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Secondary metabolites from selected marine organisms Pathirana, Induruwa Charles 1986

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SECONDARY METABOLITES FROM SELECTED MARINE ORGANISMS by INDURUWA CHARLES PATHIRANA SUBMITTED IN PARTIAL FULFILLMENT REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept this thesis as conforming to the required standard THESIS THE THE UNIVERSITY OF BRITISH COLUMBIA October 1986 © Induruwa Charles Pathirana, 1986 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemistry The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 1986 ABSTRACT Marine organisms are known to produce secondary metabolites which have novel s tructures and are often b i o l o g i c a l l y a c t i v e . Chemical studies of b i o l o g i c a l l y ac t ive metabolites from three d i f f eren t marine organisms led to the discovery of s ix new compounds and six previous ly known compounds. The brown alga Dictyota binghamiae i s f a i r l y abundant in B r i t i s h Columbia coas ta l waters. A chemical study of th i s alga y ie lded ten di terpenoids of which four are new compounds. A l l the new compounds, d ictyoxide A ( 6 6 ) , d i c t y o l G acetate ( 6 8 ) , d i c t y o t r i o l A diacetate ( 6 9 ) , and e p i d i c t y o l B acetate ( 7 0 ) contain a perhydroazulene carbon skeleton f i r s t encountered in the a l g a l metabolite pachydictyol A ( 2 9 ) . Dictyoxide A ( 6 6 ) appears to be an a r t i f a c t of i s o l a t i o n . The acetates 6 8 , 6 9 , 7 0 were found to be a n t i b a c t e r i a l and a n t i f u n g a l . Six previously known compounds pachydictyol A ( 2 9 ) , d i c t y o l C ( 3 2 ) , d ictyoxide ( 3 5 ) , a c e t y l d i c t y o l a l ( 4 9 ) and the aceta ls 6 1 a and 6 1 b were a lso i so la ted from th i s a lga . Chemical studies on an Agelas sp. of sponge c o l l e c t e d in S r i Lanka y ie lded the ant imicrob ia l metabolite desbromooroidin ( 7 9 ) . An in teres t ing in terac t ion between the s t a r f i s h Dermasterias imbricata and the sea anemone St ompi a cocci nea was observed a long time ago. When contacted by the s t a r f i s h , i i the anemone d i sp lays an unusual "swimming" response which was, according to other subsequent s tudies , caused by a s ingle chemical substance in the s t a r f i s h . A study conducted to e luc idate the s tructure of th i s s t a r f i s h metabolite led to the i s o l a t i o n of imbricat ine (91), a unique benzyl te trahydroisoquinol ine a l k a l o i d . Imbricatine (91) induced S. coccinea swimming response at a very low concentration and a l so exhib i ted antitumor a c t i v i t y . Structures of a l l the new metabolites were determined by spec tra l a n a l y s i s , and chemical degradations and chemical interconvers ions . i i i TABLE OF CONTENTS Abstract i i L i s t of f igures v L i s t of Schemes v i i L i s t of Tables v i i i L i s t of Appendices ix Acknowledgements x Abbreviat ions . . x i I . Introduction 1 I I . Secondary metabolites from the brown alga Dictyota bi nghami ae 12 A. Introduct ion . . . . 1 2 B. I so la t ion and s tructure e luc ida t ion 21 C. Discussion 45 I I I . Secondary metabolites from the S r i Lankan sponge Age I as s p 47 A. Introduction 47 B. I so la t ion and s tructure e luc ida t ion 52 C. Discuss ion . . . . 5 9 IV. A secondary metabolite of the s t a r f i s h Dermasterias imbr i cat a that e l i c i t s a swimming response in the sea anemone Stompi a cocci nea 61 A. Introduct ion .61 B. I so la t ion and s tructure e luc ida t ion 66 C. Discussion 104 V. Experimental 115 V I . Appendices .137 V I I . References 158 iv LIST OF FIGURES 1. 1 H nmr spectrum of d i c t y o l G acetate (68) 27 2. 1 3 C nmr spectrum of d i c t y o l G acetate (68) 28 3. Mass spectrum of d i c t y o l G acetate (68) 29 4. 1 H nmr spectrum of dictyoxide A (66) 30 5. 1 3 C nmr spectrum of dictyoxide A (66) 31 6. Mass spectrum of dictyoxide A (66) 32 7. Conformational drawing of d ictyoxide A (66) 34 8. 1 H nmr of spectrum of d i c t y o t r i o l A diacetate (69) 37 9. SFORD 1 3 C nmr of spectrum of d i c t y o t r i o l A diacetate (69) 38 10. Mass spectrum of d i c t y o t r i o l A diacetate (69) 39 11 . Contour p lo t of the COSY 2D nmr spectrum of d i c t y o t r i o l A diacetate (69) 40 12. 1 H nmr spectrum of e p i d i c t y o l B acetate (70) 41 13. 1 3 C nmr spectrum of e p i d i c t y o l B acetate (70) 42 14. Mass spectrum of e p i d i c t y o l B acetate (70) 43 15. *H nmr spectrum of desbromooroidin (79) 54 16. 1 3 C nmr spectrum of desbromooroidin (79) 55 17. Infrared spectrum of imbricat ine (91) 71 18. nmr spectrum of imbricat ine (91) at r . t 72 19. 1 H nmr spectrum of imbricat ine (91) at 100°C . . 73 20. 1 3 C nmr spectrum of imbricat ine (91) 74 21. Contour p lo t of HETCOR 2D nmr spectrum of imbricat ine (91) 75 22. 'H nmr spectrum of 92 76 v 23. CI mass spectrum of 92 77 24. 'H nmr spectrum of 94 at r . t 80 25. 1 H nmr spectrum of 94 at 100°C 81 26. 1 3 C nmr spectrum of 94 82 27. CI mass spectrum of 94 83 28. Interpretat ion of CI ms of 94 84 29. Contour p lo t of HETCOR 2D nmr spectrum of 99 .88 30. 1 H nmr spectrum of 99 . . . . 8 9 31. 1 3 C nmr spectrum of 99 90 32. CI mass spectrum of 99 91 33. 'H nmr spectrum of 100 92 34. 1 3 C nmr spectrum of 100 93 35. CI mass spectrum of 100 94 36. 'H nmr spectrum of 102 95 37. CI mass spectrum of 102 96 38. Interpretat ion of CI ms of 102 100 39. Interpretat ion of CI ms of 99 101 40. INAPT 1 3 C nmr spectrum of imbricat ine 91 102 41. INAPT 1 3 C nmr spectrum of imbricat ine 91 103 vi LIST OF SCHEMES 1. Chemical conversions of d i c t y o l G acetate ( 6 8 ) 26 2. Synthesis of the benzyl tetrahydroisoquinol ines 9 7 and 9 8 87 3. Proposed routes for the biosynthesis of benzyl te trahydroisoquinol ines in plants 112 vi i LIST OF TABLES 1. 1 H nmr data for dictyota binghamiae diterpenoids 25 2. 1 H nmr data for desbromooroidin (79) and keramadine (80) 57 3. 1 3 C nmr data for desbromooroidin (79) and keramadine (80) 58 4. 'H nmr data for imbricat ine (91) , 94 and 93 69 5. 1 3 C nmr data for imbricat ine ( 91 ) , 94 and 93 70 6. 1 H nmr data for 102 and 99 99 v i i i LIST OF APPENDICES 1. 1 H nmr spectrum of pachydictyol A (29) 138 2. 1 3 C nmr spectrum of pachydictyol A (29) 139 3. Mass spectrum of pachydictyol A (29) 140 4. 1 H nmr spectrum of d i c t y o l C (32) 141 5. Mass spectrum of d i c t y o l C (32) 142 6. nmr spectrum of d ictyoxide (35) 143 7. 1 3 C nmr spectrum of dictyoxide (35) 144 8. Mass spectrum of d ictyoxide (35) 145 9. 'H nmr spectrum of a c e t y l d i c t y o l a l (49) i . . . 1 4 6 10. Mass spectrum of a c e t y l d i c t y o l a l (49) 147 11. 1 H nmr spectrum of aceta l 61a 148 12. Mass spectrum of aceta l 61a 149 13. 'H nmr spectrum of aceta l 61b 150 14. Mass spectrum of aceta l 61b 151 15. SFORD 1 3 C nmr spectrum of desbromooroidin (79) 152 16. 1 3 C nmr spectrum of imbricat ine (91) ( 1 H gated decoupled) 153 17. ADEPT 1 3 C nmr spectra of imbricat ine (91) 154 18. 1 3 C nmr spectrum of 94 ( 1 H gated decoupled) 155 19. ADEPT 1 3 C nmr spectra of 94 156 20. APT 1 3 C nmr spectrum of 99 157 i x ACKNOWLEDGEMENTS I wish to s incere ly thank my research supervisor , Dr . Raymond J . Andersen for h i s patience, encouragement, meticulous guidance and f r i endsh ip during the course of th i s work. It has been a pleasure to work with him. I am great ly indebted to my wife, Nelun, for her encouragement and u n f a i l i n g support throughout. Assistance given by Mike LeBlanc by-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 acknowledged. I thank my col legues who were h e l p f u l in numerous ways. The ass is tance of the s taf f of departmental nmr and mass spectrometry laborator ies and a l so of the s taf f of Bamfield Marine Stat ion i s appreciated. x ABBREVIATIONS AcOH - a c e t i c a c i d APT - Attached Proton Test ADEPT - Automated D i s t o r t i o n l e s s Enhancement by P o l a r i z a t i o n t r a n s f e r c a l c d . - c a l c u l a t e d C D C I 3 - chloroform-d, CI - chemical i o n i z a t i o n COSY - C o r r e l a t e d SpectroscopY 2D - two-dimensional DMSO-dg - dimethyl s u l f o x i d e - d 6 ED - e f f e c t i v e dose EI - e l e c t r o n impact EtOAc - e t h y l a c e t a t e FAB - Fast Atom Bombardment gc - gas chromatography gems - gas chromatography/mass spectrometry h - hour/s HETCOR - HETeronuclear C O R r e l a t i o n h p l c - high performance l i q u i d chromatography hrms - high r e s o l u t i o n mass spectrum ( e l e c t r o n impact) i - impurity i r - i n f r a r e d INAPT - I n s e n s i t i v e N u c l e i A ssigned by P o l a r i z a t i o n T r a n s f e r MeOH - methanol xi MeONa - sodium methoxide min. - minutes mp - melting point ms - mass spectrum (low resolut ion) n-BuOH - 1-butanol 1 H nmr - proton nuclear magnetic resonance 1 3 C nmr - carbon-13 nuclear magnetic resonance nOe - nuclear Overhauser enhancement ppm - parts per m i l l i o n re f . - reference r e l . i n t . - r e l a t i v e i n t e n s i t y r . t . - room temperature s - solvent s igna l T/C - t e s t / c o n t r o l t i c - th in layer chromatography uv - u l t r a v i o l e t w - water s igna l xi i To My Par ent s x i i i 1 I.INTRODUCTION A.NATURAL PRODUCTS CHEMISTRY 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. Studies of the e x t r a o r d i n a r i l y large number of plants and animals in the marine environment were not i n i t i a t e d u n t i l the l a s t three decades when the advent of SCUBA e f f e c t i v e l y removed the b a r r i e r between man and the shallow ocean. C l a s s i c a l l y , the s tructures of natural products were solved by chemical degradations and interconvers ions , and they were confirmed by synthes is . Since these techniques required a large amount of mater ia l , often only the major metabolites were charac ter i zed . C l a s s i c a l s tructure e luc ida t ion was a tedious , time consuming process. Natural products chemistry has undergone an explosive growth during the past two decades. The rate at which new compounds are discovered is now fast enough to rap id ly outdate any compilat ion of s t ruc tures . This growth i s p a r t l y a resu l t of the development of high speed, low cost computers. Computers play a cen tra l role in X-ray crys ta l lography , mass spectrometry and Fourier transform nmr, the three most important tools in modern s tructure e l u c i d a t i o n . X-ray c r y s t a l l o g r a p h i c ana lys i s i s now highly automated 2 and i t r o u t i n e l y p r o v i d e s a f a s t a n d a c c u r a t e m e t h o d t o s o l v e s t r u c t u r e s Of compounds t h a t w o u l d be d i f f i c u l t o r i m p o s s i b l e t o h a n d l e w i t h o t h e r t e c h n i q u e s . The s t r u c t u r e d e t e r m i n a t i o n o f n o r h a l i c h o n d r i n A (1) v i a an X - r a y c r y s t a l l o g r a p h i c a n a l y s i s o f i t s p - b r o m o p h e n a c y l b r o m i d e d e r i v a t i v e 1 i s a r e c e n t e x a m p l e . Mass s p e c t r o m e t r y a n d FT ( F o u r i e r t r a n s f o r m ) nmr h a v e a l s o p l a y e d a g r e a t r o l e i n a c c e l e r a t i n g t h e p a c e o f s t r u c t u r e e l u c i d a t i o n . B o t h t h e s e t e c h n i q u e s a r e c a p a b l e o f p r o v i d i n g s t r u c t u r a l i n f o r m a t i o n on m i n u t e a m o u n t s o f m a t e r i a l . M u l t i p u l s e , one d i m e n s i o n a l a n d two d i m e n s i o n a l , nmr e x p e r i m e n t s 2 h a v e g r e a t l y e x p a n d e d t h e i n f o r m a t i o n 3 OH 3 ava i lab l e to s t r u c t u r a l chemists. Homonuclear and heteronuclear s h i f t c o r r e l a t i o n s , p o l a r i s a t i o n t r a n s f e r , and J -reso lved 2D nmr are some of the new techniques that are most useful to natura l products chemists. The assignment of a l l the protons in 1 1 j3-hydroxyprogesterone (2) using a var ie ty of one and two dimensional nmr experiments i l l u s t r a t e s the a p p l i c a t i o n of mult ipulse nmr to the study of 4 complex molecules 3 . One cannot, of course, forget the contr ibut ions of the advances in separation techniques. The advent of high performance l i q u i d chromatography (hplc) was a remarkable step forward. In p a r t i c u l a r , the a v a i l a b i l i t y of new gels and res ins that can be used for the separation of h ighly polar metabolites has encouraged the study of water soluble compounds. Modern chromatography i s in fact so sophis t icated that the d i r e c t reso lut ion of amino ac id enantiomers can now be achieved by thin layer chromatography 4 . The advent of new spectroscopic and separation techniques has given chemists the resources to tackle almost any problem they are faced with. The successful e luc ida t ion of the ' s tructure of pa'lytoxin ( 3 ) 5 i l l u s t r a t e s th i s po in t . It would not be an exaggeration to state that so lv ing s tructures such as that of palytoxin (3) would have been unimaginable, a few decades ago. B.MARINE NATURAL PRODUCTS CHEMISTRY The extensive chemical research on marine organisms during the las t two decades has brought to l i g h t a large number of extraordinary metaboli tes . In general , many of these metabolites are exclusive to marine organisms and some are i s o l a t e d only from c e r t a i n species or group of species . A d e t a i l e d coverage of the past research can be found in the ser ies of books edited by Scheuer 6 and a ser ies of review 5 a r t i c l e s by F a u l k n e r 7 . The discovery of chemically in t ere s t ing metabolites with new s tructures has not only y ie lded information about nature's molecular archi tec ture but i t has a l so revealed some i n t e r e s t i n g information about b i o l o g i c a l in terac t ions taking place in the marine environment. One of the most s t r i k i n g d i scover ie s came from the study of