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 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).

Fig. 1

Chemical structures of compounds 1–4

Results and Discussion

Compound 1 was obtained as a white amorphous powder. It had a molecular formula of C34H48O8 as determined by (+)-HRESIMS protonated ion peak at m/z 585.3433 [M+H]+ (calcd for C34H49O8, 585.3422), corresponding to 11° of unsaturation. The IR spectrum showed absorption bands for hydroxy (3436 cm−1) and carbonyl (1764 cm−1) groups. The 1H NMR spectrum of 1 (Table 1) showed the presence of seven methyl singlets at δH 0.86 (CH3-18), 1.03 (CH3-19), 1.78 (CH3-27), 0.87 (CH3-28), 0.93 (CH3-29), 0.95 (CH3-30), and 1.94 (CH3-31), and one doublet methyl at δH 1.02 (J = 6.0 Hz, CH3-21). The 13C NMR and DEPT spectra presented 34 carbons ascribable to eight methyls, eight methylenes, six methines, and twelve quaternary carbons, including four sp3 ones, four olefinic ones, three carbonyls, and a ketal carbon (δC 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 H3-31 to C-23, C-24 (δC 157.8), and C-25 (δC 124.1), from H3-27 to C-24, C-25, and C-26 (δC 172.5), from H-16 (δH 4.51) to C-23 (Fig. 2) indicated the existence of a 26,23-lactone, and a 16,23-epoxy group, which constructing a spiro ring system with C-23 as the shared carbon. The HMBC correlations from H3-19 to C-1 (δC 71.3), C-9 (δC 135.1), from H3-28, H3-29 to C-3 (δC 81.0), and from H3-30 to C-8 (δC 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 δC 167.8 (C-1′) and 169.7 (C-3′), and a methylene at δC 41.1 (C-2′) were due to a malonyl moiety attached to C-3, which was supported by the HMBC correlations from H-2′ (δH 3.46; 3.40) to C-1′, C-3′, and from H-3 (δH 4.83) to C-1′.

Table 1

1H NMR spectroscopic data for compounds 1–4 (CDCl3)

No.	1 a	2 a	3 a	4 b
1	3.82, dd (9.0, 6.4)	3.80, dd (10.0, 6.3)	4.10, dd (10.0, 6.3)	4.09, dd (10.0, 6.3)
2	1.92, overlapped	1.92, m	1.98, overlapped	1.96, overlapped
3	4.83, br t (3.0)	4.81, t (3.0)	4.85, br t (3.0)	4.80, t (3.0)
5	1.46, dd (10.0, 6.0)	1.44, dd (10.0, 6.0)	1.51, dd (12.0, 4.0)	1.49, dd (12.0, 4.0)
6	1.63, overlapped	1.64, overlapped	2.15, overlapped2.06, overlapped	2.14, dd (18.8, 14.0)2.06, overlapped
7	2.09, m2.04, m	2.10, overlapped2.04, overlapped	5.49, d (6.5)	5.49, d (6.3)
11	2.43, m2.21, br dd (19.0, 8.5)	2.44, overlapped2.23, overlapped	6.30, d (6.0)	6.30, d (6.0)
12	1.84, overlapped1.62, overlapped	1.85, overlapped1.64, overlapped	2.24, d (17.5)2.08, overlapped	2.24, d (17.7)2.09, overlapped
15	1.88, dd (12.4, 8.0)1.70, dd (12.4, 6.5)	1.88, dd (12.5, 7.8)1.71, dd (12.5, 6.5)	2.06, overlapped1.71, dd (12.5, 6.5)	2.06, overlapped1.71, dd (12.5, 6.5)
16	4.51, ddd (8.0, 8.0, 6.5)	4.51, ddd (7.8, 7.8, 6.5)	4.59, ddd (7.8, 7.8, 6.5)	4.59, ddd (7.8, 7.8, 6.5)
17	1.59, overlapped	1.59, overlapped	1.67, overlapped	1.66, m
18	0.86, s	0.86, s	0.75, s	0.75, s
19	1.03, s	1.02, s	1.07, s	1.07, s
20	1.81, overlapped	1.81, overlapped	1.82, overlapped	1.81, overlapped
21	1.02, d (6.0)	1.03, d (6.0)	1.03, d (6.5)	1.02, d (6.5)
22	1.81, overlapped	1.81, overlapped	1.82, overlapped	1.82, overlapped
27	1.78, s	1.78, s	1.78, s	1.78, s
28	0.87, s	0.87, s	0.89, s	0.88, s
29	0.93, s	0.93, s	1.01, s	1.00, s
30	0.95, s	0.94, s	0.93, s	0.92, s
31	1.94, s	1.94, s	1.93, s	1.93, s
2′	3.46, d (16.5)3.40, d (16.5)	3.43, d (16.9)3.39, d (16.9)	3.46, d (17.0)3.41, d (17.0)	3.46, d (17.0)3.41, d (17.0)
4′		3.73, s		3.70, s

