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Chloromonilinic Acids C and D, Phytotoxic Tetrasubstituted 3‑Chromanonacrylic Acids Isolated from Cochliobolus australiensis with Potential Herbicidal Activity against Buffelgrass (Cenchrus ciliaris) Marco Masi,†,‡ Susan Meyer,⊥ Suzette Clement,⊥ Gennaro Pescitelli,§ Alessio Cimmino,† Massimo Cristofaro,∥,‡ and Antonio Evidente*,† †

Dipartimento di Scienze Chimiche, Università di Napoli “Federico II”, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy ‡ BBCA onlus, Via A. Signorelli 105, 00123 Rome, Italy § Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Moruzzi 13, 56124 Pisa, Italy ⊥ U.S. Forest Service Rocky Mountain Research Station, Shrub Sciences Laboratory, 735 North 500 East, Provo, Utah 84606, United States ∥ ENEA C.R. Casaccia, SSPT-BIOAG-PROBIO, Via Anguillarese 301, 00123 Rome, Italy S Supporting Information *

ABSTRACT: The fungal pathogen Cochliobolus australiensis isolated from infected leaves of the invasive weed buffelgrass (Pennisetum ciliare) was grown in vitro to evaluate its ability to produce phytotoxic metabolites that could potentially be used as natural herbicides against this weed. Two new tetrasubstituted 3-chromanonacrylic acids, named chloromonilinic acids C (1) and D (2), were isolated from the liquid cultures of C. australiensis, together with the known chloromonilinic acid B. Chloromonilinic acids C and D were characterized by spectroscopic and chemical methods as (E)-3-chloro-3[(5-hydroxy-3-(1-hydroxy-2-methoxy-2-oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid and (Z)-3-chloro-3-[(5-hydroxy-3-(2-methoxy-2oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid, respectively. The stereochemistry of chloromonilinic acids C and D was determined using a combination of spectroscopic and computational methods, including electronic circular dichroism. The fungus produced these compounds in two different liquid media together with cochliotoxin, radicinin, radicinol, and their 3-epimers. The radicinin-related compounds were also produced when the fungus was grown in wheat seed solid culture, but chloromonilinic acids were not found in the solid culture organic extract. All three chloromonilinic acids were toxic to buffelgrass in a seedling elongation bioassay, with significantly delayed germination and dramatically reduced radicle growth, especially at a concentration of 5 × 10−3 M.

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on the biological control of weeds as an alternative approach have been initiated during recent years.10 The status of bioherbicides currently on the market and their integration in cropping systems for weed management have recently been reviewed.11 The use of natural products and in particular the use of fungal phytotoxins as natural herbicides, either alone or in combination with living fungi, could be an alternative strategy for weed control.12−14 The study objective is to develop a mycoherbicide that will provide an environmentally benign control that targets buffelgrass with minimal damage to nontarget plants. Our strategy is to first isolate, identify, and test the phytotoxins produced by buffelgrass foliar pathogens for their effects on

he invasive perennial weed Cenchrus ciliaris (syn. Pennisetum ciliare) has become a serious problem in the Sonoran Desert of southern Arizona, where it has negatively impacted the native vegetation through competition for space, nutrients, and water.1−6 Furthermore, during the summer months when buffelgrass is dry, it burns readily, causing increased fire frequency in the infested areas. Fires that kill native plants such as saguaro cactus create even more space for buffelgrass, which not only survives the fire but thrives on fire.7 The landscapes of Saguaro National Park and the Coronado and Tonto National Forests will not be the same in future years without continued efforts to control this grass. Broad-spectrum herbicides and physical removal with hand tools are the only weapons presently available to manage this pest.8 However, the use of synthesized herbicides has increased herbicidal resistance and caused environmental and toxicological problems.9 Programs based © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 6, 2017 Published: October 17, 2017 2771

