Anti-inflammatory steroids from the octocoral Dendronephthya griffini

Article abstract of DOI:10.1016/j.tet.2008.01.109

Five new steroids, griffinisterones A-E (1-5), were isolated from the octocoral Dendronephthya griffini. The structures of these compounds were elucidated by extensive spectroscopic analysis. The single-crystal X-ray crystallography on 1 allowed the determination of the 24R configuration in 1. The absolute stereochemistry of 5 was determined by the application of PGME method. Compounds 3 and 4 were found to significantly inhibit the accumulation of the pro-inflammatory iNOS protein of the LPS-stimulated RAW264.7 macrophage cells.

Full text of DOI:10.1016/j.tet.2008.01.109

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 3554e3560
www.elsevier.com/locate/tet
Anti-inflammatory steroids from the octocoral Dendronephthya griffini
Chih-Hua Chao a, Zhi-Hong Wen a, I-Ming Chen a, Jui-Hsin Su a, Ho-Cheng Huang a,b,
Michael Y. Chiang c, Jyh-Horng Sheu a,d,
*
a Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan
b Department of Chemical and Materials Engineering, Cheng Shiu University, Kaohsiung 833, Taiwan
c Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan
d Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
Received 21 December 2007; received in revised form 28 January 2008; accepted 29 January 2008
Available online 2 February 2008
Abstract
Five new steroids, griffinisterones AeE (1e5), were isolated from the octocoral Dendronephthya griffini. The structures of these compounds
were elucidated by extensive spectroscopic analysis. The single-crystal X-ray crystallography on 1 allowed the determination of the 24R con-
figuration in 1. The absolute stereochemistry of 5 was determined by the application of PGME method. Compounds 3 and 4 were found to
significantly inhibit the accumulation of the pro-inflammatory iNOS protein of the LPS-stimulated RAW264.7 macrophage cells.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Octocoral; Steroid; Griffinisterones AeE; Anti-inflammatory effect
1. Introduction
2. Results and discussion
Previous chemical investigations on the octocoral of the
genus Dendronephthya have led to the isolation of structurally
unique steroids, of which some have been shown to exhibit
cytotoxic1,2 and antifouling3,4 activities. Our current chemical
investigation on Dendronephthya griffini has led to the isolation
of five new steroids, griffinisterones AeE (1e5) (Fig. 1). The
structures of 1e5 have been established by extensive spectro-
scopic analysis, including 2D NMR (1He1H COSY, HMQC,
HMBC, and NOESY) spectroscopy. In addition to the use of
2D NMR, the structures of 1 and its 24-epimer 2 were also iden-
tified by the assistance of a single-crystal X-ray crystallography
on 1. The absolute configuration at C-20 in 5 was determined by
the application of PGME method. The ability of 1e4 to inhibit
the expression of the pro-inflammatory iNOS (inducible nitric
oxide synthase) and COX-2 (cyclooxygenase-2) proteins in
LPS (lipopolysaccharide)-stimulated RAW264.7 macrophage
cells has been evaluated.
Griffinisterone A (1) was obtained as colorless crystals. The
HRESIMS of 1 exhibited a [MþH]þ peak at m/z 451.3189 and
established a molecular formula C28H44O3, implying seven
degrees of unsaturation. The 13C NMR and DEPT spectro-
scopic data (Table 1) displayed 28 carbon signals, including
five methyls, eight methylenes, eleven methines, and four qua-
ternary carbons. The IR spectrum of 1 revealed the presence of
hydroxy (nmax 3409 cmꢀ1) and carbonyl (nmax 1675 cmꢀ1
)
groups. The latter was identified as an a,b-unsaturated ketone
from the carbon resonances at dC 200.2 (qC), 158.4 (CH), and
127.4 (CH).2,4 The above data, coupled with the characteristic
methyl signals at dH 0.83 (3H, s), 1.02 (3H, s), 0.86 (3H, d,
J¼6.9 Hz), 0.87 (3H, d, J¼6.9 Hz), and 0.98 (3H, d,
J¼6.9 Hz) (Table 2), suggested that 1 might be an ergost-1-
en-3-one derivative.2,4,5 In addition, an E geometry of 1,2-di-
substituted double bond [dH 5.73 (dd, J¼15.6, 8.7 Hz), 5.53
(dd, J¼15.6, 8.1 Hz)] was also observed in 1H NMR spectrum
of 1. The above data accounted for three of the seven degrees
of unsaturation and revealed a tetracyclic nature of 1. The
gross structure of metabolite 1 was further established by the
* Corresponding author. Tel.: þ886 7 5252000x5030; fax: þ886 7 5255020.
