Ophiobolins from the Mangrove Fungus Aspergillus ustus - Journal of

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2018, 81, 2−9

Ophiobolins from the Mangrove Fungus Aspergillus ustus Tonghan Zhu,† Zhenyu Lu,† Jie Fan,† Liping Wang,†,‡ Guoliang Zhu,† Yi Wang,† Xia Li,# Kui Hong,§ Pawinee Piyachaturawat,⊥ Arthit Chairoungdua,⊥ and Weiming Zhu*,† †

Key Laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China ‡ Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, China § Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education of China, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China # School of Ocean, Shandong University, Weihai 264209, China ⊥ Department of Physiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand S Supporting Information *

ABSTRACT: Seven new ophiobolins (1−5, 12, and 14) along with the 11 known analogues (6−11, 13, 15−18) were isolated from the ethyl acetate extracts of the liquid and solid cultures of the mangrove fungus Aspergillus ustus 094102. The structures including the absolute configurations of the seven new compounds were elucidated by spectroscopic analysis, chemical methods, and quantum ECD calculations. Compounds 4−8 and 11−15 showed cytotoxicities against the G3K, MCF-7, MD-MBA-231, MCF/Adr, A549, and HL-60 human cancer cell lines with the IC50 values ranging from 0.6 to 9.5 μM.

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refermented in liquid and solid media. A chemical investigation led to seven new ophiobolins that we named as ophiobolins X (1) and Y (2), 21-dehydroophiobolin U (3), ophiobolin Z (4), 21-epi-ophiobolin Z (5), 21-epi-ophiobolin O (12), and 21deoxyophiobolin K (14), as well as 11 known analogues being identified. The known compounds included ophiobolin K (6),8,13 6-epi-ophiobolin K (7),8,13 ophiobolin G (8),10,13 6epi-ophiobolin G (9),13 ophiobolin P (10),4 21,21-O-dihydro6-epi-ophiobolin G (11),29 ophiobolin O (13),12 ophiobolin Q (15),4 ophiobolin H (16),13 5,6-di-epi-ophiobolin H (17),29 and ophiobolin U (18).11 Compounds 2−10 and 12−15 were from the liquid culture, while compounds 1, 11, and 16−18 were from solid cultures. Compounds 4−8 and 11−15 exhibited cytotoxicities against the G3K, MCF-7, MD-MBA231, MCF-7/Adr, A549, and HL-60 human cancer cell lines.

arine microorganisms, especially marine fungi, have long been regarded as a major source of new bioactive natural products (NPs).1−3 Ophiobolins are naturally occurring sesterterpenes within an extraordinary 5−8−5 fused-ring nucleus4 and display herbicidal,5,6 nematicidal,7,8 antifungal,9 and antibacterial activities,9−11 as well as cytotoxicity against cancer cells.4,12,13 Since the first ophiobolin A was disclosed as cochliobolin in 195714 and ophiobolin in 1958,15 48 naturally occurring ophiobolins have been reported; these are ophiobolins A−M6−10,14−22 and O−W4,11,12 (in which ophiobolin U was used to name two different structures23), 6-epi-ophiobolins A,24 C,13,18 G,13 and I−O,6−9,12,13,22 3,4anhydroophiobolin A,24 3,4-anhydro-6-epi-ophiobolin A,24,25 ophiobolin A lactone,25,26 3,4-anhydro-6-hydroxyophiobolin A,27 ophiobolin B lactone,9 3,4-anhydro-6-epi-ophiobolin B,28 21,21-O-dihydro-6-epi-ophiobolin G,29 18,19,21,21-O-tetrahydro-18,19-dihydroxy-6-epi-ophiobolin G,29 21-deoxy-6-epiophiobolin G,11 16,17-dihydro-21-deoxy-6-epi-ophiobolin G,11 5-O-methylophiobolin H,29 5,6-di-epi-ophiobolin H,29 5O-methyl-5,6-di-epi-ophiobolin H,29 25-hydroxyophiobolin I,21 and 8-deoxyophiobolin J.22 As part of our ongoing study on new bioactive NPs from marine fungi,30−34 the fermentation broth of the mangrove fungus Aspergillus ustus 094102 was re-evaluated. We had previously identified 14 new cytotoxic drimane sesquiterpenoids and meroterpenoids with a benzofuran nucleus.34 This fungus was also shown by HPLC-UV to produce ophiobolins in liquid fermentation (Figure S66). To further enrich the chemical diversity of ophiobolins, A. ustus 094102 was © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 displayed an HRESIMS peak at m/z 385.2734 [M + H]+ corresponding to the molecular formula C25H36O3, indicating 8 degrees of unsaturation. The IR spectrum suggested a hydroxy (3447 cm−1) and an ester or lactone carbonyl (1744 cm−1). The 1H and 13C NMR data (Tables 1 and 2) of 1 revealed five methyl groups including a doublet methyl and four singlets, five methylenes, 10 methines including four olefinic ones, and five nonprotonated carbons Received: April 17, 2017 Published: December 29, 2017 2

DOI: 10.1021/acs.jnatprod.7b00335 J. Nat. Prod. 2018, 81, 2−9

Journal of Natural Products

Article

Chart 1

Table 1. 1H NMR (600 MHz) Data for Compounds 1−5, 12, and 14 in CDCl3 1 position 1 2 3 4 5 6 8 9

2

δH (J in Hz)

δH (J in Hz)

1.39, m 1.63, dd (14.4, 3.7) 1.96, ddd (13.0, 10.0, 3.7)

1.65, dd (14.4, 7.7) 1.07, dd (14.4, 9.6) 2.14, ddd (9.6, 7.7, 4.3)

1.85, m 2.26, brd (15.0) 4.95, brdd (6.4, 6.4) 3.57, ddd (10.0, 6.4, 2.0) 6.91, ddd (8.5, 8.5, 2.0) 2.78, brdd (13.0, 8.5) 1.88, m

