Bioactive metabolites from marine organisms.

The marine molluscs Siphonaria pectinata, S. normalis, Onchidium verruculatum and Peronia peronii were investigated because they are trail following species, that are known to possess repugnatorial skin glands. Extraction of the skin glands of these organisms led to the isolation of the compounds, pectinatone, dihydrosiphonarins A and B, ilikonapyrone and peroniatriols I and II, respectively. The marine tunicate, Lissoclinum patella was also studied because the original methanol extract of the tunicate exhibited a variety of biological activities. A series of eight cytotoxic cyclic peptides have been isolated from this tunicate, all of which contain thiazole and oxazoline amino acids as a common structural feature.

BIOACTIVE METABOLITES FROM MARINE ORGANISMS by Joseph Edward Biskupiak A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medicinal Chemistry The University of Utah June 1985 TIlE UNIVERSrTY ()F trrAH GR.ADCA.TE SCI-IO()L SUPERVISORY COrvf.~lfIT1~EE APPRO\;1"AL of a dissertation submitted by ( Joseph E. Biskupiak This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. ArJ (q /qtS" C! JYa-J Chairman: Chris r-1. Ireland Arthur D. Broom ~~~C:,~2 d,_ Martin p~~zer ,~-=--~=,-~-.::. C. Dale Poulter THE UNIVERSlTY Of· UTAH GRi\IJUATE SCH()()L FINAL READING APPROVAL To the Graduate Council of The University of Utah: I have read the dissertation of Jose:gh E. Bi skupiak in its final form and have found that () its format, citations, and bibliographic style are consistent and acceptable~ (2) its illustrative rnaterials including figures. tables~ and charts are in place; and (3) the final manuscript is satisfactory to the Supervisory Committee and is ready for submission to the Graduate SchooL Chris M. Ireland Member. Supervisory Committee Approved for the Major Department ~-zt~_ Arthur D. Broom Chairmar.: Dean Approved for the (iraduatc Council ~k'- James L" Copyright © Joseph Edward Biskupiak 1985 All Rights Reserved ABSTRACT The marine molluscs Siphonaria pectinata, ~. norrnalis, Onchidium verruculatum and Peronia peronii were investigated because they are trail following species, that are known to possess repugnatorial skin glands. Extraction of the skin glands of these organisms led to the isolation of the compounds, pectinatone, dihydrosiphonarins A and B, ilikonapyrone and peroniatriols I and II, respectively. The marine tunicate, Lissoclinum patella was also studied because the original methanol extract of the tunicate exhib­ited a variety of biological activities. A series of eight cytotoxic cyclic peptides have been isolated from this tunicate, all of which contain thiazole and oxazoline amino acids as a common structural feature. CONTENTS Page ABS TRACT. · · · • · • . • · . . • . . . . • • . . . . . . • . . • • • • . • . . . . • . . • . • . . • . •• i v LIST OF TABLES ••.•.•.••.••.•..••....••••••••.••••.•••.•••• vi LIST OF FIGURES ••••••..•..•••..•.•.•••.••..•••.•.....•••• vii ACKNOWLEDGMENTS. • . . • • . . • • • . • • . . • . • . . • • • • • • • • • • • • • . . • • • • . vii i INTRODUCTION TO SIPHONARIIDS AND ONCHIDS •••.•.••...•.••..•• 1 Biology of the Siphonariids .•.••..•••••••...•.•..•••. 13 The Chemistry of Siphonaria pectinata .••••••.••••.••. 15 The Chemistry of Siphonaria normalis .•.•••••.•..•.••. 25 Biology of the Onchids •••...••••••...•.•..•..•.•••••. 33 The Chemistry of Onchidium verruculatum •••..••••.•••• 35 The Chemistry of Peronia peronii .•••••••.•••••••.••.• 41 INTRODUCTION TO TUNICATES ••.•....•.••••••.•...••••..•...•. 57 Review of Ascidian Chemistry ••.•••.•.••••..•••••.•••• 60 The Chemistry of Lissoclinum patella ••••.••.•••••...• 79 stereochemistry of Thiazole Amino Acids •••..•.•••••.• 99 EXPERIMENTAL •.•••••••.••••.•..•••.•••••.•.•..••••.•...•.• 110 The Chemistry of siphonaria pectinata •••..•...•••... ll0 The Chemistry of Siphonaria normalis •..••••.•.••.••. 114 The Chemistry of Onchidium verruculatum .......•..•.. 116 The Chemistry of Peronia peronii .•••.•...•.•..•...•• 120 The Chemistry of Lissoclinum patella .••....••••••.•. 126 APPENDIX .••••••.•••••••.•••.••...••.•.••.......•.......•. 140 REFERENCES •..••.••••.••••••.•.•..•••.•••...•.••...••.•.•. 145 LIST OF TABLES Table Page 1. Taxonomy of siphonariids and onchids .................. 1 2. Chemical shift comparison for ~- and y-pyrones ....... 18 3. Chemical shift comparison •.................•......•.. 44 4. Oxazoline spectral comparison .....•......•........... 83 5. Thiazole stereochemistry •.........•..•.........•.•.. 103 6. L1210 results A •..................••................ 121 7. L1210 results B ....•........•..•........•...•....... 121 LIST OF FIGURES Figure Page 1. structure of a typical Siphonaria species ............ 14 2. Mass spectral fragmentation pattern for 2,4,6-trimethylnonanoic acid ..•.................. 24 3. Pathway for generation of the artifact ............... 34 4. Structure of a typical Onchidella species ............ 36 5. Mass spectral fragmentation pattern of ilikonapyrone ..................•...•••.•.....•.... 39 6. Oxidative cleavage of IKP, PT I and PT II ............ 45 7. IKP, PT I stereochemistry ......................•....• 50 8. PT II stereochemistry •....••...........•...........•. 51 9. 03 mechanism scheme ...•..•....•...........••......•.. 55 10. structure of a typical tunicate species ....•......... 58 11. Ulicyclamide's ElMS data ..........••..••............. 86 12. Patellamides' mass spectral data ..•...•....•....•.... 90 13. Lissoclinamides' and ulicyclamide's FAB MS data.......................................... 9 7 14. 102 addition to thiazoles ..• ' .........••...•......... 102 15. GC analysis of patellamide A ......•...........•..... 106 16. Trichloromethylleucine formation ...•......•......... 109 17. 1H NMR Spectrum of pectinatone ...••............•.... 141 18. 1H NMR Spectrum of dihydrosiphonarin A ........••.... 142 19. 1H NMR Spectrum of ilikonapyrone acetonide .•...•.... 143 20. 1H NMR Spectrum of patellamide A ..•...••.•••.......• 144 ACKNOWLEDGMENTS I would especially like to thank my parents for their love and encouragement throughout my life. To my sister Judy just a big thanks! To Chots, without your love and support this never would have happened; all I can say is love you and sorry for the inconvenience. A very special thanks to Chris, for without your moral support and constant guidance, this work would remain undone and I would not be the scientist that I am today. David, I will always remember the friendship that we have developed over the years in the lab. Rock and Jeffrey, you two are real special; thanks for all the good times we spent together. Finally, I would like to thank all that I have come to know over the years. Thank you all! INTRODUCTION TO SIPHONARIIDS AND ONCHIDS The gastropod molluscs are divided into three sub-classes: the Prosobranchia, the Opisthobranchia and the Pulmonata (see Table 1). Naked gastropod molluscs of the subclass Opisthobranchia have evolved various chemical defenses, which include secretion of very strong acids (pH=1) and toxic metabolites. Nudibranchs, a member of the subclass Opisthobranchia, are typically brightly colored molluscs lacking an external shell. Marine biologists have observed that nudibranchs are seldom eaten by preda­tors, despite their lack of a protective covering. 1 Origi­nally, it was thought that their cryptic coloration or habit of hiding among the rocks was responsible for their survival. 2 It was later shown, however, that molluscs Table 1 Taxonomy of Siphonariids and Onchids SUBCLASS ORDER FAMILY opisthobranchia Prosobranchia Pulmonata Onchidacea Stylommatophora Bassomatophora Siphonariidae 2 employ defensive mechanisms based on biological (e.g., stinging cells) and chemical (e.g., acidic secretions) strategies. Thompson also reported that molluscs secreted non-acidic constituents in addition to the strong acidic secretions. Johannes carried Thompson's observation one step further, by reporting that the nudibranch Phyllidia varicosa emits a mucus which killed several species of crustaceans and a molly shortly after exposure to the mucus or the nudibranch. Johannes characterized this mucus as being a volatile, heat stable, tasteless material of neutral pH, possessing a strong odor. 3 These observations prompted Scheuer to investigate the chemical nature of this mucus. An X-ray diffraction study of a derivative of the active component of the mucus defined its structure and the com­pound was named 9-isocyanopupukeanane (~).4 It was also observed while collecting P. varicosa that it fed on an off-white sponge, Hymeniacidon §R. It was shown that the active component (1) was selectively accumulated by £. vari­cosa from this sponge, the first direct evidence that nudi­branchs acccumulate defensive metabolites from their diet. 5 In addition to the isonitrile functionality, which exhibits antifeedant properties, it was thought that a furan ring can also exhibit antifeedant properties. Scheuer isolated the two furanosesquiterpenoids, nakafuran-8 (~) and nakafuran-9 (1) from two dorid nudibranchs, Hypselodoris godeffroyana and Chromodoris maridadilus, respectively 3 1 2 3 4 and also from their common prey (i.e., food source), the sponge Oysidea fragilis. 6 In support of the supposition that a furan ring can exhibit antifeedant properties, both compounds were found to possess antifeedant properties against common reef fish. Compounds based on a drimane ring system are also believed to exhibit antifeedant properties, as illustrated by compound (!) and polygodial (~) isolated from the diges­tive glands of Dendrodoris limbata by Cimino, et al. 7 Interestingly, from the terrestrial East African plant, Warburgia stuhlmanni, polygodial was isolated by Nakanishi et al. 8 as the major bioactive component. Despite these compounds being isolated from the digestive glands of the nudibranch, a chemotaxonomic study of sponges in the area (potential food sources) failed to reveal a specific prey/ predator relationship. While earlier examples showed that molluscs concentrate toxic metabolites from their diet, there exists evidence that they can biosynthesize these toxic metabolites as well. The most compelling evidence for de novo synthesis of toxic metabolites comes from the biosynthetic work of Cimino et al. with the previously mentioned Dendrodoris limbata. 9 Injection of [2-14cJmevalonic acid-dibenzylethyl­enediamine salt into the hepatopancreas of seven individual animals led to the isolation of radioactive polygodial (~) and radioactive (§), formed by the thermolysis of fatty 5 acyl esters of compound (!). Toxic secretions are not limited to nudibranchs. While the Onchidacea are not classified as nudibranchs, they are intertidial marine slugs with glands that secrete noxious metabolites. The onchid, Onchidella binneyi, pro­vides a clear example of a chemical defense mechanism. When molested, the onchid discharges a mucus that contains almost exclusively the labile compound onchidal (2).10 Onchidal was also isolated from Onchidium floridanum, a related organism, whose mucus has been reported to act as a deterrent to several possible predators including crabs and fish. ll Onchidal was also shown to be a potent inhibitor of gram-positive bacteria (specifically Staphy­lococcus aureus).lO In addition to the order Onchidacea, another member of the pulmonates, (Latin pulmo meaning lung), the Siphona­riidae have also been shown to possess bioactive compounds. Hochlowski and Faulkner have isolated diemenensin-A (~) and denticulatin-B (~) as the major bioactive components of Siphonaria diemenensis12 and Siphonaria denticulatin,13 respectively. Both compounds exhibited toxicity to goldfish with denticulatin-B being the most active. Antibacterial screening of the CHC13 extract from Siphonaria pectinata led to the discovery of pectinatone (10), a new polypropionate antibiotic. 14 Pectinatone was found to be active against gram (+) bacteria; Staphylococcus 6 HO CHO 4 5 HO 6 OCOCH3 7 OH 8 8 aureus, Bacillus subtilis, yeast Candida albicans, and Saccharomyces cerevisiae. Further from the onchid Peronia peronii, the two bis­pyrone triols, peroniatriol I (11) and II (12), have been isolated, after saponification of a complex mixture of biologically active esters. The original CHC13 extract of Peronia exhibited good in vitro cytotoxicity (0.5 micro­grams/ ml) against the L1210 cell line. Purification of the extract led to a greater than 50-fold increase in bio­logical activity. Unfortunately, the active components of the extract appear to be a complex esterified mixture of the two bispyrone triols. However, compounds (11) and (12) also exhibited good in vitro cytotoxicity against the L1210 cell line, albeit not as good as the activity exhibited by the concentrated fractions of the ester mix­tures. In addition to employing chemicals for a defensive mechanism, siphonariids and onchids also utilize chemicals for their homing mechanism. There exists a wealth of in­formation on the ability of these marine organisms to home by following a mucus trail which they have secreted. Unfor­tunately, no chemical studies have defined the chemicals involved in this process. However, the isolation of various compounds in large amounts by noninvasive methods (i.e., whole body extractions) leads one to speculate, that their biological role in the host organism may be involved with I I HO I I HO o o 11 I I HO 9 o o 10 their homing ability. Homing behavior has been reported for Siphonaria pecti­nata by Thomas. 1S Field observations by Thomas were con­ducted on a group of Siphonaria pectinata inhabiting a concrete seawall at Virginia Key, Florida. It was observed that the siphonariids would graze (feed) at low tide and when the rocks became hot and dry from the sun and wind, they would return to their home (in this case, a shell scar on the concrete seawall). No motion was observed when the siphonariids were submerged. The siphonariids studied had shell lengths between 4 and 16 mm. The great­est distance observed that a siphonariid homed from was 75 cm, though usually the distance was less than 15 cm. While grazing (leaving home to feed), the speed exhibited by a siphonariid was between 0.1 and 0.8 cm/min. No speed was given for the return trip home, though it was mentioned that the return trip was much more rapid. Field observations conducted on the onchid, Onchidium verruculatum were similar to the results for the siphona­riids. 16 This study was conducted on a group of onchids found along a rocky shore 1 km north of Messilah Beach Hotel, Kuwait. The onchids in this study were 4-5 cm long. As with the siphonariids, the outward grazing trip proceeded slowly, about 1.3 em/min. at speeds of 3.7 em/min. However, when homing they moved When the onchids were displaced from their homes by up to 2 m, they would continuously 11 spiral until they came in contact with an older trail of theirs and home on it. That a chemical trail is responsible for all this behavior, is borne out by studies conducted with Siphonaria normalis on the outer shore of Coconut Island, Oahu, Hawaii. After the siphonariids had left their homes and traversed a ways from them, the trails between them and their homes were scrubbed with a wire brush and/or treated with sodium hydroxide and rinsed with seawater. After this treatment, none of the siphonariids homed. 17 The whole body extracts of both Onchidium verrucu­latum18 and Siphonaria normalis19 have also been studied. Initial examination of the IH NMR spectra of the CHC13 extracts of these organisms displayed signals reminiscent of polypropionate metabolites. Only the CHC13 extract of Siphonaria normalis displayed biological activity in any of the tests conducted (L1210, borderline activity approx. 