Aromatic alkaloids from ascidians

Atta-ur-Rahman (Ed.) Studies in Natural Products Chemistry, Vol. 23 © 2000 Elsevier Science B.V. All rights reserved

233

AROMATIC ALKALOIDS FROM ASCIDIANS BRUCE F. BOWDEN School ofBiomedical and Molecular Sciences, James Cook University, Townsville 4811 Qld., Australia ABSTRACT: More than 200 aromatic alkaloids isolated from marine tunicates are grouped into structural types and discussed in terms of their reported pharmacological activity. The major groups of alkaloids which are discussed in detail include the benzopentathiepins and trithianes, lamellarins and related structures, ecteinascidins, Pcarboline alkaloids, and the pyridoacridines. References to reported syntheses are also included. Effects of substituent changes within groups on observed activity (structure/activity relationships) are discussed where sufficient data is available to draw such conclusions. The current state of development and potential of the ecteinascidins and lamellarins in cancer treatment and control is summarised, together with details of current knowledge regarding their modes of action. The antiviral activity of the Pcarboline alkaloids (eudistomins and eudistomidins) is discussed in structural terms. Cytotoxicity data for the benzopentathiepins (lissoclinotoxins and varacins) and for the P)n"idoacridine alkaloids are summarised, together with evidence for their modes of action. The high potential for some of these alkaloids to be developed as therapeutic agents for the treatment of cancer and viral infection is reinforced by the summarised data.

INTRODUCTION Much interest and research over the past 15 or so years has been directed towards ascidian metabolites because of the high incidence of pharmacological activity that they display. Few comprehensive reviews exist on the topic; the only general ones being those produced by Davidson in 1993 [1,2] and the Marine Natural Product literature reviews by Faulkner which always contain a section on tunicate metabolites [313]. Although a number of non-nitrogenous metabolites have been isolated from the tunicates, the majority of compounds isolated have been derived from amino acids. In particular, the two most commonly encountered classes are cyclic peptides and polycyclic aromatic alkaloids. SCOPE AND ORGANISATION OF THIS REVIEW Although the division between the peptides and other nitrogen-containing metabolites derived from ascidians is not a clear cut one, the literature published on the non-peptidic, tunicate-derived alkaloids which contain aromatic (often in combination with heteroaromatic) rings has been summarised. The few linear peptides (which contain aromatic amino acid residues) have also been included because of their apparent structural

234

BRUCE F.BOWDEN

relationship to some of the reported alkaloids. Cyclic peptides, nucleosides and simple heteroaromatic molecules such as pyrroles and polypyrroles are outside the scope of this review. The emphasis is on the reported pharmacological activities of compounds and any leads to structure-activity relationships within groups of metabolites. For this reason, alkaloids are grouped in terms of structural similarity (or apparent biogenetic origin) rather than in groups based on their source species, genera, or families. This facilitates dealing with metabolites which have been isolated from two or more genera or families and avoids the question of whether such metabolites are produced by the host or by microbes (symbiotic algae, cyanobacteria, bacteria, fungi) which may be present. Some of the compounds which are discussed are strikingly similar to reported marine sponge metabolites. Indeed, in one or two cases the same metabolite has been isolated from members of these two very distinct groups of organisms. Such examples are likely to be the product of a symbiont which can co-exist with either organism, but proof of such a hypothesis is difficult. Although in sponges, some cell separation strategies have been used to locate metabolites within cells and to pin-point the likely producer of metabolites, to my knowledge, no comparable studies have been carried out with ascidians. I am also not aware of any authenticated examples of marine microorganism cultures which have yielded any of the chemicals isolated from extracts of their normal host. Simple Derivatives of the Amino acids Phenylalanine and Tyrosine The simplest aromatic alkaloids which have been isolated from ascidians are those derived directly or indirectly from phenylalanine, tyrosine, phenylethylamine or tyramine. Phenylethylamine (1) itself has been reported [14] from a Lissoclinum sp., as has its urea derivative (2) from Didemnum ternatanum [15]. The methyl ether of 3,5-diiodotyramine (3) and the corresponding urea derivative (4) were isolated from an unidentified Didemnid tunicate from Cocos lagoon, Guam, and (3) was reported to have marginal cytotoxic activity (IC50 20 [ig/mL against L1210

YV^

.V 1

R 1. R = R = H 3. R = I. R = OCH3 ^••••••••••••••••••••••••1

^NH2

/v.^^B^p''''s^

J"1L^ R

°

2. R = R = H 4. R = I. R = OCH3

1^

VA R

AROMATIC ALKALOIDS

235

murine lymphoma cells in culture)[16] arid antifungal activity against Candida albicans. No activity data was presented for the urea (4). The genus Aplidium has afforded some metabolites where the amino group of what initially appears to have been a dopamine residue has ended up as part of a heteroaromatic ring. Three such metabolites: (5), (6), and (7), were reported from Aplidium pliciferum [17]. No activity data was presented for these metabolites, however the apparently related 1,2,3trithiane (8) reported from the New Zealand Aplidium sp. D [18], exhibited antibacterial activity against gram positive bacteria {Bacillis subtilis) and fungi {Candida albicans), but no activity against gram negative bacteria {Escherichi coli and Pseudomonas aeruginosa). The trithiane ring underwent base-induced epimerisation to (9), and both isomers were marginally cytotoxic (IC50 12-13 |Lig/mL) to P388 murine leukemia cells in vitro. No antiviral activity was observed, but both displayed cytotoxicity to the host basal cell carcinoma (BSC) cells in the antiviral assay [18].

236

BRUCE F.BOWDEN

Lissoclinotoxins, Varacins and Related Metabolites A significant number of benzopentathiepin and benzotrithiane derivatives have been reported since the first report of a trithiane. The first reported example of these metabolites was lissoclinotoxin A [19], but the literature on this group is complicated by the instability of the free bases [20], the equilibrium between the pentathiepins and the trithianes [21], and the initial incorrect positional location of substituents in lissoclinotoxin A [19]. With the exception of the N-acetylated derivatives which have been reported [22], it appears that all benzopentathiepins which have been reported were in fact isolated as their protonated amine salts. The free bases are reported to be unstable; the instability is believed to result from attack by the free amino group on the pentathiepin ring [20]. Lissoclinotoxin A was initially reported [19] as the benzotrithiane (10) but the structure was subsequently corrected [21] to the benzopentathiepin (11). The chirality of such unsymmetrically substituted benzopentathiepins was noted by two authors [14,23] and optical activity has been reported [14], Lissoclinotoxin A has been reported from Lissoclinum perforatum [19,21] from the Eastern Atlantic where it co-occurred with lissoclinotoxin B (12), and from a Lissoclinum sp. from the Great Barrier Reef (GBR), Australia [14].

Varacin (13), the methyl ether of Lissoclinotoxin A, has been reported from Lissoclinum vareau [24], and from a "Far Eastern" Polycitor sp. (where it was also isolated as its N-acetyl derivative after acetylation of the crude extract to facilitate separation) [22]. A number of syntheses of varacin have been reported [20,25,26,27]. "Desmethylvaracin" (14) was

237

AROMATIC ALKALOIDS

reported as its trifluoracetate (TFA salt) from a Eudistoma sp. from Palau [28]. The salt of the thiomethyl derivative of varacin (15) was isolated as a 2:3 inseparable mixture with its trithiane analogue (16) from a Lissoclinum sp. from Pohnpei, while the N,N-dimethyl analogues (17), (18) of these two derivatives were isolated from Lissoclinum japonicum from Palau [28]. Lissoclinotoxins C (19) and D (20) were reported from an Australian collection of ?i Lissoclinum sp [14]. 0CH3

0CH3

s

\

1

HSCCLJ

s

/

s •^N ®

OCOCF3

15. R = H 17. R»CH3 OCH3 HO

1

f T S**^SCH3

1 NH2 19.

">^®

^OCOCF3

16. R=:H 18. R=CH3

1 1

OCH3

OCH3

1

"VY^W"

r-V 1

NH2

1

20.

NH2

1

1

^••••••••••^••^•••liHHHHMBl

The N-acetyl derivatives of varacins A (21), B (22) and C (23) were reported from fractionation of the acetylated extract of a "Far Eastern'' Polycitor sp. [22]. Antimicrobial Activity As a general rule, these sulfixr-containing derivatives of tyramine exhibit good antimicrobial activity : lissoclinotoxin A is reported to be active against a wide range of both gram positive and gram negative bacteria, as well as yeasts and fimgi [21]. Varacin (13) is reported to afford a 20mm zone of inhibition against Bacillis subtilis at 0.1 |Lig / disk [22].

