Four new compounds from Neoboletus magnificus

Abstract Four new compounds, compounds 1, 2, 4, 6, along with two known compounds 3, 5, were isolated from the methanol extract of the fruiting body of Neoboletus magnificus. The structures of compounds were elucidated by HRMS and NMR spectroscopic methods. The in vitro anti-inflammatory activity of the isolated compounds was evaluated. Graphical Abstract


Introduction
Boletales are of important source of medicinal mushroom resources, particularly in the Boletaceae family, which contains natural products with major representative such as butenolide (Duncan et al. 2003), sesquiterpene (Wada et al. 1995), steroid ingredients (Toi et al. 2007), nitrogen compounds (Kahner et al. 1998;Kawagishi et al. 2006), benzene derivatives (Song et al. 2009), sugar and fatty acids. Various natural compounds have wide ranges of bioactivities, such as anticancer, cytotoxicity, antibacterial activity and free radical scavenging ability (Takahashi et al. 1992;Yun et al. 2001;Bala et al. 2011;Kamo et al. 2004). Besides, (R)-3-hydroxybutanoyl-(R)-carnitine, Carnitine-esters from the mushroom Suillus laricinus promoted hyaluronan-degradation by human skin fibroblasts (Kawagishi et al. 2006). Two new peptides, tylopeptins A and B were isolated from the fruiting body of the mushroom Tylopilus neofelleus, which were shown to be active against some Gram-positive bacteria (Lee et al. 1999). In our continuing search for novel secondary metabolites with potential bioactivity from Neoboletus magnificus, four new compounds and two new natural compounds were obtained from this mushroom. In this manuscript, we describe the isolation, structural elucidation of these compounds.

Results and discussion
Compound 1 was isolated as a yellow oil. The molecular formula of compound 1 was determined to be C 18 H 30 O 3 by positive HR-EIMS (m/z 294.2203, calcd. 294.2195. The 13 C-NMR, DEPT, and HMQC spectra of compound 1 showed signals of two methyls, 10 methylenes, four methines, and two quaternary carbons including a ester bond (d 176.0, C-1) and a ketone(d 203.9, C-7), respectively. The presence of two conjugated E, E-form enone systems was revealed by the 1 H-NMR spectrum of 1 [d 6.12 (1 H,d,J ¼ 15.6 Hz,7.23(1 H,dd,J ¼ 15.6,9.68 Hz,6.27(2 H,ddd,J ¼ 21.44,15.2,6.4,11)]. Furthermore, 1 H-1 H COSY correlations from H-12 (d H 2.20) to H-11 (d H 6.25) and H-9 (d H 7.23) to H-10 (d H 6.27) revealed the presence of the C ¼ C connected with one methylene (d C 34.1) and the C ¼ C connected directly. This further proved the trans-geometry (E) of the double bond. The UV spectrum of 1 displayed a k max at 203 nm. The 1 H-NMR spectrum showed signals at 3.64 (3H), which can be attributed to the terminal methoxyl of aliphatic chains. HMBC correlations were observed at C-1/ H-2, H-3, H-OCH 3 ; C-7/H-5, H-6, H-8, H-9. Further analysis of EI-MS data indicated that notable fragment ion peaks were observed at m/z 57, 71, 85 and 129. The locations of a carbonyl carbon (d C 203.9) at C-7 and a carbonyl carbon (d C 176.0) at C-1 were identified from the HMBC spectrum and EI-MS analysis. Based on the above data, compound 1 was determined to be (8E, 10E)-7-oxo-8, 10-heptadecadienoic acid.
Moreover the cis-geometry (Z) of the double bond was determined from the d value (23.27, 26.60) of the allylic carbon, since allylic carbon signals of Z-and E-isomers were observed at d ca.27 and ca.32, respectively (Ishii et al. 2006). Hence, the structure of compound 2 was (Z)-N-(3-hydroxybutyl)-4-decene.
Compound 3, a yellow oil was determined to have the molecular formula of C 12 H 25 NO 2 on the basis of positive HR-ESIMS (m/z 238.1775 [M þ Na] þ , calcd. 238.1778) and 13 C NMR-data, [a]25D-22.1(c 0.24, MeOH). Analysis of its 1 D NMR data established the similar structure to compound 2, especially when observing the 13 C-NMR data, compound 3 was less than a carbon carbon double bond compared to compound 2. The HMBC correlations from C-1 to H-2 and H-3 and 1 H-1 H COSY correlations from an amide proton (N-H, d H 7.71) to H-1 0 , from H-2 to H-3 further indicated amide carbonyl bond between C-1 0 and C-2. H-3' gave 1 H-1 H COSY correlations to a hydroxy proton (O-H, d H 4.46, d, J ¼ 4.62 Hz), H-4 0 and H-2', which C-2 0 was connected to C-1 0 .Accordingly, the structure of compound 3 was concluded as N-(3-hydroxybutyl) octanamide.
Compound 5 was isolated as a yellow oil having the molecular formula of C 9 H 13 NO 2 deduced from the positive HRESIMS ion at m/z 190.0830 [M þ Na] þ (calcd. 190.0838), [a]25D-26.1(c 0.28, MeOH). The NMR spectra of compound 5 obviously showed the presence of a substituted pyrrole ring. Careful comparison of the NMR data of compound 5 with those of compound 4 disclosed a less hydroxymethyl located in C-5 (d C 132.61). Therefore, the structure of compound 5 was established as 1-(3 0 -hydroxybutyl) -2-carbaldehyde-1H-pyrrole.