aMeasured at 500 MHz

bMeasured at 600 MHz

Fig. 2

Characteristic mutual HMBC (blue arrow) and 1H–1H COSY correlations for a compounds 1 and 2 (HMBC in red arrow was exclusive for 2) and b compounds 3 and 4 (HMBC in red arrow was exclusive for 4)

The ROESY correlations between H-1/H-5, H3-30/H-16 demonstrated that 1-OH was β orientation while H-16 was α orientation. The key diagnostic ROESY correlation between H3-18 and H3-31 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 β orientation for H-3, which can be seen as a consequence of the nearly same dihedral angle between H-3/H-2α, and H-3/H-2β when H-3 adopted β orientation (Fig. 3). Therefore, compound 1 was determined as shown in Fig. 1, and given the name irpexoate A.

Fig. 3

Key ROESY correlations for compounds 1–4

The white amorphous powder compound 2, named irpexoate B, possessed the molecular formula of C35H50O8 as determined by the (+)-HRESIMS sodium adduct ion peak at m/z 621.3399 [M+Na]+ (calcd for C35H50O8Na, 621.3398), indicating 11 indices of hydrogen deficiency. The 1H and 13C NMR spectra of 2 (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 δC 52.4 (C-4′) in 2, along with the HMBC correlation from H3-4′ (δH 3.72) to C-3′ (δC 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.

Table 2

13C NMR spectroscopic data for compounds 1–4 (CDCl3)

No.	1 a	2 a	3 a	4 b
1	71.3, CH	71.3, CH	71.6, CH	71.6, CH
2	33.4, CH2	33.5, CH2	33.5, CH2	33.7, CH2
3	81.0, CH	80.4, CH	81.0, CH	80.3, CH
4	36.5, C	36.6, C	36.4, C	36.5, C
5	44.4, CH	44.3, CH	43.9, CH	43.9, CH
6	17.9, CH2	17.8, CH2	23.1, CH2	23.1, CH2
7	26.0, CH2	26.1, CH2	120.2, CH	120.4, CH
8	134.8, C	134.8, C	141.8, C	141.8, C
9	135.1, C	135.2, C	144.2, C	144.3, C
10	43.0, C	43.1, C	43.4, C	43.5, C
11	23.9, CH2	23.9, CH2	119.8, CH	119.8, CH
12	31.6, CH2	31.6, CH2	37.6, CH2	37.6, CH2
13	43.4, C	43.4, C	42.8, C	42.8, C
14	47.4, C	47.4, C	47.9, C	48.0, C
15	39.5, CH2	39.5, CH2	40.0, CH2	40.1, CH2
16	75.5, CH	75.4, CH	75.2, CH	75.2, CH
17	53.0, CH	53.0, CH	53.5, CH	53.5, CH
18	18.4, CH3	18.3, CH3	18.4, CH3	18.4, CH3
19	13.8, CH3	13.7, CH3	15.2, CH3	15.2, CH3
20	26.3, CH	26.3, CH	26.1, CH	26.1, CH
21	20.8, CH3	20.8, CH3	20.6, CH3	20.6, CH3
22	37.5, CH2	37.5, CH2	37.4, CH2	37.5, CH2
23	107.7, C	107.6, C	107.6, C	107.5, C
24	157.8, C	157.8, C	157.7, C	157.7, C
25	124.1, C	124.1, C	124.1, C	124.1, C
26	172.5, C	172.5, C	172.5, C	172.3, C
27	8.3, CH3	8.3, CH3	8.3, CH3	8.3, CH3
28	27.2, CH3	27.2, CH3	27.5, CH3	27.4, CH3
29	21.5, CH3	21.5, CH3	22.2, CH3	22.2, CH3
30	25.2, CH3	25.2, CH3	25.9, CH3	25.8.CH3
31	10.9, CH3	10.9, CH3	10.8, CH3	10.9, CH3
1′	167.8, C	165.9, C	167.7, C	165.9, C
2′	41.1, CH2	41.7, CH2	40.4, CH2	41.6, CH2
3′	169.7, C	167.1, C	168.1, C	166.8, C
4′		52.4, CH3		52.4, CH3

