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Polyketide derived metabolites from the marine hydroid Garveia annulata Fahy, Eoin 1986

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POLYKETIDE DERIVED METABOLITES FROM THE MARINE HYDROID GARVEIA ANNULATA by EOIN FAHY B.Sc. University College Galway, 1981. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department of Chemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August 1986 Eoin Fahy, 1986. In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of C h E ^ I S T ^  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 2F±\ ABSTRACT The marine hydroid Garveia annulata i s a small, b r i g h t l y colored coelenterate whose crude methanol extracts exhibit potent a n t i b a c t e r i a l and antifungal a c t i v i t y . The compounds responsible for t h i s b i o l o g i c a l a c t i v i t y were p u r i f i e d and characterised as a series of related 1-[4H]-anthracenone derivatives. Twenty one metabolites have been isolated and their structures were elucidated by using a combination of spectral analysis, chemical interconversions, synthesis and single c r y s t a l X-ray d i f f r a c t i o n analysis. The structure of garveatin A (22)r the major metabolite, was determined v i a a series of NMR experiments and by an X-ray d i f f r a c t i o n analysis of i t s enol t r i a c e t a t e £2 . Garveatins B (88), C (£3.) and D (M) share a common carbon skeleton with 77. The garvin family represents a d i f f e r e n t polyketide folding pattern as seen in garvin A (124) and garvin B (126.) which contain an n-propyl group and a delta lactone f u n c t i o n a l i t y , respectively. Both the garveatins and the garvins contain oxidized analogs i n the form of 2-hydroxy derivatives, 9,10 quinones and C2,2' dimers. NMR analysis and o p t i c a l rotation experiments indicate that the C2 p o s i t i o n of the 2-hydroxy compounds i s racemic. Garvalones A (137) and B (141) represent the corresponding 2-(3-oxobutyl) derivatives of garvins A and B respectively. They occur as pairs of C2 epimers. Their structures were confirmed by spectral comparison with 2-(3-oxobutyl) garveatin A (140) which was synthesised from 77. Annulins A (144) and B (148) have degraded anthracene skeletons and they appear to be products of garveatin B metabolism. A l l four families of annulata secondary metabolites appear to be produced by straightforward polyketide biogenesis. Different folding patterns of a putative nonaketide precursor account for a l l the structures elaborated. These polyketides represent the f i r s t examples of t h i s type of metabolism i n coelenterates. i i i TABLE OF CONTENTS Abstract i i L i s t of Tables v L i s t of Schemes v i L i s t of Plates v i L i s t of Figures v i i Acknowledgements x Abbreviations x i I. INTRODUCTION A. Marine Natural Products Chemistry 1 B. Hydroid Zoology and Chemistry 8 C. Condensed Polyketides from Marine Invertebrates 16 II . SECONDARY METABOLITES FROM THE HYDROID GARVEIA ANNULATA A. The Garveatins 38 B. The Garvins 92 C. The Garvalones 135 D. The Annulins 153 I I I . DISCUSSION 169 IV. EXPERIMENTAL 176 V. BIBLIOGRAPHY 206 iv LIST OF TABLES 1. 1 3 C NMR data for garveatin A (12) and ferruginin A (£3.) .... 46 2. *H NMR data for garveatin A and ferr u g i n i n A derivatives...46 3. NMR data for garveatins A-D 62 4. C NMR data for the garveatin compounds 63 5. "^H NMR data for the garvin compounds 108 6. "'"H NMR data for the acetates of the 2-hydroxy der i v a t i v e s . 110 7. NMR data for the quinones . 112 8. 1H NMR data for the dimers 116 9. *H NMR data for the garvalones 141 10. C NMR data for the garvins and garvalones 142 11. 1H NMR data for the annulins 161 12. 1 3C NMR data for the annulins 162 13. Results of i n - v i t r o a n t i b i o t i c assays for the G. annulata metabolites 171 v LIST OF SCHEMES 1. Mass spectral fragmentation of phenol acetate 53 2. Synthesis of 2-acetoxy f 8,9-dimethyl garveatin A (101) 66 3. Proposed biogenetic pathway for the garveatins 81 4. Proposed biogenetic pathway for the garvins 119 5. Proposed route for production of garvalone A 145 6. Proposed biogenetic pathway for the annulins 164 LIST OF PLATES 1. Garveia annulata 37 v i LIST OF FIGURES 1. Phylogenetic c l a s s i f i c a t i o n of Garveia annulata 9 2. L i f e - c y c l e of the marine hydroid Obelia 11 3. I l l u s t r a t i o n of annulata 13 4. Long-range HETCOR spectrum of garveatin A (22) 48 4a. Pulse sequence for long-range HETCOR experiment on 22 49 5. Computer generated x-ray structure of the enol t r i a c e t a t e of garveatin A (£2) 55 6. 80 MHz 1H NMR spectrum of trimethyl garveatin A (&QJ 82 7. 80 MHz 1H NMR spectrum of garveatin B (M) 83 8. 80 MHz 1H NMR spectrum of garveatin C (22.) 84 9. 270 MHz 1H NMR spectrum of garveatin D (M) 85 10. 80 MHz 1H NMR spectrum of 2-hydroxy garveatin A tr i a c e t a t e (M) 86 11. 80 MHz 1H NMR spectrum of 2-hydroxy garveatin B (102) 87 12. 80 MHz 1H NMR spectrum of garveatin A quinone (1H5J 88 13. 270 MHz 1H NMR spectrum of garveatin B dimer (1M) 89 14. 100 MHz 1 3 C NMR spectrum of garveatin C (<L3J 90 1 3 15. 75 MHz C NMR spectrum of garveatin A quinone (1H5_) 91 16. 75 MHz INAPT spectra of 2-hydroxy garvin A (112.) 99 17. Computer generated X-ray structure of the acetate of garvin A quinone (123) 102 18. 400 MHz 1H NMR spectrum of 2-hydroxy garvin A (Hi) 120 18a. 400 MHz 1H NMR spin simulation for the n-propyl side chain of H £ 121 v i i 19. 300 MHz -"-H NMR spectrum of 3-methyl garvin A (125.) 122 20. 80 MHz 1H NMR spectrum of garvin A quinone (122) 123 21. 300 MHz 1H NMR spectrum of trimethyl garvin B (125.) 124 21a. 300 MHz NMR spin simulation for the lactone spin system i n 129 125 22. 270 MHz 1H NMR spectrum of 2-hydroxy garvin B (13£) 126 23. 300 MHz-'-H NMR spectrum of 2-hydroxy garvin B t r i a c e t a t e (131&) 127 24. 300 MHz^H NMR spectrum of 2-hydroxy garvin B tri a c e t a t e (131b) 128 25. 270 MHz 1H NMR spectrum of garvin B quinone (132) 129 25a. Comparison of the 400 MHz *H NMR lactone spin system of 132 with a simulated spectrum 130 26. 400 MHz 1H NMR spectrum of mixed dimer (13JI) 131 27. 100 MHz 1 3H NMR spectrum of 2-hydroxy garvin A (119) 132 28. 75 MHz 1 3 C NMR spectrum of 2-hydroxy garvin B (13j0_) 133 29. 75 MHz 1 3 C NMR spectrum of garvin B quinone (112) 134 30. 400 MHz 1H NMR spectrum of garvalone A (13_1) 146 31. 400 MHz 1H NMR spectrum of methyl garvalone A (131) 147 31a. 400 MHz 1H NMR spin simulation of the H18-19 system in 131 148 32. 400 MHz 1H NMR spectrum of 2-(3-oxobutyl)-garveatin A(14jOJ 149 33. 80 MHz 1H NMR spectrum of garvalone B (HI) 150 34. 75 MHz 1 3 C NMR spectrum of garvalone A (132) 151 35. 75 MHz 1 3 C NMR spectrum of 2-(3-oxobutyl)-garveatin A (140) 152 v i i i 36. Computer generated X-ray structure of annulin A (144)....157 37. 300 MHz 1H NMR spectrum of annulin A (144) 165 38. 80 MHz 1H NMR spectrum of annulin B (14£) 166 39. 75 MHz 1 3 C NMR spectrum of annulin A (144) 167 40. 75 MHz 1 3 C NMR spectrum of annulin B (148.) 168 ix ACKNOWLEDGEMENTS I would l i k e to extend my sincere thanks to my research supervisor, Dr. Raymond Andersen, for his advice, encouragement and friendship during a most enjoyable four years i n Vancouver. I am grateful to Mike LeBlanc for performing the bioassays and a s s i s t i n g with the hydroid c o l l e c t i o n s , and to my co-workers in the laboratory for th e i r support. The assistance of the s t a f f of the departmental NMR and Mass Spec, laboratories and also the s t a f f of Bamfield Marine Station i s greatly appreciated. x ABBREVIATIONS CDC13 = Chloroform-d 1 CHC13 = Chloroform CH3CN = A c e t o n i t r i l e DMSO = Dimethylsulfoxide EtOAc = Ethyl acetate HPLC = High performance l i q u i d chromatography HRMS = High resolution mass spectrum i = impurity peak IR = Infra red MeOH = Methanol MS = (Low resolution) mass spectrum Na2S04 = Sodium sulfate (anhydrous) ''"H NMR = Proton nuclear magnetic resonance 13 C NMR = Carbon -13 nuclear magnetic resonance nOe = nuclear Overhauser enhancement mp = melting point RT = room temperature S = solvent signal SFORD = Single frequency off resonance decoupled SCUBA = Self contained underwater breathing apparatus TLC = Thin layer chromatography TMS = Tetramethyl s i l a n e UV = Ul t r a v i o l e t w = water signal x i "We dance round a ring and suppose, But the secret s i t s i n the middle and knows." R. Frost (The Secret Sits) xii I: INTRODUCTION. A. MARINE. NATURAL PRODUCTS CHEMISTRY. Research i n natural products chemistry (the study of secondary metabolites) has t r a d i t i o n a l l y focused on t e r r e s t r i a l species, mainly from the plant and microbial kingdoms, as sources of compounds which exhibit i n t e r e s t i n g b i o l o g i c a l a c t i v i t y . Many of the pharmacologically active drugs i n use today, such as morphine, atropine and streptomycin are natural products or semi-synthetic derivatives thereof. U n t i l recently, the vast number of organisms found i n the oceans of the world have been l a r g e l y untapped as a source of b i o l o g i c a l l y active molecules. However, the great pot e n t i a l of the marine environment has been recognised for some time, as evidenced i n the results of screening tests conducted i n the early 1970's on over 1600 diverse marine s p e c i e s 1 . The results showed that 9% contained compounds with antitumor a c t i v i t y , compared with 2-3% of t e r r e s t r i a l organisms. A number of technological advances i n the l a s t 25 years have catalysed the growth of marine natural products chemistry, and attracted researchers into t h i s area of study. The advent of SCUBA diving has greatly f a c i l i t a t e d the c o l l e c t i o n of sub-tidal organisms . which were previously quite inaccesible compared to t h e i r t e r r e s t r i a l counterparts. Advances i n the use of physical techniques such as NMR spectroscopy, mass spectrometry and x-ray d i f f r a c t i o n analysis has enabled researchers to elucidate the 1 structures of compounds present in very small amounts, a si t u a t i o n often encountered with marine metabolites. Screening large numbers of marine organisms has revealed some gen e r a l i t i e s about the d i s t r i b u t i o n of b i o l o g i c a l l y active 2 substances . Considerable interest has recently been focused on members of the more primitive marine phyla as sources of secondary metabolites. Creatures p a r t i c u l a r l y r i c h in toxic chemicals include slow-moving, s h e l l - l e s s molluscs l i k e sea-hares and nudibranchs, and soft-bodied s e s s i l e organisms l i k e sponges, tunicates, sof t - c o r a l s and gorgonians. Clues about the presence of bioactive compounds can be obtained by studying the ecology and behaviour of marine organisms. For example, i n densely populated habitats such as coral reefs and tidepools, s e l e c t i o n pressures have favored the evolution of chemical defenses. Many organisms can synthesize toxic substances or concentrate them from food sources i n order to deter predators or keep competitors from approaching too c l o s e l y . Thus, many families of marine invertebrates that outwardly appear to be t o t a l l y defenseless ac t u a l l y have few predators. The potential of the marine environment as a source of new pharmaceuticals i s beginning to be rea l i s e d as a number of compounds are currently undergoing c l i n i c a l t r i a l s as a n t i b i o t i c , antitumor, analgesic and anti-inflammatory agents 3' 4. The 5 compound 1-B-D-arabino- furanosyl cytosine (ARA-C) (1) i s used i n cancer chemotherapy as an anti-leukemic drug. It i s a derivative of spongouridine (ARA-U) (2) isolated from the sponge Cryptothetia c r y p t a 6 and more recently from the gorgonian 2 Eunicella c a v o l i n i . The tunicates, comprising some 2000 species, are p a r t i c u l a r l y r i c h i n bioactive substances. Rinehart's group has isolated the c y c l i c peptide didemnin (3) from the Caribbean o tunicate Tridemnum cyanophorum . C l i n i c a l tests i n - v i t r o have shown that t h i s molecule i n h i b i t s the growth of L1210 leukemia c e l l s at a concentration of only 0.001 umole/ml. The s e s s i l e , p l a n t - l i k e , c o l o n i a l invertebrates from the phylum Bryozoa have yielded a number of interesting metabolites in recent years. P e t t i t ' s group from Arizona State have obtained a series of anti-tumor compounds named bryostatins from the bryozoan Bugula n e r i t i n a , a species that forms moss-like colonies on ships h u l l s and other marine equipment. These compounds are strongly cytotoxic and anti-leukemic, and i t has been suggested that the 20 membered lactone ring structure acts as an ionophore, making c e l l membranes porous to certain ions. Bryostatin 1 (4) i s 9-11 an example of t h i s family of metabolites A series of four bipyroles, named tambjamines A-D (5.-.8J have been isol a t e d from the bryozoan Sessibugula translucens by Faulkner's group and they show antimicrobial and cytotoxic 12 a c t i v i t y . These compounds were previously found in nudibranchs of the Tambje genus. 13 The B r i t i s h Columbia bryozoans Phidolopora p a c i f i c a and Diaperoecia c a l i f o r n i c a * 4 have yielded the purine alkaloids phidolopin (9) and desmethyl phidolopin (10). containing the rare na t u r a l l y occurring n i t r o group. These compounds are responsible for much of the antifungal and a n t i a l g a l a c t i v i t y associated with extracts of t h i s organism. 4 2 H HO PAc > ^ 0 H C 0 2 M e 4 N H OMe NH 5 X = H 6 X =Br N H OMe N / NH 7 X = H 8 X = Br 5 9 R = C H. 10 R -- H 6 The above examples serve to i l l u s t r a t e that metabolites from marine sources exhibit a wide var i e t y of b i o l o g i c a l a c t i v i t i e s . Usually , these compounds are assumed to act as chemical defenses for the host organisms, however, most of the evidence i s circumstantial, due to the d i f f i c u l t i e s of testing the defensive hypothesis. The l i t e r a t u r e on marine natural products chemistry has been extensively reviewed i n a series of books and review a r t i c l e s by Scheuer 1^ and Faulkner 1**, i l l u s t r a t i n g the rapid progress and d i v e r s i f i c a t i o n achieved i n t h i s f i e l d of research i n the l a s t twenty years. This thesis w i l l describe the i s o l a t i o n and structure elucidation of a group of novel secondary metabolites from the P a c i f i c hydroid Garveia annulata which exhibit potent i n - v i t r o a n t i b a c t e r i a l and antifungal a c t i v i t y . These compounds, which appear to be of polyketide o r i g i n , represent an inte r e s t i n g v a r i a t i o n i n coelenterate chemistry and t h i s study w i l l hopefully contribute to the growing body of research on the chemistry and ecological roles of bioactive metabolites from the ocean. 7 B. HYPRQIP ?OOLOGY AND. CHEMISTRY The marine hydroid Garveia annulata i s a member of the phylum Coelenterata (or Cnidaria), containing about 9000 l i v i n g species. In addition to hydroids, t h i s phylum also includes j e l l y f i s h e s , sea-anenomes and corals. Coelenterates show a number 17-20 of distinguishing features . They have a body wall consisting of two layers of c e l l s . The outer layer i s c a l l e d the epidermis, and the inner layer , which forms the l i n i n g of the gut i s c a l l e d the gastrodermis. Between these two layers i s a j e l l y - l i k e substance known as the mesoglea. Coelenterates exhibit two d i f f e r e n t body forms, the medusa which i s adapted to a pelagic existence, and the polyp which i s adapted to an s e s s i l e benthic existence. Most species feed on zooplankton and fi n e p a r t i c u l a t e matter. Prey i s caught with the tentacles surrounding the o r a l cavity and immobilised by specialised stinging c e l l s c a l l e d nematocysts which are unique to t h i s phylum. The mouth i s the only opening into the gut cavity, and these animals lack organs or d i f f e r e n t i a t e d muscle c e l l s . There are three classes of coelenterates: the Hydrozoa, Scyphozoa and Anthozoa (Figure 1.). The Scyphozoa are composed mainly of the large j e l l y f i s h e s , whereas the sea-anemones and most hard corals belong to the Anthozoa. The class Hydrozoa i s subdivided into two orders : the Siphonophora which contain f r e e -swimming species such as the freshwater organism Hydra, and the 8 PHYLUM COELENTERATA £LA£& ANTHOZOA (sea anemones hard corals) HYDROZOA SCYPHOZOA (hydroids,hydra) (large j e l l y f i s h ) QBHER HYDROIDEA (ses s i l e polyps) SIPHONOPHORA (free-swimming colonies) SUBORDER GYMNOBLASTEA FAMILY ACTRACTYLIDAE QEfflI£ GARVEIA SPECIES ANNULATA Figure 1. Phylogenetic c l a s s i f i c a t i o n of the marine hydroid Garveia annulata. 9 Hydroidea, composed mainly of s e s s i l e c o l o n i a l organisms known as hydroids. annulata belongs to the l a t t e r order. The hydroids are generally small, r e l a t i v e l y inconspicuous, p l a n t - l i k e organisms which are often dismissed as "sea-weed". They are wholly aquatic, being found i n both fresh and s a l t water, and they exhibit a metagenic l i f e - c y c l e composed of an asexual polypoid stage and a sexual medusa stage. A hydroid colony consists of a network of interconnected polyps or zooids which share a common ga s t r o i n t e s t i n a l cavity. The three body layers i n a hydroid colony , epidermis, mesoglea and gastrodermis are a l l continuous, making i t d i f f i c u l t to say where one indiv i d u a l begins and another ends. The organism i s attached to a rock or other substratum v i a a basal stem known as a hydrorhiza or stolon. Branches or pedicels extend from t h i s structure which in turn give r i s e to bell-shaped hydranths. A single polyp consists of a hydranth and the part of the stalk between the hydranth and the point of o r i g i n of the preceeding branch. The external surface of the colony i s protected by a chitinous layer c a l l e d the perisarc which i s expanded into bell-shaped hydrothecae to accomodate the hydranths. Around the mouth of the hydranth are located a number of tentacles which contain nematocysts at the extremities. The hydranth captures, ingests and digests food, which then i s transported to the common gastrovascular cavity. Hydroids are polymorphic organisms that contain reproductive polyps c a l l e d gonozooids i n addition to the hydranths (gastrozooids). These gonozooids produce medusae by asexual 10 11 budding. The medusae eventually become detached and leave the colony. Some . of the medusae produce eggs while others produce spermatozoa. F e r t i l i s e d eggs give r i s e to c i l i a t e d larvae, c a l l e d planulae, which become fixed to some object and form a new colony. This i s the f i r s t stage of hydroid generation. As the young polyp grows i t puts out branches that terminate i n new polyps. Eventually, reproductive polyps appear and another generation of medusae i s produced. Figure 2 i l l u s t r a t e s the l i f e -cycle of Qbelia, a t y p i c a l c o l o n i a l hydroid. G. annulata f which i s found along the P a c i f i c coast of North America from Sitka, Alaska to Santa Catalina Island, C a l i f o r n i a , 21 was i d e n t i f i e d by Nutting i n 1901 from samples obtained by the Harriman expedition i n Alaska (see Figure 3). It t y p i c a l l y inhabits exposed rocky reefs at a depth of 2-120 metres and i s most abundant in l a t e winter and early spring. Colonies of G. annulata f which are 5-10 cm t a l l and consist of 20-30 polyps, are often found growing on or through sponges and algae. The most d i s t i n c t i v e feature of t h i s organism i s i t s bright orange colour, making i t quite conspicuous compared to most other hydroids. The oval-shaped gonophores are s e s s i l e medusoid structures which on a given colony are either a l l male or a l l female. Male medusoids shed t h e i r sperm to the sea and f e r t i l i s e eggs from the female medusoids. These eggs are retained by the female gonophores and are eventually l i b e r a t e d as planula larvae, which can s e t t l e and form a new colony. L i t t l e else i s known of the species. G. annulata gets i t s name from the numerous rings or annulations on 22 the stems of the colonies 12 13 Hydro-ids seem to have received scant attention from zoologists and chemists a l i k e , possibly due to t h e i r small and inconspicuous nature and to the d i f f i c u l t i e s i n c o l l e c t i n g and i d e n t i f y i n g the d i f f e r e n t species. With the exception of t h i s t h e s i s , the limited number of hydroid chemical studies have a l l 23 been carried out on Mediterranean species. In 1980, Cimino reported the i s o l a t i o n of the novel polyhydroxylated s t e r o i d 11, which i s characterised by a C18 oxygen f u n c t i o n a l i t y , from the hydroid Eudendrium sp. More recently, Fattorusso's group 24,25 have isolated three steroids 12-14 from Eudendrium glomerulatum, a l l of which were hydroxylated at the 2, 3, and 18 positions. 2 6 E. glomerulatum has also yielded four a c y c l i c , polyhalogenated monoterpenes 15-18, which had been previously iso l a t e d from the red alga Plocamium cartilagineum and the sea-hare Aplysia limacina. 