Bioactive Dibenzo-Î±-pyrone Derivatives from the Endophytic Fungus

Article pubs.acs.org/jnp

Bioactive Dibenzo-α-pyrone Derivatives from the Endophytic Fungus Rhizopycnis vagum Nitaf22 Daowan Lai,†,‡ Ali Wang,†,‡ Yuheng Cao,† Kaiyi Zhou,† Ziling Mao,† Xuejiao Dong,† Jin Tian,†,§ Dan Xu,† Jungui Dai,⊥ Yu Peng,∥ Ligang Zhou,*,† and Yang Liu*,# †

Key Laboratory of Plant Pathology, Ministry of Agriculture/Department of Plant Pathology, College of Plant Protection, China Agricultural University, Beijing 100193, People’s Republic of China § National Centre for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China ⊥ State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100050, People’s Republic of China ∥ Technical Centre of Hunan Tobacco Industry Co. Ltd., Changsha 410014, People’s Republic of China # Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences/Key Laboratory of Agro-products Processing, Ministry of Agriculture, Beijing 100193, People’s Republic of China S Supporting Information *

ABSTRACT: Six new dibenzo-α-pyrones, rhizopycnolides A (1) and B (2) and rhizopycnins A−D (3−6), together with eight known congeners (7−14), were isolated from the endophytic fungus Rhizopycnis vagum Nitaf22 obtained from Nicotiana tabacum. The structures of the new compounds were unambiguously elucidated using NMR, HRESIMS, TDDFT ECD calculation, and X-ray crystallography data. Rhizopycnolides A (1) and B (2) feature an uncommon γ-butyrolactonefused dibenzo-α-pyrone tetracyclic skeleton (6/6/6/5), while rhizopycnin B (4) was the first amino group containing dibenzo-α-pyrone. Rhizopycnolides A (1) and B (2) are proposed to be biosynthesized from polyketide and tricarboxylic acid cycle pathways. The isolated compounds were tested for their antibacterial, antifungal, and cytotoxic activities. Among them, rhizopycnolide A (1), rhizopycnins C (5) and D (6), TMC-264 (8), penicilliumolide D (11), and alternariol (12) were active against the tested pathogenic bacteria Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas lachrymans, Ralstonia solanacearum, Staphylococcus hemolyticus, and Xanthomonas vesicatoria with MIC values in the range 25−100 μg/mL. Rhizopycnin D (6) and TMC-264 (8) strongly inhibited the spore germination of Magnaporthe oryzae with IC50 values of 9.9 and 12.0 μg/mL, respectively. TMC-264 (8) showed potent cytotoxicity against five human cancer cell lines (HCT-116, HepG2, BGC-823, NCIH1650, and A2780) with IC50 values of 3.2−7.8 μM.

D

B (2), rhizopycnins A−D (3−6), and eight known compounds (7−14) (Figure 1). The structures of the new compounds were elucidated by analysis of the NMR and HRESIMS data, and their absolute configurations were determined by quantum chemical ECD calculations (for 1−3) and X-ray crystallography (for 1). Interestingly, rhizopycnolides A (1) and B (2) feature an uncommon tetracyclic skeleton (6/6/6/5) in which a γbutyrolactone is fused to a dibenzo-α-pyrone moiety through a spiro carbon. Structurally, compounds 1 and 2 were hybrid products of polyketide and tricarboxylic acid cycle (TCA) pathways. Herein, we reported the isolation, structure elucidation, and biological activities of the isolated compounds.

ibenzo-α-pyrones are polyketides with a 6H-benzo[c]chromen-6-one tricyclic skeleton, commonly found in compounds from fungi of the genus Alternaria.1,2 These metabolites were also reported from other fungal genera, plants, and bacteria.3 Presently, 53 natural dibenzo-α-pyrones have been reported, which display diverse biological activities including cytotoxic, antimicrobial, antioxidant, and phytotoxicity, and these compounds have attracted considerable interest within the pharmaceutical community.3 Endophytic fungi have been reported to be a promising source of novel bioactive substances with unique structures.4−6 In our ongoing search for new bioactive metabolites from endophytic fungi,7,8 a chemical investigation of the endophytic Rhizopycnis vagum Nitaf22, isolated from the healthy root of Nicotiana tabacum, was carried out, and this led to the isolation of 14 polyketides, including the new rhizopycnolides A (1) and © 2016 American Chemical Society and American Society of Pharmacognosy

Received: April 12, 2016 Published: July 21, 2016 2022

DOI: 10.1021/acs.jnatprod.6b00327 J. Nat. Prod. 2016, 79, 2022−2031

Journal of Natural Products

Article

Figure 1. Structures of the isolated compounds (1−14).

Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of 1 and 2 1 (DMSO-d6) position

δC, type

1 2

69.3, C 41.6, CH2

3 4 4a 6 6a 7 8 9 10 10a 10b 11

78.3, 82.6, 145.8, 164.2, 99.8, 163.3, 100.8, 166.0, 105.3, 136.2, 119.9, 35.7,

CH C C C C C CH C CH C C CH2

12 13 1-CH3 1-OH 3-OCH3 7-OH 9-OCH3 12-OH

66.8, CH 176.3, C 27.3, CH3 57.0, CH3 56.0, CH3

δH, mult. (J in Hz) 2.33, dd (3.3, 12.8) 1.91, dd (12.8, 13.2) 3.67, dd (3.3, 13.2)

6.70, d (2.2) 7.60, d (2.2)

2.82, dd (9.3, 13.0) 2.13, dd (9.6, 13.0) 4.55, ddd (6.3, 9.3, 9.6) 1.63, s 5.59, s 3.37, s 11.30, s 3.87, s 6.25, d (6.3)

2 (DMSO-d6) δC, type 68.9, C 39.5, CH2a 77.4 CH 80.0, C 145.1, C 163.8, C 100.5, C 163.1, C 100.8, CH 165.9, C 105.3, CH 136.4, C 120.1, C 36.8, CH2 66.6, CH 176.7, C 30.0, CH3 56.9, CH3 56.0, CH3


6.71, d (2.2) 7.37, d (2.2)

2.66, dd (9.4, 13.4) 2.62, dd (9.5, 13.4) 4.62, dt (6.7, 9.4) 1.59, s 5.65, s 3.39, s 11.15, s 3.88, s 6.25, d (6.4)

