New briaranes from the octocorals Briareum excavatum (Briareidae) and Junceella fragilis (Ellisellidae)

Article abstract of DOI:10.1016/j.tet.2008.01.023

Six 12-hydroxybriaranes, including four new diterpenoids, briaexcavatins I-L (1-4), and two known metabolites, excavatolides C (5) and E (6), have been isolated from the cultured scleraxonia Briareum excavatum. In addition, the gorgonian coral Junceella fragilis yielded a new chlorinated briarane, fragilide C (10). The structures of above compounds were determined by spectroscopic methods and the structures of 5 and 6 were further confirmed by X-ray data analysis for the first time. The absolute configuration of 6 was elucidated by chemical conversion. Some of these briaranes have displayed inhibitory effects on superoxide anion generation by human neutrophils.

Full text of DOI:10.1016/j.tet.2008.01.023

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 2596e2604
www.elsevier.com/locate/tet
New briaranes from the octocorals Briareum excavatum
(Briareidae) and Junceella fragilis (Ellisellidae)
a,c
a,b
d
e
Ping-Jyun Sung a,b, , Mei-Ru Lin , Yin-Di Su , Michael Y. Chiang , Wan-Ping Hu ,
*
Jui-Hsin Su c,f, Mo-Chih Cheng c,g, Tsong-Long Hwang h, Jyh-Horng Sheu c,f,
*
a Department of Planning and Research and Coral Research Center, National Museum of Marine Biology and Aquarium, Checheng, Pingtung 944, Taiwan
b Institute of Marine Biotechnology, National Dong Hwa University, Checheng, Pingtung 944, Taiwan
c Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan
d Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan
e Faculty of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan
f Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan
g Medical and Pharmaceutical Industry Technology and Development Center, Taipei 248, Taiwan
h Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan
Received 14 December 2007; received in revised form 7 January 2008; accepted 7 January 2008
Available online 11 January 2008
Abstract
Six 12-hydroxybriaranes, including four new diterpenoids, briaexcavatins IeL (1e4), and two known metabolites, excavatolides C (5) and E
(6), have been isolated from the cultured scleraxonia Briareum excavatum. In addition, the gorgonian coral Junceella fragilis yielded a new chlo-
rinated briarane, fragilide C (10). The structures of above compounds were determined by spectroscopic methods and the structures of 5 and 6
were further confirmed by X-ray data analysis for the first time. The absolute configuration of 6 was elucidated by chemical conversion. Some of
these briaranes have displayed inhibitory effects on superoxide anion generation by human neutrophils.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Briarane; Briareum excavatum; Briaexcavatin; Junceella fragilis; Fragilide; Superoxide anion
1. Introduction
chlorinated briarane metabolite, fragilide C (10), was obtained
from the gorgonian J. fragilis (Ellisellidae). The structures of
compounds 1e6 and 10 were established by extensive spectral
data analysis; the structures of briaranes 5 and 6 were further
determined by X-ray analysis and the absolute configuration of
6 was established by chemical methods. Some of these
In our continuing research on novel natural substances ob-
tained from the marine invertebrates of Taiwanese waters, a se-
ries of interesting terpenoids and steroids have been isolated
from the octocorals Alcyonium sp.,1,2 Briareum sp.,3 Briareum
excavatum,4e7 Ellisella robusta,8e11 Junceella fragilis,12e19
Junceella juncea,14,20 Rumphella antipathies,21e26 and the tu-
nicate Eudistoma sp.27 In this paper, we report the isolation,
structure determination, and biological activity of four new
briaranes, briaexcavatins IeL (1e4), along with two known
compounds, excavatolides C (5) and E (6),28 from the cultured
scleraxonia B. excavatum (Briareidae). In addition, a new
R1
OAc
OAc
OH
AcO
AcO
15
3
4
2
1
10
16
14
13
5
6
OAc
12
11
7
R3
9
8
HO
H
H
19 O
O
O
O
O
20
17
R2
18
AcO
O
2
1: R1 = H, R2 = OAc, R3 = β-OH
5: R1 = R2 = OAc, R3 = β-OH
6: R1 = H, R2 = OH, R3 = β-OH
7: R1 = H, R2 = OAc, R3 = α-OH
* Corresponding authors. Tel.: þ886 8 8825037; fax: þ886 8 8825087.
E-mail address: pjsung@nmmba.gov.tw (P.-J. Sung).
0040-4020/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.01.023

P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
2597
Table 1
13C NMR data for diterpenoids 1e4a
OAc
OAc
R
OH
O
AcO
Position
1
2
3
4
45.7 (s)b
75.1 (d)
31.7 (t)
28.5 (t)
45.6 (s)
74.3 (d)
39.5 (t)
69.2 (d)
45.4 (s)
77.8 (d)
41.4 (t)
71.2 (d)
47.9 (s)
74.2 (d)
40.3 (t)
70.8 (d)
R
HO
1
H
H
HO
HO
O
O
O
2
AcO
AcO
3
O
O
4
3: R = α-OH
8: R = β-OCO(CH2)2CH3
4: R = OH
9: R = OAc
5
145.1 (s)
118.3 (d)
74.8 (d)
71.0 (s)
74.1 (d)
41.3 (d)
44.5 (d)
66.8 (d)
28.9 (t)
76.1 (d)
15.4 (q)
27.2 (q)
65.1 (s)
11.0 (q)
170.6 (s)
9.0 (q)
145.5 (s)
125.0 (d)
73.3 (d)
70.7 (s)
73.6 (d)
41.5 (d)
44.6 (d)
66.8 (d)
28.9 (t)
76.0 (d)
15.5 (q)
68.2 (t)
65.1 (s)
11.0 (q)
170.3 (s)
9.0 (q)
147.3 (s)
122.2 (d)
73.5 (d)
70.3 (s)
67.1 (d)
43.8 (d)
75.3 (s)
71.8 (d)
124.0 (d)
139.0 (d)
17.8 (q)
25.8 (q)
62.7 (s)
9.6 (q)
146.2 (s)
122.2 (d)
73.6 (d)
70.6 (s)
67.2 (d)
48.9 (d)
78.2 (s)
73.3 (d)
30.2 (d)
74.9 (d)
14.4 (q)
25.3 (q)
66.4 (s)
10.3 (q)
170.3 (s)
17.0 (q)
21.4 (q)
21.3 (q)
21.2 (q)
6
7
OR
OH
AcO
8
15
3
4
2
1
10
16
9
10
14
13
5
6
chair
O
12
11
Cl
7
11
12
9
H
20AcO
O
8
19 O
17
13
14
18
O
10: R = COCH2CH3
11: R = Ac
15
16
17
18
briaranes exhibited inhibitory effects on superoxide anion gen-
eration by human neutrophils.