238

BRUCE F.BOWDEN

Lissoclinotoxins A (11) and B (12) yield zones of 35mm and 30mm respectively against Staphylococcus aureus at 100 jig/disk [21], while the N,N-dimethyl metabolites (17, 18) afforded a 15 mm zone at the same concentration [28]. The reported antifungal activity of varacin against Candida albicans varies from zones of inhibition of 20 mm at 0.1 |Lig per disk [22] to 14mm at 2 |Lig/disk [24] while lissoclinotoxin A is reported to yield zones of inhibition of 27 mm at 40 jxg/disk, 19 mm at 10 |ig/disk and 8 mm at 1 ng/disk [14] with a minimum inhibitory concentration (MIC) of 40 fig/mL [21]. Lissoclinotoxin D was reported to be a little less active than A with inhibition zones of 19 mm at 40 ng/disk and 15 mm at 10 fxg/disk [14], and the N,N-dimethyl derivatives (17,18) are also reported to be active [28]. In order to produce comparable zones of inhibition to those observed with varamine using N-acetylvaramine against B, subtilis and C albicans, 10 and 100 times as much per disk respectively were required. Similar results were observed for the other N-acetylated derivatives, varacins A-C (14-16), yet the authors stated: "This series does not depend upon the presence of the free amino group for its activity "[22]. OCH3 HQCCX

AS^

OCH3 O

HOCCX

^-.^^

Q ^Q

9CH3 HQCC

Lissoclinotoxins A and B were active against ichthyopathogenic strains of Aeromonas salmonicida and Vibrio anguillarum , while lissoclinotoxin A also exhibited activity against resistant malaria, Plasmodium faciparum with an IC50 of 296 nM/L [21]. Cytotoxicity As well as having antimicrobial activity, the benzopentathiepins and benzotrithianes are cytotoxic. The cytotoxicity of these metabolites was first reported for lissoclinotoxin A in the form of a sea urchin egg assay, where a concentration of 16 |Lig/mL inhibited cell division [21], then the

AROMATIC ALKALOIDS

239

IC50 against L1210 murine leukemia cultured cells was reported in the range 1-4 fig/mL [19,21]. In in vivo screening against L1210 in mice, the survival rate of treated mice divided by controls and expressed as a percentage (T/C) was 125% at a dose of 3.125 mg/Kg, but dropped to 25% when a dose of 12.5 mg/Kg was utilised because of the toxicity of lissoclinotoxin A in mice (LD50 <50 mg/Kg) [21]. Varacin is more cytotoxic than lissoclinotoxin, with a reported ICsoof 0.05 |Lig/mL against HCT116 (human colon tumor cells) [24]. Preliminary assays suggested that the cytotoxicity was due to DNA damaging activity because of differential cytotoxicity towards the CHO cell line EM9 (chlordeoxyuridine sensitive) compared to BRl (BCNU resistant) cells [24]. The N,N-dimethyl derivatives (17, 18) were tested against the American National Cancer Institute (NCI) 60 cell line panel but no useful selectivity was found [28]. Protein Kinase C Inhibition Because Protein Kinase C-mediated phosphorylation of target proteins is thought to be critical to many aspects of cellular physiology that depend on control of mitosis or selective gene expression, inhibitors of PKC are sought as potential antineoplastic agents, as well as for their potential therapeutic value as cardiovascular, inflammatory, and CNS agents. The 2:3 mixture of 15 and 16 inhibited PKC with an IC50 of 0.3 iig/mL, and desmethyl varacin (14) showed comparable activity. The mixture of (15 and 16) was also separately tested against PKCa, PKCe and PKC^ (isozymes with different biochemical requirements); all IC50 values were in the range 0.8-2.1 |ig/mL with the highest activity shown against PKCa. Although the N,N-dimethyl analogues (17,18) had less activity than the other compounds against PKC, it was noted that the trithiane was twice as active as the pentathiepin, and this was interpreted as an indication that the activity is not merely due to release of elemental sulfur. The amino or dimethylamino group was concluded to be necessary for activity against PKC because the quaternary ammonium salt (24) which was prepared during structural elucidation was essentially inactive [28].

240

BRUCE F.BOWDEN

Other Metabolites Containing Residues of One or Two Aromatic Amino Acids Polycarpine (25), which was isolated from Polycarpa aurata collected at Chuk Atoll [29], inhibited inosine monophosphate dehydrogenase (an enzyme used to detect potential antiproliferative drugs) with an IC50 ^f 0.015^g/mL (0.03 jLiM). Inhibition was however reversible with excess dithiothreitol, which suggested polycarpine reacts with sulfhydryl groups on the enzyme.

Etzionine (26) was isolated from an unidentified Red Sea tunicate [30] and reported to exhibit antifiingal and antibacterial activity. The MIC for etzionine against C albicans was 3 ^ig/mL in RPMI-1640 broth and 12.5 ^ig/mL in Sabouraud dextrose broth. Antibacterial activity was observed against gram positive microorganisms (Aspergillis nidulans and B. subtilis), but it was inactive against gram negative bacteria. Etzionine exhibited marginal cytotoxicity (IC50 10 (xg/mL against P388 murine leukemia cells), although the crude extract of the red ascidian was more active (IC50 of 5

AROMATIC ALKALOIDS

241

^ig/mL against P388), inferring that an unidentified cytotoxic agent has yet to be isolated [30]. Polyandrocarpamides A-D (27-30), from the marine ascidian Polyandrocarpa sp. [31], are metabolites which appear to be derived from tyrosine (bromotyrosine, or iodotyrosine) and tryptophane. No activity was reported for the polyandrocarpamides.

27. 28. 29. 30.

Ri Ri Rj Ri

= Br. Ra = H = I. R2 = H = R2 = H = Br. Ra = COCH3

Similar metabolites which appear to be derived from tyrosine and brominated tyrosine include botryllamides A-D (31-34), which were isolated from brightly coloured styelid ascidians from the genus Botryllus collected in Fiji and in Australia [32]. Botryllamide D was reported to exhibit marginal cytotoxicity after 72 hour exposure to the human colon cancer cell line HCT 116 (IC50 17 |Lig/mL), but were inactive in vivo . The rubrolides, which were isolated from Riterella rubra [33] may well be derived from precursors similar to the botryllamides, however, the rubrolides are not alkaloids so have been mentioned here only for comparative purposes. Tunichromes The tunichromes are a group of reducing blood pigments from tunicates which were initially implicated in metal ion accumulation (particularly vanadium and iron), and have been recently reviewed [34]. They are dipeptides constructed from 2p(3,4,5-trihydoxyphenyl)alanine (Topa) or 2p(3,4-dihydoxyphenyl)alanine (Dopa) and a C-terminally bound 3,4dihydroxystyrylamine or 3,4,5-trihydroxy-styrylamine unit, and while it is now postulated that small proteins which are rich in Dopa and/or Topa units accumulate the metal ions rather than the tunichromes themselves.

242

BRUCE F.BOWDEN

OH

H3O

H3CO

3 1 . R = Br 32. R = H

H3CC

3 3 . R = Br 34. R = H

they are an interesting group of metabolites. Although in the strictest sense they are (decarboxylated) tripeptides, a brief overview is included in this review of alkaloids because of apparent structural similarities with rigidin, the polycitrins, polycitone A, the lukianols, ningalins, and lamellarins. The structure of Tunichrome B-1 (which is now referred to as Tunichrome An-1 because it was isolated from Ascidia nigra) was

AROMATIC ALKALOIDS

243

reported [35] to be (35), and subsequently verified by synthesis [36]. The structure of tunichrome Pm-1 (36) (from Phallusia mammosa) and two related tunichromes, Pm-2 (37) and Pm-3 (38), which contain one less double bond than Tunichrome An-1, were subsequently reported, with evidence that tunichrome in tunicate cells is present as the free peptide, not bound to vanadium [37]. A third group of tunichromes, Mm-1 (39) and Mm-2 (40) were reported [38] from the iron accumulating stolidobranch ascidian Morgula manhattensis. These are composed of two Dopa residues and either a glycine (Mm-1) or a leucine (Mm-2).

OH

HO

39. R = H 40. R = i-Bu

Related to the tunichromes are halocyamines A (41) and B (42) from Halocynthia roretzi. [39]. These are tetrapeptides which contain a Dopa residue (note: the authors have depicted a D-Dopa residue in their structure). Both halocyamines displayed antimicrobial activity against B, subtilis (MIC SOjig /mL), B. megateruine (MIC 50 fig/mL) , B, cereus (MIC 100 \k% /mL) and the yeast Cryptococcus neoformans (MIC 100 |Lig /mL). Halocyamine A was also found to be cytotoxic to neuroblastoma N18 cells at 160 ^iM for 24 hours. It caused degeneration of neurite and soma in cultured rat fetal brain cells at 100 ^xM and death of Hep-G2 cells at 200 \iM for 24 hours.

244

BRUCE F. BOWDEN

r Y ^r

.Xy V S

HO

OH

41. R = X. Ri =H 42.R = Y.Ri=X

•CH2~CH3 OH

The other modified linear peptides reported are virenamides A-E (4347) from the didemnid ascidian Diplosoma virens [40,41]. Strictly speaking, these metabolites are apparently derived from 4 amino acids so should be regarded as tetrapeptides, but are more conveniently regarded as carboxy-protected tripeptides (where the carboxyl group has been

44. R = i-Pr 45. R = CH2Ph

245

AROMATIC ALKALOIDS

converted, presumably via condensation with cysteine and subsequent decarboxylation, to a thiazole ring). While these metabolites do not contain Dopa or Topa residues they all contain at least one phenylalanine residue. The virenamides showed modest cytotoxicity towards a panel of cultured cells: virenamide A exhibited ICso's of 2.5 |ig/mL against P388 (murine leukemia), and 10 jig/mL against A549 (human lung carcinoma), HT29 (multidrug resistant human colon carcinoma) and CVl (monkey kidney) cells. It exhibited Topoisomerase II activity (IC50 of 2.5 jig/mL). Virenamides B and C both exhibited IC50 values of 5 ^ig/mL against P388, A549,HT29 and CVl cells. A number of metabolites which appear to be derived from Tyrosine, Dopa, or Topa residues and to have similar biogenetic origins to the tunichromes have been reported. Rigidin (48), isolated from the Okinawan marine tunicate Eudistoma c./ rigida exhibited calmodulin antagonistic activity with an IC50 of 5 x 10-^ M against calmodulin-activated brain phosphodiesterase [42]. Some other pyrrolo[2,3d]pyrimidine-2,4-diones which have been synthesised have shown weak affinity for the benzodiazepine receptor. ,

U

>

.