E-mail address: sheu@mail.nsysu.edu.tw (J.-H. Sheu).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.109

C.-H. Chao et al. / Tetrahedron 64 (2008) 3554e3560
3555
OH
OH
OH
to C-12, C-13, C-14, and C-17. The detailed analysis of the
1He1H COSY and HMBC correlations (Fig. 2) further estab-
lished the planar structure of 1. The NOE correlations between
H-8 and both H3-18 and H3-19, and H3-18 and both H-20 and
H-21a further established the stereochemistry of the nucleus
as shown in the formula of 1. A single-crystal X-ray diffraction
analysis (Fig. 3)6 was used to confirm the above elucidation and
determine the stereochemistry of the side chain in 1. Thus, the
24R configuration of 1 was established.
1: R =
2: R =
3: R =
R
OH
H
H
H
O
H
Griffinisterone B (2) was found to possess the same molec-
ular formula C28H44O3 as that of 1 from the HRESIMS and
NMR spectroscopic data. The 13C NMR and 1H NMR spectro-
scopic data (Tables 1 and 2) of 2 were found to be quite similar
to those of 1, except for the C-28 carbon resonating at d 17.5
(CH3) in 1 and at d 18.1 (CH3) in 2, revealing the 24R/S
isomers of both compounds. Since the 24R configuration has
been assigned for 1, the 24S configuration was then deter-
mined for 2. On the basis of above findings and by NOE cor-
relations between H3-18 and H-20 as well as between H-8 and
both H3-18 and H3-19, the structure of 2 was fully established.
Griffinisterone C (3) was obtained as an amorphous solid.
The formula of 3 was found to be C27H42O3, 14 mass units
less than that of 1, as deduced from HRESIMS and NMR
HOOC
HOOC
R
4: R =
5: R =
H
H
H
H
O
H
Figure 1. Structures of metabolites 1e5.
2D NMR studies, particularly in He1H COSY, HMQC, and
HMBC experiments. The correlations of He1H COSY re-
vealed three separated proton sequences (aec), as depicted in
Figure 2. A tertiary hydroxy group attached at C-17 [dC 86.1
(qC)] was deduced from the HMBC correlations from H3-18
1
1
1
spectroscopic data. The H NMR spectrum of 3 was found
to be quite similar to that of 1 (Table 2), but with the absence
of C-28 methyl. The E geometry of 22,23-disubstituted double
bond and the presence of C-24 methylene were elucidated by
the coupling constants and multiplicity of H-23 proton (d 5.60,
1H, ddd, J¼15.4, 8.2, 6.8 Hz). Above data and further inspec-
tion of the 2D NMR correlations (Fig. 2) established the struc-
ture of this metabolite as revealed by formula 3.
