Fungal transformation of isosteviol lactone and its biological evaluation for inhibiting the AP-1 transcription factor

Article abstract of DOI:10.1016/j.phytochem.2009.03.015

A number of hydroxylated diterpenoids were obtained from the microbial transformation of isosteviol lactone (4α-carboxy-13α-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone) (2) with Mucor recurvatus MR 36, Aspergillus niger BCRC 31130, and

Full text of DOI:10.1016/j.phytochem.2009.03.015

Phytochemistry 70 (2009) 759–764
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Fungal transformation of isosteviol lactone and its biological evaluation
for inhibiting the AP-1 transcription factor
Bo-Hon Chou a, Li-Ming Yang a,d, Shwu-Fen Chang b, Feng-Lin Hsu c, Chia-Hsin Lo a,e, Wen-Kuang Lin a,
Li-Hsuan Wang f, Pan-Chun Liu a, Shwu-Jiuan Lin a,
*
a Department of Medicinal Chemistry, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
b Division of Cell and Molecular Biology, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan
c Graduate Institute of Pharmacognosy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
d Division of Medicinal Chemistry, National Research Institute of Chinese Medicine, Taipei 112, Taiwan
e Forensic Science Center of Taipei City Police Department, Taipei 115, Taiwan
f Department of Clinical Pharmacy, College of Pharmacy, Taipei Medical University, Taipei 110, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of hydroxylated diterpenoids were obtained from the microbial transformation of isosteviol
lactone (4a-carboxy-13a-hydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone) (2) with
Received 25 October 2008
Received in revised form 21 January 2009
Available online 22 April 2009
Mucor recurvatus MR 36, Aspergillus niger BCRC 31130, and Absidia pseudocylindrospora ATCC 24169. Incu-
bation of 2 with M. recurvatus and Asp. niger led to isolation of seven known compounds (1 and 3–8).
Incubation of 2 with Abs. pseudocylindrospora produced 5 and six previously unreported compounds
(9–14). The structures of these isolated compounds were deduced by high-field NMR techniques (1H,
13C, DEPT, COSY, NOESY, HSQC, and HMBC), and those of 9 and 11 were further confirmed by X-ray crys-
tallographic analyses. Subsequently, the inhibitory effects on activator protein-1 (AP-1) activation in lipo-
polysaccharide-stimulated RAW 264.7 macrophages of all of these compounds were evaluated.
Compounds 2–5, 8, 9, 11, and 12 exhibited significant inhibitory activity, while 3 was more potent than
the reference compound of dexamethasone.
Keywords:
Microbial transformation
Isosteviol lactone
Diterpenoid
Activator protein-1
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
glucocorticoid-mediated repression through AP-1-responsive ele-
ments (Jonat et al., 1990; Smith et al., 1996; González et al.,
Activator protein-1 (AP-1) is a group of dimeric factors consti-
tuted by members of the Jun and Fos families of DNA-binding
proteins (Jochum et al., 2001). Many stimuli induce the binding
of AP-1 to the promoter region of various genes that govern cellu-
lar processes such as inflammation, proliferation, and apoptosis
(Wisdom, 1999). Thus, AP-1 proteins are recognized as regulators
of cytokine expression and important modulators in inflammatory
diseases, such as rheumatoid arthritis, psoriasis, and psoriatic
arthritis (Zenz et al., 2008). Glucocorticoids have long been used
as effective immunosuppressive agents in the treatment of condi-
tions involving T-cell- or cytokine-mediated tissue damage
(Barnes, 2006). They are also widely used to treat inflammatory
and autoimmune disorders. The major anti-inflammatory effects
of glucocorticoids appear to largely be due to interactions between
the activated glucocorticoid receptor (GR) and proinflammatory
transcription factors that mediate the expression of inflammatory
genes (Saklatvala, 2002). Many researchers have described a mech-
anism of GR-mediated transcriptional repression involving the
physical interaction of GR and AP-1. This interaction results in
2000). Thus, inhibition or modulation of AP-1 activation may repre-
sent an attractive target for developing novel therapeutic agents for
treating inflammatory-mediated conditions (Tsuchida et al., 2006).
