Campafungins: Inhibitors of Candida albicans and Cryptococcus neoformans Hyphal Growth.

Campafungin A is a polyketide that was recognized in the Candida albicans fitness test due to its antiproliferative and antihyphal activity. Its mode of action was hypothesized to involve inhibition of a cAMP-dependent PKA pathway. The originally proposed structure appeared to require a polyketide assembled in a somewhat unusual fashion. However, structural characterization data were never formally published. This background stimulated a reinvestigation in which campafungin A and three closely related minor constituents were purified from fermentations of a strain of the ascomycete fungus Plenodomus enteroleucus. Labeling studies, along with extensive NMR analysis, enabled assignment of a revised structure consistent with conventional polyketide synthetic machinery. The structure elucidation of campafungin A and new analogues encountered in this study, designated here as campafungins B, C, and D, is presented, along with a proposed biosynthetic route. The antimicrobial spectrum was expanded to methicillin-resistant Staphylococcus aureus, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Aspergillus fumigatus, and Schizosaccharomyces pombe, with MICs ranging as low as 4-8 µg mL-1 in C. neoformans. Mode-of-action studies employing libraries of C. neoformans mutants indicated that multiple pathways were affected, but mutants in PKA/cAMP pathways were unaffected, indicating that the mode of action was distinct from that observed in C. albicans.

Sphaerostilbellins, New Antimicrobial Aminolipopeptide Peptaibiotics from Sphaerostilbella toxica.

Sphaerostilbella toxica is a mycoparasitic fungus that can be found parasitizing wood-decay basidiomycetes in the southern USA. Organic solvent extracts of fermented strains of S. toxica exhibited potent antimicrobial activity, including potent growth inhibition of human pathogenic yeasts Candida albicans and Cryptococcus neoformans, the respiratory pathogenic fungus Aspergillus fumigatus, and the Gram-positive bacterium Staphylococcus aureus. Bioassay-guided separations led to the purification and structure elucidation of new peptaibiotics designated as sphaerostilbellins A and B. Their structures were established mainly by analysis of NMR and HRMS data, verification of amino acid composition by Marfey's method, and by comparison with published data of known compounds. They incorporate intriguing structural features, including an N-terminal 2-methyl-3-oxo-tetradecanoyl (MOTDA) residue and a C-terminal putrescine residue. The minimal inhibitory concentrations for sphaerostilbellins A and B were measured as 2 µM each for C. neoformans, 1 µM each for A. fumigatus, and 4 and 2 µM, respectively, for C. albicans. Murine macrophage cells were unaffected at these concentrations.

Arenicolins: C-Glycosylated Depsides from Penicillium arenicola.

During investigation of the secondary metabolism of four strains of Penicillium arenicola, two new depsides, arenicolins A (1) and B (2), were isolated and characterized. Their structures were established mainly by analysis of NMR and HRMS data and by comparison with known compounds. These depsides incorporate intriguing structural features, including dual alkyl side chains and a C-glycosyl unit, with 1 also containing an acylated 2-hydroxymethyl-4,5,6-trihydroxycyclohexenone moiety. Although the arenicolins were produced by all strains tested, arenicolin A (1) was obtained using only one of five medium compositions employed, while arenicolin B (2) was produced in all media tested. Neither compound showed antibacterial or antifungal activity, but 1 exhibited cytotoxicity toward mammalian cell lines, including colorectal carcinoma (HCT-116), neuroblastoma (IMR-32), and ductal carcinoma (BT-474), with IC50 values of 7.3, 6.0, and 9.7 µM, respectively.

Intramolecular [2 + 2] Photocycloaddition of Altrenogest: Confirmation of Product Structure, Theoretical Mechanistic Insight, and Bioactivity Assessment.

While studying the environmental fate of potent endocrine-active steroid hormones, we observed the formation of an intramolecular [2 + 2] photocycloaddition product (2) with a novel hexacyclic ring system following the photolysis of altrenogest (1). The structure and absolute configuration were established by X-ray diffraction analysis. Theoretical computations identified a barrierless two-step cyclization mechanism for the formation of 2 upon photoexcitation. 2 exhibited progesterone, estrogen, androgen, and pregnane X receptor activity, albeit generally with reduced potency relative to 1.

Environmental photochemistry of dienogest: phototransformation to estrogenic products and increased environmental persistence via reversible photohydration.

