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Three new indolocarbazole alkaloids, 3-hydroxy-4‘-N-methylstaurosporine (3), 3-hydroxy-4‘-N-demethylstaurosporine (4), and 3‘-demethoxy-3‘-hydroxy-4‘-N-demethylstaurosporine (5), were isolated from the marine ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. in addition to two known staurosporines. The structures were determined by 1D and 2D homonuclear and ¹H-detected heteronuclear NMR spectroscopy and from comparisons with published data. CD measurements for these five staurosporine derivatives, as well as the previously described seven staurosporines, are reported, confirming that all derivatives possess the 2‘S,3‘R,4‘R,6‘R configuration.

A phytochemical analysis of the leaves of Aglaia dasyclada collected in Yunnan Province (People's Republic of China) yielded five cyclopentabenzofurans (1−5) of the rocaglamide family that are common secondary metabolites of Aglaia species as well as four biogenetically related compounds of the aglain (7), aglaforbesin (8) and forbaglin (9, 10) types. In addition, the cinnamic acid amide dasyclamide (6), which is a putative biogenetic precursor of these compounds (7−10), was isolated. The structures of the new compounds (6−10) were assigned unambiguously from the combined use of 1D and 2D NMR spectroscopy and mass spectrometry.

Bioassay-guided fractionation of organic extracts of Cladosporium herbarum, isolated from the marine sponge Callyspongia aerizusa, yielded two new macrolide metabolites:  pandangolide 3 and 4 (1 and 2) and the known fungal metabolites pandangolide 2 (3), cladospolide B (4), and iso-cladospolide B (5). Also isolated were the antimicrobially active (against Bacillus subtilis and Staphylococcus aureus) furan carboxylic acids:  Sumiki's acid (6) and its new derivative, acetyl Sumiki's acid (7). All structures were elucidated by spectroscopic methods.

Bioassay-guided fractionation of an extract obtained from roots of Aglaia duperreana led to the isolation of 17 1H-cyclopenta[b]benzofurans of the rocaglamide type. Of the compounds isolated, four rocaglamide derivatives (2, 6, 11, and 16) were obtained as new natural products, and their structure elucidation was conducted by spectral methods. For bioassay-guided fractionation and determination of LC50 and EC50 values, neonate larvae of Spodoptera littoralis were employed. The results of chronic feeding assays have shown new aspects of the structure−activity relationship of rocaglamide derivatives. The substitution of a hydroxyl group at C-8b by a methoxyl substituent leads to a loss of insecticidal activity in a manner not previously documented in this compound class.

Two new indolocarbazole alkaloids, 3-hydroxy-3‘-demethoxy-3‘-hydroxystaurosporine (5) and 11-hydroxy-4‘-N-demethylstaurosporine (6), were isolated from the marine ascidian Eudistoma toealensis and its predator, the marine flatworm Pseudoceros sp. In addition, five known derivatives were isolated in their protonated states, which caused the pyran-ring system to adopt a boat conformation. The structures were determined by 1D and 2D homonuclear and ¹H-detected heteronuclear NMR spectroscopy and from comparisons with published data. The heteronuclear correlations were necessary to establish reliable data for the structure elucidation.

Three new triterpenoid saponins named silenosides A−C (1−3) were obtained from the roots of Silene vulgaris. Their structures were elucidated by spectral and chemical methods as β-d-galactopyranosyl(1→2)-β-d-glucuronopyranosyl-3β-hydroxy-23-oxoolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→3)-β-d-xylopyranosyl(1→4)-α-l-rhamnopyranosyl(1→2)-β-d-fucopyranoside; 3-O-β-d-galactopyranosyl(1→2)-β-d-glucuronopyranosyl-3β,16α-dihydroxy-23-oxoolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→4)-[β-d-glucopyranosyl(1→2)]-α-l-rhamnopyranosyl(1→2)-β-d-fucopyranoside; and 3-O-α-l-arabinopyranosyl(1→3)-[β-d-galactopyranosyl(1→2)]-β-d-glucuronopyranosyl-3β,16α-dihydroxy-23-oxoolean-12-en-28-oic acid 28-O-β-d-xylopyranosyl(1→4)-[β-d-glucopyranosyl(1→2)]-α-l-rhamnopyranosyl(1→2)-β-d-fucopyranoside, respectively.