DOI: 10.1021/acs.jnatprod.7b00583 J. Nat. Prod. 2017, 80, 2771−2777

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(IR, UV, 1H NMR, and MS) and physical (specific rotation) data were very similar to those previously reported.18,19 Chloromonilinic acid B was first isolated together with its bromine analogue, chloromonilinic acid A,17 and chloromonilicin (4, Figure 1),18 from the cherry rot fungus Monilinia f ructicola. While 4 appeared to inhibit the growth of M. f ructicola, chloromonilinic acids A and B were inactive in these tests. Chloromonilicin was recently isolated by some of the authors from Alternaria sonchi, and its absolute configuration was assigned for the first time by X-ray diffraction analysis and by computed and experimental electronic circular dichroism (ECD) data.19 Chloromonilicin has also been shown to have antibiotic activity against bacteria and yeasts.19 The preliminary investigation of the 1H and 13C NMR spectroscopic data of chloromonilinic acids C (1) and D (2) showed signal systems that were similar to each other and to those of chloromonilinic acid A and chloromonilicin. These data are also consistent with the absorption bands observed in the UV spectra for an extended aromatic chromophore20 and bands typical for hydroxy, carbonyl, aromatic, and olefinic groups observed in the IR spectra.21 Chloromonilinic acid C (1) showed a molecular formula of C16H13ClO8 as deduced by its HRESIMS and 13C NMR data consistent with 10 indices of hydrogen deficiency. Its 1H NMR data (Table 1) showed two singlets due to H-7 and H-9 of a 1,2,3,5-tetrasubstituted aromatic ring at δ 6.86 and 6.67, which as expected were coupled (J < 1 Hz) in the COSY spectrum.22 The spectrum also showed the singlets of a trisubstituted olefinic proton (H-13), a benzylic methyl (Me-8), and an ester methoxy group at δ 6.72, 2.43, and 3.75.20 The 13C NMR spectrum (Table 1) showed the singlets of an α,β-unsaturated carbonyl (C-4), an ester (C-1), and an acid (C-14) carbonyl groups at δ 182.6, 173.6, and 166.9, a tertiary oxygenated carbon (C-6) at δ 157.7, and a tertiary chlorinated carbon (C-12) at δ 126.1. The spectrum showed two aromatic (C-7 and C-9) and an olefinic protonated carbon (C-13) and a hydroxymethine (C-2), as well as the quartets of the benzylic methyl and the methoxy groups, which, also on the basis of the couplings observed in the HSQC spectrum,22 were assigned to the signals at δ 108.1, 113.2, 136.8, and 67.1 and 22.3 and 52.9, respectively.23 The couplings observed in the HMBC spectrum22 (Table 1) confirmed the assignment of the three carbonyl carbons and those of the oxygenated and chlorinated aromatic and olefinic carbons and allowed the assignment of the remaining sp2 tertiary (C-10 and C-11) and quaternary carbons (C-3, C-5, and C-8). In fact, the couplings observed between C-3 and H-2, C-5 and H-7 and H-9, C-8 and Me-8, C-10 and H-9, and C-11 and H-2 and H-13 supported the assignment for the signals at δ 162.0, 161.4, 150.0, 120.3, and 109.6 to C-11, C-10, C-8, C-3, and C-5, respectively.23 These results suggested the presence of a tetrasubstituted 4H-chromanone moiety in 1. The coupling observed in the HMBC spectrum between C-11 and H-2 and H-13, C-4 and C-3 with H-2, and C-8 with Me-8 permitted assignment of the 3′-chloroacrylic acid, the methyl 2-hydroxyethanoate, and the benzylic methyl groups to C-11, C-3, and C-8, respectively. On the basis of these findings, chloromonilinic acid C was formulated as 3-chloro-3-[(5-hydroxy-3-(1-hydroxy-2-methoxy2-oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid (1). The structure assigned to 1 was confirmed by all the couplings observed in the HMBC spectrum and the data from the HRESIMS spectrum. This latter showed [M + H]+ ions at m/z

