Metabolites from the Endophytic Fungus Curvularia sp. M12 Act as

Article pubs.acs.org/jnp

Metabolites from the Endophytic Fungus Curvularia sp. M12 Act as Motility Inhibitors against Phytophthora capsici Zoospores Muhammad Abdul Mojid Mondol,† Jannatul Farthouse,‡ Mohammad Tofazzal Islam,‡ Anja Schüffler,§ and Hartmut Laatsch*,† †

Institute for Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Tamannstrasse 2, D-37077 Göttingen, Germany ‡ Department of Biotechnology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh § Institute of Biotechnology and Drug Research, D-67663 Kaiserslautern, Germany S Supporting Information *

ABSTRACT: The endophytic fungus Curvularia sp., strain M12, was isolated from a leaf of the medicinal plant Murraya koenigii and cultured on rice medium followed by chemical screening of the culture extract. Chromatographic analysis led to the isolation of four new compounds, murranofuran A (1), murranolide A (2), murranopyrone (3a), and murranoic acid A (4a), along with six known metabolites, N-(2-hydroxy-6methoxyphenyl)acetamide (5), curvularin (6), (S)-dehydrocurvularin (7), pyrenolide A (8), modiolide A (9), and 8-hydroxy-6-methoxy-3-methylisocoumarin (10). The structures of the known compounds were confirmed by comparing ESI HR mass spectra, 1H and 13C NMR, and optical rotation data with values reported in the literature. The planar structures of the new compounds were elucidated by extensive analysis of 1D and 2D NMR and mass data. The absolute configurations of the new compounds were established by coupling constant analysis, modified Mosher’s method, and CD data. Compound 8 showed a strong motility impairing activity against Phytophthora capsici zoospores at a low concentration (100% at 0.5 μg/mL) in a short time (30 min). Compounds 2, 3a, 6, 7, 9, and 10 exhibited zoospore motility impairment activity at higher concentrations (IC50: 50−100 μg/mL).

P

increase the concentration of the fungicides and the frequency of their usage, factors that may lead to the spread of resistance in other species. In addition, preparations that in fact demonstrate activity against peronosporomycetes often contain copper, tin, or other persistent chemicals, whose toxicity represent enormous threats to the ecosystem, human health, and the environment in general.6 Therefore, there is a very urgent need for natural agents that are biodegradable and will exclusively control peronosporomycete phytopathogens. We hope to find such agents in endophytes. Endophytic fungi are ubiquitous in the plant kingdom. They produce bioactive secondary metabolites that may help plants to defend themselves against microorganisms, insects, and herbivores7−11 and thus may also have agricultural applications. In our search for natural agents to control peronosporomycetes, we studied the curry plant, Murraya koenigii. Its leaf extract is used as an antimicrobial agent in Ayurvedic medicine and also has insecticidal properties.12 On screening this plant for endophytic fungi, a Curvularia sp. strain (M12) was isolated using M2 agar medium. On the basis of prescreening results (1H NMR and TLC), a culture of the strain was scaled-up on rice medium. Chromatographic purification of the ethyl acetate

eronosporomycetes, also known as water molds, are a group of fungus-like eukaryotic microorganisms, phylogenetically related to brown algae. They include saprophytes as well as pathogens of plants, insects, crustaceans, fish, and other vertebrate animals. Saprophytic peronosporomycetes play key roles in decomposition and recycling of organic matter; however plant pathogenic species, especially those of the genus Phytophthora (the “plant destroyer” in Greek), are particularly devastating phytopathogens. They cause diseases in important crop species such as potato, tomato, pepper, soybean, cucurbits, pumpkin, squash, and eggplant as well as do environmental damage in natural ecosystems, resulting in multibillion dollar losses every year.1 Some species of Phytophthora, notably Phytophthora capsici, are highly dynamic and destructive pathogens of vegetables, with a wide range of hosts.2 One of the most distinguishing characteristics of the peronosporomycete organisms is the production of motile zoospores in their early life stages. These zoospores play an important role in the infection of plants.3−5 Peronosporomycetes were traditionally classified as fungi, but in fact have little in common with them, exhibiting marked differences in physiology, biochemistry, and genetics. While the cell walls of fungi are composed mainly of chitin, the peronosporomycetes contain glucan and cellulose. Fungicides that target chitin biosynthesis will therefore have no effect on peronosporomycetes. This lack of effect may cause farmers to © 2017 American Chemical Society and American Society of Pharmacognosy

Received: August 28, 2016 Published: February 14, 2017 347

DOI: 10.1021/acs.jnatprod.6b00785 J. Nat. Prod. 2017, 80, 347−355

Journal of Natural Products

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Chart 1

Table 1. 13C and 1H NMR Data for 1 and 2 in CD3OD 1

a

no.

