i d e n t i c a l to the corresponding substructures i n gersemolide (167). r u b i f o l i d e f170), epilophodione f171) and gersolide (176). NMR comparisons are made in Tables 15. 16. 17. 18. 25. and 26. F i n a l l y , a fourth s t r u c t u r a l fragment, a methyl substituted enol formate, was apparent from 1 3 C NMR resonances at 9.8, 119.2, 150.7 and 176.8ppm, -^H NMR resonances at 2.23("d", J=2.lHz, 3H) and 9.46(s, lH)ppm and IR bands at 1720 and 1650cm - 1. Compare, 13 for example, the appropriate C NMR resonances of the model 207 compound, norpectinatone (190) . with those of rubiformate (122), Table 27. The IR bands also compare favourably. For example, the carbon-carbon double bond s t r e t c h of the enol formate 1 9 1 2 0 6 occurs at 1650cm - 1 (run i n CgH 1 2) a n d t n e carbonyl absorption band of formates i s observed i n the region 1730-— 1 1 97 1715cm . The presence of the formate moiety was consistent with a MS fragment ion at a m/z of 315 daltons (M + -29) corresponding to acyl cleavage and the loss of CHO. The preceeding arguments indicated that rubiformate (122) possessed f i v e carbon-carbon double bonds, a ketone, a formate, a lactone carbonyl and a lactone r i n g . Since no add i t i o n a l unsaturated f u n c t i o n a l i t i e s were evident, 177 had to possess a second ring to account f o r the t o t a l of nine units of 184 Table 26. C NMR assignments for rubiformate f177) . and s p e c t r a l comparison with epilophodione f171). Chemical s h i f t , ppm. Chemical s h i f t , ppm. Carbon # 121 a Carbon # 1 2 1 ° 1 45.5 Z 1 41.8(CH) 2 46.5 Z 2 45.5(CH2) 3 204.5 3 205.5(C) 4 157.8 4 143.9(C) 2 5 125.5(br) 5 133.5(CH) 6 150.7 ./ 7 119.2 / 8 31.2 Z 9 43.6(CH2) 9 76.8 10 78.4(CH) 10 147.7 11 148.1(CH) 11 134.2 12 136.2(C) 12 29.8 Z' y 13 21.0(CH 2) 13 30.5 Z 14 33.5(CH 2) 14 145.0 15 145.4(C) 2 15 113.3 16 115.6(CH2) 16 18.4 17 17.2(CH 3) 17 23. l y 18 21.1(CH 3) y 18 9.8 19 22.3(CH 3) y 19 173.1 20 173.6(C) 20 176.8 / i n t h i s column "br" at C5 (125.5(br)) means broad s i g n a l . cont'd. 185 Table 21. continued. b i n t h i s column "/" means no advantage i n comparing, z , vassignments within a column may be interchanged. 0 191 190 i 3 Table 27. C NMR data comparison of 177 and 190. Chemical s h i f t , ppm. Chemical s h i f t , ppm. Carbon # 177 Carbon # 190a 5 125.5 9 126.1 6 150.7 10 159.2 7 119.2 11 106.5 18 9.8 19 8.7 a r e f e r e n c e 207, spectrometer frequency not reported, CDCl-j. unsaturation. This c a r b o c y c l i c ring had to accomodate f i v e 13 remaining unassigned a l i p h a t i c carbons ( C NMR: 29.8, 30.5, 31.2, 45.4 and 46.5ppm). The new rearranged degraded cembrane diterpenoid s t r u c t u r e , 177. possessing a 13-membered r i n g , 186 e f f e c t i v e l y accomodated a l l the i d e n t i f i e d s t r u c t u r a l fragments. l o g A seri e s of NOE, spin-spin decoupling and SINEPT NMR experiments v e r i f i e d that 177 represented the correct s t r u c t u r e . As discussed above, the isopropenyl and methyl substituted enone moieties could be placed at t h e i r respective positions through the observance of NOE's into one of the H2 protons (2.70-2.78ppm). I r r a d i a t i o n of the methyl protons resonating at 2.01ppm (Mel7) also induced a small NOE into the two proton m u l t i p l e t y region resonating between 2.55-2.65ppm, which presumably should be assigned to the H2 1, HI, or the H12 or H13 methylene protons. Decoupling experiments (to be discussed) demonstrated that the multiplet should be assigned to H13a, H13b. I r r a d i a t i o n of the o l e f i n i c proton resonating at 7.03ppm (H5) not only induced an NOE into Mel7 (2.01ppm), confirming the assignment of the Z configuration given to the C4-C5 double bond, but also the formate proton (H20) at 9.46ppm. The reverse r e s u l t also held true. Hence, the placement of the formate at the C6 p o s i t i o n was confirmed. I r r a d i a t i o n of the multiplet resonance at 2.20ppm (Hl2b) markedly sharpened the H12a multiplet (1.63(m, lH)ppm) situated between the water (1.56ppm) and- Mel7 resonances and as a re s u l t of a l l y l i c coupling sharpened the o l e f i n i c lactone resonance at 7.11ppm (H10), and v i a homoallylic coupling sharpened the lactone carbinol (H9) resonance at 5.29ppm. I r r a d i a t i o n of the Hl2a mu l t i p l e t (1.63ppm) markedly sharpened i t s geminal partner, H12b (2.20(m, lH)ppm) and sharpened the mult i p l e t resonating between 2.55-2.65ppm (H13a, Hl3b). I r r a d i a t i o n of the lactone carbinol resonance at 5.29ppm (H9) sharpened the o l e f i n i c H10 resonance at 7.11ppm and the two 187 methylene protons at 2.98(dd"m", J=6.6,2.7Hz, H8a) and 3.04(ddm, J=6.6,2.7Hz, H8b)ppm to two broad m u l t i p l e t s . I r r a d i a t i o n of ei t h e r of the H8 protons sharpened both the lactone H9 resonance (5.29ppm) and Mel8 (2.23ppm). The observance of coupling between the Mel8 and H8 protons was consistent with the couplings 145 reported by M. D'Ambrosio et a l . between the Mel9 and H9 protons i n the cembranoid metabolites £ 2 and ££. This presumably can be accounted for by W-coupling between the Mel8 and H8 protons. The proton connectivity about the ring was consistent with the proposed structure, 177. The assignment of an E configuration to the C6-C7 double bond was consistent with the r e s u l t that NOE's were observed between the formate and o l e f i n i c H5 resonance but not the Mel8 protons, i e . , the formate proton (H20) points toward the H5 proton and away from the methyl protons (Mel8). A molecular model possessing both; the same r e l a t i v e configuration at the CI and C9 carbons as the appropriate carbons i n gersemolide (167) and ge r s o l i d e (176? • and the s t r u c t u r a l constraints imposed by the NOE r e s u l t s , demonstrated that when the C6-C7 bond has an E configuration the Mel8 protons need not n e s s e s s a r i l y l i e i n the v i c i n t y of any other protons. However, i f the C6-C7 bond had the opposite, Z configuration the Mel8 protons would have to l i e i n close proximity to at least the lactone c a r b i n o l proton (H9). On i r r a d i a t i n g the methyl resonance at 2.23ppm (Mel8) no NOE's were observed. These r e s u l t s though only providing negative evidence are consistent with the proposal of an E configuration. The proposed arrangement also minimises the 188 s t e r i c hinderence between the "bent" formate chain and the Mel8 protons. 199 Four SINEPT experiments, optimised for p o l a r i s t i o n transfer through 7Hz coupling, provided further evidence for the proposed c o n s t i t u t i o n of rubiformate (122) and confirmed several 13 C NMR assignments. I r r a d i a t i n g the formate proton at 9.46ppm (H20) resulted i n three bond p o l a r i s a t i o n transfer to the carbon resonance at 150.7(C6)ppm and four bond transfer into the resonance at 119.2(C7)ppm. A second i r r a d i a t i o n at 7.03ppm (H5) showed two bond transfer to the carbon resonances at 157.8(C4) and 150.7(C6)ppm. I r r a d i a t i o n of the methyl resonance at 2.23ppm (Mel8) showed two bond p o l a r i s a t i o n t r a n s f e r to the carbon resonance at 119.2(C7)ppm. Hence, the SINEPT r e s u l t s confirmed the s t r u c t u r a l c o n s t i t u t i o n by connecting the enol formate and the enone moieties through the C5-C6 bond and unambiguously established the presence of a methyl group at the C7 p o s i t i o n . A f i n a l experiment involved i r r a d i a t i o n of the H10 o l e f i n i c lactone proton (7.11ppm) and resulted i n p o l a r i s a t i o n transfer into the carbon resonances at 77.8(C9), 134.2(C11) and 173.1(C19)ppm. Proposed *H and 1 3 C NMR assignments are included i n Tables 25. and 26. Several observations i n the NMR spectra indicated that rubiformate (122) e x i s t s i n two low energy conformations i n s o l u t i o n . The methyl resonance at 2.23ppm (Mel8) appeared as an apparent "doublet". However, i r r a d i a t i o n of t h i s resonance did not sharpen a sign a l with an equivalent coupling. S i m i l a r l y , sequential i r r a d i a t i o n of a l l resonances i n the spectrum did not reduce the methyl s i g n a l to a s i n g l e t . At 400MHz the methyl 189 "doublet" (Me-18) had a "coupling constant" of 2.7Hz as compared to 2.1Hz at 300MHz and at 80MHz the coupling was not observed at a l l . The o l e f i n i c lactone resonance at 7.11ppm (H10) appeared as a m u l t i p l e t possessing a large coupling of approximately 5.8Hz, the resonance was more complex and broader than that observed for metabolites 167-176. In addition to these ambiguities the H8 methylene protons at 2.98 and 3.04ppm did not decouple to simple doublets on i r r a d i a t i n g the lactone ca r b i n o l resonance at 5.29ppm (H9). In the *H NMR spectrum obtained i n deuterated benzene (CgDg) (see Figure 50.) the Hl6a isopropenyl o l e f i n i c resonance at 4.62ppm appeared as an apparent "doublet" of multiplets with j=5.7Hz. No coupling partner for J=5.7Hz could be located by decoupling experiments. The H8a and H8b protons, both appearing as two sets of two pairs of doublet of doublets, resonating between 1.69-1.80ppm (for both p a i r s J*s=7.2,5.1Hz) and between 2.11-2.22ppm (downfield dd, J=10.8,6.3Hz, and u p f i e l d dd, J=ll.l,6.3Hz), r e s p e c t i v e l y , were coupled only to t h e i r geminal H8 partner and the H9 lactone proton (4.78ppm). Ambiguities, in the methyl signals resonating between 1.46-1.58ppm and the o l e f i n i c lactone resonance at 6.52ppm (H10) were also noted. In both CgDg and CDCI2 the r a t i o of the two conformers was approximately 5:4 (evidenced by the NMR in t e g r a t i o n s ) . In the 1 3 C NMR spectrum the C5 resonance at 125.5ppm appears as a broad s i g n a l and many other resonances appear as two signals l y i n g v i r t u a l l y on top of one another. I t i s u n l i k e l y that the problem r e s u l t s from the presence of two c l o s e l y related metabolites, 190 such as a pair of diasteriomers, for one would expect the 13 chemical s h i f t s of at l e a s t some of the C NMR resonances to be s i g n i f i c a n t l y d i f f e r e n t as was noted for the two metabolites epilophodione (171) and lophodione ( 1 8 8 ) A l s o during the i s o l a t i o n there was no evidence that two compounds were present i n the rubiformate (177) f r a c t i o n . The existence of two conformations has a precedent; i t was noted that both gersemolide (167) and isoepilophodione B (173) existed i n two low energy conformations. Variable temperature "^H NMR experiments would be informative to the problem, but were precluded by the decomposition of the metabolite. Chemical studies aimed at interconverting rubiformate (177) to a c r y s t a l l i n e ene-dione or methyl enol ether, for the purpose of x-ray a n a l y s i s , were unsuccessful. Rubiformate (177) was treated, under both r e f l u x and non-reflux conditions, with potassium carbonate/"wet" methanol or "wet" acetone followed by the subsequent addition of the methylating agent, methyl iodide. Under the various conditions employed decomposition of rubiformate r e s u l t e d , to y i e l d a complex mixture of very polar compounds which were not characterised further. The structure of rubiformate (177) can only be viewed as a tentative proposal; an unambiguous s t r u c t u r a l proof, through x-ray analysis of a c r y s t a l l i n e d e r i v a t i v e or synthesis, i s required (v). Sesquiterpene 178; (+)-7?-Cubebene-3-acetate (178). obtained as a clear o i l , did not give a parent ion i n the mass spectrum. However, a carbon 192 s 15 r 160 140 120 100 80 60 40 20 PPM 0 Pigure 51. C NMR spectrum of {+) -yj-cubebene-3-acetate (122.). 13 count in the C NMR spectrum (Figure 51.) of 178 indicated the 205 presence of seventeen carbons. An APT experiment demonstrated that metabolite 178 consisted of three quaternary, s i x methine, four methylene and four methyl carbons (see Table 28.). The IR — l 13 absorption at 1732cm , the C NMR resonances at 21.6(CH 3), 76.1(CH) and 170.4(C)ppm and the resonance at 5.39(d, J=8.0Hz, lH)ppm i n the NMR spectrum of 178 (Figure 52.) indicated the presence of a secondary acetate. In the MS of 178. a fragment ion at a m/z of 202 daltons ( C 1 5 H 2 2 , observed m/z 202.1713, requires 202.1722), corresponding to the loss of a c e t i c acid v i a a McLafferty rearrangement, confirmed the presence of the acetate. I t i s known that l o s s of a c e t i c acid i s p a r t i c u l a r l y f a c i l e f o r 197 a l l y l i c acetates on s i x membered rings . The preceeding arguments indicated that (+)-/9-cubebene-3-acetate (178) had a molecular formula of ci7 H26°2* Subtraction °f the acetate moiety from t h i s molecular formula l e f t f i f t e e n carbons, thus 178 was probably a sesquiterpene acetate containing f i v e units of unsaturation. A number of other s t r u c t u r a l fragments were apparent from s p e c t r a l evidence. 