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Article Cite This: J. Nat. Prod. 2018, 81, 1578−1587

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Cytochalasans Produced by the Coculture of Aspergillus f lavipes and Chaetomium globosum Wenjing Wang,† Jiaojiao Gong,† Xiaorui Liu,† Chong Dai,† Yanyan Wang,‡ Xiao-Nian Li,§ Jianping Wang,† Zengwei Luo,† Yuan Zhou,† Yongbo Xue,† Hucheng Zhu,† Chunmei Chen,*,† and Yonghui Zhang*,†

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†

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ First College of Clinical Medical Science, China Three Gorges University & Yichang Central People’s Hospital, Yichang 443003, People’s Republic of China § State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People’s Republic of China S Supporting Information *

ABSTRACT: The cocultivation of Aspergillus f lavipes and Chaetomium globosum, rich sources of cytochalasans, on solid rice medium, resulted in the production of 13 new, highly oxygenated cytochalasans, aspochalasinols A−D (1−4) and oxichaetoglobosins A−I (5−13), as well as seven known compounds (14−20). Of these compounds, 13 is a novel cytochalasan with an unexpected 2-norindole group. The isolated compounds were characterized by NMR spectroscopy, single-crystal X-ray crystallography, and ECD experiments. Compounds 1−4 represent the first examples of Asp-type cytochalasans with C-12 hydroxy groups, which may be a result of the coculture, as hydroxylated Me-12 groups are frequently found in Chae-type cytochalasans from C. globosum. In addition, 5−10 are unusual cytochalasans with an oxygenated C-10. Interestingly, 13 is the first example of a naturally occurring cytochalasan possessing a uniquely degraded indole ring that is derived from chaetoglobosin W, with 11 and 12 both serving as its biosynthetic intermediates. In the coculture of A. f lavipes and C. globosum, most of these cytochalasans are more functionalized than normal cytochalasans, and the underlying causes may attract substantial attention from synthetic biologists. The cytotoxicities against five human cancer cell lines (SW480, HL-60, A549, MCF-7, and SMMC-7721) and the immunomodulatory activities of these new compounds were evaluated in vitro.

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The notably different colors of the coculture and the axenically grown controls of these two fungi on the PDA plate (Figure S1) suggested that under coculture conditions some silent biosynthetic pathways were triggered and generated different secondary metabolites or altered the accumulation of common metabolites. To support this hypothesis, extracts prepared from a 21 d cocultivation of A. f lavipes with C. globosum and axenic condition controls on solid rice medium were subjected to HPLC-DAD analyses (Figure 1). Significantly, we found a decrease in the accumulation of chaetoglobosin F (14) by as much as 38-fold;23 up to 25-fold increases in the contents of chaetoglobosin D (15)24 and chaetoglobosin C (17);24 no obvious change in the accumulation of armochaetoglobosin I (16)9 in the cocultivation extract compared to axenic culture of C. globosum; an up to 15-fold decrease in the content of epicocconigrone (19);25 and an up to 20-fold increase in the accumulation of epicoccine (20)26 in the extract of the coculture

ytochalasans are composed of polyketides and amino acids and are well known for having a wide variety of biological activities,1,2 showing cytotoxic,3 antifouling,4 nematicidal,5 and anti-inflammatory properties.6 To date, over 300 cytochalasans with diverse structures and bioactivities have been isolated and characterized.7 In our previous studies on this class of compounds, a series of bioactive cytochalasans with novel skeletons were isolated from Aspergillus f lavipes and Chaetomium globosum; armochaeglobine A and asperchalasine A are representatives of these compounds.8−14 These results in conjunction with literature data demonstrated that these two fungi are rich sources of cytochalasans, and they produce these compounds in a reliable manner. However, many biosynthetic genes of fungi remain silent or less active in laboratory cultivations.15 Inspired by the success in obtaining unexpected bioactive secondary metabolites from cocultivations,16−22 we applied this methodology to A. f lavipes and C. globosum to determine if there are silent genes that could be activated to produce novel cytochalasans. © 2018 American Chemical Society and American Society of Pharmacognosy

Received: February 2, 2018 Published: July 3, 2018 1578

DOI: 10.1021/acs.jnatprod.8b00110 J. Nat. Prod. 2018, 81, 1578−1587

Journal of Natural Products

Article

Figure 1. HPLC chromatograms of the EtOAc extracts from coculture experiments monitored at a wavelength of 210 nm.

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RESULTS AND DISCUSSION Aspochalasinol A (1) was isolated as colorless crystals. The molecular formula C24H35NO5, with eight degrees of unsaturation, was deduced by HRESIMS spectrum. The IR spectrum of 1 showed a strong carbonyl absorption band at 1690 cm−1 as well as an O−H stretching band at 3380 cm−1. The 1H NMR data (Table 1) of 1 showed the presence of two olefinic protons at δH 6.17 (1H, d, J = 10.9 Hz) and 5.55 (1H, brs), three oxygenated protons at δH 3.79 (1H, dd, J = 12.0, 4.3 Hz) and 4.06 (2H, s), and four methyl singlets at δH 1.56, 1.31, 0.89, and 0.89. The 13C NMR and DEPT data (Table 3) showed 24 carbon resonances, comprising three carbonyl carbons [δC 176.2 (C-1), 215.2 (C17), and 212.2 (C-21)], four olefinic carbons [δC 144.6 (C-6), 128.3 (C-7), 126.2 (C-13), and 137.2 (C-14)], four methyl groups [δC 12.8 (C-11), 23.9 (C-23), 22.1 (C-24), and 15.5 (C25)], six methylenes including an oxygenated carbon [δC 49.7 (C-10), 63.3 (C-12), 37.1 (C-15), 33.5 (C-16), 27.2 (C-19), and 35.0 (C-20)], six methines including an oxygenated carbon [δC 52.2 (C-3), 51.7 (C-4), 35.6 (C-5), 44.2 (C-8), 78.8 (C-18), and 25.8 (C-22)], and one quaternary carbon [δC 69.0 (C-9)]. These data indicated that 1 was an analogue of aspochalasin M.30 Other than the additional hydroxy moiety at Me-12, the planar structure of 1 was ultimately determined to be the same as that of aspochalasin M by analysis of its NMR spectra including HSQC, 1H−1H COSY, and HMBC data (Figure 2). The relative configuration of compound 1 was established by a NOESY experiment (Figure 2). The NOESY correlations of H-3/Me-11, H-5/H-8, and H-8/Me-25 indicated the core structure of 1 is in an identical configuration to that of aspochalasin M. In addition, the NOESY correlations of H-13/H-20b and H-18/H-20b suggested an α-orientation for H-18, which allowed us to determine the relative configuration of 1. A single-crystal X-ray diffraction pattern of 1 was obtained using Cu Kα radiation (Figure 3), and this pattern gave the absolute configuration of 1 to be 3S,4R,5S,8S,9S,18S. Aspochalasinol B (2) was also isolated as a pale,yellow, amorphous solid. Its molecular formula was determined to be C24H35NO5, which is the same as that of 1, based on its HRESIMS data. Comparison of the 13C NMR data of 2 (Table 3) with those of 1 showed that the resonances of C-16 and C-20 of 2 were shifted downfield by ΔδC 3.3 and 1.3 ppm, respectively, and the signals of C-17, C-18, and C-19 were shifted upfield by ΔδC 2.0, 2.4, and 1.7 ppm, respectively, which

