Montagnuphilones A–G, Azaphilones from Montagnulaceae sp

Article pubs.acs.org/jnp

Montagnuphilones A−G, Azaphilones from Montagnulaceae sp. DM0194, a Fungal Endophyte of Submerged Roots of Persicaria amphibia Jian-Guang Luo,†,‡ Ya-ming Xu,† Dustin C. Sandberg,§ A. Elizabeth Arnold,§ and A. A. Leslie Gunatilaka*,† †

Natural Products Center, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of Arizona, 250 E. Valencia Road, Tucson, Arizona 85706, United States ‡ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China § School of Plant Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, Arizona 85721, United States S Supporting Information *

ABSTRACT: Seven azaphilones, montagnuphilones A−G (1−7), together with previously known azaphilones 8−11, were encountered in Montagnulaceae sp. DM0194, an endophytic fungus isolated from submerged roots of Persicaria amphibia. The structures of 1−7 were elucidated on the basis of their MS and NMR spectroscopic analysis. Compounds 1−8 were evaluated for their cytotoxicity and ability to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 macrophage cells. Among these, none were found to be cytotoxic to RAW264.7 cells up to 100.0 μM, but 8, 5, and 2 showed NO inhibitory activity with IC50 values of 9.2 ± 0.9, 25.5 ± 1.1, and 39.6 ± 1.8 μM, respectively.

A

polyketide-derived metabolites, including macrolides,4,6a azaphilones,6b and terpenoids.6c

zaphilones belong to a structurally diverse class of fungal metabolites with a highly oxygenated pyranoquinone bicyclic core encountered in genera such as Aspergillus, Hypoxylon, Monascus, and Penicillium (Eurotiomycetes).1 Many azaphilones have been reported to exhibit antimicrobial, antifungal, anti-inflammatory, antioxidant, antiviral, cytotoxic, nematicidal, and nitric oxide (NO) inhibitory activities.2 As part of our ongoing search for bioactive metabolites from endosymbiotic fungi,3 we have investigated Montagnulaceae sp. DM0194 (Dothideomycetes), a fungal strain inhabiting live submerged roots of Persicaria amphibia (L.) Delarbre (water smartweed; Polygonaceae), an emergent aquatic macrophyte of the Sonoran Desert bioregion. When cultured in potato dextrose broth (PDB) containing 0.25 mM CuSO4 (PDB +Cu2+),4 this fungus produced seven new azaphilones, montagnuphilones A−G (1−7), together with previously known azaphilones 8−11. Reported herein are the structure elucidation of 1−7 and NO inhibitory activity of 1−8. Montagnulaceae is a new fungal family established in 2001,5a but many of the genera regarded as members of the Montagnulaceae remain understudied and therefore poorly understood for a systematic treatment.5b Among the most commonly recognized genera (Bimuria, Didymocrea, Kalmusia, Karstenula, Montagnula, Paraphaeosphaeria, Paraconiothyrium, and Letendraea),5b only some species of the genera Paraphaeosphaeria and Paraconiothyrium have been studied for their metabolites, leading to characterization of a variety of © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The mycelia and the culture broth derived from a PDB+Cu2+ culture of Montagnulaceae sp. DM0194, separated by filtration, were extracted with MeOH and EtOAc, respectively. The MeOH extract of the mycelia was concentrated and extracted with EtOAc. The resulting EtOAc extracts derived from mycelia and culture broth had similar TLC profiles and were therefore combined and fractionated by silica gel column chromatography (CC), Sephadex LH-20 gel-permeation chromatography, and RP-C18 CC followed by preparative HPLC to yield metabolites 1−11. Montagnuphilone A (1), obtained as a yellow, amorphous powder, was determined to have the molecular formula C21H20O9 by a combination of HRESIMS and NMR data, indicating 12 degrees of unsaturation. The 1H NMR data (Table 1) included signals due to two meta-coupled aromatic protons [δH 6.22 (d, J = 2.4 Hz) and 6.21 (d, J = 2.4 Hz)], an oxygenated methine [δH 5.67 (t, J = 3.2 Hz)], a pair of trans olefinic protons [δH 7.10 (d, J = 15.2 Hz) and 6.33 (d, J = 15.2 Hz)], an uncoupled olefinic proton [δH 6.01 (s)], two methyl singlets [δH 1.46 and 2.24], and two oxygenated methylene Received: August 2, 2016 Published: January 18, 2017 76

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Chart 1

Table 1. 1H NMR Data for Compounds 1−7 (400 MHz, J in Hz) 1a

2b

4.93, dd (13.2, 1.6) 4.76, dd (13.2, 1.6) 6.01, s

4.93, d (12.8) 4.76, d (12.8) 5.15, s

6 9 10

3.20, 2.92, 5.67, 1.46, 7.10,

2.85, 2.64, 5.52, 1.35, 2.45,

11

6.33, d (15.2)

2.45, brs

4.57, 4.29, 2.90, 2.58, 3.13, 2.83, 5.63, 1.46, 2.78, 2.54, 2.30,

12 3′ 5′ 7′ OCH3

6.22, d (2.4) 6.21, d (2.4) 2.24, s

6.12, d (2.4) 6.08, d (2.4) 2.11, s

6.22, d (2.4) 6.24, d (2.4) 2.24, s

position 1 4 5

a

brd (17.6) brd (17.6) t (3.2) s d (15.2)

