NOVEL SECONDARY METABOLITES FROM SELECTED BRITISH COLUMBIAN
MARINE INVERTEBRATES
by
STEPHEN WILLIAM AYER
A THESIS SUBMITTED IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES
Department of Chemistry
We accept this thesis as conforming
d standard
THE UNIVERSITY OF BRITISH COLUMBIA
March 1985
© Stephen William Ayer, 1985 In presenting this thesis in partial fulfilment of the re• quirements for an advanced degree at the The University of
British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my
Department or by his or her representatives. It is under• stood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.
Department of Chemistry
The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5
Date: March 1985 Abstract
Marine organisms show potential as sources for novel, biologically and pharmacologically active, secondary metabolites. Examination of three nudibranch and one bryozoan species for biologically active metabolites has led to the isolation and structural elucidation of nine new and two known secondary metabolites. The structures of all the compounds were determined by using a combination of spectral analysis, chemical interconversion, synthesis, and single-crystal X-ray diffraction analysis.
The British 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 antibacterial and antifungal activity. From
Aldisa cooperi, two A*-3-ketosteroidal acids 23 and 24, and glycerol ether 25 were isolated. Acid 23 showed feeding deterrent activity against fish. 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 like" odour of the nudibranch.
The bryozoan Phidolopora pacifica was examined in an attempt to correlate the absence of surface fouling, in the field, with the presence of biologically active secondary metabolites. The purine alkaloids 179 and 180, which
i i contain the rare naturally occurring nitro functionality, were responsible for much of the antifungal and antialgal activity of the crude extracts. Three nitrophenols 181,
189, and 209 were also isolated from P. pacifica.
Nitrophenol 181 had been previously shown to inhibit chloroplast development both in green plants and in the unicellular algae Eugl ena sp. Table of Contents
Abstract ii
List of Figures vi
List of Schemes vii
List of Tables viii
List of Appendices ix
Acknowledgements x
Dedication xi
Abbreviations xii
I . Introduction 1
A. Overview 1
B. Natural Products Chemistry 3
C. Primary and Secondary Metabolites 8
D. Chemical Ecology 11
II. Nudibranchs 16
A. Introduction 16
1. Gastropod Secondary Metabolites 16
2. Nudibranch Defense Mechanisms 21
B. Secondary Metabolites from the Dorid Nudibranch Aldisa cooperi (Robilliard and Baba, 1972) 25
1 1. Introduction 25
2. Isolation and Structure Elucidation 25
3. Biological Activities 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
1 4. Assignment of the H NMR Spectrum of Nanaimoal using One and Two-Dimensional NMR Techniques 81
5. Isoacanthodoral 99
6. Acanthodoral 112
7. Biological Activities of A. nanaimoensis Secondary Metabolites 117
8. Discussion 117
III. 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 List of Figures
1. Phylogenetic classification 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 1 11. 400 MHz H NMR spectrum of nanaimoal (CDC1 ) ... 63 (61) 3 1 12. Model systems for the H NMR chemical shifts 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
1 15. 400 MHz H 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. Partial 400 MHz *H NMR 2D /-resolved spectrum (symmetrized) of nanaimoal (61) 97 19. Slices of individual peaks shown at the top of Figure 18 to show multiplicities 98
20. Computer generated X-ray structure of isoacanthodoral's 2,4-dinitrophenylhydrazone derivative 96 110 vi 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 final X-ray model of p-bromophenacylphidolopin 194 160
vi i List 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 diacetyl 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 ....
viii List of Tables
1. Comparison of the 'H NMR data for various A"-3-ketosteroids (CDC1 ) 29 3 13 2. C 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 (CDC1 ) . 39 3 13 4. C NMR data for 2,6-dimethyl-5-heptenal (53) and citronellal (55) 51 1 5. H NMR data (400 MHz) for nanaimoal (61) and derivatives 83
6. Nudibranch sesquiterpenoids 119
7. Bryozoan metabolites 134
8. 'H NMR data (CDC1 , 80 MHz) and spectral comparisons for 3 nitrophenols isolated from Phidolopora paci fi ca 153 9. 'H NMR data and spectral comparisons for purine derivatives isolated from Phidolopora pacifica 158
ix List 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 like to gratefully acknowledge the guidance, encouragement, patience, and friendship of Dr. R.J.
Andersen.
Many people have influenced the outcome of this 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 diving, assisted with all the invertebrate collections, 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 typical cryptobranch dorid nudibranch and Ron Long kindly provided photographs of all the invertebrates studied. I thank Sandra Millen for helpful discussions on nudibranch biology.
A number of people assisted with the collection of the marine organisms, and the spectral data of the compounds isolated from them. I thank the staff of the Bamfield
Marine Station and the departmental NMR and MS laboratories for courteous and reliable assistance.
xi To Jean, Roxanne, Genevieve, Dorothy, Bill, 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 liquid chromatography
HRMS = High resolution mass spectrum
IR = Infrared
MS = Low resolution mass spectrum
'H NMR = Proton nuclear magnetic resonance
13 C 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 = Ultraviolet
W = Water signal
xi i i I. INTRODUCTION
A. OVERVIEW
The purpose of the research undertaken and reported in
this thesis was to isolate and elucidate the structures of
secondary metabolites* exhibiting interesting biological
activities "from selected British 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 ecologically important
secondary metabolites turn out to be pharmacologically
1 active (or vice versa) , therefore the isolations were
directed by in vitro screening for antifungal and antibacterial activities. Because not all potentially
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 purified fraction.
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 definition of a secondary metabolite, see Section C of this Chapter. Natural products chemistry and the chemistry of secondary metabolites are generally regarded as synonymous.
1 2
4. Unambiguous synthesis.
5. - Single crystal X-ray diffraction analysis.
Three species of nudibranchs were investigated for biologically active secondary metabolites. Nudibranchs lack
the physical protection of an external shell, but are seldom eaten. They deter predators using a variety of mechanisms,- as will 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 steroidal acids, of which one displayed fish antifeedant activity (from
Aldisa cooperi), and two are degraded monoterpenes
structurally 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 it employs an effective
antifouling agent. P. pacifica turned out to be a source of rather interesting purine alkaloids containing the
relatively rare, naturally occurring, nitro group.
Initial screens indicated that some of the isolated
metabolites exhibit profound biological activities. For ex•
ample, one of the purine alkaloids was very active against 3 the pennate diatom Cyl i ndrot heca fusiformi s .while the mixture of sesquiterpenoid aldehydes from A. nanaimoensis effectively inhibited the growth of the bacteria Bacillus subtil is. Collaboration with pharmacologists and biologists to study the full spectrum of biological activities, both pharmacological and ecological, that are exhibited by the metabolites described in this thesis would appear in order.
B. NATURAL PRODUCTS CHEMISTRY
Natural products chemistry (the chemistry of secondary metabolites) has undergone explosive growth during the last 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 structural tools were classical 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, al• 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 crystal X-ray diffraction analysis.
With the advent of high performance liquid chromatography (HPLC), routine Fourier transform NMR, and rapid computer aided X-ray analysis, the Chemist has been able to solve chemical problems previously thought 4 impossible. These include, for example, the structural
2 elucidation of pheromones available in microgram amounts ,
3 isolation and structural determination of phytohormones , and elucidation of structures, such as palytoxin (1)* and 5 brevetoxin (2) , that are so complex it is even difficult 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 biologists and pharmacologists) of biologically active isolates has provided, and will continue to provide, interesting chemistry. Analysis of relatively small water soluble molecules will become more prevalent, and this should enable the isolation and structure elucidation of physiologically interesting compounds, par•
6 ticularly oligopeptides . Finally, investigation of the enzymology and biosynthesis of secondary metabolites should provide valuable insights into why organisms produce such
7 compounds .
Marine natural products research has greatly
intensified over the last two decades. An excellent series
8 of books edited by Scheuer provide timely, exhaustive
reviews of important aspects of marine natural products
9 research. Other authoritative reviews by Baker ,
10 11 12 13 10 Christophersen , Coll , Faulkner , Fenical , Moore , 6
15 and Scheuer provide additional highlights of chemical research on the organisms found beneath the sea.
The pragmatic application of natural products isolated from terrestrial sources is well known, particularly 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 for,
16 17 practical application ' . For example; the isolation of nereistoxin (3_) from the marine worm Lumbr i coner ei s 8 heteropoday led to the development of the structurally related insecticide Padan (4) which is in common use in
19 Japan . The nucleoside 1-j3-D-arabinofuranosylcytosine
20 (Ara-C) (5) , an effective 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 potential. Didemnin B (7) appears to be the most potent didemnin in blocking the growth of L-1210 mouse leukemia cells as well as inhibiting the Herpes simplex virus. Didemnin B is currently undergoing clinical trials as an
2 antileukemia drug ".
Results such as the above clearly demonstrate how the study of marine secondary metabolites may lead to some valuable pragmatic spinoffs. Current research efforts are 7
CH.CHOHCO—N-CH-CO—MeLeu—Thr—Sta—•Hip—Leu —Pro—«-Me,Tyr —O-i U | 1_ 7
now being directed towards isolating metabolites with a
2 25 specific biological activity *' , greatly increasing the
probability that a compound with a desired pharmacological
effect will be found.
Although some researchers and one drug company*
* Recently the Roche Research Institute for Marine Pharmacology at Dee Why, Australia, established in 1974, ceased operation. 8 involved in marine natural products research have become somewhat disillusioned by a paucity of practical results
(compared to overly optimistic initial expectations), the development of a new drug requires a large investment of
26 capital and years of development . Given time, it seems likely that the marine environment will eventually yield its own "penicillin".
" For the cynic questioning the biomedical value of the sea, it 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 terrestrial resources for drugs. What is required is that more investigators, especially from the drug industry, take a dip in the oceans to seek the 17 pearls awaiting discovery."
C. PRIMARY AND SECONDARY METABOLITES
Historically the organic compounds found in living organisms were divided into two categories. Primary metabolites were defined as compounds made by an organism primarly to sustain life. ' The compounds, and the biosynthetic pathways used to generate them, were designated as common to many, if not all, living organisms. Secondary metabolites, on the other hand, were deemed not essential to the basic protoplasmic metabolism of the organism and were thus initially considered to be endproducts (waste or stor• age) of metabolism. 9
It is likely that the above historical definition of a secondary metabolite will fall into disuse. It is 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))27 and of the Mucorales (the trisporic acids 1J) and 11)2 8.
The notion that secondary metabolites are endproducts of metabolism has turned out to be a gross oversimplification.
The field of ecological biochemistry (chemical ecology) has grown rapidly over the last 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
29 metabolites. Campbell has put forward a more useful defi• nition based on the extent of taxonomic distribution of a particular compound. It states that:
Plant, fungal, bacterial (and animal) cells are
composed of two distinguishable types of molecules:
a) materials that are widely distributed in nature,
being found at least in all families in an order, often
in all orders of a class or in all classes in a phylum,
and in some instances, in all phyla in a kingdom and in
all five kingdoms; and
b) materials that occur uniquely in a single strain
or species, that are found in two or more closely
related members of a single genus, or that are found
sporadically in a limited number of evolutionarily
unrelated species in different genera, families, orders,
•classes, phyla or kingdoms.
Cell constituents of type (a) are primary
metabolites, while cell constituents of type (b) are
secondary metabolites. [N.B. Enzymes and nucleic acids,
though species-specific in distribution, are normally
excused from the primary/secondary metabolism
* Despite these advances, many secondary metabolites still have no known function. This may be due to a lack of suit• able experimentation or because the function is too complex to discern given the current state of knowledge. 11
discussion].
This thesis discusses secondary metabolites isolated from selected British 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 likely that the compounds isolated from the nudibranchs are involved in their relative immunity from predation, whereas the bryozoan metabolites may impede surface fouling and overgrowth.
D. CHEMICAL ECOLOGY
Reseacch over the last fifteen years has led to the de• velopment of a new interdisciplinary subject; chemical
30 ecology . Studies in chemical ecology have for the first time provided a rational and satisfying explanation for the roles of at least a part of the enormous proliferation 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 is considered by itself, without reference to other organisms, there is no evident reason why the organism
31 should produce them (secondary metabolites) .
There are two types of ecological interactions mediated by secondary metabolites; alleochemic effects
32 (interspecific) and intraspecific chemical effects .
Alleochemic effects are subdivided into allomones 1 2
(adaptative advantage to the producer) and kairomones
(adaptative advantage to the receiver) while intraspecific chemical effects are subdivided into autotoxins, autoinhibitors, 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 (thiols 12, 13, and disulfide 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 literature has been summarized by Harborne in his excellent book "Introduction
2 to Ecological Biochemistry" .
CH3CH=CHCH2SH 0 12 Me 0
(CH3)2CHCH2CH2SH 0 13 Me
CH3CH=CHCH2SSCH3 15 14 13
Chemical ecology in the marine environment is still in its infancy. A recent chapter by Barbier introduced,
33 summarized, and analyzed this current state of affairs .
Many compounds have been shown to be biologically active against selected screening organisms or cell lines, but little is known about the functions of the metabolites in their natural environment. This is primarly due to the difficulty 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 specific sponge upon which it feeds
(Hymeni aci don sp.) a substance that is apparently lethal to 34 fish and crustaceans . 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 specific metabolites from its diet. Subsequent to this discovery, a number of other metabolites isolated from dorid skin secretions have been shown to inhibit feeding by fish (using a standard bioassay) at concentrations of 10 ug metabolite per mg of food
35 36 pellet ' . The demonstrated antifeedant activity would appear to be ecologically significant*.
37 * Recently Gerhart demonstrated that prostaglandin A is an agent of chemical defense in the Caribbean gorgonian2 Plexaura homomalla. 1 4
The literature on nudibranch chemistry has been
1 36 38 39 extensively reviewed ' ' , and recently Faulkner has proposed that in the evolution of dorid nudibranchs, loss of the shell is correlated with the presence of defensive mechanisms based upon chemicals derived from food. Although this theory is appealing, the limited ecological studies which have been performed to date are insufficient to prove that a particular metabolite is solely 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 fish to immediately flee; squeezing L. magnifica into an aquarium containing fish 0 causes poisoning and death of the fish within minutes' .
The purified toxins were therefore assumed to play a 1 5
1 defensive role in the sponge" . It is interesting to note that the purified toxins, Latrunculins A (18) and B (19), exhibit effects on cultured mouse neuroblastoma and
42 fibroblast cells . In both cell types, submicromolar toxin concentrations (as low as 50 ng/mL) rapidly induce changes in cell morphology that are reversible upon removal of the toxin. The significance of these observations are unknown.
The above examples illustrate that compounds isolated from marine organisms show profound biological activities, which in many instances are advantageous to the producing organism. The promising pharmacological activities 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 field of research. II. NUDIBRANCHS
"Many nudibranchs, but especially the dorids, have a penetrating fruity odour that is pleasant when mild but nauseating when concentrated. Undoubtedly, this odour is one of the reasons why nudibranchs seem to 3 be let strictly alone by predatory animals
A. INTRODUCTION
1. GASTROPOD SECONDARY METABOLITES
Nudibranchs are members of the phylum Mollusca (see
Figure 1'"), a phylum that contains an estimated 75,000 living and 35,000 fossil species. This large phylum, second in size only to the phylum Arthropoda, is subdivided into seven classes. Of the seven molluscan classes, the
Gastropoda has been studied in the greatest detail for
5 natural products chemistry" .
Gastropod molluscs of the subclass Opisthobranchia are characterized by a greatly reduced, or completely absent shell. In spite of this lack of physical protection,
6 opisthobranchs have few known predators* . To account for the observed relief from predation, early investigators speculated that opisthobranchs may utilize a chemical
6 7 defense mechanism" '* . Chemical studies on opisthobranchs
8 9 were initiated on the large herbivorous aplysiomorphs" '" .
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
1 1 BULLOMORPHA APLYSIAMORPHA PLEUROBRA1SJCHOMORPH A PTEROPODA
ORDER
SACOGLASSA NUDIBRANCHIA PYRAMIDELLA
AEOLIDACEA ARMINACEA DENDRONOTACEA DORIDACEA SUBORDER
Figure 1. Phylogenetic classification of nudibranchs. N.B. Organisms classified according to Behrens'*. 18 variety of secondary metabolites some of which were toxic to
50 potential fish predators . Further investigations demonstrated that the metabolites were likely being
51 concentrated from the animals' algal diets . 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
52 53 potential. Aplysistatin (2T)) and dolatriol (21) showed
5 antileukemia activity while dactylyne (22) * produced a dose dependent prolongation of phenobarbital-induced hypnosis in animals by inhibiting the metabolism of phenobarbital. By
55 itself, enine 22 had no effect . Encouraged by the success on aplysiomorphs, marine chemists turned their attention to smaller opisthobranch molluscs, specifically the carnivorous nudibranchs.
