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REVIEW
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Fungal natural products in research and development Cite this: Nat. Prod. Rep., 2014, 31, 1425
Anja Schueffler* and Timm Anke*
Covering: 2009 to 2013 To date approximately 100 000 fungal species are known although far more than one million are expected. The variety of species and the diversity of their habitats, some of them less exploited, allow the conclusion that fungi continue to be a rich source of new metabolites. Besides the conventional fungal isolates, an increasing interest in endophytic and in marine-derived fungi has been noticed. In addition new screening strategies based on innovative chemical, biological, and genetic approaches have led to novel fungal metabolites in recent years. The present review focuses on new fungal Received 2nd May 2014
natural products published from 2009 to 2013 highlighting the originality of the structures and their biological potential. Furthermore synthetic products based on fungal metabolites as well as new
DOI: 10.1039/c4np00060a
developments in the uses or the biological activity of known compounds or new derivatives are
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discussed.
1 1.1 2
5
Introduction Current strategies in the search for new metabolites New metabolites, mode of action studies, new genetic methods, and approaches toward the generation of metabolites Compounds recently introduced into the market New developments in the uses of known compounds or new derivatives References
1
Introduction
3 4
The review covers papers and patents on fungal metabolites published from 2009–2013 with emphasis on the novelty of the structures, their bioactivities and their expected or realized uses in the Pharma and Agro industries. In part the topics highlighted here have been discussed in previous reviews like marine and endophytic natural products published from 2009–2013,1–6 industrial aspects of natural products chemistry.7 1.1
Current strategies in the search for new metabolites
The fungal sources of new metabolites have been broadened from saprophytic terrestrial strains to marine habitats and living plants with their endophytes. While by far most of the Institut f¨ ur Biotechnologie und Wirkstoff-Forschung (Institute of Biotechnology and Drug Research), Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany. E-mail: schueffl[email protected]; [email protected]
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metabolites described are produced by asexual stages of ascomycetes e.g. Penicillium and Aspergillus species, the interest in basidiomycetes and the number of metabolites derived from them has grown considerably. Zygomycetes although easily isolated and cultured seem to be less prolic producers. The test systems used in the search for new biological activities and new compounds have been adapted to the needs of medicine and agriculture. In the antibiotics eld there is an urgent need to overcome resistance of pathogenic bacteria and fungi with compounds with new structures and modes of action. In the eld of other medications a number of very interesting biochemical test systems have been developed yielding very interesting results.
2 New metabolites, mode of action studies, new genetic methods, and approaches toward the generation of metabolites Hypocreolide (1), a nonenolide, was derived from fermentations of the ascomycete Hypocrea lactea.8 1 shows moderate antibacterial, antifungal and cytotoxic activities. Since neither the relative nor the absolute stereochemistry of 1 could be initially assigned, a stereochemically exible total synthesis was developed providing 1 in nine steps and 12% overall yield.
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Japan. The metabolite inhibits Ca2+ signaling in a yeast mutant.11
The cytospolides A–Q (2–11) and decytospolides A and B (12; 13) are unusual nonalolides isolated from a Cytospora sp., an endophytic fungus derived from Ilex canariensis.9,10 Absolute congurations were assigned using the modied Mosher's method and CD/TDDFT calculations. The cytospolides B (3), E (6), and P (10) exhibited cytotoxic activities against A-549 (human lung carcinoma) cells with LD50 values of 5, 7, and 2 mg mL1, the cytospolides K, M (7), N (8), O (9), Q (11), and decytospolide B (13) showed only moderate activities with LD50 values of 11–39 mg mL1. The other metabolites were inactive.
Benzophomopsin A (14), a new benzoxepin was isolated from an endophytic Phomopsis strain derived from a cherry tree in
Anja Schueffler studied Biology at the University of Kaiserslautern. Her PhD focused on the characterisation of fungal natural products with antimicrobial activity. In 2010 she received a DFG fellowship and joined Prof. Fenical's group at the Scripps Institute for Oceanography in San Diego, where she studied antibacterial natural products from streptomycetes. In 2011 she started a research fellowship at the Institute of Biotechnology and Drug Research (IBWF, Kaiserslautern). Her scientic work focuses on bioactive natural products, the biosynthesis of secondary metabolites as well as the isolation and taxonomy of fungi of diverse habitats.
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Four new ascochlorin derivatives have been described. Cylindrol A5 (15),12 produced by Cylindrocarpon sp. FKI-4602 exhibits moderate antibacterial activities whereas 100 -deoxy10a0 -hydroxyascochlorin13 from Acremonium sp. LG0808 inhibited the migration of metastatic prostate cancer cells PC3M. LL-Z1272a epoxide (16) was derived from a mutant of Ascochyta viciae14 and seems to be the long proposed intermediate in the biosynthesis of ascochlorin (17) from the precursor LL-Z1272a.15 Recent ndings demonstrated that 17 inhibits eukaryotic respiration by binding to both active sites (Qo and Qi) of the cytochrome bc1 complex.16 This clearly distinguishes 17 from other antibiotics binding to only one site like the strobilurins and antimycins. Ascofuranone (18), a related fungal metabolite, specically inhibits the alternative oxidase of Trypanosoma brucei (TAO), the cause of human African trypanosomiasis, at subnanomolar concentrations by binding to the ubiquinol binding domain of the quinol oxidase. The chemotherapeutic efficacy of 18 both in vitro and in vivo had been demonstrated.17–19 Since TAO is essential for the parasites survival but absent in humans it offers an interesting target for the development of novel drugs. Saimoto et al.20 synthesized a number of ascofuranone derivatives and analogues. They succeeded in the identication of the pharmacophore and the binding site within the enzyme and obtained a synthetic derivative “compound 24” (19) inhibiting TAO with a lower IC50 (0.06 nM) as compared to 18.
Timm Anke received his diploma (Biochemistry) and his PhD degree from the University of T¨ ubingen. From 1973 he spent two years as a research associate with Fritz Lipmann at the Rockefeller University in New York. Back in T¨ ubingen he obtained the venia legendi in 1979. He obtained the professorship of Biotechnology at the University of Kaiserslautern in 1981 from which he retired in 2010. Since 1998 he heads the Institute of Biotechnology and Drug Research IBWF e.V. His research interests are with fungal metabolites, their bioactivities and biosyntheses.
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Review
The ascotricins A (20) and B (21) from Ascotricha chartarum were isolated in a screening for new antagonists of sphingosine-1-phosphate receptor 1 (S1P1) by Yonesu et al.21 Sphingosine-1-phosphate (Sph-1-P) bound to this receptor promotes proliferation, migration and tube formation of vascular endothelial cells. Agonists of S1P1 like ngolimod act as immunosuppressants. In S1P1 expressing cells 20 and 21 acted as S1P1 antagonists with IC50 values of 8.2 and 1.8 mM respectively, inhibited [33P] Sph-1-P binding at 120 and 39 mM, and inhibited the migration of human umbilical vein endothelial cells at IC50 values of 94 and 28 mM. Therefore the ascotricins offer the opportunity to investigate the role of Sph-1-P and its receptor in vascular endothelial cells and angiogenesis. In the course of their studies the authors also found that analogs of caloporoside22 had S1P1 antagonist activity.
Aquastatin A (22) was rst described from Fusarium aquaeductuum as an inhibitor of mammalian adenosin triphosphatases by a group at Sankyo in 1993.23 It inhibits mammalian Na+/K+-ATPase with an IC50 value of 7.1 mM, and
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porcine stomach mucous H+/K+-ATPase with an apparent IC50 value of 6.2 mM. Thus far in testing, no antimicrobial activity had been detected at a concentration of 1 mg mL1. A patent was led for the use in treatment of gastric ulcers.24 In 2003 Ondeyka et al. of Merck Laboratories25 described another interesting biological activity of 22. They isolated the structurally related exophillic acid from Exophiala pisciphila and described that both compounds inhibited the strand transfer reaction of HIV-1 integrase, an enzyme crucial for the replication and spread of HIV with a rather high IC50 value of 50 mM for 22. In 2009 22 was reisolated from cultures of a Sporothrix species in the course of a screening for inhibitors of bacterial enoyl-acyl carrier protein (ACP) reductase, a key enzyme in bacterial fatty acid biosynthesis.26 22 inhibited the enoyl-ACP reductase of Staphylococcus aureus with an IC50 of 3.2 mM. The growth of S. aureus, both wild type and a methicillin-resistant strain, was inhibited at minimum inhibitory concentrations of 16–32 mg mL1. The enoyl-ACP reductase of S. pneumoniae was inhibited with an IC50 of 9.2 mM. It is interesting that the degalactosylated aquastatin B (23), prepared by acid hydrolysis of 22, retains the inhibitory activities, while aquastatin C (24)27 is completely inactive in these assays. In 2009 22, isolated from a marine-derived Cosmospora species, was found to inhibit the protein tyrosine phosphatase 1B (PTP1B).28 22 exhibited potent activity against PTP1B with an IC50 value of 0.19 mM. Kinetic analyses of PTP1B suggested a competitive inhibition. Tests with hydrolysis products (23 and 24), suggested that the dihydroxy pentadecyl benzoic acid (corticiolic acid29) moiety is the pharmacophore. 22 showed modest but selective inhibitory activity toward PTP1B compared to other protein tyrosine phosphatases. Because PTP1B plays a central role in signal transduction, a wide variety of pharmacological effects are to be expected by inhibitors of this enzyme.
The compounds JBIR-37 (25) and -38 (26) were isolated in 2009 from the marine sponge-derived fungus Acremonium sp.30 Although these compounds do not exhibit cytotoxic activity and in addition no other bioactivities are described, they are the rst examples of glycosyl benzenes from fungi.
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Xanthepinone (27), an interesting polyketide, was isolated in a search for new antifungal agents inhibiting the spore germination of phytopathogenic fungi.31 By comparison of its ITS sequence the producing soil fungus was found to be closely related to the plant pathogen Phoma medicaginis. 27 completely inhibited the germination of Magnaporthe grisea, Phythophthora infestans, and Botrytis cinerea conidia at concentrations of 2, 5, and 10 mg mL1, respectively. Only very weak antibacterial (Proteus vulgaris and Staphylococcus aureus) and no cytotoxic activities were detected at 50 mg mL1.
The herbaridines A (28) and B (29), as well as O-phenethylherbarin (30) are new naphthoquinone derivatives isolated in a screening for cytotoxic metabolites. The producer IBWF79B-90A, a sterile lamentous ascomycete could not be identied by morphological features or by its ITS sequence.32 28 and 29 show moderate antibacterial activity at concentrations of 20–50 mg mL1. Antifungal activity against lamentous fungi was not observed. The cytotoxic activity against several human cell lines ranged from 0.5–2.5 mg mL1 and was comparable to herbarin.
Review
IC50 values of 52.8 and 41.2 mmol L1, respectively. The penicitrinols F–I (35–38) were derived from the same fungus36 and were found to exhibit no cytotoxic activities. Two interesting citrinin trimers named tricitrinols A (39) and B (40) were extracted from a Penicillium citrinum strain isolated from crater ash in China.38 Both compounds exhibited cytotoxic activity against HL60 and HCT116 cells with IC50 values of 3.2 to 8.6 mM. 40 was found to induce apoptosis, cell cycle arrest, DNA damage and inhibition of human DNA topoisomerase 2a. These results supported by computational docking analysis suggest that 40 is an intercalating topoisomerase 2a inhibitor which could serve as a lead structure for further studies.38 In 2013 the citrinin dimers penicitrinone E (41) and penicitrinol J (42) were isolated together with two citrinin monomers from a marine-derived Penicillium species.37 No signicant cytotoxicity was detected but 42 was slightly active against Staphylococcus aureus.
Herqueiazol (43), herqueioxazol (44), and herqueidiketal (45) are new polyaromatic metabolites isolated from a Penicillium sp. derived from a marine sediment.39 45 possesses a novel skeleton and exhibited moderate cytotoxicity but signicant activity (IC50 23.6 mM) against Staphylococcus aureus sortase A. This enzyme attaches proteins to the bacterial cell wall and is considered as a possible target for new antibiotics.
