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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 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 thesis as conforming MARINE INVERTEBRATES by STEPHEN WILLIAM AYER d standard THE UNIVERSITY OF BRITISH COLUMBIA March 1985 © Stephen William Ayer, 1985 In presenting t h i s thesis in p a r t i a l fulfilment of the re-quirements for an advanced degree at the The University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It i s under-stood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Chemistry The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: March 1985 Abstract Marine organisms show potential as sources for novel, b i o l o g i c a l l y and pharmacologically ac t i v e , secondary metabolites. Examination of three nudibranch and one bryozoan species for b i o l o g i c a l l y active metabolites has led to the i s o l a t i o n and s t r u c t u r a l elucidation of nine new and two known secondary metabolites. The structures of a l l the compounds were determined by using a combination of spectral a n a l y s i s , chemical interconversion, synthesis, and si n g l e - c r y s t a l X-ray d i f f r a c t i o n a n a l y s i s . The B r i t i s h Columbian dorid nudibranch Acant hodori s nanaimoensi s yielded three new sesquiterpenoids. The struc-tures of nanaimoal (6_1) , acanthodoral (64) , and isoacanthodoral (6J5) represent novel sesquiterpenoid carbon skeletons. The natural mixture of aldehydes 61, 64, and 65 exhibited a n t i b a c t e r i a l and antifungal a c t i v i t y . From Aldisa cooperi, two A*-3-ketosteroidal acids 23 and 24, and glycerol ether 25 were i s o l a t e d . Acid 23 showed feeding deterrent a c t i v i t y against f i s h . The dendronotid nudibranch Meli be leonina gave 2,6-dimethy1-5-heptenal 53 and 2,6-dimethyl -5-heptenoic acid 5_4_. The aldehyde 5_3 was responsible for the "grapefruit l i k e " odour of the nudibranch. The bryozoan Phidolopora pacifica was examined in an attempt to correlate the absence of surface f o u l i n g , in the f i e l d , with the presence of b i o l o g i c a l l y active secondary metabolites. The purine a l k a l o i d s 179 and 180, which i i c o n t a i n the r a r e n a t u r a l l y o c c u r r i n g n i t r o f u n c t i o n a l i t y , were r e s p o n s i b l e f o r much of the 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 the crude e x t r a c t s . Three n i t r o p h e n o l s 181, 189, and 209 were a l s o i s o l a t e d from P. pacifica. N i t r o p h e n o l 181 had been p r e v i o u s l y shown to i n h i b i t c h l o r o p l a s t development b o t h i n green p l a n t s and i n the u n i c e l l u l a r a l g a e Eugl ena s p . Table of Contents Abstract i i L i s t of Figures vi L i s t of Schemes v i i L i s t of Tables v i i i L i s t of Appendices ix Acknowledgements x Dedication xi Abbreviations x i i I . Introduction 1 A. Overview 1 B. Natural Products Chemistry 3 C. Primary and Secondary Metabolites 8 D. Chemical Ecology 11 I I . Nudibranchs 16 A. Introduction 16 1. Gastropod Secondary Metabolites 16 2. Nudibranch Defense Mechanisms 21 B. Secondary Metabolites from the Dorid Nudibranch Aldisa cooperi ( R o b i l l i a r d and Baba, 1972) 25 1. Introduction 1 25 2. Isolation and Structure Elucidation 25 3. B i o l o g i c a l A c t i v i t i e s of Aldisa cooperi Metabolites 42 4. Discussion 43 C. Secondary Metabolites from the Dendronotid Nudibranch Meli be leonina (Gould, 1852) 47 1. Introduction 47 2. Isolation and Structure Elucidation 47 iv 3. Discussion 53 D. Secondary Metabolites from Acant hodoris nanaimoensis (O'Donoghue, 1921) 56 1 . Introduction 56 2. Nanaimoal 59 3. Synthesis of Nanaimoal's (p-Bromophenyl)urethane Derivative. Assignment of Structure 70 4. Assignment of the 1H NMR Spectrum of Nanaimoal using One and Two-Dimensional NMR Techniques 81 5. Isoacanthodoral 99 6. Acanthodoral 112 7. Bi o l o g i c a l A c t i v i t i e s of A. nanaimoensis Secondary Metabolites 117 8. Discussion 117 I I I . Bryozoans 132 A. Introduction to the Bryozoans 132 B. Secondary Metabolites from Phidolopora pacifica (Robertson 1908) 149 1 . Discussion 163 IV. Experimental 171 V. Appendices 200 VI. Bibliography 204 v L i s t of Figures 1. Phylogenetic c l a s s i f i c a t i o n of nudibranchs 17 2. Typical cryptobranch dorid nudibranch 20 3. Aldisa cooperi 26 4. Secondary metabolites from the dorid nudibranch Aldisa cooper i 27 5. "Un-natural" 20S marine steroids 37 6. Mel i be leonina 48 7. Secondary metabolites from the dendronotid nudibranch Meli be Ieoni na 49 8. Acanthodori s nanaimoensis 57 9. Secondary metabolites from Acanthodoris nanaimoensis 60 10. GC analysis of a crude chloroform extract of A. nanaimoensis 61 11. 400 MHz 1H NMR spectrum of nanaimoal (61) (CDC13) ... 63 12. Model systems for the 1H NMR chemical s h i f t s of the gem-dimethyl group in nanaimoal (6_1) 70 13. Nanaimoane carbon skeleton showing the numbering scheme 80 14. Pulse sequence for the homonuclear COSY NMR experiment 89 15. 400 MHz 1H NMR COSY/45 spectrum of nanaimoal's (p-bromophenyl)urethane derivative 91 16. Expansion and amplification of Figure 15 to show homoallylic couplings 92 17. Pulse sequence for 2D /-resolved NMR experiment .... 93 18. P a r t i a l 400 MHz *H NMR 2D /-resolved spectrum (symmetrized) of nanaimoal (61) 97 19. S l i c e s of individual peaks shown at the top of Figure 18 to show m u l t i p l i c i t i e s 98 20. Computer generated X-ray structure of isoacanthodoral's 2,4-dinitrophenylhydrazone derivative 96 110 v i 21. Computer generated X-ray structure of acanthodoral's (p-bromophenyl )urethane derivative 114 116 22. Phidolopora pacifica 150 23. A computer-generated perspective drawing of the f i n a l X-ray model of p-bromophenacylphidolopin 194 160 v i i L i s t of Schemes 1. Interpretation of the HRMS of 3-oxo-4-cholenoic acid (23) 30 2. Interpretation of the HRMS of 3-oxo-4,22-choladienic acid (24) 33 3. Interpretation of the MS of diac e t y l derivative 44 .. 41 4. Proposed mechanism for the microbial transformation of cholesterol into 17-ketosteroids 44 5. Interpretation of the mass spectral fragmentation of 2,6-dimethyl-5-heptenal (53) 52 6. Interpretation of the MS of nanaimoal (61) 62 7. Biogenetic arguments used in support of structure 61 for nanaimoal 67 8. Retrosynthetic analysis of the postulated structure for nanaimoal 72 9. Previous synthesis of the nanaimoane carbon skeleton 73 10. Interpretation of the MS of urethane 79 11. Interpretation of the MS of isoacanthodoral (65) .. 101 12. Biogenetic arguments leading to the consideration of 100 as the structure for isoacanthodoral 103 13. Acid catalyzed rearrangement of 98 107 14. Proposed biogenesis of isoacanthodoral (65) from acanthodoral (64) .". 109 15. Interpretation of the MS fragmentation of nitrophenol 189 154 16. Interpretation of the MS of desmethylphidolopin .... v i i i L i s t of Tables 1. Comparison of the 'H NMR data for various A"-3-ketosteroids (CDC13) 29 2. 1 3C NMR data and spectral comparisons for the assignment of stereochemistry to 3-oxo-4-cholenoic acid (23) 36 3. 'H NMR data for various 20R and 20S steroids (CDC13) . 39 4. 1 3C NMR data for 2,6-dimethyl-5-heptenal (53) and c i t r o n e l l a l (55) 51 5. 1H NMR data (400 MHz) for nanaimoal (61) and derivatives 83 6. Nudibranch sesquiterpenoids 119 7. Bryozoan metabolites 134 8. 'H NMR data (CDC13, 80 MHz) and spectral comparisons for nitrophenols isolated from Phidolopora paci fi ca 153 9. 'H NMR data and spectral comparisons for purine derivatives isolated from Phidolopora pacifica 158 ix 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 guidance, encouragement, patience, and friendship of Dr. R.J. Andersen. Many people have influenced the outcome of t h i s work and a number of individuals stand out. My wife Roxanne pro-vided unrelenting love, support, and encouragement for which I am deeply indebted. Mike LeBlanc competently introduced me to SCUBA di v i n g , assisted with a l l the invertebrate c o l l e c t i o n s , and performed the bioassays. Dr. 0 . Chan and Ms. Marietta T. Austria provided friendly and helpful assistance with the 2D-NMR studies. David Behrens allowed me to reproduce his drawing of a t y p i c a l cryptobranch dorid nudibranch and Ron Long kindly provided photographs of a l l the invertebrates studied. I thank Sandra Millen for h e l p f u l discussions on nudibranch biology. A number of people assisted with the c o l l e c t i o n of the marine organisms, and the spectral data of the compounds iso l a t e d from them. I thank the staff of the Bamfield Marine Station and the departmental NMR and MS laboratories for courteous and r e l i a b l e assistance. x i To Jean, Roxanne, Genevieve, Dorothy, B i l l , and Allen Abbreviat ions AQN = Acquisition DMSO = Dimethylsulfoxide EtOAc = Ethyl acetate g = Grease peak GC = Gas chromatography GC-MS = gas chromatography - mass spectrometry HPLC = High performance l i q u i d chromatography HRMS = High resolution mass spectrum IR = Infrared MS = Low resolution mass spectrum 'H NMR = Proton nuclear magnetic resonance 1 3C NMR = Carbon-13 nuclear magnetic resonance nOe = Nuclear Overhauser enhancement mp = Melting point RD = Relaxation delay RT = Room temperature S = Solvent signal SFORD = Single frequency off resonance decoupled SCUBA = Self-contained underwater breathing apparatus TLC = Thin layer chromatography U = Unknown impurity signal UV = U l t r a v i o l e t W = Water signal xi i i I. INTRODUCTION A. OVERVIEW The purpose of the research undertaken and reported in th i s thesis was to isolate and elucidate the structures of secondary metabolites* exhibiting interesting b i o l o g i c a l a c t i v i t i e s "from selected B r i t i s h Columbian marine invertebrates. It was anticipated at the outset that some of these compounds would be important to the ecology of the source organisms. Many examples exist where e c o l o g i c a l l y important secondary metabolites turn out to be pharmacologically active (or vice v e r s a )1, therefore the i s o l a t i o n s were directed by in vitro screening for antifungal and a n t i b a c t e r i a l a c t i v i t i e s . Because not a l l p o t e n t i a l l y interesting compounds would be active against the limited number of microbes screened, thin layer chromatography (TLC) and nuclear magnetic resonance (NMR) were used to broadly characterize every p u r i f i e d f r a c t i o n . The structures of the isolated metabolites were deter-mined by one or more of the following methods: 1. Interpretation of spectral data. 2. Comparison with known compounds. 3. Chemical interconversions. * For the d e f i n i t i o n of a secondary metabolite, see Section C of t h i s Chapter. Natural products chemistry and the chemistry of secondary metabolites are generally regarded as synonymous. 1 2 4. Unambiguous synthesis. 5. - Single c r y s t a l X-ray d i f f r a c t i o n a n a l y s i s . Three species of nudibranchs were investigated for b i o l o g i c a l l y active secondary metabolites. Nudibranchs lack the physical protection of an external s h e l l , but are seldom eaten. They deter predators using a variety of mechanisms,-as w i l l be discussed in a subsequent chapter, and one of these defensive mechanisms seems to involve the employment of noxious secondary metabolites. A number of secondary metabolites were isolated from the three nudibranch species studied; three represent biogenetically related sesquiterpenoid aldehydes each with a new carbon skeleton (from Acanthodoris nanaimoensis), two are s t e r o i d a l acids, of which one displayed f i s h antifeedant a c t i v i t y (from Aldisa cooperi), and two are degraded monoterpenes s t r u c t u r a l l y similar to known insect pheromones (from Mel i be I eoni na) . One species of bryozoan (Phidolopora pacifica) was also studied. The unfouled nature of P. pacifica in the natural environment led to speculation that i t employs an e f f e c t i v e a n t i f o u l i n g agent. P. pacifica turned out to be a source of rather interesting purine a l k a l o i d s containing the r e l a t i v e l y rare, naturally occurring, n i t r o group. I n i t i a l screens indicated that some of the isolated metabolites exhibit profound b i o l o g i c a l a c t i v i t i e s . For ex-ample, one of the purine a l k a l o i d s was very active against 3 the pennate diatom Cyl i ndrot heca fusiformi s .while the mixture of sesquiterpenoid aldehydes from A. nanaimoensis e f f e c t i v e l y inhibited the growth of the bacteria Bacillus subtil is. Collaboration with pharmacologists and b i o l o g i s t s to study the f u l l spectrum of b i o l o g i c a l a c t i v i t i e s , both pharmacological and e c o l o g i c a l , that are exhibited by the metabolites described in t h i s thesis would appear in order. B. NATURAL PRODUCTS CHEMISTRY Natural products chemistry (the chemistry of secondary metabolites) has undergone explosive growth during the l a s t quarter century. In the 1950's and early 1960's natural products research was characterized by investigations of the structures of molecules available in large quantities from natural sources. The primary str u c t u r a l tools were c l a s s i c a l degradation and transformation reactions. Introduction of spectroscopic techniques such as NMR and mass spectroscopy (MS), enabled the Chemist to propose structures on the basis of spectroscopic evidence alone, a l -though these were usually supported by a few key chemical transformations. Confirmation of structure came from synthesis, conversion to a known compound, or by the rapidly evolving single c r y s t a l X-ray d i f f r a c t i o n a n a l y s i s . With the advent of high performance l i q u i d chromatography (HPLC), routine Fourier transform NMR, and rapid computer aided X-ray a n a l y s i s , the Chemist has been able to solve chemical problems previously thought 4 impossible. These include, for example, the structural elucidation of pheromones available in microgram amounts2, is o l a t i o n and structural determination of phytohormones3, and elucidation of structures, such as palytoxin (1)* and brevetoxin ( 2 )5, that are so complex i t i s even d i f f i c u l t to draw them. The days of the Natural Products Chemist are far from numbered. We are at the dawn of a new era. Investigation 5 (by collaboration 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 active i s o l a t e s has provided, and w i l l continue to provide, interesting chemistry. Analysis of r e l a t i v e l y small water soluble molecules w i l l become more prevalent, and this should enable the i s o l a t i o n and structure elucidation of p h y s i o l o g i c a l l y interesting compounds, par-t i c u l a r l y o l i g o p e p t i d e s6. F i n a l l y , investigation of the enzymology and biosynthesis of secondary metabolites should provide valuable insights into why organisms produce such compounds7. Marine natural products research has greatly i n t e n s i f i e d over the l a s t two decades. An excellent series of books edited by Scheuer8 provide timely, exhaustive reviews of important aspects of marine natural products research. Other authoritative reviews by Baker9, Christophersen1 0, C o l l1 1, F a u l k n e r1 2, F e n i c a l1 3, Moore1 0, 6 and Scheuer1 5 provide additional highlights of chemical research on the organisms found beneath the sea. The pragmatic application of natural products isolated from t e r r e s t r i a l sources i s well known, p a r t i c u l a r l y in the areas of human and veterinary medicine. The marine environment has also yielded a number of compounds (or synthetic analogs) that have found, or show potential f o r , p r a c t i c a l a p p l i c a t i o n1 6'1 7. For example; the i s o l a t i o n of nereistoxin (3_) from the marine worm Lumbr i coner ei s heteropoday8 led to the development of the s t r u c t u r a l l y related i n s e c t i c i d e Padan (4) which i s in common use in Japan1 9. The nucleoside 1-j3-D-arabinofuranosylcytosine (Ara-C) ( 5 )2 0, an e f f e c t i v e anticancer drug that has been in use for over a decade in the treatment of leukemia, was de-veloped as a result of semisynthetic manipulation of spongouridine (Ara-U) (6) isolated from the sponge t Cryptotethia crypta2^'22. A family of natural products, the didemnins, isolated by Rinehart et al. from the Caribbean tunicates Trididemnun species23, show great biomedical p o t e n t i a l . Didemnin B (7) appears to be the most potent didemnin in blocking the growth of L-1210 mouse leukemia c e l l s as well as i n h i b i t i n g the Herpes simplex v i r u s . Didemnin B is currently undergoing c l i n i c a l t r i a l s as an antileukemia drug2". Results such as the above c l e a r l y demonstrate how the study of marine secondary metabolites may lead to some valuable pragmatic s p i n o f f s . Current research 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 | 1 _ 7 now being directed towards i s o l a t i n g metabolites with a s p e c i f i c b i o l o g i c a l a c t i v i t y2* '2 5, greatly increasing the pro b a b i l i t y that a compound with a desired pharmacological effect w i l l be found. Although some researchers and one drug company* * Recently the Roche Research Institute for Marine Pharmacology at Dee Why, A u s t r a l i a , established in 1974, ceased operation. 8 involved in marine natural products research have become somewhat d i s i l l u s i o n e d by a paucity of p r a c t i c a l results (compared to overly optimistic i n i t i a l expectations), the development of a new drug requires a large investment of c a p i t a l and years of development2 6. Given time, i t seems l i k e l y that the marine environment w i l l eventually y i e l d i t s own " p e n i c i l l i n " . " For the cynic questioning the biomedical value of the sea, i t is important to point out that only a handful of laboratories and perhaps a score or two of chemists and pharmacologists have been investigating the marine resource. This compares extremely meagerly with hundreds and perhaps thousands of similar investigators who have in the course of at least a century investigated t e r r e s t r i a l resources for drugs. What is required is that more investigators, e s p e c i a l l y from the drug industry, take a dip in the oceans to seek the pearls awaiting discovery."1 7 C. PRIMARY AND SECONDARY METABOLITES H i s t o r i c a l l y the organic compounds found in l i v i n g organisms were divided into two categories. Primary metabolites were defined as compounds made by an organism primarly to sustain l i f e . ' The compounds, and the biosynthetic pathways used to generate them, were designated as common to many, i f not a l l , l i v i n g organisms. Secondary metabolites, on the other hand, were deemed not essential to the basic protoplasmic metabolism of the organism and were thus i n i t i a l l y considered to be endproducts (waste or stor-age) of metabolism. 9 It i s l i k e l y 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 secondary metabolite w i l l f a l l into disuse. It i s now ap-parent, for example, that fungal secondary metabolites are involved in the physiology of the producing organism. Perhaps two of the best known examples are the fungal sex hormones of the Achl ya (antheridiol (8) and oogoniol ( 9 ) ) 2 7 and of the Mucorales (the t r i s p o r i c acids 1J) and 1 1 ) 2 8 . The notion that secondary metabolites are endproducts of metabolism has turned out to be a gross o v e r s i m p l i f i c a t i o n . The f i e l d of ecological biochemistry (chemical ecology) has grown rapidly over the la s t 15 years, demonstrating the 10 important ecological roles secondary metabolites have in nature* (see the following section). These findings have prompted a re-evaluation of the terms primary and secondary metabolites. Campbell2 9 has put forward a more useful 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 pa r t i c u l a r compound. It states that: Plant, fungal, b a c t e r i a l (and animal) c e l l s are composed of two distinguishable types of molecules: a) materials that are widely d i s t r i b u t e d in nature, being found at least in a l l families in an order, often in a l l orders of a class or in a l l classes in a phylum, and in some instances, in a l l phyla in a kingdom and in a l l f i v e kingdoms; and b) materials that occur uniquely in a single s t r a i n or species, that are found in two or more closely related members of a single genus, or that are found sporadically in a limit e d number of evolu t i o n a r i l y unrelated species in d i f f e r e n t genera, f a m i l i e s , orders, •classes, phyla or kingdoms. C e l l constituents of type (a) are primary metabolites, while c e l l constituents of type (b) are secondary metabolites. [N.B. Enzymes and nucleic acids, though spec i e s - s p e c i f i c in d i s t r i b u t i o n , are normally excused from the primary/secondary metabolism * Despite these advances, many secondary metabolites s t i l l have no known function. This may be due to a lack of s u i t -able experimentation or because the function i s too complex to discern given the current state of knowledge. 11 dis c u s s i o n ] . This thesis discusses secondary metabolites isolated from selected B r i t i s h Columbian marine organisms. Although the function of the metabolites cannot be conclusively es-tablished, circumstantial evidence suggests the isolated metabolites are involved in interspecies communication. It seems l i k e l y that the compounds isolated from the nudibranchs are involved in their r e l a t i v e immunity from predation, whereas the bryozoan metabolites may impede surface fouling and overgrowth. D. CHEMICAL ECOLOGY Reseacch over the la s t f i f t e e n years has led to the de-velopment of a new i n t e r d i s c i p l i n a r y subject; chemical ecology3 0. Studies in chemical ecology have for the f i r s t time provided a r a t i o n a l and s a t i s f y i n g explanation for the roles of at least a part of the enormous p r o l i f e r a t i o n of secondary metabolites observed in nature. This has led to another functional yet somewhat inaccurate (see previous section) description of secondary metabolites: If the organism i s considered by i t s e l f , without reference 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 )3 1. There are two types of ecological interactions mediated by secondary metabolites; alleochemic effects ( i n t e r s p e c i f i c ) and i n t r a s p e c i f i c chemical e f f e c t s3 2. Alleochemic e f f e c t s are subdivided into allomones 1 2 (adaptative advantage to the producer) and kairomones (adaptative advantage to the receiver) while i n t r a s p e c i f i c chemical e f f e c t s are subdivided into autotoxins, aut o i n h i b i t o r s , and pheromones. Allomones and pheromones are by far the most studied chemical communicants to date. In some cases, as with the common skunk Mephitis mephitis, the chemical message may be a bouquet of compounds (t h i o l s 12, 13, and d i s u l f i d e 14), a mixture that acts as a pheromone (alarm) and an allomone (defense) simultaneously. The spray from the skunks' anal scent glands sends potential predators fleeing while at the same time warning other skunks of the potential danger nearby. In other cases a single substance, for example cantharidin (15_) from the meloid beetle Lytta vesicatoria, can act as a feeding deterrent to potential predators. The l i t e r a t u r e has been summarized by Harborne in his excellent book "Introduction to Ecological Biochemistry"2 . CH3CH=CHCH2SH 12 Me 0 0 (CH3)2CHCH2CH2SH 13 CH3CH=CHCH2SSCH3 Me 0 15 14 13 Chemical ecology in the marine environment i s s t i l l in i t s infancy. A recent chapter by Barbier introduced, summarized, and analyzed t h i s current state of a f f a i r s3 3. Many compounds have been shown to be b i o l o g i c a l l y active against selected screening organisms or c e l l l i n e s , but l i t t l e i s known about the functions of the metabolites in their natural environment. This i s primarly due to the d i f f i c u l t y of experimenting and observing in the marine ecosystem. Perhaps the best documented study in marine chemical ecology involves a dorid nudibranch. Schulte and Scheuer demonstrated that the nudibranch, Phyllidia varicosa, accumulates from a s p e c i f i c sponge upon which i t feeds (Hymeni aci don sp.) a substance that i s apparently l e t h a l to f i s h and crustaceans3 4. The substance, 9-isocyanopupukeanane (16.) , was isolated from both organisms. Interestingly, a related metabolite is present only in the sponge. Apparently the nudibranch must have some means of concentrating s p e c i f i c metabolites from i t s d i e t . Subsequent to thi s discovery, a number of other metabolites isolated from dorid skin secretions have been shown to i n h i b i t feeding by f i s h (using a standard bioassay) at concentrations of 10 ug metabolite per mg of food p e l l e t3 5'3 6. The demonstrated antifeedant a c t i v i t y would appear to be ec o l o g i c a l l y s i g n i f i c a n t * . * Recently Gerhart3 7 demonstrated that prostaglandin A2 i s an agent of chemical defense in the Caribbean gorgonian Plexaura homomalla. 