nudibranchs, commonly known as sea s lugs . These s h e l l - l e s s , often b r i g h t l y coloured animals, despite the ir v u l n e r a b i l i t y , have almost no known predators . The discovery that metabolites extracted from the ir skins exhibi ted f i s h antifeedent a c t i v i t y led to the hypothesis that they u t i l i z e d chemical antifeedents to ward off predators . A l b i c a n y l acetate (4) 8 and furodysinin ( S ) 8 ' ' 9 i so la ted from Cadi i ria I uteomargi nata, nakafuran 8 (6) and nakafuran 9 (7) from Hypselodoris godeffroyana and Chromodoris marisdadi I us 1 0 , and 9-isocyanopupukeanane (8) from Phyllidea varicosa^ are some of the fasc inat ing metabolites that are thought to be defensive substances. A large port ion of the recent marine natural products research has been d irected towards marine pharmacology. As a r e s u l t , a number of marine secondary metabolites have been found to exh ib i t promising pharmacological proper t i e s . The diterpenes ha l imadatr ia l ( 9 ) 1 2 and halimadalactone (10) 1 3 i s o l a t e d from several green a l g a l species of the genus Halimeda show a n t i b a c t e r i a l and cytotoxic a c t i v i t i e s . Spatol (11) , i s o l a t e d from the brown alga Spatoglossum schmi t tii 1 4 , and tedanolide ( 1 2 ) , i so la ted from the sponge Tadoni a ignis^5 Ac 7 are two potent cytotox ins . Agelasines (eg. agelasine A ( 1 3 ) 1 6 , D ( 1 4 ) 1 6 , E (15 ) 1 7 ) which show a n t i m i c r o b i a l and antispasmodic a c t i v i t i e s have been i so la ted from the sponge Agelas nakamurai. A nucleoside, tuberc id in (16), i so la ted from the marine alga Hypnea valendiae was found to be a muscle r e l a x a n t 1 8 . P e t t i t et al. have i so la ted a family of metabol i tes , the bryostat ins (eg. bryos ta t in 1 ( 1 7 ) 1 9 ) , which 8 are potent antitumor agents. The s t r i k i n g red egg masses of the nudibranch Hexabranchus sanguineus, though exposed and vulnerable , were observed by Roesner and Scheuer to have a high l e v e l of immunity to predators . Prompted by th i s unusual observat ion, they i so la ted ulapual ide A (18) and B (19) which both i n h i b i t e d L1210 leukemia c e l l p r o l i f e r a t i o n 2 0 . It i s a rather easy task to pick out hundreds of 9 b i o l o g i c a l l y act ive compounds s imi lar to the above examples from the marine natural products l i t e r a t u r e . Unfortunately , only a small f r a c t i o n of. the many lead compounds have turned out to be successful in more exhaustive drug t e s t s . Rinehart et al . i s o l a t e d the di demni ns from the tunicate Tri di demnum cyanophorum2 1 . These in t ere s t ing peptides showed very high antitumor a c t i v i t y . The most potent one, didemnin B ( 2 0 ) , i s now undergoing c l i n i c a l t r i a l s as an antitumor d r u g 2 2 . A second marine metabolite named manoalide ( 2 1 ) 2 3 has a lso reached c l i n i c a l t r i a l s as an antiinflammatory agent, and both compounds undoubtedly have made a- major contr ibut ion to the recent renewed interes t in the search for drugs from the sea. Another route to drug development exp lo i t s the p o s s i b i l i t y of using natural compounds as templates for synthet ic analogs which w i l l have some therapeutic use. The sponge metabolites spongothymidine ( 2 2 ) 2 4 and spongouridine ( 2 3 ) 2 5 have led to the synthet ic product 24, a powerful a n t i v i r a l agent used in the treatment of herpes v i r u s 2 6 , and the anticancer drug Ara-C ( 2 5 ) 2 7 which is used in cancer chemotherapy. It i s evident from past e f f o r t s that i t i s a rather d i f f i c u l t task to discover a metabolite with des irable therapeutic q u a l i t i e s . C l e a r l y the eventual development of a "Drug from the Sea" w i l l require a great deal of patience and perseverence. This thes is describes the research c a r r i e d out to C H 3 C H O H C O - * N - C H - C O - M e L e u - T h r - S t a - H i p - L e u - P r o - M e T y r -\ y I 2 2 0 11 i so la te and e luc idate the s tructures of secondary metabolites found in three marine organisms. The s tructures of the metabolites were determined by in terpre ta t ion of s p e c t r a l data, and by chemical degradations and interconvers ions . A chemical study of Dictyota binghamiae, a brown a l g a l species c o l l e c t e d in B r i t i s h Columbia, y ie lded ten diterpenes of which four are new compounds. A b i o l o g i c a l l y ac t ive a l k a l o i d was i so la ted as the major metabolite from an Agelas sp. of sponge c o l l e c t e d in S r i Lanka. The s t a r f i s h Dermasterias imbricata was known to induce an unususal "swimming" behavior in the sea anemone St ompi a cocci nea. It was also known that a s ingle chemical substance in the s t a r f i s h was responsible for the behavioral response. The structure of the act ive compound was solved. It turned out to be a benzyl tetrahydroisoquinol ine a l k a l o i d which has several unusual s t r u c t u r a l features . The fol lowing chapters contain a de ta i l ed account of the above mentioned research. 1 2 SECONDARY METABOLITES FROM THE BROWN ALGA Dictyota bi nghami ae A. INTRODUCTION Marine algae have been a very good source of a wide var ie ty of secondary metabolites which are in teres t ing from both chemical and b i o l o g i c a l points of view. These metabolites range from simple molecules such as the 1,3-dione (26) 2 8 to complicated macro molecules such as brev i tox in B (27) 2 9 . 27 Among a l g a l metabol i tes , the tox ins , inc luding the well known saxi tox in ( 2 8 ) 3 0 , are almost exc lus ive ly produced by e i ther phytoplankton or blue-green algae. In contras t , algae belonging to other d i v i s i o n s , i e . brown algae, red algae and 13 HN VNH, X H 28 N>=NH2 OH OH green algae, are rare ly known to produce secondary metabolites with such d r a s t i c b i o l o g i c a l a c t i v i t i e s . Brown algae, are almost exc lus ive ly found in the i n t e r t i d a l and subt ida l regions of the marine h a b i t a t . The d i s t i n g u i s h i n g chemical feature of brown algae in the order Dic tyota les i s the presence of d i t erpeno ids . C y c l i c di terpenoids 'are the t y p i c a l metabolites of the members of the family Dictyotaceae. Pachydictyol A (29), reported by H i r s c h f e l d et al . in 1973 from the brown alga Pachydi ctyon coriaceum3^ c o l l e c t e d along the P a c i f i c coast of C a l i f o r n i a , was the f i r s t member of a group of a l g a l d i terpenoids containing the perhydroazulene r ing system. A number of oxygenated der iva t ives of pachydictyol A were subsequently discovered. These include d i c t y o l A (30), B (31), C (32) and D (33) i so la ted from Dictyota dichotoma var . i m p l e x a 3 2 , 3 3 , and d i c t y o l E (34) and dictyoxide (35) from Ditophus I i gul at us 3 3 3 4 . Almost simultaneously d i c t y o l A and B were 1 4 also i so la ted from a herbivorous sea hare Aplysia depilans which was known to feed upon D. di chot oma3S. 29 R,= R 2= <uR 3 = H 31 R f < ° H H R2=<H R 3 = H 33 R 2 = < S HRr<^3 - H 3 4 R r OH R = R =<!"| 3 1 2 > l 3 7 R f < ^ c R 2 < H R 3 = H 3 0 3 2 R = H 6 0 R = 0Ac The r e la ted metabolites d i c t y o t a d i o l (36) and d i c t y o l B acetate (37) were reported from D. dichotoma c o l l e c t e d in the B r i t i s h C h a n n e l 3 6 , while d i c t y t r i o l (38), dictyone (39) and the deoxygenated metabolites d i c t y t r i e n e A (40) and B (41) were reported from a Japanese c o l l e c t i o n of the same a l g a 3 7 . Japanese workers have a lso reported the i s o l a t i o n of four new compounds, d i c t y o l F (42), e p i d i c t y o l F (43), 15 1 6 epoxypachydictyol A ( 4 4 ) , and methoxydictydiene ( 4 5 ) , thought to be an a r t i f a c t , from Dictyota dichotoma3*. Pachydictyol A epoxide ( 4 6 ) was i so la ted from D. flabellata c o l l e c t e d from the Gulf of C a l i f o r n i a 3 9 . Metabol i tes with the xenicane skeleton have been found in Dictyotaceae species belonging to the genera Dictyota and Dilophus. Xenic in ( 4 7 ) , i so la ted from soft c o r a l Xenia el ongata'0, was the f i r s t diterpene with th i s skeleton to be reported. 47 D i c t y o d i a l ( 4 8 ) , the f i r s t a l g a l metabolite having a xenicane carbon skeleton, was i so la ted by Finer et al. from D. crenulat a" 1 . This discovery was followed by the i s o l a t i o n of a number of metabolites which had minor s t r u c t u r a l v a r i a t i o n s . These include a c e t y l d i c t y o l a l(4 9 ) , hydroxyace ty ld i c tyo la l ( 5 0 ) , d i c t y o d i a c e t a l ( 5 1 ) and i sodictyohemiacetal ( 5 2 ) i so la ted from Dictyota di c hot oma1* 2 , and 4 /3-hydroxydictyodial ( 5 3 ) i so la ted from Dictyota 17 OHC 48 R = CHO R = H 1 2 49 R = CH OAc R = H 1 2 2 so R = CH OAc R = OH 1 2 2 53 R = CHO R = OH 1 2 55 61b R = H 19 So rR 18 crenul at a" 3 . Dictyota dichotoma has a lso y ie lded dictybfurans T (54) and C ( 5 5 ) a a , while compounds (56),(57),(58) and (59) with lactone formation involv ing a carbonyl group in the side chain have been reported from D. pr ol i fi cans'*5 . The study of minor metabolites of the alga Pachydictyon coriaceum, c o l l e c t e d along the Japanese coast , y ie lded a c e t y l d i c t y o l C (60) and the two acetals 61a and 61b which are reported to be a r t i f a c t s a r i s i n g from the addi t ion of methanol to d i c t y o d i a l ( 4 8 ) 4 6 . Acetylcoriacenone (62) and isoacetylcoriacenone (63) are two more in teres t ing metabolites which show further c y c l i z a t i o n of xenicane skeleton, both having a four membered r ing fused to the nine membered carbocyc l i c r i n g . Both were i s o l a t e d from Pachydictyon coriaceum'*1 . Algae, being a p r i n c i p a l component of the marine 19 ecosystem, experience constant pressure from herbivorous predators . It i s not s u r p r i s i n g , therefore , that a l g a l metabolites a lso appear in the chemical studies c a r r i e d out on herbivorous animals. D i c t y o l A (30) and B (31) were i so la ted from both Dictyota dichotoma var . implexa 5 and the sea hare Aplysia depilans3 by two I t a l i a n groups in separate s tud ies . Dictyolactone (64) 1 4 , i s o l a t e d from A. depilans and the hemiacetal of acetoxycrenulide (65) i so la ted from A. vaccaria1*6 show minor v a r i a t i o n s in s tructure compared to a l g a l metabol i tes , demonstrating the a l g a l o r i g i n of Aplysia metabol i tes . Many marine organisms are observed to employ a chemical defense mechanisms to ward off predators . Chemical studies of a few a l g a l species have demonstrated the use of th i s type of defensive strategy against h e r b i v o r e s 4 9 . 20 The foregoing section includes only selected examples of d i terpenoids with perhydroazulene and xenicane skeletons reported from members of Dictyotaceae. Since a l l the metabolites i so la ted from Dictyota bi nghami ae in the current study belong to these two fami l ies of compounds, a d e s c r i p t i o n of the many other types of diterpenes found in Dictyotaceae has not been inc luded. 21 B.ISOLATION AND STRUCTURE ELUCIDATION The brown alga Dictyota binghamiae, a member of the fami ly . Dictyotaceae, i s f a i r l y abundant in the subt ida l regions of B r i t i s h Columbia coas ta l waters. We have found, however, that i t shows a seasonal v a r i a t i o n in abundance. Its a v a i l a b i l i t y and the a n t i b a c t e r i a l a c t i v i t y shown by i t s crude extracts prompted us to i n i t i a t e a chemical study of D. bi nghami ae. The alga was c o l l e c t e d in the shallow bays near Dixon Is land and Execution Rock in Barkley Sound. I n i t i a l c o l l e c t i o n s of the alga were d r i e d in a convection oven at 50°C for 24 h. The dr i ed alga was then powdered in a Wiley m i l l and extracted with hexane in a Soxhlet ex trac ter . The hexane extract was concentrated in vacuo to give a dark o i l which was fract ionated by step gradient f lash chromatography using the fol lowing solvent sequence: ( i ) hexane ( i i ) hexane/chloroform 1:1 ( i i i ) chloroform (iv) chloroform/ethyl acetate 9:1 (v) ch loroform/ethyl acetate 1:1. It was evident from crude 1 H nmr and th in layer chromatography that the f i r s t two fract ions contained only fat ty compounds, hence, they were not studied fur ther . The f r a c t i o n e luted with chloroform looked in teres t ing by t i c and 1 H nmr examination. Further p u r i f i c a t i o n of th i s f r a c t i o n by r a d i a l t i c on s i l i c a gave crude samples of pachydictyol A (29), d ic tyoxide (35) and dictyoxide A (66). A d d i t i o n a l f rac t iona t ion by reverse phase hplc gave pure 22 samples of both compounds. The other two fract ions from the f lash chromatography were both green o i l s when concentrated in vacuo i n d i c a t i n g the presence of pigments. However, t i c revealed two compounds of terpenoid nature which were more polar than the prev ious ly i so la ted compounds. Further studies on these compounds were postponed because of the ir low concentrat ion. Later c o l l e c t i o n s of the alga were immersed in methanol immediately after c o l l e c t i o n . The methanol was decanted from the a lga , concentrated in vacuo, and p a r t i t i o n e d between hexane and water. After drying with anhydrous Na 2 SO„ and concentration /n vacuo , the hexane extract was subjected to the previously described step gradient f lash chromatography. The chloroform eluted frac t ions now contained an a d d i t i o n a l compound, aceta l (61a), which was obtained in pure form from the r a d i a l t i c f r a c t i o n a t i o n . Pure aceta l (61b) was obtained by r a d i a l t i c o f the e thy l acetate/chloroform e luted fract ions from the f lash chromatography. The same f lash fract ions a lso y ie lded crude samples of d i c t y o l G acetate (68), d i c t y o t r i o l A diacetate (69), d i c t y o l C (32) and a mixture of a c e t y l d i c t y o l a l (48) and e p i d i c t y o l B acetate (70). F i n a l p u r i f i c a t i o n of these compounds was achieved by hplc and preparat ive t i c . A number of resonances in the 1 H nmr spectrum (5 1.61(S , 3H), 1.69(s, 3H), 1.81(S , 3H) , 3.92(br d, J=8 Hz, 1H), 4.24(s, 1H), 4.74(s, 1H), 5.12(t , / = 6 H z , 1H), 5.38(br s, 1H) ppm, see appendix 1) of one of the pure compounds 23 (1113:^288.2453, required for C 2 0 H 3 2 O 288.2454) indicated that i t was pachydictyol A (29). Comparison with an authentic sample ('H nmr, t i c ) confirmed t h i s . Having i d e n t i f i e d pachydictyol A (29), i t was straightforward to i d e n t i f y dictyoxide (35) and d i c t y o l C (32) by comparing t h e i r spec tra l data with the reported values (see appendices 1,2,3 for 29; 4, 5 for 32 and 6, 7, 8 for 35). The other prev ious ly known compounds aceta ls 61a (see appendices 11, 12) and 61b (see appendices 13, 14), and a c e t y l d i c t y o l a l (49) (see appendices 9, 10) were a lso i d e n t i f i e d by comparing the i r spectra l propert ies to the l i t e r a t u r e values . D i c t y o l G acetate (68) was obtained as an o p t i c a l l y ac t ive ( [a]^ 2 + 5 0 ° ) c o l o u r l e s s o i l from preparative reverse phase h p l c . High r e s o l u t i o n mass spectrometry showed that i t had a molecular formula of C 2 2 H 3 « 0 3 . Its i r spectrum contained hydroxyl (3500 cm"1) and carbonyl (1725 cm" 1) absorption bands. A three-proton resonance at 5 2.02 ppm in the 1 H nmr spectrum and a s i g n i f i c a n t fragment ion at m/z 286 (M+ -CH 3 C0 2 H) in the mass spectrum, indicated the presence of an acetate ester (see f i g . 