Isolation, characterization and biological activity evaluation of bioactive compounds from Marine Sponge - Associated Fungi.

NELSON GONÇALO MORTÁGUA GOMES ISOLATION, CHARACTERIZATION AND BIOLOGICAL ACTIVITY EVALUATION OF BIOACTIVE COMPOUNDS FROM MARINE SPONGE-ASSOCIATED FUNGI Tese de Candidatura ao grau de Doutor em Ciências Biomédicas submetida ao Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. Orientador – Professor Doutor Anake Kijjoa Professor Catedrático Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto. The experimental work of this thesis has been carried out in the Departamento de Química, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto. The candidate performed this work with a doctoral fellowship (SFRH/BD/65671/2009) supported by FCT (Fundação para a Ciência e a Tecnologia) supported by the European Social Fund (ESF). This work was also partially funded by the Project MARBIOTECH (reference NORTE-07-0124-FED ER-000047) within the SR&TD Integrated Program MARVALOR—Building research and innovation capacity for improved management and valorization of marine resources, supported by the Programa Operacional Regional do Norte (ON.2—O Novo Norte) and by the European Regional Development. Author: Nelson Gonçalo Mortágua Gomes [email protected] +351 912 039 663 PhD Thesis in Biomedical Sciences Title: Isolation, Characterization and Biological Activity Evaluation of Bioactive Compounds from Marine Sponge-Associated Fungi Supervisor: Professor Doutor Anake Kijjoa [email protected] Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto March 2014 __________________________________ AUTHOR’S DECLARATION Under the terms of the nº2, alínea a, do artigo 31º do Decreto-Lei nº230/2009, the author hereby declared that he has afforded a major contribution to the conceptual design and technical execution of the work, interpretation of the results and manuscript preparation of the original articles included in this thesis. Under the terms of the referred Decree-Law, the author hereby declared that the following original articles/communications were prepared in the scope of this thesis. LIST OF PUBLICATIONS/COMMUNICATIONS PUBLICATIONS Articles in International Peer-Reviewed Journals Original Research Gomes, N.M., Dethoup, T., Singburaudom, N., Gales, L., Silva, A.M.S, Kijjoa, A. (2012). Eurocristatine, a new diketopiperazine dimer from the marine spongeassociated fungus Eurotium cristatum. Phytochemistry Letters 5(4), 717–720. Eamvijarn, A., Gomes, N.M., Dethoup, T., Buaruang, J., Manoch, L., Silva, A., Pedro, M., Marini, I., Roussis, V., Kijjoa, A. (2013). Bioactive meroditerpenes and indole alkaloids from the soil fungus Neosartorya fischeri (KUFC 6344), and the marine-derived fungi Neosartorya laciniosa (KUFC 7896) and Neosartorya tsunodae (KUFC 9213). Tetrahedron 69(40), 8583–8591. Gomes, N.M., Bessa, L.J., Buttachon, S., Costa, P.M., Buaruang, J., Dethoup, T., Silva, A.M.S., Kijjoa, A. (2014). Antibacterial and Antibiofilm Activities of Tryptoquivalines and Meroditerpenes Isolated from the Marine-Derived Fungi Neosartorya paulistensis, N. laciniosa, N. tsunodae, and the Soil Fungi N. fischeri and N. siamensis. Marine Drugs 12(2), 822-839. Book Chapters Theoretical Background Gomes, N.M., Buttachon, S., Kijjoa, A. Meroterpenoids from Marine Microorganisms: Potential Scaffolds for New Chemotherapy Leads. In: Anticancer Drugs from Marine Origins (Ed. Kim, S.-K.) (Accepted). COMMUNICATIONS Posters Gomes, N.G.M., Dethoup, T., Gales, L., Silva, A.M.S., Kijjoa, A. Bioactive Secondary Metabolites from the Marine Sponge-Associated Fungus Eurotium cristatum. 2nd International Symposium of Modern Medicine, Traditional Chinese Medicine and Uighur Medicine and 12th National Symposium of Integrative Medicine in Prevention and Treatment of Respiratory Diseases. Urumqi, China, September 15-20, 2012. Gomes, N.G.M., Dethoup, T., Gales, L., Silva, A.M.S., Kijjoa, A. Bioactive Secondary Metabolites from Marine Sponge-Associated Fungi Collected from the Gulf of Thailand. 14th International Symposium on Marine Natural Products & 8th European Conference on Marine Natural Products. La Toja, Galicia, Spain, September 15-20, 2013. vii INDEX INDEX AKNOWLEDGMENTS…………………………………………………….. xxi ABSTRACT…………………………………………………………………. xxv RESUMO…………………………………………………………………….. xxvii LIST OF ABBREVIATIONS……..………………………………………… xxix STATEMENT OF THE OBJECTIVES……………………………………. xxxv Chapter I. Introduction…………………………………….......... 1 1.1. General Introduction……………………………………………………. 3 1.1.1. Natural Products: An Established and Continuing Source of Novel Drug Leads…………………………………………………………... 3 1.2. Ocean as an Unlimited Source for Novel Drugs………………....... 5 1.2.1. Clinical Pipeline of Marine Pharmaceuticals…………………....... 7 1.2.2. Statistical Perspective on Marine Natural Products Research..... 13 1.2.3. Marine Microbial Consortia as the True Metabolic Source of Bioactive Metabolites………………………………………………………. 15 1.2.4. Marine Natural Products Research and the “Supply Problem”.... 16 1.3. The Potential of Marine Microbial Diversity……………………....... 18 1.3.1. Marine Microorganisms as a Source of Novel Drug Candidates. 19 1.3.2. Marine Microorganisms: Cultivating the Uncultured and Activating Silent Clusters…………………………………………………... 21 1.4. Fungi from Marine Habitats as Producers of Relevant Bioactive Secondary Metabolites………………………………………………………. 22 1.4.1. Definition of Marine Fungi and Taxonomy………………………... 24 1.4.2. Distribution of Marine-Derived Fungi and Chemical Studies…… 25 1.4.3. Marine Sponge-Associated Fungi – A Promising Source of Bioactive Secondary Metabolites…………………………………………. 27 1.4.4. Biologically Active Metabolites of Marine Sponge-Associated Fungi in Preclinical Development……………………………………… 29 viii INDEX Chapter II. Chemistry of the Genera Emericella, Eurotium and Neosartorya...................................................................... 33 2.1. An Overview of Secondary Metabolites…………………………….. 35 2.2. The Genus Emericella………………………………………………….. 36 2.2.1. Emericella aurantio-brunnea……………………………………….. 38 2.2.2. Emericella desertorum……………………………………………… 38 2.2.3. Emericella falconensis……………………………………………… 39 2.2.4. Emericella foveolata………………………………………………… 40 2.2.5. Emericella heterothallica……………………………………………. 41 2.2.6. Emericella nidulans…………………………………………………. 41 2.2.7. Emericella purpurea………………………………………………… 42 2.2.8. Emericella quadrilineata……………………………………………. 42 2.2.9. Emericella rugulosa…………………………………………………. 44 2.2.10. Emericella striata………………………………………………....... 44 2.2.11. Emericella unguis………………………………………………….. 45 2.2.12. Emericella variecolor………………………………………………. 46 2.2.13. Unspecified Emericella species………………………………...... 48 2.3. The Genus Eurotium……………………………………………………. 51 2.3.1. Eurotium chevalieri………………………………………………….. 51 2.3.2. Eurotium cristatum………………………………………………...... 51 2.3.3. Eurotium herbariorum………………………………………………. 52 2.3.4. Eurotium repens…………………………………………………...... 53 2.3.5. Eurotium rubrum.......................................................................... 54 2.4. The Genus Neosartorya………………………………………………... 55 2.4.1. Neosartorya fischeri…………………………………………………. 56 2.4.2. Neosartorya glabra………………………………………………….. 58 2.4.3. Neosartorya pseufischeri………………………………………....... 59 2.4.4. Neosartorya quadricincta………………………………………....... 60 2.4.5. Neosartorya siamensis…………………………………………....... 60 2.4.6. Neosartorya tsunodae…………………………………………… … 61 2.4.7. Unspecified Neosartorya species………………………………….. 62 ix INDEX Chapter III. Results and Discussion………………………...... 63 3.1. Chemical Investigation of the Marine Derived Fungi……………... 65 3.1.1. Structure Elucidation of Triterpenoid Steroids……………………. 68 3.1.1.1. Ergosterol (EV1)……………………………………………… 68 3.1.1.2. Ergosterol peroxide (EV2)………………………………....... 73 3.1.2. Structure Elucidation of Orcinol and 1H-Indole-3-carboxylic acid………………………………………………………………………….... 77 3.1.2.1. Orcinol (EV3)………………………………………………….. 77 3.1.2.2. 1H-Indole-3-carboxylic acid (EV4)………………………….. 78 3.1.3. Structure Elucidation of Anthraquinones………………………….. 81 3.1.3.1. Erythroglaucin (EC1)…………………………………………. 81 3.1.3.2. Physcion (EC2) ………………………………………………. 84 3.1.3.3. Catenarin (EC3) ………………………………………........... 87 3.1.3.4. Emodin (EC4) ……………………………………………....... 90 3.1.3.5. Questin (EC5) ……………………………………………....... 95 3.1.4. Structure Elucidation of Diketopiperazine Derivatives………...... 100 3.1.4.1. cyclo-(L-Tryptophyl-L-phenylalanyl) (EV5)……………....... 100 3.1.4.2. Neoechinulin A (EC8) ……………………………………….. 104 3.1.4.3. Neoechinulin E (EC9) ……………………………………….. 110 3.1.4.4. Neoechinulin (EC7) ………………………………………….. 113 3.1.4.5. Echinulin (EC6) ………………………………………………. 116 3.1.4.6. Eurocristatine (EC10) ……………………………………...... 121 3.1.5. Structure Elucidation of Meroditerpenes………………………….. 127 3.1.5.1. Chevalone B (NL1) ………………………………………...... 127 3.1.5.2. Aszonapyrone A (NL2)………………..……………………... 133 3.1.5.3. Aszonapyrone B (NL3) …………………………………....... 136 3.1.5.4. Sartorypyrone C (NP1) …………………………………....... 139 3.1.6. Structure Elucidation of Quinazolinone Alkaloids……………...... 145 3.1.6.1. 4(3H)-Quinazolinone (NP4) ………………………………… 145 3.1.6.2. 3’-(4-Oxoquinazolin-3-yl)spiro[1H-indole-3,5’-oxolane]- 2,2’-dione (NP5/NL5) …………………………………………………. 147 3.1.6.3. Tryptoquivaline L (NP2/NL4) ……………………………….. 151 3.1.6.4. Tryptoquivaline H (NP3) …………………………………….. 157 xvi FIGURES INDEX Figure 76. Structures of the compounds tested for antifungal and antibacterial activities………………………………………………………….. 174 Figure 77. Structures of the compounds tested for in vitro cytotoxic activity against human cancer cell lines………………………………….….. 175 Figure 78. Structures of the compounds tested for antibacterial and antibiofilm activities…………………………………………………………….. 177 Figure 79. Biomass quantification of biofilms of Gram-positive bacteria formed in the presence of concentrations ranging from 2x MIC to 1/4 MIC of aszonapyrone A (NL2)………………………………………………... 180 Figure 80. Evaluation of S. aureus ATCC 25953 biofilm formation. Live/dead viability staining images after 24h. Control (A); Biofilm formation in the presence of the MIC (B) and in the presence of 1/2 of the MIC (C) of aszonapyrone A (NL2)……………………………………….. 181 Figure 81. Structures of the meroditerpenes tested for antibacterial activity………………………………………………………………………....... 182 Figure 82. Clatria reinwardti (a). E. variecolor KUFC 7092. Colony on MEA with 70% sea water after 14 days (b). Conidiophores and conidia (c). SEM image of ascospores (d, e)………………………………………… 186 Figure 83. Mycale sp. (a). E. cristatum KUFC 7356. Colony on PDA with 70% sea water after 14 days (b). Conidiophores and conidia (c). SEM image of ascospores (d, e)……………………………………………………. 187 Figure 84. Diseased-coral (Porites lutea)…………………………………… 188 Figure 85. N. laciniosa KUFC 7896. Colonies incubated for 14 days at 28ºC on CZA (a), CYA (b) and MEA (c) media. Ascomata (d), Conidia (e). SEM image of ascospores (f)……………………………………………. 189 Figure 86. Chondrilla australiensis (a). N. paulistensis KUFC 7897. Colony on PDA with 70% sea water after 14 days (b). Ascomata (c). Conidia (d). SEM image of ascospores (d, e)………………………………. 190 xvii TABLES INDEX TABLES INDEX Table 1. Marine pharmaceuticals: Current pipeline………………………... 9 Table 2. 1H and 13C NMR data (CDCl3, 300.13 and 75.47 MHz) of EV1…………………………………………………………………………........ 68 Table 3. 1H and 13C NMR data (DMSO-d6, 500.13 and 125.77 MHz) of EV2………………………………………………………………………........... 74 Table 4. 1H and 13C NMR data (CDCl3, 300.13 and 75.47 MHz) of EV3... 77 Table 5. 1H and 13C NMR data (DMSO-d6, 500.13 and 125.77 MHz) of EV4………………………………………………………………………………. 79 Table 6. 1H and 13C NMR data (CDCl3, 500.13 and 125.77 MHz) of EC1. 82 Table 7. 1H and 13C NMR data (CDCl3, 500.13 and 125.77 MHz) of EC2. 85 Table 8. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC3…………………………………………………………………………........ 88 Table 9. 1H and 13C NMR data (DMSO-d6, 500.13 and 125.77 MHz) of EC4………………………………………………………………………........... 90 Table 10. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC5………………………………………………………………………........... 96 Table 11. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EV5…………………………………………………………………………….... 100 Table 12. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC8………………………………………………………………………........... 105 Table 13. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC9………………………………………………………………………........... 110 Table 14. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC7…………………………………………………………………………........ 114 Table 15. Comparison of 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC7 and EC9…………………………………………………. 115 Table 16. Comparison of 1H and 13C NMR data (300.13 and 75.47 MHz) of EC6 (CDCl3) and EC8 (DMSO-d6)………………………………………… 117 Table 17. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC10…………………………………………………………………………...... 121 Table 18. 1H and 13C NMR data (CDCl3, 300.13 and 75.47 MHz) of NL1. 128 xviii TABLES INDEX Table 19. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NL2…………………………………………………………………………….... 133 Table 20. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz, T = 45 ºC) of NL3…………………………………………………………………… 137 Table 21. Comparison of the 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NL2 and NL3…………………………………………...... 138 Table 22. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NP1…………………………………………………………………………........ 140 Table 23. 1H and 13C NMR data (DMSO-d6, 500.13 and 125.77 MHz) of .NP4…………………………………………………………………………....... 145 Table 24. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NP5/NL5……………………………………………………………………....... 148 Table 25. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NP2/NL4……………………………………………………………………....... 152 Table 26. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NP3………………………………………………………………………........... 158 Table 27. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NP6…………….......................................................................................... 163 Table 28. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of NL6…………………………………………………………………………….... 167 Table 29. Growth inhibitory effect of diketopiperazine alkaloids EC7EC10 and meroditerpenes NL2 and NL3………………………………….... 176 Table 30. Antibacterial activity, expressed in μg/mL of compounds NP1NP6 and NL2-NL6 against reference strains (a) and of NL2 against multidrug-resistant isolates (b)……………………………………………….. 177 Table 31. Antibacterial efficacy of combined effect of antibiotics with compounds NP1-NP6 and NL2-NL6 (15 μg/disc) against three multidrug-resistant isolates, using the disc diffusion method……………... 178 Table 32. FIC Index results obtained with aszonapyrone A (NL2) and antibiotic combinations by checkerboard method………………………...... 179 xix SCHEMES INDEX SCHEMES INDEX Scheme 1. Schematic diagram of the research process…………………… xxxv Scheme 2. Biosynthetic pathway of the anthraquinones EC1-EC5………. 99 Scheme 3. Proposed biosynthetic pathway for eurocristatine (EC10)……. 127 Scheme 4. Proposed biosynthetic pathway for sartorypyrone C (NP1), aszonapyrones A (NL2) and B (NL3) and chevalone B (NL1)…………….. 143 Scheme 5. Biosynthetic pathways for 3-(4-oxoquinazolin-3-yl)spiro[1Hindole-3,5-oxolane]-2,2’-dione (NP5/NL5) and tryptoquivalines L (NP2/NL4), H (NP3) and F (NP6)…………………………………………… 171 xx xxi ACKNOWLEDGMENTS ACKNOWLEDGMENTS I would like to acknowledge some people without whom this work would not have been made possible. I wish to express my deep gratitude and admiration to Professor Dr. Anake Kijjoa, my supervisor, mentor and friend, for his unwavering support, constant encouragement but mainly for his supervision, characterized by seriousness and accuracy. I have nothing else than to be proud and considered to be an enormous privilege to have performed this study under his supervision. My sincere thanks to Professor Dr. Madalena Pinto, Head of Department of the Departamento de Química Orgânica, Faculdade de Farmácia, Universidade do Porto, CEQUIMED – Centro de Química Medicinal da Universidade do Porto, for providing logistical support. I am indebt to Professor Dr. Artur Silva, Departamento de Química, Universidade de Aveiro, for the 1H and 13C NMR measurements, and to Dr. Mick Lee, Department of Chemistry, Leicester University, UK, for HRMS measurements. Special thanks are given to Professor Dr. Luís Gales, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, for the X-ray crystallography analysis. I am deeply indebt to Professor Dr. Tida Dethoup and Professor Dr. Leka Manoch, from the Department of Plant Pathology, Kasetsart University, Bangkok, Thailand, and to Mr. Jamrearn Buaruang, Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand, for collection, isolation, identification and culture of the marine fungi, as well as preparation of the extracts. Also a thankful word for Professor Dr. Paulo Costa, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, and Dr. Lucinda Bessa, CIIMAR – Centro Interdisciplinar de Investigação Marinha e Ambiental, xxii ACKNOWLEDGMENTS Universidade do Porto, for performing the antibacterial and antibiofilm assays, to to Professor Dr. Madalena Pedro, CEQUIMED – Centro de Química Medicinal da Universidade do Porto and Instituto Superior de Ciências da Saúde do Norte (CESPU), for the cytotoxic activity assays, and to Professor Dr. Eugénia Pinto, CEQUIMED – Centro de Química Medicinal da Universidade do Porto, Faculdade de Farmácia, Universidade do Porto, for antifungal assays. A very special thanks goes to Mrs. Júlia Bessa not only for her technical assistance, but also for the constant encouragement and friendship. My sincere thanks goes also to Ms. Sara Cravo, Ms. Sonia Pereira and Mrs. Isabel for their technical assistance. Special thanks to my neighbor and friend, Professor Dr. Augusto Faustino for introducing me to Professor Dr. Anake Kijjoa. A sincere and eternal thank to my lab mates Amnat, Ângela, Chadaporn, Clayton, Decha, Ioulia, Madalena, Renato, Suradet, Sutsawat and Tida for all the shared knowledge, for providing an amazing working atmosphere, and above all for their friendship. I would like also to express my gratitude to my hometown friends Anaís, Ricardo, Roberto and Tânia and to my long time friends André, Daniel and Francisco from being always there when I need. A kind and eternal thank to my brother Luís, to my sister-in-law Cláudia, and to my nephews, Kiko and Bá, for all the love and support during these four years. An additional special thank to my brother Luís, for the eternal encouragement and for being not only a brother, but also a father and a friend. I would like to express my gratitude to my parents whom words cannot describe their existence. Thanks to my father Nelson, for providing me all the support (including financial when needed) to achieve my goals, for his encouragement, and for all the sacrifices to make my life better. xxiii ACKNOWLEDGMENTS Finally, I would like to thank to my mother Elisabete, not only for all the support, patience and love, but essentially from her constant fighting attitude against all the unfair life adversities, that always inspired me. Mom, this thesis is dedicated to you. xxiv xxv ABSTRACT ABSTRACT The marine environment is a tremendous source of biologically active metabolites, representing a valuable source with great potential for the development of pharmaceuticals. Marine-derived fungi are known to produce structurally unique secondary metabolites, and since the beginning of the 1990s, a sharp and exponential increase in the number of reported relevant biologically active metabolites occurred. The aim of this study was to isolate and evaluate the biological activity of secondary metabolites produced by marine derived-fungi collected from the Gulf of Thailand. To achieve this aim, three marine sponge-associated fungi, Emericella variecolor KUFC 7092, Eurotium cristatum KUFC 7356 and Neosartorya paulistensis KUFC 7897, and the diseased coral-derived fungus Neosartorya laciniosa KUFC 7896, were investigated. Chemical investigation of the ethyl acetate extract of the culture of E. variecolor KUFC 7092, isolated from the marine sponge Clathria reinwardti, resulted in the isolation of five known compounds, ergosterol, ergosterol peroxide, orcinol, 1H-indole-3-carboxylic acid and cyclo-(L-tryptophyl-L-phenylalanyl). A new diketopiperazine dimer, eurocristatine, was isolated in addition to nine known metabolites including the anthraquinones erythroglaucin, physcion, catenarin, emodin and questin, and the diketopiperazine alkaloids echinulin, neoechinulin, neoechinulin A and neoechinulin E, from the ethyl acetate extract of the culture of the sponge-associated fungus E. cristatum KUFC 7356. The diketopiperazine derivatives, including the new diketopiperazine dimer, were evaluated for their in vitro growth inhibitory activity against MCF-7 (breast adenocarcinoma), NCI-H-460 (non-small lung cancer) and A375-C5 (melanoma) cell lines, and the results showed that they were inactive against the three cell lines. Both anthraquinones and diketopiperazine alkaloids were also evaluated for their antifungal activity against the human pathogenic fungi Candida albicans, Aspergillus fumigatus and Trichophyton rubrum, as well as for their antibacterial xxxii LIST OF ABBREVIATIONS Me2CO Acetone MEA Malt Extract Agar MeOH Methanol MetAP2 Methionine aminopeptidase-2 MH Mueller-Hinton Agar MHB Mueller-Hinton Broth MHz Mega hertz MIC Minimum Inhibitory Concentration MIP Macrophage Inflammatory Protein MKK6 Mitogen-activated protein kinase kinase 6 MKK7 Mitogen-activated protein kinase kinase 7 mm Millimeter MMAE Monomethyl Auristatin E MMAF Monomethyl Auristatin F MMP-2 Matrix Metalloproteinase 2 MMP-9 Matrix Metalloproteinase 9 MOPS 3-(N-morpholino)propanesulfonic acid mp Melting point in ºC MPP+ 1-Methyl-4-phenylpyridinium MRSA Methicillin-resistant Staphylococcus aureus MS Mass Spectrometry NA Not Available Na2SO4 Sodium sulfate NAD(P)H Nicotinamide Adenine Dinucleotide Phosphate NaHCO3 Sodium bicarbonate NCE New Chemical Entities NCI National Cancer Institute NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NK-1 Human Neurokinin Receptor 1 nm Nanometer NMR Nuclear Magnetic Resonance NO Nitric oxide NOESY Nuclear Overhauser Effect Spectroscopy xxxiii LIST OF ABBREVIATIONS NRPS Nonribossomal Peptide Synthetase NSCLC Non-Small Cell Lung Cancer ºC Celsius degrees OD595 Optical Density at 595 nm ORTEP Oak Ridge Thermal Ellipsoid Plot OX Oxacillin PBS Phosphate Buffered Saline pcPTase Polycyclic polyketide prenyltransferase PEPCK Phosphoenolpyruvate carboxykinase PGE2 Prostaglandin E2 P-gp P-glycoprotein 1 PKCα Protein Kinase C α PTLC Preparative Thin Layer Chromatography PTP1B Protein-Tyrosine Phosphatase 1B Py Pyridine q Quartet QToF Quantum Time of Flight RAPD Random Amplified Polymorphic DNA ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute s Singlet S Streptomycin SAR Structure-Activity Relationship SEM Scanning Electron Microscope SEM Standard error of the mean Si gel Silica gel SP Substance P sp. Species (singular) spp. Species (plural) SRB Sulforhodamine B SSF Solid State Fermentation t Triplet TGF-β1 Transforming Growth Factor β1 xxxiv LIST OF ABBREVIATIONS TLC Thin Layer Chromatography TNF-α Tumor Necrosis Factor α TSB Tryptic Soy Broth UPLC Ultra Performance Liquid Chromatography US United States UV Ultraviolet VA Vancomycin VEGF Vascular Endothelial Growth Factor VRE Vancomycin-resistant Enterococcus δ Chemical shift value in ppm ε Molar absorptivity (molar extinction coefficient) ΣFIC FIC index xxxv STATEMENT OF THE OBJECTIVES STATEMENT OF THE OBJECTIVES The main objective of the present study is to investigate the secondary metabolites produced by cultures of the marine sponge and coral-associated fungi for evaluation of their anticancer, antibacterial and antifungal activities. As our group has been focusing mainly on the constituents of the genus Neosartorya, investigation of the secondary metabolites of the marine-derived Neosartorya species will allow us to compare the chemical profiles of the marine strains with their terrestrial counterparts. Besides, this comparative study will provide an additional tool, useful for a taxonomic characterization of the genus, through a chemotaxonomic analysis (Frisvad et al., 2008). To achieve these objectives, the work was divided according to Scheme 1: Scheme 1. Schematic diagram of the research process. I. Collection, Isolation, Identification and Culture of the Marine Fungi The marine sponges and coral were collected by scuba diving in the Gulf of Thailand and the Andaman Sea. Identification of the fungi was based on macroand microscopic characteristics observed under light and scanning electron microscopes (SEM) as xxxvi STATEMENT OF THE OBJECTIVES well as with molecular data such as DNA GC content and RAPD (random amplified polymorphic DNA) fingerprint (Glass and Donaldson, 1995). II. Isolation and Identification of the Secondary Metabolites The crude ethyl extracts of the culture of the marine fungi were fractionated by column chromatography, and the purification of the metabolites from the column fractions was carried out by PTLC, and/or crystallization. The structures of the pure compounds were established by 1D and 2D NMR spectral analysis (1H, 13C, DEPT 90º and 135º, COSY, HSQC, HMBC, NOESY), HRMS, IR, UV, and Xray crystallography when the compounds exist in crystalline form. III. Biological Activity Evaluation a) Screening of antitumor activity: The effects of the compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by the National Cancer Institute (NCI, US) in the “In vitro Anticancer Drug Discovery Screen” using the protein-binding dye sulforhodamine B to assess cell growth (Skehan et al., 1990). b) Screening of antifungal activity: The compounds were evaluated for their growth inhibitory effects against yeasts, filamentous fungi and filamentous dermatophytes by a dilution method (Wayne, 2002a; 2002b). c) Screening of antibacterial activity: The compounds were tested for their antibacterial activity against human pathogenic bacteria (including multidrugresistant isolates) (Johnson et al., 2002; Odds, 2003; Wayne, 2011). 