δ Ca

type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

157.6 118.7 51.8 112.9 47.3 212.9 29.8 13.1 194.8 59.3 30.7 8.7 20.7 176.3 56.7 53.0

CH C CH CH CH C CH3 CH3 C C CH2 CH3 CH3 C CH3 CH3

2 δH, mult. (J in Hz)b 7.36, d (1.3) 3.20, ddd (3.9, 2.6, 1.3) 5.48, d (2.6) 3.12, qd (7.1, 3.9) 2.15, s 1.00, d (7.1)

1.89, qd (7.5, 1.6) 0.77, t (7.5) 1.32, s

δ Ca

type

172.1 123.9 156.9 111.5 41.6 76.9 60.9 58.2 67.0 20.4 51.6 58.2

C CH CH C CH2 CH CH CH CH CH3 CH3 CH3

δH, mult. (J in Hz)c 6.27, d (5.7) 7.37, d (5.7) 2.19, 2.97, 3.04, 2.79, 3.55, 1.24, 3.19, 3.31,

2.15, ABX (15.2, 8.5, 2.1) ddd (8.5, 8.6, 2.2) dd (8.6, 4.4) dd (8.2, 4.4) dq (8.2, 6.4) d (6.4) s s

3.47, s 3.66, s

Measured at 125 MHz. bMeasured at 300 MHz. cMeasured at 600 MHz.

HRMS, consistent with the molecular formula C16H24O6. This was confirmed by 13C and 1H NMR data (Table 1). In the 13C NMR spectrum, 16 resonances were observed and ascribed to five sp2 carbons (three carbonyl and two olefinic carbons) and 11 sp3 carbons with the help of phase-sensitive HSQC. The planar structure of compound 1 (Figure 1) was determined via extensive analysis of 2D NMR data. The olefinic methine H-1 (δC 157.6, δH 7.36) showed HMBC

extract led to the isolation of four new (1, 2, 3a, and 4a) and six known compounds (5−10). The isolation, structure elucidation, and antizoospore activity of these compounds are discussed here.

■

RESULTS AND DISCUSSION Compound 1 was isolated as an optically active, amorphous solid with a pseudomolecular ion peak [M + H]+ in the ESI 348



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Figure 1. COSY (bold bonds) and HMBC (arrows) correlations for 1−3a, 4a, and 5.

a high level of theory (see Experimental Section). The calculated ECD spectra are dominated by the stereoelectronic effects of C-3 and C-4. When these are in the S-configuration, both centers are contributing a positive but different Cotton effect (3S < 4S) to the CD spectrum (see Supporting Information, Figures S1 and S2). This is also the reason that, in the calculations, not only the four trans-(3S,4S,5R*,10R*) isomers but also the four cis-(3R,4S,5R*,10R*) dihydrofurans showed a positive Cotton effect and a similar curve shape to that observed for compound 1. The influences of C-5 and C-10 on the ECD shape are minor and appear at shorter wavelength (compare Figures S2 and S3, Supporting Information). Additionally, a positive optical rotation at 589 nm was predicted for all eight (4S)-isomers, as found experimentally for 1. Due to the trans-orientation of the substituents at C-3,4, murranofuran A must have the absolute (3S,4S) configuration, as the (3R,4R) isomers would show a negative Cotton effect. The ECD spectra calculated for the (3S,4S,5R,10R) and (3S,4S,5R,10S) isomers are a better fit with the experimental CD spectrum of 1 (Figure 2). However, a further confirmation

couplings with C-2, C-3, and C-4. H-3 showed a COSY correlation with H-4 and H-5. Additional HMBC correlations with C-1, C-2, and C-4 (Figure 1) indicated a dihydrofuran moiety. The methoxy carbon C-15 showed an HMBC correlation with the dihydrofuran carbon C-4 (δC 112.9), forming an acetal moiety. The resonance of the keto carbonyl at 212.9 (C-6), a COSY correlation between H-5 and H3-8, and HMBC correlations between H3-7/C-5,6 and H-5/C-3,6 identified a butan-2-one fragment that was connected via C-5 with C-3 of the dihydrofuran, as confirmed by the HMBC correlations of H-3 with C-5 and C-8 and of H-5 and H-8 with C-3. The remaining partial formula, C7H11O3, is accounting for a carbomethoxy group, a carbonyl, a methyl, an ethyl fragment, and a quaternary carbon, forming a 2-ethyl-2-methyl-3ketopropionate (Figure 1). The alternative 2-keto-3-ethyl-3methylpropionate was excluded on the basis of the HMBC correlations and calculated NMR data. In the HMBC spectrum, the methyl singlet at δH 1.32 (H3-13) correlated with both carbonyls and with the ethyl residue, so that all fragments must be connected via the remaining quaternary carbon at δC 59.3 (C-10). Only two natural products with this propionate fragment seem to be known: specifernin13 and berkelic acid.14 Taking into account the different type of the ketone (aliphatic instead of conjugated), their respective NMR data are in good agreement with the data measured for the 1 fragment. We did not find examples of the isomeric 3-ethyl-3methylpyruvic acid fragment in the natural product literature.15 We observed only a weak HMBC correlation between H-1 of the dihydrofuran and C-9 of this ketopropionate ester fragment, but not between C-9 and H-3. There was, however, only one way to connect both fragments via free valences, resulting in structure 1, which we named murranofuran A with respect to the origin of the endophyte. According to the coupling constant between H-3 and H-4 (J = 2.6 Hz), the dihydrofuran protons of 1 should be in a transposition; DFT calculations predicted correspondingly a dihedral angle of ∼120° and a coupling constant of 2.6 Hz, as in the experiment. The syn-facial orientation of H-4 and the butanone side chain was further confirmed by strong NOE signals between H-4/H-5 and H-4/H3-8, so that a (3R,4R) or a (3S,4S) configuration, respectively, resulted. To further investigate the configuration, the electronic circular dichroism (ECD) spectra of all eight pairs of enantiomers were calculated for 1, using ab initio methods on

