Armochaetoglobins A–J: Cytochalasan Alkaloids from Chaetomium

Article pubs.acs.org/jnp

Armochaetoglobins A−J: Cytochalasan Alkaloids from Chaetomium globosum TW1-1, a Fungus Derived from the Terrestrial Arthropod Armadillidium vulgare Chunmei Chen,† Jianping Wang,† Junjun Liu,† Hucheng Zhu,† Bin Sun,† Jing Wang,† Jinwen Zhang,‡ Zengwei Luo,† Guangmin Yao,† Yongbo Xue,*,† and Yonghui Zhang*,† †

Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, and Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: Ten new cytochalasan alkaloids, termed armochaetoglobins A−J (1−10), and four known chaetoglobosins (11−14) were isolated from a methanol extract of Chaetomium globosum TW1-1, a fungus isolated from the medicinal terrestrial arthropod Armadillidium vulgare. Their structures were elucidated by a combination of spectroscopy, single-crystal Xray crystallography, and ECD calculations. Armochaetoglobins A−E (1−5) represented the first examples of seco-chaetoglobosins arising from an oxidative cleavage of C-19 and C-20. Among these compounds, armochaetoglobin A (1) features an unusual pyrrole ring. The cytotoxic activities of 2−10 were evaluated, and armochaetoglobin H (8) showed moderate inhibitory activities against five human cancer cell lines, with IC50 values ranging from 3.31 to 9.83 μM. species. Armochaetoglobins A−E (1−5) represent the first examples of 19,20-seco-chaetoglobosins. Among these compounds, armochaetoglobin H (8) exhibited moderate inhibitory activities toward five human cancer cell lines, with IC50 values ranging from 3.31 to 9.83 μM. Herein, we report the fermentation, isolation, structure elucidation (including the absolute configuration analysis), and in vitro cytotoxic activities of these new compounds.

I

n addition to plants, animals are also used in traditional Chinese medicine. Approximately 15 species of arthropods, such as Malaphis chinensis and Bombyx mori, are currently used in Chinese medicine on a regular basis.1,2 Natural products from arthropod-derived fungi have attracted considerable attention because they are rich sources of chemically interesting and biologically potent secondary metabolites.3−6 Secondary metabolites from medicinal arthropod-derived fungi provide an interesting field of inquiry for chemists and pharmacologists.7−10 Until now, only a small fraction of medicinal arthropods and medicinal arthropod-derived fungi have been analyzed chemically or explored for their potential biological activities.11−13 As part of our program to discover structurally unique and biologically active secondary metabolites from medicinal arthropod-derived fungi, Chaetomium globosum TW1-1 was isolated for the first time from the terrestrial isopod Armadillidium vulgare, a traditional Chinese medicine used to treat chronic bronchitis, amenorrhea, and malaria epidemics.14 The chemical constituents of C. globosum have been widely investigated, and a large number of cytochalasans have been isolated and reported.15−18 To the best of our knowledge, however, no report has characterized cytochalasans of arthropod-derived fungal origin. Our current research resulted in the isolation of 10 new cytochalasan alkaloids, termed armochaetoglobins A−J (1−10), and four known chaetoglobosins (11−14)16,19,20 from the methanol extract of this © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION C. globosum culture broth was extracted with methanol. The whole extract was partitioned and purified by repeated column chromatography involving silica gel, reversed-phase silica gel C18, Sephadex LH-20, and semipreparative HPLC to yield 10 new compounds, termed armochaetoglobins A−J (1−10), and four known chaetoglobosins (11−14). By comparison of the NMR and MS data with published data, the known compounds were identified as cytoglobosin E (11),16 isochaetoglobosin J (12),19 cytoglobosin C (13),16 and chaetoglobosin W (14).20 Compound 1 was obtained as a colorless powder. The molecular formula C32H37N3O3 was deduced by the positive high-resolution electrospray ionization mass spectra (HRESIMS), indicating 16 degrees of unsaturation. Detailed analysis Received: August 6, 2014

A

DOI: 10.1021/np500626x J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

of its 1H and 13C NMR spectra (Tables 1 and 2) revealed 32 carbons, including four methyl groups [δH 0.58 (3H, d, J = 7.3 Hz, Me-11), 0.96 (3H, d, J = 6.7 Hz, 16-Me), 1.19 (3H, s, Me12), 1.68 (3H, d, J = 1.1 Hz, 18-Me)], two methylenes, 18 methines [one aldehyde carbon (δC 197.7), six sp3 and 11 sp2 carbons], and eight quaternary carbons [one amide carbonyl (δC 179.4), two sp3 and five sp2 carbons]. Examination of the 1 H and 13C NMR spectra of 1 showed the presence of a 3substituted indolyl group [δH 6.87 (1H, s, H-2′), 7.10 (1H, d, J = 7.9 Hz, H-4′), 6.94 (1H, m, H-5′), 7.05 (1H, m, H-6′), and 7.29 (1H, d, J = 8.1 Hz, H-7′); δC 124.3 (CH, C-2′), 111.8 (C, C-3′), 129.0 (C, C-3′a), 119.4 (CH, C-4′), 119.8 (CH, C-5′), 122.3 (CH, C-6′), 112.2(CH, C-7′), and 137.9 (C, C-1′a)]. The core structure of 1, an isoindolone, was elucidated by the 1 H−1H COSY and HMBC spectra (Figure 1). The 1H−1H COSY cross-peak of H-3/H-4 and the long-range HMBC correlations from H-3 to C-1 and C-4 and from H-4 to C-1 and C-9 revealed the presence of a pyrrolidine-2-one moiety. The cyclohexane ring was deduced from the 1H−1H COSY crosspeaks of H-4/H-5 and H-7/H-8 and confirmed by the following HMBC correlations: from H-4 to C-5 and C-6; from H-5 to C-4, C-6, and C-9; and from H-7 to C-6 and C-8. Me-11 and Me-12 were assigned to C-5 and C-6 on the basis of the HMBC correlations from Me-11 to C-4, C-5, and C-6 and from Me-12 to C-5, C-6, and C-7, respectively. The coexistence of an epoxide was elucidated by the characteristic chemical shifts of C-6 (δC 59.3) and C-7 (δC 64.5). The aliphatic chain (C-13−C-19) at C-8 was readily elucidated by a combination of the 1H−1H COSY crosspeaks of H-8/H-13/H-14/H-15/H-16/H-17 and the HMBC correlations from 16-Me to C-15, C-16, and C-17 and from 18Me to C-17, C-18, and C-19. The 1H−1H COSY cross-peaks of H-20/H-21/H-22 coupled with the HMBC correlations from

