Guaiane Sesquiterpenes from Biscogniauxia nummularia Featuring Potent Antigerminative Activity

Séverine Amand†, Aude Langenfeld†, Alain Blond†, Joëlle Dupont‡, Bastien Nay†, and Soizic Prado*†

† Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS/MNHN, Muséum National d’Histoire Naturelle, 57 Rue Cuvier (CP54), 75231 Paris Cedex 05, France

‡ Laboratoire de Mycologie, UMR 7205 CNRS/MNHN, Muséum National d’Histoire Naturelle, 57 Rue Cuvier (CP39), 75231 Paris Cedex 05, France

J. Nat. Prod., Article ASAP

DOI: 10.1021/np2009913

Publication Date (Web): April 9, 2012

Copyright © 2012 The American Chemical Society and American Society of Pharmacognosy

Abstract

Xylaranone, a previously unreported guaiane sesquiterpene along with the known terpenoid xylaranol B and the two mellein derivatives 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin and 3,5-dimethyl-8-hydroxy-3,4-dihydroisocoumarin were isolated from Biscogniauxia nummularia. Pogostol was also isolated from this fungus, and in light of our spectroscopic data, its structure was revised and corrected. This fungus, which was isolated as an endophyte from the plum yew Cephalotaxus harringtonia, is also suspected of being a pathogen. Interestingly, we report here the potent antigerminative activity of xylaranone and xylaranol B against seeds of Raphanus sativus at concentrations comparable to glyphosate, a commonly used herbicide. This effect suggests a role for these metabolites in the latent fungal pathogenesis of B. nummularia.

In the course of a program on the structure determination and ecological studies of metabolites from endophytic fungi, we are currently working on Biscogniauxia nummularia (Bull.) Kuntze (6E2a). This is an apparent endophytic fungus isolated from the plum yew Cephalotaxus harringtonia (Siebold & Zucc.) Koidz. and identified by the species-level molecular marker of fungi, the ITS rDNA sequences. Biscogniauxia belongs to the xylariaceous genus represented in Europe by 10 known species growing on the bark of trees and shrubs, preferably on dead or dying branches and more rarely on trunks. It is currently suspected of being pathogenic to certain trees.(1) Although it is usually accepted that endophytes can have profound effects on plant ecology, fitness, and evolution, some examples attest that under certain conditions endophytes act as parasites and cause disease or reduce the fitness of their host plants.(2, 3) Indeed, endophytes and pathogens share some virulence factors such as phytotoxic mycotoxins.(4) For example, Biscogniauxia mediterranea (De Not.) Kuntz, an endophytic fungus widespread in Sardinian oak forests, is considered as the main causes of cork oak (Quercus suber L.) decline. From this fungus, Evidente et al. have isolated and characterized a pyrano derivative, biscopyran, as the main phytotoxic metabolite.(5) Recently, B. nummularia has been reconsidered as a pathogenic agent of the European beech (Fagus sylvatica L.) in Sicily and Calabria (Italy).(6) Nevertheless, the role of secondary metabolites produced by this ascomycete had not yet been studied with regard to their role in plant decay.

We report here the isolation and characterization, based on extensive 2D-heteronuclear NMR studies, of the new guaiane sesquiterpene 1 along with a previously known terpenoid, xylaranol B,(7) and two mellein derivatives, 3,5-dimethyl-8-hydroxy-3,4-dihydroisocoumarin (5-methymellein)(8) and 3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin (8-methoxy-5-methylmellein).(9) Moreover, the terpenoid previously reported as pogostol(10-12) was also isolated. In view of our analytical data, we suggest the revised structure 2 for this compound. Moreover, remarkable antigerminative activity against the seeds of Raphanus sativus L. was noted for compound 1 and xylaranol B. Such an effect, which is comparable to the herbicide glyphosate, could explain the role of these metabolites in the suspected phytopathogenesis of B. nummularia.

Compound 1 was obtained as a colorless oil. Its HRESIMS showed the protonated molecular ion [M + H]⁺ corresponding to the formula C₁₅H₂₄O₂, which implies four degrees of unsaturation. The IR spectrum exhibited absorption bands at ν_max 3421 and 1732 cm^–1 indicative of the presence respectively of a hydroxy and a carbonyl group. The ¹³C J-modulated NMR spectrum (CD₃OD) exhibited the resonances of 15 carbons including three methyls, five methylenes including an sp² (═CH₂) at δ_C 108.6, four methines, three quaternary carbons including a carbonyl at δ_C 223.8, and an ethylenic carbon at δ_C 153.3 (Table 1). Taking into account the four degrees of unsaturation, the molecule 1 should include two rings. The ¹H NMR spectrum displayed typical signals of three methyls, two tertiary (δ_H 1.73, s, H-12 and δ_H 1.15, s, H-14) and one secondary (δ_H 1.02, d, H-15), and an olefinic methylene (δ_H 4.62 and 4.70, m, H-13). The ¹H–¹H COSY spectrum (CD₃OD) identifies three main substructures, which are represented in Figure 1. The HMBC correlations were indicative of a cyclopentanone ring moiety. Methines H-1 (δ_H 2.57) and H-4 (δ_H 2.01), methylene H-2 (δ_H 2.72 and 2.32), and methyl CH₃-15 (δ_H 1.02) were correlated with carbonyl C-3 at δ_C 223.8. The chemical shift of the quaternary carbon (C-10) at δ_C 75.3 indicated that it was linked to an oxygen. Moreover, it was correlated to the two methines H-1 (δ_H 2.57) and H-5 (δ_H 2.13), the methylenes H-9 (δ_H 1.94; 1.70) and H-2, and the methyl group CH₃-14 at δ_H 1.15. These HMBC correlations with those observed in the ¹H–¹H COSY allowed the construction of a seven-membered ring. Finally, the olefinic moiety was linked to C-7 because of the correlation between the methine H-7 (δ_H 2.20) and the C-11 at δ_C 153.3. The relative stereochemical assignments of carbons C-1, C-4, C-5, C-7, and C-10 were resolved using the ¹H–¹H NOESY experiments in CD₃OD and C₅D₅N (Figure 1). These displayed correlations in CD₃OD between H-1 and H-5, indicating a cis ring fusion. Moreover, the spatial correlations of CH₃-14 (δ_H 1.15) with H8-b (δ_H 1.56) and H9-a (δ_H 1.94) clearly indicated that they are on the same face of the molecule. On the other hand, the correlations of H-1 with H-9b (δ_H 1.70) suggested that CH₃-14 and H-1 are placed on opposite faces. Moreover, in C₅D₅N, a correlation between the hydroxy (δ_H 5.74) on C-10 and H-1 confirmed that CH₃-14 and H-1 are on opposite sides. In addition, H-7 (δ_H 2.20) showed a cross-peak with H-8a (δ_H 1.80) and H-9b (δ_H 1.70). These results thereby placed the C-7 isoprenyl group on the α-face. Similarly, NOE correlations between H-4 and H-1 indicated that the methyl CH₃-15 is also on this face. Consequently, the structure of 1 was assigned as depicted and named xylaranone. It is noteworthy that a trans-fused isomer of 1 was synthesized by Piers et al. as an intermediate in the stereoselective synthesis of α-bulnesene.(13)

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