Seco-tremulane Sesquiterpenoids from the Cultures of the Medicinal Fungus Irpex lacteus HFG1102

Abstract

Abstract

Six previously undescribed 5,6-seco-tremulane analogues, together with two known ones, were isolated from the culture broth of the medicinal fungus Irpex lacteus HFG1102. The structures of the new compounds were elucidated via extensive spectroscopic methods, including NMR and HRMS spectroscopic analyses.

Graphical Abstract

Electronic supplementary material

The online version of this article (10.1007/s13659-018-0157-y) contains supplementary material, which is available to authorized users.

Introduction

Higher fungi are valuable and irreplaceable resources of natural product-derived lead compounds for drug research and development [1]. The fungus Irpex lacteus has been used as a medicinal fungus for decades in China for the treatment of inflammation, bacterial/fungal infection, and urinary retention [2]. The polysaccharide products of the submerged culture of I. lacteus have been developed and approved by SFDA as a clinically used drug for curing chronic glomerulonephritis [2]. However, although this fungus has been widely used as an industrial source to obtain polysaccharide products, its secondary metabolites remained unknown.

The 5,6-seco-tremulane skeleton, which was regarded as the resultant of Baeyer–Villiger oxidation of tremulane, was first and mainly reported from the basidiomycete Conocybe siliginea [3, 4]. To the best of our knowledge, the distribution of this type of sesquiterpenes only reported from three species, the basidiomycete Flavodon flavus BCC 17421 [5], and the endophytic fungi Colletotrichum capsici [6] and Ceriporia lacerate [7]. Notably, this type of sesquiterpenes always presented in inseparable mixtures. This study, as part of our ongoing search for promising lead compounds from higher fungi, concentrated on the secondary metabolites of the culture broth of the medicinal fungus I. lacteus, led to the isolation of six undescribed 5,6-seco-tremulane congeners (1–6) and two known tautomeric isomers (7, 8) (Fig. 1). Herein, we report the isolation, structure elucidation of the new compounds.

Fig. 1.

Structures of compounds 1–8

Results and Discussion

Compounds 1 and 2 were obtained as an inseparable colourless mixture with the ratio of approximately 1:0.9 deduced from the intensity of ¹H NMR resonance. The ¹H and ¹³C NMR spectra of the mixture displayed paired signals, indicating the similar structures of 1 and 2. Nevertheless, comprehensive analysis of the 1D and 2D NMR spectra expedited the structural elucidation of 1 and 2 unambiguously based on the relatively less overlapped signals. Elucidating of the HSQC, HMBC, and ¹H–¹H COSY spectra facilitated the assignments of two class of signals corresponding to 1 and 2.

The signals assigned for compound 1 consisted of a methyl singlet, six methylenes, four methines (including a ketal carbon), and four quaternary carbons (two olefinic, one carbonyl) (Tables 1, 2). The ¹H–¹H COSY of H-13/H-6/H-7/H-8, and H-4/H-3/H-12 enabled the connection of C-13–C-6–C-7–C-8 and C-12–C-3–C-4, respectively. The HMBC correlations from H₃-14 (δ_H 1.09) and H-15 (δ_H 3.22, 3.19) to C-9 (δ_C 44.6), C-10 (δ_C 42.7), and C-8 (δ_C 44.4), along with from H-10 (δ_H 2.31, 2.00) to C-1 (δ_C 139.4), C-2 (δ_C 133.2), and C-7 (δ_C 49.3) enabled the completion of a five-membered ring constructed by C-1–C-7–C-8–C-9–C-10 and an exocyclic double bond located at C-1 (Part A) (Fig. 2). Moreover, the HMBC correlations from oxygen-bearing methylene H-11 (δ_H 4.49, 4.39) to C-1, C-2, C-3 (δ_C 42.4), and C-12 (δ_C 110.6), and from H-12 (δ_H 6.09) and H-4 (δ_H 2.92, 2.49) to the carbonyl C-5 (δ_C 178.3) led to construction of tetrahydrofuro[2,3-b]furan-2(3H)-one moiety which connected with part A through the double bond C-1–C-2 (Fig. 2). The above evidences led to the establishment of the planar structure of 1, which also in accordance with the molecular formula deduced from HRESIMS result which gave a sodium adduct ion peak at m/z 287.1258 [M+Na]⁺ (calcd for C₁₅H₂₀O₄Na, 287.1254).

Table 1.

