Sesquiterpenes from an Endophytic Aspergillus flavus - Journal of

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Sesquiterpenes from an Endophytic Aspergillus flavus Zhen Liu,† Jing-Yi Zhao,† Sen-Feng Sun,† Yong Li,† Jing Qu,† Hai-Tao Liu,‡ and Yun-bao Liu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, People’s Republic of China

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S Supporting Information *

ABSTRACT: Eight new cadinene-sesquiterpenes (1−8), one eudesmane-sesquiterpene (9), and three known compounds (10−13) were isolated from an endophytic fungus, Aspergillus f lavus, which was isolated from a toxic medicinal plant, Tylophora ovata. Their structures were elucidated by interpretation of spectroscopic data, and absolute configurations determined according to the specific rotation and electron circular dichroism methods. Compounds 4−8, 11, and 12 exhibited latent hepatic protection effects at 10 μM, and compound 12 selectively inhibited the proliferation of MCF-7 breast cancer cells with an IC50 values of 2.6 μM. two double-bond protons at δH 7.25 (H-5) and 6.60 (H-5′), eight oxygenated methylene protons at δH 5.19 (H-3a), 5.04 (H-3b), 5.11 (H-14a), 4.59 (H-14b), 4.52 (H-3′a), 4.50 (H3′b), 4.82 (H-14′a), and 4.79 (H-14′b), and four methyl protons at δH 0.98 (H-12), 0.91 (H-13), 1.02 (H-12′), and 0.92 (H-13′). In the 13C NMR data (Table 1), 30 resonance signals were observed, including four carbonyls and six olefinic carbons. The 1H−1H COSY and HSQC spectra indicated the presence of two spin systems (Figure 1). The extensive analysis of HMBC data indicated 1 was a dimer of two sesquiterpenes (units A and B in Figure 1).8 The structure of unit A was deduced according to the HMBC correlations (Figure 1) from H-9′ to C-1′/7′/10′, H-14′ to C-1′/2′/10′, H-6′ to C-1′, H-5′ to C-1′/3′/4′/15′, and H-3′ to C-4′/5′/15′. Similarly, the HMBC correlations from H-9 to C-1/7/10/14, H-14 to C-1/ 9/10, H-1 to C-2/5/7/9/10, H-5 to C-1/3/4/7/15, and H-3 to C-2/4/5/15 indicated that unit B possessed a sevenmembered lactone ring fused with a hexatomic ring via C-1 and C-6. The connection of those two moieties through an ester linkage was deduced by the HMBC correlation from H14 to C-15′. There are six stereocenters and one exocyclic double bond in 1, and the relative configurations were deduced according to the coupling constants and NOESY spectrum data. H-6′ and H-7′ in unit A were assigned as trans based on the large coupling constants (JH‑6′, H‑7′ = 10.2 Hz), which were identical to those of 3-[(4S,5R)-1,3,4,5,6,7-hexahydro-5-(1-methylethyl)-3-oxo-4-isobenzofuranyl]-2-(hydroxymethyl)-(2E)-2-propenoic acid.9 The geometry of Δ4′,5′ was deduced to be E

T

he Aspergillus genus is well known for its medical and commercial importance. Some Aspergillus fungi produce devastating toxins and cause severe diseases, but they are also a rich source of bioactive compounds such as the cholesterollowing drug lovastatin.1 In our long-term studies of endophytic fungi found in toxic medicinal plants, Aspergillus f lavus was isolated from Tylophora ovata. Although A. f lavus is well kown for producing aflatoxins, carcinogenic and mutagenic secondary metabolites,2 a number of novel bioactive compounds have been reported from this fungus over the last two decades, including cyclopiazonic acid (CPA), kojic acid (KA), and indole diterpenes.3−6 Some metabolites showed significant bioactivities, including the antifungal aspirochlorines.7 Chemical screening of an ethyl acetate extract by HPLCDAD-UV and HPLC-MS indicated that the endophytic A. f lavus contained large quantities of sesquiterpenoids. Further systematic studies led to the isolation of eight new cadinenesesquiterpenes (1−8), one eudesmane-sesquiterpene (9), and three known compounds (10−13). Some of these compounds, 4−8, 11, and 12, revealed significant hepatic protection effects and cytotoxic activities to the HCT-116, HepG2, BGC-823, A549, and MCF7 cancer cells. Herein, the isolation, structure elucidation, and biological activities of 1−12 are reported.

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RESULTS AND DISCUSSION Compound 1 was isolated as a white, amorphous powder. Its molecular formula was C30H40O10 with 11 degrees of unsaturation, as deduced from its HRESIMS data. It exhibited a UV absorption band at 211 nm, while IR absorption bands were observed at 3398 and 1687 cm−1 for hydroxy and carbonyl, respectively. The 1H NMR data (Table 1) displayed © XXXX American Chemical Society and American Society of Pharmacognosy

Received: December 29, 2018

A

DOI: 10.1021/acs.jnatprod.8b01084 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

to the HRESIMS and 1H NMR data, compound 1a was determined to be 3-[(4S,5R)-1,3,4,5,6,7-hexahydro-5-(1-methylethyl)-3-oxo-4-isobenzofuranyl]-2-(hydroxymethyl)-(2E)-2propenoic acid. The molecular formula of 1b was determined to be C15H24O7, possessing four double-bond equivalents by HRESIMS spectrometry. Considering the δH‑5 was shifted from 7.25 (d, 4.0 Hz) in 1 to 6.25 (d, 10.5 Hz) in 1b, a ringopening reaction may have occurred in the seven-membered lactone during the hydrolysis experiment (Figure 2). The absolute configurations of 1a and 1b were established by electron circular dichroism (ECD). The Cotton effects of 1a were identical to the published data; therefore, the absolute configurations of C-6 and C-7 were determine to be S and R, respectively, and the configuration of 1b was deduced to be 1S, 6S, 7R, 10S because its ECD spectrum was similar to that of the structurally related compound xylaric acid B.9 Thus, the structure of 1 was deduced and named (4′E,1S,6S,7R,10S,6′S,7′R)-aspergilloid A. Compound 2 exhibited a sodium adduct ion peak at m/z 289.1419 in the HRESIMS spectrum, corresponding to the molecular formula C15H22O4. The IR spectrum displayed absorption bands for an OH group (3397 cm−1) and an unsaturated carboxyl group (1694 cm−1). Two methyl signals at δH 1.33 (H-12) and 0.97 (H-14) and one olefinic signal at 6.87 (H-5) were observed in the 1H NMR data (Table 2). The 13 C NMR data (Table 3) and HSQC correlations showed the presence of two methyl groups, five CH2 groups including one O-bearing CH2 group, four CH groups, and four carbons (one carboxyl carbon and two O-bearing carbons). The doublebond group and carboxyl group accounted for two unsaturation degrees, which indicated compound 2 possessed a three-ring system. The connection of CH3, CH2, and CH carbons was determined by the 1H−1H COSY experiment (Figure 1). The HMBC correlations from H-3/5 to C-1, H-2 to C-4, H-3/H-1 to C-5, H-1/2 to C-6, and H-5 to C-15 led to the establishment of a cyclohex-4-ene-4-carboxylic acid group in 2. In addition, a six-membered ring A fused with the