OH

0

fl

rfi 48.

1

/"

1

Lukianols, Polycitrins, Lamellarins and Related Compounds Lukianols A and B (49 and 50) were isolated from an unidentified ascidian collected at Palmyra atoll (N.E. of the Solomon Islands) [43]. Lukianol A exhibited moderate cytotoxicity (IC50 of 1 |Lig/mL against KB cell cultures), but lukianol B was 100 times less active [43]. The lukianols are closely related to polycitrins A and B (51 and 52), which were isolated from a Polycitor sp. [44]. Polycitrin A has been synthesised [45] by a proposed biomimetic route (based on known slime mould metabolites), but no activity data for the polycitrins has been published. These metabolites are some of the simplest of a group of highly substituted pyrrole derivatives

246

BRUCE F.BOWDEN

which have been isolated from prosobranch molluscs, ascidians and sponges. The apparent biogenetic origin of these metabolites is from two, three or more tyrosine, Dopa or Topa moieties.

49. R=»H 50. R r: I

51.R = H 52. R = CH3

The lukianols appear to arise by the same biogenetic pathways as for two metabolites (53 and 54) which were isolated from the southern Australian marine sponge Dendrilla cactos and called lamellarins O and P [46]. The choice of names is unfortunate, as these apparently tyrosinederived tetracyclic metabolites are structurally closer to the lukianols and polycitrins than to the Dopa and Topa derived hexacyclic lamellarins. Some evidence for restricted rotation of the phenolic aromatic rings in these metabolites was reported. Subsequently, lamellarins Q and R (55, 56) were reported from a sample oi Dendrilla cactos from southern New South Wales (Australia) [47]. Although both sponge extracts displayed mild antibiotic activity, the activity was not due to the lamellarins. Syntheses of lamellarin-0, lamellarin-Q, and lukianol-A have been reported [48]. Although lamellarins O - R were isolated from sponges the close structural association between these metabolites and those reported from ascidians is unquestionable. The ningalins were recently reported from an undescribed dark purple ascidian collected at Ningaloo Reef, Western Australia [49]. Ningalin A (57) appears to be a Dopa analogue closely allied to the tyrosine dimer lamellarin Q , while the pentacyclic ningalin B (58) fits into the biogenetic gap between the tetracyclic polycitrins, lamellarins O and P, and the more highly condensed hexacyclic

247

AROMATIC ALKALOIDS

lamellarins. No pharmacological activity has been reported for ningalins A and B.

COOCH3 55.

B3. X = H 54. X = OH

OH

57. COOCH3

OH

58.

248

BRUCE F.BOWDEN

Lamellarins A - D (59-62) were the first reported members [50] of what is now a fairly extensive group of alkaloids. Lamellarins A - D were initially isolated from prosobranch molluscs (which presumably fed on either ascidians or sponges). The structure of lamellarin A (59) was confirmed by x-ray analysis, and hindered rotation of the (oxygenated) phenyl ring was predicted [50]. Lamellarins E - H (63 - 66) were reported from the didemnid ascidian Didemnum chartaceum collected from the Republic of the Sechelles, atoll of Aldabra in the Indian Ocean [51], and two reports on the n.m.r. assignments for 5,6-dihydrolamellarin H (67) by 1,1-, l,n-, n,l- and n,n-ADEQUATE experiments have been published [52,53]. No information on the origin of 5,6-dihydrolamellarin has been given other than that it was "from a marine ascidian of the genus Didemnum ." Presumably it wasfromthe same source as lamellarins E-H. The synthesis of lamellarin-G trimethyl ether has been reported [54].

59. Rj = R3 = H. Ra = R4 = R5 = Re = CH3, X = OCH3. Y = O H ol. Rj — R3 =5 1 = H, R2 — R4 — R5 ~ R5 — Cri3, X s= O C H 3

63. Rj = R4 = I = H, R2 — R3 ^ R5 = Rg — CH3, X 5= O H 64. Ri = Y = H, R2 = R3 = R4 = R5 = Re = CH3. X = O H 60. Rj = R3 = R5 = CH3, R2 = R4 — Re = X = Y = H 67. Rj = R2 = R3 — R4 — R5 — Re ~ X ss Y = H 68. Rj = Y = H, R2 = R3 = R4 — R5 = Re ~ CH3, X = OCH3 69. Rj = Re = X = Y =H, R2 = R3 — R4 = R5 = CH3 70. Ri = R3 = Y = H. R2= R4 = R5 = Re= CH3. X = O H 71. Rj = R4 = Re = X = Y = H, R2 — R3 — R5 ''^ C H 3 74. Rj = R2 = R3 — R4 — Re '''^ X = Y = H, Rg = C H 3 75. Rj = R4 = Y = H, R2 = R3 = R5 = Re ~ OH3, X = O C H 3 76. RJ = R4 = X = Y s= H, R2 = R3 — R5 = Re ~ OH3

77. 80. 81. 82. 83.

Ri = R4 = H, R2 = R3 = R5 = Re = CH3. X = OCH3. Y = OH Ri = S03Na. R2 = R3 = R5=Re = X = OCH3. R4 = Y = H Ri = S03Na, R2=R3 = R5=Re = CH3. R4 = X = Y = H Ri = S03Na. R2 = R3 = R5 = Re = CH3, R4 = H. X = OCH3. Y = OH RJ = S03Na. Rg = R3 = Re = OCH3. R4 = R5 = X = Y = H

AROMATIC ALKALOIDS

249

Lamellarins I - N (68 - 73) were isolated along With the known lamellarins A - D from Didemnum chartaceum collected at Lihou Reef in the Coral Sea [55], while a sample of the same organism from the GBR yielded lamellarins A, B, C, I, K and M [55], Lamellarin S (74) was isolated along with lamellarin K fbom a Didemnum sp. collected from the southern coast of N. S. W. (Australia) [56]. Although x-ray data and molecular modelling indicate a barrier of around 85 kJ/mol to rotation of the phenyl ring [50,56], no optical activity had been observed for the lamellarins until the transient atropism reported for lamellarin S, where the half-life for optical activity was estimated at around 90 days [56]. Lamellarin K reisolated from fresh material collected at Lihou Reef had no observable rotation despite the rotation being measured 17 days after collection of the living organism (author's unpublished results). A fiirther five optically inactive lamellarins, lamellarins T-Y (75-79) were recently reported from an unidentified ascidian collected from the Arabian Sea. In addition, the 20sulfate esters of lamellarins T (80), U (81), V(82) and the 20-sulfate of lamellarin Y(83) were reported [57]. Lamellarin N (73) was also isolated and characterised; it had previously only been characterised as its triacetate [55].

1 ^\

y

R30—^

)

j^ hv^ T 1\ L«--K^ r^ 1

OH

R iQ

~ \

0

\ _ 0

1 60. 62. 66. 72. 73. 78. 79.

Rj = R3 = R4 = R5= CH3, R2 = H, X = OCH3 R| = R3 — R4 = Cri3, R2 — R5 = X = H Rj — R2 ~ R3 — R4 — R5 = X = H Ri = R3 = R4 = R5 = CH3. R2 = H. X = OH Rj = R2 — R4 = CH3^ R3 = R5 = H, X = H Ri « R2 » R4 = R6= CH3, R3 « H. X » OCH3 Ri = R2 = R4 = R5= CH3' R3 = H. X = OH

1 1 1 1 1 1 1

^ mmammmmmmmaammmmmmmmmmmmmmmmmmmmmmmmmamM

Antimicrobial Activity and Cytotoxicity The lamellarins generally do not exhibit much antimicrobial activity, but do show significant differential cytotoxicity. Lamellarin D was reported to cause 78% inhibition of development of fertilised sea urchin eggs at 19 |ig/mL, while lamellarin C reportedly caused only 15% inhibition, and lamellarins A and B were inactive at the same concentration [50], No activity data has been reported for lamellarins E - H, while from results of preliminary testing, lamellarins I, K and L were reported to show comparable and significant cytotoxicity against three different cell lines in culture with IC50 values of around 0.25 |ig/mL [55]. Lamellarins K and L

250

BRUCE F. BOWDEN

were also reported to exhibit moderate immunomodulatory activity [55]. A recent more extensive cytotoxicity assessment of a number of the lamellarins [58] is summarised in Table (1). Lamellarin D- triacetate and lamellarin K-triacetate displayed high potency against lung carcinoma cells (A549) in culture. Lamellarin N (73) was reported to show some selectivity in the NCI 60 cell line panel towards the melanoma cell lines SK-MEL-5 (LC50 1.87 x lO-^ M) and UACC-62 (LC50 9.88 x 10-^ M), but the lamellarin sulfates decomposed before they could be tested [57]. The selective cytotoxicity exhibited by several of the lamellarins makes them good candidates for further development as potential anticancer agents. Table 1.