¹H NMR spectroscopic data of compounds 1–6 (δ in ppm)

No.	1 a	2 a	3 a	4 a	5 a	6 b
3	3.70, m	3.03, dd (11.1, 2.5)	3.74, m	3.07, dd (11.4, 2.8)	3.87, m	3.78, m
4	2.92, dd (19.0, 11.0) 2.49, dd (19.0, 4.1)	2.56, dd (16.5, 2.5) 2.25, dd (16.5, 11.1)	2.94, dd (19.2, 11.0) 2.49, dd (19.2, 4.0)	2.56, dd (16.6, 2.8) 2.26, dd (16.6, 11.4)	2.63, dd (17.6, 9.4) 2.57, dd (17.6, 9.3)	2.90, dd (17.8, 9.6) 2.65, dd (17.8, 2.0)
6	5.78, ddd (17.0, 9.7, 7.2)	5.75, ddd (17.8, 9.5, 9.5)	5.73, ddd (17.0, 9.7, 7.2)	5.74, dd (17.8, 9.5, 9.5)	3.70, qd (6.0, 6.0)	4.21, m
7	3.35, m	3.35, m	3.43, m	3.44, m	2.95, m	2.82, m
8	1.97, overlapped 1.31, dd (13.2, 8.5)	1.97, overlapped 1.34, dd (13.4, 8.5)	1.67, dd (12.7, 8.2) 1.55, dd (12.7, 9.3)	1.64, dd (12.4, 8.3) 1.54, dd (12.4, 8.7)	1.61, dd (13.2, 8.9) 1.38, dd (13.2, 6.9)	1.62, dd (12.9, 10.4) 1.58, dd (12.9, 8.3)
10	2.31, br d (16.2) 2.00, br d (16.2)	2.19, br d (16.2) 1.94, br d (16.2)	2.22, d (14.3) 1.99, d (14.3)	2.16, br d (14.9) 1.88, br d (14.9)	2.31, br d (14.5) 2.02, br d (14.5)	2.03, br d (16.8) 1.83, br d (16.8)
11	4.49, d (12.4) 4.39, d (12.4)	4.41, br d (12.1) 4.24, br d (12.1)	4.47, d (11.6) 4.35, d (11.6)	4.40, br d (12.1) 4.24, br d (12.1)	4.22, d (12.1) 4.16, d (12.1)	4.56, br d (12.3) 4.46, br d (12.3)
12	6.09, d (5.6)	5.19, s	6.10, d (5.7)	5.21, s	4.40, dd (8.8, 8.8) 4.28, dd (8.8, 8.8)	6.20, d (5.5)
13	5.09, dd (17.0, 1.3) 5.00, dd (9.7, 1.3)	5.06, br d (17.8) 4.90, overlapped	5.12, dd (17.0, 1.5) 5.01, dd (9.7, 1.5)	5.09, br d (17.7) 4.90, overlapped	1.09, d (6.0)	1.17, d (7.0)
14	1.09, s	1.09, s	3.41, d (12.0) 3.40, d (12.0)	3.40, d (12.0) 3.39, d (12.0)	1.12, s	1.12, s
15	3.22, d (11.0) 3.19, d (11.0)	3.26, d (12.0) 3.24, d (12.0)	0.92, s	0.95, s	0.86, s	0.85, s
16		3.65, s		3.66, s

^aRecorded in CD₃OD, recorded in 600 MHz

^bRecorded in CDCl₃, recorded in 800 MHz

Table 2.

¹³C NMR spectroscopic data of compounds 1–6 (150 MHz, δ in ppm)

No.	1 a	2 a	3 a	4 a	5 a	6 b
1	139.4, C	138.0, C	139.1, C	137.6, C	148.7, C	138.1, C
2	133.2, C	133.8, C	133.5, C	134.1, C	130.6, C	130.0, C
3	42.4, CH	46.6, CH	42.4, CH	46.5, CH	39.3, CH	41.5, CH
4	37.2, CH2	37.1, CH2	37.3, CH2	37.2, CH2	34.0, CH2	34.5, CH2
5	178.3, C	174.3, C	178.3, C	174.3, C	180.5, C	174.3, C
6	144.2, CH	144.5, CH	144.0, CH	144.3, CH	71.4, CH	66.3, CH
7	49.3, CH	48.7, CH	48.3, CH	48.8, CH	49.2, CH	47.9, CH
8	44.4, CH2	44.3, CH2	44.1, CH2	44.2, CH2	42.5, CH2	39.9, CH2
9	44.6, C	44.7, C	44.8, C	44.8, C	38.4, C	37.4, C
10	42.7, CH2	42.6, CH2	42.9, CH2	42.6, CH2	46.5, CH2	48.5, CH2
11	70.2, CH2	69.4, CH2	70.2, CH2	69.5, CH2	60.1, CH2	71.5, CH2
12	110.6, CH	102.7, CH	110.5, CH	102.7, CH	73.9, CH2	109.8, CH
13	115.0, CH2	114.5, CH2	115.1, CH2	114.5, CH2	19.4, CH3	20.7, CH3
14	69.2, CH2	69.3, CH2	23.1, CH3	23.2, CH3	29.6, CH3	27.4, CH3
15	24.5, CH3	24.5, CH3	70.9, CH2	71.1, CH2	28.6, CH3	28.6, CH3
16		52.3, CH3		52.3, CH3

