Chaetoglobosins from Chaetomium globosum, an Endophytic Fungus


Apr 7, 2014 - ABSTRACT: In preceding studies, cultivation of Chaetomium globosum, an endophytic fungus in Ginkgo biloba, produced five cytochalasan ...
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Chaetoglobosins from Chaetomium globosum, an Endophytic Fungus in Ginkgo biloba, and Their Phytotoxic and Cytotoxic Activities He Li,∥ Jian Xiao,∥ Yu-Qi Gao, Jiang−Jiang Tang, An-Ling Zhang, and Jin-Ming Gao* Shaanxi Engineering Center of Bioresource Chemistry and Sustainable Utilization, College of Science, Northwest A&F University, Yangling 712100, Shaanxi, People’s Republic of China S Supporting Information *

ABSTRACT: In preceding studies, cultivation of Chaetomium globosum, an endophytic fungus in Ginkgo biloba, produced five cytochalasan mycotoxins, chaetoglobosins A, G, V, Vb, and C (1−5), in three media. In the present work, five known chaetoglobosins, C, E, F, Fex, and 20-dihydrochaetoglobosin A (5−9), together with the four known compounds (11−14), were isolated from the MeOH extracts of the solid culture of the same endophyte. The structures of these metabolites were elucidated on the basis of spectroscopic analysis. Treatment of chaetoglobosin F (7) with (diethylamino)sulfur trifluoride (DAST) in dichloromethane afforded an unexpected fluorinated chaetoglobosin, named chaetoglobosin Fa (10), containing an oxolane ring between C-20 and C-23. The phytotoxic effects of compounds 1, 3−8, and 10 were assayed on radish seedlings; some of these compounds (1, 3, and 6−8) significantly inhibited the growth of radish (Raphanus sativus) seedlings with inhibitory rates of >60% at a concentration of 50 ppm, which was comparable or superior to the positive control, glyphosate. In addition, the cytotoxic activities against HCT116 human colon cancer cells were also tested, and compounds 1 and 8−10 showed remarkable cytotoxicity with IC50 values ranging from 3.15 to 8.44 μM, in comparison to the positive drug etoposide (IC50 = 2.13 μM). The epoxide ring between C-6 and C-7 or the double bond at C-6(12) led to a drastically increased cytotoxicity, and chaetoglobosin Fa (10) displayed a markedly increased cytotoxicity but decreased phytotoxicity. KEYWORDS: Ginkgo biloba, endophytes, Chaetomium globosum, chaetoglobosins, phytotoxicity, cytotoxicity, mycotoxins



medium, afforded chaetoglobosins A (1) and C (5);15 fermentation of this fungus using Sabouraud’s medium led to the production of chaetoglobosins G (2), V (3), and Vb (4);16 recultivation of the same fungus on modified PDA medium provided chaetoglobosin G (2) and chaetoglobosin C (5).17 In continuation of our search for biologically active metabolites generated by Chaetomium species,18 the fractionation of the MeOH extract of the cultures of the endophytic C. globosum using rice medium led to the isolation of nine alkaloids including five known chaetoglobosins (5−9) and four known compounds 11−14 (Figure 1). Additionally, we semisynthesized a new fluorinated chaetoglobosin F derivative, chaetoglobosin Fa (10), starting from chaetoglobosin F (7) with DAST. Herein, we report the isolation of these isolated fungal metabolites, preparation of chaetoglobosin Fa (10), and phytotoxic and cytotoxic evaluation of the semisynthesized and natural compounds.

INTRODUCTION Fungi are an important source of bioactive meatbolites that exhibit a wide range of bioactivities, such as antitumor, phytotoxic, fungistatic, and bacteriostatic properties.1 These metabolites offer interesting templates for potential agricultural and medicinal uses. Minor changes in molecules enhance specific activity and improve bioactivity.1 Fungi of the Chaetomium species, which belong to the family Chaetomiaceae, are the largest genus of saprophytic ascomycetes, with more than 350 Chaetomium species.2 Up to now, more than 200 metabolites with a wide range of bioactivities have been isolated from the genus Chaetomium, but compared with its richness of species, more bioactive secondary metabolites might be found in this fungus.2 Chaetoglobosins, well-known mycotoxins biosynthesized by a hybrid polyketide synthase−nonribosomal peptide synthetase, represent an important subfamily of 10-(indol-3-yl)-[13]cytochalasan natural products that have attracted considerable interest because of a large variety of biological activities, such as cytotoxic, antifungal, phytotoxic, and nematicidal activities.2−13 To date, more than 40 chaetoglobosins have been reported from the cultures of some fungi, most belonging to the genus Chaetomium.2 On the basis of the knowledge that the biosyntheses of secondary metabolites in endophytes are dependent on the culture parameters and available nutrition (one strain−many compounds, OSMAC) to improve the search for potent bioactive substances,14 previous investigations of Chaetomium globosum, an endophytic fungus isolated from the fresh healthy leaves of Ginkgo biloba using potato dextrose agar (PDA) © 2014 American Chemical Society



MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on an Autopol III automatic polarimeter (Rudolph Research Analytical). IR spectra were run on a Bruker TENSOR27 spectrophotometer. UV spectra were measured by a Thermo Scientific Evolution-300 UV−visible spectrophotometer. NMR experiments were carried out on a Bruker Avance III 500 spectrometer, with

