Hartmut Laatsch

Interactions of melatonin and its metabolites with the ABTS cation radical: extension of the radical scavenger cascade and formation of a novel class of oxidation products, C2-substituted 3-indolinones

Abstract:  Melatonin had previously been shown to reduce up to four 2,2‘‐azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulfonic acid) cation radicals (ABTS•+) via a scavenger cascade ending with N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK). However, when melatonin is added to the reaction system in much lower quantities than ABTS•+, the number of radicals scavenged per melatonin molecule is considerably higher and can attain a value of ten. Under conditions allowing for such a stoichiometry, novel products have been detected which derive from AFMK (1). These were separated by repeated chromatography and the major compounds were characterized by spectroscopic methods, such as mass spectrometry (HPLC‐MS, EI‐MS and ESI‐HRMS), 1H nuclear magnetic resonance (NMR) and 13C NMR, heteronuclear multiple bond connectivity (HMBC) correlations. The identified substances are formed by re‐cyclization and represent 3‐indolinones carrying the side chain at C2; the N‐formyl group can be maintained, but deformylated analogs seem to be also generated, according to MS. The primary product from AFMK (1) is N‐(1‐formyl‐5‐methoxy‐3‐oxo‐2,3‐dihydro‐1H‐indol‐2‐ylidenemethyl)‐acetamide (2), which is obtained after purification as E‐ and Z‐isomers (2a, 2b); a secondary product has been identified as N‐(1‐formyl‐2‐hydroxy‐5‐methoxy‐3‐oxo‐2,3‐dihydro‐1H‐indol‐2‐ylmethyl)‐acetamide (3). When H2O2 is added to the ABTS•+ reaction mixture in quantities not already leading to substantial reduction of this radical, compound 3 is isolated as the major product, whereas 2a and 2b are virtually absent. The substances formed differ from all previously known oxidation products which derive from melatonin and are, among these, the first 3‐indolinones. Moreover, the aliphatic side chain at C2 is reminiscent of other substances which have been synthesized in the search for melatonin receptor ligands.

Evolving Trends in the Dereplication of Natural Product Extracts: New Methodology for Rapid, Small-Scale Investigation of Natural Product Extracts

The use of an HPLC bioactivity profiling/microtiter plate technique in conjunction with capillary probe NMR instrumentation and access to appropriate databases effectively short-circuits conventional dereplication procedures, necessarily based on multimilligram extracts, to a single, more rapid submilligram operation. This approach to dereplication is illustrated using fungal or bacterial extracts that contain known compounds. In each case the dereplication steps were carried out on microgram quantities of extract and demonstrate the discriminating power of (1)H NMR spectroscopy as a definitive dereplication tool.

Reactions of the melatonin metabolite AMK (N1-acetyl-5-methoxykynuramine) with reactive nitrogen species: Formation of novel compounds, 3-acetamidomethyl-6-methoxycinnolinone and 3-nitro-AMK

Abstract:  The melatonin metabolite N1‐acetyl‐5‐methoxykynuramine (AMK) was found to be unstable in air when adsorbed on a thin‐layer silica gel chromatography plate, a result that is in good agreement with the relatively high reactivity of this compound. Three novel main products were separated from the reaction mixture and identified by mass spectrometry and nuclear magnetic resonance data as: (i) 3‐acetamidomethyl‐6‐methoxycinnolinone (AMMC), (ii) 3‐nitro‐AMK (AMNK, N1‐acetyl‐5‐methoxy‐3‐nitrokynuramine), and (iii) N‐[2‐(6‐methoxyquinazolin‐4‐yl)‐ethyl]‐acetamide (MQA). AMMC and AMNK are shown to be nonenzymatically formed also in solution, by nitric oxide (NO) in the first case, and by a mixture of peroxynitrite and hydrogen carbonate, in the second one. The use of three different NO donors, PAPA‐NONOate, S‐nitroso‐N‐acetylpenicillamine and sodium nitroprussiate led to essentially the same results, with regard to a highly preferential formation of AMMC; AMNK was not detected in these reaction systems. Competition experiments with the NO scavenger N‐acetylcysteine indicate a somewhat lower reactivity compared with the competitor. Peroxynitrite led to AMNK formation in the presence of physiological concentrations of hydrogen carbonate at pH 7.4, but not in its absence, indicating that nitration involves a mixture of carbonate radicals and NO2, formed from the peroxynitrite‐CO2 adduct. No AMMC was detected after AMK exposure to peroxynitrite. Both AMNK and AMMC exhibited a much lower reactivity toward 2,2′‐azino‐bis‐(3‐ethylbenzthiazoline‐6‐sulfonic acid) (ABTS) cation radicals than did AMK. In a competition assay for hydroxyl radicals, AMMC showed prooxidant properties, whereas AMNK was a moderate antioxidant. AMMC and AMNK should represent relatively stable physiological products, although their rates of synthesis are still unknown and may be low. Formation of these compounds may contribute to the disappearance of AMK from tissues and body fluids.

