Cadinane sesquiterpenoids of Phomopsis cassiae, an endophytic fungus associated with Cassia spectabilis (Leguminosae) Geraldo Humberto Silva a , Helder Lopes Teles a , Lisine ´ia Maria Zanardi a , Maria Claudia Marx Young b , Marcos Nogueira Eberlin c , Renato Hadad c , Ludwig H. Pfenning d , Claudio Miguel Costa-Neto e , Ian Castro-Gamboa a , Vanderlan da Silva Bolzani a ,A ˆ ngela Regina Arau ´jo a, * a NuBBE-Nu ´ cleo de Bioensaio, Biossı ´ntese e Ecofisiologia de Produtos Naturais, Instituto de Quı ´mica, Universidade Estadual Paulista, CP 355, CEP 14801-970, Araraquara, SP, Brazil b Secc ¸a ˜o de Fisiologia e Bioquı ´mica de Plantas, Instituto de Bota ˆnica, CP 4009, CEP 01061-970, Sa ˜o Paulo, SP, Brazil c Thompson Mass Spectroscopy Laboratory, Instituto de Quı ´mica, Universidade Estadual de Campinas, CP 6154, CEP 13083-970, Campinas, SP, Brazil d Departamento de Fitopatologia, Universidade Federal de Lavras, CP 3037, CEP 37200-000, Lavras, MG, Brazil e Departamento de Bioquı ´mica e Imunologia, Faculdade de Medicina de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, CEP 14049-900, Ribeira ˜o Preto, SP, Brazil Received 8 February 2006; received in revised form 27 April 2006 Available online 20 July 2006 Abstract Five cadinane sesquiterpenes derivatives were isolated by bioassay-guided fractionation from Phomopis cassiae, an endophytic fungus isolated from Cassia spectabilis. The structures of the two diastereoisomeric 3,9,12-trihydroxycalamenenes (1, 2); 3,12-dihydroxycalam- enene (3); 3,12-dihydroxycadalene (4) and 3,11,12-trihydroxycadalene (5) were established on the basis of analyses of 1D and 2D NMR and HRTOFMS experiments. Antifungal activity of the isolates was evaluated against Cladosporium sphaerospermum and Cladosporium cladosporioides, revealing 5 as the most active compound. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Phomopsis; Endophytic fungi; Cadinane Sesquiterpenes; Antifungal 1. Introduction Endophytic fungi are organisms living within the tissues of hosts plants (Andrews and Hirano, 1991). These organ- isms are known as potential sources of biologically active secondary metabolites (Dreyfuss and Chapela, 1994; Tan and Zou, 2001; Strobel et al., 2004) and relatively little is known about the potential of these microorganisms from Brazilian endemic plant species (Azevedo et al., 2000, 2004). As part of our studies on bioactive agents from metabo- lites produced by endophytic fungi associated with species of native plants from the Brazilian Cerrado, we initially selected 16 species of plants, which had already been phy- tochemically profiled by our group. Those species were submitted for isolation of endophytic fungi and 82 pure isolates were obtained, preserved, cultivated in liquid media and extracted with ethyl acetate to obtain crude extracts. Some metabolites produced by fungi were already published by our group (Teles et al., 2005; Cafe ˆu et al., 2005; Silva et al., 2005). The crude EtOAc extract of Phomopsis cassiae isolated from Cassia spectabilis showed moderate antifungal activ- ity against Cladosporium sphaerospermum and Cladospo- rium cladosporioides, stimulating further studies. Bioassay guided fractionation of this extract afforded five new ses- quiterpenes (1–5). 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2006.06.004 * Corresponding author. Tel.: +55 016 3301 6658; fax: +55 016 3322 7932. E-mail address: araujoar@iq.unesp.br (A ˆ . Regina Arau ´ jo). www.elsevier.com/locate/phytochem Phytochemistry 67 (2006) 1964–1969 PHYTOCHEMISTRY