3a

d (19.2) d (19.2) t (2.8) s brs

4a

d (14.4) d (14.4) brd (18.0) brd (18.0) brd (20.6) brd (20.6) t (3.2) s m m m

5c

4.42,brd (2.0) 2.90, 2.58, 3.13, 2.83, 5.67, 1.45, 2.78, 2.54, 2.30,

brd (18.0) brd (18.0) brd (20.6) brd (20.6) t (3.6) s m m m

6.21, d (2.4) 6.25, d (2.4) 2.29

4.43, 4.30, 2.72, 2.18, 2.89, 2.65, 5.50, 1.44, 2.06, 1.79, 2.09, 1.93, 3.93, 6.10, 6.01, 2.14,

6c

d (16.0) d (16.0) d (19.6) d (19.6) brd (18.0) brd (18.0) t (2.8) s m m m m t (6.8) d (1.8) d (1.8) s

4.53, 4.27, 2.04, 1.81, 2.85, 2.65, 5.59, 1.43, 2.20, 2.65, 2.09, 1.94, 3.95, 6.20, 6.09, 2.16,

7c

d (16.0) d (16.0) d d brd (19.2) brd (19.2) t (2.4) s m m m m t (7.2) d (2.0) d (2.0) s

5.01, d (12.4) 4.79, d (12.4) 5.19, s 2.91, 2.72, 5.61, 1.43, 2.54,

brd (19.2) brd (19.2) t (2.4) s m

2.54, m

6.21, 6.10, 2.15, 3.66,

d (2.0) d (2.0) s s

In acetone-d6. bIn CDCl3+CD3OD (50:1). cIn CDCl3.

Table 2. 13C NMR Data for Compounds 1−7 (100 MHz) position 1 3 4 4a 5 6 7 8 8a 9 10 11 12 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ OCH3 a

1a 64.6, 157.6, 112.0, 146.3, 32.4, 77.9, 75.2, 196.9, 118.2, 23.7, 137.2, 123.4, 167.0, 105.5, 165.8, 101.6, 163.5, 112.5, 144.8, 24.5, 171.5,

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

2b 64.0, 167.2, 101.4, 147.9, 31.9, 76.1, 74.0, 195.8, 111.8, 23.6, 29.0, 30.7, 174.3, 104.2, 164.9, 100.7, 162.2, 111.8, 143.7, 24.0, 170.7,

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

3a 60.2, 105.6, 38.3, 147.6, 34.6, 77.5, 75.1, 198.0, 127.2, 23.7, 28.4, 34.1, 176.2, 105.6, 166.3, 101.6, 163.5, 112.5, 144.8, 24.4, 171.6,

4a

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

60.5, 105.4, 38.3, 147.8, 35.0, 77.8, 75.3, 198.3, 127.2, 23.2, 28.4, 34.0, 176.1, 105.7, 166.6, 101.6, 163.6, 112.5, 145.1, 24.6, 171.8,

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

5c 58.2, 103.8, 37.7, 148.2, 35.0, 76.2, 74.5, 198.2, 127.4, 23.6, 36.8, 23.5, 67.9, 105.1, 165.6, 101.0, 160.6, 111.4, 144.4, 24.2, 170.8,

6c CH2 C CH2 C CH2 CH C C C CH3 CH2 CH2 CH2 C C CH C CH C CH3 C

57.7, 103.7, 36.8, 148.1, 34.4, 76.1, 74.3, 197.7, 127.4, 23.8, 37.6, 23.5, 68.1, 105.2, 165.6, 101.3, 160.8, 111.5, 144.0, 24.3, 170.7,

7c CH2 C CH2 C CH2 CH C C C CH3 CH2 CH2 CH2 C C CH C CH C CH3 C

64.2, 167.0, 101.6, 147.8, 32.1, 76.2, 74.2, 195.8, 111.9, 24.1, 29.2, 30.9, 172.3, 105.3, 165.6, 101.3, 160.6, 111.4, 144.1, 24.2, 170.7, 51.9,

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

In acetone-d6. bIn CDCl3+CD3OD (50:1). cIn CDCl3.

protons [δH 4.93 (dd, J = 13.2, 1.6 Hz) and δH 4.76 (dd, J = 13.2, 1.6 Hz)]. The 13C NMR spectrum of 1 (Table 2) assigned

with the help of its DEPT data revealed the presence of two methyls, two methylenes (of which one was oxygenated), six 77

