Antifungal metabolites from the plant endophytic fungus Pestalotiopsis foedan

Article abstract of DOI:10.1021/np070590f

Pestafolide A (1), a new reduced spiro azaphilone derivative, and pestaphthalides A (2) and B (3), two new isobenzofuranones, have been isolated form solid cultures of an isolate of Pestalotiopsis foedan. The structures of these compounds were determined mainly by analysis of their NMR spectroscopic data. The relative configuration of 1-3 was assigned by analysis of ¹H NMR 7-values and NOESY data, and the absolute configuration was determined by application of the CD excitation chirality and modified Mosher method. Compounds 1-3 showed modest antifungal activity.

Full text of DOI:10.1021/np070590f

J. Nat. Prod. 2008, 71, 615–618
615
Antifungal Metabolites from the Plant Endophytic Fungus Pestalotiopsis foedan
Gang Ding,†,‡ Shuchun Liu,† Liangdong Guo,† Yuguang Zhou,† and Yongsheng Che*,†
Key Laboratory of Systematic Mycology and Lichenology, and Center for Bio-Energy and Industrial Biotechnology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing, 100080, People’s Republic of China, and Graduate School of Chinese Academy of Sciences,
Beijing, 100039, People’s Republic of China
ReceiVed October 23, 2007
Pestafolide A (1), a new reduced spiro azaphilone derivative, and pestaphthalides A (2) and B (3), two new
isobenzofuranones, have been isolated form solid cultures of an isolate of Pestalotiopsis foedan. The structures of these
compounds were determined mainly by analysis of their NMR spectroscopic data. The relative configuration of 1-3
1
was assigned by analysis of H NMR J-values and NOESY data, and the absolute configuration was determined by
application of the CD excitation chirality and modified Mosher method. Compounds 1-3 showed modest antifungal
activity.
Endophytic fungi that inhabit normal tissues of the host plants
without causing apparent pathogenic symptoms have been dem-
onstrated to be rich sources of bioactive natural products.1–3 As
one class of the most widely distributed endophytic fungi, Pesta-
lotiopsis spp. are prolific producers of different types of bioactive
metabolites.4–12 During our ongoing chemical investigations of
endophytic fungi as sources of new bioactive natural products, a
subculture of an isolate of Pestalotiopsis foedan was grown in solid-
substrate fermentation culture. Its organic solvent extract displayed
antifungal activity against Candida albicans (ATCC 10231),
Geotrichum candidum (AS2.498), and Aspergillus fumigatus (ATCC
10894). Bioassay-guided fractionation of this extract led to the
isolation of one new reduced azaphilone derivative and two new
isobenzofuranones that have been named pestafolide A (1) and
pestaphthalides A (2) and B (3). Details of the isolation, structure
elucidation, and stereochemical assignments of these compounds
are presented here.
Table 1. NMR Spectroscopic Data for Pestafolide A (1) in
CDCl3
HMBC
(H f C#)
a
position
δH (J in Hz)
δCb, mult.
NOESYa
8b
1a
1b
4.46, br, d (16)
4.05, br, dd (16, 2.1)
57.2, CH2 2, 7, 9
3
13
2
3
4
127.4, qC
199.3, qC
77.3, qC
5
6a
6b
4.00, dd (10, 5.8)
2.54, br, dd (18, 5.8)
2.38, br, d (18, 10)
72.6, CH
36.1, CH2 2, 5, 7, 8
3, 6, 7, 15
8a
8b, 15
7
150.7, qC
8a
8b
2.32, br, d (18)
2.20, br, d (18)
41.3, CH2 2, 6, 7, 9
6a, 10a, 10b
10b
9
95.2, qC
10a
10b
11a
11b
12a
12b
13
1.69, br, d (11)
1.50, m
1.90, m
1.61, m
1.61, m
1.18, m
3.76, m
1.09, d (6.0)
1.27, s
33.9, CH2 9, 11, 12
8a
8b
8
19.0, CH2 10, 12, 13
9
32.3, CH2 10, 11, 13, 14
67.1, CH
11, 12, 14
1b
6b
14-CH3
15-CH3
21.8, CH3 12, 13
17.9, CH3 3, 4, 5
a Recorded at 400 MHz. b Recorded at 100 MHz.