Potent trienone and dienone steroid hormones undergo a coupled photohydration (in light)-thermal dehydration (in dark) cycle that ultimately increases their environmental persistence. Here, we studied the photolysis of dienogest, a dienone progestin prescribed as a next-generation oral contraceptive, and used high resolution mass spectrometry and both 1D and 2D nuclear magnetic resonance spectroscopy to identify its phototransformation products. Dienogest undergoes rapid direct photolysis (t1/2 ∼ 1-10 min), forming complex photoproduct mixtures across the pH range examined (pH 2 to 7). Identified products include three photohydrates that account for ∼80% of the converted mass at pH 7 and revert back to parent dienogest in the absence of light. Notably, we also identified two estrogenic compounds produced via the A-ring aromatization of dienogest, evidence for a photochemically-induced increase in estrogenic activity in product mixtures. These results imply that dienogest will undergo complete and facile photolytic transformation in sunlit surface water, yet exhibit greater environmental persistence than might be anticipated by inspection of kinetic rates. Photoproduct mixtures also include transformation products with different nuclear receptor binding capabilities than the parent compound dienogest. These outcomes reveal a dynamic fate and biological risk profile for dienogest that must also take into account the composition and endocrine activity of its transformation products. Collectively, this study further illustrates the need for more holistic regulatory, risk assessment, and monitoring approaches for high potency synthetic pharmaceuticals and their bioactive transformation products.

Anti-Cryptococcus Phenalenones and Cyclic Tetrapeptides from Auxarthron pseudauxarthron.

Auxarthrones A-E (1-5), five new phenalenones, and two new naturally occurring cyclic tetrapeptides, auxarthrides A (7) and B (8), were obtained from three different solvent extracts of cultures of the coprophilous fungus Auxarthron pseudauxarthron. Auxarthrones C (3) and E (5) possess an unusual 7a,8-dihydrocyclopenta[a]phenalene-7,9-dione ring system that has not been previously observed in natural products. Formation of 1-5 was found to be dependent on the solvent used for culture extraction. The structures of these new compounds were elucidated primarily by analysis of NMR and MS data. Auxarthrone A (1) was obtained as a mixture of chromatographically inseparable racemic diastereomers (1a and 1b) that cocrystallized, enabling confirmation of their structures by X-ray crystallography. The absolute configurations of 7 and 8 were assigned by analysis of their acid hydrolysates using Marfey's method. Compound 1 displayed moderate antifungal activity against Cryptococcus neoformans and Candida albicans, but did not affect human cancer cell lines.

Pestaloporonins: Caryophyllene-Derived Sesquiterpenoids from a Fungicolous Isolate of Pestalotiopsis sp.

Three new sesquiterpenoids (pestaloporonins A-C; 1-3) related to the caryophyllene-derived punctaporonins were isolated from cultures of a fungicolous isolate of Pestalotiopsis sp. The structures of 1-3 were determined by analysis of NMR and HRMS data, and the structure of 1, including its absolute configuration, was confirmed by X-ray crystallographic analysis. Compounds 1 and 2 contain new bicyclic and tricyclic ring systems, respectively.

Diplodiatoxin, chaetoglobosins, and diplonine associated with a field outbreak of Stenocarpella ear rot in Illinois.

Stenocarpella maydis causes a fungal dry-rot of maize ears and is associated with diplodiosis, a neuromycotoxicosis in cattle grazing harvested maize fields in southern Africa and Argentina. There have been no reports of Stenocarpella metabolites in maize crop residues. Chemical investigations of S. maydis-infected grain from ears exhibiting different levels of ear rot severity following a 2010 field outbreak of Stenocarpella ear rot in Illinois led to the detection of diplodiatoxin and chaetoglobosins M and O as major components in the ethyl acetate extracts by LC-MS. Following post-harvest moist incubation of the S. maydis-infected grain, the amounts of each compound increased (approx. tenfold) and chaetoglobosin K was detected as a dominant toxin. In separate (1)H NMR-based analyses, the neurotoxin diplonine was detected as a minor component in methanol extracts of S. maydis-infected grain as well as cultures of S. maydis isolates from Midwest corn. Proline betaine (=stachydrine) and glycine betaine were also detected in these extracts as major components. This constitutes the first report of chaetoglobosin M, chaetoglobosin O, proline betaine, or glycine betaine from S. maydis, and the first record of diplodiatoxin, diplonine, proline betaine, glycine betaine, or chaetoglobosins M, O, or K being associated with a natural field outreak of S. maydis ear rot.

Isolation, characterization, and antioxidant activity of bromophenols of the marine red alga Rhodomela confervoides.

