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Novel secondary metabolites from selected British Columbian marine invertebrates Ayer, Stephen William 1985

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NOVEL SECONDARY METABOLITES FROM SELECTED BRITISH COLUMBIAN MARINE INVERTEBRATES  by STEPHEN WILLIAM AYER  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in THE FACULTY OF GRADUATE STUDIES Department of Chemistry  We accept t h i s t h e s i s as conforming d standard  THE UNIVERSITY OF BRITISH COLUMBIA March 1985  ©  Stephen W i l l i a m A y e r , 1985  In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t of quirements  for  an advanced degree at the The  B r i t i s h Columbia, I agree that the freely  available  Library  f o r r e f e r e n c e and  University  shall  study. of  this  scholarly  by  the  may  be  Department or by h i s or her stood  that  copying  f i n a n c i a l gain  shall  or not  granted  representatives.  publication be  allowed  permission.  Department of Chemistry The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date: March  1985  of  make  reof it  I f u r t h e r agree  that permission f o r extensive copying purposes  the  thesis Head  It  this  without  is  of  my  under-  thesis my  for  for  written  Abstract Marine organisms show p o t e n t i a l as sources b i o l o g i c a l l y and p h a r m a c o l o g i c a l l y metabolites.  for novel,  a c t i v e , secondary  Examination of three nudibranch and one  bryozoan s p e c i e s f o r b i o l o g i c a l l y a c t i v e m e t a b o l i t e s to  has l e d  the i s o l a t i o n and s t r u c t u r a l e l u c i d a t i o n of nine new and  two known secondary m e t a b o l i t e s .  The s t r u c t u r e s of a l l the  compounds were determined by u s i n g a combination of s p e c t r a l a n a l y s i s , chemical i n t e r c o n v e r s i o n , s y n t h e s i s , and s i n g l e - c r y s t a l X-ray d i f f r a c t i o n The  analysis.  B r i t i s h Columbian d o r i d nudibranch Acant hodori  nanaimoensi  s y i e l d e d three new s e s q u i t e r p e n o i d s .  t u r e s of nanaimoal (6_1) , acanthodoral isoacanthodoral skeletons.  (6J5) represent  novel  The s t r u c -  (64) , and s e s q u i t e r p e n o i d carbon  The n a t u r a l mixture of aldehydes 6 1 , 64, and 65  e x h i b i t e d a n t i b a c t e r i a l and a n t i f u n g a l a c t i v i t y . Aldisa  cooperi,  g l y c e r o l ether  From  two A * - 3 - k e t o s t e r o i d a l a c i d s 23 and 24, and 25 were i s o l a t e d .  deterrent a c t i v i t y against Meli be leonina  s  fish.  A c i d 23 showed feeding The dendronotid  nudibranch  gave 2 , 6 - d i m e t h y 1 - 5 - h e p t e n a l 53 and  2 , 6 - d i m e t h y l - 5 - h e p t e n o i c a c i d 5_4_.  The aldehyde 5_3 was  r e s p o n s i b l e f o r the " g r a p e f r u i t l i k e " odour of the nudibranch. The  bryozoan Phidolopora  pacifica  was examined i n an  attempt to c o r r e l a t e the absence of s u r f a c e f o u l i n g , i n the f i e l d , with the presence of b i o l o g i c a l l y a c t i v e secondary metabolites.  The purine a l k a l o i d s 179 and 1 8 0 , which  ii  contain  the  rare  were r e s p o n s i b l e activity 189,  of  and 209  the  naturally  occurring  f o r much of crude  the  extracts.  were a l s o  isolated  Nitrophenol  181  chloroplast  development  unicellular  a l g a e Eugl ena  sp.  functionality,  a n t i f u n g a l and  antialgal  Three n i t r o p h e n o l s from P.  had been p r e v i o u s l y both  nitro  shown t o  i n green  plants  181,  pacifica. inhibit and i n  the  Table of Contents Abstract  ii  List  of F i g u r e s  vi  List  of Schemes  vii  L i s t of Tables List  viii  of Appendices  ix  Acknowledgements  x  Dedication  xi  Abbreviations  xii  I . Introduction  1  A. Overview  1  B. N a t u r a l Products C. Primary and  Chemistry  3  Secondary M e t a b o l i t e s  8  D. Chemical Ecology  11  I I . Nudibranchs  16  A. I n t r o d u c t i o n  16  1. Gastropod Secondary M e t a b o l i t e s  16  2. Nudibranch Defense Mechanisms  21  B. Secondary M e t a b o l i t e s from the D o r i d Nudibranch Aldisa cooperi (Robilliard Baba, 1972)  25 1  1. I n t r o d u c t i o n 2. I s o l a t i o n and  and  Structure Elucidation  3. B i o l o g i c a l A c t i v i t i e s Metabolites  of Aldisa  cooperi  4. D i s c u s s i o n  25 42 43  C. Secondary M e t a b o l i t e s from the Dendronotid Nudibranch Meli be leonina (Gould, 1852) 1. I n t r o d u c t i o n 2. I s o l a t i o n and  25  47 47  Structure Elucidation iv  47  3. D i s c u s s i o n  53 from Acant  D. Secondary M e t a b o l i t e s nanaimoensis  hodoris  (O'Donoghue, 1921)  56  1 . Introduction  56  2. Nanaimoal 3. Synthesis of Nanaimoal's (p-Bromophenyl)urethane D e r i v a t i v e . Assignment of S t r u c t u r e  59 70  1  4. Assignment of the H NMR Spectrum of Nanaimoal using One and Two-Dimensional Techniques 5. Isoacanthodoral  81 99  6. Acanthodoral  112  7. B i o l o g i c a l A c t i v i t i e s Secondary M e t a b o l i t e s  of A.  nanaimoensis  8. D i s c u s s i o n  117 117  I I I . Bryozoans  132  A. I n t r o d u c t i o n to the Bryozoans B. Secondary M e t a b o l i t e s pacifica  NMR  (Robertson  from  1908)  1 . Discussion  132 Phidolopora 149 163  IV. Experimental  171  V. Appendices  200  VI. Bibliography  204  v  L i s t of F i g u r e s  1.  Phylogenetic c l a s s i f i c a t i o n  2.  T y p i c a l cryptobranch d o r i d nudibranch  20  3.  Aldisa  26  4.  Secondary m e t a b o l i t e s from the d o r i d nudibranch  cooper  of nudibranchs  17  cooperi  Aldisa  i  27  5.  "Un-natural" 20S marine s t e r o i d s  37  6.  Mel i be leonina  48  7.  Secondary m e t a b o l i t e s from the d e n d r o n o t i d nudibranch  Meli  be Ieoni  8.  Acanthodori  9.  Secondary m e t a b o l i t e s from Acanthodoris  10.  na  49 s  nanaimoensis  57  nanaimoensis  60  GC a n a l y s i s of a crude c h l o r o f o r m e x t r a c t of A.  nanaimoensis  11. 400 MHz  61 1  H NMR  (61)  spectrum of nanaimoal  (CDC1 3 ) . . . 63  1  12. Model systems f o r the H NMR chemical s h i f t s of the gem-dimethyl group i n nanaimoal (6_1) 13.  Nanaimoane carbon s k e l e t o n showing the numbering  14.  Pulse sequence f o r the homonuclear  COSY NMR  70  scheme 80  experiment 89  1  15. 400 MHz H NMR COSY/45 spectrum of nanaimoal's (p-bromophenyl)urethane d e r i v a t i v e  91  16. Expansion and a m p l i f i c a t i o n of F i g u r e 15 to show homoallylic couplings  92  17.  Pulse sequence f o r 2D / - r e s o l v e d NMR  experiment  18. P a r t i a l 400 MHz *H NMR 2D / - r e s o l v e d spectrum (symmetrized) of nanaimoal (61)  . . . . 93 97  19. S l i c e s of i n d i v i d u a l peaks shown at the top of F i g u r e 18 to show m u l t i p l i c i t i e s 98 20. Computer generated X-ray s t r u c t u r e of i s o a c a n t h o d o r a l ' s 2,4-dinitrophenylhydrazone d e r i v a t i v e 96 110 vi  21. Computer generated X-ray s t r u c t u r e of acanthodoral's (p-bromophenyl )urethane d e r i v a t i v e 114 116 22.  Phidolopora  pacifica  150  23. A computer-generated p e r s p e c t i v e drawing of the f i n a l X-ray model of p-bromophenacylphidolopin 194 160  vi i  L i s t of Schemes  1.  (23)  I n t e r p r e t a t i o n of the HRMS of 3-oxo-4-cholenoic a c i d 30  2. I n t e r p r e t a t i o n of the HRMS of 3-oxo-4,22-choladienic a c i d (24) 3.  33  I n t e r p r e t a t i o n of the MS of d i a c e t y l d e r i v a t i v e 44 . . 41  4. Proposed mechanism f o r the m i c r o b i a l c h o l e s t e r o l into 17-ketosteroids  t r a n s f o r m a t i o n of 44  5. I n t e r p r e t a t i o n of the mass s p e c t r a l fragmentation of 2,6-dimethyl-5-heptenal (53) 6.  I n t e r p r e t a t i o n of the MS of nanaimoal (61)  7. Biogenetic nanaimoal  62  arguments used i n support of s t r u c t u r e 61 for 67  8. Retrosynthetic nanaimoal 9.  52  a n a l y s i s of the p o s t u l a t e d  Previous s y n t h e s i s  structure  for 72  of the nanaimoane carbon skeleton  10.  I n t e r p r e t a t i o n of the MS of urethane  11.  I n t e r p r e t a t i o n of the MS of i s o a c a n t h o d o r a l  73 79  (65) . .  101  12. B i o g e n e t i c arguments l e a d i n g t o the c o n s i d e r a t i o n of 100 as the s t r u c t u r e f o r i s o a c a n t h o d o r a l 103 13.  Acid catalyzed  rearrangement of 98  14. Proposed b i o g e n e s i s of i s o a c a n t h o d o r a l acanthodoral (64) .". 15.  107 (65) from  109  189  I n t e r p r e t a t i o n of the MS fragmentation of n i t r o p h e n o l 154  16.  I n t e r p r e t a t i o n of the MS of d e s m e t h y l p h i d o l o p i n  viii  ....  L i s t of Tables  1. Comparison of the 'H NMR data f o r v a r i o u s A " - 3 - k e t o s t e r o i d s (CDC1 3 )  29  13  2. C NMR data and s p e c t r a l comparisons f o r the assignment of s t e r e o c h e m i s t r y to 3-oxo-4-cholenoic a c i d (23) 36 3. 'H NMR  data f o r v a r i o u s 20R and 20S s t e r o i d s  (CDC1 3 ) . 39  13  4. C NMR data f o r 2,6-dimethyl-5-heptenal (53) and c i t r o n e l l a l (55) 1  5. H NMR data  (400 MHz) f o r nanaimoal  51  (61) and d e r i v a t i v e s 83  6.  Nudibranch s e s q u i t e r p e n o i d s  119  7.  Bryozoan m e t a b o l i t e s  134  8. 'H NMR data (CDC1 3 , 80 MHz) and s p e c t r a l comparisons f o r n i t r o p h e n o l s i s o l a t e d from Phidolopora paci fi ca 153 9. 'H NMR data and s p e c t r a l comparisons f o r p u r i n e d e r i v a t i v e s i s o l a t e d from Phidolopora pacifica  ix  158  L i s t of Appendices  1. 400 MHz  NMR  spectrum of 75  201  2. 400 MHz  NMR  spectrum of 98  202  3. 400 MHz  NMR  spectrum of 114  203  x  Acknowledgements  I would l i k e to g r a t e f u l l y acknowledge the encouragement, p a t i e n c e , and  f r i e n d s h i p of Dr.  guidance, R.J.  Andersen. Many people have i n f l u e n c e d the outcome of t h i s work and a number of i n d i v i d u a l s stand o u t .  My wife Roxanne pro-  v i d e d u n r e l e n t i n g l o v e , s u p p o r t , and encouragement f o r which I am deeply me  indebted.  Mike LeBlanc competently  to SCUBA d i v i n g , a s s i s t e d with a l l the  c o l l e c t i o n s , and performed the b i o a s s a y s .  invertebrate Dr. 0 . Chan and  Ms. M a r i e t t a T. A u s t r i a p r o v i d e d f r i e n d l y and a s s i s t a n c e with the 2D-NMR s t u d i e s . me  introduced  helpful  David Behrens allowed  to reproduce h i s drawing of a t y p i c a l cryptobranch d o r i d  nudibranch and Ron  Long k i n d l y p r o v i d e d photographs of a l l  the i n v e r t e b r a t e s s t u d i e d .  I thank Sandra M i l l e n f o r  h e l p f u l d i s c u s s i o n s on nudibranch b i o l o g y . A number of people a s s i s t e d with the c o l l e c t i o n of the marine organisms, and the s p e c t r a l data of the compounds i s o l a t e d from them.  I thank the s t a f f of the Bamfield  Marine S t a t i o n and the departmental f o r courteous and  reliable  NMR  assistance.  xi  and MS  laboratories  To J e a n , Roxanne, G e n e v i e v e , Dorothy, B i l l , and A l l e n  Abbreviat ions  AQN  = Acquisition  DMSO  = Dimethylsulfoxide  EtOAc  = Ethyl acetate  g  = Grease peak  GC  = Gas chromatography  GC-MS  = gas chromatography  HPLC  = High performance l i q u i d  HRMS  = High r e s o l u t i o n mass spectrum  IR  = Infrared  MS  = Low r e s o l u t i o n mass spectrum  'H NMR  = Proton nuclear magnetic  13  - mass spectrometry chromatography  resonance  C NMR = Carbon-13 nuclear magnetic  resonance  nOe  = Nuclear Overhauser enhancement  mp  = Melting point  RD  = R e l a x a t i o n delay  RT  = Room temperature  S  = Solvent s i g n a l  SFORD  = S i n g l e frequency o f f resonance decoupled  SCUBA  = S e l f - c o n t a i n e d underwater  TLC  = Thin l a y e r  U  = Unknown  UV  = Ultraviolet  W  = Water s i g n a l  chromatography  impurity  signal  xi i i  b r e a t h i n g apparatus  I.  INTRODUCTION  A. OVERVIEW The  purpose of the r e s e a r c h undertaken and reported i n  t h i s t h e s i s was to i s o l a t e and e l u c i d a t e the s t r u c t u r e s of secondary m e t a b o l i t e s * e x h i b i t i n g  interesting  biological  a c t i v i t i e s "from s e l e c t e d B r i t i s h Columbian marine invertebrates. It was a n t i c i p a t e d at the outset that some of these compounds would be important organisms.  t o the ecology of the source  Many examples e x i s t where e c o l o g i c a l l y  important  secondary m e t a b o l i t e s turn out t o be p h a r m a c o l o g i c a l l y active  (or vice  1  v e r s a ) , t h e r e f o r e the i s o l a t i o n s were  d i r e c t e d by in vitro  s c r e e n i n g f o r a n t i f u n g a l and  antibacterial activities.  Because not a l l p o t e n t i a l l y  i n t e r e s t i n g compounds would be a c t i v e a g a i n s t the l i m i t e d number of microbes screened, t h i n l a y e r chromatography (TLC) and nuclear magnetic resonance (NMR) were used t o broadly c h a r a c t e r i z e every p u r i f i e d The  fraction.  s t r u c t u r e s of the i s o l a t e d m e t a b o l i t e s were deter-  mined by one or more of the f o l l o w i n g methods: 1.  I n t e r p r e t a t i o n of s p e c t r a l  data.  2.  Comparison with known compounds.  3.  Chemical i n t e r c o n v e r s i o n s .  * For the d e f i n i t i o n of a secondary m e t a b o l i t e , see S e c t i o n C of t h i s Chapter. N a t u r a l products chemistry and the chemistry of secondary m e t a b o l i t e s are g e n e r a l l y regarded as synonymous. 1  2  4.  Unambiguous s y n t h e s i s .  5. - S i n g l e c r y s t a l X-ray d i f f r a c t i o n  Three s p e c i e s of nudibranchs  analysis.  were i n v e s t i g a t e d f o r  b i o l o g i c a l l y a c t i v e secondary m e t a b o l i t e s .  Nudibranchs lack  the p h y s i c a l p r o t e c t i o n of an e x t e r n a l s h e l l , but are seldom eaten.  They deter predators u s i n g a v a r i e t y of mechanisms,-  as w i l l be d i s c u s s e d in a subsequent c h a p t e r , and one  of  these d e f e n s i v e mechanisms seems to i n v o l v e the employment of noxious secondary m e t a b o l i t e s .  A number of secondary  m e t a b o l i t e s were i s o l a t e d from the three nudibranch s t u d i e d ; three represent b i o g e n e t i c a l l y  related  s e s q u i t e r p e n o i d aldehydes each with a new (from Acanthodoris  nanaimoensis),  carbon s k e l e t o n  two are s t e r o i d a l  of which one d i s p l a y e d f i s h a n t i f e e d a n t a c t i v i t y Aldisa  cooperi),  and  species  acids,  (from  two are degraded monoterpenes  s t r u c t u r a l l y s i m i l a r to known i n s e c t pheromones (from Mel i be I eoni na) .  One  s p e c i e s of bryozoan  studied.  The  (Phidolopora  unfouled nature of P.  pacifica)  pacifica  was  i n the n a t u r a l  environment l e d to s p e c u l a t i o n that i t employs an a n t i f o u l i n g agent. rather  P.  pacifica  also  effective  turned out to be a source of  i n t e r e s t i n g purine a l k a l o i d s c o n t a i n i n g the  relatively rare, naturally occurring, nitro Initial  screens  i n d i c a t e d that some of the  m e t a b o l i t e s e x h i b i t profound ample, one  group. isolated  biological activities.  of the p u r i n e a l k a l o i d s was  For  ex-  very a c t i v e a g a i n s t  3 the pennate diatom Cyl i ndrot heca mixture  of s e s q u i t e r p e n o i d aldehydes  effectively subtil  is.  fusiformi  s .while the  from A.  nanaimoensis Bacillus  i n h i b i t e d the growth of the b a c t e r i a C o l l a b o r a t i o n with pharmacologists and  to study the f u l l  biologists  spectrum of b i o l o g i c a l a c t i v i t i e s ,  both  p h a r m a c o l o g i c a l and e c o l o g i c a l , that are e x h i b i t e d by  the  m e t a b o l i t e s d e s c r i b e d i n t h i s t h e s i s would appear i n o r d e r .  B. NATURAL PRODUCTS CHEMISTRY N a t u r a l products chemistry  (the chemistry of  secondary  m e t a b o l i t e s ) has undergone e x p l o s i v e growth d u r i n g the quarter century. products  In the  r e s e a r c h was  1960's n a t u r a l  c h a r a c t e r i z e d by i n v e s t i g a t i o n s of the  s t r u c t u r e s of molecules natural sources.  1950's and e a r l y  available  The primary  c l a s s i c a l degradation and  in large quantities  from  s t r u c t u r a l t o o l s were  transformation r e a c t i o n s .  I n t r o d u c t i o n of s p e c t r o s c o p i c techniques such as NMR mass spectroscopy  last  and  (MS), enabled the Chemist to propose  s t r u c t u r e s on the b a s i s of s p e c t r o s c o p i c evidence a l o n e , a l though these were u s u a l l y supported by a few transformations.  key  chemical  C o n f i r m a t i o n of s t r u c t u r e came from  s y n t h e s i s , c o n v e r s i o n to a known compound, or by the e v o l v i n g s i n g l e c r y s t a l X-ray d i f f r a c t i o n With the advent of h i g h performance  rapidly  analysis. liquid  chromatography (HPLC), r o u t i n e F o u r i e r transform NMR,  and  r a p i d computer a i d e d X-ray a n a l y s i s , the Chemist has been able to s o l v e chemical problems p r e v i o u s l y thought  4  impossible. elucidation  These i n c l u d e ,  f o r example, the s t r u c t u r a l  of pheromones a v a i l a b l e  2  i n microgram amounts ,  i s o l a t i o n and s t r u c t u r a l d e t e r m i n a t i o n of and e l u c i d a t i o n brevetoxin  of s t r u c t u r e s , 5  3  phytohormones ,  such as p a l y t o x i n  ( 2 ) , that are so complex  (1)*  and  i t i s even d i f f i c u l t to  draw them.  The days of the N a t u r a l Products Chemist are f a r from numbered.  We are at the dawn of a new  era.  Investigation  5 (by c o l l a b o r a t i o n with b i o l o g i s t s and pharmacologists) of b i o l o g i c a l l y a c t i v e i s o l a t e s has p r o v i d e d , and w i l l to p r o v i d e , i n t e r e s t i n g c h e m i s t r y . small water s o l u b l e molecules and  continue  A n a l y s i s of r e l a t i v e l y  w i l l become more p r e v a l e n t ,  t h i s should enable the i s o l a t i o n and s t r u c t u r e  e l u c i d a t i o n of p h y s i o l o g i c a l l y  i n t e r e s t i n g compounds, par-  6  t i c u l a r l y o l i g o p e p t i d e s . F i n a l l y , i n v e s t i g a t i o n of the enzymology and b i o s y n t h e s i s of secondary  m e t a b o l i t e s should  provide v a l u a b l e i n s i g h t s i n t o why organisms produce  such  7  compounds .  Marine n a t u r a l products r e s e a r c h has g r e a t l y i n t e n s i f i e d over the l a s t two decades. of books e d i t e d by Scheuer  8  An e x c e l l e n t  series  p r o v i d e t i m e l y , exhaustive  reviews of important a s p e c t s of marine n a t u r a l products research.  9  Other a u t h o r i t a t i v e reviews by B a k e r , 10  Christophersen , C o l l  1 1  , Faulkner  12  , Fenical  1 3  , Moore  10  ,  6  and S c h e u e r  15  p r o v i d e a d d i t i o n a l h i g h l i g h t s of chemical  r e s e a r c h on the organisms  found beneath the s e a .  The pragmatic a p p l i c a t i o n of n a t u r a l p r o d u c t s i s o l a t e d from t e r r e s t r i a l sources i s w e l l known, p a r t i c u l a r l y areas of human and v e t e r i n a r y m e d i c i n e . environment  i n the  The marine  has a l s o y i e l d e d a number of compounds (or  s y n t h e t i c analogs) that have found, or show p o t e n t i a l f o r , practical a p p l i c a t i o n  '  1 7  .  For example; the i s o l a t i o n of  (3_) from the marine worm Lumbr i coner ei s  nereistoxin heteropoda  1 6  8  l e d to the development of the s t r u c t u r a l l y  y  r e l a t e d i n s e c t i c i d e Padan (4) which Japan  19  .  i s i n common use i n  The n u c l e o s i d e 1-j3-D-arabinofuranosylcytosine  (Ara-C) ( 5 )  2 0  , an e f f e c t i v e a n t i c a n c e r drug that has been i n  use f o r over a decade i n the treatment of leukemia, was developed as a r e s u l t of s e m i s y n t h e t i c m a n i p u l a t i o n of spongouridine  (Ara-U)  (6) i s o l a t e d from the sponge  t  Cryptotethia  crypta ^' . 2  22  A f a m i l y of n a t u r a l p r o d u c t s , the  didemnins, i s o l a t e d by Rinehart et al. from the Caribbean t u n i c a t e s Trididemnun potential. didemnin  species , 23  show great b i o m e d i c a l  Didemnin B (7) appears t o be the most potent  i n b l o c k i n g the growth of L-1210 mouse leukemia  c e l l s as w e l l as i n h i b i t i n g the Herpes  simplex  Didemnin B i s c u r r e n t l y undergoing c l i n i c a l antileukemia  virus. t r i a l s as an  2  drug ".  R e s u l t s such as the above c l e a r l y demonstrate study of marine  how the  secondary m e t a b o l i t e s may l e a d t o some  v a l u a b l e pragmatic s p i n o f f s .  Current r e s e a r c h e f f o r t s are  7  C H . 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 —Pro—«-Me,Tyr — O - i  U 7  |  1  now being d i r e c t e d towards i s o l a t i n g specific  2  biological a c t i v i t y * '  2 5  m e t a b o l i t e s with a  , greatly  i n c r e a s i n g the  p r o b a b i l i t y that a compound with a d e s i r e d pharmacological e f f e c t w i l l be found. Although  some r e s e a r c h e r s and one drug company*  * Recently the Roche Research I n s t i t u t e f o r Marine Pharmacology a t Dee Why, A u s t r a l i a , e s t a b l i s h e d i n 1974, ceased o p e r a t i o n .  _  8 i n v o l v e d i n marine n a t u r a l products  r e s e a r c h have become  somewhat d i s i l l u s i o n e d by a p a u c i t y of p r a c t i c a l (compared to o v e r l y o p t i m i s t i c  initial  results  e x p e c t a t i o n s ) , the  development of a new drug r e q u i r e s a l a r g e investment of 26  c a p i t a l and years of d e v e l o p m e n t . likely own  Given time, i t seems  t h a t the marine environment w i l l e v e n t u a l l y y i e l d i t s  "penicillin".  " For the c y n i c q u e s t i o n i n g the biomedical value of the s e a , i t i s important t o p o i n t out that only a handful of l a b o r a t o r i e s and perhaps a score or two of chemists and pharmacologists have been i n v e s t i g a t i n g the marine r e s o u r c e . T h i s compares extremely meagerly with hundreds and perhaps thousands of s i m i l a r i n v e s t i g a t o r s who have i n the course of at l e a s t a century i n v e s t i g a t e d t e r r e s t r i a l resources f o r d r u g s . What i s r e q u i r e d i s that more i n v e s t i g a t o r s , e s p e c i a l l y from the drug i n d u s t r y , take a d i p i n the 1oceans t o seek the 7 p e a r l s awaiting d i s c o v e r y . "  C. PRIMARY AND SECONDARY METABOLITES H i s t o r i c a l l y the organic compounds found i n l i v i n g organisms were d i v i d e d i n t o two c a t e g o r i e s .  Primary  m e t a b o l i t e s were d e f i n e d as compounds made by an organism p r i m a r l y t o s u s t a i n l i f e . ' The compounds, and the b i o s y n t h e t i c pathways used t o generate them, were designated as common to many, i f not a l l , l i v i n g organisms.  Secondary  m e t a b o l i t e s , on the other hand, were deemed not e s s e n t i a l t o the b a s i c protoplasmic thus i n i t i a l l y age)  metabolism of the organism and were  c o n s i d e r e d t o be endproducts (waste or s t o r -  of metabolism.  9 It i s l i k e l y secondary  that the above h i s t o r i c a l d e f i n i t i o n of a  metabolite w i l l  fall  into disuse.  p a r e n t , f o r example, that fungal secondary i n v o l v e d i n the p h y s i o l o g y of the producing Perhaps two  and of the Mucorales  (8)  m e t a b o l i t e s are organism.  n o t i o n that secondary  (9))  and o o g o n i o l  (the t r i s p o r i c a c i d s 1J) and  11)  2  8  2 7  .  m e t a b o l i t e s are endproducts of  metabolism has turned out to be a gross The  ap-  of the best known examples are the fungal sex  hormones of the Achl ya ( a n t h e r i d i o l  The  I t i s now  oversimplification.  f i e l d of e c o l o g i c a l b i o c h e m i s t r y (chemical ecology)  grown r a p i d l y over the l a s t 15 y e a r s , demonstrating  the  has  10 important e c o l o g i c a l r o l e s secondary m e t a b o l i t e s have i n nature*  (see the f o l l o w i n g s e c t i o n ) .  These f i n d i n g s have  prompted a r e - e v a l u a t i o n of the terms primary and secondary metabolites.  Campbell  29  has put forward  a more u s e f u l d e f i -  n i t i o n based on the extent of taxonomic d i s t r i b u t i o n of a p a r t i c u l a r compound.  It states that:  Plant, fungal, bacterial  (and animal) c e l l s are  composed of two d i s t i n g u i s h a b l e types of molecules: a) m a t e r i a l s that are widely d i s t r i b u t e d i n n a t u r e , being  found at l e a s t  i n a l l f a m i l i e s i n an o r d e r , o f t e n  i n a l l orders of a c l a s s or i n a l l c l a s s e s i n a phylum, and  i n some i n s t a n c e s , i n a l l phyla  all  f i v e kingdoms; and  i n a kingdom and i n  b) m a t e r i a l s t h a t occur uniquely  in a single  strain  or s p e c i e s , that are found i n two or more c l o s e l y r e l a t e d members of a s i n g l e genus, or that are found sporadically  i n a l i m i t e d number of e v o l u t i o n a r i l y  u n r e l a t e d s p e c i e s i n d i f f e r e n t genera, f a m i l i e s , o r d e r s , • c l a s s e s , phyla or kingdoms. C e l l c o n s t i t u e n t s of type  (a) are primary  m e t a b o l i t e s , while c e l l c o n s t i t u e n t s of type secondary m e t a b o l i t e s . [N.B. though s p e c i e s - s p e c i f i c  (b) are  Enzymes and n u c l e i c a c i d s ,  i n d i s t r i b u t i o n , are normally  excused from the primary/secondary metabolism  * D e s p i t e these advances, many secondary m e t a b o l i t e s s t i l l have no known f u n c t i o n . T h i s may be due t o a lack of s u i t able experimentation or because the f u n c t i o n i s too complex to d i s c e r n given the c u r r e n t s t a t e of knowledge.  11 discussion]. T h i s t h e s i s d i s c u s s e s secondary m e t a b o l i t e s  isolated  from s e l e c t e d B r i t i s h Columbian marine organisms.  Although  the f u n c t i o n of the m e t a b o l i t e s cannot be c o n c l u s i v e l y est a b l i s h e d , c i r c u m s t a n t i a l evidence  suggests  the  isolated  m e t a b o l i t e s are i n v o l v e d i n i n t e r s p e c i e s communication.  It  seems l i k e l y that the compounds i s o l a t e d from the nudibranchs  are i n v o l v e d i n t h e i r  relative  immunity from  p r e d a t i o n , whereas the bryozoan m e t a b o l i t e s may surface f o u l i n g and  impede  overgrowth.  D. CHEMICAL ECOLOGY Reseacch over the l a s t f i f t e e n years has velopment of a new ecology  30  .  l e d to the  de-  i n t e r d i s c i p l i n a r y s u b j e c t ; chemical  S t u d i e s i n chemical ecology have f o r the  time provided a r a t i o n a l and  first  s a t i s f y i n g e x p l a n a t i o n for the  r o l e s of at l e a s t a part of the enormous p r o l i f e r a t i o n of secondary m e t a b o l i t e s observed another  in nature.  T h i s has  l e d to  f u n c t i o n a l yet somewhat i n a c c u r a t e (see previous  s e c t i o n ) d e s c r i p t i o n of secondary m e t a b o l i t e s :  If the  organism i s c o n s i d e r e d by i t s e l f , without r e f e r e n c e to other organisms, there i s no evident reason why  the organism  should produce them (secondary m e t a b o l i t e s ) There are two  Alleochemic  .  types of e c o l o g i c a l i n t e r a c t i o n s mediated  by secondary m e t a b o l i t e s ; a l l e o c h e m i c ( i n t e r s p e c i f i c ) and  31  effects  i n t r a s p e c i f i c chemical  effects  e f f e c t s are s u b d i v i d e d i n t o allomones  3 2  .  1 2  ( a d a p t a t i v e advantage to the producer)  and  kairomones  ( a d a p t a t i v e advantage to the r e c e i v e r ) while  intraspecific  chemical e f f e c t s are s u b d i v i d e d i n t o a u t o t o x i n s , a u t o i n h i b i t o r s , and pheromones.  Allomones and pheromones  are by f a r the most s t u d i e d chemical communicants to d a t e . In mephitis,  some c a s e s , as with the common skunk the chemical message may  ( t h i o l s 12, 13, and d i s u l f i d e  be a bouquet of compounds  14), a mixture  pheromone (alarm) and an allomone (defense) The  Mephitis  t h a t a c t s as a simultaneously.  spray from the skunks' anal scent glands sends p o t e n t i a l  p r e d a t o r s f l e e i n g while at the same time warning other skunks of the p o t e n t i a l danger nearby.  In other cases a  s i n g l e substance, f o r example c a n t h a r i d i n meloid b e e t l e Lytta  vesicatoria,  (15_) from the  can act as a f e e d i n g  d e t e r r e n t to p o t e n t i a l p r e d a t o r s .  The  l i t e r a t u r e has been  summarized by Harborne i n h i s e x c e l l e n t book " I n t r o d u c t i o n to E c o l o g i c a l B i o c h e m i s t r y "  2  .  CHCH=CHCHSH 3  2  Me 0  12  0  (CH)CHCHCHSH 3 2  2  2  Me 0  13  CHCH=CHCHSSCH 3  2  14  3  15  13 Chemical ecology its  infancy.  i n the marine environment i s s t i l l i n  A recent chapter by B a r b i e r i n t r o d u c e d ,  summarized, and analyzed t h i s c u r r e n t s t a t e of a f f a i r s Many compounds have been shown t o be b i o l o g i c a l l y a g a i n s t s e l e c t e d s c r e e n i n g organisms or c e l l little  3 3  .  active  l i n e s , but  i s known about the f u n c t i o n s of the m e t a b o l i t e s i n  t h e i r n a t u r a l environment.  T h i s i s p r i m a r l y due to the  d i f f i c u l t y of experimenting  and o b s e r v i n g i n the marine  ecosystem. Perhaps the best documented study i n marine chemical ecology  i n v o l v e s a d o r i d nudibranch.  S c h u l t e and Scheuer  demonstrated that the nudibranch, Phyllidia accumulates from a s p e c i f i c  sponge upon which i t feeds  (Hymeni aci don sp.) a substance f i s h and c r u s t a c e a n s  34  .  The  varicosa,  that i s a p p a r e n t l y l e t h a l to substance,  9-isocyanopupukeanane (16.) , was i s o l a t e d from both organisms.  I n t e r e s t i n g l y , a related metabolite  present only i n the sponge.  is  Apparently the nudibranch  must  have some means of c o n c e n t r a t i n g s p e c i f i c m e t a b o l i t e s from its diet.  Subsequent t o t h i s d i s c o v e r y , a number of other  m e t a b o l i t e s i s o l a t e d from d o r i d s k i n s e c r e t i o n s have been shown to i n h i b i t feeding by f i s h  (using a standard  bioassay)  at c o n c e n t r a t i o n s of 10 ug m e t a b o l i t e per mg of food pellet  3 5  '  3 6  .  The demonstrated a n t i f e e d a n t a c t i v i t y would  appear t o be e c o l o g i c a l l y 37  significant*.  * Recently G e r h a r t demonstrated that p r o s t a g l a n d i n A 2 i s an agent of chemical defense i n the Caribbean gorgonian Plexaura  homomalla.  1 4  The l i t e r a t u r e on nudibranch chemistry has been 1  extensively r e v i e w e d '  36  '  38  , and r e c e n t l y F a u l k n e r  39  has  proposed that i n the e v o l u t i o n of d o r i d nudibranchs, l o s s of the s h e l l i s c o r r e l a t e d with the presence of d e f e n s i v e mechanisms based upon chemicals d e r i v e d from f o o d . t h i s theory i s a p p e a l i n g , the l i m i t e d e c o l o g i c a l which have been performed  Although  studies  t o date a r e i n s u f f i c i e n t to prove  that a p a r t i c u l a r m e t a b o l i t e i s s o l e l y r e s p o n s i b l e f o r an "observed" l a c k of p r e d a t i o n . A second example of marine chemical e c o l o g y , that i n v o l v e s a much l e s s q u a n t a t i v e b i o a s s a y , concerns the Red Sea  sponge Latrunculia  magnifica,  sponges that grow exposed. d i v e r s in situ  one of the few Red Sea  Squeezing of the sponge by SCUBA  causes c u r i o u s f i s h to immediately  squeezing L. magnifica  i n t o an aquarium c o n t a i n i n g  causes p o i s o n i n g and death of the f i s h w i t h i n  flee; fish 0  minutes' .  The p u r i f i e d t o x i n s were t h e r e f o r e assumed to p l a y a  15 1  d e f e n s i v e r o l e i n the sponge" .  I t i s i n t e r e s t i n g to note  that the p u r i f i e d t o x i n s , L a t r u n c u l i n s A (18) and B ( 1 9 ) , e x h i b i t e f f e c t s on c u l t u r e d mouse neuroblastoma and fibroblast c e l l s  4 2  .  In both c e l l  t y p e s , submicromolar t o x i n  c o n c e n t r a t i o n s (as low as 50 ng/mL) r a p i d l y induce changes in c e l l morphology toxin.  that are r e v e r s i b l e upon removal of the  The s i g n i f i c a n c e of these o b s e r v a t i o n s are unknown.  The above examples  illustrate  that compounds i s o l a t e d  from marine organisms show profound b i o l o g i c a l  activities,  which i n many i n s t a n c e s are advantageous to the producing organism.  The promising pharmacological a c t i v i t i e s shown by  some of the m e t a b o l i t e s i s o l a t e d from marine organisms on the  b a s i s of a presumed e c o l o g i c a l r o l e augurs w e l l 'for the  acceptance of marine chemical ecology as a v i a b l e f i e l d of research.  II.  NUDIBRANCHS  "Many nudibranchs, but e s p e c i a l l y the d o r i d s , have a p e n e t r a t i n g f r u i t y odour that i s p l e a s a n t when m i l d but nauseating when c o n c e n t r a t e d . Undoubtedly, t h i s odour i s one of the reasons why nudibranchs seem3 to be l e t s t r i c t l y alone by predatory animals  A. INTRODUCTION  1. GASTROPOD SECONDARY METABOLITES Nudibranchs are members of the phylum M o l l u s c a  (see  F i g u r e 1'"), a phylum that c o n t a i n s an estimated 75,000 living  and  35,000 f o s s i l s p e c i e s .  T h i s l a r g e phylum, second  i n s i z e only to the phylum Arthropoda, i s s u b d i v i d e d i n t o seven c l a s s e s . Gastropoda  Of the seven molluscan c l a s s e s , the  has been s t u d i e d i n the g r e a t e s t d e t a i l f o r  n a t u r a l products Gastropod  5  chemistry" .  molluscs of the s u b c l a s s O p i s t h o b r a n c h i a are  c h a r a c t e r i z e d by a g r e a t l y reduced, or completely shell.  In s p i t e of t h i s lack of p h y s i c a l  opisthobranchs have few the observed  relief  6  s p e c u l a t e d that opisthobranchs may 6  7  defense mechanism" '* . were i n i t i a t e d I t was  protection,  known p r e d a t o r s * .  from p r e d a t i o n , e a r l y  Chemical  absent  To account f o r investigators  u t i l i z e a chemical s t u d i e s on  on the l a r g e herbivorous  opisthobranchs 8  soon d i s c o v e r e d t h a t the sea hares  (Aplysia  spp.)  were capable of s t o r i n g i n t h e i r d i g e s t i v e gland a l a r g e  16  9  aplysiomorphs" '" .  MOLLUSCA  PHYLUM  GASTROPODA  CLASS  OPISTHOBRANCHIA  SUBCLASS  1 BULLOMORPHA  1  APLYSIAMORPHA  PLEUROBRA1SJCHOMORPHA  PTEROPODA ORDER  SACOGLASSA  AEOLIDACEA  NUDIBRANCHIA  ARMINACEA  Figure  DENDRONOTACEA  1. P h y l o g e n e t i c c l a s s i f i c a t i o n Organisms c l a s s i f i e d a c c o r d i n g  PYRAMIDELLA  DORIDACEA  of n u d i b r a n c h s . to Behrens'*.  N.B.  SUBORDER  18 v a r i e t y of secondary  m e t a b o l i t e s some of which were t o x i c to  potential fish predators  50  .  Further  investigations  demonstrated that the m e t a b o l i t e s were l i k e l y c o n c e n t r a t e d from the animals' a l g a l d i e t s of the m e t a b o l i t e s i s o l a t e d  5 1  being  .  The m a j o r i t y  from the sea hares' d i g e s t i v e  glands were monoterpenoids, s e s q u i t e r p e n o i d s or d i t e r p e n o i d s , many of which c o n t a i n e d a c o v a l e n t l y bound bromine atom.  Halogenated acetogenins and n i t r o g e n  c o n t a i n i n g compounds were a l s o  represented.  Three sea hare m e t a b o l i t e s have shown pharmacological potential.  Aplysistatin  (2T))  52  and d o l a t r i o l  (21)  53  showed  5  a n t i l e u k e m i a a c t i v i t y while d a c t y l y n e ( 2 2 ) * produced a dose dependent p r o l o n g a t i o n of p h e n o b a r b i t a l - i n d u c e d hypnosis i n animals by i n h i b i t i n g the metabolism of p h e n o b a r b i t a l . i t s e l f , enine 22 had no e f f e c t  5 5  .  on aplysiomorphs, marine chemists  By  Encouraged by the success turned t h e i r a t t e n t i o n to  smaller opisthobranch m o l l u s c s , s p e c i f i c a l l y the c a r n i v o r o u s nudibranchs. Nudibranchs have a body which i n c o r p o r a t e s the v i s c e r a l mass, mantle, and f o o t , and i s e x t e r n a l l y symmetrical  bilaterally  with a s l u g - l i k e f l a t t e n e d form (see F i g u r e 2 ) .  They completely lack a s h e l l i n the a d u l t form.  Nudibranchs  range i n s i z e from 3 t o 300 mm and a r e found throughout the world.  Over 100 s p e c i e s have been d e s c r i b e d * " on the west  coast of North America.  A l l nudibranchs  f e e d i n g on a wide range of i n v e r t e b r a t e s . Doridacea)  are predominately  are p r e d a t o r s , Dorids  (suborder  a s s o c i a t e d with sponges,  19  bryozoans ( e c t o p r o c t s ) , and other  three  Aeolidacea)  t u n i c a t e s , while members of  the  suborders (Dendronotacea, Arminacea, and are p r i m a r l y a s s o c i a t e d with  Nudibranchs are h e r m a p h r o d i t i c ,  56  .  they possess a c t i v e sex  organs of both sexes s i m u l t a n e o u s l y . evolutionary adaptation  coelenterates  as i t a l l o w s  T h i s may  be  for greater  an probability  of f i n d i n g a mate s i n c e every i n d i v i d u a l of the  same species  i s an e l i g i b l e p a r t n e r .  how  nudibranchs f i n d and  Little  recognize  i s known about each o t h e r .  It i s possible  that some of the compounds i s o l a t e d from nudibranch s k i n s e c r e t i o n s may rhinophores,  57  act as r e c o g n i t i o n or sex pheromones .  which are chemosensory organs on  The  the  nudibranchs' head, c o u l d act as the pheromone r e c e p t o r s  56  .  20  Branchial Plume  F i g u r e 2. T y p i c a l cryptobranch d o r i d nudibranch. From Behrens"", used with p e r m i s s i o n .  The study of secondary nudibranchs  metabolites isolated  from  has proven to be a r i c h and rewarding  Numerous novel secondary  area.  m e t a b o l i t e s have been i s o l a t e d by  groups working i n C a l i f o r n i a , H a w a i i , I t a l y and B r i t i s h Columbia.  Most of the compounds i s o l a t e d have been  s e s q u i t e r p e n o i d aldehydes, f u r a n s , or i s o n i t r i l e s ,  although  mono-, d i - , and t r i t e r p e n o i d s , s t e r o i d s , a l k a l o i d s , a c e t y l e n e s , and purine r i b o s i d e s are a l s o S e v e r a l Gastropods that s t i l l  retain a v i s i b l e external  s h e l l have been examined by n a t u r a l products chemists l a s t few y e a r s .  58  represented .  i n the  I t was known f o r some time that s h e l l e d  molluscs possessed  s e c r e t o r y glands s i m i l a r to those 46  occurring in nudibranchs .  When c o n s i d e r e d i n c o n j u n c t i o n  with Faulkner and G h i s e l i n s ' e v o l u t i o n a r y h y p o t h e s i s that  21 the e v o l u t i o n of a chemical  defense mechanism was  p r e a d a p t i v e , e n a b l i n g nudibranchs shell  3 9  ,  to dispense with  i t i s not s u r p r i s i n g that i n t e r e s t i n g  secondary  m e t a b o l i t e s have been i s o l a t e d from nudibranchs' cousins.  their  shelled  C e r t a i n pulmonates ( s u b c l a s s Pulmonata) are a r i c h  source of unusual secondary m e t a b o l i t e s with biological a c t i v i t i e s  5 9  .  interesting  U n l i k e the m e t a b o l i t e s  isolated  from nudibranchs, however, the pulmonate m e t a b o l i t e s are polyketides.  Two examples are shown below:  R e c e n t l y , chemical opisthobranch  examination  of a member of the  order Pleurobranchomorpha has y i e l d e d  i n t e r e s t i n g , b i o l o g i c a l l y a c t i v e , nitrogenous  60  compounds .  2. NUDIBRANCH DEFENSE MECHANISMS In order to deter p r e d a t o r s  (fishes,  c r u s t a c e a n s , and other opisthobranchs nudibranchs  56  '  61  starfish,  are known to prey on  ) i t has been suggested  t h a t these o f t - t i m e s  22 h i g h l y c o n s p i c u o u s , slow moving, s o f t bodied animals a bimodal d e f e n s i v e s t r a t e g y defense  61  .  utilize  The primary l e v e l of  ( u s e f u l only a g a i n s t v i s u a l p r e d a t o r s ) i s e i t h e r to  evade d e t e c t i o n  ( c r y p s i s ) or once having been d e t e c t e d , to  discourage the predator from i n i t i a t i n g an a t t a c k .  In other  words, i n the l a t t e r , nudibranchs u t i l i z e aposematism; the d e f e n s i v e a d a p t a t i o n i n which an animal  blatantly  a d v e r t i z e s , u s i n g conspicuous warning c o l o u r a t i o n , that i t i s not a s u i t a b l e food s o u r c e .  The r o l e of c o l o u r a t i o n i n  nudibranch defense has been debated  f o r many y e a r s  6 2  6  ' " . It  now appears probable that most nudibranchs are c r y p t i c a l l y or d i s r u p t i v e l y c o l o u r e d (the animal has a p a t t e r n  that  breaks up i t s body o u t l i n e on i t s usual substratum), while some well-defended  63  ( d i s t a s t e f u l ) species are aposematic .  More work is- r e q u i r e d before the complex nature of nudibranch c o l o u r a t i o n  is fully  39  understood .  The second l e v e l of d e f e n s e , u s e f u l i n the event of first  l e v e l f a i l u r e , or i n cases of p r e d a t o r s that hunt  chemically  (by f o l l o w i n g mucous t r a i l s  57  f o r example )  i n v o l v e s a d a p t a t i o n s that may be b e h a v i o r a l , m o r p h o l o g i c a l , or chemical i n n a t u r e  61  .  Nematocysts, s p i c u l e s , and  b e h a v i o r a l responses have a l l been i m p l i c a t e d , t o v a r y i n g d e g r e e s , i n nudibranch d e f e n s e . s t o r i n g u n f i r e d nematocysts  The b i z a r r e p r a c t i c e of  ( s t i n g i n g c e l l s ) , o b t a i n e d from  t h e i r c o e l e n t e r a t e d i e t , c h a r a c t e r i z e s many a e o l i d 6  nudibranchs ".  L i k e l y an a d a p t a t i o n that a l l o w s the a e o l i d s  to s a f e l y feed on c o e l e n t e r a t e s and m o d i f i e d to p r o v i d e a  23 cheap means of defense, nematocysts  probably combine to work  with chemical s e c r e t i o n s to give a e o l i d s t h e i r predators.  immunity  from  Not a l l nudibranchs feed on c o e l e n t e r a t e s  ( d o r i d s feed e x c l u s i v e l y on sponges, bryozoans t u n i c a t e s ) , consequently nematocysts  and  are unimportant  i n the  defense of these s p e c i e s . A few -dorid s p e c i e s have hard c a l c a r e o u s s p i c u l e s c o n t a i n e d w i t h i n t h e i r mantles.  Obtained from t h e i r  sponge  d i e t s , they give the nudibranch a r i g i d shape and p r o v i d e some p r o t e c t i o n from p r e d a t o r s .  A v o r a c i o u s nudibranch  p r e d a t o r , the opisthobranch Navoanax  inermis,  does not eat  s p i c u l e - c o n t a i n i n g d o r i d s , while r e a d i l y consuming non-spiculose o n e s content  65  .  Todd has suggested that the high ash  (most of which i s a t t r i b u t a b l e to s p i c u l e s ) and  calorific  low  content of some d o r i d s makes these nudibranchs of  poor n u t r i t i o n a l value to p r e d a t o r s Many nudibranchs w i l l  66  .  swim i n response to being  d i s t u r b e d or threatened by a p r e d a t o r , and t h i s may  be con67  s i d e r e d the most b a s i c b e h a v i o r a l defense mechanism . Nudibranchs  with l a r g e c e r a t a  (finger-like  respiratory  d i g e s t i v e s t r u c t u r e s o c c u r r i n g i n groups of p a r a l l e l along the dorsum, suborders Dendronotacea,  and  series  A e o l i d a c e a and  Arminacea) tend to autotomize the c e r a t a at o n l y the  slight-  est p r o v o c a t i o n , e n a b l i n g a captured nudibranch t o make good an escape  (the l o s t c e r a t a are r e g e n e r a t e d ) .  Nudibranchs the e p i d e r m i s *  6  have a l a r g e number of s e c r e t o r y glands i n from which some d o r i d s s e c r e t e s u l f u r i c  acid  24 when d i s t u r b e d  68  .  Species of a l l four nudibranch  are known to s e c r e t e non-acid noxious lar conditions.  substances  suborders under  Johannes found the d o r i d Phyllidia  s e c r e t e d a t o x i n that was  l e t h a l to f i s h and  simivaricosa  crustaceans*  7  and subsequent chemical a n a l y s i s of t h i s s e c r e t i o n showed i t to c o n t a i n 9-isocyanopupukeanane ( 1 £ ) as p r e v i o u s l y d i s c u s s e d . 'Recent s t u d i e s have shown of  d o r i d nudibranchs  38  that a wide v a r i e t y  produce organic compounds that were  e a s i l y s o l u b l i z e d by e x t r a c t i o n of the whole animals non-polar  solvents.  with  I t i s l i k e l y these m e t a b o l i t e s were  coming from the s e c r e t o r y g l a n d s , not the g u t s  36  , and a num-  ber of these m e t a b o l i t e s e x h i b i t e d f i s h a n t i f e e d a n t activity. In c o n c l u s i o n , d e f e n s i v e secondary been u n e q u i v o c a l l y proven to e x i s t  m e t a b o l i t e s have not  i n nudibranchs, nor have  other p o s s i b l e r o l e s f o r the m e t a b o l i t e s been  adequately  investigated.  evidence,  On  the b a s i s of c i r c u m s t a n t i a l  e v o l u t i o n a r y t h e o r y , and may  f i s h antifeedant a c t i v i t i e s ,  hypothesize that d e f e n s i v e s e c r e t i o n s make an  c o n t r i b u t i o n to nudibranch the r e l a t i v e  importance of any  mechanism and combination eaten.  defense.  important  L i t t l e i s known about  s p e c i f i c nudibranch  i t seems a nudibranch  one  defense  will utilize a  of d i f f e r e n t d e f e n s i v e s t r a t e g i e s to a v o i d being  More s t u d i e s are needed to determine the  relative  importance of the d i f f e r e n t d e f e n s i v e mechanisms and  to  i d e n t i f y the p r e d a t o r s that provided the s e l e c t i v e p r e s s u r e s l e a d i n g to t h e i r  56  development .  25 ALDISA  B. SECONDARY METABOLITES FROM THE DORID NUDIBRANCH COOPERI  (ROBILLIARD AND BABA, 1972)  1. INTRODUCTION Aldisa  cooperi  (Aldisa  3) i s an orange d o r i d usually  sanguinea  cooperi ,  see F i g u r e  69  (suborder D o r i d a c e a , f a m i l y  Aldisidae)  found deeply embedded i n i t s p r e f e r r e d p r e y , the  r e d d i s h orange association  sponge Ant hoarcuat  i s l i k e l y cryptic  0  a graceae . 7  The  t o p o t e n t i a l p r e d a t o r s , even  though SCUBA d i v e r s c a n , from a d i s t a n c e , e a s i l y A. cooperi  nudibranch on the sponge.  l o c a t e the  may grow t o 20 - 25 mm  in l e n g t h and has been r e p o r t e d from Washington, B r i t i s h Columbia, and Japan.  U n l i k e many d o r i d s c o l l e c t e d  B a r k l e y Sound, B.C., A. cooperi  from  d i d not have a d e t e c t a b l e  odour.  2. ISOLATION AND STRUCTURE ELUCIDATION The nudibranchs were c o l l e c t e d by hand (SCUBA, depths of  1 t o 10 m) and immediately  After was  immersed whole i n methanol.  one t o three days at room temperature, the methanol  decanted and saved.  additional  The animals were washed an  four times with methanol.  The methanol e x t r a c t s  were combined and vacuum f i l t e r e d t o give an aqueous methanolic s u s p e n s i o n .  The suspension was p a r t i t i o n e d be-  tween b r i n e and e t h y l a c e t a t e , and the e t h y l a c e t a t e s o l u b l e m a t e r i a l was f r a c t i o n a t e d by f l a s h Chromatographically s i m i l a r  chromatography.  f r a c t i o n s were pooled t o give a  F i g u r e 3. Aldisa cooperi. Fraser U n i v e r s i t y ,  Photographer:  Ron Long, Simon  27 mixture of s t e r o i d a l a c i d s !23 and 24 mg/animal), g l y c e r o l ether ketones  (see F i g u r e 4 ) .  25,  7 1  (10:3, 2.8  and a mixture of s t e r o i d a l  Additional fractions  containing  f a t s and s t e r o l s were not s t u d i e d f u r t h e r .  F i g u r e 4.  Secondary m e t a b o l i t e s from the d o r i d nudibranch Aldi  sa  cooperi  The s t e r o i d a l a c i d f r a c t i o n was p r e p a r a t i v e t h i n l a y e r chromatography give a white c r y s t a l l i n e relative the  solid.  f u r t h e r p u r i f i e d by ( p r e p a r a t i v e TLC) to  I t was  apparent from the  i n t e n s i t i e s of the peaks i n the o l e f i n i c  'H NMR  region of  spectrum of t h i s m a t e r i a l that a mixture of  28 c l o s e l y r e l a t e d compounds had been o b t a i n e d .  Additional  p u r i f i c a t i o n by reverse-phase p r e p a r a t i v e TLC  yielded  3-oxo-4-cholenoic  a c i d 2_3 and  i t s unsaturated a n a l o g ,  3-oxo-4,22-choladienic a c i d 24 * . The major m e t a b o l i t e of the m i x t u r e , acid  ( 2 3 ) , mp  3-oxo-4-cholenoic  = 178-179 °C, had a molecular formula  C2«H3603  (HRMS, m/z observed 372.2658, r e q u i r e d 372.2664) that demanded seven u n i t s of u n s a t u r a t i o n . 1  the H NMR 3H), and  spectrum at 6 0.74 1.21  ( s , 3H)  Methyl resonances i n  ( s , 3H), 0.94  ( d , / = 5.8  i n combination with the  requirement  for 24 carbon atoms i n d i c a t e d the molecule was degraded  steroid.  The chemical s h i f t of the  possibly a  methyl  s i n g l e t s , the presence of a one proton s i n g l e t at 8 ppm,  an u l t r a v i o l e t  Hz,  (UV) a b s o r p t i o n at Xm-ax 236 nm  5.74 (e  6,200), and a p o s i t i v e 2,4-dinitrophenylhydrazone t e s t suggested a A * - 3 - k e t o s t e r o i d nucleus of the type shown i n 26. Comparison of the  1  H NMR  chemical s h i f t s of the methyl  s i n g l e t s and the o l e f i n i c proton to the corresponding s i g n a l s i n cholestenone Table 1 ) .  (2_7) showed e x c e l l e n t agreement (see  The presence of A " - 3 - k e t o s t e r o i d nucleus  f u r t h e r s u b s t a n t i a t e d by a l o s s of ketene  (CH2CO) i n the  mass spectrum of 23_ to give a peak at m/z 330.2564 It i s w e l l known that molecules that  was  (31%).  incorporate  s u b s t r u c t u r e 26 lose ketene v i a a process which may  * I t was a l s o found that the mixture c o u l d be r e a d i l y p u r i f i e d by normal phase HPLC of the methyl e s t e r s .  be  29  26  1  Table 1. Comparison of the H NMR A ' - 3 - k e t o s t e r o i d s (CDC1 3 )  data  for various  chemical s h i f t , 5  H on C#  23°  4 18 19  a  24°  5.74 0.74 1.21  400 MHz.  b  5.74 0.77 1.20  b  5.74 0.72 1.19  80 MHz.  v i s u a l i z e d as shown i n Scheme 1 a diagnostic  2 1  for A"-3-ketosteroids  287.2028 ( 1 1 % ) , 249.1861  72  73  .  Other fragment  ions  were a l s o observed at m/z  ( 3 8 % ) , and 124.0897 (96%) (see  Scheme 1 b ) . The  s t r u c t u r e of the C 1 7  s i d e chain s u b s t i t u e n t was  deduced from the f o l l o w i n g s p e c t r a l evidence:  i ) The pres-  ence of a methyl doublet at 6 0.94 ( d , / = 5.8 Hz, 3H)  1.  Interpretation  of the HRMS of a c i d (23).  3-oxo-4-cholenoic  31 indicated  the C-21 methyl was not o x i d i z e d .  a d d i t i o n a l methyl resonances, i n a d d i t i o n  i i ) Absence of  to those assigned  to the C-18 and C-19 carbons of the s t e r o i d a l n u c l e u s , i n d i cated the side c h a i n was not branched and must i n c o r p o r a t e a c a r b o x y l group at C-24 ester  1  (IR 1700 cm"  and formation of methyl  28 upon treatment with diazomethane).  mass spectrum of 23, a s i g n i f i c a n t fragment at m/z 271.2047  (required  i i i ) In the i o n was detected  f o r C 1 9 H 2 7 0 , 271.2062) which  corresponded to a l o s s of the 1-methyl-4-butanoic a c i d chain from 23 as i n d i c a t e d  side  i n Scheme 1b.  The s t e r o i d a l a c i d , 3-oxo-4,22-choladienic a c i d  (24) ,  o c c u r r e d as the minor component of the b i n a r y s t e r o i d a l a c i d  32 mixture i n a v a r y i n g r a t i o depending upon the c o l l e c t i o n . The  mass spectrum of 24 i n d i c a t e d a m o l e c u l a r  C «H3fl03 2  formula  (HRMS, m/z observed 370.2507, r e q u i r e d 370.2508),  t h e r e f o r e 24 d i f f e r e d from the major s t e r o i d by possessing one  l e s s u n i t of u n s a t u r a t i o n .  (s,  3H) and 1.20 ( s , 3H), a one proton  and  a UV a b s o r p t i o n  A"-3-ketosteroid  Methyl resonances at 6 0.77 s i n g l e t at 6 5.74,  at 241 nm (MeOH) d i c t a t e d a  nucleus the same as t h a t found f o r 23.  T h i s assignment was supported by fragment ions at m/z 328.2403 ( 4 5 % ) , 285.1862 ( 2 4 % ) , 247.1720 ( 5 % ) , and 124.0895 (100%) i n the HRMS of 24 (Scheme 2 ) .  The 'H NMR spectrum  showed resonances a t 6 6.96 (dd, / = 15.5,8.7 Hz, 1H) and 5.76  (d, / = 15.5 Hz, 1H) ppm.which suggested that the  a d d i t i o n a l u n i t of u n s a t u r a t i o n unsaturated  carboxyl carbonyl  was i n c o r p o r a t e d  i n an a,/3  of type 2j}. The E  c o n f i g u r a t i o n of the double bond f o l l o w e d  from the 15.5 Hz  1  v i c i n a l c o u p l i n g constant i n the H NMR spectrum.  The HRMS  of 24 showed an abundant ion at m/z 271.2058 (70%, r e q u i r e d for  C 1 9 H 2 7 0 , 271.2062) a r i s i n g  fragmentation  from the a l l y l i c  of the 1-methyl-4-but-2-enoic a c i d side chain  s u b s t i t u e n t i n 24 (Scheme 2 ) . Due t o the s t e r e o s p e c i f i c i t y of t h e enzymes i n v o l v e d i n s t e r o i d b i o s y n t h e s i s one would expect that a c i d 23 and i t s unsaturated  3-oxo-4-cholenoic  analog 24 each would e x i s t as  only one of the p o s s i b l e 128 s t e r e o i s o m e r s * .  It i s well  *The side chain double bond i n 24 was a l r e a d y the E c o n f i g u r a t i o n .  known tO' have  33  0  m/z  328 (45%)  m/z 124 (100%) + 2H  Scheme 2. I n t e r p r e t a t i o n of the HRMS of 3-oxo-4,22-choladienic acid (24).  known from s t u d i e s on both t e r r e s t i a l and a q u a t i c that  s t e r o i d s are b i o s y n t h e s i z e d 7  of S-squalene 2 , 3 - o x i d e * . (30) i s the i n i t i a l algae i n i t i a l l y  from an i n i t i a l  organisms cyclization  In animals and f u n g i , l a n o s t e r o l  intermediate formed, while p l a n t s and  produce c y c l o a r t e n o l  (31).  The  34 intermediates  30 and  biosynthetically tively.  The  3_1 are then transformed  i n t o the s t e r o i d s or p h y t o s t e r o i d s ,  absolute  stereochemistry  r i n g system i s remarkably constant;  of the ABCD s t e r o i d a l  the few  u s u a l l y show the 5/3 i n s t e a d of the 5a C h o l i c a c i d (32),  stereochemistry. i s an example. and  I t was 1  supported by  (Table  H NMR  1 ) , that the a b s o l u t e  C  NMR  has  known  of the b i l e a c i d s ,  from b i o g e n e t i c  reasoning  comparisons to cholestenone  r i n g systems in 23 and 13  exceptions  absolute  one  postulated  respec-  (27)  s t e r e o c h e m i s t r i e s of the ABCD  2_4 were as shown.  been shown to be an e x c e l l e n t method f o r  comparing the r e l a t i v e s t e r e o c h e m i s t r i e s of s i m i l a r molecules.  The  l a r g e data  a l l o w e d comparison of the 23 to cholestenone  13  base f o r 13  C  NMR  C  NMR  s p e c t r a of s t e r o i d s  s p e c t r a of s t e r o i d a l a c i d  (27) and methyl 3-oxo-4-cholenoate 75  p r e p a r e d from 23^) to methyl 5/3-cholan-24-oate (3_3) . comparisons are summarized in Table 2.  The  of the  cholestenone  (21)  24.  be-  supported the assignment  r e g u l a r s t e r o i d a l ABCD r i n g s t e r e o c h e m i s t r y  n a t u r a l products 23 and  The  close  correspondence f o r the resonances of the r i n g carbons tween a c i d 23_ and  (28,  to both  Comparison of the s i d e chain  carbon resonances of e s t e r 2_8 to those of methyl 5/3-cholan-24-oate (3_3) i n d i c a t e d i d e n t i c a l s t e r e o c h e m i s t r i e s and stereochemistry stereochemistry.  relative  allowed the assignment of  at C-20  as R, the  regular s t e r o i d a l  absolute  35  Further s p e c t r a l evidence supporting the 20R c o n f i g u r a t i o n f o r 2_3 and 24 was a v a i l a b l e from the literature.  A number of s t e r o i d s i s o l a t e d from the marine  environment have the " u n - n a t u r a l " 20S c o n f i g u r a t i o n as shown in F i g u r e 5  76  '  77  stereochemistry  .  In order t o confirm the assignment of 20S  to compounds 34, 35, 36 and 3_7, Vanderah and  D j e r a s s i unambiguously  s y n t h e s i z e d both C-20 epimers of the  a c e t a t e e s t e r s of each compound.  Comparison  1  of the H NMR  s p e c t r a of the epimeric p a i r s showed that the C-21 methyl  36  13  Table 2 . C NMR data and s p e c t r a l comparisons f o r the assignment of s t e r e o c h e m i s t r y to 3-oxo-4-cholenoic a c i d  (23)  chemical s h i f t , 6 fl  Carbon #  27  (CDC1 3 )  28 C  (CDCl3)  rf  23  (CD2Cl2)  e  (CDC1 3 )/  1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19  35.7 33.9 198.9 123.6 171.0 32.9 32. 1 35.7 53.8 38.6 21.0 39.4? 42.4 55.9 24. 1 28. 1 56. 1 12.0 17.4  35.8? 34.0 1 99.4 123.9 171.4 32.9 32. 1 35.4? 53.9 38.6 21.1 39.7 42.5 55.9 24.2 28. 1 55.9 12.0 17.4  37.9 21.6 27.4? 27.8? 44. 1 27.5? 26.9 36.2 40.8 35.8 21.1 40.6 43.0 56.9 24.4 28.4 56.4 12.1 24.2  35.7? 34.0 199.6 1 23.8 171.5 33.0 32.0 35.3? 53.9 38.6 21.1 39.7 42.5 55.9 24.2 28.0 55.9 12.0 17.4  20 21 22 23 24 25 26 27  35.7 18.7 36. 1 23.8 39.6? 27.6 22.5 22.8  35.8? 18.3 31 . 3 ^h 31 . \ 1 74.6  35.6 18.3 31 . 2 h 31 . 1 175.0  35.7? 18.2 30.8 30.8  51.5  51 .3  -OCH3  a  33*  h  From r e f e r e n c e 7 5 , compound 2 0 7 . " From r e f e r e n c e  compound 2 5 6 . MHz.  e  c  20.1 MHz.  switched.  ' Not  Spectrometer frequency not g i v e n . f 20.1 MHz. observed due  ?'  h  Assignments may  to slow  relaxation.  d  be  75, 100.6  37  F i g u r e 5. "Un-natural" 20S marine  resonance was compounds. the C-21  s h i f t e d = 0.1  ppm  sterols.  t o higher f i e l d  i n the  C a r e f u l examination of the chemical s h i f t s of  methyl groups  i n e s t e r s 28 and 38  (prepared from 24  with diazomethane) proved that they both have the more common 20R  20S  configuration  (Table 3 ) .  To r a t i o n a l i z e  the  38 ^  0.1  20R,  ppm  s h i e l d i n g of the  Vanderah and  "un-natural"  20S  20S  s i d e chain  epimer e x i s t s p r e f e r e n t i a l l y  s h i e l d i n g anisotropy  cone of C-16  side chain  the  of  to C-17  bond and  p r o j e c t s to the in 39.  a p r e f e r r e d conformation  in 20R  the  in that  methyl group r e s i d e s in  r e l a t i v e to C-13), as d e p i c t e d against  methyl group over  D j e r a s s i suggested the  conformation wherein the C-21  remainder of the  C-21  left  the  the (cis  Although an argument s t e r o i d s had  been  r a i s e d , the  l a r g e d i f f e r e n c e s i n the observed GC m o b i l i t i e s  of the C-20  epimers suggested the  of the C-20  epimers e x i s t s in a p r e f e r r e d r o t a t i o n a l  conformation. epimeric at C20  Vanderah and but  of  side chain  Djerassi felt  of one  that  or  both  sterols,  i d e n t i c a l conformer composition  should e x h i b i t i d e n t i c a l gas  chromatographic  C l e a r l y , more work i s r e q u i r e d i n t e r e s t i n g , conformational  behavior.  to s e t t l e t h i s s u b t l e , yet  question.  0  Both a c i d s 2 3 and  78  and  the major a c i d 23 has  24  7 9  are  known s y n t h e t i c compounds  a l s o appeared i n the  literature  as  39 1  Table 3. H NMR data (CDC1 3 )  40  Compound  Chemical s h i f t of C-21, 6 a b  f o r v a r i o u s 20R and 20S s t e r o i d s  0.84  41  G  fl  0.94  28  0.93  42  43  fl  b  3_8  6  1.00  1 . 1 0 ° 1.10  100 MHz, see r e f e r e n c e 76 400 Mz  40  R=  V  ^  v  ^ 0 C H  3  ,—'  the r e s u l t of m i c r o b i a l t r a n s f o r m a t i o n of other steroids  8 0  .  precursor  The p h y s i c a l data f o r both compounds are i n  agreement with the l i t e r a t u r e  values.  N e i t h e r a c i d has been  p r e v i o u s l y reported from n a t u r a l s o u r c e s , and they the f i r s t  isolation  of b i l e a c i d s from a marine  represent  40 invertebrate. In order to e s t a b l i s h a d i e t a r y o r i g i n and 2 4 , a methanolic e x t r a c t  of Ant hoarcuat  examined (see experimental f o r d e t a i l s ) . usually derive  with A. graceae  found a s s o c i a t e d  i t s pigmentation from i t . The  neither  2 3 or 2 4 but i t d i d c o n t a i n  A ' - 3 - k e t o s t e r o i d s that were found Cholestenone  (22)  was  a graceae  was  A. cooperi  was  and appears  to  sponge c o n t a i n e d  the same mixture of  i n A. cooperi  .  the major component of both m i x t u r e s .  In a d d i t i o n , two oxygenated  compounds, seemingly s t e r o i d a l  (molecular formulas: C 2 9 H<, O  in nature  f o r the a c i d s 23_  0  been i s o l a t e d from A. graceae.  The  4  and C 2 sHu «Oi,) , have  s t r u c t u r e s of these  m e t a b o l i t e s are unknown at t h i s t i m e , but they do not  appear  to be A " - 3 - k e t o s t e r o i d s .  OAc AcO 44  The  t h i r d m e t a b o l i t e i s o l a t e d from A.  1-0-hexadecyl-glycerol  ( 2 5 ) , was  diacetyl derivative 4 4 : C23HjMl05 400.3141, r e q u i r e d  400.3189).  