Figure 2. Experimental ECD curve of murranofuran A (1) and calculated curve (thin line) for the (3S,4S,5R,10R) isomer.

of the configuration at C-5 and C-10 based on ECD and NMR data was not possible, and therefore only the (3S,4S) configuration is reliable. Further details are discussed in the Supporting Information. It is worth mentioning that 2,3dihydrofurans are very rare in nature, with most of those reported to date being fungal metabolites related to aflatoxins.16 Murranolide A (2) was obtained as an optically active, amorphous solid. The molecular formula of C12H18O6 was assigned on the basis of (+)-ESI HRMS and NMR data (Table 1). The IR absorptions at νmax 3442 and 1768 cm−1 together 349



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with resonances in the 13C NMR spectrum at 172.1 and 67.0 were indicative of a butenolide and hydroxy groups, respectively. In the COSY data, two spin systems from H-2 to H-3 and from H2-5 to H3-10 were identified. The carbonyl signal at δC 172.1 (C-1), along with the acetal resonance at δC 111.5 (C-4), the COSY correlation between H-2 and H-3, and the HMBC correlations between H-2,3/C-1 and H-2,3/C-4, suggested the presence of an α,β-unsaturated five-membered lactone ring. The second spin system was connected to C-4 of the butenolide, as confirmed by an HMBC correlation of H2-5 with C-3,4 (Figure 1). One of the methoxy groups (δH 3.19, δC 51.6, OMe-11) was attached to C-4, while the other one at δH 3.31 (δC 58.2, OMe-12) was connected to C-6, as determined by HMBC correlations of H3-11 and H3-12 with C-4 and C-6, respectively. In addition to the butenolide there must be an additional ring to account for the remaining double-bond equivalent. Using the carbon shifts, this was assigned as an epoxide at C-7,8. The planar structure of 2 was further established by comprehensive analysis of 2D NMR data. Compound 2 is a new oxygenated polyketide, assigned as murranolide A. The related plakilactones are produced by marine sponges.17 The relative configuration of the 7,8-epoxide in 2 was derived from the magnitude of the coupling constant between H-7 and H-8, whose value (J = 4.4 Hz) indicated a cis disubstitution. This was confirmed by the nuclear Overhauser signals between H3-10/H2-5 and H3-10/H-6. The absolute configuration of C-9 in 2 was addressed by the modified Mosher’s method. The positive ΔδH value for H3-10 and negative ΔδH values for the remaining protons (Figure 3) indicated a (9S)-configuration in

Figure 4. Experimental and calculated (thin line) ECD curves of murranolide A (2).

one CH3, eight CH (including six olefinic), and one carbonyl carbon by analysis of the HSQC spectrum. In the COSY spectrum, a continuous coupling sequence was found from H-2 to H3-10 (Figure 1). This spin system was connected with a carbonyl group (δC 163.1), as indicated by an HMBC correlation of H-2 and H-3 with C-1. The double-bond geometry at C-6 and C-8 in 3a was determined as E based on the coupling constants of H-6 (J = 15.5 Hz) and H-9 (J = 14.4 Hz) (Table 2). The sequence of three double bonds was interrupted by two oxymethine carbons. As one double-bond equivalent was left, the carbons C-4 and C-5 could be bridged by the remaining oxygen atom, forming epoxide 11. This option is, however, clearly excluded by the experimental 13C NMR shifts, which are far away from those expected for an epoxide in an aliphatic chain (δC ∼55−60 ppm). If we take compound 11, however, into account as a biosynthetic intermediate, 2,3-dihydropyran2-one 3a/3b or butenolide 12 could be formed easily by attack of the carboxylate on C-5 or C-4 in 11, respectively. As no suitable HMBC correlations were found to differentiate between these alternatives, the 13C NMR data of 3a/3b and 12 (both diastereomers) were calculated using ab initio methods. The results (see Supporting Information, Table S1) verified the cis-dihydropyrone 3a. Corresponding compounds from the literature are the isomers musacin D19 and phomalactone;19 the complete agreement with the data of the latter confirmed structure 3a additionally (see Supporting Information, Table S1). A conformation analysis of the diastereomers predicted for cis-3a a coupling constant between H-4/H-5 of J = 1.5 Hz. For trans-3b, a biaxial orientation of H-4 and H-5 dominated in the conformer mixture, and a value of J = 8.2 Hz was calculated. The close similarity of experimental and calculated shifts and the coupling constant between H-4 and H-5 (J = 3.0 Hz) indicated therefore a (S*,S*)-cis-dihydropyrane, 3a. This was additionally confirmed by NOESY contacts between H-4 and H-6, while the cross signal between H-4 and H-7 as expected for 3b was missing. Very recently diplopyrone B (3c) has been published, a stereoisomer of 3a with a (Z)-Δ6 double bond.20 The NMR data of 3a and 3c are clearly different, but also for 3c, we reproduced the carbon shifts by ab initio calculations within the experimental error limits (see Supporting Information, Table S1). For 3c only the configuration at C-5 had been determined, but on the basis of the shifts and the H-4,5 coupling constant of J = 3.1 Hz, from our calculations a (4S,5S)-configuration (corresponding to the cis-orientation) resulted for 3c as well. As for compounds 1 and 2, the absolute configuration of 3a was assigned from CD (Figure 5) and ORD calculations (found

Figure 3. ΔδH (δH = δS − δR) values for MTPA esters 2a and 2b of 2 and 4c and 4d of 4b.