10 micrograms/ml). Isolation of the major metabo­lites from both organisms yielded ilikonapyrone (13) (from Onchidium verruculatum) and dihydrosiphonarins A (14) and B (15) (both from Siphonaria normalis). The biological role of these compounds has yet to be demonstrated, despite their abundance in their representative organisms. o ,I HO 13 14: R = Me IS: R = Et 12 o 13 Biology of the Siphonariids Siphonaria, an early and deviant offshoot from the main pulmonate stock that never left the sea shore, has been a taxonomic problem. Siphonaria has adapted well to the aquatic environment (Figure 1); the mantle cavity is filled with sea water and a secondary gill has developed in place of the ctenidia. There can be up to 30 lamellae in the secondary gill hanging down from the roof of the mantle cavity. As with the prosobranch limpets, the res­piratory water current passes between the gill lamallae in the opposite direction to the flow of blood, which pro­vides the maximum efficiency for the respiratory process. This is in marked contrast to other members of the pulmo­nates, where the mantle cavity has been converted into a pulmonary sac. By means of a siphon (from which the family has earned its name), the mantle cavity connects to the exterior on the right side of the shell. A ridge, which crosses the floor of the mantle cavity and enters the siphon, divides the dorsally disposed mantle cavity into an anterior inha­lent and posterior exhalent channels. The rectum is situ­ated between the channels and discharges the faeces outside the shell. When submerged, the siphonariid is inactive and remains firmly clamped down to the surface of the rock or seawall it claims as its home. When the siphonariid becomes exposed by the falling tide, it becomes mobile Mantle Edge------J­She 11 -------' Faa t --------I-----\-­Mantle Cavity -----+-"'"'\----~ 14 -J--+----- Head Inhalant Current Exhalant Current Lamellae Figure 1. Structure of a typical Siphonaria species 15 and grazes on encrusting algae and diatoms. It returns to its home (a shell scar on the rocks or seawalls) when the rocks become hot and dry due to the sun and wind. Because these grazing trips are of short duration and only at low tide, Siphonaria differs greatly in its eating habits from the prosobranch limpets which it otherwise resembles rather closely.20,21 The Chemistry of Siphonaria pectinata Interest in Siphonaria pectinata was generated because it is known to be a trail following (homing) species that possess repugnatorial glands responsible for a chemical defense system. 11 ,15,16 Siphonaria pectinata was collected from a sea wall at the entrance to Key Biscayne, Florida. The organisms were soaked for 5 days in MeOH. The MeOH extract was concentrated and partitioned between brine and CHCI3. Examination of the CHCl3 whole body extract of the organisms showed potent antibiotic activity against gram (+) bacteria: Staphylococcus aureus, Bacillus subtilis, and yeast Candida albicans and Saccharomyces cerevisiae. Inspection of the silica gel thin-layer chromatography (tIc) plate (3:2 isooctane/ethyl acetate) of the extract revealed a single UV absorbing spot that streaked. Because of this streaking behavior, it was felt that I was dealing with a polar acidic functionality that could be methylated by treatment with diazomethane [IR also indicated the 16 presence of acidic hydroxyl group(s) (3163 br)]. A portion of the extract was treated with diazomethane in diethyl ether. Inspection of the tlc of the reaction mixture indi­cated that there were now 2 UV active spots that did not streak. Column chromatography (silica geli 3:2 isooctanej ethyl acetate) of the original CHC13 extract led to the isolation of 181 mg of pectinatone (10), which crystallized on standing in CH2C12. Further, HPLC of the diazomethane reaction mixture (Partisil 10i 7:3 isooctanejethyl acetate) led to the isolation of the isomeric methyl ethers, y-O­methylpectinatone (16) and a-O-methylpectinatone (17) in equal amounts. The electron impact high resolution mass spectrum (El HRMS) of pectinatone revealed that its molecular formula was C21H3403' which requires 5 degrees of unsaturation. Spectral data indicated the presence of a y-hydroxy-a-pyrone further conjugated to a trisubstituted olefin, accounting for the five degrees of unsaturation: IR 3163 br, 1685, 1649 br, 1624 cm-1 ; UVmax 301 nm (E 5063); 13C NMR 0166.5 s, 166.2 S, 159.2 s, 142.9 d, 14.3 q, 11.6 q, 8.7 q; 1H NMR 126.2 s, 107.0 s, 98.8 s, 02.0 (s, 6H), 1.89 (d, 3H, J=l Hz), 5.38 (dq, 1H, J=10, 1 Hz), 8.8 (br s, 1H). That I was dealing wi th a y-hydroxy- Cl-pyrone moiety was further borne out by the results obtained from the diazomethane reaction. The isolation of the two isomeric methyl ethers was consistent with the known mechanism for 17 16 o Me 17 18 the methylation with diazomethane. The first step of the reaction is abstraction of the most acidic proton, which would be the proton of the y-hydroxy group thus giving the anion on oxygen. Equilibration of the charge between the alpha and gamma oxygens results in an 1:1 mixture of the two methyl ether products. Further evidence for pectinatone being a y-hydroxy­a- pyrone and not an a-hydroxy-y-pyrone comes from the spec-tral data of compounds (16) and (17) and the two known compounds nectriapyrone (18)22 and tridachione (19) .23 The assignment of a- and y-pyrones can be accomplished by comparison of 1) the 13C chemical shift of the carbonyl of the pyrone (Table 2) and 2) the UVmax for the pyrone ring system. 24 A comparison of 13C chemical shifts for a- and y­pyrones indicates that compound (16) is an a-pyrone and compound (17) is a y-pyrone. Therefore, pectinatone is an a-pyrone. Similarily, the UV spectra of a- and Y-pyrones are equally informative. The UVmax for compound Table 2 Chemical Shift Comparison for a- and Y-Pyrones Carbon # Cmpd (16) Nectriapyrone Cmpd (17) Tridachione 2 3 4 5 6 168.6 109.8 165.5 108.6 159.3 166.4 101.6 165.4 91.8 160.3 161.9 99.3 181.6 117.6 158.6 161.0 97.8 181.6 118.4 160.1 19 18 20 (16) was 313 nm, which is characteristic for a-pyrones. y­Pyrones, on the other hand, exhibit UVmax at 260 nm as il­lustrated by (17) whose UVmax was 261 nm. The UVmax of pec­tinatone (301 nm) was typical of a-pyrone. All that remained to be accomplished was determining the nature of the side chain present in pectinatone. The y-hydroxy-a-pyrone conjugated to an olefin accounts for a C10HI103 portion which leaves a C11H23 portion. The IH NMR spectrum of pectinatone contained signals for 3 secondary and a primary methyl groups suggestive of a polypropionate chain (20). Proton decoupling studies at 500 MHz allowed assignment of the side chain. Irradiation of the allylic proton at 62.61 collapsed the olefinic proton at 5.38, the methyl at 0.98 and the diastereotopic methylenes protons at 1.31 and 1.06, thus defining the first two propionate units. Irradiation of the methylene multiplet at 1.23 collapsed the triplet methyl signal at 0.86 and the second set of diastereotopic methylene protons at 1.31 and 1.06 fixing the end unit. The remaining 2 methine protons coallesced at 1.47 ppm. Irradiation of them collapsed the doublet methyls at 0.85 and 0.80 as well as methylene protons at 1.17 and 0.93. Ozonolysis of the trisubstituted olefin, followed by oxidative work-up with Jones' reagent gave 2,4,6-tri­methylnonanoic acid. The electron impact mass spectrum of the acid exhibited a weak parent ion at 200.1764 which 21 20 22 matched for C12H2402 (calc 200.1776). Further, the spectrum contained additional peaks which corresponded to fragmenta­tion of the acid in either direction (Figure 2). Esterifi­cation of the acid with diazomethane gave the methyl ester, which had a rotation [a]D +34.20 (c 0.038, Et20) and was assigned the 2S,4R,6S absolute configuration (lit. value +35.20 ).25 In addition to pectinatone, a minor metabolite was isolated on examination of the original CHC13 extract by HPLC Silica gel HPLC with diisopropyl ether gave a UV absorbing fraction, which on further HPLC purification (RP-18, 20%H20/MeOH) gave 8.6 mg of a clear oil (21). EI HRMS of (21) indicated that the molecular formula was C20H3403' obsd 322.2504, calc 322.2509, thus indicating that there were 4 degrees of unsaturation. The 13C NMR spectrum of (21) exhibited signals for a ketone (203.3 ppm), a hemiketal carbon (106.3 ppm), an olefin bearing oxygen (181.6 and 101.0 ppm) and a trisub­stituted olefin (147.7 and 125.8 ppm). Therefore, compound (21) must contain 1 ring. These data, along with 1H NMR signals at 01.98 and 1.81 for the vinyl methyls and the signal at 1.55 for the methyl of the hemiketal carbon, gave the partial structure (22). The spectral data for (22) gave a satisfactory correlation for the published literature values for (22) .26 Decoupling studies at 500 MHz indicated that the side chain in compound (21) was 23 OH OH 22 HO 45 87 115 129 157 Figure 2. Mass spectral fragmentation pattern for 2,4,6-trimethylnonanoic acid 24 25 identical to the side chain in pectinatone. Because of the inability to affect separation of the epimeric mixture of hemiketals (epimeric at the hemiketal carbon as indicated by the doubling of certain signals in both the 13C and IH NMR spectra), the stereochemistry was not assigned. The Chemistry of siphonaria normalis Interest in Siphonaria normalis grew out of the results with siphonaria pectinata. Siphonaria normalis is also a trail following species that is thought to possess repug-natorial glands. Collections of Siphonaria normalis were made at Diamond Head Beach, Oahu, Hawaii. The animals were stored in isopropanol at SoC for a week. The isopro­panol extract was filtered and solvent removed in vacuo to yield an oily watery residue which was partitioned be­tween CHC13 and brine. The CHC13 extract displayed border­line in vitro cytotoxicity against the L1210 cell line (10 micrograms/ml). The extract was chromatographed on silica gel with EtOAc. The UV active fractions were com­bined and subjected to HPLC on ODS-3 and Partisil 10 to yield dihydrosiphonarin A (14, 0.21 mg/animal) and dihydro­siphonarin B (15, 0.05 mg/animal) and the degradation pro­ducts (~) and (24). Dihydrosiphonarin A (14), [uJo -24.90 (c 0.99, CH2C12), C2SH440a (high resolution FAB mass measurement of MH+, obsd 509.3136, for C2sH450a requires 509.3114), exhibited HO 23: R = Me 24: R = Et o 26 R 27 spectral data for a fully sUbstituted y-pyrone bearing 3 methyl groups: [IR 1646, 1595 cm-1 i 13C NMR 0179.8 s, 162 . 0 s , 161 . 4 s , 12 1 . 9 s , 118. 2 s i 1 H NMR 82 . 3 5 ( s , 3 H) , 1.98 (s 3H), 1.93 (s, 3H)], a saturated ketone (IR 1717 cm-1 i 13C NMR 0208.1 s), 2 ketal groups (13C NMR 0105.2 s, 102.7 s), a secondary ether (1 3C NMR 073.4 d) and 2 secondary hydroxyl groups (13C NMR 074.0 d and 73.9 d). Further, proton decoupling studies at 500 MHz defined the following spin systems: (25) [lH NMR 83.73 (dd, 1H, J= 10.5, 1 Hz) , 3.23 (dt, 1H, J=7, 3 Hz), 2.61 (q, 1H, J=7 HZ) , 2.35 (dq, 1H, J=10.5, 7 Hz) , 1.61 (ddq, 1H, J=14, 7 , 3 Hz) , 1.42 (ddq, 1H, J=7, 3 , 1 Hz) , 1.32 (ddq, 1H, J=l4, 7, 7 Hz) , 1.10 (d, 3H, J=7 Hz) , 0.88 (t, 3H, J=7 Hz) , 0.72 (d, 3H, J=7 Hz) ] i (£2) [lH NMR 0 3.68 (dd, 1H, J=3, 2.4 HZ), 2.04 (dq, 1H, J=7, 3 Hz), 1.93 (dq, 1H, J=7, 2 . 4 Hz), 1.26 (d, 3H, J=7 Hz), 1.25 (d, 3H, J=7 Hz)]; (27) [lH NMR 83.25 (q, 1H, J=7 Hz), 1.23 (d, 3H, J=7 Hz)]. All of these data were consistent with the proposed struc­ture (14) for dihydrosiphonarin A. Dr. John Faulkner, in a concurrent study with Sipho­naria zelandica had isolated the corresponding compounds, siphonarins A (28) and B (29), which were the 3-keto analogs of dihydrosiphonarins A (14) and B (15). An X-ray diffrac­tion experiment, performed by Dr. Jon Clardy with siphonarin A, defined the relative stereochemistry of the molecule. Because of the availability of these data, the selective 28 o 25 V OH 26 27 29 oxidation at C-3 could be helpful in determining relative stereochemistry of dihydrosiphonarins A and B. The oxi­dation of dihydrosiphonarin A with PCC (pyridinium chlo­rochromate) in refluxing benzene afforded siphonarin A (identical in all respects to an authentic sample) and the isomeric compound (30), thus establishing the rela­tive stereochemistry at 10 of 11 asymetric centers. The C-3 hydroxyl of (14) was assigned the Q* stereochemistry on the basis of coupling constants and conformational analy­sis. X-ray analysis showed that the side chain of siphona­rin A is positioned such that the terminal methyl group is in the shielding cone of the pyrone ring by hydrogen bonding between the C-13 hydroxyl group and the C-3 ketone. A similar solution conformation is confirmed by IH NMR data for both siphonarin A (28) and dihyrosiphonarin A (14). The terminal methyl groups in the side chain of (28, 00.94) and (14, 0 0.88) resonate at higher field than the comparable signal in denticulatin A (dl, 0 1.03) due to shielding by the pyrone ring. The adjacent methylene protons are diastereotopic indicating that the side chain exists in a preferred (rigid) conformation and the coupling between protons at C-4 and C-5 [J=1 Hz for both (28) and (14)] is that predicted from the X-ray conformation. There­fore, the sUbstituents at C-3 and C-4 must exist in a prior­ity antireflective (parf) relationship to obtain a 7 Hz coupling between the protons at C-3 and C-4. OH 28: R = Me 29: R '" Et 30 30 31 Dihydrosiphonarin B (15), [a]D -32.60 (c 0.33, CH2C12), C29H46 08 (high resolution FAB mass measurement of MH+, obsd 523.3284, C29H470a requires 523.3271) indicating that (15) was a methylene homolog of (14). In support of this view was the 1H NMR spectrum of (15) which was nearly super­imposable on that of (14) except that the methyl signal at 02.35 in (14) was replaced by ethyl signals at 1.12 (t, 3H, J=7 Hz) and 2.78 (q, 2H, J=7 Hz), indicating the comparable relationship between (14) and (15) as in siphona­rin A (28) and siphonarin B (29). The two minor metabolites (23, C17H2406) and (24, C18H2606) were also homologs. The 1H NMR spectra of (23) and (24) contained signals assignable to a 2,3,5-trimethyl­y- pyrone [lH NMR 6 2.26 (s, 3H), 2.14 (s, 3H), 1.95 (s, 3H)] and a 2-ethyl-3,5-dimethyl-y-pyrone [lH NMR 0 2.60 (q, 2H, J=7 Hz), 2.16 (s, 3H), 1.95 (s, 3H), 1.17 (t, 3H, J=7 Hz)] ,respectively. The remaining signals in the 1H NMR spectra of (23) and (Ai) were identical and assigned to the spin system (32): [lH NMR 0 4.15 (q, 1H, J=7 Hz), 3 . 63 (dd, 1H, J=2. 5, 2. 5 Hz), 2. 87 (dq, IH, J=7, 2. 