Anserinones A and B: New Antifungal and Antibacterial Benzoquinones from the Coprophilous Fungus Podospora anserina Hui-juan Wang, Katherine B. Gloer, and James B. Gloer* Department of Chemistry, University of Iowa, Iowa City, Iowa 52242 James A. Scott and David Malloch Department of Botany, University of Toronto, Toronto, Ontario M5S 1A1, Canada Received January 31, 1997 X Two new benzoquinones with antifungal, antibacterial, and cytotoxic activities have been isolated from liquid cultures of the coprophilous fungus Podospora anserina. The structures of anserinones A (1) and B (2) were assigned on the basis of MS and NMR results, and the absolute stereochemistry of 2 was deduced by analysis of 1 H-NMR data for its (R)- and (S)-2- phenylbutyryl ester derivatives. Antagonistic interactions among coprophilous (dung- colonizing) fungi often involve the production of a chemical agent by one species that inhibits the growth of another. 1 Our studies of these fungi have led to the discovery of a variety of new antifungal agents. 1,2 As part of a continuing study of coprophilous fungi, we investigated the chemistry of an isolate of Podospora anserina (Cesati) Niessl (Lasiosphaeriaceae) that dis- played antifungal activity. This investigation led to the isolation of two new benzoquinones, which we named anserinones A (1) and B (2). This report describes the isolation, structure elucidation, and biological activities of these metabolites. A subculture of an isolate of P. anserina (JS 162), originally obtained from mouse dung, inhibited the growth of coprophilous fungal competitors in direct competition assays, and the EtOAc extract of the filtered culture broth also exhibited antifungal effects. This extract was fractionated by radial chromatography on Si gel, and the resulting fractions were separated further by reversed-phase HPLC or preparative TLC to afford anserinones A (1) and B (2). Analysis of 1 H- and 13 C-NMR data for anserinone A (Table 1) revealed the presence of one aliphatic ketone carbonyl, two conjugated ketone units, one oxygenated olefinic carbon, three non-oxygenated olefinic carbons, one methylene unit, one OCH 3 group, and two ad- ditional methyl groups. The three carbonyls, together with the oxygenated sp 2 carbon and the OCH 3 group, required at least four oxygen atoms. The 1 H-NMR spectrum (Table 1) contained five signals that integrated for a total of 12 protons. On the basis of these data, and on HREIMS analysis, anserinone A was assigned the formula C 11 H 12 O 4 . The NMR data suggested a trisubstituted benzo- quinone-type structure for 1, and the quinone unit, together with the aliphatic ketone group, accounted for all six degrees of unsaturation. Irradiation of the methyl singlet at δ 2.26 (H 3 -10) and the methylene singlet at δ 3.65 (H 2 -8) in selective INEPT experiments (Table 1) revealed the connection of both of these groups to the aliphatic ketone carbonyl to give an acetonyl group. On the basis of chemical shift and selective INEPT data, the olefinic carbon at δ 158.3 must be linked to the OCH 3 group and to the upfield-shifted olefinic methine (δ 107.5/5.92). The substituted olefinic quinone carbons at δ 136.2 and δ 144.1 must, therefore, bear the remaining methyl and acetonyl substituents. Selective INEPT correlations of the vinyl methyl signal at δ 1.97 to C-1 (δ 186.9) and of the acetonyl methylene signal at δ 3.65 to C-4 (δ 181.4) required a 1,4- benzoquinone structure. Although additional selective INEPT correlations (Table 1) supported the assignment of structure 1 for anserinone A, the position of the OCH 3 group could not be unambiguously determined. Because of its expected upfield-shift effect on -carbon signals, 3 however, this group was assigned the position adjacent to C-4 (δ 181.4) rather than to C-1 (δ 186.9). Analysis of the 1 H-, 13 C-, and DEPT NMR spectra for anserinone B (2) indicated the molecular formula C 11 H 14 O 4 . This formula was confirmed by HREIMS, and differs from that of anserinone A (1) by addition of two hydrogen atoms. The EIMS also contained a * To whom correspondence should be addressed. Phone: (319) 335- 1361. FAX: (319) 335-1270. E-mail: james-gloer@uiowa.edu. X Abstract published in Advance ACS Abstracts, June 1, 1997. Table 1. NMR Data for Anserinones A (1) and B (2) anserinone A (1) anserinone B (2) position δH (mult.) a δC b selective INEPT (C no.) b δH (mult., JHH) a,c δC b 1 186.9 187.3 2 144.1 143.4 3 136.2 139.6 4 181.4 182.9 5 158.3 158.3 6 5.92 (s) 107.5 1, 2, 4, 5 5.90 (s) 107.3 7 1.97 (s) 12.8 1, 2, 3 2.08 (s) 12.8 8 3.65 (s) 40.9 2, 3, 4, 9 2.68 (d, 6.6) 35.9 9 203.0 3.96 (m) 67.6 10 2.26 (s) 30.2 9 1.25 (d, 6.2) 24.0 11 3.79 (s) 56.2 5 3.78 (s) 56.1 a Recorded at 300 MHz. b Recorded at 75 MHz. c JHH ) Hz. 629 J. Nat. Prod. 1997, 60, 629-631 S0163-3864(97)00071-2 CCC: $14.00 © 1997 American Chemical Society and American Society of Pharmacognosy