both H-20 and H-21 to C-22 and C-23 indicated the presence of a pyrrole ring. In addition, the linkage of the pyrrole ring to C-9 was established by the HMBC correlations from H-4 and H-8 to C-23 and from H-22 to C-9. Finally, the location of the indolyl group was assigned to C-3 through C-10 by the HMBC correlations from H-10 to C-2′, C-3′, and C-3′a. The relative configuration of 1 was deduced by a NOESY experiment (Figure 2). The E-geometry of Δ13 and Δ17 double bonds was deduced from the large coupling constants of J13,14 (15.4 Hz) and the NOESY correlation of H-16/18-Me, respectively. The strong NOESY correlation of H-5 with H-8 suggested the boat conformation of the cyclohexane ring. The NOESY correlations of Me-11/H-3, H-3/Me-12, and Me-12/ H-7 indicated that they were cofacial, and they were assigned as being in the α-orientation. Additional NOESY interactions of H-4 with H-10 and H-22 revealed the β-orientation of H-4 and the cis ring junction of the pyrrolidine-2-one and the cyclohexane rings. Previous studies have revealed that the essential elements of most cytochalasans have the same configuration.21,22 To the best of our knowledge, the relative configuration of 16-Me in all naturally occurring cytochalasans is reported to possess an α-orientation in the macrocycle.23 Taking the biosynthetic pathway of cytochalasans into consideration,8,24 the configuration of 16-Me in 1 should be consistent with the reported cytochalasan analogues but requires verification by X-ray crystallography when a suitable crystal is available. Therefore, the relative configuration of all chiral centers in 1 with the exception of C-16 was elucidated. These data confirmed compound 1, named armochaetoglobin A, as a new unprecedented 19,20-seco-chaetoglobosin alkaloid characterized by a bicyclic isoindolone integrated with an aliphatic chain (C-13−C-19), a pyrrole ring, and a benzopyrrole. B

DOI: 10.1021/np500626x J. Nat. Prod. XXXX, XXX, XXX−XXX

C

a

6.72 dd (2.6, 1.6) 6.03 dd (3.4, 2.6)

6.00 dd (3.4, 1.6)

6.87 7.10 6.94 7.05 7.29 0.96 1.68

20 21

22

2′ 4′ 5′ 6′ 7′ 16-CH3 18-CH3 20-OCH3

dt (7.9, 3.8) m m d (4.7) m m

m br d (9.4) d (12.7) d (12.7) m m

7.00 7.53 7.13 7.20 7.38 0.87 1.57

s d (7.7) m m d (8.1) d (6.7) s

2.41 dt (18.2, 6.4)

2.54 dt (18.2, 6.9)

2.42 4.87 3.82 3.58 3.47 1.69

2.76 dd (14.6, 7.9) 1.30 d (7.5) 1.31 s 5.38 dd (15.3, 8.8) 5.54 ddd (15.3, 8.5, 5.2) 2.13 dt (14.7, 4.9) 1.84 dt (14.7. 9.3)

3.85 3.13 2.36 2.95 3.46 3.13

2b

In CD3OD. bIn CDCl3. cIn DMSO-d6.

s d (7.9) m m d (8.1) d (6.7) d (1.1)

2.66 m 6.27 br d (8.6) 9.28 s

3.77 m 2.60 br d (6.1) 1.87 m 2.84 d (5.7) 2.50 dd (8.5, 5.7) 2.86 dd (13.7, 5.5) 2.55 dd (13.7, 9.8) 0.58 d (7.3) 1.19 s 6.04 dd (15.4, 8.5) 5.21 ddd (15.4, 7.6, 6.8) 2.09 m 2.01 m

1a

16 17 19

15

14

11 12 13

3 4 5 7 8 10

position

7.03 7.52 7.16 7.23 7.40 1.01 1.69

br s d (7.8) m m d (8.1) d (6.7) s

2.58 m

3.57 m 1.71 m

2.75 m 6.18 br d (9.8) 9.22 s

3.80 m 3.00 m 2.25 m 2.93 d (5.0) 3.21 dd (8.5, 5.0) 3.09 dd (14.4, 3.7) 2.75 dd (14.4, 8.4) 1.25 d (7.5) 1.30 s 5.72 dd (15.3, 8.6) 5.54 ddd (15.3, 7.1, 6.8) 2.13 m

3b

7.15 7.55 7.03 7.09 7.34 1.05 1.70

s d (7.7) m m d (8.0) d (6.7) d (1.1)

2.35 dt (20.0, 5.8)

2.70 m

2.29 m

2.77 m 6.39 br d (8.8) 9.32 s

3.82 m 2.74 m 1.82 m 2.81 d (5.3) 2.70 m 2.95 dd (14.2, 5.0) 2.86 dd (14.2, 7.1) 0.79 d (7.2) 1.20 s 6.01 dd (15.4, 8.8) 5.46 ddd (15.4, 7.1, 6.7) 2.19 m 2.13 m