Table 1. 1H NMR (800 MHz) and 13C NMR (200 MHz) Data in MeOH-d4 for 1 no.

δC

1 2 3

54.1, CH 174.4, C 62.8, CH2

4 5 6

130.7, C 146.6, CH 41.1, CH

7

49.5, CH

8

22.2, CH2

9

36.0, CH2

10 11

74.0, C 28.7, CH

12 13 14

21.6, CH3 15.6, CH3 66.5, CH2

15

168.5, C

δH (J in Hz) 3.60, d (11.2) 5.19, d (14.4) 5.04, d (14.4) 7.25, d (4.0) 2.67, td (11.2, 4.0) 1.58−1.54, overlap 1.75−1.70, overlap 1.34, m 2.27, dt (13.3, 3.4) 1.43, td (13.3, 3.9) 2.10, m 0.98, 0.91, 5.11, 4.59,

d d d d

(6.4) (6.4) (12.5) (12.5)

no.

δC

1′ 2′ 3′

125.9, C 176.1, C 57.4, CH2

4′ 5′ 6′

135.0, C 146.6, CH 35.7, CH

7′

46.8, CH

8′

21.5, CH2

9′

23.8, CH2

10′ 11′

167.3, C 29.1, CH

12′ 13′ 14′

21.9, CH3 18.0, CH3 73.4, CH2

15′

168.5, C

δH (J in Hz)

4.52, d (12.8) 4.50, d (12.8) 6.60, d (10.8) 3.57, tt (10.2, 2.0) 1.58−1.54, overlap 1.97, m 1.62, m 2.49, dt (11.0, 6.0) 2.44, m

1.75−1.70, overlap 1.02, d (6.4) 0.92, d (6.4) 4.82, d (2.4) 4.79, d (2.4)

based on the cross-peak between H-3′ and H-6′ in the NOESY spectrum. In addition, the correlations between H-14 (δH 4.59) and H-6, H-14 (δH 4.59) and H-8b (δH 1.34), and H-1 and H-7 as well as the large coupling constants (J = 11.2 Hz) between H-6 and H-1/7 indicated that H-14 and H-6 were on the same side, while H-1 and H-7 were on the opposite side. In order to determine the absolute configuration of 1, a basic hydrolysis experiment was conducted. After hydrolysis with LiOH, two compounds, 1a and 1b, were obtained. According B



Article

Figure 1. 1H−1H COSY (blue) and key HMBC correlations (red) of compounds 1−9.

Figure 2. Hydrolysis of compound 1.

Table 2. 1H NMR Data in MeOH-d4 for 2−6 δH (J in Hz) a

b

no.

2

1 2

1.5, dt (11.5, 4.0) 1.89−1.82, m

2.68, t (11.6)

3

2.22, m 2.04, m 6.87, m

3.28, 2.96, 7.28, 2.24, 1.45, 1.85, 1.15, 2.15, 1.37, 2.48, 2.28, 1.04, 0.83,

5 6 7 8 9 10 11 12 13 14

2.09, t (6.5) 1.74, m 1.59−1.55, overlap 1.59−1.55, overlap 1.24−1.19, overlap 1.29, m 1.33, 3.74, 3.70, 0.97,

s d (9.5) d (9.5) d (6.5)

4a

3

d (21.3) d (21.3) br s m tt (12.0, 3.0) dq (13.3, 3.1) qd (13.0, 3.5) dq (12.8, 3.5) td (13.0, 4.0) td (12.6, 3.8) m d (7.0) d (7.0)

1.44, qd (11.0, 3.0) 1.89−1.80, overlap 1.32, m 2.39, dd (17.7, 4.0) 2.17, m 7.13, br s 1.89−1.80, overlap 1.25−1.16, overlap 1.89−1.80, overlap 1.25−1.16, overlap 2.04, m 1.53 qd (12.6, 3.6) 2.10, td (11.0, 3.3) 2.24, m 0.98, d (6.6) 0.84, d (6.6)

5b 3.84 d (10.7)

2.13, d (12.0)

3.42 d (16.5) 3.34, d (16.5) 6.72, d (10.7) 2.25, q (10.7) 1.53, tt (10.7, 2.5) 1.74, overlap 1.10, pd (13.1, 3.9) 2.47, dt (13.3, 3.2) 2.05, td (13.3, 3.5)

4.82, d (12.2) 4.59, d (12.2) 6.80, d (11.0) 2.62, q (12.0) 1.36, tt (12.0, 3.0) 1.81−1.76, m 1.27, m 2.02, dt (13.5, 3.4) 1.66, dd (13.5, 4.5) 1.74−1.70, m 1.81−1.76, m 0.97, d (6.8) 0.75, d (6.8)

1.74, overlap 0.93, d (6.9) 0.69, d (6.9) 5.03, br s 4.80, br s

17

6b

4.45, d (10.0) 4.06, d (10.0) 2.00, s

a

Data were recorded at 600 MHz. bData were recorded at 500 MHz.

C


Journal of Natural Products Table 3.

