Paraminabeolides A-F, cytotoxic and anti-inflammatory marine withanolides from the soft coral Paraminabea acronocephala

Article abstract of DOI:10.1021/np2000705

(Chemical Equation Presented) Six new withanolides, paraminabeolides A-F (1-6), along with five known compounds, minabeolides-1, -2, -4, -5, and -8 (7-11), were isolated from a Formosan soft coral, Paraminabea acronocephala. The structures of these compounds were elucidated by extensive spectroscopic analysis and chemical transformation. The absolute configuration of 4 was determined by the application of Mosher's method. Compounds 1 and 7 were cytotoxic toward Hep G2 cancer cells. Compounds 1-4 and 7-10 were found to significantly inhibit the accumulation of the pro-inflammatory iNOS protein. Compounds 7-10 also could effectively reduce the expression of COX-2 protein.

Full text of DOI:10.1021/np2000705

ARTICLE
pubs.acs.org/jnp
Paraminabeolides AꢀF, Cytotoxic and Anti-inflammatory Marine
Withanolides from the Soft Coral Paraminabea acronocephala
Chih-Hua Chao,† Kuei-Ju Chou,† Zhi-Hong Wen,†,‡ Guey-Horng Wang,§ Yang-Chang Wu,^
Chang-Feng Dai,|| and Jyh-Horng Sheu*,†,‡
†Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan, Republic of China
‡Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan, Republic of China
§Department of Cosmetic Science, Chia-Nan University of Pharmacy and Science, Tainan 717, Taiwan, Republic of China
^College of Chinese Medicine, China Medical University, Taichung 404, Taiwan, Republic of China
Institute of Oceanography, National Taiwan University, Taipei, 106, Taiwan, Republic of China
S Supporting Information
b
ABSTRACT:
Six new withanolides, paraminabeolides AꢀF (1ꢀ6), along with five known compounds, minabeolides-1, -2, -4, -5, and -8 (7ꢀ11),
were isolated from a Formosan soft coral, Paraminabea acronocephala. The structures of these compounds were elucidated by
extensive spectroscopic analysis and chemical transformation. The absolute configuration of 4 was determined by the application of
Mosher’s method. Compounds 1 and 7 were cytotoxic toward Hep G2 cancer cells. Compounds 1ꢀ4 and 7ꢀ10 were found to
significantly inhibit the accumulation of the pro-inflammatory iNOS protein. Compounds 7ꢀ10 also could effectively reduce the
expression of COX-2 protein.
ithanolides are a group of naturally occurring steroidal
lactones, in which a C-22/C-26 δ-lactone is generally
up-regulation of the pro-inflammatory iNOS (inducible nitric
oxide synthase) and COX-2 (cyclooxygenase-2) proteins in LPS
(lipopolysaccharide)-stimulated RAW264.7 macrophage cells
were evaluated.
W
present in the side chain, although in some cases a C-23/C26
γ-lactone might occur. Withaferin A,1 isolated from the leaves of
Withania somnifera, was discovered as the first member of this
group. Withanolides have received considerable attention due to
their versatile biological activities, such as antitumor,2 cytotoxic,3
immunosuppresive,3,4 antimicrobial,5 anti-inflammatory,6 and
chemopreventive7 activities. The minabeolides, isolated from
the soft coral Minabea sp.,8 represent the first class of marine
withanolides. Although they were discovered more than two
decades ago, no biological activities have been reported for the
minabeolides. This prompted us to investigate new structures
and the biological activities for compounds of this class from
marine organisms. In the course of this study, six new with-
anolides (1ꢀ6) and five known compounds, minabeolides-1, -2,
-4, -5, and -8 (7ꢀ11), were isolated from the Formosan soft coral
Paraminabea acronocephala. The new structures were established
by extensive spectroscopic analysis. The absolute configuration
at C-22 of 4 was determined by the application of Mosher’s
method. The cytotoxicity of compounds 1ꢀ11 against human
liver carcinoma (HepG2 and HepG3), human breast carcinoma
(MCF-7 and MDA-MB-231), and human lung carcinoma
(A-549) cell lines and the ability of 1ꢀ5 and 7ꢀ11 to inhibit
’ RESULTS AND DISCUSSION
The ethanolic extract of the soft coral P. acronocephala was
partitioned between EtOAc and H2O to afford the EtOAc-
soluble fraction, which was subjected to silica gel column
chromatography. The fractions containing steroids were selected
1
on the basis of characteristic peaks of methyl groups in the H
NMR spectra. These fractions were subsequently subjected to a
series of chromatographic separations to afford six new with-
anolides, paraminabeolides AꢀF (1ꢀ6), and five known com-
pounds (7ꢀ11).8 The single-crystal X-ray diffractions analysis
(Figure 1)9 for both known compounds 9 and 11 further
confirmed the structures of these two metabolites.
The HRESIMS spectrum of paraminabeolide A (1) exhibited
a pseudomolecular ion peak at m/z 459.2509 [M þ Na]þ,
consistent with a molecular formula of C28H36O4, appropriate
for 11 degrees of unsaturation. The 13C NMR and DEPT
Received: January 25, 2011
Published: March 22, 2011
r
Copyright
2011 American Chemical Society and
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Figure 1. X-ray ORTEP drawings of compounds 9 and 11.
are close to those of 7, suggesting that both compounds have
the same configuration at C-22. This was further evidenced by
a comparison of the CD data of 1 with those of parasorbic acid
and related analogues.11 The CD spectrum of 1 exhibited a
positive Cotton effect at 248 nm, indicating that 1 should have a
22R configuration.11
HRESIMS analysis of paraminabeolide B (2) provided a
molecular formula of C30H42O5. The NMR spectroscopic data
of 2 resembled those of minabeolide-2 (8), with differences in the
side chain moiety. The appearance of an IR absorption band at
1736 cmꢀ1, two doublets of secondary methyls (δ 1.02 and 1.22)
in the 1H NMR spectrum (Table 2), and the carbon signals from
C-22 to C-28 (Table 1) suggested the presence of a saturated
δ-lactone in 2. The relative configuration of C-20/C-22 was
elucidated on the basis of analysis of the 3JH,H coupling constants
and NOE correlations. As illustrated in Figure 2, a small coupling
constant (3.2 Hz) between H-20 and H-22 suggested a gauche
conformation for both protons. Furthermore, NOE correlations
between H3-21/H-23ax, H-17/H-23eq, H2-16/H-22, and H-22/
H-20 suggested a 20S,22R configuration, as revealed in Figure 2.