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methines (one oxygenated, three olefinic, and two aromatic), 11 nonprotonated carbons (one oxygenated, three olefinic, four aromatic, two ketone carbonyl, and one carboxylic acid carbonyl carbons). These 1H and 13C NMR data suggested that 1 contained an azaphilone and methylated orsellinic acid moieties. Comparison of these data with those of entonaemin A7,8 suggested their close resemblance except that the CH2OH12 in entonaemin A was replaced by a CO2H moiety (δC 167.0) in 1 (Table 2). The observed HMBC correlations (Figure S29, Supporting Information) from H-10 to C-4/C-12 and from H11 to C-3/C-12 further supported this deduction. The relative configurations of the two chiral centers (C-6 and C-7) in 1 were found to be cis by the observed NOE correlation between H-6 and H3-9 (Figure S27, Supporting Information). On the basis of the above evidence, the structure of montagnuphilone A was elucidated as (E)-3-[(6R*,7R*)-6-(2′,4′-dihydroxy-6′methylbenzoyloxy)-7-hydroxy-7-methyl-8-oxo-5,6,7,8-tetrahydro-1H-isochromen-3-yl]acrylic acid (1). Montagnuphilone B (2), also obtained as a yellow, amorphous powder, gave the molecular formula C21H22O9 with 11 degrees of unsaturation by the analysis of its HRESIMS and 13C NMR data, suggesting that it may be a saturated analogue of 1. Comparison of 1H and 13C NMR data of 1 and 2 confirmed that the double bond at C-10(11) of 1 was replaced by a CH2−CH2 moiety [δH 2.45 (4H, br s); δC 30.7 (CH2) and 29.0 (CH2)] (Tables 1 and 2) in 2. This was further supported by the HMBC data (Figure S29), which showed a correlation of H-4 with C-10. The relative configuration at C-6 and C-7 of 2 was also deduced to be cis by the observed correlation between H-6 and H3-9 in its 1D selective NOESY spectrum (Figure S27, Supporting Information). Thus, the structure of montagnuphilone B was deduced as 3-[(6R*,7R*)-6-(2′,4′-dihydroxy-6′methylbenzoyloxy)-7-hydroxy-7-methyl-8-oxo-5,6,7,8-tetrahydro-1H-isochromen-3-yl]propanoic acid (2). Montagnuphilones C (3) and D (4) were determined to have the same molecular formula of C21H24O10 from their HRESIMS and NMR data, indicating that they contain two hydrogen atoms and one oxygen atom more than 2. The 1H NMR and 13C NMR data of 3 and 4 were almost identical except for the chemical shift data for H2-1 [δH 4.57 (d, J = 14.4 Hz) and 4.29 (d, J = 14.4 Hz) for 3; 4.42 (2H, brd, J = 2.0 Hz) for 4]. Detailed analysis of the NMR data (Tables 1 and 2) of 3 and 4 also suggested that structures of 3 and 4 were similar to that of 2. However, signals for the 3(4)-double bond of 2 were found to be absent in the NMR spectra of 3 and 4. Instead, a methylene [δH 2.90 (brd, J = 18.0 Hz), 2.58 (brd, J = 18.0 Hz); δC 38.3 for 3; δH 2.90 (brd, J = 18.0 Hz), 2.58 (brd, J = 18.0 Hz); δC 38.3 for 4] and hemiketal carbon (δC 105.6 for 3; 105.4 for 4) were observed. These data suggested that a molecule of H2O was added to the 3(4)-double bond of 2 to form a hemiketal at C-3 in 3 and 4. Although 3 contained a peak due to [M − H]− in its negative ion HRESIMS, the presence of a prominent peak in the MS of 3 and 4 due to loss of a molecule of H2O leading to a fragment ion at m/z 417 with a conjugated dienone moiety provided additional evidence for the presence of OH-3. The hemiketal at C-3 in 3 was further confirmed by the 3J-HMBC correlations observed for 3 from H2-1 to C-3, from H2-4 to C-5, C-8a, and C-10, from H2-10 to C-4 and C12, and from H2-11 to C-3 (Figure 1). In the NOESY data of 3 (Figure S27, Supporting Information), the NOE correlations of H-6 to H3-9 indicated the cis configuration for H-6 and CH3-9 in 3. The NOE correlations of H-5β (δH 3.13)/H-4β and H4β/H-10a (δH 2.54) suggested that these were on the same face

Figure 1. Selected HMBC and NOESY correlations of 3.