1 4 The l i t e r a t u r e on nudibranch chemistry has been extensively r e v i e w e d1'3 6'3 8, and recently Faulkner3 9 has proposed that in the evolution of dorid nudibranchs, loss of the s h e l l i s correlated with the presence of defensive mechanisms based upon chemicals derived from food. Although this theory i s appealing, the limit e d ecological studies which have been performed to date are 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 metabolite i s sol e l y responsible for an "observed" lack of predation. A second example of marine chemical ecology, that involves a much less quantative bioassay, concerns the Red Sea sponge Latrunculia magnifica, one of the few Red Sea sponges that grow exposed. Squeezing of the sponge by SCUBA divers in situ causes curious f i s h to immediately f l e e ; squeezing L. magnifica into an aquarium containing f i s h causes poisoning and death of the f i s h within minutes'0. The p u r i f i e d toxins were therefore assumed to play a 1 5 defensive role in the sponge"1. It i s interesting to note that the p u r i f i e d toxins, Latrunculins A (18) and B (19), exhibit effects on cultured mouse neuroblastoma and fi b r o b l a s t c e l l s4 2. In both c e l l types, submicromolar toxin concentrations (as low as 50 ng/mL) rapidly induce changes in c e l l morphology that are reversible upon removal of the tox i n . The significance of these observations are unknown. The above examples i l l u s t r a t e that compounds isolated from marine organisms show profound b i o l o g i c a l a c t i v i t i e s , which in many instances are advantageous to the producing organism. The promising pharmacological a c t i v i t i e s shown by some of the metabolites isolated from marine organisms on the basis of a presumed ecological role augurs well 'for the acceptance of marine chemical ecology as a viable f i e l d of research. I I . NUDIBRANCHS "Many nudibranchs, but especially the dorids, have a penetrating f r u i t y odour that i s pleasant when mild but nauseating when concentrated. Undoubtedly, th i s odour i s one of the reasons why nudibranchs seem to be l e t s t r i c t l y alone by predatory animals 3 A. INTRODUCTION 1. GASTROPOD SECONDARY METABOLITES Nudibranchs are members of the phylum Mollusca (see Figure 1'"), a phylum that contains an estimated 75,000 l i v i n g and 35,000 f o s s i l species. This large phylum, second in size only to the phylum Arthropoda, is subdivided into seven c l a s s e s . Of the seven molluscan c l a s s e s , the Gastropoda has been studied in the greatest d e t a i l for natural products chemistry"5. Gastropod molluscs of the subclass Opisthobranchia are characterized by a greatly reduced, or completely absent s h e l l . In spite of t h i s lack of physical protection, opisthobranchs have few known predators*6. To account for the observed r e l i e f from predation, early investigators speculated that opisthobranchs may u t i l i z e a chemical defense mechanism"6'*7. Chemical studies on opisthobranchs were i n i t i a t e d on the large herbivorous aplysiomorphs"8'"9. It was soon discovered that the sea hares (Aplysia spp.) were capable of storing in their digestive gland a large 16 MOLLUSCA PHYLUM GASTROPODA CLASS OPISTHOBRANCHIA SUBCLASS BULLOMORPHA 1 APLYSIAMORPHA PLEUROBRA1S JCHOMORPHA 1 PTEROPODA ORDER SACOGLASSA NUDIBRANCHIA PYRAMIDELLA AEOLIDACEA ARMINACEA DENDRONOTACEA DORIDACEA SUBORDER Figure 1. Phylogenetic c l a s s i f i c a t i o n of nud i b r a n c h s . N.B. Organisms c l a s s i f i e d according to Behrens'*. 18 variety of secondary metabolites some of which were toxic to potential f i s h p r e d a t o r s5 0. Further investigations demonstrated that the metabolites were l i k e l y being concentrated from the animals' algal d i e t s5 1. The majority of the metabolites isolated from the sea hares' digestive glands were monoterpenoids, sesquiterpenoids or diterpenoids, many of which contained a covalently bound bromine atom. Halogenated acetogenins and nitrogen containing compounds were also represented. Three sea hare metabolites have shown pharmacological p o t e n t i a l . A p l y s i s t a t i n (2T))52 and d o l a t r i o l (21)5 3 showed antileukemia a c t i v i t y while dactylyne (22)5* produced a dose dependent prolongation of phenobarbital-induced hypnosis in animals by i n h i b i t i n g the metabolism of phenobarbital. By i t s e l f , enine 22 had no e f f e c t5 5. Encouraged by the success on aplysiomorphs, marine chemists turned their attention to smaller opisthobranch molluscs, s p e c i f i c a l l y the carnivorous nudibranchs. Nudibranchs have a body which incorporates the v i s c e r a l mass, mantle, and foot, and i s externally b i l a t e r a l l y symmetrical with a s l u g - l i k e flattened form (see Figure 2). They completely lack a s h e l l in the adult form. Nudibranchs range in size from 3 to 300 mm and are found throughout the world. Over 100 species have been described*" on the west coast of North America. A l l nudibranchs are predators, feeding on a wide range of invertebrates. Dorids (suborder Doridacea) are predominately associated with sponges, 19 bryozoans (ectoprocts), and tunicates, while members of the other three suborders (Dendronotacea, Arminacea, and Aeolidacea) are primarly associated with c o e l e n t e r a t e s5 6. Nudibranchs are hermaphroditic, they possess active sex organs of both sexes simultaneously. This may be an evolutionary adaptation as i t allows for greater p r o b a b i l i t y of finding a mate since every individual of the same species i s an e l i g i b l e partner. L i t t l e i s known about how nudibranchs find and recognize each other. It i s possible that some of the compounds isolated from nudibranch skin secretions may act as recognition or sex pheromones5 7. The rhinophores, which are chemosensory organs on the nudibranchs' head, could act as the pheromone r e c e p t o r s5 6. 20 Branchial Plume Figure 2. Typical cryptobranch dorid nudibranch. From Behrens"", used with permission. The study of secondary metabolites isolated from nudibranchs has proven to be a r i c h and rewarding area. Numerous novel secondary metabolites have been isolated by groups working in C a l i f o r n i a , Hawaii, Italy and B r i t i s h Columbia. Most of the compounds isolated have been sesquiterpenoid aldehydes, furans, or i s o n i t r i l e s , although mono-, d i - , and tr 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 , acetylenes, and purine ribosides are also represented5 8. Several 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 natural products chemists in the last few years. It was known for some time that shelled molluscs possessed secretory glands similar to those occurring in nudibranchs4 6. When considered in conjunction with Faulkner and Ghiselins' evolutionary hypothesis that 21 the evolution of a chemical defense mechanism was preadaptive, enabling nudibranchs to dispense with their s h e l l3 9, i t i s not surprising that interesting secondary metabolites have been isolated from nudibranchs' shelled cousins. Certain pulmonates (subclass Pulmonata) are a r i c h source of unusual secondary metabolites with interesting b i o l o g i c a l a c t i v i t i e s5 9. Unlike the metabolites isolated from nudibranchs, however, the pulmonate metabolites are polyketides. Two examples are shown below: Recently, chemical examination of a member of the opisthobranch order Pleurobranchomorpha has yielded 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 compounds60. 2. NUDIBRANCH DEFENSE MECHANISMS In order to deter predators ( f i s h e s , s t a r f i s h , crustaceans, and other opisthobranchs are known to prey on nudibranchs5 6'6 1) i t has been suggested that these oft-times 22 highly conspicuous, slow moving, soft bodied animals u t i l i z e a bimodal defensive s t r a t e g y6 1. The primary le v e l of defense (useful only against v i s u a l predators) i s either to evade detection (crypsis) or once having been detected, to discourage the predator from i n i t i a t i n g an attack. In other words, in the l a t t e r , nudibranchs u t i l i z e aposematism; the defensive adaptation in which an animal blatantly advertizes, using conspicuous warning colouration, that i t is not a suitable food source. The role of colouration in nudibranch defense has been debated for many y e a r s6 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 coloured (the animal has a pattern that breaks up i t s body outline on i t s usual substratum), while some well-defended ( d i s t a s t e f u l ) species are aposematic6 3. More work is- required before the complex nature of nudibranch colouration i s f u l l y understood3 9. The second le v e l of defense, useful in the event of f i r s t l e v e l f a i l u r e , or in cases of predators that hunt chemically (by following mucous t r a i l s for example5 7) involves adaptations that may be behavioral, morphological, or chemical in n a t u r e6 1. Nematocysts, sp i c u l e s , and behavioral responses have a l l been implicated, to varying degrees, in nudibranch defense. The bizarre practice of storing unfired nematocysts (stinging c e l l s ) , obtained from their coelenterate d i e t , characterizes many aeolid nudibranchs6". Likely an adaptation that allows the aeolids to safely feed on coelenterates and modified to provide a 23 cheap means of defense, nematocysts probably combine to work with chemical secretions to give aeolids t h e i r immunity from predators. Not a l l nudibranchs feed on coelenterates (dorids feed exclusively on sponges, bryozoans and t u n i c a t e s ) , consequently nematocysts are unimportant in the defense of these species. A few -dorid species have hard calcareous spicules contained within their mantles. Obtained from th e i r sponge d i e t s , they give the nudibranch a r i g i d shape and provide some protection from predators. A voracious nudibranch predator, the opisthobranch Navoanax inermis, does not eat spicule-containing dorids, while readily consuming non-spiculose ones6 5. Todd has suggested that the high ash content (most of which i s attr i b u t a b l e to spicules) and low c a l o r i f i c content of some dorids 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 s6 6. Many nudibranchs w i l l swim in response to being disturbed or threatened by a predator, and t h i s may be con-sidered the most basic behavioral defense mechanism6 7. Nudibranchs with large cerata ( f i n g e r - l i k e respiratory and digestive structures occurring in groups of p a r a l l e l series along the dorsum, suborders Dendronotacea, Aeolidacea and Arminacea) tend to autotomize the cerata at only the s l i g h t -est provocation, enabling a captured nudibranch to make good an escape (the lost cerata are regenerated). Nudibranchs have a large number of secretory glands in the epidermis*6 from which some dorids secrete s u l f u r i c acid 24 when d i s t u r b e d6 8. Species of a l l four nudibranch suborders are known to secrete non-acid noxious substances under simi-lar conditions. Johannes found the dorid Phyllidia varicosa secreted a toxin that was l e t h a l to f i s h and crustaceans*7 and subsequent chemical analysis of t h i s secretion showed i t to contain 9-isocyanopupukeanane (1£) as previously discussed. 'Recent studies have shown38 that a wide variety of dorid nudibranchs produce organic compounds that were easily solublized by extraction of the whole animals with non-polar solvents. It is l i k e l y these metabolites were coming from the secretory glands, not the g u t s3 6, and a num-ber of these metabolites exhibited f i s h antifeedant a c t i v i t y . In conclusion, defensive secondary metabolites have not been unequivocally proven to exist in nudibranchs, nor have other possible roles for the metabolites been adequately investigated. On the basis of circumstantial evidence, evolutionary theory, and f i s h antifeedant a c t i v i t i e s , one may hypothesize that defensive secretions make an important contribution to nudibranch defense. L i t t l e i s known about the r e l a t i v e importance of any s p e c i f i c nudibranch defense mechanism and i t seems a nudibranch w i l l u t i l i z e a combination of d i f f e r e n t defensive strategies to avoid being eaten. More studies are needed to determine the r e l a t i v e importance of the d i f f e r e n t defensive mechanisms and to identify the predators that provided the selective pressures leading to th e i r development5 6. 25 B. SECONDARY METABOLITES FROM THE DORID NUDIBRANCH ALDISA  COOPERI (ROBILLIARD AND BABA, 1972) 1. INTRODUCTION Aldisa cooperi (Aldisa sanguinea cooperi69, see Figure 3) i s an orange dorid (suborder Doridacea, family Aldisidae) usually found deeply embedded in i t s preferred prey, the reddish orange sponge Ant hoarcuat a graceae70. The association i s l i k e l y cryptic to potential predators, even though SCUBA divers can, from a distance, e a s i l y locate the nudibranch on the sponge. A. cooperi may grow to 20 - 25 mm in length and has been reported from Washington, B r i t i s h Columbia, and Japan. Unlike many dorids c o l l e c t e d from Barkley Sound, B.C., A. cooperi did not have a detectable odour. 2. ISOLATION AND STRUCTURE ELUCIDATION The nudibranchs were co l l e c t e d by hand (SCUBA, depths of 1 to 10 m) and immediately immersed whole in methanol. After one to three days at room temperature, the methanol was decanted and saved. The animals were washed an additional four times with methanol. The methanol extracts were combined and vacuum f i l t e r e d to give an aqueous methanolic suspension. The suspension was parti t i o n e d be-tween brine and ethyl acetate, and the ethyl acetate soluble material was fractionated by fl a s h chromatography. Chromatographically similar fractions were pooled to give a Figure 3. Aldisa Fraser University, cooperi. Photographer: Ron Long, Simon 27 mixture of st e r o i d a l acids !23 and 247 1 (10:3, 2.8 mg/animal), glycerol ether 25, and a mixture of steroidal ketones (see Figure 4). Additional fractions containing fats and sterols were not studied further. Figure 4. Secondary metabolites from the dorid nudibranch Aldi sa cooperi The st e r o i d a l acid f r a c t i o n was further p u r i f i e d by preparative thin layer chromatography (preparative TLC) to give a white c r y s t a l l i n e s o l i d . It was apparent from the r e l a t i v e i n t e n s i t i e s of the peaks in the o l e f i n i c region of the 'H NMR spectrum of t h i s material that a mixture of 28 closely related compounds had been obtained. Additional p u r i f i c a t i o n by reverse-phase preparative TLC yielded 3-oxo-4-cholenoic acid 2_3 and i t s unsaturated analog, 3-oxo-4,22-choladienic acid 24 * . The major metabolite of the mixture, 3-oxo-4-cholenoic acid (23), mp = 178-179 °C, had a molecular formula C2« H3 603 (HRMS, m/z observed 372.2658, required 372.2664) that demanded seven units of unsaturation. Methyl resonances in the 1H NMR spectrum at 6 0.74 (s, 3H), 0.94 (d, / = 5.8 Hz, 3H), and 1.21 (s, 3H) in combination with the requirement for 24 carbon atoms indicated the molecule was possibly a degraded s t e r o i d . The chemical s h i f t of the methyl s i n g l e t s , the presence of a one proton singlet at 8 5.74 ppm, an u l t r a v i o l e t (UV) absorption at Xm-ax 236 nm (e 6,200), and a positive 2,4-dinitrophenylhydrazone test suggested a A*-3-ketosteroid nucleus of the type shown in 26. Comparison of the 1H NMR chemical s h i f t s of the methyl singlets and the o l e f i n i c proton to the corresponding signals in cholestenone (2_7) showed excellent agreement (see Table 1). The presence of A"-3-ketosteroid nucleus was further substantiated by a loss of ketene (CH2CO) in the mass spectrum of 23_ to give a peak at m/z 330.2564 (31%). It i s well known that molecules that incorporate substructure 26 lose ketene v i a a process which may be *It was also found that the mixture could be readily p u r i f i e d by normal phase HPLC of the methyl esters. 29 26 Table 1. Comparison of the 1H NMR data for various A'-3-ketosteroids (CDC13) chemical s h i f t , 5 H on C# 23° 24° 2 1 b 4 5.74 5.74 5.74 18 0.74 0.77 0.72 19 1.21 1.20 1.19 a 400 MHz. b 80 MHz. v i s u a l i z e d as shown in Scheme 1a7 2. Other fragment ions diagnostic for A"-3-ketosteroids7 3 were also observed at m/z 287.2028 (11%), 249.1861 (38%), and 124.0897 (96%) (see Scheme 1b). The structure of the C1 7 side chain substituent was deduced from the following spectral evidence: i) The pres-ence of a methyl doublet at 6 0.94 (d, / = 5.8 Hz, 3H) 1. Interpretation of the HRMS of 3-oxo-4-cholenoic acid (23). 31 indicated the C-21 methyl was not oxidized. i i ) Absence of additional methyl resonances, in addition to those assigned to the C-18 and C-19 carbons of the st e r o i d a l nucleus, i n d i -cated the side chain was not branched and must incorporate a carboxyl group at C-24 (IR 1700 cm"1 and formation of methyl ester 28 upon treatment with diazomethane). i i i ) In the mass spectrum of 23, a s i g n i f i c a n t fragment ion was detected at m/z 271.2047 (required for C1 9H2 70, 271.2062) which corresponded to a loss of the 1-methyl-4-butanoic acid side chain from 23 as indicated in Scheme 1b. The st e r o i d a l a c i d , 3-oxo-4,22-choladienic acid (24) , occurred as the minor component of the binary steroidal acid 32 mixture in a varying r a t i o depending upon the c o l l e c t i o n . The mass spectrum of 24 indicated a molecular formula C 2 « H 3 f l 0 3 (HRMS, m/z observed 370.2507, required 370.2508), therefore 24 d i f f e r e d from the major s t e r o i d by possessing one less unit of unsaturation. Methyl resonances at 6 0.77 (s, 3H) and 1.20 (s, 3H), a one proton singlet at 6 5.74, and a UV absorption at 241 nm (MeOH) dict a t e d a A"-3-ketosteroid nucleus the same as that found for 23. This assignment was supported by fragment ions at m/z 328.2403 (45%), 285.1862 (24%), 247.1720 (5%), and 124.0895 (100%) in the HRMS of 24 (Scheme 2). The 'H NMR spectrum showed resonances at 6 6.96 (dd, / = 15.5,8.7 Hz, 1H) and 5.76 (d, / = 15.5 Hz, 1H) ppm.which suggested that the additional unit of unsaturation was incorporated in an a,/3 unsaturated carboxyl carbonyl of type 2j}. The E configuration of the double bond followed from the 15.5 Hz v i c i n a l coupling constant in the 1H NMR spectrum. The HRMS of 24 showed an abundant ion at m/z 271.2058 (70%, required for C1 9H2 70, 271.2062) a r i s i n g from the a l l y l i c fragmentation of the 1-methyl-4-but-2-enoic acid side chain substituent in 24 (Scheme 2). Due to the s t e r e o s p e c i f i c i t y of the enzymes involved in steroid biosynthesis one would expect that 3-oxo-4-cholenoic acid 23 and i t s unsaturated analog 24 each would exist as only one of the possible 128 stereoisomers*. It is well *The side chain double bond in 24 was already known tO' have the E configuration. 33 0 m/z 328 (45%) m/z 124 (100%) + 2H Scheme 2. Interpretation of the HRMS of 3-oxo-4,22-choladienic acid (24). known from studies on both t e r r e s t i a l and aquatic organisms that steroids are biosynthesized from an i n i t i a l c y c l i z a t i o n of S-squalene 2,3-oxide7*. In animals and fungi, lanosterol (30) i s the i n i t i a l intermediate formed, while plants and algae i n i t i a l l y produce cycloartenol (31). The 34 intermediates 30 and 3_1 are then transformed b i o s y n t h e t i c a l l y into the steroids or phytosteroids, respec-t i v e l y . The absolute stereochemistry of the ABCD steroidal ring system is remarkably constant; the few exceptions known usually show the 5/3 instead of the 5a absolute stereochemistry. Cholic acid (32), one of the b i l e acids, i s an example. It was postulated from biogenetic reasoning and supported by 1H NMR comparisons to cholestenone (27) (Table 1), that the absolute stereochemistries of the ABCD ring systems in 23 and 2_4 were as shown. 1 3C NMR has been shown to be an excellent method for comparing the r e l a t i v e stereochemistries of similar mole-c u l e s . The large data base for 1 3C NMR spectra of steroids allowed comparison of the 1 3C NMR spectra of s t e r o i d a l acid 23 to cholestenone (27) and methyl 3-oxo-4-cholenoate (28, prepared from 23^ ) to methyl 5/3-cholan-24-oate (3_3)7 5. The comparisons are summarized in Table 2. The close correspondence for the resonances of the ring carbons be-tween acid 23_ and cholestenone (21) supported the assignment of the regular s t e r o i d a l ABCD ring stereochemistry to both natural products 23 and 24. Comparison of the side chain carbon resonances of ester 2_8 to those of methyl 5/3-cholan-24-oate (3_3) indicated i d e n t i c a l r e l a t i v e stereochemistries and allowed the assignment of stereochemistry at C-20 as R, the regular s t e r o i d a l absolute stereochemistry. 35 Further spectral evidence supporting the 20R configuration for 2_3 and 24 was available from the l i t e r a t u r e . A number of steroids isolated from the marine environment have the "un-natural" 20S configuration as shown in Figure 57 6'7 7. In order to confirm the assignment of 20S stereochemistry to compounds 34, 35, 36 and 3_7, Vanderah and Djerassi unambiguously synthesized both C-20 epimers of the acetate esters of each compound. Comparison of the 1H NMR spectra of the epimeric pairs showed that the C-21 methyl 36 Table 2. 1 3C NMR data and spectral comparisons for the assignment of stereochemistry to 3-oxo-4-cholenoic acid (23) chemical s h i f t , 6 Carbon # 27fl 28 33* 23 (CDC13)C (CDCl3)r f ( C D2C l2)e (CDC13)/ 1 35.7 35.8? 37.9 35.7? 2 33.9 34.0 21.6 34.0 3 198.9 1 99.4 27.4? 199.6 4 123.6 123.9 27.8? 1 23.8 5 171.0 171.4 44. 1 171.5 6 32.9 32.9 27.5? 33.0 7 32. 1 32. 1 26.9 32.0 8 35.7 35.4? 36.2 35.3? 9 53.8 53.9 40.8 53.9 10 38.6 38.6 35.8 38.6 1 1 21.0 21.1 21.1 21.1 1 2 39.4? 39.7 40.6 39.7 1 3 42.4 42.5 43.0 42.5 1 4 55.9 55.9 56.9 55.9 15 24. 1 24.2 24.4 24.2 1 6 28. 1 28. 1 28.4 28.0 1 7 56. 1 55.9 56.4 55.9 18 12.0 12.0 12.1 12.0 19 17.4 17.4 24.2 17.4 20 35.7 35.8? 35.6 35.7? 21 18.7 18.3 18.3 18.2 22 36. 1 31 .3^ 31 . 2 h 30.8 23 23.8 31 . \h 31 . 1 h 30.8 24 39.6? 1 74.6 175.0 25 27.6 26 22.5 27 22.8 -OCH3 51.5 51 .3 a From reference 75, compound 207. " From reference 75, compound 256. c Spectrometer frequency not given. d 100.6 MHz. e 20.1 MHz. f 20.1 MHz. ?'h Assignments may be switched. ' Not observed due to slow relax a t i o n . 37 Figure 5. "Un-natural" 20S marine s t e r o l s . resonance was shifted = 0.1 ppm to higher f i e l d in the 20S compounds. Careful examination of the chemical s h i f t s of the C-21 methyl groups in esters 28 and 38 (prepared from 24 with diazomethane) proved that they both have the more common 20R configuration (Table 3 ) . To r a t i o n a l i z e the 38 ^ 0.1 ppm shielding of the 20S C-21 methyl group over the 20R, Vanderah and Djerassi suggested the side chain of the "un-natural" 20S epimer exists p r e f e r e n t i a l l y in that conformation wherein the C-21 methyl group resides in the shielding anisotropy cone of C-16 to C-17 bond and the remainder of the side chain projects to the l e f t (cis r e l a t i v e to C-13), as depicted in 39. Although an argument against a preferred conformation in 20R steroids had been rais e d , the large differences in the observed GC m o b i l i t i e s of the C-20 epimers suggested the side chain of one or both of the C-20 epimers ex i s t s in a preferred rotational conformation. Vanderah and Djerassi f e l t that s t e r o l s , epimeric at C20 but of i d e n t i c a l conformer composition should exhibit i d e n t i c a l gas chromatographic behavior. C l e a r l y , more work i s required to s e t t l e t h i s subtle, yet i n t e r e s t i n g , conformational question. 0 Both acids 2 37 8 and 2 4 7 9 are known synthetic compounds and the major acid 23 has also appeared in the l i t e r a t u r e as 39 Table 3. 1H NMR data for various 20R and 20S steroids (CDC13) Compound 40 41 28 42 43 3_8 Chemical s h i f t 0.84G 0.94fl 0.93b 1.00fl 1.10° 1.106 of C-21, 6 a 100 MHz, see reference 76 b 400 Mz 40 R= V ^ v ^ 0 C H 3 ,—' the result of microbial transformation of other precursor s t e r o i d s8 0. The physical data for both compounds are in agreement with the l i t e r a t u r e values. Neither acid has been previously reported from natural sources, and they represent the f i r s t i s o l a t i o n of b i l e acids from a marine 40 invertebrate. In order to establish a dietary o r i g i n for the acids 23_ and 2 4 , a methanolic extract of Ant hoarcuat a graceae was examined (see experimental for d e t a i l s ) . A. cooperi was usually found associated with A. graceae and appears to derive i t s pigmentation from i t . The sponge contained neither 23 or 24 but i t did contain the same mixture of A'-3-ketosteroids that were found in A. cooperi . Cholestenone (22) was the major component of both mixtures. In addition, two oxygenated compounds, seemingly steroidal in nature (molecular formulas: C2 9H<, 0O 4 and C2 sHu «Oi,) , have been isolated from A. graceae. The structures of these metabolites are unknown at this time, but they do not appear to be A"-3-ketosteroids. The t h i r d metabolite isolated from A. cooperi, 1-0-hexadecyl-glycerol ( 2 5 ) , was characterized as i t s d i a c e t y l derivative 4 4 : C2 3HjM l05 (HRMS, m/z observed 400.3141, required 400.3189). Isolation as the d i a c e t y l derivative greatly 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 and s t r u c t u r a l assignment. The mass spectrum of 4 4 showed peaks assignable by the fragmentation pathway outlined in Scheme 3 OAc AcO 44 41 and allowed assignment of the ether linkage to C-3. The 1H NMR spectrum supported this assignment; 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 to the protons on C - l , the multiplet at 6 5.19 (m, IH) was assigned to the C-2 methine proton, and the doublet at 6 3.55_(d, / = 5.4 Hz, 2H) was assigned to the methylene protons on the carbon bearing the ether f u n c t i o n a l i t y . 