Two samples of the marine sponge Stylissa carteri collected in Indonesia yielded two new bromopyrrole alkaloids:  debromostevensine (1) and debromohymenin (2), as well as nine other known congeners (3−11). The structures of the new compounds were unambiguously established on the basis of their NMR and mass spectra.

An undescribed Philippine marine sponge of the genus Strongylophora yielded a new meroditerpenoid−strongylophorine dimer (1) and the known meroditerpenoids, strongylophorine-2 (2), strongylophorine-3 (3), and strongylophorine-4 (4). The structures of the compounds were established on the basis of NMR spectroscopic and mass spectrometric data. The position of the inter-unit linkage in the new compound was elucidated after methylation and 1D ¹H NOE difference experiments. This is the first report wherein the¹H and ¹³C NMR data of the strongylophorine congeners are fully and unambiguously assigned on the basis of 2D NMR spectroscopy. Compounds 2 and 3 exhibited slight activity against Micrococcus luteus and Salmonella typhii, respectively. Compound 3 was active against the phytopathogenic fungus Cladosporium cucumerinum and also against the neonate larvae of the polyphagous pest insect Spodoptera littoralis (EC50 of 69 [±0.48 (S.E.)] ppm) when incorporated into artificial diet. Compound 1 was found to be the most active in the brine shrimp lethality test with a LC50 of 10.5 [±0.43 (S.E.)] μg/mL.

The marine soft coral Lobophytum pauciflorum collected from Mindoro Island, Philippines, yielded four new lobane diterpene derivatives:  the acetate congeners of epoxylobatrienol and lobatrienediol (2 and 7, respectively), a methoxyl congener of lobatetraene (10), and an oxepin congener of lobatrienetriol (11), and six known derivatives (1, 3−6, and 8). The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (¹H, ¹³C, COSY, ¹H-detected direct, and long-range ¹³C−¹H correlations) and mass spectrometric (EIMS) data. All of the compounds were active against the phytopathogenic fungus Cladosporium cucumerinum. Compound 1 was found to be active against the Gram-positive bacteria Bacillus subtilis and the yeast Saccharomyces cerevisiae. The isolated lobane diterpenes were also active in the brine shrimp lethality test. In the latter bioassay, compounds 8 and 10 were the most active congeners with LC50's of 0.64 and 4.18 μg/mL, respectively.

The Micronesian sponge Oceanapia sp. afforded three pyridoacridine alkaloids:  the known compounds kuanoniamine C (1) and kuanoniamine D (2), as well as the new N-deacyl derivative (3) of the kuanoniamines. Compounds 1 and 2 exhibited insecticidal activity toward neonate larvae of the polyphagous pest insect Spodoptera littoralis (LC50 of 156 and 59 ppm, respectively), when incorporated into artificial diet. Both compounds also showed toxicity in the brine shrimp lethality test with a LC50 of 37 μg/mL (compound 1) and 19 μg/mL (compound 2), respectively. The N-deacyl derivative did not show any remarkable effect in both bioassays. Cytotoxicity of the alkaloids was studied in vitro, using two human cell lines. The new derivative (3) appeared to be active in the same range of concentrations as kuanoniamine C (1) and D (2). The IC50 of 3 was 1.2 μg/mL toward HeLa cells and 2.0 μg/mL toward MONO-MAC 6 cells. In receptor binding assays compound 2 showed affinity to A1- and A2A-adenosine receptors with Ki values of 2.94 and 13.7 μM, respectively. Compound 1 was less active than compound 2, whereas compound 3 showed no affinity toward adenosine receptors. In addition, compounds 1−3 exhibited moderate affinity to benzodiazepine binding sites of GABAA receptors.