5 Hz), 2.74 (dq, 1H, J=7, 2.5 Hz), 1.38 (d, 3R, J=7 Hz), 0.97 (d, 3H, J=7 Hz), 0.93 (d, 3H, J=7 Hz)]. supporting these assignments were IR bands at 3600, 1723 and 1700 cm-1 attributable to the alcohol, ketone and acid functionali­ties, respectively. Careful reexamination of the crude extract of Siphonaria normalis indicated that (~) and 32 32 33 (~) were artifacts of silica gel chromatography using ethyl acetate. It is believed that the ketal rings of (14) and (15) undergo acid catalyzed ring opening to give -diketones which can then undergo a retro-Claisen reaction to generate (~) and (24) (Figure 3). In support of this theory, acid catalyzed hydrolysis of dihydrosiphonarin A (14) gave the acid (~) in good yield. Biology of the Onchids Onchidella is an opisthobranch mollusc. As with sipho­naria, its taxonomy has been a bit of a problem. Because the stomach of the onchid closely resembles that of the pulmonates, Onchidel1a is perhaps better thought of as an opisthobranch, whose development of a lung parallels that of the Pulmonata. Onchide11a has a very small posterior mantle cavity, which receives the anus and the excretory aperture. The mantle cavity serves mainly as an excretory channel. In marked contrast to the siphonariids, the mantle cavity contains no ctenidium or secondary gill. The development of an entirely new pulmonary chamber allows this opistho­branch to breathe air. In the anterior, close to the head lies the female genital aperture. As with all the opistho­branchs, they are hermaphrodites and there is usually a reciprocal fertilization between copulating pairs. Onchids occur in the high intertidal zone on rocky ,I HO HO OH ,f OH o Figure 3. Pathway for generation of the artifact 34 o 35 shores, where they feed on young algae and encrusting di­atoms. Debris (sand, pieces of sponge, etc.) carries the microorganisms into the mouth. This material passes into the gut by muscular action and the stomach contents are digested during high tide, when the onchid is inactive. Like the siphonariids, Onchidella dominate the high inter­tidal zone when the tide is low. At this time, they can be found grazing on the available food sources. They home into a rock crevice, where they live with other onchids, when the tide returns (Figure 4).21 The Chemistry of Onchidium verruculatum Onchidium verruculatum collected at Portlock, Oahu, Hawaii were stored in acetone for 24 h. The acetone fil­trate was evaporated and the residue partitioned between ether and H20 to give 0.53 g of an organic oil. Chromato­graphy of the oil on Sephadex LH-20 (CH2Cl2/hexane, 4:1) and Bio-sil A (EtOAc) gave an inseparable mixture of UV absorbing esters (lR of the ester carbonyl 1735 em-I). Saponification of the mixed esters, however, yielded a single triol, ilikonapyrone (13). llikonapyrone (13, C32H4S07; HR ElMS obsd 544.335, cale 544.340) exhibited spectral data corresponding to 2 fully substituted ~pyrone rings bearing methyl groups at the e carbons [lR 1660, 1610 em-1 ; UVmax 260 nm (E 12700); 13c NMR 0180.1 s, 180.0 s, 165.5 s, 165.3 s, 164.7 36 Anterior edge of mantle l~rO,(:;"Il-Eye Mouth --------.."t!.I.--.(!:r Foot--------------~~- ~~-~-......---Anus Female Aperture Pneumostome Figure 4. Structure of a typical Onchidella species 37 33 HO OH 34 ~ OH 35 ~ 36 38 s, 164.6 s, 119.3 s, 119.2 s, 118.0 s, 117.3 s, 9.7 q, 9.6 q (3C) i 1H NMR ~ 1.93 (S, 3H), 1.92 (s, 3H), 1.91 (s, 3H), 1.89 (s, 3H)] and the isolated spin systems (11)-(36): (33) [lH NMR 05.60 (dq, 1H, J=9, 1 Hz), 3.90 (dq, 1H, J=9, 7 Hz), 1.70 (d, 3H, J=l Hz), 1.2 (d, 3H, J=7 Hz)], (ll) [lH NMR 0 4.20 (dd, 1H, J=8, 1 Hz), 4.04 (d, 1H, J=7 Hz), 3.13 (dq, 1H, J=8, 7 Hz), 1.88 (m, 1H), 1.10 (d, 3H, J=7 Hz), o. 8 7 ( d , 3 H , J = 7 Hz)], ( 1.2) [ 1 H NMR <5 3 . 60 (m , IH), 2.96 (dq, 1H, J=7, 7 Hz), 1.48 (ddq, 1H, J=14, 7, 3 Hz), 1.30 (ddq, 1H, J=14, 7, 7 Hz), 1.18 (d, 3H, J=7 Hz), 0.89 (t, 3H, J=7 Hz)] and (36) [lH NMR 02.62 (q, 2H, J=7 Hz), 1.20 (t, 3H, J=7 Hz)]. The electron-impact mass spectrum of ilikonapyrone was equally informative (Figure 5). Abun­dant ions at m/z 486 and 180, resulting from consecutive McLafferty rearrangements, indicated that spin systems (34) and (35) were attached to the same pyrone ring. Oxida­tion of the allylic alcohol with Mn02 in CH2Cl2 yielded the a,a-unsaturated ketone (37) [IR 1675, 1660, 1600 cm-1 ; UV 242 nm (E 13800), 257 (E 13300); 1H NMR 0 1.76 (d, 3H, J=l HZ), 6.43 (dq, 1H, J=9, 1 Hz)] indicating that spin systems (11) and (34) were also connected. oxidative cleav­age of the double bond in ilikonapyrone with the Lemieux-von Rudloff reagent (Os04/NaI04), followed by a reductive work­up with NaBH4' gave the monopyrones (~) and (39). Com­pounds (~) and (39) both exhibited spectral data for single pyrone rings and the side chains were determined by proton o f I HO 1 m/z 486 m/z 180 • I o o Figure 5. Mass spectral fragmentation pattern of ilikonapyrone 39 I I HO o I I HO I I HO 37 38 o 39 ,I OH 40 o decoupling studies: 41 (38) [lR 1660, 1610 cm- l ; UVmax 258 nm (€ 6000); 1 H NMR 0 3. 78 (m , 2 H), 3. 2 3 (m , 1 H) , 2. 61 ( q , 2H, J=7 Hz), 1.96 (s, 3H), 1.92 (s, 3H), 1.20 (m, 6H)] and (39) [lR 1660, 1610 cm-1 ; UVmax 260 (€ 7200); 1H NMR 04.12 (dd, 1H, J=9, 3 Hz), 3.68 (m, 3H), 3.14 (dq, 1H, J=9, 7 Hz), 3.03 (dq, 1H, J=7, 7 Hz), 1.96 (s, 3H), 1.95 (s, 3H), 1.94 (m, 1H), 1.60-1.44 (m, 2H), 1.26 (d, 3H, J=7 HZ), 1.15 (d, 3H, J=7 Hz), 0.99 (t, 3H, J=7 Hz), 0.95 (d, 3H, J=7 HZ)]. The trans geometry of the double bond was assigned by a 13C-1H correlated 2D NMR experiment, which assigned the proton olefinic methyl resonance at 1.70 ppm to a carbon quartet at 12.2 ppm. 27 The relative stereochemistry of the 7 asymmetric cen-ters of ilikonapyrone was determined by an X-ray analysis of the acetonide derivative. The X-ray solution did not distinguish between the enantiomeric pair. The 3R* , 4R* , 10S*, 11S*, 12S*, 13S*, 16S* enantiomer depicted is an arbitary choice. The Chemistry of Peronia peronii Peronia peronii were collected in Guam and the animals were freeze dried. The freeze dried animals were subjected to cold extraction with CHC13' The CHC13 extract showed very good cytotoxicity against the L1210 cell line (lC50 0.5 micrograms/ml). The L1210 screen was used to guide 42 the fractionation of the extract on silica gel with EtOAc. Active fractions were further purified by HPLC (Partisil la, EtOAc) to yield several fractions which exhibited fur­ther enhancement of activity. Two of these fractions, when subjected to RP-18 HPLC yielded the 4 peroxysterols (40) through (43) (structures assigned by HR ElMS, I H- NMR spectra and comparison to known compounds when applicable). Interestingly compounds (41) and (42) were previously iso­lated by Schmitz and Djerassi28 from several sponges and no cytotoxicity data were reported. sesin and Ireland have also isolated several of these sterol peroxides from a Dolabella species and an unidentified tunicate species. 29 Because on recrystallization from MeOH/H20 the activity diminished, these sterols were not pursued further. One fraction, which was devoid of steroids, exhibited good activity which was enhanced on further purification. This fraction contained a family of esters, all of which had the same UV chromophore (UVmax 260 nm) reminiscent of ili­konapyrone. Further fractionation increased the activity, but similar to the situation with ilikonapyrone, these esters could not be separated. Saponification, however, led to the isolation of 2 triols that had formulae isomeric with ilikonapyrone, peroniatriol I (11) and II (12). Peroniatriol I and II exhibited spectral data very similar to ilikonapyrone (IKP) as illustrated by the follow­ing comparison of their 13C_1H correlated 20 spectral data 43 40 41 R 42: R = CaH15 43: R = C1 0 H2 1 44 (Table 3). Not all the carbons and protons could be detect­ed in this experiment; however, 13C NMR data suggested the presence of 2 fully substituted y-pyrone rings: peronia­triol I [13C NMR 0179.8 s (2C), 164.8 s (2C), 164.5 s (2C), 119.7 s (2C), 118.6 s (2C») and peroniatriol II r13C NMR 0180.1 s (2C), 165.7 s, 165.1 s, 164.9 s, 164.5 s, 120.1 S, 118.5 S, 117.4 s, 116.1 s). The double bond in peroniatriol I and II was found to possess the trans geometry as defined by the correlated spectra which showed that the olefinic methyl resonances at 1.70 ppm and 1.63 ppm correlated to the carbon resonances at 11.9 ppm and 13.9 ppm, respectively.27 The location of the double bond in peroniatriol I and II was determined in analogous fashion with ilikonapyrone by oxidative clea-vage followed by reductive work-up (Figure 6). Oxidation Table 3 Chemical Shift Comparison C f IKP C i PERONIATRIOL I PERONIATRIOL II IH-13C IH-13C lH-13c 15 5.60 127.1 11 5.59 127.1 5.86 125.4 11 4.20 71.9 15 4.14 72.2 3.72 73.0 13 4.04 79.6 13 4.06 79.5 4.05 80.1 16 3.90 34.8 10 3.90 34.5 3.90 34.5 3 3.60 75.9 3 3.75 75.2 3.57 75.2 10 3.13 39.5 16 3.15 39.3 3.03 39.8 4 2.96 41.2 4 2.90 41. 5 2.84 42.0 22 2.62 25.1 22 2.56 24.6 2.51/2.28 24.7 12 1.88 37.2 14 1.84 36.3 1. 87 34.3 27 1. 70 12.2 28 1. 70 11. 9 1. 63 13.9 , t HO • I HO HO•• ,I HO ,I HO • I HO o o o o o o , I OH 17ikonapyrone f I HO Peroniatriol I Peroniatriol II o o o Figure 6. Oxidative cleavage of IKP, PT I and PT II 45 o o 46 was affected by ozonolysis rather than using the Lemieux­von Rudloff reagent (Os04' Nal04) because of the inability to obtain the needed reagents. A CH2C12 solution that was saturated with 03 was added to each compound in CH2Cl2 at -70 oC. The reaction mixtures were allowed to warm to room temperature and solvent was removed in vacuo to yield white solids. The solid mixtures were then subjected to HPLC on Partisil 10 with EtOAc. Because chromatography failed to affect purification, solvent was removed in vacuo and the white solid mixtures were dissolved in MeOH and NaBH4 added to each. Removal of solvent and HPLC with the same system as above led to the isolation of pyrones (44) and (46) from PT I and pyrones (45) and (46) from PT II. The identical pyrone (46) was isolated from both PT I and II ozonolysis. The ElMS exhibited a parent ion at m/z 310 and a prominent fragment at m/z 250 corresponding to loss of acetic acid from the parent. The 500 MHz proton NMR exhibited signals for all the protons present in the molecule. Proton decoupling studies defined the following isolated spin systems: [IH NMR 02.63 (q, 2H, J=7.6 Hz), 1.23 (t, 3H, J=7.6 Hz)] and [IH NMR 04.27 (m,IH), 4.00 (m, IH), 3.87 (m, IH), 3.14 (dq, 1H, J=8.8, 7 Hz), 2.10 (m, IH), 1.15 (d, 3H, J=7 Hz), 0.98 (d, 3H, J=7 Hz)]. In addition to these spin systems, the 1H NMR spectrum exhibited signals at 02.10 (s, 3H) for an acetate methyl, I I HO ,I HO I I OH 47 o 44 o 45 o 46 48 2.00 and 1.96 for the 2 pyrones methyls. All of these data led us to structure (46). Pyrone (44) isolated from ozonolysis of PT I exhibited a parent ion at m/z 268 in the ElMS. The 1H NMR spectrum exhibited singlets at 62.00 and 1.99 for the two pyrone methyls. A 1-methyl-2-hydroxybutyl side chain [lH NMR <53 .75 (m, 1H, J=7, 7, 3 Hz), 3. 01 (dq, 1H, J=7, 7 Hz), 1.54 (m, 1H, J=10.5, 7.4, 3 Hz), 1.40 (m, 1H, J=10.5, 7.4, 7 Hz), 1.30 (d, 3H, J=7 Hz), 1. 00 (t, 3H, J=7. 4 Hz)] and a 1-methyl-2-hydroxyethyl side chain [1H NMR 03.81 (m, 2H), 3. 23 (m, 1H, J=7, 3. 5, 3. 5 Hz), 1.23 (d, 3H, J=7 Hz)] were defined by proton decoupling studies at 500 MHz. From these data, structure (44) was assigned for the other pyrone from PT I ozonolysis. The remaining pyrone from PT II ozonolysis was assigned structure (45) on the basis of the following data: ElMS: M+ 268; 1H NMR: singlets at 1.98 and 1.97 ppm for the pyrone methyls and the same spin systems as in (44): [lH NMR 03.73 (m, 1H, J=7, 7, 3 Hz), 3.05 (dq, 1H, J=7, 7 Hz), 1.61 (m, 1H, J=14, 7.4, 3 Hz), 1.38 (m, 1H, J=14, 7.4, 7 Hz), 1. 31 (d, 3H, J=7 Hz), 1. 02 (t, 3H, J=7. 4 Hz)] and [lH NMR 03.87 (m, 1H, J=14, 3.5 Hz), 3.80 (m, 1H, J=14, 10.5 Hz), 3.24 (m, 1H, J=10.5, 7, 3.5 Hz), 1.21 (d, 3H, J=7 Hz)]. pyrones (44) and (45) were shown to be epimeric as illustrated by proton decoupling studies. If one locks 49 the l-methyl-2-hydroxyethyl side chain into a 6-membered H-bonded ring to the pyrone oxygen the coupling is readily explained on the basis of the to the pyrone proton being axial in one case and equatorial in the other. It should be mentioned that the monopyrone (38) from cleavage of the double bond in IKP also contains this 1-methyl-2- hydroxyethyl side chain and the stereochemistry is known. Both monopyrones from IKP and PT I (38 and 44, respectively) exhibited equal coupling constants in this side chain, suggesting that the same stereochemistry was present. The methine proton exhibits equal coupling (3.5 Hz) to both methylene protons, suggesting a dihedral angle of 60 0 to each proton. This requires the methine proton to be in an equatorial position (Figure 7). Pyrone (45) from PT II exhibits coupling of 10.5 and 3.5 Hz, suggesting that the methine proton is in an axial position (Figure 8). The consequences of these coupling constants are that the asymmetric center between the pyrone and olefin is the same in IKP and PT I and the opposite in PT II. The spin system containing the 1,3-diol was found to be the same in IKP and PT I, while the allylic hydroxyl in PT II was found to possess the opposite stereochemistry. This stereochemical result is based on proton decoupling studies conducted with IKP, PT I and PT II at 500 MHz and verified by isolation of the identical monopyrone (46) from ozonolysis of both PT I and II. Spin system (47) J 12 3.5 Hz J13 3.5 Hz Figure 7. IKP, PT I stereochemistry 50 51 J 10.5 Hz 12 J13 3.5 Hz H o Figure 8. PT II stereochemistry I I HO OH 47 48 52 53 exhibited coupling constants, JII,12= 1 Hz, J12,13= 7 Hz in IKP which suggests that it adopts a 6-membered H-bonded ring (48) with H12 and H13 in an axial arrangement and HII equatorial. The same coupling constants are observed in PT I (J14,15= 1 Hz and J13,14= 7 Hz), suggesting it has the same relative configuration as IKP. Coupling con­stants in a 6-membered ring are indicative of the interac-tion being observed. A 7 Hz coupling constant requires an axial-axial interaction, while a 2 Hz coupling constant requires an axial-equatorial interaction. PT II, on the other hand, exhibits coupling constants (J14,15= 2 Hz and J13,14= 1 Hz) (49) which requires 2 axial-equatorial inter­actions (50). This means that the allylic alcohol exhibits a stereochemical difference in these molecules. This was verified by ozonolysis of PT I and II, because pyrone (46), isolated from both, which fortuitously no longer contained the allylic alcohol, was identical from both PT I and II. Isolation of this pyrone (46), as an acetate ester, can be rationalized by examination of the reaction mechanism for the ozonolysis (Figure 9). Initial attack of the double bond results in the first intermediate shown which rear­ranges to the molozonide. Decomposition of the molozonide in a normal fashion leads to the epimeric pyrones plus a new intermediate which can rearrange to the hydroperoxide epoxide, shown. Attack of hydride, from NaBH4 during work­up, results in cleavage of the C-C bond rather than the 54 ~ I I I I HO OH 49 50 ss "Other Monopyrone" o Figure 9. 03 Mechanism scheme 56 c-o bond, resulting in loss of the a11y1ic hydroxyl center and isolation of the pyrone as its acetate ester. The driving force for the cleavage of the C-C bond appears to be expulsion of hydroxide ion analogous to the Dakin and Baeyer-Vi11iger reactions. The stereochemistry of the 1-methyl-2-hydroxybuty1 side chain was found to be the same in all 3 molecules on the basis of decoup1ing studies at 500 MHz, which showed that the coupling constants were all equivalent. Therefore, structures (11) and (12) have been arrived at for PT I and II, respectively_ INTRODUCTION TO TUNICATES Tunicates (ascidians, sea squirts) encompass a hetero­genous group of animals, which possess a myriad of families, genera and species (Phylum Chordata; Subphylum Urochordata; Class Ascidacea). These sedentary marine organisms are found throughout the world in coastal waters. There are 2 kinds of sessile ascidians, the simple and the compound or colonial ascidians. The simple ascidians are individual animals whereas the compound ascidians consist of colonies of individuals produced by budding from a parent individual. These colonies can be of brillant color and/or massive proportions (e.g., Amaroucium and Fragarium), while others form thin encrusting expansions on surfaces of marine shells and plants (e.g., Botryllus and Leptoclinum). Some compound ascidians (e.g., Clavelina and Perophora) are solely con­nected together by a common creeping stolon from which new buds are produced. The structure of a simple ascidian (Figure 10) is representative of a compound one as well. simple ascidians have been likened to a leather bottle with 2 spouts, occurr­ing in the form of 2 funnellike projections. These 2 spouts represent their incurrent or buccal and excurrent or atrial apertures. Atrial siphon Anus Genital -----++-'1, duct S tornach ----++-M;:--"7 ~- .rw...."