2 (CDCl3) δC, type 71.0, C 39.9, CH2 77.7, CH 81.1, C 145.5, C 164.2, Cb 100.96, C 164.3, Cb 101.00, CH 166.5, C 105.5, CH 135.7, C 119.5, C 36.2, CH2 67.9, CH 176.6, C 30.5, CH3 57.1, CH3 55.9, CH3


6.58, d (1.9) 7.25, d (1.9)c

3.06, dd (9.0, 13.1) 2.60, dd (9.4, 13.1) 4.86, t (9.1) 1.78, s 3.47, s 11.21, s 3.90, s

a c

Signal overlapped with the solvent peak and deduced from the HMQC and HMBC spectra. bAssignments within a column may be interchanged. Signal partially overlapped with the solvent peak.

peak at m/z 391.10249 [M − H]− in the HRESIMS spectrum, suggesting a molecular formula of C19H20O9, with 10 degrees of unsaturation. The IR spectrum indicated the presence of hydroxy (3446 cm−1), ester carbonyl (1788, 1772, 1668 cm−1), and benzene ring (1630, 1570 cm−1) groups. The 13C NMR spectrum (Table 1) showed a total of 19 signals that could be classified into two ester carbonyls (δC 176.3, 164.2), eight sp2hybridized carbons, four oxygenated aliphatic carbons (δC 82.6, 78.3, 69.3, 66.8), two aliphatic methylenes (δC 41.6, 35.7), two methoxyl groups (δC 57.0, 56.0), and one methyl group (δC

In addition, a plausible biosynthetic pathway for 1 and 2 was proposed.

■

RESULTS AND DISCUSSION

The fungal EtOAc extract was successively subjected to repeated column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC to afford compounds 1−14 (Figure 1). Rhizopycnolide A (1) was isolated as a colorless crystal (CH2Cl2-MeOH). It exhibited a prominent pseudomolecular 2023



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presence of the aforementioned tetrasubstituted benzene ring but also positioned the methoxyl group (δH 3.87) at C-9. Moreover, H-10 showed HMBC correlation to an olefinic carbon (C-10b, δC 119.9), while one oxygenated olefinic carbon (δC 145.8) remained to be assigned. Considering the chemical shift of the ester carbonyl (C-6, δC 164.2), this unassigned sp2carbon should be connected to C-6 via an ester bond to furnish a 1H-isochromen-1-one moiety, which is also present in the coisolated compounds 2−14. This was supported by the similar NMR data observed for this unit in these compounds (Tables 1 and 2). In addition, two oxygenated methine protons (δH 3.67, 4.55), two couples of methylene protons (δH 2.33, 1.91; δH 2.82, 2.13), one methoxyl (δH 3.37, s), and one methyl group (δH 1.63, s) were unassigned in the upfield region of the 1H NMR spectrum. Analysis of the 1H−1H COSY spectrum allowed the establishement of two isolated spin systems: −CH2 (2)−CH (3) and −CH2 (11)−CH (12)−OH (Figure 2). These moieties were connected by analysis of the HMBC spectrum. The HMBC correlations from 1-CH3 (δH 1.63) to C-1 (δC 69.3), C10b (δC 119.9), and C-2 (δC 41.6); from H2-2 (δH 2.33, 1.91) to C-1, C-10b, and C-4 (δC 82.6); and from H-3 (δH 3.67) to C-4 and C-4a (δC 145.8) allowed the construction of a C-1−C4 unit that linked to the 1H-isochromen-1-one moiety via C10b and C-4a. The correlation between the second methoxyl group (δH 3.37) and C-3 (δC 78.3) suggested this group was located at C-3. Further correlations from H2-11 (δH 2.82, 2.13) to C-3, C-4, and C-4a and from H-3 (δH 3.67) to C-11 (δC 35.7) allowed the establishment of a σ bond between C-11 and C-4. Similarly, the correlations from H2-11, H-12 (δH 4.55), and 12-OH (δH 6.25) to the ester carbonyl (δC 176.3, C-13) indicated that C-13 was directly bonded to C-12. The above

27.3) by analysis of DEPT-135 and HMQC spectra. In the downfield region of the 1H NMR spectrum, signals for two meta-coupled aromatic protons at δH 6.70 (d, J = 2.2 Hz) and 7.60 (d, J = 2.2 Hz) and for a chelated phenolic hydroxy group at δH 11.30 (s) were discerned (Table 1), which suggested the presence of a 1-carbonyl-2-hydroxyl-4,6-substituted benzene ring in the molecule when the 13C NMR data were taken into consideration. This was corroborated by analysis of the HMBC spectrum, in which correlations from the phenolic hydroxy group OH-7 (δH 11.30) to C-6a (δC 99.8), C-7 (δC 163.3), and C-8 (δC 100.8); from the aromatic proton H-8 (δH 6.70) to C6a, C-7, C-9 (δC 166.0), and C-10 (δC 105.3); from the other aromatic proton H-10 (δH 7.60) to C-6 (δC 164.2), C-6a, C-8, C-9, and C-10a (δC 136.2); and from one methoxyl (δH 3.87, s) to C-9 were observed (Figure 2). This not only confirmed the

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of 1.

Table 2. 1H (400 MHz) and 13C (100 MHz) NMR Data of 3−6 3 (DMSO-d6) position

δC, type

1 2 3 4

70.7, 120.0, 145.0, 61.4,

C C C CH

4a 6 6a 7 8

148.0, 164.6, 99.8, 163.5, 100.4,

C C C C CH

9 10

166.0, C 104.8, CH

10a 10b 1-CH3 1-OH 2-NH2 3-OCH3 /3OH 4-OH

136.3, C 116.8, C 27.3, CH3

7-OH 9-OCH3 a

δH, mult. (J)

5.17, d (8.4)

6.69, d (2.0) 7.49, d (2.0)

1.67, s 5.94, s

4 (DMSO-d6) δC, type

4 (CD3OD)

δH, mult. (J)

δC, type

δH, mult. (J)


δH, mult. (J)