Nudibranchs have a body which incorporates the visceral mass, mantle, and foot, and is externally bilaterally symmetrical with a slug-like flattened form (see Figure 2).
They completely lack a shell 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. All 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
56 Aeolidacea) are primarly associated with coelenterates .
Nudibranchs are hermaphroditic, they possess active sex organs of both sexes simultaneously. This may be an evolutionary adaptation as it allows for greater probability of finding a mate since every individual of the same species is an eligible partner. Little is known about how nudibranchs find and recognize each other. It is possible that some of the compounds isolated from nudibranch skin
57 secretions may act as recognition or sex pheromones . The rhinophores, which are chemosensory organs on the
56 nudibranchs' head, could act as the pheromone receptors . 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 rich and rewarding area.
Numerous novel secondary metabolites have been isolated by groups working in California, Hawaii, Italy and British
Columbia. Most of the compounds isolated have been sesquiterpenoid aldehydes, furans, or isonitriles, although mono-, di-, and triterpenoids, steroids, alkaloids,
58 acetylenes, and purine ribosides are also represented .
Several Gastropods that still retain a visible external shell 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
46 occurring in nudibranchs . 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
39 shell , it is not surprising that interesting secondary metabolites have been isolated from nudibranchs' shelled cousins. Certain pulmonates (subclass Pulmonata) are a rich source of unusual secondary metabolites with interesting
59 biological activities . 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
60 interesting, biologically active, nitrogenous compounds .
2. NUDIBRANCH DEFENSE MECHANISMS
In order to deter predators (fishes, starfish, crustaceans, and other opisthobranchs are known to prey on
56 61 nudibranchs ' ) it has been suggested that these oft-times 22 highly conspicuous, slow moving, soft bodied animals utilize
61 a bimodal defensive strategy . The primary level of defense (useful only against visual predators) is either to evade detection (crypsis) or once having been detected, to discourage the predator from initiating an attack. In other
words, in the latter, nudibranchs utilize aposematism; the
defensive adaptation in which an animal blatantly
advertizes, using conspicuous warning colouration, that it
is not a suitable food source. The role of colouration in
62 6 nudibranch defense has been debated for many years '" . It
now appears probable that most nudibranchs are cryptically
or disruptively coloured (the animal has a pattern that
breaks up its body outline on its usual substratum), while
63 some well-defended (distasteful) species are aposematic .
More work is- required before the complex nature of
39 nudibranch colouration is fully understood .
The second level of defense, useful in the event of
first level failure, or in cases of predators that hunt
57 chemically (by following mucous trails for example )
involves adaptations that may be behavioral, morphological,
61 or chemical in nature . Nematocysts, spicules, and
behavioral responses have all been implicated, to varying
degrees, in nudibranch defense. The bizarre practice of
storing unfired nematocysts (stinging cells), obtained from
their coelenterate diet, characterizes many aeolid
6 nudibranchs ". 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 their immunity from predators. Not all nudibranchs feed on coelenterates
(dorids feed exclusively on sponges, bryozoans and tunicates), consequently nematocysts are unimportant in the defense of these species.
A few -dorid species have hard calcareous spicules contained within their mantles. Obtained from their sponge diets, they give the nudibranch a rigid shape and provide some protection from predators. A voracious nudibranch predator, the opisthobranch Navoanax inermis, does not eat spicule-containing dorids, while readily consuming
65 non-spiculose ones . Todd has suggested that the high ash content (most of which is attributable to spicules) and low calorific content of some dorids makes these nudibranchs of
66 poor nutritional value to predators .
Many nudibranchs will swim in response to being disturbed or threatened by a predator, and this may be con•
67 sidered the most basic behavioral defense mechanism .
Nudibranchs with large cerata (finger-like respiratory and digestive structures occurring in groups of parallel series along the dorsum, suborders Dendronotacea, Aeolidacea and
Arminacea) tend to autotomize the cerata at only the slight• 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
6 the epidermis* from which some dorids secrete sulfuric acid 24
68 when disturbed . Species of all four nudibranch suborders are known to secrete non-acid noxious substances under simi• lar conditions. Johannes found the dorid Phyllidia varicosa 7 secreted a toxin that was lethal to fish and crustaceans* and subsequent chemical analysis of this secretion showed it to contain 9-isocyanopupukeanane (1£) as previously
38 discussed. 'Recent studies have shown 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 likely these metabolites were
36 coming from the secretory glands, not the guts , and a num•
ber of these metabolites exhibited fish antifeedant activity.
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 fish antifeedant activities, one
may hypothesize that defensive secretions make an important
contribution to nudibranch defense. Little is known about
the relative importance of any specific nudibranch defense
mechanism and it seems a nudibranch will utilize a
combination of different defensive strategies to avoid being
eaten. More studies are needed to determine the relative
importance of the different defensive mechanisms and to
identify the predators that provided the selective pressures
56 leading to their development . 25
B. SECONDARY METABOLITES FROM THE DORID NUDIBRANCH ALDISA
COOPERI (ROBILLIARD AND BABA, 1972)
1. INTRODUCTION
Aldisa cooperi (Aldisa sanguinea cooperi69, see Figure 3) is an orange dorid (suborder Doridacea, family Aldisidae) usually found deeply embedded in its preferred prey, the
0 reddish orange sponge Ant hoarcuat a graceae7 . The association is likely cryptic to potential predators, even
though SCUBA divers can, from a distance, easily locate the
nudibranch on the sponge. A. cooperi may grow to 20 - 25 mm in length and has been reported from Washington, British
Columbia, and Japan. Unlike many dorids collected from
Barkley Sound, B.C., A. cooperi did not have a detectable odour.
2. ISOLATION AND STRUCTURE ELUCIDATION
The nudibranchs were collected 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 filtered to give an aqueous
methanolic suspension. The suspension was partitioned be•
tween brine and ethyl acetate, and the ethyl acetate soluble
material was fractionated by flash chromatography.
Chromatographically similar fractions were pooled to give a Figure 3. Aldisa cooperi. Photographer: Ron Long, Simon Fraser University, 27
7 1 mixture of steroidal acids !23 and 24 (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 steroidal acid fraction was further purified by
preparative thin layer chromatography (preparative TLC) to
give a white crystalline solid. It was apparent from the
relative intensities of the peaks in the olefinic region of
the 'H NMR spectrum of this material that a mixture of 28 closely related compounds had been obtained. Additional purification by reverse-phase preparative TLC yielded
3-oxo-4-cholenoic acid 2_3 and its 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 C «H 0 2 36 3
(HRMS, m/z observed 372.2658, required 372.2664) that demanded seven units of unsaturation. Methyl resonances in
1 the H 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 steroid. The chemical shift of the methyl
singlets, the presence of a one proton singlet at 8 5.74
ppm, an ultraviolet (UV) absorption at X - 236 nm m ax (e 6,200), and a positive 2,4-dinitrophenylhydrazone test
suggested a A*-3-ketosteroid nucleus of the type shown in
26.
1 Comparison of the H NMR chemical shifts of the methyl
singlets and the olefinic 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 (CH CO) in the 2
mass spectrum of 23_ to give a peak at m/z 330.2564 (31%). It is well known that molecules that incorporate
substructure 26 lose ketene via a process which may be
*It was also found that the mixture could be readily purified by normal phase HPLC of the methyl esters. 29
26
1 Table 1. Comparison of the H NMR data for various A'-3-ketosteroids (CDC1 ) 3
chemical shift, 5
H on C# 23° 24° 21b
4 5.74 5.74 5.74 18 0.74 0.77 0.72 19 1.21 1.20 1.19
a b 400 MHz. 80 MHz.
72 visualized as shown in Scheme 1a . Other fragment ions
73 diagnostic for A"-3-ketosteroids were also observed at m/z 287.2028 (11%), 249.1861 (38%), and 124.0897 (96%) (see
Scheme 1b).
The structure of the C side chain substituent was 17 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. ii) Absence of additional methyl resonances, in addition to those assigned to the C-18 and C-19 carbons of the steroidal nucleus, indi• cated the side chain was not branched and must incorporate a
1 carboxyl group at C-24 (IR 1700 cm" and formation of methyl ester 28 upon treatment with diazomethane). iii) In the mass spectrum of 23, a significant fragment ion was detected at 271.2047 (required for C H 0, 271.2062) which m/z 19 27 corresponded to a loss of the 1-methyl-4-butanoic acid side chain from 23 as indicated in Scheme 1b.
The steroidal acid, 3-oxo-4,22-choladienic acid (24) ,
occurred as the minor component of the binary steroidal acid 32 mixture in a varying ratio depending upon the collection.
The mass spectrum of 24 indicated a molecular formula
C 0 (HRMS, observed 370.2507, required 370.2508), 2«H3fl 3 m/z therefore 24 differed from the major steroid 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) dictated 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
1 vicinal coupling constant in the H NMR spectrum. The HRMS of 24 showed an abundant ion at m/z 271.2058 (70%, required for C H 0, 271.2062) arising from the allylic 19 27 fragmentation of the 1-methyl-4-but-2-enoic acid side chain substituent in 24 (Scheme 2).
Due to the stereospecificity of the enzymes involved in steroid biosynthesis one would expect that 3-oxo-4-cholenoic acid 23 and its 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 terrestial and aquatic organisms that steroids are biosynthesized from an initial cyclization
7 of S-squalene 2,3-oxide *. In animals and fungi, lanosterol
(30) is the initial intermediate formed, while plants and algae initially produce cycloartenol (31). The 34 intermediates 30 and 3_1 are then transformed biosynthetically into the steroids or phytosteroids, respec• tively. 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 bile acids, is an example. It was postulated from biogenetic reasoning
1 and supported by H NMR comparisons to cholestenone (27)
(Table 1), that the absolute stereochemistries of the ABCD ring systems in 23 and 2_4 were as shown.
13 C NMR has been shown to be an excellent method for comparing the relative stereochemistries of similar mole•
13 cules. The large data base for C NMR spectra of steroids
13 allowed comparison of the C NMR spectra of steroidal acid
23 to cholestenone (27) and methyl 3-oxo-4-cholenoate (28,
75 prepared from 23^) to methyl 5/3-cholan-24-oate (3_3) . 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 steroidal 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 identical relative stereochemistries and allowed the assignment of stereochemistry at C-20 as R, the regular steroidal absolute stereochemistry. 35
Further spectral evidence supporting the 20R configuration for 2_3 and 24 was available from the
literature. A number of steroids isolated from the marine environment have the "un-natural" 20S configuration as shown
76 77 in Figure 5 ' . 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
1 acetate esters of each compound. Comparison of the H NMR
spectra of the epimeric pairs showed that the C-21 methyl 36
13 Table 2. C NMR data and spectral comparisons for the assignment of stereochemistry to 3-oxo-4-cholenoic acid (23)
chemical shift, 6
fl Carbon # 27 28 33* 23
C rf e (CDC1 ) (CDCl ) (CD Cl ) (CDC1 )/ 3 3 2 2 3
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 30.8 h .2 hh 23 23.8 31 .\ 31 . 1 30.8 24 39.6? 1 74.6 175.0 25 27.6 26 22.5 27 22.8
-OCH 51.5 51 .3 3
a From reference 75, compound 207. " From reference 75, compound 256. c Spectrometer frequency not given. d 100.6 h MHz. e 20.1 MHz. f 20.1 MHz. ?' Assignments may be switched. ' Not observed due to slow relaxation. 37
Figure 5. "Un-natural" 20S marine sterols.
resonance was shifted = 0.1 ppm to higher field in the 20S compounds. Careful examination of the chemical shifts 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 rationalize 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 preferentially 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 left (cis
relative to C-13), as depicted in 39. Although an argument against a preferred conformation in 20R steroids had been
raised, the large differences in the observed GC mobilities
of the C-20 epimers suggested the side chain of one or both
of the C-20 epimers exists in a preferred rotational
conformation. Vanderah and Djerassi felt that sterols,
epimeric at C20 but of identical conformer composition
should exhibit identical gas chromatographic behavior.
Clearly, more work is required to settle this subtle, yet
interesting, conformational question.
0
78 Both acids 23 and 2479 are known synthetic compounds
and the major acid 23 has also appeared in the literature as 39
1 Table 3. H NMR data for various 20R and 20S steroids (CDC1 ) 3
Compound 40 41 28 42 43 3_8
G fl fl 6 Chemical shift 0.84 0.94 0.93b 1.00 1.10° 1.10 of C-21, 6
a 100 MHz, see reference 76 b 400 Mz
V v 40 R= ^ ^0CH3
,—'
the result of microbial transformation of other precursor
80 steroids . The physical data for both compounds are in agreement with the literature values. Neither acid has been previously reported from natural sources, and they represent the first isolation of bile acids from a marine 40 invertebrate.
In order to establish a dietary origin for the acids 23_ and 24, a methanolic extract of Ant hoarcuat a graceae was examined (see experimental for details). A. cooperi was usually found associated with A. graceae and appears to derive its pigmentation from it. The sponge contained neither 23 or 24 but it 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: C H and C sHu ) , have 29 <,0O4 2 «Oi, 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.
OAc AcO
44
The third metabolite isolated from A. cooperi,
1-0-hexadecyl-glycerol (25), was characterized as its diacetyl derivative C Hj 0 (HRMS, observed 44: 23 Ml 5 m/z 400.3141, required 400.3189). Isolation as the diacetyl derivative greatly facilitated both the purification and structural assignment. The mass spectrum of 44 showed peaks assignable by the fragmentation pathway outlined in Scheme 3 41
1 and allowed assignment of the ether linkage to C-3. The H
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 functionality.
357 (0.56%)
327 (2%) I ACO-/
P^OCH2(CH2)uCH3 255 (29%)
Scheme 3. Interpretation of the MS of diacetyl derivative 44
Glycerol ether 25 is a known natural product, commonly referred to as chimyl alcohol. It has been shown to slight• ly inhibit the progress of the murine Ehrlich ascites
81 tumor . Subsequent to its isolation from A. cooperi, 2_5 was isolated in our laboratory from the dorid nudibranch
8 2 Arc hi dor is mont ereyensi 5 , and was shown to have potent in vitro antibiotic activity against Staphylococcus aureus and
Bacillus subtil is. Interestingly, 2_5 was also shown to have 42
1 antifeedant activity (18 ug mg~ of food pellet) against the tide pool sculpin Oligocottus maculosus.
3. BIOLOGICAL ACTIVITIES OF ALDISA COOPERI METABOLITES Because the steroidal acids 23 and 24 were present in such high concentration relative to the animals body weight, it seemed reasonable to assume that they were involved in the chemical defense of the nudibranch. To gain some evidence in support of this hypothesis, a standard goldfish
36 bioassay was used to test the antifeedant properties of the steroidal acid 23. The results showed that 23 effec• tively inhibited feeding at < 15 ug/mg of food pellet, while cholestenone (2J_) was totally inactive at > 100 ug/mg of food pellet. The nudibranch was apparently obtaining an inactive metabolite from its diet and chemically modifying it to produce an active antifeedant. Verification of this supposition would require injection of A. cooperi with radiolabelled cholestenone followed by isolation of radioactive steroidal acids. It should be stated that, al• though the steroidal acids were not isolated from the sponge, the possibility exists that they were present in very small amounts and were not detected by the isolation procedure. This possibility 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 collection of
A. cooperi made in the Queen Charlotte Islands, B.C., gave 43 steroidal acids 23 and 24 in a ratio similar to that found,
in the Barkley Sound collections. This finding supported the hypothesis that acids 2_3 and 2_4 are produced de novo by
A. cooperi.
4. DISCUSSION
Biogenetically, one can envisage the steroidal acids being produced from cholestenone via a series of conventional fatty 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 is
83 an intermediate . The evidence suggested the degradation pathway shown in Scheme 4. The conversion of sitosterol
(45) and campesterol (46) into andro.st-4-ene-3,17-dione (47 )
has also been studied and both of these compounds are also
8 0 degraded ( Mycobact eri urn sp.) via the steroidal acid 23 . Thus, it is not unreasonable to believe that the nudibranch
ingests cholestenone (and related steroidal ketones) from
its sponge diet, and modifies them to the steroidal acids 23_ and 24, via a mechanism similar to that shown in Scheme 4.
These steroidal acids might then be useful as defensive
allomones.
The use of steroids for chemical defense is not limited
to A. cooperi. The great diving beetle (Dytiscus
marginal is) utilizes an array of steroidal ketones to ward off potential predators. The major component of the mixture
8 was shown to be 11-deoxycorticosterone (Cortexone) (48) ". 44
47
Scheme 4. Proposed mechanism for the microbial 83 transformation of cholesterol into 17-ketosteroids .