Microbial transformation is an area of great interest for prepar-
ing products which are difficult to obtain by conventional chemical
methods (Arantes and Hanson, 2007). There is an increasing body
of information about use of biocatalysis for selective conversion
of synthetic and natural products to intensify either their biological
properties or to lead to new biological activities (Buchanan and Re-
ese, 2001; Gładkowski et al., 2007; Venisetty and Ciddi, 2003). Spe-
cial attention has been paid to filamentous fungi because they are
capable of catalyzing regio- and stereoselective hydroxylation of a
variety of nonfunctionalized hydrocarbon centers of a great variety
of substrates (Lehmann and Stewart, 2001). Isosteviol lactone (2),
an ent-beyerane tetracyclic diterpenoid, was prepared by reacting
isosteviol (1) with m-chloroperbenzoic acid, and its activity on
mitochondrial metabolism has been described (Braguini et al.,
2003). Ghisalberti (1997) indicated that some highly oxygenated
diterpenoids usually have higher levels of biological activity than
their less-hydroxylated precursors. Previously, we studied the
microbial transformation of 2 and evaluated the transformation
products on the androgen response element (Chou et al., 2008).
* Corresponding author. Tel.: +886 2 27361661x6133; fax: +886 2 28264276.
E-mail address: shwu-lin@tmu.edu.tw (S.-J. Lin).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.03.015

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B.-H. Chou et al. / Phytochemistry 70 (2009) 759–764
In order to obtain additional hydroxylated compounds, the present
investigation was carried out to continue studying the ability of
other microorganisms to accomplish structural modifications of 2
and to develop new biological activities. Since tetracyclic diterpe-
noids possess a formal similarity to steroids (Hanson, 1992) and
are still being used to develop immunoinflammatory agents
(Chang et al., 2006, 2008), an AP-1-mediated luciferase reporter
gene assay was used to evaluate the modified compounds with re-
spect to the parent compound. The production, isolation, and struc-
tural characterization as well as the inhibitory effects on AP-1
activation in lipopolysaccharide (LPS)-stimulated macrophages of
these metabolites are reported herein. Additionally, the stereo-
chemical characteristics of 9 and 11 were also confirmed by X-
ray crystallographic analyses.
the multiplicity of the H-12 signal in the 1H NMR spectrum, which
is a broad singlet in 9 and a double-doublet in 10, indicated that
the proton was in an
a-orientation in 9 and a b-orientation in 10
(de Oliveira and Strapasson, 1996). The NOESY spectra also showed
cross-peaks of d 4.01 with H-11 and CH3-17 in 9; and d 3.68 with
H-9b, and H-11, H-14 in 10. In addition, an X-ray crystallographic
experiment was also carried out to confirm the structure of 9
(Fig. 2). Thus, structures of 9 and 10 were determined to be 4
carboxy-12b,13 -dihydroxy-13,16-seco-ent-19-norbeyeran-16-oic
acid 13,16-lactone and -carboxy-12 ,13 -dihydroxy-13,16-
a-
a
4a
a
a
seco-ent-19-norbeyeran-16-oic acid 13,16-lactone, respectively.
Compound 11 had a quasi-molecular ion at m/z 351.2178
[M+H]+ (calc. 351.2171) in the HRESIMS, consistent with a molec-
ular formula of C20H31O5. The 13C NMR spectrum displayed reso-
nances for 20 carbons, while the DEPT spectrum showed the
presence of three methyl, eight methylene, three methine, and
six quaternary carbons. Comparison of the 13C NMR and DEPT
spectra with 2 suggested that 11 contains one more oxygen atom
than 2. The HSQC spectrum showed a new proton geminal to an
alcohol resonating at dH 3.53 (dC 77.2). In the HMBC spectrum, dC
77.2 exhibited cross-peaks with dH 1.24 (H-5b), 2.36-2.45 (H-6
and H-14), and 3.33 (H-15). Thus, hydroxylation had occurred at
2. Results and discussion
In a continuing investigation of the microbial transformation of
isosteviol lactone (2) (Chou et al., 2008), the bioconversion of 2
with Mucor recurvatus, Aspergillus niger, and Absidia pseudocylind-
rospora was undertaken and led to isolation of compounds 3–14
(Fig. 1). Incubation of 2 with M. recurvatus yielded four known
compounds of 1 and 3–5, whereas incubation of 2 with Asp. niger
afforded the five known compounds 1, 4, and 6–8. The structures
of known metabolites were determined by comparison of the
NMR spectroscopic data with values in the literature (de Oliveira
and Strapasson, 1996; Chou et al., 2008). On the other hand, incu-
bation of 2 with Abs. pseudocylindrospora yielded 5 and six new
compounds of 9–14. Compounds 9 and 10 displayed similar 1H
and 13C NMR spectra. They also showed quasi-molecular ions
[M+H]+ at m/z 351.2174 and 351.2173, respectively, in the HRE-
SIMS, corresponding to the molecular formula of C20H31O5 (calc.