1H–1H COSY NMR data led to the identification of two isolated
proton spin-systems corresponding to the C-5–C-6 and C-10–C-
14 subunits of structure 1. HMBC correlations of the isolated methyl
protons H3-15 with the oxygenated quaternary carbon C-4, the
oxymethine carbon C-5, and the ketone carbon C-3 indicated that
C-4 was connected to C-3, C-5, and C-15. Correlations from H-5
to C-7 and from H-6 to C-2 and C-7 led to the connection of C-7
to both C-2 and C-6. Further HMBC correlations from H2-1 to C-2
and C-3 revealed the connectivities of C-2 to both C-1 and the
ketone carbon C-3, thereby completing the cyclohexenone subunit
of 1. HMBC correlations from H2-1 to C-9 and from H-8 to C-2,
C-6, C-7, and C-9 permitted completion of the dihydropyran moiety
that was fused to the cyclohexenone unit at C2/C7. Correlations of
H2-10 with C-8 and of H2-11 with C-9 established the connectivity
between C-9 and C-10. Since no HMBC correlation was observed
from H-13 to either C-4, C-5, or C-9, the location of the required
ring remained to be determined. Treatment of pestafolide A (1)
with acetic anhydride resulted in formation of a diacetate,13 and
the 1H NMR spectrum of the product revealed two additional acetate
methyl singlets at δH 2.01 and 2.11, respectively. The signal
corresponding to H-5 (δH 4.00) was shifted downfield in the
spectrum of the diacetate to δH 5.99, indicating that one hydroxy
group was attached to C-5 in 1. However, the remaining hydroxy
Results and Discussion
Pestafolide A (1) was assigned the molecular formula C15H22O5
(five unsaturations) on the basis of HRESI analysis [m/z 305.1363
(M + Na)+; ∆ +0.4 mmu] and NMR data (Table 1). Analysis of
the 1H, 13C, and HMQC NMR data for 1 revealed the presence of
two methyl groups, six methylene units (one of which was
oxygenated), two oxymethines, two oxygenated quaternary carbons,
two olefinic carbons, and one ketone carbon (δC 199.6). These data
accounted for all 1H and 13C resonances except two exchangeable
protons and required compound 1 to be tricyclic. Analysis of the
* To whom correspondence should be addressed. Tel: 86 10 82618785.
Fax: 86 10 82618785. E-mail: cheys@im.ac.cn.
† Institute of Microbiology, Chinese Academy of Sciences.
‡ Graduate School of Chinese Academy of Sciences.
10.1021/np070590f CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/21/2008

616 Journal of Natural Products, 2008, Vol. 71, No. 4
Ding et al.
Table 2. NMR Spectroscopic Data for Pestaphthalides A (2)
and B (3) in CD3OD
pestaphthalide A (2)
pestaphthalide B (3)
a
a
position
δH (J in Hz)
δCb, mult.
δH (J in Hz)
δCb, mult.
1
3
3a
4
5
6
7
7a
173.5, qC
85.4, CH 5.23, d (4.3)
148.3, qC
102.0, CH 6.71, s
164.7, qC
112.7, qC
173.4, qC
85.9, CH
148.2, qC
102.3, CH
164.7, qC
112.8, qC
156.8, qC
104.5, qC
5.32, d (2.4)
6.67, s
156.5, qC
104.9, qC
8
4.19, qd (6.3, 2.4) 68.8, CH 3.98, qd (6.3, 4.3) 69.8, CH
9-Me
10-Me 2.08, s
1.17, d (6.3)
18.5, CH3 1.21, d (6.3)
7.8, CH3 2.18, s
18.0, CH3
7.8, CH3
a Recorded at 400 MHz. b Recorded at 100 MHz.
Figure 1. Key NOESY correlations of pestafolide A (1).