The marine sponge Dysidea herbacea collected from Indonesia yielded four new polybrominated diphenyl ether congeners 2−5 and the known derivatives 1, 6, and 7. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (¹H, ¹³C, COSY, ¹H-detected direct and long-range ¹³C−¹H correlations) and mass spectrometric (EIMS) data. All of the compounds were active against the Gram-positive bacteria Bacillus subtilis and the phytopathogenic fungus Cladosporium cucumerinum. The isolated polybrominated compounds were also active in the brine shrimp lethality test. In the latter bioassay, compounds 1 and 6 were the most active with LC50's of 0.96 [SE ± 0.19] and 0.94 [SE ± 0.70] μg/mL, respectively.

Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 micromol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting Km values for Mg2+-ATP, GSH, and spermidine of 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0. 09 mM, respectively, and a kcat of 415 +/- 78 min-1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (Ki = 0. 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).

The proteins on metaphase chromosomes theoretically may be distributed ubiquitously throughout the karyotype, may be present uniquely on individual chromosomes or classes of chromosomes, or may exist in any combination of the above. Separation of chromosomes according to size using sucrose velocity gradients in high capacity zonal centrifuge rotors allows sufficient fractionation of the genome to indicate the distribution of proteins within the karyotype. Flow cytometric analysis and direct microscopic analysis were used to evaluate qualitatively the types of chromosomes present in the fractions obtained. This report is the first quantitative evidence that some of the chromosomal proteins are not distributed ubiquitously on all of the chromosomes of the karyotype.

Stacking, Relaxation Time, Tryptamine, 5′-Adenosine Monophosphate Complex formation between 5′-adenosine monophosphate (5′-AMP) and tryptamine was in­ vestigated by measuring 13C chemical shifts and spin-lattice relaxation times. The chemical shift changes observed were attributed to ring current effects originating in the stacking of the two respective aromatic moieties and to puckering changes of the AMP ribose. Differences in the magnitude of the shifts of the aromatic carbons were related to the geometry of the complex and compared with those found for AMP selfassociation. Upon complex formation the relaxation times of especially the tryptamine indole carbons were greatly reduced, this was explained by an in­ crease in the particle size. Small changes found for the AMP carbons in solutions without tryptamine result from AMP selfassociation. Deviations from isotropic motion observed for the non-aromatic moieties are discussed.

A cell suspension culture of Catharanthus roseus was treated during the growth phase with the nucleoside analogue 5-azacytidine, a powerful inducer of cell differentiation, as evidenced with animal and human cell lines. After induction the cell culture was further incubated in a production medium in the absence of the inducer for 10 days. The extraction of the freeze dried cells and analysis by high performance liquid chromatography showed an additional intense peak in the elution profile, not present in extracts of untreated cells. The structure of the newly synthesized metabolite, elucidated by mass spectrometry and 1H and 13C NMR, was a lignan type compound, lirioresinol B mono-β-D-glucoside. This compound has hitherto not been found in Catharanthus roseus plants or cell cultures.

C -13 NMR data for linamarin and lotaustralin, obtained after feeding of either Zygaena Jilipendulae or Heliconius melpomone (Lepidoptera) with C-13 enriched valine and isoleucine respectively, indicate that intact units of amino acid with loss of the carboxyl group are incorporated during the biosynthesis of these cyanogenic glycosides.

