Griseaketides A–D, New Aromatic Polyketides from the Pathogenic Fungus Magnaporthe grisea

Magnaporthe grisea is the causal agent of rice blast disease, which is the most serious disease of cultivated rice. Aromatic polyketides are its typical metabolites and are involved in the infection process. In the search for novel lead compounds, chemical investigation of the fungus M. grisea M639 has led to the isolation of four new aromatic polyketides (salicylaldehyde skeleton bearing an unsaturated side chain), griseaketides A–D (1–4), as well as 15 known compounds (5–19). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analyses, including HR-MS, 2D NMR. Compound 12 showed prominent activity that killed 94.5% of C. elegans at 400 ppm and 66.9% at 200 ppm over 24 h. This is the first report describing the nematicidal activity of this type aromatic polyketide.


Introduction
Exploration of natural sources of novel bioactive compounds as drugs or lead compounds has been an emerging field over the past decades, and exciting evidence has been provided by the isolation of microbe-derived metabolites [1]. The fungal kingdom includes many species with unique and unusual biochemical pathways, which results in lots of structurally fascinating secondary metabolites with promising biological and pharmacological properties, such as penicillin, cyclosporine and statins [2].
A group of structurally and actively diverse metabolites were produced by plant pathogenic strains [3][4][5], which are responsible for function as essential determinants of pathogenicity or virulence. The rice blast fungus Magnaporthe grisea (imperfect stage of Pyricularia grisea Sacc.) causes a serious disease on agriculturally significant plants including rice, wheat, and barley. Each year rice blast causes losses of 10-30% of the rice harvest and therefore poses a threat to the world's most important food security crop [6]. Previous, studies have shown that aromatic polyketide phytotoxins [7], O-nitrophenol derivatives [8], and naphthalenones [9] are the typical secondary metabolites of M. grisea. Some secondary metabolites (such as melanin, siderophores and fumonisin) of pathogenic fungi often function as essential determinants of pathogenicity and are involved in the infection process [10,11]. Thus, examining their secondary metabolism leads to the discovery of novel compounds with interesting structures or modes of action that could be useful for agrochemistry or pharmacology [5]. In the work, Table 1. 1 H and 13 C NMR Data of Compounds 1 and 2 (δ in ppm, J in Hz).

