Pestalotheols A-D, bioactive metabolites from the plant endophytic fungus Pestalotiopsis theae

Article abstract of DOI:10.1021/np700744t

Pestalotheols A-D (1-4), four new metabolites, have been isolated from cultures of an isolate of the plant endophytic fungus Pestalotiopsis theae. The structures of these compounds were determined by NMR spectroscopy, and 1 is further confirmed by X-ray crystallography. The absolute configurations of compounds 1 and 3 were assigned by application of the modified Mosher method. Pestalotheol C (3) displayed an inhibitory effect on HIV-1_LAI replication in C8166 cells with an EC₅₀ value of 16.1 μM.

Full text of DOI:10.1021/np700744t

664
J. Nat. Prod. 2008, 71, 664–668
Pestalotheols A-D, Bioactive Metabolites from the Plant Endophytic Fungus Pestalotiopsis theae
Erwei Li,† Renrong Tian,‡ Shuchun Liu,† Xulin Chen,*,‡ Liangdong Guo,† 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 State Key Laboratory of Virology, Wuhan Institute of
Virology, Chinese Academy of Sciences, Wuhan 430071, People’s Republic of China
ReceiVed December 24, 2007
Pestalotheols A-D (1–4), four new metabolites, have been isolated from cultures of an isolate of the plant endophytic
fungus Pestalotiopsis theae. The structures of these compounds were determined by NMR spectroscopy, and 1 is further
confirmed by X-ray crystallography. The absolute configurations of compounds 1 and 3 were assigned by application
of the modified Mosher method. Pestalotheol C (3) displayed an inhibitory effect on HIV-1LAI replication in C8166
cells with an EC50 value of 16.1 µM.
Plant endophytic fungi are well-known as sources of bioactive
secondary metabolites.1,2 Pestalotiopsis species are very common
in their distribution, occurring on a wide range of substrata, and
many are saprobes, while others are either pathogenic or endophytic
on living plant leaves and twigs.3 Chemical investigations of some
Pestalotiopsis spp. have afforded a variety of bioactive natural
products.4–14 Pestalotiopsis theae is known as a causal fungus for
tea gray blight disease, and chemical studies of this fungus have
led to the identification of phytotoxins and plant growth regu-
lators.15–17 During an ongoing search for new bioactive metabolites
from plant endophytic fungi, a subculture of an isolate of P. theae
(LN560), obtained from branches of an unidentified tree on Jianfeng
Mountain, Hainan Province, People’s Republic of China, was grown
in solid-substrate fermentation culture. Its organic solvent extract
displayed an inhibitory effect on HIV-1LAI replication in C8166
cells. Bioassay-guided fractionation of this extract afforded four
new compounds, which have been named pestalotheols A-D (1–4).
Details of the isolation, structure elucidation, and biological activity
of these compounds are reported herein.
led to the identification of three isolated proton spin-systems
corresponding to the C-2-C-3, C-5-C5-OH, and C-8-C-9
fragments of structure 1. HMBC correlations from H-2 to C-3 and
C-9, from H-3a to C-2 and C-4, and from H-3b to C-4 and C-9
established the tetrahydrofuran subunit. In turn, correlations from
H-5 to C-4, C-6, C-7, and C-9, the exchangeable proton at 4.95
ppm to C-4, C-5, and C-6, H2-8 to C-4, C-6, C-7, and C-9, and
H-9 to C-4 and C-5 led to the identification of a reduced
furanocyclohexene moiety with a hydroxy group attached to C-5.
Other correlations of H3-16 and H3-17 with C-2 and C-3 indicated
that C-2, C-16, and C-17 are all connected to C-15, while those
from H3-13 and H3-14 to C-11 and C-12, H2-11 to C-7 and C-10,
and H2-8 to C-10 completed the C-10-C-14 substructure of 1,with
C-10 directly attached to C-7. Key HMBC correlations from the
second exchangeable proton (δH 3.86) to C-3, C-4, C-5, and C-9
and the third exchangeable proton (δH 3.16) to C-2, C-15, C-16,
and C-17 indicated that C-4 and C-15 are attached to free hydroxy
groups. Considering the chemical shifts of C-6 (δC 167.5) and C-12
(δC 80.6), as well as the unsaturation requirement for 1, C-6 and
C-12 have to be connected to the same oxygen atom to form a
dihydropyranone moiety. On the basis of these data, the gross
structure of pestalotheol A was established as 1.