characterized  cooperi, as i t s  (HRMS, m/z observed  I s o l a t i o n as the d i a c e t y l  d e r i v a t i v e g r e a t l y f a c i l i t a t e d both the p u r i f i c a t i o n s t r u c t u r a l assignment. assignable  and  The mass spectrum of 4 4 showed peaks  by the fragmentation pathway o u t l i n e d  i n Scheme 3  41 and allowed assignment of the ether l i n k a g e to C-3. NMR  spectrum  supported t h i s assignment;  1  The H  thus resonances  at 6  4.36 (dd, J = 3.9,11.7 Hz, IH) and 4.14 (dd, J = 6.7,11.7 Hz,  1H) ppm were assigned t o the protons on C - l , the  multiplet  at 6 5.19 (m, IH) was a s s i g n e d t o the C-2 methine  p r o t o n , and the doublet at 6 3.55_(d, / = 5.4 Hz, 2H) was a s s i g n e d to the methylene protons on the carbon b e a r i n g the ether f u n c t i o n a l i t y .  357  327  (2%)  I  (0.56%)  ACO-/ P^OCH (CH ) CH 2  2  u  3  255 (29%)  Scheme 3.  Interpretation  Glycerol  of the MS of d i a c e t y l 44  derivative  ether 25 i s a known n a t u r a l p r o d u c t , commonly  r e f e r r e d t o as chimyl a l c o h o l .  I t has been shown to s l i g h t -  l y i n h i b i t the progress of the murine E h r l i c h a s c i t e s tumor  81  .  Subsequent t o i t s i s o l a t i o n from A. cooperi,  was i s o l a t e d i n our l a b o r a t o r y from the d o r i d Arc hi dor is mont ereyensi vitro Bacillus  is.  nudibranch  82  5 , and was shown to have potent in  a n t i b i o t i c a c t i v i t y a g a i n s t Staphylococcus subtil  2_5  aureus  and  I n t e r e s t i n g l y , 2_5 was a l s o shown t o have  42  antifeedant a c t i v i t y  1  (18 ug mg~  t i d e pool s c u l p i n Oligocottus  of food p e l l e t ) a g a i n s t the maculosus.  3. BIOLOGICAL ACTIVITIES OF ALDISA  COOPERI  METABOLITES  Because the s t e r o i d a l a c i d s 23 and 24 were present i n such high c o n c e n t r a t i o n r e l a t i v e to the animals body weight, it  seemed reasonable t o assume that they were i n v o l v e d i n  the chemical defense evidence bioassay  of the nudibranch.  i n support of t h i s h y p o t h e s i s , a standard 36  goldfish  was used to t e s t the a n t i f e e d a n t p r o p e r t i e s of  the s t e r o i d a l a c i d 23. tively  To gain some  The r e s u l t s showed that 23 e f f e c -  i n h i b i t e d f e e d i n g at < 15 ug/mg of food p e l l e t , while  cholestenone  (2J_) was t o t a l l y  food p e l l e t .  The nudibranch  i n a c t i v e at > 100 ug/mg of was apparently o b t a i n i n g an  i n a c t i v e m e t a b o l i t e from i t s d i e t and c h e m i c a l l y modifying i t t o produce an a c t i v e a n t i f e e d a n t .  Verification  s u p p o s i t i o n would r e q u i r e i n j e c t i o n of A. cooperi r a d i o l a b e l l e d cholestenone  of t h i s with  f o l l o w e d by i s o l a t i o n of  radioactive steroidal acids.  I t should be s t a t e d t h a t , a l -  though the s t e r o i d a l a c i d s were not i s o l a t e d from the sponge, the p o s s i b i l i t y e x i s t s that they were present i n very small amounts and were not detected by the i s o l a t i o n procedure.  T h i s p o s s i b i l i t y would be very remote, however,  as the' nudibranch  would have t o consume l a r g e amounts of  sponge i n order t o o b t a i n the l a r g e q u a n t i t i e s of s t e r o i d a l a c i d s found A.  cooperi  i n the s k i n of each a n i m a l .  One c o l l e c t i o n of  made i n the Queen C h a r l o t t e I s l a n d s , B.C., gave  43  s t e r o i d a l a c i d s 23 and 24 i n a r a t i o s i m i l a r to that in the Barkley Sound c o l l e c t i o n s .  This finding  supported  the h y p o t h e s i s that a c i d s 2_3 and 2_4 are produced A.  found,  de novo  by  cooperi.  4. DISCUSSION B i o g e n e t i c a l l y , one can envisage the s t e r o i d a l being produced  acids  from cholestenone v i a a s e r i e s of  c o n v e n t i o n a l f a t t y a c i d type 0 - o x i d a t i o n r e a c t i o n s .  I t has  been shown that i n the m i c r o b i a l t r a n s f o r m a t i o n of c h o l e s t e r o l i n t o 17-keto an i n t e r m e d i a t e  83  .  s t e r o i d s , 3-oxo-4-cholenoic  acid i s  The evidence suggested the degradation  pathway shown i n Scheme 4. (45) and campesterol  The c o n v e r s i o n of  sitosterol  (46) i n t o andro.st-4-ene-3,17-dione  (47 )  has a l s o been s t u d i e d and both of these compounds are a l s o degraded  ( Mycobact  eri urn sp.) v i a the s t e r o i d a l a c i d 23  8 0  .  Thus, i t i s not unreasonable to b e l i e v e that the nudibranch i n g e s t s cholestenone  (and r e l a t e d s t e r o i d a l ketones)  from  i t s sponge d i e t , and m o d i f i e s them to the s t e r o i d a l a c i d s 23_ and 24, v i a a mechanism s i m i l a r to that shown i n Scheme 4. These s t e r o i d a l a c i d s might then be u s e f u l as d e f e n s i v e allomones. The use of s t e r o i d s f o r chemical defense i s not to A. cooperi. marginal  is)  The  great d i v i n g b e e t l e  (Dytiscus  u t i l i z e s an a r r a y of s t e r o i d a l ketones to ward  off potential predators. was  limited  shown to be  The major component of the mixture  1 1 - d e o x y c o r t i c o s t e r o n e (Cortexone)  8  (48) ".  44  47 Scheme 4. Proposed mechanism f o r the m i c r o b i a l 83 t r a n s f o r m a t i o n of c h o l e s t e r o l i n t o 1 7 - k e t o s t e r o i d s .  T h i s substance has the same stunning natural secretion.  e f f e c t on f i s h as the  Numerous other water b e e t l e s produce the  45  same or  r e l a t e d compounds.  s t e r o i d a l skin toxins s k i n of the  halothurin  ( f o r example: b u f o t a l i n  ,  (50_))  8 5  , and  a neurotoxin of  oxygenated s t e r o i d s nudibranchs.  lineal  a  6 1  (49)),  The  sea  F i n i a l l y , two  have been i s o l a t e d from  and  while Adalaria  Flabellina  affinis,  seen that s t e r o i d s  isolated  steroidal 88  example . are  a group  of s t r u c t u r a l l y s i m i l a r compounds that e x h i b i t  a wide  variety  oft-times  of b i o l o g i c a l a c t i v i t i e s , depending on  s u b t l e molecular changes.  46  R=H  it  Coryphella  and  p e r o x i d e s of which 5a, 5 a - e p i d i o x y s t e r o i d 5_2 i s an be  use  dorid  sp. produces a mixture of  From these examples i t can  (for  additional  h i g h l y oxygenated s t e r o i d 51 was  peregrina,  the  cucumbers s y n t h e s i z e  steroidal structure  to deter t h e i r f i s h p r e d a t o r s .  from Hervia  toads u t i l i z e  salamander c o n t a i n s d e f e n s i v e s e c r e t i o n s  example: samandarin 86  Frogs and  46  47 C. SECONDARY METABOLITES FROM THE DENDRONOTID.NUDIBRANCH MELI BE LEONINA  (GOULD,  1852)  1. INTRODUCTION The dendronotid nudibranch Meli be leonina  (see F i g u r e  6) has one of the most unusual f e e d i n g b e h a v i o r s of any member of the phylum m o l l u s c a . M. leonina  U n l i k e many other nudibranchs,  i s not a predator of s e s s i l e bottom d w e l l i n g  a n i m a l s , r a t h e r , i t feeds upon zooplankton by m a j e s t i c a l l y sweeping the sea with i t s l a r g e o r a l Our chemical s t u d i e s on M. leonina  hood  89  .  were prompted by a  90  r e p o r t that the nudibranchs' primary means of defense was  an  o d i f e r o u s substance known to be repugnant to p o t e n t i a l predators  91  observation  and were i n i t i a t e d 92  after a fortuitous  that allowed c o l l e c t i o n of enough animals f o r  chemical a n a l y s i s .  Specimens of M. leonina  were c o l l e c t e d  d u r i n g a r e p r o d u c t i v e congregation of the nudibranchs i n a shallow k e l p bed  (depths of 1 to 5 m)  at Cates  Vancouver, B.C.  The number of nudibranchs was  Park, extremely  2  high  (= 50 animals/m ) and t h e i r odour c o u l d be d e t e c t e d in  situ  by SCUBA d i v e r s .  2. ISOLATION AND  STRUCTURE ELUCIDATION  F r e s h l y c o l l e c t e d whole specimens (38 animals) were immediately immersed i n c h l o r o f o r m and e x t r a c t e d shaker) f o r 1.5  hours.  (wrist a c t i o n  The c h l o r o f o r m s o l u b l e m a t e r i a l  f i l t e r e d , d r i e d over sodium s u l f a t e , and c o n c e n t r a t e d in  was  49 vacuo 1.51  to give an orange mg/animal).  NMR  " g r a p e f r u i t " s m e l l i n g o i l (57.4  mg;  a n a l y s i s of the crude o i l showed the  presence of two major components, 2,6-dimethyl-5-heptenal (53) and 2,6-dimethyl-5-heptenoic a c i d relative  r a t i o of  3.3:1* that was  small amount of f a t . The  (54) ( F i g u r e 7) i n a  contaminated with a  i n d i v i d u a l components were  subsequently r e s o l v e d by s i l i c a  g e l column chromatography  ( g r a d i e n t hexane/chloroform).  8  F i g u r e 7.  The  9  Secondary m e t a b o l i t e s from the dendronotid nudibranch Mel i be leonina  l e a s t p o l a r m e t a b o l i t e , 2,6-dimethyl-5-heptenal  (J53) , had a molecular formula of C 9 H 1 6 0 (M+, m/z v e r i f i e d by the appearance spectrum.  1  A H NMR  of 9 carbons i n the  13  140), C  spectrum of t h i s substance d i s p l a y e d  resonances a p p r o p r i a t e f o r three methyl groups at 8 (bs,  3H), 1.61  NMR  ( b s , 3H) and  1.11  ( d , / = 7.1  1.70  Hz, 3H), f o r a  * The r e l a t i v e r a t i o was obtained by measuring the peaks h e i g h t s of the C-2 methyl doublets at 6 1.11 f o r 53 and 5 1.20 ppm f o r 54.  50 s i n g l e o l e f i n i c proton at 6 5.10 proton at 9.62  ( d , / = 1.9 Hz,  (m), and f o r an  1H) ppm.  The  aldehyde  aldehyde  f u n c t i o n a l i t y a l s o d i s p l a y e d an IR a b s o r p t i o n at 1723 and a  13  C  NMR  resonance at 6 205.0 (d) ppm  cm"  (see Table 4 ) .  55  In  the mass spectrometer, aldehyde 53 r e a d i l y underwent  a M c L a f f e r t y rearrangement  to g i v e the base peak at m/z 82  as i n d i c a t e d i n Scheme 5.  Other  fragment ions at m/z 41 ,  55, 67, and 69 supported an a c y c l i c degraded skeleton f o r 5_3.  Comparison of the  to that of c i t r o n e l l a l the  13  C  NMR  resonances  spectrum of aldehyde assignment  13  C  NMR  monoterpenoid spectrum of 53_  (5_5) allowed the t o t a l (Table 4 ) .  5_3 was  The 400 MHz  assignment 1  H  of  NMR  w e l l r e s o l v e d and allowed  of a l l the protons i n the m o l e c u l e , the chemical  s h i f t s and assignments  are l i s t e d i n the experimental  (Chapter 4 ) . All  the s p e c t r a l evidence suggested that the non-polar  compound r e s p o n s i b l e f o r the f r a g r a n t odour of the nudibranch was 5-heptenal  the degraded monoterpene  (53).  Aldehyde  2,6-dimethyl-  53 has been r e p o r t e d , as a  51 Table 4.  13  C  NMR  Citronellal  data f o r 2,6-Dimethyl-5-heptenal  (55)  93  .  2,6-Dimethyl-5-heptenal  Carbon  #  (53)  Chemical  C i t r o n e l l a l (55)  Carbon #  s h i f t , 6° (mult)  1  G  (53) and  Chemical 8  shift,  c  b  (mult)  205.0 (d)  c  1  202.2 (d)  2  51 .0 (t)  2  45.9  (d)  3  27.8  (d)  3  30.7  (t)  4  37.0  (t)  4  25.4  (t)  5  25.4  (t)  5  1 23. 5 (d)  6  124. 1 (d)  6  132.7  (s)  7  131.5  (s)  7  25.7  (q)  8  25.6  (q)  8  17.7  (q)  9  17.6  (q)  9  13.3  (q)  10  19.8  (q)  100.6 MHz.  b  25  MHz.  c  M u l t i p l i c i t y , determined from a s i n g l e  frequency o f f resonance  (SFORD) experiment.  n a t u r a l p r o d u c t , from numerous s o u r c e s .  Identifications  g e n e r a l l y have been made s o l e l y on the b a s i s of GC-MS analysis.  Aldehyde  5_3 has been r e p o r t e d as a pheromonal  component of the ant Lasius specifically  carniolicus,  originating 9  from the i n s e c t s ' h e a d " , as a chemical  52 Eucalyptus  component of the e s s e n t i a l o i l from the l e a v e s of sp.  95  and Carphephorous  corymbosus  e x t r a c t e d from Jasminum  sambac  9 6  ,  and as a component  flowers  97  .  Interestingly, a  r e l a t e d secondary m e t a b o l i t e , 2,6-dimethyl-1,5-heptadien-3-ol a c e t a t e ( 5 6 ) , i s an i n s e c t 9 8  (Ps eudococcus  sex phermone  comsl ocki )  .  The  odiferous  c h a r a c t e r o f s y n t h e t i c 2,6-dimethyl-5-heptenal has not escaped the a t t e n t i o n of the perfume i n d u s t r y where racemic aldehyde _53 has been produced on a g r e a t e r than one ton per year  basis  9 9  .  m/z 82 m/z 55  m/z 69  Scheme 5.  I n t e r p r e t a t i o n of the mass s p e c t r a l fragmentation of 2,6-dimethyl-5-heptenal ( 5 3 ) .  The more p o l a r m e t a b o l i t e from M. leonina molecular formula of C 9 H 1 6 0 2 required  (HRMS, m/z observed  156.1151) and i t s 'H NMR  spectrum  was c l o s e l y r e l a t e d to aldehyde 5_3. -1  (3500 - 2200 and 1700 cm )  had a 156.1151,  i n d i c a t e d that i t  IR a b s o r p t i o n bands  c h a r a c t e r i s t i c of a c a r b o x y l i c  53 a c i d and the absence of an aldehyde proton i n the 'H NMR spectrum suggested that the p o l a r m e t a b o l i t e was 2, 6-dimethyl-5-heptenoic a c i d showed fragment  (5_4) .  The mass spectrum  ions at m/z 83 (100%) and 82 ( 4 6 % ) , as w e l l  as intense peaks at 69, 67, 55, and 41 i n f u l l the proposed s t r u c t u r e . diazomethane  support of  Treatment of 54 with e t h e r e a l  generated the methyl e s t e r 5J7, c o n f i r m i n g the  presence of the c a r b o x y l i c a c i d moiety.  Although a c i d 5_4  had not p r e v i o u s l y been i s o l a t e d as a n a t u r a l product* i t i s a known s y n t h e t i c compound  100  .  3. DISCUSSION To the best of the author's knowledge, i s o l a t i o n of aldehyde 5_3 and a c i d 54 represent only the second examples of  i s o l a t i o n of nonhalogenated monoterpenes  degraded monoterpenoids)  from a marine  (granted  invertebrate.  * Subsequent to the p u b l i c a t i o n of our paper d e s c r i b i n g the M. leonina m e t a b o l i t e s , Burger et al. r e p o r t e d a c i d 54 as a c o n s t i t u e n t from the sex a t t r a c t i n g s e c r e t i o n of the dung b e e t l e Kheper  lamarchi.  54 Halogenated monoterpenoids are w e l l known from the herbivorous sea hares  (Aplysia  s p p . ) , and were  t r a c e d to t h e i r red a l g a l d i e t s . sponge Plakortis  zygompha  The pleasant s m e l l i n g  produces ( p o s s i b l y by  b i o g e n e s i s ) two degraded monoterpenes 58 and which c o n t a i n h a l o g e n s the halogenated and  101  subsequently  .  The  symbiotic  5jJ, n e i t h e r of  b i o l o g i c a l s i g n i f i c a n c e of  monoterpenoids ( i s o l a t e d from the red algae  sea hares) and the degraded monoterpenes from the sponge  are unknown.  Many authors suggest  that the compounds are  i n v o l v e d i n the organisms' apparent r e l i e f  from p r e d a t i o n .  Much l i k e the tannins i s o l a t e d from t e r r e s t r i a l t r e e s and shrubs, perhaps the halogenated  monoterpenoids are a general  l e v e l d e f e n s i v e mechanism to which only s p e c i a l i s t s  (sea  hares) have adapted. In view of the p o s t u l a t e d d e f e n s i v e r o l e f o r the 91  o d i f e r o u s compound , we activity  t e s t e d 53 and  54 f o r a n t i f e e d a n t  in a standard g o l d f i s h b i o a s s a y  36  .  The  a c i d 54 showed no a c t i v i t y at 100 wg/(mg of food while the aldehyde 5_3 was  too v o l a t i l e  carboxylic pellet),  for r e l i a b l e  testing.  I t would be an i n t e r e s t i n g b i o l o g i c a l p r o j e c t to s y n t h e s i z e racemic  2,6-dimethyl-5-heptenal  (53) and e v a l u a t e i t s  t o x i c i t y and a n t i f e e d a n t a c t i v i t y a g a i n s t a v a r i e t y of p o t e n t i a l nudibranch  predators.  It i s q u i t e p o s s i b l e that e i t h e r aldehyde 5_3, a c i d or the n a t u r a l l y o c c u r r i n g mixture aggregation or sex pheromones. gregate  act as M.  M. leonina  54,  leonina  i s known to ag-  i n great numbers and to then suddenly  completely  55 d i s a p p e a r , only to reappear weeks or months l a t e r  9 1  .  It i s  tempting t o suggest that the major l y p o p h i l i c compounds i s o l a t e d from the skin e x t r a c t s of t h i s animal are i n v o l v e d in the dynamic p o p u l a t i o n  jumps observed.  More  c o l l a b o r a t i o n with marine b i o l o g i s t s w i l l be needed to t e s t these e c o l o g i c a l  hypotheses.  58  59  56 D. SECONDARY METABOLITES FROM ACANTHODORIS  NANAIMOENSIS  (O'DONOGHUE, 1921)  1. INTRODUCTION The chemical s t u d i e s  on Acant  hodoris  nanaimoensis  (see  F i g u r e 8 ) , prompted by an o b s e r v a t i o n that the nudibranch had a f r a g r a n t odour, were i n i t i a t e d by J o c e l y n T. Hellou  1 0 2  .  The a s s o c i a t i o n  nudibranch  of odour with  interesting  chemistry was w e l l known from the p i o n e e r i n g  s t u d i e s on Phyllidia  varicosa  mentioned i n the preceeding  chapter and were supported  by the i s o l a t i o n of luteone (60)  from Cadii  nata .  na Iuteomargi  103  H e l l o u had shown that the o d o u r i f e r o u s p r i n c i p l e c o u l d be e f f e c t i v e l y e x t r a c t e d from the whole animals the specimens i n methanol immediately  by immersing  after c o l l e c t i o n ,  soaking a t room temperature f o r three days, and then d e c a n t i n g the s u p e r n a t a n t . in  vacuo  The supernatant  was evaporated  t o give a c o n c e n t r a t e d MeOH-H20 s o l u t i o n  partitioned  between b r i n e and c h l o r o f o r m .  that was  The orange  58 organic  phase was  d r i e d over anhydrous sodium s u l f a t e  evaporated to give a sweet s m e l l i n g o i l y Silica  thin  residue.  l a y e r chromatography (TLC)  a n a l y s i s of  crude e x t r a c t showed the presence of a non-polar designated  10  as n a n a i m o a l ' , that was  p r e p a r a t i v e TLC.  The  1  H NMR  spectrum of the  The  aldehydic  major component appeared at 6 9.83 analogous proton 9.71  by  purified closely  resonance of  ( t , / = 3. Hz)  and  the the  of the minor c o n s t i t u e n t resonated at 6  ( t , J = 3 Hz)  a s s o c i a t i o n with  proton  the  metabolite,  readily purified  odouriferou's o i l i n d i c a t e d the presence of two related aldehydes.  and  ppm.  Subsequent s p e c t r a l a n a l y s i s i n  biogenetic  reasoning  allowed H e l l o u to  suggest three p o s s i b l e s t r u c t u r e s f o r nanaimoal, the major component: namely 61, 62 or  63.  S p e c t r a l arguments d i d not allow an unambiguous c h o i c e of any  of the above s t r u c t u r e s f o r nanaimoal, t h e r e f o r e  H e l l o u prepared d e r i v a t i v e s of the molecule i n hopes that a s u i t a b l e c r y s t a l l i n e compound c o u l d be found for s t r u c t u r a l  59 e l u c i d a t i o n by s i n g l e c r y s t a l X-ray d i f f r a c t i o n  analysis.  Of the e i g h t d e r i v a t i v e s prepared, none p r o v i d e d crystals.  At t h i s p o i n t Ms.  H e l l o u wrote up her  p r e l i m i n a r y s t u d i e s on A. nanaimoensis the  project  1 0 2  suitable  and ceased work on  .  Subsequent work on A. nanaimoensis^ '^ 05  that the TLC spot corresponding f i v e c l o s e l y r e l a t e d compounds.  06  has shown  t o nanaimoal i s a mixture of The s t r u c t u r e s of the three  most abundant m e t a b o l i t e s have been solved and were shown to be the isomeric aldehydes:  nanaimoal (61), acanthodoral  ( 6 4 ) , and i s o a c a n t h o d o r a l (65) (see F i g u r e 9 ) * .  The s t r u c -  t u r e s of the two l e a s t abundant m e t a b o l i t e s have not yet been deduced.  2. NANAIMOAL > In February, 1982, a c o l l e c t i o n  (SCUBA, depths of 1 to  10 m, Barkley Sound) of 120 specimens of A. nanaimoensis  was  worked up f o l l o w i n g H e l l o u ' s procedure to p r o v i d e 2.8 gms (23 mg/animal) of an orange o i l that contained the characteristic  nudibranch  fragrance.  presence of a non-polar  spot  A n a l y t i c a l TLC showed the  (Rj- 0.25; 50% Hexane/CHCl 3 )  that c h a r r e d l i g h t p u r p l e and gave a p o s i t i v e t e s t 2,4-dinitrophenylhydrazine. other non-polar  In a d d i t i o n t o t h i s s p o t , three  and one p o l a r spots were a l s o  These were subsequently  with  present.  shown t o be f a t s and s t e r o l s .  * The a b s o l u t e s t e r e o c h e m i s t r i e s of the A. m e t a b o l i t e s are as shown.  nanaimoensis  60  F i g u r e 9. nanaimoens  M a t e r i a l which remained at the o r i g i n run  Acanthodoris  Secondary M e t a b o l i t e s from i s.  in a l l the TLC p l a t e s  (brown c h a r , p o s s i b l y f a t t y a c i d s ) was  not i n v e s t i g a t e d  further. Column chromatography y i e l d e d 218.6  mg  (silica  (1.8 mg/animal) of m a t e r i a l  to the f r a g r a n t aldehyde f r a c t i o n Hz);  6 9.72  ( t , / = 3 Hz)  a n a l y s i s , Figure compounds.  for  1  [ H NMR Gas  corresponding  6 9.84  (t, / = 3  chromatographic  (GC)  10, i n d i c a t e d the presence of three major  (65,  the remaining  seen.  ppm].  These were designated  isoacanthodoral  g e l ) of the crude e x t r a c t  two  as nanaimoal (61,  2 0 % ) , and acanthodoral  (64,  79%),  1%).  Peaks  l e a s t abundant m e t a b o l i t e s were a l s o  The mixture c o u l d r e a d i l y be separated  by p r e p a r a t i v e  61  GC or by high performance l i q u i d chromatography  (HPLC).  For  l a r g e s c a l e p u r i f i c a t i o n HPLC was found to be the most convenient .  Figure  10.  GC a n a l y s i s of a crude c h l o r o f o r m e x t r a c t of A.  Nanaimoal  nanai  mo ens  is.  (6_1) had a molecular formula of C i 5 H 2 « 0  (HRMS, m/z observed 220.1836, r e q u i r e d 220.1827). infrared  The  (IR) spectrum of 61_ showed a l d e h y d i c C-H and C=0 1  s t r e t c h i n g bands at 2750 and 1710 cm"  respectively.  The  assignment of the lone oxygen i n nanaimoal to an a l d e h y d i c c a r b o n y l was supported by a resonance at 6 203.3 (d) ppm i n its  13  C NMR  spectrum.  Partial  s t r u c t u r e 66 was 1  from an ABX spin system i n the H NMR  inferred  spectrum ( F i g u r e 11);  the a l d e h y d i c proton at 8 9.83 ( t , J = 3.2 Hz, 1H) ppm was coupled t o protons at 6 2.29 (dd, J = 14.5,3.2 Hz, 1H) and 2.24  (dd, J = 14.5,3.2 Hz, 1H) ppm.  In a d d i t i o n , an intense  62 ion at m/z 176 i n the mass spectrum of nanaimoal,  fragment resulting  from l o s s of ethanal v i a a M c L a f f e r t y  rearrangement, supported t h i s assignment  Scheme 6.  1  The  I n t e r p r e t a t i o n of the MS of nanaimoal (61)  H NMR spectrum of nanaimoal a l s o  resonances f o r a gem-dimethyl 1  (Scheme 6 ) .  1380 cm" )  contained  a t 6 0.98 ( s , 6H) (IR 1395 and  ( s u b s t r u c t u r e 6 7 ) , an i s o l a t e d t e r t i a r y methyl at  1  Figure 11. 400 MHz H NMR spectrum of nanaimoal (61) i n CDC1 3 . a) e n t i r e spectrum, b) expansion of the a l i p h a t i c r e g i o n , c) expansion of the aldehydic proton resonance. CO  64 1.05 ( s , 3H) ( s u b s t r u c t u r e 6_8), an i s o l a t e d a l l y l i c  AB spin  system at 5 1.77 ( d , / = 17.3 Hz, 1H) and 1.85 ( d , / = 17.3 Hz, 1H) ( s u b s t r u c t u r e 6_9) , four a l l y l i c  protons at 1.81  (m,  2H) and 2.02 (m, 2H) ppm, and a s i x proton a l i p h a t i c m u l t i p l e t s i t u a t e d between 8 1.41 and 1.66 ppm.  The  13  C  NMR  spectrum of aldehyde 61 c o n t a i n e d resonances at 6 133.8 and 123.3  (both- s i n g l e t s i n a SFORD experiment) a p p r o p r i a t e f o r  a tetrasubstituted  olefin.  The s p e c t r a l data i n d i c a t e d that nanaimoal was a bicyclic  s e s q u i t e r p e n o i d that c o n t a i n e d three  tertiary  methyl groups, an ethanal s i d e c h a i n , a t e t r a s u b s t i t u t e d o l e f i n , and s i x a l l y l i c i s o l a t e d AB spin system.  protons of which two comprised an The presence of s i x a l l y l i c  protons r u l e d out 62 as a p o s s i b l e s t r u c t u r e f o r nanaimoal. The remaining s t r u c t u r a l f e a t u r e s to be d e f i n e d were the pos i t i o n s of the three a d d i t i o n a l a l i p h a t i c methylene c a r b o n s , the  s i z e of the b i c y c l i c r i n g system and the s u b s t i t u t i o n  p a t t e r n of s u b s t r u c t u r e s 66, 6_7, 68, and 69. The mass spectrum of nanaimoal m/z  (6_1) showed an ion at  122 ( C 9 H 1 3 ) which suggested fragmentation i n v o l v i n g the  l o s s of a methyl group f o l l o w e d by a r e t r o D i e l s - A l d e r r e a c t i o n as shown i n Scheme 6.  The fragment ion was much  more intense i n the mass spectrum of nanaimool 10.4°  (70) [ a ] D +  (prepared from nanaimoal by NaBHu r e d u c t i o n ) and  allowed measurement of i t s exact mass (HRMS, m/z observed 121.1016, r e q u i r e d f o r C 9 H 1 3 , 121.1017).  I t was concluded  from t h i s r e s u l t that the r i n g system i n nanaimoal must be  65 b i c y c l o [ 4.4.0]dec-1 (6)-ene  (7_1).  consistent  with only four p o s s i b l e  namely 61,  63,  12 or  The  s p e c t r a l data  structures  was  f o r nanaimoal;  73.  71  72 Spectroscopically, differentiating possible  structures  spectroscopy was Particularly  useful  in e l i m i n a t i n g  structures the  s i m i l a r i t y between 61 and  favour of  1.  structure  structure  Biogenetic  61  f o r nanaimoal.  'H  NMR  7_2 and  73.  inherent  63_.  I t was  b l e , at t h i s p o i n t , to b u i l d an extremely strong as the c o r r e c t  four  f o r nanaimoal proved c h a l l e n g i n g .  i n t e r e s t i n g however, was  spectroscopic  between the  The  possi-  case f o r 6_1  arguments in  f o r nanaimoal were:  reasoning.  Working with the assumption that a sesguiterpenoid  (the presence of  nanaimoal  15 carbons  was  and  66 only one  oxygen made the p o s s i b i l i t y that nanaimoal  was  an acetogenin  fit  the b i o g e n e t i c  non-biogenetic  remote) only s t r u c t u r e s JS1 and isoprene  rule  1 0 7  ,  6_3  therefore  i s o p r e n o i d compounds T2 and  7_3 c o u l d  be e l i m i n a t e d from c o n s i d e r a t i o n on t h i s b a s i s . Numerous s e s q u i t e r p e n o i d m e t a b o l i t e s  have been d i s 1 0 6  covered that v i o l a t e the  isoprene  rule  [see for  example; i s o a c a n t h o d o r a l  (6j>) and  9-isocyano-  pupukeanane ( 1 6 ) ] , however, on c l o s e r i n s p e c t i o n these m e t a b o l i t e s being  can  u s u a l l y be r a t i o n a l i z e d  formed from a b i o g e n e t i c  isoprenoid  namely f a r n e s y l pyrophosphate, and by d e f i n i t i o n , the b i o g e n e t i c  as  precurser,  t h e r e f o r e obey,  isoprene  rule.  Scheme 7 o u t l i n e s the b i o g e n e t i c arguments used in support of e i t h e r s t r u c t u r e £ 1 or s t r u c t u r e 6_3 for nanaimoal.  There were b i o g e n e t i c precedents f o r  the proposed f i r s t c y c l i z a t i o n of both compounds. d e r i v e d from an  step in the  S t r u c t u r e 61 c o u l d be  intermediate  belonging  formation rationally  to the  known monocyclofarnesane f a m i l y (Scheme 7a), s t r u c t u r e 6_3 c o u l d be d e r i v e d from an that had  the carbon s k e l e t o n of the  metabolite  pleraplysillin-1  proposed second c y c l i z a t i o n  (7_4)  well while  intermediate  sponge  (Scheme 7b).  The  r e a c t i o n i n the  formation  of s t r u c t u r e 61  i s analogous to  formation  of r i n g C i n the pimerane  B i o g e n e t i c a l l y , s t r u c t u r e 61 was  the  diterpenes  109  the most l i k e l y  .  67 s t r u c t u r e f o r nanaimoal.  The l a r g e number of  n a t u r a l l y o c c u r r i n g monocyclofarnesane s e s q u i t e r p e n o i d s o c c u r r i n g i n both t e r r e s t r i a l and marine organisms vs only one example of a m e t a b o l i t e r e l a t e d t o the p l e r a p l y s i l l i n - 1 this  s k e l e t o n supported  contention.  74  Scheme 7.  B i o g e n e t i c arguments used i n support of s t r u c t u r e 61 f o r nanaimoal.  68 2.  1  H NMR  Double-resonance experiments. 1  In the H NMR  of nanaimoal, s e l e c t i v e  i r r a d i a t i o n of the a l l y l i c 2.02  (see F i g u r e  methylene protons at 6  11) i n a double-resonance  experiment caused a s i m p l i f i c a t i o n of the broad geminal a l l y l i c  methylene proton m u l t i p l e t at 5  1.81, an i n d i c a t i o n  of h o m o a l l y l i c c o u p l i n g * .  This  r e s u l t supported the e l i m i n a t i o n of h y p o t h e t i c a l s t r u c t u r e s 12 and 73 from c o n s i d e r a t i o n the  same c o n c l u s i o n reached by b i o g e n e t i c reasoning)  as c o u p l i n g between these two a l l y l i c the  (supporting  methylenes, i n  analogous spin systems, was p r e d i c t e d to be  negligible.  3.  Chemical s h i f t of the gem-dimethyl The chemical s h i f t , 6 0.98  group.  ( s , 6H) ppm,  and  chemical s h i f t e q u i v a l e n c e of the gem-dimethyl in nanaimoal functionality  (6_1) ( s u b s t r u c t u r e 6_7) i n d i c a t e d was  Comparison  systems supported t h i s assignment chemical s h i f t assignment of the was  this  a t t a c h e d to the t e t r a s u b s t i t u t e d  carbon-carbon double bond.  functionality  group  to model  (Figure  12).  gem-dimethyl  supported by the o b s e r v a t i o n  upon r e d u c t i o n to nanaimool s h i f t s of the gem-dimethyl  The  that  (7J3), the chemical groups were only  * Decoupling experiments o u t l i n e d i n a subsequent allowed the assignment of these p r o t o n s .  section  69 marginally  affected.  On  the other hand, the  chemical s h i f t of the t h i r d quaternary methyl ( s u b s t r u c t u r e 68) u p f i e l d by  6 0.17  s u b s t a n t i a l l y a f f e c t e d (moved  ppm).  Unfortunately be  was  no reasonable model system could  found f o r the chemical s h i f t s of a gem-dimethyl  group in a s t r u c t u r e of type 6_3 that would r e i n f o r c e t h i s argument. 4.  Reduction of the a l d e h y d i c  carbonyl.  Reduction of the a l d e h y d i c  carbonyl a f f e c t e d  the chemical s h i f t of the quaternary m e t h y l , as mentioned above, as w e l l as causing (average + 0.14  ppm)  of the  isolated  methylene protons ( s u b s t r u c t u r e 69).  an u p f i e l d  shift  allylic This  result  i n d i c a t e d a c l o s e s p a t i a l r e l a t i o n s h i p between the c a r b o n y l and Of  the  isolated a l l y l i c  methylene  protons.  the s t r u c t u r e s remaining under c o n s i d e r a t i o n ,  namely 6_1 and ef f e c t .  63_, only 61 c o u l d account f o r such an  70  6 1.00 ( s , 6H)  Figure  1  12. Model systems f o r the H NMR chemical s h i f t s of the gem-dimethyl group i n nanaimoal.  3. SYNTHESIS OF NANAIMOAL'S (P-BROMOPHENYL)URETHANE DERIVATIVE.  ASSIGNMENT OF STRUCTURE  The s t r u c t u r e of nanaimoal (61) had been p o s t u l a t e d using a combination of s p e c t r a l and b i o g e n e t i c reasonings as discussed  i n the p r e v i o u s  section.  s p e c t r o s c o p i c or degradative  In the absence of any  scheme that would  unambigously  allow assignment of either* s t r u c t u r e 61 or 63 t o nanaimoal,  71  s y n t h e s i s of the molecule r e p r e s e n t i n g  the most l i k e l y  s t r u c t u r e , compound 6 1 , was  The  in o r d e r .  d e r i v a t i v e of nanaimoal, was s i n c e t h i s d e r i v a t i v e had  urethane 75, a  chosen as a s y n t h e t i c  already  target  been prepared in an  attempt to generate a s u i t a b l e c r y s t a l l i n e d e r i v a t i v e f o r X-ray a n a l y s i s .  