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Lyngbyabellin A (1), a significantly cytotoxic compound with unusual structural features, was isolated from a Guamanian strain of the marine cyanobacterium Lyngbya majuscula. This novel peptolide is structurally related to dolabellin (2) in that both depsipeptides bear a dichlorinated beta-hydroxy acid and two functionalized thiazole carboxylic acid units. Its gross structure has been elucidated by spectral analysis, including 2D NMR techniques. The absolute stereochemistry of 1 was determined by chiral HPLC analysis of hydrolysis products and by characterization of the degradation products methyl 7,7-dichloro-3-hydroxy-2,2-dimethyloctanoate (3) and the corresponding acid 4. The total structure was further supported by molecular modeling studies. The isolation of 1 from L. majuscula once more supports the proposal that many compounds originally isolated from the sea hare Dolabella auricularia are of cyanobacterial origin. Lyngbyabellin A (1) was shown to be a potent disrupter of the cellular microfilament network.

Studies on the biosynthesis of cylindrospermopsin (1), a potent hepatotoxin associated with the cyanobacterium Cylindrospermopsis raciborskii, indicate that 1 is an acetogenin with guanidinoacetic acid serving as the starter unit of the polyketide chain. Feeding experiments show that C14 and C15 of 1 are derived from C1 and C2 of glycine, respectively, and C4 through C13 arise from five contiguous acetate units attached head to tail. The methyl carbon on C13 originates from the C(1) pool. The starter unit, established by the incorporation of [guanidino-(13)C,alpha-(15)N]-guanidinoacetic acid into N16 and C17 of 1, does not appear to be formed from glycine by known amidination pathways. The origin of the NH-CO-NH segment in the uracil ring is also unknown.

A synthesis of cryptophycin 52 (2) is reported using a Sharpless asymmetric dihydroxylation (AD) strategy to install the epoxide moiety. The high stereoselectivity of the AD reaction that allows for an efficient means of preparing the epoxide is in contrast to the standard direct epoxidation of cryptophycin substrates, which proceeds with poor diastereoselectivity. Methodology for conversion of the diol AD product to the requisite epoxide is disclosed. The transformation has been optimized to proceed in high yield in the presence of base sensitive functionality.

Lyngbyastatin 2 (1) and norlyngbyastatin 2 (2), new cytotoxic analogues of dolastatin G (3) and nordolastatin G (4), respectively, have been isolated as constituents from a Guamanian variety of the marine cyanobacterium Lyngbya majuscula. Structure elucidation of these cyclic depsipeptides relied on extensive application of 2D NMR techniques. The finding of these new metabolites further supports the proposal that many compounds originally isolated from the sea hare Dolabella auricularia are most likely of cyanobacterial origin.

3-Hydroxy-N-methylwelwitindolinone C isonitrile (3), 3-hydroxy-N-methylwelwitindolinone C isothiocyanate (4), and the novel cyclic ether N-methylwelwitindolinone D isonitrile (6) are three new alkaloids from two terrestrial Fischerella spp. belonging to the Stigonemataceae. Photooxidation of N-methylwelwitindolinone C isonitrile (1) leads to isonitriles 3 and 6. Isonitrile 3 is readily hydrated to 3-hydroxy-N-methylwelwitindolinone C formamide (5), an artifact produced during the isolation procedure.

Five new metabolites have been isolated from a lyngbyastatin 1- and dolastatin 12-producing assemblage of Lyngbya majuscula and Schizothrix calcicola collected at Tumon Bay, Guam. Structure elucidation employed 2D NMR techniques and chemical derivatization. These compounds have been assigned the trivial names tumonoic acids A (2), B (1), and C (5); methyl tumonoate A (3), and methyl tumonoate B (4). Compounds 1 and 4 were also found in a lyngbyastatin 1-producing strain of L. majuscula from Guam.