This substance has the same stunning effect on fish as the natural secretion. Numerous other water beetles produce the 45 same or related compounds. Frogs and toads utilize steroidal skin toxins (for example: bufotalin (49)), the skin of the salamander contains defensive secretions (for
8 5 example: samandarin (50_)) , and sea cucumbers synthesize
86 halothurin , a neurotoxin of steroidal structure and use it to deter their fish predators. Finially, 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 a61 while Adalaria sp. produces a mixture of steroidal 88 peroxides of which 5a, 5a-epidioxysteroid 5_2 is an example .
From these examples it can be seen that steroids are a group of structurally similar compounds that exhibit a wide variety of biological activities, depending on oft-times subtle molecular changes.
46 R = H 46 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 is not a predator of sessile bottom dwelling animals, rather, it feeds upon zooplankton by majestically
89 sweeping the sea with its large oral hood .
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
91 predators and were initiated after a fortuitous
92 observation that allowed collection of enough animals for
chemical analysis. Specimens of M. leonina were collected 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
2 high (= 50 animals/m ) and their odour could be detected in
situ by SCUBA divers.
2. ISOLATION AND STRUCTURE ELUCIDATION
Freshly collected whole specimens (38 animals) were im•
mediately immersed in chloroform and extracted (wrist action
shaker) for 1.5 hours. The chloroform soluble material was
filtered, dried over sodium sulfate, and concentrated in
49 vacuo to give an orange "grapefruit" smelling oil (57.4 mg; 1.51 mg/animal). NMR analysis of the crude oil 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 relative ratio of 3.3:1* that was contaminated with a small amount of fat. The individual components were subsequently resolved by silica 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 C H 0 (M+, 140), 9 16 m/z 13 verified by the appearance of 9 carbons in the C NMR
1 spectrum. A H NMR spectrum of this 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 relative ratio 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 olefinic proton at 6 5.10 (m), and for an aldehyde proton at 9.62 (d, / = 1.9 Hz, 1H) ppm. The aldehyde functionality also displayed an IR absorption at 1723 cm"
13 and a C 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 acyclic degraded monoterpenoid
13 skeleton for 5_3. Comparison of the C NMR spectrum of 53_ to that of citronellal (5_5) allowed the total assignment of
13 1 the C NMR resonances (Table 4). The 400 MHz H NMR spectrum of aldehyde 5_3 was well resolved and allowed assignment of all the protons in the molecule, the chemical shifts and assignments are listed in the experimental
(Chapter 4).
All 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
13 Table 4. C NMR data for 2,6-Dimethyl-5-heptenal (53) and 93 Citronellal (55) .
2,6-Dimethyl-5-heptenal (53) Citronellal (55)
Carbon # Chemical Carbon # Chemical shift, 6° shift, 8b c c (mult) (mult)
1 202.2 (d) 1 205.0 (d) 2 51 .0 (t) 2 45.9 (d) 3 27.8 (d) 3 30.7 (t) 4 37.0 (t) 4 25.4 (t) 5 25.4 (t) 5 1 23. 5 (d) 6 124. 1 (d) 6 132.7 (s) 7 131.5 (s) 7 25.7 (q) 8 25.6 (q) 8 17.7 (q) 9 17.6 (q) 9 13.3 (q) 10 19.8 (q)
G c 100.6 MHz. b25 MHz. Multiplicity, determined from a single frequency off resonance (SFORD) experiment.
natural product, from numerous sources. Identifications generally have been made solely on the basis of GC-MS analysis. Aldehyde 5_3 has been reported as a pheromonal component of the ant Lasius carniolicus, originating
9 specifically from the insects' head ", as a chemical 52 component of the essential oil from the leaves of Eucalyptus
95 96 sp. and Carphephorous corymbosus , and as a component
97 extracted from Jasminum sambac flowers . Interestingly, a related secondary metabolite,
2,6-dimethyl-1,5-heptadien-3-ol acetate (56), is an insect
98 sex phermone (Ps eudococcus comsl ocki ) . 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
99 year basis .
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 C H 0 (HRMS, observed 156.1151, 9 16 2 m/z required 156.1151) and its 'H NMR spectrum indicated that it
was closely related to aldehyde 5_3. IR absorption bands
-1 (3500 - 2200 and 1700 cm ) characteristic 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 full 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* it is
100 a known synthetic compound .
3. DISCUSSION To the best of the author's knowledge, isolation of
aldehyde 5_3 and acid 54 represent only the second examples of isolation 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 attracting 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 algal diets. The pleasant smelling sponge Plakortis zygompha produces (possibly by symbiotic biogenesis) two degraded monoterpenes 58 and 5jJ, neither of
101 which contain halogens . The biological 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 relief from predation.
Much like the tannins isolated from terrestrial trees and shrubs, perhaps the halogenated monoterpenoids are a general level defensive mechanism to which only specialists (sea hares) have adapted.
In view of the postulated defensive role for the
91 odiferous compound , we tested 53 and 54 for antifeedant
36 activity in a standard goldfish bioassay . The carboxylic acid 54 showed no activity at 100 wg/(mg of food pellet), while the aldehyde 5_3 was too volatile for reliable testing.
It would be an interesting biological project to synthesize racemic 2,6-dimethyl-5-heptenal (53) and evaluate its toxicity and antifeedant activity against a variety of potential nudibranch predators.
It is quite possible that either aldehyde 5_3, acid 54, or the naturally occurring mixture act as M. leonina aggregation or sex pheromones. M. leonina is known to ag• gregate in great numbers and to then suddenly completely 55
91 disappear, only to reappear weeks or months later . It is tempting to suggest that the major lypophilic compounds isolated from the skin extracts of this animal are involved in the dynamic population jumps observed. More collaboration with marine biologists will 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 initiated by Jocelyn T.
102 Hellou . 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 isolation of luteone (60)
103 from Cadii na Iuteomargi nata .
Hellou had shown that the odouriferous principle could be effectively extracted from the whole animals by immersing the specimens in methanol immediately after collection, soaking at room temperature for three days, and then decanting the supernatant. The supernatant was evaporated
to give a concentrated MeOH-H 0 solution that was in vacuo 2 partitioned between brine and chloroform. The orange
58 organic phase was dried over anhydrous sodium sulfate and evaporated to give a sweet smelling oily residue.
Silica thin layer chromatography (TLC) analysis of the crude extract showed the presence of a non-polar metabolite,
10 designated as nanaimoal ', that was readily purified by
1 preparative TLC. The H NMR spectrum of the purified odouriferou's oil 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 crystalline compound could be found for structural 59 elucidation by single crystal X-ray diffraction analysis.
Of the eight derivatives prepared, none provided suitable crystals. At this point Ms. Hellou wrote up her preliminary studies on A. nanaimoensis and ceased work on 102 the project .
Subsequent work on A. nanaimoensis^05'^06 has shown that the TLC spot corresponding to nanaimoal is a mixture of
five closely 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 collection (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 oil that contained the charac•
teristic nudibranch fragrance. Analytical TLC showed the
presence of a non-polar spot 0.25; 50% Hexane/CHCl ) (Rj- 3 that charred light purple and gave a positive test with
2,4-dinitrophenylhydrazine. In addition to this spot, three
other non-polar and one polar spots were also present.
These were subsequently shown to be fats and sterols.
* 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 origin in all the TLC plates run (brown char, possibly fatty acids) was not investigated further.
Column chromatography (silica gel) of the crude extract yielded 218.6 mg (1.8 mg/animal) of material corresponding
1 to the fragrant aldehyde fraction [ H NMR 6 9.84 (t, / = 3
Hz); 6 9.72 (t, / = 3 Hz) ppm]. Gas chromatographic (GC) analysis, 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 liquid chromatography (HPLC). For large scale purification HPLC was found to be the most con• venient .
Figure 10. GC analysis of a crude chloroform extract of A. nanai mo ens is.
Nanaimoal had a molecular formula of Ci H «0 (6_1) 5 2
(HRMS, m/z observed 220.1836, required 220.1827). The infrared (IR) spectrum of 61_ showed aldehydic C-H and C=0 1 stretching bands at 2750 and 1710 cm" respectively. The assignment of the lone oxygen in nanaimoal to an aldehydic carbonyl was supported by a resonance at 6 203.3 (d) ppm in
13 its C NMR spectrum. Partial structure 66 was inferred 1 from an ABX spin system in the H 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 addition, an intense 62 fragment ion at m/z 176 in the mass spectrum of nanaimoal, resulting from loss of ethanal via a McLafferty rearrangement, supported this assignment (Scheme 6).
Scheme 6. Interpretation of the MS of nanaimoal (61)
1 The H NMR spectrum of nanaimoal also contained resonances for a gem-dimethyl at 6 0.98 (s, 6H) (IR 1395 and
1 1380 cm" ) (substructure 67), an isolated tertiary methyl at 1 Figure 11. 400 MHz H NMR spectrum of nanaimoal (61) in CDC1 . a) entire spectrum, b) expansion of the aliphatic region3 , c) expansion of the aldehydic proton resonance.
CO 64
1.05 (s, 3H) (substructure 6_8), an isolated allylic AB spin
system at 5 1.77 (d, / = 17.3 Hz, 1H) and 1.85 (d, / = 17.3
Hz, 1H) (substructure 6_9) , four allylic protons at 1.81 (m,
2H) and 2.02 (m, 2H) ppm, and a six proton aliphatic
13 multiplet situated between 8 1.41 and 1.66 ppm. The C NMR
spectrum of aldehyde 61 contained resonances at 6 133.8 and
123.3 (both- singlets in a SFORD experiment) appropriate for a tetrasubstituted olefin.
The spectral data indicated that nanaimoal was a
bicyclic sesquiterpenoid that contained three tertiary methyl groups, an ethanal side chain, a tetrasubstituted
olefin, and six allylic protons of which two comprised an
isolated AB spin system. The presence of six allylic
protons ruled out 62 as a possible structure for nanaimoal.
The remaining structural features to be defined were the po•
sitions of the three additional aliphatic methylene carbons,
the size of the bicyclic 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
122 (C H ) which suggested fragmentation involving the m/z 9 13 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 NaBH reduction) and u
allowed measurement of its exact mass (HRMS, m/z observed 121.1016, required for C H , 121.1017). It was concluded 9 13 from this 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, differentiating between the four possible structures for nanaimoal proved challenging. 'H NMR spectroscopy was useful in eliminating structures 7_2 and 73. Particularly interesting however, was the inherent spectroscopic similarity between 61 and 63_. It was possi• ble, at this 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 possibility that nanaimoal was an acetogenin remote) only structures JS1 and 6_3
107 fit the biogenetic isoprene rule , therefore non-biogenetic isoprenoid compounds T2 and 7_3 could be eliminated from consideration on this basis.
Numerous sesquiterpenoid metabolites have been dis•
106 covered that violate the isoprene rule [see for example; isoacanthodoral (6j>) and 9-isocyano- pupukeanane (16)], however, on closer inspection these metabolites can usually be rationalized as being formed from a biogenetic isoprenoid precurser, namely farnesyl pyrophosphate, and therefore obey, by definition, the biogenetic isoprene rule.
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 first cyclization step in the formation
of both compounds. Structure 61 could be rationally
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 pleraplysillin-1 (7_4) (Scheme 7b). The
proposed second cyclization reaction in the
formation of structure 61 is analogous to the
109 formation of ring C in the pimerane diterpenes .
Biogenetically, structure 61 was the most likely 67 structure for nanaimoal. The large number of naturally occurring monocyclofarnesane
sesquiterpenoids occurring in both terrestrial and marine organisms vs only one example of a metabolite related to the pleraplysillin-1 skeleton supported
this contention.
74
Scheme 7. Biogenetic arguments used in support of structure 61 for nanaimoal. 68
1 2. H NMR Double-resonance experiments.
1 In the H NMR of nanaimoal, selective
irradiation of the allylic methylene protons at 6
2.02 (see Figure 11) in a double-resonance
experiment caused a simplification of the broad
geminal allylic 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 allylic methylenes, in
the analogous spin systems, was predicted to be
negligible.
3. Chemical shift of the gem-dimethyl group.
The chemical shift, 6 0.98 (s, 6H) ppm, and
chemical shift equivalence of the gem-dimethyl group
in nanaimoal (6_1) (substructure 6_7) indicated this functionality was attached to the tetrasubstituted
carbon-carbon double bond. Comparison to model
systems supported this assignment (Figure 12). The
chemical shift assignment of the gem-dimethyl
functionality was supported by the observation that
upon reduction to nanaimool (7J3), the chemical
shifts 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 shift of the third quaternary methyl
(substructure 68) was substantially affected (moved upfield by 6 0.17 ppm).
Unfortunately no reasonable model system could
be found for the chemical shifts of a gem-dimethyl
group in a structure of type 6_3 that would reinforce
this argument.
4. Reduction of the aldehydic carbonyl.
Reduction of the aldehydic carbonyl affected
the chemical shift of the quaternary methyl, as
mentioned above, as well as causing an upfield shift
(average + 0.14 ppm) of the isolated allylic
methylene protons (substructure 69). This result
indicated a close spatial relationship between the
carbonyl and the isolated allylic methylene protons.
Of the structures remaining under consideration,
namely 6_1 and 63_, only 61 could account for such an
ef fect. 70
6 1.00 (s, 6H)
1 Figure 12. Model systems for the H NMR chemical shifts 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 likely structure, compound 61, was in order. The urethane 75, a derivative of nanaimoal, was chosen as a synthetic target since this derivative had already been prepared in an attempt to generate a suitable crystalline derivative for
X-ray analysis.
H
75
The most likely structure for nanaimoal (6_1) had a relatively simple carbon skeleton, therefore retrosynthetic analysis provided a straightforward synthetic strategy
(Scheme 8). Antithetic 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 facile in the conversion of triene 7_7 to diene
110 78 (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 cyclization precurser. The problem was thus reduced to finding the appropriate dienophile.
Methyl methacrylate (81L) seemed a logical choice for the dienophile initially. However, examination of the 72
Scheme 8. Retrosynthetic analysis of the postulated structure for nanaimoal.
111 literature (as well as theoretical considerations) revealed that this reaction would likely provide a good yield of the wrong regioisomer. If the correct regioisomer
112 could be obtained , the neopentyl nature of the ester carbonyl would pose a problem in any reactions designed to
113 extend the side chain by the required one carbon . These 73 two factors ruled out 81 as a suitable dienophile.
Scheme 9. Previous synthesis of the nanaimoane carbon skeleton.
Isoprene (B2) had been shown to react with myrcene, generating a mixture of regioisomers 7J7 and 83 in a ratio of 1 1 0 2:3 , in low yield*. As discussed above, cyclization of a 77 generated diene 7_B.' compound having the required carbon skeleton of structure 6_1. To finish a synthesis via this route, selective hydroboration of the monosubstituted olefin would be required to generate alcohol JSL' oxidation of which should provide (i)-aldehyde 6_1. This overall reaction se• quence suffers,, frorn one serious drawback, a plethora of products were shown (and were expected) to be formed upon
110 reaction of myrcene and isoprene . Although it would be relatively easy to vacuum distill the mixture and obtain a somewhat purified mixture of olefins 11_ and 8_3, it was
* It was realized from the outset that unambiguous synthesis of structure 61 or its derivative was the goal of the synthesis, therefore high yield was not an absolute requirement. 74 reasoned that a readily available dienophile such as
3-methyl-3-buten-1 -ol (8J)) would give a cleaner reaction product that could be easily purified by flash chromatography. The alcohol 80 could be used in large ex• cess to suppress the appearance of products arising from the dimerization of myrcene. It was hoped that 80 would give a
ratio of regioisomeric products 7_6 and 84 similar to that obtained with isoprene as the dienophile, that is 2:3.
11 In the event *, the Diels-Alder reaction (225 °C
sealed tube, 8 h, neat, 4:1 alcohol:myrcene) proceeded in a
very low but adequate yield (polymerization of starting materials was the major side reaction) to generate, in one
step after purification by flash chromatography, a mixture
of regioisomers that occurred in a ratio of 2:3 (76:84).
Separation of the regioisomeric alcohols proved
moderately difficult, however, adequate purification could
be achieved by recycling radial 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 olefinic 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 olefinic proton respective• ly. The C-2 olefinic ring proton resonated at 6 5.35 (bs,
1 1H) ppm. The H NMR spectral data were very similar for each regioisomer, the major difference was in the chemical shifts of the cyclohexene ring olefinic protons (6 5.29 for
84 vs 5 5.35 for 76) .
With the mixture of regioisomeric alcohols in hand, methods for affecting 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 cyclization reaction that generated carbocation character at
C-10, leading to the undesired formation of cyclic ethers 8_5 and £36. Preliminary cyclization experiments proved this concern to be warranted. Attempted cyclization of the mixture of alcohols 76 and 84 using BF •EtOEt in refluxing 3 anhydrous ether failed to generate any of the desired cyclized products 7_0 or j5_7. Instead, a non-polar compound, assumed to be a mixture of ethers Ji_5 and 86 was produced 115 with total consumption of the starting alcohols .
