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Abstract

A new steroid, chaetglotone (1), together with 3 known compounds (2-4), were isolated from Chaetomium globosum, which is an endophytic fungus isolated from the root of Coptis chinensis Franch. The new compound was characterized by one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometer. The relative configuration and absolute configuration of 1 were further determined via the DP4 + and Early Childhood Development protocols, separately.

Keywords

endophytic fungus secondary metabolites chaetglotone

Introduction

Chaetomium Kunze, the largest genus of Chaetomiaceae, is important for the microbial control of plant diseases. The species within this genus are cosmopolitan and are widespread in soil, air, and plants. The genus Chaetomium has been recognized as an important source of structurally diverse natural products. Approximately 200 metabolites, such as alkaloids, steroids, flavonoids, and terpenoids, have been extracted from Chaetomium spp.¹ Therefore, research on structurally new biologically active products originating from Chaetomium species is continuously increasing.

In recent years, extensive studies on the bioactive chemical constituents of C globosum have been performed by many research groups. Six new indole alkaloids with antibacterial activity were isolated from marine-fish-derived C globosum.² The anti-inflammatory effects have been explored of the secondary metabolites from C globosum, including chaetomugilin I, chaetomugilin J, and 11-epi-chaetomugilin.³ Armochaetoglasin B, chaetoglobosin V, and chaetoglobosin J, isolated from C globosum TW1-1, showed weak cytotoxic activity.⁴ Equisetin, with antimicrobial activities, was isolated from the solid fermentation culture of C globosum.⁵ Chaephilone E, also isolated from C globosum, showed remarkable growth inhibitory activity against brine shrimp.⁶ Three epipolythiodioxopiperazine alkaloids and a pyridine benzamide isolated from C globosum 7951 inhibited the growth of MCF-7, MDA-MB-231, H460, and HCT-8 cells.⁷

A new steroid, chaetglotone (1), and three known compounds (2-4) present in C globosum were found in Coptis chinensis Franch (Figure 1). In this paper, the isolation and structural elucidation of these compounds are described.

Figure 1.

Structures of compounds 1 to 4.

Results and Discussion

Chaetglotone (1) was obtained as a yellowish powder. Based on the high-resolution electrospray ionization mass spectrometer (HR-ESI-MS) ion peak at m/z 497.2872 [M + Na]⁺ (calculated for 497.2874), its molecular formula was identified as C₂₈H₄₂O₆, with 8 degrees of unsaturation. The ¹H-NMR, ¹³C-NMR, and HSQC spectral data indicated that compound 1 had 6 methyl groups (δ_H 0.75 [3H, d, J = 6.9 Hz, H-28], 0.76 [3H, d, J = 6.9 Hz, H-26], 0.93 [3H, d, J = 6.9 Hz, H-27], 1.09 [3H, d, J = 7.0 Hz, H-21], 1.17 [3H, s, H-18], 1.36 [3H, s, H-19]; δ_C 10.4 [C-28], 15.6 [C-26], 22.2 [C-27], 15.0 [C-21], 17.1 [C-18], 22.2 [C-19]), 5 methylene groups (δ_H 1.28 [1H, dd, J = 14.1, 4.3 Hz, H-1], 2.70 [1H, td, J = 13.1, 2.8 Hz, H-1], 1.60 [1H, m, H-2], 1.81 [1H, m, H-2], 2.31 [2H, overlap, H-4], 1.96 [1H, dd, J = 13.3, 6.2 Hz, H-15], 2.41 [1H, dd, J = 13.6, 7.4 Hz, H-15], 2.89 [1H, dd, J = 24.5, 3.9 Hz, H-7], 3.09 [1H, td, J = 24.5, 2.8 Hz, H-7]; δ_C 35.6 [C-1], 32.3 [C-2], 42.4 [C-4], 45.5 [C-15], 28.3 [C-7]), 10 methine groups (δ_H 1.64 [1H, m, H-24], 2.11 [1H, m, H-25], 2.31 [1H, overlap, H-17], 2.60 [1H, m, H-20], 3.30 [1H, overlap, H-23], 3.43 [1H, m, H-3], 3.77 [1H, s, H-12], 4.20 [1H, dd, J = 7.7, 1.2 Hz, H-22], 4.51 [1H, q, J = 6.9 Hz, H-16], 5.45 [1H, t, J = 3.0 Hz, H-6]; δ_C 14.7 [C-24], 27.2 [C-25], 57.9 [C-17], 39.6 [C-20], 74.7 [C-23], 72.2 [C-3], 80.7 [C-12], 85.0 [C-22], 83.7 [C-16], 117.8 [C-6]), 6 quaternary carbons (δ_C 140.3 [C-5], 153.9 [C-8], 135.8 [C-9], 39.1 [C-10], 52.2 [C-13], 83.7 [C-14]), and 1 carbonyl carbon (δ_C 201.3 [C-11]) (Supplemental Table S1). Analysis of the ¹H-¹H correlation spectroscopy (COSY) spectrum indicated strong correlations between H-1/H-2/H-3/H-4, H-6/H-7, H-15/H-16/H-17/H-20/H-22/H-23/H-24/H-25/H-26, H-20/H-21, H-24/H-28, and H-5/H-27 (Figure 2). The ¹H detected heteronuclear multiple bond correlation (HMBC) spectrum revealed correlations between H-19 and C-1/C-5/C-9/C-10, H-6 and C-8, H-7 and C-8, H-15 and C-8, H-12 and C-11, H-18 and C-12/C-13/C-14/C-17, and H-21 and C-20 (Figure 2). The molecular formula revealed that C-16 and C-22 comprise rings with oxygen. Further, the planar structure of compound 1 was confirmed.