buffelgrass. In particular, three foliar pathogens, Cochliobolus australiensis, Nigrospora sphaerica, and Pyricularia grisea, identified on buffelgrass in its North American range have been selected as potential sources of candidate phytotoxins for buffelgrass control. From the organic extract of P. grisea grown on liquid culture two monosubstituted hex-4-ene-2,3-diols, named pyriculins A and B, together with (10S,11S)(−)-epipyriculol, trans-3,4-dihydro-3,4,8-trihydroxy-1(2H)-napthalenone, and (4S)-(+)-isosclerone, were isolated. When bioassayed in a buffelgrass coleoptile and radicle elongation test, (10S,11S)-(−)-epipyriculol proved to be the most toxic compound, and further studies are in progress.15 Recently, a new phytotoxin, named cochliotoxin, was isolated from the liquid culture of C. australiensis together with radicinin, radicinol, and their 3-epimers. When the phytotoxic activity of these compounds was evaluated against buffelgrass as well as co-occurring nontarget native grasses of the Sonoran Desert, cochliotoxin exhibited some selective phytotoxicity against buffelgrass relative to native grasses. However, further experiments to evaluate its possible use as a natural herbicide against buffelgrass at a range of concentrations and across a wider array of nontarget native species are needed.16 In an effort to optimize production of cochliotoxin for this purpose, C. australiensis was later grown in two liquid media and also in wheat seed solid culture. This paper reports on the chemical characterization and phytotoxic activity of two new tetrasubstituted 3-chromanonacrylic acids, named chloromonilinic acids C (1) and D (2), isolated from the culture filtrates of C. australiensis grown in PDB (potato dextrose broth) together with the known chloromonilinic acid B. Chloromonilicin was isolated from its mycelium. The production of these metabolites was also evaluated under different culture conditions.

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RESULTS AND DISCUSSION The phytotoxic organic extract obtained from PDB liquid culture of C. australiensis was purified as detailed in the Experimental Section. Two new tetrasubstituted chromanonacrylic acids were isolated and named chloromonilinic acids C and D (1 and 2, Figure 1). They were isolated together with known

Figure 1. Structures of chloromonilinic acids C (1), D (2), and B (3) and chloromonilicin (4).

metabolites including chloromonilinic acid B (3, Figure 1),17 cochliotoxin, radicinin, radicinol, and their 3-epimers.16 Compound 3, cochliotoxin, radicinin, and radicinol and their 3-epimers were identified by comparing their spectroscopic (IR, UV, 1H NMR, and MS) and physical (specific rotation) data with those previously reported.16,17 From the mycelium of the same fungus the known compound chloromonilicin (4, Figure 1) was isolated for the first time. Its spectroscopic 2772

DOI: 10.1021/acs.jnatprod.7b00583 J. Nat. Prod. 2017, 80, 2771−2777

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Table 1. 1H and 13C NMR Data of Chloromonilinic Acids C, D, and B, (1, 2, and 3), Recorded in Methanol-d4a,b 1 position

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Me-8 OMe

173.6 s 67.1 d 120.3 s 182.6 s 109.6 s 157.7 s 108.1 d 150.0 s 113.2 d 161.4 s 162.0 s 126.1 s 136.8 d 166.9 s 22.3 q 52.9 q

d

δH (J in Hz)

HMBC

δC

H-2, OMe

172.8 s 31.6 t 116.4 s 183.6 s 108.8 s 157.3 s 108.5 d 149.9 s 113.0 d 161.2 s 160.5 s 124.2 s 134.6 d 167.9 s 22.4 q 52.8 q

5.15 (1H) s

6.86 (1H) se 6.67 (1H) se

H-2 H-2 H-7, H-9 H-7 H-9 Me-8 H-7, Me-8 H-9 H-2, H-13 H-13

6.72 (1H) s 2.43 (3H) s 3.75 (3H) s

3c

2

H-13 H-7, H-9

d

δH (J in Hz)

HMBC

δC

H2-2, OMe

172.1 s 31.2 t 116.3 s 183.3 s 109.1 s 157.7 s 108.7 d 149.5 s 112.9 d 161.2 s 160.6 s 132.4 s 134.0 d 167.9 s 22.3 q 52.6 q