H  75  The  most l i k e l y  s t r u c t u r e f o r nanaimoal (6_1) had  a  r e l a t i v e l y simple carbon s k e l e t o n , t h e r e f o r e r e t r o s y n t h e t i c a n a l y s i s provided (Scheme 8 ) . followed  by  78  110  synthetic  A n t i t h e t i c cleavage of the C-4  strategy  to C-5  bond  f u n c t i o n a l group i n t e r c o n v e r s i o n generated  compound 7_6. shown to be  a straightforward  The facile  (Scheme 9 ) .  c o r r e s p o n d i n g s y n t h e t i c r e a c t i o n had i n the conversion Retrosynthetic  of t r i e n e 7_7 to diene  a n a l y s i s of compound 7_6  r e v e a l e d a simple D i e l s - A l d e r r e a c t i o n between myrcene and  a d i e n o p h i l e of type 80,  The  reduced to f i n d i n g the a p p r o p r i a t e  the d i e n o p h i l e  initially.  (79)  f o r example, would generate  required c y c l i z a t i o n precurser.  Methyl methacrylate  been  problem was  thus  dienophile.  (81L) seemed a l o g i c a l choice However, examination of  the  for  the  72  Scheme 8. R e t r o s y n t h e t i c  literature  1 1 1  a n a l y s i s of the p o s t u l a t e d s t r u c t u r e f o r nanaimoal.  (as well as t h e o r e t i c a l  considerations)  revealed t h a t t h i s r e a c t i o n would l i k e l y provide  a good  y i e l d of the wrong r e g i o i s o m e r .  regioisomer  could be o b t a i n e d carbonyl  112  ,  I f the c o r r e c t  the n e o p e n t y l nature of the e s t e r  would pose a problem i n any r e a c t i o n s designed to  extend the s i d e chain by the r e q u i r e d one c a r b o n  113  .  These  73 two  f a c t o r s r u l e d out  Scheme 9.  81 as a s u i t a b l e d i e n o p h i l e .  Previous  Isoprene ( B 2 ) had generating 2:3  1 10  s y n t h e s i s of the nanaimoane carbon skeleton.  been shown to react with myrcene,  a mixture of regioisomers  , i n low y i e l d * .  77 generated diene 7_B.'  7J7 and  As d i s c u s s e d above, c y c l i z a t i o n a  To  f i n i s h a synthesis via this  r o u t e , s e l e c t i v e hydroboration  of the monosubstituted o l e f i n  would be r e q u i r e d to generate a l c o h o l JSL' ( i ) - a l d e h y d e 6_1.  quence suffers,, frorn one  o x i d a t i o n of which  T h i s o v e r a l l r e a c t i o n se-  s e r i o u s drawback, a p l e t h o r a of  products were shown (and were expected) to be r e a c t i o n of myrcene and  of  compound having the r e q u i r e d carbon  s k e l e t o n of s t r u c t u r e 6_1.  should provide  83 in a r a t i o of  isoprene  r e l a t i v e l y easy to vacuum d i s t i l l  110  .  formed upon  Although i t would be  the mixture and  somewhat p u r i f i e d mixture of o l e f i n s 11_ and  8_3,  obtain a  i t was  * I t was r e a l i z e d from the outset that unambiguous s y n t h e s i s of s t r u c t u r e 61 or i t s d e r i v a t i v e was the goal of the s y n t h e s i s , t h e r e f o r e high y i e l d was not an a b s o l u t e requirement.  74  reasoned that a r e a d i l y  a v a i l a b l e d i e n o p h i l e such as  3-methyl-3-buten-1 - o l (8J))  would give a c l e a n e r r e a c t i o n  product that c o u l d be e a s i l y p u r i f i e d by chromatography.  The  flash  a l c o h o l 80 c o u l d be used in l a r g e  cess to suppress the appearance of products a r i s i n g d i m e r i z a t i o n of myrcene.  It was  with  isoprene  In the e v e n t  11  from the  hoped that 80 would give a  r a t i o of r e g i o i s o m e r i c products 7_6 and obtained  ex-  84 s i m i l a r  to that  as the d i e n o p h i l e , that i s  2:3.  * , the D i e l s - A l d e r r e a c t i o n (225 °C  sealed t u b e , 8 h, neat, 4:1  alcohol:myrcene) proceeded i n a  very  ( p o l y m e r i z a t i o n of  low  but adequate y i e l d  m a t e r i a l s was step a f t e r  starting  the major s i d e r e a c t i o n ) to generate, i n  p u r i f i c a t i o n by f l a s h chromatography, a mixture  of regioisomers  that occurred  Separation  i n a r a t i o of 2:3  (76:84).  of the r e g i o i s o m e r i c a l c o h o l s proved  moderately d i f f i c u l t , however, adequate p u r i f i c a t i o n be achieved  by r e c y c l i n g r a d i a l TLC  EtOAc/petroleum e t h e r ) .  The  1.68  (Chromatotron:  could 12%  major isomer 84 d i s p l a y e d peaks  for the t h r e e methyl groups at 6 0.91 3H), and  one  ( s , 3H),  (bs, 3H), c a r b i n o l protons at 3.73  1.60 (m,  (bs, 2H)  and  75 two  (tm, 3 = 1  o l e f i n i c protons at 5.08  (bs, 1H) ppm. at 6 0.92 2H), and  1.60  The C-2  1H) ppm.  1  H NMR  1H)  1.68  (m,  f o r the methyl groups, respective-  r i n g proton resonated at 6 5.35  (bs,  s p e c t r a l data were very s i m i l a r f o r  each r e g i o i s o m e r , the major d i f f e r e n c e was s h i f t s of the cyclohexene 84 vs 5 5.35  5.29  (bs, 3H), 3.73  s i d e chain o l e f i n i c proton  olefinic  The  (bs, 3H),  ( b t , J = 1 Hz,  c a r b i n o l protons and ly.  1H) and  The minor isomer 76 d i s p l a y e d s i m i l a r peaks  ( s , 3H), 5.08  Hz,  i n the  r i n g o l e f i n i c protons  chemical  (6 5.29  for  f o r 76) .  With the mixture  of r e g i o i s o m e r i c a l c o h o l s i n hand,  methods f o r a f f e c t i n g the formation of the r e q u i r e d C-4  to  C-5  side  bond were e x p l o r e d .  The  chain h y d r o x y l group was  n u c l e o p h i l i c nature of the  expected  to pose a problem i n any  c y c l i z a t i o n r e a c t i o n that generated c a r b o c a t i o n c h a r a c t e r at C-10, and  l e a d i n g to the undesired formation of c y c l i c e t h e r s 8_5  £36.  P r e l i m i n a r y c y c l i z a t i o n experiments proved  concern  to be warranted.  mixture  of a l c o h o l s 76 and  Attempted c y c l i z a t i o n of the 84 using BF 3 •EtOEt i n r e f l u x i n g  anhydrous ether f a i l e d to generate c y c l i z e d products 7_0 or j5_7. assumed to be a mixture  any  of the d e s i r e d  I n s t e a d , a non-polar  of ethers Ji_5 and  with t o t a l consumption of the s t a r t i n g  86 was  alcohols  group would be r e q u i r e d .  compound,  produced 1 1 5  To a l l e v i a t e t h i s problem, p r o t e c t i o n of the  considered  this  . hydroxyl  A number of p r o t e c t i n g groups were  ( f o r example, the a c e t y l or benzyl  esters),  however, to f a c i l i t a t e p u r i f i c a t i o n of the i n t e r m e d i a t e s  (UV  76  chromophore) and to allow d i r e c t comparison of the p r o t e c t e d , c y c l i z e d , s y n t h e t i c products to the d e r i v a t i z e d n a t u r a l product* the (p-bromophenyl)urethane was chosen as the p r o t e c t i n g The German w o r k e r s  110  functionality  group.  had c y c l i z e d the mixture of  o l e f i n s 77 and 8_3 with formic a c i d  (85%, 12 h r , 100 °C) to  give a 48% y i e l d of c y c l i z e d products 7j5 and 88. a c i d c y c l i z a t i o n was  Formic  t h e r e f o r e attempted on the mixture of  urethanes 89 and 90, prepared by r e a c t i n g the a l c o h o l s 7_6 and 84 with 4-bromophenyl i s o c y a n a t e .  Heating the mixture  * One of the d e r i v a t i v e s of nanaimoal prepared was the p-bromophenylurethane d e r i v a t i v e 75. I t f a i l e d t o provide c r y s t a l s s u i t a b l e f o r s i n g l e - c r y s t a l X-ray a n a l y s i s from a l l the s o l v e n t s t r i e d . D e r i v a t i v e 7_5 was q u i t e s t a b l e and c o u l d be s t o r e d at 4 °C, i n the d a r k , without s i g n i f i c a n t decomposition.  77 of urethanes i n 98-100% formic a c i d at 60 °C f o r 12 hours gave one major, n o n - p o l a r , TLC spot that had an i d e n t i c a l Rj to the d e r i v a t i z e d n a t u r a l product 7_5. products  Two  more p o l a r  ( p o s s i b l y formate e s t e r 91 and t e r t i a r y a l c o h o l  92)  and brown, l i k e l y p o l y m e r i c , m a t e r i a l that remained at the o r i g i n of the p r e p a r a t i v e TLC plate.were a l s o found in minor amounts.  The more p o l a r products were not  investigated  further.  92  R =H 400 MHz  product was  'H NMR  a n a l y s i s of the major, non-polar,  devoid of resonances f o r o l e f i n i c protons and  o l e f i n i c methyl groups, and showed the appearence new  a l i p h a t i c methyl resonances.  the non-polar product was (±)-7J5 and 93.  This result  indicated  a mixture of c y c l i z e d  Comparison of the new  methyl  resonances to those of d e r i v a t i z e d nanaimoal  of four that  urethanes  singlet 75 showed a  d i r e c t correspondence between those of the s y n t h e t i c minor  78 regioisomer to those of the d e r i v a t i z e d n a t u r a l product Encouraged  by t h i s r e s u l t , a t t e n t i o n was  assignment  of s t r u c t u r e to the u n c y c l i z e d  urethanes  75.  turned to the regioisomeric  (89 and 90), to be f o l l o w e d by c o n v e r s i o n of pure  regioisomer 89 to d e r i v a t i z e d  nanaimoal.  D e r i v a t i z a t i o n of the major r e g i o i s o m e r i c a l c o h o l j}_4 with 4-bromophenyl isocyanate p r o v i d e d urethane 90 i n good yield.  A n a l y s i s of the 400 MHz  *H NMR  a broadened AB spin system at 6 1.77 and  (bd, J = 17.6 Hz,  8 1.90  on C-3.  spectrum of 90 showed (bd, / = 17.6 Hz,  1H) due  to the a l l y l i c  1H)  protons  Most of the broadening c o u l d be removed by  irradiation  of the C-2  cyclohexene proton at 6 5.29.  c o n f i r m the c o u p l i n g was  v i c i n a l and not a l l y l i c  To  (as would  be the case f o r regioisomer 89) a d i f f e r e n c e nuclear Overhauser  enhancement (nOe)  I r r a d i a t i o n of the C-2 for  90 at 6 5.29  allylic on C-3  experiment  performed.  cyclohexene o l e f i n i c proton observed  resulted  in an nOe  p r o t o n s on carbons C-3 appeared  was  enhancement of the four  and C-7.  The a l l y l i c  (bd, J =  as two broadened d o u b l e t s at 8 1.77 (bd, J = 17.6 Hz)  17.6 Hz) and  8 1.90  experiment.  Since the major regioisomer c o u l d now  in this difference be  f i n e d as 90, i t f o l l o w e d that the minor regioisomer urethane The  nOe  de-  was  89. 400 MHz  1  H NMR  spectrum of 89 showed resonances f o r  three methyl groups at 8 0.94 1.68  protons  ( s , 3H), 1.60  ( s , 3H), c a r b i n o l protons at 4.24  protons at 5.09  (tm, J = 7 Hz,  ( s , 3H), and  (m, 2H), and  1H) and 5.37  (bs', 1H)  olefinic ppm.  79 Resonances f o r the urethane moiety were observed at 6 6.51 (bs, 1H, NH), 7.26 Hz, 2H). fragment  ( d , / = 8.4 Hz, 2H) and 7.40  ( d , / = 8.4  In the mass spectrum of urethane 89, intense ions were observed at m/z 204 (48%), 136 (22%), 69  (100%), 55 (52%) and 41 ( 9 0 % ) . c o u l d a r i s e from a M c L a f f e r t y  The fragment  ion at m/z 204  fragmentation of the type  shown i n Scheme 10, while the i o n at m/z 136 (HRMS, m/z observed 136.1239, r e q u i r e d f o r C 1 0 H 1 6  136.1252) l i k e l y r e -  s u l t s from a r e t r o D i e l s - A l d e r r e a c t i o n (Scheme 10). The fragment  ions at m/z 69, 55, and 41 are t y p i c a l of a  4-methyl-3-pentenyl s u b s t i t u e n t .  Scheme 10.  Br  I n t e r p r e t a t i o n of the MS of urethane 89.  Heating urethane 89 i n 98-100% formic a c i d at 60 °C f o r 12 hours accomplished the r e q u i r e d c y c l i z a t i o n t o give urethane  (±)-7_5 i n e x c e l l e n t y i e l d .  i d e n t i c a l by HPLC, MS and 'H NMR  Synthetic  (±)-75 was  comparison to the  80 p-bromophenylurethane d e r i v a t i v e prepared from nanaimoal. Nanaimoal has a new We  s e s q u i t e r p e n o i d carbon s k e l e t o n .  propose to name t h i s s k e l e t o n nanaimoane and  as shown in F i g u r e nanaimoal was  13.  The  i nfra)  13.  of  (64) and  isoacanthodoral  (65)  .  14  Figure  stereochemistry  proposed on the b a s i s of i t s b i o g e n e t i c  r e l a t i o n s h i p to acanthodoral (vide  absolute  to number i t  13  Nanaimoane carbon s k e l e t o n showing the numbering scheme.  81 4. ASSIGNMENT OF THE *H NMR SPECTRUM OF NANAIMOAL USING ONE AND TWO-DIMENSIONAL NMR TECHNIQUES Nanaimoal (6_1) had a new carbon  skeleton, therefore 1  s t u d i e s d i r e c t e d towards the t o t a l assignment of the H and 13  C  NMR s p e c t r a of t h i s novel m e t a b o l i t e were  Close examination (Figure  initiated.  }  of nanaimoal's 400 MHz H NMR spectrum  11) revealed t h a t , because of the f l o p p y nature of  the b i c y c l o f 4 . 4 . 0 ] d e c - 1 ( 6 ) - e n e r i n g system, most of the geminal r i n g methylene protons were chemical lent.  equiva-  Because they were n o t , i n most c a s e s , a l s o magnetic 1  e q u i v a l e n t , the 400 MHz order and consequently  H NMR spectrum was h i g h l y second  complex.  In s p i t e of t h i s  c o m p l i c a t i n g f a c t o r , c a r e f u l use of s e l e c t e d 1  shift  H NMR double-resonance  (decoupling)  one-dimensional  experiments,  two-dimensional homonuclear c o r r e l a t i o n  spectroscopy  (COSY/45), and two-dimensional J spectroscopy (2D 1  ./-resolved) allowed complete assignment of nanaimoal's H NMR spectrum (chemical s h i f t s but not a l l c o u p l i n g cons t a n t s , see Table 5 ) . usefulness niques  116  T h i s study demonstrates the  (and l i m i t a t i o n s ) of s e l e c t e d modern NMR techt o a r e a l problem i n n a t u r a l products  chemistry.  From the outset of t h i s NMR s t u d y , i t was envisaged that upon completion  1  of the H NMR s p e c t r a l  t o t a l assignment of the  13  C NMR spectrum would a l s o be  completed using h e t e r o n u c l e a r spectroscopy  (CSCM*).  assignments,  1  13  H- C  correlation  However, the r e s u l t s of the CSCM  * CSCM = chemical s h i f t c o r r e l a t i o n map  82 experiments c l e a r l y demonstrated that the l a r g e s t  limitation  to o b t a i n i n g a reasonable spectrum was sample s i z e .  Not  enough n a t u r a l m a t e r i a l was a v a i l a b l e f o r a s u i t a b l e CSCM experiment to be adequately c a r r i e d o u t * . proton broad-band  13  (BB) decoupled  Even though a  C NMR spectrum could  r e a d i l y be obtained on 35 mg of urethane 75 with as few as 256 scans [ 3 0 ° f l i p a n g l e , 2.0 s r e l a x a t i o n delay absence of BB proton decoupling  (except during  (RD)], the  acquisition)  in the CSCM experiment d i d not allow a s u i t a b l e spectrum to be o b t a i n e d even a f t e r RD = 2.0 s ) .  18 hours ( A , = 3.3 ms, A  2  = 1.67 ms,  A number of s p e c t r a l parameters must be  o p t i m i z e d to get good CSCM r e s u l t s with a small sample s i z e (namely the RD, and the AT and A  2  values), this  requires  perhaps a number of experiments to be r u n . A l a r g e sample size  i s the s i n g l e most important f a c t o r r e q u i r e d f o r  o b t a i n i n g a s u i t a b l e CSCM spectrum i n a reasonable amount of magnet t i m e . a.  One-Dimensional  One-dimensional nanaimoal  1  1  H NMR Experiments  H NMR experiments were conducted on  ( 6 1 ) , nanaimool  (7fj) and the (p-bromophenyl)-  urethane d e r i v a t i v e 75 which, f o r comparison, was run i n two different  s o l v e n t s , CDC13 and benzene-d 6 .  1  The H NMR data  f o r these four experiments i s summarized i n Table 5, along with s e l e c t e d r e s u l t s from the 2D / - r e s o l v e d experiment on  * Although the s t r u c t u r e of nanaimoal (61) was deduced by s y n t h e s i s , the low y i e l d and t e d i o u s p u r i f i c a t i o n procedure d i d not allow g e n e r a t i o n of racemic urethane 75 on a l a r g e s c a l e (> 10 mg).  83 1  Table 5. H NMR derivatives.  data  (400 MHz)  f o r nanaimoal  chemical  (61)  and  shift a, c  fl  75°  25*  61  1  1 .79 (bm)  1 .77 (bm)  1.81  2  4 5  1 .55- 1 .62 1 .52- 1 .63 1 .56- 1 .66 (m) (m) (m) 1.41- 1 .49 1.41- 1 .48 1.41- 1 .49 (m) (m) (m) --  6  1 .98 (bm)  7  1 .40 ( t , 7)  8  -  9  1 .62 (bd, 1 .53 (bd, 1 .77 (bd, 17) 17) 17)  1 .76  (s)  1 .59 (bd, 17)  1 .76 (bd, 1 .68 (bd, 1 .85 (bd, 17) 17) 17)  1 .84  (s)  10  -  -  -  1 .75 (bd, 17) -  1 1  1 .56 (m)  1 .54 (m)  H on  C#  3  13 14 15  2.02  (m)  1 .23- 1 . 37 1 .44 (m) (m) 1 .56 (m) -  -  d  19  6.51 7.39  19, 21  7.27  a  1 .94 (bm)  (m)  2.24 (dd, 3, 14 .5) 1 .63 (m) 1 .63 (m) 2.29 (dd, 3, 14 .5) 4.23 (m). 4.19 (m) 9.84 ( t , 3) 0.98 (s)<J 1 .02 ( s ) ^ 0.98 ( s ) ^ 0.97 ( s ) ^ 1 .00 (s)<* 0.98 (s) 0.91 (s) 0.84 (s) 1 .05 (s)  12  NH 18,  -  61  fl  CDC1 3 . onto F 2 .  b d  (bs) 5.83 6.83 (d) 7.34 V (d)  c  70  1 .80  (s)  1 .60  (s)  1 .44  (s)  -  -  2. 01  1 .78 (m)  (s)  -  -  1 .97 (bm)  -  2. 22  (s)  2. 29  (s) 3.72  0. 98 0. 98 1 .05  (s)<{ 0.97 ( s ) ^ 0.98 (s) 0.88  (m) (s)^ (s)  d  (s)  (bs)  -  benzene-d 6 . 2D / - r e s o l v e d spectrum, p r o j e c t i o n May be r e v e r s e d .  84 nanaimoal  (6_1).  S e l e c t e d proton s p i n - s p i n decoupling  experiments allowed a number of proton assignments made, and these assignments of  to be  were l a t e r confirmed by the duet  homonuclear two-dimensional  experiments.  I r r a d i a t i o n of the two proton broad s i n g l e t at 6 1.98 (bs,  2H) i n urethane 75 (CDC1 3 ) caused a two proton  ( t , J = 6 Hz, 2H) ppm t o c o l l a p s e to a  centered at 6 1.40 singlet.  triplet  T h e r e f o r e , the two protons r e s p o n s i b l e f o r t h i s  t r i p l e t must be part of a s u b s t r u c t u r e of type 94, a f a c t only accountable f o r i f they were the chemical s h i f t equival e n t C-7 methylene protons ( f o r numbering scheme see F i g u r e 13).  Consequently, the broad m u l t i p l e t at 5 1.98 ppm must  a r i s e from the a l l y l i c  C-6 methylene p r o t o n s .  Upon  i r r a d i a t i o n of the C-6 methylene protons a number of changes were a l s o seen i n the three proton a l l y l i c r e s o n a t i n g between 8 1.72 and 1.84 ppm a c t u a l l y one h a l f of an a l l y l i c  multiplet  ( t h i s m u l t i p l e t was  AB q u a r t e t at 8 1.76  overlapped by a two proton broadened m u l t i p l e t c e n t e r e d at 6 1.79 ppm).  The two proton broadened m u l t i p l e t at 8 1.79  collapsed to a t r i p l e t at  6 1.76 sharpened  q u a r t e t at 6 1.62  (/ = 7 Hz) and the overlapped doublet  up.  The other h a l f of the a l l y l i c  (J = 17 Hz) was a l s o n o t i c a b l y  in t h i s double-resonance  experiment.  both the C-1 8 1.76  [6 1.79 (bm)] and C-9 [6 1.62  (bd, / = 17 Hz) ] a l l y l i c  one double-resonance  sharpened  This result  that the C-6 methylene protons were a l l y l i c a l l y  AB  indicated  coupled to  (bd, / = 17 H z ) ,  proton p a i r s .  experiment, a l l the proton  Thus, using resonances  85 of s u b s t r u c t u r e 95 c o u l d be r e a d i l y a s s i g n e d .  When the 'H NMR  spectrum  of urethane  7!) was obtained i n  benzene~d 6 a number of changes were noted i n the resonances of s p i n system  95.  The C-7 methylene protons were no longer  chemical s h i f t e q u i v a l e n t and a complex m u l t i p l e t centered about 6 1.30 ppm was observed I r r a d i a t i o n of the a l l y l i c  f o r these p r o t o n s .  C-6 methylene protons at 6 1.94  (bm) c o l l a p s e d t h i s m u l t i p l e t t o an AB doublet of d o u b l e t s , a l l o w i n g measurement of the geminal c o u p l i n g constant (J = 13 H z ) .  The i s o l a t e d AB doublet of doublet resonances f o r  the C-9 protons were a l s o s h i f t e d r e l a t i v e t o t h e i r i n CDC13 and now appeared 1.53  at 6 1.68  ( d , / = 17 Hz, 1H) ppm.  position  ( d , / = 17 Hz, 1H) and  The C-1 methylene protons  resonated at 6 1.77 i n benzene-d 6 and were again c o l l a p s e d to a t r i p l e t  (/ = 6 Hz) upon i r r a d i a t i o n of the C-6  methylene protons at 6 1.94.  86 Spin system  1  95 was a l s o c l e a r l y  evident i n the H  NMR  spectrum  (CDC1 3 ) of nanaimoal (6_1) , although d e s h i e l d i n g  effects,  l i k e l y due to the diamagnetic  a l d e h y d i c c a r b o n y l , were noted. i n nanaimoal (61) were c l e a r l y  The C-7 methylene protons d e s h i e l d e d with respect to  t h e i r chemical s h i f t i n urethane chemical s h i f t e q u i v a l e n t .  a n i s o t r o p y of the  7j> and were no-longer  Because the C-7 methylene  resonances overlapped with the resonances of the C-2 and C-3 protons, direct  measurement of the chemical s h i f t s of the  C-7 protons was not p o s s i b l e .  However, i r r a d i a t i o n  C-6 protons at 6 2.01 ppm i n a double-resonance  of the  experiment  c o l l a p s e d the C-7 protons m u l t i p l e t to an AB doublet of doublets.  One C-7 proton resonated at 8 1.44  and the other at 6 1.56 with respect to t h e i r  ( d , / = 16 H z ) .  chemical s h i f t s  ( d , J = 16 Hz)  Also deshielded  i n urethane  7_5 was the  C-9 AB q u a r t e t which i n nanaimoal (61) resonated at 6 1.77 (d,  J  = 17.3 Hz, 1H) and 1.85  (d,  J  = 17.3 Hz, 1H) ppm.  The  chemical s h i f t s of the C-6 and C-1 methylene protons were only s l i g h t l y d e s h i e l d e d with r e s p e c t to the analogous resonances  i n urethane 75 (see Table 5 ) .  With s p i n system  95 c l e a r l y  d e f i n e d , a l l that remained  was assignment of the C-2, C-3, and C-11 methylene p r o t o n s . The chemical s h i f t s of the methyl groups c o u l d be assigned by analogy t o model systems as was p r e v i o u s l y d i s c u s s e d i n S e c t i o n II.D.2.  In nanaimoal, and i n a l l the d e r i v a t i v e s  s t u d i e d , a complex two proton m u l t i p l e t was always observed between 5 1.49 and 6 1.41 ppm.  Irradiation  of the C-1  87 methylene protons at 5 1.79* resonance  experiment  of urethane  75 i n a double  d i d not r e s u l t i n any  s i m p l i f i c a t i o n of  t h i s m u l t i p l e t , t h e r e f o r e , i t c o u l d not be due to the protons. spectrum  Since the m u l t i p l e t was of nanaimoal (6_1)  in p a r t , to the C-11 m u l t i p l e t was It was  C-2 1  a l s o present i n the H  i t c o u l d not be due,  methylene p r o t o n s .  NMR  i n whole or  Therefore, this  a s s i g n e d to the two geminal protons at  C-3.  not p o s s i b l e , because of the second order nature of  t h i s m u l t i p l e t to determine  the exact chemical s h i f t of each  of the C-3  methylene p r o t o n s , however, the 2D  /-resolved  experiment  (to be d i s c u s s e d subsequently) was  able to show  t h a t , i n nanaimoal ( 6 1 ) , s h i f t e q u i v a l e n t and The c o u l d now urethane  the two C-3  protons are chemical  resonate at 6 1.44  l o c a t i o n of the C-2  methylene protons  be e a s i l y e x t r a c t e d from the 75  ppm.  (CDC1 3 ) by e l i m i n a t i o n .  1  H NMR  spectrum  of  They resonated as a  complex two proton m u l t i p l e t between 6 1.62 Again the 2D / - r e s o l v e d experiment  resonances  was  and  6 1.55  useful in assigning  the chemical s h i f t s of these protons i n nanaimoal  (61).  They were chemical s h i f t e q u i v a l e n t and resonated at 8 ppm  (see subsequent s e c t i o n ) .  one-dimensional  1  H NMR  The  experiments  ppm.  1.60  r e s u l t s of the on nanaimoal and i t ' s  d e r i v a t i v e s are summarized i n Table  5.  * As p r e v i o u s l y mentioned, one of the C-9 proton resonances overlapped the m u l t i p l e t a r i s i n g from the C-1 p r o t o n s . Any i r r a d i a t i o n of the C-9 protons would not a f f e c t the argument however.  88 b.  1  Two-Dimensional  H NMR Experiments  The past 5 years has seen two-dimensional NMR spectroscopy  (2D-NMR)* r a p i d l y e x p l o i t e d as a u s e f u l and  powerful t o o l f o r both b i o c h e m i c a l ( s t r u c t u r e  elucidation  and b i o s y n t h e s i s ) and s y n t h e t i c n a t u r a l product s t u d i e s .  As  a review and indepth d i s c u s s i o n of 2D-NMR i s beyond the scope of t h i s t h e s i s , i t w i l l be assumed that most readers are  f a m i l i a r with the b a s i c concepts and theory of 2D-NMR.  Reviews by B e r n s t e i n Bax  116  118  , Benn and G u n t h e r  119  ,  and a book by  , when combined, p r o v i d e a more than adequate  intro-  d u c t i o n t o both the p r a c t i c a l and t h e o r e t i c a l a s p e c t s of 2D-NMR. One  of the s i m p l e s t , and i n f a c t the very  proposed two-dimensional two-dimensional  experiment  117  , was the  homonuclear chemical s h i f t  (COSYt) e x p e r i m e n t  120  .  The experiment  correlation  employs the  r a d i o f r e q u e n c y pulse sequence shown i n F i g u r e The  first  14  121  .  p r e p a r a t o r y p u l s e (90°x) i s f o l l o w e d by the  evolution period detection  first  (t 2 ) .  ( « , ) and the second mixing p u l s e (90°), and The p r e p a r a t o r y p u l s e has constant phase  and the phase of the mixing p u l s e i s incremented  in 90°  steps ( ^ T ) . The r e c e i v e r phase chosen, $ 2 or $ 3 , s e l e c t s the coherence  t r a n s f e r echo or a n t i - e c h o , r e s p e c t i v e l y , and  c a n c e l s the a x i a l peaks a t F , = 0. are  I f two n u c l e i , A and X,  spin c o u p l e d , two s e t s of c r o s s peaks w i l l occur i n the 117  * In 1971, J e e n e r f i r s t suggested the p o s s i b l i t y of two-dimensional NMR experiments, t COSY = c o r r e l a t e d s p e c t r o s c o p y  89  RD - 9 0 ° (x) - J , - 90°(*,) - AQN  RD = r e l a x a t i o n  ( f 2 ; #2  or 4>3)  delay  AQN = a c q u i s i t i o n  Figure  14. Pulse sequence  f o r the homonuclear exper imen,t.  COSY NMR  two-dimensional spectrum centered at ( 5 a , 6 X ) and ( 5 x F ^ ) , A  in a d d i t i o n , diagonal peaks at ( § a , 6 a ) and ( 6 X , 6 X ) w i l l a l s o be s e e n .  T h i s p u l s e sequence r e s u l t s i n a  two-dimensional spectrum (both dimensions showing  proton  chemical s h i f t s ) that i n d i c a t e s c o n n e c t i v i t y p a t t e r n s between coupled p r o t o n s . The COSY/45  122  spectrum of nanaimoal's  urethane d e r i v a t i v e 75 i  s  shown i n F i g u r e  (p-bromophenyl)15.  With  refer-  ence t o Table 5 (proton assignments based on 1D-NMR doubleresonance experiments) most of the expected c o n n e c t i v i t y r e l a t i o n s h i p s are indeed represented by c r o s s peaks i n the COSY/45 spectrum.  The C-1,C-2,C-3; the C-6,C-7; and the  C-9a,C-9b spin systems were r e a d i l y mapped o u t . The low i n t e n s i t i e s of the C-1,C-2 c r o s s peaks may i n d i c a t e a short t r a n s v e r s e r e l a x a t i o n time ( T 2 ) f o r one or both of the C-2 methylene p r o t o n s  123  , or p o s s i b l y , small v i c i n a l  coupling.  90 N o t i c e a b l y absent  i n F i g u r e 15 are c r o s s peaks due to the  long range h o m o a l l y l i c c o u p l i n g s seen i n the 1D doubleresonance experiments.  These c r o s s peaks may be seen  however, upon expansion and dropping of the contour threshold.  F i g u r e 16 shows a s e c t i o n of F i g u r e 15 expanded  and with more contour l e v e l s .  Cross peaks f o r a l l the ex-  pected long range h o m o a l l y l i c c o u p l i n g s are now The COSY/45 experiment  visible.  allowed c o n f i r m a t i o n of the spin  systems and c o u p l i n g p a t t e r n s deduced from the one-dimensional  1  H NMR experiments, however, t h i s  d i d not y i e l d any a d d i t i o n a l assignment  experiment  i n f o r m a t i o n regarding the  of chemical s h i f t s t o the s t r o n g l y coupled C-2  and C-3 methylene p r o t o n s * .  To a s s i s t  i n the assignments of  these p r o t o n s , and to separate the o v e r l a p p i n g proton resonance m u l t i p l e t s f o r c o u p l i n g constant a n a l y s i s , a homonuclear 2D / - r e s o l v e d nanaimoal  1  H NMR experiment  was performed on  (61^).  Homonuclear two-dimensional an NMR experiment  / - r e s o l v e d spectroscopy i s  t h a t , when a p p l i e d to protons f o r example,  a l l o w s s e p a r a t i o n of the chemical s h i f t  information  (8) from  the s c a l a r proton-proton c o u p l i n g s ( / ) . Obviously a u s e f u l 1  technique when a p p l i e d to molecules whose H NMR c o n s i s t s of many o v e r l a p p i n g r e s o n a n c e s spectroscopy has found a p p l i c a t i o n the  1  H NMR  125  spectrum  , 2D / - r e s o l v e d  i n the i n t e r p r e t a t i o n of  s p e c t r a of a number of complex m o l e c u l e s  1  126  .  * An experiment does e x i s t whereby a H NMR COSY spectrum 1 may be o b t a i n e d with what amounts H broad-band 12 1 t o complete d e c o u p l i n g i n the F , d i m e n s i o n ' .  4N 1a,b/2a,b 9a/9b^o  |e3a.b/2a.b  %  ess*6a,b/7a,b  2.0  1.8  1.6  1.4  1.2  1.0  0.8  ppm  F i g u r e 15. 400 MHz 'H NMR COSY/45 spectrum of nanaimoal' (p-bromophenyl)urethane d e r i v a t i v e (not symmetrized).  92  F i g u r e 16. Expansion and homoallylic couplings.  amplification  of F i g u r e  15 to show  93 The  pulse sequence used f o r the homonuclear 2D  / - r e s o l v e d experiment i s the simple Hahn spin-echo sequence shown i n F i g u r e  17  127  .  T h i s pulse sequence leads to a  J-modulated spin-echo.  By using a step-wise  of t , the 2D data matrix transformation Vector  incrementation  i s b u i l t up and the F o u r i e r  i n the t , dimension w i l l be a f u n c t i o n of / .  diagrams have been found to be q u i t e u s e f u l as a  conceptual experiment  a i d f o r e x p l a i n i n g the 2D / - r e s o l v e d 128  .  RD - 90°(*,) - 0.5/, - 1 8 0 ° ( $ 2 ) - 0.5*, - AQN($ 3 )  Figure  17.  Pulse sequence f o r 2D / - r e s o l v e d NMR  experiment.  A number of c o m p l i c a t i n g f a c t o r s must be taken consideration /-resolved  1  i n order  H NMR  peak i n t e n s i t i e s ,  to c o r r e c t l y i n t e r p r e t a 2D  spectrum.  These a r e :  1) v a r i a t i o n i n  2) second order e f f e c t s , and  appearance of a r t i f a c t s . below:  into  3) the  These three f a c t o r s are d i s c u s s e d  94  1.  The i n t e n s i t i e s of the peaks i n the 0 ° p r o j e c t i o n *  onto F 2 w i l l vary a c c o r d i n g t o the amount of c o u p l i n g the proton e x p e r i e n c e s .  Thus a h i g h l y coupled  proton  w i l l have a r e l a t i v e l y low i n t e n s i t y while methyl s i n g l e t s w i l l g e n e r a l l y be q u i t e intense (see F i g u r e 18).  Because of t r a n s v e r s e r e l a x a t i o n  ( c h a r a c t e r i z e d by  the s p i n - s p i n r e l a x a t i o n time T 2 ) , the magnitude of the magnetization present at the beginning of a c q u i s i t i o n i n a 2D / - r e s o l v e d experiment w i l l have decreased factor exp(-t!/T2)  by a  i n comparison to the i n i t i a l  m a g n e t i z a t i o n , t h a t i s , a f t e r the 9 0 ° p u l s e . I n t e n s i t i e s of p r o j e c t i o n peaks a r i s i n g with l a r g e T 2 times reduced.  from protons  (fast relaxation) w i l l  thus be  In g e n e r a l , the i n t e n s i t i e s of the peaks w i l l  not a f f e c t the i n t e r p r e t a t i o n of the spectrum, unless of c o u r s e , e i t h e r the i n t e n s i t y drops to zero or a small peak i s obscured  2.  