2.51, 1.66, 4.20, 2.45,

dd (15.1, 7.3) m m brd (4.3)

2.93, m 2.73, dd (18.6, 6.4) 2.18, dd (18.6, 1.7)

12

14

δH (J in Hz)

δH (J in Hz)

1.44, m 1.67, dd (13.3, 3.2) 1.94, ddd (13.1, 10.6, 3.2)

1.47, m 1.65, dd (13.2, 3.4) 1.70, ddd (11.8, 11.8, 3.4)

1.38, m 1.56, m 2.17, m

δH (J in Hz)

1.36, m 1.66, m 2.32, ddd (12.4, 9.9, 2.7)

δH (J in Hz)

2.24, d (14.0) 2.00, d (14.0)

2.26, d (14.3) 1.91, d (14.3)

2.28, d (13.5) 1.94, d (13.5)

2.37, d (19.1) 2.51, d (19.1)

3.13, dd (10.6, 1.7)

3.26, brd (11.8)

3.25, brd (11.0)

3.39, brd (9.9)

5.92, br. dd (8.4, 5.59, dd (8.2, 8.6) 8.4) 2.60, brdd (13.7, 2.37, m 8.7) 1.72, m 1.76, m

5.68, m

5.77, brs

2.18, m

1.90, m,

2.43, m

2.41, m

2.94, m

1.85, m

1.84, m

1.67, m

1.60, m

1.53, m

1.53, m 1.39, ddd (12.0, 12.0, 8.3) 1.89, m 1.66, m 2.10, m 2.62, m 5.19, dd (10.2, 10.2) 6.04, dd (11.5, 10.2) 5.92, d (11.5) 1.26, d (7.2) 9.47, s 0.92, s 0.90, d (6.7) 1.73, s 1.79, s

2.36, ddd (13.6, 9.9, 2.29, ddd (13.6, 2.7) 10.0, 2.7) 1.44, m 1.44, m 1.46, m 1.46, m

1.35, m, 1.41, m

1.40, m 1.39, m

1.64, 1.22, 1.85, 2.51, 5.16,

1.75, 1.54, 2.05, 2.66, 5.16,

1.56, 1.25, 2.03, 2.68, 5.18,

1.73, m

12

1.41, m 1.43, m

1.36, m 1.37, m

13 14 15 16

1.55, 1.79, 2.13, 2.70, 5.23,

1.53, 1.46, 2.22, 2.83, 5.22,

17

6.01, m

6.02, dd (11.6, 10.1)

18 20 21 22 23 24 25 26 27 3-OH

6.00, m 1.26, s

6.07, 1.27, 6.13, 0.96, 0.87, 1.74, 1.82, 3.25,

s d (6.7) s s

δH (J in Hz) 2.04, d (12.5) 2.60, d (12.5)

5

7.09, dd (8.2, 8.2)

10

0.99, 0.91, 1.74, 1.81,

4

3.40, dd (10.1, 6.1)

1.60, ddd (14.1, 10.1, 10.1) 1.32, m

m m m m dd (9.1, 9.1)

3

m m m m dd (10.1, 10.1)

d (11.6) s s s d (6.8) s s s

m m m m dd (9.5, 9.5)

1.63, 1.20, 1.85, 2.48, 5.11,

m m m m dd (9.9, 9.9)

m, m m m m

m m m m m

6.06, dd (11.6, 9.5)

6.03, dd (11.8, 9.9)

5.95, m

5.97, m

6.02, 1.26, 5.35, 0.91, 0.96, 1.74, 1.82, 3.28, 3.36,

5.97, 1.26, 5.33, 0.88, 0.95, 1.73, 1.79, 3.34, 3.45,

5.95, 1.23, 5.34, 0.89, 0.87, 1.71, 1.79, 3.35, 3.45, 2.39,

5.97, 1.41, 1.68, 0.93, 0.88, 1.73, 1.80,

d (11.6) s s s d (6.7) s s s s

d (11.8) s s s d (6.7) s s s s

m s s s d (6.7) s s s s s

m s s s d (6.7) s s

resembled those of ophiobolin K (6),8,13 with the exception that an aldehyde carbonyl signal (δC/H 196.2/9.22) and a

that were classified as a carbonyl, an oxygenated and two olefinic carbons, and a quaternary carbon. These data closely 3


Journal of Natural Products Table 2.

13

Article

C NMR (150 MHz) Data for Compounds 1−5, 12, and 14 in CDCl3 1

2

3

4

5

12

14

position

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

δC, type

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

35.1, CH2 51.8, CH 80.4, C 46.7, CH2 81.5, CH 44.5, CH 130.3, C 140.7, CH 25.3, CH2 54.4, CH 43.5, C 43.3, CH2 26.7, CH2 47.4, CH 35.5, CH 137.5, CH 122.0, CH 120.2, CH 135.4, C 25.9, CH3 171.7, C 18.3, CH3 20.3, CH3 18.1, CH3 26.5, CH3

38.0, CH2 36.9, CH 86.5, C 50.5, CH2 75.5, CH 43.9, CH 110.1, C 82.4, CH 30.0, CH2 47.9, CH 44.2, C 44.7, CH2 23.8, CH2 48.1, CH 33.0, CH 136.0, CH 122.1, CH 120.3, CH 135.3, C 21.1, CH3 141.7, CH 19.0, CH3 17.9, CH3 18.1, CH3 26.4, CH3 55.4, CH3

40.5, CH2 181.3, C 39.2, CH 43.5, CH2 205.7, C 135.9, C 136.5, C 158.0, CH 24.5, CH2 51.2, CH 41.7, C 40.7, CH2 27.5, CH2 45.6, CH 35.2, CH 136.9, CH 122.6, CH 119.9, CH 136.2, C 19.2, CH3 192.1, CH 19.2, CH3 20.2, CH3 18.1, CH3 26.5, CH3