3.70 (2H) s

6.85 (1H) se 6.67 (1H) se

H2-2 H2-2, H-7 H-7, H-9 H-7 H-9, Me-8 Me-8 H-7, Me-8 H-9 H2-2, H-13 H-13

6.80 (1H) s 2.44 (3H) s 3.73 (3H) s

H-13 H-7, H-9

d

δH (J in Hz) 3.54 (2H) s

6.83 (1H) se 6.66 (1H) se

HMBC H2-2, OMe OMe H2-2 H2-2, H-7, H-9 H-7, H-9 H-7 H-9, Me-8 Me-8 H-7, Me-8 H-9 H2-2, H-13 H-13

6.65 (1H) s 2.42 (3H) s 3.66 (3H) s

H-13 H-7, H-9

a 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. cThese assignments although recorded in methanol-d4 were similar to those recorded in acetone-d6 and dioxane-d8 for 1H and 13C NMR spectra, respectively, as previously reported.17 dMultiplicities were assigned by DEPT spectrum. eA metacoupling (1% at 300 K were found whose computational data were averaged using Boltzmann weights at 300 K. The linear fits between weighted-average calculated shielding values and experimental chemical shifts were then plotted.27 Linear fits were generally good (shown in the Supporting Information), with R values of 0.996−0.997 for both 1H and 13 C (excluding the problematic C-12 linked to chlorine)27 and mean absolute errors (MAEs) of 0.12 ppm for 1H and 3.0 ppm for 13C. In particular, for the E isomer the MAE was 0.09 ppm for all 1H’s, and for the diagnostic H-13 the error was 0.04 ppm; for the Z isomer the MAE was 0.16 ppm for all 1H’s and −0.42 ppm for H-13. 13C NMR spectroscopic data were less useful, as they displayed similar MAEs of 2.8 and 3.2 ppm for the E and Z isomers, respectively. Further confirmation of the (12E) geometry came from a more detailed analysis of the molecular modeling results. For the Z isomer, in fact, most DFT low-energy conformers showed a distance of ca. 2.8 Å 2773

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recorded under the same conditions (Table 1). To support the assignment of 2 and 3, NMR DFT GIAO calculations were performed on the two compounds using the same procedure as described for 1. In Figure 3, the differences in the chemical

Figure 3. Observed and calculated chemical shift differences (ppm) for diagnostic 1H and 13C signals of 2 and 3. Calculations run at the B3LYP/pcS-2/PCM (methanol) level on DFT geometries (see text).

Figure 2. UV−vis absorption (top) and ECD spectra (bottom) of 1 measured in acetonitrile (solid lines, 4.6 mM, 0.02 cm cell) compared with spectra calculated for (S)-1 with TDDFT at the B3LYP/def2TZVP/PCM level as a Boltzmann average of 10 conformers at 300 K (dotted lines). Calculated spectra were obtained as sums of Gaussian bands with 0.3 eV exponential half-width, red-shifted by −5 nm. The ECD spectrum was scaled by a factor 0.5.

shifts of the four diagnostic signals for the experimental (δ2 − δ3) and calculated (δZ − δE) spectra are shown. For all signals, the calculations consistently reproduce the observed shielding or deshielding, and the absolute values are also within the MAE ranges (see above). The geometry of the Δ12,13 double bond is thus assigned as (Z)-2 and (E)-3. The phytotoxic activity of the three chloromonilinic acids against buffelgrass was demonstrated in seedling elongation bioassays at a range of concentrations (see Experimental Section). The compounds had no direct negative effect on seed germination percentage, but delayed germination was observed for all three compounds, and this delay increased progressively with concentration (Figure 4). Chloromonilinic acid C (1) caused germination delay at concentrations as low as 10−4 M, and all three compounds delayed germination for 4 days at 5 × 10−3 M. Radicle elongation was also essentially completely suppressed at this concentration for all three compounds, and a significant decrease in radicle length was seen even at 5 × 10−4 M. For coleoptile elongation, a large negative impact was seen only at the highest concentration. Both radicle and coleoptile elongation were strongly suppressed at this concentration, with only the coleorhiza emerging from the germinated seed in most cases. Only chloromonilinic acid C (1) significantly suppressed coleoptile elongation at concentrations of 10−3 and 5 × 10−4 M. When the fungus was grown in M1D liquid medium, chloromonilinic acids B (3), C (1), and D (2), cochliotoxin, radicinin, radicinol, and their 3-epimers were detected in the organic extract, while chloromonilicin was isolated from the mycelium. When the fungus was grown in wheat seed solid culture, chloromonilinic acids and chloromonilicin were not found in the organic extract, while cochliotoxin, radicinin, radicinol, and their 3-epimers were produced in this culture condition. These results are not surprising because it is known that some fungi are able to produce metabolites belonging to disparate classes of natural compounds when grown in different culture conditions. Recently, the seed pathogen Pyrenophora semeniperda showed the ability to produce cytochalasins and pyrenophoric acids when grown in cheatgrass and wheat seed