2.18 The configuration of the remaining stereocenters was determined by computational methods as for 1. Due to the (9S)-configuration and the cis-configured oxirane, only two series with four (4R*,6R*,7R,8S,9S)- and four (4R*,6R*,7S,8R,9S)-configured diastereomers had to be calculated. It was expected that the butenolide was responsible for the Cotton effect at ∼250 nm. The calculations predicted a negative signal for the (4R)-configuration, while the (6S)configuration is responsible for the trough at 220 nm, as was observed experimentally for 2. Finally, the (4R,6S,7R,8S,9S)configuration was assigned for 2 based on experimental versus calculated ECD curves (Figure 4 and Supporting Information, Figure S4). Compound 3a was obtained as white crystals with the molecular formula C10H12O3 determined by (+)-ESI HRMS. The presence of hydroxy and carbonyl groups was inferred by IR absorptions at νmax 3411 and 1714 cm−1, respectively. The signals in the 1H and 13C NMR spectra of 3a were ascribed to 350



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Table 2. 13C and 1H NMR Data for 3a, 4a, and 5 3a

4a

no.

δ Ca

type

δH, mult. (J in Hz)b

1 2 3 4 5 6 7

163.1 122.9 144.5 63.0 80.9 121.8 135.9

C CH CH CH CH CH CH

6.11, 6.97, 4.20, 4.89, 5.69, 6.44,

8

130.1

9 10

132.9 18.2

δ Cc

type

d (9.4) dd (9.4, 5.3) dd (5.3, 3.0)f dd (6.4, 3.0) dd (15.5, 6.4) dd (15.5, 10.5)

170.1f 119.5 145.3 127.3 147.1 73.1 34.5

C CH CH CH CH CH CH2

CH

6.08, dd (14.4, 10.5)f

36.2

CH2

CH CH3

5.82, dq (14.4, 6.4) 1.77, d (7.0)

68.7 23.7

CH CH3

5 δH, mult. (J in Hz)d 5.64, 6.63, 7.46, 6.05, 4.16, 1.55, 1.67, 1.45, 1.55, 3.72, 1.15,

d (12.0) t (12.0) dd (15.4, 12.0) dd (15.4, 6.2) m m m m m m d (6.0)

δ Cc

type

115.6 155.8 111.6 128.7 103.8 153.6

C C CH CH CH C

173.4

C

22.8 56.5

δH, mult. (J in Hz)e

6.51, dd (8.3, 2.0) 7.06, t (8.3) 6.53, dd (8.3, 2.0)

CH3 CH3

2.18, s 3.81, s

a

Measured in CDCl3 at 125 MHz. bMeasured in CDCl3 at 300 MHz. cMeasured in CD3OD at 125 MHz. dMeasured in CD3OD at 600 MHz. Measured in CD3OD at 300 MHz. fVery weak signal in CD3OD at 300 MHz.

e

assigned by COSY experiments. The ΔδH values (ΔδH = δS − δR) are shown in Figure 3. Negative ΔδH values for H-2 to H2-6 and positive values for H2-8 to H3-10 corresponding to the ΔδH pattern for diesters of anti-1,4-diols (type C system) reported by Riguera21 indicated the absolute configuration of C-6 and C9 in 4a as (R,R). The structure of 4a was therefore conclusively determined as (2Z,4E,6R,9R)-6,9-dihydroxydeca-2,4-dienoic acid. The molecular formula of 5 was determined as C9H11NO3 based on (+)-ESI HRMS and supported by the 13C NMR spectrum (Table 2). The IR spectrum exhibited an absorption at 1645 cm−1, indicating the presence of an amide group. The 1 H and 13C NMR spectra revealed methyl, methoxy, and aromatic signals. The 1H NMR data showed the typical pattern of a 1,2,3-trisubstituted benzene ring. This was confirmed via the observed coupling constants and the COSY correlations (Figure 1 and Table 2). The structure of compound 5 was conclusively determined as N-(2-hydroxy-6-methoxyphenyl)acetamide, based on spectroscopic data analysis. Compound 5 has been synthesized previously,10 but as no spectroscopic data are available in the literature, they are presented herein. To the best of our knowledge, this is the first reported isolation of compound 5 from nature. The structures of further known compounds were confirmed by ESI HRMS, 1H and 13C NMR, and optical rotation data, along with database analysis,16 as curvularin (6),22,23 (S)dehydrocurvularin (7),24,25 pyrenolide A (8),26 modiolide A (9), 26,27 and 8-hydroxy-6-methoxy-3-methylisocoumarin (10).28 Compounds 2, 3a, and 8−10 are likely derived from hydroxylated and/or unsaturated decanoic acids such as 4a, which are also potential precursors of 3-methylisocoumarins, phthalides, pyrones, and the respective ring-opened acids. Most of their aliphatic derivatives belong to either the decarestrictines or cephalosporolides. Although several hundred C10-lactones were reported, less than 50 of the respective unbranched free C10-acids (type 4a) seem to be known. The 2,3-unsubstituted butenolides of type 2 are especially rare in nature. We found less than 20 of these in the literature.15,16 A certain degree of similarity is observed only with ramariolides A and B.29 Compounds 1, 2, 3a, 4a, and 5−10 were subjected to motility inhibitory and zoosporicidal activity tests against P. capsici. The concentration and time-course activities are presented in Table 3. Clearly, zoospore motility halting activity

83.3°, calcd 151.2°) as a (4S,5S) isomer, which we named murranopyrone.