compared to what was seen in the axenic culture of A. f lavipes. In addition, some unexpected peaks were observed at retention times between 22 and 32 min; the corresponding compounds were then isolated and identified as 13 new cytochalasans including aspochalasinols A−D (1−4) and oxichaetoglobosins A−I (5−13) together with the known compound chaetomugilide B (18).27 Compounds 1−4 represent the first examples of Asp-type cytochalasans possessing hydroxylated Me-12 groups, which may be a result of the coculture, as hydroxy groups at the Me-12 are often found in Chae-type cytochalasans from C. globosum.8,9,28,29 In addition, compounds 5−10 are unusual cytochalasans with oxygenated C-10. Moreover, compound 13 is the first example of a naturally occurring cytochalasan possessing a uniquely degraded indole ring that is derived from chaetoglobosin W with 11 and 12 both serving as biosynthetic intermediates (Scheme 1). In the coculture of A. f lavipes and C. Scheme 1. Plausible Biogenetic Pathway of 13

globosum, most of these cytochalasans are heavily modified compared with normal cytochalasans, and the underlying causes may attract substantial attention from synthetic biologists. As shown in Figure S2, chaetomugilide B (18) appears deep red, and the accumulation of 18 might be responsible for this color change under cocultivation conditions (Figure S1). Herein, the detailed comparison of the HPLC chromatograms of the coculture and axenic cultures and the isolation and structural elucidation of these new compounds (1−13) in addition to their cytotoxic and immunomodulatory activities are described. 1579



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Chart 1

biosynthetic reports for chaetoglobosin A,2,32 we propose that these isomers were possibly generated by oxygenase activity from a common intermediate. Oxichaetoglobosin A (5) was isolated as a white, amorphous powder, and its molecular formula was established to be C32H38N2O6, which indicates 14 degrees of unsaturation based on its HRESIMS spectrum. The IR spectrum of 5 showed an absorption band at 3446 cm−1 assignable to a hydroxy group and a strong carbonyl absorbance at 1689 cm−1. The 1H NMR data of 5 (Table 2) showed signals corresponding to four methyl groups at δH 1.79, 1.14, 1.06, and 0.41 and eight olefinic or aromatic protons including five aromatic protons at δH 7.50 (1H, d, J = 6.8 Hz), 7.48 (1H, d, J = 6.7 Hz), 7.30 (1H, s), 7.24 (1H, t, J = 7.6 Hz), and 7.12 (1H, t, J = 7.5 Hz), which were attributed to a 3-substituted indolyl group.1,9 Analysis of the 13C NMR (Table 3) data of 5 revealed 32 carbon signals, including three carbonyl groups [δC 176.5 (C-1), 203.6 (C-19), and 209.2 (C-23)], 12 olefinic or aromatic carbons, one sp3 quaternary carbon [64.8 (C-9)], three sp3 methylene groups, and eight sp3 methine groups including three oxygenated carbons [δC 62.0 (C-7), 71.0 (C-10), and 72.2 (C-20)]. These data suggested that compound 5 is likely a Chae-type cytochalasan.8,9 Further analyses of the 1 H−1H COSY and HMBC data (Figure 2) of 5 revealed that its planar structure was similar to that of chaetoglobosin F33 with the exception of the C-10 position in 5 being oxygenated. Hence, the planar structure of 5 was established as shown in Figure 2. The relative configuration of 5 was established by evaluation of its NOESY spectrum. Interactions of H-3/Me-11, H-3/Me12, H-7/H-13, H-13/H-15b, H-15b/H-17, and H-17/H-19

suggested that the configuration of C-18 in 2 may be opposite that of 1. The configuration of H-18 was further confirmed by the NOESY correlation of H-18/Me-25. The relative configurations of the remaining chiral centers of 2 were consistent with those of 1 based on their NOESY spectra (Figure S20). Compound 2 was ultimately determined to be the C-18 epimer of 1 based on the similarities in their ECD (electronic circular dichroism) curves (Figure 4) and optical rotation values. Aspochalasinols C (3) and D (4) shared the same molecular formula (C24H35NO5) as compound 1. The 1H and 13C NMR data (Tables 1 and 3) of 3 and 4 closely resembled those of 1 and 2, indicating that they are structurally similar. 1H−1H COSY correlations of H-20/H-19 and H-17/H-16/H-15 together with the chemical shifts of the C-17 (δC 74.6 and 77.0) and C-18 (δC 209.5 and 215.6) for 3 and 4 placed the carbonyl group at C-18 and the hydroxy group at C-17. The relative configurations of C17 in 3 and 4 were established by analyses of their NOESY spectra (Figures S29 and S38) as well as a comparison of their chemical shifts and coupling constants with those of 1. The NOESY correlation of H-13/H-17 in 3 suggested H-17 of 3 was α-oriented, and the correlations of H-13/H-19a and H-17/H19b in 4 suggested H-17 of 4 was β-oriented. Thus, the 17epimeric relationship between 3 and 4 was unambiguously defined. The experimental ECD spectra of 3 and 4 were almost the same as that of 1 (Figure 4), thus confirming the absolute configurations of 3 and 4 as shown. Compounds 1 and 2 are C-18 epimers, while compounds 3 and 4 are C-17 epimers, and these compounds are isomers between each other, which frequently occurs in the cytochalasans, as illustrated by aspochalasins M, P, S, and T.30,31 Based on 1580



