Immunosuppressive Isopimarane Diterpenes From Cultures of the Endophytic Fungus Ilyonectria robusta

Five new isopimarane diterpenes, robustaditerpene A-E (1–5), which include 19-nor-isopimarane skeleton and isopimarane skeleton, were isolated from the liquid fermentation of the endophytic fungus Ilyonectria robusta collected from Bletilla striata. The structure elucidation and relative configuration assignments of all compounds were accomplished by interpretation of NMR and HRESIMS spectrometric analyses and 13C NMR calculation. And the absolute configuration of 1-5 were identified by single-crystal X-ray diffraction and ECD calculation. Compound 3 inhibited lipopolysaccharide-induced B lymphocytes cell proliferation with an IC50 value at 17.42 ± 1.57 μM while compound 5 inhibited concanavalin A-induced T lymphocytes cell proliferation with an IC50 value at 75.22 ± 6.10 μM. These data suggested that compounds 3 and 5 may possess potential immunosuppressive prospect.


INTRODUCTION
Immunosuppressive agents are a significant class of clinical drugs for organic transplantation and treatment of autoimmune diseases, such as rheumatoid arthritis, systemic lupus, multiple sclerosis, myasthenia gravis, and pemphigus (Dangroo et al., 2016). Although excellent advantages were taken, these immunosuppressive agents have some inevitable and serious side effects including the renal and liver toxicity, infection, malignancy, and others (Smith et al., 2003). Therefore, it is a significant requirement to develop new, efficient, and safe immunosuppressive agents.
Natural products play a highly important role in the drug discovery and development process. Many clinically applied drugs derived from or were natural products (Newman and Cragg, 2016). Several immunosuppressive agents, such as mycophenolic acid, cyclosporin A, tacrolimus, sirolimus, corticosteroids and so on, were also generated from natural products (Kahan, 2003;Wang et al., 2019).
Endophytic fungi are a rich source of natural products (Ye et al., 2021). The number of natural products from endophytic fungi are more than any other endophytic microorganism class, and they have various structures and a broad spectrum of bioactivities. Furthermore, natural products from endophytic fungi have various, even novel structures, which can be grouped into several types including alkaloids, steroids, terpenoids, quinones and so on (Tan and Zou, 2001;Zhang et al., 2006). One of the largest groups of bioactive natural products that have been identified is the terpenoids. Diterpenoids derived from C20 precursor (E, E, E)-geranylgeranyl diphosphate with more than 12,000 described compounds (Quin et al., 2014;Zi et al., 2014). Some isopimarane diterpenes with immunosuppressive activity have been reported (Chen et al., 2020).
For searching the leading compounds with immunosuppressive activity, we studied an endophytic fungus Ilyonectria robusta, isolated from the medicinal plant Bletilla striata. Five new isopimarane diterpenes (1-5) ( Figure 1) were obtained from the crude extract of this fungus. Biological evaluation suggested that some compounds displayed immunosuppressive activity against T and B lymphocytic cell proliferation. Herein, we describe isolation, elucidation, and biological activity of these isopimarane diterpenes.

Fungal Material
The strain Ilyonectria robusta was isolated from the rhizome of Bletilla striata collected from Enshi, Hubei province, and was identified as Ilyonectria robusta via 18S rDNA-sequence and deposited at South-Central University for Nationalities, China. The sequence data for this strain had been submitted to the DDBJ/EMBL/Genbank with accession No. JX045819.1.

Fungal Fermentation, Extraction and Isolation
The strain Ilyonectria robusta was cultured on potato dextrose agar medium at 25°C for 5 days to prepare the seed culture. The agar plugs were cut into small pieces and inoculated to 500 ml Erlenmeyer flasks each containing 300 ml of liquid medium (5% glucose, 0.15% peptone from porcine meat, 0.5% yeast extract, 0.05% KH 2 PO 4 , and 0.05% MgSO 4 . Total volume was 50 L) and fermented on a rotatory shaker at 25°C and 160 rpm for a month.
The culture broth (50 L) of Ilyonectria robusta was centrifuged to separate the mycelium and liquid cultures. The liquid layer was concentrated under reduced pressure to afford 8 L of extract and further partitioned with ethyl acetate 24 L for three times at room temperature. The mycelia were soaked with MeOH (total 6 L) at room temperature. The extract was evaporated under reduced pressure, resolved in water (2 L), and further extracted with ethyl acetate 6 L three times.

