Stachybotrysins AâG, Phenylspirodrimane Derivatives from the

Article pubs.acs.org/jnp

Stachybotrysins A−G, Phenylspirodrimane Derivatives from the Fungus Stachybotrys chartarum Jinlian Zhao,† Jiamin Feng,† Zhen Tan,† Jimei Liu,† Jianyuan Zhao,‡ Ridao Chen,† Kebo Xie,† Dewu Zhang,‡ Yan Li,† Liyan Yu,‡ Xiaoguang Chen,† and Jungui Dai*,†,§ †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, §Key Laboratory of Biosynthesis of Natural Products of National Health and Family Planning Commission, Institute of Materia Medica, and ‡Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Seven new phenylspirodrimane derivatives named stachybotrysins A−G (2−8), together with five known compounds (1, 9−12), were isolated from Stachybotrys chartarum CGMCC 3.5365. Stachybotrysin D (5) is the first reported example of a naturally occurring alcoholic O-sulfation of a phenylspirodrimane, and stachybotrysins F and G (7 and 8) are the first examples possessing an isobenzotetrahydrofuran ring with an acetonyl moiety attached. The structures of these compounds were elucidated on the basis of extensive spectroscopic data analysis and by comparison with reported data. The absolute configurations of 1−8 were determined by X-ray single-crystal diffraction, electronic circular dichroism (ECD), and calculated ECD. Compounds 1 and 8 displayed anti-HIV activity with IC50 values of 15.6 and 18.1 μM, respectively, and 2, 7, 9, and 11 showed inhibitory effect on influenza A virus with IC50 values ranging from 12.4 to 18.9 μM. Stachybotrys is a genus of filamentous fungi comprising approximately 100 species and is widely distributed, generally found in soil and various decaying plant substrates.1 These fungi are known for the production of a variety of secondary metabolites including trichothecene mycotoxins,2,3 diterpenes,4 isochroman,5 cochlioquinones,6 and the prenylated phenol derivatives ranging from chartarutines,7 S. microspora triprenyl phenols,8 stachybotrins,9 kampanols,10 stachyflins,11 and bisabosquals12 to phenylspirodrimanes.13,14 These metabolites exhibit a wide range of bioactivities including the inhibition of pancreatic cholesterol esterase,15 anticomplement16 and antiviral activity,13,14 enhancers of fibrinolytic plasminogen,8 and inhibitors of TNF-α liberation and cytotoxicity.17 In the course of our search for new phenylspirodrimane derivatives with structural novelty and pharmacological potency, a 70 L fermentation of S. chartarum CGMCC 3.5365 from China General Microbiological Culture Collection Center (CGMCC) was performed. The HPLC-UV-guided fractionation and purification of the EtOAc extract led to the isolation of seven new phenylspirodrimane derivatives, stachybotrysins A−G (2− 8), along with five known compounds, F1839-I (1),15 stachybotrysin (9),17,18 Mer-NF5003E (10),19 stachybotrylactone acetate (11),2 and stachybotryslactone B (12).17,18 The structures of these compounds were determined by spectroscopic data analysis and comparison with reported data. Herein, we report the detailed isolation, structure elucidation, and biological activities of these compounds. © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was isolated as a white, amorphous powder and identified as known compound F1839-I on the basis of HRESIMS and NMR spectroscopic analysis.15 The X-ray single-crystal diffraction using the anomalous scattering of Cu Kα radiation confirmed its absolute configuration to be 3R, 5S, 8R, 9R, and 10S (Figure 1 and Figure S2). The electronic circular dichrosim (ECD) spectrum showed a positive Cotton effect (CE) at 218 nm and negative CEs at 279 and 339 nm (Figure S3), and this distinctive CE feature provided evidence to elucidate the absolute configuration of this class of compounds. Received: January 5, 2017 Published: May 22, 2017 1819