Figure 2.

Key HMBC and ¹H-¹H COSY data of 1.

The relative configuration of compound 1 was assigned based on the ROESY spectrum and coupling constants (Figure 3). The ROESY spectrum showed correlations between H-19 and H_b-1/H-12, H-18 and H_b-15/H-20, H-16 and H_a-15, and H-21 and H-17. In addition, the coupling constants of H-22/H-23 and H-23/H-24 were 1.2 and 8.2 Hz, respectively, indicating that H-22/H-23 were in a gauche conformation and H-23/H-24 were in a staggered conformation. Compound 1 is very similar to the known compound asperfloroid⁸ except that the position of C-7 in compound 1 is not substituted by carbonyl. The chirality of the chiral carbons is assumed to be 3S, 10S, 12R, 13S, 16S, 17R, 20S, 22S, 23R, and 24R. However, the chirality of the carbon at position 14 is uncertain because it is a quaternary carbon with inadequate coupling information. Therefore, the DP4 + protocol was chosen for chiral analysis. The conformers were optimized at the B3LYP/6-31 + G(d, p) level, and the optimized representative conformations were subjected to NMR GIAO calculations at the B3LYP/6-311 + G(d, p) level.^9,10 The chirality of C-14 was found to be S*. Therefore, the relative configuration of compound 1 can be determined as 3S*, 10S*, 12R*, 13S*, 14S*, 16S*, 17R*, 20S*, 22R*, 23R*, and 24R*.

Figure 3.

Key nuclear overhausereffect spectroscopy (NOESY) correlations and coupling constants of 1.

The absolute configuration of compound 1 was further determined by comparing the experimental and calculated ECD spectra. The calculated ECD data were obtained using time-dependent density functional theory (TDDFT) at the B3LYP/6-311 + G(d, p) level. The predicted ECD spectra of (3S, 10S, 12R, 13S, 14S, 16S, 17R, 20S, 22R, 23R, 24R)-1 and (3R, 10R, 12S, 13R, 14R, 16R, 17S, 20R, 22S, 23S, 24S)-1 were compared with the experimental ECD spectra. The predicted ECD curves of (3S, 10S, 12R, 13S, 14S, 16S, 17R, 20S, 22R, 23R, 24R)-1 were similar to the experimental ECD curve (Supplemental Figure S3). Thus, the absolute configuration of compound 1 was confirmed to be 3S, 10S, 12R, 13S, 14S, 16S, 17R, 20S, 22R, 23R, and 24R. Compound 1 was named chaetglotone.

The known compounds were identified as 13-epi-higginsianin C (2),¹¹ higginsianin B (3),¹² and (22E, 24R) -ergoster-5, 7, 22-triene-3β-ol (4).¹³

Conclusions

In this study, a new compound, chaetglotone (1), and 3 previously reported compounds (2-4) were isolated, purified, and characterized from the endophytic fungus C globosum isolated from the root of Coptis chinensis Franch.

Experimental Section

General Experimental Procedures

NMR spectra in CD₃OD were recorded on a Bruker DXR-600 instrument (600 and 150 MHz), and optical rotation on an Autopol IV automatic polarimeter (RUDOLPH). HR-ESI-MS data were obtained using a UPLC-Q Exactive MS system (Thermo Fisher), and IR spectra with an IRT racer-100 (Shimadzu) with KBr pellets. Column chromatography (CC) was performed using silica gel (300-400 mesh, Qingdao Haiyang Chemical Co., Ltd) or a Sephadex LH-20 (Aladdin). Semipreparative HPLC was carried out on an Agilent Technologies 1260 Infinity II system with a diode array detector. A C-18 reversed phase column was used (5 μm, 10 × 250 mm) (Agela).

