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New Metabolites from Endolichenic Fungus Pleosporales sp. by Yang Jiao a ) 1), Gang Li a )1), Hai-Ying Wang b ), Jing Liu b ), Xiao-Bin Li a ), Lu-Lu Zhang b ), Zun-Tian Zhao* b ), and Hong-Xiang Lou* a ) a

) Department of Natural Products Chemistry, Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, No. 44 West Wenhua Road, Jinan 250012, P. R. China (phone: þ 86-531-88382012; fax: þ 86-531-88382019; e-mail: [email protected]) b ) College of Life Sciences, Shandong Normal University, No. 88 East Wenhua Road, Jinan 250014, P. R. China (phone: þ 86-531-86180719; fax: þ 86-531-86180749; e-mail: [email protected])

Eight new metabolites were obtained from the culture of an endolichenic fungus, Pleosporales sp. Their structures were determined as three terphenyl derivatives, cucurbitarins A – C (1 – 3, resp.), two structurally related compounds, cucurbitarins D and E (4 and 5, resp.), two benzocoumarins, 3,10dihydroxy-4,8-dimethoxy-6-methylbenzocoumarin (6) and 3,8,10-trihydroxy-4-methoxy-6-methylbenzocoumarin (7), as well as one cyclohexenone, (5R)-5-hydroxy-2,3-dimethylcyclohex-2-en-1-one (8), based on the spectroscopic data.

Introduction. – As special symbiotic organisms of photosynthetic algae or cyanobacteria (photobiont), and a fungal partner (mycobiont), lichens also harbor numerous endolichenic fungi which represent a largely untapped resource of smallmolecule natural products [1]. Examples including eleven heptaketides from Corynespora sp. [1] [2], six ambuic acid derivatives and one torreyanic acid analog from Pestalotiopsis sp. [3], three allenyl phenyl ethers and three alkynyl phenyl ethers from Neurospora terricola [4], several chromone derivatives (polyketides) from Coniochaeta sp. [5] [6], Preussia africana [7], and Ulocladium sp. [8], two cyclic pentapeptides from Xylaria sp. [9], twelve perylenequinones from Phaeosphaeria sp. [10], and five entkaurane diterpenoids from Geopyxis aff. majalis and Geopyxis sp. AZ0066 [11] have been reported. As part of our ongoing research on active ingredients of fungi inhabiting lichens and liverworts [12 – 14], we investigated an endolichenic fungus, Pleosporales sp. Since different culture conditions are known to afford different metabolites [15 – 19], and in an attempt to maximize chemical diversity of the metabolites produced by this fungus, we used potato dextrose broth (PDB) and rice as culture media. Chemical investigation of the AcOEt extract from the culture filtrate of fermentations on PDB led to the isolation and characterization of four new metabolites, cucurbitarins A – C (1 – 3, resp.) and (5R)-5-hydroxy-2,3-dimethylcyclohex-2-en-1-one (8), as well as two known steroids, dankasterone A (10) and (17R)-4hydroxy-17-methylincisterol (11). The AcOEt extract of cultures grown on rice medium afforded cucurbitarins D and E (4 and 5, resp.), 3,10-dihydroxy-4,81)

These authors contributed equally to this work. Õ 2015 Verlag Helvetica Chimica Acta AG, Zîrich

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dimethoxy-6-methylbenzocoumarin (6), 3,8,10-trihydroxy-4-methoxy-6-methylbenzocoumarin (7), and 2,5-dimethoxy-3,6-bis(4-methoxypheny1)-1,4-benzoquinone (9), of which 4 – 7 were new compounds. Herein, we describe the fermentation, isolation, and structure elucidation of the new compounds.

Results and Discussion. – Compounds 1 – 11 were isolated from the fermentation of Pleosporales sp. on two culture media, using a combination of size-exclusion, normalphase, and reversed-phase chromatography. Structure Elucidation. Compound 1, white amorphous solid, had the molecular formula C19H18O3 as determined by HR-ESI-MS (m/z 295.1336 ([M þ H] þ ; calc. 295.1329)), implying eleven degrees of unsaturation. Analysis of 1H- and 13C-NMR, and HSQC spectra of 1 (Table 1) revealed the presence of one MeO, one CH2 , one CH, and one CH¢O group, 14 aromatic/olefinic C-atoms (including ten H-bearing and one O-bearing C-atom), and one a,b-unsaturated ketone. The molecular formula and

a

3.79 (s)