69.5, 161.8, 128.2, 167.4,

C C C C

71.4, 166.4, 130.4, 170.4,

C C C C

70.6, 134.1, 143.1, 171.9,

C C C C

135.3, 119.8, 154.5, 101.9,

C C C CH

142.9, 164.4, 100.5, 163.5, 101.3,

C C C C CH

144.5, 166.2, 102.1, 165.7, 102.8,

C C C C CH

140.8, 163.3, 101.0, 163.5, 102.7,

C C C C CH

150.1, 163.9, 97.8, 164.2, 101.3,

C Ca C Ca CH

165.9, C 104.5, CH 135.7, C 122.8, C 30.5, CH3

6.74, d (2.2) 7.63, d (2.2)

56.2, CH3

3.81, s

58.5, CH3

1.72, 6.46, 7.15, 3.61,

s s s s

56.0, CH3

6.37, d (8.4) 11.42, s 3.88, s

56.0, CH3

11.47, s 3.89, s

168.2, C 105.8, CH

6.66, d (2.3) 7.76, d (2.3)

136.9, C 125.3, C 31.7, CH3

1.83, s

59.7, CH3

3.73, s

56.5, CH3

3.92, s

166.0, C 106.6, CH 134.9, C 130.4, C 28.3, CH3

6.86, d (2.2) 7.67, d (2.2)

1.75, s 6.47, s

165.3, C 105.3, CH 137.3, C 110.5, C 21.0, CH3

δH, mult. (J)

6.84, s

6.40, d (1.6) 7.18, d (1.6)

2.78, s

10.19, s

56.2, CH3

11.34, s 3.92, s

11.60, s

Assignments within a column may be interchanged. 2024



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moieties account for nine of the 10 required degrees of unsaturation; thus the ester carbonyl (C-13) has to connect to the oxygenated quaternary carbon (C-4) via an ester bond to complete the gross structure of 1. The relative configuration was established by analysis of the 1 H−1H coupling constants and NOESY correlations. The large vicinal coupling constant (3JH‑2b,H‑3 = 12.8 Hz) between H-2b (δH 1.91, dd) and H-3 (δH 3.67, dd) indicated that both protons were axial, while H-2a (δH 2.33, dd) was equatorial. In the NOESY spectrum, cross-peaks were found between 1-CH3 (δH 1.63), H-2a, and H-3, suggesting the co-orientation of these protons to the same face (tentatively as β), while the correlations between H-11a (δH 2.82, dd) and H-2b indicated that both protons were on the opposite face (α). Moreover, H12 (δH 4.55, ddd) showed correlations to H-11a and 3-OMe (δH 3.37), implying the spatial proximity of these protons. Hence, the relative configuration was established (Figure 2). The absolute configuration of 1 was established by quantum chemical ECD calculation.9 The calculated ECD spectrum of (1S,3R,4S,12R)-1 at the B3LYP/6-31+G(d) level with the conductor polarizable continuum model (CPCM) in MeOH fit well with the experimental spectrum of 1 (Figure 3), suggesting

Figure 4. X-ray crystal structure of 1.

found in 1, suggesting that a different conformation (Figure 5 and Figure S2 in the Supporting Information) was adopted for

Figure 3. Experimental ECD spectrum of 1 and the calculated ECD spectrum of (1S,3R,4S,12R)-1. Figure 5. Selected NOESY correlations of 2.

the 1S, 3R, 4S, 12R configuration of 1. In order to confirm this deduction, a crystal of 1 was obtained through repeated recrystallization from CH2Cl2−MeOH, and a single-crystal Xray diffraction experiment was carried out using Cu Kα radiation, allowing an explicit assignment of the absolute structure as 1S, 3R, 4S, 12R on the basis of the Flack parameter of −0.04(6) (CCDC 1440494) (Figure 4).10 Rhizopycnolide B (2) was isolated as a colorless, amorphous solid. Its molecular formula was determined to be the same as that of 1. Analysis of the 1D and 2D NMR spectra (Table 1) indicated that 2 shared the same gross structure as 1; however the NMR data for C-2, C-3, C-4, 1-Me, and C-11 were notably different. For example, large 13C chemical shift differences (ΔδC = δC(2) − δC(1), ppm) were observed for C-2 (−2.1), C-3 (−0.9), C-4 (−2.6), 1-Me (+2.7), and C-11 (+1.1), suggesting a different stereochemistry for 2. The relative configuration was determined by analysis of the coupling constants and NOESY spectrum (DMSO-d6). Assuming 1-CH3 was β-oriented, as found in the coisolated congeners (1, 7, and 8), the correlation seen between 1-OH (δH 5.65, s) and the axial H-3 (δH 3.85, dd) revealed that these protons were directed to the α-face, while 1-CH3 (δH 1.59, s) was equatorial, which was in contrast to the axial orientation as

2. The correlation between H-3 and H2-11 (δH 2.66, 2.62, each dd) permitted the assignment of CH2-11 to the α-face. The weak correlation seen between H-12 and H-3 was in agreement with the proposed stereochemistry, as depicted in Figure 1. Thus, the relative stereochemistry of 2 was different from that of 1 only at C-3. The absolute configuration of 2 was determined by TDDFT ECD calculations. The calculated ECD spectrum of (1S,3S,4S,12R)-2 at the B3LYP/6-31+G(d) level with the CPCM in MeOH fit well with the experimental spectrum of 2 (Figure 6); thus the absolute configuration of 2 was determined as 1S, 3S, 4S, 12R. The different ECD spectra between 2 and 1 were consistent with their different conformations in the cyclohexene ring. Recently, a structrually related metabolite, penicilliumolide A, was isolated from the endophytic fungus Penicillium chermesinum obtained from the mangrove tree Heritiera littoralis.11 Rhizopycnolides A (1) and B (2) and penicilliumolide A were the only examples of the rare γ-butyrolactone-fused dibenzo-αpyrones. However, the configurations of C-1 and the spiro carbon (C-4) in penicilliumolide A were tentatively assigned 2025



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method at the B3LYP/6-31+G(d) level (CPCM = MeOH). The predicted ECD spectra of these structures are shown in Figures 8 and S4, among which the calculated spectrum for (1R,4R)-3 matched the experimental spectrum. Therefore, the absolute configuration of 3 was established as 1R, 4R.