The relative configuration of pestalotheol A (1) was assigned
by analysis of 1H-1H coupling constants and NOESY data (Table
1). The large trans-diaxial-type coupling constant observed between
H-8b and H-9 (11 Hz) indicated that both protons are in pseudoaxial
orientations with respect to the corresponding cyclohexene ring.
NOESY correlations from the exchangeable proton OH-4 with H-2
and H-8b indicated that these protons are all on the same face of
the ring system, whereas correlations from H-9 to OH-5 were used
to place them on the opposite face of the molecule, thereby
establishing the relative configuration of pestalotheol A as 1.
Ultimately, the structure of 1 was confirmed by X-ray crystal-
lographic analysis, and a perspective ORTEP plot is shown in Fig-
ure 1.
The absolute configuration of pestalotheol A (1) was assigned
by application of the modified Mosher method.18,19 Treatment of
1 with (S)-MTPA Cl and (R)-MTPA Cl afforded the (R)-MTPA
ester (1a) and (S)-MTPA ester (1b), respectively. The difference
in chemical shift values (∆δ ) δS - δR) for the diastereomeric
esters 1b and 1a was calculated in order to assign the absolute
configuration at C-5. Calculations for all of the relevant signals
except one (H3-16) suggested the R absolute configuration at C-5.
Therefore, the 2R, 4R, 5R, and 9S absolute configuration was
proposed for compound 1 on the basis of the ∆δ results summarized
in Figure 2.
Results and Discussion
The molecular formula of pestalotheol A (1) was determined to
be C16H24O6 (five degrees of unsaturation) by analysis of its
HRESIMS (m/z 335.1466 [M + Na]+; ∆ +0.4 mmu) and NMR
data (Table 1). Analysis of the 1H, 13C, and HMQC NMR
spectroscopic data of 1 revealed the presence of three exchangeable
protons, four methyl groups, three methylene units, three oxyme-
thines, three oxygenated quaternary carbons, two olefinic carbons,
and one R,ꢀ-conjugated carbonyl carbon (δC 192.6). These data
1
accounted for all H and 13C NMR resonances and required the
compound to be tricyclic. Analysis of the 1H-1H COSY NMR data
* To whom correspondence should be addressed. Tel: 86 10 82618785.
Fax: 86 10 82618785. E-mail: (Y.C.) cheys@im.ac.cn; (X.C.) chenxl@
wh.iov.cn.
† Institute of Microbiology.
Pestalotheol B (2) was assigned a molecular formula of C16H24O5
(five degrees of unsaturation) on the basis of its HRESIMS (m/z
‡ Wuhan Institute of Virology.
10.1021/np700744t CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/28/2008

BioactiVe Metabolites from Pestalotiopsis theae
Journal of Natural Products, 2008, Vol. 71, No. 4 665
Table 1. NMR Spectroscopic Data of Pestalotheol A (1) in Acetone-d6
a
position
δH (J in Hz)
δCb, mult.
HMBC (H f C#)
NOESY
2
3a
3b
4
5
6
7
8a
8b
9
10
11a
11b
12
13
14
15
16
17
OH-4
OH-5
OH-15
4.12, dd (10, 6.4)
2.49, dd (13, 10)
1.76, dd (13, 6.4)
85.8, CH
35.2, CH2
3, 9, 15, 16, 17
2, 4, 15
4, 9
4-OH, 16, 17
5, 5-OH, 9, 16, 17
4-OH, 5
80.1, qC
72.8, CH
167.5, qC
108.6, qC
22.7, CH2
4.08, d (5.7)
4, 6, 7, 9
3a, 3b
2.63, dd (15, 5.7)
2.12, dd (15, 11)
3.90, dd (11, 5.7)
4, 6, 7, 9, 10
4, 6, 7, 9, 10
4, 5
4-OH
3a, 5-OH, 16, 17
77.0, CH
192.6, qC
47.7, CH2
2.54, d (16)
2.37, d (16)
7, 10, 12, 13, 14
7, 10, 12, 13, 14
80.6, qC
1.41, s
1.33, s
27.8, CH3
24.5, CH3
72.1, qC
26.7, CH3
25.6, CH3
10, 11, 12, 14
11, 12, 13
1.18, s
1.09, s
3.86, s
4.95, d (5.7)
3.16, s
2, 15, 17
2, 15, 16
3, 4, 5, 9
4, 5, 6
2, 3a, 9
2, 3a, 9
2, 3b, 8b
3a, 9
2, 15, 16, 17
3a
a Recorded at 400 MHz. b Recorded at 100 MHz.