Several citrinin dimers and derivatives from different halotolerant or marine Penicillium strains were described.33–37 Pennicitrinone D (31), a new citrinin dimer, was isolated from Penicillium notatum along with the known pennicitrinone A (or dicitrinin A, penicitrinone A), citrinin, and mycophenolic acid. When tested for cytotoxic activities 31 was found inactive.34 The penicitrinols C–E (32–34) were isolated from a marine derived P. citrinum35 32 and 34 were slightly active against HL-60 cells, with
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Chrysoxanthone (46) is an unusual heterodimer of the tetrahydroxanthone blennolide A and the anthraquinone 2-hydroxychrysophanol linked through a diaryl ether bridge. It was isolated from mycelia of the sterile lamentous ascomycete IBWF11-95A. Its structure was elucidated by two-dimensional NMR spectroscopy. 46 shows antibacterial and antifungal activity with MIC values between 2.5 and 20 mg mL1. The cytotoxicity was moderate with IC50-values of 50 mg mL1 for Jurkat, L-1210, and Colo-320 cells.40
Isochaetochromin A1 (47), a new bis-naphtho-g-pyrone isomer was isolated together with the known isochaetochromins B1 (48) und B2 (49) from fermentations of Penicillium sp. FKI-4942 in a search for new inhibitors of mammalian triacylglycerol (TG) synthesis.41,42 In an assay using CHO-K1 (Chinese hamster ovary) microsomes the incorporation of 14C-palmitoyl-CoA into 1,2-dioleoyl-sn-glycerol was inhibited by 47, 48, and 49 with IC50 values of 33 mM, 11 mM, and 5.6 mM respectively. Inhibitors of TG synthesis are possible candidates for the development of therapeutic agents to treat obesity.
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AC1, AC2, AD1, AD2, AE1 and AE2, were isolated from the culture broth of Talaromyces pinophilus FKI-3864 by Kawaguchi et al.46 Dinapinone AB2 showed potent inhibition of TG synthesis in intact mammalian cells with an IC50 value of 1.17 mM, whereas the other dinapinones exhibited weaker activities.
The xanthoradones A (52) and B (53) are products of Penicillium radicum FKI-3765-2.47,48 Despite a moderate activity against Staphylococcus aureus and Bacillus subtilis these compounds reduce the MIC value for the b-lactam imipenem from 16 to 0.06 mg mL1 (52) and 0.03 mg mL1 (53) against methicillin-resistant Staphylococcus aureus (MRSA) whereas xanthoradones themselves are not active against MRSA. Xanthoradone C (54), described in 2010 49 showed weaker effects with a reduction of imipenem MIC value to 0.5 mg mL1. Interestingly xanthoradones seems to inhibit FtsZ, the bacterial tubulin homolog which is essential for formation of the septum and recruiting of other proteins for cell division. A patent which claims the concurrent use of FtsZ inhibitors and b-lactam antibiotics was led in 2011 by Merck Sharp & Dohme Corp.50 and several natural products were listed for their ability to inhibit FtsZ. Among others the xanthoradones as well as the structurally related viriditoxin (55)51 are named as lead structures. For 55 the inhibition of FtsZ was conrmed.52
The dinapinones were isolated from strain FKI-3864 (named Penicillium pinophilum43–45/Talaromyces pinophilus46) in a search for inhibitors of TG-synthesis. Dinapinone A (50), a mixture of two stereoisomers A1 und A2 inhibited TG- and cholesteryl ester synthesis with an IC50 value of 0.097 mM and 0.31 mM respectively.43 It was observed that the inhibitory activity on TG synthesis of the separated compounds was substantially reduced. Dinapinones A1 and A2 are atropisomers consisting of two monomers with the same planar structure of dihydronaphthopyranone as monapinone A (51).44 The monapinones were only produced when P. pinophilum was grown in seawater supplemented medium.45 They showed rather weak inhibition of TG synthesis. Eight new dinapinones, AB1, AB2,
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Xanalteric acids I (56) and II (57) are metabolites of an endophytic Alternaria sp. isolated from leaves of the mangrove Sonneratia alba.53 These compounds are the rst fungal compounds with a 10H-phenaleno[1,2,3-de]chromene skeleton. They exhibit modest cytotoxic activity against L5178Y cells at 10 mg mL1 and weak antibacterial activity against Staphylococcus aureus.
Botrytis cinerea (sexual state Botryotinia fuckeliana) is a very important pathogen affecting many crops and a prolic producer of structurally diverse phytotoxins. A very concise and readable review on the chemistry and structural determination of botcinolides, botcinins, and botcinic acids produced by Botrytis cinerea strains has appeared in 2009.54 The regulation of the biosyntheses of the Botrytisphytotoxins has been studied in detail. Gene knock-out and complementation studies revealed that the transcriptional regulator bcreg1 is required for pathogenicity, conidiogenesis, and the production of secondary metabolites. A bcreg1 mutant was able to penetrate plant tissue but was not able to cause necrotic lesions. In addition, the mutant did not produce detectable levels of the sesquiterpene botrydial (58) and the polyketide botcinic acid (59).55 BcAtf1 controls a diversity of cellular processes and has broad regulatory functions. It also controls secondary metabolism, a Dbcatf1 mutant contained signicantly elevated levels of the phytotoxins botrydial, botryendial (60), and botcinin A (61).56
Emervaridione (62) and varioxiranediol (63),57 two new metabolites have been derived from fermentations of an Emericella variecolor (Aspergillus stellatus) strain. Despite of their interesting structures no cytotoxic or antimicrobial activities have been detected so far for these metabolites.
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Ceramidastin (64) was described as a metabolite of a Penicillium sp. isolated from a soil sample in a screening for inhibitors of a cloned ceramidase from Pseudomonas aeruginosa.58 This enzyme is thought to aggravate atopic dermatitis in humans and inhibitors might therefore alleviate this condition. 64 inhibited the bacterial ceramidase with an IC50 value of 6.25 mg mL1. 64 did not inhibit human ceramidase and no antimicrobial and cytotoxic activities were detected up to concentrations of 200 and 100 mg mL1, respectively. 64 is similar to the toxic rubratoxins, e.g. rubratoxin A (65). This metabolite, however, did not inhibit the bacterial ceramidase at 100 mg mL1.
Fimetarone A (66) was isolated from cultures of a Fimetariella sp. (Sordariales) isolated from a sample of “Chinese caterpillar fungus” used in traditional Chinese medicine for all kinds of medications.59 It consists of a preparation of fruiting bodies of Ophiocordyceps sinensis, still connected to the caterpillar larva of the moth Thitarodes sp. (Hepialidae) which it colonizes. 66 has a new spiro[chroman-3,70 -isochromene]-4,60 (80 H)-dione skeleton and was obtained as 1 : 1 atropdiastereomeric mixture. 66 showed modest cytotoxic activity against T24 cells (human bladder carcinoma) with an IC50 value of 40.2 mM.
The epicoccolides A (67) and B (68) are new polyketides derived from an endophytic Epicoccum strain of Theobroma cacao. They were isolated together with epicolactone and other compounds in a search for metabolites inhibiting the growth of Pythium ultimum, Aphanomyces cochlioides, and
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Rhizoctonia solani, all three important plant pathogens. Beside antifungal activities 67 and 68 exhibit modest antibacterial activities.60
Chloropestolide A (69) was isolated from Pestalotiopsis ci derived from branches of Camellia sinensis. This highly functionalized spiroketal with a very unusual ring system was found to inhibit the growth of HeLa and HT 29 cells at concentrations of 0.7 and 4.2 mM. The replication of HIV-1 in C8166 cells was inhibited with an IC50 value of 64.9 mM.61 From the same fungus. grown on solid substrates six new chloropestolides B–G were isolated featuring chlorinated spiro[benzo[d][1,3]dioxine2,7-bicyclo[2.2.2]octane]-4,8-dione (e.g. 69) and spiro[benzo-[d][1,3]dioxine-2,1-naphthalene]-2,4-dione skeletons (e.g. chloropestolide E 70).62 The biosynthesis of the chloropestolides has been proposed to start from the precursors 71 and 72. Diels– Alder reactions with 71 as diene and 72 as dienophile or with 71 as dienophile and 72 as diene should yield e.g. 69 or 70, respectively.
Decalpenic acid (73) was isolated from Penicillium verruculosum in a screening for compounds inducing early osteoblastic markers in a pluripotent mouse mesenchymal cell line. Osteoblasts are responsible for bone formation in embryonic development and adult life and arise from mesenchymal stem cells. It is assumed that compounds
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inducing osteoblastogenesis might become valuable tools to elucidate steps in osteoblast differentiation or might be developed into therapies for bone diseases. 73 signicantly induced early markers such as alkaline phosphatase activity and osteopontin mRNA in C3H10T1/2 cells at concentration of 1 mM.63 Sakamota et al.64 have shown that 73 acts as a new retinoid and induces early osteoblastic markers in pluripotent mesenchymal cells through activation of retinoic acid receptor g.
The haplofungins A–H (74–81) from Lauriomyces bellulus are composed of an arabinonic acid moiety linked by an ester bond to a long modied alkyl chain.65 The absolute conguration of 74 has been determined.66 The haplofungins inhibit the inositol phosphorylceramide (IPC) synthase of Saccharomyces cerevisiae with IC50 values from 1.5–65 ng mL1.67 The IPC synthase of Aspergillus fumigatus is less sensitive. Since fungal IPC is essential for growth and differs substantially from the human glycosphingolipid, IPC synthase is considered an interesting target for selective fungicides. Unfortunately, however, with the exception of Candida glabrata, no activity against C. albicans, C. parapsilosis, C. tropicalis, Cryptococcus neoformans and A. fumigatus was observed in vivo. The same was found for khafrefungin,68,69 an IPC inhibitor with a similar structure. In this respect the haplofungins differ from the cyclic depsipeptide pleofungin A70,71 an IPC inhibitor which exhibits also very strong antifungal activities. Two new caloporosides, G (82) and H (83) were isolated from the basidiomycete Gloeoporus dichrous in a search for inhibitors of spore germination of Magnaporthe grisea and Fusarium graminearum.72 Caloporoside A (84) was rst isolated as a potent and selective inhibitor of phospholipase C of pig brain.22 Other interesting biological activities have been described for caloporoside derivatives: inhibition of the binding of 35S-TBPS to the GABAA/benzodiazepine chloride channel receptor complex in vitro,73,74 inhibition of the binding of hyaluronic acid to its receptor CD44,75 as well as S1P1 antagonist activity.22 From fruiting bodies of the basidiomycete Hericium erinaceus three new metabolites, hericenone I (85), hericene D (86),76 and isohericenone (87)77 were isolated. 85 and 86 exhibit very low antibacterial and cytotoxic activities while 87 exhibited potent cytotoxic activity against A549, SK-OV-3, SK-MEL-2 and HCT-15 cell lines with IC50 values of 2.6, 3.1, 1.9 and 2.9 mM respectively.
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Caripyrin (88) is a new pyridyloxirane from cultures of the basidiomycete Caripia montagnei.78 88 was found to inhibit conidial germination and appressorium formation in the rice blast fungus Magnaporthe oryzae, whereas the infection-related morphogenesis in several other phytopathogenic fungi was not affected. In plant assays on rice, 88 was found to protect plants efficiently against infection with Magnaporthe oryzae. Contrary to the structurally related fusaric acid (89), 88 was neither cytotoxic, antibacterial, nor nematicidal.
The udalactaranes A (90), B (91), and udasterpurenol A (92) were the rst secondary metabolites described from Phlebia (Mycoacia) uda.79 The new sesquiterpenoids were isolated together with the known hyphodontal and sterpuric acid in a screening of basidiomycetes for antifungal antibiotics. 90 was isolated as a mixture A (90a) and epi-A (90b) in a diastereomeric ratio of 1.6 : 1. 91 is a disesquiterpene consisting of an isolactarane and a sterpurene part. As found for 90, 91 was isolated as a mixture of two epimers in a 3 : 1 ratio (91a/b). When tested for antibiotic activities 90a and 90b (1.6 : 1) and 91a and 91b (3 : 1) showed modest antifungal activities inhibiting the spore germination of Fusarium graminearum at 10 and 5 mg mL1, respectively.
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prevented. The same effect was observed for marasmal C (102) at 50 mg mL1. Conidial germination of B. cinerea was not inhibited by any of the compounds at concentrations up to 100 mg mL1.81
From three basidiomycetes, Xeromphalina sp., Stereum sp., and Pleurocybella porrigens, six triquinane sesquiterpenes with unprecedented modications and a rearranged sesquiterpene related to coriolin C have been isolated.80 Metabolites lacking the exocyclic enone moiety exhibited no or reduced antibiotic activities against fungi and bacteria. The cytotoxicities (IC50 value) against Jurkat cells of xeromphalinone A (93), B (94), F (98) and chlorostereone (99) ranged from 1 to 5 mg mL1 (2 to 20 mM). For xeromphalinones C (95), D (96), and E (97) no cytotoxic activities were observed at concentrations up to 50 mg mL1 (above 100 mM). The formation of cysteine adducts was observed by HPLC-MS for 93, 94, and 98 as well as for 99, the rst example for a chlorinated triquinane from a terrestrial fungus.
