Chinese Chemical Letters  2020, Vol. 31 Issue (6): 1406-1409   PDF    
Anti-inflammatory spirobisnaphthalene natural products from a plant-derived endophytic fungus Edenia gomezpompae
Yingzi Tana,1, Zhikai Guob,d,1, Mengyue Zhua, Jing Shia, Wei Lia, Ruihua Jiaoa, Renxiang Tana,c, Huiming Gea,*     
a State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, School of Life Sciences, Nanjing University, Nanjing 210023, China;
b Hainan Key Laboratory of Tropical Microbe Resources, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China;
c State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China;
d Hainan Academy of Tropical Agricultural Resource, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
Abstract: Spirobisnaphthalenes comprise a relatively rare family of natural products that are normally isolated from fungi and occasionally from plants. Here we reported the discovery of seven new preussomerintype spirobisnaphthalenes, preussomerins YT1-YT7 (1-7), and seven known ones (8-14), from the endophytic fungus Edenia gomezpompae, enriching the structural diversity of this family of natural products. Their structures were established by 1D and 2D NMR spectroscopy, HRESIMS analysis and comparison with previously reported compounds, with the absolute configurations of compounds 1 and 2 being further confirmed by single-crystal X-ray diffraction using Cu Kα radiation. The antiinflammatory activities of all isolates were assessed by measuring the production of NO in LPS-induced RAW264.7 macrophage cells. Among them, compounds 8 and 13 exhibited potent inhibitory activities on the production of NO, with IC50 values of 2.61 and 1.32 μmol/L, respectively.
Keywords: Endophytic fungus    Spirobisnaphthalene natural products    Preussomerin-type    Structure elucidation    Anti-inflammatory activity    

Spirobisnaphthalenes are a growing family of rare natural products containing two naphthalene-derived C10 units bridged through a spiroketal linkage [1]. Members of this family include preussomerins, decaspirones, palmarumycins and spiro-nonadienes which have been mainly isolated from fungi and although occasionally from plant sources [1-3]. The preussomerins belong to a relatively rare class of spirobisnaphthalene possessing a unique structural feature. They consist of two unsaturated bicyclo [4.4.0]decane units connected via three oxygen bridges through two spiroketal carbons [1]. This class of compounds have been reported to possess a variety of biological activities such as antifungal, antibacterial, cytotoxic, anti-inflammatory and antileishmanial activities [2, 4-6]. Microbes derived from unexplored or underexplored ecological niches are becoming to be a potential source for discovering structurally interesting and biologically active natural products [7-10]. During our ongoing search for new bioactive metabolites from plant endophytic fungi [11-13], a fungus strain E. gomezpompae was isolated from an unidentified plant collected from Hainan Province of China. LC–MS analysis of its metabolites revealed a series of compounds with molecular weights ranging from 300 to 400 Dalton and a common maximum UV absorption at around 225 nm. Subsequent chemical investigation of this fungus afforded seven new preussomerin-type spirobisnaphthalenes, preussomerins YT1-YT7 (1-7), and seven known compounds, including preussomerin EG1 (8) [14], preussomerin EG2 (9) [14], preussomerin EG4 (10) [15], palmarumycin SA1 (11) [2], MB7056B (Ⅱ) (12) [16], CJ-12, 371 (13) [17], and palmarumycin CP17 (14) [18] (Fig. 1). In this paper, we report the details of isolation, structural elucidation, and anti-inflammatory activity of all isolates (1-14).

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Fig. 1. Structures of compounds 1–14.

A 40-liter fermentation of the fungus E. gomezpompae was conducted and the broth was filtered and extracted with ethyl acetate. The afforded extract was subjected to silica gel column, Sephadex LH-20 column, reversed-phase ODS column chromatography and further purified by reversed-phase HPLC with a gradient CH3CN-H2O system to afford compounds 1–14.