DOI: 10.1021/acs.jnatprod.7b00014 J. Nat. Prod. 2017, 80, 1819−1826

Journal of Natural Products

Article

Stachybotrysin B (3) was obtained as a white, amorphous powder. Its molecular formula was determined to be C25H34O6 by HRESIMS, requiring nine degrees of unsaturation. The 1H, 13 C, DEPT, and HSQC spectroscopic data (Table 1) were extremely similar to those of 2, except for the absence of a methyl group (δH 2.45/δC 21.5) and the appearance of an additional oxymethylene unit (δH 4.66/δC 64.3) in 3. This oxymethylene moiety was assigned as H2-7′ positioning at C-4′ by the HMBC correlations of H-3′/C-7′ and H2-7′/C-3′, C-4′, and C-5′ (Figure 2). Thus, the planar structure of 3 was elucidated. Its relative configuration was determined to be the same as that of 2 with 1D NOE correlations of H3-15/H3-14, H-11b, and H-8, H-3/H3-14, H3-12/H-11a, and H-5/H3-13 (Figure S22). The positive CE at 225 nm and negative CEs at 288 and 324 nm in its ECD spectrum (Figure S26) further established that 3 possesses the same 3R, 5S, 8R, 9R, and 10S configuration as 2. Stachybotrysin C (4) was obtained as a pale brown, amorphous powder. Its molecular formula was determined as C25H34O7 on the basis of HRESIMS, corresponding to nine degrees of unsaturation. The 1H, 13C, DEPT, and HSQC spectroscopic data (Table 1) were closely similar to those of 3, except that an extra oxymethine (δH 4.04, ddd, J = 12.1, 7.0, 2.5 Hz; δC 65.1) was observed in 4. It was assigned to be located at C-2 due to the 1H−1H COSY correlations of H-2/H2-1 and H2/H-3 as well as the HMBC correlations of H-2/C-3 and H-3/ C-2 (Figure 2). The small coupling constant of JH‑3/H‑2 (J = 2.5 Hz) indicated that H-3 and H-2 were on the same face of the molecule, and the 1D NOEs (H3-15/H3-14, H-11b, and H-2; H3-14/H-3 and H-2; H3-12/H-11a; H-5/H3-13, Figure S33) along with the ECD spectrum (negative CEs at 288 and 323 nm, Figure S37) suggested that H3-15, H-8, H2-11, H-3, and H2 were β-oriented, while H-5 and H3-12 were α-oriented. The 2α-OH or 2β-OH in 4 can also be determined by the rule of the γ-gauche effect. Because 4 has 15β-CH3 attached to C-10, the γ-gauche effect of 2β-OH should induce upfield shifts of C4 and C-10 compared with those of compound 3 (δC‑4 37.4 and δC‑10 42.4), but 4 actually displayed δC‑4 38.9 and δC‑10 44.4, suggesting the OH-2 was not β-oriented. Thus, the absolute configuration of 4 was identified as 2R, 3S, 5S, 8R, 9R, and 10S. Stachybotrysin D (5) was obtained as a yellowish, amorphous powder. It has a molecular formula of C23H32O8S as determined by HRESIMS, with eight degrees of unsaturation. The daughter ion peak at m/z 371.2210 [M − SO3 − H2O + H]+ suggested the existence of a sulfate moiety in 5. Its 1H, 13 C, DEPT, and HSQC spectroscopic data (Table 1) were quite similar to those of the known compound Mer-NF5003E (10).19 The difference was that the aromatic proton (H-3′) and the oxymethylene (H2-7′) were shifted downfield to δH 6.88 (from δH 6.62) and δH 5.30 (from δH 4.71), respectively. Two hydroxy signals (δH 9.66 and δH 3.25) were established with the aid of a D2O exchange experiment (Figures S37 and S38). The one at δH 3.25 was assigned as OH-3 due to the 1H−1H COSY interaction of OH-3/H-3 (Figure 2), while no 1H−1H COSY and HMBC correlations were detected for the one at δH 9.66; however the highly downfield shift implied its location at C-2′ compared with 10. These NMR data together with HRESIMS data indicated that the oxymethylene (H2-7′) was O-sulfated, and it was further proved by the downfield shift of C-7′ (δC 64.0 → δC 67.7) and the upfield shift of C-4′ (δC 147.0 → δC 142.5) (Table 1). This is the first reported example of a naturally occurring alcoholic O-sulfation of a phenylspirodrimane. The 1D NOE interactions (H3-15/H3-14, H-11b, and H-

Figure 1. Structure of 1 (F1839-I) resulting from single-crystal X-ray diffraction.

Stachybotrysin A (2) was obtained as a white, amorphous powder. It has a molecular formula of C25H34O5 as determined by HRESIMS, corresponding to nine degrees of unsaturation. The IR spectrum showed absorption bands for hydroxy (3262 cm−1) and carbonyl (1674 cm−1) groups. The UV spectrum displayed absorptions at 231, 284, and 328 nm. The 1H NMR and HSQC spectroscopic data (Table 1) displayed an aldehyde proton (δH 10.41, 1H, s, H-8′), an aromatic proton (δH 6.20, 1H, s, H-3′), an oxymethine proton (δH 4.56, 1H, t-like, J = 2.8 Hz, H-3), a pair of methylene protons (δH 3.15, 1H, d, J = 16.3 Hz, H-11b; δH 2.83, 1H, d, J = 16.3 Hz, H-11a), a methine proton (δH 2.23, 1H, dd, J = 12.3, 2.6 Hz, H-5), and six methyls (δH 2.45, 3H, s, H3-7′; δH 1.94, 3H, s, H3-2″; δH 1.08, 3H, s, H315; δH 0.96, 3H, s, H3-14; δH 0.91, 3H, s, H3-13; and δH 0.79, 3H, d, J = 6.4 Hz, H3-12). The 13C NMR and DEPT spectroscopic data (Table 1) indicated 25 carbon resonances, including six methyl carbons (δC 28.4, 22.1, 21.5, 21.0, 16.3, and 16.0), five methylene carbons (δC 32.0, 31.2, 25.6, 22.9, and 21.5), five methine carbons (δC 187.7, 112.0, 78.1, 41.9, and 37.5, including one aldehyde, one aromatic, and one oxygenated), one carbonyl carbon (δC 170.3), five nonprotonated aromatic carbons (δC 168.9, 159.0, 142.2, 111.8, and 111.7, including two oxygenated), one oxygenated tertiary carbon (δC 99.7), and two quaternary carbons (δC 43.2 and 37.4). The above data revealed that compound 2 has a typical phenylspirodrimane skeleton. However, the presence of an additional acetyl group (δC‑1″ 170.3, δC‑2″ 21.0/δH‑2″ 1.94) was observed in 2, which was further supported by the daughter ion peak at m/z 355.2262 [M − CH3COOH + H]+ in its HRESIMS spectrum. The downfield shift of H-3 (δH 3.17 → δH 4.56) as well as the HMBC correlation between H-3 and C-1″ (Figure 2) unambiguously established the acetyl group to be linked to C-3 through an ester bond. Thus, the planar structure of 2 was elucidated. The 1D NOE correlations of H3-15/H3-14, H-11b, and H-8, H-3/H3-14, H3-12/H-11a, and H-5/H3-13 (Figure 2 and Figure S11) indicated that H3-15, H-8, H2-11, and H-3 were syn-oriented, while H-5 and H3-12 were opposite, identical to those of 1. Moreover, the ECD spectrum of 2 displayed similar CEs (positive CE at 219 nm and negative CEs at 284 and 323 nm, Figure S15) to those of 1, and its optical rotation ([α]25D −98.5, c 0.52, MeOH) was also in accordance with that of 1 ([α]25D −96.0, c 0.40, MeOH), suggesting a 3R, 5S, 8R, 9R, and 10S configuration of 2. 1820