...,Im _______ Bucca 1 siphon --l~r------ Mant 1 e ~L-..m--H---- Intestine Wll..LtU-H-----Gonad Figure 10. Structure of a typical tunicate species 58 59 The tunic or test, which surrounds the body, is the only other notable external feature of ascidians. The tunic varies from species to species. It may be of a carti­laginous, coriaceous, fibrous or membraneous consistency, is usually opaque, but sometimes appears hyaline and trans­parent (e.g., Corella and Salpa). The outer surface may be smooth, wrinkled or rough, capillated, papillated or mammillated. The tunic is largely composed of cellulose. Underneath the tunic is the inner musclar mantle which surrounds the cavity containing the visceral organs. The visceral anatomy includes a branchial sac which is attached to the stomach via the oesophagus. The stomach leads to intestines which terminates at the rectum. The rectum opens by the anus into the atrial cavity. The excre­ment is carried to the exterior by a constant outward flow of water through the cloacal aperture. The ascidians are hermaphrodites and the reproductive glands lie between the loops of the intestine. Despite their hermaphroditism not all are self-fertilising. Ascidians bear a central nervous system and a circulatory system with a heart and blood sinuses. They also have renal organs which consist of bladder-like vesicles. Products of excretion are depos­ited inside the vesicles because of a lack of an excretory duct. 31 60 Review of Ascidian Chemistry The current interest in tunicates is a result of anti­tumor screening, both in vitro32 (L1210) and in vivo33 (P3BB), which demonstrates that approximately 13% of the tunicate species studied produce cytotoxic metabolites. In comparison, only 3% of terrestrial plant species have been found to possess cytotoxic metabolites. 32 Despite the abundance of cytotoxic constituents, until only very recently few chemical studies of tunicates have been under­taken. Cytotoxic constituents have been encountered in three families, Polyclinidae, Styelidae and Didemnidae. The majority of compounds thus isolated have been amino acid derivatives. Nearly all the compounds isolated have been found to exhibit biological activity. The spectrum of biological activities includes antibiotic, antineoplastic and antiviral activity. Of the amino acid derivatives isolated, the peptides are the largest group. These peptides are cyclic and con­tain both normal peptide linkages as well as ester linkages (i.e., depsipeptides). There are only 3 examples of cyclic depsipeptides, the didemnins A (51), B (52) and C (22) isolated from a Caribbean tunicate of the family Didem­nidae (species of the genus Trididemnum) by Rinehart. 34 The didemnins spectrum of activities includes inhibition of the growth of Herpes simplex (HS), types 1 and 2 (DNA 61 .ll: R = H 52: R = H 53: R = COCH(OH) CH3 62 are highly cytotoxic to L1210 leukemia cells and protect mice against P388 leukemia (T/C values up to 199) and B16 melanoma (T/C values up to 160).33 Interesting structural features of the didemnins are the presence of hydroxyiso­valerylpropionate, a new structural unit for depsipeptides and the appearance of a new stereoisomer of the uncommon amino acid statine. The cyclic peptides are the larger of the 2 groups of peptides and contain 9 examples. A unique structural feature of this group is that they all contain thiazole amino acids, which are believed to arise biosynthetically from the dehydrative cyclization of a cysteinyl peptide such that C-2 of the thiazole originates from the carbonyl of an amino acid on the N-terminal side of cysteine. 36 The first report of a cyclic peptide occurred in 1980,37 when Ireland and Scheuer reported the isolation and gross structures of ulicyclamide (54) and ulithiacy­clamide (55), lipophilic peptides from Lissoclinum patella. Following this initial report, Ireland in 198238 reported the biological activity of ulicyclamide, ulithiacyclamide, and patellamides A (56), B (57) and C (58), 3 additional metabolites from Lissoclinum patella. All 5 of the cyclic peptides exhibited good in vitro activity against the L12l0 leukemia cell line with IC50 values ranging from 0.35 to 7.2 micrograms/mI. Ireland et al. at this time also re-ported on the absolute configurations for all the amino 63 54 o o I Y o 55 64 56 ,..-Ph I 57 65 acids except the thiazoles by chiral GC of N-trifluoro­acetyl methyl ester (TFA-ME) derivatives. Shortly there­after, Biskupiak and Ireland reported a general method for determining the absolute configuration of 2-(1'-amino­alkyl) thiazole-4-carboxylic acids, based on the reaction of thiazoles with singlet oxygen (102) .36 More recent­ly, Ireland in 1983 reported the isolation, structure deter­mination and biological activity of 3 additional cyclic peptides, the lissoclinamides 1 (59), 2 (60) and 3 (61) from Lissoclinum patella, which exhibited borderline cyto­toxicity (lCSO> 10 micrograms/ml) against the L1210 cell line. 39 Hamamoto in 1983,40 aided by cell culture assay techni­ques, isolated ulithiacyclamide and the new cyclic peptide ascidiacyclamide (62) from an unidentified tunicate species collected at Rodda Reef, Queensland, Australia. Ascidia­cyclamide, which has a symmetric structure, exhibited good in vitro activity against PV1 cultured cells (polyoma virus) . The next group of amino acid derivatives consists of modified amino acid residues where 1 or 2 amino acids are utilized along with some other precursor. The largest group of compounds originates from the colonial tunicate Eudistoma olivaceum41 ,42 collected in the Caribbean. Two families of compounds derived from the oxathiazepine ring system 63 or the I-pyrrolinyl-6-carboline ring system 64 o~ , I H H H N 58 59 66 r-Ph I 60: 61: 67 68 have been isolated, both of which had not been reported. Compounds based on the condensed oxathiazepine ring system 63 are the most active antiviral agents (Herpes simplex virus, type 1, HSV-1) and will be discussed first. Biosynthetically, the condensed oxathiazepine ring system can be thought of as arising from the amino acids tryptophan (N-2 thru C-9a) and cysteine (C-1, C-10, C-11 and S-12). There are 4 sites of sUbstitution in this system, with 3 sites on the aromatic ring (C-S, C-6 and C-7) and 1 site on the amine nitrogen located at C-10 of the condensed oxathiazepine ring. SUbstituents encountered in these positions include bromine, hydroxyl, acetyl and acetoxy. The 4 most potent antiviral agents are eUdistomins C (65), E (66), K (67) and L (68). The eUdistomins based on the 1-pyrrolinyl-S-carboline ring system 64 show borderline activity against HSV-l, Saccharomyces cerevisiae (yeast) and Bacillus subtilis (gram-positive bacterium). The I-pyrrolinyl-S-carbolines are thought to be biosynthetically derived from trypthophan and glutamate. There are 5 sites of sUbstitution in this system, 3 sites on the aromatic carbons (C-S, C-6 and C-7), 1 site on the pyrrole nitrogen and 1 site at C-1 of the pyridine ring. The sUbstituents are the same as encountered in the condensed oxathiazepine ring system. The next example is the compound dendrodoine (69, 5-(3-N-dimethylamino)-1,2,4-thiadiazolyl-3-indanylmethanone, 69 R R R' R" RJ I I C (65): H OH Br H E (66): Br OH H H K (67): H H Br H L (68): H Br H H 63 64 70 which was isolated from the tunicate Dendrodoa grossularia (family Styelidae) found off the coast of Northern Bre­tagne, France. 43 Dendrodoine can be thought of as also arising biosynthetically from the amino acid tryptophan with further modification. An interesting structural fea­ture of this molecule is the l,2,4-thiadiazole moiety. Dendrodoine exhibited very potent in vitro L1210 cytotoxi­city. Because of its activity, dendrodoine has been the subject of synthetic interest. The synthesis of dendrodoine has recently been described. 44 Aplidiasphingosine (70) is an example of a metabolite from a tunicate, which is biosynthetically derived from an amino acid (serine with loss of its carboxyl) and a diterpenic acid. 45 Aplidiasphingosine was isolated as the active constituent of an orange-flecked compound tuni­cate (Aplidium species) collected from the Gulf of Califor­nia. The original crude extract possessed antibiotic, antineoplastic and antiviral activity. Pure aplidiasphingo­sine was shown to possess a myriad of activities including: antibiotic activity (against Bacillus subtilis, Klebsiella pneumonia, Bacteroides fragilis, Mycobacterium avium, Sar­cina lutea, Clostridium perfringens, Candida albicans (yeast) and Pencillium oxalicum in vitro) and cytotoxicity (against KB and L1210 tumor cells cultured in vitro). The antiviral activity originally possessed by the crude extract was not associated with aplidiasphingosine. 71 o 69 OH 72 The polyandrocarpidines are another example of a modi­fied amino acid nucleus. The polyandrocarpidines may be thought of as being biosynthetically derived from arginine with modifications. Andersen and Faulkner first reported that a red encrusting colonial tunicate (Polyandrocarpa species) possessed antibiotic activity.46 Cheng and Rine­hart in 1978 proposed structures for the 2 major bioactive components from the same tunicate and named them the poly­androcarpidines I (71) and II (72). In 1982, Carte' and Faulkner reported revised structures for the polyandro­carpidines; Rinehart in 1983 confirmed these new struc­tures. 47 ,48 Polyandrocarpidines I and II were originally found to be a 9:1 mixture; however, Faulkner subsequently showed that 71 and 72 were a mixture of 2 isomers each. Thus from what was thought to be polyandrocarpidine I come polyandrocarpidines A (73) and B (74) and from polyandrocar­pidine II come polyandrocarpidines C (75) and D (76). Thus, the polyandrocarpidines are N-alkyl-y-alkylidene­y- lactams rather than amides of a cyclopropenyl acid. Not only do these compounds possess antibiotic activity, they have also been shown to be cytotoxic to L12l0 and KB cells in vitro and also possess slight antiviral activity (HSV-l) as well. The remaining 4 compounds to be discussed under the heading of modified amino acid derivatives are examples of metabolism products of amino acids. Halocynine (77) H ,.. N HN~NANH2 H 71: n = 5 72: n = 4 73: n = 5 74: n = 4 75: n = 5 76: n = 4 o 73 74 is a betaine isolated from muscle of the tunicate Halo­cynthia roretzi. 49 Although halocynine is the first example of a 2-hydroxylated betaine, the 2-oxo derivative is a known biosynthetic intermediate in the pathway from lysine to betaines. 50 Despite being found in large amounts in the muscle of the tunicate, no biological role for halocy­nine is known at this time. From the marine ascidian Didemnum ternatanum, N,N'­diphenethylurea (7S) was isolated as the sole secondary metabolite. 51 N,N'-diphenethylurea can be assumed to be a product of phenylalanine metabolism. That N,N'-diphen­ethyl urea is not an artifact of the work-up procedure is assured, because the frozen tunicate was extracted by 3 different methods, which all gave the same results. The only biological activity associated with the urea is that it is a weak depressant. sesin and Ireland isolated 2 similar iodinated phenethylamine derivatives from an uniden­tified Didemnum species. 52 3,5-Diiodo-4-methoxyphenethyl­amine (79) and the corresponding urea, N,N'-3,5- diiodo-4- methoxyphenethylurea (SO) were isolated from a tunicate collected at Cocos Lagoon, Guam. These 2 secondary meta­bolites appear to be metabolism products of the amino acid tyrosine. In contrast to Didemnum ternatanum, which is known to harbor procaryotic algal symbionts, this particular Didemnum species is devoid of algal symbionts. This finding means that this tunicate may be capable of de novo synthesis 75 +~- Me/4 77 78 OMe 80 76 of amino acids, which was previously assumed possible only by the algal symbionts. 3,5-Diiodo-4-methoxyphenethyl­amine showed in vitro activity against the yeast Candida albicans and was also weakly cytotoxic (L1210). I will now focus my attention on secondary metabolites from ascidians, namely quinones, steroids and terpenes, which can be biosynthesized from acetyl-CoA. Except for the 2 quinone derivatives, these metabolites are complete­ly devoid of biological activity. Geranyl hydroquinone (81) was isolated by Fenical from an Aplidium §R. collected in the Gulf of California. 53 The related prenylhydroquinone (82) and corresponding chromenol (83) were isolated by Howard from Aplidium californicum collected near the San Francisco Bay.54 Both hydroquinones exhibited good in vivo activity against P-388 in mice. There are 2 variations on the steroids which have been reported to date. The first sterol is 24-hydroperoxy- 24-vinylcholesterol (84), which has been isolated from 2 tunicates, Phallusia mamillata and Ciona intestinalis by Guyot and Davoust. 55 From these same 2 tunicates, Guyot and Durgeat also isolated 4 different 9(11)-unsaturated sterol peroxides, (85), (86), (87) and (88) .56 As previously mentioned, these compounds did not exhibit any biological activity. The last 3 compounds to be discussed under this heading are 3 new carotenoids isolated from the marine tunicates 77 OH 81 OH 82 83 84 85: R = H 86: R = CH2 22 87: R = Me, !J. 88: R = Et 78 R 79 Halocynthia roretzi and Sidnyum argus. Two carotenoids were isolated from Halocynthia roretzi by Matsuno et al., halocynthiaxanthin57 (89) (5,6-epoxy-3,3'-dihydroxy-7',8'­didehydro- 5,6,7,8-tetrahydro-S,8-caroten-8-one) and my til­oxanthinone58 (90) (3,8'-dihydroxy-7,8-didehydro-8,K-caro­tene- 3',6'-dione). Belaud and Guyot isolated sidnyaxanthin (91) from the bright orange colonial tunicate, Sidnyum argus. 59 A structural- feature of all 3 of these carote­noids is that they contain a carbon-carbon triple bond. The Chemistry of Lissoclinum patella A study of didemnid tunicates from Palau of the Western Caroline Islands has been initiated. The didemnids tuni­cates were chosen because it is known they harbor unicell­ular prokaryotic algae. 60 ,61 It is believed that these algal symbionts are capable of nitrogen fixation. Because of this ability, these tunicates are likely candidates for possessing novel nitrogenous metabolites, which may exhibit antineoplastic activity as is the case with some terrestrial alkaloids. 62 Lissoclinum patella, a dark green compound didemnid tunicate, was collected from Eil Malk Island and Iwayama Bay, Korror Island. The crude extract exhibited in vitro activity against the CEM, L1210, HELA and P388 cell lines. The extract also exhibited in vivo activity against the P388 cell line in mouse. From these multiple collections, 80 OH 0 .:pa 81 a total of 8 cytotoxic cyclic peptides has been isolated. Samples were collected in an average depth of 2 m by snor­keling. Typically, the frozen animal was lyophilized and ground to a powder. The powdered tissue was extracted exhaustively in a Soxhlet apparatus with petroleum ether, CCl4 and CHCI3. Because tIc (thin-layer chromatography) revealed overlapping fractions in the organic layers, they were combined and chromatographed on silica gel. Three peptide fractions were eluted with EtOAc. A fourth peptide fraction was eluted with EtOAc/MeOH (95:5). Each band was subsequently resolved by high-performance liquid chroma­tography (RP-18i 8:2 MeOH/H20) into 2 components. The first peptide band (150 mg) was a 7:3 mixture of ulithiacy­clamide (55) and patellamide A (56). The second band (70 mg) contained patellamide B (57) and lissoclinamide 1 (59) in an 8:2 ratio. Band three (230 mg) contained ulicyclamide (54) and patellamide C (58) in a 9:1 ratio. The fourth and most polar band (624 mg) contained lissoclinamides 2 (60) and 3 (61) in a 2:1 ratio. Previously, workers in this laboratory identified the structures of 5 of these cyclic peptides. 