4a

Table 1. 1H NMR Data for Compounds 1−10 (400 MHz, J in Hz)

7.15 7.55 7.03 7.09 7.33 1.06 1.70 3.65

s d (7.8) m m d (8.0) d (6.7) br s s

2.24 m

2.67 m

2.26 m

2.79 m 6.39 br d (9.4) 9.32 s

3.84 m 2.73 m 1.83 m 2.81 d (5.2) 2.69 m 2.91 dd (14.3, 4.6) 2.88 dd (14.3, 6.9) 0.82 d (7.4) 1.22 s 5.99 dd (15.5, 8.8) 5.45 ddd (15.5, 7.2, 6.8) 2.17 m

5a dd (7.1, 3.6) m m br s m dd (14.7, 4.0)

m br d (8.9) d (14.8) d (14.8)

1.59 ddd (16.4, 10.6, 5.7) 2.88 ddd (19.2, 10.5, 3.7) 1.00 ddd (19.2, 10.6, 5.7) 6.97 s 7.53 d (6.7) 7.03 m 7.00 m 7.32 d (6.8) 0.89 d (6.8) 1.50 s

2.11 m

2.39 5.08 3.00 2.63

1.35 d (7.3) 4.10 s 5.92 ddd (15.1, 10.2, 1.7) 5.00 ddd (15.1, 11.0, 3.3) 2.18 m 1.82 dt (13.5, 11.5)

2.75 dd (14.7, 3.5)

3.58 2.62 2.50 5.58 2.77 3.00

6a dt (8.4, 3.4) m m br s m dd (14.4, 3.4)

6.98 7.50 7.13 7.20 7.37 1.01 1.82

br s d (8.0) m m d (8.0) d (6.7) s

1.67 m

1.81 m

4.72 t (5.1) 2.91 ddd (17.9, 8.9, 5.7) 2.42 m

2.70 m 6.19 br d (9.0)

1.30 d (7.2) 1.74 s 6.30 dd (14.3, 11.0) 5.16 ddd (14.3, 11.0, 2.7) 2.37 m 2.06 m

2.65 dd (14.4, 8.5)

3.37 2.77 2.41 5.37 2.76 2.98

7b m m m br s m dd (14.4, 4.4)

7.01 7.45 6.93 6.97 7.24 0.93 1.26

d (2.3) d (7.8) m m d (7.9) d (6.7) d (0.7)

6.08 d (16.7)

7.73 d (16.7)

2.39 m 5.42 br d (9.7) 4.88 d (4.2)

1.08 d (7.1) 3.94 br s 5.98 dd (15.2, 10.3) 5.06 ddd (15.2, 10.7, 3.5) 2.20 m 1.90 dt (13.9, 11.3)

2.67 dd (14.4, 5.1)

3.36 2.79 2.28 5.40 2.45 2.76

8c m m m s m dd (14.4, 3.9)

2.40 ddd (19.0, 13.5, 2.4) 0.48 ddd (19.0, 12.0, 3.8) 7.03 d (2.1) 7.52 d (7.8) 6.94 m 7.01 m 7.27 d (7.9) 0.84 d (6.7) 1.49 s

0.72 dt (13.2, 12.0)

3.23 dt (11.6, 3.8) 0.99 m

1.14 d (7.2) 1.70 s 5.82 dd (15.1, 10.2) 4.49 ddd (15.1, 9.1, 3.6) 2.06 m 1.66 dd (12.8, 11.6) 2.24 m 4.80 br d (8.8) 3.39 s

2.58 dd (14.4, 3.4)

3.28 2.46 2.24 5.25 2.49 2.84

9c s q (7.0) d (10.2) m d (14.9)

2.85 ddd (19.2, 12.4, 3.1) 1.49 ddd (19.2, 5.1, 2.8) 7.21 s 7.60 d (7.9) 7.05 m 7.11 m 7.37 d (8.0) 1.00 d (6.8) 1.82 d (1.2)

1.89 m

2.70 m

2.75 m 6.11 br d (9.9)

1.22 d (7.0) 1.28 s 5.27 ddd (15.0, 9.5, 1.6) 5.12 ddd (15.0, 10.9, 2.6) 2.37 m 1.85 m

3.23 d (14.9)

2.44 2.16 3.40 2.72 3.37

10a

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DOI: 10.1021/np500626x J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR for Compounds 1−10 (100 MHz, J in Hz) position 1 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2′ 3′ 3′a 4′ 5′ 6′ 7′ 1′a 16-CH3 18-CH3 20-OCH3 a

1a 179.4 55.2 53.0 37.4 59.3 64.5 49.3 53.8 34.2 12.6 19.5 131.7 130.8 40.2 35.0 162.1 139.0 197.7 118.8 107.1 108.1 134.2 124.3 111.8 129.0 119.4 119.8 122.3 112.2 137.9 19.7 9.3

2b s d d d s d d s t q q d d t d d s d d d d s d s s d d d d s q q

174.4 54.7 41.1 32.0 58.8 60.8 43.7 63.5 32.7 14.8 21.5 125.5 135.3 40.8 31.7 131.1 133.7 68.4 61.4 26.6 35.4 206.7 123.9 110.5 127.4 118.1 119.6 122.1 111.6 136.4 21.2 13.9

3b s d d d s d d s t q q d d t d d s t t t t s d s s d d d d s q q

174.2 54.0 44.1 33.4 58.3 60.9 44.5 63.7 33.5 14.3 20.9 127.8 132.6 39.4 33.4 159.2 138.2 195.6 61.5 26.4 36.0 207.2 123.4 110.8 127.1 118.2 120.0 122.4 111.6 136.4 19.4 9.4