351.2171). The 13C NMR spectra of 9 and 10 displayed the same
resonances for 20 carbons, while the DEPT spectra showed the
presence of three methyl, eight methylene, three methine, and
six quaternary carbons. The HSQC spectra of 9 and 10 showed
new resonances at dH 4.01 (dC 71.7) and dH 3.68 (dC 75.8), respec-
tively, in comparison with those of 2. This indicated that 9 and
10 contain one more oxygen atom than does 2. Analysis of the
HSQC and HMBC spectra of 9 and 10 and their comparison with
2 demonstrated that dH 4.01 showed connectivities with dC 25.6
(C-17), 42.5 (C-14), 48.8 (C-9), and 82.3 (C-13) in 9, and dH 3.68
had connectivities with dC 28.7 (C-11), 82.6 (C-13), and 24.4 (C-
17) in 10. Thus, the additional oxygen present in both molecules
was located at C-12. The orientation of the 12-OH follows from
C-7. The a-orientation of the 7-OH was suggested from the cross-
peaks of d 3.53 (H-7) with H-5b, H-9b, H-6, and H-14 in the NOESY
experiment. Moreover, the structure of 11 was confirmed by X-ray
crystallographic analysis (Fig. 3). Thus, 11 was established as 4
carboxy-7 ,13 -dihydroxy-13,16-seco-ent-19-norbeyeran-16-oic acid
13,16-lactone.
a-
a
a
Compound 12 showed a quasi-molecular ion peak at m/z
351.2193 [M+H]+ (calc. 351.2171) in the HRESIMS, corresponding
to a molecular formula of C20H31O5. The DEPT spectrum showed
the disappearance of one CH3 signal and the presence of one new
CH2 signal at d 69.3 in comparison with those of 2. Analysis of
the 1H, 13C NMR, HSQC, and HMBC spectra and comparison with
2 indicated that the resonances of C-13 and C-16 had shifted
downfield from d 79.8 to 83.5 and d 171.8 to 172.7, respectively.
The resonances of C-12 and C-14 had shifted upfield from d 38.7
to 34.5 and 47.5 to 43.4, respectively, suggesting that an OH might
reside at C-17. Additionally, two new protons resonating at d 3.81
and 3.91 and showing connectivities to C-12 (d 34.5), C-13 (d 83.5),
and C-14 (d 43.4) also suggested the presence of an OH at C-17. On
the basis of the above evidence, 12 was assigned to be 4
carboxy-13 ,17-dihydroxy-13,16-seco-ent-19-norbeyeran-16-oic
acid 13,16-lactone.
a-
a
Compound 13 had a quasi-molecular ion peak at m/z 389.1955
[M+Na]+ in the HRESIMS, corresponding to the molecular formula
of C20H30O6Na. The DEPT, HSQC, and HMBC spectra showed the
disappearance of one CH3 and one CH signal, and the presence of
R1
O
O
O
R1
COOH
COOH
3: R1 = H
8: R1 = OH
1: R1 = H
4: R1 = OH
2: R1 = R2 = R3 = R4 = R5 = R6 = H
R4
17
5: R1 = β-OH, R2 = R3 = R4 = R5 = R6 = H
6: R1 = β-OH, R2 = R3 = OH, R4 = R5 = R6 = H
7: R1 = R3 = R4 = R5 = R6 = H, R2 = OH
9: R1 = R2 = R3 = R5 = R6 = H, R4 = β-OH
10: R1 = R2 = R3 = R5 = R6 = H, R4 = α-OH
11: R1 = α-OH, R2 = R3 = R4 = R5 = R6 = H
12: R1 = R2 = R3 = R4 = R6 = H, R5 = OH
13: R1 = R2 = R3 = R4 = H, R5 = R6 = OH
14: R1 = α-OH, R5 = OH, R2 = R3 = R4 = R6 = H
R5
R3
R2
12
14
11
O
9
1
4
8
2
3
16
5 R6
O
15
6
R1
COOH
18
19
Fig. 1. Structures of compounds 1–14.
Fig. 2. Perspective drawing of the X-ray structure of 9.

B.-H. Chou et al. / Phytochemistry 70 (2009) 759–764
761
Table 1
Data of compounds showing significant results on an AP-1-mediated luciferase
reporter gene assaya.
