Furan Derivatives and Polyketides from the Fungus Irpex lacteus

Meng Wang·Zheng-Hui Li·Masahiko Isaka·Ji-Kai Liu·Tao Feng

Abstract Eight new furan derivatives, irpexins A-H (1- 8), two new polyketides, irpexins I and J (9 and 10), together with nine known compounds were isolated from the fermentation of Irpex lacteus.The structures and absolute confi gurations were elucidated on the basis of extensive spectroscopic methods and Mosher ester reaction.All compounds shows no cytotoxicity to human MCF-7 and Hela cancer cell lines at the concentration of 10 μM.

Keywords Irpex lacteus· Furan derivatives · Polyketides

1 Introduction

Fungi are important sources of natural products for the discovery of drugs and agricultural chemicals [1- 5], which have attracted great attention from pharmaceutical chemists and pharmacologists.Irpex lacteusis a pathogenic wooddecaying fungus belonging to the family Polyporaceae [6].Its crude polysaccharide fraction has long been used as a traditional Chinese medicine for the treatment of chronic glomerulonephritis in clinic [7].Chemists have researched the fermentation products of this fungus and the chemical constituents of fruiting bodies.In 1981, Hayashi et al.isolated three new nematicidal metabolites including two furan derivatives from their lactose liquid medium [8].Our previous chemical investigations on the liquid fermentation of this fungus reported new carbon skeletons irlactins A—D that featured a 6/6 backbone [9], as well as a series of tremulane sesquiterpenoids [10,11].Later, studies on the fruiting bodies reported a series of triterpenes, irpeksins A—E, which inhibit the production of NO by RAW264.7 macrophages induced by LPS, and a new skeletal triterpenoid irpexolidal with a 6/5/6/5/6/5-fused polycyclic skeletal system [12,13].Recently, Duan et al.isolated four new bioactive metabolites from the endophytic fungusIrpex lacteusDR10-1, including one tremulane sesquiterpene irpexlacte A and three new furan derivatives irpexlactes B-D [14].In the current study, eight new furan derivatives, irpexins A-H (1- 8), two new polyketides, irpexins I and J (9 and 10), together with nine known compounds were obtained from cultures ofI.lacteusin rice medium.Their structures have been established by extensive spectroscopic methods, as well as modified Mosher’s method.All new compounds were evaluated for their cytotoxic activities against two human MCF-7 and Hela cell lines.

2 Results and Discussion

Compound 1 was isolated as a colorless oil.Its molecular formula C10H14O3was determined on the basis of positive high resolution (HR) ESI—MS atm/z183.10158 [M + H] + (calcd for C10H15O3+, 183.10157), corresponding to four degrees of unsaturation.In the 1 H NMR spectrum (Table 1), one methyl group atδH0.96 (3H, t,J= 7.5 Hz, H-10), one aldehyde proton atδH9.50 (1H, s, H-1), and two olefi nic protons atδH7.19 (1H, d,J= 3.5 Hz, H-3) and 6.28 (1H, d,J= 3.5 Hz, H-4) were readily identified.The 13 C NMR and DEPT data (Table 2) revealed ten carbon resonances ascribable for one CH3, three CH2, four CH, and two C.Of them, signals atδC151.8 (s, C-2), 124.1 (d, C-3), 108.9 (d, C-4), 163.8 (s, C-5) were suggested to establish a furan moiety conjugated to a aldehyde group atδC177.0 (d, C-1).These data were very similar to these of irpexlacte C [14].The main difference was that the position of the hydroxyl group at C-7 in irpexlacte C was moved to C-8 in 1, which was verified by the 1 H- 1 H COSY correlation ofδH1.51 (2H, m, H-9) withδH3.56 (2H, m, H-8) and of H-8 with H-10, together with the HMBC correlations from H-10 toδC30.3 (t, C-9) and 72.2 (d, C-8).Therefore, a planar structure of 1 was fi gured out as shown in Fig.1.The absolute confi guration of 1 was determined by the Mosher reaction.Compound 1 reacted withR-Mosher reagent andS-Mosher reagent to obtain compounds 1a and 1b (for their 1 H NMR spectra, see Figs.S8 and S9 in the Supporting Information), respectively.In the 1 H NMR spectrum, the chemical shifts ofδH2.76—2.71 (2H, m, H-6) andδH 2.07—2.03 (2H, m, H-7) in 1a were higher thanδH2.63—2.56 (2H, m, H-6) andδH2.02—1.96 (2H, m, H-7) in 1b, while the chemical shifts ofδH1.67—1.65 (2H, m, H-9) andδH0.85—0.83 (3H, m, Me-10) were lower thanδH1.74—1.73 (2H, m, H-9) andδH0.95—0.92 (3H, m, Me-10) in 1b.Due to the shielding effect of phenyl, this result suggested that —C9-C10— in 1a was on the same side as phenyl inR-Mosher, and —C9-C10— in 1b is not on the same side as phenyl inS-Mosher corresponding to A-type and B-type in Fig.2, respectively.At this point, the absolute confi guration of C-8 was determined asR.Compound 1 was, therefore, established as irpexin A, as depicted.

