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Dec 15, 2015 - Marco Masi,. † ... Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte Sant'Angelo, Vi...
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Bioactive Secondary Metabolites Produced by the Oak Pathogen Diplodia corticola Marco Masi,† Lucia Maddau,§ Benedetto Teodoro Linaldeddu,§ Alessio Cimmino,† Wanda D’Amico,† Bruno Scanu,§ Marco Evidente,† Angela Tuzi,† and Antonio Evidente*,† †

Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte Sant’Angelo, Via Cintia 4, 80126 Napoli, Italy § Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy S Supporting Information *

ABSTRACT: Three new lactones and a new fatty acid ester, named sapinofuranones C and D, diplopyrone B, and diplobifuranylone C, respectively, were isolated from Diplodia corticola, together with sphaeropsidins A and C, diplopyrone, diplobifuranylones A and B, diplofuranone A, and the (S,S)-enantiomer of sapinofuranone B. Sapinofuranones C and D, diplopyrone B, and diplobifuranylone C were characterized as (5S)-5-((1,S-1,6-dihydroxyhexa-2,4-dienyl)-dihydrofuran-2-one, 4,5-dihydroxy-deca-6,8-dienoic acid methyl ester, (5S)-5-hydroxy-6-(penta-1,3-dienyl)-5,6-dihydro-pyran-2-one, and 5′-((1R)-1hydroxyethyl)-2′,5′-dihydro-2H-[2,2′]bifuranyl-5-one by spectroscopic and chemical methods, respectively. The relative configuration of sapinofuranone C was assigned by X-ray diffraction analysis, whereas its absolute configuration was determined by applying the advanced Mosher’s method to its 11-O-p-bromobenzoyl derivative. The same method was used to assign the absolute configuration to C-5 of diplopyrone B and to that of the hydroxyethyl of the side chain of diplobifuranylone C, respectively. The metabolites isolated were tested at 1 mg/mL on leaves of cork oak, grapevine cv. ‘Cannonau’, and tomato using the leaf puncture assay. They were also tested on tomato cuttings at 0.2, 0.1, and 0.05 mg/mL. Each compound was tested for zootoxic activity on Artemia salina L. larvae. The efficacy of sapinofuranone C and diplopyrone B on three plant pathogens, namely, Athelia rolfsii, Fusarium avenaceum, and Phytophthora nicotianae was also evaluated. In all phytotoxic assays only diplopyrone B was found to be active. It also showed strong inhibition on the vegetative growth of A. rolfsii and P. nicotianae. All metabolites were inactive in the assay performed for the zootoxic activity (A. salina) even at the highest concentration used (200 μg/mL). Diplopyrone B showed a promising antioomycete activity for the control of Phytophthora spp. also taking into account the absence of zootoxic activity. KEYWORDS: Botryosphaeriaceae, phytotoxins, furanones, α-pyrone, unsaturated carboxylic acids, sapinofuranones C and D, diplopyrone B, diplobifuranylone C

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INTRODUCTION

suber in Sardinia. The biological activities of these new metabolites were also examined.

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Several studies have pointed out the important role played by Diplodia and Neof usicoccum species in the complex etiology of oak decline worldwide.1−5 Among these, the infections of Diplodia corticola A.J.L. Phillips, A. Alves & J. Luque have caused serious and negative impacts on oak ecosystems, where it limits both the vitality and the productivity of these trees.6−10 D. corticola is well-known for its ability to produce a broad array of secondary metabolites in vitro, some of which showed phytotoxic, antifungal, and antibacterial activities, including diplopyrone, diplofuranones A and B, diplobifuranylones A and B, and the (S,S)-entantiomer of sapinofuranone B.11−13 D. corticola also produces the well-known phytotoxin sphaeropsidin A,14 which also showed antimicrobial15−17and anticancer18−20 activities. Here, we report the isolation and characterization of three new lactones, named sapinofuranone C, diplopyrone B, and diplobifuranylone C, and a new dihydroxydecadienoic acid methyl ester, named sapinofuranone D, from culture filtrates of a D. corticola strain, isolated from symptomatic tissue of Quercus © XXXX American Chemical Society

MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured in CHCl3 on a P-1010 digital polarimeter (Jasco, Tokyo, Japan); IR spectra were recorded as deposit glass film on a 5700 FT-IR spectrometer (Thermo Electron Corp. Nicolet, Madison, WI, USA), and UV spectra were measured in MeCN on a V-530 spectrophotometer (Jasco, Tokyo, Japan); 1H and 13C NMR spectra were recorded at 400/100 or 500/125 MHz in CDCl3 on Bruker spectrometers (Karlsruhe, Germany). The same solvent was used as internal standard. Carbon multiplicities were determined by DEPT spectra.21 DEPT, COSY-45, HSQC, HMBC, and NOESY experiments21 were performed using Bruker microprograms. HRESI and ESI spectra were recorded on Q-TOF Micro Mass (Waters, Milford, MA, USA) and 6120 quadrupole LC/MS instruments (Agilent Technologies, Milan, Italy), respectively. Analytical and preparative TLC were performed on silica gel (Kieselgel Received: October 26, 2015 Revised: December 15, 2015 Accepted: December 15, 2015

A

DOI: 10.1021/acs.jafc.5b05170 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. 1H and 13C NMR Data of Sapinofuranones C, 1, and D, 3, and Diplopyrone B, 2a,b 1 position

δ Cc

2

δH (J in Hz)

1 2

178.1 s

3

28.4 t

4

23.3 t

5

83.1 d

6

69.2 d

7

126.9 d

2.53 (1H) ddd (18.0, 10.5, 6.1) 2.45 (1H) ddd (18.0, 9.7, 7.5) 2.17 (1H) m 2.06 (1H) m 4.45 (1H) ddd (7.5, 6.0, 5.3) 4.55 (1H) dd (10.9, 5.3) 5.38 (1H) t (10.9)

8

132.3 d

6.17 (1H) t (10.9)

9

124.6 d

10

136.0 d

11

62.2 t

6.52 (1H) br ddd (15.3, 10.9) 5.97 (1H) dt (15.3, 4.8) 4.13 (2H) br dd (4.8)

HMBC H-5, H2-4, H2-3 H2-4

δ Cc

3

δH (J in Hz)

163.1 s 122.8 d

HMBC H-3

6.14 (1H) d (9.9)

H-4, H-5

δCc

δH (J in Hz)

174.7 s 30.4 t

2.48 (2H) t (7.2) 1.85 (1H) m

27.6 t

HMBC OMe, H2-2, H2-3 H-4, H2-3 H-5, H2-2

1.69 (1H) m H-6, H2-3

144.5 d

6.99 (1H) dd (9.9, 5.1)

H-5, H-6, H-3

74.1 d

3.46 (1H) m

H-5, H2-3, H2-2

H-8, H2-4

63.0 d

133.2 d

6.28 (1H) dd (10.4, 8.6) 6.26 (1H) br ddd (14.3, 10.4) 5.90 (1H) dq (14.3, 7.1) 1.81 (3H) br dd (7.1)

H-6, H-7, H-9

126.2 d

H-7, H-8, H-10, Me-11 Me-11, H-9

133.0 d

4.32 (1H) t (7.6) 5.26 (1H) dd (10.8, 7.6) 6.1 5 (1H) t (10.8) 6.33 (1H) t (10.8) 5.80 (1H) dq (10.8, 6.5) 1.78 (3H) d (6.5)

H-4, H-7

77.2 d

H-3, H-4, H-6, H-7 H-4, H-5, H-7, H-8 H-6, H-8

71.0 d

H2-4, H2-3

4.24 (1H) dd (5.1, 3.1) 5.31 (1H) dd (8.6, 3.1) 5.58 (1H) t (8.6)