37 ,38 Because the structure elucidation of these compounds is germane to the present discussion, they will be summarized. My involvement with this project was twofold: 1) isolation of 3 additional peptides, the lissoclinamides, which were sequenced using FAB MS. As a result of this work, the 82 structure of a peptide was revised; 2)the development of a method for determining the chirality of thiazole amino acids. That I was dealing with lipophilic cyclic peptides was deduced from the IR spectra. The IR spectra of these peptides were transparent in the OH and CO OR regions but exhibited intense absorptions at 3300, 1670 and 1650 cm-l , indicative of peptide linkages. Ulicyclamide (54) has the molecular formula C33H39N705- 52; obsd 677.2446, calc 677.2439. The EI mass spectrum contained peaks at m/z 620 (M+-C4H9) and 586 (M+-C7H7), corresponding to loss of isoleucine and phenylalanine side chains, respectively. All 33 carbons were observed in the 13c NMR spectrum of 54. Four singlet resonances between 0171.9 and 170.5 were indicative of a tetrapeptide. 1H NMR signals at o 8.08 (s) and 8.03 (s) along with sp2 carbon resonances at 161.1 (s), 160.5 (s), 151.4 (s), 148.9 (s), 124.3 (d) and 123.8 (d) were assignable to 2 thiazole rings. A sin­glet at 07.30 (5 H) and 2 methylene protons at 3.25 and 2.93 in the 1H NMR spectrum, along with carbon signals at 136.8 (s), 130.6 (d, 2 C), 129.5 (d, 2 C), 128.2 (d) and 41.8 (t), were indicative of phenylalanine. 1H and 13C signals for proline were also readily assignable: [13C signals: 857.5 Cd), 54.4 (t), 29.8 (t) and 25.9 (t); IH signals: 04.52 (t, J=8 Hz), 3.25 (m), 2.1 (m) and 1.9 (m)]. 83 In addition to the above assignments, signals, were observed in the 220-MHz IH NMR spectrum for 3 isolated spin systems: ( 9 2 ) [0 9. 06 ( d , 1 H, J=5 Hz), 5 . 38 ( dq, 1 H, J=7, 5 Hz), 1. 71 (d, 3 H, J=7 Hz)] i (93 ) [8 7 .85 (d, 1 H, J=10 Hz), 5.26 (dd, 1 H, J=10, 7 Hz), 1.30 (m, 1 H), 1.20 (m, 2 H), 0.85 (t, 3H,J=7 Hz), 0.73 (d, 3 H, J=7 Hz) and (94) [8 4.82 (dq, 1 H, J=7, 4 Hz), 4.26 (d, 1 H, J=4 Hz), 1.44 (d, 3 H, J=7 Hz)]. Partial structure (94) along with 13C signals at 167.7 (s), 83.3 (d) and 76.3 (d) indicated the presence of an oxazoline ring which ultimately must be derived from threonine. This was supported by 13C spectral comparison (Table 4) with (95) prepared as described. 63 Amino acid assignments were confirmed by total hydroly­sis and analysis of the TFA-ME derivatives by GC-MS. The thiazole structures were determined by their characteristic mass spectral fragmentation pattern (i.e., M+ and/or M+-side chain). Alanine thiazole exhibited an M+ ion at 282, while isoleucine thiazole exhibited ions at 324 and 267 for M+ The absolute configurations Table 4 Oxazoline Spectral Comparison Carbon # 2 5 4 6 (94) 167.7 83.3 76.3 22.9 (95) 167.0 78.7 74.7 21.0 84 for all the amino acids except the alanine and isoleucine thiazoles were established by GC retention correlation of the N-trifluoroacetyl methyl ester (TFA-ME) derivatives on a column coated with an optically active phase (SP-300, Supelco) .66 Due to the low temperature ceiling of the column (140oC), the thiazoles do not elute off the column. Total acid hydrolysis of (54) with 6 N HCI followed by derivatization and GC analysis led to the following assign­ments: L-threonine, L-proline and L-phenylalanine. Selective hydrolysis of the oxazoline ring of (54) (5% H2S04/MeOH, followed by acetylation) gave the linear peptide (96). This reaction is a 2 step process which initially proceeds to the depsipeptide. The mechanism of oxazoline hydrolysis has been examined64 and is known to proceed to the amino ester salt in acid. The amino acid sequence of (96) was established by high-resolution electron impact mass spectroscopy (HREIMS) (Figure 11) and originally led to structure (97) for ulicyclamide. ulithiacyclamide (55) has the molecular formula C32H42- Nso6S4: obsd 762.2105, calc 762.2101. The UV maximum (MeOH) at 247 nm (E 7000) was virtually identical to ulicyclamide [UV Amax (MeOH) 248 nm (e 7900)], indicating that (55) was also a cyclic peptide with two thiazole rings. Ulithiacyc1amide was found to be a symmetrical molecule as indicated by the 1H and 13c NMR spectra which exhibited signals for only half the molecule. The NMR data indicated 85 92 93- 96 86 677.2451 622.2034 497.1575 257.0737 453.1416 Figure 11. Ulicyclamidels ElMS data 87 the presence of a leucine thiazole [ 13C: 8160.1 (s), 149.2(s), 124.1 (d), 48.5 (d), 46.5 (t), 25.3 (d), 22.8 (q) and 22.7 (q); 1H: 87.72 (s), 7.70 (d, J=9 Hz), 5.24 (m), 1.66 (m), 1.35 (m, 2 H), 0.90 (d, 3 H, J=7 Hz) and 0.78 (d, 3 H, J=7 Hz)], cystine [13c: 0 170.5 (s), 48.4 (d) and 46.5 (t); 1H: 0 8. 50 (d, J=9 Hz), 5 . 36 (m), 3 . 22 (dd, J=14, 6 Hz) and 3.02 (dd, J=14, 4 Hz)] and an oxazoline ring [ 13 C : 0 16 7 . 3 ( s), 8 1 . 7 ( d), 7 4 • 3 ( d ) and 2 2 . 1 ( q); 1 H : 84.71 (m), 4.05 (dd, J=8, 2 Hz) and 1.1 (d, 3 H, J=7 Hz)]. Cystine must be condensed with threonine to form the oxazoline because decoupling studies indicated that the cystine proton at C-15 was homoallylically coupled to the oxazoline proton at C-11 (J=2 Hz). This homoallylic coupling was observed in the oxazoline (95) and has been reported previously for 6 2oxazolines. 64 Therefore, leucine thiazole was on the C-side of threonine, which is in agreement with the proposed structure (55) for ulithiacyclamide. Total acid hydrolysis of (55) furnished 2 L-threonines, L-cystine and 2 leucine thiazoles (M+ 324 and M+-C4H9 267). Patellamide A (56) has the molecular formula C35H50NS- 06S2; obsd 742.3280, calc 742.3297. IR data (3395,1675, 1655 cm-1), 1H NMR data [07.83 (s, 2 H)] and 13C NMR data [ 0 160 . 5 ( s , 2 C), 149. 4 ( s , 2 C), 123. 0 ( d , 2 C)] were indicative of a cyclic peptide with 2 thiazole amino acids. Acid hydrolysis corroborated these assignments yielding L-serine, L-threonine, L-isoleucine and valine thiazole 88 (GC-MS; m/z 310 (M+), 268 (M+-C3H6) in a 1:1:2:2 ratio. In support of the acid hydrolysis yielding L-serine and L-threonine, the 13C and 1H spectra of (56) contained signals for a 82,3-oxazoline and a 5-methyl-82,3-oxazoline: [ 0 169 . 1 (s), 72. 2 (t), 67. 4 (d);o 4 .30 (dd, 1 H, J=8, 4 Hz), 4.80 (m, 2 H) and 0168.5 (s), 81.6 (d), 73.6 (d); 04 . 3 0 ( d , 1 H , J = 4 Hz), 4 • 89 (m , 1 H), 1 . 4 7 ( d , 3 H , J = 6 Hz)], respectively. The absence of homoallylic coupling in the 1H spectrum of (56) allowed assignment of the fused oxazolines-thiazoles (98) and (99), plus 2 isoleucines accounted for all the atoms in (56). As before, 5% H2S04/MeOH caused the hydrolysis of the 2 oxazolines yielding 2 linear tripeptide derivatives, (100) and (~). High-resolution mass spectra of (100) and (101) confirmed their molecular formulae as well as the serine and threonine N terminal, thus determining that valine thiazole is the C terminal in both cases. High resolution mass spectral fragmentations for (100) and (101) are shown in Figure 12. Patellamide B (57) has the molecular formula C38H48N8- 06S2' obsd 776.3128, calc 776.3138. The 6 N Hel hydrolysis of pate11amide B yielded L-threonine, L-isoleucine, L-Ieu­cine, alanine thiazole and phenylalanine thiazole (GC-MSi m/z 358 (M+), 267 (M+-C7H7) in approximately a 2:1:1:1:1 ratio. Signals in the 1H and 13c NMR spectra of (57) fur­ther verified the presence of the 2 thiazoles: 1H NMR R )IN H 98: R = H 99: R:: Me Me 100: R:: H 101: R = Me 89 B \ o +. R c +. 90 102 470.2196 (0.2) ~ 284.1071 (0.2) ~ 411.2099 (3.4) £ 268.1485 (0.2) ~ 116.0710 (0.1) 103 456.2043 (0.1) A 284.1071 (0.2) ~ 397.1905 (0.4) £ 268.1485 (0.2) Q 102.0555 107 442.1880 (0.5) ~ 284.1070 (0.1) ~ 383.1755 (0.3) £ 240.1174 (0.4) Q 116.0711 108 518.2174 (2.4) ~ 284.1071 (0.2) ~ 459.2063 (0.2) £ 299.1218 (0.1) Q 116.0711 109 504.2034 (0.7) ~ 270.0915 (0.2) ~ 445.1907 (0.1) £ 285.1064 (0.4) Q 116.0711 Figure 12. Patellamides' mass spectral data 91 87.62 (d, 1 H, J=10 Hz), 7.40 (m, 5 H), 5.50 (ddd, 1 H,J=10, 10, 7 HZ), 3.45 (dd, 1 H, J=14, 10 Hz), 3.31 (dd, 1 H, J=14, 7 Hz); 13C NMR 0136.3(s), 129.2 (d, 2 C), 128.7 (d, 2 C), 127.1 (d), 53.3 (d), 40.7 (t). The 1H and 13C NMR spectra of (57) contained resonances for 2 threonine derived oxazolines (102), which was consis­tent with the isolation of 2 equivalents of threonine: 13C NMR 816 8 . 2 ( s), 16 8 . 0 ( s), 8 2 . 5 ( d), 82 . 1 ( d), 7 3 . 8 ( d , 2 C), 2 3 • 2 ( q), 2 1 • 8 ( q); 1 H NMR 0 5. 0 1 ( m , 2 H), 4 . 3 8 (d, 1 H, J=4 Hz), 4.29 (d, 1 H, J=4 Hz), 1.47 (d, 3 H, J=7 Hz), 1.45 (d, 3 H, J=7 Hz). Again the absence of homo­allylic coupling for the signals at 0 4. 38 and 4.29 allowed assignment of the fused oxazolines-thiazoles (103) and (104), plus isoleucine and leucine accounted for all the atoms in (57). Again, as before, selective hydrolysis of (57) yielded 2 linear tripeptides, (105) and (106). Partial structures (105) and (106) along with high-resolu­tion mass spectral fragmentation data showed that each tripeptide contained a thiazole at the C terminal and threo­ine at the N terminal (Figure 12). Because of the inability of mass spectral data to distinguish between leucine and isoleucine, 1H NMR was utilized. Signals at 8 0.97 (d, 3 H, J=7 Hz) and 0.95 (t, 3 H, J=7 HZ) in the spectrum of (105) defined isoleucine. Leucine was defined by a pair of methyl doublets at 00.98 and 0.96 (J=7 Hz) for the isopropyl group in the spectrum of (106). 92 93 Patellamide C (58) exhibited a parent ion at 762.2973 for C37H46N806S2 which requires 762.2981; therefore, (58) differs from patellamide B by only a methylene. Total acid hydrolysis confirmed this, by yielding 2 L-threonines, L-isoleucine, L-valine (replacing leucine), alanine thiazole and phenylalanine thiazole. Resonances for valine, isoleu­cine and the identical oxazolines (102) were observed in the 1H and 13c spectra of (58). Further, resonances for the latter 3 were superimposable on the corresponding sig­nals in the spectra of (57). These data led to the proposed structure for patellamide C (58), which was verified, as before, by degradation to the linear tripeptides (105) and (107) followed by high-resolution mass spectral analysis (Figure 12). Lissoclinamides 2 (60) and 3 (61) had identical molec­ular formulae C33H41N,05S2; obsd 679.2575 and 679.2588, respectively; calc 679.2610. Because the 1H and 13C were so similar, it was assumed that the peptides had identical amino acid compositions and differed by conformation or stereochemistry. A dihydro relationship to ulicyclamide (C33H39N705S2) was suggested by the striking similarity of spectral data. Proton decoupling studies of (60) and (61) confirmed the presence of proline, phenylalanine, isoleucine and alanine side chains as well as a threonine oxazoline analogous to ulicyclamide. Each spectrum con­tained resonances for a single thiazole at 0 7.97 and 8.07, 94 OMe OMe o ~N H Ph 95 respectively. Resonances for the second thiazole were replaced by an ABX spin system [(60): 0 5.21 (ddd, 1 H, J=ll, 7, 1 Hz), 3.64 (dd, 1 H, J=ll, 11 Hz), 3.42 (dd, 1 H, J=ll, 7 Hz); (61): 05.25 (dd, 1 H, J=ll, 7 Hz), 3.73 (dd, 1 H, J=ll, 7 Hz), 3.64 (dd, 1 H, J=ll, 11 Hz)] charac-teristic of a ~2-thiazoline.36 GC/MS analysis of the total acid hydrolysis products from (60) and (61) as TFA-ME deri­vatives confirmed NMR assignments yielding equimolar amounts of L-threonine, L-proline, D-isoleucine, L- phenylalanine, alanine thiazole and 1/2 equivalent of L-cystine (due to oxidation of cysteine during work-up). Cystine was assigned an L configuration after conversion to alanine with Raney Ni. Selective hydrolysis of (60) and (61) with 5% H2S04/ MeOH (followed by work-up) gave the same linear peptide (108) in both cases, suggesting that the peptides were epimeric at the alanine side chain of the thiazole. Thia­zoles are known to racemize at the a-carbon in acid (see page 101 of this thesis). The fast atom bombardment mass spectra of peptides have been found useful in the sequencing of peptides. Cleavages for each peptide link are observed because the lower energy used in FAB results in the cleavage of only high energy bonds. 64 The fast atom bombardment (FAB) mass spectra of (108) (positive and negative ion modes) were consistent with the amino acid sequence shown for (108). The positive ion FAB spectrum (Figure 13) showed 96 (M+H)+ and ions at m/z 615 and 472, corresponding to losses of carbomethoxyproline-CO and N-acetylthreonine, respec­tively, thus fixing the C and N termini. The central tri­peptide fragment (m/z 472) underwent further fragmentation from the N terminal side, losing in succession, an isoleu­cine side chain (m/z 387) and the thiazoline ring (m/z 257), placing isoleucine at C-2 of the thiazoline ring. The negative ion FAB spectrum of (108) showed (M-H)- and both the N terminal and C terminal series of ions from sequential cleavage of each peptide. I was initially surprised when the FAB spectra showed this result since the EI spectra of the hydrolysis products were similar to the spectrum for the hydrolysis product from ulicyclamide. Because of this finding, the original structure assignment for ulicyclamide was reexamined. The absence of homoallylic coupling for the oxazoline in the IH NMR spectrum of ulicyclamide (the C-4 proton of the oxazoline and protons on a carbon attached at C-2), led to a structure (97), where the isoleucine thiazole was fused to C-2 of the oxazoline. As mentioned earlier, this coupling was observed in ulithiacyclamide, where a cystine is fused to C-2 of the oxazoline. Therefore, structure hypothesis (97) seemed to be most compatible with the EI mass spectral data. However, with the avail­ability of FAB data and a reexamination of the EI data for the hydrolysis product (109) of ulicyclamide, (97) y o HO 629 472 627 180 470 415 385 N H 257 257 COOCHJ 643 Figure 13. Lissoclinamides· and ulicyclamide's FAB MS data 97 98 was revised to (54). The FAB mass spectrum of the hydroly­sis product (109) (Figure 13) was identical to the hydro­lysis product obtained from lissoclinamides A and ~ with a 2 mass unit shift for fragments containing the isoleucine thiazole. Further, these assignments were verified by examination of the FAB spectrum of the CD3 ester derivative of (109) (preparation of the CD3 ester was necessary because a fragment of interest was isobaric with a glycerol peak). The ions at m/z 627, 415, 277 and 130 in the spectrum of the (109) showed a 3 mass unit shift in the spectrum of the CD3 ester, verifying that they were generated by sequen­tial fragmentation from the N terminal side. Lissoclinamide 1 (59) has the molecular formula C35H43- N705S2; obsd 705.2835, calc 705.2771. Proton decoupling studies at 500 MHz, as well as GC and GC/MS analysis of the 6 N HCI hydrolysis products (as TFA-ME derivatives) determined the presence of L-threonine, L-proline, L-phenyl­alanine, valine thiazole and isoleucine thiazole in equi­molar amounts. All the atoms in the formula were account­ed for with these amino acids and threonine in the form of an oxazoline. Therefore, selective hydrolysis of the oxazoline ring with 5% H2S04 in MeOH gave the linear peptide (110) after derivatization. The positive ion FAB mass spectrum (Figure 13) of (110) exhibited ions at m/z 641 and 498 fixing carbomethoxyproline and N-acetylthreonine at the respective termini. The central piece (m/z 498) 99 underwent further fragmentation (m/z 427 and 299) analogous to peptides (108) and (109), positioning valine thiazole at the N-terminal side of the central peptide. The negative ion FAB spectrum had abundant fragment ions which corrobo­rated the sequence. stereochemistry of Thiazole Amino Acids The stereochemistry of all the amino acids except the thiazoles present in the cyclic peptides had been deter­mined by chiral GC. Due to the low temperature ceiling of the chiral column (140oC), the thiazoles would not elute. Therefore, it was necessary to devise a degradation scheme for the thiazoles, which would allow analysis by chiral GC. At the time, the only method available to determine the stereochemistry of thiazole amino acids in peptides was X-ray analysis. Thiazoles are unusual sulfur containing amino acids, which are believed to arise biosynthetically from the dehy­drative cyclization of a cysteinyl peptide such that C-2 of the thiazole originates from the carboxyl of an amino acid on the N terminal side of cysteine. 