Figure 6. Experimental ECD spectrum of 2 and the calculated ECD spectrum of (1S,3S,4S,12R)-2.

based on a biosynthetic assumption along with NOESY interpretation. Rhizopycnin A (3) was isolated as a white, amorphous solid, whose molecular formula was established as C16H15ClO7 by HRESIMS, bearing two more protons than that of TMC-264 (8).12 A comparison of their NMR data (Table 2) indicated that the ketone signal (C-4) in 8 was replaced by an oxygenated methine group (δC 61.4, C-4; δH 5.17, d, J = 8.4 Hz, H-4) in 3. The methine proton was coupled to a D2O-exchangeable proton (δH 6.37, d, J = 8.4 Hz), thus indicating the presence of a hydroxy-bearing methine at C-4. This was confirmed by analysis of the HMBC spectrum, in which correlations were seen from H-4 (δH 5.17, d) to C-2 (δC 120.0), C-3 (δC 145.0), C-4a (δC 148.0), and C-10b (δC 116.8) (Figure 7). Meanwhile, the NOESY correlation observed between H-4 and 3-OCH3 (δH 3.81, s) also corroborated the proposed structure (Figure 7).

Figure 8. Experimental ECD spectrum of 3 and the calculated ECD spectra of (1R,4R)-3 and (1S,4S)-3.

A literature search indicated that penicilliumolide C has the same planar structure as that of 3; however, the configuration of C-4 in penicilliumolide C was not assigned, and both compounds differed in specific rotations ([α]33D +12.4 (c 0.0625, CHCl3) for 3; [α]27D −42.7 (c 0.78, acetone) for penicilliumolide C11). Rhizopycnin B (4) was isolated as a yellow, amorphous solid, whose molecular formula was determined to be C16H15NO7. Its NMR data were similar to the coisolated TMC-264 (8),12 except that an amino group (δH 7.15, s, 2H) in 4 replaced the chlorine atom of 8, suggesting that 4 was a 2-amino derivative of 8. This was verified by analysis of the HMBC and NOESY spectra (Figure 7). In the HMBC spectrum, correlations were observed from 2-NH2 (δH 7.15) to C-1 (δC 69.5) and C-3 (δC 128.2), from 1-CH3 (δH 1.72, s) to C-1, C-2 (δC 161.8), and C10b (δC 122.8), and from 3-OMe (δH 3.61, s) to C-3. Meanwhile, NOESY cross-peaks were seen between 1-OH (δH 6.46, s)/1-CH3 and 2-NH2 and between 2-NH2 and 3-OMe. The absolute configuration of 4 was determined as shown in Figure 1 by comparison of the optical rotation with 8 ([α]25.7D −108.0 (c 0.125, MeOH) for 4; [α]25D −43.8 (c 0.5, CHCl3) for 812). Rhizopycnin B (4) is the first amino group containing dibenzo-α-pyrone to our knowledge. Rhizopycnin C (5) was isolated as a yellowish, amorphous powder. Its molecular formula was determined as C15H11ClO7, which was a CH2 unit less than that of TMC-264 (8). Their NMR data (Table 2) were similar, except that one methoxyl group in 8 was replaced by a hydroxy group (δH 10.19, s) in 5. In the HMBC spectrum, this hydroxy group (3-OH, δH 10.19) showed correlations to C-2 (δC 134.1), C-3 (δC 143.1), and C-4 (δC 171.9), indicating the presence of 3-OH in 5. This was consistent with the NOESY spectrum (Figure 7). Therefore, 5 was elucidated as the 3-demethyl derivative of 8. The absolute configuration of 5 was determined as 1R, the same as that of 8,13 based on their similar specific specific rotation data ([α]33D −18.5 (c 0.1, CHCl3) for 5; [α]25D −43.8 (c 0.5, CHCl3) for 812). Rhizopycnin D (6), a yellow, amorphous powder, has a molecular formula of C14H9ClO5 as determined by HRESIMS, which was one less CH2 unit than that of palmariol B (9).14

Figure 7. Key HMBC and NOESY correlations of 3−5.

Theoretically, there are four possible structures for 3 (i.e., (1R, 4R)-3, (1R, 4S)-3, (1S, 4S)-3, and (1S, 4R)-3), as it has two chiral centers (C-1, C-4) in the molecule. However, since H-4 was distant from 1-OH or 1-CH3, no clear correlations were found between these protons in the NOESY spectrum; thus the relative configuration could not be resolved by NOESY. The quantum chemical ECD computations for these possible structures were then carried out by using the TDDFT 2026



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Scheme 1. Plausible Biosynthetic Pathway Proposed for 1−5, 7, and 8

Table 3. Antibacterial Activities of the Isolated Compounds [MIC (IC50), μg/mL]a

a

bacterium

1

A. tumefaciens B. subtilis P. lachrymans R. solanacearum S. hemolyticus X. vesicatoria

100 (56.7) 75 (45.5) 100 (44.7) >200 (nd) >200 (nd) >200 (nd)

5 100 100 25 50 25 100

(47.3) (44.6) (4.3) (36.5) (7.0) (52.5)

6

8

50 (16.3) 50 (18.6) >200 (nd) 50 (12.4) >200 (nd) 75 (22.7)

>200 (ndc) 50 (21.6) >200 (nd) >200 (nd) >200 (nd) >200 (nd)

11 50 50 50 50 50 50

(15.0) (22.4) (16.4) (24.2) (26.7) (15.9)

12 50 50 25 25 50 25

(11.4) (7.0) (3.5) (5.5) (7.9) (4.9)

streptomycin sulfateb 5.0 5.0 7.5 5.0 5.0 10

(1.4) (1.4) (2.3) (1.2) (1.4) (2.6)

Compounds 2, 4, 7, and 10 were inactive (MIC > 200 μg/mL). bPositive control. cnd: not determined.

