Pulvinulin A, graminin C, and cis-gregatin B--new natural furanones from Pulvinula sp. 11120, a fungal endophyte of Cupressus arizonica.

In continuing our search for bioactive and/or novel secondary metabolites from endosymbiotic microorganisms of the Sonoran desert [1], we have investigated an EtOAc extract derived from the solid (potato dextrose agar, PDA) culture of the endophytic fungal strain, Pulvinula sp. 11120, aff. Pulvinula archeri (Pyronemataceae, Pezizoymycetes, Pezizomycotina, Ascomycota) isolated from the healthy leaf tissue of the Arizona cypress, Cupressus arizonica (Cupressaceae). Herein we report the isolation and structure elucidation of three new natural furanone metabolites, pulvinulin A (1), graminin C (2), and cis-gregatin B (3), together with graminin B (4), and 10-norparvulenone (5) from Pulvinula sp. 11120 (Figure 1). This constitutes the first report of metabolites from a fungal strain of the genus Pulvinula. 10-Norparvulenone (5) with antiinfluenza viral activity [2] has previously been reported from an endophyte of a mangrove plant [3]. Figure 1 Structures of pulvinulin A (1), graminin C (2), cis-gregatin B (3), graminin B (4), and 10-norparvulenone (5) from Pulvinula sp. 11120. Natural furanones constitute a rare and a comparatively small class of biologically active fungal metabolites previously encountered in the fungal genera, Aspergillus [4,5], Cephalosporium [6], and Penicillium [7]. Despite its simplicity, the central core of these metabolites has undergone several structural revisions over the past three decades. The core structure, 3-acyl-4-methoxyfuran-2(3H)-one, the first proposed for these metabolites [8,9], was later revised to 4-acyl-3-methoxyfuran-3(2H)-one [10], and finally to the currently accepted 4-(methoxycarbonyl)furan-3(2H)-one [11]. Several fungal-derived furanones have been reported to have phytotoxic [12], antibacterial and antifungal activities [9] and implicated as the causal agents of stripe disease of wheat [12] and brown stem rot of adzuki beans [9]. Pulvinulin A (1), [α]D25 +108, was obtained as a colorless oil, the molecular formula of which was determined to be C18H28O4 on the basis of its HRESIMS (m/z 331.1851 [M+Na]+; calcd 331.1880), accounting for five degrees of unsaturation. 1H and 13C NMR spectra together with HSQC data of 1 indicated the presence of a tertiary methyl [δH 1.37 (s); δC 21.6], two primary methyls, one methoxyl, five methylenes, two methines of which one is oxygenated [δH 3.28 (d, J = 10.4 Hz); δC 75.9], four olefinics, an oxygenated quaternary carbon (δC 83.6), one ketone carbonyl carbon (δC 208.3) and an ester carbonyl carbon (δC 167.6). The presence of two spin systems, CH3CH2CH2CH2CH2CH(O)CH– and CH3CH2CH=CH–CH=CH– in 1 was evident from its 1H–1H COSY data (Figure 2). The connectivity of these spin systems, tertiary methyl, methoxycarbonyl, and non-protonated carbons was established by analysis of the HMBC data (Figure 2). HMBC correlations of H-1'/C-4 and H-1'/C-5 established the connectivity between C-5 and C-1'. Tertiary methyl protons (δH 1.37) showed HMBC correlations to the ketone carbonyl (C-4) and C-1' placing the tertiary methyl at C-5. HMBC correlations of H-3/C-4, H-2/C-3, and H-2/C-4 established the connectivity between C-3 and C-4. Placement of the methoxycarbonyl group at C-3 was supported by the HMBC correlations of H-2 and H-3 to each other and to this carbonyl carbon (C-1'"). Since two double bonds, and ketone and ester carbonyls accounted for four degrees of unsaturation, 1 should possess a monocyclic framework. Based on its molecular formula and the presence of a ketone and an ester functionality accounting for 3 of the 4 oxygen atoms in 1 suggested that the two oxygenated carbons C-2 and C-5 were linked to each other through a common oxygen atom to form a furan ring system. The E configuration for Δ1',2' and the Z configuration for Δ3',4' double bonds in 1 were assigned based on their 3JHH values of 15.2 Hz and 11.2 Hz, respectively. These data indicated that 1 possessed a structure similar to that of graminin B (4), also encountered in this study (see below) and recently reported as a metabolite of the fungus, Paraconiothyrium sp. [13]. The only difference between 1 and 4 was found to be the absence of a Δ2,3 double bond in 1. Thus, 1 was suspected to be a 2,3-dihydro derivative of graminin B. The ROESY spectrum of 1 showed cross peaks among 5-CH3, H-2 and H-3 suggesting that these have co-facial orientation (Figure 1). Catalytic hydrogenation (H2/Pd-C) and acetylation (Ac2O/pyridine) of pulvinulin A (1) afforded its tetrahydro derivative 6 and the enol acetate 7 (Figure 3), respectively, further supporting the proposed structure 1 for this metabolite. The absolute configuration of the only chiral center (C-5) in graminin B (4) was assumed to be R by comparison of its [α]D data with known furanones [13]. However, the presence of three chiral centers (C-2, C-3, and C-5) in pulvinulin A (1) precluded the use of its [α]D value for this purpose. Biosynthetically, 1 may share a common pathway with 2–4 co-occurring in Pulvinula sp. 11120 suggesting that these would bear the same absolute configuration at C-5. Since 2–4 have been determined to have R configuration at C-5 (see below), pulvinulin A (1) may be assumed to have the same absolute configuration at this position. Thus, the structure of pulvinulin A was established as methyl (2S,3R,5R)-5-[(1'E,3'Z)-hexa-1,3-dienyl]-5-methyl-4-oxo-2-pentyl-2,3,4,5-dihydrofuran-3 carboxylate (1). Figure 2 1H–1H COSY, and selected HMBC and ROESY correlations of 1. Figure 3 Structures of chemical transformation products 6–9 obtained from metabolites 1–3. Metabolite 2, [α]D25 +159, was obtained as a colorless oil. A molecular formula of C18H24O4 was determined for 2 on the basis of its HRESIMS (m/z 305.1746 [M+H]+, calcd C18H25O4: 305.1747) and 13C NMR data, accounting for seven degrees of unsaturation. The 1H and 13C NMR data of 2 were almost identical with those of graminin B (4) [13] except that 2 contained an additional double bond which was placed at C-1"(2") based on its NMR data (see Experimental). Because of its structural relationship to graminin B (4), metabolite 2 was given the trivial name graminin C. Catalytic hydrogenation of 2 afforded 8 (Figure 3), which was shown to be identical with the hydrogenation product of 4, further supporting their structural relationship. The optical rotation value of 8 ([α]D25 −64) was found to be almost identical to that of the hydrogenation product of 4 ([α]D25 −61) suggesting both 2 and 4 may be assumed to have the same absolute configuration at the only chiral center (C-5) of these molecules. Since the absolute configuration at C-5 of 4 has been assumed to be R [13], the absolute configuration at C-5 of 2 may tentatively be assigned as R. Thus, the structure of graminin C was established as methyl (5R)-5-[(1'E,3'Z)-hexa-1,3-dienyl]-5-methyl-4-oxo-2-[(E)-pent-1-enyl]-4,5-dihydrofuran-3 carboxylate (2). Comparison of the 1H and 13C NMR data of the metabolite 3, [α]D25 +168, with those reported for synthetic cis-gregatin B [11] suggested that they are identical with each other. The absolute configuration at the only chiral center (C-5) in synthetic 3 was established as R based on the absolute configuration of the starting materials used for its synthesis, but its [α]D value has not been determined due to the lack of sufficient material [11]. Catalytic hydrogenation of 3 afforded its new derivative, 1'(2'),3'(4')- tetrahydro-gregatin B (9) (Figure 3), [α]D25 −57. Metabolites 4 and 5 were identified as graminin B [13] and 10-norparvulenone [3] by comparison of their spectroscopic data with those reported. Antimicrobial activity of furanones 1–4 against Escherichia coli ATCC 25922 was evaluated using the agar diffusion method [14]. Diameters of the inhibition zones were measured after incubation for 24 h at 37°C. Ampicillin was used as the positive control. The activity data (Table 1) suggested that the antibacterial activity of these metabolites depended on the number of double bonds that are in conjugation with the carbonyl moiety of the furanone ring. Table 1 Antibacterial activity of metabolites 1–4 against E. coli ATCC 25922.