4a s d d d s d d s t q q d d t d d s d t t t s d s s d d d d s q q

176.5 55.0 48.2 36.8 59.1 62.7 47.1 65.6 34.9 13.3 20.2 130.0 133.1 40.4 34.9 161.6 139.4 197.5 176.5 28.8 36.3 208.8 125.4 110.8 129.0 119.2 120.1 122.5 112.5 138.1 19.6 9.3

5a s d d d s d d s t q q d d t d d s d s t t s d s s d d d d s q q

176.4 55.0 48.1 36.8 59.1 62.7 47.3 65.5 33.7 13.4 20.2 130.0 133.2 40.4 34.9 161.6 139.4 197.5 174.7 28.4 36.1 208.6 125.5 110.6 129.1 119.2 120.1 122.5 112.5 138.1 19.6 9.3 52.1

6a s d d d s d d s t q q d d t d d s d s t t s d s s d d d d s q q q

176.5 55.7 51.5 35.8 144.0 128.4 49.0 68.6 32.4 13.0 63.5 131.2 132.9 42.7 33.6 138.7 128.9 53.9 213.6 37.2 38.3 210.6 126.1 109.9 129.3 119.8 122.4 120.2 112.7 137.8 21.6 16.2

7b s d d d s d d s t q t d d t d d s t s t t s d s s d d d d s q q

174.6 53.9 50.7 34.7 139.6 125.9 46.5 67.7 34.2 13.9 20.0 131.3 131.3 40.7 33.4 149.6 134.0 203.7 71.9 38.7 31.8 208.8 123.2 110.8 127.2 118.3 119.7 122.2 111.5 136.4 19.7 12.1

8c s d d d s d d s t q q d d t d d s s d t t s d s s d d d d s q q

172.0 53.3 47.6 33.5 143.8 125.0 46.1 65.8 32.5 12.4 61.5 130.8 130.7 41.3 31.5 138.6 132.6 81.0 200.4 135.9 131.2 198.7 124.3 109.1 127.5 118.5 118.3 120.7 111.2 136.1 20.9 10.6

9c s d d d s d d s t q q d d t d d s d s d d s d s s d d d d s q q

173.5 52.8 49.5 34.9 139.3 125.3 47.7 66.1 31.6 13.1 19.8 131.0 129.7 41.6 30.6 133.6 134.1 77.4 71.1 26.8 36.4 210.7 125.4 108.3 127.9 118.9 118.4 120.6 111.1 136.0 21.2 12.4

10a s d d d s d d s t q q d d t d d s d d t t s d s s d d d d s q q

175.8 98.8 54.2 37.1 91.7 77.7 47.1 62.6 34.8 14.6 18.2 129.3 135.6 40.9 34.5 156.4 132.4 197.0 205.2 32.9 36.2 206.2 126.0 110.7 128.7 119.7 120.1 122.5 112.5 137.8 19.8 10.9

s d d d s d d s t q q d d t d d s s s t t s d s s d d d d s q q

In CD3OD. bIn CDCl3. cIn DMSO-d6.

Figure 1. Key 1H−1H COSY (bold) and HMBC (arrows) correlations establishing the planar structure of armochaetoglobin A (1). Figure 2. Key NOESY correlations of armochaetoglobin A (1).

To determine the absolute configuration of C-3−C-9 in the isoindolone core and C-16 in the side chain of 1, the theoretical calculated electronic circular dichroism (ECD; for detailed procedures see the Supporting Information, SI) spectra of two possible models 1A (3S,4R,5S,6R,7S,8R,9R,16S) and 1B (3S,4R,5S,6R,7S,8R,9R,16R) were performed using timedependent density functional theory (TDDFT). The optimized conformations of 1A and 1B (Figures S1 and S3, SI) were obtained and further used for the ECD calculation at the B3LYP/6-311++G** level. The overall pattern of the

experimental ECD spectrum was in reasonable agreement with the calculated ECD spectra of 1A and 1B (Figure 3). Thus, the absolute configuration of the isoindolone core structure of 1 was established as 3S,4R,5S,6R,7S,8R,9R, but that of C-16 remained to be determined. Compound 2 was obtained as a colorless powder. On the basis of the positive HRESIMS data, the molecular formula C32H42N2O5 was elucidated with 13 degrees of unsaturation. A comparison of the spectroscopic data for 2 and 1 indicated D

DOI: 10.1021/np500626x J. Nat. Prod. XXXX, XXX, XXX−XXX

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Compound 5 was obtained as a colorless powder. The molecular formula C33H40N2O6 was assigned by the positive HRESIMS. The main difference between 5 and 4 was the appearance of a new methoxyl group (Table 1). Thus, compound 5 was assumed to be a methyl ester of 4, as indicated by the HMBC correlation from 20-OMe (δH 3.65 and δC 52.1) to C-20. The relative and absolute configuration of 5 was shown to be the same as that of 2 by careful analysis of their NOESY and experimental ECD spectra (Figure S8, SI). Compound 6 was obtained as colorless crystals. The molecular formula of 6 was assigned as C32H38N2O4 by the positive HRESIMS. Interpretation of the 1H and 13C NMR data of 6 revealed that the structure of 6 was quite similar to that of cytoglobosin E (11).16 The only difference in the 13C NMR was the absence of the carbonyl carbon signal at δC 204.9 in 11 and the presence of a methylene at δC 53.9 in 6, which was supported by HMBC correlations from H-19 to C-20 and C-21. The relative configuration of 6 was elucidated as the same as that of 11 by inspection of the NOESY experiment (Figure 5).