Bioactive metabolites produced by Chaetomium globosum, an endophytic fungus isolated from Ginkgo biloba

A novel cytotoxic chlorinated azaphilone derivative named chaetomugilin D (1), together with three known metabolites, chaetomugilin A (2), chaetoglobosins A (3) and C (4), has been isolated by a bioassay-guided fractionation from the EtOAc extract of the cultures of Chaetomium globosum, an endophytic fungus found in the leaves of Ginkgo biloba. Structure of 1 was established by analyses of spectroscopic methods, including 2D-NMR experiments (COSY, NOESY, HMQC, and HMBC). Compounds 1-4 displayed significant growth inhibitory activity against the brine shrimp (Artemia salina) and Mucor miehei.

In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones.

A series of monomeric and dimeric naphthoquinones with potential for treatment of Leishmania infections was identified in vitro using both a direct cytotoxicity assay against extracellular promastigotes of Leishmania donovani, L. infantum, L. enriettii, and L. major and a test against intracellular amastigote L. donovani residing within murine macrophages. Several naphthoquinones proved to be active at concentrations in the microgram range (EC(50) 0.9-17.0 microg/ml). When tested against a panel of human cancer cell lines (KB, SKMel, A549, MDA) and murine bone marrow culture-derived macrophages (BMMPhi) as mammalian host cell controls, compounds with anti-Leishmania-activity showed moderate (EC(50)>25 microg/ml) to pronounced (EC(50)<10 microg/ml) toxic effects.

Essramycin : A First Triazolopyrimidine Antibiotic Isolated from Nature

In the course of our screening program for new bio-active compounds, a novel triazolopyrimidine antibiotic, essramycin (1), was obtained from the culture broth of the marine Streptomyces sp., isolate Merv8102. Structure 1 was established by intensive NMR studies and by mass spectra. The compound is antibacterially active with MIC of 2 to 8 μg/ml against Gram-positive and Gram-negative bacteria, while it showed no antifungal activity. The fermentation and isolation, as well as the structure elucidation and biological activity of 1 are described.

Integrated approach to explore the potential of marine microorganisms for the production of bioactive metabolites

During the last 10 years marine organisms have provided a large number of new natural products. Interesting compounds have mainly been derived from macroorganisms such as sponges, ascidians, corals and bryozoans. The number of secondary metabolites from marine microorganisms is smaller, but rapidly increasing. Because of the enormous difficulties involved in harvesting products from marine animals, and the fact that some of the bioactive compounds are produced by associated bacteria, the advantages of sustainable production of bioactive metabolites by bacteria or fungi, under the protection of natural resources, seem to be very attractive for the future. This review describes current progress in the isolation and identification of novel marine microorganisms, the discovery of new secondary metabolites, the biotechnological approaches to overproduce them, as well as the evaluation and characterization of their bioactivity.

Metabolic products of microorganisms. 185. The anthraquinones of the Aspergillus glaucus group. I. Occurrence, isolation, identification and antimicrobial activity.

The occurrence of emodin, erythroglaucin, physcion, physcion-9-anthrone, questin, catenarin, and catenarin-8-methyl ether in different species of the Aspergillus glaucus group (genus Eurotium) was investigated. So far catenarin-8-methyl ether (1, 4, 6-trihydroxy-8-methoxy-3-methylanthraquinone) has not been described as a natural product; it was therefore given the name rubrocristin. The chemical and physical properties of rubrocristin are reported. In addition a new violet pigment (C16H12O5) was isolated and characterized by its MS-, IR- and UV-spectra.The antimicrobial properties of all substances were examined in the agar diffusion assay. Gram-positive bacteria were the most sensitive organisms and catenarin was the most active naturally occurring substance. Synthetically obtained 1, 4, 6, 8-tetrahydroxy-anthraquinone was slightly more active than catenarin, whereas rubrocristin showed no antibacterial activity.

Mansouramycins A-D, cytotoxic isoquinolinequinones from a marine streptomycete.

Chemical screening of the ethyl acetate extract from the marine-derived Streptomyces sp. isolate Mei37 resulted in five isoquinolinequinones, four new derivatives, mansouramycin A-D (1, 3-5), and the known 3-methyl-7-(methylamino)-5,8-isoquinolinedione (2). Their structures were elucidated by NMR and MS techniques and by comparison with related compounds. Cytotoxicity profiling of the mansouramycins in a panel of up to 36 tumor cell lines indicated significant cytotoxicity of several derivatives, with pronounced selectivity for non-small cell lung cancer, breast cancer, melanoma, and prostate cancer cells.

1-Hydroxy-1-norresistomycin and Resistoflavin Methyl Ether: New Antibiotics from Marine-derived Streptomycetes † , ††

Cultivation of the marine-derived streptomycete isolate B8005 delivered three known antibiotics, resistomycin (1), resistoflavin (3a) and tetracenomycin (4), and a further member of the rare resistomycin class, the weakly antibiotically active 1-hydroxy-1-norresistomycin (2). From a related marine strain B4842, 1 and resistoflavin methyl ether (3b) have been isolated. The formation of 2 is of interest from a biosynthetic point of view.