In the NOESY spectrum of 2, correlations between H-22/H3-28,
H-23ax/H-25, and H-24/H2-23 suggested that the two methyl
groups (H3-27 and H3-28) are cis to H-22, an orientation
discovered for the first time in natural withanolides.12 In order
to further confirm the above elucidation, unsaturated δ-lactone 8
was hydrogenated to yield the 1,2,4,5β,24,25-hexahydro deriva-
tive 8a (Scheme 1),13 of which the configurations at C-5 and the
lactone moiety were deduced by the analysis of NOE correlations
and comparison of CD data with known analogues. The two
lactonic methyls H3-27 and H3-28 in 8a were deduced as trans to
H-22 by the analysis of NOE correlations as shown in Figure 2.
The CD data of 8a showed negative Cotton effects at 290 and
215 nm,14 confirming the 5R and the 22R,24R,25R configura-
tions, respectively. Consequently, the side chain moiety in 2 was
determined as depicted.
spectroscopic data (Table 1) displayed 28 carbon signals,
including four methyls, seven methylenes, 10 methines, and
seven quaternary carbons. The IR spectrum revealed the pre-
sence of carbonyl (1705 and 1659 cmꢀ1) groups. The presence
of an aldehyde was evidenced by the downfield singlet of a proton
at δ 9.86 (Table 2), which correlates to a carbon signal at δ 205.9
in the HSQC spectrum. The carbon resonances at δ 186.3 (C),
155.3 (CH), 127.7 (CH), 124.1 (CH), and 168.4 (C) as well as
the proton resonances at δ 7.01 (1H, d, J = 10.4 Hz), 6.23 (1H, d,
J = 10.4 Hz), and 6.08 (1H, s) were characteristic signals of
steroids with a 1,4-dien-3-one moiety in ring A.10 In addition,
proton resonances atδ 4.35 (1H, dt, J = 13.6, 4.0Hz), 1.94 (3H, s),
and 1.88 (3H, s) and carbon resonances at δ 166.5 (C), 148.5
(C), 122.2 (C), 77.7 (CH), 20.5 (CH3), and 12.3 (CH3) were
characteristic of an R,β-unsaturated δ-lactone, as compared to
minabeolide-1 (7).8 The Hꢀ H COSY correlation between
H-1/H-2 and HMBC correlations from H3-19 to C-1, C-5, C-9,
and C-10 further confirmed the 1,4-dien-3-one moiety in ring A.
The HMBC correlations from H3-27 to C-24, C-25, and C-26 as
well as from H3-28 to C-23, C-24, and C-25 corroborated
the presence of an unsaturated δ-lactone moiety. A comparison
of the NMR spectroscopic data of 1 with those of 78 disclosed
that the methyl group attached at C-13 in 7 was replaced by
an aldehyde in 1. This was further supported by the more
downfield shift of C-13 (δ 59.5, C) and the HMBC correlation
from the aldehyde proton to this carbon. The coupling
constants and splitting patterns of the side chain moiety of 1
1
1
Analysis of the HREIMS and 13C NMR spectroscopic data of
paraminabeolide C (3) suggested a molecular formula of
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Table 1. 13C NMR Spectroscopic Data of Compounds 1ꢀ6
position
1,a δC, mult.
2,a δC, mult.
3,b δC, mult.
3,c δC, mult.
4,a δC, mult.
4,d δC, mult.
5,a δC, mult.
6,b δC, mult.
1
155.3, CH
127.7, CH
186.3, C
155.5, CH
127.6, CH
186.3, C
155.5, CH
127.6, CH
186.3, C
155.7, CH
127.8, CH
185.7, C
155.9, CH
127.5, CH
186.4, C
156.1, CH
127.7, CH
185.9, C
155.9, CH
127.5, CH
186.5, C
155.9, CH
127.5, CH
186.4, C
2
3
4
124.1, CH
168.4, C
124.0, CH
168.6, C
124.0, CH
168.7, C
124.2, CH
168.8, C
123.8, CH
169.3, C
124.0, CH
169.3, C
123.8, CH
169.3, C
123.9, CH
169.3, C
5
6
32.4, CH2
33.5, CH2
37.5, CH
52.0, CH
43.3, C
32.6, CH2
33.6, CH2
35.6, CH
52.3, CH
43.4, C
32.7, CH2
33.5, CH2
35.7, CH
52.0, CH
43.4, C
32.7, CH2
33.8, CH2
35.7, CH
52.5, CH
43.6, C
32.9, CH2
33.5, CH2
35.6, CH
52.1, CH
43.6, C
32.8, CH2
33.8, CH2
35.5, CH
52.5, CH
43.7, C
32.9, CH2
33.5, CH2
35.6, CH
52.0, CH
43.6, C
32.7, CH2
33.7, CH2
34.6, CH
52.2, CH
43.7, C
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
OAc
23.8, CH2
33.4, CH2
59.5, C
22.6, CH2
34.5, CH2
45.9, C
22.7, CH2
34.5, CH2
45.9, C
23.0, CH2
35.0, CH2
46.3, C
22.8, CH2
39.2, CH2
43.0, C
23.0, CH2
39.5, CH2
43.1, C
22.8, CH2
39.2, CH2
43.0, C
21.8, CH2
33.9, CH2
51.3, C
54.9, CH2
24.8, CH2
28.2, CH2
51.5, CH
205.9, CH
18.6, CH3
39.5, CH
13.3, CH3
77.7, CH
29.9, CH2
148.5, C
54.7, CH2
24.2, CH2
26.8, CH2
52.3, CH
62.0, CH2
18.7, CH3
39.5, CH
12.7, CH3
78.7, CH
28.8, CH2
30.2, CH
40.8, CH
175.1, C
54.3, CH2
24.3, CH2
27.5, CH2
52.5, CH
62.4, CH2
18.8, CH3
42.6, CH
13.6, CH3
76.1, CH
82.8, CH
42.6, CH
42.6, CH
178.9, C
54.6, CH2
24.5, CH2
28.1, CH2
53.3, CH
62.7, CH2
18.7, CH3
43.5, CH
14.4, CH3
75.9, CH
83.9, CH
43.4, CH
42.9, CH
179.1, C
54.6, CH2
24.5, CH2
27.8, CH2
51.6, CH
11.8, CH3
18.7, CH3
42.6, CH
13.4, CH3
70.4, CH
79.0, CH
37.0, CH
40.0, CH
179.2, C
54.9, CH2
24.7, CH2
28.3, CH2
52.2, CH
11.9, CH3
18.6, CH3
43.3, CH
13.9, CH3
69.6, CH
80.1, CH2
37.6, CH
40.2, CH
179.5, C
54.6, CH2
24.5, CH2
27.8, CH2
51.7, CH
11.9, CH3
18.7, CH3
42.8, CH
11.9, CH3
70.4, CH
79.3, CH
40.1, CH
42.2, CH
179.6, C
55.2, CH2
24.8, CH2
22.6, CH2
50.3, CH
172.5, C
18.8, CH3
28.8, CH
16.2, CH3
81.3, CH
84.8, CH
40.0, CH
42.9, CH
177.7, C
122.2, C
166.5, C
12.3, CH3
20.5, CH3
13.7, CH3
14.1, CH3
171.2, C
13.6, CH3
17.6, CH3
171.2, C
13.9, CH3
17.9, CH3
170.9, C
9.7, CH3
8.3, CH3
10.1, CH3
8.4, CH3
14.0, CH3
13.6, CH3
13.4, CH3
17.5, CH3
21.1, CH3
21.2, CH3
20.9, CH3
a Spectra were measured in CDCl3 (100 MHz). b Spectra were measured in CDCl3 (125 MHz). c Spectra were measured in pyridine-d5 (125 MHz).