Received: Revised: Accepted: Published: 3734

November 14, 2013 March 19, 2014 April 7, 2014 April 7, 2014 dx.doi.org/10.1021/jf500390h | J. Agric. Food Chem. 2014, 62, 3734−3741

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Figure 1. Structures of compounds 1−14: chaetoglobosins A (1), G (2), V (3), Vb, (4), C (5), E (6), F (7), and Fex (8); 20-dihydrochaetoglobosin A (9); and chaetoglobosin Fa (10), as well as fumigaclavine B (11), 5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (12), 2′-O-methyladenosine (13), and 5′-deoxy-5′-methylamino-adenosine (14). tetramethylsilane (TMS) or CF3COOH as internal and external standards, respectively. Electrospray ionization mass spectrometry (ESI-MS) was obtained on a Bruker Esquire 6000 instrument, and high-resolution ESI-MS was recorded on a Thermo Fisher Scientific Q-TOF mass spectrometer. Thin-layer chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd.). Column chromatography (CC) was performed on MCI gel (75−150 mm, Mitsubishi Chemical Corp., Tokyo, Japan), 200−300 mesh silica gel (Qingdao Marine Chemical Factory, China), Sephadex LH-20 (Pharmacia), and RP-18 (Merck). Fractions were monitored by TLC, and spots were visualized by spraying with 5% H2SO4 in ethanol, followed by heating. All other chemicals used in this study were of analytical grade. Five cytochalasans, chaetoglobosins A, G, V, Vb, and C (1−5), were isolated and identified previously from the cultures of C. globosum, an endophyte residing in the leaves of G. biloba, in three different media.15−17 Fungal Material. The fungal strain C. globosum was isolated from the sterilized leaves of G. biloba, a medicinal plant growing in Linyi,

Shandong province, China, authenticated on the basis of morphological studies,15 and has been deposited at the College of Science, Northwest A&F University. Preparative Scale Culture. This fungus was grown on a sterilized moistened rice medium in Roux flasks (100 g/flask × 200) at 25 °C for 21 days to give green moldy rice. Extraction and Isolation. The fungal cultures were ultrasonically extracted four times with MeOH. The solvent was removed under reduced pressure to give a crude extract. The extract was dissolved in 90% MeOH/H2O (2 L) and treated four times with petroleum ether to give 0.5 g of residue. The remaining layer was adjusted to 50% aqueous methanol and partitioned with CHCl3. The chloroformsoluble portion (20 g) was subjected to column chromatography on silica gel with CHCl3/MeOH (100:1, 50:1, 20:1, 10:1) to give four fractions A−D. Fraction A (4.5 g) was subjected to MCI column chromatography (CC) and Sephadex LH-20 (CHCl3/MeOH 1:1), followed by purification with PTLC to yield compounds 5 (12 mg) and 7 (50 mg). 3735

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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data of Compounds 7 and 10a compound 10b

compound 7 no.

a

δC

c

δH (mult, J in Hz)

c

1 2 3 4 5 6 7 8 9 10

173.64

11 12 13 14 15 16 17 18 19 20 21

12.11 19.13 128.52 132.85 40.20 32.80 147.71 134.97 209.10 70.29 32.74

0.61 (3H, 1.09 (3H, 6.33 (1H, 5.17 (1H, 1.97, 2.37 2.75 (1H, 6.19 (1H,

22 23 1′ 2′ 3′ 3′a 4′ 5′ 6′ 7′ 1′a 16-Me 18-Me

36.41 204.06

2.11, 2.61 (2H, m)

52.16 47.89 35.66 56.94 61.22 46.85 63.66 29.88

124.27 109.02 127.52 117.92 120.84 118.42 111.44 136.00 19.63 11.98

δC

b

175.23 8.29 3.63 2.34 1.57

(1H, (1H, (1H, (1H,

s) m) m) overlap)

2.64(1H, m) 2.18 (1H, m) 1.57 (2H, overlap) d, 7.1) s) m) dd, 14.4, 11.7) (2H, m) m) d, 9.5)

4.58 (1H, m) 2.58 (2H, m)

10.88 (1H, br s) 7.09 (1H, s)

7.43 7.05 6.97 7.32

(1H, (1H, (1H, (1H,

d, 7.8) m) m) d, 8)

0.98 (3H, d, 6.5) 1.69 (3H, s)

53.39 48.36 37.03 57.55 62.45 49.80 66.11 31.85

δC (mult, J in Hz)

δH (mult, J in Hz)

174.21 53.71 50.45 36.91 57.37 62.62 45.79 56.86 (JC,F = 28.8) 35.69

12.85 19.65 129.94 133.61 41.01 33.51 148.47 135.84 209.75 71.95 34.84

13.99 20.48 128.96 133.77 41.23 34.23 153.30 130.75 202.62 84.46 24.25

38.07 205.03

36.23 (JC,F = 30.4) 123.66 (JC,F = 224.7)

124.98 110.81 128.51 118.91 121.95 119.54 112.29 137.63 19.78 12.43

124.75 111.74 128.30 118.80 122.08 119.62 112.36 137.89 19.29 11.80

9.13 4.20 2.72 2.14

(1H, (1H, (1H, (1H,

s) m) m) m)