^aRecorded in CD₃OD, recorded in 150 MHz

^bRecorded in CDCl₃, recorded in 200 MHz

Fig. 2.

Key ¹H–¹H COSY and HMBC correlations of compounds 1–6

A ROESY experiment determined the relative configuration of compound 1. The ROESY cross peaks of H-7/H-8β/H-15 led to the assignment of H-7 and 15-CH₂OH as β orientation. The significant ROESY correlation between H-3 and H-12, H-10 and H-11 suggested that H-3 and H-12 were at the same side, and the C-1–C-2 double bond were Z configuration. Therefore, the structure of compound 1 was established as 11,12-epoxy-15-hydroxy-5,6-seco-tremula-1,6(13)-dien-5,12-olide (Fig. 1).

The other set of signals for compound 2 showed resemblance with those of compound 1, but with the presence of an additional methoxy group (δ_H 3.65, δ_C 52.3). Careful analysis of the 2D NMR led to the conclusion that the only difference between 1 and 2 was the lactone group in 1 opened in 2 and the carbonyl group was methyl esterified, which was confirmed by the HMBC correlation from the methoxy (δ_H 3.65) to the carbonyl C-5 (δ_C 174.3) as well as the absence of HMBC correlation from H-12 to C-5. The relative configuration of 2 was identical with that of 1 by interpretation of the ROESY spectrum. The HRESIMS result of the mixture also gave a molecular formula of C₁₆H₂₄O₅ (m/z 319.1529 [M+Na]⁺, calcd for C₁₆H₂₄O₅Na, 319.1516) which further reinforced the above assignment. Thus, compound 2 was deduced as methyl 12β,15-dihydroxy-5,6-seco-tremula-1,6(13)-dien-5-oate (Fig. 1).

Compounds 3 and 4 were isolated as an inseparable mixture with the ratio of 1:0.6. The 1D NMR data of the mixture showed high similarities with those of the 1/2 mixture, implying that compounds 3 and 4 were analogues of 1 and 2. Elucidating the 2D NMR spectra of the mixture promoted the completion of 3 and 4 which possessing the same planar structures with those of 1 and 2, respectively. Considering of the above information, a conclusion could be drawn which compounds 3 and 4 differed from 1 and 2 in spatial configuration. Analysis of the ROESY spectrum of 3/4, in combination with ChemBio3D simulations confirmed the speculation. The ROESY spectrum displayed cross peaks of H-14 (δ_H 3.41, 3.40 for 3; 3.40, 3.39 for 4)/H-8 (δ_H 1.67, 1.55 for 3; 1.64, 1.54 for 4), H-14/H-10 (δ_H 2.22, 1.99 for 3; 2.16, 1.88 for 4). As shown in Fig. 3, the minimized energy optimized molecular models of 3 and 4 suggested that only when the 14-CH₂OH adopted the equatorial direction, the above ROESY correlation could be seen. Therefore, compounds 3 and 4 were C-9 epimers of compounds 1 and 2, respectively. This conclusion was also evidenced by the large discrimination of ¹³C NMR chemical shifts of C-14 and C-15 between 1 and 3, 2 and 4. Thus, compounds 3 and 4 were identified as shown in Fig. 1, and were given the respective names 11,12-epoxy-14-hydroxy-5,6-seco-tremula-1,6(13)-dien-5,12-olide, and methyl 12β,14-dihydroxy-5,6-seco-tremula-1,6(13)-dien-5-oate.

Fig. 3.