1 CHAPTER I. INTRODUCTION CHAPTER I INTRODUCTION 2 CHAPTER I. INTRODUCTION 3 CHAPTER I. INTRODUCTION 1.1. General Introduction Since ancient times, Nature played a major role as the main source of medicinal extracts used, as well as the source of several natural products that led to the development of various drugs currently used for the treatment of a wide spectrum of diseases. Based on empirical knowledge, natural product extracts, mainly plant-derived, have formed the basis of traditional medicine systems. However, it was only in the 20th century that research focused on the identification and characterization of the compounds responsible for the medicinal properties. The urgent need of new therapeutic alternatives, mainly for the treatment of cancer and infectious diseases, as well as the new chemical entities (NCE) decline in drug development pipelines, led to the rekindling of interest in “rediscovering natural products” focusing in alternative sources such as the oceans. It is clear that Nature will continue to be a major source of new structural leads (Cragg and Newman, 2013). Berkowitz’s comment in Rouhi’s report clearly states the potential and essential role of natural products in therapeutics: “We would not have the topselling drug class today, the statins; the whole field of angiotensin antagonists and angiotensin converting-enzyme inhibitors; the whole area of immunosuppressives; nor most of the anticancer and antibacterial drugs. Imagine all of those drugs not being available to physicians or patients today.” (Rouhi, 2003). 1.1.1. Natural Products: An Established and Continuing Source of Novel Drug Leads Despite the millenary use of natural medicinal products, research concerning the isolation and use of pure substances in therapeutics dates from the post-Industrial Revolution period in the 19th century. The emergence of natural products clearly reached its climax in the last century, with the discovery of several drugs covering a wide range of diseases (Cragg and Newman, 2013). The discovery of penicillin G (1) (Figure 1) from the filamentous fungus Penicillium notatum by Fleming in the late 1920s, and the observation of the broad 4 CHAPTER I. INTRODUCTION therapeutic use of this agent in the 1940s, was a landmark discovery that ushered in a new Era in medicine, “The Golden Age of Antibiotics”, and promoted the intensive investigation of Nature as a source of novel bioactive agents (Walsh, 2004). The opium poppy (Papaver somniferum) is another classic and valuable example being the source of papaverine (2) (Figure 1), which formed the basis for the antihypertensive agent verapamil. However this plant is better known as a source of the opiate analgesic alkaloid morphine (3) (Figure 1). Most of the cholesterol lowering agents (statins) are synthetic analogues derived from the fungal metabolites mevastatin (4) (Figure 1), isolated from Penicillium citrinum (Endo et al., 1976), as well as lovastatin (5) (Figure 1) isolated from Monascus ruber and Aspergillus terreus (Buckland et al., 1989; Negishi et al., 1986). Two lipid-regulating drugs of this class, atorvastatin and simvastatin, feature prominently in the top ten drugs by cost, led by Lipitor® (atorvastatin) with an annual sale reaching 7.2 billion US dollars in 2010, only in the US (Kleinrock, 2011). 1 2 3 4 R = H 5 R = Me 6 Figure 1. Some examples of natural drug leads approved for use in Medicine. 5 CHAPTER I. INTRODUCTION Paclitaxel (6) (Taxol®) (Figure 1), the exciting plant-derived chemotherapy drug was originally isolated from the Pacific yew tree Taxus brevifolia. The discovery in 1979 of its mode of action, through promotion of the assembly of tubulin into microtubules by Schiff and Horwitz and its report in 1980 (Schiff and Horwitz, 1980), was a key milestone in the lengthy development process, being approved for clinical use against ovarian cancer in 1992 and against breast cancer in 1994. Recently, a new semi-synthetic derivative, cabazitaxel (Jevtana®) developed by Sanofi-Aventis, was approved by the FDA for the treatment of hormone-refractory prostate cancer (http://www.cancer.gov/cancertopics/druginfo/fda-cabazitaxel, accessed on 24th November 2013). During the last century, natural products research led to the development of several drugs currently used in Medicine against a wide range of diseases. In fact, the most recent survey by Newman and Cragg (2012) analyzing the sources of new drugs over the period 01/1981 – 12/2010, indicates that while 66% of the 1073 small molecule NCEs are formally synthetic, only 36% can be classified as truly synthetic or devoid of natural inspiration. Furthermore, when considering disease categories, for the 1073 small molecules, 69% of anti-infectives and 75% of anticancer drugs introduced into the market can be traced back as naturally derived or inspired. The role of natural sources in the finding of novel drug leads is unquestionable and since there are still many unexplored resources from Nature, an enormous chance for finding new or less investigated organisms, and thereby new lead structures still exists (Aly et al., 2011; Hill, 2013). 1.2. Ocean as an Unlimited Source for Novel Drugs Comprising almost three quarters of the Earth’s surface, oceans represent a rich source of both biological and chemical diversity, containing nearby 200 000 catalogued species, representing only ~9% from the estimated value of 2.2 million of species dwelling in ocean depths (Mora et al., 2011). Some marine habitats are known to be particularly numerous in species, especially tropical marine reefs, 12 CHAPTER I. INTRODUCTION Lovaza®, a highly purified and concentrated ω-3 polyunsaturated fatty acids preparation obtained from fish oils mainly comprised of EPA (20:5(n-3)) (17) and DHA (22:6(n-3)) (18) (Figure 3b), was approved in 2004 by FDA as an adjunct to diet in adults with high triglyceride levels (≥500 mg/dL) (Koski, 2008). It was commercialized in EU under the tradename Omacor®, and is also used in coronary arteriosclerosis, familial combined hyperlipidemia as well as for secondary prevention after myocardial infarction (Martindale, 2012). 20 19 17 18 Figure 3b. Marine natural products or derivatives thereof approved for use by FDA or EMA. Brentuximab vedotin is a CD30-directed antibody-drug conjugate (ADC) consisting of three components: the chimeric IgG1 antibody cAC10 which is specific for human CD30, the microtubule disrupting agent monomethyl auristatin E (19) (Figure 3b) (MMAE), and a protease-cleavable linker that covalently attaches MMAE (19) to cAC10. The microtubule disrupting agent MMAE (19) binds to tubulin, disrupting the microtubule network which leads to cell cycle arrest 13 CHAPTER I. INTRODUCTION and apoptotic death of the cells (Watanabe et al., 2007). Brentuximab vedotin (Adcentris®) was approved by FDA for the treatment of anaplastic large T-cell systemic malignant lymphoma and Hodgkin’s disease (Martindale, 2012). MMAE (19) is a synthetic analog of dolastatin 10 (20) (Figure 3b) originally isolated from the sea hare Dolabella auricularia, and recently from a marine cyanobacterium Symploca sp. (Luesch et al., 2001). It is expected that the number of candidates to enter clinical trials in a near future is vast. Only referring to the period from 2009 to 2011, the preclinical pharmaceutical pipeline consists of over 250 marine compounds that will enrich the marine clinical pharmaceutical pipeline with novel lead compounds (Mayer et al., 2013). Despite some limitations, the current success rate of discovery from the marine world, namely seven clinically approved drugs from 22 000, is significantly better than the industry average (Gerwick and Moore, 2012). 1.2.2. Statistical Perspective on Marine Natural Products Research Over 22 000 structurally diverse marine metabolites have been isolated and characterized over the last five decades, with 1152 new compounds only for the year 2011 (Blunt et al., 2013). Figure 4. Temporal trend in the number of novel products obtained from marine organisms (Hu et al., 2011). 14 CHAPTER I. INTRODUCTION Before the 1980s, marine natural products discovered annually were less than 100, however there was an outstanding development since the mid-1980s (Figure 4), coincident with the development and progress of structure elucidation techniques (Hu et al., 2011). Regarding to its source, majority of novel compounds from marine organisms, approximately 75% were isolated from invertebrates mainly from the phyla Porifera (mostly sponges) but also from Coelenterate phyla (mostly coral) (Figure 5). Additionally, algae and microorganisms are also major sources of novel metabolites, with a special focus on microorganisms due to the increasing focus from marine natural product chemists (Hu et al., 2011). In contrast to macroorganisms, microorganisms represent promising natural product sources due to the feasible and sustainable production of large quantities of secondary metabolites with reasonable cost, by large-scale cultivation and fermentation of the source organisms (Waites et al., 2001; Xiong et al., 2013). Figure 5. Temporal trends in the number of novel compounds isolated from different marine organisms between 1985 and 2008. ▲ Marine invertebrate; ■ Marine algae; Marine microorganisms (including phytoplankton) (Hu et al., 2011) 15 CHAPTER I. INTRODUCTION 1.2.3. Marine Microbial Consortia as the True Metabolic Source of Bioactive Metabolites The symbiotic microbial consortia also prove to be a source of bioactive compounds with pharmaceutical potential, and there is a growing recognition that marine invertebrates and marine plants surface are usually populated with enormous quantities of associated or symbiotic microorganisms (Gerwick and Fenner, 2013). According to Gerwick and Fenner (2013), at the end of the year 2012, there were twenty-one marine derived or marine-inspired agents in the clinic or clinical trials (outdated data). Pie chart A (Figure 6) summarizes the collected sources of organisms that have yielded these agents, and reveals that marine invertebrates are the richest collected source (18 of 21, 86%). Figure 6. Pie charts showing A: the collected sources of the twenty-one marinederived or marine-inspired agents in the clinic or clinical trial, and B: the same agents with their demonstrated or predicted metabolic sources (Gerwick and Fenner, 2013). 16 CHAPTER I. INTRODUCTION However, there are strong evidences that some promising marine natural compounds in clinical trials, as well as some approved marine-derived agents supposedly produced by invertebrates, are in fact metabolic products of their associated microorganisms or derived from a diet of prokaryotic microorganisms (Gerwick and Fenner, 2013; Gerwick and Moore, 2012; Haefner, 2003; Simmons et al., 2008). For example, dolastatin 10 (20) (Figure 3b) which was first reported as a metabolite of the Indian Ocean sea hare Dolabella auricularia (Pettit et al., 1987), was later found to be produced by the gastropod’s diet cyanobacteria of the genera Simploca and Lyngbya (Luesch et al., 2001; Williamson et al., 2000). Another example refers to a recent report by Rath and co-workers on metagenomic sequencing of total DNA from Ecteinascidia turbinata and associated microorganisms, suggesting that the individual genes responsible for trabectedin (14) (Figure 3a) production has microbial origin from the associated-γproteobacterium Candidatus Endoecteinascidia frumentensis (Rath et al., 2011). As noted above, the collected source has oftentimes been shown or is strongly suspected of harboring or feeding upon microorganisms that are the actual producers of the bioactive agent. Analysis of pie chart B (Figure 6), displaying the actual or suspected metabolic source of the twenty-one marine derived or marineinspired agents in the clinic or clinical trials by the time of the end of 2012, reveals that marine microbes are the real metabolic jewels of the world’s oceans, accounting for 90% of the twenty-one marine derived agents in clinical trials and approved pharmaceutical agents by the end of 2012 (Gerwick and Fenner, 2013). The realization that microbes are the true metabolic sources of several relevant bioactive compounds raised hope of obtaining a sustainable and less limited supply of compounds for testing and drug development. 1.2.4. Marine Natural Products Research and the “Supply Problem” The fact that virtually no marine natural product can so far be found on the shelves of pharmacies is certainly not due to a limited chemical diversity of marine organisms. The last fifty years of research on marine natural products led to the development of seven agents that have entered the clinic as approved drugs, plus 17 CHAPTER I. INTRODUCTION a substantial number of candidates progressing through the development process. However, despite the success stories, several factors contribute to the lack of the development in the area of marine pharmaceuticals. Biologically active natural products are often produced in relatively small amounts, and often by rare animals whose natural populations cannot sustain the extensive collections required for clinical trials. Insufficient quantities of material to allow for study completion, difficulties of retrieving a sustained and reliable harvest of a marine organism, limited amounts of compounds for pre-clinical development, and difficulties in culturing marine organisms, are limiting factors that need to be overcome (Bhadury et al., 2006). To overcome such limitations, several approaches can be used like the chemical synthesis, mass cultivation of the producer organisms, or through genomic engineering, but the large scale production of metabolites with potential clinical relevance to meet the demand for clinical trials and drug development, continues to be a major challenge. Despite the vast number of examples of successfully synthesized natural products with medicinal relevance (Nicolaou et al., 2012), not always chemical synthesis may be a solution due to the economically non viable approach. Trabectedin (ecteinascidin-743) (14) and halichondrin B (15) (Figure 3a) examples demonstrate that some marine natural products have such complex structures leading to high costs associated with their chemical synthesis (Cuevas et al., 2000). A notable exception is ziconotide (12) (Figure 3a), which due to its peptide nature can be obtained in virtually unlimited amount through synthesis (Olivera, 2000). Mariculture of macroorganims is another alternative mean for the production of large amounts of metabolites, but rarely can be considered to be cost-effective. The first example of mass cultivation of sponge species for pharmaceutical purposes was the large-scale culture of Lissodendoryx sp. to obtain sufficient amount of halichondrin B (15) (Figure 3a) for clinical trials. From one metric ton of the sponge, only ca. 300 mg of a mixture of two halichondrin analogues could be obtained (Hart et al., 2000). Even with a more favorable ratio of compound production vs. biomass, to obtain approximately 1 g of trabectedin (14) (Figure 3a), close to one metric ton (wet weight) of Ecteinascidia turbinata has to be collected and extracted, which is economically unfeasible (Mendola, 2000). 18 CHAPTER I. INTRODUCTION Organisms that could easily be cultured in large amounts would thus be ideal. Such organisms might well be marine microorganisms. 1.3. The Potential of Marine Microbial Diversity Whereas in the last decades the focus in marine natural product research was mainly on macroorganisms such as sponges, sea weeds and others, nowadays it is clearly evident that microorganisms from the marine habitat are equally rich sources of novel constituents, and often the true metabolic producers of bioactive metabolites whose production was originally attributed to a macroorganism (Gerwick and Fenner, 2013; Gerwick and Moore, 2012; Haefner, 2003; Simmons et al., 2008). Additionally, microorganisms are far more accessible through sustainable production by fermentation than many marine macroorganisms that usually have to be collected from limited wild stocks (Proksch et al., 2010). These two main factors reveal that microbes are truly the treasure troves of new marine pharmaceuticals. As microorganisms occupy almost every niche on Earth, scientists speculate that each drop of water taken from the ocean will contain microbial species unknown to humans in a ratio 9:1 (Colwell, 2002). To maximize the chemical diversity available from marine microbes, other sources like the deepocean and geothermal vents are becoming the focus of considerable interest from natural product research chemists (Bhatnagar and Kim, 2010; Pettit, 2011). In fact, diverse bioactive secondary metabolites have been reported from cultured extreme-tolerant microorganisms, extremophiles, and deep-sea microbes. Due to the extreme physical and chemical conditions at deep-sea, hydrothermal vents sites conditions are constantly fluctuating, representing a nearly inexhaustible source of genomic innovation (Pettit, 2011). Although numerous natural products have been identified from marine microorganisms during the last decades, it is obvious that a plethora of compounds still await discovery. This assumption results not only from the fact that only a small number of microorganisms have been cultivated and discovered yet but also from recent genome sequencing projects (Brakhage and Schroeckh, 2011). 19 CHAPTER I. INTRODUCTION 1.3.1. Marine Microorganims as a Source of Novel Drug Candidates Microorganisms are a prolific source of structurally diverse bioactive metabolites and have yielded some of the most important products of the pharmaceutical industry, including antibiotics and blockbuster medicines with tremendous economic importance. These include antibacterial agents such as the penicillins and cephalosporins, aminoglycosides and tetracyclines, immunosuppressive agents, cholesterol lowering agents such as mevastatin and lovastatin, and antihelmintic and antiparasitic drugs such as ivermectins. These tremendous successful stories of drug discovery from microorganisms, revolutionized the therapy concepts, pushing the academia and pharmaceutical industry to develop programs on natural product discovery, emphasized on marine microbial fermentation based technologies (Debbab et al., 2010). Current marine clinical pharmaceutical pipeline includes one microbial metabolite in phase I of clinical trials, but several drug leads from marine microbiota in pre-clinical development are expected to advance clinical development to be pharmaceutically relevant drugs (Bhatnagar and Kim, 2010; Gerwick and Fenner, 2013; Newman and Hill, 2006). Salinosporamide A (NPI-0052) (21) (Figure 7), a β-lactone produced by fermentation cultures of Salinispora tropica, is a powerful 20S proteasome inhibitor and one of the most promising candidates in chemotherapy (Feling et al., 2003). Nereus Pharmaceuticals has licensed this compound under the name Marizomib®, which is now in Phase I clinical trials as a single agent in patients with advanced solid tumors, and also in combination with vorinostat, a histone deacetylase inhibitor, for the treatment of advanced pancreatic carcinoma, non-small cell lung carcinoma and melanoma (Hamlin et al., 2009; Potts et al., 2011). Interestingly, S. tropica belongs to the recently described and exclusively marine genus of Streptomycete bacteria known as Salinispora, of which three species are recognized and all inhabit marine sediments. From over 100 cultivated Salinispora spp. strains, more than 80% inhibited human tumor cell growth, and 35% showed antibacterial properties toward a wide range of drug-resistant human pathogens, 20 CHAPTER I. INTRODUCTION representing one of the most recent and promising sources for novel drug leads (Fenical and Jensen, 2006). Plinabulin (NPI-2358) (22) (Figure 7) is a potent and selective tumor vascular disrupting agent, and one of over 200 synthetic analogues in the series that were prepared following the discovery of the parent compound phenylahistidin (also known as halimide) (23) (Figure 7). Phenylahistidin (23) was isolated from cultures of the marine alga-derived fungus Aspergillus ustus, and displayed potent cytotoxic activity toward human cancer cell lines and tubulin polymerization inhibition (Kanoh et al., 1997; 1999). Two phase I trials have been reported, as a single agent in patients with advanced solid tumors or lymphomas (Mita et al., 2010), or in combination with docetaxel in patients with non-smal cell lung cancer (NSCLS) (Millward et al., 2012). The favorable results led to a phase II ADVANCE clinical trial of plinabulin (22) in combination with docetaxel in patients with NSCLS, that was recently completed, but no further information was provided (http://clinicaltrials.gov/show/NCT00630110 accessed on 18th October 2013). 21 23 22 Figure 7. Chemical structures of novel drug candidates produced by marine microorganisms. Finally, the most recent and exciting discovery is seriniquinone, produced by a unique marine bacterium of the genus Serinicoccus, recovered from shallow marine sediments. In vitro screening in the NCI 60 cell line panel showed pronounced selectivity toward all melanoma cell lines tested, particularly hostile to a dangerous form of metastatic melanoma. Seriniquinone has a unique way of 21 CHAPTER I. INTRODUCTION inducing apoptosis, targeting the recently discovered protein dermcidin, overexpressed in melanoma, activating apoptosis and the induction of autophagocytosis leading to efficient cell death (Fenical et al., 2013). 1.3.2. Marine Microorganisms: Cultivating the Uncultured and Activating Silent Clusters Until recently, the inability to cultivate most naturally occurring microorganisms has severely limited the exploration of the potential of both terrestrial and marine microbes. In fact, it has been estimated that much less than 1% of microorganisms seen microscopically have been cultured. However, marine microorganisms low culturability may reflect the artificial conditions inherent in most culture media, e.g., the lack of specific nutrient required for growth. Energy sources, nutrients and proper physicochemical conditions are necessary for microbial growth, and culture mimicking the natural environment is critical to recover the uncultivated microorganisms (Kjer et al., 2010; Zengler et al., 2002). 24 14 Figure 8. Structures of cyanosafracin B (24) and the hemisynthesis product trabectedin (14). Alternatively, these compounds could be produced in bulk, by total or semisynthetic pathways, through fermentation technologies. A widely known example is the production of trabectedin (14) (Figure 8). Due to the supply 28 CHAPTER I. INTRODUCTION antimicrobial polybrominated biphenyl ethers which might keep the sponge free of other bacteria (Unson et al., 1994). Marine sponges have been demonstrated to represent one of the richest sources of fungal diversity. Several cultivation-based studies with marine sponges have shown a vast biological diversity, yielding the greatest taxonomic diversity in comparison with other marine sources (Figure 13) (Bugni and Ireland, 2004). Figure 13. The number of distinct fungal genera based on the marine source (Bugni and Ireland, 2004). For some given species of sponges, analogously to sponge-associated bacteria, some of the marine-derived strains appear to be unique and at least partially sponge specific and fairly stable in both space and time (Hentschel et al., 2002; Taylor et al., 2007). However, opposite to bacteria, it seems that many of the isolated fungi from sponges are of suspected terrestrial origin, due to the similarity to typical terrestrial strains. Interestingly of 681 fungal strains isolated from sixteen sponges worldwide, most belonged to Aspergillus and Penicillium genera, ubiquitous in terrestrial habitats (Höller et al., 2000). The isolation of several metabolites from terrestrial microorganisms originally reported from marine sponges, also supports and provides experimental evidence to this hypothesis, however unlike bacteria, so far there is no report on 29 CHAPTER I. INTRODUCTION fungi as source of any natural product previously ascribed to a marine sponge (König et al., 2006; Thomas et al., 2010). A plausible and widely accepted explanation for the fact that many of the fungi isolated from sponges belong to genera ubiquitous from terrestrial habitats, is that fungal spores may be washed into the sea and sequestered by sponges through filter feeding and retained in the inhalant canals. Furthermore, so far there is no evidence indicating that fungi actively grow inside sponges suggesting that their presence is in fact limited to spores. However, many of the compounds reported from marine isolates of the genera Aspergillus or Penicilium are often remarkably different from those of terrestrial counterparts suggesting that at least, on a biosynthetic level, many sponge-associated fungi are distinguished from their terrestrial analogues (Proksch et al., 2010). 