Table 1
13C NMR spectroscopic data for compounds 1e5
Position 1a
2a
3a
4b
5b
1
158.4 (CH)c 158.4 (CH) 158.4 (CH) 158.3 (CH) 158.3 (CH)
127.4 (CH) 127.4 (CH) 127.4 (CH) 127.4 (CH) 127.4 (CH)
200.2 (qC) 200.2 (qC) 200.2 (qC) 200.2 (qC) 200.2 (qC)
41.0 (CH2) 41.0 (CH2) 41.0 (CH2) 40.9 (CH2) 40.9 (CH2)
2
Griffinisterone D (4) was isolated as an amorphous solid. The
HRESIMS of 4 established a molecular formula C28H42O3, im-
plying eight degrees of unsaturation. The comparison of NMR
spectroscopic data of 4 with those of dendronesterone B2 re-
vealed that 4 might be a derivative of dendronesterone B. The
C-21 carboxyl functionality in 4 was assigned on the basis of
the carbon resonance at d 181.7 (qC)7 and the IR absorption at
1699 cmꢀ1. The gross structure of 4 was further established
by the 2D NMR studies, particularly in 1He1H COSY,
3
4
5
44.2 (CH)
44.2 (CH) 44.2 (CH) 44.3 (CH)
44.3 (CH)
6
27.6 (CH2) 27.6 (CH2) 27.6 (CH2) 27.5 (CH2) 27.5 (CH2)
31.2 (CH2) 31.2 (CH2) 31.3 (CH2) 31.3 (CH2) 31.2 (CH2)
7
8
35.9 (CH)
49.5 (CH)
38.9 (qC)
35.9 (CH) 35.9 (CH) 35.6 (CH)
49.5 (CH) 49.5 (CH) 50.0 (CH)
35.6 (CH)
49.9 (CH)
38.9 (qC)
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
a
38.9 (qC)
38.9 (qC)
39.0 (qC)
21.1 (CH2) 21.1 (CH2) 21.1 (CH2) 21.2 (CH2) 21.2 (CH2)
32.0 (CH2) 32.0 (CH2) 32.0 (CH2) 37.5 (CH2) 37.5 (CH2)
1
HMQC, and HMBC experiments. The correlations of He1H
47.6 (qC)
50.8 (CH)
47.6 (qC)
50.8 (CH) 50.8 (CH) 55.9 (CH)
47.6 (qC)
42.3 (qC)
42.3 (qC)
55.7 (CH)
COSY revealed four separated proton sequences, as shown in
Figure 2. The HMBC correlations from H3-19 to C-1, C-5,
C-9, and C-10, and from H3-18 to C-12, C-13, C-14, and
C-17, as well as other correlations illustrated in Figure 2 estab-
lished the planar structure of 4. In the NOESY spectrum of 4
(Fig. 4), the NOE correlations between H-8 and both H3-18
and H3-19, and H-5 and H-9 revealed that the stereochemistry
of the nucleus is the same as that of dendronesterone B. Similar
to those found in 1e3, H3-18 was found to show NOE response
with H-20, but not with H-17, suggesting the a position of the
carboxyl functionality.
Griffinisterone E (5) was obtained as an amorphous solid,
which gave a [MþNa]þ peak at m/z 437.3027 in the HRESIMS.
Thus, a molecular formula of C27H42O3 was established. The
NMR spectra of 5 were almost identical with those of 4 except
that the NMR data of 5 showed the absence of an sp2 methylene
23.4 (CH2) 23.4 (CH2) 23.4 (CH2) 23.4 (CH2) 23.5 (CH2)
39.0 (CH2) 39.2 (CH2) 39.1 (CH2) 27.2 (CH2) 27.1 (CH2)
86.1 (qC)
86.1 (qC)
86.1 (qC)
52.5 (CH)
52.5 (CH)
14.9 (CH3) 14.9 (CH3) 14.9 (CH3) 12.3 (CH3) 12.3 (CH3)
13.0 (CH3) 13.0 (CH3) 13.0 (CH3) 13.0 (CH3) 13.0 (CH3)
50.0 (CH)
50.0 (CH) 49.9 (CH) 47.6 (CH)
47.0 (CH)
64.4 (CH2) 64.4 (CH2) 64.4 (CH2) 181.7 (qC) 181.2 (qC)
127.1 (CH) 127.3 (CH) 132.9 (CH) 30.8 (CH2) 32.1 (CH2)
138.5 (CH) 138.6 (CH) 129.4 (CH) 32.2 (CH2) 25.0 (CH2)
43.2 (CH)
32.9 (CH)
43.5 (CH) 42.1 (CH2) 155.3 (qC) 38.8 (CH2)
33.1 (CH) 28.4 (CH) 33.6 (CH) 27.8 (CH)
19.8 (CH3) 19.7 (CH3) 22.2 (CH3) 21.8 (CH3) 22.3 (CH3)
19.9 (CH3) 20.2 (CH3) 22.4 (CH3) 21.9 (CH3) 22.7 (CH3)
17.5 (CH3) 18.1 (CH3)
106.9 (CH2)
Spectra recorded at 75 MHz in CDCl3 at 25 ꢁC.
b
Spectra recorded at 125 MHz in CDCl3 at 25 ꢁC.