Three new drimane sesquiterpenoids were discovered in a screening for inhibitors of spore germination of phytopathogenic fungi. The mixture of marasmene B and epi-marasmene B (100) inhibited the germination Magnaporthe oryzae spores completely at a concentration of 100 mg mL1, while Fusarium graminearum, Phytophthora infestans, and Botrytis cinerea were not affected. In the conidial germination assay, Marasmal B (101) was found to be the most active secondary metabolite tested. At a concentration of 5 mg mL1, conidial germination in M. oryzae, P. infestans, and F. graminearum was completely
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Ganodermycin (103), a sesquiterpenoid, was isolated in a search for new inhibitors of CXCL10 expression in a reporter gene assay with transiently transfected human MonoMac6 cells.82 CXCL10 (inducible protein 10) is a chemoattractant which contributes to the recruitment of inammatory cells such as macrophages and T-lymphocytes and thereby plays an important role in chronic inammation. 103 Inhibited CXCL10 synthesis and secretion with IC50 values around 40 mg mL1. In addition, 103 exhibited modest antibacterial and antifungal activities.
In recent years several sesquiterpenes with a chamigrane skeleton and an endoperoxide have been described from fungi. The merulins A–D (104–107) were isolated from an unidentied basidiomycete belonging to the family Meruliaceae isolated from the plant Xylocarpus granatum.83,84 At the same time the steperoxides A–D (108, 104, 109, 110) were described as metabolites from Steccherinum ochraceum (family Meruliaceae).85,86 It later turned out that steperoxide B and merulin A are identical (104). For 110 antibacterial activity against Staphylococcus aureus has been detected. When tested against two cancer cell lines 104 and 106 proved to be cytotoxic with IC50 values between 1.57 and 5 mg mL1, respectively. 106 was not cytotoxic up to concentrations of 10 mg mL1. Compounds 104 to 108 were tested for antiangiogenic effects.84 In a rat aortic sprouting assay 106 showed strong (2.5 mM) and 108 weaker activities (25 mM) whereas all others had no effect. In following experiments 106 showed suppression of endothelial cell proliferation and migration mediated through the Erk1/2 signaling pathway and inhibited neovessel formation ex vivo and in vivo in a mouse matrigel plug assay. The talaperoxides A–D (111–114) together with 104 were isolated from a mangrove-derived Talaromyces avus strain.87 All ve compounds were toxic to Artemia salina (LD50 < 10 ppm) and cytotoxic against a panel of human cancer cell lines with
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IC50 values between 0.7 and 19.8 mg mL1. No antimicrobial activity was observed. Like artemisinin, 104–106 were found to be active against Trypanosoma brucei at concentrations of 0.8 to 8 mM. In addition, SAR studies as well as semisynthetic modications of 104 were described88 and claimed in a patent which was led in July 2012.89 The semi-synthetic modications yielded several candidates which exhibit pronounced activity. Two compounds (115 and 116) displayed an IC50 of 60 nM and a selectivity index greater than 300-fold against cell lines. Phomopsene (117) and methyl phomopsenate (118), two new diterpenes, were isolated from Phomopsis amygdali in a “geneguided screening approach”90 Using degenerated primers a geranylgeranyl diphosphate synthase gene required for diterpene biosynthesis was identied in Phomopsis amygdali. Consequently the phomopsene synthase gene was identied and heterologous expressed in E. coli. Isolation of the protein and incubation with geranylgeranyl diphosphate lead to the identication of 117 which could then be detected in extracts of Phomopsis amygdali. Further NMR studies in extracts of the producing fungus identied 118 as a related metabolite. No information on the biological activities was given for both compounds.
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The diterpene wickerol A (119) isolated from Trichoderma atroviride FKI-3737 was described in a patent as inhibitor of inuenza virus replication with an IC50 of 70 ng mL1 whereas the proliferation of the human cell line used for the inuenza assay was affected only at 7 mg mL1.91 At 10 mg mL1 no pronounced antimicrobial activity was detected in an agar diffusion assay against a panel of bacteria and fungi. In 2012 the wickerols A (119) and B (120) were isolated from a different Trichoderma atroviride strain FKI-3849.92,93 When tested for antiinuenza activity 120 proved to be less active. Trichodermanin A which is identical to 120, was isolated from an endophytic T. atroviride S361 derived from Cephalotaxus fortune.94 Interestingly the wickerols are the rst examples of a 6-5-6-6 ring skeleton.
From an endolichenic strain of Nodulisporium two new 3,4seco-4-methyl-progesteroids named nodulisporisteriod A (121) and B (122)95 were isolated together with demethoxyviridin and inoterpene B, previously described from Inonotus obliquus.96 This is the rst report of 3,4-seco-4-methyl-pregnan steroids. 121 and 122 inhibited the amyloid b aggregation at 100 mM approximately 50%. The aggregation of amyloid b into brillar plaques is thought to be one of the causes of Alzheimer's disease.
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The atlantinones A (123) and B (124) were isolated from the guttate of Penicillium citreonigrum cultured on solid media in a study to investigate the changes of secondary metabolite production in the presence of 5-azacytidine.97 The new meroterpenes were not detected when the fungus was grown in the absence of the mutagen. No antibacterial and antifungal activities have been detected.
In 1993 the pyripyropenes A–D (125–128) consisting of a pyridine, a-pyrone and sesquiterpene moiety were isolated from Aspergillus fumigatus FO-1289. They inhibited a key enzyme catalyzing the esterication of cholesterol, the acylCoA cholesterol acyltransferase (ACAT) in the nM range without exhibiting antimicrobial and cytotoxic activity. When 125 was injected into mice no apparent toxicity was detected.98–100 In 1998 Cases et al.101 discovered that ACAT exists in two isoforms and later 125 was found to selectively inhibit ACAT-2 and therefore was used as model compound.102–108 In 2011 Ohshiro et al.109 demonstrated that atherogenic mice treated with 125 (10 to 100 mg kg1) showed decreased cholesterol absorption as well as lower cholesterol and cholesteryl oleate levels resulting in atherosclerosis protection. Ohshiro & Tomoda110 reviewed specic ACAT inhibitors focusing on ACAT-2-specic inhibitors like 125. In 2013 Ohtawa et al.111–113 developed ACAT-2 inhibitors based on 125 and established structure–activity relationships. They identied several derivatives with higher ACAT-2 inhibition, e.g. a 7-O-p-cyanobenzoyl PPPA derivative (129), which seems to be the most potent ACAT-2 inhibitor to date with an IC50 of 0.9 nM and a selectivity index of 4622 (IC50 ACAT-1/IC50 ACAT-2).111 In recent years several patents were led for the preparation of pyripyropenes.114–119 Besides ACAT-2 inhibition, anti-angiogenic effects and anti-proliferative activity against HUVEC cells were described for 125, 126, and 128 (IC50 0.1–1.8 mM)120 and a growth inhibition of corn earworm larvae (Helicoverpa zea) for 125.121 Based on the insecticidal activity several patents were led in recent years.122–124
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Allantofuranone (130), a new and uncommon g-lactone, was isolated from the plant pathogenic fungus Allantophomopsis lycopodina.125 It is a strong and quite selective antifungal antibiotic especially against Paecilomyces and Penicillium species. In pure media 130 is slowly converted into the inactive allantopyrone B (131) while Magnaporthe grisea detoxies 130 into the inactive allantopyrones C and D (132, 133). The interesting biosynthesis of 130 starts from two L-phenylalanines with polyporic acid (134) as a key intermediate.126 Allantopyrone A (135) isolated from a different A. lycopodina exhibited cytotoxic activity against HL60 cells with an IC50 value of 0.32 mM and induced apoptosis.127
In 2009 and 2010 several styrylpyrones named phellinins A1 (136), A2 (137), B, and C were described from Phellinus sp.128–130 Phenillins B and C are reported to be mixtures of inseparable components. All phellinins were able to scavenge DPPH (a,adiphenyl-b-picrylhydrazyl), ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and superoxide radicals.129,130 From Inonotus hispidus Zan et al.131 reported antioxidant hispidin derivatives named inonotusin A (138) and B (139). In addition, 139 exhibited cytotoxic activity at 19
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mM (IC50) against the cell line MCF-7. The bishispidins phaeolschidins A–D (140–143) and phaeolschidin E (144), all exhibiting antioxidative activity were isolated from fruiting bodies of Phaeolus schweinitzii.132 These compounds did not show any cytotoxic effect up to 200 mM against the cell lines HCT116 and SW480. Several styrylpyrones with other than antioxidative activities were isolated in the specied period. The trimeric hispidin derivative phellinstatin (145) produced by Phellinus linteus inhibits the enoyl-ACP reductase (FabI) a key enzyme of fatty acid biosynthesis of Staphylococcus aureus with an IC50 value of 6 mM. The MICs for two S. aureus strains were considerably higher with 128 mg mL1.133 In 2011 ve new hispidin derivatives, the phellibaumins A–E (146–150), together with known related compounds were described134 from fruiting bodies of Phellinus baumii. Effects on NF-kB activation (indication e.g. inammation) in prostate cancer cells (PC-3 cells) were tested and IC50 values for those ve compounds ranged from 41 (for 147) to 74 mM. Recently Lee et al.135 isolated penstyrylpyrone (151) from a marine-derived Penicillium exhibiting protein tyrosine phosphatase 1B (PTP1B) inhibiting activity with an IC50 value of 5.28 mM. This enzyme is considered to be a
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negative regulator in insulin signaling and inhibitors might be useful in treating type 2 diabetes and obesity (for review on natural products:136). In addition 151 was able to suppress the production of pro-inammatory mediators like NO and prostaglandin E2 with IC50 values of 12.32 mM and 9.35 mM respectively.135 In 2011 a concisive review on styrylpyrones and their potential in medicinal applications has been published.137 In 2009 JBIR-12 (152) and JBIR-25 (153), two compounds with DPPH radical scavenging activity comparable to atocopherol (IC50 ¼ 50 mM) were isolated.138 152 from Penicillium sp. NBRC 103941, is a highly oxygenated tetrahydronaphthalene with an acyl chain with an DPPH radical scavenging activity at IC50 values of 75 mM. Its methylated and acetylated derivatives did not show any activity up to 200 mM. 153 139 was isolated from the hyphomycete CR28109. Its DPPH radical scavenging activity was comparable with 152. Coincidentally Ingavat et al.140 described 153 from a marine-derived Aspergillus aculeatus and named it Aspergillusol A. In their assays 153 inhibited the a-glucosidase of Saccharomyces cerivisiae and Bacillus stearothermophilus with IC50 values of 465 and 1060 mM respectively and showed weak cytotoxicity
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against HuCCA-1, A549, and MOLT-3 cells with IC50 values of 50, 74, and 19 mM, respectively. 153 is the rst fungal example of a tyrosine derivative with an oxime moiety and its total synthesis was published in 2010.141
The asperparalines A–C (154–156) had been described as metabolites of Aspergillus japonicus causing paralysis of silkworms (Bombyx mori, at 3 mg per g body weight).142,143 In very elegant mode of action studies Hirata et al.144 now have proven that 154 selectively blocks insect acetylcholine receptors while vertebrate (chicken) receptors were hardly affected. This opens the way to investigate the mechanism of this selectivity, which could lead to new insecticides with very low toxicity.
Penicillium sp. CNL-338 isolated from the red alga Laurencia sp. from the Bahamas Islands produced the lumazine peptide penilumamide (157)145 The peptide has 1,3dimethyl-lumazine-6-carboxylic acid as an unusual starter unit which is coupled to methionine sulfoxide and anthranilic acid methyl ester. Neither antibacterial nor antifungal activity was described.