7.23 (d, J ¼ 7.2) 7.38 (t, J ¼ 7.2) 7.30 (t, J ¼ 7.2)

7.37 (d, J ¼ 7.8) 7.45 (t, J ¼ 7.8) 7.36 – 7.38 (m)

57.3

139.0 128.0 129.4 128.2 132.6 130.9 128.2 127.5

(d, J ¼ 3.6) (dt, J ¼ 9.0, 4.2) (dd, J ¼ 16.8, 4.8, Ha ), (dd, J ¼ 16.8, 9.6, Hb )

7.77 (d, J ¼ 7.2) 7.43 (t, J ¼ 7.2) 7.27 (t, J ¼ 7.2)

7.70 (d, J ¼ 7.8) 7.36 (t, J ¼ 7.8) 7.27 (t, J ¼ 7.8)

4.95 3.85 3.13 3.70 142.0 128.4 129.6 127.4 135.6 132.3 129.0 127.1

74.0 45.0 36.1

182.5 116.4 188.7

d(C )

) Assignments were based on 1H,1H-COSY, HSQC, HMBC, and NOESY experiments.

1’ 2’,6’ 3’,5’ 4’ 1’’ 2’’,6’’ 3’’,5’’ 4’’ 1-OH 2-OH 3-MeO 4-OH

67.9 43.9 36.1

4.73 (d, J ¼ 2.4) 3.60 (dt, J ¼ 13.2, 3.6) 2.64 (dd, J ¼ 15.6, 3.6, Ha ), 3.29 (dd, J ¼ 15.6, 13.2, Hb )

4 5 6

d( H )

197.2 120.7 169.0

2

d(H )

d(C )

1

1 2 3

Position

7.45 – 7.46 (m) 7.34 – 7.35 (m) 7.54 – 7.55 (m)

8.00 (d, J ¼ 7.5) 7.51 – 7.52 (m) 7.54 – 7.55 (m)

7.56 (s)

d( H )

4

128.6 129.4 129.3 132.6 136.3 126.2 129.1 129.3

157.3 200.0

78.3 198.9 140.5

d(C )

6.04 (s)

7.41 (d, J ¼ 7.6) 7.35 (dd, J ¼ 7.6, 7.2) 7.30 (t, J ¼ 7.2) 6.49 (s) 5.07 (d, J ¼ 5.0)

7.48 (d, J ¼ 7.6) 7.37 (dd, J ¼ 7.6, 7.2) 7.28 (t, J ¼ 7.2)

4.04 (q, 4.8) 2.66 (dd, J ¼ 13.8, 5.1, Ha ), 2.14 (dd, J ¼ 13.8, 5.1, Hb )

d( H )

5

143.4 126.0 127.0 127.2 139.2 128.1 127.9 127.0

78.9 215.2

82.6 74.4 44.5

d(C )

Table 1. 1H- and 13C-NMR Data (600 and 150 MHz, resp.) of 1 and 4 (in CDCl3 ), 2 (in ( D5 )pyridine), and 5 (in ( D6 )DMSO ) a ). d in ppm, J in Hz. Arbitrary atom numbering as indicated in the Formulae.

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eleven degrees of unsaturation accounted for all 1H- and 13C-NMR resonances but one exchangeable H-atom, and suggested that 1 was a terphenyl derivative with two monosubstituted benzene rings [20]. Interpretation of the 1H,1H-COSY spectrum led to the identification of the fragment C(4)¢C(5)¢C(6) (Fig.). The HMBCs (Fig.) of H¢C(4) (d(H) 4.73) with C(2) (d(C) 120.7) and C(3) (169.0), of Ha¢C(6) (d(H) 2.64) with C(1) (d(C) 197.2) and C(2) established the fragment C(6)¢C(1)¢C(2)¼ C(3)¢C(4). The HMBC of MeO (d(H) 3.79 (s)) with C(3) (d(C) 169.0) indicated that the MeO group was located at C(3). Further HMBCs H¢C(4)/C(1’), H¢C(5)/ C(1’), H¢C(5)/C(2’), H¢C(5)/C(6’), and CH2(6)/C(1’) disclosed the linkage of one benzene ring to C(5). Another benzene ring was located at C(2), which was confirmed by the HMBCs of H¢C(2’’) and H¢C(6’’) with C(2). Accordingly, the planar structure of 1 was unambiguously determined as depicted. The relative configuration of 1 was assigned by analysis of coupling constants and NOESY data. The trans-coupling constant (J ¼ 13.2) between H¢C(5) and Hb¢C(6), and the small coupling constant (J ¼ 2.4) between H¢C(4) and H¢C(5) indicated a cofacial arrangement of the OH group at C(4) and the benzene ring at C(5). This conclusion was further confirmed by the key NOESY correlations of H¢C(5) and Hb¢C(6) with H¢C(2’) or H¢C(6’). The absolute configuration of 1 was assigned on the basis of the CD spectrum and the back-octant rule [21]. An obvious negative Cotton effect at 341 nm (Demax ¢ 1.44) for n ! p* transition of the a,b-unsaturated ketone allowed us to propose the absolute configuration (4S,5R). Compound 2 was also obtained as white amorphous solid with a molecular formula of C18H16O3 as determined by HR-ESI-MS (m/z 281.1184 ([M þ H] þ ; calc. 281.1172)). Its 1H- and 13C-NMR spectra (Table 1) suggested structural features similar to those of