Compounds
Luciferase activity
Compounds
Luciferase activity
2
3
4
5
7
8
2.38 0.18
1.88 0.39
2.69 0.92
2.39 0.64
5.46 1.53
2.66 0.99
9
11
12
Control
Dex
2.73 0.58
3.02 0.71
2.58 0.86
3.95 0.53
2.33 0.36
a
The concentration of each test compound was 10
l
M. All firefly luciferase
activities were normalized to Renilla luciferase activity. The data were expressed as
multiples of luciferase activity compared to the no-treatment (control) group.
Dexamethasone (Dex) was the reference compound as the positive control. Each
value is the average of the firefly/Renilla luciferase ratio and is presented as the
mean SEM (n = 3). *Significantly different at p < 0.05, using Student’s t-test for
paired samples.
Fig. 3. Perspective drawing of the X-ray structure of 11.
one new CH2 signal at d 69.4 and one quaternary carbon at d 77.4
when compared with 2. In the HMBC spectrum, two new proton
resonances at d 3.86 (d, J = 11.5 Hz) and 3.94 (d, J = 11.5 Hz) show-
ing connectivities to C-12 (d 30.5), C-13 (d 83.9), and C-14 (d 38.5)
suggested the presence of an OH at C-17. On the other hand, the
lack of a carbinol–methine resonance in the 1H NMR spectrum
and the disappearance of one tertiary carbon in the DEPT spectrum
suggested that another OH might have been introduced at C-5 or C-
9. In the HMBC spectrum, the resonance of dC 77.4 showed connec-
tivities with H-7 (dH 1.32), H-14 (dH 1.67), H-11 (dH 1.87), H-12 (dH
1.99), H-15 (dH 3.75), CH3-20 (dH 1.41), and OH (dH 5.57), thus con-
firming the presence of an OH at C-9. Thus, 13 was assigned to be
but also regio- and stereoselective hydroxylation. In addition, re-
sults showed that 2–5, 8, 9, 11, and 12 exhibited significant inhib-
itory activities toward AP-1 activation. In particular, 3 exhibited
greater inhibition than dexamethasone. Further work is ongoing
to investigate 3 functioning on other transcriptional factor-regu-
lated pathways, such as NF-jB and Sp-1. This investigation also
demonstrates that biohydroxylation represents a powerful tool
for the regio- and stereoselective introduction of hydroxyl groups
into organic compounds.
4. Experimental
4a-carboxy-9b,13a,17-trihydroxy-13,16-seco-ent-19-norbeyeran-
16-oic acid 13,16-lactone.
4.1. General
Compound 14 displayed a quasi-molecular ion peak at m/z
367.2132 [M+H]+ (calc. 367.2121) in the HRESIMS, corresponding
to the molecular formula of C20H31O6. The 1H, 13C NMR, and HSQC
spectra were similar to those of 12 except for new signals at dH
3.63 (1H, m) and dC 77.9. This indicated that 14 contains one more
oxygen atom than 12. The location of the OH was confirmed by a
detailed analysis of the HMBC data. The chemical shift of dH 3.63
showed connectivities with C-5 (dC 54.8), C-8 (dC 41.5), C-14 (dC
39.5), and C-15 (dC 32.4). Thus, hydroxylation had occurred at C-
Melting points were determined using a Yanagimoto micro-
melting point apparatus and are uncorrected. Optical rotations
were determined on a JASCO DIP-1020 digital polarimeter. 1H,
13C NMR, DEPT, and 2D-NMR (COSY, NOESY, HSQC, and HMBC)
spectra were recorded on a Bruker Avance-500 spectrometer.
Chemical shifts are reported in parts per million (ppm) with re-
spect to the corresponding solvent as the internal standard, and
coupling constants (J) are in Hertz (Hz). Low- and high-resolution
ESI mass spectra were recorded using a VG Platform Electrospray
ESI/MS spectrometer. X-ray single-crystal diffraction was mea-
sured on a Siemens SMART CCD XRD. Column chromatography
(CC) was performed with Kieselgel silica (70–230 and 230–400
mesh, Merck, Darmstadt, Germany). HPLC was performed on a Hit-
achi L-2130 apparatus equipped with a refractometer detector (L-
2490). Purification by means of HPLC was conducted using a Beta-
7. An
a-orientation of the OH at C-7 was suggested from the
cross-peaks of dH 3.63 with H-5 (dH 1.31), H-6 (dH 2.46-2.52),
H-9b (dH 1.07-1.14), and H-14 (dH 2.91), whereas no effect was
observed between dH 3.63 and 1.16 (CH3-20) in the NOESY exper-
iment. Thus, 14 was established to be 4a-carboxy-7a,13a,17-trihy-
droxy-13,16-seco-ent-19-norbeyeran-16-oic acid 13,16-lactone.