Cytotoxicity of Different Lamellarins Against a Panel of Tumor Cells [58] Mean IC50 (M.M) Cell line:

1

A549

HT29

MEL28

0.71

0.9

2.1

0^93

5.5

18

5.2

>10

10.1 1

0.14

0.05

0.06

0.008

0.8

0.16 1

4.9

4.8

0.38

2

5

4.7

5 1

Lamellarin I-acetate

9

9.2

4.1

9

9.3

>10

9.1

1

Lamellarin J

2.9

3.9

0.58

1.2

0.6

5.8

2.9

1

Lamellarin K

0.19

0.017

0.19

0.75

0.18

0.38

0.4

1

Lamellarin K-triacetate

0.09

0.16

0.15

0.16

0.005

0.47

0.93 1

Lamellarin L

1.2

1.4

0.8

1.3

0.6

6

1.2

1

Lamellarin L-triacetate

2.4

2.4

2.2

2.5

1.1

>3

2.3

1

Lamellarin M

0.15

0.17

0.07

0.17

0.06

0.56

0.54 1

Lamellarin M-triacetate

0.91

1.1

0.76

3.1

0.22

>1

0.9

1

Lamellarin N-triacetate

0.32

0.3

0.1

0.16

0.02

3.2

1.6

1

P388

Schabel

Lamellarin A

0.89

0.91

0.36

Lamellarin B

10.1

10.4

Lamellarin D-triacetate

0.11

Lamellarin I

AUXBl CCH^C5

Perhaps even more interesting than the direct cytotoxicity is the reported ability of certain of the lamellarins to act on multidrug-resistant (MDR) cell lines, and to display resistance modifier activity [58]. Lamellarin I (68) in particular showed the highest chemosensitising activity of the lamellarins tested. Lamellarin I was 9-16 times more effective than verapamil (a well known chemosensitising agent), effectively increasing the cytotoxicity of doxorubin, vinblastine and daunorubicin in a concentration dependent manner in MDR cells. Results of in vitro measurements of rhodaminel23 accumulation in multidrug-resistant Lo Vo/Dx cells indicated that lamellarin I reversed MDR by directly inhibiting

251

AROMATIC ALKALOIDS

the P-glycoprotein-mediated drug efflux [58]. Further studies to assess the potential of the lamellarins as therapeutic agents for the treatment of cancer and for their activity as chemosensitising agents in MDR cells are in progress.

OH

OH

OH

HO

HO

Among the most highly substituted of the pyrrole derivatives reported from ascidians to date, is polycitone-A (84), which was isolated along with polycitrins A and B from a tunicate of the genus Polycitor [44], The biosynthetic processes which lead to (substituted) phenacyl substituents

252

BRUCE F.BOWDEN

at C2 and C5 of the pyrrole ring (in place of a carboxylic acid functionality, or hydrogen) are unclear. The penta-0-methyl derivative of polycitone A exhibited marginal cytotoxicity by inhibiting the growth of SV40 transformed fibroblast cells at 10 jig/mL. Polycitone A appears to be structurally similar to ningalins C (85) and D (86) as well as several reported sponge metabolites. Ningalins C and D co-occurred with ningalins A and B in the purple Didemnum sp. collected at Ningaloo Reef in Western Australia [49]. The origin of the additional carbon atoms needed to progress from a structure like ningalin B to the hexacyclic ningalin C or the octacyclic ningalin D has not been determined. The carbon skeleton for ningalin D is the same as for the sponge metabolite purpurone (87) which was reported [59] from an lotrochota sp. Also apparently structurally related are stomiamides A - D (88-91) from the Patagonian burrowing sponge, Cliona sp [60]. No pharmacological activity has been reported for any ningalins although speculation of the potential role these metabolites may play in iron accumulation is included in the report [49]. Purpurone is an inhibitor of ATP-citrate lyase with an IC50 value of 7 jig/mL [59], while the stomiamides exhibited antibiotic activity against gram positive bacteria (5. aureus, B. subtilis, Micrococcus luteus) at 50 fig/disk, but no activity against gram negative bacteria or the yeast C albicans [60].

OH

HO

HO

OH

HO

88. 89. 90. 91.

Ri = OH. R2 = R3 = H R, aR3=OH. R2 = H R i = H . R2 = R3 = OH Ri=R2=R2 = OH

253

AROMATIC ALKALOIDS

Tetrahydroisoquinoline Alkaloids - Ecteinascidins This group of alkaloids is reasonably uncommon in ascidians, but the sole group of representatives, the ecteinascidins, are potentially one of the most useful group of anticancer agents found to datefrommarine sources. The ecteinascidins are tetrahydroisoquinoline alkaloids which have been isolated from Ecteinascidia turbinata. Reports of the extraordinary cytotoxicity of extracts of Ecteinascidia turbinata date back to 1969 when in vivo screening of the crude extract against P388 leukemia in mice yielded T/C values of 272 with 4/6 cures. It was not however until 1990 that the first structures of the unstable cytotoxic constituents were eludidated and simulataneously published by two groups [61,62]. The ecteinascidins are similar in structure to the dimeric isoquinolinequinone alkaloids, the safracins and saframycins, which are microbial derived [63], and the sponge-derived renieramycins [64, 65]. The ecteinascidins have been named according to dominant ions in their mass spectra. Ecteinascidin 729 (92) = Et 729, Ecteinascidin 743 (93) = Et 743, Ecteinascidin 745 (94) = Et 745, Ecteinascidin 759A (95) = Et 759A, Ecteinascidin 759B (96) = Et 759B, and Ecteinascidin 770 (97) = Et 770. Several of the ecteinascidins have molecular weights 18 mass units higher than their names would suggest as a result of water loss in the mass spectrometer: the molecular weight of Et 729 is 747, while Et 743 and the two isomeric Et 759 structures have molecular weights of 761 and 777 respectively. X-ray OCH3

1

HQ^ OCOCH3 HgC^

"if

jl y A

H

^l

II

ijiRiT

1

1

\

H3CC

if' ^ ^ HO X \ ^

92. = H, R 2 = OH 93. Ri = CH3. R2 = OH 94. Ri = CH3. R2 = H 95. RI = CH3. R2 = OH 1 96. Ri = CH3. R2 = OH N-oxides 97. Ri -CH3. R2 = CN ^

1

254

BRUCE F. BOWDEN

Structures for 21-0-methyl-N12-formylecteinascidin 729, and for Et 759, the N-oxide of ecteinascidin 743 [66,67], confirmed the proposed relative stereochemistry. All members of this original group of ecteinascidins are composed of three tetrahydroisoquinoline units (A-C). A further six additional ecteinascidins have subsequently been reported. Two, ecteinascidin 722 (97) and ecteinascidin 736 (98), contain two tetrahydroisoquinoline units (A and B) but the C unit is a tetrahydro-Pcarboline residue [66]. The remaining four ecteinascidins, Et 597 (99), Et 583 (100), Et 594 (101) and Et 596 (102) are also biologically active and have been described as "putative biosynthetic precursors'* of the other ecteinascidins [68]. These ecteinascidins also have tetrahydroisoquinoline units A and B, but L-cysteine or its a-oxo analogue is the C unit. Identification of the absolute stereochemistry of that unit has led to confirmation that the absolute stereochemistries of the ecteinascidins are the same as for the safracins and saframycins [68].

CHa

H,C

98.R = H 9 9 . Rj s= CH 3

100. R = CH3 101. R = H

102. R = CH3. XY = OCH2O 103. R s CH3, X = OCH3. Y = OH

AROMATIC ALKALOIDS

255

Biogenesis of the Ecteinascidins Some biosynthetic studies involving incorporation of labelled tyrosine, cysteine, methionine, glycine and tryptophane [68,69], have already been performed on the ecteinascidins, to partially confirm the proposed biosynthetic origin of the A and B units from Dopa units. Further studies are in progress to test the hypothesis that the electrophilic ketone in an intermediate such as Et 594 or Et 596 can be condensed in a PictetSpengler reaction with a Dopa derivative to form the third tetrahydroisoquinoline group (C unit) in Et 729 or Et 743 [68]. Cytotoxicity The ecteinascidins show very good in vitro and in vivo activity, and have been reported to have activity against P388 lymphoma, B16 melanoma, M5076 ovarian sarcoma, Lewis lung carcinoma, and the LX-1 and MX-1 human lung and human mammary xenografts [70]. Et 743 exhibited IC50 values of 1.3 and 0.5 ng/mL against P388 [62] and L1210 [61] respectively in vitro , and a T/C of 167 at 15 ^ig/Kg against P388 in vivo [61]. Et 729 exhibited an IC50 value of 0.93 ng/mL against P388 [62] and afforded T/C values of 214 at 3.8 |ig/Kg against P388 and 246 at 10 fxg/Kg against B16 melanoma in vivo [61] . An approximately 1:1 mixture of Et729 and 743 afforded a T/C value of 188 against B16 melanoma at O.lmg/Kg administered daily for 9 days, and a T/C value of 147 against colon carcinoma 26 with the same dose regime [62]. Et 745 afforded an IC50 of 88 ng/mL against L1210 in vitro , and a T/C of only 111 at 250 mg/Kg against P388 in vivo [61]. Ecteinascidins 722 and 736 are both reported to be "highly active" against L1210 cells in vitro , and Et 722 is reported to be highly active in vivo against P388, B16 melanoma and Lewis lung carcinoma [71]. Comparative studies of Et 597, Et 583 and Et 594 against Et 743 and Et 729 reveal lower activities (4 to 100 times less) against P388 (murine lymphoma), A549 (human lung carcinoma), and HT29 (human colon carcinoma) [68]. Comparable activities were observed for Et 597 and Et 583 against MEL 28 (human melanoma) and for Et 597 against CV-1 (monkey kidney) cells. All strongly inhibit protein, DNA, and RNA synthesis, as well as RNA polymerase activity. The ecteinascidins also exhibit antimicrobial activity (5. subtilis) with MIC values (|Lig/disk) of 0.02 (Et 743), 0.08 (Et 729), 0.14 (Et 597), 0.74 (Et 583) and 0.37 (Et 594) [68]. Structure/activity relationships for the ecteinascidins are not clear at this point in time, but a comparison of reported activities for Et 743 and Et 745 clearly demonstrates the importance of the hydroxyl group to the cytotoxicity: its removal results in more than two orders of magnitude loss in activity. "The potent anticancer activity of ecteinascidins may, at least in part, be attributed to unit C, since the

256

BRUCE F. BOWDEN

related saframycin A (104), for example, which lacks the C unit, has lower efficacy than Et 729 in comparable tumor models" [68].