d Spectra were measured in pyridine-d5 (100 MHz).
C30H42O6, representing one more oxygen atom than the molec-
ular formula of 2. Compounds 2 and 3 were found to have the
same substituent patterns in rings AꢀD, as concluded by
comparison of their 1D and 2D NMR spectroscopic data.
Furthermore, it was found that the chemical shift of the lactone
carbonylcarbon of3 was shifted downfield (Table 1) as compared
to that of 2, suggesting the presence of a five-membered lactone
ring in 37 rather than a six-membered lactone ring. This was
further confirmed by the IR absorption band at 1772 cmꢀ1. All of
the evidence indicated that compound 3 varied from 2 only in
their respective side chains. The C-22 to C-28 moiety of 3 was
found to be quite similar to that of subtrifloralactones F and G,
isolated previously from the plant Deprea subtriflora.7 Interpreta-
tion of the 2D NMR spectroscopic data of 3 confirmed the above
elucidation and thus established its planar structure.
The relative configuration of 3 was elucidated on the basis of
analysis of 3JH,H coupling constants and NOE correlations. In the
NOESY spectrum of 3, both H3-19 and H-18a show NOE
correlations with H-8, suggesting they are all β-oriented. In
addition, NOE correlations between H-12β/H-17, H-12R/H-17,
and H-14/H-17 revealed that the relative configuration for the
steroidal nucleus of 3 is the same as the known minabeolides
(7ꢀ11).8 A small coupling constant between H-20 and H-22
(2.0 Hz, CDCl3) suggested a gauche conformation of these two
protons (rotamer a, Figure 3). The 20S,22S configuration was
suggested according to the diagnostic NOE correlations between
H-20/22-OH, H3-21/22-OH, H-23/H3-21, H-17/H-23, and
H2-16/H-22 (Figure 3). In the same manner, a large 3JH,H value
(8.0 Hz) between H-22 and H-23 led to the establishment
of two possible rotamers, b (22S,23R) and c (22S,23S), as illus-
trated in Figure 3. Among these two rotamers, only rotamer b
coincidentally satisfies the NOE correlations between H-22/
H-24, H3-28/22-OH, and H-23/H-17. In addition, NOE cor-
relations between H-23/H-25 and H-23/H3-28 suggested the
R-orientation of H3-28 and the β-orientation of H3-27 (Figure 4).
Consequently, the 20S,22S,23R,24R,25R configuration was deter-
mined for 3.
Paraminabeolide D (4) was assigned a molecular formula of
C28H40O4, according to the HRESIMS and NMR spectroscopic
data (Tables 1 and 3). A comparison of spectroscopic data of 4
with those of 7 indicated that they differ only in the nature of the
side chains. In addition, the substituent pattern in the side chain
moiety of 4 was similar to that of 3, but with the differences in
the 13C shifts (C5D5N, Table 1) of the C22ꢀC28 moiety, such as
C-22 (69.6 for 4; 75.9 for 3), C-23 (80.1 for 4; 83.9 for 3), C-27
(10.1 for 4; 13.9 for 3), and C-28 (8.4 for 4; 17.9 for 3). The two
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1
Table 2. H NMR Spectroscopic Data of Compounds 1 and 2
methyl groups (C-27 and C-28) of the lactone ring in 4 displayed
carbon chemical shifts smaller than 10.1 ppm, revealing a cis
geometry of these two protons.15 Also, the large upfield shift (9.5
ppm, C5D5N, Table 1) of the C-28 signal in 4, relative to that in
3, might arise from the steric proximity of C-28 to both the C-27
and C-22 groups, as the C-27 signal in 4 was upfield-shifted by
only 3.8 ppm. Thus, both H3-27 and H3-28 were suggested to be
position
1, δH (J in Hz)a
2, δH (J in Hz)a
1
2
4
6
7.01, d (10.4)
6.23, d (10.4)
6.08, s
7.03, d (10.4)
6.24, d (10.4)
6.08, s
a: 2.45, m
a: 2.47, m
b: 2.36, m
a: 2.03, m
b: 2.37, m
a: 1.97, m
7
Scheme 1. Chemical Conversions of 8 and 4a
b: 1.04, m
1.58, m
b: 1.02, m
1.72, m
8
9
1.07, m
1.08, m
11
a: 1.80, m
a: 1.74, m
b: 1.60, m
a: 2.67, dt (13.2, 3.2)
b: 1.60, m
a: 2.41, m
12
b: 1.12, m
1.44, m
a: 1.90, m
b: 1.10, m
1.20, m
a: 1.70, m
14
15
b: 1.82, m
a: 2.02, m
b: 1.19, m
a: 1.77, m
16
b: 1.70, m
1.61, m
9.86, s
b: 1.55, m
1.21, m
a: 4.33, d (12.0)
17
18
b: 3.89, d (12.0)
1.24, s
2.09, m
1.05, d (6.8)
4.60, dt (12.4, 3.2)
a: 1.86, ddd (13.2, 13.2, 3.2)
19
20
21
22
23
1.14, s
1.94, m
1.02, d (7.2)
4.35, dt (13.6, 4.0)
a: 2.40, m
b: 1.93, m
b: 1.58, m
2.18, m
24
25
2.54, m
27
28
OAc
1.88, s
1.94,z s
1.22, d (7.2)
1.02, d (7.2)
2.10, s
a Conditions: (a) Pd/C, H2, 1 atm, pyridine; (b) Me2C(OMe)2, catalytic
p-TsOH, MeOH, rt; (c) Me2C(OMe)2, catalytic p-TsOH, MeOH, 60 ꢀC.
a Spectra were measured in CDCl3 (400 MHz).
