Isoprenylated Chromone DeriVatiVes from Pestalotiopsis fici
Journal of Natural Products, 2009, Vol. 72, No. 8 1485
Hz, H-7), 2.87 (1H, dd, J ) 15, 11 Hz, H-5a), 2.80 (1H, dd, J ) 16,
6.5 Hz, H-9a), 2.55 (1H, d, J ) 17 Hz, H-3a), 2.50 (1H, d, J ) 17 Hz,
H-3b), 2.43 (1H, dd, J ) 15, 11 Hz, H-5b), 2.42 (1H, dd, J ) 16, 6.5
Hz, H-9b), 1.63 (3H, s, H3-13), 1.58 (3H, s, H3-12), 1.31 (3H, s, H3-
15), 1.28 (3H, s, H3-14).
a different fusion pattern between the cyclohexane and the furan
rings. Compounds 5 and 6 are new analogues of the known
compound 11,19 whereas the heterodimer 7 could be derived from
6 and isosulochrin (9).13,17 Compounds 4-6 could be derived from
1-3 via reactions including oxidation, reduction, and cyclization.
The discovery of these new bioactive metabolites further expanded
the structural diversity of the bioactive products produced by the
fungus P. fici.
Another sample of 1 (1.0 mg, 0.004 mmol), (R)-MTPA Cl (2.0 µL,
0.011 mmol), and pyridine-d5 (0.5 mL) was processed as described
1
above for 1a to afford 1b: H NMR (pyridine-d5, 400 MHz) δ 5.79
(1H, ddd, J ) 11, 7.2, 2.0 Hz, H-6), 5.26 (1H, t, J ) 6.5 Hz, H-10),
3.53 (1H, d, J ) 2.0 Hz, H-7), 2.90 (1H, dd, J ) 16, 6.5 Hz, H-9a),
2.80 (1H, dd, J ) 15, 11 Hz, H-5a), 2.53 (1H, d, J ) 17 Hz, H-3a),
2.49 (1H, d, J ) 17 Hz, H-3b), 2.44 (1H, dd, J ) 16, 6.5 Hz, H-9b),
2.30 (1H, dd, J ) 15, 11 Hz, H-5b), 1.64 (3H, s, H3-13), 1.60 (3H, s,
H3-12), 1.30 (3H, s, H3-15), 1.27 (3H, s, H3-14).
Experimental Section
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a
Shimadzu Biospec-1601 spectrophotometer. IR data were recorded
Pestaloficiols G and H (2 and 3): pale yellow oil; [R]25 +136 (c
1
using a Nicolet Magna-IR 750 spectrophotometer. H and 13C NMR
D
0.1, CH3OH); UV (CH3OH) λmax (log ε) 340 (4.09) nm; IR (neat) νmax
3383, 2975, 2929, 1627, 1583, 1418, 1370, 1247, 1152 cm-1; 1H NMR
(acetone-d6, 400 MHz) 2: δ 7.22 (1H, d, J ) 12 Hz, H-9), 6.41 (1H,
d, J ) 12 Hz, H-10), 4.77 (1H, br d, J ) 2.0 Hz, H-7), 3.93 (1H, br
s, OH-7), 3.71 (1H, br s, OH-6), 3.62 (1H, ddd, J ) 11, 5.4, 2.0 Hz,
H-6), 2.69 (1H, dd, J ) 16, 5.4 Hz, H-5a), 2.51 (1H, d, J ) 16 Hz, H-3a),
2.42 (1H, d, J ) 16 Hz, H-3b), 2.26 (1H, dd, J ) 16, 11 Hz, H-5b), 1.91
(3H, s, H3-13), 1.88 (3H, s, H3-12), 1.43 (3H, s, H3-15), 1.39 (3H, s, H3-
14); 3: δ 7.08 (1H, d, J ) 12 Hz, H-10), 6.74 (1H, d, J ) 12 Hz, H-9),
4.26 (1H, br d, J ) 1.6 Hz, H-7), 3.93 (1H, br s, OH-7), 3.81 (1H, br
s, OH-6), 3.80 (1H, ddd, J ) 11, 5.4, 1.6 Hz, H-6), 2.69 (1H, dd, J )
16, 5.4 Hz, H-5a), 2.50 (1H, d, J ) 16 Hz, H-3a), 2.45 (1H, d, J ) 16
Hz, H-3b), 2.26 (1H, dd, J ) 16, 11 Hz, H-5b), 1.87 (3H, s, H3-13),
1.84 (3H, s, H3-12), 1.46 (3H, s, H3-15), 1.44 (3H, s, H3-14); 13C NMR
(acetone-d6, 100 MHz) 2: δ 191.4 (C, C-4), 160.6 (C, C-8a), 144.6 (C,
C-11), 130.8 (C, C-8), 130.0 (CH, C-9), 121.8 (CH, C-10), 109.4 (C,
C-4a), 79.8 (C, C-2), 69.6 (CH, C-6), 67.8 (CH, C-7), 47.9 (CH2, C-3),
27.2 (CH3, C-15), 26.7 (CH3, C-13), 25.4 (CH3, C-14), 24.8 (CH2, C-5),
18.7 (CH3, C-12); 3: δ 191.8 (C, C-4), 162.1 (C, C-8a), 143.2 (C, C-11),
133.3 (CH, C-9), 129.1 (C, C-8), 124.5 (CH, C-10), 110.3 (C, C-4a),
80.3 (C, C-2), 76.4 (CH, C-7), 69.6 (CH, C-6), 47.8 (CH2, C-3), 27.0
(CH3, C-13), 26.5 (CH3, C-15), 26.1 (CH2, C-5), 25.4 (CH3, C-14),
17.9 (CH3, C-12); HMBC data (acetone-d6, 400 MHz) H2-3 f C-2, 4,
4a, 14, 15; H2-5 f C-4, 4a, 6, 7, 8a; H-7 f C-5, 6, 8, 8a, 9; H-9 f
C-7, 8, 8a, 10, 11; H-10 f C-8, 9, 12, 13; H3-12 f C-10, 11, 13;
H3-13 f C-10, 11, 12; H3-14 f C-2, 3, 15; H3-15 f C-2, 3, 14;
NOESY correlations (acetone-d6, 500 MHz) 2: H-7 f H-10; H-10 f
H-7; 3: H-7 f H-9; H-9 f H-7; HRESIMS m/z 301.1402 (calcd for
C16H22O4Na, 301.1410).