13

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Compound 3 was obtained as a white, amorphous powder. Its molecular formula, C15H20O5, was deduced by HRESIMS data. The UV spectrum showed an absorption peak at 206 nm, and the IR spectrum displayed absorptions for a carboxyl group (1694 cm−1). The 1H NMR data (Table 2) displayed 18 resonances including two methyl protons at δH 1.04 (H-12) and δH 0.83 (H-13) and one olefinic proton at δH 7.28 (H-5). The 13C NMR data (Table 3) together with HSQC data showed that compound 3 possessed 15 signals for two methyl carbons, three CH2 carbons, six CH carbons, and four nonprotonated carbons. The characteristic signals for isopropyl and a trisubstituted double bond, δH 0.83 (3H, d, J = 7.0 Hz, H-13), 1.04 (3H, d, J = 7.0 Hz, H-12), 2.28 (1H, m, H-11), and 7.28 (1H, br s, H-5) in the 1H NMR spectrum and δC 15.5 (C-13), 21.7 (C-12), 28.0 (C-11), 130.1 (C-4), 140.7 (C-5), and 169.2 (C-15) in the 13C NMR spectrum, implied compound 3 was a cadinene sesquiterpene.10 It was further confirmed by the detailed analysis of 2D NMR data. The correlations (Figure 1) in the 1H−1H COSY and HSQC spectra led to the establishment of an isopropylcyclohexane group in 3. In the HMBC spectrum, correlations from H-1 to C-2/5, H-3 to C-2/4/5, and H-5 to C-1/3/4/7 indicated a cyclohex-4-en-2-one unit connected to an isopropylcyclohexane group through C-1 and C-6. The two carbonyl groups linked at C-4 and C-10 were determined by the correlations from H-9/10 to C-14 and from H-5 to C-15. Thus, the planar structure of 3 was elucidated. The relative configuration of 3 was assigned by correlations in the NOSEY spectrum and coupling constants. The crosspeaks of H-1 and H-7 and H-6 and H-10 implied that H-1 and H-7 (Figure 3) were in α-orientations and H-6 and H-10 were in β-orientations. The large coupling constants of H-1/H-6 (J = 11.6 Hz) and H-1/10 (J = 11.6 Hz) as well as H-6/H-7 (J = 12.0 Hz) also confirmed the connection of this configuration. The ECD method was used to determine the absolute configuration of 3. According to the octant rule of saturated cyclohexanone, a negative Cotton effect at 287 nm indicated 3 had a stereochemistry of 1S, 6S, and 7R (Figure 4). The configuration was confirmed by the calculated ECD spectrum of 3a, which was similar to the experimental spectrum (Figure 5). Thus, the structure of compound 3 was elucidated and named (1S,6S,7R)-aspergilloid C.

C NMR Data in MeOH-d4 for 2−6

no.

2a

3b

4a

5b

6b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

42.5, CH 21.1, CH2 20.8, CH2 134.1, C 142.6, CH 83.1, C 55.7, CH 22.3, CH2 31.5, CH2 29.0, CH 81.8, C 23.2, CH3 78.6, CH2 20.7, CH3 170.8, C

54.0, CH 209.9, C 40.4, CH2 130.1, C 140.7, CH 42.6, CH 47.2, CH 24.7, CH2 30.2, CH2 43.5, CH 28.0, CH 21.7, CH3 15.5, CH3 179.3, C 169.2, C

42.3, CH 28.5, CH2 25.7, CH2 132.1, C 140.5, CH 44.0, CH 46.8, CH 25.2, CH2 31.1, CH2 50.9, CH 27.5, CH 21.6, CH3 15.5, CH3 179.7, C 171.1, C

75.4, CH 175.8, C 34.5, CH2 130.4, C 147.7, CH 52.1, CH 48.2, CH 26.4, CH2 34.5, CH2 151.0, C 29.8, CH 22.0, CH3 16.4, CH3 105.9, CH2 171.2, C

53.8, CH 178.8, C 59.2, CH2 130.8, C 148.7, CH 42.0, CH 46.4, CH 22.0, CH2 32.9, CH2 76.3, C 29.5, CH 21.9, CH3 15.9, CH3 76.2, CH2 169.3, C 172.5, C 20.9, CH3

a

Data were recorded at 150 MHz. bData were recorded at 125 MHz.

cyclohex-4-ene-4-carboxylic acid group (ring B) via C-1 and C6 was established by the HMBC correlations from H-1/2/8 to C-10, H-7/14 to C-1, H-1/8 to C-6, and H-5/9 to C-7. The HMBC correlations from H-12 to C-7/13, H-13 to C-6/7/12, and H-7 to C-13 indicated that ring A was fused with a tetrahydrofuran ring system. Therefore compound 2 was determined to be a tricyclic-cadinene sesquiterpene. The relative configuration of 2 was elucidated from the NOSEY data. The correlations (Figure 3) between H-1 and H12/13b (δH 3.70), H-5 and H-7, and H-7 and H-13a (δH 3.74) indicated ring A is cis-fused with ring B and ring C, respectively. The cross-peaks between H-1/2 and H-14 and between H-3b (δH 2.04) and H-10/14 as well as the large coupling constant value of H-1 and H-10 confirmed that H-1 and H-14 were in a β-orientation. The absolute configuration was determined by the calculated ECD method. The theoretical ECD spectrum of 2a with 1S, 6S, 7R, 11R, 14R configuration was consistent with the experimental ECD spectrum (Figure 5). Thus, the structure of compound 2 was established as shown.

Figure 3. Key NOE correlations (blue) of compounds 2−9. D



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Figure 4. Empirical rules for compounds 3, 5, and 9.

Figure 5. Experimental and calculated ECD of compounds 2−5.

The relative configuration of 4 was proposed to be the same as 3 based on the analysis of NOESY data and the coupling constant values. The large coupling constants of H-1/H-6 (J = 11.0 Hz) and H-1/H10 (J = 11.0 Hz) and the NOE correlation of H-1 and H-7 (Figure 3) indicted the H-1 and H-7 were on the same side, and H-6 and 10 were on opposite sides. Similarly, the absolute configuration of 4 was elucidated by the ECD method. The negative Cotton effect at 251 nm, belonging to the absorption of the α,β-unsaturated carboxyl group, in the ECD spectrum of 4, was consistent with that of 3 (a negative shoulder peak in the ECD spectrum), which indicated the stereochemistry at C-1/6/7/10 was the same as that of 3. The calculated ECD spectrum of 4a, with the 1S, 6R, 7R, and 10S configuration, supported this assignment (Figure 5). Thus, the structure of compound 4 was established and named (1S,6R,7R,10S)-aspergilloid D. Compound 5 was isolated as a white powder, with the molecular formula C15H22O5 (five double-bond equivalents) as