Key ROESY correlations of compounds 1–4

Compound 5 was isolated as colourless oil. The HRESIMS result presented a molecular formula of C₁₅H₂₄O₄ as indicated by the sodium adduct ion peak at m/z 291.1572 [M+Na]⁺ (calcd for C₁₅H₂₄O₄Na, 291.1567). The 1D NMR data of 5 (Tables 1, 2) were classified into three methyls, five methylenes (two oxygenated), three methines (one oxygenated), and four quaternary carbons (two olefinic ones, and a carbonyl). These data closely related to the co-isolate conocenolide A (7) [3], suggesting compound 5 was a congener of 7. The main discrepancy between 5 and 7 was that the absence of the C-6–C-13 double bond while the presence of an oxygen-bearing methine and a methyl doublet in 5 compared to those of 7, suggested that the C-6 in 7 was oxygenated in 5, which was verified by the ¹H–¹H COSY correlations of H-6/H-7, H-6/H-13 (Fig. 2). Unfortunately, the inadequate sample of 5 (0.8 mg) precluded the determination of the absolute configuration of 6-OH. Therefore, compound 5 was identified as 6,11-dihydroxy-5,6-seco-tremul-1-en-5,12-olide (Fig. 1).

Compound 6 was obtained as colourless oil. The ¹H and ¹³C NMR spectra exhibited signals which ascribable to three methyls, four methylenes (one oxygenated), four methines (two oxygenated), and four quaternary carbons (a tetrasubstituted double bond, a carbonyl). These data were reminiscent of those of compounds 1 and 3 (Tables 1, 2). Compared the NMR data of 6 with those of 1 revealed that the large differences located at C-6, C-13, and C-15. Detailed analysis of the 2D NMR spectra of 6 indicated that the C-6–C-13 double bond was oxygenated, while C-15 remained unoxygenated. This postulation was validated by the ¹H–¹H COSY correlations of H-6/H₃-13, and H-6/H-7, and HMBC correlation from H₃-14 to H₃-15 (Fig. 2). Due to scarcity of samples, 6-OH remained unassigned. The above assignments were consistent with the HRESIMS data, which gave a sodium adduct ion peak at m/z 289.1416 [M +Na]⁺, suggesting a molecular formula of C₁₅H₂₂O₄ (calcd for C₁₅H₂₂O₄Na, 289.1410). Thus, the structure of 6 was established as shown in Fig. 1 and was named as 11,12-epoxy-6-hydroxy-5,6-seco-tremul-1-en-5,12-olide.

Conocenolides A (7) and B (8) [3] were two abundant and separable but tautomeric isomers accompanied in this study. Their structures were identified compared with the literature data.

In conclusion, six hitherto unknown 5,6-seco-tremulanes were isolated from the culture broth of the medicinal fungus I. lacteus. All these 5,6-seco-tremulane skeletons were proposed to be biosynthesized via Baeyer–Villiger oxidation, rearrangement, elimination, oxidation, and esterification of the corresponding 5-oxo-tremulane. This study represents the first report of 5,6-seco-tremulanes from the fungus I. lacteus, and also expands the distribution of 5,6-seco-tremulane-type sesquiterpenoids as fungal metabolites.

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 a Bruker Avance III 600 MHz spectrometer (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, dimensions 150 mm × i.d. 9.4 mm, flow rate 7 ml min⁻¹) and a DAD detector (Agilent Technologies, Santa Clara, CA, USA).

Fungal Material

The fungus Irpex lacteus was collected from Changbai Mountain Nature Reserve in 2012. The strain of I. lacteus in this study was isolated from the fresh fruiting bodies and kept on potato, dextrose, and agar (PDA) culture medium. A voucher specimen (No. CGBWSHFG1102) 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%), KH₂PO₄ (0.05%) and MgSO₄ (0.05%). Sixty Erlenmeyer flasks (500 ml) each containing 350 ml of above-mentioned culture medium were inoculated with I. lacteus 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 I. lacteus HFG1102 was filtered and concentrated to 3 l followed by partitioned between EtOAc and water for four times to give an EtOAc layer. Meanwhile, the mycelia were extracted by EtOH (95%) for three times. The EtOAc layer together with the mycelium extract were concentrated under reduced pressure to afford a crude extract (wt 16.0 g). This residue was separated by MPLC (MeOH/H₂O, 0–100%) to give sixteen main fractions (A–P).

Subfraction I was separated by Sephadex LH-20 CC (MeOH) to afford three subfractions I1–I3. Subfraction I3 was further purified by prep-HPLC (MeCN/H₂O, 30–50%, 25 min) to give two isolated but interchangeable compounds 7 (t_R = 16.5 min) and 8 (t_R = 17.8 min) with a totally yield 22 mg.

Subfraction J was separated by Sephadex LH-20 CC (MeOH) to remove colourants, and further separated by Sephadex LH-20 (acetone) to afford four subfractions J1–J4. Subfraction J2 was purified by prep-HPLC (MeCN/H₂O, 26–46%, 25 min) to give 1/2 (t_R = 16.5 min) with a yield 2.0 mg. Subfraction J3 was purified by prep-HPLC (MeCN/H₂O, 26–46%, 25 min) to give 3/4 (t_R = 18.2 min) with a yield 1.8 mg.