14 OH 15 C. CONDENSED POLYKETIDES FROM HAfilRB. INVERTEBRATES Introduction A l l of the organic soluble metabolites discussed i n t h i s thesis appear to be derived from li n e a r polyketide precursors which are subsequently modified by a variety of condensation, methylation, a l k y l a t i o n and oxidation reactions. In order to place the array of metabolites obtained from G_«_ annulata i n perspective, a review of polyketide -derived compounds from marine organisms i s presented below. Although none of these metabolites were obtained from organisms belonging to the same phylum (Coelenterata) as G.annulata, many st r u c t u r a l s i m i l a r i t i e s were evident, and the l i t e r a t u r e reviewed provided many helpful insights into the structure elucidation of the G_«_ annulata metabolites. The marine environment has long been recognised as an abundant source of b r i g h t l y coloured organisms, e s p e c i a l l y from the phyla Echinodermata and Coelenterata. The Echinoderms, many of which have b r i l l i a n t l y pigmented outer surfaces, have attracted considerable attention from chemists over the l a s t 100 years due to t h e i r r e l a t i v e ease of c o l l e c t i o n . Echinoderms are divided into f i v e d i s t i n c t classes: Crinoidea ( s e a - l i l i e s ) , Asteroidea (sea-stars), Ophiuroidea ( b r i t t l e - s t a r s ) , 17 Holothuroidea (sea-cucumbers) and Echinoidea (sea-urchins) . A l l of these classes have been shown to elaborate pigments of 16 polyketide o r i g i n but researchers in t h i s area have concentrated on sea-urchins (Echinoidea) and the crinoids (Crinoidea). Echinoidal Pigments Sea-urchins are protected by numerous calcareous spines of varied hues which protrude from an oval s h e l l or t e s t . The spines and tests contain polyhydroxylated naphthoquinone pigments bound as s a l t s that are released upon d i s s o l u t i o n of the calcareous material with strong acid. The compounds isolated to date can be divided into two groups based on the presence of juglone (19) or naphthazarin (20) skeletons. A s e m i - t r i v i a l nomenclature based on 27 these structures has been adopted. The study of echinoidal pigments was i n i t i a t e d by Mc Munn in 2 8 1885 when he reported the presence of a red compound c a l l e d "Echinochrome A" in the sea-urchin Echinus esculentus. The structure was shown to be 6-ethyl- 2,3,7- trihydroxynaphthazarin (21) by wallenfels i n 1939 2 9, and v e r i f i e d by s y n t h e s i s 3 0 . Prior to the 1960's, research i n t h i s area had been greatly hindered by inadequate methods for purifying these pigments, then known as "spinochromes", and by misleading combustion analyses, resu l t i n g i n d i f f e r e n t structure proposals for the same compound by d i f f e r e n t groups. The advent of NMR spectroscopy and improved mass-spectral insruments, allowed groups led by Scheuer in Hawaii, Sutherland in A u s t r a l i a and Thomson in Scotland to c l a r i f y the s i t u a t i o n by c o r r e c t l y assigning the structures of the spinochromes A-E (22-26). These pigments are widely 31-33 d i s t r i b u t e d i n sea-urchins , and the synthesis of several spinochromes has been reported by Scheuer and Thomson^^"^8# 17 OH 0 24 COCH 3 OH OK 25 OH H OH 26 OH OH OH 18 By 1970, about twenty pigments containing substituted juglone and naphthazarin skeletons had been i d e n t i f i e d . The range of substituents included a c e t y l , e t h y l , hydroxyl and methoxyl groups. The co-occurrence of methoxylated naphthazarins (22,28.) in Diadema antillarum P h i l l i p s raised the p o s s i b i l i t y that some of the isolated naphthoquinones are a r t i f a c t s formed by hydrolysis of methylated natural products in the 6N HC1 solution used i n the extraction procedure 4 0. It i s quite possible that a 2,3-dimethyl precursor of 22 or 22. could be monodemethylated during the HC1 digestion process, considering how rapidly t h i s type of reaction occurs in refluxing ethanol-HCl 4 1. An interesting feature of the naphthazarin-type compounds i s the p o s s i b i l i t y of tautomerism due to the presence of hydroxyl groups at the 5- and 8- positions. NMR studies and transacylation reactions have been u t i l i s e d by Moore and Scheuer to decide which i s the predominant tautomer i n s o l u t i o n . When naphthazarin i s monosubstituted with methoxy, hydroxy, ethyl or acetoxy groups, the structure was shown to be 22. i n chloroform s o l u t i o n . This assignment was based on the observation that the C3 proton resonates further u p f i e l d than the C6 and C7 aromatic protons i n model juglones and naphthoquinones. Also, when R=Et, the C3 proton i s present as a sharp t r i p l e t due to a l l y l i c coupling with the methylene protons of the side-chain, indicating the presence of a fixed double bond in the quinone ring rather than a delocalised bond. In contrast, 2-acetylnaphthazarin has structure 30, indicating an 'aromatic at t r a c t i o n * of the acetyl group. Thus in 19 OH 0 R = OH, OCH 3 , OAc or Et OH 0 0 30 31 20 disubstituted naphthazarins containing one acetyl group, the nature of the predominant tautomer i s determined by the degree of •quinoidal a t t r a c t i o n ' or 'aromatic a t t r a c t i o n ' of the other substituent. Mass spectrometry has also proved quite useful i n structure 43 determination of these molecules. Djerassi and Scheuer have proposed a set of empirical rules for analysing the mass-spectral fragmentation of naphthoquinones. Their rules are based on results from a large number of natural and synthetic compounds. As would be expected of b r i g h t l y coloured pigments, UV-visible spectroscopy i s a valuable tool i n the gross s t r u c t u r a l analysis of the naphthoquinones and in the determination of t h e i r 39 substitution patterns . A number of more unusual compounds, in addition to the simple juglone and naphthazarin derivatives outlined above, have been isolated from echinoids. The pigment 2-methyl-8-hydroxy-2H-pyrano[3,2g]naphthazarin (31) isolated from Echinothrix diadema repesented the f i r s t sea-urchin compound with a side-chain greater than two carbon atoms. Thomson's group has reported the binaphthoquinone ethylidene -3,3-bis(2,6,7- trihydroxynaphthazarin) (32) and i t s anhydro 40 derivative 22 from the spines of Spatangus purpureus . Compound 22 did not exhibit a molecular ion i n the mass spectrum. Instead, the spectrum contained a base peak at m/z 238 and the fragmentation pattern was e s s e n t i a l l y the same as that displayed by 2Ar a compound present i n minor amounts i n the same extract. Methylation of 22. with diazomethane yielded the hexamethyl ether 21 35 which gave a weak molecular ion at m/z 586 in d i c a t i v e of a dimeric structure. Structure 12 was confirmed by condensing trihydroxynaphthazarin (34) with acetaldehyde to give a biquinone i d e n t i c a l by TLC, UV and IR with 12. The i n s t a b i l i t y of the biquinones 12 and 15. on acid-treated s i l i c a gel i s noteworthy since one of the breakdown products i s compound 14. However, the authors argue that 14 i s a genuine natural product since treatment of 12 with cold concentrated HC1 for 12 hours did not promote decomposition. Dehydration of 12 with hot sulphuric acid yielded an anhydro compound i d e n t i c a l with 33. The role of the naphthoquinone pigments i n the metabolism and ecology of the echinoids remains an open question. Reports of the absence of p a r t i c u l a r compounds from d i f f e r e n t specimens of 39 the same species indicate that a central metabolic role i s 45 un l i k e l y . Vevers has proposed that these compounds may function as a l g i s t a t s , which i s i n keeping with the defensive role of a number of natural products from various other marine invertebrates 4* 5' 4^ such as sponges, corals and nudibranchs. Inspection of the s t r u c t u r a l formulae of the naphthoquinone compounds suggests a polyketide o r i g i n and support for t h i s 48 biosynthetic pathway has been provided by Lederer's group who have shown that [2- 1 4C] l a b e l l e d acetate i s incorporated into 6-e t h y l - 2,3,7- trihydroxynaphthazarin (21) by the sea-urchin Arabacia pustulosa. It i s noteworthy that several plant products are c l o s e l y related to the echinoidal pigments, and 2,7-dihydroxy naphthazarin (36). a constituent of several sea-urchins, has also 23 been found i n the fungus Helicobasidium mompa Tanaka . Crin o i d a l Pigments The crinoids are the most ancient class of the phylum Echinodermata with an extensive f o s s i l record consisting mainly of stalked crinoids ( s e a - l i l i e s ) , some of which s t i l l e x i s t . The majority of the l i v i n g species are comprised of about 550 fr e e -swimming comulatids, also known as feather-stars. In contrast to the echinoids, the crinoids generally do not contain naphthazarin or juglone derivatives, but instead produce compounds based on anthraquinone (37) and naphthopyrone (38) structures. These compounds are e a s i l y extracted from the organisms with acetone or other organic solvents. Studies of the chemistry of the highly coloured crinoids started with the work of Moseley on board the Challenger i n the 52 East Indies i n 1874 . Moseley described spectroscopically the pigments "purple pentacrinin", "red pentacrinin" and "antedonin" from a number of deep-sea species. Subsequently i n 1890, Mc Munn examined the extract of a c r i n o i d which i s now believed to be 53 Ptilometra a u s t r a l i s Wilton As was the case for the echinoidal pigments, the molecular structure of these compounds remained a mystery u n t i l the 1960*s when Sutherland's group i n Queensland, A u s t r a l i a published the structures of three anthraquinones isolated from the bright red 54 cr i n o i d Comatula pectinata . These are c a l l e d rhodocomatulm 6-methyl- (39) and 6,8-dimethyl ethers (40) and rubrocomatulin 7-methyl ether (41). Evidence for the butyryl side-chain i n the rhodocomatulins 24 25 was provided by the formation of 1,3,6,8 - t e t r a -hydroxyanthraquinone (42) and butyric acid on treatment of 3_9_ or 40 with refluxing hydrobromic-acetic acid. This reaction i s apparently f a c i l i t a t e d by s t e r i c hindrance between the butyryl group and the CIO carbonyl, forcing the side-chain out of the plane of the r i n g . It i s interesting to note the s i m i l a r i t y of the lichen pigment s o l o r i n i c acid (43) with the above 55 compounds . In 1972, Thomson reported the structure of rhodolamprometrin (44) from specimens of Lamprometra klunzingeri co l l e c t e d in the Red Sea5**. It d i f f e r s from 3_9_ only in the replacement of the butyryl side-chain with an acetyl group and the absence of the C6 methoxyl group. Compound Al* which contains an extra hydroxyl group, has a UV-visible spectrum t y p i c a l of 1,4,5-trihydroxyquinones and i t s infrared spectrum indicates that both carbonyls are hydrogen-bonded. The three compounds 39-41 have also been isolated as t h e i r water-soluble C2-sulfate S 7 R ft esters ' . Sutherland l a t e r isolated a group of three pigments from the c r i n o i d Ptilometra a u s t r a l i s Wilton and named them rhodoptilometrin (45). isorhodoptilometrin (46) and 59 rhodoptilometric acid (47) These compounds d i f f e r from the Comatula pigments i n that they contain side-chains i n the beta positions rather than i n the alpha p o s i t i o n s . NMR evidence and the observed o p t i c a l a c t i v i t y of £5. indicated the presence of a CHOHCH^CHj group. This assignment was further validated by the i d e n t i f i c a t i o n of propionic acid as one of the products of Kuhn-Roth degradation of 45. The isomeric compound isorhodoptilometrin (46) d i f f e r s from 45 only i n the nature of the hydroxypropyl side-chain. 26 OH 0 OH 4 4 0 45 R ^ H R 2 =(S)-CHOHEt 46 R 1 = H R 2 = C H 2 C H O H C H 3 47 R ^ C O O H R 2 = C H 2 C H 2 C H 3 27 Isorhodoptilometrin 6-methyl ether (48) has previously been described as nalgiovensin, iso l a t e d from the mould Pe n i c i l l i u m  nalgiovensis L a x a 6 0 . It i s inter e s t i n g to note that O-methylation c h a r a c t e r i s t i c of the Comatula compounds does not seem to occur in the Ptilometra pigments. Studies by Scheuer's group on the P a c i f i c c r i n o i d Comanthus  bennetti provide further examples (49-51) of anthraquinones alkylated at the C3 p o s i t i o n 6 1 . Compound A3, was previously known as a degradation product of the corresponding trimethyl ether, ptil o m e t r i c acid, while rhodoptilometrin (45) could be oxidised to y i e l d 50. As well as the anthraquinones, crinoids also contain a varie t y of inter e s t i n g substituted naphthapyrones. Sutherland's group isol a t e d the l i n e a r naphthapyrone comantherin s u l f a t e (52) 62 from the species Comanthera perplexa . The su l f a t e ester f u n c t i o n a l i t y makes t h i s compound water-soluble, and acid hydrolysis y i e l d s the hydrophobic comantherin (53). A second compound, neocomantherin (54). which d i f f e r s from 51 only i n i t s C3 substituent, was isolated from the acid hydrolysate. It i s presumably present i n the c r i n o i d as the su l f a t e ester 55. Methylation of 51 yielded a compound i d e n t i c a l with the known rubrofusarin dimethyl ether (56). The 5-dimethyl derivative of neocomantherin (54) was subsequently isol a t e d from Comantheria  briarus B e l l in 1980 6 3. Green specimens of the c r i n o i d Comanthus parvicurrus  timorensis yielded the cl o s e l y related angular naphthopyrones 57-59, present as sulf a t e e s t e r s 6 4 , i t was noticed that i f the 28 OMe OMe 52 X = s o 3 - R 53 X = H R 54 X = H R 55 X = s o 3 - R 56 X = CH 3 R = CH = CH = CH R 57 R = H R<] = H 58 R=OCH 3 R ^ H 59 -R=OCH 3 R 1 = C H 3 29 crinoids were not transferred d i r e c t l y from the sea to acetone but were allowed to autolyse , the three corresponding s u l f a t e -free compounds were detected, presumably formed by the action of a sulfatase. Sulfation of these phenols with sulfamic acid i n pyridine yielded products of i d e n t i c a l with those observed for the water-soluble compounds 57-59. The l i s t of polyketide pigments from crinoids has recently 6 5 been extended to include three 10,10'-bianthrones 60-62 derived from the known compounds A4. and A6_. These bianthrones t y p i c a l l y contain a p r i n c i p a l ion corresponding to the anthrone formed by cleavage of the central bond and transfer of a H r a d i c a l . The r e l a t i v e sterochemistry at the 10, 10' bond was ascertained to be as shown i n jLQ. or i t s enantiomer, as opposed to the meso form, by comparison with the model compound emodin bianthrone £3_. Natural 60 i s o p t i c a l l y active, indicating that the organism s e l e c t i v e l y produces one enantiomer. Minor amounts of the meso form were also detected i n the extracts, possibly as an a r t i f a c t . The r e l a t i v e stereochemistry of £1 and £2 has not yet been ascertained. A minor component from the same c r i n o i d was i d e n t i f i e d as the bianthraquinone £4. which i s quite similar to the known bianthrone skyrin (65) The authors believe that £A may be formed by a e r i a l oxidation of £1 i n the acetone extract, since there i s a precedent for t h i s type of reaction i n bianthrones. S i m i l a r l y , the phenanthroperylenequinone ££ (also from the c r i n o i d Lampometra palmata gyges) may also be an a r t i f a c t , but the presence of compound £2 i n the absence of bianthrones and biquinones i n Himerometra robustipinna seems to indicate that the 30 OH 0 OH 60 R=Pr R ^ P r 61 R = p r R^CHOHEt 62 R = CHOHEt R^CHOHEt 63 R = C H 3 R 1 =CH 3 OH 0 OH OH 0 OH 64 R = Pr R ^ C H O H E t 65 R = C H 3 R t = C H 3 31 crinoids are capable of synthesising these compounds. It i s inte r e s t i n g to note that a series of hydroxylated phenanthroperylenequinones (68) have been found i n the f o s s i l i s e d remains of a stalked c r i n o i d Apiocrinus ( M i l l e r i c r i n u s ) by 67 Blumer . These compounds, known as f r i n g e l i t e s , may be genuine natural products or a l t e r n a t i v e l y might be derived by geochemical modification of the corresponding bianthrones. Attempts to correlate the presence of p a r t i c u l a r pigments with the taxonomy of the c r i n o i d species studied so far has led to confusing r e s u l t s . Sutherland 6 5 has postulated that the 'non-chemotaxonomic* d i s t r i b u t i o n of polyketides i n comatulid crinoids i s due to the potential a v a i l a b i l i t y of a whole range of polyketides to a l l or many species, so that each species can adjust to i t s p a r t i c u l a r ecological environment by elaborating a d i f f e r e n t s e l e c t i o n of compounds. 57 Experiments carried out by Sutherland's group indicate that the sulfated polyketides might provide crinoids with a chemical defense mechanism since they have been shown to have an i n h i b i t o r y e f f e c t on feeding by f i s h . Surveys of the stomach contents of f i s h c o l l e c t e d from areas where crinoids are known to be present revealed that c r i n o i d fragments were t y p i c a l l y absent, supporting the theory that these compounds render the crinoids unpalatable to predators. With regard to the source of polyketide pigments in c r i n o i d a l t i s s u e , i t i s u n l i k e l y that they are obtained from t h e i r d i e t since the two species Ptilometra a u s t r a l i s and Comatula cratera have been found i n the same habitat but contain 32 OH 0 OH 66 R = Pr R^CHOHEt 67 R 1 = C H 3 R 1 =CH 3 OH 0 OH 68 R'='H or OH 33 completely d i f f e r e n t types of anthraquinones. In the absence of evidence of symbiotic organisms i t i s reasonable to assume that the c r i n o i d a l polyketides are of endogenous o r i g i n . The s i m i l a r i t y of c r i n o i d a l anthraquinones with compounds isolat e d from plants and lichens indicate that polyketide condensation patterns s i m i l a r to those observed i n the biosyntheses of plant anthraquinones may also occur i n c r i n o i d s 6 8 . As Sutherland 5 9 remarked: "Crinoids, which are so p l a n t - l i k e i n external form have some synthetic c a p a b i l i t i e s which are t y p i c a l l y manifest i n plants rather than i n animals". Miscellaneous The other four classes of the phylum Echinodermata have yielded pigments similar to those described above from echinoderms. For example, a monomethyl ether of 3J[ has been 50 isolated from the holothuroid Polycheria rufescens Brandt , 51 while Singh and co-workers have isol a t e d two dimethyl ethers of 26 from the asteroid Acanthaster pla n c i Linn., the compounds 22 and 3JL from an unidentified Antedon c r i n o i d and several naphthoquinones including 21 and 22 from the ophiuroids Ophiocoma  erinaceus M i l l and Trosch and 0 . i n s u l a r i a Lyman. Although the echinoderms have been the most p r o l i f i c source of polyketide-derived metabolites to date, a number of other marine organisms contain examples of these compounds. The sea-worm Halla parthenopeia was found by I t a l i a n r e s e a r c h e r s 6 9 t o contain 7-hydroxy-8-methoxy-6-methyl-l,2-anthraquinone (69) known as hallochrome, which i s responsible for the bright red colour of the epidermal c e l l s of t h i s species. Cimino has recently isolated 34 the corresponding hydroquinone l f2,7-trihydroxy-8-methoxy-6-methyl anthracene (70) from the butanol extracts of the same organism, and has proposed that t h i s compound i s the biogenetic 7 0 precursor of hallochrome . Evidence that marine microorganisms also produce aromatic compounds of polyketide o r i g i n was provided by a Japanese 71 72 group ' in 1975. In the course of screening for a n t i b i o t i c s produced by actinomycetes from marine environments, a Chainia species was isolated from a sea mud and was shown to contain the benz(a)-anthraquinone 21 which was responsible for the a n t i b i o t i c a c t i v i t y . This compound was very unstable to heat and l i g h t , being converted to the l i n e a r naphthacenequinone 72. 35 0 OH OH OH 0 72 36 Plate 1. Garveia annulata (Nutting). I I : SECONDARY METABOLITES FROM THE MARINE HYDROID GARVEIA ANNULATA INTRODUCTION The choice of a suitable marine organism for a natural products study i s dictated by a number of factors, the most important being s u f f i c i e n t a v a i l a b i l i t y , i n t e r e s t i n g b i o l o g i c a l a c t i v i t y of i t s extracts and uniqueness with respect to previous studies. The hydroid Garveia annulata f u l f i l l s these c r i t e r i a as outlined below. G. annulata was collected by hand using SCUBA during the winter and early spring months on exposed rocky reefs , at depths of 2 to 20 metres, in the Deer group of islands i n Barkley Sound, Vancouver Island, B.C. Upon immersion i n methanol, the organism immediately imparts i t s bright orange coloration to the solvent, and i n large quantities gives a dense brownish sol u t i o n . Typical c o l l e c t i o n s have yielded 12-16g of methanol-soluble material from 400g dry weight of hydroid, making i t an extremely r i c h source of secondary metabolites r e l a t i v e to other cold-water organisms studied in t h i s laboratory. The f i r s t c o l l e c t i o n s of G^ . annulata were made in 1983. B i o l o g i c a l screening conducted on the crude methanol extract indicated the presence of substantial i n - v i t r o a n t i b a c t e r i a l and antifungal a c t i v i t y . Therefore, t h i s organism represented an obvious target for i s o l a t i o n and structure elucidation of the compound(s) responsible for the observed b i o l o g i c a l a c t i v i t y . In addition, the extreme paucity of data on natural-products from hydroids (only one novel metabolite from a hydroid had ever been 38 reported at that stage ), increased the p r o b a b i l i t y that G.  annulata extracts might well contain d i s t i n c t i v e l y d i f f e r e n t chemistry from that of previously studied invertebrate species. Examination of the extracts of G_*_ annulata has revealed the presence of four families of polyketide-derived metabolites. The p u r i f i c a t i o n and structure elucidation of each family w i l l be discussed separately. They are as follows: A: The garveatins. B: The garvins. C: The garvalones. D: The annulins. A. THE GARVEATINS (i) Garveatin A Preliminary investigations of the crude methanol extract of G. annulata by s i l i c a - g e l thin layer chromatography (TLC) using 1:50:50 acetic acid/ethyl acetate/hexane as an eluant (this solvent system w i l l be referred to as the "standard TLC system" in the remainder of t h i s thesis) indicated that the major component of the mixture was a yellow-orange compound with an R f of 0.37. This major band had a dark yellow long-wave UV fluorescence. When acetic acid was omitted from the solvent system, t h i s component showed considerable streaking on s i l i c a -g e l , giving a bright yellow long-wave UV fluorescence. These observations indicated the presence of an a c i d i c molecule containing a highly conjugated chromophore. The p u r i f i c a t i o n 73 scheme used to p u r i f y t h i s compound, l a t e r named garveatin A , 39 i s outlined below. Freshly collected G.annulata was immersed i n methanol i n 4-l i t r e p l a s t i c jars and stored at room temperature for 3-7 days. The densely pigmented methanol extract, t y p i c a l l y about 8 l i t r e s , was f i l t e r e d through C e l i t e and evaporated i n vacuo to give an aqueous suspension (400ml). This aqueous phase was successively extracted with hexane(3x400ml), methylene chloride(3x400ml) and ethyl acetate(2x400ml). The i n i t i a l f r a c t i o n a t i o n involved vacuum-filtration chromatography using a 3.5 cm thick s i l i c a pad i n a sintered-glass funnel (10cm dia.) attached to a f i l t e r - f l a s k . This method was found to be preferable to the more conventional f l a s h column setup, since the larger diameter of the funnel permitted higher concentrations of crude extract, and the shorter "column" length minimized i r r e v e r s i b l e adsorption to the s i l i c a g e l . Stepwise eluti o n of the methylene chloride phase of the hydroid extract(4g) from the above-mentioned suction-flash column, using ethyl acetate/hexane mixtures, yielded a f r a c t i o n (1.5g) eluting with 100% ethyl acetate. This f r a c t i o n was further subjected to Sephadex LH-20 chromatography using 90% MeOH/CH2Cl2 as the eluant. A bright yellow-orange band, which was strongly retarded and eluted as the f i n a l component of the mixture, proved to be almost pure garveatin A. F i n a l p u r i f i c a t i o n of garveatin A was obtained by t r i t u r a t i o n with 80% CHCl^/hexane and c r y s t a l l i z a t i o n from acetone to give orange needles (300mg) (m.p.236-240°C). HRMS analysis of garveatin A established the molecular formula as C 20 H20°5 m / / z 3 4 0 * 1 3 1 7 O D S ' / 340.1311 calc'd.). The intense molecular ion suggested an aromatic compound, and t h i s 40 evidence was supported by i t s UV spectrum (X m = „ 232, 282, 323(sh), 432nm in MeOH) which was t y p i c a l of a p o l y c y c l i c aromatic chromophore. The *H NMR spectrum (CDCl^) contained aromatic resonances at 7.10 (brs,lH) and 7.15ppm (s,lH) whose up f i e l d s h i f t ( r e l a t i v e to benzene) suggested that they were ortho or para to a phenol group. Additional evidence for the presence of phenol f u n c t i o n a l i t i e s was provided by proton si n g l e t s at 10.56 and 17.34 ppm, th e i r downfield chemical s h i f t s indicating a strong H-bonding e f f e c t . Observation of a green color on spraying with f^Cl-j solution also pointed to the presence of a phenol group chelated to a carbonyl . 13 Analysis of the C NMR spectrum of garveatin A (75 MHz, acetone- d^) indicated the presence of ten aromatic resonances at 106.0(s), 110.1(d), 113.9(8), 118.3(d), 120.6(8), 136.9(s), 138.5(s), 149.l(s), 161.6(s), and 170.5 ppm(s), the two most downfield resonances being appropriate for carbons bearing 13 phenols. Additional C NMR signals at 204.l(s) and 32.7 ppm(q), in conjunction with a H^ NMR s i n g l e t at 2.68 ppm(3H), suggested 13 the presence of an aromatic acetyl group. C NMR data for the model compound o-hydroxyacetophenone (73) gives a value of 204.4 75 1 ppm for the carbonyl carbon i n CDClg solution , while the H nmr s h i f t of the aromatic acetyl group in the juglone derivative (74) 7 6 i s exactly the same as i n garveatin A A broad s i n g l e t at 2.40 ppm (3H) in the "*"H NMR spectrum was assigned to an aromatic methyl group exhibiting long-range coupling to the aromatic proton at 7.10 ppm (brs, 1H). This coupling pattern was confirmed by performing a decoupling 41 75 76 42 experiment on the trimethyl derivative JLQ. of garveatin A, where i r r a d i a t i o n of the broad s i n g l e t at 7.41ppm produced a sharpening of the methyl s i n g l e t at 2.38ppm. .In addition , a difference nOe experiment on the same derivative i n which the broad aromatic s i n g l e t at 7.41 ppm was i r r a d i a t e d produced an enhancement i n the i n t e n s i t i e s of both the aromatic proton at 7.65ppm and the aromatic methyl at 2.38ppm. These experiments established that the two aromatic protons were p e r i with respect to each other and that the aromatic methyl group was ortho to one of the aromatic protons. The above data led us to propose p a r t i a l structure 7JL for garveatin A, which i s analagous to the known compound musizin (76) . Musizin i s a constituent of wood from a species of an African tree and contains an IR band at 1630 cm"*, due to the 77 strongly chelated carbonyl group . This compares favorably to the carbonyl stretching frequency of 1610 cm""* for garveatin A. In addition, the UV spectrum ( l i g h t petroleum) of musizin contains maxima at 219, 266 and 402 nm, which i s quite similar to the spectrum of garveatin A (MeOH) in acid solution ( m a x 238, 253, 298, 402 nm). The remaining part of the molecule has the formula C^H^C^r and nine of the hydrogen atoms can be accounted for by si n g l e t s at 1.63 (6H) and 1.98ppm(3H) in the 1H NMR spectrum. The former resonance was assigned to a gem-dimethyl and t h i s was supported by 1 3 C NMR signals at 28.9 (q, 2C) and 40.8ppm (s). The downfield po s i t i o n of the methyl group at 1.98ppm suggested that i t was attached to an sp^ carbon, either an o l e f i n or an enol group. 