C-5, C-7, and C-8 each bearing a hydroxy group. On the basis of
these data, the gross structure of pestaphthalide A was characterized
as 5,7-dihydroxy-3-(1-hydroxyethyl)-6-methyl-3H-isobenzofuran-
1-one as depicted in 2.
group in 1 could not be located by acetylation experiment, and
C-13 could form an ether linkage with either C-4 or C-9 because
of the lack of HMBC correlations from H-13 to any of these
carbons. Although the 13C chemical shift of C-9 (δC 95.2) matched
well with that reported for a hemiketal carbon,14–17 the different
chemical environment for C-9 in 1 and those hemiketal carbons
reported in the literature would not favor this assignment. In
addition, geometric constraints would preclude linkage of C-13 to
the oxygen at C-4.18 Comparison of the 13C NMR chemical shifts
for C-4 (δC 77.3) and C-5 (δC 72.6) of 1 with those for
corresponding carbons (δC 77.6 and 72.7, respectively) of the model
compound monascusone A19 revealed the connectivity of the two
hydroxy groups to C-4 and C-5; therefore C-9 and C-13 must be
attached to the same oxygen to complete the third six-membered
ring in 1.
The relative configuration of pestafolide A (1) was assigned by
analysis of 1H NMR J-values and NOESY data (Table 1). The large
coupling constant (10 Hz) observed between H-5 and H-6b indicated
that both protons were trans to each other with respect to the
corresponding six-membered ring. NOESY correlations of H-6b
with H3-15 and H-8b and of H-8b with H-1a and H-10b placed
these protons on the same face of the ring system, whereas
correlation from H-1b to H-13 placed these two protons on the
opposite face of the ring sytem. On the basis of these data, the
relative configuration of pestafolide A was established as shown
in 1 (Figure 1).
Pestaphthalide B (3) was assigned the same molecular formula
of C11H12O5 as pestaphthalide A (2) on the basis of HRESIMS
analysis [m/z 247.0557 (M + Na)+] and NMR data (Table 2).
Analysis of the 1H and 13C NMR data for 3 revealed the presence
of nearly identical structural features to those found in 2, except
that the chemical shifts for the two oxymethines C-3 (δH/δC 5.32/
85.4 in 2; 5.23/85.9 in 3) and C-8 (δH/δC 4.19/68.8 in 2; 3.98/69.8
in 3) were different, as well as the 1H–1H coupling constant
observed between H-3 and H-8 (2.4 Hz in 2 and 4.3 Hz in 3,
respectively). These data implied that 3 was a stereoisomer of 2,
and this conclusion was supported by analysis of its COSY and
HMBC data.
The relative configuration of compounds 2 and 3 was assigned
by NOED data. Upon irradiation of H-3, enhancement was observed
for H-8 in the NOE difference spectrum of 2, suggesting a cis
relationship between H-3 and H-8, whereas enhancement was
observed for H3-9 in the spectrum of 3, indicating a trans
relationship between H-3 and H-8.
The absolute configuration at C-3 for pestaphthalides A (2) and
B (3) was again determined by application of the CD excitation
chirality method. The CD spectrum of 3 showed the same chirality
as that reported for spirolaxine,21 and the CD curve of 2 displayed
the opposite chirality of that for spirolaxine and 3, suggesting the
3S absolute configuration for 2 and the 3R absolute configuration
for 3. On the basis of the relative configuration established for 2
and 3 by NOED experiments, the absolute configuration at C-8 for
these was assigned as S for these compounds. Considering the
relative configuration established by NOED data, the absolute
configuration at C-8 for 2 and 3 was assigned as S. As confirmation,
the absolute configuration at C-8 was also assigned by application
of the modified Mosher method.22 Treatment of 2 with (S)-MTPACl
and (R)-MTPACl afforded the R-MTPA ester (2a) and S-MTPA
ester (2b), respectively. The difference in chemical shift values (∆δ
) δS - δR) for the diastereomeric esters 2b and 2a was calculated
in order to assign the absolute configuration at C-8 (Figure 2).
Calculations for all of the relevant signals suggested the S absolute
configuration at C-8 in 2. In a similar fashion, the absolute
configuration at C-8 was also determined to be S in 3 (Figure 2).
The above assignments were further supported by comparison of
the 1H NMR data and specific rotation values of 2 and 3 with those
reported for their closest related synthetic analogues 5 and 6.23
Pestafolide A (1) and pestaphthalides A (2) and B (3) were
evaluated for antifungal activity against Candida albicans (ATCC
10231), Geotrichum candidum (AS2.498), and Aspergillus fumi-
gatus (ATCC 10894) in agar diffusion assays. Pestafolide A (1)
displayed antifungal activity against Aspergillus fumigatus (ATCC
10894), affording a zone of inhibition of 10 mm at 100 µg/disk.