Figure 5. Experimental ECD curve of murranopyrone (3a, bold line) and calculated curve for the (4S,5S) (thin line) and (4R,5S) isomer (dotted line).

Murranoic acid A (4a) was obtained as a colorless oil. In the positive ion mode, the ESI HR mass spectrum showed a pseudomolecular ion peak at m/z 223.0941 [M + H]+, indicating the molecular formula C10H16O4. This was supported by the 13C NMR data (Table 2). The IR spectrum displayed a broad absorption band centered at 3350 cm−1 and a sharp band at 1694 cm−1, suggesting the presence of hydroxy and carbonyl functionalities, respectively. The 1H and 13C NMR resonances were ascribed by the HSQC and HMBC spectra to one methyl (sp3-connected), one surprisingly weak carbonyl, two methylene (sp3), two aliphatic, and four olefinic methine carbons (Table 2). In the COSY spectrum, a consecutive proton coupling sequence was observed from H-2 to H3-10 (Figure 1). The geometry of the double bonds at C-2 and C-4 was determined as Z and E, respectively, based on the coupling constants of H-2 (J = 12.0 Hz) and H-5 (J = 15.4 Hz), respectively. The gross structure of 4a was derived by 2D NMR data analysis (Figure 1). The absolute configuration of 4a was determined by the modified Mosher’s method.18 All 1H signals of the (S,S)- and (R,R)-bis-MTPA ester derivatives of the methyl ester 4b were 351



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Table 3. Motility Inhibitory and Zoosporicidal Activities of 1, 2, 3a, 4a, and 5−10 against the Late Blight Phytopathogen P. capsici motility inhibitory and zoosporicidal activities over control (% ± SE)a 15 min no.

dose (μg/mL)

1

100 400 500 50 100 300 500 100 200 300 500 100 400 500 200 400 50 100 400 25 50 100 200 400 0.5 1.0 2.5 5 10 25 50 100 50 100 200 400 500

2

3a

4a

5 6

7

8

9 10

inhibition 0 0 0 0 0 0 0 0 10 43 60 0 0 5 0 5 0 0 0 0 0 0 57 100 81 90 100 100 100 100 0 55 0 49 66 81 100

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0 0 0 0 0 0 0 0 0 9 4 0 0 1 0 1 0 0 0 0 0 0 4 0 5 7 0 0 0 0 0 4 0 4 6 5 0

30 min lysis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 50 0 0 0 0 52 85 0 0 0 0 0 0 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

inhibition 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 9 7 0 0 0 0 0 0 0

0 0 78 0 0 10 35 14 28 52 100 0 5 100 0 45 5 12 58 0 0 89 100 100 100 100 100 100 100 100 0 65 5 65 78 90 100

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

45 min lysis

0 0 5 0 0 0 4 3 4 4 0 0 1 0 0 3 2 1 3 0 0 5 0 0 0 0 0 0 0 0 0 7 1 7 6 4 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 52 0 0 5 33 64 99 0 0 0 0 0 0 0

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

inhibition 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 0 0 1 5 6 1 0 0 0 0 0 0 0

0 0 100 0 27 35 48 50 62 81 100 0 11 100 5 59 9 22 85 0 43 100 100 100 100 100 100 100 100 100 5 91 54 72 81 93 100

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0 0 0 0 4 5 6 6 5 9 0 0 3 0 2 8 1 3 7 0 6 0 0 0 0 0 0 0 0 0 1 5 6 4 5 6 0

60 min lysis 0 0 0 0 0 0 0 0 0 0 5 0 0 6 0 0 0 0 0 0 0 0 17 55 4 5 20 45 82 100 0 0 0 0 0 0 34

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

inhibition 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 2 7 1 1 3 4 0 0 0 0 0 0 0 0 6

0 25 100 0 50 58 73 71 82 87 100 18 32 100 7 70 51 69 100 11 83 100 100 100 100 100 100 100 100 nt 10 95 68 75 83 95 100

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0 4 0 0 8 4 5 6 7 9 0 3 5 0 1 5 6 8 0 2 9 0 0 0 0 0 0 0 0

± ± ± ± ± ± ±

1 4 7 6 7 4 0

lysis 0 0 5 0 0 0 0 0 0 0 7 0 0 8 0 0 0 0 0 0 0 0 40 59 5 7 22 53 94 nt 0 6 0 0 0 0 50

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0 0 1 0 0 0 0 0 0 0 2 0 0 2 0 0 0 0 0 0 0 0 6 6 1 2 7 8 5

± ± ± ± ± ± ±

0 1 0 0 0 0 6

Data presented here are average values ± SE of at least three replications in each dose of compound. nt, not tested. Fluazinam 500F was used as positive control, which impaired motility (100%) and caused subsequent lysis of >50% stopped zoospores within 60 min at 0.04 μg/mL.