43 although i t s C NMR s h i f t (7.1ppm) was quite shielded. 13 Consideration of the remaining three C NMR resonances at 198.6 (s), 103.3(s) and 175.9 ppm(s) i n garveatin A led to the proposal of a keto -enol system containing a methyl group on the central carbon atom. In order to s a t i s f y the molecular formula, the keto-enol and gem-dimethyl f u n c t i o n a l i t i e s must be incorporated into a six-membered ring fused to the naphthalene moiety to give structure 12 for garveatin A. Garveatin A bears a close resemblance to fer r u g i n i n A (83). a pigment isol a t e d from the berries of a group of t r o p i c a l trees belonging to the genus Vismia that grow i n Central and South 78 1 America . The H NMR spectrum of ferruginin A (acetone d g) contains phenol resonances at 10.35 (s) and 17.55ppm(s), which compare cl o s e l y to those of garveatin A (10.56 and 17.34 ppm). Also, there are two aromatic protons at 7.26 and 7.02 ppm, the l a t t e r resonance showing long-range coupling to the aromatic methyl group at 2.40 ppm, as observed with garveatin A. The enolised beta-diketone system i s also present, however, the C2 pos i t i o n i s unsubstituted and a gem-diprenyl group, instead of a gem-dimethyl group, exists at C4. Comparison of the spectral data of various methylated and acetylated derivatives of garveatin A with the corresponding analogs of fer r u g i n i n A provided further proof for the proposed structure (see Table 2). Treatment of garveatin A with diazomethane produced a mono-methyl derivative Z8_, i d e n t i f i e d by a molecular ion at 354 daltons i n the mass spectrum, and a methyl s i n g l e t (3H) at 4.04 ppm i n the NMR spectrum. This compound was much more soluble in CDC1., than garveatin A and was also more stable i n solution. 0R 1 0R 2 0 12 13 77 R ,R^R 2 = H 78 R = C H 3 R 1 f R 2= H 79 R , R V R 2 = C H 3 80 R ,R 2 =CH 3 R 1 = H 81 R,R v .R 2 = Ac 82 R,R 2=Ac R 1 = H 83 R , R 1 , R 2 = H 84 R = C H 3 R V R 2 = H 85 R ,R 2 = C H 3 R ^ H 86 R l R 1 =CH 3 R2=H 45 Table 1: C NMR data for Garveatin A (acetone dg) and 79 Ferruginin A (dioxane d g ) . Chemical s h i f t s i n ppm from TMS. Carbon# Garveatin A (77) Ferruginin A (83) 1 198.64 192.6 2 103.25 105.9 3 175.89 180.8 4 45.36 51.3 4a 149.09 142.6 5 118.31 124.5 6 138.53 140.8 7 120.61 123.5 8 161.63 155.8 8a 113.94 109.7 9 170.47 164.9 9a 105.95 112.7 10 110.09 116.2 10a 136.88 137.9 Table 2: *H NMR (CDCl-j) data for derivatives of Garveatin A and 79 Ferruginin A Chemical s h i f t s in ppm from TMS. H on C# 22 7_a 22. M £2 £A &5_ M 5 7.02 7.01 7.41 7.30 7.0 6.94 7.23 7.06 10 7.15 7.13 7.65 7.20 7.1 7.01 7.09 7.06 80R 10.56 10.40 3.86* 3.91 10.1 10.10 3.88 10.23 90R 17.34 17.25 3.97*15.53 16.5 16.85 15.48 4.00 * indicates assignments may be reversed. 46 The presence of two chelated OH protons at 10.40 and 17.25 ppm indicated that the C8 and C9 OH groups were unsubstituted, and th i s was supported by the fact that the aromatic proton s h i f t s ( 7.01 and 7.13 ppm) were almost i d e n t i c a l to those of garveatin A (7.02 and 7.15 ppm). Thus, methylation had occured at the C3 pos i t i o n , and t h i s was confirmed by an nOe experiment where i r r a d i a t i o n of the gem-dimethyl group produced an enhancement of the C3 methoxy (4.04 ppm) and the C10 aromatic (7.13 ppm) protons. The "'"H NMR and UV data i s in excellent agreement with that of the methylated fe r r u g i n i n A derivative 8_4_ prepared by 79 diazomethane treatment of f£3_ by Marini-Bettolo and co-workers . 13 Assignment of the C NMR spectrum of garveatin A was assisted by carrying out a long - range HETCOR (heteroscalar correlated spectroscopy) 2-D NMR experiment on a 400 mg sample. The HETCOR experiment ' establishes d i r e c t c o nnectivities l l between two d i f f e r e n t bonded nuclei (in t h i s case H and C) and 13 therefore allows one type of nucleus (e.g. C) to be assigned from the known assignment of the other type. The pulse sequence used to produce the HETCOR spectrum (see Figure 4a) produces a transfer of p o l a r i z a t i o n from the protons to the carbon nuclei and therefore leads to a four - f o l d enhancement in s e n s i t i v i t y 13 in the C channel. Coupled nuclei y i e l d signals with the coordinates (H), (C) in the 2-dimensional contour p l o t . Using appropriate delay times D3 and D4, the experiment can be 2 3 optimised to detect long - range J ^ H and J ^ H couplings while suppressing the much larger ^Jru coupling. This approach was 47 PPM I I 20 40 . 60 H 80 ^100 | i 20 1 40 J 60 |200 H J CH.H5 C9a.H,10 . C 8 q , H 5 H 1 0 ~ - C10.H5 — C7.H5 C12.H13.-C1 3.H12 -CA.H12.H13 C2.H11 C5.HH ' C 7 . H U - C 6 . H U C4a,H12.H13 7 6 5 4 3 Figure 4. Long-range HETCOR spectrum of 22 in acetone d, Figure 4a. Pulse Sequence for Long-range HETCOR Experiment on garveatin A (12). 9.0° 90° D2 , D3 DECOUPLE 180< 90° D4 Status INSTRUMENT: Varian XL -300. PARAMETERS: 500 mg i n 10 mm tube (acetone d g ) . 13 1 C sweep width: 16,000 Hz. -"-H NMR sweep width: 2250 Hz. Delay time: 2 sec. Acquisi t i o n time: 64 msec. Number of increments: 128. 144 transients per increment. Number of points in t 2:2048. Number of points i n 512. J1XH set at 130 Hz. JNXH set at 10 Hz. Gives: D3 = 0.05 sec, D4 = 0.033 sec. Total a c q u i s i t i o n time: l l h o u r s . 49 adopted for the HETCOR experiment on 22. since several of the aromatic carbons were not d i r e c t l y bonded to protons and therefore would not give correlations i f a one - bond experiment was employed. With the Varian XL-300 software, one enters a value of JNXH which corresponds to the long - range coupling constant that one wishes to observe, and the D3 (1/2J) and D4 (1/3J) values are then calculated automatically. A value of 10 Hz was used i n t h i s 3 experiment even though J C H couplings involving aromatic 82 hydrogens are usually 6-8 Hz . Acquisition of the data took 11 hours and the resultant spectrum i s shown i n Figure 4, represented as a contour p l o t . It can be seen that both H5 and H10 protons transfer magnetisation to the C resonance at 113.9 ppm, i d e n t i f y i n g i t as C8a. There i s also a c o r r e l a t i o n between H10 (6.97 ppm) and the resonance at 106.0 ppm, assigned to C9a. Three-bond correlations between H5 (6.87 ppm) and C14, CIO and C7 can also be i d e n t i f i e d . Two-bond connectivities between C2-H11 2 and C4a-Hl2, H13 are present since J C H ( t y p i c a l l y l-4Hz) i s i n 3 the order of J C H « It i s somewhat discouraging to f i n d a number of spurious peaks i n the spectrum, e s p e c i a l l y i n the upper r i g h t -hand region. Also, no c o r r e l a t i o n between H10 and C5 i s observed which i s surprising since these two nuclei should couple with a J value of about 7 Hz. These imperfections may be a consequence of 1 3 the fact that compromise values of J C H and J C H were used to calculate the a c q u i s i t i o n parameters, since the molecule contains both aromatic and a l i p h a t i c substructures. Ideally, i t may be necessary to use a d i f f e r e n t set of parameters to optimise p o l a r i s a t i o n transfer through the d i f f e r e n t types of bonds i n the 50 molecule. The p r e f e r e n t i a l methylation of garveatin A at the C3 posi t i o n with diazomethane indicates that the C3 OH group i s more nucleophilic or perhaps more a c i d i c than the C8 and C9 OH groups. Evidence for the existence of an enolate anion at C3 i s supported by the hypsochromic UV s h i f t of garveatin A in acid (no s h i f t was observed i n base) and the absence of a C3 OH proton resonance i n the *H NMR spectrum. In addition, the compound streaks considerably when analysed by TLC i n neutral solvents, but migrates as a ti g h t spot on addition of 1% acetic acid, indicating a negatively charged species. Methylation of garveatin A with dimethyl su l f a t e and K 2 C 0 3 in refluxing acetone yielded the trimethyl derivative ££. as well as smaller amounts of the 3,8-dimethyl derivative 7J9_. Assignment of a methoxy group at the C8 rather than the C9 position of the l a t t e r compound i s based on the fact that the broad C5 aromatic proton i s s h i f t e d farther downfield than the the CIO s i n g l e t on methylation. Also, comparison with the corresponding dimethyl analog of fer r u g i n i n A JL5_ substantiates t h i s assignment. Treatment of garveatin A with pyridine and acetic anhydride at room temperature overnight formed the t r i a c e t a t e JH and lesser amounts of the 3,8-diacetate £2, analogous to the dimethyl s u l f a t e methylation reaction. Three si n g l e t s i n the *H NMR spectrum (80 MHz, CDClj) of £1 at 2.35, 2.54 and 2.55 ppm were assigned to the acetate methyl groups, the most shielded resonance being appropriate for the C3 po s i t i o n . The aromatic protons were shi f t e d downfield to 7.59 (H5) and 7.84 ppm(HlO) as 51 a consequence of the deshielding e f f e c t of acetylation of the para groups. The mass spectrum of £1 shows a molecular ion at m/z 466 with successive loss of three 42 dalton fragments corresponding to the three acetate groups. Loss of 42 mass units i s a very f a c i l e cleavage for aromatic and enol acetates and can be envisaged as a Mc L a f f e r t y rearrangement res u l t i n g i n loss of ketene and keto-enol tautomerisation of the resu l t i n g ketone to give the phenol (Scheme 1). As was the case with the dimethyl derivative 7_9_ of garveatin A, the d i a c e t y l derivative £2 i s acetylated at the C8 pos i t i o n , causing substantial deshielding of the C5 proton. Garveatin A t r i a c e t a t e (£1) , on slow evaporation in 1:1 CHClj/nexane at 4° C c r y s t a l l i s e d as yellow needles. However, X-ray d i f f r a c t i o n analysis of one of the cry s t a l s which was performed by He Cun-heng and Jon Clardy of Cornell University revealed the presence of the isomeric structure (£Z)i which contains an enol acetate f u n c t i o n a l i t y instead of an aromatic acetyl group, and a phenolic OH group at C9. Subsequent TLC examination of the sample of cr y s t a l s used i n the X-ray analysis indicated the presence of a second compound, which gave a dark spot when exposed to I 2 vapour. S i l i c a - g e l chromatography was used to pu r i f y t h i s compound, present in a 1:3 r a t i o with the t r i a c e t a t e . The *H NMR spectrum of the minor compound contained two doublets at 5.00 and 5.38 ppm (1H each) with 2 Hz coupling, i n d i c a t i v e of an o l e f i n i c methylene group. There was also a phenolic proton resonance at 15.33 ppm and the aromatic CIO proton (7.24 ppm) was shielded r e l a t i v e to the corresponding signal in the spectrum of the t r i a c e t a t e {SOL), proving that the 52 0 87 53 C9 OH was not acetylated. The mass spectrum gave a molecular ion at 466 daltons, and was sim i l a r i n many respects to that of the t r i a c e t a t e £1 . Therefore , t h i s minor compound corresponded to structure £2 obtained by X-ray analysis, and i t evidently was formed by rearrangement of £1 during the c r y s t a l l i s a t i o n process. In order to investigate the rearrangement reaction more f u l l y , a sample of pure t r i a c e t a t e £1 was heated at 60°C in benzene containing a c r y s t a l of para-toluenesulfonic acid. Quantitative conversion to the enol-acetate isomer £2 occurred after 12 hours, indicating that the l a t t e r i s the thermodynamically more stable structure. The computer-generated perspective drawing of £2 (Figure 5) indicates that both the C8 and C15 O-acetates are rotated roughly perpendicular to the plane of the aromatic nucleus to avoid serious s t e r i c repulsions. Triacetate £1 would be expected to give r i s e to strong s t e r i c repulsion between the C8 and C9 O-acetate groups and t h i s s t r a i n could be relieved to some extent on rearrangement. It i s possible that a trace of acid i n the CHCl^ used i n the c r y s t a l l i s a t i o n of 81 catalysed the rearrangment i n the sample submitted for X-ray d i f f r a c t i o n analysis. ( i i ) Garveatin B_ The f r a c t i o n (500 mg) obtained by elution of the suction-f l a s h column with 50% ethyl acetate/hexane was subjected to LH-20 chromatography i n 90% MeOH/CH^^j followed by preparative TLC i n 50% ethyl acetate/hexane to give a p a r t i a l l y p u r i f i e d orange component with an Rf of 0.48 in the standard TLC system. Further 54 Figure 5. Computer generated X-Ray structure of the enol triacetate of garveatin A (£2). p u r i f i c a t i o n of th i s component on HPLC (normal-phase, ethyl acetate/hexane gradient) gave pure garveatin B (&8J (15mg) as an orange yellow o i l . The 1H NMR spectrum (80 MHz, CDC13) of garveatin B 8 3 revealed that i t was very c l o s e l y related to garveatin A. Resonances at 1.62 (s,6H), 1.99 (s,3H), 2.46 (brs, 3H), 7.04 (brs, 1H), and 7.12 (s,lH) were v i r t u a l l y i d e n t i c a l to the corresponding resonances for the protons at C12 and 13, C l l , C14, C5 and CIO respectively in garveatin A. The appearance of additional resonances at 1.18 (t,J=7Hz, 3H), and 2.80 ppm (q,J=7Hz, 2H) combined with the absence of an acetyl methyl resonance around 2.70 ppm i n the spectrum of £8_ indicated that garveatin B contained an ethyl side-chain at C7 instead of an acetyl group. A molecular formula of C 20 H22°4' established by HRMS was consistent with t h i s assignment and the UV (in MeOH) maxima of 240, 260, 317 and 417nm were quite s i m i l a r to those of garveatin A. An inter e s t i n g feature of the *H NMR spectrum of garveatin B in CDCI3 solution i s the presence of the diketo tautomer (2H) i n approximately 1:3 r a t i o with the keto-enol form. As a consequence, the phenolic protons appear as doubled peaks at 9.90, 10.25 ppm (1:3) and 16.23, 17.15 ppm (1:3), and there are weak resonances at 1.50(d,J=7Hz) and 3.98 ppm(q,J=7Hz) due to the protons at C l l and C2 respectively i n the diketo tautomer. The u p f i e l d s h i f t of the phenolic protons i n the diketo form r e l a t i v e to the keto-enol form suggests a weaker H-bonding in t e r a c t i o n with the CI carbonyl group. This i s l i k e l y due to 56 0 OH OH 88 R =H 89 R = C H 3 0 OH OH 91 92 57 non-planarity of the CI oxygen with the aromatic nucleus, or to a decrease i n the electron density of the CI oxygen i n the diketo form due to the absence of resonance form 22. as indicated on page 57. The keto-enol tautomerism observed in the spectrum of garveatin B i s quite s i m i l a r to that reported for f e r r u g i n i n A 79 (83) in CDC1.J solution , where i t was observed that the proportion of the diketo form increased with increasing temperature. In addition, only the keto-enol form was present i n acetone dg solution, as was the case for garveatin B. Treatment of garveatin B with with diazomethane yielded 3-methyl garveatin B (8JL) as the major product. In t h i s compound, the keto-enol tautomer i s "frozen" as a consequence of methylation of the C3 OH group, precluding any contribution from a diketo form. As a re s u l t , the ^H NMR spectrum of 8_9_ does not show any s p l i t t i n g of the resonances corresponding to the phenol protons. ( i i i ) Garveatin £ TLC analysis of the f l a s h f r a c t i o n eluting with 20% ethyl acetate/hexane (140 mg) indicated the presence of a component which had a white fluorescence under long-wave UV. LH-20 chromatography i n 90% MeOH/CH-^C^, followed by preparative TLC in 40% ethyl acetate/hexane gave pure garveatin C(iLi) (20 mg) which was responsible for the UV fluorescence. Garveatin C i s more non-polar than garveatins A and B (R^ 0.54 in standard TLC system) and gave orange c r y s t a l s (mp 125 C) on slow evaporation of a hexane solution at 4°C. 58 93 R = H R ^ C H 94 R=Ac R 1 = CH 95 R,R 1 = C H 3 96 R f R 1 = H 97 R f R 1 = H 98 R , R 1 = A c 99 R = Ac R ^ H 59 Comparison of the H NMR spectrum (80 MHz, CDC13) of 22 with that of garveatin A indicated many s i m i l a r i t i e s , and resonances at 2.38 (brs, 3H), 2.62(s,3H), 1.58(s,6H), 7.19(s,lH) and 7.35(brs,lH) could be assigned to an aromatic methyl at C6, an aromatic acetyl at C7, a gem-dimethyl at C4 and aromatic protons at CIO and C5, by analogy. The remaining resonances were assigned to another gem-dimethyl group ( 1.49 (s,6H) ppm), an aromatic methyl ether (3.92, s,3H) and a phenolic proton (14.45 s,lH) The downfield s h i f t of the C5 proton i n 22 r e l a t i v e to i t s p o s i t i o n in the spectrum of garveatin A (7.35 vs. 7.10 ppm) indicated that the aromatic methyl ether was at the C8 p o s i t i o n . This follows from the deshielding influence of a methoxy group r e l a t i v e to a hydroxyl group on ortho and para positions i n an aromatic 84 nucleus . Consideration of the biogenesis of garveatin C indicated that the second gem-dimethyl group was at the C2 position and hypsochromic s h i f t s i n a l l the UV peaks r e l a t i v e to those i n 22 was consistent with a less extensively conjugated chromophore. 13 The C NMR spectrum (see Table 4) of 22. contains three ketone carbonyl resonances at 211.6, 205.8 and 204.9 ppm and two 3 quaternary sp carbons at 48.1 and 55.2 ppm which support the proposed structure for the a l i c y c l i c r i n g . The most deshielded resonance at 211.6 ppm i s appropriate for the unconjugated ketone at C3. Acetylation of garveatin C (acetic anhydride/pyridine) gave a quantitative y i e l d of the monoacetate (2A). The chemical s h i f t of the C2 gem-dimethyl (1.35 ppm) i s 0.14 ppm upf i e l d from that 60 i n the H NMR spectrum of the parent compound, which suggests that introduction of an acetoxy group at the C9 position a l t e r s the anisotropic e f f e c t of the neighboring carbonyl group, resu l t i n g in a shielding of the C2 gem-dimethyl protons. F i n a l proof of the structure of 22 was achieved by converting both garveatins A and C to the dimethoxy derivative 25.. Treatment of 22 with either diazomethane or dimethyl su l f a t e f a i l e d to y i e l d any of the desired product, but methylation with methyl iodide and K 2C0 3 i n refluxing acetone gave a quantitative y i e l d of 23.» S i m i l a r l y , methylation of garveatin A with methyl iodide gave 23. as the major product. This reaction involves C-methylation at the C2 position of the diketo tautomer of 22, as well as O-methylation at the C8 and C9 positions. (iv) Garveatin p_ The f l a s h f r a c t i o n eluting with 100% ethyl acetate was further p u r i f i e d on LH-20 (90% MeOH/CH2Cl2) resu l t i n g i n a f r a c t i o n containing a mixture of minor metabolites , which were subjected to preparative TLC (a: 10% ethyl acetate/CHCl 3, b: 25% ethyl acetate/hexane) to give garveatin D (21L) (8 mg) as a yellow o i l . The mass spectrum of 2JL showed a parent ion at m/z 354 daltons, appropriate for a molecular formula of C2i H20°5* T ^ e l f l NMR spectrum (270 MHz, CDC13) of 23. showed a s t r i k i n g resemblance to that of 22t the only noticeable difference being the absence of a methoxy group around 3.9 ppm, an u p f i e l d s h i f t i n the po s i t i o n of the C5 proton (7.15 vs. 7.35 ppm) r e f l e c t i n g the lack of methylation at C8, and the presence of two phenolic resonances 61 Table 3j_ 1H NMR data (CDC13) for Garveatins A-D Chemical s h i f t s i n ppm from TMS. H on C# A (77) B (88) C (93) D (96) 5 7.02 7.04 7.35 7.05 80R 10.65 10.25 3.92 10.20 90H 17.34 17.15 14.45 16.15 10 7.15 7.12 7.15 7.12 11 1.98 1.99 1.49 1.50 11a - - 1.49 1.50 12 1.63 1.62 1.58 1.58 13 1.63 1.62 1.58 1.58 14 2.40 2.46 2.38 2.41 15 - 2.80 q - -16 2.68 1.18 t 2.62 2.63 A l l resonances are s i n g l e t s unless otherwise s p e c i f i e d . 62 13 Table AJL C NMR data for Garveatin compounds. Chemical s h i f t s in ppm from TMS. :# A(77) a C(93) b D(96) b A quinone * * * 1 198.6 205.8 205.4 159.5 2 103.3 55.3 54.7 112.4* 3 175.9 211.6 211.6 200.0 $ 4 45.4 48.1 48.0 48.3 4a 149.1 142.8 142.7 154.3 5 118.3 125.2 120.2 123.1 6 138.5 140.0* 141.0* 147.2 7 120.6 135.0 125.2 127.7 8 161.6 155.9 156.0 159.1* 8a 113.9 116.3 110.8 118.6* 9 170.5 163.7 166.0 193.0 9a. 106.0 108.8 107.1 136.3 & 10 110.1 114.7 115.2 180.4 10a 136.9 138.0* 139.4* 131.9 & 11 7.7 24.6 24.6 8.1 11a - 24.6 24.6 -12 30.2 28.5 28.6 26.4 13 30.2 28.5 28.6 26.4 14 21.3 19.5 20.7 20.5 * * 15 204.1 204.9 204.2 202.0 16 32.7 32.4 32.3 31.9 80Me - 64.1 - -*,#,$, & indicates assignments may be reversed. a: run in acetone D 6. b: run in CDCl 3* 63 (10.20, 16.15 ppm) instead of just one as in 22.. The structure 23. was proposed for garveatin D on the basis of the above data. (v) 2-Hydroxyaarveatin A.. Examination of the most polar f l a s h chromatography fractions (eluted with 100% ethyl acetate and 20% MeOH/ethyl acetate) revealed the presence of two major metabolites which were yellow-orange i n colour and had R f's of 0.1 and 0.28 i n the standard TLC system. LH-20 chromatography ( 90% MeOH/CH2Cl2) of th i s material produced a f r a c t i o n containing approximately lOOmg of an orange s o l i d , a portion of which was fractionated on a reversed phase preparative TLC plate ( 80% MeOH/H20 ) to give 10 mg of pure 2-hydroxy garveatin A (22.) • Compound 22 was poorly soluble i n organic solvents, and i t was therefore characterised as i t s tri a c e t a t e 2ILr prepared by treatment with acetic anhydride and pyridine. Triacetate 9_8_ was a colourless s o l i d with a molecular formula of C26 H26°9 ^ HRMS m/z observed 482.1567, req'd 482.1577). Comparison of i t s "'"H NMR spectrum (80 MHz, CDC13) to that of t r i a c e t y l garveatin A (8JL) indicated the presence of id e n t i c a l aromatic rings i n both molecules. However, a l i p h a t i c methyl resonances at 1.45(s, 3H), 1.57(s, 3H), and 1.79ppm(s,3H), and an acetyl methyl resonance at 2.20 ppm suggested a non-symmetrical a l i c y c l i c ring s i m i l a r to that proposed for the diacetate of 2-hydroxy garvin A (120), a metabolite whose structure had previously been elucidated (see page 91) using a combination of nOe and X-ray data. Methyl 64 resonances at 1.50, 1.60 and 1.70 ppm i n the H NMR spectrum of 120 correspond c l o s e l y to those in .9_8_, leading to the proposed structure for 22. An attempt to confirm t h i s assignment by ] 3 analysis of the C spectrum (75 MHz, CDC13) of the t r i a c e t a t e 2£ led to considerable confusion, since only two of the three ketone carbons and six of the eight methyl carbons could be observed. Changing solvents (DMSO-dg, c g D 6 ' acetone-d g), temperature and delay times did not a l l e v i a t e t h i s problem. However, treatment of 98 with pyridine at room temperature converted i t to the 1 13 diacetate (j)9_), whose H and C NMR spectra were e n t i r e l y consistent with the proposed structure. Reacetylation of the diacetate with acetic anhydride/pyridine regenerated the t r i a c e t a t e 98. 2-Hydroxygarveatin A (22) appears to be formed by C2 hydroxylation of garveatin A (22)• It i s interesting to speculate whether t h i s reaction occurs by a e r i a l oxidation during the storage period and p u r i f i c a t i o n steps, or whether the process i s enzymatically controlled in-vivo. The presence of r e l a t i v e l y large amounts of 2-hydroxygarveatin A i n fresh extracts of G.  annulata f p r i o r to any chromatography, seems to indicate that the compound i s not an a r t i f a c t produced by oxidation of garveatin A, and that i t occurs as a metabolite in the intact organism. In order to carry out an i n - v i t r o conversion of garveatin A to 2-hydroxygarveatin A, i t was decided to make use of lead tetraacetate to introduce an oxygen atom i n the form of an acetoxy group at the C2 position of garveatin A . F i r s t , the C8 and C9 OH groups were protected as t h e i r methyl ethers (Scheme 2). This was achieved by treatment of 22 with dimethyl s u l f a t e to 65 Scheme 2. 