357 (0.56%) 327 (2%) I ACO-/ P^OCH 2 (CH 2 ) u CH 3 255 (29%) Scheme 3. Interpretation of the MS of di a c e t y l derivative 44 Glycerol ether 25 i s a known natural product, commonly referred to as chimyl alcohol. It has been shown to s l i g h t -ly i n h i b i t the progress of the murine E h r l i c h ascites tumor8 1. Subsequent to i t s i s o l a t i o n from A. cooperi, 2_5 was isolated in our laboratory from the dorid nudibranch Arc hi dor is mont ereyensi 5 8 2 , and was shown to have potent in vitro a n t i b i o t i c a c t i v i t y against Staphylococcus aureus and Bacillus subtil is. I n t e r e s t i n g l y , 2_5 was also shown to have 42 antifeedant a c t i v i t y (18 ug mg~1 of food p e l l e t ) against the tide pool sculpin Oligocottus maculosus. 3. BIOLOGICAL ACTIVITIES OF ALDISA COOPERI METABOLITES Because the st e r o i d a l acids 23 and 24 were present in such high concentration r e l a t i v e to the animals body weight, i t seemed reasonable to assume that they were involved in the chemical defense of the nudibranch. To gain some evidence in support of thi s hypothesis, a standard goldfish bioassay3 6 was used to test the antifeedant properties of the stero i d a l acid 23. The results showed that 23 effec-t i v e l y i n h i b i t e d feeding at < 15 ug/mg of food p e l l e t , while cholestenone (2J_) was t o t a l l y inactive at > 100 ug/mg of food p e l l e t . The nudibranch was apparently obtaining an inactive metabolite from i t s diet and chemically modifying i t to produce an active antifeedant. V e r i f i c a t i o n of thi s supposition would require inje c t i o n of A. cooperi with radio l a b e l l e d cholestenone followed by i s o l a t i o n of radioactive s t e r o i d a l acids. It should be stated that, a l -though the s t e r o i d a l acids were not isolated from the sponge, the p o s s i b i l i t y exists that they were present in very small amounts and were not detected by the i s o l a t i o n procedure. This p o s s i b i l i t y would be very remote, however, as the' nudibranch would have to consume large amounts of sponge in order to obtain the large quantities of steroidal acids found in the skin of each animal. One c o l l e c t i o n of A. cooperi made in the Queen Charlotte Islands, B.C., gave 43 s t e r o i d a l acids 23 and 24 in a r a t i o similar to that found, in the Barkley Sound c o l l e c t i o n s . This finding supported the hypothesis that acids 2_3 and 2_4 are produced de novo by A. cooperi. 4. DISCUSSION Bio g e n e t i c a l l y , one can envisage the ster o i d a l acids being produced from cholestenone v i a a series of conventional fa t t y acid type 0-oxidation reactions. It has been shown that in the microbial transformation of cholesterol into 17-keto steroids, 3-oxo-4-cholenoic acid i s an intermediate8 3. The evidence suggested the degradation pathway shown in Scheme 4. The conversion of s i t o s t e r o l (45) and campesterol (46) into andro.st-4-ene-3,17-dione (47 ) has also been studied and both of these compounds are also degraded ( Mycobact eri urn sp.) via the ster o i d a l acid 238 0 . Thus, i t is not unreasonable to believe that the nudibranch ingests cholestenone (and related s t e r o i d a l ketones) from i t s sponge d i e t , and modifies them to the ster o i d a l acids 23_ and 24, via a mechanism similar to that shown in Scheme 4. These s t e r o i d a l acids might then be useful as defensive allomones. The use of steroids for chemical defense i s not limited to A. cooperi. The great diving beetle (Dytiscus marginal is) u t i l i z e s an array of s t e r o i d a l ketones to ward off potential predators. The major component of the mixture was shown to be 11-deoxycorticosterone (Cortexone) (48)8". 44 47 Scheme 4. Proposed mechanism for the microbial transformation of cholesterol into 17-ketosteroids8 3. This substance has the same stunning effect on f i s h as the natural secretion. Numerous other water beetles produce the 45 same or related compounds. Frogs and toads u t i l i z e s t e r o i d a l skin toxins (for example: bufotalin (49)), the skin of the salamander contains defensive secretions (for example: samandarin (50_))8 5, and sea cucumbers synthesize h a l o t h u r i n8 6, a neurotoxin of s t e r o i d a l structure and use i t to deter their f i s h predators. F i n i a l l y , two additional oxygenated steroids have been isolated from dorid nudibranchs. The highly oxygenated steroid 51 was isolated from Hervia peregrina, Flabellina affinis, and Coryphella lineal a 6 1 while Adalaria sp. produces a mixture of steroidal peroxides of which 5a, 5a-epidioxysteroid 5_2 i s an example8 8. From these examples i t can be seen that steroids are a group of s t r u c t u r a l l y similar compounds that exhibit a wide variety of b i o l o g i c a l a c t i v i t i e s , depending on oft-times subtle molecular changes. 46 R = H 4 6 47 C. SECONDARY METABOLITES FROM THE DENDRONOTID.NUDIBRANCH  MELI BE LEONINA (GOULD, 1852) 1. INTRODUCTION The dendronotid nudibranch Meli be leonina (see Figure 6) has one of the most unusual feeding behaviors of any mem-ber of the phylum mollusca. Unlike many other nudibranchs, M. leonina i s not a predator of s e s s i l e bottom dwelling animals, rather, i t feeds upon zooplankton by majestically sweeping the sea with i t s large oral hood8 9. Our chemical studies on M. leonina90 were prompted by a report that the nudibranchs' primary means of defense was an odiferous substance known to be repugnant to potential predators9 1 and were i n i t i a t e d after a fortuitous observation9 2 that allowed c o l l e c t i o n of enough animals for chemical a n a l y s i s . Specimens of M. leonina were collecte d during a reproductive congregation of the nudibranchs in a shallow kelp bed (depths of 1 to 5 m) at Cates Park, Vancouver, B.C. The number of nudibranchs was extremely high (= 50 animals/m2) and their odour could be detected in situ by SCUBA div e r s . 2. ISOLATION AND STRUCTURE ELUCIDATION Freshly c o l l e c t e d whole specimens (38 animals) were im-mediately immersed in chloroform and extracted (wrist action shaker) for 1.5 hours. The chloroform soluble material was f i l t e r e d , dried over sodium s u l f a t e , and concentrated in 49 vacuo to give an orange "grapefruit" smelling o i l (57.4 mg; 1.51 mg/animal). NMR analysis 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 acid (54) (Figure 7) in a r e l a t i v e r a t i o of 3.3:1* that was contaminated with a small amount of f a t . The individual components were subsequently resolved by s i l i c a gel column chromatography (gradient hexane/chloroform). 8 9 Figure 7. Secondary metabolites from the dendronotid nudibranch Mel i be leonina The least polar metabolite, 2,6-dimethyl-5-heptenal (J53) , had a molecular formula of C9H1 60 (M+, m/z 140), v e r i f i e d by the appearance of 9 carbons in the 1 3C NMR spectrum. A 1H NMR spectrum of t h i s substance displayed resonances appropriate for three methyl groups at 8 1.70 (bs, 3H), 1.61 (bs, 3H) and 1.11 (d, / = 7.1 Hz, 3H), for a * The r e l a t i v e r a t i o was obtained by measuring the peaks heights of the C-2 methyl doublets at 6 1.11 for 53 and 5 1.20 ppm for 54. 50 single o l e f i n i c proton at 6 5.10 (m), and for an aldehyde proton at 9.62 (d, / = 1.9 Hz, 1H) ppm. The aldehyde fu n c t i o n a l i t y also displayed an IR absorption at 1723 cm" and a 1 3C NMR resonance at 6 205.0 (d) ppm (see Table 4). 55 In the mass spectrometer, aldehyde 53 readily underwent a McLafferty rearrangement to give the base peak at m/z 82 as indicated in Scheme 5. Other fragment ions at m/z 41 , 55, 67, and 69 supported an a c y c l i c degraded monoterpenoid skeleton for 5_3. Comparison of the 1 3C NMR spectrum of 53_ to that of c i t r o n e l l a l (5_5) allowed the t o t a l assignment of the 1 3C NMR resonances (Table 4). The 400 MHz 1H NMR spectrum of aldehyde 5_3 was well resolved and allowed assignment of a l l the protons in the molecule, the chemical s h i f t s and assignments are l i s t e d in the experimental (Chapter 4). A l l the spectral evidence suggested that the non-polar compound responsible for the fragrant odour of the nudibranch was the degraded monoterpene 2,6-dimethyl-5-heptenal (53). Aldehyde 53 has been reported, as a 51 Table 4. 1 3C NMR data for 2,6-Dimethyl-5-heptenal (53) and C i t r o n e l l a l ( 5 5 )9 3. 2,6-Dimethyl-5-heptenal (53) C i t r o n e l l a l (55) Carbon # Chemical s h i f t , 6° (mult)c Carbon # Chemical s h i f t , 8b (mult)c 1 205.0 (d) 2 45.9 (d) 3 30.7 (t) 4 25.4 (t) 5 1 23. 5 (d) 6 132.7 (s) 7 25.7 (q) 8 17.7 (q) 9 13.3 (q) 1 202.2 (d) 2 51 .0 (t) 3 27.8 (d) 4 37.0 (t) 5 25.4 (t) 6 124. 1 (d) 7 131.5 (s) 8 25.6 (q) 9 17.6 (q) 10 19.8 (q) G100.6 MHz. b25 MHz. cM u l t i p l i c i t y , determined from a single frequency off resonance (SFORD) experiment. natural product, from numerous sources. I d e n t i f i c a t i o n s generally have been made solely on the basis of GC-MS ana l y s i s . Aldehyde 5_3 has been reported as a pheromonal component of the ant Lasius carniolicus, o r i g i n a t i n g s p e c i f i c a l l y from the insects' head9", as a chemical 52 component of the essential o i l from the leaves of Eucalyptus sp.9 5 and Carphephorous corymbosus 9 6, and as a component extracted from Jasminum sambac f l o w e r s9 7. Interestingly, a related secondary metabolite, 2,6-dimethyl-1,5-heptadien-3-ol acetate (56), is an insect sex phermone (Ps eudococcus comsl ocki ) 9 8. The odiferous character of synthetic 2,6-dimethyl-5-heptenal has not escaped the attention of the perfume industry where racemic aldehyde _53 has been produced on a greater than one ton per year b a s i s9 9. m/z 82 m/z 55 m/z 69 Scheme 5. Interpretation of the mass spectral fragmentation of 2,6-dimethyl-5-heptenal (53). The more polar metabolite from M. leonina had a molecular formula of C9H1 602 (HRMS, m/z observed 156.1151, required 156.1151) and i t s 'H NMR spectrum indicated that i t was closely related to aldehyde 5_3. IR absorption bands (3500 - 2200 and 1700 cm-1) c h a r a c t e r i s t i c of a carboxylic 53 acid and the absence of an aldehyde proton in the 'H NMR spectrum suggested that the polar metabolite was 2, 6-dimethyl-5-heptenoic acid (5_4) . The mass spectrum showed fragment ions at m/z 83 (100%) and 82 (46%), as well as intense peaks at 69, 67, 55, and 41 in f u l l support of the proposed structure. Treatment of 54 with ethereal diazomethane generated the methyl ester 5J7, confirming the presence of the carboxylic acid moiety. Although acid 5_4 had not previously been isolated as a natural product* i t i s a known synthetic compound1 0 0. 3. DISCUSSION To the best of the author's knowledge, i s o l a t i o n of aldehyde 5_3 and acid 54 represent only the second examples of i s o l a t i o n of nonhalogenated monoterpenes (granted degraded monoterpenoids) from a marine invertebrate. * Subsequent to the publication of our paper describing the M. leonina metabolites, Burger et al. reported acid 54 as a constituent from the sex a t t r a c t i n g secretion of the dung beetle Kheper lamarchi. 54 Halogenated monoterpenoids are well known from the herbivorous sea hares (Aplysia spp.), and were subsequently traced to their red a l g a l d i e t s . The pleasant smelling sponge Plakortis zygompha produces (possibly by symbiotic biogenesis) two degraded monoterpenes 58 and 5jJ, neither of which contain h a l o g e n s1 0 1. The b i o l o g i c a l significance of the halogenated monoterpenoids (isolated from the red algae and sea hares) and the degraded monoterpenes from the sponge are unknown. Many authors suggest that the compounds are involved in the organisms' apparent r e l i e f from predation. Much l i k e the tannins isolated from t e r r e s t r i a l trees and shrubs, perhaps the halogenated monoterpenoids are a general l e v e l defensive mechanism to which only s p e c i a l i s t s (sea hares) have adapted. In view of the postulated defensive role for the odiferous compound91, we tested 53 and 54 for antifeedant a c t i v i t y in a standard goldfish b i o a s s a y3 6. The carboxylic acid 54 showed no a c t i v i t y at 100 wg/(mg of food p e l l e t ) , while the aldehyde 5_3 was too v o l a t i l e for r e l i a b l e t e s t i n g . It would be an interesting b i o l o g i c a l project to synthesize racemic 2,6-dimethyl-5-heptenal (53) and evaluate i t s t o x i c i t y and antifeedant a c t i v i t y against a variety of potential nudibranch predators. It i s quite possible that either aldehyde 5_3, acid 54, or the naturally occurring mixture act as M. leonina aggregation or sex pheromones. M. leonina i s known to ag-gregate in great numbers and to then suddenly completely 55 disappear, only to reappear weeks or months l a t e r9 1. It is tempting to suggest that the major l y p o p h i l i c compounds isolated from the skin extracts of this animal are involved in the dynamic population jumps observed. More collaboration with marine b i o l o g i s t s w i l l be needed to test these ecological hypotheses. 58 59 56 D. SECONDARY METABOLITES FROM ACANTHODORIS NANAIMOENSIS  (O'DONOGHUE, 1921) 1. INTRODUCTION The chemical studies on Acant hodoris nanaimoensis (see Figure 8), prompted by an observation that the nudibranch had a fragrant odour, were i n i t i a t e d by Jocelyn T. H e l l o u1 0 2. The association of odour with interesting nudibranch chemistry was well known from the pioneering studies on Phyllidia varicosa mentioned in the preceeding chapter and were supported by the i s o l a t i o n of luteone (60) from Cadii na Iuteomargi nata103. Hellou had shown that the odouriferous p r i n c i p l e could be e f f e c t i v e l y extracted from the whole animals by immersing the specimens in methanol immediately after c o l l e c t i o n , soaking at room temperature for three days, and then decanting the supernatant. The supernatant was evaporated in vacuo to give a concentrated MeOH-H20 solution that was partitioned between brine and chloroform. The orange 58 organic phase was dried over anhydrous sodium sulfate and evaporated to give a sweet smelling o i l y residue. S i l i c a thin layer chromatography (TLC) analysis of the crude extract showed the presence of a non-polar metabolite, designated as nanaimoal1 0', that was readily p u r i f i e d by preparative TLC. The 1H NMR spectrum of the p u r i f i e d odouriferou's o i l indicated the presence of two closely related aldehydes. The aldehydic proton resonance of the major component appeared at 6 9.83 ( t , / = 3. Hz) and the analogous proton of the minor constituent resonated at 6 9.71 ( t , J = 3 Hz) ppm. Subsequent spectral analysis in association with biogenetic reasoning allowed Hellou to suggest three possible structures for nanaimoal, the major component: namely 61, 62 or 63. Spectral arguments did not allow an unambiguous choice of any of the above structures for nanaimoal, therefore Hellou prepared derivatives of the molecule in hopes that a suitable c r y s t a l l i n e compound could be found for structural 59 elucidation by single c r y s t a l X-ray d i f f r a c t i o n analysis. Of the eight derivatives prepared, none provided suitable c r y s t a l s . At this point Ms. Hellou wrote up her preliminary studies on A. nanaimoensis and ceased work on the p r o j e c t1 0 2. Subsequent work on A. nanaimoensis^05'^06 has shown that the TLC spot corresponding to nanaimoal i s a mixture of fiv e c l o s e l y related compounds. The structures of the three most abundant metabolites have been solved and were shown to be the isomeric aldehydes: nanaimoal (61), acanthodoral (64), and isoacanthodoral (65) (see Figure 9)*. The struc-tures of the two least abundant metabolites 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 following Hellou's procedure to provide 2.8 gms (23 mg/animal) of an orange o i l that contained the charac-t e r i s t i c nudibranch fragrance. A n a l y t i c a l TLC showed the presence of a non-polar spot (Rj- 0.25; 50% Hexane/CHCl3) that charred light purple and gave a positive test with 2,4-dinitrophenylhydrazine. In addition to t h i s spot, three other non-polar and one polar spots were also present. These were subsequently shown to be fats and s t e r o l s . * The absolute stereochemistries of the A. nanaimoensis metabolites are as shown. 60 Figure 9. Secondary Metabolites from Acanthodoris nanaimoens i s. Material which remained at the o r i g i n in a l l the TLC plates run (brown char, possibly fatty acids) was not investigated further. Column chromatography ( s i l i c a gel) of the crude extract yielded 218.6 mg (1.8 mg/animal) of material corresponding to the fragrant aldehyde f r a c t i o n [1H NMR 6 9.84 ( t , / = 3 Hz); 6 9.72 ( t , / = 3 Hz) ppm]. Gas chromatographic (GC) a n a l y s i s , Figure 10, indicated the presence of three major compounds. These were designated as nanaimoal (61, 79%), isoacanthodoral (65, 20%), and acanthodoral (64, 1%). Peaks for the remaining two least abundant metabolites were also seen. The mixture could readily be separated by preparative 61 GC or by high performance l i q u i d chromatography (HPLC). For large scale p u r i f i c a t i o n HPLC was found to be the most con-venient . Figure 10. GC analysis of a crude chloroform extract of A. nanai mo ens i s . Nanaimoal (6_1) had a molecular formula of C i5H2« 0 (HRMS, m/z observed 220.1836, required 220.1827). The infrared (IR) spectrum of 61_ showed aldehydic C-H and C=0 stretching bands at 2750 and 1710 cm"1 r e s p e c t i v e l y . The assignment of the lone oxygen in nanaimoal to an aldehydic carbonyl was supported by a resonance at 6 203.3 (d) ppm in i t s 1 3C NMR spectrum. P a r t i a l structure 66 was inf e r r e d from an ABX spin system in the 1H NMR spectrum (Figure 11); the aldehydic proton at 8 9.83 ( t , J = 3.2 Hz, 1H) ppm was coupled to 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 fragment ion at m/z 176 in the mass spectrum of nanaimoal, result i n g from loss of ethanal via a McLafferty rearrangement, supported this assignment (Scheme 6). Scheme 6. Interpretation of the MS of nanaimoal (61) The 1H NMR spectrum of nanaimoal also contained resonances for a gem-dimethyl at 6 0.98 (s, 6H) (IR 1395 and 1380 cm"1) (substructure 67), an isolated t e r t i a r y methyl at Figure 11. 400 MHz 1H NMR spectrum of nanaimoal (61) in CDC13. a) entire spectrum, b) expansion of the a l i p h a t i c region, c) expansion of the aldehydic proton resonance. CO 64 1.05 (s, 3H) (substructure 6_8), an isolated 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) (substructure 6_9) , four a l l y l i c protons at 1.81 (m, 2H) and 2.02 (m, 2H) ppm, and a six proton a l i p h a t i c multiplet situated between 8 1.41 and 1.66 ppm. The 1 3C NMR spectrum of aldehyde 61 contained resonances at 6 133.8 and 123.3 (both- singlets in a SFORD experiment) appropriate for a tetrasubstituted o l e f i n . The spectral data indicated that nanaimoal was a b i c y c l i c sesquiterpenoid that contained three t e r t i a r y methyl groups, an ethanal side chain, a tetrasubstituted o l e f i n , and six a l l y l i c protons of which two comprised an isolated AB spin system. The presence of six a l l y l i c protons ruled out 62 as a possible structure for nanaimoal. The remaining structural features to be defined were the po-s i t i o n s of the three additional a l i p h a t i c methylene carbons, the size of the b i c y c l i c ring system and the substitution pattern of substructures 66, 6_7, 68, and 69. The mass spectrum of nanaimoal (6_1) showed an ion at m/z 122 (C9H1 3) which suggested fragmentation involving the loss of a methyl group followed by a retro Diels-Alder reaction as shown in Scheme 6. The fragment ion was much more intense in the mass spectrum of nanaimool (70) [ a ]D + 10.4° (prepared from nanaimoal by NaBHu reduction) and allowed measurement of i t s exact mass (HRMS, m/z observed 121.1016, required for C9H1 3, 121.1017). It was concluded from t h i s result that the ring system in nanaimoal must be 65 bicyclo[ 4.4.0]dec-1 (6)-ene (7_1). The spectral data was consistent with only four possible structures for nanaimoal; namely 61, 63, 12 or 73. 71 72 Spectroscopically, d i f f e r e n t i a t i n g between the four possible structures for nanaimoal proved challenging. 'H NMR spectroscopy was useful in eliminating structures 7_2 and 73. P a r t i c u l a r l y interesting however, was the inherent spectroscopic s i m i l a r i t y between 61 and 63_. It was possi-ble, at t h i s point, to build an extremely strong case for 6_1 as the correct structure for nanaimoal. The arguments in favour of structure 61 for nanaimoal were: 1. Biogenetic reasoning. Working with the assumption that nanaimoal was a sesguiterpenoid (the presence of 15 carbons and 66 only one oxygen made the p o s s i b i l i t y that nanaimoal was an acetogenin remote) only structures JS1 and 6_3 f i t the biogenetic isoprene r u l e1 0 7, therefore non-biogenetic isoprenoid compounds T2 and 7_3 could be eliminated from consideration on t h i s basis. Numerous sesquiterpenoid metabolites have been di s -covered that v i o l a t e the isoprene r u l e1 0 6 [see for example; isoacanthodoral (6j>) and 9-isocyano-pupukeanane (16)], however, on closer inspection these metabolites can usually be r a t i o n a l i z e d as being formed from a biogenetic isoprenoid precurser, namely farnesyl pyrophosphate, and therefore obey, by d e f i n i t i o n , the biogenetic isoprene r u l e . Scheme 7 outlines the biogenetic arguments used in support of either structure £1 or structure 6_3 for nanaimoal. There were biogenetic precedents for the proposed f i r s t c y c l i z a t i o n step in the formation of both compounds. Structure 61 could be r a t i o n a l l y derived from an intermediate belonging to the well known monocyclofarnesane family (Scheme 7a), while structure 6_3 could be derived from an intermediate that had the carbon skeleton of the sponge metabolite p l e r a p l y s i l l i n - 1 (7_4) (Scheme 7b). The proposed second c y c l i z a t i o n reaction in the formation of structure 61 i s analogous to the formation of ring C in the pimerane d i t e r p e n e s1 0 9. Biogenetically, structure 61 was the most l i k e l y 67 structure for nanaimoal. The large number of naturally occurring monocyclofarnesane sesquiterpenoids occurring in both t e r r e s t r i a l and marine organisms vs only one example of a metabolite related to the p l e r a p l y s i l l i n - 1 skeleton supported this contention. 74 Scheme 7. Biogenetic arguments used in support of structure 61 for nanaimoal. 68 2. 1H NMR Double-resonance experiments. In the 1H NMR of nanaimoal, selec t i v e i r r a d i a t i o n of the a l l y l i c methylene protons at 6 2.02 (see Figure 11) in 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 multiplet at 5 1.81, an indication of homoallylic coupling*. This result supported the elimination of hypothetical structures 12 and 73 from consideration (supporting the same conclusion reached by biogenetic reasoning) as coupling between these two a l l y l i c methylenes, in the analogous spin systems, was predicted to be n e g l i g i b l e . 3. Chemical s h i f t of the gem-dimethyl group. The chemical s h i f t , 6 0.98 (s, 6H) ppm, and chemical s h i f t equivalence of the gem-dimethyl group in nanaimoal (6_1) (substructure 6_7) indicated t h i s f u n c t i o n a l i t y was attached to the tetrasubstituted carbon-carbon double bond. Comparison to model systems supported th i s assignment (Figure 12). The chemical s h i f t assignment of the gem-dimethyl fu n c t i o n a l i t y was supported by the observation that upon reduction to nanaimool (7J3), the chemical s h i f t s of the gem-dimethyl groups were only * Decoupling experiments outlined in a subsequent section allowed the assignment of these protons. 69 marginally affected. On the other hand, the chemical s h i f t of the t h i r d quaternary methyl (substructure 68) was substantially affected (moved upf i e l d by 6 0.17 ppm). Unfortunately no reasonable model system could be found for the chemical s h i f t s of a gem-dimethyl group in a structure of type 6_3 that would reinforce t h i s argument. 4. Reduction of the aldehydic carbonyl. Reduction of the aldehydic carbonyl affected the chemical s h i f t of the quaternary methyl, as mentioned above, as well as causing an u p f i e l d s h i f t (average + 0.14 ppm) of the isolated a l l y l i c methylene protons (substructure 69). This result indicated a close s p a t i a l r e l a t i o n s h i p between the carbonyl and the isolated a l l y l i c methylene protons. Of the structures remaining under consideration, namely 6_1 and 63_, only 61 could account for such an ef f e c t . 70 6 1.00 (s, 6H) Figure 12. Model systems for the 1H NMR chemical s h i f t s of the gem-dimethyl group in nanaimoal. 