No. 1 (in CDCl 3 ) 2 (in CDCl 3 )
δ H δ C HMBC δ H δ C HMBC  Compound 2 was obtained as optically active amorphous powder ([α] 18 D -16.0 c 0.16, MeOH) and its molecular formula of C14H14O3 was established by the negative HR-ESI-MS, revealing 8 degrees of unsaturation. Analysis of the 1 H and 13 C NMR (Table 1) data of 2 revealed the presence of a monooxygenated 1,2,3-trisubstituted aromatic ring, two pairs of double bonds, a primary alcohol, a carbonyl group, an oxymethine and one methyl ( Figure 1). These signals revealed that 2 is similar to 1 and suggested to be an aromatic polyketide [13]. The lower-field shifting of carbon signal (δC-8 84.4) in 2 and the observed HMBC correlations ( Figure 2) from H-1 to C-8 suggested that C-1 and C-8 are adjacent substituents on the aromatic ring and form a 1,3-dihydroisobenzofuran system. One branch was deduced to be −C-8−C-9−C-10−C-11−C-12 from complete interpretations of key cross-peaks in the COSY spectrum (H-8/H-9/H-10/H-11/H-12) together with key cross-peaks (from H-8 to C-9 and C-10; from H-10 to C-8, C-11 and C-12; from H-11 to C-9, C-10 and C-13; from H-14 to C-12 and C-13) in the HMBC spectrum to determinate the side chain moiety ( Figure 2). The configuration of conjugated double bonds was elucidated as E geometries by their coupling constants (J9−10 = 15.1 Hz, J11−12 = 15.7 Hz) [14]. The configurations of C-8 cannot be determined by the present data. Thus, the structure of 2 was determined as shown in Figure 1 and named as griseaketide B.
Compound 3 was obtained as a pale yellow oil. HR-ESI-MS analysis of 3 provided a molecular formula of C14H16O3, corresponding to an unsaturation number of 6. The UV absorption maxima at 226 and 271 nm suggested the presence of a conjugated system in the molecule. Compound 3 was very similar to compound 2, but the ketone (δC 198.8) of 2 was replaced by one hydroxyl in 3 ( Table  2). The 1    correlations from H-1 to C-8 to suggest that C-1 and C-8 are adjacent substituents on the aromatic ring and formed a 1,3-dihydroisobenzofuran system ( Figure 2). The E geometries of the two double bonds was inferred by coupling constants (J9−10 = 15.1 Hz, J11−12 = 15.2 Hz) [14]. The configurations of C-8 and 13-OH cannot be determined by the present data. Thus, the relative configuration of 3 is shown as Figure 1 and named as griseaketide C.   correlations from H-1 to C-8 to suggest that C-1 and C-8 are adjacent substituents on the aromatic ring and formed a 1,3-dihydroisobenzofuran system ( Figure 2). The E geometries of the two double bonds was inferred by coupling constants (J9−10 = 15.1 Hz, J11−12 = 15.2 Hz) [14]. The configurations of C-8 and 13-OH cannot be determined by the present data. Thus, the relative configuration of 3 is shown as Figure 1 and named as griseaketide C.  correlations from H-1 to C-8 to suggest that C-1 and C-8 are adjacent substituents on the aromatic ring and formed a 1,3-dihydroisobenzofuran system ( Figure 2). The E geometries of the two double bonds was inferred by coupling constants (J9−10 = 15.1 Hz, J11−12 = 15.2 Hz) [14]. The configurations of C-8 and 13-OH cannot be determined by the present data. Thus, the relative configuration of 3 is shown as Figure 1 and named as griseaketide C.    (Table 1) data of 2 revealed the presence of a monooxygenated 1,2,3-trisubstituted aromatic ring, two pairs of double bonds, a primary alcohol, a carbonyl group, an oxymethine and one methyl ( Figure 1). These signals revealed that 2 is similar to 1 and suggested to be an aromatic polyketide [13]. The lower-field shifting of carbon signal (δ C-8 84.4) in 2 and the observed HMBC correlations (Figure 2) from H-1 to C-8 suggested that C-1 and C-8 are adjacent substituents on the aromatic ring and form a 1,3-dihydroisobenzofuran system. One branch was deduced to be −C-8−C-9−C-10−C-11−C-12 from complete interpretations of key cross-peaks in the COSY spectrum (H-8/H-9/H-10/H-11/H-12) together with key cross-peaks (from H-8 to C-9 and C-10; from H-10 to C-8, C-11 and C-12; from H-11 to C-9, C-10 and C-13; from H-14 to C-12 and C-13) in the HMBC spectrum to determinate the side chain moiety (Figure 2). The configuration of conjugated double bonds was elucidated as E geometries by their coupling constants (J 9−10 = 15.1 Hz, J 11−12 = 15.7 Hz) [14]. The configurations of C-8 cannot be determined by the present data. Thus, the structure of 2 was determined as shown in Figure 1 and named as griseaketide B.
Compound 3 was obtained as a pale yellow oil. HR-ESI-MS analysis of 3 provided a molecular formula of C 14 H 16 O 3 , corresponding to an unsaturation number of 6. The UV absorption maxima at 226 and 271 nm suggested the presence of a conjugated system in the molecule. Compound 3 was very similar to compound 2, but the ketone (δ C 198.8) of 2 was replaced by one hydroxyl in 3 ( Table 2) Figure 2). The HMBC experiment showed correlations from H-1 to C-8 to suggest that C-1 and C-8 are adjacent substituents on the aromatic ring and formed a 1,3-dihydroisobenzofuran system (Figure 2). The E geometries of the two double bonds was inferred by coupling constants (J 9−10 = 15.1 Hz, J 11−12 = 15.2 Hz) [14]. The configurations of C-8 and 13-OH cannot be determined by the present data. Thus, the relative configuration of 3 is shown as Figure 1 and named as griseaketide C. The molecular formula of griseaketide D (4) was found to be C 14 H 18 O 4 by HR-ESI-MS and 13 C NMR analysis. Its 1 H and 13 C NMR (Table 2) signals were similar to that of 1, except for the carbonyl group in 1 that was replaced by a hydroxyl group at C-11. On the basis of the consecutive COSY correlations from H-8 to H-14 ( Figure 2) and the coupling constants (J 12−13 = 14.6 Hz), the side chain was elucidated as 1',3',4'-trihydroxy-5'E-heptaenyl. The observed HMBC (Figure 2) correlations of H-1/C-10 suggested a seven membered ether ring was fused to the trisubstituted benzene ring through an oxygen bridge between C-1 and C-10. The relative configuration of 4 was deduced from ROESY correlations ( Figure 3) and comparisons with data reported in the literature [15]. The ROESY cross-peaks of H-8/H-9β and H-10 indicated that they are all cofacial and assigned as β-oriented which were consistent with xylarinol B [15]. The configuration of 11-OH cannot be determined by the present data. Accordingly, the structure and relative configuration of 4 was established as shown. Nematicidal activity of compounds. The pure compounds 1 and 5-12 were tested for their nematicidal activity. Compounds 5-8 showed weak nematicidal activities against Caenorhabditis elegans at 400 ppm over 48 h, but compound 12 showed prominent activity that killed 94.5% of C. elegans at 400 ppm and 66.9% at 200 ppm over 24 h.