Nygerone A (158) was produced only when Aspergillus niger ATCC 1015 was cultured in the presence of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat).146 To date no bioactivity was published for 158. Several reviews on “unlocking” silent gene clusters by applying several methods have been published in recent years.147–150
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Protuboxepins A (159) and B (160), two oxepin-containing pyrimidine alkaloids and protubonines A (161) and B (162), two diketopiperazine-type alkaloids were isolated from a marinederived Aspergillus sp. SF-5044.151 From the same Aspergillus strain two new tetraketides named protolactone A (163) and B (164) with a new ring system had been published earlier.152 Data concerning bioactivities of these two compounds were not presented. 159, 161, and 162 were tested against a panel of cell lines. Only 159 exhibited weak cytotoxicity with IC50 values between 75 and 250 mM.151 159 binds to a,b-tubulin which causes metaphase arrest and promotes apoptosis at 80 and 160 mM.153 Since the structure of 159 differs substantially from known tubulin inhibitors it might become a useful lead structure. In 2012 154 a Korean patent was published which claims protuboxepins and protubonines for the treatment of cancer. The oxepinamides D–G (165–168) from Aspergillus puniceus showed transactivation effects on the Liver X Receptor alpha (LXRa) in a reporter gene assay.155 This nuclear receptor is thought to be a pharmacological target for metabolic disorders, chronic inammation and neurodegenerative diseases, and cancer.156 165–168 increased the LXRa controlled gene activity at concentrations between 1 to 30 mM with 2.8 to 4.1 fold induction. At 50 mM cytotoxic effects were observed.155 The indole alkaloid dimer brevianamide J (169) and the diketopiperazine alkaloids brevianamides K–N and later O–R, were all produced by the same Aspergillus versicolor.157,158 Additional brevianamides S (170) to V were isolated from a different Aspergillus versicolor strain with 170 being the second example of a dimeric diketopiperazine besides 169.159 170 had a MIC of 6.25 mg mL1 against the Bacille Calmette–Gu´ erin, which is an attenuated strain of the bovine tuberculosis bacillus Mycobacterium bovis used in screenings for antitubercular compounds.159 Calpinactam (FKI-4905, 171), isolated from the zygomycete Mortierella alpina is a highly selective antimycobacterial agent. It inhibits the growth of Mycobacterium smegmatis and M. tuberculosis with MIC values of 0.78 and 12.5 mg mL1, respectively. None of the other tested bacteria and fungi were affected.160 The structure of 171 was determined by spectroscopic methods and total synthesis. 171 is a hexapeptide with a caprolactam ring at its C-terminus. Its absolute stereochemistry was determined by amino acid analysis and total synthesis to D -phenylalanyl-L-leucyl-L-histidyl-D-glutaminyl-D-allo-isoleucyl-Lcaprolactam.161 It is interesting that the mycobacterial siderophores (Mycobactins)162 also contain a C-terminal caprolactam ring which, however, bears an hydroxyl group at the ring nitrogen. To explore the structure–activity relationships of 171, derivatives with different amino acids were synthesized utilizing solid-phase peptide synthesis. Among them only a
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peptide with an activity.163
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D-ala
in place of
D-glu
showed moderate
Apicidin (172) was rst described in 1996 by a group of Merck as a Fusarium metabolite with very strong inhibitory activity against the parasitic protozoa Plasmodium berghei, P. falciparum, Eimeria tenella, Neospora caninum, Caryospora bigenetica. Mode of action (MOA) studies revealed that 172 is a very potent inhibitor of the parasites histone deacetylases (HDACs) inducing hyperacetylation of histones thus interfering with a central pathway in eukaryotic transcriptional control. The apicidins share antiprotozoal activity and MOA with other fungal cyclic tetrapeptides like HC-toxin, trapoxin A and chlamydocin.164 Their strong cytotoxic activities at low nanomolar concentrations and their MOA indicate that the selectivity of antiparasitic and antineoblastic activity had to be addressed with regard to minimize in vivo toxicity. In the meantime a number of syntheses of 172 and variants with higher activity and selectivity against protozoa have been patented and published.165,166 An excellent survey on natural products as modulators of histone acetylation and their uses in antineoblastic therapy has been compiled by Salvador and Luesch in 2012.167
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Recently, as an alternative to chemical synthesis, a genetic approach to obtain new apicidins through modication of the nonribosomal apicidinsynthase (AS 1) by Jin et al.168 resulted in the production of the apicidins B (173), D2 (174), and E (175) in F. semitectum. The sequencing of complete genomes offers the opportunity to detect genes coding for enzymes leading to hitherto unknown metabolites. Von Bargen et al.169 discovered an AS 1-like gene cluster in F. fujikuroi, a fungus from which so far no HDAC inhibitor had been described. By overexpression of the pathway specic transcription factor they were able to produce a new metabolite, apicidin F (176). Here, L-isoleucine is replaced by L-phenylalanine and L-2-amino-8-oxodecanoic acid by L-2-aminooctanedioic acid. 176 exhibited good activity against Plasmodium falciparum with an IC50 of 670 nM.169
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Heterozygote deletion mutants of the pathogen Candida albicans with strain-specic bar codes were used in co-cultured screenings (yeast haploid deletion approach). Using nonlethal concentrations of extracts or pure compounds individual mutants show an altered tness and proling could reveal the mode of action. Using this screening strategy phaeofungin (177) and phomafungin (178), two novel lipodepsipeptides were isolated.170,171 The 25-membered cyclic depsipeptide 177 isolated from a Phaeosphaeria sp. contains seven amino acids and ab,gdihydroxy-g-methylhexadecanoic acid.170 177 causes the release of ATP in wild-type C. albicans strains, the MICs for C. albicans, Aspergillus fumigatus and Trichophyton mentagrophytes were determined to 16 mg mL1, 8 mg mL1 and 4 mg mL1 respectively. Pronounced antifungal activities were described for 178 with MICs of 2–8 mg mL1 against the above mentioned human pathogens.171 The 28 member ring consisting of eight amino acids and a b-hydroxy-g-methyl-hexadecanoic acid was isolated from an unspecied Phoma species.
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Scopulariopsis brevicaulis isolated from a marine sponge was found to produce the depsipeptides Scopularides A (179) and B (180).172 They showed activity against several cancer cell lines at 10 mg mL1. In 2009 a patent was published claiming these cyclic peptides for the treatment of cancer.173
Verticilide A1 (181) from a Verticillium sp. was found to inhibit the ryanodine binding to its receptors in cockroaches with an IC50 value of 4.2 mM. The homologous receptor in mice was more than 10 times less sensitive.174,175 The stereochemistry and the total synthesis were published in 2006.176 Against the nematode C. elegans and the brine shrimp A. salina the MICs were found to be 20 mg mL1 (23 mM) and no cytotoxic or antimicrobial activities were detected. In 2012 Ohshiro et al.177 isolated 181 and three new verticilides named A2 (182), A3 (183), and B1 (184) from a different Verticillium species. These compounds were tested against two isozymes of acyl-CoA cholesterol acyltransferase (ACAT-1 and -2). With IC50 values between 0.23 and 1.3 mM ACAT-2 was more sensitive than ACAT1 with IC50 values between 2.5 and 11 mM In contrast to 181, 182, 183, and 184 did not inhibit the ryanodine receptor (up to 100 mM). 184 exhibited moderate toxicity towards Artemia salina and broad antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, and Mucor racemosus.
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In 2012 a solid-phase synthesis for the stepwise coupling of N-methyl amino acids and hydroxycarboxylic acids on solid support was described for the related anthelmintic cyclodepsipeptide PF1022A (185) and its commercial analogue emodepside (186) which could be applied to other cyclodepsipeptides and synthetic screening libraries.178,179 A concise review on anthelmintic cyclcooctadepsipeptides with emphasis on the mode of action of 185 and 186 appeared in 2012.180 The Omphalotins A–D were isolated in 1997 and 1998 from fermentations of Omphalotus olearius (Basidiomycetes), as strong and selective nematicides acting preferably on Meloidogyne incognita, an important plant pathogenic root not nematode.181–184 During continuing efforts to obtain better producers a monokaryotic (haploid) strain was found to yield not only more of omphalotins A–D but also ve new nematicidal derivatives. The omphalotins E–I (187–191) are oxidatively modied cyclic dodecapeptides with four (188–191) of them containing an unprecedented N-hydroxylated tricyclic tryptophan derivative.185 While no antibacterial, antifungal, and cytotoxic activities were detected, the nematicidal activity against M. incognita compared favorably with ivermectin with LD90 values between 2–10 mg mL1.
NBRI23477 A (192) and B (193), two new atpenins, have been isolated from Penicillium atramentosum as inhibitors of human prostate cancer cells (DU-145) at concentrations of 0.95 and 0.71 mg mL1.186 The stereoselective total synthesis of 193, together with atpenins A4, 193, and harzianopyridone has been achieved by Ohtawa et al.187 The atpenins, rst described by Omura's group as antifungal antibiotics interfering with the ATP generating system in Raji cells188,189 are potent and specic inhibitors of mitochondrial complex II succinate ubiquinol oxidoreductase.190 In anaerobic parasitic eukaryotes, complex II functions as a terminal oxidase in the NADH-fumarate pathway. Since this pathway is essential for growth and survival of anaerobic parasitic helminths and some cancer cells it offers an interesting target for the development of new chemotherapeutics.191
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Three new metabolites were discovered from a marine derived Phialocephala species.192 The sorbicillin dimer, dihydrotrichodermolide (194), the sorbicillin monomer dihydromethylsorbicillin (195), and the benzofuran phialofurone (196) exhibited cytotoxic activity against the P388 cell line with IC50 values of 11.5 mM, 0.1 mM, and 0.2 mM respectively. The sorbifuranones A, B, and C (196–198) were isolated from Penicillium strains derived from Mediterranean sponges.193 These structures are a new class of sorbicillinol-derivatives. Unfortunately, no information about biological activities were given.194 Sorbiterrin A (199) was described from Penicillium terrestre isolated from marine sediments. 199 showed moderate acetylcholinesterase inhibition with an IC50 value of 25 mg mL1. 199 is the rst example of a sorbicillinoid with a bridged [3.3.1] ring system. The sorbicillamines A–E (200–204) were reported by Guo et al. in 2013.195 They were isolated from a deep sea derived Penicillium species and did not exhibit cytotoxic effects against a panel of cell lines up to 10 mM. Interestingly, 204 is the rst sorbicillin trimer containing a nitrogen atom. The sorbicillinoids JBIR-59 (205) and JBIR-124 (206) were described from a Penicillium citrinum strain isolated from a marine sponge.196,197 The radical scavenging potential of these two compounds was reported. Li et al.198 described chloctanspirones A (207) and B (208), two chlorinated sorbicillinoids with a bicyclo[2.2.2]octane-2-spiro cyclohexane skeleton, both isolated from a marine sediment derived Penicillium terrestre. 207 and 208 showed cytotoxic activity against HL-60 with IC50 values of 9.2 and 39.7 mM. A review on the biosynthesis, bioactivities, and synthetic approaches covering all sorbicillinoids until 2010 was published in 2011 by Harned and Volp.199 Guignardia bidwellii causes black rot in grapes. The ascomycete was found to produce besides the known guignardic acid (209)200 phenguignardic acid (210), a new phytotoxin.201 209 and 210 are phytotoxic in a non-host-specic manner. No cytotoxic or antimicrobial activities could be detected. In 2013 seven new derivatives (211–217) were described202 of which alaguignardic acid (211), and guignardianones A (212), E (216), and F (217) exhibited phytotoxic properties. The absolute conguration of this series of guignaridic acid derivatives was determined by vibrational circular dichroism (VCD) spectroscopy.203 The total synthesis of 210 has been achieved by Stoye et al.204 Pleurone (218) was isolated from fruiting bodies of Pleurotus eryngii var. ferulae in the course of a screening for inhibitors of human neutrophil elastase, an enzyme which is considered to play a role in skin ageing.205 However, the IC50 value of 49.4 mM compared unfavorably with epigallocatechin gallate (IC50 8.8 mM) used as standard.
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SMTPs (Stachybotrys microspora triprenyl phenols) are interesting compounds which activate plasminogen and enhance its binding to brin. This stimulation of endogenous brinolytic activity is considered an interesting approach to develop antithrombotic agents. The rst metabolite of this series, stablapin (219), was isolated from Stachybotrys microspora IFO30018 in 1996.206 In 2000 Hu et al.207 described two dimeric analogs SMTP-7 (220) and -8 (221) which exhibited an enhanced activity as compared to the monomers at concentrations of 80 to 200 mM. In recent years SMTP derivatives with different N-linked side chains were published. This side chain can be controlled by feeding amines which was done by
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Hasegawa et al.,208 resulting in 11 new analogs and by Koide et al.209,210 resulting in 22 SMTP derivatives with a phenylamine- or phenylglycine-based side chain. Some of them show plasminogen-modulating activity comparable to 220. 220 got increased attention in recent years especially because of its thrombolytic, anti-inammatory, and antioxidant effects. The treatment of cerebral infarcts and thrombosis were studied.211–215 Hu et al.216 tested 220 in vivo, in mice it enhances plasmin generation at 5–10 mg kg1. The effects on bleeding and rebleeding were tested (up to 30 mg kg1) and no prolonged or increased bleeding was detected. In rats 220 was tested in a pulmonary embolism model (5 mg kg1) and an increase of clot clearing was observed (20% in 20 min). It was therefore concluded that compounds of this type might be leads for the development of antithrombotic medications.216
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lymphocytes and in the CNS. 223 inuences processes in neurogenesis, neural cell function, and migration (for review see ref. 220). These results support ongoing research to identify new modulators for sphingosine-1-phosphate receptors.