Preussomerin YT1 (1) was obtained as pale yellow crystals, and the molecular formula was determined to be C22H20O7, corresponding to the [M+Na]+ ion at m/z 419.1133 in its HRESIMS, requiring thirteen degrees of unsaturation. The NMR spectra contained characteristic signals for a trisubstituted aromatic ring [δH 7.09 (d, J =8.1 Hz, H-7'), 7.40 (t, J =8.0 Hz, H-8') and 7.50 (d, J =7.6 Hz, H-9'); δC 121.0 (C-5'), 151.2 (C-6'), 121.0 (C-7'), 130.4 (C-8'), 118.8 (C-9') and 130.8 (C-10')], a tetrasubstituted aromatic ring [δH 6.68 (d, J=8.8 Hz, H-7) and 6.78 (d, J =8.8 Hz, H-8); δC 118.5 (C-5), 141.6 (C-6), 116.0 (C-7), 118.1 (C-8), 151.0 (C-9), and 122.2 (C-10)] and one phenolic group [br s, δH 8.19 (9-OH)] (Tables 1 and 2). Analysis of the 13C NMR and HSQC spectra revealed two methoxyl groups at δC 56.3 and 58.5, one carbonyl carbon of a ketone at δC 193.3, two spiroketal carbons at δC 94.1 and 94.9, three methylenes at δC 40.6, 22.3 and 28.6 and two oxygenated methines at δC 71.6 and 79.6 (Table 2). The partial structures H (1)-H (2)-H (3), H (7)- H (8), H (2')- H (3') and H (7')-H (8')-H (9') were established by their 1H–1H COSY correlations (Fig. 2). The key HMBC correlations from H-1 to C-2, C-3, C-5, C-9 and C-10; from H-2 to C-4; and from H-3 to C-4 and C-5 further confirmed the partial structure a (Fig. 3). Similarly, the partial structure b (Fig. 3) was verified by the HMBC correlations of H-9' with C-1' and of H-2', H-3', H-7' and H-9' with C-4'. Clearly, there were two unsaturations left. Thus, substructures a and b must be combined by three oxygens and the structure of 1 could be tentatively determined as shown in Fig. 1. The observation of the coupling constants of 2.4 Hz and 4.6 Hz between H-1 and H- 2 and the coupling constants of 2.8 Hz between H-2' and H-3' proved that H-1 and H-3' both occupied pseudoequatorial orientation. Ultimately, these deductions were strongly proven by single-crystal X-ray diffraction experiment using Cu Kα radiation with the absolute configurations being determined as 1S, 4R, 3'R, 4'S (Fig. 4).

Table 1
1H NMR (400 MHz) spectroscopic data for compounds 1–7 in acetone-d6.

Table 2
13C NMR (100 MHz) spectroscopic data for compounds 1–7 in acetone-d6.

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Fig. 2. Key 1H-1H COSY and HMBC correlations of compounds 1, 3, 5, 7.

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Fig. 3. Partial structures a and b.

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Fig. 4. X-ray crystallographic analysis of 1 and 2.

Preussomerin YT2 (2) was isolated as pale yellow crystals and had the same molecular formula of C22H20O7 as that of preussomerin YT1 (1) deduced from HRESIMS and NMR data (Tables 1 and 2). Analysis of the 1H, 13C NMR, 1H-1H COSY, HSQC and HMBC spectra of 2 revealed that all structural units appeared identical with those of 1. Thus, compound 2 should have the same planar structure as 1 based on the above data. However, compounds 1 and 2 exhibited an epimeric relationship at a center, which is further supported by a comparison of their notable chemical shifts of the asymmetric center C-1. Meanwhile, the large 3J1H, 2H of 8.0 Hz suggested the methoxy group at C-1 adopted the pseudoaxial position. Finally, the absolute configuration of 2 was confirmed as 1R, 4R, 3'R, 4'S by single-crystal X-ray diffraction using Cu Kα radiation (Fig. 4), which only differs from 1 at C-1.

Preussomerin YT3 (3) and preussomerin YT4 (4) were determined to have the same molecular formula of C21H18O7 on the basis of HRESIMS (m/z 405.0953 [M + Na]+). They had characteristic bisspirobisnaphthalene NMR signals as found in preussomerin YT1 (1). Their NMR spectra closely resembled those of 1 except the absence of the methoxy group on C-1 in 1. Furthermore, the presence of a hydroxyl group [δH 4.79 (d) and 5.34 (d)] instead of a methoxyl group at C-1 established a downfield shift of H-1 (δH 5.20, dd, J = 3.2, 6.4 Hz and 5.14, t, J = 7.3, 9.3 Hz) and a upfield shift of C-1 (δC 63.0 and 67.2). Their structures were confirmed by 1H-1H COSY correlations of H-1 with H-2 and of H-1 with OH-1 and the key HMBC correlations of OH-1 with C-1, C-2, C-10 (Fig. 2). For the stereochemistry of C-1, the hydroxyl group OH-1 in 3 was inferred as being placed in the equatorial position of the pseudochair ring owing to the small coupling constant observed between H-1 and H-2 (3J1H, 2H = 3.2 and 6.4 Hz), which were similar to those of 1. Meanwhile, comparison of the 13C NMR data (Table 2) of 3 at C-4, C-3' and C-4' with those of 1 indicated the same configurations as 1. For the stereochemistry of 4, the large coupling constants of H-1 suggested that the hydroxyl group OH-1 was located in the axial position of the pseudochair ring. The 13C NMR data (Table 2) of 4 at C-4, C-3' and C-4' were same with those of 2, implying these carbons have same configurations. Thus, combining with the biogenetic consideration, the absolute configurations of 3 and 4 were tentatively assigned as 1S, 4R, 3'R, 4'S and 1R, 4R, 3'R, 4'S, respectively (Fig. 1).