42.3, CH2 51.9, CH 81.5, C 51.5, CH2 119.6, C 51.7, CH 136.1, C 127.5, CH 28.7, CH2 42.5, CH 44.0, C 44.8, CH2 27.8, CH2 52.1, CH 32.8, CH 136.3, CH 123.4, CH 120.4, CH 135.4, C 24.5, CH3 110.1, CH 23.5, CH3 21.3, CH3 18.2, CH3 26.5, CH3 49.8, CH3 54.7, CH3



34.5, CH2 50.2, CH 77.6, C 54.4, CH2 219.8, C 52.8, CH 129.6, C 132.3, CH 24.2, CH2 55.2, CH 43.5, C 42.9, CH2 26.6, CH2 47.0, CH 35.8, CH 137.6, CH 121.7, CH 120.2, CH 135.1, C 27.0, CH3 21.5, CH3 18.8, CH3 20.4, CH3 18.1, CH3 26.5, CH3

and of H3-22 (δH 0.99) with H-15 (δH 2.70) and H-6 (δH 3.57), indicating cis-configurations of these hydrogen atoms. In the NOE difference experiment of 1, H-2, H-5 (δH 4.95), and H3-22 were enhanced after irradiation of H-6 (δH 3.57), while H-10 (δH 1.73) was enhanced after irradiation of H-14 (δH 2.13). The NOE enhancements of H-2, H-6, and H-15 were also observed after H3-22 was irradiated, but H-10 and H-14 were not enhanced. These data indicated the cis-orientation of H-2, H-5, H-6, H3-20, and H3-22 and the trans-orientation of H3-22 with both H-10 and H-14, which are cis-fused A/B and trans-fused B/C rings. The relative shielded chemical shift of C-1 (δC 35.1) further supported the cis-fused A/B system.9,13 The NOESY correlation of H-15 (δH 2.70) to H-18 (δH 6.00) (Figures 2 and S8) along with the relatively small J16,17 value (9.1 Hz) supported the Z-configuration of the Δ16-double bond. The absolute configuration of 1 was determined by electronic circular dichroism (ECD) measurement and calculation. The ECD spectra of 1 and ent-1 were calculated using the time-dependent density functional theory (TD-DFT) at the B3LYP/6-31G(d) level.35,36 The measured ECD is consistent with the calculated ECD of 1 but opposite that of ent-1 (Figure 3), indicating a (2S,3R,5S,6S,10S,11R,14R,15S)configuration. Therefore, the structure of 1, namely, ophiobolin X, was identified as shown. The molecular formula of ophiobolin Y (2) was deduced as C26H40O3 on the basis of HRESIMS, indicating 8 degrees of unsaturation. The 1H, 13C, and DEPT NMR data (Tables 1 and 2, Figures S10−S12) were similar to those of 1. The major differences were the disappearance of the lactone carbonyl signal (δC 171.7) and the appearance of an oxygenated methine signal (δC/H 82.4/3.40) along with a methoxy signal

ketone carbonyl signal (δC 217.6) were absent and an additional lactone carbonyl (δC 171.7) and oxygenated methine (δC/H 81.5/4.95) signals were present. In addition, the obvious shielded shifts for C-4, C-6, C-7, and C-8 were observed (Tables 2 and S2), suggesting a five-membered lactone nucleus between rings A and B. The key HMBC correlations of H-5 (δH 4.95), H-6 (δH 3.57), and H-8 (δH 6.91) to C-21 (δC 171.7) as well as the COSY correlations of H-4 (δH 1.85)/H-5/H-6/H-2 (δH 1.96)/H-1 (δH 1.39 and 1.63) confirmed the structure (Figures 1, S6, and S7). The same relative configuration as 6 was deduced from the NOESY (Figures 2 and S8) and NOE difference (Figures 2 and S9) spectra. The NOESY data of 1 displayed connections of H-2 (δH 1.96) with H3-20 (δH 1.26) and H3-22 (δH 0.99)

Figure 1. Key COSY and HMBC correlations of compounds 1−5, 12, and 14. 4



Article

Figure 2. Key NOE correlations of compounds 1−5 and 12.

(2S,3R,5S,6S,8S,10S,11R,14R,15S) from the same sign of specific rotation and ECD Cotton effect around λmax 210 nm. Compound 3 showed an HRESIMS peak at m/z 367.2637 [M + H]+, indicating a molecular formula of C25H34O2 and 8 degrees of unsaturation. The similarity of 1H, 13C, and DEPT NMR data (Figures S18−S20) with ophiobolin U11 revealed 3 as an analogue of ophiobolin U (18). The difference is the substitution of the hydroxymethyl (δC/H 66.8/4.00) in ophiobolin U (18)11 by an aldehyde (δC/H 192.1/9.47) in 3. This deduction was further evidenced by the key HMBC correlations from H-21 (δH 9.47) to C-6 (δC 135.9) and C-8 (δC 158.0) (Figure S23). The NOESY spectrum (Figure S24) showed correlations of H3-22 (δH 0.92) to H-3 (δH 2.93) and H-15 (δH 2.62), H-10 (δH 1.67) to H-1a (δH 2.04), H3-20 (δH 1.26) to H-1b (δH 2.60), and H-15 to H-18 (δH 5.92), suggesting the same relative configuration and Z-configuration of the Δ16-double bond as in ophiobolin U. The same (3R,10S,11R,14R,15S) absolute configuration of 3 was assumed from the same biosynthetic origin and the same sign of specific rotation to ophiobolin U. Thus, compound 3 was identified as 21-dehydroophiobolin U. Compound 4, ophiobolin Z, was deduced to have the molecular formula C27H42O4 from the HRESIMS. The similarity of the NMR data to those of 1 suggested 4 as a derivative of 1. The obvious difference included the absence of the lactone carbonyl carbon (δC 171.7) and oxygenated methine (δH/C 4.95/81.5), but the addition of two methoxy signals (δH/C 3.28/49.8, 3.36/54.7), a ketal signal (δC 119.6), and an acetal signal (δH/C 5.35/110.1). These data possibly suggested that H-5 and the 21-carbonyl in 1 were substituted by the corresponding methoxy and methoxylated methine, respectively. This deduction was also supported by the key HMBC from the 21-methoxy proton (δH 3.36) to C-21 (δC 110.1) and the 5-methoxy proton (δH 3.28) to C-5 (δC 119.6), along with the correlations of H-21 (δH 5.35) to C-5, C-6 (δC 51.7), C-8 (δC 127.5), and the 21-methoxy carbon (δC 54.7), H-8 (δH 5.68) to C-6, H-6 (δH 3.13) to C-5, C-7 (δC 136.1),