H-6, H-5

119.6 d

H-10, H-9, H6, H2-11 H-6, H2-11

134.8 d

H-8

134.8 d

H-10, H-9

125.7 d

18.3 q

126.1 d

18.0 q

H-5, H-8, H-9 H-6, Me-10 H-7, Me-10 H-9

H-9, H-10

OMe

51.6 q

3.66 (3H) s

The chemical shifts are in δ values (ppm) from TMS. b2D 1H, 1H (COSY), and 2D 13C, 1H (HSQC) NMR experiments delineated the correlations of all the protons and the corresponding carbons; cMultiplicities were assigned by DEPT spectrum; dAn allylic coupling ( 2σ(I)], R1 = 0.0733, wR2 = 0.1581; R indice (all data), R1 = 0.1359, wR2 = 0.1910; largest diff peak and hole (e/Å3), 0.208 and −0.240. Biological Assays. Leaf Puncture Assay. Young cork oak, grapevine, and tomato leaves were utilized for this assay. Each compound was assayed at 1.0 mg/mL. Compounds were first dissolved in MeOH, and then a stock solution with sterile distilled water was made. A droplet (20 μL) of test solution was applied on the adaxial sides of leaves that had previously been needle punctured. Droplets (20 μL) of MeOH in distilled water (4%) were applied on leaves as control. Each treatment was repeated three times. The leaves were kept in a moistened chamber to prevent the droplets from drying. Leaves were observed daily and scored for symptoms after 7 days. The effect of the toxins on the leaves was observed for up to 10 days. Lesions were estimated using APS Assess 2.0 software following the tutorials in the user’s manual.24 The lesion size was expressed in mm2. Tomato Cutting Assay. Tomato cuttings were taken from 21-day-old seedlings, and each compound was assayed at 0.05, 0.1, and 0.2 mg/mL. Cuttings were placed in the test solutions (2 mL) for 72 h and then transferred to distilled water. Symptoms were visually evaluated up to 7 days. Sphaeropsidin A was used as positive control, and MeOH in distilled water (4%) was used as negative control. Antifungal Assays. Sapinofuranone C, 1, and diplopyone B, 2, were also preliminariliy tested on three different plant pathogens including two fungal species (Athelia rolfsii and Fusarium avenaceum) and one Oomycete (Phytophthora nicotianae). The sensitivity of all species to these compounds was evaluated on carrot agar medium (CA) as inhibition of the mycelial radial growth. In brief, mycelial plugs (6 mm diameter) were cut from the margin of actively growing 4-day-old colonies using a flamed cork borer. One plug was placed in the center of a 9 cm diameter Petri dish with the mycelia in contact with the medium. Then 20 μL of the test solution at different concentrations (50, 100, and 200 μg/plug) was applied on the top of each plug. The negative control was obtained by applying 20 μL of MeOH. The solvent was evaporated in a laminar flow cabinet, and the plates were incubated at 20 °C for 4−7 days depending on the fungal species until the target fungi used as negative control covered the plate’s surface. The antifungal activity of D

DOI: 10.1021/acs.jafc.5b05170 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry the compounds was also compared with metalaxyl-M (mefenoxam; p.a. 43.88%) (Syngenta, Milan, Italy), a synthetic fungicide to which the Oomycete are sensitive, whereas pentachloronitrobenzene (PCNB) was used as positive control for Ascomycetes and Basidiomycetes. Colony diameters were measured in two perpendicular directions for all treatments. Each treatment consisted of three replicates, and the experiment was repeated twice. Artemia salina Bioassay. All compounds were assayed on brine shrimp larvae (A. salina L.). The assay was performed in cell culture plates with 24 cells (Corning) as previously described. The metabolites were dissolved in methanol and tested at 10, 50, 100, and 200 μg/mL. Tests were performed in quadruplicate. The percentage of larval mortality was determined after incubation for 24 and 36 h at 27 °C in the dark.