1 3 C NMR resonances at 109.0(CH2) and 152.8(C)ppm and the two broad one proton s i n g l e t resonances at 5.01 and 5.05ppm i d e n t i f i e d an exocyclic methylene. A shielded one proton mu l t i p l e t resonating at 0.58ppm t e n t a t i v e l y suggested the presence of a cyclopropyl group. In the MS, a base peak was observed at a m/z of 159 daltons corresponding to the loss of a C 3Hg fragment (m/z 43) from the C 1 5 H 2 2 fragment ion. The three methyl signals resonating between 0.90-0.96ppm i n the *H NMR spectrum a l l appeared as doublets, and hence the C^Hg fragment 193Table 28. C NMR assignments for ( + )-/?-cubebene-3-acetate (178). Chemical s h i f t , ppm. Chemical s h i f t ppm. Carbon # Carbon # 1 37.4(C) 10 30.4(CH) Y 2 38.9(CH 2) Z 11 109.0(CH2) 3 76.KCH) 12 26.5(CH) Y 4 152.8(C) 13 18.8(CH 3) X 5 44.2(CH) Y 14 19.8(CH 3) X 6 34.6(CH) Y 15 20.0(CH 3) X 7 33.5(CH) Y 16 170.4(C) 8 31.1(CH 2) Z 17 21.6(CH 3) X 9 31.2(CH 2) Z Assignments may be interchanged. probably resulted from the l o s s of an isopropyl residue. Two units of unsaturation were accounted for by the acetate carbonyl and the o l e f i n i c e x o c y c l i c methylene. Hence, (+)-/?-cubebene-3-acetate (178) had to possess three carbocyclic rings, one of which was assigned to a cyclopropyl r i n g . The sesquiterpenoid cubebene structure 178 e f f e c t i v e l y accomodated a l l the i d e n t i f i e d s t r u c t u r a l fragments. The connectivity from the H2 methylene protons through to the H l l protons was established by a s e r i e s of decoupling experiments. I r r a d i a t i o n of the "^H NMR carbinol resonance at 5.39(d, J=8.0Hz, H3)ppm sharpened 195 the exocyclic methylene s i n g l e t resonances (5.01 (Hlla) and 5.05ppm (Hllb)) and reduced the one proton doublet of doublets (J=14.4f8.0Hz) at 2.38ppm (H2b) to a doublet (J=14.4Hz). These results demonstrated an a l l y l i c coupling between H3 and the exocyclic methylene protons (Hlla and H l l b ) . I r r a d i a t i o n of H2b (2.38ppm) reduced the carbinol resonance at 5.39ppm (H3) to a s i n g l e t and collapsed the part of the multiplet resonating between 1.55-1.67ppm possessing the large doublet coupling of 14.4Hz. The magnitude of the coupling suggested that H2b possessed a geminal partner. The geminal partner, H2a, had only a small coupling (<0.5Hz) to the v i c i n y l H3. On i r r a d i a t i n g at 1.62ppm (H2a) the carbinol doublet at 5.39ppm (H3) sharpened and the H2b doublet of doublets (2.38ppm) s i m p l i f i e d to a doublet (J=8.0Hz). Thus, (+)-/?-cubebene-3-acetate (178) possessed the above described f i v e - s p i n system and the exocyclic methylene and acetate moieties were placed at t h e i r appropriate p o s i t i o n s i n structure 178. (+)-^-Cubebene-3-acetate (178) rapidly decomposed to give a complex mixture of very polar material when i t was exposed to NMR solvents, other manipulations, or when l e f t standing. Hydrogenation of 178 resulted i n a mixture of two dihydro epimers that proved to be very stable compounds. The epimers, which were not readily seperable, were obtained i n a r a t i o of 4:1 as evidenced by the 1H NMR spectrum (Figure 53.). The fragmentation patterns of the two epimers were v i r t u a l l y i d e n t i c a l on GCMS analysis, however, neither epimer gave a parent ion. In the EIMS and FAB MS of the mixture, very low i n t e n s i t y M + peaks ( r e l . , i n t e n s i t y <0.6) at m/z of 264 daltons were observed. Fragment 197 peaks at m/z of 204 and 161 (100%) daltons corresponded to the loss of acetic acid followed by the subsequent loss of the isopropyl residue. AcO. AcO v 192 193 A seri e s of NOE and spin-spin decoupling experiments v e r i f i e d that 178 represented the correct structure for the natural product (+)-f?-cubebene-3-acetate and that the dihydro epimers 192 and 193 represented the major and minor products, respec t i v e l y . I r r a d i a t i o n of the H3 t r i p l e t resonance (J=7.2Hz), of the major epimer 192. at 5.12ppm reduced H2b at 2.33(dd, J=14.8f 7.2Hz, lH)ppm to a doublet (J=14.8Hz) and sharpened the H4 multiplet at 2.47ppm. Equivalent decouplings were apparent for the minor epimer, 193. upon i r r a d i a t i n g at 4.67ppm (H3). I r r a d i a t i o n of the cyclopropyl methine resonance at 0.54ppm (H6) markedly sharpened the obscured H5 "multiplet" at 0.83ppm and sharpened the H7 mul t i p l e t at 1.38ppm. I r r a d i a t i o n at 0.83ppm (H5) sharpened both the H6 multiplet at 0.54ppm and the methine H4 multiplet resonance at 2.47ppm. As expected, i r r a d i a t i o n of H4 (2.47ppm) markedly sharpened H5 (0.83ppm), s i m p l i f i e d the carbinol H3 t r i p l e t (5.12ppm) to a doublet and collapsed the Mell doublet (J=6.0Hz) at 0.97ppm to a s i n g l e t . Hence, connectivity was extended from the methylene H2 protons to the methine H7 199 proton for both epimers. Subsequent NOE experiments (the r e s u l t s are summarized i n Table 29.) allowed the r e l a t i v e stereochemistry from the H2b proton through to the H5 proton to be defined f o r both the major and minor dihydro epimers 192 and 1£3_, r e s p e c t i v e l y , and hence that of (+)-/?-cubebene-3-acetate (178) was also established. Table 29. NOE r e s u l t s observed for the dihydro epimers, 192 and 122.. Protons with observed NOE i n : Proton i r r a d i a t e d 192 193 H2b H3 H3 Mel5 ? H3 H2b H2b H4 X H4 H3 X H5 ? Mell ? ?-not possible to say whether NOE resulted. X-no equivalent NOE resulted for 193. A H NMR spectrum of the epimer mixture, 192 and 193. was obtained i n deuterated benzene (CgDg) (see Figure 54.). Many resonances were s h i f t e d , p a r t i c u l a r l y the H5 and H6 cyclopropyl methine resonances at 0.64(m, 1H) and 0.50(m, lH)ppm, r e s p e c t i v e l y . I r r a d i a t i o n of the methyl resonance at 0.86(d, J=7.2Hz)ppm (Mel5) induced NOE's into the H5 resonance at 200 s Figure 54. H NMR spectrum of the mixture of dihydro epimers 192 and 121 in C CD C. 0.64ppm, the methylene H2b resonance at 2.18ppm and into part of the two multiplet regions resonating between 1.35-1.40 and 1.48-1.66ppm. The H2b proton possessed a /9-configuration as did H5 and hence, the r e l a t i v e configuration at CIO was defined, i . e . , Mel5 also possessed a ft-configuration. I r r a d i a t i o n of the methine H6 resonance at 0.50ppm induced an NOE, after c a r e f u l inspection, into the methine H4 resonance of the minor epimer 193 at 2.20ppm. The r e s u l t was consistent with the observation that no NOE existed between the two cyclopropyl protons, H5 and H6, i e . , H6 possessed an a-configuration and hence, the two protons were disposed i n a trans arrangement. The preceeding arguments were t o t a l l y consistent with the gross s t r u c t u r a l c o n s t i t u t i o n of 178. NOE experiments and coupling constant arguments established the r e l a t i v e configuration at a l l of the c h i r a l centers except C7. The r e l a t i v e configuration given to the C7 c h i r a l carbon and represented i n structure 178 was consistent with the re s u l t that no NOE was observed between the methine H6 resonance, centered at 0.54ppm, and the H7 methine mu l t i p l e t at 1.38ppm (assignment based on a decoupling r e s u l t , see above). The configurational assignment at C7 was also consistent with the observation that the three 1 3 C NMR methyl resonances of C13, C14 and C15 were v i r t u a l l y i d e n t i c a l with the appropriate carbon resonances reported for (-)-epicubebol (121) 2 0 8, (+)-cubebol (125J 2 0 9 and 210 (±)-/?-cubebene-nor-ketone (196) (see Table 30.). Since 13 . C NMR i s an excellent method for comparing the r e l a t i v e stereochemistries of s i m i l a r molecules, one would expect that i f the r e l a t i v e stereochemistry at either the C7 or CIO carbons were 202 194 195 196 3 3 Table 30. C NMR data comparison of the methyl resonances of H f i r ISA, 1£5_ and 19_£. Chemical s h i f t , ppm. Carbon # 12£ 194 a 195 b 196 c 13 18.8 Z 1 9 . l z 18.7 Z 18.9 Z 14 19.8 Z 19.8 Z 19.6 Z 19.5 Z 15 20.0 Z 20.0 Z 20.0 Z 20.0 Z areference 208, 100MHz, CDC1 3. ^reference 209, spectrometer frequency not reported, CDCl^. c a u t h e n t i c sample kindly provided by Dr. E. Piers of the University of B r i t i s h Columbia. Assignments within a column may be interchanged. d i f f e r e n t the chemical s h i f t s of the C13, C14 and C15 methyl resonances would be s h i f t e d by a ppm or two. Sesquiterpenes i s o l a t e d from s o f t corals and i n fact from coelenterates in general tend to exi s t as the o p t i c a l antipod of the form found i n t e r r e s t r i a l and marine plants. This i s exemplified by the two metabolites 194 and 195. i s o l a t e d from the 203 197 198 199 brown alga D i c t v o p t e r i s d i v a r i c a t a z u o and a s o f t c o r a l , a 209 C e s p i t u l a r i a species , r e s p e c t i v e l y . They e x i s t as the o p t i c a l antipodes of one another (ignoring the stereochemistry at C-4, 211 (-)-cubebol i s known from t e r r e s t r i a l plants ) and are s p e c t r o s c o p i c a l l y i d e n t i c a l . S i m i l a r l y , a - and /?-cubebene (197 and 198. respectively) i s o l a t e d from the o i l of Piper cubeba L. 212 both showed negative o p t i c a l rotations and (+)-a-cubebene (199) i s o l a t e d from gorgonians of the genus Pseudoplexaura 98 possesses a p o s i t i v e o p t i c a l r o t a t i o n . Thus, since (+)-/?-cubebene-3-acetate f178) has a p o s i t i v e o p t i c a l r o t a t i o n and i s derived from a s o f t c o r a l i t i s suggested that i t s absolute configuration i s as represented i n structure 178. There were a few anomalies i n the NMR spectra (Figures 52-54., and Table 31.). A number of the coupling patterns could not r e a d i l y be r a t i o n a l i s e d . An example i s provided by the 21 cyclopropyl H6 m u l t i p l e t (0.58ppm). An authentic sample of 196 had an i d e n t i c a l coupling pattern for H6. The complexity was probably the r e s u l t of observed long range (3 and 4 bond) couplings. These couplings may be a t t r i b u t e d to the extreme r i g i d i t y of (+)-/?-cubebene-3-acetate (178). e a s i l y demonstrable by a molecular model, which r e s u l t s i n a v a r i e t y of possible W-204 Table 31. H NMR assignments for (+)-/?-cubebene-3-acetate (178) and the dihydro epimer 192. Chemical s h i f t , ppm. Carbon # 122. 122a 2a 1.55-1.67(m) 1.52-1.65(m) b 2.38(dd, J=14.4, 8.0Hz, IH) 2.33(dd, J=14.8,7.2Hz, IH) 3 5.39(d, J=8.0Hz, IH) 5.12(t, J=7.2Hz, IH) 4 / 2.47(m, IH) 5 0.80-0.90(obscured) 0.83(m, IH) 6 0.58(m, IH) 0.54(m, IH) 7 1.55-1.67(m) z , y 1.38(m, IH) 8 1.37-1.48(m, 2H) y ? 9 1.05-1.15(m, 2H) y ? 10 1.27(m, l H ) z ' y ? 11a 5.01(bs, IH) 0.97(d, J=6.0Hz, 3H) b 5.05(bs, IH) / 12 1.74(m, IH) 1.69(m, IH) 13 0.90-0.96(3 x d's) 0.88-1.03(m) 14 0.90-0.96(3 x d's) 0.88-1.03(m) 15 0.90-0.96(3 x d's) 0.94(d, J=6.7Hz, 3H) 17 2.01(s, 3H) 2.01(s, 3H) a w i t h i n t h i s column could not assign "?". z , yassignments within a column may be interchanged. coupling arrangements. In a r i g i d system W-coupling can be as 197 1 13 large as 7Hz A . Proposed H and x C NMR assignments for (+)-/?-205 cubebene-3-acetate (178) are included in Tables 28. and 31.. The proposed NMR assignments for the major dihydro epimer 192. are also included i n Table 31. (vi) . Pukalide (£1) : Pukalide (£3.) , obtained as a white c r y s t a l l i n e s o l i d , was i d e n t i f i e d by comparison of i t s physical and spec t r a l data with l i t e r a t u r e v a l u e s 1 1 4 and authentic IR and 1H NMR spectra k i n d l y provided by Dr. P. J . Scheuer of the University of Hawaii. ( v i i ) . B r i a r e i n d i t e r p e n o i d s 1 4 8 , U3. and. 1 M : Ptilosarcenone (179). obtained as an amorphous s o l i d , was i d e n t i f i e d by comparison of i t s physical and spec t r a l data with 213 1 l i t e r a t u r e values and an authentic H NMR spectrum kindly provided by Dr. D. J . Faulkner of the Scripps I n s t i t u t i o n of Oceanography. Butanoate 178 was obtained as a glass that showed a parent ion i n the EIMS at a m/z of 508.1873 daltons corresponding to a molecular formula of C 2gH 3 3ClOg (requires a m/z of 508.1865 daltons) which required ten units of unsaturation. Spectral data, p a r t i c u l a r l y the 1H NMR spectrum (Figure 55.), indicated that 180 was c l o s e l y related to ptilosarcenone (12SJ (see Table 32). However, one of the acetate resonances i n the 1H NMR spectrum of 179 was replaced i n the spectrum of 180 by resonances at 0.99(t, J=7.2Hz, 3H), 1.71(m, 2H) and 2.38(t, J=7.2Hz, 2H)ppm which were assigned to a butanoate ester. A series of NOE experiments demonstrated that the butanoate 206 207 Table 32. H NMR assignments for butanoate 180 and s p e c t r a l comparison with 179. Chemical s h i f t , ppm. Carbon # lfifl. 