data were acquired with Varian Mercury-400 and -500 spectrometers
using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1; pyridine-d5:
δH 7.21, 7.58, 8.73) as references. The HMQC and HMBC experiments
were optimized for 145.0 and 8.0 Hz, respectively. ESIMS data were
recorded on a Bruker Esquire 3000plus spectrometer, and HRESIMS
data were obtained using Bruker APEX III 7.0 T and APEXII FT-ICR
spectrometers, respectively.
Fungal Material. The culture of P. fici was isolated from branches
of Camellia sinensis in the suburb of Hangzhou, in April 2005. The
isolate was identified as P. fici by one of authors (L.G.) based on
sequence (GenBank Accession number DQ812914) analysis of the
ITS region of the rDNA and assigned the accession number AS 3.9138
() W106-1) in China General Microbial Culture Collection (CGMCC)
at the Institute of Microbiology, Chinese Academy of Sciences, Beijing.
The fungal strain was cultured on slants of potato dextrose agar (PDA)
at 25 °C for 10 days. Agar plugs were used to inoculate 250 mL
Erlenmeyer flasks, each containing 50 mL of media (0.4% glucose,
1% malt extract, and 0.4% yeast extract), and the final pH of the media
was adjusted to 6.5 before sterilization. Flask cultures were incubated
at 25 °C on a rotary shaker at 170 rpm for 5 days. Fermentation was
carried out in twelve 500 mL Erlenmeyer flasks each containing 80 g
of rice. Spore inoculum was prepared by suspension in sterile, distilled
H2O to give a final spore/cell suspension of 1 × 106/mL. Distilled H2O
(100 mL) was added to each flask, and the contents were soaked
overnight before autoclaving at 15 lb/in.2 for 30 min. After cooling to
room temperature, each flask was inoculated with 5.0 mL of the spore
inoculum and incubated at 25 °C for 40 days.
Extraction and Isolation. The fermented material was extracted
with EtOAc (4 × 1.0 L), and the organic solvent was evaporated to
dryness under vacuum to afford the crude extract (10 0.0 g), which
was fractionated by silica gel vacuum liquid chromatography (VLC)
using petroleum ether-EtOAc gradient elution. The fraction (136 mg)
eluted with 32% EtOAc was separated by Sephadex LH-20 column
chromatography (CC) eluting with 1:1 CHCl3-CH3OH. The resulting
subfractions were combined and further purified by semipreparative
RP HPLC (Agilent Zorbax SB-C18 column; 5 µm; 9.4 × 250 mm; 45%
CH3OH in H2O for 2 min, followed by 45- 60% for 28 min; 2 mL/
min) to afford a mixture of 2 and 3 (4.3 mg). The fraction eluted with
8% EtOAc (50 mg) was purified by HPLC (70% CH3OH in H2O for
2 min, followed by 70-75% for 25 min; 2 mL/min) to afford 1 (4.0
mg). Fractions eluted with 10-33% EtOAc were combined (480 mg)
and fractionated again by Sephadex LH-20 CC using CHCl3-CH3OH
(1:1). Purification of resulting subfractions with different gradients
afforded pestaloficiols I (4; 2.4 mg; 70% CH3OH in H2O for 2 min,
followed by 70-79% for 25 min), J (5; 3.0 mg; 60% CH3OH in H2O
for 2 min, followed by 60-90% for 23 min), K (6; 2.0 mg; 70% CH3OH
in H2O for 2 min, followed by 70-89% for 20 min), and L (7; 1.4 mg;
45% CH3OH in H2O for 2 min, followed by 45-60% for 28 min).