General Experimental Procedures
Optical rotations were measured on a Rudolph Autopol IV polarimeter. UV spectra were obtained on a UH5300 UV-VIS Double Beam Spectrophotometer. IR spectra were obtained by using a Shimadu Fourier Transform Infrared spectrophotometer with KBr pellets. 1D and 2D NMR spectra were run on Bruker Avance III 600 MHz and Bruker Avance NEO 500 MHz Frontiers in Pharmacology | www.frontiersin.org January 2022 | Volume 12 | Article 766441 2 spectrometer with TMS as an internal standard. X-ray crystallographic analysis was on the Bruker D8 QUEST. High Resolution Electrospray Ionization Mass Spectra (HRESIMS) were recorded on a Thermo scientific Q Exactive Orbitrap MS system. Column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People's Republic of China), RP-18 gel (20-45 μm, Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Sweden). Medium Pressure Liquid Chromatography (MPLC) was performed on a Biotage SP1 equipment, and columns packed with RP-18 gel. Preparative High Performance Liquid Chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 μm, 9.4 × 150 mm or 21.2 × 150 mm, flow rate 4 ml/min) and a DAD detector. Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd. Qingdao), and spots were visualized by heating silica gel plates sprayed with 10% H 2 SO 4 in EtOH. A Cell Counting Kit-8 assay kit was used to measure cell viability (Dojindo, Kumamoto, Japan).

Quantum Chemical Calculation Methods
Systematic conformational analyses were performed by SYBYL-X 2.1.1 based on molecular mechanics with MMFF94s force field (Goto and Osawa, 1989). The conformers with a distribution higher than 1% will be further optimized. The optimization and frequency of conformers were calculated on B3LYP/6-31G(d) level of theory with the IEF-PCM solvent model (MeOH) in the Gaussian09 software package (Frisch et al., 2013).

C NMR Calculation
After analyzing and optimizing the conformer candidates of (2′R*)-4 and (2′S*)-4, the conformers within 4 kcal/mol of the global minimum were selected for further 13 C NMR calculations. Gauge-independent atomic orbital (GIAO) calculations of the shielding values of (2′R*)-4 and (2′S*)-4 were accomplished by the time-dependent density functional theory (TDDFT) at the ωB97x-D/6-31G(d) level with IEF-PCM solvent model (MeOH) in the Gaussian09 software package (Frisch et al., 2013). The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energies. The linear correlation coefficient (R 2 ) and root-mean-square deviation (RMSD) were calculated for the evaluation of the deviations between the experimental and calculated results. The calculation results were processed by an in-house Excelbased program.

ECD Calculation
The optimized conformers were selected for further electronic circular dichroism (ECD) calculation. The ECD (TDDFT) calculation of all conformations was performed on B3LYP/6-311G(d) Level of theory with IEF-PCM solvent model (MeOH). All the DFT calculations were performed by Gaussian09 software package (Turney et al., 2012). The calculated and weighted ECD curve were all generated by SpecDisv 1.71 (Bruhn et al., 2013), respectively.

T and B Cell Proliferation Inhibitory Activity Assay
All the compounds were subjected to evaluate their inhibition on the proliferation of T and B lymphocytes. Fresh spleen cells were obtained from female BALB/c mice (18-20 g). Spleen cells (1 × 10 6 cells) were seeded in triplicate in 96well flat plates and cultured at 37°C for 48 h in 96-well flat plates, in the presence or absence of various concentrations of
The molecular formula of Robustaditerpene C (3), a white powder, was determined as C 20 H 30 O 5 by (-)-HRESIMS (m/z 349.20373 [M-H] -). Analysis of the 1D NMR data ( Table 1) of 3 suggested that it was an isopimarane derivative bearing the structural similarities to 2, except that the oxygenated methylene at δ c 59.1(C-16) in two became a carboxyl group at δ c 176.0 (C-16) in 3. This finding was supported by the HMBC correlations ( Figure 4) from H-15 (δ H 2.15) to C-16 (δ c 176.0), and from Me-17 (δ H 0.90) to C-12 (δ c 38.0), C-13 (δ c 34.6), C-14 (δ c 47.8), and C-15 (δ c 49.9). The ROESY analysis ( Figure 4) indicated relative configuration of 3 was the same as that of 2. Biosynthetically, compounds 2 and 3 shared the same isopimarane skeleton, and the absolute configuration was determined based on the comparison of calculated ECD and experimental CD spectra ( Figure 5). The structure of 3 was assigned as shown.
Robustaditerpene D (4), white powder, gave a molecular formula C 23 H 36 O 6 , as established by HRESIMS data (m/z 431.24045 [M + Na] + ). The 1D NMR data ( Table 2) were closely related to those of 2, except for three additional carbons at δ c 176.4 (C-1′), δ c 67.9 (C-2′), and δ c 20.5 (C-3′), and the downfield-shifted carbon C-16 (δ c 62.8 in 4, δ c 59.1 in 2). In combination with comparison of molecular mass of 2 and 4, the HMBC correlations from H-16 to C-1′ and H-3′ to C-1′ and the 1 H-1 H COSY correlations of H-2′/H-3′ ( Figure 6) indicated that the three additional carbons (C-1′ C-2′, and C-3′) were a lactic acid esterified with the hydroxyl at C-16. Further analysis of 2D NMR data suggested other parts of 4 were the same as those of 2. In accordance with the biogenetic pathway rules, the absolute configuration of 2 would be the same as the other parts of 4, which indicated other of 4 to be the isopimarane skeleton. Therefore, 4 consisted of esterification of the hydroxyl at C-16 of 2 and a lactic acid. However, the R/S configuration of C-2′ can't be determined by the ROESY signals. To further confirm the relative configuration of 4, especially at C-2′, 13 C NMR calculations of two possible conformers (2′R*)-4 and (2′S*)-4 were performed. As shown in Figure 7, the correlation coefficient (R 2 ), mean absolute deviation (MAE), and the largest deviations (Δ max ) of the (2′R*)-4 isomer [R 2 0.9991, MAE 1.16 ppm, Δ max 3.2 ppm (C-19)] were better than (2′S*)-4 isomer [R 2 0.9985, MAE 1.58 ppm, Δ max 3.4 ppm (C-19)] (Supplementary Material), which suggested that the relative configuration of C-2′ is R. The absolute configuration of 4, the R/S configuration of the lactic acid part, was determined by the comparison of calculated ECD and experimental CD spectra (Figure 7). Compound 4 was characterized as shown.
Robustaditerpene E (5) was isolated as a yellow amorphous solid, and its molecular formula C 23 H 34 O 7 (m/z 445.21960 [M + Na] + ), was established by HRESIMS. The 1D NMR data ( Table 2) were similar to those of 3, but three additional carbons at δ c 176.0 (C-1′), δ c 68.0 (C-2′), and δ c 20.6 (C-3′) and downfield-shifted C-2 (δ c 70.8 in 5, δ c 65.3 in 3). According to the HMBC correlations from H-2 to C-1′ (δ c 176.0) and H-3′ to C-1′, the 1 H-1 H COSY signals ( Figure 6) of H-2′/H-3′, and the comparison of molecular mass of 3 and 5, these additional carbons also belonged to a lactic acid esterified with the hydroxyl at C-2. Other parts of 5 were identical to those of 3 after the analysis of 2D NMR spectra. Thus, 5 consisted of esterification of the hydroxy at C-2 of 3 and a lactic acid. Biogenetically, other parts of 5 and 3 shared the same isopiamarane skeleton, and absolute configuration. The absolute configuration of 5, in particular the R/S configuration of lactic acid part was based on the comparison of calculated ECD and experimental CD spectra ( Figure 8). Thus, compound 5 was determined as shown.