3.00 (1H, m) 3.02 (1H, m) 3.30 (1H, 3.20 (1H, 1.16 (3H, 1.30 (3H, 5.35 (1H, 7.02 (1H, 2.04, 2.37 2.70 (1H, 6.73 (1H,

m, H-10a) m, H-10b) d, 7.4) s) m) dd, 15.3, 9.2) (2H, m) m) d, 9.8)

5.40 (1H, m) 2.69 (1H, m, H-21a) 2.21 (1H, m, H-21b) 2.36−2.45 (2H, m) 12.08 (1H, br s) 7.57 (1H, s)

7.94 7.36 7.34 7.67

(1H, (1H, (1H, (1H,

d, 7.5) m) m) d, 7.5)

0.92 (3H, d, 6.7) 1.85 (3H, s)

Assignments were aided by a combination of 1H−1H COSY, HSQC, and HMBC experiments. bMeasured in C5D5N. cMeasured in DMSO-d6. m/z 529.17 [M − H]−; 1H and 13C NMR (DMSO-d6) data were identical to those recorded in the literature.5 20-Dihydrochaetoglobosin A (9): white amorphous powder; [α]D15 = −56.2 (c 0.10 in CHCl3) (lit.5 [α]D = −52.0 (MeOH)); 1 H NMR (500 MHz, DMSO-d6) δ 10.84 (1H, s, H-2), 8.15 (1H, s, H1′), 7.38 (1H, d, J = 7.9 Hz, H-4′), 7.31 (1H, d, J = 8.1 Hz, H-7′), 7.04 (1H, m, H-5′), 7.01 (1H, s, H-2′), 6.94 (1H, m, H-6′), 6.74 (1H, dd, J = 15.2, 3.3 Hz, H-21), 6.42 (1H, d, J = 15.1 Hz, H-22), 6.06 (1H, m, H-13), 5.10 (1H, m, H-14), 4.98 (1H, d, J = 4.5 Hz, 20-OH), 4.87 (1H, d, J = 5 Hz, 19-OH), 4.60 (1H, d, J = 9.9 Hz, H-17), 4.44 (1H, br s, H-20), 4.01(1H, br s, H-19), 3.62 (1H, m, H-3), 2.76 (1H, dd, J = 14.0, 5.2 Hz, H-10a), 2.66 (1H, d, J = 5.4 Hz, H-7), 2.32−2.42 (3H, m, H-4, 8, 10b), 2.52 (1H, m, overlap, H-16), 2.22 (1H, m, H-15a), 1.70 (2H, m, H-5, 15b), 1.63 (3H, s, 18-Me), 1.06 (3H, s, H-12), 0.88 (3H, d, J = 6.5 Hz, 16-Me), 0.52 (3H, d, J = 7.2 Hz, H-11); 13C NMR (125 MHz, DMSO-d6) δ 198.03 (C-23), 173.35 (C-1), 147.81 (C-21), 136.08 (C-1a′), 134.74 (C-14), 133.93 (C-18), 131.40 (C-17), 127.30 (C-3a′), 126.07 (C-13), 123.51 (C-5′), 123.10 (C-22), 120.92 (C-2′), 118.43 (C-6′), 117.95 (C-4′), 111.39 (C-7′), 109.78 (C-3′), 78.14 (C19), 76.04 (C-20), 63.21 (C-9), 61.07 (C-7), 56.82 (C-6), 52.13 (C3), 46.74 (C-8), 45.63 (C-4), 38.82 (C-15), 35.78 (C-5), 32.96 (C10), 31.73 (C-16), 21.26 (16-Me), 19.01 (C-12), 13.58 (18-Me),

Fraction B (1.8 g) was applied to MCI CC using MeOH/H2O (10:90−100:0) to provide fractions B1, B2, B3, and B4. Fraction B3 was subjected to Sephadex LH-20 (CHCl3/ MeOH 1:1), then followed by silica gel with CHCl3/acetone (8:2) and CHCl3/MeOH (35:1) to give compounds 6 (18 mg) and 8 (10 mg). Fraction C (6.1 g) was applied to MCI CC using MeOH/H2O (10:90−100:0) to provide fractions C1, C2, C3, C4, and C5. Fraction C2 was purified by repeated CC on Sephadex LH-20 (CHCl3/MeOH 1:1) and silica gel (CHCl3/MeOH, 95:5; CHCl3/acetone 1:1) to give compound 14 (4.7 mg). Fraction C4 was chromatographed over Sephadex LH-20 with MeOH and repeatedly purified by CC with CHCl3/acetone (8:2) and CH2Cl2/MeOH (98:2) to give compound 9 (5 mg). Fraction D (2.9 g) was purified by CC on MCI, Sephadex LH-20 (MeOH), and silica gel (petroleum ether/acetone, 1:1; CHCl3/ acetone, 2:1; CHCl3/MeOH, 10:1), to give compounds 11 (1.8 mg), 12 (10 mg), and 13 (4 mg). Chaetoglobosin F (7): white amorphous powder; [α]D15 = −67.5 (c 0.10 in CHCl3) (lit.12 [α]D = −69.0 (CHCl3)); 1H and 13C NMR data, see Table 1; ESI-MS (negative) m/z 529.17 [M − H]−. These data were identical to those recorded in the literature.4,19,20 Chaetoglobosin Fex (8): white amorphous powder; [α]D15 = +62.5 (c 0.10 in CHCl3) (lit.5 [α]D = +63.8 (MeOH)); ESI-MS (negative) 3736

