Strobiloscyphones A-F, 6-Isopentylsphaeropsidones and Other Metabolites from Strobiloscypha sp. AZ0266, a Leaf-Associated Fungus of Douglas Fir.

Six new 6-isopentylsphaeropsidones, strobiloscyphones A-F (1-6), and a new hexadecanoic acid, (2Z,4E,6E)-8,9-dihydroxy-10-oxohexadeca-2,4,6-trienoic acid (7), together with sphaeropsidone (8) and its known synthetic analogue 5-dehydrosphaeropsidone (9) were isolated from Strobiloscypha sp. AZ0266, a fungus inhabiting the leaf litter of Douglas fir (Pseudotsuga menziesii). The structures of 1-7 were established on the basis of their high-resolution mass and 1D and 2D NMR spectroscopic data, and their relative and/or absolute configurations were determined by NOE, comparison of experimental and calculated ECD spectra, and application of the modified Mosher's ester method. Of these, strobiloscyphone F (6) contains a novel highly oxygenated tetracyclic oxireno-octahydrodibenzofuran ring system. Natural products 1, 6, and 9 and the semisynthetic analogue 12 derived from 8 exhibited cytotoxic activity, whereas 9 and 12 showed antimicrobial activity. Possible biosynthetic pathways to 1-6, 8, and 9 are proposed.

Chlorinated Dehydrocurvularins and Alterperylenepoxide A from Alternaria sp. AST0039, a Fungal Endophyte of Astragalus lentiginosus.

Investigation of Alternaria sp. AST0039, an endophytic fungus obtained from the leaf tissue of Astragalus lentiginosus, led to the isolation of (-)-(10E,15S)-4,6-dichloro-10(11)-dehydrocurvularin (1), (-)-(10E,15S)-6-chloro-10(11)-dehydrocurvularin (2), (-)-(10E,15S)-10(11)-dehydrocurvularin (3), and alterperylenepoxide A (4) together with scytalone and α-acetylorcinol. Structures of 1 and 4 were established from their spectroscopic data, and the relative configuration of 4 was determined with the help of nuclear Overhauser effect difference data. All metabolites were evaluated for their cytotoxic activity and ability to induce heat-shock and unfolded protein responses. Compounds 2 and 3 exhibited cytotoxicity to all five cancer cell lines tested and increased the level of the pro-apoptotic transcription factor CHOP, but only 3 induced the heat-shock response and caused a strong unfolded protein response.

Selective inhibition of p97 by chlorinated analogues of dehydrocurvularin.

The ATPase p97 is a ubiquitin targeted segregase that uses the energy of ATP binding and hydrolysis to extract ubiquitylated substrates from biological membranes, from other proteins, or from protein complexes to carry out myriad tasks in eukaryotes. Increased p97 activity has been linked to a poor prognosis in cancer patients, making p97 an anti-neoplastic target. In the present study, we show that dehydrocurvularin (DHC) and its chlorinated variants are covalent inhibitors of p97, interfering with its ATPase activity. Interestingly, cellular studies revealed both DHC and its monochloro analogue interfere with both the proteasome and p97, whereas its dichloro analogue showed p97 specificity.

Altertoxins with potent anti-HIV activity from Alternaria tenuissima QUE1Se, a fungal endophyte of Quercus emoryi.

Screening of a small library of natural product extracts derived from endophytic fungi of the Sonoran desert plants in a cell-based anti-HIV assay involving T-cells infected with the HIV-1 virus identified the EtOAc extract of a fermentation broth of Alternaria tenuissima QUE1Se inhabiting the stem tissue of Quercus emoryi as a promising candidate for further investigation. Bioactivity-guided fractionation of this extract led to the isolation and identification of two new metabolites, altertoxins V (1) and VI (2) together with the known compounds, altertoxins I (3), II (4), and III (5). The structures of 1 and 2 were determined by detailed spectroscopic analysis and those of 3-5 were established by comparison with reported data. When tested in our cell-based assay at concentrations insignificantly toxic to T-cells, altertoxins V (1), I (3), II (4), and III (5) completely inhibited replication of the HIV-1 virus at concentrations of 0.50, 2.20, 0.30, and 1.50 µM, respectively. Our findings suggest that the epoxyperylene structural scaffold in altertoxins may be manipulated to produce potent anti-HIV therapeutics.

Geopyxins A-E, ent-kaurane diterpenoids from endolichenic fungal strains Geopyxis aff. majalis and Geopyxis sp. AZ0066: structure-activity relationships of geopyxins and their analogues.

