Tagetolone and Tagetenolone: Two Phytotoxic Polyketides from Alternaria tagetica M. Marcela Gamboa-Angulo, † Karlina Garcı ´a-Sosa, † Fa ´ tima Alejos-Gonza ´ lez, † Fabiola Escalante-Erosa, † Guillermo Delgado-Lamas, ‡ and Luis M. Pen ˜ a-Rodrı ´guez* ,† Grupo de Quı ´mica Orga ´ nica, Unidad de Biotecnologı ´a, Centro de Investigacio ´n Cientı ´fica de Yucata ´n A.C., Calle 43 No. 130, Colonia Chuburna ´, Me ´rida, Yucata ´n, Me ´xico 97200 and Instituto de Quı ´mica, Universidad Nacional Auto ´noma de Me ´xico, Circuito Exterior, Ciudad Universitaria, Me ´xico D.F. 04510 Two new phytotoxic polyketides, tagetolone (1) and tagetenolone (2), in addition to tyrosol and p-hydroxybenzoic acid, have been isolated from the organic crude extract of culture filtrates from the fungal pathogen Alternaria tagetica. Complete characterization of all structures was carried out following a careful analysis of their spectroscopic data (IR, MS, 1 H and 13 C NMR, and 2D NMR experiments). Keywords: Alternaria tagetica; Tagetes erecta; phytotoxins; marigold; tagetolone; tagetenolone; tyrosol; p-hydroxybenzoic acid INTRODUCTION The fungal pathogen Alternaria tagetica severely infects marigold (Tagetes erecta L.) plants, causing necrotic spots on leaves, stems, and petals and, in some cases, resulting in plant death (1). As part of our search for the bioactive metabolites produced by A. tagetica, we have previously reported the isolation and identification of the nonhost-specific toxin zinniol and two new dimeric nonphytotoxic me- tabolites, bis-7-O-7′′,8-O-8′′- and bis-7-O-8′′,8-O-7′′- zinniol (2). Although these metabolites were obtained from a low-polarity (hexane) fraction resulting from a solvent partition of the original crude extract, testing the medium-polarity fraction (ethyl acetate) in the leaf spot assay produced necrotic damage which gave a clear indication of the presence of a second group of phytotoxic metabolites biosynthesized by A. tagetica. We report here the bioassay-guided purification of the medium- polarity fraction and the identification of the phytotoxic metabolites tagetolone (1), tagetenolone (2), and tyrosol, in addition to a fourth, nonphytotoxic, metabolite iden- tified as p-hydroxybenzoic acid (Figure 1). MATERIALS AND METHODS UV spectra were determined in a DU-65 Beckman UV-vis spetrophotometer using MeOH as solvent. IR spectra were measured using a FT-IR Nicolet Magna 750 or a FT-IR Protege 460. LREIMS and HRFABMS were recorded at 70 eV on a JEOL-JMS-SX102A mass spectrometer and HREIMS was recorded at 70 eV on a JEOL-JMS-AX-505HA mass spectrom- eter. GC-MS analyses were performed on a Hewlett-Packard 5890 gas chromatograph coupled to a Hewlett-Packard 5971A mass selective detector. Following are the conditions used for GC analyses: 0.5 µL of sample; Ultra 1 column (Hewlett- Packard, cross-linked methyl silicone gum, 25 m long, 0.32 mm i.d., 0.52 µm fill thickness); flow rate, 1 mL/min; temperature programs, (a) T 1 ) 150 °C, T2 ) 290 °C, (b) T1 ) 70 °C, T2 ) 290 °C, gradient ) 10 °C/min. NMR spectra were recorded on Varian Unity-300, Bruker AMX-400, and Varian Unity Plus- 500 spectrometers, at 300, 400, and/or 500 MHz for 1 H, and 75, 100, or 125 MHz for 13 C, using CDCl3 or (CD3)2CO (Aldrich Chemical Co., Milwakee, WI) as solvents and tetramethyl- silane (TMS; Aldrich) as internal standard. Industrial-grade solvents were glass-distilled in the laboratory prior to use. Flash column (flash CC) and vacuum liquid chromatography (VLC) purifications were carried out using silica gel (200-400 mesh and TLC-grade, respectively) from Aldrich. Preparative * To whom correspondence should be addressed. Phone: +52-9-981-3923. Fax: +52-9-981-3900. E-mail: lmanuel@ cicy.mx. † Grupo de Quı ´mica Orga ´ nica, Unidad de Biotecnologı ´a, Centro de Investigacio ´n Cientı ´fica de Yucata ´n A.C.. ‡ Instituto de Quı ´mica, Universidad Nacional Auto ´noma de Me ´xico, Circuito Exterior, Ciudad Universitaria. Figure 1. Metabolites of A. tagetica: tagetolone (1), tag- etenolone (2), and zinniol (3). 1228 J. Agric. Food Chem. 2001, 49, 1228-1232 10.1021/jf000872k CCC: $20.00 © 2001 American Chemical Society Published on Web 02/10/2001