3) . The *H and 1 3 C nmr spectra of acetate 68 (see f i g s . 1 and 2) also c l e a r l y indicated that i t had the same subst i tuted b i c y c l i c r ing system as pachydictyol A (29) (see Table 1) and thus the acetoxy substituent had to be attached to the side chain.- A deshielded resonance at 5 5.57 ppm (dt, J=8,4 Hz) in the 1 H nmr spectrum of 68, which was not present in the 1 H nmr spectrum of 29, could be 24 assigned to a methine proton a to the acetoxy f u n c t i o n a l i t y . The chemical sh i f t of the methine proton implied that i t must also be a l l y l i c , and a double resonance experiment showed that i t was spin coupled to the o l e f i n i c proton on C14 (5 5.12, br d, J=8 Hz) . Thus the acetoxy f u n c t i o n a l i t y in 68 must be attached to C13. Reduction of d i c t y o l G acetate (68) with l i t h i u m in ethylamine gave pachydictyol A (29) which was i d e n t i c a l to an authentic sample by t i c and 1 H nmr comparison (see Scheme 1). The react ion was stopped before completion to prevent further reduction of products that re su l t ed in a lowered y i e l d . Dictyoxide A (66) was obtained as an o p t i c a l l y ac t ive ([a]^ 2 - 5 . 6 ° ) pale yellow o i l that had the molecular formula C 2 o H 3 0 0 . I ts i r spectrum showed an absence of e i ther hydroxyl or carbonyl absorptions and i t s 1 3 C nmr spectrum (see f i g . 5) contained two resonances at 5 79.5 and 68.6 ppm, appropriate for the carbon atoms of an ether f u n c t i o n a l i t y . 66 Table 1. 'H nmr data for Dictyota binghamiae diterpenoids (400 MHz, CDC13 ) Chemical S h i f t , 6 ppm Carbon Pachydictyol A (29) Dictyoxide A (66) Dictyol G acetate D i c t y o t r i o l A Epidictyol B acetate (68) diacetate (69) (70) 1 2 . 67 = 10 Hz 2 .62 q,J=9 Hz 2 . 79 q,J=10 Hz 2 .87 q,J=10 Hz 2 2 .62 m - 2 .49 m 2 . 49 m 2 . 25 m 3 5 . 33 br s 5 . 37 br s 5 .33 br s 5 . 34 br s 5 . 34 br s 5 2 . 32 br t 2 .75 br t,J=10 Hz 2 .33 br t 2 .33 m 2 .49 br t 6 3 .92 br d,J=8 Hz 3 .74 dd,J=10,4 Hz 3 .93 br d,J=8 Hz 3 .87 dd,J=8,3 Hz 3 .92 dd,J=8,4 I 7 - - - 2 .02 dd,J=10,3 Hz -8 1 1 .92 . 76 ddd,J=15,10,2Hz dd,J=15,6 Hz -9 - - - 5 . 59 dd,J=6,2 Hz 5 .59 dd,J=6,2 I 1 1 - - - 1 , .86 m -12 - 1 . . 14 q,c/=12 Hz 1 . ,93 m 4 , .92 m -13 - 4 .45 ddd,J=12,8,4 Hz 5. .57 dt, J=8,4 Hz - -14 5 . 12 br t,J=6 Hz 5, .20 br d,J =8 Hz 5. , 12 br d ,L/ = 8 Hz 5. . 10 t , J =7 Hz 5. . 10 m 16 1 . 61 br s 1 , .65 br s 1 . 72 d,J= 1.5 Hz 1 , .64 br s 17 1 . 81 br s 1 . 79 br s 1 . 82 d,J= 1 Hz 1 . 82 d,J=1 Hz 1 . 81 dd,J = 1.5 18 4 . , 75 s 4 . 82 s 4 . 75 s 5. IO s 5 . 10 s 4 . 74 br s 4 . 79 s 4 . 73 s 5. 03 s 5 . 04 19 1 . 00 d, J-= 7 Hz 0. 95 d,J= 7 Hz 1 . 04 d,J = 7 Hz 0. 94 d,J= 7 Hz 0. 93 d,J=7 Hz 20 1 . 69 br s 1 . 70 br s 1 . 74 d,J= 1.5 Hz 1 . 70 br s 1 . 69 br s Scheme 1. Chemical c o n v e r s i o n s of d i c t y o l G a c e t a t e (68) CXi 68 150 i i«Jnlirlr»U tin1--' •-— 160 120 80 40 PPm 1 3 F i g . 2 . C nmr spect rum of d i c t y o l G a c e t a t e (68) ( 1 0 0 . 6 MHz , CDC 1 3 ) -90 80 70 60 50 40 30 20 10 0 243 - W i |-| I'I i i i VI [ 240 . 258 260 2d8 286 I. I., , .. I I I I I | I / 280 l l l l l l 02 I I 1 I II M 68 34G 300 I ' 1 ' 1 ' 1 | 1 1 1 1 1 1 t I I I I I I I I I II I I I I I I I | | | | I | | , | , 320 340 380 380 F i g . 3 . Mass spect rum of d i c t y o l G a c e t a t e (68) 30 >-66 S i i W 4# * * • i i i i i 1 1 1 : 1 » r • • — 160 120 80 40 ppm F i g . 5 . i J C nmr spec t rum of d i c t y o x i d e A (66) ( 1 0 0 . 6 MHz, CDC1. , ) . F i g . 6. M a s s s p e c t r u m o f d i c t y o x i d e A ( 6 6 ) oo 33 Six a d d i t i o n a l carbon resonances at 8 152.0, 141.0, 133.5, 126.8, 124.9 and 106.8 ppm could.be a s s i g n e d to three o l e f i n f u n c t i o n a l i t i e s . Since the molecular formula of 66 r e q u i r e s s i x s i t e s of u n s a t u r a t i o n , i t was concluded that 66 must be t r i c y c l i c . The 1H nmr spectrum of 6 6 (see f i g . 4) showed methyl resonances at 8 0.95 (d, 3H, J=7 Hz), 1.65 (br s, 3H), 1.79 (br s, 3H) and 1.70 (br s, 3H) ppm that were v i r t u a l l y i d e n t i c a l to those observed f o r the three o l e f i n i c and one a l i p h a t i c methyl groups i n p a c h y d i c t y o l A (29) (see Table 1). In a d d i t i o n , o l e f i n i c proton resonances at 5 5.37 (br s, 1H), 4.82 (br s, 1H) and 4.79 (br s, 1H) ppm were s i m i l a r to those observed f o r t h e i r c o u n t e r p a r t s on C3 and C18 i n 29. The above evidence l e d to the hy p o t h e s i s that d i c t y o x i d e A ( 6 6 ) had the same carbon s k e l e t o n as p a c h y d i c t y o l A (29) but that the a l c o h o l f u n c t i o n a l i t y of 29 was r e p l a c e d by a c y c l i c ether i n 6 6 . I t was assumed t h a t one p o i n t of attachment of the c y c l i c ether was C6 and a resonance at 8 3.74 (dd, 1H, J=10,4 Hz) ppm was a s s i g n e d to the methine proton on t h a t carbon. A d o w n f i e l d resonance at 8 4.45 (ddd, 1H, /= 12, 8, 4 Hz) ppm was a s s i g n e d to a methine proton on the second ether carbon. T h i s methine proton was shown v i a a double resonance experiment to be s p i n coupled to the o l e f i n i c proton on C14 (8 5.20 ppm, br d, 1H, J=8 Hz) and hence C13 had to be the second p o i n t of attachment of the c y c l i c e t h e r . The c o n s t i t u t i o n of d i c t y o x i d e A ( 6 6 ) was confirmed by treatment of d i c t y o l G a c e t a t e ( 6 8 ) with aqueous a c e t i c a c i d 34 which gave a 20% y i e l d of 66 (see Scheme 1) and a very low y i e l d of a second compound which was presumed to have the structure 67. An analys i s of the coupl ing constants observed for H13 and H12a in ether 66 revealed that the tetrahydropyran r ing assumed a chair conformation and allowed us to assign the r e l a t i v e conf igurat ion at C13. H13 shows a 8 Hz v i c i n a l coupling to H14, a 4 Hz a x i a l - e q u a t o r i a l coupl ing to H12j3 and a 12 Hz a x i a l - a x i a l coupl ing to H12a (see table 1). Therefore HI 3 must be a x i a l and the v i n y l residue must be e q u a t o r i a l . H12a appears as quartet at 5 1.14 ppm with a 12 Hz coupling constant. It must therefore be coupled to H12/3 with a 12 Hz geminal coupl ing and to an a x i a l proton on C11. Thus the C19 methyl must be equator ia l and cis to the C13 v i n y l subst i tuent . Since the r e l a t i v e stereochemistry at C11 in pachydictyol A (29) i s known, i t can be concluded that dictyoxide A (66) adopts the conformation shown in F i g . 7 . F i g . 7. Conformational drawing of dictyoxide A (66). 35 The epimer 67 could not be adequately character ized due to a very l imi t ed sample s i z e , but i t s 1 H nmr spectrum was consistent with the proposed s tructure . D i c t y o t r i o l A d iacetate (69) was obtained as an o p t i c a l l y ac t ive ( [a]^ 2 + 1 2 . 5 ° ) co lour less o i l that had a molecular formula of C 2 a H 3 6 0 5 . Its i r spectrum showed hydroxyl (3450 c m - 1 ) and carbonyl (1730 cm"1) absorption bands. Two s ing le t methyl resonances at 6 2.06 and 2.07 ppm in the 'H nmr spectrum of 69 (see f i g . 8) indicated that i t was a d iace ta te . The remainder of the 'H nmr spectrum suggested that the diacetate 69 had the same carbon skeleton as pachydictyol A (29) and that the A 3 ' 4 , A 1 0 - 1 1 8 , A 1 4 1 1 5 and C6 hydroxy f u n c t i o n a l i t i e s were intact (see Table 1). Two resonances at 5 4.92 (m, 1H) and 5.59 (dd, 1H, J=2,6 Hz) ppm could be assigned to methine protons a to the acetoxy f u n c t i o n a l i t i e s . This implied that the acetates were both esters of secondary a lcoho l s , one of them a l l y l i c . I r r a d i a t i o n of the a l l y l i c methine proton (5 5.59 ppm) d id not change e i ther the C13 or C14 proton resonances (5 5.34 and 5.10 ppm) but i t d i d s impl i fy the H8a resonance to a broad doublet (8 1.76 ppm, J=15 Hz) and the H8/3 resonance to a doublet of doublets (5 1.92 ppm, J=15,8 Hz) . Decoupling further demonstrated a 1.5 Hz W coupling between H8a and H6, confirming . the assignment of the former resonance. The a l l y l i c acetate must therefore be on C9 and i t was assigned the a conf igurat ion because both the m u l t i p l i c i t y and chemical s h i f t of the a acetoxy proton were s u b s t a n t i a l l y 36 di f f erent than those reported for d i c t y o l B acetate (37) in which the C9 acetate i s j3 (12: H9a, 5 5.16 ppm, t , J=7 Hz) . The stereochemistry at C9 was confirmed by demonstrating a d i f ference nOe in H5, H8/3 and H8a when H9 was i r r a d i a t e d . Placement of the second acetoxy f u n c t i o n a l i t y on C12 followed from the fact that i t had to be on a n o n - a l l y l i c secondary carbon, which according to decoupling experiments, was not v i c i n a l to the C9 acetate . Further support for the pos i t ions of both acetoxy f u n c t i o n a l i t i e s could be found in a 1 H 2D nmr COSY 5 0 experiment (see f i g . 11). C l e a r l y v i s i b l e are the (14/13,13') and (12/13,13') o f f -d iagona l peaks which would be expected i f diacetate 69 contains a C12 acetoxy. It i s a lso poss ib le to follow the required coupl ing sequence from the C9 acetoxy a methine proton to the C6 carb ino l methine proton. Thus s t a r t i n g at C9, we f ind the o f f -d iagonal elements (9/8a), (9/8/3), (7/8/3) and (6/7) . 69 R = OAc 70 R =H E p i d i c t y o l B acetate (70) was obtained as an o p t i c a l l y F i g . 9 . SFORD 1 J C nmr spect rum of d i c t y o t r i o l A d i a c e t a t e (69) ( 1 0 0 . 6 MHz, CDC1~). w ^ CO F i g . 10. Mass spect rum of d i c t y o t r i o l A d i a c e t a t e ( 6 9 ) . to VO F i g . 1 1 . C o n t o u r p l o t o f t h e COSY 2D nmr s D e c t r u m o f d i c t y o t r i o l A d i a c e t a t e ( 6 9 ) ( 4 0 0 M H z , C D C 1 3 ) . O f f - d i a g o n a l r e s p o n s e s e s t a b l i s h i n g p r o t o n s p i n - c o u p l i n g c o n n e c t i v i t i e s a re l a b e l l e d w i t h t h e p r o t o n s i n v o l v e d , t h e d o w n f i e l d r e s o n a n c e l i s t e d f i r s t . O n l y t h e c o n n e c t i v i t i e s r e q u i r e d t o d e m o n s t r a t e t h e p o s i t i o n s o f t h e a c e t a t e f u n c t i o n a l i t i e s a r e shown. CT^S'0 A c HO \ ^ J ^ 70 JLJUUI . A A * K J^ UUIA^ JM, J , » J » • 1 r 5 4 3 2 1 PPm F i g . 12 . H nmr spect rum of e p i d i c t y o l B a c e t a t e (70) (400 MHz, C D C 1 - ) . .» 70 — r — 160 120 80 40 ppm Fig. 13. C nmr spectrum of epidictyol B acetate (70) (100.6 MHz, CDCI-). F i g . 14 . Mass s p e c t r u m of e p i d i c t y o l B a c e t a t e (70). 44 ac t ive .([a]^ 2 + 4 3 . 6 ° ) co lour l e s s o i l which had a molecular formula of C 2 2 H 3 f t 0 3 . Its i r spectrum contained hydroxyl (3500 cm"1) and carbonyl (1740 c m - 1 ) absorption bands. A major fragment ion at m/z 286 (M+ - CH 3 C0 2 H) in the mass spectrum (see f i g . 14) and a s ing le t methyl resonance at 8 2.02 ppm in the 1 H nmr spectrum (see f i g . 12) indicated the presence of an acetate e s ter . The 1 H nmr resonances 5 5.59 (dd, 1H, J=6,2 Hz) , 5.34 (br s, 1H), 5.10 (m, 1H), 5.10 (br s, 1H) and 5.04 (br s, 1H) ppm of 70 were i d e n t i c a l to those of 69 i n d i c a t i n g that the acetoxy f u n c t i o n a l i t y i s attached to C9 (see table 1). The assignment of the a conf igurat ion to the acetate in 7.0 i s p a r a l l e l e d the stereochemical assignment at C9 in 69. 45 C.DISCUSSION The secondary metabolites d i c t y o l G acetate (68), d i c t y o t r i o l A diacetate (69) 1 and e p i d i c t y o l B acetate (70) represent three new addit ions to the group of metabolites with perhydroazulene carbon skeletons commonly found in members of family Dictyotaceae. It i s i n t e r e s t i n g to note that a l l three new compounds contain an acetoxy f u n c t i o n a l i t y . Pachydictyol A (29),d i c t y o l C (32), d i c t y o l F (42) and e p i d i c t y o l F (43) are reported to have a n t i m i c r o b i a l a c t i v i t y while d ic tyoxide (35), epoxypachydictyol A (44) and methoxydictydiene (45) do not show any a n t i b a c t e r i a l a c t i v i t y 3 1 ' 3 8 . A standard i n - v i t r o disc bioassay on the three new acetates 68, 69 and 70 showed mild ant i fungal a c t i v i t y 'Two diterpenoids i so la ted from Dictyota i ndi ca were named d i c t y o t r i o l A (71) and B ( 7 2 ) 5 1 . 