179 a 2 5.79(d f J=8.7Hzf IH) 5.72(d r J=9Hz, IH) 3 5.60(dd, J=12.4,8.7Hz, IH) 5.63(dd, J=12,9Hz, IH) 4 S ^ - S ^ f m ) 1 5.99(d, J=12Hzr IH) 6 5.23(dm, J=3.9Hz,lH) 5.26(ddd, J=4,2,lHz, IH) 7 4.98(d r J=3.9Hz, IH) 5.01(d, J=4Hz, IH) 9 5.46(d, J=7.5Hzf IH) 5.49(d, J=7Hzf IH) 10 2.73(m, IH) 2.82(m, IH) 11 2.79(mf IH) 2.82(m, IH) 13 5.87(d, J=10.4,0.9Hz, IH) 5.90(d, J=llHz, IH) 14 6.58(d,J=10.4Hz, IH) 6.61(d, J = l l H z f IH) 15 1.17(s, 3H) 1.18(s, 3H) 16a S ^ - S ^ d i i ) 1 5.96(dd, J=2 f lHz flH) b 6.12(dm, J=0.9Hz, IH) 6.12(dd f J=l,lHz, IH) 17 2.36(q, J=7.2Hzf IH) 2.41(q f J=7Hz, IH) 18 1.20(d, J=7.2Hzf 3H) 1.21(d, J=7Hz, 3H) 20 1.30(d f J=7.2Hz, 3H) 1.30(d f J=7Hzf 3H) 22 2.38(t, J=7.2Hz, 2H) 2.15(s, 3H) j 23 1.71(m, 2H) / 24 0.99(t, J=7.2Hzf 3H) / 26 2.20(s, 3H) 2.20(s, 3H)^ -OH 3.42(s, IH) 3.43(s, IH) cont'd. 208 Table 32. continued. a r e f e r e n c e 213a, 220MHz, solvent not reported. 1 s i g n a l s l i e p a r t i a l l y on top of one another. •'chemical s h i f t s obtained from my spectra, and may be interchanged. The assignment of the resonance at 2.20ppm should be to the Me24 protons in metabolite 179. ester i n 180 was attached to C2, while the acetate ester was attached to C9. I r r a d i a t i o n of the acetate methyl resonance at 2.20ppm (Me26) induced NOE's into the Mel5, H3 and H7 protons resonating at 1.17, 5.60 and 4.98ppm, res p e c t i v e l y . Simultaneous i r r a d i a t i o n of the butanoate a-methylene (H22's) proton resonance at 2.38ppm and the H17 resonance at 2.36ppm induced NOE's into the resonances at 4.98 (H7, from H17), 5.46 (H9, from H17) and 1.20ppm (Mel8, from H17). The i r r a d i a t i o n also induced NOE's into the resonances at 6.58 (H14, from the butanoate a-CH 2), 1.71 (methylene H23, from the butanoate a-CH2) and 0.99ppm (Me24, from the butanoate e*-CH2). Hence, the butanoate and the acetate had to be placed at the C2 and C9 p o s i t i o n s , r e s p e c t i v e l y . I r r a d i a t i o n of the hydroxyl -OH proton (3.42ppm) attached to C8, demonstrated s t r u c t u r a l consistencies by inducing NOE's into the methyl resonance at 1.20ppm (Mel8) and the H10 and H l l methine m u l t i p l e t s at 2.73 and 2.79ppm, re s p e c t i v e l y . Proposed 1H NMR assignments are included i n Table 32. 209 D_. B i o l o g i c a l A c t i v i t i e s . I n i t i a l i n t e r e s t i n the chemistry of Gersemia rubiformis was sparked by the potent i n v i t r o antimicrobial a c t i v i t i e s shown by crude methanolic e x t r a c t s . None of the pure sesquiterpenes and diterpenes, which were abundant enough to be tested, showed appreciable a n t i m i c r o b i a l a c t i v i t y . It was not p o s s i b l e to t e s t the very minor or unstable and quite p o s s i b l y the most in t e r e s t i n g metabolites i n any of the bioassays ( A 4 , 5 Z , A 7 ' 1 7 -isogersemolide (168). A 4 , 5 E , A 7' 1 7-isogersemolide (169) . the unknown diterpene, gersolide (176) and rubiformate (177) were not tested). Rubifolide (170) and epilophodione (171) showed no i n s e c t i c i d a l a c t i v i t i e s i n a confused beetle bioassay (Tribolium  confusum), and although both 170 and 171 showed mild h e r b i c i d a l a c t i v i t y i n a Lemna paucicostata (duck weed) bioassay, when tested on higher plants no a c t i v i t y was observed. The metabolites 165-177 i s o l a t e d from Gersemia rubiformis were present i n r e l a t i v e l y high concentrations (see Experimental for comprehensive d e t a i l s ) . The t o t a l percentage y i e l d of —2 terpenoid metabolites was 2.8 x 10 %. Hence, i t seems reasonable to expect that a number, i f not a l l of these metabolites would exh i b i t a b i o l o g i c a l r o l e / a c t i v i t y . Of the metabolites tested, dihydrotochuinyl acetate (166) exhibits f i s h antifeedant a c t i v i t y . Metabolite 166 completely i n h i b i t e d feeding at a concentration of 0.15mg/pellet (0.0056mg/mg of food p e l l e t ) though there was a marked preference for the control at concentrations as low as 0.005mg/pellet (0.0002mg/mg of food p e l l e t ) . Since the concentration of metabolite 166 per mg of £. 210 rubiformis was O.Ollmg and the average concentration obtained from Toauina tetraquetra per animal was 0.35mg (C.00004mg/mg of nudibranch), the experimental i n h i b i t o r y concentration i s very comparable. Thus, i t would appear that £. rubiformis u t i l i s e s a defensive allomone and the highly s p e c i a l i s e d feeder, the nudibranch, T_. tetraquetra has learned to ingest and sequester metabolite 166 for the purpose of i t s own defense. This observation p o s s i b l y provides an explanation for the marked 122a dietary dependency of the nudibranch 203 Diterpenes, such as pseudopterolide (185) , k a l l o l i d e A (1122) 1 5 7 and lophotoxin ( 2 M ) 1 0 9 , that are s t r u c t u r a l l y related to metabolites 167-177 exhibit diverse b i o a c t i v i t i e s . 203 Pseudopterolide (185) shows potent cytotoxic a c t i v i t y , 157 k a l l o l i d e A (107) e x h i b i t s potent antiinflammatory a c t i v i t y and lophotoxin (200) f co-occurring with pukalide (£3 . ) , i s a i n o neuromuscular toxin . The diterpenes 167-177 w i l l l i k e l y y i e l d s i m i l a r r e s u l t s . The preliminary i n v i t r o cytotoxic a c t i v i t i e s of r u b i f o l i d e (170) and epilophodione (171) are reported i n Table 33. Epilophodione (171) i s currently undergoing i n vivo screens. CH0 211 Table 33. Results of in. v i t r o c y t o t o x i c i t y assays for r u b i f o l i d e (HQ.) and epilophodione (171) . (IC-50, ng/mL of active d i l u t i o n ) . C e l l l i n e s : Metabolite A549-human lung murine melanoma human colon 170 na na na 171 27 24 5.8 Moser-human lung SWl271-human lung 170 na na 171 6.8 6.6 na-no s i g n i f i c a n t a c t i v i t y at maximum concentrated tested. B i o l o g i c a l a c t i v i t i e s of the novel a l l y l i c acetate, (+)-/?-cubebene-3-acetate (178) were precluded due to i t s i n s t a b i l i t y , and for s i m i l a r reasons pukalide (£3_), ptilosarcenone (179) and the butanoate 180 were not test e d . £.. Biosvnthetic Proposals. A biogenetic pathway for tochuinyl acetate (167) and dihydrotochuinyl acetate (166), proceeding through the epoxide containing intermediate 201. i s proposed in Scheme 1. The pathway involves the conversion of farnesylpyrophosphate (2112) to the bisabolene intermediate 203. Generation of the parent alcohol 204 followed by a c e t y l a t i o n , leads to lfLfc. Dehydrogenation followed by a c e t y l a t i o n r e a d i l y gives 1£5_. It may be that the 212 aromatic metabolite 165 i s an a r t i f a c t . The cembrane skeleton f205) can obviously be generated from geranylgeranylpyrophosphate f206), as i l l u s t r a t e d i n Scheme 2. 213 Scheme 2. Biogenetic proposals for cembrane, pseudopterane, gersolane and degraded 13-membered ring diterpenoids, 167-177. 214 B i o l o g i c a l oxidations and other secondary transformations would eventually give metabolites 171-175. and subsequent reduction of an ene-dione moiety followed by condensation would lead to a furan-containing skeleton as required by r u b i f o l i d e f170). The co-occurrence of both ene-dione and furano- f u n c t i o n a l i t i e s was previously noted i n the gorgonian Lophogorgia alba from which the ene-dione metabolites lophodione (188) and isolophodione f189) and the furan-containing metabolites pukalide (£3_) and lophotoxin (200) were i s o l a t e d 1 ^ . I t has been suggested that the metabolites 188 and 189 may represent possible precursors to the furan-containing cembrenolides. 203 In t h e i r report of pseudopterolide (185). Fenical et a l . suggested that the pseudopterane carbon skeleton, which s u p e r f i c i a l l y looks l i k e a simple dimerization of two geranyl residues, might instead a r i s e from ring contraction of a cembranoid precursor. The gersolane skeleton of 176 and the proposed skeleton of rubiformate (180), l i k e the pseudopterane skeleton of 185, also appear s u p e r f i c i a l l y to have been formed by coupling of two geranyl units with subsequent secondary transformations. Since Gersemia rubiformis contains s i m i l a r l y f u c t i o n a l i z e d diterpenoids having cembrane, pseudopterane and gersolane skeletons etc., i t seems more l i k e l y that the l a t t e r two (or three) skeletons a r i s e from rearrangements of a cembrane p r e c u r s o r * 9 0 * 3 ' 2 0 3 , as i l l u s t r a t e d i n Scheme 2. Epilophodione (171) and gersemolide (167), for example, are i n p r i n c i p l e d i r e c t l y related by such a contraction (through an a l l y l i c rearrangement), although i t i s l i k e l y that the contraction would take place before complete f u n c t i o n a l i z a t i o n of the hydrocarbon 215 skeleton (see Scheme 2.). The presence of both ene-dione and furano- f u n c t i o n a l i t i e s i n pseudopterolides was noted i n the k a l l o l i d e s A-C (107. 1B6P 1 £ 2 ) 1 5 7 , and hence the interconversion of pseudopterane metabolites to r u b i f o l i d e (170) or v i s a versa also appears f e a s i b l e . (+)-/?-Cubebene-3-acetate (17g) could be generated by f o l d i n g of a farnesylpyrophosphate precursor with subsequent oxidation at the appropriate carbon i n the manner represented i n Figure 56. However, the precursor from which cubebanes are considered to be derived i s the cadinane skeleton (207), which i s i t s e l f derived 214 from either the bisabolene or germacrene carbonium ions ( 2 M and 209. respectively) shown i n Scheme 3.. Figure 56. Biogenetic speculation for (+)-/?-cubebene-3-acetate (12fi>. £. Further Observations and Discussion. Tochuinyl acetate (165) and dihydrotochuinyl acetate (166) represent, to the best of our knowledge, the f i r s t examples of cuparane sesquiterpenes i s o l a t e d from a soft c o r a l . The cuparane sesquiterpene skeleton, however, i s well documented in the 216 Scheme 3. Biogenetic proposal for (+) -f?-cubebene-3-acetate 210 marine environment, p a r t i c u l a r l y from red algae of the genus 215 Laurencia . In nearly a l l cases the metabolites are 216 halogenated, most often with bromine . a-Bromocuparene 217 (210) . for example was i s o l a t e d from L . g l a n d u l i f e r a . Cuparanes i s o l a t e d from molluscs, for example the sea hare Aplysia d a c t y l o m e l a 6 2 b . are known, but these are probably derived from a l g a l dietary sources. The i s o l a t i o n of diterpenes 167-177 from Gersemia rubiformis 217 c o l l e c t e d o f f the west coast of Canada i s of i n t e r e s t for a number of reasons. The pseudopterane diterpenoids, only recently 157 203 discoverd by Fenical's group ' , represent a family of Alcyonarian metabolites that have a previously unknown carbon skeleton. To date, the pseudopteranes have been taxonomically r e s t r i c t e d to the order Gorgonacea. Hence, the pseudopteranes 167-169 are the f i r s t examples is o l a t e d from any source other than a gorgonian. Secondly, G_. rubiformis represents the f i r s t organism to contain both pseudopterane and cembrane diterpenes, a f a c t that has biosynthetic implications (see preceeding s e c t i o n ) . F i n a l l y , fi. rubiformis contains gersolide (176) and rubiformate (177). the f i r s t examples of diterpenes (though 177 would appear to be a degraded diterpene) possessing new rearranged carbon skeletons with 13-membered rings. The B r i t i s h Columbian specimens of Gersemia rubiformis were co l l e c t e d on four occasions. The c o l l e c t i o n s were made i n May 1986, A p r i l 1987 (spring), August 1985 (summer) and October 1986 ( f a l l ) . On each occasion, a l l the metabolites 165-177 were present. The unknown diterpene was i s o l a t e d only from the May c o l l e c t i o n . The percentage y i e l d s of the metabolites were at a maximum for the c o l l e c t i o n s made i n August and October (quoted i n the Experimental). A l l percentage y i e l d s were at l e a s t halved i n the two spring c o l l e c t i o n s . The y i e l d s of the two pseudopterolides, 168 and 169. isoepilophodione A (122)t r u b i f o l (175), gersolide (176) and rubiformate (177) were reduced to trace q u a n t i t i e s (<0.2mg). Such observations imply that there i s a "slow down" in metabolic prossesses during the winter months, 218 and that either the metabolites are simply degraded and excreted, or are used for a b i o l o g i c a l purpose. Rubifolide f 1 7 0 ) , the major secondary metabolite i s o l a t e d from G_. rubiformis. i s i n a c t i v e i n a l l the b i o l o g i c a l screens undertaken thus far (see preceeding s e c t i o n ) . The p o s i t i o n of 170 at the end of possible converging biosynthetic pathways implies that i t i s the biosynthetic end or "waste" product of the diterpenoid biosyntheses. (see preceeding 1 57 s e c t i o n ) . However, Fenical et a l . have demonstrated that the furan-containing metabolite k a l l o l i d e A (107) can be converted to k a l l o l i d e C ( 1 8 7 ) through s i n g l e t photooxidation of 107. This may mean therefore that 187 i s not a true secondary metabolite, or on the other hand i t may mean that 107 and thus r u b i f o l i d e ( 1 7 0 ) are the i n i t i a l b iosynthetic precursors for a l l the other diterpenoids present i n the respective organisms (see Scheme 2 . ) . The two pseudopteranes 168 and 1 6 9 . the four isomers 1 7 1 -174. r u b i f o l (175) and the unknown diterpene are a l l probably true secondary metabolites. The metabolites 168. 