of the ring system in 3, which led to assignment of the relative configuration of the hemiketal carbon (C-3) as shown in Figure 1. The NOE correlation of H-6 to H3-9 in 4 (Figure S28, Supporting Information) was helpful again in establishing the cis configuration for H-6 and CH3-9. The foregoing suggested that montagnuphilones C (3) and D (4) were epimeric at C-3. In their 1H NMR spectra (Table 1), CH2-11 appeared as a single set of signals at δH 2.30 (m), supporting the open-chain structures for 3 and 4 and not the alternate oxaspirolactone structures resulting from lactonization between CO2H and OH3.9 Thus, the structures of montagnuphilones C and D were assigned as 3-[(3R*,6R*,7R*)-6-(2′,4′-dihydroxy-6′-methylbenzoyloxy)-3,7-dihydroxy-7-methyl-8-oxo-3,4,5,6,7,8-hexahydro-1H-isochromen-3-yl]propanoic acid (3) and 3[(3S*,6R*,7R*)-6-(2′,4′-dihydroxy-6′-methylbenzoyloxy)-3,7dihydroxy-7-methyl-8-oxo-3,4,5,6,7,8-hexahydro-1H-isochromen-3-yl]propanoic acid (4), respectively. The HRMS and NMR data for montagnuphilones E (5) and F (6) suggested that they had the same molecular formula, C21H26O9. The NMR data of 5 and 6 showed close similarities, only with small but diagnostic differences for H2-1 [δH 4.43 (d, J = 16.0 Hz) and 4.30 (d, J = 16.0 Hz) for 5; δH 4.53 (d, J = 16.0 Hz) and 4.27(d, J = 16.0 Hz) for 6] (Tables 1 and 2). These data suggested that 5 and 6 are also a pair of C-3 epimers similar to 3 and 4. Furthermore, analysis of the 1H NMR and 13 C NMR data of 5 and 6 (Tables 1 and 2) indicated close structural similarities to 3 and 4. A notable difference in the NMR spectra was that the CO2H group attached to C-11 of 3 and 4 was replaced by a CH2OH in 5 and 6 [δH 3.93 (2H, J = 6.8 Hz), δC 67.9 for 5; δH 3.95 (2H, J = 7.2 Hz), δC 68.1 for 6]. The HMBC correlations observed for 6 (Figure S29, Supporting Information) from H-12 to C-10 and C-11 confirmed the location of this CH2OH group. The NOESY spectra of 5 and 6 showed strong correlations between H-6 and H3-9 (Figures S27 and S28, Supporting Information), confirming the cis configuration of H-6 and CH3-9 in both of these metabolites, as was found in 1−4. In addition, the NOESY correlation observed between H-4β and H-10 suggested that 3-OH in 5 had an α orientation. In their 1H NMR spectra (Table 1), CH2-12 of 3 and 4 appeared as a single set of signals at δH 3.93 (t, 6.8 Hz) and 3.95 (t, 7.2 Hz), respectively, supporting the open-chain structures for these metabolites and not the alternate oxaspiroketal structures.10 Therefore, the structures of montagnuphilones E and F were elucidated as (3R*,6R*,7R*)-3,7-dihydroxy-3-(3-hydroxyprop78

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1.5 mL microcentrifuge tubes.17 Tubes were incubated under ambient light/dark conditions at room temperature (ca. 21.5 °C) for four months. Emergent fungi were isolated into pure culture on 2% MEA, vouchered in sterile water, and deposited as living vouchers at the Robert L. Gilbertson Mycological Herbarium of the University of Arizona. One endophytic fungus, isolate DM0194, was obtained from submerged root tissue and was used for the present study. This strain has been accessioned at the Robert L. Gilbertson Mycological Herbarium at the University of Arizona as accession DM0194. Total genomic DNA was isolated from fresh mycelium of DM0194,17 and the nuclear ribosomal internal transcribed spacers and 5.8s gene (ITS rDNA; ca. 600 base pairs [bp]) were amplified with the first portion of the nuclear ribosomal large subunit as a single fragment by PCR (ITSrDNA-partial LSUrDNA).17 Positive amplicons were sequenced bidirectionally as described previously.17 A consensus sequence was assembled, and basecalls were made by phred18 and phrap19 with orchestration by Mesquite,20 followed by manual editing in Sequencher (Gene Codes Corp.). The sequence was submitted to GenBank under accession KF673734. Because the isolate DM0194 did not produce diagnostic fruiting structures in culture, three methods were used to tentatively identify isolate DM0194 using molecular sequence data. First, the LSUrDNA portion of the sequence was ̈ Bayesian classifier for fungi21 available evaluated using the naive through the Ribosomal Database Project (http://rdp.cme.msu.edu/). The Bayesian classifier estimated placement within Dothideomycetes with high support, but placement at finer taxonomic levels was not possible. Therefore, the entire sequence was compared against the GenBank database using BLAST.22 The top BLAST matches were primarily to cultured and uncultured Coniothyrium, Paraconiothyrium, and related taxa within the Pleosporales (Dothideomycetes) and to unknown Ascomycota, cloned fungi from diverse environments, and unidentified endophytes. To clarify the phylogenetic placement and taxonomic assignment of DM0194, we downloaded the top 100 BLAST matches from GenBank and aligned DM0194 and the resulting data set automatically using MUSCLE (http://www.ebi.ac. uk/Tools/msa/muscle/) with default parameters. The alignment was trimmed so that starting and ending points were consistent, and ambiguous regions were excluded manually in Mesquite18 prior to analysis. The data set was analyzed using maximum likelihood in GARLI23 using the GTR+I+G model of evolution as determined by ModelTest.24 This analysis placed DM0194 in close proximity to various Coniothyrium and Paraconiothyrium species, including voucher specimens, as well as diverse endophytes from Arizona reported previously but not yet identified.17,25 We therefore examined the literature for current phylogenetic hypotheses regarding these fungal taxa. Archived alignments from two studies26,27 were used to guide taxon sampling for a third analysis, which sought to determine the placement of DM0194 with regard to known diversity in the clade containing Coniothyrium and relatives. The final data set included 24 terminal taxa and was trimmed to consistent starting and ending points based on the availability of sequences in GenBank (326 characters). The alignment was assembled in MUSCLE, checked by eye in Mesquite, and analyzed using maximum parsimony in PAUP 4.0a14928 and maximum likelihood in GARLI, as above. Support for the topology was assessed using 1000 bootstrap replicates in the parsimony and likelihood frameworks in PAUP and GARLI, respectively. The analysis unequivocally placed the sequence with strong support within a clade containing sequences of well-identified specimens Coniothyrium, Paraconiothyrium, Neosetophoma, and Microsphaeropsis (the anamorphic form of Paraphaeosphaeria michotii), as well as unidentified endophytes from aquatic and terrestrial plants in Arizona (see Figure S30). DM1094 is most closely related to other endophytes from aquatic plants and was not placed clearly in any currently recognized genus. The clade identified here is generally consistent with the Paraconiothyrium/Paraphaeosphaeria clade identified previously,26 which was confirmed within the Montagnulaceae in subsequent work.29 Therefore, we designate this strain as Montagnulaceae sp. DM0194, pending morphological and multilocus analyses.