Compound 2 was isolated as a colorless oil.Its molecular formula C10H12O3was determined on the basis of HRESI-MS, corresponding to fi ve degrees of unsaturation.The13C NMR and DEPT data of 2 were very similar to these of irpexlacte C [14], except for one double bond in 2.In1H-1H COSY spectrum, the correlation ofδH6.84 (1H, m, H-9) withδH2.21 (1H, m, H-8) andδH5.06 (2H, m, H-10) revealed a double bond was formed between C-9 and C-10 in 2.The optical rotation of 2 ([α] = - 38.0), contrary to that of 1 ([α] = + 44.8) and those analogues reported in the literature [15], indicated that the absolute confi guration of the C-6 in the side chain wasS.Compound 2 was, therefore, established as irpexin B, as depicted.

Compound 3 was isolated as a colorless oil.Its molecular formula C10H16O3was determined on the basis of HR-ESIMS, corresponding to three degrees of unsaturation.The13C NMR and DEPT data (Table 2) of 3 were very similar to these of irpexlacte C [14], except that the aldehyde unit was replaced by a hydroxymethyl (δC57.5) in 3, as supported by the HMBC correlations fromδH4.59 (1H, s, H-1) toδC153.3 (s, C-2) and 108.4 (d, C-3).The optical rotation of 3 ([α] = - 16.1) suggested C-6 to beSform.Compound 3 was, therefore, established as irpexin C, as depicted.

Compound 4 was isolated as a colorless oil.Its molecular formula C10H20O4was determined on the basis of HR-ESI—MS atm/z251.12544 [M + Na] + (calcd for C12H20O4Na + , 251.12538), corresponding to three degrees of unsaturation.The 13 C NMR and DEPT data of 4 were very similar to these of 1 (Table 2), except that the aldehyde unit in 1 was replaced by a dimethoxymethine group as supported by the HMBC correlations fromδH5.38 (1H, s, H-1) toδC149.0 (s, C-2), 109.1 (d, C-3) and two OCH3(δC52.8).The optical rotation of 4 ([α] = + 37.5), related to that of 1, revealed the absolute confi guration of C-8 to beR.Compound 4 was, therefore, established as irpexin D, as depicted.

Compound 5 was isolated as a colorless oil.Its molecular formula C12H18O3was determined on the basis of the HR-ESI—MS data atm/z211.13280 [M + H] + (calcd for C12H19O3+, 211.13287), corresponding to four degrees of unsaturation.The 13 C NMR and DEPT data of 5 were very similar to these of 1 (Table 2), except for two additional carbon resonances linked to a carbonyl carbon forming a propionyl group, replacing the formyl group in 1.It was supported by the 1 H- 1 H COSY correlation ofδH1.20 (3H, t,J= 7.4 Hz, Me-1) withδH2.80 (2H, m, H-2), together with the HMBC correlations fromδH7.11 (1H, d, H-5), H-1, and H-2 toδC189.7 (s, C-3) and 151.2 (s, C-4).The optical rotation data ([α] = + 14.6) indicated C-10 to beRform.Compound 5 was, therefore, established as irpexin E, as depicted.