Figure 3. Measured ECD curve of 1 and calculated ECD curves of 1 and ent-1.

(δC/H 55.4/3.25). In addition, the 5-methine signals and the 7nonprotonated carbon signal were obviously shielded. The key COSY correlation of the oxygenated methine proton signal (δH‑8 3.40) to H2-9 (δH 2.18/1.60) as well as the key HMBC correlations from the methoxy signal (δH 3.25) to C-8 (δC 82.4) and from the olefinic methine signal (δH‑21 6.13) to C-5 (δC 75.5), C-6 (δC 43.9), C-7 (δC 110.1), and C-8 were observed. These data suggested that the Δ7-double bond of 1 flipped to a Δ7,21-double bond in 2, and the additional methoxy-substituted methine group was at C-8. The similar NOESY coupled patterns (Figures 2 and S16) to 1 indicated the same relative configuration and Z-Δ16-double bond in 2. The cis-orientation of the CH3O-8 and H-5 can be deduced from a key NOESY correlation of H-5 (δH 4.20) to the 8methoxy proton (δH 3.25) as well as the NOE enhancement of CH3O-8 after irradiation of H-5 (Figures 2 and S17). Assuming the same biosynthetic origin, the absolute configuration of ophiobolin Y (2) could be deduced as 5



Article

Figure 4. ORTEP drawings of compounds 6 and 9 (Cu Kα).

and C-21, H-4a (δH 2.24) to C-3 (δC 81.5), C-5, and C-6 (δC 51.7), and H-4b (δH 2.00) to C-20 (δC 24.5) (Figure S30). The NOESY data (Figure S31) showed correlations of H-2 (δH 1.94) to H3-20 (δH 1.26) and H3-22 (δH 0.91), H-6 to H10 (δH 2.36) and H-21 (δH 5.35), H-4a (δH 2.24) to CH3O-5 (δH 3.28), and H-4b (δH 2.00) to CH3O-21 (δH 3.36). When H-6 and H-10 were irradiated, the proton signals of H-10, H21, and CH3O-5, and H-6 and H-14 were enhanced, respectively. The signals of H-2 and CH3O-21 were enhanced after irradiation of H3-22 (Figure S32). These data combined with there being no NOE effect between H-6 and H-2 (Figure S32) suggested both trans-fused A/B and B/C rings and the cis-orientation of H-2/H3-20/H3-22/CH3O-21 and CH3O-5/ H-6/H-10/H-14. The Δ16-double bond was assigned the Zconfiguration on the basis of the NOESY correlations of H-15 (δH 2.51) to H-18 (δH 6.02) as well as the relatively small J16,17 (9.5 Hz) value. Compound 5 also has the same molecular formula of C27H42O4 as compound 4 from the HRESIMS, indicating it is an isomer of 4. The 1H and 13C NMR data were almost the same as those of 4, except for the shielded shifts of the 2methine signal and the deshielded shifts of the 6-methine and the two methoxy signals, suggesting a stereoisomer of 4. The NOESY data showed correlations of H-2 (δH 1.70) to H3-20 (δH 1.26) and H3-22 (δH 0.88), H-6 (δH 3.26) to H-10 (δH 2.29), H-4a (δH 2.26), and CH3O-5 (δH 3.34), H-4b (δH 1.91) to H-2 (δH 1.70), and H-15 (δH 2.48) to H-18 (δH 5.97). In addition, NOESY correlations of H-21 (δH 5.33) to H-2 and H-4b were observed. These observations combined with NOE enhancement of H-6 and H-14 signals after irradiation of H-10 (Figure S40) indicated 5 as the 21-epimer of 4, namely, 21-epiophiobolin Z. The absolute configurations of 4 and 5 were determined by chemical transformation from 6-epi-ophiobolin K (7).8,13 After acidic methanolysis, 6-epi-ophiobolin K (7) was converted to compounds 4 and 5, implying the same configurations at C-2, C-3, C-6, C-10, C-11, C-14, and C-15. Thus, the absolute configurations of compounds 4 and 5 were deduced to be (2S,3R,5R,6R,10S,11R,14R,15S,21R) and (2S,3R,5R,6R,10S,11R,14R,15S,21S), respectively. Compound 12 showed an HRESIMS peak at m/z 453.2962 [M + Na]+, indicating the same molecular formula of C27H42O4 as compound 13; compound 12 is an isomer of 13. The 1H and 13C NMR data were similar to those of 13, except for the shielded shifts of the 2-methine and 4-methylene signal and the deshielded shifts of 6-methine and the 26methoxy signals, suggesting a stereoisomer of 13. The NOESY spectrum showed correlations of H-2 (δH 2.17) to H3-20 (δH 1.23) and H3-22 (δH 0.89) and of H3-22 (δH 0.89) to H-15 (δH 2.66) and H-6 (δH 3.25). These observations combined with the NOE enhancement of H-6 (δH 3.25) and CH3O-21 (δH 3.45) signals after irradiation of CH3O-5 (δH 3.35) and NOE enhancement of H-10 (δH 1.60) after irradiation of H-14 (δH