19
20
171.1 (s)
28.1 (q)
21.7 (q)
21.1 (q)
Acetate methyls
21.5 (q)
21.4 (q)
21.2 (q)
21.4 (q)
21.4 (q)
21.1 (q)
21.0 (q)
171.4 (s)
170.7 (s)
170.7 (s)
168.4 (s)
2. Results and discussion
2.1. Isolation and structure determination of briaranes from
B. excavatum
Acetate carbonyls
170.5 (s)
170.4 (s)
168.2 (s)
170.5 (s)
169.6 (s)
170.2 (s)
170.2 (s)
168.2 (s)
The briaexcavatins and excavatolides were isolated by con-
ventional methods as outlined in Section 3. Briaexcavatin I (1)
was obtained as a white powder and the molecular formula of
1 was determined to be C26H36O10 (nine degrees of unsatura-
tion) by analysis of 13C and 1H NMR data (Tables 1 and 2) in
conjunction with DEPT results; this conclusion was further
confirmed by HRESIMS (m/z calcd: 531.2206; found:
531.2209 [MþNa]þ). Comparison of the 1H NMR and
DEPT data with the molecular formula indicated that there
must be an exchangeable proton, requiring the presence of
a hydroxy group, and this deduction was supported by a broad
absorption in the IR spectrum at 3497 cmꢀ1. The IR spectrum
of 1 also showed strong bands at 1775 and 1742 cmꢀ1, consis-
tent with the presence of g-lactone and ester groups, respec-
tively. From the 13C NMR data of 1 (Table 1), the presence
of a trisubstituted olefin group was deduced from the signals
of two carbons resonating at d 145.1 (s, C-5) and 118.3 (d,
CH-6), and was further supported by an olefin proton signal
a
Spectra recorded at 100 MHz in CDCl3 at 25 ꢁC.
Multiplicity deduced by DEPT and HMQC spectra and indicated by the
b
usual symbol.
singlet (d 1.19, 3H, s, H3-15), a methyl doublet (d 1.04, 3H,
d, J¼7.2 Hz, H3-20), two aliphatic protons (d 2.40, 1H, d,
J¼4.0 Hz, H-10; 2.05, 1H, m, H-11), five oxymethine protons
(d 5.17, 1H, d, J¼8.4 Hz, H-7; 5.05, 1H, d, J¼7.6 Hz, H-2;
4.97, 1H, br s, H-9; 4.79, 1H, dd, J¼3.2, 2.8 Hz, H-14; 4.06,
1H, m, H-12), and three pairs of aliphatic methylene protons
(d 2.58, 1H, td, J¼14.8, 5.6 Hz, H-3b; 1.60, 1H, m, H-3a;
2.47, 1H, br d, J¼14.0 Hz, H-4b; 1.94, 1H, td, J¼14.8,
1
4.4 Hz, H-4a; 1.82, 2H, m, H2-13) were observed in the H
NMR spectrum of 1.
1
The structure and all of the assignments made from the H
and 13C NMR data of 1 were determined with the assistance of
2D NMR studies. From the 1He1H COSY spectrum of 1
(Fig. 1), it was possible to establish the proton sequences
from H-2/H2-3, H2-3/H2-4, H2-4/H-6 (by allylic coupling),
H-6/H-7, and H-9/H-10. These data, together with the
HMBC correlations between H-2/C-1, 4, 10; H2-3/C-1, 4, 5;
H2-4/C-2, 3, 5, 6; H-6/C-4; H-7/C-5, 6; H-9/C-8; and H-10/
C-1, 8, 9 (Fig. 1), established the connectivity from C-1 to
C-10 within the 10-membered ring. The vinyl methyl attached
at C-5 was confirmed by the HMBC correlations between
H3-16/C-4, 5; H2-4/C-16; and H-6/C-16, and was further con-
firmed by the allylic coupling between H3-16/H-6. The meth-
ylcyclohexane ring, which is fused to the 10-membered ring at
1
at d 5.23 (1H, d, J¼8.4 Hz, H-6) in the H NMR spectrum
of 1 (Table 2). An 8,17-epoxide group was confirmed from
the signals of two quaternary oxygenated carbons at d 71.0
(s, C-8) and 65.1 (s, C-17), and from the chemical shift of
the C-18 tertiary methyl (dH 1.63, 3H, s; dC 11.0, q). More-
over, four carbonyl resonances appeared at d 170.6 (s, C-
19), 170.5 (s, ester carbonyl), 170.4 (s, ester carbonyl), and
168.2 (s, ester carbonyl), confirming the presence of a g-lac-
1
tone and three ester groups in 1. In the H NMR spectrum
of 1, three acetate methyls (d 2.22, 3H, s; 2.01, 3H, s; 1.99,
3H, s) were observed. Thus, from the NMR data, five degrees
of unsaturation were accounted for and 1 must be tetracyclic.