(4), that is, (12E,2S)-1. It is interesting to note that the ECD spectra of (12E,2S)-1 and (2S)-(4) differ strongly in terms of overall appearance and intensity (the spectrum of 4 is 1 order of magnitude more intense).19 Because the chromophoric systems are similar in 1 and 4, the differences are mostly due to the conformational flexibility of 1 and to the well-known sensitivity of ECD to molecular conformation.35 Chloromonilinic acid D (2) had a molecular formula of C16H13ClO7 deduced by its HRESIMS and 13C NMR data and consistent with 10 indices of hydrogen deficiency; thus it differed from 1 by one oxygen atom. The 1H and 13C NMR spectra of 2 showed the absence of C-2 oxymethine signals present in 1 and the signals typical of a methylene α-positioned with respect to both an unsaturated carbonyl and ester groups at δ 3.70 (2H, s)/31.6 (t). Couplings observed in the COSY, HSQC, and HMBC (Table 1) data permitted assignment of the chemical shifts of the protons and carbons. Thus, the structure of 2 was defined as 3-chloro-3-[(5-hydroxy-3-(2-methoxy-2oxoethyl)-7-methyl-4-oxo-4H-chromen-2-yl)]acrylic acid (2). It appeared to be the (12Z) geometrical isomer of the known chloromonilinic acid B (3). This compound was previously isolated as reported above from M. f ructicola; however, its 1H and 13C NMR spectra were only partially elucidated.17 The structure of 2 was confirmed by the couplings observed in the HMBC spectrum and the data from the HRESIMS spectrum, which showed the [M + H]+ ions at m/z 355.0407 and 353.0432, again reminiscent of the presence of the two isotopes of the chlorine atom. The different geometry of the double bond of the 3-chloroacrylic acid residue of 2 compared to 3 was indicated by the deshielded shifts (Δδ 0.16, 0.15, and 0.07, respectively) of H-2, H-13, and OMe and the shielding (Δδ 8.2) of C-12, observed when comparing the 1H and the 13C NMR data of 2 and 3 2774

DOI: 10.1021/acs.jnatprod.7b00583 J. Nat. Prod. 2017, 80, 2771−2777

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Figure 4. Buffelgrass seedling elongation bioassay results for three chloromonilinic acid compounds at concentrations from 0 (control) to 5 × 10−3 M: (1) chloromonilinic acid C, (2) chloromonilinic acid D, (3) chloromonilinic acid B. Response variables were radicle length 3 days postgermination, coleoptile length 3 days postgermination, and days to germination. Within each panel, means headed by the same letter are not significantly different at p < 0.05 based on an LSMeans test from mixed model analysis of variance. Error bars represent standard error of the mean. following parameters: mixing time, 200−500 ms; band-selective pulse, 5.74 ms; f1 pulse bandwidth, 700 Hz. HRESI and ESI spectra were recorded on a Waters Q-TOF Micro Mass and on an Agilent 6120 Quadrupole LC/MS instrument, respectively. Analytical and preparative TLC were performed on silica gel (Kieselgel 60, F254, 0.25 and 0.5 mm, respectively) and on reversed-phase (Merck, Kieselgel 60 RP-18, F254, 0.20 mm) plates. The spots were visualized by exposure to UV radiation (253 nm), or iodine vapor, or by spraying first with 10% H2SO4 in MeOH and then with 5% phosphomolybdic acid in EtOH, followed by heating at 110 °C for 10 min. Column chromatography was performed using silica gel (Merck, Kieselgel 60, 0.063−0.200 mm). Fungal Strain. The Cochliobolus strain used in this study (LJ-4B) was obtained from leaf spot lesions on a buffelgrass leaf tissue collection made near La Joya, Hidalgo County in south Texas, USA, in September 2014. It was identified as closely similar to C. australiensis by Sanger sequencing of the ITS region as previously described.16 Extraction and Purification of Cochliobolus australiensis Secondary Metabolites. Liquid Cultures. The strain of Cochliobolus was grown in PDB as previously reported.16 The culture filtrate (3 L) was extracted exhaustively with EtOAc. The combined organic extracts were dried with Na2SO4 and evaporated under reduced pressure. The brown-red oil residue recovered (877.9 g) was fractioned by column chromatography on silica gel eluted with CHCl3−i-PrOH (9:1). Eleven fractions were collected and pooled on the basis of similar TLC profiles. The residues of the first five fractions were further purified following the reported procedures,16 and radicinin, 3-epi-radicinin, cochliotoxin, radicinol, and 3-epi-radicinol were obtained as pure compounds. The residues of the eight (45.3 mg) and ninth (136.8 mg) fractions were combined and further purified by column chromatography on silica gel eluted with EtOAc−MeOH− H2O (85:10:5), yielding six homogeneous fractions. The residue of the second fraction (58.6 mg) of this column was further purified by TLC eluted with EtOAc−MeOH−H2O (85:10:5), yielding chloromonilinic acid B (3, 44.7 mg, Rf 0.42) as an amorphous solid. The residue of the third fraction (39.8 mg) was purified by TLC eluted with EtOAc−MeOH−H2O (85:10:5), affording an additional amount of