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Table 1. 1H and 13C NMR Data of Compounds 2−6 in Acetone-d6 2a position

δC, type

1

25.6, CH2

δH (J in Hz) 1.14, m

3a

22.9, CH2

3

78.1, CH

4 5

37.4, C 41.9, CH

6

21.5, CH2

7

32.0, CH2

8

37.5, CH

9 10 11

99.7, C 43.2, C 31.2, CH2

12

15.9, CH3

13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′

28.4, CH3 22.1, CH3 16.3, CH3 111.8, C 159.0, C 112.0, CH 146.8, C 111.7, C 168.9, C 21.5, CH3 187.4, CHO 170.3, C 21.0, CH3

1″ 2″

1.57, overlapped 1.90, overlapped 4.56, t-like (2.8) 2.23, dd (12.3, 2.6) 1.55, overlapped 1.56, overlapped 1.56, overlapped 1.63, overlapped 1.90, overlapped

2.83, d (16.3, Ha) 3.15, d (16.3, Hb) 0.79, d (6.4) 0.91, s 0.96, s 1.08, s

6.20, s

2.45, s 10.41, s

1.94, s

5b

6a

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

δH (J in Hz)

δC, type

25.6, CH2

1.08, overlapped 1.69, m

34.9, CH2

1.40, dd (12.1, 7.0) 1.74, t-like (12.1)

25.0, CH2

25.5, CH2

1.51, overlapped 1.87, overlapped 4.56, brs

65.1, CH

4.04, ddd (12.1, 7.0, 2.5)

26.0, CH2

1.06, overlapped 1.91, overlapped 1.51, overlapped 1.91, overlapped 3.32, brs

1.71, m 2

4b

23.0, CH2

78.1, CH 37.5, C 41.9, CH 21.5, CH2

2.21, dd (11.3, 2.8) 1.55, overlapped

80.8, CH

4.85, d (2.5)

38.9, C 41.4, CH 21.2, CH2

2.13, dd (12.1, 2.5) 1.50, overlapped

75.3, CH 38.3, C 41.9, CH 21.7, CH2

1.59, overlapped 32.0, CH2

37.5, CH 99.8, C 43.2, C 31.5, CH2

16.0, CH3 28.4, CH3 22.1, CH3 16.2, CH3 112.6, C 162.2, C 110.1, CH 146.5, C 109.8, C 169.6, C 64.3, CH2 188.1, CHO 170.3, C 21.0, CH3

1.54, overlapped 1.62, overlapped 1.87, overlapped

2.84, d (16.4, Ha) 3.15, d (16.4, Hb) 0.79, d (6.4) 0.90, s 0.95, s 1.05, s

6.56, s

4.66, s 10.28, s

1.93, s

32.0, CH2

1.55, overlapped

32.1, CH2

1.62, overlapped 37.2, CH

1.94, overlapped

100.0, C 44.4, C 31.3, CH2


2.89, d (16.4, Ha) 3.22, d (16.4, Hb) 0.80, d (6.6) 0.91, s 0.99, s 1.13, s

6.67, s

4.73, s 10.33, s

1.97, s

37.7, CH 100.2, C 43.2, C 31.3, CH2

16.0, CH3 29.1, CH3 22.8, CH3 16.4, CH3 112.3, C 160.0, C 107.7, CH 142.5, C 109.5, C 168.9, C 67.7, CH2 187.8, CHO

2.24, dd (12.0, 2.8) 1.51, overlapped 1.56, overlapped 1.51, overlapped 1.61, overlapped 1.92, overlapped

2.79, d (16.3, Ha) 3.14, d (16.3, Hb) 0.76, d (6.5) 0.98, s 0.87, s 1.05, s

6.88, s

5.30, s 10.26, s

δH (J in Hz) 1.12, overlapped 1.97, overlapped

26.5, CH2

1.59, overlapped 1.97, overlapped

75.2, CH

3.37, brs

38.6, C 40.9, CH

2.45, dd (13.2, 3.2)

23.5, CH2

1.48, qd (13.2, 4.5)

33.9, CH2

1.72, ddt (13.2, 4.5, 3.2) 2.36, ddd (13.2, 4.5, 2.4, Ha) 2.58, td (13.2, 4.5, Hb)

147.4, C 100.9, C 43.7, C 27.0, CH2

3.10, d (16.7, Ha) 3.34, d (16.7, Hb)


4.98, brs, Ha; 4.99, brs, Hb 1.02, s 0.88, s 0.91, s

6.53, s

5.37, s 10.10, s

2.07, s

a

Data were recorded at 400 MHz for proton and at 100 MHz for carbon in acetone-d6. bData were recorded at 600 MHz for proton and at 150 MHz for carbon in acetone-d6.