Compound 4, a white, amorphous powder, was found to have the molecular formula C15H22O4 as deduced from HRESIMS, in combination with 13C NMR spectroscopy. The UV and IR spectra were similar to those of 3, indicating that these two compounds possessed the same core skeleton. Comparison of the 1H NMR and 13C NMR data (Tables 2 and 3) showed the absence of a carbonyl (δC 209.9 in 3) and an additional methylene (δC 28.5 in 4) and two upfield carbons signals (δC 54.0 and 40.4 in 3, while δC 42.3 and 25.7 in 4). Those data together with the degrees of unsaturation suggested that the carbonyl at C-2 in 3 was changed to a methylene in 4. The detailed analysis of the resonances in the 2D NMR spectra of 4 supported this speculation. The connections of C-1/2/3/ 5/6/7/8/9/10/11/12/13 were assigned as shown according to the 1H−1H COSY data (Figure 1). The HMBC correlations from H-2 to C-4/6, from H-3 to C-4/5/15, and from H-9/10 to C-14 finally led to the establishment of the planar structure of 4. E



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Table 4. 1H NMR and 13C NMR Data in MeOH-d4 for 7−9 7e no.

δC

8a δH (J in Hz)

δC

9a δH (J in Hz)

130.5, C 156.3, C

δC

1 2

132.0, C 156.6, C

3

113.8, CH

7.23, br s

114.0, CH

7.23, br s

40.8, CH2

4 5 6

132.0, C 121.9, CH 143.4, C

7.49, br s

130.5, C 123.4, CH 143.2, C

7.37, br s

71.3, C 53.4, CH 27.6, CH2

7 8 9

44.9, CH 18.6, CH2 23.5, CH2

10 11 12

36.9, CH 32.1, CH 21.4, CH3

13 14

16.8, CH3 64.7, CH2

15

171.8, C

2.77, m 1.64−1.57, m 2.17, dt (13.5, 3.4) 1.66, dd (13.5,4.5) 3.27, m 2.41, m 1.06, d (7.0)

44.5, CH 20.2, CH2 22.4, CH2

0.68, d (7.0) 3.87, dd (10.4, 5.2) 3.49, t (10.4)

19.9, CH3 64.4, CH2

216.8, C 35.9, CH2

36.8, CH 34.6, CH 22.4, CH3

171.8, C

δH (J in Hz) 2.66, ddd (15.8, 11.0, 5.1) 2.37, ddd (15.8, 6.6, 5.1) 2.01, dd (6.6, 4.2) 1.88−1.83, overlap 1.81, m 1.88−1.83, overlap 1.57, q (12.6) 1.93, m 3.70, td (11.0, 4.3) 2.04, dd (12.6, 4.2) 1.30, t (6.6)

2.53, br s 1.82, overlap 2.07−1.99, overlap 1.82, overlap 3.26, m 2.07−1.99, overlap 1.00, d (6.8)

55.6, CH 68.7, CH 45.3, CH2

0.85, d (6.8) 3.82, dd (10.2, 4.0) 3.35, t (10.2)

20.0, CH3 19.2, CH3

4.83, 4.82, 1.78, 1.18,

23.5, CH3

1.29, s

48.7, C 148.1, C 112.4, CH2

br s br s s s

a

Data were recorded at 600 MHz for 1H NMR and 150 MHz for 13C NMR. eData were recorded at 800 MHz for 1H NMR and 200 MHz for 13C NMR.

determined from the HRESIMS and NMR data. The 1H NMR data (Table 2) displayed 19 resonances including two methyl protons at δH 0.93 (H-12) and 0.69 (H-13) as well as three double-bond protons at δH 6.72 (H-5), 5.03 (H-14a), and 4.80 (H-14b). The 13C NMR (Table 3) data and 2D NMR spectra indicated that 5 contained 15 resonances for two CH3 carbons, four CH2 carbons including one sp2 carbon, five CH carbons, and four nonprotonated carbons including two carboxyl groups. Inspection of the 1H−1H COSY and HSQC correlations (Figure 1) led to the assignment of a spin system containing H-1/5/6/7/8/9/11/12/13. The HMBC correlations from H-5/9/14 to C-1, from H-1/8/9/14 to C-10, and from H-14 to C-9 indicated a terminal double bond located at C-10 and C-14. In addition, the HMBC correlations from H-3 to C-2/4/5/15, from H-5 to C-1/3/4/15, and from H-6 to C4 implied a 2-methylenesuccinic acid group linked at C-6. Thus, the planar structure was elucidated as shown.11 The relative configuration of 5 was elucidated on the basis of spectroscopic analysis. In the 1H NMR spectrum, H-6 displayed a quartet signal with a large coupling constant value (J = 10.7 Hz) at 2.25 ppm, suggesting the trans configurations of H-6 and H-1/H-7. These assignments were further confirmed by the NOESY correlations (Figure 3) between H-1 and H-5/7 and between H-6 and H-14a/14b. Thus, the relative configurations of three stereocenters were established. The E-geometry of Δ4,5 was deduced by the NOE cross-peak between H-6 and H-3. The absolute configuration of 5 was determined by the ECD method. The ECD spectrum displayed a negative chirality, which indicated the counterclockwise orientation from an α,βunsaturated carboxyl to Δ10,14 (Figure 4). In addition, the calculated ECD spectrum of 5a was consistent with the experimental spectrum of 5, indicating that the absolute configuration of 5 was 1S, 6S, 7R (Figure 5). Therefore, the