Preussomerin YT5 (5) and preussomerin YT6 (6) were shown to have the same molecular formula of C21H18O7 based on their HRESIMS data. The 1H and 13C NMR spectra of 5 and 6 (Tables 1 and 2) showed similar chemical shifts to those of preussomerin YT1 (1). Comparison of the 1H and 13C NMR spectra of 5 and 6 with those of 1 indicated the absence of a methoxy group and the presence of an additional hydroxyl group on C-3'. This was confirmed by the correlations (Fig. 2) of the hydroxyl group with its vicinal protons (indicated by 1H-1H COSY and HMBC spectra). Through analysis of the 13C NMR data of these chiral carbons and the protons coupling constants and from the biogenetic consideration, the absolute configurations of 5 and 6 were established to be the same as compounds 1 and 2, respectively (Fig. 1).

Analysis of preussomerin YT7 (7) by HRESIMS and 13C NMR suggested the molecular formula of C20H16O7. Likewise, the 1H and 13C NMR data (Tables 1 and 2) of 7 displayed a similar pattern to those of 1 except for the absence of two oxygenated methyls on C-1 and C-3'. Comprehensive analysis of the 1H, 13C NMR, HSQC, 1H-1H COSY and HMBC spectra of 7 permitted the establishment of the structure of 7 as shown in Fig. 1. The absolute configuration could be deduced from the comparison of the 13C NMR data and the protons coupling constants of 7 with those of 6, established to be 1R, 4R, 3'R, 4'S.

Compounds 8-14 were identified as preussomerin EG1 (8) [14], preussomerin EG2 (9) [14], preussomerin EG4 (10) [15], palmarumycin SA1 (11) [2], MB7056B (Ⅱ) (12) [16], CJ-12, 371 (13) [17] and palmarumycin CP17 (14) [18] by comparing their NMR spectroscopic data with those reported in the literature.

Spirobisnaphthalenes have been reported to have anti-inflammatory activity [2, 19]. Thus, all compounds (1-14) were firstly evaluated for their anti-inflammatory activity by monitoring their inhibitory effects on the production of NO in LPS-induced RAW264.7 macrophage cells. Among them, compounds 8 and 13 displayed significant anti-inflammatory activity at concentrations down to 3 μmol/L with IC50 values of 2.61 μmol/L and 1.32 μmol/L, with no obvious cytotoxicity, whereas the cell survival of RAW264.7 was very low in toxicity studies for other compounds as shown in Table 3.

Table 3
Inhibitory effects of compounds 1–14 on LPS-induced NO production in RAW264.7.

In conclusion, we have identified seven new preussomerin-type spirobisnaphthalenes, preussomerins YT1-YT7 (1-7), and seven known compounds (8-14), from the endophytic fungus E. gomezpompae. Their structures were unambiguously determined by analysis of their NMR and HRESIMS data, with the absolute configurations of compounds 1 and 2 being confirmed by singlecrystal X-ray diffraction. The new modifications of these isolated new compounds expand the chemical diversity of the spirobisnaphthalene family of natural products. Furthermore, antiinflammatory activity assays have showed that compounds 8 and 13 exhibited significant inhibitory activities on the production of NO in LPS-induced RAW264.7 macrophage cells with no obvious cytotoxicity, presenting us with a great opportunity to discover promising natural agents for new anti-inflammatory drugs.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was financially supported by MOST (Nos. 2018YFA0902000 and 2018YFC1706200), and National Natural Science Foundation of China (Nos. 81925033, 21861142005, 81773591, 21761142001, 81673333 and 21661140001), the Central Public-interest Scientific Institution Basal Research Fund for CATAS-ITBB (Nos. 1630052020032, 19CXTD-32 and 1630052019011) and the Hainan Provincial Basic and Applied Basic Research Fund for High-Level Talents in Natural Science (No. 2019RC306).

Appendix A. Supplementary data

Supplementary material related to this article canbefound, in the online version, at doi:https://doi.org/10.1016/j.cclet.2020.03.059.

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