Fungal Material

The endophytic fungus was identified as C globosum. The coverage and maximum similarities were 100%. The GenBank accession number was KU720060.1. The strain was stored in the School of Pharmaceutical Sciences, South Central University for Nationalities.

Extraction and Isolation

The fermentation material was solid rice medium (100 g of rice and 100 mL of water in each 500 mL culture flask) and culture was at 28 °C for one month. The culture was extracted with MeOH (10 L × 4) to obtain 423 g of crude extract. This was mixed with hot water in a ratio of 1:1, and then extracted with light petroleum (2 L × 4) and EtOAc (2 L × 4). The ethyl acetate extract weighed 61.16 g. The crude extracts were separated using positive-phase column chromatography. The eluent was composed of light petroleum: EtOAc (15:1-0:1) and EtOAc: MeOH (15:1 to 0:1). Subsequently, 18 fractions (Fr. 1-Fr. 18) were obtained based on the similarity of the chromatographic behavior. Fr. 8 was recrystallized to yield compound 6 (14.3 mg). Fr. 10 was separated into five fractions (Fr. 10-1-Fr. 10-5) by ODS MPLC. The eluent was composed of MeOH: H₂O (1:9-1:0, stepwise). Fr. 10-3 was separated by Sephadex LH-20 eluting with MeOH and purified by HPLC (acetonitrile: H₂O = 35:65) to obtain compound 1 (5.3 mg, t_R = 21.7 min). Fr. 10-4 was fractionated via Sephadex LH-20 eluting with MeOH, and purified by HPLC (acetonitrile: H₂O = 64:36) to obtain compound 2 (2.8 mg, t_R = 24.5 min) and compound 3 (7.9 mg, t_R = 47.8 min).

Chaetglotone (1): yellowish powder $[α]_{D}^{25}$ = + 52°(c = 0.25, MeOH). UV (MeOH) λmax (logε): 210 (3.49), 255 (3.35) HR-ESI-MS m/z 497.2872 [M + Na]⁺ (calculated for C₂₈H₄₂O₆, 497.2874). ¹H and ¹³C-NMR data see Supplemental Table S1.

Supplemental Material

sj-tif-2-npx-10.1177_1934578X211044574 - Supplemental material for Secondary Metabolites of the Endophytic Fungus Chaetomium globosum Isolated From Coptis chinensis

Supplemental material, sj-tif-2-npx-10.1177_1934578X211044574 for Secondary Metabolites of the Endophytic Fungus Chaetomium globosum Isolated From Coptis chinensis by Jia-Cheng Ji, Pan-Pan Wei, Xiao-Yang Han, Zheng-Hui Li, Hong-Lian Ai and Xin-Xiang Lei in Natural Product Communications

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Supplemental material, sj-png-3-npx-10.1177_1934578X211044574 for Secondary Metabolites of the Endophytic Fungus Chaetomium globosum Isolated From Coptis chinensis by Jia-Cheng Ji, Pan-Pan Wei, Xiao-Yang Han, Zheng-Hui Li, Hong-Lian Ai and Xin-Xiang Lei in Natural Product Communications

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Supplemental material, sj-png-8-npx-10.1177_1934578X211044574 for Secondary Metabolites of the Endophytic Fungus Chaetomium globosum Isolated From Coptis chinensis by Jia-Cheng Ji, Pan-Pan Wei, Xiao-Yang Han, Zheng-Hui Li, Hong-Lian Ai and Xin-Xiang Lei in Natural Product Communications

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Supplemental material, sj-png-10-npx-10.1177_1934578X211044574 for Secondary Metabolites of the Endophytic Fungus Chaetomium globosum Isolated From Coptis chinensis by Jia-Cheng Ji, Pan-Pan Wei, Xiao-Yang Han, Zheng-Hui Li, Hong-Lian Ai and Xin-Xiang Lei in Natural Product Communications

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Supplemental material, sj-png-13-npx-10.1177_1934578X211044574 for Secondary Metabolites of the Endophytic Fungus Chaetomium globosum Isolated From Coptis chinensis by Jia-Cheng Ji, Pan-Pan Wei, Xiao-Yang Han, Zheng-Hui Li, Hong-Lian Ai and Xin-Xiang Lei in Natural Product Communications

Footnotes

Acknowledgments

The authors thank the Analytical & Measuring Center, School of Pharmaceutical Sciences, SCUN for their help with NMR measurements.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China (grant nos. 31870513, 2019CFA072).

ORCID iD

Hong-Lian Ai

Supplemental Material

Supplemental material for this article is available online.

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