4 New developments in the uses of known compounds or new derivatives The complex prenylated indole alkaloid waikialoid A (222) was isolated from an Aspergillus species from a soil sample collected near Waikiki Beach, Hawaii.217 222 was able to inhibit biolm formation of Candida albicans with an IC50 value of 1.4 mM and inhibited hyphal morphogenesis but was not able to disrupt preformed biolms. No direct antifungal or cytotoxic activity was detected up to concentrations of 200 mM. This is remarkable because the structurally related stephacidin B shows pronounced cytotoxicity against several cell lines with IC50 values
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REVIEW
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Fungal natural products in research and development Cite this: Nat. Prod. Rep., 2014, 31, 1425
Anja Schueffler* and Timm Anke*
Covering: 2009 to 2013 To date approximately 100 000 fungal species are known although far more than one million are expected. The variety of species and the diversity of their habitats, some of them less exploited, allow the conclusion that fungi continue to be a rich source of new metabolites. Besides the conventional fungal isolates, an increasing interest in endophytic and in marine-derived fungi has been noticed. In addition new screening strategies based on innovative chemical, biological, and genetic approaches have led to novel fungal metabolites in recent years. The present review focuses on new fungal Received 2nd May 2014
natural products published from 2009 to 2013 highlighting the originality of the structures and their biological potential. Furthermore synthetic products based on fungal metabolites as well as new
DOI: 10.1039/c4np00060a
developments in the uses or the biological activity of known compounds or new derivatives are
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discussed.
1 1.1 2
5
Introduction Current strategies in the search for new metabolites New metabolites, mode of action studies, new genetic methods, and approaches toward the generation of metabolites Compounds recently introduced into the market New developments in the uses of known compounds or new derivatives References
1
Introduction
3 4
The review covers papers and patents on fungal metabolites published from 2009–2013 with emphasis on the novelty of the structures, their bioactivities and their expected or realized uses in the Pharma and Agro industries. In part the topics highlighted here have been discussed in previous reviews like marine and endophytic natural products published from 2009–2013,1–6 industrial aspects of natural products chemistry.7 1.1
Current strategies in the search for new metabolites
The fungal sources of new metabolites have been broadened from saprophytic terrestrial strains to marine habitats and living plants with their endophytes. While by far most of the Institut f¨ ur Biotechnologie und Wirkstoff-Forschung (Institute of Biotechnology and Drug Research), Erwin-Schroedinger-Str. 56, 67663 Kaiserslautern, Germany. E-mail: schueffl[email protected]; [email protected]
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metabolites described are produced by asexual stages of ascomycetes e.g. Penicillium and Aspergillus species, the interest in basidiomycetes and the number of metabolites derived from them has grown considerably. Zygomycetes although easily isolated and cultured seem to be less prolic producers. The test systems used in the search for new biological activities and new compounds have been adapted to the needs of medicine and agriculture. In the antibiotics eld there is an urgent need to overcome resistance of pathogenic bacteria and fungi with compounds with new structures and modes of action. In the eld of other medications a number of very interesting biochemical test systems have been developed yielding very interesting results.
2 New metabolites, mode of action studies, new genetic methods, and approaches toward the generation of metabolites Hypocreolide (1), a nonenolide, was derived from fermentations of the ascomycete Hypocrea lactea.8 1 shows moderate antibacterial, antifungal and cytotoxic activities. Since neither the relative nor the absolute stereochemistry of 1 could be initially assigned, a stereochemically exible total synthesis was developed providing 1 in nine steps and 12% overall yield.
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Japan. The metabolite inhibits Ca2+ signaling in a yeast mutant.11
The cytospolides A–Q (2–11) and decytospolides A and B (12; 13) are unusual nonalolides isolated from a Cytospora sp., an endophytic fungus derived from Ilex canariensis.9,10 Absolute congurations were assigned using the modied Mosher's method and CD/TDDFT calculations. The cytospolides B (3), E (6), and P (10) exhibited cytotoxic activities against A-549 (human lung carcinoma) cells with LD50 values of 5, 7, and 2 mg mL1, the cytospolides K, M (7), N (8), O (9), Q (11), and decytospolide B (13) showed only moderate activities with LD50 values of 11–39 mg mL1. The other metabolites were inactive.
Benzophomopsin A (14), a new benzoxepin was isolated from an endophytic Phomopsis strain derived from a cherry tree in
Anja Schueffler studied Biology at the University of Kaiserslautern. Her PhD focused on the characterisation of fungal natural products with antimicrobial activity. In 2010 she received a DFG fellowship and joined Prof. Fenical's group at the Scripps Institute for Oceanography in San Diego, where she studied antibacterial natural products from streptomycetes. In 2011 she started a research fellowship at the Institute of Biotechnology and Drug Research (IBWF, Kaiserslautern). Her scientic work focuses on bioactive natural products, the biosynthesis of secondary metabolites as well as the isolation and taxonomy of fungi of diverse habitats.
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Four new ascochlorin derivatives have been described. Cylindrol A5 (15),12 produced by Cylindrocarpon sp. FKI-4602 exhibits moderate antibacterial activities whereas 100 -deoxy10a0 -hydroxyascochlorin13 from Acremonium sp. LG0808 inhibited the migration of metastatic prostate cancer cells PC3M. LL-Z1272a epoxide (16) was derived from a mutant of Ascochyta viciae14 and seems to be the long proposed intermediate in the biosynthesis of ascochlorin (17) from the precursor LL-Z1272a.15 Recent ndings demonstrated that 17 inhibits eukaryotic respiration by binding to both active sites (Qo and Qi) of the cytochrome bc1 complex.16 This clearly distinguishes 17 from other antibiotics binding to only one site like the strobilurins and antimycins. Ascofuranone (18), a related fungal metabolite, specically inhibits the alternative oxidase of Trypanosoma brucei (TAO), the cause of human African trypanosomiasis, at subnanomolar concentrations by binding to the ubiquinol binding domain of the quinol oxidase. The chemotherapeutic efficacy of 18 both in vitro and in vivo had been demonstrated.17–19 Since TAO is essential for the parasites survival but absent in humans it offers an interesting target for the development of novel drugs. Saimoto et al.20 synthesized a number of ascofuranone derivatives and analogues. They succeeded in the identication of the pharmacophore and the binding site within the enzyme and obtained a synthetic derivative “compound 24” (19) inhibiting TAO with a lower IC50 (0.06 nM) as compared to 18.
Timm Anke received his diploma (Biochemistry) and his PhD degree from the University of T¨ ubingen. From 1973 he spent two years as a research associate with Fritz Lipmann at the Rockefeller University in New York. Back in T¨ ubingen he obtained the venia legendi in 1979. He obtained the professorship of Biotechnology at the University of Kaiserslautern in 1981 from which he retired in 2010. Since 1998 he heads the Institute of Biotechnology and Drug Research IBWF e.V. His research interests are with fungal metabolites, their bioactivities and biosyntheses.
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The ascotricins A (20) and B (21) from Ascotricha chartarum were isolated in a screening for new antagonists of sphingosine-1-phosphate receptor 1 (S1P1) by Yonesu et al.21 Sphingosine-1-phosphate (Sph-1-P) bound to this receptor promotes proliferation, migration and tube formation of vascular endothelial cells. Agonists of S1P1 like ngolimod act as immunosuppressants. In S1P1 expressing cells 20 and 21 acted as S1P1 antagonists with IC50 values of 8.2 and 1.8 mM respectively, inhibited [33P] Sph-1-P binding at 120 and 39 mM, and inhibited the migration of human umbilical vein endothelial cells at IC50 values of 94 and 28 mM. Therefore the ascotricins offer the opportunity to investigate the role of Sph-1-P and its receptor in vascular endothelial cells and angiogenesis. In the course of their studies the authors also found that analogs of caloporoside22 had S1P1 antagonist activity.
Aquastatin A (22) was rst described from Fusarium aquaeductuum as an inhibitor of mammalian adenosin triphosphatases by a group at Sankyo in 1993.23 It inhibits mammalian Na+/K+-ATPase with an IC50 value of 7.1 mM, and
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porcine stomach mucous H+/K+-ATPase with an apparent IC50 value of 6.2 mM. Thus far in testing, no antimicrobial activity had been detected at a concentration of 1 mg mL1. A patent was led for the use in treatment of gastric ulcers.24 In 2003 Ondeyka et al. of Merck Laboratories25 described another interesting biological activity of 22. They isolated the structurally related exophillic acid from Exophiala pisciphila and described that both compounds inhibited the strand transfer reaction of HIV-1 integrase, an enzyme crucial for the replication and spread of HIV with a rather high IC50 value of 50 mM for 22. In 2009 22 was reisolated from cultures of a Sporothrix species in the course of a screening for inhibitors of bacterial enoyl-acyl carrier protein (ACP) reductase, a key enzyme in bacterial fatty acid biosynthesis.26 22 inhibited the enoyl-ACP reductase of Staphylococcus aureus with an IC50 of 3.2 mM. The growth of S. aureus, both wild type and a methicillin-resistant strain, was inhibited at minimum inhibitory concentrations of 16–32 mg mL1. The enoyl-ACP reductase of S. pneumoniae was inhibited with an IC50 of 9.2 mM. It is interesting that the degalactosylated aquastatin B (23), prepared by acid hydrolysis of 22, retains the inhibitory activities, while aquastatin C (24)27 is completely inactive in these assays. In 2009 22, isolated from a marine-derived Cosmospora species, was found to inhibit the protein tyrosine phosphatase 1B (PTP1B).28 22 exhibited potent activity against PTP1B with an IC50 value of 0.19 mM. Kinetic analyses of PTP1B suggested a competitive inhibition. Tests with hydrolysis products (23 and 24), suggested that the dihydroxy pentadecyl benzoic acid (corticiolic acid29) moiety is the pharmacophore. 22 showed modest but selective inhibitory activity toward PTP1B compared to other protein tyrosine phosphatases. Because PTP1B plays a central role in signal transduction, a wide variety of pharmacological effects are to be expected by inhibitors of this enzyme.
The compounds JBIR-37 (25) and -38 (26) were isolated in 2009 from the marine sponge-derived fungus Acremonium sp.30 Although these compounds do not exhibit cytotoxic activity and in addition no other bioactivities are described, they are the rst examples of glycosyl benzenes from fungi.
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Xanthepinone (27), an interesting polyketide, was isolated in a search for new antifungal agents inhibiting the spore germination of phytopathogenic fungi.31 By comparison of its ITS sequence the producing soil fungus was found to be closely related to the plant pathogen Phoma medicaginis. 27 completely inhibited the germination of Magnaporthe grisea, Phythophthora infestans, and Botrytis cinerea conidia at concentrations of 2, 5, and 10 mg mL1, respectively. Only very weak antibacterial (Proteus vulgaris and Staphylococcus aureus) and no cytotoxic activities were detected at 50 mg mL1.
The herbaridines A (28) and B (29), as well as O-phenethylherbarin (30) are new naphthoquinone derivatives isolated in a screening for cytotoxic metabolites. The producer IBWF79B-90A, a sterile lamentous ascomycete could not be identied by morphological features or by its ITS sequence.32 28 and 29 show moderate antibacterial activity at concentrations of 20–50 mg mL1. Antifungal activity against lamentous fungi was not observed. The cytotoxic activity against several human cell lines ranged from 0.5–2.5 mg mL1 and was comparable to herbarin.
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IC50 values of 52.8 and 41.2 mmol L1, respectively. The penicitrinols F–I (35–38) were derived from the same fungus36 and were found to exhibit no cytotoxic activities. Two interesting citrinin trimers named tricitrinols A (39) and B (40) were extracted from a Penicillium citrinum strain isolated from crater ash in China.38 Both compounds exhibited cytotoxic activity against HL60 and HCT116 cells with IC50 values of 3.2 to 8.6 mM. 40 was found to induce apoptosis, cell cycle arrest, DNA damage and inhibition of human DNA topoisomerase 2a. These results supported by computational docking analysis suggest that 40 is an intercalating topoisomerase 2a inhibitor which could serve as a lead structure for further studies.38 In 2013 the citrinin dimers penicitrinone E (41) and penicitrinol J (42) were isolated together with two citrinin monomers from a marine-derived Penicillium species.37 No signicant cytotoxicity was detected but 42 was slightly active against Staphylococcus aureus.
Herqueiazol (43), herqueioxazol (44), and herqueidiketal (45) are new polyaromatic metabolites isolated from a Penicillium sp. derived from a marine sediment.39 45 possesses a novel skeleton and exhibited moderate cytotoxicity but signicant activity (IC50 23.6 mM) against Staphylococcus aureus sortase A. This enzyme attaches proteins to the bacterial cell wall and is considered as a possible target for new antibiotics.