In the search for endogenous cardiac glycosides, two different inhibitors of the sodium pump have been isolated from bovine adrenals. Inhibitor A with a molecular mass of 600 Da and a UV maximum at 250 nm was purified from 16 kg of bovine adrenals. The pure substance (<1 ng) inhibited the sodium pump of human red blood cells with an affinity similar to that of ouabain, yet it cross-reacted with antibodies against the bufadienolide proscillaridin A but not against the cardenolide ouabain. Inhibitor A was slightly more hydrophilic than ouabain on RP-C18 high pressure liquid chromatography. Hence, it showed properties similar to the proscillaridin A immunoreactivity (Sich, B., Kirch, U., Tepel, M., Zideck, W., and Schoner, W. (1996) Hypertension 27, 1073-1078) that increased in humans with systolic blood pressure and pulse pressure. Inhibitor B of the sodium pump with a molecular mass of 584 Da was purified 10(6)-fold from 20 kg of bovine adrenals. It cross-reacted with antibodies against ouabain but not with antibodies against proscillaridin A and inhibited the sodium pump of human and rat red blood cells with the same affinity as ouabain. All other properties, such as the retention time in a C18-reversed phase chromatography, molecular mass determination by electrospray mass spectrometry and fragmentation pattern, and UV and 1H NMR spectroscopic data, were identical to ouabain. Hence, sodium pump inhibitor B from bovine adrenals is the cardenolide ouabain.

A fraction of a methanolic extract of aerial parts of Anthyllis sericeayielded vicenin 2 and the two new compounds isorhamnetin 3-O-(2-O-β-glucopyranosyl-6-O-α-rhamnopyranosyl)-β-galactopyranoside and isorhamnetin 3-O-(2-O-β-glucopyranosyl)-βl-galactopyranoside-7-O-β-glucopyranoside.

Genistein-8-C-glucoside and the so far unknown genistein-6-C-glucoside have been isolated from Genista cinerea ssp cinerea. Their NMR characteristics and distribution within the section Spartioides of the genus Genista are reported.

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-alpha-L-rhamnopyranosyl-alpha-L-rhamnopyranosyl-beta-hydroxytetradec anoyl-beta-hydroxytetradecanoate (Rha-Rha-C14-C14), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.

A survey of alkaloids from Cytisophyllum sessilifoliumhas shown the presence of several quinolizidine and dipiperidine alkaloids. The investigation afforded a new alkaloid, 7-hydroxylupanine. The structure of this alkaloid was assigned spectroscopically, α-Isosparteine, sparteine, β-isosparteine, ammodendrine, lusitanine, isolupanine, 5,6-dehydrolupanine, lupanine, a tentative dihydroxylupanine, cis-adenocarpine and trans-adenocarpine have been found. The co-occurrence of quinolizidine alkaloids and adenocarpine appears to be a good chemotaxonomic character of the genus Cytisophyllum.

Analysis of the Philippine marine sponge Xestospongia ashmorica afforded four new manzamine congeners 1−4 and four known compounds 5 and 7−9. Compound 1 is the 6-deoxy derivative of manzamine X, while 2−4 are the N-oxides of manzamine J (5), 3,4-dihydromanzamine A (6), and manzamine A (7), respectively. The structures of the new compounds were unambiguously established on the basis of NMR spectroscopic (¹H, ¹³C, COSY, ¹H-detected direct, and long-range ¹³C−¹H correlations) and mass spectrometric (EI, FAB-MS, and electrospray ionization) data. Alkaloid N-oxide structures were confirmed by conversion to the corresponding tertiary bases by reduction with Zn/HCl. This is the first report of the occurrence of bioactive manzamine N-oxides in marine sponges. Compound 7 exhibited insecticidal activity toward neonate larvae of the polyphagous pest insect Spodoptera littoralis (with an ED50 of 35 ppm) when incorporated in artificial diet and offered to larvae in a chronic feeding bioassay. Compound 7 was also active against the Gram-positive bacteria Bacillus subtilis and Staphylococcus aureus. Cytotoxicity was studied in vitro using L1578y mouse lymphoma cells. From the alkaloids studied, the N-oxides 3 and 4 were the most active (ED50 = 1.6 μg/mL) followed by compound 7 (ED50 = 1.8 μg/mL).