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contain an additional terminal double bond (7: δC 150.0, 114.1, δH 5.16, s, 4.93, s; 8: δC 150.8, 114.2, δH 5.13, s, 4.97, s) and a methoxy group (7: δC 57.2, δH 3.24, s; 8: δC 57.0, δH 3.19, s) that were not present in 5. Further analysis of their HMBC data placed the methoxy group at C-10 and the terminal double bond between C-6 and C-12 from the correlations of Me-26/C-10, H12/C-5, and H-12/C-7. The relative configurations of 7 and 8 were consistent with those of 5, and the configurations of H-7 in 7 and 8 were determined based on their NOESY data. Therefore, compounds 7 and 8 were a pair of C-10 epimers. However, similar to the challenges faced with 5 and 6, their relative configurations at C-10 could not be determined from their NOESY spectra, and the absolute configuration of each compound was elucidated from their ECD spectra (Figure 5). Oxichaetoglobosins E (9) and F (10) have the same molecular formula (C33H40N2O6) as that of 7 and 8 based on their HRESIMS data. In addition, the 1H and 13C NMR data of 9 and 10 were similar to those of 7 and 8 except for the absence of the terminal double bond in 9 and 10. Analysis of their HMBC data revealed that the terminal double bonds in 7 and 8 were replaced by endocyclic double bonds between C-5 and C-6 in both 9 and 10. The relative configurations of all the chiral centers of 9 and 10, except for C-10, were established to be the same as those of 7. Thus, compounds 9 and 10 were a pair of C10 epimers. The absolute configuration of each of these two compounds was elucidated by comparing their ECD spectra with those of 7 and 8 (Figure 5). Oxichaetoglobosins G (11) and H (12) were isolated as pale yellow, amorphous solid. Their molecular formulas (C32H38N2O7), which suggested 14 degrees of unsaturation, were determined from their HRESIMS data. Comparison of the 1 H and 13C NMR data of 11 and 12 (Tables 2 and 3) with those of compounds 5−10 indicated that 11 and 12 are highly oxidized cytochalasans from the chaetoglobosin family. Detailed analyses of their 1H−1H COSY and HMBC spectra revealed that their structures were similar to chaetoglobosin W34 with the exception of an epoxide from C-3 to C-6. The only difference between 11 and 12 and chaetoglobosin W was the presence of an additional epoxide between C-2′ and C-4′ in the former, which was supported by HMBC correlations from H-2′ to C-1′ and C3′a and from H-10 to C-2′, C-3′, and C-3′a. Based on their NOESY spectra, the relative configurations of the cores of compounds 11 and 12 were the same as chaetoglobosin W. The difference between compounds 11 and 12 was the stereochemistry of the C-2′/C-3′-epoxide. Unfortunately, because of the lack of solid evidence such as a single-crystal X-ray diffraction pattern, the configuration of the epoxides could not be determined. According to our previous research and because chaetoglobosins share the same biosynthetic pathway,1,2,32 the absolute configurations of 11 and 12 were preliminarily assigned as 3R,4R,5S,6S,7S,8R,9S,16S,20S. Oxichaetoglobosin I (13) has a molecular formula of C31H38N2O7, which contains one carbon atom fewer than regular Chae-type cytochalasans. In contrast to the 1H and 13C NMR spectra of 5−12, which contain resonances characteristic of a 3-substituted indolyl moiety, the absence of the proton and carbon signals from H-2′ and C-2′ and the presence of an additional carbonyl (C-3′, δC 199.7) suggested the loss of a methine from the indole moiety of 13. The above structural assignment was confirmed by the HMBC correlations from H-4′ (δH 7.71) and H-10 to C-3′ (δC 199.7) (Figure 6). Hence, the planar structure of 13 was established as shown. The relative configuration of compound 13 was identical to those of 11 and

Table 1. 1H NMR Data for Compounds 1−4 (400 MHz, CD3OD) position

1

3

3.28 m

4

2.73 m

5 7 8 10a

15b 16a

2.56 m 5.55 brs 2.97 brd (10.9) 1.17 ddd (13.7, 8.8, 5.2) 1.10 ddd (13.7, 8.7, 5.1) 1.31 d (7.2) 4.06 s 6.17 d (10.9) 2.14 dd (12.2, 5.7) 2.77 m 3.23 m

16b

2.02 m

10b 11 12 13 15a

2 3.29 dd (5.3, 2.1) 2.68 m 2.59 m 5.55 brs 3.02 brd (9.9) 1.16 ddd (13.6, 8.7, 5.5) 1.09 ddd (13.6, 8.6, 5.4) 1.31 d (7.2) 4.06 s 6.21 d (10.9) 2.22 dd (11.8, 4.9) 2.71 m 2.13 ddd (13.2, 6.1, 2.7) 2.98 td (13.2, 2.3)

17

19a

3.79 dd (12.0, 4.3) 1.98 m

4.17 dd (7.7, 2.4) 2.04 m

19b

1.52 m

1.94 m

20a 20b 22 23 24 25

1.96 m 3.43 m 1.58 m 0.89 d (6.6) 0.89 d (6.6) 1.56 s

2.04 m 3.41 m 1.57 m 0.89 d (6.6) 0.89 d (6.6) 1.61 s

18

3

4

3.32 (overlap)

3.31 (overlap)

2.46 dt (5.9, 2.7) 2.63 m 5.55 brs 3.18 m 1.24 ddd (13.9, 9.2, 5.0) 1.14 ddd (13.9, 9.0, 4.7) 1.29 d (7.2) 4.07 s 6.22 d (10.7) 2.15 m

2.45 dd (6.1, 2.4) 2.63 m 5.55 brs 3.19 brd (11.0) 1.22 ddd (13.7, 8.9, 5.0) 1.13 ddd (13.7, 8.9, 4.9) 1.29 d (7.2) 4.06 s 6.27 d (10.9) 1.87 brd (12.7)

2.21 m 2.28 m

2.63 (overlap) 2.35 brt (13.8)

1.88 m

1.78 m

4.18 dd (8.9, 2.1)

4.02 dd (6.1, 1.8)

3.75 m

4.26 ddd (19.9, 11.9, 3.9) 2.56 ddd (19.9, 8.0, 2.9) 2.64 (overlap) 3.00 m 1.63 m 0.91 d (6.6) 0.90 d (6.6) 1.33 s