Figure. 1H,1H-COSY (— —) and key HMB (H ! C) correlations of 1 and 3 – 8

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1, except for the absence of the MeO group (d(H) 3.79 (s), d(C) 57.3) in 2, which was further supported by comprehensive analysis of 2D-NMR data (Fig.). The absolute configuration of 2 was also assigned by the CD spectrum and the back-octant rule [21]. Since an obvious negative Cotton effect at 322 nm (Demax ¢ 0.27) was observed to be the same as that of 1, the absolute configuration was determined as (4S,5R). Compound 3 was obtained as white amorphous solid. HR-ESI-MS (m/z 491.1671 ([M þ Na] þ ; calc. 491.1676)) provided its molecular formula as C26H28O8 . The 1H- and 13 C-NMR, and HSQC spectra of 3 (Table 2) revealed that 3 also had a typical terphenyl-type substructure [20]. Detailed analysis of the 1D-NMR data of 3 in the upfield region indicated the presence of two MeO groups and a glucose moiety. Strong HMBCs of MeO¢C(5) (d(H) 3.60) with C(5) (d(C) 146.5), of MeO¢C(3) (d(H) 3.64) with C(3) (d(C) 152.8), and of H¢C(1’’’) (d(H) 5.66) with C(2) indicated a pentasubstituted benzene ring with C(3) and C(5) bearing MeO groups, and the location of the glucose moiety at C(2). The chemical shift of C(1’’’) (d(C) 103.1) and the coupling constant of H¢C(1’’’) (J ¼ 7.8) suggested b-configuration for the glucose unit. Therefore, the structure of 3 was determined as shown. Compound 4 was obtained as yellow solid and had the molecular formula C17H12O3 as deduced from HR-ESI-MS (m/z 265.0862 ([M þ H] þ ; calc. 265.0859)). The 1H- and 13 C-NMR, and HSQC spectra of 4 (Table 1) revealed the presence of one O-bearing Cqatom, 14 aromatic/olefinic C-atoms (eleven H-bearing), and two C¼O groups. The molecular formula and twelve degrees of unsaturation accounted for all 1H- and 13 C-NMR resonances but one exchangeable H-atom, which indicated a tricyclic framework with two monosubstituted benzene rings as in 1. However, detailed analysis of 13C-NMR data indicated that the central ring of 4 was different from that of 1. The HMBC cross-peaks of H¢C(3) (d(H) 7.56) with C(1) (d(C) 78.3), C(2) (198.9), C(4) (157.3), and C(5) (200.0) revealed a five-membered ring with a C¼C bond, two C¼O groups, and a Cq-atom [22]. The HMBCs of H¢C(2’)/H¢C(6’) (d(H) 8.00 (d, J ¼ 7.5)) with C(4), as well as of H¢C(2’’)/H¢C(6’’) (d(H) 7.45 – 7.46 (m)) with C(1) indicated that the two benzene rings were linked to C(4) and C(1) of the central ring, respectively. Table 2. 1H- and 13C-NMR Data (600 and 150 MHz, resp.; in (D5 )pyridine) of 3 a ). d in ppm, J in Hz. Position 1 2 3 4 5 6 1’ 2’,6’ 3’,5’ 4’ 1’’ 2’’,6’’ a

d(H )

7.63 (s) 7.87 (d, J ¼ 7.8) 7.41 (t, J ¼ 7.8) 7.30 (t, J ¼ 7.8) 7.90 (d, J ¼ 7.8)

d(C )