Figure 3. Experimental and calculated ECD of 1 (1A and 1B) in MeOH.

similarities except that an aldehyde group (δH 9.28, δC 197.7) in 1 was replaced by a hydroxymethyl group (δH 3.82, 3.58; δC 68.4) in 2, and an aliphatic chain (C-20−C-23) was located at C-9 in 2 instead of the pyrrole ring in 1. This information was supported by the HMBC correlations from H2-19 to C-17 and Me-18, from H-21 and H-22 to C-20 and C-23, and from H-8 to C-23, as well as the 1H−1H COSY spin system of H2-20/H221/H2-22. The relative configuration of 2 was in agreement with that of 1 based on the similar NOESY spectra and NMR patterns, as well as biosynthetic considerations.8,23,24 As shown in Figure S4 (SI), the Cotton effects in the experimental ECD spectrum of 2 were consistent with those in the calculated ECD curves. Therefore, the absolute configuration of 2 was deduced with the exception of C-16. Consequently, the structure of 2 was characterized by a bicyclic isoindolone decorated with two unusual aliphatic chains (C-13−C-19 and C-20−C-23) and has been accorded the trivial name armochaetoglobin B. Compound 3 was found to possess the molecular formula C32H40N2O5, as evidenced by its positive HRESIMS. The NMR data of 3 closely resembled those of 2. The only difference observed in the 13C NMR spectra was that the signal corresponding to the hydroxymethyl group (δC 68.4) at C-18 in 2 was replaced by an aldehyde (δC 195.6) in 3. This difference was confirmed by the HMBC correlations from H-19 to C-17 and Me-18 and further supported by the downfield shift of C-17 by 4.5 ppm and C-18 by 28.1 ppm. The relative configurations of all stereocenters of 3 were identical to those of 2, as established using the information from the NOESY spectrum and by comparison of the NMR data of the two compounds. In addition, the ECD spectrum of 3 was measured and was almost identical with that of 2, indicating the same chiral centers in the two compounds (Figure S8, SI). Based on the positive HRESIMS of 4, its molecular formula C32H38N2O6 was deduced, containing 14 mass units more than that of compound 3. The signals in the 1H and 13C NMR spectroscopic data of 4 strikingly matched those of 3, indicating that they shared the same 19,20-seco-chaetoglobosin backbone. The only significant difference was the lack of a hydroxymethyl carbon signal at C-20 (δC 61.5) in 3 and the presence of a new carboxyl acid moiety at δC 176.5 in 4. This deduction was further confirmed by the HMBC correlations of H2-21 and H222 to C-20. The observed NOESY correlations of H-3/Me-11, H-3/Me-12, Me-12/H-7, H-7/H-13, H-4/H-10, and H-8/H-22 closely resembled those of 3, indicating the same relative configuration. The absolute configuration of 4 was also determined through comparison of its ECD spectrum with that of 2 (Figure S8, SI).

Figure 4. X-ray structure of compound 6.

After many attempts, a suitable crystal for X-ray diffraction was obtained (MeOH−H2O, 20:1), and the structure and absolute configuration of 6 were unambiguously confirmed by singlecrystal X-ray diffraction analysis with Cu Kα radiation (Figure 4, CCDC 988146). The chiral centers of C-3, C-4, C-5, C-8, C9, and C-16 were assigned as 3S, 4R, 5S, 8S, 9S, 16S on the basis of the refined Flack parameter [0.20 (4)].25 The molecular formula of armochaetoglobin F (7) was deduced from the HRESIMS data. Examination of the 1H and 13 C NMR data and the DEPT (distortionless enhancement by polarization transfer) analysis of 7 showed that this compound bore a close resemblance to isochaetoglobosin J (12).19 The only difference between 7 and 12 was that the keto carbonyl group in 12 was replaced by a hydroxy group at C-20 in 7. This conclusion was supported by the presence of an oxygenated methine signal at δH 4.72 (H-20) and δC 71.9 (C-20). The HMBC correlations of H-20 with C-18 and C-22 and of H2-21 and H2-22 with C-20 confirmed the above deduction. The relative configuration of the isoindolone moiety of 7 was determined by its NOESY spectrum (Figure 5) and shown to be equal to that of 12.19 On the basis of the X-ray structures of 6 and chaetoglobosins E and Fex,15 conformations of the macrocycle ring were relatively stabilized. Subsequently, the NOESY correlations of H-15α with H-13 and H-18, of H-14 with H-8 and H-16, and of H-18 with H-20 established the αorientation of 16-Me and β-orientation of 20-OH in the E

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Figure 5. Key NOESY correlations of compounds 6−10.

HMBC cross-peaks from H-19 to C-17 and C-20 and from H20 to C-18 and C-21. The relative configuration of 9 was deduced by the NOESY (Figure 5) correlations of H-15α with H-13 and H-17, H-19/H-17, and H-17/H-20 and further confirmed by comparing its NMR data with those of cytoglobosin G.16 The absolute configuration of 9 was determined as shown by analogy to that of 6 on the basis of their similar ECD curves recorded for both compounds (Figure S9, SI). The molecular formula of 10 was deduced from the HRESIMS data, which was in accordance with the 13C NMR data. The 1H and 13C NMR spectra of 10 (Tables 1 and 2) showed remarkable similarities to those of chaetoglobosin W (14),20 with differences that resulted from an altered substituent at C-20. A comprehensive comparison of the NMR data showed that the only difference between 10 and 14 was the disappearance of an oxygenated methine carbon (δC 72.2, C-20) in 14 and the presence of a carbonyl group (δC 205.2, C-20) in 10. This conclusion was confirmed by the HMBC correlations from H-21 and H-22 to C-20. The relative configuration of 10 was elucidated as the same as 14 based on the coupling constants and NOESY data (Figure 5). All of the chiral centers of 10 were in complete agreement with those of 14, as demonstrated by the key correlations observed in the NOESY spectra of 10 and their closely related 1H and 13C NMR. Because compound 10 showed nearly the same ECD spectrum as that of 6, its absolute configuration was determined as 3S, 4R, 5S, 6S, 7S, 8R, 9S, 16S (Figure S9, SI). Armochaetoglobins B−J (2−10) were tested for their cytotoxic activities against five human tumor cell lines, including a myeloid leukemia line (HL-60 cells), a hepatocellular carcinoma line (SMMC-7721), a lung carcinoma line (A-549), a breast cancer line (MCF-7), and a colon cancer line (SW480). cis-Platin and paclitaxel were used as positive