The absolute configuration of pestafolide A (1) was determined
by application of the CD excitation chirality method. The CD
spectrum of 1 showed a positive Cotton effect at 217 (∆ꢀ +43)
nm and a negative Cotton effect at 248 (∆ꢀ -24) nm, which was
nearly identical to that of (R)-2-acetyl-3,6-dihdroxycyclohex-2-
enone-4,20 suggesting the R absolute configuration at C-4, which
was exactly the same as that of monascusone A.19 Therefore, the
4R, 5S, 9S, and 13S absolute configuration was assigned for
pestafolide A (1).
The molecular formula of pestaphthalide A (2) was determined
to be C11H12O5 (six unsaturations) on the basis of HRESIMS
analysis [m/z 247.0558 (M + Na)+; ∆ +0.1 mmu] and was
supported by 1H and 13C NMR data (Table 2). Analysis of the 1H,
13C, and HMQC NMR data for 2 revealed the presence of two
methyl groups, two oxymethines, six aromatic carbons (one
protonated), and one carboxyl carbon. These data accounted for
all but three exchangeable protons and required the presence of
1
two rings for 2. Analysis of H–1H COSY NMR data led to the
identification of one isolated proton spin-system corresponding to
the C-3–C-9 subunit of structure 2. HMBC correlations of H3-10
with three nonprotonated aromatic carbons C-5 (δC 164.7), C-6 (δC
112.7), and C-7 (δC 156.5) indicated that C-6 was connected to
C-5, C-7, and C-10. Correlations from H-4 to C-3, C-5, C-6, and
C-7a and from H-3 to C-1, C-3a, C-4, C-7a, and C-8 permitted
completion of the 5,6,7-trisubstituted isobenzofuranone ring with

Antifungal Metabolites from Pestalotiopsis foedan
Journal of Natural Products, 2008, Vol. 71, No. 4 617
Extraction and Isolation. The fermented rice substrate was freeze-
dried and extracted with MEK (3 × 500 mL), and the organic solvent
was evaporated to dryness under vacuum to afford 5.0 g of crude extract.
The crude extract was fractionated by silica gel VLC using petroleum
ether-EtOAc gradient elution. The fraction (100 mg) that was eluted
with 25% EtOAc was further separated by Sephadex LH-20 column
chromatography (CH2Cl2-n-C6H14, 4:1) to afford a subfraction of 30
mg, and purification of this fraction by semipreparative reversed-phase
HPLC (Kramosil C18 column; 10 µm; 10 × 250 mm, 2 mL/min)
afforded pestafolide A (1; 1.9 mg, tR 26.0 min; 45% MeOH in H2O
over 2 min, 45–53% over 40 min). The fraction (150 mg) that was
eluted with 40% EtOAc was fractionated again by Sephadex LH-
20 column chromatography using the same solvent system as
described above to give a subfraction of 40 mg, and further
purification by semipreparative reversed-phase HPLC afforded
pestaphthalides A (2; 2.0 mg, tR 30.3 min; 30% MeOH in H2O over
5 min, 30–60% over 60 min) and B (3; 2.3 mg, tR 32.8 min; same
gradient as in purification of 2).
Figure 2. ∆δ values (in ppm) ) δS - δR obtained for (R)- and
(S)-MTPA esters 2a, 2b, 3a, and 3b.
Pestaphthalide A (2) showed activity against Candida albicans
(ATCC 10231), causing a zone of inhibition of 13 mm, and
pestaphthalide B (3) showed activity against Geotrichum candidum
(AS2.498) with a 11 mm zone of inhibition when tested at the same
level (fluconazole: 18–28 mm zones of inhibition for C. albicans,
A. fumigatus, and G. candidum at 100 µg/disk).
Pestafolide A (1): colorless oil; [R]D -19 (c 0.05, CH3OH); UV
(CH3OH) λmax 203 (ꢀ 18 600), 255 (ꢀ 15 800) nm; CD (c 1.0 × 10-4
M, CH3OH) λmax (∆ꢀ) 248 (-24), 217 (+43) nm; IR (neat) νmax 3413
(br), 2937, 1681, 1434 cm-1; 1H, 13C NMR, HMBC, and NOESY data,
see Table 1; HRESIMS obsd m/z 305.1363 (M + Na)+, calcd for
C15H22O5 Na, 305.1359.