a

A (polyketide, 88% at 10 μg/mL), cyclo-(L-Phe-L-Leu-L-Leu-LLeu-L-Leu) (oligopeptide, 88% at 10 μg/mL),33 and gageotetrin B (lipopeptide, 63% at 10 μg/mL).34 Thus, pyrenolide A (8) is one of the most potent zoospore motility inhibitory metabolites reported to date. Its potent activity is probably due to the presence of the epoxide in the molecule: the trichothecenes (which are also epoxide-containing cyclic lactones) showed pronounced inhibitory and zoosporicidal activities.35 There is evidence to suggest that motile zoospores locate hosts and aggregate at the sites of infection in response to specific chemical signals released from the host.36,37 Flagellar motility is critical for the life cycle and pathogenicity of zoosporic phytopathogens, and it is likely that protein kinase is involved in maintaining flagellar motility.30 Any disruption of the motility of zoospores considerably decreases potential pathogenesis caused by peronosporomycete phytopatho-

was increased with increasing dose and time after the treatment. The most noticeable zoospore motility inhibitory activity was exhibited by compound 8, where the highest activity (100%) was achieved at a very low concentration (0.5 μg/mL) within a short time (30 min) (Table 3). Interestingly, 53% of stopped zoospores lysed after treatment with compound 8 at 5.0 μg/ mL. Compounds 2, 3a, 6, 7, 9, and 10 also showed more than 50% motility inhibitory activity (IC50) at a concentration of 50−100 μg/mL. The corresponding activity of the remaining compounds was negligible. Motility inhibitory activity against phytopathogenic peronosporomycete zoospores has been reported for different classes of compounds isolated from plants and microbes. The following inhibitory activity against zoospores was reported: staurosporine (indolocarbazole, 12% at 1 μg/mL),30 dipropylphloroglucinol (phloroglucinol, 90% at 2 μg/mL),31 quebracho (polyflavonoid tannin, 26% at 0.5 μg/mL),32 cryptosporiopsin 352



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gens.38,39 Thus, the notorious peronosporomycete phytopathogens can be controlled by halting the motility of zoospores, thereby preventing their aggregation at the host infection sites.30 Pyrenolide A (8) showed remarkable motility inhibitory activity against P. capsici zoospores. As this metabolite is small in size, is biodegradable, and can be easily synthesized in the laboratory, this secondary metabolite could be used as a biopesticide or as a lead compound in the design of a novel environmentally friendly agrochemical agent against P. capsici and related phytopathogens.

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in CH2Cl2 (SF76−91), and 20% MeOH in CH2Cl2 (SF92, 93). Compounds 2 (1.7 mg) and 5 (1.0 mg) were purified from combined fractions SF7 + 8 by PTLC, eluting with CHCl3−n-hexane−MeOH (1:0.3:0.2). Similarly, fractions 28 and 29 were combined and subfractionated by silica column chromatography, eluting with 100% CH2Cl2 (SF1−15), 2% MeOH in CH2Cl2 (SF16−23), 4% MeOH in CH2Cl2 (SF24−36), and 20% MeOH in CH2Cl2 (SF37−50). Compounds 1 (1.1 mg), 6 (16.0 mg), and 8 (2.5 mg) were purified from SF14 + 15, SF25, and SF12 + 13 by PTLC, eluting with CHCl3− MeOH−n-hexane (2:0.4:0.6), EtOAc−n-hexane (2.1:0.9), and acetone−n-hexane (1:2), respectively, whereas 9 (13.3 mg) was obtained from SF26 by crystallization. Similarly, fractions 30 and 31 were combined and subfractionated, eluting with 100% CH2Cl2 (SF1−123), 2% MeOH in CH2Cl2 (SF124−152), 8% MeOH in CH2Cl2 (SF153− 162), and 14% MeOH in CH2Cl2 (SF163−172). Compounds 4a (13.0 mg), 3a (3.6 mg), 7 (8.6 mg), and 10 (2.0 mg) were purified from SF160−164, SF2, SF3, and SF127 + 128 by PTLC, eluting with CHCl3−n-hexane−MeOH (1:0.3:0.2), EtOAc−n-hexane (0.1:1.4), CHCl3−n-hexane−MeOH (1:0.4:0.1), and acetone−n-hexane (2:1), respectively. Murranofuran A (1): colorless, amorphous solid; [α]20 D +81.8 (c 0.11, MeOH); CD spectrum in MeOH, see Figure 2; IR νmax 2927, 1724, 1647, 1604, 1455, 1377, 1236, 1104 cm−1; 1H and 13C NMR (CD3OD), see Table 1; (+)-ESI HRMS m/z 313.1646 [M + H]+ (calcd for C16H25O6, 313.1645). Murranolide A (2): colorless, amorphous solid; [α]20 D −9.4 (c 0.17, MeOH); CD spectrum in MeOH, see Figure 4; IR νmax 3442, 2930, 1768, 1457, 1104 cm−1; 1H and 13C NMR (CD3OD), see Table 1; (+)-ESI HRMS m/z 281.0997 [M + Na]+ (calcd for C12H18O6Na, 281.0995). Murranopyrone (3a): colorless crystals; [α]20 D +83.3 (c 0.36, MeOH); CD spectrum in MeOH, see Figure 5; IR νmax 3411, 2923, 1714, 1380, 1260, 1082, 990 cm−1; 1H and 13C NMR (CDCl3), see Table 2; (+)-ESI HRMS m/z 181.0859 [M + H]+ (calcd for C10H13O3, 181.0859). Murranoic acid A (4a): colorless oil; [α]20 D −43.4 (c 1.3, MeOH); IR νmax 3350, 2924, 1694, 1431, 1205 cm−1; 1H and 13C NMR data (CD3OD), see Table 2; (+)-ESI HRMS m/z 223.0941 [M + Na]+ (calcd for C10H16O4Na, 223.0940). N-(2-Hydroxy-6-methoxyphenyl)acetamide (5): colorless, amorphous solid; IR νmax 2929, 1645, 1599, 1536, 1475, 1380, 1250, 1091 cm−1; 1H and 13C NMR (CD3OD), see Table 2; (+)-ESI HRMS m/z 204.0631 ([M + Na]+) (calcd for C9H11NO3Na, 204.0631). Preparation of (S)-MTPA Ester 2a. Compound 2 (0.5 mg) was dissolved in 300 μL of pyridine, 10 μL of (R)-(−)-MTPA-Cl was added, and the mixture was stirred at 20 °C for 16 h and dried under air flow. The reaction mixture was fractionated first by PTLC, eluting with CHCl3−n-hexane−MeOH (1:0.4:0.6), and then purified on LH20, eluting with 20% MeOH in CH2Cl2, to yield the (S)-MTPA ester (2a, 0.7 mg). Compound 2a: colorless oil; 1H NMR (CD3OD, 600 MHz) δH 6.32 (1H, d, J = 5.7 Hz, H-2), 7.41 (1H, d, J = 5.7 Hz, H-3), 2.20 (2H, m, H2-5), 3.14 (1H, m, H-6), 3.15 (1H, dd, J = 6.9, 4.0 Hz, H-7), 2.98 (1H, dd, J = 9.0, 4.0 Hz, H-8), 5.12 (1H, m, H-9), 1.45 (3H, d, J = 6.5 Hz, H3-10), 3.21 (3H, s, H3-11), 3.35 (3H, s, H3-12), 3.54 (3H, s, OMe), 7.54−7.41 (5H, m); (+)-ESI HRMS m/z 497.1395 [M + Na]+ (calcd for C22H25F3O8Na, 497.1393). Preparation of (R)-MTPA Ester 2b. The (R)-MTPA ester (2b) of compound 2 (0.5 mg) was prepared from (S)-(+)-MTPA-Cl and purified in the same way as 2a to obtain 2b (0.7 mg). Compound 2b: colorless oil; 1H NMR (CD3OD, 600 MHz) δH 6.32 (1H, d, J = 5.7 Hz, H-2), 7.41 (1H, d, J = 5.7 Hz, H-3), 2.21 (2H, m, H2-5), 3.17 (1H, m, H-6), 3.20 (1H, dd, J = 8.3, 4.2 Hz, H-7), 3.07 (1H, dd, J = 9.1, 4.2 Hz, H-8), 5.09 (1H, dd, J = 9.1, 6.5 Hz, H-9), 1.32 (3H, d, J = 6.5 Hz, H3-10), 3.21 (3H, s, H3-11), 3.35 (3H, s, H3-12), 3.57 (3H, s, OMe), 7.55−7.40 (5H, m, Phe); (+)-ESI HRMS m/z 497.1400 [M + Na]+ (calcd for C22H25F3O8Na, 497.1393). Preparation of Methyl Ester 4b. Compound 4a (6.0 mg) was dissolved in MeOH, and an excess of diazomethane solution was added. After a few seconds at room temperature, the mixture was