OCH3

°vV

CH3

1

*° 1

H3C

T

1 H3C0 T0 X \ ^i"^

^ V ^

NH

^ ^ ^ - ^ '^

^"

CN

CH3 104. ^•••^•^••••i^B

Mode of Action Ecteinascidin 743 has been demonstrated to bind in the minor groove of DNA by covalent attachment to the exocyclic amino group at position 2 of guanine. Comparison with anthramycin, another minor groove alkylating agent which alkylates at guanine N2 showed Et 743 covered 3-5 base pairs and exhibited a different sequence selectivity to anthramycin [72]. Computer modelling of the covalent adduct of Et 743 to DNA using the reactive carbinolamine group and N2 of guanine in the minor groove of the DNA double helix suggests that the A and B units stack against the DNA backbone [67]. Clinical Trials and Drug Supplies The preclinical pharmacology of Et 729 has been reported [73]. The current clinical plan for the more available ecteinascidin, Et 743, calls for three 0.5 mg doses per patient [74]. Ecteinacidin 743 is currently in phase 1 clinical trials in three European countries and in the United States [68]. The ecteinascidins have currently been extracted from harvested tunicate. Some partial syntheses of the ecteinascidin structure have been reported [70, 75], and one enantioselective total synthesis of ecteinascidin 743 has been published [74].

AROMATIC ALKALOIDS

257

Indole Derived Alkaloids These are perhaps the most diverse group of aromatic alkaloids isolated from ascidians. Their structures span a range of complexity, starting with 6-bromoindole-3-aldehyde (105) (previously reported from a marine Pseudomonas sp.), and the brominated quinazolinedione (106) from Pyura sacciformis [76]. CHO /

.Xx^ 105.

o II

1 1

rl

1

106.

1

6-Bromotryptamine (107) has been reported [14] from a collection of a Lissoclinum sp. from the GBR, and from Didemnum candidum from the Gulf of Mexico [77], while 5-bromo-N,N-dimethyl tryptamine (108) was reported from a New Caledonian collection of Eudistoma fragum where it co-occurred with woodinine [78]. 5-Bromo-N,N-dimethyltryptamine exhibited antimicrobial activity (with 12 mm and 17 mm zones of inhibition reported at 100 ^g per 6 mm disk against S.aureus and E coli resp.; 17mm and 22 mm zones at 200 |Lig per disk against the same microorganisms). A synthesis of 5-bromo-N,N-dimethyltryptamine has been reported [79]. H3C NHo y

6

C\H KJ

^ ^

Brv 107,

nK} 6H

1

N^— PHo 1

108.

1

Dendrodoine (109) [80,81], the related aminoimidazole (110) analogue [82] and the recently reported oxadiazinone, alboinon (111) [83] were reported from Dendrodoa grossularia. There are structural similarities between these tryptophane-derived metabolites, the phenylalanine-derived metabolites (5), (6) and (7), and the tyrosine-derived metabolite

258

BRUCE F. BOWDEN

r^C \c\ \KJw H

109.

0 IK:

Cr>

^'^^>^^'^{

"

110.

1

"1-

v^mmmmm

polycarpine (25). Dendrodoine, whose synthesis has also been reported [84], is cytotoxic (ID50 lOjig/mL against L1210 lymphoma cells) [85] and inhibits DNA synthesis in vitro, measured by incorporation of ^Hthymidine by cultured L1210 cells [81]; no activity data has been reported for (110) but its structural relationship to methyl aplysinopsin, a powerful antidepressant, has been noted [82]. Didemnimides A-D (112115) were isolated from the Caribbean mangrove ascidian, Didemnum conchyliatum [86]. The structure of didemnimide A was confirmed by xray analysis and the didemnimides were reported to be predator deterrents against carnivorous fish. Urochordamines A (116) and B (117) are larval settlement/metamorphosis-promoting alkaloids from the tunicate Ciona savignyi [87]. They also exhibited antibacterial activity against the gram positive marine microorganism Bacillus marinus but were inactive against gram negative marine bacteria and fungi.

112. 113. 114. 115.

R=Ri = H R = Br. Ri = H R = H. Ri = C H 3 R = Br. Ri = C H 3

1 1 6 . Ri = X. R 2 = Y 117. R i = Y . R 2 = X

259

AROMATIC ALKALOIDS

The symmetrical dimeric 6-bromoindole derivatives (118) and (119) have been reported from the Gulf of California tunicate Didemnum candidum, but no stereochemistry or activity data were reported [77]. Wakayin (120), a novel pyrroloiminoquinone alkaloid from an ascidian Clavelina sp., was reported to be cytotoxic (IC50 of 0.5|Lig/mL against HCTl 16) [88]. It inhibits topoisomerase II at 250 (xM and shows a 3-fold differential in cytotoxicity towards CHO cell line EM (which is DNA repair deficient) compared to BRl, indicating it exhibits cytotoxicity by interfering with or damaging DNA. It also e^diibits antimicrobial activity (MIC 0.3|Lig/mL against 5. subtilis). The structural similarity between wakayin and some sponge metabolites such as isobatzelline C and the discorhabdins/prianosines has been noted.

118.

119.

OCH3

120. NHCH3

121. R=:H 122. R»OH

11-Hydroxystaurosporine (121), another dimeric indole structure which was isolated from an unidentified tunicate [89], was reported to be a highly cytotoxic, powerful protein kinase C inhibitor. Cytotoxicity was characterised by an IC50 of 0.7 nM against KB cells, while the IC50 for

260

BRUCE F.BOWDEN

PKC inhibition was 2.2 nM. In a LoVo (MDR cytotoxicity) screen it exhibited 75% activity at O.OSfiM. The 3,11-diol (122) was also reported to co-occur, but was incompletely characterised. a-Carbolines a-Carbolines are not common tunicate metabolites. The grossularines [80,85] from Dendrodoa grossularia are the sole representatives of this structural class. The initial structure reported for grossularine (= grossularine-1) was incorrect [80], but it was subsequently revised when data for both grossularine-1 (123) and -2 (124) were reported [85]. Both grossularines are cytotoxic (ID50 = 6ng/mL for grossularine-1 and 4^g/mL for grossularine-2 against L1210 murine lymphoma), and both grossularines cause accumulation of cells in the Gl phase (at lOjig/mL and 1.5|ig/mL resp. by cell-flow cytofluorimetric analysis). Both are active down to 10 ng/mL in a cloning bioassay against WiDr (colon) and MCF7 (breast) solid human cancer cells. Grossularine-2 appears to act on DNA as a mono-intercalating agent [85]. Desmethyl grossularine-1 (125) has been reported from Polycarpa aurata from Chuk Atoll, but no activity data was presented [29].

OH 123. R=:CH3 125. R = H

124.

P-Carbolines and Tetrahydro- P-Carbolines These form one of the largest group of reported metabolites from ascidians, and are notable for the antiviral activity which is displayed by

261

AROMATIC ALKALOIDS

some members. The majority of these alkaloids are either named as eudistomins or eudistomidins. For the purposes of this review, these have been grouped into Cl-unsubstituted P-carbolines, P-carbolines with a Clpyrrolyl group, P-carbolines with a Cl-pyrrolinyl group, p-carbolines Structures and Sources of Some ^-Carboline Metabolites and Derivatives

Table 2.

1

Name

Source [Reference]

Substituents

Structure

Simple P-carbolines which are unsubstituted at CI

CS C6 C7 C8

N9

1

p-Carboline

126

H

H

H

H

H

Ritereliasigimiloides[\ 19]

1

Eudistomin N

127

H

Br

H

H

H

Eudistoma olivaceum [91,120]

1

Eudistomin 0

128

H

H

Br H

H

Eudistoma olivaceum [91,120] Riterella siginilloides [119]

1

Eudistomin D

129

Br OH H

H

H

Eudistoma olivaceum [91,120] Eudistoma glaucus [96]

1

Eudistomidin D

130

Br OH H

H

CH3

Eudistoma glaucus [96]

1

Eudistomin J

131

H

OH Br H

H

Eudistoma olivaceum [91]

1 Bromoeudistomin D

132

Br OH Br H

H

Synthetic [120]

1 Bromoeudistomidin D

133

Br OH Br H

CH3

Synthetic [123]

1

P-Carbc)lines with a 2-pyrrolyl substi tuent at CI

1

1

Eudistomin M

134

H

OH H

H

H

Eudistoma olivaceum [91,120]

1

1

Eudistomin A

135

H

OH Br H

H

Eudistoma olivaceum [91,120]

1

P-Carboline;s with a 2-(A-pyrrolinyl) su bstituentatCl Eudistomin I

136

H

H

H

H

H

1

Eudistoma olivaceum [91,120,99] Eudistoma glaucus [96]

1

Eudistomin G

137

H

H

Br H

H

Eudistoma olivaceum [91,120,99]

Eudistomin H

138

H

Br

H

H

H

Eudistoma olivaceum [91,120,99] Eudistoma glaucus [96]

1 1

Eudistomin Q

139

H

OH H

H

H

Eudistoma olivaceum [91,120]

Eudistomin P

140

H

OH Br H

H

Eudistoma olivaceum [91,120,99]

Eudistomidin A

141

H

Br

H

Eudistoma glaucus [116]

H OH

1

262

BRUCE F. BOWDEN

with other CI substituents, tetrahydro-P-carbolines and dihydro-Pcarbolines. The structures and sources of isolated p-carboline metabolites and their pharmacologically active derivatives for Cl-unsubstituted pcarbolines, P-carbolines with a Cl-pyrrolyl group, and p-carbolines with a Cl-pyrrolinyl group are tabulated in Table (2). Syntheses have been reported for eudistomins D [90, 91], H [91,92], I [91,93,92,94], N [91], M [94], O [91] and Q [91].