15; and δH 0.88, 3H, s, H3-14). The biggest difference was the methyl doublet in the upfield region was absent and an extra olefinic methylene (δH 4.99, brs/δH 4.98, brs, H2-12; δC 110.5) was present. This methylene was established to be located at C8 through a double bond with evidence from the 1H−1H COSY interaction of H 2 -12/H-7b (δ 2.58) and the HMBC correlations of H2-12/C-8, C-9, and C-7 as well as H2-7/C-8 and C-12 (Figure 2). In addition, an acetyl group (δC‑1″ 170.5, δC‑2″ 20.8/δH‑2″ 2.07) was recognized in 6, and the downfield shift of H2-7′ (δH 5.37) suggested the acetyl group was linked to C-7′ through an ester bond. This conclusion was confirmed with the HMBC correlation between H2-7′ and C-1″ (Figure 2). Moreover, the upfield shift of C-4′ from around δC 147.0 (in 3 and 4, Table 1) to δC 143.2 (in 6, Table 1), which was similar to that of compound 5 with the oxymethylene (H2-7′) sulfated,

8; H-3/H3-14; H3-12/H-11a; H-5/H3-13, Figure S45) in association with the similar CEs to those observed for 1 in its ECD spectrum (negative CEs at 279 and 339 nm, Figure S49) revealed the β-orientations of H3-15/H-8/H2-11/H-3 and α-orientations of H-5/H3-12. Thus, the stereochemistry of 5 was determined as 3R, 5S, 8R, 9R, and 10S. Stachybotrysin E (6) was obtained as a white, amorphous powder. Its molecular formula was identified as C25H32O6 according to HRESIMS, corresponding to 10 degrees of unsaturation. The 1H, 13C, DEPT, and HSQC spectroscopic data (Table 1) were similar to those of compound 5, revealing a phenylspirodrimane skeleton with typical proton signals (δH 6.53, 1H, s, H-3′; δH 3.37, 1H, brs, H-3; δH 3.34, 1H, d, J = 16.7 Hz, H-11b/δH 3.10, 1H, d, J = 16.7 Hz, H-11a; δH 2.45, 1H, dd, J = 13.2, 3.2 Hz, H-5; δH 1.02, 3H, s, H3-13; δH 0.91, 3H, s, H31821



Article

Figure 2. Key 1H−1H COSY (blue lines), HMBC (red →), and NOE (↔) correlations of 2−8.

theoretical ECD of four stereoisomers, (3R,5S,8R,9R,10S,8′R)I, (3R,5S,8R,9R,10S,8′S)-II, (3S,5R,8S,9S,10R,8′S)-III, and (3S,5R,8S,9S,10R,8′R)-IV, were calculated using the TD-DFT method at the B3LYP/6-31G(d) level, and the comparison of the calculated and experimental ECD curves (Figure 3A) identified a 3R, 5S, 8R, 9R, 10S, and 8′R configuration of 7. Stachybotrysin G (8) was obtained as a greenish, amorphous powder. Its molecular formula was determined to be the same as that of 7 (C26H36O5) based on HRESIMS, requiring nine degrees of unsaturation. Its 1D (Table 2) and 2D NMR data identified an identical planar structure to 7 with similar 1H/13C chemical shifts and the same 1 H− 1 H COSY/HMBC correlations. Only a small difference was observed in the 1H NMR data, which was that the chemical shifts of H2-9′ were δH 2.93 (1H, dd, J = 15.3, 4.4 Hz, H-9′b)/δH 2.73 (1H, dd, J = 15.3, 8.3 Hz, H-9′a) in 8, while they were δH 3.21 (1H, dd, J = 16.3, 3.4 Hz, H-9′b)/δH 2.72 (1H, dd, J = 16.3, 8.3 Hz, H-9′a) in 7. This change might be attributable to different configurations of C-8′. This conclusion was confirmed with its 1D NOE interactions (H3-15/H3-14, H-8, and H-11b; H-3/ H3-14; H3-13/H-5 and OH-3; H-11a and H-9′a/H3-12, Figure 2 and S78) and different ECD CE from that of 7 (a positive CE was observed at 276 nm in 7, while a negative CE was detected at 286 nm in 8, Figures S71 and S82). Based on a comparison of the theoretically calculated ECD curves of (3R,5S,8R,9R,10S,8′R)-I, (3R,5S,8R,9R,10S,8′S)-II, (3S,5R,8S,9S,10R,8′S)-III, and (3S,5R,8S,9S,10R,8′R)-IV with the experimental ECD curve of 8 (Figure 3B), the best match of the absolute configuration of 8 was 3R, 5S, 8R, 9R, 10S, and 8′S, which further proved that 8 is an 8′-epimer of 7. Metabolites 7 and 8 are the first examples of phenylspirodrimanes possessing an isobenzotetrahydrofuran ring with an acetonyl moiety attached. Compounds 1−12 are polyketide−terpenoid hybrid molecules. Compounds 1−6 and 10−12 are proposed to be derived from a common intermediate, ilicicolin B, that was proved at

further proved the linkage of the acetyl group. The 1D NOE interactions of H3-15/H3-14 and H2-11, H3-14/H-3, and H-5/ H3-13 (Figure S56) indicated that H3-15, H2-11, and H-3 were on the same face of the molecule, while H-5 was oppositely oriented. The closely similar NOE interactions of 6 to those of 2−5 suggested a 3R, 5S, 9R, and 10S configuration of 6. Stachybotrysin F (7) was obtained as a greenish, amorphous powder. It possesses a molecular formula of C26H36O5 as established by HRESIMS, corresponding to nine degrees of unsaturation. The 1H, 13C, DEPT, and HSQC spectroscopic data (Table 2) showed characteristic signals of a phenylspirodrimane scaffold. Apart from these, additional signals for an oxymethine (δH‑8′ 5.57/δC‑8′ 78.8), a methylene (δH‑9′ 3.21, 2.72/δC‑9′ 49.8), and a methyl (δH‑11′ 2.14/δC‑11′ 30.7) were observed. Among them, the methylene and methyl groups were assigned to an acetonyl moiety by the HMBC correlations from H3-11′/H2-9′ to C-10′ and C-9′ (Figure 2), and the existence of an acetonyl was also confirmed with the daughter ion peak at m/z 371.2210 [M − CH3COCH3 + H]+ in the HRESIMS spectrum. It was noted that the phenylspirodrimane skeleton along with acetonyl unit accounted for eight degrees of unsaturation, suggesting the presence of another ring system to correspond to nine degrees of unsaturation for 7. The downfield shifts of H2-7′ (from around δH 4.70 in 3 and 4 to δH 4.90/4.81 in 7) and C-7 (from around δC 64.0 to δC 73.3) as well as the HMBC correlations of H2-7′/C-3′, C-4′, C-5′, and C-8′ (Figure 2) indicated a furan ring to be fused to the phenyl at C-4′/C-5′. The acetonyl was connected to the furan ring through the C-8′/C-9′ single bond based on the 1H−1H COSY interactions of H-8′/H2-9′ and the HMBC correlations from H2-9′ to C-8′ and C-5′ (Figure 2). Thus, the planar structure of 7 was identified. The 1D NOE spectra displayed interactions of H3-15/H3-14 and H-11b, H-3/H3-14, H3-13/H-5 and OH-3, and H3-12/H-11a and H-8′ (Figures 2 and S67), suggesting the syn-orientation of H3-15/H-8/H2-11/H-3 and cis-orientation of H-5/H3-12. To elucidate the absolute configuration of 7, the 1822