Table 2. 1H NMR Spectroscopic Data of Pestalotheols B-D
(2–4) in Acetone-d6
pestalotheol
pestalotheol
pestalotheol
B (2)
C (3)
D (4)
a
a
position
δH (J in Hz)
δH (J in Hz)
δH (J in Hz)
2
4.10, dd (8.6, 7.5) 4.07, dd (9.4, 6.7) 4.59, dd (8.8, 8.6)
3a
3b
2.53, dd (13, 8.6)
2.00, dd (13, 7.5)
2.54, dd (13, 9.4)
1.71, dd (13, 6.7)
3.28, dd (17, 8.6)
3.18, dd (17, 8.8)
4
5a
5b
6
7
8a
8b
9
10
2.13, d (10)
2.07, d (10)
3.74, d (2.7)
6.79, s
6.95, s
2.66, s
Figure 1. Thermal ellipsoid representation of 1.
4.63 br, d (8.5)
2.64, dd (15, 5.6)
2.12, dd (15, 11)
3.67, dd (11, 5.6)
2.49, t (12)
2.21, dd (12, 4.8)
3.85, dd (12, 4.8)
6.14, d (11)
11a
11b
12
2.49, d (16)
2.37, d (16)
6.24, d (11)
13
14
1.39, s
1.34, s
1.78, s
1.73, s
1.40, s
1.40, s
15
Figure 2. ∆δ values (in ppm) ) δS - δR obtained for (S)- and
(R)-MPTA esters 1a and 1b.
16
17
1.19, s
1.06, s
3.75, s
1.08, s
1.23, s
1.19, s
1.25, s
OH-4
OH-6
OH-15
a Recorded at 400 MHz.
3.87, d (8.7)
3.31, s
319.1520 [M + Na]+; ∆ +0.1 mmu) and NMR data (Tables 2 and
3) Analysis of the 1H and 13C NMR data for 2 revealed the presence
of structural features similar to those found in 1, except that the
oxymethine (δH/δC 4.08/72.8) was replaced by signals for a
methylene unit (δH/δC 2.13, 2.07/41.2) in the NMR spectra of 2,
and this observation was supported by HMBC correlations from
these newly observed methylene protons to C-4 and C-7. Therefore,
the gross structure of pestalotheol B was proposed as 2. The relative
3.19, s
3.62, s
isopropyl side chain attached at C-2, like that appearing in 1 and
2, was established. Four isolated proton spin-systems corresponding
to the C-2-C-3, C-5-C-6 (including C-6-OH), C-8-C-9, and
C-10-C-11 subunits of structure 3 were established by analysis of
its COSY NMR data. HMBC correlations from H-5 to C-3 and
C-4, H-6 to C-7 and C-8, and H-8b to C-4, C-6, C-7, and C-9
permitted completion of a cyclohexane unit. Key HMBC correla-
tions from H3-13 and H3-14 to C-11 and C-12 and from the olefinic
proton H-10 to C-6 and C-8 enabled the completion of the C-7-C-
14 partial structure of 3, with C-7 directly attached to C-10.
Correlations from the exchangeable proton (δH 3.87) to C-5, C-6,
and C-7 established the connectivity between C-6 and a hydroxy
group. Considering the chemical shifts of C-4 (δC 69.5) and C-5
(δC 60.1), as well as the unsaturation requirement for 3, C-4 and
C-5 have to be connected to one oxygen atom to form an epoxide
moiety. On the basis of these data, the planar structure of
pestalotheol C was established as depicted in 3.
1
configuration of 2 was assigned by analysis of H-1H coupling
constants, NOESY data, and comparison of its 1H NMR data with
those of pestalotheol A (1). The absolute configuration of pesta-
lotheol B (2) was assigned as shown by analogy to pestalotheol
A (1).