126. 127. 128. 129. 130. 131. 132. 133.

R = Ri = R2 =R3 = H R = Ri =: R3 = H, R2 = Br R = Ri = R2 = H. R3 = Br R = R3 = H, Ri = Br, R2 = OH R = CH3. Ri = Br. R2 = OH. R3 = H R = Ri = H. R2 = OH. R3 = Br R = H. Ri = R3 = Br. R2 = OH R = CH3, Ri = R3 = Br. Rj = OH

136. 137. 138. 139. 140. 141.

134. R = Ri = H 135. R = H. Ri=:Br

R = Ri = R2 = H R = R2=H. R i = B r R = Br. Rj = R2 = H R = OH. Ri = R2 = H R = OH. Ri = Br. R2 = H R = Br. Ri = H. R2 = OH

p-Carbolines which have other substituents at CI include eudistomin U (142)fromthe Caribbean didemnid Lissoclinumfragile[95], eudistomidins C (143) [96,97], E (144) [98] and F (145) [98]fromthe Okinawan ascidian Eudistoma glaucus , eudistomins R (146), S (147) and T (148) from the

263

AROMATIC ALKALOIDS

Caribbean tunicate Eudistomd dlivaceum [99], and eudistalbins A (149) and B (150) from the New Caledonian ascidian Eudistoma album [100]. The syntheses of eudistomins S [101], T [93,94,101,102,103,104] and U [90,105] have been reported.

SCHo

142.

S-^CHg

145.

146. R = H . R i = B r 147. R = Br. R i = H 148. R » Ri = H

CHo

149. R = H , Ri =NH2 150. RRi = 0

A number of tetrahydro-p-carbolines with various substituents at CI have been reported. They include woodenine (151) [78], from the New Caledonian ascidian Eudistoma fragum, eudistomidin B (152) from Eudistoma glaucus [96,97] and lissoclin C (153) from a Lissoclinum sp. collected on the GBR [14]. The latter, together with the report of eudistomin U and isoeudistomin U from Lissoclinum fragile collected in the Caribbean [95], are the sole reports of isolation of p-carboline alkaloids from didemnid ascidians; they are more generally found in the families Polycitoridae and Polyclinidae. The structure of woodenine has been confirmed by total synthesis [92,102]. Arborescidimes A-D (154-157),

BRUCE F. BOWDEN

264

examples of tetrahydro-p-carbolines which are brominated at C7, were reported from Pseudodistoma arborescens [106].

151

HaC

N—CHo

155. 156. R = H, Rj = OH 157. R = OH. Rj = H

The group of tetrahydro-P-carbolines that have an oxathiazepine ring include eudistomins C (158), E (159), F (160), K (161), and L (162), Ksulfoxide (163), and debromoeudistomin K (164). The occurrence of these eudistomins is summarised in Table (3), and the highest levels of antiviral activity have been exhibited by members of this group. The stereochemistry of the N-0 bond was initially suggested to be p [91], but later shown to be a [107]. The structure and absolute stereochemistry of eudistomin K has been confirmed by solution of its x-ray structure [108], while syntheses have been reported for eudistomin F [109], eudistomin L [110], N(10)-acetyleudistomin L [111,112] and debromoeudistomin L (= debromoeu-distomin K) [110, 113]. (-) 0-Methyldebromoeudistomin E has also been synthesised [113].

AROMATIC ALKALOIDS

265

NHR 158. 159. 160. 161. 162. 164.

Table 3.

R = Ri = H. R2 = OH. R3 = Br R = R3 = H, Ri = Br. R2 = OH R = COCH3, Rj = H. R2 = OH. R3 = Br R = Rj = R2 = H. R3 = Br R = Ri = R3 = H. R2 = Br R = Ri = R2 = R3 = H

163.

Reported Occurrence of Eudistomins that Contain an Oxathiazepine Ring Occurrence

1

Compound

Structure

1 Location [References]

1

Eudistomin C

158

Eudistoma olivaceum Riterella sigillinoides

Caribbean [91,125] New Zealand [119]

1 |

1

Eudistomin E

159

Eudistoma olivaceum Eudistoma album

Caribbean [91,125] New Caledonia [100]

|

1

Eudistomin F

160

Eudistoma olivaceum

Caribbean [91]

1

Eudistomin K

161

Eudistoma olivaceum Riterella sigillinoides

Caribbean [91,125] New Zealand [119]

Eudistomin K sulfoxide

163

Riterella sigillinoides

New Zealand [126,119]

Eudistomin L

162

Eudistoma olivaceum

Caribbean [91.125]

Debromoeudistomin K

164

Riterella sigillinoides

New Zealand [119]

|

There are only two reported examples of dihydro-p-carbolines. The first was isoeudistomin U which was initially reported to be a 4substituted dihydro-a-carboline derivative [95], but whose structure was revised after total synthesis to 3,4-dihydroeudistomin U (165) [114]. Recently, the dihydro—P-carboline (166) was reported from an undescribed ascidian of the genus Eudistoma [115].

-Q'

-R

165. R =: H 166. R = Br

1

266

BRUCE F.BOWDEN

Biosynthetic Studies Biogenetic work to date has concerned only those P-carbolines which have a 5-membered N-containing ring substituted at CI. Although the biogenesis of eudistomidin A (141) was speculated to be from condensation of tryptophane with glutamate [116], biosynthetic studies with eudistomins H (138) and I (136) indicate a more direct route from tryptophane and proline [117,118]. Data which indicate a proposed order for bromination and decarboxylation of tryptophane have also been presented. Activity of P-Carbolines, Tetrahydro-^-Carbolines and Dihydro-pCarbolines p-Carboline (126) is reported to inhibit HSV-1 and polio vaccine type 1 at a concentration of 2 ^g per disk [119]. In preliminary antiviral assays eudistomins D (129), N (127), and O (128) displayed moderate inhibition of HSV-1 [120], and eudistomin O was subsequently reported to inhibit HSV-1 and polio vaccine type 1 in vitro at a concentration of 500 ng/disk[l 19]. Eudistomins D, N, and O also exhibited antibacterial activity against B. subtilis , but although N and O were initially reported to also inhibit the yeast Saccharomyces cerevisiae [120], the activity was not supported in a subsequent report [91]. Remarkable synergism was however observed with mixtures of N and O which produced antibiotic activity while none was observed with either of the pure compounds [91]. An investigation of the photoactive properties of N and O showed that N was the most active (similar to harmine) in the presence of UVA light (long wavelength UV), and that O was moderately phototoxic [121]. Eudistomidin D (130) is cytotoxic with IC50 values of 2.4 |xg/mL and 1.8 [ig/mL against the murine leukemias L1210 and L5178Y respectively. It also induces Cd?^ releasefromthe sarcoplasmic reticulum, and is 10 times more potent than caffeine. Eudistomin J (131) was not reported to display any activity. The synthetic bromoeudistomin D (132) was reported to be a novel inducer of calcium releasefromfragmentedsarcoplasmic reticulum that causes contraction of skinned muscle fibres at >10mM[122]. Contractions were completely blocked by lOmM procaine. It was reported that bromoeudistomin D may induce Ca^"^ release from the sarcoplasmic reticulum through physiologically-relevant Ca^^ channels. Eudistomins D, J, N, and O were also tested; only D and J stimulated Cd?^ release but required 7 times higher concentrations than bromo eudistomin D, whose calcium-releasing effect was 400 times more potent than caffeine [91]. Results suggest that the 5-Br, 6-OH and 7-Br are all important for activity. Another semi-synthetic eudistomin derivative, N-methyl bromoeudistomin D (i.e. 7-bromo-eudistomidin D), was also subsequently

AROMATIC ALKALOIDS

267

reported to be a potent inducer of calcium release from the sarcoplasmic reticulum of skeletal muscle [123]. No activities have been observed for those eudistomins substituted at CI with a pyrrole residue: eudistomins A (135) and M (134) [120,91], although eudistomin M was reported to be moderately phototoxic [121]. Eudistomins G (137), H (138), and I (136) in preliminary antiviral assays exhibited moderate inhibition of HSV-1, and an investigation of the photoactive properties of H and I showed that the presence of UVA (long wavelength LFV) light had little effect on their activity [121]. Eudistomin I also displayed antibacterial activity against B, subtilis [120,91]. Eudistomins H and P (140) were reported to inhibit the yeast S, cerevisiae , while eudistomins P and Q (139) displayed antibacterial activity against B. subtilis [120,91]. Eudistomidin A (141) is a calmodulin agonist with an IC50 of 2 X 10-^ M against calmodulin-activated brain phosphodiesterase [116]. Eudistomin U (142) was not cytotoxic to CEM human leukemia lymphoblasts, nor did it exhibit antimicrobial activity against marine bacterial strains, although it exhibited strong activity against Agrobacterium tumefaciens [95]. In contrast to the activity of eudistomin A, eudistomidin C (143) is cytotoxic with IC50 values of 0.36 |Lig/mL and 0.42 ^ig/mL against L1210 and L5178Y cells in vitro and is a calmodulin antagonist, with an IC50 of 3 x 10-^ M [96]. No activities have been reported for eudistomidins E (144) and F (145) [98] . The original report of the structures of eudistomins R (146), S (147) and T (148) [99] contained no bioactivity data, but subsequent papers reporting the syntheses of eudistomin T have commented on its antimicrobial activity [124]. Eudistalbin A (149) is cytotoxic to KB human buccal carcinoma cells (ED50 3.2 |Lig/mL) while eudistalbin B (150) is inactive in the same screen [100], suggesting that the amino group, a feature also present in eudistomidin C, is necessary for cytotoxicity. Activity ofTetrahydro-^Carbolines The tetrahydro-P-carbolines generally exhibit higher levels of biological activity than their fully aromatic relatives. Antiviral activity is especially evident in the tetrahydro-P-carbolines that have an oxathiazepine ring, speculated to have been derived from cysteine [125]. Woodinine (151) displayed antimicrobial activity against S. aureus and Exoli, with zones of inhibition of 16mm and 8mm resp. at 100 |xg per 6 mm disk, and 18mm and 11mm resp. at 200 |ig / disk [78]. Eudistomidin B (152) was cytotoxic to both L1210 and L5178Y cells with reported IC50 values of 3.4 |Lig/mL and 3.1 \iglmL respectively. Eudistomidin B also activated rabbit heart muscle actomyosis ATPase by 93% at 3 x 10-^ M [96]. No activity data was reported for lissoclin C (153) [14]. The only arborescidime which was reported to be active was arborescidime D (157),