amino derivative (4). Meanwhile, reduction of 7 could afford the intermediates S3 and S4, which could further react with oxaloacetic acid, a TCA cycle intermediate that had been reported to be involved in the biosynthesis of sorokinianin (sesquiterpene/TCA pathways),25 followed by lactonization and hydrogenation at C-12 to afford 1 and 2, respectively. The isolated compounds were tested for their antibacterial activities. Rhizopycnin C (5), penicilliumolide D (11), and alternariol (12) were active against the six tested bacteria, including Agrobacterium tumefaciens, Bacillus subtilis, Pseudomonas lachrymans, Ralstonia solanacearum, Staphylococcus hemolyticus, and Xanthomonas vesicatoria, with MICs in the range 25− 100 μg/mL. Rhizopycnolide A (1) was active against A. tumefaciens, B. subtilis, and P. lachrymans, with MIC values of 100, 75, and 100 μg/mL, respectively, whereas its 3-epimer, rhizopycnolide B (2), was inactive against the tested bacteria (MIC > 200 μg/mL). Rhizopycnin D (6) showed inhibition against A. tumefaciens, B. subtilis, and R. solanacearum, with an equal MIC value of 50 μg/mL, and against X. vesicatoria, with an MIC value of 75 μg/mL. TMC-264 (8) selectively inhibited the growth of B. subtilis (MIC value of 50 μg/mL) (Table 3). Palmariol B (9) and alternariol 9-methyl ether (13) were reported to have antibacterial activities against A. tumefaciens, B. subtilis, P. lachrymans, R. solanacearum, and X. vesicatoria, with IC50 values in the range 16.7−34.3 μg/mL.21 The isolated compounds were evaluated for their antifungal activities on Magnaporthe oryzae. Among them, rhizopycnin D (6) and TMC-264 (8) strongly inhibited the spore germination of M. oryzae with IC50 values of 9.9 and 12.0 μg/mL,

Comparison of their NMR data (Table 2) revealed their great similarity, except that the signals for the methoxyl group in 9 were missing in 6, suggesting that 9-OMe in 9 was replaced by 9-OH in 6. This could explain their differences in molecular formula, and their chemical shifts differed only in carbons/ protons around C-9. This conclusion was secured by analysis of the HMBC spectrum. Thus, compound 6 was elucidated as the demethyl derivative of 9. The known compounds were identified by comparing their physical and spectroscopic data with those reported in the literature and included penicilliumolide B (7),11 TMC-264 (8),12,15 palmariol B (9),14,16 graphislactone A (10),17,18 penicilliumolide D (11),11,13 alternariol (12),19,20 alternariol 9-methyl ether (13),21,22 and graphislactone E (14).23 Previously, there was no phytochemical investigation on the Rhizopycnis fungi to our knowledge; thus compounds 1−14 represented the first secondary metabolites reported from this genus. Dibenzo-α-pyrones are heptaketides biosynthesized through the polyketide pathway,23,24 while rhizopycnolides A (1) and B (2) are proposed to be mixed biosynthesis products of polyketide and TCA pathways (Scheme 1). The condensation of one acetyl CoA and six malonyl CoA could give rise to a heptaketide (S1). Oxidation of this heptaketide at C-4 and Omethylation at 9-OH and/or 3-OH with S-adenosyl methionine (SAM) would yield the intermediates S2 and 7, which could produce 5 and 8 by chlorination at C-2, respectively. Subsequent hydrogenation of the ketone group in 8 would produce 3. Similarly, amination at C-2 of 7 could afford the 22027



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(Beijing Ke Yi Instrument Factory, Beijing, China). UV spectra were recorded on a TU-1810 UV−vis spectrophotometer (Beijing Persee General Instrument Co., Ltd., Beijing, China). Specific rotations were recorded on a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, NJ, USA). Circular dichroism (CD) spectra were recorded on a JASCO J-810 CD spectrometer (JASCO Corp., Tokyo, Japan). Infrared (IR) spectra were measured on a Thermo Nicolet Nexus 470 FT-IR spectrometer (Thermo Electron Scientific Instrument Crop., WI, USA). High-resolution electrospray ionization mass spectrometry (HRESIMS) spectra were recorded on a Bruker Apex IV FTMS instrument (Bruker Daltonics, Bremen, Germany) or an LC 1260-Q-TOF/MS 6520 machine (Agilent Technologies, CA, USA). 1 H, 13C, and 2D NMR (HMQC, COSY, HMBC, NOESY) spectra were measured on an Avance 400 NMR spectrometer (Bruker BioSpin, Zürich, Switzerland). Chemical shifts are expressed in δ (ppm) referring to the solvent residual peaks at δH 2.50, δC 39.5 for DMSO-d6 or to the inner standard tetramethylsilane for CDCl3 and CD3OD, and coupling constants (J) are in hertz. Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) and Sephadex LH-20 (Pharmacia Biotech, Uppsala, Sweden) were used for column chromatography. Medium-pressure liquid chromatography (MPLC) separation was carried out on an Eyela-VSP-3050 instrument (Tokyo Rikakikai Co., Tokyo, Japan). HPLC-DAD analysis was performed using a Shimadzu LC-20A instrument with an SPD-M20A photodiode array detector (Shimadzu Corp., Tokyo, Japan) and an analytic C18 column (250 mm × 4.6 mm i.d., 5 μm; Phenomenex Inc., Torrance, CA, USA). Semipreparative HPLC separation was carried out on a Lumtech instrument (Lumiere Tech. Ltd., Beijing, China) equipped with a K-501 pump (flow rate: 3 mL/min) and a K-2501 UV detector using a Luna-C18 column (250 mm × 10 mm i.d., 5 μm, Phenomenex Inc.). Precoated silica gel GF-254 plates (Qingdao Marine Chemical Inc.) were used for analytical TLC. Spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating. Fungal Source. The endophytic fungus (strain Nitaf22) was isolated from the inner tissue of the healthy root of a three-year-old tobacco N. tabacum L. grown in the greenhouse of the campus at China Agricultural University (CAU) in July 2014. The fungus was identified by morphological inspection and sequencing of the ITSrDNA region (GenBank accession no. KM095527), which showed a 100% similarity to that of R. vagum JN859316.1.28 A voucher specimen was deposited in the Department of Plant Pathology, CAU. Fermentation and Extraction. The fungus was cultured on PDA (potato dextrose agar) medium for 5 days at 25 °C, and then a slice of agar containing fungal hyphae was transferred to a 250 mL Erlenmeyer flask containing 150 mL of PDB (potato dextrose broth). The cultivation was performed in a rotatory shaker for another 7 days at 150 rpm and 25 °C to produce the seed culture, which was used to inoculate the autoclaved rice media in 1 L Erlenmeyer flasks each

respectively, which were comparable with that of the positve control carbendazim (IC50 = 6.9 μg/mL). Penicilliumolide B (7) showed a slight inhibition (IC50 = 82.1 μg/mL), while the other tested compounds were inactive (IC50 > 200 μg/mL) (Table 4). In a previous report, palmariol B (9) and alternariol Table 4. Inhibitory Activities against the Spore Germination of M. oryzae

a b

compounda

IC50 (μg/mL)

6 7 8 carbendazimb

9.9 82.1 12.0 6.9

Compounds 1 and 10−12 were inactive (IC50 > 200 μg/mL). Positive control.