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12.02 (C-11); ESI-MS (negative) m/z 529.17 [M − H]−. These data were identical to those recorded in the literature.5,21 Semisynthesis of Chaetoglobosin Fa (10).22 To a solution of compound 7 (37 mg, 0.070 mmoL) in CH2Cl2 (2 mL) was added DAST (28 μL, 0.21 mmoL), and the mixture was stirred at room temperature for 1.5 h. Water (2 mL) was added to the solution, and the mixture was diluted with CH2Cl2 (30 mL). The solution was washed sequentially with water (2 × 15 mL) and brine (15 mL), dried over Na2SO4, and filtered, and the solvent was removed in vacuo. The residue was chromatographed over silica gel (petroleum ether/acetone 7:3) to give compound 10 (20 mg, 53.9%). Chaetoglobosin Fa (10): white amorphous powder; [α]D29 = −98.5 (c 0.20 in CHCl3); UV (CHCl3) λmax (log ε) = 227(3.55), 242 (3.74), 282 (3.40), 290 (3.30); 1H and 13C NMR data, see Table 1; IR (KBr) υ 3390, 2955, 2923, 1686, 1638, 1073 cm−1; HR ESI-MS (negative) m/z 531.2656 [M − H]− (calcd for C32H36N2O4F, 531.2665). Fumigaclavine B (11): white amorphous powder; 1H NMR (500 MHz, CD3OD) δ 7.13 (1H, d, J = 8.1 Hz, H-12), 7.09 (1H, m, H-13), 6.93 (1H, d, J = 6.9 Hz, H-14), 6.90 (1H, s, H-2), 4.54 (1H, m, H-9), 3.44 (1H, dd, J = 14, 4.3 Hz, H-4a), 3.21(1H, d, J = 10.2 Hz, H-7a), 2.95 (1H, dd, J = 11.4, 2.6 Hz, H-7b), 2.68 (1H, m, H-5), 2.64 (1H, m, H-4b), 2.63 (1H, H-10), 2.47 (3H, s, NMe), 2.16 (1H, m, H-8), 1.28 (3H, d, J = 7.4 Hz, H-17); 13C NMR (125 MHz, CD3OD) δ 135.39 (C-15), 130.53 (C-11), 123.52 (C-13), 123.52 (C-16), 119.25 (C-2), 113.92 (C-12), 109.51 (C-14), 109.51 (C-3), 69.62 (C-9), 62.39 (C5), 58.36 (C-7), 43.89 (N-Me), 41.83 (C-10), 36.88 (C-8), 26.99 (C4), 17.12 (C-17); ESI-MS (positive) m/z 257.0 [M + H]+. These data were identical to those reported in the literature.23 5-(Hydroxymethyl)-1H-pyrrole-2-carbaldehyde (12): white amorphous powder; 1H NMR (500 MHz, CD3OD) δ 9.36 (1H, s, H-7), 6.98 (1H, d, J = 3.6 Hz, H-3), 6.26 (1H, d, J = 3.5 Hz, H-4), 4.60 (2H, s, H-6); 13C NMR (125 MHz, CD3OD) δ 180.59 (C-7), 143.38 (C-5), 133.95 (C-2), 123.42 (C-3), 110.41 (C-4), 57.84 (C-6); ESI-MS (negative) m/z 124.08 [M − H]−. These data were identical to those described in the literature.24 2′-O-Methyladenosine (13): white amorphous powder; 1H NMR (500 MHz, DMSO-d6) δ 8.39 (1H, s, H-2), 8.14 (1H, s, H-8), 7.37 (2H, s, NH2), 6.00 (1H, d, J = 5.97 Hz, H-1′), 4.34 (1H, m, H-3′), 4.33 (1H, m, H-2′), 3.98 (1H, m, H-4′), 3.67 (1H, m, H-5′), 3.30 (3H, s, OMe); 13C NMR (125 MHz, DMSO-d6) δ 156.20 (C-6), 152.56 (C-8), 149.01 (C-4), 139.72 (C-2), 119.28 (C-5), 86.47 (C-4′), 85.82 (C-2′), 82.45 (C-1′), 68.81 (C-3′), 61.51 (C-5′), 57.48 (C-OMe); ESI-MS (positive) m/z 282.04 [M + H]+. These data were identical to those described in the literature.25 5′-Deoxy-5′-methylamino-adenosine (14): white amorphous powder; 1H NMR (500 MHz, CD3OD) δ 8.48 (1H, s, H-2), 8.40 (1H, s, H-8), 6.05 (1H, m, H-1′), 4.77 (1H, m, H-2′), 4.47 (1H, m, H4′), 4.37 (1H, m, H-3′), 3.45 (1H, m, H-5′a), 3.20 (1H, m, H-5′b), 2.67 (3H, s, N−CH3); 13C NMR (125 MHz, CD3OD) δ 153.37 (C6), 150.87 (C-4), 146.42 (C-2), 144.83 (C-8), 121.76 (C-5), 91.23 (C1′), 79.58 (C-4′), 75.79 (C-2′), 75.39 (C-3′), 57.91 (C-5′), 40.19 (N− CH3); ESI-MS (positive) m/z 281.07 [M + H]+. These data were identical to those described in the literature.26 Phytotoxicity Bioassay. Phytotoxicity was assayed by the method reported previously.27,28 Briefly, radish seedlings were washed by running water for 120 min and then soaked in 0.3% KMnO4 for 15 min and flushed to colorless. An acetone solution containing a sample at a defined concentration (200 or 50 ppm) was poured on two sheets of filter paper in a 12-well microplate. After removal of the solvent in vacuo, 200 μL of aseptic water was added on it. Radish seedlings of uniform shape and size were placed on the filter paper. In this experiment the seedlings were grown for 4 days at 25 °C under completely dark conditions. The inhibitory effects were observed after 96 h. Glyphosate was used as the positive control. Acetone served as a blank control. Each experiment was conducted three times and presented as the mean ± standard deviation of three replicates. Cytotoxicity Bioassay. Cytotoxicity in vitro was assessed by the SRB colorimetric assay, which estimates cell number indirectly by measuring total basic amino acids of cultured cells.29 Briefly, 100 μL aliquots of the exponentially growing HCT116 cells containing 2.5 ×