2.05) (Figure S48) indicated 12 as the 21-epimer of 13, namely, 21-epi-ophiobolin O (12). The absolute configuration of 12 was determined by chemical transformation from ophiobolin K (6).8,13 After acidic methanolysis, ophiobolin K (6) was converted to compounds 12 and 13, implying the same configurations at C-2, C-3, C-6, C-10, C-11, C-14, and C15. Thus, the absolute configuration of 12 was deduced to be (2S,3R,5S,6S,10S,11R,14R,15S,21R). Compound 14 showed an HRESIMS peak at m/z 371.2949 [M + H]+, indicating a molecular formula of C25H38O2 and 7 degrees of unsaturation. The 1H, 13C, and DEPT NMR data (Figures S49−S51) were very similar to those of 6, except for the appearance of additional olefinic methyl signals (δC/H 21.5/1.68) and the disappearance of an aldehyde signal (δC/H 196.2/9.22), suggesting that the 21-CHO of 6 might be changed to 21-CH3 with the same relative configuration. This deduction was further supported by key HMBC correlations from H3-21 (δH 1.68) to C-6 (δC 52.8) and C-7 (δC 129.6) (Figure S53). The relative shielded chemical shift of C-1 (δC 34.5) indicated the cis-fused A/B system.9,13 The same sign of specific rotation as 6 implied the same absolute configuration. Thus, compound 14 was identified as 21deoxyophiobolin K (14). By comparison of spectroscopic, ECD, and specific rotation data (Supporting Information) with those reported, the known ophiobolins 6−11, 13, and 15−18 were identified as ophiobolin K (6),8,13 6-epi-ophiobolin K (7),8,13 ophiobolin G (8),10,13 6-epi-ophiobolin G (9),13 ophiobolin P (10),4 21,21-O-dihydro-6-epi-ophiobolin G (11),29 ophiobolin O (13),12 ophiobolins Q (15),4 ophiobolin H (16),13 5,6-diepi-ophiobolin H (17),29 and ophiobolin U (18),11 respectively. For the first time the structures of ophiobolin K (6) and 6-epi-ophiobolin G (9) were confirmed by X-ray single-crystal diffraction with Cu Kα radiation. After oxidation by 2iodoxybenzoic acid, compound 11 was transformed to 9. Thus, compounds 11 and 9 possessed the same (2S,6R,10S,11R,14R,15S) configuration. The absolute configuration of compound 7 was confirmed by acidic dehydration of 7 to 9. Based on the above results, we further resolved the absolute configuration of the known 21,21-O-dihydro-6-epiophiobolin G (11). To further investigate the chemical diversity of the ophiobolins, A. ustus 094102 was fermented in liquid medium and solid medium. The solid cultivation produced similar ophiobolins to the liquid cultivation except for 1 and 16−18 (Figure S67). The liquid cultivation was carried out in static and shaking modes, and ophiobolins were only produced in the static cultivation. The liquid cultivation times were investigated under static conditions for 20, 33, 42, 51, 61, 74, and 100 d, respectively. The results showed that the cultivation time has little impact on the diversity of the ophiobolins in liquid static cultivation. 6



Article

Table 3. Cytotoxicities against Tumor Cells for 1−15, 17, and 18 (IC50, μM) G3K MD-MBA231 MCF-7 MCF-7/Adr A549 HL-60 MCF-10A

1

2

3

4

5

6

7

8

9

10

11

12

13

17

18

adriamycin

>20 >40

>20 >40

>20 39.8

19.1 18.5

>20 21.4

>20 5.9

6.8 4.1

9.5 14.1

16.8 11.6

>20 >40

1.9 >20

/b /

/ /

/ /

/ /

>20 >40

>20 >40

45.0a 3.0

>40 >40 >50 >50 /

>40 >40 / / /

>40 >40 / / /

13.2 5.4 / / >40

9.4 7.9 / / >40

5.1 4.1 / / 8.4

12.8 26.1 / / >40

22.9 34.6 / / >40

15.4 35.4 / / >40

>40 >40 / / /

>40 >40 >50 >50 /

/ / 0.6 0.8 /

/ / 2.4 1.7 /

/ / 15.1 7.0 /

/ / 33.8 7.2 /

>40 >40 / / /

>40 >40 / / /

0.1 2.9 1.3 0.02 0.2

14

15

a

Gemcitabin was the positive control for the G3K cancer cell line. b/ Cytotoxicities were not evaluated.