Several citrinin dimers and derivatives from different halotolerant or marine Penicillium strains were described.33–37 Pennicitrinone D (31), a new citrinin dimer, was isolated from Penicillium notatum along with the known pennicitrinone A (or dicitrinin A, penicitrinone A), citrinin, and mycophenolic acid. When tested for cytotoxic activities 31 was found inactive.34 The penicitrinols C–E (32–34) were isolated from a marine derived P. citrinum35 32 and 34 were slightly active against HL-60 cells, with
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Chrysoxanthone (46) is an unusual heterodimer of the tetrahydroxanthone blennolide A and the anthraquinone 2-hydroxychrysophanol linked through a diaryl ether bridge. It was isolated from mycelia of the sterile lamentous ascomycete IBWF11-95A. Its structure was elucidated by two-dimensional NMR spectroscopy. 46 shows antibacterial and antifungal activity with MIC values between 2.5 and 20 mg mL1. The cytotoxicity was moderate with IC50-values of 50 mg mL1 for Jurkat, L-1210, and Colo-320 cells.40
Isochaetochromin A1 (47), a new bis-naphtho-g-pyrone isomer was isolated together with the known isochaetochromins B1 (48) und B2 (49) from fermentations of Penicillium sp. FKI-4942 in a search for new inhibitors of mammalian triacylglycerol (TG) synthesis.41,42 In an assay using CHO-K1 (Chinese hamster ovary) microsomes the incorporation of 14C-palmitoyl-CoA into 1,2-dioleoyl-sn-glycerol was inhibited by 47, 48, and 49 with IC50 values of 33 mM, 11 mM, and 5.6 mM respectively. Inhibitors of TG synthesis are possible candidates for the development of therapeutic agents to treat obesity.
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AC1, AC2, AD1, AD2, AE1 and AE2, were isolated from the culture broth of Talaromyces pinophilus FKI-3864 by Kawaguchi et al.46 Dinapinone AB2 showed potent inhibition of TG synthesis in intact mammalian cells with an IC50 value of 1.17 mM, whereas the other dinapinones exhibited weaker activities.
The xanthoradones A (52) and B (53) are products of Penicillium radicum FKI-3765-2.47,48 Despite a moderate activity against Staphylococcus aureus and Bacillus subtilis these compounds reduce the MIC value for the b-lactam imipenem from 16 to 0.06 mg mL1 (52) and 0.03 mg mL1 (53) against methicillin-resistant Staphylococcus aureus (MRSA) whereas xanthoradones themselves are not active against MRSA. Xanthoradone C (54), described in 2010 49 showed weaker effects with a reduction of imipenem MIC value to 0.5 mg mL1. Interestingly xanthoradones seems to inhibit FtsZ, the bacterial tubulin homolog which is essential for formation of the septum and recruiting of other proteins for cell division. A patent which claims the concurrent use of FtsZ inhibitors and b-lactam antibiotics was led in 2011 by Merck Sharp & Dohme Corp.50 and several natural products were listed for their ability to inhibit FtsZ. Among others the xanthoradones as well as the structurally related viriditoxin (55)51 are named as lead structures. For 55 the inhibition of FtsZ was conrmed.52
The dinapinones were isolated from strain FKI-3864 (named Penicillium pinophilum43–45/Talaromyces pinophilus46) in a search for inhibitors of TG-synthesis. Dinapinone A (50), a mixture of two stereoisomers A1 und A2 inhibited TG- and cholesteryl ester synthesis with an IC50 value of 0.097 mM and 0.31 mM respectively.43 It was observed that the inhibitory activity on TG synthesis of the separated compounds was substantially reduced. Dinapinones A1 and A2 are atropisomers consisting of two monomers with the same planar structure of dihydronaphthopyranone as monapinone A (51).44 The monapinones were only produced when P. pinophilum was grown in seawater supplemented medium.45 They showed rather weak inhibition of TG synthesis. Eight new dinapinones, AB1, AB2,
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Xanalteric acids I (56) and II (57) are metabolites of an endophytic Alternaria sp. isolated from leaves of the mangrove Sonneratia alba.53 These compounds are the rst fungal compounds with a 10H-phenaleno[1,2,3-de]chromene skeleton. They exhibit modest cytotoxic activity against L5178Y cells at 10 mg mL1 and weak antibacterial activity against Staphylococcus aureus.
Botrytis cinerea (sexual state Botryotinia fuckeliana) is a very important pathogen affecting many crops and a prolic producer of structurally diverse phytotoxins. A very concise and readable review on the chemistry and structural determination of botcinolides, botcinins, and botcinic acids produced by Botrytis cinerea strains has appeared in 2009.54 The regulation of the biosyntheses of the Botrytisphytotoxins has been studied in detail. Gene knock-out and complementation studies revealed that the transcriptional regulator bcreg1 is required for pathogenicity, conidiogenesis, and the production of secondary metabolites. A bcreg1 mutant was able to penetrate plant tissue but was not able to cause necrotic lesions. In addition, the mutant did not produce detectable levels of the sesquiterpene botrydial (58) and the polyketide botcinic acid (59).55 BcAtf1 controls a diversity of cellular processes and has broad regulatory functions. It also controls secondary metabolism, a Dbcatf1 mutant contained signicantly elevated levels of the phytotoxins botrydial, botryendial (60), and botcinin A (61).56
Emervaridione (62) and varioxiranediol (63),57 two new metabolites have been derived from fermentations of an Emericella variecolor (Aspergillus stellatus) strain. Despite of their interesting structures no cytotoxic or antimicrobial activities have been detected so far for these metabolites.
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Ceramidastin (64) was described as a metabolite of a Penicillium sp. isolated from a soil sample in a screening for inhibitors of a cloned ceramidase from Pseudomonas aeruginosa.58 This enzyme is thought to aggravate atopic dermatitis in humans and inhibitors might therefore alleviate this condition. 64 inhibited the bacterial ceramidase with an IC50 value of 6.25 mg mL1. 64 did not inhibit human ceramidase and no antimicrobial and cytotoxic activities were detected up to concentrations of 200 and 100 mg mL1, respectively. 64 is similar to the toxic rubratoxins, e.g. rubratoxin A (65). This metabolite, however, did not inhibit the bacterial ceramidase at 100 mg mL1.
Fimetarone A (66) was isolated from cultures of a Fimetariella sp. (Sordariales) isolated from a sample of “Chinese caterpillar fungus” used in traditional Chinese medicine for all kinds of medications.59 It consists of a preparation of fruiting bodies of Ophiocordyceps sinensis, still connected to the caterpillar larva of the moth Thitarodes sp. (Hepialidae) which it colonizes. 66 has a new spiro[chroman-3,70 -isochromene]-4,60 (80 H)-dione skeleton and was obtained as 1 : 1 atropdiastereomeric mixture. 66 showed modest cytotoxic activity against T24 cells (human bladder carcinoma) with an IC50 value of 40.2 mM.
The epicoccolides A (67) and B (68) are new polyketides derived from an endophytic Epicoccum strain of Theobroma cacao. They were isolated together with epicolactone and other compounds in a search for metabolites inhibiting the growth of Pythium ultimum, Aphanomyces cochlioides, and
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Rhizoctonia solani, all three important plant pathogens. Beside antifungal activities 67 and 68 exhibit modest antibacterial activities.60
Chloropestolide A (69) was isolated from Pestalotiopsis ci derived from branches of Camellia sinensis. This highly functionalized spiroketal with a very unusual ring system was found to inhibit the growth of HeLa and HT 29 cells at concentrations of 0.7 and 4.2 mM. The replication of HIV-1 in C8166 cells was inhibited with an IC50 value of 64.9 mM.61 From the same fungus. grown on solid substrates six new chloropestolides B–G were isolated featuring chlorinated spiro[benzo[d][1,3]dioxine2,7-bicyclo[2.2.2]octane]-4,8-dione (e.g. 69) and spiro[benzo-[d][1,3]dioxine-2,1-naphthalene]-2,4-dione skeletons (e.g. chloropestolide E 70).62 The biosynthesis of the chloropestolides has been proposed to start from the precursors 71 and 72. Diels– Alder reactions with 71 as diene and 72 as dienophile or with 71 as dienophile and 72 as diene should yield e.g. 69 or 70, respectively.
Decalpenic acid (73) was isolated from Penicillium verruculosum in a screening for compounds inducing early osteoblastic markers in a pluripotent mouse mesenchymal cell line. Osteoblasts are responsible for bone formation in embryonic development and adult life and arise from mesenchymal stem cells. It is assumed that compounds
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inducing osteoblastogenesis might become valuable tools to elucidate steps in osteoblast differentiation or might be developed into therapies for bone diseases. 73 signicantly induced early markers such as alkaline phosphatase activity and osteopontin mRNA in C3H10T1/2 cells at concentration of 1 mM.63 Sakamota et al.64 have shown that 73 acts as a new retinoid and induces early osteoblastic markers in pluripotent mesenchymal cells through activation of retinoic acid receptor g.
The haplofungins A–H (74–81) from Lauriomyces bellulus are composed of an arabinonic acid moiety linked by an ester bond to a long modied alkyl chain.65 The absolute conguration of 74 has been determined.66 The haplofungins inhibit the inositol phosphorylceramide (IPC) synthase of Saccharomyces cerevisiae with IC50 values from 1.5–65 ng mL1.67 The IPC synthase of Aspergillus fumigatus is less sensitive. Since fungal IPC is essential for growth and differs substantially from the human glycosphingolipid, IPC synthase is considered an interesting target for selective fungicides. Unfortunately, however, with the exception of Candida glabrata, no activity against C. albicans, C. parapsilosis, C. tropicalis, Cryptococcus neoformans and A. fumigatus was observed in vivo. The same was found for khafrefungin,68,69 an IPC inhibitor with a similar structure. In this respect the haplofungins differ from the cyclic depsipeptide pleofungin A70,71 an IPC inhibitor which exhibits also very strong antifungal activities. Two new caloporosides, G (82) and H (83) were isolated from the basidiomycete Gloeoporus dichrous in a search for inhibitors of spore germination of Magnaporthe grisea and Fusarium graminearum.72 Caloporoside A (84) was rst isolated as a potent and selective inhibitor of phospholipase C of pig brain.22 Other interesting biological activities have been described for caloporoside derivatives: inhibition of the binding of 35S-TBPS to the GABAA/benzodiazepine chloride channel receptor complex in vitro,73,74 inhibition of the binding of hyaluronic acid to its receptor CD44,75 as well as S1P1 antagonist activity.22 From fruiting bodies of the basidiomycete Hericium erinaceus three new metabolites, hericenone I (85), hericene D (86),76 and isohericenone (87)77 were isolated. 85 and 86 exhibit very low antibacterial and cytotoxic activities while 87 exhibited potent cytotoxic activity against A549, SK-OV-3, SK-MEL-2 and HCT-15 cell lines with IC50 values of 2.6, 3.1, 1.9 and 2.9 mM respectively.
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Caripyrin (88) is a new pyridyloxirane from cultures of the basidiomycete Caripia montagnei.78 88 was found to inhibit conidial germination and appressorium formation in the rice blast fungus Magnaporthe oryzae, whereas the infection-related morphogenesis in several other phytopathogenic fungi was not affected. In plant assays on rice, 88 was found to protect plants efficiently against infection with Magnaporthe oryzae. Contrary to the structurally related fusaric acid (89), 88 was neither cytotoxic, antibacterial, nor nematicidal.
The udalactaranes A (90), B (91), and udasterpurenol A (92) were the rst secondary metabolites described from Phlebia (Mycoacia) uda.79 The new sesquiterpenoids were isolated together with the known hyphodontal and sterpuric acid in a screening of basidiomycetes for antifungal antibiotics. 90 was isolated as a mixture A (90a) and epi-A (90b) in a diastereomeric ratio of 1.6 : 1. 91 is a disesquiterpene consisting of an isolactarane and a sterpurene part. As found for 90, 91 was isolated as a mixture of two epimers in a 3 : 1 ratio (91a/b). When tested for antibiotic activities 90a and 90b (1.6 : 1) and 91a and 91b (3 : 1) showed modest antifungal activities inhibiting the spore germination of Fusarium graminearum at 10 and 5 mg mL1, respectively.
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prevented. The same effect was observed for marasmal C (102) at 50 mg mL1. Conidial germination of B. cinerea was not inhibited by any of the compounds at concentrations up to 100 mg mL1.81
From three basidiomycetes, Xeromphalina sp., Stereum sp., and Pleurocybella porrigens, six triquinane sesquiterpenes with unprecedented modications and a rearranged sesquiterpene related to coriolin C have been isolated.80 Metabolites lacking the exocyclic enone moiety exhibited no or reduced antibiotic activities against fungi and bacteria. The cytotoxicities (IC50 value) against Jurkat cells of xeromphalinone A (93), B (94), F (98) and chlorostereone (99) ranged from 1 to 5 mg mL1 (2 to 20 mM). For xeromphalinones C (95), D (96), and E (97) no cytotoxic activities were observed at concentrations up to 50 mg mL1 (above 100 mM). The formation of cysteine adducts was observed by HPLC-MS for 93, 94, and 98 as well as for 99, the rst example for a chlorinated triquinane from a terrestrial fungus.