1 6 9 . 171-174 and 175 were always present from r a p i d l y processed extracts. Also gersemolide (167) only p a r t i a l l y isomerised to A 4 , 5 Z , A 7 ' 1 7 -isogersemolide (168) af t e r extensive reflux i n the presence of a c a t a l y t i c quantity of formic a c i d , no reaction was observed at room temperature with 48 hours of s t i r r i n g . Rubifol (175) and the unknown diterpene could not be prepared by treatment of eplilophodione ( 1 7 1 ) , or isoepilophodiones A ( 1 7 2 ) , B (173) and C (174) with water or methanol i n the presence of c a t a l y t i c amounts of formic a c i d . None of the metabolites, 1 6 5 - 1 7 7 . i s o l a t e d from west coast Gersemia rubiformis were found in specimens c o l l e c t e d on the east 219 coast of Canada. Instead, the s t e r o i d a l metabolites JL5£.-1£1 186 previously reported by Kingston et a l . were noted, along with the rather unstable sesquiterpene acetate, (+) -/?-cubebene-3-acetate f178), possessing the cubebane skeleton. Cubebane sesquiterpenoids have been previously reported i n other 209 Alcyonarian species, for example, (+)-cubebol (195) and (+)-a-9 8 cubebene (199) were i s o l a t e d from soft coral and gorgonian species, r e s p e c t i v e l y . The skeleton i s also well represented i n both t e r r e s t r i a l and marine plants, though generally i n these sources they e x i s t as the o p t i c a l antipod of the metabolites 218 noted i n coelenterates . Oxygenation at C3 has, as far as we know, not previously been reported. The role of metabolite 178 i n the host organism remains unknown. Its i n s t a b i l i t y precluded bioassay studies. Since the west coast specimens of £. rubiformis possess a chemical defense i t i s l i k e l y that the east coast animals u t i l i z e a s i m i l a r defensive strategy. Hence, sesquiterpene 178 may have a defensive r o l e . Gersemia rubiformis i s a species of the family Naphtheidae. The chemistry of t h i s family was reviewed on page 70.. The chemistry of £. rubiformis bears l i t t l e resemblance to that previously noted for the family. The discovery of i n t e r e s t i n g metabolites i n a cold water s o f t c o r a l , some of which are v i r t u a l l y i d e n t i c a l to those previously reported from t r o p i c a l s o f t corals and gorgonians, suggests that although not many species of Gorgonacea and Alcyonacea grow in cold temperate waters the species that do might reasonably be expected to have chemistry s i m i l a r to t h e i r t r o p i c a l r e l a t i v e s . 220 A l l of the metabolites present i n the skin extracts of Toouina tetraquetra. with the exception of pukalide ( £3J , can be d i r e c t l y traced to the coelenterates that make up i t s d i e t . Tochuinyl acetate f165), dihydrotochuinyl acetate f166) and r u b i f o l i d e (170) are a l l metabolites of the sof t c o r a l Gersemia rubiformis which i s the major dietary organism of the Port Hardy 122a animals . Pukalide has been reported as a metabolite of the t r o p i c a l soft c o r a l , Sinularia flbrupta114r and the t r o p i c a l gorgonians, Lophooorgia r i g i d a 1 0 9 ' 1 1 0 . and L. a l b a 1 1 1 . However, £. rubiformis i s the only s o f t coral reported i n B r i t i s h 32 Columbian waters and we have not been able to f i n d pukalide (63) in fi. rubiformis extracts. Thus, the exact o r i g i n of pukalide (£3.) remains unknown. It could be an extremely minor metabolite that has eluded us thus f a r , i t could be a metabolite of an as yet unreported s o f t c o r a l , or i t could r e s u l t from a metabolic transformation carri e d out by the nudibranch on some £. rubiformis metabolite. Of i n t e r e s t here i s the fa c t that C o l l has demonstrated convincingly that i n a S i n u l a r i a species of s o f t c o r a l , pukalide (£1) can be found only during one or two days of 219 the year when animals are spawning . Perhaps, therefore, i t i s not s u r p r i s i n g that we f a i l e d to i s o l a t e pukalide (£1) from extracts of G_. rubiformis. Also, with t h i s precedent i t i s not so s u r p r i s i n g to f i n d that the unknown diterpene was i s o l a t e d only from the May c o l l e c t i o n . Further studies into these two observations should be undertaken. Ptilosarcenone (179) has been reported as one of the major metabolites of the sea pen (order Pennatulacea), P t i l o s a r c u s 213 gurneyi . which i s a component of the diet of Bamfield 221 189 specimens of Toquina t e t r a q u e t r a x . An exhaustive study of the metabolites of £. gurneyi c o l l e c t e d at Seattle, Washington and Sidney, B r i t i s h Columbia f a i l e d to uncover the butanoate analogue 180 or any metabolite which could be viewed as a precursor to i t . Probably the chemistry of the Bamfield population of £. gurneyi d i f f e r s s l i g h t l y from S t r a i t of Georgia and Puget Sound populations. Ptilosarcenone (179) was reported to be generated t 213a f a c i l e elimination of butanoic acid from ptilosarcone (211) • To discount the p o s s i b i l i t y that 179 and 180 were i s o l a t i o n a r t i f a c t s , a rapid workup of a chloroform extract of f r e s h l y c o l l e c t e d T_. tetraquetra specimens was ca r r i e d out. Only diterpenes 179 and 180 were found with no trace of the putative parent compounds, for example ptilosarcone (211). It appears that ptilosarcenone ( 1 7 9 ) and i t s butanoate analogue, 180. are the metabolites a c t u a l l y present i n the skin extracts of I . 34 tetraquetra. Nudibranchs possess a c i d i c dietary t r a c t s , j u s t the conditions required f o r the f a c i l e elimination of butanoic acid from ptilosarcone ( 2 1 1 ) . Thus, i t may be that the 2 2 2 metabolites o r i g i n a l l y ingested by the nudibranch were ptilosarcone (211) and the butanoate 180. S p e c i a l i s t feeders, i t was suggested i n chapter I I , can ingest large quantities of otherwise t o x i c or " d i s t a s t f u l " metabolites i n order to gain the benefits of an exclusive food source, etc.. The findings reported here are consistent with t h i s and other suggestions that some dentronotoid nudibranchs feed ex c l u s i v e l y on p a r t i c u l a r coelenterates. Findings indicate that T_. tetraquetra ingests and sequesters the sesquiterpene f i s h antifeedant, dihydrotochuinyl acetate (166). Since J_. tetraquetra c o l l e c t e d at Port Hardy possess a chemical defense i t i s l i k e l y that animals at Bamfield possess a s i m i l a r defensive strategy. Ptilosarcenone (179) i s a known i n s e c t i c i d e and an i n h i b i t o r 213a of enzyme esterases . Perhaps i t i s u t i l i s e d as a defensive allomone by the nudibranch. Ptilosarcenone (179) needs to be tested for f i s h antifeedant a c t i v i t y . The i n s t a b i l i t y of butanoate 180 precluded b i o l o g i c a l assays, but one would suspect due to i t s s t r u c t u r a l s i m i l a r i t y with 179 that i t might possess s i m i l a r a c t i v i t i e s . 223 YI. EXPERIMENTAL. The H NMR spectra were recorded on Bruker WP-80, Varian XL-300 and Bruker WH-400 spectrometers. C NMR spectra were recorded on Varian XL-300 and Bruker WH-400 spectrometers. Tetramethylsilane was used as an in t e r n a l standard for the *H NMR spectra. Deutero-chloroform was used as the NMR solvent unless otherwise stated, and as an i n t e r n a l standard (6=77.Oppm) i n the 13 13 C NMR spectra. The hydrogen attachment i n the C NMR spectra i s given only when i t has been determined by an NMR experiment. Low re s o l u t i o n mass spectra were recorded on an AEI MS902 spectrometer and high r e s o l u t i o n mass spectra were recorded on an AEI MS50 instrument. Low resolution gas chromatograph mass spectra were run on a Kratos MS80RFA spectrometer and CarloErba 4160 gas chromatograph. Infrared spectra were recorded on a Perkin-Elmer 1710 Fourier Transform spectrometer. U l t r a v i o l e t -v i s i b l e spectra were recorded on a Bausch and Lomb Spectronic-2000 spectrophotometer. O p t i c a l rotation measurements were recorded on a Perkin-Elmer 141 polarimeter using a 10cm m i c r o c e l l . A Fisher-Johns apparatus was used to determine melting points and these values are uncorrected. Gas chromatography was performed on a Hewlett Packard 5890A instrument and a flame i o n i s a t i o n detector was used. High performance l i q u i d chromatography (HPLC) was performed on Perkin-Elmer s e r i e s 2 or Waters 501 l i q u i d chromatograph instruments, and Perkin-Elmer LC55 spectrophotometer or Waters 440 absorbance detector and/or Perkin-Elmer LC-25 r e f r a c t i v e index detectors were employed for peak detection. Whatman Magnum-9 P a r t i s i l 10 or 224 Magnum-9 ODS-3 P a r t i s i l 10 columns were used for the preparative HPLC. The HPLC solvents were BDH OmniSolv grade or Fisher HPLC grade; water was glass d i s t i l l e d ; a l l other solvents were reagent grade. Merck S i l i c a Gel 60 PF-254 and pre-coated 60 F 254 s n e e t s were used for preparative t h i n layer and th i n layer chromatography (TLC), r e s p e c t i v e l y . Merck S i l i c a Gel 230-400 Mesh was used for f l a s h chromatography and Merck S i l i c a Gel 60 PF-254 with CaS0 4.l/2H 20 was employed in r a d i a l t h i n layer chromatrography (Harrison Research chromatotron model 7924, FMI lab pump model R PG-150). Sephadex LH-20 res i n was used f o r molecular exclusion chromatography. To prevent p o t e n t i a l l y i n t e r e s t i n g metabolites from being discarded a l l p a r t i a l l y p u r i f i e d f r a c t i o n s were broadly characterised using TLC and NMR spectroscopic a n a l y s i s . When a metabolite i s stated as being pure i t consists of a 1 13 single peak on HPLC, and i t s s p e c t r a l data (MS, H and C NMR etc..) i s consistent with t h i s . In the f i s h antifeedant bioassays g o l d f i s h (Carassius  auratus) were fed both treated (metabolites H, 12, 165. 166. 167. 170. 171. 173 and 175 were dissolved i n dichloromethane and the required volume transferred onto the surface of a p e l l e t (Wardleys shrimp p e l l e t s ) , average weight of a p e l l e t , 27mg) and control p e l l e t s (treated with IOJJL of dichloromethane) at the same time. The f i s h were then observed u n t i l a l l the control had been consumed and for 15-20 minutes thereafter. I f the treated p e l l e t had only been nosed or spat out during t h i s period i t was 225 considered to be unpalatable. The experiments were repeated three times and the reported r e s u l t s were duplicated. 