When evaluated by a paper disk-agar diffusion method,37 compounds 1, 4, 5, 7, and 9−13 were inactive against Candida albicans (ATCC 10231), Pseudomonas aeruginosa (ATCC10145), Escherichia coli (ATCC 11775), Enterobacter aerogenes (ATCC 13048), and Bacillus subtilis (ATCC 6051) (Figure S1). However, cytotoxicity was observed and evaluated against the human gemcitabine-resistant pancreatic cancer cell line (G3K), the breast adenocarcinoma cell line (MCF-7), triple-negative breast cancer cells (MD-MBA-231), the adriamycin-resistant human breast cancer cell line (MCF-7/ Adr), the nontumorigenic breast epithelial cell line (MCF10A), human non-small-cell lung cancer cells (A549), and human promyelocytic leukemia cells (HL-60) by the MTT method.38 The results showed that compounds 7, 8, and 11 were active against G3K, 6 and 7 against MD-MBA-231, 5 and 6 against MCF-7, 4−6 against MCF-7/Adr, 12 and 13 against A549, and 12−15 against the HL-60 cell line, respectively (Table 3). Compounds 7 and 11 were most active against the MD-MBA-231 and G3K cell lines with IC50 values of 4.1 and 1.9 μM, respectively, indicating the trans-fused α,β-unsaturated cyclopentanone with cyclooctenyl methanol is a key structural feature for cytotoxicity against the G3K cell line. Compound 6 was the most active against the MCF-7 and MCF/Adr cancer cell lines, with IC50 values of 5.1 and 4.1 μM, respectively. Except for 6, the compounds showed no toxicity against normal breast cells. The results suggested that the 2,5dimethoxyl-2H,3H,5H-furan moiety (4, 5) or H-6β and HO3 (6) are required for cytotoxicity against the MCF-7 and MCF-7/Adr cell lines, while HO-3 and the α,β-unsaturated aldehyde moiety (6, 7) are required for cytotoxicity against the MD-MBA-231 cell line, and the 2,5-dimethoxyl-2H,3H,5Hfuran moiety (12, 13) is required for cytotoxicity against the A549 and HL-60 cell lines. This is the first report on the cytotoxicities of ophiobolins 4−8 and 11 against the G3K, MD-MBA-231, and MCF-7/Adr cell lines, ophiobolin 5 against the MCF-7 cell line, and the ophiobolins 12 and 13 against the A549 and HL-60 cancer cell lines.

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Q-TOF LC/MS. Semipreparative HPLC was performed using an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5 μm, 4 mL/min) and a π NAP column (COSMOSIL-pack, 10 × 250 mm, 5 μm, 4 mL/ min). Column chromatography (CC), vacuum-liquid chromatography (VLC), and thin-layer chromatography (TLC) were performed on Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), silica gel H, and plates precoated with silica gel GF254 (Qingdao Marine Chemical Factory, Qingdao, China), respectively. Sea salt was from Weifang Haisheng Chemical Factory. Triethylamine, trifluoroacetic acid, 4-dimethylaminopyridine, and 2-iodoxybenzoic acid were from Tianjin Fuyu Fine Chemical Co., Ltd., Wujiang Chuangwei Chemical Co., Ltd., Sinopharm Chemical Reagent Co., Ltd., and J&K Scientific Ltd., respectively. Fungus. The mangrove fungal strain A. ustus 094102 was isolated from the rhizosphere soil of Bruguiera gymnorrhiza and was identified according to the morphological characteristics and the ITS sequences.34 Fermentation and Extraction. The fungus A. ustus 094102 was statically cultured at 25 °C for 28 days in one hundred 1000 mL conical flasks, each containing 300 mL of liquid medium that was composed of 20 g of maltose, 20 g of mannitol, 20 g of CaCO3, 10 g of glucose, 10 g of monosodium glutamate, 0.5 g of KH2PO4, 0.3 g of MgSO4·7H2O, 3 g of yeast extract, 1 g of corn steep liquor, 33 g of sea salt, and 1 L of tap water (pH 7.0). The whole fermentation broth (30 L) was filtered by cheesecloth to separate the mycelia from the filtrate. The mycelia were extracted three times by 80% volume aqueous acetone. The acetone solution was concentrated under reduced pressure to give an aqueous solution. The aqueous solution was extracted three times with an equivalent volume of EtOAc, while the filtrate was extracted three times with an equivalent volume of EtOAc. The combined EtOAc extracts were concentrated under vacuum to give crude gum 1 (40.0 g). Spores were also directly inoculated into eighty 500 mL conical flasks each containing 10 g of broken corn kernels, 1.5 g of yeast extract, 5 g of monosodium glutamate, 0.25 g of KH2PO4, 0.15 g of MgSO4, and 30 mL of tap water. The fungus A. ustus 094102 was grown under static conditions at 28 °C for 20 days. The solid-state fermented products were extracted three times with 3-fold volumes of EtOAc−MeOH (10:1, v/v). The extracted solution was concentrated under vacuum to give crude gum 2 (140.3 g). Purification. The gum 1 (40.0 g) was separated into seven fractions on a silica gel VLC column using stepwise gradient elution of CH2Cl2−petroleum ether (50−100%) followed by MeOH−CH2Cl2 (0−50%). Fraction 3 (5.2 g) was separated into four subfractions by VLC on RP-18 silica using stepwise gradient elution of 5−90% MeOH−H2O. Subfraction 3-2 (1.3 g) was fractionated into five subfractions on semipreparative HPLC (83% MeOH−H2O) with an ODS column. Subfraction 3-2-2 (230 mg) was further purified by semipreparative HPLC over a π NAP column (83% MeOH−H2O) to yield compounds 10 (8.8 mg, tR 18.9 min) and 7 (54.0 mg, tR 24.0 min). Subfraction 3-2-3 (61 mg) was purified by semipreparative HPLC over a π NAP column (84% MeOH−H2O) to yield compounds 6 (6.7 mg, tR 25.3 min) and 8 (5.3 mg, tR 31.4 min). Subfraction 3-2-4 (223 mg) was purified by semipreparative HPLC over a π NAP column (84% MeOH−H2O) to yield compounds 3 (4.2 mg, tR 33.1 min) and 9 (59.4 mg, tR 39.1 min). Subfraction 3-3

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were measured on a Waters 2487 dual λ absorbance detector. ECD spectra were collected using a JASCO J-715 spectropolarimeter. IR spectra were recorded on a Nicolet Nexus 470 spectrophotometer as KBr disks. 1H, 13C, DEPT, HMQC, HMBC, COSY, NOESY, and NOE difference NMR spectra were recorded on a JEOL JNM-ECP 600 spectrometer, a Bruker Avance 600 spectrometer, or an Agilent 500 MHz DD2 spectrometer. The chemical shifts were recorded as δ values using tetramethylsilane as an internal standard (1−6, 8, 10, 11, 13−18) or CDCl3 signals for reference (δH/C 7.26/77.16) (7, 9, 12). MS data were measured using a Q-TOF ULTIMA GLOBAL GAA076 LC mass spectrometer and Agilent Technologies 6530 Accurate-Mass 7