71 14 R or S 72 14 S or R The Chinese work on D. indica, and our work on D. bi nghami ae52 appeared almost simultaneously in the l i t e r a t u r e . It i s obvious that the assignment of c i s - f u s e d rings for 71 and 72 i s an e r r o r . When corrected to include trans-fused r i n g s , 71 i s the same compound described e a r l i e r as d i c t y t r i o l ( 6 8 ) 3 7 . (The name d i c t y t r i o l B for the C-14 epimer of d i c t y t r i o l (68) has the precedence over the name i s o d i c t y t r i o i which appeared la t er in the l i t e r a t u r e 5 3 ) . 46 against Pythium ultimum, Rhisoctonia solani, and Helmi nt hospori um sativum. D i c t y o l G acetate (42) and e p i d i c t y o l B acetate (70) also showed mild a n t i b a c t e r i a l a c t i v i t y against Staphylococcus aureus and Bacillus subtilis. D i c t y o t r i o l A diacetate (69) d id not show any observable a c t i v i t y against these two b a c t e r i a . Dictyoxide A (66) was found to be present only in the oven-dried samples of Dictyota binghamiae. The observed absence of (66) in the methanol extracted alga seems to indicate that i t i s an a r t i f a c t formed from d i c t y o l G acetate (68) during oven d r y i n g . The two acetals 61a and 61b also appear to be a r t i f a c t s a r i s i n g from the react ion of d i c t y o d i a l (48) with methanol as was reported e a r l i e r by Japanese workers" 6 . It i s quite common to f ind more than one c la s s of diterpenes in a s ing le Dictyotaceae species . This phenomenon i s a lso true for Dictyota binghamiae which contains metabolites with both perhydroazulene and xenicane carbon skeletons. 47 SECONDARY METABOLITES FROM THE SRI LANKAN SPONGE Agelas sp. A.INTRODUCTION Sp o n g e s ( p h y l u m P o r i f e r a ) a r e p r i m i t i v e m a r i n e i n v e r t e b r a t e s w h i c h a r e c o n s i d e r e d t o be t h e s i m p l e s t m u l t i c e l l u l a r a n i m a l s . C h e m i c a l s t u d i e s on t h e s e a n i m a l s have r e v e a l e d t h a t t h e y a r e a p r o l i f i c s o u r c e o f s e c o n d a r y m e t a b o l i t e s w i t h u n u s u a l s t r u c t u r e s . I t i s i n t e r e s t i n g t o n o t e t h a t most o f t h e s e compounds do n o t h a v e a n y t e r r e s t r i a l a n a l o g s . The m a j o r i t y o f sp o n g e m e t a b o l i t e s a r e t e r p e n o i d s . 73 The b r o m o p y r r o l e a l k a l o i d , o r o i d i n ( 7 3 ) , i s o l a t e d f r o m Agel as oroides by F o r e n z a et al . i n 1 9 7 1 5 a i s a t y p i c a l e x a m p l e o f p e r h a p s t h e b e s t known f a m i l y o f a l k a l o i d s f r o m m a r i n e s p o n g e s . O r o i d i n ( 7 3 ) , h a s s i n c e a p p e a r e d a s t h e m a j o r m e t a b o l i t e o f s e v e r a l Agel as s p e c i e s . F o r e n z a et al. a l s o r e p o r t e d t h e i s o l a t i o n o f b r o m o p y r r o l e s 74, 7 5 , 76 a n d 77, b u t t h e i r s t r u c t u r a l p r o p o s a l f o r o r o i d i n was c o n t r o v e r s i a l a n d t h e c o r r e c t s t r u c t u r e was l a t e r p r o p o s e d by G a r c i a et al. 48 and v e r i f i e d v i a s y n t h e s i s 5 5 . A recent inves t iga t ion of the sponge Agelas sceptrum (Lamarck) by Walker et al . produced a s ingle c r y s t a l X-ray d i f f r a c t i o n analys i s of o r o i d i n (73), confirming the correct s t r u c t u r e 5 6 . The s tructure of sceptr in (78), the major a n t i m i c r o b i a l const i tuent of A. sceptrum, was a lso secured by X-ray crys ta l lography . Attempts to prove the hypothesis that 78 is the [2+2] cyc lo adduct of desbromooroidin (79) were u n s u c c e s s f u l 5 6 . Keramadine (80), which has the Z geometry, was found in a study of an unknown Agelas species c o l l e c t e d in J a p a n 5 7 . Chevolot et al. reported the i s o l a t i o n of midpakamide (81) from the Hawaiian sponge Agelas mauritiana50 . 74 75 R*NH 2 7 6 OH 7 7 R = OMe 49 M e t a b o l i t e s which are b i o l o g i c a l l y r e l a t e d to o r o i d i n (73) have been a l s o i s o l a t e d from sponges i n the fami l y Axinellidae. Sharma and Burkholder i s o l a t e d monobromophakellin (82) and d i b r o m o p h a k e l l i n (83) from Phakellia f l a b e l l a t a 5 9 . Sharma et al. l a t e r r e p o r t e d the i s o l a t i o n of 84, which shows f u r t h e r c y c l i s a t i o n of the o r o i d i n ( 7 3 ) s k e l e t o n , from the same s p o n g e 6 0 . The brominated 50 51 compound 85 has been i so la t ed from Axinella verrucosa and Acanthella aurant iaca6 1 , while stevensine (86) has been found in an u n i d e n t i f i e d sponge 6 2 . The presence of s imi lar metabolites in these sponges of d i f f e r e n t fami l ies indicates a chemical r e l a t i o n s h i p between them and perhaps indicates errors in the taxonomic c l a s s i f i c a t i o n s . The b i o l o g i c a l a c t i v i t y exhib i ted by the metabolites of the ' o r o i d i n group' i s noteworthy. A l l the members of the genus Agelas examined in a study of Caribbean sponges by Walker et al. were shown to have a n t i m i c r o b i a l a c t i v i t y 5 6 . Faulkner, even af ter studying the metabolites of Verongia species for several years , concluded in an e a r l i e r report that the most ac t ive a n t i b i o t i c of those sponges had not been d e s c r i b e d 6 3 . This may be true for Agel as species as w e l l . 52 B.ISOLATION AND STRUCTURE ELUCIDATION An un ident i f i ed Agelas sp. was c o l l e c t e d by hand using SCUBA on shallow rocky reefs off Mt. Lav in ia and Hikkaduwa, S r i Lanka. Freshly c o l l e c t e d animals were immersed in methanol and kept at low temperatures u n t i l work up. The aqueous suspension obtained af ter removing methanol from th i s extract was extracted sequent ia l ly with hexane, chloroform and e thy l acetate . The r e s u l t i n g aqueous phase was f r e e z e - d r i e d . The combined organic extracts were chromatographed on Sephadex LH-20 to obtain pure desbromooroidin (79). LH-20 chromatography of the methanol so luble port ion of the freeze-dr ied aqeuous phase gave more of pure 79. Pure 79 was insoluble in less polar organic solvents but f a i r l y soluble in methanol. This general i n s o l u b i l i t y sometimes created unexpected problems. When the i n i t i a l aqueous suspension obtained after removal of methanol from another batch of the sponge c o l l e c t i o n was extracted with organic s o l v e n t s , , a brown p r e c i p i t a t e was obtained. This brown p r e c i p i t a t e , upon p u r i f i c a t i o n by LH-20 chromatography gave 79. Desbromooroidin (79) was found to be unstable, and there fore , i t was kept away from l i g h t and solvents whenever p o s s i b l e . Its i r spectrum showed a broad N-H absorption (3600-2800 cm"1) and an amide carbonyl s treching band (1680 c m - 1 ) . The uv spectrum of 79 exhibi ted a maximum at 269 nm, 53 s i m i l a r to the metabolites of the o r o i d i n group. The 1 H nmr spectrum of 79 in CD3OD was rather simple. Several decoupling experiments revealed the i s o l a t e d spin system -CH=CH-CH 2- [5 3.97(br d,2H, /=5 Hz) , 6.17(dt, 1H, 7=16,5 Hz) , 6.23 (d, 1H, 7=16 Hz) ppm]. The 16 Hz coupl ing between the o l e f i n i c protons indicated an E conf igurat ion for the double bond. The 'H nmr spectrum also contained three aromatic resonances [6 6.74 ( s , ' 1 H ) , 6.82(d, 1H, y=1.5 Hz) , 6.92(d, 1H, 7=1.5 Hz) ppm J . The 1 H nmr spectrum of 79 in DMSO-d6 ( f i g . 15) showed, in addi t ion to the previous ly observed resonances, six exchangeable protons [6 7.43(br s, 2H), 8.48(t , 1H, 7=5 Hz), 11.85(br s, 1H), 12.0(br, 1H), 12.55(br, 1H) ppm]. The methylene protons in DMSG~d6 appeared as a t r i p l e t , showing a d d i t i o n a l coupl ing to the exchangeable proton at 6 8.48 (t , 1H, 7=5 Hz) ppm. This es tabl i shed the subunit -CH=CH-CH 2-NH-. The aromatic resonances [6 6.90(dd, 1H, 7=2.5,1.5 Hz) , 6.95(dd, 1H, 7=2.8,1.5 Hz) ppm] and the exchangeable proton at 5l1.85(br s, 1H) ppm could be assigned to a 2 ,4 -d i subs t i tu ted pyrro le r ing which was indicated by a p o s i t i v e E h r l i c h test (red c o l o u r ) . The remaining 1 H nmr resonances [6 7.43(br s, 2H), I2 .0(br , 1H), 12.55(br, 1H) ppm] indicated an imidazolinium moiety suggesting the presence of ' o r o i d i n skeleton' in 79. The electron-impact mass spectrum of 79 d id not show a molecular ion and was of no use. A FAB mass spectrum showed molecular ions at m/z 310 (M++H) and m/z 312 (M++2+H) F i g . 1 5 . *H nmr spec t rum of d e s b r o m o o r o i d i n (79) (400 MHz, D M S 0 - d g ) . at H 0 79 1 6 0 1 4 0 1 2 0 1 0 0 8 0 6 0 4 0 2 0 ppm 0 F i g . 16. C nmr spect rum of d e s b r o m o o r o i d i n (79) ( 1 0 0 . 6 MHz, DMSO-dg) . 56 ind ica t ing that 7 9 had the molecular formula C, ^ ^ N j O B r . The 1 3 C nmr spectrum of 7 9 was in agreement with th i s assignment (see f i g . 16 and appendix 15). Further support for the proposed s tructure came from the comparison of spec tra l data with that of the previous ly reported metabolite keramadine ( 8 0 ) (see tables 2 and 3). Mass spectrometry of other fract ions obtained from the LH-20 chromatography indicated the presence of o r o i d i n ( 7 3 ) [FAB: m/z 388(M++H), 390(M++2+H), 392(M++4+H)], sceptr in ( 7 8 ) [FAB: m/z 619(M++H), 62l(M++2+H), 623(M++4+H)], and 4,5-dibromopyrrole-2-carboxamide ( 7 5 ) [EI ms: m/z 266(M + ) , 268(M++2), 270(M ++4)], but the trace amounts i so la ted were not s u f f i c i e n t for a complete i d e n t i f i c a t i o n of i n d i v i d u a l compounds. Due to the l i m i t e d a v a i l a b i l i t y of the sponge m a t e r i a l , work on these metabolites was not pursued.. 57 Table 2. 1 H nmr data for desbromooroidin (79) and keramadine (80). Chemical s h i f t , 8 ppm Pos i t ion 7 9 ° 80 1 12.55 br 3 12.00 br 11.96 br s 4 6.86 br s 7.02 s 6 6.23 d, 7=16 Hz 6.23 7=11 Hz 7 6.17 d t , 7=16,5 Hz 5.81 dt , 7= i1 ,5 .6 Hz 8 3.97 br t , 7=5 Hz 4.01 t , 7=5.6 Hz 9 8.48 t , 7=5 Hz 8.22 t , 7=5.6 Hz 12 11.85 br s 11 .58 br s 13 6.95 dd, 7=2.8,1.5 Hz 6.92 dd, 7=2.9,1.5 Hz 15 6.90 dd, 7=2.5,1.5 Hz 6.80 dd, 7=2.9,1.5 Hz N(2) 7.43 br s 7.59 br s N(1)CH 3 - 3.38 s a. 400 MHz, DMSC-d 6 , b. ref .57 58 Table 3. 1 3 C nmr data for desbromooroidin (79) and keramadine (80) . Chemical s h i f t , 5 ppm Carbon # 79 a 80* 2 147.6 s 1 46.9 s 4 1 16.4 d c 112.0 5 1 24.9 s 1 23.7 s 6 1 27. 1 d 1 33.3 d 7 110.9 d c 113.8 d 8 40.0 38.6 t 10 159.6 s 1 59.6 s 1 1 1 26.7 s 1 26.7 s 1 3 121.3 d 121.3 s 14 95. 1 s 95.0 s 15 111.9 d 111.7 d N( 1)CH 3 29.2 q a. 100.6 MHz, DMSO-d 6 , assignments are based on comparison, b. r e f . 5 7 , c. may be interchanged. 59 C.DISCUSSION Desbromooroidin (79) i s the la tes t addi t ion to the well known o r o i d i n group of a lka lo ids found in marine sponges. Members of t h i s family possess both pyrro le and imidazolinium moiet ies . The natura l occurrence of 79 was foreseen by Faulkner et al. when they i so la ted sceptr in (78) which is conceptual ly the [2+2] cyclo adduct of 7 9 5 6 . Although attempts in Faulkner ' s laboratory f a i l e d to provide any evidence favouring that concept, the natural occurrence of desbromooridin (79) may be regarded as a favourable i n d i c a t i o n . However, the question whether desbromooroidin (79) i s the biogenetic precursor of 78 s t i l l remains to be answered. Many of the halogenated he terocyc l i c compounds of the o r o i d i n group have been reported to possess a n t i m i c r o b i a l a c t i v i t y . The a n t i m i c r o b i a l const i tuent of Agelas oroidas was i d e n t i f i e d as 4 ,5-dibromo-2-cyanopyrrole ( 7 4 ) 6 " . Sceptr in (78) was reported to have considerable a n t i b a c t e r i a l a c t i v i t y as well as some ant i fungal a c t i v i t y 5 6 . The b i o l o g i c a l a c t i v i t y reported for keramadine (80) i s i n t e r e s t i n g . It was i so la ted in a study of p h y s i o l o g i c a l l y act ive substances of marine sponges by a Japanese group and was discovered to be an antagonist of serotonergic r e c e p t o r s 5 7 . Not s u r p r i s i n g l y , desbromooroidin (79) was found to exh ib i t s i g n i f i c a n t a n t i b a c t e r i a l a c t i v i t y against Staphylococcus aureus and Bacillus subtilis and ant i fungal 60 a c t i v i t y against Phyt hi um ul t i mum, Rhisoctonia solani and He I minthospori um sativum. 61 I I I . A SECONDARY METABOLITE OF THE STARFISH Dermasterias imbri cat a, THAT ELICITS A SWIMMING RESPONSE IN THE SEA ANEMONE Stompi a cocci nea A.1.INTRODUCTION S t a r f i s h e s , or sea s tar s , are i n t e r t i d a l marine invertebrates belonging to the c lass Asteroidea of the phylum Echinodermata. These fasc inat ing animals have rather heavy arms which project from a cen tra l disc with an imperfect r a d i a l symmetry. Most species have 5-6 arms but the number may be as high as 4 0 6 5 . The arms usual ly increase in width toward the base. The average diameter of a grown s t a r f i s h is about 12 to 24 cm but there are some sea s tars which.are less than 2 cm in diameter. Asteroids are ac t ive predators , feeding upon a l l sorts of invertebrates and even f i s h 6 5 . Chemical studies on asteroids have led to the i s o l a t i o n of polyhydroxylated steroids and s a p o n i n s 6 6 . Saponins were previous ly known only from a large number of t e r r e s t r i a l p lant s . However, we now know that saponins are the t y p i c a l metabolites of echinoderms. The general s tructure of saponins cons i s t s of an aglycone with a carbohydrate side cha in . Saponins der ived from s tarf i shes are known as asterosaponins and they contain s t e r o i d a l aglycones. The presence of saponins in s t a r f i s h was discovered in 1960 by Yasumoto and Hashimoto 6 7 . In 1965 they i so la ted the 62 8 7 two major saponins of Asterias amurensis. The s tructure of one of them, asterosaponin A (87), was determined in 1973 by Ikegami et a / . 