Irpexoates A–D, Four Triterpenoids with Malonyl Modifications from the Fruiting Bodies of the Medicinal Fungus Irpex lacteus

Four eburicane-type triterpenoids with malonyl modifications, namely irpexoates A–D (1–4), were isolated from the fruiting bodies of the medicinal fungus Irpex lacteus. The structures of the new compounds were established by extensive spectroscopic methods, including 1D and 2D NMR, HRESIMS spectroscopic analysis. Irpexoate B (2) displayed weak cytotoxicity against four human cancer cell lines (A-549, SMMC-7721, MCF-7, SW480) with IC50 values varying from 22.9 to 34.0 μM, and irpexoate D (4) showed weak cytotoxicity against the human cancer cell line SW480 with an IC50 value of 35.2 μM.


Introduction
Natural products from higher fungi (mushroom) are regarded as a vital source of lead compounds in the field of drug research and development [1]. The fungus Irpex lacteus is widely used in China as a folk medicine [2]. Previously chemical investigation on this fungus major focused on secondary metabolites from culture broth [3,4], while the study on the fruiting bodies remained untapped.
Triterpenoids are one of the largest groups of secondary metabolites from higher fungi. To the best of our knowledge, lanostane-type triterpenoids are by far the most diverse category of higher fungi-derived triterpenoids. This type of triterpenes always encounters in the genus Ganoderma and the fungus Poria cocos [1]. However, eburicane-type triterpenes, which also can be considered as 24-methyl lanostanes, have rarely been found from fungi. Triterpenoids with malonyl modification are not prevalent Electronic supplementary material The online version of this article (https://doi.org/10.1007/s13659-018-0160-3) contains supplementary material, which is available to authorized users. in fungal metabolites. From biosynthetic point, the malonyl moieties were introduced by the precursor malonyl-CoA, which was a key intermediate in the pathways of fatty acid biosynthesis and fatty acid elongation, and also a key signaling molecule in mammalian cells [5]. As our continuous efforts to search promising lead compounds from mushroom, four malonyl modified eburicanes, namely irpexoates A-D (1-4) (Fig. 1), were isolated from the fruiting bodies of the medicinal fungus I. lacteus. Herein, we report the isolation, structure elucidation, and cytotoxicity against five human cancer lines of the isolates irpexoates A-D (1-4).  3 -21). The 13 C NMR and DEPT spectra presented 34 carbons ascribable to eight methyls, eight methylenes, six methines, and twelve quaternary carbons, including four sp 3 ones, four olefinic ones, three carbonyls, and a ketal carbon (d C 107.7, C-23). All these spectroscopic features implied the presence of an eburicane skeleton in compound 1. Exhaustive analysis of the 2D NMR spectra furnished the establishment of the structure of 1. The HMBC correlations from H 3 -31 to C-23, C-24 (d C 157.8), and C-25 (d C 124.1), from H 3 -27 to C-24, C-25, and C-26 (d C 172.5), from H-16 (d H 4.51) to C-23 (Fig. 2) indicated the existence of a 26,23-lactone, and a 16,23epoxy group, which constructing a spiro ring system with C-23 as the shared carbon. The HMBC correlations from H 3 -19 to C-1 (d C 71.3), C-9 (d C 135.1), from H 3 -28, H 3 -29 to C-3 (d C 81.0), and from H 3 -30 to C-8 (d C 134.8) (Fig. 2) revealed that C-1 and C-3 were oxygenated, and a double bond was assigned at the positions C-8-C-9. Apart from the signals assigned to the eburicane skeleton, the remained three carbon signals, i.e. two carbonyls at d C 167.8 (C-1 0 ) and 169.7 (C-3 0 ), and a methylene at d C 41.1 (C-2 0 ) were due to a malonyl moiety attached to C-3, which was supported by the HMBC correlations from H-2 0 (d H 3.46; 3.40) to C-1 0 , C-3 0 , and from H-3 (d H 4.83) to C-1 0 .

Results and Discussion
The ROESY correlations between H-1/H-5, H 3 -30/H-16 demonstrated that 1-OH was b orientation while H-16 was a orientation. The key diagnostic ROESY correlation between H 3 -18 and H 3 -31 revealed that C-23 was R* configuration (Fig. 3). As for the configuration of C-3, the broad triplet of H-3 with a small coupling constant (3.0 Hz) indicated the b orientation for H-3, which can be seen as a consequence of the nearly same dihedral angle between H-3/H-2a, and H-3/H-2b when H-3 adopted b orientation (Fig. 3). Therefore, compound 1 was determined as shown in Fig. 1, and given the name irpexoate A.
The white amorphous powder compound 2, named irpexoate B, possessed the molecular formula of C 35 (Tables 1, 2) showed highly similarities to those of 1, indicating that these two compounds possessed identical carbon scaffold and substitution patterns. Compared to 1, the presence of an additional methoxy group at d C 52.4 (C-4 0 ) in 2, along with the HMBC correlation from H 3 -4 0 (d H 3.72) to C-3 0 (d C 167.1) (Fig. 2) revealed that the terminal carboxylic acid group was methyl-esterified in 2. Other characteristic ROESY correlations suggested that the stereochemistry of chiral centers in 2 was identical with those of 1. As describe above, compound 2 was determined as shown in Fig. 1  13 C NMR data of 3 to those of 1 (Tables 1 , 2) revealed that 3 was an analogue of 1. Carefully analysis of the HMBC and 1 H-1 H COSY spectra of 3 revealed the existence of a conjugated diene locating at C-7-C-8-C-9-C-11, which was ascertainable by the HMBC correlations from the olefinic protons H-7 (d H 5.49) and H-11 (d H 6.30) to C-8 (d C 141.8), C-9 (d C 144.2) as well as the 1 H-1 H COSY cross peaks between H-6/H-7, H-11/H-12 (Fig. 2). The stereochemistry of 3 was assigned by a ROESY experiment and coupling constants analysis. The diagnostic ROESY correlations between H-1/H-5, H 3 -18/H-20, H 3 -18/H 3 -31, H 3 -30/H-16/H-17 indicated that both H-1 and H-16 were a orientation, and the spiro carbon C-23 was R* configuration (Fig. 3). The triplet peak of H-3 (d H 4.85) with small coupling constant (3.0 Hz) suggested the malonate moiety at the axial position. Therefore, compound 3 was established as shown in Fig. 1 (Tables 1, 2) to those of 3 showed that they were closely related analogues featuring identical carbon frameworks. 2D NMR spectra analysis of 4 indicated that the main distinction was attributable to the substituent group at C-3. The HMBC correlations from H-3 (d H 4.80) to C-1 0 (d C 165.9), from H-2 0 (d H 3.41, 3.46) to C-1 0 and C-3 0 (d C 166.8), and CH 3 -4 0 (d H 3.70) to C-3 0 allowed a methyl malonate moiety linked at C-3. The relative     (Table 3). However, compounds 1 and 3 were devoid of cytotoxicity. Thus, the conclusion could be drawn that the absence of methoxy group in the malonyl moiety attenuated the cytotoxicity.

Fungal Material
The