2.67 m 3.10 m 1.64 m 0.91 d (6.6) 0.91 d (6.6) 1.33 s

suggested that these protons were cofacial and were assigned to be α-oriented. Furthermore, the NOESY cross-peaks of H-4/H8, H-5/H-8, H-8/H-14, H-14/H-16, and H-16/18-Me indicated that these protons were on the opposite face of the ring system and thus β-oriented. Due to the free rotation of the C-3/C-10 single bond, the relative configuration at C-10 could not be determined from the NOESY spectrum. Thus, the relative configuration of 5 was determined except for the C-10 position. As chaetoglobosins share the same biosynthetic pathway,1,2,32 the identical ECD curves of 5 and armochaetoglobin M8 allowed us to assign the absolute configuration of 5 as 3R,4R,5S,6R,7S,8R,9R,16S,20S (Figure 5). Oxichaetoglobosin B (6) was isolated as a white, amorphous powder and had the same molecular formula as 5 based on their HRESIMS data. Its planar structure was confirmed to be the same as that of 5 by a comparison of their 1H−1H COSY and HMBC spectra. The relative configurations of all the chiral centers on the rings of 6 were the same as those of 5 based on their NOESY spectra (Figure S56). Therefore, 6 was the C-10 epimer of 5, and the absolute configuration of 6 was identical to that of 5 based on the similarities of their ECD curves (Figure 5) and optical rotation values. The molecular formulas of oxichaetoglobosins C (7) and D (8) were determined to be C33H40N2O6 from their HRESIMS data; this formula contains 14 mass units (CH2) more than that of 5. Subsequent comparison of the 1H and 13C NMR data of 7 and 8 (Tables 2 and 3) with those of 5 confirmed that 7 and 8 1581



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Table 2. 1H NMR Data for Compounds 5−13 (400 MHz) position

5b

6a

7a

8a

3

3.74 brs

3.74 t (2.0)

3.40 t (2.4)

4

2.89 m

2.73 m

5

1.85 (overlap)

1.61 m

2.76 dd (5.7, 2.0) 2.66 m

7 8

2.80 d (5.8) 2.21 dd (9.9, 5.8) 4.77 s

2.72 d (6.0) 2.11 dd (9.9, 6.0) 4.99 d (2.2)

3.80 d (10.3) 2.47 t (10.0)

3.82 d (10.1) 2.58 m

4.49 d (2.7)

1.07 d (6.7) 1.15 s

0.41 d (7.2) 1.14 s

15a 15b

6.36 dd (14.9, 10.4) 5.20 ddd (14.9, 10.9, 2.6) 2.33 d (13.1) 1.99 d (13.1)

6.35 ddd (15.3, 10.1, 1.7) 5.19 ddd (15.3, 11.1, 2.8) 2.43 brd (15.3) 2.05 m

16

2.61 (overlap)

2.80 m

17

5.75 brs

6.25 d (9.3)

20

4.29 brs

21a 21b

1.67 m 1.85 (overlap)

22a

2.95 m

10a 10b 11 12a 12b 13 14

24 25 26 2′ 4′ 5′ 6′ 7′

10a

11a

12a

13a

3.22 d (3.9)

3.60 d (7.9)

3.12 brs

2.91 brs

3.06 s

2.74 s

3.24 s

3.80 d (9.9) 2.10 m

2.11 dd (14.1, 7.1) 3.33 d (8.5) 2.45 (overlap)

2.24 q (7.2)

3.57 t (7.5) 1.92 t (9.9)

2.04 dd (14.0, 7.2) 3.23 d (9.2) 2.31 t (9.4)

4.17 d (6.3)

4.23 d (4.9)

4.03 d (7.9)

0.49 d (6.3) 5.16 s 4.93 s 6.11 dd (15.2, 9.7) 5.17 ddd (15.2, 11.1, 2.7) 2.41 brd (13.6) 2.00 brd (13.6)

0.66 d (6.7) 5.13 s 4.97 s 6.20 ddd (15.2, 9.5, 1.8) 5.26 ddd (15.2, 11.1, 2.7) 2.45 brd (13.6) 2.04 m

1.01 s 1.50 s

1.16 s 1.61 s

2.69 d (13.4) 2.64 d (13.4) 0.94 d (6.9) 0.99 s

2.55 m 2.49 m 0.98 d (7.0) 1.25 s

3.91 d (17.1) 3.25 d (17.1) 1.07 d (7.2) 1.22 s

6.23 dd (15.2, 10.1) 5.00 ddd (15.2, 10.1, 2.5) 2.34 d (11.9) 1.99 m

6.31 m

5.65 ddd (14.9, 9.6, 1.5) 5.34 ddd (14.3, 11.1, 2.6) 2.45 (overlap) 2.04 m

2.76 m (overlap) 6.22 d (9.4)

2.81 m

2.70 m

5.20 ddd (15.2, 11.1, 2.7) 2.47 brd (13.5) 2.10 m (overlap) 2.81 m.

5.67 ddd (15.0, 9.6, 1.8) 5.35 ddd (14.7, 10.8, 2.3) 2.50 m 2.10 m 2.89 m

2.83 m

5.59 ddd (14.9, 9.5, 1.6) 5.34 ddd (14.9, 11.1, 2.6) 2.46 brd (13.9) 2.02 dt (13.9, 11.4) 2.83 m

6.28 d (9.6)

6.30 d (8.6)

6.33 d (9.5)

6.36 d (9.1)

6.35 d (8.9)

4.74 dd (7.1, 4.4) 1.53 m 1.84 dd (14.5, 7.1) 2.76 m

4.70 dd (7.5, 4.0) 1.79 m 1.46 m

4.82 dd (7.6, 4.1) 1.82 m 1.79 m

4.74 m

4.90 (overlap)

4.98 t (5.4)

5.02 t (5.2)

1.86 m 1.62 m

2.00 m 1.87 m

6.42 dd (9.8, 1.2) 5.00 dd (6.7, 5.2) 1.79 m

1.80 m 1.62 m

2.69 m

2.85 m

2.83 m

3.02 t (6.6)

2.94 m

2.78 m

2.65 m

2.31 m

2.68 m

0.91 d (6.6) 1.76 s

1.06 d (6.7) 1.79 s

7.30 s 7.49 d (6.7) 7.12 t (7.5) 7.49 t (6.7) 7.24 d (7.7)

7.11 s 7.56 d (7.8) 7.03 t (8.1) 7.10 t (8.1) 7.34 d (8.1)

1.04 d (6.6) 1.79 s 3.24 s 7.09 s 7.56 d (7.9) 7.04 t (7.5) 7.12 t (7.2) 7.36 d (8.1)