The molecular formula of pestalotheol C (3) was established as
C16H24O4 (five degrees of unsaturation) on the basis of HRESIMS
analysis (m/z 303.1569 [M + Na]+; ∆ +0.3 mmu) and the NMR
1
data (Tables 2 and 3). The H and 13C NMR data for 3 differed
significantly from those of 1 and 2, suggesting a substantial
structural change. Upon analysis of COSY and HMBC data, the
same partial structure of a tetrahydrofuran ring with a 2-hydroxy-

666 Journal of Natural Products, 2008, Vol. 71, No. 4
Li et al.
Table 3. 13C NMR Spectroscopic Data of Pestalotheols B–D (2–
4) in Acetone-d6
compounds showed noticeable in vitro antimicrobial or antifungal
activity when tested at 100 µg/disk.
Pestalotheols A (1), B (2), and D (4) are new members of the
chromenone type of metabolites. Biogenetically, these compounds
could be derived from two units of isoprenoids and a polyketide,
whereas pestalotheol C (3) could be their biosynthetic precursor.
Natural products containing a reduced tetrahydro-2H-furo[3,2-
g]chromene unit have been reported previously, such as visamminol
(5).20 However, pestalotheols A (1), B (2), and D (4) differ
significantly from their naturally occurring precedents in the identity
of the substituents and, most notably, in the fused pattern between
the cyclohexene (aromatic in 4) moiety and the furan ring.
Pestalotheol C (3) is closely related to illifunone E (6)21 and
illicinone E,22 but differs in the identity of the substituents as well
as the presence of an epoxide moiety cis-fused to the cyclohexane
ring.
pestalotheol B (2) pestalotheol C (3) pestalotheol D (4)
position
δCa, mult.
δCa, mult.
δCa, mult.
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
85.9, CH
39.8, CH2
81.3, qC
86.0, CH
31.1, CH2
69.5, qC
90.2, CH
31.6, CH2
139.5, qC
115.6, CH
155.7, qC
119.9, qC
103.5, CH
155.3, s qC
191.9, qC
49.0, CH2
79.5, qC
41.2, CH2
167.4, qC
108.4, qC
22.4, CH2
76.8, CH
191.3, qC
47.7, CH2
80.6, qC
60.1, CH
64.8, CH
134.3, qC
35.4, CH2
77.8, CH
128.7, CH
121.9, CH
136.2, qC
26.4, CH3
18.0, CH3
71.9, qC
27.4, CH3
25.2, CH3
72.0, qC
26.7, CH3
26.5, CH3
71.4, qC
Experimental Section
26.9, CH3
25.6, CH3
25.8, CH3
26.7, CH3
26.0, CH3
25.6, CH3
General Experimental Procedures. Optical rotations were measured
on a Perkin-Elmer 241 polarimeter, and UV data were recorded on a
Hitachi U-2800 spectrophotometer. IR data were recorded using a
Bruker Vertex 70 spectrophotometer. 1H and 13C NMR data were
acquired with a Bruker Avance-400 spectrometer using solvent signals
(acetone-d6; δH 2.05/δC 29.8, 206.1) 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 a Bruker APEX III 7.0 T
spectrometer.
Fungal Material. The culture of P. theae was isolated by one of
the authors (L.G.) from the branches of an unidentified tree near the
Jianfeng Mountain, Hainan Province, in April 2005. The isolate was
identified and assigned the accession number LN560 in L.G.’s 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. Fermentation was carried
out in four 500 mL Fernbach flasks each containing 100 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.23 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 rice substrate was extracted
repeatedly with MEK (3 × 500 mL), and the organic solvent was
evaporated to dryness under vacuum to afford 2.51 g of crude extract.
The extract was fractionated by silica gel VLC using petroleum
ether-EtOAc gradient elution. The fraction (26 mg) eluted with 15%
EtOAc was further separated by semipreparative reversed-phase HPLC
(Kramosil C18 column; 10 µm; 10 × 250 mm; 2 mL/min) to afford
pestalotheol D (4; 4.5 mg, tR 18.4 min; 60 to 80% CH3OH in water
over 30 min). The fractions eluted with 30% (50 mg), 50% (100 mg),
55% (140 mg), and 60% (150 mg) EtOAc were fractionated again by
Sephadex LH-20 column chromatography using CHCl3-CH3OH (1:1)
as eluents. Purification of these fractions with different gradients afforded
pestalotheols A (1; 30.0 mg, tR 11.8 min; 30 to 60% CH3OH in water
over 35 min), B (2; 4.5 mg, tR 16.0 min; 40 to 55% CH3OH in water
over 25 min), and C (3; 9.0 mg, tR 17.2 min; 55 to 75% CH3OH in
water over 30 min).
a Recorded at 100 MHz.