9-methyl ether (13) were found to have slight activities against this pathogenic fungus, with IC50 values of 118.7 and 123.2 μg/ mL, respectively.21 The isolated compounds were also tested for their cytotoxicities against human cancer cells (Table 5). TMC-264 (8) showed notable cytotoxicity against five human cancer cell lines, namely, HCT-116, HepG2, BGC-823, NCI-H1650, and A2780, with respective IC50 values of 4.2, 5.9, 7.8, 3.2, and 3.6 μM. In the literature, TMC-264 was reported to be cytotoxic against several cancer cells including HuCCA-1, HepG2, A549, MOLT-3, HeLa, T47D, MDA-MB231, and MRC-5, with IC50 values in the range 1.1−12.6 μM.11 Rhizopycnin C (5) showed weak effects against A549 and HCT116 cell lines, with IC50 values of 25.5 and 37.3 μM, respectively. Alternariol 9-methyl ether (13) displayed only very weak cytotoxicity against A549 cells (IC50 = 70.4 μM), while alternariol (12) was inactive at the tested concentration of 10 μM. In contrast, these two compounds were reported to exhibit genotoxic potential in cultured mammalian cells.26 Interestingly, Bensassi et al. reported that the individual compound (i.e., 12 and 13) exhibited only moderate cytotoxicity against HCT116 cells when tested alone, but their toxic potential were synergistic.27 The other tested compounds were inactive.

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EXPERIMENTAL SECTION

General Experimental Procedure. Melting points (uncorrected) were measured on an XT4-100B microscopic melting-point apparatus

Table 5. Cytotoxicity of the Isolated Compounds (IC50, μM)

a

compound

HCT-116

HepG2

BGC-823

NCI-H1650

A2780

6 8 10 11 12 paclitaxela compound

>10 4.2 >10 >10 >10 4.0 × 10−3 A375

>10 5.9 >10 >10 >10 1.2 × 10−2 A549

>10 7.8 >10 >10 >10 3.3 × 10−3 MCF-7

>10 3.2 >10 >10 >10 6.8 × 10−2 HCT116

>10 3.6 >10 >10 >10 3.8 × 10−2 Capan2

1 5 7 9 13 paclitaxela

>50.0 >50.0 >50.0 >50.0 >50.0 2.2 × 10−2

>50.0 25.5 >50.0 >50.0 70.4 2.3 × 10−2

>50.0 >50.0 >50.0 >50.0 >50.0 0.6 × 10−3

>50.0 37.3 >50.0 >50.0 >50.0 0.9 × 10−3

>50.0 >50.0 >50.0 >50.0 >50.0 1.7 × 10−2

Positive control. 2028



Article

391.1048 [M − H]− (calcd for C19H19O9, 391.1035), 427.0817 [M + Cl]− (calcd for C19H20ClO9, 427.0801). Rhizopycnin A (3): white, amorphous solid; [α]33D +12.4 (c 0.0625, CHCl3); UV (MeOH) λmax (log ε) 247 (3.81), 328 (3.17), 360 (2.34) nm; ECD (c = 7.05 × 10−4 M, MeOH) λ (Δε) 213 (+7.91), 235 (−0.73), 244 (+0.06), 253 (−0.66), 288 (+1.65), 304 (+0.13), 329 (+0.48) nm; IR (KBr) νmax 3421, 2943, 1688, 1619, 1569, 1500, 1462, 1435, 1376, 1322, 1256, 1203, 1162, 1076, 1026, 1006, 966, 854, 800, 732, 683, 667 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) see Table 2; HRESIMS m/z 353.04256 [M − H]− (calcd for C16H14ClO7, 353.04226). Rhizopycnin B (4): yellow, amorphous solid; [α]25.7D −108.0 (c 0.125, MeOH); UV (MeOH) λmax (log ε) 247 (4.57), 343 (3.67), 389 (3.82) nm; ECD (c = 7.50 × 10−4 M, MeOH) λ (Δε) 218 (−5.20), 248 (+2.24), 262 (+0.63), 290 (+2.92), 312 (+2.25), 388 (−2.47) nm; IR (KBr) νmax 3367, 3181, 2984, 2939, 1681, 1617, 1571, 1529, 1440, 1376, 1336, 1252, 1199, 1160, 1075, 1033, 1007, 975, 925, 886, 851, 800, 779, 760, 672, 650, 621 cm−1; 1H NMR (DMSO-d6 or CD3OD, 400 MHz), 13C NMR (DMSO-d6 or CD3OD, 100 MHz) see Table 2; HRESIMS m/z 332.0779 [M − H]− (calcd for C16H14NO7, 332.0776), 368.0546 [M + Cl]− (calcd. for C16H15ClNO7, 368.0543). Rhizopycnin C (5): yellow, amorphous powder; [α]33D −18.5 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 237 (4.28), 266 (4.15), 360 (3.79) nm; ECD (c = 7.38 × 10−4 M, MeOH) λ (Δε) 216 (−4.05), 229 (+2.02), 238 (+2.41), 259 (−1.41),267 (−0.94), 276 (−1.62), 319 (+0.49), 347 (−0.48), 381 (+0.27) nm; IR (KBr) νmax 3431, 1682, 1621, 1566, 1499, 1435, 1335, 1253, 1204, 1163, 1106, 1027, 1004, 917, 862, 785, 762, 672 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) see Table 2; HRESIMS m/z 337.01103 [M − H]− (calcd for C15H10ClO7, 337.01096). Rhizopycnin D (6): yellow, amorphous powder; UV (MeOH) λmax (log ε) 256 (3.90), 344 (3.49) nm; IR (acetone) νmax 3357, 2925, 2853, 1672, 1620, 1599, 1569, 1499, 1457, 1437, 1368, 1211, 1163, 1110, 1072, 1024, 848, 800, 762, 658 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) see Table 2; HRESIMS m/z 291.00624 [M − H]− (calcd. for C14H8ClO5, 291.00657). Computation Details. Molecular Merck force field (MMFF) and DFT/TDDFT calculations were performed with the SYBYL-X 2.0 software package and the Gaussian 09 program package, respectively, using default grids and convergence criteria. MMFF conformational search-generated low-energy conformers within a 10 kcal/mol energy window were subjected to geometry optimization using the DFT method at the B3LYP/6-31G(d) level, and solvent effects were taken into account by using the conductor polarizable continuum model for MeOH. Frequency calculations were run at the same level of theory to verify that each optimized conformer was a true minimum. TDDFT ECD calculations of the low-energy conformers (≥1%) without imaginary frequencies were performed at the B3LYP/6-31+G(d) level with CPCM for MeOH. The number of excited states per conformer was 20 for 1 and 3 and 30 for 2. The ECD spectrum of each conformer was simulated by the program SpecDis29 using a Gaussian band shape with 0.3 eV exponential half-width from dipole-length dipolar and rotational strengths. The equilibrium population of each conformer at 298.15 K was calculated from its relative Gibbs free energies using Boltzmann statistics. The Boltzmann-averaged ECD spectra for (1S,3R,4S,12R)-1, (1S,3S,4S,12R)-2, (1R,4R)-3, and (1R,4S)-3 were generated according to the Boltzmann distributions of the lowest energy conformers for each structure, while the ECD spectra for (1S,4S)-3 and (1S,4R)-3 were simulated by simply mirroring the spectra of their enantiomers, respectively. Theoretical ECD spectra were then compared with the experimental ones to determine the absolute configuration. The experimental ECD absorption values (y-axes) were multiplied by 2 for 1 (Figure 3) and 3 (Figure 8) to match the calculated data. X-ray Single-Crystallographic Analysis. Rhizopycnolide A (1) was crystallized from MeOH−CH2Cl2 at room temperature. The Xray crystallographic data were obtained on an Agilent Gemini E singlecrystal diffractometer using Cu Kα radiation. Crystal data: C19H20O9, M = 392.35, hexagonal, a = 10.9360(4) Å, c = 25.7965(6) Å, U = 2671.83(19) Å3, T = 103.1, space group P65 (no. 170), Z = 6, μ(Cu