General experimental procedures
Optical rotations were measured with a Horiba SEPA-300 polarimeter. IR spectra were obtained on a Tensor 27 spectrophotometer with KBr pellets. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. 1 D ( 1 H, 13 C, and DEPT) and 2 D ( 1 H-1 H COSY, HSQC, HMBC, and ROESY) NMR spectra were collected on a Bruker Avance III 500, 600 and 800 spectrometer. ESIMS and HRESIMS spectra were acquired on an Agilent G6230 spectrometer. EI and HREI spectra were acquired were obtained on Waters AutoSpec Premier P776.Semi-preparative HPLC separations were performed on an Agilent 1100 liquid chromatograph with a Waters X-Bridge Prep Shield RP18 (10 Â 150 mm) column. Column chromatography (CC) was performed using silica gel (100-200 and 200-300 mesh, Qingdao Marine Chemical Co.Ltd., Qingdao, China) and Sephadex LH-20 (40-70 lm, Amersham Pharmacia Biotech AB, Uppsala, Sweden).Fractions were monitored by TLC (GF 254 , Qingdao Marine Chemical Co. Ltd., Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 5% H 2 SO 4 in EtOH. All solvents were distilled prior to use.

Fungal materials
Fresh fruiting bodies of Neoboletus magnificus were collected from Yunnan Province, People's Republic of China, in July 2016. The fungal samples were identified by Research Fellow Zhu-Liang Yang from Kunming Institute of Botany, Chinese Academy of Science (CAS). A voucher specimen (KM20160702) was deposited in the school of Faculty of Life Science and Technology, Kunming University of Science and Technology, China.

Anti-inflammatory activity assays
RAW 264.7 cells were used to evaluate the inhibitory activity toward NO production by LPS-activated macrophages Baek et al. 2015). The RAW264.7 cells were seeded into 96-well plates, stimulated with 1 lg/ml LPS, and the test compound (final concentration 50 lM) was added. The drug-free group and L-NMMA-positive drug group were set as controls. After the cells were cultured overnight, the medium was assayed for NO production, and the absorbance was measured at 570 nm. MTS was added to the remaining medium for cell viability detection, eliminating the toxic effects of the compounds on the cells.

Conclusions
In this study, four new compounds, compounds 1, 2, 4, 6, along with two known compounds 3, 5, were isolated from the methanol extract of the fruiting body of Neoboletus magnificus. The structures of compounds were elucidated by extensive spectroscopic analyses including 1D-and 2D-NMR techniques.

Disclosure statement
No potential conflict of interest was by the authors.