Non-isoprenoid botryane sesquiterpenoids from basidiomycete Boletus edulis and their cytotoxic activity

Introduction

Botryane sesquiterpenoids possess a non-isopreniod system skeleton, which have been found limited to several fungi such as Botrytis cinerea¹ and Daldinia concentrica². The representative botryane sesquiterpenoids are botrydial^1a and its derivatives, which are characterized from phytopathogenic fungus B. cinerea. These sesquiterpenoids showed a wide range of biological activities. For instance, they were responsible for the typical lesions associated with B. cinerea infection, and they played an important role in the pathogenicity of the organism in vivo.^1c,3 Botryane sesquiterpenoids attracted great interests of chemists to carry out a large number of investigations including chemical transformations,^3b structure-activity relationships,^1c synthesis,⁴ and biosynthesis⁵. Our group has long been focused on the chemical study on higher fungi. Recently, three new botryane sesquiterpeniods, boledulins A-C (1–3), have been isolated from cultures of Boletus edulis Bull, an edible basidiomycete collected from southwest of China. The structures were established by extensive spectroscopic data. It is noted that compound 3 is a 15-nor-botryane sesquiterpenoid which was seldom found previously. In addition, compounds 1–3 were evaluated for their cytotoxicity against five human cancer cell lines. This paper reports the isolation, structural elucidation, and cytotoxicity of these compounds.

Results and Discussion

Compound 1 was isolated as optical active white solid ([α]_D²⁰ + 16.8). HRESIMS displayed an [M + Na]⁺ peak at m/z 337.1990 (calcd 337.1990 for C₁₇H₃₀O₅Na) indicating a molecular formula C₁₇H₃₀O₅ corresponding to three degrees of unsaturation. IR spectrum revealed the existence of hydroxy and carbonyl groups due to absorption bands at 3439 and 1729 cm^-1, respectively. The ¹³C NMR spectrum gave 17 carbon resonances (Table 1). Besides two methoxy signals at δ_C 51.6 and 59.4, 15 resonances can be ascribable to four methyls, three sp³ methylenes, four sp³ methines, three sp³ quaternary carbons, and one sp² quaternary carbon at δ_C 174.6. These data suggested that compound 1 might be a bicyclic sesquiterpenoid.

In the HMBC spectrum, the correlation of a methoxy signal at δ_H 3.70 (3H, s) with δ_C 174.6 (s, C-10) established a methyl ester group. A key correlation of δ_H 2.45 (1H, d, J = 12 Hz, H-1) with C-10 suggested the linkage of C-10 to the methine. Starting from this methine, a structural fragment was established by the analysis of ¹H-¹H COSY spectrum as shown in Figure 1. The HMBC correlations of H-1 and δ_H 1.56 (1H, d, J = 11.0 Hz, H-5) with the oxygen-containing quaternary carbon signal at δ_C 87.8 (s, C-9) revealed the connections of C-9 to C-1 and C-5, respectively. Hence, ring A was established as shown in Figure 1. Ring B was readily built due to the rest of carbon and proton resonances leading to the only perfect linkage, which was supported further by the HMBC correlations (Figure 1). In addition, a methoxy group placed at C-15 was also deduced from the HMBC correlation (Figure 1).

Open image in new window

In the ROESY spectrum, the observed cross peaks of H-1/H-5, H-2/H-4, H-1/CH₃-11, H-4/CH₃-12 and H-4/CH₃-14 suggested H-1, H-5, and Me-11 in the same side, while H-2, H-4, Me-12, and Me-14 in the opposite side. Further, these above ROESY cross peaks limited OH-9 to be the same side with H-1. Structurally, compound 1 should be a derivative of botrydial ([α]_D²⁰ + 34),^1a the first botryane sesquiterpenoid isolated from Botrytis cinerea,^1a whose absolute configuration has been identified on the basis of synthesis and biosynthesis^4-6. Accordingly, the absolute configuration of 1 could be determined as 1S, 2R, 4S, 5R, 8S, 9S. Therefore, the structure of 1 (boledulin A) was established.