Three new drimane sesquiterpenoids were discovered in a screening for inhibitors of spore germination of phytopathogenic fungi. The mixture of marasmene B and epi-marasmene B (100) inhibited the germination Magnaporthe oryzae spores completely at a concentration of 100 mg mL1, while Fusarium graminearum, Phytophthora infestans, and Botrytis cinerea were not affected. In the conidial germination assay, Marasmal B (101) was found to be the most active secondary metabolite tested. At a concentration of 5 mg mL1, conidial germination in M. oryzae, P. infestans, and F. graminearum was completely
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Ganodermycin (103), a sesquiterpenoid, was isolated in a search for new inhibitors of CXCL10 expression in a reporter gene assay with transiently transfected human MonoMac6 cells.82 CXCL10 (inducible protein 10) is a chemoattractant which contributes to the recruitment of inammatory cells such as macrophages and T-lymphocytes and thereby plays an important role in chronic inammation. 103 Inhibited CXCL10 synthesis and secretion with IC50 values around 40 mg mL1. In addition, 103 exhibited modest antibacterial and antifungal activities.
In recent years several sesquiterpenes with a chamigrane skeleton and an endoperoxide have been described from fungi. The merulins A–D (104–107) were isolated from an unidentied basidiomycete belonging to the family Meruliaceae isolated from the plant Xylocarpus granatum.83,84 At the same time the steperoxides A–D (108, 104, 109, 110) were described as metabolites from Steccherinum ochraceum (family Meruliaceae).85,86 It later turned out that steperoxide B and merulin A are identical (104). For 110 antibacterial activity against Staphylococcus aureus has been detected. When tested against two cancer cell lines 104 and 106 proved to be cytotoxic with IC50 values between 1.57 and 5 mg mL1, respectively. 106 was not cytotoxic up to concentrations of 10 mg mL1. Compounds 104 to 108 were tested for antiangiogenic effects.84 In a rat aortic sprouting assay 106 showed strong (2.5 mM) and 108 weaker activities (25 mM) whereas all others had no effect. In following experiments 106 showed suppression of endothelial cell proliferation and migration mediated through the Erk1/2 signaling pathway and inhibited neovessel formation ex vivo and in vivo in a mouse matrigel plug assay. The talaperoxides A–D (111–114) together with 104 were isolated from a mangrove-derived Talaromyces avus strain.87 All ve compounds were toxic to Artemia salina (LD50 < 10 ppm) and cytotoxic against a panel of human cancer cell lines with
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IC50 values between 0.7 and 19.8 mg mL1. No antimicrobial activity was observed. Like artemisinin, 104–106 were found to be active against Trypanosoma brucei at concentrations of 0.8 to 8 mM. In addition, SAR studies as well as semisynthetic modications of 104 were described88 and claimed in a patent which was led in July 2012.89 The semi-synthetic modications yielded several candidates which exhibit pronounced activity. Two compounds (115 and 116) displayed an IC50 of 60 nM and a selectivity index greater than 300-fold against cell lines. Phomopsene (117) and methyl phomopsenate (118), two new diterpenes, were isolated from Phomopsis amygdali in a “geneguided screening approach”90 Using degenerated primers a geranylgeranyl diphosphate synthase gene required for diterpene biosynthesis was identied in Phomopsis amygdali. Consequently the phomopsene synthase gene was identied and heterologous expressed in E. coli. Isolation of the protein and incubation with geranylgeranyl diphosphate lead to the identication of 117 which could then be detected in extracts of Phomopsis amygdali. Further NMR studies in extracts of the producing fungus identied 118 as a related metabolite. No information on the biological activities was given for both compounds.
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The diterpene wickerol A (119) isolated from Trichoderma atroviride FKI-3737 was described in a patent as inhibitor of inuenza virus replication with an IC50 of 70 ng mL1 whereas the proliferation of the human cell line used for the inuenza assay was affected only at 7 mg mL1.91 At 10 mg mL1 no pronounced antimicrobial activity was detected in an agar diffusion assay against a panel of bacteria and fungi. In 2012 the wickerols A (119) and B (120) were isolated from a different Trichoderma atroviride strain FKI-3849.92,93 When tested for antiinuenza activity 120 proved to be less active. Trichodermanin A which is identical to 120, was isolated from an endophytic T. atroviride S361 derived from Cephalotaxus fortune.94 Interestingly the wickerols are the rst examples of a 6-5-6-6 ring skeleton.
From an endolichenic strain of Nodulisporium two new 3,4seco-4-methyl-progesteroids named nodulisporisteriod A (121) and B (122)95 were isolated together with demethoxyviridin and inoterpene B, previously described from Inonotus obliquus.96 This is the rst report of 3,4-seco-4-methyl-pregnan steroids. 121 and 122 inhibited the amyloid b aggregation at 100 mM approximately 50%. The aggregation of amyloid b into brillar plaques is thought to be one of the causes of Alzheimer's disease.
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The atlantinones A (123) and B (124) were isolated from the guttate of Penicillium citreonigrum cultured on solid media in a study to investigate the changes of secondary metabolite production in the presence of 5-azacytidine.97 The new meroterpenes were not detected when the fungus was grown in the absence of the mutagen. No antibacterial and antifungal activities have been detected.
In 1993 the pyripyropenes A–D (125–128) consisting of a pyridine, a-pyrone and sesquiterpene moiety were isolated from Aspergillus fumigatus FO-1289. They inhibited a key enzyme catalyzing the esterication of cholesterol, the acylCoA cholesterol acyltransferase (ACAT) in the nM range without exhibiting antimicrobial and cytotoxic activity. When 125 was injected into mice no apparent toxicity was detected.98–100 In 1998 Cases et al.101 discovered that ACAT exists in two isoforms and later 125 was found to selectively inhibit ACAT-2 and therefore was used as model compound.102–108 In 2011 Ohshiro et al.109 demonstrated that atherogenic mice treated with 125 (10 to 100 mg kg1) showed decreased cholesterol absorption as well as lower cholesterol and cholesteryl oleate levels resulting in atherosclerosis protection. Ohshiro & Tomoda110 reviewed specic ACAT inhibitors focusing on ACAT-2-specic inhibitors like 125. In 2013 Ohtawa et al.111–113 developed ACAT-2 inhibitors based on 125 and established structure–activity relationships. They identied several derivatives with higher ACAT-2 inhibition, e.g. a 7-O-p-cyanobenzoyl PPPA derivative (129), which seems to be the most potent ACAT-2 inhibitor to date with an IC50 of 0.9 nM and a selectivity index of 4622 (IC50 ACAT-1/IC50 ACAT-2).111 In recent years several patents were led for the preparation of pyripyropenes.114–119 Besides ACAT-2 inhibition, anti-angiogenic effects and anti-proliferative activity against HUVEC cells were described for 125, 126, and 128 (IC50 0.1–1.8 mM)120 and a growth inhibition of corn earworm larvae (Helicoverpa zea) for 125.121 Based on the insecticidal activity several patents were led in recent years.122–124
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Allantofuranone (130), a new and uncommon g-lactone, was isolated from the plant pathogenic fungus Allantophomopsis lycopodina.125 It is a strong and quite selective antifungal antibiotic especially against Paecilomyces and Penicillium species. In pure media 130 is slowly converted into the inactive allantopyrone B (131) while Magnaporthe grisea detoxies 130 into the inactive allantopyrones C and D (132, 133). The interesting biosynthesis of 130 starts from two L-phenylalanines with polyporic acid (134) as a key intermediate.126 Allantopyrone A (135) isolated from a different A. lycopodina exhibited cytotoxic activity against HL60 cells with an IC50 value of 0.32 mM and induced apoptosis.127
In 2009 and 2010 several styrylpyrones named phellinins A1 (136), A2 (137), B, and C were described from Phellinus sp.128–130 Phenillins B and C are reported to be mixtures of inseparable components. All phellinins were able to scavenge DPPH (a,adiphenyl-b-picrylhydrazyl), ABTS (2,20 -azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and superoxide radicals.129,130 From Inonotus hispidus Zan et al.131 reported antioxidant hispidin derivatives named inonotusin A (138) and B (139). In addition, 139 exhibited cytotoxic activity at 19
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mM (IC50) against the cell line MCF-7. The bishispidins phaeolschidins A–D (140–143) and phaeolschidin E (144), all exhibiting antioxidative activity were isolated from fruiting bodies of Phaeolus schweinitzii.132 These compounds did not show any cytotoxic effect up to 200 mM against the cell lines HCT116 and SW480. Several styrylpyrones with other than antioxidative activities were isolated in the specied period. The trimeric hispidin derivative phellinstatin (145) produced by Phellinus linteus inhibits the enoyl-ACP reductase (FabI) a key enzyme of fatty acid biosynthesis of Staphylococcus aureus with an IC50 value of 6 mM. The MICs for two S. aureus strains were considerably higher with 128 mg mL1.133 In 2011 ve new hispidin derivatives, the phellibaumins A–E (146–150), together with known related compounds were described134 from fruiting bodies of Phellinus baumii. Effects on NF-kB activation (indication e.g. inammation) in prostate cancer cells (PC-3 cells) were tested and IC50 values for those ve compounds ranged from 41 (for 147) to 74 mM. Recently Lee et al.135 isolated penstyrylpyrone (151) from a marine-derived Penicillium exhibiting protein tyrosine phosphatase 1B (PTP1B) inhibiting activity with an IC50 value of 5.28 mM. This enzyme is considered to be a
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negative regulator in insulin signaling and inhibitors might be useful in treating type 2 diabetes and obesity (for review on natural products:136). In addition 151 was able to suppress the production of pro-inammatory mediators like NO and prostaglandin E2 with IC50 values of 12.32 mM and 9.35 mM respectively.135 In 2011 a concisive review on styrylpyrones and their potential in medicinal applications has been published.137 In 2009 JBIR-12 (152) and JBIR-25 (153), two compounds with DPPH radical scavenging activity comparable to atocopherol (IC50 ¼ 50 mM) were isolated.138 152 from Penicillium sp. NBRC 103941, is a highly oxygenated tetrahydronaphthalene with an acyl chain with an DPPH radical scavenging activity at IC50 values of 75 mM. Its methylated and acetylated derivatives did not show any activity up to 200 mM. 153 139 was isolated from the hyphomycete CR28109. Its DPPH radical scavenging activity was comparable with 152. Coincidentally Ingavat et al.140 described 153 from a marine-derived Aspergillus aculeatus and named it Aspergillusol A. In their assays 153 inhibited the a-glucosidase of Saccharomyces cerivisiae and Bacillus stearothermophilus with IC50 values of 465 and 1060 mM respectively and showed weak cytotoxicity
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against HuCCA-1, A549, and MOLT-3 cells with IC50 values of 50, 74, and 19 mM, respectively. 153 is the rst fungal example of a tyrosine derivative with an oxime moiety and its total synthesis was published in 2010.141
The asperparalines A–C (154–156) had been described as metabolites of Aspergillus japonicus causing paralysis of silkworms (Bombyx mori, at 3 mg per g body weight).142,143 In very elegant mode of action studies Hirata et al.144 now have proven that 154 selectively blocks insect acetylcholine receptors while vertebrate (chicken) receptors were hardly affected. This opens the way to investigate the mechanism of this selectivity, which could lead to new insecticides with very low toxicity.
Penicillium sp. CNL-338 isolated from the red alga Laurencia sp. from the Bahamas Islands produced the lumazine peptide penilumamide (157)145 The peptide has 1,3dimethyl-lumazine-6-carboxylic acid as an unusual starter unit which is coupled to methionine sulfoxide and anthranilic acid methyl ester. Neither antibacterial nor antifungal activity was described.