by the t a i l i n g  of a l a r g e one.  Strong c o u p l i n g i n t r o d u c e s a very important c o m p l i c a t i o n i n t o the i n t e r p r e t a t i o n of a homonuclear 2D / - r e s o l v e d 1  H NMR  spectrum  129  '  130  .  The 2D / - r e s o l v e d spectrum of a  s t r o n g l y coupled system w i l l be much more complex than i t s weakly coupled e q u i v a l e n t because many a d d i t i o n a l second order peaks may be p r e s e n t .  F o r t u n a t e l y , second  * P r o j e c t i o n of the t i l t e d 2D / - r e s o l v e d spectrum i s assumed in t h i s d i s c u s s i o n u n l e s s otherwise s t a t e d .  95 order peaks are f r e q u e n t l y not the t i l t e d  spectrum, and  e l i m i n a t e them*. a second order  symmetrization may  Care must be e x e r c i s e d i n i n t e r p r e t i n g  be v e r i f i e d with the a i d of a  two-dimensional s i m u l a t i o n  The  t h i r d complicating  17).  inaccurate  program  If  one  pulse  1 3 2  (see  be p a r t l y e l i m i n a t e d  procedure and  180° refocussing  Artifacts  1 8 0 ° r e f o c u s s i n g pulse  c y c l i n g the phases of p u l s e s and 131  .  / - r e s o l v e d spectrum as a re-  These a r t i f a c t s can  Exorcycle  130  factor i s a r t i f a c t s .  sometimes a r i s e i n the 2D s u l t of an  be used to  2D / - r e s o l v e d spectrum, i f doubt e x i s t s ,  assignments may  3.  symmetrical about F, i n  by  r e c e i v e r using  the  by the use of a composite  .  i s aware of the above l i m i t a t i o n s and  r e c t l y account for them, i n t e r p r e t a t i o n of a 2D spectrum should  Figure  can  cor-  /-resolved  be a r e l a t i v e l y s t r a i g h t f o r w a r d e x e r c i s e .  If the spectrum i s l a r g e l y f i r s t o r d e r , and  the appearence  of a r t i f a c t s can be minimized, a 2D / - r e s o l v e d spectrum can y i e l d a great deal of i n f o r m a t i o n about the s t r u c t u r e of an unknown n a t u r a l p r o d u c t .  * An AB s p i n system g i v e s r i s e to second order peaks that are symmetrical about F, in the t i l t e d spectrum. They give r i s e to a peak midway between A and B in the 0 ° p r o j e c t i o n onto F 2 and cannot be removed by s y m m e t r i z a t i o n .  96 F i g u r e 18 shows the 400 MHz nanaimoal  (61).  I t was  2D ./-resolved spectrum of  r e a d i l y apparent by i n s p e c t i o n of  the F 2 p r o j e c t i o n that most of the protons p r e v i o u s l y 1  a s s i g n e d by i n t e r p r e t a t i o n of the 1D H NMR present.  The C-1  spectrum were  a l l y l i c methylene protons at 5 1.80  were  c l e a r l y r e s o l v e d from o v e r l a p by the C-9 AB doublet of d o u b l e t s (6 1.76 lent.  The two  and 6 1.84)  and were chemical s h i f t  intense peaks at 6 1.60  and 6 1.44  equiva-  c o u l d be  a s s i g n e d to the C-2 and C-3 methylene protons r e s p e c t i v e l y , each methylene p a i r being chemical s h i f t  equivalent.  A number of second order and/or a r t i f a c t peaks were present i n the spectrum. resonance was  The most n o t i c a b l e second order  the peak at 6 2.25, midway between the  resonances a r i s i n g  from the C-11  AB methylene p r o t o n s .  This  peak was expected based on an e x p l a n a t i o n of t h i s second order e f f e c t by B a x  133  .  Other smaller second order and/or  a r t i f a c t peaks c o u l d not be r a t i o n a l i z e d , however, and except f o r obscuring one of the C-7  resonances at 6 1.56  they  d i d not i n t e r f e r e with the i n t e r p r e t a t i o n of the spectrum. S l i c e s of s e l e c t e d 19.  i n d i v i d u a l peaks are shown i n F i g u r e  They c l e a r l y demonstrate the second order c o m p l e x i t y of  the c o u p l i n g s .  Because  i n s t r o n g l y coupled systems the  o b s e r v a b l e resonances may  not be t r u e l y d e p i c t e d i n F , no  i n t e r p r e t a t i o n of the c o u p l i n g s was attempted.  The geminal  c o u p l i n g s of the methylene protons on C-9 and the geminal and v i c i n a l c o u p l i n g s of the methylene protons on C-11  were  r e a d i l y e x t r a c t e d from the spectrum, and t h e i r v a l u e s are  97  - -30  F  2  (PPM)  1  F i g u r e 18. P a r t i a l 400 MHz H NMR 2D / - r e s o l v e d spectrum (symmetrized) of nanaimoal (61) showing (from bottom to top) the contour p l o t and the "proton decoupled" spectrum d e r i v e d from the 2D / spectrum; X denotes second order t r a n s i t i o n s of the 11a, 11b geminal p a i r ; Y denotes other second order and/or a r t i f a c t peaks.  98 listed  i n Table 5.  2 a,b  F i g u r e 19. S l i c e s of i n d i v i d u a l peaks shown at the top of F i g u r e 18 t o show m u l t i p l i c i t i e s .  99  5. ISOACANTHODORAL (65) MS, M+ 220, C 1 5 H 2 f l O c o n s t i t u t e d  Isoacanthodoral 20%  of the s e s q u i t e r p e n o i d aldehyde f r a c t i o n  the s k i n e x t r a c t of Acanthodoris Figure  i s o l a t e d from  nanaimoensis  (see GC t r a c e ,  10). Aldehyde ( £ 5 ) c o u l d be p u r i f i e d by p r e p a r a t i v e  GC or HPLC, however, the small q u a n t i t y of i s o a c a n t h o d o r a l extracted  (400ug/animal), i t s h i g h v o l a t i l i t y , and i t s  s u s c e p t a b i l i t y to o x i d a t i o n ( t o form a c a r b o x y l i c a c i d ) made i s o l a t i o n and c h a r a c t e r i z a t i o n d i f f i c u l t . convenient to convert  isoacanthodoral  c r y s t a l l i n e 2,4-dinitrophenylhydrazone  I t was found most  to e i t h e r i t s d e r i v a t i v e 96 o r ,  a f t e r r e d u c t i o n to a l c o h o l 9 7 , converted  to i t s  (p-bromophenyl)urethane d e r i v a t i v e 9 8 .  96  H  100 1  (65) was s i m i l a r  . The H NMR spectrum of i s o a c a n t h o d o r a l  to that of nanaimoal ( 6 1 ) . Resonances f o r three methyl groups, one of which was o l e f i n i c , were seen at 8 0.91 ( s , 3H), 0.99 ( s , 3H) and 1.64 ( b s , 3H) ppm.  An ABX s p i n system  was observed at 6 2.13 (dd, / = 3.3,14.8 Hz, 1H), 2.70 ( d d , J = 3.3,14.8 Hz, 1H), and 9.73 ( t , / = 3.3 Hz, 1H) ppm which c o u l d be a t t r i b u t e d to an e t h a n a l quaternary carbon  s u b s t i t u e n t attached  (substructure 6 6 ) .  to a  T h i s assignment was  supported by an intense fragment ion a t m/z 177 i n the mass spectrum which c o u l d a r i s e by l o s s of the e t h a n a l s i d e c h a i n via  an a l l y l i c  fragmentation  as shown i n Scheme 11a. A  fragment ion at m/z 176 i n d i c a t e d that l o s s of the ethanal s i d e c h a i n by a M c L a f f e r t y facile  (Scheme 11b). A  13  type fragmentation  C NMR  was a l s o  resonance at 6 204.6 (d)  supported the assignment of the lone oxygen i n isoacanthodoral  to an a l d e h y d i c c a r b o n y l .  Comparison of the  chemical s h i f t s of the AB resonances i n the ethanal chain of i s o a c a n t h o d o r a l responding AB proton  (65) (6 2.13 and 2.70) to the c o r -  resonances i n nanaimoal (61) (8 2.29  and  2.22 ppm) showed a marked d i f f e r e n c e i n chemical  and  i n d i c a t e d the e t h a n a l  vs  shift,  s i d e c h a i n was i n a n o t i c a b l y  d i f f e r e n t chemical environment. corresponding  side  carbon i n the  13  The chemical s h i f t of the  C NMR  spectrum, 8 57.1 f o r 65  8 53.7 f o r nanaimoal, supported t h i s argument. The  presence of s u b s t r u c t u r e 99 i n i s o a c a n t h o d o r a l  i n f e r r e d from the presence of an o l e f i n i c (bs, W ] / 2 ~ 6.4 Hz) coupled  was  proton a t 5 5.24  t o the o l e f i n i c  methyl resonance  101  at 6 1.64 ( b s , 3H), t h i s assignment was supported by a double-resonance experiment.  The  13  C NMR  spectrum of  isoacanthodoral  showed resonances at 6 137.4 (s) and 6 131.9  (d) a p p r o p r i a t e  f o r a t r i s u b s t i t u t e d double bond, and was  a s s i g n a b l e to s u b s t r u c t u r e 99. The s p e c t r a l data bicyclic  i n d i c a t e d that i s o a c a n t h o d o r a l  s e s q u i t e r p e n o i d that c o n t a i n e d  was a  three methyl groups,  one of which was o l e f i n i c , and an ethanal side c h a i n attached  to a quaternary c a r b o n .  I t was d i f f i c u l t t o  a s c e r t a i n the number of a l l y l i c methylene or methine protons 1  from the H NMR of i s o a c a n t h o d o r a l , however, from the integration  1  i n the H NMR  spectrum of (p-bromophenyl)-  urethane d e r i v a t i v e 98 (see appendix) i t was l i k e l y  that no  more than three a l l y l i c methylene and/or methine p r o t o n s  1 02  were present i n the m o l e c u l e . From s p e c t r a l evidence i t seemed c o n c e i v a b l e isoacanthodoral  was  b i o g e n e t i c a l l y r e l a t e d to nanaimoal  which l e d to c o n s i d e r a t i o n of aldehyde 100 structure for isoacanthodoral.  (61)  as a p o s s i b l e  Nanaimoal had  i s o p r e n o i d type carbon s k e l e t o n which one might be  that  a regular  could  imagine  formed by a b i o g e n e t i c type c y c l i z a t i o n of f a r n e s y l  pyrophosphate 101  v i a (formally) carbocation  12).  102  Carbocation  102  (see Scheme  c o u l d c o l l a p s e v i a a number of  pathways which c o u l d , a f t e r s i d e chain o x i d a t i o n , generate at l e a s t 4 p o s s i b l e p r o d u c t s . by l o s s of proton proton  or H c  Ha,  One  of the p r o d u c t s ,  would be nanaimoal (6_1) .  would generate aldehydes 103  formed  Loss of  or 104  respec-  t i v e l y , molecules both l a c k i n g an o l e f i n i c methyl group and n e i t h e r of which c o u l d t h e r e f o r e represent isoacanthodoral. functionality  the s t r u c t u r e of  To generate the r e q u i r e d o l e f i n i c methyl  from c a r b o c a t i o n  102  would r e q u i r e a molecular  1 03 rearrangement. a  Thus, a 1,2-hydride s h i f t of H-5  1,2-methyl m i g r a t i o n  and  l o s s of proton  followed  c o u l d generate  aldehyde 100, a s t r u c t u r e that would i n c o r p o r a t e many of s p e c t r a l f e a t u r e s of  by  the  isoacanthodoral.  Scheme 12. B i o g e n e t i c arguements l e a d i n g to the c o n s i d e r a t i o n of 100 as the s t r u c t u r e f o r i s o a c a n t h o d o r a l .  Although s t r u c t u r e 100 was  appealing  for  i s o a c a n t h o d o r a l , four p i e c e s of s p e c t r a l evidence and  one  chemical i n t e r c o n v e r s i o n were used to e l i m i n a t e i t from further consideration.  As p r e v i o u s l y mentioned, there was  a  marked d i f f e r e n c e between the chemical s h i f t s of the protons  104 alpha and  to the a l d e h y d i c c a r b o n y l  i n nanaimoal (61) (6 2.29  2.23 ppm) and the chemical s h i f t s of the analogous  protons i n i s o a c a n t h o d o r a l  (6_5) ( 6 2.70 and 2.13 ppm).  This  d i f f e r e n c e c o u l d not r e a d i l y be r a t i o n a l i z e d i f i s o a c a n t h o d o r a l had s t r u c t u r e 1 0 0 . Even i f the s i d e chain was a x i a l l y o r i e n t a t e d , as i n 1 0 5 , there d i d not seem to be any  good reason why one of the a-methylene protons should be  s e l e c t i v e l y deshielded. isoacanthodoral allylic  The mass spectrum of  i n d i c a t e d the ethanal  side chain may be  (see Scheme 11), implying the presence of e i t h e r of  s u b s t r u c t u r e s 106 or 1 0 7 .  0 H  105  CH  H  106  The  107  diamagnetic a n i s o t r o p y of the o l e f i n i c double bond could  p o s s i b l y d e s h i e l d one of the a-methylene protons i f there was a p r e f e r r e d rotamer p o p u l a t i o n  f o r the s i d e c h a i n i n  1 05 e i t h e r of s u b s t r u c t u r e s 106 The  o l e f i n i c proton  sharp (W^2  =  6.4  or  107.  in i s o a c a n t h o d o r a l was  unusually  H z ) , i n d i c a t i v e of an o l e f i n i c  proton  which l a c k e d v i c i n a l c o u p l i n g ( s u b s t r u c t u r e 106). isoacanthodoral  (65) was  When  reduced to i s o a c a n t h o d o r o l  o l e f i n i c proton was  s h i f t e d u p f i e l d by 0.14  the double bond and  oxygen f u n c t i o n a l i t i e s might be  p r o x i m i t y to each o t h e r .  ppm,  of the protons  c a r b i n o l carbon i n i s o a c a n t h o d o r o l s e l e c t i v e l y d e s h i e l d e d , 6 2.04  the  al pha to the  (97) continued  to be  ( d t , J = 13.5,7.2 Hz,  implying that the aldehyde c o u l d not be the In s t r u c t u r e 100  the chemical  nanaimoal (thus the ABX chemical  fc  be expected to be wider than 6.4 The concrete.  chemical  evidence  In 1974  ^  e  to  o l e f i n i c proton would  a g a i n s t s t r u c t u r e 100  was  more  Minale and co-workers i s o l a t e d a v e r o l  ether 109,  underwent a molecular compound 110,  origin  Hz.  from the marine sponge Disidea  of a v a r o l , dimethyl  and  s p i n systems would have s i m i l a r  the W-\/2 °f  s h i f t s ) and  1H)  environment  of the s i d e chain would be expected to be s i m i l a r  (108)  in c l o s e  Although r e d u c t i o n of the aldehyde  carbon-oxygen double bond, one  of t h i s e f f e c t .  the  suggesting  removes the a n i s o t r o p i c d e s h i e l d i n g i n f l u e n c e of  6 1.41,  (97)  that had  avcrc  13  ".  upon treatment with  rearrangement to g i v e a the same  r i n g system as nanaimoal (61).  A derivative acid  substance,  bicyclof4.4.0jdec-1(6)-ene I t was  i s o a c a n t h o d o r a l c o u l d be represented  reasoned that i f  by s t r u c t u r e  100,  treatment of i t s (p-bromophenyl)urethane d e r i v a t i v e  (98),  106 with a c i d , should generate the urethane d e r i v a t i v e of nanaimoal. 100%  When t h i s r e a c t i o n was c a r r i e d out with 98 -  formic a c i d at 70 °C f o r 10 hours, 98 was converted  q u a n t i t a t i v e l y to a new compound 112 which r e t a i n e d the olefinic  methyl f u n c t i o n a l i t y  the o l e f i n i c  proton  was now  1  ( H NMR  11.6 Hz.  5 1.59).  o  } 2  f  T h i s chemical evidence  i m p l i e d that the double bond had isomerized olefinic  The W /  about the  methyl group to give a new compound c o n t a i n i n g  substructure impossible  113 (see Scheme 1.3).  T h i s i s o m e r i z a t i o n was  for structure 100.  F o r t u n a t e l y , at t h i s p o i n t , the c r y s t a l s t r u c t u r e of acanthodoral's solved  {vide  p-bromophenylurethane d e r i v a t i v e (114) was infra)  a l l o w i n g a c o r r e c t proposal  s t r u c t u r e of isoacanthodoral  t o be made.  f o r the  The proposal  was  1 07 OBPU  OBPU  based upon the s p e c t r a l data reasoning.  i n combination  Scheme 14 i n d i c a t e s a proposed (6j5)  isoacanthodoral intermediate.  Proton  induced  i n acanthodoral  nanaimoal (61_) .  Proton  cyclobutane utility  (115)  (64)  could  induced c y c l o b u t a n e  from ketones  136  has  pivitol  generate fragmentations  are of  f o r the formation of f i v e membered  induced  synthetic  rings  1 3 5  .  shown that when f o r m y l c y c l o b u t a n e  i s t r e a t e d with a c i d  products.  as the  are known s y n t h e t i c a l l y , and proton  fragmentations  Venus-Danilova  biogenesis for  fragmentation of the  cyclobutane r i n g  from aldehydes  (64)  with a c a n t h o d o r a l  with b i o g e n e t i c  i t fragments to g i v e a number of  The major product  i s aldehyde  b i o g e n e t i c v e i w p o i n t , a proton induced  (116).  From a  cyclobutane  fragmentation has not p r e v i o u s l y been documented.  108  114  Proton  induced  fragmentation  occur by e i t h e r of two the C-10 C-10  to C-11  to C-11  of acanthodoral  or C-8  to C-11  bonds.  or 117  bond cleavage  of  Fragmentation of  bond, .followed by l o s s of proton H x  to C-11  may  pathways, that i s , fragmentation  e r a t e nanaimoal ( 6 1 ) , whereas l o s s of proton quent to C-8  (6_4)  the  would genor H z  subse-  could generate 65  (-Hy)  (-H z ), compounds that would i n c o r p o r a t e a l l the  s p e c t r a l f e a t u r e s of both i s o a c a n t h o d o r a l and i t s rearrangement p r o d u c t . given  Although the b i o g e n e t i c argument  i n Scheme 14 i s s p e c u l a t i v e , and must be t e s t e d  e x p e r i m e n t a l l y , use of b i o g e n e t i c theory allowed t u r e of i s o a c a n t h o d o r a l to be formulated as 65, absolute stereochemistry.  struc-  including  V e r i f i c a t i o n of the proposed  s t r u c t u r e f o r i s o a c a n t h o d o r a l was t a l X-ray d i f f r a c t i o n  the  obtained by a s i n g l e c r y s -  study, performed by He Cun-heng  Jon C l a r d y at C o r n e l l U n i v e r s i t y .  and  F i g u r e 20 r e p r e s e n t s a  computer-generated drawing, i n c l u d i n g  absolute  s t e r e o c h e m i s t r y , of i s o a c a n t h o d o r a l ' s 2 , 4 - d i n i t r o p h e n y l hydrazone d e r i v a t i v e  JJ6 .  109  Scheme 14. Proposed b i o g e n e s i s of i s o a c a n t h o d o r a l (65) from acanthodoral (64). C r y s t a l s of 9_6 belonged 6.027 (1)°.  t o space group P2, with a =  ( 1 ) , b = 31.613 ( 8 ) , c = 8.910 (1) n r  1 0  , and 0 = 80.89  The asymmetric u n i t c o n s i s t e d of two molecules of  composition C 2 i H , O N i , . 3  t  A f t e r c o l l e c t i o n of d i f f r a c t i o n  d a t a , s o l u t i o n by d i r e c t methods was r o u t i n e and l e a s t - s q u a r e s refinements converged  to a standard  c r y s t a l l o g r a p h i c r e s i d u a l of 0.048. A number of f e a t u r e s should be noted about the s t r u c t u r e i s o a c a n t h o d o r a l (65):  F i g u r e 20. Computer generated X-ray s t r u c t u r e of i s o a c a n t h o d o r a l ' s 2,4-dinitrophenylhydrazone d e r i v a t i v e  111 1.  I t i s a non-isoprenoid  s e s q u i t e r p e n o i d , that i s ,  does not obey the isoprene 2.  it  rule.  L i k e nanaimoal, one of the methyl groups o r i g i n a l l y present  i n the b i o g e n e t i c f a r n e s y l pyrophosphate  p r e c u r s e r has undergone c y c l i z a t i o n and i s now contained 3.  in a carbocyclic ring.  The- d e c a l i n r i n g system i s cis (stereochemistry  fused  b i o g e n e t i c a l l y a r i s i n g from the  e q u a t o r i a l nature of the C 5 s i d e c h a i n p r i o r to cyclization  to a c a n t h o d o r a l ) .  ample of a cis  T h i s i s the f i r s t ex-  fused d e c a l i n r i n g system t o be  i s o l a t e d from a n u d i b r a n c h . 4.  The d e s h i e l d i n g of one of the methylene protons alpha  to the c a r b o n y l may be r a t i o n a l i z e d by the  f o l l o w i n g argument; i n s o l u t i o n , a h i g h l y rotamer e x i s t s  populated  ( l i k e l y the same conformer as i n the  s o l i d s t a t e , see F i g u r e 20) whereby one of the a-methylene protons l i e s w i t h i n the d e s h i e l d i n g region of the carbon-carbon double bond.  This  causes s e l e c t i v e d e s h i e l d i n g of only one of the a-methylene  protons.  Isoacanthodoral  (65) has a new  sesquiterpenoid  carbon s k e l e t o n f o r which we propose the name isoacanthodorane and to number i t as shown i n F i g u r e 2 0 .  1 12 6. ACANTHODORAL Acanthodoral  (64) o c c u r r e d as the l e a s t abundant  s e s q u i t e r p e n o i d m e t a b o l i t e from Acanthodoris which a s t r u c t u r e has been a s s i g n e d * . showed a parent was  nanaimoensis  to  GC-MS of aldehyde 6_4  i o n at m/z 220 which suggested  acanthodoral  isomeric with nanaimoal (6_1) and i s o a c a n t h o d o r a l ( 6 5 ) .  The base peak at m/z 84 was d i a g n o s t i c f o r acanthodoral (64),  as n e i t h e r 61 or 65 showed s i g n i f i c a n t  ( > 5%)  ion  i n t e n s i t y at m/z 84. Intense fragment ions at m/z 176 and 161  suggested  the presence of an e t h a n a l s u b s t i t u e n t as was  found f o r nanaimoal (61) and i s o a c a n t h o d o r a l (65)t» I s o l a t i o n of pure acanthodoral  (64) was extremely  difficult  owing t o i t s t r a c e abundance (70 ug/animal) and h i g h volatility.  I t was t h e r e f o r e i s o l a t e d as i t s c r y s t a l l i n e  (p-bromophenyl)urethane d e r i v a t i v e 114. The most e f f i c i e n t i s o l a t i o n procedure aldehydes  was t o reduce  the n a t u r a l mixture of  t o the c o r r e s p o n d i n g a l c o h o l s and t o separate  nanaimool (6_1) from the mixture of i s o a c a n t h o d o r o l (65) and acanthodorol  (118) by r a d i a l TLC (100% CHC1 3 ).  D e r i v a t i z a t i o n of the isomeric a l c o h o l s 97 and 118 with 4-bromophenyl isocyanate f o l l o w e d by HPLC p u r i f i c a t i o n y i e l d e d pure samples of (p-bromophenyl)urethane d e r i v a t i v e s  *As p r e v i o u s l y mentioned, two t r a c e m e t a b o l i t e s , at l e a s t one of which i s isomeric (GC-MS) with the major s e s q u i t e r p e n o i d aldehydes, were present i n the s k i n e x t r a c t of A.  nanaimoensis.  t The GC-MS spectrum of nanaimoal showed intense fragment ions at m/z 176 and 161 (100%), i s o a c a n t h o d o r a l showed a fragment i o n at m/z 176 but d i d not e x h i b i t any i o n i n t e n s i t y a t m/z 161.  OH  1  1  118  4  Acanthodoral's c r y s t a l l i n e  (p-bromophenyl)urethane  d e r i v a t i v e 114 (mp 109-110 °C, hexane), had a molecular formula C 2 2 H 3 0 B r N 0 2  (HRMS, m/z observed 421.1441, 419.1438;  r e q u i r e d 421.1439, 419.1460) v e r i f y i n g an isomeric r e l a t i o n s h i p f o r a c a n t h o d o r a l with nanaimoal isoacanthodoral  (65).  1  The H NMR  (6_1) and  spectrum of urethane 114  showed three a l i p h a t i c , quaternary methyl resonances at 6 0.81  ( s , 3H), 0.89 ( s , 3H), and 0.96 ( s , 3H).  Although the  GC-MS spectrum of aldehyde 64 suggested the presence of an e t h a n a l s i d e chain s u b s t i t u e n t , the c a r b i n o l region i n the 1  H NMR  spectrum of urethane 114 (see Appendix) showed only  the AB p o r t i o n of an ABX s p i n system, i n d i c a t i v e of a s u b s t r u c t u r e of type 1 1 9 .  1  A l s o present i n the H  NMR  1 14 spectrum of 114 were resonances f o r an i s o l a t e d AX tem  at 6 1.09  (d,  J  = 9.4  Hz,  1H)  and  1.84  spin sys-  (d, / =  Hz,1H), from which s u b s t r u c t u r e 120 c o u l d be  9.4  inferred.  H H  120  119  Due  to the magnitude of the geminal c o u p l i n g constant  Hz)  s u b s t r u c t u r e 120 was  membered r i n g NMR  1 3 7  .  The  spectrum were due  6 6.50 protons  (bs, 1H), 7.27  (9.4  l i k e l y c o n f i n e d w i t h i n a 4 or 5  remainder of the s i g n a l s i n the to the  1  H  (p-bromophenyl)urethane moiety;  (d, 2H), 7.40  (d, 2H)  ppm,  and  to  12  that appeared as a s e r i e s of complex m u l t i p l e t s  r e s o n a t i n g between 6 1.2 the remaining  protons  absence of a l l y l i c  and  1.7  that c o u l d be assigned  of the t e r p e n o i d f u n c t i o n a l i t y .  proton  acanthodoral  (64)  The  The  resonances suggested that the  t e r p e n o i d p o r t i o n of d e r i v a t i v e 114 contained carbocyclic rings.  to  three  s p e c t r a l data suggested that because  c o n t a i n e d only three methyl groups i t was  l i k e l y b i o g e n e t i c a l l y r e l a t e d to both nanaimoal ( £ 1 ) , and isoacanthodoral  (65) .  No  known s e s q u i t e r p e n o i d carbon  s k e l e t o n c o u l d account f o r the observed s p e c t r a l  data,  1 15 t h e r e f o r e the s t r u c t u r e of acanthodoral  (64) , i n c l u d i n g the  a b s o l u t e c o n f i g u r a t i o n , was solved by s i n g l e - c r y s t a l X-ray diffraction analysis.  The s t r u c t u r a l d e t e r m i n a t i o n was  performed by He Cun-heng and Jon C l a r d y at C o r n e l l U n i v e r s i t y on acanthodoral's  (p-bromophenyl)urethane  d e r i v a t i v e 114. C r y s t a l s of 114 belonged  to the common monoclinic space  group Ply with a = 9.581 ( 1 ) , b = 6.406 ( 1 ) , c = 34.45 (1) 10  m~ ,  and /3 = 85.85 ( 1 ) ° .  C22H3002NBr  Two molecules  formed the asymmetric u n i t .  of composition  A f t e r c o l l e c t i o n of  the d i f f r a c t i o n d a t a , a phasing model was found by standard heavy atom methods, and l e a s t - s q u a r e s refinements with heavy atoms and i s o t r o p i c hydrogens converged crystallographic reflections.  t o a standard  r e s i d u a l of 0.078 f o r the observed  Both molecules  i n the asymmetric u n i t had the  same s t e r e o s t r u c t u r e , and a computer generated p e r s p e c t i v e drawing i s given i n F i g u r e 21. The average bond angles f o r the four membered r i n g Acanthodoral  i n 114 was 8 6 ° .  64 has a new s e s q u i t e r p e n o i d carbon  s k e l e t o n f o r which we propose the name acanthodorane and t o number i t as shown i n F i g u r e 21.  1 16  F i g u r e 21. Computer generated X-ray s t r u c t u r e of a c a n t h o d o r a l ' (p-bromophenyl)urethane d e r i v a t i v e 114.  1 17 7. BIOLOGICAL ACTIVITIES OF A. nanaimoensi  s SECONDARY  METABOLITES In view of the b i o l o g i c a l a c t i v i t i e s nudibranch m e t a b o l i t e s  shown by known  (see Table 6 ) , the n a t u r a l  s e s q u i t e r p e n o i d mixture from A. nanaimoensis  was evalua-  ted f o r i t s a n t i b a c t e r i a l and a n t i f u n g a l a c t i v i t i e s . The mixture showed a n t i b a c t e r i a l a c t i v i t y Baci  I I us subtilis  and Staphylococcus  a n t i f u n g a l a c t i v i t y a g a i n s t Pythiam Rhizoctonia mum  solani  aureus  ultimum  a l l at 670 ug/O/4  against as w e l l as  and  inch d i s k ) (mini-  i n h i b i t o r y c o n c e n t r a t i o n s were not  determined).  Due to the small amounts i s o l a t e d , and the need f o r l a r g e amounts of m e t a b o l i t e s  f o r s t r u c t u r a l work ( i n an  attempt to generate enough nanaimoal urethane d e r i v a t i v e 7 5 f o r 2D-NMR experiments) the s e s q u i t e r p e n o i d aldehydes were not t e s t e d f o r f i s h a n t i f e e d a n t  activity.  8. DISCUSSION The  co-occurrence  i n A. nanaimoensis  of three  s e s q u i t e r p e n o i d s each with a new carbon s k e l e t o n was g r a t i f y i n g , however, i t was not s u r p r i s i n g i n l i g h t of prev i o u s s t u d i e s on nudibranch s e s q u i t e r p e n o i d s .  Table  6 isa  summary of a l l the nudibranch s e s q u i t e r p e n o i d s arranged a c c o r d i n g t o s k e l e t a l type with comments as t o t h e i r and  biological  source  activities.  It can be seen from Table  6 that the drimane carbon  s k e l e t o n makes up a l a r g e m a j o r i t y of the s e s q u i t e r p e n o i d s  1 18  i s o l a t e d from nudibranchs and they show, with a few exceptions, antifeedant a c t i v i t y against  fish.  Although the  s i g n i f i c a n c e of the carbon s k e l e t o n i s not c l e a r , drimane s e s q u i t e r p e n o i d s have a l s o been shown to possess i n s e c t antifeedant a c t i v i t y The  1 3 8  , and in vivo  antifungal a c t i v i t y .  m a j o r i t y of s e s q u i t e r p e n o i d s  isolated f a l l  into  three groups: i ) f u r a n s , i i ) aldehydes (dialdehydes) and iii)  isonitriles.  or produced de novo  They may be d e r i v e d from d i e t a r y by the nudibranch (see Table  i n a b i l i t y to f i n d a d i e t a r y s o u r c e o c c u r r e n c e , and r a t i o , of the A. sesquiterpenoids  ,  and the constant  13  biosynthesis.  was  i n c o r p o r a t e d i n t o the s e s q u i t e r p e n o i d a l d e h y d e s .  activity.  This re-  t e n a t i v e however, as the  r a d i o l a b e l l e d metabolites stant s p e c i f i c  Initial  C l a b e l e d mevalonic a c i d  i n j e c t e d i n t o the stomach of A. nanaimoensis  s u l t must be c o n s i d e r e d  6 ) . Our  nanaimoensis  suggested de novo  biosynthe'tic s t u d i e s showed that  102  sources  have not been c r y s t a l l i z e d  to con-  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS  SKELETON  METABOLITES  COMMENTS  A. Acarbocyclic  1.  apofarnesane  ^  ^  ^  ^  ^  ^  ^  Isolated frcm Anisodoris n o b i l i s , dihydroapofarnesal (121) was responsible for 38D the nudibranch's f r u i t y c d o r .  dihydroapofarnesal (121)  2.  farnesane  marislin (122) ,  0CH  3  124  a  b  The major metabolite o f Chromcdoris marislae was m a r i s l i n (122), cctrpounds 123a, 123b, 124a and 124b were present as minor 13y c o n s t i t u e n t s . The isomeric p a i r s 123a, 123b and I24a, 124b are formally related by a [3,3] sigmatropic rearrangement. The authors suggest 123a and 123b are a r t i f a c t s o f the i s o l a t i o n procedure.  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON  METABOLITES  COMMENTS  famesane (cont' d)  0  1  1  0R 125  2  R1 = H, R2 = H  126  R1 = Ac, R2 = H  127  R1 = Ac, R2 =Ac  i  i  0  r  Archidoris odhneri yielded a series o f related farnesic acid glycerides 140 1 (125-128) . '•C-labeled mevalonic a c i d injected i n t o the stomach o f A. odhneri was incorporated i n t o the farnesic 38 a c i d moiety of 125 *\ 125 showed moderate i n v i t r o a n t i b i o t i c a c t i v i t y against Staphylococcus aureus, but had no demonstrable f i s h antifeedant a c t i v i t y .  0H  128 Dendrolasin (129), a known sponge metabolite has been i s o3 l6 a t e d from both Cadlina leutomarginata and Hypselodoris 141 ghiselini collected i n California.  dendrolasin (129)  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON  METABOLITES  COMMENTS A known sponge metabolite, l o n g i f o l i n (130) was i s o l1a4 t2 e d from the nudibranch Glossodoris gracilis .  farnesane (cont'd)  l o n g i f o l i n (130)  Monocyclof arnesane metabolite 131 was i s o l a t e d3 form Archidoris montereyensis and A. odhneri 8k. I t i s b i o g e n e t i c a l l y related to farnesic a c i d glyceride 125 by a proton induced c y c l i z a t i o n .  B. Monocarbocyclic  1. Monocyclofarnesane  *  %%%  1  0 131  132  Furan 132, whose structure was postulated on the basis o f mass and *H NMR spectral data 36 was i s o l a t e d from Cadlina luteomarginata . The structure has not been confirmed by synthesis or interconversion t o a known compound.  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON 2.  METABOLITES  COMMENTS  microcionin-2  Microcionin-2 (133), a known sponge metabolite was 3i5 s o l a t e d from Cadlina luteomarginata . 133 has been proposed as an intermediate i n the biogenesis of 44 nakafuran-8 and nakafuran-91 .  microcionin-2 (133)  3.  P l e r a p l y s i l l i n - 1 (134) a known sponge metabolite, was i s o l a t e d from Cadlina 35 luteomarginata .  pleraplysillin-1  p l e r a p l y s i l l i n - 1 (134) C.  Bicarbocyclic  1.  OH  drimane  135  R= H  136  R = Ac  Isolated from both Archidoris montereyensis 38 5 and A. odhneri * , drimane rretabolite 135 showed feeding deterent a c t i v i t y against the t i d e p c o l skulpin Oligocottus maculosus at 18 14 g/(mg o f food p e l l e t ) . C l abeled u mevalonic a c i d fed t o A. montereyensis was incorporated i n t o the terpenoid p o r t i o n of 135.  