66 give trimethylgarveatin A (8J1) as previously described. The 3-methoxy group was then hydrolised p r e f e r e n t i a l l y , by refluxing i n 50% aqueous acetic acid to y i e l d the 8,9-dimethyl derivative 100. When t h i s compound was s t i r r e d with Pb(OAc)^ i n g l a c i a l acetic acid at room temperature overnight, the major reaction product was the 2-acetoxy derivative 101. The "^H NMR spectrum (80 MHz, CDC13) of 101 contained methyl singlets at 1.47, 1.57 and 1.76 ppm as well as an acetate resonance at 2.23 ppm, which had almost i d e n t i c a l chemical s h i f t s to the corresponding resonances i n 2-hydroxygarveatin A t r i a c e t a t e (23.) • The UV maxima of both compounds were very s i m i l a r , indicating the presence of the same type of chromophore. (vi) 2-Hvdroxyaarveatin £• The less polar metabolite present i n the f l a s h f r a c t i o n eluted with 100% ethyl acetate was p u r i f i e d by LH-20 chromatography (90% MeOH/C^C^) to give 40 mg of a yellow s o l i d , whose molecular formula was determined to be C20 H22°5 b ^ H R M S (m/z observed 342.1467, req'd 342.1468). The chemical s h i f t s of the three methyl groups in the "'"H NMR (80 MHz, CDCl^) spectrum (1.52, 1.65, 1.81ppm) corresponded clo s e l y to those in the a l i c y c l i c ring of 2-hydroxygarvin A (119) (1.52, 1.66, 1.81ppm)(see page 95). Furthermore, the remaining proton resonances bore a close resemblance to those for the aromatic nucleus and i t s substituents i n garveatin B (88) . This '''H NMR data, in conjunction with the molecular formula, suggested that t h i s compound was 2-hydroxygarveatin B (102). Evidently, 67 RO' 0 102 R,R 1= H 103 R = H R 1 = A c 104 R , R 1 = A c OH 0 OH 105 R = C H 3 C 0 -106 R = C H 3 C H 2 -107 68 of an acetyl group with an ethyl side-chain causes a substantial increase i n hydrophobicity, r e s u l t i n g in a higher value and greater s o l u b i l i t y i n chloroform of 102 compared to j£2. A similar difference i n p o l a r i t y was noticed between garveatin A (12) and garveatin B (£8J • Treatment of ±Q2 with acetic anhydride and pyridine yielded a mixture of the diacetate (103) and the t r i a c e t a t e (104) as the major reaction products. In t h i s case the diacetate contained acetoxy groups at the C8 and C9 positions, as evidenced by the chemical s h i f t s of the aromatic protons at 7.62 and 7.66 ppm i n the *H NMR spectrum (80 MHz, CDC1 3), as well as a broad s i n g l e t at 4.63 ppm, due to the C2 OH group. This i s i n contrast to the substitution pattern of the diacetate of 2-hydroxy garveatin A (97) which contains acetoxy groups at the C2 and C8 positions. ( v i i ) Quinone derivatives of. the Garveatins A second class of oxidized derivatives of the garveatins i s represented by the substituted naphthoquinones, garveatin A quinone (105) and garveatin B quinone (106). These compounds are present in low concentration (<5%) r e l a t i v e to the parent garveatins, but because of t h e i r bright red colour , they are e a s i l y detected i n the crude extracts. The ethyl acetate phase of the annulata extract was subjected to suction-flash chromatography using an ethyl acetate/CH 2Cl 2 gradient to give a deep red f r a c t i o n which was further p u r i f i e d by preparative TLC (2% MeOH/CH2Cl2) to give llmg of garveatin A quinone (10JL) as a red o i l . This compound was also 69 present i n the hexane and CH 2C1 2 phases , but was more d i f f i c u l t to p u r i f y from these extracts due to the presence of other components with approximately the same values. Quinone 105 was shown by HRMS to have a molecular formula of C20 H18°6 ^ m / / z o b s ' d 354.1108, req'd 354.1103) and i t s UV spectrum (in MeOH) and bright red colour was t y p i c a l of anthraquinone-type 62 1 compounds . The H NMR spectrum of 105 suggested a close rel a t i o n s h i p to garveatin A (22) and resonances at 1.98 (s,3H), and 1.63ppm (s,6H) could be assigned to a methyl group at C2 and a gem-dimethyl at C4 respectively. In addition, s i n g l e t s at 2.60 (3H) and2.41 ppm (brs, 3H) were appropriate for an aromatic acetyl at C7 and an aromatic methyl substituent at C6. However, only one aromatic proton was present i n the ^H NMR spectrum of 22 and i t s chemical s h i f t ( 7.55 ppm) corresponded c l o s e l y to that of the C4 proton ( 7.57 ppm) i n the anthraquinone (107) prepared 8 6 by Marini-Bettolo's group . The 1 3 C NMR spectrum (75 MHz, CDC13) of 1£5_ contained resonances at 193.0 and 180.4 ppm which were appropriate for the quinone carbonyls of the proposed structure. The presence of two phenolic proton resonances at 11.45 and 11.66 ppm indicated that 105 exists as the 3-keto tautomer i n CDClg. In th i s form, the 1-OH group can form a H-bond with the C9 quinone carbonyl oxygen atom, and th i s chelating e f f e c t i s responsible for the downfield s h i f t of the 1-OH proton. Garveatin A quinone (105) was present i n fresh extracts (< 48 hours) of annulata, indicating that i t may well be a true metabolite, and not just an oxidation a r t i f a c t of garveatin A. Treatment of garveatin A under a stream of a i r for 16 hours i n a 1:1 mixture of IN NaOH and MeOH gave a s u r p r i s i n g l y low y i e l d of the quinone 103. (10%), proving that even under vigorous oxidation conditions, t h i s conversion i s not very f a c i l e . S i m i l a r l y , when 22 was allowed to stand overnight i n a methanolic s l u r r y of s i l i c a - g e l i n the presence of either HC1 or NaOH, very low y i e l d s (<5%) of the quinone were obtained. Garveatin B quinone (106) has also been detected i n extremely small (<1 mg) amounts i n samples of garveatin B which have been allowed to stand i n sol u t i o n . This compound has not been observed i n the crude G_«_ annulata extracts so there i s no firm evidence that i t i s a bona f i d e natural product. However, oxidation of garveatin B to garveatin B quinone occurs i n CDC13 solution at room temperature. Treatment of pure garveatin B (2 mg) with CDCl^ (1 ml), and analysis of t h i s sample on normal phase HPLC (50% EtOAc/Hexane) at various time-intervals showed the formation of garveatin B quinone, the concentration reaching a maximum (68% of to t a l ) after 50 minutes. Quinone 106 was obtained from t h i s reaction as a red o i l with an of 0.63 i n the standard TLC system and i t had a molecular formula of C20 H20°5 ( l t l / / z 3 4 0 ' 1 3 0 6 ' req'd 340.1311). Its "4 NMR (400MHz, CDCl^) spectrum was almost i d e n t i c a l to that of garveatin A quinone (105). except for the presence of resonances at 1.16 (t,J=7Hz, 3H) and 2.78 ppm (q,J=7Hz,2H) due to an aromatic ethyl group, and the absence of an aromatic acetyl resonance around 2.6 ppm. t h i s compound also appears to ex i s t as the 3-keto tautomer (in chloroform solution) since both the OH protons (11.79 and 11.84 ppm) are quite far downfield. 71 ( v i i i ) Garveatin Dimers The f i r s t evidence of the presence of dimers in the G.  annulata extracts came when analysis of the "'"H NMR spectrum (400 MHz, CDCl^) of garveatin B (££.) indicated the presence of a second set of resonances whose in t e n s i t y increased with time to give a 1:2 r a t i o to ££ after 24 hours. Subsequent r e p u r i f i c a t i o n of the NMR sample by preparative TLC (40% ethyl acetate/hexane) yielded a second less polar compound (R^ 0.67 in standard TLC system) whose NMR spectrum (CDCl^) contained signals corresponding to the "extra" resonances i n that of 8J1. Thus, 8_8_ had p a r t i a l l y decomposed to another compound i n CDCl^ s o l u t i o n . On standing in acetone-dg or CgDg solutions, no such decomposition was detected, either by NMR or TLC. The decomposition product, which had previously been i d e n t i f i e d as a very minor constituent of the f l a s h f r a c t i o n containing garveatin B, had a UV spectrum which showed strong s i m i l a r i t i e s to that of 2-hydroxy garveatin B (102) in both neutral and basic so l u t i o n . The '''H NMR spectrum (400 MHz, CDCl^) contained resonances at 1.18 (t,J=7Hz, 3H), 2.80 (q,J=7Hz, 2H), 2.43 (brs, 3H), 6.98 (s,2H), 9.38 (s,lH) and 15.83 (s,lH) ppm, appropriate for aromatic ethyl , aromatic methyl, two aromatic and two phenolic protons respectively. This suggested that the aromatic portion of garveatin B was present i n the molecule, but i t was inte r e s t i n g to note that a l l the above resonances were shielded i n comparison to those i n garveatin B, by amounts varying from 0.03 ppm for the C14 methyl to 0.40 ppm for the C9 OH proton. The remaining resonances i n the 1H NMR spectrum were methyl singlets at 1.38, 72 1.66 and 1.88 ppm, indicating that the a l i c y c l i c ring now contained an asymmetric centre. Due to the lim i t e d amount of 13 sample, i t was not possible to obtain good C spectral data, but a very weak spectrum (75 MHz, cgDg) did indicate resonances at 202.4 and 209.0 ppm, appropriate for ketone carbons at CI and C3 respectively. These correspond c l o s e l y to the CI and C3 carbons in garveatin C (JLl) (205.8 and 211.6 ppm). A resonance at 63.0 ppm i n the spectrum of the decomposition product has almost the same chemical s h i f t as the C2 carbon (64.1 ppm) i n .9_3_. This suggests that the C2 pos i t i o n i n t h i s compound i s also d i a l k y l a t e d . The EI mass spectrum of the decomposition product gave a highest mass ion at 326 amu, suggesting that i t was a s t r u c t u r a l isomer of f££. However, the major product obtained by acetylation with acetic anhydride/ pyridine, which contained acetoxy groups at the C8 and C9 positions as evidenced by the chemical s h i f t s of the two aromatic protons (7.61, brs,lH and 7.75, s,lH), gave a weak molecular ion at 818 amu i n the mass spectrum. It was concluded that the decomposition product had structure 108, produced by dimeric association of two garveatin B monomers. The mass spectrum of the acetate (109) also showed weak fragment ions at 776 and 734 amu, corresponding to successive losses of two acetate units from the parent ion. A much more intense peak at 410 daltons corresponds to cleavage of the 2,2* carbon-carbon bond and transfer of a hydrogen to form an ion corresponding to the diacetate of garveatin B. Further losses of 42 daltons from t h i s daughter ion gave intense peaks at 368 and 326 daltons. By 73 108 R = H 109 R = Ac analogy, the peak at 326 daltons i n the mass spectrum of the underivatized decomposition product 108 was i n fact the daughter ion formed by an extremely f a c i l e cleavage of the central bond and transfer of a hydrogen atom. No peak at 650 daltons could be detected i n the EI mass spectrum of 108. Evidently, the acetylated derivative of the dimer i s more stable (or more v o l a t i l e ) i n the mass spectrum and i t can therefore can detected at a high gain s e t t i n g . There i s ample precedent for the above observations, since i t i s well established that p r i n c i p a l ions i n the mass spectra of bianthrones are daughter ions of mass M/2+1 for symmetrical dimers 6 5. The corresponding dimer of garveatin A was not observed when th i s compound was allowed to stand i n CDCl^ sol u t i o n . In an attempt to prepare t h i s dimer, more vigorous conditions were employed. Treatment of 22 with cone. HC1 i n CH^CN solution under a stream of a i r gave i n low y i e l d (10%) a product which was p u r i f i e d by preparative TLC i n 5% MeOH/CHCl3. The highest mass ion i n the EI mass spectrum occured at 340 daltons, appropriate for cleavage at the 2,2'bond to generate the monomer, as previously discussed for 10$.. The "*"H NMR spectrum (300 MHz, CDClj) contained resonances assignable to the naphthalene portion of garveatin A, and as i n the case of garveatin B dimer, t h e i r chemical s h i f t s were u p f i e l d compared to the value's i n the spectrum of 22. Three-proton s i n g l e t s at 1.47, 1.59 and 1.94 ppm, due to methyl groups at the C2 and C4 (2) positions, completed the *H NMR spectrum. More concrete proof of the dimeric nature of garveatin A 75 dimer(HH) was obtained by acetylation with acetic anhydride and pyridine to y i e l d the tetraacetate H I . A weak peak at 846 daltons was observed i n the mass spectrum of 111, in addition to peaks at 804, 762, 720 and 678 daltons due to successive losses of the four acetate groups. As was the case for the tetraacetate of garveatin B dimer (10j9_), resonances at 7.64 (brs, IH) and 7.79 ppm (s,lH) i n the *H NMR spectrum of H i r e f l e c t e d acetylation at the C8 (C8') and C9 (C9 1) positions of H £ . In p r i n c i p l e , dimerisation at the C2 pos i t i o n of garveatin A could lead to the meso form 112 and also to a racemic (+,-) mixture of the R,R and S,S forms 113. Since the meso form i s diastereomeric with either enantiomer of the racemate, i t would be expected to have d i f f e r e n t chemical and spectral properties and also a d i f f e r e n t R f value from that of the racemic form. 87 Cameron and co-workers have separated the meso and (+,-) diastereomers of 10,10' emodin bianthrone (114) and i d e n t i f i e d each by using a c h i r a l NMR s h i f t reagent. They found that on addition of tris[3-heptafluoropropylhydroxymethylene-(+) camphorato] europium III to a solution of the hexaacetate (115) of one diastereomeric form, the 10,10* proton s i n g l e t in the *H NMR spectrum was s p l i t into two l i n e s of equal i n t e n s i t y . Since the two enantiomers could display d i f f e r e n t chemical s h i f t s when in contact with the c h i r a l reagent, t h i s diastereomer was formulated as the racemic mixture. Addition of the s h i f t reagent to the hexaacetate of the other diastereomer did not induce any s p l i t t i n g i n the *H NMR l i n e s , implying that t h i s was the meso form of the bianthrone. It was anticipated that treatment of the tetraacetate (111) OR 0 OR 1U R =H 115 R = Ac 0 OH OH 77 of garveatin A dimer with the same c h i r a l s h i f t reagent would enable us to (a): determine whether one or both stereoisomeric forms were present in the sample, and (b): d i s t i n g u i s h between the meso and (+,-) forms i f only one type of stereoisomer was present. However, on t i t r a t i o n of 111 with the s h i f t reagent, no changes in the chemical s h i f t s or m u l t i p l i c i t y of the proton signals was observed. Thus, i t appeared that the reagent was not complexing with the dimer and consequently no conclusions could be drawn from the experiment. (ix) Desacetyl derivatives Treatment of garveatin A (21) with strong acid provided an indicat i o n of the l a b i l i t y of the aromatic acetyl group under these conditions. When H was refluxed for 10 minutes i n the presence a 1:1 (v/v) mixture of cone. HCl and g l a c i a l a c e tic acid, a less polar product was obtained in approx. 50% y i e l d . This compound had a molecular weight of 298 daltons corresponding to a loss of 42 daltons from H . Its 1H NMR spectrum (300 MHz, CDCl^) contained three aromatic protons, and a methyl s i n g l e t i n the region of 2.60 ppm was absent. The rest of the spectrum was almost i d e n t i c a l to that of H , indicating that the aromatic acetyl group had been l o s t to give structure 116. Two of the aromatic protons at 7.00 and 6.68 ppm were broad singlets which collapsed to doublets with 1.6 Hz coupling on i r r a d i a t i o n of the aromatic methyl group at 2.43 ppm. Therefore, these resonances were assigned to the protons at C5 and C7 respectively, the C7 resonance being more shielded due to i t s beta p o s i t i o n on the 78 naphthalene nucleus. Also present in the H NMR spectrum of H £ were resonances corresponding to the diketo tautomer i n a 2:5 r a t i o with those of the keto-enol form. It i s inter e s t i n g to note that no keto-enol tautomerism was evident in the CDCl^ spectrum of 12/ t h i s molecule being present only as the 1-keto, 2,3-enol form. When garveatin A quinone (105) was treated with CH3COOH/HCl, desacetyl garveatin A quinone (117) was formed, as evidenced by a molecular ion at 312 daltons in the mass spectrum, and the appearance of another aromatic proton (7.11 ppm,brs,IH) in the *H NMR spectrum (300 MHz, CDCl 3) i n place of an aromatic acetyl resonance. As in the previous case, i r r a d i a t i o n of the aromatic methyl group at 2.48 ppm collapsed the broad C5 and C7 protons to sharp doublets (J=1.6 Hz), and established the meta orientation of these hydrogens. In contrast to desacetyl garveatin A (116). compound H I did not exhibit any of the diketo form i n the NMR spectrum, suggesting a very strong H-bonding inte r a c t i o n between the CI OH group and the quinone carbonyl. The hydrolysis of the aromatic acetyl group i s analagous to the hydrolysis of the n-butyryl group of rhodocomatulin dimethyl ether (10.), a pigment isolated from a species of marine c r i n o i d 54 by Sutherland's group . In t h i s case, butyric acid was also iso l a t e d and i d e n t i f i e d by gas and paper chromatography. Sutherland postulated that the elimination occurs v i a a reverse F r i e d e l Crafts acylation reaction, and by analogy, a l i k e l y intermediate i n the case of the garveatin A reaction would be structure H8_, res u l t i n g from attack of a proton at C7. 79 (x) pic-syntheses The garveatins are t y p i c a l examples of polyketide - derived metabolites. I t appears l i k e l y that a l l the garveatins are derived from the same nonaketide precursor which has been dimethylated at C8 and either mono- or dimethylated at CIO (Scheme 3). The folding pattern shown can lead to garveatins A and D v i a multiple a l d o l condensations and dehydrations. 0-Methylation of garveatin D can give garveatin C, whereas reduction of the aromatic acetyl group of garveatin A produces garveatin B, although i t i s conceivable the l a t t e r step could occur before c y c l i s a t i o n . Another possible route to garveatin B may involve a precursor containing a butyric acid s t a r t e r unit. Also, i n view of the fact that the C2 pos i t i o n of garveatin A has been shown to be highly susceptible to a l k y l a t i o n , i t i s possible that methylation of garveatin A ( i . e . after c y c l i s a t i o n ) could be the f i n a l step i n garveatin D biosynthesis. 80 CH 3 c=o S-Enz 88 93 Scheme 3. 81 •rt o m 84 16.15 Figure 9. 270 MHz H^ NMR spectrum of M in CDC1 86 87 88 0 105 I ! | I I I I | I I I I | I I I • | i 200 130 1 i ' 1 1 1 I 150 1 1 1 1 1 1 1 1 1 1 1 1 ' 1 1 1 1 1 1 1 1 1 1 1 ^ ' ' ' i m '' i ' 1 ' ' i ' 40 120 100 80 l i > i i I i < i i i i 60 i 40 ' 1 1 I ' 20 I • ' ' ' | 0 PPM. Figure 15. 75 MHz 1 3 C NMR spectrum of 1£5_ in CDC13, B. THE GARVINS (i) 2-hvdroxvqarvin A A second family of anthracenones whose carbon skeleton d i f f e r e d from that of the garveatins was discovered on further examination of the methanol extracts of G_t annulata. This new family, named the garvins, was f i r s t recognised through a detailed study of 2-hydroxygarvin A (H9_). This metabolite was obtained i n reasonably large amounts (lOOmg/collection), was r e l a t i v e l y easy to p u r i f y and was highly soluble i n CDC13, making i t well suited to NMR studies. TLC analysis of the crude hexane and methylene chloride phases of the G_«. annulata extracts indicated the presence of a component which had a white fluorescence under long-wave UV. This compound eluted with 50% ethyl acetate/hexane from the s i l i c a - g e l vacuum f l a s h column and was further p u r i f i e d on LH-20 (90% MeOH/CH 2Cl 2)• A f i n a l f r a c t i o n a t i o n on preparative TLC (10% ethyl acetate/CHCl 3) gave pure 2-hydroxygarvin A ( H i ) , which c r y s t a l l i s e d from d i e t h y l ether as yellow needles (m.p. 195-197°C). Compound 119 had a molecular formula of C23 H26°7 (M+414.1677, calc'd 414.1679) and a UV spectrum with maxima at 226, 278, 318, 332, and 385 nm, in d i c a t i v e of an anthracenone chromophore. The *H NMR spectrum (400 MHz,CDCl3) contained resonances at 6.98 and 7.21 ppm, which corresponded clo s e l y to the chemical s h i f t s of the H5 and H10 protons in garveatins A-D. 92 A s i n g l e t (IH) at 14.25 ppm was appropriate for a phenol proton hydrogen-bonded to a carbonyl oxygen. Acetylation of (119) with acetic anhydride and pyridine formed the diacetate 119 which contained new *H NMR resonances at 2.25(s,3H) and 2.48 ppm(s,3H) that could be assigned to one a l i p h a t i c and one phenolic acetate respectively. The downfield s h i f t of the aromatic proton at 7.21 i n 119 to 7.73 ppm on acetylation required that t h i s proton be ortho or para to the phenol. Also, the absence of carbinol methine or methylene protons i n the *H NMR spectrum of 119 indicated that the 13 a l i p h a t i c alcohol was t e r t i a r y . A C NMR resonance at 81.6 ppm (s), r e f l e c t i n g the deshielding influence of an alcohol oxygen atom, supported t h i s assignment. The presence of an n-propyl group was inferred from the *H NMR spectrum of H9_. Resonances at 1.04 (3H,t,7Hz), 1.73 (m,2H) and 3.15 (m,2H) were shown by decoupling experiments to comprise an iso l a t e d seven spin system appropriate for a three carbon chain attached to an aromatic nucleus. I r r a d i a t i o n of the two-proton multiplet at 1.73 ppm collapsed the t r i p l e t at 1.04 ppm to a s i n g l e t , and the multiplet at 3.15 ppm to an AB system with 12 Hz coupling. This showed that the two benzylic protons were not chemical s h i f t equivalent, d i f f e r i n g by 0.05 ppm. I r r a d i a t i o n of the three-proton t r i p l e t at 1.10 ppm collapsed the multiplet at 1.73 ppm to a doublet of doublets (J=7,8Hz) whereas i r r a d i a t i o n of the multiplet at 3.15 collapsed the same resonance to a quartet (J=7Hz). Resonances at 36.0 ( t ) , 25.3 (t) and 14.7 ppm(q) 13 in the gated decoupled C NMR spectrum were assigned to the three carbons of the n-propyl side-chain. Methyl resonances at 93 3.98 and 3.99 ppm (both 3H fs) were assigned to an aromatic methyl ether ( 1 3C NMR: 55.9,q) and a methyl ester ( 1 3C NMR: 52.3,q; 168.3,s). Further evidence for the methyl ester came from a prominent mass spectral fragment at m/z 381 (M+-31) due to loss of an OCH3 group. The remaining 1H NMR resonances at 1.52, 1.66 and 1.81 ( a l l 3H,s) could be attributed to three a l i p h a t i c methyl l 3 groups. A gated decoupled C NMR spectrum (100MHz, CDC13) of 119 contained two quartets of quartets (J= 160, 4 Hz) at 28.2 and 30.6 ppm, indicating that two of the a l i p h a t i c methyls were attached to the same carbon. The chemical s h i f t s of these methyl groups were close to those of the C12 and C13 carbons (28.5 ppm) 13 in the C spectrum of garveatin C (23J , which suggested that 119 might also contain a gem-dimethyl substituent at C4. This assignment was supported by the presence of a s i n g l e t at 46.9 ppm comparable to the C4 resonance at 48.1 ppm i n the spectrum of garveatin C. A resonance at 209.9 ppm (s) was assigned to an unconjugated ketone, which accounted for the remaining oxygen atom in the molecular formula. The above spectral data was consistent with a 1-anthracenone skeleton containing phenol, t e r t i a r y alcohol, n-propyl, methyl ester, methyl ether, ketone, methyl (3x) and aromatic proton (2x) f u n c t i o n a l i t i e s . A combination of NMR experiments and biogenetic arguments was employed to determine the placement of substituents on the anthracenone nucleus. A difference nOe experiment i n which the aromatic s i n g l e t at 7.21 ppm i n the "'"H NMR spectrum of H9_ was i r r a d i a t e d , produced nOe enhancements i n the other aromatic proton at 6.98 ppm and also i n the methyl resonances at 1.52 and 94 12 13 . 119 R = H 120 R = Ac 124 R = H 125 R = CH 3 95 1.81 ppm. This result i s consistent with placement of the gem-dimethyl group at C4, the phenolic proton at C9 and aromatic protons at CIO (7.21ppm) and C5 (6.98ppm). Ir r a d i a t i o n of the methyl s i n g l e t at 1.52 ppm generated nOe's in the methyl s i n g l e t at 1.81 and the aromatic proton at 7.21 ppm. S i m i l a r l y , i r r a d i a t i o n at 1.81 ppm produced an enhancement of the resonances at 1.52 and 7.21 ppm. Neither of the l a s t two i r r a d i a t i o n s produced an observable enhancement i n the methyl s i n g l e t at 1.66 ppm. On the basis of these experiments a ketone was placed at C3, and a l i p h a t i c methyl and t e r t i a r y alcohol substituents at C2. The 13 a l i p h a t i c methyl resonance at 29.0 ppm i n the gated C NMR spectrum did not show any long-range coupling which was consistent with attachment at the C2 carbon. A close correspondence between the C3 ketone resonance at 209.9 ppm and the C3 resonance at 211.6 ppm i n the spectrum of garveatin C provided further evidence for a six-membered a l i c y c l i c ring in 112.. The remaining n-propyl, methyl ether and methyl ester substituents had to be attached to carbons C6-C8. There are only two arrangements of these groups, represented by structures 119 and 121/ which are consistent with straight-forward polyketide biogenesis. I r r a d i a t i o n of the aromatic proton at 6.98 ppm (H5) induced an nOe in the methyl resonance at 3.98 ppm, which led us to propose 119 as the correct structure. Furthermore, acetylation of the C9 OH group s h i f t e d the C15 methylene protons of the n-propyl side-chain u p f i e l d by 0.31 ppm (3.15,m, in H9_; 2.94,m, in 121). This re s u l t was consistent with placement of the n-propyl group at C8, where i t i s in close s p a t i a l proximity to the C9 OH 96 group. 