1.06 d (6.7) 1.82 s 3.19 s 7.12 s 7.54 d (7.9) 7.03 brt (7.5) 7.12 brt (8.1) 7.37 d (8.1)

22b

3.45 dd (6.3, 1.6) 2.61 m

9c

2.71 m

1.08 d (6.7) 1.83 s

1.07 d (6.7) 1.82 s

1.96 m 1.75 ddd (14.4, 11.8, 6.1) 3.15 ddd (16.6, 7.9, 6.5) 2.73 dt (16.6, 6.5) 1.05 d (6.8) 1.83 s

5.22 s 7.25 d (7.5) 6.78 t (7.5) 7.12 t (7.9) 6.65 d (7.9)

5.22 s 7.23 d (7.4) 6.77 t (7.4) 7.11 t (7.9) 6.68 d (7.9)

7.71 d (7.6) 6.59 t (7.6) 7.24 t (7.7) 6.73 d (6.8)

2.59 m 1.00 d (6.1) 1.71 s 3.09 s 7.16 s 7.46 d (7.9) 6.99 t (7.6) 7.08 t (7.3) 7.37 d (8.1)

1.07 d (6.7) 1.82 s 3.15 s 7.12 s 7.60 d (7.9) 7.02 t (7.1) 7.12 t (7.1) 7.38 d (8.2)

3.39 d (9.6) 2.61 t (9.6)

a

In CD3OD. bIn CDCl3. cIn DMSO-d6.

The growth inhibitory effects of compounds 1−13 against five cancer cell lines (SW480, HL-60, A549, MCF-7, and SMMC7721) were evaluated.35,36 Compounds 8 and 11 showed weak antiproliferative activities against MCF-7 with IC50 values of 20.7 and 27.9 μM, respectively (Table S1). These compounds were also evaluated in the ConA/LPS-induced splenocyte proliferation assay (Table S2);37,38 all the tested compounds (other than 3, 10, and 11) inhibited the proliferation of ConAinduced murine T cells, and compounds 3, 6, 7, 8, 9, and 10 inhibited the proliferation of LPS-induced murine B cells. None of the compounds evaluated were active against Staphylococcus aureus and Colon bacillus.39,40

12 based on their NOESY data. As chaetoglobosins share the same biosynthetic pathway,1,2,32 the absolute configuration of 13 was assigned to be 3R,4R,5S,6S,7S,8R,9S,16S,20S. Compound 13 is the first example of a naturally occurring cytochalasan possessing a uniquely degraded indole ring, and 13 was proposed to be generated from chaetoglobosin W with 11 and 12 both serving as biosynthetic intermediates (Scheme 1). Notably, the hydroxylation of the C-12 methyl group is frequently observed in Chae-type cytochalasans from C. globosum but has never been reported in the Asp-type cytochalasan from A. f lavipes. In our coculture assay, four C12 hydroxylated Asp-type cytochalasans (compounds 1−4) were identified, which suggests that C. globosum may contain an oxygenase capable of hydroxylating the C-12 methyl group of compounds 1−4, whereas in A. f lavipes, the same oxygenase was missing or inactivated when cultured axenically. An unexpected oxygenase responsible for the hydroxylation of the C-10 of compounds 5−10 was activated by the coculturing of C. globosum and A. f lavipes, which led to a series of Chae-type cytochalasans that underwent additional modifications.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were obtained in a 0.7 mL cell on a Rudolph Autopol IV automatic polarimeter (Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra were recorded with a PerkinElmer Lambda 35 spectrophotometer (PerkinElmer, Inc., USA). ECD data were acquired with a JASCO-810 instrument (JASCO Co., Ltd., Tokyo, Japan). IR spectra were measured by a Bruker Vertex 70 FT-IR spectrophotometer

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Table 3. 13C NMR Data for Compounds 1−13 (400 MHz) position

1a

2a

3a

4a

5b

6a

7a

8a

9c

10a

11a

12a

13a

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

176.2 52.2 51.7 35.6 144.6 128.3 44.2 69.0 49.7 12.8 63.3 126.2 137.2 37.1 33.5 215.2 78.8 27.2 35.0 212.2 25.8 23.9 22.1 15.5

176.6 52.0 52.2 35.6 144.4 128.1 44.4 68.9 49.7 12.7 63.3 126.0 136.6 38.2 36.8 213.2 76.4 25.5 36.3 212.9 25.8 23.8 22.2 15.2

177.3 52.4 55.4 35.9 144.0 128.6 43.3 69.7 50.0 12.8 63.3 125.8 138.6 36.2 31.9 74.6 209.5 36.5 37.6 211.8 25.8 23.9 22.1 16.3

177.5 52.3 56.0 35.9 143.8 129.0 43.2 70.4 50.1 12.8 63.3 127.4 138.3 34.8 31.9 77.0 215.6 35.7 38.3 212.4 25.8 23.9 22.1 15.0

176.5 56.4 48.0 36.5 57.7 62.0 49.2 64.8 71.0 12.9 19.5 128.8 133.4 41.2 33.1 149.5 136.7 203.6 72.2 31.5 39.6 209.2 19.8 12.3

178.2 58.5 46.1 37.5 59.1 63.3 50.2 66.0 71.4 12.3 19.5 129.6 134.6 42.0 34.5 149.5 136.8 205.4 72.8 31.6 40.0 210.8 20.3 12.5

175.0 61.4 46.2 126.0 133.4 67.5 52.4 61.7 79.6 16.5 14.6 128.8 132.9 40.9 33.0 147.8 135.1 203.9 70.6 30.7 37.7 208.7 19.9 12.2 56.5 136.5 123.7 111.2 125.9 118.6 118.9 121.2 111.8

177.4 63.3 49.8 127.8 134.4 69.9 53.4 63.9 82.3 17.4 14.7 129.6 135.9 42.4 34.6 150.4 136.8 205.5 72.3 32.1 38.8 210.8 20.2 12.4 56.8 138.5 125.7 112.4 127.1 120.5 120.4 122.9 112.7

173.2 105.3 56.9 36.8 91.0 78.4 48.2 67.7 50.4 13.6 18.6 130.8 135.5 42.0 34.7 150.2 136.1 205.9 72.5 32.7 39.2 207.6 20.1 12.3