T and B Cell Proliferation Inhibitory Activity
The relatively abundant compounds 1-5 were evaluated for their inhibitory activity against cell proliferation of concanavalin A (Con A)-induced T lymphocytes and lipopolysaccharide (LPS)induced B lymphocytes. As shown in Table 3, robustaditerpene C 3) and E (5) showed the suppressive activity against the cell proliferation of LPS-induced B lymphocytes with IC 50 value at 17.42 ± 1.57 μM and Con A-induced T lymphocytes with IC 50 value at 75.22 ± 6.10 μM, respectively. These data indicate that compounds 3 and 5 have certain research value in immunosuppression.
Isopimarane diterpenes are biosynthetically formed from the conversion of geranylgeranyl pyrophosphate into (+)-copalyl cation and then in turn into sandaracopimaraneyl cation (Dewick, 2009). Interestingly, these diterpenes are generally obtained from plants and fungi, which showed diverse bioactivity (Reveglia et al., 2018). Until now, isopimarane diterpenes with immunosuppressive activity were first reported  in 2020 (Chen et al., 2020). This research provides several isopimarane scaffolds with potential immunosuppressive activity and enriched structure diversity. These compounds give more choices to develop new agents with inhibition on the steps of immune response. And these isopimarane scaffold compounds will be modified by organic synthesis to improve the immunosuppressive activity.

CONCLUSION
Herein, five new isopimarane diterpenes, which possess 19-norisopimarane skeleton or isopimarane skeleton, were obtained from the endophytic fungus Ilyonectria robusta. According to 1D and 2D NMR analysis, X-ray single crystal diffraction analysis, 13 C NMR calculation, and ECD calculation, the absolute configuration of these compounds was confirmed. Biological evaluation for immunosuppressive activity indicated that compound 3 and 5 displayed some activity against cell proliferation of B lymphocytes and T lymphocytes, respectively. Among these compounds, single crystal of compound 1 was obtained.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
The studies involving animals were reviewed and approved by Animal Ethics Committee of South-Central University for Nationalities [SYXK (Wuhan) 2016-0089, No.2021-SCUEC-AEC-033].

AUTHOR CONTRIBUTIONS
H-LA isolated and provided the fungus. H-LA, J-KL, and D-KZ designed experiments. KY was responsible for compounds isolation and elucidated the structures. KY, XL, XZ, and P-PW performed chemical calculation. Z-HL was responsible for the evaluation of immunosuppressive activity. KY wrote the paper. All authors read and approved the final manuscript.

FUNDING
This work was supported by the National Natural Science Foundation of China (31870513, 32000011, 31960082).