1.4.4. Biologically Active Metabolites from Marine Sponge-Associated Fungi in Preclinical Development Despite the absence of marine fungal metabolites in the current marine clinical pharmaceutical pipeline, marine sponge-associated fungi are known for being an excellent reservoir of bioactive metabolites with potent activities covering a wide range of biological functions (Bugni and Ireland, 2004; Rateb and Ebel, 2011; Saleem et al., 2007). The previously referred phenylahistidin (23) (Figure 7), the lead structure of plinabulin (22) (Figure 7) whose phase II clinical trials have been recently completed, is the most important representative of marine fungalderived natural products so far. The first metabolite reported from a sponge-associated fungus is the polyketide trichoharzin (32) (Figure 14), which was isolated from an imperfect fungus Trichoderma harzianum Rifai isolated from a marine sponge Mycale cecilia (Kobayashi et al., 1993). Interestingly, the trichoharzin analog, deoxynortrichoharzin (33) (Figure14) was also obtained from the sponge derivedfungus Paecilomyces cf. javanica (Rahbaek et al., 1998). Sorbicillactone A (34) (Figure 14), an alkaloid possessing a unique bicyclic lactone structure isolated from the sponge Ircinia fasciculata-associated fungus Penicillium chrysogenum, is known to have antiviral and neuroprotective 30 CHAPTER I. INTRODUCTION properties (Bringmann et al., 2005; 2007). Owing to its highly selective cytostatic activity against murine leukemic lymphoblasts, this compound has been recently qualified for human trials. The heterodimer asperazine (35), isolated from the saltwater culture of the Caribbean Hyrtios sponge-associated Aspergillus niger, also displayed significant differential cytotoxicity against a mouse leukemia cell line (L1210) (Varoglu et al., 1997). 38 R = MeOH 39 R = Me 32 R = EtOH 33 R = Me 34 35 36 R = Et 37 R = H Figure 14. Examples of marine sponge-derived fungi compounds with potential for preclinical development. Moreover, scopularides A (36) and B (37) (Figure 14), natural cyclodepsipeptides isolated from the marine sponge-derived fungus Scopulariopsis brevicaulis were recently patented due to their potent inhibitory 31 CHAPTER I. INTRODUCTION activity against several tumor cell lines, including pancreatic and colon tumor cell lines (Yu et al., 2008). The fungus Paraconiothyrium cf sporulosum, isolated from the Caribbean marine sponge Ectyplasia perox, was found to produce a new class of sesquiterpene epoxycyclohexenones with an unprecedented distinctive substitution and oxidation patters (Mohamed et al., 2009). Strikingly, epoxyphomalin A (38) (Figure 14) displayed not only remarkable cytotoxicity at nanomolar concentrations, but also an intriguing activity profile in COMPARE analyses that did not correlate with those of reference anticancer agents, suggesting a different mode of action from that of the reference compounds. Furthermore, it was also found that incubation of purified human 20S proteasome with epoxyphomalins A (38) and B (39) (Figure 14) led to a dose-dependent inhibition of chymotrypsin-, caspase-, and trypsin-like proteasome activities, indicating a potent inhibition of 20S proteasome. Consequently, epoxyphomalins could be considered as new promising chemotherapeutic alternatives for the treatment of several tumors, due to their capacity to inhibit the proteasome (Mohamed et al., 2010). There is a vast number of bioactive marine sponge-derived fungal metabolites with a wide set of bioactivities, and many of them are potential candidates for preclinical development (Blunt et al., 2013; Proksch et al., 2010; Rateb and Ebel, 2011; Thomas et al., 2010; Xiong et al., 2013). Thus, sponge-fungal association is a potential chemical and ecological phenomenon which provides sustainable resource for developing novel pharmaceutical leads for a promising and rational target in drug discovery. 32 33 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA CHAPTER II CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 34 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 35 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA Four marine-derived fungi selected for chemical investigation in this thesis belong to three distinct genera, namely Emericella (Emericella variecolor KUFC 7092), Eurotium (Eurotium cristatum KUFC 7356) and Neosartorya (Neosartorya paulistensis KUFC 7897 and N. laciniosa KUFC 7896). The four fungal species are the sexual counterparts of the genus Aspergillus, a relevant and efficient saprophytic genus widely found in diverse environments, including Oceans. Aspergillus genus consists of several hundred highly aerobic mold species and has been the target of several studies mainly due to several human pathogenic species responsible for infections leading to a high rate of morbidity and mortality, but also due to the relevant economic impact, mainly in developing countries, associated with the destructive effect in agriculture. On the other hand, this genus includes several species widely used in the food and beverage industry, being classified as a biotechnological “cell factory” (Gibbons and Rokas, 2013). Additionally, the genus Aspergillus was the source of several drugs currently used in therapeutics as referred previously, having revolutionized Medicine, yielding several blockbuster drugs and drug leads of enormous potential (Aly et al., 2011). The biotechnological potential of Aspergillus relies not only on economic relevance of microbial fermentations, but also on the genomic diversity leading to the production of several bioactive metabolites, classifying the genus as an attractive and valuable source for drug discovery, which is the main focus of the present study (Lee et al., 2013). Also the fact that ascomycetous fungi can be grown easily in culture and their teleomorphic states induced in the laboratory, led to the selection of these genera as a target for chemical and biological studies. 2.1. An Overview of Secondary Metabolites Contrary to Aspergillus genus, only a few species of teleomorphs have been investigated for their secondary metabolites and associated biological activities. However, the number of bioactive compounds reported so far is exponentially increasing, both the previously reported from other species and novel chemical structures. Due to the highly specific profiles of secondary metabolites, especially from Aspergillus and Penicillium teleomorphs, it is not 36 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA surprising the isolation of several common extrolites produced by a limited number of fungal species from the same genus. The distinct morphological features from fungal sexual stages allowed a dual nomenclature permitting separate names for anamorphs of fungi with a pleomorphic life-cycle, causing several equivoques regarding the correct and unequivocal taxonomic classification of several species. The increased number of mycologists recognizing the urgent need of a transition to a single-name nomenclatural system for fungi, resulted in the preparation of “Amsterdam Declaration on Fungal Nomenclature”, agreed under the auspices of the International Commission on the Taxonomy of Fungi (ICTF) during the symposium “One Fungus = One Name” held in Amsterdam in April 2011 (Hawksworth et al., 2011a; 2011b). The discontinuance of the dual nomenclature system was later approved and adopted in the 18th International Botanical Congress in Melbourne in July 2011, and the Vienna edition of the “International Code of Botanical Nomenclature” has been replaced by the “The International Code of Nomenclature for algae, fungi, and plants (The Melbourne Code)” published in December 2012. According to the “Melbourne Code”, after 1 January 2013 one fungus can only have one name (Hawksworth, 2011a; 2011b; McNeill et al., 2011). The following literature review will focus strictly on the teleomorphs from the genera Emericella, Eurotium and Neosartorya, following the taxonomic classification of the species adopted by the authors by the date of the publication, not necessarily adopting “Melbourne Code” recent recommendations. Despite the chemotaxonomic importance of secondary metabolites, the aim of this review is not to give an extensive and exhaustive report of all the secondary metabolites isolated from the referred genera. Instead, an overview on novel secondary metabolites and their associated bioactivities will be presented as an insight on the potential of the selected species for the isolation of novel bioactive secondary metabolites. 2.2. The Genus Emericella The genus Emericella was described by Berkeley (1857) based on E. variecolor Berk. & Broome, and comprises thirty-four species, corresponding to the 37 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA sexual state of Aspergillus species, notably the A. nidulans group among others. The genus Emericella comprises a well-studied group of secondary metabolite-rich fungi that are of concern in food and feed processing and storage, because of many sterigmatocystin producing species. Additionally, the genus was proved to be a prolific source of relevant bioactive metabolites with E. nidulans being the source of echinocandin B (40) (Figure 15), the starting molecule for the semisynthetic antifungal anidulafungin (Eraxistm®) (41) (Figure 15) approved for the treatment of candidiasis of the esophagus and disseminated candidiasis, intraabdominal and peritonitis (Hof and Dietz, 2009; Martindale, 2012). 40 41 Figure 15. Structures of echinocandin B (40) and semi-synthetic derivative anidulafungin (41). 44 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 2.2.9. Emericella rugulosa New prenylated xanthones, reguloxanthones A-C (91-93), 14methoxytajixanthone (94), and tajixanthone ethanoate (95) (Figure 24) were isolated from the fungus E. rugulosa, along with several known xanthones. Additionally, regulosone (96) (Figure 24) was also isolated from E. rugulosa and exhibited the in vitro antimalarial activity against Plasmodium falciparum, antimycobacterial activity, as well as strong cytotoxic activity against BC1, KB, and NCI-H187 cancer cell lines (Moosophon et al., 2009). 95 91 96 92 93 94 Figure 24. New constituents of E. rugulosa. 2.2.10. Emericella striata Chemical analysis of E. striata strain 80-NE-22 resulted in the isolation of emestrin (97), emestrin B (98) and related dethiodiketopiperazine derivatives lacking polysulfide internal bond, namely aurantioemestrin (99) and dethiosecoemestrin (100) (Figure 25) (Kawahara et al., 1986; Nozawa et al., 1987b; Seya et al., 1985; 1986a; 1986b). 45 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 97 x = 2 98 x = 3 99 R = S 100 R = O 101 Figure 25. New constituents of E. striata strain 80-NE-22. Both emestrin (97) and emestrin B (98) were found to possess antibacterial activity against Escherichia coli and Bacillus subtilis, while dethiosecoemestrin (100) was active against E.coli (Seya et al., 1986b; Nozawa et al., 1987b). Additionally, emestrin (97) displayed also antifungal activity, inhibiting the growth of Gibberella zeae and Penicillium expansum (Seya et al., 1986a). Recently, emestrin (97) was also isolated from E. nidulans MFW39, isolated from marine ascidia, and was found to exhibit relevant cytotoxicity against T47D, HepG2, C28, and HeLa cell lines (Nursid et al., 2011). Also from E. striata strain 80-NE-22, the new xanthone cycloisoemericellin (101) was later reported (Figure 25) along with the known arugosins (Kawahara et al., 1988b). 2.2.11. Emericella unguis E. unguis strain IFM 42017 was found to produce four new depsidones, emeguisins A-C (102-104) and 2-chlorounguinol (105), along with the new phthalide 3-ethyl-5,7-dihydroxy-3,6-dimethylphthalide (106) (Figure 26). Although the EtOAc extract of this fungus was found to have monoamine oxidase (MAO) inhibitory activity, the isolated depsidones displayed only a moderate activity. Additionally, emeguisin A (102) was found to inhibit the growth of Bacillus subtilis (Kawahara et al., 1988c; 1988d). 46 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 102 R1 = R2 = R3 = H 103 R1 = R3 = H, R2 = Me 104 R1 = Me, R2 = H, R3 = Cl 105 107 106 108 R1 = Ph, R2 = Me 109 R1 = i-Pr, R2 = Me 110 R1 = Ph, R2 = CH2OH Figure 26. New constituents of E. unguis. Later, the new emerguisins-related depside guisinol (107) (Figure 26) was reported from a marine isolate originated from the jellyfish Stomolophus meleagris collected in the Paria Bay, Venezuela, by Nielsen et al. (1999). Guisinol (107) exhibited antibacterial activity against Staphylococcus aureus. Unguisins A-C (108-110) (Figure 26), the first cyclic heptapeptides with GABA incorporated in the ring, were also isolated from the same extract. Unguisin A (108) and B (109) were found to exhibit moderate antibacterial activity against S. aureus and Vibrio parahaemolyticus (Malmström, 1999; Malmström et al., 2002a). 2.2.12. E. variecolor E. variecolor, the perfect state of Aspergillus variecolor (syn. A. stellatus) is one of the most studied species from the genus Emericella. It was found to produce a variety of secondary metabolites, including prenylated xanthones, sesterterpenes and meroterpenoids. 47 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA Emervaridione (111) and varioxiranediol (112) (Figure 27) were isolated from the EtOAc extract of an endophytic E. variecolor (Liangsakul et al., 2011). The marine E. variecolor strain M75-2, isolated from a Caribbean sponge, was found to produce three new terrein derivatives, varitriol (113), varioxirane (114) and dihydroterrein (115), along with the new xanthone varixanthone (116) (Figure 27). Varitriol (113) was tested in the NCI 60-cell line in vitro panel, and was found to exhibit strong cytotoxic activity against several cancer cell lines, with remarkable potency toward renal cancer cell line RXF 393 (GI50 = 0.163 µM) and breast cancer cell line T-47D (GI50 = 0.210 µM). While varixanthone (116) displayed potent antimicrobial activity against Escherichia coli, Proteus sp. and Bacillus subtilis, with a minimal inhibitory concentration (MIC) value of 12.5 µg/mL (Malmström et al., 2002b). Another marine sponge-derived strain isolated from the marine sponge Haliclona valliculata collected in Elba, Italy, yielded the new prenylated xanthone isoemericellin (117) and a new anthraquinone evariquinone (118) (Figure 27). Evariquinone (118) exhibited strong antiproliferative activity towards KB and NCIH460 cells at a concentration of 3.16 µg/mL (Bringmann et al., 2003). Two new sesterterpenes, 6-epi-ophiobolin G (119) and 6-epi-ophiobolin N (120) (Figure 27), along with six previously reported ophiobolins, were isolated by Wei et al. (2004) from the EtOAc extract of E. variecolor GF10 which was isolated from a marine sediment collected in Gokasyo Gulf, Japan. All ophiobolins, including 6-epi-ophiobolin G (119) and 6-epi-ophiobolin N (120) displayed cytotoxic activity against neuroblastoma cell line Neuro 2A (Wei et al., 2004). Later, a new polyketide, shimalactone A (121) (Figure 27) was discovered from the same extract, and was shown to induce neuritogenesis in neuroblastoma cell line Neuro 2A at 10 µg/mL (Wei et al., 2005). The toxic metabolite asteltoxine (122) (Figure 27) was isolated from the moldy rice artificially infected with E. variecolor NHL 2881 (Maebaxashi et al., 1983). 48 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 111 112 113 116 118 119 120 121 122 117 114 115 Figure 27. New constituents of E. variecolor. 2.2.13. Unspecified Emericella species Bioassay guided-fractionation of the endophytic Emericella sp. HK-ZJ, isolated from the mangrove plant Aegiceras corniculatum, resulted in the discovery of six new isoindolone derivatives, emerimidines A (123) and B (124), and the meroterpenoids emeriphenolicins A-D (125-128) (Figure 28). Only emerimidines A (123) and B (124) showed moderate in vitro antiviral activity against H1N1, with IC50 values of 42.07 µg/mL and 62.05 µg/mL, respectively (Zhang et al., 2011). 49 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 123 R1 = H, R2 = Me 124 R1 = Me, R2 = H 125 R1 = R2 = OMe, R3 = Cl, R4 = OH 126 R1 = OMe, R2 = R4 = OH, R3 = Cl 127 R1 = R2 = OMe, R3 = R4 = OH 128 Figure 28. New constituents of Emericella sp. HK-ZJ. Competing co-culture of the marine-derived fungus Emericella sp. (strain CNL-665) with the marine actinomycete Salinispora arenicola, yielded two new cyclic depsipeptides, emericellamides A (129) and B (130) (Figure 29), both of which displayed modest antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) (Oh et al., 2007). 129 130 Figure 29. Structures of emericellamides A (129) and B (130). Recently, Xu et al. (2013a) reported the isolation of the new epitetrathiodiketopiperazine secoemestrin D (136), along with five new sesterterpenoids with an unprecedented carbon skeleton, emericellenes A-E (131135) (Figure 30), from the endophytic fungal strain Emericella sp. AST0036, isolated from a leaf of Astragalus lentiginosus. Secoemestrin D (136) exhibited strong cytotoxic activity against six cancer cell lines with apparent selectivity to 50 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA human glioma (SF-268) and metastatic breast adenocarcinoma (MDA-MB-231) cell lines. 131 R = CHO 132 R = COOH 133 134 R = α - CHO 135 R = β - CHO 136 Figure 30. New constituents of Emericella sp. AST0036. The strain 25379 of Emericella sp., isolated from the surface of a coral collected at Marietas Islands, Mexico, produced the new xanthones 15chlorotajixanthone hydrate (137) and 14-methoxytajixanthone (138) (Figure 31). Both compounds inhibited the activation of the calmodulin-sensitive cAMP phosphodiesterase, with 14-methoxytajixanthone (138) displaying comparable effect with that of chlorpromazine (Figueroa et al., 2009). 137 138 Figure 31. New xanthones of Emericella sp. 25379 strain. 51 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 2.3. The Genus Eurotium Eurotium species are the sexual states of Aspergillus species, notably the A. glaucus group among others which is closely related to Emericella, and are also widely found in the environment (Geiser, 2009). Some Eurotium species have been used for manufacturing the traditional Japanese fermented food “dried bonito” (katsuobushi) and “karebushi” (Miyake et al., 2010). Although only a few species of Eurotium have been investigated for their bioactive secondary metabolites, there are already some recent reports on the isolation of numerous types of compounds, such as alkaloids, phenolic compounds and anthraquinones. 2.3.1. Eurotium chevalieri The strain E. chevalieri EuCO1 collected from rhizosphere soil at Surathani Province, Thailand was found to produce five new meroditerpenoids, chevalones A-D (139-142) and aszonapyrone B (143), as well as the new sesquiterpene alkaloid eurochevalierine (144) (Figure 32). Chevalone D (142) and eurochevalierine (144) exhibited antimalarial activity against Plasmodium falciparum with IC50 values of 3.1 and 3.4 µg/mL, while chevalone C (141) displayed strong antibacterial activity against Mycobacterium tuberculosis, with a MIC value of 6.3 µg/mL. Additionally, chevalones B-D (140-142) and eurochevalierine (144) were found to be cytotoxic against BC1, KB and NCI-H187 cancer cell lines, with IC50 values ranging from 2.9 to 9.8 µg/mL (Kanokmedhakul et al., 2011). 2.3.2. Eurotium cristatum Three new diketopiperazine indole alkaloids and a new diketopiperazine dimer, cristatumins A-D (145-148) (Figure 33) have been reported from the culture extract of the endophytic fungus E. cristatum EN-220, which was isolated from the marine alga Sargassum thunbergii. While cristatumin A (145) exhibited potent 52 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA inhibitory activity against Escherichia coli and Staphylococcus aureus, cristatumin B (146) displayed moderate lethal activity against brine shrimp (Artemia salina) (Du et al., 2012). 139 R = H 140 R = Ac 141 142 143 144 Figure 32. New constituents of E. chevalieri EuCO1. 2.3.3. Eurotium herbariourum A new diketopiperazine dimer, cristatumin E (149) (Figure 33), was recently isolated from a marine alga-associated E. herbariorum strain HT-2. Cristatumin E (149) was found to be moderately cytotoxic against K562 tumor cell line with an IC50 value of 8.3 µM, however it displayed weak antibacterial activity against Enterobacter aerogenes and Escherichia coli (Li et al., 2013a). 53 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 2.3.4. Eurotium repens Benzyl derivatives have frequently been reported from several Eurotium species. Two new additional benzyl derivatives, (E)-2-(hept-1-enyl)-3- (hydroxymethyl)-5-(3-methylbut-2-enyl)benzene-1,4-diol (150) and (E)-4-(hept-1enyl)-7-(3-methylbut-2-enyl)-2,3-dihydrobenzofuran-2,5-diol (151) (Figure 34) have been isolated from the fungus E. repens (UM-031509), along with several known benzyl derivatives. 145 147 149 146 148 Figure 33. Structures of cristatumins A-E (145-149). Compound 150 showed good binding affinity for human opioid or cannabinoid receptors (Gao et al., 2011a). It also exhibited antimicrobial activity against Staphylococcus aureus and Candida glabrata, and moderate antimalarial activity against chloroquine-sensitive and –resistant Plasmodium falciparum (Gao et al., 2012). 60 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 2.4.4. Neosartorya quadricincta A new prenylated dihydroisocoumarin derivative, PF1223 (188) (Figure 39), was reported from the culture of N. quadricincta strain PF1223. 188 Figure 39. Structure of PF1223 (188). PF1223 (188) inhibited [3H]ethynylbicycloorthobenzoate ([3H]EBOB) binding by 65% at the concentration of 2.2 µM, exhibiting binding activity for the insect GABA receptor and proving to be a lead compound for the development of novel insecticides (Ozoe et al., 2004). 2.4.5. Neosartorya siamensis The EtOAc extract of the culture of the new specie N. siamensis KUFC 6349 collected from forest soil at Samaesarn Island, Chonburi Province (Thailand), yielded two new quinazolinone derivatives, tryptoquivaline O (189) and 3’-(4oxoquinazolin-3-yl)spiro[1H-indole-3,5’-oxolane]-2,2’-dione (190), four new fiscalin analogues, epi-fiscalin C (191), epi-fiscalin A (192), neofiscalin A (193) and, epineofiscalin A (194), as well as the new indoloazepinone derivative, sartorymensin (195) (Figure 40). Sartorymensin (195) exhibited moderate in vitro growth inhibitory activity on the human U373 and Hs683 glioblastoma, the A549 non-small cell lung cancer, the MCF-7 breast cancer and the SKMEL-28 melanoma cell lines, with IC50 values ranging from 39 to 73 µM (Buttachon et al., 2012). 61 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 189 191 R1 = i-Pr, R2 = H, R3 = R4 = Me 192 R1 = i-Pr, R2 = R4 = H, R3 = Me 193 R1 = R3 = H, R2 = i-Pr, R4 = Me 194 R1 = i-Pr, R2 = R3 = H, R4 = Me 195 190 Figure 40. New constituents of N. siamensis KUFC 6349. 2.4.6. Neosartorya tsunodae The only report on the chemical investigation of N. tsunodae was recently published by Kijjoa’s group. The marine sponge-associated fungus N. tsunodae KUFC 9213, yielded sartorypyrone B (196) (Figure 41), a new analogue of chevalone C. Sartorypyrone B (196) displayed strong growth inhibitory activity against MCF-7, NCI-H460 and A375-C5 cancer cell lines with GI50 values of 17.8 ± 7.4, 20.5 ± 2.4 and 25.0 ± 4.4 µM, respectively (Eamvijarn et al., 2013). 196 Figure 41. Structure of sartorypyrone B (196). 62 CHAPTER II.CHEMISTRY OF THE GENERA EMERICELLA, EUROTIUM AND NEOSARTORYA 2.4.7. Unspecified Neosartorya species Chemical investigation of an unspecified Neosartorya sp. from soil sample led to the isolation of a novel angiogenesis inhibitor, azaspirene (197) (Figure 42). Azaspirene (197) completely inhibited VEGF (vascular endothelial growth factor) induced cell migration in human umbilical vein endothelial cells (HUVECs) at the concentration of 27.1 µM, without significant cell toxicity (Asami et al., 2002). Further studies showed that the anti-angiogenic activity was associated with the suppression of Raf-1 activation (Asami et al., 2008). Another endothelial-cell growth inhibitor RK-805 (6-oxo-6-deoxyfumagillol) (198) was later isolated from the same culture. Additionally, RK-805 (198) also selectively inhibited methionine aminopeptidase-2 (MetAP2), supporting the potential of the compound for the development of new angiogenesis inhibitors (Asami et al., 2004). Marine-derived Neosartorya sp. HN-M-3, collected from marine mud in the intertidal zone of Hainan Province (China), was found to produce four new tryptoquivaline analogues, tryptoquivalines P-S (199-202) (Figure 42) (Sun et al., 2012; Xu et al., 2013b). 