Benzoic Acid Derivatives from Piper Species and Their Fungitoxic Activity against Cladosporium cladosporioides and C. sphaerospermum Joa ˜ o Henrique G. Lago, † Cle ´cio Sousa Ramos, † Diego Campos C. Casanova, † Andreia de A. Morandim, ‡ Debora Cristina B. Bergamo, ‡ Alberto J. Cavalheiro, ‡ Vanderlan da S. Bolzani, ‡ Maysa Furlan, ‡ Elsie F. Guimara ˜ es, § Maria Claudia M. Young, ⊥ and Massuo J. Kato* ,† Instituto de Quı ´mica, Universidade de Sa ˜ o Paulo, C.P. 26077-05599-970, Sa ˜ o Paulo, SP, Brazil, Instituto de Quı ´mica, Universidade Estadual Paulista, CP 355, 14800-900, Araraquara, SP, Brazil, Instituto de Pesquisas do Jardim Bota ˆ nico, CEP 22460-030, Rio de Janeiro, RJ, Brazil, and Sec ¸ a ˜ o de Fisiologia e Bioquı ´mica de Plantas, Instituto de Bota ˆ nica, CP 4005, 01051, Sa ˜ o Paulo, SP, Brazil Received December 18, 2003 Piper crassinervium, P. aduncum, P. hostmannianum, and P. gaudichaudianum contain the new benzoic acid derivatives crassinervic acid (1), aduncumene (8), hostmaniane (18), and gaudichaudianic acid (20), respectively, as major secondary metabolites. Additionally, 19 known compounds such as benzoic acids, chromenes, and flavonoids were isolated and identified. The antifungal activity of these compounds was evaluated by bioautographic TLC assay against Cladosporium cladosporioides and C. sphaerospermum. Piperaceae species have been extensively investigated as a source of new natural products with potential anti- tumoral, antimicrobial, antifungal, and insecticidal activi- ties. 1-3 The phytochemical profile in Piper species is characterized by the production of typical classes of com- pounds such as amides, benzoic acids, and chromenes in addition to lignans, neolignans, and a few alkaloids. 4-6 As part of our research aiming to uncover new antifungal compounds in Brazilian Piperaceae species, we have previ- ously reported the occurrence of pyrrolidine amides in P. hispidum 7 and piperidine, isobutyl, and pyrrolidine amides in P. tuberculatum 8 and P. arboreum. 9 Additionally, anti- fungal prenylated hydroquinones and flavanones from leaves of P. crassinervium 10 have been described as well. The search for new antifungal compounds will contribute to establish a model for further investments in this field in order to preserve, to study, and to exploit rationally the remaining flora of Sa ˜ o Paulo State. In the course of the Biota Program (http://www.biota.sp), a large number of Piper species collected in the state of Sa ˜ o Paulo had their extracts assayed by means of bioau- tography using Cladosporium cladosporioides (Fresen) de Vries and C. sphaerospermum (Perzig). Since the extracts of Piper crassinervium Kunth, P. aduncum L., P. hostman- nianum (Miquel) C. DC., and P. gaudichaudianum Kunth showed high growth inhibitory activity, they were subjected to dereplication procedures using chromatographic tech- niques associated with the bioautographic assay. Thus, the major aim of this paper is to describe the isolation and structural determination of the new antifungal compounds from Piperaceae species. Results and Discussion The MeOH extract from leaves of P. crassinervium yielded three