13 Assignment of the rest of the C NMR spectrum of 119 was 88 aided by analysis of the gated decoupled spectra run i n CDC13 and i n CDCl^ with added D2O. A doublet of doublets at 105.0 ppm, showing a large one-bond coupling of 160 Hz and a small three-bond coupling of 6Hz (to the H10 proton) was assigned to the C5 pos i t i o n , since t h i s resonance i s shielded by the ortho methoxy group at C6. The other doublet of doublets at 116.5 ppm (160,6Hz) was assigned to the CIO carbon which showed three-bond coupling to the C5 proton. The planar geometry of the C-H and C-C bonds i n aromatic molecules gives r i s e to three-bond C-C-C-H couplings i n the 6-8 Hz range, which are usually much larger than two-bond C-C-H couplings. The resonance at 107.3 ppm, assigned to the C9a p o s i t i o n , appeared as a doublet of doublets (J=8,4.8Hz) i n the gated spectrum run i n CDCl-j, but collapsed to a doublet on addition of D2O. This indicated that the C9 OH proton i s coupled to C9a (J=4.8Hz), and t h i s coupling i s removed on exchange of that proton with D2O. This leaves a three-bond 8Hz coupling to the H10 proton. The resonance at 116.7 ppm, assigned to the C8a po s i t i o n , appeared as a broad, distorted doublet in the gated spectrum i n CDCl^f but collapsed to a broad s i n g l e t on addition of D2O. As i n the previous case, t h i s implies a three-bond coupling to the C9 OH proton. The broadness of the resonance in the D 20 exchanged spectrum probably r e f l e c t s three-bond coupling to both H5 and H10 protons, as well as to one or both H15 protons on the n-propyl side-chain. F i n a l l y , the resonances at 199.9 and 209.9 ppm, assigned to the CI and C3 carbonyl groups respectively collapsed 97 from broad doublets (J=2.9, 2.0Hz resp.) to broad singlets on addition of D2O. This indicates a long-range coupling to the C2 OH group, and the smaller J value may be a consequence of the non-planarity of the H-O-C-C bond sequence. At t h i s stage there was s t i l l some ambiguity over the assignment of C5 and CIO (105.0 or 116.5 ppm) and also C8a and on nn C9a (107.3 or 116.7 ppm). The use of a novel experiment ' cal l e d INAPT (Insensitive Nuclei Assigned by Po l a r i s a t i o n Transfer) provided the c r u c i a l assignment information. This technique, developed recently by Bax, provides long-range heteronuclear connectivity information by using a set of low int e n s i t y radiofrequency pulses to i r r a d i a t e a preselected proton resonance. This pulse sequence can then transfer proton 13 magnetisation to any C nucleus that has a s i g n i f i c a n t long-range scalar interaction with t h i s proton. The major advantage of t h i s technique over other methods i s that t h i s long-range connectivity i s obtained with high s e n s i t i v i t y , on the order of the normal *H decoupled spectrum. Therefore the INAPT experiment can be performed on much more d i l u t e samples than i s currently 91 possible using the 2-D HETCOR technique. INAPT also requires a much smaller data base since i t i s a "l-D" experiment. Another advantage of the INAPT technique i s i t s s e l e c t i v i t y , only one proton resonance i s i r r a d i a t e d at a time and consequently only one set of correlations are observed. Figure 16 shows the decoupled spectra obtained with INAPT transfers from H5 and H10. Since jCH couplings i n aromatic molecules are i n the order of 6-8 Hz, the experiment was optimised for transfer through 7Hz 98 99 couplings. I r r a d i a t i o n of the H5 proton (6.98 ppm) resulted in transfer of magnetisation to resonances at 116.5, 116.7 and 126.9 ppm. Since the resonance at 126.9 ppm had previously been assigned to C7, i t follows that the CIO and C8a resonances are at 116.5 and 116.7 ppm respectively. S i m i l a r l y , i r r a d i a t i o n of H10 (7.21 ppm) produced a transfer to resonances at 46.9 (C4), 107.3, 116.7 (C8a) and 105.0 ppm. Therefore, C5 (105.0) and C9a(107.3) were p o s i t i v e l y assigned. The low-intensity peak at 142.1 ppm in t h i s spectrum i s probably due to the presence of a s i g n i f i c a n t four-bond long-range coupling between H10 and C8 (see ref. 90). A similar argument can be used to explain the presence of a weak resonance at 107.3 ppm (C9a) when H5 i s i r r a d i a t e d . Both spectra were acquired with 800 scans, requiring approximately 30 minutes each, with a 60 mg sample s i z e . Support for the proposed structure of 2-hydroxygarvin A came from i t s s i m i l a r i t y to garvin A quinone (122), whose structure was solved by an X-ray d i f f r a c t i o n analysis on i t s monoacetate (122.) . ( i i ) garvjn A. quinone Close examination of the fractions eluting with 50% ethyl acetate/hexane and 100% ethyl acetate by f l a s h chromatography enabled us to i d e n t i f y a minor compound, whose bright red color was reminiscent of garveatin A quinone (105). Sequential p u r i f i c a t i o n by LH-20 (90% MeOH/CH2Cl2) chromatography, preparative TLC (50%ethyl acetate/hexane) and normal-phase HPLC (ethyl acetate/hexane) yielded 10 mg of an orange-red s o l i d . Mass spectrometry indicated that i t had a molecular formula of 100 C23 H24°7 4 1 2 « 1 5 1 8 ' c a l c x d 412.1522). 1H NMR analysis (see Table 7) showed that i t contained the n-propyl, methyl ether and methyl ester substituents present i n 2-hydroxygarvin A (119). Singlets at 1.61 (6H) and 1.98 ppm (3H) were almost i d e n t i c a l to the resonances assigned to the Cll-13 methyl groups i n the ''"H NMR spectrum of garveatin A (22), suggesting the presence of the same a l i c y c l i c ring i n both compounds. Acetylation with acetic anhydride and pyridine produced a monoacetate which c r y s t a l l i s e d from acetone, and whose structure was shown to be 123 by single -c r y s t a l X-ray d i f f r a c t i o n analysis, performed by Van Duyne and Clardy at Co r n e l l . Therefore the parent compound, named garvin A quinone, had structure 122. A computer-generated perspective drawing of 123 i s given i n Figure 17, and exhibits a pronounced non-planarity of the quinone ring which adopts a boat-like conformation with the C9 and CIO carbons above the plane of the rin g . An enolic resonance at 12.15 ppm(s,lH) i n the *H NMR spectrum of 122 indicates hydrogen-bonding between the CI OH proton and the C9 carbonyl oxygen. Thus, 122 exists as the 3-keto tautomer i n chloroform solution, as i s the case for garveatin A quinone (105) and garveatin B quinone (106). ( i i i ) Garvin A By analogy with the garveatin family, i t was anticipated that the annulata extracts might contain a garvin-type compound having a keto-enol f u n c t i o n a l i t y i n the a l i c y c l i c portion of the molecule, similar to that i n garveatins A and B. This prediction was v e r i f i e d by p u r i f i c a t i o n of garvin A (124.) 101 Figure 17. Computer generated X-ray structure of the acetate of garvin A quinone (123.) • using LH-20 chromatography (90% MeOH/CH2Cl2) and preparative TLC (10% ethyl acetate/CHCl 3) from the f l a s h f r a c t i o n s eluted with 100% ethyl acetate. Compound 121 was obtained as a bright yellow s o l i d that gave a parent ion i n the mass spectrum at m/z 398, appropriate for a molecular formula of C23 H26°6* I t s l f l N M R spectrum showed resonances for the n-propyl (1.03, t, J=7Hz, 3H; 1.73, m, 2H; 3.18,m, 2H), methyl ether, methyl ester (3.95,s, 3H; 3.79, s, 3H), H5 (6.91, s, 1H) and H10 (7.17, s, 1H) f u n c t i o n a l i t i e s found i n 2-hydroxygarvin A (Hi) . Additional resonances at 1.64 (s,6H), 1.99 (s,1.5H), 1.50 (d,J=7Hz,1.5H) and 4.01 (q,J=7Hz,0.5Hz) corresponded c l o s e l y to the signals assigned to the tautomeric forms of ring A i n garveatin B (J3J1) . A simple combination of the two portions indicated by ''"H NMR allowed the assignment of structure 124 to garvin A. The tautomeric forms were present i n approximately 1:1 r a t i o as measured by peak i n t e n s i t i e s i n the ''"H NMR spectrum. Treatment of garvin A with diazomethane yielded 3-methyl garvin A (125.) as the major reaction product. A *H NMR resonance at 3.93 ppm (s,3H) was appropriate for a methyl ether, and the presence of the hydrogen-bonded C9 OH s i n g l e t at 15.87 ppm indicated that the C3 OH group had been methylated. This res u l t i s analagous to the methylation of garveatin B (&&) at the C3 OH po s i t i o n with diazomethane. (iv)Garvjn B_ The second most abundant polyketide-type metabolite present in the G^ . annulata extracts was found to be garvin B (12£)• This 103 was an o p t i c a l l y active yellow s o l i d which co-eluted with garveatin A when fractionated by f l a s h chromatography (ethyl acetate/hexane). Further p u r i f i c a t i o n on LH-20 (90% MeOH/CH2Cl2) yielded s l i g h t l y impure garvin B, which was poorly soluble i n polar organic solvents such as acetone and methanol. T r i t u r a t i o n of the impure garvin B sample with acetone was used as the f i n a l p u r i f i c a t i o n step. Garvin B (126.) was shown by HRMS to have a molecular formula of C 21 H20°6 (obsv'd 368.1269, req'd 368.1260) and i t gave a bright yellow fluorescence under long-wave UV when TLC'd on s i l i c a - g e l . Methyl resonances at 1.54, 1.58 and 1.85 ppm ( a l l s,3H) in the 1H NMR spectrum (300 MHz, DMSO d,,) of 126. showed a D close resemblance to the resonances for the methyl groups i n the a l i c y c l i c ring of garveatin A (12) (DMSO d g : 1.59,s,6H; 1.90,s,3H), indicating that the two molecules had th i s substructure i n common. Resonances at 7.16 and 7.35 ppm could be assigned to aromatic protons at C5 and CIO of an anthracenone skeleton by analogy with other Garveia metabolites. The presence of two phenolic groups i n the molecule was indicated by si n g l e t s at 11.14 and 17.85 ppm in the *H NMR spectrum and v e r i f i e d by acetylation with acetic anhydride/pyridine to y i e l d the 3,6-diacetate (122) and the 3,6,9-triacetate (128.) as the major reaction products. A six proton spin system (1.50, d, J=7Hz, 3H; 4.77, m, IH; 3.23, dd, J=19,13Hz, IH; 4.45, dd, J=19,3Hz, IH) was assigned to an n-propyl residue which contained an oxygen substituent on the central carbon and an a r y l substituent at one of the terminal carbons. Decoupling experiments were performed on the trimethyl derivative (122.) r formed by treatment of garvin B 104 126 R ,R 1 = H 127 R=Ac R1 = H 128 R, R 1 = Ac 105 with methyl iodide, since t h i s compound was soluble i n CDCl^ and showed better resolution i n the ''"H NMR spectrum. In addition, a spectrum of the above-mentioned spin-system i n 129 was generated using a spin-simulation program (Figure 21a), and the simulated spectrum c l o s e l y matched the appropriate resonances i n the actual *H NMR spectrum. 13 The C NMR spectrum (75 MHz, DMSO d g) of garvin B contained resonances at 20.4, 75.4, 34.0 ppm which were assigned to the substituted propyl residue, the most deshielded resonance being appropriate for an oxygen-bearing methine carbon. A resonance at 170.6 ppm indicated an ester f u n c t i o n a l i t y i n the molecule. Modification of the structure of garvin A (12A) by replacing the methoxy substituent at C6 with a phenol, and formation of a delta lactone between an alcohol at C16 and a carboxylic acid group at C14 leads to structure 126 which e f f e c t i v e l y accounts for a l l the properties of garvin B. This molecule contains a c h i r a l center at C16 and i s o p t i c a l l y active, as evidenced by the s p e c i f i c rotation measurement of [«c]D +172.69° ( c 0.26, CHC13) for the trimethyl derivative 129. This c h i r a l i t y accounts for the non-equivalence of the gem-dimethyl groups i n the NMR spectra of garvin B. Compound 129 has been methylated at the C2 p o s i t i o n as well as at the C6 and C9 OH groups, analogous to formation of 2,8,9-trimethyl garveatin A (9_5_) from garveatin A with methyl iodide. 106 Table 5_L H NMR data (CDC13) for the Garvins. Chemical s h i f t s i n ppm from TMS. H on C# Garvin A 2-OH Garvin A Garvin B a 2-OH Garvin B 124. 112. 126. 120 20H — — — 4.02 br s 5 6.91 6.98 7.16 7.11(7.11) * * 60R 3.95 3.98 11.14 11.44(11.45) 90R 15.14 14.25 17.85* 14.61(14.62) 10 7.17 7.21 7.35 7.11(7.11) 11 1.99 1.66 1.85 1.65(1.66) 12 1.64 1.52 1.54 1.50(1.54) 13 1.64 1.81 1.58 1.80(1.82) 140R 3.97 3.99* - -15 3.18 m 3.15 ra 3.23 dd 3.41(3.44)dd 4.35 dd 4.38(4.38)dd 16 1.73 m 1.73 m 4.77 m 4.77(4.77)m 17 1.03 t 1.04 t 1.50 d 1.64(1.65)d * indicates assignments may be reversed. 130 exists as a mixture of diastereomers. 107 (v) 2-Hvdroxyaarvin B. In p a r a l l e l with the garvin A s e r i e s , the 2-hydroxy derivative of garvin B (130) was isol a t e d as a minor constituent from the £L_ annulata extracts. P u r i f i c a t i o n by LH-20 chromatography and preparative TLC (10% ethyl acetate/CHCl^) yielded a component which migrated as a single spot on TLC (R^ 0.35 i n standard system). The •'"H NMR spectrum (300MHz, CDC13) of 130f however, indicated a 1:1 mixture of diastereomers, since seven of the eleven resonances were doubled at t h i s f i e l d strength. 13 The C NMR spectrum (see Table 10) also confirmed the presence of a diastereomeric mixture, since f i f t e e n of the twenty one carbon resonances were doubled at a f i e l d strength of 75 MHz. The resonances for the a l i c y c l i c ring carbons and the three a l i p h a t i c methyls were i n excellent agreement with the corresponding signals in the spectrum of 2-hydroxygarvin A (119). S i m i l a r l y , the resonances for the remaining naphthalenic and la c t o n i c substructures of 130 could be matched with those i n the 13 C NMR spectrum of garvin B (126). We have not determined which of the two c h i r a l centers in 2-hydroxygarvin B i s epimeric in the two naturally-occurring diastereomers. However, the fact that garvin B i s o p t i c a l l y active implies the existence of only one configuration at C16 and suggests that b i o l o g i c a l hydroxylation at C2 has occurred i n non-s t e r e o s p e c i f i c fashion. Acetylation of 2-hydroxygarvin B with acetic anhydride and pyridine yielded a 1:1 mixture of the epimeric t r i a c e t a t e s 131a 108 130 R=H 131 R=Ac 0 132 R = H 133 R = Ac 109 Table £: H NMR data for the Acetates of 2-hydroxy derivatives. Chemical s h i f t s i n ppm from TMS . A l l spectra run in CDC1 H on C# 21 104 120, 131b 20Ac 2.20 2.22 2.25 2.22 2.24 5 7.61 7.55 7.06 7.50 7.50 60R - - 3.99 * 2.39 2.41 80Ac * 2.34 2.45 - - -90AC * 2.41 2.45 2.48 * 2.45 2.51 10 7.79 7.70 7.73 7.80 7.80 11 1.45 1.44 1.50 1.47 1.49 12 1.57 1.55 1.60 1.58 1.63 13 1.79 1.78 1.83 1.84 1.81 14 2.41 2.50 - - -140Me - - 3.99 - -15 16 2.50 2.70 q 1.13 t 2.94 1.70 m m 3.20 m 3.92 m 4.56 m 3.27 3.68 4.58 17 — — 1.03 d 1.58 d 1.55 3* * indicates assignments may be reversed. 110 and 131b as the major reaction products. It was possible to separate these t r i a c e t a t e s by preparative TLC since t h e i r R f values d i f f e r e d by 0.03 i n 50% ethyl acetate/hexane. Both 13ia and 131b gave a molecular ion of 510 daltons in the mass spectrum and had almost i d e n t i c a l UV spectra. However, the i r 1H NMR spectra showed some d i s t i n c t differences, e s p e c i a l l y the chemical s h i f t s of the a l i p h a t i c methyl resonances. Both compounds contained three methyl singlets i n the 2.2-2.5 ppm region corresponding to the three acetate methyls, and also two aromatic singlets (1H each) at 7.50 and 7.80 ppm. (vi) Garyjn Quinone Using the same fr a c t i o n a t i o n steps described for the p u r i f i c a t i o n of garvin A quinone (122) (LH-20 chromatography, preparative TLC and normal phase HPLC), another bright red compound was p u r i f i e d and i d e n t i f i e d as garvin B quinone (132). Quinone 132 had a molecular formula of C2i Hi8°7 a s determined by HRMS (obt'd. 382.1055, req'd. 382.10xx). The base induced bathochromic s h i f t of the peak at 397nm (to 463nm) in the UV spectrum was t y p i c a l of a naphthoquinone substructure. The "^H NMR spectrum (400MHz, CDCl^) contained resonances at 1.58 (s,3H), 1.60 (s,3H), 1.95 (s,3H) and 7.64 (s,lH) ppm which corresponded cl o s e l y to those of H l l , H12, H13 and H5 in the spectrum of garvin A quinone (122). The six proton spin system [1.63 ( d, J=7Hz, 3H), 3.23 (dd, J=19,12 Hz,lH), 3.99 (dd, J=19,3 Hz, 1H), 4.72 (m,lH)] indicated the presence of a delta lactone system, similar to that i n the spectrum of garvin B (12A) • One of the methylene protons at C15 has been s h i f t e d u p f i e l d by 0.36 ppm on 111 Table 2: -""H NMR data (CDC13) for the quinones. Chemical s h i f t s i n ppm from TMS. on C# Garveatin A Garveatin B Garvin A Garvin B quinone (IH5J quinone (1M) quinone(122) quinone (13_2) 10R 11.66* 11.84* 12.15 12.28* 5 7.55 7.51 7.56 7.64 60R - - 4.03* 11.85* 80R 11.45* * 11.79 - -11 1.98 1.97 1.98 1.95 12 1.63 1.61 1.61 1.58* 13 1.63 1.61 1.61 1.60* 14 2.41 2.45 - -140R - - 3.98* -15 - 2.78 q 2.95 m 3.23 dd 3.99 dd 16 2.60 1.16 t 1.60 m 4.72 m 17 — — 1.05 t 1.63 d *,# indicates assignments may be reversed. 112 replacement of a phenol group with a ketone (3.99 ppm i n 122., 4.35 ppm i n 125.). This suggests that t h i s proton may be oriented such that i t l i e s i n the shielding cone of the C9 ketone i n 132. Singlets at 11.85 and 12.28 ppm are appropriate for hydrogen-bonded phenol protons at CI and C6 f providing support for the existence of the 3-keto tautomer i n chloroform solution, as i s 13 also the case for garvin A quinone. The C NMR spectrum of 132 (75 MHZrCDCl^) i s consistent with the proposed structure. Acetylation of 132 with acetic anydride/pyridine yielded the diacetate 133 as a yellow o i l . A molecular ion of 466 daltons i n the mass spectrum was appropriate for a molecular formula of C25 H22°9 a n d a s ^ n 9 l e t a t 2.38 ppm (6H) i n the "^H NMR spectrum (300MHz, CDCl^) was assigned to the two aromatic acetate methyl groups. ( v i i ) Garvin h djmey During the p u r i f i c a t i o n of garvin A (124.) on s i l i c a g e l , a minor, more non-polar component which had not previously been detected i n the crude extracts was characterised and i d e n t i f i e d as garvin A dimer (134). In p a r a l l e l with the mass spectra of the dimers of garveatins A and B (110 and 108), compound 134 did not give an observable molecular ion i n the EI spectrum, the highest-mass peak occuring at 398 daltons corresponding to a monomeric garvin A unit. The *H NMR spectrum of 134 indicated the presence of the same naphthalenic subunit as i n garvin A and 2-hydroxygarvin A. The n-propyl, methyl ether, methyl ester and aromatic protons were a l l shielded, r e l a t i v e to the corresponding chemical s h i f t s i n 119. This shielding e f f e c t of the naphthalenic 113 134 R = H 135 R=Ac 135 114 substituents was also observed i n the H NMR spectra of 108 and Hf i and appears to be a feature of 2,2' dimers of t h i s type. Singlets at 1.58, 1.71 and 1.94 ppm ( a l l 3H) were assigned to methyl groups at C2 and C4 (2) of the a l i c y c l i c ring and 1 3 C NMR resonances at 202.5, 63.4, 209.9 and 47.9 ppm were almost i d e n t i c a l to those assigned to C1-C4 respectively i n the spectrum of garveatin B dimer (108). Therefore the monomeric subunits are connected at the C2 position of ±2Ar s i m i l a r to the other dimers studied. I r r a d i a t i o n of the methyl si n g l e t s at 1.47 and 1.59 ppm induced an nOe i n the H10 proton at 7.05 ppm, which assigned these resonances as the gem-dimethyl protons at C12 and C13. Confirmation of the dimeric nature of 134 was obtained by acetylation with acetic anhydride/pyridine to give the diacetate (13_5J . This derivative gave a molecular ion at 878 daltons i n the EI mass spectrum corresponding to a molecular formula of C50 H54°14* Resonances at 2.32 (s,3H) and 7.72 ppm (s,1H(H10)) i n the 1H NMR spectrum indicated acetylation at the C9 and C9*OH positions. Subsequently, various reaction conditions were investigated for the conversion of garvin A to the dimer 134. It was found that treatment of a methanolic solution of garvin A with s i l v e r oxide under a stream of a i r produced the dimer 134 in reasonable y i e l d . Furthermore, the dimer could be reconverted to the monomer by hydrogenolysis in, the presence of palladium on charcoal, ind i c a t i n g that the 2,2' bond i s susceptible to reductive cleavage. However, dimerisation of garvin A did not appear to occur to any s i g n i f i c a n t extent i n CDC1, solution as was the case 115 for the dimerisation of garveatin B. ( v i i i ) Garveatin B-aarvin £ dimer Analysis of the less polar fractions obtained by LH-20 chromatography of the G.annulata extracts revealed the presence of a compound with a s l i g h t l y lower R f (0.63) than garveatin B dimer (108) in the standard TLC system. P u r i f i c a t i o n by preparative TLC (10% ethyl acetate/CHCl 3) and reverse phase HPLC (95% CH3CN/H20) yielded 7mg of an orange o i l whose 1H NMR spectrum (400MHz,CDC13) indicated sets of resonances corresponding to both garveatin B and garvin B substructures. A FAB mass spectrum showed a molecular ion at 693 daltons (M ++l), appropriate for a molecular formula of C4i H4o°io a n <^ strong fragment ions at m/z 326/327 and 368/369 corresponding to garveatin B and garvin B monomeric units respectively. This data indicated that the compound was a mixed dimer with structure 136. The "^H NMR spectrum of 136 was consistent with attachment of the monomeric units at the 2,2' po s i t i o n , s i m i l a r to the other dimers previously characterised. In addition, many of the resonances showed u p f i e l d s h i f t s r e l a t i v e to the corresponding resonances i n the "*"H NMR spectra of the monomers. In the case of the up f i e l d H15 resonance i n 136, the chemical s h i f t (2.35 ppm) d i f f e r e d by more than 1 ppm from that in 2-hydroxygarvin B (3.41 ppm). The phenol proton resonances were assigned on the basis of s i m i l a r i t y of two of them (9.10,15.70 ppm) to the phenolic resonances i n the spectrum of garveatin B dimer (lUfl) . When equal amounts of garveatin B and garvin B were s t i r r e d for 24 hours in a chloroform solution containing s i l i c a g e l , the 116 Table j£ j_ H NMR Data (CDC13) for the Dimers, Chemical s h i f t s i n ppm from TMS. Compound Garveatin A Garveatin B Garvin A Garveatin B-Garvin B dimerlHILL dimer (108) d i m e r H M l dimer (136) H on C# 5 10 11 12 13 14 15 16 17 60R 80H 90H 140Me 6.98 7.05 1.94 1.47 1.59 2.38 2.58 10.34 15.81 6.98 6.98 1.88 1.38 1.66 2.43 2.53(m) 2.70(m) 1.07(t) 9.37 15.79 6.89 7.11 1.94 1.58 1.71 2.90(m) 1.60(m) 0.93(t) 3.94 14.65 3.94 6.90:6.92 6.90:7.02 1.80:1.82* 1.24:1.20* 1.78:1.81* 2.42: -2.42(m):2.35(dd) 2.77(m):3.83(dd) 1.00(t):4.40(m) I. 51(d) II . 56 14.85 9.10 15.70 A l l resonances are si n g l e t s unless indicated otherwise. * indicates assignment may be reversed. 117 symmetrical dimer 108 and the mixed dimer 136 were formed i n approximately equal amounts. Therefore, i t seems l i k e l y that both these compounds are a r t i f a c t s formed during f r a c t i o n a t i o n of the G. annulata extracts. Surprisingly, no evidence of a symmetrical garvin B dimer was detected in the extracts, even though garvin B i s one of the most abundant anthracenone metabolites present. This seems to suggest that garveatin B may be the i n i t i a t o r of the dimerisation reaction to produce 136. probably v i a a free-r a d i c a l mechanism. In view of the fact that garveatin B tautomerises r e a d i l y i n chloroform solution to the diketo form, generation of a f r e e - r a d i c a l at the C2 position of the diketo tautomer would be a l i k e l y i n i t i a t i n g step i n the reaction sequence. Biogenesis It appears l i k e l y that the same C18 polyketide precursor proposed i n the biogenesis of the garveatin series i s u t i l i s e d to form the garvins, v i a an alt e r n a t i v e folding pattern (Scheme 4). Thus, monomethylation and dimethylation of the nonaketide precursor at C8 and CIO respectively, followed by c y c l i s a t i o n could generate an anthracenone containing a 2-oxopropyl group at C7. Reduction of t h i s f u n c t i o n a l i t y to an n-propyl group, as well as methylation at the C6 OH and C14 COOH positions y i e l d s garvin A (12A). Al t e r n a t i v e l y , reduction of the 2-oxopropyl group to a secondary alcohol, followed by condensation with a carboxylic acid group at C14 to produce a delta lactone can be invisaged in the biogenesis of garvin B (126). Further oxidations at C2 and 118 119 122 S P I N S I M U L A T I O N : 2 - H Y 0 R 0 X Y G A R V I N A A 3 B C D E S P I N S Y S T E M ( 4 0 0 M H Z ) A3B1C1D1E1 LW 1 . 000« i C O U P L I N G CONST MINF i i JAB 7.0000 MAXF 1500.01 i JAC 7.0000 THS 0. IOOI ' J AD 0 J A E 0 C H E M I C A L S H I F T J B C 0 A 416.0c i JBD 7 . O O O O B 692. Oi i J B E 8.0000 C 672.0c i JCD 8.0000 D 1 254 . Oi i J C E E 1266.0c > J D E 12.0000 to 3 2 1 ppm. Figure 18a. 400 MHz spin simulation for the n-propyl side-chain of H I . 