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation values were measured on a PerkinElmer polarimeter, model 241, at the sodium D line (λ = 589 nm). CD spectra were recorded by a Jasco J-810 spectropolarimeter. Mass spectra were measured with a Bruker micrOTOF 10237. The NMR spectra were measured on a Mercury300 (300.141 MHz), a Varian Inova 500 (125.707 MHz for 13C NMR spectra), and a Varian Inova 600 (599.740 MHz) spectrometer. Chemical shifts (δ) were referenced to CH3OH at 3.30 for 1H and 49.0 for 13C and to CHCl3 at 7.24 for 1H and 77.0 for 13C. IR spectra were taken on a Jasco FT/IR-4100 type A instrument. Mosher’s reagent was bought from Sigma-Aldrich, Germany. Size exclusion chromatography was performed on Sephadex LH-20 (Lipophilic Sephadex, Amersham Biosciences, Ltd.; purchased from Sigma-Aldrich Chemie, Steinheim, Germany). Column chromatography was done on silica gel 60 (0.063 to 0.20 nm) and preparative thin-layer chromatography (PTLC) on MN-silica gel/p UV254 (both obtained from Macherey-Nagel GmbH & Co. KG, Düren, Germany). All solvents used were either analytical grade or distilled prior to use. Fungal Material and Identification. The healthy leaves of the curry plant (Murraya koenigii) were collected from the garden of medicinal plants, Department of Pharmacy, Rajshahi University, Bangladesh. The leaves were kept in a plastic zipper pack at 4 °C for two months. A leaf was washed with tap water to remove dust and debris. The surface of the leaf was then sterilized as described previously.40 The excess NaOCl was removed by washing the leaves three times with sterilized water. They were then air-dried on sterile filter paper. The leaf was cut into ca. 1 × 1 cm pieces using sterilized scissors and placed on M2 agar (in a Petri dish, with agar prepared from 10 g glucose, 4 g yeast extract, 4 g malt extract, 18 g agar, and 1 L tap water). The plate was incubated at 25 °C, and the outgrowing fungal strain M12 was isolated and maintained on M2 agar. The fungal strain M12 was identified as Curvularia sp. based on the sequence of its internal transcribed spacer region (ITS) and morphology. Its ITS sequence shows 100% identity to C. spicifera strain CBS 274.52, as well as to C. spicifera strain CBS 199.31 (GenBank Accession No. JN192387 and HF934915, respectively). This strain is currently deposited in the Microbial Culture Collection at the Institute of Organic and Biomolecular Chemistry, Georg-August-University Göttingen, Germany, under the curatorship of Prof. Laatsch. Fermentation and Isolation. The fungal strain M12 was subcultured on M2 agar medium and incubated at 25 °C for 3 days. Rice (200 g) and tap water (130 mL) were added into each of 24 Pflasks and then sterilized (121 °C for 15 min). The P-flasks were inoculated with well-grown agar cultures of the strain M12 (pieces 1 × 1 cm) and incubated at 30 °C for 25 days. The whole culture medium was extracted with EtOAc and then filtered through Celite (diatomaceous earth). The filtrate was concentrated to dryness in vacuo at 40 °C to obtain a reddish gum. This extract was treated with cyclohexane to remove fats. The remainder (4.0 g) was then divided into 70 fractions (F1−70, 15 mL for each fraction) by Sephadex LH20 column chromatography, eluting with CH2Cl2−MeOH (1:1). Fractions F32 and F33 were combined and again subfractionated by silica column chromatography, eluting with 200 mL of each of the following solvent mixtures: 100% CH2Cl2 (SF1−31), 4% MeOH in CH2Cl2 (SF32−53), 8% MeOH in CH2Cl2 (SF54−75), 12% MeOH 353