Multiplicity deduced by DEPT. The chemical shifts referenced to residual
signal of CDCl3 at d 77.0 ppm.
c

3556
C.-H. Chao et al. / Tetrahedron 64 (2008) 3554e3560
Table 2
1H NMR spectroscopic data for compounds 1e5
Position
1a
2a
3a
4b
5b
1
7.15 d (10.0)c
5.86 d (10.0)
2.37 dd (18.0, 14.0)
2.22 dd (18.0, 4.5)
1.94 m
7.15 d (10.0)
5.86 d (10.0)
2.37 dd (18.0, 14.0)
2.22 dd (18.0, 4.5)
1.94 m
7.15 d (10.0)
5.86 d (10.0)
2.37 dd (18.0, 14.0)
2.22 dd (18.0, 4.5)
1.94 m
7.07 d (10.0)
5.81 d (10.0)
2.36 dd (17.5, 14.0)
2.22 dd (17.5, 3.5)
1.88 m
7.08 d (10.0)
5.83 d (10.0)
2.36 dd (17.5, 14.5)
2.22 dd (17.5, 3.5)
1.91 m
2
4b
4a
5
6
1.42 m
1.73 m
1.42 m
1.73 m
1.42 m
1.73 m
1.42 m
1.72 m
1.42 m
1.72 m
7
1.01 m
1.50 m
1.01 m
1.50 m
1.02 m
1.50 m
0.94 m
1.45 m
0.96 m
1.45 m
8
9
11
1.06 m
1.84 m
1.06 m
1.84 m
1.05 m
1.84 m
0.96 m
1.68 m
0.98 m
1.70 m
1.48 m
1.80 m
1.48 m
1.80 m
1.49 m
1.80 m
1.38 m
1.79 m
1.40 m
1.74 m
12b
12a
14
1.72 m
1.79 m
1.72 m
1.80 m
1.72 m
1.81 m
1.15 m
1.09 m
1.17 m
1.10 m
15
1.65 m
1.09 m
1.65 m
1.10 m
1.70 m
1.13 m
1.62 m
1.10 m
1.64 m
1.10 m
16
1.83 m
1.47 m
1.83 m
1.48 m
1.86 m
1.50 m
1.90 m
1.30 m
1.89 m
1.30 m
17
18
1.67 m
0.76 s
1.67 m
0.76 s
0.83 s
1.02 s
0.82 s
1.02 s
0.83 s
1.02 s
19
20
0.98 s
2.25 m
0.99 s
2.26 br dd (7.5, 3.5)
2.33 m
4.01 m
2.32 m
4.01 m
2.36 m
4.02 m
21a
21b
22
3.79 m
5.73 dd (15.6, 8.7)
5.53 dd (15.6, 8.1)
3.77 m
5.75 dd (15.6, 9.3)
5.49 dd (15.6, 8.4)
3.78 m
5.79 dd (15.4, 8.7)
5.60 ddd (15.4,8.2, 6.8)
1.63 m
2.03 m
1.97 m
1.48 m
1.27 m
23
24
25
26
27
28
1.99 m
1.53 m
1.99 m
1.54 m
1.97 m
1.65 m
1.17 m
1.51 m
2.20 m
0.86 d (6.9)
0.87 d (6.9)
0.98 d (6.9)
0.86 d (6.9)
0.88 d (6.9)
0.98 d (6.9)
0.90 d (5.7)
0.90 d (5.7)
1.00 d (6.5)
1.01 d (6.5)
4.75 s
0.855 d (7.0)
0.862 d (7.0)
4.67 s
a
Spectra recorded at 300 MHz in CDCl3 at 25 ꢁC.
Spectra recorded at 500 MHz in CDCl3 at 25 ꢁC.
J values (in Hz) in parentheses. The chemical shifts referenced to TMS at d 0.0 ppm.
b
c
substituent at C-24 and revealed the association of two protons
with C-24 in 5. The careful inspection of the 2D NMR spectro-
scopic data of 5 has led to the establishment of its planar struc-
ture. The relative stereochemistry of compound 5 was also
established by comparison of NOESY correlations (Fig. 4)
with those of 4. To determine the absolute configuration at
C-20 for 5, it was treated with (S)- or (R)-PGME (phenylglycine
methyl ester) in the presence of 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide hydrochloride (EDC$HCl), 4-(dimethyl-
amino)pyridine (4-DMAP), and 4-(dimethylamino)pyridine
hydrochloride (4-DMAP$HCl)8 to yield the corresponding
(S)- or (R)-PGME amide (5a or 5b), respectively. The chemical
shift differences of (S)-PGME amide (5a) and (R)-PGME
amide (5b) were summarized in Figure 5 and established the
R-configuration at C-20.9,10
that compounds 1, 2, and 4 reduced the levels of the iNOS pro-
tein at this concentration to 49.7ꢂ8.6%, 48.9ꢂ16.2%, and
29.8ꢂ7.4%, respectively, relative to the control cells stimu-
lated with LPS. Moreover, at the same concentration com-
pound 3 showed more potent inhibition of iNOS as the
expression of this pro-inflammatory protein was reduced to
a level of 8.1ꢂ5.9% (Fig. 6). The toxicity of 1e4 to
RAW264.7 cells was also assessed by trypan blue staining.