Ambiguine G nitrile is a new indole alkaloid from the terrestrial blue-green alga Hapalosiphon delicatulus (UH isolate IC-13-1). It is the first nitrile to be found in the Stigonemataceae.

Four monocyclic precursors were assembled in the total synthesis of the proposed structure 1-A of (+)-tolyporphin A O,O-diacetate (X=Ac). Comparison of the spectroscopic data demonstrated that synthetic tolyporphin O,O-diacetate did not match the O,O-diacetate prepared from natural (+)-tolyporphin A (X=H), calling for a structural revision of this class of natural products. On the basis of a series of NMR experiments including synthetic intermediates, the structure of tolyporphin A is concluded to be 1-B, in which the configurations of quaternary centers C7 and C17 are opposite to those in the originally proposed structure.

A new solid tumor selective cytotoxic analogue of dolastatin 10 (1) has been isolated from the marine cyanobacterium Symploca hydnoides, collected near Guam. This metabolite has been assigned the trivial name symplostatin 1 (2). This discovery supports the proposal that many compounds isolated from the seahare Dolabella auricularia, the original source of the dolastatins, are of dietary origin.

Cryptophycin 1 is a remarkably potent antiproliferative compound that shows excellent antitumor activity against mammary, colon, and pancreatic adenocarcinomas in mouse xenographs. At picomolar concentrations, cryptophycin 1 blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. The compound binds to tubulin, inhibits microtubule polymerization, and depolymerizes preformed microtubules in vitro. Its exceptionally powerful antitumor activity (many-fold greater than paclitaxel or the vinca alkaloids) raises important questions about its mechanism of action. By quantitative video microscopy, we examined the effects of cryptophycin 1 on the dynamics of individual microtubules assembled to steady state from bovine brain tubulin. At low nanomolar concentrations, in the absence of net microtubule depolymerization, cryptophycin 1 potently stabilized microtubule dynamics. It reduced the rate and extent of microtubule shortening and growing and increased the frequency of rescue. The results suggest that cryptophycin 1 exerts its antiproliferative and antimitotic activity by binding reversibly and with high affinity to the ends of microtubules, perhaps in the form of a tubulin-cryptophycin 1 complex, resulting in the most potent suppression of microtubule dynamics yet described.

Cryptophycin 46 (2), -175 (3), and -176 (4) have been identified as three new trace constituents of Nostoc sp. GSV 224. Cryptophycin-46 is an epimer of cryptophycin-3 (5) and to date is the only naturally occurring analogue having the S configuration at C-10 (C-2 in Unit B). Cryptophycins-175 and -176 also differ in unit B where 3 is the O-methyl analogue of cryptophycin-45 (6) and 4 is the O-desmethyl analogue of cryptophycin-21 (8). The relative and absolute stereochemistries of the three new analogues have been related to known cryptophycins by synthesis.

Historically, many new anticancer agents were first detected in a prescreen; usually consisting of a molecular/biochemical target or a cellular cytotoxicity assay. The agent then progressed to in vivo evaluation against transplanted human or mouse tumors. If the investigator had a large drug supply and ample resources, multiple tests were possible, with variations in tumor models, tumor and drug routes, dose-decrements, dose-schedules, number of groups, etc. However, in most large programs involving several hundred in vivo tests yearly, resource limitations and drug supply limitations have usually dictated a single trial. Under such restrictive conditions, we have implemented a flexible in vivo testing protocol. With this strategy, the tumor model is dictated by in vitro cellular sensitivity; drug route by water solubility (with water soluble agents injected intravenously); dosage decrement by drug supply, dose-schedule by toxicities encountered, etc. In this flexible design, many treatment parameters can be changed during the course of treatment (e.g., dose and schedule). The discovery of two active agents are presented (Cryptophycin-1, and Thioxanthone BCN 183577). Both were discovered by the intravenous route of administration. Both would have been missed if they were tested intraperitoneally, the usual drug route used in discovery protocols. It is also likely that they would have been missed with an easy to execute fixed protocol design, even if injected i.v.