structure of compound 5 was elucidated and named (1S,6S,7R)-aspergilloid E. Compound 6, a white powder, was found to possess the molecular formula C17H24O7 as deduced from the HRESIMS and NMR data. The UV absorption and characteristic 1H NMR signals (Table 2) at δH 0.97 (3H, d, J = 6.8, H-12), 0.75 (3H, d, J = 6.8, H-13), and 6.80 (1H, d, J = 11.0 Hz, H-5) suggested that 6 might share the same skeleton structure with hydroheptelidic acid.12 The additional carbonyl at δC 172.5 ppm and singlet methyl at δC 20.9 ppm (Table 3) as well as the downfield shifts of C-3 and H-3a/3b revealed that compound 6 was an acetyl product of hydroheptelidic acid.12 This speculation was supported by the correlations (Figure 1) from H-3 to C-16 and H-17 to C-16 in the HMBC data. The relative and absolute configurations of 6 were determined by the NOESY and ECD data, respectively. The NOSEY resonances (Figure 3) of H-1 and H-5/7 and H-6 and H-14 indicated that H-1/7 were in α-orientations while H-6 and H-14 were β-oriented. In addition, the NOE correlation between H-3 and H-6 led to the determination of the Econfiguration of Δ4,5. The Cotton effects at 231 (−) and 205 (+) nm in the ECD spectra were identical to those of hydroheptelidic acid, indicating that compound 6 possesses the same absolute configuration as hydroheptelidic acid.12 Thus, compound 6 was named (1S,6S,7R,10S)-aspergilloid E. Compound 7 was isolated as a colorless oil. The molecular formula C15H20O4 was determined based on HRESIMS data. The UV spectrum displayed characteristic absorptions (212, 248, and 296 nm) for benzene. The analysis of the IR spectrum indicated the presence of hydroxy (3407 cm−1) and carbonyl (1684 cm−1) groups in 7. The 1H NMR data (Table 4) showed 17 resonances including two aromatic protons at δH 7.23 (H-3) and 7.49 (H-5), two oxygenated protons at δH 3.87 (H-14a) and 3.49 (H-14b), and two methyl protons at δH 1.06 (H-12) and 0.68 (H-13). The 13C NMR and 2D NMR spectra F



Article

302 nm suggested the 4R, 5R, 7R, 8S,10R configurations of 9. Thus, the structure of compound 9 was established and named 4α,8α-dihydroxyeudesman-11-en-1-one. Three known compounds, 3-[(4S,5R)-1,3,4,5,6,7-hexahydro5-(1-methylethyl)-3-oxo-4-isobenzofuranyl]-2-(hydroxymethyl)-(2E)-2-propenoic acid (10),9 sporogen AO-1 (11),15 and 3β,4α-dihydroxy-26-methoxyergosta-7,24(28)-dien-6-one (12),16 were also isolated from A. f lavus. Compounds 1−8 and 10 were classified as cadinene derivatives. Their plausible biosynthesis pathway is shown in Scheme 1 (Supporting Information). The hepatic protection effects of compounds 2−12 were evaluated on the acetaminophen (APAP)-induced damage model of HepG2 cells at 10.0 μM. Compounds 4−8, 11, and 12 were found to significantly improve the HepG2 cell survival rates from 24.0% (APAP, 8 mM) to 33.2−41.6%, while the positive control bicyclol, a clinically used hepatic protectant, increased the survival rate to 31.9% (Figure 6). According to

showed 7 possessed 15 carbons including one carbonyl and six aromatic carbons. The 1H−1H COSY and HSQC spectra exhibited the presence of a spin system, CH2−CH−CH2− CH2−CH−CH(CH3)−CH3 (Figure 1). The HMBC correlations from H-3/5/7/14 to C-1, H-7/8 to C-6, and H-3/5 to C15 indicated that compound 7 was a cadinene derivative with an aromatic ring B.13 The relative configuration of 7 was deduced based on the correlations in the NOESY spectrum. The cross-peak of δH 0.68 (H-13) and δH 3.49 (H-14b) indicated the cis configuration of C-7 and C-10 (Figure 3). The absolute configuration was determined by the sign of the specific rotation value. According to Yan’s method, the sign of the specific rotation value should be governed by the absolute configuration of C-7 (7R: +; 7S: −). Thus, compound 7 was assumed to be 7R, 10R due to the positive specific rotation.13 Therefore, the structure of compound 7 was established and named (7R,10R)-aspergilloid G. Compound 8, a colorless oil, was determined to possess a molecular formula identical to 7. The UV and IR spectra of 8 also displayed similar absorption peaks to those of 7. Comparison of the 1H NMR and 13C NMR data of these two compounds (Table 4) led to the assignment of the same planar structure. However, in the NOSEY spectrum, there was no correlation between H-14 and H-13, which implied the isopropyl at C-7 and the hydroxymethyl at C-10 were in a trans configuration (Figure 3). Similarly, 7R, 10S were assigned by the dextrorotation of compound 8. Thus, compound 8 was determined and named (7R,10S)-aspergilloid H. Compound 9 was obtained as a white, amorphous powder with an end absorption at 202 nm. Its HRESIMS showed a sodium adduct ion at m/z 275.1627 ([M + Na]+), suggesting a molecular formula of C 15H24O 3 with four degrees of unsaturation. The 1H NMR spectrum (Table 4) displayed three singlet methyl protons at δH 1.78 (H-13), 1.18 (H-14), and 1.29 (H-15) as well as two double-bond protons at δH 4.83 (H-12a) and 4.82 (H-12b). The 13C NMR data revealed 15 carbons including a carbonyl carbon (δC 216.8), two oxygenated carbons (δC 68.7, 71.3), and two double-bond carbons (δC 148.1, 112.4). Analysis of the 1H−1H COSY and HSQC spectra led to the establishment of two spin systems, [CH2−CH2] and [CH2−CH−CH−CH2−CH] (Figure 1). The assigned spin systems as well as the HMBC correlations from H-2 to C-1/4, H-3 to C-1/4, H-5 to C-9/10, H-6 to C-8/ 10, H-9 to C-1/7/10, H-14 to C-5/9, and H-15 to C-3 indicated the presence of a 4,8-dihydroxy-4,10-dimethyldecahydronaphthalen-1-one group. One 2-propene group located at C-7 was elucidated based on the HMBC correlations from H-12 to C-7/13, H-13 to C-7/12, and H-6/8 to C-11. Thus, compound 9 was established to be an eudesmane-sesquiterpene.14 The relative configuration of 9 was determined by the coupling constant values and NOSEY correlation data. The large coupling constant between H-7 and H-8 (J = 11.0 Hz) indicated these two protons were on opposite sides. The NOE cross-peaks of H-2a (δH 2.66) and H-14/15, H-15 and H-6b (δH 1.57), H-14 and H-8/H-6b (δH 1.57), and H-5 and H-7/ H-9b (δH 1.30) revealed that H-14, H-15, and H-8 were in the β-configurations, while H-5 and H-7 were in α-configurations (Figure 3). The absolute configuration of 9 was determined by the ECD spectrum according to the octant rule for saturated cyclohexanone. As shown in Figure 4, the negative Cotton effect at