To alleviate this problem, protection of the hydroxyl group would be required. A number of protecting groups were considered (for example, the acetyl or benzyl esters), however, to facilitate purification of the intermediates (UV 76
chromophore) and to allow direct comparison of the protected, cyclized, synthetic products to the derivatized natural product* the (p-bromophenyl)urethane functionality was chosen as the protecting group.
110 The German workers had cyclized the mixture of olefins 77 and 8_3 with formic acid (85%, 12 hr, 100 °C) to give a 48% yield of cyclized products 7j5 and 88. Formic acid cyclization 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 failed to provide crystals suitable for single-crystal X-ray analysis from all the solvents tried. Derivative 7_5 was quite stable and could be stored at 4 °C, in the dark, without significant 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 identical Rj to the derivatized natural product 7_5. Two more polar products (possibly formate ester 91 and tertiary alcohol 92) and brown, likely polymeric, material that remained at the origin 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 olefinic protons and olefinic methyl groups, and showed the appearence of four new aliphatic methyl resonances. This result indicated that the non-polar product was a mixture of cyclized 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 this result, 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.
Derivatization of the major regioisomeric alcohol j}_4 with 4-bromophenyl isocyanate provided urethane 90 in good yield. 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 allylic protons on C-3. Most of the broadening could be removed by irradiation of the C-2 cyclohexene proton at 6 5.29. To confirm the coupling was vicinal and not allylic (as would be the case for regioisomer 89) a difference nuclear
Overhauser enhancement (nOe) experiment was performed.
Irradiation of the C-2 cyclohexene olefinic proton observed for 90 at 6 5.29 resulted in an nOe enhancement of the four allylic protons on carbons C-3 and C-7. The allylic 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 this difference nOe experiment. Since the major regioisomer could now be de• fined as 90, it followed that the minor regioisomer was urethane 89.
1 The 400 MHz H 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 olefinic 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 C H 136.1252) likely re• 10 16 sults from a retro Diels-Alder reaction (Scheme 10). The fragment ions at m/z 69, 55, and 41 are typical 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 cyclization to give urethane (±)-7_5 in excellent yield. Synthetic (±)-75 was
identical 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 this skeleton nanaimoane and to number it as shown in Figure 13. The absolute stereochemistry of nanaimoal was proposed on the basis of its biogenetic
relationship 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
1 studies directed towards the total assignment of the H and
13 C NMR spectra of this novel metabolite were initiated.
} 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 shift equiva•
lent. Because they were not, in most cases, also magnetic
1 equivalent, the 400 MHz H NMR spectrum was highly second
order and consequently complex. In spite of this
complicating factor, careful use of selected one-dimensional
1 H NMR double-resonance (decoupling) experiments,
two-dimensional homonuclear correlation spectroscopy
(COSY/45), and two-dimensional J spectroscopy (2D
1 ./-resolved) allowed complete assignment of nanaimoal's H
NMR spectrum (chemical shifts but not all coupling con•
stants, see Table 5). This study demonstrates the
usefulness (and limitations) of selected modern NMR tech•
116 niques to a real problem in natural products chemistry.
From the outset of this NMR study, it was envisaged
1 that upon completion of the H NMR spectral assignments,
13 total assignment of the C NMR spectrum would also be
1 13 completed using heteronuclear H- C correlation
spectroscopy (CSCM*). However, the results of the CSCM
* CSCM = chemical shift correlation map 82 experiments clearly demonstrated that the largest limitation to obtaining a reasonable spectrum was sample size. Not enough natural material was available for a suitable CSCM experiment to be adequately carried out*. Even though a
13 proton broad-band (BB) decoupled C NMR spectrum could readily be obtained on 35 mg of urethane 75 with as few as
256 scans [30° flip 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 = 3.3 ms, = 1.67 ms, (A, A2 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 and values), this requires AT A2 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.
1 a. One-Dimensional H NMR Experiments
1 One-dimensional H NMR experiments were conducted on nanaimoal (61), nanaimool (7fj) and the (p-bromophenyl)- urethane derivative 75 which, for comparison, was run in two
1 different solvents, CDC1 and benzene-d . The H NMR data 3 6 for these four experiments is 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 yield and tedious purification procedure did not allow generation of racemic urethane 75 on a large scale (> 10 mg). 83
1 Table 5. H NMR data (400 MHz) for nanaimoal (61) and derivatives.
chemical shift
fl H on C# 75° a, c 25* 61fl 61 70
1 1 .79 (bm) 1 .77 (bm) 1.81 (m) 1 .8 0 (s) 1 .78 (m) 2 1 .55- 1 .62 1 .52- 1 .63 1 .56- 1 .66 1 .6 0 (s) (m) (m) (m) 3 1.41- 1 .49 1.41- 1 .48 1.41- 1 .49 1 .4 4 (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 .7 6 (s) 1 .59 (bd, 17) 17) 17) 17)
1 .76 (bd, 1 .68 (bd, 1 .85 (bd, 1 .8 4 (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) 3 ) 1 3 0.98 (s) 14 0.97 (s)^ 1 .00 (s)<* 0.98 d 0. (s) 98 (s)^ 0.98 (s)d 1 5 0.91 (s) 0.84 (s) 1 .05 (s) 1 .0 5 (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 b c CDC1 . benzene-d . 2D /-resolved spectrum, projection d onto F 3. May be reversed6 . 2 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 (CDC1 ) caused a two proton triplet 3 centered at 6 1.40 (t, J = 6 Hz, 2H) ppm to collapse to a singlet. Therefore, the two protons responsible for this triplet must be part of a substructure of type 94, a fact only accountable for if they were the chemical shift 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 allylic C-6 methylene protons. Upon irradiation of the C-6 methylene protons a number of changes were also seen in the three proton allylic multiplet resonating between 8 1.72 and 1.84 ppm (this multiplet was actually one half of an allylic 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 triplet (/ = 7 Hz) and the overlapped doublet at 6 1.76 sharpened up. The other half of the allylic AB quartet at 6 1.62 (J = 17 Hz) was also noticably sharpened in this double-resonance experiment. This result indicated that the C-6 methylene protons were allylically 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) ] allylic proton pairs. Thus, using one double-resonance experiment, all the proton resonances 85 of substructure 95 could be readily assigned. When the 'H NMR spectrum of urethane 7!) was obtained in benzene~d a number of changes were noted in the resonances 6 of spin system 95. The C-7 methylene protons were no longer chemical shift equivalent and a complex multiplet centered about 6 1.30 ppm was observed for these protons. Irradiation of the allylic 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 shifted relative to their position in CDC1 and now appeared at 6 1.68 (d, / = 17 Hz, 1H) and 3 1.53 (d, / = 17 Hz, 1H) ppm. The C-1 methylene protons resonated at 6 1.77 in benzene-d and were again collapsed 6 to a triplet (/ = 6 Hz) upon irradiation of the C-6 methylene protons at 6 1.94. 86 1 Spin system 95 was also clearly evident in the H NMR spectrum (CDC1 ) of nanaimoal , although deshielding 3 (6_1) effects, likely due to the diamagnetic anisotropy of the aldehydic carbonyl, were noted. The C-7 methylene protons in nanaimoal (61) were clearly deshielded with respect to their chemical shift in urethane 7j> and were no-longer chemical shift equivalent. Because the C-7 methylene resonances overlapped with the resonances of the C-2 and C-3 protons, direct measurement of the chemical shifts of the C-7 protons was not possible. However, irradiation 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 shifts 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 shifts of the C-6 and C-1 methylene protons were only slightly deshielded with respect to the analogous resonances in urethane 75 (see Table 5). With spin system 95 clearly defined, all that remained was assignment of the C-2, C-3, and C-11 methylene protons. The chemical shifts 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 all 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 simplification of this multiplet, therefore, it could not be due to the C-2 1 protons. Since the multiplet was also present in the H NMR spectrum of nanaimoal (6_1) it could not be due, in whole or in part, to the C-11 methylene protons. Therefore, this multiplet was assigned to the two geminal protons at C-3. It was not possible, because of the second order nature of this multiplet to determine the exact chemical shift 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 shift equivalent and resonate at 6 1.44 ppm. The location of the C-2 methylene protons resonances 1 could now be easily extracted from the H NMR spectrum of urethane 75 (CDC1 ) by elimination. They resonated as a 3 complex two proton multiplet between 6 1.62 and 6 1.55 ppm. Again the 2D /-resolved experiment was useful in assigning the chemical shifts of these protons in nanaimoal (61). They were chemical shift equivalent and resonated at 8 1.60 ppm (see subsequent section). The results of the 1 one-dimensional H NMR experiments on nanaimoal and it's derivatives are summarized in Table 5. * As previously mentioned, one of the C-9 proton resonances overlapped the multiplet arising from the C-1 protons. Any irradiation of the C-9 protons would not affect the argument however. 88 1 b. Two-Dimensional H 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 is beyond the scope of this thesis, it will be assumed that most readers are familiar with the basic concepts and theory of 2D-NMR. 118 119 Reviews by Bernstein , Benn and Gunther , and a book by 116 Bax , when combined, provide a more than adequate intro• duction to both the practical and theoretical aspects of 2D-NMR. One of the simplest, and in fact the very first 117 proposed two-dimensional experiment , was the two-dimensional homonuclear chemical shift correlation 120 (COSYt) experiment . The experiment employs the 121 radiofrequency pulse sequence shown in Figure 14 . The first preparatory pulse (90°x) is followed by the evolution period («,) and the second mixing pulse (90°), and detection (t ). The preparatory pulse has constant phase 2 and the phase of the mixing pulse is incremented in 90° steps (^ ). The receiver phase chosen, $ or $ , selects T 2 3 the coherence transfer echo or anti-echo, respectively, and cancels the axial peaks at F, = 0. If two nuclei, A and X, are spin coupled, two sets of cross peaks will occur in the 117 * In 1971, Jeener first suggested the possiblity of two-dimensional NMR experiments, t COSY = correlated spectroscopy 89 RD - 90° (x) - J, - 90°(*,) - AQN (f ; # or 4> ) 2 2 3 RD = relaxation delay AQN = acquisition Figure 14. Pulse sequence for the homonuclear COSY NMR exper imen,t. two-dimensional spectrum centered at (5 , 6 ) and (5 a X xF ^A), in addition, diagonal peaks at ( § , 6 ) and (6 , 6 ) will a a X X also be seen. This pulse sequence results in a two-dimensional spectrum (both dimensions showing proton chemical shifts) that indicates connectivity patterns be• tween coupled protons. 12 2 The COSY/45 spectrum of nanaimoal's (p-bromophenyl)- s urethane derivative 75 i shown in Figure 15. With refer• ence to Table 5 (proton assignments based on 1D-NMR double- resonance experiments) most of the expected connectivity relationships 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 intensities of the C-1,C-2 cross peaks may indicate a short transverse relaxation time (T ) for one or both of the C-2 2 123 methylene protons , or possibly, small vicinal 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 levels. Cross peaks for all the ex• pected long range homoallylic couplings are now visible. The COSY/45 experiment allowed confirmation of the spin systems and coupling patterns deduced from the 1 one-dimensional H NMR experiments, however, this experiment did not yield any additional information regarding the assignment of chemical shifts to the strongly coupled C-2 and C-3 methylene protons*. To assist in the assignments of these protons, and to separate the overlapping proton resonance multiplets for coupling constant analysis, a 1 homonuclear 2D /-resolved H NMR experiment was performed on nanaimoal (61^). Homonuclear two-dimensional /-resolved spectroscopy is an NMR experiment that, when applied to protons for example, allows separation of the chemical shift information (8) from the scalar proton-proton couplings (/). Obviously a useful 1 technique when applied to molecules whose H NMR spectrum 125 consists of many overlapping resonances , 2D /-resolved spectroscopy has found application in the interpretation of 1 126 the H NMR spectra of a number of complex molecules . 1 * An experiment does exist whereby a H NMR COSY spectrum 1 may be obtained with what amounts to complete H broad-band 12 1 decoupling in the F, dimension ' . 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 is the simple Hahn spin-echo sequence 127 shown in Figure 17 . This pulse sequence leads to a J-modulated spin-echo. By using a step-wise incrementation of t, the 2D data matrix is built up and the Fourier transformation in the t, dimension will be a function of /. Vector diagrams have been found to be quite useful as a conceptual aid for explaining the 2D /-resolved 128 experiment . RD - 90°(*,) - 0.5/, - 180°($ ) - 0.5*, - AQN($ ) 2 3 Figure 17. Pulse sequence for 2D /-resolved NMR experiment. A number of complicating factors must be taken into consideration in order to correctly interpret a 2D 1 /-resolved H NMR spectrum. These are: 1) variation in peak intensities, 2) second order effects, and 3) the appearance of artifacts. These three factors are discussed below: 94 1. The intensities of the peaks in the 0° projection* onto F will vary according to the amount of coupling 2 the proton experiences. Thus a highly coupled proton will have a relatively low intensity while methyl singlets will generally be quite intense (see Figure 18). Because of transverse relaxation (characterized by the spin-spin relaxation time T ), the magnitude of the 2 magnetization present at the beginning of acquisition in a 2D /-resolved experiment will have decreased by a factor exp(-t!/T ) in comparison to the initial 2 magnetization, that is, after the 90° pulse. Intensities of projection peaks arising from protons with large T times (fast relaxation) will thus be 2 reduced. In general, the intensities of the peaks will not affect the interpretation of the spectrum, unless of course, either the intensity drops to zero or a small peak is obscured by the tailing of a large one. 2. Strong coupling introduces a very important complication into the interpretation of a homonuclear 2D /-resolved 1 129 130 H NMR spectrum ' . The 2D /-resolved spectrum of a strongly coupled system will be much more complex than its weakly coupled equivalent because many additional second order peaks may be present. Fortunately, second * Projection of the tilted 2D /-resolved spectrum is assumed in this discussion unless otherwise stated. 95 order peaks are frequently not symmetrical about F, in the tilted spectrum, and symmetrization may be used to eliminate them*. Care must be exercised in interpreting a second order 2D /-resolved spectrum, if doubt exists, assignments may be verified with the aid of a 130 two-dimensional simulation program . 3. The third complicating factor is artifacts. Artifacts sometimes arise in the 2D /-resolved spectrum as a re• sult of an inaccurate 180° refocussing pulse (see Figure 17). These artifacts can be partly eliminated by cycling the phases of pulses and receiver using the 131 Exorcycle procedure and by the use of a composite 132 180° refocussing pulse . If one is aware of the above limitations and can cor• rectly account for them, interpretation of a 2D /-resolved spectrum should be a relatively straightforward exercise. If the spectrum is largely first order, and the appearence of artifacts can be minimized, a 2D /-resolved spectrum can yield a great deal of information about the structure of an unknown natural product. * An AB spin system gives rise to second order peaks that are symmetrical about F, in the tilted spectrum. They give rise to a peak midway between A and B in the 0° projection onto F and cannot be removed by symmetrization. 2 96 Figure 18 shows the 400 MHz 2D ./-resolved spectrum of nanaimoal (61). It was readily apparent by inspection of the F projection that most of the protons previously 2 1 assigned by interpretation of the 1D H NMR spectrum were present. The C-1 allylic methylene protons at 5 1.80 were clearly resolved from overlap by the C-9 AB doublet of doublets (6 1.76 and 6 1.84) and were chemical shift equiva• lent. The two intense peaks at 6 1.60 and 6 1.44 could be assigned to the C-2 and C-3 methylene protons respectively, each methylene pair being chemical shift equivalent. A number of second order and/or artifact peaks were present in the spectrum. The most noticable second order resonance was the peak at 6 2.25, midway between the resonances arising from the C-11 AB methylene protons. This peak was expected based on an explanation of this second 133 order effect by Bax . Other smaller second order and/or artifact peaks could not be rationalized, 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. Slices of selected individual peaks are shown in Figure 19. They clearly 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 vicinal couplings of the methylene protons on C-11 were readily extracted from the spectrum, and their values are 97 - -30 F2 (PPM) 1 Figure 18. Partial 400 MHz H 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 transitions of the 11a, 11b geminal pair; Y denotes other second order and/or artifact peaks. 