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON  METABOLITES  COMMENTS Albicanol (137) and albicanol acetate (138) were i s o l a t e d from Cadlina l u t e a r a r g i n a t a ^ c o l l e c t e d i n B r i t i s h Columbia. A l b i c a n o l acetate (138) showed antifeedant a c t i v i t y against g o l d f i s h at 10 ug/(mg o f food p e l l e t ) while a l b i c a n o l (137) was inactive (50 pg/(mg of food p e l l e t ) ) . A l b i c a n o l (137) had been previously i s o l a t e d from the liverwort 1 3 Diplophyllum a l b i c a n s .  drimane (cont'd)  albicanol (137) R = H a l b i c anol acetate (138) R = Ac  38a  Isolated from Chromodoris a l b o n o t a t a , pu'ulenal (139) was r e a d i l y hydrolyzed t o a 5:2 mixture o f polygodial (140), a known f i s h antifeedant, and 9-epipolygodial a compound devoid of antifeedant a c t i v i t y .  pu'ulenal (139)  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON  METABOLITES  drimane (cont'd)  COMMENTS Polygodial (140) was i s o l a t e d from 11 5 Dendrodoris Lurtbata * , D. k r e b s i i , D nigra 1 6 and D. tuberculosa *+ . A known insect antifeedant, polygodial a l s o has f i s h antifeedant a c t i v i t y . L a b e l l i n g studies with 1 ^C-labeled mevalonic a c i d showed 1401 1 was 7 biosynthesized de novo by D. limbata * .  141  E s t e r f i e d t o a series o f f a t t y acids with varying degrees o f unsaturation, sesquiterpenoid d e r i v a t i v e 141 (stereochemistry a t C-11 unknown) was l o c a l i z e d i n the d i g e s t il vh 8e gland o f 11 5 Dendrodoris limbata * • . 141 could a l s o be r e a d i l y i s o l a t e d from acetone skin extracts o f D o r i o p s i l1 1l a6 albopunctata and D o r i o p s i l l a j a n a i n a l * . The esters 141 d i d not show f i s h antifeedant a c t i v i t y and i t was suggested they are 1d1 +e t o x i f i c a t i o n products o f polygodial (140). C-labeled mevalonic acid was incorporated i n t o the sesquiterpenoid 147 moiety o f 1 4 1 . S t i r r i n g 141 i n the presence o f a c i d y i e l d e d euryfuran (144), a known nudibranch metabolite.  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON drinane  METABOLITES  (cont'd)  AcO,  ^ ^ ^ ^ • O A c olepupuane (142)  COMMENTS Olepupuane (142), a metabolite b i o g e n e t i c a l l y related t o polygodial (140) occured i n Dendrodoris n i g r a , D. tuberculosa, D. k r e b s i i , D o r i s p s Ji ll+t> l a albopunctata and D o r i o p s i l l a janaina - . I t was shown t o i n h i b i t feeding o f the p a c i f i c damsel f i s h (Dascyllus aruanus). The ED 5 Q was found t o be 15-20 ug/(mg o f p e l l e t ) , comparable t o that o f polygodial (140). The methoxy acetal 143 was i s o l a t e d from one c o l l e c t i o n o f D o r i o p s i l l a albopunctata, stored i n methanol.  AcC\  143  euryfuran (144)  Euryfuran (144), a known synthetic compound, was i s o l a t e d from Hypselodoris porterae, H. c a l i fo r n i e n s i s , and the 141 i n t e r t i d a l sponge Euryspongia s p . . An antifeedant r o l e f o r 144 i s suggested although no quantitative tests were performed.  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON 2.  METABOLITES  COMMENTS 36  pallescensin-A  Isolated from Cadlina luteomarginata , pallescensin-A (145) i s b i o g e n e t i c a l l y related t o euryfuran (144). 145 was t o x i c t o goldfish and showed antifeedant a c t i v i t y against the skulpin Clinocottus a n a l i s .  pallescensin-A (145)  3.  agassizin 141  Obtained from Hyselodoris a g a s s i z i , agassizin (146) i s s t r u c t u r a l l y related t o 4 9 pallescensin-G, a known sponge m e t a b o l i t e 1 . An antifeedant r o l e f o r 146 was implied. agassizin (146) 4.  spiniferin-2  rrv spiniferin-2 (147)  A known sponge metabolite, s p i n i f e r i n - 2 (147) was i s o l a t e d from Hypselodoris danielae 1h collected i n Hawaii  TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON 5.  COMMENTS  METABOLITES  Nanaimoal (61) was i s o l a t1 0e5 d from Acanthodoris nanaimoensis (see t e x t ) .  nanaimoane  H  nanaimoal (61)  6.  isoacanthodorane  0  Isoacanthodral (65) was 1i0s6 o l a t e d from Acanthodoris nanaimoensis (see t e x t ) .  isoacanthodoral (65)  7.  nakafuran-8  nakafuran-8 (148)  The biogenetic precurser t o nakafuran-9 (152), nakafuran-8 (148) had antifeedant properties against the common reef fishes Chaetodon spp. Furan 148 was i s o l a t e d from Chronodoris maridadilus, Hypsilodoris llt i H1 godeffroyana \ H. c a l i f o r n i e n s i s , and the sponge Dysidea f r a g i l i s .  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON  METABOLITES  nakafuran-8 (cont'd)  COMMENTS Three oxidation products of nakafuran-8 have been reported. Butenolide 149 i s believed t o be an a r t i f a c t formed by a i r oxidation of 1 4 8 w h i l e 150 and 151 were i s o l a t e d from H. zebra and i t s dietary sponge 50 D. e t h e r i a l .  149  RO  8.  nakafuran-9  V  150  R=Ac  151  R=H  nakafuran-9 (152)  A s t r u c t u r a l l y i n t e r e s t i n g sesquiterpenoid, nakafuran-9 (152) has been i s o l a t e d from Chromodoris 11maridadilus, Hypselodoris 11 1 godeffroyana *'*, and H. g h i s e l i n i * . A metabolite of the sponge Dysidea f r a g i l i s , 152 shows antifeedant a c t i v i t y against the common reef fishes Chaetodon spp. The methoxy butenolide of nakafuran-9, was assumed t o have antifeedant properties against p o t e n t i a l nudibranch predators. I t was i s o l a t e d from 1 H. g h i s e l i n i and oo-occured 4T with nakafuran-9 .  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON  9;  METABOLITES  furodysin  Furodysin (153), a known sponge metabolite, 35 was i s o l a t e d from Cadlina luteomarginata .  furodysin  10.  COMMENTS  (153)  furodysinin  furodysinin  (154)  The metabolites i s o l a t e d from Cadlina luteomarginata showed marked variations between c o l l e c t i o n s and c o l l e c t i n g s i t e s , i n d i c a t i n g a dietary source. This hypothesis was supported by the i s o l a t i o n of many of the C. luteomarginata metabolites from the sponges upon which i t preys. The only metabolite common to both C a l i f o r n i a and B r i t i s h Columbia c o l l e c t i o n s of C. 35 3 6 luteomarginata was furodysinin (154) » . Furodysinin (154) i s b i o g e n e t i c a l l y related t o furodysin (153). Furodysinin (154) was a l s o i s o l a t e d from Hypselodorisi b 0 zebra c o l l e c t e d i n Bermudian w a t e r s .  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS  SKELETON  cont'd  METABOLITES  COMMENTS  11.  Two nudibranch metabolites, 155 and3 6 156, i s o l a t e d from Cadlina luteomarginata , were traced t o the sponge A x i n e l l a sp. Both 155 and 156 were t o x i c t o g o l d f i s h and showed antifeedant a c t i v i t y against g o l d f i s h and the skulpin Clinocottus a n a l i s .  155  R  =  -NC  156  R  =  -NCS  P h y l l i d i a p u l i t z e r i concentrates a x i s o n i t r i l e - l (157) from the sponge upon 11 5 which i t feeds, A x i n e l l a cannabina * . I s o n i t r i l e 157 was inactive as an antifeedant but was t o x i c t o f i s h at a concentration as low as 8 ppm.  12.  NC  X  a x i s o n i t r i l e - l (157)  TABLE 6:  NUDIBRANCH SESQUITERPENOIDS cont'd  SKELETON  METABOLITES  D. T r i c a r b o c y c l i c  COMMENTS 9-isocyanopupukeanane (158) was i s o l a t e d from P h y l l i d i a v a r i c o s a . See Sections I.D and I I . A. 2.  1. pupukeanane  7 9-isocyanopupukeanane (158)  2. acanthcdorane  Acanthodoral (64) was i s o l1 0a6 t e d from Acanthodoris nanaimoensis (see t e x t ) .  acanthodoral (64)  132 III.  BRYOZOANS  A. INTRODUCTION TO THE BRYOZOANS Marine bryozoans have proven t o be a r i c h source of novel, b i o l o g i c a l l y a c t i v e , natural products.  One c r i t e r i o n  used by marine n a t u r a l p r o d u c t s chemists i n s e l e c t i n g potential  s e s s i l e marine organisms f o r i n v e s t i g a t i o n  i s the  a b i l i t y of the organism to compete f o r the l i m i t e d ecological are  space a v a i l a b l e  for growth  important f o u l i n g o r g a n i s m s  152  151  .  Since bryozoans  , they have the a b i l i t y to  out-compete other organisms f o r growing space, and to s u r v i v e under very t r y i n g c o n d i t i o n s  ( f o r example, some  s p e c i e s a r e very r e s i s t a n t t o the a n t i f o u l i n g p a i n t s used on ships).  In s p i t e of these i n t e r e s t i n g e c o l o g i c a l  i s t i c s bryozoans have been l a r g e l y chemists, u n t i l Bryozoans  character-  ignored by organic  recently. (phylum Bryozoa) are c o l o n i a l animals of  which approximately 4000 l i v i n g  s p e c i e s are known.  Most are  marine organisms, although a few freshwater s p e c i e s have been documented, mainly i n the c l a s s P h y l a c t o l a e m a t a . The bryozoan c o l o n i e s vary i n h e i g h t and width and occur i n a variety  of m o r p h o l o g i c a l forms; hence the names  f a l s e - c o r a l s , sea-mats, and moss animals a r e commonly used to d e s c r i b e them.  In B r i t i s h Columbian waters a l l three  forms a r e common.  Examples a r e  pacifica Bugula  and Phi dol opora  pacifica  s p . , and the sea-mat  1 5 3  : c o r a l l i k e - Het  eropora  , moss-animal type -  type - Membranipora  membranacea.  1 33 The  small members of a colony  (zooids) have body w a l l s  that a r e c a l c a r e o u s , g e l a t i n o u s or c h i t i n o u s and a r e u s u a l l y l e s s than 0.5 mm i n l e n g t h .  A few t o many m i l l i o n  may make up a bryozoan c o l o n y .  zooids  Part of the zooid body w a l l  i s a c i r c u l a r or horseshoe-shaped s t r u c t u r e c a l l e d the lophophore; i t bears c i l i a t e d t e n t a c l e s which may be protruded  out of an o r i f i c e t o gather the small  plankton  ( c h i e f l y diatoms and other phyloplankton) that make up the bryozoans' d i e t .  The gut i s U shaped and the anus opens  j u s t o u t s i d e the lophophore.  The zooids are u s u a l l y  connected through gaps or pores i n the body w a l l s and some are m o d i f i e d  f o r other  s p e c i a l i z e d f u n c t i o n s such as  c l e a n i n g , p r o t e c t i o n , or brooding the young. C o l o n i e s are h e r m a p h r o d i t i c ,  both male and female  zooids can occur i n the same c o l o n y .  The f e r t i l i z e d egg may  develop i n t o a free-swimming l a r v a capable of f e e d i n g , or as in the case with most s p e c i e s , the f e r t i l i z e d eggs pass a brood chamber.  into  The brooded l a r v a e have a s h o r t e r  free-swimming l i f e and do not f e e d .  Most l a r v a e a t t a c h  themselves to a s u r f a c e and change i n t o a zooid from which the colony  develops by asexual budding.  Only s i x bryozoan s p e c i e s have been i n v e s t i g a t e d f o r t h e i r n a t u r a l products chemistry t o d a t e .  The r e s u l t s of  these s t u d i e s are summarized i n Table 7. The neritina  f i r s t bryozoan to be s t u d i e d c h e m i c a l l y was ( L i n n a l u s ) from which V i l l e l a  adenochrome-like pigment i n 1948  1 54  .  i s o l a t e d an  Bugula  TABLE 7:  BRYOZOAN METABOLITES ORGANISMS AND METABOLITES ISOLATED  Alcyonidium gelatinosum (L.) (Ref..155)  H „^S -CH CH OH  STRUCTURE ELUCIDATION  COMMENTS AND BIOLOGICAL ACTIVITIES  Structure 159 was proposed onl the basis 13 of C and E NMR spectral comparisons t o model compounds and was confirmed by synthesis.  Sulfoxonium ions had never been encountered i n nature previous t o the i s o l a t i o n of 159. Isolated i n 5 ppm y i e l d based on the animal wet weight, 159 i s the c a u s i t i v e agent o f "Dogger Bank I t c h " , an eczematous a l l e r g i c contact dermatitus caused by exposure t o A. gelatinosum. A severe occupational disease, "Dogger Bank Itch" i s widely d i s t r i b u t e d among trawlermen working i n the Dogger Bank area of the North Sea.  +  C  3  2  2  (2-hydroxyethyl) dimethylsulfonium ion (159)  Alcyonidium hirsutum (Ref. 156) The water extract o f A. hirsutum showed s i g n i f i c a n t t o x i c i t y t o mice at 1000 mg/kg, and showed an i n h i b i t i o n zone against Escherichia c o l i . The chloroform extract was t o x i c to the herpes virus (HSV-1).  An extract o f A. convoluta showed a n t i neoplastic a c t i v i t y (PS system).  Amathia convoluta (Ref. 157T"  1  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED Bugula n e r i t i n a (Sinnaeus) (Ref. 157-161)  R'O  4 /  CH3O  ^s^s^J*  ^  R"0  ° ^ Y H 1  STRUCTURE ELUCIDATION  COMMENTS AND BIOLOGICAL ACTIVITIES  The structure o f bryostatin 1 (160) was obtained by single-crystal X-ray crystallography at -100°C. Residual R factor = 0.07. The enantiomer shown was selected on the basis of anomalous scattering effects due to oxygen and carbon for CuKa radiation. Apparently, e f f o r t s t o determine the absolute configuration o f bryostatin 1 are continuing.  The antineoplastic a c t i v i t y of a B. n e r i t i n a extract was f i r s t documented i n 1970. Bryostatin 1 (160) was i s o l a t e d from 500 kg o f wet animals. In the murine P388 lymphocyte leukemia (PS system) macrolide 160 showed 52-96% l i f e extensions at 10-70 ug/kg i n j e c t i o n dose l e v e l s and an ED 5 Q o f 0.89 ug/mL against the P388 i n v i t r o c e l l l i n e . I t also shows potent a c t i v i t y against the L1210 (lymphocyte leukemia, 34-51% l i f e extension a t 37.5-150 u g A g ) and M5 (M5076 ovarian carcinoma, 40-48% l i f e extension a t 5-20 ug/kg and 20-65% curative i n the tumor regression model a t 20-40 ug/kg) experimental tumor systems. A polyketide biogenesis i s suggested f o r bryostatin 1. The oxygen atoms a t 01, 03, 05, 011, 019A, and 019B are a l l i n the i n t e r i o r o f the large oxygen r i c h cavity suggesting the molecule may have cation binding c a p a b i l i t i e s .  >  S  OH  R= COCH3  R"=H bryostatin 1 (160)  0  R  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  STRUCTURE ELUCIDATION  B. n e r i t i n a (cont'd)  R = H  R"=H bryostatin 2 (161)  R= C0CH3 ^  ^  R"= COCH3 (162)  ^  ^  COMMENTS AND BIOLOGICAL ACTIVITIES  The structure of Bryostatin 2 (161) showed a 60% increase i n l i f e span a t 30 u.g/kg i n the murine P388 PS bryostatin 2 (161) was 13 system. based on C NMR and a 400 MHz H NMR studies i n comparison t o bryostatin 1. Selective acetylation of 160 or 161 gave i d e n t i c a l acetate (TLC) 162, which upon careful deacetylation gave a mixture o f bryostatins 1 (160) and 2 (161).  TABLE 7:  BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  B. n e r i t i n a (cont'd)  0  R'=  R"=H bryostatin 4 (163)  STRUCTURE ELUCIDATION  COMMENTS AND BIOLOGICAL ACTIVITIES  The structure o f bryostatin 4 (163) was 13 assigned using C NMR l and 400 MHz E NMR, solution phase secondary ion MS, and selective hydrolysis experiments. The substituent pattern of the butyrate and isovalerate esters were based on the assumption that s t e r i c compression at C-20 compared t o C-7 would favour hydrolysis of the less hindered ester (C-7), as was found for bryostatin 1.  44.5 mg o f 163 was i s o l a t e d from 50 kg wet weight o f B. n e r i t i n a . Apparently, b r y o s t a t i n 4 (163) i s much less cytotoxic than bryostatin 1. I t was suggested that substituents common t o bryostatins 1-4 (160, 161, 163 and 164) constitute the unique requirements f o r anticancer a c t i v i t y while the ester substituents at C-7 and C-20 influence the degree o f c y t o t o x i c i t y and antineoplastic e f f e c t s . Bryostatin 4 (163) showed 62% increase i n l i f e span at 46 ug/kg i n the murine P388 PS system and substantial c e l l growth i n h i b i t o r y (PS c e l l l i v e E D 5 Q , 14 10-3-10- ug/mL) a c t i v i t y .  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED B. n e r i t i n a  (cont'd)  & ' \ ^ f ^ y c  R"°  STRUCTURE ELUCIDATION  S r  \  H  •  COMMENTS AND BIOLOGICAL ACTIVITIES  The structure o f 72.2 mg o f 164 was i s o l a t e d from 500 kg o f bryostatin 3 (164) was B. n e r i t i n a . B r y o s t a t i n 3 (164) shows 63% extension at 30 p,g/kg i n the P388 assigned using1 3FAB mass l i f e spectrometry, C NMR, lymphocytic leukemia (PS system). OCH 400 MHz hi NMR, and IR spectroscopy (IR band -1 at 1785 cm indicated a possible 5 or 6-membered lactone carbonyl group with an a electronegative substituent).  OH R=  COCH3  R" = H  bryostatin 3 (164) F l u s t r a f o l i a c e a (L.) (Ref. 156, 162-169)  c i s - c i t r a l (165)  The structures 165169 were assigned on the basis o f GC-MS comparisons t o authentic samples.  The a n t i b i o t i c a c t i v i t y (vs Staphylococcus aureus) o f t h i s bryozoan was most pronounced i n the older parts o f the fronds, and was correlated with a c h a r a c t e r i s t i c strong lemonlike odor. The compounds apparently responsible f o r the a n t i b i o t i c a c t i v i t y were monoterpenoids 165-169. Freeze dried samples of F. f o l i a c e a were devoid o f a n t i b a c t e r i a l a c t i v i t y , however, they d i d show a n t i v i r a l a c t i v i t y and i n h i b i t i o n o f the guinea-pig ileum i n v i t r o .  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  STRUCTURE ELUCIDATION  COMMENTS AND BIOLOGICAL ACTIVITIES  F. f o l i a c e a (cont'd)  1  18  X  X  geraniol (166)  t r a n s - c i t r a l (167)  1  JL  nerol (168)  c i t r o n e l l o l (169)  Y B r ^ ^ j  H  3  A flustramine A (170)  The structures o f the No b i o l o g i c a l a c t i v i t i e s were reported f o r alkaloids 170-178 metabolites 170 t o 178, however the crude isolated from F. petroleum ether extract (from freeze dried f o l i a c e a , were material) and the p u r i f i e d alkaloids 170 and determined by detailed 171 exhibited muscle-relaxant a c t i v i t y both interpretation of the i n vivo and i n v i t r o . spectral data.  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  STRUCTURE ELUCIDATION  F. f o l i a c e a (cont'd)  X flustraraine B (171)  Y  flustramine C (172) OH  r ^ V v N — C H  3  flustraminol A (173)  COMMENTS AND BIOLOGICAL ACTIVITIES  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  STRUCTURE ELUCIDATION  F. f o l i a c e a (cont'd)  OH _  B r  I  AjC  y N  ^CH  3  flustraminol B (174)  A flustramide A (175) 0  6-brcm>-^-rnethyl-Nb-for^TiyltryptarTi Lne (176)  COMMENTS AND BIOLOGICAL ACTIVITIES  TABLE 7:  BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  F . f o l i a c e a (cont'd)  177  STRUCTURE ELUCIDATION  COMMENTS AND BIOLOGICAL ACTIVITIES  Quinoline derivative 177 i s l i k e l y an a r t i f a c t as ethanol was used i n the extraction procedure. The authors speculate that i f the ethoxy group was introduced during the i s o l a t i o n procedure, i t must have replaced a s t r i k e n l y reactive group.  Amide 178 exists as mixture of E and Z rotamers about the amide bond.  Nugula n e r i t a (Ref. 157T  The aqueous 2-propanol extract o f N. n e r i t a , i n several doses, l e d t o 168-200% l i f e extension i n the PS system.  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED Phidolcpora (Ref. 170)  STRUCTURE ELUCIDATION  For s t r u c t u r a l e l u c i d a t i o n and b i o l o g i c a l a c t i v i t i e s , see t e x t . Synthetic studies toward the synthesis o f phidolopin 179, desmethylphidolopin 180, and a number o f s t r u c t u r a l analogues are currently underway.  pacifica  1 phidolcpin (179)  N  N  1  CH  3  COMMENTS AND BIOLOGICAL ACTIVITIES  desmethy lphid(Dlcpin (180)  OH  dr °  N 2  ^0H  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  STRUCTURE ELUCIDATION  Sessibugula translucens (Ref. 171)  0CH  3  NHR 182  X = H  Y = H  R = H  183  X = Br  Y = H  R = H  184  X = H  Y = H  R = iBu  185  X = H  Y = Br  R = iBu  The structures 182185 were elucidated using spectral methods and comparison t o the known compound, 4-methoxy-2,21bipyrrole-5carboxaldehyde (186).  COMMENTS AND BIOLOGICAL ACTIVITIES  Sessibugula translucens i s preyed upon by the nembrothid nudibranchs Tambje abdere, T. e l i o r a and Roboastra t i g r i s . A mixture o f enamines 182 and 183 (182, 40%; 183, 60%) i n h i b i t e d c e l l devision i n the f e r t i l i z e d sea urchin egg assay a t 1 pig/mL i n seawater and showed moderate a n t i m i c r o b i a l a c t i v i t y a t 50p,g/disk against E s c h e r i c i a c o l i , Staphylococcus aureus, B a c i l l u s s u b t i l i s , and V i b r i o anguillarum.  OCH.  186  The isobutylamines 184 and 185 i n h i b i t e d c e l l d i v i s i o n at 1 |j.g/mL, showed antimicrobial a c t i v i t y against Candida albicans, B. s u b t i l i s , S_. aureus and V. anguillarum a t 5 jlg/disk, and shewed mild a c t i v i t y against E. c o l i at 50 |j.g/disk.  TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED  STRUCTURE ELUCIDATION  Thalamaporella gothica floridana (Ref. 157)  An extract o f T. gothica f l o r i d a n a showed antineoplastic a c t i v i t y (PS system).  Zoobotryon v e r t i c i l l a n t u m (Delle Chiaja, 1828) (Ref. 172)  N(CH )  2  N(CH )  2  3  187  3  188  COMMENTS AND BIOLOGICAL ACTIVITIES  Proposed on the basis Compound 187 i n h i b i t e d c e l l d i v i s i o n o f the of spectral data, f e r t i l i z e d sea urchin egg (ED 5 Q = 16 ^g/mL). structures 187 and 188 Complete pharmacological evaluations o f 187 were confirmed by and 188 are i n progress. synthesis. Reduction of compound 188 (Zn-HoAc) yielded the free amine 187, oxidation of 187 T30% H202/MeOH) yielded 188 i n quantative yield.  1 46 In 1970  et al.  Pettit  1 5 7  r e p o r t e d that a number of bryozoans  contained a n t i c a n c e r c o n s t i t u e n t s .  B.  neritina  was  the  f i r s t bryozoan s p e c i e s to y i e l d a pure compound, designated as b r y o s t a t i n  1 (160).  B r y o s t a t i n 1 was  a c t i v e a g a i n s t the  murine P388 lymphocyte leukemia (PS system) both in in and  vitro,  and  i t s s t r u c t u r e was  spectroscopic t e c h n i q u e s  bryostatin  1 was  .  .  crystallographic  Since the s t r u c t u r e of  the s t r u c t u r e s determined.  bryostatin 2 (161) 161  secured by  and  r e p o r t e d , three a d d i t i o n a l b r y o s t a t i n s have  been i s o l a t e d and  4 (163)  158  vivo  15 9  , bryostatin 3 (164)  160  They were  , and  bryostatin  A l l three are s t r u c t u r a l v a r i a n t s of the same  basic bryopyran  macrolide  ring skeleton.  Whether the b r y o s t a t i n s are endogenous, or are d e r i v e d from common bryozoan food sources phytoplankton,  such as b a c t e r i a  remains to be determined.  ecological role b r y o s t a t i n 4 was  ( i f any)  As does the  of the m e t a b o l i t e s .  i s o l a t e d from B.  neritina  and  Because  collected  in both  the Gulf of Mexico (U.S.) and Gulf of Sagami (Japan), P e t t i t has s p e c u l a t e d that the m e t a b o l i t e may product  of B.  neritina  rather than  be a b i o s y n t h e t i c  from a d i e t a r y  source.  " . . . a d e f i n i t e c o n c l u s i o n regarding t h i s b i o c h e m i c a l q u e s t i o n w i l l r e q u i r e c a r e f u l chemical examination of the1 microorganisms ingested by B. neritina and/or a c e t a t e b i o s y n t h e t i c feeding 1 6 1 ( 'C) experiments."  Demonstrating the c l o s e r e l a t i o n s h i p between ecology and pharmacological  chemical  research, Christophersen  and  147  Carle,  in a series  o f p a p e r s , have d e s c r i b e d t h e i r Flustra  the marine bryozon by  a  1977  report  titled  Produced  by M a r i n e  isolated  and  foliacea  (L.)  "Anti-fouling  A l g a e and  elucidated  1 6 3 - 1 6 7  Role of  Bryozoans"  the s t r u c t u r e s  1 6 8  ,  of  m e t a b o l i t e s , o f w h i c h n i n e r e p r e s e n t e d new and  five  were known m o n o t e r p e n o i d s .  apparently  responsible  bryozoan*.  The  physostigmine, elucidated and  the  structures  i n d o l e , or q u i n o l i n e  solely  on  the b a s i s  configurations.  difference  nOe  structural  assignments.  Although  marine ecosystem, Nudibranchs,  not  p r e d a t o r s and feeding  on  one  nudibranchs bryozoan and  182  some have become h i g h l y bryozoan.  -  185  from  both  have  the  the  not was  interpretation represent made of  the bryozoan  from  predation.  of t h e main specialized,  upon  bryozoan only the their  pacifica' ).  nudibranch-  a number o f  Sessibugula  Carte  173  interesting  isolated  i n the  In some c a s e s  ( f o r example see Corambe  association  bearing  when f e e d i n g  F a u l k n e r , i n s t u d y i n g s u c h an  bryozoan  of  were  s y s t e m s were  no means f r e e  s u r p r i s i n g l y , a r e one  particular  alkaloids  s p e c t r o s c o p y i n the  are uncannily c r y p t i c  host  secondary  seem t o be w e l l a d a p t e d  t h e y a r e by  have  activity  of s p e c t r a l  E x t e n s i v e use  enhancement NMR  bryozoans  ring  i n T a b l e 7 do  Antibiotics  marine  of t h e a l k a l o i d s  on  Prompted  monoterpenoids  f o r the a n t i b i o t i c  s t r u c t u r e s drawn  absolute  The  .  they  14  work  bipyrroles  t ransIucens  * The n o n - v o l a t i l e m e t a b o l i t e s e x t r a c t e1 5d6 from F. were d e v o i d o f a n t i b a c t e r i a l a c t i v i t y .  Foliacea  and  148 the d e f e n s i v e  secretion  abdere  and T.  eliora^ \  varied  biological  isolated  of i t s nudibranch predators The s t r u c t u r a l  7  activities  from bryozoans  Tambje  d i v e r s i t y and  of the secondary  metabolites  l e a d s o n e . t o wonder what u n i q u e  type  o f m o l e c u l e s have y e t t o be d i s c o v e r e d a s more a n d more bryozoans are i n v e s t i g a t e d chemistry*. chemical potential  for their  natural products  I t i s r e a s o n a b l e t o s u g g e s t , from t h e l i m i t e d  studies  performed  so f a r , t h a t  i n the u n r e l e n t i n g  search  bryozoans  f o r drugs  show  great  from t h e s e a .  * Numerous s p e c i e s o f b r y o z o a n s c o l l e c t e d i n New Z e a l a n d w a t e r s show a n t i v i r a l a c t i v i t y . The s t r u c t u r e s o f t h e compounds r e s p o n s i b l e f o r t h i s a c t i v i t y a r e c u r r e n t l y under 2 investigation *.  1 49 B. SECONDARY METABOLITES FROM PHIDOLOPORA  PACIFICA  (ROBERTSON 1908) Phidolopora  pacifica  (see F i g u r e 22), u s u a l l y r e f e r r e d  to as the " l a c y bryozoan", i s commonly found on rocky outcrops  (depths of 3 to 15 m) i n Barkley  Columbia. P.  Classified  pacifica  Sound, B r i t i s h  i n the bryozoan order C h e i l o s t o m a t a ,  has a h i g h l y i n t r i c a t e , i n f l e x i b l e  calcium  carbonate s k e l e t o n b u i l t up i n t o a r u f f l e d l a c y network. I t i s one of about 75 genera of Bryozoa common t o the P a c i f i c Northeast  17  Our a t t e n t i o n was drawn to P. pacifica  ".  by the  absence of f o u l i n g organisms on i t s s k e l e t o n and the strong in  vitro P.  a n t i f u n g a l and a n t i a l g a l a c t i v i t y of i t s e x t r a c t s . pacifica  was f i r s t c o l l e c t e d i n May 1982 near  Diceman I s l a n d i n the Broken Group of I s l a n d s , Sound, B.C.  The bryozoan  Barkley  (143 gms d r i e d weight a f t e r  e x t r a c t i o n ) was immediately soaked i n methanol a f t e r c o l l e c t i o n and s t o r e d at room temperature f o r two days.  At  the end of t h i s time, the m a t e r i a l was ground i n a Waring blender with methanol, and f i l t e r e d .  The combined  greenish  brown methanol e x t r a c t s were c o n c e n t r a t e d t o about one quarter  of the o r i g i n a l volume and the r e s u l t i n g aqueous  methanolic suspension was p a r t i t i o n e d between b r i n e and ethyl acetate. now c o n t a i n e d  The e t h y l a c e t a t e  s o l u b l e m a t e r i a l , which  most of the d a r k i s h green c o l o r , was d r i e d  over anhydrous sodium s u l f a t e .  The sodium s u l f a t e was  f i l t e r e d o f f and the sample c o n c e n t r a t e d in vacuo 769  mg (0.54%) of a dark g r e e n i s h  brown o i l .  to give  Flash  151 chromatography gave a p o l a r f r a c t i o n which, a f t e r (179)  p u r i f i c a t i o n by p r e p a r a t i v e TLC, y i e l d e d p h i d o l o p i n (1.4 mg, (<1 mg)  (180)  0.001%, mp = 225°C) and desmethylphidolopin as l i g h t yellow c r y s t a l l i n e s o l i d s .  180 represent new  and  n a t u r a l products that c o n t a i n the  relatively rare, naturally occurring, nitro  179  functionality.  180  A second c o l l e c t i o n Sound, B.C.)  Both 179  of P.  pacifica  (Deer Group of I s l a n d s , Barkley was  in order to e x t r a c t more 179 and  worked up i n the usual 180, and  to i s o l a t e  way  any  b i o l o g i c a l l y a c t i v e compounds of l e s s e r p o l a r i t y .  After  f l a s h chromatography of the crude" organic e x t r a c t ,  fractions  having s i m i l a r chromatographic p o l a r i t i e s were pooled submitted a c t i v e and  for bioassay.  and  Three f r a c t i o n s were moderately  these were coded P1, P2 and P3.  The  least polar  1 52 of these f r a c t i o n s , f r a c t i o n P1, a f t e r p r e p a r a t i v e y i e l d e d 8.5 mg (189).  (0.15%) of  4-methoxymethyl-2-nitrophenol  S i m i l a r l y , f r a c t i o n P2 gave of  2-nitrophenol  (181)  The  l e a s t p o l a r n i t r o p h e n o l , 4-methoxymethyl( 1 8 9 ) , had a molecular formula of C e H 9 N O «  (HRMS, m/z observed 183.0534; r e q u i r e d f i v e u n i t s of u n s a t u r a t i o n .  7.59  The  1  ( d d , / = 2.0, 8.4  183.0532) demanding spectrum of 189  H NMR  8) showed resonances at 5 7.16  suggesting ring.  (179) and desmethyl-  (180).  2-nitrophenol  (Table  4-hydroxymethyl-  and f r a c t i o n P3, the most p o l a r  f r a c t i o n , gave a mixture of p h i d o l o p i n phidolopin  TLC  ( d , J = 8.4 Hz,  Hz, 1H), and 8.09  1H),  ( d , / = 2.0 Hz,  1H)  the presence of a 1 , 2 , 4 - t r i s u b s t i t u t e d benzene  A d d i t i o n a l resonances at 8 3.41  ( s , 3H) and 4.43  (s,  153 Table 8. 'H NMR data (CDC1 3 , 80 MHz) and s p e c t r a l comparisons f o r n i t r o p h e n o l s i s o l a t e d from Phidolopora pacifi  ca.  chemical s h i f t , 6  on C#  181  189  209  190  3  8.09 2.0)  (d,  8.14 2.0)  (d,  8.09 2.1)  5  7.59 2.0,  (dd, 8.4)  7.63 2.2,  (dd, 8.5)  7.59 (dd, 2.1, 8.9)  7.40  6  7.16 8.4)  (d,  7.18 8.5)  (d,  7.14 8.9)  (d,  7.02  7  4.43  (s)  4.71  (s)  4.48  (s)  0-OH  10.58  (s)  OH OMe  OEt  Me  10.58 1.61  3.41  (s)  10.57  (d,  (s)  7.82  1 0. 38  (bs)  (s) 1.28 ( t , 6.9) 3.57 ( t , 6.9) 2.31  1 54 2H) were a s s i g n e d to a b e n z y l i c methyl ether while an exchangable s i n g l e t at 6 10.58 p h e n o l i c hydrogen.  The  sharpness of the p h e n o l i c  resonance suggested i t was i n t r a m o l e c u l a r hydrogen NMR  (s) was a s s i g n e d to a hydrogen  p o s s i b l y i n v o l v e d i n an  bond.  Comparison  of the observed  chemical s h i f t s f o r 4-methoxymethyl-2-nitrophenol  to the c a l c u l a t e d v a l u e s comparison  175  (189)  gave good agreement, as d i d  to the l i t e r a t u r e v a l u e s f o r 4-hydroxy-  3-nitrotoluene  ( 190 )  1 7 6  .  