3. SYNTHESIS OF NANAIMOAL'S (P-BROMOPHENYL)URETHANE  DERIVATIVE. ASSIGNMENT OF STRUCTURE The structure of nanaimoal (61) had been postulated using a combination of spectral and biogenetic reasonings as discussed in the previous section. In the absence of any spectroscopic or degradative scheme that would unambigously allow assignment of either* structure 61 or 63 to nanaimoal, 71 synthesis of the molecule representing the most l i k e l y structure, compound 6 1 , was in order. The urethane 75, a derivative of nanaimoal, was chosen as a synthetic target since t h i s derivative had already been prepared in an attempt to generate a suitable c r y s t a l l i n e derivative for X-ray a n a l y s i s . The most l i k e l y structure for nanaimoal (6_1) had a r e l a t i v e l y simple carbon skeleton, therefore retrosynthetic analysis provided a straightforward synthetic strategy (Scheme 8). Anti t h e t i c cleavage of the C-4 to C-5 bond followed by functional group interconversion generated compound 7_6. The corresponding synthetic reaction had been shown to be f a c i l e in the conversion of triene 7_7 to diene 781 1 0 (Scheme 9). Retrosynthetic analysis of compound 7_6 revealed a simple Diels-Alder reaction between myrcene (79) and a dienophile of type 80, for example, would generate the required c y c l i z a t i o n precurser. The problem was thus reduced to finding the appropriate dienophile. Methyl methacrylate (81L) seemed a l o g i c a l choice for the dienophile i n i t i a l l y . However, examination of the H 75 72 Scheme 8. Retrosynthetic analysis of the postulated structure for nanaimoal. l i t e r a t u r e1 1 1 (as well as t h e o r e t i c a l considerations) revealed that t h i s reaction would l i k e l y provide a good y i e l d of the wrong regioisomer. If the correct regioisomer could be o b t a i n e d1 1 2, the neopentyl nature of the ester carbonyl would pose a problem in any reactions designed to extend the side chain by the required one c a r b o n1 1 3. These 73 two factors ruled out 81 as a suitable dienophile. Scheme 9. Previous synthesis of the nanaimoane carbon skeleton. Isoprene ( B 2 ) had been shown to react with myrcene, generating a mixture of regioisomers 7J7 and 83 in a r a t i o of 2:3 1 1 0 , in low y i e l d * . As discussed above, c y c l i z a t i o n of 77 generated diene 7_B.' a compound having the required carbon skeleton of structure 6_1. To f i n i s h a synthesis via this route, selective hydroboration of the monosubstituted o l e f i n would be required to generate alcohol JSL' oxidation of which should provide (i)-aldehyde 6_1. This o v e r a l l reaction se-quence suffers,, frorn one serious drawback, a plethora of products were shown (and were expected) to be formed upon reaction of myrcene and i s o p r e n e1 1 0. Although i t would be r e l a t i v e l y easy to vacuum d i s t i l l the mixture and obtain a somewhat p u r i f i e d mixture of o l e f i n s 11_ and 8_3, i t was * It was realized from the outset that unambiguous synthesis of structure 61 or i t s derivative was the goal of the synthesis, therefore high y i e l d was not an absolute requirement. 74 reasoned that a readily available dienophile such as 3-methyl-3-buten-1 - o l (8J)) would give a cleaner reaction product that could be easily p u r i f i e d by fla s h chromatography. The alcohol 80 could be used in large ex-cess to suppress the appearance of products a r i s i n g from the dimerization of myrcene. It was hoped that 80 would give a r a t i o of regioisomeric products 7_6 and 84 similar to that obtained with isoprene as the dienophile, that is 2:3. In the event1 1*, the Diels-Alder reaction (225 °C sealed tube, 8 h, neat, 4:1 alcohol:myrcene) proceeded in a very low but adequate y i e l d (polymerization of starting materials was the major side reaction) to generate, in one step afte r 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 in a r a t i o of 2:3 (76:84). Separation of the regioisomeric alcohols 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 could be achieved by recycling r a d i a l TLC (Chromatotron: 12% EtOAc/petroleum ether). The major isomer 84 displayed peaks for the three methyl groups at 6 0.91 (s, 3H), 1.60 (bs, 3H), and 1.68 (bs, 3H), carbinol protons at 3.73 (m, 2H) and 75 two o l e f i n i c protons at 5.08 (tm, 3 = 1 Hz, 1H) and 5.29 (bs, 1H) ppm. The minor isomer 76 displayed similar peaks at 6 0.92 (s, 3H), 1.60 (bs, 3H), 1.68 (bs, 3H), 3.73 (m, 2H), and 5.08 (bt, J = 1 Hz, 1H) for the methyl groups, carbinol protons and side chain o l e f i n i c proton respective-l y . The C-2 o l e f i n i c ring proton resonated at 6 5.35 (bs, 1H) ppm. The 1H NMR spectral data were very similar for each regioisomer, the major difference was in the chemical s h i f t s of the cyclohexene ring o l e f i n i c protons (6 5.29 for 84 vs 5 5.35 for 76) . With the mixture of regioisomeric alcohols in hand, methods for affe c t i n g the formation of the required C-4 to C-5 bond were explored. The nucleophilic nature of the side chain hydroxyl group was expected to pose a problem in any c y c l i z a t i o n reaction that generated carbocation character at C-10, leading to the undesired formation of c y c l i c ethers 8_5 and £36. Preliminary c y c l i z a t i o n experiments proved this concern to be warranted. Attempted c y c l i z a t i o n of the mixture of alcohols 76 and 84 using BF3•EtOEt in refluxing anhydrous ether f a i l e d to generate any of the desired cy c l i z e d products 7_0 or j5_7. Instead, a non-polar compound, assumed to be a mixture of ethers Ji_5 and 86 was produced with t o t a l consumption of the s t a r t i n g a l c o h o l s1 1 5. To a l l e v i a t e t h i s problem, protection of the hydroxyl group would be required. A number of protecting groups were considered (for example, the acetyl or benzyl e s t e r s ) , 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 intermediates (UV 76 chromophore) and to allow d i r e c t comparison of the protected, c y c l i z e d , synthetic products to the derivatized natural product* the (p-bromophenyl)urethane f u n c t i o n a l i t y was chosen as the protecting group. The German workers1 1 0 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 acid (85%, 12 hr, 100 °C) to give a 48% y i e l d of c y c l i z e d products 7j5 and 88. Formic acid c y c l i z a t i o n was therefore attempted on the mixture of urethanes 89 and 90, prepared by reacting the alcohols 7_6 and 84 with 4-bromophenyl isocyanate. Heating the mixture * One of the derivatives of nanaimoal prepared was the p-bromophenylurethane derivative 75. It f a i l e d to provide c r y s t a l s suitable for s i n g l e - c r y s t a l X-ray analysis from a l l the solvents t r i e d . Derivative 7_5 was quite stable and could be stored at 4 °C, in the dark, without s i g n i f i c a n t decomposition. 77 of urethanes in 98-100% formic acid at 60 °C for 12 hours gave one major, non-polar, TLC spot that had an i d e n t i c a l Rj to the derivatized natural product 7_5. Two more polar products (possibly formate ester 91 and t e r t i a r y alcohol 92) and brown, l i k e l y polymeric, material that remained at the o r i g i n of the preparative TLC plate.were also found in minor amounts. The more polar products were not investigated further. 92 R = H 400 MHz 'H NMR analysis of the major, non-polar, product was devoid of resonances for o l e f i n i c protons and o l e f i n i c methyl groups, and showed the appearence of four new a l i p h a t i c methyl resonances. This result indicated that the non-polar product was a mixture of c y c l i z e d urethanes (±)-7J5 and 93. Comparison of the new methyl singlet resonances to those of derivatized nanaimoal 75 showed a direct correspondence between those of the synthetic minor 78 regioisomer to those of the derivatized natural product 75. Encouraged by th i s r e s u l t , attention was turned to the assignment of structure to the uncyclized regioisomeric urethanes (89 and 90), to be followed by conversion of pure regioisomer 89 to derivatized nanaimoal. Deriv a t i z a t i o n of the major regioisomeric alcohol j}_4 with 4-bromophenyl isocyanate provided urethane 90 in good y i e l d . Analysis of the 400 MHz *H NMR spectrum of 90 showed a broadened AB spin system at 6 1.77 (bd, / = 17.6 Hz, 1H) and 8 1.90 (bd, J = 17.6 Hz, 1H) due to the a l l y l i c protons on C-3. Most of the broadening could be removed by i r r a d i a t i o n of the C-2 cyclohexene proton at 6 5.29. To confirm the coupling was v i c i n a l and not a l l y l i c (as would be the case for regioisomer 89) a difference nuclear Overhauser enhancement (nOe) experiment was performed. Irrad i a t i o n of the C-2 cyclohexene o l e f i n i c proton observed for 90 at 6 5.29 resulted in an nOe enhancement of the four a l l y l i c protons on carbons C-3 and C-7. The a l l y l i c protons on C-3 appeared as two broadened doublets at 8 1.77 (bd, J = 17.6 Hz) and 8 1.90 (bd, J = 17.6 Hz) in t h i s difference nOe experiment. Since the major regioisomer could now be de-fined as 90, i t followed that the minor regioisomer was urethane 89. The 400 MHz 1H NMR spectrum of 89 showed resonances for three methyl groups at 8 0.94 (s, 3H), 1.60 (s, 3H), and 1.68 (s, 3H), carbinol protons at 4.24 (m, 2H), and o l e f i n i c protons at 5.09 (tm, J = 7 Hz, 1H) and 5.37 (bs', 1H) ppm. 79 Resonances for the urethane moiety were observed at 6 6.51 (bs, 1H, NH), 7.26 (d, / = 8.4 Hz, 2H) and 7.40 (d, / = 8.4 Hz, 2H). In the mass spectrum of urethane 89, intense fragment ions were observed at m/z 204 (48%), 136 (22%), 69 (100%), 55 (52%) and 41 (90%). The fragment ion at m/z 204 could arise from a McLafferty fragmentation of the type shown in Scheme 10, while the ion at m/z 136 (HRMS, m/z observed 136.1239, required for C1 0H1 6 136.1252) l i k e l y re-sults from a retro Diels-Alder reaction (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 substituent. Br Scheme 10. Interpretation of the MS of urethane 89. Heating urethane 89 in 98-100% formic acid at 60 °C for 12 hours accomplished the required c y c l i z a t i o n to give urethane (±)-7_5 in excellent y i e l d . Synthetic (±)-75 was i d e n t i c a l by HPLC, MS and 'H NMR comparison to the 80 p-bromophenylurethane derivative prepared from nanaimoal. Nanaimoal has a new sesquiterpenoid carbon skeleton. We propose to name th i s skeleton nanaimoane and to number i t as shown in Figure 13. The absolute stereochemistry of nanaimoal was proposed on the basis of i t s biogenetic re l a t i o n s h i p to acanthodoral (64) and isoacanthodoral (65) (vide i nfra) . 14 13 Figure 13. Nanaimoane carbon skeleton 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 studies directed towards the t o t a l assignment of the 1H and 1 3C NMR spectra of t h i s novel metabolite were i n i t i a t e d . Close examination of nanaimoal's 400 MHz }H NMR spectrum (Figure 11) revealed that, because of the floppy nature of the bicyclof4.4.0]dec-1(6)-ene ring system, most of the geminal ring methylene protons were chemical s h i f t equiva-l e n t . Because they were not, in most cases, also magnetic equivalent, the 400 MHz 1H NMR spectrum was highly second order and consequently complex. In spite of t h i s complicating factor, careful use of selected one-dimensional 1H NMR double-resonance (decoupling) experiments, two-dimensional homonuclear c o r r e l a t i o n spectroscopy (COSY/45), and two-dimensional J spectroscopy (2D ./-resolved) allowed complete assignment of nanaimoal's 1H NMR spectrum (chemical s h i f t s but not a l l coupling con-stants, see Table 5). This study demonstrates the usefulness (and limi t a t i o n s ) of selected modern NMR tech-n i q u e s1 1 6 to a real problem in natural products chemistry. From the outset of this NMR study, i t was envisaged that upon completion of the 1H NMR spectral assignments, t o t a l assignment of the 1 3C NMR spectrum would also be completed using heteronuclear 1H-13C c o r r e l a t i o n spectroscopy (CSCM*). However, the results of the CSCM * CSCM = chemical s h i f t correlation map 82 experiments c l e a r l y demonstrated that the largest l i m i t a t i o n to obtaining a reasonable spectrum was sample s i z e . Not enough natural material was available for a suitable CSCM experiment to be adequately c a r r i e d out*. Even though a proton broad-band (BB) decoupled 1 3C NMR spectrum could readily be obtained on 35 mg of urethane 75 with as few as 256 scans [30° f l i p angle, 2.0 s relaxation delay (RD)], the absence of BB proton decoupling (except during acquisition) in the CSCM experiment did not allow a suitable spectrum to be obtained even after 18 hours ( A , = 3.3 ms, A 2 = 1.67 ms, RD = 2.0 s ) . A number of spectral parameters must be optimized to get good CSCM results with a small sample size (namely the RD, and the AT and A 2 values), this requires perhaps a number of experiments to be run. A large sample size is the single most important factor required for obtaining a suitable CSCM spectrum in a reasonable amount of magnet time. a. One-Dimensional 1H NMR Experiments One-dimensional 1H NMR experiments were conducted on nanaimoal (61), nanaimool (7fj) and the (p-bromophenyl)-urethane derivative 75 which, for comparison, was run in two di f f e r e n t solvents, CDC13 and benzene-d6. The 1H NMR data for these four experiments i s summarized in Table 5, along with selected results from the 2D /-resolved experiment on * Although the structure of nanaimoal (61) was deduced by synthesis, the low y i e l d and tedious p u r i f i c a t i o n procedure did not allow generation of racemic urethane 75 on a large scale (> 10 mg). 83 Table 5. 1H NMR data (400 MHz) for nanaimoal (61) and d e r i v a t i v e s . chemical s h i f t H on C# 75° 25* 61 f l 61 a, c 70fl 1 1 .79 (bm) 1 .77 (bm) 1.81 (m) 1 . 80 (s) 1 .78 (m) 2 1 .55-1 .62 1 .52- 1 .63 1 .56- 1 .66 1 . 60 (s) (m) (m) (m) 3 1.41- 1 .49 1.41- 1 .48 1.41- 1 .49 1 . 44 (s) (m) (m) (m) 4 - - - - -5 - - - - -6 1 .98 (bm) 1 .94 (bm) 2.02 (m) 2. 01 (s) 1 .97 (bm) 7 1 .40 ( t , 1 .23-1 . 37 1 .44 (m) 7) (m) 1 .56 (m) 8 - - - - -9 1 .62 (bd, 1 .53 (bd, 1 .77 (bd, 1 . 76 (s) 1 .59 (bd, 17) 17) 17) 17) 1 .76 (bd, 1 .68 (bd, 1 .85 (bd, 1 . 84 (s) 1 .75 (bd, 17) 17) 17) 17) 10 - - - - -1 1 1 .56 (m) 1 .54 (m) 2.24 (dd, 2. 22 (s) 3, 14 .5) 1 .63 (m) 1 .63 (m) 2.29 (dd, 2. 29 (s) 3, 14 .5) 1 2 4.23 (m). 4.19 (m) 9.84 ( t , 3.72 (m) 1 3 0.98 (s)<J 1 .02 ( s ) ^ 3 ) 0.98 ( s ) ^ 0. 98 (s)<{ 0.97 ( s ) ^ 14 0.97 ( s ) ^ 1 .00 (s)<* 0.98 (s)d 0. 98 ( s ) ^ 0.98 (s)d 1 5 0.91 (s) 0.84 (s) 1 .05 (s) 1 . 05 (s) 0.88 (s) NH 6.51 (bs) 5.83 (bs) 18, 19 7.39 (d) 6.83 -7.34 19, 21 7.27 (d) V a CDC13. b benzene-d6. c 2D /-resolved spectrum, projection onto F2. d May be reversed. 84 nanaimoal (6_1). Selected proton spin-spin decoupling experiments allowed a number of proton assignments to be made, and these assignments were later confirmed by the duet of homonuclear two-dimensional experiments. Irradiation of the two proton broad singlet at 6 1.98 (bs, 2H) in urethane 75 (CDC13) caused a two proton t r i p l e t centered at 6 1.40 ( t , J = 6 Hz, 2H) ppm to collapse to a s i n g l e t . Therefore, the two protons responsible for t h i s t r i p l e t must be part of a substructure of type 94, a fact only accountable for i f they were the chemical s h i f t equiva-lent C-7 methylene protons (for numbering scheme see Figure 13). Consequently, the broad multiplet at 5 1.98 ppm must arise from the a l l y l i c C-6 methylene protons. Upon i r r a d i a t i o n of the C-6 methylene protons a number of changes were also seen in the three proton a l l y l i c multiplet resonating between 8 1.72 and 1.84 ppm (this multiplet was actually one half of an a l l y l i c AB quartet at 8 1.76 overlapped by a two proton broadened multiplet centered at 6 1.79 ppm). The two proton broadened multiplet at 8 1.79 collapsed to a t r i p l e t (/ = 7 Hz) and the overlapped doublet at 6 1.76 sharpened up. The other half of the a l l y l i c AB quartet at 6 1.62 (J = 17 Hz) was also noticably sharpened in t h i s double-resonance experiment. This result indicated that the C-6 methylene protons were a l l y l i c a l l y coupled to both the C-1 [6 1.79 (bm)] and C-9 [6 1.62 (bd, / = 17 Hz), 8 1.76 (bd, / = 17 Hz) ] a l l y l i c proton p a i r s . Thus, using one double-resonance experiment, a l l the proton resonances 85 of substructure 95 could be readily assigned. When the 'H NMR spectrum of urethane 7!) was obtained in benzene~d6 a number of changes were noted in the resonances of spin system 95. The C-7 methylene protons were no longer chemical s h i f t equivalent and a complex multiplet centered about 6 1.30 ppm was observed for these protons. Ir r a d i a t i o n of the a l l y l i c C-6 methylene protons at 6 1.94 (bm) collapsed this multiplet to an AB doublet of doublets, allowing measurement of the geminal coupling constant (J = 13 Hz). The isolated AB doublet of doublet resonances for the C-9 protons were also s h i f t e d r e l a t i v e to their position in CDC13 and now appeared at 6 1.68 (d, / = 17 Hz, 1H) and 1.53 (d, / = 17 Hz, 1H) ppm. The C-1 methylene protons resonated at 6 1.77 in benzene-d6 and were again collapsed 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 95 was also c l e a r l y evident in the 1H NMR spectrum (CDC13) of nanaimoal (6_1) , although deshielding e f f e c t s , l i k e l y due to the diamagnetic anisotropy of the aldehydic carbonyl, were noted. The C-7 methylene protons in nanaimoal (61) were c l e a r l y deshielded with respect to their chemical s h i f t in urethane 7j> and were no-longer chemical s h i f t equivalent. 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 possible. However, i r r a d i a t i o n of the C-6 protons at 6 2.01 ppm in a double-resonance experiment collapsed the C-7 protons multiplet to an AB doublet of doublets. One C-7 proton resonated at 8 1.44 (d, J = 16 Hz) and the other at 6 1.56 (d, / = 16 Hz). Also deshielded with respect to their chemical s h i f t s in urethane 7_5 was the C-9 AB quartet which in 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 deshielded with respect to the analogous resonances in urethane 75 (see Table 5). With spin system 95 c l e a r l y defined, a l l that remained was assignment of the C-2, C-3, and C-11 methylene protons. The chemical s h i f t s of the methyl groups could be assigned by analogy to model systems as was previously discussed in Section II.D.2. In nanaimoal, and in a l l the derivatives studied, a complex two proton multiplet was always observed between 5 1.49 and 6 1.41 ppm. Irradiation of the C-1 87 methylene protons at 5 1.79* of urethane 75 in a double resonance experiment did not result in 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 , therefore, i t could not be due to the C-2 protons. Since the multiplet was also present in the 1H NMR spectrum of nanaimoal (6_1) i t could not be due, in whole or in part, to the C-11 methylene protons. Therefore, th i s multiplet was assigned to the two geminal protons at C-3. It was not possible, because of the second order nature of t h i s multiplet to determine the exact chemical s h i f t of each of the C-3 methylene protons, however, the 2D /-resolved experiment (to be discussed subsequently) was able to show that, in nanaimoal (61) , the two C-3 protons are chemical s h i f t equivalent and resonate at 6 1.44 ppm. The location of the C-2 methylene protons resonances could now be e a s i l y extracted from the 1H NMR spectrum of urethane 75 (CDC13) by elimination. They resonated as a complex two proton multiplet between 6 1.62 and 6 1.55 ppm. Again the 2D /-resolved experiment was useful in assigning the chemical s h i f t s of these protons in nanaimoal (61) . They were chemical s h i f t equivalent and resonated at 8 1.60 ppm (see subsequent s e c t i o n ) . The results of the one-dimensional 1H NMR experiments on nanaimoal and i t ' s derivatives are summarized in Table 5. * As previously mentioned, one of the C-9 proton resonances overlapped the multiplet a r i s i n g from the C-1 protons. Any i r r a d i a t i o n of the C-9 protons would not af f e c t the argument however. 88 b. Two-Dimensional 1H NMR Experiments The past 5 years has seen two-dimensional NMR spectroscopy (2D-NMR)* rapidly exploited as a useful and powerful tool for both biochemical (structure elucidation and biosynthesis) and synthetic natural product studies. As a review and indepth discussion 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 familiar with the basic concepts and theory of 2D-NMR. Reviews by B e r n s t e i n1 1 8, Benn and Gunther1 1 9, and a book by Bax1 1 6, when combined, provide a more than adequate intro-duction to both the p r a c t i c a l and theoretical aspects of 2D-NMR. One of the simplest, and in fact the very f i r s t proposed two-dimensional experiment1 1 7, was the two-dimensional homonuclear chemical s h i f t c o r r e l a t i o n (COSYt) experiment1 2 0. The experiment employs the radiofrequency pulse sequence shown in Figure 141 2 1. The f i r s t preparatory pulse (90°x) i s followed by the evolution period («,) and the second mixing pulse (90°), and detection (t 2) . The preparatory pulse has constant phase and the phase of the mixing pulse i s incremented in 90° steps ( ^T ) . The receiver phase chosen, $2 or $3, selects the coherence transfer echo or anti-echo, re s p e c t i v e l y , and cancels the a x i a l peaks at F, = 0. If two n u c l e i , A and X, are spin coupled, two sets of cross peaks w i l l occur in the * In 1971, Jeener1 1 7 f i r s t suggested the p o s s i b l i t y of two-dimensional NMR experiments, t COSY = correlated spectroscopy 89 RD - 90° (x) - J , - 90°(*,) - AQN ( f2; #2 or 4>3) RD = relaxation delay AQN = acq u i s i t i o n Figure 14. Pulse sequence for the homonuclear COSY NMR exper imen,t. two-dimensional spectrum centered at ( 5a, 6X) and ( 5xF ^ A ) , in a d d i t i o n , diagonal peaks at ( §a, 6a) and ( 6X, 6X) w i l l also be seen. This pulse sequence results in a two-dimensional spectrum (both dimensions showing proton chemical s h i f t s ) that indicates connectivity patterns be-tween coupled protons. The COSY/45 1 2 2 spectrum of nanaimoal's (p-bromophenyl)-urethane derivative 75 is shown in Figure 15. With refer-ence to Table 5 (proton assignments based on 1D-NMR double-resonance experiments) most of the expected connectivity r e l a t i o n s h i p s are indeed represented by cross peaks in 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 readily mapped out. The low i n t e n s i t i e s of the C-1,C-2 cross peaks may indicate a short transverse relaxation time (T2) for one or both of the C-2 methylene p r o t o n s1 2 3, or possibly, small v i c i n a l coupling. 90 Noticeably absent in Figure 15 are cross peaks due to the long range homoallylic couplings seen in the 1D double-resonance experiments. These cross peaks may be seen however, upon expansion and dropping of the contour threshold. Figure 16 shows a section of Figure 15 expanded and with more contour l e v e l s . Cross peaks for a l l the ex-pected long range homoallylic couplings are now v i s i b l e . The COSY/45 experiment allowed confirmation of the spin systems and coupling patterns deduced from the one-dimensional 1H NMR experiments, however, this experiment did not y i e l d any additional information regarding the assignment of chemical s h i f t s to the strongly coupled C-2 and C-3 methylene protons*. To a s s i s t in the assignments of these protons, and to separate the overlapping proton resonance multiplets for coupling constant ana l y s i s , a homonuclear 2D /-resolved 1H NMR experiment was performed on nanaimoal (61^). Homonuclear two-dimensional /-resolved spectroscopy i s an NMR experiment that, when applied to protons for example, allows separation of the chemical s h i f t information (8) from the scalar proton-proton couplings (/). Obviously a useful technique when applied to molecules whose 1H NMR spectrum consists of many overlapping resonances1 2 5, 2D /-resolved spectroscopy has found application in the interpretation of the 1H NMR spectra of a number of complex mo l e c u l e s1 2 6. * An experiment does exist whereby a 1H NMR COSY spectrum may be obtained with what amounts to complete 1H broad-band decoupling in the F, dimension1 2'1. 4 -N 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 Figure 15. 400 MHz 'H NMR COSY/45 spectrum of nanaimoal' (p-bromophenyl)urethane derivative (not symmetrized). 92 Figure 16. Expansion and amplification of Figure 15 to show homoallylic couplings. 93 The pulse sequence used for the homonuclear 2D /-resolved experiment i s the simple Hahn spin-echo sequence shown in Figure 1 71 2 7. This pulse sequence leads to a J-modulated spin-echo. By using a step-wise incrementation of t , the 2D data matrix is b u i l t up and the Fourier transformation in the t , dimension w i l l be a function of / . Vector diagrams have been found to be quite useful as a conceptual aid for explaining the 2D /-resolved experiment1 2 8. RD - 90°(*,) - 0.5/, - 180°($2) - 0.5*, - AQN($3) Figure 17. Pulse sequence for 2D /-resolved NMR experiment. A number of complicating factors must be taken into consideration in order to co r r e c t l y interpret a 2D /-resolved 1H NMR spectrum. These are: 1) var i a t i o n in peak i n t e n s i t i e s , 2) second order e f f e c t s , and 3) the appearance of a r t i f a c t s . These three factors are discussed below: 94 1. The i n t e n s i t i e s of the peaks in the 0° projection* onto F2 w i l l vary according to the amount of coupling the proton experiences. Thus a highly coupled proton w i l l have a r e l a t i v e l y low intensity while methyl singlets w i l l generally be quite intense (see Figure 18). Because of transverse relaxation (characterized by the spin-spin relaxation time T2) , the magnitude of the magnetization present at the beginning of acquisition in a 2D /-resolved experiment w i l l have decreased by a factor exp(-t!