aMeasured at 125 MHz

bMeasured at 150 MHz

Compound 3 gave a molecular formula of C34H46O8 according to the 13C NMR data and the (+)-HRESIMS sodium adduct ion peak at m/z 605.3058 [M+Na]+ (calcd for C34H46O8Na, 605.3085). A comparison of the 1H and 13C 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 1H–1H 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 (δH 5.49) and H-11 (δH 6.30) to C-8 (δC 141.8), C-9 (δC 144.2) as well as the 1H–1H 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, H3-18/H-20, H3-18/H3-31, H3-30/H-16/H-17 indicated that both H-1 and H-16 were α orientation, and the spiro carbon C-23 was R* configuration (Fig. 3). The triplet peak of H-3 (δH 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 and given the trivial name irpexoate C.

A molecular formula of C35H48O8 was assigned to compound 4 as determined by the (+)-HRESIMS sodium adduct ion peak at m/z 619.3250 [M+Na]+ (calcd for C35H48O8Na, 619.3241). Comparison of the 1D NMR data of 4 (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 (δH 4.80) to C-1′ (δC 165.9), from H-2′ (δH 3.41, 3.46) to C-1′ and C-3′ (δC 166.8), and CH3-4′ (δH 3.70) to C-3′ allowed a methyl malonate moiety linked at C-3. The relative configuration of 4 was consistent with that of 3 as established by the ROESY spectrum. The structure of 4 was consequently assigned as shown in Fig. 1, and named as irpexoate D.

Irpexoates A–D (1–4) were evaluated for the cytotoxicity against five human cancer cell lines (the human myeloid leukemia cell line HL-60, the human hepatocellular carcinoma cell line SMMC-7721, the lung cancer cell line A549, the breast cancer cell line MCF-7, and the human colon cancer cell line SW-480). The results showed that compound 2 exhibited weak cytotoxicity against the four human cancer cell lines (A-549, SMMC-7721, MCF-7, SW480) with IC50 values varying from 22.9 to 34.0 μM, while compound 4 only showed weak cytotoxicity against the human colon cancer cell line SW480 with an IC50 value of 11.2 ± 0.25 μM (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.

Table 3

Cytotoxicity of compounds 2 and 4 against five human cancer cell lines (IC50 ± SD in μM)

Compound	HL-60	A-549	SMMC-7721	MCF-7	SW480
2	> 40	22.9 ± 0.92	30.1 ± 0.43	24.2 ± 1.11	34.0 ± 1.43
4	> 40	> 40	> 40	> 40	35.2 ± 1.15
Cisplatin		14.1 ± 0.11	11.9 ± 0.49	14.1 ± 1.00	11.2 ± 0.25

Experimental

General Experimental Procedures

Optical rotations were obtained on a JASCO P-1020 digital polarimeter (Horiba, Kyoto, Japan). UV spectra were recorded on a Shimadzu UV-2401PC UV–visible recording spectrophotometer (Shimadzu, Kyoto, Japan). A Tenor 27 spectrophotometer (Bruker Optics GmbH, Ettlingen, Germany) was used for scanning IR spectroscopy using KBr pellets. 1D and 2D NMR spectra were obtained on Bruker DRX 500 MHz and Avance III 600 MHz spectrometers (Bruker Corporation, Karlsruhe, Germany). HRESIMS were recorded on an Agilent 6200 Q-TOF MS system (Agilent Technologies, Santa Clara, CA, USA). Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden) and silica gel (Qingdao Haiyang Chemical Co., Ltd, Qingdao, China) were used for column chromatography (CC). Medium pressure liquid chromatography (MPLC) was performed on a Büchi Sepacore System equipped with pump manager C-615, pump modules C-605 and fraction collector C-660 (Büchi Labortechnik AG, Flawil, Switzerland), and columns packed with Chromatorex C-18 (dimensions 450 mm × i.d. 14 mm, particle size: 40–75 μm, Fuji Silysia Chemical Ltd., Kasugai, Japan). Preparative high performance liquid chromatography (prep-HPLC) were performed on an Agilent 1260 liquid chromatography system equipped with a Zorbax SB-C18 column (particle size 5 μm, dimension 150 mm × i.d. 9.4 mm, flow rate 7 ml min−1, respectively) and a DAD detector (Agilent Technologies).