Article

Chemical Transformation of 7 to 4 and 5. To the solution of 7 (10 mg) in absolute MeOH (10 mL) were added dried molecular sieves (70 mg) and Dowex 50WX2 cation resin (50 mg) under stirring and a nitrogen atmosphere at room temperature (rt), and the resulting solution was stirred for 3 h. Then the reaction mixture was poured into H2O (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was then purified by semipreparative HPLC (ODS, 84% MeOH−H2O) to yield compounds 4 (1.3 mg, 11.6% yield) and 5 (1.9 mg, 17.0% yield). The two compounds were identified from the same MS, 1H NMR (Figure S62), optical rotation, and co-HPLC (Figure S61) with the natural ones. Chemical Transformation of 6 to 12 and 13. To the solution of 6 (12.4 mg) in absolute MeOH (10 mL) were added dried molecular sieves (80 mg) and Dowex 50WX2 cation resin (60 mg) under stirring and a nitrogen atmosphere at rt, and the resulting solution was stirred for 3 h. Then the reaction mixture was poured into H2O (100 mL) and extracted with CH2Cl2 (3 × 100 mL). The combined organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was then purified by semipreparative HPLC (π-NAP, 85% MeOH−H2O) to yield fraction 1 and fraction 2. Fraction 1 was then purified by semipreparative HPLC (ODS, 85% MeOH−H2O) to yield 12 (2.1 mg, tR 26.8 min, 15.1% yield). Fraction 2 was purified by semipreparative HPLC (ODS, 90% MeOH−H2O) to yield 13 (2.6 mg, tR 18.1 min, 18.7% yield). The two products were identified from the same MS, 1H NMR (Figure S63), optical rotation, and co-HPLC (Figure S61) as the natural ones. Chemical Transformation of 11 to 9. 2-Iodoxybenzoic acid (67 mg) was dissolved in dry DMSO (10 mL) and stirred under a nitrogen atmosphere at rt for 30 min. Compound 11 (30 mg) in CH2Cl2 (3 mL) was then added, and the resulting solution was continuously stirred for 20 min under the same conditions. The reaction mixture was then poured into CH2Cl2 (20 mL) and washed with saturated NaHCO3 solution followed by brine, and the CH2Cl2 phase was combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained residue was purified by a silica gel VLC column eluting with petroleum ether−EtOAc (v/v 4:1) to give compound 9 (18 mg, 60.3% yield), which was identified by the same MS, 1H NMR, and optical rotation as the natural one (Figure S64). Chemical Transformation of 7 to 9. 4-Dimethylaminopyridine (5 mg) and triethylamine (0.12 mL) were added to compound 7 (32 mg) in CH2Cl2 (10 mL), and the resulting solution was stirred under a nitrogen atmosphere at rt. Then trifluoroacetic acid (0.06 mL) was added drop by drop. The reaction mixture was stirred under the same conditions for another 10 min and then was poured into 60 mL of a saturated NaHCO3 solution at 0 °C to quench the reaction. The reaction mixture was extracted with EtOAc (3 × 50 mL). The organic phase was combined and washed with brine, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The obtained residue was purified by a silica gel VLC column eluting with petroleum ether−EtOAc (v/v 4:1) and further by Sephadex LH-20 eluting with MeOH−CH2Cl2 (v/v 1:1) to give compound 9 (11 mg, 36.1% yield), which was identified by co-HPLC and co-TLC with the natural one (Figure S65). X-ray Crystal Data for 6 and 9. Compounds 6 and 9 were obtained as colorless crystals from MeOH. Crystal data of 6 and 9 were obtained on a Bruker Smart CCD area detector diffractometer with graphite-monochromated Cu Kα radiation (λ = 1.541 78 Å). The structures were solved by direct methods (SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of full-matrix least-squares refinement for 6 was based on 3229 unique reflections (2θ < 50°) and 260 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0471, wR2 = 0.1161, and R = 0.0567 for I > 2σ(I) data. The final refinement for 9 was based on 2636 unique reflections (2θ < 50°) and 251 variable parameters and converged with unweighted and weighted agreement factors of R1 = 0.0564, wR2 = 0.1241, and R = 0.0931 for I > 2σ(I) data. Crystal 6 was orthorhombic with a molecular formula of