124 S P I N S I M U L A T I O N : T R I M E T H Y L G A R V I N B 3 0 0 M H Z A 3 B C D S P I N S Y S T E M X L - 3 0 0 IH OBSERVE STANDARD PARAMETERS A3B1C1D1 LW 2 . 0 0 0 0 C O U P L I N G CONST MINF 0 J A B 0 MAXF 2 0 0 0 . 0 0 J A C 0 T H S 0. 1000 JAD 7.0000 J B C 1 9 . 0 0 0 0 C H E M I C A L S H I F T J B D 1 3 . 0 0 0 0 A 4 6 5 . 0 0 J C D 3 . 0 0 0 0 B 9 8 1 . 0 0 C 1 2 3 9 . 0 0 D 1 3 3 5 . 0 0 to I i i i ' | i i i i | i i i 1 1 1 1 1 1 1 1 1 r 1 1 1 1 1 r 4-5 4.0 3.5 3.0 2.5 2.0 Figure 21a. 300 MHz spin simulation for the lactone system of 12_9_. 126 127 J 129 Figure 25a. Comparison of lactone spin system in 400 MHz H NMR spectrum of 122 with a simulated spectrum. 131 132 133 134 C. THE GARVALONES Closely related to the garvin family are garvalones A (137) and B (141), two minor metabolites which contain the unusual 3-92 oxobutyl f u n c t i o n a l i t y at the C2 position . (i) Garvalone A. Garvalone A (137), the more abundant of the two, was detected as a minor component of the LH-20 fractions containing 2-hydroxygarvin A (112.) . It had a white long-wave UV fluorescence, s i m i l a r to 119, and was p u r i f i e d by preparative TLC (50% ethyl acetate/hexane) to give 20 mg of a l i g h t yellow o i l . Compound 137, which was o p t i c a l l y i n a c t i v e , was shown by HRMS to have a molecular formula of C27 H32°7 (obsv'd 468.2162, req'd 468.2149). A close relationship between the aromatic portion of the previously reported 2-hydroxygarvin A (119) and a fragment of 137 was immediately apparent from a comparison of thei r NMR and UV data. The UV spectrum ( m a x 228, 279, 317, 330, and 382 nm) of 121 was v i r t u a l l y i d e n t i c a l to that of H9_ (X „ =„ 226, 278, IUciX 318, 332, and 385 nm) and i t s 1H NMR spectrum (Table 9) contained resonances that were appropriate for the n-propyl (1.11, t , J=7Hz, 3H; 1.75, m, 2H; 3.15, m, 2H), methyl ester, methyl ether (3.93, s, 3H; 3.96, s, 3H), H5 (6.93, s, IH) and H10 (7.13, s, IH) f u n c t i o n a l i t i e s also found i n H9_. A nearly exact 13 correspondence of the C NMR resonances assigned to the carbons in the naphthalene nucleus, the propyl side chain, the methyl ester and methyl ether f u n c t i o n a l i t i e s i n the two compounds 135 (Table 10) confirmed the presence of the naphthalenic substructure 13_& in compound 137. Functionality which accounted for the remaining atoms ^ C11 H16°3^ °^ 9 a r v a l o n e A (137) could be r e a d i l y i d e n t i f i e d upon 1 13 further consideration of i t s H and C NMR spectra. Four methyl si n g l e t s at 1.47, 1.55, 1.59, and 2.04, and a complex multiplet at 2.23 (4H) ppm located the remaining hydrogen atoms. Carbon resonances at 39.1 (t) and 31.4 (t) required that the protons resonating at 2.23 ppm be attached to two methylene carbons. Carbonyl resonances at 211.9, 207.0, and 203.9 showed that the three oxygens were part of ketone f u n c t i o n a l i t i e s and resonances at 58.3 (s) and 47.9 (s) ppm i d e n t i f i e d quarternary carbons. A comparison of the carbon resonances at 203.9, 58.3, 211.9, and 47.9 ppm i n the spectrum of 137 to the resonances exhibited by carbons 1 to 4 i n garveatin C (9_3_) (CI, 204.9; C2, 55.3; C3, 211.6; C4, 48.1) showed that they were v i r t u a l l y i d e n t i c a l . This correspondence implied that both metabolites had the 1-oxo, 2-d i a l k y l , 3-oxo, 4-dialkyl f u n c t i o n a l i t i e s in common. Demonstration of an nOe between the aromatic proton resonance at 7.13(H10) and two methyl resonances at 1.55 and 1.59 ppm i n the '''H NMR of 137 provided evidence for a gem-dimethyl group at C4. The "^H NMR resonance at 1.47 (s, 3H) was assigned to a methyl substituent at C2 by analogy with the other Garveia metabolites, and a methyl ketone residue was indicated by a "^H NMR resonance at 2.04(s, 3H) and 1 3 C NMR resonances at 207.0(s) and 30.5(q) ppm. Insertion of the two methylene carbons between the carbonyl carbon of the methyl ketone residue and the remaining unsa t i s f i e d 136 18 0 OR 'OCH OCH. 137 R = H 139 R = CH. OCH OCH. 138 137 valence at C2 of the anthracenone skeleton gave the proposed structure 137 for garvalone A. In the mass spectrum, 137 shows intense fragment ions at 398 (M+ + H - (CH3COCH2CH2-) :100%) and 397 (M+ - (CH3COCH2CH2-) :89%) as would be expected. Treatment of garvalone A (137) with K 2C0 3 and methyl iodide in refluxing acetone gave the monomethyl derivative (125.) i n nearly quantitative y i e l d . The resonances for the four methylene protons, which had appeared as a complex multiplet at 2.23 ppm in the *H NMR of 137, were more extensively dispersed i n the spectrum of 121 (2,48, m, IH; 2.24, m, IH; 1.95 to 2.10, m, 2H). A series of decoupling experiments and spin simulations (Figure 31a) showed that these four protons constituted an ABCD spin system, which confirmed the linkage of the two methylene carbons as postulated. In order to provide a suitable model compound for the cyclohexadione system i n 137, a synthesis of 2-(3-oxobutyl) garveatin A (140) was undertaken, u t i l i s i n g a Michael reaction 93 between garveatin A (22) and methyl v i n y l ketone . I n i t i a l attempts to carry out t h i s reaction in the presence of polar solvents such as methanol and anhydrous ethanol (K 2C0 3,reflux) were unsuccessful and only led to formation of material which remained at the o r i g i n when chromatographed on s i l i c a - g e l . However, using benzene i n the presence of K 2C0 3 and a c r y s t a l of 18-crown-6, at reflux under N 2 for 24 hours produced the desired product i n 80% y i e l d . Apparently, the carbanion (at C2) of garveatin A i s more unstable and consequently more reactive i n a non-polar solvent such as benzene. The use of crown 138 ether also appears to f a c i l i t a t e the reaction by chelating the K counter-ion r and thereby enabling the "naked" anion to be alkylated more e f f i c i e n t l y . (ii)Garvalone B_ Garvalone B (141), an extremely minor component of the crude extracts, was iso l a t e d as an o p t i c a l l y active ([°*]D +136.92°, c. 0.39, CHCl^) l i g h t yellow o i l . In the mass spectrum, 141 showed a parent ion at m/z 438 appropriate for a molecular formula of C26 H28°6* T n e 1 h N M R ° f ( t a D l e also showed a number of resonances that were doubled, again indicating the presence of two diastereomers (ratio 2:3) which we were unable to separate. The major isomer displayed signals at 1.46(s, 3H), 1.55(s, 3H), 1.57(s, 3H), 2.06(s, 3H) and 2.24(m, 4H) ppm that corresponded to the "'"H NMR resonances displayed by the f u n c t i o n a l i t y i n the a l i c y c l i c ring of garvalone A (137). The remaining "'"H NMR resonances in the spectrum of H I (1.62(d, J=7HZ, 3H), 3.42(dd, J=12, 18Hz, IH), 4.39(dd, J=18, 3Hz, IH), 4.72(m, IH), 7.06 (s, IH), 7.10(s, IH), 11.50(s, IH) and 15.44(s, lH)ppm) showed a close correspondence to the observed H^ NMR resonances for the aromatic and l a c t o n i c portions of garvin B (141). Combining the two s t r u c t u r a l fragments resulted in the proposed structure 141 for garvalone B. Garvalone B was quant i t a t i v e l y converted to the diacetate (142) in accordance with the proposed structure. A synthesis of a mixture of garvalone B diastereomers (141) (ratio 2:3) was achieved v i a a Michael reaction between garvin B (126) and methyl v i n y l ketone. The major isomer formed i n the Michael reaction was the minor naturally occurring isomer. Once again, 139 Table 9: -""H NMR Data (CDCl-j) . Chemical s h i f t s are i n ppm from TMS. Compound Carbon # 10R 20R 5 60R 80R 9 OR 10 11 11a 12 13 14 140R 15 16 17 18 19 21 Garvalone A (122) Garvalone B (111) 6.93 3.96& 15.10 7.13 1.47 1.55* 1.59* 3.93& 3.15 1.75,m l . l l , t 2.23,m 2.23fm 2.04 7.06 11.50 15. 44* 7.10 1.46 1.55 1.57" # 3.42,dd 4.39,dd 4.72,m 1.62,d 2.24,m 2.24,m 2.06 2-(3-oxobutyl) garveatin A(14H) 6.65 10.13 16.29 7.12 1.48 1.54* 1.58 2.34 # 2.55 2.24 m 2.24 m 2.04 m *.&,# indicate assignments may be reversed, 140 Table 10_: 1 J c NMR (CDCl 3)data. Chemical s h i f t s are i n ppm from TMS. Compound 2-Hydroxy garvin A11121 Garvalone (137) A 2-(3-oxobutyl) garveatin A(140) 2-Hydroxy garvin B(130) Carbon # 1 199.9 203.9 204.1 199.71(199.55) 2 81.6 58.3 58.0 81.64(81.57) 3 209.9 211.9 211.3 209.83 4 46.9 47.9 47.9 47.14(47.05) 4a 142.9 143.0 142.5* 145.40(145.26) 5 105.0 $ 105.0 $ 115.5 111.81 6 157.7 157.4 141.2* 161.27(161.16) 7 126.9 126.5 125.3 115.94 8 142.1 142.5 156.0 144.58(144.54) 8a 116.7* 116.8* 110.8 110.81(110.77) 9 166.0 166.6 166.1 167.39(167.17) 10 116.5 $ 116.8 $ 120.2 116.03 9a 107.3* 108.7* 107.8 107.00(106.88) 10a 142.0 142.8 139.5* 143.84 11 - 24.2 24.1 28.43(27.99)* 12 - 29.9* 29.9* 29.15(29.04)* 13 - 27.0* 27.1* 30.70(30.44)* 14 168.3 168.6 20.7 169.85(169.80) 15 36.0 36.1 204.2 34.60(34.36) 16 25.3 25.4 32.3 75.43(75.35) 17 14.7 14.8 - 20.83 18 - 31.4 31.3 -19 — 39.1 38.9 141 Table 10 cont'd. 20 - 207.0 206.9 21 - 30.5 30.7 60R 55.9 55.9 _ 140R 52.3 52.5 *,# indicate that the assignments may be interchanged. 141 R = H K 2 R=Ac since garvin B occurs as a single enantiomer, i t seems reasonable to assume that i t i s the c h i r a l center at C2 which i s epimeric in the natural garvalone B stereoisomers. This assignment i s consistent with the lack of o p t i c a l a c t i v i t y found for garvalone A (137) which implies a nonstereospecific a l k y l a t i o n at C2 in both molecules. The p o s s i b i l i t y that the garvalones are a r t i f a c t s due to contamination of solvents by methyl v i n y l ketone was ruled out by the following evidence: (a) the garvalones were found repeatedly i n G_«_ annulata c o l l e c t i o n s ; (b) i f methyl v i n y l ketone was present i n any of the solvents used i n the extraction and p u r i f i c a t i o n procedures, i t would be reasonable to expect that the major a l k y l a t i o n product would be 2-(3-oxobutyl) garveatin A (140), since garveatin A i s by far the most abundant polyketide metabolite. Compound 140 has never been detected i n any of the G.  annulata c o l l e c t i o n s to date, even though i t can be r e a d i l y synthesised v i a the Michael reaction discussed previously. Biogenesis: The s t r u c t u r a l s i m i l a r i t y of the garvalones to the garvins suggests a common biogenetic o r i g i n from a nonaketide precursor as outlined i n the previous chapter. A l k y l a t i o n at the CIO position of the precursor (either before or after c y c l i s a t i o n ) by an activated four-carbon moiety such as an acetoacetyl derivative (Scheme 5), followed by reduction of one of the ketone groups seems to be a plausible pathway for the biosynthesis of the 2-(3-oxobutyl) side-chain of the garvalones. The obvious lack of s t e r e o s p e c i f i c i t y of t h i s a l k y l a t i o n raises serious doubts as to 143 whether t h i s step i s subject to enzymatic control. A possible explanation may be that garvins A and B are acting as f r e e -r a d i c a l or e l e c t r o p h i l i c scavengers i a vivo by reacting with a four-carbon compound such as a methyl v i n y l ketonyl or acetoacetyl moiety v i a a Michael addition at C2. S i m i l a r l y , the 2-hydroxygarvin derivatives may have been formed from the garvins by f r e e - r a d i c a l attack of molecular oxygen at C2, suggesting an antioxidant role for these compounds. 144 145 . 146 S P I N S I M U L A T I O N : M E T H Y L G A R V A L O N E A B C D S P I N S Y S T E M ( 4 0 0 A 1 B 1 C 1 D 1 LW M I N F 0 MAXF 1 5 0 0 . 0 0 T H S 0 . 1 0 0 0 C H E M I C A L S H I F T A 7 9 2 . 0 0 B 8 2 0 . 0 0 C 9 0 0 . 0 0 D 9 9 2 . 0 0 M H Z 2 . 0 0 0 0 C O U P L I N G C O N S T 2A 22 2.0 ppm ( s ) Figure 31a. Spin simulation of H18-19 system in 400 MHz spectrum of 13_9_. 149 150 137 OMe OMe 1 r — T — I — I — I — I — I — I — I l — l — 1 — i T—1—I—I—I—I—t—I—I—r—1 1—r—r—i—i—^ 1—i 1 i i i—I i • - i | ' 1 ' ' I ' ' ' ' | ' ' ' ' | ' I ' l | ' ' ' ' I I I I ' | I I I I | I I I I | I I I I | I I I I | I I I I [ I I I I | I I I I | I I I I | I I I I | . I I I | I i i I | i I t I j I 1 M , I I i 1 | i i i I \ I I 1 1 | I I 20 200 180 160 140 120 100 80 60 40 20 0 PPM 13 Figure 34. 75 MHz NMR spectrum of H I i n CDC13, 152 D. THE ANNULINS. The fourth and f i n a l class of metabolites obtained from G. 94 annulata, c a l l e d the annulins , are characterised by a degraded anthracene skeleton in which the a l i c y c l i c six-membered ring has been cleaved. TLC analysis of the f l a s h f r a c t i o n eluted with 50% ethyl acetate/hexane indicated the presence of two unidentified compounds with R f's close to 0.5 in the standard system, and which gave a p o s i t i v e color reaction with I 2 vapour. This f r a c t i o n (500 mg) was chromatographed on LH-20 (90% MeOH/CH2Cl2) to give a major peak which was further p u r i f i e d by preparative TLC and normal phase HPLC to y i e l d two pure compounds, annulin A (141) (20 mg) and annulin B (Hfi.) (12 mg). When spotted on a TLC plate and subjected to the vapours of a 25% aqueous NH^ solution, annulin A went dark purple while annulin B turned bright pink. This colour reaction indicated the presence of a quinone f u n c t i o n a l i t y i n these molecules, and i t i s l i k e l y that the NH^ c p reacts with the carbonyl group of the quinone to form an imine . (i) Annulin A Annulin A (144), obtained from 95% ethanol as bright orange c r y s t a l s , was shown by HRMS to have a molecular formula of C19 H20°7 * M + 3 6 0 * 1 2 2 1 ' req'd 360.1209). The 1H NMR spectrum of annulin A contained resonances which could be assigned to aromatic methyl (2.44, s, 3H), and ethyl (1.14, t, 3H; 2.76, q, 153 154 2H) substituents, a methyl ether or ester (3.87, s, 3H), and two a l i p h a t i c methyl groups (1.63, s, 3H; 1.72, s, 3H). Subtracting the six carbon atoms required by the methyl and ethyl resonances observed i n the "*"H NMR of annulin A from i t s molecular formula leaves a residue of thirteen carbons, indicating that the fourteen carbon anthracene-type skeleton of the previously reported Garveia metabolites could not be present. Additional resonances in the *H NMR spectrum of annulin A could be assigned to a single aromatic proton (7.47,s,lH) and a phenolic proton (12.12, s, IH). The chemical s h i f t s of the resonances assigned to the aromatic proton, the phenolic proton, and the aromatic methyl and ethyl groups bore a s t r i k i n g resemblance to the chemical s h i f t s of the corresponding resonances i n the NMR spectrum of garveatin B quinone (106), an a r t i f a c t formed during s i l i c a gel p u r i f i c a t i o n of garveatin B (88). This s i m i l a r i t y (see Table 11) suggested that annulin A contained a napthaquinone nucleus 143 with substituents on the aromatic ring which were i d e n t i c a l to those present i n garveatin B quinone (106). Consistent with t h i s assignment was the observation of H-bonded (1616 cm - 1) and non H-bonded (1657 cm"1) quinone carbonyl stretching bands i n the IR, a base induced bathochromic s h i f t to 559nm c h a r a c t e r i s t i c of napthaquinones in the UV, and two carbonyl resonances at 180.91 and 185.97 ppm i n 13 the C NMR spectrum of annulin A (table 12). The remaining f u n c t i o n a l i t y i n annulin A could be read i l y 13 i d e n t i f i e d from i t s spectral data. C NMR resonances at 169.35 and 54.04, in conjunction with the -^H NMR resonance at 3.87 ppm 155 and an IR band at 1750 cm , indicated a methyl ester. A C NMR resonance at 101.45 and a 1H NMR resonance at 4.92 (bs, IH) were 1 3 assigned to a hemiketal, while a C NMR resonance at 89.08ppm was assigned to a t e r i a r y ether carbon. Four candidate structures (144-147) could be constructed from the i d e n t i f i e d fragments. The expected e q u i l i b r a t i o n of the hemiketal f u n c t i o n a l i t y i n structures 145. and 147 would give in both cases a mixture of two diastereomers which would be expected to have quite d i f f e r e n t "^H NMR spectra. Careful examination of the NMR spectrum of annulin A f a i l e d to uncover any evidence for a mixture of diastereomers, suggesting that structures 145. and 147 were improbable candidates. E q u i l i b r a t i o n of the hemiketal f u n c t i o n a l i t y i n structures 144 and 146 would e f f e c t racemization. Our i n i t i a l attempt to measure the o p t i c a l a c t i v i t y of annulin A was p o s i t i v e ([ ] D +24.9°;c 0.35), casting doubt on the v a l i d i t y of candidate structures 144 and 146.. A l i m i t e d supply of annulin A (<4mg) precluded a spectroscopic or chemical resolution of t h i s dilemma. The structure of annulin A (144) was solved by s i n g l e - c r y s t a l x-ray d i f f r a c t i o n analysis by Xu and Clardy at C o r n e l l . A computer generated drawing of the f i n a l x-ray model of annulin A (144.) i s given in Figure 36. Annulin A i s a naturally occurring racemate, and the enantiomer shown i s a r b i t r a r y . The only c h i r a l center, CI, i s a hemiketal which presumably epimerizes as argued above. The five-membered ring i s e s s e n t i a l l y planar; a l l int e r n a l t o r s i o n a l angles are less than 10°. Thus the t r i c y c l i c portion of annulin A i s planar within experimental error. There appear to be intramolecular hydrogen bonds between 156 Figure 36. Computer generated X-ray structure of Annulin A (1AA.). i 157 H05. As expected, the ethyl sidechain i s rotated out of the molecular plane by roughly 90°. A re-examination of the o p t i c a l a c t i v i t y of annulin A (144), using a larger sample obtained from a second i s o l a t i o n , demonstrated that our i n i t i a l observation of o p t i c a l a c t i v i t y was erroneous. ( i i ) Annulin B_ Annulin B (148) was obtained as an o p t i c a l l y active [oC]D+8.0;c 0.2) orange o i l that was shown by HRMS to have a molecular formula of C 2 1 H 2 2 0 7 (M+ 386.1361, req'd 386.1366). The 1H NMR spectrum of annulin B again revealed the presence of a napthaquinone substructure containing hydroxyl, ethy l , and methyl substituents on the aromatic ring as i n annulin A (144) (Table 11). Support for t h i s fragment came from IR bands at 1657 and 1638 cm 1 assigned to the quinone carbonyl stretching v i b r a t i o n s , 13 a base induced bathochromic s h i f t to 530nm in the UV, and C NMR resonances at 181.14 and 178.62 ppm assigned to the quinone carbonyl carbons. Functionality accounting for the remaining atoms i n annulin 13 B (CgH-^O^) was i d e n t i f i e d from i t s spectral data. C NMR resonances at 167.68 and 53.45, a *H NMR resonance at 3.76 ppm (s, 3H), and an IR band at 1757 cm"1 i d e n t i f i e d a methyl ester. 1H NMR resonances at 1.49 (s, 3H), 1.51 (s, 3H), and 1.85 (s, 3H) ppm could be assigned to three a l i p h a t i c methyl groups and a 13 gated decoupled C NMR spectrum of 148 contained resonances at 23.78 and 25.90 ppm that both appeared as quartets of quartets t y p i c a l of a gem-dimethyl moiety. An IR band at 1738 cm"1 and a 158 MeO 148 159 C NMR resonance at 203.02 were assigned to a ketone, and C NMR resonances at 43.64 and 84.48ppm were assigned to quarternary and oxygen bearing t e r t i a r y carbons respectively. The presumption of a common biogenesis for annulin B (148) and the rest of the Garveia metabolites led us to situate the ketone and the gem-dimethyl array at positions corresponding to C3 and C4 i n the anthracene skeleton of the other metabolites. The chemical s h i f t of the quarternary carbon resonance i n the spectrum of 148 (43.64 ppm), which was close to that assigned to C4 (46.9 ppm) in 2-hydroxygarvin A (119.), supported t h i s placement. The chemical s h i f t of the t e r t i a r y carbon (84.48 ppm) in the spectrum of annulin B was quite si m i l a r to the chemical s h i f t of the C2 carbon i n 119 (81.6 ppm), implying that i t too was attached to an oxygen, a single a l k y l , and two carbonyl carbons. Thus the t e r t i a r y carbon i n annulin B had to be attached to the ketone carbonyl at C3, the ester carbonyl, the remaining a l i p h a t i c methyl, and the remaining oxygen atom. The f i n a l s i t e of unsaturation required by the molecular formula of annulin B could be generated by forming an ether linkage between the oxygen atom on C2 and an unsatisfied valence at C9a resulting i n the proposed structure 148 for annulin B. 13 A complete C NMR assignment for annulin B i s given in Table 12. The assignments were made from empirical calculations 75 95 using juglone and rugulosin as model compounds. Of pa r t i c u l a r note are the resonances at 163.8 and 119.1 ppm assigned to C9a and C4a respectively which r e f l e c t the influence of the ether oxygen on the o l e f i n i c carbons of the quinone. Thus, 160 Table 11: 1H NMR (CDC13) data. Chemical s h i f t s are i n ppm from TMS. Proton Annulin A Annulin B Garveatin B on Carbon (144) (148) quinone (106) no. 1-OH 4.92s 11.79s 3 3.87s 5 7.47s 7.31s 7.51s 8-OH 12.12s 12.35s 11.84s 11 1.85s 1.97s 12 1.63s* 1.49s* 1.61s 13 1.72s* 1.51s* 1.61s 14 2.44s 2.42s 2.45s 15 2.76q 2.73q 2.78q 16 1.14t 1.15t 1.16t 17 3.76s * assignments may be reversed. 161 Table 12: C NMR (75 MHz,CDCl3) data. Chemical s h i f t s are i n ppm from TMS. Carbon Annulin AX1AA) Annulin B(148) ns. 1 101.45 167.68 2 169.35 84.48 3 54.04 203.02 4 89.08 43.64 4a 154.29 119.11 5 122.10 120.65 6 145.63 147.64 7 139.81* 136.06 8 160.36 160.43 8a 113.40 111.11 9 185.97 181.14 9a 140.76* 163.79 10 180.91 178.62 10a 130.37 127.59 11 20.38 12 26.43$ 23.78* 13 27.94$ 25.90* 14 20.08 20.40 15 19.50 19.10 16 12.73 12.77 17 53.45 *,$ : assignments may be reversed. 162 C9a i s strongly deshielded by the electron withdrawing e f f e c t of the oxygen atom, whereas C4a experiences a shielding e f f e c t due to a resonance contribution a r i s i n g from d e l o c a l i s a t i o n of the lone-pair on the ether oxygen atom. A similar e f f e c t i s seen i n 13 the C NMR spectrum of stemphone (JJL9_) , a pigment from the 96 fungal pathogen Stemphylium sarcinaeforme . This compound also contains a six-membered ether ring fused to a quinone , and resonances at 151.6 and 119.2 ppm for C5 and C6 respectively are comparable to those of C9a and C4a i n annulin B. Biogenesis; Annulins A (144) and B (148) both appear to be degradation products of garveatins. The conversion of garveatin B (&8J to annulin B (148) requires oxidation of the central ring to a quinone, hydroxylation at C2, cleavage of the Cl-C9a bond, and oxidation of the CI carbon to a carboxylic acid. 2-Hydroxygarveatin B (102), a co-occuring metabolite, i s a possible intermediate i n t h i s pathway. Conversion of any p o t e n t i a l garveatin precursor to annulin A (144) requires the removal of at le a s t one carbon atom (C3) i n addition to oxidation state transformations (see scheme 6). Somewhat s u r p r i s i n g l y , no corresponding degraded analogs of garveatin A were detected i n the extracts of G_». annulata. In view of the fact that garveatin A i s present i n much higher concentration than garveatin B, i t seems un l i k e l y that garveatin A and i t s derivatives are metabolised to annulin-type products. 163 Scheme 6. 