It was found that 1e4 were not cytotoxic, as the cell survival
was not changed in the presence of 1e4 at the concentration of
10 mM (Fig. 6).
It has to be noted that the 24R/S isomers, 1 and 2, represent
the first two examples of 17,21-dihydroxyergost-1,22-dien-3-
one derivatives. The NMR spectroscopic data of these two iso-
mers were found to have the following main differences,
which might be useful for the differentiation of isomers of
this type: (1) in the case of 24R isomer 1, the value of chemical
shift difference between H-22 and H-23 (D¼dH-22ꢀdH-23) was
found to be 0.20 ppm, which was changed to 0.26 ppm as ob-
served for 24S isomer 2; (2) the carbon resonance of C-28 was
observed at d 17.5 for 1 and 18.1 for 2, as measured in CDCl3.
The in vitro anti-inflammatory effect of the sterones 1e4
was tested. In this assay, the up-regulation of the pro-inflam-
matory iNOS and COX-2 proteins of the LPS-stimulated
RAW264.7 macrophage cells was evaluated using the immu-
noblot analysis. Although these four compounds did not
inhibit the COX-2 expression at 10 mM, however, it was found

C.-H. Chao et al. / Tetrahedron 64 (2008) 3554e3560
3557
OH
c
18
OH
19
20
a
17
b
9
8
1 and 2
O
O
14
5
7
4
OH
4
OH
3
HOOC
c
d
a
b
4
5
O
1H–1 H COSY
HMBC
Figure 4. Selective NOSEY correlations of 4 and 5.
Figure 2. Selective He1H COSY and HMBC correlations of 1e4.
1
PGME
+ 0.034
O
-0.135
+ 0.086
+ 0.098
+ 0.086
We also applied a modified method for the preparation of the
PGME amides of 5 by treating 5 with PGME, EDC$HCl,
4-DMAP, and 4-DMAP$HCl. Comparing with the original
method to prepare the PGME amide with PGME in the pres-
ence of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexa-
fluorophosphate (PyBop), 1-hydroxybenzotriazole (HOBt),
and N-methylmorpholine, the cost of our modified method is
less expensive.
-0.032
-0.042
-0.060
-0.014
H
H
H
O
H
Δδ = δ (S)-δ (R) PGME amide
5a. R = (S)-PGME amide
5b. R = (R)-PGME amide
Figure 5. 1H NMR chemical shift differences of PGME amides of 5.
77.0 ppm). LRMS and HRMS were obtained by ESI on a Bruker
APEX II mass spectrometer. Silica gel 60 (Merck, 230e400
mesh) was used for column chromatography. Precoated Silica
gel plates (Merck Kieselgel 60 F254 0.2 mm) were used for
analytical TLC. High-performance liquid chromatography
(HPLC) was performed on a Shimadzu LC-10ATVP apparatus
equipped with a Shimadzu SPD-10AVP UV detector. The
columns used in HPLC separation are YMC-Pack Pro C18
(reverse-phase column, 250ꢃ10 mm, 5 mm) and Varian Dyna-
3. Experimental
3.1. General experimental procedures
Optical rotations were measured on a Jasco P-1020 polari-
meter. IR spectra were recorded on Jasco FT/IR-4100 fourier
transform infrared spectrophotometer. The NMR spectra were
recorded on Varian Mercury-Plus 300 FT-NMR(or Varian Unity
INOVA 500 FT-NMR) instrument at 300 MHz (or 500 MHz) for
1H (referenced to TMS, dH 0.0 ppm) and 75 MHz (or 125 MHz)
for 13C in CDCl3 (referenced to the center line of CDCl3, dC
˚
max, Si-60 (normal-phase column, 250ꢃ21.4 mm, 100 A,
5 mm). (S)-(þ)-2-Phenylglycine methyl ester hydrochloride,
Figure 3. X-ray ORTEP drawing of 1.

