Microporotriol, a new cadinane-type sesquiterpenoid from the cultures of the wood-decay fungus Microporus affinis HFG829

Abstract A new cadinane-type sesquiterpenoid, microporotriol (1), together with four known compound, 5-methylresorcinol (2), (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one (3), (22E,24R)-ergosta-5,7,22-trien-3β-ol (4), (22E,24R)-5α,8α-epidioxy-ergosta-6,22-dien-3β-ol (5), were isolated from the fermentation broth of the wood decaying fungus Microporus affinis HFG829. The structures of the compounds were established by extensive spectroscopic methods, including 1D & 2D NMR, along with HRMS spectroscopic analysis. The relative configuration of 1 was confirmed by NMR calculation. Compound 1 was evaluated for the cytotoxicity against five human cancer cell lines. Graphical abstract


Introduction
Secondary metabolites from fungi are of medical, industrial and/or agricultural importance which have attracted, and continue to attract much attention from the scientific community (Schueffler and Anke 2014;Chen and Liu 2017). Fungal secondary metabolites are proved to be associate with fungal sporulation processes and development (Calvo et al. 2002), for example, zearalenone from Fusarium graminearum played an inducing role in sporulation and an enhancing role in perithecial formation (Wolf and Mirocha 1973), and butyrolactone I from Aspergillus terreus induces the sporulation and increases the production of lovastatin (Schimmel et al. 1998).
The wood-decay fungi is a class of fungi possessing the ability to produce laccases that digest moist woods and then cause them to rot. This process triggers the degradation of complex molecules to return nutrients to the soil in nature (Riley et al. 2014). However, sometimes this kind of fungi also can serve as the role of pathogenic factor for forest and mushroom-cultivation industry. The special roles of wood-decay fungi make them of great importance to be chemically investigated. The fungus Microporus affinis falls into the family of Polyporaceae, and always grows on the fallen woods in tropical and subtropical regions (Bi et al. 1993). However, this fungus has never been chemically studied. In an ongoing program to explore biological active substances from fungi, the culture broth of M. affinis HFG829 was investigated, which led to the isolation of a new cadinane-type sesquiterpenoid, together with four known compounds. Cadinane-type sesquiterpenoid is quite common as fungal metabolites, such as (þ)-10a-hydroxy-4-muurolen-3-one isolated from Favolaschia sp. 87129 with inhibition of leukotriene biosynthesis (Zapf et al. 1996); stereumins C and D exhibiting potent nematicidal activities against Panagrellus redivivus (Li et al. 2008). We, herein, report the isolation, structure elucidation, and biological evaluation of the new compound.