104 cells/mL were added to each well of a 96-well flat-microtiter plate and cells allowed to attach for 24 h. Then the medium was replaced by fresh medium, and cells were incubated with various amounts of the test compound for an additional 72 h. Four replicate wells were used in each point in the experiments. After incubation at 37 °C, culture medium was moved, cells were fixed in situ with 100 μL aliquots of cold 10% trichloroacetic acid (TCA), and plates were incubated for 1 h at 4 °C. Thereafter, supernatant was discarded and plates were washed five times with distilled water and air-dried. Sulforhodamine B solution at 0.4% (w/v) in 1% acetic acid was added to each well, and plates were incubated for 20−30 min at room temperature. The unbound dye was removed by washing five times with 1% acetic acid, and plates were air-dried. Bound sulforhodamine B was subsequently solubilized with 10 mM Tris base, and the absorbance was read at 560 nm using an Epoch (Bio-Tek) microplate reader. The percentage of cell viability was calculated relative to control wells designated as 100% viable cells. All of the results were expressed as the mean ± standard deviation of triplicate determinations. The data analysis was performed using Origin 8.0 software.



RESULTS AND DISCUSSION Cultivation, Isolation, and Structure Elucidation. The fungal strain was cultured in rice medium at 28 °C for 21 days. The extract of the culture was partitioned between chloroform and 50% MeOH/H2O. The chloroform extract was repeatedly purified by RP-18 gel, silica gel, and Sephadex LH-20 CC to afford nine compounds. The structures of the known compounds were characterized by means of spectroscopic comparisons with literature data as chaetoglobosins C (5), E (6), F (7),4,19,20 and Fex (8),5 20-dihydrochaetoglobosin A (9),5,21 fumigaclavine B (11),23 5-(hydroxymethyl)-1H-pyrrole2-carbaldehyde (12),24 2′-O-methyladenosine (13),25 and 5′deoxy-5′-methylamino-adenosine (14) (Figure 1).26 Some of the isolated chaetoglobosins (A, C, E, and F) are well-known metabolites isolated from different fungal strains of C. globosum,2 whereas 20-dihydrochaetoglobosin A (9), which was reported for the first time from the plant endophytic C. globosum, had been isolated before from C. subaffine.5 A search of known C. globosum secondary metabolites from the CrossFire Beilstein database revealed that these four compounds, 11−14, had not been previously described in C. globosum. Semisynthesis of Chaetoglobosin Fa (10). Selective introduction of fluorine atom(s) to bioactive natural and unnatural products is of extreme interest in pharmaceutical research30 because fluorine atom(s) are well-known as pharmacophore(s). (Diethylamino)sulfur trifluoride (DAST) is often used for the preparation of fluorinated compounds. This reagent can convert alcohols into fluorides or ketones into gem-difluoro compounds under quite mild conditions.22,31 As a consequence, we made attempts to obtain 20-fluorochaetoglobosin F through treatment of chaetoglobosin F (7) with DAST (3 equiv) in dichloromethane at room temperature for 1.5 h; unexpectedly, however, this process failed to directly give 20fluorinated product but provided 23-fluorinated product chaetoglobosin Fa (10) in 53.9% yield, which possesses an oxolane ring between C-20 and C-23, as shown in Scheme 1. HR-ESIMS of compound 10 gave a pseudomolecular ion at m/z 531.2656 [M − H]− (calcd for C32H36N2O4F, 531.2665). These data, coupled with 13C NMR spectroscopic data, established the molecular formula of 10 as C32H37N2O4F (15 degrees of unsaturation), suggesting 2 amu more than the mass of chaetoglobosin F (7). The 1H-decoupled 19F NMR spectroscopic data also confirmed the existence of the fluorine atom (δF −100) in 10 (see Figure S3 in the Supporting 3737

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Scheme 1. Chemical Transformation of 7 into 10