67 In spite of the probability that thiazoles are derived from chiral precursors, uncertainty existed in the literature for sev­eral years regarding the chirality of the C-2 sUbstituent. For example, the first thiazole amino acids isolated from 6 N HCI hydrolysis of thiostrepton68 and bottromycin69 100 108 109 H \~ 110 101 were racemic. This result has been affirmed with the lissoclinum peptides, finding that the thiazoles isolated from 6 N Hel hydrolysis were racemic. Subsequent to degra­dation studies, the structure of thiostrepton was determined by X-ray studies, and the thiazoles were shown to have the S absolute configuration, corresponding to an L-amino acid as the C-2 substituent. 70 It is not suprising that acid hydrolysis of a peptide results in racemization at C-1' of a thiazole amino acid of the general formula (111). The initial inclination was that protonation of the ring nitrogen of the thiazole facilitates racemization at C-l' via a relay type mechanism involving the 2,3-double bond and that if the aromaticity of the system were destroyed prior to hydrolysis, the C-2 side chain could be carved out with retention of chirality. The desired product of such a scheme is an a-amino acid. The chirality of the a-amino acid could then be determined by chiral GC. Wasserman reported several years ago that thiazoles undergo a 4+2 cycloaddition with singlet oxygen (102) to give a thioozonide, which decomposes in MeOH to an amide (Figure 14) .71 This procedure was attractive for several reasons, in addition to disrupting the aromaticity of the system: the desired oxidation state is retained at C-2 and acid hydrolysis should give the desired amino acid directly. 102 Ph '02 ~='\ CH30H .p "S1Ph ... --0 Figure 14. 102 addition to thiazoles 103 To test the utility of the reaction, leucine thiazole (112) obtained from acid hydrolysis of ulithiacyclamide (55) was allowed to react with 102 (generated from the thermal decomposition of triphenyl phosphite ozonide), followed by acid hydrolysis and derivitization to the TFA-ME derivative. The product was nearly a 50:50 mixture of D- and L-Ieucine as shown by chiral GC. Interestingly, the thiazole (112) exhibited an optical rotation [a]D +5.3 0 , but was confirmed to be racemic by IH NMR spectroscopy using the chiral shift reagent, Eu(hfc)3 (doubling of cer­tain peaks were observed, e.g., the thiazole proton). How­ever, reaction of ulithiacyclamide (55) with 102 followed by hydrolysis gave 2 molar equivalents of D-Ieucine (Table 5) , indicating that both thiazoles in (55) are chiral and possess an B absolute configuration. This method has been used to determine the stereochemistry of all the thia-zole amino acids present in the lissoclinum peptides. To illustrate the simplicity of this new method, a comparison Peptide ulithiacyclamide ulicyclamide patellamide A patellamide B patellamide C lissoclinamide 1 lissoclinamide 2 lissoclinamide 3 Table 5 Thiazole stereochemistry Results two D-Ieucine thiazoles D-alanine and L-isoleucine thiazoles two D-valine thiazoles D-alanine and D-phenylalanine thiazoles D-alanine and D-phenylalanine thiazoles L-valine and D-isoleucine thiazoles D-alanine thiazole L-alanine thiazole 104 111 105 of the GC traces from the analysis of patellamide A is shown (Figure 15). The lower trace A is the GC trace of the total hydrolysis of patellamide A. The 2 peaks corre­spond to the amino acids L-threonine and L-isoleucine. The middle trace ~ is the GC trace of the hydrolysis mixture after pretreatment with 102 . The appearance of a new peak corresponding to a valine amino acid is observed. Finally, the upper trace ~ is the GC trace with coinjection of stan­dards, which determines the chirality of the amino acids derived from 102 degradation of the thiazoles. Patellamide A was found to contain 2 D-valine thiazoles. This newly developed method for determining the abso­lute configuration of 2-(l'-aminoalkyl)thiazole-4-carboxylic acids in peptides was utilized to revise the absolute con­figurations of dysidenin and isodysidenin, 2 thiazole con­taining peptides, from the sponge Dysidea herbacea. The sponge Q. herbacea is the source of a wide variety of natu­ral products including halogenated phenylethers72 , sesqui­terpenes73 and a series of modified peptides, all of which possess a trichloromethyl group.74 Dysidin, the first such peptide isolated from Dysidea herbacea, was assigned structure (113) with an B absolute configuration at the trichloromethyl bearing carbon, by X-ray (heavy atom method).74 Shortly thereafter, 2 addi­tional metabolites, dysidenin75 (114) and isodysidenin,76 (115) were isolated. The structure of isodysidenin with 25 A L lie L lie c 20 LVal OVal L Thr 15 Tmin 10 o Vol 5 o Figure 15. GC analysis of patel1amide A 106 107 an ~ absolute configuration at both trichloromethyl bearing carbons was again established by X-ray_ Dysidenin was shown to be the C-5 epimer of isodysidenin by a chemical correlation. 77 Further, from the acid hydrolysis of both dysidenin and isodysidenin, thiazole (116) was isolated, which exhibited a very small positive rotation [aJD +0.650 and +0.770 • These very small positive rotations were used as fUrther proof that the pair were epimeric at c-s. It was felt this result warranted reinvestigation, since stud­ies with lissoclinum peptides indicated that 2-(I'-amino­alkyl) thiazoles racemize upon acid hydrolysis. Further, it was intriguing that two X-ray studies indicated opposite absolute configurations at the trichloromethyl bearing carbons, since a common precursor, trichloromethylleucine (Figure 16), could be postulated for all 3 compounds. Treatment of both isodysidenin and dysidenin with 102' followed by work-up and GC analysis, indicated that the thiazoles have the ~ absolute configurations, not the B configurations as assigned by x-ray, due to the isolation of L-alanine in both cases. Therefore, both dysidenin and isodysidenin have the opposite absolute configurations as reported earlier and trichloromethyl bearing carbons of dysidin, dysidenin and isodysidenin have the same abso­lute configuration, namely the B absolute configuration. 78 113 Me YlCC1 , H 108 116 YYCOOH CI~ ~H2 III 1 SCoA cO2 yyCOOH \...,,:P ~ ClP 0 ~CoA CJ~ 0 Figure 16. Tr;chloromethylleuc;ne formation 109 EXPERIMENTAL SECTION Infrared spectra were recorded on a Beckman 620 MX spectrophotometer. Electron ionization mass spectra were recorded on an AEI MS-902 or on photographic plates by using a CEC 110B spectrometer or a MAT 112S spectrometer. FAB mass spectra were recorded with a MAT 731 instrument fitted with an Ion Tech neutral atom gun. Samples were dissolved in (1:1) dimethylsulfoxide/glycerol for FAB analy­sis. 1H and 13C NMR spectra were recorded on a Bruker WM-500 spectrometer or a JEOL FX270 spectrometer. Chemical shifts are reported relative to Me4Si. Low resolution GC-EI mass spectra were recorded on a HP-5985 spectrometer. Gas chromatograms were recorded on a Varian Model 3700 gas chromatograph. HPLC separations were performed on a waters Model 201 system with a Model 441 refractive index detector. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter. The Chemistry of Siphonaria pectinata Siphonaria pectinata was collected from a sea wall at the entrance to Key Biscayne, Florida. The animals were soaked in MeOH for 5 days at oOC. The MeOH extract was gravity filtered thru Whatman #1 filter paper, solvent removed in vacuo and the residue was partitioned between 111 brine and CHC13. The CHC13 was removed in vacuo to give 530 mg of a yellow-brown organic oil. Silica gel column chromatography of 244 mg of the oil eluting with isooctane/ EtOAc (3:2) gave 181 mg of pectinatone (10) that crystalliz­ed on standing in CH2C12 at OOC. Pectinatone (10). mp 127-90 C; [a.]D +62 0 (c 0.184, CHC13); IR (CH2C12) 3163 br, 2951, 1685-1649 br, 1624, 1543, 1218 cm-1 ; UVmax 301 nm (E 5060); 13C NMR (CDC13) 8 166.5 s, 166.2 s, 159.2 s, 142.9 d, 126.2 s, 107.0 s, 98.9 s, 45.8 t, 44.7 t, 39.3 t, 30.5 d, 29.7 d, 28.3 d, 21.1 q, 20.2 q, 20.1 q, 19.9 t, 14.7 q, 14.3 q, 11.6 q, 8.7 qi 1H NMR (COC13) 8 8.8 (s, 1H), 5.38 (dq, 1H, J=10, 1 Hz), 2.64 (m, 1H), 2.0 (s, 6H), 1.89 (d, 3H, J=l Hz), 1 . 4 7 ( m , 2 H), 1 . 3 1 ( m , 2 H), 1 . 2 3 ( m, 2 H), 1 . 1 7 (m , IH), 1.06 (m, 2H), 0.98 (d, 3H, J=6 Hz), 0.93 (m, 1H), 0.85 Cd, 3H, J=6 Hz), 0.80 (d, 3H, J=6 Hz) i EI HRMS C21H3403, obsd 334.2503, calc 334.2509. Formation of the isomeric methyl ethers. (16) and ilZl. A portion (76.5 mg) of the CHC13 extract was subjected to dropwise addition of diazomethane in ether. Reaction was terminated when the yellow-green color of the diazo­methane persisted. Solvent was removed in vacuo and the oil was subjected to HPLC (Partisil 10, isooctane/EtOAc, 7:3) to yield the two isomeric methyl ethers (16) and (17) as oils in equivalent amount (25 mg) . 1l2l. [a]O +69.6 (c 1.13, CH2C12)i IR (CH2C12) 2965, 112 1698, 1570 cm-1 i UVmax 313 nm (E 6.99X10 3)i 13C NMR (CDCl3) o 168.6 s, 165.5 s, 159.3 s, 142.8 d, 126.4 s, 109.8 s, 108.6 s, 60.1 q, 45.8 t, 44.7 t, 39.2 t, 30.5 d, 29.6 d, 28.3 d, 21.1 q, 20.2 q, 20.1 q, 19.9 t, 14.8 q, 14.3 q, 11.8 q, 10.1 qi 1H NMR (CDCl3) <55.38 (dq, 1H, J=10, 1 Hz) , 3.82 (s, 3H) , 2.65 (m, 1H) , 2.06 (s, 3H) , 1.96 (s, 3H) , 1.90 (d, 3H, J=l Hz) , 1.48 (m, 2H) , 1.33 (m, 2H) , 1.26 (m, 2H) , 1.16 (m, 1H) , 1.07 (m, 2H) , 0.99 (d, 3H, J=7 Hz), 0.95 (m, 1H), 0.88 (t, 3H, J=7 HZ), 0.86 (d, 3H, J=7 H) , 0.82 (d, 3H, J=7 HZ)i EI HRMS C22H3603 cbsd 348.267, calc 348.266. 1660, 1595 cm-1 ; UVmax 261 nm (E 7.66X10 3 ); 13C NMR (CDCl3) o 181.6 s, 161.9 s, 158.6 s, 143.5 d, 129.8 s, 117.6 s, 99.3 s, 55.1 q, 45.8 t, 44.7 t, 39.2 t, 30.5 d, 29.6 d, 28.3 d, 21.1 q, 20.3 q, 20.1 q, 19.9 t, 14.6 q, 14.3 q, 11.7 q, 6.8 qi 1H NMR (CDCl3) (} 5.46 (br d, 1H, J=10, 1 Hz), 3. 95 ( s , 3 H), 2. 68 (m , 1H), 2. 0 ( s , 3 H), 1 • 94 (d, 3H, J=l HZ), 1.89 (s, 3H), 1.50 (m, 2H), 1.37 (m, 2H), 1.26 (m, 2H), 1.18 (m, 1H), 1.10 (m, 2H), 1.03 (d, 3H, J=7 Hz), 0 • 98 (m , 1H), 0 • 88 ( d , 3 H , J = 7 Hz), 0 • 87 ( t , 3 H , J=7 Hz), 0.82 (d, 3H, J=7 Hz); EI HRMS C22H3603 obsd 348.266, calc 348.266. 2,4,6-Trimethy1nonanoic acid. Pectinatone (10 mg) was dissolved in 10 ml CH2C12 at -70°C. Dropwise addition of an ozone saturated solution of CH2C12 was added. The 113 reaction was terminated when it was judged complete by tIc. The solution was allowed to warm to room temperature and solvent removed in vacuo. Acetone (10 ml) was added and Jones' reagent added by dropwise addition. Addition was terminated when the orange color of the reagent persist­ed. H20 (10 ml) added and the solution extracted 3X with Et20. Et20 layers dried over Na2S04, filtered and solvent removed in vacuo to give 2 mg of 2,4,6-trimethylnonanoic acid (HR ElMS 200.1764 for C12H2402 required 200.1776). The methyl ester was prepared by addition of diazomethane and exhibited an optical rotation [a]O + 34.20 (c 0.038, Et20 and was assigned the 2S,4R,6S absolute configuration (lit. + 35.20 ) .25 Isolation of the hemiketal (21). A portion (77.7 mg) of the original CHCl3 subjected to Partisil 10 HPLC with diisopropylether to give 4 UV active fractions, which were further subjected to RP-18 HPLC with 20% H20/MeOH to give 8.6 mg of a clear oil (21). (21) . IR (CH2CI2) 3570, 3350 br, 2930, 1690, 1670, 1620, 1570 cm-1 ; 13C NMR cS 203.3 9, 181.6 s, 147.7 d, 125.8 s, 106.3 9, 101.1 s, 45.5 t, 44.7 t, 39.3 t, 30.9 d, 29.7 d, 28.4 d, 22.3 q, 20.9 t, 20.5 q, 20.1 q, 19.9 q, 14.4 q, 13.4 q, 7.6 q; 1H NMR (CDCI3) 06.1 (dq, 1H, J=10, 1 Hz), 3.9 (br s, 1H), 2.73 (m, 1H), 1.98 (s, 3H), 1.81 (d, 3H, J=l Hz), 1.55 (s, 3H), 1.47 (m, 2h), 1.32 (m, 2H), 1 . 22 (m , 2 H), 1 . 1 7 (m , 2 H), 1 . 07 (m , 2 H), 0 . 98 ( d , 3H, 114 J=7 Hz), 0.94 (m, 1H), 0.87 (t, 3h, J=7 Hz), 0.86 (d, 3H, J=6.5 Hz), O.SO (d, 3H, J=6.5 Hz); EI HRMS C20H3403, obsd 322.2504, calc 322.2509. The Chemistry of Siphonaria normalis Siphonaria normalis (200 specimens) were collected at Diamond Head Beach, Oahu, Hawaii and stored in isopro­panol at 50C for one week. The resulting extract was con­centrated in vacuo, partitioned between brine (100 ml) and chloroform (3x100 ml) and the combined chloroform layers dried over magnesium sulfate and evaporated to give 1.517 g of an organic oil. Column chromatography (silica gel 62: EtoAc) gave a UV absorbing oil (128 mg). HPLC [ODS-3, H20/ MeOH (3:7) and Partisil 10 isooetane/EtOAc (3:7)] of a portion (64 mg) of this oil gave dihydrosiphonarin A (14, 21.3 mg), dihydrosiphonarin B (15, 5.2 mg) and the degradation products (23, 1.9 mg and 24, 0.9 mg). Dihydrosiphonarin A (14). C28H450S (HR FAB mass mea­surement, obsd 509.3136, calc 509.3114), [a]D -24.9 (e, 0.9925, CH2CI2); IR (CH2CI2) 3460, 3370, 2933, 1716, 1646, 1595 em- 1 ; 13C NMR (CDCI3) 0 208.1 s, 179.8 s, 162.0 s, 161.4 s, 121.9 s, 118.2 s, 105.2 s, 102.7 s, 74.0 d, 73.9 d, 73.4 d, 50.4 d, 45.6 d, 42.7 d, 39.6 d, 39.5 d, 37.9 d, 27.4 t, 17.6 q, 12.9 q, 12.8 q, 12.1 q, 11.0 q, 9.8 q, 9.4 q (2C) , 9.1 q, (2C) i 1H NMR (CDCI3) 0 5.49 (br s, 1H) , 3.95 (br s, 1H) , 3.73 (dd, 1H, J=10.5, 1 Hz), 3.68 115 (dd, 1H, J=3, 2.4 Hz), 3.25 (q, 1H, J=7 Hz), 3.23 (dt, 1H, J=7, 3 Hz), 2.61 (q, 1H, J=7 Hz), 2.35 (s, 3H), 2.35 (dq, 1H, J=7, 2.5 Hz), 1.61 (ddq, 1H, J=14, 7, 3 Hz), 1.42 (ddq, 1H, J=7, 3, 1 Hz), 1.32 (ddq, 1H, J=14,7, 7 Hz), 1.26 (d, 3H, J=7 Hz), 1.25 (d, 3H, J=7 Hz), 1.23 (d, 3H, J=7 Hz), 1.10 (d, 3H, J=7 Hz), 0.89 (d, 3H, J=7 Hz), 0.88 (t, 3H, J=7 Hz), 0.72 (d, 3H, J=7 Hz). Dihydrosiphonarin B (15). C29H4708 (HR FAB mass mea-surement, obsd 523.3289, calc 523.3271); [aJD -32.60 (c 0.331, CH2Cl2); IR (CH2Cl2) 3463, 3367,2935,1717,1645, 1593 cm-1 ; 1H NMR (CDCl3) 03.72 (dd, 1H, J=10.5, 2.5 Hz), 3.68 (dd, 1H, J=3, 2.4 Hz), 3.34 (q, 1H, J=7 Hz), 3.24 (dt, 1H, J=7, 3 Hz), 2.78 (q, 2H, J=7 Hz), 2.61 (q, 1H, J=7 Hz), 2.34 (dq, 1H, J=10.5, 7 Hz), 2.05 (dq, 1H, J=7, 3 Hz), 1.99 (s, 3H), 1.95 (s, 3H), 1.92 (dq, 1H, J=7, 2.4 Hz), 1.62 (ddq, 1H, J=14, 7, 3 Hz), 1.40 (ddq, 1H, J=7, 3, 2.5 Hz), 1.32 (ddq, 1H, J=14, 7,7 Hz), 1.26 (d, 3H, J=7 Hz), 1.25 (d, 3H, J=7 Hz), 1.24 (d, 3H, J=7 Hz), 1.22 (t, 3H, J=7 Hz), 1.10 (d, 3H, J=7 Hz), 0.89 (d, 3H, J=7 Hz), 0.88 (t, 3H, J=7 Hz). ll1l. C17H240 6 (HREI mass measurement, obsd 324.15673, calc 324.15729)1 [ a.] D -116. 