Antifungal Clerodane Diterpenes from Macaranga monandra (L) Muell. et Arg. (Euphorbiaceae) MARTIN A. SALAH, ² ERDAL BEDIR, ² NGEH J. TOYANG, ‡ IKHLAS A. KHAN, ² M. DEWAYNE HARRIES, § AND DAVID E. WEDGE* ,§ National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, Heifer Project International, NW Province, Cameroon, and United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, Mississippi 38677 Hexane and ethyl acetate phases of the methanol extract of Macaranga monandra showed fungal growth inhibition of Colletotrichum acutatum, C. fragariae and C. gloeosporioides, Fusarium oxysporum, Botrytis cinerea, Phomopsis obscurans, and P. viticola. Bioassay-guided fractionation led to the isolation of two active clerodane-type diterpenes that were elucidated by spectroscopic methods (1D-, 2D-NMR, and MS) as kolavenic acid and 2-oxo-kolavenic acid. A 96-well microbioassay revealed that kolavenic acid and 2-oxo-kolavenic acid produced moderate growth inhibition in Phomopsis viticola and Botrytis cinerea. KEYWORDS: Fungicide; antifungal activity; natural products; phytopathogenic fungi; microtiter assay; bioautography INTRODUCTION Owing to the continuing development of microbial resistance in medicine and agriculture, discovery of new antimicrobial substances is an important, if not urgent, research objective. In addition, the desire for safer agrochemicals with less environ- mental and mammalian toxicity is a major concern. Particularly desirable is the discovery of novel prototype antimicrobial agents representing new chemical classes that operate by different modes of action than existing antifungal agents, and conse- quently, lack cross-resistance to chemicals currently used (1, 2). Following natural product leads offers an efficient approach to discovering and optimizing new agrochemicals for disease control. After screening more than 75 different plants that were initially selected for their frequent application by indigenous people in Cameroon for medicinal needs, only two were active in the assays (Macaranga mondra and Pterygota kamerunensis). Therefore, we began a study to evaluate several natural products from Macaranga monandra for their potential use as disease control agents for filamentous pathogenic fungi of the genera Colletotrichum, Botrytis, Phomopsis, and Fusarium. Macaranga monandra (L) Muell. et Arg. is a tree found in equatorial rain forest of Cameroon and other central African countries. The genus Macaranga is represented by 300 species (3), distributed throughout the tropical and subtropical regions. Previously reported phytochemicals from the genus include a prenylstilbene (4-6), flavonoids (7-10), a diterpene (11), tannins (12), and sterols (13, 14). The presence of diterpene clerodanes or antifungal activity has not been previously reported in Macaranga monandra. On the basis of the growth inhibition of C. acucatum, C. fragariae, and C. gloeosporioides in preliminary screens using direct bioautography, we selected to study in detail the hexane and EtOAc phases of the MeOH extract of M. monandra. We now describe the bioassay-guided isolation, antifungal activity using a novel 96-well microbioassay method, and the structure elucidation of kolavenic acid (1)(15) and 2-oxo-kolavenic acid (2)(16), which are neo-clerodane-type diterpenes previously isolated from Aristolochia species and Eperua purpurea (15, 16). MATERIALS AND METHODS General Experimental. The 1D- and 2D Nuclear Magnetic Reso- nance (NMR) spectra were obtained on a Bruker Avance DRX 500 FT spectrometer operating at 500 and 125 MHz for 1 H and 13 C measurements, respectively. The chemical shift values are reported as parts per million (ppm) units relative to tetramethylsilane (TMS) for 1 H- and 13 C-; coupling constants are in Hz. For the 13 C NMR spectra, multiplicities were determined by a DEPT experiment. LC-ESIMS were obtained using a Bruker BioApex FT-MS in ESI mode. Chromatographic Conditions. Thin layer chromatography (TLC), precoated Si 250F plates (Merck); developing system, hexanes/EtOAc/ MeOH (10:10:2); developing temperature, 22 °C; visualization, vanillin/ H 2SO4 (1 g vanillin in 100 mL of 20% H2SO4 (in EtOH)). Column chromatography, silica gel 230-400 mesh (Baker). Plant Material. Macaranga monandra (L) Muell. et Arg. (Euphor- biaceae) stem bark was collected in Cameroon in the dry season of 1998. The voucher specimen was identified and authenticated by Dr. * Corresponding author. Tel.: 662 915-1137. E-mail: dwedge@ olemiss.edu. ² National Center for Natural Products Research. ‡ Heifer Project International. § United States Department of Agriculture. J. Agric. Food Chem. 2003, 51, 7607-7610 7607 10.1021/jf034682w CCC: $25.00 © 2003 American Chemical Society Published on Web 11/15/2003