In addition, a vinyl methyl (d 2.00, 3H, s, H3-16), a methyl
C-1 and C-10, was elucidated by He1H COSY correlations
from H-10/H-11, H-11/H3-20, H-11/H-12, H-12/H2-13,
and H2-13/H-14; and by the HMBC correlations between
1

2598
P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
Table 2
1H NMR data for diterpenoids 1e4a
Position
1
2
3
4
2
3a
5.05 d (7.6)b
1.60 m
4.80 d (8.0)
2.02 m
4.53 d (6.4)
2.08 m
4.95 d (10.4)
1.98 m
3b
4a
2.58 td (14.8, 5.6)
1.94 td (14.8, 4.4)
2.47 br d (14.0)
5.23 d (8.4)
5.17 d (8.4)
4.97 br s
2.80 dd (15.2, 12.0)
4.19 dd (12.0, 4.8)
2.77 dd (15.2, 12.4)
4.24 dd (12.4, 4.8)
2.89 dd (15.6, 12.0)
4.13 dd (12.0, 5.2)
4b
6
5.55 d (8.0)
5.81 d (8.0)
4.96 br s
5.50 d (9.6)
6.15 d (9.6)
5.80 d (4.4)
2.55 d (4.4)
5.33 ddd (8.8, 1.6, 1.2)
5.81 d (8.8)
5.77 d (1.2)
7
9
10
2.40 d (4.0)
2.05 m
4.06 m
2.33 d (3.6)
2.04 m
4.06 m
2.12 d (1.2)
11
12
3.75 d (5.6)
5.79 dd (10.0, 5.6)
3.71 ddd (11.2, 2.8, 2.8)
1.67 ddd (14.6, 12.8, 2.0)
2.03 m
13a
13b
1.82 m (2H)
1.82 m (2H)
14
15
4.79 dd (3.2, 2.8)
1.19 s
2.00 s
4.83 dd (3.2, 3.2)
1.24 s
5.53 d (10.0)
1.23 s
2.02 s
4.81 dd (2.2, 2.0)
1.24 s
2.08 d (1.2)
16a
16b
4.65 d (14.8)
4.75 d (14.8)
1.66 s
18
20
1.63 s
1.61 s
1.39 s
2.23 s
2.11 s
1.77 s
1.17 s
2.25 s
2.01 s
1.99 s
1.04 d (7.2)
2.22 s
2.01 s
1.04 d (7.2)
2.25 s
2.09 s
Acetate methyls
1.99 s
1.98 s
1.97 s
a
Spectra recorded at 400 MHz in CDCl3 at 25 ꢁC.
J values (in Hz) in parentheses.
b
H
O
15AcO
2S*
H
H
O
H
O
16
H
H
1S*
O
H
15
14S*
OAc
H
5Z
16
20OAc 10S*
9S*
H
H
5
7S*
1
11R*
8R*
H
H
10
HO
O
H
12S*
17R*
HO
8
H
: NOE 18
O
O
O
20
O
O
17
19
:
1H-1H COSY
C)
Figure 2. Selective NOESY correlations of 1.
: HMBC (H
18
O
Figure 1. The 1He1H COSY and HMBC correlations of 1.
on same face of the structure; these were assigned as the a pro-
tons, as the C-15 methyl is the b-substituent at C-1. Thus, the
hydroxy group at C-12 is at the b face and is cis to the C-20
methyl group. H-9 was found to exhibit strong NOE responses
with H-11, H3-18, and H3-20. From the consideration of
molecular models, H-9 was found to be reasonably close to
H-11, H3-18, and H3-20 when H-9 was a-oriented in the 10-
membered ring and H3-18 was placed on the b face in the
g-lactone moiety. H-14 showed an NOE response with H3-
15 but not with H-10, showing that this proton was positioned
on the equatorial direction and has a b-orientation at C-14.
Furthermore, one proton of the C-3 methylene (d 2.58, H-3b)
showed strong NOE correlations with H3-15 and H-7, suggest-
ing that these protons are on the b face of 1. The NOE corre-
lation between H-6 and H3-16 suggested that the D5 double
bond exists in the Z form. Based on the above findings, the
structure, including the relative stereochemistry of 1, was elu-
cidated and the configurations of all chiral centers of 1 were
assigned as 1S*, 2S*, 5Z, 7S*, 8R*, 9S*, 10S*, 11R*, 12S*,
14S*, 17R*. By detailed analysis, the spectral data of 1 were
found to be very similar to those of a known briarane metab-
H-2/C-14; H-10/C-11, 20; H-11/C-1, 10, 12; H-12/C-13, 20;
H2-13/C-1, 11, 12, 14; H-14/C-10, 12; and H3-20/C-10, 11,
12. The ring junction C-15 methyl group was positioned at
C-1 from the HMBC correlations between H3-15/C-1, 10,
14; H-2/C-15; and H-10/C-15. The HMBC correlations also
indicated that the acetoxy groups are attached at C-2 and C-
14. The remaining acetoxy and hydroxy groups were posi-
tioned at C-9 and C-12, as indicated by analysis of key
1He1H COSY correlations and characteristic NMR signals.
These data, together with the HMBC correlations between
H-7/C-19 and H3-18/C-8, 17, 19, were used to establish the
molecular framework of 1.
Based on previous surveys, all the naturally occurring briar-
ane-type metabolites have the C-15 methyl group that is
trans to H-10, and these two groups are assigned as b- and
a-oriented in most briarane derivatives.29,30 The relative con-
figuration of 1 was elucidated from the NOE interactions
observed in an NOESY experiment (Fig. 2). In the NOESY
experiment of 1, H-10 gives strong NOE responses with H-
2, H-11, and H-12, indicating that these protons are situated
1
olite, briareolide F (7).31 However, by comparison of the H

P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
2599
and 13C NMR chemical shifts of the C-12 methine of 1 (dH
4.06, 1H, m; dC 66.8, d) with those of 7 (dH 3.70, 1H, m; dC
71.2, d), it was shown that the hydroxy group in 1 attached
at C-12 is b-oriented.
