Published: April 21, 2011 Copyright r 2011 American Chemical Society and American Society of Pharmacognosy 1353 dx.doi.org/10.1021/np1005983 | J. Nat. Prod. 2011, 74, 1353–1357 ARTICLE pubs.acs.org/jnp Xylarenones CE from an Endophytic Fungus Isolated from Alibertia macrophylla Camila Martins de Oliveira, † Geraldo Humberto Silva, § Luis Oct  avio Regasini, † Ot  avio Flausino, Jr., † Silvia Noelí L opez, || B  arbara Marcondes Abissi, † Roberto Gomes de Souza Berlinck, ^ Lara Dur ~ aes Sette, ‡ Rafaella Costa Bonugli-Santos, ‡ Andr  e Rodrigues, O Vanderlan da Silva Bolzani, † and Angela Regina Araujo* ,† † NuBBE, N ucleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais, Departamento de Química Org ^ anica, Instituto de Química, Universidade Estadual Paulista, Rua Professor Francisco Degni s/n, Araraquara, S ~ ao Paulo, Brazil § Departamento de Química, Universidade Federal de Sergipe, Avenida Vereador Olimpio Grande, SN, Itabaiana, Sergipe, Brazil ^ Instituto de Química de S ~ ao Carlos, Universidade de S ~ ao Paulo, CP 780, S ~ ao Carlos, S ~ ao Paulo, Brazil ‡ Divis ~ ao de Recursos Microbianos, Centro Pluridisciplinar de Pesquisas Químicas Biol ogicas e Agrícolas, Universidade Estadual de Campinas, Rua Alexandre Cazsellato, 999, Paulinia, S~ ao Paulo, Brazil O Centro de Estudos de Insetos Sociais, Universidade Estadual de S~ ao Paulo, Avenida 24A, 1515, Bela Vista, Rio Claro, S ~ ao Paulo, Brazil ) Farmacognosia, Facultad de Ciencias Bioquímicas y Farmac euticas, Universidad Nacional de Rosario, 2000-Rosario, Argentina b S Supporting Information ABSTRACT: Xylarenones CE(24), three new eremophilane sesquiterpenes, have been isolated from solid substrate cultures of a Camarops- like endophytic fungus isolated from Alibertia macrophylla. The structures were elucidated by analysis of spectroscopic data. Compounds were evaluated in subtilisin and pepsin protease assays, and compound 2 showed potent inhibitory activity against both proteases. B oth laboratory and field studies have demonstrated that wild plant species require fungal endophytes for stress tolerance and survival. 1 Therefore, each of the nearly 300 000 species of terrestrial plants on Earth may host one or more fungal endophytes. 2 Some endophyteplant associations reportedly confer benefits to host plants, including tolerance to herbivory, heat, salinity, diseases, and drought. 1 Moreover, the production of bioactive secondary metabolites by fungal endophytes could in some cases enhance a plants ability to avoid microbial infections, overpredation, and space competition by other invasive plant species. 3 Thus, the study of fungal endophytes is currently considered a reasonable approach to the discovery of novel, bioactive natural products. 3,4 Proteases are important enzymatic targets because these proteins control the formation of functional peptides that participate in physiological processes. 5 As part of our ongoing search for new protease inhibitors from endophytic fungi of the Cerrado and of the Atlantic Rain Forest of Brazil, four fungal strains were isolated from Alibertia macrophylla (Rubiaceae). 6 One strain (AM-02) yielded a crude extract with protease inhibitory activity and was chosen for chemical, biological, and taxonomic investigations. Herein we report the isolation of four eremophilane sesquiterpenes, three of which are new, and all of them displayed inhibitory activity toward pepsin. Compound 2 also inhibited subtilisin. ’ RESULTS AND DISCUSSION The sesquiterpene xylarenone A (1) was identified by com- parison of its IR, MS, 1 H NMR, and 13 C NMR data with literature values for the same compound previously isolated from Xylaria sp. NCY2. ([R] 20 D = þ144.1, c 4.614, CHCl 3 ). 7 Xylarenone C (2) was isolated as an optically active, colorless oil ([R] 28 D þ14). Analysis by HRESIMS indicated a [M þ Na] þ ion at m/z 455.2773, corresponding to the formula C 26 H 40 NaO 5 . The IR spectrum of 2 showed a broad band at 3442 cm 1 (ν OH ), as well as bands assigned to ester (1730 cm 1 ) and R,β-unsaturated carbonyl (1679 cm 1 ) functionalities. Analysis of 13 C (BBD and DEPT 135) NMR spectra indicated the presence of six methyl, seven methylene, seven methine, and six nonprotonated carbons, two of which were assigned to carbonyl groups, at δ 195.2 (R,β- unsaturated ketone) and δ 175.0 (ester). The 1 H NMR spectrum of 2 showed the presence of a deshielded oxymethine proton at Received: August 24, 2010