197 198 199 R1 = H, R2 = OMe 200 R1 = H, R2 = OH 201 R1 = OH, R2 = OMe 202 Figure 42. New constituents of unspecified Neosartorya species. 63 CHAPTER III. RESULTS AND DISCUSSION CHAPTER III RESULTS AND DISCUSSION 64 CHAPTER III. RESULTS AND DISCUSSION 65 CHAPTER III. RESULTS AND DISCUSSION 3.1. Chemical Investigation of the Marine Derived Fungi Chemical investigation of the marine sponge-associated fungus Emericella variecolor KUFC 7092, resulted in isolation of ergosterol (EV1), ergosterol peroxide (EV2), orcinol (EV3), 1H-indole-3-carboxylic acid (EV4) and cyclo-(Ltryptophyl-L-phenylalanyl) (EV5) (Figure 43). EV1 EV4 EV5 EV2 EV3 Figure 43. Constituents of Emericella variecolor KUFC 7092. The EtOAc extract of the marine sponge-derived fungus Eurotium cristatum KUFC 7356 furnished five known anthraquinones (EC1-EC5), four previously reported indolopolyketopiperazine alkaloids (EC6-EC9) and a new diketopiperazine dimer (EC10) (Figure 44). 66 CHAPTER III. RESULTS AND DISCUSSION EC8 EC9 EC10 EC1 R1 = OH, R2 = CH3, R3 = H EC2 R1 = R3 = H, R2 =CH3 EC3 R1 =OH, R2 = R3=H EC4 R1 = R2 = R3 = H EC5 R1 = R2 = H, R3 = CH3 EC6 EC7 Figure 44. Constituents of Eurotium cristatum KUFC 7356. Two marine-derived Neosartorya species were investigated namely, the diseased coral-derived N. laciniosa KUFC 7896 and the marine sponge associated N. paulistensis KUFC 7897. From the EtOAc extract of the culture of N. laciniosa KUFC 7896, a new tryptoquivaline analogue, tryptoquivaline T (NL6) , was isolated, in addition to five known metabolites including the meroditerpenes chevalone B (NL1), aszonapyrones A (NL2) and B (NL3), tryptoquivaline L (NL4) and 3’-(4oxoquinazolin-3-yl)spiro[1H-indole-3,5’-oxolane]-2, 2’-dione (NL5) (Figure 45). Examination of the EtOAc extract of the culture of N. paulistensis KUFC 7897 yielded a new aszonapyrone analogue, sartorypyrone C (NP1), in addition to five previously reported metabolites including tryptoquivalines L (NP2), H (NP3) and F (NP6), 4(3H)-quinazolinone (NP4) and 3’-(4-oxoquinazolin-3-yl)spiro[1Hindole-3,5’-oxolane]-2, 2’-dione (NP5) (Figure 46). 67 CHAPTER III. RESULTS AND DISCUSSION NL2 R = Ac NL3 R = H NL1 NL5 NL4 R = OH NL6 R = CHO Figure 45. Constituents of Neosartorya laciniosa KUFC 7896. NP4 NP5 NP1 NP2 R1 = OH, R2 = Me NP3 R1 = OH, R2 = H NP6 R1 = H, R2 = H Figure 46. Constituents of Neosartorya paulistensis KUFC 7897. 68 CHAPTER III. RESULTS AND DISCUSSION 3.1.1. Structure Elucidation of Triterpenoid Steroids 3.1.1.1. Ergosterol (EV1) EV1 was isolated as white amorphous solid. The 13C NMR, DEPT and HSQC spectra (Table 2) revealed the presence of two quaternary sp2 (δC 141.3, 139.7), four methine sp2 (δC 135.5, 131.9, 119.5, 116.3), two quaternary sp3 (δC 42.0, 37.0), seven methine sp3 (δC 70.3, 55.7, 54.5, 46.2, 42.8, 40.4, 33.0), seven methylene sp3 (δC 40.7, 39.0, 38.3, 31.9, 29.5, 28.3, 23.0) and six methyl (δC 21.1, 19.9, 19.6, 17.6, 16.2, 12.0) carbons. Table 2. 1H and 13C NMR data (CDCl3, 300.13 and 75.47 MHz) of EV1. Position δC, type δH, mult. (J in Hz) COSY HMBC 1α β 38.3, CH2 1.29, m 2.01, m H-2a H-2b C-3 --- 2a b 31.9, CH2 2.01, m 1.51, m H-1α H-1β, 3 --- --- 3 70.3, CH 3.63, m H-2b, 4a, 4b --- 4a b 40.7, CH2 2.44, m 2.28, m H-3, 4b, 6 H-3, 4a, 6 C-2, 3, 5, 6, 10 --- 5 139.7, C --- --- --- 6 119.5, CH 5.57, dd (5.6, 2.3) H-4a, 4b, 7 C-4, 8, 10 7 116.3, CH 5.38, m H-6, 14 C-5, 9, 14 8 141.3, C --- --- --- 9 46.2, CH 1.96, m --- C-5 10 37.0, C --- --- --- 11a b 23.0, CH2 1.69, m 1.33, m H-11b, 12a H-11a, 12b --- --- 12a b 39.0, CH2 2.04, m 1.24, m H-11a, 12b H-11b, 12a C-14 C-18 13 42.0, C --- --- --- 14 54.5, CH 2.02, m H-7 C-9, 13, 17 15 28.3, CH2 1.74, m H-16b --- 16a b 29.5, CH2 1.76, m 1.29, m --- H-15 --- --- 17 55.7, CH 1.26, m H-20 C-12, 13, 14, 15, 16, 20, 21 18 12.0, CH3 0.63, s --- C-12, 13, 14, 17 19 16.2, CH3 0.94, s --- C-1, 5, 9, 10 20 40.4, CH 2.03, m H-17, 21, 22 C-13 21 21.1, CH3 1.04, d (6.7) H-20 C-17, 20, 22, 23 22 135.5, CH 5.16, dd (15.0, 7.5) H-20, 23 C-20, 22, 24 23 131.9, CH 5.23, dd (15.0, 7.0) H-22, 24 C-20, 22, 24, 25, 28 24 42.8, CH 1.82, m H-23, 25, 28 C-22, 23, 25, 26/27 25 33.0, CH 1.48, m H-24, 26/27 C-22, 23, 26/27 26/27 19.9, CH3 19.6, CH3. 0.84, d (6.7) 0.82, d (6.7) H-25 H-25 C-24, 25, 26/27 C-24, 25, 26/27 28 17.6, CH3 0.92, d (6.7) H-24 C-23, 24, 25 69 CHAPTER III. RESULTS AND DISCUSSION The presence of six sp2 carbons (δC 141.3, 139.7, 135.5, 131.9, 119.5 and 116.3), together with the four olefinic protons at δH 5.57, dd (J = 5.6, 2.3 Hz), 5.38, m, 5.23, dd (J = 15.0, 7.0 Hz) and 5.16, dd (J = 15.0, 7.5 Hz), suggested the existence of three double bonds in the molecule. The COSY spectrum (Table 2) showed that the proton at δH 5.16, dd (J = 15.0, 7.5 Hz; δC 135.5) was coupled to the proton at δH 5.23, dd (J = 15.0, 7.0 Hz; δC 131.9), suggesting that they belong to a trans substituted double bond. The COSY spectrum also displayed correlations between the olefinic proton at δH 5.23, dd (J = 15.0, 7.0 Hz) and the methine proton at δH 1.82, m (δC 42.8). In turn, the multiplet of the methine proton at δH 1.82 gave cross peaks to the signals of the methine proton at δH 1.48, m (δC 33.0) and the doublet of the methyl protons at δH 0.92 (J = 6.7 Hz; δC 17.6), whereas the multiplet of the methine proton at δH 1.48 (δC 33.0) also gave cross peaks with the two methyl doublets at δH 0.84 (J = 6.7 Hz; δC 19.9) and δH 0.82 (J = 6.7 Hz; δC 19.6). These correlations suggested the presence of the coupling system of: 5.16 dd (15.0, 7.5) 135.5 131.9 5.23 dd (15.0, 7.0) 0.82 d (6.7) 0.92 d (6.7) 1.82 m 0.84 d (6.7) 1.48 m 19.9 33.0 42.8 17.6 19.6 On the other hand, the olefinic proton at δH 5.16, dd (J = 15.0, 7.5 Hz; δC 135.5) gave cross peaks to the multiplet of the methine proton at δH 2.03 (δC 40.4) which, in turn, gave cross peaks to the methyl doublet at δH 1.04 (J = 6.7 Hz; δC 21.1) as well as the multiplet of the methine proton at δH 1.26 (δC 55.7), suggesting the presence of the coupling system of 2,5,6-trimethyl-3-hepten-2-yl (A). 76 CHAPTER III. RESULTS AND DISCUSSION Ergosterol (EV1) and ergosterol peroxide (EV2) belong to the subclass of modified triterpenoid steroids, which include several widely known drugs such as corticosteroids and cardioactive glycosides, several biological and physiologically relevant hormones, as well as phytosterols. Isolation of ergosterol and ergosterol peroxide from the extract of E. variecolor KUFC 7092 was not surprising since ergosterol is the predominant fungal sterol, representing approximately 70% of the sterols present in fungi. As a component of fungal membranes, ergosterol is involved in several biological functions such as membrane fluidity regulation, activity and distribution of integral proteins and control of the cellular cycle (Bard et al., 1993). Analogously, isolation of ergosterol peroxide is not unexpected since it has been reported as the H2O2-dependent enzymatic oxidation product of ergosterol (Bates et al., 1976). Despite their wide distribution, both fungal steroids have been a focus of several reports regarding to their therapeutic potential. Apart from being the major precursor for the synthesis of vitamin D2 (Holick, 2003), there are a number of reports on biologically active properties of ergosterol as well as of ergosterol peroxide. Both ergosterol and its peroxide were reported as anti-inflammatory agents, suppressing LPS-induced inflammation in RAW 246.7 macrophages through the inhibition of NF-κB and C/EBPβ, as well as phosphorylation of MAPK’s (Kobori et al., 2007). Kuo et al. (2011) have found that the anti-inflammatory action of ergosterol was also associated with the inhibition of COX-2 expression. In the previous study by Kobori et al. (2007), ergosterol peroxide was found to display growth inhibitory activity against colorectal adenocarcinoma HT29 cells, presumably as a result of the accumulation of ROS. In spite of the fact that antitumor molecular mechanisms of ergosterol and ergosterol peroxide are not clarified yet, there are several reports on their cytotoxic activity against a wide spectrum of cancer cell lines. While ergosterol displayed growth inhibitory activity against MCF-7 (breast adenocarcinoma) cell line (Subbiah and Abplanalp, 2003) and an inhibitory effect on rat urinary bladder carcinogenesis (Yazawa et al., 2000), ergosterol peroxide was shown to be cytotoxic against human hepatocellular carcinoma Hep 3B (Chen et al., 2009a) and human multiple myeloma U266 cells (Rhee et al., 2012), and against human gastric tumor (SNU1), human hepatoma (SNU-354), human colorectal tumor (SNU-C4) and murine 77 CHAPTER III. RESULTS AND DISCUSSION sarcoma-180 cell lines (Nam et al., 2001). Additionally, ergosterol peroxide also exhibited inhibitory effect against androgen-sensitive (LNCaP) and –insensitive (DU-145) human prostate cancer cells at micromolar concentrations (Russo et al., 2010), as well as against several chemoresistant tumor cells (Wu et al., 2012). Interestingly, while ergosterol acts as a modulator of the cholesterol regulatory effect in cell cycle progression in human cells (Suárez et al., 2002), its peroxide displayed the anti-atherosclerotic activity, through the ihbition of hACAT-1 and LpPLA2 (Kim et al., 2005). The biological properties of ergosterol peroxide include also antibacterial (Duarte et al., 2007) and trypanocidal activity (Ramos-Lignorio et al., 2012) against Mycobacterium tuberculosis and Trypanosoma cruzi, respectively, as well as renal fibrosis preventive effect (Zhu et al., 2013). 3.1.2. Structure Elucidation of Orcinol and 1H-Indole-3carboxylic acid 3.1.2.1. Orcinol (EV3) EV3 was isolated as colorless mass. The 13C NMR, DEPT and HSQC spectra (Table 4) revealed the presence of three quaternary sp2 (δC 157.8, 156.3, 141.0), three methine sp2 (δC 112.1, 111.3, 103.4) and one methyl (δC 21.4) carbon. The 1H NMR spectrum revealed the presence of three meta-coupled aromatic protons at δH 6.19, dd, J = 2.0, 2.0 Hz (δC 103.4), δC 6.30, d, J = 2.0 Hz, 2H (δC 112.1 and 111.3), and the aromatic methyl protons at δH 2.13, s (δC 21.4) (Table 4). Table 4. 1H and 13C NMR data (CDCl3, 300.13 and 75.47 MHz) of EV3. Position δC, type δH, mult. (J in Hz) HMBC 1 157.8, C --- --- 2 103.4, CH 6.19, dd (2.0, 2.0) C-1, 3, 4, 6 3 156.3, C --- --- 4 112.1, CH 6.30, d (2.0) C-2, 3, 6, 7 5 141.0, C --- --- 6 111.3, CH 6.30, d (2.0) C-1, 2, 4, 7 7 21.4, CH3 2.13, s C-4, 5, 6 The low frequencies of the aromatic protons, together with high frequencies of the quaternary sp2 carbons (δC 157.8 and 156.3) suggested the 1,3,5- 78 CHAPTER III. RESULTS AND DISCUSSION trissubstituted methylphenol. The HMBC spectrum (Table 4) showed cross peaks of the methyl singlet at δH 2.13 (δC 21.4) to the carbon at δC 141.0, 112.1 and 111.3; of the doublet at δH 6.30 (J = 2.0 Hz) to the carbons at δC 156.3, 112.1, 111.3, 103.4 and 21.4; and of the double doublet at δH 6.19 (J = 2.0, 2.0 Hz; δC 103.4) to the carbon at δC 157.8, 156.3, 112.1 and 111.3, thus suggesting the structure of EV3 as 5-methylbenzene 1,3-diol. 157.8 2.13 s 21.4 6.19 dd (2.0, 2.0) 6.30 d (2.0) 156.3 103.4 112.1 111.3 141.0 The 1H and 13C NMR data of EV3 were in agreement with those reported for orcinol (Figure 49) by Gavin and Tabacchi (1975). Orcinol (EV3) has been also reported from the lichens Evernia prunasti (L.) ARCH (Nomura et al., 2012) and Parmotrema grayana and was found to have antioxidant activity (Thadhani et al., 2011). 1 2 3 4 5 6 7 Figure 49. Structure of orcinol (EV3). 3.1.2.2. 1H-Indole-3-carboxylic acid (EV4) EV4 was isolated as white amorphous solid. The 13C NMR, DEPT and HSQC spectra (Table 5) revealed the presence of four quaternary sp2 (δC 165.9, 79 CHAPTER III. RESULTS AND DISCUSSION 136.4, 126.0, 107.3) and five methine sp2 (δC 132.3, 122.1, 121.0, 120.6, 112.2) carbons. Table 5. 1H and 13C NMR data (DMSO-d6, 500.13 and 125.77 MHz) of EV4. Position δC, type δH, mult. (J in Hz) COSY HMBC 2 132.3, CH 8.00, s --- C-3, 8, 9 3 107.3, C --- --- --- 4 120.6, CH 8.00, d (7.6) H-5 C-3, 6, 8 5 121.0, CH 7.15, ddd (7.6, 7.6, 1.3) H-4, 6 C-7, 9 6 122.1, CH 7.19, ddd (7.6, 7.6, 1.3) H-5, 7 C-4, 8 7 112.2, CH 7.46, d (7.6) H-6 C-5, 9 8 136.4, C --- --- --- 9 126.0, C --- --- --- 10 165.9, CO --- --- --- NH --- 11.81, s --- --- OH --- 11.95, brs --- --- The 1H NMR spectrum (Table 5) displayed two doublets at δH 8.00 (J = 7.6 Hz) and δH 7.46 (J = 7.6 Hz), two double double doublets at δH 7.19 (J = 7.6, 7.6, 1.3 Hz) and δH 7.15 (J = 7.6, 7.6, 1.3 Hz), two singlets at δH 8.00 and 11.81, besides a broad singlet at δH 11.95. The COSY spectrum (Table 5) exhibited cross peaks of the doublet at δH 8.0 (J = 7.6 Hz) to the double double doublet at δH 7.15 (J = 7.6, 7.6, 1.3 Hz), and of the doublet at δH 7.46 (J = 7.6 Hz) to the double double doublet at δH 7.19 (J = 7.6, 7.6, 1.3 Hz). In turn, the two double double doublets at δH 7.15 (J = 7.6, 7.6, 1.3 Hz) and 7.19 (J = 7.6, 7.6, 1.3 Hz) were correlated with each other, as well as with the doublets at δH 8.00 (J = 7.6 Hz) and 7.46 (J = 7.6 Hz), respectively, suggesting the presence of a 1,2-disubstituted benzene ring (A). This was confirmed by the correlations displayed by the HMBC spectrum (Table 5) which showed cross peaks of the doublet at δH 7.46 (J = 7.6 Hz; δC 112.2) to the sp2 methine carbon at δC 121.0 and the sp2 quaternary carbon at δC 126.0, and of the doublet at δH 8.0 (J = 7.6 Hz; 120.6) to the sp2 methine carbon at δC 122.1 and the sp2 quaternary carbon at δC 136.4. Furthermore, the HMBC spectrum also exhibited cross peaks of the doublet at δH 8.00 (J = 7.6 Hz; δC 120.6) to the quaternary sp2 carbon at δC 107.3, and of the singlet at δH 8.00 (δC 132.3) to the quaternary sp2 carbons at δC 107.3 and 126.0, suggesting that one of the substituents of the 1,2-dissubstituted benzene ring was an ethenyl fragment (B). Since the singlet at δH 8.00 (δC 132.3) also 80 CHAPTER III. RESULTS AND DISCUSSION showed the HMBC cross peak to quaternary sp2 carbon at δC 136.4, the cyclic nature of the substituents on the carbons at δC 126.0 and 136.4 was suggested (B). Moreover, the chemical shift value of the substituted aromatic carbon at δC 136.4 and of the olefinic carbon at δC 132.3 (δH 8.00, s) were characteristics of the nitrogen bearing sp2 carbons. A 132.3 B 7.19 ddd (7.6, 7.6, 1.3) 7.15 ddd (7.6, 7.6, 1.3) 8.00 d (7.6) 8.00 s 8.00 d (7.6) 11.81 s 136.4 7.46 d (7.6) 120.6 107.3 120.6 126.0 112.2 122.1 121.0 Since the 1H NMR spectrum displayed a singlet at δH 11.81, the presence of the 2,3-dihydro-1H-indole rings system was proposed. That the substituent on C-3 (δC 107.3) of the indole moiety was the carboxyl group was supported by the presence of the carbon at δC 165.9, characteristic of the conjugated carbonyl, and a broad singlet at δH 11.95 in the 1H NMR spectrum. This hypothesis was also supported by a low chemical shift value of C-3 (δC 107.3) which is characteristic of the sp2 carbon with the electron withdrawing substituent. Consequently the structure of EV4 was proposed as: 1 2 3 4 5 6 7 8 9 10 Figure 50. Structure of 1H-indole-3-carboxylic acid (EV4). 81 CHAPTER III. RESULTS AND DISCUSSION Literature survey revealed that the structure of EV4 corresponded to the monoindole alkaloid 1H-indole-3-carboxylic acid (Figure 50) (Wang et al., 2012) previously reported not only from fungi (Wu et al., 2013) but also from several other sources such as Streptomyces sp. (Kavitha et al., 2010, Shaaban et al., 2008), plant pathogens (Tan et al., 2004) and marine sponges (Wang et al., 2012). 1H-Indole-3-carboxylic acid proved to be a promising antimicrobial agent, displaying good antibacterial activity not only against the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermis, but also against the Gramnegative Escherichia coli and Xanthomonas campestris. Additionally, the monoindole alkaloid exhibited also good antifungal activity against dermatophytes such as Candida albicans and Epidermophyton flocosum, as well as strong growth inhibitory activity against Fusarium oxysporum (Kavitha et al., 2010). 3.1.3. Structure Elucidation of Anthraquinones 3.1.3.1. Erythroglaucin (EC1) EC1 was isolated as red needles. The 1H NMR spectrum (Table 6) exhibited the signals of three hydrogen-bonded hydroxyl groups at δH 13.75, s, 12.46, s and 12.38, s, in addition to three doublets of the aromatic protons at δH 7.41 (J = 2.5 Hz), 7.14 (J = 0.8 Hz) and 6.71 (J = 2.5 Hz), and a methyl doublet at 2.36 (J = 0.8 Hz). The 13C NMR spectrum (Table 6) displayed sixteen carbon signals which, through DEPT (90º and 135º) and HSQC spectra (Table 6), were categorized into two conjugated ketone carbonyls (δC 188.9, 186.5), nine quaternary sp2 (δC 166.4, 165.2, 157.9, 157.2, 140.7, 135.1, 111.8, 110.6, 110.5), three methine sp2 (δC 129.3, 105.4, 106.9), one methoxy (δC 56.2) and one methyl (δC 16.5) carbons. The COSY spectrum (Table 6) showed correlations between the metacoupled aromatic protons at δH 6.71, d (J = 2.5 Hz; δC 106.9) and δH 7.41, d (J = 2.5 Hz; δC 105.4) on the one hand, and between the aromatic proton at δH 7.14, d (J = 0.8 Hz; δC 129.3) and the methyl protons at δH 2.36, d (J = 0.8 Hz; δC 16.5) on the other hand, suggesting the presence of one 1,2,3,4,6-pentasubstituted and one 1,2,3,5-tetrasubstituted benzene rings in the molecule. 82 CHAPTER III. RESULTS AND DISCUSSION 129.3 106.9 105.4 7.41 d (2.5) 2.36 d (0.8) 16.5 7.14 d (0.8) 6.71 d (2.5) Table 6. 1H and 13C NMR data (CDCl3, 500.13 and 125.77 MHz) of EC1. Position δC, type δH, mult. (J in Hz) COSY HMBC 1 157.2, C --- --- --- 2 129.3, CH 7.14, d (0.8) H-11 C-4, 9a, 11 3 140.7, C --- --- --- 4 157.9, C --- --- --- 4a 111.8, C --- --- --- 5 105.4, CH 7.41, d (2.5) H-7 C-7, 8a, 10 6 166.4, C --- --- --- 7 106.9, CH 6.71, d (2.5) H-5 C-5, 8, 8a 8 165.2, C --- --- --- 8a 110.5, C --- --- --- 9 188.9, CO --- --- --- 9a 110.6, C --- --- --- 10 186.5, CO --- --- --- 10a 135.1, C --- --- --- 11 16.5, CH3 2.36, d (0.8) H-2 C-2, 3, 4 OCH3-6 56.2, CH3 3.95, s --- C-6 OH-1 --- 12.38, s --- C-1, 2, 9a OH-4 --- 13.75, s --- C-3, 4, 4a OH-8 --- 12.46, s --- C-7, 8, 8a The HMBC spectrum (Table 6) showed correlations of the hydrogenbonded hydroxyl proton at δH 12.38, s to the carbons at δC 157.2, 129.3, and 110.6, of another hydrogen-bonded hydroxyl proton at δH 13.75, s, to the carbons at δC 157.9, 140.7 and 111.8, of the methyl protons at δH 2.36, d (J = 0.8 Hz; δC 16.5) to the carbons at δC 140.7, 129.3 and 157.9, as well as of the aromatic proton at δH 7.14, d (J = 0.8 Hz; δC 129.3) to the carbons at δC 110.6 and 157.9, suggesting that the 1,2,3,4,6-pentasubstituted benzene was part of the system shown below: 83 CHAPTER III. RESULTS AND DISCUSSION 157.2 12.38 s 110.6 16.5 13.75 s 140.7 129.3 111.8 157.9 2.36 d (0.8) 7.14 d (0.8) That three of the substituents of the 1,2,3,5-tetrasubstituted benzene ring were hydroxyl, methoxy and the carbonyl groups was substantiated by the HMBC correlations of the aromatic proton at δH 6.71, d (J = 2.5 Hz; δC 106.9) to the carbons at δC 110.5 and 105.4, of the aromatic proton at δH 7.41, d (J = 2.5 Hz; δC 105.4) to the carbons at δC 186.5, 110.5 and 106.9, of the hydrogen-bonded hydroxyl proton at δH 12.46, s to the carbons at δC 165.2, 106.9 and 110.5, and of the methoxy proton at δH 3.95, s (δC 56.2) to the carbon at δC 166.4. 165.2 110.5 12.46 s 3.95 s 56.2 186.5 7.41 d (2.5) 166.4 105.4 106.9 6.71 d (2.5) Combining these two partial structures led to the structure of EC1 as 1,4,8trihydroxy-6-methoxy-3-methylanthracene-9,10-dione. The NMR data of EC1 were compatible with those reported for erythroglaucin (Figure 51), the anthraquinone isolated from different species of the genus Eurotium (Anke et al., 1980). Erythroglaucin has not only been reported as a metabolic product from different genus of microfungi, namely Alternaria (Andersen et al., 2008; Suemitsu et al., 1977), but also from macrofungi (Beattie et al., 2010) and several lichens of different genera (Manojlović et al., 2000; Sochting and Frödén, 2002). 84 CHAPTER III. RESULTS AND DISCUSSION 1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 11 Figure 51. Structure of erythroglaucin (EC1). Regarding to its biological properties, this anthraquinone was reported to have antibacterial activity against Pseudomonas fluorescens and Pseudomonas glicinea, and antifungal activity against human pathogenic fungi including Aspergillus niger, Doratomyces stemonitis, Penicillium verucosum and Trichoderma viride (Manojlović et al., 2000). Interestingly, erythroglaucin was found to cause DNA cleavage through photo-induced production of ROS (Rajendran et al., 2004). It displayed also moderate radical-scavenging activity against DPPH (Wang et al., 2006). Erythroglaucin was shown to display structureuncoupling activity using rat liver mitochondria (Betina and Kuzela, 1987), as well as iron-chelating capability (Engstrom et al., 1982). 3.1.3.2. Physcion (EC2) EC2 was isolated as orange needles, and the general features of its 1H and 13C NMR spectra closely resembled those of EC1. However, the 1H NMR spectrum (Table 7) revealed the presence of two hydrogen-bonded hydroxyl groups at δH 12.13, s and δH 12.33, s, four aromatic protons at δH 6.69, d (J = 2.5 Hz), 7.09, d (J = 0.8 Hz), 7.38, d (J = 2.5 Hz) and 7.64, d (J = 0.8 Hz), one methoxy group (δH 3.94, s) and one aromatic methyl group (δH 2.46, s). The 13C NMR spectrum (Table 7) displayed also sixteen carbon signals which were categorized, according to DEPT (90º and 135º) and HSQC (Table 7), as two ketone carbonyls (δC 195.9, 182.1), eight quaternary sp2 (δC 166.5, 165.2, 162.5, 148.5, 135.2, 133.2, 114.0, 110.3), four methine sp2 (δC 125.5, 121.3, 108.2, 85 CHAPTER III. RESULTS AND DISCUSSION 106.8), one methoxy (δC 56.1) and one methyl (δC 22.2) carbon, suggesting the anthraquinone nucleus. Table 7. 1H and 13C NMR data (CDCl3, 500.13 and 125.77 MHz) of EC2. Position δC, type δH, mult. (J in Hz) HMBC 1 162.5, C --- --- 2 125.5, CH 7.09, d (0.8) C-4, 9a, 11 3 148.5, C --- --- 4 121.3, CH 7.64, d (0.8) C-2, 9a, 10, 11 4a 133.2, C --- --- 5 108.2, CH 7.38, d (2.5) C-7, 8a, 10 6 166.5, C --- --- 7 106.8, CH 6.69, d (2.5) C-5, 8, 8a 8 165.2, C --- --- 8a 110.3, C --- --- 9 195.9, CO --- --- 9a 114.0, C --- --- 10 182.1, CO --- --- 10a 135.2, C --- --- 11 22.2, CH3 2.46, s C-2, 3, 4 OCH3-6 56.1, CH3 3.94, s C-6 OH-1 --- 12.13, s C-1, 2, 9a OH-4 --- --- --- OH-8 --- 12.33, s C-7, 8, 8a That one of the 1,2,3,5-tetrasubstituted benzene rings was a 1-hydroxy-3methoxybenzene ring of the anthraquinone was corroborated not only by the chemical shift values of the aromatic protons and their corresponding carbons (δH 6.69, d, J = 2.5 Hz; δC 106.8 / δH 7.38, d, J = 2.5 Hz; δC 108.2) and the hydrogenbonded hydroxyl group at δH 12.33, s, but also by the HMBC correlations of the hydroxyl proton at δH 12.33, s to the carbons at δC 165.2, 110.3 and 106.8, of the aromatic proton at δH 6.69, d (J = 2.5 Hz; δC 106.8) to the carbon at δC 165.2, 108.2 and 110.3, and of the aromatic proton at δH 7.38, d (J = 2.5 Hz; δC 108.2) to the carbons at δC 110.3 and 106.8 and to the carbonyl carbon at δC 182.1. That another part of the anthraquinone was a 1-hydroxy-3-methylbenzene was supported by the presence of the hydrogen-bonded hydroxyl proton at δH 12.13, s, which gave HMBC correlations to the carbons at δC 162.5, 125.5 and 114.0, and the methyl singlet at δH 2.46 (δC 22.2) which, in turn, gave HMBC correlations to the carbons at δC 148.5, 125.5, and 121.3, as well as the HMBC correlations of the doublet of the aromatic proton at δH 7.64 (J = 0.8 Hz; δC 121.3) to the carbonyl at 92 CHAPTER III. RESULTS AND DISCUSSION Therefore, the structure of EC4 was proposed as 1,6,8-trihydroxy-3methylanthracene-9,10-dione, or commonly known as emodin (Figure 54). The 1H and 13C NMR data of EC4, as well as its physical constants were compatible with those of the anthraquinone emodin, reported earlier from several species of the lichens of the genus Xanthoria (Manojlović et al., 2000). 