H
15AcO
1S*H
H
H
OH
13Z
4R*
H
2S*H
OAc
16
OH
5Z
11R*
10S*
H
9S*
H
7S*
8R*
H
12R*
The new briarane diterpene, briaexcavatin J (2), had a mo-
lecular formula of C28H38O13 as deduced by HRESIMS (m/z
calcd: 605.2210; found: 605.2209 [MþNa]þ). The IR spec-
trum of 2 indicated the presence of hydroxy (3439 cmꢀ1),
g-lactone (1775 cmꢀ1), and ester (1735 cmꢀ1) groups. From
the 13C NMR data of 2 (Table 1), a trisubstituted olefin (d
145.5, s, C-5; 125.0, d, CH-6) and five carbonyl resonances
(d 171.4, 170.7, 170.7, 168.4, 4ꢂs, ester carbonyls; 170.3, s,
C-19) were observed. The four esters were identified as ace-
HO 20
H
H
O
17R*
18
: NOE
O
Figure 4. Selective NOESY correlations of 3.
positions of two acetoxy groups at C-2 and C-9 were con-
firmed by the correlations between the oxymethine protons
at dH 4.53 (H-2) and 5.80 (H-9) with the acetate carbonyls
at dC 170.5 (s) and 169.6 (s), respectively, in the HMBC spec-
trum of 3. The relative configuration of 3 was confirmed as be-
ing similar to that of a known metabolite, briaexcavatolide W
(8),32 by comparison of the NMR chemical shifts and coupling
constants analysis for the chiral centers C-1, -2, -4, -7, -8, -9,
-10, and -17. In the NOESY experiment of 3 (Fig. 4), H3-20
was found to exhibit NOE correlations with H-9, H-10, and
H-12, but not with H3-15, indicating that the C-20 methyl
was a-oriented in 3. However, H-12 was assigned on the
b face by a strong NOE correlation between H-12 and
H3-20, but not between H-10 and H3-15. By consideration of
molecular models, H-12 was found to be reasonably close to
H3-20, but not to H-10 and H3-15, when H-12 and CH3-20
were b- and a-oriented, respectively, and these two groups
should be positioned on the equatorial direction in the cyclo-
hexene ring. The cis geometry of the C-13/C-14 double
bond was indicated by a 10.0 Hz coupling constant between
H-13 (d 5.79, 1H, dd, J¼10.0, 5.6 Hz) and H-14 (d 5.53,
1H, d, J¼10.0 Hz). Based on the above findings, the structure
of 3 was established and the chiral centers for this compound
were assigned as 1S*, 2S*, 4R*, 5Z, 7S*, 8R*, 9S*, 10S*,
11R*, 12R*, 13Z, 17R*.
1
tates by the presence of four methyl resonances in the H
NMR spectrum of 2 at d 2.25 (3H, s), 2.09 (3H, s), 1.98
(3H, s), and 1.97 (3H, s) (Table 2). The planar structure of 2
was determined by 2D NMR experiments. The coupling infor-
1
mation in the He1H COSY experiment of 2 enabled identifi-
cation of the C-2/3/4, C-4/6 (by allylic coupling), C-6/7, C-6/
16 (by allylic coupling), C-9/10/11/12/13/14, and C-11/20
units. From these data, together with the results of an
HMBC experiment of 2, the molecular framework of 2 could
be further established. The HMBC correlations also revealed
that the acetate groups are attached at C-2, C-9, C-14, and
C-16; thus, the remaining hydroxy groups should be posi-
tioned at C-4 and C-12.
The relative stereochemistry of 2 was elucidated from the
NOE interactions observed in an NOESY experiment
(Fig. 3) and the configurations of all chiral centers except
C-1 and C-4 of 2 were confirmed as being the same as those
of 1 by comparison of the proton chemical shifts, coupling
constants, and NOE correlations. The hydroxy group at C-4
was assigned the b-configuration primarily due to the NOE
correlation between H-4 and H-3a, and this led to the assign-
ment of the R*-configuration at C-1. The relative configura-
tions of all chiral centers of 2 were assigned as 1R*, 2S*,
4R*, 5Z, 7S*, 8R*, 9S*, 10S*, 11R*, 12S*, 14S*, 17R*.
Briaexcavatin K (3) had the molecular formula C24H32O10,
as established by HRESIMS (m/z calcd: 503.1893; found:
503.1895 [MþNa]þ). Its IR spectrum exhibited a broad OH
Briaexcavatin L (4), was isolated as a white powder, and had
the molecular formula C26H36O12 on the basis of HRESIMS.
The IR spectrum of 4 showed bands at 3428, 1759, and
1728 cmꢀ1, consistent with the presence of hydroxy, g-lactone,
and ester carbonyl groups, respectively. Based on detailed spec-
stretch at 3438 cmꢀ1
, a g-lactone carbonyl group at
tral data analysis and by comparison of the H and 13C NMR
1
1773 cmꢀ1, and ester carbonyl groups at 1728 cmꢀ1. Carbonyl
data of 4 with those of other briarane diterpenoids reported
previously, it was found that diterpenoid 4 is the 4-O-deacetyl
derivative of a known briarane metabolite, briaexcavatolide U
(9),33 and should possess a structure as represented by formula
4. The structure of 4 was further confirmed by 2D NMR exper-
iments and the chiral centers for this compound were assigned
resonances in the 13C NMR spectrum of 3 confirmed the pres-
1
ence of a g-lactone and two other esters (Table 1). In the H
NMR spectrum of 3 (Table 2), two acetate methyls were ob-
served at d 2.23 (3H, s) and 2.11 (3H, s). The planar structure
of 3 was proposed with the assistance of 2D NMR studies. The
H
H
15AcO
15AcO
H
H
OH
OH
4R*
4R*
2S*H
2S* H
16
16
H
H
1R*
1S*
H
H
14S*
14S*
OAc
H
H
H
H
OAc
OAc
5Z
5Z
H
7S*
H
20 OAc 10S*
20OAc 10S*
9S*
9S*
H
H
H
7S*
8R*
17R*
H
11R*
11S*
H
H
8R*
H
H
HO
HO
O
O
12S*
H
17R*
12S*
HO
18
: NOE
: NOE18
O
O
Figure 3. Selective NOESY correlations of 2.