Article

Table 2. 1H and 13C NMR Data of Compounds 7 and 8 in Acetone-d6a 7 position

δC, type

1

25.0, CH2

2

26.0, CH2

3 4 5 6

75.5, CH 38.3, C 40.7 CH 21.7, CH2

7

32.1, CH2

8 9 10 11

37.3, 98.6, 43.3, 32.0,

12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′

16.0, CH3 29.1, CH3 22.7, CH3 16.2, CH3 112.6, C 154.6, C 99.9, CH 142.3, C 113.7, C 156.4, C 73.2, CH2

8′ 9′

78.8, CH 49.8, CH2

10′ 11′

206.7, C 30.7, CH3

CH C C CH2

8

δH (J in Hz) 0.99, 1.85, 1.50, 1.93, 3.33,

overlapped overlapped overlapped m brs

2.16, dd (11.3, 3.1) 1.47, overlapped 1.53, overlapped 1.47, overlapped 1.57, overlapped 1.85, overlapped

2.81, 3.13, 0.75, 0.96, 0.86, 1.02,

d (16.3, Ha) d (16.3, Hb) d (6.5) s s s

6.19, s

4.81, d (12.0) 4.90, d (12.0) 5.57, m 2.72, dd (16.3, 8.3, Ha) 3.21, dd (16.3, 3.4, Hb) 2.15, s

δC, type 25.0, CH2 26.1, CH2 75.4, 38.3, 40.8, 21.7,

CH C CH CH2

32.3, CH2 38.0, 98.7, 43.0, 32.0,

CH C C CH2

16.1, CH3 29.0, CH3 22.9, CH3 16.5, CH3 112.8, C 154.6, C 100.0, CH 142.4, C 113.7, C 156.5, C 73.2, CH2 79.3, CH 49.9, CH2

206.2, C 30.9, CH3

δH (J in Hz) 1.03, 1.90, 1.51, 1.91, 3.31,

overlapped overlapped overlapped overlapped brs

2.14, dd (12.1, 2.6) 1.49, overlapped 1.51, overlapped 1.49, overlapped 1.55, overlapped 1.82, m

2.77, 3.15, 0.72, 0.96, 0.86, 1.02,

d (16.1, Ha) d (16.1, Hb) d (6.5) s s s

6.19, s

4.92, d (12.0) 4.82, d (12.0) 5.53, m 2.73, dd (15.3, 8.2, Ha) 2.93, dd (15.3, 4.4, Hb)

Figure 3. Calculated ECD spectra of I (3R, 5S, 8R, 9R, 10S, 8′R), II (3R, 5S, 8R, 9R, 10S, 8′S), III (3S, 5R, 8S, 9S, 10R, 8′S), and IV (3S, 5R, 8S, 9S, 10R, 8′R) and the experimental ECD spectra of 7 and 8.

the positive control, IC50 = 2.0 nM). Cytotoxicity results (Table S2) revealed that 1, 2, and 8 were weakly active against the HepG2 cell line, with IC50 values of 18.4, 24.7, and 24.6 μM, respectively (paclitaxel as the positive control, IC50 = 6.3 nM), and 1 was also weakly active against the NCI-H460 and BGC823 cell lines with IC50 values of 15.8 and 21.9 μM, respectively (paclitaxel as the positive control, IC50 = 1.0 and 0.8 nM, respectively).

2.21, s

a

Data were recorded at 600 MHz for proton and at 150 MHz for carbon in acetone-d6.

the molecular level to originate from orsellinic acid and farnesyldiphosphate (Figure S4A),20 whereas 7−9 were structurally differentiated with an extra C3 unit connected to the phenolic moiety, indicating that they might be derived from a non-orsellinic acid biosynthetic precursor. A plausible biogenetic pathway suggested that they shared a polyketone precursor (1a) originating from one acetyl-CoA starter and five malonyl-CoA extenders. Then 1a was farnesylated, oxidized, and decarboxylated to yield the intermediate 1b, followed by several steps of enzymatic cyclization to generate 9 and a subsequent modification of oxidation and nonstereoselective cyclization to further form 7 and 8 (Figure S4B). All compounds were evaluated for their antiviral activity against HIV-1 virus and influenza A virus (IAV), as well as cytotoxicity against five human tumor cell lines: HCT116, NCIH460, BGC823, Daoy, and HepG2. Compounds 1−3, 6, and 8 displayed low anti-HIV activity with IC50 values in the range 15.6−20.5 μM (Table S1: efavirenz as the positive control, IC50 = 4.0 nM), and 2, 7−9, and 11 showed an inhibitory effect on IAV with IC50 values in the range 12.4−23.4 μM (ribavirion as