Pestafolide A (1) possesses the same spiro azaphilone skeleton
as that appearing in decipinin A and the daldinins.18,24–26 However,
pestafolide A differs significantly from these known compounds
by having a tetrahydro-1H-isochromen-8(5H)-one moiety instead
of the common 3,4-dihydro-7H-isochromene-6,8-dione unit and by
the absence of a substituent next to the ketal carbon on the
tetrahydropyran ring. The tetrahydroisochromenone partial structure
in 1 resembles the dihydroisochromenone core structure presented
in monascusone A,19 but 1 differs from monascusone A by the
presence of an additional tetrahydropyran ring spirally joined to
the tetrahydroisochromenone moiety at C-9. Isobenzufuranoes have
been isolated frequently from microbial sources,21,27–32 such as
acetophthalidin (4),32 which is the most closely related natural
product to pestaphthalides A (2) and B (3). However, 2 and 3 differ
from 4 by having a methyl group at C-6 and a hydroxyethyl
substituent at C-3. Acetophthalidin derivatives 5 and 6 were
prepared due to instability of acetophthalidin,23 and these two
synthetic analogues closely resembled 2 and 3, with the only
difference of the methyl group presented at C-6 for 2 and 3.
Pestafolide A (1) and pestaphthalides A (2) and B (3) are the first
secondary metabolites to be reported from P. foedan.
Pestaphthalide A (2): brown oil; [R]D +51 (c 0.05, CH3OH); UV
(CH3OH) λmax 215.5 (ꢀ 13 800), 256 (ꢀ 10 400) nm; CD (c 1.0 × 10-4
M, CH3OH) λmax (∆ꢀ) 226 (+13), 290 (-2.8) nm; IR (neat) νmax 3363
(br), 2927, 1717, 1612 cm-1 1H and 13C NMR data, see Table 2;
;
HRESIMS obsd m/z 247.0558 (M + Na)+, calcd for C11H12O5 Na,
247.0557.
Pestaphthalide A (3): brown oil; [R]D -41 (c 0.05, CH3OH); UV
(CH3OH) λmax 214 (ꢀ 17 500), 255 (ꢀ17 500) nm; CD (c 1.0 × 10-4
M, CH3OH) λmax (∆ꢀ) 222 (-4.5), 260 (+0.3) nm; IR (neat) νmax 3360,
2924, 1717, 1631 cm-1; 1H and 13C NMR data, see Table 2; HRESIMS
obsd m/z 247.0557 (M + Na)+, calcd for C11H14O3 Na, 247.0557.
Acetylation of Pestafolide A (1). A solution of pestafolide A (1.0
mg), 4-N,N-(dimethylamino)pyridine (catalytic amount), and acetic
anhydride (0.5 mL) was stirred at room temperature for 24 h. The
resulting solution was dried to afford the diacetate (0.8 mg): 1H NMR
(CDCl3, 400 MHz) δ 5.99 (1H, dd, J ) 10, 6.5 Hz, H-5), 4.30 (1H, br
d, J ) 16 Hz, H-1a), 4.13 (1H, br d, J ) 16 Hz, H-1b), 3.78 (1H, m,
H-13), 2.50 (1H, m, H-6a), 2.34 (1H, m, H-6b), 2.14 (1H, m, H-8a),
2.11 (3H, s, CH3COO-), 2.08 (1H, m, H-8b), 2.01 (3H, s, CH3COO-),
1.90 (1H, m, H-11a), 1.70 (1H, m, H-10a), 1.60 (1H, m, H-12a, H-11b),
1.50 (1H, m, H-10b), 1.25 (3H, s, 15-Me), 1.17 (1H, m, H-12b), 1.09
(3H, d, J ) 6.5, 14-Me); ESIMS m/z [M + Na]+ 389.2.
Preparation of (R)-MTPA Ester (2a) and (S)-MTPA Ester (2b).