226 D i a u l u l a sandieaensis. C o l l e c t i o n Data. Specimens of D_. sandiegensis (152 animals, 42.6g dry weight, af t e r extraction) were c o l l e c t e d by hand using SCUBA (-1 to -15m) on several exposed rocky reefs located i n the Deer Group of Islands, Bamfield, B r i t i s h Columbia. Freshly c o l l e c t e d animals were immediately immersed i n methanol and allowed to extract at 2°C. Fxtraction and Chromatographic Separation. A number of c o l l e c t i o n s of D_. sandieoensis were made. The following represents t y p i c a l y i e l d s and i s o l a t i o n precedures used for a September c o l l e c t i o n (when the concentration of metabolites 41 and AZ was at a maximum). After storage at 2°C f o r two days the methanol was decanted and replaced with fresh solvent and the sample was l e f t to extract at 2°C f o r a further two days. The f i r s t and second methanol extracts were combined and evaporated i n vacuo. The r e s u l t i n g residue was p a r t i t i o n e d between brine (200mL) and ethyl acetate (5 x 125mL). The combined ethyl acetate extracts were dried over sodium sulphate for 1 hour and evaporated i n vacuo to give 1.4g of brown o i l . Flash chromatography of the brown o i l (1:1 acetone/methylene chloride) gave a f r a c t i o n (90mg) which contained d i a u l u s t e r o l s A (11) i B (12) and f a t s . Sephadex LH-20 chromatography (80% methanol/chloroform, 2.5 x 100cm column) removed most of the 227 f a t s . The r e s u l t i n g enriched mixture of compounds Al and A2 (47mg) was p u r i f i e d v i a reverse phase HPLC (70% methanol/water) to y i e l d pure steroids as clear o i l s (41: 24.0mg; 42: 3.2mg. 0.16mg (0.06% dry weight) and 0.02mg (0.007% dry weight) per animal, r e s p e c t i v e l y ) . When spotted on TLC both ster o i d s could be v i s u a l i s e d by short wave UV l i g h t , iodine absorptions, and on exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat green chars turning bright red were observed. P h y s i c a l . Spectral and Chemical Conversion D_a±a.. Dia u l u s t e r o l A (Al) : clear o i l ; [a] 2 5 D+29.0°(c 1.15, CH 2C1 2); Rf0.18 (EtOAc); UV(MeOH) X m a x 265nm (c 9830); IR(CH 2C1 2) 3380, 2957, 2875, 1722, 1664, 1626, 1459, 1377, 878, and 753cm~1; AH NMR (300MHz) 0.65(s, 3H), 0.96(d, J=6.8Hz, 3H), 1.06(tm, J=9.2Hz, IH), 1.17(s, 3H) , 1.21(d, J=7.0Hz, 3H), 1.30-1.42(m region), 1.46(s, 6H), 1.51-2.0(m region), 2.08(ddm, J=12.0,7.2Hz, IH), 2.19(dm, J=13.6Hz, IH), 2.34(dd, J=16.4,9.0Hz, IH), 2.41(m, IH), 2.42(dd, J=16.4,3.8Hz, IH), 2.66(very bs, Why2=27Hz, IH), 2.87(very bs, Wh^2=27Hz, IH), 3.25(very bs, Wh^2=15Hz, IH), 3.88(dt, J=10.7,4.9Hz, IH), 4.15(m, IH), 4.28(t, J=4.9Hz, IH), 5.87(t, J=0.9Hz, IH), 6.54(d, J=4.9Hz, lH)ppm; 1 3 C NMR (75MHz) 12.5(CH 3), 18.7(CH 3), 20.4(CH 2), 21.1(CH 3), 21.8(CH 2), 22.3(CH 3), 22.6(CH 2), 26.1(CH 3), 26.2(CH 3), 27.6(CH 2), 35.8(CH 2), 35.9(CH), 38.5(CH 2), 38.6(CH 2), 40.6(C), 41.1(CH 2), 43.7(CH 2), 44.5(C), 47.8(CH), 56.0(CH), 56.2(CH), 64.4(CH), 65.2(CH), 66.7(CH), 83.6(C), 123.6(CH), 128.KCH), 146.2(C), 166.9(C), 172.4(C), 188.7(C)ppm. 228 Preparation o£ Dia u l u s t e r o l A, t r i a c e t a t e (41): 41 (24.0mg) was s t i r r e d at room temperature for 13 hours under nitrogen i n a solution of p y r i d i n e / a c e t i c anhydride (3:1; 8mL). Evaporation of the reagents under high vacuum generated a residue that was p u r i f i e d v i a preparative TLC (ethyl acetate) to give pure t r i a c e t a t e 41 (25.0mg). Compound 41: clear o i l ; [ct] 2 5 D + 7 0 . 0 ° (c 1.35, CH 2C1 2); Rf0.56 (EtOAc); UV(MeOH) ><max 264nm (« 12800); IR(CH 2C1 2) 2960, 2880, 1740, 1667, 1628, 1460, 1371, 1249, 1061, 740cm - 1; XH NMR (300MHz) 0.65(s, 3H), 0.96(d, J=7.2Hz, 3H), 1.04(tm, J=9.0Hz), 1.23(s, 3H), 1.29(d, J=7.6Hz, 3H), 1.42(s, 6H), 1.46-1.98(m, region), 2.03(s, 3H), 2.06(s, 3H), 2.10(s, 3H), 2.19(dm, J=13.5Hz, 1H) 2.42(dd, J=16.2,6.3Hz, 1H), 2.57(dd, J=16.2,7.7Hz, 1H), 5.10(dt, J=13.8,4.8Hz, 1H), 5.23(m, 1H), 5.60(m, 1H), 5.89(bs, 1H) , 6.43(d, J=5.4Hz, lH)ppm; 1 3 C NMR (75MHz) 12.5, 18.6, 19.8, 20.4, 20.7, 20.9, 21.0, 21.2, 21.8, 22.5, 25.9, 26.0, 27.5, 35.8, 35.8, 35.9, 38.5, 40.5, 41.1, 42.1, 44.4, 47.7, 55.9, 56.2, 64.7, 67.5, 67.6, 83.1, 123.7, 124.2, 147.6, 166.5, 169.4, 170.0, 170.1, 170.1, 187.6ppm; HRMS, observed m/z 642.3779, C37 H54°g requires 642.3768; LRMS, m/z ( r e l . , i n t e n s i t y ) 642 (M +, 3), 582 (4), 522 (10), 496 (9), 494 (10), 376 (100), 361 (55), 265 (33), 249 (23), 171 (37), 147 (41), 129 (38), 119 (75). Attempts Ifi. Make Ths. 2/l-Acetonjd,e of Diaulusterol A. (41) : To 41 (5.0mg) was added 3mL 2,2-dimethoxypropane or acetone, lmL dimethylformamide and one small c r y s t a l of p-toluene sulphonic acid monohydrate. The reaction mixture was refluxed for 2 hours, 20ml of CH 0C1- was added and the reaction quenched and extracted 229 with 5% sodium bicarbonate solution (2 x 15raL) and water (15mL). The organic extract was dried over. anhydrous sodium sulphate for 1 hour f i l t e r e d and concentrated in vacuo to y i e l d 3.5mg of yellowy o i l . The o i l was fractionated by preparative TLC (100% ethyl acetate, c o l l e c t e d two short wave UV active bands with R f's of 0.80 and 0.70). The two bands c o l l e c t e d (1.4mg and 1.6rag, respectively) were not r e a d i l y i d e n t i f i a b l e . 300MHz XH NMR analysis indicated that they were not the expected acetonides. The Mel8 resonance observed at 0.65(s, 3H)ppm i n 41 was not apparent and would not be expected to s h i f t . The char a c t e r i s a t i o n of the two products was not pursued further. Diaulusterol fi (42): clear o i l ; [a] 2 5 D-84.7°(c 0.32, CH 2C1 2); Rf0.15 (EtOAc); DV(MeOH) X m a x 265nm (*4319); IR(CH 2C1 2) 3410, 2966, 2877, 1664, 1626, 1458, 1384, 1064, 1029, 879cm"1; XH NMR (300MHz) 0.65(s, 3H), 0.97(d, J=7.2Hz, 3H), 1.09(tm, J=9.5Hz, IH), 1.17(s, 3H), 1.23(s, 6H), 1.30-1.87(m region), 1.95(m, IH), 2.07(m, IH), 2.19(m, IH), 2.29(m, IH), 2.42(m, IH), 3.88(m, IH), 4.27(t, J=5.2Hz, IH), 5.87(bs, IH), 6.54(d, J=5.2Hz, lH)ppm; HRMS, observed m/z 430.3055, C 2 7 H 4 2 0 4 requires 430.3083; LRMS, m/z ( r e l . , i n t e n s i t y ) 430 (M +, 14), 412 (79), 397 (49), 394 (28), 379 (27), 339 (23), 301 (20), 283 (20), 275 (37), 173 (54), 105 (64), 95 (61), 91 (57), 81 (72), 69 (87), 59 (100). Preparation c_f_ Djaulvsterpl a diacetate (52): 42 (3.2mg) was s t i r r e d for 11 hours at room temperature under nitrogen i n a solution of p y r i d i n e / a c e t i c anhydride (3:1; 3mL). Evaporation of the reagents under high vacuum generated a residue that was p u r i f i e d v i a preparative TLC (ethyl acetate) to give pure diacetate 53_. Compound 52: clear o i l ; Rf0.55 (EtOAc); DV(MeOH) 230 xmax 2 6 5 n m ( £ 7121); IR(CH 2C1 2) 2967, 2880, 1742, 1668, 1628, 1458, 1371, 1262, 1044cm"1; 1H NMR (300MHz) 0.65(s, 3H), 0.98(d, J=7.lHz, 3H), 1.09(m, 1H), 1.24(s, 9H), 1.30-2.00(m region), 2.04(s, 3H), 2.08(s, 3H), 2.20(m, 1H), 2.42(tm, J=9.5Hz, 1H), 5.11(dt, J=13.4,3.9Hz, 1H), 5.60(m, 1H), 5.89(bs, 1H)), 6.43 (d, J=6.9Hz, lH)ppm; HRMS, observed m/z 514.3257, C 3 1H 4gOg requires 514.32958; LRMS, m/z ( r e l . , i n t e n s i t y ) 514 (M +, 2), 496 (2), 454 (9), 412 (27), 394 (55), 379 (50), 376 (36), 361 (33), 265 (29), 171 (42), 119 (100), 105 (67), 95 (27), 91 (39). 231 Gersemia rubiformis. Collection Data. £. rubiformis was co l l e c t e d both on the West and East coasts of Canada. Specimens were co l l e c t e d by hand using SCUBA on and around several exposed rocky reefs (-1 to -15m) located within the Gordon Group of Islands, B r i t i s h Columbia, and at Admiral's Cove, Cape Broyle and Bay B u l l s , Newfoundland (south of St. Johns) Extraction and Chromatographic Separation For B r i t i s h Columbian Specimens. Four c o l l e c t i o n s of £. rubiformis were made. The following represents t y p i c a l i s o l a t i o n procedures and the maximum y i e l d s observed are quoted (these varied greatly with season). Freshly c o l l e c t e d animals (1.5Kg dry weight, a f t e r extraction) were immediately immersed i n methanol and allowed to extract at room temperature for 2-3 days. They were homogenised in a Waring blender and vacuum f i l t e r e d through C e l i t e . The homogenised material was extracted i n fresh methanol at room temperature for a further two days. The f i r s t and second methanol extracts were combined and concentrated i n vacuo to lOOOmL (approximately 1/3 of the o r i g i n a l volume). The yellow brown so l u t i o n was sequentially extracted with hexanes (4 x 250mL), methylene chloride (2 x 250mL), ethyl acetate (2 x 250mL) and brine (500mL). The combined organic extract was concentrated to 232 500mL and dried over anhydrous sodium sulphate for 3 hours. Vacuum f i l t r a t i o n followed by evaporation i n vacuo gave a dark red o i l (33g). The o i l was fractionated by f l a s h chromatography (50mm diameter columns, 20cm s i l i c a g e l , step gradient of 5% ethyl acetate/hexanes to 10% methanol/ethyl acetate, run r e p e t i t i v e l y with 4g samples) to y i e l d f r a c t i o n s containing f a t s , steroids and carotenoids. Fractions e l u t i n g with 5%, 25% ethyl acetate/hexanes and two fr a c t i o n s eluting with 100% ethyl acetate (collected as t e s t tube f r a c t i o n s and combined according to TLC (5% methanol/methylene chloride) results) gave f r a c t i o n s A (2.1g), B (3.25g), C (2.3g) and D (2.2g), r e s p e c t i v e l y . Subsequent f r a c t i o n a t i o n of these f r a c t i o n s followed. ( i ) . Fraction A; P u r i f i c a t i o n of f r a c t i o n A by r a d i a l TLC (step gradient consi s t i n g of two solvent systems, 100% hexanes followed by 100% methylene chloride) gave a crude f r a c t i o n which contained f a t s , tochuinyl acetate (1£5J and dihydrotochuinyl acetate (!££.) (250mg). This f r a c t i o n was further p u r i f i e d by reverse phase EPLC (80%/15%/5% methanol/water/methylene chloride) to y i e l d crude samples of 165 (42mg) and 166 (31.8mg). Repetitive reverse phase HPLC (80%/15%/5% methanol/water/methylene chloride) resulted i n _3 pure samples of 165 (18.7mg, 1.3 x 10 % dry weight) and 1££ _3 (16.7mg, 1.1 x 10 % dry weight), both as clear o i l s . When spotted on TLC both sesquiterpenes could be v i s u a l i s e d by short wave UV l i g h t , iodine absorptions, and on exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat brown 233 chars turning to purple were observed. ( i i ) . FractJPP E: Impurities ( f a t s , s t e r o i d s , etc..) i n f r a c t i o n B were p a r t i a l l y removed v i a preparative TLC (2% methanol/methylene chloride, c o l l e c t e d short wave UV active band, R^O.64) and Sephadex LH-20 column chromatography (70% methanol/chloroform, 2.5 x 100cm column). The re s u l t i n g enriched sample of r u b i f o l i d e (120.) (350mg) was further p u r i f i e d by a combination of normal phase HPLC (3:3:1 d i e t h y l ether/hexanes/methylene chloride) and reverse phase HPLC (7:2:1 methanol/water/methylene chloride) to y i e l d pure 170 (220mg, 1.5 x 10~ 2% dry weight) as a white c r y s t a l l i n e s o l i d (by slow evaporation from 50% methanol/methylene chloride at 4°C). When spotted on TLC 170 could be v i s u a l i s e d by short wave UV l i g h t , iodine absorption, and on exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat a yellow char turning bright yellow brown then to purple was observed. ( i i i ) . Fraction £: P u r i f i c a t i o n of f r a c t i o n C commenced with preparative TLC (5% methanol/methylene ch l o r i d e , c o l l e c t e d a series of close running short wave UV active bands, Rf's0.71-0.83) to give a fra c t i o n which contained f a t s , carotenoids, gersemolide f167). A 4 ' 5 z , A 7' 1 7-isogersemolide (lfifi.), A 4 , 5 E , A 7' 1 7-isogersemolide (1££) , isoepilophodiones A (122), B (123.) and C (124), r u b i f o l (125.), an unknown diterpene and gersolide (176) (485mg). 234 Fractionation of t h i s f r a c t i o n by r a d i a l TLC (step gradient consisting of two solvent systems, 50% methylene chloride/hexanes followed by 1:1:1 d i e t h y l ether/hexanes/methylene chloride) gave two f r a c t i o n s . The l e a s t polar one contained 167, 168. 169. 173. 174. 175. an unknown diterpene, 176 and f a t s (314.6mg). The other consisted of 172 and fa t s (23.8mg) which was combined with f r a c t i o n D aft e r the preparative TLC p u r i f i c a t i o n (see below). Normal phase HPLC (3:3:1 diethylether/hexanes/methylene choride) on the more complex mixture gave six f r a c t i o n s three of which contained ju s t f a t s , the other three subfractions consisted of: (i) 167. 169. an unknown diterpene and f a t s (41.6mg); ( i i ) 168. 