Four new ent-kaurane diterpenoids, geopyxins A-D (1-4), were isolated from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, whereas Geopyxis sp. AZ0066 inhabiting the same host afforded two new ent-kaurane diterpenoids, geopyxins E and F (5 and 6), together with 1 and 3. The structures of 1-6 were established on the basis of their spectroscopic data, while the absolute configurations were assigned using modified Mosher's ester method. Methylation of 1-3, 5, and 6 gave their corresponding methyl esters 7-11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14 and diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7-10, 12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds 7-9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure-activity perspective, the electrophilic α,ß-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be necessary but not sufficient for both cytotoxicity and heat-shock activation.

Smardaesidins A-G, isopimarane and 20-nor-isopimarane diterpenoids from Smardaea sp., a fungal endophyte of the moss Ceratodon purpureus.

Five new isopimarane diterpenes, smardaesidins A-E (1- 5) and two new 20-nor-isopimarane diterpenes, smardaesidins F (6) and G (7), together with sphaeropsidins A (8) and C-F (10-13) were isolated from an endophytic fungal strain, Smardaea sp. AZ0432, occurring in living photosynthetic tissue of the moss Ceratodon purpureus . Of these, smardaesidins B (2) and C (3) were obtained as an inseparable mixture of isomers. Chemical reduction of sphaeropsidin A (8) afforded sphaeropsidin B (9), whereas catalytic hydrogenation of 8 yielded 7-O-15,16-tetrahydrosphaeropsidin A (14) and its new derivative, 7-hydroxy-6-oxoisopimara-7-en-20-oic acid (15). The acetylation and diazomethane reaction of sphaeropsidin A (8) afforded two of its known derivatives, 6-O-acetylsphaeropsidin A (16) and 8,14-methylenesphaeropsidin A methyl ester (17), respectively. Methylation of 10 yielded sphaeropsidin C methyl ester (18). The planar structures and relative configurations of the new compounds 1-7 and 15 were elucidated using MS and 1D and 2D NMR experiments, while the absolute configurations of the stereocenters of 4 and 6-8 were assigned using a modified Mosher's ester method, CD spectra, and comparison of specific rotation data with literature values. Compounds 1-18 were evaluated for their potential anticancer activity using several cancer cell lines and cells derived from normal human primary fibroblasts. Of these, compounds 8, 11, and 16 showed significant cytotoxic activity. More importantly, sphaeropsidin A (8) showed cell-type selectivity in the cytotoxicity assay and inhibited migration of metastatic breast adenocarcinoma (MDA-MB-231) cells at subcytotoxic concentrations.

Maximizing chemical diversity of fungal metabolites: biogenetically related Heptaketides of the endolichenic fungus Corynespora sp. (1).

In an attempt to explore the biosynthetic potential of the endolichenic fungus Corynespora sp. BA-10763, its metabolite profiles under several culture conditions were investigated. When cultured in potato dextrose agar, it produced three new heptaketides, 9-O-methylscytalol A (1), 7-desmethylherbarin (2), and 8-hydroxyherbarin (3), together with biogenetically related metabolites scytalol A (4), 8-O-methylfusarubin (5), scorpinone (6), and 8-O-methylbostrycoidin (7), which are new to this organism, and herbarin (8), a metabolite previously encountered in this fungal strain. The use of malt extract agar as the culture medium led to the isolation of 6, 8, 1-hydroxydehydroherbarin (9), and 1-methoxydehydroherbarin (10), which was found to be an artifact formed during the extraction of the culture medium with methanol. The structures of all new compounds were determined by interpretation of their spectroscopic data and chemical interconversions.

Cytotoxic Cytochalasins and Other Metabolites from Xylariaceae sp. FL0390, a Fungal Endophyte of Spanish Moss.

Two new metabolites, 6-oxo-12-norcytochalasin D (1) and 4,5-di-isobutyl-2(1H)-pyrimidinone (2), together with seven known metabolites, cytochalasins D (3), Q (4), and N (5), 12-hydroxyzygosporin G (6), heptelidic acid chlorohydrin (7), (+)-heptelidic acid (8), and trichoderonic acid A (9), were isolated from Xylariaceae sp. FL0390, a fungal endophyte inhabiting Spanish moss, Tillandsia usneoides. Metabolite 1 is the first example of a 12-norcytochalasin. All metabolites, except 2 and 9, showed cytotoxic activity in a panel of five human tumor cell lines with IC50S of 0.2-5.0 µM.

Inhibition of HIV-1 Replication by Secondary Metabolites From Endophytic Fungi of Desert Plants.