and H-7), a broad double doublet (J = 15.3 and 10.9 Hz), and a double triplet (J = 15.3 and 4.8 Hz) at δ 6.52 (H-9) and 5.97 (H10), respectively. The proton H-10 also coupled in the COSY spectrum with the proton of a terminal hydroxyl methylene group (H2C-11) resonating as a broad doublet (J = 4.8 Hz) at δ 4.13, whereas H-7 coupled with the proton of a hydroxylated methine (HC-6) appearing as a double doublet (J = 10.9 and 5.3 Hz) at δ 4.55. In fact, being the head of the 1,6-dihydroxy-2,4hexadienyl side chain, it (H-6) coupled with the proton of another secondary hydroxylated methine (HC-5), resonating as a doublet of double doublets (J = 7.5, 6.0, and 5.3 Hz) at δ 4.45. H-5, in turn, coupled with the protons of the adjacent methylene group (H2C-4) observed as two multiplets at δ 2.17 and 2.06. These latter complex systems were also coupled with the protons of the adjacent methylene group (H2C-3) resonating as two doublets of double doublets (J = 18.0, 10.5, and 6.1 Hz and J = 18.0, 9.7, and 7.5 Hz) at δ 2.53 and 2.45, typical chemical shift values for protons adjacent to a carbonyl group.27 The couplings observed in the HSQC spectrum (Table 1)21 allowed us to assign the signals at δ 136.0, 132.3, 126.9, 124.6, 83.1, 69.2, 62.2, 28.4, and 23.3 to C-10, C-8, C-7, C-9, C-5, C-6, C-11, C-3, and C-4, respectively. The 13C NMR spectrum (Table 1) also showed the presence of a typical singlet of an ester carbonyl group at δ 178.1 (C-1).28,29 Considering the total of four unsaturations, sapinofuranone C should contain a furanone ring. This result was also confirmed by the long-range coupling observed in the HMBC spectrum (Table 1)21 between C-2 and H-5, H2-4, and H2-3. The couplings observed in the same spectrum between C-6 with H2-4 and H2-3 and C-5 with H-8 also allowed us to locate the 1,6-dihydroxy-2,4-hexadienyl side chain at C-5 of 3,4dihydro-furan-2-one ring. On the basis of these findings, sapinofuranone C could be formulated as 5-(1,6-dihydroxyhexa-2,4-dienyl)-dihydrofuran-2-one, 1. The structure assigned to 1 was further supported by preparing the corresponding 6,11-O,O′-diacetyl derivative, 5 (Figure 1), with pyridine and acetic anhydride. Its IR spectrum significantly did not show bands accounting for hydroxy groups.26 Its 1H NMR (Table 2) differed from that of 1 for the expected downfield shifts (Δδ 1.18 and 0.51) of H-6 and H2-11 observed, respectively, as double doublet (J = 9.6 and 5.4 Hz) and a broad doublet (J = 5.7 Hz) at δ 5.73 and 4.64 and for the presence of the two singlets of the acetyl groups at δ 2.08 and 2.07. Its ESI-MS spectrum showed the sodiated dimer form [2M + Na]+ and the sodium cluster [M + Na]+ at m/z 586 and 305, respectively. The relative configuration of sapinofuranone C was determined by X-ray diffractometric analysis of a suitable crystal obtained by a slow evaporation of EtOAc/n-hexane (1:3, v/v) solution. This stereochemistry, reported in the ORTEP view of 1 (Figure 2), allowed us to locate the hydrogens at C-5 and C-6 in the trans configuration and to assign the relative configuration R/

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RESULTS AND DISCUSSION The EtOAc extract of D. corticola culture filtrate was fractionated by silica gel column chromatography. Further preparative TLC separations yielded four new metabolites, named herein sapinofuranone C, 1 (2.12 mg/L), diplopyrone B, 2 (1.21 mg/ L), sapinofuranone D, 3 (0.70 mg/L), and diplobifuranylone C, 4 (0.66 mg/L) (Figure 1), along with the well-known

Figure 1. Structures of sapinofuranones C, 1, and D, 3, diplopyrone B, 2, diplobifuranylone C, 4, 6,11-O,O′-diacetyl, 5, 6,11-O,O′-di-p-bromobenzoyl, 6, 11-O-p-bromobenzoyl, 7, and 6-O-p-bromobenzoyl, 8, esters of sapinofuranone C, 6-O-S-, 9, and 6-O-R-MTPA, 10, esters of 11-O-pbromobenzoyl sapinofuranone C, 5-O-acetyl-, 11, 5-O-S-, 12, and 5-OR-MTPA, 13, esters of diplopyone B, 6′-O-acetyl, 14, 6′-O-S-, 15, and 6′O-R-MTPA, 16, esters of diplobifuranylone C.

sphaeropsidins A and C, diplopyrone, diplobifuranylones A and B, diplofuranone A, and the (S,S)-enantiomer of sapinofuranone B. The identification of all the already known compounds was carried out by comparison of their spectroscopic data (1H and 13C NMR and ESI-MS) and specific optical properties with those previously reported in the literature.11−14,25 Preliminary 1H and 13C NMR investigation, consistent with the bands observed in the IR and UV spectra,26,27 showed that the new sapinofuranone C was closely related with sapinofuranones A and B.28 Sapinofuranone C, 1, showed a molecular formula of C10H14O4, as deduced from its HRESI-MS, which was consistent with four hydrogen deficiencies. Detailed investigation of its 1H NMR spectrum (Table 1) and COSY spectrum21 showed the presence of four olefinic protons belonging to cis and trans adjacent disusbstituted double bonds resonating as two triplets (J = 10.9 Hz) at δ 6.17 and 5.38 (H-8

Figure 2. ORTEP view of sapinofuranone C, 1, showing atomic labeling. Displacement ellipsoids are drawn at the 30% probability level. E