prenylated 4-hydroxybenzoic acids [4-hy- droxy-3-(3′,7′-dimethyl-3′-hydroxy-1′-oxo-6′-octenyl)benzo- ic acid (crassinervic acid, 1), 4-hydroxy-(3′,7′-dimethyl-1′- oxo-octa-2′-E-6′-dienyl)benzoic acid 11 (2), and 4-hydroxy- (3′,7′-dimethyl-1′-oxo-octa-2′-Z-6′-dienyl)benzoic acid 11 (3)], 3,4,5-trimethoxydihydrocinnamic acid 12 (4), 1,4-dihydroxy- 2-(3′,7′-dimethyl-1′-oxo-octa-2′-E-6′-dienyl)benzene 10 (5), and two flavanones [5,7-dihydroxy-4′-methoxyflavanone 13 (nar- ingenin 4′-methyl ether, 6) and 5,4′-dihydroxy-7-methoxy- flavanone 14 (sakuranetin, 7)]. The CH 2 Cl 2 - and hexane-soluble parts of the MeOH extract of the leaves of P. aduncum yielded one new prenylated benzoic acid [methyl 2-methoxy-5-(3′-methyl- 1′-oxo-2′-butenyl)benzoate (aduncumene, 8)], four known chromenes [methyl 2,2-dimethyl-8-(3′-methyl-2′-butenyl)- 2H-1-benzopyran-6-carboxylate (9), methyl 2,2-dimeth- yl-2H-1-benzopyran-6-carboxylate (10), methyl 8-hydroxy- 2,2-dimethyl-2H-1-benzopyran-6-carboxylate (11), and 2,2-dimethyl-2H-1-benzopyran-6-carboxylic acid 15,16 (12)], 4-methoxy-3-(3′,7′-dimethylocta-2′-E-6′-dienyl)benzoic acid 15 (13), and two dihydrochalcones [2′,6′-dihydroxy-4′-meth- oxydihydrochalcone 15 (14) and piperaduncine B 17 (15)]. The CH 2 Cl 2 -MeOH (2:1) extract of the leaves of P. hostmannianum yielded methyl 2,2-dimethyl-2H-1-ben- zopyran-6-carboxylate 16 (10), three prenylated methyl hy- droxybenzoate derivatives [methyl 4-hydroxy-3-(3′-methyl- 2′-butenyl)benzoate 18,19 (16), methyl 4-hydroxy-3-(3′-methyl- 2′-hydroxy-3′-butenyl)benzoate 16 (17), and the new derivative methyl 4-hydroxy-3-(2′,3′-dihydroxy-3′-methylbutyl)ben- zoate (hostmaniene, 18)], and pinocembrin 20,21 (19). The CH 2 Cl 2 -MeOH (2:1) extracts of the leaves and roots of P. gaudichaudianum yielded two chromenes [2-methyl- 2-(4′-methyl-3′-pentenyl)-8-(3′′-methyl-2′′-butenyl)-2H-1- benzopyran-6-carboxylic acid (gaudichaudianic acid, 20) and methyl 2,2-dimethyl-1-oxo-2H-1-benzopyran-6-carbox- ylate 22 (21)], three prenylated benzoic acids [4-methoxy-3- (3′,7′-dimethylocta-2′-E-6′-dienyl)benzoic acid 15 (13), 4-hy- droxy-3-(3′,7′-dimethylocta-2′-E-6′-dienyl)benzoic acid 23 (22), and methyl 4-hydroxy-3-(3′-methyl-1′-oxo-2′-butenyl)ben- zoate 24 (methyl taboganate, 23)], and pinocembrin 20,21 (19). Compound 1 was isolated from the leaves of P. crassin- ervium as a white amorphous solid. Its molecular formula was established as C 17 H 22 O 5 by analysis of its HREIMS spectrum (m/z 306.1459, calcd 306.1467). The IR spectrum indicated the presence of hydroxyl groups (3380 cm -1 ), two carbonyl groups (1696, 1611 cm -1 ), and an aromatic ring (1489, 1421 cm -1 ). The 1 H NMR spectrum exhibited signals * To whom correspondence should be addressed. Tel/Fax: +55-11-3091- 3813. E-mail: majokato@iq.usp.br. † Instituto de Quı ´mica, Universidade de Sa ˜ o Paulo. ‡ Instituto de Quı ´mica, Universidade Estadual Paulista. § Instituto de Pesquisas do Jardim Bota ˆ nico. ⊥ Sec ¸ a ˜ o de Fisiologia e Bioquı ´mica de Plantas, Instituto de Bota ˆ nica. 1783 J. Nat. Prod. 2004, 67, 1783-1788 10.1021/np030530j CCC: $27.50 © 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 10/26/2004