Position

d( H )

d(C )

136.1 152.3 152.8 126.5 146.5 113.2 135.0 132.2 128.4 127.7 139.3 130.2

3’’,5’’ 4’’ 1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 6’’’

7.48 (t, J ¼ 7.8) 7.37 (t, J ¼ 7.8) 5.66 (d, J ¼ 7.8) 4.58 (t, J ¼ 8.4) 4.25 (dd, J ¼ 9.6, 3.6) 4.55 (d, J ¼ 3.0) 4.20 (t, J ¼ 6.0) 4.40 (dd, J ¼ 11.4, 5.4, Ha ), 4.51 (dd, J ¼ 11.4, 6.6, Hb ) 3.64 (s) 3.60 (s)

129.1 128.1 103.1 72.4 76.1 70.8 78.1 62.9

3-MeO 5-MeO

) Assignments were based on 1H,1H-COSY, HSQC, HMBC, and NOESY experiments.

61.2 61.2

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The absolute configuration of 4 was assigned on the basis of the CD spectrum and the back-octant rule [21]. An obvious negative Cotton effect at 316 nm (Demax ¢ 2.20) for n ! p* transition of the a,b-unsaturated ketone allowed us to propose the configuration at C(1) as (R). Compound 5 was obtained as colorless crystalline powder (MeOH). The molecular formula of C17H16O4 was determined by HR-ESI-MS (m/z 307.0953 ([M þ Na] þ ; calc. 307.0941)), implying ten degrees of unsaturation. The 1H- and 13C-NMR data (Table 1) of 5 displayed structural similarity to 4 with a five-membered ring at the center. The HMBCs (Fig.) of HO¢C(1) (d(H) 6.49) with C(1) (d(C) 82.6), C(2) (74.4), and C(5) (215.2), of HO¢C(2) (d(H) 5.07) with C(1), C(2), and C(3) (d(C) 44.5), as well as of HO¢C(4) (d(H) 6.04) with C(4) (d(C) 78.9) and C(5) evidenced that three OH groups were located at C(1), C(2), and C(4), respectively. The HMBCs of H¢C(2’)/H¢C(6’) (d(H) 7.48 (d, J ¼ 7.6)) with C(4), as well as of H¢C(2’’)/H¢C(6’’) (d(H) 7.41 (d, J ¼ 7.6)) with C(1) suggested that, as in 4, the two benzene rings were linked to the central ring via C(1) and C(4). The relative configuration of 5 was assigned by analysis of its NOESY data. In the NOESY spectrum, Hb¢C(3) correlated with HO¢C(4) but not with H¢C(2’), indicating that the OH group at C(4) was b-oriented. Ha¢C(3) correlated with HO¢C(1), but not with H¢C(2’’), suggesting that the OH group at C(1) was a-oriented. Strong NOESY correlations of H¢C(2) with HO¢C(1) and H¢C(2’) indicated b-orientation of the OH group at C(2). The absolute configuration of 5 was assigned on the basis of the CD spectrum and the back-octant rule [21]. An obvious negative Cotton effect at 337 nm (Demax ¢ 5.76) for n ! p* transition of the a,bunsaturated ketone allowed us to propose the absolute configuration (1S,2R,4R). Compound 6, white solid, had the molecular formula C16H14O6 as determined by HR-ESI-MS (m/z 303.0864 ([M þ H] þ ; calc. 303.0863)), indicating ten degrees of unsaturation. The 1H- and 13C-NMR, and HSQC spectra (Table 3) of 6 revealed the presence of one exchangeable H-atom, one Me group, two MeO groups, twelve aromatic/olefinic C-atoms (including three aromatic CH and five O-bearing C-atoms), and one C¼O group. In the HMBC spectrum, correlations of the aromatic H-atoms H¢C(5) (d(H) 6.73) with C(4a) (d(C) 131.8), C(10b) (140.0), C(6) (127.4), and C(6a) (111.5), and of H¢C(7) (d(H) 7.28) with C(6a), C(10a) (d(C) 99.2), C(8) (166.7), and C(9) (99.4) implied the presence of a naphthalene skeleton. Since the naphthalene moiety accounted for seven degrees of unsaturation, and the additional C¼C bond and the C¼O group for further two degrees, 6 was assumed to possess a tricyclic framework to meet the requirement of ten degrees of unsaturation. The relatively low chemical shift of the C¼O group (d(C) 164.7) indicated the presence of a lactone in the third ring, which was fused to the naphthalene moiety. These data accounted for all 1H- and 13 C-NMR resonances except for one exchangeable H-atom, and the above indicated a benzocoumarin skeleton [23]. The HMBCs (Fig.) of MeO¢C(4) (d(H) 3.98) with C(4) (d(C) 147.0), of MeO¢C(8) (d(H) 3.92) with C(8), and of Me¢C(6) (d(H) 2.79) with C(5) (d(C) 112.4), C(6), and C(6a), indicated that two MeO groups were located at C(4) and C(8), respectively, while a Me group was at C(6). The HMBCs of HO¢C(10) (d(H) 11.86) with C(10) (d(C) 165.5) and C(10a), suggested that the OH group was linked to C(10). According to the molecular formula, a second OH group had to be placed at C(3) (d(C) 138.2). The structure of 6 was unambiguously determined as depicted.