3558
C.-H. Chao et al. / Tetrahedron 64 (2008) 3554e3560
2004. The location was about 60 km west of Singda Harbor,
iNOS
Kaohsiung County, Taiwan. A voucher specimen was depos-
ited in the Department of Marine Biotechnology and Re-
sources, National Sun Yat-sen University (specimen no.
041206A).
β-actin
120
100
80
60
40
20
0
A
3.3. Extraction and separation
The octocoral (15 kg fresh wt) was collected and freeze-
dried. The freeze-dried material was minced and extracted
exhaustively with EtOH (3ꢃ10 L). The organic extract was
concentrated to an aqueous suspension and was further parti-
tioned between EtOAc and water. The EtOAc extract (100 g)
was fractionated by open column chromatography on silica
gel using n-hexaneeEtOAc and EtOAceMeOH mixtures of
increasing polarity to yield 19 fractions. Fraction 7, eluted
with n-hexaneeEtOAc (2:1), was further subjected to silica
gel column chromatography with gradient elution (n-hexa-
neeacetone, 90:10 to 85:15), and followed by normal-phase
HPLC (n-hexaneeEtOAc, 4:1) to afford a mixture of 4 and
5. The final purification was carried out by reverse-phase
HPLC (CH3CNeH2O, 4:1) to obtain compounds 4 (1.5 mg)
and 5 (4.5 mg). Fraction 8, eluted with n-hexaneeEtOAc
(1:1), was further subjected to silica gel column chromatogra-
phy with gradient elution (n-hexaneeacetone, 9:1 to 8:2),
Sephadex LH-20 (acetone) and followed by normal-phase
HPLC (n-hexaneeEtOAc, 85:15) to yield a mixture of 1e3.
Finally, this mixture was purified by reverse-phase HPLC
(CH3CNeH2O, 4:1) to yield compounds 1 (3.0 mg), 2
(2.5 mg), and 3 (3.0 mg), and 1 (24R epimer) was eluted after
2 (24S epimer).
∗
∗
∗
∗
a
-
1b
2c
3d
4e
COX-2
β-actin
B
140
120
100
80
60
40
20
3.3.1. Griffinisterone A (1)
Colorless crystals; mp 184e185 ꢁC; [a]D22 ꢀ20 (c 0.24,
1H NMR data, see Tables 1 and 2; ESIMS m/z 451 [MþNa]þ;
HRESIMS m/z 451.3186 [MþNa]þ (calcd for C28H44O3Na,
451.3188).
0
a
-
1b
2c
3d
4e
CHCl3); IR (KBr) vmax 3409, 1675, and 1382 cmꢀ1 13C and
;
Figure 6. Effect of compounds 1e4 on iNOS and COX-2 protein expression of
RAW264.7 macrophage cells by immunoblot analysis. (A) Immunoblots of
iNOS and b-actin; (B) immunoblots of COX-2 and b-actin. The values are
meanꢂSEM (n¼6). Relative intensity of the LPS alone stimulated group
was taken as 100%. Under the same experimental condition CAPE (caffeic
acid phenylethyl ester, 10 mM) reduced the levels of the iNOS and COX-2
to 2.5ꢂ3.7% and 67.2ꢂ13.4%, respectively. *: Significantly different from
LPS alone stimulated group (P<0.05); a: stimulated with LPS alone; b: stim-
ulated with LPS in the presence of 1 (10 mM); c: stimulated with LPS in the
presence of 2 (10 mM); d: stimulated with LPS in the presence of 3
(10 mM); e: stimulated with LPS in the presence of 4 (10 mM).
3.3.2. Griffinisterone B (2)
Amorphous solid; [a]2D2 þ3 (c 0.45, CHCl3); IR (KBr) vmax
3360, 1674, and 1382 cmꢀ1 13C and 1H NMR data, see Tables
;
1 and 2; ESIMS m/z 451 [MþNa]þ; HRESIMS m/z 451.3189
[MþNa]þ (calcd for C28H44O3Na, 451.3188).