Figure 6. Hepatic protection effects of compounds 2−12 (*P < 0.001, compared with the solvent control group; #P < 0.05, ##P < 0.01, compared with APAP group).

the hepatic protection activities of the cadinene-sesquiterpenes, a preliminary structure−activity relation can be summarized as follows: the protective efficacy of compounds 4−8 and 10, which have various substitutions at C-1/2/3, suggested ring B seems to contribute little to cell proliferation. The isopropyl unit at C-7 and the substituted groups at C-14 in ring A might dominate their activities. In addition, compound 12 selectively inhibited the proliferation of MCF-7 breast cancer cells with an IC50 value of 2.6 μM. In 2016, Lee isolated three 2,3-seco-cadinene sesquiterpenes from the capsaicin-containing culture medium metabolized by A. oryzae, a filamentous fungus closely allied to A. f lavus but widely utilized in food fermentation industries.17 Comparative analysis of genome sequence revealed that the two species share similar secondary metabolite gene clusters.2 The isolation of compounds 1−8 and 10 in our research further demonstrated that both species have cadinene sesquiterpene biosynthesis-related enzymes.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Rudolph Autopol V automatic polarimeter at 20 °C. UV spectra were acquired by a Jasco V-670 UV−visible/NIR spectrophotometer. ECD spectra were measured with a JASCO J815 spectropolarimeter. IR analysis was performed using a Nicolet 5700 FT-IR spectrometer. NMR spectra were acquired with a Bruker AVIIIHD-600, Agilent Technologies DD2-500, or Bruker 800 spectrometer. Chemical shift values were referenced to internal signals of residual proton or carbon (δH 3.31 and δC 49.0 for MeOH-

G



Article

d4). HRESIMS data were obtained by an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS spectrometer or Thermo Q Exactive Focus LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-20A detector, using a YMC-Pack ODS-A (250 × 10 mm, 5 μm) or YMC-Pack C8 (250 × 10 mm, 5 μm) column. ODS (45−70 μm, Merck) and silica gel (200−300 mesh, Qingdao Marine Chemical Inc. China) were used for column chromatography (CC). TLC was conducted on glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc. China). Plant Material. The leaves of T. ovata were collected in Guangxi Province, China, and identified by Prof. Song-Ji Wei at Guangxi College of Chinese Traditional Medicine, in May 2015. The specimen was assigned accession No. S2505 and deposited in the herbarium of the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Fungal Material. The fungus isolated from the surface-sterilized leaves of Tylophora ovata using a PDA plate was assigned the accession number WET-7. This fungus strain was identified as Aspergillus f lavus according to the ITS sequence (GenBank accession no. KP418791) by the Beijing Sunbiotech Co., Ltd. The fungus was kept in Liu’s lab at the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, China. Fermentation, Extraction, and Isolation. A. f lavus was cultivated in 200 mL of modified Martin broth containing glucose (20 g/L), peptone (5 g/L), yeast extract (2 g/L), KH2PO4 (0.5 g/L), and MgSO4·7H2O (0.3 g/L) in a 500 mL conical flask. The pH of the medium was adjusted to 6.4 using hydrochloric acid. Then the broth was incubated on a rotary shaker (110 rpm) at 28 °C for 3 days and transferred as a seed culture into 1000 mL flasks containing 200 g of rice and 200 mL of water. After culturing for 30 days at room temperature, the fermented materials were extracted with EtOAc. The EtOAc solution was evaporated under reduced pressure to yield the extract (50.8 g). The extract was distributed in water and partitioned with petroleum ether and ethyl acetate, sequentially. The EtOAc fraction (12.0 g) was applied to a silica gel column and eluted with a CH2Cl2−MeOH gradient solvent (100:1−1:1) to afford three fractions (A, 0.3 g; B, 1.4 g; C, 2.7 g). Fraction A (0.3 g) was purified by RP-HPLC (YMC-Pack ODS-A) using 25% acetonitrile in water to afford 11 (8 mg, tR = 37 min). Fraction B (1.4 g) was separated over an ODS column and eluted with a gradient of increasing methanol in water (10−100%), to obtained four subfractions (B1, 162 mg; B2, 128 mg; B3, 239 mg; B4, 49 mg). Fraction B1 was subjected to RPHPLC (YMC-Pack ODS-A) with 25% acetonitrile−water to afford 3 (10 mg, tR = 66 min). Fraction B2 was chromatographed on RPHPLC (YMC-Pack ODS-A) eluted with 25% acetonitrile−water (ACN−H2O) to afford 10 (9 mg, tR = 87 min). Fraction B3 was separated by RP-HPLC (YMC-Pack ODS-A) using 40% acetonitrile in water to afford 4 (2 mg, tR = 23 min). Fraction B4 was purified by RP-HPLC (YMC-Pack ODS-A) with 60% acetonitrile−water to afford 12 (2 mg, tR = 48 min). Fraction C (2.7 g) was chromatographed on an ODS column using a gradient eluent of MeOH−H2O to yielded six subfractions (C1, 230 mg; C2, 167 mg, C3, 142 mg; C4, 137 mg; C5, 50 mg; C6, 70 mg). Then, the purification of C2 with petroleum−EtOAc (2:1) gave three subfractions (C2.1, C2.2, C2.3). Fraction C2.2 (10 mg) was purified by RP-HPLC (YMC-Pack C8) using 20% acetonitrile in water to afford 9 (2 mg, tR = 23 min). Fraction C3 was subjected to RP-HPLC (YMC-Pack ODS-A) using 25% acetonitrile in water to afford 6 (8 mg, tR = 37 min). Fraction C4 was separated by RP-HPLC (YMCPack ODS-A) with 20% ACN−H2O to afford 2 (4 mg, tR = 66 min). Fraction C5 was purified using RP-HPLC (YMC-Pack ODS-A) with 40% ACN−H2O to afford 5 (2 mg, tR = 52 min), 8 (2 mg, tR = 40 min), and 7 (2 mg, tR = 42 min). Fraction C6 was separated over a silica gel column to obtain six subfractions (C6.1, C6.2, C6.3, C6.4, C6.5, C6.6). Fraction C6.4 (5 mg) was purified by RP-HPLC (YMCPack ODS-A) using 45% ACN−H2O to afford 1 (1 mg, tR = 45 min). Hydrolysis of Compound 1. Compound 1 (1 mg) was dissolved in 200 μL of LiOH (1 mg/mL) and reacted at room temperature for 2 h. Then, compounds 1a (0.5 mg, tR = 46 min) and 1b (0.2 mg, tR =

