Drimane sesquiterpenoids from a wetland soil-derived fungus Aspergillus calidoustus TJ403-EL05

Sitian Zhang, Shuyuan Mo, Fengli Li, Yaxin Zhang, Jianping Wang, Zhengxi Hu and Yonghui Zhang

Abstract Soil-derived fungi represent an insufficiently tapped reservoir for discovering new and bioactive natural products(NPs), and despite an ever-increasing number of unknown NPs have been discovered over the past few decades,much of the hidden biosynthetic potential is still in an urgent need to be disclosed.In this research, a chemical investigation was performed on a wetland soil-derived fungus Aspergillus calidoustus TJ403-EL05, leading to the isolation of a total of fourteen drimane sesquiterpenoids (1–14), incorporating three new ones, namely ustusols F–H (1–3).Their structures, comprising absolute configurations, were completely authenticated by widespread spectroscopic data,quantum chemical 13C NMR and ECD calculations, and X-ray crystallography experiments.Compound 14 exhibited moderate anti-inflammatory activity by inhibiting the LPS-induced NO release (IC50 = 25.6 μM).

Keywords: Aspergillus calidoustus, Drimane sesquiterpenoids, Structure elucidation, Anti-inflammatory activity

1 Introduction

Over the past few decades, a large proportion of medicines originate from various natural resources, especially from the field of microbiology [1].Terrestrial microorganisms have a huge biosynthetic capacity to produce structurally diverse and pharmacologically active NPs,which have become important chemical entities in drug discovery [2].For example, cyclosporine, isolated from the soil-derivedTolypocladiuminflatum, was the first immunosuppressive agent to enable selective immune regulation of T cells, without excessive toxicity [3].The discovery of cyclosporine in 1971 initiated a new era in the immunopharmacology field, and is still widely used in clinical practice.Therefore, soil-derived fungi have attracted, and will attract increasing attention in the NPsrelated research fields.

Aimed at searching for structurally unique and pharmacologically attractive NPs from the soil-derived fungi[4–6], strainA.calidoustusTJ403-EL05 that was separated from a wetland soil collected from East Lake in Wuhan City, caught our attention and was thus chemically investigated, which afforded three new drimane sesquiterpenoids, namely ustusols F–H (1–3), and eleven known congeners (4–14).In this paper, the isolation, structural characterization, and anti-inflammatory activity of these drimane sesquiterpenoids (Fig.1) were elaborated.

Fig.1 Chemical structures of compounds 1–14

2 Results and discussion

Compound 1 was purified as a colorless crystal.According to the HRESIMS analysis showing a sodium adduct ion atm/z303.1567 (calcd for 303.1567), its molecular formula was determined as C16H24O4,implying 5 degrees of unsaturation.The1H NMR data(Table 1) of 1 showed obvious signals as three methyl protons (δH1.08, 1.04, and 0.90), four methylene protons (δH4.97/4.70, 1.62/2.08, 1.42/1.33, and 1.58),three methine protons (δH6.05, 3.97, and 1.98) and one methoxy proton (δH3.31).With the help of DEPT and HSQC spectroscopic analyses, the13C NMR data(Table 1) of 1 demonstrated the existence of 16 carbon signals that were attributable to three methyls (δC35.0,23.1 and 18.5), one methoxy (δC54.4), four methylenes(δC69.0, 43.1, 30.5, and 18.4), three methines (δC125.2,76.3, and 46.1), four quaternary carbons (δC135.6, 74.6,41.7 and 33.8) and one ester carboxyl carbon (δC175.2).The 1D and 2D NMR data of 1 were highly similar to those of the known 9α-hydroxy-5α-drim-7-ene-6-one-11,12-olide (6) [7], uncovering 1 and 6 to possess the same drimane sesquiterpenoid core skeleton.The significant difference of 1 and 6 was the existence of one methoxy group linked at C-6 in 1 instead of a conjugated ketone carbonyl (C-6) in 6, as further supported based on the key HMBC correlations (Fig.2) of 6-OMe(δH3.31) with C-6 (δC76.3) and of H-6 (δH3.97) with C-5 (δC46.1), C-7 (δC125.2), and C-8 (δC135.6).In the NOESY experiment (Fig.3), the key NOE correlations of H-6 with Me-14 (δH1.04)/Me-13 (δH0.90) and of H-5 (δH1.98) with Me-15 (δH1.08) suggested that H-6, Me-13 and Me-14 should all beβ-oriented, while H-5 and Me-15 were allα-oriented.However, no useful NOE signals could be applied to verify the configuration of C-9.Fortunately, a suitable crystal of 1 was acquired by recrystallization and then furnished for X-ray crystallographic experiment (Fig.4).According to a Flack parameter of 0.01(3), the absolute configuration of 1 was unequivocally confirmed as 5S, 6S, 9S, and 10S.Accordingly, the absolute structure of 1, named as ustusol F, was defined.