Figure 5. Selective NOESY correlations of 4.

2600
P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
Figure 6. Computer-generated ORTEP plots of 5 and 6 showing the relative configurations.
Table 3
1H and 13C NMR data for diterpenoid 10
OAc
AcO-0.001
+0.006
1H NMRa
13C NMRb
-0.030
Position
6a: R = (S)-MTPA
6b: R = (R)-MTPA
Δ = (S)– (R) ppm
1
47.1 (s)d
72.5 (d)
40.5 (t)
97.2 (s)
137.9 (s)
55.3 (d)
78.6 (d)
81.4 (s)
71.7 (d)
40.7 (d)
56.2 (s)
29.7 (t)
24.7 (t)
73.9 (d)
15.5 (q)
117.9 (t)
50.0 (d)
7.2 (q)
+0.062
RO
5.32 d (7.6)c
1.53 d (16.0); 3.38 dd (16.0, 7.6)
H
2
O
O
+0.160 HO
3a/b
4
+0.015
O
5
Figure 7. 1H NMR chemical shift difference [d(S)-MTPAꢀd(R)-MTPA] of the
MTPA esters of 6.
6
4.90 m
4.33 d (2.8)
7
8
9
10
5.64 d (2.0)
2.80 br s
as 1S*, 2S*, 4R*, 5Z, 7S*, 8R*, 9S*, 10S*, 11S*, 12S*, 14S*,
17R* by its NOESY experiment (Fig. 5).
11
The known briaranes, excavatolides C (5) and E (6), were
first isolated from a wild-type Taiwanese octocoral B. excava-
tum and their structures were elucidated by spectral data anal-
ysis.28 The absolute configuration of 5 was then determined by
chemical methods in a later study.7 In this study, we report the
X-ray structures of excavatolides C (5) and E (6) for the first
time (Fig. 6). In order to determine the absolute configuration
of briarane 6, this compound was treated with (ꢀ) or (þ)-
MTPA chloride to yield the (S)- and (R)-MTPA esters 6a
12a/b
13/130
14
2.22 m; 1.25 m
1.92 m; 1.84 m
5.02 dd (3.2, 3.2)
1.23 s
15
16a/b
17
18
5.94 d (2.0); 5.64 dd (2.0, 1.6)
2.76 q (6.8)
1.32 d (6.8)
19
174.2 (s)
51.2 (t)
20a/b
OH-4
Acetates
2.42 dd (3.2, 2.4); 2.64 d (2.4)
6.61 br s
2.23 s
21.6 (q)
169.3 (s)
20.8 (q)
169.9 (s)
8.6 (q)
1
and 6b, respectively. Comparison of the H NMR chemical
shifts for 6a and 6b (D values shown in Fig. 7) led to the as-
signment of the S-configuration at C-12. Therefore, the abso-
lute configurations of all chiral centers of 6 were assigned as
1S, 2S, 5Z, 7S, 8R, 9S, 10S, 11R, 12S, 14S, 17R.
2.09 s
Propionate
1.14 t (7.6)
2.36 m
27.8 (t)
176.6 (s)
a
Spectra recorded at 400 MHz in CDCl3 at 25 ꢁC.
Spectra recorded at 100 MHz in CDCl3 at 25 ꢁC.
J values (in Hz) in parentheses.
b
c
d
2.2. Isolation and structure determination of fragilide C from
J. fragilis
Multiplicity deduced by DEPT and HMQC spectra and indicated by usual
symbol.
The new chlorinated briarane, fragilide C (10), was isolated
as a white solid. The molecular formula of C27H35ClO11 (10
degrees of unsaturation) was established from the mass ions
at m/z 593 (MþNa)þ and 595 (Mþ2þNa)þ in the ESIMS
spectrum and was further supported by HRESIMS (m/z calcd:
593.1765; found: 593.1767 [MþNa]þ). The IR spectrum of 10
also showed strong bands at 3385, 1789, and 1740 cmꢀ1, con-
sistent with the presence of hydroxy, g-lactone, and ester

P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
2601
O
H
2S*
EtOCO
O
Ha
H
O
O
OH
Hb
H
HO
4R*
O
1R*
15
H
H
14S*
16
4
6S* H
Cl
OAc
H
10S*
O H
OAc
H
5
H
1
10
9S*
7R*
O
11R*
H
H
8R*
H
11
O
Cl
H
O
H
O
8
17R*
20
O
: NOE
O
19
O
:
1H-1H COSY
: HMBC (H C)
18
O
Figure 9. Selective NOESY correlations of 10.
Figure 8. The 1He1H COSY and HMBC correlations of 10.