98 listed in Table 5. 2 a,b Figure 19. Slices of individual peaks shown at the top of Figure 18 to show multiplicities. 99 5. ISOACANTHODORAL Isoacanthodoral MS, M+ 220, C H flO constituted (65) 15 2 20% of the sesquiterpenoid aldehyde fraction isolated from the skin extract of Acanthodoris nanaimoensis (see GC trace, Figure 10). Aldehyde (£5) could be purified by preparative GC or HPLC, however, the small quantity of isoacanthodoral extracted (400ug/animal), its high volatility, and its susceptability to oxidation (to form a carboxylic acid) made isolation and characterization difficult. It was found most convenient to convert isoacanthodoral to either its crystalline 2,4-dinitrophenylhydrazone derivative 96 or, after reduction to alcohol 97, converted to its (p-bromophenyl)urethane derivative 98. 96 H 100 1 . The H NMR spectrum of isoacanthodoral (65) was similar to that of nanaimoal (61). Resonances for three methyl groups, one of which was olefinic, 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 66). 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 allylic 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 13 facile (Scheme 11b). A C NMR resonance at 6 204.6 (d) supported the assignment of the lone oxygen in isoacanthodoral to an aldehydic carbonyl. Comparison of the chemical shifts 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 shift, and indicated the ethanal side chain was in a noticably different chemical environment. The chemical shift of the 13 corresponding carbon in the C NMR spectrum, 8 57.1 for 65 vs 8 53.7 for nanaimoal, supported this argument. The presence of substructure 99 in isoacanthodoral was inferred from the presence of an olefinic proton at 5 5.24 (bs, W]/2 ~ 6.4 Hz) coupled to the olefinic methyl resonance 101 at 6 1.64 (bs, 3H), this assignment was supported by a 13 double-resonance experiment. The C NMR spectrum of isoacanthodoral showed resonances at 6 137.4 (s) and 6 131.9 (d) appropriate for a trisubstituted double bond, and was assignable to substructure 99. The spectral data indicated that isoacanthodoral was a bicyclic sesquiterpenoid that contained three methyl groups, one of which was olefinic, and an ethanal side chain attached to a quaternary carbon. It was difficult to ascertain the number of allylic methylene or methine protons 1 from the H NMR of isoacanthodoral, however, from the 1 integration in the H NMR spectrum of (p-bromophenyl)- urethane derivative 98 (see appendix) it was likely that no more than three allylic methylene and/or methine protons 1 02 were present in the molecule. From spectral evidence it 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 cyclization of farnesyl pyrophosphate 101 via (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 H , would be nanaimoal (6_1) . Loss of a proton or H would generate aldehydes 103 or 104 respec• c tively, molecules both lacking an olefinic methyl group and neither of which could therefore represent the structure of isoacanthodoral. To generate the required olefinic methyl functionality from carbocation 102 would require a molecular 1 03 rearrangement. Thus, a 1,2-hydride shift 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 it from further consideration. As previously mentioned, there was a marked difference between the chemical shifts of the protons 104 alpha to the aldehydic carbonyl in nanaimoal (61) (6 2.29 and 2.23 ppm) and the chemical shifts of the analogous protons in isoacanthodoral (6_5) ( 6 2.70 and 2.13 ppm). This difference could not readily be rationalized if isoacanthodoral had structure 100. Even if the side chain was axially orientated, as in 105, there did not seem to be any good reason why one of the a-methylene protons should be selectively deshielded. The mass spectrum of isoacanthodoral indicated the ethanal side chain may be allylic (see Scheme 11), implying the presence of either of substructures 106 or 107. 0 H 105 CH H 106 107 The diamagnetic anisotropy of the olefinic double bond could possibly deshield one of the a-methylene protons if there was a preferred rotamer population for the side chain in 1 05 either of substructures 106 or 107. The olefinic proton in isoacanthodoral was unusually = sharp (W^2 6.4 Hz), indicative of an olefinic proton which lacked vicinal coupling (substructure 106). When isoacanthodoral (65) was reduced to isoacanthodorol (97) the olefinic proton was shifted upfield by 0.14 ppm, suggesting the double bond and oxygen functionalities 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 selectively deshielded, 6 2.04 (dt, J = 13.5,7.2 Hz, 1H) and 6 1.41, implying that the aldehyde could not be the origin of this effect. 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 fc e chemical shifts) and the W-\/2 °f ^ olefinic 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 13 (108) from the marine sponge Disidea avcrc ". A derivative of avarol, 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 if isoacanthodoral could be represented by structure 100, treatment of its (p-bromophenyl)urethane derivative (98), 106 with acid, 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 1 olefinic methyl functionality ( H NMR 5 1.59). The of W}/2 the olefinic proton was now 11.6 Hz. This chemical evidence implied that the double bond had isomerized about the olefinic methyl group to give a new compound containing substructure 113 (see Scheme 1.3). This isomerization was impossible for structure 100. Fortunately, at this point, the crystal 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 pivitol intermediate. Proton induced fragmentation of the cyclobutane ring in acanthodoral (64) could generate nanaimoal (61_) . Proton induced cyclobutane fragmentations from aldehydes are known synthetically, and proton induced cyclobutane fragmentations from ketones are of synthetic 135 utility for the formation of five membered rings . 136 Venus-Danilova has shown that when formylcyclobutane (115) is treated with acid it fragments to give a number of products. The major product is 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 is, 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 H would gen• x erate nanaimoal (61), whereas loss of proton or H subse• z quent to C-8 to C-11 bond cleavage could generate 65 (-Hy) or 117 (-H ), compounds that would incorporate all the z spectral features of both isoacanthodoral and its rearrangement product. Although the biogenetic argument given in Scheme 14 is speculative, and must be tested experimentally, use of biogenetic theory allowed the struc• ture of isoacanthodoral to be formulated as 65, including absolute stereochemistry. Verification of the proposed structure for isoacanthodoral was obtained by a single crys• tal X-ray diffraction study, performed by He Cun-heng and Jon Clardy at Cornell University. 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 = 10 6.027 (1), b = 31.613 (8), c = 8.910 (1) nr , and 0 = 80.89 (1)°. The asymmetric unit consisted of two molecules of composition C . After collection of diffraction 2 iH3 ,OtNi, 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 is a non-isoprenoid sesquiterpenoid, that is, it does not obey the isoprene rule. 2. Like nanaimoal, one of the methyl groups originally present in the biogenetic farnesyl pyrophosphate precurser has undergone cyclization and is now contained in a carbocyclic ring. 3. The- decalin ring system is cis fused (stereochemistry biogenetically arising from the equatorial nature of the C sidechain prior to 5 cyclization to acanthodoral). This is the first 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 rationalized by the following argument; in solution, a highly populated rotamer exists (likely the same conformer as in the solid state, see Figure 20) whereby one of the a-methylene protons lies within the deshielding region of the carbon-carbon double bond. This causes selective 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 it 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 significant ( > 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 difficult owing to its trace abundance (70 ug/animal) and high volatility. It was therefore isolated as its crystalline (p-bromophenyl)urethane derivative 114. The most efficient isolation 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 radial TLC (100% CHC1 ). 3 Derivatization of the isomeric alcohols 97 and 118 with 4-bromophenyl isocyanate followed by HPLC purification yielded pure samples of (p-bromophenyl)urethane derivatives *As previously mentioned, two trace metabolites, at least one of which is 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 114 118 Acanthodoral's crystalline (p-bromophenyl)urethane derivative 114 (mp 109-110 °C, hexane), had a molecular formula C H BrN0 (HRMS, observed 421.1441, 419.1438; 22 30 2 m/z required 421.1439, 419.1460) verifying an isomeric relationship for acanthodoral with nanaimoal (6_1) and 1 isoacanthodoral (65). The H NMR spectrum of urethane 114 showed three aliphatic, 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 1 H NMR spectrum of urethane 114 (see Appendix) showed only the AB portion of an ABX spin system, indicative of a 1 substructure of type 119. Also present in the H 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 inferred. H H 119 120 Due to the magnitude of the geminal coupling constant (9.4 Hz) substructure 120 was likely confined within a 4 or 5 137 1 membered ring . The remainder of the signals in the H 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 functionality. The absence of allylic 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 it was likely biogenetically related to both nanaimoal (£1), and isoacanthodoral (65) . No known sesquiterpenoid carbon skeleton could account for the observed spectral data, 1 15 therefore the structure of acanthodoral (64) , including the absolute configuration, was solved by single-crystal X-ray diffraction analysis. 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) 10 m~ , and /3 = 85.85 ( 1 )°. Two molecules of composition C 0 NBr formed the asymmetric unit. After collection of 22H30 2 the diffraction 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 reflections. Both molecules in the asymmetric unit had the same stereostructure, and a computer generated perspective drawing is given in Figure 21. The average bond angles for the four membered ring in 114 was 86°. Acanthodoral 64 has a new sesquiterpenoid carbon skeleton for which we propose the name acanthodorane and to number it 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 biological activities shown by known nudibranch metabolites (see Table 6), the natural sesquiterpenoid mixture from A. nanaimoensis was evalua• ted for its antibacterial and antifungal activities. The mixture showed antibacterial activity against Baci I I us subtilis and Staphylococcus aureus as well as antifungal activity against Pythiam ultimum and Rhizoctonia solani all at 670 ug/O/4 inch disk) (mini• mum inhibitory concentrations were not determined). Due to the small amounts isolated, and the need for large amounts of metabolites for structural work (in an attempt to generate enough nanaimoal urethane derivative 75 for 2D-NMR experiments) the sesquiterpenoid aldehydes were not tested for fish antifeedant activity. 8. DISCUSSION The co-occurrence in A. nanaimoensis of three sesquiterpenoids each with a new carbon skeleton was gratifying, however, it was not surprising in light of pre• vious studies on nudibranch sesquiterpenoids. Table 6 is a summary of all the nudibranch sesquiterpenoids arranged according to skeletal type with comments as to their source and biological activities. It can be seen from Table 6 that the drimane carbon skeleton makes up a large majority of the sesquiterpenoids 1 18 isolated from nudibranchs and they show, with a few exceptions, antifeedant activity against fish. Although the significance of the carbon skeleton is not clear, drimane sesquiterpenoids have also been shown to possess insect 138 antifeedant activity , and in vivo antifungal activity. The majority of sesquiterpenoids isolated fall into three groups: i) furans, ii) aldehydes (dialdehydes) and iii) isonitriles. They may be derived from dietary sources or produced de novo by the nudibranch (see Table 6). Our 102 inability to find a dietary source , and the constant occurrence, and ratio, of the A. nanaimoensis sesquiterpenoids suggested de novo biosynthesis. Initial 13 biosynthe'tic studies showed that C 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 radiolabelled metabolites have not been crystallized to con• stant specific activity. TABLE 6: NUDIBRANCH SESQUITERPENOIDS SKELETON METABOLITES COMMENTS A. Acarbocyclic 1. apofarnesane ^^^^^^^ Isolated frcm Anisodoris nobilis, dihydroapofarnesal (121) was responsible for 38D the nudibranch's fruity cdor . dihydroapofarnesal (121) 2. farnesane The major metabolite of Chromcdoris marislae was marislin (122), cctrpounds 123a, 123b, 124a and 124b were present as minor 13y constituents . The isomeric pairs 123a, 123b and I24a, 124b are formally related by a marislin (122) , [3,3] sigmatropic rearrangement. The authors suggest 123a and 123b are artifacts of the isolation procedure. 0CH3 124 a b TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS famesane (cont' d) Archidoris odhneri yielded a series of related farnesic acid glycerides 140 1 1 1 0 (125-128) . '•C-labeled mevalonic acid injected into the stomach of A. odhneri was incorporated into the farnesic 38 acid moiety of 125 *\ 125 showed moderate 0R 2 in vitro antibiotic activity against Staphylococcus aureus, but had no 125 R1 = H, R2 = H demonstrable fish antifeedant activity. 126 R1 = Ac, R2 = H 127 R1 = Ac, R2 =Ac i i 0 r0H 128 Dendrolasin (129), a known sponge metabolite has been isolated from both 36 Cadlina leutomarginata and Hypselodoris 141 ghiselini collected in California. dendrolasin (129) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS farnesane (cont'd) A known sponge metabolite, longifolin (130) was isolated from the nudibranch Glossodoris 142 gracilis . longifolin (130) B. Monocarbocyclic Monocyclof arnesane metabolite 131 was isolated form Archidoris montereyensis and A. 3 odhneri 8k. It is biogenetically related 1. Monocyclofarnesane to farnesic acid glyceride 125 by a proton induced cyclization. %%% * 1 0 131 Furan 132, whose structure was postulated on the basis of mass and *H NMR spectral data 36 was isolated from Cadlina luteomarginata . The structure has not been confirmed by synthesis or interconversion to a known 132 compound. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 2. microcionin-2 Microcionin-2 (133), a known sponge metabolite was isolated from Cadlina 35 - luteomarginata . 133 has been proposed as an intermediate in the biogenesis of 44 nakafuran-8 and nakafuran-91 . microcionin-2 (133) 3. pleraplysillin-1 Pleraplysillin-1 (134) a known sponge metabolite, was isolated from Cadlina 35 luteomarginata . pleraplysillin-1 (134) C. Bicarbocyclic OH 1. drimane Isolated from both Archidoris montereyensis 38 5 and A. odhneri * , drimane rretabolite 135 showed feeding deterent activity against the tidepcol skulpin Oligocottus maculosus at 18 14 g/(mg of food pellet). C-labeled u mevalonic acid fed to A. montereyensis was incorporated into the terpenoid portion of 135. 135 R = H 136 R = Ac TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS drimane (cont'd) Albicanol (137) and albicanol acetate (138) were isolated from Cadlina luteararginata^ collected in British Columbia. Albicanol acetate (138) showed antifeedant activity against goldfish at 10 ug/(mg of food pellet) while albicanol (137) was inactive (50 pg/(mg of food pellet)). Albicanol (137) had been previously isolated from the liverwort 1 3 Diplophyllum albicans . albicanol (137) R = H albic anol acetate (138) R = Ac 38a Isolated from Chromodoris albonotata , 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 activity. pu'ulenal (139) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS drimane (cont'd) Polygodial (140) was isolated from 11 5 Dendrodoris Lurtbata * , D. krebsii, D nigra 1 6 and D. tuberculosa *+ . A known insect antifeedant, polygodial also has fish antifeedant activity. Labelling studies with 1 ^C-labeled mevalonic acid showed 140 was 11 7 biosynthesized de novo by D. limbata * . Esterfied to a series of fatty acids with varying degrees of unsaturation, sesquiterpenoid derivative 141 (stereochemistry at C-11 unknown) was localized in the digestive gland of 11 5 lh 8 Dendrodoris limbata * • . 141 could also be readily isolated from acetone skin extracts of Doriopsilla albopunctata and 11 6 Doriopsilla janainal * . The esters 141 did not show fish antifeedant activity and it was suggested they are detoxification products of 11+ polygodial (140). C-labeled mevalonic acid 141 was incorporated into the sesquiterpenoid 147 moiety of 141 . Stirring 141 in the presence of acid yielded euryfuran (144), a known nudibranch metabolite. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS drinane (cont'd) Olepupuane (142), a metabolite biogenetically related to polygodial (140) AcO, occured in Dendrodoris nigra, D. tuberculosa, D. krebsii, Dorispsilla albopunctata and J l+t> Doriopsilla janaina - . It was shown to inhibit feeding of the pacific damsel fish (Dascyllus aruanus). The ED was found to 5Q ^^^^•OAc be 15-20 ug/(mg of pellet), comparable to that of polygodial (140). The methoxy acetal 143 was isolated from one collection of olepupuane (142) Doriopsilla albopunctata, stored in methanol. AcC\ 143 Euryfuran (144), a known synthetic compound, was isolated from Hypselodoris porterae, H. calif orniensis, and the 141 intertidal sponge Euryspongia sp. . An antifeedant role for 144 is suggested although no quantitative tests were performed. euryfuran (144) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 36 2. pallescensin-A Isolated from Cadlina luteomarginata , pallescensin-A (145) is biogenetically related to euryfuran (144). 145 was toxic to goldfish and showed antifeedant activity against the skulpin Clinocottus analis. pallescensin-A (145) 3. agassizin 141 Obtained from Hyselodoris agassizi , agassizin (146) is structurally related to 49 pallescensin-G, a known sponge metabolite1 . An antifeedant role for 146 was implied. agassizin (146) 4. spiniferin-2 A known sponge metabolite, spiniferin-2 rrv (147) was isolated from Hypselodoris danielae 1h collected in Hawaii spiniferin-2 (147) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 5. nanaimoane Nanaimoal (61) was isolated from 105 Acanthodoris nanaimoensis (see text). H nanaimoal (61) 6. isoacanthodorane Isoacanthodral (65) was isolated from 0 106 Acanthodoris nanaimoensis (see text). isoacanthodoral (65) 7. nakafuran-8 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 llt iH1 godeffroyana \ H. californiensis , and nakafuran-8 (148) the sponge Dysidea fragilis. TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS nakafuran-8 (cont'd) Three oxidation products of nakafuran-8 have been reported. Butenolide 149 is believed to be an artifact formed by air oxidation of 148while 150 and 151 were isolated from H. zebra and its dietary sponge 50 D. etherial . 149 RO V 150 R=Ac 151 8. nakafuran-9 R = H A structurally interesting sesquiterpenoid, nakafuran-9 (152) has been isolated from Chromodoris maridadilus, Hypselodoris 11 11 1 godeffroyana *'*, and H. ghiselini * . A metabolite of the sponge Dysidea fragilis, 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 14T with nakafuran-9 . nakafuran-9 (152) TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 9; furodysin Furodysin (153), a known sponge metabolite, 35 was isolated from Cadlina luteomarginata . furodysin (153) 10. furodysinin 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 it preys. The only metabolite common to both California and furodysinin (154) British Columbia collections of C. 35 3 6 luteomarginata was furodysinin (154) » . Furodysinin (154) is biogenetically related to furodysin (153). Furodysinin (154) was also isolated from Hypselodoris zebra ib0 collected in Bermudian waters . TABLE 6: NUDIBRANCH SESQUITERPENOIDS cont'd SKELETON METABOLITES COMMENTS 11. Two nudibranch metabolites, 155 and 156, 36 isolated from Cadlina luteomarginata , 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. 155 R = -NC 156 R = -NCS 12. Phyllidia pulitzeri concentrates axisonitrile-l (157) from the sponge upon 11 5 which it feeds, Axinella cannabina * . Isonitrile 157 was inactive as an antifeedant but was toxic to fish at a concentration as low as 8 ppm. NC X axisonitrile-l (157) 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 106 Acanthodoris nanaimoensis (see text). acanthodoral (64) 132 III. BRYOZOANS A. INTRODUCTION TO THE BRYOZOANS Marine bryozoans have proven to be a rich source of novel, biologically active, natural products. One criterion used by marine natural products chemists in selecting potential sessile marine organisms for investigation is the ability of the organism to compete for the limited 151 ecological space available for growth . Since bryozoans 152 are important fouling organisms , they have the ability to out-compete other organisms for growing space, and to survive under very trying conditions (for example, some species are very resistant to the antifouling paints used on ships). In spite of these interesting ecological character• istics bryozoans have been largely ignored by organic chemists, until recently. Bryozoans (phylum Bryozoa) are colonial animals of which approximately 4000 living 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 false-corals, sea-mats, and moss animals are commonly used to describe them. In British Columbian waters all three 153 forms are common. Examples are : coral like - 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 million zooids may make up a bryozoan colony. Part of the zooid body wall is a circular or horseshoe-shaped structure called the lophophore; it bears ciliated tentacles which may be protruded out of an orifice to gather the small plankton (chiefly diatoms and other phyloplankton) that make up the bryozoans' diet. 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 fertilized egg may develop into a free-swimming larva capable of feeding, or as in the case with most species, the fertilized eggs pass into a brood chamber. The brooded larvae have a shorter free-swimming life 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 results of these studies are summarized in Table 7. The first bryozoan to be studied chemically was Bugula neritina (Linnalus) from which Villela isolated an 154 adenochrome-like pigment in 1948 . TABLE 7: BRYOZOAN METABOLITES ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED Alcyonidium gelatinosum (L.) Structure 159 was Sulfoxonium ions had never been encountered (Ref..155) proposed on the basis in nature previous to the isolation of 159. 13 l of C and E NMR Isolated in 5 ppm yield based on the animal spectral comparisons to wet weight, 159 is the causitive agent of model compounds and was "Dogger Bank Itch", an eczematous allergic confirmed by contact dermatitus caused by exposure to A. synthesis. gelatinosum. A severe occupational disease, + "Dogger Bank Itch" is widely distributed CH3„^S -CH2CH2OH among trawlermen working in the Dogger Bank area of the North Sea. (2-hydroxyethyl) dimethylsulfonium ion (159) Alcyonidium hirsutum (Ref. 156) The water extract of A. hirsutum showed significant toxicity to mice at 1000 mg/kg, and showed an inhibition zone against Escherichia coli. The chloroform extract was toxic to the herpes virus (HSV-1). Amathia convoluta An extract of A. convoluta showed anti• (Ref. 157T" neoplastic activity (PS system). 1 TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED > Bugula neritina (Sinnaeus) The structure of The antineoplastic activity of a B. (Ref. 157-161) bryostatin 1 (160) was neritina extract was first documented in obtained by 1970. Bryostatin 1 (160) was isolated from single-crystal X-ray 500 kg of wet animals. In the murine P388 R'O 4/ ^s^s^J* crystallography at lymphocyte leukemia (PS system) macrolide 160 -100°C. Residual R showed 52-96% life extensions at 10-70 ug/kg factor = 0.07. injection dose levels and an ED of 0.89 5Q The enantiomer shown ug/mL against the P388 in vitro cell line. was selected on the It also shows potent activity against the CH3O ^ basis of anomalous L1210 (lymphocyte leukemia, 34-51% life scattering effects due extension at 37.5-150 ugAg) and M5 (M5076 to oxygen and carbon ovarian carcinoma, 40-48% life extension at for CuKa radiation. 5-20 ug/kg and 20-65% curative in the tumor R"0 °^YH1S0R Apparently, efforts to regression model at 20-40 ug/kg) experimental OH determine the absolute tumor systems. configuration of A polyketide biogenesis is suggested for bryostatin 1 are bryostatin 1. The oxygen atoms at 01, 03, R= COCH3 continuing. 05, 011, 019A, and 019B are all in the interior of the large oxygen rich cavity suggesting the molecule may have cation binding capabilities. R"=H bryostatin 1 (160) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED B. neritina (cont'd) The structure of Bryostatin 2 (161) showed a 60% increase in bryostatin 2 (161) was life span at 30 u.g/kg in the murine P388 PS 13 based on C NMR and system. a R = H 400 MHz H NMR studies in comparison to bryostatin 1. Selective acetylation of 160 or 161 gave identical acetate (TLC) 162, which upon careful R"=H deacetylation gave a mixture of bryostatins bryostatin 2 (161) 1 (160) and 2 (161). C0CH R= 3 ^^^^ R"= COCH3 (162) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED B. neritina (cont'd) The structure of 44.5 mg of 163 was isolated from 50 kg wet bryostatin 4 (163) was weight of B. neritina. Apparently, 13 assigned using C NMR bryostatin 4 (163) is much less cytotoxic l and 400 MHz E NMR, than bryostatin 1. It was suggested that solution phase substituents common to bryostatins 1-4 (160, secondary ion MS, and 161, 163 and 164) constitute the unique selective hydrolysis requirements for anticancer activity while experiments. The the ester substituents at C-7 and C-20 0 substituent pattern of influence the degree of cytotoxicity and the butyrate and antineoplastic effects. Bryostatin 4 (163) isovalerate esters were showed 62% increase in life span at 46 ug/kg based on the assumption in the murine P388 PS system and substantial that steric compression cell growth inhibitory (PS cell live ED , 14 5Q R'= at C-20 compared to C-7 10-3-10- ug/mL) activity. would favour hydrolysis of the less hindered R"=H ester (C-7), as was found for bryostatin bryostatin 4 (163) 1. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED B. neritina (cont'd) The structure of 72.2 mg of 164 was isolated from 500 kg of bryostatin 3 (164) was B. neritina. Bryostatin 3 (164) shows 63% assigned using FAB mass life extension at 30 p,g/kg in the P388 13 spectrometry, C NMR, lymphocytic leukemia (PS system). &'\^f^yc \ OCH40 0 MHz hi NMR, and IR spectroscopy (IR band -1 at 1785 cm indicated a possible 5 or 6-membered lactone carbonyl group with an a electronegative Sr substituent). R"° H • OH R= COCH3 R" = H bryostatin 3 (164) Flustra foliacea (L.) The structures 165- The antibiotic activity (vs Staphylococcus (Ref. 156, 162-169) 169 were assigned on aureus) of this bryozoan was most pronounced the basis of GC-MS in the older parts of the fronds, and was comparisons to correlated with a characteristic strong authentic samples. lemonlike odor. The compounds apparently responsible for the antibiotic activity were monoterpenoids 165-169. Freeze dried samples of F. foliacea were devoid of antibacterial activity, however, they did show antiviral activity and inhibition of the guinea-pig cis-citral (165) ileum in vitro. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED F. foliacea (cont'd) 1 1 8 X X geraniol (166) trans-citral (167) JL 1 nerol (168) citronellol (169) Y The structures of the No biological activities were reported for alkaloids 170-178 metabolites 170 to 178, however the crude isolated from F. petroleum ether extract (from freeze dried Br^^j H 3 foliacea, were material) and the purified alkaloids 170 and determined by detailed 171 exhibited muscle-relaxant activity both interpretation of the in vivo and in vitro. spectral data. A 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^VvN— CH3 flustraminol A (173) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED F. foliacea (cont'd) OH _ yN BrAjC ^CH3 flustraminoI l B (174) A flustramide A (175) 0 6-brcm>-^-rnethyl-Nb-for^TiyltryptarTi Lne (176) TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED F. foliacea (cont'd) Quinoline derivative 177 is likely an artifact as ethanol was used in the extraction procedure. The authors speculate that if the ethoxy group was introduced during the isolation procedure, it 177 must have replaced a strikenly reactive group. Amide 178 exists as mixture of E and Z rotamers about the amide bond. Nugula nerita The aqueous 2-propanol extract of N. (Ref. 157T nerita, in several doses, led to 168-200% life extension in the PS system. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED Phidolcpora pacifica For structural elucidation and biological (Ref. 170) activities, see text. Synthetic studies toward the synthesis of phidolopin 179, desmethylphidolopin 180, and a number of structural analogues are currently underway. 1 phidolcpin (179) desmethy lphid(Dlcpi n (180) N1 N CH3 OH drN°2 ^0H TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED Sessibugula translucens The structures 182- Sessibugula translucens is preyed upon by (Ref. 171) 185 were elucidated the nembrothid nudibranchs Tambje abdere, T. using spectral methods eliora and Roboastra tigris. A mixture of and comparison to the enamines 182 and 183 (182, 40%; 183, 60%) 0CH3 known compound, inhibited cell devision in the fertilized sea 4-methoxy-2,21- urchin egg assay at 1 pig/mL in seawater and bipyrrole-5- showed moderate antimicrobial activity at carboxaldehyde (186). 50p,g/disk against Eschericia coli, NHR Staphylococcus aureus, Bacillus subtilis, and Vibrio anguillarum. 182 X = H Y = H R = H 183 X = Br Y = H R = H OCH. 184 X = H Y = H R = iBu 185 X = H Y = Br R = iBu 186 The isobutylamines 184 and 185 inhibited cell division at 1 |j.g/mL, showed antimicrobial activity against Candida albicans, B. subtilis, S_. aureus and V. anguillarum at 5 jlg/disk, and shewed mild activity against E. coli at 50 |j.g/disk. TABLE 7: BRYOZOAN METABOLITES cont'd ORGANISMS AND METABOLITES STRUCTURE ELUCIDATION COMMENTS AND BIOLOGICAL ACTIVITIES ISOLATED Thalamaporella gothica An extract of T. gothica floridana showed floridana antineoplastic activity (PS system). (Ref. 157) Zoobotryon verticillantum Proposed on the basis Compound 187 inhibited cell division of the (Delle Chiaja, 1828) of spectral data, fertilized sea urchin egg (ED = 16 ^g/mL). 5Q (Ref. 172) structures 187 and 188 Complete pharmacological evaluations of 187 were confirmed by and 188 are in progress. synthesis. Reduction of compound 188 (Zn-HoAc) N(CH ) 3 2 yielded the free amine 187, oxidation of 187 T30% H 0 /MeOH) yielded 2 2 188 in quantative 187 yield. N(CH3)2 188 1 46 157 In 1970 Pettit et al. reported that a number of bryozoans contained anticancer constituents. B. neritina was the first bryozoan species to yield 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 its structure was secured by crystallographic 158 and spectroscopic techniques . Since the structure of bryostatin 1 was reported, three additional bryostatins have been isolated and the structures determined. They were 15 9 160 bryostatin 2 (161) , bryostatin 3 (164) , and bryostatin 161 4 (163) . All three are structural 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 (if any) of the metabolites. Because bryostatin 4 was isolated from B. neritina collected in both the Gulf of Mexico (U.S.) and Gulf of Sagami (Japan), Pettit has speculated that the metabolite may be a biosynthetic product of B. neritina rather than from a dietary source. "... a definite conclusion regarding this biochemical question will require careful chemical examination of the microorganisms ingested by 1 B. and/or ( 'C) acetate biosynthetic feeding neritina 161 experiments." Demonstrating the close relationship between chemical ecology and pharmacological research, Christophersen and 147 Carle, in a series of papers, have described their work on 163-167 the marine bryozon Flustra foliacea (L.) . Prompted by a 1977 report titled "Anti-fouling Role of Antibiotics 168 Produced by Marine Algae and Bryozoans" , they have isolated and elucidated the structures of 14 secondary metabolites, of which nine represented new marine alkaloids and five were known monoterpenoids. The monoterpenoids were apparently responsible for the antibiotic activity of the bryozoan*. The structures of the alkaloids bearing the physostigmine, indole, or quinoline ring systems were elucidated solely on the basis of spectral interpretation and the structures drawn in Table 7 do not represent absolute configurations. Extensive use was made of difference nOe enhancement NMR spectroscopy in the structural assignments. Although bryozoans seem to be well adapted in the marine ecosystem, they are by no means free from predation. Nudibranchs, not surprisingly, are one of the main bryozoan predators and some have become highly specialized, only feeding on one particular bryozoan. In some cases the nudibranchs are uncannily cryptic when feeding upon their bryozoan host (for example see Corambe pacifica'173). Carte and Faulkner, in studying such an interesting nudibranch- bryozoan association have isolated a number of bipyrroles 182 - 185 from both the bryozoan Sessibugula t ransIucens and * The non-volatile metabolites extracted from 156 F. Foliacea were devoid of antibacterial activity . 148 the defensive secretion of its nudibranch predators Tambje abdere and T. eliora^7\ The structural diversity and varied biological activities of the secondary metabolites isolated from bryozoans leads one.to wonder what unique type of molecules have yet to be discovered as more and more bryozoans are investigated for their natural products chemistry*. It is reasonable to suggest, from the limited chemical studies performed so far, that bryozoans show great potential in the unrelenting search for drugs from the sea. * Numerous species of bryozoans collected in New Zealand waters show antiviral activity. The structures of the compounds responsible for this activity are currently under 2 investigation *. 