Compound 6 was isolated as a colorless oil.Its molecular formula C12H18O3was determined on the basis of HR-ESIMS data.The 1 H and 13 C NMR spectra were very close to those of 5 (Tables 1 and 2), except for the hydroxyl group at C-10 (δC72.3) in 5 moved to the C-11 (δC67.7) in 6, as verified by the 1 H- 1 H COSY correlation ofδH3.84 (1H, m, H-11) withδH1.51 (2H, m, H-10) andδH1.21 (3H, d,J= 6.2 Hz, Me-12).The calculated optical rotation data ([α] = - 10.7) matched well with the experimental data([α] = - 14.3) for 6 suggested C-10 to beRform (see Supporting Information).Compound 6 was, therefore, established as irpexin I, as depicted.

Table 1 1 H NMR (600 MHz) Data of 1 - 10 (δin ppm and Jin Hz)

Table 2 13C NMR (150 MHz) Data of 1- 10 (δ in ppm and J in Hz)

Fig.1 Chemical structures of compounds 1- 19

Compound 7 was isolated as a colorless oil.Its molecular formula C12H16O4was determined on the basis of the positive high resolution (HR) ESI-MS at m/z 225.11211 [M + H] + (calcd for C12H17O4+, 225.11214), corresponding to fi ve degrees of unsaturation.The13C and DEPT spectra displayed 12 carbon resonances which were closely related to that of 5 (Table 2), except that the signals atδC31.3 (t, C-2) andδC72.3 (d, C-10) in 5 were replaced by signals ofδC69.3 (d, C-2) andδC209.0 (s, C-10) in 7, respectively.The 1 H- 1 H COSY correlation ofδH 1.47 (3H, d,J= 7.0 Hz, Me-1) withδH 4.83 (1H, q,J= 7.0 Hz, H-2) suggested that C-2 in 5 was substituted by a hydroxyl group.In addition, the1H-1H COSY correlation ofδH3.03 (2H, t,J= 7.3 Hz, H-8) withδH2.84 (2H, t,J= 7.3 Hz, H-9), andδH2.46 (2H, q,J= 7.3 Hz, H-11) withδH1.08 (3H, t,J= 7.3 Hz, Me-12), as well as the HMBC correlation from H-12 to C-10 andδC35.9 (t, C-11) suggested that C-10 in 5 was substituted by a ketone group.The optical rotation ([α] = - 14.0), comparing with those analogues reported in the literature [16], indicated C-2 to beSform.Therefore, the structure of 7 was established as irpexin G, as shown.

Fig.2 Four types of products of compound 1 reacting with R- and S-type Mosher reagents

Compound 8 was isolated as a colorless oil.Its molecular formula C13H18O4was determined on the basis of HR-ESIMS.The 13 C NMR and DEPT data of 8 were very similar to these of 7 (Table 2), except that the hydroxy at C-2 was replaced by a methoxy in 8, as supported by a HMBC correlation fromδH3.23 (3H, s,OCH3) toδC81.4 (d, C-2).Detailed analysis of 2D NMR data suggested that other parts of 8 were the same to those of 7.Therefore, compound 8 was established as irpexin K, as depicted.