1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 11 Figure 54. Structure of emodin (EC4). Despite its isolation from the fungi of the genus Eurotium (Anke et al., 1980), emodin has been widely reported from several plant genera including Cassia (Kim et al., 2004; Lee et al., 2010), Frangula (Kremer et al., 2012) and Polygonum (Hsiang and Ho, 2008; Kuo et al., 1997; Leu et al, 2008), and as a major constituent of rhubarb, Rheum palmatum L. (Chen et al., 2002; 2010a; Choi et al., 2013). Emodin is the most extensively studied anthraquinone which displayed a vast number of pharmacological properties such as antitumor, immunosuppressive, antimicrobial, anti-inflammatory and antidiabetic. Literature survey reveals several reports on its cytotoxic activity against several human cancer cell lines through several different mechanisms (Hsu and Chung, 2012). Emodin exhibited antiproliferative effect against Raji, HeLa, Calu-1 Wish, Vero, SW620 and K562 cell lines (Choi et al., 2007; Kuo et al., 1997; Lin et al.,2001), with a notable antitumor effect in human leukemia K562 cell line, both in vitro and in vivo, leading to a significant decrease of tumor volume and weight in nude mice inoculated with K562 cells (Chun-Guang et al., 2010). However, the majority of reports refer to the anticancer potential of emodin against human pancreatic and liver cancer cell lines. Emodin demonstrated antiproliferative and antimetastatic 93 CHAPTER III. RESULTS AND DISCUSSION effect on human pancreatic cancer cell line SW 1999 (Liu et al., 2011a) and induced Panc-1 cells apoptosis through the decline of the mitochondrial membrane potential (Liu et al., 2012). Furthermore, it also potentiated the antitumor effects of gemcitabine in human pancreatic cancer cell line SW 1999, which was related to the downregulation of NF-κB, leading to an enhancement on the effect of gemcitabine and overcoming resistance (Liu et al., 2011b). Interestingly, emodin displayed the synergistic property of overcoming chemoresistance in several cancer cell lines, significantly enhancing the DNA damage caused by oxaliplatin and inducing oxaliplatin resistance reversal in hepatocellular carcinoma HepG2 cells, through the inhibition of ERCC1 expression by the FGFR2/ERK1/2 signaling pathway (Chen et al., 2013). Notably, emodin was reported to display antiproliferative activity against other human liver cancer cells such as SK-HEP-1, Bel-7402 and PLC/PRF/5, through several complementary mechanisms including the activation of caspase-8 and -9, p53 and p21 upregulation, NF-κB downregulation, and intracellular ROS production (Shieh et al., 2004; Yu et al., 2013). Emodin was also proved to be an effective apoptosis inducer in human promyeloleukemic HL-60 and in C6 glioma cells through the activation of the caspase-3 cascade (Chen et al., 2002; Kuo et al., 2009), and the antiproliferative activity against prostate cancer cell lines LNCaP and PC-3, as well as against the human lung (A549) and bone (MG-63) cell lines, also seems to be partially dependent on the generation of ROS (Masaldan and Iyer, 2014). The antimetastatic activity was also observed in prostate DU 145 and lung A549 cancer cell lines by downregulation of the expression of the chemokine receptor CXCR4 (Ok et al., 2012). Zhang et al. (1999) reported that emodin was a potent tyrosine kinase inhibitor and repressed cellular transformation and metastasis in breast cancer cells (Zhang et al., 1999; Zhang and Hung, 1996). Recently, the antiproliferative effect on breast cancer cell lines was reported as being associated with the downregulation of the estrogen receptor α (ERα) protein levels, thereby suppressing ERα transcriptional activation in MCF-7 and MDA-MB-453 cell lines (Huang et al., 2013). These findings led us to hypothesize that the in vitro anticancer activity of the crude extract of Eurotium cristatum KUFC 7356 against MCF-7 cancer cell line (Almeida et al., 2010) may be in part derived from emodin cytotoxic activity. Additionally, emodin was found to inhibit the migration and invasion in human tongue cancer SCC-4 cells through the inhibition of gene 94 CHAPTER III. RESULTS AND DISCUSSION expression of MMP-9 and MMP-2 (Chen et al., 2010a) and by inducing DNA damage and inhibiting DNA repair gene expression (Chen et al., 2010b). Recently, emodin was also classified as a potential new therapeutic drug for head and neck squamous cell carcinoma cancer due to its in vivo inhibitory activity of TWIST1induced invasions, and inhibition of the β-catenin and Akt pathways (Way et al., 2014). Immunosuppressive properties of emodin have also been reported demonstrating a helpful effect for the modulation of immune suppression and induction of immune tolerance through the inhibition of the differentiation and maturation of dendritic cells and increased production of CD4+CD25+ T cells (Zhang et al., 2012). Emodin immunosuppressive properties were confirmed by the immunosuppressive effect mediated through the generation of hydrogen peroxide from semiquinone and regulation by arachidonic acid metabolites (Huang et al., 1992), and the in vivo effect alleviating acute rejection following liver transplantation in rats as prolonging liver allograft survival (Tong et al., 2011). Administration of emodin against LPS-induced mouse mastitis revealed a protective effect against LPS-induced mammary gland injury and inflammatory cell infiltration by decreasing the transcriptional production of proinflammatory cytokines TNF-α, IL-6 and IL-1β (Li et al., 2013b). The anti-inflammatory properties of emodin were also observed in other in vivo models, displaying an effective antiinflammatory response in collagen-induced arthritic mice through the inhibition of the NF-κB pathway and subsequent MMP production (Hwang et al., 2013) as well as a significant reduction of oedema volume in carrageenan-induced rat-paw model through iNOS inhibition (Gosh et al., 2010). Recently, the in vitro antiinflammatory activity of emodin on LPS-induced RAW264.7 macrophages was also reported, supposedly due to the inhibition of P-gp function caused by the decreased expression of COX-2 protein through the MAPK/AP1 pathway (Choi et al., 2013). Emodin revealed also therapeutic potential in the prevention or treatment of glucose-induced structural and functional abnormalities, due to the amelioration of glucose-induced TGF-β1 and matrix synthesis in human peritoneal mesothelial cells by inhibiting PKCα activation and phosphorylation of CREB (Chan et al., 2003). Furthermore, emodin exhibited potent and selective in vitro and in vivo inhibitory activity against 11β-hydroxysteroid dehydrogenase type 1, and its oral administration in diet-induced obese mice antagonized insulin resistance and lowered blood glucose and hepatic PEPCK (Feng et al., 2010; Liu 95 CHAPTER III. RESULTS AND DISCUSSION et al., 2009). Recently, it was also reported to cause a significant decrease of collagen IV and fibronectin production (Yang et al., 2013a), revealing a potential use not only in diabetic nephropathy but also generally in metabolic syndrome or type 2 diabetes. Its potential in the treatment of progressive renal diseases was also confirmed by the amelioration of renal failure in nephrectomized rats treated with emodin, due to the suppression of IL-1β induced mesangial cells proliferation (Wang et al., 2007). The potent in vivo anti-angiogenic effect through the inhibitory function toward VEGF-A –induced angiogenesis by blocking receptor-2 (KDR/Flk1) phosphorylation (Kwak et al., 2006) as well as the in vitro anticoagulation effect of emodin-eluting biodegradable polymer coating stents (Pan et al., 2010), turn emodin as an interesting candidate for the development of alternative drugs for the treatment of cardiovascular diseases. When administered orally in IgE-sensitized mice, emodin exhibited anti-allergic activity, suppressing IgE-mediated anaphylactic reaction and mast cell activation (Lu et al., 2011). Emodin was also demonstrated to be a potential candidate for the development of antipigmentation agents due to its inhibition of phosphorylation of Kit and other transmembrane tyrosine kinases, and subsequent melanin synthesis (Lee et al., 2010). Emodin exhibited antimicrobial activity against several human pathogenic fungi and bacteria such as Aspergillus niger, Doratomyces stemonitis, Trichoderma viride and Penicillium verucosum, and against the bacteria Pseudomonas fluorescens, Pseudomonas glicinea, Pseudomonas phaseolicola (Manojlović et al., 2000), Streptococcus iniae (Nakano et al., 2012) and Helicobacter pylori strains SS1 and ATCC 43504 (Chen et al., 2009b), respectively. Emodin displayed also antiviral activity against herpes simplex virus (HSV) type 1, through the inhibition of HSV-1 UL12 alkaline nuclease (Hsiang and Ho, 2008). 3.1.3.5. Questin (EC5) EC5 was isolated as orange-yellow amorphous solid and its 1H and 13C NMR spectra (Table 10) were very similar to those of EC2. The 1H spectrum (Table 10) displayed the signals of one hydrogen-bonded hydroxyl proton at δH 13.24, s, two pairs of meta-coupled aromatic protons at δH 7.42, d (J = 1.6 Hz) / 7.12, d (J = 1.6 Hz), and at δH 7.20, d (J = 2.3 Hz) / 6.84, d (J = 2.3 Hz), one methoxy group at δH 3.90, s and one methyl group at δH 2.39, s. The 13C NMR, 96 CHAPTER III. RESULTS AND DISCUSSION DEPT and HSQC spectra (Table 10) revealed the presence of one hydrogenbonded carbonyl (δC 186.3), one non hydrogen-bonded carbonyl (δC 182.3), eight quaternary sp2 (δC 164.4, 163.4, 161.7, 146.6, 136.8, 132.0, 114.4, 112.6), four methine sp2 (δC 124.2, 119.1, 106.9, 104.9), one methoxy (δC 56.3) and one methyl (δC 21.4) carbon. Table 10. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC5. Position δC, type δH, mult. (J in Hz) HMBC 1 161.7, C --- --- 2 124.2, CH 7.12, d (1.6) C-4, 9a, 11 3 146.6, C --- --- 4 119.1, CH 7.42, d (1.6) C-2, 9a, 10, 11 4a 132.0, C --- --- 5 106.9, CH 7.20, d (2.3) C-7, 8a, 10 6 164.4, C --- --- 7 104.9, CH 6.84, d (2.3) C-5, 6, 8a 8 163.4, C --- --- 8a 112.6, C --- --- 9 186.3, CO --- --- 9a 114.4, C --- --- 10 182.3, CO --- --- 10a 136.8, C --- --- 11 21.4, CH3 2.39, s C-2, 3, 4 OCH3-8 56.3, CH3 3.90, s C-8 OH-1 --- 13.24, s --- OH-6 --- ND --- ND Not detected The chemical shift values of the meta-coupled protons (and their corresponding carbons) at δH 7.42, d (J = 1.6 Hz; δC 119.1) and 7.12, d (J = 1.6 Hz; δC 124.2), and the aromatic methyl protons at δH 2.39, s (δC 21.4) were similar to those of the 1-hydroxy-3-methylphenyl portion of EC2. This was corroborated by the HMBC correlations of the aromatic proton at δH 7.42, d (J = 1.6 Hz; δC 119.1) to the carbonyl carbon at δC 182.3, and the carbons at δC 124.2, 114.4, 21.4, of the aromatic proton at δH 7.12, d (J = 1.6 Hz; δC 124.2) to the carbons at δC 119.1, 114.4, 21.4, and of the methyl protons at δH 2.39, s (δC 21.4) to the carbons at δC 146.6, 124.2 and 119.1. 97 CHAPTER III. RESULTS AND DISCUSSION 13.24 s 182.3 161.7 114.4 21.4 7.42 d (1.6) 146.6 124.2 119.1 2.39 s 7.12 d (1.6) That another part of the anthraquinone was 6-hydroxy-8-methoxyphenyl instead of 8-hydroxy-6-methoxyphenyl as in EC2 was supported by the presence of only one hydrogen-bonded hydroxyl group, as well as by the HMBC correlations of the aromatic proton at δH 7.20, d (J = 2.3 Hz; δC 106.9) to the carbons at δC 182.3, 112.6, 104.9, of the aromatic proton at δH 6.84, d (J = 2.3 Hz; δC 104.9) to the carbons at δC 164.4, 112.6, 106.9, as well as of the methoxy protons at δH 3.90, s (δC 56.3) to the carbon at δC 163.4. 56.3 3.90 s 163.4 112.6 182.3 7.20 d (2.3) 164.4 106.9 104.9 6.84 d (2.3) Consequently, the structure of EC5 was established as 1,6-dihydroxy-8methoxy-3-methyl-9,10-anthracene-9,10-dione, commonly known as questin (Figure 55) (Fujimoto et al., 1999). Like erythroglaucin, physcion, catenarin and emodin, questin has been also previously reported from several species of Eurotium (Anke et al., 1980; Slack et al, 2009), as well as from plants belonging to the genera Cassia (Hyun et al., 2009) and Polygonum (Choi et al., 2007). 98 CHAPTER III. RESULTS AND DISCUSSION 1 2 3 4 4a 5 6 7 8 8a 9 9a 10 10a 11 Figure 55. Structure of questin (EC5). Like emodin, questin also displayed high immunosuppressive activity against Con A (T cells) and LPS-induced (B cells) proliferation of mouse splenic lymphocytes (Fujimoto et al., 1999). Questin was also proved to be cytotoxic against human colon cancer cell SW620 and exhibited inhibitory activity against Cdc25 B (Choi et al., 2007). Anthraquinones are aromatic polyketides produced by repetitive Claisen condensations of an acyl CoA starting unit with malonyl-CoA elongations units. Biosynthesis of erythroglaucin (EC1), physcion (EC2), catenarin (EC3), emodin (EC4) and questin (EC5) is shown in Scheme 2 (Dewick, 2009). Anthraquinones are commonly isolated from plants of the Polygonaceae family, e.g., the genus Rheum and Polygonum (Choi et al., 2007; 2013; Feng et al., 2013; Hsiang and Ho, 2008; Kuo et al., 1997; Leu et al., 2008; Qiu et al., 2013), however they have been also reported from several fungal species of the genus Eurotium (Anke et al., 1980; Du et al., 2008; Li et al., 2009a; Slack et al., 2009). Although erythroglaucin, physcion and catenarin have been previously reported from E. cristatum (Almeida et al., 2010; Anke et al., 1980), this study reports the presence of emodin and questin for the first time from this species (Gomes et al., 2012). 99 CHAPTER III. RESULTS AND DISCUSSION 7 Hypothetical poly-β-keto ester Aldol reactions, enolizations Emodin (EC4) -CO2 -H2O [O] Catenarin (EC3) Questin (EC5) O2 O-Methylation O-Methylation Physcion (EC2) Erythroglaucin (EC1) O-Methylation Scheme 2. Biosynthetic pathway of the anthraquinones EC1-EC5. 100 CHAPTER III. RESULTS AND DISCUSSION 3.1.4. Structure Elucidation of Diketopiperazine Derivatives 3.1.4.1. cyclo-(L-Tryptophyl-L-phenylalanyl) (EV5) EV5 was isolated as white solid. The 13C NMR, DEPT and HSQC spectra (Table 11) revealed the presence of six quaternary sp2 (δC 166.9, 166.2, 136.5, 136.1, 127.6, 108.8), ten methine sp2 (δC 129.7, 129.7, 128.1, 128.1, 126.4, 124.5, 121.0, 118.8, 118.5, 111.4), two methine sp3 (δC 55.6, 55.3) and two methylene sp3 (δC 39.7, 29.7) carbons. Table 11. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EV5. Position δC, type δH, mult. (J in Hz) COSY HMBC 1 --- 10.9, s --- C-3, 8, 9 2 124.5, CH 6.94, s --- C-3, 8, 9 3 108.8, C --- --- --- 4 118.8, CH 7.47, d (7.5) H-5 C-3, 6, 8 5 118.5, CH 6.97, dd (7.5, 7.5) H-4, 6 C-7, 9 6 121.0, CH 7.06, dd (7.5, 7.5) H-5, 7 C-4, 8 7 111.4, CH 7.31, d (7.5) H-6 C-5, 9 8 136.1, C --- --- --- 9 127.6, C --- --- --- 10a b 29.7, CH2 2.78, dd (14.4, 4.3) 2.50, dd (14.4, 5.9) H-10b, 11 H-10a, 11 C-2, 3, 11, 16 C-2, 3, 11, 16 11 55.3, CH 3.96, m H-10, 12 C-16 12 --- 7.94, d (2.3) H-11 C-14, 16 13 166.2, CO --- --- --- 14 55.6, CH 3.83, m H-15, 17 C-13 15 --- 7.73, d (2.3) H-14 C-11, 13 16 166.9, CO --- --- --- 17a b 39.7, CH2 2.43, dd (13.4, 4.6) 1.80, dd (13.4, 7.1) H-14, 17b H-14, 17a C-13, 14, 18, 19, 23 C-13, 14, 18, 19, 23 18 136.5, C --- --- --- 19 129.7, CH 6.68, d (6.9) H-20 C-17, 21 20 128.1, CH 7.17, m H-19, 21 C-18, 22 21 126.4, CH 7.17, m H-20, 22 C-19, 23 22 128.1, CH 7.17, m H-21, 23 C-18, 20 23 129.7, CH 6.67, d (6.9) H-22 C-17, 21 The 1H NMR spectrum in combination with the HSQC spectrum (Table 11), displayed the signals of four aromatic protons at δH 7.47, d, J = 7.5 Hz (δC 118.8), 6.97, dd, J = 7.5, 7.5 Hz (δC 118.5), 7.06, dd, J = 7.5, 7.5 Hz (δC 121.0), and 7.31, d, J = 7.5 Hz (δC 111.4). The COSY spectrum (Table 11) showed cross peaks of the doublet at δH 7.47 (J = 7.5 Hz) to the double doublet at δH 6.97 (J = 7.5, 7.5 101 CHAPTER III. RESULTS AND DISCUSSION Hz), as well as of the doublet at δH 7.31 (J = 7.5 Hz) to the double doublet at δH 7.06 (J = 7.5, 7.5 Hz), with the coupling constant values indicating an orthocoupling between the two pairs of protons. Furthermore, the two double doublets at δH 6.97 (J = 7.5, 7.5 Hz) and δH 7.06 (J = 7.5, 7.5 Hz) were correlated with each other indicating the presence of a 1,2-disubstituted benzene ring. Analogously to EV4, the HMBC cross peaks of the methine singlet at δH 6.94 (δC 124.5) to the quaternary sp2 carbon at δC 136.1 and 127.6, of the doublet at δH 7.47 (J = 7.5 Hz; δC 118.8) to δC 108.8 and δC 136.1, and of the doublet at δH 7.31 (J = 7.5 Hz; δC 111.4) to δC 127.6, revealed the presence of a 2,3-dihydro-1H-indole moiety (A). A 124.5 7.06 dd (7.5, 7.5) 6.97 dd (7.5, 7.5) 7.47 d (7.5) 6.94 s 10.90 s 7.31 d (7.5) 136.1 118.8 108.8 127.6 111.4 121.0 118.5 The 1H NMR spectrum also displayed the signals of additional five aromatic protons at δH 6.68, d, J = 6.9 Hz, δH 7.17, m, δH 7.17, m, δH 7.17, m and δH 6.67,d, J = 6.9 Hz, which, in combination with the HSQC spectrum, were assigned to the monosubstituted benzene ring (B). This was supported by the COSY cross peaks of the two doublets at δH 6.68 (J = 6.9 Hz; δC 129.7) and δH 6.67 (J = 6.9 Hz; δC 129.7) to a multiplet at δH 7.17, as well as by the HMBC cross peaks of the doublets at δH 6.68 (J = 6.9 Hz; δC 129.7) and δH 6.67 (J = 6.9 Hz; δC 129.7) to the carbon at δC 126.4, and of the multiplet at δH 7.17 to the carbons at δC 129.7 and 136.5. 108 CHAPTER III. RESULTS AND DISCUSSION 6.96 s 102.8 110.2 143.8 Therefore, the structure of EC8 was proposed as (3Z)-3-[[2-(1,1dimethylallyl)-1H-indol-3-yl]methylene]-6-methyl-piperazine-2,5 dione which is commonly known as neoechinulin A (Figure 57) (Li et al., 2004). 20 1 10 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 3a 7a Figure 57. Structure of neoechinulin A (EC8). Neoechinulin A has been widely reported from several species of fungi, both from marine and terrestrial environments, essentially from species of the genus Aspergillus (Dossena et al., 1974; Li et al., 2004; Marchelli et al., 1977; Nagasawa et al., 1975; Yagi and Do, 1999) and Eurotium (Du et al., 2012; Kim et al., 2013; Li et al., 2008b; Slack et al., 2009). Interestingly, most of the Aspergillus spp. reported to produce neoechinulin A, refer to Eurotium anamorphs. Additionally, the diketopiperazine alkaloid was also found to be a metabolic product of several marine derived species of the genus Chaetomium (Wang et al., 2006), Microsporum (Dewapriya et al., 2013; Wijesekara et al., 2014) and 109 CHAPTER III. RESULTS AND DISCUSSION Penicillium (Zhou et al., 2010), as well as from the higher plants Bridelia ferruginea (Pettit et al., 2008) and Cyrtomium fortumei (Yang et al., 2013b). Neoechinulin A was reported as a potential candidate for the development of new drugs for the treatment of neurodegenerative diseases like Parkinson and Alzheimer’s disease. Neoechinulin A can protect neuronal PC12 cells from cytotoxicity due to oxidative / nitrosative stress induced by the superoxide / nitric oxide co-generator 3-morpholinosydronimine (SIN-1), inducing activation of caspase-3-like proteases and by elevating the cellular reserve capacity for NAD(P)H generation (Akashi et al., 2012; Kimoto et al., 2007; Kuramochi et al., 2008; Maruyama et al., 2004). Subsequent studies revealed that neoechinulin A can also protect PC12 cells from the Parkinson disease-inducing neurotoxins MPP+ and rotenone (Akashi et al., 2011; Kajimura et al., 2008). Interestingly, a SAR study performed to examine the cytoprotective activity of neoechinulin A and several synthetic derivatives, against cytotoxicity induced by SIN-1 in NGFdifferentiated PC12 cells, revealed that the C-8/C-9 double bond plays a key role in the cytoprotective activity (Aoki et al., 2010). Recently, neoechinulin A revealed potential to be developed as a modulator of neuro-inflammatory process in Alzheimer’s disease due to its anti-inflammatory activity, presumably dependent on the regulation of p38 and ASK-1 kinases, and nuclear translocation of the NFκB p65 and p50 subunits (Dewapriya et al., 2013). The anti-inflammatory effect was also observed in LPS-stimulated RAW 264.7 macrophages through the inhibition of the NF-κB and p38 MAPK pathways. Additionally, neoechinulin A also markedly suppressed the production of NO and PGE2 and the expression of iNOS and COX-2 (Kim et al., 2013). In other recent study, neoechinulin A antiproliferative effect against human cervical carcinoma HeLa cells has been tested. It was found to exhibit significant cytotoxic activity by inducing cell apoptosis in association with the activation of the expression of caspase family enzymes and p53, followed by Bax and Bcl-2 regulation (Wijisekara et al., 2014). Neoechinulin A also displayed significant radical scavenging activity against DPPH, having higher antioxidant activity than α-tocopherol, and its UV-A protecting activity being more effective than the sunscreen oxybenzone (Li et al., 2004; Yagi and Do, 1999). 110 CHAPTER III. RESULTS AND DISCUSSION 3.1.4.3. Neoechinulin E (EC9) EC9 was isolated as an orange-red solid. The 13C NMR (Table 13) displayed signals for eighteen carbons, which were categorized as two amide carbonyls (δC 160.5, 152.3), six quaternary sp2 (δC 157.3, 145.6, 135.2, 126.1, 123.4, 103.8), six methine sp2 (δC 144.9, 121.1, 119.8, 119.7, 116.0, 111.6), one methylene sp2 (δC 112.0), one quaternary sp3 (δC ~39.0) and two methyl (δC 27.7, 27.7) carbons. The 1H and 13C NMR spectra of EC9 (Table 13) closely resembled those of EC8, except for the lack of one methine (δH 4.14, dd, J = 6.9, 1.7 Hz; δC 50.6) and one methyl (δH 1.45, d, J = 6.9 Hz; δC 19.8), and the presence of the additional quaternary sp2 carbon at δC 157.3. Table 13. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC9. Position δC, type δH, mult. (J in Hz) COSY HMBC 1 --- 11.23, s --- C-2, 3, 3a 2 145.6, C --- --- --- 3 103.8, C --- --- --- 3a 126.1, C --- --- --- 4 119.7, CH 7.43, d (7.6) H-5 C-6, 7a 5 119.8, CH 7.02, dd (7.6, 7.6) H-4, 6 C-3a, 7 6 121.1, CH 7.11, dd (7.6, 7.6) H-5, 7 C-4, 7a 7 111.6, CH 7.43, d (7.6) H-6 C-3a, 5 7a 135.2, C --- --- --- 8 116.0, CH 7.19, d (1.0) --- C-2, 3a, 10 9 123.4, C --- --- --- 10 160.5, CO --- --- --- 11 --- 11.97, brs --- --- 12 157.3, C --- --- --- 13 152.3, CO --- --- --- 14 --- 9.85, brs --- C-10, 12 15 ~39.0, C --- --- --- 16 144.9, CH 6.07, dd (17.5, 10.6) H-17a, 17b --- 17a b 112.0, CH2 5.06, d (17.5) 5.08, d (10.6) H-16 H-16 C-15, 16 C-15, 16 18 27.7, CH3 1.48, s --- C-2, 15, 16, 19 19 27.7, CH3 1.48, s --- C-2, 15, 16, 18 The 1H NMR and HSQC spectra (Table 13) exhibited the signals of four aromatic protons at δH 7.43, d (J = 7.6 Hz, 2H; δC 119.7 and 111.6), 7.11, dd (J = 7.6, 7.6 Hz; δC 121.1) and 7.02, dd (J = 7.6, 7.6 Hz; δC 119.8) which, through the COSY correlations, revealed the presence of a 1,2-disubstituted benzene ring, similar to that of EC8. Furthermore, the singlet at δH 11.23, which gave HMBC cross peaks to the carbon of the benzene ring at δC 126.1 and the substituted olefinic carbons at δC 103.8 and 145.6, suggested that the 1,2-disubstituted 111 CHAPTER III. RESULTS AND DISCUSSION benzene ring is part of the 2,3-dihydro-1H-indole ring system. Similar to EC8, the presence of the 1-methylbuten-3-yl fragment was evidenced by the coupling of the two doublets at δH 5.06 (J = 17.5 Hz; δC 112.0) and 5.08 (J = 10.6 Hz; δC 112.0) to the double doublet at δH 6.07 (J = 17.5, 10.6 Hz; δC 144.9), as well as the presence of the two methyl groups at δH 1.48, s (δC 27.7). Like EC8, the 1methylbuten-3-yl on C-2 (δC 145.6) of the indole ring was substantiated by the HMBC cross peak between the singlet of the methyl protons at δH 1.48 (δC 27.7) and the carbon at δC 145.6 of the indole moiety. 135.2 11.23 s 111.6 119.8 121.1 7.11 dd (7.6, 7.6) 7.43 d (7.6) 7.02 dd (7.6, 7.6) ~39 7.43 d (7.6) 119.7 144.9 103.8 126.1 27.7 145.6 6.07 dd (17.5, 10.6) 1.48 s 112.0 5.06 d (17.5) 5.08 d (10.6) Contrary to EC8, the olefinic proton of a trisubstituted double bond and the broad singlets of the amide hydrogens appeared at higher frequencies (δH 7.19, J = 1.0 Hz; δC 116.0) / (δH 9.85; δH 11.97, respectively). Interestingly, the broad singlet at δH 9.85 showed HMBC cross peaks to the quaternary carbons at δC 160.5 and 157.3, while the broad singlet at δH 11.97 did not exhibit any cross peaks. Similar to EC8, the doublet of the olefinic proton at δH 7.19 (J = 1.0 Hz; δC 116.0) exhibited HMBC cross peaks to the carbons at δC 126.1 and 145.6 of the 2,3-dihydro-1H-indole moiety, in addition to the quaternary sp2 carbon at δC 160.5. Considering the chemical shift value of the carbons and protons, as well as the HMBC correlations, this portion of EC9 must be: 112 CHAPTER III. RESULTS AND DISCUSSION 7.19 d (1.0) 9.85 brs 157.3 152.3 123.4 116.0 160.5 Thus, the structure of EC9 was proposed as: 1 10 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 3a 7a Figure 58. Structure of the enol form of neoechinulin E (EC9). After a literature search, it was found that neoechinulin E, the prenylated indolopiperazine derivative, isolated from Aspergillus amstelodami (Marchelli et al., 1977) presented the same 1H and 13C data as that of EC9. However, the structure of neoechinulin E was reported as: 1 10 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 3a 7a 113 CHAPTER III. RESULTS AND DISCUSSION It is important to note that the structure of EC9 and neoechinulin E are tautomers, which EC9 represents an enol form (Figure 58). Contrary to neoechinulin A, neoechinulin E has not widely been reported. Except for its identification from the fungi Aspergillus amstelodami and the plant Cyrtomium fortumei, (Marchelli et al., 1977 and Yang et al., 2013b respectively), neoechinulin E was reported so far, from the genus Eurotium (Li et al., 2008b; Slack et al., 2009). To the best of our knowledge, the only reported biological property of neoechinulin E refers to its strong radical scavenging activity against DPPH (Li et al., 2008b). 