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Table 3. 1H- and 13C-NMR Data of 6 – 8 (600 and 150 MHz, resp.; in CDCl3 ) a ). d in ppm, J in Hz. Position

6

7

d( H ) 1 2 3 4 4a 5 6 6a 7 8 9 10 10a 10b 4-MeO 6-Me 8-MeO 10-OH a

d(C )

d( H )

164.7 138.2 147.0 6.73 (s)

7.28 (s) 6.57 (s)

3.98 (s) 2.79 (s) 3.92 (s) 11.86 (s)

131.8 112.4 127.4 111.5 105.3 166.7 99.4 165.5 99.2 140.0 56.4 25.6 55.9

8 d(C ) 165.8 139.7 148.7

6.93 (s)

7.37 (d, J ¼ 1.7) 6.46 (d, J ¼ 1.7)

3.94 (s) 2.76 (s)

133.7 113.6 127.4 112.1 105.8 166.2 102.2 165.9 99.1 141.4 56.6 25.4

d( H )

2.65 (dd, J ¼ 17.4, 2.4, Ha ), 2.45 (dd, J ¼ 17.4, 7.8, Hb )

d(C ) 197.1 131.5 151.2 41.5

4.21 – 4.26 (m) 2.74 (dd, J ¼ 16.2, 3.6, Ha ), 2.47 (dd, J ¼ 16.2, 9.0, Hb )

66.2 46.6

1.78 (s) 1.97 (s)

10.8 21.6

11.96 (s)

) Assignments were based on 1H,1H-COSY, HSQC, HMBC, and NOESY experiments.

Compound 7 was also obtained as white solid. Its molecular formula was determined as C15H12O6 by HR-ESI-MS (m/z 289.0708 ([M þ H] þ ; calc. 289.0707)). The 1H-NMR data of 7 (Table 3) were closely related to those of 6, except for the absence of one MeO group. A comprehensive analysis of the HMBC spectrum (Fig.) indicated that a OH instead of a MeO group was attached to C(8) in 7. The structure of 7 was elucidated as 3,8,10-trihydroxy-4-methoxy-6-methylbenzocoumarin. Compound 8 was also obtained as white amorphous solid. The 1H-NMR spectrum of 8 showed two Me signals d(H) 1.78 (s) and 1.97 (s)), signals of two CH2 groups (2.45 (dd, J ¼ 17.4, 7.8), 2.47 (dd, J ¼ 16.2, 9.0), 2.65 (dd, J ¼ 17.4, 2.4), and 2.74 (dd, J ¼ 16.2, 3.6)), and of a CH¢O group (4.21 – 4.26 (m)). The 13C-NMR spectrum revealed that 8 contained eight C-atoms, including those of an a,b-unsaturated ketone moiety (d(C) 131.5, 151.2, and 197.1), and of two Me (10.8 and 21.6), two CH2 (41.5 and 46.6), and a CH¢O group (66.2). These data consisted of all 1H- and 13C-NMR resonances. Therefore, its molecular formula of C8H12O2 (three degrees of unsaturation) was assigned by the above mentioned data and ESI-MS (m/z 141.3 ([M þ H] þ )). Three degrees of unsaturation indicated that 8 should consist of a six-membered ring containing an a,b-unsaturated ketone group and two Me groups located at the C¼C bond. The connection between C(3) and C(6) was confirmed by the 1H,1H-COSY correlations H¢C(5)/CH2(4) and H¢C(5)/CH2(6), and the key HMBCs Me(8)/C(4), CH2(4)/C(2), and CH2(4)/C(3). The C(1)¢C(6) linkage was verified by the HMBCs CH2(6)/C(1) and CH2(6)/C(2). The absolute configuration of 8 was assigned by the