Blue-green algae (cyanobacteria) are among the most primitive of organisms, having an evolutionary history spanning some four billion years. These procaryotic, photosynthetic microorganisms, which have the morphology and physiology of both bacteria and plants, are ubiquitous and adaptive to a wide variety of environments, including hot springs and polar ice caps. Although the most conspicuous species are found in freshwater and terrestrial habitats, it is estimated that the total number of autonomous marine species is more than double that of strictly terrestrial ones.

Lipophilic and hydrophilic extracts of over 900 strains of cultured blue-green algae (cyanophyta) were examined in vitro for their ability to inhibit the reverse transcriptases (RT) of avian myeloblastosis virus (AMV) and human immunodeficiency virus, type 1 (HIV-1). Eighteen (2.0%) aqueous extracts showed activity against AMV and HIV RTs. The maximal level of RT inhibition achieved by some of the active extracts was equivalent to that measured for 3'-azido-2',3'-di-deoxythymidine (AZT) at 668 ng/ml. Examination of partially purified fractions prepared by C18 column chromatography demonstrated that the RT inhibition observed could not be attributed entirely to the degradation of transcript DNA, template RNA, or enzyme protein in the reaction mixture. Thus, these results indicate that cultured blue-green algae may represent a novel source of compounds that inhibit RT activity, including that of HIV-1.

Laxaphycins A and B are the major components in an antifungal mixture of cyclic peptides from the terrestrial blue-green alga Anabaena laxa FK-1-2. NMR and MS spectral studies coupled with amino acid analysis indicate that the gross structures of laxaphycins A and B are cyclic (Aoc-Hse-E-Dhb-Hyp-Hse-Phe-Leu-Ile-Ile-Leu-Gly) where Aoc is a 3-aminooctanoic acid residue and cyclic (Ala-Hleu-Gln-N-MeIle-Hasn-Thr-Pro-Leu-Thr-Ade-Val- Hleu) where Ade is a 3-aminodecanoyl unit, respectively. Laxaphycin E, a minor cyclic undecapeptide, differs in gross structure from laxaphycin A in possessing a 3-aminodecanoic acid unit (Ade) in lieu of Aoc, whereas laxaphycin D, a minor cyclic dodecapeptide, differs from laxaphycin B in possessing a 3-aminooctanoyl unit (Aoc) instead of an Ade unit.

Mouse monoclonal and rabbit polyclonal antibodies were produced against conjugates of keyhole limpet hemocyanin and chemically defined palytoxin haptens. Palytoxin haptens were produced by derivatization of the primary amino group with sulfosuccinimidyl 4-( N -maleimidomethyl)cyclohexane-l-carboxylate or succinimidyl 3-(2-pyridyldithio)propionate. Selected antibodies were used to develop five palytoxin-specific enzyme-linked immunoassay formats for the quantitation of palytoxin in biological matrices, including crude extracts of Palythoa tuberculosa . The formats developed include an indirect competitive inhibition enzyme-linked immunoassay, two types of direct competitive inhibition enzyme-linked immunoassays, and both indirect and direct sandwich enzyme-linked immunosorbent assays. The sandwich enzyme-linked immunosorbent assays are capable of detecting as little as 10 pg palytoxin per test, but may be subject to matrix interference. The direct competitive inhibition enzyme-linked immunoassays detect as little as 30 pg palytoxin per test with a total assay time of only 4 hr. The enzyme-linked immunoassays do not cross-react with the other marine toxins tested, but do cross-react with certain non-toxic, treated preparations of palytoxin. The enzyme-linked immunoassays were used to quantitate palytoxin in P. tuberculosa extracts and to monitor toxin isolation. These enzyme-linked immunoassay systems can substitute for the mouse bioassay of palytoxin, providing a rapid, sensitive, and accurate means of toxin detection.