(R)-(ꢀ)-2-Phenylglycine methyl ester hydrochloride, and 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDC$HCl) were purchased from Aldrich and 4-(dimethylami-
no)pyridine (4-DMAP) was purchased from Lancaster. 4-
DMAP$HCl was prepared by 4-DMAP and concd HCl in THF.
3.3.3. Griffinisterone C (3)
Amorphous solid; [a]2D2 ꢀ5 (c 0.58, CHCl3); IR (KBr) vmax
3383, 1674, and 1382 cmꢀ1 13C and 1H NMR data, see Tables
;
1 and 2; ESIMS m/z 437 [MþNa]þ; HRESIMS m/z 437.3033
[MþNa]þ (calcd for C27H42O3Na, 437.3031).
3.2. Organism
3.3.4. Griffinisterone D (4)
Amorphous solid; [a]2D2 ꢀ7 (c 0.32, CHCl3); IR (KBr) vmax
The octocoral D. griffini was collected unexpectedly by
a bottom trawl net at depths ranged from 200 to 100 m during
an ecological investigation at Taiwan Straight in December
1699 and 1682 cmꢀ1 13C and 1H NMR data, see Tables 1 and
;
2; ESIMS m/z 449 [MþNa]þ; HRESIMS m/z 449.3029
[MþNa]þ (calcd for C28H42O3Na, 449.3031).

C.-H. Chao et al. / Tetrahedron 64 (2008) 3554e3560
3559
3.3.5. Griffinisterone E (5)
Amorphous solid; [a]2D2 ꢀ8 (c 0.38, CHCl3); IR (KBr) vmax
3.5. In vitro anti-inflammatory assay
1700 and 1683 cmꢀ1
;
13C and 1H NMR data, see Tables 1 and
The anti-inflammatory assay was modified from Ho et al.11
and Park et al.12 Murine RAW264.7 macrophages were ob-
tained from the American Type Culture Collection (ATCC,
no. TIB-71) and cultured in Dulbecco’s modified essential me-
dium (DMEM) containing 10% heat-inactivated fetal bovine
serum, at 37 ꢁC in a humidified 5% CO2e95% air incubator
under standard conditions. The macrophage cells were plated
in 60 mm culture dishes (3ꢃ106 cells), and test compounds
were added to the culture medium for 16 h. Then, cells were
washed with ice-cold PBS, lysed in ice-cold lysis buffer, and
then centrifuged at 20,000g for 30 min at 4 ꢁC. The superna-
tant was decanted from the pellet and retained for Western
blot analysis. Protein concentrations were determined by the
DC protein assay kit (Bio-Rad) modified by the method of
Lowry et al.13 Samples containing equal quantities of protein
were subjected to SDSepolyacrylamide gel electrophoresis,
and the separated proteins were electrophoretically transferred
to polyvinylidene difluoride membranes (PVDF; Immobilon-P,
Millipore, 0.45 mm pore size). The resultant PVDF membranes
were incubated with blocking solution and incubated for
180 min with antibody against inducible nitric oxide synthase
(iNOS; 1:1000 dilution; Transduction Laboratories) and cyclo-
oxygenase-2 (COX-2; 1:1000 dilution; Cayman Chemical)
protein. The blots were detected using ECL detection reagents
(PerkineElmer, Western Blot Chemiluminescence Reagent
Plus) according to the manufacturer instructions. The mem-
branes were reprobed with a monoclonal mouse anti-b-actin
antibody (1:2500, Sigma) as the loading control. For the im-
munoreactivity data, the intensity of each drug-treated band
is expressed as the integrated optical density (IOD), calculated
with respect to the average optical density of the correspond-
ing control (LPS-only treatment) band. For statistical analysis,
all the data were analyzed by a one-way analysis of variance
(ANOVA), followed by the StudenteNewmaneKeuls post
hoc test for multiple comparisons. A significant difference
was defined as a P value of <0.05.
2; ESIMS m/z 437 [MþNa]þ; HRESIMS m/z 437.3027
[MþNa]þ (calcd for C27H42O3Na, 437.3031).