DOI: 10.1021/acs.jafc.5b05170 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry R at C-5/C-6 stereogenic centers. Compound 1 crystallizes in the orthorhombic P212121 space group with one molecule in the asymmetric unit. All bond lengths and angles are in the normal range. The molecule consists of a γ-lactone ring substituted at C5 with a 1,6-dihydroxyhexa-2,4-hexadienyl group. The fivemembered ring is in the enveloped conformation, with C-4 at the flap. In the 2,4-hexadienyl group all of the carbon atoms are coplanar within 0.02 Å and the cis/trans/trans configuration at the C-7-C-8/C-8-C-9/C-9-C-10 bonds is observed. The molecule is strongly bent with an angle of 65.2(2)° between the mean planes of the five-membered ring and the 2,4-exadienyl group. In the crystal packing the hydroxy groups are involved in strong intermolecular OH···H hydrogen bonds that wrap around the screw axes. The absolute configuration of 1 was assigned by applying the advanced Mosher’s method30 to the hydroxylated methine (C6). Due to 1 also having a primary hydroxyl group at C-11, it was first converted into the corresponding 11-O-p-bromobenzoyl derivative, 7 (Figure 1) by reaction with the p-bromobenzoyl chloride carried out in basic conditions; the same reaction afforded the other 6-O-p-bromobenzoyl, 8, and the 6,11-O,O′-dip-bromobenzoyl, 6, derivatives (Figure 1). The IR, UV, and 1H NMR (Table 2) data of derivatives 6−8 were consistent with the structure assigned to 1. The 11-O-p-bromobenzoyl derivative, 7, was converted into the corresponding S-MTPA, 9, and R-MTPA, 10, monoesters at C-6 (Figure 1) by reaction with R-(−)-αmethoxy-α-trifluoromethyl-α-phenylacetyl (MTPA) and S(+)-MTPA chloride, the spectroscopic data of which were consistent with the structure assigned to 1. Subtracting the chemical shifts of the protons (Table 2) of 6-O-R-MTPA of 7, 10, from those of 6-O-S-MTPA of 7, 9, esters, the Δδ (9−10) values for all of the protons were determined as reported in Figure 3.

tail of the side chain in 2 instead of the hydroxymethylene group present in 1. Furthermore, the region of the olefinic protons appeared more complex due to the signals of two further olefinic methines at δ 122.8 (d)/6.14 (J = 9.9 Hz) and 144.5 (d)/6.99 (dd, J = 9.9 and 5.1 Hz), whereas the signals of the two aliphatic methylene groups disappeared. The new olefinic methines could be HC-3 and HC-4, which in 1 were the methylene groups H2C3 and H2C-4, as shown by the couplings observed in the HMBC spectrum (Table 1) and in particular those of C-2 with H-3, C-3 with H-4 and H-5, and C-4 with H-3, H-5, and H-6. However, when diplopyrone B was acetylated, an unexpected monoacetyl derivative, 11 (Figure 1), was obtained. In fact, when its 1H NMR spectrum (Table 3) was compared to that of 2, the singlet of the acetyl group at δ 2.08 was observed together with the downfield shift of H-5 (Δδ 1.19), resonating as a multiplet at δ 5.43, instead of that expected for H-6. Its ESI-MS spectrum showed the sodiated dimer form [2M + Na]+ and the sodium cluster [M + Na]+ at m/z 466 and 245, respectively. On the basis of these findings 2 appeared to be a 5,6-disubstituted-5,6dihydropyran-2-one. The couplings observed in the COSY, HSQC, and HMBC spectra allowed us to assign the chemical shift to all of the protons and carbons as reported in Table 1, and 2 was formulated as 5-hydroxy-6-(penta-1,3-dienyl)-5,6-dihydro-pyran-2-one. Thus, diplopyrone B is a new α-pyrone related to diplopyrone, and both should be shunt products in the biosynthesis of furan-2-ones, the other main metabolites produced by D. corticola. The Z and E configurations of the double bonds located between C(7)−C(8) and C(9)−C(10) were deduced from the typical constant values measured for the coupling of their geminal protons,27 whereas the absolute configuration at C-5 was determined by applying the above-cited advanced Mosher’s method.30 Thus, 2 was converted into the corresponding SMTPA, 12, and R-MTPA, 13, monoesters at C-5 (Figure 1) by reaction with R-(−)-α-methoxy-α-trifluoromethyl-α-phenylacetyl (MTPA) and S-(+)-MTPA chloride, the spectroscopic data of which were consistent with the structure assigned to 2. Subtracting the chemical shifts of the protons (Table 3) of 5O-R-MTPA of 2, 13, from those of 5-O-S-MTPA of 2, 12, esters, the Δδ (12−13) values for all of the protons were determined as reported in Figure 3. The positive Δδ values were located on the right-hand side and the negative values on the left-hand side of model A.30 This model allowed us to assign the S configuration to C-5. Thus, 2 was formulated as (5S)-5-hydroxy-6-(penta-1,3dienyl)-5,6-dihydro-pyran-2-one. Sapinofuranone D, 3, had a molecular formula of C11H18O4 as deduced from its HRESI-MS and consistent with three hydrogen deficiencies. The comparison of its 1H and 13C NMR spectra (Table 1) with those of 1 showed the presence of a terminal methyl group (Me-10) [δ 18.0 (q)/1.78 (d, J = 6.5 Hz)] instead of the hydroxymethylene group in the side chain and also for the presence of the signals of a methoxy group at δ 51.6 (q)/3.66 (s). This latter was identified as a methyl ester group as deduced from its coupling in the HMBC spectrum (Table 1) with C-1. Thus, 3 could be the methyl ester of the acid generated from the hydrolysis of the furan-2-one ring of 1. The analysis of COSY, HSQC, and HMBC spectra confirms this hypothesis and allowed us to assign the chemical shifts to all of the protons and carbons of 3 as reported in Table 1. Compound 3 was therefore formulated as 4,5-dihydroxy-deca-6,8-dienoic acid methyl ester. It was not possible to assign the absolute configuration to sapinofuranone D by applying the adavanced Mosher’s method30 for the presence of two hydroxylated adjacent methines (HC-4