80 (c 0.113, CH2Cl2) ; IR (CH2- C12) br 3600-2200, 2937, 1723, 1653, 1578 cm- 1 ; 1H NMR (CDCl 3) <5 4.15 ( q, 1H, J=7 Hz), 3.63 (dd, 1H, J=2.5, 2.5 HZ) , 2.87 (dq, 1H, J=7, 2.5 Hz) , 2.74 (dq, 1H, J=7, 2.5 Hz) , 2.26 (s, 3H) , 2.14 (s, 3H) , 1.95 (s, 3H) , 1.38 (d, 116 3H, J=7 Hz), 0.97 (d, 3H, J=7 Hz), 0.93 (d, 3H, J=7 Hz). il!l. C18H2606 (HREI mass measurement, obsd 338.17315, calc 338.17294), [a]D - 86.50 (c 0.052, CH2C12)i IR (CH2C12) br 3600-2200, 2933, 1722, 1647, 1578 cm-l ; lH NMR (CDC13) <54.17 (q, lH, J=7 Hz), 3.63 (dd, lH, J=7, 2.5 HZ), 2.78 (q, 2H, J=& Hz), 1.99 (s, 3H), 1.95 (s, 3H), 1.39 (d, 3H, J=7 Hz), 1.36 (d, 3H, J=7 Hz), 1.17 (t, 3H, J=7 Hz), 0.92 (d, 3H, J=7 Hz). oxidation of dihydrosiphonarin A (14). PCC (2 mg) and sodium acetate (10 mg) were added to benzene (5 ml) and the mixture brought to reflux. A solution of (14) (4 mq) in benzene (2 ml) was added and reflux continued for 4.5 h. The reaction mixture was evaporated in vacuo and filtered through a silica gel pad to give an oil. HPLC of the oil (Partisil 10, isooctane/EtOAc, 3:7) gave (28) (1 mg, 25% yield), [a]n +19.40 (c 0.108, CH2C12) and (30) (1 mg, 25% yield). HCl hydrolysis of (14) to (23). A portion of (14) (2 mg) was stirred in 1 ml of 30% HClaq in 5 ml of THF to give 1 mg of (~). The Chemistry of Onchidium verruculatum Onchidium verruculatum (N = 400) were collected at Portlock, Oahu, Hawaii and stored in acetone at soc for 24 h. The acetone extract was gravity filtered through Whatman #1 paper and concentrated in vacuo to an oily watery 117 residue. The residue was partitioned between brine (100 ml) and ether (3x 100 ml). The combined organic layers were dried over MgS04' filtered and evaporated to give 0.53 g of an organic oil. Chromatography of the oil on Sephadex LH-20 (CH2C12/hexane, 4:1) and Bio-sil A (EtOAc) gave a mixture of esters (60 mg) and 2 mg of the 11,13- dipropionate ester of ilikonapyrone: IR (CH2C12) 3550, 1735, 1660, 1370 cm-1 ; ElMS 656 (M+) , 598 (M-CH3CH2CHO)+, 524 (598-CH3CH2C02H)+, 450 (524-CH3CH2C02H)+; HR ElMS obsd 656.391, calc 656.392 for C38H5609. Saponification of the ester mixture. The mixture of esters (60 mg) was stirred in 1% KOH/MeOH (25 ml) at room temperature for 2 h. water (25 ml) was added to the reaction and the mixture extracted with CHC13 (3x 50 ml). The combined organic layers were dried over Na2S04' filtered and solvent removed in vacuo to give 45 mg of IKP (13). IKP (13). HR ElMS C32H4807, obsd 544.335, calc 544.- 340; mp 96-98oC; [a.]D -160 (c 1.5, CH2C12); lR (CH2C12) 3550, 1660, 1610 cm-1 ; UVmax (MeOH) 260 nm (e: 12700); 1H NMR (CDC13) 0 5.60 (dq, 1H, J=9, 1 Hz), 4.20 (dd, 1H, J=8, 1 Hz), 4.04 (d, 1H, J=7 Hz), 3.90 (dq, 1H, J=9, 7 Hz), 3.60 (m, 1H), 3.13 (dq, 1H, J=8, 7 Hz), 2.96 (dq, 1H, J=7, 7 Hz), 2.62 (q, 2H, J=7 Hz), 1.93 (5, 3H), 1.92 (5, 3H), 1.91 (5, 3H), 1.89 (5, 3H), 1.88 (m, 1H), 1.70 (d, 3H, J=l Hz), 1.48 (ddq, 1H, J=14, 7, 3 Hz), 1.30 (ddq, 1H, J=14, 7, 7 Hz), 1.25 Cd, 3H, J=7 Hz), 1.20 (t, 3H, J=7 118 Hz), 1.lS Cd, 3H, J=7 HZ), 1.10 (d, 3H, J=7 Hz), 0.89 (t, 3H, J=7 Hz), 0.S7 (d, 3H, J=7 HZ); 13C NMR (CDCI3) 81S0.1 s, lS0.0 s, 165.5 s, 165.3 s, 164.7 s, 164.6 s, 137.5 s, 127.1 d, 119.3 s, 119.2 s, 11S.0 s, 117.3 5, 79.6 d, 75.9 d, 71.9 d, 41.2 d, 39.5 d, 37.2 d, 34.S d, 2S.0 t, 25.1 t, lS.6 q, 15.9 q, 15.8 q, 12.2 q, 11.5 q, 9.9 q, 9.S q, 9.7 q, 9.6 q (3C). Mn02 oxidation of IKP to (37). Mn02 (30 mg) and IKP (10 mg) were stirred in CH2Cl2 (20 ml) at room temperature for 24 h. The reaction was filtered through Whatman #1 paper and the chloroform removed in vacuo to yield the crude product. Partisil 10 HPLC (EtOAc) gave (27) (5 mg) as the only product. llll. IR (CH2CI2) 3550, 1675, 1660, 1600, 1380 cm-1 ; UV (MeOH) 242 nm (E 13800), 275 nm (e 13300); 1H NMR (CDCI3) o 6.34 (dq, 1H, J=9, 1 Hz), 4.0 (dd, 1H, J=S, 1 Hz), 3.90 (m, 1H), 3.60 (m, 2H), 2.54 (q, 2H, J=7 Hz), 1.92 (s, 9H), 1.SS (s, 3H), 1.76 (d, 3R, J=l Hz), 1.34 (d, 3H, J=7 Hz), 1.26 (d, 6H, J=7 Hz), 1.lS (d, 3H, J=7 Hz), 1.05 (m, 6H). Os04/NaI04 oxidative cleavage of IKP to (38) and (39). IKP (13, 45 mg) was dissolved in dioxane (25 ml) and H20 (5 ml). Os04 (1 crystal) was added and the reaction stirred at room temperature. After 30 min NaI04 (100 mg) was added and stirring continued for 24 h. H20 (25 ml) was added to the reaction and the mixture extracted with ether (3x 25 ml). The combined organic layers were dried over MgS04' 119 filtered and evaporated to give 30 mg of an oil. The oil was dissolved in MeOH (20 ml) and NaBH4 (50 mg) added. The reaction was stirred for 1 h at room temperature and then terminated by addition of H20 (10 ml). The mixture was extracted with ether (3x 25 ml), the combined organic layers dried over MgS04' filtered and concentrated in vacuo to yield 25 mg of oil. Partisil 10 HPLC (EtoAc) gave (~) (8 mg) and (~) (12 mg). nll· 1660, 1610 cm- l ; UVmax (MeOH) 258 nm (£ 6000); IH NMR (CDC1 3) 0 3.78 (m, 2H) , 3.23 (m, IH) , 2.61 (q, 2H, J=7 Hz) , 1.96 (s, 3H) , 1.92 (s, 3H) , 1.20 (ro, 6H) ; ElMS 210 (M+) , 193, 179, 122. 1.1ll. [aJD -16.70 (c 0.3, CH2C12) ; lR (CH2C12) 3610, 1660,1610 cm- l ; UVmax (MeOH) 260 nm ( £ 7200) ; IH NMR (CDCl 3) 04.12 (dd, IH, J=9, 3 Hz), 3.68 (m, 3H), 3.14 (dq, 1H, J=9, 7 Hz), 3.03 (dq, IH, J=7, 7 Hz), 1. 96 (s, 3 H), 1 • 9 5 ( s , 3 H), 1. 9 4 (m , IH), 1. 60 -1. 44 (m , 2 H), 1. 2 6 (d, 3H, J=7 Hz), 1.15 (d, 3H, J=7 Hz), 0.99 (t, 3H, J=7 Hz), 0.95 (d, 3H, J=7 Hz); ElMS 326 (M+) , 268, 180. lKP acetonide formation. lKP (13, 14 mg) was dissolved in 2,2-dimethoxypropane (4 ml). A single crystal of TsOH was added and the reaction refluxed for 3 h. The reaction was diluted with dry benzene (10 ml). The organic layer was evaporated to give the acetonide derivative (10 mg): crystals from hexane; mp 134-1350 C; HR ElMS obsd 584.372, 120 calc 584.3715 for C3SH5207; 1H NMR (CnC1 3) a 5.56 (dq, 1H, J=9, 1 Hz) , 3.90 (dd, 1H, J=10.5, 4.4 Hz), 3.88 (dq, 1H, J=9, 7 Hz) , 3.70 (ddd, 1H, J=7, 7, 3 Hz) , 3.64 (d, 1H, J=7.1 Hz), 3.14 (dq, 1H, J=10.5, 7 Hz), 3.07 (dq, 1H, J=7, 7 Hz), 2.62 (q, 2H, J=7 Hz), 2.00 (s, 3H), 1.99 (s, 3 H), 1 . 9 8 ( s , 3 H), 1 • 95 ( s , 3 H), 1 . 88 (m , 1H , J = 7 . 1 , 4 . 4 HZ), 1 . 7 4 ( d , 3 H , J = 1 Hz), 1. 6 7 ( m , 1 H), 1 . 4 3 ( m , 1H), 1.25 (d, 3H, J=7 Hz), 1.23 (s, 6H), 1.20 (t, 3H, J=7 Hz), 1.18 (d, 3H, J=7 Hz), 1.10 (d, 3H, J=7 Hz), 0.89 (t, 3H, J=7 Hz), 0.87 (d, 3H, J=7 Hz). The Chemistry of Peronia peronii Peronia peronii (N = 60) were collected at Merizo Pier and Pago Bay, Guam. The animals were freeze dried after collection. Upon receipt of the freeze dried ani­mals, they were subjected to cold (SoC) extraction with CHC13 for 4 days. The resulting CHC13 extract was grav­ity filtered thru Whatman #1 paper and solvent removed in vacuo to yield 8.0 9 of extract. The CHC13 extract exhibited good activity (IC50 0.5 micrograms/ml) against the L1210 cell line. The CHC13 extract was subjected to silica qel 62 column chromatography with EtOAc as eluting solvent and gave numerous active fractions (see Table 6). Fractions #3 and 4 were then subjected to further purifica­tion (HPLC, Partisil 10, EtOAc) and led to the isolation of the fractions shown (see Table 7). Further purification 121 of fractions #31 and 32 led to the previously mentioned peroxysterols as well as cholesterol. Because these com­pounds were not responsible for the activity associated with the extract, they were not pursued further. Fraction #4, however, contained no steroids, but instead a family of esters, all of which had the same UV chromophore (260 nm). An IR spectrum of fraction #4 contained absorptions at 1660 and 1600 cm-1 • Furthermore, a 1H NMR spectrum of fraction #4 exhibited prominent peaks around 2.0 ppm Table 6 L1210 Results A Fraction # 1 2 3 4 5 6 7 8 9 Fraction # 31 32 33 41 42 43 44 45 46 Activity (lla/mll Table 7 L1210 Results B >10 1.0 0.2 0.2 0.5 1.0 3.0 5.0 3.0 Activity (uq/mll 0.5 0.07 >10 0.3 0.07 0.4 1.85 >10 0.07 122 for pyrone methyls. Also, upon more careful reexamination of fractions #5-9 from the initial chromatography, small amounts of these esters were observed. All of these data suggested the presence of a y-pyrone ring analogous to the situation observed with ilikonapyrone. Saponification of a mixture of the UV active esters from P. peronii. A portion (140 mg) of the mixed UV active ester fraction was subjected to 30 ml of 1% KOH/MeOH with stirring at room temperature for 16 h. H20 (50 ml) was added to the reaction mixture and extracted with CH2C12 (3x 50 ml). The combined organic layers were dried over MgS04, filtered and solvent removed in vacuo to yield 60 mg of an organic oil. HPLC (RP-18, 30% H20/MeOH) of this oil led to the isolation of the isomeric saponification products, peroniatriol I (11) and peroniatriol II (12) in equivalent amounts (22 mg each). Peroniatriol I (11). HR FAB 545.346738 (MH+) C32H49- 07 calc 545.3478; UVmax (CH2CI2) 259 nm (£ 13000); 13C NMR ( CDC 13 ) <5 1 7 9 • 8 s ( 2 C), 16 4 . 8 s ( 2 C), 164 . 5 s ( 2 C) , 137.5 s, 127.0 d, 119.7 s (2C) , 118.6 s (2C) , 79.5 d, 75.2 d, 72.2 d, 41.5 d, 39.3 d, 36.3 d, 34.5 d, 27.9 t, 24.7 t, 18.6 q, 14.4 q, 14.0 q, 11.9 q, 11.3 q, 10.2 q, 9.6 q (2 C) , 9.5 q (3C) ; IH NMR ( CDC13) <5 5.59 (dq, IH, J=9, 1 Hz) , 4.14 (dd, IH, J=8, 1 HZ), 4.06 (d, IH, J=7 HZ), 3.90 (dq, IH, J=9, 7 Hz), 3.75 em, IH, J=7, 7, 7 Hz), 3.15 (dq, IH, J=8, 7 Hz), 2.90 (dq, J=7, 7 Hz), 2.76 (OH) , 2.56 123 (m, 2H, J=7 Hz), 1.97 (s, 3H), 1.96 (s, 3H), 1.93 (s, 3H), 1.89 (s, 3H), 1.84 (m, 1H), 1.70 (d, 3H, J=l Hz), 1.57 (m, 1H), 1.35 (m, 1H), 1.30 (d, 3h, J=7 Hz), 1.29 (d, 3H, J=7 Hz), 1.20 (t, 3H, J=7 Hz), 1.18 (d, 3H, J=7 Hz), 0.99 (t, 3H, J=7 Hz), 0.95 (d, 3H, J=7 Hz). Peroniatriol II (12). HR FAB 545.346738 (M+) C32H49- 07 calc 545.3478; UVmax (CH2Cl2) 259 (£ 13000); 13C NMR ( CDC 13 ) 0 18 0 • 1 s ( 2 C), 165 • 7 s , 165 • 1 s, 164 • 9 s , 164 • 5 s, 137.3 s, 125.4 d, 120.1 s, 118.5 s, 117.4 s, 116.1 s, 80.11 d, 75.21 d, 73.0 d, 42.04 d, 39.77 d, 34.5 d, 34.33 d, 27.6 t, 24.65 t, 19.82 q, 15.65 q, 14.12 q, 13.88 q, 10.8 q, 9.96 q (4C), 9.53 q (2C); 1H NMR (CDC13) <5 5.86 (dq, 1H, J=9, 1 Hz), 4 • 40 (OH) , 4. 05 (d, 1H, J=2 Hz), 3. 90 ( dq, 1H, J=9 ,7Hz), 3 • 72 ( dd , 1H, J=8 ,1Hz), 3. 57 ( ddd , 1H, J=7, 7, 3 Hz), 3.03 (dq, 1H, J=8, 7 Hz), 2.84 (dq, 1H, J=7, 7 Hz), 2.51 (m, 1H, J=14, 7 Hz), 2.28 (m, 1H, J=14, 7 Hz), 2.04 (s, 3H), 1.90 (s, 3H), 1.87 (s, 3H), 1.87 (m, 1H), 1.82 (s, 3H), 1.63 (d, 3H, J=7 Hz), 1.61 (m, IH), 1.38 (m, 1H), 1.31 (d, 3H, J=7 Hz), 1.16 (d, 3H, J=7 HZ), 1.14 (d, 3H, J=7 Hz), 1.02 (d, 3H, J=7 Hz), 0.99 (t, 3H, J=7 Hz), 0.87 (t, 3H, J=7 Hz). Peroniatriol I acetonide formation. Peroniatriol I (11, 5 mg) was dissolved in 2,2-dimethoxypropane (4 ml). A single crystal of TsOH was added and the reaction refluxed for 3 h. The reaction was diluted with dry benzene (10 ml). The solvent was evaporate to give 4 mg of a clear 124 oil. This oil was subjected to HPLC on Partisil 10 with EtOAc to give the acetonide (3 mg): IH NMR (CDCI3) 0 5.53 (dq, IH, J=9, 1 Hz), 3.96 (dd, IH, J= 10.5, 4.4 Hz), 3.87 ( d q , 1 H , J = 9, 7 Hz), 3. 7 4 ( d d d , IH, J = 7, 7, 7 Hz), 3. 61 (d, IH, J=7.1 Hz), 2.64 (m,lH, J=14, 7 Hz), 2.58 (m, 1H J=14, 7 Hz), 2. 00 (s, 3H), 1 • 97 (s , 3H), 1.965 (s, 3H), 1.96 (s, 3H), 1.96 (m, 1H), 1.73 (d, 3H, J=l Hz), 1.57 (m, 1H), 1.35 (m, 1H), 1.30 (d, 3H, J=7 Hz), 1.29 (d, 3H, J=7 Hz), 1,26 (s, 3H), 1.24 (s, 3H), 1.20 (t, 3H, J=7 Hz), 1.14 (d, 3H, J=7 Hz), 0.99 (t, 3H, J=7 Hz), 0.95 (d, 3H, J=7 Hz). Peroniatriol II acetonide formation. Peroniatriol II (12, 5 mg) was dissolved in 2,2-dimethoxypropane (4 ml). A single crystall of TsOH was added and the reaction refluxed for 12 h. The reaction was diluted with dry ben­zene (10 ml). The solvent was evaporated to give 3 mg of a clear oil. This oil was subjected to HPLC on Partisil 10 with EtOAc to give the acetonide (1 mg): 1H NMR (CDCI3) <5 5.53 (dq, 1H, J=9, 1 Hz), 3.96 (dd, 1H, J=10.5, 4.4 Hz), 3.84 (dq, 1H, J=9, 7 Hz), 3.69 (ddd, 1H, J=7, 7,3 Hz), 3.63 (d, 1H, J=7.1 Hz), 3.10 (dq, 1H, J=10.5, 7 Hz), 3.04 (dq, 1H, J=7, 7 Hz) , 2.64 (m, 1H, J=14, 7 Hz) , 2.58 (m, 1H, J=14, 7 Hz), 2.03 (s, 3H), 1.98 (s, 3H) , 1.97 (s, 3H) , 1.96 (s, 3H) , 1.95 (m, 1H) , 1.67 (m, 1H) , 1.65 (d, 3H, J=l Hz), 1.43 (m, 1H), 1.30 (d, 3H, J=7 Hz), 1.29 (d, 3H, J=7 Hz), 1.26 (s, 6H), 1.20 (t, 3H, J=7 Hz), 1.18 (d, 3H, 125 J=7 Hz), 1.14 (d, 3H, J=7 Hz), 0.99 (t, 3H, J=7 Hz). OzonolYsis of peroniatriol I. Peroniatriol I (5 mg, 11) was dissolved in CH2C12 at -70oC. Dropwise addition of a solution containing 03 in CH2C12 was continued until tIc indicated that starting material was no longer present. The flask was removed from the acetone/dry ice bath, allowed to warm to room temperature and solvent removed in vacuo. The solid white compound was subjected to Partisil 10 HPLC with EtOAc. Resulting solid was dissolved in 20 ml of MeOH and 100 mg of NaBH4 added. Reaction terminated by addition of 10 ml of H20 and extracted with CH2C12 (3x 25 ml). Combined organics dried over MgS04' filtered and solvent removed in vacuo to give 3 mg of a clear oil. HPLC of this oil on Partisil 10 with EtOAc gave the mono­pyrones (44) (1 mg) and (46) (1 mg) . (44) • ElMS 268 (M+); 1H NMR (CDC13) 03.81 (m, 2H, J=10.5, 3.5, 3.5 Hz), 3.75 (m, 1H, J=7, 7, 7 Hz), 3.23 (m, 1H, J=7, 3. 5, 3.5 Hz), 3. 01 (dq, 1H, J=7, 7 Hz), 2. 0 (s, 3H), 1.99 (s, 3H), 1.54 (m, 1H, J=10.5, 7.4, 7 Hz), 1.40 (m, 1H, J=10.5, 7.4,7 Hz), 1.30 (d, 3H, J=7 Hz), 1.23 (d, 3H, J=7 Hz), 1.00 (t, 3H, J=7.4 Hz). 1.lll. ElMS 310 (M+)i 1H NMR (CDC13) 04.27 (m, 1H), 4.00 (m, 1H), 3.87 (m, 1H), 3.14 (dq, 1H, J=8.8, 7 Hz), 2.63 (q, 2H, J=7.6 Hz), 2.10 (s, 3H), 2.10 (m, 1H), 2.00 ( s , 3 H), 1 . 96 ( s , 3 H), 1 . 2 3 (t , 3 H , J=7. 6 Hz), 1. 15 (d, 3H, J=7 Hz), 0.98 (d, 3H, J=7 Hz). 126 Ozonolysis of peroniatriol II. Peroniatriol II (5 mg, 12) was dissolved in CH2Cl2 at -70oC. Dropwise addition of a solution containing 03 in CH2Cl2 was continued until tlc indicated that starting material was no longer present. The flask was removed from the acetone/dry ice bath, allowed to warm to room temperature and solvent removed in vacuo. The solid white compound was subjected to Partisil 10 HPLC with EtOAc. The resulting solid was dissolved in 20 ml of MeOH and 100 mg of NaBH4 added. Reaction terminated by addition of 10 ml of H20 and extracted with CH2Cl2 (3x 25 ml). Combined organics dried over MgS04, filtered and solvent removed in vacuo to give 3 mg of a clear oil. HPLC of this oil on Partisil 10 with EtOAc gave the mono­pyrones (45) (1 mg) and (46) (1 mg). (45) . EIMS 268 (M+); IH NMR (CDCl3) 0 3.87 (m, IH, J=14, 3.5 HZ), 3.80 (m, 1H, J=14, 10.5 Hz), 3.73 (m, 1H, J=7, 7, 3 Hz), 3.24 (m, 1H, J=10.5, 7, 3.5 Hz), 3.05 (dq, IH, J=7, 7 Hz), 1.98 (s, 3H), 1.97 (s, 3H), 1.61 (m, IH, J=14, 7.4, 3 Hz), 1.38 (m, IH, J=14, 7.4, 7 Hz), 1.31 (d, 3H, J=7 Hz), 1.21 (d, 3H, J=7 Hz), 1.02 (t, 3H, J=7. 4 Hz) . The Chemistry of Lissoclinum patella Collection of Lissoclinum patella. Colonies of Lisso­clinum patella were collected in June 1981 by snorkeling (-2 m) in Iwayama Bay (near the Continental Hotel dock), 127 Korror Island, Western Caroline Islands. The frozen animals were lyophilized and ground to a powder (413 g) in a Wiley mill. The powdered tissue was extracted exhaustively in a Soxhlet apparatus with petroleum ether, CC14 and CHC13. Rotoevaporation of the solvent gave 570 mg, 460 mg and 500 mg of oil, respectively. The combined petroleum ether, CC14 and CHC13 extracts (1.530 g) were chromatographed on silica gel 60 (Merck, 60 x 2.5 cm). Three peptide frac­tions eluted with EtOAc. A fourth band eluted in EtoAcl MeOH (95:5). Each of the four bands was subjected to HPLC (RP-18; MeOHI H20, 80:20) and resolved into 2 peptides. The first band (150 mg) gave ul i thiacyclamide (55) (95 mg) and patellamide A (56) (40 mg). The second band (70 mg) gave patellamide B (57) (55 mg) and lissoclinamide 1 (59) (14 mg). Band three (230 mg) gave ulicyclamide (54) (190 mg) and patellamide C (58) (20 mg). The fourth and most polar band (624 mg) gave lissoclinamide ~ (60) (400 mg) and 1 (61) (200 mg). Ulicyclamide (54). IR (CH2C12) 3300, 1670, 1650 cm-1 ; high resolution EI mass measurement, obsd 677.2446, C33H39N705S2 requires 677.2439; 13C-NMR (CDC13) o171.9(3C) (s), 170.5 (s), 167.7 (s), 161.1 (s), 160.5 (s), 151.4 (s), 148.9 (s), 136.8 (s), 130.6 (2 C) (d) I 129. 