Subsequently, the inhibitory effects on AP-1 activation in LPS-
stimulated RAW 264.7 macrophages of all of these compounds
were evaluated to search for potential novel immunoinflammatory
agents. Results of evaluation of inhibitory effects using an
AP-1-mediated luciferase reporter gene assay established that
compounds 2–5, 8, 9, 11, and 12 showed significant suppression
sil Silica-100 column (250 ꢀ 10 mm, 5
lm, at a flow rate of 2 mL/
min) (Thermo Scientific, Waltham, MA, USA). TLC plates (Si 60 with
F254) were purchased from Merck (Darmstadt, Germany). All spots
were detected by spraying with 10% H2SO4, followed by heating.
of the expression of AP-1 target genes, with
greater inhibition than the reference compound of dexamethasone
(Table 1).
3 exhibiting
4.2. Substrate 2
Isosteviol lactone (2) was prepared as previously reported
(Braguini et al., 2003).
3. Conclusions
4.3. Microorganisms
Thirteen compounds, 1 and 3–14, were obtained from the pre-
parative-scale fermentation of 2 with M. recurvatus MR 36, Asp. ni-
ger BCRC 31130, and Abs. pseudocylindrospora ATCC 24169.
Compounds 9–14 have not previously been reported. The results
indicated that these three selected fungal strains also possess the
characteristics of reaction selectivity on the ent-beyerane skeleton
as we previously reported (Chang et al., 2008; Chou et al., 2008).
The reactions involved not only isomerization of the lactone ring
Fungal cultures of M. recurvatus MR 36, Asp. niger BCRC 31130,
and Abs. pseudocylindrospora ATCC 24169 were obtained from the
Division of Medicinal and Natural Products Chemistry, College of
Pharmacy, University of Iowa, Iowa City, IA, USA, and Bioresources
Collection and Research Center, Hsinchu, Taiwan. These fungi were
maintained on Sabouraud-maltose and Czapek’s agar slants, and
stored in a refrigerator at 4 °C.

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B.-H. Chou et al. / Phytochemistry 70 (2009) 759–764
4.4. Preparation of medium
4.5.2. X-ray diffraction analysis of 9
C20H30O5, M 350.44, monoclinic, C2, a 44.8056 (11) Å, b 7.2959
(2) Å, c 11.8141 (3) Å, V 3825.76 (17) Å3; Z 8, Dcalc 1.217 g cmꢂ3
F(0 0 0) 1520, k (Mo K ) 0.71073 Å, T 295(2) K, 15643 reflection
collected. Final GooF 0.958, final R indices R1 0.0574, wR2 0.1375,
478 parameters,
0.086 mmꢂ1, R indices based on 8427 reflections
with I > 2 (I) absorption corrections applied. Complete crystallo-
Fungal fermentations were carried out in medium as described
previously (Chang et al., 2008). The pH of the medium was ad-
justed to 7.0 with 6 N HCl before sterilization by autoclaving at
121 °C for 15 min.
,
a
l
r
graphic data of 9 were deposited in the Cambridge Crystallographic
Data Centre (CCDC 706276). These data can be obtained free of
charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or
from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44
1223 336 033; e-mail: data_request@ccdc.cam.ac.uk).