/T2) in comparison to the i n i t i a l magnetization, that i s , after the 90° pulse. Intensities of projection peaks a r i s i n g from protons with large T2 times (fast relaxation) w i l l thus be reduced. In general, the i n t e n s i t i e s of the peaks w i l l not affect the interpretation of the spectrum, unless of course, either the intensity drops to zero or a small peak is obscured by the t a i l i n g of a large one. 2. Strong coupling introduces a very important complication into the interpretation of a homonuclear 2D /-resolved 1H NMR s p e c t r u m1 2 9'1 3 0. The 2D /-resolved spectrum of a strongly coupled system w i l l be much more complex than i t s weakly coupled equivalent because many additional second order peaks may be present. Fortunately, second * Projection of the t i l t e d 2D /-resolved spectrum is assumed in t h i s discussion unless otherwise stated. 95 order peaks are frequently not symmetrical about F, in the t i l t e d spectrum, and symmetrization may be used to eliminate them*. Care must be exercised in interpreting a second order 2D /-resolved spectrum, i f doubt e x i s t s , assignments may be v e r i f i e d with the aid of a two-dimensional simulation program1 3 0. 3. The t h i r d complicating factor i s a r t i f a c t s . A r t i f a c t s sometimes arise in the 2D /-resolved spectrum as a re-sult of an inaccurate 180° refocussing pulse (see Figure 17). These a r t i f a c t s can be partly eliminated by c y c l i n g the phases of pulses and receiver using the E x o r c y c l e1 3 1 procedure and by the use of a composite 180° refocussing p u l s e1 3 2. If one i s aware of the above limi t a t i o n s and can cor-r e c t l y account for them, interpretation of a 2D /-resolved spectrum should be a r e l a t i v e l y straightforward exercise. If the spectrum is largely f i r s t order, and the appearence of a r t i f a c t s can be minimized, a 2D /-resolved spectrum can y i e l d a great deal of information about the structure of an unknown natural product. * An AB spin system gives 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° projection onto F2 and cannot be removed by symmetrization. 96 Figure 18 shows the 400 MHz 2D ./-resolved spectrum of nanaimoal (61) . It was readily apparent by inspection of the F2 projection that most of the protons previously assigned by interpretation of the 1D 1H NMR spectrum were present. The C-1 a l l y l i c methylene protons at 5 1.80 were c l e a r l y resolved from overlap by the C-9 AB doublet of doublets (6 1.76 and 6 1.84) and were chemical s h i f t equiva-l e n t . The two intense peaks at 6 1.60 and 6 1.44 could be assigned to the C-2 and C-3 methylene protons respe c t i v e l y , each methylene pair 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 in the spectrum. The most noticable second order resonance was the peak at 6 2.25, midway between the resonances a r i s i n g from the C-11 AB methylene protons. This peak was expected based on an explanation of t h i s second order effect by Bax1 3 3. Other smaller second order and/or a r t i f a c t peaks could not be r a t i o n a l i z e d , however, and ex-cept for obscuring one of the C-7 resonances at 6 1.56 they did not interfere with the interpretation of the spectrum. S l i c e s of selected individual peaks are shown in Figure 19. They c l e a r l y demonstrate the second order complexity of the couplings. Because in strongly coupled systems the observable resonances may not be truely depicted in F, no interpretation of the couplings was attempted. The geminal couplings of the methylene protons on C-9 and the geminal and v i c i n a l couplings of the methylene protons on C-11 were readily extracted from the spectrum, and their values are 97 - -30 F 2 ( P P M ) Figure 18. P a r t i a l 400 MHz 1H NMR 2D /-resolved spectrum (symmetrized) of nanaimoal (61) showing (from bottom to top) the contour plot and the "proton decoupled" spectrum derived from the 2D / spectrum; X denotes second order tra 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 l i s t e d in Table 5. 2 a,b Figure 19. S l i c e s of indi v i d u a l peaks shown at the top of Figure 18 to show m u l t i p l i c i t i e s . 9 9 5. ISOACANTHODORAL Isoacanthodoral (65) MS, M+ 220, C1 5H2flO constituted 20% of the sesquiterpenoid aldehyde fr a c t i o n isolated from the skin extract of Acanthodoris nanaimoensis (see GC trace, Figure 10). Aldehyde ( £ 5 ) could be p u r i f i e d by preparative GC or HPLC, however, the small quantity of isoacanthodoral extracted (400ug/animal), i t s high v o l a t i l i t y , and i t s susceptability to oxidation (to form a carboxylic acid) made i s o l a t i o n and characterization d i f f i c u l t . It was found most convenient to convert isoacanthodoral to either i t s c r y s t a l l i n e 2,4-dinitrophenylhydrazone derivative 96 or, after reduction to alcohol 97, converted to i t s (p-bromophenyl)urethane derivative 98. 96 H 100 . The 1H NMR spectrum of isoacanthodoral (65) was similar to that of nanaimoal ( 61 ). Resonances for 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 (bs, 3H) ppm. An ABX spin system was observed at 6 2.13 (dd, / = 3.3,14.8 Hz, 1H), 2.70 (dd, J = 3.3,14.8 Hz, 1H), and 9.73 ( t , / = 3.3 Hz, 1H) ppm which could be attributed to an ethanal substituent attached to a quaternary carbon (substructure 6 6 ) . This assignment was supported by an intense fragment ion at m/z 177 in the mass spectrum which could arise by loss of the ethanal side chain via an a l l y l i c fragmentation as shown in Scheme 11a. A fragment ion at m/z 176 indicated that loss of the ethanal side chain by a McLafferty type fragmentation was also f a c i l e (Scheme 11b). A 1 3C NMR resonance at 6 204.6 (d) supported the assignment of the lone oxygen in isoacanthodoral to an aldehydic carbonyl. Comparison of the chemical s h i f t s of the AB resonances in the ethanal side chain of isoacanthodoral (65) (6 2.13 and 2.70) to the cor-responding AB proton resonances in nanaimoal (61) (8 2.29 and 2.22 ppm) showed a marked difference in chemical s h i f t , and indicated the ethanal side chain was in a noticably d i f f e r e n t chemical environment. The chemical s h i f t of the corresponding carbon in the 1 3C NMR spectrum, 8 57.1 for 65 vs 8 53.7 for nanaimoal, supported th i s argument. The presence of substructure 99 in isoacanthodoral was inferred from the presence of an o l e f i n i c proton at 5 5.24 (bs, W ] / 2 ~ 6.4 Hz) coupled to the o l e f i n i c methyl resonance 101 at 6 1.64 (bs, 3H), this assignment was supported by a double-resonance experiment. The 1 3C NMR spectrum of isoacanthodoral showed resonances at 6 137.4 (s) and 6 131.9 (d) appropriate for a t r i s u b s t i t u t e d double bond, and was assignable to substructure 99. The spectral data indicated that isoacanthodoral was a b i c y c l i c sesquiterpenoid that contained three methyl groups, one of which was o l e f i n i c , and an ethanal side chain attached to a quaternary carbon. It was d i f f i c u l t to ascertain the number of a l l y l i c methylene or methine protons from the 1H NMR of isoacanthodoral, however, from the integration in the 1H NMR spectrum of (p-bromophenyl)-urethane derivative 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 protons 1 02 were present in the molecule. From spectral evidence i t seemed conceivable that isoacanthodoral was biogenetically related to nanaimoal (61) which led to consideration of aldehyde 100 as a possible structure for isoacanthodoral. Nanaimoal had a regular isoprenoid type carbon skeleton which one could imagine might be formed by a biogenetic type c y c l i z a t i o n of farnesyl pyrophosphate 101 v i a (formally) carbocation 102 (see Scheme 12). Carbocation 102 could collapse via a number of pathways which could, after side chain oxidation, generate at least 4 possible products. One of the products, formed by loss of proton Ha, would be nanaimoal (6_1) . Loss of proton or Hc would generate aldehydes 103 or 104 respec-t i v e l y , molecules both lacking an o l e f i n i c methyl group and neither of which could therefore represent the structure of isoacanthodoral. To generate the required o l e f i n i c methyl f u n c t i o n a l i t y from carbocation 102 would require a molecular 1 03 rearrangement. Thus, a 1,2-hydride s h i f t of H-5 followed by a 1,2-methyl migration and loss of proton could generate aldehyde 100, a structure that would incorporate many of the spectral features of isoacanthodoral. Scheme 12. Biogenetic arguements leading to the considera-tion of 100 as the structure for isoacanthodoral. Although structure 100 was appealing for isoacanthodoral, four pieces of spectral evidence and one chemical interconversion were used to eliminate i t from further consideration. As previously mentioned, there was a marked difference between the chemical s h i f t s of the protons 104 alpha to the aldehydic carbonyl in nanaimoal (61) (6 2.29 and 2.23 ppm) and the chemical s h i f t s of the analogous protons in isoacanthodoral (6_5) ( 6 2.70 and 2.13 ppm). This difference could not readily be rati o n a l i z e d i f isoacanthodoral had structure 100. Even i f the side chain was a x i a l l y orientated, as in 105, there did 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. The mass spectrum of isoacanthodoral indicated the ethanal side chain may be a l l y l i c (see Scheme 11), implying the presence of either of substructures 106 or 107. H 0 105 CH H 106 107 The diamagnetic anisotropy of the o l e f i n i c double bond could possibly deshield one of the a-methylene protons i f there was a preferred rotamer population for the side chain in 1 05 either of substructures 106 or 107. The o l e f i n i c proton in isoacanthodoral was unusually sharp (W^2 = 6.4 Hz), ind i c a t i v e of an o l e f i n i c proton which lacked v i c i n a l coupling (substructure 106). When isoacanthodoral (65) was reduced to isoacanthodorol (97) the o l e f i n i c proton was shifted u p f i e l d by 0.14 ppm, suggesting the double bond and oxygen f u n c t i o n a l i t i e s might be in close proximity to each other. Although reduction of the aldehyde removes the anisotropic deshielding influence of the carbon-oxygen double bond, one of the protons al pha to the carbinol carbon in isoacanthodorol (97) continued to be s e l e c t i v e l y deshielded, 6 2.04 (dt, J = 13.5,7.2 Hz, 1H) and 6 1.41, implying that the aldehyde could not be the o r i g i n of t h i s e f f e c t . In structure 100 the chemical environment of the side chain would be expected to be similar to nanaimoal (thus the ABX spin systems would have similar chemical s h i f t s ) and the W-\/2 °f fc^e o l e f i n i c proton would be expected to be wider than 6.4 Hz. The chemical evidence against structure 100 was more concrete. In 1974 Minale and co-workers isolated averol (108) from the marine sponge Disidea a v c r c1 3" . A derivative of a v a r o l , dimethyl ether 109, upon treatment with acid underwent a molecular rearrangement to give a substance, compound 110, that had the same bicyclof4.4.0jdec-1(6)-ene ring system as nanaimoal (61). It was reasoned that i f isoacanthodoral could be represented by structure 100, treatment of i t s (p-bromophenyl)urethane derivative (98), 106 with a c i d , should generate the urethane derivative of nanaimoal. When this reaction was carried out with 98 -100% formic acid at 70 °C for 10 hours, 98 was converted quantitatively to a new compound 112 which retained the o l e f i n i c methyl fu n c t i o n a l i t y (1H NMR 5 1.59). The W}/2 o f the o l e f i n i c proton was now 11.6 Hz. This chemical evidence implied that the double bond had isomerized about the o l e f i n i c methyl group to give a new compound containing substructure 113 (see Scheme 1.3). This isomerization was impossible for structure 100. Fortunately, at t h i s point, the c r y s t a l structure of acanthodoral's p-bromophenylurethane derivative (114) was solved {vide infra) allowing a correct proposal for the structure of isoacanthodoral to be made. The proposal was 1 07 OBPU OBPU based upon the spectral data in combination with biogenetic reasoning. Scheme 14 indicates a proposed biogenesis for isoacanthodoral (6j5) with acanthodoral (64) as the p i v i t o l intermediate. Proton induced fragmentation of the cyclobutane ring in acanthodoral (64) could generate nanaimoal (61_) . Proton induced cyclobutane fragmentations from aldehydes are known s y n t h e t i c a l l y , and proton induced cyclobutane fragmentations from ketones are of synthetic u t i l i t y for the formation of f i v e membered r i n g s1 3 5. Venus-Danilova1 3 6 has shown that when formylcyclobutane (115) i s treated with acid i t fragments to give a number of products. The major product i s aldehyde (116). From a biogenetic veiwpoint, a proton induced cyclobutane fragmentation has not previously been documented. 108 114 Proton induced fragmentation of acanthodoral (6_4) may occur by either of two pathways, that i s , fragmentation of the C-10 to C-11 or C-8 to C-11 bonds. Fragmentation of the C-10 to C-11 bond, .followed by loss of proton Hx would gen-erate nanaimoal (61), whereas loss of proton or Hz subse-quent to C-8 to C-11 bond cleavage could generate 65 (-Hy) or 117 (-Hz), compounds that would incorporate a l l the spectral features of both isoacanthodoral and i t s rearrangement product. Although the biogenetic argument given in Scheme 14 i s speculative, and must be tested experimentally, use of biogenetic theory allowed the struc-ture of isoacanthodoral to be formulated as 65, including absolute stereochemistry. V e r i f i c a t i o n of the proposed structure for isoacanthodoral was obtained by a single crys-t a l X-ray d i f f r a c t i o n study, performed by He Cun-heng and Jon Clardy at Cornell U n i v e r s i t y . Figure 20 represents a computer-generated drawing, including absolute stereochemistry, of isoacanthodoral's 2,4-dinitrophenyl-hydrazone derivative JJ6 . 109 Scheme 14. Proposed biogenesis of isoacanthodoral (65) from acanthodoral (64). Crystals of 9_6 belonged to space group P2, with a = 6.027 (1), b = 31.613 (8), c = 8.910 (1) n r1 0, and 0 = 80.89 ( 1 ) ° . The asymmetric unit consisted of two molecules of composition C2 i H 3 , O t N i , . After c o l l e c t i o n of d i f f r a c t i o n data, solution by direct methods was routine and least-squares refinements converged to a standard crystallographic residual of 0.048. A number of features should be noted about the struc-ture isoacanthodoral (65): Figure 20. Computer generated X-ray structure of isoacanthodoral's 2,4-dinitrophenylhydrazone derivative 111 1. It i s a non-isoprenoid sesquiterpenoid, that i s , i t does not obey the isoprene r u l e . 2. Like nanaimoal, one of the methyl groups o r i g i n a l l y present in the biogenetic farnesyl pyrophosphate precurser has undergone c y c l i z a t i o n and i s now contained in a carbocyclic r i n g . 3. The- decalin ring system i s cis fused (stereochemistry biogenetically a r i s i n g from the equatorial nature of the C5 sidechain prior to c y c l i z a t i o n to acanthodoral). This i s the f i r s t ex-ample of a cis fused decalin ring system to be isolated from a nudibranch. 4. The deshielding of one of the methylene protons alpha to the carbonyl may be rati o n a l i z e d by the following argument; in s o l u t i o n , a highly populated rotamer exists ( l i k e l y the same conformer as in the s o l i d state, see Figure 20) whereby one of the a-methylene protons l i e s within the deshielding region of the carbon-carbon double bond. This causes se l e c t i v e deshielding of only one of the a-methylene protons. Isoacanthodoral (65) has a new sesquiterpenoid carbon skeleton for which we propose the name isoacanthodorane and to number i t as shown in Figure 20. 1 12 6. ACANTHODORAL Acanthodoral (64) occurred as the least abundant sesquiterpenoid metabolite from Acanthodoris nanaimoensis to which a structure has been assigned*. GC-MS of aldehyde 6_4 showed a parent ion at m/z 220 which suggested acanthodoral was isomeric with nanaimoal (6_1) and isoacanthodoral (65). The base peak at m/z 84 was diagnostic for acanthodoral (64), as neither 61 or 65 showed s i g n i f i c a n t ( > 5%) ion intensity at m/z 84. Intense fragment ions at m/z 176 and 161 suggested the presence of an ethanal substituent as was found for nanaimoal (61) and isoacanthodoral (65)t» Isolation of pure acanthodoral (64) was extremely d i f f i c u l t owing to i t s trace abundance (70 ug/animal) and high v o l a t i l i t y . It was therefore 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 derivative 114. The most e f f i c i e n t i s o l a t i o n procedure was to reduce the natural mixture of aldehydes to the corresponding alcohols and to separate nanaimool (6_1) from the mixture of isoacanthodorol (65) and acanthodorol (118) by ra d i a l TLC (100% CHC13). Derivatization of the isomeric alcohols 97 and 118 with 4-bromophenyl isocyanate followed by HPLC p u r i f i c a t i o n yielded pure samples of (p-bromophenyl)urethane derivatives *As previously mentioned, two trace metabolites, at least one of which i s isomeric (GC-MS) with the major sesquiterpenoid aldehydes, were present in the skin extract of A. nanaimoensis. t The GC-MS spectrum of nanaimoal showed intense fragment ions at m/z 176 and 161 (100%), isoacanthodoral showed a fragment ion at m/z 176 but did not exhibit any ion intensity at m/z 161. OH 1 1 4 118 Acanthodoral's c r y s t a l l i n e (p-bromophenyl)urethane derivative 114 (mp 109-110 °C, hexane), had a molecular formula C2 2H3 0BrN02 (HRMS, m/z observed 421.1441, 419.1438; required 421.1439, 419.1460) ve r i f y i n g an isomeric relati o n s h i p for acanthodoral with nanaimoal (6_1) and isoacanthodoral (65) . The 1H NMR 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 ethanal side chain substituent, the carbinol region in the 1H NMR spectrum of urethane 114 (see Appendix) showed only the AB portion of an ABX spin system, in d i c a t i v e of a substructure of type 119. Also present in the 1H NMR 1 14 spectrum of 114 were resonances for an isolated AX spin sys-tem at 6 1.09 (d, J = 9.4 Hz, 1H) and 1.84 (d, / = 9.4 Hz,1H), from which substructure 120 could be i n f e r r e d . Due to the magnitude of the geminal coupling constant (9.4 Hz) substructure 120 was l i k e l y confined within a 4 or 5 membered r i n g1 3 7. The remainder of the signals in the 1H NMR spectrum were due to the (p-bromophenyl)urethane moiety; 6 6.50 (bs, 1H), 7.27 (d, 2H), 7.40 (d, 2H) ppm, and to 12 protons that appeared as a series of complex multiplets resonating between 6 1.2 and 1.7 that could be assigned to the remaining protons of the terpenoid f u n c t i o n a l i t y . The absence of a l l y l i c proton resonances suggested that the terpenoid portion of derivative 114 contained three carbocyclic rings. The spectral data suggested that because acanthodoral (64) contained only three methyl groups i t was l i k e l y biogenetically related to both nanaimoal ( £ 1 ) , and isoacanthodoral (65) . No known sesquiterpenoid carbon skeleton could account for the observed spectral data, H H 119 120 1 15 therefore the structure of acanthodoral (64) , including the absolute configuration, was solved 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 a n a l y s i s . The structural determination was performed by He Cun-heng and Jon Clardy at Cornell University on acanthodoral's (p-bromophenyl)urethane derivative 114. Crystals of 114 belonged to the common monoclinic space group Ply with a = 9.581 (1), b = 6.406 (1), c = 34.45 (1) m~10, and /3 = 85.85 ( 1 ) ° . Two molecules of composition C22 H 3002N B r formed the asymmetric u n i t . After c o l l e c t i o n of the d i f f r a c t i o n data, a phasing model was found by standard heavy atom methods, and least-squares refinements with heavy atoms and isotropic hydrogens converged to a standard crystallographic residual of 0.078 for the observed r e f l e c t i o n s . Both molecules in the asymmetric unit had the same stereostructure, and a computer generated perspective drawing i s given in Figure 21. The average bond angles for the four membered ring in 114 was 8 6 ° . Acanthodoral 64 has a new sesquiterpenoid carbon skeleton for which we propose the name acanthodorane and to number i t as shown in Figure 21. 1 16 Figure 21. Computer generated X-ray structure of acanthodoral' (p-bromophenyl)urethane derivative 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 shown by known nudibranch metabolites (see Table 6), the natural sesquiterpenoid mixture from A. nanaimoensis was evalua-ted for i t s a n t i b a c t e r i a l and antifungal 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 against Baci I I us subtilis and Staphylococcus aureus as well as antifungal a c t i v i t y against Pythiam ultimum and Rhizoctonia solani a l l at 670 ug/O/4 inch disk) (mini-mum in h i b i t o r y concentrations were not determined). Due to the small amounts i s o l a t e d , and the need for large amounts of metabolites for structural work (in an attempt to generate enough nanaimoal urethane derivative 7 5 for 2D-NMR experiments) the sesquiterpenoid aldehydes were not tested for f i s h antifeedant a c t i v i t y . 8. DISCUSSION The co-occurrence in A. nanaimoensis of three sesquiterpenoids each with a new carbon skeleton was g r a t i f y i n g , however, i t was not surprising in l i g h t of pre-vious studies on nudibranch sesquiterpenoids. Table 6 i s a summary of a l l the nudibranch sesquiterpenoids arranged according to skeletal type with comments as to th e i r source and b i o l o g i c a l a c t i v i t i e s . It can be seen from Table 6 that the drimane carbon skeleton makes up a large majority of the sesquiterpenoids 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 f i s h . Although the sig n i f i c a n c e of the carbon skeleton i s not c l e a r , drimane sesquiterpenoids have also been shown to possess insect antifeedant a c t i v i t y1 3 8, and in vivo antifungal a c t i v i t y . The majority of sesquiterpenoids isolated f a l l into three groups: i) furans, i i ) aldehydes (dialdehydes) and i i i ) i s o n i t r i l e s . They may be derived from dietary sources or produced de novo by the nudibranch (see Table 6). Our i n a b i l i t y to find a dietary s o u r c e1 0 2, and the constant occurrence, and r a t i o , of the A. nanaimoensis sesquiterpenoids suggested de novo biosynthesis. I n i t i a l biosynthe'tic studies showed that 1 3C labeled mevalonic acid injected into the stomach of A. nanaimoensis was incorporated into the sesquiterpenoid aldehydes. This re-sult must be considered tenative however, as the radiol a b e l l e d metabolites have not been c r y s t a l l i z e d to con-stant s p e c i f i c a c t i v i t y . TABLE 6: NUDIBRANCH SESQUITERPENOIDS SKELETON METABOLITES COMMENTS A. Acarbocyclic 1. apofarnesane 2. farnesane ^ ^ ^ ^ ^ ^ ^ dihydroapofarnesal (121) marislin (122) , 0 C H 3 124 a b Isolated frcm Anisodoris nobilis, dihydroapofarnesal (121) was responsible for the nudibranch's fruity c d o r3 8 D. The major metabolite of Chromcdoris marislae was marislin (122), cctrpounds 123a, 123b, 124a and 124b were present as minor constituents1 3 y. The isomeric pairs 123a, 123b and I24a, 124b are formally related by a [3,3] sigmatropic rearrangement. The authors suggest 123a and 123b are artifacts of the isolation procedure. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS famesane (cont' d) 1 1 0 0 R 2 125 R1 = H, R2 = H 126 R1 = Ac, R2 = H 127 R1 = Ac, R2 =Ac i i 0 r0H 128 dendrolasin (129) Archidoris odhneri yielded a series of related farnesic acid glycerides (125-128)14 0. 1 '•C-labeled mevalonic acid injected into the stomach of A. odhneri was incorporated into the farnesic acid moiety of 12538*\ 125 showed moderate i n vitro antibiotic ac t i v i t y against Staphylococcus aureus, but had no demonstrable fi s h antifeedant a c t i v i t y . Dendrolasin (129), a known sponge metabolite has been isolated from both Cadlina leutomarginata3 6 and Hypselodoris g h i s e l i n i1 4 1 collected in California. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS farnesane (cont'd) longifolin (130) A known sponge metabolite, longifolin (130) was isolated from the nudibranch Glossodoris g r a c i l i s1 4 2. B. Monocarbocyclic 1. Monocyclofarnesane *% % % 1 0 131 Monocyclof arnesane metabolite 131 was isolated form Archidoris montereyensis and A. odhneri3 8k. It i s biogenetically related to farnesic acid glyceride 125 by a proton induced cyclization. 132 Furan 132, whose structure was postulated on the basis of mass and *H NMR spectral data was isolated from Cadlina luteomarginata3 6. The structure has not been confirmed by synthesis or interconversion to a known compound. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 2. microcionin-2 microcionin-2 (133) Microcionin-2 (133), a known sponge metabolite was isolated from Cadlina luteomarginata3 5. 133 has been-proposed as an intermediate i n the biogenesis of nakafuran-8 and nakafuran-914 4. 3. pleraplysillin-1 pleraplysillin-1 (134) Pleraplysillin-1 (134) a known sponge metabolite, was isolated from Cadlina luteomarginata 3 5. C. Bicarbocyclic 1. drimane OH 135 R = H 136 R = Ac Isolated from both Archidoris montereyensis and A. odhneri3 8*5, drimane rretabolite 135 showed feeding deterent activity against the tidepcol skulpin Oligocottus maculosus at 18 ug/(mg of food p e l l e t ) . 1 4C-labeled mevalonic acid fed to A. montereyensis was incorporated into the terpenoid portion of 135. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS drimane (cont'd) albic albicanol (137) R = H anol acetate (138) R = Ac pu'ulenal (139) Albicanol (137) and albicanol acetate (138) were isolated from Cadlina luteararginata^ collected i n Bri t i s h Columbia. Albicanol acetate (138) showed antifeedant act i v i t y against goldfish at 10 ug/(mg of food pellet) while albicanol (137) was inactive (50 pg/(mg of food p e l l e t ) ) . Albicanol (137) had been previously isolated from the liverwort Diplophyllum albicans1 3. Isolated from Chromodoris albonotata3 8 a, pu'ulenal (139) was readily hydrolyzed to a 5:2 mixture of polygodial (140), a known fish antifeedant, and 9-epipolygodial a compound devoid of antifeedant a c t i v i t y . TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES drimane (cont'd) 141 COMMENTS Polygodial (140) was isolated from Dendrodoris Lurtbata11*5, D. kre b s i i , D nigra and D. tuberculosa1 *+6. A known insect antifeedant, polygodial also has fis h antifeedant a c t i v i t y . Labelling studies with 1^C-labeled mevalonic acid showed 140 was biosynthesized de novo by D. limbata1 1*7. Esterfied to a series of fatty acids with varying degrees of unsaturation, sesquiterpenoid derivative 141 (stereochemistry at C-11 unknown) was localized i n the digestive gland of Dendrodoris limbata11*5 •l h 8. 141 could also be readily isolated from acetone skin extracts of Doriopsilla albopunctata and Doriopsilla janainal1 1*6. The esters 141 did not show fis h antifeedant activity and i t was suggested they are detoxification products of polygodial (140). 11+C-labeled mevalonic acid was incorporated into the sesquiterpenoid moiety of 1411 4 7. Stirring 141 i n the presence of acid yielded euryfuran (144), a known nudibranch metabolite. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS drinane (cont'd) A c O , ^ ^ ^ ^ • O A c olepupuane (142) A c C \ Olepupuane (142), a metabolite biogenetically related to polygodial (140) occured i n Dendrodoris nigra, D. tuberculosa, D. krebsii, Dorispsilla albopunctata and Doriopsilla janainaJ-l+t>. It was shown to inhibit feeding of the pacific damsel fis h (Dascyllus aruanus). The ED5Q was found to be 15-20 ug/(mg of p e l l e t ) , comparable to that of polygodial (140). The methoxy acetal 143 was isolated from one collection of Doriopsilla albopunctata, stored i n methanol. 143 Euryfuran (144), a known synthetic compound, was isolated from Hypselodoris porterae, H. c a l i f orniensis, and the inter t i d a l sponge Euryspongia s p .1 4 1. An antifeedant role for 144 i s suggested although no quantitative tests were performed. euryfuran (144) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 2. pallescensin-A pallescensin-A (145) Isolated from Cadlina luteomarginata3 6, pallescensin-A (145) i s biogenetically related to euryfuran (144). 145 was toxic to goldfish and showed antifeedant activity against the skulpin Clinocottus analis. 3. agassizin Obtained from Hyselodoris a g a s s i z i1 4 1, agassizin (146) i s structurally related to pallescensin-G, a known sponge metabolite14 9. An antifeedant role for 146 was implied. agassizin (146) 4. spiniferin-2 rrv A known sponge metabolite, spiniferin-2 (147) was isolated from Hypselodoris danielae collected in Hawaii 1 h spiniferin-2 (147) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 5. nanaimoane H Nanaimoal (61) was isolated from Acanthodoris nanaimoensis105 (see text). nanaimoal (61) 6. isoacanthodorane 0 isoacanthodoral (65) Isoacanthodral (65) was isolated from Acanthodoris nanaimoensis106 (see text). 7. nakafuran-8 nakafuran-8 (148) The biogenetic precurser to nakafuran-9 (152), nakafuran-8 (148) had antifeedant properties against the common reef fishes Chaetodon spp. Furan 148 was isolated from Chronodoris maridadilus, Hypsilodoris godeffroyanal l t\ H. c a l i f o r n i e n s i si H 1, and the sponge Dysidea f r a g i l i s . TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS nakafuran-8 (cont'd) 149 RO V 150 R = A c Three oxidation products of nakafuran-8 have been reported. Butenolide 149 i s believed to be an art i f a c t formed by a i r oxidation of 1 4 8 w h i l e 150 and 151 were isolated from H. zebra and i t s dietary sponge D. e t h e r i a l5 0. 8. nakafuran-9 151 R = H nakafuran-9 (152) A structurally interesting sesquiterpenoid, nakafuran-9 (152) has been isolated from Chromodoris maridadilus, Hypselodoris godeffroyana11*'*, and H. g h i s e l i n i1 1*1. A metabolite of the sponge Dysidea f r a g i l i s , 152 shows antifeedant activity against the common reef fishes Chaetodon spp. The methoxy butenolide of nakafuran-9, was assumed to have antifeedant properties against potential nudibranch predators. It was isolated from H. ghiselini and oo-occured with nakafuran-91 4 T. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 9; furodysin 10. furodysinin furodysin (153) furodysinin (154) Furodysin (153), a known sponge metabolite, was isolated from Cadlina luteomarginata3 5. The metabolites isolated from Cadlina luteomarginata showed marked variations between collections and collecting sites, indicating a dietary source. This hypothesis was supported by the isolation of many of the C. luteomarginata metabolites from the sponges upon which i t preys. The only metabolite common to both California and British Columbia collections of C. luteomarginata was furodysinin (154)3 5» 3 6. Furodysinin (154) is biogenetically related to furodysin (153). Furodysinin (154) was also isolated from Hypselodoris zebra collected i n Bermudian watersi b 0. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 11. 155 R = -NC 156 R = -NCS Two nudibranch metabolites, 155 and 156, isolated from Cadlina luteomarginata3 6, were traced to the sponge Axinella sp. Both 155 and 156 were toxic to goldfish and showed antifeedant activity against goldfish and the skulpin Clinocottus analis. 12. NC X axisonitrile-l (157) Phyllidia 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 which i t feeds, Axinella cannabina11*5. Isonitrile 157 was inactive as an antifeedant but was toxic to fish at a concentration as low as 8 ppm. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS D. Tricarbocyclic 9-isocyanopupukeanane (158) was isolated from Phyllidia varicosa. See Sections I.D 1. pupukeanane and II. A. 2. 7 9-isocyanopupukeanane (158) 2. acanthcdorane Acanthodoral (64) was isolated from Acanthodoris nanaimoensis106 (see text). acanthodoral (64) 132 I I I . BRYOZOANS A. INTRODUCTION TO THE BRYOZOANS Marine bryozoans have proven to 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 natural products chemists in selecting p o t e n t i a l s e s s i l e marine organisms for investigation i s the a b i l i t y of the organism to compete for the limited e c o l o g i c a l space available for growth1 5 1. Since bryozoans are important fouling organisms1 5 2, they have the a b i l i t y to out-compete other organisms for growing space, and to survive under very trying conditions (for example, some species are very resistant to the a n t i f o u l i n g paints used on sh i p s ) . In spite of these interesting ecological character-i s t i c s bryozoans have been largely ignored by organic chemists, u n t i l recently. Bryozoans (phylum Bryozoa) are c o l o n i a l animals of which approximately 4000 l i v i n g species are known. Most are marine organisms, although a few freshwater species have been documented, mainly in the class Phylactolaemata. The bryozoan colonies vary in height and width and occur in a variety of morphological forms; hence the names f a l s e - c o r a l s , sea-mats, and moss animals are commonly used to describe them. In B r i t i s h Columbian waters a l l three forms are common. Examples a r e1 5 3: coral l i k e - Het eropora pacifica and Phi dol opora pacifica , moss-animal type -Bugula sp., and the sea-mat type - Membranipora membranacea. 1 33 The small members of a colony (zooids) have body walls that are calcareous, gelatinous or chitinous and are usually less than 0.5 mm in length. A few to many m i l l i o n zooids may make up a bryozoan colony. Part of the zooid body wall i s a c i r c u l a r or horseshoe-shaped structure c a l l e d the lophophore; i t bears c i l i a t e d tentacles which may be protruded out of an o r i f i c e to 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 is U shaped and the anus opens just outside the lophophore. The zooids are usually connected through gaps or pores in the body walls and some are modified for other specialized functions such as cleaning, protection, or brooding the young. Colonies are hermaphroditic, both male and female zooids can occur in the same colony. The f e r t i l i z e d egg may develop into a free-swimming larva capable of feeding, or as in the case with most species, the f e r t i l i z e d eggs pass into a brood chamber. The brooded larvae have a shorter free-swimming l i f e and do not feed. Most larvae attach themselves to a surface and change into a zooid from which the colony develops by asexual budding. Only six bryozoan species have been investigated for their natural products chemistry to date. The res u l t s of these studies are summarized in Table 7. The f i r s t bryozoan to be studied chemically was Bugula neritina (Linnalus) from which V i l l e l a isolated an adenochrome-like pigment in 1948 1 5 4. TABLE 7: BRYOZOAN METABOLITES ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES Alcyonidium gelatinosum (L.) (Ref..155) C H 3 „ ^S + -CH 2 CH 2 OH Structure 159 was proposed on the basis of 1 3C and lE NMR spectral comparisons to model compounds and was confirmed by synthesis. (2-hydroxyethyl) dimethylsulfonium ion (159) Alcyonidium hirsutum (Ref. 156) Amathia convoluta (Ref. 157T" Sulfoxonium ions had never been encountered i n nature previous to the isolation of 159. Isolated in 5 ppm yield based on the animal wet weight, 159 i s the causitive agent of "Dogger Bank Itch", an eczematous allergic contact dermatitus caused by exposure to A. gelatinosum. A severe occupational disease, "Dogger Bank Itch" i s widely distributed among trawlermen working i n the Dogger Bank area of the North Sea. The water extract of A. hirsutum showed significant toxicity to mice at 1000 mg/kg, and showed an inhibition zone against Escherichia c o l i . The chloroform extract was toxic to the herpes virus (HSV-1). An extract of A. convoluta showed anti-neoplastic ac t i v i t y (PS system). 1 TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED > STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES Bugula neritina (Sinnaeus) (Ref. 157-161) R'O 4 / ^s^s^J* CH3O ^ R " 0 ° ^ Y H 1 S 0 R OH R= COCH3 R " = H bryostatin 1 (160) The structure of 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, efforts to determine the absolute configuration of bryostatin 1 are continuing. The antineoplastic ac t i v i t y of a B. neritina extract was f i r s t documented i n 1970. Bryostatin 1 (160) was isolated from 500 kg of 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 injection dose levels and an ED5Q of 0.89 ug/mL against the P388 i n vi t r o c e l l l i n e . It also shows potent activity against the L1210 (lymphocyte leukemia, 34-51% l i f e extension at 37.5-150 ugAg) and M5 (M5076 ovarian carcinoma, 40-48% l i f e extension at 5-20 ug/kg and 20-65% curative i n the tumor regression model at 20-40 ug/kg) experimental tumor systems. A polyketide biogenesis i s suggested for bryostatin 1. The oxygen atoms at 01, 03, 05, 011, 019A, and 019B are a l l i n the interior of the large oxygen rich cavity suggesting the molecule may have cation binding capabilities. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES B. neritina (cont'd) R = H R"=H bryostatin 2 (161) The structure of bryostatin 2 (161) was based on 1 3C NMR and 400 MHz aH NMR studies i n comparison to bryostatin 1. Selective acetylation of 160 or 161 gave identical acetate (TLC) 162, which upon careful deacetylation gave a mixture of bryostatins 1 (160) and 2 (161). Bryostatin 2 (161) showed a 60% increase i n l i f e span at 30 u.g/kg i n the murine P388 PS system. R= C0CH3 ^ ^ ^ ^ R"= COCH3 (162) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES B. neritina (cont'd) 0 R'= R " = H bryostatin 4 (163) The structure of bryostatin 4 (163) was assigned using 1 3C NMR and 400 MHz lE 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 steric compression at C-20 compared to C-7 would favour hydrolysis of the less hindered ester (C-7), as was found for bryostatin 1. 44.5 mg of 163 was isolated from 50 kg wet weight of B. neritina. Apparently, bryostatin 4 (163) i s much less cytotoxic than bryostatin 1. It was suggested that substituents common to bryostatins 1-4 (160, 161, 163 and 164) constitute the unique requirements for anticancer ac t i v i t y while the ester substituents at C-7 and C-20 influence the degree of cytotoxicity and antineoplastic effects. Bryostatin 4 (163) showed 62% increase i n l i f e span at 46 ug/kg in the murine P388 PS system and substantial c e l l growth inhibitory (PS c e l l l i v e ED5 Q, 10-3-10-14 ug/mL) ac t i v i t y . TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES B. neritina (cont'd) & ' \ ^ f ^ y c \ O C H R " ° S r H • OH R= C O C H 3 R " = H bryostatin 3 (164) Flustra foliacea (L.) The structure of bryostatin 3 (164) was assigned using FAB mass spectrometry, 13C NMR, 400 MHz hi NMR, and IR spectroscopy (IR band at 1785 cm-1 indicated a possible 5 or 6-membered lactone carbonyl group with an a electronegative substituent). The structures 165-169 were assigned on the basis of GC-MS comparisons to authentic samples. 72.2 mg of 164 was isolated from 500 kg of B. neritina. Bryostatin 3 (164) shows 63% l i f e extension at 30 p,g/kg i n the P388 lymphocytic leukemia (PS system). The antibiotic a c t i v i t y (vs Staphylococcus (Ref. 156, 162-169) c i s - c i t r a l (165) aureus) of this bryozoan was most pronounced i n the older parts of the fronds, and was correlated with a characteristic strong lemonlike odor. The compounds apparently responsible for the antibiotic activity were monoterpenoids 165-169. Freeze dried samples of F. foliacea were devoid of antibacterial a c t i v i t y , however, they did show antiviral activity and inhibition of 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. foliacea (cont'd) 1 1 8 X X geraniol (166) trans-citral (167) JL 1 nerol (168) citronellol (169) Y B r ^ ^ j H 3 A The structures of the alkaloids 170-178 isolated from F. foliacea, were No biological a c t i v i t i e s were reported for metabolites 170 to 178, however the crude petroleum ether extract (from freeze dried material) and the purified alkaloids 170 and determined by detailed interpretation of the spectral data. 171 exhibited muscle-relaxant activity both i n vivo and i n v i t r o . flustramine A (170) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED F. foliacea (cont'd) X flustraraine B (171) Y flustramine C (172) OH r ^ V v N — C H 3 flustraminol A (173) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES F. foliacea (cont'd) OH _ B r A j C y N ^ C H 3 I flustraminol B (174) A flustramide A (175) 0 6-brcm>-^-rnethyl-Nb-for^TiyltryptarTi Lne (176) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES F. foliacea (cont'd) 177 Quinoline derivative 177 i s lik e l y an artifact as ethanol was used in the extraction procedure. The authors speculate that i f the ethoxy group was introduced during the isolation procedure, i t must have replaced a strikenly reactive group. Amide 178 exists as mixture of E and Z rotamers about the amide bond. Nugula nerita (Ref. 157T The aqueous 2-propanol extract of N. nerita, i n several doses, led to 168-200% l i f e extension in the PS system. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES Phidolcpora pacifica (Ref. 170) 1 phidolcpin (179) N N desmethy lphid( 1 C H 3 OH drN°2 ^ 0 H Dlcpin (180) For structural elucidation and biological a c t i v i t i e s , see text. Synthetic studies toward the synthesis of phidolopin 179, desmethylphidolopin 180, and a number of structural analogues are currently underway. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES Sessibugula translucens (Ref. 171) 0 C H 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 182- 185 were elucidated using spectral methods and comparison to the known compound, 4-methoxy-2,21-bipyrrole-5-carboxaldehyde (186). OCH. 186 Sessibugula translucens i s preyed upon by the nembrothid nudibranchs Tambje abdere, T. eliora and Roboastra t i g r i s . A mixture of enamines 182 and 183 (182, 40%; 183, 60%) inhibited c e l l devision i n the f e r t i l i z e d sea urchin egg assay at 1 pig/mL i n seawater and showed moderate antimicrobial activity at 50p,g/disk against Eschericia c o l i , Staphylococcus aureus, Bacillus s u b t i l i s , and Vibrio anguillarum. The isobutylamines 184 and 185 inhibited c e l l division at 1 |j.g/mL, showed antimicrobial activity against Candida  albicans, B. s u b t i l i s , S_. aureus and V. anguillarum at 5 jlg/disk, and shewed mild activity against E. c o l i at 50 |j.g/disk. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES ISOLATED STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES Thalamaporella gothica floridana (Ref. 157) Zoobotryon verticillantum (Delle Chiaja, 1828) (Ref. 172) N ( C H 3 ) 2 187 N ( C H 3 ) 2 188 Proposed on the basis of spectral data, structures 187 and 188 were confirmed by synthesis. Reduction of compound 188 (Zn-HoAc) yielded the free amine 187, oxidation of 187 T30% H202/MeOH) yielded 188 in quantative yi e l d . An extract of T. gothica floridana showed antineoplastic activity (PS system). Compound 187 inhibited c e l l division of the fe r t i l i z e d sea urchin egg (ED5Q = 16 ^ g/mL). Complete pharmacological evaluations of 187 and 188 are in progress. 1 46 In 1970 P e t t i t et al. 1 5 7 reported that a number of bryozoans contained anticancer constituents. B. neritina was the f i r s t bryozoan species to y i e l d a pure compound, designated as bryostatin 1 (160). Bryostatin 1 was active against the murine P388 lymphocyte leukemia (PS system) both in vivo and in vitro, and i t s structure was secured by crystallographic and spectroscopic t e c h n i q u e s1 5 8. Since the structure of bryostatin 1 was reported, three additional bryostatins have been isolated and the structures determined. They were bryostatin 2 (161)1 5 9, bryostatin 3 (164)1 6 0 , and bryostatin 4 ( 1 6 3 )1 6 1. A l l three are st r u c t u r a l variants of the same basic bryopyran macrolide ring skeleton. Whether the bryostatins are endogenous, or are derived from common bryozoan food sources such as bacteria and phytoplankton, remains to be determined. As does the ecological role ( i f any) of the metabolites. Because bryostatin 4 was isolated from B. neritina c o l l e c t e d in both the Gulf of Mexico (U.S.) and Gulf of Sagami (Japan), P e t t i t has speculated that the metabolite may be a biosynthetic product of B. neritina rather than from a dietary source. "... a d e f i n i t e conclusion regarding t h i s biochemical question w i l l require careful chemical examination of the microorganisms ingested by B. neritina and/or (1'C) acetate biosynthetic feeding experiments."1 6 1 Demonstrating the close relat i o n s h i p between chemical ecology and pharmacological research, Christophersen and 147 C a r l e , i n a s e r i e s of papers, have d e s c r i b e d t h e i r work on the marine bryozon Flustra foliacea ( L . )1 6 3 - 1 6 7. Prompted by a 1977 r e p o r t t i t l e d " A n t i - f o u l i n g Role of A n t i b i o t i c s Produced by Marine Algae and B r y o z o a n s "1 6 8, they have i s o l a t e d and e l u c i d a t e d the s t r u c t u r e s of 14 secondary m e t a b o l i t e s , of which nine represented new marine a l k a l o i d s and f i v e were known monoterpenoids. The monoterpenoids were a p p a r e n t l y r e s p o n s i b l e f o r the a n t i b i o t i c a c t i v i t y of the bryozoan*. The s t r u c t u r e s of the a l k a l o i d s b e aring the physostigmine, i n d o l e , or q u i n o l i n e r i n g systems were e l u c i d a t e d s o l e l y on the b a s i s of s p e c t r a l i n t e r p r e t a t i o n and the s t r u c t u r e s drawn i n Table 7 do not represent a b s o l u t e c o n f i g u r a t i o n s . E x t e n s i v e use was made of d i f f e r e n c e nOe enhancement NMR spectroscopy i n the s t r u c t u r a l assignments. Although bryozoans seem to be w e l l adapted in the marine ecosystem, they are by no means f r e e from p r e d a t i o n . Nudibranchs, not s u r p r i s i n g l y , are one of the main bryozoan p r e d a t o r s and some have become h i g h l y s p e c i a l i z e d , only f e e d i n g on one p a r t i c u l a r bryozoan. In some cases the nudibranchs are uncannily c r y p t i c when f e e d i n g upon t h e i r bryozoan host ( f o r example see Corambe pacifica'173). C a r t e and F a u l k n e r , i n studying such an i n t e r e s t i n g nudibranch-bryozoan a s s o c i a t i o n have i s o l a t e d a number of b i p y r r o l e s 182 - 185 from both the bryozoan Sessibugula t ransIucens and * 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 e d from F. Foliacea were d e v o i d of a n t i b a c t e r i a l a c t i v i t y1 5 6. 148 the d e f e n s i v e s e c r e t i o n of i t s nudibranch p r e d a t o r s Tambje abdere and T. eliora^7\ The s t r u c t u r a l d i v e r s i t y and v a r i e d b i o l o g i c a l a c t i v i t i e s of the secondary m e t a b o l i t e s i s o l a t e d from bryozoans leads one.to wonder what unique type of molecules have yet to be d i s c o v e r e d as more and more bryozoans are 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 c h e m i s t r y * . I t i s reasonable to suggest, from the l i m i t e d chemical s t u d i e s performed so f a r , that bryozoans show great p o t e n t i a l i n the u n r e l e n t i n g search f o r drugs from the sea. * Numerous s p e c i e s of bryozoans c o l l e c t e d i n New Zealand waters 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 of the 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 are c u r r e n t l y under i n v e s t i g a t i o n2 * . 1 49 B. SECONDARY METABOLITES FROM PHIDOLOPORA PACIFICA  (ROBERTSON 1908) Phidolopora pacifica (see Figure 22), usually referred to as the "lacy bryozoan", i s commonly found on rocky outcrops (depths of 3 to 15 m) in Barkley Sound, B r i t i s h Columbia. C l a s s i f i e d in the bryozoan order Cheilostomata, P. pacifica has a highly i n t r i c a t e , i n f l e x i b l e calcium carbonate skeleton b u i l t up into a r u f f l e d lacy network. It is one of about 75 genera of Bryozoa common to the P a c i f i c Northeast1 7". Our attention was drawn to P. pacifica by the absence of fouling organisms on i t s skeleton and the strong in vitro antifungal and a n t i a l g a l a c t i v i t y of i t s extracts. P. pacifica was f i r s t c o l l e c t e d in May 1982 near Diceman Island in the Broken Group of Islands, Barkley Sound, B.C. The bryozoan (143 gms dried weight after extraction) was immediately soaked in methanol after c o l l e c t i o n and stored at room temperature for two days. At the end of th i s time, the material was ground in a Waring blender with methanol, and f i l t e r e d . The combined greenish brown methanol extracts were concentrated to about one quarter of the o r i g i n a l volume and the resulting aqueous methanolic suspension was partitioned between brine and ethyl acetate. The ethyl acetate soluble material, which now contained most of the darkish green c o l o r , was dried over anhydrous sodium s u l f a t e . The sodium sulfate was f i l t e r e d off and the sample concentrated in vacuo to give 769 mg (0.54%) of a dark greenish brown o i l . Flash 151 chromatography gave a polar fraction which, after p u r i f i c a t i o n by preparative TLC, yielded phidolopin (179) (1.4 mg, 0.001%, mp = 225°C) and desmethylphidolopin (180) (<1 mg) as l i g h t yellow c r y s t a l l i n e s o l i d s . Both 179 and 180 represent new natural products that contain the r e l a t i v e l y rare, naturally occurring, n i t r o f u n c t i o n a l i t y . 179 180 A second c o l l e c t i o n (Deer Group of Islands, Barkley Sound, B.C.) of P. pacifica was worked up in the usual way in order to extract more 179 and 180, and to i s o l a t e any b i o l o g i c a l l y active compounds of lesser p o l a r i t y . After flas h chromatography of the crude" organic extract, fractions having similar chromatographic p o l a r i t i e s were pooled and submitted for bioassay. Three fractions were moderately active and 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, after preparative TLC yielded 8.5 mg (0.15%) of 4-methoxymethyl-2-nitrophenol (189 ) . S i m i l a r l y , f r a c t i o n P2 gave of 4-hydroxymethyl-2-nitrophenol (181) and fraction P3, the most polar f r a c t i o n , gave a mixture of phidolopin (179) and desmethyl-phidolopin (180) . The least polar nitrophenol, 4-methoxymethyl-2-nitrophenol (189), had a molecular formula of CeH9N O « (HRMS, m/z observed 183.0534; required 183.0532) demanding f i v e units of unsaturation. The 1H NMR spectrum of 189 (Table 8) showed resonances at 5 7.16 (d, J = 8.4 Hz, 1H), 7.59 (dd, / = 2.0, 8.4 Hz, 1H), and 8.09 (d, / = 2.0 Hz, 1H) suggesting the presence of a 1,2,4-trisubstituted benzene r i n g . Additional resonances at 8 3.41 (s, 3H) and 4.43 (s, 153 Table 8. 