The mushroom Tyromyces chioneus belongs to the family Polyporaceae,which has a wide distribution in most parts of China,such as Hebei,Shanxi and Heilongjiang provinces. Previous chemical investiga­tion on this fungus only reported a cadinane sesquiter­penoid,which was demonstrated to exhibit significant anti-HIV-1 activity^[1]. In the course of searching for more biologically active compounds^[2,3,4,5],two new sesquiterpenoids,tyromol A (1) and tyromol B (2),together with two previously known sesquiterpenoids 3 and 4,were obtained (Figure 1) from cultures of T. chioneus. Herein,we report the isolation and structural elucidation of these compounds.

Figure 1 Structures of compounds 1-4

Compound 1 was isolated as white powder. Its molecular formula C₁₅H₂₂O₃ was deduced from HR- ESI-MS at m/z 273.145 9 [M+Na]⁺ (calcd. 273.146 6),requiring for five degrees of unsaturation. The IR spectrum showed absorption bands for OH (3 428 cm^-1),C=O (1 770 cm^-1) and C=C (1 632 cm^-1) moieties. The ¹H NMR spectrum (Table 1) exhibited two secondary methyls (δ_H 1.14,d,J = 7.2 Hz; δ_H 0.81,d,J = 6.8 Hz),

Table 1

Table 1 1H and 13C NMR spectroscopic data of 1-2 at 600/150 MHz,respectively. Chemical shift values δ in ppm,coupling constants J in Hz (in parentheses) Position 1 2 1H (CDCl3) 13C (CDCl3) 1H (CDCl3) 13C (CDCl3) 1H (CD3OD) 1 55.0,qC 1.59,overlap 46.1,CH 1.43,m 2 2.12,m 32.3,CH2 1.66,m 20.4,CH2 1.52,m 1.96,m 1.59,overlap 1.42,m 3 2.34,m 32.2,CH2 2.12,m 26.8,CH2 1.96,m 2.08,m 1.90,m 4 148.7,qC 137.5,qC 5 5.48,br s 123.2. CH 5.84,d (5.5) 125.6,CH 5.70,d (5.4) 6 4.04,d (3.4) 85.9,CH 2.45,m 33.4,CH 2.35,m 7 2.36,m 38.5,CH 1.59,overlap 38.7,CH 1.45,m 8 1.68,m 23.3,CH2 1.48,m 19.7,CH2 1.39,m 1.27,m 1.08,m 9 1.48,m 29.3,CH2 1.53,m 34.3,CH2 1.37,m 1.49,m 1.29,m 10 1.69,m 34.2,CH 72.2,qC 11 2.75,m 42.8,CH 2.01,m 35.0,CH 1.83,m 12 179.6,qC 4.01,br s 67.3,CH2 3.21,m 13 1.14,d (7.2) 9.1,CH3 0.89,d (7.0) 10.4,CH3 0.75,d (7.0) 14 0.81,d (6.8) 16.0,CH3 1.23,s 29.5,CH3 1.07,s 15 4.23,dd (14.5,10.0) 62.1,CH2 3.50,dd (7.5,3.2) 67.0,CH2 3.76,d (5.4) OH-10 3.96,s OH-12 4.37,t (5.2) OH-15 4.65,t (5.7)

Table 1 1H and 13C NMR spectroscopic data of 1-2 at 600/150 MHz,respectively. Chemical shift values δ in ppm,coupling constants J in Hz (in parentheses)

three protons attached to two oxygenated carbons separately (δ_H 4.23,2H,dd,J = 14.5,10.0 Hz; δ_H 4.04,1H,d,J = 3.4 Hz),and one olefinic proton (δ_H 5.48,br s). The ¹³C NMR and DEPT spectra (Table 1) revealed the presence of one double bond (δ_C 148.7,C-4; δ_C 123.2,C-5),one oxygenated methine carbon (δ_C 85.9,C-6),one oxygenated methylene carbon (δ_C 62.1,C-15) and one carbonyl carbon (δ_C 179.6,C-12),as well as two methyl groups (δ_C 16.0,C-14; δ_C 9.1,

C-13),four methylenes (δ_C 32.3,C-2; δ_C 32.2,C-3; δ_C 29.3,C-9; δ_C 23.3,C-8),three methines (δ_C 42.8,C-11; δ_C 38.5,C-7; δ_C 34.2,C-10),and one quaternary carbon (δ_C 55.0,C-1).

The ¹H-¹H COSY spectrum displayed signals establishing two fragments as shown in Figure 2. The HMBC correlations from H-10 and H-6 to C-1 established a substituted hexatomic ring A,while the HMBC correlations from H-3 and H-15 to C-4 and from H-2 and H-5 to C-1 constructed a five-membered carbon ring B. In addition,the HMBC correlations

Figure 2 Key 1H-1H COSY,HMBC and ROESY correlations of 1

from H-6,H-11,and H-13 to C-12 suggested a five- membered lactone ring C. Therefore,the planar structure of 1 was established,which was similar to that of 15-hydroxy-6α,12-epoxy-7β,10αH,11βH-spiroax-4- ene (3)^[6],except that C-12 was oxygenated to a carbonyl carbon,as supported by the HMBC correlations from H-6,H-7,and H-13 to δ_C 179.6 (s,C-12). The relative configuration of 1 was assigned on the basis of an ROESY experiment (Figure 2). ROESY correlations of H-5/H-6,H-5/H-14,and H-6/H-7 suggested that H-5,H-6,H-7,and Me-14 were at the same side,while the ROESY correlations of H-7/H-11 suggested that H-11 should be at the same side with H-7. Thus,the structure of compound 1 was established and named as tyromol A.