Figure 3. Structures of 6-O-S-, 9, and 6-O-R-MTPA, 10, esters of 11-Op-bromobenzoyl sapinofuranone C, 5-O-S-, 12, and 5-O-R-MTPA, 13, esters of diplopyrone B, and O-S-, 15, and O-R-MTPA, 16, esters of diplobifuranylone C reporting the Δδ values obtained by comparison of each proton system.

The positive Δδ values are located on the right-hand side and the negative values on the left-hand side of model A as reported previously.30 This model allowed us to assign the S configuration to C-6 and consequently the S configuration to C-5. Thus, 1 was formulated as (5S)-5-((1S)-1,6-dihydroxyhexa-2,4-dienyl)-dihydrofuran-2-one, 1. Diplopyrone B, 2, had a molecular formula of C10H12O3, deduced from its HRESI-MS spectrum and consistent with five hydrogen deficiencies. The comparison of the 1H and 13C NMR spectra of 1 and 2 (Table 1) showed that they are closely related but that 2 differed in some functionalities. In fact, a methyl group (Me-11), resonating at δ 18.1 (q)/1.81 (d, J = 7.1 Hz), was the F

DOI: 10.1021/acs.jafc.5b05170 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

spectroscopic data of which were consistent with the structure assigned to 4. Subtracting the chemical shifts of the protons (Table 4) of 6′-O-R-MTPA of 4, 16, from those of 6′-O-S-MTPA of 4, 15, esters, the Δδ (15−16) values for all of the protons were determined as reported in Figure 3. The positive Δδ values were located on the right-hand side and the negative values on the lefthand side of model A.30 The model allowed us to assign the R configuration to the 1-hydroxyethyl side chain. Thus, 4 was formulated as 5′-((1R)-1-hydroxyethyl)- 2′,5′-dihydro-2H[2,2′]bifuranyl-5-one. Two bioassays were used to investigate the phytotoxic activity of sapinofuranones C, 1, and D, 3, diplopyrone B, 2, and diplobifuranylone, 4. In the leaf-puncture bioassay, the phytotoxicity was evaluated on cork oak, grapevine, and tomato leaves. None of these compounds, except 2, were active in this assay. Specifically, diplopyrone B, 2, caused necrotic lesions on cork oak, grapevine, and tomato leaves (area lesion sizes of 11.9, 28.39, and 57.6 mm2, respectively). In tomato cuttings bioassay, compound 2 was also active. Stewing on stem and/or wilting symptoms were observed at 0.2, 0.1, and 0.05 mg/mL. None of the other compounds caused any visible symptoms in this bioassay even at the highest concentration used. The lack of phytotoxicity of sapinofuranones C, 1, and D, 3, when compared to the toxic related sapinofuranones A and B,28 was due to the modification of the side chain at C-5 in 1 and the furan-2-one ring in 3, respectively. Diplobifuranylone C, 4, was inactive as were the closely related diplobifuranylones A and B13 previously isolated from the same fungus, which, compared to sapinofuranones A and B, showed the cyclization of the side chain at C-5 into the corresponding 2,5-disubstituted dihydrofuran. Furthermomore, 4 also showed the conversion of the furan-2-one ring into the corresponding butenolide. Finally, diplopyrone B, 2, was as phytotoxic as the related diplopyrone.11 These results showed that both pyran-2-one and furan-2-one rings are important features imparting phytotoxicity and that in the pyran-2-one subgroup the nature of the side chain is less important than in the furan-2-one subgroup. Sapinofuranone C, 1, and diplopyrone B, 2, were also preliminarily screened for antifungal and antioomycete activities in vitro against three plant pathogens at 50, 100, and 200 μg/ plug. Compound 2 displayed good efficacy for the growth inhibition of the pathogens tested. Among these, P. nicotianae seems to be the most sensitive target organisms followed by A. rolfsii, whereas F. avenaceum was shown to be the most resistant one (Figure 4). In particular, diplopyrone B completely inhibited the mycelial growth of P. nicotianae at each of the concentrations used in this study, showing the same effectiveness as metalaxyl-M (data not shown). It also inhibited A. rolfsii (93.0, 85.2, and 43.8% at 200, 100, and 50 μg/plug, respectively). In the A. salina bioassay, none of the compounds showed activity at the highest concentration used (200 μg/L). In conclusion, two new sapinofuranones, C and D, the new diplopyrone B, and diplobifuranylone C were isolated along with sphaeropsidins A and C, diplopyrone, diplobifuranylones A and B, diplofuranone A, and the S,S-enantiomer of sapinofuranone B from D. corticola, a fungal pathogen belonging to the family Botryosphaeriaceae recognized as one of the most important pathogens involved in the decline of cork oak forests.5,8−10 Sapinofuranone C and diplobifuranylone C belong to butanolide and butenolide groups, which are well-known as plant, fungal, and lichen metabolites that also exhibit interesting biological activities.36 Sapinofuranone C is closely related to sapinofuronones A and B produced by Diplodia sapinea isolated