Compound 9 was isolated as a colorless oil.Its molecular formula C10H14O4was determined on the basis of HR-ESIMS atm/z221.07843 [M + Na] + (calcd for C10H14O4Na+, 221.07843), corresponding to four degrees of unsaturation.In the 1 H NMR spectrum (Table 1), one methyl group atδH1.06 (t,J= 7.3 Hz, Me-9), one methoxy group atδH3.20 (s,OCH3), two olefi nic protons atδH6.22 (d,J= 5.7 Hz, H-2) and 7.12 (d,J= 5.7 Hz, H-3) were identified.The 13 C and DEPT spectra (Table 2) displayed 10 carbon resonances corresponding to one CH3, one OCH3, three CH2, two CH (two olefi nic carbon atδC153.6 and 124.7), and three C (one ketone carbon atδC210.0, one ester carbonyl atδC169.7, one oxygenated sp3quaternary carbon atδC110.4) (Table 2).Considering one double bond, one ketone carbon and ester carbonyl, compound 9 should possess a single ring framework.Primary analysis of 2D NMR data indicated a fi ve-membered unsaturated lactone as built by C-1 to C-4 [17], as well as a 3-pentone moiety link to C-4 (Fig.3).In addition, one HMBC correlation from OMe toδC110.4 (s, C-4) indicated one methoxy at C-4.The optical rotation ([α] = 0) indicated that 9 should be racemic.Compound 9 was, therefore, identifed as irpexin I, as depicted.

Fig.3 Key HMBC and 1H- 1 H COSY correlations of compounds 1,9, and 10

Compound 10 was isolated as a colorless oil.Its molecular formula C12H20O2was determined on the HR-ESIMS atm/z197.15361 [M + H] + (calcd for C12H21O2+, 197.15361), corresponding to three degrees of unsaturation.In the 1 H NMR spectrum (Table 1), two methyl group atδH0.98 (t,J= 7.6 Hz, Me-1) and 0.97 (t,J= 7.6 Hz, Me-12), one oxygenated proton atδH3.56 (1H, m, H-10) were identified.The13C and DEPT spectra (Table 2) displayed 12 carbon resonances corresponding to two CH3, six CH2, one CH (one oxygenated methine atδC72.9), and three C (one ketone carbon atδC210.0, two olefi nic carbon atδC173.2 and 142.0) (Table 2).Considering one double bond and one ketone carbon, compound 10 should possess a single ring framework.The gross structure of 10 was deduced by comprehensive analysis of its 2D NMR spectra.In the1H-1H COSY spectrum, three fragments were revealed as shown with bold lines in Fig.3.In the HMBC spectrum, the correlation fromδH2.21 (2H, q,J= 7.6 Hz, H-2) toδC142.0 (s, C-3), 210.0 (s, C-4), and 173.2 (s, C-7), and fromδH2.60 (1H, m, H-8a) and 2.52 (1H, m, H-8b), as well as fromδH2.51 (2H, m, H-6) to C-4 established a fi ve-membered ring by C-3, C-4, C-5, C-6, and C-7.At the same time, the ethyl branch was connected to C-3, and the other branch composed by C8, C9, C10, C11, and C12 was connected to C-7.According to 1 H- 1 H COSY spectrum, a hydroxy attached to C-10 (δC72.9) was established.The optical rotation data ([α] = - 9.7) suggestedSform of C-10.Compound 10 was, therefore, identified as irpexin J, as depicted.

Other known compounds were identified by comparison of their NMR spectroscopic data with the literature, namely irpexlacte B (11) [14], irpexlacte C (12) [14], irpexlacte D (13) [14], 1-(5-(hydroxymethyl)-2-furanyl)-pentanone (14) [18], 5-(3-oxopentyl)-2-furancarboxaldehyde (15) [19], (1R,2R)-1-(5-(pent-4-en-1-yl)furan-2-yl)propane-1,2-diol (16) [20], (1R,2R)-1-(5-pentylfuran-2-yl)propane-1,2-diol (17) [20], 2,5,8-decanetrione (21) [19], 2-butyl-3hydroxy-2-cyclopenten-1-one (19) [22].

All new compounds were evaluated for their cytotoxicity against human MCF-7 and Hela cancer cell lines.However, no compounds showed cytotoxicity against two cell lines at the concentration of 10 μM.