Table 4
Inhibitory effects of briaranes 1, 4e6, and 10 on superoxide anion generation
by human neutrophils in response to fMet-Leu-Phe/cytochalastin B
groups. From the 13C NMR data of 10 (Table 3), the presence
of an exocyclic carbonecarbon double bond was deduced
from the signals of two carbons resonating at d 137.9 (s, C-
5) and 117.9 (t, CH2-16), and further supported by two olefin
proton signals at d 5.94 (1H, d, J¼2.0 Hz, H-16a) and 5.64
Compound
Superoxide generation inhibitiona (%)
1
2.38ꢃ1.06
3.04ꢃ3.30
15.47ꢃ2.92
4.88ꢃ5.09
11.61ꢃ2.80
4
5
1
6
(1H, dd, J¼2.0, 1.6 Hz, H-16b) in the H NMR spectrum of
10
a
10 (Table 3). Moreover, four carbonyl resonances appeared
at d 176.6 (s), 174.2 (s), 169.9 (s), and 169.3 (s), confirming
the presence of a g-lactone and three ester groups in 10. In
the 1H NMR spectrum of 10, two acetate methyl signals
were observed (d 2.23, 3H, s; 2.09, 3H, s). The additional
acyl group was confirmed as a propionyloxy group based on
1H NMR studies, including a 1He1H COSY experiment
(Fig. 8), which revealed five contiguous protons (d 1.14, 3H,
t, J¼7.6 Hz; 2.36, 2H, m). The carbon signal observed at
d 176.6 (s) was correlated with the signal of the methylene
protons at d 2.36 in the HMBC spectrum and was thus as-
signed as the carbon atom of the propionate carbonyl
(Fig. 8). Also, it was found that the NMR data of 10 were sim-
ilar to those of a known diterpene, juncin ZI (11), which was
isolated previously from a South China Sea gorgonian coral
J. juncea,34 except that an acetoxy group of compound 11
was replaced by a propionyloxy group in 10. The main prob-
lem was to locate the propionate group at C-2, -9, or -14, and
the two acetates at the remaining two positions. The propio-
Percentage of inhibition (Inh %) at 10 mg/mL concentration. Results are
presented as meansꢃSEM (n¼3).
(d 2.22), suggesting that these protons (H-2, H-9, H-10,
H-12a, and H3-18) are located on the same face and can be as-
signed as a protons, as the C-15 methyl group is b-oriented.
H-14 was found to exhibit a strong NOE response with H3-
15, but not with H-10, showing that this proton is of b-orien-
tation. H-9 was found to show NOE correlations with H-10
and H-17, and, from molecular models, was found to be rea-
sonably close to H-10 and H-17; therefore, H-9 should
be placed on the a face in 10, and H-17 is b-oriented in the
g-lactone moiety. However, no NOE response was observed
between OH-4 and any proton in the NOESY experiment of
10, so the stereochemistry of the hydroxy group at C-4 cannot
be determined by this way. Fortunately, by comparing the 13C
NMR chemical shifts for C-4 (d 97.2, s) and C-8 (d 81.4, s) of
10 with those of the known briarane 11 (d 97.2, s, C-4; 81.4, s,
C-8), the 4-hydroxy group in 10 should be b-oriented. Further-
more, H-7 exhibited strong NOE correlations with H-17 and
H-6, suggesting that these protons are on the b face of 7.
Based on the above findings, the configurations of all chiral
centers of 10 were assigned to be 1R*, 2S*, 4R*, 6S*, 7R*,
8R*, 9S*, 10S*, 11R*, 14S* and 17R*.
1
nate ester was positioned at C-2 from the He13C long-range
correlations observed between H-2 (d 5.32) and the carbonyl
carbon (d 176.6) of the propionate in the HMBC spectrum
of 10 (Fig. 8), suggesting that fragilide C (10) is the 2-deace-
toxy-2-propionyloxy derivative of compound 11.
The chemical shifts of exocyclic 11,20-epoxy groups in
briarane derivatives have been summarized, and although the
13C NMR peaks for C-11 and C-20 appear at d 55e61 and
47e52 ppm, respectively, the epoxy group is a-oriented
(11R*), and the cyclohexane ring is of a chair conformation.17
Based on the above observations, the configuration of the
11,20-epoxy group in 10 (d 56.2, s, C-11; 51.2, t, CH2-20)
should be a-oriented and the cyclohexane ring in 10 should
be of a chair conformation. The relative stereochemistry of
10 was elucidated from the NOE interactions observed in an
NOESY experiment (Fig. 9). Due to the a orientation of H-
10, the ring junction C-15 methyl group should be b-oriented,
as no NOE correlation was observed between H-10 and H3-15.
In the NOESY spectrum of 10, H-10 gives NOE correlations
with H-2, H-9, H3-18, and one proton of the C-12 methylene
2.3. Biological activity
In biological activity experiments, excavatolide C (5) and
fragilide C (10) were found to show 15.47 and 11.61% inhib-
itory effects on superoxide anion generation by human neutro-
phils at 10 mg/mL, respectively (Table 4).
3. Experimental
3.1. General experimental procedures
Melting points were determined on FARGO apparatus and
were uncorrected. Optical rotation values were measured with

2602
P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
a JASCO P-1010 digital polarimeter at 25 ꢁC. Infrared spectra
were obtained on a VARIAN DIGLAB FTS 1000 FT-IR spec-
trophotometer. NMR spectra were recorded on a VARIAN
MERCURY PLUS 400 FT-NMR at 400 MHz for 1H and
100 MHz for 13C, in CDCl3. Proton chemical shifts were ref-
erenced to the residual CHCl3 signal (d 7.26 ppm). 13C NMR
spectra were referenced to the center peak of CDCl3 at
d 77.1 ppm. ESIMS and HRESIMS data were recorded on
a BRUKER APEX II mass spectrometer. Column chromatog-
raphy was performed on silica gel (230e400 mesh, Merck,
Darmstadt, Germany). TLC was carried out on precoated Kie-
selgel 60F254 (0.25 mm, Merck, Darmstadt, Germany) and
spots were visualized by spraying with 10% H2SO4 solution
followed by heating. HPLC was performed using a system
comprising a HITACHI L-7100 pump, a HITACHI photo
diode array detector L-7455, and a RHEODYNE 7725 injec-
tion port. A semi-preparative normal phase column (Hibar
250e25 mm, LiChrospher Si 60, 5 mm) and a semi-preparative
reverse phase column (Hibar 250e10 mm, Purospher STAR
RP-18e, 5 mm) were used for HPLC.
acetone to afford briarane 1 (3.4 mg, 20:1). Fraction C9 was
separated by normal phase HPLC, using mixtures of CH2Cl2
and acetone to afford fractions from C9-1 to C9-8. Fraction
C9-6 was repurified by reverse phase HPLC, using mixtures
of CH3CN and H2O to afford briaranes 4 (4.3 mg, 4:1), 3
(2.1 mg, 1:2), and 2 (2.6 mg, 1:3).