164 165 166 167 168 E. CONCLUSION. A var i e t y of polyketide - derived secondary metabolites have been obtained from the methanol extracts of the marine hydroid Garveia annulata, collected o ff Vancouver Island, B.C. These have been divided into four d i s t i n c t groups based on th e i r carbon skeletons: the garveatins, the garvins, the garvalones and the annulins. The garveatins and garvins both contain oxidized metabolites i n the form of 2-hydroxy analogs, 9,10 quinones and 2,2* dimers. The observance of diastereomeric forms of 2-hydroxy garvin B i n a 1:1 r a t i o , combined with the lack of o p t i c a l a c t i v i t y i n any of the other 2-hydroxy derivatives tested, suggests a non-enzymatic route for conversion of the parent keto-enol compounds to the corresponding 2-hydroxy derivatives. Likewise, the quinones and dimers appear to be produced by a e r i a l oxidation processes. However, the presence of considerable amounts of the 2-hydroxy compounds and quinones i n the fresh extracts indicates that these compounds may well be present i n the inta c t organism. The low yi e l d s of oxidation products obtained on vigorous oxidation of garveatin A i n the laboratory tends to support t h i s hypothesis. The apparent lack of c h i r a l i t y at the C2 center in the garvalones also casts serious doubts as to whether the in s e r t i o n of the 3-oxobutyl group i s subject to b i o l o g i c a l c o n t r o l . The fact that garvalone A has been found repeatedly i n d i f f e r e n t c o l l e c t i o n s of GJ_ annulata precludes i t s formation from garvin A 169 by contamination of the solvents with methyl v i n y l ketone. Addition of 1 ml of methyl v i n y l ketone to a 4 l i t r e jar containing a fresh methanol extract of annulata did not produce any noticeable increase i n the y i e l d of garvalone A on subsequent work-up. This evidence seems to indicate that the garvalones are present i n the intact organism and are derived from the parent garvins A and B by a non-stereospecific addition of a C4 unit to the C2 p o s i t i o n . The annulins represent degraded forms of garveatin B where the central ring has been converted to a quinone moiety and the a l i c y c l i c ring has been subjected to oxidative cleavage. It i s not apparent why the corresponding degraded analogs of the much more abundant garveatin A have not been detected in the extracts. The Qj_ annulata metabolites exhibit considerable a n t i b i o t i c a c t i v i t y as seen i n the results of the i n - v i t r o a n t i b a c t e r i a l and antifungal assays (Table 13). Minimum i n h i b i t o r y concentrations of 1 ug or less were recorded for some of the compounds tested i n the a n t i b a c t e r i a l assays, i n general, these metabolites appear to be more toxic to bacteria than to fungi. It i s i n t e r e s t i n g to note that no a n t i b i o t i c a c t i v i t y was detected i n the crude aqueous extracts of annulata. neither was there a noticeable orange color c h a r a c t e r i s t i c of the polyketides. It had been anticipated that some of the hydroid metabolites might be present as water-soluble su l f a t e esters, analogous to those reported by S u t h e r l a n d ^ ' ^ from various species of c r i n o i d s . However, no anthracenone-type compounds were 57 detected i n the aqueous extracts. The c r i n o i d a l s u l f a t e esters 170 Table 13. Results of i n - v i t r o a n t i o b i o t i c assays for the G. annulata metabolites. Minimum i n h i b i t o r y concentrations reported i n ug/disc. COMPOUND Be. St. Pyth. Rhiz. Helm. Garveatin A 20 20 nt. Garveatin B 10 10 80 80 80 Garveatin C 30 30 na. 30 nt. Garveatin D 2.5 2.5 na. na, nt. 2-hydroxy garveatin A 372 31 124 31 nt. 2-hydroxy garveatin B 13 13 na. 64 na. Garveatin A quinone 50 50 nt. Garveatin B quinone 10 10 40 40 126 Garvin A 20 20 80 80 nt. 2-hydroxy garvin A 22 109 190 430 1300 Garvin A quinone 12 na. nt, Garvin B 30 na. 160 na. 171 Table 13, cont'd. COMPOUND Be. St. Pyth. Rhiz. Helm. 2-hydroxy 29 29 348 348 348 garvin B Garvin B 10 10 na. na. nt. quinone Garvalone A 85 255 na. na. nt. Garvalone B 60 na. 60 180 nt. Annulin A 2 0.5 40 40 80 Annulin B 1.5 7.5 na. na. nt. Bacteria; Be.: B a c i l l u s s u b t i l i s . St.: Staphylococcus aureus. £unai: Pyth.: Pythium ultimum. . Rhiz.: Rhizoctonja, s o l a n i . Helm.: Helminthosporium sativum. na.: not active at maximum concentration tested, nt.: not tested. 172 were shown to have strong f i s h anti-feedant a c t i v i t y when tested on a number of l o c a l marine species at the concentrations found in the c r i n o i d s . A similar type of fish-antifeedant bioassay was conducted i n t h i s laboratory using g o l d f i s h as the test species and employing food p e l l e t s containing varying amounts of garveatin A (22) up to 500 ug. The f i s h did not discriminate between test and control p e l l e t s , suggesting that the annulata polyketides probably don't play a defensive role against f i s h predators i n the marine environment. The 1-anthracenone skeleton elaborated by many of the G.annulata polyketides i s t y p i c a l of several potent a n t i b i o t i c 97 and antineoplastic drugs such as mithramycin , the g o g o chromomycins and the olivomcins , is o l a t e d from various fungal species. They also share some st r u c t u r a l s i m i l a r i t i e s with the anthracyclines daunomycin and adriamycin produced by a Streptomyces species" 1" 0 0. A l l of the above fungal metabolites contain sugar residue(s) linked to the aglycone portion, i n contrast, no evidence of carbohydrate-containing polyketides has been found in the aqueous or organic phases of G_»_ annulata. The only compound characterised from the aqueous phase of the G.annulata extracts was homarine (N-methylpicolinic acid) (150), a metabolite which has previously been found in several marine invertebrates^" 0^". This substance proved to be the most abundant low molecular - weight component and was characterised by 1 13 comparison of i t s H and C NMR data with those obtained from 102 103 another source . There i s evidence that homarine acts as a transmethylating agent in shrimp muscle homogenates and i s 173 capable of trans f e r r i n g i t s methyl group to various acceptor molecules. Netherton and Gurin speculate that homarine i s not only a methyl donor (analagous to S-adenosyl methionine) but may serve as a reservoir of methyl groups i n Cru s t a c e a . This raises the i n t r i g u i n g p o s s i b i l i t y that homarine may be involved in the methylation of the G.annulata polyketides. The true metabolic role of the polyketide metabolites i n the l i f e - c y c l e of Gj_ annulata remains a mystery. Apart from th e i r potent i n - v i t r o a n t i b i o t i c a c t i v i t y which points to a p o s s i b l l e defensive role against marine micro-organisms, the demonstrated r e a c t i v i t y of the keto-enol compounds suggests that they might act as anti-oxidants or f r e e - r a d i c a l scavengers in-vivo. Considering the high concentrations of polyketides present i n the hydroid, they would be expected to serve a d i s t i n c t b i o l o g i c a l role since the organism obviously expends a large amount of metabolic energy in synthesising them. These compounds appear to be endogenous since there i s no evidence of a symbiotic relationship with any microbial species, and no indicati o n that they might be derived from a dietary source. 174 ^rjr^coo' C H 3 150 175 I l l : EXPERIMENTAL The H NMR spectra were recorded on Bruker WP-80r N i c o l e t -13 Oxford 270, Varian XL-300 and Bruker WH-400 spectrometers. C NMR spectra were recorded on Bruker WP-80, Bruker WH-400 and Varian XL-300 spectrometers. TMS was used as an inter n a l standard. Low resolution mass spectra were recorded on an AEI MS902 spectrometer and high resolution mass spectra on an AEI MS50 instrument. IR spectra were recorded on BOMEM Fourier Transform and Perkin-Elmer 710B spectrometers. UV-Visible spectra were recorded on a Bausch and Lomb Spectronic-2000 spectrometer. Optical rotation measurements were recorded on a Perkin-Elmer 141 polarimeter using a 10cm mi c r o c e l l . A Fisher-Johns apparatus was used to determine melting points and these values are uncorrected. Merck S i l i c a gel 230-400 mesh was used for f l a s h and preparative thin layer chromatography and Whatman Magnum-9 P a r t i s i l - 1 0 and P a r t i s i l - 1 0 ODS 3 columns were used for preparative HPLC. Sephadex LH-20 resin was used for molecular exclusion chromatography. R^'s are l i s t e d for a l l compounds i n an a n a l y t i c a l TLC system using a 1:50:50 acetic acid/ethyl acetate/hexane eluent. The HPLC solvents were Fisher grade or BDH HPLC grade. Water was glass- d i s t i l l e d and a l l other solvents were reagent grade. 176 C o l l e c t i o n Data Garveia annulata was coll e c t e d by hand using SCUBA (-2 to -9m) on exposed rocky reefs i n Barkley Sound, B r i t i s h Columbia during the winter and spring months. Extraction and Chromatography Freshly co l l e c t e d whole specimens were immediately placed in methanol and stored at room temperature. The methanol extract was decanted and f i l t e r e d after three days. The f i l t r a t e was rotary-evaporated to give an aqueous suspension that was dil u t e d to 400ml with d i s t i l l e d water and extracted successively with hexane(3x400ml ), methylene chloride(3x400ml ) and ethyl acetate(2x400ml ). The hexane(600mg), methylene chloride(1.5g) and ethyl acetate(700mg) phases were fractionated separately by step-gradient vacuum f l a s h chromatography using a 3.5cm thick s i l i c a pad in a sintered-glass funnel (10cm diameter). Fractions eluting with the same solvent composition from each separation were combined. Elution with 20% ethyl acetate/hexane, 50% ethyl acetate/hexane, 100% ethyl acetate, and 20% methanol/ethyl acetate gave fractions A(140mg), B(500mg), C(1.5g) and D(700mg) respectively. Fraction A: P u r i f i c a t i o n of f r a c t i o n A on a Sephadex LH-20 column (3.5cm di a . x 900cm) using 90% MeOH/CH2Cl2 as the eluant gave subfractions (i) and ( i i ) in order of e l u t i o n . 177 Fractionation of (i) by preparative TLC (40% ethyl acetate/ hexane) gave garveatin C (23J (20mg). P u r i f i c a t i o n of ( i i ) by preparative TLC (10% ethyl acetate/ hexane) and reverse-phase HPLC (95% CH3CN/H20) yielded garveatin B- garvin B dimer (136) (6mg). Fraction B_: P u r i f i c a t i o n of f r a c t i o n B by LH-20 chromatography (90% MeOH/CH2Cl2) gave subtractions ( i i i ) to ( v i i i ) , in order of elu t i o n . Subtraction ( i i i ) was p u r i f i e d by preparative TLC (50% ethyl acetate/ hexane) to give garvalone A (137) (20mg). Subtraction (iv) was p u r i f i e d by preparative TLC (10% ethyl acetate/ CHC13) to y i e l d 2-hydroxygarvin A (119) (lOOmg). Subfraction (v) was fractionated by preparative TLC (50% ethyl acetate/ hexane) to give impure annulin A and pure annulin B (148) (12mg). Chromatography on normal phase HPLC (40% ethyl acetate/ hexane) gave pure annulin B (144) (12mg). When spotted on TLC plates and exposed to the vapours of a 25%aq. NH3 solution, annulin A turned dark purple while annulin B gave a bright pink spot. Subfraction (vi) had a deep red color and was p u r i f i e d by preparative TLC (50% ethyl acetate/ hexane) and normal phase HPLC (ethyl acetate/ hexane gradient) to give garvin A quinone (122) (lOmg) and garvin B quinone (132) (8mg). Subfraction ( v i i ) was p u r i f i e d by preparative TLC (25% ethyl acetate/ hexane) to y i e l d garveatin B dimer (108) (3mg) and 178 impure garveatin B. The l a t t e r was p u r i f i e d on normal phase HPLC (ethyl acetate/ hexane gradient) to give garveatin B (££) (15mg). Subtraction ( v i i i ) was p u r i f i e d by preparative TLC (10% ethyl acetate/ hexane) to y i e l d 2-hydroxygarvin B (130) (14mg) as a mixture of diastereomers. Fraction £: P u r i f i c a t i o n of f r a c t i o n C by LH-20 chromatography (90% MeOH/CH2Cl2) yielded subtractions (ix) to (xiv), i n order of elu t i o n . Subtraction (ix) was p u r i f i e d by preparative TLC (50% ethyl acetate/ hexane) to give 2-hydroxy garveatin B (102) (40mg). Subfraction (x) was p u r i f i e d by preparative TLC (10% ethyl acetate/ CHC13) to y i e l d garvin A dimer (134) (5mg) and garvin A (12A) (35mg). Subfraction (xi) was p u r i f i e d by preparative TLC (5% MeOH/CHCl^ and 25% ethyl acetate/ hexane) to give garvalone B (141) (3mg) and garveatin D (M) (8mg). Subfraction ( x i i ) was p u r i f i e d by preparative TLC (2% MeOH/ CHCl.j) to give garveatin A quinone (105) (llmg). Subfraction ( x i i i ) eluted late as a very broad yellow band and contained almost pure garveatin A. F i n a l p u r i f i c a t i o n by t r i t u r a t i o n with 80% CHCl^/ hexane and c r y s t a l l i s a t i o n from acetone gave pure garveatin A (22) (300mg). Subfraction (xiv) contained garvin B mixed with a small amount of garveatin A. The l a t t e r was removed by t r i t u r a t i o n with acetone to give pure garvin B (12£.) (lOOmg) . 179 Fraction D_: Fraction D contained a mixture of r e l a t i v e l y polar compounds which were applied on LH-20 (90% MeOH/CH2Cl2) to give subfraction (xv). P u r i f i c a t i o n of a portion of (xv) by reversed phase preparative TLC (80% MeOH/H20) gave 2-hydroxy garveatin A (97) (12mg). 180 Spectral Data; Garveatin A. (77); obtained as yellow-orange needles; m.p. 236-240°C (cryst. from acetone) ; UV (MeOH) neutral/basic 232 ( 18000), 282 ( 10000), 323(sh) ( 5000), 432 ( 12000), a c i d i c 238 ( 17000), 253(sh) ( 12000), 298 ( 7000), 402 nm ( 7000); IR (CHC13) 3010, 1725 (sh), 1680, 1610 cm"1; 1H NMR (80 MHz,CDCl3) 1.63 (s,6H), 1.98 (s,3H), 2.40 (br s,3H), 2.69 (s,3H), 7.02 (br S,1H), 7.15 (s,lH), 10.56 (s,lH), 17.34 ppm (s,lH); 1H NMR (300MHz,acetone d g) 1.58 (s,6H), 1.99 (s,3H), 2.41 (br s,3H), 2.70 (s,3H), 6.87 (br s,lH), 6.97 ppm (s,lH); 1 3 C NMR (75 MHz,acetone d 6) 204.12 (s), 198.64 (s), 175.89 (s), 170.47 (s), 161.63 (s), 149.09 (s), 138.53 (s), 136.88 (s), 120.61 (s), 118.32 (d), 113.94 (s), 110.09 (d), 105.95 (s), 103.25 (s), 45.36 (s), 32.69 (q,2C), 30.17 (q), 21.28 (q), 7.70 (q) ppm; HRMS 340.1317, calc'd for C2QE20O5 340.1311; LRMS m/z ( r e l . intensity) 340 (19), 325 (38), 310 (5), 283 (5). Preparation of 3-methyl garveatin A. (78): Garveatin A (22.) (16mg) was dissolved in di e t h y l ether (3 ml,anhyd.) and treated with diazomethane (generated by adding 0.6 ml 5M NaOH to N-methylnitro-nitroso-guanidine). After standing for 3 hours the reaction mixture was p u r i f i e d by preparative chromatography (10% ethyl acetate/CHCl 3) to y i e l d 3-methyl garveatin A (22.) (3 mg) as the major reaction product. Compound (78); obtained as an orange o i l . R f 0.54; UV (MeOH) neutral 238, 286, 405 nm, basic 238, 286, 429 nm; XH NMR (80 MHz,CDCl3) 1.58 (s,6H), 2.08 (s,3H), 2.41 (br s,3H), 2.66 (s,3H), 4.04 (s,3H), 7.01 (br S,1H), 7.13 (s,lH), 10.40 (s,lH), 17.25 ppm 181 (s,lH); HRMS 354.1468, calc,d for C 2 ] H 2 2 0 5 354.1468; LRMS m/z ( r e l . intensity) 354 (M+ 80), 339 (75), 323 (10). Preparation pJL Dimethyl (79) and Trimethyl Garveatin A (8Q): A suspension of 37 mg of garveatin A (12), 550 mg K 2C0 3 and 400 u l (4.2 mmoles) of dimethyl sul f a t e in 3 ml acetone was heated at reflux under an N 2 atmosphere for 21 hours. After cooling, 4 mis H 20 and 400 u l 1M KOH were added. After two hours the mixture was extracted with CH 2C1 2 (4x4ml). The aqueous phase was a c i d i f i e d to pH 2 and extracted with 2x5ml CH 2C1 2. The CH 2C1 2 extracts were dried over Na^O^ and concentrated. Preparative chromatography using 5% ethyl acetate/hexane and 1% acetic a c i d / C H 2 C l 2 yielded dimethyl garveatin A (22.) (6 mg) and trimethyl garveatin A (M) (15 mg). Compound (22.): obtained as a yellow o i l . R f 0.53; UV (MeOH) 206, 235, 286, 494 nm; 1H NMR (80 MHz,CDCl3) 1.59 (s,3H), 2.08 (s,3H), 2.38 (br s,3H), 2.63 (s,3H), 3.91 (s,3H), 4.01 (s,3H), 7.20 (s,lH), 7.30 (br s,lH), 15.83 ppm (S,1H); HRMS 368.1625, calc'd for C 2 2 H 2 4 0 5 368.1624; LRMS m/z (re l . i n t e n s i t y ) 368 (93), 353 (100), 337 (18). Compound (JL0J : obtained as a yellow-orange o i l . R f 0.43; UV (MeOH) 230, 265, 306 (sh), 360 nm; IR (CHC13) 3600, 3000, 2410, 1690, 1620, 1425, 1320, 1210 cm"1; 1H NMR (80 MHz,CDCl3) 1.63 (s,6H), 2.08 (s,3H), 2.38 (br s,3H), 2.63 (s,3H), 3.86 (s,3H), 3.97 (s,3H), 4.03 (s,3H), 7.41 (br s,lH), 7.65 (s,lH) ppm; HRMS 382.1787, calc'd for C 2 3 H 2 6 0 5 382.1780; LRMS m/z ( r e l . intensity) 382 (76), 367 (65), 352 (13), 351 (9), 336 (15), 335 (39). 182 Preparation oJL Tyjaqetyl GaKVeatin A (81): A solution of garveatin A (12) (6.6mg) i n 500 u l pyridine and 500 u l acetic anhydride was s t i r r e d overnight at room temperature and evaporated under high vacuum. The reaction mixture was p u r i f i e d by preparative TLC (10% ethyl acetate/CHCl 3) to y i e l d d i a c e t y l garveatin A (£2) (1 mg) and t r i a c e t y l garveatin A (£1) (5.2 mg) as the. major reaction products. Compound ( £2 ) : obtained as a bright yellow o i l . R^ 0.50 i n 10% ethyl acetate/hexane; UV (MeOH) 226, 261, 305, 357 nm(sh); IR (CHC13) 3050, 2930, 1765, 1708, 1655, 1630 cm"1; "hi NMR (80 MHZ,CDC13) 1.53 (s,6H), 1.85 (s,3H), 2.35 (s,3H), 2.38 (s,6H), 2.53 (s,3H), 7.24 (s,lH), 7.40 (br s,lH), 10.96 (s,lH) ppm; HRMS 424.1512, calc'd for C 2 4 H 2 4 ° 7 424.1522; LRMS m/z ( r e l . intensity) 424 (16), 382 (17), 340 (100), 325 (35), 311 (12). Compound (£1) : obtained as c o l o r l e s s , feathery c r y s t a l s (from E t 2 0 ) ; R^ 0.3 i n 10 % ethyl acetate/hexane); UV (MeOH) 226, 264, 305, 317 (sh), 355 (sh) nm; ^ -H NMR (80 MHz,CDCl3) 1.60 (s,6H), 1.80 (s,3H), 2.35 (s,3H), 2.43 (br s,3H), 2.54 (s,3H), 2.55 (s,3H), 7.59 (br s,lH), 7.84 (s,lH) ppm; HRMS 466.1628, calc'd for C 2 6H 2 6O g 466.1628; LRMS m/z ( r e l . intensity) 466 (4), 424 (24), 382 (24), 340 (100), 325 (22), 311 (11), 297 (3). Preparation of Enol Garveatin A tEiapetate (87); T r i a c e t y l garveatin A (£1) (1 mg) was dissolved i n benzene (1 ml) i n a screw-cap v i a l and 2 cry s t a l s of p-toluenesulfonic acid were added. The solution was s t i r r e d at 60°C for 4 hours and parti t i o n e d between 5% K 2C0 3 and ethyl acetate. The ethyl acetate phase was dried with Na2SO^ and rotary evaporated to produce 183 enol garveatin A t r i a c e t a t e (JLD in quantitative y i e l d . Compound (S2): obtained as long yellow c r y s t a l s (from 1:1 CHCl 3/hexane); R f 0.52 i n 10% ethyl acetate/CHCl 3; UV (MeOH) 203, 238, 275 (sh), 403nm; IR (CHC13) 1763, 1670, 1630, 1605 cm"1; XH NMR (400MHz,CDC13) 1.53 (S,6H), 1.90 (s,3H), 2.05 (s,3H), 2.33 (s,3H), 2.38 (s,3H), 2.55 (br s,3H), 5.00 (d,J=2Hz,lH), 5.38 (d,J=2Hz,lH), 7.24 (s,lH), 7.45 (br s,lH), 15.33 (s,lH) ppm; HRMS 466.1631 calc'd. for C 2 g H 2 6 0 8 466.1628; LRMS m/z ( r e l . intensity) 466 (6), 424 (21), 382 (37), 340 (100), 325 (56), 311 (12), 296 (14). Garveatin B_ (88): Obtained as a yellow o i l . R f 0.50; UV (MeOH) neutral 240 (34200), 260 (16200), 317 (9000), 417 (10600) nm, basic 223 (29800), 243 (40100), 325 (10000), 341 (8900), 417 (14300) nm; IR (CHC13) 2960, 1710, 1625, 1600, 1430 cm"1; "4 NMR (CDCL3) 1.18(t, J=7Hz, 3H), 1.62(s, 3H), 2.46(brs, 3H), 2.80(q, J=7HZ, 2H), 7.04(brs, IH), 7.12(s, IH), 10.25(s, IH), 17.15(s, IH), (diketo tautomer: 1.50(d, J=7Hz, 3H), 3.98(q, J=7HZ, IH), 9.90(s, IH), 16.23(s, IH)); 1H NMR (acetone-d g) 1.15(t, J=7Hz, 3H), 1.65(s, 6H), 1.98(s, 3H), 2.43(s,3H), 2.78(q, J=7Hz, 2H), 7.10(s, IH), 7.31(s, IH); HRMS 326.1526, calc'd for C 2 ( )H 2 20 4 326.1514; LRMS m/z (r e l . i n t e n s i t y ) 326 (60), 311 (100), 296 (9), 283 (18), 269 (10), 255 (13). Preparation of 3-Methyl garveatin 1 (89): Garveatin B (88) (5 mg) i n d i e t h y l ether was treated with excess diazomethane for 2 hr. Chromatography on s i l i c a gel 184 yielded 3-methyl garveatin B (89) (3 mg). Compound £9_ : obtained as an orange o i l . R f 0.6 in 80% CHCl 3/hexane; UV (MeOH) 210(12,400), 238(16,100), 265(9,300), 287(8,500), 320sh(3,900), 426(3,800) nm; 1H NMR (CDC13) 1.18(t, J=7Hz, 3H), 1.55(s, 6H) , 2.05(s, 3H), 2.44(brs, 3H), 2.80(q, J=7Hz, 2H), 4.00(s, 3H), 7.03(brs, IH), 7.10(s, IH), 10.19(s, IH), 17.08(s, IH) ppm; LRMS m/z ( r e l . intensity) 340 (M+ 56), 325 (100), 309 (24). Garveatin £ (93); Obtained as orange c r y s t a l s (hexane) m.p. 125°C; R f 0.54; UV MeOH), 228(13,700), 274(19,700), 305sh(3,300), 389(4,400) nm; IR (CHC13) 2930, 1700, 1620, 1455, 1390 cm"1; 1H NMR (CDC13) 1.49(s, 6H), 1.58(s, 6H), 2.38(brs, 3H), 2.62(s, 3H), 3.92(s, 3H), 7.19(s, IH), 7.35(brs, IH), 14.45(s, IH); 1 3 C NMR (CDC13) 211.6(8), 205.8(8), 204.9(s), 163.7(s), 155.9(s), 142.8(s), 140.0(s), 138.0(s), 135.0(s), 125.2(d), 116.3(s), 114.7(d), 108.8(s), 64.1(q), 55.3(s), 48.1(s), 32.4(q), 28.5(q, 2C), 24.6(q, 2C), 19.5(q); HRMS 368.1616, calc'd for C 2 2 H 2 4 0 5 368.1624. LRMS m/z ( r e l . intensity) 368 (70), 353 (29), 298 (15), 283 (26). Preparation pj. Garveatin £ acetate (94): Garveatin C (93) (5mg) was added to 250 u l of acetic anhydride and 250 u l of pyridine and the reaction mixture was s t i r r e d overnight at room temperature. The reagents were removed in-vacuo f and the residue was p u r i f i e d v i a preparative s i l i c a TLC(50% ethyl acetate/hexane) to give garveatin C acetate (94) (4 mg) . Compound 24.: obtained as a pale yellow o i l ; UV (MeOH) 224, 257, 295, 350 nm; """H NMR (CDC13) 1.35(s, 6H) , 1.55(s, 6H) , 185 2.37(brs, 3H), 3.84(s, 3H), 7.48(brs, IH), 7.66(s, IH); HRMS 410.1735, calc'd for C 2 4 H 2 g 0 6 410.1730; LRMS m/z (r e l . i n t e n s i t y ) 410 (11), 368 (100), 353 (44), 325 (10), 298 (12). Preparation pJL 9~Methyl gagyeatin £ (95); Garveatin A (77) (8 mg) was dissolved i n acetone (10 ml) to which K 2 C 0 3 a n ^ methyl iodide (500 ul) had been added. The reaction mixture was refluxed overnight. The residue obtained after f i l t r a t i o n and concentration in-vacuo. was p u r i f i e d by preparative TLC (50% ethyl acetate/hexane) to give 9-methyl garveatin C (95) as the major product (2 mg). Compound^: obtained as a pale yellow o i l . R f 0.50; UV (MeOH) 225, 255, 295sh, 351 nm; 1H NMR (CDC13) 1.38(s, 6H), 1.54(s, 6H), 2.38(bs, 3H), 2.61(s, 3H), 3.84(s, 3H), 4.01(s, 3H), 7.45(bs, IH), 7.52(s, lH)ppm; LRMS m/z ( r e l . intensity) 382 (M + 100), 367 (23), 339 (12), 335 (20), 312 (64), 297 (36). Garveatin C (93) (10 mg) i n 10 ml of HPLC grade acetone was treated with K 2C0 3 (anhydrous, 130mg) and methyl iodide (500ul). The reaction mixture was refluxed for 7 h. The reaction mixture was f i l t e r e d and evaporated to give a single product which was i d e n t i c a l by TLC, UV, 1 H NMR, and mass spectral comparison to the 9-methyl garveatin C sample prepared from garveatin A as described above. Garveatin D_ (96) ; obtained as a yellow s o l i d . R f 0.40; UV(MeOH) 238, 269, 417nm; 1H NMR(300 MHz,CDCl3) 1.50(s,6H), 1.58(s,6H), 2.41(br s,3H), 2.63(s,3H), 7.05(br s,lH), 7.12(s,lH), 186 10.20(s,lH), 16.15(s,lH)ppm; HRMS 354.1465, calc'd for C 2 1 H 2 2 0 5 354.1468; LRMS m/z ( r e l . intensity) 354(M +,53), 339(59), 321(30), 311(15), 297(13), 293(16). Preparation of 2-Hydroxyaarveatin A. t r i a c e t a t e (98): Fraction D (200mg) obtained from f l a s h chromatography of the methylene chloride phase of the extract was dissolved i n pyridine(3ml) and acetic anhydride(7ml) and s t i r r e d at room temperature for 48 hours. After evaporation of the reagents, the residue was p u r i f i e d by f l a s h chromatography (methanol/methylene chloride gradient) and preparative TLC (50% ethyl acetate/hexane) to give 2-hydroxygarveatin A t r i a c e t a t e (98) (90mg). Triacetate  (98); obtained as a colourless s o l i d . Rf0.25; UV (MeOH) 226, 260, 303, 355nm; •''H NMR (400 MHz,CDCl3) 1.45(s,3H), 1.57(s,3H), 1.79(s,3H), 2.20(s,3H), 2.34(s,3H), 2.41(br s,3H), 2.50(s,3H), 7.61(s,lH), 7.79(s,lH)ppm; HRMS 482.1567, calc'd. for C 2 g H 2 g 0 9 482.1577; LRMS m/z ( r e l . intensity) 482(M +,3), 440(25), 398(100), 356(20), 338(32), 314(83), 313(77). Preparation of diacetate (99) ; On standing i n py r i d i n e - d g i n an NMR tube overnight, t r i a c e t a t e 23 p a r t i a l l y decomposed to diacetate 23. (yield 40%) which was p u r i f i e d i n v i a f l a s h chromatography (ethyl acetate/hexane gradient). Diacetate 99; Rf0.40; UV (MeOH) 226, 272, 306(sh), 385nm; "hi NMR(80 MHz,CDCl3) 1.58(s,3H), 1.63(s,3H), 1.74(s,3H), 2.24(s,3H), 2.40(s,3H), 2.45(br s,3H), 2.55(s,3H), 7.26(1H), 7.47(s,lH), 9.26(s,lH)ppm; 1 3 C NMR(75 MHz,CDCl3) 206.5, 202.7, 198.4, 169.7, 163.7, 145.0, 142.8, 187 139.6, 137.9, 134.1, 126.9, 116.3, 115.1, 108.1, 82.6, 47.4, 32.0, 31.2, 29.3, 23.4, 21.1, 20.0, 19.7ppm; MS m/z(rel. intensity) 440(M+,14), 398(71), 356(9), 340(14), 338(25), 314(90), 313(100), 295(31). Preparation oJL 8.9-Dimethyl Garveatin A (1QQ): Trimethyl garveatin A (79) (10 mg) was dissolved i n 5 mis 50% aq. acetic acid i n a round-bottomed flask and refluxed for 10 minutes. The reaction mixture was d i l u t e d with H^ O and partitioned with C H 2 C l 2 . The CH 2C1 2 phase was dried with Na 2S0 4 and rotary-evaporated to give a quantitative y i e l d of 8,9-dimethyl garveatin A (100) . Compound 100: obtained as a l i g h t yellow o i l . R f 0.54; 1H NMR (300 MHz,CDCl3) 1.54 (s,6H), 2.01 (s,3H), 2.39 (br s,3H), 2.61 (s,3H), 3.83 (s,3H), 4.03 (s,3H), 7.45 (br s,lH), 7.63 (s,lH) ppm; MS m/z ( r e l . intensity) 368 (M+ 100), 353 (51), 342 (10), 341 (11), 325 (80). Preparation of. 2-Acetoxy.8.9-dimethyl Garveatin A f 1011 : 8,9-dimethyl garveatin A (100) (10 mg) was s t i r r e d i n g l a c i a l a c e tic acid (10 ml) containing excess Pb(OAc) 4 (60 mg) at room temperature. After 16 hours the reaction mixture was d i l u t e d with H 20 and partitioned with C H 2 C l 2 . The organic phase was dried with Na 2S0 4 and p u r i f i e d by preparative TLC (5% ethyl acetate/hexane) to y i e l d 2-acetoxy,8,9-methyl garveatin A (101) (6 mg) as the major reaction product. Compound 101: obtained as a c o l o r l e s s o i l . R f 0.44; UV (MeOH) 261, 300 (sh), 356 nm; 1H NMR (80 MHz,CDCl3) 1.47 (s,3H), 1.57 (s,3H), 1.76 (s,3H), 2.23 188 (s,3H), 2.37 (br s,3H), 2.58 (s,3H), 3.80 (s,3H), 4.00 (s,3H), 7.43 (br s,lH), 7.57 (s,lH) ppm; MS m/z ( r e l . intensity) 426 (M+ 48), 398 (5), 384 (42), 368 (14), 353 (9), 342 (100), 341 (92), 327 (56). 