175.9 95.1 55.0 37.1 90.5 78.4 48.2 63.9 46.7 14.5 18.2 130.9 135.4 42.0 34.6 150.0 136.1 205.9 72.5 32.6 38.9 208.2 20.1 12.4

138.2 124.0 116.0 126.9 119.6 120.2 122.6 112.6

177.2 58.1 47.4 32.6 150.8 72.4 50.2 64.2 82.3 13.4 114.2 129.4 135.8 42.1 34.7 150.3 136.9 205.6 72.1 31.7 38.3 210.7 20.2 12.4 57.0 138.3 125.2 112.3 127.7 120.0 120.4 122.8 112.7

173.5 103.6 57.3 36.8 90.7 77.9 48.9 66.8 51.7 13.7 18.2 130.8 135.6 41.9 35.0 150.8 136.2 205.6 72.5 32.7 40.6 207.7 20.1 12.2

136.7 122.8 116.2 125.2 118.6 120.1 122.8 112.6

177.6 57.9 45.4 32.8 151.0 72.1 51.4 63.9 81.9 13.0 114.1 129.2 135.4 42.2 34.6 149.8 136.8 205.5 72.4 31.6 39.5 210.8 20.3 12.5 57.2 138.2 124.9 111.4 127.6 120.0 120.5 122.7 112.7

150.9 79.8 92.4 132.4 124.9 120.3 130.8 111.5

149.9 82.3 92.4 132.5 124.1 120.5 130.8 111.7

153.0 199.7 118.2 132.1 116.1 135.7 118.7

a

In CD3OD. bIn CDCl3. cIn DMSO-d6.

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of compounds 1, 3, and 5. shifts are expressed in ppm with reference to the CDCl3 (δH 7.26/δC 77.0), DMSO-d6 (δH 2.50/δC 39.52), and CD3OD (δH 3.31/δC 49.0) signals. The crystallographic data were recorded on a Bruker APEX DUO diffractometer equipped with graphite-monochromatized Cu Kα radiation (λ = 1.541 78 Å). Packing materials for column chromatog-

(Bruker, Karlsruhe, Germany). NMR spectra were recorded on a Bruker AM-400 NMR spectrometer (Bruker, Karlsruhe, Germany). HRESIMS data were obtained on a Bruker micrOTOF II spectrometer. Compounds were purified by an Agilent 1260 HPLC system semipreparative HPLC equipped with a DAD detector. Chemical 1583



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Wuhan, Hubei Province, China. The sequence data for these strains have been submitted to the DDBJ/EMBL/GenBank under accession nos. KF993614 and KP339510. Two voucher samples, CCM20121113 and QM507, were preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology. Coculture Analysis. Each fungus was cultured on potato dextrose agar at 28 °C for 7 d to prepare the seed culture. Agar plugs were cut into small pieces (approximately 0.4 × 0.4 × 0.4 cm3) and inoculated into Erlenmeyer flasks (1 L). The flasks had been sterilized by autoclaving, and each one contained 200 g of rice and 200 mL of distilled water. The cocultivation and axenic condition controls were performed in tripartite. As fungus A. f lavipes grew more quickly than C. globosum, the inoculum of C. globosum was 3-fold greater than that of A. f lavipes to avoid overwhelming accumulation of A. f lavipes in the flasks. After inoculation, all flasks were incubated at 28 °C for 21 d. At the time of harvest, CH3CH2OH (600 mL/flask) was added, and the culture was subjected to ultrasonication and then extracted with an equal volume of EtOAc. A solution of 20 μL of the extract in 2 mL of MeOH was subjected to HPLC-DAD analysis (Welch XB-C18 column, 4.6 × 250 mm, 5 μm, 210 nm, 1 mL/min gradient elution from 90% H2O/MeCN to 100% MeCN over 60 min). Fermentation and Isolation. The spores of C. globosum and A. f lavipes on PDA plates were inoculated into 200 × 1 L Erlenmeyer flasks, each one containing 200 g of rice and 200 mL of distilled water, incubated at 28 °C for 21 days. The separation of the EtOAc extract (100 g) was carried out by silica gel column chromatography (CC, 80− 120 mesh) eluting with CH2Cl2−MeOH (100:1−0:1, v/v) to afford seven fractions (Fr. A−Fr. G). Fr. C (4.0 g) was subjected to silica gel CC (200−300 mesh) eluting with CH2Cl2−MeOH (100:1−5:1, v/v) to afford five fractions (Fr. C1−Fr. C5). Fr. C3 was separated by MPLC (MeOH−H2O, 20:80− 100:0) and further purified by semipreparative HPLC (MeOH−H2O, 67:33, v/v) to yield 17 (11.5 mg). Fr. C4 was separated with Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and further purified by semipreparative HPLC (MeCN−H2O, 52:48, v/v) to yield 14 (5.0 mg), 15 (8.5 mg), and 16 (7.2 mg). Fr. D (7.5 g) was subjected to MPLC (MeOH−H2O, 20:80−100:0) to give seven fractions (D1−D7). Fraction D3 was separated with Sephadex LH-20 (MeOH) and further purified by semipreparative HPLC (MeOH−H2O, 60:40, v/v) to yield 3 (7.0 mg) and 4 (3.3 mg). Fraction D4 was separated with Sephadex LH-20 (MeOH) and further purified by semipreparative HPLC (MeCN−H2O, 31:69, v/v) to yield 1 (7.2 mg) and 2 (5.2 mg). Fraction D5 was separated with Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and chromatographed on silica gel eluting with a step gradient of CH2Cl2−MeOH (100:1−5:1, v/v) to yield 18 (25 mg). Fr. E (4.5 g) was subjected to MPLC (MeOH−H2O, 20:80−100:0) to give six fractions (E1−E6). Fraction E3 was separated with Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and further purified by semipreparative HPLC (MeCN−H2O, 55:45, v/v) to yield 11 (3.8 mg). Fraction E4 was separated with Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and further purified by semipreparative HPLC (MeCN−H2O, 56:44, v/v) to yield 12 (2.7 mg) and 13 (3.0 mg). Fr. F (6.0 g) was subjected to silica gel CC [CH2Cl2−MeOH (70:1− 5:1, v/v)] to yield five fractions (F1−F5). Of these, Fr. F3 was subjected to MPLC (MeOH−H2O, 20:80−100:0) to give seven fractions (F3.1−

Figure 3. X-ray ORTEP drawing of compound 1.

Figure 4. Experimental ECD spectra of aspochalasinols A−D (1−4) in MeOH.