yl)-7-methyl-8-oxo-3,4,5,6,7,8-hexahydro-1H-isochromen-6-yl 2′,4′-dihydroxy-6′-methylbenzoate (5) and (3S*,6R*,7R*)-3,7dihydroxy-3-(3-hydroxypropyl)-7-methyl-8-oxo-3,4,5,6,7,8-hexahydro-1H-isochromen-6-yl 2′,4′-dihydroxy-6′-methylbenzoate (6), respectively. Montagnuphilone G (7), obtained as a yellow, amorphous powder, was suspected to be the methyl ester of 2 by direct comparison of their NMR data. The 1H and 13C NMR data of 2 and 7 (Tables 1 and 2) were almost identical except that 7 showed the presence of a methoxy group (δH 3.66, δC 51.9) that was not found in 2. The upfield shift of C-12 from δC 174.3 in 2 to δC 172.3 in 7 revealed the location of the methoxy group, which was further confirmed by correlations between OCH3 and C-12 in the HMBC spectrum (Figure S29, Supporting Information). Thus, montagnuphilone G was identified as (6R*,7R*)-7-hydroxy-3-(3-methoxy-3-oxopropyl)-7-methyl-8-oxo-5,6,7,8-tetrahydro-1H-isochromen-6-yl 2′,4′-dihydroxy-6′-methylbenzoate. In addition to the metabolites 1−7, four known azaphilones, rubiginosins A (10)8 and B (8)8 and comazaphilones A (9)11 and D (11),11 were also isolated and identified by comparison of their 1H and 13C NMR data with those reported. Previous studies have suggested that some azaphilones exhibit anti-inflammatory activities by inhibiting nitric oxide production.12−15 NO is a multifunctional signaling molecule that is known to regulate various physiological and pathophysiological processes, such as vascular and neurological functions, and NO production has also been related to the progression of tumors.16 Thus, metabolites 1−6 and 8, representing all three structural types encountered in this study, were selected for evaluation of their cytotoxic activity and ability to inhibit NO production. A preliminary MTTbased cytotoxicity assay revealed that these azaphilones were not cytotoxic (over 90% cell survival) to lipopolysaccharideactivated RAW264.7 cells used for NO inhibitory assay at concentrations up to 100 μM. In the NO inhibitory assay, rubiginosin B (8) and montagnuphilones B (5) and E (2) showed activity with IC50 values of 9.2 ± 0.9, 25.5 ± 1.1, and 39.6 ± 1.8 μM, respectively. The positive control, NG-nitro-Larginine methyl ester (L-Name), had an IC50 of 30.6 ± 2.0 μM in this assay (see Table S1, Supporting Information).

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a Jasco Dip-370 polarimeter using MeOH as the solvent. UV spectra were recorded with a Shimadzu UV 2601 spectrophotometer. 1D and 2D NMR spectra were recorded with a Bruker Avance III 400 NMR instrument at 400 MHz for 1H NMR and 100 MHz for 13C NMR. Chemical shift values (δ) are given in parts per million (ppm), and the coupling constants are in Hz. Lowresolution and high-resolution MS were recorded on Shimadzu LCMS-QP8000α and JEOL HX110A spectrometers, respectively. HPLC purifications were carried out on a 10 × 250 mm Phenomenex Luna 5 μm C18 (2) column with a Waters Delta Prep system equipped with a PDA 996 detector. Fungal Isolation and Identification. In June 2012, a healthy individual of Persicaria amphibia (L.) Delarbre was collected from shallow water (ca. 17 cm) at Willow Creek Reservoir in Prescott, Arizona, USA (N 34°36.127, W 112°26.251, 1564 m.a.s.l.).17 The entire plant was washed in tap water, separated into roots, shoots, and leaves, and cut into ca. 2 mm2 segments that were surface-sterilized by agitating sequentially in 95% EtOH for 30 s, 0.5% NaOCl for 2 min, and 70% EtOH for 2 min.17 A total of 96 segments of each tissue type were surface-dried under sterile conditions and then placed individually onto 700 μL of 2% malt extract agar in individual, sterile, 79