The relative configuration of pestalotheol C (3) was assigned
by analysis of 1H-1H coupling constants and NOESY correlations
(Figure 3). The large coupling constant observed between H-8a
and H-9 (12 Hz) and between H-2 and H-3a (9.4 Hz) suggested a
trans relationships between adjacent protons. NOESY correlations
from H-8a to H-2 and OH-6 and from H-5 to H-3b placed these
protons on the same face of the ring system, and correlations of
H-9 with H-3a and H-6 indicated that they are on the opposite face
of the ring system. NOESY correlations of H-6 with H-11 and of
H-8b with H-10 established the Z-geometry of the C-7/C-10 double
bond. On the basis of these data, the relative configuration of
pestalotheol C was established as shown in 3.
The absolute configuration of pestalotheol C (3) was also
assigned by application of the modified Mosher method. The
differences in chemical shift values (∆δ ) δS - δR) for the
diastereomeric esters 3b and 3a were calculated in order to assign
the absolute configuration at C-6. Calculations for all of the relevant
signals suggested the R absolute configuration at C-6, and the 2R,
4R, 5S, 6R, and 9S absolute configuration was proposed for
compound 3 on the basis of the results summarized in Figure 4.
The molecular formula of pestalotheol D (4) was determined to
be C16H20O4 (seven unsaturations) by analysis of its HRESIMS (m/z
299.1252 [M + Na]+; ∆ +0.2 mmu) and NMR data (Tables 2 and
3). Analysis of its NMR data revealed the presence of the same
2-hydroxyisopropyl side chain and dihydropyranone moiety as those
present in 1 and 2, except that the signals for four sp3 carbons,
C-4, C-5, C-8, and C-9, were replaced by four aromatic resonances
in the NMR spectra of 4, indicating that the cyclohexene ring
occurring in 1 and 2 was aromatized. HMBC correlations from H-5
to C-3, C-6, C-7, and C-9 and from H-8 to C-4, C-6, C-9, and
C-10 further confirmed the presence of an aromatic ring in 4. The
13C NMR chemical shift of C-9 (δC 155.3) indicated that it was
oxygenated, and an HMBC correlation of H-2 with C-9 established
the connectivity of C-2 and C-9 via an oxygen to form a
dihydrofuran ring. On the basis of these data, the structure of
pestalotheol D was established as depicted in 4.
Pestalotheol A (1): colorless needles, mp 223–225 °C; [R]D +122
(c 1.17, CH3OH); UV (CH3OH) λmax 278 (ꢀ 6500) nm; IR (neat) νmax
3391 (br), 2976, 1650, 1595, 1373, 1258, 1165, 1085, 1052 cm-1; 1H,
13C NMR, HMBC, and NOESY data, see Table 1; HRESIMS obsd
m/z 335.1466 [M + Na]+ (calcd for C16H24O6Na, 335.1470).
Pestalotheols A-D (1–4) were tested for in vitro activity against
HIV-1LAI. Pestalotheol C (3) showed an inhibitory effect on HIV-1
replication in C8166 cells, with EC50 and CC50 values of 16.1 and
163 µM, respectively (selectivity index, SI ) 10.1; the positive
control indinavir sulfate showed an EC50 value of 8.18 nM).