3.3.6. Preparation of (S) and (R)-PGME amides of 5
To a stirred solution of compound 5 (1 mg) and (S)-PGME
(2 mg) in a 1 mL mixture of CHCl3eDMF (10:1) were succes-
sively added DMAP (2 mg) and 4-DMAP$HCl (2 mg). After
the mixture was stirred at 0 ꢁC for 5 min, EDC$HCl (2 mg)
was added. The reaction mixture was then moved to a freezer
at 4 ꢁC for overnight. The mixture was then stirred at room
temperature for 3 h. Subsequently, ethyl acetate was added,
and the resulting solution was successively washed with 5%
HCl, saturated NaHCO3(aq), and brine. The organic layer
was dried over anhydrous Na2SO4 and concentrated to give
a residue, which was chromatographed on silica gel using
n-hexaneeEtOAc (8:1) as eluent to afford the (S)-PGME
amide (5a) (0.8 mg). The same procedure was used to prepare
the (R)-PGME amide (5b) (0.6 mg from 1 mg of 5) with (S)-
1
PGME. Selective H NMR (CDCl3) of 5a: dH 7.355 (5H, br
s, Ph), 7.044 (1H, d, J¼10.0 Hz, H-1), 6.467 (1H, d,
J¼7.2 Hz, NH), 5.831 (1H, d, J¼10.0 Hz, H-2), 5.610 (1H,
d, J¼7.2 Hz, CHeN), 3.735 (3H, s, OCH3), 1.650 (1H, m,
H-17), 1.443 (2H, m, H-22), 1.479 (1H, m, H-25), 0.946
(3H, s, H3-19), 0.834 (6H, d, J¼6.3 Hz, H3-26 and H3-27),
1
0.620 (3H, s, H3-18). Selective H NMR (CDCl3) of 5b: dH
7.339 (5H, br s, Ph), 7.104 (1H, d, J¼10.0 Hz, H-1), 6.460
(1H, d, J¼7.2 Hz, NH), 5.845 (1H, d, J¼10.0 Hz, H-2),
5.653 (1H, d, J¼7.2 Hz, CHeN), 3.749 (3H, s, OCH3),
1.682 (1H, m, H-17), 1.409 (2H, m, H-22), 1.381 (1H, m,
H-25), 0.988 (3H, s, H3-19), 0.755 (3H, s, H3-18), 0.748
(6H, d, J¼6.3 Hz, H3-26 and H3-27).
3.4. X-ray diffraction analysis of griffinisterone A (1)
A suitable colorless crystal (0.8ꢃ0.8ꢃ0.2 mm3) of 1 was
grown by slow evaporation of the acetone containing few drops
of benzene at 0 ꢁC. Diffraction intensity data were acquired
with a Rigaku AFC7S single-crystal X-ray diffractometer
with graphite-monochromated Mo Ka radiation (l¼
Acknowledgements
Financial support was provided by Ministry of Education
(95C030313) and National Science Council of Taiwan (NSC
95-2113-M-110-011-MY3) awarded to J.-H.S.
˚
0.71073 A). Crystal data for 1: C28H44O3 (formula weight
428.63), approximate crystal size, 0.8ꢃ0.8ꢃ0.2 mm3, mono-
˚
clinic, space group, P21 (#4), T¼298(2) K, a¼12.467(4) A, b¼
3
ꢁ
References and notes
˚
˚
˚
7.636(2) A, c¼14.244(5) A, b¼110.18(3) , V¼1272.7(7) A ,
Dc¼1.118 Mg/m3, Z¼2, F(000)¼472, m(Mo Ka)¼0.070 mmꢀ1
.
1. Yoshikawa, K.; Kanekuni, S.; Hanahusa, M.; Arihara, S.; Ohta, T. J. Nat.
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A total of 3426 reflections were collected in the range
1.88ꢁ<q<26.00ꢁ, with 2695 independent reflections [R(int)¼
0.0420], completeness to qmax was 99.9%; psi-scan empirical
absorption correction applied; full-matrix least-squares refine-
ment on F2, the number of data/restraints/parameters were
2695/1/293; goodness-of-fit on F2¼1.019; final R indices
[I>2s(I)], R1¼0.0436, wR2¼0.1106; R indices (all data), R1¼
0.0656, wR2¼0.1225, largest difference peak and hole, 0.139
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3
˚
and ꢀ0.227 e/A .

3560
C.-H. Chao et al. / Tetrahedron 64 (2008) 3554e3560
Copies of the data can be obtained, free of charge, on application to the
Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: þ44
1223 336033 or e-mail: deposit@ccdc.cam.ac.uk).
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