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final volume of 400 μL and then quickly mixed with a glass rod; 1% aqueous DMSO was used as control. The motility of zoospores was observed under a light microscope at 10-fold magnification. Quantification of time-course changes of motility and lysis of zoospores were carried out as described previously.32

evaporated to dryness and then purified by PTLC, eluting with CHCl3−n-hexane−MeOH (1:0.4:0.1), to deliver the methyl ester 4b (4.0 mg). Compound 4b: colorless, amorphous solid; 1H NMR (CD3OD, 300 MHz) δH 5.67 (1H, d, J = 10.0 Hz, H-2), 6.67 (1H, d, J = 10.0 Hz, H-3), 7.48 (1H, dd, J = 15.0, 10.0 Hz, H-4), 6.09 (1H, dd, J = 15.0, 6.0 Hz, H-5), 4.18 (1H, m, H-6), 1.55 (1H, m, H-7b), 1.66 (1H, m, H-7a), 1.46 (1H, m, H-8b), 1.55 (1H, m, H-8a), 3.72 (1H, m, H-9), 1.15 (3H, d, J = 6.0 Hz, H-10), 3.69 (3H, s, OMe); (+)-ESI HRMS m/z 237.1097 [M + Na]+ (calcd for C11H18O4Na, 237.1097). Preparation of (S,S)-Bis-MTPA Ester 4c. The methyl ester 4b (2.0 mg) was dissolved in 1.0 mL of pyridine; 15 μL of (R)(−)-MTPA-Cl was added to the reaction vial, stirred at 20 °C for 16 h, dried under air flow, redissolved in EtOAc, washed with H2O, and purified by PTLC, eluting with CHCl3−n-hexane−MeOH (1:0.3:0.4), to obtain 4c (3.0 mg). Compound 4c: colorless oil; 1H NMR (CD3OD, 600 MHz) δH 5.74 (1H, d, J = 11.4 Hz, H-2), 6.58 (1H, t, J = 11.4 Hz, H-3), 7.47 (1H, overlapped, H-4), 5.83 (1H, dd, J = 15.5, 6.7 Hz, H-5), 5.48 (1H, m, H-6), 1.58 (1H, m, H-7b), 1.63 (1H, m, H-7a), 1.60 (2H, m, H-8), 5.12 (1H, m, H-9), 1.32 (3H, d, J = 6.2 Hz, H-10), 7.48−7.33 (10H, m), 3.70, 3.54, 3.49 (3 × 3H, s, 3 OMe); (+)-ESI HRMS m/z 669.1894 [M + Na]+ (calcd for C31H32F6O8Na, 669.1893). Preparation of (R,R)-Bis-MTPA Ester 4d. The (R,R)-bis-MTPA ester 4d was prepared from 4b (2.0 mg) and (S)-(+)-MTPA-Cl in the same way as for 4c to obtain 4d (3.4 mg). Compound 4d: colorless oil; 1H NMR (CD3OD, 600 MHz) δH 5.76 (1H, d, J = 11.3 Hz, H-2), 6.66 (1H, t, J = 11.3 Hz, H-3), 7.61 (1H, dd, J = 15.6, 11.3 Hz, H-4), 6.08 (1H, dd, J = 15.6, 6.3 Hz, H-5), 5.60 (1H, m, H-6), 1.70 (1H, m, H-7b), 1.76 (1H, m, H-7a), 1.47 (2H, m, H-8), 5.03 (1H, m, H-9), 1.13 (3H, d, J = 6.2 Hz, H-10), 7.34 (10H, m), 3.70, 3.57, 3.47 (3 × 3H, s, 3 OMe); (+)-ESI HRMS m/z 669.1894 [M + Na]+ (calcd for C31H32F6O8Na, 669.1893). Computational Methods. Conformer distributions were searched using a systematic approach with MMFF, implemented in Spartan’14.41 The geometries of all resulting conformers within an energy range of

Metabolites from the Endophytic Fungus Curvularia sp. M12 Act as

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