Noreupenifeldin, a Tropolone from an Unidentified Ascomycete ⊥ Sloan Ayers, † Deborah L. Zink, † Joanne S. Powell, ‡ Christine M. Brown, ‡ Alan Grund, § Gerald F. Bills, 4 Gonzalo Platas, 4 Donald Thompson, ‡ and Sheo B. Singh* ,† Natural Products Chemistry, Merck Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065, Merck and Co., Inc., Branchburg Farm, 203 RiVer Road, SomerVille, New Jersey 08876, Bio-Technical Resources, 1035 South SeVenth Street, Manitowoc, Wisconsin 54220, and CIBE, Merck, Sharp & Dohme de Espana, S.A., Josefa Valcarcel 38, 28027 Madrid, Spain ReceiVed September 24, 2007 Noreupenifeldin (2), a new monotropolone derivative of the bistropolone eupenifeldin (1), was isolated from an unidentified ascomycete by bioassay-guided fractionation as part of our search for new anthelmintics. The structure of 1 was confirmed by comparison with literature data. The structure of 2 was elucidated from MS and 1D and 2D NMR data. Compounds 1 and 2 are diastereomers of pycnidione (3) and epolone A (4), respectively. Compounds 1-3 were evaluated for their anthelmintic activity against the parasitic worm Hemonchus contortus. Compounds 1 and 3 exhibited modest in Vitro activity, showing EC 90 50 and 83 µg/mL, respectively, in reducing motility of L3 larvae of H. contortus. Compound 2 was inactive, indicating that the second tropolone moiety is required for activity. Great success has been achieved in the development of anthel- mintic drugs in the last 50 years. During this period all current classes of anthelmintic synthetic drugs were developed, including the benzimidazoles and imidazothiazoles (such as levamisole). The discovery and development of the macrolactone natural product avermectin from Streptomyces aVermitilis was the most significant advance made during the 1980s, leading to the regulatory approval of ivermectin for treatment of parasitic infections. Ivermectin exhibited superior potency and excellent broad-spectrum activity. However, resistance to ivermectin and all other classes of anti- parasitic drugs has been observed, and infection by internal parasites such as Hemonchus contortus remains one of the greatest economic threats to the sheep and goat industry. Increased emergence of resistance to current drugs necessitates the continued need for further research to discover new classes of anthelmintics, especially those with novel modes of action. 1 Our strategy to discover new anthelmintics has been to screen extracts of terrestrial plants 2–4 and bacterial 5 and fungal fermenta- tions using in Vitro activity against H. contortus, 6 which is one of the most prevalent parasitic worms that infect small ruminants. Evaluation of in ViVo activity was accomplished using Heligmo- somoides polygyrus (a related organism) in mice. 7 An acetone extract of the liquid fermentation of an ascomycetous fungus showed in Vitro activity and was selected for further study. Bioassay-guided fractionation using the in Vitro H. contortus assay led to the isolation of a known bistropolone, eupenifeldin (1), and a new monotropolone derivative of 1 named noreupenifeldin (2). The isolation, structure elucidation, and anthelmintic activities of these compounds are described. An organic extract of the liquid fermentation was partitioned with methylene chloride and ethyl acetate. The methylene chloride and ethyl acetate extracts were combined and chromatographed on aC 18 preparative HPLC column to yield compounds 1 (57.6 mg/ L) and 2 (10.5 mg/L) (see Experimental Section for details). Compound 1 was obtained as an off-white solid. Low-resolution MS analysis indicated a molecular weight of 548. 1 H NMR data were essentially identical to literature data reported for eupenife- ldin, 8 allowing assignment of 1 as eupenifeldin rather than pycnidione (3). 9 Noreupenifeldin (2) was also obtained as an off-white solid. Its formula, C 32 H 40 O 6 , was deduced from the molecular ion of 521.2877 [M + H] + obtained by HRESIFTMS. This formula differs from eupenifeldin (1) by a loss of CO. The carbon count was confirmed by the 13 C NMR spectrum, which showed 32 signals. The 1 H NMR data of 2 were very similar to eupenifeldin (1), except for a few significant chemical shift differences. The aromatic resonances H-1′ and H-5′ were shifted upfield by approximately 1 ppm, and the aromatic methyl singlet H 3 -1′′ was shifted upfield by 0.34 ppm. These shifts were consistent with the substitution of the tropolone with a benzene ring, supported by the absence of an electron-withdrawing carbonyl group in the ring. The substitution of a benzene ring for a tropolone ring was also evident from the 13 C NMR spectrum, where two carbonyl signals were present from δ C 169–171 in 1, while only one signal was present in this region for 2. The skeleton of 2 was confirmed by the COSY, HMQC, and HMBC spectroscopic data. The relative configuration of 2 was confirmed to be the same as that of eupenifeldin (1) by the ROESY spectrum. Selected ROESY correlations are shown in Figure 1. The key correlations included H-8 to H-25, H-16, and H-11, confirming the cis-ring fusion at C8-C19. Similar key ROESY correlations were from H-18 to H-9 and H-24. These correlations suggested that the relative configuration of 2, at these positions, was identical to that of eupenifeldin (1) and that 2 was a diastereomer of epolone A(4). 6 The numbering scheme used here is different than in ref 6 ⊥ Dedicated to Dr. G. Robert Pettit of Arizona State University for his pioneering work on bioactive natural products. * To whom correspondence should be addressed. Tel: (732) 594-3222. Fax: (732) 594-6880. E-mail: sheo_singh@merck.com. † Natural Products Chemistry, MRL. ‡ Branchburg Farm. § Bio-Technical Resources. 4 CIBE. J. Nat. Prod. 2008, 71, 457–459 457 10.1021/np070513k CCC: $40.75  2008 American Chemical Society and American Society of Pharmacognosy Published on Web 12/21/2007