3 Experimental Section

3.1 General Experimental Procedures

Optical rotations were measured with a Horiba SEPA-300 polarimeter.IR spectra were obtained with a Tenor 27 spectrophotometer using KBr pellets.1D and 2D spectra were run on a Bruker Avance III 600 MHz spectrometer with TMS as an internal standard.Chemical shifts (δ) were expressed in ppm with reference to the solvent signals.Mass spectra were recorded on an Agilent 6200 Q-TOF MS system.Column chromatography (CC) was performed on silica gel (200—300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People’s Republic of China), RP-18 gel (20—45 μm, Fuji Silysia Chemical Ltd., Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Sweden).Medium Pressure Liquid Chromatography (MPLC) was performed on a Biotage SP1 equipment, and columns packed with RP-18 gel.Preparative High Performance Liquid Chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 μm, 9.4 mm × 150 mm or 21.2 mm × 150 mm) and a DAD detector.Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd.Qingdao), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4in EtOH.

3.2 Fungal Material

Irpex lacteuswas collected from the Wangtianshu Scenic Area, Xishuangbanna, Yunnan Province in July 2014, and authenticated by Prof.Yu-Cheng Dai of Beijing Forestry University.A voucher specimen ofI.lacteuswas deposited at the Higher Fungi Chemistry Group of School of Pharmaceutical Sciences, South-Central University for Nationalities (No.HFG 201407-SCUN201804.2).

3.3 Fermentation Condition

This strain was cultured on PDA medium for 8 days, and then was cut into small pieces to incubate on solid rice medium.The rice medium was inoculated in 500-mL Erlenmeyer flasks, each containing 100 g of rice medium and 100 mL of water.Flask cultures were put into a high pressure sterilizing pot and sterilized at 121 ℃ for 15 min.Then, the strain ofI.lacteuswas cultured in the rice medium, and two hundred 500-mL Erlenmeyer flasks were dark incubated fi xedly at 25 °C for 30 days.

3.4 Extraction and Isolation

The culture ofI.lacteusin rice medium (20 kg) was extracted fi ve times with Me2CO to give a crude extract, which was partitioned into water and EtOAc layers.The EtOAc layer (72 g) was subjected to CC over silica gel (80-100 mesh) eluted with a solvent system of CHCl3/MeOH (from 1:0 to 0:1) to obtain nine fractions (A-I).Fraction C (6 g) was separated by MPLC over RP-18 silica gel column eluted with MeOH/H2O (from 5:95 to 100:0, v/v) to give fi ve sub-fractions (C1-C5).Fraction C1was repeatly separated by CC over silica gel eluted with petroleum ether/ethyl acetate (15:1) to gain 18 (4.6 mg), and then purified by prep-HPLC (CH3CN/H2O from 5:95 to 25:75 in 25 min, v/v) to give 13 (0.8 mg,tR= 21.6 min).Fraction C2was separated by CC over Sephadex LH-20 (methanol), and then purified by prep-HPLC (CH3CN/H2O = 16:84, v/v) to give 2 (2.2 mg,tR= 20.1 min),12 (23.7 mg,tR= 18.0 min) and 19 (2.0 mg,tR= 24.1 min).Fraction C3was repeatly separated by CC over silica gel eluted with petroleum ether/ ethyl acetate (15:1), and then purified by prep-HPLC (CH3CN/H2O from 8:92 to 20:80 in 40 min, v/v) to give 7 (4.5 mg,tR= 20.8 min) and 15 (0.6 mg,tR= 17.3 min).Fraction C4was separated by CC over Sephadex LH-20 (methanol), and then purified by prep-HPLC (MeOH/H2O from 25:75 to 55:45 in 30 min, v/v) to give 1 (24.8 mg,tR= 22.6 min) and 4 (2.5 mg,tR= 17.4 min).