174 and fa t s (30.4mg); ( i i i ) HI, 123., 12£ and f a t s (161.6mg). Reverse phase HPLC (60% methanol/water) on subfraction (i) —4 yielded pure 167 (14.2mg, 9.5 x 10 % dry weight), pure 1£9_ (3.0mg, 2.0 x 10~ 4% dry weight), both as clear c r y s t a l l i n e needles, and a pure sample of an unknown diterpene (2.2mg, 1.5 x —4 10 % dry weight) as a clear o i l . Reverse phase HPLC (60% methanol/water) on subfraction ( i i ) yielded pure 1£8_ (l.Omg, 6.7 —5 x 10 % dry weight) as clear c r y s t a l l i n e needles and pure 174 (17.4mg, 1.2 xlO % dry weight) as a clear o i l . Reverse phase HPLC (60% methanol/water) on subfraction ( i i i ) yielded pure 173 (77.7mg, 5.1 xlO % dry weight) as a pale yellow o i l , pure 175 (7.6mg, 5.1 x 10~ 4% dry weight) and pure 176 (1.5mg, 1.0 x 10~ 4% dry weight), both as clear c r y s t a l l i n e needles. (iv) . Fraction D_: P u r i f i c a t i o n of f r a c t i o n D commenced with preparative TLC (5% methanol/methylene chloride, c o l l e c t e d a serie s of close 235 running short wave DV acti v e bands, *sO.50-0.63) to y i e l d a f r a c t i o n (179.3mg) containing epilophodione (171). isoepilophodiones A (122) and B (173), rubiformate (177), f a t s and carotenoids. Radial TLC on t h i s f r a c t i o n (step gradient consisting of two solvent systems, 50% methylene chloride/hexanes followed by 1:1:1 d i e t h y l ether/hexanes/methylene chloride) gave two f r a c t i o n s . The l e a s t polar f r a c t i o n (28.4mg) contained 173 and f a t s , and was combined with f r a c t i o n C after the preparative TLC p u r i f i c a t i o n (see above). The other consisted of 171. 172. 177 and f a t s (66.9mg). Reverse phase HPLC (60% methanol/water) on the more polar f r a c t i o n yielded pure 171 (16.0mg, 1.1 x 1 0 - 3 % dry weight) as cl e a r c r y s t a l l i n e needles, pure 172 (8.9mg, 5.9 x 10~ 4 % dry weight) as a pale yellow o i l and pure 177 (3.6mg, 2.4 x —4 10 % dry weight) as a clear o i l . When spotted on TLC a l l the diterpenes i s o l a t e d from f r a c t i o n s C and D could be v i s u a l i s e d by short wave UV l i g h t , iodine absorptions, and on exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat yellow/browm chars turning to grey/black were observed. The i n t e n s i t y of the spots varied somewhat from one diterpene to another. Extraction, chromatographic separation Hydroqenation £££ Newfoundland Specimens. Specimens of £. rubiformis were co l l e c t e d on a number of occasions between October 1986 and January 1987. The following represents t y p i c a l i s o l a t i o n procedures. 236 Freshly c o l l e c t e d animals (250g dry weight, a f t e r extraction) were immediately frozen and stored at -5°C. In February 1987 the specimens were defrosted, homogenised i n a Waring blender and extracted i n methanol at room temperature for 3 days. After vacuum f i l t r a t i o n through c e l i t e the homogenised material was extracted i n fresh methanol at room temperature for a further 3 days, and then vacuum f i l t e r e d . The two extracts were kept seperated and concentrated i n  vacuo to 300mL (approximately 1/10 of the o r i g i n a l volumns). The yellow brown solutions were p a r t i t i o n e d between ethyl acetate (6 x 175mL) and brine (200mL). The combined organic extracts i n each case were concentrated to 200mL and dried over anhydrous sodium sulphate for 2 hours. Vacuum f i l t r a t i o n followed by evaporation in vacuo gave two dark orangy brown o i l s (approximately 2g i n each case). The two o i l s were fractionated by f l a s h chromatography (40mm diameter colums, 20cm s i l i c a g e l , step gradient of 100% hexanes to 5% ethyl acetate/hexanes) to y i e l d a f r a c t i o n , e l u t i n g with 5% ethyl acetate/hexanes s o l u t i o n , containing f a t s and (+)-/?-cubebene-3-acetate (178). Crude samples of ste r o i d s 158-161 x . elu t i n g with 25%-100% ethyl acetate/hexanes s o l u t i o n s , were evidenced by 300MHz 1H NMR s p e c t r a l comparison to the l i t e r a t u r e 186 data . These were not p u r i f i e d further. Fractionation of the l e a s t polar f r a c t i o n by r a d i a l TLC (step gradient consisting of two solvent systems, 100% hexanes followed by 2 1/2% ethyl acetate/hexanes) gave a f r a c t i o n which i n the case of the f i r s t methanol extract was p u r i f i e d by normal phase HPLC (2 1/2% ethyl acetate/hexanes) to y i e l d 253.4mg of crude 178. 237 P u r i f i c a t i o n of t h i s f r a c t i o n by reverse phase HPLC (80%/10%/10% methanol/water/methylene chloride) gave pure 178 (4.7mg, 1.88 x —3 10 % dry weight) as a clear o i l , which on standing r a p i d l y decomposed to a yellow/black o i l c o n s i s t i n g of a complex mixture of very polar materials. When spotted on 178 could be v i s u a l i s e d by exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat to give a d i s t i n c t i v e bright yellow char fading to f a i n t brown (a f a i n t iodine absorption was also observed). In the case of the second methanol extract, a f t e r the r a d i a l TLC p u r i f i c a t i o n , the crude (+)-/?-cubebene-3-acetate (178) was subjected to hydrogenation. lOmL of methanol and a c a t a l y t i c amount of palladium (on activated charcoal) was added to the crude 178 (75.3mg, mixture of fa t s and 178). The so l u t i o n was saturated with hydrogen for 15 mins and l e f t under hydrogen at balloon pressure for 13hrs with s t i r r i n g . The palladium c a t a l y s t was f i l t e r e d o f f (on f i n e scintered glass) and the volume reduced to dryness to y i e l d 61.3mg of white s o l i d . This s o l i d was p u r i f i e d on normal phase HPLC (2 1/2% ethyl acetate/hexanes) to y i e l d a f r a c t i o n containing the two dihydro epimers 192 and 123. (1.2mg) i n a r a t i o of 4:1, r e s p e c t i v e l y , as evidenced by NMR and GC a n a l y s i s . The two epimers 192 and 193 were not seperable by by HPLC, but the mixture did give two d i s t i n g u i s h a b l e peaks on GC analysis using a 15 meter DB-1 column at a temperature of 140°C. When spotted on TLC the dihydro epimer mixture could be v i s u a l i s e d by iodine absorption, and sulphuric acid spray (40% sulphuric acid/methanol). In both cases nothing was i n i t i a l l y observed, after a period of 15 mins, a f a i n t brown/yellow char 238 turning bright orange yellow then to purple. A strong iodine absorption was also observed. p h y s i c a l . spectral and Chemical Interconvecsion Data. Tochuinyl acetate (1£5J: c l e a r o i l ; [a]25D-42.5°(c 1.09, C H 2 C l 2 ) ; Rf0.80 (5% MeOH/CH 2Cl 2); UV(MeOH) \ m a x 219nm (« 5047); IR(CH 2C1 2) 2963, 2882, 1734, 1516, 1480, 1466, 1379, 1245, and 1034cm"1; XH NMR (300MHz) 1.13(s, 3H), 1.33(s, 3H), 1.58(m, 1H), 1.75-1.88(m, 4H), 1.94(s, 3H) , 2.30(s, 3H) , 2.48(m, 1H), 3.36(d, J = l l . l H z , 1H), 3.59(d, J = l l . l H z , 1H), 7.08(d, J=8.1Hz, 2H), 7.22(d, J=8.1Hz, 2H)ppm; 1 3 C NMR (75MHz) 19.5(CH 3), 20.2(CH 2), 20.8(CH 3), 20.9(CH 3), 25.0(CH 3), 34.8(CH 2), 37.6(CH 2), 47.4(C), 49.8(C), 70.6(CH 2), 126.7(CH), 128.6(CH), 135.3(C), 142.9(C), 171.2(C)ppm; HRMS, observed m/z 260.1777, C 1 7 H 2 4 0 2 requires 260.1777; LRMS, m/z ( r e l . , i n t e n s i t y ) 260 (M +, 23), 200(12), 185 (13), 171 (9), 158 (51), 143 (61), 132 (100), 119 (77), 105 (48), 91 (39), 77 (13), 65 (10), 55 (12). 25 o Dihydrotochuinyl acetate (16JL) : cl e a r o i l ; [a] D~29.3 (c 1.11, CH 2C1 2); Rf0.84 (5% MeOH/CH 2Cl 2); DV(MeOH) \ m a x 224nm (* 371); IR(CH 2C1 2) 2963, 2882, 2820, 1731, 1653(very weak), 1468, 1379, 1238, 1034, 985, and 952cm"1; 1H NMR (300MHz) 1.04(s, 3H), 1.07(s, 3H), 1.42-1.55(m, 2H), 1.65(s, 3H) , 1.65-1.80(m, 3H), 2.02(s, 3H), 2.20(m, 1H), 2.61(m, 2H), 2.70(m, 2H), 3.74(d, J=11.0Hz, 1H), 3.81(d, J=11.0Hz, 1H), 5.39(m, 1H), 5.53(bs, lH)ppm; 1 3 C NMR (75MHz) 19.5, 20.1, 21.0, 22.7, 22.8, 28.4, 31.9, 35.3, 37.0, 46.9, 50.3, 70.1, 119.0, 119.1, 130.5, 138.5, 171.4ppm; HRMS, observed ra/z 262.1926, C,7H9,-0~ requires 239 262.1934; LRMS, m/z ( r e l . f i n t e n s i t y ) 262(M + f 24), 260 (7), 219 (2), 218 (1), 217 (1), 202 (21), 200 (10), 187 (26), 185 (11), 173 (17), 171 (6), 159 (31), 145 (55), 132 (85), 119 (100), 105 (78), 91 (70), 77(39), 67 (33), 61 (41), 55 (42). Oxidation oL Dihydrotochuinyl acetate (1&£): 3.8mg of 166 were refluxed for 18 hours on palladium (on activated charcoal) i n 95% ethanol (3mL). The palladium cat a l y s t was f i l t e r e d o f f (on f i n e scintered glass) and the volume reduced to dryness to y i e l d 3.5mg of 165 (in quantitive y i e l d ) . Gersemolide (167); c l e a r c r y s t a l l i n e needles (50% methanol/diethyl ether at 0°C); mp 138-140°; [ a ] 2 5 D-31.8°(c 0.90, CH 2C1 2); Rf0.77 (5% MeOH/CH 2Cl 2); UV(MeOH) \ m a x 233nra (* 9008); IR(CH 2C1 2) 3072, 2992, 2943, 2873, 1762, 1699, 1678, 1648, 1604, 1442, 1221, 1135, 1065, and 911cm"1; "'"H NMR (300MHz) 1.28(m, 2H) , 1.64(bs, 3H), 1.73(bs, 3H), 1.83(d, J=1.2Hz, 3H), 2.01(m, IH), 2.26(m, IH), 2.32-2.45(m, IH), 2.38(dd, J=15.1,9.5Hz, IH), 2.53(dd, J=15.1,4.5Hz, 1H) , 3.29(d, J=0.6Hz, IH), 4.76(m, 2H), 5.20(bs, IH), 5.38(m, IH), 5.47(bs,lH), 6.33(q, J=1.2Hz, IH), 7.12(bs, lH)ppm; 1 3 C NMR (75MHz) 19.8(CH 3), 21.9(CH 3), 22.0(CH 2), 23.1(CH 3), 31.4(CH 2), 41.2(CH), 44.9(CH 2), 62.4(CH), 79.0(CH), 112.3(CH 2), 116.1(CH 2), 123.8(CH), 134.2(C), 138.1(C), 146.4(C), 150.0(CH), 156.2(C), 173.7(C), 197.6(C), 208.3(C)ppm; HRMS, observed m/z 328.1686, C 2 n H 2 4 0 4 requires 328.1675 ; LRMS, ra/z ( r e l . , i ntensity) 328 (M +, 31), 283 (21), 268 (13), 246 (33), 232 (10), 229 (14), 213 (11), 201 (16), 189 (23), 178 (62), 164 (42), 159 (32), 151 (85), 135 (52), 121 (43), 119 (43), 105 (79), 91 (100), 82 (86), 68 (64), 53 (62). Tsomerisation o_f Gersemolide with Formic AC id, (167) : 167 240 (2.0mg) and 3 drops of formic acid were l e f t s t i r r i n g under gentle reflux for 16 hours i n 4mL of ethyl acetate. A f t e r the addition of a further 6ml of ethyl acetate the reaction was quenched and extracted with 5% sodium bicarbonate (2 x lOraL) and water (lOmL). The organic extract was dried over anhydrous sodium sulphate for 20 mins, f i l t e r e d through scintered glass and concentrated i n vacuo to y i e l d a 30:3:20 mixture (1.5mg) of 167. 168 and one other u n i d e n t i f i e d compound, re s p e c t i v e l y (evidenced by 300MHz *H NMR a n a l y s i s ) . Distinguishable 1H 300MHz NMR data for u n i d e n t i f i e d compound (presumably a A^'^E isomer of 167): 1.15(m), 1.74(bs, 3H), 1.93 (bs, 3H), 2.18(d, J=1.5Hz, 3H), 2.78(dd, J=13.5,3.lHz, 1H), 3.69(s, 1H), 4.94(ra, 1H), 5.09(bs, 2H), 5.17(m, 1H), 5.33(m, 1H) , 6.05(q, J=1.5Hz, 1H), 7.25(bs r lH)ppm. A 4 F 5 z . A 7 ' 1 7 - i s o a e r s e m o l i d e (168): clear c r y s t a l l i n e needles (50% d i e t h y l ether/methanol at room temperature); [a] D~27.3 (c 0.02, CH 2C1 2); R f0.75 (5% MeOH/CH 2Cl 2); UV(MeOH) X M A X 218 (« 3047), shoulder at 237nm; XH NMR (300 and 400MHz) 1.18(m, 1H) , 1.26(m, l H ) r 1.73(bs f 3H), 1.85(s f 3H), 1.93(s r 3H), 1.95-2.06(m region), 2.14(d f J=1.6Hz, 3H), 2.31-2.49(m region), 2.72(dd, J=13.6,3.8Hz, 1H), 4.94(m, 1H), 5.07(bs, 1H), 5.93(bs, 1H), 6.11(q, J=1.6Hz, 1H), 7.10(bs, 1H); HRMS, observed 328.1684, C20 H24°4 r e < 3 u i r e s 328.1675; LRMS, m/z ( r e l . , i n t e n s i t y ) 328 (M +, 100), 310 (25), 300 (21), 283 (60), 267 (22), 255 (17), 239 (20), 201 (38), 189 (38), 173 (50), 164 (45), 150 (60), 136 (37), 121 (49), 105 (46), 95 (68), 91 (65), 82 (51), 79 (53), 67 (75), 55 (35), 41 (77). 241 A 4 Y 5 E . A 7 ' 1 7 - l s o g e r s e m o l i d e (l£i): clear c r y s t a l l i n e needles (methanol at 0°C); mp 121-124°C; [a] 2 5 D-225.3°(c 0.37, C H 2 C l 2 ) ; R.0.77 (5% Me0H/CH oCl o); UV(MeOH) \ „ 205 (* 4759), shoulder at 242nm; IR(CH 2Cl 2) 3017, 2960, 2930, 2855, 1761, 1695, 1672, 1645, 1607, 1458, 1379, 1175, 1045, and 871cm"1; 1H NMR (300MHz) 1.26-1.41(m region, 2H), 1.72(bs, 3H), 2.01(s, 3H), 2.06(s, 3H), 2.08(d, J=1.2Hz, 3H), 2.17(tdm, J=12.2,1.5Hz, IH), 2.33(m, IH), 2.49-2.57(m region, 2H), 2.71(dd, J=14.0,5.7Hz, IH), 4.76(bs, IH) 4.86(q, J=0.9Hz, IH) , 5.92(d, J=0.6Hz,. IH) , 6.21(q, J=1.2Hz, IH) , 7.02(t, J=0.6Hz, lH)ppm; 1 3 C NMR (75MHz) 14.2, 18.4, 21.9, 23.1, 23.8, 28.4, 43.5, 44.3, 77.6, 112.7, 130.8, 131.4, 135.1, 145.3, 147.3, 148.1, 150.0, 172.4, 193.8, 203.3ppm; HRMS, observed m/z 328.1669, C 2 o H 2 4 ° 4 r e < 3 u i r e s 328.1675; LRMS, m/z ( r e l . , i n t e n s i t y ) 328 (M +, 25), 310 (10), 300 (15), 283 (27), 267 (19), 255 (13), 241 (20), 201 (35), 189 (38), 173 (54), 159 (42), 149 (65), 135 (52), 121 (59), 105 (68), 91 (95), 79 (78), 67 (100). Isomerisation ojL A 4 ' 5 E . A 7 , 1 7 - l s o a e r s e m o l i d e (1££) Kith. Iodine; To a s t i r r i n g s o l u t i o n of 169 (1.9rag) i n 2mL of ethyl acetate was added 5 drops of a solution of one small c r y s t a l of iodine i n lOmL benzene. The reaction was quenched a f t e r 16 hours with 5mL of aqueous sodium b i s u l p h i t e s o l u t i o n ( s t i r r e d for 10 mins), and p a r t i t i o n e d with ethyl acetate (3 x 5mL). The combined organic extracts were dried over anhydrous sodium sulphate f o r 30 mins, f i l t e r e d through scintered glass and concentrated in vacuo to y i e l d a 3:1 mixture (1.3rag) of 169 and 168, respectively, as evidenced by 3 00MHz 1H NMR an a l y s i s . Rubifolide (120.): white c r i s t a l l i n e s o l i d (50% methanol/ methylene chloride at 4°C); mp 159-160°C; [a] 2 5 D+31.7°(c 0.39, 242 CH 2C1 2); Rf0.80 (5% MeOH/CH 2Cl 2); DV(MeOH) \ m a x 279nm [e 16433); IR(CH 2Cl 2) 3076, 2998, 2931, 2867, 1753, 1644, 1606, 1447, 1199, 1069, 902, and 863cm"1; 2H NMR (300 and 400MHz) 1.18(td, J=14.0,2.7Hz, 1H), 1.64(m, 1H), 1.74(bs, 3H), 1.92(bs, 3H), 1.98(bs, 3H), 2.08(dm, J=14.0Hz, 1H), 2.36(td, J=12.6,4.2Hz, 1H), 2.43(td, J=14.0,2.7Hz, 1H) , 2.55(m, 2H), 2.68(dd, J=ll.4,4.2Hz, 1H), 3.22(t, J=11.4Hz, 1H), 4.88(bs, 1H), 4.90(m, 1H), 4.95(dm, J=11.4Hz, 1H), 5.99(bs, 1H), 6.07 (bs, 1H), 6.86(bs, lH)ppm; 1 3 C MMR (75MHz) 9.5(q), 19.2(q), 20.0(t), 25.7(q), 30.5(t), 31.2(t), 39.5(t), 43.3(d), 78.7(d), 112.9(t), 113.8(d), 117.l(s), 117.4(d), 127.0(s), 132.8(s), 145.4(s), 149.4(s), 149.9(s), 152.1(d), 174.5(s)ppm; HRMS, observed m/z 312.1275, ^•20YL2A°2 requires 312.1276; LRMS, m/z ( r e l . , intensity) 312 (M +, 41), 216 (12), 201 (11), 173 (6), 148 (100), 133 (44), 120 (22), 105 (34), 91 (19), 79 (13), 77 (15), 69 (7), 68 (11), 67 (11), 65 (9), 55 (5), 53 (12). 