Figure 2. Selected NOE correlations of C-20/C-22 rotamer of 2 (left) and the δ-lactone moieties of 2 and 8a (right).
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Figure 3. Selected NOE correlations (top) and Newman projections (bottom) for (a) C-20/C-22 and (b and c) C-22/C-23 rotamers of compound 3.
trans oriented to H-23 (Figure 4). This was further confirmed by
the comparison of NOE correlations for the side chain moieties
in both 3 and 4, as shown in Figure 4. The absolute configuration
at C-22 was determined by the application of Mosher’s
method.16 The (S)- and (R)-MTPA esters of 4 (4a and 4b,
respectively) were prepared using the corresponding (R)- and
(S)-MTPA chlorides, respectively. The determination of chemi-
cal shift differences for the protons neighboring C-22 led to the
assignment of the 22S configuration in 4 (Figure 4). Further-
more, the significant differences between the NOE correlations
of 3 and 4 could be observed for those between H-23/H-24 and
H-22/H3-28 in 4, in contrast to those between H-22/H-24 and
H-23/H3-28 in 3 (Figure 4). Accordingly, the 22S,23R,24S,25R
configuration was suggested for 4.
Paraminabeolide E (5) gave the same molecular formula as
that of 4, based on the interpretation of the HRESIMS and 13C
NMR spectroscopic data (Table 1). A comparison of NMR
spectroscopic data of 5 with those of 4 revealed that they possess
an epimeric lactone ring. Likewise, the trans location for the two
methyls, C-27 (δ 14.0) and C-28 (δ 13.6), in 5 was readily
deduced according to their more downfield chemical shifts, as
compared to the relevant data in 4 and in the literature.15 The 13C
NMR spectroscopic data for the lactone moiety in 5 were
different from those in 3, although the H3-27 and H3-28 are in
trans location in both compounds. The differences in relative
configurations for the lactone moieties between compounds 3
and 5 could be characterized by the analysis of their NOE
correlations. In the NOESY spectrum of 5, NOE correlations
between H-23/H3-27, H-23/H3-21, H-23/H-24, and H-22/H3-
28 suggested the β-orientations of H-22/H3-28 and the
R-orientations of H3-21, H-23, and H3-27 (Figure 4). Further-
more, the absolute configuration for compound 5 was deduced
by an unexpected but reasonable chemical transformation from 4
to 5 in an attempt to prepare the acetonide derivative of 4 by a
known reaction under acidic reaction conditions.17 The reaction
to obtain the desired compound 4c was unsuccessful at room
temperature (Scheme 1). By raising the reaction temperature to
60 ꢀC, we obtained an unexpected product, which is identical
to 5. Accordingly, compound 5 was determined as the C-25
epimer of 4.
The molecular formula of paraminabeolide F (6) was found to
be C28H36O5, as deduced from HRESIMS and 13C NMR data,
appropriate for 11 degrees of unsaturation. The IR absorption
bands showed the absence of hydroxy groups and the presence of
an ester carbonyl (1722 cmꢀ1) and a γ-lactone (1779 cmꢀ1).
The γ-lactone unit was confirmed by the carbon resonances at δ
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Figure 4. Comparison of NOE correlations for the side chain moieties of 3ꢀ5 and 1H NMR chemical shift differences of MTPA esters of 4.
84.8 (C-23, CH) and 177.7 (C-26, C) (Table 2). A comparison
of NMR spectroscopic data of 6 with those of 3ꢀ5 revealed that
6 has a similar steroidal nucleus to those in 3ꢀ5; however,
differences in the carbon resonances of C-18 (δ 172.5, C) and
C-22 (δ 81.3, C) in 6 disclosed the presence of a δ-lactone
linkage between these two carbons. This was further confirmed
by an IR absorption band at 1722 cmꢀ1. The presence of NOE
correlations between H3-19/H-8 and H-14/H-17 and the ab-
sence between H3-19/H-9 suggested a common configuration
for the steroidal nucleus (Figure 5). The 13C NMR shifts for the
methyl groups (H3-27 and H3-28) of the γ-lactone ring in 6 were
found to be consistent with those in 3, which was further
confirmed by an NOE correlation between H-23/H-25
(Figure 5). This suggested that the relative configuration of the
γ-lactone ring in 6 is the same as that in 3. A large coupling
constant (9.0 Hz) between H-22 and H-20 suggested the
pseudoaxial orientation for these two protons. In addition, the
presence of NOE correlations between H-17/H-20 and H3-21/
H-22 and the absence of correlations between H-22/H-20
suggested the 20S,22S configuration. The absolute configuration
at C-23 of 6 could be suggested to be the same as that of 3 by
biogenetic considerations.
selective cytotoxicity toward HepG2 cancer cells with IC50 values
of 8.0 and 5.2 μM, respectively, while 7 showed weak cytotoxicity
toward MCF-7 cancer cells with an IC50 value of 18.7 μM. Also, 2
showed weak cytotoxicity toward MDA-MB-231 and MCF-7
cancer cells with IC50 values of 19.3 and 14.9 μM, respectively.
We also investigated the inhibition of compounds 1ꢀ5 and
7ꢀ11 toward LPS-induced pro-inflammatory protein (iNOS
and COX-2) expression in RAW264.7 macrophage cells by
Western blot analysis (Figure 6). At a concentration of 10 μM,
compounds 1ꢀ4 and 7ꢀ10 significantly reduced the levels of
iNOS to 11.0 ( 7.7%, 7.3 ( 1.0%, 37.9 ( 9.9%, 43.4 ( 9.5%,
9.6 ( 1.9%, 45.7 ( 7.7%, 23.2 ( 4.6%, and 6.3 ( 1.5%, respe-
ctively, while compounds 7ꢀ10 significantly reduced the levels
of COX-2 to 18.3 ( 7.2%, 51.2 ( 11.5%, 22.4 ( 9.9%, and
31.3 ( 10.7%, respectively, in comparison with those of
control cells stimulated with LPS only (100% for both iNOS
and COX-2). However, a decrease of β-actin (66.5 ( 11.4%
relative to the control group) occurred at 10 μM of compound 1,
revealing that it may exhibit cytotoxicity against the tested
macrophage cells.