3.1.4.4. Neoechinulin (EC7) EC7 was isolated as a purple solid. The 13C NMR (Table 14) displayed twenty-three carbon signals which were categorized, according to HSQC and DEPT (135º and 90º), as ten quaternary sp2 (δC 160.5, 157.3, 152.3, 145.3, 135.5, 134.4, 131.1, 124.2, 123.0, 103.6), six methine sp2 (δC 145.0, 124.1, 120.8, 119.6, 116.2, 110.6), one methylene sp2 (δC 111.9), one quaternary sp3 (δC ~39.0), one methylene sp3 (δC 33.8) and four methyl (δC 27.7, 27.7, 25.6, 17.7) carbons. The 13C NMR spectrum (Table 14) was very similar to that of EC9 except for the presence of additional two methyl carbons at δC 17.7 and 25.6, one methylene sp3 at δC 33.8, one methine sp2 at δC 124.1 and one quaternary sp2 at δC 131.1. The COSY spectrum (Table 14) displayed correlations of the olefinic proton at δH 5.35, t (J = 7.4 Hz; δC 124.1) to the methylene protons at δH 3.39, d (J = 7.4 Hz, 2H; δC 33.8) and the methyl protons at δH 1.73, s (2CH3, δC 17.7 and 25.6), suggesting that the additional five carbons was 3-methyl-2-butenyl moiety. 3.39 d (7.4) 3.39 d (7.4) 131.1 124.1 25.6 17.7 1.73 s 1.73 s 33.8 5.35 t (7.4) 114 CHAPTER III. RESULTS AND DISCUSSION This was confirmed by the HMBC cross peaks of the doublet of the methylene protons at δH 3.39 (J = 7.4 Hz, 2H; δC 33.8) to the methine sp2 carbon at δC 124.1 and the quaternary sp2 carbon at δC 131.1, of the triplet at δH 5.35 (J = 7.4 Hz; δC 124.1) to the methyl carbons at δC 17.7 and 25.6, and of the singlet at δH 1.73 (2CH3, δC 17.7 and 25.6) to the quaternary sp2 carbon at δC 131.1 and the methine sp2 carbon at δC 124.1. Table 14. 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC7. Position δC, type δH, mult. (J in Hz) COSY HMBC 1 --- 11.11, s --- C-2, 3, 3a 2 145.3, C --- --- --- 3 103.6, C --- --- --- 3a 124.2, C --- --- --- 4 119.6, CH 7.34, d (8.1) H-5 C-3, 6, 7a 5 120.8, CH 6.87, d (8.1) H-4, 20 C-3a, 7, 20 6 134.4, C --- --- --- 7 110.6, CH 7.21, s H-20 C-3a, 5, 20 7a 135.5, C --- --- --- 8 116.2, CH 7.19, s --- C-2, 3a, 10 9 123.0, C --- --- --- 10 160.5, CO --- --- --- 11 --- 11.98, brs --- C-9, 13 12 157.3, CO --- --- --- 13 152.3, CO --- --- --- 14 --- 9.77, brs --- C-10 15 ~39.0, C --- --- --- 16 145.0, CH 6.07, dd (17.3, 10.5) H-17a, 17b C-15, 18, 19 17a b 111.9, CH2 5.06, d (17.3) 5.09, d (10.5) H-16, 17b H-17, 17a C-15, 16 C-15, 16 18 27.7, CH3 1.48, s --- C-2, 16, 19 19 27.7, CH3 1.48, s --- C-2, 16, 18 20 33.8, CH2 3.39, d (7.4) H-21 C-5, 6, 7, 21, 22 21 124.1, CH 5.35, t (7.4) H-20, 23, 24 C-23, 24 22 131.1, C --- --- --- 23 17.7, CH3 1.73, s H-21 C-21, 22, 24 24 25.6, CH3 1.73, s H-21 C-21, 22, 23 Unlike the 1H NMR spectrum of EC9, the 1H NMR spectrum (Table 14) of EC7 displayed three aromatic protons at δH 7.34, d (J = 8.1 Hz; δC 119.6), δH 7.21, s (δC 110.6) and δH 6.87, d (J = 8.1 Hz; δH 120.8), suggesting the presence of a substituent on C-5 or C-6 of the benzene ring of the 2,3-dihydro-1H-indole portion. Since the doublet at δH 7.34 (J = 8.1 Hz; δC 119.6) gave HMBC cross peaks to the carbons at δC 103.6 and 135.5, which were assigned to C-3 and C-7a of the indole portion, and also to the quaternary sp2 carbon at δC 134.4, the substituent was on C-6 (δC 134.4) of the benzene ring. That the substituent on C-6 (δC 134.4) was the 115 CHAPTER III. RESULTS AND DISCUSSION 3-methyl-2-butenyl group was supported by the HMBC cross peaks of the doublet at δH 6.87 (J = 8.1 Hz; δC 120.8) and the singlet at δH 7.21 (δC 110.6) to the methylene carbon signal at δC 33.8, as well as of the doublet at δH 3.39 (J = 7.4 Hz; δC 33.8) to the aromatic carbons at δC 120.8 and 110.6. 3.39 d (7.4) 33.8 110.6 134.4 120.8 7.21 s 6.87 d (8.1) Table 15. Comparison of 1H and 13C NMR data (DMSO-d6, 300.13 and 75.47 MHz) of EC7 and EC9. Position EC7 EC9 δC, type δH, mult. (J in Hz) δC, type δH, mult. (J in Hz) 1 --- 11.11, s --- 11.23, s 2 145.3, C --- 145.6, C --- 3 103.6, C --- 103.8, C --- 3a 124.2, C --- 126.1, C --- 4 119.6, CH 7.34, d (8.1) 119.7, CH 7.43, d (7.6) 5 120.8, CH 6.87, d (8.1) 119.8, CH 7.02, dd (7.6, 7.6) 6 134.4, C --- 121.1, CH 7.11, dd (7.6, 7.6) 7 110.6, CH 7.21, s 111.6, CH 7.43, d (7.6) 7a 135.5, C 135.2, C --- 8 116.2, CH 7.19, s 116.0, CH 7.19, d (1.0) 9 123.0, C --- 123.4, C --- 10 160.5, CO --- 160.5, CO --- 11 --- 11.98, brs --- 11.97, brs 12 157.3, CO --- 157.3, C --- 13 152.3, CO --- 152.3, CO --- 14 --- 9.77, brs --- 9.85, brs 15 ~39.0, C --- ~39.0, C --- 16 145.0, CH 6.07, dd (17.3, 10.5) 144.9, CH 6.07, dd (17.5, 10.6) 17a b 111.9, CH2 5.06, d (17.3) 5.09, d (10.5) 112.0, CH2 5.06, d (17.5) 5.08, d (10.6) 18 27.7, CH3 1.48, s 27.7, CH3 1.48, s 19 27.7, CH3 1.48, s 27.7, CH3 1.48, s 20 33.8, CH2 3.39, d (7.4) --- --- 21 124.1, CH 5.35, t (7.4) --- --- 22 131.7, C --- --- --- 23 17.7, CH3 1.73, s --- --- 24 25.6, CH3 1.73, s --- --- 116 CHAPTER III. RESULTS AND DISCUSSION The rest of the 13C and 1H signals were very similar to those of EC9 (Table 15). Therefore, EC7 corresponded to EC9 with a prenyl group on C-6 (δC 134.4). The structure established for EC7 corresponded to neoechinulin (Figure 59), a prenylated indolopiperazine derivative previously reported from the fungi Aspergillus amstelodami (Marchelli et al., 1977) and Aspergillus ruber (Nagasawa et al., 1975). 1 10 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 3a 7a Figure 59. Structure of neoechinulin (EC7). 3.1.4.5. Echinulin (EC6) EC6 was isolated as white amorphous solid. The 13C NMR spectrum (Table 16) revealed the presence of two amide carbonyls (δC 168.7, 167.9), five methine sp2 (δC 145.7, 124.5, 122.8, 122.8, 115.1), eight quaternary sp2 (δC 141.3, 133.8, 132.9, 132.2, 131.5, 129.0, 123.4, 104.1), one methylene sp2 (δC 112.3), two methine sp3 (δC 54.6, 50.7), one quaternary sp3 (δC 38.9), three methylene sp3 (δC 34.6, 31.3, 29.5) and seven methyl (δC 27.9, 27.8, 25.8, 25.7, 19.8, 17.9, 17.9) carbons. The 1H NMR spectrum (Table 16) displayed signals of one amine proton at δH 8.08, s, two aromatic protons at δH 7.16, s and 6.83, s, two amide protons at δH 6.80, brs and 5.74, brs, five olefinic protons at δH 6.13, dd (J = 17.5, 10.6 Hz), 5.43, t (J = 6.1 Hz), 5.39, m, 5.19, d (J = 17.5 Hz) and 5.18, d (J = 10.6), two methine protons at δH 4.43, d (J = 10.0 Hz) and 4.12, m, six methylene protons at δH 3.67, dd (J = 14.7, 2.9 Hz), 3.56, d (J = 6.1 Hz; 2H), 3.42, d (J = 6.8 Hz; 2H) 117 CHAPTER III. RESULTS AND DISCUSSION and 3.21, dd (J = 14.7, 11.7 Hz) and seven methyl groups at δH 1.89, s, 1.83, s, 1.77, s (6H), 1.57, d (J = 7.3 Hz) and 1.54, s (6H). Table 16. Comparison of the 1H and 13C NMR data (300.13 and 75.47 MHz) of EC6 (CDCl3) and EC8 (DMSO-d6). Position EC6 EC8 δC, type δH, mult. (J in Hz) δC, type δH, mult. (J in Hz) 1 --- 8.08, s --- 10.95, s 2 141.3, C --- 143.8, C --- 3 104.1, C --- 102.8, C --- 3a 129.0, C --- 125.6, C --- 4 115.1, CH 7.16, s 118.5, CH 7.20, d (7.3) 5 133.8, C --- 119.3, CH 7.02, ddd (7.3, 7.3, 1.2) 6 122.8, CH 6.83, s 120.6, CH 7.08, ddd (7.3, 7.3, 1.2) 7 123.4, C --- 111.5, CH 7.43, dd (7.3, 1.2) 7a 132.2, C --- 134.9, C --- 8a b 29.5, CH2 3.21, dd (14.7, 11.7) 3.67, dd (14.7, 2.9) 110.2, CH 6.96, s 9 54.6, CH 4.43, d (10.0) 124.5, C --- 10 168.7, CO --- 159.0, CO --- 11 --- 6.80, brs --- 8.35, d (1.7) 12 50.7, CH 4.12, m 50.6, CH 4.14, dd (6.9, 1.7) 13 167.9, CO --- 166.0, CO --- 14 --- 5.74, brs --- 8.27, brs 15 38.9, C --- ~39.0, C --- 16 145.7, CH 6.13, dd (17.5, 10.6) 144.7, CH 6.08, dd (17.3, 10.4) 17a b 112.3, CH2 5.19, d (17.5) 5.18, d (10.6) 111.4, CH2 5.05, d (17.3) 5.06, d (10.4) 18 27.9, CH3 1.54, s 27.7, CH3 1.51, s 19 27.8, CH3 1.54, s 27.7, CH3 1.51, s 20 34.6, CH2 3.42, d (6.8) 19.8, CH3 1.45, d (6.9) 21 124.5, CH 5.39, m --- --- 22 131.5, C --- --- --- 23 17.9, CH3 1.77, s --- --- 24 25.8, CH3 1.77, s --- --- 25 31.3, CH2 3.56, d (6.1) --- --- 26 122.8, CH 5.43, t (6.1) --- --- 27 132.9, C --- --- --- 28 17.9, CH3 1.89, s --- --- 29 25.7, CH3 1.83, s --- --- 30 19.8, CH3 1.57, d (7.3) --- --- Analysis of the 1H and 13C NMR spectra of EC6 revealed that, except for the substituted benzene ring, another part of the molecule was similar to that of EC8 differing only in the single bond on C-8/C-9 in the former, instead of the double bond in the latter. 220 REFERENCES Bringmann, G., Lang, G., Steffens, S., Günther, E., Schaumann, K. 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Structure determination of six fungal metabolites, tryptoquivaline E, F, G, H, I and J from Aspergillus fumigatus. Chemical & Pharmaceutical Bulletin. 26(1), 111-117. Yamazaki, M., Okuyama, E., Maebayashi, Y. (1979). Isolation of some new tryptoquivaline-related metabolites from Aspergillus fumigatus. Chemical & Pharmaceutical Bulletin 27(7), 1611-1617. 268 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS 269 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.1. Ergosterol (EV1) I.1.1. 1H NMR spectrum (300.13 MHz) I.1.2. 13C NMR spectrum (75.47 MHz) 270 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.2. Ergosterol peroxide (EV2) I.2.1. 1H NMR spectrum (500.13 MHz) I.1.2. 13C NMR spectrum (125.77 MHz) 271 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.3. Orcinol (EV3) I.3.1. 1H NMR spectrum (300.13 MHz) I.3.2. 13C NMR spectrum (75.47 MHz) 272 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.4. 1H-Indole-3-carboxylic acid (EV4) I.4.1. 1H NMR spectrum (500.13 MHz) I.4.2. 13C NMR spectrum (125.77 MHz) 273 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.5. cyclo-(L-Tryptophyl-L-phenylalanyl) (EV5) I.5.1. 1H NMR spectrum (300.13 MHz) I.5.2. 13C NMR spectrum (75.47 MHz) 274 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.6. Erythroglaucin (EC1) I.6.1. 1H NMR spectrum (500.13 MHz) I.6.2. 13C NMR spectrum (125.77 MHz) 275 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.7. Physcion (EC2) I.7.1. 1H NMR spectrum (500.13 MHz) I.7.2. 13C NMR spectrum (125.77 MHz) 276 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.8. Catenarin (EC3) I.8.1. 1H NMR spectrum (300.13 MHz) I.8.2. 13C NMR spectrum (75.47 MHz) 277 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.9. Emodin (EC4) I.9.1. 1H NMR spectrum (500.13 MHz) I.9.2. 13C NMR spectrum (125.77 MHz) 284 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.16. Chevalone B (NL1) I.16.1. 1H NMR spectrum (300.13 MHz) I.16.2. 13C NMR spectrum (75.47 MHz) 285 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.17. Aszonapyrone A (NL2) I.17.1. 1H NMR spectrum (300.13 MHz) I.17.2. 13C NMR spectrum (75.47 MHz) 286 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.18. Aszonapyrone B (NL3) I.18.1. 1H NMR spectrum (300.13 MHz) I.18.2. 13C NMR spectrum (75.47 MHz) 287 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.19. Tryptoquivaline L (NP2/NL4) I.19.1. 1H NMR spectrum (300.13 MHz) I.19.2. 13C NMR spectrum (75.47 MHz) 288 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.20. 3’-(4-Oxoquinazolin-3-yl)spiro[1H-indole-3,5’-oxolane]-2,2’- dione (NP5/NL5) I.20.1. 1H NMR spectrum (300.13 MHz) I.20.2. 13C NMR spectrum (75.47 MHz) 289 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.21. Tryptoquivaline T (NL6) I.21.1. 1H NMR spectrum (300.13 MHz) I.21.2. 13C NMR spectrum (75.47 MHz) 290 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.22. Sartorypyrone C (NP1) I.22.1. 1H NMR spectrum (300.13 MHz) I.22.2. 13C NMR spectrum (75.47 MHz) 291 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.23. Tryptoquivaline H (NP3) I.23.1. 1H NMR spectrum (300.13 MHz) I.23.2. 13C NMR spectrum (75.47 MHz) 292 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.24. 4(3H)-Quinazolinone (NP4) I.24.1. 1H NMR spectrum (500.13 MHz) I.24.2. 13C NMR spectrum (125.77 MHz) 293 APPENDIX I. NMR SPECTRA OF THE ISOLATED COMPOUNDS I.25. Tryptoquivaline F (NP6) I.25.1. 1H NMR spectrum (300.13 MHz) I.25.2. 13C NMR spectrum (75.47 MHz) to yield 53 mg of echinulin (Smetanina et al., 2007) and the mother liquor (247 mg) was combined with frs. 191–201 (201 mg) and purified by TLC (Si Gel, CHCl 3 :Me 2 CO:HCO 2 H, 95:5:1) to give 5 mg of variecolorin J (Wang et al., 2007). Frs. 209–216 were combined (216 mg) and recrystallized in Me 2 CO to give 51 mg of echinulin and the mother liquor (159 mg) was purified by TLC (Si Gel, CHCl 3 :Me 2 CO:HCO 2 H, 95:5:1) to yield 13 mg of neoechinulin A (Li et al., 2008a). Frs. 217–234 were combined (228 mg) and recrystallized in Me 2 CO to give 79 mg of echinulin. Frs. 235–247 were combined (302 mg) and recrystallized in Me 2 CO to give 26 mg of echinulin and the mother liquor (272 mg) was recrystallized in a mixture of CH 2 Cl 2 and petrol to give 17 mg of neoechinulin E (Li et al., 2008a). Frs. 303–321 were combined (338 mg) and recrystallized in MeOH to give 22 mg of eurocristatine (1). The mother liquor of frs 303– 321 was then combined with frs. 277–301 (1.18 g) and applied on a column chromatography of LiChroprep Si60 (0.04– 0.063 mm Merck, 45 g) and eluted with mixtures of CHCl 3 – petrol, CHCl 3 –Me 2 CO, CHCl 3 –MeOH, 100 mL subfrs. were collected as follows: subfrs. 1–20 (CHCl 3 –petrol, 1:4), 21–96 (CHCl 3 –petrol, 3:7), 97–118 (CHCl 3 –petrol, 2:3), 119–157 (CHCl 3 –petrol, 1:1), 158–179 (CHCl 3 –petrol, 3:2), 180–290 (CHCl 3 –petrol, 7:3), 291–325 (CHCl 3 –petrol, 4:1), 326–349 (CHCl 3 –petrol, 9:1), 350–368 (CHCl 3 ), 369–379 (CHCl 3 –Me 2 CO, 9:1), 380–403 (CHCl 3 –Me 2 CO, 7:3), 404–413 (CHCl 3 –Me 2 CO, 1:1), 414–416 (CHCl 3 –MeOH, 1:1). Subfrs. 230–326 were combined (263 mg) and recrystallized in CHCl 3 to give more 11 mg of eurocristatine (1). 3.3. Eurocristatine (1) White crystals; mp 244–245 8C (CHCl 3 /MeOH); [ a ] 20D = +300 (c 0.02, MeOH); UV (MeOH) l max (log e ) 212 (4.5), 241 (4.2), 302 (3.9) nm; IR (KBr) y max 3450, 3190, 2961, 1662, 1453, 1326 cm 1 ; 1 H and 13 C NMR see Table 1; HRESIMS m/z 569.2905 [M+H] + (calculated for C 32 H 37 N 6 O 4 , 569.2876). 3.4. X-ray crystal structure of eurocristatine (1) Crystals suitable for X-ray diffraction were obtained by slow evaporation of a solution in CHCl 3 /MeOH for eurocristatine (1). Diffraction data were collected at 100 K with a Gemini PX Ultra equipped with Cu K a radiation ( l = 1.54184 A ˚). The structure was solved by direct methods using SHELXS-97 and refined with SHELXL-97. Crystals were monoclinic, space group P2 1 , cell volume 2308.37(5) A ˚ 3 and unit cell dimensions a = 10.3549(2) A ˚, b = 9.9862(2) A ˚and c = 22.7778(3) A ˚and b = 101.464(2)8 (uncertainties in parenthesis). Carbon, oxygen and nitrogen atoms were refined anisotropically. Hydrogen atoms were refined freely with isotropic displacement parameters. The flack x parameter was refined with SHELXL-97 by means of TWIN and BASF to yield 0.031(18). Tables containing the final fractional coordinates, temperature parameters, bond distances, and bond angles were deposited with the Cambridge Crystallographic Data Centre: CCDC reference number 879091. Acknowledgements This work was partially supported by the project PEst-C/MAR/ LA0015/2011 funded by Fundac¸a ˜o para a Cie ˆncia e a Tecnologia (FCT) and the European Regional Development Fund (ERDF) through the COMPETE – Operational Competitiveness Programme as part of the National Strategic Reference Framework. NMG thanks Fundac¸a ˜o para a Cie ˆncia e Tecnologia (FCT) for the scholarship (SFRH/BD/65671/2009) supported by the European Social Fund (ESF). We thank Mrs. Ju ´lia Bessa, Ms. Sara Cravo for technical support and Dr. Mick Lee of the Department of Chemistry, Leicester University, UK for HRMS. 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Bioactive meroditerpenes and indole alkaloids from the soil fungus Neosartorya fischeri (KUFC 6344), and the marine-derived fungi Neosartorya laciniosa (KUFC 7896) and Neosartorya tsunodae (KUFC 9213). Tetrahedron 69(40), 8583-8591. 302 APPENDIX III. EAMVIJARN ET AL., 2013 Bioactive meroditerpenes and indole alkaloids from the soil fungus Neosartorya fischeri (KUFC 6344), and the marine-derived fungi Neosartorya laciniosa (KUFC 7896) and Neosartorya tsunodae (KUFC 9213) Amnat Eamvijarn a , b , Nelson M. Gomes a , Tida Dethoup a , b , Jamrearn Buaruang c , Leka Manoch b , Artur Silva d , Madalena Pedro e , f , Ioulia Marini g , Vasilios Roussis g , Anake Kijjoa a , * a ICBAS eInstituto de Ci^ encias Biom edicas de Abel Salazar and CIIMAR, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal b Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand c Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand d Departamento de Química, Universidade de Aveiro, 4810-1933 Aveiro, Portugal e CEQUIMED eCentro de Química Medicinal da Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal f Grupo de Biologia Molecular e Celular (GBMC), Centro de Investigac¸ ~ ao em Ci^ encias da Sa ude (CICS), Instituto Superior de Ci^ encias da Sa ude do Norte, CESPU, Rua Central de Gandra 1317, 4585-116 Gandra, Portugal g Department of Pharmacognosy and Chemistry of Natural Products, School of Pharmacy, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece article info Article history: Received 8 June 2013 Received in revised form 18 July 2013 Accepted 22 July 2013 Available online 31 July 2013 Keywords: Neosartorya fischeri (KUFC 6344) Neosartorya laciniosa (KUFC 7896) Neosartorya tsunodae (KUFC 9213) Meroditerpenes Sartorypyrones Aszonalenin abstract Two new metabolites including a new aszonalenin analogue (1c) and a new meroditerpene (3) were isolated, together with aszonalenin (1a), acetylaszonalenin (1b), 13-oxofumitremorgin B (2), aszonapyrone A (4b) and helvolic acid, from the culture of the soil fungus Neosartorya fischeri (KUFC 6344). While the ethyl acetate extract of the culture of the diseased coral-derived fungus Neosartorya laciniosa (KUFC 7896) furnished aszonapyrone B (4a), aszonapyrone A (4b), tryptoquivaline L and 3 0 -(4oxoquinazolin-3-yl) spiro[1H-indole-3,5 0 -oxolane]-2,2 0 -dione, the ethyl acetate extract of the culture of the marine sponge-associated fungus Neosartorya tsunodae (KUFC 9213) yielded a new analogue of chevalone C (5) and helvolic acid. The structures of the new compounds were established based on 1D and 2D NMR spectral analysis as well as HR-ESIMS. Compounds 1aec,2,3,4a,4b and 5were evaluated for their in vitro growth inhibitory activity on the MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines by the protein binding dye SRB method. Ó2013 Elsevier Ltd. All rights reserved. 1. Introduction Neosartorya species (Trichocomaceae) are sexual forms of the Aspergillus species, notably the section Fumigati. While the Aspergillus species only produce conidospores, Neosartorya species produce both a sexual state with ascospores and an asexual state with conidiospores. 1 Unlike Aspergillus,Neosartorya species have not been extensively investigated for their secondary metabolites. Consequently, we started to investigate the secondary metabolites of the fungi of this genus. We have previously reported isolation and structure elucidation of three new reverse prenylated indole derivatives sartoryglabrins AeC from the culture of a Thai collection of Neosartorya glabra. 2 We later reported isolation of cadenene sesquiterpene, the indole derivative eurochevalierine, brasiliaminde B, pyripyropene A and three new metabolites including 1,4diacetyl-2,5-dibenzylpiperazine 3,7 00 -oxide, a quinazolinonecontaining indole derivative and a phenyl ester of 2,4-dihydroxy6-methylbenzoic acid from the culture of the soil fungus Neosartorya pseudofisheri, as well as the in vitro cytostatic activity of the cadenene sesquiterpene and eurochevalierine in human cancer cancer cells. 3 Recently, we reported isolation of a new indole alkaloid sartorymensin, and new analogues of tryptoquivaline and fiscalins, produced by a Thai collection of the soil fungus Neosartorya siamensis (KUFC 6349), as well as their in vitro growth *Corresponding author. Tel.: þ351 220428331; fax: þ351 222062232; e-mail addresses: anki[email protected],[email protected] (A. Kijjoa). Contents lists available at SciVerse ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet 0040-4020/$ esee front matter Ó2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.07.078 Tetrahedron 69 (2013) 8583e8591 inhibitory activity on the human U373 and Hs683 glioblastoma, the A549 non-small cell lung cancer, the MCF-7 breast cancer and the SKMEL-28 melanoma cell lines by MTT colorimetric assay. 4 In our ongoing search for bioactive compounds produced by soil and marine-derived fungi of the genus Neosartorya, we have investigated the cultures of the soil fungus Neosartorya fischeri and the marine-derived fungi Neosartorya tsunodae and Neosartorya laciniosa. Although N. fischeri has been extensively investigated for its secondary metabolites, 5e9 to the best of our knowledge, there is no report on the secondary metabolites produced by N. tsunodae and N. laciniosa. Examination of a collection of N. fischeri (KUFC 6344), isolated from the coastal forest soil in Thailand, resulted in isolation of a new aszonalenin analogue (1c) and a new meroditerpene sartorypyrone A (3), in addition to five known metabolites including aszonalenin (1a), 10 acetylaszonalenin (1b), 10 13oxofumitremorgin B (2), 11 aszonapyrone A (4b) 12 (Fig. 1) and helvolic acid, 13 from the ethyl acetate extract of its culture. In order to investigate the secondary metabolites profile of the marine-derived fungi of this genus, we also examined Thai collections of N. laciniosa (KUFC 7896), isolated from a diseased coral (Porites lutea) from the Gulf of Thailand, and N. tsunodae (KUFC 9213), isolated from the marine sponge Aka coralliphaga, collected from the coral reef of the Similan islands, Phagna province, southern Thailand. The ethyl acetate extract of the culture of N. laciniosa (KUFC 7896) furnished aszonapyrone B (4a), 14 aszonapyrone A (4b) 12 (Fig. 1), tryptoquivaline L 4 and 3 0 -(4-oxoquinazolin-3-yl) spiro[1H-indole-3,5 0 -oxolane]-2,2 0 -dione, 4 while the ethyl acetate extract of the culture of N. tsunodae (KUFC 9213) yielded, besides helvolic acid, 13 sartorypyrone B (5), a new analogue of chevalone C (Fig. 1). Additionally, we also evaluated the in vitro growth inhibitory activity of compounds 1aec,2,3,4a,band 5(Fig. 1) on MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer) and A375-C5 (melanoma) cell lines by the protein binding dye SRB method. 2. Results and discussion Compound 1c was isolated as pale yellow semisolid, and its molecular formula C 24 H 23 N 3 O 4 was established on the basis of the (þ)-HR-ESIMS m/z418.1767 [MþH] þ , indicating fifteen degrees of unsaturation. The general features of the 1 H and 13 C NMR spectra of 1c closely resembled those of aszonalenin (1a) and acetylaszonalenin (1b). The IR spectrum showed absorption bands for hydroxyl (3377 cm 1 ), amine (3242 cm 1 ), aromatic (3089, 1485 cm 1 ), amide carbonyl (1680,1657 cm 1 ) and olefin (1605 cm 1 g) groups. The 13 C NMR, DEPT and HSQC spectra (Table 1) revealed two amide carbonyls ( d C 169.1 and 166.9), one N-formyl ( d C 162.4), five quaternary sp 2 ( d C 153.1,136.1.133.6,133.1,127.0), eight methine sp 2 ( d C 142.6, 132.7, 131.3, 125.7, 120.5, 118.4, 115.5, 112.1), one methylene sp 2 ( d C 115.0), two quaternary sp 3 ( d C 59.4, 41.2), two methine sp 3 ( d C 80.9, 57.1), one methylene sp 3 ( d C 32.7) and two methyl ( d C 22.8, 22.4) carbons. The coupling system of the aromatic protons observed in the COSY spectrum (Table 1) revealed the presence of one 1,2-disubstituted and one 1,2,4-trisubstituted benzene rings. Analysis of the HMBC spectrum (Table 1) indicated that the 1,2,41 1a: R1 = H, R2 = H b: R1 = H, R2 = OAc c: R1 = OH, R2 = CHO 2 3 4a: R = H b: R = Ac 17 15 23 16 24 18 21 1 22 19 20 6 7 26 3 4 5 25 12 13 14 9 10 11 2 15 16 17 13 14 26 18 13 5 8 19 20 21 12 10 9 6 3 4 5 7 22 25 11 8 24 23 2 19 20 21 14 15 18 3' 5' 1 2 2' 1' 4' 13 8 4 5 2 3 9 6 12 17 16 7 10 11 14 6 10 11 3 4 5 12 1 13 27 7 8 9 26 17 18 19 20 16 15 21 25 28 29 22 23 24 14 26 25 11 12 15 19 20 21 16 17 18 4 5 10 1 2 3 9 24 23 22 6 7 8 Fig. 1. Secondary metabolites of Neosartorya fischeri (KUFC 6344), N. laciniosa (KUFC 7896) and N. tsunodae (KUFC 9213). A. Eamvijarn et al. / Tetrahedron 69 (2013) 8583e85918584 trisubstituted benzene ring was part of a 2,3-dihydro-1H-indole moiety while the 1,2-disubstituted benzene ring was part of a 3,4dihydro-1H-1,4-benzodiazepine-2,5-dione portion. That the 2,3dihydro-1H-indole and the 3,4-dihydro-1H-1,4-benzodiazepine2,5-dione portions were linked together through a pyrrolidine ring was corroborated by the HMBC cross peaks of H-2 ( d H 5.99, s) to C11 ( d C 57.1); H-10 a ( d H 3.38, dd, J¼13.9, 8.6 Hz) to C-3 ( d C 59.4), C-9 ( d C 136.1), C-17 ( d C 169.1), as well as of N16-H ( d H 8.27, s) to C-11 and C-14 ( d C 127.0) (Fig. 2a). Additionally, the coupling system observed in the COSY spectrum (Table 1) also revealed the presence of a 2methylbut-3-en-2-yl substituent, which was corroborated by the HMBC cross peaks (Table 1)ofH 3 -5 0 ( d H 1.15,s)toC-2 0 ( d C 142.6), C3 0 ( d C 41.2), CH 3 -4 0 ( d C 22.8); H-4 0 ( d H 1.02, s) to C-2 0 , C-3 0 ,CH 3 -5 0 ( d C 22.4); H-2 0 ( d H 5.90, dd, J¼17.3, 10.8 Hz) to CH 3 -5 0 ; H-1 0 a and H-1 0 b ( d H 5.11, d, J¼17.3 Hz and 5.15, d, J¼10.8 Hz) to C-2 0 and C-3 0 . That the 2-methylbut-3-en-2-yl substituent was on C-3 was substantiated by the HMBC cross peaks of H-2 to C-3 0 ; H-10 a to C-3 0 as well as of H 3 -4 0 and H 3 -5 0 to C-3 (Fig. 2a). These 1 H and 13 C NMR data were very similar to those reported for aszonalenin, 5 and since the HMBC spectrum (Fig. 2a) also displayed cross peak between the proton signal at d H 8.95, s, to the signal of C-8 ( d C 133.6), the formyl group was placed on N-1. In order to verify the relative configuration of C-2, C-3 and C-11, the NOESY experiment was carried out. The NOESY spectrum (Fig. 2b) displayed correlations of H-2 to H 3 -4 0 ,H 3 -5 0 , H-10 a ; and of H-11 ( d H 4.04, t, J¼8.4 Hz) to H-10 b ( d H 2.43, dd, J¼13.9, 8.2 Hz); however no correlation between H-2 and H-11 was observed. Consequently, the relationship between H-2 and H-11 was trans whereas the relationship between H-2 and the 2-methylbut-3-en2-yl substituent on C-3 was cis. Thus, the relative configuration of C-2, C-3 and C-11 of 1c was the same as that of the corresponding carbons of aszonalenin. 