268

BRUCE F. BOWDEN

which was cytotoxic to KB human buccal carcinoma cells (IC50 3|ig/mL) [106]. The group of eudistomins which contain an oxathiazapine ring exhibit the highest levels of antiviral activity. Those with a phenolic hydroxyl are active against HSV-1 down to 5-lOng/ disk [91]. The order of antiviral activity has been reported to be eudistomin E (150) (which strongly inhibited HSV-1 at 25-50 ng/12.5 mm disk) [125,91] followed by eudistomin C (158), (which has been reported to strongly inhibit HSV-1 [125,119] and Polio vaccine type 1 [119] at 40-50 ng /disk), followed by L (162) (some inhibition of HSV-1 at 100 ng/ disk) [125,91] and K (161) (some inhibition of HSV-1 at 250 ng/ disk [125,91] or both HSV-1 and polio vaccine type 1 at 40-50 ng/disk) [119]. These eudistomins generally also exhibit antimicrobial activity; C against B. subtilis , E. coli and Penicillium atrovenetum ; E against B. subtilis ; K and L against B. subtilis, E. coli, S, cerevisiae and P. atrovenetum [91]. No activity of eudistomin F (151) has been reported [91]. Eudistomin E was also reported to be cytotoxic to KB human buccal carcinoma cells with an ED50 of <5.0 ng/mL (100% cytotoxicity at 5 ng/mL) [100] , while eudistomin K was reported to be cytotoxic both in vitro against P388 cells (IC50 0.01 fig/mL) and in vivo against L1210 and P388 murine leukemias as well as HCT-8, a human colon tumor. The T/C value of 137 at 100 |xg/Kg was reported for P388 [119]. Debromoeudistomin K (164) is active against HSV-1 and Polio vaccine type 1 at a concentration of 400 ng/disk, while eudistomin K sulfoxide (163) was reported to be active at the same concentration [119] and also at 200 ng/disk [126]. The bioactivity of the dihydro-p-carboline isoeudistomin U (165) was reported to be similar to that of eudistomin U: it was not cytotoxic to CEM human leukemia lymphoblasts, nor did it exhibit antimicrobial activity against marine bacterial strains, although it exhibited strong activity against Agrobacterium tumefaciens [95]. No activity data was reported for (166) [115]. Quinolines Two metabolites which were called trididemnic acids A (167) and B (168), were isolated from a Northeast Pacific ascidian Trididemnum sp. [127]. Xanthurenic acid (169) co-occurred with the trididemnic acids suggesting biosynthesis from tryptophane; no activity data was reported. Pyridoacridine Alkaloids Members of this group of alkaloids have been reported from ascidians (Urochordata), sponges (Porifora), anemones (Cnidaria) and one prosobranch (MoUusca), although the latter example is likely to be due to

269

AROMATIC ALKALOIDS

OH

O R^

OH

"A Ky lOl S^

^ ^

f^

- N ^ ""COOH

HO^

167. R = H 168. R = OH

OH

"S

T

"^COOH 1

OH 169.

L ^ ^ ^ ^ ^ ^

accumulation of the alkaloids from dietary intake. In one or two cases the same metabolite has been reported from both tunicates and sponges. The discussion here will deal only with those alkaloids which are tunicatederived, but is cross-referenced in cases where the same metabolite has also been isolated from another organism. The structure, synthesis and biological chemistry of marine-derived pyridoacridine alkaloids were reviewed by Molinski in 1993 [128]. Tetracyclic Pyridoacridine Alkaloids The known tetracyclic pyridoacridine alkaloids from marine sources are dominated by those isolated from tunicates. All reported tetracyclic pyridoacridine alkaloids to date have oxygenfimctionalityat C8, and the alkaloids can readily be divided into a group that has a carbonyl group at NH«

170.

171. 172. 173. 174. 175. 176. 177. 178.

R = X.Ri=H R = Y. R i = H R = Z. Ri = H R = X,Ri =OH R = Y, R, = OH R = X.Ri=OCH3 R = Y. Ri = OCHo R = X, R i = OOC(CH2)7CH=CH(CH2)7CH3

179. R = Y. Ri = 0 0 C ( C H 5 7 C H = C H ( C H 2 ) 7 C H 3

180. R = CH3. Ri a H

270

BRUCE F. BOWDEN

C8 (8//-pyrido[4,3,2-w« Jacridones) and a group that has an ether at C8 (the tautomeric, 11//-pyrido[4,3,2-w« Jacridines). The first group contains pantherinine (170) [129], cystodytins A-J (171-180) [130,131,132], diplamine (181) [133] and lissoclins A (182) and B (183) [14]. The second group contains norsegoline (184) [134,135], and varamines A (185) and B (186) [136]. Pantherinine (170), the simplest example to date of the first group, was isolated from the South Australian ascidian Aplidium pantherinum [129]. Cystodytins A -1 (171-179) were isolated from Cystodytes dellechiajei collected in Okinawa [130,131,137]. In each case the isomeric P,Pdimethyl acrylate and tiglate amides (R = X and R = Y) could not be separated and were characterised as mixtures. Cystodytin A was also reported from a Fijian Cystodytes sp., which in addition contained cystodytin J (180) along with a number of pentacyclic pyridoacridines [132]. Diplamine (181) was reported from a Diplosoma sp. [133], while lissoclins A (182) and B (183) (which only differ from diplamine by the nature of the acyl group in the amide side chain) were reported from a collection of a Lissoclinum sp. in Australia [14].

181. R = CH3 182. R = i-Bu 183. R = X

A

The structure of norsegoline (184), which was isolated from a Eudistoma sp. from the Red Sea, was disclosed in two reports [134,135], while varamines A (185) and B (186) were isolatedfroma Fijian collection of Lissoclinum vareau [136]. Syntheses of cystodytin J [138], other cystodytins [139], diplamine [138,140,141], and norsegoline [142,143] have been reported.

AROMATIC: AtKALOIDS

271

,

HN^R

COOCH3

f^ Y ^

J-Vi 184

•f**^^V'''^OCH3

^

'%/'^

^*^

T

11 II

1

v^*

1

1

185. R = CH2CH3 186. R = CH3

1 1

wmmmm^ammmmmmmmm^mmtmmmmmS

Biological Activity of the Tetracyclic Pyridoacridines The cytotoxicity of some of the tetracyclic pyridoacridines is summarised in Table (4). Despite the published statement [136] that "the varamines are about 1 order of magnitude more cytotoxic than the cystodytins which contain the same aromatic nucleus but lack the S-methyl group'', a comparison of the data for diplamine with that for cystodytin J does not support any significant cytotoxicity enhancement due to the thiomethyl group. The observed cytotoxicity difference between the varamines and comparable cystodytins is presumably due to their tautomeric aromatic nuclei. Based on limited data, among the cystodytins, the nature of the amide functionality on the side chain seems to have little effect on the cytotoxicity, but the presence of a hydroxyl group at the side chain benzylic position appears to marginally decrease the cytotoxicity while an ether functionality at the same position significantly enhances the activity. A detailed assessment of the antileukemic properties of norsegoline has been published [144], but no activity data has been reported for lissoclins A and B [14]. As well as their cytotoxicity, varamines A and B were also reported to exhibit potent antifungal activity [136], while the cystodytin A/B mixture and cystodytin C were 36 times and 13 times repectively more powerful than caffeine in calcium-releasing activity in sarcoplasmic reticulum [130]. Mode of Action Incorporation studies on a number of these alkaloids showed that pyridoacridines disrupt DNA and RNA synthesis with little effect on protein synthesis [132]. Results were consistent with DNA being the

272

BRUCE F. BOWDEN

Table 4.