'H NMR data (CDC13, 80 MHz) and spectral comparisons for nitrophenols isolated from Phidolopora pacifi ca. chemical s h i f t , 6 on C# 189 181 209 190 3 8.09 (d, 2.0) 8.14 (d, 2.0) 8.09 (d, 2.1) 7.82 5 7.59 (dd, 2.0, 8.4) 7.63 (dd, 2.2, 8.5) 7.59 (dd, 2.1, 8.9) 7.40 6 7.16 (d, 8.4) 7.18 (d, 8.5) 7.14 (d, 8.9) 7.02 7 4.43 (s) 4.71 (s) 4.48 (s) 0-OH 10.58 (s) 10.58 (s) 10.57 (s) 1 0. 38 OH 1.61 (bs) OMe 3.41 (s) OEt 1.28 ( t , 6.9) 3.57 ( t , 6.9) Me 2.31 1 54 2H) were assigned to a benzylic methyl ether while an exchangable singlet at 6 10.58 (s) was assigned to a phenolic hydrogen. The sharpness of the phenolic hydrogen resonance suggested i t was possibly involved in an intramolecular hydrogen bond. Comparison of the observed 'H NMR chemical s h i f t s for 4-methoxymethyl-2-nitrophenol (189) to the calculated v a l u e s1 7 5 gave good agreement, as did comparison to the l i t e r a t u r e values for 4-hydroxy-3-nitrotoluene ( 190 )1 7 6 . The chemical s h i f t assignments for both 189 and 190 are given in Table 8. OH m/z Scheme 15. Interpretation of the MS fragmentation of nitrophenol 189. The mass spectrum of 189 f u l l y supported the assigned structure (Scheme 15). Fragment ions at m/z 137 (C8H902, 10.4%) and 106 (C7H60, 38.4%) indicated the presence of an aromatic n i t r o f u n c t i o n a l i t y . These fragment ions correspond to losses of N02 and [CH30 + N02] from the molecular i o n1 7 7. Fragmentation involving the loss of the methyl ether f u n c t i o n a l i t y via a benzylic cleavage gives a 155 fragment ion at m/z 152 (C7H6N03, 100%), the base peak in the spectrum. To the best of the author's knowledge, i s o l a t i o n of 4-methoxymethyl-2-nitrophenol (189) i s only the second exam-ple of a n i t r o containing phenol to be isolated from the marine environment. The f i r s t example, 2-methoxy-4,6-dinitrophenol (191) , was isolated as an antimicrobial constituent from the red alga Mar gi ni s por urn aberrans^76. Surprisingly, nitrophenol 189 has not previously been reported in the l i t e r a t u r e . Isolated from fraction P2, 4-hydroxymethyl-2-nitrophenol (181) had a molecular formula C7H7NOtt (HRMS, m/z observed 169.0379; required 169.0375). The 1H NMR spectrum of 181 was very similar to that of 4-methoxymethy1-2-hitrophenol (189) except for the absence of the methyl ether resonance. This indicated that 181 was the hydroxy derivative of 189 (see Table 8). Treatment of 4-hydroxymethyl-2-nitrophenol (181) with p-toluenesulfonic acid in methanol resulted in the formation of methyl ether 189, thereby co r r e l a t i n g the two structures. Phenol 181 had previously been reported as a synthetic compound with interesting b i o l o g i c a l a c t i v i t y (vide infra). Phidolopin (179) had a molecular formula C1 0H1 3N5O5 (HRMS, m/z observed 331.0917; required 331.0917) demanding 11 degrees of unsaturation. Resonances at 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 Hz), and 10.56 (s, 1H, exchanges with D20) 156 in the 1H NMR spectrum of phidolopin (179) indicated that the molecule contained the nitrophenol residue, substructure 192. A benzylic cleavage in the mass spectrum of phidolopin, which resulted in the nitrophenol residue giving r i s e to the observed base peak at m/z 152 (C7H6N03) supported th i s assignment. The chemical s h i f t of the benzylic methylene protons, 6 5.46, indicated substructure 192 was l i k e l y attached to either a nitrogen or oxygen atom in 179. The remainder of phidolopin had to consist of a CyHyNaC^ fragment which contained six degrees of unsaturation. 1H NMR resonances at 6 3.39 (s, 3H) and 3.59 (s, 3H) indicated two methyl groups attached to either oxygen or nitrogen atoms and the IR spectrum suggested the presence of at least one amide carbonyl (1657 cm"1). A purine nucleus containing oxygen, methyl and 4-hydroxy-3-nitrobenzyl substituents could account for a l l the 157 s t r u c t u r a l requirements of phidolopin. Comparison of the methyl singlet resonances to the two high f i e l d methyl resonances in the 1H NMR of caffeine (193) showed a correspondence, as did the C-8 resonance at 6 7.63 (s, 1H) (see Table 9). It was not possible however, on the basis of spectral c o r r e l a t i o n s , to unambigously establish the substitution pattern of the substituents on the purine r i n g . The structure of phidolopin (179) was therefore solved via 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 i t s p-bromophenacyl derivative (194). Derivative 194 was prepared in good y i e l d by reacting phidolopin with p-bromophenacyl bromide in the presence of KHC03 and l8-crown-6. The X-ray structural determination was performed by He Cun-heng and Jon Clardy at Cornell U n i v e r s i t y . CH CH 3 193 0 0 Br N N CH 3 194 158 Table 9. 'H NMR data and spectral comparisons for purine derivatives isolated from Phidol opora pacifica. chemical s h i f t , 6 H on C or 179 f l 180* 193 c 195^ 196^ N# 1 1 1 . 16 (s, 1 1 . 18 (s, 1H) 1H) 3 11.90 (s, 1H) 8 7.63 (s, 8.26 (s, 7.48 (s, 8.20 (s, 8.11 (s, 1H) 1H) 1H) 1H) 1H) 10 5.46 (s, 5.41 (s, 5.46 (s, 5.45 (s, 2H) 2H) 2H) 2H) N1 Me 3.39 (s, 3.39 (s, 3.16 (s, 3H)e 3H)e 3H) N3 Me 3.59 (s, 3.35 (s, 3.57 (s, 3.35 (s, 3H)e 3H) 3H)e 3H) N7 Me 3.99 (s, 3H)e 270 MHz, CDC13 0 400 MHz, DMSO-d6 c CDC13, Sadtler Standard NMR Spectra No. 10393M d 60 MHz, DMSO-d6, Ref. 179 e May be reversed C H 3 1 8 ° 159 A c r y s t a l of 194 suitable for X-ray d i f f r a c t i o n was grown by slow evaporation of an acetone-methanol-a c e t o n i t r i l e s o l u t i o n . Preliminary X-ray photographs showed t r i c l i n i c symmetry, and accurate l a t t i c e constants of a = 9.549(3), b = 9.514(1), c = 15.212(3) lO-10m, a = 72.69(1), j3 = 82.96(2), and 7 = 81.87 (2)° were determined by a least squares f i t of f i f t e e n d i f f r a c t i o n measured 20-values. After c o l l e c t i o n and correction of the d i f f r a c t i o n data, a phasing model was obtained by standard heavy atom methods, and refinements converged to a standard crystallographic residual of 0.0939 for the observed r e f l e c t i o n s . Figure 23 i s a computer generated perspective drawing of the f i n a l X-ray model of the p-bromophenacyl derivative of phidolopin less hydrogens. Bond distances and angles generally agreed well with anticipated values. With the structure of phidolopin (179) in hand, attention was turned to the more polar metabolite, des-methylphidolopin (180) . Desmethylphidolopin (180) had a molecular formula C13H,,N505 (HRMS, m/z observed 317.0777; required 317.0760). Fragment ions observed at m/z 166 (100%) and 152 (75%) in the mass spectrum of 180 indicated that desmethylphidolopin was similar in structure to phidolopin, except for the absence of one of the purine ring methyl f u n c t i o n a l i t i e s . Resonances at 6 5.41 (s, 2H), 7.12 (d, J = 8.7 Hz, 1H), 7.58 (dd, / = 8.7,2.6 Hz, 1H), 8.00 (d, J = 2.6 Hz, 1H) and 11.05 (s, 1H) in the 'H NMR spectrum (DMSO-d6) of 180 suggested the presence of the nitrophenol 160 161 residue 192. Attachment of th i s f u n c t i o n a l i t y to N-7 of the purine nucleus was assigned by analogy to phidolopin, and was supported by comparisons of the chemical s h i f t of the N-8 proton in desmethylphidolopin to suitable model compounds {vide infra). The only remaining st r u c t u r a l feature to be determined for desmethylphidolopin was the location of the methyl group on the purine nucleus. Comparison of the 1H NMR spectrum of desmethylphidolopin to those of both 7-benzyl-3-methylxanthine (195) and 7-benzyl-1-methylxanthine ( 196 ) 1 7 9 (Table 9) showed that the methyl group in des-methylphidolopin 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 in both des-methylphidolopin and 7-benzyl-3-methylxanthine were i d e n t i -cal and resonated at 8 3.35 (DMSO-d6). S i m i l a r l y , the purine ring NH proton resonances were of v e r t i a l l y equiva-lent chemical s h i f t , 6 11.16 for 180 vs 11.18 for 195. Comparison of the 1H NMR resonances for the N-1 methyl and NH protons in 7-benzyl-1-methylxanthine 196 to the analogous resonances in desmethylphidolopin showed vast differences in chemical s h i f t s . Additional evidence for an N-3 methyl group came from the mass spectrum of desmethylphidolopin. The base peak at m/z 166 could be assigned to the 3-methylxanthine fragment ion 197. The pos i t i o n of methylation in xanthines can be readily obtained from their mass spectral fragmentation1 8 0. A major fragmentation i s via a retro-Diels-Alder reaction 162 0 0 CH 3 N N CH 3 ^ N N -> 195 196 involving the N-1 and C-2 atoms such that 1-methylxanthine gives a base peak at m/z 109 [M+(166) - CH3NCO] while 3-methylxanthine gives 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, required for C5H5N30 123.0432) in the mass spectrum of desmethylphidolopin could arise via loss of HCNO from fragment ion 197 (Scheme 16), supporting the assignment of the methyl group to N-3. The spectroscopic evidence, in comparison to suitable model compounds, allowed the proposal of structure 180 for desmethylphidolopin. To confirm the proposed structure, a synthesis of 180 has been planned by Andersen and T i s c h l e r1 8 1. At the present time, a synthetic sample of desmethylphidolopin has not yet been secured. 163 0 CL H \ A H T* H T' A ) - V» * HNCO 0 ™ N L M 3 CH 3 197 m/z 166 (100%) m / z 123 (27%) Scheme 16. Interpretation of the MS fragmentation of desmethylphidolopin 180. 1. DISCUSSION Phidolopin (179) represents a new addition to the very small but important group of naturally occurring purine derivatives based on the xanthine nucleus that includes caffeine (193), theophylline (198), and theobromine (199) . It represents only the second example of a naturally occurring xanthine derivative to be isolated from a marine organism, the f i r s t was a report that caffeine had been isolated from the Chinese gorgonian Echinogorgia pseudossapoy 8 2. Phidolopin i s a unique xanthine derivative in that i t contains the r e l a t i v e l y rare naturally occurring n i t r o f u n c t i o n a l i t y . Purine derivatives based on nuclei other than xanthine are well known from the marine organisms and in most 1 64 instances they have pronounced physiological a c t i v i t i e s . Purine ribosides such as doridosine (200) are the most common. Doridosine (1-methylisoguanosine) was isolated from the digestive gland of the dorid nudibranch Anisodoris nobilis and exhibits a variety of pharmacological a c t i v i t i e s . Perhaps one of the most dramatic was the observation that 200 caused the heart rate of anesthetized mice to be reduced by up to 50 percent for many hours, after which the animals completely r e c o v e r1 8 3. Doridosine has also been isolated from the Australian sponge Tedania digit at a1 8 *. Isoguanosine (201) , isolated from the dorid nudibranch Di aul ul a sandi egensis 1 8 5, and spongosine [2-methoxyadenosine (202)], isolated from the sponge Tedania digitata**6 are two other examples of b i o l o g i c a l l y active 165 purine ribosides from marine organisms. F i n a l l y , 9-/3-D-arabinofuranosyladenine (203) and i t s 3'-acetate (204) , compounds well known as potent synthetic a n t i v i r a l agents, were isolated as natural products from the I t a l i a n gorgonian Eunice! I a cav ol i ni 1 8 7 . 205 Other purine derivatives other than ribosides have been isolated from the marine organisms. Ageline A (205) and B (206), mild ichthyotoxins that possess moderate antimicrobial a c t i v i t y , were isolated from the P a c i f i c 166 sponge Agelas s p .1 8 8, while hokupurine (207) has been isolated from both the nudibranch Phestilla melanobrachi a and the coral upon which i t feeds, Tubastrea coccinea}89. Rather s u r p r i s i n g l y , in contrast to the other marine purine d e r i v a t i v e s , 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 could be found for hokupurine. CH 3 On the basis of the examples given i t was not unexpected to find that phidolopin had 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 in the limited b i o l o g i c a l testing performed in our laboratory. It showed in vitro antifungal a c t i v i t y against Pyt hi am ultimum, Rhizoctonia solani and He I mint hosporium satiurn with a minimum in h i b i t o r y concentration of 70 ug per one-quarter inch d i s k , for a l l three species. It 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 and Staphlococcus aureus and i t showed a n t i a l g a l a c t i v i t y against the pennate diatom Cylindrotheca fus i formi s 1 9 0 . The a n t i a l g a l a c t i v i t y shown by phidolopin could i n d i -cate that 179 plays an active role in the chemical ecology of P. pacifica by i n h i b i t i n g the growth of epiphytes. A similar role had been postulated for homarine (208), a metabolite which was isolated from the gorgonians Leptogorgia virgulata and L. setacea. Homarine (208) showed in vitro a n t i a l g a l a c t i v i t y against the benthic pennate diatom, Navicula s al i ni c ol o1 9 1 . CH 3 208 Phenolic compounds are well known to be a n t i b i o t i c and p h y t o t o x i c1 9 2, and phenols or brominated phenols from both marine r e d1 9 3 and brown algae1 9* have been shown to i n h i b i t the growth of pelagic u n i c e l l u l a r algae. Price and Wain1 9 5 have shown that nitrophenols, including 4-hydroxymethyl-2-nitrophenol (181) , i n h i b i t chloroplast development in both 168 green plants 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 relationships indicated that for substituted toluenes, a n i t r o group in the 3-position and a hydroxyl group or ether linkage in the 4-position were essential for a c t i v i t y , whereas the nature of the functional group in the benzylic position could vary considerably. It appears that the nitrophenols isolated from P. pacifica may be i d e a l l y suited to i n h i b i t epiphyte growth by i n h i b i t i n g chloroplast development. Further b i o l o g i c a l work is re-quired to substantiate the above hypothesis. Xanthine based alkaloids are generally of plant orgin, therefore i s o l a t i o n of xanthine derived metabolites from animal sources raises the question of their biogenetic o r i g i n . While gorgonians are well known to incorporate symbiotic zooxanthellae within their tissues (and therefore plant c e l l s could be the ultimate source of the caffeine found in Echenogorgia ps e udos s a po 1 8 2 ) .bryozoans are generally regarded as symbiont f r e e . It is perhaps l i k e l y that P. pacifica obtains the xanthine based metabolites from i t s phytoplankton d i e t . This hypothesis was supported by the i s o l a t i o n of phidolopin (179) from two additional species of bryozoans, Diaperoecia call for nica and Hippodi pi osi a i nscul pi a1 9 6, which were also c o l l e c t e d in Barkley Sound. If phidolopin i s indeed diet derived, the dietary source must be stable to both seasonal and l o c a t i o n a l v a r i a t i o n (within Barkley Sound) as 179 was isolated from every c o l l e c t i o n of P. pacifica examined for 169 secondary metabolites. The dietary hypothesis outlined above remains to be supported by i s o l a t i o n of phidolopin from an a l g a l source. The co-occurrence of phidolopin 179, 4-hydroxymethyl-2-nitrophenol (181), and 4-methoxymethyl-2-nitrophenol (189) in a methanolic extract of P. pacifica led us to examine whether or not benzyl methyl ether 189 and benzyl alcohol 181 were a r t i f a c t s of the i s o l a t i o n procedure. A number of benzyl methyl ethers have been isolated from marine organisms, many of which are believed to be a r t i f a c t s formed by methylation of the precurser benzylic alcohols with the methanol used for e x t r a c t i o n1 9 7. To test i f methyl ether 189 was indeed a natural product, one c o l l e c t i o n of P. pacifica was extracted with ethanol. The ethanol extract was worked up in the usual way with care being taken not to expose the extract to methanol, and 4-ethoxymethyl-2-nitrophenol (209) was isolated after chromatography on s i l i c a gel (for 1H NMR data of 209 see Table 8). No traces of methyl ether 189 were found. Extraction of another c o l l e c t i o n of P. pacifica with acetone gave phidolopin (179), desmethylphidolopin (180) and benzyl alcohol 181 but no benzyl ethers. It could be concluded from these results that both 189 and 209 were a r t i f a c t s of the i s o l a t i o n procedure. Since treatment of 181 with acid generated 189 in reasonable y i e l d (vide supra), i t seemed reasonable that acid catalyzed ether formation from benzyl alcohol 181 was also the source of the benzyl ethers in the natural 170 extracts. OH 0 OCH 2CH 3 N02 C H 3 \ 209 210 A r e q u i s i t e , precursor natural product that could be converted to benzylic alcohol 181 under laboratory conditions was not found in the ethyl acetate soluble extracts of P. pacifica. Attempts to cleave phidolopin to give 181 and/or 189 and theophylline (210) using p-toluenesulfonic acid in methanol gave only unreacted starting material. The p o s s i b i l i t y remains that cleavage of phidolopin to generate 181 may be catalyzed by a nucleophile such as Br", however th i s p o s s i b i l i t y was not explored in the laboratory. IV. EXPERIMENTAL The 1H and 1 3C NMR spectra were recorded on Bruker WH-400, Bruker WP-80, Nicolet-Oxford 270 and Varian XL-100 spectrometers. Tetramethylsilane (6 = 0) was employed as an internal standard for the 'H NMR spectra and CDC13 (5 = 77.00) was used, as both an internal standard for the 1 3C NMR spectra, and as solvent, unless otherwise indicated. Low-resolution and high-resolution electron impact MS were measured on an A.E.I. MS-902 and MS-50 spectrometers, re-spectively. Infrared spectra 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 Spectronic 2000 spectrophotometer. Optical rotations were measured on a Perkin-Elmer model 141 polarimeter using a 10 cm m i c r o c e l l . A Fisher-Johns apparatus was used to determine melting points and these values are uncorrected. Gas chromatography and high performance l i q u i d chromatography were performed on Hewlett-Packard 5830A and Perkin-Elmer Series 2 instruments, res p e c t i v e l y . A Perkin-Elmer LC55 UV detector and/or a Perkin-Elmer LC-25 re f r a c t i v e index detector were employed for peak detection during HPLC. A thermal conductivity or flame ionization detector was used for GC. A Whatman Magnum-9 P a r t i s i l 10 or a Magnum-9 ODS 10 column were used for preparative HPLC. The HPLC solvents were Fisher HPLC grade or Caledon HPLC 171 172 grade; water was glass d i s t i l l e d ; a l l other solvents were reagent grade. Merck S i l i c a Gel 60 PF-254 was used for preparative TLC, Merck S i l i c a Gel 230-400 Mesh was used for flash chromatography, and Merck S i l i c a Gel 60 PF-254 with CaSOa-1/2 H20 was employed in ra d i a l TLC. 173 Aldi s a cooperi C o l l e c t i o n Data Aldisa cooperi was col l e c t e d at various locations in 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 various locations in the Queen Charlotte Islands, B.C. Immediately after c o l l e c t i o n , the animals were immersed whole in methanol and stored at room temperature for one to three days. If the animals were not worked up immediately, they were stored at low temperature (4 to -5 °C) u n t i l used (usually within two months). Extraction and Chromatographic Separation As a number of c o l l e c t i o n s of A. cooperi were made and no va r i a t i o n in metabolites is o l a t e d was observed (except r e l a t i v e amounts) the following represents a ty p i c a l i s o l a t i o n procedure. After storage at -5 °C for two months the methanol used for extraction of the whole animals was decanted and saved. The 129 whole animals (37.7 g dry weight after extraction) were soaked an additional four times (0.5 h each) with methanol and the combined methanol extracts were vacuum f i l t e r e d to give an aqueous methanolic suspension. The suspension was partitioned between water (100 mL) and ethyl acetate (5 x 100 mL). The ethyl acetate was washed with 174 br ine ( 2 x 100 mL), dried over sodium s u l f a t e , and evaporated in vacuo to give 2.1 g (5.6%, 16 mg/animal) of an orange o i l containing a white s o l i d . Fractionation of the material by column chromatography (chloroform) gave crude fractions which contained f a t s , s t e r o l s , s t e r o i d a l ketones, the steroid acids 2_3 and 2 4 , and glycerol ether 2 5 . Combination of the fractions containing the s t e r o i d a l acids followed by preparative TLC (5:95 methanol/chloroform then, on a second p l a t e , with 10:90 methanol/chloroform) gave 359 mg (1.0%, 2.8 mg/animal) of a mixture containing 3-oxo-4-cholenoic acid (2_3) and 3-oxo~4,22-choladienic acid ( 2 4 ) in a r a t i o of 10:3. The r a t i o was determined by the measuring the peak heights of the C-18 methyl resonances in the 1H NMR spectrum. The acids 2 3 and 24 could be separated 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 (acetic acid/water), reverse phase preparative TLC (30% water/ethanol + 15 drops acetic acid/100 mL) or normal phase HPLC of their methyl esters ( 2 . 4 % isopropanol/hexane). Reverse phase preparative TLC was found to be the most convenient. Combination of the column fractions containing the s t e r o i d a l ketones followed by preparative TLC (chloroform) gave 10.0 mg (0.03%, 0.1 mg/animal) of a mixture of s t e r o i d a l ketones consisting mainly of cholestenone ( 2 7 ) . Fractions containing the glycerol ether were combined and p u r i f i e d by preparative TLC to give 1-0-hexadecyl-glycerol ( 2 5 ) . To aid in the p u r i f i c a t i o n and characterization of 25 i t was converted to i t s d i a c e t y l derivative 4 4 . 1 75 3-Oxo-4-cholenoic acid (23): mp 178-179 °C; UV (CH3CN) Xm a v 1 I l i a A 236 nm (e 6200); IR (KBr) 3600 - 2400, 1720, 1700 and 1640 cm"1; 1H NMR (400 MHz, CDC13) 6 5.74 (bs, 1H, C„ H), 2.63 -0.81 (m, 25H), 1.21 (s, 3H, C1 9 Me), 0.94 (d, J = 6 Hz, 3H, C2 1 Me), 0.74 (s, 3H, C1 B Me); 1 3C NMR, see Table 2; HRMS, observed m/z 372.2658, C2i,H3 60 3 requires 372.2664; MS, m/z ( r e l intensity) 373 (21), 372 (82), 229 (22), 124 (100), 121 (21), 107 (29), 95 (25), 93 (23), 91 (26), 81 (25), 79 (24), 67 (22), 55 (66) , 44 (54), 41 (31). 3 -OXO-4,22-choladienic acid (24): 1H NMR (80 MHz, CDC13) 6 6.96 (dd, J = 8.8,15.4 Hz, 1H, C2 2 H), 5.76 (d, / = 15.4 Hz, 1H, C2 3 H), 5.74 (bs, 1H, C„ H), 2.60 - 0.80 (m, 21H), 1.20 (s, 3H, C , g Me), 1.11 (d, J = 6.4 Hz, 3H, C2, Me), 0.77 (s, 3H, C1 8 Me); HRMS, observed m/z 370.2507, required for C2„ H3, 03 370.2508; MS, m/z ( r e l intensity) 371 (24), 370 (94), 328 (34), 271 (55), 229 (42), 147 (30), 133 (24), 124 (100), 121 (23), 119 (24), 109 (20), 107 (28), 105 (27), 95 (28), 93 (30), 91 (30), 81 (28), 79 (35), 67 (26), 55 (34), 43 (30), 41 (23). Preparation of di a c e t y l derivative 44. To 8 mg (0.025 mmole) of crude 1-0-hexadecyl-glycerol was added 1 mL of pyridine and 0.5 mL of acetic anhydride. After s t i r r i n g at room temperature overnight, the reaction mixture was evaporated to dryness in vacuo and p u r i f i e d by 176 preparative TLC to give 8 mg (0.020 mmole, 79%) of diac e t y l derivative 44: *H NMR (80 MHz, CDC13) 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 (bs, 28H), 0.88 (m, 3H); HRMS, observed m/z 400.3141, C23 H „a05 requires 400.3189; MS, m/z ( r e l intensity) 400 (0.28), 297 (55), 255 (45), 159 (77), 117 (100), 103 (48), 101 (33), 100 (84), 97 (55), 96 (34), 83 (72), 82 (44), 71 (55), 69 (44), 57 (78), 55 (52). Steroidal ketones from A. cooperi. The s t e r o i d a l ketone f r a c t i o n , which consisted mainly of cholestenone (27_) , was not separated further. In combination, the high resolution mass spectrum and the 1H NMR spectrum of the mixture indicated the presence of four steroidal ketones, m/z observed 412.3728, required for C2 9H „80 412.3705; m/z observed 396.3384, required for C28HailO 396.3392; m/z observed 384.3396, required for C27H,,ftO 384.3392; m/z observed 382.3246, required for C2 7H4 20 382.3235; 1H NMR 6 5.75 ( s ) , 5.21 (m), 4.73 (bs, C=CH2), 4.67 (bs, C=CH2), 1.57 (bs), 1.18 ( s ) , 0.94 (d, J = 6.4 Hz), 0.71 (s); UV (Hexane) Xm a x 229 nm. Steroidal ketones from Anthoarcuata graceae. 1 77 Ant hoarcuat a graceae was coll e c t e d at various locations in Barkley Sound, B.C. at depths of 1 to 15m. Immediately after c o l l e c t i o n the sponge (592 g dry weight after extraction) was immersed whole in methanol and stored at room temperature for 2 days. At the end of thi s time the sponge was homogenized in a Waring blender and vacuum f i l t e r e d in the presence of C e l i t e . The resulting crude extract was concentrated to about 1.8 L and partitioned be-tween brine and ethyl acetate (5 x 400 mL). The combined ethyl acetate layers were washed with brine, dried over sodium s u l f a t e , f i l t e r e d , and concentrated in vacuo to give 5.2 g (0.88%) of a gummy orange o i l . The o i l was fractionated by column chromatography (gradient of EtOEt in CHC13) to give 61.8 mg (0.010%) of a mixture of steroidal ketones whose 'H NMR spectrum was es s e n t i a l l y i d e n t i c a l to that of the mixture of steroidal ketones obtained from Al di s a cooperi. Methylation of 3-oxo-4-cholenoic acid (23). To 2 mg (0.0054 mmole) of (23) in 1 mL ether was added 2 mL of freshly prepared ethereal diazomethane (Aldrich's MNNG - Diazomethane apparatus). After completion of the reaction (TLC), the ether was removed i.n vacuo to give 2 mg (96%) of ester 28: UV Xm a x (MeOH) 241 nm (e 15,000); IR (CHC13 cast) 2940, 1740, 1675, 1190, 1170 cm"1; 'H NMR (400 MHz, CDC13) 6 5.74 (bs, 1H, C» H), 3.68 (s, 3H, OMe), 1.19 178 (s, 3H, C1 9 Me), 0.93 (d, J = 6.8 Hz, 3H, C 2 i Me), 0.7 2 (s, 3H, C1 B Me); 1 3C NMR see Table 2; HRMS, observed m/z 386.2821, required for C25 H3 80 3 386.2821; LRMS, m/z ( r e l intensity) 386 (85), 263 (23), 229 (38), 147 (30), 133 (25), 124 (100), 107 (41), 105 (32), 93 (44), 91 (44), 81 (56), 79 (45), 67 (42), 55 (96), 43 (58), 41 (61). Methylation of a mixture of 3-oxo-4-cholenoic acid (23) and  3 -QXO-4,22-choladienic acid (24) 26.3 mg (0.071 mmole) of a crude mixture of 23 and 24 were treated with diazomethane as previously described for 23.' Evaporation of the solvent gave 26.0 mg (95%) of a mixture of esters 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 ester 3_8: UV Xm a x (MeOH) 239, 214 nm; IR (CHC13 cast) 2930, 2850, 1723, 1674, 1270, 1240 cm"1; 1H NMR (400 MHz, CDC13) 6 6.84 (dd, / = 16,9 Hz, 1H, C2 2 H), 5.76 (d, J = 16 Hz, 1H, C2 3 H), 5.74 (s, 1H, C„ H), 3.74 (s, 3H, OCH3), 1.19 (s, 3H, C1 9 Me ), 1.10 (d, J = 7 Hz, 3H C2 1 Me), 0.75 (s, 3H, C1 8 Me); HRMS, observed m/z 384.2655, required for C2 5H3 603 384.2664; LRMS, m/z ( r e l intensity) 384 (72), 342 (22), 272 (21), 271 (81), 269 (20), 253 (21), 225 (46), 201 (20), 175 (22), 147 (24), 124 (53), 107 (21), 105 (21), 95 (21), 93 (21), 91 (22), 81 (29), 79 (100), 77 (23), 55 (30), 41 (22). 