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same strategy as described above. An obvious negative Cotton effect at 316 nm (Demax ¢ 0.59) for n ! p* transition of the a,b-unsaturated ketone allowed us to propose the configuration at C(5) as (R). Three known compounds were isolated and identified as 2,5-dimethoxy-3,6-bis(4methoxypheny1)-1,4-benzoquinone (9) [24], dankasterone A (10) [25], and (17R)-4hydroxy-17-methylincisterol (11) [26] by comparison of their NMR and MS data with those reported in literature. This work was supported by the National Natural Science Foundation of China (Nos. 30730109 and 30925038).

Experimental Part General. TLC: Precoated silica-gel GF254 plates (SiO2 ; Qingdao Marine Chemical Industry); visualization by UV light at 254 nm or by spraying with 10% H2SO4/EtOH, followed by heating. Column chromatography (CC): SiO2 (200 – 300 mesh; Qingdao Marine Chemical Industry) and Sephadex LH-20 gel (Pharmacia Biotech). Semi-prep. HPLC: Agilent 1100-G1310A isopump equipped with a G1322A degasser, a G1314A VWD detector (210 nm), and a ZORBAX SB-C18 column (9.4   250 mm, 5 mm). Optical rotations: PerkinElmer 241MC polarimeter. UV Spectra: Shimadzu UV-2450 spectrophotometer; lmax (log e) in nm. CD Spectra: Chirascan spectropolarimeter; lmax (De) in nm. IR Spectra: Nicolet iN 10 Micro FT-IR spectrometer; ˜n in cm ¢ 1. 1H- and 13C-NMR spectra: Bruker Avance-DRX-600 spectrometer (600 and 150 MHz, resp.); d in ppm rel. to Me4Si as internal standard, J in Hz. 2D-NMR Spectra: recorded with standard pulse programs and acquisition parameters. ESI-MS: API-4000 triplestage quadrupole instrument; in m/z. HR-ESI-MS: Finnigan LCQdeca mass spectrometer; in m/z. Fungal Strain. The fungus was isolated from an unknown species of lichen collected from Qingliang Mountain, Zhejiang Province, P. R. China. The fresh lichen thallus was washed under tap water, surfacesterilized in 75% EtOH for 30 s, 2.5% NaClO for 2 min, and 75% EtOH for 30 s, aseptically, followed by washing with sterile water. The sterilized thallus was cut into 2   2 mm pieces. Pieces were plated aseptically on potato dextrose agar (PDA) and malt extract agar (MEA) in Petri dishes, and incubated at 258. The emergent fungus was subcultured and purified on PDA. The strain was assigned the Accession No. 4791a, and a sample was deposited with the Key Laboratory of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan. The fungus was identified by J. L. based upon the nuclear ITS rDNA sequences compared to GenBank sequence database in NCBI. The ITS sequence of strain 4791a showed 99% homology to Pleosporales sp. (GenBank: KC871044.1). Further taxonomic identification at a narrower level based on sequence comparison seemed unreliable. Fermentation. The fungal strain was cultured on slants of PDA at 258 for 15 d. Then, the fungus was cultured in five Erlenmeyer flasks (500 ml) each containing 200 ml of PDB and was incubated at 228 on a rotary shaker (110 rpm) for 7 d to obtain the seed culture. Finally, seed broth (70 ml) was added to 70 Erlenmeyer flasks (500 ml), each containing 200 ml of PDB, and these flasks were maintained at 258 for 30 d in a shaker incubator (110 rpm). Additionally, seed broth (200 ml) was added to 50 Erlenmeyer flasks (500 ml), each containing solid rice medium made of rice (80 g) and H2O (120 ml). These flasks were maintained at 258 for 30 d in a constant temp. incubator. Extraction and Isolation. The fermented material (PDB) was filtered to remove mycelia. The filtrate was concentrated under reduced pressure to 1 l and then extracted with AcOEt (5   1 l). The obtained crude extract (3 g) was separated by CC (SiO2 ; petroleum ether (PE; 60 – 908)/acetone 100 : 0 ! 0 : 100) to give nine fractions, Frs. 1 – 9. Fr. 4 (238 mg) was separated by CC (SiO2 ; CH2Cl2/MeOH 5 : 2) to give three subfractions, Frs. 4.1 – 4.3. Fr. 4.1 (30 mg) was subjected to CC (Sephadex LH-20; CH2Cl2/MeOH 1 : 1) and then separated by HPLC (MeOH/H2O 85 : 15, 1.8 ml min ¢ 1) to provide 10 (tR 37.7 min; 5.1 mg) and 11 (tR 39.1 min; 2.1 mg). Fr. 5 (148 mg) was also subjected to CC (Sephadex LH-20; CH2Cl2/MeOH 1 : 1) to furnish two subfractions, Frs. 5.1 and 5.2. Fr. 5.1 (35 mg) was filtered to obtain 1 (8.4 mg) as white amorphous solid. Fr. 5.2 (16 mg) was purified by HPLC (MeOH/H2O 30 : 70, 1.8 ml min ¢ 1) to afford 8 (tR