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer model-343 digital polarimeter (PerkinElmer Inc., Waltham, MA, USA). The ECD and UV absorption spectra were recorded on a JASCO J-815 spectropolarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were acquired on a Nicolet 5700 FT-IR microscope spectrometer (FTIR Microscope Transmission, Thermo Electron Scientific Instrument Corp., Madison, WI, USA). 1D and 2D NMR spectra were obtained at 600 or 400 MHz for 1 H NMR and 150 or 100 MHz for 13C NMR on a VNOVA SYSTEM600 spectrometer (Varian Inc., Palo Alto, CA, USA) or Bruker AVIII 400 spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts (δ) are given in ppm, and coupling constants (J) are given in hertz (Hz). HRESIMS data were measured using an LTQ-FT Ultra ESI-FTICR-MS spectrometer (ThermoFisher Scientific, CA, USA). Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, PR China), Sephadex LH-20 gel (Amersham Biosciences, Uppsala, Sweden), and MCI gel (Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography (CC). 1823



Article

four fractions (Fr8.5.1−Fr8.5.4). Fr8.5.1 (102 mg) was further separated via RP semipreparative HPLC eluting with CH3OH−H2O (50:50, v/v) at 3 mL/min to yield 5 (13 mg, tR 15 min) and another peak (7.5 mg), which afforded 11 (4.0 mg, tR 24 min) through RP semipreparative HPLC eluting with CH3OH−H2O (70:30, v/v) at 3 mL/min. Stachybotrysin A (2): white, amorphous powder; [α]25D −98.5 (c 0.52, MeOH); UV (MeOH) λmax (log ε) 231 (3.10), 284 (3.07), and 328 (2.71) nm; ECD (MeOH) λmax (Δε) 219 (2.53), 284 (−2.62), and 322 (−1.80) nm; IR (λmax) 3262, 2963, 1674, 1591, 1457, 1375, 1283, and 1242 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 415.2473 [M + H]+ (calcd for C25H35O5, 415.2479), m/z 437.2289 [M + Na]+ (calcd for C25H34O5Na, 437.2299), and m/z 355.2262 [M − CH3COOH + H]+ (calcd for C23H31O3, 355.2268). Stachybotrysin B (3): white, amorphous powder; [α]25D −96.2 (c 0.52, MeOH); UV (MeOH) λmax (log ε) 212 (3.23), 230 (3.10), 286 (2.97), and 332 (2.81) nm; ECD (MeOH) λmax (Δε) 225 (2.02), 288 (−2.08), and 324 (−1.83) nm; IR (λmax) 3377, 2940, 1713, 1651, 1611, 1435, 1376, and 1252 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 453.2236 [M + Na]+ (calcd for C25H34O6Na, 453.2248) and m/z 413.2318 [M − H2O + H]+ (calcd for C25H33O5, 413.2323). Stachybotrysin C (4): pale brown, amorphous powder; [α]25D −68.9 (c 0.72, MeOH); UV (MeOH) λmax (log ε) 212 (3.19), 230 (3.01), 286 (2.86), and 331 (2.62) nm; ECD (MeOH) λmax (Δε) 288 (−1.71) and 323 (−1.72) nm; IR (λmax) 3390, 2942, 1716, 1652, 1612, 1435, 1376, and 1257 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 469.2186 [M + Na]+ (calcd for C25H34O7Na, 469.2197) and m/z 429.2271 [M − H2O + H]+ (calcd for C25H33O6, 413.2272). Stachybotrysin D (5): yellowish, amorphous powder; [α]25D −30.7 (c 0.60, MeOH); UV (MeOH) λmax (log ε) 230 (3.12), 283 (2.99), and 332 (2.66) nm; ECD (MeOH) λmax (Δε) 280 (−0.90) and 339 (−1.27) nm; IR (λmax) 3444, 2936, 1658, 1612, 1439, 1307, 1253, and 959 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 469.1880 [M + H]+ (calcd for C23H33O8S, 469.1891), m/z 451.1775 [M − H2O + H]+ (calcd for C23H31O7S, 451.1785), and m/z 371.2210 [M − SO3 − H2O + H]+ (calcd for C23H31O4, 371.2217). Stachybotrysin E (6): white, amorphous powder; [α]25D −47.1 (c 0.34, MeOH); UV (MeOH) λmax (log ε) 216 (3.20), 245 (2.96), and 304 (3.16) nm; ECD (MeOH) λmax (Δε) 208 (3.02), 238 (−3.25), and 297 (−1.40) nm; IR (λmax) 3550, 2951, 1738, 1642, 1444, 1338, 1265, and 964 cm−1; 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 429.2263 [M + H]+ (calcd for C25H33O6, 429.2272), m/z 411.2157 [M − H2O + H]+ (calcd for C25H31O5, 411.2166), and m/z 369.2049 [M − CH3COOH + H]+ (calcd for C23H29O4, 369.2060). Stachybotrysin F (7): greenish, amorphous powder; [α]25D +76.3 (c 0.32, MeOH); UV (MeOH) λmax (log ε) 212 (3.39) and 282 (2.11) nm; ECD (MeOH) λmax (Δε) 209 (2.67), 226 (−1.16), and 276 (1.12) nm; IR (λmax) 3392, 2934, 1707, 1624, 1389, 1356, 1258, and 1044 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z 451.2445 [M + Na]+ (calcd for C26H36O5Na, 451.2455) and m/z 371.2210 [M − CH3COCH3 + H]+ (calcd for C23H31O4, 371.2217). Stachybotrysin G (8): greenish, amorphous powder; [α]25D −90.9 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 212 (3.46) and 282 (2.17) nm; ECD (MeOH) λmax (Δε) 207 (8.43), 228 (−1.85), and 286 (−0.84) nm; IR (λmax) 3389, 2934, 1710, 1640, 1388, 1359, 1045, and 896 cm−1; 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z 451.2443 [M + Na]+ (calcd for C26H36O5Na, 451.2455) and m/z 371.2213 [M − CH3COCH3 + H]+ (calcd for C23H31O4, 371.2217). X-ray Crystallographic Analysis. Compound 1 was obtained as colorless transparent crystals from DMSO−acetone (1:1) using the vapor diffusion method. Crystal data were obtained on a Rigaku MicroMax 002+ single-crystal X-ray diffractometer with Cu Kα radiation using the ω and κ scan technique to a maximum 2θ value of