The chemical s h i f t assignments f o r  both 189 and 190 are given i n Table 8.  OH  m/z  Scheme 15. I n t e r p r e t a t i o n of the MS n i t r o p h e n o l 189.  fragmentation of  The mass spectrum of 189 f u l l y supported the a s s i g n e d structure 10.4%) and  'H  (Scheme 15). 106  Fragment ions at m/z 137  ( C 7 H 6 0 , 38.4%) i n d i c a t e d the presence of an  aromatic n i t r o f u n c t i o n a l i t y . correspond to l o s s e s of N0 2 molecular i o n  (C8H902,  1 7 7  .  and  These  fragment  [CH 3 0 + N0 2 ]  ions from the  Fragmentation i n v o l v i n g the l o s s of the  methyl ether f u n c t i o n a l i t y v i a a b e n z y l i c cleavage g i v e s a  155 fragment  ion a t m/z 152 ( C 7 H 6 N 0 3 , 100%), t h e base peak i n  the spectrum. To the best of the author's knowledge, i s o l a t i o n of (189) i s only the second exam-  4-methoxymethyl-2-nitrophenol  p l e of a n i t r o c o n t a i n i n g phenol t o be i s o l a t e d marine environment. 4,6-dinitrophenol  The f i r s t  (191),  from the  example, 2-methoxy-  was i s o l a t e d as an a n t i m i c r o b i a l  c o n s t i t u e n t from the red a l g a Mar gi ni s por urn  aberrans^ . 76  S u r p r i s i n g l y , n i t r o p h e n o l 189 has not p r e v i o u s l y been reported i n the l i t e r a t u r e . I s o l a t e d from f r a c t i o n P2, 4-hydroxymethyl2-nitrophenol  (181) had a m o l e c u l a r formula C7H7NOtt (HRMS,  m/z observed 169.0379; r e q u i r e d  169.0375).  1  The H NMR  spectrum of 181 was very s i m i l a r t o that of 4-methoxymethy12-hitrophenol  (189) except f o r the absence of the methyl  ether resonance.  T h i s i n d i c a t e d that 181 was the hydroxy  d e r i v a t i v e of 189 (see Table 8 ) . 4-hydroxymethyl-2-nitrophenol a c i d i n methanol  Treatment of  (181) with p - t o l u e n e s u l f o n i c  r e s u l t e d i n the formation of methyl ether  189, thereby c o r r e l a t i n g  the two s t r u c t u r e s .  Phenol 181 had  p r e v i o u s l y been reported as a s y n t h e t i c compound with interesting biological activity Phidolopin  (vide  infra).  (179) had a m o l e c u l a r formula C 1 0 H 1 3 N 5 O 5  (HRMS, m/z observed 331.0917; r e q u i r e d 331.0917) demanding 11 degrees of u n s a t u r a t i o n .  Resonances a t 6 5.46 ( s ,  2H),  7.16  ( d , J = 8.6 Hz, 1H), 7.61 (dd, / = 2.2,8.6 Hz, 1H),  8.08  ( d , / = 2.2 H z ) , and 10.56 ( s , 1H, exchanges  with D 2 0)  156 1  i n the  H NMR  spectrum of p h i d o l o p i n  the molecule c o n t a i n e d 192.  (179)  the n i t r o p h e n o l  i n d i c a t e d that  residue,  substructure  A b e n z y l i c cleavage i n the mass spectrum of  p h i d o l o p i n , which r e s u l t e d i n the n i t r o p h e n o l residue g i v i n g r i s e to the observed base peak at m/z supported t h i s assignment.  The  b e n z y l i c methylene p r o t o n s ,  6 5.46,  192  was  in  179.  l i k e l y attached  The  ( s , 3H)  H NMR  i n d i c a t e d two  s i x degrees of ( s , 3H)  methyl groups attached  presence of at l e a s t one  and  3.59  to e i t h e r  the IR spectrum suggested  amide carbonyl  (1657  nucleus c o n t a i n i n g oxygen, methyl and  3-nitrobenzyl  substructure  to c o n s i s t of a  resonances at 6 3.39  oxygen or n i t r o g e n atoms and  purine  indicated  the  to e i t h e r a n i t r o g e n or oxygen atom  CyHyNaC^ fragment which contained 1  (C 7 H 6 N0 3 )  chemical s h i f t of  remainder of p h i d o l o p i n had  unsaturation.  152  1  cm" ).  the A  4-hydroxy-  s u b s t i t u e n t s c o u l d account f o r a l l the  157 s t r u c t u r a l requirements of p h i d o l o p i n . methyl s i n g l e t resonances to the two resonances i n the  1  H NMR  It was  high  of c a f f e i n e (193)  correspondence, as d i d the C-8 (see Table 9 ) .  Comparison of  the  f i e l d methyl showed a  resonance at 6 7.63  (s,  1H)  not p o s s i b l e however, on the b a s i s of  s p e c t r a l c o r r e l a t i o n s , to unambigously e s t a b l i s h the s u b s t i t u t i o n p a t t e r n of the s u b s t i t u e n t s on the purine The  s t r u c t u r e of p h i d o l o p i n  (179)  was  s i n g l e - c r y s t a l X-ray d i f f r a c t i o n on derivative  (194).  D e r i v a t i v e 194  ring.  t h e r e f o r e solved v i a  i t s p-bromophenacyl  was  prepared i n good y i e l d  by r e a c t i n g p h i d o l o p i n with p-bromophenacyl bromide i n the presence of KHC03 and determination  was  l8-crown-6.  performed by He  The  X-ray s t r u c t u r a l  Cun-heng and  Jon Clardy  Cornell University.  CH  CH  3  193  0  Br  N N CH  0  3  194  at  158  Table 9. 'H NMR data and s p e c t r a l comparisons f o r purine d e r i v a t i v e s i s o l a t e d from Phidol opora pacifica.  chemical  H on C or  179  180*  fl  193  shift, 6  195^  c  196^  N#  1  1 1 . 16 ( s , 1H)  1 1 . 18 ( s , 1H)  3  11.90 ( s , 1H)  8  7.63 ( s , 8.26 ( s , 7.48 ( s , 1H) 1H) 1H)  8.20 ( s , 8.11 ( s , 1H) 1H)  10  5.46 ( s , 5.41 ( s , 2H) 2H)  5.46 ( s , 5.45 ( s , 2H) 2H)  N1 Me  3.39 (s, e 3H)  3.39e ( s , 3H)  N3 Me  3.59 ( s , 3.35 ( s , 3.57e ( s , e 3H) 3H) 3H)  N7 Me  270 MHz, CDC13  3.35 ( s , 3H)  3.99 (s, e 3H)  0  400 MHz, DMSO-d6  Standard NMR Spectra No. 10393M e  3.16 ( s , 3H)  d  c  60 MHz, DMSO-d6, R e f . 179  May be r e v e r s e d  CH  3  1  CDC1 3 , S a d t l e r  8  °  159 A c r y s t a l of 194 s u i t a b l e f o r X-ray d i f f r a c t i o n  was  grown by slow evaporation of an acetone-methanolacetonitrile solution. triclinic  P r e l i m i n a r y X-ray photographs showed  symmetry, and accurate l a t t i c e c o n s t a n t s of a = 10  9.549(3), b = 9.514(1), c = 15.212(3) lO- m, a = j3 = 82.96(2), and 7 = 81.87 squares  ( 2 ) ° were determined  f i t of f i f t e e n d i f f r a c t i o n measured  72.69(1), by a l e a s t  20-values.  A f t e r c o l l e c t i o n and c o r r e c t i o n of the d i f f r a c t i o n d a t a , a phasing model was and  obtained by standard heavy atom methods,  refinements converged  to a standard c r y s t a l l o g r a p h i c  r e s i d u a l of 0.0939 f o r the observed  reflections.  F i g u r e 23  i s a computer generated p e r s p e c t i v e drawing of the  final  X-ray model of the p-bromophenacyl d e r i v a t i v e of p h i d o l o p i n l e s s hydrogens.  Bond d i s t a n c e s and angles g e n e r a l l y  agreed  w e l l with a n t i c i p a t e d v a l u e s . With the s t r u c t u r e of p h i d o l o p i n (179) a t t e n t i o n was  turned to the more p o l a r m e t a b o l i t e , des-  methylphidolopin molecular  i n hand,  (180) .  Desmethylphidolopin  formula C 1 3 H,,N 5 0 5  (180) had a  (HRMS, m/z observed  317.0777;  r e q u i r e d 317.0760).  Fragment ions observed at m/z  (100%) and  i n the mass spectrum of 180  152  (75%)  that desmethylphidolopin was p h i d o l o p i n , except  Hz,  J = 2.6 Hz,  1H) and  (DMSO-d6) of 180  indicated  i n s t r u c t u r e to  f o r the absence of one of the p u r i n e r i n g  methyl f u n c t i o n a l i t i e s . (d, J = 8.7  similar  166  Resonances at 6 5.41  1H), 7.58 11.05  suggested  (dd, / = 8.7,2.6 Hz, ( s , 1H)  i n the  the presence  'H NMR  ( s , 2H),  7.12  1H), 8.00  (d,  spectrum  of the n i t r o p h e n o l  160  161  r e s i d u e 192.  Attachment of t h i s f u n c t i o n a l i t y t o N-7 of the  p u r i n e nucleus was a s s i g n e d by analogy was supported N-8 proton  by comparisons of the chemical s h i f t of the  i n desmethylphidolopin  compounds {vide The  to p h i d o l o p i n , and  t o s u i t a b l e model  infra).  only remaining  s t r u c t u r a l f e a t u r e to be determined  for desmethylphidolopin  was the l o c a t i o n of the methyl group  on the p u r i n e n u c l e u s .  Comparison of the H NMR  desmethylphidolopin 3-methylxanthine ( 196 )  17 9  1  spectrum of  to those of both 7 - b e n z y l -  (195) and  7-benzyl-1-methylxanthine  (Table 9) showed that the methyl group i n des-  m e t h y l p h i d o l o p i n must be attached to N-3 of the purine r i n g . The chemical  s h i f t s of the N-3 methyl groups i n both des-  m e t h y l p h i d o l o p i n and 7-benzyl-3-methylxanthine c a l and resonated at 8 3.35 (DMSO-d 6 ). purine r i n g NH proton l e n t chemical  shift,  resonances  S i m i l a r l y , the  were of v e r t i a l l y  1  resonances  f o r the N-1 methyl and  NH protons  i n 7-benzyl-1-methylxanthine  resonances  i n desmethylphidolopin  196 to the analogous  showed v a s t d i f f e r e n c e s i n  shifts.  Additional  evidence  f o r an N-3 methyl group came from  the mass spectrum of d e s m e t h y l p h i d o l o p i n . m/z  The base peak at  166 c o u l d be a s s i g n e d to the 3-methylxanthine  ion 197.  equiva-  6 1 1 . 1 6 f o r 180 vs 11.18 f o r 195.  Comparison of the H NMR  chemical  were i d e n t i -  The p o s i t i o n  of m e t h y l a t i o n i n xanthines can be  r e a d i l y obtained from t h e i r mass s p e c t r a l A major fragmentation  fragment  fragmentation  i s via a retro-Diels-Alder  180  reaction  .  162  0 N  CH ^  0  3  N  ->  N  CH  N  3  196  195  i n v o l v i n g the N-1 and C-2 atoms such that 1-methylxanthine g i v e s a base peak at m/z 109 [M+(166) - CH3NCO] while 3-methylxanthine g i v e s a corresponding peak at m/z 123 [M+(166) - HNCO].  A major fragment ion at m/z 123 (27%,  HRMS, m/z obser.ved  123.0434, r e q u i r e d f o r C 5 H 5 N 3 0 123.0432)  in the mass spectrum of desmethylphidolopin  could arise v i a  l o s s of HCNO from fragment ion 197 (Scheme 16), supporting the assignment of the methyl group to N-3. The  s p e c t r o s c o p i c e v i d e n c e , i n comparison t o s u i t a b l e  model compounds, allowed the p r o p o s a l of s t r u c t u r e 180 f o r desmethylphidolopin.  To c o n f i r m the proposed s t r u c t u r e , a  s y n t h e s i s of 180 has been planned Tischler  1 8 1  .  by Andersen and  At the present time, a s y n t h e t i c sample of  d e s m e t h y l p h i d o l o p i n has not yet been  secured.  163  0  A  H\  A) 0 ™ L  M  CL  T*  H  H  T'  V» N  -  CH  3  *  HNCO  3  197 m/z  166 (100%)  Scheme  16.  m  /  123 (27%)  z  I n t e r p r e t a t i o n of the MS fragmentation of desmethylphidolopin 1 8 0 .  1. DISCUSSION Phidolopin  (179) r e p r e s e n t s a new a d d i t i o n t o the very  small but important group of n a t u r a l l y o c c u r r i n g purine d e r i v a t i v e s based on the xanthine  nucleus  that  includes  ( 1 9 3 ) , t h e o p h y l l i n e ( 1 9 8 ) , and theobromine  caffeine  (199).  I t r e p r e s e n t s only the second example of a n a t u r a l l y o c c u r r i n g xanthine organism, the f i r s t  d e r i v a t i v e t o be i s o l a t e d  was a r e p o r t that c a f f e i n e had been  i s o l a t e d from the Chinese gorgonian pseudossapo  y  8 2  .  Echinogorgia  P h i d o l o p i n i s a unique xanthine  i n that i t c o n t a i n s the r e l a t i v e l y nitro  from a marine  derivative  rare n a t u r a l l y occurring  functionality. Purine d e r i v a t i v e s based on n u c l e i other than  xanthine  are w e l l known from the marine organisms and i n most  1 64  i n s t a n c e s they have pronounced p h y s i o l o g i c a l Purine r i b o s i d e s such as d o r i d o s i n e common.  activities.  (200) a r e the most  D o r i d o s i n e (1-methylisoguanosine) was i s o l a t e d  the d i g e s t i v e gland of the d o r i d nudibranch  from  Anisodoris  and e x h i b i t s a v a r i e t y of pharmacological  nobilis  activities.  Perhaps one of the most dramatic was the  o b s e r v a t i o n that 200 caused the heart r a t e of a n e s t h e t i z e d mice t o be reduced by up t o 50 percent f o r many hours, a f t e r which the animals completely r e c o v e r  183  .  D o r i d o s i n e has  a l s o been i s o l a t e d from the A u s t r a l i a n sponge digit  at  a  1 8  nudibranch  *.  Isoguanosine  (201) , i s o l a t e d from the d o r i d 1 8 5  Di aul ul a  sandi  Tedania  egensis  , and spongosine  [2-methoxyadenosine ( 2 0 2 ) ] , i s o l a t e d from the sponge digitata**  6  are two other examples of b i o l o g i c a l l y  Tedania  active  165 p u r i n e r i b o s i d e s from marine organisms. 9-/3-D-arabinofuranosyladenine  (203) and  Finally, i t s 3'-acetate  (204) , compounds w e l l known as potent s y n t h e t i c  antiviral  a g e n t s , were i s o l a t e d as n a t u r a l products from the gorgonian Eunice!  I a cav ol i ni  1 8  7  Italian  .  205 Other purine d e r i v a t i v e s other than r i b o s i d e s have been i s o l a t e d from the marine organisms.  A g e l i n e A (205) and B  (206), m i l d i c h t h y o t o x i n s that possess moderate a n t i m i c r o b i a l a c t i v i t y , were i s o l a t e d from the  Pacific  166 sponge Agelas isolated and  sp.  1 8 8  , while hokupurine (207)  from both the nudibranch Phestilla  has  been  melanobrachi  the c o r a l upon which i t f e e d s , Tubastrea  Rather s u r p r i s i n g l y , derivatives,  a  coccinea . }89  i n c o n t r a s t to the other marine  no s i g n i f i c a n t b i o l o g i c a l a c t i v i t y c o u l d  purine be  found f o r hokupurine.  CH  On  3  the b a s i s of the examples given  unexpected to f i n d that p h i d o l o p i n had  i t was  significant  b i o l o g i c a l a c t i v i t y in the l i m i t e d b i o l o g i c a l performed in our activity  laboratory.  I t showed in  a g a i n s t Pyt hi am ultimum,  He I mint hosporium concentration three s p e c i e s .  satiurn  Rhizoctonia  with a minimum  not  testing  vitro  antifungal solani  and  inhibitory  of 70 ug per one-quarter i n c h d i s k , f o r a l l I t had  a n t i b a c t e r i a l a c t i v i t y against  167  Bacillus  subtilis  antialgal fus i formi  and Staphlococcus  aureus  s  19 0  . a c t i v i t y shown by p h i d o l o p i n c o u l d  c a t e that 179 p l a y s an a c t i v e r o l e of P. pacifica  by i n h i b i t i n g  role  Leptogorgia vitro  virgulata antialgal  diatom, Navicula  indi-  i n the chemical ecology  the growth of e p i p h y t e s .  had been p o s t u l a t e d f o r homarine  m e t a b o l i t e which was  in  Cylindrotheca  a c t i v i t y a g a i n s t the pennate diatom  The a n t i a l g a l  similar  and i t showed  isolated  A  (208), a  from the gorgonians  and L. setacea.  Homarine  (208) showed  a c t i v i t y a g a i n s t the b e n t h i c pennate  s al i ni c ol o  CH  191  .  3  208  P h e n o l i c compounds are w e l l known to be a n t i b i o t i c phytotoxic marine r e d  192  1 9 3  and  , and phenols or brominated phenols from both and brown a l g a e  19  *  the growth of p e l a g i c u n i c e l l u l a r  have been shown t o i n h i b i t algae.  P r i c e and W a i n  195  have shown that n i t r o p h e n o l s , i n c l u d i n g 4-hydroxymethyl2-nitrophenol  (181),  i n h i b i t c h l o r o p l a s t development  i n both  168 green p l a n t s and  the u n i c e l l u l a r algae Eugl ena  sp.  S t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s i n d i c a t e d that f o r s u b s t i t u t e d t o l u e n e s , a n i t r o group i n the 3 - p o s i t i o n and hydroxyl  group or ether  a  l i n k a g e i n the 4 - p o s i t i o n were  e s s e n t i a l f o r a c t i v i t y , whereas the nature of the f u n c t i o n a l group i n the b e n z y l i c p o s i t i o n c o u l d vary c o n s i d e r a b l y . appears that the n i t r o p h e n o l s be  i s o l a t e d from P.  i d e a l l y s u i t e d to i n h i b i t epiphyte  c h l o r o p l a s t development.  Further  q u i r e d to s u b s t a n t i a t e the above  pacifica  growth by  It may  inhibiting  b i o l o g i c a l work i s rehypothesis.  Xanthine based a l k a l o i d s are g e n e r a l l y of p l a n t o r g i n , t h e r e f o r e i s o l a t i o n of xanthine d e r i v e d m e t a b o l i t e s animal sources r a i s e s the q u e s t i o n origin.  of t h e i r  While gorgonians are w e l l known to  symbiotic  zooxanthellae  from  biogenetic incorporate  w i t h i n t h e i r t i s s u e s (and  therefore  p l a n t c e l l s c o u l d be the u l t i m a t e source of the c a f f e i n e found in Echenogorgia  ps e udos s a po  18 2  ) .bryozoans are  g e n e r a l l y regarded as symbiont f r e e . that P.  pacifica  T h i s hypothesis  i s o l a t i o n of p h i d o l o p i n  s p e c i e s of  Hippodi Barkley  (179)  b r y o z o a n s , Diaperoecia  pi osi a i nscul pi a Sound.  likely  o b t a i n s the xanthine based m e t a b o l i t e s  i t s phytoplankton d i e t . the  It i s perhaps  1 9 6  ,  was  from  supported by  from two a d d i t i o n a l call  for nica  and  which were a l s o c o l l e c t e d i n  If p h i d o l o p i n i s indeed d i e t d e r i v e d , the  d i e t a r y source must be s t a b l e to both seasonal locational variation  (within Barkley  i s o l a t e d from every c o l l e c t i o n of P.  and  Sound) as 179 pacifica  was  examined f o r  169 secondary  metabolites.  The  dietary hypothesis outlined  above remains to be supported by i s o l a t i o n of p h i d o l o p i n from an a l g a l  source.  The co-occurrence 2-nitrophenol  of p h i d o l o p i n 179,  (181), and  4-methoxymethyl-2-nitrophenol  e x t r a c t of P.  in a methanolic  4-hydroxymethyl-  pacifica  (189)  l e d us to examine  whether or not benzyl methyl ether 189 and benzyl a l c o h o l 181 were a r t i f a c t s of the i s o l a t i o n procedure. benzyl methyl e t h e r s have been i s o l a t e d  A number of  from marine  organisms, many of which are b e l i e v e d to be a r t i f a c t s  formed  by m e t h y l a t i o n of the p r e c u r s e r b e n z y l i c a l c o h o l s with the methanol used f o r e x t r a c t i o n 189 was P. was  1 9 7  .  To t e s t  indeed a n a t u r a l p r o d u c t , one c o l l e c t i o n of  pacifica  was  e x t r a c t e d with e t h a n o l .  worked up i n the u s u a l way  2-nitrophenol  of  (209) was 1  g e l ( f o r H NMR  methyl ether 189  c o l l e c t i o n of P.  data of 209  were found.  procedure.  4-ethoxymethyl-  see Table 8 ) .  E x t r a c t i o n of  from these  209 were a r t i f a c t s of the  Since treatment (vide  another  (180) and benzyl a l c o h o l 181  I t c o u l d be concluded  i n reasonable y i e l d  No t r a c e s  with acetone gave p h i d o l o p i n  (179), d e s m e t h y l p h i d o l o p i n  that both 189 and  ethanol e x t r a c t  i s o l a t e d a f t e r chromatography on  pacifica  no benzyl e t h e r s .  The  with care being taken not to  expose the e x t r a c t to methanol, and  silica  i f methyl ether  results  isolation  of 181 with a c i d generated  supra),  but  i t seemed reasonable  a c i d c a t a l y z e d ether formation from benzyl a l c o h o l 181 a l s o the source of the benzyl e t h e r s i n the n a t u r a l  189 that was  170 extracts.  OH  0  CH \  N0  3  2  OCH CH 2  3  210  209  A r e q u i s i t e , precursor  n a t u r a l product that c o u l d  converted to b e n z y l i c a l c o h o l 181 c o n d i t i o n s was  not  e x t r a c t s of  pacifica.  P.  starting material. phidolopin  theophylline  (210)  The  possibility  to  using  a c i d in methanol gave only  to generate 181 may  laboratory.  soluble  Attempts to c l e a v e p h i d o l o p i n  unreacted  remains that cleavage of  be c a t a l y z e d by a  such as Br", however t h i s p o s s i b i l i t y was the  laboratory  found in the e t h y l a c e t a t e  give 181 and/or 189 and p-toluenesulfonic  under  be  not  nucleophile  explored  in  IV.  The  1  H and  13  C NMR  EXPERIMENTAL  s p e c t r a were recorded on Bruker  WH-400, Bruker WP-80, N i c o l e t - O x f o r d 270 and V a r i a n XL-100 spectrometers.  T e t r a m e t h y l s i l a n e (6 = 0) was employed as an  i n t e r n a l standard f o r the 'H NMR  s p e c t r a and CDC1 3  (5 =  77.00) was used, as both an i n t e r n a l standard f o r the NMR  s p e c t r a , and as s o l v e n t , u n l e s s otherwise  L o w - r e s o l u t i o n and h i g h - r e s o l u t i o n e l e c t r o n measured on an A.E.I. spectively.  13  C  indicated.  impact MS were  MS-902 and MS-50 s p e c t r o m e t e r s , r e -  I n f r a r e d s p e c t r a were recorded on a  Perkin-Elmer model 710B spectrometer and u l t r a v i o l e t absorbances  were measured with a Cary-14 or Bausch and Lomb  S p e c t r o n i c 2000 spectrophotometer.  O p t i c a l r o t a t i o n s were  measured on a Perkin-Elmer model 141 p o l a r i m e t e r using a 10 cm m i c r o c e l l .  A F i s h e r - J o h n s apparatus was used to  determine m e l t i n g p o i n t s and these values are u n c o r r e c t e d . Gas chromatography and h i g h performance chromatography were performed  liquid  on Hewlett-Packard  Perkin-Elmer S e r i e s 2 i n s t r u m e n t s , r e s p e c t i v e l y .  5830A and A  Perkin-Elmer LC55 UV d e t e c t o r and/or a Perkin-Elmer LC-25 refractive  index d e t e c t o r were employed f o r peak d e t e c t i o n  d u r i n g HPLC.  A thermal c o n d u c t i v i t y or flame  d e t e c t o r was used f o r GC. a Magnum-9 ODS  ionization  A Whatman Magnum-9 P a r t i s i l  10 column were used  10 or  f o r p r e p a r a t i v e HPLC.  The HPLC s o l v e n t s were F i s h e r HPLC grade or Caledon HPLC  171  172 grade; water was g l a s s d i s t i l l e d ;  a l l other s o l v e n t s were  reagent g r a d e . Merck S i l i c a Gel 60 PF-254 was used f o r p r e p a r a t i v e TLC, Merck S i l i c a Gel 230-400 Mesh was used f o r f l a s h chromatography, and Merck S i l i c a Gel 60 PF-254 with CaSOa-1/2 H 2 0 was employed i n r a d i a l TLC.  173 Aldi  Collection  sa  cooperi  Data  Aldisa  cooperi  was c o l l e c t e d at v a r i o u s l o c a t i o n s i n  Barkley Sound, B r i t i s h Columbia  at depths of 1 to 15m.  One  c o l l e c t i o n was made from v a r i o u s l o c a t i o n s i n the Queen C h a r l o t t e I s l a n d s , B.C.  Immediately  a f t e r c o l l e c t i o n , the  animals were immersed whole i n methanol and s t o r e d at room temperature  f o r one t o three d a y s .  I f the animals were not  worked up immediately, they were s t o r e d at low temperature (4 to -5 °C) u n t i l used  ( u s u a l l y w i t h i n two months).  E x t r a c t i o n and Chromatographic S e p a r a t i o n  As a number of c o l l e c t i o n s of A. cooperi no v a r i a t i o n  were made and  i n m e t a b o l i t e s i s o l a t e d was observed (except  r e l a t i v e amounts) the f o l l o w i n g r e p r e s e n t s a t y p i c a l isolation  procedure.  A f t e r storage at -5 °C f o r two months the methanol used f o r e x t r a c t i o n of the whole animals was decanted and saved. The  129 whole animals (37.7 g dry weight a f t e r  extraction)  were soaked an a d d i t i o n a l four times (0.5 h each) with methanol and the combined methanol e x t r a c t s were vacuum f i l t e r e d t o g i v e an aqueous methanolic s u s p e n s i o n . The suspension was p a r t i t i o n e d between water (100 mL) and e t h y l acetate  (5 x 100 mL). The e t h y l a c e t a t e was washed with  174 br ine  ( 2 x 100 mL), d r i e d over sodium s u l f a t e ,  evaporated in  vacuo  and  to give 2.1 g (5.6%, 16 mg/animal) of an  orange o i l c o n t a i n i n g a white s o l i d . m a t e r i a l by column chromatography  F r a c t i o n a t i o n of the  (chloroform) gave crude  f r a c t i o n s which c o n t a i n e d f a t s , s t e r o l s , s t e r o i d a l  ketones,  the s t e r o i d a c i d s 2_3 and 2 4 , and g l y c e r o l ether 2 5 . Combination  of the f r a c t i o n s c o n t a i n i n g the s t e r o i d a l  followed by p r e p a r a t i v e TLC  (5:95 methanol/chloroform  on a second p l a t e , with 10:90 mg  (24)  (2_3) and  i n a r a t i o of 10:3.  measuring the  1  H NMR  The  phase p r e p a r a t i v e TLC  (2.4%  gave  359  containing  r a t i o was  acid  determined by the methyl  resonances i n  The a c i d s 2 3 and 2 4 c o u l d be separated  spectrum.  acid/100 mL)  then,  3-oxo~4,22-choladienic  the peak h e i g h t s of the C-18  by f r a c t i o n a l c r y s t a l l i z a t i o n  was  methanol/chloroform)  (1.0%, 2.8 mg/animal) of a mixture  3-oxo-4-cholenoic a c i d  acids  ( a c e t i c a c i d / w a t e r ) , reverse  (30% water/ethanol + 15 drops  or normal phase HPLC of t h e i r methyl  isopropanol/hexane).  acetic  esters  Reverse phase p r e p a r a t i v e  found to be the most c o n v e n i e n t .  Combination  TLC  of the  column f r a c t i o n s c o n t a i n i n g the s t e r o i d a l ketones f o l l o w e d by p r e p a r a t i v e TLC  (chloroform) gave 10.0 mg  mg/animal) of a mixture of s t e r o i d a l ketones mainly of cholestenone  (27).  (0.03%,  0.1  consisting  F r a c t i o n s c o n t a i n i n g the  g l y c e r o l ether were combined and p u r i f i e d by p r e p a r a t i v e to give  TLC  1 - 0 - h e x a d e c y l - g l y c e r o l ( 2 5 ) . To a i d i n the  p u r i f i c a t i o n and c h a r a c t e r i z a t i o n of 25 i t was its diacetyl derivative 4 4 .  converted to  1 75 3-Oxo-4-cholenoic a c i d 1  (23): mp 178-179 °C; UV (CH3CN) X m a v  Ilia A  236 nm (e 6200); IR (KBr) 3600 - 2400, 1720, 1700 and 1640 1  cm" ; 0.81 C21  1  H NMR (400 MHz, CDC1 3 ) 6 5.74 ( b s , 1H, C „ H ) , 2.63 -  (m, 25H), 1.21 ( s , 3H, C 1 9 Me), 0.94 ( d , Me), 0.74 ( s , 3H, C 1 B Me);  13  2  (21),  = 6 Hz, 3H,  C NMR, see Table 2; HRMS,  observed m/z 372.2658, C i,H3 6 0 3 r e q u i r e s (rel  J  372.2664; MS, m/z  i n t e n s i t y ) 373 ( 2 1 ) , 372 ( 8 2 ) , 229 ( 2 2 ) , 124 (100), 121 107 ( 2 9 ) , 95 ( 2 5 ) , 93 ( 2 3 ) , 91 ( 2 6 ) , 81 ( 2 5 ) , 79 ( 2 4 ) ,  67 ( 2 2 ) , 55 (66) , 44 ( 5 4 ) , 41 ( 3 1 ) .  3-OXO-4,22-choladienic 6.96  acid  1  (24): H NMR (80 MHz, CDC1 3 ) 6  (dd, J = 8.8,15.4 Hz, 1H, C 2 2 H ) , 5.76 ( d , / = 15.4 Hz,  1H, C 2 3 H ) , 5.74 ( b s , 1H, C „ H ) , 2.60 - 0.80 (m, 21H), 1.20 ( s , 3H, C , g Me), 1.11 ( d ,  J  = 6.4 Hz, 3H, C 2 , Me), 0.77  (s,  3H, C 1 8 Me); HRMS, observed m/z 370.2507, r e q u i r e d f o r C2„H3,03  370.2508; MS, m/z ( r e l i n t e n s i t y ) 371 ( 2 4 ) ,  370  (94), 328 ( 3 4 ) , 271 ( 5 5 ) , 229 ( 4 2 ) , 147 ( 3 0 ) , 133 ( 2 4 ) , 124 (100), 121 ( 2 3 ) , 119 ( 2 4 ) , 109 ( 2 0 ) , 107 ( 2 8 ) , 105 ( 2 7 ) ,  95  (28), 93 ( 3 0 ) , 91 ( 3 0 ) , 81 ( 2 8 ) , 79 ( 3 5 ) , 67 ( 2 6 ) , 55 ( 3 4 ) , 43 ( 3 0 ) , 41 ( 2 3 ) .  P r e p a r a t i o n of d i a c e t y l d e r i v a t i v e 44.  To 8 mg (0.025 mmole) of crude 1 - 0 - h e x a d e c y l - g l y c e r o l was added 1 mL of p y r i d i n e After  and 0.5 mL of a c e t i c  s t i r r i n g a t room temperature  anhydride.  o v e r n i g h t , the r e a c t i o n  mixture was evaporated t o dryness in vacuo  and p u r i f i e d by  176 p r e p a r a t i v e TLC t o give 8 mg (0.020 mmole, 79%) of d i a c e t y l d e r i v a t i v e 44:  *H NMR (80 MHz, CDC1 3 ) 6 5.19 (m, 1H), 4.36  (dd, J = 3.9,11.7 Hz, IH), 4.14 (dd, J = 6.7,11.7 Hz, 1H), 3.55  (d,  J  = 5.4 Hz, 2H), 3.44 ( t , / = 6.2 Hz, 2H), 2.09 ( s ,  3H), 2.07 ( s , 3H), 1.27 ( b s , 28H), 0.88 (m, 3H); HRMS, observed m/z 400.3141, C 2 3 H „ a 0 5 r e q u i r e s 400.3189; MS, m/z ( r e l i n t e n s i t y ) 400 (0.28), 297 ( 5 5 ) , 255 ( 4 5 ) , 159 ( 7 7 ) , 117 (100), 103 ( 4 8 ) , 101 ( 3 3 ) , 100 ( 8 4 ) , 97 ( 5 5 ) , 96 ( 3 4 ) , 83 ( 7 2 ) , 82 ( 4 4 ) , 71 ( 5 5 ) , 69 ( 4 4 ) , 57 ( 7 8 ) , 55 ( 5 2 ) .  S t e r o i d a l ketones  from A.  The s t e r o i d a l ketone of cholestenone  cooperi.  f r a c t i o n , which c o n s i s t e d mainly  (27_) , was not separated f u r t h e r . In 1  combination, the high r e s o l u t i o n mass spectrum and the H NMR spectrum  of the mixture i n d i c a t e d the presence of four  s t e r o i d a l ketones, m/z observed 412.3728, r e q u i r e d f o r C29H„80  412.3705; m/z observed 396.3384, r e q u i r e d f o r  C 2 8 H a i l O 396.3392; m/z observed 384.3396, r e q u i r e d f o r C27H,,ftO 384.3392; m/z observed 382.3246, r e q u i r e d f o r 1  C 2 7 H 4 2 0 382.3235; H NMR 6 5.75 ( s ) , 5.21 (m), 4.73 ( b s , C=CH 2 ), 4.67 ( b s , C=CH 2 ), 1.57 ( b s ) , 1.18 ( s ) , 0.94 ( d , J = 6.4 H z ) , 0.71 ( s ) ; UV (Hexane) X m a x 229 nm.  S t e r o i d a l ketones from Anthoarcuata  graceae.  1 77 Ant hoarcuat  a graceae  was c o l l e c t e d a t v a r i o u s l o c a t i o n s  in Barkley Sound, B.C. at depths of 1 to 1 5 m .  Immediately  a f t e r c o l l e c t i o n the sponge (592 g dry weight a f t e r e x t r a c t i o n ) was immersed whole i n methanol and s t o r e d at room temperature f o r 2 days.  At the end of t h i s time the  sponge was homogenized i n a Waring blender and vacuum filtered  i n the presence of C e l i t e .  e x t r a c t was concentrated  The r e s u l t i n g crude  to about 1.8 L and p a r t i t i o n e d be-  tween b r i n e and e t h y l acetate  (5 x 400 mL).  The combined  e t h y l a c e t a t e l a y e r s were washed with b r i n e , d r i e d over sodium s u l f a t e , f i l t e r e d , and concentrated 5.2 g (0.88%) of a gummy orange o i l .  in vacuo  t o give  The o i l was  f r a c t i o n a t e d by column chromatography (gradient of EtOEt i n CHC1 3 ) t o give 61.8 mg (0.010%) of a mixture of s t e r o i d a l ketones whose 'H NMR spectrum was e s s e n t i a l l y  i d e n t i c a l to  that of the mixture of s t e r o i d a l ketones obtained Al di s a  from  cooperi.  M e t h y l a t i o n of 3-oxo-4-cholenoic a c i d ( 2 3 ) .  To 2 mg (0.0054 mmole) of (23) i n 1 mL ether was added 2 mL of f r e s h l y prepared  e t h e r e a l diazomethane  MNNG - Diazomethane a p p a r a t u s ) . reaction (96%)  A f t e r completion  (TLC), the ether was removed i.n vacuo  of e s t e r 28: UV X m a x  (Aldrich's of the  t o give 2 mg  (MeOH) 241 nm (e 15,000); IR 1  (CHC13 c a s t ) 2940, 1740, 1675, 1190, 1170 cm" ; 'H NMR (400 MHz, CDC1 3 ) 6 5.74 ( b s , 1H, C» H ) , 3.68 ( s , 3H, OMe), 1.19  178 ( s , 3H, C 1 9 Me), 0.93 ( d , J 3H, C 1 B Me);  13  =  6.8 Hz, 3H,  C i 2  Me), 0.7 2  (s,  C NMR see Table 2; HRMS, observed m/z  386.2821, r e q u i r e d f o r C 2 5 H 3 8 0 3  386.2821; LRMS, m/z  (rel  i n t e n s i t y ) 386 ( 8 5 ) , 263 ( 2 3 ) , 229 (38), 147 ( 3 0 ) , 133 124  (25),  (100), 107 ( 4 1 ) , 105 ( 3 2 ) , 93 (44), 91 ( 4 4 ) , 81 ( 5 6 ) , 79  (45), 67 ( 4 2 ) , 55 ( 9 6 ) , 43 ( 5 8 ) , 41  (61).  M e t h y l a t i o n of a mixture of 3-oxo-4-cholenoic a c i d 3-QXO-4,22-choladienic  26.3  (23) and  a c i d (24)  mg (0.071 mmole) of a crude mixture of 23 and 24  were t r e a t e d with diazomethane as p r e v i o u s l y d e s c r i b e d f o r 23.' E v a p o r a t i o n of the s o l v e n t gave 26.0 mg (95%) of a mixture of e s t e r s 28_ and 3_8.  P u r i f i c a t i o n by normal phase  HPLC (2.4% isopropanol/hexane) gave pure e s t e r 3_8: UV X m a x (MeOH) 239, 214 nm; IR (CHC13 c a s t ) 2930, 2850, 1723, 1270, 16,9  1  1674,  1  1240 cm" ; H NMR (400 MHz, CDC1 3) 6 6.84 (dd, / = Hz, 1H, C 2 2 H ) , 5.76 ( d , J  =  16 Hz, 1H, C 2 3 H ) ,  5.74  ( s , 1H, C „ H ) , 3.74 ( s , 3H, OCH 3 ), 1.19 ( s , 3H, C 1 9 Me ) , 1.10 ( d , J = 7 Hz, 3H C 2 1 Me), 0.75 ( s , 3H, C 1 8 Me); HRMS, observed m/z 384.2655, r e q u i r e d f o r C 2 5 H 3 6 0 3  384.2664; LRMS,  m/z  ( r e l i n t e n s i t y ) 384 ( 7 2 ) , 342 (22), 272 ( 2 1 ) , 271  (81),  269  ( 2 0 ) , 253 ( 2 1 ) , 225 ( 4 6 ) , 201 ( 2 0 ) , 175 ( 2 2 ) , 147  (24),  124  ( 5 3 ) , 107 ( 2 1 ) , 105 ( 2 1 ) , 95 ( 2 1 ) , 93 ( 2 1 ) , 91 ( 2 2 ) , 81  ( 2 9 ) , 79 (100), 77 ( 2 3 ) , 55 ( 3 0 ) , 41  (22).  