Pestalotheols A-D (1–4) were also evaluated for activity against
a panel of bacteria includingStaphylococcus aureus (ATCC 6538),
Streptococcus mutans (ATCC 25175), Enterococcus faecalis (ATCC
19433), and Sarcina lutea (CMCC B28001), and the fungi
Geotrichum candidum (AS2.498), Candida albicans (ATCC 10231),
and Aspergillus fumigatus (ATCC 10894). However, none of these
24
X-ray Crysrallographic Analysis of Pestalotheol A (1). Upon
crystallization from MeOH-H2O (10:1) using the vapor diffusion
method, colorless crystals were obtained for 1, a crystal (0.26 × 0.24
× 0.18 mm) was separated from the sample and mounted on a glass
fiber, and data were collected using a Bruker SMART 1000 CCD
diffractometer with graphite-monochromated Mo KR radiation, λ )
0.71073 Å at 294(2) K. Crystal data: C16H24O6, M ) 312.35, space
group orthorhombic, P212121; unit cell dimensions a ) 6.465(2) Å, b
) 10.184(4) Å, c ) 25.525(9) Å, V ) 1680.6(10) Å3, Z ) 4, Dcalcd
)
1.234 mg/m3, µ ) 0.094 mm-1, F(000) ) 672. The structure was solved

BioactiVe Metabolites from Pestalotiopsis theae
Journal of Natural Products, 2008, Vol. 71, No. 4 667
Figure 3. Key NOESY correlations for pestalotheol C (3).
MHz) H-2 f C-3, 4, 16, 17; H-3a f C-4, 9; H-3b f C-2, 4, 9, 15;
H2-5 f C-4, 7; H-8a f C-4, 6, 7, 9, 10; H-8b f C-4, 7; H2-11 f
C-7, 10, 12, 13, 14; H3-13 f C-11, 12, 14; H3-14 f C-11, 12, 13;
H3-16 f C-2, 15, 17; H3-17 f C-2, 15, 16; OH-4 f C-3, 4, 5, 9;
OH-15 f C-2, 15, 16, 17; key NOESY correlations (acetone-d6, 400
MHz) H-2 T OH-4, H3-16, H3-17; H-3a T H-5a, H-9; H-5a T H-3a,
9; H-9 T H-3a, H-5a, H3-16, H3-17; OH-4 T H-2, H-3b, H-5b;
HRESIMS obsd m/z 319.1520 [M + Na]+ (calcd for C16H24O5Na,
319.1521).
Pestalotheol C (3): colorless powder; [R]D +230 (c 0.60, CH3OH);
Figure 4. ∆δ values (in ppm) ) δS - δR obtained for (S)- and
(R)-MPTA esters 3a and 3b.
UV (CH3OH) λmax 247 (ꢀ 16 600) nm; IR (neat) νmax 3435 (br), 2972,
2922, 1376, 1053, 1018 cm-1 1H and 13C NMR data, see Tables 2 and
;
3; HMBC data (acetone-d6, 400 MHz) H-2 f C-3, 9, 15, 16, 17; H-3a
f C-2, 4, 5, 15; H-3b f C-4, 9; H-5 f C-3, 4, 6, 7, 10; H-6 f C-7,
8, 10; H-8b f C-4, 6, 7, 9, 10; H-9 f C-4; H-10 f C-6, 8; H3-13 f
C-10, 11, 12, 14; H3-14 f C-10, 11, 12, 13; H3-16 f C-2, 15, 17;
H3-17 f C-2, 15, 16; OH-6 f C-5, 6, 7; OH-15 f C-2, 15, 16, 17;
key NOESY correlations (acetone-d6, 400 MHz) H-2 T H-8a, H3-16,
H3-17; H-3a T H-9; H-3b T H-5; H-5 T H-3b; H-6 T H-9, H-11;
H-8a T H-2, OH-6; H-8b T H-10; H-9 T H-3a, H-6, H3-16;
HRESIMS obsd m/z 303.1569 [M + Na]+ (calcd for C16H24O4Na,
303.1572).
by direct methods using SHELXL-9725 and refined using full-matrix
least-squares difference Fourier techniques. All non-hydrogen atoms
were refined with anisotropic displacement parameters, and all hydrogen
atoms were placed in idealized positions and refined as riding atoms
with the relative isotropic parameters. Absorption corrections were
applied with the Siemens Area Detector Absorption Program
(SADABS).26 The 9269 measurements yielded 3477 independent
reflections after equivalent data were averaged, and Lorentz and
polarization corrections were applied. The final refinement gave R1 )
0.0524 and wR2 ) 0.1156 [I > 2σ(I)].
Preparation of (R)-MTPA Ester (3a) and (S)-MTPA Ester (3b).