23 min) were purified by RP-HPLC (YMC-Pack ODS-A) with 30% ACN−H2O. Compound 1: white, amorphous powder; [α]20 D +70.8 (c 0.09 MeOH); UV (MeOH) λmax (log ε) 211 (4.37) nm; IR νmax 3398, 2960, 2879, 1687, 1202, 1138 cm−1; 1H NMR and 13C NMR data see Table 1; (+)-HRESIMS m/z 583.2501 [M + Na]+ (calcd C30H40NaO10, 583.2514). Compound 1a: white, amorphous powder; 1H NMR (500 MHz, MeOH) δH 4.47 (2H, s, H-3a), 6.25 (1H, d, J = 10.8 Hz, H-5), 3.64 (1H, m, H-6), 1.59 (1H, m, H-7), 1.94 (1H, m, H-8a); 1.48 (1H, m, H-8b), 2.42 (2H, m, H-9), 1.73 (1H, m, H-11), 1.01 (1H, d, J = 6.4 Hz, H-12), 0.90 (3H, d, J = 6.4 Hz, H-13), 4.84 (3H, dd, J = 15.5, 2.4 Hz, H-14a), 4.75 (1H, dd, J = 15.5, 2.4 Hz, H-14b); (−)-HRESIMS m/z 279.12369 [M − H]− (calcd C15H19O9, 279.12270). Compound 1b: white, amorphous powder; 1H NMR (500 MHz, MeOH) δH 2.33 (1H, d, J = 10.5 Hz, H-1), 4.39 (1H, d, J = 11.5 Hz, H-3a), 4.30 (1H, d, J = 11.5 Hz, H-3b), 6.25 (1H, d, J = 10.5 Hz, H5), 2.83 (1H, m, H-6), 1.19−1.33 (1H, overlap, H-7b), 2.20 (1H, m, H-8a), 1.19−1.33 (1H, overlap, H-8b), 1.59 (1H, m, H-9a), 1.19− 1.33 (1H, overlap, H-9b), 1.78 (1H, m, H-11), 0.89 (3H, d, J = 6.7 Hz, H-12), 0.79 (3H, d, J = 6.7 Hz, H-13) 3.64 (2H, s, H-14); (+)-HRESIMS m/z 339.14102 [M + Na]+ (calcd C15H24NaO7, 339.14252). Compound 2: white, amorphous powder; [α]20 D +72.4 (c 0.50 MeOH); UV (MeOH) λmax (log ε) 212 (4.04) nm; IR νmax 3397, 2933, 1694, 1241, 1033 cm−1; 1H NMR and 13C NMR data see Tables 2 and 3; (+)-HRESIMS m/z 289.1419 [M + Na]+ (calcd C15H22NaO4, 289.1410). Compound 3: white, amorphous powder; [α]20 D −12.5 (c 0.16 MeOH); UV (MeOH) λmax (log ε) 206 (3.77) nm; IR νmax 2972, 2936, 1694, 1250, 1188 cm−1; 1H NMR and 13C NMR data see Tables 2 and 3; (−)-HRESIMS m/z 279.1248 [M − H]− (calcd C15H20NaO5, 279.1238). Compound 4: white, amorphous powder; [α]20 D +14.3 (c 0.70 MeOH); UV (MeOH) λmax (log ε) 219 (4.00) nm; IR νmax 2936, 2856, 1694, 1424, 1288, 1200 cm−1; 1H NMR and 13C NMR data see Tables 2 and 3; (−)-HRESIMS m/z 265.1452 [M − H]− (calcd C15H21O4, 265.1445). Compound 5: white, amorphous powder; [α]20 D −9.2 (c 0.25 MeOH); UV (MeOH) λmax (log ε) 204 (3.96), 217 sh (3.96) nm; IR νmax 3131, 2947, 1698, 1401, 1198 cm−1; 1H NMR and 13C NMR data see Tables 2 and 3; (+)-HRESIMS m/z 305.1353 [M + Na]+ (calcd C15H22NaO5, 305.1359). Compound 6: white, amorphous powder; [α]20 D −58.97 (c 0.68 MeOH); UV (MeOH) λmax (log ε) 211 (4.28) nm; IR νmax 2957, 2923, 1457, 1377 cm−1; 1H NMR and 13C NMR data see Tables 2 and 3; (+)-HRESIMS m/z 363.14075 [M + Na] + (calcd C17H24NaO7, 363.14142). Compound 7: colorless oil; [α]20 D +20.0 (c 0.21 MeOH); UV (MeOH) λmax (log ε) 212 (4.09), 248 (3.54), 296 (3.12) nm; IR νmax 3407, 2967, 2936, 1684, 1425, 1203, 1141, 843 cm−1; 1H NMR and 13 C NMR data see Table 4; (+)-HRESIMS m/z 265.1419 [M + Na]+ (calcd C15H21O4, 265.1434). Compound 8: colorless oil; UV (MeOH) [α]20 D +26.4 (c 0.17 MeOH); λmax (log ε) 210 (4.01), 248 (3.43), 297 (3.01) nm; IR νmax 3383, 2957, 1685, 1424, 1205, 1142, 842, 801 cm−1; 1H NMR and 13 C NMR data see Table 4; (+)-HRESIMS m/z 287.1262 [M + Na]+ (calcd C15H20NaO4, 287.1254). Compound 9: white, amorphous powder; [α]20 D −1.6 (c 0.25 MeOH); UV (MeOH) λmax (log ε) 202 (3.47) nm; IR νmax 3324, 2972, 2943, 1696, 1441 cm−1; 1H NMR and 13C NMR data see Table 4; (+)-HRESIMS m/z 275.1627 [M + Na]+ (calcd C15H24NaO3, 275.1618). ECD Calculation. All calculations were performed using the Gaussian 09 software. The conformation analysis was performed in DS (Discovery Studio) 2018 with the MMFF94s force field. The stable conformers subjected to ECD calculation were optimized by the time-dependent density functional theory (TDDFT) method at the B3LYP/6-31G(d) level with the polarized continuum (PCM) model in MeOH. Eighty lowest conformers were calculated using H