5 ( 2 C) (d) I 128. 2 (d), 124. 3 (d) I 123. 8 (d), 83.3 (d) I 76.3 (d) I 57.5 (d), 54.4 (2C) (dt) I 49.6 Cd), 48.1 (d), 41.8 (t), 39.0 (d), 29.8 (t), 26.0 (t), 25.9 128 (t), 25.4 (q), 22.9 (q), 16.2 (q), 10.9 (q); IH-NMR (CDC13) o 9. 06 (d, 1 H, J=5 Hz), 8 • 67 (d, 1 H, J=7 Hz), 8 • 08 (s, 1 H), 8. 03 ( s , 1 H), 7. 8 5 ( d, 1 H, J= 10Hz), 7. 3 0 ( s , 5 H), 5.38 (dq, 1 H, J=5,7 Hz), 5.26 (dd, 1 H, J=10,7 Hz), 4.89 (m, 1 H), 4.82 (dq, 1 H, J=4,7 Hz), 4.52 (t, 1 H, J=8 Hz), 4.26 (d, 1 H, J=4 HZ), 3.25 (m, 3 H), 2.93 (dd, 1 H, J=14,10 Hz), 2.60 (m, 1 H), 2.1 (m, 2 H), 1.9 (m, 2 H), 1.71 (d, 3 H, J=7 Hz), 1.44 (d, 3 H, J=7 Hz), 1.30 (m, 1 H), 1.20 (m, 2 H), 0.85 (t, 3 H, J=7 Hz), 0.73 (d, 3 H, J=7 Hz). Ulithiaeye1amide (55). IR (CH2C12) 3300, 1670, 1650 em-1 ; high resolution EI mass measurement, obsd 762.2105, C32H42N806S4 requires 762.2101; 13 C - NMR ( CDC 13 ) 0 170. 5 (s), 170 • 0 (s), 167. 3 (s), 160 • 1 (s), 149.2 (s), 124.1 (d), 81.7 (d), 74.3 (d), 48.5 (d), 48.4 (d), 46.5 (t)(2), 25.3 (d), 22.8 (q), 22.7 (q), 22.1 ( q); 1 H - NMR ( CDC 13 ) 0 8. 5 0 ( d , J = 9 Hz), 7. 7 2 ( s), 7. 7 0 (d, J=9 Hz), 5. 36 (m), 5. 24 (m), 4. 71 (m), 4. 05 (dd, J=8, 2 Hz), 3.22 (dd, J=14,6 Hz), 3.02 (dd, J=14,4 Hz), 1.66 (m), 1.35 (m, 2 H), 1.1 (d, 3 H, J=7 Hz), 0.90 (d, 3 H, J=7 Hz), 0.78 (d, 3 H, J=7 Hz). Pate11amide A (56). [ a ] D + 113 • 90 ( cO. 27, CH 2 C 12) ; IR ( CH 2 C 12 ) 3 395 I 313 0 , 3 055 , 2 969 I 2940 , 2885 , 1675 , 1655 , 1535, 1510, 1489 em-1 ; high resolution EI mass measurement, obsd 742.3280, C35H50N806S2 requires 742.3297; 1 3C-NMR ( CDC 13 ) 0 1 71 • 8 ( s), 1 71 • 5 ( 2 C) (s), 169 • 5 ( s), 169 • 1 ( s) , 129 168.5 (8), 160.5 (2C) (8), 149.4 (2C) (s), 123.0 (2C) (d), 81.6 (d), 73.6 (d), 72.1 (t), 67.4 (d), 54.9 (2C) (d), 52.4 (d), 52.1 (d), 37.1 (d), 36.8 (d), 33.3 (2C) (d), 24.9 (t), 24. 7 (t), 21. 7 (q), 19 • 2 (2 C) (q), 1 7 • 9 ( 2 C) (q), 15 • 0 (q), 14.9 (q), 11.1 (q), 10.6 (q); 1H-NMR (CDCl3) <5 7.95 (m, 2 H), 7.83 (s, 2 H), 7.41 (d 2 H, J=10 Hz), 5.22 (m, 2 H), 4.89 (dq, 1 H, J=6,4 Hz), 4.80 (dd, 1 H, J=8,4 Hz), 4.65 (dd, 1 H, J=8,6 Hz), 4.56 (dd, 1 H, J=10,8 Hz), 4.30 (dd, 1 H, J=8,4 Hz), 4.30 (d, 1 H, J=4 Hz), 2.32 (m, 2 H), 1. 96 (m, 2 H), 1 • 47 (d, 3 H, J=6 Hz), 1. 13 (d, 3 H, J=7 Hz), 1.08 (d, 3 H, J=7 Hz), 0.81 (d, 3 H, J=7 Hz), 0.75 (t, 3 H, J=7 Hz), 0.73 (t, 3 H, J=7 Hz). Patellamide B (57). IR (CH2C12) 3374, 3330, 1662, 1480 em-1 ; high resolution EI mass measurement, obsd 776.3128, C38H48N806S2 requires 77 6 • 3 13 8; 13 C - NMR ( CDC 13 ) <5 1 7 3 • 3 ( s), 1 7 3 • 0 ( s), 172 • 8 (s), 170.8 (s), 168.2 (s), 168.0 (9), 161.8 (s), 161.6 (s), 147. 6 (s), 147. 2 (9), 136. 3 (9), 129 • 2 (2C) (d), 128. 7 (2C) (d), 127.1 (d), 123.6 (2C) (d), 82.5 (d), 82.1 (d), 73 • 8 (d) (2), 53 • 3 (d), 52.5 (d), 47. 8 (d), 46. 7 (d), 40. 7 (t) , 39.0 (t) , 32.9 (d) , 25.1 (t) , 25.1 (q) , 23.2 (q) , 21.8 (q) , 21.0 (3C) (q) , 15.0 (q) , 8.8 (q) ; 1H-NMR (CDC13) 0 7.62 (d, 1 H, J=9 HZ), 7.62 (d, 1 H, J=10 Hz), 7.62 (d, 1 H, J=ll Hz) , 7.62 (m, 1 H), 7.49 (s, 1 H), 7.39 (s, 1 H) , 7.30 (m, 5 H) , 5.50 (ddd, 1 H, J=10,lO,7 Hz) , 5.39 (dq, 1 H, J=9 / 7 HZ), 5.01 (m, 3 H) I 4.77 (dd, 1 H, J=11/7 130 Hz) , 4.38 (d, 1 H, J=4 Hz) , 4.29 (d, 1 H, J=4 Hz), 3.45 (dd, 1 H, J=14,10 Hz) , 3.31 (dd, 1 H, J=14,7 Hz) , 2.24 (m, 1 H) , 2.08 (m, 1 H) , 1.74 (d, 3 H, J=7 HZ), 1.61 (m, 2 H) , 1.47 (d, 3 H, J=7 Hz) , 1.45 (d, 3 H, J=7 Hz) , 1.09 (d, 1 H, J=7 Hz) , 1.07 (d, J=6 Hz), 1.05 (d, J=7 Hz), 0.93 (t, J=6 HZ). Patel1amide C (5S). IR (CH2C12) 3380, 1675, 1655, 1535, 1510 cm-1 j high resolu-tion EI mass measurement, obsd 762.2973, C37H46NS06S2 requires 762.2981; 13C-NMR (CDC13) o 173.2 (s) , 173.0 (s) , 172.6 (s) , 170.8 (s) , 16S.0 (s) , 167.9 (s) , 161.S (2C) (s) , 147.5 (s) , 147.2 (s) , 136.1 (s) , 129.1 (2C) (d) , 128.6 (2C) (d) , 127.0 (d) , 123.7 (d) , 123.5 (d) , 82.3 (2C) (d), 73.6 (2C) (d), 55.8 (d), 53.1 (d), 52.3 (d), 46.5 (d), 40.7 (t), 32.6 (d), 27.7 (d), 24.S (t), 21.0 (q), 20.S (q), 20.6 ( q), 19 • 6 ( q), 19 • 1 ( q), 15 • 0 ( q), 8. 5 ( q); 1 H - NMR ( CDC 13 ) o 7.62 (m, 4 H), 7.50 (s, 1 H), 7.44 (s, 1 H), 7.39 (m, 5 H), 5.50 (ddd, 1 H, J=9,9,7 Hz), 5.36 (dq, 1 H, J=10,7 Hz), 4.98 (m, 2 H), 4.77 (dd,l H, J=11,8 Hz), 4.53 (dd, 1 H, J=11,8 Hz), 4.35 (d, 1 H, J=4 Hz), 4.26 (d, 1 H, J=4 Hz), 3.43 (dd, 1 H, J=14,9 Hz), 3.28 (dd, 1 H, J=14,7 Hz), 2.24 (m, 2 H), 1.74 (d, 3 H, J=7 Hz), 1.62 (m 2 H), 1.44 (d, 3 H, J=7 Hz), 1.41 (d, 3 H, J=7 Hz), 1.09 (d, 3 H, J=7 HZ), 1.07 (d, 3 H, J=7 Hz), 1.04 (d, 3 H, J=7 Hz), 0.S9 (t, 3 H, J=7 Hz). Lissoc1inamide 1 (59). IR (CH2C12) 3390, 3320, 2940, 131 2820, 1672, 1640, 1542, 1500 em-I; high resolution EI mass measurement, obsd 705.2835, C35H43N705S2 requires 705.2771; 1H-NMR (CDC13) 0 8.97 (d, 1 H, J=6 Hz), 8.73 (d, 1 H, J=6 Hz), 8.05 (s, 1 H) , 8.03 (s, 1 H), 7.97 (d, 1 H, J=10 Hz), 7.31 (m, 5 H) , 5.35 (dd, 1 H, J=9,6 Hz) , 5.15 (t, 1 H, J=10 Hz) , 4.87 (dq, 1 H, J=4,6 Hz), 4.86 (m, 1 H) , 4.58 (t, 1 H, J=8 Hz) , 4.30 ( d, 1 H, J=4 Hz), 3.28 (dd, 1 H, J=13,5 Hz) , 3.27 (m, 1 H) , 2.91 (dd, 1 H, J=13,10 Hz), 2.78 (dqq, 1 H, J=10,7,7 Hz), 2.78 (m, 1 H), 2.22 (m, 1 H), 1.87 (m, 2 H), 1.65 (m, 2 H), 1.64 (m, 2 H), 1.46 (d, 3 H, J=6 Hz), 1.08 (d, 3 H, J=7 Hz), 1.04 (d, 3 H, J=7 HZ), 0.92 (t, 3 H, J=7 Hz), 0.75 (d, 3 H, J=7 Hz). Lissoe1inamide 2 (60). IR (CH2C12) 3380, 3315, 2985, 2935, 2860, 1674, 1638, 1530, 1440 em-I; high resolution EI mass measurement, obsd 679.2575, C33H41N705S2 requires 679 • 2 610; 13 C - NMR ( CDC 13 ) 0 18 2 • 6 ( s), 1 71. 8 ( s), 171. 8 (s), 171.7 (s), 169.1 (s), 169.1 (s), 159.6 (s), 148.3 (s), 135.6 (s), 129.9 (2C) (d), 128.2 (2C) (d), 126.7 (d), 123.7 (d), 80.8 (d), 78.7 (d), 75.6 (d), 56.7 (d), 54.2 (d) I 52.0 (d), 47.3 (t), 47.0 (d), 38.5 (d), 37.8 (t), 36.3 (t) , 28.7 (t) , 26.6 (t) , 25.5 (t) , 22.0 (q) , 21.7 (q) , 14.0 (q) , 11.7 (q) i 1H-NMR (CDC1 3 ) o 7.99 (s, 1 H) , 7.98 (d, 1 H, J=6 Hz) , 7.56 (d, 1 H, J=10 Hz), 7.18 (m, 5 H) , 6.70 ( d, 1 H, J=8 Hz) , 5.33 (dq, 1 H, J=7,8 Hz), 5.21 (ddd, 1 H, J=11,7,1 Hz) , 5.14 (m, 2 H) , 4.81 (dq, 1 H, J=5,6 Hz), 4.62 (t, 1 H, J=7 Hz), 4.27 (d, 1 H, J=5 132 Hz), 3.80 (m, 1 H), 3.48 (dd, 1 H, J=ll,ll Hz), 3.42 (dd, 1 H, J=11,7 Hz), 3.42 (m, 1 H), 3.17 (dd, 1 H, J=14,6 Hz), 3.02 (dd, 1 H, J=14,6 Hz), 2.45 (m, 1 H), 2.34 (m, 1 H), 2 • 14 ( m , 1 H), 2. 01 (m , 2 H), 1 • 56 ( d , 1H , J = 7 Hz), 1 • 51 (d, 3 H, J=6 Hz), 1.43 (m, 1 H), 1.32 (m, 1 H), 1. 02 (d, 3 H, J=7 Hz), 0.93 (t, 3 H, J=8 Hz). Lissoelinamide 3 (61). IR (CH2CI2) 3380, 3320, 3020, 2980, 2940, 1665, 1635, 1510, 1410 em-1 ; high resolution EI mass measurement, obsd 679.2588, C33H41N705S2 requires 679.2610; 13C-NMR (CDCI3) 0174.6 (s), 170.7 (s), 170.6 (s) , 170.6 (s) , 169.9 (s) , 169.0 (s) , 159.1 (s) , 148.0 (s) , 135.5 (s) , 129.6 (2C) (d) , 128.2 (2C) (d) , 126.9 (d) , 123.3 (d) , 81.7 (d) , 78.8 (d) , 74.9 (d) , 56.3 (d) , 53.2 (d) , 52.8 (d) , 47.3 (d) , 46.8 (t) , 40.3 (t) , 38.9 (d) , 35.2 (t) , 28.5 (t) , 25.0 (t) , 24.9 (t) , 24.4 (q) , 21.6 (q) , 14.8 (q) , 10.2 (q) ; 1H-NMR (CDCI3) 0 8.58 (d, 1 H, J=8 Hz), 8.24 (d, 1 H, J=7 Hz), 8.07 (s, 1 H), 7.70 (d, 1 H, J=10 Hz), 7.30 (m, 5 H), 5.37 (dq, 1 H, J=7,7 Hz), 5.21 (dd, 1 H, J=11,7 Hz), 4.95 (ddd, 1 H, J=9,8,5 Hz), 4.81 (m, 1 H), 4.77 (dq, 1 H, J=5,6 Hz), 4.59 (t, 1 H, J=8 Hz), 4.23 (d, 1 H, J=5 Hz), 3.73 (dd, 1 H, J=ll,ll HZ), 3.64 (dd, 1 H, J=11,7 Hz), 3.38 (m, 1 H), 3.21 (dd, 1 H, J=13,5 Hz), 2.95 (dd, 1 H, J=13,9 Hz), 2.35 (m, 2 H), 2.16 (m, 1 H), 1.82 (m, 1 H), 1.81 (m, 1 H), 1.75 (m, 1 H), 1.60 (m, 1 H), 1.56 (d, 3 H, J=7 Hz), 1.46 (d, 3 H, J=6 Hz), 1.32 (m, 1 H), 0.95 (t, 3 H, J=7 HZ), 0.91 133 (t, 3 H, J=7 HZ). Total acid hydrolysis of Lissoclinum peptides. The peptides (5 mg) and 6 N HCl (5 ml) were heated at 1000C for 18 h in a Pyrex threaded bomb sealed with a Teflon screw cap. The cooled reaction mixture was transferred to a 25 ml round-bottom flask and rotoevaporated to dry­ness. The hydrolysate was dissolved in MeOH (25 ml). Anhydrous HCl (gas) was bubbled through the solution for 1 min. Afterwards the solution was refluxed for 1 h. Upon cooling, the solvent was rotoevaporated and the residue suspended in CH2Cl2 (5 ml) with trifluoroacetic anhydride (1 ml) in a Pyrex threaded bomb sealed with a Teflon screw cap. The mixture was heated at 1500C for 10 min. The cooled reaction vessel was placed in ice and the solvent evaporated in a stream of nitrogen gas. The residues were resuspended in CH2Cl2 and used for GC and GC-MS analysis. The data below are presented as follows: (A) GC-EIMS analysis (fragmentation data; 3% OV-17, 6 ft x 1/8 in; program 60-250oC at 10°C/min, 5 min delay at 60oC); (B) GC retention for 0 and L isomers (12% SP-300, 12 ft x 1/8 in; program 110-140oC at 2°C/min, 30 min delay at 110°C). Ulicyclamide (54). Ulicyclamide contained L-threo-nine, L-proline, L-phenylalanine, isoleucine thiazole and alanine thiazole. Ulithiacyclamide (55). Ulithiacyclamide contained 2 L-threonines, L-cysteine and 2 leucine thiazoles. 134 Patellamide A (56). Patellamide A contained L-serine, L-threonine, 2 L-isoleucines and 2 valine thiazoles [m/z 310(M+), 279(M+-OCH3)' 268, 267, 235, 208, 166, 69]. Patellamide B (57). Patellamide B contained L-Ieucine, 2 L-threonines, L-isoleucine, an alanine thiazole[m/z 282 (M+), 2 51 (M+ - OCH 3)' 2 3 5 , 2 2 2, 2 07, 18 5 , 1 7 a , 15 3, 14 a , 138, 69] and a phenylalanine thiazole[m/z 358 (M+), 327(M+­OCH3)' 267, 235, 91, 69]. Patellamide C (58). Patellamide contained 2 L-threo-nines, L-isoleucine, L-valine, an alanine thiazole and a phenylalanine thiazole. Lissoclinamide 1 (59). Lissoclinamide 1 contained L-proline, L-threonine, L- phenylalanine, valine thiazole and isoleucine thiazole[m/z 324 (M+), 267 (M+-C4H9) , 236, 235, 208, 171, 139, 69]. Lissoclinamide 2 (60). Lissoclinarnide 2 contained L-threonine, L-proline, L- phenylalanine, D-isoleucine, L-cysteine and alanine thiazole. Lissoclinamide 3 (61). Lissoclinamide 3 contained L-threonine, L-proline, L- phenylalanine, D-isoleucine, L-cysteine and alanine thiazole. General procedure for reaction of peptides with singlet oxygen (~Q21. Triphenylphosphite (2.0 g, 6.4 mmol) was dissolved in CH2Cl2 (100 m1) in a round-bottom flask and cooled to -70oC in a dry ice/acetone bath. A stream of ozone was bubbled through the solution until a deep blue 135 ozone color persisted. A stream of N2 was bubbled through the solution, still at -70oC, to remove excess ozone. Immediately afterward, the round-bottom flask was fitted with a connecting hose adapter outleted to a second flask through Tygon tubing and a pipet. The second flask contain­ed the peptide (5-10 mg) dissolved in CH2Cl2 (30 ml) at room temperature. The flask containing triphenylphosphite ozonide was removed from the -70oC bath and allowed to warm slowly to room temperature. Vigorous bubbling was observed almost immediately in both flasks. After gas evolution ceased the progress of the reaction was moni­tored by TLC. The process was repeated if starting material remained. After the reaction was completed the CH2Cl2 was removed under a stream of nitrogen and the crude product treated directly with 6 N HCI (6 ml) for 20 h at 1000C in a Pyrex threaded bomb sealed with a Teflon screw cap. Afterwards the HCI was removed in vacuo to give a mixture of amino acid hydrochlorides. The mixture of amino acid hydrochlorides was dissolv­ed in MeOH (50 ml) in a round-bottom flask and anhydrous HCI bubbled through the solution for 1 min. The flask was fitted with a reflux condenser and refluxed for 1 h. Upon cooling the solvent was removed in vacuo, and the residue suspended in CH2Cl2 (5 ml) and trifluoroacetic anhydride (1 ml) in a Pyrex threaded bomb and heated to 136 1500 C for 10 min. The cooled reaction mixture was placed in a ice bath and carefully evaporated under a stream of nitrogen. The residue was taken up in CH2C12 (1 ml) and injected on the GC (In the case of alanine thiazoles, the ethyl ester was prepared instead of the methyl ester because of the presence of an ubiquitous peak in the GC trace of D- and L-alanine which complicated their identification). Determination of the chirality of thiazole amino acids. The chirality of all thiazoles amino acids was determined by GC retention times of the a-amino acid degradation products from 102 reaction on a chiral GC column (SP-300, 12 ft x 1/8 in; program 110-1400C at 2°C/min, 30 min delay at 1100 C). The general protocol called for the comparison of GC traces for the 102 reaction and the acid hydrolysis of the corresponding peptide without singlet oxygen treat­ment to define which peaks correspond to amino acids gene­rated by thiazole degradation. The chirality of the newly formed amino acids was determined by comparison or coinjec­tion with D,L amino acid standards. Ulicyclamide (54). Ulicyclamide contained L-isoleu­cine thiazole and D-alanine thiazole. Ulithiacyclamide (55). Ulithiacyclamide contained 2 D-leucine thiazoles. Patellamide A (56). thiazoles. Patellamide B (57). Patellamide contained 2 D-valine Patellamide B contained D-ala- 137 nine thiazo1e and D-phenylalanine thiazole. Patellamide C (58). Patellamide C contained D-ala­nine thiazole and D-phenylalanine thiazole. Lissoclinamide 1 (59). Lissoclinamide 1 contained D-isoleucine thiazole and L-valine thiazole. Lissoclinamide 2 (60). Lissoclinamide 2 contained D-alanine thiazole. Lissoclinamide 3 (61). Lissoclinamide 3 contained L-alanine thiazole. Dysidenin (114). Dysidenin contained L-alanine thia-zole. Isodysidenin (115). Isodysidenin contained L-alanine thiazole. Selective hydrolysis of the oxazoline ring. The pep­tide (10 mg) was refluxed in 5% H2S04/MeOH (20 ml) for 1 h. The reaction was basified to pH 12 with 10% aqueous NaOH and immediately extracted with CH2C12 (3 x 20 ml). The combined organic layers were dried over Na2s04 and evaporated to give an oil. The oil was N-acetylated by stirring in pyridine (100 microliters) and acetic anhydride (500 microliters) at room temperature for 24 h. Excess reactants were removed under vacuum. The residue was sapon­ified by stirring with 1% KOH/MeOH at room temperature for 1 h. After acidification to pH 1 with 10% aqueous HCl the product was extracted with CH2C12 (3 x 20 ml). The CH2Cl2 layers were dried over Na2so4 and evaporated, 138 and the residue was methylated with CH2N2 or by ref1uxing for 1 h in CD30D/HC1 to obtain the CD3 ester. The crude product was purified by HPLC (silica gel, EtOAc/MeOH, 95:5). High-resolution mass spectra (El and/or FAB) were obtained for the hydrolysis products. (100). Electron impact mass spectrum exhibited frag­ments at m/z: 751.2801, 677.2451, 622.2034, 594.2069, 497.1575, 453.1416, 257.0737, 223.0649, 195.0610, 180.0321, 138.0007. (101). Electron impact mass spectrum exhibited frag­ments at m/z: 470.2196, 411.2099, 284.1071, 268.1485, 116.0710. (105). Electron impact mass spectrum exhibited frag­ments at m/z: 442.1880, 383.1755, 284.1070, 240.1174, 116.0711. (106). Electron impact mass spectrum exhibited frag­ments at m/z: 518.2174, 459.2063, 299.1218, 284.1071, 116.0711. (107). Electron impact mass spectrum exhibited frag­ments at m/z: 504.2034, 445.1907, 285.1064, 270.0915, 116.0711. (108). The positive ion FAB mass spectrum exhibited fragments at m/z: 772.3202 (MH+) , 643.2384, 615.2439, 472.1843, 470.1848, 415.1584, 415.0925, 387.2011, 387.0951, 257.0753, 643, 629, 615, 472, 470, 431, 415, 387, 277, 257, 130. The negative ion FAB mass spectrum exhib