Information). The IR spectrum of 10 exhibited absorption bands at 3390 (NH and/or OH), 1686 (CO), and 1638 (CO) cm−1. The 13C NMR and DEPT spectra of 10 (Table 1) displayed 32 carbon signals, including 4 methyls, 4 sp3 methylenes, 7 sp3 and 8 sp2 methines, 3 sp3 and 4 sp2 quaternary carbons, and 1 ketone carbon (δC 202.62), as well as an amide carbonyl (δC 174.21). The 1H NMR spectrum of 10 showed the presence of an indole moiety, including 1,2disubstituted benzene [δH 7.94 (t, J = 7.5 Hz, H-4′), 7.36 (m, H-5′), 7.34 (m, H-6′), and 7.67 (t, J = 7.5 Hz, H-7′)], an olefinic proton at δH 7.57 (H-2′), and a broad NH singlet at δH 12.08 (H-1′), along with the 13C NMR spectrum, which gave six aromatic carbons at δC 137.89 (s, C-1′a), 128.30 (s, C-3′a), 119.62 (d, C-6′), 122.08 (d, C-5′), 118.80 (d, C-4′), and 112.36 (d, C-7′) and two olefinic carbons at δC 124.75 (d, C2′) and 111.74 (s, C-3′). In addition, the 1H NMR spectrum showed the presence of two tertiary methyls (δH 1.30, 1.85), two secondary methyls (δH 0.92, 1.16), three olefinic protons (δH 5.35, m; 6.73, d; 7.02, dd), and an amide proton (δH 9.13, s). Comparison of its 13C NMR data with those of 7 revealed that an additional sp3 quaternary carbon C-23 (δC 123.66) with a huge coupling constant (JC, F = 224.7 Hz) in 10 appeared and that a corresponding ketone carbon at δC 204.06 in 7 disappeared. Meanwhile, two carbons adjacent to C-23, an sp3 methylene carbon C-22 signal (δC 36.23, JC, F = 30.4 Hz) and a quaternary carbon C-9 (δC 56.86, JC, F = 28.8 Hz), were also observed. These typical splitting patterns indicated that this fluorine atom was attached to C-23, as evidenced by HMBC correlations from H-21 and H-22 to C-23. The downfield chemical shift of sp3 hybridized C-23 (δC 123.66) revealed the connection with the additional oxygen atom. Considering degrees of unsaturation, this molecule should contain an oxolane ring between C-20 and C-23. This deduction was supported by HMBC correlations of H-20 (δH 5.40) with C-23 and H-21 with C-20 as well as 1H−1H COSY correlations of H-20/H-21/H-22 (Figure 2). This gave rise to significant differences in the chemical shifts measured in C5D5N of C-17 to C-23 between 7 and 10 (Table 1). Finally, comprehensive 1H−1H COSY and HMBC analysis (Figure 2) allowed the complete assignment of the proton and carbon signals for 10 (see the Supporting Information). Rearrangement reactions are sometimes observed in DAST fluorination.32−34 For example, preparation of γ-fluorobutyrolactones from the reaction of γ-ketoacids with DAST was

Figure 2. Key HMBC, 1H−1H COSY, and ROESY correlations in 10.

reported by Chen et al.34 In the current work, similarly, the mechanism of oxolane ring formation between C-20 and C-23 in 10 is postulated to occur via the pathway (Figure 3). The

Figure 3. Potential mechanism of chemical transformation of compound 7 into 10.

first step is the nucleophilic displacement of fluorine on sulfur by oxygen of the 20-OH accompanied by elimination of hydrogen fluoride to give the intermediate 7a. Then, the addition of fluorine to C-23 of 7a was accompanied by O-23 attached to C-20 from the backside and elimination of Et2NFSO and F−.35,36 Consequently, an inversion of the configuration of C-20 in the resulting 10 took place, which was further confirmed by the cross-peak between H-8 and H-20 in the ROESY spectrum of 10 (Figure 2). 3738