6 8 . Thornasteroside A (88) i s another example of an asterosaponin which was i s o l a t e d from the s t a r f i s h Acant hast er pianci 6 9 . The presence of a su l fate group at C-3 and the attachment of an o l igosacchar ide moiety to C-6 of the aglycone are general features of asterosaponins. Sepsitoside A (89) i s one of a group of unusual saponins discovered from Echinaster saposit u s 7 0 . The absence of the su l fate group and the c y c l i z a t i o n of the carbohydrate chain between C-3 and C-6 of the aglycone are the d i s t i n c l y d i f f erent features possessed by E. sapositus saponins. 64 OH Holothurins , saponins i s o l a t e d from sea cucumbers, (for example, holothurin B ( 9 0 ) 7 1 ) are d i f f eren t from asterosaponins. The presence of a s t ero id aglycone in asterosaponins and a t r i t e r p e n o i d aglycone in holothurins i s the basic d i f f erence . In both s tar f i shes and sea cucumbers, the saponins are bel ieved responsible for the general t o x i c i t y of the a n i m a l s 6 6 . It has been known for a long time that various marine invertebrates such as sea anemones, b r i t t l e s t a r s , sea urchins and molluscs d i sp lay 'avoidance react ions ' and 'escape responses' when they are in the presence of, or when they are contacted by, s t a r f i s h e s 7 2 . According to some s tudies , th i s 65 unusual behavior of prey organisms is caused by a s t e r o s a p o n i n s 7 3 , but i t has a lso been found that some prey organisms react to substances other than a s t e r o s a p o n i n s 7 4 . 2.THE LEATHER STAR, Dermast erias imbricata B i o l o g i s t s long ago discovered an in tere s t ing i n t e r a c t i o n between the Northeast P a c i f i c sea anemone, St ompia cocci nea, and the s t a r f i s h , Dermasteri as imbricata1*. As Ward d i s c r i b e s i t , "when contacted by the s t a r f i s h , the sea anemone responds in a s t r i k i n g and extraordinary manner: the anemone releases i t s basal d isc from the substratum and then propels i t s e l f through the water by means of a ser ies of whip- l ike mot ions" 7 6 . He also reported that a s ingle chemical substance was responsible for e l i c i t i n g the unusual response in the anemone and suggested that the chemical was an aminopolysaccharide. Subsequent work aimed at e luc ida t ing the s tructure of the act ive metabolite of Dermast eri as imbricata was c a r r i e d out by the groups of D. Ross and W. Ayer at the U n i v e r s i t y of A l b e r t a . Using a bioassay guided f rac t ionat ion scheme they were able to i so la te the pure metabolite and to show that i t was a su l fur conta in ing , 24-carbon compound 7 4 ' ' 7 7 . 66 B.ISOLATION AND STRUCTURE ELUCIDATION In the present study, the s t a r f i s h Dermasterias imbricata was c o l l e c t e d from B r i t i s h Columbia coas ta l waters where i t i s commonly a v a i l a b l e . Dermasterias imbricata has f ive arms with an average diameter of about 15 cm in a grown animal . Adult specimens are usua l ly orange-brown in co lour . The animals were immersed in methanol immediately a f ter c o l l e c t i o n , and usual ly a f ter 2-3 days, the methanol was decanted and f i l t e r e d through C e l i t e . The r e s u l t i n g f i l t r a t e was concentrated in vacuo, d i s so lved in water, and passed through XAD-4 r e s i n . Imbricatine (91) was adsorbed onto the XAD-^ 4 which was then washed repeatedly with d i s t i l l e d water p r i o r to e lu t ion with hot methanol. The golden-yellow eluate was concentrated to dryness in vacuo and passed through Biogel P2 using 1% acet ic a c i d in water as the e luent . The c o l l e c t e d fract ions were monitored by uv spectroscopy, and those with uv absorptions at 283 and 292 nm were pooled together and f r e e z e - d r i e d . The yellow s o l i d obtained was further p u r i f i e d by chromatography on Sephadex LH-20 using methanol/water 4:1 as the eluent to give pure imbricat ine (91) as an amorphous white s o l i d . This p u r i f i c a t i o n procedure was a modif icat ion of the one o r i g i n a l l y employed by the Un ivers i ty of Alberta groups. Imbricatine (91) i s a water-soluble compound whose i r spectrum (see f i g . 17) had a broad absorption in the N-H and O-H region (3600-2800 c m - 1 ) and carboxylate s tre tch ing bands 67 at 1630 and 1595 cm" 1 . The molecular formula of imbricat ine ( 9 1 ) , C 2 U H 2 6 N 4 0 7 S , was determined from the mass spectra of the parent compound [FAB: 515(M++H)] and the dimethylpentaacetyl d e r i v a t i v e 9 2 [FAB: 753(M++H); hrms: (M +-OAcbenzyl) observed 603.1756, required for C a y ^ ^ G - T o S 603.1761]. Its amino ac id nature was indicated by a p o s i t i v e colour react ion with n i n h y d r i n 2 and uv absorptions at 283(e 2300) and 292(e 2100) nm. Eighteen protons attached to carbons were shown by 1 H and 1 3 C ADEPT2 nmr spectra of imbricat ine ( 9 1 ) (see f ig .18 and appendix 17). The presence of three - C H - C H 2 - un i t s [ 1 H nmr: 5 4.67(dd, 1H, 7=8,4 Hz) , 3.22(dd, 1H, 7=13.9,4.5 Hz) , 2.85(dd, 1H, 7=13.9,8.4 Hz); 3.76(dd, 1H, 7=12.5,5 Hz) , 4.03(dd, 1H, 7=16,5 Hz) , 2.71(dd, 1H, 7=16,12.5 Hz); 3.60(dd, 1H, 7=9,5 Hz) , 3.30(dd, 1H, 7=15,9 Hz) , 3.02(dd, 1H, 7=15,5 Hz) ppm] in 9 1 was es tabl i shed by 1 H nmr decoupling experiments ' and a HETCOR experiment 2 (a ' H / 1 3 C nmr c o r r e l a t i o n experiment, see f i g . 21). A 1 ,4 -d i subst i tu ted phenyl r ing was indicated by 1 H nmr resonances at 5 6.71(d, 2H, 7=8 Hz) and 7.04(d, 2H, 7=8 Hz) ppm. Comparison of the 1 3 C nmr spectrum of 9 1 (5 170.5, 169.7, 159.7, 156.4, 155.8, 137.8, 137.7, 131.1, 130.5, 126.8, 126.5, 115.3, 112.7, 110.0, 104.0, 54.9, 52.9, 52.3, 37.3, 32.3, 27.2, 25.1 ppm, see f i g . 19) with that of tyros ine (5 170.4, 156.9, 130.7, 124.8, 115.6, 53.7, 35.0 ppm) i d e n t i f i e d a benzyl ic fragment 2 ( 9 1 ) gave a brown colour with ninhydrin and that turned bright blue with H 2 S 0 4 and heat. A s imi lar colour react ion was observed for N-methylhist idines and tyros ine . 68 91 R=R=H 1 2 92 R=Ac R =Me 1 . 2 in imbricat ine ( 9 1 ) , and the the 1 3 C resonances of 9 1 at 5 37.3 (t) and 52.9 (d) ppm were assigned to C-12 and C-1 r e s p e c t i v e l y . An a d d i t i o n a l 1 H nmr resonance at 5 6.42 (s) ppm indicated the presence of a polysubst i tuted aromatic r i n g . The 1 3 C nmr spectrum of ( 9 1 ) a lso pointed to the presence of two carboxy l i c ac id f u n c t i o n a l i t i e s [5 169.7(s) and 170.5(s) ppm]. A 5- th io-N-methylhis t id ine fragment in 9 1 could be i d e n t i f i e d by comparing 1 H and 1 3 C nmr spec tra l data to the values reported for the symmetrical d i s u l f i d e 9 3 (see tables 4 and 5) i s o l a t e d from echinoderm e g g s 7 8 . Table 4. 'H nmr data for imbricatine (91), 94 and 93. Chemical s h i f t , 6 ppm Carbon # 9 1 a 94 a 7 1 3 4 5 7 12 14 15 2' 6' 7' 4 . 67 3.76 4.03 2.71 42 22 85 04 71 73 30 3 .02 3.34 3.60 4.31 3.66 dd,J=8.4,4.5 Hz dd,J=12.5,5 Hz dd,J=16,5 Hz dd, J=12.5,16 Hz dd, J=13.9,4.5 dd, J13.9,8 . 4 d, J=8 Hz d, J=8 Hz s:8.73 s* dd, J=15,9 Hz, dd, J=15,5 Hz: d, 2H, d=8 Hz" dd, J=5,9 Hz: t, J=8 Hz* s : 3.87 s* Hz Hz 4.57 dd , L / = 8,4 Hz 3.20 dd,J= 12,4 Hz 2.90 dd.,J = 16,4 Hz 2.71 dd,' J= 12, 16 Hz 6.08 d, d=2 Hz 6.22 d, J=2 Hz 3.12 dd, J=14,4 Hz 2.80 dd, J=14,8 Hz 7.04 d, J= 8 Hz 6.70 d, J= 8 Hz 8.95 s 3.44 d, 2H, J=7.5 Hz" 4.29 t, J= 7.5 Hz 3.94 s* a. spectra run in D M S O - d s *. chemical s h i f t s in D z O . <0 Table 5. 1 3 C nmr data for imbricat ine ( 9 1 ) , 9 4 and 9 3 . Chemical s h i f t , 5 ppm Carbon # 9 1 * 9 4 a 9 3 * 1 52.9 d 53.0 d 3 54.9 d 55.5 d 4 27.2 t 30.0 t 5 110.0 s 106.0 d 6 1 59.7 s 157.0 s 7 104.0 d 100.9 d 8 1 55.8 s 154.7 s 9 112.7 s 110.8 s 10 137.7 s 1 36.0 s -1 1 169.7 s 1 69.2 s 1 2 37.3 t 37.8 t 13 126.5 s 126.9 s 1 4 1 30.5 d 1 30.2 d 1 5 115.3 d 115.0 d 1 6 156.4 s 1 56.0 s 2' 1 37.8 d 141.1 d 4' 126.8 s 130.0 s 5' 131.1 s 134.8 s 6' 25.1 t 25.5 t 7' 52.3 d 54.5 d 8' 1 70.5 s 173.3 s 9' 32.3 q 34.0 r q a. spectra run in DMSO-d6 * . Chemical s h i f t s in D 2 0 . nmr spect rum of i m b r i c a t i n e (91) a t 100 C (400 MHz, D M S 0 - d r ) . 0 75 F i g . 2 1 . Contour p l o t of HETCOR 2D nmr s p e c t r u m of ( 9 1 ) . 1 13 Only the H and C nmr chemical s h i f t regions required to demonstrate the C-H correlat ions of three -CH-CH„-systems were included. ^ F ig . 23. CI mas s s p e c t r u m o f 92. 78 The remaining eight protons required by the molecular formula could be located by a sequence of d e r i v a t i z a t i o n reac t ions . Imbricatine (91) could be methylated (MeOH/HCl, r e f l u x , 2.5h) and acety lated ( A c 2 0 / p y r i d i n e , - r . t . , 2 0 h ) to give the dimethylpentaacetate (92), the 1 H nmr spectrum of which indicated the presence of three phenolic acetates (5 2.19, 2.29, 2.42 ppm; 3H each), one N a l k y l and one N,N , d i a l k y l amide (5 1.74, 1.91 ppm; 3H each), and two methyl esters (see f i g . 22). The above f u n c t i o n a l i t i e s account for a l l eight exchangeable protons. A methine proton resonance at 5 4.73(q, /=8 Hz) ppm showed coupling to a methylene proton resonance at 5 3.l8(m) ppm, and a resonance at 5 6 . l 8 ( d , /=8 Hz) ppm e s t a b l i s h i n g the presence of -CH 2 -CH-NH- unit in 92, which in turn required the presence of - C H 2 - C H - N H 2 in 91. This unit was assigned to the N-methylhist idine moiety in 91. C a t a l y t i c hydrogenation of 91 (Pd on act ivated carbon, 24 h) f a i l e d to cleave the su l f ide l inkage . Reduction of imbricat ine (91) with Ra-Ni (aqueous MeOH/reflux, 2.5h) d i d cleave the sulphide bond to give the te trahydroisoquinol ine 94, and N-methylh is t id ine , which was i d e n t i c a l to 3-methylhist idine and d i f f erent from 1-methylhist idine by t i c comparison. 3 This was supported by gc and gems comparison of N - t r i f l u o r o a c e t y l , n-butyl ester der iva t ives of N - m e t h y l h i s t i d i n e s 7 9 . The EI mass spectrum of 94 d id not show 3 1-methylhist idine, Rf =0.13; 3 -methylh is t id ine , Rf =0.10 in n-BuOH/AcOH/water 5 .5:2:2.5 . 79 a parent ion but the CI mass spectrum (see f i g s . 27 and 28) showed a parent ion at m/z 316 (M++H) which was consistent with the molecular formula C 1 7 H 1 7 N 0 5 (hrms: (M +-OHbenzyl) observed 208.1611, required for C 1 0 H 1 0 N O « : 208.1610). The CI mass spectrum also showed fragment ions at m/z 270 (M*-C0 2 H), 242 ( M + - NH=CHC02H v i a re tro D ie l s A l d e r ) , 208 (M +-OHbenzyl) and 164 (M +-(OHbenzyl+C0 2)) t y p i c a l of tetrahydro - i s o q u i n o l i n e s 8 0 (see f i g s . 27 and 28). 1 H nmr resonances at 5 6.70(d, 1H, J=8 Hz) and 7.04(d, 1H, /=8=Hz) ppm required that the benzyl residue in 94 and also in 91, contain a para hydroxyl substituent (see f i g s . 17, 24). In fac t , the 1 H and 1 3 C nmr data for p-hydroxybenzyl substituent and C-1 are nmr spect rum of 94 at r . t . (270 MHz, DMS0-1 | 1 1 1 1 I 1 1 1 1 I i l 'i i l _ r - r H ' i l l F i g . 2 6 . C nmr spect rum of 94 (75 MHz, DMSO-d,) . j ^ , ^ ^ r , , , I i I I I 1 I 280 300 CIO 340 360 330 400 420 440 4 6 0 480 5 0 0 F i g . 2 7 . CI mass spect rum of 9 4 . CO OJ 84 m/z 162 F i g . 2 8 . I n t e r p r e t a t i o n of CI ms of 94. 85 v i r t u a l l y i d e n t i c a l in both 9 1 and 94 i n d i c a t i n g that th i s region i s least af fected in the Ra-Ni reduction (see tables 4 and 5). 1 H nmr resonances at 5 6.08(d, 1H, 7=2 Hz) and 6.22(d, 1H, 7=2 Hz) ppm in the spectrum of 9 4 were assigned to two meta protons on the aromatic r ing of the te trahydroisoquinol ine moeity, requir ing that the two hydroxyl subst i tuents on th i s r ing a lso be meta disposed. Imbricatine ( 9 1 ) has only a s ingle proton (5 6.42(s) ppm) on the aromatic r ing of the i soquinol ine moeity, the other non-phenolic p o s i t i o n being occupied by the sulphide l inkage . Compound 97 and i t s trans isomer 9 8 were synthesized as model compounds for the Ra-Ni reduction product 9 4 . A biogenet ic- type synthetic procedure invo lv ing the react ion between Dopa methyl ester and epoxide 9 6 was used to prepare the model compounds 8 1 (see scheme 2). The c i s conf igurat ion of the C1 and C3 substituents in 97 was confirmed by the observation of nuclear Overhauser enhancements in the 1 H nmr resonances of H3 (5 3.48(dd, 7=11,5 Hz) ppm), H12 (53.2l (dd, 7=14,4 Hz) ppm) and H12' (5 (dd, 7=14,8 Hz) ppm) when the H1 resonance at 6 4.06(dd, 7=8,4 Hz) ppm was i r r a d i a t e d . Both isomers. 9 7 and 9 8 were converted to t h e i r respective CHC1 3 soluble d e r i v a t i v e s 9 9 and 1 0 0 . N-Acetylated te trahydroisoquinol ines are known to d i sp lay a doubling of the ir 1 H nmr resonances 8 2 . The minor trans isomer 1 0 0 showed the presence of two forms in almost equal amounts as determined by 1 H nmr (see f i g . 33). One form predominates in the case of the major c i s isomer 9 9 , the 86 ioo R.-Ac ^ - ^ O M e r a t i o being 1:8 (see f i g . 30). The coupling constants displayed by the protons on the he terocyc l i c r ing of the te trahydroisoquinol ine moiety of the two d i f f erent forms of 100 were d i f f e r e n t (for example, in 100 the two resonances for H3: 5 4.63(dd, /=3,6 Hz) and 4.86(dd, /=4,8 Hz) , see f i g . 33) i n d i c a t i n g that doubling of the nmr resonances i s due to the existence of two conformers, not to the presence of two acetate rotamers as reported for 1 0 1 8 2 . Comparison of the 1 H nmr spectra of the model compound Scheme 2. Synthesis of the benzyltetrahydroisoquino!ines 97 and 98 88 g . 2 9 . Contour p l o t of HETCOR 2D nmr s p e c t r u m of 99. 1 13 Only the H and C nmr chemical s h i f t regions required to demonstrate the C-H corre lat ions of two -CH-CH ? -systems were included. 180 1 6 0 1 4 0 1 2 0 1 0 0 ~r~n 8 0 6 0 4 0 2 0 P P M F i g . 