Compound 2 was also obtained as white powder. Its molecular formula was determined to be C₁₅H₂₆O₃ by HR-ESI-MS,corresponding to three degrees of unsaturation. The IR spectrum showed absorption bands for a hydroxy group (3 427 cm^-1) and double bonds (1 634 cm^-1). The ¹³C NMR and DEPT spectra (Table 1) showed 15 carbon signals that attributed to two methyls,six methylenes,five methines,and two quaternary carbons. The ¹H and ¹³C NMR spectra of 2 were similar to those of agripilol C (4)^[7]. Careful comparison of their NMR data indicated that they were a pair of isomers,with the only difference assigned to the configuration of H₃-14. The HMBC spectrum suggested that its planner structure was identical to that of compound 4. In the ROSEY spectrum,correlations of H₃-14/H-1/H-7 and OH-10/H-6 indicated the α- orientation of H₃-14 and β-orientation of OH-10. Thus,compound 2 (tyromol B) was established as shown.

The known compounds 3 and 4 were purified as white powders,and characterized as 15-hydroxy-6α,12- epoxy-7β,10αH,11βH-spiroax-4-ene (3)^[6],and agripilol C (4)^[7] by comparison of their ¹H and ¹³C NMR data with those reported in the literature. Compounds 3 and 4 were isolated from this fungus for the first time. Experimental section General experimental procedures

Optical rotations were measured on a Jasco-P-1020 polarimeter. UV spectra were measured on a Shimadzu UV-2401 PC spectrophotometer. IR spectra were obtained by using a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. NMR spectra were acquired with an Avance III 600 MHz instrument. HR-ESI-MS were measured on an API QSTAR Pulsar mass spectrometer. Silica gel (200-300 mesh and 80-100 mesh,Qingdao Marine Chemical Inc.,China),RP-18 gel (40-75 µm,Fuji Silysia Chemical Ltd.,Japan)and Sephadex LH-20 (Amersham Biosci­ences,Sweden) were used for column chromatography (CC). Fractions were monitored by TLC (Qingdao Marine Chemical Inc.,China) and spots visualized by heating silica gel plates immersed in vanillin-H₂SO₄ in EtOH. Fungal material and cultivation conditions

The fungus T. chioneus was collected from the Ailao Mountain in Yunnan Province,People’s Republic of China,in July 2003. The fungus was identified by Prof. Mu Zang at the Kunming Institute of Botany. The voucher specimen was deposited at the Herbarium of Kunming Institute of Botany (No. HFC 20110812X). Culture medium: glucose (5%),pork peptone (0.15%),yeast (0.5%),KH₂PO₄ (0.05%),MgSO₄ (0.05%). The fermentation was carried out on a shaker at 24 ℃ and 150 r·min^-1 for 20 d. Extraction and isolation The culture broth (20 L) was extracted three times with EtOAc (3 × 10 L). The combined EtOAc extracts were evaporated in vacuo to give a residue (10.0 g). The residue was subjected to silica gel CC with a gradient elution system of petroleum ether-acetone (30∶1→0∶1) to obtain six fractions (A-F). Fraction C was eluted with petroleum ether-acetone (5∶1). It was then subjected to Sephadex LH-20 CC (CHCl₃-MeOH,1∶1) and silica gel CC with petroleum ether-acetone (4∶1) to afford 2 (2.0 mg) and 4 (3.0 mg). Fraction E was separated by repeated silica gel CC (petroleum ether-acetone,10∶1→0∶1) to yield fractions E01-E03. Fraction E02 was chromatographed on a RP-18 column (MeOH− H₂O,30%) and then purified by CC on silica gel with petroleum ether-acetone (2∶1) to give 1(1.0 mg) and 3 (2.0 mg).

Tyromol A (1): C₁₅H₂₂O₃,white powder; [α] +16.7 (c 0.10,CHCl₃); UV (CHCl₃) λ_max (log ε) 240 (2.1),199 (1.8) nm; IR (KBr) ν_max 3 428,2 923,1 770,1 632,1 169 cm^-1; ¹H NMR (CDCl₃,600 MHz) and ¹³C NMR (CDCl₃,150 MHz) data,see Table 1; HR- ESI-MS m/z 273.145 9 [M+Na]⁺ (calcd. for C₁₅H₂₂O₃Na 273.146 6).

Tyromol B (2): C₁₅H₂₆O₃,white powder; [α] +8.8 (c 0.10,CHCl₃); UV (CHCl₃) λ_max (log ε) 239 (1.9) nm; IR (KBr) ν_max 3 427,2 926,1 634,1 384,1 035 cm^-1; ¹H NMR (CDCl₃ and CD₃OD,600 MHz) and ¹³C NMR (CDCl₃,150 MHz) data,see Table 1; ESI-MS (positive) m/z 277 [M+Na]⁺; HR-ESI-MS m/z 277.177 4 [M+Na]⁺ (calcd. for C₁₅H₂₆O₃Na 277.177 9).