(186 mg) was further purified by semipreparative HPLC over an ODS column (84% MeOH−H2O) to yield compounds 11 (5.3 mg, tR 25.2 min), 4 (3.8 mg, tR 31.2 min), 5 (3.9 mg, tR 38.2 min), and 2 (3.0 mg, tR 42.3 min). Subfraction 3-4 (390 mg) was fractionated into five subfractions on semipreparative HPLC (83% MeOH−H2O) with an ODS column. Subfraction 3-4-2 (57 mg) was further purified by semipreparative HPLC over an ODS column (83% MeOH−H2O) to yield compound 15 (9.5 mg, tR 13.4 min). Subfraction 3-4-3 (39 mg) was further purified by semipreparative HPLC over an ODS column (85% MeOH−H2O) to yield compound 14 (6.7 mg, tR 24.8 min). Subfraction 3-4-4 (46 mg) was further purified by semipreparative HPLC over a π NAP column (85% MeOH−H2O) to yield compounds 12 (3.5 mg, tR 20.2 min) and 13 (3.6 mg, tR 30.0 min). The gum 2 (140.3 g) was separated into three fractions on a silica gel VLC column using stepwise gradient elution of CH2Cl2− petroleum ether (50−100%) followed by MeOH−CH2Cl2 (0− 50%). Fraction 2 (5.3 g) was separated into five subfractions on Sephadex LH-20, eluting with MeOH−CH2Cl2 (1:1, v/v). Subfraction 2-2 (30 mg) was further purified by semipreparative HPLC over a π NAP column (84% MeOH−H2O) to yield compound 16 (2.7 mg, tR 17.0 min). Subfraction 2-3 (40 mg) was further purified by semipreparative HPLC with an ODS column (85% MeOH−H2O) to give compound 18 (3.3 mg, tR 19.7 min). Subfraction 2-4 (2.1 g) was further purified by semipreparative HPLC with an ODS column (84% MeOH−H2O) to give compounds 1 (87.1 mg, tR 16.8 min), 11 (286.6 mg, tR 24.9 min), and 17 (93.2 mg, tR 26.9 min). Ophiobolin X (1): colorless solid; [α]20D +24 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 236 (4.48) nm; ECD (0.0017 M, MeOH) λmax (Δε) 209 (+0.6), 239 (−2.1), and 263 (+0.7) nm; IR (KBr) νmax 3447, 2962, 2928, 2871, 1744, 1682, 1651, 1455, 1376, 1220, 1187, 1107, 1030, 1010, 761 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 385.2734 [M + H]+ (calcd for C25H37O3, 385.2737). Ophiobolin Y (2): colorless solid; [α]20D +60 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 238 (4.27) nm; ECD (0.0025 M, MeOH) λmax (Δε) 212 (+4.8) nm; IR (KBr) νmax 3431, 2956, 2925, 2853, 1655, 1457, 1381, 1210, 1183, 1146, 1088 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 423.2861 [M + Na]+ (calcd for C26H40O3Na, 423.2870). 21-Dehydroophiobolin U (3): colorless oil; [α]20D +137 (c 0.5, MeOH); UV (MeOH) λmax (log ε) 238 (4.56) nm; ECD (0.0014 M, MeOH) λmax (Δε) 209 (−6.9), 245 (+23.5), and 313 (−3.4); IR (KBr) νmax 2962, 2929, 2874, 1705, 1650, 1621, 1456, 1376, 1276 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 367.2637 [M + H]+ (calcd for C25H35O2, 367.2632). Ophiobolin Z (4): colorless oil; [α]20D +72 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 235 (4.27) nm; ECD (0.0012 M, MeOH) λmax (Δε) 204 (−4.4) and 239 (+19.3) nm; IR (KBr) νmax 3484, 2957, 2917, 2858, 1744, 1685, 1644, 1452, 1376, 1314, 1182, 1112, 1078, 1013 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 453.2979 [M + Na]+ (calcd for C27H42O4Na, 453.2975). 21-epi-Ophiobolin Z (5): colorless oil; [α]20D +143 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 235 (4.27) nm; ECD (0.0012 M, MeOH) λmax (Δε) 202 (−5.5), and 240 (+21.4) nm; IR (KBr) νmax 3499, 2950, 2927, 2850, 1744, 1687, 1650, 1452, 1375, 1315, 1186, 1158, 1089, 1013, 977 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 453.2968 [M + Na]+ (calcd for C27H42O4Na, 453.2975). 21-epi-Ophiobolin O (12): colorless oil; [α]20D +23 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 235 (4.32) nm; ECD (0.0012 M, MeOH) λmax (Δε) 206 (+16.2), and 225 (−5.7) nm; IR (KBr) νmax 3481, 2960, 2927, 1457, 1376, 1332, 1314, 1294, 1260, 1103, 1087, 979 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 453.2962 [M + Na]+ (calcd for C27H42O4Na, 453.2975). 21-Deoxyophiobolin K (14): colorless solid; [α]20D +58 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (2.90) nm; IR (KBr) νmax 3434, 2928, 1735, 1630, 1457, 1376, 1235, 1202, 1157, 1142, 1126, 1102, 980 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/ z 371.2949 [M + H]+ (calcd for C25H39O2, 371.2945) and 393.2763 [M + Na]+ (calcd for C25H38O2Na, 393.2764). 8



Article

C25H36O3, space group P2(1)2(1)2(1) with a = 11.8802(5) Å, b = 13.0342(6) Å, c = 14.3874(6) Å, V = 2227.87(17) Å3, Z = 45, Dcalcd = 1.146 mg/m3, μ = 0.572 mm−1, and F(000) = 840. Crystal size: 0.43 × 0.38 × 0.35 mm3, T = 293(2) K. Absolute structure parameter: 0.0(4). A total of 4827 unique reflections (2θ < 50°) were collected. Crystal 9 was a monoclinic with the molecular formula C25H34O2, space group P2(1) with a = 10.4439(12) Å, b = 8.0181(11) Å, c = 13.5427(17) Å, V = 1127.0(2) Å3, Z = 2, Dcalcd = 1.080 mg/m3, μ = 0.511 mm−1, and F(000) = 400. Crystal size: 0.40 × 0.18× 0.08 mm3, T = 293(2) K. Absolute structure parameter: 0.0(7). A total of 3504 unique reflections (2θ < 50°) were collected. These data have been deposited in the Cambridge Crystallographic Data Centre as supplementary publication nos. CCDC 1037864 and 1037865. Copies of these data can be obtained free of charge, on application to the CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax +44 (0)-1223-336033; e-mail [email protected]).

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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.7b00335. Bioassay protocols used and the NMR spectra of compounds 1−11 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-532-82031268. Fax: +86-532-82031268. E-mail: [email protected]. ORCID

Pawinee Piyachaturawat: 0000-0002-1677-0777 Weiming Zhu: 0000-0002-7591-3264 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by grants from the NSFC (Nos. 81561148012, U1501221, 41376148, and 81373298) and Qingdao Postdoctoral Applied Research Projects (No. 861605040089).

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REFERENCES

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