Most antiretroviral drugs currently in use to treat an HIV-1 infection are chemically synthesized and lead to the development of viral resistance, as well as cause severe toxicities. However, a largely unexplored source for HIV-1 drug discovery is endophytic fungi that live in a symbiotic relationship with plants. These fungi produce biologically active secondary metabolites, which are natural products that are beneficial to the host. We prepared several hundred extracts from endophytic fungi of desert plants and evaluated the inhibitory effects on HIV-1 replication of those extracts that showed less than 30% cytotoxicity in T-lymphocytes. Those extracts that inhibited viral replication were fractionated in order to isolate the compounds responsible for activity. Multiple rounds of fractionation and antiviral evaluation lead to the identification of four compounds, which almost completely impede HIV-1 replication. These studies demonstrate that metabolites from endophytic fungi of desert plants can serve as a viable source for identifying potent inhibitors of HIV-1 replication.

Tricinonoic acid and tricindiol, two new irregular sesquiterpenes from an endophytic strain of Fusarium tricinctum.

Two new rare irregular sesquiterpenes, tricinonoic acid (1) and tricindiol (2), and the known furanopyrrolidones, NG-391 (3) and NG-393 (4), have been isolated from an EtOAc extract of Fusarium tricinctum, a fungus endophytic in the root tissue of the Sonoran desert plant, Rumex hymenosepalus. The structures of 1 and 2 were elucidated on the basis of their high-resolution mass, 1D and 2D NMR spectroscopic data. A possible biosynthetic route to 1 and 2 from farnesyl diphosphate is proposed.

Zinagrandinolides A-C, cytotoxic delta-elemanolide-type sesquiterpene lactones from Zinnia grandiflora.

Three new delta-elemanolide-type sesquiterpene lactones, zinagrandinolides A-C (1-3), and the known delta-elemanolide 4 have been isolated by a bioassay-guided fractionation of a cytotoxic hexane extract of the aerial parts of Zinnia grandiflora. The structures of 1-3 were determined on the basis of high-resolution mass and NMR data. All compounds exhibited strong cytotoxicity against the cancer cell lines NCI-H460, MCF-7, SF-268, and MIA Pa Ca-2 and the normal human fibroblast cell type WI-38, but none showed significant selectivity.

Globosumones A-C, cytotoxic orsellinic acid esters from the Sonoran desert endophytic fungus Chaetomium globosum.

Three new esters of orsellinic acid, globosumones A-C (1-3), and three known compounds, orsellinic acid (4), orcinol, and trichodion (5), were isolated from Chaetomium globosum endophytic on Ephedrafasciculata (Mormon tea). The structures of the new compounds 1-3 were established spectroscopically, which included 2D NMR experiments and 1H NMR studies on Mosher's ester derivatives. All compounds were evaluated for inhibition of cell proliferation in a panel of four cancer cell lines, NCI-H460 (non-small cell lung cancer), MCF-7 (breast cancer), SF-268 (CNS glioma), and MIA Pa Ca-2 (pancreatic carcinoma), and normal human fibroblast cells (WI-38). Only globosumones A (1) and B (2) were found to be moderately active.

Cytotoxic and other metabolites of Aspergillus inhabiting the rhizosphere of Sonoran desert plants.

In a study to discover potential anticancer agents from rhizosphere fungi of Sonoran desert plants cytotoxic EtOAc extracts of four Aspergillus strains have been investigated. Two new metabolites, terrequinone A (1) and terrefuranone (2), along with Na-acetyl aszonalemin (LL-S490beta) (3) were isolated from As. terreus occurring in the rhizosphere of Ambrosia ambrosoides, whereas As. terreus inhabiting the rhizosphere of an unidentified Brickellia sp. afforded dehydrocurvularin (4), 11-methoxycurvularin (5), and 11-hydroxycurvularin (6). As. cervinus isolated from the rhizosphere of Anicasanthus thurberi contained two new compounds, 4R*,5S*-dihydroxy-3-methoxy-5-methylcyclohex-2-enone (7) and 6-methoxy-5(6)-dihydropenicillic acid (8), in addition to penicillic acid (9). Penicillic acid was also isolated from As. wentii occurring in the rhizosphere of Larrea tridentata. The structures of 1-9 were elucidated by spectroscopic methods and chemical derivatizations. Acetylation of 2 afforded 14-acetylterrefuranone (13) and 14-deoxy-13(14)-dehydroterrefuranone (14). Metabolites 1-9, the dienone 14, and 5(6)-dihydropenicillic acid (16) were evaluated for cytotoxicity in a panel of four human cancer cell lines and in normal human primary fibroblast cells. Compounds 4 and 5 displayed considerable cytotoxicity, whereas 1, 6, 9, and 14 were found to be moderately active, with 6 and 9 exhibiting selective cytotoxicity against cancer cell lines compared with the normal fibroblast cells.