Results and discussion
Compound 1 was isolated as a colourless oil. The HREIMS spectrum of 1 showed an ion peak at m/z 256.2038 [M] þ , corresponding to the molecular formula of C 15 H 28 O 3 (calcd for C 15 H 28 O 3 , 256.2038). Compound 1 could not be detected by the UV detector during the separation process implying the absence of any chromophores, like the carbonyl or double bond. The 13 C NMR and DEPT data (Table S1) presented carbon resonances which were classified into three methyls, five methylenes (one oxygenated), six methines (one oxygenated), and one oxygenated quaternary carbons. The planar structure of 1 was established by analysis of the HSQC, HMBC, and 1 H-1 H COSY spectra. The 1 H-1 H COSY spectrum confirmed the presences of a six-membered ring (C 1 ÀC 2 ÀC 3 ÀC 4 ÀC 5 ÀC 6 ) with a hydroxy group at C-2 and a moiety (C 6 -C 7 -C 8 -C 9 ) which kept accordance with correlations of H-1ÀH-2ÀH 2 -3ÀH-4ÀH 2 -5ÀH-6ÀH-1 and H-6-H-7-H 2 -8-H 2 -9. The HMBC correlations from H-14 (d H 1.22, 3H) and 10-OH (d H 4.82) to C-1 (d C 57.8), C-10 (d C 73.8), and C-9 (d C 42.6) enabled the establishment of another six-membered ring (C 1 -C 6 -C 7 -C 8 -C 9 -C 10 ), of which C-10 is substituted by a methyl and a hydroxy group. The aforementioned spectroscopic evidence revealed the existence of a decahydronaphthalene skeleton. Besides, shared HMBC correlations of H-12 (d H 0.89, 3H) and H-13 (d H 0.71, 3H) to C-11 (d C 27.2)/C-7 (d C 48.7) suggested that an isopropanyl group attached to C-7. The 1 H-1 H COSY correlations between H-4 (d H 1.97)/H-15 (d H 3.52, 2H)/15-OH (d H 3.60) indicated that a hydromethyl group is connected to C-4. Thus, the planar structure of 1 was established as shown in Figure 1.
Computational methods have accelerated the structural determination of natural products (Lodewyk et al. 2012). NMR calculation showed superiority over traditional methods in discriminating stereoisomers of natural products (Micco et al. 2010;Grimblat and Sarotti 2016). Due to the 1 H NMR of most of the terpenoids always presented overlapping signals which posed challenges in determining the relative configuration only by the ROESY spectrum. Therefore, the relative configuration of 1 was confirmed by NMR calculation. A conformation search of 1 by MMFF94s force field gave 20 conformers with population higher than 1% Osawa 1989, 1993). All these conformers were optimized at B3LYP/6-31G(d) level of theory and were further subjected to NMR calculation on PCM-B972/pcSseg-1 level of theory in acetone on Gaussian 09 program (Frisch et al. 2010). As shown in Figure 2, the results showed that the correlation coefficient (R 2 ) between the calculated and experimental data from linear regression analysis was 0.9945. There was only one outlier higher than 3 ppm between the calculated and experimental data (Dd ¼ 3.1 (C-6)), the mean absolute deviation (MAD) and the root-mean-square deviation (RMSD) were 0.47 and 0.93 ppm, respectively (Supplementary material). Thus, the NMR calculation results supported the conclusion of the ROESY spectrum in assigning the relative configuration of 1.
To establish the absolute configuration of 1, the specific rotations calculations on possible structure of (1R,2R,4S,6R,7R,10S)-1 was calculated at b3lyp/6-311g(d,p) level of theory with PCM model in methanol based on previous B3LYP/6-31G(d)-optimized geometries on Gaussian 09 program. As a result, the calculated specific rotation was þ 67.7 , while the experimental value is þ 17.5 . Although the calculated specific rotation and the experimental one showed same sign, however, it was insufficient to determine the absolute configuration not only by the large magnitude discrepancy, but also by the possible racemic nature of terpenoids (Finefield et al. 2012). In order to verify if compound 1 was optically pure or not, we should plan to carry out chiral HPLC analysis of 1. However, the shortage of sample as well as the absence of any chromophores hampered the plan.
In conclusion, compound 1 was determined as a cadinane-type sesquiterpenoid substituted by three hydroxy groups while devoid of any chromophores. Finally, compound 1 was trivially named as microporotriol.
Compound 1 was evaluated for its cytotoxicity against the five human cancer cell lines, human myeloid leukaemia cell line (HL-60), human hepatocellular carcinoma cell line (SMMC-7721), lung cancer cell line (A549), breast cancer cell line (MCF-7), and human colon cancer (SW-480). However, compound 1 displayed no significant cytotoxicity against the five human cancer cell lines.

Fungal material
The fungus M. affinis HFG829 was collected from Mang Mountain National Forest Park, Chenzhou City, Hunan Province in 2006. The strain of M. affinis HFG829 in this study was isolated from the fresh fruiting bodies and kept on potato-dextrose-agar (PDA) culture medium. The strain was identified by Prof. Yu-cheng Dai, who is a mushroom specialist of Beijing Forestry University. A voucher specimen (No. Dai2006_2) was deposited in the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences. The culture medium to ferment this fungus consisted of glucose (5%), peptone from porcine meat (0.15%), yeast powder (0.5%), KH 2 PO 4 (0.05%), and MgSO 4 (0.05%). Sixty Erlenmeyer flasks (500 ml) each containing 350 mL of above-mentioned culture medium were inoculated with M. affinis HFG829 strains, respectively. Fermentation were carried out on rotatory shakers at 25 C and 150 rpm for 25 days in dark environment.

Extraction and isolation
The culture broth (20 L) of M. affinis HFG829 was filtered and concentrated to 3 liters followed by partitioned between EtOAc and water four times to give an EtOAc layer. Meanwhile, the mycelia were extracted with EtOH (95%) for three times. The EtOAc layer together with the mycelium extract were concentrated under reduced pressure to afford a crude extract (8.0 g). This residue was separated by silica gel CC eluting with CHCl 3 /MeOH (0:100!100:0) to give ten main fractions (A-J).

Cytotoxicity assay
Compound 1 was evaluated for cytotoxicity against five human cancer cell lines by MTS (an analogue of MTT) method. The five human cancer cell lines included human myeloid leukaemia cell line (HL-60), human hepatocellular carcinoma cell line (SMMC-7721), lung cancer cell line (A549), breast cancer cell line (MCF-7), and human colon cancer (SW-480). The cell line SMMC-7721 was bought from China Infrastructure of Cell Line Resources (Beijing, China), and the remaining others from American Type Culture Collection (ATCC, Manassas, VA, USA). In these tests, DDP and taxol were used as the positive controls. Each tumour cell line was exposed to the test compound at a concentration of 40 lM in triplicates for 48 h.

Conclusions
In conclusion, the secondary metabolites of the culture broth of the wood decaying fungus Microporus affinis have been investigated for the first time. A new cadinanetype sesquiterpenoid, along with four known compounds are reported in this research. Compound 1 was devoid of cytotoxicity against the five human cancer cell lines.