4.5. Biotransformation of isosteviol lactone (2) by M. recurvatus, Asp.
niger, and Abs. pseudocylindrospora
Using 24-h-old stage II cultures, a solution of 2 (1.06 g dissolved
in 10.6 mL DMF) was evenly distributed among 50 flasks contain-
ing stage II cultures. Substrate-containing cultures were incubated
for 144 h. Extraction as previously described (Chang et al., 2008)
produced 4.2 g of brown oil, 3.1 g of black oil, and 4.7 g of brown
oil after respective bioconversions with M. recurvatus, Asp. niger,
and Abs. pseudocylindrospora. The crude residue from M. recurvatus
(4.2 g) was subjected to CC over silica gel (70–230 mesh,
5 ꢀ 90 cm). In total, four fractions (1–4) were obtained by eluting
with mixtures of CH2Cl2–EtOH (600 mL each of 20:1, 15:1, and
10:1). The elutes were monitored using TLC. The fractions were
combined on the basis of similar TLC profiles. Further chromatog-
raphy of fraction 2 (910 mg) over silica gel (230–400 mesh,
3 ꢀ 50 cm) eluted with n-hexane–EtOAc (1:1) yielded three frac-
tions (2-1 to 2-3), and 760 mg of 2 was recovered from fraction
2-1. Fractions 2-2 (9 mg) and 2-3 (90 mg) were recrystallized with
EtOAc to give 4.5 mg of 3 and 80 mg of 4 as white prisms and white
needles, respectively. Fraction 3 (602 mg) was applied to a silica
gel column (230–400 mesh, 3 ꢀ 55 cm) eluted with n-hexane–
EtOAc (1:2) to give three fractions (3-1 to 3-3). Fraction 3-1
(570 mg) was recrystallized with n-hexane–EtOAc (1:2) to give 1
(66 mg). The mother liquid of fraction 3-1 was evaporated in vac-
uum and then subjected to CC over silica gel eluted with n-hex-
ane–EtOAc (1:2) to give 3 (490 mg). After recrystallization of
fraction 3-3 (15 mg) with EtOAc, 5 mg of 5 was obtained as white
prisms. The crude residue from Asp. niger (3.1 g) was subjected to
CC over silica gel (70–230 mesh, 4 ꢀ 60 cm). In total, 5 fractions
(1–5) were obtained by eluting with mixtures of CH2Cl2–MeOH
(500 mL each of 20:1, 15:1, and 10:1). With further chromatogra-
4.5.3. Compound 10
White powder; ½a 2D5
ꢁ
–76.8 (c 0.5, MeOH); for 1H and 13C NMR
spectra, see Tables 2 and 3; HRESIMS m/z 351.2173 [M+H]+
(C20H31O5, calc. 351.2171).
4.5.4. Compound 11
White crystals, m.p. > 300 °C; ½a 2D5
ꢁ
–10.4 (c 0.5, MeOH); for 1H
and 13C NMR spectra, see Tables 2 and 3; HRESIMS m/z 351.2178
[M+H]+ (C20H31O5, calc. 351.2171).
4.5.5. X-ray diffraction analysis of 11
C20H30O5, M 350.44, orthorhombic, P212121, a 11.1996 (2) Å, b
12.2087 (2) Å, c 13.4538 (2) Å, V 1839.57 (5) Å3;
Z
4, Dcalc
) 0.71073 Å, T 295(2) K,
12063 reflection collected. Final GooF 1.026, final R indices R1
0.0443, wR2 0.1200, 227 parameters,
0.089 mmꢂ1, R indices
based on 4156 reflections with I > 2 (I) absorption corrections ap-
1.265 g cmꢂ3, F(0 0 0) 760, k (Mo K
a
l
r
plied. Complete crystallographic data of 11 were deposited in the
Cambridge Crystallographic Data Centre (CCDC 706277). These
data can be obtained free of charge via http://www.ccdc.cam.a-
c.uk/conts/retrieving.html, or from the CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK (fax: +44 1223 336 033; e-mail:
data_request@ccdc.cam.ac.uk).
4.5.6. Compound 12
White powder; ½a 2D5
ꢁ
–7.2 (c 0.5, MeOH); for 1H and 13C NMR
spectra, see Tables 2 and 3; HRESIMS m/z 351.2193 [M+H]+
phy of fraction
2 (360 mg) over silica gel (230–400 mesh,
(C20H31O5, calc. 351.2171).
3 ꢀ 55 cm) eluted with n-hexane–EtOAc (150 mL each of 3:1, 2:1,
and 1:1), 1 (60 mg) was obtained. After recrystallization of frac-
tions 3 and 4 with EtOAc and MeOH, 7 (17 mg) and 5 (21 mg) were
respectively obtained. The mother liquid of fraction 4 (650 mg)
was evaporated in vacuum and then applied to a silica gel column
(230–400 mesh, 2 ꢀ 55 cm) eluted with n-hexane–EtOAc (2:1) to
yield 3. After recrystallization with EtOAc–MeOH, 500 mg of 3
was obtained as white crystals. Fraction 5 (32 mg) was purified
by semi-preparative HPLC (CH2Cl2–isopropanol, 12:1) to give 8
(10 mg) and 9 (6 mg). The crude residue from Abs. pseudocylindros-
pora (4.7 g) was purified by CC over silica gel using mixtures of
n-hexane, EtOAc, CH2Cl2, and MeOH with increasing polarity to
obtain five fractions (1–5). Fraction 3 (321 mg) was subjected to
CC over silica gel eluted with n-hexane–EtOAc to yield a white so-
lid (279 mg). The white solid was further purified by HPLC on a
semi-preparative column (CH2Cl2–isopropanol, 12:1) to give 6
(80 mg), 9 (90 mg), 10 (8 mg), 11 (68 mg), and 12 (2.5 mg). Fraction
4 (90 mg) was subjected to repeated semi-preparative HPLC sepa-
ration (CH2Cl2–isopropanol, 12:1) to give 13 (8 mg) and 14
(16 mg).