Figure 5. Experimental ECD spectra of oxichaetoglobosins A−F (5− 10) in MeOH. raphy were silica gel (80−120 mesh, 100−200 mesh, and 200−300 mesh, Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), ODS (50 μm, YMC, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Fungal Material. The sample of the fungus Chaetomium globosum TW1-1 was isolated from Armadillidium vulgare in November 2012 at Tongji Medical College, Hubei Province, China, and Aspergillus flavipes (507) was collected from the intertidal zone of the Yangtze River,

Figure 6. Key 2D NMR correlations of 13. 1584



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Oxichaetoglobosin F (10): white, amorphous powder (MeOH); [α]25D +34.9 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 204 (4.43), 220 (4.60), 281 (3.72) nm; CD (MeOH) λmax (Δε) 216 (13.57), 234 (−2.57) nm; IR (KBr) νmax 3403, 2954, 2928, 2869, 1690.0, 1435, 1382, 1257, 1101, 1033 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 583.2764 [M + Na]+ (calcd for C33H40N2O6Na, 583.2784). Oxichaetoglobosin G (11): pale yellow, amorphous solid (MeOH); [α]25D −67.3 (c 0.17, MeOH); UV (MeOH) λmax (log ε) 204 (4.43), 240 (4.07), 292 (3.28) nm; CD (MeOH) λmax (Δε) 209 (−20.50), 220 (−4.95), 237 (−15.15), 267 (−0.21) nm; IR (KBr) νmax 3414, 2928, 1684, 1438, 1385, 1208, 1142 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 585.2573 [M + Na] + (calcd for C32H38N2O7Na, 585.2577). Oxichaetoglobosin H (12): pale yellow, amorphous solid (MeOH); [α]25D +51.3 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 205 (4.36), 236 (3.97), 294 (3.15) nm; CD (MeOH) λmax (Δε) 203 (−5.05), 215 (12.23), 227 (−18.00), 247 (12.22) nm; IR (KBr) νmax 3398, 2971, 2930, 1683, 1417, 1384, 1205, 1139, 1039 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 563.2774 [M + H]+ (calcd for C32H39N2O7, 563.2757). Oxichaetoglobosin I (13): pale yellow, amorphous powder (MeOH); [α]25D −39.4 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 202 (4.21), 228 (4.34), 369 (3.55) nm; CD (CH2Cl2/MeOH, 1:1) λmax (Δε) 229 (11.27), 244 (−3.66) nm; IR (KBr) νmax 3445, 3358, 2964, 2928, 2869, 1700, 1415, 1207, 1035 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 573.2598 [M + Na]+ (calcd for C31H39N2O7Na, 573.2577). X-ray Crystal Structure Analysis. Crystals of 1 were obtained from CH2Cl2/MeOH/H2O. The intensity data were collected at 100 K on a Bruker APEX DUO diffractometer outfitted with an APEX II CCD using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structures were determined by direct methods using SHELXS-97. Crystallographic data for the reported structure have been deposited with the Cambridge Crystallographic Data Center (CCDC) as supplementary publication no. CCDC 1561327. Crystal data for aspochalasinol A (1): C24H35NO5·2(H2O), M = 453.56, a = 8.0540(5) Å, b = 12.5480(7) Å, c = 14.0945(8) Å, α = 115.8600(10)°, β = 94.7020(10)°, γ = 102.4350(10)°, V = 1226.37(12) Å3, T = 100(2) K, space group P1, Z = 2, μ(Cu Kα) = 0.731 mm−1, 15 234 reflections measured, 6970 independent reflections (Rint = 0.0254). The final R1 values were 0.0559 (I > 2σ(I)). The final wR(F2) values were 0.1587 (I > 2σ(I)). The final R1 values were 0.0569 (all data). The final wR(F2) values were 0.1626 (all data). The goodness of fit on F2 was 1.072. Flack parameter = 0.14(4). Biological Assays. Cytotoxicity against Cancer Cell Lines. Cytotoxicity of the isolated compounds against the five cancer cell lines (SW480, HL-60, A549, MCF-7, and SMMC-7721) was evaluated using the MTT method. Doxorubicin (Beijing Solarbio Science & Technology Co., Ltd., Beijing, People’s Republic of China) was used as positive control. All cells were cultured in RPMI.1640 medium (Hangzhou Jinuo Biology Technology Co., Ltd., Hangzhou, People’s Republic of China), supplemented with 10% fetal bovine serum (ExCell Biology Co., Ltd., Taicang, People’s Republic of China), 100 units/mL penicillin, and 100 μg/mL streptomycin (Solarbio) at 37 °C in a humidified atmosphere with 5% CO2. Tumor cells were seeded in 96well microtiter plates at 5000 cells/well. After 24 h, test compounds were added to the wells. After incubation for 48 h, 20 μL of 5 mg/mL 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Solarbio) was added, and the incubation was continued for 4 h at 37 °C. The cell viability was determined by measuring the metabolic conversion of MTT into purple formazan precipitate by viable cells. The medium was then removed, and cells were then lysed with 100 μL of triplex solution of 10% sodium dodecyl sulfate, 5% isopropyl alcohol, and 12 mM HCl. The MTT assay results were obtained using a microplate spectrophotometer (SpectraMax I3, Molecular Devices, USA) plate reader at 570 nm. Compounds were tested at five concentrations (50, 25, 12.5, 6.25, 3.12, and 1.56 μM) in 100% DMSO with a final concentration of DMSO of 0.5% (v/v) in each well. Each