1 49 B. SECONDARY METABOLITES FROM PHIDOLOPORA PACIFICA (ROBERTSON 1908) Phidolopora pacifica (see Figure 22), usually referred to as the "lacy bryozoan", is commonly found on rocky outcrops (depths of 3 to 15 m) in Barkley Sound, British Columbia. Classified in the bryozoan order Cheilostomata, P. pacifica has a highly intricate, inflexible calcium carbonate skeleton built up into a ruffled lacy network. It is one of about 75 genera of Bryozoa common to the Pacific 17 Northeast ". Our attention was drawn to P. pacifica by the absence of fouling organisms on its skeleton and the strong in vitro antifungal and antialgal activity of its extracts. P. pacifica was first collected 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 collection and stored at room temperature for two days. At the end of this time, the material was ground in a Waring blender with methanol, and filtered. The combined greenish brown methanol extracts were concentrated to about one quarter of the original 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 color, was dried over anhydrous sodium sulfate. The sodium sulfate was filtered off and the sample concentrated in vacuo to give 769 mg (0.54%) of a dark greenish brown oil. Flash 151 chromatography gave a polar fraction which, after purification by preparative TLC, yielded phidolopin (179) (1.4 mg, 0.001%, mp = 225°C) and desmethylphidolopin (180) (<1 mg) as light yellow crystalline solids. Both 179 and 180 represent new natural products that contain the relatively rare, naturally occurring, nitro functionality. 179 180 A second collection (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 isolate any biologically active compounds of lesser polarity. After flash chromatography of the crude" organic extract, fractions having similar chromatographic polarities 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 fractions, fraction P1, after preparative TLC yielded 8.5 mg (0.15%) of 4-methoxymethyl-2-nitrophenol (189). Similarly, fraction P2 gave of 4-hydroxymethyl- 2-nitrophenol (181) and fraction P3, the most polar fraction, gave a mixture of phidolopin (179) and desmethyl- phidolopin (180). The least polar nitrophenol, 4-methoxymethyl- 2-nitrophenol , had a molecular formula of C H NO« (189) e 9 (HRMS, m/z observed 183.0534; required 183.0532) demanding 1 five units of unsaturation. The H 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 ring. Additional resonances at 8 3.41 (s, 3H) and 4.43 (s, 153 Table 8. 'H NMR data (CDC1 , 80 MHz) and spectral 3 comparisons for nitrophenols isolated from Phidolopora pacifi ca. chemical shift, 6 on C# 189 181 209 190 3 8.09 (d, 8.14 (d, 8.09 (d, 7.82 2.0) 2.0) 2.1) 5 7.59 (dd, 7.63 (dd, 7.59 (dd, 7.40 2.0, 8.4) 2.2, 8.5) 2.1, 8.9) 6 7.16 (d, 7.18 (d, 7.14 (d, 7.02 8.4) 8.5) 8.9) 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 it was possibly involved in an intramolecular hydrogen bond. Comparison of the observed 'H NMR chemical shifts for 4-methoxymethyl-2-nitrophenol (189) 175 to the calculated values gave good agreement, as did comparison to the literature values for 4-hydroxy- 17 6 3-nitrotoluene ( 190 ) . The chemical shift 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 fully supported the assigned structure (Scheme 15). Fragment ions at 137 (C H 0 , m/z 8 9 2 10.4%) and 106 (C H 0, 38.4%) indicated the presence of an 7 6 aromatic nitro functionality. These fragment ions correspond to losses of N0 and [CH 0 + N0 ] from the 2 3 2 177 molecular ion . Fragmentation involving the loss of the methyl ether functionality via a benzylic cleavage gives a 155 fragment ion at m/z 152 (C H N0 , 100%), the base peak in 7 6 3 the spectrum. To the best of the author's knowledge, isolation of 4-methoxymethyl-2-nitrophenol (189) is only the second exam• ple of a nitro containing phenol to be isolated from the marine environment. The first 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 literature. Isolated from fraction P2, 4-hydroxymethyl- 2-nitrophenol had a molecular formula C H NO (HRMS, (181) 7 7 tt 1 m/z observed 169.0379; required 169.0375). The H 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 correlating the two structures. Phenol 181 had previously been reported as a synthetic compound with interesting biological activity (vide infra). Phidolopin ( ) had a molecular formula C H N O 179 10 13 5 5 (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), .08 (d, / = 2.2 Hz), and 10.56 (s, 1H, exchanges with D 0) 8 2 156 1 in the H 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 rise to the observed base peak at 152 (C H N0 ) m/z 7 6 3 supported this assignment. The chemical shift of the benzylic methylene protons, 6 5.46, indicated substructure 192 was likely 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 1 unsaturation. H 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 1 presence of at least one amide carbonyl (1657 cm" ). A purine nucleus containing oxygen, methyl and 4-hydroxy- 3-nitrobenzyl substituents could account for all the 157 structural requirements of phidolopin. Comparison of the methyl singlet resonances to the two high field methyl 1 resonances in the H 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 correlations, to unambigously establish the substitution pattern of the substituents on the purine ring. The structure of phidolopin (179) was therefore solved via single-crystal X-ray diffraction on its p-bromophenacyl derivative (194). Derivative 194 was prepared in good yield by reacting phidolopin with p-bromophenacyl bromide in the presence of KHC0 and l8-crown-6. The X-ray structural 3 determination was performed by He Cun-heng and Jon Clardy at Cornell University. CH CH3 193 0 0 Br N N 194 CH3 158 Table 9. 'H NMR data and spectral comparisons for purine derivatives isolated from Phidol opora pacifica. chemical shift, 6 H on C or 179fl 180* 193c 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, e e 3H) 3H) 3H) N3 Me 3.59 (s, 3.35 (s, 3.57 (s, 3.35 (s, e e 3H) 3H) 3H) 3H) N7 Me 3.9e9 (s, 3H) 0 c 270 MHz, CDC1 400 MHz, DMSO-d CDC1 , Sadtler 3 6 3 d Standard NMR Spectra No. 10393M 60 MHz, DMSO-d , Ref. 179 6 e May be reversed 18 CH3 ° 159 A crystal of 194 suitable for X-ray diffraction was grown by slow evaporation of an acetone-methanol- acetonitrile solution. Preliminary X-ray photographs showed triclinic symmetry, and accurate lattice constants of a = 10 9.549(3), b = 9.514(1), c = 15.212(3) lO- m, a = 72.69(1), j3 = 82.96(2), and 7 = 81.87 (2)° were determined by a least squares fit of fifteen diffraction measured 20-values. After collection and correction of the diffraction 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 reflections. Figure 23 is a computer generated perspective drawing of the final 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 C H,,N 0 (HRMS, observed 317.0777; 13 5 5 m/z 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 functionalities. 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-d ) of 180 suggested the presence of the nitrophenol 6 160 161 residue 192. Attachment of this functionality to N-7 of the purine nucleus was assigned by analogy to phidolopin, and was supported by comparisons of the chemical shift of the N-8 proton in desmethylphidolopin to suitable model compounds {vide infra). The only remaining structural feature to be determined for desmethylphidolopin was the location of the methyl group 1 on the purine nucleus. Comparison of the H NMR spectrum of desmethylphidolopin to those of both 7-benzyl- 3-methylxanthine (195) and 7-benzyl-1-methylxanthine 17 9 ( 196 ) (Table 9) showed that the methyl group in des• methylphidolopin must be attached to N-3 of the purine ring. The chemical shifts of the N-3 methyl groups in both des• methylphidolopin and 7-benzyl-3-methylxanthine were identi• cal and resonated at 8 3.35 (DMSO-d ). Similarly, the 6 purine ring NH proton resonances were of vertially equiva• lent chemical shift, 6 11.16 for 180 vs 11.18 for 195. 1 Comparison of the H 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 shifts. 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 position of methylation in xanthines can be 180 readily obtained from their mass spectral fragmentation . A major fragmentation is via a retro-Diels-Alder reaction 162 0 0 N CH3^ N N -> N CH3 195 196 involving the N-1 and C-2 atoms such that 1-methylxanthine gives a base peak at 109 [M+(166) - CH NCO] while m/z 3 3-methylxanthine gives a corresponding peak at m/z 123 [M+(166) - HNCO]. A major fragment ion at m/z 123 (27%, HRMS, obser.ved 123.0434, required for C H N 0 123.0432) m/z 5 5 3 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 181 Tischler . At the present time, a synthetic sample of desmethylphidolopin has not yet been secured. 163 0 CL H H\ A T* H T' A) - V» * HNCO 0 ™ N LM 3 CH3 197 m/z 166 (100%) 123 (27%) m/z 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 first was a report that caffeine had been isolated from the Chinese gorgonian Echinogorgia y 82 pseudossapo . Phidolopin is a unique xanthine derivative in that it contains the relatively rare naturally occurring nitro functionality. 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 activities. 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 activities. 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 183 which the animals completely recover . Doridosine has also been isolated from the Australian sponge Tedania 18 digit at a *. Isoguanosine (201) , isolated from the dorid 185 nudibranch Di aul ul a sandi egensis , and spongosine [2-methoxyadenosine (202)], isolated from the sponge Tedania 6 digitata** are two other examples of biologically active 165 purine ribosides from marine organisms. Finally, 9-/3-D-arabinofuranosyladenine (203) and its 3'-acetate (204) , compounds well known as potent synthetic antiviral agents, were isolated as natural products from the Italian gorgonian Eunice! I a cav ol i ni 18 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 activity, were isolated from the Pacific 166 188 sponge Agelas sp. , while hokupurine (207) has been isolated from both the nudibranch Phestilla melanobrachi a and the coral upon which it feeds, Tubastrea coccinea}89. Rather surprisingly, in contrast to the other marine purine derivatives, no significant biological activity could be found for hokupurine. CH3 On the basis of the examples given it was not unexpected to find that phidolopin had significant biological activity in the limited biological testing performed in our laboratory. It showed in vitro antifungal activity against Pyt hi am ultimum, Rhizoctonia solani and He I mint hosporium satiurn with a minimum inhibitory concentration of 70 ug per one-quarter inch disk, for all three species. It had antibacterial activity against 167 Bacillus subtilis and Staphlococcus aureus and it showed antialgal activity against the pennate diatom Cylindrotheca fus i formi s 1 9 0 . The antialgal activity shown by phidolopin could indi• cate that 179 plays an active role in the chemical ecology of P. pacifica by inhibiting 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 antialgal activity against the benthic pennate 1 9 1 diatom, Navicula s al i ni c ol o . CH3 208 Phenolic compounds are well known to be antibiotic and 192 phytotoxic , and phenols or brominated phenols from both 193 19 marine red and brown algae * have been shown to inhibit 195 the growth of pelagic unicellular algae. Price and Wain have shown that nitrophenols, including 4-hydroxymethyl- 2-nitrophenol (181), inhibit chloroplast development in both 168 green plants and the unicellular algae Eugl ena sp. Structure-activity relationships indicated that for substituted toluenes, a nitro group in the 3-position and a hydroxyl group or ether linkage in the 4-position were essential for activity, 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 ideally suited to inhibit epiphyte growth by inhibiting chloroplast development. Further biological work is re• quired to substantiate the above hypothesis. Xanthine based alkaloids are generally of plant orgin, therefore isolation of xanthine derived metabolites from animal sources raises the question of their biogenetic origin. While gorgonians are well known to incorporate symbiotic zooxanthellae within their tissues (and therefore plant cells could be the ultimate source of the caffeine 1 8 2 found in Echenogorgia ps e udos s a po ) .bryozoans are generally regarded as symbiont free. It is perhaps likely that P. pacifica obtains the xanthine based metabolites from its phytoplankton diet. This hypothesis was supported by the isolation of phidolopin (179) from two additional species of bryozoans, Diaperoecia call for nica and 196 Hippodi pi osi a i nscul pi a , which were also collected in Barkley Sound. If phidolopin is indeed diet derived, the dietary source must be stable to both seasonal and locational variation (within Barkley Sound) as 179 was isolated from every collection of P. pacifica examined for 169 secondary metabolites. The dietary hypothesis outlined above remains to be supported by isolation of phidolopin from an algal 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 artifacts of the isolation procedure. A number of benzyl methyl ethers have been isolated from marine organisms, many of which are believed to be artifacts formed by methylation of the precurser benzylic alcohols with the 197 methanol used for extraction . To test if methyl ether 189 was indeed a natural product, one collection 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 1 silica gel (for H NMR data of 209 see Table 8). No traces of methyl ether 189 were found. Extraction of another collection 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 artifacts of the isolation procedure. Since treatment of 181 with acid generated 189 in reasonable yield (vide supra), it 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 CH3\ N02 OCH2CH3 209 210 A requisite, 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 possibility remains that cleavage of phidolopin to generate 181 may be catalyzed by a nucleophile such as Br", however this possibility was not explored in the laboratory. IV. EXPERIMENTAL 1 13 The H and C 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 CDC1 (5 = 3 13 77.00) was used, as both an internal standard for the C 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 ultraviolet 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 microcell. A Fisher-Johns apparatus was used to determine melting points and these values are uncorrected. Gas chromatography and high performance liquid chromatography were performed on Hewlett-Packard 5830A and Perkin-Elmer Series 2 instruments, respectively. A Perkin-Elmer LC55 UV detector and/or a Perkin-Elmer LC-25 refractive index detector were employed for peak detection during HPLC. A thermal conductivity or flame ionization detector was used for GC. A Whatman Magnum-9 Partisil 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 distilled; all other solvents were reagent grade. Merck Silica Gel 60 PF-254 was used for preparative TLC, Merck Silica Gel 230-400 Mesh was used for flash chromatography, and Merck Silica Gel 60 PF-254 with CaSOa-1/2 H 0 was employed in radial TLC. 2 173 Aldi s a cooperi Collection Data Aldisa cooperi was collected at various locations in Barkley Sound, British Columbia at depths of 1 to 15m. One collection was made from various locations in the Queen Charlotte Islands, B.C. Immediately after collection, 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) until used (usually within two months). Extraction and Chromatographic Separation As a number of collections of A. cooperi were made and no variation in metabolites isolated was observed (except relative amounts) the following represents a typical isolation 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 filtered 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 sulfate, and evaporated in vacuo to give 2.1 g (5.6%, 16 mg/animal) of an orange oil containing a white solid. Fractionation of the material by column chromatography (chloroform) gave crude fractions which contained fats, sterols, steroidal ketones, the steroid acids 2_3 and 24, and glycerol ether 25. Combination of the fractions containing the steroidal acids followed by preparative TLC (5:95 methanol/chloroform then, on a second plate, 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 (24) in a ratio of 10:3. The ratio was determined by the measuring the peak heights of the C-18 methyl resonances in 1 the H NMR spectrum. The acids 23 and 24 could be separated by fractional crystallization (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 steroidal ketones followed by preparative TLC (chloroform) gave 10.0 mg (0.03%, 0.1 mg/animal) of a mixture of steroidal ketones consisting mainly of cholestenone (27). Fractions containing the glycerol ether were combined and purified by preparative TLC to give 1-0-hexadecyl-glycerol (25). To aid in the purification and characterization of 25 it was converted to its diacetyl derivative 44. 1 75 3-Oxo-4-cholenoic acid (23): mp 178-179 °C; UV (CH CN) X 3 mav 1 Ilia A 236 nm (e 6200); IR (KBr) 3600 - 2400, 1720, 1700 and 1640 1 1 cm" ; H NMR (400 MHz, CDC1 ) 6 5.74 (bs, 1H, C„ H), 2.63 - 3 0.81 (m, 25H), 1.21 (s, 3H, C Me), 0.94 (d, = 6 Hz, 3H, 19 J 13 C Me), 0.74 (s, 3H, C Me); C NMR, see Table 2; HRMS, 21 1B observed 372.2658, C ,H 0 requires 372.2664; MS, m/z 2i 3 6 3 m/z (rel 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). 1 3 -4,22-choladienic acid (24): H NMR (80 MHz, CDC1 ) 6 -OXO 3 6.96 (dd, = 8.8,15.4 Hz, 1H, C H), 5.76 (d, / = 15.4 Hz, J 22 1H, C H), 5.74 (bs, 1H, C„ H), 2.60 - 0.80 (m, 21H), 1.20 23 (s, 3H, C Me), 1.11 (d, = 6.4 Hz, 3H, C , Me), 0.77 (s, ,g J 2 3H, C Me); HRMS, observed 370.2507, required for 18 m/z C „H ,0 370.2508; MS, (rel intensity) 371 (24), 370 2 3 3 m/z (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 diacetyl 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 stirring at room temperature overnight, the reaction mixture was evaporated to dryness in vacuo and purified by 176 preparative TLC to give 8 mg (0.020 mmole, 79%) of diacetyl derivative 44: *H NMR (80 MHz, CDC1 ) 6 5.19 (m, 1H), 4.36 3 (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 400.3141, C 3H„ 0 requires 400.3189; MS, m/z 2 a 5 m/z (rel 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 steroidal ketone fraction, which consisted mainly of cholestenone (27_) , was not separated further. In 1 combination, the high resolution mass spectrum and the H NMR spectrum of the mixture indicated the presence of four steroidal ketones, m/z observed 412.3728, required for C H„ 0 412.3705; observed 396.3384, required for 29 8 m/z C H O 396.3392; observed 384.3396, required for 28 ail m/z C H,, O 384.3392; observed 382.3246, required for 27 ft m/z 1 C H 0 382.3235; H NMR 6 5.75 (s), 5.21 (m), 4.73 (bs, 27 42 C=CH ), 4.67 (bs, C=CH ), 1.57 (bs), 1.18 (s), 0.94 (d, 2 2 J = 6.4 Hz), 0.71 (s); UV (Hexane) X 229 nm. max Steroidal ketones from Anthoarcuata graceae. 