Fig.2 Key 1H–1H COSY and HMBC correlations of compounds 1–3

Fig.3 Key NOESY correlations (dashed black arrows) of compounds 1–3

Fig.4 X-ray crystallographic structures of 1, 11, and 14

Compound 2, obtained as a white powder, was determined to possess a molecular formula of C15H24O3, as evidenced by its positive HRESIMS data atm/z275.1618(calcd for C15H24O3Na+, 275.1618).By comparing its1H,13C, and DEPT NMR data (Table 1) with those of the known 6-epi-pereniporin A (4) [8], we could speculate that both compounds were structural analogues, with the only distinction being that one hydroxy group linked at C-11 was absent in 2, as fully supported by the HMBC correlations (Fig.2) of H2-11 (δH3.45) with C-8 (δC138.2), C-9 (δC74.6), and C-12 (δC61.5).Similar NOESY data (Fig.3) and ECD curves (Fig.5) between 1 and 2 proved that these two compounds possessed the identical absolute configuration.Accordingly, the absolute structure of 2, named as ustusol G, was defined.

Table 1 NMR data of 1–3 (δ in ppm, J in Hz)

Fig.5 Experimental ECD curves of compounds 1 and 2 in MeOH

Fig.6 Linear correlation between the experimental and calculated 13C NMR chemical shifts for 3

Fig.7 Experimental and calculated ECD spectra of compound 3

Compound 3 was deduced to have a molecular formula of C15H24O3, as evidenced via its HRESIMS data.By comparing the 1D NMR data (Table 1) of 3 to those of the known ustusol D (11) [9] (Fig.1) whose absolute structure was verified via crystallography experiment(Fig.4), it revealed that both compounds possessed the identical drimane sesquiterpenoid core skeleton, with the only exception that a hydroxy group was linked at C-2 in 11 by C-3 in 3.This conclusion was further corroborated via the key1H–1H COSY cross-peaks of H2-1/H2-2/H-3, as well as the HMBC correlations of both Me-14 and Me-15 with C-3, C-4, and C-5 (Fig.2).The NOE cross-peaks (Fig.3) of Me-15α/H-3/H-5 and H2-11/Me-13βdemonstrated that OH-3 wasβ-oriented in 3.To validate this speculation, the13C NMR chemical shifts of 3 were predicted at the B972/pcSseg-2 level showing the correlation coefficient (R2) value of 0.9985(Fig.6), which completely supported our proposed relative structure.Lastly, the quantum chemical electronic circular dichroism (ECD) calculation was employed for 3.To our expectation, the calculated ECD plot was closely similar to the experimental one (Fig.7), proclaiming its absolute configuration as 3S, 5S, 9R, and 10S, and this compound was named as ustusol H.

Apart from new compounds 1–3, eleven known congeners were also isolated fromA.calidoustusTJ403-EL05 and identified as 6-epi-pereniporin A (4) [8], 6-epi-O-methyl-pereniporin A (5) [8], 9a-hydroxy-5a-drim-7-ene-6-one-11,12-olide (6) [7], 6-dehydroxy-6-oxopereniporin A (7) [8], strobilactone A (8) [10], pereniporin B (9) [11],dendocarbin C (10) [12], ustusol D (11) [9], 9a,11,12-trihydroxydrim-7-en-6-one (12) [13], 12-hydroxyalbrassitriol (13) [14] and drim-8-en-6β,7a,11-triol (14) [15], by comparison of their HRESIMS and NMR data with those reported in the literature.

In the bioactivity assay, due to the limited amounts of 3, other compounds (1–2 and 4–14) were tested for anti-inflammatory activity by using LPS-induced murine macrophages RAW264.7 cells.As a result,only compound 14 was found to show an inhibitory effect against the NO release (IC50= 25.6 μM), and the remaining compounds did not exhibit significant activity with IC50values of > 40 μM (positive control MG132:IC50= 0.32 μM).

Supplementary Information

The online version contains supplementary material available at https:// doi.org/ 10.1007/ s13659- 022- 00349-w.

Additional file 1.Supplementary figures and tables.

Acknowledgements

This project was supported financially by the National Natural Science Foundation for Distinguished Young Scholars (No.81725021), the National Natural Science Foundation of China (Nos.81573316 and 31870326), the Innovative Research Groups of the National Natural Science Foundation of China (No.81721005), the Fundamental Research Funds for the Central Universities (No.2020kfyXJJS083), the National Key R&D Program of China (No.2021YFA0910500), the Research and Development Program of Hubei Province(No.2020BCA058), and the Chinese Medicine Research Foundation of Health Commission of Hubei Province (No.ZY2021Z019).

Author contributions

All authors read and approved the final manuscript.

Funding

The National Natural Science Foundation for Distinguished Young Scholars,81725021, Yonghui Zhang, the National Natural Science Foundation of China,Nos.81573316, 31870326, the Innovative Research Groups of the National Natural Science Foundation of China, No.81721005,Yonghui Zhang, the Fundamental Research Funds for the Central Universities, No.2020kfyXJJS083,Zhengxi Hu, the National Key R&D Program of China, No.2021YFA0910500,Yonghui Zhang, the Research and Development Program of Hubei Province,No.2020BCA058, Jianping Wang, the Chinese Medicine Research Foundation of Health Commission of Hubei Province, No.ZY2021Z019, Jianping Wang.

Declarations

Competing interests

The authors declare no conflict of interest.

Received: 20 April 2022 Accepted: 24 May 2022