F3.7). Fr. F3.3 was purified by semipreparative HPLC (MeOH−H2O, 55:45, v/v) to yield 5 (1.5 mg) and 6 (2.0 mg). Fr. F3.4 was purified by semipreparative HPLC (MeCN−H2O, 47:53, v/v) to yield 9 (3.2 mg) and 10 (2.5 mg). Fraction F3.5 was purified by repeated CC on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) and then purified by semipreparative HPLC (MeOH−H2O, 62:38, v/v) to yield 7 (3.1 mg) and 8 (2.3 mg). Fr. G (5.4 g) was subjected to silica gel CC [CH2Cl2−MeOH (50:1− 5:1, v/v)] to yield three fractions (G1−G3). Of these, Fr. G1 was subjected to MPLC (MeOH−H2O, 20:80−100:0) to give seven fractions (G3.1−G3.7). Fr. G3.4 was purified by semipreparative HPLC (MeOH−H2O, 40:60, v/v) to yield 19 (2.5 mg). Fr. G2 was subjected to MPLC (MeOH−H2O, 20:80−100:0) and then purified by repeated CC on Sephadex LH-20 (CH2Cl2−MeOH, 1:1) to yield 20 (15.2 mg). Aspochalasinol A (1): colorless crystals; (MeOH), [α]25D −58.9 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 203 (4.26) nm; CD (MeOH) λmax (Δε) 222 (5.06), 292 (−6.37), 322 (−2.03) nm; IR νmax 3381, 2965, 1689, 1448, 1384, 1049 cm−1; 1H and 13C NMR data see Tables 1 and 3; HRESIMS m/z 440.2407 [M + Na]+ (calcd for C24H35NO5Na, 440.2413). Aspochalasinol B (2): pale yellow, amorphous solid (MeOH); [α]25D −61.4 (c 0.58, MeOH); UV (MeOH) λmax (log ε) 204 (4.22) nm; CD (MeOH) λmax (Δε) 224 (11.19), 280 (−2.74), 316 (2.60) nm; IR νmax 3376, 2949, 1688, 1448, 1386, 1028 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 440.2409 [M + Na]+ (calcd for C24H35NO5Na, 440.2413). Aspochalasinol C (3): pale yellow, amorphous solid (MeOH); [α]25D −76.2 (c 0.53, MeOH); UV (MeOH) λmax (log ε) 205 (4.20) nm; CD (MeOH) λmax (Δε) 224 (5.95), 282 (−3.24), 316 (−1.01) nm; IR νmax 3359, 2956, 1685, 1446, 1385, 1026 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 440.2412 [M + Na]+ (calcd for C24H35NO5Na, 440.2413). Aspochalasinol D (4): pale yellow, amorphous solid (MeOH); [α]25D −68.4 (c 0.33, MeOH); UV (MeOH) λmax (log ε) 202 (4.19) nm; CD (MeOH) λmax (Δε) 228 (4.23), 292 (−6.71), 318 (−3.03) nm; IR νmax 3400, 2955, 1683, 1446, 1385, 1018 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 440.2408 [M + Na]+ (calcd for C24H35NO5Na, 440.2413). Oxichaetoglobosin A (5): white, amorphous powder (MeOH); [α]25D −71.8 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 202 (4.09), 220 (4.16), 281 (3.17) nm; CD (MeOH) λmax (Δε) 232 (−6.49) nm; IR νmax 3397, 2949, 2837, 1680, 1449, 1386, 1023 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 547.2796 [M + H]+ (calcd for C32H39N2O5, 547.2808). Oxichaetoglobosin B (6): white, amorphous powder (MeOH); [α]25D +81.2 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 202 (4.07), 221 (4.17), 282 (3.57) nm; CD (MeOH) λmax (Δε) 228 (−3.75) nm; IR νmax 3346, 2962, 2923, 1679, 1437, 1019 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 569.2608 [M + Na]+ (calcd for C32H38N2O5Na, 569.2628). Oxichaetoglobosin C (7): white, amorphous powder (MeOH); [α]25D +59.6 (c 0.12, CH3OCH3); UV (MeOH) λmax (log ε) 203 (4.39), 220 (4.49), 280 (3.75) nm; CD (MeOH) λmax (Δε) 222 (−3.82) nm; IR (KBr) νmax 3398, 2925, 2668, 1686, 1437, 1381, 1267, 1105, 1025 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 583.2789 [M + Na]+ (calcd for C33H40N2O6Na, 583.2784). Oxichaetoglobosin D (8): white, amorphous powder (MeOH); [α]25D +64.5 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 203 (4.49), 220 (4.56), 280 (3.68) nm; CD (MeOH) λmax (Δε) 212 (−2.82) nm; IR (KBr) νmax 3404, 2962, 2926, 2202, 2060, 1686, 1435, 1380, 1266, 1115, 1093, 1027 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 583.2794 [M + Na]+ (calcd for C33H40N2O6Na, 583.2784). Oxichaetoglobosin E (9): white, amorphous powder (MeOH); [α]25D +37.4 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 203 (4.67), 220 (4.77), 280 (3.89) nm; CD (MeOH) λmax (Δε) 222 (−10.08) nm; IR (KBr) νmax 3416, 2956, 2922,2853, 1691, 1459, 1378, 1254, 1098, 1030 cm−1; 1H and 13C NMR data, see Tables 2 and 3; HRESIMS m/z 583.2760 [M + Na]+ (calcd for C33H40N2O6Na, 583.2784). 1585



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concentration of the compounds was tested in three parallel experiments. IC50 values were calculated using the means ± SEM using GraphPad Prism 5. Effects on T and B Lymphocyte Proliferation. Three male KM (25 g) mice (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., China) were sacrificed, and their spleens were removed under aseptic conditions. Splenic lymphocytes were prepared as previously described.37 A 180 μL cell suspension was seeded in 96-well plates at 5 × 105 cells/well. The cells were stimulated by 5 μg/mL ConA (Solarbio) or 10 μg/mL lipopolysaccharide (LPS, Solarbio), proliferated, and differentiated into T cells and B cells, respectively, with 20 μL of test compounds added to each wells. Test compounds were dissolved in DMSO with a final concentration of 40 μM in each well. The cells were cultured for 48 h and detected by MTT at 570 nm with a microplate reader. Controls with and without ConA and LPS were used to establish the baseline proliferation for stimulated and unstimulated cells. Antimicrobial Activity. New compounds were evaluated for their antimicrobial activities against two representative microbes, S. aureus (a Gram-positive bacterium) and C. bacillus (a Gram-negative bacterium), using an agar-well diffusion method with cefotaxime sodium and tetracycline as positive controls and the solvent (DMSO) as the negative control.39

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00110.

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HRESIMS, IR, UV, 1D and 2D NMR, CD spectra of 1− 13, and bioassay data for 1−13 (PDF) X-ray crystallographic data of 1 (CIF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C. Chen). *E-mail: [email protected] (Y. Zhang). ORCID

Yonghui Zhang: 0000-0002-7222-2142 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (No. T2016088); National Natural Science Foundation for Distinguished Young Scholars (No. 81725021), Innovative Research Groups of the National Natural Science Foundation of China (81721005); the National Natural Science Foundation of China (Nos. 81573316, 31570361, 21602068, 81773597, and 21772048); the Academic Frontier Youth Team of HUST; and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in testing of ECD, UV, and IR.

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