and HC-5), which could negatively affect the change of proton shifts in the two diastereomers of MTPA, on which this method is based. The application of chiroptical methods (ORD, ECD, and VCD) combined with molecular mechanical calculations, which recently has often been used to assign the absolute configuration to several natural bioactive compounds,31−35 could also be very difficult, due to the high flexibility of the molecule and also the very small available amounts of 3, just sufficient for its structure determination and biological characterization. Sapinofuranone D is not an artifact of the extraction and purification processes as in both steps MeOH was not used. Furthermore, the methyl esters, which could be generated by the transesterification of the other lactones (sphaeropsidin A, diplopyrone, sapinofuranones, diplofuranone A, and diplobifuranylones), were not isolated. Diplobifuranylone C, 4 (Figure 1), had a molecular formula of C10H12O4, as deduced from its HRESI-MS spectrum and consistent with five hydrogen deficiencies. The preliminary investigation of its 1H and 13C NMR spectra (Table 4), consistent with IR and UV absorptions, showed that it is closely related to diastereomeric diplobifuranylones A and B, previuosly isolated from D. corticola.13 However, a more detailed investigation of these spectra (Table 4) showed the presence of a further two signals in the olefinic regions δ 152.9 (d)/7.40 (br d, J = 6.1 Hz) and 123.3 (d)/6.18 (br d, J = 6.1 Hz) and the disappearance of those of two aliphatic methylene groups. Thus, the furan-2-one ring appeared converted into a butenolide ring in 4 as shown by the couplings observed in its HMBC spectrum (Table 4) between C-2 and the protons H-3 and H-4, between C3 and H-4, and between C-4 and H-3. Through the analysis of the COSY, HSQC, and HMBC spectra the chemical shifts to all of the protons and carbons were assigned, as reported in Table 4. Thus, 4 was formulated as 5′-(1-hydroxyethyl)-2′,5′-dihydro2H-[2,2′]bifuranyl-5-one. This structure was further confirmed by preparing the corresponding monoacetyl derivative, 14, by acetylation with pyridine and acetic anydride. Its IR spectrum did not show bands for hydroxy groups.26 The 1H NMR spectrum of 14 (Table 4) differed from that of 4 essentially for the downfield shifts (Δδ 1.04) of the methine proton of the 1-hydroxyethyl group attached to C-5′ of the tetrahydofuran ring, which resonated as a double quartet (J = 7.0 and 3.2 Hz) at δ 4.92 and for the presence of the singlet of the acetyl group at δ 2.05. Its ESI-MS showed the dimeric sodiated form [2M + Na]+, the potassium [M + K]+, and the sodium [M + Na]+ clusters at m/z 499, 277, and 261, respectively. The relative configuration of 4 was assigned by comparing its proton system with those previously reported for both diplobifuranylones A and B.13 In particular, the chemical shifts and multiplicities of the unchanged dihydrofuran ring, namely, H-2′, H-3′, H-4′, and H-5′, and those of the 1-hydroxyethyl side chain at C-5′ are very close to those of diplobifuranylone B and substantially differed from the data of the same protons in diplobifuranylone A. Thus, diplobifuranylone C should have the same relative configuration as diplobifuranylone B. This was also confirmed by the correlation observed in the NOESY spectrum between H-4′ and the protons of the 1-hydroxyethyl side chain at C-5′. In the same spectrum the expected correlation between H3 and H-4 was also observed. The absolute configuation of the 1hydroxyethyl side chain was assigned by applying the above-cited advanced Mosher’s method.30 Thus, 4 was converted into the corresponding S-MTPA, 15, and R-MTPA, 16, monoesters (Figure 1) by reaction with R-(−)-α-methoxy-α-trifluoromethylα-phenylacetyl (MTPA) and S-(+)-MTPA chloride, the G