Semipreparative HPLC was performed on a Shimadzu HPLC instrument equipped with a Shimadzu RID-10A detector (Shimadzu Corporation, Tokyo, Japan) and a Grace Adsorbosphere C18 column (250 mm × 10 mm, i.d., 5 μm, W.R. Grace Co., Columbia, MD, USA) or a Grace Allsphere silica gel column (250 mm × 10 mm, i.d., 5 μm, W.R. Grace Co.). Analytical TLC was carried out on precoated silica gel GF254 plates (Qingdao Marine Chemical Industry, Qingdao, PR China), and spots were visualized under UV light or by spraying with 10% H2SO4 in 90% EtOH followed by heating at 120 °C. Fungal Material, Fermentation, Extraction, and Isolation. The fungal strain S. chartarum CGMCC 3.5365 was purchased from China General Microbiological Culture Collection Center (CGMCC, Beijing, PR China). It was maintained on slants of modified potato dextrose agar (PDA) medium (potato 200 g, glucose 20 g, distilled water 1 L, KH2PO4 3 g, MgSO4 0.75 g, vitamin B1 10 mg, agar 8.0 g, pH 6.0; the media were autoclaved at 115 °C for 30 min) at 4 °C. Seed cultures were performed in Erlenmeyer flasks (250 mL) containing 100 mL of PDA liquid medium on a shaker at 150 rpm at 25 °C for 2 days; after that 5 mL seed cultures were inoculated into each 1000 mL flask with 300 mL of medium and cultivated for 14 days (150 rpm, 25 °C). The cultures (70 L) were filtered under reduced pressure to afford the filtrate and mycelia. The filtrate was applied to an Amberlite XAD16 macroporous adsorbent resin column (Rohm & Haas Co., Philadelphia, PA, USA) and then eluted with H2O and 90% EtOH successively to give 25 g of residue under reduced pressure. The dried mycelia (490 g) were extracted with methanol by ultrasonic extraction to afford 202 g of residue. The two residues were partitioned successively with petroleum ether, EtOAc, and n-butanol to yield 11 and 126 g of EtOAc extracts, respectively. The two parts (137 g) were combined for further separation based on their identical HPLC and TLC profiles. It was initially separated on silica gel CC eluting with a petroleum ether−acetone gradient (100:0, 5:1, 3:1, 1:1, 1:3, 1:5, and 0:100) as well as 100% methanol to give nine fractions based on TLC analysis. Fraction 3 (1.93 g) was further separated on silica gel CC eluting with a detailed petroleum ether−acetone gradient (100:0, 20:1, 10:1, 8:1, 6:1, 4:1, 2:1, and 0:100) to obtain seven fractions (Fr3.1−Fr3.7). Fr3.3 (200 mg) gave 2 (5.4 mg, tR 34 min) and 6 (5.0 mg, tR 43 min) via RP semipreparative HPLC eluting with CH3OH−H2O (70:30, v/ v) at 3 mL/min. Fr3.4 (250 mg) afforded 1 (76.0 mg, tR 28 min) through RP semipreparative HPLC eluting with CH3OH−H2O (68:32, v/v) at 3 mL/min. Fr3.5 (154 mg) yielded 3 (31.2 mg, tR 29 min) by RP semipreparative HPLC eluting with CH3OH−H2O (56:44, v/v) at 3 mL/min. Fr3.6 (39 mg) gave 10 (6.1 mg, tR 21 min) via RP semipreparative HPLC eluting with CH3OH−H2O (53:47, v/ v) at 3 mL/min. Fraction 5 (21.5 g) was first subjected to silica gel CC eluting with a dichloromethane−MeOH gradient with 0.1% NH4OH (200:1, 100:0, 80:1, 50:1, 30:1, 20:1, 10:1, 1:1, and 0:100) to obtain eight fractions (Fr5.1−Fr5.8). Fr5.5 (4.4 g) was then further separated on MCI CC to generate seven fractions (Fr5.5.1−Fr5.5.7). Fr5.5.2 (154 mg) was then separated via normal-phase semipreparative HPLC eluting with n-hexane−EtOAc (1:2, v/v) at 4 mL/min to yield Fr5.5.2.1−Fr5.5.2.6. Fr5.5.2.1 (6.6 mg) gave 8 (1.1 mg, tR 36 min) by RP semipreparative HPLC eluting with CH3CN−H2O (45:55, v/v) at 4 mL/min. Fr5.5.2.3 (39 mg) afforded 12 (3.8 mg, tR 27 min) through normal-phase semipreparative HPLC eluting with n-hexane−EtOAc (1:1, v/v) at 4 mL/min. Fr5.5.2.5 (45 mg) yielded 4 (28.7 mg, tR 22 min) by RP semipreparative HPLC eluting with CH3OH−H2O (65:35, v/v) at 3 mL/min. Fr5.5.2.6 (7 mg) gave 9 (2.1 mg, tR 26 min) via RP semipreparative HPLC eluting with CH3CN−H2O (47:53, v/ v) at 3 mL/min. Fr5.5.3 (351 mg) was separated through normalphase semipreparative HPLC eluting with n-hexane−EtOAc (1:1, v/v) at 4 mL/min to yield seven fractions (Fr5.5.3.1−Fr5.5.3.7), and Fr5.5.3.2 (93 mg) was further subjected to RP semipreparative HPLC eluting with CH3CN−H2O (58:42, v/v) at 4 mL/min to yield 7 (4.5 mg, tR 17 min). Fraction 8 (5.49 g) was first subjected to Sephadex LH-20 CC to obtain six fractions (Fr8.1−Fr8.6). Fr8.5 (693 mg) was then separated on silica gel CC eluting with a dichloromethane− MeOH gradient with 0.1% NH4OH (10:1, 8:1, 6:1, and 3:1) to give 1824