The isolation and structural elucidation of westiellamide [1] from the terrestrial blue-green alga Westiellopsis prolifica is described. This moderately cytotoxic cyclic peptide appears to be identical with a bistratamide-type marine natural product from the aplouso-branch ascidian Lissoclinum bistratum.

Tolytoxin [1], the major cytotoxin associated with Scytonema mirabile strain BY-8-1, Scytonema burmanicum strain DO-4-1 and Scytonema ocellatum strains DD-8-1, FF-65-1, and FF-66-3, has been shown to be 6-hydroxy-7-O-methylscytophycin B. Minor amounts of three other new cytotoxic scytophycins, 6-hydroxyscytophycin B [2], 19-O-demethylscytophycin C [3], and 6-hydroxy-7-O-methylscytophycin E [4], have also been isolated from these cyanophytes. The gross structures and stereochemistry are based on nmr and cd analysis and on comparison with scytophycins A-E.

Microcystin-LA accounts for the protein phosphatase (types 1, 2A and 3) inhibitory activity of the terrestrial blue-green alga Hapalosiphon hibernicus BZ-3-1.

Scytophycin B, 6-hydroxyscytophycin B, scytophycin E, and 6-hydroxy-7-O-methylscytophycin E account for the cytotoxicity and fungicidal activity of the terrestrial blue-green algaCylindrospermum muscicola.

(2S,4S)-4-Hydroxyarginine is an intermediate in the biosynthesis of anatoxin-a(s) from L-arginine.

Dihydroteleocidin B, which is a derivative of teleocidin from Streptomyces, showed potent tumor-promoting activity in vivo when painted on mouse skin. Although the chemical structure of dihydroteleocidin B is entirely different from those of phorbol esters, the tumor-promoting activity of dihydroteleocidin B was found to be comparable to that of 12-O-tetradecanoylphorbol 13-acetate (TPA) in vivo. Teleocidin from Streptomyces and lyngbyatoxin A and debromoaplysiatoxin from the marine blue-green alga Lyngbya majuscula induced ornithine decarboxylase activity when painted on mouse skin, their effects being similar to those of dihyroteleocidin B and TPA. 13-cis-Retinoic acid inhibited this ornithine decarboxylase induction when painted on the skin 1 hr before these natural products. These three compounds produced adhesion of human promyelocytic leukemia cells (HL-60) to the flasks and inhibited differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. The in vitro biological potencies of teleocidin and lyngbyatoxin A were almost as great as those of dihydroteleocidin B and TPA, but that of debromoaplysiatoxin was much weaker.

The gross structures of fragilamide and martensines A and E, the first basic indole alkaloids to be isolated from a marine eukaryotic plant, are reported.

Several new halogenated monoterpenes (3–6,10–15) have been isolated from Hawaiian Chondrococcus hornemanni which suggest that the halogenated myrcenes are formed by the enzymatic addition of BrCl followed by dehydrohalogenation.

Nine halogenated acetic acids and nine halogenated acrylic acids have been identified in the aqueous extract of Hawaiian Asparagopsis taxiformis .

A status report of a program initiated in early 1981 to evaluate extracts of cultured blue-green algae for pharmacological activity and to isolate and identify active agents is presented. Novel macrolides and nucleosides, for example, have been shown to be responsible for the cytotoxic and fungicidal activities of many blue-green algae belonging to the Scytonemataceae. In the last two years this program has been expanded to include studies on marine animals that harbor symbiotic algae. Macrolides, for example, have been found to account for the potent cytotoxicity of an Indonesian sponge Hyattella sp. and a Guamanian tunicate Lissoclinum patella.