3.3.2. Briaexcavatin I (1)
White powder; mp 273e275 ꢁC; [a]D25 þ50 (c 0.15,
CHCl3); IR (neat) nmax 3497, 1775, 1742 cmꢀ1; for 13C
(CDCl3, 100 MHz) and 1H (CDCl3, 400 MHz) NMR data,
see Tables 1 and 2; ESIMS m/z 531 (MþNa)þ; HRESIMS
m/z 531.2209 (calcd for C26H36O10þNa, 531.2206).
3.3.3. Briaexcavatin J (2)
White powder; mp 130e132 ꢁC; [a]D25 þ21 (c 0.13,
CHCl3); IR (neat) nmax 3439, 1775, 1735 cmꢀ1; for 13C
(CDCl3, 100 MHz) and 1H (CDCl3, 400 MHz) NMR data,
see Tables 1 and 2; ESIMS m/z 605 (MþNa)þ; HRESIMS
m/z 605.2209 (calcd for C28H38O13þNa, 605.2210).
3.2. Animal material
3.3.4. Briaexcavatin K (3)
White powder; mp 154e156 ꢁC; [a]D25 þ67 (c 0.11,
3.2.1. B. excavatum
CHCl3); IR (neat) nmax 3438, 1773, 1728 cmꢀ1
;
13C (CDCl3,
Specimens of the cultured octocoral B. excavatum were col-
lected by hand in a 0.6-ton cultivating tank located in the Na-
tional Museum of Marine Biology and Aquarium (NMMBA),
Taiwan, in December 2006. This organism was identified by
comparison with previous descriptions.35e37 Living reference
specimens are being maintained in the authors’ marine organ-
ism cultivating tank and a voucher specimen was deposited in
the NMMBA, Taiwan.
1
100 MHz) and H (CDCl3, 400 MHz) NMR data, see Tables
1 and 2; ESIMS m/z 503 (MþNa)þ; HRESIMS m/z
503.1895 (calcd for C24H32O10þNa, 503.1893).
3.3.5. Briaexcavatin L (4)
White powder; mp 180e182 ꢁC; [a]D25 þ72 (c 0.22,
CHCl3); IR (neat) nmax 3428, 1759, 1728 cmꢀ1
;
13C (CDCl3,
1
100 MHz) and H (CDCl3, 400 MHz) NMR data, see Tables
1 and 2; ESIMS m/z 563 (MþNa)þ; HRESIMS m/z
563.2101 (calcd for C26H36O12þNa, 563.2104).
3.2.2. J. fragilis
Specimens of the gorgonian coral J. fragilis were collected
by hand using SCUBA off the coast of southern Taiwan in Au-
gust 2006, at a depth of 20 m. This organism was identified by
comparison with previous descriptions.36,38 Living reference
specimens are being maintained in the authors’ marine organ-
ism cultivating tank and a voucher specimen was deposited in
the NMMBA, Taiwan.
3.3.6. Excavatolide C (5)
The related physical (mp, optical rotation value) and spec-
tral (IR, 1H, and 13C NMR) data of 5 are in full agreement with
those reported previously.28
3.3.7. Excavatolide E (6)
The related physical (mp, optical rotation value) and spec-
tral (IR, 1H, and 13C NMR) data of 6 are in full agreement with
those reported previously.28
3.3. Extraction and isolation
3.3.1. B. excavatum
The freeze-dried and minced material of the octocoral B.
excavatum (wet weight 672 g, dry weight 270 g) was extracted
with a mixture of MeOH and CH2Cl2 (1:1) at room tempera-
ture. The residue was partitioned between EtOAc and H2O.
The EtOAc layer was separated on Sephadex LH-20 and
eluted using MeOH/CH2Cl2 (2:1) to yield three fractions,
AeC. Fraction C was separated on silica gel and eluted using
hexane/EtOAc (stepwise, 20:1epure EtOAc) to yield fractions
1e9. Fraction C8 was separated by gravity column with silica
gel and eluted using hexane/EtOAc to afford briaranes 5
(35 mg, 3:1) and 6 (130 mg, 2:1). A mixture from C8 was pu-
rified by normal phase HPLC, using a mixture of CH2Cl2 and
3.3.8. J. fragilis
The freeze-dried and minced material of the gorgonian
coral J. fragilis (wet weight 628 g, dry weight 206 g) was ex-
tracted with a mixture of MeOH and CH2Cl2 (1:1) at room
temperature. The residue was partitioned between EtOAc
and H2O. The EtOAc layer was separated on silica gel and
eluted using hexane/EtOAc (stepwise, 50:1dpure EtOAc) to
yield 17 fractions AeQ, and one of these fractions (fraction
K) was further separated by gravity column with silica gel
and eluted using CH2Cl2/EtOAc (stepwise, 20:1dpure
EtOAc) to afford 23 fractions K1eK23. Fraction K7 was

P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
2603
purified by normal phase HPLC, using a mixture of CH2Cl2
and EtOAc to afford briarane 10 (0.9 mg, 15:1).
by monitoring the superoxide dismutase-inhibitable reduction
of ferricytochrome c.
3.3.9. Fragilide C (10)
Acknowledgements
White powder; mp 274e275 ꢁC; [a]D25 þ28 (c 0.05, CHCl3);
IR (neat) nmax 3385, 1789, 1740 cmꢀ1; for 13C (CDCl3,
100 MHz) and 1H (CDCl3, 400 MHz) NMR data, see Table 3;
ESIMS m/z 593 (MþNa)þ, 595 (Mþ2þNa)þ; HRESIMS m/z
593.1767 (calcd for C27H35ClO11þNa, 593.1765).
This research work was supported by grants from the
National Science Council (NSC 95-2320-B-291-001-MY2)
and by intramural funding from the NMMBA, Taiwan,
awarded to P.-J.S.