179 Melibe  Leoni  na  C o l l e c t i o n , e x t r a c t i o n and chromatographic  Meli be leonina  (38 animals) was  Park, North Vancouver, B.C.  separation.  c o l l e c t e d at Cates  on October 6,  1981.  Immediately  a f t e r c o l l e c t i o n , the nudibranchs were immersed whole in chloroform  ( 2 L ) , and  upon r e t u r n i n g to the l a b o r a t o r y were  e x t r a c t e d on a w r i s t a c t i o n shaker f o r 1.5 chloroform  was  separatory  f u n n e l , d r i e d over sodium s u l f a t e ,  concentrated mg  separated  h.  in  vacuo  The  from the water l a y e r i n a and  (dry i c e t h i m b l e , < 20°C) to give  of an orange " g r a p e f r u i t " s m e l l i n g o i l .  column chromatography  (step g r a d i e n t  d i e t h y l ether/chloroform)  of  Silica  100%  f a t s , s t e r o l s , 2,6-dimethyl-5-heptenal (53_) 2,6-dimethyl-5-heptenoic a c i d ( 5 4 ) .  of 5_3.  gave 19 mg  and  P o o l i n g of  the  (33% of choloform s o l u b l e e x t r a c t )  ( d i e t h y l ether) gave 3 mg  s o l u b l e e x t r a c t ) of  (400 MHz,  CDC1 3 ) 6 9.62  H ) , 2.36  1H, C 3  H),  (m, 1.70  (5% of  by  chloroform  54.  2,6-dimethyl-5-heptenal (53): IR  (m,  TLC  Combination of f r a c t i o n s c o n t a i n i n g 5_4 followed  p r e p a r a t i v e TLC  1H, C 5  40%  hydrocarbons,  f r a c t i o n s c o n t a i n i n g 5_3, followed by p r e p a r a t i v e (chloroform)  gel  hexane to  gave s t r a i g h t c h a i n  57.4  (CHC1 3 ) 1723  ( d , J = 1.9  1H, C 2  H), 2.05  (bs, 3H, C 8  Me),  Hz,  2  cm" ;  1  NMR  1H, C, H), 5.10  (m, 2H, C „ 2H), 1.61  H  (bs, 3H, C 7  (m,  1.77 Me),  180 3  1.41 (m, 1H, C 3 H ) , 1.11 ( d , / = 7.1 Hz, 3H, C 9 Me); ' C NMR see Table 4; GC-MS, m/z ( r e l i n t e n s i t y )  140 ( 2 . 2 ) , 125 (0.3)  82 (100), 69 (26.9), 67 (67.0), 55 (26.3), 41 (73.4).  2,6-dimethyl-5-heptenoic a c i d 1  (54): IR (CHC13) 3500 - 2200,  1  1700 cm" ; H NMR (400 MHz, CDC1 3 ) 8 5.13 (m, 1H), 2.49 (m, 1H), 2.05 (m, 2H), 1.76 (m, IH), 1.70 ( b s , 3H), 1.62 3H),  1.48 (m, 1H), 1.20 ( d , J = 7.1 Hz, 3H); HRMS, m/z  observed (rel  (bs,  156.1151, r e q u i r e d f o r C 9 H 1 6 0 2  intensity  156.1151; MS, m/z  ) 156 ( 2 2 ) , 138 ( 3 . 1 ) , 83 (100), 82 (46.4),  74 (56.1), 69 (65.5), 67 (32.4), 55 (49.3), 41 (95.0).  181 Acanthodoris  nanaimoensis  C o l l e c t i o n Data  Acanthodoris  nanaimoensis  was c o l l e c t e d at v a r i o u s  l o c a t i o n s i n Barkley Sound, and from Sunset Beach near Horseshoe Bay, B r i t i s h Columbia. separate  No attempts were made to  the small f r a c t i o n of A. hudsoni  c o - c o l l e c t e d with A. nanaimoensis had  that was  at some s i t e s .  shown that the s k i n e x t r a c t of A. hudsoni  nanaimoal 61. variation  Hellou  198  a l s o contained  In a l l the c o l l e c t i o n s made there was l i t t l e  i n the r a t i o of nanaimoal (61): i s o a c a n t h o d o r a l  (6_5): acanthodoral  (6_4) i s o l a t e d .  Immediately a f t e r  c o l l e c t i o n the animals were immersed whole i n methanol and s t o r e d at room temperature f o r one to three days.  I f the  animals were not worked up immediately they were s t o r e d at low  temperature (4 t o -5 °C), i n the dark, u n t i l used  ( u s u a l l y w i t h i n two months).  E x t r a c t i o n and Chromatographic  Separation  As a number of c o l l e c t i o n s of A. nanaimoensis and  no v a r i a t i o n  in metabolites  were made  i s o l a t e d was observed, the  f o l l o w i n g r e p r e s e n t s a t y p i c a l procedure.  A l l weights of  the v o l a t i l e a l d e h y d i c components are i n e r r o r as i t was i m p o s s i b l e to remove a l l t r a c e s of s o l v e n t under high vacuum without s u b s t a n t i a l l o s s e s of the s e s q u i t e r p e n o i d s .  182 A.  nanaimoensis  F e b r u a r y , 1982,  (120 animals) was c o l l e c t e d i n  i n B a r k l e y Sound, B r i t i s h Columbia.  They  were immediately immersed whole in methanol and stored at room temperature  f o r two d a y s .  At the end of t h i s time the  methanol used f o r e x t r a c t i o n of the whole animals decanted and saved.  was  The nudibranchs were soaked an  a d d i t i o n a l f i v e times (0.5 h each) with methanol and the methanol e x t r a c t s were combined and c o n c e n t r a t e d in  vacuo  (<  20°C, dry i c e / a c e t o n e t h i m b l e ) . t o one-quarter of the o r i g i n a l volume.  The aqueous methanolic e x t r a c t  then p a r t i t i o n e d between b r i n e 100 mL).  (100 mL)  vacuo  was  and c h l o r f o r m  (4 x  The combined o r g a n i c l a y e r s were then washed with  b r i n e and d r i e d over sodium s u l f a t e . in  (700 mL)  gave 2.3  Removal of the s o l v e n t  g (16 mg/animal) of an o d o u r i f e r o u s orange  oil. Column chromatography ( g r a d i e n t of 100% hexane to 10% ethyl acetate/chloroform) yielded f r a c t i o n s containing s t e r o l s , and the s e s q u i t e r p e n o i d aldehydes nanaimoal acanthodoral  (64),  and  i s o a c a n t h o d o r a l (6_5) .  fats,  (61),  P o o l i n g of the  a l d e h y d i c f r a c t i o n s f o l l o w e d by removal of the s o l v e n t gave 218.6  mg  (1.8 mg/animal, 10% of the crude c h l o r o f o r m  e x t r a c t ) of a s l i g h t l y yellow o i l y mixture c o n s i s t i n g of 61 (1.4 mg/animal), 64 (0.2 mg/animal), and 65 (0.4 mg/animal) in a r a t i o of 79:1:20 as determined by a n a l y t i c a l GC. most abundant aldehydes p r e p a r a t i v e GC  (3% OV-17  The  (61 and 65) c o u l d be separated by on Chromosorb (HP) 80/100 Mesh,  183  i n i t i a l temperature R  t  65 17.6 min.)  140°C, rate  1°C/min., R  t  61 16.2 min.,  or HPLC (50:50 hexane/methylene c h l o r i d e ) .  HPLC was found to be the most c o n v e n i e n t , a l t h o u g h i t was not p o s s i b l e  to p u r i f y acanthodoral (64) adequately by t h i s  method.  Nanaimoal (61): c o l o u r l e s s  o i l , [ a ] D - 7 ° (c 3.0, CHC1 3 ); IR  (CHC1 3 ) 2920, 2750, 1730, 1470, 1395, 1380, 930, 870, 800 1  and 750 cm" ; 'H NMR (Table 5); [ m u l t i p l i c i t i e s determined 133.8  13  C NMR (100.6 MHz, CDC13)  by SFORD experiment]  6 203.3  ( s ) , 125.3 ( s ) , 53.7 ( t ) , 43.7 ( t ) , 39.8 ( t ) ,  (d),  34.8  ( t ) , 33.6 ( s ) , 31.6 ( t ) , 28.1 ( s ) , 27.9 ( q ) , 27.9 ( q ) , 25.9 ( q ) , 21.3 ( t ) , 19.4 ( t ) ; HRMS, m/z observed 220.1836,  re-  q u i r e d f o r C 1 5 H 2 l t O 220.1827; GC-MS, m/z ( r e l i n t e n s i t y ) 220 ( 4 ) , 177 ( 6 ) , 176 ( 4 9 ) , 162 ( 1 6 ) , 161 (100), 121 ( 6 ) , 105 (33), 69 ( 7 ) , 55 ( 12) , 41  Acanthodoral  (64): c o l o u r l e s s  r e s i d u a l CHC13 (d,  J  0.88  (24).  1  o i l ; H NMR (400 MHz, CDC1 3 ,  (8 7.25) used as i n t e r n a l r e f e r e n c e ) 6 9.59  = 2.8 Hz, 1H), 1.82 ( d , / = 9.2 Hz, 1H), 1.06 ( s ,  3H),  ( s , 3H), 0.82 ( s , 3H). GC-MS, m/z ( r e l i n t e n s i t y ) 220  ( 1 ) , 205 ( 1 4 ) , 187 ( 9 ) , 177 ( 1 0 ) , 176 ( 4 8 ) , 161 ( 4 8 ) , 137 (28),  121 (24), 109 ( 1 8 ) , 107 ( 2 6 ) , 105 ( 3 8 ) , 97 ( 1 4 ) , 95  (44), 93 (29), 91 ( 2 4 ) , 84 (100), 81 (54), 79 ( 2 8 ) , 69 (43), 67  ( 2 2 ) , 55 (35), 41  (76).  Isoacanthodoral (65): c o l o u r l e s s  o i l ; *H NMR (400 MHz,  1 84 CDC1 3 ) 6 9.72  (dd, / = 3.3,3.2 Hz,  (dd, / = 14.8,3.3 Hz,  1H), 2.14  (dd, / = 14.8,3.2 Hz, 13  ( b s , 3H), 1.00  MHz,  CDC1 3 ) 6 204.6, 135.4, 129.9, 57.1, 46.3, 29.0,  26.8,  23.6,  0.91  (bs, 1H),  1.65  32.3,  ( s , 3H)  1H), 5.23  20.0,  ( s , 3H);  19.3  C  (the two  NMR  1H),  (100.6  40.1,  38.4,  quaternary  carbons c o u l d not be c o n f i d e n t l y a s s i g n e d due to the sample s i z e ) ; GC-MS, m/z ( r e l i n t e n s i t y ) 176  ( 3 5 ) , 121  (97), 74  (26), 107  178  (88), 95 ( 9 2 ) , 93  ( 2 8 ) , 69 (100), 55  ( 3 4 ) , 41  2.71  limited  (12), 177  ( 2 3 ) , 91  (77),  ( 2 4 ) , 81  (67).  Reduction of the mixture of aldehydes  61, 64, and  65.  In order to reduce the v o l a t i l i t y of the s e s q u i t e r p e n o i d aldehydes, to s i m p l i f y t h e i r s e p a r a t i o n , and to o b t a i n c r y s t a l l i n e d e r i v a t i v e s , the s e s q u i t e r p e n o i d aldehyde  fraction  from A. nanaimoensis  sodium b o r o h y d r i d e . 57.3  mg  (1.51  To a 50 mL  round bottom f l a s k was  A t o t a l of 161.7  mixture of crude 6_1, 64, and 6_5 was isopropyl alcohol  mg  (0.735 mmol) of the  ( s o l u t i o n turned cloudy) and added  to the sodium borohydride with s t i r r i n g .  added and s t i r r e d  (50 mL)  The  A f t e r 24 h r , 30 mL of  f o r an a d d i t i o n a l 2 h r .  end of t h i s time the r e a c t i o n mixture was tween water  added  d i s s o l v e d i n 30 mL of  s o l u t i o n c l e a r e d a f t e r a few minutes. water was  reduced with  mmol) of sodium borohydride and 2 mL of  isopropyl alcohol.  dropwise  was  partitioned  and c h l o r o f o r m (4 x 25 mL).  At the be-  The com-  bined c h l o r o f o r m l a y e r s were d r i e d over magnesium s u l f a t e  185 and f i l t e r e d .  Evaporation of the solvent p r o v i d e d 82.8 mg  (0.373 mmol, 51%) of a mixture of 61 (56% by GC, 3% SP2250 on Chromosorb (HP) 80/100 Mesh, 160°C f o r 10 min then 10 °C/min, R  7.71 m i n ) , 64 ( 4 % , R  t  {  5.79 min) and 65 (36%, R  t  8.14 m i n ) .  The remaining 4% was due to the minor  metabolites  (1%, R  t  4.08 min; 3%, R  not c h a r a c t e r i z e d f u r t h e r .  {  11.65 min) that were  The low y i e l d  i s l i k e l y due to weighing e r r o r  f o r the aldehydes ( t r a c e s of  s o l v e n t were not removed on h i g h vacuum). TLC  (silica  nanaimool  i n the r e d u c t i o n  Repetitive  radial  g e l , 100% CHC1 3 ) p r o v i d e d 25.8 mg of pure  (70): o i l ;  [ o ] D + 1 0 . 4 ° (c 0.61, MeOH); IR (CHC1 3 ) 1  3600, 3400, 2920, 1460, 1380, 1360, 1020, 910, and 740 cm" ; 1  H NMR  (400 MHz, CDC1 3 ) 5 3.72 (m, 2H), 1.97 ( b s , 2H), 1.78  (bs, 2H), 1.7 5 (bd,  J  =  17 Hz, 1H), 1.59 (bd,  J  1H), 0.98 ( s , 3H), 0.97 ( s , 3H), 0.88 ( s , 3H);  = 13  17 Hz,  C NMR  (100.1 MHz, CDC1 3 ) 6 133.4, 125.5, 59.7, 44.0, 39.9,  34.8,  31.8, 30.8, 28.0, 27.9, 24.9, 21.5, 19.5; HRMS, m/z observed 222.1988, r e q u i r e d f o r C 1 5 H 2 6 0 222.1984; MS, m/z  (rel  i n t e n s i t y ) 222 ( 4 0 ) , 207 (100), 189 ( 3 0 ) , 179 ( 2 4 ) ,  177  ( 3 3 ) , 121 (20); and 13.0 mg of a mixture of 97 and 118 (87:13 by GC) which was used d i r e c t l y the (p-bromophenyl)urethane  f o r the p r e p a r a t i o n of  derivatives.  P r e p a r a t i o n of the (p-bromophenyl)urethane d e r i v a t i v e s of 97 and 118.  186 A t o t a l of 13.0 mg (0.0586 mmole) of the mixture of 97 and  118 i n 3 mL of carbon t e t r a c h l o r i d e was added to a 5 mL  r e a c t i o n v i a l c o n t a i n i n g 67.9 mg (0.343 mmole) of 4-bromophenyl isocyanate  i n 1.5 mL of carbon  tetrachloride.  The  v i a l was sealed and heated with s t i r r i n g  at 60°C f o r 20  h.  At the end of t h i s time the r e a c t i o n was cooled to room  temperature; t r a n s f e r r e d to a 25 mL f l a s k and the excess 4-bromophenyl isocyanate was destroyed with methanol. Evaporation (100%  of the s o l v e n t , followed by p r e p a r a t i v e TLC  c h l o r o f o r m ) , gave 24 mg (0.0571 mmole, 98%) of a  mixture of 98 and 114. ration  P r e p a r a t i v e reverse phase HPLC sepa-  (15% w a t e r / a c e t o n i t r i l e ) gave pure samples of 98 and  114. ,  98: o i l , [ a ] D - 3 9 ° (c 0.88, hexane);  'H NMR  (400 MHz, CDC1 3 )  6 7.40 ( d , 2H), 7.27 ( d , 2H), 6.50 (br s, 1H), 5.06 (br s, = 6.4 Hz, 1H), 4.18 (m, 2H) , 2.10 (ddd,  J  =  13.0,6.4,9.0 Hz, 1H), 1.48 (ddd, J = 13.0,6.7,8.9 Hz, 1H), 1.61 (bs, 3H), 1.01 ( s , 3H), 0.90 ( s , 3H);  13  C NMR  MHz, CDC1 3 ) only t e r p e n o i d carbons are l i s t e d ,  (100.6  6 134.2,  131.0, 63.3, 45.6, 42.5, 40.4, 38.0, 37.5, 34.1, 32.3, 29.0, 26.5,  23.3, 20.0, 19.3; MS, m/z ( r e l i n t e n s i t y ) 421 ( 1 ) , 419  (1),  244 ( 1 ) , 242 ( 2 ) , 217 ( 9 ) , 215 ( 9 ) , 204 (31), 189 ( 1 8 ) ,  177 (31),  114:  (100), 107 (39), 105 (13), 95 (31), 93 (13), 91 (16), 81 69 (32), 55 ( 1 4 ) , 41 ( 1 9 ) .  mp 109-110 °C (hexane);  1  H NMR  (400 MHz, CDC1 3 ) 5 7.40  187 (d,  2H), 7.27 ( d , 2H), 6.50 ( b s , 1H), 4.17 (dd, J=  11.1,7.7  Hz, 1H), 4.14 (dd, J = 11.1,6.9 Hz, 1H), 1.84 ( d , J = 9.2 Hz, 1H), 1.09 ( d ,  = 9.2 Hz, 1H), 0.96 ( s , 3H), 0.89 ( s ,  J  3H), 0.81 ( s , 3H); HRMS, m/z observed 421.1441 and 419.1438, required- f o r C 2 2 H 3 0 B r N 0 2  421.1439 and 419.1416; MS, m/z  i n t e n s i t y ) 204 ( 6 6 ) , 189 (100), 161 ( 2 0 ) , 95 ( 2 3 ) , 81  (rel  (30),  69 ( 2 4 ) .  A c i d c a t a l y z e d i s o m e r i z a t i o n of 98. P r e p a r a t i o n of 1 1 2 .  A t o t a l of 2.5 mg of 9ji was placed i n a 2 mL r e a c t i o n vial,  1.5 mL of 98-100% formic a c i d was added and the v i a l  was capped.  A f t e r h e a t i n g at 70 °C o v e r n i g h t , the r e a c t i o n  was cooled and concentrated brownish r e s i d u e .  (in  vacuo)  The r e s i d u e was d i s s o l v e d i n ether (25  mL) and washed with 5% b i c a r b o n a t e x 10 mL).  to give a l i g h t  ( 2 x 1 0 mL) and water (1  The ether l a y e r was d r i e d over sodium s u l f a t e ,  f i l t e r e d and the s o l v e n t removed  in  vacuo  to give, after  p r e p a r a t i v e TLC ( c h l o r o f o r m ) , 1.1 mg of. 1 1 2 .  No t r a c e s of  1  98 were d e t e c t e d by H NMR or HPLC (reverse phase, 10:90 water/acetonitrile). 7.41  1 1 2 : o i l ; 'H NMR (400 MHz, CDC1 3 ) 5  (m, 2H), 7.27 (m, 2H) , 6.49 ( b s , 1H) , 5.29 ( b s ,  11.6 Hz, 1H), 1.59 ( b s , 3H), 0.88 ( s , 3H), 0.80 ( s , HRMS, m/z observed 421.1463, r e q u i r e d f o r C 2 2 H 3 0  81  = 3H);  BrN02  421.1439; MS, m/z ( r e l i n t e n s i t y ) 421 ( 1 ) , 419 ( 1 ) ,  217  (23),  215 ( 2 3 ) , 205 ( 1 3 ) , 204 ( 7 7 ) , 189 (100), 177 ( 4 3 ) ,  (17),  161 ( 2 7 ) , 133 ( 1 4 ) , 121 ( 1 9 ) , 119 ( 2 8 ) , 107 ( 2 8 ) ,  175 106  188 (39), 79  105 (54), 95 ( 1 8 ) , 94 ( 2 6 ) , 93 (28), 91 ( 3 1 ) , 81  (23),  (18); 69 (22), 55 ( 2 2 ) .  P r e p a r a t i o n of nanaimoal's  (p-bromophenyl)urethane  d e r i v a t i v e 75.  A t o t a l of 16.2 mg (0.073 mmol) of 70 was r e a c t e d with 4-bromophenyl i s o c y a n a t e (65.2 mg, 0.329 mmol) by the method d e s c r i b e d f o r 97 and 118 to g i v e , a f t e r p r e p a r a t i v e TLC 1  ( c h l o r o f o r m ) , 31 mg (0.073 mmol, 100%) of 75: o i l ; H NMR (see  Table 5 ) ,  13  C NMR  (100.6 MHz, CDC13) [ m u l t i p l i c i t i e s  determined by SFORD experiment]  153.5 ( s ) , 137.1 ( s ) ,  133.4  ( s ) , 132.1 ( d ) , 125.3 ( s ) , 120.2 ( d ) , 115.8 ( s ) , 62.8 ( t ) , 43.7  ( t ) , 39.8 ( t ) , 39.5 ( t ) , 34.4 ( t ) , 33.5 ( s ) , 31.68 ( t ) ,  30.71  ( s ) , 28.0 ( q ) , 27.8 ( q ) , 24.7 ( q ) , 21.3 ( t ) , 19.4 ( t ) ;  HRMS, m/z observed 421.1439, r e q u i r e d f o r C 2 2 H 3 0  81  BrN02  421.1439; MS, m/z ( r e l i n t e n s i t y ) 216 (10), 214 ( 1 0 ) , 204 (33),  189 (100), 176 (27), 161 ( 4 0 ) , 105 ( 3 2 ) , 91 (21), 55  (23) .  P r e p a r a t i o n of the 2,4-dinitrophenylhydrazone d e r i v a t i v e s of 61, 65, and 64.  78.6 mg (0.357 mmol) of the aldehyde mixture was d i s s o l v e d i n 2 mL of methanol and 10 mL of a mixture containing  108 mg of 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e i n 10 mL of  methanol was added.  The mixture was s t i r r e d  f o r three hours  189 a f t e r which i t was p a r t i t i o n e d between water chloroform  (4 x 25 mL).  The chloroform  (50 mL) and  l a y e r s were com-  b i n e d , d r i e d over sodium s u l f a t e , f i l t e r e d , and evaporated to g i v e  131.1 mg (0.328 mmol, 92%) of a mixture of  2,4-dinitrophenylhydrazone  d e r i v a t i v e s 211, 212 and 96.  211  Separation  212  by normal phase p r e p a r a t i v e HPLC (10% 1  chloroform/hexane) gave pure 9 6 : H NMR 10.9  ( b s , 1H), 9.08 ( d ,  J  =  (270 MHz, CDC1 3 ) 6  2.5 Hz, 1H), 8.25 (dd,  J  =  2.5,9.4 Hz, 1H), 7.88 ( d , / = 9.4 Hz, 1H), 7.42 (dd, / = 6.0,6.0 Hz, 1H), 5.11 ( b s , 1H), 2.79 (dd, / = 6.0,14.0 Hz, 1H), 2.24 (dd, / = 6.0,14.0 Hz, 1H), 1.95 (m, 3H), 1.66 ( b s , 3H), 1.03 ( s , 3H), 0.90 ( s ,  3H).  P r e p a r a t i o n of r e g i o i s o m e r i c a l c o h o l s 76 and 84.  190 Following T i s c h l e r ' s p r o c e d u r e  115  21.3 g (0.247 mol) of  3-methyl-3-buten-1-ol (80) was combined with 8.4 g (0.062 mol) of myrcene K a r i u s tubes.  (7j)) and d i v i d e d e q u a l l y among three 37 cm Nitrogen gas was bubbled through the  s o l u t i o n s f o r 15 min a f t e r which the tubes were sealed and p l a c e d in an oven.  The r e a c t i o n mixture was heated to  230 °C f o r 8 h and then allowed  to c o o l t o RT  Upon c o o l i n g , two l a y e r s s e p a r a t e d . ed l i t t l e  overnight.  A n a l y t i c a l TLC  indicat-  c o m p o s i t i o n a l d i f f e r e n c e s between the l a y e r s .  The  m a t e r i a l from the three tubes was combined, d i s s o l v e d in ether  (150 mL) and washed with water (50 mL).  The  light  yellow ether l a y e r was d r i e d over sodium s u l f a t e and the ether was removed in hexane  vacuo.  The r e s i d u e was taken  up i n  (100 mL), added to the top s u r f a c e of s i l i c a g e l (250  g) i n a 14 cm Buchner f u n n e l , and the hexane was drawn through the s i l i c a  g e l by s u c t i o n .  A d d i t i o n a l hexane  mL) was added and drawn through the s i l i c a . was repeated  with a d d i t i o n a l hexane  50% chloroform/hexane ethyl acetate  (500 mL), and  The mixture of r e g i o i s o m e r i c  a l c o h o l s , J59 and 90, was present wash.  The procedure  (500 mL), followed by  (500 mL), c h l o r o f o r m  (2 x 500 mL).  (200  i n the f i r s t  ethyl acetate  Removal of the s o l v e n t gave 7.58 g of a mixture that  contained at l e a s t three components as i n d i c a t e d by a n a l y t i c a l TLC. a l c o h o l iBO.  The major component was recovered  starting  F r a c t i o n a t i o n of a 2.48 g p o r t i o n of t h i s  mixture by f l a s h chromatography (5 cm diameter column, 6 inches s i l i c a  g e l , 10% e t h y l acetate/petroleum  ether) gave  191 264  mg  (1.19 mmole, 6% c a l c u l a t e d  alcohol  f r a c t i o n ) of crude r e g i o i s o m e r s 76 and  (400 MHz,  CDC1 3 ) 6 5.35  H z ) , 1.68  (bs, C H 3 ) ,  ( s , CH 3 );  MS,  179 93  ( 1 5 ) , 177 ( 5 0 ) , 81  (95).  y i e l d based on  m/z  1.61  ( b s ) , 5.29  0.915  ( r e l i n t e n s i t y ) 222  ( 1 4 ) , 161  (23), 79  (21), 135  (25), 69  84:  ( 1 7 ) , 109  6.4  0.911  ( 1 ) , 189  ( 2 3 ) , 43  (12% e t h y l  NMR  ( 3 5 ) , 107  Separation of the a l c o h o l s 76 and 84 was  r e c y c l i n g r a d i a l chromatography  H  ( s , CH3),  ( 3 ) , 207  (100), 55  1  (bt, J =  ( b s ) , 5.08  (bs, C H 3 ) ,  entire  (3),  (45),  ( 2 0 ) , 41 achieved  by  acetate/petroleum  e t h e r , flow = 3.5 mL/min) to g i v e pure samples of 7_6 and These were converted d i r e c t l y i n t o the urethane d e r i v a t i v e s  8_9 and  P r e p a r a t i o n of a mixture derivatives  89 and  (p-bromophenyl)-  90.  (p-bromophenyl)urethane  90.  A t o t a l of 16.6 r e g i o i s o m e r s 76 and mg  of  mg  (0.0748 mmol) of the crude  .  84 were r e a c t e d i n the usual way  (0.379 mmol) of 4-bromophenyl isocyanate i n a 10  reaction  flask.  Methanol was  4-bromophenyl isocyanate and p r e p a r a t i v e TLC  of a mixture  7.40  ( d ) , 7.27  H z ) , 4.24 CH3),  the mixture  of 89 and 90:  ( d ) , 5.37  (m), 1.68  1  H NMR  ( b s ) , 5.30  (bs, CH3),  0.933 ( s , C H 3 ) .  with  1.60  was mg  excess  p u r i f i e d by (0.0174 mmol,  (400 MHz,  75  mL  added to decompose the  (chloroform) to g i v e 7.3  23%)  84.  CDC1 3 ) 6  ( b s ) , 5.09  (bt,  (bs, C H 3 ) ,  0.943 ( s ,  J  =  1  1 92 P r e p a r a t i o n of a mixture of (±)-75 and 93•  A t o t a l of 5.0 mg (0.012 mmol) of the mixture of r e g i o i s o m e r s 89 and 90 were combined with 5 mL of 98-100% formic a c i d stir  i n a 10 mL round bottom f l a s k equiped with a  bar and condenser.  The s o l u t i o n was heated, with  s t i r r i n g , at" 70 °C f o r 12 h.  At the end of t h i s p e r i o d the  r e a c t i o n mixture had turned p u r p l i s h brown. evaporated to dryness in vacuo,  The mixture was  taken up i n e t h e r , washed  with 5% sodium bicarbonate (2 x 5 mL), and d r i e d magnesium s u l f a t e .  over  Evaporation of the s o l v e n t , f o l l o w e d by  p r e p a r a t i v e TLC ( c h l o r o f o r m ) , gave three products (Ry 0.43, 0.22,  and 0.03).  The major product  (Rj- 0.43) was 3.1 mg 1  (0.007 mmol, 62%) of a mixture of 75 and 93: H NMR (400 MHz, CDC1 3 ) 6 7.40 ( d ) , 7.27 ( d ) , 4.23 (m), 0.98 ( s , C H 3 ) , 0.97  ( s , C H 3 ) , 0.94 ( s , C H 3 ) , 0.935 ( s , C H 3 ) , 0.92 ( s , C H 3 ) ,  0.91  (s, CH3).  P r e p a r a t i o n of (p-bromophenyl)urethane  d e r i v a t i v e 90.  A t o t a l of 8.2 mg (0.0369 mmol) of a l c o h o l regioisomer 84 was r e a c t e d with 49.5 mg (0.250 mmol) of 4-bromophenyl i s o c y a n a t e by the usual procedure to g i v e , a f t e r TLC  preparative  ( c h l o r o f o r m ) , 11.9 mg (0.0283 mmol, 77%) of urethane 90:  'H NMR  (400 MHz, CDC1 3 ) 6 7.41 ( d , 2H), 7.27 ( d , 2H), 6.51  (bs, 1H), 5.29 ( b s , 1H), 5.08 ( b t , 7 = 7 2H), 1.90 (bd,  J  Hz, 1H), 4.24 (m,  = 17.6 Hz, 1H), 1.77 (bd,  J  = 17.6 Hz, 1H),  193 1.68 ( b s , 3H), 1.60 ( b s , 3H), 0.93 ( s , 3H); HRMS, m/z observed 419.1447, r e q u i r e d f o r C 2 2 H 3 0  79  BrN02  419.1460; MS,  m/z  ( r e l i n t e n s i t y ) 421 ( 7 ) , 419 ( 7 ) , 217 ( 1 4 ) , 215 ( 1 4 ) ,  204  ( 4 9 ) , 176 ( 4 3 ) , 161 ( 4 0 ) , 135 ( 2 5 ) , 107 (100), 93 ( 5 2 ) ,  91 ( 3 0 ) , 79 ( 3 6 ) , 69 ( 9 1 ) , 55 ( 3 3 ) , 41  (92).  P r e p a r a t i o n of (p-bromophenyl)urethane d e r i v a t i v e 89.  A t o t a l of 4.7 mg (0.021 mmol) of a l c o h o l 76 was reacted with  regioisomer  44.1 (0.223 mmol) of 4-bromophenyl  isocyanate by the usual procedure t o g i v e , a f t e r  preparative  TLC ( c h l o r o f o r m ) , 4.6 mg (0.011 mmol, 52%) of urethene 89: 1  H NMR (400 MHz, C D C l 3 ) 6 7.40 ( d , 2H), 7.26 ( d , 2H),  6.51  (bs, 1H), 5.37 ( b s , 1H), 5.09 ( b t , / = 7 Hz, 1H), 4.24 (m, 2H),  1.82 (bd, / = 17 Hz, 1H), 1.68 ( b s , 3H), 1.60 ( b s ,  3H),  0.94 ( s , 3H); HRMS, m/z observed 419.1443, r e q u i r e d f o r C22H30  79  BrN02  419.1460; MS, m/z ( r e l i n t e n s i t y ) 421 (11),.  419 ( 1 1 ) , 217 ( 2 5 ) , 215 ( 2 5 ) , 204 ( 4 8 ) , 161 ( 4 4 ) , 135 ( 4 5 ) , 107 ( 4 7 ) , 93 ( 5 8 ) , 81 ( 4 4 ) , 69 (100), 55 ( 5 2 ) , 43 ( 4 6 ) , (90).  P r e p a r a t i o n of (±)-75 .  A t o t a l of 2.9 mg (0.0069 mmol) of urethane 8_9 was c y c l i z e d f o l l o w i n g the same procedure d e s c r i b e d f o r the mixture of 89 and 90. (chloroform)  P u r i f i c a t i o n by p r e p a r a t i v e TLC  gave 2.0 mg (0.0048 mmol, 69%) of (±)-7_5  41  1 94 i d e n t i c a l by  *H NMR,  MS,  product d e r i v a t i v e 7 5 .  and HPLC r e t e n t i o n  time to n a t u r a l  195 Phi  Collection  dol opora  pacifica  Data.  Phidolopora  pacifica  was c o l l e c t e d by hand using SCUBA  from Diceman I s l a n d i n the Broken Group and from a v a r i e t y of  sites  i n the Deer Group of I s l a n d s , Barkley Sound,  B r i t i s h Columbia. 20 m.  C o l l e c t i o n s were made at depths of 5 to  Samples were immediately immersed i n methanol,  ethanol or acetone, s t o r e d at RT f o r 1-3 days and then at 4 to  -5 °C, i n the dark, u n t i l  used.  E x t r a c t i o n and Chromatographic  1.  Methanol  Separation.  Extraction  A number of c o l l e c t i o n s were e x t r a c t e d with methanol. The same i s o l a t i o n scheme was used f o r each e x t r a c t i o n . f o l l o w i n g represents a t y p i c a l e x t r a c t i o n and  The  isolation  procedure. The bryozoan (143 g dry weight a f t e r e x t r a c t i o n ) ground i n a Waring blender with the methanol e x t r a c t i o n of the whole a n i m a l s .  was  (1L) used f o r  Vacuum f i l t r a t i o n  of the  crude e x t r a c t i n the presence of C e l i t e gave a g r e e n i s h brown methanolic f i l t r a t e which was c o n c e n t r a t e d to about 250 mL and p a r t i t i o n e d between b r i n e and e t h y l a c e t a t e 150 mL).  (3 x  The combined e t h y l a c e t a t e e x t r a c t s were washed  with 200 mL of b r i n e and d r i e d over sodium s u l f a t e .  The  1 96 e t h y l a c e t a t e was  evaporated to give 796  dark greenish-brown crude o i l . f l a s h chromatography (40 mm gel, 20%  The  mg  (0.56%) of a  o i l was  fractionated  diameter column, 6 inches  step g r a d i e n t of 5% e t h y l acetate/petroleum methanol/ethyl a c e t a t e ) to y i e l d  (181),  ether  (189),  phidolopin (179),  bryozoan  (720  g dry weight a f t e r e x t r a c t i o n )  ground i n a Waring blender  with the ethanol  e x t r a c t i o n of the whole a n i m a l s . c o n c e n t r a t i o n of the suspension  that was  ethyl acetate  filtrate  mL).  The  s u l f a t e to give  1.29  vacuo)  ethanol-water (200 mL)  (300 mL)  and  and d r i e d over sodium Flash  diameter column, 6 inches s i l i c a  u s i n g a step g r a d i e n t of 5% e t h y l acetate/petroleum methanol/ethyl a c e t a t e gave d i f f e r e n t  containing  and  combined e t h y l a c e t a t e  g (0.18%) of a dark o i l .  chromatography (40 mm  (in  L of an  p a r t i t i o n e d between b r i n e  (4 x 200  was  (4L) used f o r  Filtration  gave 1.5  l a y e r s were washed with b r i n e  gel)  ether  to  fractions  f a t s , s t e r o l s , 4-ethoxymethyl-2-nitrophenol  (209) , p h i d o l o p i n ( 1 7 9 ) , and  desmethylphidolopin  F r a c t i o n s from the column that c o n t a i n e d and  and  Ethanol E x t r a c t i o n The  20%  to  (180).  desmethylphidolopin  2.  silica  fractions containing  f a t s , s t e r o l s , 4-methoxymethyl-2-nitrophenol 4-hydroxymethyl-2-nitrophenol  by  (180).  209 were combined  f u r t h e r p u r i f i e d by p r e p a r a t i v e r e v e r s e phase TLC  water/(95% e t h a n o l ) , Rj- ^ 0.6] ( c h l o r o f o r m , Rj-  0.3)  f o l l o w e d by p r e p a r a t i v e  to give 3.5  mg  of  [20:80 TLC  4-ethoxymethyl-  2 - n i t r o p h e n o l as a yellow  oil.  1  4-methoxy-2-nitrophenol (189): H NMR (80 MHz, CDC13) 6 10.58 ( s , 1H), 8.09 ( d ,  J  =  2.0 Hz, 1H), 7.59 (dd,  J  =  2.0,8.4 Hz, 1 H ) , 7.16 ( d , / = 8.4 Hz, IH), 4.43 ( s , 3.41  ( s , 3H); HRMS, observed m/z 183.0534, C 8 H 9 N O „  183.0532; MS, m/z ( r e l i n t e n s i t y )  2H),  requires  183 ( 4 1 ) , 182 (20),  152  (100), 141 ( 4 0 ) , 136 ( 2 0 ) , 127 ( 3 1 ) , 123 ( 6 1 ) , 106 ( 6 6 ) ,  105  (33), 78 ( 2 8 ) , 77 ( 5 1 ) , 65 ( 2 9 ) , 53 ( 2 8 ) , 51 ( 5 6 ) , 45 ( 3 3 ) , 41 (20) , 39 (42) , 31 ( 5 1 ) , 29 (71 ) .  4-hydroxymethyl-2-nitrophenol  (181): 'H NMR (80 MHz, CDC1 3 )  6 10.58 ( s , 1H), 8.14 ( d , / = 2.2, 1H), 7.63 (dd, / = 2.2,8.5 Hz,' 1H), 7.18 ( d , / = 8.5 Hz, 1H), 4.71 ( s , 2H), 1.61 ( b s , OH + H 2 0 ) ; HRMS, observed m/z 169.0379, C 7 H 7 N O « requires  169.0375; MS, m/z ( r e l i n t e n s i t y )  169 (100), 123  ( 3 6 ) , 122 ( 3 2 ) , 106 ( 2 6 ) , 105 ( 2 0 ) , 95 ( 2 6 ) , 94 ( 2 4 ) , 77 (28), 66 ( 2 2 ) , 65 ( 5 1 ) , 53 ( 3 0 ) , 51 ( 3 0 ) , 39 ( 4 4 ) .  Phidolopin  (179): mp 226-227 °C (CH 3 CN); UV (CH3CN) 351 nm  (e 3,300), 275 ( 16,800); IR (CHC13 c a s t ) 3300 ( b ) ,  1697,  1  1657, 1626, 1532 cm" ; 'H NMR (270 MHz, CDC1 3 ) 10.56 ( s , 1H, exchanges with D 2 0 ) , 8.08 ( d , / = 2.2 Hz, IH), 7.63 ( s , 7.61  (dd,  5.46  ( s , 2H), 3.59 ( s , 3H), 3.39 ( s , 3H); HRMS observed m/z  J  = 2.2, 8.6 Hz, IH), 7.16 ( d ,  331.0917, r e q u i r e d f o r C 1 f l H l 3 N 5 0 5  J  = 8.6 Hz,  1H),  331.0917; MS, m/z  i n t e n s i t y ) 331 ( 2 0 ) , 313 (17), 180 ( 7 5 ) , 150 (100).  1H),  (rel  198 Desmethylphidolopin  1  (180) : H NMR (400 MHz, DMSO-d6) 5 11.16  ( s , 1H), 11.05 ( s , 1H), 8.26 ( s , 1H), 8.00 ( d , 1H), 7.58 (dd, 5.41  J  =  2.6, 8.7 Hz, 1H), 7.12 ( d ,  J  J  = 2.6 Hz, = 8.7,  1H),  ( s , 2H), 3.35 ( s , 3H); HRMS observed m/z 317.0777,  re-  q u i r e d f o r C ^ H ^ N j O s 317.0760; MS, m/z ( r e l i n t e n s i t y ) 317 (37), 299 ( 3 5 ) , 177 ( 3 3 ) , 166 (100), 152 ( 7 5 ) , 123 ( 2 7 ) , (21), 106 ( 3 6 ) , 105 ( 2 7 ) , 95 ( 3 7 ) , 77 ( 3 0 ) , 69 ( 2 7 ) , (24), 55 ( 2 6 ) , 51  107  57  (20).  4-Ethoxy-2-nitrophenol  (209):  1  H NMR (80 MHz, CDC1 3 ) 8 10.57  ( s , 1H), 8.09 ( d , J = 2.1 Hz, 1H), 7.59 (dd, / = 2.1,8.9 Hz, 1H), 7.14 ( d ,  J  =  8.9 Hz, 1H), 4.48 ( s , 2H), 3.57 ( q ,  J  =  6.9 Hz, 2H), 1.28 ( t , J = 6.9 Hz, 3H); HRMS observed m/z 197.0696, r e q u i r e d f o r CsH^NO, 197.0688; MS, m/z intensity)  (rel  197 ( 4 9 ) , 153 ( 2 2 ) , 152 (100), 135 ( 2 0 ) ,  123  (31), 106 ( 4 3 ) , 105 ( 2 0 ) , 77 ( 2 4 ) , 51 ( 2 5 ) , 29 ( 2 3 ) .  P r e p a r a t i o n of p-bromophenacyl d e r i v a t i v e 1 9 4 .  To 2 mg (0.006 mmol) of p h i d o l o p i n (179) d i s s o l v e d mL of a c e t o n i t r i l e  in 1  (heated t o d i s s o l v e , gave a yellow  s o l u t i o n ) was added 27.9 mg (0.279 mmol) of potassium b i c a r b o n a t e and 4 mL of a stock s o l u t i o n made up of 100.6 mg (0.362 mmol) p-bromophenacyl bromide, 14.1 mg (0.053 mmol) l8-Crown-6, and 25 mL of a c e t o n i t r i l e .  The mixture was  s t i r r e d a t 75°C f o r 1 h f o l l o w e d by an a d d i t i o n a l hour a t RT.  At the end of t h i s time the r e a c t i o n mixture was  199 p a r t i t i o n e d between water (20 mL) and e t h y l a c e t a t e (3 x 30 mL).  Most of the l i g h t yellow c o l o u r was present  organic l a y e r .  i n the  The e t h y l a c e t a t e was d r i e d over magnesium  s u l f a t e , and concentrated  in  vacuo.  P u r i f i c a t i o n by  p r e p a r a t i v e TLC (5% methanol/chloroform) gave 3 mg (0.006 mmol, 100%) of d e r i v a t i v e 194; o f f white s o l i d ; mp 197 °C dec; 7.79  1  H NMR  (400 MHz, CDC1 3 ) 5 7.80 ( d , J = 2.3 Hz, IH),  ( d , / = 8 Hz, 2H), 7.68 ( s , IH),  7.59 ( d , / = 8 Hz,  2H), 7.49 (dd, J = 2.3,8.4 Hz, 1H), 6.91 ( d , J= 8.4 Hz, 1H), 5.45  ( s , 2H), 5.38 ( s , 2H), 3.57 ( s , 3H), 3.37 ( s , 3H).  V.  APPENDICES  200  Hi/cm  *  Appendix 1. 400 MHz  3  NMR  2  spectrum of 75 i n CDC1 3 .  f  1  0  Hi/cm M.  10 000  8 000  mo  Hi  5 000  4 000  50  Hi  2 500  2 000  2000 1000 500  11 PPM (6) 0  Appendix 2. 400 MHz  NMR  spectrum of 98 i n CDC1 to o to  VI.  BIBLIOGRAPHY  1. For example see: F a u l k n e r , D.J. 1983, 14, 61.  Rev.  Latinoamer.  Quim.  2. For reviews see: (a) Baker, R.; Evans, D.A. Chem. Brit. 1980, 16, 412. (b) R i t t e r , F . J . , Ed. "Chemical Ecology: Odour Communication i n Animals"; E l s e v i e r : Amsterdam, 1979. 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