macrocyclic ring of 7, which closely resembled those recorded for chaetoglobosin Fex.15 In addition, the absolute configuration of 7 was found to be identical to that of 6, as revealed by their similar rotation values and ECD curves (Figure S9, SI). Compound 8 was obtained as a yellow powder, with the molecular formula C32H36N2O5, as established from a quasimolecular ion peak in the positive HRESIMS. A side-by-side comparison of the NMR data of 8 with those of 6 suggested that compound 8 was structurally related to 6. The differences between 8 and 6 can be explained by replacement of three aliphatic methylene carbons by an oxygenated methine carbon and a pair of olefinic carbons. This information, combined with the HMBC correlations from H-19 to C-18 and C-20 and from H-21 to C-20, C-22, and C-23, along with 1H−1H COSY correlation between H-21 and H-22, indicated the presence of a hydroxy group at C-19 and a double bond between C-21 and C-22 in 8. The relative configuration of 8, determined by a NOESY experiment (Figure 5), was shown to be the same as that of 6, except for the chiral center of C-19. The βconfiguration of 19-OH was deduced by the NOESY correlations from H-17 to H-19 and H-13. In addition, the ECD curve (Figure S9, SI) of 8 was similar to that of 6, which confirmed the absolute configuration of 8. Therefore, the structure of 8 was established as shown. Compound 9 was obtained as a white powder, and its molecular formula of C32 H40 N2 O 4 was established by HRESIMS. A comparison of the 1 H and 13 C NMR spectroscopic data of 9 with those of 1219 revealed that they were similar. However, the appearance of two additional oxygenated methines at δH 3.39 (1H, s, H-19) and 3.23 (1H, dt, J = 11.6, 3.8 Hz, H-20) in 9 suggested that the C-19 and C-20 in 9 were substituted by hydroxy groups, rather than carbonyl groups in 12. This conclusion was further supported by the 1 H−1H COSY correlations of H-19/H-20/H-21/H-22 and the F

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Table 3. Cytotoxic Activities of Compounds 2−9 compound

HL-60

SMMC-7721

A-549

MCF-7

SW480

BEAS-2B

2 3 4 5 6 7 8 9 10 DDP Taxol

16.47 11.86 >40 13.98 >40 12.58 3.31 >40 18.21 1.33 40 13.84 9.83 >40 17.43 5.19 40 19.78 >40 14.26 8.01 >40 17.68 8.98 40 >40 >40 22.70 >40 14.81 4.06 >40 19.14 11.36 40 >40 >40 38.17 >40 20.55 3.90 >40 35.28 13.30 40 5.68 >40 16.03 3.34 >40 16.58 11.11 0.58

eluting with CH2Cl2−MeOH (10:1−3:1) progressively to obtain four fractions (Fr. 1−Fr. 4). Fr. 1 was subjected to CC over silica gel to yield five subfractions (1a−1e). Subfraction 1b was separated with Sephadex LH-20 (MeOH) and further purified by semipreparative RPC18 HPLC to afford 1 (1.5 mg, tR 32.7 min, 65% MeCN in H2O), 4 (6.9 mg, tR 25.8 min, 65% MeOH in H2O), and 12 (6.4 mg, tR 31.4 min, 68% MeOH in H2O). Subfraction 1c was fractionated on Sephadex LH-20 eluted with MeOH, ODS (40% to 100%, MeOH in H2O), and semipreparative RP-C18 HPLC to afford 3 (3.5 mg, tR 42.7 min, 60% MeOH in H2O), 5 (3.8 mg, tR 51.4 min, 50% MeCN in H2O), 6 (4.3 mg, tR 46.4 min, 80% MeOH in H2O), and 7 (14.3 mg, tR 47.0 min, 52% MeCN in H2O). Compound 2 (11.1 mg) was obtained from subfraction 1d by semipreparative RP-C18 HPLC (tR 36.8 min, 47% MeOH in H2O). Fr. 2 was subjected to ODS CC (25% to 100%, MeOH in H2O) to give three subfractions (2a−2c). Compounds 8 (6.1 mg), 9 (7.0 mg), 11 (4.1 mg), and 14 (6.7 mg) were obtained from subfraction 2b by silica gel CC (PE−acetone, 3:1), Sephadex LH-20 (MeOH), and semipreparative RP-C18 HPLC (tR 12.0 min, 60% MeCN in H2O, tR 40.1 min, 47% MeCN in H2O, and tR 21.3 min, 68% MeOH in H2O, respectively). Subfraction 2c was purified by semipreparative HPLC (tR 26.0 and 37.4 min, 48% MeCN in H2O, respectively) to yield compounds 10 (11.0 mg) and 13 (18.2 mg). Armochaetoglobin A (1): white powder; [α]20 D +16.2 (c 0.04, CD3OD); UV (MeOH) λmax (log ε) 204 (5.05) nm; IR νmax 3428, 2925, 1677, 1632, 1454, 1384 cm−1; ECD (MeOH) λmax (Δε) 206 (−0.6), 225 (+0.6), 238 (−0.3) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + H]+ m/z 512.2893, calcd for C32H38N3O3, 512.2913). Armochaetoglobin B (2): white powder; [α]20 D +33.6 (c 0.40, CD3OD); UV (MeOH) λmax (log ε) 203 (4.73), 225 (4.49), 283 (3.67) nm; IR νmax 3401, 2924, 1685, 1632, 1454, 1431, 1383 cm−1; ECD (MeOH) λmax (Δε) 212 (−1.4), 223 (−1.3), 239 (−0.4), 274 (+1.6) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 557.2969, calcd for C32H42N2O5Na, 557.2991). Armochaetoglobin C (3): white powder; [α]20 D +24.8 (c 0.11, CD3OD); UV (MeOH) λmax (log ε) 205 (4.48), 223 (4.62), 281 (3.71) nm; IR νmax 3419, 2927, 1682, 1455, 1384 cm−1; ECD (MeOH) λmax (Δε) 235 (−2.7), 275 (+1.6) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 555.2815, calcd for C32H40N2O5Na, 555.2835). Armochaetoglobin D (4): white powder; [α]20 D +12.0 (c 0.57, CD3OD); UV (MeOH) λmax (log ε) 204 (4.00), 223 (4.23), 276 (3.18) nm; IR νmax 3349, 2925, 1678, 1453, 1385 cm−1; ECD (MeOH) λmax (Δε) 221 (−3.7), 236 (−3.4), 272 (+0.9) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 569.2603, calcd for C32H38N2O6Na, 569.2627). Armochaetoglobin E (5): white powder; [α]20 D +25.5 (c 0.13, CD3OD); UV (MeOH) λmax (log ε) 202 (4.35), 223 (4.56), 281 (3.51) nm; IR νmax 3349, 2925, 1678, 1453, 1385 cm−1; ECD (MeOH) λmax (Δε) 225 (−10.4), 233 (−7.6) nm; for 1H NMR (400