cultures but spirostaphylotrichins when grown in liquid culture (PDB).36−39 In conclusion, two new tetrasubstituted chromanonacrylic acids, named chloromonilinic acids C (1) and D (2), were isolated together with chloromonilinic acid B (3), cochliotoxin, radicinin, radicinol, and their 3-epimers from the culture filtrates of C. australiensis grown on PDB. While chromanones are reported as plant and microbial metabolites,40 and acrylic acids such as 3-methylthiopropenoic acid were isolated from Xanthomonas campestris pv hortoceras responsible for lettuce crown rot,41 the isolation of chromanonacrylic acid derivatives as bioactive natural products is unusual. In fact, chloromonilinic acids A and B from M. f ructicola were reported to be inactive, although the related compound chloromonilicin showed antibiotic activity.17 This is the first time that chloromonilinic acids have been reported as phytotoxic fungal metabolites with potential herbicidal activity. Whether these compounds are directly implicated in the etiology of leaf spot disease caused by C. australiensis on buffelgrass and therefore potentially useful as mycoherbicides remains to be determined.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH on a Jasco P-1010 digital polarimeter; IR spectra were recorded as deposited glass film on a Thermo Nicolet 5700 FTIR spectrometer; UV spectra were measured in MeOH on a Jasco V-530 spectrophotometer; ECD spectra were recorded on a Jasco J-815 spectropolarimeter in MeCN; 1H and 13C NMR spectra were recorded at 500 and 400 and at 125 and 100 MHz, respectively, in methanol-d4 or CDCl3 on Varian and Bruker spectrometers. The same solvents were used as internal standard. Carbon multiplicities were determined by DEPT spectra.22 DEPT, COSY-45, HSQC, and HMBC experiments22 were performed using Bruker and Varian microprograms. Band-selective ROESY experiments were performed with the 2775