DOI: 10.1021/acs.jafc.5b05170 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05170. Crystallographic data for the structures have also been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 1414884. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving. htmlor from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: + 44-1223/336-033 UV, IR, NMR, and HRESI MS spectra of compounds 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.E.) Phone: +39 081 2539178. Fax: +39 081 674330. E-mail: [email protected]. Figure 4. In vitro effects of diplopyrone B, 2, on vegetative growth of three plant pathogens at concentrations ranging from 50 to 200 μg/plug.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Italy, for recording NMR spectra. A.E. is associated with Istituto di Chimica Biomolecolare del CNR, Pozzuoli, Italy.

from infected cypress trees. Diplopyone B belongs to the αpyrone group of naturally occurring compounds, which are broadly distributed, being produced by plants, animals, marine organisms, and microbes, and having interesting biological properties.36−38 These include metabolites containing the pyran2-one moiety produced by fungi belonging to several genera and showing antibiotic, antifungal, cytotoxic, neurotoxic, and phytotoxic activities.39 Sapinofuranone D belongs to the class of unsaturated fatty acids, which are very common as naturally occurring compounds.40 Finally, diplobifuranylone C as well as the closely related diplobifuranylones A and B produced from the same fungus belongs to bifuranyls and are the first examples of naturally occurring compounds containing two linked furan rings.13 Our findings extend the knowledge on the secondary metabolite pattern of D. corticola and confirm the high chemical diversity of the species, both phylogenetically closely related and not, belonging to this genus.28,41−44 Despite the efforts made in the past few years regarding the isolation and characterization of fungal secondary metabolites from forest pathogens, their role in the infection strategies of these fungi remains largely unknown. However, the screening of D. corticola organic extracts resulted in the discovery of a new natural compound, diplopyrone B, 2, which completely inhibited the vegetative growth of P. nicotianae. These data appear relevant considering that Phytophthora spp. infections are effectively controlled with few synthetic fungicides in both agriculture and forestry. Additionally, the use of these types of compounds has led to a number of problems, including the development of fungicide resistance and potentially harmful effects to human health.45,46 Increasing public concerns over fungicide safety and possible damage to the environment have resulted in increased attention given to natural products for use in the control and prevention of the disease caused by oomycetes. In this context, the strong antioomycete activity shown by diplopyrone B, 2, suggests that this compound could be a promising antioomycete drug for the control of Phytophthora spp. also taking into account the absence of zootoxic activity. 28

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REFERENCES

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DOI: 10.1021/acs.jafc.5b05170 J. Agric. Food Chem. XXXX, XXX, XXX−XXX