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18.7 min; 2.7 mg). Fr. 6 (300 mg) was filtered to get 2 (15.2 mg) as white amorphous solid. Fr. 8 (86 mg) was separated by HPLC (MeOH/H2O 70 : 30, 1.8 ml min ¢ 1) to afford 3 (tR 13.6 min; 1.1 mg). The rice culture medium containing the mycelium was cut into small pieces and extracted with AcOEt. The AcOEt extract was dried under reduced pressure using a rotary evaporator at 388. The crude extract (8 g) was separated by CC (SiO2 ; PE (60 – 908)/acetone 100 : 0 ! 0 : 100) to give five fractions, Frs. 1 – 5. An orange crystalline solid obtained from Fr. 1 (15 mg) was purified by CC (Sephadex LH-20; MeOH) to yield 9 (1.4 mg). Fr. 3 (45 mg) was subjected to CC (Sephadex LH-20; CHCl3/MeOH 1 : 1) to provide three subfractions, Frs. 3.1 – 3.3. Fr. 3.3 (21 mg) was purified by HPLC (MeOH/H2O 60 : 40, 1.8 ml min ¢ 1) to obtain 4 (tR 27.0 min; 13.5 mg, ). Separation of Fr. 4 (70 mg) by CC (Sephadex LH-20; MeOH) gave three subfractions, Frs. 4.1 – 4.3. Compound 5 (16.3 mg) crystallized from Fr. 4.2 (28 mg). Fr. 4.3 (36 mg) was subjected to HPLC (MeOH/H2O 72 : 38, 1.8 ml min ¢ 1) to give 7 (tR 21.0 min; 6.1 mg). Fr. 5 (76 mg) was separated by repeated CC (Sephadex LH-20; CHCl3/MeOH 1 : 1) to afford two subfractions, Frs. 5.1 and 5.2. From Fr. 5.2 (57 mg), a white solid was filtered to give 6 (33.7 mg). Cucurbitarin A ( ¼ (4S,5R)-4-Hydroxy-3-methoxy-2,5-diphenylcyclohex-2-en-1-one; 1). White amorphous solid. [a] 22 D ¼ ¢ 94 (c ¼ 0.08, CH2Cl2 ). UV (MeCN): 264 (4.31). CD (MeCN): 341 ( ¢ 1.44). IR: 3463, 3028, 2950, 1598, 1589, 1493, 1466, 1380, 1360, 1246, 1086. 1H- and 13C-NMR: see Table 1. HR-ESIMS (pos.): 295.1336 ([M þ H] þ , C19H19O þ3 ; calc. 295.1329). Cucurbitarin B ( ¼ (4S,5R)-3,4-Dihydroxy-2,5-diphenylcyclohex-2-en-1-one; 2). White amorphous solid. [a] 22 D ¼ ¢ 43 (c ¼ 0.15, MeOH). UV (MeOH): 269 (4.16). CD (MeOH): 268 ( ¢ 0.26), 322 ( ¢ 0.27). IR: 3058, 2679, 1614, 1593, 1495, 1373, 1304, 1106. 1H- and 13C-NMR: see Table 2. HR-ESI-MS (pos.): 281.1184 ([M þ H] þ , C18H17O þ3 ; calc. 281.1172). Cucurbitarin C ( ¼ 3’,5’-Dimethoxy-1,1’:4’,1’’-terphenyl-2’-yl b-d-Glucopyranoside; 3). White amorphous solid. [a] 22 D ¼ ¢ 19 (c ¼ 0.1, MeOH). UV (MeOH): 224 (4.47), 258 (4.28). CD (MeOH): 269 ( ¢ 3.03). IR: 3378, 2932, 1488, 1392, 1069. 