Withanolides with a saturated lactone ring in the side chain are
common in plants.7,12,13 The withanolides with a saturated
δ-lactone ring from plants have all been found to possess
24R,25β-dimethyl substituents. Some semisynthetic withanolides
with a saturated δ-lactone, prepared by hydrogenation of the
The cytotoxicities of compounds 1ꢀ11 against HepG2,
Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cells were
evaluated. The data revealed that compounds 1 and 7 showed
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Journal of Natural Products
ARTICLE
1
Table 3. H NMR Spectroscopic Data of Compounds 3ꢀ6
position
3, δH (J in Hz)a
3, δH (J in Hz)b
4, δH (J in Hz)c
4, δH (J in Hz)d
5, δH (J in Hz)c
6, δH (J in Hz)a
1
2
4
6
7.02, d (10.5)
6.23, dd (10.5, 2.0)
6.08, s
7.01, d (10.0)
6.42, dd (10.0, 2.0)
6.30, s
7.05, d (10.0)
6.23, dd (10.0, 1.6)
6.07, s
7.01, d (10.4)
6.43, dd (10.4, 1.6)
6.28, s
7.05, d (10.4)
6.23, dd (10.4, 2.0)
6.07, s
7.05, d (10.0)
6.24, dd (10.0, 2.0)
6.08, s
a: 2.46, td (13.0, 4.5)
b: 2.37, m
a: 1.94, m
b: 1.03, m
1.71, m
a: 2.28, m
a: 2.46, td (13.2, 4.8)
b: 2.36, dt (13.2, 1.8)
a: 1.93, m
a: 2.29 td (13.6, 4.4)
b: 2.19, dt (13.6, 2.4)
a: 1.70, m
a: 2.45, td (13.2, 4.4)
b: 2.35, dt (13.2, 2.0)
a: 1.93, m
a: 2.55, td (13.5, 4.5)
b: 2.37, dt (13.5, 3.5)
a: 2.02, m
b: 2.19, m
7
a: 1.68, m
b: 0.84, m
b: 1.03, m
b: 0.84, m
1.40, m
b: 1.03, m
b: 0.97, m
8
1.54, m
1.60, m
1.61, m
2.70, qd (11.0, 4.0)
1.07, m
9
1.10, m
0.93, td (11.0, 3.5)
1.62, m
1.05, m
0.86, m
1.04, m
11
a: 1.73, m
b: 1.58, m
a: 2.40, m
b: 1.18, m
1.25, m
1.69, m
a: 1.52, m
1.69, m
2.03, m
b: 1.49, m
a: 1.97, m
1.80, m
12
a: 2.47, dt (13.0, 3.0)
b: 1.09, m
1.02, m
a: 2.00, m
b: 1.25, m
1.10, m
a: 2.01, m
b: 1.26, m
1.08, m
a: 2.14, dt (13.5, 3.5)
b: 1.25 m
b: 1.10, m
0.80, m
14
15
1.08, m
a: 1.70, m
b: 1.16, m
a: 2.02, m
b: 1.50, m
1.67, m
a: 1.50, m
b: 1.01, m
a: 2.17, m
b: 1.56, m
1.88, m
a: 1.62, m
b: 1.12, m
a: 1.90, m
b: 1.36, m
1.63, m
a: 1.44, m
a: 1.61, m
b: 1.07, m
a: 1.79, m
b: 1.39, m
1.62, m
a: 1.83 m
b: 1.02, m
a: 2.05, m
b: 1.26 m
16
a: 1.80 m
b: 1.36, m
1.85, m
b: 1.61 m
17
18
1.79 m
a: 4.31, d (12.0)
b: 3.95, d (12.0)
1.23, s
a: 4.44, d (12.0)
b: 4.06, d (12.0)
1.06, s
0.75, s
0.67, s
0.75, s
19
1.23, s
1.08, s
1.22, s
1.30, s
20
1.79, m
2.19, m
1.77, m
2.10, m
1.76, m
2.23, m
21
1.15, d (6.5)
3.79, dd (8.0, 2.0)
3.97, t (8.0)
2.06, m
1.46, d (6.5)
4.12, m
1.08, d (6.8)
3.85, br d (9.6)
4.22, dd (9.6, 4.4)
2.61, m
1.31, d (6.4)
4.14, br d (9.6)
4.60, dd (9.6, 4.4)
2.83, m
1.10, d (6.8)
3.86, br d (9.6)
4.39, dd (9.6, 6.0)
2.29, m
1.11, d (6.5)
4.36, dd (9.0, 4.0)
4.10, dd (11.0, 4.0)
2.16, m
22
23
4.27, t (8.0)
2.32, m
24
25
2.16, m
2.32, m
2.72, m
2.91, m
2.26, m
2.24, m
27
1.25, d (7.0)
1.25 d (7.0)
2.11, s
1.27, d (6.5)
1.39, d (6.5)
2.02, s
1.16, d (7.2)
0.94, d (6.8)
1.17, d (6.8)
1.09, d (6.8)
1.27, d (7.2)
1.14, d (7.2)
1.28, d (6.5)
1.28, d (6.5)
28
OAc
OH
6.70, d (5.0)
a Spectra were measured in CDCl3 (500 MHz). b Spectra were measured in pyridine-d5 (500 MHz). c Spectra were measured in CDCl3 (400 MHz).
d Spectra were measured in pyridine-d5 (400 MHz).
corresponding compounds with an unsaturated δ-lactone using a
palladium catalyst, have 24β,25β-dimethyl substituents.13 Our
present study reports a novel saturated withanolide (2) where
the two methyl groups at C-24 and C-25 are both R-oriented.
Likewise, withanolides possessing a 24R,25β-dimethyl-γ-lactone
ring in the side chain, such as compounds 3 and 6, have been
discovered previously only from the plants Physalis philadelphic18
and Deprea subtriflora.7 Both compounds 4 (24β,25β-dimethyl-γ-
lactone) and 5 (24β,25R-dimethyl-γ-lactone) represent the first
withanolides that have a different configuration in the γ-lactone
ring compared to the common analogues (24R,25β-dimethyl-γ-
lactone).
NMR spectra were recorded on a Bruker AVANCE 300 FT-NMR (or
Varian 400 MR NMR/Varian Unity INOVA 500 FT-NMR) instrument
at 300 MHz (or 400/500 MHz) for H (referenced to TMS, δH 0.00
1
ppm, for both CDCl3 and C5D5N) and 75 MHz (or 100/125 MHz) for
13C (referenced to δC 77.0 for CDCl3 and to internal TMS at δC 0.0
ppm for C5D5N). ESIMS were recorded on a Bruker APEX II mass
spectrometer. Silica gel 60 (Merck, 230ꢀ400 mesh) and LiChroprep
RP-18 (Merck, 40ꢀ63 μm) were used for column chromatography.
Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) and
precoated RP-18 F254S plates (Merck, 1.05560) were used for TLC
analysis. High-performance liquid chromatography (HPLC) was per-
formed on a Hitachi L-7100 pump equipped with a Hitachi L-7400 UV
detector at 210 nm and a semipreparative reversed-phase column
(Merck, Hibar Purospher RP-18e, 5 μm, 250 ꢁ 10 mm).
Animal Material. The soft coral Paraminabea acronocephala was
collected by hand using scuba off the western coast of Pingtung county, in
May 2009, at a depth of 10 m, and was stored in a freezer until being
extracted. This soft coral was identified by one of the authors (C.-F.D.). A
voucher specimen (specimen no. 200905PA) was deposited in the
Department of Marine Biotechnology and Resources, National Sun Yat-
sen University.
’ EXPERIMENTAL SECTION
General Experimental Procedures. The melting points were
determined using a Fisher-Johns melting point apparatus. Optical
rotations were determined with a JASCO P1020 digital polarimeter.
The CD and IR spectra were obtained on a JASCO V-650, a JASCO
J-815, and a JASCO FT/IR-4100 spectrophotometer, respectively. The
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Journal of Natural Products
ARTICLE
Figure 5. Selected NOE correlations for compound 6.
Extraction and Isolation. The frozen bodies of the soft coral P.
acronocephala (3.8 kg fresh wt) were minced and extracted exhaustively
with EtOH(6 ꢁ 2 L). The organic extractwas concentratedtoanaqueous
suspension and was further partitioned between EtOAc and H2O. The
EtOAc extract (30 g) was fractionated by open column chromatography
on silica gel using n-hexaneꢀEtOAc and EtOAcꢀMeOH mixtures of
increasing polarity to yield 28 fractions. Fraction 21 (3.6 g), eluted with
n-hexaneꢀEtOAc (1:6), was further separated by silica gel column
chromatography with gradient elution (n-hexaneꢀacetone, 5:1 to 2:1)
to yield eight subfractions (21A to 21H). Subfraction 21E was separated
by RP-18 open column chromatography (MeOHꢀH2O, 50% to 100%)
to yield 11 subfractions (21E1 to 21E11). Subfraction 21E6 was subjected
to RP-18 HPLC (CH3CNꢀMeOHꢀH2O, 5:64:31) to obtain com-
pounds 3 (0.9 mg), 1 (1.8 mg), and 6 (0.6 mg). Subfraction 21E7 was
further fractionated by silica gel column chromatography using gradient
elution (n-hexaneꢀacetone, 6:1 to 3:1) to afford 10 subfractions (21E7A
to E21E7J). Subfraction 21E7G, which was identified to contain steroids
by the analysis of the 1H NMR spectrum, was subjected to RP-18 HPLC
(CH3CNꢀMeOHꢀH2O, 5:63:32) to afford compounds 5 (0.9 mg), 4
(1.9 mg), 2 (1.5 mg), 10 (15.6 mg), 8 (3.2 mg), and 11 (2.8 mg).
Compounds 9 (200 mg) and 7 (2.0 mg) were obtained from fraction 19
by repeated column chromatography over silica gel (n-hexaneꢀacetone,
8:1 to 5:1) and RP-18 HPLC (MeOHꢀH2O, 85:15).
1375, 1241, 1183, 1044, 1000 cmꢀ1
;
13C NMR and 1H NMR data, see
Tables 1 and 3; ESIMS m/z 521 [M þ Na]þ; HRESIMS m/z 521.2876
[M þ Na]þ (calcd for C30H42O6Na, 521.2879).
Paraminabeolide D (4): amorphous solid; [R]24 þ28 (c 0.09,
D
CHCl3); IR (KBr) νmax 3464, 2938, 2859, 1773, 1656, 1618, 1451,
1380, 1340, 1241, 1168, 1050 cmꢀ1 13C NMR and 1H NMR data, see
;
Tables 1 and 3; ESIMS m/z 463 [M þ Na]þ; HRESIMS m/z 463.2825
[M þ Na]þ (calcd for C28H40O4Na, 463.2824).
Paraminabeolide E (5): amorphous solid; [R]24 ꢀ33 (c 0.12,
D
CHCl3); IR (KBr) νmax 3455, 2938, 2859, 1776, 1658, 1613, 1454,
1380, 1294, 1241, 1201, 1171, 1041 cm ;
ꢀ1 13C NMR and 1H NMR data,
see Tables 1 and 3; ESIMS m/z 463 [M þ Na]þ; HRESIMS m/z
463.2822 [M þ Na]þ (calcd for C28H40O4Na, 463.2824).
Paraminabeolide F (6): amorphous solid; [R]24 þ70 (c 0.06,
D
CHCl3); IR (KBr) νmax 2957, 2932, 2876, 2855, 1779, 1722, 1662,
1621, 1455, 1369, 1294, 1240, 1131, 1011 cmꢀ1
;
13C NMR and H
1
NMR data, see Tables 1 and 3; ESIMS m/z 475 [M þ Na]þ; HRESIMS
m/z 475.2459 [M þ Na]þ (calcd for C28H36O5Na, 475.2460).
Minabeolide-1 (7): colorless oil; [R]24 þ35 (c 0.20, CHCl3).
Minabeolide-2 (8): colorless oil; [R]24DD þ45 (c 0.32, CHCl3).
Minabeolide-4 (9): colorless prism; mp 261ꢀ262 ꢀC; [R]24 þ18
D
(c 4.81, CHCl3).
Minabeolide-5 (10): colorless oil; [R]24D þ13 (c 1.56, CHCl3).
Minabeolide-8 (11): colorless prism; mp 260ꢀ261 ꢀC; [R]24D þ37
(c 0.28, CHCl3).
Paraminabeolide A (1): amorphous solid; [R]24 þ83 (c 0.18,
D
CHCl3); CD (1.1 ꢁ 10ꢀ4 M, MeOH) λmax (Δε) 270 (ꢀ1.74), 248
(þ6.96), and 232 (þ11.1); IR (KBr) νmax 2933, 2863, 1705, 1659, 1621,
Crystallographic Data and X-ray Structure Analysis of 9. A
suitable colorless crystal (0.5 ꢁ 0.3 ꢁ 0.3 mm3) of 9 was grown by slow
evaporation from a MeOHꢀacetoneꢀn-hexane (1:3:10) solution.