Subfraction K was separated by Sephadex LH-20 CC (MeOH) to remove colourants, and further separated by Sephadex LH-20 (acetone) to afford five subfractions K1–K5. Subfraction K1 was purified by prep-HPLC (MeCN/H₂O, 25–45%, 25 min) to give 5 (t_R = 17.5 min) with a yield 1.0 mg. Subfraction K3 was purified by prep-HPLC (MeCN/H₂O, 26–46%, 25 min) to give 6 (t_R = 18.9 min) with a yield 0.8 mg.

Spectroscopic Data of Compounds

11,12-Epoxy-15-hydroxy-5,6-seco-tremula-1,6(13)-dien-5,12-olide (1) and methyl 12β,15-dihydroxy-5,6-seco-tremula-1,6(13)-dien-5-oate (2)

Colourless oil; ${[\alpha ]}_{\text{D}}^{26}$ − 56.9 (c 0.09, MeOH); UV (c 0.0306 mg ml⁻¹, MeOH) λ_max nm (Abs): 205 (0.7693), 232 (0.3060); IR (KBr) ν_max 3425, 2927, 2858, 1632, 1384, 1031 cm⁻¹; ¹H NMR (600 MHz, CD₃OD) data, see Table 1; ¹³C NMR (150 MHz, CD₃OD) data, see Table 2; HRESIMS 1: C₁₅H₂₀O₄ m/z 287.1258 [M+Na]⁺ (calcd for C₁₅H₂₀O₄Na, 287.1254); 2: C₁₆H₂₄O₅ m/z 319.1529 [M+Na]⁺ (calcd for C₁₆H₂₄O₅Na, 319.1516).

11,12-Epoxy-14-hydroxy-5,6-seco-tremula-1,6(13)-dien-5,12-olide (3) and methyl 12β,14-dihydroxy-5,6-seco-tremula-1,6(13)-dien-5-oate (4)

Colourless oil; ${[\alpha ]}_{\text{D}}^{26}$ − 77.5 (c 0.10, MeOH); UV (c 0.015 mg ml⁻¹, MeOH) λ_max nm (Abs): 205 (0.4599), 238 (0.1222); IR (KBr) ν_max 3425, 2927, 2860, 1728, 1631, 1032 cm⁻¹; ¹H NMR (600 MHz, CD₃OD) data, see Table 1; ¹³C NMR (150 MHz, CD₃OD) data, see Table 2; HRESIMS 3: C₁₅H₂₀O₄ m/z 287.1250 [M+Na]⁺ (calcd for C₁₅H₂₀O₄Na, 287.1254); 4: C₁₆H₂₄O₅ m/z 319.1513 [M+Na]⁺ (calcd for C₁₆H₂₄O₅Na, 319.1516).

6,11-Dihydroxy-5,6-seco-tremul-1-en-5,12-olide (5)

Colourless oil; C₁₅H₂₄O₄; ${[\alpha ]}_{\text{D}}^{26}$ + 14.6 (c 0.07, MeOH); UV (MeOH) λ_max nm (log ε): 206 (4.13), 254 (2.97); IR (KBr) ν_max 3422, 2952, 2930, 2868, 1750, 1632, 1383, 1193, 1022 cm⁻¹; ¹H NMR (600 MHz, CD₃OD) data, see Table 1; ¹³C NMR (150 MHz, CD₃OD) data, see Table 2; HRESIMS m/z 291.1572 [M +Na]⁺ (calcd for C₁₅H₂₄O₄Na, 291.1567).

11,12-Epoxy-6-hydroxy-5,6-seco-tremul-1-en-5,12-olide (6)

Colourless oil; C₁₅H₂₂O₄; ${[\alpha ]}_{\text{D}}^{26}$ + 47.8 (c 0.07, MeOH); UV (MeOH) λ_max nm (log ε): 205 (3.46), 245 (2.18); IR (KBr) ν_max 3432, 2927, 2859, 1631, 1385, 1031 cm⁻¹; ¹H NMR (800 MHz, CDCl₃) data, see Table 1; ¹³C NMR (200 MHz, CDCl₃) data, see Table 2; HRESIMS m/z 289.1416 [M+Na]⁺ (calcd for C₁₅H₂₂O₄Na, 289.1410).

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Compliance with Ethical Standards

Conflict of interest

The authors declare no conflict of interest.