DOI: 10.1021/acs.jnatprod.7b00583 J. Nat. Prod. 2017, 80, 2771−2777

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chloromonilinic acid B (3, 5.7 mg, 16.8 mg/L), chloromonilinic acid C (1, 9.1 mg, 3.0 mg/L, Rf 0.34), and chloromonilinic acid D (2, 18.7 mg, 6.2 mg/L, Rf 0.25) as amorphous solids. The harvested mycelium was lyophilized (13.7 g from 3 L of culture filtrate) and macerated with EtOAc (3 × 100 mL) for 24 h at room temperature in the dark. The organic extracts were combined, dried with anhydrous Na2SO4, and evaporated under reduced pressure, yielding a brown oil (62.5 mg). The latter was purified by TLC eluted with n-hexane−EtOAc (6:4), yielding chloromonilicin (4, 6.7 mg, 2.23 mg/L, Rf 0.36) as a yellowish compound. The strain of Cochliobolus was also grown in M1D culture at room temperature (22 °C) by inoculating 500 mL of sterile broth in 1 L Erlenmeyer flasks with fragments of mycelial mat produced on PDA (potato dextrose agar) and incubating in shaker culture for 14 days. The mycelium was removed from the medium by centrifugation and filtering, and the resulting filtrates were lyophilized and frozen at −20 °C until extraction and analysis. The lyophilized culture filtrate (1 L) was dissolved in distilled H2O (1/10 of its original volume) and extracted with EtOAc (3 × 100 mL). The organic extracts were combined and dried on Na2SO4, and the solvent was evaporated under reduced pressure, yielding a brown-red oil (121.2 mg). The presence of compounds 1−3 and radicinin-related metabolites in the organic extract was ascertained by TLC analysis carried out using EtOAc− MeOH−H2O (85:10:5) and CHCl3−i-PrOH (93:7) as solvent systems for the elution. The harvested mycelium was lyophilized (3.7 g from 1 L of culture filtrate) and macerated with EtOAc (3 × 30 mL) for 24 h at room temperature in the dark. The organic extracts were combined, dried with anhydrous Na2SO4, and evaporated under reduced pressure, yielding a brown oil (27.7 mg). The presence of chloromonilicin (4) in this organic extract was ascertained by TLC analysis carried out using n-hexane−EtOAc (6:4) as solvent system. Solid Culture. For solid culture on wheat seeds, C. australiensis conidia were produced on sterile buffelgrass leaf segments plated onto water agar. Approximately 4 mg of conidia suspended in sterile H2O was added to 200 g of soaked, autoclaved wheat seeds, and the mixture was placed in a sterile 1 L Erlenmeyer flask with an aluminum foil cap at 22 °C. The flask was hand-shaken periodically during the 4-week incubation period to prevent caking together of the grains. The culture was spread in pans and air-dried for at least several weeks prior to extraction. The dried material (162 g) was minced using a laboratory mill and extracted with 500 mL of MeOH−H2O (1% NaCl) (1:1). The mixture was centrifuged for 1 h at 7000 rpm. The pellet was extracted again with the same solvent mixture under the same conditions, and the two supernatants were pooled and defatted with n-hexane (2 × 500 mL). The resulting aqueous phase was extracted with CH2Cl2 (3 × 500 mL). The combined organic extracts were dried over Na2SO4 and evaporated under reduced pressure to yield a brown solid residue (159.0 mg). The chromatographic profile by TLC analysis, using EtOAc−MeOH−H2O (85:10:5) and CHCl3−i-PrOH (93:7) as solvent systems, showed the presence of radicinin, 3-epiradicinin, cochliotoxin, radicinol, and 3-epi-radicinol when compared in mixture with standard samples. Chloromonilinic acid C (1): UV λmax (log ε) 329 (3.97), 263 (4.55), 247 (4.56) nm; IR νmax 3460, 1745, 1657, 1610, 1440 cm−1; 1H and 13 C NMR, see Table 1; HRESIMS (+) m/z 371.0358 [M + 2 + H]+ and 369.0380 [M + H]+ (calcd for C16H14ClO8 371.0358 and 369.0377). Chloromonilinic acid D (2): UV λmax (log ε) 333 (4.03), 260 (4.68) nm; IR νmax 3360, 1732, 1656, 1607, 1435 cm−1; 1H and 13C NMR, see Table 1; HRESIMS (+) m/z 355.0407 [M + 2 + H]+ and 353.0432 [M + H]+ (calcd for C16H14ClO7 355.0399 and 353.0428). Chloromonilinic acid B (3): UV λmax (log ε) 333 (3.69), 263 (sh. 3.25), 247 (4.34) nm [lit.17 UV λmax (MeOH) (log ε) 331 (3.59), 260 (sh. 3.20), 244 (4.25) nm]; IR νmax 3352, 1737, 1703, 1658, 1608, 1438 cm−1 [lit.17 IR νmax 3350, 1738, 1703, 1657, 1601, 1435 cm−1]; 1 H and 13C NMR, see Table 1; HRESIMS (+) m/z 355.0398 [M + 2 + H]+ and 353.0432 [M + H]+ (calcd C16H14ClO7 355.0399 and 353.0428). Computational Methods. MMFF and preliminary DFT calculations were run with Spartan’16 (Wavefunction, Irvine, CA, USA, 2016) with standard parameters and convergence criteria; DFT and