The thiolase involved in biosynthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera generates an acetyl-enzyme species during catalysis. Up to 0.86 [14C] acetyl eq/subunit of this homotetrameric enzyme is accumulated by acid precipitation in the presence of [14C]acetyl-CoA. Gel filtration of the same solutions produced only 7% acetyl-enzyme suggesting hydrolytic lability of the acetyl-enzyme during the 10-min isolation at 4 degrees C. In an effort to identify active site residues which may function as basic groups to deprotonate at C-2 of acetyl-CoA to generate the required nucleophilic equivalent in carbon-carbon bond formation, we have prepared and tested haloacetyl-thioesters, oxoesters, and amides in the panthetheine pivalate series (Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., and Walsh, C. T. (1987) J. Biol. Chem. 262, 82-89). The [14C]bromoacetyl-oxoester alkylatively inactivates thiolase irreversibly with stoichiometric incorporation of four labels/tetramer. Determination of amino acid composition of the radiolabeled tryptic peptide indicated trapping of Cys-89 (Peoples, O. P., Masamune, S., Walsh, C. T., and Sinskey, A. J. (1987) J. Biol. Chem. 262, 97-102), the same residue modified by iodoacetamide. When the bromoacetyl-thioester was used, inactivation was pH-dependent. The data are consistent with the competition of two processes, acylation, and alkylation. Direct (rather than secondary) alkylation of thiolase by the inactivator accounts for the significant 14C incorporation into thiolase with the thioester labeled with [14C] in the pantetheine pivalate moiety. It appears likely that the haloacetyl analogs described herein should be generally useful for affinity labeling other enzymes using acetyl-CoA as a substrate.

The major cytotoxic metabolites of Lissoclinum patella from Pohnpei have been isolated and identified. Most of the cytotoxicity is attributed to a new cyclic peptide, ulithiacyclamide B [4], which is closely related to ulithiacyclamide [1]. The proposed structure of 4 is based on spectroscopic analysis and chemical degradation. Compound 4 exhibits an IC50 of 17 ng/ml against the KB cell line but does not show selective cytotoxicity against solid tumor cells in the Corbett assay.

The structure of phaseolotoxin, a toxin produced by cultured Pseudomonas syringae pv. phaseolicola , the causal agent of halo blight disease in bean plants is revised to 3. The structure of octicidin, isolated from leaves of bean plants infected with pv. phaseolicola . has been determined to be 4, a protease degradation product of 3 which is formed in the plant.

The preparation of [3H]lyngbyatoxin A by catalytic triton-proton exchange of lyngbyatoxin A and [3H]debromoaplysiatoxin by tritiation-debromination of aplysiatoxin is described. The dose-response curves for the binding of [3H]lyngbyatoxin A and [3H]debromoaplysiatoxin to a mouse epidermal particulate fraction are virtually the same as the one previously described for [3H]12-O-tetradecanoylphorbol-13-acetate ([3H]TPA). The specific binding of [3H]TPA, [3H]-lyngbyatoxin A and [3H]debromoaplysiatoxin to the mouse epidermal particulate fraction is inhibited to the same degree by unlabeled TPA, teleocidin, lyngbyatoxin A, aplysiatoxin and debromoaplysiatoxin. This study indicates that TPA, lyngbyatoxin A and debromoaplysiatoxin bind to the same high affinity receptor in mouse skin.

Majusculamides A and B are major lipophilic constituents of the blue-green alga Lyngbya majuscula Gomont. Detailed spectral analysis, chemical degradation, and x-ray crystallographic studies show that majusculamide A is N-((2R)-2-methyl-3-oxodecanoyl)-D-N,O-dimethyltyrosyl-L-N-methylvalinamide (6a) and that majusculamide B is N-((2S)-2-methyl-3-oxodecanoyl)-D-N,O-dimethyltyrosyl-L-N-methylvalinamide (6b). Majusculamide B is epimerized into a mixture of majusculamides A and B and then degraded into D-N,O-dimethyltyrosyl-L-N-methylvaline lactam (2) and racemic 2-methyl-3-oxodecanoic amide (1) at 140/sup 0/C in anhydrous dimethyl sulfoxide.