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417 [M − H − H2O]− (45), 267 (40), 167 (100); HRESIMS m/z 435.1292 [M − H]− (calcd for C21H23O10, 435.1297). Montagnuphilone D (4): pale yellow, amorphous powder; [α]25 D +153 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 267 (4.1), 303 (3.8), 1 13 356 (3.3) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/z 417 [M − H − H2O]− (35), 267 (30), 167 (100); HRESIMS m/z 419.1339 [M − H − H2O]− (calcd for C21H21O9, 435.1342). Montagnuphilone E (5): faint yellow, amorphous powder; [α]25 D +171 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 264 (4.2), 303 (3.8), 352 (3.2) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 403 [M − H − H2O]− (30), 253 (28), 167 (100); HRESIMS m/z 403.1380 [M − H − H2O]− (calcd for C21H23O8, 403.1393). Montagnuphilone F (6): pale yellow, amorphous powder; [α]25 D +85 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 265 (4.1), 302 (3.7) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 403 [M − H − H2O]− (32), 215 (52), 167 (100); HRESIMS m/z 403.1372 [M − H − H2O]− (calcd for C21H23O8, 403.1393). Montagnuphilone G (7): yellow, amorphous powder; [α]25 D +171 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 270 (4.0), 305 (4.0), 352 (3.9) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 431 [M − H]− (95), 281 (62), 263 (28), 167 (100); positive HRESIMS m/z 455.1307 [M + Na]+ (calcd for C22H24O9Na, 455.1313). Bioassays. Methods used for cytotoxicity (MTT) and NO production assays involved those described previously.30 All experiments were performed in triplicates.

Cultivation and Isolation of Metabolites. A seed culture of Montagnulaceae sp. DM0194 grown on PDA for 2 weeks was used for inoculation. Mycelia were scraped out, mixed with sterile water, and filtered through a 100 μm filter to separate spores from the mycelia. Absorbance of the spore solution was measured (at 600 nm) and adjusted to between 0.3 and 0.5. This spore solution was used to inoculate 4 × 2 L Erlenmeyer flasks, each holding 1 L of the medium (PDB) containing 0.25 mM CuSO4 and incubated at 160 rpm and 28 °C. The glucose level in the medium was monitored using glucose strips (URISCAN glucose strips). On day 28, the strip gave a green color for the glucose test, indicating the absence of glucose in the medium. Mycelia were then separated by filtration. The filtrate (4 L) was extracted with EtOAc (3 × 2000 mL). The combined EtOAc layer was washed with water, dried over anhydrous Na2SO4, and evaporated under reduced pressure to give a crude extract of the culture broth (450 mg). The mycelia were exhaustively extracted with MeOH in an ultrasonic bath for 1 h at 25 °C, and the resulting extract was filtered through a layer of Celite 545. The filtrate was concentrated to about one-third of its volume in vacuo below 40 °C and was extracted with EtOAc (3 × 250 mL). After removing the solvent, a crude extract of mycelia (516 mg) was obtained. Since the TLC and HPLC profiles of the two extracts were nearly identical, they were combined and used for fractionation and isolation of metabolites. The combined extract (966 mg) was partitioned between hexane and 80% aqueous MeOH. The 80% aqueous MeOH fraction was diluted with H2O to 50% aqueous MeOH and extracted with CHCl3. Evaporation of solvents under reduced pressure yielded hexane (108 mg) and CHCl3 (760 mg) fractions. The CHCl3 fraction (760 mg) was then subjected to gel-permeation chromatography over a column of Sephadex LH-20 (50 g) made up in hexanes−CH2Cl2 (1:4, 300 mL) and eluted with 300 mL each of CH2Cl2−acetone (4:1), CH2Cl2−acetone (2:3), and MeOH, and the resulting fractions were combined based on their TLC (SiO2; CHCl3−MeOH, 10:1) profiles to afford three combined fractions, A−C. Of these, fractions A and B were found to be major and were therefore subjected to further fractionation. Fraction A (345 mg) was separated by reversed-phase (RP) C18 (40 μm; 18 g) CC and by sequential elution with 60% aqueous MeOH, 70% aqueous MeOH, and 80% aqueous MeOH to afford three fractions, A1−A3. TLC (SiO2; CHCl3−MeOH, 95:5) investigation of these suggested that fractions A1 and A2 contained major and interesting metabolites. Thus, fraction A1 (81 mg) was separated by RP-HPLC using 50% aqueous MeOH to give 6 (1.5 mg, tR = 38 min), 5 (1.2 mg, tR = 40.5 min), 7 (1.9 mg, tR = 49 min), 9 (4.2 mg, tR = 64 min), and 10 (4.1 mg, tR = 75 min). Fraction A2 (105.5 mg) on purification by silica gel (20 g) CC and elution with CHCl3−MeOH (30:1) yielded 11 (32.7 mg). Fraction B (277 mg) was further fractionated on a column of silica gel (50 g) by elution with CHCl3−MeOH (97:3, 90:10, 85:15) to give four subfractions (B1−B4). Fraction B1 (11.9 mg) was purified by RP-HPLC using 38% aqueous MeOH to give 4 (1.8 mg, tR = 39 min) and 3 (2 mg, tR = 44 min). Fraction B2 (188.3 mg) on purification by silica gel (20 g) CC and elution with CHCl3−MeOH (96:4) afforded 8 (152.9 mg), and a mixture of compounds, which was further purified by HPLC (C18; 50% aqueous MeOH) to give 2 (5.8 mg, tR = 27 min). Further separation of fraction B3 (58.1 mg) by preparative TLC (C18) using 50% aqueous MeOH containing 0.5% HCOOH afforded 1 (4.9 mg, Rf = 0.14). Montagnuphilone A (1): yellow, amorphous powder; [α]25 D +198 (c 0.36, MeOH); UV (MeOH) λmax (log ε) 266 (4.1), 304 (3.8), 386 (4.0) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 415 [M − H]− (100), 265 (52), 249 (20), 167 (40); HRESIMS m/z 415.1035 [M − H]− (calcd for C21H19O9, 415.1035). Montagnuphilone B (2): yellow, amorphous powder; [α]25 D +101 (c 0.39, MeOH); UV (MeOH) λmax (log ε) 266 (4.0), 305 (3.7), 355 (3.6) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 417 [M − H]− (95), 267 (64), 249 (58), 167 (100); HRESIMS m/z 417.1207 [M − H]− (calcd for C21H21O9, 417.1186). Montagnuphilone C (3): pale yellow, amorphous powder; [α]25 D +103 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 266 (2.9), 302 (3.8), 353 (4.1) nm; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z