Diffraction intensity data were acquired with a CCD area detector with
graphite-monochromated Mo KR radiation (λ = 0.71073 Å). Crystal
data for 9: C27H38O3 (formula weight 410.57), approximate crystal size,
0.5 ꢁ 0.3 ꢁ 0.3 mm3, orthorhombic, space group, P212121 (#19), T =
298(2) K, a = 6.5336(5) Å, b = 14.5778(10) Å, c = 23.6095(16) Å, β =
90.00(3)ꢀ, V = 2248.7(3) Å3, Dc = 1.213 Mg/m3, Z = 4, F(000) = 896,
μ(Mo KR) = 0.077 mmꢀ1. A total of 8990 reflections were collected
in the range 2.22ꢀ < θ < 25.00ꢀ, with 3586 independent reflections
1440, 1388, 1291, 1147, 1120, 1027 cm ;
ꢀ1 13C NMR and 1H NMR data,
see Tables 1 and 2; ESIMS m/z 459 [M þ Na]þ; HRESIMS m/z
459.2509 [M þ Na]þ (calcd for C28H36O4Na, 459.2511).
Paraminabeolide B (2): amorphous solid; [R]24 þ8 (c 0.09,
D
CHCl3); IR (KBr) νmax 2936, 2873, 1736, 1662, 1609, 1454, 1370,
1240, 1183, 1084, 1039 cm ;
ꢀ1 13C NMR and 1H NMR data, see Tables 1
and 2; ESIMS m/z 505 [M þ Na]þ; HRESIMS m/z 505.2927 [M þ
Na]þ (calcd for C30H42O5Na, 505.2930).
Paraminabeolide C (3): amorphous solid; [R]24 þ13 (c 0.09,
D
CHCl3); IR (KBr) νmax 3456, 2936, 2873, 1772, 1738, 1662, 1454,
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Journal of Natural Products
ARTICLE
Hydrogenation of Compound 8. Compound 8 (5 mg) was
hydrogenated with 10 mg of 10% Pd/C in pyridine (1 mL) at room
temperature and atmospheric pressure for 24 h. After the catalyst was
removed by filtration, the filtrate was concentrated to give a residue,
which was chromatographed on silica gel using n-hexaneꢀEtOAc (3:1)
as eluent to afford 8a (4 mg). The relative configuration was established
by analysis of NOE correlations observed in an NOESY experiment. CD
(1.9 ꢁ 10ꢀ4 M, MeOH) λmax (Δε) 290 (ꢀ0.35) and 215 (ꢀ2.04); 1H
NMR (CDCl3, 400 MHz) of 8a, δ 4.48 (1H, br d, J = 12.0 Hz, H-22),
4.25 (1H, d, J = 12.0 Hz, H-18a), 3.87 (1H, d, J = 12.0 Hz, H-18b), 2.71
(1H, m, H-25), 2.65 (1H, m, H-4a), 2.43 (1H, br d, J = 12.8 Hz, H-12a),
2.30 (1H, m, H-2a), 2.22 (1H, m, H-24), 2.20 (1H, m, H-2b), 2.08 (3H,
s, OCOCH3), 2.04 (2H, m, H-4b and H-20), 2.00 (1H, m, H-1a), 1.89
(1H, m, H-6a), 1.85 (1H, m, H-5), 1.79 (2H, m, H-16a and H-23a), 1.70
(1H, H-15a), 1.54 (1H, m, H-8), 1.53 (2H, m, H2-7), 1.52 (1H, m,
H-16b), 1.51 (1H, m, H-9), 1.50 (1H, m, H-11a), 1.40 (1H, m, H-1b),
1.37 (1H, m, H-23b), 1.30 (2H, m, H-11b and H-14), 1.27 (1H, m,
H-6b), 1.26 (1H, m, H-17), 1.16 (3H, d, J = 6.8 Hz, H3-27), 1.15 (2H, m,
H-12b, and H-15b), 1.08 (3H, d, J = 6.4 Hz, H3-21), 1.03 (3H, s, H3-19),
0.94 (3H, d, J = 6.4 Hz, H3-28); 13C NMR (CDCl3, 100 MHz) of 8a, δ
213.0 (C, C-3), 176.2 (C, C-26), 171.2 (C, OCOCH3), 79.8 (CH,
C-22), 62.3 (CH2, C-18), 55.6 (CH, C-14), 52.7 (CH, C-17), 45.9 (C,
C-13), 43.9 (CH, C-5), 42.2 (CH2, C-4), 41.0 (CH, C-9), 39.2 (CH,
C-20), 38.2 (CH, C-25), 37.1 (CH2, C-2), 36.8 (CH2, C-1), 35.7 (CH,
C-8), 35.1 (CH2, C-12), 34.8 (C, C-10), 29.2 (CH, C-24), 27.2 (CH2,
C-23), 27.1 (CH2, C-16), 26.4 (CH2, C-6), 25.9 (CH2, C-7), 24.0 (CH2,
C-15), 22.6 (CH3, C-19), 21.1 (CH3, OCOCH3), 21.0 (CH2, C-11),
18.2 (CH3, C-28), 12.9 (CH3, C-21), 12.1 (CH3, C-27); ESIMS m/z
509 [M þ Na]þ.
Preparation of (S)- and (R)-MTPA Esters of 4. To a solution of
4 (0.5 mg) in pyridine (0.4 mL) was added (R)-MTPA chloride (25 μL),
and the mixture was allowed to stand for 3 h at room temperature. The
reaction was quenched by the addition of H2O (1.0 mL), and the
mixture was subsequently extracted with EtOAc (3 ꢁ 1.0 mL). The
EtOAc-soluble layers were combined, dried over anhydrous MgSO4, and
evaporated. The residue was subjected to short silica gel column
chromatography using n-hexaneꢀacetone (5:1) to yield the (S)-MTPA
ester, 4a (0.4 mg). The same procedure was used to prepare the (R)-
MTPA ester, 4b (0.5 mg from 0.5 mg of 4), with (S)-MTPA chloride.
Selected 1H NMR (CDCl3, 300 MHz) of 4a: δ 7.380ꢀ7.600 (5H, m,
Ph), 7.031 (1H, d, J = 10.0 Hz, H-1), 6.228 (1H, d, J = 10.0 Hz, H-2),
6.072 (1H, s, H-4), 5.200 (1H, br d, J = 9.8 Hz, H-22), 4.409 (1H, dd, J =
9.8, 4.0 Hz, H-23), 3.526 (3H, s, OMe), 1.223 (3H, s, H3-19), 1.141 (3H,
d, J = 7.1 Hz, H3-27), 0.966 (3H, d, J = 6.8 Hz, H3-21), 0.772 (3H, d, J =
6.8 Hz, H3-28), 0.713 (3H, s, H3-18); ESIMS m/z 679 [M þ Na]þ. 1H
NMR (CDCl3, 300 MHz) of 4b: δ 7.380ꢀ7.600 (5H, m, Ph), 7.050
(1H, d, J = 10.0 Hz, H-1), 6.245 (1H, d, J = 10.0 Hz, H-2), 6.091 (1H, s,
H-4), 5.199 (1H, br d, J = 10.2 Hz, H-22), 4.374 (1H, dd, J = 10.2, 3.9 Hz,
H-23), 3.546 (3H, s, OMe), 1.225 (3H, s, H3-19), 1.107 (3H, d, J = 7.1
Hz, H3-27), 1.016 (3H, d, J = 6.8 Hz, H3-21), 0.727 (3H, s, H3-18), 0.696
(3H, d, J = 7.1 Hz, H3-28); ESIMS m/z 679 [M þ Na]þ.