5 Taking together the 1 H and 13 C NMR data, the HMBC and NOESY correlations and the molecular formula, the structure of 1c was established as 1-formyl-5-hydroxyaszonalenin. Although several analogues of aszonalenin have been previously isolated, 15 to our knowledge, this is the first report of isolation of the 5-hydroxyl analogue of aszonalenin. Compound 3was isolated as brown viscous mass and its molecular formula C 28 H 40 O 5 was established on the basis of the (þ)-HRESIMS m/z457.2954 [MþH] þ , indicating nine degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3434 cm 1 ), olefinic (3080, 1585 cm 1 ) and carbonyl (1730, 1684 cm 1 ) groups. The 13 C NMR, DEPT and HSQC spectra (Table 2) revealed the presence ofone estercarbonyl ( d C 171.1),oneconjugated ester carbonyl ( d C 166.8), six quaternary sp 2 ( d C 166.1, 159.9, 146.6, 138.2, 135.4, 101.7), three methine sp 2 ( d C 124.1, 120.5, 101.1), one methylene sp 2 ( d C 109.2), one quaternary sp 3 ( d C 39.1), two methine sp 3 ( d C 78.9, 51.0), seven methylene sp 3 ( d C 39.7, 38.2, 31.7, 28.6, 26.3, 23.6, 22.4) and six methyl ( d C 26.0, 21.3,19.6,17.5,16.3,15.9) carbons. The existence of a 4-hydroxy-6-methyl-2H-pyran-2-one moiety was supported not only by the cross peak between H-19 ( d H 6.00, d, J¼0.7 Hz) and H 3 -21 ( d H 2.19, s) in the COSY spectrum but also by HMBC cross peaks of H-19 to C-17 ( d C 101.7), C-18 ( d C 166.1), C-20 ( d C 159.9), C-21 ( d C 19.6) and H 3 -21 to C-19 ( d C 101.1) and C-20 (Fig. 3a). That another portion of the molecule 1-acetoxy-2,2-dimethyl-4methylidenecyclohexyl derivative was evidenced by the correlations of H-1 ( d H 4.66, dd, J¼8.9, 4.1) to H-6 b ( d H 1.55, m) and H-6 a ( d H 1.85, m) in the COSY spectrum, as well as by HMBC crosspeaks of H-1 to C-2 ( d C 39.1), C-3 ( d C 51.0), CH 3 -26 ( d C 17.5) and CO ( d C 170.1); H-7a ( d H 4.88, br s) and H-7b ( d H 4.62, br s) to C-3 and C-5 ( d C 31.7); H-5 b ( d H 2.31, dt, J¼13.5, 5.6 Hz) to C-1 ( d C 78.9), C-3, C-4 ( d C 146.6) and C-7 ( d C 109.2); H 3 -25 ( d H 0.94, s), and H 3 -26 ( d H 0.79, s) to C-1, C-2, C-3 (Fig. 3a). Since H-1 appeared as a double doublet with the coupling constants of 8.9 and 4.1 Hz, the position of the acetoxyl group on C-1 was b . Moreover, the COSY spectrum also exhibited correlations of H11 ( d H 5.05, dd, J¼5.6, 6.5 Hz) to H 3 -23 ( d H 1.58, s) and H-12 ( d H 2.08, m), as well as of H-15 ( d H 5.29,ddd, J¼7.2, 7.2,1.0 Hz) to H 3 -24 ( d H 1.77, Table 1 1 H and 13 C NMR (CDCl 3 , 300.13 and 75.47 MHz) and HMBC assignments for 1c Position d C , type d H (Jin Hz) COSY HMBC 2 80.9, CH 5.99, s C-3 0 , 8, 9, 11, NCHO 3 59.4, C d 4 112.1, CH 6.79, d (2.5) H-6 C-5, 6, 8 5 153.1, C d 6 115.5, CH 6.74, dd (8.5, 2.5) H-4, 7 C-4, 8 7 118.4, CH 7.76, d, (8.5) H-6 C-5, 9 8 133.6, C d 9 136.1, C d 10 a 32.7, CH 2 3.38, dd (13.9, 8.6) H-10 b , 11 C-3, 3 0 ,9,17 b 2.43, dd (13.9, 8.2) H-10 a , 11 C-2, 3 0 ,9 11 57.1, CH 4.04, t (8.4) H-10 a ,10 b C-10 13 166.9, CO d 14 127.0, C d 15 133.1, C d 16 d8.27, s C-11, 14 17 169.1, CO d 18 120.5, CH 6.92, d (7.7) H-19 C-14, 20 19 132.7, CH 7.43, ddd (7.7, 7.7, 1.5) H-18, 20 C-15, 21 20 125.7, CH 7.21, dd (7.7, 7.7) H-19, 21 C-14, 18 21 131.3, CH 7.75, d (7.7) H-20 C-13, 19 1 0 a 115.0, CH2 5.11, d (17.3) H-2 0 C-2 0 ,3 0 b 5.15, d (10.8) H-2 0 2 0 142.6, CH 5.90, dd (17.3, 10.8) H-1 0 a, 1 0 b C-5 0 3 0 41.2, C d 4 0 22.8, CH 3 1.02, s C-2 0 ,3,3 0 ,5 0 5 0 22.4, CH 3 1.15, s C-2 0 ,3,3 0 ,4 0 NCHO 162.4, CO 8.95, s C-8 (a) (b) 1c 3' 2' 21 19 20 18 17 4' 1' 5' 10 12 4 6 5 15 14 16 11 13 1c 1 8 7 9 2 3 3' 2' 21 19 20 18 17 4' 1' 5' 10 12 4 6 5 15 14 16 11 13 1 8 7 9 2 3 Fig. 2. Key HMBC (a) and NOESY (b) correlations of compound 1c. A. Eamvijarn et al. / Tetrahedron 69 (2013) 8583e8591 8585 s) and H-16 ( d H 3.20, d, J¼7.2 Hz), suggesting the presence of two trisubstituted double bonds in the molecule. These coupling systems were corroborated by HMBC cross peaks of H-11 signal to the signals of C-9 ( d C 38.2) and C-23 ( d C 15.9); and H 3 -23 to C-9, C-10 ( d C 135.4) and C-11 ( d C 124.1), as well as cross peaks of H-15 signal to the signals of C-13 ( d C 39.7) and C-24 ( d C 16.3). Additionally, the HMBC spectrum also exhibited a cross peak between the signals of H-12 and C-14 ( d C 138.2), suggesting that the two trisubstituted double bonds were connected through C-12 and C-13, thus another partial structure was a 3,7-dimethylnona-2,6-diene. That the 3,7-dimethylnona-2,6diene portion and the 1-acetoxy-2,2-dimethyl-4-methylidenecyclohexyl derivative were connected through C-8 of the former and C-3 of the latter was substantiated by cross peaks observed between the signals of H-3 ( d H 1.72, dd, J¼9.1, 3.1) and H-8 ( d H 1.59, m) in the COSY spectrum. Moreover, the HMBC cross peaks of H-16 signal to the signals of C-17, C-18 and C-22 ( d C 166.8) provided evidence that another substituent of the 3,7-dimethylnona-2,6-diene portion was the 4-hydroxy-6-methyl-2H-pyran-2-one moiety, connected through C-16 of the former and C-17 of the latter. In order to determine the relative configuration of the stereogenic carbons and the configuration of the double bonds at C-10 and C-14, the NOESY experiment was carried out. As the NOESY spectrum (Fig. 3b) exhibited cross peaks of H-3 signal to the signals of H-1 and H 3 -25, the relationship between H-1 and H-3 was cis. Consequently, the relative configuration of C-1 and C-3 was 1S*, 3S*. Furthermore, since the NOESY spectrum also exhibited cross peaks of H-11 to H-12 and H-9 ( d H 1.80, m), as well as of H-15 to H-13 ( d H 2.04, m) and H-16 (Fig. 3b), the configuration of the double bonds at C-10 and C-14 was 10E,14E. Thus, compound 3was a new monocyclic meroditerpene, which we have named sartorypyrone A. Compound 5was isolated as yellow viscous mass and its molecular formula C 30 H 42 O 7 was established on the basis of the (þ)-HRESIMS m/z515.3038 [MþH] þ (calculated 515.3009), indicating ten degrees of unsaturation. The IR spectrum showed absorption bands for ester (1734 cm 1 ), conjugated ketone (1670 cm 1 ) and olefinic (1631, 1596 cm 1 ) groups. The 13 C NMR, DEPT and HSQC spectra (Table 3) revealed the presence of one carbonyl of a conjugated ketone ( d C 180.8), two ester carbonyls ( d C 170.7 and 170.2), three quaternary sp 2 ( d C 162.7, 160.7, 98.5), one methine sp 2 ( d C 111.8), four quaternary sp 3 ( d C 84.2, 37.4, 37.3, 36.9), five methine sp 3 ( d C 77.7, Table 2 1 H and 13 C NMR (CDCl 3 , 300.13 and 75.47 MHz) and HMBC assignments for 3 Position d C , type d H (Jin Hz) COSY HBMC 1 78.9, CH 4.66, dd (8.9, 4.1) H-6 a ,6 b C-2, 3, 5, 26, CO (Ac) 2 39.1, C d 3 51.0, CH 1.72, dd (9.1, 3.1) H-8 C-2, 4, 8 4 146.6, C d 5 a 31.7, CH 2 2.03, m H-5 b ,6 a ,6 b b 2.31, dt, (13.5, 5.6) H-5 a ,6 a ,6 b C-1, 3, 4, 7 6 a 28.6, CH 2 1.85, m H-5 a ,5 b ,6 b b 1.55, m H-5 a ,5 b ,6 a 7a 109.2, CH 2 4.88, br s H-7b C-3, 5 b 4.62, br s H-7a C-3, 5 8 23.6, CH 2 1.59, m H-3, 9 9 38.2, CH 2 1.80, m H-8 10 135.4, C d 11 124.1, CH 5.05, dd (5.6, 6.5) H-12, 23 C-9, 23 12 26.3, CH 2 2.08, m H-11, 13 13 39.7, CH 2 2.04, m H-12 14 138.2, C d 15 120.5, CH 5.29, ddd (7.2, 7.2, 1.0) H-16, 24 C-13, 24 16 22.4, CH 2 3.20, d (7.2) H-15 C-14, 15, 17, 18, 22 17 101.7, C d 18 166.1, C d 19 101.1, CH 6.00, d (0.7) H-21 C-17, 18, 20, 21 20 159.9, C d 21 19.6, CH 3 2.19, s H-19 C-19, 20 22 166.8, CO d 23 15.9, CH 3 1.58, s C-9, 10, 11 24 16.3, CH 3 1.77, s C-13, 14, 15 25 26.0, CH 3 0.94, s C-1, 2, 3, 26 26 17.5, CH 3 0.79, s C-1, 2, 3, 25 OAc 171.1, CO d 21.3, CH 3 2.07, s CO (OAc) (b) (a) 1 1 3 13 14 15 12 10 11 22 19 18 16 17 5 6 4 2 3 26 25 9 7 8 24 23 20 21 3 13 14 15 12 10 11 22 19 18 16 17 5 6 4 2 3 26 25 9 7 8 24 23 20 21 Fig. 3. Key HMBC (a) and NOESY (b) correlations of compound 3. Table 3 1 H and 13 C NMR (CDCl 3 , 300.13 and 75.47 MHz) and HMBC assignments for 5 Position d C , type d H (Jin Hz) COSY HMBC 1 a 41.8, CH 2 1.36, dd (14.8, 3.8) H-2 b 2.08, dd (14.8, 3.8) H-2 2 69.4, CH 5.36, ddd (4.0, 3.8, 3.8) H-1, 3 C-3, 4, CO (Ac-2) 3 77.7, CH 4.61, d (4.0) H-2 C-2, 4, 22, 23, CO (Ac-3) 4 37.4, C d 5 55.3, CH 1.04, m H-6 6 17.6, CH 2 1.59, m H-5, 7 a , b 7 a 40.8, CH 2 1.92, ddd (12.8, 3.0, 3.0) H-6, 7 b b 1.12, m H-6, 7 a 8 37.3, C d 9 60.8, CH 0.95, m H-11 10 36.9, C d 11 18.9, CH 2 1.39, m H-9, 12 1.42, m 12 40.0, CH 2 1.68, m H-11 2.03, m 13 84.2, C d 14 52.4, CH 1.51, dd (12.7, 4.9) H-15 a , b C-8, 13 15 a 15.3, CH 2 2.55, dd (16.4, 4.9) H-14, 15 b C-13, 14, 16, 17, 21 b 2.15, dd (16.4, 12.7) H-14, 15 a 16 98.5, C d 17 180.8, CO d 18 111.8, CH 6.05, br s H-20 C-16, 19, 20 19 160.7, C d 20 19.3, CH 3 2.22, s H-18 C-18, 19 21 162.7, C d 22 29.0, CH 3 0.90, s C-3, 4, 5, 23 23 17.4, CH 3 1.05, s C-3, 4, 5, 22 24 17.0, CH 3 1.12, s C-1, 5, 9, 10 25 16.3, CH 3 0.92, s C-7, 8, 9, 14 26 20.5, CH 3 1.29, s C-12, 13, 14, Ac-2 Ac-2 170.2, CO d 21.3, CH 3 2.06, s CO (Ac-2) Ac-3 170.7, CO d 20.9, CH 3 2.03, s CO (Ac-3) A. Eamvijarn et al. / Tetrahedron 69 (2013) 8583e85918586 69.4, 60.8, 55.3, 52.4), six methylene sp 3 ( d C 41.8, 40.8, 40.0,18.9,17.6, 15.3) and eight methyl ( d C 29.0, 21.3, 20.9, 20.5, 19.3, 17.4, 17.0, 16.3) groups. Except for the presence of an additional acetoxyl group, the 1 H and 13 C NMR data (Table 3), revealed the existence of a perhydrophenanthrene moiety connected to the 2-methyl-4H-pyran-4one portion through the methylene group and the ethereal bridge, similar to those of chevalone C, a meroditerpenoid isolated from Eurotium chevalieri. 14 The COSY spectrum (Table 3) revealed the coupling of the oxymethine proton ( d H 5.36, ddd, J¼4.0, 3.8, 3.8, H-2) to another oxymethine proton ( d H 4.61, d, J¼4.0, H-3) and to the methylene protons at d H 1.36, dd, J¼14.8, 3.8 and d H 2.08, dd, J¼14.8, 3.8 ( d C 41.8). That the acetoxyl groups were on C-2 ( d C 69.4) and C-3 ( d C 77.7) were corroborated by the HMBC cross peaks of H-2 signal and the methyl protons signal at d H 2.06, s ( d C 21.3) to the carbonyl signalat d C 170.2, as well as of the HMBC cross peaks of H-3 signal and the signal of the methyl protons at d H 2.03, s ( d C 20.9) to the carbonyl signal at d C 170.7, respectively (Fig. 4a). The HMBC spectrum also exhibited cross peaks of H-3 signal to the signals of CH 3 -23 ( d C 17.4) and CH 3 -22 ( d C 29.0), C-4 ( d C 37.4) and C-2, as well as cross peaks of H-2 to the signals of C-3 and C-4 (Fig. 4a). Since H-3 and H-2, respectively, appeared as a doublet at d H 4.61 (J¼4.0 Hz) and a double of double doublet at d H 50.36(J¼4.0, 3.8, 3.8), the positions of the acetoxyl groups on C-2 and C-3 were both b oriented. 16 The positions of the methyl groups on C-8, C-10 and C-13 were determined by a NOESY experiment. The NOESY spectrum (Fig. 4b) exhibited cross peaks of the methyl protons signal of the b -acetoxyl group on C-2 ( d H 2.06, s) to the signals of CH 3 -23 ( d H 1.05, s) and CH 3 -24 ( d H 1.12, s), implying that CH 3 -24 was b oriented. That CH 3 - 25 and CH 3 -26 were both b oriented was evidenced by the cross peaks between the signals of CH 3 -24 and CH 3 -25 ( d H 0.92, s), as well as between the signals of CH 3 -25 and CH 3 -26 ( d H 1.29, s). The NOESY spectrum also displayed cross peaks of the signal of H-15 a ( d H 2.55, dd, J¼16.4, 4.9) to the signals of H-14 ( d H 1.51, dd, J¼12.7, 4.9) and H7 a ( d H 1.92, ddd, J¼12.8, 3.0, 3.0), suggesting that the latters were a oriented. In turn, the signal of H-7 a also exhibited cross peaks to H-5 signal ( d H 1.04, m), implying that it was also a oriented. Since H3 signal was found to exhibit cross peak with the proton signal at d H 1.36, dd, J¼14.8, 3.8, the latter was assigned for H-1 a . Thus, compound 5is a 2 b -acetoxyl analogue of chevalone C, and since it is a new compound, we have named it sartorypyrone B. To our knowledge, this is the first report of isolation of meroditerpenes from N. fischeri, even though xanthone derivatives including ergochrome and indole alkaloids, such as aszonalenin derivatives, tryptoquivalines, fiscalins and fumitremorgins, have been previously reported from this fungus. 5e9 The biosynthetic pathways leading to the formation of sartorypyrone A (3), aszonapyrone B (4a), aszonapyrone A (4b)andsartorypyrone B (5) can be hypothesized to originate from the reaction of the triketide derivative (I) with GPP to form the meroditerpene intermediate (II). Enolization of II leads to the formation of the intermediate III, which can undergo different modes of cyclization. Cyclization via pathway awould yield the monocyclic diterpene intermediate (IV), which upon acetylation, produces sartorypyrone A (3), while cyclization via pathway bwould lead to the formation of aszonapyrone B (4a); and after acetylation of the b -hydroxyl group on C-3, yields aszonapyrone A (4b). On the other hand, formation of the pyran ring (pathway c) would lead to the hexacyclic derivative (VI), which gives chevalone C upon acetylation of the b -hydroxyl group on C-3. Hydroxylation of C-2 of chevalone C, followed by acetylation of this b -hydroxyl group would finally form sartorypyrone B (5). Isolation of sartorypyrone A (3), together with aszonapyrone B (4a), aszonapyrone A (4b) and sartorypyrone B (5) provides evidence that extension of the diterpene unit by the triketide moiety occurs prior to cyclization of GPP, and that different modes of cyclization of GPP can operate to produce these meroditerpenoids (Fig. 5). Aszonalenin derivatives (1aec), 13-oxofumitremorgin B (2), sartorypyrone A (3), aszonapyrone B (4a), aszonapyrone A (4b)and sartorypyrone B (5) were evaluated for their capacity to inhibit the in vitro growth of MCF-7 (breast adenocarcinoma), NCI-H460 (nonsmall cell lung cancer) and A375-C5 (melanoma) cell lines, using the protein binding dye SRB method. Results (Table 4) showed that, amongthe meroditerpenes tested, aszonapyrone A (4b) was the most active,showingstronggrowthinhibitoryactivityagainst thethreecell lines, with GI 50 ¼13.60.9 m M,11.61.5 m Mand10.21.2 m M, for MCF7, NCI-H460 and A375-C5, respectively, while aszonapyrone B (4a), whose structure corresponds to 3-deacetyl aszonapyrone A (4b), was inactiveatthehighestconcentrationtested(150 m M).SartorypyroneB (5) also exhibited strong growth inhibitory activity, although less active than aszonapyrone A (4b), having GI 50 ¼17.87.4 m M, 20.52.4 m M and 25.04.4, respectively, for MCF-7, NCI-H460 A375C5. Interestingly, sartorypyrone A (3), which possesses a monocyclic diterpene core, was more selective, exhibiting similar inhibitory activity to sartorypyrone B (5) against A375-C5(GI 50 ¼21.51.9 m M), but less active against MCF-7 (GI 50 ¼46.37.6 m M) and NCI-H460 (GI 50 ¼37.34.0 m M) cell lines. On the contrary, all the three aszonaleninderivatives(1aec) were foundtobeinactiveagainstallthethree cell lines at the highest concentration tested (150 m M), whereas 13oxofumitremorgin B (2) exhibited only weak inhibitory activity against all the three cell lines (GI 50 ¼115.020.0 m M, 123.311.5 m M and 68.612.9 m M, respectively, for MCF-7, NCI-H460 and A375-C5). 3. Experimental section 3.1. General experimental procedures Melting points were determined on a Bock monoscope and are uncorrected. Optical rotations were determined on an ADP410 Polarimeter. Infrared spectra were recorded on an ATT Mattson (b) (a) 3 2 1 6 7 10 4 5 14 19 13 18 20 5 26 24 25 12 23 11 8 916 17 21 22 15 3 2 1 6 7 10 4 5 14 19 13 18 20 5 26 24 25 12 23 11 8 916 17 21 22 15 Fig. 4. Key HMBC (a) and NOESY (b) correlations of compound 5. A. Eamvijarn et al. / Tetrahedron 69 (2013) 8583e8591 8587 Genesis Series FTIRÔusing WinFIRST Software. 1 H and 13 C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument operating at 300.13 and 75.4 MHz, respectively. High resolution mass spectra were measured with a Waters Xevo QToF mass spectrometer coupled to a Waters Aquity UPLC system. A Merck silica gel GF 254 was used for preparative TLC, and a Merck Si gel 60 (0.2e0.5 mm) was used for analytical chromatography. 3.2. Fungal material The strain KUFC 6344 was isolated from coastal forest soil at Samaersarn island (altitude 12  34 0 23 00 N,100  57 0 23 00 E), Chonburi Province, Thailand, in November 2008. Briefly, 1 g of the soil (collected from the surface) was placed in 65% ethanol for 10e20 min, after which the liquid was drained off. The soil particles were placed into sterile Petri dishes, mixed with GAN and streptomycin sulfate and incubated at 28  C. The fungus was identified as N. fischeri by the morphological features, including characteristic of ascospores, conidiogenesis and colonies. The identification was supported by sequence analysis of the b -tubulin, calmodulin and actin genes and homologies of those gene sequences between the strain and N. fischeri NRRL 181 T were 100%, 100% and 99.7% (GeneBank accession No.; EF669796, EF669865 and DQ094863), respectively. The pure cultures were deposited as KUFC 6344 at the 2 A cetylation c VI I _ H + a V III b H 2 O 1) C-2 hydroxyation 2) OH-2 acetylation chevalone C 5 II IV b a 3 4a A cetylation 4b GPP A cetylation Fig. 5. Proposed biogenesis of meroditerpenes sartorypyrone A (3), aszonapyrone B (4a), aszonapyrone A (4b) and sartorypyrone B (5). A. Eamvijarn et al. / Tetrahedron 69 (2013) 8583e85918588 Mycology Laboratory, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand, and as IFM 59696 at the Medical Mycology Research Center, Chiba University, Japan. The strain KUFC 7896 was isolated from a diseased coral (P. lutea ulcerative white spot) at Ao Nuan Lan island (altitude 12  53 0 56.85 00 N, 100  46 0 39.66 00 E), Chonburi Province, in the Gulf of Thailand, in May 2010. The diseased coral was washed with 0.06% sodium hypochlorite for 1 min, followed by sterilized sea water for three times. The diseased coral was cut into 0.50.5 cm pieces and placed on malt extract agar (MEA) with 70% sea water and incubated at 28  C for 5e7 days. The fungus was identified as N. laciniosa by morphological characteristics, such as colony growth rate and growth pattern on standard media namely Czapek’s agar (CZA), Czapek yeast autolysate agar (CYA) and malt extract agar (MEA). Microscopic characteristics including size, shape, ornamentation of ascospores and Aspergillus laciniosus anamorph were examined under light and scanning electron microscopes. This identification was supported by sequence analysis of the b -tubulin gene as described in the previous report 17 and the pure cultures were deposited as KUFC 7896 at Kasetsart University Fungal Collection, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand, and as MMERU 01 at Microbes Marine Environment Research Unit, Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand. N. tsunodae Yaguchi, Abliz & Y. Horie was isolated from the marine sponge A. coralliphaga, which was collected from the coral reef of the Similan islands, Phagna province, Thailand, by scuba diving at 10 m depth, in April 2010 and the sponge was identified by J. Buaruang. Briefly, the sponge tissue was cut into a piece of 0.50.5 cm, placed on the malt extract agar (MEA) with 70% sea water and incubated for 28  C for 7 days. The fungus was identified by one of us (T.D.), by morphological features, including the characteristic of ascospores and colonies. The identification was supported by sequence analysis of the b -tubulin gene described in the previous report 18 and the pure cultures were deposited as KUFC 9213 at Kasetsart University Fungal Collection, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand. 3.3. Extraction of the metabolites N. fischeri (KUFC 6344) was cultured for one week in five 90 mm Petri dishes with 25 ml of potato dextrose agar per dish. Twenty five 1000 ml Erlenmeyer flasks each containing cooked rice (300 g), were autoclaved, incubated with two mycelia plugs of N. fischeri (KUFC 6344) and incubated at 28  C for 30 days. The moldy rice was macerated in ethyl acetate (20 L total) for three days and then filtered. The two layers were separated using a separatory funnel and the ethyl acetate solution was concentrated at a reduced pressure to yield 62.5 g of crude ethyl acetate extract, which was dissolved in 500 ml of a 4:1 mixture of CHCl 3 and EtOAc and then washed with H 2 O(3500 ml). The organic layer was dried with anhydrous Na 2 SO 4 ,filtered and evaporated under reduced pressure to give 50 g of the crude extract, which was applied on a column chromatography of Silica gel (350 g) and eluted with mixtures of petroleCHCl 3 and CHCl 3 eMe 2 CO, 250 ml fractions were collected as follows: Frs 1e89 (petroleCHCl 3 , 1:1), 90e179 (petroleCHCl 3 , 3:7), 180e288 (petroleCHCl 3 , 1:9), 289e358 (CHCl 3 ), 359e430 (CHCl 3 eMe 2 CO, 9:1), 431e451 (CHCl 3 eMe 2 CO, 7:3). Frs 107e131 were combined (2.8 g) and applied on a Silica gel column (30 g) and eluted with mixtures of CHCl 3 epetrol, 100 ml fractions were collected as follows: sfrs 1e38 (petroleCHCl 3 , 7:3), 39e64 (petroleCHCl 3 , 1:1). Sfr 41 (286 mg) was crystallized in a mixture of petrol and CHCl 3 to give 39.4 mg of aszonalenin (1a). Sfrs 46e64 were combined (50.9 mg) and purified by TLC (Si Gel, CHCl 3 /petrol, 9:1) to give 24 mg of sartorypyrone A (3). Frs 132e159 were combined (1.86 g) and applied on a Silica gel column (30 g) and eluted with mixtures of CHCl 3 epetrol, 100 ml fractions were collected as follows: sfrs 1e66 (petroleCHCl 3 , 7:3), 67e112 (petroleCHCl 3 , 1:1). Sfrs 49e64 were combined (188 mg) and purified by TLC (Si Gel, CHCl 3 /Me 2 CO/HCO 2 H, 9:1:0.1) to give an additional 18 mg of sartorypyrone A (3). Sfrs 65e80 were combined (530 mg) and purified by TLC (Si Gel, CHCl 3 /EtOAc/petrol/HCO 2 H, 8:1:1:0.1) to give 40 mg of 13-oxofumitremorgin B (2). Frs 175e189 were combined (890.9 mg) and crystallized in a mixture of petrol and CHCl 3 to give 320 mg of aszonapyrone A (4b). Frs 190e225 were combined (975 mg) and crystallized in a mixture of petrol and CHCl 3 to give 140 mg of aszonapyrone A (4b) and the mother liquor was applied on a Silica gel column (28 g) and eluted with mixtures of CHCl 3 epetrol, 100 ml fractions were collected as follows: sfrs 1e8 (petroleCHCl 3 , 1:1), 9e43 (petroleCHCl 3 , 3:7), 44e53 (petroleCHCl 3 , 1:9). Sfrs 9e16 were combined and crystallized in a mixture of petrol and CHCl 3 to give an additional 53 mg aszonapyrone A (4b). Frs 226e236 were combined (1.75 g) and crystallized in a mixture of petrol and CHCl 3 to give 214.4 mg of acetylaszonalenin (1b). Frs 237e293 were combined and crystallized in a mixture of petrol and CHCl 3 to give helvolic acid (153 mg). Frs 294e332 were combined (1.15 g) and applied on a Silica gel column (30 g) and eluted with mixtures of petroleCHCl 3 ,100ml fractions were collected as follows: sfrs 1e7 (petroleCHCl 3 , 1:1), 8e66 (petroleCHCl 3 , 3:7), 67e81 (petroleCHCl 3 , 1:9). Sfrs 35e40 were combined (70.9 mg) and purified by TLC (Si Gel, CHCl 3 /EtOAc/ Me 2 CO/HCO 2 H, 15:6:1:0.1) to give 34 mg of 1-formyl-5-hydroxyaszonalenin (1c). N. laciniosa (KUFC 7896) was cultured for one week in five 90 mm Petri dishes with 25 ml of malt extract agar (MEA) with 70% sea water per dish. Thirty 1000 ml Erlenmeyer flasks, each containing 200 g rice and 200 ml water, were autoclaved, inoculated with ten mycelia plugs of the fungus and incubated at 28  C for 30 days. The moldy rice was macerated in ethyl acetate (12 L) for five days and then filtered. The two layers were separated using a separatory funnel, and the ethyl acetate solution was concentrated at a reduced pressure to yield 72 g of crude ethyl acetate extract, which was applied on a column chromatography of Silica gel (800 g) and eluted with mixtures of petroleCHCl 3 and CHCl 3 eMe 2 CO, 250 ml fractions were collected as follows: frs 1e19 (CHCl 3 epetrol, 3:7), 20e114 (CHCl 3 epetrol, 1:1), 115e188 (CHCl 3 epetrol, 7:3), 189e340 (CHCl 3 epetrol, 9:1), 341e484 (CHCl 3 eMe 2 CO, 9:1), 485e547 (CHCl 3 eMe 2 CO, 7:3), 548e603 (CHCl 3 eMe 2 CO, 1:1). Frs 365e396 were combined (3.35 g) and recrystallized in Me 2 CO to give 24.1 mg Table 4 Growth inhibitory effect of aszonalenin derivatives (1aec), 13-oxofumitremorgin B (2), sartorypyrone A (3), aszonapyrone B (4a), aszonapyrone A (4b) and sartorypyrone B (5) in three human tumour cell lines Compounds GI 50 ( m M) MCF-7 NCI-H460 A375-C5 1a >150 >150 >150 1b >150 >150 >150 1c >150 >150 >150 2115.220.0 123.311.5 68.612.9 346.37.6 37.34.0 21.51.9 4a >150 >150 >150 4b 13.60.9 11.61.5 10.21.2 517.87.4 20.52.4 25.04.4 Results are given as the lowest concentration causing 50% of cell growth inhibition (GI 50 ) after a continuous exposure to the compounds for 48 h, and are expressed as meanSEM of three independent experiments performed in duplicate. Doxorubicin was used as positive control, GI 50 : MCF-7¼60.31.2 nM; NCI-H460¼19.61.9 nM; A375-C5¼130.025.2 nM. A. Eamvijarn et al. / Tetrahedron 69 (2013) 8583e8591 8589 