1 77 Ant hoarcuat a graceae was collected at various locations in Barkley Sound, B.C. at depths of 1 to 15m. Immediately after collection 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 this time the sponge was homogenized in a Waring blender and vacuum filtered in the presence of Celite. 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 sulfate, filtered, and concentrated in vacuo to give 5.2 g (0.88%) of a gummy orange oil. The oil was fractionated by column chromatography (gradient of EtOEt in CHC1 ) to give 61.8 mg (0.010%) of a mixture of steroidal 3 ketones whose 'H NMR spectrum was essentially identical 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 X (MeOH) 241 nm (e 15,000); IR max 1 (CHC1 cast) 2940, 1740, 1675, 1190, 1170 cm" ; 'H NMR (400 3 MHz, CDC1 ) 6 5.74 (bs, 1H, C» H), 3.68 (s, 3H, OMe), 1.19 3 178 (s, 3H, C Me), 0.93 (d, 6.8 Hz, 3H, C Me), 0.7 2 (s, 19 J = 2i 13 3H, C Me); C NMR see Table 2; HRMS, observed 1B m/z 386.2821, required for C 5H 03 386.2821; LRMS, (rel 2 38 m/z 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. Purification by normal phase HPLC (2.4% isopropanol/hexane) gave pure ester 3_8: UV X max (MeOH) 239, 214 nm; IR (CHC1 cast) 2930, 2850, 1723, 1674, 3 1 1 1270, 1240 cm" ; H NMR (400 MHz, CDC1 ) 6 6.84 (dd, / = 3 16,9 Hz, 1H, C H), 5.76 (d, 16 Hz, 1H, C H), 5.74 22 J = 23 (s, 1H, C„ H), 3.74 (s, 3H, OCH ), 1.19 (s, 3H, C Me ), 3 19 1.10 (d, = 7 Hz, 3H C Me), 0.75 (s, 3H, C Me); HRMS, J 21 18 observed 384.2655, required for C H 0 384.2664; LRMS, m/z 25 36 3 m/z (rel 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 Collection, extraction and chromatographic separation. Meli be leonina (38 animals) was collected at Cates Park, North Vancouver, B.C. on October 6, 1981. Immediately after collection, 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 sulfate, and concentrated in vacuo (dry ice thimble, < 20°C) to give 57.4 mg of an orange "grapefruit" smelling oil. Silica gel column chromatography (step gradient of 100% hexane to 40% diethyl ether/chloroform) gave straight chain hydrocarbons, fats, sterols, 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 1 2,6-dimethyl-5-heptenal (53): IR (CHC1 ) 1723 cm" ; H NMR 3 (400 MHz, CDC1 ) 6 9.62 (d, = 1.9 Hz, 1H, C, H), 5.10 (m, 3 J 1H, C H ), 2.36 (m, 1H, C H), 2.05 (m, 2H, C„ 2H), 1.77 5 2 (m, 1H, C H), 1.70 (bs, 3H, C Me), 1.61 (bs, 3H, C Me), 3 8 7 180 3 1.41 (m, 1H, C H), 1.11 (d, / = 7.1 Hz, 3H, C Me); ' C NMR 3 9 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 (CHC1 ) 3500 - 2200, 3 1 1 1700 cm" ; H NMR (400 MHz, CDC1 ) 8 5.13 (m, 1H), 2.49 (m, 3 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 C H 0 156.1151; MS, 9 16 2 m/z (rel intensity ) 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 Collection Data Acanthodoris nanaimoensis was collected at various locations in Barkley Sound, and from Sunset Beach near Horseshoe Bay, British Columbia. No attempts were made to separate the small fraction of A. hudsoni that was 198 co-collected with A. nanaimoensis at some sites. Hellou had shown that the skin extract of A. hudsoni also contained nanaimoal 61. In all the collections made there was little variation in the ratio of nanaimoal (61): isoacanthodoral (6_5): acanthodoral (6_4) isolated. Immediately after collection 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, until used (usually within two months). Extraction and Chromatographic Separation As a number of collections of A. nanaimoensis were made and no variation in metabolites isolated was observed, the following represents a typical procedure. All weights of the volatile aldehydic components are in error as it was impossible to remove all traces of solvent under high vacuum without substantial losses of the sesquiterpenoids. 182 A. nanaimoensis (120 animals) was collected in February, 1982, in Barkley Sound, British Columbia. They were immediately immersed whole in methanol and stored at room temperature for two days. At the end of this 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 origi• 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 sulfate. Removal of the solvent in vacuo gave 2.3 g (16 mg/animal) of an odouriferous orange oil. Column chromatography (gradient of 100% hexane to 10% ethyl acetate/chloroform) yielded fractions containing fats, sterols, 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 slightly yellow oily mixture consisting of 61 (1.4 mg/animal), 64 (0.2 mg/animal), and 65 (0.4 mg/animal) in a ratio of 79:1:20 as determined by analytical GC. The most abundant aldehydes (61 and 65) could be separated by preparative GC (3% OV-17 on Chromosorb (HP) 80/100 Mesh, 183 initial temperature 140°C, rate 1°C/min., 61 16.2 min., Rt 65 17.6 min.) or HPLC (50:50 hexane/methylene chloride). Rt HPLC was found to be the most convenient, although it was not possible to purify acanthodoral (64) adequately by this method. Nanaimoal (61): colourless oil, [a] -7° (c 3.0, CHC1 ); IR D 3 (CHC1 ) 2920, 2750, 1730, 1470, 1395, 1380, 930, 870, 800 3 1 13 and 750 cm" ; 'H NMR (Table 5); C NMR (100.6 MHz, CDC1 ) 3 [multiplicities 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 C H O 220.1827; GC-MS, (rel intensity) 220 15 2lt m/z (4), 177 (6), 176 (49), 162 (16), 161 (100), 121 (6), 105 (33), 69 (7), 55 ( 12) , 41 (24). 1 Acanthodoral (64): colourless oil; H NMR (400 MHz, CDC1 , 3 residual CHC1 7.25) used as internal reference) 9.59 3 (8 6 (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 (rel 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 oil; *H NMR (400 MHz, 1 84 CDC1 ) 6 9.72 (dd, / = 3.3,3.2 Hz, 1H), 5.23 (bs, 1H), 2.71 3 (dd, / = 14.8,3.3 Hz, 1H), 2.14 (dd, / = 14.8,3.2 Hz, 1H), 13 1.65 (bs, 3H), 1.00 (s, 3H) 0.91 (s, 3H); C NMR (100.6 MHz, CDC1 ) 6 204.6, 135.4, 129.9, 57.1, 46.3, 40.1, 38.4, 3 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 size); GC-MS, m/z (rel 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 volatility of the sesquiterpenoid aldehydes, to simplify their separation, and to obtain crystalline derivatives, 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 alcohol. A total 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 stirring. The solution cleared after a few minutes. After 24 hr, 30 mL of water was added and stirred for an additional 2 hr. At the end of this 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 filtered. 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, 7.71 min), 64 (4%, 5.79 min) and 65 (36%, Rt R{ Rt 8.14 min). The remaining 4% was due to the minor metabolites (1%, 4.08 min; 3%, 11.65 min) that were Rt R{ not characterized further. The low yield in the reduction is likely due to weighing error for the aldehydes (traces of solvent were not removed on high vacuum). Repetitive radial TLC (silica gel, 100% CHC1 ) provided 25.8 mg of pure 3 nanaimool (70): oil; [o] +10.4° (c 0.61, MeOH); IR (CHC1 ) D 3 1 3600, 3400, 2920, 1460, 1380, 1360, 1020, 910, and 740 cm" ; 1 H NMR (400 MHz, CDC1 ) 5 3.72 (m, 2H), 1.97 (bs, 2H), 1.78 3 (bs, 2H), 1.7 5 (bd, J = 17 Hz, 1H), 1.59 (bd, J = 17 Hz, 13 1H), 0.98 (s, 3H), 0.97 (s, 3H), 0.88 (s, 3H); C NMR (100.1 MHz, CDC1 ) 6 133.4, 125.5, 59.7, 44.0, 39.9, 34.8, 3 31.8, 30.8, 28.0, 27.9, 24.9, 21.5, 19.5; HRMS, m/z observed 222.1988, required for C H 0 222.1984; MS, (rel 15 26 m/z 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 directly for the preparation of the (p-bromophenyl)urethane derivatives. Preparation of the (p-bromophenyl)urethane derivatives of 97 and 118. 186 A total 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 vial containing 67.9 mg (0.343 mmole) of 4-bromophenyl isocyanate in 1.5 mL of carbon tetrachloride. The vial was sealed and heated with stirring at 60°C for 20 h. At the end of this 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: oil, [a] -39° (c 0.88, hexane); 'H NMR (400 MHz, CDC1 ) D 3 6 7.40 (d, 2H), 7.27 (d, 2H), 6.50 (br s, 1H), 5.06 (br s, = 6.4 Hz, 1H), 4.18 (m, 2H) , 2.10 (ddd, J = 13.0,6.4,9.0 Hz, 1H), 1.48 (ddd, J = 13.0,6.7,8.9 Hz, 1H), 13 1.61 (bs, 3H), 1.01 (s, 3H), 0.90 (s, 3H); C NMR (100.6 MHz, CDC1 ) only terpenoid carbons are listed, 6 134.2, 3 131.0, 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 (rel 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). 1 114: mp 109-110 °C (hexane); H NMR (400 MHz, CDC1 ) 5 7.40 3 187 (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 C H BrN0 421.1439 and 419.1416; MS, (rel 22 30 2 m/z intensity) 204 (66), 189 (100), 161 (20), 95 (23), 81 (30), 69 (24). Acid catalyzed isomerization of 98. Preparation of 112. A total of 2.5 mg of 9ji was placed in a 2 mL reaction vial, 1.5 mL of 98-100% formic acid was added and the vial was capped. After heating at 70 °C overnight, the reaction was cooled and concentrated (in vacuo) to give a light brownish residue. The residue was dissolved in ether (25 mL) and washed with 5% bicarbonate (2x10 mL) and water (1 x 10 mL). The ether layer was dried over sodium sulfate, filtered and the solvent removed in vacuo to give, after preparative TLC (chloroform), 1.1 mg of. 112. No traces of 1 98 were detected by H NMR or HPLC (reverse phase, 10:90 water/acetonitrile). oil; 'H NMR (400 MHz, CDC1 ) 5 112: 3 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); 81 HRMS, observed 421.1463, required for C H BrN0 m/z 22 30 2 421.1439; MS, m/z (rel 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 total 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 1 (chloroform), 31 mg (0.073 mmol, 100%) of 75: oil; H NMR 13 (see Table 5), C NMR (100.6 MHz, CDC1 ) [multiplicities 3 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); 81 HRMS, observed 421.1439, required for C H BrN0 m/z 22 30 2 421.1439; MS, m/z (rel 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 stirred for three hours 189 after which it was partitioned between water (50 mL) and chloroform (4 x 25 mL). The chloroform layers were com• bined, dried over sodium sulfate, filtered, and evaporated to give 131.1 mg (0.328 mmol, 92%) of a mixture of 2,4-dinitrophenylhydrazone derivatives 211, 212 and 96. 211 212 Separation by normal phase preparative HPLC (10% 1 chloroform/hexane) gave pure H NMR (270 MHz, CDC1 ) 6 96: 3 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. 190 115 Following Tischler's procedure 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. Analytical TLC indicat• ed little 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 light 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 silica gel (250 g) in a 14 cm Buchner funnel, and the hexane was drawn through the silica gel by suction. Additional hexane (200 mL) was added and drawn through the silica. 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 first ethyl acetate wash. Removal of the solvent gave 7.58 g of a mixture that contained at least three components as indicated by analytical TLC. The major component was recovered starting alcohol iBO. Fractionation of a 2.48 g portion of this mixture by flash chromatography (5 cm diameter column, 6 inches silica gel, 10% ethyl acetate/petroleum ether) gave 191 264 mg (1.19 mmole, 6% calculated yield based on entire 1 alcohol fraction) of crude regioisomers 76 and 84: H NMR (400 MHz, CDC1 ) 6 5.35 (bs), 5.29 (bs), 5.08 (bt, = 6.4 3 J Hz), 1.68 (bs, CH ), 1.61 (bs, CH ), 0.915 (s, CH ), 0.911 3 3 3 (s, CH ); MS, (rel intensity) 222 (3), 207 (1), 189 (3), 3 m/z 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 radial chromatography (12% ethyl acetate/petroleum ether, flow = 3.5 mL/min) to give pure samples of 7_6 and 84. These were converted directly into the (p-bromophenyl)- urethane derivatives 8_9 and 90. Preparation of a mixture of (p-bromophenyl)urethane derivatives 89 and 90. A total 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 flask. Methanol was added to decompose the excess 4-bromophenyl isocyanate and the mixture was purified by preparative TLC (chloroform) to give 7.3 mg (0.0174 mmol, 1 23%) of a mixture of 89 and 90: H NMR (400 MHz, CDC1 ) 6 3 7.40 (d), 7.27 (d), 5.37 (bs), 5.30 (bs), 5.09 (bt, J = 1 Hz), 4.24 (m), 1.68 (bs, CH ), 1.60 (bs, CH ), 0.943 (s, 3 3 CH ), 0.933 (s, CH ). 3 3 1 92 Preparation of a mixture of (±)-75 and 93• A total 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 stir bar and condenser. The solution was heated, with stirring, 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 sulfate. 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 1 (0.007 mmol, 62%) of a mixture of 75 and 93: H NMR (400 MHz, CDC1 ) 6 7.40 (d), 7.27 (d), 4.23 (m), 0.98 (s, CH ), 3 3 0.97 (s, CH ), 0.94 (s, CH ), 0.935 (s, CH ), 0.92 (s, CH ), 3 3 3 3 0.91 (s, CH ). 3 Preparation of (p-bromophenyl)urethane derivative 90. A total 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, CDC1 ) 6 7.41 (d, 2H), 7.27 (d, 2H), 6.51 3 (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 79 observed 419.1447, required for C H BrN0 419.1460; MS, 22 30 2 m/z (rel 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 total 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: 1 H NMR (400 MHz, CDCl ) 6 7.40 (d, 2H), 7.26 (d, 2H), 6.51 3 (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 79 C H BrN0 419.1460; MS, (rel intensity) 421 (11),. 22 30 2 m/z 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 total of 2.9 mg (0.0069 mmol) of urethane 8_9 was cyclized following the same procedure described for the mixture of 89 and 90. Purification by preparative TLC (chloroform) gave 2.0 mg (0.0048 mmol, 69%) of (±)-7_5 1 94 identical by *H NMR, MS, and HPLC retention time to natural product derivative 75. 195 Phi dol opora pacifica Collection Data. Phidolopora pacifica was collected by hand using SCUBA from Diceman Island in the Broken Group and from a variety of sites in the Deer Group of Islands, Barkley Sound, British 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, until used. Extraction and Chromatographic Separation. 1. Methanol Extraction A number of collections were extracted with methanol. The same isolation scheme was used for each extraction. The following represents a typical extraction and isolation 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 filtration of the crude extract in the presence of Celite gave a greenish brown methanolic filtrate 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 sulfate. The 1 96 ethyl acetate was evaporated to give 796 mg (0.56%) of a dark greenish-brown crude oil. The oil was fractionated by flash chromatography (40 mm diameter column, 6 inches silica gel, step gradient of 5% ethyl acetate/petroleum ether to 20% methanol/ethyl acetate) to yield fractions containing fats, sterols, 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. Filtration (in vacuo) and concentration of the filtrate 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 oil. Flash chromatography (40 mm diameter column, 6 inches silica gel) using a step gradient of 5% ethyl acetate/petroleum ether to 20% methanol/ethyl acetate gave different fractions containing fats, sterols, 4-ethoxymethyl-2-nitrophenol (209) , phidolopin (179), and desmethylphidolopin (180). Fractions from the column that contained 209 were combined and further purified 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 oil. 1 4-methoxy-2-nitrophenol (189): H NMR (80 MHz, CDC1 ) 6 3 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 183.0534, C H NO„ requires m/z 8 9 183.0532; MS, m/z (rel 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, CDC1 ) 3 6 10.58 (s, 1H), 8.14 (d, / = 2.2, 1H), 7.63 (dd, / = 2.2,8.5 Hz,' 1H), 7.18 (d, / = 8.5 Hz, 1H), 4.71 (s, 2H), 1.61 (bs, OH + H 0); HRMS, observed 169.0379, C H NO« 2 m/z 7 7 requires 169.0375; MS, m/z (rel 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 (CH CN); UV (CH CN) 351 nm 3 3 (e 3,300), 275 ( 16,800); IR (CHC1 cast) 3300 (b), 1697, 3 1 1657, 1626, 1532 cm" ; 'H NMR (270 MHz, CDC1 ) 10.56 (s, 1H, 3 exchanges with D 0), 8.08 (d, / = 2.2 Hz, IH), 7.63 (s, 1H), 2 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 C H N 0 331.0917; MS, (rel 1fl l3 5 5 m/z intensity) 331 (20), 313 (17), 180 (75), 150 (100). 198 1 Desmethylphidolopin : H NMR (400 MHz, DMSO-d ) 5 11.16 (180) 6 (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 (rel 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). 1 4-Ethoxy-2-nitrophenol (209): H NMR (80 MHz, CDC1 ) 8 10.57 3 (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 (rel 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 acetonitrile (heated to dissolve, 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 acetonitrile. The mixture was stirred 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 light yellow colour was present in the organic layer. The ethyl acetate was dried over magnesium sulfate, and concentrated in vacuo. Purification by preparative TLC (5% methanol/chloroform) gave 3 mg (0.006 mmol, 100%) of derivative 194; off white solid; mp 197 °C 1 dec; H NMR (400 MHz, CDC1 ) 5 7.80 (d, = 2.3 Hz, IH), 3 J 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 Hi/cm * 3 2 1 0 Appendix 1. 400 MHz NMR spectrum of 75 in CDC1 . 3 f Hi/cm M. 10 000 8 000 4 2000 mo Hi 5 000 000 1000 50 Hi 2 500 2 000 500 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) Ritter, F.J., Ed. "Chemical Ecology: Odour Communication in Animals"; Elsevier: Amsterdam, 1979. (c) Rockstein, M., Ed. "Biochemistry of Insects"; Academic Press: New York, 1978. (d) Harborne, J.B. 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