Figure 6. Effect of compounds 1ꢀ5 and 7ꢀ11 at 10 μM on the LPS-
induced pro-inflammatory iNOS and on COX-2 protein expression of
RAW264.7 macrophage cells by immunoblot analysis. (A) Quantifica-
tion of immunoblots of iNOS. (B) Quantification of immunoblots of
COX-2. The values are means ( SEM (n = 6). The relative intensity of
the LPS alone stimulated group was taken as 100%. *Significantly
a
different from LPS alone stimulated group (*p < 0.05). Stimulated
with LPS. bStimulated with LPS in the presence of 1ꢀ5 and 7ꢀ11 (10
μM). (C) Quantification of immunoblots of β-actin.
[R(int) = 0.0277]; completeness to θmax was 97.4%; psi-scan absorption
correction applied; full-matrix least-squares refinement on F2, the
number of data/restraints/parameters were 3586/0/275; goodness-
of-fit on F2 = 1.046; final R indices [I > 2σ(I)], R1 = 0.0370 wR2 =
0.0886; R indices (all data), R1 = 0.0385, wR2 = 0.0894, largest difference
peak and hole, 0.198 and ꢀ0.175 e/Å3.
Crystallographic Data and X-ray Structure Analysis of 11.
A suitable colorless crystal (0.6 ꢁ 0.5 ꢁ 0.4 mm3) of 11 was grown by
slow evaporation from a MeOHꢀacetoneꢀn-hexane (1:3:10) solution.
Diffraction intensity data were acquired with a CCD area detector with
graphite-monochromated Mo KR radiation (λ = 0.71073 Å). Crystal
data for 11: C29H42O5 (formula weight 470.65), approximate crystal
size, 0.6 ꢁ 0.5 ꢁ 0.4 mm3, monoclinic, space group, P21 (#4), T =
150(2) K, a = 13.846(4) Å, b = 6.1634(17) Å, c = 15.497(4) Å, β =
105.886(15)ꢀ, V = 1272.1(6) Å3, Dc = 1.229 Mg/m3, Z = 2, F(000) =
512, μ(Mo KR) = 0.082 mmꢀ1. A total of 8917 reflections were
collected in the range 1.37ꢀ < θ < 25.16ꢀ, with 3933 independent
reflections [R(int) = 0.0170]; completeness to θmax was 98.8%; psi-scan
absorption correction applied; full-matrix least-squares refinement on
F2, the number of data/restraints/parameters were 3933/1/312; good-
ness-of-fit on F2 = 1.065; final R indices [I > 2σ(I)], R1 = 0.0285 wR2 =
0.0692; R indices (all data), R1 = 0.0301, wR2 = 0.0725, largest difference
peak and hole, 0.241 and ꢀ0.153 e/Å3.
Cytotoxicity Testing. Cell lines were purchased from the Amer-
ican Type Culture Collection (ATCC). Cytotoxicity assays were
performed using the MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-
tetrazolium bromide] colorimetric method.19,20 Doxorubicin was em-
ployed as positive control, which exhibited cytotoxic activity toward
HepG2, Hep3B, MDA-MB-231, MCF-7, and A-549 cancer cell lines
with IC50 values of 0.5, 0.7, 2.2, 1.2, and 2.2 μM, respectively.
Compounds were considered to be inactive with IC50 values > 20
μM/mL.
In Vitro Anti-inflammatory Assay. Macrophage (RAW264.7)
cell line was purchased from ATCC. In vitro anti-inflammatory activity
of tested compounds was measured by examining the inhibition of
lipopolysaccharide (LPS)-induced upregulation of iNOS (inducible
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Journal of Natural Products
ARTICLE
nitric oxide synthetase) and COX-2 (cyclooxygenase-2) proteins in
macrophage cells using Western blotting analysis.21
(15) The 13C NMR chemical shifts for the cis- and trans-methyls in
γ-lactone compounds are summarized in Table S1, which can be found
in the Supporting Information.
(16) (a) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092–4096. (b) Randazzo, A.; Bifulco, G.;
Giannini, C.; Bucci, M.; Debitus, C.; Cirino, G.; Gomez-Paloma, L.
J. Am. Chem. Soc. 2001, 123, 10870–10876.
(17) F€urstner, A.; Nagano, T.; M€uller, C.; Seidel, G.; M€uller, O.
Chem. Eur. J. 2007, 13, 1452–1462.
(18) (a) Gu, J. -Q.; Li, W.; Kang, Y. -H.; Su, B. -N.; Fong, H. H. S.;
Van Breemen, R. B.; Pezzuto, J. M.; Kinghorn, A. D. Chem. Pharm. Bull.
2003, 51, 530–539. (b) Su, B.-N.; Misico, R.; Park, E. J.; Santarsiero,
B. D.; Mesecar, A. D.; Fong, H. H. S.; Pezzuto, J. M.; Kinghorn, A. D.
Tetrahedron 2002, 58, 3453–3466.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra for
S
b
1ꢀ6 and 8a and 13C NMR chemical shifts for the cis- and trans-
methyls in γ-lactone compounds from the literature. This
material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
(19) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.;
Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker,
R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589–601.
(20) Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.;
Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R.
Cancer Res. 1988, 48, 4827–4833.
Corresponding Author
*Tel: 886-7-5252000, ext. 5030. Fax: 886-7-5255020. E-mail:
sheu@mail.nsysu.edu.tw.
(21) Jean, Y.-H.; Chen, W.-F.; Sung, C.-S.; Duh, C.-Y.; Huang, S.-Y.;
Lin, C.-S.; Tai, M.-H.; Tzeng, S.-F.; Wen, Z.-H. Br. J. Pharmacol. 2009,
158, 713–725.
’ ACKNOWLEDGMENT
This work was supported by grants from the National Science
Council of Taiwan (NSC98-2113-M-110-002-MY3) and Minis-
try of Education (97C031702) awarded to J.-H.S.
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