Mar. Drugs 2014, 12 823 their antibacterial activity against four reference strains (Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa), as well as the environmental multidrug-resistant isolates. Only aszonapyrone A (4a) and sartorypyrone A (8) exhibited significant antibacterial activity as well as synergism with antibiotics against the Gram-positive multidrug-resistant strains. Antibiofilm assays of aszonapyrone A (4a) and sartorypyrone A (8) showed that practically no biofilm was formed in the presence of their 2× MIC and MIC. However, the presence of a sub-inhibitory concentration of ½ MIC of 4a and 8 was found to increase the biofilm production in both reference strain and the multidrug-resistant isolates of S. aureus. Keywords: antibacterial; antibiofilm; multidrug-resistant; tryptoquivalines; meroditerpenes; Neosartorya; marine-derived fungi 1. Introduction Infectious diseases are leading health problems with high morbidity and mortality in the developing countries. Although the introduction of penicillin and other antibiotics ushered in an era of effective treatment of microbial infection, their overuse has caused acquired resistance of pathogens to antimicrobial agents. Since the mid-1970s, resistance to antimicrobial agents has become an escalating problem [1]. In the last 30 years, treatment of infections caused by Gram-positive bacteria has been more problematic than ever, with infections being caused by multidrug-resistant organisms, particularly methicillin-resistant staphylococci, penicillinand erythromycin-resistant pneumococci, and vancomycin-resistant enterococci [2]. The development of resistance to multiple drugs is therefore a worldwide problem in the treatment of these infectious diseases caused by clinically relevant pathogenic microorganisms and must be approached in a large variety of strategies [3]. Although there is a continuing effort in the pharmaceutical industry to develop new antimicrobial agents for the treatment of resistant infections, pursuing new antibiotic drugs is still a fair and necessary strategy to combat the multidrug-resistant bacteria that are spreading both in the community and clinical setting [4]. Since the marine environment is a prolific source of bioactive compounds with extraordinary chemical and biological diversity [5–7], it has become a potential target in the search for new antibiotics. Specifically, the marine-derived fungi which have been reported as producers of bioactive metabolites with antiviral [8,9], antitumor [10,11] and antibacterial [12–14] activities. In the pursuit for bioactive secondary metabolites produced by marine and soil fungi of the genus Neosartorya, we have recently reported isolation and structure elucidation of sartorypyrone A (8), aszonapyrone A (4a), aszonalenin, acetylaszonalenin, 1-formyl-5-hydroxyaszonalenin and 13-oxofumitremorgin B, from the culture of the soil fungus Neosartorya fischeri (KUFC 6344), sartorypyrone B (7b) from the marine sponge-associated fungus N. tsunodae, as well as aszonapyrone A (4a), aszonapyrone B (4b), tryptoquivaline L (1a) and 3′-(4-oxoquinazolin-3-yl) spiro[1H-indole-3,5′-oxolane]-2,2′-dione (2) from N. laciniosa isolated from a diseased coral [15]. Mar. Drugs 2014, 12 824 Figure 1. Secondary metabolites from Neosartorya paulistensis, N. laciniosa, N. siamensis, N. tsunodae and N. fischeri. Examination of a collection of N. paulistensis (KUFC 7897), isolated from the marine sponge Chondrilla australiensis, collected from the Gulf of Thailand, resulted in isolation of a new aszonapyrone analogue which we have named sartorypyrone C (5), in addition to five known metabolites including tryptoquivalines L (1a), H (1b), F (1c), 3′-(4-oxoquinazolin-3-yl) spiro [1H-indole-3,5′-oxolane]-2, 2′-dione (2) and 4(3H)-quinazolinone (3) (Figure 1). Reexamination of the 24 3 8 6 7a: R = H 7b: R = OAc 1 1a: R1 = OH, R2 = Me 1b: R1 = OH, R2 = H 1c: R1 =H, R2 = H 1d: R1 = CHO, R2 = Me 2 4a: R = Ac 4b: R = H 5 6 10 7 9 8 5 1 14 2 4 3 19 18 17 21 22 20 16 15 13 12 11 23 26 24 25 15 14 8 13 27 16 7 4 3 2 6 5 9 12 21 20 18 23 22 19 17 10 11 25 26 Mar. Drugs 2014, 12 825 column fractions left over from our previous work of N. laciniosa (KUFC 7896) [15] led to isolation of a new tryptoquivaline analogue, tryptoquivaline T (1d), whereas reexamination of the nonpolar fractions from the column chromatography of N. siamensis (KUFC 6349) [16] furnished chevalone B (6) and chevalone C (7a) (Figure 1). The isolated compounds were evaluated, together with sartorypyrone B (7b) previously isolated from N. tsunodae and sartorypyrone A (8) (Figure 1) previously isolated from N. fischeri, for antibacterial activity against the Gram-positive (Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 6633) and Gram-negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacteria, as well as multidrug-resistant isolates from the environment. The potential synergism between these fungal metabolites and antibiotics was evaluated against multidrug-resistant bacteria, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococci (VRE). Since aszonapyrone A (4a) and sartorypyrone A (8) exhibited interesting antibacterial activity against both Gram-negative reference strains and the environmental multidrug-resistant isolates, their capacity to inhibit biofilm formation was also studied. Table 1. 1H and 13C NMR (DMSO, 300.13 and 75.47 MHz) and HMBC assignment for tryptoquivaline T (1d). Position δC, Type δH, (J in Hz) COSY HMBC 1 --- --- 2 81.0, CH 6.10, s H-29 C-3, 13, 14 3 84.2, C --- 4 133.5,C --- 5 126.8, CH 8.04, dd (8.0, 1.0) H-6 C-3, 7, 9 6 126.7, CH 7.46, ddd (8.0, 8.0, 1.0) H-5 C-4, 8 7 131.8, CH 7.61, ddd (8.0, 8.0, 1.0) H-6, 8 C-5, 9 8 116.4, CH 7.56, ddd (8.0, 1.0) H-7 C-4, 6 9 138.4, C --- 11 171.1, CO --- 12 57.7, CH 5.47, dd (10.7, 8.6) H-13 C-11, 18 13a 33.8, CH2 3.30, dd (14.0, 8.6) H-12, 13b C-2, 3, 4 b 3.40, dd (14.0, 10.7) H-12, 13a C-2, 3, 4 14 172.5, CO --- 15 64.1, C --- 18 160.0, CO --- 19 121.4, C --- 20 126.2, CH 8.26, d (8.0, 1.0) H-21 C-18, 22, 24 21 127.6, CH 7.63, ddd (8.0, 8.0, 1.0) H-20, 22 C-19, 23 22 135.1, CH 7.93, ddd (8.0, 8.0, 1.0) H-21, 23 C-20, 24 23 127.4, CH 7.76, d (8.0) H-22 C-19, 21 24 147.8, C --- 26 148.1, CH 8.62, s C-12, 18, 24 27 26.6, CH3 1.72, s C-14, 15, 28 28 25.5, CH3 1.55, s C-14, 15, 27 29 162.3, CHO 8.73, d (0.9) H-2 C-2 Mar. Drugs 2014, 12 826 2. Results and Discussion Compound 1d was isolated as white solid, and its molecular formula C24H20N4O5 was established on the basis of the (+)-HRESIMS m/z 445.1512 [M + H]+, indicating 17 degrees of unsaturation. The IR spectrum showed absorption bands for aromatic (3010, 1582, 1450 cm−1) and carbonyls of ester/amide groups (1700 cm−1). The general features of the 1H and 13C spectra of 1d (Supplementary Information, Figures S1 and S2) closely resembled those of tryptoquivaline L (1a). The 13C NMR, DEPT and HSQC spectra (Table 1) revealed three amide/ester carbonyls (δC 172.5, 171.1 and 160.0), one N-formyl (δC 162.3), four quaternary sp2 (δC 147.8, 138.4, 133.5, 121.4), nine methine sp2 (δC 148.1, 135.1, 131.8, 127.6, 127.4, 126.8, 126.7, 126.2, 116.4), two quaternary sp3 (δC 84.2 and 64.1), two methine sp3 (δC 81.0 and 57.7), one methylene sp3 (δC 33.8) and two methyl (δC 26.6 and 25.5) carbons. Analysis of the 1H, 13C NMR, HSQC, COSY and HMBC (Table 1) revealed the presence of the N-substituted quinazolin-4-one and the 6-5-5 gem-dimethyl imidazoindole ring systems which were connected via a five membered spirolactone as in tryptoquivaline L [16]. However, the only difference between 1d and tryptoquivaline L (1a) is the presence of the formyl group on N-16 of the gem-dimethyl imidazoindole moiety in the former and a hydroxyl group in the latter. On the other hand, the structure of 1d differs from that of tryptoquivaline O, previously reported from N. siamensis by Buttachon et al. [16], in that there are two methyl groups on C-15 of the imidazoindole ring in the former instead of one methyl group in the latter. The assignments of the proton and carbon chemical shifts for CH3-27 and CH3-28 were based on the NOESY correlation between the signals of H-2 (δH 6.10, s) and CH3-27(δH 1.72, s). Thus, 1d is a new tryptoquivaline analogue which we have named tryptoquivaline T. Since the chemical shift values of H-2 and H-12 of tryptoquivaline T (1d) are similar of those of the corresponding protons of tryptoquivaline O, we assume that the stereochemistry of tryptoquivaline T (1d) is the same as that of tryptoquivaline O, i.e., C-2S, C-3S and C-12R. This assumption was also supported by the negative value of the rotation tryptoquivaline T (1d). Compound 5 was also isolated as white solid (mp, 200–202 °C) and its molecular formula C26H38O4 was established on the basis of the (+)-HRESIMS m/z 415.2836 [M + H]+ (calculated 415.2848), indicating eight degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3445 cm−1), conjugated ester carbonyl (1668 cm−1) and olefin (1649, 1636 cm−1) groups. The 13C NMR, DEPT and HSQC spectra (Table 2) exhibited the signals of one conjugated ester carbonyl (δC 164.6), five quaternary sp2 (δC 164.3, 159.3, 136.6, 126.2. 101.4), one methine sp2 (δC 99.9), three quaternary sp3 (δC 38.8, 38.4 and 36.8), one oxymethine sp3 (δC 76.9), two methine sp3 (δC 56.0, 54.8), seven methylene sp3 (δC 38.0, 37.9, 34.2, 27.1, 22.1, 18.2, 17.7), and six methyl (δC 28.1, 21.2, 20.3, 19.2, 16.3 and 15.7) carbons. Except for the presence of one more methyl group instead of an exocyclic methylene group, and a tetra-substituted double bond (δC 126.2 and 136.6), the 1H and 13C data (Table 2, Supplementary Information, Figures S3 and S4) revealed the existence of the perhydrophenanthrene moiety connected to the 4-hydroxy-6-methyl-2H-pyran-2-one portion through the methylene group, similar to those of aszonapyrone B (4b) [15]. That the double bond in the perhydrophenanthrene moiety was on C-13 and C-14 was supported by the HMBC correlations of H3-26 (δH 1.55) to C-12 (δC 34.2), C-13 (δC 126.2), C-14 (δC 136.6); H-15 (δH 3.02, brs) to C-8 (δC 38.8), C-13, C-14, C-16 (δC 101.4), C-17 (δC 164.3) and C-21 (δC 164.6). That the hydroxyl group on C-3 was β is supported by the chemical shift value (δC 15.7) of the C-4 axial methyl (CH3-23) which suffered a γ-gauche interaction [17]. Thus, compound 5 is a new analogue of aszonapyrones which we have named sartorypyrone C. Mar. Drugs 2014, 12 827 Table 2. 1H and 13C NMR (DMSO, 300.13 and 75.47 MHz) and HMBC assignment for sartorypyrone C (5). Position δC, Type δH, (J in Hz) COSY HMBC 1 37.9, CH2 1.65, m H-2 2 27.1, CH2 1.46, m H-1, 3 3 76.9, CH 2.97, m H-2 4 38.4, C --- --- 5 54.8, CH 0.67, brd (9.6) H-6 6 17.7, CH2 1.35, m H-5, 7 7 38.0, CH2 1.96, m H-6 8 38.8, C -- 9 56.0, CH 0.97, brd (11.4) H-11 CH3-24 10 36.8, C --- 11 18.2, CH2 1.46, m H-9, 12 12 34.2, CH2 1.94, m H-11 13 126.2, C --- --- 14 136.6, C --- --- 15 22.1, CH2 3.02, brs --- C-8, 13, 14, 16, 17, 21 16 101.4, C --- --- 17 164.3, C --- --- 18 99.9, CH 5.90, s CH3-20, C-1617, 19, 20 19 159.3, C --- --- 20 19.2, CH3 2.12, s H-18 C-18, 19 21 164.6, C --- --- C-3, 4, 5, 23 22 28.1, CH3 0.86, s --- C-3, 4, 5, 22 23 15.7, CH3 0.66, s --- 24 16.3, CH3 0.77, s --- C-1, 5, 9, 10 25 21.2, CH3 0.89, s --- C-8, 9, 14 26 20.3, CH3 1.55, s --- C-12, 13, 14 Compounds 1–8 (Figure 1) were tested for their antibacterial activity against bacterial reference strains and environmental multidrug-resistant isolates, and their MIC and MBC (when determined) values are shown in Table 3A. It is interesting to note that neither of the indole alkaloids (1a–d, 2) exhibited relevant antibacterial activity. However, within the meroditerpene group, only aszonapyrone A (4a) and sartorypyrone A (8) presented significant MIC values against Gram-positive bacteria. Aszonapyrone A (4a) showed the MIC values of 8 µg/mL against S. aureus ATCC 25923 and B. subtilis ATCC 6633, while sartorypyrone A (8) showed the MIC values of 32 and 64 µg/mL, respectively, against the same reference strains. Based on these results, the MIC values of these two compounds were further determined against Gram-positive multidrug-resistant strains. While aszonapyrone A (4a) was found to be active against both S. aureus MRSA and Enterococcus spp. VRE isolates, sartorypyrone A (8) did not show any inhibition on the growth of Enterococcus spp. VRE isolates in the range of concentrations tested (Table 3B). MBC values were only achieved for aszonapyrone A (4a) against Gram-positive reference strains, and since sartorypyrone A (8) did not exhibit any bactericidal effect against any strain, its MBC values could not be determined. Mar. Drugs 2014, 12 828 Table 3. Antimicrobial activity, expressed in µg/mL of 1–8 against references strains (A) and of 4a and 8 against multidrug-resistant isolates (B). (A) Compounds S. aureus ATCC 25923 P. aeruginosa ATCC 27853 B. subtilis ATCC 6633 E. coli ATCC 25922 MIC MBC MIC MBC MIC MBC MIC MBC 1a 128 − 128 256 128 − 128 − 1b 128 − 128 256 128 − 128 − 1c 128 − 128 128 128 − 128 − 1d − − 128 − 128 − 128 − 2 256 − 128 256 128 − 128 − 3 128 − 128 256 128 − 128 − 4a 8 64 128 256 8 16 128 − 4b 256 − 128 256 128 − 128 − 5 128 − 128 256 128 − 128 − 6 − − − − − − − − 7a − − − − − − − − 7b − − − − − − − − 8 32 − − − 64 − − − (B) Compounds S. aureus B1 S. aureus B1 E. faecalis W1 E. faecium W5 MIC MBC MIC MBC MIC MBC MIC MBC 4a 8 − 8 − 16 − 16 − 8 32 − 32 − − − − − (−): >256 µg/mL. Mar. Drugs 2014, 12 829 Table 4. Antibacterial efficacy (halos, mm) of combined effect of antibiotics with compounds 1–8 (15 µg/disc) against three multidrug-resistant isolates, using the disc diffusion method. E. coli G1 S. aureus B1 E. faecium W5 Antibiotics Compounds CIP AMP CTX S OX AMP CTX VA AMP E 1a 7 7 = 7.5 = = = = = = 1b 7 7 = 7.5 = = = = = = 1c 7 7 = 7.5 = = = = = = 1d 7 7 = 7.5 = = = = = = 2 7 7 = 7.5 = = = = = = 3 7 7 = 7.5 = = = = = = 4a 8 8 = 8 12.5 13 13 11 12 13.5 4b 7 7 = 7.5 11 10 10.5 11 12 13.5 5 7 7 = 7.5 = = = = = = 6 7 7 = 7.5 = 9 = = = = 7a 8 8 = 8 9.5 10 9.5 8.5 9 8 7b 7 7.5 = 7.5 8.5 9.5 8.5 = = = 8 7.5 7.5 = 7.5 10 10 10 8.5 7 7 Control 0 0 14 0 0 7 0 8 0 0 Control: Antibiotic with no compounds; CIP: Ciprofloxacin; AMP: Ampicillin; CTX: Cefotaxime; S: Streptomycin; OX: Oxacillin; VA: Vancomycin; E: Erythromycin. (=): Indicates no influence of the compound; same result as obtained with no compound. Table 5. Fractional inhibitory concentration (FIC) index results obtained with 4a/8 and antibiotic combinations by checkerboard method. Bacterial Isolate 4a-OX 8-OX 4a-VA 8-Va 4a-AMP 8-AMP ΣFIC Activity a ΣFIC Activity ΣFIC Activity ΣFIC Activity ΣFIC Activity ΣFIC Activity S. aureus B1 0.562 I 0.516 I − − − − 2 I 0.516 I S. aureus B2 2 I 0.625 I − − − − 2 I 0.625 I E. faecalis W1 − − − − 0.312 S − − 0.75 I − − E. faecium W5 − − − − 0.312 S − − 0.75 S − − AMP: Ampicillin; OX: Oxacillin; VA: Vancomycin; a S = synergism; I = indifference; (−): Not determined. Mar. Drugs 2014, 12 830 The disc diffusion method (Table 4) revealed a small synergistic association between all the compounds tested and the antibiotics to which E. coli G1 was resistant. Even though only a few compounds showed synergism against S. aureus B1 and E. faecium W5, association of aszonapyrone A (4a) with the antibiotics was found to produce the biggest halos, whereas sartorypyrone A (8) increased the antibiotic inhibition halos against S. aureus B1 and, to a lesser extent, against E. faecium W5. Interestingly, although chevalone C (7a) alone did not show antibacterial activity at the highest concentration tested (MIC > 256 mg/mL), it demonstrated a synergistic effect with antibiotics against all three multidrug-resistant isolates. The results of the Checkerboard method, represented by the FIC index, are shown in Table 5. The combination effect of aszonapyrone A (4a) with oxacillin (OX) and ampicillin (AMP) against MRSA and VRE isolates, respectively, was found to be indifferent (ΣFIC > 0.5); however, aszonapyrone A (4a) was found to lower the MIC of each antibiotic tested, thus, it may be considered a partially synergist effect. The association of aszonapyrone A (4a) with vancomycin (VA) showed a clear synergistic effect (ΣFIC < 0.5) against the two VRE isolates tested. The combination of sartorypyrone A (8) with OX and AMP against MRSA isolates was found to be also indifferent. Since the MIC of sartorypyrone A (8) against VRE was higher than 256 µg/mL, no checkerboard method was performed for this compound against VRE isolates. The effect of aszonapyrone A (4a) and sartorypyrone A (8), at different concentrations (ranging from 2× MIC to 1/4× MIC), on the biofilm formation of S. aureus ATCC 25923, B. subtilis ATCC 6633 and S. aureus B1, and also E. faecalis W1 (in the case of 4a) was also assessed using the biomass quantification, and the results are shown in Figure 2. All the strains tested showed no biofilm formation in the presence of 2xMIC and MIC of aszonapyrone A (4a) and sartorypyrone A (8). However, S. aureus ATCC 25923 and S. aureus B1 formed more biofilm in the presence of a sub-inhibitory concentration (1/2× MIC) of aszonapyrone A (4a) (Figure 2A). Moreover, S. aureus ATCC 25923 was found to produce a significantly (P < 0.05) higher amount of biomass in the presence of 1/2× MIC of sartorypyrone A (8), when compared to the control (Figure 2B). In order to confirm the effect of these compounds on biofilm formation, the microscopic visualization of the biofilm produced by S. aureus ATCC 25923 was carried out using a Live/Dead staining. After 24 h, the majority of the cells within the biofilm were viable and large aggregates embedded in a matrix could be observed (Figure 3A). In the presence of aszonapyrone A (4a), at a concentration equal to the MIC, no biofilm was formed and also no growth was observed (Figure 3B). However, at the concentration of 1/2× MIC, it was possible to observe more biofilm in comparison to the control (Figure 3C). These results are in agreement with those obtained in the biomass quantification for the same experimental conditions. However, this result is not unexpected since there are several reports of the increase in biofilm formation in both Gram-positive and Gram-negative bacteria in the presence of sub-inhibitory concentrations of antibiotics [18–20]. Interestingly, the BIC value of aszonapyrone A (4a) was found to be higher than 12× MIC against mature biofilms of both S. aureus B1 (BIC > 96 µg/mL) and E. faecalis W1 (BIC > 192 µg/mL). However, the exact BIC value could not be determined due to the limited quantity of this compound available to perform all these biological assays. These very high BIC values may reflect the difficulty of 4a in penetrating the extracellular biofilm matrix, thus hampering the eradication of the pre-established biofilm. Examination of the structures of the meroditerpenes tested (Figure 1) suggested the existence of some common features necessary for the antibacterial activity of this class of compounds. Although Mar. Drugs 2014, 12 831 aszonapyrone A (4a), aszonapyrone B (4b), sartorypyrone C (5) and sartorypyrone A (8), all contain the 4-hydroxy-6-methyl-2H-pyran-2-one ring, only aszonapyrone A (4a) and sartorypyrone A (8) have the β-acetoxyl group on C-3. On the other hand, this 4-hydroxy-6-methyl-2H-pyran-2-one ring is connected to the perhydrophenanthrene portion by the ethereal bridge, forming a more rigid pentacyclic structure in chevalone B (6). On the other contrary, both chevalone C (7a) and sartorypyrone B (7b) contain the 6-methyl-4H-pyran-4-one ring connected to the perhydrophenanthrene portion by an ethereal bridge. Therefore, it is apparent that the presence of a free 4-hydroxy-6-methyl-2H-pyran-2-one ring on C-15 and the β-acetoxyl group on C-4 of the perhydrophenanthrene portion are required for the antibacterial activity of this series of meroditerpenes. Figure 2. Biomass quantification of biofilms of Gram-positive bacteria formed in the presence of different concentrations (ranging from 2× MIC to 1/4× MIC) of 4a (A) and 8 (B). Figure 3. Evaluation of S. aureus ATCC 25953 biofilm formation. Live/dead viability staining images after 24 h. Control (A); Biofilm formation in the presence of the MIC (B) and in the presence of ½ of the MIC (C) of 4a. 3. Experimental Section 3.1. General Experimentation Procedures Melting points were determined on a Bock monoscope and are uncorrected. Optical rotations were determined on an ADP410 Polarimeter (Bellingham+Stanley Ltd., Tunbridge Wells, Kent, UK) Infrared spectra were recorded on an ATT Mattson Genesis Series FTIR™ using WinFIRST Software. 1H and 13C NMR spectra were recorded at ambient temperature on a Bruker AMC instrument (Bruker Mar. Drugs 2014, 12 832 Biosciences Corporation, Billerica, MA, USA) operating at 300.13 and 75.4 MHz, respectively. High resolution mass spectra were measured with a Xevo QToF mass spectrometer (Waters Corporations, Milford, MA, USA) coupled to the Aquity UPLC system (Waters Corporations, Milford, MA, USA). A Merck silica gel GF254 was used for preparative TLC, and a Merck Si gel 60 (0.2–0.5 mm) was used for analytical chromatography. 3.2. Extraction and Isolation Extraction and Isolation of Secondary Metabolites from the Culture of Neosartorya paulistensis (KUFC 7897) Neosartorya paulistensis (KUFC 7897) was isolated from the marine sponge Chondrilla australiensis which was collected from Mu Kho Lan Beach, Chonburi Province, Thailand in May 2010. After rinsing with sterile sea water, the sponge was dried on sterile filter papers and cut into small pieces (5 × 5 mm) and placed on the plates containing malt extract agar (MEA, 30 g of malt extract, 15 g of bacto agar, distilled water 1000 mL and adjusted to the final pH at 5.5) with 70% sea water and incubated at 28 °C under 12 h light/12 h dark cycle for 7 days. The fungus was identified by Prof. Dr. Leka Manoch (Department of Plant Pathology, Kasetsart University, Bangkok, Thailand), by morphological features, including the characteristic of ascospores and colonies. The identification was supported by sequence analysis of the β-tubulin gene described in the previous report [21] and the pure cultures were deposited as KUFC 7897 at Kasetsart University Fungal Collection, Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok, Thailand, and as MMERU 02 at Microbes Marine Environment Research Unit, Division of Environmental Science, Faculty of Science, Ramkhamhaeng University, Bangkok, Thailand. The fungus was cultured for two weeks at 28 °C in 10 Petri dishes (i.d. 90 mm) containing 25 mL of MEA with 70% sea water per dish. Fifty 1000 mL Erlenmeyer flasks each containing rice (200 g), water (30 mL), and sea water (70 mL), were autoclaved, inoculated with five mycelia plugs of N. paulistensis and incubated at 28 °C for 30 days, after which the mouldy rice was macerated in ethyl acetate (15 L total) for 10 days and then filtered. The two layers were separated using a separatory funnel and the ethyl acetate solution was concentrated at a reduced pressure to yield crude ethyl acetate extract, which was washed with 5% of NaHCO3 solution (2 × 500 mL) and H2O (3 × 500 mL). The organic layer was dried with anhydrous Na2SO4, filtered and evaporated under reduced pressure to give 51 g of crude extract which was applied on a column chromatography over 610 g of Si gel (0.2–0.5 mm, Merck KGaA, Darmstadt, Germany) and eluted with mixtures of CHCl3–petrol and CHCl3–Me2CO, wherein 250 mL fractions were collected as follows: fractions 1–25 (CHCl3–petrol, 1:4), 26–149 (CHCl3–petrol, 3:7), 150–177 (CHCl3–petrol, 2:3), 178–278 (CHCl3–petrol, 1:1), 279-399 (CHCl3–petrol, 3:2), 400–534 (CHCl3–petrol, 4:1), 535–549 (CHCl3–petrol, 9:1), 550–592 (CHCl3), 593–837 (CHCl3–Me2CO, 9:1), 838–976 (CHCl3–Me2CO, 8:2), 977–1047 (CHCl3–Me2CO, 7:3), 1048–1090 (CHCl3–Me2CO, 1:1) and 1091–1112 (CHCl3–Me2CO, 1:4). Frs 299–307 (526 mg) were combined and recrystallized in petrol to give 12 mg of sartorypyrone C (5). Frs 627–696 were combined (1.50 g) and crystallized in a mixture of CHCl3 and acetone to give 70 mg of tryptoquivaline L (1a). Frs 739–774 were combined (400 mg) and crystalized in Me2CO to give 95 mg of tryptoquivaline H (1b). The mother liquor of Mar. Drugs 2014, 12 839 23. Franklin, R.; Cockerill, M.D., III. Performance Standards for Antimicrobial Susceptibility Testing, Twenty-First Informational Supplement M100-S21; Clinical and Laboratory Standards Institute (CLSI): Wayne, PA, USA, 2011. 24. Odds, F.C. Synergy, antagonism, and what the chequerboard puts between them. J. Antimicrob. Chemother. 2003, 52, doi:10.1093/jac/dkg301. 25. Johnson, S.A.; Goddard, P.A.; Iliffe, C.; Timmins, B.; Rickard, A.H.; Robson, G.; Handley, P.S. Comparative susceptibility of resident and transient hand bacteria to para-chloro-meta-xylenol and triclosan. J. Appl. Microbiol. 2002, 93, 336–344. © 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). 333 APPENDIX V. GOMES ET AL. (ACCEPTED) APPENDIX V Gomes, N.M., Buttachon, S., Kijjoa, A. Meroterpenoids from Marine Microorganisms: Potential Scaffolds for New Chemotherapy Leads. In: Anticancer Drugs from Marine Origins (Ed. Kim, S.-K.) (Accepted). 334 APPENDIX V. GOMES ET AL. (ACCEPTED) 335 APPENDIX V. GOMES ET AL. (ACCEPTED)