1R Lanthanide S h i f t ME Experj.ment On RtifrjfPljde (Hfl) : 6.5 —4 x 10 moles (674.Omg) of Eu(FOD) 3 were dissolved i n CDC13 (1.0ml). 400MHz '''H NMR spectra were run with a 5mm sample tube —5 containing 6.5 x 10 moles (20.3mg) of 170. The subsequent addition of 10/JL (6.5 x 10~ 6moles), followed by two further 10^ /L additions of s h i f t reagent solution resulted i n the spectra given 3 + in Figures 30a, b and c , corresponding to [Eu ]/[substrate (170)1 r a t i o s of 1:10, 2:10 and 3:10, respectively. A l l manipulations were performed under nitrogen i n a glove box with pre-dried (75°C) glassware. Epilophodione (171): clear c r y s t a l l i n e needles (methanol at 243 0°C); mp 153-155°C; [a] 2 5 D+136.2°(c 0.42, CH 2Cl 2);R f0.67 (5% MeOH/CH-Cl-); UV(MeOH) X „ 264 (* 10893), 211nra (e 14133); IR(CH 2C1 2) 3077, 2940, 2865, 1756, 1683, 1645, 1619, 1607, 1443, 1201, 1065, 948, and 900cm"1; 1H NMR (300MHz) 1.38(m, IH), 1.61(bs, 3H), 1.86(m, IH), 1.91(d, J=1.6Hz, 3H), 2.19(d, J=1.2Hz, 3H), 2.20-2.47(m, 2H), 2.51(m, 2H), 2.67(dd, J=13.3,4.5Hz, IH), 2.82(dd, J=13.3,4.8Hz, IH), 4.92(m, 2H), 5.27(m, IH), 6.12(bs, IH), 6.38(q, J=1.6Hz, IH), 7.15(bd, J=0.9Hz, lH)ppm; 1 3 C NMR (75MHz) 17.2(CH 3), 21.0(CH 2), 21.1(CH 3), 22.3(CH 3), 33.5(CH 2), 41.8(CH), 43.6(CH 2), 45.5(CH 2), 78.4(CH), 115.6(CH 2), 127.1(CH), 133.5(CH), 136.2(C), 143.9(C), 145.4(C), 148.1(CH), 151.8(C), 173.6(C), 192.1(C), 205.5(C)ppm; HRMS, observed m/z 328.1680, C20 H24°4 r e < 3 u i r e s 328.1675; LRMS, m/z ( r e l . , i n t e n s i t y ) 328 (9), 310 (3), 295 (1), 285 (2), 267 (3), 232 (7), 219 (9), 201 (6), 178 (50), 151 (100), 135 (55), 133 (32), 107 (31), 105 (31), 95 (31), 91 (40), 82 (95), 79 (45), 77 (36), 67 (48), 53 (39). Isomerization o_f. Epilophodione (HI) With Iodine: To a s t i r r i n g s o l u t i o n of 171 (6.7mg) in 2mL ethyl acetate was added 5 drops of a so l u t i o n of one small c r y s t a l of iodine dissolved i n lOmL benzene. P a r t i a l conversion of 171 was noted a f t e r 12 hours by TLC (5% methanol/methylene chloride) examination. The reaction was quenched with 5mL of aqueous sodium b i s u l p h i t e s o l u t i o n ( l e f t s t i r r i n g for 35mins) and p a r t i t i o n e d using ethyl acetate (3 x 5mL). The combined organic extracts were dried over sodium sulphate for 15 rains, f i l t e r e d through scintered glass and concentrated i n vacuo to y i e l d a 2:1:4:1 mixture (5.5rag) of 121, 172. 173 and one other u n i d e n t i f i e d compound, respectively (evidenced by 300MHz 1H NMR a n a l y s i s ) . 244 Isoepilophodione A (112): pale yellow o i l ; [ct] 2 5 D+137.6°(c 0.55, CH 2C1 2); R f0.71 (5% MeOH/CH 2Cl 2); UV(MeOH) \ m a x 246 (* 12818), 208nm (* 16167); IR(CH 2Cl 2) 3070, 2985, 2936, 2845, 1761, 1681, 1648, 1619, 1605, 1445, 1380, 1276, 1065, and 1026cm"1; 1H NMR (300MHz) 1.66(bs, 3H), 1.99(bs, 3H), 2.06(m, 1H), 2.16(d, J=1.2Hz, 3H), 2.33-2.45(m region, 3H), 2.50(m, 2H), 2.98(dd, J=13.5,5.1Hz, 1H), 3.04(dd, J=13.5,4.5Hz, 1H), 4.54(bs, 1H), 4.76(q, J=0.6Hz, 1H), 5.23(bm, 1H), 6.07(bs, 1H), 6.68(q, J=1.2Hz, 1H), 7.19(bs, lH)ppm; 1 3 C NMR (75MHz) 14.3, 18.3, 22.1, 23.4, 29.0, 38.9, 41.5, 42.8, 78.1, 113.2, 130.2, 132.3, 134.1, 140.0, 144.9, 147.8, 149.8, 172.2, 194.6, 202.6ppm; HRMS, observed m/z 328.1686, C 2 Q H 2 4 0 4 requires 328.1675; LRMS, m/z ( r e l . , i n t e n s i t y ) 328 (M +, 4), 310 (3), 295 (2), 282 (1), 267 (3), 232 (8), 219 (6), 178 (21), 151 (26), 135 (25), 121 (28), 107 (30), 95 (26), 91 (35), 82 (100), 67 (43). Tsomerisation Q£_ Isoepilophodione A. (112) With iMinfi.: To a s t i r r i n g s o l u t i o n of 172 (2.5mg) i n 2mL ethyl acetate was added 5 drops of a s o l u t i o n of one small c r y s t a l of iodine dissolved i n lOmL benzene. The reaction was quenched a f t e r 8 hours with 5mL of aqueous sodium b i s u l p h i t e s o l u t i o n ( l e f t s t i r r i n g for 35 mins) and p a r t i t i o n e d using ethyl acetate (3 x 5mL). The combined organic extracts were dried over sodium sulphate for 15 rains, f i l t e r e d through scintered glass and concentrated i n vacuo to y i e l d a 1:3:2 mixture (1.9mg) of 171. 172 and 173. respectively (evidenced by 300MHz 1H NMR a n a l y s i s ) . Isomerisation Q£ Isoepilophodione A, (172) W±til Formic Acid: 172 (1.5mg) and 3 drops of formic acid were l e f t s t i r r i n g under 245 gentle reflux for 5 hours i n 3mL of methanol. After the addition of lOmL of ethyl acetate the reaction was quenched and extracted with 5% sodium bicarbonate (2 x lOmL) and water (lOmL). The organic extract was dried over anhydrous sodium sulphate for 20 mins, f i l t e r e d through cintered glass and concentrated i n vacuo to y i e l d a 3:1:1 mixture (0.9mg) of 122/ 173 and 174. resp e c t i v e l y (evidenced by 300MHz XH NMR a n a l y s i s ) . Tsoepilophodione £ (122): pale yellow o i l ; [ a ] 2 5 D + 2 9 8 ° ( c 0.40, CH 2C1 2); Rf0.77 (5% MeOH/CH 2Cl 2); UV(MeOH) X m a x 267 (* 14429), 212nm (* 13111); IR(CH 2Cl 2) 2982, 2935, 2875, 1758, 1683, 1643, 1620, 1610, 1443, 1381, 1260, 1120, 1105, 1064, 1026, and 882cm"1; 1H NMR (300MHz) 1.41(very broad, IH), 1.70(bs, 3H), 1.88(bs, 3H), 2.06(m, 3H), 2.20-2.62(broad m region, 3H), 2.68-2.92(very broad, 2H), 4.03(very broad, IH), 4.64(bs, IH), 4.83(bm, IH), 5.27(very broad, IH), 6.21(bm, IH), 6.25(bs, IH), 7.16(very broad, lH)ppm; HRMS, observed m/z 328.1670, C 20 H24°4 requires 328.1675; LRMS, m/z ( r e l . , i n t e n s i t y ) 328 (M +, 11), 312 (3), 232 (7), 219 (10), 201 (6), 178 (49), 151 (95 ), 135 (45), 122 (42), 107 (33), 95 (31), 91 (40), 82 (100), 67 (48). Isomerisation oj. Isoepilophodione B. (173) With Iodine: To a s t i r r i n g s o l u t i o n of 173 (8.2mg) i n 2mL of ethyl acetate was added 5 drops of a s o l u t i o n of one small c r y s t a l of iodine dissolved i n lOmL benzene. P a r t i a l conversion of 173 was noted a f t e r 14 hours by TLC (5% methanol/methylene chloride) examination. The reaction was quenched with 5mL of aqueous sodium b i s u l p h i t e s o l u t i o n ( s t i r r e d f o r 30 mins) and p a r t i t i o n e d using ethyl acetate (3 x 5mL). The combined organic extracts were dried over anhydrous sodium sulphate for 30 mins, f i l t e r e d through 246 scintered glass and concentrated i n vacuo to y i e l d a 1:1:10:1 mixture (7.6mg) of 171 f 172. 173 and one other un i d e n t i f i e d compound, respectively (evidenced by 300MHz XH NMR a n a l y s i s ) . Isoepilophodione C. ( H i ) : clear o i l ; [«] 2 5 D-170°(c 0.26, CH 2C1 2); Rf0.75 (5% MeOH/CH 2Cl 2); DV(MeOH) X m a x 230nm (« 2426); IR(CH 2C1 2) 2982, 2942, 2868, 1756, 1689, 1668, 1643, 1614, 1441, 1380, 1335, 1102, 1091, 1042, and 958cm""1; 1H NMR (300 and 400MHz) 1.61(bs, 3H), 1.70-1.82(m ,2H), 1.85-2.06(m, 3H), 1.95(d, J=1.2Hz, 3H), 2.14(m, IH), 2.20(m, 2H), 3.05(dd, J = l l . l r l . 8 H z , IH), 3.08(d, J=15.0Hz, IH), 3.73(d r J=15.0Hz, IH), 4.69(bs, IH), 4.81(m, IH), 5.06(m, IH), 5.90(d, J=0.9Hz, IH), 6.08(s, IH), 6.29(q, J=1.2Hz, IH), 6.79(d, J=1.8Hz, lH)ppm; 1 3 C NMR (75MHz) 18.1, 21.2, 27.3, 27.6, 35.7, 41.6, 41.7, 48.3, 80.0, 113.0, 125.3, 128.3, 132.9, 143.7, 145.8, 148.0, 154.3, 173.9, 196.9, 202.0ppm; HRMS, observed m/z 328.1671, C 20 H24°4 requires 328.1675; LRMS, m/z ( r e l . , intensity) 328 (M +, 8), 310 (6), 300 (3), 283 (6), 267 (4), 259 (4), 178 (19), 161 (17), 150 (100), 135 (14), 121 (27), 109 (29), 91 (28), 82 (60). Rubifol (175): clear c r y s t a l l i n e needles (methanol at room temperature); mp 127-130°C; [ a ] 2 5 D -41.3°(c 0.38, CH 2C1 2); Rf0.76 (5% MeOH/CH 2Cl 2); DV(MeOH) \ m a x 230 (*6658), 207nm (« 10284); IR(CH 2C1 2) 3509(broad), 2980, 2934, 2869, 1756, 1700, 1671, 1644, 1610, 1447, 1382, 1338, 1200, 1094, and 1042cm"1; XE NMR (300MHz) 1.20(s, 3H), 1.67-1.77(m, IH), 1.70(bs, 3H), 1.82(bt, J=10.7Hz, IH), 2.06(bs, 3H), 2.09-2.19(m region, 2H), 2.21-2.37(m region, 2H), 2.47(d, J=17.3Hz, IH), 2.59(dd, J=10.8,3.9Hz, IH), 3.34(bd, J=10.7Hz, IH), 3.37(d, J=17.3Hz, IH), 3.91(bt, J=10.8Hz, IH), 247 4.82(bs f 1H), 4.83(m, 1H), 4.84(bs, IE), 5.1(very broad W h / 2 = 4 6 H Z ' ' 6 * 3 1 ( b s ' l H ) ' 6.64(d f J=0.6Bz, lH)ppm; 1 3 C NMR (75MHz) 18.2(CB 3) V 21.3(CH 2), 24.9(CH 3), 26.6(CH 2), 26.7(CH 3), 37.6(CH 2), 39.3(CH 2), 40.7(CH), 50.2(CH 2) f 78.3(CH), 80.7(C), 112.9(CH 2), 126.3(CH), 132.3(C), 145.2(C), 147.2(CH), 155.2(C), 173.6(C), 202.9(C), 217.3(C)ppm; HRMS, observed m/z 346.1785, C20 H26°5 r e c 3 u i r e s 346.1781; LRMS, m/z ( r e l . , intensity) 346(M +, 2), 328 (5), 310 (3), 303 (10), 285 (5), 243 (6), 215 (14), 178 (17), 161 (16), 151 (89), 140 (45), 133 (33), 121 (33), 109 (54), 91 (62), 82 (100), 67 (67). Unknown Diterpene: clear o i l ; Rf0.79 (5% MeOH/CH 2Cl 2); 1H NMR (300MHz) 1.43(bs, 3H), 1.62(bs, 3H), 1.64(m, 1H), 1.80(td, J=11.7,0.3Hz, 1H), 1.97(d, J=1.5Hz, 3H), 2.02-2.11(m, 3H), 2.19-2.29(m, 2H), 2.84(bd, J=17.9Hz, 1H), 3.00(bd, J=17.9Hz, 1H), 3.15(d, J=11.7Hz, 1H), 3.18(s, 3H), 3.83(baseline bump, 1H), 4.58(bs, 1H), 4.79(bs, 1H) , 5.17(very bs, 1H), 6.19(bs, 1H), 6.98(bs, lH)ppm; P a r t i a l 1 3 C NMR (75MHz) 17.8, 20.4, 27.2, 35.9, 39.9, 41.0, 50.6, 80.9, 112.8, 126.1, 132.0, 143.9, 148.6, 174.1ppm; HRMS, observed m/z 360.1949, C2i H28°5 r e < 3 u i r e s 360.1937; LRMS, m/z ( r e l . , i n t e n s i t y ) 360 (M +, 2), 345 (1), 332 (5), 328 (1), 317 (1), 301 (2), 288 (2), 242 (2), 205 (6), 178 (7), 165 (18), 161 (14), 154 (37), 137 (18), 123 (25), 109 (15), 99 (20), 93 (16), 82 (100), 67 (20). Attempts l£ Chemically Tnterconvert EpilPPhodjpne (171) and Tsoepilophodiones A. (122) , £ (121) £ (111) IP. Rubjfpl (125.) a M tiifi Unknown Diterpene: Metabolites 121, 122, 122 and 121 (1-3mg), and 3 drops of formic acid were l e f t s t i r r i n g or under reflux for upto 16 hours (shorter reaction times were t r i e d ) i n 248 3mL of methanol (for methanolysis to generate unknown diterpene) or 3mL of 5:1 acetone/water or 3mL 5:1 tetrahydrofuran/water (for hydration to generate 175). After the addition of lOraL of ethyl acetate the reaction was quenched and extracted with 5% sodium bicarbonate (2 x lOmL) and water (lOmL). The organic extract was dried over anhydrous sodium sulphate for 20 mins f i l t e r e d through scintered glass and concentrated i n vacuo to y i e l d : with water and formic acid as reagents, 171 gave a 1:2 mixture of 171 and 173, res p e c t i v e l y , 173 was apparently uneffected, and 174 gave a 5:1 mixture of 174 and an u n i d e n t i f i e d compound that had previously been noted as a decomposition product of 174 (might presumably be 4 5 the A ' E isomer of 174): with methanol and formic acid as reagents, 171 gave a 3:1 mixture of two uni d e n t i f i e d compounds, po s s i b l y methyl ethers but not the unknown diterpene, for 172 the re s u l t s are reported on p. 245., and 173 gave a 1:2:3:3 mixture of 173, the two u n i d e n t i f i e d compounds obtained i n the reaction of 171 (the major product from previous, was know the minor product) and one further u n i d e n t i f i e d compound, respectively. A l l the above r e s u l t s were evidenced by 300MHz 1H NMR analysis. Distinguishable •'"H 300MHz NMR data for the uni d e n t i f i e d decomposition and "isomerisation" product of 174: 1.70(bs, 3H), 1.77-2.26(m regions), 2.22(bs, 3H), 2.93(dd, J=12.0, 5.2Hz, 1H), 3.07(m, 1H), 