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B3LYP/6-31+G(d,p) (for compound 2, 4, and 5) or Cam-B3LYP/631+G(d,p) (for compound 3) with the half-bandwidth no more than 0.35 eV. The final calculated ECD spectra were obtained according to the Boltzmann-calculated contribution of each conformer. Hepatic Protection Assay. The MTT method was used to test the hepatic protection effects of the isolated compounds. HepG2 cells were cultured in 96-well plates. The isolated compounds (2−12) and acetaminophen were added in the culture medium to give final concentrations of 10 μM and 10 mM, respectively, and incubated for 48 h. In addition, bicyclol and DMSO were used as positive and negative control, respectively. Then, 100 μL of MTT (0.5 mg/mL) was added to each well and incubated for 4 h at 37 °C. After adding 150 μL of DMSO, absorbances at 570 nm were measured. The survival rate of HepG2 cells = the mean OD of the medicated group/ the mean OD of the solvent control group. Cytotoxicity Assay. The cytotoxic activities of compounds 2−12 were measured using the MTT method. The cell lines (HCT-116, HepG2, BGC-823, A549, and MCF7) were cultured in the RRMI 1640 medium containing 10% fetal bovine serum and 100 U/mL penicillin at 37 °C. After incubating the cells in a 96-well plate for 24 h, the compounds were added to give final concentrations of 0.1, 1, and 10 μM, respectively, and experiments were carried out in triplicate. Then 100 μL of MTT (0.5 mg/mL) was added to each well after 72 h of culturing. Four hours later, 200 μL of DMSO was added to dissolve the formazan crystals. Absorbances at 570 nm were measured. The inhibition rates were calculated by the following formula: Inhibition rate = 1 − the mean OD of the medicated group/ the mean OD of the solvent control group. Paclitaxel and DMSO were used as positive and negative control, respectively.

IC50 (μmol/L), 72 h HCT-116

HepG2

BGC-823

A549

MCF7

12 Taxol

>10 1.8 nM

>10 1.9 nM

>10 0.4 nM

>10 13.7 nM

2.6 0.2 nM

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REFERENCES

(1) Sanchez, J. F.; Somoza, A. D.; Keller N, P.; Wang, C. C. C. Nat. Prod. Rep. 2012, 29, 351−371. (2) Amaike, S.; Keller, N. P. Annu. Rev. Phytopathol. 2011, 49, 107− 133. (3) Vadlapudi, V.; Borah, N.; Yellusani, K. R.; Gade, S.; Reddy, P.; Rajamanikyam, M.; Vempati, L. N. S.; Gubbala, S. P.; Chopra, P.; Upadhyayula, S. M.; Amanchy, R. Sci. Rep. 2017, 7, 1−10. (4) Lee, Y. J.; Hagler, W. M. J. Food Sci. 1991, 56, 871−872. (5) Saldan, N. C.; Almeida, R. T. R.; Avincola, A.; Porto, C.; Galuch, M. B.; Magon, T. F. S.; Pilau, E. J.; Svidzinski, T. I. E.; Oliveira, C. C. Food Chem. 2018, 241, 113−121. (6) Wicklow, D. T.; Dowd, P. F. J. Org. Chem. 1990, 55, 5299−5301. (7) Klausmeyer, P.; McCloud, T. G.; Tucker, K. D.; Cardellina, J. H.; Shoemaker, R. H. J. Nat. Prod. 2005, 68, 1300−1302. (8) Yamaguchi, Y.; Mantita, D.; Takeuchi, T.; Kuramochi, K.; Kuriyama, I.; Sugawara, F.; Yoshida, H.; Mizushina, Y. Biosci., Biotechnol., Biochem. 2010, 74, 793−801. (9) Yan, S.; Li, S.; Wu, W.; Zhao, F.; Bao, L.; Ding, R.; Gao, H.; Wen, H.; Song, F.; Liu, H. Chem. Biodiversity 2011, 8, 1689−1700. (10) Hosozawa, S.; Miura, I.; Kido, M.; Munoz, O.; Castillo, M. Phytochemistry 1985, 24, 2317−2323. (11) Hu, Z. X.; Shi, Y. M.; Wang, W. G.; Tan, J. W.; Zhou, M.; Du, X.; Zhang, Y. H.; Pu, J. X.; Sun, H. D. Org. Lett. 2016, 18, 2284−2287. (12) Calhoun, L. A.; Findlay, J. A.; Miller, J. D.; Whitney, N. Mycol. Res. 1992, 96, 281−286. (13) Salmoun, M.; Braekman, J. C.; Ranarivelo, Y.; Rasamoelisendra, R.; Ralambomanana, D.; Dewelle, J.; Darro, F.; Kiss, R. Nat. Prod. Res. 2007, 21, 111−120. (14) Granados, G. A.; Gutierrez, M. C.; Rivas, F.; Arias, J. M. Phytochemistry 2001, 58, 891−895. (15) Yurchenko, A. N.; Smetanina, O. F.; Kalinovskii, A. I.; Kirichuk, N. N.; Yurchenko, E. A.; Afiyatullov, S. S. Chem. Nat. Compd. 2013, 48, 996−998. (16) Qiao, M. F.; Ji, N. Y.; Miao, F. P.; Yin, X. L. Magn. Reson. Chem. 2011, 49, 366−369. (17) Lee, M.; Cho, J. Y.; Lee, Y. G.; Lee, H. J.; Lim, S.; So-Young Lee, S. Y.; Young-Do Nam, Y. D.; Moon, J. H. Food Sci. Biotechnol. 2016, 25, 1259−1264.

Table 5. Cytotoxicity of Compound 12 no.

Article

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01084. UV, IR, MS, 1D and 2D NMR spectra for compounds 1−9 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-83162679. Fax: +86-10-63017757. ORCID

Zhen Liu: 0000-0002-1763-4953 Yong Li: 0000-0003-1672-6589 Yun-bao Liu: 0000-0002-1338-0271 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by grants from the National Natural Science Foundation of China [No. 21572276], the CAMS Innovation Fund for Medical Sciences [No. 2016-I2M-1-010], and the Fundamental Research Funds for the Central Institutes (2018RC350007). I


Sesquiterpenes from an Endophytic Aspergillus flavus - Journal of

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