containing 100 g of rice and 110 mL of distilled water. The scale-up fermentation was carried out using a total of 4.5 kg of rice under static conditions at room temperature (RT) in the dark for 35 days. The cultivation was stopped by adding EtOAc to each flask and extracted at RT for 5 days. The extraction was repeated three times, and the resulting EtOAc extracts were combined and subsequently condensed under vacuum using a rotatory evaporator to obtain a brownish residue (65 g). Isolation. The EtOAc extract (60 g) was subjected to vacuum liquid chromatography (8 × 10 cm, i.d × l) over silica gel eluting with a gradient of CH2Cl2−MeOH (100:0, 99:1, 98:2, 97:3, 95:5, 90:10, 80:20, 50:50, 0:100, v/v) to obtain 10 fractions (Fr. A−J). Fr. E (2.8 g) was subjected to MPLC (3.2 × 50 cm, i.d × l) over silica gel using a mixture of petroleum ether and acetone with increasing polarity (20:1, 20:2, 20:3, 20:4, 20:5, 20:10, 20:20, 0:20, v/v) as eluents to yield eight subfractions (Frs. E-1−E-8). Compound 13 (4 mg) was crystallized from Fr. E-3, while 1 was precipitated from Fr. E-6, and purified by recrystallization from CH2Cl2−MeOH to give a colorless crystal (6.0 mg). Compound 2 (4.0 mg) was isolated from Fr. E-7 by semipreparative HPLC using 50% MeOH−H2O as eluent. Fr. F (4.6 g) was chromatographed over silica gel (5.5 × 50 cm, i.d × l) eluting with a gradient of CH2Cl2−CH3COCH3 (20:1, 20:2, 20:3, 20:4, 20:5, 20:10, 20:20, 0:20, v/v) to obtain eight subfractions (Frs. F-1−F-8) after pooling the similar fractions as monitored by TLC. Fr. F-5 was further subjected to gel permeation chromatography over Sephadex LH-20 (CHCl3−MeOH, 1:1, v/v) to yield six subfractions (Frs. F-51−F-5-6), among which Fr. F-5-2 was purified by semipreparative HPLC using 60% MeOH−H2O as eluent to produce 8 (3.0 mg). Fr. F-6 was processed in a similar manner by chromatography over Sephadex LH-20, followed by purification by HPLC (65% MeOH− H2O) to yield 6 (3.2 mg). Similarly, Fr. F-7 was subjected size exclusion chromatography over Sephadex LH-20 (CHCl3−MeOH, 1:1) to obtain seven subfractions (Frs. F-7-1−F-7-7). Fr. F-7-1 was purified by semipreparative HPLC using 60% MeOH−H2O as eluent to afford 7 (3.0 mg). Likewise, Frs. F-7-4 and F-7-6 were purified by semipreparative HPLC eluting with 45% and 60% MeOH−H2O, respectively, to yield 4 (5.0 mg) and 3 (2.0 mg) from Fr. F-7-4 and 14 (2.0 mg) and 12 (4.2 mg) from Fr. F-7-6. Fr. G (9.6 g) was subjected to VLC (10 × 12 cm, i.d × l) over silica gel eluting with a mixture of petroleum ether−acetone (5:1, 4:1, 3:1, 2:1, 1:1, v/v) with increasing polarity to obtain five subfractions (Frs. G-1−G-5). Fr. G-3 was first subjected to chromatography over Sephadex LH-20 (CHCl3−MeOH, 1:1) then purified by HPLC (65% MeOH−H2O) to afford 10 (1.0 mg). Fr. G-4 was chromatographed over Sephadex LH-20 (CHCl3− MeOH, 1:1), followed by purification with semipreparative HPLC (55% MeOH−H2O), to obtain 5 (1.8 mg). Fr. H (6.2 g) was chromatographed over silica gel eluting with a gradient of petroleum ether−acetone (20:1−0:1, v/v) to give six subfractions (Fr. H-1−H6). Among them, Fr. H-2 and Fr. H-3 were purified by HPLC using 75% and 70% MeOH−H2O as eluents to afford 11 (2.8 mg) and 9 (2 mg), respectively. Rhizopycnolide A (1): colorless crystal (CH2Cl2−MeOH); mp 256−257 °C; [α]33D −28.5 (c 0.2, CH3COCH3); UV (MeOH) λmax (log ε) 246 (4.53), 332 (3.76) nm; ECD (c = 3.19 × 10−4 M, MeOH) λ (Δε) 209 (+1.33), 232 (−7.82), 268 (+0.24), 278 (+0.03), 294 (+0.45), 319 (+0.63), 332 (+0.60) nm; IR (KBr) νmax 3446, 3141, 2983, 2950, 2837, 1788, 1772, 1668, 1630, 1570, 1438, 1384, 1356, 1288, 1261, 1240, 1203, 1166, 1107, 1036, 1016, 962, 929, 846, 835, 801, 768, 717 cm−1; 1H NMR (DMSO-d6, 400 MHz), 13C NMR (DMSO-d6, 100 MHz) see Table 1; HRESIMS m/z 391.10249 [M − H]− (calcd for C19H19O9, 391.10236). Rhizopycnolide B (2): colorless, amorphous solid; [α]25.3D −49.9 (c 0.125, MeOH); UV (MeOH) λmax (log ε) 247 (4.29), 331 (3.47) nm; ECD (c = 6.37 × 10−4 M, MeOH) λ (Δε) 208 (−8.83), 220 (−6.58), 224 (−6.97), 246 (+10.78), 286 (−0.40), 296 (−0.24), 318 (−1.09) nm; IR (KBr) νmax 3456, 2919, 2850, 1786, 1681, 1617, 1569, 1504, 1463, 1436, 1391, 1348, 1206, 1162, 1105, 1033, 969, 939, 843, 799, 738, 685, 666 cm−1; 1H NMR (DMSO-d6 or CDCl3, 400 MHz), 13C NMR (DMSO-d6 or CDCl3, 100 MHz) see Table 1; HRESIMS m/z 2029