A sample of 3 (1.0 mg, 0.003 mmol), (S)-MPTA Cl (5.0 µL, 0.026
mmol), and pyridine-d5 (0.5 mL) were allowed to react in an NMR
Preparation of (R)-MTPA Ester (1a) and (S)-MTPA Ester (1b).
A solution of 1 (2.0 mg, 0.006 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)-MTPA Cl (9.5 µL, 0.050 mmol)
were quickly added, and all contents were mixed thoroughly by shaking
the NMR tube carefully. The reaction was performed at room
1
tube at ambient temperature for 24 h, with the H NMR data of the
R-MTPA ester derivative (3a) were obtained directly on the reaction
mixture: 1H NMR (pyridine-d5, 400 MHz) δ 6.10 (1H, br s, H-6), 6.00
(1H, d, J ) 12 Hz, H-11), 5.80 (1H, d, J ) 12 Hz, H-10), 4.19 (1H,
dd, J ) 9.2, 6.8 Hz, H-2), 4.08 (1H, d, J ) 2.8 Hz, H-5), 3.93 (1H, dd,
J ) 12, 4.8 Hz, H-9), 2.67 (1H, dd, J ) 12, 9.2 Hz, H-3a), 2.64(1H,
t, J ) 12 Hz, H-8a), 2.17 (1H, dd, J ) 12, 4.8 Hz, H-8b), 1.76 (1H,
dd, J ) 12, 6.8 Hz, H-3b), 1.53 (3H, s, H-13), 1.39 (3H, s, H-14), 1.30
(3H, s, H-16), 1.07 (3H, s, H-17).
1
temperature and monitored by H NMR at each 24 h. The reaction
was found to be complete at 48 h. The mixture was evaporated to
dryness and purified by semipreparative reversed-phase HPLC (Kra-
mosil C18 column; 10 µm; 10 × 250 mm; 2 mL/min, 30 to 100%
CH3OH in H2O over 35 min, then 100% CH3OH over 5 min) to afford
1
1a (1.9 mg, tR 37.4 min): white powder; H NMR (acetone-d6, 400
Similarly, the reaction mixture from another sample of 3 (1.0 mg,
0.003 mmol), (R)-MPTA Cl (5.0 µL, 0.026 mmol), and pyridine-d5
MHz) δ 5.65 (1H, s, H-5), 4.55 (1H, s, OH-12), 4.11 (1H, dd, J ) 9.2,
6.8 Hz, H-2), 3.73 (1H, dd, J ) 11, 5.5 Hz, H-9), 3.13 (1H, s, OH-15),
2.69 (1H, dd, J ) 16, 5.6 Hz, H-8a), 2.66 (1H, d, J ) 16 Hz, H-11a),
2.48 (1H, d, J ) 16 Hz, H-11b), 2.18 (1H, dd, J ) 16, 11 Hz, H-8b),
1.91 (1H, dd, J ) 12, 9.2 Hz, H-3a), 1.88 (1H, dd, J ) 12, 6.8 Hz,
H-3b), 1.48 (3H, s, H-13), 1.32 (3H, s, H-14), 1.09 (3H, s, H-16), 0.94
(3H, s, H-17); ESIMS m/z [M + Na]+ 551.2.
1
(0.5 mL) was processed as described above for 3a to afford 3b: H
NMR (pyridine-d5, 400 MHz) δ 6.18 (1H, br s, H-6), 6.13 (1H, d, J )
12 Hz, H-11), 5.93 (1H, d, J ) 12 Hz, H-10), 4.15 (1H, dd, J ) 8.4,
7.2 Hz, H-2), 4.05 (1H, d, J ) 2.0 Hz, H-5), 3.95 (1H, dd, J ) 12, 4.8
Hz, H-9), 2.68 (1H, dd, J ) 14, 9.2 Hz, H-3a), 2.64(1H, t, J ) 14 Hz,
H-8a), 2.24 (1H, dd, J ) 14, 4.8 Hz, H-8b), 1.71 (1H, dd, J ) 14, 6.4
Hz, H-3b), 1.51 (3H, s, H-13), 1.46 (3H, s, H-14), 1.29 (3H, s, H-16),
1.06 (3H, s, H-17).