controls for antitumor activity. Among the compounds tested, armochaetoglobin H (8) exhibited moderate cytotoxicity, with IC50 values ranging from 3.31 to 9.83 μM (Table 3). Compounds 2, 3, 5, 7, and 10 showed weak cytotoxic activities. Interestingly, a drastic difference was observed between compounds 4 and 5, suggesting that the seco-chaetoglobosin with a carbonyl moiety may exert better cytotoxic activity. Of particular note, compound 4 profoundly decreased cell viability in the hepatocellular carcinoma SMMC-7721 cells, while having minimal effects on the other tumor cells or the noncancerous BEAS-2B cells. It is likely that compound 4 selectively inhibits signaling events occurring in hepatocellular carcinoma cells.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were determined with a PerkinElmer 341 polarimeter. The UV, ECD, and FT-IR spectra were measured using a Varian Cary 50 instrument, a JASCO-810 ECD spectrometer, and a Bruker Vertex 70 instrument, respectively. The NMR spectra were recorded on a Bruker AM-400 spectrometer, and the 1H and 13C NMR chemical shifts were referenced to the solvent or solvent impurity peaks for CDCl3 (δH 7.26 and δC 77.0), CD3OD (δH 3.31 and δC 49.0), and DMSO-d6 (δH 2.50 and δC 39.5). HRESIMS data were obtained in the positive ion mode on a Thermo Fisher LC-LTQ-Orbitrap XL spectrometer. Semipreparative HPLC was carried out using a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 autosampler injector, and an Ultimate 3000 DAD detector controlled by Chromeleon software (version 6.80), using a reversed-phase C18 column (5 μm, 10 × 250 mm, Welch Ultimate XB-C18). Column chromatography (CC) was performed using silica gel (100−200 and 200−300 mesh; Qingdao Marine Chemical Inc., China), ODS (50 μm, YMC Co. Ltd., Japan), and Sephadex LH-20 (GE Healthcare BioSciences AB, Sweden). Thin-layer chromatography was performed on silica gel 60 F254 (Yantai Chemical Industry Research Institute) and RP-C18 F254 plates (Merck, Germany). Fungal Material. A sample of the fungus Chaetomium globosum TW1-1 was isolated from Armadillidium vulgare in November 2012 at Tongji Medical College, Hubei Province, China. The sequence data for this strain have been submitted to the DDBJ/EMBL/GenBank under accession no. KF993614. A voucher sample, CCM20121113, was preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology. Fermentation and Isolation. The strain was cultured on potato dextrose agar at 28 °C for 7 days to prepare the seed culture. Agar plugs were cut into small pieces (approximately 0.4 × 0.4 × 0.4 cm3) and inoculated into 200 Erlenmeyer flasks (1 L), previously sterilized by autoclaving, each containing 200 g of rice and 200 mL of distilled water. All flasks were incubated at 28 °C for 21 days. At this time, the growth of cells was stopped by adding 300 mL of EtOAc to each flask, followed by ultrasonic extraction with methanol. The methanol was removed under reduced pressure to yield a brown extract (213.0 g). The extracts were subjected to chromatography on a silica gel column G