Reported Cytotoxicity of Some Tetracyclic Pyridoacridine Alkaloids

1

Compound

Structure

IC50

Cell line

Reference

1

Pantherinine

170

4.5 ng/mL

P388

129

1

Cystodytin J

180

0.5 \ig/mL

HCT

132

CystodytinA/B

171/172

0.22 ng/mL

L1210

130

Cystodytin C

173

0.24 \ig/mL

L1210

130

Cystodytin D/E

174/175

1.1 ng/mL 1.4fig/mL

L1210 KB

131 131

CystodytinsF/G

176/177

0.068 |xg/mL 0.078 ng/mL

L1210 KB

131 131

Cystodytin H/I

178/179

0.080 \igfmL 0.092 fig/mL

L1210 KB

131 131

Diplamine

181

<0.5 ^ig/mL 0.02 jig/mL

HCT L1210

132 133

Varamine A

185

0.03 jig/mL

L1210

136

Varamine B

186

0.05 ng/mL

L1210

136

Shermilamine B

188

5ng/mL 0.3-0.4 jig/mL

KB P388

148 145

Shermilamine C

189

7|ig/mL 0.3-0.4 jig/mL

HCT P388

132 145

Kuanoniamine A

190

l.OjLig/mL

KB

148

Kuanoniamine B

191

lOfig/mL

KB

148

Kuanoniamine D

193

5 fig/mL

KB

148

Dehydrokuanoniamine B

194

3.3 jig/mL

HCT

132

1

primary target of the pyridoacridine alkaloids, which appeared to be DNA intercalators. The effect of diplamine on Topoisomerase-I (Topo-I) was assessed using the supercoiled DNA relaxation assay and diplamine inhibited Topo-I at a much higher concentration than the TopoisomeraseII (Topo-II) decatenation reaction. The IC90 value for the Topo II decatenation assay was 9.2|iM whereas Topo-I activity was still present at 14 mM. All tested pyridoacridines inhibited the Topo II -mediated decatenation of kinoplast DNA in a dose-dependent manner. In another study, shermilamine B was reported to inhibit Topo-II at 30 |LiM (compared to the reported [132] IC90 value of 118 |LIM) while shermilamine A was found to be inactive [145]. Disruption of the function of Topo II subsequent to intercalation is a probable mechanism by which

273

AROMATIC ALKALOIDS

pyridoacridines inhibit the proliferation of HCT (human colon tumor) cells. Pentacyclic Pyridoacridine Alkaloids Shermilamines A (187) [146,147] , B (188) [147,148], C (189) [132], kuanoniamines A-D (190-193) [148] and dehydrokuanoniamine B (194) [132] can be envisaged to arise from similar precursors to the cystodytins or varamines. Kuanoniamine C is identical to dercitamide which had previously been isolated from a sponge, Dercitus sp [149].

190.

fi

°

187. R = Br.Ri = CH3 188. R = H. Ri = CH3 189. R = H. Ri =X 191. 192. 193. 194.

R = i-Bu ^ R = CHj^H3 RsCHa R=X

Shermilamines A and B were isolated from a purple Trididemnum sp. [146,147], while shermilamine C was reported from a purple fleshy Cystodytes sp.fromFiji [132]. Both shermilamines A and B were reported in a paper which described metabolites from Amphicarpa meridiana and Leptoclinides sp. [145], while shermilamine B was also reported under the name debromoshermilamine from a Red Sea Eudistoma sp. [135]. Biosynthetic studies on shermilamine B have been reported [150]. The

274

BRUCE F. BOWDEN

kuanoniamines were isolated from the lamellarid mollusc, Chelynotus semperi and their unidentified tunicate food source from Pohnpei [148]. Dercitamide (191) and kuanoniamine D were subsequently reported from dark purple tunicates of the genus Cystodytes collected in Pohnpei [152], while dehydrokuanoniamine B [132] was isolated from a Fijian Cystodytes sp. Available cytotoxicity data on these metabolites, which is included in Table (4), indicate they are significantly less active than their tetracyclic analogues. The remaining pentacyclic pyridoacridine alkaloids can be divided into two structural groups: those with an additional angular ringfiisedat C9,10 of the acridine ring C include ascididemnin (195), 11-hydroxyascididemnin (196), and 2-bromoleptoclinidinone (197), while those with a linear ring fiision at C8,9 of ring C include amphimedine (198), meridine (199) and cystodamine (200).

^CHq

1 9 5 . Ri = R2 = H 1 9 6 . Ri = H. R2 = OH 1 9 7 . Ri = Br. R2 = H

198.

^^' 1 9 9 . R = OH 2 0 0 . R = NH2

Ascididemnin (195), initially isolated from an Okinawan Didemnum sp., was reported to be cytotoxic (IC50 of 0.39 |Lig/mL against L1210 murine leukemia), and to have calcium releasing activity in the sarcoplasmic reticulum (7 times more powerfiil than caffeine) [151]. It was

275

AROMATIC ALKALOIDS

subsequently reported from a Eudistoma sp. [153], and from a Leptoclinides sp. [145]. The latter confirmed the cytotoxicity (IC50 of 0.3 0.4 |ig/mL against P388 murine leukemia cells), and reported Topoisomerase-II inhibition at 75|LIM for ascididemnin. In addition, the isolation of 11-hydroxyascididemnin (196) was reported, and said to be cytotoxic although no data was supplied to support the assertion. The structure of 2-bromoleptoclinidinone, isolated from an ascidian tentatively identified as a Leptoclinides sp., was initially incorrectly reported [154], but subsequently corrected to (197) [155]. 2-Bromoleptoclinidinone was reported to be cytotoxic with an IC50 of 0.4 |Lig/mL against P388 (PS) cells [154]. "Leptoclinidinone" is synonymous with ascididemnin. The synthesis of 2-bromoleptoclinidinone has been reported [156]. Amphimedine (198) was a known metabolite from an Amphimedon sp. of sponge [157] but it has also subsequently been reported from a Didemnum collected in Okinawa [151], andfroma Mediterranean ascidian, Cystodytes delle chiajei [158]. Evidence was presented for the binding of amphimedine to DNA and it was proposed that inhibition of DNA synthesis (by intercalation) rather than inhibition of Topoisomerases caused the cytotoxicity [159]. A number of total syntheses of amphimedine have been reported [160,161,162,163,164,165]. The structure of meridine (199), which was isolated from the South Australian tanicatQ Amphicarpa meridiana [145, 166] and subsequently also reported from a sponge Corticum sp. [167], is supported by an x-ray structure. Meridine is reported to have antifiingal properties [167] and two total syntheses have been reported [168,169]. A stable tautomer of meridine (201) was also reported to be cytotoxic to P388 (PS) cells (IC50 of 0.3-0.4 ^ig/mL) but to be inactive against Topoisomerase II [145]. Cystodamine (200) was reportedfroma Mediterranean ascidian, Cystodytes delle chiajei [158]. It has an amino group in place of the hydroxy 1 group in meridine and was reported to be cytotoxic (IC50 of 1.0 |Lig/mL against CEM human leukemic lymphoblasts). .

1***^

Y""

^

'^0

(T

^

^

1

201. mm^mmmmm^mi^md

276

BRUCE F. BOWDEN

More Complex Polycyclic Pyridoacridines Among the more complex pyridoacridines from ascidians, the symmetrical dibenzotetraazaperylene eilatin (202) and eudistones A (203) and B (204) all are ring fiised at both C8,9 and C9,10 of the acridine C-ring, while segoline A (205) and isosegoline A (206) represent yet another site of fusion, Eilatin (202) was isolated from a Eudistoma sp. from the Red Sea, along with segoline A (205), isosegoline A (206) and norsegoline (184) [170,135]. Eilatin was also subsequently reported from a Fijian Cystodytes sp. [132], where it was reported to be responsible for the positive biochemical prophage induction assay which was being used as a rapid test for antitumor agents that interact with DNA, and to be moderately cytotoxic. Segoline A, isosegoline and eilatin have been reported to be potent regulators of cellular growth and differentiation, and to affect cyclic

AROMATIC ALKALOIDS

277

AMP-mediated processes [171]. No activity data has been published for eudistones A and B. Ungrouped Alkaloids Among those alkaloids whose biogenetic origins are not obvious are the polycarpamines. Polycarpamines A-F (207-212) were isolated from only one of a number of collection of Polycarpa aurata [172]. Only polycarpamine B exhibited significant antifungal activity {S. cerevisiae and C. albicans); the lack of optical activity for polycarpamine A and other polycarpamines with aliphatic methoxy groups suggests they may be artefacts of the isolation process.

H3CL

^CH3 N

HX(

H3(l

^CH3 N

H3CO'

" ^

A s ^^

1Y ^ ^s

OCH3

H3ca 207. 208. 209. 210.

R = H. Ri = OCH3 R,Ri = O RRi = S R = COCH3. Ri = OCH3

211. R = H 2 1 2 . R - COCHj1

CONCLUDING REMARKS Aromatic alkaloids from ascidians have provided a number of very promising leads especially in the area of cancer antiproliferative agents. The cytotoxic activity of the ecteinascidins and their application to clinical trials as anticancer agents stand out, but the potential of the lamellarins has yet to be fiilly investigated and exploited. A number of other metabolite groups also clearly hold potential: the antiviral activity of the eudistomins will undoubtedly be used as biochemical tools, whether or not they become clinical antiviral agents, and the mechanism of cytotoxicity of pyridoacridine alkaloids by suppression of DNA/RNA/protein synthesis will continue to be investigated.

278

BRUCE F.BOWDEN

With current emphasis on marine microorganism research it is conceivable that we may soon know the source of some of the metabolites which are common to certain ascidians and sponges. ABBREVIATIONS BSC Dopa Et GBR HCT ID50/IC50 LD50 MDR MIC NCI T/C TEA Topa

Basal cell carcinoma 3,4-Dihydroxyphenylalanine Ecteinascidin Great Barrier Reef, Queensland, Australia Human colon tumor cell culture Dose/Concentration required to reduce activity (or proliferation) by 50% Lethal dose for 50% of treated individuals Multi-Drug Resistant Minimum inhibitory concentration American National Cancer Institute Treated (usually days survival) divided by controls, expressed as% Trifluoracetic acid 3,4,5-Trihydroxyphenylalanine

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AROMATIC ALKALOIDS

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