Fungal Material

The fungus Irpex lacteus was collected from the Wangtianshu Scenic Area, Xishuangbanna, Yunnan Province in July 2014, and authenticated by Prof. Yu-Cheng Dai (Beijing Forestry University), who is a mushroom specialist. A voucher specimen of I. lacteus was deposited at the Mushroom Bioactive Natural Products Research Group in Kunming Institute of Botany (No. HFG 201407).

Extraction and Isolation

The dry fruiting bodies of I. lacteus (1.47 kg) was pulverized and macerated five times with 95% EtOH at room temperature. The extract was evaporated under reduced pressure and partitioned between EtOAc and water for three times to give an EtOAc layer (53 g). The crude extract was eluted on MPLC with a stepwise gradient of MeOH/H2O (20–100%) to afford eight fractions (A–H).

Fraction E was applied to silica gel column chromatography eluting with petroleum ether/acetone (5:1–2:1) to give ten subfractions (E1–E10). Subfraction E5 was purified by prep-HPLC (MeCN/H2O: 45–65%, 7 mL min−1, 20 min) to yield compound 4 (1.8 mg, t = 13.8 min). Fraction F was subjected to normal silica gel CC (CHCl3/MeOH, 40:1–1:1) to furnish seven subfractions (F1–F7). Subfraction F6 was purified on prep-HPLC (MeCN/H2O: 65%, isocratic, 18 mL min−1, 20 min) to yield compound 1 (26.6 mg, t = 9.5 min). Compound 2 (17.0 mg, t = 12.9 min) was purified from subfraction F1 by prep-HPLC (MeCN/H2O: 55–75%, 7 mL min−1, 20 min). Subfraction F4 was fractionated into five subfractions (F4a–F4e) by Sephadex LH-20, eluting with CHCl3–MeOH (1:1). Subfraction F4e was separated using silica gel CC (CHCl3/MeOH, 100:1 –10:1) to afford eight subfractions (F4e1–F4e8). Subfraction F4e5 was fractionated by Sephadex LH-20 (acetone) to give six subfractions (F4e5a–F4e5f). Subfraction F4e5c was further purified using prep-HPLC with a solvent gradient of 45–65% MeCN in H2O over 20 min, flow rate 7 ml·min−1, to yield compound 3 (3.0 mg, t = 12.9 min).

Spectroscopic Data of Compounds

Irpexoate A (1)

Amorphous white powder; −24.6 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 206.4 (4.23); IR (KBr) νmax 3436, 2960, 2933, 2878, 1764, 1612, 1381, 1314, 1158, 1036, 958 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Tables 1 and 2; HRESIMS m/z 585.3433 [M+H]+ (calcd for C34H49O8, 585.3422).

Irpexoate B (2)

Amorphous white powder; −23.5 (c 0.15, MeOH); IR (KBr) νmax 3439, 2959, 2878, 1753, 1631, 1439, 1313, 1033, 957 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Tables 1 and 2; HRESIMS m/z 621.3399 [M+Na]+ (calcd for C35H50O8Na, 621.3398).

Irpexoate C (3)

Amorphous white powder; −4.0 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 209.0 (4.17), 232.6 (4.05), 242.0 (4.06) and 250.0 (3.93); IR (KBr) νmax 3438, 2963, 2932, 2883, 1765, 1633, 1315, 1156, 1038, 958 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, see Tables 1 and 2; HRESIMS m/z 605.3058 [M+Na]+ (calcd for C34H46O8Na, 605.3085).

Irpexoate D (4)

Amorphous white powder; −4.8 (c 0.09, MeOH); IR (KBr) νmax 3444, 2961, 2928, 2884, 1752, 1636, 1444, 1314, 1153, 1036, 958 cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Tables 1 and 2; HRESIMS m/z 619.3250 [M+Na]+ (calcd for C35H48O8Na, 619.3241).

Cytotoxicity Assays

Human myeloid leukemia HL-60 cells, lung cancer A-549 cells, hepatocellular carcinoma SMMC-7721 cells, breast cancer MCF-7 cells, and colon cancer SW480 cell lines were used in the cytotoxic assay. All the cells were cultured in DMEM or RPMI-1640 medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2 at 37 °C. The cytotoxicity assay was performed according to the MTS (3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium) method in 96-well microplates. Briefly, 100 μL of adherent cells was seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded just before drug addition to an initial density of 1 × 105 cells ml−1. Each tumor cell line was exposed to the tested compounds dissolved in DMSO at various concentrations in triplicate for 48 h, with cisplatin (Sigma, USA) as a positive control. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC50 values were calculated by Reed and Muench’s method [6].

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