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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.6b00714. 1 H, 13C, and HSQC NMR spectra of 1−7, HMBC spectra of 1−3, 6, and 7, 1D NOESY spectra of 1−6, key HMBC correlations for 1, 2, 6, and 7, results of phylogenetic analyses confirming placement of the fungus DM0194, NO production inhibitory activity for 1−6 and 8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (A. A. L. Gunatilaka): (520) 621-9932. Fax: (520) 6218378. E-mail: [email protected]. ORCID

A. A. Leslie Gunatilaka: 0000-0001-9663-3600 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support for this work was provided by Grant R01 CA090265 funded by the National Cancer Institute (NCI) and Grant P41 GM094060 funded by National Institute of General Medical Sciences (NIGMS). We thank China Scholarship Council for awarding a Postdoctoral Fellowship (to J.-G.L.) and L. Battista and P. Espinosa-Artiles respectively for their assistance in collecting plant material in the field and largescale culturing of the fungal strain.

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REFERENCES

(1) Gao, J. M.; Yang, S. X.; Qin, J. C. Chem. Rev. 2013, 113, 4755− 4811. (2) Osmanova, N.; Schultze, W.; Ayoub, N. Phytochem. Rev. 2010, 9, 315−342. (3) (a) Wijeratne, E. M. K.; He, H.; Franzblau, S. G.; Hoffman, A. M.; Gunatilaka, A. A. L. J. Nat. Prod. 2013, 76, 1860−1865. (b) Xu, Y.; 80