1H- and 13C-NMR: see Table 1. HR-ESI-MS (pos.): 486.2121 ([M þ NH4 ] þ , C26H32NO þ8 ; calc. 486.2122), 491.1671 ([M þ Na] þ , C26H28NaO þ8 ; calc. 491.1676). Cucurbitarin D ( ¼ (2R)-2-Hydroxy-2,4-diphenylcyclopent-4-ene-1,3-dione; 4). Yellow solid. [a] 22 D ¼ þ 70 (c ¼ 0.1, CH2Cl2 ). UV (MeCN): 227 (4.38), 303 (4.31). CD (CH2Cl2 ): 211 ( þ 1.71), 233 ( ¢ 1.26), 251 ( þ 1.28), 316 ( ¢ 2.20), 367 ( þ 0.91). IR: 3403, 3065, 1747, 1700, 1563, 1306, 1067. 1H- and 13C-NMR: see Table 1. HR-ESI-MS (pos.): 265.0862 ([M þ H] þ , C17H13O þ3 ; calc. 265.0859), 282.1127 ([M þ NH4 ] þ , C17H16NO þ3 ; calc. 282.1125), 287.0680 ([M þ Na] þ , C17H12NaO þ3 ; calc. 287.0679). Cucurbitarin E ( ¼ (2S,3R,5R)-2,3,5-Trihydroxy-2,5-diphenylcyclopentanone; 5). Colorless crystalline powder (MeOH). [a] 22 D ¼ þ 340 (c ¼ 0.1, MeOH). UV (MeOH): 217 (4.20). CD (MeOH): 236 ( þ 2.43), 337 ( ¢ 5.76). IR: 3402, 3198, 1755, 1494, 1341, 1091. 1H- and 13C-NMR: see Table 1. HR-ESIMS (pos.): 302.1401 ([M þ NH4 ] þ , C17H20NO þ4 ; calc. 302.1387), 307.0953 ([M þ Na] þ , C17H16NaO þ4 ; calc. 307.0941). 3,10-Dihydroxy-4,8-dimethoxy-6-methylbenzocoumarin ( ¼ 3,10-Dihydroxy-4,8-dimethoxy-6-methyl-2H-naphtho[1,2-b]pyran-2-one; 6). White solid. UV (MeCN): 235 (4.17), 259 (4.30). IR: 3453, 2991, 2968, 1614, 1353. 1H- and 13C-NMR: see Table 3. HR-ESI-MS (pos.): 303.0864 ([M þ H] þ , C16H15O þ6 ; calc. 303.0863), 320.1129 ([M þ NH4 ] þ , C16H18NO þ6 ; calc. 320.1129). 3,8,10-Trihydroxy-4-methoxy-6-methylbenzocoumarin ( ¼ 3,8,10-Trihydroxy-4-methoxy-6-methyl2H-naphtho[1,2-b]pyran-2-one; 7). White solid. UV (MeCN): 234 (5.17), 258 (4.30). IR: 3554, 3464, 3408, 1605, 1467, 1336. 1H- and 13C-NMR: see Table 3. HR-ESI-MS (pos.): 289.0708 ([M þ H] þ , C15H13O þ6 ; calc. 289.0707), 306.0973 ([M þ NH4 ] þ , C15H16NO þ6 ; calc. 306.0972), 311.0527 ([M þ Na] þ , C15H12NaO þ6 ; calc. 311.0526). (5R)-5-Hydroxy-2,3-dimethylcyclohex-2-en-1-one (8). White amorphous solid. [a] 22 D ¼ ¢ 8 (c ¼ 0.3, CH2Cl2 ). UV (MeOH): 233 (3.70). CD (MeOH): 260 ( ¢ 0.34), 316 ( ¢ 0.59). IR: 3400, 2925, 1718, 1654, 1381, 1058. 1H- and 13C-NMR: see Table 3. ESI-MS (pos.): 141.3 ([M þ H] þ , C8H13O þ2 ; calc. 141.1).

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New metabolites from endolichenic fungus Pleosporales sp.

Eight new metabolites were obtained from the culture of an endolichenic fungus, Pleosporales sp. Their structures were determined as three terphenyl d...
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