TDDFT calculations were run with Gaussian’16 with default grids and convergence criteria.42 Conformational searches were run with the Monte Carlo algorithm implemented in Spartan’16 using the Merck molecular force field (MMFF). All structures thus obtained were first optimized with the DFT method at the ωB97X-D/6-31G(d) level in vacuo, then reoptimized at the ωB97X-D/6-311+G(d,p) level including the polarizable continuum model for MeOH or MeCN in its IEF formulation. The procedure afforded from 10 to 15 minima for compounds 1, 2, and 3 and only two minima for compound 4. TDDFT calculations were run with various functionals (B3LYP, CAM-B3LYP, M06) and the def2-TZVP basis set and including PCM for MeCN. Average ECD spectra were computed by weighting component ECD spectra with Boltzmann factors at 300 K estimated from DFT internal energies. ECD spectra were generated using the program SpecDis,33,34 using dipole-length rotational strengths; the difference with dipole-velocity values was negligible in all cases. NMR calculations were run with the GIAO method, B3LYP functional, pcS-2 basis set, and including PCM for MeOH. Average shielding values were computed by the same averaging procedure described above. The whole sets of 1H and 13C shieldings for compounds 1−4 were combined and plotted against the respective 1H and 13C chemical shifts recorded in methanol-d4; then a linear fit was applied to estimate the fitting R parameter and the mean absolute errors. Seedling Elongation Bioassay. Seeds of buffelgrass (Cenchrus ciliaris) with the enclosing burr stripped off were used for this assay. Each compound (1−3) was first dissolved in DMSO and then brought to one of four concentrations with distilled H2O (final DMSO concentration 2%): 5 × 10−3, 10−3, 5 × 10−4, and 10−4 M. For each compound at each concentration, 250 μL of the solution was pipetted into each of three 3.5 cm Petri dishes onto the surface of one filter paper. Seeds were incubated in 2% DMSO in the control treatment. Six buffelgrass seeds were arranged onto the surface of each filter paper in a pattern that made it possible to track individual seeds. Petri dishes were sealed with Parafilm to retard moisture loss and incubated at 25 °C with a 12:12 h photoperiod. Germination, defined as protrusion of at least the coleorhiza, was scored each day for 10 days, and germination day was tracked individually for each seed. Three days after germination, the coleoptile and radicle length for each seedling was recorded using electronic calipers. Unlike earlier buffelgrass seedling bioassays with other compounds, a common observation in this test was that seeds initiated germination with the protrusion of the coleorhiza, but subsequent radicle and coleoptile development was completely suppressed. Seeds that produced neither a radicle nor a coleoptile but did produce a coleorhiza were scored as germinated, with both radicle and coleoptile lengths of zero. Germination time was scored as the day that the coleorhiza emerged rather than the day the radicle emerged in these cases. Seeds that produced just a coleorhiza and coleoptile were scored as germinated with a measured coleoptile length but a radicle length of zero. Data from the seedling elongation bioassay were analyzed individually for each compound using mixed model analysis of variance (SAS 9.4, Proc Mixed). Block (a set of five Petri dishes, one at each concentration) was the random effect, and compound concentration was the fixed treatment effect. The DMSO control was included in each analysis. Data for individual seeds were considered within-block replicates for each treatment. For each compound, the effect of concentration on germination time and coleoptile and radicle length was evaluated using LSMeans separations from analysis of variance on log-transformed data. Untransformed values are reported.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00583. NMR and HRESIMS spectra of compounds 1−3 (PDF) 2776

DOI: 10.1021/acs.jnatprod.7b00583 J. Nat. Prod. 2017, 80, 2771−2777

Journal of Natural Products

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 081 2539178. Fax: +39 081 674330. E-mail: [email protected]. ORCID

Gennaro Pescitelli: 0000-0002-0869-5076 Antonio Evidente: 0000-0001-9110-1656 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded in part through the U.S. Forest Service State and Private Forestry Biological Control of Invasive Plants Grant Program. The authors gratefully acknowledge the support of R. Reardon and A. White of the U.S. Forest Service throughout the course of this project. K. Wahl-Villarreal of the U.S. Fish and Wildlife Service made the original pathogen field collection. A.E. is associated with “Istituto di Chimica Biomolecolare del CNR”, Pozzuoli, Italy. Prof. L. Di Bari and Dr. M. Górecki are thanked for help with NMR spectroscopy and ECD measurements.

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DOI: 10.1021/acs.jnatprod.7b00583 J. Nat. Prod. 2017, 80, 2771−2777