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145.0°. The structure was solved by direct methods with SHELXS-97, and all non-hydrogen atoms were refined anisotropically using the least-squares method. All hydrogen atoms were positioned by geometric calculations and difference Fourier overlapping calculations. Crystal data of 1: C23H32O4 (M = 372.48); orthorhombic crystal (0.67 × 0.56 × 0.18 mm); space group P212121; unit cell dimensions a = 11.014(3) Å, b = 13.291(4) Å, c = 13.869(5) Å, V = 2030.3(11) Å3; Z = 4; ρcalcd = 1.219 g/cm3; a total of 3964 unique reflections [R(int) = 0.1579 (inf-0.9 Å)] was measured, of which 3463 were observed (|F|2 ≥ 2σ|F|2); the final refinement gave R1 = 0.0733, wR2 = 0.1881, and S = 1.082; Flack parameter = 0.0(2). Crystallographic data for the structure of 1 have been submitted to the Cambridge Crystallographic Data Centre as supplementary publication CCDC 1524534. ECD Calculations of 7 and 8. Since the relative configurations of 7 and 8 were established by 1D NOE spectra as syn-oritentaion of H315/H-8/H2-11/H-3 and cis-orientation of H-5/H3-12 with undetermined H-8′, there were four stereoisomers of 7 and 8: (3R,5S,8R,9R,10S,8′R)-I, (3R,5S,8R,9R,10S,8′S)-II, (3S,5R,8S,9S,10R,8′S)-III, and (3S,5R,8S,9S,10R,8′R)-IV. The resultant conformers were further optimized and checked as the true minima of the potential energy surface by the density functional theory method at the B3LYP/6-31G(d) level, and the 100 lowest electronic transitions were calculated. ECD spectra of different conformers were simulated using a Gaussian function with a half-bandwidth of 0.3 eV. The overall theoretical ECD spectra were given on the basis of the Boltzmann weighting of each conformer. The theoretically calculated ECD spectra of I−IV were compared with the experimental ECD data of 7 and 8, which led to the assignment of the absolute configurations of 7 and 8. Antiviral Activity Bioassay.21 Antiviral activity of the compounds against HIV and IAV was performed. 293T cells (2 × 105) were cotransfected with 0.6 μg of pNL-Luv-E−-Vpu− and 0.4 μg of pHIT/ G. After 48 h, the VSV-G pseudo-typed viral supernatant was harvested by filtration through a 0.45 μm filter, and the concentration of viral capsid protein was determined by p24 antigen capture ELISA (Biomerieux). SupT1 cells were exposed to VSV-G pseudo-typed virus (MOI = 1) at 37 °C for 48 h in the absence or presence of test compounds at different concentrations (efavirenz and ribavirion were used as positive controls for HIV and IAV, respectively). Each concentration of the compounds was tested in three parallels. The inhibitory rate was determined by using a firefly luciferase assay system (Promega). IC50 values were calculated using Microsoft Excel software. Cytotoxicity Bioassay.22 Cytotoxicity of the compounds against five human tumor cell lines (HCT116, NCI-H460, BGC823, Daoy, and HepG2) was measured using the MTT assay. The cells were maintained in a RRMI S7 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin, and 100 g/mL streptomycin. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2. Tumor cells were seeded in 96-well microliter plates at 1200 cells per well. After 24 h, compounds were added to the wells. After incubation for 96 h, cell viability was determined by measuring the metabolic conversion of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) into purple formazan crystals by viable cells. The MTT assay results were read using an MK3 Wellscan (Labsystem Dragon, Helsinki, Finland) plate reader at 570 nm. All compounds were tested at five concentrations (10−4, 10−5, 10−6, 10−7, and 10−8 M) in 100% DMSO with a final concentration of DMSO of 0.1% (v/v) in each well. Paclitaxel was used as a positive control. Each concentration of the compounds was tested in three parallels. IC50 values were calculated using Microsoft Excel software.

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12; 1D/2D NMR, HRESIMS, IR, UV, and ECD spectra for 2−8 (PDF) X-ray crystallographic data (CIF)

AUTHOR INFORMATION

Corresponding Author

*Tel: 86-10-63165195. Fax: 86-10-63017757. E-mail: jgdai@ imm.ac.cn. ORCID

Dewu Zhang: 0000-0001-6289-0617 Liyan Yu: 0000-0002-8861-9806 Jungui Dai: 0000-0003-2989-9016 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by CAMS Initiative for Innovative Medicine (CAMS-I2M-2-002) and the National Infrastructure of Microbial Resources (No. NIMR-2015-3).

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REFERENCES

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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.7b00014. Antiviral and cytotoxicity data for 1−12; ECD spectrum for 1; proposed biosynthetic pathway of compounds 1− 1825



Article

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