179 Melibe Leoni na C o l l e c t i o n , extraction and chromatographic separation. Meli be leonina (38 animals) was c o l l e c t e d at Cates Park, North Vancouver, B.C. on October 6, 1981. Immediately after c o l l e c t i o n , the nudibranchs were immersed whole in chloroform (2L), and upon returning to the laboratory were extracted on a wrist action shaker for 1.5 h. The chloroform was separated from the water layer in a separatory funnel, dried over sodium s u l f a t e , and concentrated in vacuo (dry ice thimble, < 20°C) to give 57.4 mg of an orange "grapefruit" smelling o i l . S i l i c a gel column chromatography (step gradient of 100% hexane to 40% di e t h y l ether/chloroform) gave straight chain hydrocarbons, f a t s , s t e r o l s , 2,6-dimethyl-5-heptenal (53_) and 2,6-dimethyl-5-heptenoic acid (54). Pooling of the fractions containing 5_3, followed by preparative TLC (chloroform) gave 19 mg (33% of choloform soluble extract) of 5_3. Combination of fractions containing 5_4 followed by preparative TLC (diethyl ether) gave 3 mg (5% of chloroform soluble extract) of 54. 2,6-dimethyl-5-heptenal (53): IR (CHC13) 1723 cm"2; 1H NMR (400 MHz, CDC13) 6 9.62 (d, J = 1.9 Hz, 1H, C, H), 5.10 (m, 1H, C5 H ), 2.36 (m, 1H, C2 H), 2.05 (m, 2H, C„ 2H), 1.77 (m, 1H, C3 H), 1.70 (bs, 3H, C8 Me), 1.61 (bs, 3H, C7 Me), 180 1.41 (m, 1H, C3 H), 1.11 (d, / = 7.1 Hz, 3H, C9 Me); '3C NMR see Table 4; GC-MS, m/z (rel intensity) 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 acid (54): IR (CHC13) 3500 - 2200, 1700 cm"1; 1H NMR (400 MHz, CDC13) 8 5.13 (m, 1H), 2.49 (m, 1H), 2.05 (m, 2H), 1.76 (m, IH), 1.70 (bs, 3H), 1.62 (bs, 3H), 1.48 (m, 1H), 1.20 (d, J = 7.1 Hz, 3H); HRMS, m/z observed 156.1151, required for C9H1 602 156.1151; MS, m/z (rel i n t e n s i t y ) 156 (22), 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 collecte d at various locations in Barkley Sound, and from Sunset Beach near Horseshoe Bay, B r i t i s h Columbia. No attempts were made to separate the small f r a c t i o n of A. hudsoni that was co-collected with A. nanaimoensis at some s i t e s . H e l l o u1 9 8 had shown that the skin extract of A. hudsoni also contained nanaimoal 61. 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 v a r i a t i on in the rat i o of nanaimoal (61): isoacanthodoral (6_5): acanthodoral (6_4) i s o l a t e d . Immediately after c o l l e c t i o n the animals were immersed whole in methanol and stored at room temperature for one to three days. If the animals were not worked up immediately they were stored at low temperature (4 to -5 °C), in the dark, u n t i l used (usually within two months). Extraction and Chromatographic Separation As a number of c o l l e c t i o n s of A. nanaimoensis were made and no va r i a t i o n in metabolites isolated was observed, the following represents a t y p i c a l procedure. A l l weights of the v o l a t i l e aldehydic components are in error as i t was impossible to remove a l l traces of solvent under high vacuum without substantial losses of the sesquiterpenoids. 182 A. nanaimoensis (120 animals) was c o l l e c t e d in February, 1982, in Barkley Sound, B r i t i s h Columbia. They were immediately immersed whole in methanol and stored at room temperature for two days. At the end of th i s time the methanol used for extraction of the whole animals was decanted and saved. The nudibranchs were soaked an additional five times (0.5 h each) with methanol and the methanol extracts were combined and concentrated in vacuo (< 20°C, dry ice/acetone thimble).to one-quarter of the o r i g i -nal volume. The aqueous methanolic extract (700 mL) was then partitioned between brine (100 mL) and chlorform (4 x 100 mL). The combined organic layers were then washed with brine and dried over sodium s u l f a t e . Removal of the solvent in vacuo gave 2.3 g (16 mg/animal) of an odouriferous orange o i l . Column chromatography (gradient of 100% hexane to 10% ethyl acetate/chloroform) yielded fractions containing f a t s , s t e r o l s , and the sesquiterpenoid aldehydes nanaimoal (61) , acanthodoral (64) , and isoacanthodoral (6_5) . Pooling of the aldehydic fractions followed by removal of the solvent gave 218.6 mg (1.8 mg/animal, 10% of the crude chloroform extract) of a s l i g h t l y yellow o i l y mixture consisting 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. The most abundant aldehydes (61 and 65) could be separated by preparative GC (3% OV-17 on Chromosorb (HP) 80/100 Mesh, 183 i n i t i a l temperature 140°C, rate 1°C/min., Rt 61 16.2 min., Rt 65 17.6 min.) or HPLC (50:50 hexane/methylene c h l o r i d e ) . HPLC was found to be the most convenient, although i t was not possible to purify acanthodoral (64) adequately by this method. Nanaimoal (61): colourless o i l , [ a ]D -7° (c 3.0, CHC13); IR (CHC13) 2920, 2750, 1730, 1470, 1395, 1380, 930, 870, 800 and 750 cm"1; 'H NMR (Table 5); 1 3C NMR (100.6 MHz, CDC13) [ m u l t i p l i c i t i e s determined by SFORD experiment] 6 203.3 (d), 133.8 ( s ) , 125.3 ( s ) , 53.7 ( t ) , 43.7 ( t ) , 39.8 ( t ) , 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-quired for C15H2ltO 220.1827; GC-MS, m/z ( r e l intensity) 220 (4), 177 (6), 176 (49), 162 (16), 161 (100), 121 (6), 105 (33), 69 (7), 55 ( 12) , 41 (24). Acanthodoral (64): colourless o i l ; 1H NMR (400 MHz, CDC13, residual CHC13 (8 7.25) used as internal reference) 6 9.59 (d, J = 2.8 Hz, 1H), 1.82 (d, / = 9.2 Hz, 1H), 1.06 (s, 3H), 0.88 (s, 3H), 0.82 (s, 3H). GC-MS, m/z ( r e l intensity) 220 (1), 205 (14), 187 (9), 177 (10), 176 (48), 161 (48), 137 (28), 121 (24), 109 (18), 107 (26), 105 (38), 97 (14), 95 (44), 93 (29), 91 (24), 84 (100), 81 (54), 79 (28), 69 (43), 67 (22), 55 (35), 41 (76). Isoacanthodoral (65): colourless o i l ; *H NMR (400 MHz, 1 84 CDC13) 6 9.72 (dd, / = 3.3,3.2 Hz, 1H), 5.23 (bs, 1H), 2.71 (dd, / = 14.8,3.3 Hz, 1H), 2.14 (dd, / = 14.8,3.2 Hz, 1H), 1.65 (bs, 3H), 1.00 (s, 3H) 0.91 (s, 3H); 1 3C NMR (100.6 MHz, CDC13) 6 204.6, 135.4, 129.9, 57.1, 46.3, 40.1, 38.4, 32.3, 29.0, 26.8, 23.6, 20.0, 19.3 (the two quaternary carbons could not be confidently assigned due to the limited sample s i z e ) ; GC-MS, m/z ( r e l intensity) 178 (12), 177 (77), 176 (35), 121 (26), 107 (88), 95 (92), 93 (23), 91 (24), 81 (97), 74 (28), 69 (100), 55 (34), 41 (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 sesquiterpenoid aldehydes, to simplify t h e i r separation, and to obtain c r y s t a l l i n e d e r i v a t i v e s , the sesquiterpenoid aldehyde fraction from A. nanaimoensis was reduced with sodium borohydride. To a 50 mL round bottom flask was added 57.3 mg (1.51 mmol) of sodium borohydride and 2 mL of isopropyl a l c o h o l . A t o t a l of 161.7 mg (0.735 mmol) of the mixture of crude 6_1, 64, and 6_5 was dissolved in 30 mL of isopropyl alcohol (solution turned cloudy) and added dropwise to the sodium borohydride with s t i r r i n g . The solution cleared after a few minutes. After 24 hr, 30 mL of water was added and s t i r r e d for an additional 2 hr. At the end of t h i s time the reaction mixture was partitioned be-tween water (50 mL) and chloroform (4 x 25 mL). The com-bined chloroform layers were dried over magnesium sulfate 185 and f i l t e r e d . Evaporation of the solvent provided 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 for 10 min then 10 °C/min, Rt 7.71 min), 64 (4%, R{ 5.79 min) and 65 (36%, Rt 8.14 min). The remaining 4% was due to the minor metabolites (1%, Rt 4.08 min; 3%, R{ 11.65 min) that were not characterized further. The low y i e l d in the reduction is l i k e l y due to weighing error for the aldehydes (traces of solvent were not removed on high vacuum). Repetitive r a d i a l TLC ( s i l i c a g e l , 100% CHC13) provided 25.8 mg of pure nanaimool (70): o i l ; [ o ]D + 1 0 . 4° (c 0.61, MeOH); IR (CHC13) 3600, 3400, 2920, 1460, 1380, 1360, 1020, 910, and 740 cm"1; 1H NMR (400 MHz, CDC13) 5 3.72 (m, 2H), 1.97 (bs, 2H), 1.78 (bs, 2H), 1.7 5 (bd, J = 17 Hz, 1H), 1.59 (bd, J = 17 Hz, 1H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H); 1 3C NMR (100.1 MHz, CDC13) 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, required for C1 5H2 60 222.1984; MS, m/z (rel intensity) 222 (40), 207 (100), 189 (30), 179 (24), 177 (33), 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 for the preparation of the (p-bromophenyl)urethane d e r i v a t i v e s . Preparation of the (p-bromophenyl)urethane derivatives 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 in 3 mL of carbon tetrachloride was added to a 5 mL reaction v i a l containing 67.9 mg (0.343 mmole) of 4-bromophenyl isocyanate in 1.5 mL of carbon t e t r a c h l o r i d e . The v i a l was sealed and heated with s t i r r i n g at 60°C for 20 h. At the end of t h i s time the reaction was cooled to room temperature; transferred to a 25 mL flask and the excess 4-bromophenyl isocyanate was destroyed with methanol. Evaporation of the solvent, followed by preparative TLC (100% chloroform), gave 24 mg (0.0571 mmole, 98%) of a mixture of 98 and 114. Preparative reverse phase HPLC sepa-ration (15% water/acetonitrile) gave pure samples of 98 and 114. , 98: o i l , [ a ]D -39° (c 0.88, hexane); 'H NMR (400 MHz, CDC13) 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); 1 3C NMR (100.6 MHz, CDC13) only terpenoid carbons are l i s t e d , 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 intensity) 421 (1), 419 (1), 244 (1), 242 (2), 217 (9), 215 (9), 204 (31), 189 (18), 177 (100), 107 (39), 105 (13), 95 (31), 93 (13), 91 (16), 81 (31), 69 (32), 55 (14), 41 (19). 114: mp 109-110 °C (hexane); 1H NMR (400 MHz, CDC13) 5 7.40 187 (d, 2H), 7.27 (d, 2H), 6.50 (bs, 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, J = 9.2 Hz, 1H), 0.96 (s, 3H), 0.89 (s, 3H), 0.81 (s, 3H); HRMS, m/z observed 421.1441 and 419.1438, required- for C2 2H3 0BrN02 421.1439 and 419.1416; MS, m/z ( r e l intensity) 204 (66), 189 (100), 161 (20), 95 (23), 81 (30), 69 (24). Acid catalyzed isomerization of 98. Preparation of 112. A t o t a l of 2.5 mg of 9ji was placed in a 2 mL reaction v i a l , 1.5 mL of 98-100% formic acid was added and the v i a l was capped. After heating at 70 °C overnight, the reaction was cooled and concentrated (in vacuo) to give a l i g h t brownish residue. The residue was dissolved in ether (25 mL) and washed with 5% bicarbonate ( 2 x 1 0 mL) and water (1 x 10 mL). The ether layer was dried over sodium s u l f a t e , f i l t e r e d and the solvent removed in vacuo to give, after preparative TLC (chloroform), 1.1 mg of. 112. No traces of 98 were detected by 1H NMR or HPLC (reverse phase, 10:90 wate r / a c e t o n i t r i l e ) . 112: o i l ; 'H NMR (400 MHz, CDC13) 5 7.41 (m, 2H), 7.27 (m, 2H) , 6.49 (bs, 1H) , 5.29 (bs, = 11.6 Hz, 1H), 1.59 (bs, 3H), 0.88 (s, 3H), 0.80 (s, 3H); HRMS, m/z observed 421.1463, required for C2 2H3 08 1BrN02 421.1439; MS, m/z ( r e l intensity) 421 (1), 419 (1), 217 (23), 215 (23), 205 (13), 204 (77), 189 (100), 177 (43), 175 (17), 161 (27), 133 (14), 121 (19), 119 (28), 107 (28), 106 188 (39), 105 (54), 95 (18), 94 (26), 93 (28), 91 (31), 81 (23), 79 (18); 69 (22), 55 (22). Preparation of nanaimoal's (p-bromophenyl)urethane  derivative 75. A to t a l of 16.2 mg (0.073 mmol) of 70 was reacted with 4-bromophenyl isocyanate (65.2 mg, 0.329 mmol) by the method described for 97 and 118 to give, after preparative TLC (chloroform), 31 mg (0.073 mmol, 100%) of 75: o i l ; 1H NMR (see Table 5), 1 3C 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, required for C2 2H3 08 1BrN02 421.1439; MS, m/z ( r e l intensity) 216 (10), 214 (10), 204 (33), 189 (100), 176 (27), 161 (40), 105 (32), 91 (21), 55 (23) . Preparation of the 2,4-dinitrophenylhydrazone derivatives of 61, 65, and 64. 78.6 mg (0.357 mmol) of the aldehyde mixture was dissolved in 2 mL of methanol and 10 mL of a mixture containing 108 mg of 2,4-dinitrophenylhydrazine in 10 mL of methanol was added. The mixture was s t i r r e d for three hours 189 after which i t was parti t i o n e d between water (50 mL) and chloroform (4 x 25 mL). The chloroform layers were com-bined, dried over sodium s u l f a t e , f i l t e r e d , and evaporated to give 131.1 mg (0.328 mmol, 92%) of a mixture of 2,4-dinitrophenylhydrazone derivatives 211, 212 and 96. Separation by normal phase preparative HPLC (10% chloroform/hexane) gave pure 9 6 : 1H NMR (270 MHz, CDC13) 6 10.9 (bs, 1H), 9.08 (d, J = 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 (bs, 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 (bs, 3H), 1.03 (s, 3H), 0.90 (s, 3H). Preparation of regioisomeric alcohols 76 and 84. 211 212 190 Following T i s c h l e r ' s procedure1 1 5 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 (7j)) and divided equally among three 37 cm Karius tubes. Nitrogen gas was bubbled through the solutions for 15 min after which the tubes were sealed and placed in an oven. The reaction mixture was heated to 230 °C for 8 h and then allowed to cool to RT overnight. Upon cooling, two layers separated. A n a l y t i c a l TLC indicat-ed l i t t l e compositional differences between the layers. The material from the three tubes was combined, dissolved in ether (150 mL) and washed with water (50 mL). The l i g h t yellow ether layer was dried over sodium sulfate and the ether was removed in vacuo. The residue was taken up in hexane (100 mL), added to the top surface of s i l i c a gel (250 g) in a 14 cm Buchner funnel, and the hexane was drawn through the s i l i c a gel by suction. Additional hexane (200 mL) was added and drawn through the s i l i c a . The procedure was repeated with additional hexane (500 mL), followed by 50% chloroform/hexane (500 mL), chloroform (500 mL), and ethyl acetate (2 x 500 mL). The mixture of regioisomeric alcohols, J59 and 90, was present in the f i r s t ethyl acetate wash. Removal of the solvent gave 7.58 g of a mixture that contained at least three components as indicated by a n a l y t i c a l TLC. The major component was recovered sta r t i n g alcohol iBO. Fractionation of a 2.48 g portion of t h i s mixture by flash chromatography (5 cm diameter column, 6 inches s i l i c a g e l , 10% ethyl acetate/petroleum ether) gave 191 264 mg (1.19 mmole, 6% calculated y i e l d based on entire alcohol fraction) of crude regioisomers 76 and 84: 1H NMR (400 MHz, CDC13) 6 5.35 (bs), 5.29 (bs), 5.08 (bt, J = 6.4 Hz), 1.68 (bs, CH3), 1.61 (bs, CH3), 0.915 (s, CH3), 0.911 (s, CH3); MS, m/z ( r e l intensity) 222 (3), 207 (1), 189 (3), 179 (15), 177 (14), 161 (21), 135 (17), 109 (35), 107 (45), 93 (50), 81 (23), 79 (25), 69 (100), 55 (23), 43 (20), 41 (95). Separation of the alcohols 76 and 84 was achieved by recycling r a d i a l chromatography (12% ethyl acetate/petroleum ether, flow = 3.5 mL/min) to give pure samples of 7_6 and 84. These were converted d i r e c t l y into the (p-bromophenyl)-urethane derivatives 8_9 and 90. Preparation of a mixture of (p-bromophenyl)urethane derivatives 89 and 90. A t o t a l of 16.6 mg (0.0748 mmol) of the crude . regioisomers 76 and 84 were reacted in the usual way with 75 mg (0.379 mmol) of 4-bromophenyl isocyanate in a 10 mL reaction f l a s k . Methanol was added to decompose the excess 4-bromophenyl isocyanate and the mixture was p u r i f i e d by preparative TLC (chloroform) to give 7.3 mg (0.0174 mmol, 23%) of a mixture of 89 and 90: 1H NMR (400 MHz, CDC13) 6 7.40 (d), 7.27 (d), 5.37 (bs), 5.30 (bs), 5.09 (bt, J = 1 Hz), 4.24 (m), 1.68 (bs, CH3), 1.60 (bs, CH3), 0.943 (s, CH3), 0.933 (s, CH3). 1 92 Preparation of a mixture of (±)-75 and 93• A t o t a l of 5.0 mg (0.012 mmol) of the mixture of regioisomers 89 and 90 were combined with 5 mL of 98-100% formic acid in a 10 mL round bottom flask equiped with a s t i r bar and condenser. The solution was heated, with s t i r r i n g , at" 70 °C for 12 h. At the end of this period the reaction mixture had turned purplish brown. The mixture was evaporated to dryness in vacuo, taken up in ether, washed with 5% sodium bicarbonate (2 x 5 mL), and dried over magnesium s u l f a t e . Evaporation of the solvent, followed by preparative TLC (chloroform), gave three products (Ry 0.43, 0.22, and 0.03). The major product (Rj- 0.43) was 3.1 mg (0.007 mmol, 62%) of a mixture of 75 and 93: 1H NMR (400 MHz, CDC13) 6 7.40 (d), 7.27 (d), 4.23 (m), 0.98 (s, CH3), 0.97 (s, CH3), 0.94 (s, CH3), 0.935 (s, CH3), 0.92 (s, CH3), 0.91 (s, CH3). Preparation of (p-bromophenyl)urethane derivative 90. A t o t a l of 8.2 mg (0.0369 mmol) of alcohol regioisomer 84 was reacted with 49.5 mg (0.250 mmol) of 4-bromophenyl isocyanate by the usual procedure to give, after preparative TLC (chloroform), 11.9 mg (0.0283 mmol, 77%) of urethane 90: 'H NMR (400 MHz, CDC13) 6 7.41 (d, 2H), 7.27 (d, 2H), 6.51 (bs, 1H), 5.29 (bs, 1H), 5.08 (bt, 7 = 7 Hz, 1H), 4.24 (m, 2H), 1.90 (bd, J = 17.6 Hz, 1H), 1.77 (bd, J = 17.6 Hz, 1H), 193 1.68 (bs, 3H), 1.60 (bs, 3H), 0.93 (s, 3H); HRMS, m/z observed 419.1447, required for C2 2H3 07 9BrN02 419.1460; MS, m/z ( r e l intensity) 421 (7), 419 (7), 217 (14), 215 (14), 204 (49), 176 (43), 161 (40), 135 (25), 107 (100), 93 (52), 91 (30), 79 (36), 69 (91), 55 (33), 41 (92). Preparation of (p-bromophenyl)urethane derivative 89. A t o t a l of 4.7 mg (0.021 mmol) of alcohol regioisomer 76 was reacted with 44.1 (0.223 mmol) of 4-bromophenyl isocyanate by the usual procedure to give, after preparative TLC (chloroform), 4.6 mg (0.011 mmol, 52%) of urethene 89: 1H NMR (400 MHz, CDCl3) 6 7.40 (d, 2H), 7.26 (d, 2H), 6.51 (bs, 1H), 5.37 (bs, 1H), 5.09 (bt, / = 7 Hz, 1H), 4.24 (m, 2H), 1.82 (bd, / = 17 Hz, 1H), 1.68 (bs, 3H), 1.60 (bs, 3H), 0.94 (s, 3H); HRMS, m/z observed 419.1443, required for C2 2H3 07 9BrN02 419.1460; MS, m/z ( r e l intensity) 421 (11),. 419 (11), 217 (25), 215 (25), 204 (48), 161 (44), 135 (45), 107 (47), 93 (58), 81 (44), 69 (100), 55 (52), 43 (46), 41 (90). Preparation of (±)-75 . A t o t a l of 2.9 mg (0.0069 mmol) of urethane 8_9 was cy c l i z e d following the same procedure described for the mixture of 89 and 90. P u r i f i c a t i o n by preparative TLC (chloroform) gave 2.0 mg (0.0048 mmol, 69%) of (±)-7_5 1 94 i d e n t i c a l by *H NMR, MS, and HPLC retention time to natural product derivative 7 5 . 195 Phi dol opora pacifica C o l l e c t i o n Data. Phidolopora pacifica was co l l e c t e d by hand using SCUBA from Diceman Island in the Broken Group and from a variety of s i t e s in the Deer Group of Islands, Barkley Sound, B r i t i s h Columbia. Collections were made at depths of 5 to 20 m. Samples were immediately immersed in methanol, ethanol or acetone, stored at RT for 1-3 days and then at 4 to -5 °C, in the dark, u n t i l used. Extraction and Chromatographic Separation. 1. Methanol Extraction A number of c o l l e c t i o n s were extracted with methanol. The same i s o l a t i o n scheme was used for each extraction. The following represents a t y p i c a l extraction and i s o l a t i o n procedure. The bryozoan (143 g dry weight after extraction) was ground in a Waring blender with the methanol (1L) used for extraction of the whole animals. Vacuum f i l t r a t i o n of the crude extract in the presence of C e l i t e gave a greenish brown methanolic f i l t r a t e which was concentrated to about 250 mL and partitioned between brine and ethyl acetate (3 x 150 mL). The combined ethyl acetate extracts were washed with 200 mL of brine and dried over sodium s u l f a t e . The 1 96 ethyl acetate was evaporated to give 796 mg (0.56%) of a dark greenish-brown crude o i l . The o i l was fractionated by f l a s h chromatography (40 mm diameter column, 6 inches s i l i c a g e l , step gradient of 5% ethyl acetate/petroleum ether to 20% methanol/ethyl acetate) to y i e l d fractions containing f a t s , s t e r o l s , 4-methoxymethyl-2-nitrophenol (189) , 4-hydroxymethyl-2-nitrophenol (181) , phidolopin (179) , and desmethylphidolopin (180). 2. Ethanol Extraction The bryozoan (720 g dry weight after extraction) was ground in a Waring blender with the ethanol (4L) used for extraction of the whole animals. F i l t r a t i o n (in vacuo) and concentration of the f i l t r a t e gave 1.5 L of an ethanol-water suspension that was partitioned between brine (200 mL) and ethyl acetate (4 x 200 mL). The combined ethyl acetate layers were washed with brine (300 mL) and dried over sodium sulfate to give 1.29 g (0.18%) of a dark o i l . Flash chromatography (40 mm diameter column, 6 inches s i l i c a gel) using a step gradient of 5% ethyl acetate/petroleum ether to 20% methanol/ethyl acetate gave d i f f e r e n t fractions containing f a t s , s t e r o l s , 4-ethoxymethyl-2-nitrophenol (209) , phidolopin (179), and desmethylphidolopin (180) . Fractions from the column that contained 209 were combined and further p u r i f i e d by preparative reverse phase TLC [20:80 water/(95% ethanol), Rj- ^ 0.6] followed by preparative TLC (chloroform, Rj- 0.3) to give 3.5 mg of 4-ethoxymethyl-2-nitrophenol as a yellow o i l . 4-methoxy-2-nitrophenol (189): 1H 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, 2H), 3.41 (s, 3H); HRMS, observed m/z 183.0534, C8H9N O „ requires 183.0532; MS, m/z ( r e l intensity) 183 (41), 182 (20), 152 (100), 141 (40), 136 (20), 127 (31), 123 (61), 106 (66), 105 (33), 78 (28), 77 (51), 65 (29), 53 (28), 51 (56), 45 (33), 41 (20) , 39 (42) , 31 (51), 29 (71 ) . 4-hydroxymethyl-2-nitrophenol (181): 'H NMR (80 MHz, CDC13) 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 (bs, OH + H20); HRMS, observed m/z 169.0379, C7H7N O « requires 169.0375; MS, m/z ( r e l intensity) 169 (100), 123 (36), 122 (32), 106 (26), 105 (20), 95 (26), 94 (24), 77 (28), 66 (22), 65 (51), 53 (30), 51 (30), 39 (44). Phidolopin (179): mp 226-227 °C (CH3CN); UV (CH3CN) 351 nm (e 3,300), 275 ( 16,800); IR (CHC13 cast) 3300 (b), 1697, 1657, 1626, 1532 cm"1; 'H NMR (270 MHz, CDC13) 10.56 (s, 1H, exchanges with D20), 8.08 (d, / = 2.2 Hz, IH), 7.63 (s, 1H), 7.61 (dd, J = 2.2, 8.6 Hz, IH), 7.16 (d, J = 8.6 Hz, 1H), 5.46 (s, 2H), 3.59 (s, 3H), 3.39 (s, 3H); HRMS observed m/z 331.0917, required for C1flHl3N505 331.0917; MS, m/z ( r e l intensity) 331 (20), 313 (17), 180 (75), 150 (100). 198 Desmethylphidolopin (180) : 1H NMR (400 MHz, DMSO-d6) 5 11.16 (s, 1H), 11.05 (s, 1H), 8.26 (s, 1H), 8.00 (d, J = 2.6 Hz, 1H), 7.58 (dd, J = 2.6, 8.7 Hz, 1H), 7.12 (d, J = 8.7, 1H), 5.41 (s, 2H), 3.35 (s, 3H); HRMS observed m/z 317.0777, re-quired for C^H^NjOs 317.0760; MS, m/z ( r e l intensity) 317 (37), 299 (35), 177 (33), 166 (100), 152 (75), 123 (27), 107 (21), 106 (36), 105 (27), 95 (37), 77 (30), 69 (27), 57 (24), 55 (26), 51 (20). 4-Ethoxy-2-nitrophenol (209): 1H NMR (80 MHz, CDC13) 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, required for CsH^NO, 197.0688; MS, m/z ( r e l intensity) 197 (49), 153 (22), 152 (100), 135 (20), 123 (31), 106 (43), 105 (20), 77 (24), 51 (25), 29 (23). Preparation of p-bromophenacyl derivative 194. To 2 mg (0.006 mmol) of phidolopin (179) dissolved in 1 mL of a c e t o n i t r i l e (heated to di s s o l v e , gave a yellow solution) was added 27.9 mg (0.279 mmol) of potassium bicarbonate and 4 mL of a stock solution 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 at 75°C for 1 h followed by an additional hour at RT. At the end of this time the reaction mixture was 199 partitioned between water (20 mL) and ethyl acetate (3 x 30 mL). Most of the l i g h t yellow colour was present in the organic layer. The ethyl acetate was dried 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 preparative TLC (5% methanol/chloroform) gave 3 mg (0.006 mmol, 100%) of derivative 194; off white s o l i d ; mp 197 °C dec; 1H NMR (400 MHz, CDC13) 5 7.80 (d, J = 2.3 Hz, IH), 7.79 (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 H i / c m * 3 2 1 0 Appendix 1. 400 MHz NMR spectrum of 75 in CDC13. f Hi/cm mo 50 M. 10 000 Hi 5 000 Hi 2 500 8 000 4 000 2 000 2000 1000 500 11 PPM (6) 0 Appendix 2. 400 MHz NMR spectrum of 98 in CDC1 to o to VI. BIBLIOGRAPHY 1. For example see: Faulkner, D.J. Rev. Latinoamer. Quim. 1983, 14, 61. 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 in Animals"; E l s e v i e r : Amsterdam, 1979. (c) Rockstein, M., Ed. "Biochemistry of Insects"; Academic Press: New York, 1978. (d) Harborne, J.B. 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