A solution of 2 (1.0 mg, 0.005 mmol) in CH3OH was transferred to a
clean NMR tube, and then the solvent was completely removed under
vacuum. Pyridine-d5 (0.5 mL) and (S)-MTPACl (4.5 µL, 0.025 mmol)
were quickly added into the NMR tube, and all contents were mixed
thoroughly by shaking the NMR tube carefully. The reaction was
performed at room temperature, and the solution was allowed to stand
for 24 h. 1H NMR data of the R-MTPA ester (2a) were obtained without
purification: 1H NMR (pyridine-d5, 400 MHz) δ 6.99 (1H, s, H-4), 5.70
(1H, dq, J ) 7.5, 2.6 Hz, H-8), 5.36 (1H, br s, H-3), 2.32 (3H, s, 10-
Me), 1.35 (3H, d, J ) 7.5 Hz, 9-Me); EIMS m/z 440 (M+; 3%), 189
(88%), 105 (100%), 77 (68%).
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. The CD spectra were
recorded on a JASCO J-815 spectropolarimeter, using CH3OH as
solvent. IR data were recorded using a Nicolet Magna-IR 750
1
spectrophotometer. H and 13C NMR data were acquired with Varian
Mercury-400 and -500 spectrometers using solvent signals (CDCl3; δH
7.26/δC 77.7) 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. HRESIMS and EIMS data
were obtained using a Bruker APEX III 7.0 T spectrometer and APEX
II FT-ICR, respectively.
Fungal Material. The culture of P. foedan was isolated by one of
authors (L.G.) from the branches of an unidentified tree near Dongzai,
Hainan Province, on April 2, 2005. The isolate was identified (by L.G.)
and assigned the accession number L436 in the culture collection 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. The 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 four 500 mL Fernbach flasks each containing 75 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.33 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.
In a similar fashion, compound 2 (1.0 mg, 0.005 mmol), (R)-
MPTACl (4.5 µL, 0.025 mmol), and pyridine-d5 (0.5 mL) were allowed
to react in an NMR tube at ambient temperature for 24 h and processed
1
as described above for 2a to afford 2b: H NMR (pyridine-d5, 400
MHz) δ 7.01 (1H, s, H-4), 5.64 (1H, m, H-8), 5.45 (1H, br s, H-3),
2.36 (3H, s, 10-Me), 1.22 (3H, d, J ) 7.5 Hz, 9-Me); EIMS m/z 440
(M+; 16%), 206 (55%), 179 (100%).
Preparation of (R)-MTPA Ester (3a) and (S)-MTPA Ester (3b).
The preparation of 3a and 3b followed the same procedure as described
above for 2a and 2b. Compound 3a: 1H NMR (pyridine-d5, 400 MHz)
δ 7.03 (1H, s, H-4), 5.92 (1H, m, H-8), 5.57 (1H, br s, H-3), 2.51 (3H,
s, 10-Me), 1.52 (3H, d, J ) 7.5 Hz, 9-Me); EIMS m/z 440 (M+; 13%),
1
189 (100%), 105 (38%). Compound 3b: H NMR (pyridine-d5, 400
MHz) δ 7.05 (1H, s, H-4), 5.85 (1H, m, H-8), 5.68 (1H, br s, H-3),
2.53 (3H, s, 10-Me), 1.42 (3H, d, J ) 7.5 Hz, 9-Me); EIMS m/z 440
(M+; 9%), 189 (100%), 105 (29%).

618 Journal of Natural Products, 2008, Vol. 71, No. 4
Ding et al.
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Antifungal Bioassays. Antifungal bioassays were conducted ac-
cording to a literature procedure.34 The yeasts Candida albicans (ATCC
10231) and Geotrichum candidum (AS2.498) were grown on Sabouraud
dextrose agar, and the fungus Aspergillus fumigatus (ATCC 10894)
was grown on potato dextrose agar. Test compounds were absorbed
onto individual paper disks (6 mm diameter) at 100 µg/disk and placed
on the surface of the agar. The assay plates were incubated at 25 °C
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Acknowledgment. We gratefully acknowledge financial support
from the Key Program of National Hi-Tech Research and Development
(Grant 2007AA021506), the Key Project of Chinese Academy of
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1
Supporting Information Available: H NMR, 13C NMR, and CD
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material is available free of charge via the Internet at http://pubs.acs.org.
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