3 1 . C nmr spect rum of 99 (75 MHz, CDC 1 Q ) F i g . 32 . CI mass spect rum of 99. F i g . 3 3 . H nmr spect rum of 100 (400 MHz, C D C 1 ~ ) . vo A c O ^ ^ ^ C C L M e A c O ^ J ^ N A c 100 OMe ' M i l 1 7 0 165 i 6 0 1 5 5 I 5 0 1 4 5 1 4 0 1 3 5 P P M ISO'' 1 6 0 I 4 0 120 1 0 0 8 0 T 7 6 0 i | i i i 4 0 2 0 P P M F i g . 34. C nmr spect rum of 1 0 0 (75 MHz, C D C l J Fig . 35. CI mass spectrum of 1 0 0 . F i g . 3 6 . *H nmr spect rum of 102 (400 MHz, CDC 1 0 ) . 3 l£> Ul 96 97 103 99 and i t s t r a n s isomer 100 to t h a t of the m e t h y l t e t r a a c e t y l d e r i v a t i v e 102 (see t a b l e 6, f i g s . 30, 33 and 36 ) confirmed the b e n z y l t e t r a h y d r o i s o q u i n o l i n e nature of 94 and i n d i c a t e d that the C1 and C3 s u b s t i t u e n t s were c i s as shown (see f i g s . 38 and 39 f o r i n t e r p r e t a t i o n s of mass s p e c t r a of 99 and 102). The 1H nmr resonance a s s i g n e d to H-3 (5 3.20 , (dd, /=12,4 Hz) ppm) i n 94 showed a 4 Hz c o u p l i n g to H4e and a 12 Hz c o u p l i n g to H4a ( t a b l e 4). T h i s i n d i c a t e d that H3 i s a x i a l and the C3 c a r b o x y l s u b s t i t u e n t i s e q u a t o r i a l i n 94. F u r t h e r support f o r t h i s assignment came from the observed c o u p l i n g c o n s t a n t s f o r H3 i n model compounds 97 (H3: 5 3.48(dd, /=11,5 Hz) ppm) and 103 (H3: 5 4.26(dd, 7=11,7 Hz) ppm) 8 3. 98 H4e and H3a are both strongly deshielded in imbricat ine (91) r e l a t i v e to the ir chemical s h i f t s in the Ra-Ni reduction product 94 (table 4). The di f ference in chemical s h i f t s observed for these two protons in 94 and 91 can best be explained i f the t h i o h i s t i d i n e su l f ide l inkage in imbricat ine (91) i s attached to C5 of the i soquinol ine nucleus, which in turn requires that there be a proton meta to i t at C7 and hydroxyl substituents at C6 and C8. An INAPT experiment 8 *, which showed three bond couplings between H1 (5 4.67 ppm) and both C10(5 137.7 ppm) and C8 (5 155.8 ppm) of imbricat ine (91) , confirmed th i s assignment (see f i g . 40). A second INAPT experiment showed three bond couplings between H7 (5 6.42 ppm) and both C5 (5 110.0 ppm) and C9 (5 112.7 ppm). It a lso showed two bond couplings between H7 and both C6 (6 159.7 ppm) and C8 (5 155.8 ppm). This resu l t confirmed the placement of a proton on C7 which is ortho to both C6 and C8 which bear hydroxyl subst i tuents and i t a lso confirmed the placement of t h i o h i s t i d i n e su l f ide l inkage on C5 (see f i g . 41 ) . T a b l e 6 . 'H nmr da ta f o r 102 and 99 (400 MHz, CDC I s ) . Chemica l s h i f t . 6 ppm P o s i t i o n 102 99 1 5 .01 dd , J=8,6 Hz 4 .73 dd, J=8,6 Hz 3 4 .48 t , J=10 Hz 4 .49 t , J=10 Hz 4 3 .25 m 3 .21 m 5 6 * .93 d, J = 2 Hz 6 . 6 7 + s 7 6 .89* d, J= 2Hz -8 - - 7. . 10 + s 12 3 .32 dd , J=14,8 Hz 3 . 28 dd , J=14,8 Hz 2 .88 dd, J=14,6 Hz 2 .88 dd, J=14,6 Hz 14 7 . 29 d, J=8 HZ 7. . 15 d , J=8 HZ 15 7 .04 d, J = 8 HZ 6 .84 d, J=8 Hz - O C H 3 3. .85 s 3. .86 s, 3.80 s - O C O C H s 2 .21 s 2 . 25 s, 2.28 s 2 . 30 s, 6 H - N C O C H 3 1 . .79 s 1 . .85 s * , + . maybe i n t e r c h a n g e d . 100 AcO m/z 498 OAc \ AcO 0 Me NAc + + AcO OAc m/z 343 -CH2=CO m/z 456 CH. HO CH. m/z 149 m/z 107 -CH=CO m/z 306 -CH=CO m/z 264 -CH=CO" 2 HO C0 2 Me -0 m/z 222 OH (CO Me m/z 162 F i g . 3 9 . I n t e r p r e t a t i o n of CI ms of 102. OAc m/z 348 m]_z 121 J-CH=CO m/z 306 j-CH=CO m/z 162 F i g . 3 9 . I n t e r p r e t a t i o n of CI ms of 99. F ig . 40. INAPT C nmr spectrum of imbricatine (91 ) with se lect i ve i r r a d i a t i o n of HI (above). 13 . C nmr spectrum of imbricatine (91 ) (below). (75 MHz, DMS0-dA) Fig. 41. INAPT 1 3C nmr spectrum of imbricatine (91) with selective irradiation of H7 (above). 1 3C nmr spectrum of imbricatine (91 ) (below). (75 MHz, DMS0-dfi) 1 04 C.DISCUSSION Marine organisms have thus far been a poor source of a l k a l o i d s r e l a t i v e to the ir t e r r e s t r i a l counterparts . U n t i l the late 1960's, i t was commonly bel ieved that marine organisms were devoid of a l k a l o i d s . Recently, however, a number of a l k a l o i d s have been i so la ted from marine p lants and invertebrates . Because of i t s extraordinary s t r u c t u r a l features and also i t s impact on publ ic hea l th , the marine a l k a l o i d , saxi toxin ( 2 8 ) 3 0 , holds a key place among marine natural products. It i s one of the toxins that cause p a r a l y t i c s h e l l f i s h poisoning and i t i s a metabolite of the dinof la'gellate Gonyaulex cat enel I a. Lingbyatoxin A (104), i so la ted by C a r d e l l i n a et al. in 1 97 9 8 5 7 represents the f i r s t indole a l k a l o i d from a marine source. Since then, a few more indole a l k a l o i d s have been i so la ted from marine species . Most of the simple indoles (eg. 105) come from marine algae. Tribromoderivat ive 105 was i so la ted from the red alga Laurencia brongniarti i 8 6 . B is indoles represented by 106 have been found in the blue-green alga Rivularia firma37. Tyr ian purple (107) i s a wel l known metabolite i so la ted from various mollusc s p e c i e s 8 8 . The ant ineoplas t i c metabolite ap lys inops in (108), i so la ted from f ive Thorecta s p e c i e s 8 9 and Verongia spengelIi 9 0 , the cytotoxic compound dendroine (109) i so la t ed from the marine tunicate dendrodoa grossulari a 9 1 , and 110 i so la ted from the sponge Halichondria mel anodoci a 9 2 are three 1 05 examples of simple indole d e r i v a t i v e s . Caulerpin (111), the s tructure of which was recently r e v i s e d 9 3 , has been i so la ted as the orange-red pigment from several Caul erpa species . A ser i e s of l inear peptide a lka lo ids inc luding the celenamides (eg. celenamide A (112)) and clionamide (113) have been i s o l a t e d from the P a c i f i c sponge Cliona eel at a ( G r a n t ) 9 4 , 9 5 . Cypridine l u c i f e r i n (114) and s i m i l a r compounds 1 06 1 07 are responsible for the bioluminescence exhibi ted by marine a n i m a l s 9 6 . The unusual carbazoles (eg. Hye l lazo le (115)) i so la ted from the blue green alga Hyella caespitosa 9 7 are , not s u r p r i s i n g l y , d i f f erent from the carbazoles known from t e r r e s t r i a l sources. Flustramine (116) i s t y p i c a l of the t r i c y c l i c physostigmine a l k a l o i d s that are abundant in the bryozoan Flustra foliacea9*. Phidolopin (117), a purine a l k a l o i d i s o l a t e d from the bryozoan Phi dol ophora paci fi ca, contains the r a r e l y found n i t r o g r o u p 9 9 . It was la ter observed that many Northeast P a c i f i c bryozoans contained metabolites re la ted to phidolopin ( 1 1 7 ) 1 0 0 . Zoanthoxanthin (118) and pseudozoanthoxanthin (119) represent in t ere s t ing metabolites known as zoanthoxanthins that have been i so la ted from the Mediterranean Zoanthidae species , Epizoanthus arenaceus and Parazoanthus axineI I a e 1 0 1 . A novel group of a l k a l o i d s , which includes zoanthamine (120), have been i so la ted from' an unknown Zoanthus species c o l l e c t e d from the coast of I n d i a 1 0 2 . The s tructure of the macrocyclic a l k a l o i d asc idiacylamide (121), i so la ted from an un ident i f i ed tunicate c o l l e c t e d in A u s t r a l i a , was confirmed recently by s y n t h e s i s 1 0 3 . Malingamides (eg. malingamide A ( 1 2 2 ) 1 0 a ) , majusculamides (eg. majusculamide A ( 1 2 3 ) 1 0 5 ) , and pukeleimides (eg. pukeleimide A ( 1 2 4 ) 1 0 s ) are three in t ere s t ing c lasses of compounds i so la ted from the blue-green alga Lingbya majuscula. The unusual t h i o h i s t i d i n e moiety was found in a group of metabol i tes , adenochromines A-C (125-127) i so la ted by Prota 108 et al. from the cephalopod Octopus vulgaris^07 . Later , the d isulphide 93 was discovered from the u n f e r t i l i z e d eggs of the echinoderm Par acent rot us l i v i d u s 7 9 . The above examples are a s e l ec t ive sample of the marine a l k a l o i d s reported to date. It i s not a complete l i s t of a l l the nitrogenous metabolites known from marine organisms. Members of the benzyl te trahydroisoquinol ine a lka lo ids have thus far been i so la ted only from t e r r e s t r i a l p lant s . Two poss ib le pathways have been postulated for the biosynthesis of benzy l te trahydro isoquino l ines . Both involve a condensation react ion of dopamine (see scheme 3 ) 1 0 8 . The involvement of dopamine as a precursor leads to products with the C-6 and C-7 oxygen f u n c t i o n a l i t y that i s found in a l l na tura l ly occurring benzyl te trahydroisoquinol ines (eg. coc laurine (128), laudanosine (129)). The Dopa carboxyl group is los t in the biogenesis of a l l known benzyl te trahydroisoquinol ine a l k a l o i d s . A few te trahydroisoquinol ines containing a carboxyl group at C3 have been i s o l a t e d . Compound 103, i so la ted from Mucuna seeds, i s an example 8 3 . Imbricatine (91) represents the f i r s t example of a benzyl tetrahydroisoquinol ine a l k a l o i d from a marine organism, and i t i s apparently the f i r s t example from a non-plant source. The C3 carboxyl subst i tuent , the C6/C8 hydroxylation pat tern , and the t h i o h i s t i d i n e subst i tuent represent s t r u c t u r a l features not previous ly encountered in th i s family of a l k a l o i d s . Furthermore, the t h i o h i s t i d i n e moiety having the methyl group at N-3 is d i f f e r e n t from the previously 1 09 1 10 111 OH X X sx HO C7 NH 2 2 125 HO C NH 2 2 126 127 X= RN NH R = H or Me C0 2 H i so la t ed marine N-methylhis t id ines . The s t r u c t u r a l s i m i l a r i t i e s between imbricat ine ( 9 1 ) and the adenochromines ( 1 2 5 - 1 2 7 ) i s noteworthy. Although there are d i f ferences in the subs t i tu t ion pat tern , a l l these compounds contain a phenyl r ing with two phenols and a methy l th ioh i s t id ine subst i tuent . Evidence from model experiments indicated that Dopa is a biogenetic precursor for the adenochromines 1 0 9 . S c h e m e 3 . P r o p o s e d r o u t e s f o r t h e b i o s y n t h e s i s o f b e n z y l t e t r a h y d r o i s o -q u i n o l i n e s i n p l a n t s , ( e x c e r p t e d f r o m r e f . 1 0 8 ) 1 13 OH 130 In p r i n c i p l e , a condensation of adenochromines A (125) or B (126) with a su i tab le substrate could lead to te trahydroisoquinol ines having the t r a d i t i o n a l C6, C7 hydroxylat ion pat tern . Imbricatine (91), which contains C6, C8 hydroxylation pat tern , may be b iogene t i ca l ly derived from a h i therto unknown amino ac id 130. In 13.0 both meta disposed OH f u n t i o n a l i t i e s ac t ivate C-2'. Therefore, the OH groups in 130 are better s i tuated to promote a condensation react ion leading to benzyl tetrahydroisoquinol ines than are the hydroxyl groups in Dopa. Is is poss ib le that imbricat ine (91) i s the f i r s t example of a c lass of benzyl - te trahydroisoquinol ines that are b iogene t i ca l ly derived from 1 1 4 the amino ac id 130. Imbricatine (91) induced S\ cocci nea "swimming" behavior at very low concentrations in laboratory tests ." It a lso d i sp lays s i g n i f i c a n t a c t i v i t y in the L1210 (ED <1 jug/ml) and P388 (T/C 139 at 0.5 mg/kg) ant ineoplas t i c a s s a y s 1 1 0 . " The swimming behavior i s very d i f f i c u l t to quant i fy , however, a p p l i c a t i o n of one to two drops of a 1 mg/ml so lut ion of 91 in sea water cons i s tent ly e l i c i t s the response. 1 1 5 V. EXPERIMENTAL The 1 H and 1 3 C nmr spectra were recorded on Bruker WH-400, Nico le t Oxford 270, Bruker HXS-270, Bruker WP-80 and Varian XL-300 spectrometers. Tetramethyls i lane (6=0 ppm) was employed as an in terna l standard for 1 H nmr spectra and CDC13 (5=77.0 ppm) or DMSO-d6 (5=39.5 ppm) were used as both in terna l standards and solvents for 1 3 C nmr unless otherwise ind ica ted . Low-resolution e lectron impact and FAB mass spectra were recorded on an A . E . I . MS-902 spectrometer and h igh-reso lut ion mass spectra were recorded on an A . E . I . MS-50 spectrometer. CI mass spectra were recorded on a Nermag R 10-10 C spectrometer. Infrared spectra were recorded on Perkin-Elmer 710B and 1710 spectrometers. A Bausch and Lomb Spectronic 2000 spectrometer was used to record uv absorption spectra . O p t i c a l rotat ions were measured on a Perkin-Elmer model 141 polarimeter using a 10 cm c e l l and a Fisher-Johns apparatus was used to determine melting points which are uncorrected. Gas chromatography was performed on a Hewlett-Packard 5830A instrument. A flame ion i sa t ion detector was used for the detect ion of peaks. A Perkin-Elmer Series 2 instrument was used for hplc where a Perkin-Elmer LC-55 uv detector and/or a Perkin-Elmer LC-25 r e f r a c t i v e index detector were employed for peak de tec t ion . A Whatman Magnum-9 P a r t i s i l 10 or a Magnum-9 ODS 10 column was used for preparat ive h p l c . The hplc solvents were BDH Omni-solv grade or F isher hplc 1 1 6 grade; water was glass d i s t i l l e d . A l l other chromatography solvents were reagent grade. Merck S i l i c a Gel 60 PF-254 was used for preparat ive t i c , Merck S i l i c a Gel 230-400 mesh was used for f la sh chromatography and Merck S i l i c a Gel 60 PF-254 with CaSO(,.l/2 H 2 0 was used in r a d i a l t i c . 