2-Hydroxygarveatin B_ (102) ; obtained as a yellow s o l i d . Rf0.28; UV (MeOH) 224, 275, 423nm; 1H NMR(80 MHz,CDCl3) 1.18(t,3H, J=7Hz), 1.50(s,3H), 1.65(s,3H), 1.78(s,3H), 2.47(br s,3H), 2.80(q,2H,J=7Hz), 4.60(bs, 1H0, 7.08(br s,lH), 7.13(s,lH), 9.68(s,lH), 15.15(s,lH)ppm; HRMS 342,1467, calcd. for C 2 ( )H 2 20 5 342.1468; LRMS m/z ( r e l . intensity) 342(M +,15), 327(5), 300(26), 299(30), 285(13). Preparation of. 2-HY<3roxygarveatin fi acetates; A solution of 2-Hydroxygarveatin B (102) (16mg), pyridine(0.25ml), and acetic anhydride(0.5ml) was s t i r r e d overnight at room temperature. After removal of the reagents i n - vacuo, the residue was chromatographed on s i l i c a (2% methanol/methylene chloride) to give 2-hydroxygarveatin B diacetate (103) (3mg), and 2-hydroxygarveatin B t r i a c e t a t e (104) (llmg). Diacetate 103: obtained as a l i g h t yellow o i l . R^0.30; UV (MeOH) 213, 250, 307(sh), 319, 363nm; 1H NMR(80 MHz,CDCl3) 1.15(t,3H,J=7Hz), 1.26(s,3H),1.40(s,3H), 1.83(s,3H), 2.45(s,3H), 2.47(s,3H), 2.52(br s,lH), 2.71(q,lH J=7 Hz), 4.63(br s,lH), 7.62(br s,lH), 7.66(s,lH)ppm; MS m/z(rel. intensity) 426(M+,10), 384(19), 342(47), 326(18), 324(33), 300(84), 299(100), 282(77). Triacetate (104): obtained as a colo r l e s s o i l . Rf0.40; UV (MeOH) 189 217, 260, 304, 316, 359nm; -""H NMR(80 MHz,CDCl3) 1.13(t,3H,J=7Hz), 1.44(s,3H), 1.55(s,3H), 1.78(s,3H), 2.22(s,3H), 2.45(s,6H), 2.50(br s,3H), 2.70(q,2H,J=7Hz), 7.55(br s,lH), 7.70(S,1H)ppm; HRMS 468.1776, calcd. for C 2 6H 2gOg 468.1784; LRMS m/z ( r e l . intensity) 468(M +,4), 426(20), 384(100), 342(19), 324(28), 300(44), 299(82), 282(26). Garveatin A. quinone (105) ; obtained as a red o i l . R f 0.49; UV(MeOH) 218, 265, 280(sh) and 410nm; 1H NMR(CDC13, 80MHz) 1.63(s, 6H), 1.98(s, 3H), 2.41(bs, 3H), 2.60(s, 3H), 7.55(bs, IH), 11.45(S, IH), 11.66(s, IH); 1 3 C NMR (CDC13, 75MHz) 202.1, 200.0, 193.0, 180.4, 159.5, 159.1, 156.2, 147.2, 136.3, 131.9, 127.7, 123.0, 118.6, 112.4, 48.3, 31.9, 26.4(2 carbons), 20.5, 8.1; HRMS 354.1108, calc'd for C 2 0H 1 8O g 354.1103; LRMS m/z ( r e l . intensity) 354 (M + 95), 339 (13), 326 (31), 311 (44), 297 (63), 283 (66), 269 (23). Garveatin B quinone (106); Obtained as a red o i l ; R^O.63; UV (MeOH) 208, 231, 283, 416nm; 1H NMR (400 MHz,CDCl3) 1.16(t,J=7Hz,3H), 1.61(s,3H), 1.97(s,3H), 2.45(s,3H), 2.78(q,J=7Hz,2H), 7.51(s,lH), 11.79(s,lH), 11.84(s,lH) ppm; HRMS 340.1306, calc'd. for c 2 o H 2 0 ° 5 t 3 4 0 * 1 3 1 1 ' * L R M S m / z ( r e l . intensity) 340(M,100), 326(20), 312(44), 297(38), 284(51), 269(55), 266(42). Preparation of garveatin B_ dimer (108): A solution of garveatin B (88) i n CDC13 was allowed to stand overnight i n a closed NMR tube at room temperature. The sample 190 was then chromatographed on preparative TLC (20% ethyl acetate/hexane) to give garveatin B dimer (108) i n approx. 30% y i e l d . Dimer 108; obtained as an orange o i l ; 0.66; UV (MeOH) 225, 275, 320 sh, 434 nm; XH NMR (270 MHz, CDC13) 1.07 (t,J=7Hz,3H), 1.38 (s,3H), 1.66(s,3H), 1.88 (s,3H), 2.43 (s,3H), 2.53 (m,lH), 2.70 (m,lH), 6.98( s,2H), 9.37 (s,lH), 15.79 (s,lH) ppm;13C NMR (75 MHz,CgDg) 208.9, 202.4, 167.3, 155.6, 143.5, 140.0, 137.7, 126.4, 119.9, 115.7, 110.9, 107.9, 63.0, 48.6, 33.6, 25.9, 21.2, 20.4, 19.8, 13.8 ppm; MS m/z (r e l . i n t e n s i t y ) 326 (57), 311 (100), 283 (20), 269 (11) (molecular ion not seen in EI spectrum). Preparation pJL garveatin B_ dimer tetraacetate (109) ; Garveatin B dimer (108) (2 mg) was treated with 100 u l of acetic anhydride and 100 u l of pyridine overnight. The reaction mixture was then chromatographed on preparative TLC (50% ethyl acetate/hexane) to give the tetraacetate 109 i n quantitative y i e l d . Tetraacetate 109; obtained as a l i g h t yellow o i l ; R^ 0.60; •"•H NMR (300 MHz, CDCl-j) 1.15 (t,J=7Hz,3H) , 1.56 (s,3H), 1.71 (s,3H), 1.85 (s,3H), 2.30 (s,3H), 2.42 (s,3H), 2.52 (s,3H), 2.55 (m,lH), 2.75 (m,lH), 7.61 (s,lH), 7.75 (s,lH) ppm; MS m/z ( r e l . intensity) 818 (M+,0.02), 776 (0.30), 734 (0.31), 716 (0.44), 674 (2), 410 (3), 368 (12), 326 (100), 311 (67). Preparation of garveatin A. dimer (110): Garveatin A (77) (10 mg) was dissolved i n CH3CN (25 ml) i n a screw-cap v i a l and 0.5 ml of cone. HCl was added. The mixture was 191 s t i r r e d overnight under a stream of a i r at room temp. Preparative TLC of the reaction mixture (5% MeOH/CHCl3) gave garveatin A dimer (110) (6 mg) as the major product. Dimer (110): obtained as a l i g h t yellow o i l ; R f 0.38; UV (MeOH) 231, 270, 422 nm (neutral), 245 sh, 270, 428 nm (basic); "4 NMR (80 MHz, CDC1 3) 1.47 (s,3H), 1.59 (s,3H), 1.94 (s,3H), 2.38 (br s,3H), 2.58 (s,3H), 6.98 (br S,1H), 7.05 (s,lH), 10.34 (s,lH), 15.81 (s,lH) ppm; MS m/z ( r e l . intensity) 340 (51), 325 (100), 310 (12), 297 (13) (no molecular ion was detected i n the EI spectrum). Preparation oL garveatin h dimer tetraacetate H i l l : Garveatin A dimer (110) (3 mg) was treated with 500 u l of acetic anhydride and 500 u l of pyridine and s t i r r e d overnight at room temperature. The tetraacetate 111 (1 mg ) was iso l a t e d as the major reaction product. Tetraacetate H I : obtained as a co l o r l e s s o i l ; R f 0.15; 1H NMR (300 MHz, CDC1 3) 1.72 (s,3H), 1.85 (s,3H), 2.32 (s,3H), 2.35 (s,3H), 2.44(s,3H), 2.51 (s,3H), 7.64 (br s,lH), 7.79 (s,lH) ppm; MS m/z ( r e l . intensity) 846 (M+ 0.14), 804 (0.56), 762 (0.60), 720 (0.42), 424 (4), 382 (16), 340 (94), 325 (74). Preparation of. Desacetyl Garveatin A (116) : Garveatin A (22) (20 mg) was refluxed i n a solution of 1:1 g l a c i a l acetic acid/conc. HC1 (10 ml) for 10 minutes. The reaction mixture was d i l u t e d with HjO and extracted with CH2CI2. The organic phase was washed with 5% aq. bicarbonate, concentrated and p u r i f i e d by preparative TLC (10% ethyl acetate/CHCl 3) to y i e l d desacetyl garveatin A (116) (10 mg) and 192 unreacted garveatin A (9 mg). Compound (116); obtained as a yellow o i l . R f 0.50; UV (MeOH) neutral/basic 239, 320, 343 (sh), 410, 430 nm (sh), a c i d i c 234, 256, 294 (sh), 319 (sh), 385 nm (sh); 1H NMR (300 MHz,CDCl3) keto-enol tautomer: 1.63 (s,6H), I. 98 (s,3H), 2.43 (s,3H), 6.68 (br d,J(meta)=1.6Hz,lH), 7.00(d,J(meta)=1.6Hz,lH), 7.16 (s,lH), 9.98 (s,lH), 17.00 (s,lH), diketo tautomer: 1.50 (d,J=7Hz,3H), 1.61 (s,3H), 1.62 (s,3H), 2.44 (S,3H), 4.00 (q,J=7Hz,IH), 6.75 (d,J=l.6Hz,lH), 7.01 (d,J=1.6Hz,lH), 7.15 (S,1H0, 9.62 (s,lH), 16.00 (s,lH) ppm; MS m/z (r e l . i n t e n s i t y ) 298 (M + 100), 283 (94), 255 (69), 242 (29), 214 (34). Preparation of Desacetyl Garveatin A. Qu i n c e (117) : Garveatin A quinone (105) (10 mg) was refluxed in 10 mis of 1:1 g l a c i a l acetic acid/conc.HCl for 10 minutes and worked up as for the previous reaction. The reaction mixture was p u r i f i e d by preparative TLC (10% ethyl acetate/CHCl 3) to y i e l d desacetyl garveatin A quinone (117) (6 mg). Compound (117): obtained as a red o i l . R f 0.55; UV (MeOH) neutral 210, 233, 255 (sh), 282 (sh), 422 nm, basic 270, 307, 490 nm; 1H NMR (400 MHz,CDCl3) 1.62 (s,6H), 1.98 (s,3H), 2.48 (br s,3H), 7.11 (br s,J(meta)=1.6Hz,lH), 7.52 (br s,J(meta)=1.6Hz,lH), 11.38 (s,lH), I I . 71 (s,lH) ppm; MS m/z ( r e l . intensity) 312 (M+ 78), 297 (6), 284 (44), 269 (92), 256 (100), 241 (61), 228 (30), 227 (30), 213 (35). 2-hydroxygarvin h (119): Obtained as pale yellow needles 193 (diethyl ether) mp 195°C; R f 0.33;UV (MeOH) neutral 226, 278, 318, 332, 385 nm, basic 226, 278, 332, 407 nm; IR (CHC13) 3490, 2990, 1720, 1700sh, 1610, 1380, 1210 cm - 1; 1H NMR (CDC13) 1.04(t, J=7Hz, 3H), 1.52(s, 3H), 1.66(s, 3H), 1.73(s, m, 2H), 1.81(s, 3H), 3.15(m, 2H), 3.98(s, 3H), 3.99(s, 3H), 6.98(s, IH), 7.21(s, 3H), 14.25(s, IH); 1 3 C NMR (CDCl 3) 209.9(s), 199.9(s), 168.3(s), 166.0(s), 157.7(s), 142.9(s), 142.l(s), 141.9(s), 126.9(s), 116.7(s), 116.5(d), 107.3(s), 105.0(d), 81.6(s), 55.9(q), 52.3(q), 46.9(s), 36.0(t), 30.6(q), 28.9(q), 28.2(q), 25.3(t), 14.7(q); HRMS observed 414.1677, calc'd for C 2 3 H 2 g 0 7 414.1679; LRMS m/z ( r e l . intensity) 414 (28), 398 (12), 383 (14), 372 (79), 371 (100), 357 (39), 339 (29). Preparation oj. 2-hydroxygarvin A diacetate (12Q): 2-hydroxygarvin A (119) (5 mg) was dissolved i n 500 ul of acetic anhydride and 500 u l of pyridine and s t i r r e d overnight at room temperature. The reagents were removed in-vacuo and the residue was p u r i f i e d v i a s i l i c a gel prep TLC (10% ethyl acetate/chloroform) to y i e l d 2-hydroxygarvin A diacetate (120) (4 mg). Compoundl20; obtained as a pale yellow o i l ; R^ 0.35; UV (MeOH) 226, 268, 327, 356(sh) nm; 1H NMR (CDC13) 1.03(t, J=7Hz, 3H0, 1.50(s, 3H), 1.60(s, 3H), 1.70(m, 2H), 1.83(s, 3H), 2.25(s, 3H), 2.94(m, 2H), 3.99(s, 6H), 7.06(s, IH) 7.73(s, IH); HRMS 498.1889, calc'd for C 2 7 H 3 Q 0 9 498.1890; LRMS m/z ( r e l . intensity) 498 (M + 6), 456 (48), 396 (32), 372 (8), 371 (10), 344 (12). Garvin A quinone (122)t Obtained as a red o i l ; R f 0.50; UV (MeOH) 216, 276, 384 nm; 1H NMR (CDC13) 1.05(t, J=7Hz, 3H), 1.60(m, 2H), 194 1.61(s, 6H), 1.98(s, 3H), 2.95(mf 2H), 3.98(s, 3H), 4.03(s f 3H), 7.56(s, 1H), 12.15(s, IH); HRMS observed M + m/z 412.1518, C23 H24°7 r e < 3 u i r e s 412.1522. LRMS m/z ( r e l . intensity) 412 (100), 398 (6), 397 (6), 384 (41), 369 (15) 341 (21). Garvin A quinone acetate (123): Garvin A quinone (122) was dissolved i n acetic anhydride (250 ul) and pyridine (250 ul) and s t i r r e d overnight at room temperature. The reagents were removed in-vacuo and the residue was p u r i f i e d by preparatory TLC (50% ethyl acetate/hexane) to y i e l d garvin A quinone acetate (123) (3 mg). Compound 121: obtained as l i g h t yellow needles (acetone); R f 0.55; UV (MeOH) 224, 276, 299sh, 344sh nm; "'"H NMR (CDC13) 1.02(t, J=7Hz, 3H) , 1.59(m, 2H), 1.61(s, 6H), 1.94(s, 3H), 2.37(s, 3H), 2.83(bs, 2H), 3.94(s, 3H)< 3.96(s, 3H), 7.48(s, IH); HRMS 454.1632, calc'd for C25 H26°8 4 5 4 « 1 6 2 8 ' LRMS m/z ( r e l . intensity) 454 (35), 423 (6), 412 (64), 397 (20), 384 (31), 341 (12). Garvin A (124): obtained as a yellow s o l i d . R f 0.41; UV (MeOH) 220, 247, 274, 321, 383 nm; XH NMR(300 MHz,CDCL3) 1.03(t,3H,J=7Hz), 1.64(s,3H), 1.73(m,2H), 1.99(s,3H), 3.18(m,2H), 3.95(s,3H), 3.97(s,3H), 6.91(s,lH), 7.17(s,lH), 15.14(s,IH),also 1.50(d,3H,J=7Hz), 4.01(q,lH,J=7Hz) due to diketo tautomer; HRMS 398.1724, calc'd for C 23 H26°6 398.1730; LRMS m/z ( r e l . intensity) 398(M +,100), 383(30), 367(21), 351(17). 195 Preparation of. Monomethyl Garvin A (125) ; Garvin A (124) (14mg) was dissolved i n d i e t h y l ether (3 ml, anhyd.) and treated with diazomethane (generated by adding 0.6 ml 5M NaOH to lOOmg N-methylnitro-nitroso-guanidine). After standing overnight, the reaction mixture was p u r i f i e d by preparative TLC (80% CHCl-j/nexane) to give monomethylgarvin A (125) (6.4mg) as the major product. Compound (125); Rf0.60; UV (MeOH) 220, 250, 275, 293(sh), 322, 334(sh), 390nm. XH NMR (300 MHz,CDCl3) 1.02(t,3H), 1.55(s,6H), 1.73(m,2H), 2.06(s,3H), 3.19(m,2H), 3.93(s,3H), 3.96(s,3H), 3.99(s,3H), 6.90(s,lH), 7.14(s,lH), 15.87(s,lH)ppm; MS m/z ( r e l . intensity) 412(M +,44), 397(32), 381(15), 365(24), 337(10). Garvin J3 (126) : obtained as a yellow s o l i d . Rf0.43; UV (MeOH) 229, 256, 289, 327, 389nm; 1H NMR(300 MHz, DMSO-dg) 1.50(d,3H,J=7Hz), 1.54(s,3H), 1.58(s,3H), 1.85(s,3H), 3.23(dd,lH,J=19,13Hz), 4.35(dd,lH, J=19,3Hz), 4.77(m,lH), 7.16(s,lH), 7.35(S,1H), 11.14(s,lH), 17.85(s,IH)ppm; 1 3C NMR(75 MHz, DMSO d g) 191.05, 177.97, 170.58, 166.33, 159.05, 149.09, 145.56, 142.55, 115.00, 114.40, 110.53, 110.50, 107.70, 107.65, 75.40, 48.24, 33.97, 29.24, 28.93, 20.43, 7.60ppm; HRMS 368.1269, calc'd. for C2i H20°6 368.1260; LRMS m/z ( r e l . intensity) 368(M +,6), 353(3), 325(3), 311(3). Preparation of Trimethyl Garvin B (129): A solution of garvin B (126) (15mg), K 2C0 3(lOOmg), and methyl iodide(0.5ml) i n 10ml acetone was was refluxed under a stream of N 2 for 6 hrs and then partitioned between water and 196 methylene chloride. The methylene chloride soluble material was chromatographed on s i l i c a (50% ethyl acetate/hexane) to y i e l d trimethylgarvin B (129) (lOmg) as the major reaction product. 22 Compound (129); obtained as a pale yellow s o l i d . Rf0.27; [<*JD +172.69° (c0.26,CHCl 3); UV (MeOH) 232, 275, 330nm; H^ NMR(300 MHz,CDCl3) 1.37(s,3H), 1.42(s,3H), 1.54(s,3H), 1.55(d,3H,J=7Hz), 1.57(s,3H), 3.27(dd,IH,J=19,13Hz), 3.89(s,3H), 4.04(s,3H), 4.13(dd,lH,J=19,3Hz), 4.45(m,lH), 7.13(s,lH), 7.50(s,lH)ppm; MS m/z(rel. intensity) 410(M+,100), 395(11), 377(13), 367(12), 340(49), 339(40). Preparation o_£ Garvin E. diacetate (127) and t r i a c e t a t e (128) ; Garvin B (126) (30mg) was acetylated at room temperature with pyridine(0.5ml) and acetic anhydride(0.5ml). After evaporation of the reagents, the residue was p u r i f i e d by preparative TLC (10% ethyl acetate/methylene chloride) and HPLC (10% ethyl acetate/hexane) to give garvin B diacetate (127) (5mg) and garvin B t r i a c e t a t e (128) (15mg). Compound 127; obtained as a l i g h t yellow o i l . Rf0.38; UV (MeOH) 245, 286, 389nm. 1H NMR(80 MHZ,CDC13) 1.55(s,3H), 1.57(s,3H), 1.58(d,3H,J=7Hz), 1.88(s,3H), 2.38(s,3H), 2.41(s,3H), 3.40(dd,IH,J=19,13Hz), 4.32(dd,lH,J=19,3Hz), 4.60(m,lH), 7.25(s,lH), 7.30(s,lH), 15.75(s,lH)ppm; MS m/z ( r e l . intensity) 452(M +,2), 451(5), 409(15), 367(76), 352(23). Compound 12fi: obtained as a pale yellow o i l . Rf0.24; UV (MeOH) 222, 237, 281, 292, 305(sh), 345, 365(sh), 384nm;1H NMR(400 MHz,CDCl3) 1.57(d,3H, J=7Hz), 1.58(s,3H), 1.64(s,3H), 1.81(s,3H), 2.36(s,3H), 2.40(s,3H), 197 2.57(s,3H), 3.28(dd,lH,J=19,13Hz), 3.85(ddr1H,J=19,3Hz), 4.58(m,lH), 7.48(s,lH), 7.86(s,lH)ppm; MS m/z ( r e l . intensity) 494(M +,1), 493(1), 451(13), 409(22), 367(68), 352(16). 2-Hydroxyqarvin B_ (130): obtained as a yellow s o l i d . Rf0.35; UV (MeOH) 234, 287, 386nm, (neutral), 253, 310, 417nm (basic)'; XH NMR(400MHz,CDC13) 1.50, 1.54(s,3H), 1.64, 1.65(d,J=7HZ,3H), 1.65, I. 66(s,3H), 1.80, 1.82(s,3H), 3.41, 3.44(dd,J=12,19Hz,lH), 4.02(br s,lH), 4.38(dd,J=3,19Hz,lH), 4.74(m,lH), 7.11(s,2H), I I . 44, 11.45(S,1H), 14.61, 14.62(s,IH)ppm; 1 3 C NMR(75MHz,CDCl3) 209.83, 199.71(199.55), 169.85(169.80), 167.39(167.17), 161.27(167.16), 145.40(145.26), 144.58(144.54), 143.84, 116.03, 115.94, 111.81, 110.81(110.77), 107.00(106.88), 81.64(81.57), 75.43(75.35), 47.14(47.05), 34.60(34.36), 30.70(30.44), 29.15(29.04), 28.43(27.99), 20.83ppm; HRMS 384.1214, calc'd for C21 H20°7 3 8 4 « 1 2 0 9 ' L R M S m / z ( r e l . intensity) 384(M+,32), 368(22), 353(9), 342(90), 341(100), 327(61), 309(26). Preparation of Triacetates (131a.b) of 2-hydroxygarvin E: 2-hydroxygarvin B (130) (10 mg) was treated with 300ul of acetic anhydride and 300ul of pyridine and s t i r r e d at room temperature for 16 hours. After evaporation of the solvents, the reaction mixture was p u r i f i e d by repeated preparative TLC (x3) i n 50% ethyl acetate/hexane to y i e l d the diastereomeric t r i a c e t a t e s 121a. (4 mg) and 122h (4mg). Triacetate JJH&: obtained as a col o r l e s s o i l . R f 0.42; UV (MeOH) 230, 269, 358 nm; -^H NMR (270 MHZ,CDC13) 1.47 (s,3H), 1.58 (s,3H), 1.58 (d,J=7Hz,3H), 1.84 (s,3H), 2.22 (s,3H), 2.39 (s,3H), 2.45 (s,3H), 3.20 (m,lH), 3.92 198 (m,lH), 7.50 (s,lH), 7.80 (s,lH) ppm;MS m/z ( r e l . intensity) 510 (M+ 2), 468 (19), 426 (100), 384 (10), 366 (40), 342 (28), 341 (42). Triacetate 131b; obtained as a colo r l e s s o i l . R f 0.39; UV (MeOH) 230, 268, 357 nm; 1H NMR (270 MHz,CDCl3) 1.49 (s,3H), 1.55 (d,J=7Hz,3H), 1.63 (s,3H), 2.24 (s,3H), 2.41 (s,3H), 2.50 (s,3H), 3.27 (m,lH), 3.68 (m,lH), 4.58 (m,lH), 7.50 (s,lH), 7.80 (s,lH) ppm; MS m/z ( r e l . intensity) 510 (M + 1), 468 (18), 426 (100), 384 (10), 366 (43), 342 (31), 341 (43). Garvin B quinone (132): obtained as a red o i l . R f 0.48; UV (MeOH) neutral 225 (41400), 292 (30800), 397 (7300) nm, basic 237 (37700), 270 (30000), 310 (25000), 354 (sh) (9900), 463 (8000) nm; XH NMR (80 MHz,CDCl3) 1.58 (s,3H), 1.60 (s,3H), 1.63 (d,J=7Hz,3H), 1.95 (s,3H), 3.23 (dd,J=19,12 Hz,lH), 3.99 (dd,J=19,3 Hz,lH), 4.72 (m,lH), 7.64 (s,lH), 11.85 (s,lH), 12.28 (S,1H) ppm; 1 3 C NMR (75 MHz,CDCl3) 200.04, 189.51, 180.43, 169.07, 167.25, 159.92, 153.71, 145.65, 139.11, 128.72, 118.96, 118.73, 116.22, 112.58, 75.44, 47.70, 33.35, 26.54, 25.57, 20.72, 8.11 ppm; HRMS 382.1055, calc'd for C 2 1 H 1 8 0 7 482.1052; LRMS m/z ( r e l . intensity) 382 (M + 100), 354 (47), 339 (29), 326 (10), 325 (6), 311 (18), 308 (22). Preparation pJL Garvin 1 Quinone diacetate (133): Garvin B quinone (132) (7 mg) was reacted with 500ul of acetic anhydride and 500ul of pyridine and s t i r r e d overnight at room temperature. Evaporation of the solvents under high vacuum gave the diacetate 122 i n quantitative y i e l d . Diacetate 133: 199 obtained as a pale yellow o i l . R f 0.47; UV (MeOH) neutral 215, 245 (sh), 296 (sh), 335 nm, basic 228, 249, 288, 426 nm; 1H NMR (300 MHz,CDCl3) 1.56 (d,J=7 Hz,3H), 1.59 (s,3H), 1.68 (s,3H), 1.99 (s,3H), 2.38 (s,6H), 3.12 (dd,J=18,11 Hz,IH), 3.50 (m,lH), 4.60 (m,lH), 7.75 (s,lH) ppm; MS m/z ( r e l . intensity) 466 (M + 0.1), 424 (23), 382 (37), 354 (27), 339 (15), 308 (10). Preparation oj. garvin A. djmey Hi.: Garvin A (124) (4 mg) was dissolved i n MeOH (10 ml ) and 50 mg Ag 20 was added. The mixture was s t i r r e d under a stream of a i r for 24 hours and p u r i f i e d by preparative TLC (50% ethyl acetate/hexane) to give garvin A dimer (134) (2 mg) as the major product. Dimer 134: obtained as a yellow o i l ; R^ 0.43; UV (MeOH) 228, 277, 332, 393 nm; -""H NMR (300 MHz, CDC13) 0.93 (t, J=7Hz, 3H) , 1.58 (s,3H), 1.60 (m,2H), 1.71 (s,3H), 1.94 (s,3H), 2.90 (m,2H), 3.94 (s,6H), 6.89 (S,1H), 7.11 (s,lH), 14.65 (s,lH) ppm; 1 3 C NMR (75 MHZ,CDC13) 209.9, 202.5, 168.5, 166.9, 157.2, 142.9, 142.6, 141.7, 125.2, 116.4, 115.2, 108.0, 104.7, 63.4, 55.8, 52.3, 47.9, 35.9, 32.1, 28.2, 24.9, 21.7, 14.8 ppm; MS m/z ( r e l . intensity) 398 (100), 383 (51), 367 (36), 351 (34) (molecular ion not seen in EI spectrum). Hydroaenolvsis Q±_ garvin h dimer: Garvin A dimer (134) (4 mg) was dissolved i n 95% EtOH (20ml) i n an erlenmeyer f l a s k . A spatula of palladium on activated charcoal was added, and the flask was sealed under an atmosphere of hydrogen gas and s t i r r e d overnight. A quantitative y i e l d of garvin A (124) was obtained. 200 Preparation pJL aarvin A djper diacetate 13_5_: Treatment of garvin A dimer (134) (5 mg) with 250 u l of acetic anhydride and 250 u l of pyridine overnight at room temp, yielded the diacetate 13J5_ (3 mg) as the major reaction product. Diacetate 135; obtained as a c o l o r l e s s o i l ; R f 0.61; UV (MeOH) 211, 268, 324, 355 (sh) nm; 1H NMR (300 MHz,CDCl3) 1.02 (t,J=7Hz,3H), 1.55 (s,3H), 1.60 (m,2H), 1.70 (s,3H), 1.86 (s,3H), 2.32 (s,3H), 2.91 (m,2H), 3.95 (s,3H), 3.96 (s,3H), 7.08 (s,lH0, 7.72 (s,lH) ppm; MS m/z ( r e l . intensity) 878 (M+,0.24), 836 (2), 794 (2), 398 (100), 383 (17), 369 (32). Garveatin B-garvin B_ dimer 136: Obtained as an orange o i l ; R^ 0.65; UV (MeOH) 283, 400 nm (neutral), 282, 305(sh) 414 nm (basic); 1H NMR (400 MHz,CDCl3) 1.00 (t,J=7Hz,3H), 1.20 (s,3H), 1.24 (s,3H), 1.51 (d,J=7Hz,3H), 1.78 (s,3H), 1.80 (s,3H), 1.81 (s,3H), 1.82 (s,3H), 2.35 (dd,J=19,12Hz,IH), 2.42 (s,3H), 2.42 (m,lH), 2.77 (m,lH), 3.83 (dd,J=19,3Hz,lH), 4.40 (m,lH), 6.90 (s,2H), 6.92 (S,1H), 7.02 (s,lH), 9.10 (s,lH), 14.85 (s,lH), 15.70 (s,lH) ppm; MS m/z r e l . intensity) 368 (100), 353 (53), 326 (53), 311 (88), 297 (20) (no molecular ion was seen i n the EI spectrum); FAB MS (MeOH solution i n thioglycerol) m/z 693(M+l ion), 649, 369, 368, 327, 326, 311, 297. Garvalone A (137): obtained as a l i g h t yellow o i l . Rf0.42; UV (MeOH) 228(15,000), 279(20,100), 317(4,900), 330(4,100), 382(4,400) nm; IR (CHCl,) 1570, 1610, 1718, 1737 cm"1; "hi NMR(300 201 MHz,CDCL3) l.ll(t,3H,J=7Hz), 1.47(s,3H), 1.55(s,3H), 1.59(s,3H), 1.75(m,2H), 2.04(s,3H), 2.23(m,4H), 3.15(m,2H), 3.93(s,3H), 3.96(s,3H), 6.93(s,lH), 7.13(s,lH), 15.10(s,IH)ppm; 1 3 C NMR (75 MHz,CDCL3) 211.90(s) f 207.04(s), 203.88(s), 168.61(s), 166.59(s), 157.35(s) r 142.97(s) f 142.50(s), 141.82(s), 126.45(s), 116.78(s), 115.28(d), 108 70(s), 104.96(d), 58.30(s), 55.94(q), 52.49(q), 47.85(s), 39.06(t), 36.14(t), 31.43(t), 30.52(q), 29.86(q), 27.01(q), 25.42(t), 24.21(q), 14.80(q)ppm; HRMS 468.2162, calc'd for C 2 7 H 3 2 0 7 468.2149; LRMS m/z ( r e l . intensity) 468(M +,94), 453(14), 437(14), 398(100), 397(89), 383(43). Preparation pj. Methyl GaEValone A (139): Garvalone A (137) (20mg), K 2 C 0 3 ( 1 5 0 M<3' anhyd.), and methyl iodide (0.6ml) were dissolved i n 10ml of acetone and the mixture was s t i r r e d at reflux overnight under an atmosphere of N 2. The reaction mixture was rotary-evaporated and par t i t i o n e d between water and methylene chloride. The methylene chloride phase yielded methyl garvalone A (139) (20mg). Compound (139); obtained as a pale yellow o i l . Rf0.39; UV (MeOH) 225, 263, 314nm; 1H NMR(400 MHz,CDCl3) 0.94(t,3H,J=7HZ), 1.36(s,3H), 1.52(s,3H), 1.57(s,3H), 1.6(m,2H), 1.95-2.10(m,2H), 2.05(s,3H), 2.24(m,2H), 2.48(m,2H), 2.87(m,lH), 3.14(m,lH), 3.95(s,3H), 3.97(s,3H), 7.02(s,lH), 7.49(s,IH)ppm; MS m/z ( r e l . intensity) 482(M +,57), 467(11), 451(9), 426(23), 412(38), 411(37), 397(62), 383(18). 202 Preparation of. 2-(3-oxobutyl) garveatin A (14Q) : To a solution of garveatin A (77) (15 mg) i n benzene (7ml) was added anhydrous K 2C0 3 (lOOmg), methyl v i n y l ketone (50 u l ) , and 18-crown-6 (one c r y s t a l ) . The mixture was refluxed under N 2 for 24 h after which i t was f i l t e r e d and the f i l t r a t e was concentrated in-vacuo. Preparative TLC of the residue ( s i l i c a g e l; a cetic acid/methanol/chloroform, 1:5:94) gave 2-(3-oxobutyl)garveatin A (140) (12mg) and unreacted garveatin A (2mg) . Compound 1 M : obtained as a yellow o i l . R f 0.39; UV (MeOH) neutral 231, 271, 415 nm, basic 240, 268, 420 nm; "^H NMR(CDC13, 400MHz) 1.48(s, 3H), 1.54(s, 3H), 1.58(s, 3H), 2.04(s, 3H), 2.24(m, 4H), 2.34(s, 3H), 2.55(s, 3H), 6.65(s, IH), 7.12(s, IH), 10.13(S, IH), 16.29(s, IH); 1 3 C NMR (CDCl-j) 20.7, 24.1, 27.1, 29.9, 30.7, 31.3, 32.3, 38.9, 47.9, 58.0, 107.8, 110.8, 115.5, 120.2, 125.3, 139.5, 141.2, 142.5, 156.0, 166.1, 204.1, 204.2, 206.9, 211.3 ppm; MS m/z ( r e l . intensity) 410 (M+ 18), 341 (16), 340 (32), 326 (25), 325 (100), 310 (9), 297 (12). Garvalone B (141): obtained as a l i g h t yellow o i l (3:2 mixture of diastereomers). R f 0.44; [ o c ] D +136.92° (c 0.39, CHC1 3); UV (MeOH) 236, 290, 385 nm; 1H NMR (300 MHz, CDC13) 1.464, 1.470(s, 3H), 1.566, 1.533(s, 3H), 1.584, 1.611(s, 3H), 1.646(d, J=7Hz, 3H), 2.063, 2.042 (s, 3H), 2.234 (m, 4H), 3.419, 3.424 (dd, J=18, 12Hz, IH), 4.387 (dd, J=18, 3Hz, IH), 4.732(m, IH), 7.060(s, IH), 7.102(s, IH), 11.503(s, IH), 15.442(s, IH) ppm; HRMS 438.1680, calc'd for C 2 5 H 2 g 0 7 438.1679; LRMS m/z ( r e l . intensity) 438 (M+, 12), 368(10), 353(5), 325(3). 203 Garvalone B diacetate (142); -""H NMR (300MHz, CDC13) 2.40(s, 3H), 2.47(s, 3H), 7.53(s, IH), 7.75(s, IH) ppm. Synthesis of garvalone £ (141): Garvin B (126) (15 mg) was dissolved i n benzene (8 ml ) containing 100 mg of K 2C0 3 (anhyd.) and a c r y s t a l of 18-crown-6. Excess methyl v i n y l ketone (100 ul) was added and the mixture was s t i r r e d at reflux overnight, under N 2. The reaction mixture was then f i l t e r e d and the f i l t r a t e concentrated in-vacuo. Preparative TLC (5% MeOH/CHCl3) p u r i f i c a t i o n gave garvalone B (141) (8 mg) as a 2:3 mixture of diastereomers and unreacted garvin B (4 mg). Annulin & (144): Obtained as orange c r y s t a l s from 95% ethanol, mp 174-176°C; Rf0.51; UV (MeOH) neutral 217,247 439nm, basic 286, 559nm; IR (CHCl 3) 1749.9, 1657.2, 1616.1 cm"1; 1H NMR (300 MHz,CDCl3) 1.14(t,3H,J=7HZ), .1.63(8,3H), 1.72(s,3H), 2.44(s,3H), 2.76(q,2H,J=7Hz), 3.87(s,3H), 4.92(br S,1H), 7.47(S,1H), 12.12(8,1H) ppm; 1 3 C NMR (75 MHz,CDCl3) 185.97, 180.92, 169.35, 160.32, 154.29, 145.63, 140.76, 139.81, 130.37, 122.23, 122.10, 113.40, 101.45, 89.08, 54.04, 27.94, 26.43, 20.09, 19.50, 12.73 ppm; HRMS 360.1221, calcd. for C 1 9H 2 ( )0 7: 360.1209; LRMS m/z ( r e l . intensity) 360(M +,6), 342(2), 327(1), 301(45), 283(100), 255(14). Annulin £ (148) t Obtained as an orange o i l ; Rf0.51; [°<-]D +8.0°(c.0.2,CHCl 3); UV (MeOH) neutral 208, 255, 293, 425nm, basic 235, 270(sh), 307(sh), 530nm; IR (CHC13) 1757, 1736, 1657, 1638 204 cm -L;-LH NMR (300 MHz,CDCl3) 1.15 (t, 3H, J=7Hz) , 1.49(s,3H), 1.51(s,3H), 1.85(s,3H), 2.42(s,3H), 2.73(q,2H,J=7Hz), 3.76(s,3H), 7.31(s,lH), 12.35(s,lH) ppm; 1 3 C NMR (75 MHz,CDCl3) 203.02(s), 181.14(s), 178.62(s), 167.68(s), 163.79(s), 160.43(s), 147.64(s) r 136.06(s) f 127.59(s) f 120.65(d), 119.11(s), l l l . l l ( s ) , 84.48(s), 53.45(q), 43.64(s), 25.90(q) r 23.78(q), 20.40(q), 20.38(q), 19.10(t), 12.77(q) ppm; HRMS 386.1361, calc'd. for C 2 ] H 2 2 0 7 : 386.1366; LRMS m/z ( r e l . intensity) 386(M +,17) f 358(25), 343(100), 283(17). Antimicrobial assays. A standard i n - v i t r o d i s c (0.25 in.) bioassay was used to the an t i b a c t e r i a l and antifungal a c t i v i t y of the £L«_ annulata metabolites. 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