Compound 2 was isolated as a colorless oil, that gave an [M + Na]⁺ peak at m/z 305.1720 (calcd for C₁₆H₂₆O₄Na, 305.1728) in the positive ion HRESIMS, consistent with the molecular formula C₁₆H₂₆O₄ indicating four degrees of unsaturation. The 1D NMR spectroscopic data (Tables 1 and 2) suggested that the backbone of 2 was the same as that of 1. Differences between them were identified to be a new double bond (δ_C 136.4 and 150.9) and the loss of a methoxy group at C-15 in 2. The HMBC correlations of δ_H 0.93 (3H, s, Me-14) with δ_C 136.9 (s, C-9) and δ_H 1.16 and 1.34 (each 3H, s, Me-12 and Me-13, respectively) with δ_C 150.9 (s, C-5) suggested the double bond placed between C-5 and C-9. The HMBC correlations between H-1, H-4 and C-5, C-9 were also observed. Detailed analysis of other 2D NMR data confirmed that the other parts of 2 were the same as those of 1. Therefore, the structure of 2 (boledulin B) was established.

Compound 3 was isolated as white solid. The molecular formula C₁₄H₂₄O₂ was established by HREIMS at m/z 224.1765 [M]⁺ (calcd for C₁₄H₂₄O₂ at m/z 244.1776 [M]⁺) indicating three degrees of unsaturation. The ¹H NMR spectrum displayed similar patterns to those of 1 and 2 including clear signals for four methyl signals (three singlets and one doublet) (Table 2). The ¹³C NMR spectrum revealed 14 carbon resonances ascribable to four sp³ methyls, three sp³ methylenes, four sp³ methines, one sp³ quaternary carbon, and two sp² quaternary carbons (Table 1). These data suggested that compound 3 might be a bicyclic nor-sesquiterpenoid.

The ¹³C NMR signal at δ_C 60.5 (t, C-10) allowed the existence of an oxygen-containing methylene, and starting from which, two structural fragments of CH₃CH-and CH₂CHCHCH₂CHCH-were established by the ¹H–¹H COSY spectrum as shown in Figure 1. In the HMBC spectrum, the key correlations of δ_H 1.83 (1H, m, H-1) with δ_C 131.4 (s, C-9) and δ_H 2.04 (1H, d, J = 6.4 Hz, H-5) with δ_C 131.4 (s, C-9) suggested the link of C-9 to C-1 and C-5, respectively. Hence, the structure of ring A was established. The HMBC correlation of δ_H 2.05 and 2.19 (each 1H, d, J = 16.8 Hz, H-7a and H-7b, respectively) with δ_C 129.4 (s, C-8) suggested a double bond between C-8 and C-9, which was derived from the degradation of a carbon at C-8. Analysis of other 1D and 2D NMR data established ring B as depicted in Figure 1. Therefore, compound 3 was established to be a 15-nor-botryane sesquiterpenoid and named as boledulin C.

All compounds were evaluated for their cytotoxicities against five human cancer cell lines using the MTT method as reported previously.⁷ The result displayed that compound 1 showed moderate cytotoxicity against five human cancer cell lines using cisplatin as the positive control, while compounds 2–3 were inactive against all the tested cell lines with IC₅₀ values of more than 40 μM (Table 3).

Experimental Section

General Experimental Procedures. Optical rotations were measured with a Jasco P-1020 polarimeter. IR spectra were obtained on a Bruker FT-IR Tensor 27 spectrometer using KBr pellets. 1D and 2D NMR spectra were run on an AV-400 MHz or a Bruker avance Ⅲ-600 MHz spectrometer with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to solvent signals. HREIMS were recorded on a Waters Auto Premier P776 spectrometer. HRESIMS were recorded on an API QSTAR Pulsar i spectrometer. Column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao Marine Chemical Ltd., Qingdao, People's Republic of China). An Agilent 1100 series instrument equipped with Agilent ZORBAX SB-C18 column (5 μm, 4.6 mm × 150 mm) was used for high-performance liquid chromatography (HPLC) analysis, and a semipreparative Agilent ZORBAX SB-C18 column (5 μm, 9.4 mm × 150 mm) was used for the sample preparation. Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co., Ltd. Qingdao), and spots were visualized by 10% H₂SO₄ in ethanol.