4.5.7. Compound 13
White powder; ½a 2D5
ꢁ
–34.0 (c 0.5, MeOH); for 1H and 13C NMR
spectra, see Tables 2 and 3; HRESIMS m/z 389.1955 [M+Na]+
(C20H30O6Na, calc. 389.1940).
4.5.8. Compound 14
White powder; ½a 2D5
ꢁ
–52.4 (c 0.5, MeOH); for 1H and 13C NMR
spectra, see Tables 2 and 3; HRESIMS m/z 367.2132 [M+H]+
(C20H31O6, calc. 367.2121).
4.6. Cell culture, transfection, and reporter gene assays
Twenty-four hours before transfection, about 1 ꢀ 105 mouse
RAW 264.7 macrophages per well were seeded in 96-well white
plates. The reporter plasmid, pAP-1-Luc plasmid, and an internal
control plasmid, pGL-hRluc, were transfected into RAW 264.7 cells
using lipofectamine plus (Invitrogen, San Diego, CA, USA) accord-
ing to the manufacturer’s instructions. At 48 h post-transfection,
lipopolysaccharide (LPS) from Escherichia coli (serotype 0111:B4)
(Sigma, St. Louis, MO, USA) at a final concentration of 100 ng/mL
was added to the transfected cells for 6 h. After LPS stimulation,
4.5.1. Compound 9
White crystals, m.p. > 300 °C; ½a 2D5
ꢁ
–34.8 (c 0.5, MeOH); for 1H
a final concentration of 10 lM of each test compound including
the reference compound, dexamethasone (Sigma, St. Louis, MO,
USA), in DMSO was added to the cells. Cells were harvested 24 h
and 13C NMR spectra, see Tables 2 and 3; HRESIMS m/z 351.2174
[M+H]+ (C20H31O5, calc. 351.2171).

B.-H. Chou et al. / Phytochemistry 70 (2009) 759–764
763
Table 2
1H NMR chemical shifts of compounds 9–14 (C5D5N, d values in ppm)a,b
.
Position
1
9
10
11
12
13
14
1.55-1.72c (m)
0.89 (td, J = 13.2, 4.0)
2.10-2.21c (m)
1.38 (m)
1.66 (d, J = 12.4)
0.82 (td, J = 13.2, 4.0)
2.08-2.18c (m)
1.37-1.45c (m)
2.39 (d, J = 13.2)
0.99-1.06c (m)
0.99-1.06c (m)
2.08-2.18c (m)
1.96 (m)
1.64 (d, J = 12.4)
0.80 (td, J = 13.2, 4.4)
2.16 (qt, J = 13.2, 3.6)
1.43 (m)
1.69 (br d, J = 13.5)
0.85 (td, J = 13.0, 3.5)
2.14-2.26c (m)
1.48-1.64c (m)
2.46 (d, J = 13.0)
1.06-1.08c (m)
1.06-1.08c (m)
2.14-2.26c (m)
1.95-1.98c (m)
1.48-1.64c (m)
1.24-1.32c (m)
0.96 (dd, J = 12.5, 2.5)
1.48-1.64c (m)
1.24-1.32c (m)
2.03 (d, J = 13.0)
1.48-1.64c (m)
1.95-1.98c (m)
1.48-1.64c (m)
3.30 (dd, J = 18.5, 2.0)
2.14-2.26c (m)
3.91 (d, J = 11.5)
3.81 (d, J = 11.5)
1.36 (s)
2.17 (m)
1.62-1.73c (m)
1.51 (d, J = 12.0)
2.24-2.44c (m)
1.56 (d, J = 13.5)
2.24-2.44c (m)
1.12 (td, J = 13.0, 4.0)
2.24-2.44c (m)
2.24-2.44c (m)
2.06 (d, J = 14.0)
2.21 (dd, J = 13.5, 3.0)
1.32 (m)
0.87 (td, J = 13.0, 3.5)
2.23 (m)
2
3
1.49-1.57c (m)
2.46-2.52c (m)