Pestaloficiol F (1): colorless oil; [R]25D +293 (c 0.1, CH3OH); UV
(CH3OH) λmax (log ε) 278 (4.11) nm; IR (neat) νmax 3407 (br), 2977,
2925, 1660, 1609, 1403, 1250, 1167 cm-1; 1H, 13C NMR, HMBC, and
NOESY data see Table 1; HRESIMS m/z 301.1412 (calcd for
C16H22O4Na, 301.1410).
Pestaloficiol I (4): pale yellow oil; [R]25 -79 (c 0.1, CH3 OH);
D
UV (CH3OH) λmax (log ε) 304 (4.04) nm; IR (neat) νmax 3378 (br),
1
2975, 2932, 1643, 1574, 1424, 1165 cm-1; H NMR (acetone-d6, 400
MHz) δ 6.36 (1H, d, J ) 1.5 Hz, H-9), 4.86 (1H, dd, J ) 4.0, 3.3 Hz,
H-7), 4.76 (1H, dd, J ) 4.0, 1.5 Hz, H-10), 4.35 (1H, br s, OH-6),
4.19 (1H, ddd, J ) 3.3, 3.0, 2.5 Hz, H-6), 4.13 (1H, s, OH-11), 2.71
(1H, dd, J ) 18, 2.5 Hz, H-5a), 2.57 (1H, d, J ) 17 Hz, H-3a), 2.44
(1H, d, J ) 17 Hz, H-3b), 2.32 (1H, dd, J ) 18, 3.0 Hz, H-5b), 1.44
(3H, s, H3-15), 1.38 (3H, s, H3-14), 1.23 (6H, s, H3-12/H3-13); 13C
NMR (acetone-d6, 100 MHz) δ 192.3 (C, C-4), 157.0 (C, C-8a), 135.0
(C, C-8), 128.5 (CH, C-9), 109.2 (C, C-4a), 94.6 (CH, C-10), 80.9 (C,
C-2), 86.6 (CH, C-7), 72.1 (C, C-11), 66.8 (CH, C-6), 48.1 (CH2, C-3),
29.6 (CH2, C-5), 27.4 (CH3, C-13), 27.0 (CH3, C-15), 26.4 (CH3, C-12),
25.5 (CH3, C-14); HMBC data (acetone-d6, 400 MHz) H2-3 f C-2, 4,
4a, 14, 15; H2-5 f C-4, 4a, 6, 7, 8a; H-6 f C-4a, 7, 8; H-7 f C-10;
H-9 f C-7, 8a, 10; H-10 f C-8, 9, 12, 13; H3-12 f C-10, 11, 13;
H3-13 f C-10, 11, 12; H3-14 f C-2, 3, 15; H3-15 f C-2, 3, 14; OH-
11 f C-12; NOESY correlations (acetone-d6, 500 MHz) H-5a f H-7;
H-7 f H-5a; H-6 f H3-12; H3-12 f H-6; HRESIMS m/z 317.1362
(calcd for C16H22O5Na, 317.1359).
Preparation of (R)-MTPA Ester (4a) and (S)-MTPA Ester (4b).
A sample of 4 (1.0 mg, 0.003 mmol), (S)-MTPA Cl (2.0 µL, 0.011
mmol), and pyridine-d5 (0.5 mL) were allowed to react in an NMR
1
tube at ambient temperature for 24 h, with the H NMR data of the
Preparation of (R)-MTPA Ester (1a) and (S)-MTPA Ester (1b).
A sample of 1 (1.0 mg, 0.004 mmol), (S)-MTPA Cl (2.0 µL, 0.011
mmol), and pyridine-d5 (0.5 mL) were allowed to react in an NMR
(R)-MTPA ester derivative (4a) obtained directly on the reaction
mixture: 1H NMR (pyridine-d5, 400 MHz) δ 6.92 (1H, d, J ) 1.5 Hz,
H-9), 5.92 (1H, ddd, J ) 3.3, 3.0, 2.5 Hz, H-6), 5.09 (1H, dd, J ) 4.0,
3.3 Hz, H-7), 4.96 (1H, dd, J ) 4.0, 1.5 Hz, H-10), 2.78 (1H, dd, J )
18, 2.5 Hz, H-5a), 2.77 (1H, d, J ) 17 Hz, H-3a), 2.74 (1H, d, J ) 17
Hz, H-3b), 2.63 (1H, dd, J ) 18, 3.0 Hz, H-5b), 1.44 (3H, s, H3-15),
1.35 (3H, s, H3-14), 1.32 (3H, s, H3-13), 1.09 (3H, s, H3-12).
1
tube at ambient temperature for 24 h. The H NMR data of the (R)-
MTPA ester derivative (1a) were obtained directly on the reaction
1
mixture: H NMR (pyridine-d5, 400 MHz) δ 5.77 (1H, ddd, J ) 11,
7.2, 2.0 Hz, H-6), 5.22 (1H, t, J ) 6.5 Hz, H-10), 3.52 (1H, d, J ) 2.0