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The configuration of the core skeleton, 10-(indol-3-yl)[13]cytochalasan, in these chaetoglobosins could be assigned on the basis of the biosynthesis with those of chaetoglobosin A (1)21 as well as on a ROESY experiment. The absolute configuration of 20-dihydrochaetoglobosin A (9) was established previously by X-ray single-crystal analysis as 3S, 4R, 5S, 6R, 7S, 8R, 9R, 16S, 19R, and 20S,21 so the absolute configuration of 7 was assigned as 3S*, 4R*, 5S*, 6R*, 7S*, 8R*, 9R*, 16S*, and 20S*. The members of the chaetoglobosin family possess structures that are chirally dense at six (C-3, C-4, C-5, C-8, C-9, C-16) or eight vicinal positions (C-3, C-4, C-5, C-6, C-7, C-8, C-9, C-16) and one remote site (C-19/C-20). Interestingly, the sign of the rotation is not sensitive to the minor structural changes. For example, chaetoglobosin A (1), 20-dihydrochaetoglobosin A (9), and chaetoglobosin Fa (10) reported here and prochaetoglobosin I21 all showed negative specific rotations, despite a large variance in structure at C-6, C7, C-19, and C-20 and configuration at C-5 and C-8. In addition, the co-occurrence of these isolated metabolites 1−9 in the title fungus suggests that they shared the same biogenetic pathway, and biogenetic insights previously published3,21 have suggested that the configuration at C-4, C-5, C-8, and C-9 is set via a stereospecific enzyme-mediated Diels−Alder reaction.11,21 After several attempts, unfortunately, no good crystals suitable for X-ray analysis of chaetoglobosin Fa (10) were obtained because this compound is easily changeable in proton solvents. Consequently, the absolute stereochemistry of the new semisynthetic derivative 10 was proposed as 3S*, 4R*, 5S*, 6R*, 7S*, 8R*, 9R*, 16S*, and 20R*. Thus, the total structure of 10 was elucidated as shown in Figure 1. It should be mentioned that the absolute configuration at the new chrial center C-23 of 10 remains undetermined. Phytotoxic Activity of Selected Compounds. The phytotoxic effects of eight selected compounds 1, 3−8 and 10, except for 2 and 11−14 (due to insufficient samples) on radish seedlings were examined, and the results are listed in Table 2. Chaetoglobosin A (1) darkened the roots at two concentrations of 200 and 50 ppm, these results being consistent with those obtained with alfalfa seedlings,27 and other compounds also showed similar inhibitory effects. At a low concentration of 50 ppm, compounds 1, 3−8, and 10 exhibited weak to strong phytotoxic activities against the roots with inhibition rates in the range of 26.4−67.5% and against hypocotyl elongation with inhibition rates ranging from 33.3 to 71.6% (except 10). Among the tested compounds, at 50 ppm, inhibition rates of chaetoglobosins A (1), V (3), E, F, and Fex (6−8) were >60% toward the roots, as active as positive control, glyphosate (63.5%); chaetoglobosins A (1), C, E, F, and Fex (5−8) displayed phytotoxic activities to hypocotyls with inhibition rates of almost ≥60%, which were about 2-fold of the control. In comparison to 7, its semisynthetic fluorinated derivative, chaetoglobosin Fa (10) weakly inhibited the root growth of the seedlings with 26.4 and 59.4% of inhibition at 50 and 200 ppm, respectively, but had no phytotoxicity to hypocotyls at either concentration. In addition, at low concentrations, >60% inhibition of chaetoglobosin V (3) on radish seedlings was >33−46% that of Vb (4). It seemed that the configurations of C-17 and C-21 in both molecules could influence phytotoxicity potency. At 200 ppm, chaetoglobosins A (1) and C−F (5−7) exhibited phytotoxicity to roots with inhibition rates from 71.7 to 76%, which were slightly weaker than the control (81.5%). Meanwhile, chaetoglobosins A (1) and C−F (5−7) presented

Table 2. Inhibitory Effects of Compounds 1, 3−8, and 10 on the Growth of Radish Seedlings inhibition ratioa (%) compd

a

concn (ppm)

radish root

radish hypocotyl

1

200 50

74.0 ± 3.1 63.5 ± 3.8

64.4 ± 3.0 60.0 ± 3.1

3

200 50

36.3 ± 2.8 62.3 ± 2.1

55.1 ± 3.0 71.6 ± 2.8

4

200 50

65.2 ± 2.9 46.8 ± 4.3

48.1 ± 2.8 33.3 ± 2.4

5

200 50

73.4 ± 2.9 50.0 ± 1.4

71.2 ± 4.0 71.0 ± 3.8

6

200 50

76.0 ± 3.2 65.5 ± 2.5

64.3 ± 4.1 69.0 ± 2.5

7

200 50

71.7 ± 2.9 67.5 ± 2.1

60.7 ± 3.7 64.4 ± 2.4

8

200 50

67.0 ± 2.6 64.6 ± 2.3

52.9 ± 2.49 57.9 ± 2.6

10

200 50

59.4 ± 2.3 26.4 ± 2.8

2.5 ± 2.0 4.3 ± 2.8

glyphosate

200 50

81.5 ± 3.0 63.5 ± 3.2

67.0 ± 2.3 33.9 ± 2.6

Mean ± SD.

phytotoxic effects on hypocotyls with inhibition rates from 60.7 to 71.2%, almost as active as glyphosate (67.1%). These findings demonstrated that radish seedlings were highly sensitive to these tested chaetoglobosins at low concentrations but only slightly sensitive at high concentrations. It was interesting that the dose−response relationship of these tested compounds was not apparent in our bioassay. It has been proved that some chaetoglobosins have been found to be phytotoxic to different plants. Chaetoglobosin A (1) and its 19-O-acetylchaetoglobosin A, isolated from the culture of Calonectria morganii, inhibited the root and shoot growth of cresson seedlings;37 chaetoglobosins A (1), C (5), and O, phytotoxic metabolites from Cylindrocladium floridanum, a causal fungus of alfalfa black rot disease, caused potent growth inhibition on alfalfa seedlings.27 Furthermore, chaetoglobosin K showed growth-inhibiting effects on wheat coleoptiles, isolated from cultures of Diplodia macrospora, a corn pathogen causing ear and stalk rot.38 This is the first report of phytotoxicity of these test chaetoglobosins to radish seedlings. In Vitro Cytotoxic Evaluation of Compounds 1−10. All 10 chaetoglobosins (1−10) were evaluated for their cytotoxic activities against the HTC116 cell line except for chaetoglobosin C (5), which has low solubility in DMSO, and the results are presented in Table 3. Four of them, chaetoglobosins A (1) and Fex (8), 20-dihydrochaetoglobosin A (9), and chaetoglobosin Fa (10), displayed high cytotoxic activity with IC50 values of 3.15, 4.43, 8.44, and 5.85 μM, respectively, as compared to the antitumoral drug etoposide (IC50 = 2.13 μM), whereas chaetoglobosins G (2), V (3), Vb (4), E (6), and F (7) were found to be weak (IC 50 > 17 μM). Among them, 3739