1.07-1.14c (m)
1.31 (m)
2.36 (d, J = 12.8)
0.99 (td, J = 13.2, 4.0)
1.10 (m)
2.36-2.45c (m)
1.02 (dd, J = 13.2, 4.0)
1.24 (m)
5b
6
2.10-2.21c (m)
1.92 (m)
2.36-2.45c (m)
2.46-2.52c (m)
7
1.49 (m)
1.51-1.56c (m)
1.16 (td, J = 13.4, 3.6)
0.99-1.06c (m)
2.08-2.18c (m)
1.51-1.56c (m)
b 3.68 (dd, J = 11.2, 4.8)
b 3.53 (dd, J = 8.4, 6.8)
b 3.63 (m)
1.25 (dd, J = 13.6, 3.6)
1.55-1.72c (m)
1.83 (dt, J = 14.0, 2.4)
1.55-1.72c (m)
9b
11
0.92 (dd, J = 11.6, 3.6)
1.56 (m)
OH 5.57 (s)
1.87 (2H, m)
1.07-1.14c (m)
1.62-1.73c (m)
1.49-1.57c (m)
2.09 (d, J = 13.0)
1.62-1.73c (m)
2.91 (dd, J = 13.5, 2.0)
1.49-1.57c (m)
3.47 (d, J = 18.5)
3.12 (d, J = 18.5)
3.95 (d, J = 11.5)
3.84 (d, J = 11.5)
1.36 (s)
1.32-1.39c (m)
1.85 (m)
12
14
15
17
a
4.01 (br s)
2.24-2.44c (m)
1.99 (d, J = 13.0)
2.71 (dd, J = 13.0, 2.5)
1.67 (d, J = 13.0)
3.75 (dd, J = 18.5, 2.5)
2.24-2.44c (m)
3.94 (d, J = 11.5)
3.86 (d, J = 11.5)
1.37 (s)
1.32-1.39c (m)
2.36-2.45c (m)
1.32-1.39c (m)
3.33 (d, J = 18.8)
2.99 (dd, J = 18.8, 2.4)
1.28 (s)
2.01 (dd, J = 13.6, 2.8)
1.32 (m)
1.51-1.56c (m)
1.37-1.45c (m)
3.25 (dd, J = 18.4, 3.5)
2.08-2.18c (m)
1.58 (s)
2.10-2.21c (m)
1.52 (s)
18-CH3
20-CH3
1.30 (s)
1.07 (s)
1.32 (s)
1.03 (s)
1.31 (s)
1.09 (s)
1.10 (s)
1.41 (s)
1.16 (s)
a
Assignments based on DEPT, HSQC, and HMBC.
Signal multiplicity and coupling constants (Hz) are in parentheses.
Overlapping signals.
b
c
Table 3
Iowa City, IA, for kindly providing the bacterial strains. We also
thank Mr. Yi-Hung Liu, Instrumentation Center of National Taiwan
University, for conducting of the X-ray crystallography. This re-
search was supported by grants, NSC96-2320-B-038-013 and
NSC97-2320-B-038-009, from the National Science Council of
Taiwan.
13C NMR chemical shifts of compounds 9–14 (C5D5N, d values in ppm)a.
No.
9
10
40.0
11
39.9
12
40.6
13
33.1
14
40.5
1
2
3
4
5
6
7
8
39.8
19.5
38.4
43.8
57.2
20.2
43.4
35.2
48.8
37.7
26.9
71.7
82.3
42.5
39.4
19.5
38.4
43.8
56.8
20.1
42.9
35.2
54.7
38.1
28.7
75.8
82.6
46.7
39.3
171.9
24.4
29.2
179.7
14.0
19.5
38.3
43.6
54.2
30.1
77.2
41.2
54.6
38.2
18.6
38.5
79.4
43.6
31.5
173.4
28.5
29.1
179.7
13.9
20.1
39.0
44.3
57.6
20.7
44.3
35.4
56.8
38.7
18.9
34.5
83.5
43.4
40.0
172.7
69.3
29.7
180.4
14.4
20.2
39.0
44.6
49.5
20.8
38.5
39.9
77.4
44.2
24.3
30.5
83.9
38.5
42.3
172.9
69.4
30.0
180.8
17.3
20.1
38.9
44.2
54.8
30.7
77.9
41.5
55.9
38.8
18.7
34.4
83.2
39.5
32.4
174.3
69.5
29.7
180.3
14.5
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