Discussion
Rice blast, caused by infection of the rice blast fungus, Magnaporthe grisea, is the most destructive pathogen of rice worldwide. Many metabolites from rice blast fungus have been identified and they show different activities. Pyriculol caused a necrotic lesion in a rice wounding assay and showed inhibition in a spore germination bioassay [28]. Compound pyricuol inhibited shoot growth showing a stronger effect than pyriculol and dihydropyriculol [28,29]. In our experiment, part salicylaldehyde-type products showed nematicidal activity, and among them aldehyde group-containing compounds showed nematicidal activity, which is consistent with the literature [30], while pyricuol (12) showed a prominent nematicidal activity, which was distinguished by a different substitute on the side chain, and this further provides us with an indication of the nematicidal active compounds.

Microbial Material
The fungal strain of Magnaporthe grisea M639 used in this study was isolated from the leaf spot lesions of rice collected from Yunnan province, China, in August 2012. The strain has been preserved Nematicidal activity of compounds. The pure compounds 1 and 5-12 were tested for their nematicidal activity. Compounds 5-8 showed weak nematicidal activities against Caenorhabditis elegans at 400 ppm over 48 h, but compound 12 showed prominent activity that killed 94.5% of C. elegans at 400 ppm and 66.9% at 200 ppm over 24 h.

Discussion
Rice blast, caused by infection of the rice blast fungus, Magnaporthe grisea, is the most destructive pathogen of rice worldwide. Many metabolites from rice blast fungus have been identified and they show different activities. Pyriculol caused a necrotic lesion in a rice wounding assay and showed inhibition in a spore germination bioassay [28]. Compound pyricuol inhibited shoot growth showing a stronger effect than pyriculol and dihydropyriculol [28,29]. In our experiment, part salicylaldehyde-type products showed nematicidal activity, and among them aldehyde group-containing compounds showed nematicidal activity, which is consistent with the literature [30], while pyricuol (12) showed a prominent nematicidal activity, which was distinguished by a different substitute on the side chain, and this further provides us with an indication of the nematicidal active compounds.

Microbial Material
The fungal strain of Magnaporthe grisea M639 used in this study was isolated from the leaf spot lesions of rice collected from Yunnan province, China, in August 2012. The strain has been preserved in the State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University.

Cultivation, Extraction and Isolation
The strain M. grisea M639 was cultured on PDA solid medium at 26 • C for 7 days, and then it was inoculated into 1 L Erlenmeyer flasks each containing 200 mL of sticky rice-glucose liquid medium, which were cultivated at 26 • C for 14 days. The obtained culture filtrates were extracted by EtOAc three times to give a crude extract (1.32 g). The residual H 2 O portion was extracted with n-butyl alcohol to yield a residue (14.11 g). The EtOAc fraction was separated by CC on RP-18 (MeOH-H

Nematicidal Activity
The saprophytic nematode C. elegans was cultured on oatmeal medium (20 g of oatmeal in 80 mL of H 2 O) at 25 • C for 7 days. Then the cultured nematodes were separated from the culture medium using the Baerman funnel technique, and an aqueous suspension of the nematode was prepared as a working stock. Compounds 1 and 5-12 were dissolved in methanol and then diluted to different concentrations (400 and 200 ppm) with sterile water. The nematicidal activity against C. elegans was assayed according to the method based on references [31,32].