In a similar fashion, a sample of compound 1 (2.0 mg, 0.006 mmol),
pyridine-d5 (0.5 mL), and (R)-MPTA Cl (9.5 µL, 0.050 mmol) were
allowed to react in an NMR tube at ambient temperature for 48 h, and
the reaction mixture was processed as described above for 1a to afford
Pestalotheol D (4): gum; [R]D -11 (c 0.20, CH3OH); UV (CH3OH)
max 237 (ꢀ 7800), 264 (ꢀ 2800), 366 (ꢀ 2000) nm; IR (neat) νmax 3436,
1
λ
1b (1.5 mg): gum; H NMR (acetone-d6, 400 MHz) δ 5.62 (1H, s,
2974,1680, 1628, 1448, 1371, 1256, 1165, 897 cm-1; 1H and 13C NMR
data, see Tables 2 and 3; HMBC data (acetone-d6, 400 MHz) H-2 f
C-4, 9, 16, 17; H2-3 f C-2, 4, 5, 9, 15; H-5 f C-3, 6, 7, 9; H-8 f
C-4, 6, 9, 10; H2-11 f C-7, 10, 12, 13, 14; H3-13 f C-10, 11, 12, 14;
H3-14 f C-10, 11, 12, 13; H3-16 f C-2, 15, 17; H3-17 f C-2, 15, 16;
OH-15 f C-2, 15, 16, 17; HRESIMS obsd m/z 299.1252 [M + Na]+
(calcd for C16H20O4Na, 299.1259).
H-5), 4.54 (1H, s, OH-12), 4.15 (1H, dd, J ) 9.5, 6.4 Hz, H-2), 3.88
(1H, dd, J ) 11, 5.5 Hz, H-9), 3.34 (1H, s, OH-15), 2.69 (1H, dd, J )
16, 5.6 Hz, H-8a), 2.60 (1H, d, J ) 16 Hz, H-11a), 2.32 (1H, d, J )
16 Hz, H-11b), 2.23 (1H, dd, J ) 12, 9.6 Hz, H-3a), 2.16 (1H, dd, J
) 16, 11 Hz, H-8b), 1.94 (1H, dd, J ) 12, 6.4 Hz, H-3b), 1.37 (3H, s,
H-13), 1.23 (3H, s, H-14), 1.04 (3H, s, H-16), 0.97 (3H, s, H-17);
ESIMS m/z [M + Na]+ 551.1.
Antimicrobial and Antifungal Bioassays. Antimicrobial and an-
tifungal bioassays were conducted according to a literature procedure.27
The bacterial strains were grown on Mueller-Hinton agar, the yeasts
Candida albicans (ATCC 10231) and Geotrichum candidum (AS2.498)
Pestalotheol B (2): colorless needles, mp 214–216 °C; [R]D +86 (c
0.30, CH3OH); UV (CH3OH) λmax 276 (ꢀ 8000) nm; IR (neat) νmax
1
3435 (br), 2975, 1650, 1591, 1412, 1372, 1259, 1166, 1085 cm-1; H
and 13C NMR data, see Tables 2 and 3; HMBC data (acetone-d6, 400

668 Journal of Natural Products, 2008, Vol. 71, No. 4
Li et al.
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Wuhan, People’s Republic of China), and incubated at 37 °C for 1 h.
After washing 5 times with PBST, and the HRP-labeled anti-p24
antibody (provided by Dr. Bin Yan) was added and incubated at 37 °C
for 1 h. The plate was washed 5 times with PBST, followed by adding
OPD reaction mixture. The assay plate was read at 490 nm using a
microplate reader within 30 min. The inhibition rate and the EC50 based
on p24 antigen expression level were calculated, and the selective index
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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
Sciences (Grant KSCX2-Y-G-013), and the National Natural Science
Foundation of China (Grant 30670055).
(24) Crystallographic data for compound 1 have been deposited with the
Cambridge Crystallographic Data Centre (deposition number CCDC
665253). Copies of the data can be obtained, free of charge, on
application to the director, CCDC 12 Union Road, Cambridge CB2
1EZ, UK (fax: +44 1223 336033 ore-mail: deposit@ccdc.cam.ac.uk).
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1
Supporting Information Available: H and 13C NMR spectra of
pestalotheols A-D (1-4). This material is available free of charge
via the Internet at http://pubs.acs.org .
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