1 1 7 Dictyota binghamiae C o l l e c t i o n Data Dictyota binghamiae was c o l l e c t e d by hand using SCUBA at depths of 3 to 5 m in shallow bays off Dixon Is land and Execution Rock in Barkley Sound, B r i t i s h Columbia. Extract ion and Chromatographic Separation I n i t i a l c o l l e c t i o n s of the alga were d r i e d in a convection oven at 50°C for 24 h . The dr i ed alga was powdered in a Wiley m i l l p r i o r to Soxhlet ex tract ion with hexane. Evaporation of hexane extract in vacuo gave a dark green o i l (0.02%). Later c o l l e c t i o n s of the alga were immersed in methanol immediately a f ter the c o l l e c t i o n . Usual ly within 2-3 days, the methanol was decanted and the residue was homogenised in an commercial blender with fresh methanol. The combined methanol extracts were concentrated in vacuo to give an aqueous suspension which was exhaustively extracted with hexane. Removal of the hexane in vacuo gave a dark green o i l . The c o l l e c t i o n s that were not worked up immediately were stored at low temperature [ 4 - ( - 5 ) ° C ] u n t i l used. The crude hexane extracts were frac t ionated by step gradient f lash chromatography using the fo l lowing solvent sequence: ( i ) hexane ( i i ) hexane/chloroform 1:1 1 18 ( i i i ) c h l o r o f o r m (iv) chloroform/ethyl acetate 9:1 (v) ch loroform/ethyl acetate 1:1. The chloroform eluted components from the f lash chromatography were. further p u r i f i e d by r a d i a l t i c (hexane /e thy l acetate 95:5) to give crude samples of pachydictyol A (29), dictyoxide (35), d ictyoxide A (66) and a pure sample of d i a c e t a l 61a. F i n a l p u r i f i c a t i o n of 29, 35 and 66 was accomplished by reversed phase hplc (29 and 35; w a t e r / a c e t o n i t r i l e 20:80, 66; water/ethanol 20:80). The ch loroform/ethyl acetate eluted components from the f lash chromatography were further p u r i f i e d by r a d i a l t i c (hexane/ethyl acetate 85:15) to give a pure sample of d i a c e t a l (61b) and crude samples of d i c t y o l G acetate (68), d i c t y o t r i o l A diacetate (69), d i c t y o l C (32) and a mixture of a c e t y l d i c t y o l a l (49) and e p i d i c t y o l B acetate (70). Crude 69 and 68 were p u r i f i e d by reversed phase hplc (water/ethanol 20:80) to give pure compounds. F i n a l p u r i f i c a t i o n of 32 was achieved by preparat ive t i c ( a c e t o n i t r i l e / chloroform 20:80). The mixture of 49 and 70 was separated by preparat ive t i c (chloroform/ hexane/methanol 48:50:2) and hplc (hexane/ethyl acetate 85:15) to give pure compounds. Pachydictyol A (29): obtained as a pale yellow o i l (ca. 0.013%); i r ( C H C l 3 ) : 3300, 3050-2850, 1460, 1400 cm" 1 ; 'H nmr(400 MHz, CDC1 3 ) : 6 5.33(br s, 1H),5.12(br t , J=6 Hz, 1H),4.75(s, 1H), 4.74(br s, 1H),3.92(br d, 7=8 Hz, 1H),2.67(q, 7=10 Hz, 1H), 2.62(m, 1H),2.49(m, 1H),2.32(m, 119 1H) ,1 .8Km, 3H),1.69(br s, 3H), 1 .6l(br s, 3H),1.00(d, J=l Hz, 3H) ppm; 1 3 C nmr (100.6MHz, CDC1 3 ) : 5 152.60, 141.48, 131.46, 124.81, 124.02, 107.15, 75.23, 60.58, 47.79, 46.24, 40.54, 35.17, 34.93, 34.01, 25.71, 25.66, 23.61, 17.66, 17.54, 15.80 ppm; ms m/z ( r e l . i n t . ) : 288(M + ,47) , 270(19), 255(5), 203(20), 175(13), 159(56), 109(25), 91(32), 69(73), 41(100), 28(93), Exact mass c a l c d . for C 2 o H 3 2 0 : 288.2454; found (hrms): 288.2453. D i c t y o l C ( 3 2 ) : obtained as a white c r y s t a l l i n e compound (ca. 0.005%). m.p. 57 °C; 1 H nmr (400 MHz, CDC1 3 ) : 5 5.26(br s, 1H),5.12(br t , J=8 Hz, 1H),3.87(dd, /=8,4 Hz, 1H), 2.76(br t , J=Q Hz, 1H),2.21(m, 1H),2.15(m, 1H),2.05(m, 1H), 1.93(m, 1H),1.89(br dd, /=16,6 Hz, 1H),1.82(br s, 3H),1.68 (s, 3H),1.75-1.40(m, 5H),1.60(s, 3H),1.35-1.10(m, 3H),1.20 (s, 3H),0.98(d, J=7 Hz, 3H) ppm; ms m/z ( r e l . i n t . ) : 306(M + ,12), 288(77), 270(14), 255(30), 177(50), 81(53), 69(81), 43(100), 41(80). Exact mass c a l c d . for C 2 0 H 3 a 0 2 : 306.2560; found (hrms): 306.2564. Dictyoxide ( 3 5 ) : obtained as a pale yellow o i l (ca.0.008%). 1 H nmr(400 MHz, CDC1 3 ) : 5 5.50(br s, 1H),5 .09(t , J=Q Hz, 1H),3.97(m, 1H),2.63(br d, /=12 Hz, 1H), 2 .41(dt , /=12,6 Hz, 1H),2.04(m, 2H),2.00-1.85(m, 2H),1.82-1.72(m, 1H), 1.78(br s, 3H),1.68(br s, 3H),1.60(br s, 3H),1.65-1.45(m, 5H), 1.37(m, 1H),1.25(s, 3H),1. lO(m, 1H),0.90(d, / = 7 H z , 3H) ppm; 1 3 C nmr(l00.6 MHz, CDC1 3 ) : 5 141.37, 130.57, 124.87, 123.66, 1 20 77.33, 74.19, 63.12, 61.30, 38.79, 38.38, 37.29, 34.51, 29.82, 26.32, 25.55, 22.23, 20.41, 17.63, 16.44, 15.91 ppm; ms m/z ( r e l . i n t . ) : 288(M + , 18), 270(3), 207(16), 178(17), 159(14), 121(39),- 107(46), 69(50), 55(36), 43(54), 41(64), 28(100). Exact mass c a l c d . for C 2 0 H 3 2 O : 288.2454; found (hrms): 288.2465. A c e t y l d i c t y o l a l (49): obtained as a pale yellow o i l (ca. 0.007%). uv ( C H 3 O H ) : 230.5 nm (e 6000); [ a ] 2 3 - 1 5 4 ° (c 0.16, C H 3 O H ) ; i r (f i lm) : 2930, 2870, 1740, 1680, 1640, 1400, 1380, 1240, 1060 cm" 1 ; 'H nmr (270 MHz, CDC1 3 ) : 6 9.40(d, 7=1 Hz, 1H),6.77(dd, 7=4,8 Hz, 1H), 5.37(br d, 7=11 Hz, 1H),5.09(t , 7=7 Hz, 1H),4.6(m, 2H), 3.12(m, 1H),3.00(br dd, 7=10,.15 Hz, 1H),2.76(br t , 7=8 Hz, 1H),2.27(br d, 7=10 Hz, 1H),2.17(m, 1H),2.00(s, 3H), 2.00-1.60(m, 8H),1.78(s , 3H),1.67(s, 3H),1.57(s , 3H), 0.87(d, J=7 Hz, 3H) ppm; ms m/z ( r e l . i n t . ) : 346(M + ,56), 328(53), 303(15), 301(12), 286(13), 109(30), 82(60), 69(76), 55(49), 43(100), 41(90). Exact mass c a l c d . for C 2 2 H 3 4 0 3 : 346.2509; found (hrms): 346.2517. D iace ta l 61a: obtained as a pale yellow o i l ; 1 H nmr (400 MHz, C D C I 3 ) : 5 5.85(d, 7=8 Hz, 1H),5.45(dd, 7=3,12 Hz, 1H),5.16(s, 1H),5.13(t , 7=6 Hz, 1H),5.08(s, 1H),3.38(s, 3H),3.32(s, 3H) ,3 .07(br dd, 7=12,16 Hz, 1H),2.64(ddd, 7=3,8,16 Hz, 1H), 2.27(br s, 1H),2.21(br d, 7=12 Hz, 1H),2.05-1.85(m, 5H), 1.72(br s, 3H),1.67(br s, 3H),1.60(br s, 3H),1.70-1.55 (m, 2H),1 .22(q, 7=8 Hz, 2H),0.99(d, 7=7 Hz, 3H) ppm; ms m/z ( r e l . 121 i n t . ) : 348(M + ), 3.16(14), 284(18), 201 (18), 159(57), 145(95), 109(90), 69(85), 41(100). Exact mass c a l c d . for C 2 2 H 3 6 O 3 :348.2666; found (hrms): 348.2649. D i a c t a l 61b: i r ( f i l m ) : 3000-2850, 1480, 1460, 1390, 1120, 960 cm" 1 ; 'H nmr (400 MHz, CDC13): 5 5 .9 l (br dd, 7=8,2 Hz, 1H),5.34(br s, 1H), 5.15(s, 1H),5 .14(t , 7=6 Hz, 1H),3.52(s, 3H),3.35(s , 3H), 3.11(br dd, 7=16,12 Hz, 1H),2.74(ddd, 7=16,9,4 Hz, 1H), 2.44(br s, 1H),2.27(m, 1H),1.75(br s, 3H),1.72(br s, 3H), 1.60(s, 3H),0.95(d, 7=7 Hz, 3H) ppm; ms m/z ( r e l . i n t . ) : 348(M + ,33), 316(71), 284(75), 109(48), 97( 100), 69(54), 41 (44). Exact mass c a l c d . for C 2 i H 3 6 0 3 : 348.2660; found (hrms): 348.2669. Dictyoxide A (66 ) : obtained as a pale yellow o i l (ca.0.010%); [a]^ 2 - 5 . 6 ° (c 0.16, C H C I 3 ) ; i r ( f i l m ) : 2980, 2920, 1620, 1520, 1470, 1350, 1240 cm" 1 ; 1 H nmr(400 MHz, CDC13): 5 5.37(br s, 1H),5.20(br d, 7=8 Hz, 1H),4.82(s, 1H), 4.79(s, 1H),4.45(ddd, 7=12,8,4 Hz, 1H),3.74(dd, 7=10,4 Hz, 1H), 2.75(br t , 7=10 Hz, 1H),2.62(q, 7=9 Hz, 1H),2.58-2.18(m, 5H), 1.84(m, 1H),1.79(br s, 3H),1.70(br s, 3H),1.65(br s, 3H), 1.70-1.50(m, 3H),1.14(q, 7=12 Hz, 1H),0.95(d, 7=7 Hz, 3H), ppm; 1 3 C nmr(l00.6 MHz, CDC13): 5 151.95, 142.97, 133.46, 126.84, 124.90, 106.81, 79.49, 68.55, 53.59, 46.64, 45.53, 39.67, 34.34, 34.21, 29.45, 25.78, 25.09, 20.21, 18.34, 17.26 ppm; ms m/z ( r e l . i n t . ) : 286(M + ), 268(23), 177(23), 159(20), 245(20), 120(40), 109(63), 91(40), 82(66), 69(57), 67(54), 1 22 43(49), 41(91), 28(100). Exact mass c a l c d . for C 2 0 H 3 0 0 : 286.2298; found (hrms): 286.2298. D i c t y o l G acetate (68): obtained as a co lour less o i l (ca.0.015%); [a]^ 2 + 5 0 . 0 ° (c 0.13, CHC1 3 ) ; i r ( f i lm) : 3500(OH), 3100-2850, 1725(00) , 1460, 1386, 1250 cm" 1 ; 1 H nmr(400 MHz, CDC1 3 ) : 5 5.57(dt, 7=4,8 Hz, 1H),5.33(br s, 1H) ,5 . l2 (br s, 7=8 Hz, 1H), 4.75(s, 1H),4.73(s, 1H),3.93(br d, 7=8 Hz, 1H),2.70-1.45(m, 12H) ,2 .02(s , 3H),1.82(d, 7=1 Hz, 3H),1 .74(d, 7=1.5 Hz, 3H), 1.72(d, 7=1.3 Hz, 3H),1.27(m, 1H),1.04(d, 7=7 Hz, 3H) ppm; 1 3 C nmr (100.6 MHz, CDC1 3 ) : 5 170.48, 152.38, 141,36, 136.33, 124.51, 124.13, 107.21, 74.93, 70.58, 60.47, 48.03, 46.21, 40.45, 40.37, 33.99, 32.11, 25.62, 23.84, 21.25, 18.25, 18.12, 15.82 ppm; ms m/z ( r e l . i n t . ) : 346(M +), 286(30), 268(20), 186(26), 159(21), 145(16), 133(17), 120(35), 109(50), 91(21), 82(100), 69(21), 55(23), 43(40). Exact mass c a l c d . for C 2 2 H 3 „ 0 3 : : 346.2509; found (hrms): 346.2487. D i c t y o t r i o l A diacetate (69): obtained as a co lour less o i l (ca.0.031%). [a] 2 , 2 + 1 2 . 5 ° (c 0.11, CHC1 3 ) ; i r ( f i lm) : 3450(OH), 3050-2850, 1730-1700(OO), 1450, 1380, 1250 1030, 1000, 980 cm" 1 ; 1 H nmr(400 MHz, CDC1 3 ) : 5 5.59(dd, 7=2,6 Hz, 1H) r 5.34(br s, 1H ) ,5 .10(S , 1 H ) , 5 . l O ( t , 7=7 Hz, 1H), 5..03(s, 1H),4.92(m, 1H),3.87(dd, 7=8,3 Hz, 1H),2.95(br s, 1H), 2.79(q, 7=10 Hz, 1H),2.49(m, IH),2.40-2.15(m, 4H),2.07(s , 3H), 2.06(s, 3H),2.02(dd, 7=10,3 Hz, 1H),1.92(ddd, 7=15,10,2 123 Hz, 1H), 1.86(m, 1H),1.82(m, 3H),1.76(dd, J=15,6 Hz, 1H),1.70(br s, 3H), 1.64(br s, 3H),0.94(d, J=7 Hz, 3H) ppm; 1 3 C nmr (100.6 MHz, CDC1 3 ) : 5 172.26(s), 170.13(s), 149.95(s), 142.10(S), 134.67(s), 123.84(d), 120.04(d), 113.56(t), 77.00(d), 76.48(d), 74.09(d), 59.85(d), 42.81(d), 37.69(d), 37.32(d), 33.98(t) , 28 .40(t ) , 27.94(t) , 26.08(q), 2 l . 85 (q ) , 21.53(q), I8.26(q), 16.03(q), 12.62(q) ppm; ms m/z ( r e l . i n t . ) : 404(M +), 344(10), 284(23), 266(8), 157(35), 109(33), 69(42), 43(100). Exact mass c a c l d . for C 2 4 H 3 6 0 6 : 404.2564; found (hrms) 404.2570. E p i d i c t y o l B acetate (70): obtained as a co lour less o i l (ca. 0.009%). [ a ] J 2 + 4 3 . 6 ° (c 0.3, CHC1 3 ) ; i r ( f i lm) : 3500(OH), 2950, 2870, 1740, 1460, 1380, 1280-1250 cm" 1 ; 1 H nmr (400 MHz, CDC1 3 ) : 5 5.59(dd, 7=6,2 Hz, 1H),5.34(br s, 1H),5.lO(m, 1H),5 . lO(br s, 1H),5.04(br s, 1H),3.92(dd, 7=8,4 Hz, 1H),2.87(q, 7=10 Hz, 1H), 2.49(m, 1H),2.25(m, 2H),2.09(m, 1H),2.03(s, 3H),1.97(m, 2H), 1.90-1.60(m, 2H) ,1 .8 l (dd , 7=1.5 Hz, 3H),1.69(br s, 3H), 1 .6l(br s, 3H),1.52(m, 2H),1.20(m, 1H),0.93(d, 7=7 Hz, 3H) ppm; 1 3 C nmr (100.6 MHz, CDC1 3 ) : 5 169.83, 149,86, 141.16, 131.61, 124.63, 124.00, 113.02, 76.83, 74.78, 60.91, 42.72, 39.48, 35.04, 34.27, 33.70, 28.04, 25.64, 25.57, 21.44, 17.70, 17.33, 15.61 ppm; ms m/z ( r e l . i n t . ) : 346(M +), 286(26), 268(13), 186(18), 109(36), 82(100), 69(68), 43(89), 41(93). Exact mass c a l c d . for C 2 2 H 3 ( l 0 3 : 346.2509; found (hrms): 346.2502. 1 24 Ac id Hydrolys is of d i c t y o l G acetate ( 6 8 ) D i c t y o l G acetate ( 6 8 ) (5 mg) was d i sso lved in aqueous ace t i c ac id (30%, 1 ml) containing a few drops of CH 3CN and the react ion mixture was s t i r r e d at room temperature for 24 h. It was then freeze-dr ied and the products were separated by preparative t i c (hexane/ e thy l acetate 85:15) to give d ic tyoxide A ( 6 6 ) (0.9 mg, R^ 0.56) and i t s epimer ( 6 7 ) . Compound 67 -: 1 H nmr (400 M H z , C D C l 3 ) : 5 1.16(d, J=7 Hz, 1H),1.68(s, 3H ) , 1 . 7 1 (S , 3H),3.71 (dd, / = 9 , 4 H z , 1H),4.22(ddd, /=12,8,4 Hz, 1H),4.69(br s, 2H), 5.14(br d, /=8 Hz, 1H),5.35(br s, 1H) ppm. L i / E t N H 2 reduction of d i c t y o l G acetate ( 6 8 ) D i c t y o l G acetate ( 6 8 ) (20 mg) was d i s so lved in ethylamine (1.5 ml, anhydrous). Freshly cut L i was added in excess and the react ion mixture was s t i r r e d for 20 min. The solvent was then evaporated i n vacuo and the products , inc luding 7.0 mg of unreacted 6 8 , were separated by t i c (hexane/ethyl acetate 95:5). The product, pachydictyol A (29) (2.9 mg) was i d e n t i f i e d by 1 H nmr and "tic. 1 25 Agelas sp. C o l l e c t i o n Data The sponge was c o l l e c t e d by hand using SCUBA at 10-15 m depths from reefs along the west coast near Colombo, S r i Lanka. The animals were soaked in methanol immediately after the c o l l e c t i o n and stored at low temperature [ 4 - ( - 5 ) ° C ] u n t i l used. Extrac t ion and Chromatographic Separation Methanol was decanted from the c o l l e c t i o n s and the residue was blended -in a commercial blender with fresh methanol. The homogenate was f i l t e r e d and the residue was washed with methanol u n t i l the f i l t r a t e was c o l o u r l e s s . The combined methanol extracts were concentrated in vacuo to obtain an aqueous suspension which could be fract ionated by e i ther of the fol lowing methods: (1) The aqueous suspension (from 30 g of sponge, dry weight, a f ter extract ion) was extracted with hexane, chloroform, and e thyl acetate separately . The three extracts were combined to give 1.2 g of a brown o i l a f ter removal of the solvent . The remaining aqueous suspension was f reeze -dr ied to obtain 4.6 g of pale yellow s o l i d . 126 The organic extract was chromatographed on Sephadex LH-20 using methanol/chloroform 1:1 as the e luant . B i o l o g i c a l l y act ive f rac t ions from th i s i n i t i a l separation were rechromatographed on the same column using f i r s t methanol/chloroform 4:6 and then methanol/dichloromethane 9:1 as the eluants to give 38 mg of the pure desbromooroidin (79). The methanol soluble f rac t ion of the f reeze -dr i ed mater ia l was- p u r i f i e d on Sephadex LH-20 with methanol/ dichloromethane 9:1 to y i e l d an a d d i t i o n a l 178 mg of 79. (2) The aqueous suspension (from 50 g of sponge, dry weight, a f ter extract ion) was sequent ia l ly extracted with hexane and ethyl acetate . A brown mater ia l which p r e c i p i t a t e d during the extract ion was separated from the aqueous suspension by c a r e f u l l y withdrawing the supernatant. The methanol soluble port ion of the brown p r e c i p i t a t e (2.8 g) was chromatographed on Sephadex LH-20 e lu t ing with methanol to obtain crude 79. This was rechromatographed on the same column using methanol/chloroform 1:1 as the eluant to give 600 mg of pure 79. Desbromooroidin (79): gave co lour les s c r y s t a l s of the hydrochlor ide , mp 2 5 0 - 2 6 0 ° C dec; uv (CH 3OH): 269.1 nm (e 18000); i r ( f i lm) : 3600-2800(br), 1680, 1620, 1570, 1520,. 1330 cm" 1 ; 'H nmr (300 MHz, DMSO-d s ): 5 3.97(br t , /= 5 Hz, 127 2H),6.17(dt , /=5,16 Hz, 1H),6.23(d, /= 16 Hz, 1H), 6.86(br s, 1H),6.90(dd, 7=2.5,1.5 Hz, IH) f 6 .95(dd, /= 2 .8 ,1 .5 Hz, 1H),7.43(s, 2H) ,8 .48(t , / = 5 H z , 1H),11.85(br s, 1H),12.00(br, 1H), 12.55(br, 1H) ppm; 1 3 C nmr (100.6 MHz, DMS0-d 6 ): 5 159.58(s), 147.60(s), 127.08(d), 126.74(S), 124.93(S), 121.26(d), 116.25(d), 111.91(d), 110.83(d), 95.10(S), 39.95 ppm; ms (FAB) m/z: 310 (M ++1), 312 (M++3). 