Fraction D (8 g) was separated by MPLC over RP-18 silica gel column eluted with MeOH/H2O (from 5:95 to 100:0, v/v) to give eleven sub-fractions (D1-D11).Fraction D2was purified by prep-HPLC (CH3OH/H2O = 26:74, v/v) to give 3 (2.5 mg,tR= 18.2 min) and 9 (1.0 mg,tR= 14.6 min).Fraction D3was separated by CC over Sephadex LH-20 (methanol), and then purified by prep-HPLC (CH3CN/H2O = 21:79, v/v) to give 6 (2.4 mg,tR= 20.6 min) and 10 (5.0 mg,tR= 25.0 min), and by prep-HPLC (CH3CN/H2O = 26:73, v/v) to give 11 (14.1 mg,tR= 16.2 min) and 14 (2.4 mg,tR= 18.9 min).Fraction D4was repeatly separated by CC over silica gel eluted with petroleum ether/acetone (10:1), and then purified by prep-HPLC (CH3CN/H2O = 32:68, v/v) to give 7 (3.3 mg,tR= 15.4 min).Fraction D6was separated by CC over silica gel eluted with petroleum ether/acetone (10:1), and then purified by prep-HPLC (CH3CN/H2O = 32:68, v/v) to give 16 (2.2 mg,tR= 17.5 min) and 17 (12.8 mg,tR= 19.8 min).Fraction E (7.2 g) was separated by MPLC over RP-18 eluted with MeOH/H2O (from 5/95 to 100/0, v/v) to give fi ve subfractions (E1-E5).Fraction E3was subjected to CC over silica gel (200—300 mesh) and Sephadex LH-20 (methanol), and then purified by prep-HPLC (CH3CN/H2O from 20:80 to 70:30 in 20 min, v/v) to give 8 (3.2 mg,tR= 8.9 min).

3.5 Spectroscopic Data of Compounds

3.5.1 Irpexin A (1)

3.5.2 Irpexin B (2)

3.5.3 Irpexin C (3)

3.5.4 Irpexin D (4)

3.5.5 Irpexin E (5)

3.5.6 Irpexin F (6)

3.5.7 Irpexin G (7)

3.5.8 Irpexin H (8)

3.5.9 Irpexin I (9)

3.5.9 .1 Irpexin J (10)Colorless oil;- 9.7 (c0.12, MeOH); UV (MeOH) λmax(log ε) 235 (3.18) nm.IR (KBr)νmax: 3338, 2943, 2831, 1647, 1452, 1114, 1031 cm-1;1H NMR (600 MHz,CDCl3) and13C NMR (150 MHz,CDCl3) spectroscopic data, see Tables 1 and 2; HR-ESI—MS:m/z197.15361 [M + H]+(calcd for C12H21O2+, 197.15361).

3.6 Mosher Reaction

Compound 1 (25 μmol) was added to a 10 mL round bottom flask, then 2.5 mL of dichloromethane and 50 μmol of pyridine were added.Finally, 50 μmol of (R) or (S) -MTPA-Cl was added dropwise, which was detected by the thin-layer chromatography.After 3 h of stirring at room temperature, the reaction was terminated, and the reaction solution was washed twice with 1 M diluted hydrochloric acid.The organic phase was concentrated and separated by HPLC to obtain compounds 1a and 1b respectively.

3.7 Cytotoxicity Assay

Two human cancer cell lines, breast cancer MCF-7 and Hela, were used in the cytotoxic assay.All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA) in 5% CO2at 37 ℃.The cytotoxicity assay was performed according to the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method in 96-well microplates [23].Briefl y, 100 μL adherent cells were 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 with initial density of 1 × 10 5 cells/mL.Each tumor cell line was exposed to the test compound at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μM in triplicates for 48 h, with taxol as a positive control.After compound treatment, cell viability was detected and cell growth curve was graphed.IC50value was calculated by Reed and Muench’s method [24].

AcknowledgementsThis work was financially supported by the joint research project from the National Natural Science Foundation of China (Grant No.21961142008) and Thailand Research Fund (Grant No.DBG6280008).The authors thank Analytical & Measuring Centre, South-Central University for Nationalities for the spectra measurements.

Compliance with Ethical Standards

Conflict of interestThe authors declare no confl ict of interest.

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