Nygerone A (158) was produced only when Aspergillus niger ATCC 1015 was cultured in the presence of the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA/Vorinostat).146 To date no bioactivity was published for 158. Several reviews on “unlocking” silent gene clusters by applying several methods have been published in recent years.147–150
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Protuboxepins A (159) and B (160), two oxepin-containing pyrimidine alkaloids and protubonines A (161) and B (162), two diketopiperazine-type alkaloids were isolated from a marinederived Aspergillus sp. SF-5044.151 From the same Aspergillus strain two new tetraketides named protolactone A (163) and B (164) with a new ring system had been published earlier.152 Data concerning bioactivities of these two compounds were not presented. 159, 161, and 162 were tested against a panel of cell lines. Only 159 exhibited weak cytotoxicity with IC50 values between 75 and 250 mM.151 159 binds to a,b-tubulin which causes metaphase arrest and promotes apoptosis at 80 and 160 mM.153 Since the structure of 159 differs substantially from known tubulin inhibitors it might become a useful lead structure. In 2012 154 a Korean patent was published which claims protuboxepins and protubonines for the treatment of cancer. The oxepinamides D–G (165–168) from Aspergillus puniceus showed transactivation effects on the Liver X Receptor alpha (LXRa) in a reporter gene assay.155 This nuclear receptor is thought to be a pharmacological target for metabolic disorders, chronic inammation and neurodegenerative diseases, and cancer.156 165–168 increased the LXRa controlled gene activity at concentrations between 1 to 30 mM with 2.8 to 4.1 fold induction. At 50 mM cytotoxic effects were observed.155 The indole alkaloid dimer brevianamide J (169) and the diketopiperazine alkaloids brevianamides K–N and later O–R, were all produced by the same Aspergillus versicolor.157,158 Additional brevianamides S (170) to V were isolated from a different Aspergillus versicolor strain with 170 being the second example of a dimeric diketopiperazine besides 169.159 170 had a MIC of 6.25 mg mL1 against the Bacille Calmette–Gu´ erin, which is an attenuated strain of the bovine tuberculosis bacillus Mycobacterium bovis used in screenings for antitubercular compounds.159 Calpinactam (FKI-4905, 171), isolated from the zygomycete Mortierella alpina is a highly selective antimycobacterial agent. It inhibits the growth of Mycobacterium smegmatis and M. tuberculosis with MIC values of 0.78 and 12.5 mg mL1, respectively. None of the other tested bacteria and fungi were affected.160 The structure of 171 was determined by spectroscopic methods and total synthesis. 171 is a hexapeptide with a caprolactam ring at its C-terminus. Its absolute stereochemistry was determined by amino acid analysis and total synthesis to D -phenylalanyl-L-leucyl-L-histidyl-D-glutaminyl-D-allo-isoleucyl-Lcaprolactam.161 It is interesting that the mycobacterial siderophores (Mycobactins)162 also contain a C-terminal caprolactam ring which, however, bears an hydroxyl group at the ring nitrogen. To explore the structure–activity relationships of 171, derivatives with different amino acids were synthesized utilizing solid-phase peptide synthesis. Among them only a
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peptide with an activity.163
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D-ala
in place of
D-glu
showed moderate
Apicidin (172) was rst described in 1996 by a group of Merck as a Fusarium metabolite with very strong inhibitory activity against the parasitic protozoa Plasmodium berghei, P. falciparum, Eimeria tenella, Neospora caninum, Caryospora bigenetica. Mode of action (MOA) studies revealed that 172 is a very potent inhibitor of the parasites histone deacetylases (HDACs) inducing hyperacetylation of histones thus interfering with a central pathway in eukaryotic transcriptional control. The apicidins share antiprotozoal activity and MOA with other fungal cyclic tetrapeptides like HC-toxin, trapoxin A and chlamydocin.164 Their strong cytotoxic activities at low nanomolar concentrations and their MOA indicate that the selectivity of antiparasitic and antineoblastic activity had to be addressed with regard to minimize in vivo toxicity. In the meantime a number of syntheses of 172 and variants with higher activity and selectivity against protozoa have been patented and published.165,166 An excellent survey on natural products as modulators of histone acetylation and their uses in antineoblastic therapy has been compiled by Salvador and Luesch in 2012.167
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Recently, as an alternative to chemical synthesis, a genetic approach to obtain new apicidins through modication of the nonribosomal apicidinsynthase (AS 1) by Jin et al.168 resulted in the production of the apicidins B (173), D2 (174), and E (175) in F. semitectum. The sequencing of complete genomes offers the opportunity to detect genes coding for enzymes leading to hitherto unknown metabolites. Von Bargen et al.169 discovered an AS 1-like gene cluster in F. fujikuroi, a fungus from which so far no HDAC inhibitor had been described. By overexpression of the pathway specic transcription factor they were able to produce a new metabolite, apicidin F (176). Here, L-isoleucine is replaced by L-phenylalanine and L-2-amino-8-oxodecanoic acid by L-2-aminooctanedioic acid. 176 exhibited good activity against Plasmodium falciparum with an IC50 of 670 nM.169
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Heterozygote deletion mutants of the pathogen Candida albicans with strain-specic bar codes were used in co-cultured screenings (yeast haploid deletion approach). Using nonlethal concentrations of extracts or pure compounds individual mutants show an altered tness and proling could reveal the mode of action. Using this screening strategy phaeofungin (177) and phomafungin (178), two novel lipodepsipeptides were isolated.170,171 The 25-membered cyclic depsipeptide 177 isolated from a Phaeosphaeria sp. contains seven amino acids and ab,gdihydroxy-g-methylhexadecanoic acid.170 177 causes the release of ATP in wild-type C. albicans strains, the MICs for C. albicans, Aspergillus fumigatus and Trichophyton mentagrophytes were determined to 16 mg mL1, 8 mg mL1 and 4 mg mL1 respectively. Pronounced antifungal activities were described for 178 with MICs of 2–8 mg mL1 against the above mentioned human pathogens.171 The 28 member ring consisting of eight amino acids and a b-hydroxy-g-methyl-hexadecanoic acid was isolated from an unspecied Phoma species.
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Scopulariopsis brevicaulis isolated from a marine sponge was found to produce the depsipeptides Scopularides A (179) and B (180).172 They showed activity against several cancer cell lines at 10 mg mL1. In 2009 a patent was published claiming these cyclic peptides for the treatment of cancer.173
Verticilide A1 (181) from a Verticillium sp. was found to inhibit the ryanodine binding to its receptors in cockroaches with an IC50 value of 4.2 mM. The homologous receptor in mice was more than 10 times less sensitive.174,175 The stereochemistry and the total synthesis were published in 2006.176 Against the nematode C. elegans and the brine shrimp A. salina the MICs were found to be 20 mg mL1 (23 mM) and no cytotoxic or antimicrobial activities were detected. In 2012 Ohshiro et al.177 isolated 181 and three new verticilides named A2 (182), A3 (183), and B1 (184) from a different Verticillium species. These compounds were tested against two isozymes of acyl-CoA cholesterol acyltransferase (ACAT-1 and -2). With IC50 values between 0.23 and 1.3 mM ACAT-2 was more sensitive than ACAT1 with IC50 values between 2.5 and 11 mM In contrast to 181, 182, 183, and 184 did not inhibit the ryanodine receptor (up to 100 mM). 184 exhibited moderate toxicity towards Artemia salina and broad antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa, and Mucor racemosus.
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In 2012 a solid-phase synthesis for the stepwise coupling of N-methyl amino acids and hydroxycarboxylic acids on solid support was described for the related anthelmintic cyclodepsipeptide PF1022A (185) and its commercial analogue emodepside (186) which could be applied to other cyclodepsipeptides and synthetic screening libraries.178,179 A concise review on anthelmintic cyclcooctadepsipeptides with emphasis on the mode of action of 185 and 186 appeared in 2012.180 The Omphalotins A–D were isolated in 1997 and 1998 from fermentations of Omphalotus olearius (Basidiomycetes), as strong and selective nematicides acting preferably on Meloidogyne incognita, an important plant pathogenic root not nematode.181–184 During continuing efforts to obtain better producers a monokaryotic (haploid) strain was found to yield not only more of omphalotins A–D but also ve new nematicidal derivatives. The omphalotins E–I (187–191) are oxidatively modied cyclic dodecapeptides with four (188–191) of them containing an unprecedented N-hydroxylated tricyclic tryptophan derivative.185 While no antibacterial, antifungal, and cytotoxic activities were detected, the nematicidal activity against M. incognita compared favorably with ivermectin with LD90 values between 2–10 mg mL1.
NBRI23477 A (192) and B (193), two new atpenins, have been isolated from Penicillium atramentosum as inhibitors of human prostate cancer cells (DU-145) at concentrations of 0.95 and 0.71 mg mL1.186 The stereoselective total synthesis of 193, together with atpenins A4, 193, and harzianopyridone has been achieved by Ohtawa et al.187 The atpenins, rst described by Omura's group as antifungal antibiotics interfering with the ATP generating system in Raji cells188,189 are potent and specic inhibitors of mitochondrial complex II succinate ubiquinol oxidoreductase.190 In anaerobic parasitic eukaryotes, complex II functions as a terminal oxidase in the NADH-fumarate pathway. Since this pathway is essential for growth and survival of anaerobic parasitic helminths and some cancer cells it offers an interesting target for the development of new chemotherapeutics.191
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Three new metabolites were discovered from a marine derived Phialocephala species.192 The sorbicillin dimer, dihydrotrichodermolide (194), the sorbicillin monomer dihydromethylsorbicillin (195), and the benzofuran phialofurone (196) exhibited cytotoxic activity against the P388 cell line with IC50 values of 11.5 mM, 0.1 mM, and 0.2 mM respectively. The sorbifuranones A, B, and C (196–198) were isolated from Penicillium strains derived from Mediterranean sponges.193 These structures are a new class of sorbicillinol-derivatives. Unfortunately, no information about biological activities were given.194 Sorbiterrin A (199) was described from Penicillium terrestre isolated from marine sediments. 199 showed moderate acetylcholinesterase inhibition with an IC50 value of 25 mg mL1. 199 is the rst example of a sorbicillinoid with a bridged [3.3.1] ring system. The sorbicillamines A–E (200–204) were reported by Guo et al. in 2013.195 They were isolated from a deep sea derived Penicillium species and did not exhibit cytotoxic effects against a panel of cell lines up to 10 mM. Interestingly, 204 is the rst sorbicillin trimer containing a nitrogen atom. The sorbicillinoids JBIR-59 (205) and JBIR-124 (206) were described from a Penicillium citrinum strain isolated from a marine sponge.196,197 The radical scavenging potential of these two compounds was reported. Li et al.198 described chloctanspirones A (207) and B (208), two chlorinated sorbicillinoids with a bicyclo[2.2.2]octane-2-spiro cyclohexane skeleton, both isolated from a marine sediment derived Penicillium terrestre. 207 and 208 showed cytotoxic activity against HL-60 with IC50 values of 9.2 and 39.7 mM. A review on the biosynthesis, bioactivities, and synthetic approaches covering all sorbicillinoids until 2010 was published in 2011 by Harned and Volp.199 Guignardia bidwellii causes black rot in grapes. The ascomycete was found to produce besides the known guignardic acid (209)200 phenguignardic acid (210), a new phytotoxin.201 209 and 210 are phytotoxic in a non-host-specic manner. No cytotoxic or antimicrobial activities could be detected. In 2013 seven new derivatives (211–217) were described202 of which alaguignardic acid (211), and guignardianones A (212), E (216), and F (217) exhibited phytotoxic properties. The absolute conguration of this series of guignaridic acid derivatives was determined by vibrational circular dichroism (VCD) spectroscopy.203 The total synthesis of 210 has been achieved by Stoye et al.204 Pleurone (218) was isolated from fruiting bodies of Pleurotus eryngii var. ferulae in the course of a screening for inhibitors of human neutrophil elastase, an enzyme which is considered to play a role in skin ageing.205 However, the IC50 value of 49.4 mM compared unfavorably with epigallocatechin gallate (IC50 8.8 mM) used as standard.
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SMTPs (Stachybotrys microspora triprenyl phenols) are interesting compounds which activate plasminogen and enhance its binding to brin. This stimulation of endogenous brinolytic activity is considered an interesting approach to develop antithrombotic agents. The rst metabolite of this series, stablapin (219), was isolated from Stachybotrys microspora IFO30018 in 1996.206 In 2000 Hu et al.207 described two dimeric analogs SMTP-7 (220) and -8 (221) which exhibited an enhanced activity as compared to the monomers at concentrations of 80 to 200 mM. In recent years SMTP derivatives with different N-linked side chains were published. This side chain can be controlled by feeding amines which was done by
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Hasegawa et al.,208 resulting in 11 new analogs and by Koide et al.209,210 resulting in 22 SMTP derivatives with a phenylamine- or phenylglycine-based side chain. Some of them show plasminogen-modulating activity comparable to 220. 220 got increased attention in recent years especially because of its thrombolytic, anti-inammatory, and antioxidant effects. The treatment of cerebral infarcts and thrombosis were studied.211–215 Hu et al.216 tested 220 in vivo, in mice it enhances plasmin generation at 5–10 mg kg1. The effects on bleeding and rebleeding were tested (up to 30 mg kg1) and no prolonged or increased bleeding was detected. In rats 220 was tested in a pulmonary embolism model (5 mg kg1) and an increase of clot clearing was observed (20% in 20 min). It was therefore concluded that compounds of this type might be leads for the development of antithrombotic medications.216
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lymphocytes and in the CNS. 223 inuences processes in neurogenesis, neural cell function, and migration (for review see ref. 220). These results support ongoing research to identify new modulators for sphingosine-1-phosphate receptors.
4 New developments in the uses of known compounds or new derivatives The complex prenylated indole alkaloid waikialoid A (222) was isolated from an Aspergillus species from a soil sample collected near Waikiki Beach, Hawaii.217 222 was able to inhibit biolm formation of Candida albicans with an IC50 value of 1.4 mM and inhibited hyphal morphogenesis but was not able to disrupt preformed biolms. No direct antifungal or cytotoxic activity was detected up to concentrations of 200 mM. This is remarkable because the structurally related stephacidin B shows pronounced cytotoxicity against several cell lines with IC50 values