4.66(bs, 1H), 4.84(bs, 1H) , 5.09(bm, 1H), 5.93(bs, 1H), 6.07(bs, 1H), 6.19(bs, 1H), 6.90(bs, lH)ppm. flersolide (176): clear c r y s t a l l i n e needles (methanol at room temperature); mp 176-178°C; [a]25D-54.9°(c 0.31, C H 2 C l 2 ) ; R f0.76 (5% MeOH/CH0Cl,); UV(MeOH) X _ . Y 209 (*4213), shoulder at 237nra; 249 IR(CH 2C1 2) 3091, 3052, 3037, 2995, 2937, 1758, 1683, 1679, 1609, 1480, 1446, 1394, 1333, 1242, 1198, 1084, and 1064cm"1; 1H NMR (300MHz) 0.98(dd, J=8.3,5.8Hz, 1H), 1.25-1.36(m, 1H), 1.30(s, 3H), 1.43 ( t , J=5.8Hz, 1H), 1.72(d, J=0.6Hz, 3H), 1.83(dd, J=8.3,5.8Hz, 1H), 1.98-2.13(m, 1H), 2.06(d, J=0.6Hz, 3H), 2.18(t, J=12.3Hz, 1H), 2.31(tdm, J=12.3,5.2Hz, 1H), 2.33-2.48(m region, 2H), 2.81(bd, J=12.3Hz, 1H), 4.97(bs, 1H), 5.14(d, J=0.6Hz, 1H), 5.22(bs, 1H), 5.96(q, J=0.6Hz, 1H), 6.76(bs, lH)ppm; HRMS, observed m/z 328.1674, C2o H24°4 r e ( 3 u i r e s 328.1675; LRMS, m/z ( r e l . , i n t e n s i t y ) 328 (M +, 5), 313 (6), 300 (3), 285 (5), 267 (4), 253 (4), 232 (6), 205 (16), 177 (66), 159 (29), 149 (100), 135 (41), 123 (75), 105 (50), 97 (60), 91 (79), 79 (69), 67 (61). Rubiformate (122): c l e a r o i l ; [a]25D+194.5°(c 0.06, CH 2C1 2); R f 0.69 (5% MeOH/CH 2Cl 2); DV(MeOH) \ m a x 297 (« 16780), 207nm (* 14295); IR(CH 2C1 2) 3064, 2931, 2872, 2842, 1758, 1720, 1676, 1650, 1606, 1520, 1413, 1368, 1339, 1164, 1142, and 1065cm"1; 2H NMR (300MHz) 1.63(m, 1H), 1.67(bs, 3H), 2.01(s, 3H), 2.20(m, 1H), 2.23("d", 3H), 2.55-2.65(m region, 2H), 2.68(m, 1H), 2.70-2.78(m region, 2H), 2.98(ddm, J=6.6,2.7Hz, 1H), 3.04(ddm, J=6.6,2.7Hz, 1H), 4.69(bs, 1H) , 4.78(q, J=0.9Hz, 1H), 5.29(ra, 1H), 7.03(S, 1H), 7.11(m, 1H), 9.46(s, lH)ppm; 1 3 C NMR (75MHz) 9.8, 18.4, 23.1, 29.8, 30.5, 31.2, 45.5, 46.5, 76.8, 113.3, 119.2, 125.5, 134.2, 145.0, 147.7, 150.7, 157.8, 173.1, 176.8, 204.5ppm; HRMS, observed m/z 344.1623, C20 H24°5 requires 344.1624; LRMS, m/z ( r e l . , i ntensity) 344 (M +, 55), 326(3), 315 (6), 297 (3), 283 (3), 255 (2), 221 (6), 191 (34), 177 (54), 173 (36), 161 (36), 133 (47), 124 (89), 123 (100), 105 (26), 95 (43), 91 (29), 79 (20), 67 (36). 250 Attempts &t converting Rubiformate ( H i ) T_C_ h Crystall ine D e r i v a t i v e . An. Ene-Dione Qr_ L Methvl Enol Eikex: To a s t i r r i n g s o l u t i o n of 177 (1.6mg), i n 3mL of undried acetone or methanol, was added 5 drops of a 1:5 s o l u t i o n of potassium carbonate i n the appropriate solvent. A f t e r 40 mins 170 L of methyl iodide was added. After s t i r r i n g or r e f l u x i n g for 2 hours the reaction was quenched with water (10mL), and extracted with methylene chloride (4 x 5mL) and two drops of a c e t i c a c i d . The organic extract was dried over anhydrous sodium sulphate, f i l t e r e d through cintered f i n e glass and evaporated in vacuo. In each case a complex mixture of polar compounds was obtained (evidenced by TLC) which were not i d e n t i f i a b l e by 300MHz AH NMR analysis and were not characterised f u r t h e r . (+)-£-Cubefrene-2-acetate (lift): clear o i l ; [a]25D+28.9°(c 0.24, CH 2C1 2); Rf0.34 (5% EtOAc/hexanes); DV(MeOH) \ m a x 207nm (* 6139); IR(CH 2C1 2) 3057, 2962, 2934, 2875, 1732, 1609, 1464, 1374, 1246, and 1023cm"1; 1 H NMR (300MHz) 0.58(m, IH), 0.80-0.90(obscured by methyl doublets, IH), 0.90-0.96(3 x methyl doublets, 9H), 1.05-1.15(m region, 2H), 1.27(m, IH), 1.37-1.48(m region, 2H), 1.55-1.67(m region, 2H), 1.74(m, IH), 2.01(s, 3H), 2.38(dd, J=14.4, 8.0Hz, IH) , 5.01(bs, IH), 5.05(bs, IH), 5.39(d, J=8.0Hz, lH)ppm; 1 3 C NMR (75MHz) 18.8(CH 3), 19.8(CB 3), 20.0(CH 3), 21.6(CH 3), 26.5(CH), 30.4(CH), 31.1(CH 2), 31.2(CH 2), 33.5(CH), 34.6(CH), 37.4(C), 38.9(CH 2), 44.2(CH), 76.1(CH), 109.0(CH 2), 152.8(C), 170.4(C)ppm; HRMS, no parent ion observed -C 2H 40 2 m/z 202.1713, C 1 5 H 2 2 requires 202.1722; LRMS m/z ( r e l . , intensity) 202 (M+-60, 18), 159 (100), 157 (74), 142 (38), 131 (59), 117 251 (50) , 105 (56), 91 (69), 77 (38), 69 (29), 60 (26), 54 (41). Pihydrp Epjper Mixture Qf_ f +) -ft-Cubebene-3-acetate f 178) (122 SM 132.): For * H NMR (300MHz) see Figure 54., and Table 31; LRMS of mixture, m/z ( r e l . , intensity) 264 (M +, 0.6), 204 (12), 161 (100), 133 (10), 119 (30), 105 (50), 91 (23), 81 (24), 69 (11), 55 (23), 43 (41). 252 Tochuina tetraquetra. C o l l e c t i o n D_ai&. Specimens of X« tetraquetra (8.6g average dry weight per animal, a f t e r extraction) were c o l l e c t e d by hand using SCOBA i n several exposed rocky channels (-5 to -15m) located within the Gordon Group of Islands, Port Hardy, B r i t i s h Columbia (found feeding on, or near by, Gersemia rubiformis-two animals c o l l e c t e d ) , and t y p i c a l l y sandy bottoms located within the Deer Group of Islands, Bamfield, B r i t i s h Columbia ("Bamfield animals" three animals c o l l e c t e d ) . Freshly c o l l e c t e d animals were immediately immersed i n methanol and allowed to extract at room temperature for 2-3 days before decanting the methanol to give a f i r s t skin extract. The animals were again immersed i n fresh methanol and l e f t for a further two days. The second methanol extract was decanted and the two skin extracts combined. Extraction and. chromatographic separation for Port Hardy Animals The methanol extract (lOOOmL) was concentrated i n vacuo to 50mL and p a r t i t i o n e d between brine (lOOmL) and ethyl acetate (4 150mL). After drying over sodium sulphate for 2 hours the organi extract was reduced to dryness to y i e l d 0.5g of an orangy o i l . The o i l was fra c t i o n a t e d by preparative TLC (2% methanol/methylene chloride) to y i e l d two short wave UV active bands. The l e a s t polar band (R^0.66, consisting of a serie s of close running short wave UV ac t i v e bands) contained 10.7mg of a 253 mixture of tochuinyl acetate (165), dihydrotochuinyl acetate (166). r u b i f o l i d e (170) and f a t s . The other band contained pukalide (£2) and fats (Rf0.28) and was fractionated by reverse phase HPLC (70% methanol/water) to y i e l d pure £2. (15.7mg (7.85mg (0.09% dry weight) per animal) as a white s o l i d (powdery). When spotted on TLC fL3_ could be v i s u a l i s e d by short wave UV l i g h t , iodine absorption, and on exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat a purple char turning a d i r t y green/brown was observed. The second, the l e a s t polar, UV active band was fr a c t i o n a t e d on reverse phase HPLC (80%/15%/5% methanol/water/methylene chloride) to y i e l d crude samples of tochuinyl acetate (165), dihydrotochuinyl acetate (166) and r u b i f o l i d e (170). Repetitive reverse phase HPLC p u r i f i c a t i o n s (80%/15%/5% methanol/water/methylene chloride) on these crude samples y i e l d e d both pure 165 (1.4mg, 0.7mg (0.008% dry weight) per animal) and 166 (0.7mg, 0.35mg (0.004% dry weight) per animal) as clear o i l s , and pure 170 as a white c r y s t a l l i n e s o l i d (4.2mg, 2.1mg (0.02% dry weight) per animal). Extraction and chromatographic separation f o r Bamfjeld Animals. The methanol extract (lOOOmL) was concentrated to 200mL and extracted with brine (200mL) and ethyl acetate (4 x lOOmL). Af t e r drying over sodium sulphate for 3 hours the organic extract was reduced to dryness to y i e l d 0.6g of an orangy o i l . The o i l was fractionated by preparative TLC (2% methanol/methylene chloride) to y i e l d a short wave UV active band 254 (RfO.25). This band was further p u r i f i e d on reverse phase HPLC (70% methanol/water) to y i e l d ptilosarcenone (179) (7.2mg, 2.4rag (0.03% dry weight) per animal), as an amorphous s o l i d , and i t s butanoate analogue, 1 £ £ , (5.1mg, 1.7mg (0.02% dry weight) per animal) as a glass. When spotted on TLC both 179 and 180 could be v i s u a l i s e d by short wave UV l i g h t , f a i n t iodine absorptions, and on exposure to sulphuric acid spray (40% sulphuric acid/methanol) followed by heat reddy chars turning a reddy/purple brown were observed. In the rapid workup f r e s h l y c o l l e c t e d animals were immediately immersed i n chloroform and l e f t standing at room temperature for one day. The chloroform was decanted o f f and the volume (600mL) reduced to 200mL and extracted with brine (200mL) and ethyl acetate (4 x 200mL). After drying over sodium sulphate the organic extract was reduced to dryness. The orangy o i l obtained was fractionated by non-activated preparative layer chromatography (8% ethyl acetate/methylene c h l o r i d e ) . The short wave UV active band obtained (R^O.23) was further fractionated on normal phase HPLC (15% ethyl acetate/methylene chloride) to y i e l d ptilosarcenone (179) and i t s butanoate analogue, 180. only. Total workup took less than three days. physical and Spectral Data. Pukalide (63): white powdery s o l i d (methanol at room temperature); mp 203-205°C; [a] 2 5 D+40°(c 1.0, CH 2C1 2); R f0.39 (2 1/2% MeOH/CH 2Cl 2); UV(MeOH) X m a x 249nm {c 5175); IR(CH 2C1 2) 3140, 3019, 1761, 1715, 1580, 1267, 1230, 1082, 906, 891, 869, 255 and 830cm ,• """H NMR (80MHz) 1.05(s f 3H) , 1.18(m, 1H) , 1.65(m, 1H), 1.79(bs, 3H), 2.00-2.75(m region, 4H), 2.95(m, 2H), 3.58(td, J=11.0,3.5Hz, 1H), 3.76(s, 3H) , 4.13(s, 1H), 4.90(bs, 1H), 5.18(bs, 2H), 6.38(bs, IE), 7.08(bs, lH)ppm; 1 3 C NMR (100MHz) 18.7, 19.8, 22.8, 32.4, 32.5, 40.0, 40.7, 51.2, 55.0, 57.0, 77.8, 106.4, 112.9, 113.9, 137.3, 145.8, 148.2, 148.3, 160.0, 163.8, 173.7ppm; HRMS, observed m/z 372.1563, C2i H24°6 r e ( 3 u i r e s 372.1573; LRMS, m/z ( r e l . , i n t e n s i t y ) 372 (M +, 39), 340 (29), 315 (13), 276 (17), 208 (100), 204 (47), 168 (65), 165 (89). Tochuinyl acetate (165), Dihydrotochuinyl acetate (166) and  Rubifolide (170): Data reported above. Ptilosarcenone (179): amorphous s o l i d (methanol at room temperature); mp 150-152°C; [a]25D~67.0°(c 0.57, C H 2 C l 2 ) ; Rf0.68 (5% MeOH/CH 2Cl 2); UV(MeOH) X m a x 224nm {e 12532); IR(CH 2C1 2) 3538, 2978, 1789, 1741, 1689, 1379, 1371, 1218, and 1019cm"1; XH NMR (300MHz) 1.18(s, 3H) , 1.21(d, J=7.2Hz, 3H), 1.30(d, J=7.2Hz, 3H), 2.15(s, 3H), 2.20(s, 3H), 2.41(q, J=7.2Hz, 1H), 2.69-2.84(m region, 2H), 3.43(s, 1H), 5.01(d, J=4.0Hz, 1H), 5.26(dm, J=4.0Hz, 1H), 5.49(d, J=7.5Hz, 1H), 5.63(dd, J=12.4,8 .7HZ, 1H), 5.72(d, J=8.7Hz, 1H), 5.90(dd, J=10.5,0.9Hz, 1H), 5.93-5.96(m region, 2H), 6.12(d, J=0.9Hz, 1H), 6.61(d, J=10.5Hz, lH)ppm; 1 3 C NMR (75MHz) 6.2(CH 3), 14.7(CH 3), 14.9(CH3) 21.0(CH 3), 21.8(CH 3), 38.9(CH), 43.4(C), 45.1(CH), 45.8(CH), 62.KCH), 68.9(CH), 76.6(CH), 77.7(CH), 84.0(C), 118.8(CH 2), 124.KCH), 128.6(CH), 129.6(CH), 136.5(C), 154.1(CH), 169.7(C), 170.0(C), 174.3(C), 202.2(C)ppm; HRMS, no parent ion observed -C 2H 20 m/z 438.1423, C 2 2 H 2 7 C 1 0 7 requires 438.1446; LRMS, m/z 256 ( r e l . , i n t e n s i t y ) 480 (M +, <1), 438 (5), 402 (3), 378 (3), 360 (4), 344 (5), 342 ( 5 ) f 325 (4), 324 (4), 314 (3), 236 (6), 226 (4), 219 (15), 191 (21), 175 (17), 147 (23), 135 (34), 123 (100), 107 (32), 95 (30), 91 (29), 79 (30), 69 (19). Butanoate Analogue. 18JQ: clear glass; [a] 2 5 D~49.2°(c 0.41, CH 2C1 2); Rf0.71 (5% MeOH/CH 2Cl 2); UV(MeOH) \ m a x 220nm {c 4857); IR(CH 2C1 2) 3590, 2940, 2880, 1790, 1735, 1689, 1371, 1218, 1176, 1034, and 1020cm"1; AH NMR (300 and 400MHz) 0.99(t, J=7.2Hz, 3H) , 1.17(s, 3H), 1.20(d, J=7.2Hz, 3H), 1.30(d, J=7.2Hz, 3H), 1.71(m, 2H), 2.20(s, 3H), 2.36(q, J=7.2Hz, IH), 2.38(t, J=7.2Hz, 2H), 2.73(m, IH), 2.79(m, IH), 3.42(s, IH), 4.98(d, J=3.9Hz, IH), 5.23(dm, J=3.9Hz, IH), 5.46(d, J=7.5Hz, IH), 5.60(dd, J=12.4,8.7Hz, IH), 5.79(d, J=8.7Hz, IH), 5.87(dd, J=10.4, 0.9Hz, IH), 5.93-5.96(m region, 2H), 6.12(dm, J=0.9Hz, IH), 6.58(d, J=10.4Hz, IH), HRMS, observed m/z 508.1873, C 2 g H 3 3 C l 0 8 requires 508.1865; LRMS, m/z ( r e l . , i ntensity) 508 (M +, 0.2), 438 (5), 422 (3) , 402 (3), 378 (4), 360 (4), 342 (4), 324 (4), 307 (4), 297 (4) , 287 (4), 279 (5), 271 (4), 269 (7), 255 (5), 253 (5), 251 (7), 231 (14), 219 (12), 203 (10), 191 (18), 175 (15), 147 (17), 135 (32), 123 (72), 107 (27), 95 (17), 81 (14), 71 (95), 55 (13), 43 (100). 257 211. 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The presence of a f a c i l e leaving group, bromine, and an array of s t r u c t u r a l l y related metabolites such as the lauranes (eg., 181 and 183) led to a proposed biogenetic pathway involving chamigrene intermediates. See: A. G. Gonzalez, J . Darias, A. Diaz, J . D. Fourneron, J . D. Martin, C. Perez. Tetrahedron. L e t t . , 3051, 1976. 217. T. Suzuki, M. Suzuki, E. Kurosawa. Tetrahedron. L e t t . , 3057, 1975. 218. For examples see refences 208, 211 and 212. 219. Private communication to Dr. R. J . Andersen from Dr. P. J . Scheuer of the University of Hawaii. 272 

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