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had toxic effects to mouse and rat cells and chick embryos.44,45 To our knowledge, this is the first report on the cytotoxicity of these chaetoglobosins to the HCT116 cell line. In summary, in this study, nine known metabolites including chaetoglobosins C, E, F, and Fex, and 20-dihydrochaetoglobosin A (5−9) and four known compounds (11−14) were identified from the solid culture of the endophytic C. globosum, and a new fluorinated chaetoglobosin, chaetoglobosin Fa (10), was semisynthesized from chaetoglobosin F. The phytotoxic compounds (1, 3, and 6−8) displayed marked phytotoxic activity against radish seedlings at 50 ppm, which were comparable to the positive control. Compounds 1 and 8−10 exhibited pronounced cytotoxicity against the HCT116 colon cancer cell, in contrast to the positive drug. The epoxide ring between C-6 and C-7 or the double bond at C-6(12) appeared to be important for cytotoxicity, and chaetoglobosin Fa (10) presented a markedly increased cytotoxicity but decreased phytotoxicity. On the basis of results in both bioassays, there was no relationship between cytotoxicity and phytotoxicity. The mechanisms of action of some bioactive compounds we have identified will need to be studied.

Table 3. Cytotoxicity of Compounds 1−10 against Human Colorectal HCT116 Cell Line IC50 (μM)a

improve cytotoxicity

1

3.15 ± 0.45

epoxide ring;b 20-carbonyl group

2

65.6 ± 5.9

3

29.5 ± 2.7

C-17/C-21 junction

4

18.4 ± 2.0

C-17/C-21 junction

compd

a

5 6

ntc >100

7 8 9 10

17.8 4.43 8.44 5.85

etoposide

2.13 ± 0.45

± ± ± ±

1.9 0.52 0.89 0.65

epoxide ringb double bond at C-6(12) epoxide ringb epoxide ring;b fluorine atom, furan ring

decrease cytotoxicity

epoxide ring disappearsb epoxide ring disappearsb epoxide ring disappearsb epoxide ring disappearsb 20-OH 20-OH 20-OH

Mean ± SD. bEpoxide ring at C-6/C-7. cNot tested.



chaetoglobosin A (1) was about 2.5 times more active than 20dihydrochaetoglobosin A (9) on HCT116 cells, suggesting that reduction of the carbonyl group at C-20 lowered cytotoxicity. This deduction was consistent with that of chaetoglobosin G (2) (IC 50 = 65.6 μM), showing higher toxicity than chaetoglobosin E (6) (IC50 > 100 μM), despite their lower toxicity. Chaetoglobosin F (7) exhibited much higher cytotoxic abilities than chaetoglobosin E (6), with IC50 values of 17.8 and >100 μM, respectively. These results suggested that the introduction of an epoxide ring at C6/C7 greatly improved cytotoxicity. Chaetoglobosin Fex (8) (IC50 = 4.43 μM) showed much stronger cytotoxic effects than chaetoglobosin E (6), implying that the presence of a double bond at C6(12) was favorable for imparting cytotoxicity. Especially, chaetoglobosin Fa (10) (IC50 = 5.85 μM) was about 3 times more active than the parent 7 (IC50 = 17.8 μM) toward HCT116 cells. This indicated that the introduction of F and the formation of the furan ring in 10 could enhance activity. Chaetoglobosins V (3) and Vb (4), incorporating a five-membered ring between C-17 and C-21, were found to be weak, with IC50 values of 29.5 and 18.4 μM, respectively, but stronger than the parental compound 2 (IC50 = 65.6 μM), indicating the C-17/C-21 junction in both molecules appeared to improve toxicity. These findings suggested that an epoxide ring at C-6−C-7 or a double bond at C-6(12) is an important feature responsible for activity. Reportedly, among chaetoglobosins is chaetoglobosin A (1), which has a unique inhibitory activity against actin polymerization in mammalian cells.3 Chaetoglobosins displayed cytotoxicity on a panel of human cancer cell lines.2,3,39,40 Chaetoglobosins A (1), C (5), E (6), and G (2) exhibited acute toxic effects against HeLa cells (IC50 = 3.2−20 μM).20,41 Compound 1 possessed significant cytotoxicity against P388 murine leukemia cell line.6 Compounds 5 and 2 showed cytotoxicities against human breast cancer (BC1) cell lines (IC50 = 6.27−20.0 μM).10 Recently, chaetoglobosins C (5) and F (7) exhibited moderate cytotoxic activity against SKOV-3 cells (IC50 = 2−8 μM).42 Chaetoglobosins A (1), G (2), V (3), C (5), E (6), F (7), and Fex (8) exhibited moderate cytotoxic activities against four human cancer cell lines (KB, K562, MCF7, and HepG2).43 Compounds 1 and 5 presented significant growth inhibitory activity against the brine shrimp15 and also

ASSOCIATED CONTENT

S Supporting Information *

1D and 2D NMR spectra of compound 10. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*(J.-M.G.) Phone: +86-29-87092515. E-mail: [email protected] nwsuaf.edu.cn. Author Contributions ∥

H.L. and J.X. contributed equally to this work.

Funding

This work was financially supported by the National Natural Science Foundation of China (31371886). Notes

The authors declare no competing financial interest.



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