A total of 19 naturally occurring bromophenols, with six new and 13 known structures, were isolated and identified from the methanolic extract of the marine red alga Rhodomela confervoides. The new compounds were identified by spectroscopic methods as 3,4-dibromo-5-((methylsulfonyl)methyl)benzene-1,2-diol (1), 3,4-dibromo-5-((2,3-dihydroxypropoxy)methyl)benzene-1,2-diol (2), 5-(aminomethyl)-3,4-dibromobenzene-1,2-diol (3), 2-(2,3-dibromo-4,5-dihydroxyphenyl)acetic acid (4), 2-methoxy-3-bromo-5-hydroxymethylphenol (5), and (E)-4-(2-bromo-4,5-dihydroxyphenyl)but-3-en-2-one (6). Each compound was evaluated for free radical scavenging activity against DPPH (α,α-diphenyl-ß-dipicrylhydrazyl) and ABTS [2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt] radicals. Most of them exhibited potent activities stronger than or comparable to the positive controls butylated hydroxytoluene (BHT) and ascorbic acid. The results from this study suggest that R. confervoides is an excellent source of natural antioxidants, and inclusion of these antioxidant-rich algal components would likely help prevent the oxidative deterioration of food.

Biological activity of peanut (Arachis hypogaea) phytoalexins and selected natural and synthetic Stilbenoids.

The peanut plant (Arachis hypogaea L.), when infected by a microbial pathogen, is capable of producing stilbene-derived compounds that are considered antifungal phytoalexins. In addition, the potential health benefits of other stilbenoids from peanuts, including resveratrol and pterostilbene, have been acknowledged by several investigators. Despite considerable progress in peanut research, relatively little is known about the biological activity of the stilbenoid phytoalexins. This study investigated the activities of some of these compounds in a broad spectrum of biological assays. Since peanut stilbenoids appear to play roles in plant defense mechanisms, they were evaluated for their effects on economically important plant pathogenic fungi of the genera Colletotrichum, Botrytis, Fusarium, and Phomopsis. We further investigated these peanut phytoalexins, together with some related natural and synthetic stilbenoids (a total of 24 compounds) in a panel of bioassays to determine their anti-inflammatory, cytotoxic, and antioxidant activities in mammalian cells. Several of these compounds were also evaluated as mammalian opioid receptor competitive antagonists. Assays for adult mosquito and larvae toxicity were also performed. The results of these studies reveal that peanut stilbenoids, as well as related natural and synthetic stilbene derivatives, display a diverse range of biological activities.

Pterocarpenes elicited by Aspergillus caelatus in peanut (Arachis hypogaea) seeds.

The substituted pterocarpenes named aracarpene-1 (1) and aracarpene-2 (2) were isolated from wounded peanut seeds challenged by a strain of Aspergillus caelatus. The structures of these putative phytoalexins were determined by interpretation of NMR and MS data. The aracarpenes were investigated for their antifungal and antibacterial activities as well as antioxidant, anti-inflammatory, and cytotoxic activities in mammalian cells. Aracarpene-2 demonstrated high antibacterial properties against tested gram-positive and gram-negative bacteria, whereas aracarpene-1 displayed low antibacterial properties against the same bacteria. Both compounds had no antifungal activity against Aspergillus flavus. Together with peanut stilbenoids that are also produced in the challenged seeds, these compounds may represent a class of low-molecular weight peanut metabolites with a defensive role(s) against pathogenic microorganisms.

Cicadapeptins I and II: new Aib-containing peptides from the entomopathogenic fungus Cordyceps heteropoda.

Fermentation extracts of Cordyceps heteropoda (ARSEF #1880), an entomopathogenic fungus isolated from an Australian cicada, yielded a known antifungal compound, myriocin, and a complex microheterogeneous family of novel nonribosomal peptides, each containing two residues of alpha-aminoisobutyric acid (Aib). Structure elucidation of two major components of the peptide mixture, cicadapeptins I and II (1 and 2), was accomplished by amino acid analysis and various MS, 1-D NMR, and 2-D NMR experiments. Both compounds are acylated at the N-terminus by n-decanoic acid and amidated at the C-terminus by 1,2-diamino-4-methylpentane. The amino acid sequence of cicadapeptin I is N-terminus-Hyp-Hyp-Val-Aib-Gln-Aib-Leu-C-terminus. Ile substitutes for Leu in cicadapeptin II. To our knowledge, this is the first report from fungi of consecutive Hyp or Pro residues in a nonribosomal linear peptide. ROESY data indicated that the cicadapeptins adopt a helical conformation. Cicadapeptins I and II displayed antibacterial activity and limited antifungal activity.

Lowdenic acid: a new antifungal polyketide-derived metabolite from a new fungicolous Verticillium sp.

Lowdenic acid (1), a new antifungal metabolite with an unusual structure and a mixed biogenetic origin, has been obtained from nonsporulating cultures of a previously undescribed Verticillium sp. (MYC-406 = NRRL 29280) that was isolated as a colonist of polypore basidiomata. The gross structure of 1 was proposed by analysis of NMR data and confirmed by X-ray diffraction analysis, which enabled assignment of relative stereochemistry. Compound 1 occurs as an equilibrium E/Z mixture. The known antifungal metabolite canescin A (2) was also isolated.