Fungal Material and Cultivation Conditions. The fungi Boletus edulis Bull. were collected from Ailao Mountain, Yunnan province, China. A voucher specimen was deposited at State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences. The mycelial cultures were derived from tissue plugs. Culture PDA medium: potato (peeled), 200 g, glucose, 20 g, KH₂PO₄, 3 g, MgSO₄, 1.5 g, citric acid, 0.1 g, and thiamin hydrochloride, 10 mg, in 1 L of deionized H₂O. The pH was adjusted to 6.5 before autoclaving, and the fermentation was carried out on a shaker at 25 ℃ and 150 rpm for 20 days.

Extraction and Isolation. The culture broth (20 L) was extracted three times with EtOAc. The EtOAc lay was evaporated in vacuo to yield an extract (8.6 g). The latter was subjected to a silica gel column eluted with petroleum etheracetone (1:0 to 0:1) to afford fractions1–5. Fraction 2 (1.8 g) was separated by silica gel CC (petroleum ether-Me₂CO, 10:1 → 3:1) to afford two subfractions a and b. Fraction a (100 mg) was separated repeatedly by silica gel CC (petroleum etherEtOAc, 7:1) to afford 3 (10.5 mg). Fraction b (30 mg) was separated further by HPLC (acetonitrile-H₂O, 40:60 to 60:40) to yield 1 (2.2 mg) and 2 (1.1 mg).

Boledulin A (1): white solid; [α]_D²⁰ + 16.8 (c 0.19, CHCl₃); IR (KBr) ν_max 3439, 2924, 2855, 1729, 1629, 1177, 1088 cm^-1; ¹³C (150 MHz) and ¹H NMR (600 MHz) data (CDCl₃), see Tables 1 and 2, respectively; positive ion HRESIMS m/z 337.1990 (calcd for C₁₇H₃₀O₅Na [M + Na]⁺, 337.1990).

Boledulin B (2): colorless oil; [α]_D²⁰ + 10.3 (c 0.17, CHCl₃); ¹³C (150 MHz) and ¹H NMR (600 MHz) data (CDCl₃), see Tables 1 and 2, respectively; positive ion HRESIMS m/z 305.1720 (calcd for C₁₆H₂₆O₄Na [M + Na]⁺, 305.1728).

Boledulin C (3): white solid; [α]_D²⁰ -1.0 (c 0.18, CHCl₃); IR (KBr) ν_max 3251, 2977, 2828, 1699, 1442, 1361, 1037 cm^-1; ¹³C (100 MHz) and ¹H NMR (400 MHz) data (CDCl₃), see Tables 1 and 2, respectively; HREIMS m/z 224.1765 (calcd for C₁₄H₂₄O₂ [M]⁺, 224.1776).

Cytotoxicity Assay. Five human cancer cell lines, breast cancer MCF-7, hepatocellular carcinoma SMMC-7721, human myeloid leukemia HL-60, colon cancer SW480, and lung cancer A-549 cells, 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% CO₂ at 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.⁷ Briefly, 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 × 10⁵ cells/mL. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μmol in triplicates for 48 h, with cisplatin (Sigma, USA) and taxol (National Institute for the Control of Pharmaceutical and Biological Products, P. R. China) as positive controls. After compound treatment, cell viability was detected and a cell growth curve was graphed. IC₅₀ values were calculated by Reed and Muench's method.⁸

Notes

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-011-0005-9 and is accessible for authorized users.

Acknowledgments

This work was supported in part by the National Natural Sci This project was supported by the National Basic Research Program of China (973 Program, 2009CB522300), the National Natural Sciences Foundation of China (30830113), and MOST (2009ZX09501-029).