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MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 583.2762, calcd for C33H40N2O6Na, 583.2784). Armochaetoglobin F (6): colorless crystals; [α]20 D −231.3 (c 0.14, CD3OD); UV (MeOH) λmax (log ε) 205 (4.41), 222 (4.43), 282 (3.43) nm; IR νmax 3425, 2923, 1693, 1455 cm−1; ECD (MeOH) λmax (Δε) 227 (−8.5), 295 (−8.5) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/ z 537.2709, calcd for C32H38N2O4Na, 537.2729). Armochaetoglobin G (7): white powder; [α]20 D −10.9 (c 0.57, CD3OD); UV (MeOH) λmax (log ε) 205 (4.54), 222 (4.64), 283 (3.80) nm; IR νmax = 3414, 2930, 1674, 1439, 1381 cm−1; ECD (MeOH) λmax (Δε) 205 (+4.8), 231 (−7.5) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 537.2708, calcd for C32H38N2O4Na, 537.2729). Armochaetoglobin H (8): yellow powder; [α]20 D −214.7 (c 0.18, CD3OD); UV (MeOH) λmax (log ε) 203 (4.44), 221 (4.52), 276 (3.66) nm; IR νmax 3403, 2921, 1682, 1455, 1382 cm−1; ECD (MeOH) λmax (Δε) 217 (−39.6), 273 (−2.9) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 551.2510, calcd for C32H38N2O4Na, 551.2522). Armochaetoglobin I (9): white powder; [α]20 D −20.2 (c 0.23, CD3OD); UV (MeOH) λmax (log ε) 205 (4.42), 221 (4.37), 283 (3.58) nm; IR νmax = 3415, 2927, 1603, 1447, 1384 cm−1; ECD (MeOH) λmax (Δε) 232 (−3.7), 273 (+1.1) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 539.2830), calcd for C32H40N2O4Na, 539.2886). Armochaetoglobin J (10): white powder; [α]20 D −4.5 (c 0.46, CD3OD); UV (MeOH) λmax (log ε) 203 (4.46), 221 (4.55), 283 (3.91) nm; IR νmax 3406, 2927, 1706, 1626, 1456, 1420 cm−1; ECD (MeOH) λmax (Δε) 241 (−2.4), 295 (−2.0), nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS ([M + Na]+ m/z 567.2407, calcd for C32H36N2O6Na, 567.2471). Computational Details. The conformational analyses were carried out for compounds 1 and 2 by using both the programs BALLOON26 and confab.27 The BALLOON program searches conformational space using a genetic algorithm, whereas the confab program systematically generates diverse low-energy conformations that are supposed to be close to crystal structures. The conformations generated by both programs were grouped together by removing the duplicated conformations in which the root-mean-square (RMS) distance was less than 0.5 Å. The semiempirical PM3 quantum mechanical geometry optimizations were performed on the conformations by using the program Gaussian 09.28 The duplicated conformations after geometry optimization were then identified and removed. The remaining conformations were further optimized at the B3LYP/6-31G* level of theory in methanol solvent with the IEFPCM329 solvation model by using the program Gaussian 09, and the duplicated conformations emerging after these calculations were removed according to the same RMS criteria above. The harmonic vibrational frequencies were calculated to confirm the stability of the final conformers obtained (Figures S1, S3, S5, and S7, SI). The oscillator strengths and rotational strengths of the 20 weakest electronic excitations of each conformer were calculated using the TDDFT methodology at the B3LYP/6-311++G** level of theory with methanol as solvent, using the IEFPCM solvation model implemented in Gaussian 09. The ECD spectra for each conformer were then simulated by using a Gaussian function with a bandwidth σ of 0.45 eV. The calculated spectra for each conformation were combined after Boltzmann weighting according to their population contribution (Tables S1 and S3). The molecular orbital analyses of the major conformations of 1A and 2A were performed at the B3LYP/6-311+ +G(d,p) level (Figures S2 and S6, SI).30,31 Cytotoxic Assay. Five human cancer cell lines (HL-60, SMMC7721, A-549, MCF-7, and SW-480), together with one noncancerous cell line, the human bronchial epithelial cells Beas-2B, were used in the cytotoxic activity assay. All cells were cultured in DMEM or RPMI1640 medium (HyClone, Logan, UT, USA), supplemented with 10% fetal bovine serum (HyClone) at 37 °C in a humidified atmosphere with 5% CO2. The cell viability was assessed by conducting colorimetric measurements of the amount of insoluble formazan

formed in living cells based on the reduction of MTS (3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium) (Sigma, St. Louis, MO, USA).32 Briefly, 100 μL of a suspension of the adherent cells was seeded into each well of the 96well culture plates and allowed to adhere for 12 h before addition of the test compounds. The suspended cells were seeded at an initial density of 1 × 105 cells/mL immediately before the addition of the drug. Each tumor cell line was exposed for 48 h in triplicate to the test compounds at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM, with DDP (cis-platin, Sigma) and paclitaxel as positive controls. After incubation, 20 μL of MTS (5 mg/mL) was added to each well, and the incubation was continued for 4 h at 37 °C. The medium was then removed, and cells were then lysed with 200 μL of 10% SDS. The optical density of the lysate was measured at 595 nm in a 96-well microtiter plate reader (Bio-Rad 680). The IC50 value of each compound was calculated using the method of Reed and Muench.33

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ASSOCIATED CONTENT

S Supporting Information *

ECD computational details of compounds 1 and 2, CD spectra of compounds 3−10, MS and NMR spectra of compounds 1− 10, and X-ray crystallographic data of 6 in CIF format are included. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/np500626x.

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AUTHOR INFORMATION

Corresponding Authors

*Tel/fax: +86-27-83692892. E-mail: [email protected]. cn (Y. Zhang). *E-mail: [email protected] (Y. Xue). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Y. Shi for the molecular orbital analyses and the Analysis and Measurement Centre at HUST for the IR and ECD analyses. This work was financially supported by the Program for New Century Excellent Talents in University, State Education Ministry of China (2008-0224), the National Natural Science Foundation of China (Nos. 31200258, 31370372, 31270395, and 81202423), and National Science and Technology Project of China (Nos. 2011ZX09102004 and 2013ZX09103001-020).

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REFERENCES

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DOI: 10.1021/np500626x J. Nat. Prod. XXXX, XXX, XXX−XXX