DOI: 10.1021/acs.jnatprod.6b00714 J. Nat. Prod. 2017, 80, 76−81

Journal of Natural Products

Article

Espinosa-Artiles, P.; Liu, M. X.; Arnold, A. E.; Gunatilaka, A. A. L. J. Nat. Prod. 2013, 76, 2330−2336. (c) Luo, J.-G.; Wang, X.-B.; Xu, Y.; U’Ren, J. M.; Arnold, A. E.; Kong, L.-Y.; Gunatilaka, A. A. L. Org. Lett. 2014, 16, 5944−5947. (d) Wijeratne, E. M. K.; Gunaherath, G. M. K. B.; Chapla, V. M.; Tillotson, J.; de la Cruz, F.; Kang, M.-J.; U’Ren, J. M.; Araujo, A. R.; Arnold, A. E.; Chapman, E.; Gunatilaka, A. A. L. J. Nat. Prod. 2016, 79, 340−352. (4) Paranagama, P. A.; Wijeratne, E. M. K.; Gunatilaka, A. A. L. J. Nat. Prod. 2007, 70, 1939−1945. (5) (a) Barr, M. E. Mycotaxon 2001, 77, 193−200. (b) Ariyawansa, H. A.; Tanaka, K.; Thambugala, K. M.; Phookamsak, R.; Tian, Q.; Camporesi, E.; Hongsanan, S.; Monkai, J.; Wanasinghe, D. N.; Mapook, A.; Chukeatirote, E.; Kang, J.-C.; Xu, J.-C.; McKenzie, E. H. C.; Jones, E. B. G.; Hyde, K. D. Fungal Divers. 2014, 68, 69−104. (6) (a) Seo, C.; Oh, H.; Lee, H. B.; Kim, J. K.; Kong, I. S.; Ahn, S. C. Bull. Korean Chem. Soc. 2007, 28, 1803−1806. (b) Shao, M.-W.; Kong, L.-C.; Jiang, D.-H.; Zhang, Y.-L. Rec. Nat. Prod. 2016, 10, 326−331. (c) Zhang, L.-H.; Feng, B.-M.; Chen, G.; Li, S.-G.; Sum, Y.; Wu, H.-H.; Bai, J.; Hua, H.-M.; Wang, H.-F.; Pei, Y.-H. RSC Adv. 2016, 6, 42361− 42366. (7) Buchanan, M. S.; Hashimoto, T.; Yasuda, A.; Takaoka, S.; Kan, Y.; Asakawa, Y. 37th Symposium on the Chemistry of Natural Products; Symposium Papers; Tokushima, Japan, 1995; pp 409−414. (8) Quang, D. N.; Hashimoto, T.; Stadler, M.; Asakawa, Y. J. Nat. Prod. 2004, 67, 1152−1155. (9) Davis, D. C.; Walker, K. L.; Hu, C.; Zare, R. N.; Waymouth, R. M.; Dai, M. J. Am. Chem. Soc. 2016, 138, 10693−10699. (10) (a) Macedo, F. C.; Porto, A. L. M.; Marsaioli, A. J. Tetrahedron Lett. 2004, 45, 53−55. (b) Qian, W.-J.; Wei, W.-G.; Zhang, Y.-X.; Yao, Z.-J. J. Am. Chem. Soc. 2007, 129, 6400−6401. (c) Wu, S.-H.; Chen, Y.M.; Qin, S.; Huang, R. J. Basic Microbiol. 2008, 48, 140−142. (d) Clark, R. C.; Lee, S. Y.; Searcey, M.; Boger, D. L. Nat. Prod. Rep. 2009, 26, 465−477. (e) Sun, J.; Zhu, Z.-X.; Song, Y.-L.; Ren, Y.; Dong, D.; Zheng, J.; Liu, T.; Zhao, Y.-F.; Tu, P.-F.; Li, J. Nat. Prod. Res. 2016, doi.org/10.1080/14786419.2016.1207075. (11) Gao, S. S.; Li, X. M.; Zhang, Y.; Li, C. S.; Cui, C. M.; Wang, B. G. J. Nat. Prod. 2011, 74, 256−261. (12) Hsu, Y.-W.; Hsu, L.-C.; Liang, Y.-H.; Kuo, Y.-H.; Pan, T.-M. J. Agric. Food Chem. 2011, 59, 4512−4518. (13) Cheng, M.-J.; Wu, M.-D.; Su, Y.-S.; Yuan, G.-F.; Chen, Y.-L.; Chen, I.-S. Phytochem. Lett. 2012, 5, 262−266. (14) Quang, D. N.; Stadler, M.; Fournier, J.; Tomita, A.; Hashimoto, T. Tetrahedron 2006, 62, 6349−6354. (15) Quang, D. N.; Harinantenaina, L.; Nishizawa, T.; Hashimoto, T.; Kohchi, C.; Soma, G.; Asakawa, Y. Biol. Pharm. Bull. 2006, 29, 34− 36. (16) Fukumura, D.; Kashiwagi, S.; Jain, R. K. Nat. Rev. Cancer 2006, 6, 521−534. (17) Sandberg, D. C.; Battista, L. J.; Arnold, A. E. Microb. Ecol. 2014, 67, 735−747. (18) Ewing, B.; Green, P. Genome Res. 1998, 8, 186−194. (19) Ewing, B.; Hillier, L.; Wendl, M. C.; Green, P. Genome Res. 1998, 8, 175−185. (20) Maddison, W. P.; Maddison, D. R. Mesquite, 2011, www. mesquiteproject.org. (21) Liu, K. L.; Porras-Alfaro, A.; Kuske, C. R.; Eichorst, S. A.; Xie, G. Appl. Environ. Microbiol. 2012, 78, 1523−1533. (22) Altschul, S. F.; Gish, W.; Miller, W.; Myers, E. W.; Lipman, D. J. J. Mol. Biol. 1990, 215, 403−410. (23) Zwickl, D. J. Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. Ph.D. Dissertation, The University of Texas at Austin, Austin, TX, 2008; pp 1−114. (24) Posada, D.; Crandall, K. A. Bioinformatics 1998, 14, 817−818. (25) U’Ren, J. M.; Lutzoni, F.; Miadlikowska, J.; Laetsch, A. D.; Arnold, A. E. Am. J. Bot. 2012, 99, 898−914. (26) Verkley, G.; Silva, M.; Wicklow, D.; Crous, P. W. Stud. Mycol. 2004, 50, 323−335.

(27) Damm, U.; Verkley, G. J. M.; Crous, P. W.; Fourie, P. H.; Haegi, A.; Riccioni, L. Persoonia 2008, 20, 9−17. (28) Swofford, D. L. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4; Sinauer Associates: Sunderland, MA, 2002. (29) Verkley, G.; Dukik, K.; Göker, M.; Stielow, J. B. Persoonia 2014, 32, 25−51. (30) (a) Yang, M. H.; Wang, J. S.; Luo, J. G.; Wang, X. B.; Kong, L. Y. Bioorg. Med. Chem. 2011, 19, 1409−1417. (b) Xu, X. M.; Guan, Y. Z.; Shan, S. M.; Luo, J. G.; Kong, L. Y. Phytochem. Lett. 2016, 15, 1−6.

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