Article

Notes

Kα) = 1.001, 12 449 reflections measured, 3430 unique (Rint = 0.0225), which were used in all calculations. The final wR(F2) was 0.0733 (all data). The structure was solved by direct methods using SHELXS-97 and refined with full-matrix least-squares calculations on F2 using SHELXL-97.30 Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Centre (deposition number: CCDC 1440494). Copies of these data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www. ccdc.cam.ac.uk/getstructures. Antibacterial Assay. The antibacterial activities of 1, 2, 4−8, and 10−12 were evaluated against six pathogenic bacteria, A. tumefaciens ATCC 11158, B. subtilis ATCC 11562, P. lachrymans ATCC 11921, R. solanacearum ATCC11696, S. hemolyticus ATCC 29970, and X. vesicatoria ATCC 11633. Streptomycin sulfate was used as the positive control. The minimum inhibitory concentrations (MIC) and median inhibitory concentrations (IC50) of the tested substances were determined in sterile 96-well plates by the modified broth dilution colorimetric assay.31 The antibacterial effects of 9 and 13 were reported previously by our group,21 while 3 and 14 were obtained in a limited amount; hence these compounds were not tested. Antifungal Assay. The antifungal activities of the selected compounds (1, 6−8, and 10−12) were tested against M. oryzae using a spore germination assay as described previously.7 The spores were prepared from 7-day-old cultures of M. oryzae. Each tested compound dissolved in 10% aqueous ethanol (25 μL) was mixed with an equal volume of fungal spore suspension (containing 2 × 106 spores per mL) on a concave glass slide. Slides containing the spores were incubated in a dark moist chamber at 25 °C for 8 h. Each slide was then observed under a microscope for spore germination status. Carbendazim was used as the positive control, while 10% ethanol was used as the negative control. The percentage (%) of spore germination inhibition was determined as [(Gc − Gt)/Gc] × 100, where Gc is the average of the germinated spore numbers in the negative control (n = 3), and Gt is the average of the germinated spore numbers in the treated sets (n = 3). Three replicates were used for each treatment. The median inhibitory concentration (IC50) of each sample was determined by linear regression. Cytotoxic Assay. Cytotoxicity of the compounds was tested against human carcinoma cells using the microculture tetrazolium (MTT) assay as described previously.31 The tested cell lines included colon cancer cells (HCT-116), liver hepatocellular carcinoma cells (HepG2), gastric cancer cells (BGC-823), non-small-cell lung carcinoma cells (NCI-H1650), ovarian cancer cells (A2780), melanoma cells (A375), lung adenocarcinoma cells (A549), breast cancer cells (MCF-7), and pancreatic adenocarcinoma cells (Capan2). Selected compounds were divided into two groups, and each group was tested against five different cancer cell lines. Taxol was used as the positive control. The results are shown in Table 5.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Hi-Tech R&D Program of China (2011AA10A202), the National Basic Research Program of China (2013CB127805), the Special Fund for Agro-Scientific Research in the Public Interest of China (201203037), the Chinese Universities Scientific Fund (2016QC047 and 2014RC020), and the Project of Science and Technology of Hunan Tobacco Industry Co. Ltd (KY2012YC0001). We thank Dr. W. Lin (State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing, China) for help in ECD calculation.

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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.6b00327.

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REFERENCES

ECD calculation data for 1−3 and (1D, 2D) NMR, IR, and HRESIMS spectra of 1−6 (PDF) X-ray crystallographic data for 1 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*Tel (L. Zhou): +86 10 62731199. Fax: +86 10 6273 1062. Email: [email protected]. *Tel (Y. Liu): +86 10 62815874. E-mail: [email protected]. Author Contributions ‡

D. Lai and A. Wang contributed equally. 2030



Article

(27) Bensassi, F.; Gallerne, C.; Sharaf el dein, O.; Rabeh Hajlaoui, M.; Bacha, H.; Lemaire, C. Toxicol. Mech. Methods 2015, 25, 56−62. (28) Zhou, K.; Wang, W.; Peng, Y.; Yu, R.; Yue, Y.; Lai, D.; Zhou, L. Nat. Prod. Res. Dev. 2015, 27, 1847−1852. (29) Bruhn, T.; Schaumloeffel, A.; Hemberger, Y.; Bringmann, G. Chirality 2013, 25, 243−249. (30) Sheldrick, G. M. SHELXL-97, Program for X-ray Crystal Structure Solution and Refinement; University of Göttingen: Göttingen, Germany, 1997. (31) Lu, S.; Sun, W.; Meng, J.; Wang, A.; Wang, X.; Tian, J.; Fu, X.; Dai, J.; Liu, Y.; Lai, D.; Zhou, L. J. Agric. Food Chem. 2015, 63, 3501− 3508.

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Bioactive Dibenzo-Î±-pyrone Derivatives from the Endophytic Fungus

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