1 28 Dermasterias imbricata C o l l e c t i o n Data Dermasterias imbricata was c o l l e c t e d by hand using SCUBA (-10 to -20 m) in Barkley Sound and Copper Cove in B r i t i s h Columbia. The animals were immersed in methanol immediately after c o l l e c t i o n . Extrac t ion and Chromatographic Separation The methanol was decanted from the s t a r f i s h a f t er 2-3 days and the animals (wet weight 8 kg) were re-immersed in fresh methanol to obtain a second ex trac t . The decanted methanol extracts were vacuum f i l t e r e d through C e l i t e . The f i l t r a t e was concentrated in vacuo r e s u l t i n g in a brown aqueous suspension which was d i l u t e d with water and passed through short columns of XAD-4 which had been e q u i l i b r a t e d with water. The f i r s t l i q u i d coming through these columns was c o l l e c t e d and reappl ied to a fresh column of XAD-4. A l l the XAD-4 columns were washed with water u n t i l the eluant was co lour less and then with hot methanol. The golden yellow band eluted with methanol was c o l l e c t e d and evaporated to dryness in vacuo. The r e s u l t i n g brown s o l i d was d i s so lved in water and chromatographed on a Biogel P2 column using 1% AcOH in water as the e luant . The frac t ions from t h i s column were monitored by uv spectroscopy (^ m a v 283 and 292 nm) and t i c 129 (R f 0.29 in n-BuOH/AcOH/water 5.5:2:2.5) for the presence of imbricat ine (91). The fract ions containing crude imbricat ine (91) were combined and freeze-dr ied to obtain a pale yellow s o l i d which was chromatographed on Sephadex LH-20 using methanol/ water 8:2 as the eluant to give pure imbricat ine (91) as a white s o l i d (ca. 0.004%). Imbricatine (91): i r (KBr d i s c ) : 3700-2500(br), 1630, 1595, 1515, 1447, 1397, 1244, 1175, 1080, 840 c m ' 1 ; uv (X): 228(e 10100), 248(sh, e 5200), 283U 2300), 292(e 2100) nm; 1 H nmr (400 MHz, DMSO-d 6 ): 5 7.73(s, 1H),7.04(d, /=8 Hz, 2 H ) , 6 . 7 l ( d , 7=8 Hz, 2H), 6.42(s, 1H),4.67(dd, 7=4.5,8.4 Hz, 1H),4.03(dd, 7=5,16 Hz, 1H),3.76(dd, /=12.5,5 Hz, 1H), 3.66(s,' 3H), 3.60(dd, ' / =9,5 Hz, 1H), 3.30(dd, 7=15,9 Hz, 1H), 3.22(dd, 7=13.9,4.5 Hz, 1H), 3.02(dd, 7=15,5 Hz, 1H), 2.85(dd, 7=13.9,8.4 Hz, 1H) ,2 .7 l (dd, 7=12.5,16 Hz, 1H) ppm; , 3 C nmr (75 MHz, DMSO-d 6 ): 5 170.5(s), 169.7(s), 159.7(s), 156.4(s), 155.8(s) , 137.8(d), 137.7(s), 131.1(S) , 130.5(d), 126.8(s), 126.5(s), 115.3(d), 112.7(s), 110.00(S), 104.00(d), 54.9(d), 52.9(d) , 52.3(d) , 37 .3( t ) , 32.3(q), 27 .2 ( t ) , 2 5 . l ( t ) ppm; ms (FAB) m/z: 515 [ C 2 « H 2 6 N « 0 7 S + + H ] . Preparation of d e r i v a t i v e (92) Imbricatine (91) (9 mg) was d isso lved in methanol (saturated with HC1) and refluxed for 2.5 h. The solvent was then evaporated to dryness in vacuo and into that , acet ic 130 anhydride (3 ml) and pyr id ine (3 ml) were added and the mixture was s t i r r e d at room temperature for 20 h. The crude react ion mixture obtained af ter removal of the solvents in vacuo was separated by reverse phase hplc (CH 3CN/water 3:7) to obtain 9 mg of the major product, compound 92. Compound 92: 'H nmr (400 MHz, CDC1 3 ) : 5 7.34( br s, 1H), 7.29(d, 7=8 Hz, 2H), 7.02(d, /=8 Hz, 2H), 6.89(br s, 1H), 6.15(br d, 7=8 Hz, 1H), 5.06(br t , 7=6 Hz, 1H), 4.73(br dd, 7=8,16 Hz, 1H), 4.64(br dd, 7=6,16 Hz, 1H), 4.40(br dd, 7=6,12 Hz, 1H), 3.87(s, 3H), 3.75(s, 3H) , 3 .61(S , 3H), 3.36 Odd, 7=6,14 Hz, 1H), 3.17(m, 1H), 3 . l 0 (br d, 7=16 Hz, 1H), 2.89(dd, 7=6.14 Hz, 1H), 2.41(S , 3H) , 2.28(s, 3H), 2.18(S , 3H), 1.90(s, 3H), 1.74(s, 3H) ppm; ms m/z ( r e l . i n t . ) : 603(4), 561(30), 543(25), 519(24), 501(18), 477(15), 459(10), 394(50), 352(60), 310(100), 268(100), 264(20), 222(55), 208(50), 193(30), 162(20), 127(47), 107(50); ms (FAB, CI) m/z: 753 [ C 3 6 H 4 o N o O , 2 S + + H ] ; hrms: (M +-OAcbenzyl) observed 603.1756, exact mass c a l c d . for C 2 7 H 3 1 N a 0 1 0 S 603.1761. Analys i s of N-methylhist idines by Gas Chromatography 1-Methylhist idine (1 mg) was dr i ed i n vacuo at 100°C for 0.5 h, n-BuOH (1.5 ml, saturated with HC1) added and s t i r r e d at 100°C for 15 min. The solvent was then evaporated to dryness at 100°C under a stream of n i trogen . T r i f l u o r o a c e t i c anhydride (0.1 ml) and dichloromethane (0.3 ml) were added into th i s and s t i r r e d at 125°C for 5 min. The der ivat ive 131 obtained a f ter removal of the solvents in vacuo was d isso lved in dichloromethane and used for the gc a n a l y s i s . 3 -Methy lh i s t id ine and the N-methylhist idine obtained from the Ra-Ni reduction of 91 were treated s i m i l a r l y . A column packed with SP 2250 coated on 100-120 mesh Supelcoport was used for gc. I n i t i a l temperature 125°C; 5 min. ho ld; rate 5 ° C / m i n . ; f i n a l temperature 2 2 5 ° C . N - t r i f l u o r o a c e t y l , n-butyl ester of 1-methylhist idine: R f c 26.27 m i n . ; gems m/z ( r e l . i n t . ) : 321(M + , 10), 220(88), 95(100). N - t r i f l u o r o a c e t y l , n-butyl ester of 3 -methylhis t id ine: R f c 25.29 m i n . ; gems m/z ( r e l . i n t . ) : 321(2), 220(5), 208(16), 95(100). N - t r i f l u o r o a c e t y l , n-butyl ester of N-methylhis idine from the Ra-Ni reduction of 91: R t = 25.29 min . ; gems m/z ( r e l . i n t . ) : 220(4), 208(10), 95(100). Ra-Ni D e s u l f u r i z a t i o n of imbricatine (91) Imbricatine (91) (16 mg) was d i s so lved in methanol (15 ml) and water (2 ml) . Ra-Ni suspension (0.5 ml) was added and the react ion mixture was refluxed for 2.5 h under n i trogen . The react ion mixture was then vacuum f i l t e r e d and hot methanol was used to repeatedly wash the res idue. The f i l t r a t e , a f ter removal of methanol in vacuo, was chromatographed on Sephadex LH-20 using methanol/water 9:1 as the eluant to give crude compound 94 which was further 132 p u r i f i e d by hplc (methanol/water 20:80) to obtain 8 mg of pure 94. Compound 94 : 'H nmr (400 MHz, DMSO-d 6 ): 5 7.04(d, 7=8 Hz, 2H), 6.71(d, J=8 Hz, IH), 6;27(d, 7=2 Hz, 1H), 6.13(d, J=2 Hz, 1H),4.57(dd, 7=4,8 Hz, 1H), 3.20(dd, /=4,12 Hz, 1H) , 3.12(dd, 7=4,14 Hz, IH), 2.90(dd, 7=16,4 Hz, 1H), 2.80(dd, 7=14,8 Hz, 1H) ,2 .7 l (dd, 7=12,16 Hz, 1H) ppm; 1 3 C nmr (100.6 MHz, DMSG~d 6): 5 169.2(s), 157.0(s), 156.0(s), 154.7(s), 136.0(s), 130.2(d), 126.9(s) , 115.0(d), 110.8(s), 106.0(d), 100.9(d), 55.5(d), 53.0(d) , 37 .8 ( t ) , 30.0(t) ppm; ms (CI) m/z ( r e l . i n t . ) : 316 (M++H, 1), 272(0.8), 242(1), 270(2), 208(6), 198(1), 164(6), 162(4), 152(10), 137(6), 124(13), 121(10), 109(35), 108(35), 107(21); hrms: (M +-OHbenzyl) observed 208.1611, exact mass c a l c d . for C 1 0 H 1 0 N O a : 208.1610. Preparation of compound 102 Compound 94 (3 mg) was ref luxed with MeOH (saturated with HC1) for 2.5 h. The methanol was evaporated to dryness in vacuo. Acet ic anhydride (1 ml) and pyr id ine (1 ml) were added to the residue and the mixture was s t i r r e d at r . t . for 20 h. The crude product obtained af ter removal of solvents was p u r i f i e d by hplc (EtOAc/hexane 3:2) to give 2 mg of pure 1 0 2 . Compound 1 0 2 : obtained as a white s o l i d ; 1 H nmr (400 MHz, CDC1 3 ) : 5 7.29 (d, 7=8 Hz, 2H), 7.04(d, 7=8 Hz, 2H), 6.95(d, 7=2 Hz, 1H), 6.89(d, 7=2 Hz, 1H), 5.06(dd, 7= 8,7 Hz, 1H), 4.48(dd, 7=11,9 Hz, 1H), 3.85(s, 3H) , 3.32(dd, 7=14,8 1 33 Hz, 1H), 3.25(m, 2H), 2.88(dd, /=14,7 Hz, 1H), 2.30(s, 3H), 2 .21(S , 3H), 1.79(s, 3H) ppm; ms (CI) m/z ( r e l . i n t . ) : 498(M ++H,14), 456(15), 348(56), 306(52), 264(46), 222(52), 162(19), 107(100); hrms: (M +-OAcbenzyl) observed 348.1084, exact mass c a l c d . for C 1 7 H 1 8 N 0 7 : 348.1083. Preparat ion of methyl-3-(4-methoxyphenyl)qlycidate 95 Anisaldehyde (5 ml) and methylchloroacetate (5 ml) were added slowly over a period of 2.5 h into a co ld so lut ion [ 0 - ( - 5 ) ° C ] of Na (1.5 g) in MeOH (25 ml) . The react ion mixture was s i r r e d for another 2.5 h on an ice/water bath and then for 3 h at r . t . It was then poured onto ice-water, f i l t e r e d and the f i l t r a t e was washed several times with i c e - c o l d water to give 7 g of 95 as a pale yellow powder. Compound 95: 1 H nmr (270 MHz, DMSO-d 6 ): 5 7.29(d, /=8 Hz, 2H), 6.93(d, /=8 Hz, 2H), 4 . l0 (br s, 1H), 3.79(br s, 1H), 3.78(s, 3H), 3.75(s, 3H) ppm; ms m/z ( r e l . i n t . ) : 208(M +, 10), 192(10), 161(18), 151(54), 135(12), 121(100), 105(9), 91(33), 77(36). Synthesis of compounds 97 and 98 DL-Dopa methyl ester hydrochloride (580 mg, prepared from DL-Dopa, MeOH/HCl, re f lux , 2.5 h) in 10 ml of MeOH was added to a so lut ion of 610 mg of sodium (4-methoxyphenyl)glycidate (96) (prepared by the hydrolys i s 134 of 95 with MeONa) in 15 ml of water. The pH of the so lut ion was adjusted to 4 with AcOH. The react ion mixture was then s t i r r e d at 35°C for 36 h. At the end of th i s p e r i o d , methanol was removed in vacuo to obtain an aqueous suspension which was p a r t i t i o n e d between EtOAc and 10% HC1. The aqueous layer was next neutra l i zed with K 2 C 0 3 and extracted with EtOAc. This EtOAc extract was washed with water, d r i e d over anhydrous Na 2 SO a and concentrated /n vacuo to give a mixture of compounds 97 and 98 which was separated by f lash chromatography (EtOAc/CHCl 3 3:2) on s i l i c a to obtain 145 mg of 97 and 45 mg of 98. Compound 97: obtained as a white s o l i d . 1 H nmr (270 MHz, DMSO-dg): 8 7 . l 7 ( d , 7=8' Hz, 2H), 6.84(d, 7=8 Hz, 2H), 6.70(s, 1H), 6.43(s, 1H), 4.00(dd, /=8,4 Hz, 1H), 3.70(s, 3H), 3.58(s, 3H), 3.48(dd, 7=11,5 Hz, 1H), 3 .2 l (dd , 7=14,4 Hz, 1H), 2.71(dd, 7=16,5 Hz, 1H), 2.56(dd, 7=14,8 Hz, 1H) ppm; ms (CI) m/z ( r e l . i n t . ) : 344(M++H, 16), 284(1), 222(100), 162(11), 134(1), 121(13); hrms: observed 343.1403, exact mass c a l c d . for C 1 7 H 2 1 N 0 5 : 343.1420. Compound 98: obtained as a pale yellow o i l . 1 H nmr (400 MHz, DMSO-d 6 ): 8 7.18(d, 7=8 Hz, 2H), 6.88(d, 7=8 Hz, 2H), 4.00(dd, 7=10,4 Hz, IH), 3.87(dd, 7=9,5 Hz, 1H), 3.74(s, 3H), 3.63(s, 3H), 2.86(dd, 7=14,4 Hz, 1H), 2.79(dd, 7=16,5 Hz, 1H), 2.74(dd, 7=10,4 Hz, 1H), 2.64(dd, 7=16,9 Hz, 1H) ppm. 1 35 Preparation of 99 Acet ic anhydride (2 ml) and pyr id ine (2 ml) were added to 25 mg of 97 and s t i r r e d at r . t . for 20 h. Pure 99 (38 mg) was obtained after removal of solvent from the react ion mixture. Compound 99: obtained as a white s o l i d . 1 H nmr (400 MHz, C D C l 3 ) : 5 7 . l 5 ( d , 7=8 Hz, 2H), 7.10(S , IH), 6.84(d, 7=8 Hz, 2H), 6.67(s, 1H), 4.73(dd, 7=8,7 Hz, 1H), 4.49(t , 7=10 Hz, 1H), 3.84(s, 3H), 3.79(s, 3H), 3.27(dd, 7=14,8 Hz, 1H), 3.2l(m, 2H), 2.88(dd, 7=14,7 Hz, 1H), 2.28(s, 3H), 2.25(s, 3H), 1.85(s, 3H) ppm; 1 3 C nmr (75 MHz, CDC1 3 ) : 5 172.7(s), 170.6(s), 168.1(S) , 168.0(s), 158.5(s), 141.2(s), 140.4(s), 135.5(s), 130.6(d), 129.3(s), 122.7(d), 121.7(d), 114.0(d), 61.2(d) , 55.4(d), 55.1(q), 52.3(q), 42 .5( t ) , 29 .7 ( t ) , 21.2(q) , 20.4(q), 20.5(g) ppm; ms (CI) m/z ( r e l . i n t . ) : 470(M++H, 42), 428(42), 348(100), 306(55), 264(64), 222(19), 162(24), 121(36). hrms: (M +-OMebenzyl) 348.1093, exact mass c a l c d . for C 1 7 H 1 8 N 0 7 : 348.1083. Preparation of 100 Compound 100 was prepared from 20 mg of 98 by the method described for the preparation of 99 from 97. The crude product obtained was p u r i f i e d by hplc (EtOAc/hexane 3:2) to obtain 20 mg of pure 100 as an o i l . Compound 100: 1 H nmr (400 MHz, CDC1 3 ) : 8 6.98(s, . 9H), 1 36 6.89(s, 1H), 6.87(d, J=8 Hz, 1.8H), 6.79(d, /=8 Hz, 1.8H), 6.70(s, 4H), 6.69(s, .9H), 6.53(s, 1H), 5.36(t, / = 5 H z , 1H), 4.92(t , J=l Hz, .9H), 4.86(dd, /=8,4 Hz, .9H), 4.63(dd, /=6,3 Hz, 1H), 3.78(s, 2.7H), 3.75, 3H) , 3 .58(s, 2.7H), 3.51(S, 3H), 3.3(m, 2H), 3.10-2.85(m, 4.4H), 2.43(dd, /=16,6 Hz, 1H), 2.26(s, 2.7H), 2 .25(s, 3H) , 2 .24(s, 2.7H), 2.23(s, 3H), 2 .13(s, 3H), 2.06(s, 2.7H) ppm; ms (Cl) m/z ( r e l . i n t ) : 470(M++H, 26), 428(17), 348(100), 306(39), 264(41), 222(13), 162(18), 134(1), 121(23); hrms: (M+-OMebenzyl) observed 348.1090, exact mass c a l c d . for C 1 7 H 1 8 N 0 7 348.1083. APPENDICES 2 9 Appendix 1 . 1 H nmr spect rum of p a c h y d i c t y o l A (29) (409 MHz, C D C 1 - ) . w o oo 29 s ^ ^ ^ ^ 160 120 80 40 PPm Appendix 2. C nmr spectrum of pachydictyol A (29) ( 100.6 MHz , CDCl - ) . w J VO Appendix 3. Mass spect rum of p a c h y d i c t y o l A (29). O Appendix 4 . H nmr spect rum of d i c t y o l C (32) (400 MHz, C D C 1 0 ) . 142 Appendix 5. Mass spectrum of d i c t y o l C (32 ) . w (If 160 r" T -120 80 40 PPm 13 Appendix 7. C nmr spect rum of d i c t y o x i d e (35) ( 1 0 0 . 6 MHz, C D C l j ) . Appendix 3 . Mass spec t rum of d i c t y o x i d e ( 3 5 ) . Appendix 9 . l H nmr spect rum of a c e t y l d i c t y o l a l (49) (400 MHz, C D C 1 3 ) . 147 Append ix 10. Mass s p e c t r u m of a c e t y l d i c t y o l a l (49). OMe Appendix 12. Mass spect rum of a c e t a l 6 1 a . vo IBB Appendix 14. Mass spectrum of a c e t a l (61b) B r n •N' H 0 I I 79 H H s R - r - p 100 Appendix 15 . SFORD C nmr spect rum of d e s b r o m o o r o i d i n ( 7 9 ) (75 MHz, DMSO-d.) i x 16. C nmr spect rum of i m b r i c a t i n e (91) { l H ga ted d e c o u p l e d , 75 MHz, D M S 0 - d 6 ) . -^NMe NH., «1 CHj carbons ca rbons CH carbons * i » in w »0m.0*»i »Li« i l f i r i 1 v n - r r p r - n T | T T r i IT 180 160 140 T"l—r-1—|—t 7—l"i 1 ~\ 1 t-T-J 1-1 - r r - r - i - t - r 80 T"i—r r_ i — r •1 - t-i j T i - i — r | " r n T | T - r T T t--r-T—t—r~j 60 <I0 PPM 20 Appendix 17. ADEPT C nmr s p e c t r a of i m b r i c a t i n e ( 9 1 ) (75 MHz, DMS0-d f i ) Appendix 19. ADEPT 1 3 C nmr spectra of 94 (75 MHz, DMS0-d c ) . w b cr. Appendix 2 0 . APT C nmr spect rum of 99. ( 7 5 MHz, C D C l - ) 1 58 V I I . REFERENCES Uemura, D . ; Takahashi, K . ; Yamamoto, T . ; Tanaka, T . ; Katayama, C ; Okumura, Y . ; Hi r a t a , Y. / . Amer. Chem. Soc., 1985, 107, 4796. See, for a review, Benn, R . ; Gunther, H. Angew. Chem. Int. Ed. Engl., 1983, 22, 350. H a l l , L . D . ; Sanders, K. M. / . Org. Chem., 1981, 46, 1 1 32. Gunther, K . ; Martens, J . ; Schickedanz, M. Angew. Chem. 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