3.4. Single-crystal X-ray crystallography of excavatolide C
(5)39
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tet.2008.01.023.
Suitable colorless prisms of 5 were obtained from a solution
of EtOAc. The crystal (0.2ꢂ0.5ꢂ0.8 mm) belongs to the
monoclinic system, space group P21 (# 4), with a¼
ꢁ
˚
˚
˚
8.999(2) A, b¼14.718(3) A, c¼10.882(2) A, b¼94.91(3) , V¼
References and notes
3
3
˚
˚
1435.9(5) A , Z¼2, Dcalcd¼1.310 g/cm , l (Mo Ka)¼0.71073 A.
Intensity data were measured on a Rigaku AFC7S diffracto-
meter up to 2qmax of 52ꢁ. All 4363 reflections were collected.
The structure was solved by direct methods and refined by
a full-matrix least-squares procedure. The refined structural
model converged to a final R1¼0.0353; wR2¼0.0854 for
2935 observed reflections [I>2s(I)] and 369 variable
parameters.
1. Chen, W.-C.; Sheu, J.-H.; Fang, L.-S.; Hu, W.-P.; Sung, P.-J. Nat. Prod.
Res. 2006, 20, 748e753.
2. Chen, W.-C.; Chuang, L.-F.; Sheu, J.-H.; Hu, W.-P.; Chen, Y.-P.; Lin,
M.-R.; Fang, L.-S.; Fan, T.-Y.; Li, J.-J.; Sung, P.-J. Platax 2006, 3, 9e16.
3. Sung, P.-J.; Hu, W.-P.; Fang, L.-S.; Fan, T.-Y.; Wang, J.-J. Nat. Prod. Res.
2005, 19, 689e694.
4. Sung, P.-J.; Hu, W.-P.; Wu, S.-L.; Su, J.-H.; Fang, L.-S.; Wang, J.-J.; Sheu,
J.-H. Tetrahedron 2004, 60, 8975e8979.
5. Sung, P.-J.; Chao, C.-H.; Chen, Y.-P.; Su, J.-H.; Hu, W.-P.; Sheu, J.-H.
Tetrahedron Lett. 2006, 47, 167e170.
3.5. Single-crystal X-ray crystallography of excavatolide E
6. Sung, P.-J.; Chen, Y.-P.; Hwang, T.-L.; Hu, W.-P.; Fang, L.-S.; Wu, Y.-C.;
Li, J.-J.; Sheu, J.-H. Tetrahedron 2006, 62, 5686e5691.
7. Chen, Y.-P.; Wu, S.-L.; Su, J.-H.; Lin, M.-R.; Hu, W.-P.; Hwang, T.-L.;
Sheu, J.-H.; Fan, T.-Y.; Fang, L.-S.; Sung, P.-J. Bull. Chem. Soc. Jpn.
2006, 79, 1900e1905.
(6)39
Suitable colorless prisms of 6 were obtained from a solution of
EtOAc. The crystal (0.68ꢂ0.55ꢂ0.50 mm) belongs to the ortho-
˚
8. Sung, P.-J.; Tsai, W.-T.; Chiang, M. Y.; Su, Y.-M.; Kuo, J. Tetrahedron
2007, 63, 7582e7588.
9. Su, Y.-M.; Fan, T.-Y.; Sung, P.-J. Nat. Prod. Res. 2007, 21, 1085e1090.
10. Sung, P.-J.; Chiang, M. Y.; Tsai, W.-T.; Su, J.-H.; Su, Y.-M.; Wu, Y.-C.
Tetrahedron 2007, 63, 12860e12865.
11. Sung, P.-J.; Tsai, W.-T.; Lin, M.-R.; Su, Y.-D.; Pai, C.-H.; Chung, H.-M.;
Su, J.-H.; Chiang, M. Y. Chem. Lett. 2008, 37, 88e89.
12. Sung, P.-J.; Fan, T.-Y. Heterocycles 2003, 60, 1199e1202.
13. Sung, P.-J.; Fan, T.-Y.; Fang, L.-S.; Wu, S.-L.; Li, J.-J.; Chen, M.-C.;
Cheng, Y.-M.; Wang, G.-H. Chem. Pharm. Bull. 2003, 51, 1429e1431.
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Biochem. Syst. Ecol. 2004, 32, 111e113.
rhombic system, space group P212121 (# 19), with a¼6.799(3) A,
3
˚
˚
˚
b¼18.512(6) A, c¼19.594(6) A, V¼2466.4(14) A , Z¼4,
3
˚
Dcalcd¼1.256 g/cm , l (Mo Ka)¼0.71073 A. Intensity data
were measured on a Rigaku AFC7S diffractometer up to 2qmax
of 52ꢁ. All 4132 reflections were collected. The structure was
solved by direct methods and refined by a full-matrix least-
squares procedure. The refined structural model converged to a
final R1¼0.0352; wR2¼0.0885 for 3365 observed reflections
[I>2s(I)] and 310 variable parameters.
3.6. (S)- and (R)-MTPA esters of excavatolide E (6)
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was added (ꢀ)-a-methoxy-a-(tri-fluoromethyl)phenylacetyl
(MTPA) chloride at room temperature for 6 h. The reaction
mixture was concentrated to dryness under reduced pressure
and purified by a short silica gel column with hexane/EtOAc
(4:1) to give (S)-MTPA ester 6a (2.4 mg). The (R)-MTPA ester
6b (1.8 mg) was prepared from (þ)-MTPA chloride using the
same method. Selected Dd values are shown in Figure 7.
18. Sung, P.-J.; Fang, L.-S.; Chen, Y.-P.; Chen, W.-C.; Hu, W.-P.; Ho, C.-L.;
Yu, S.-C. Biochem. Syst. Ecol. 2006, 34, 64e70.
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3.7. Human neutrophil superoxide generation
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Human neutrophils were obtained by means of dextran sed-
imentation and Ficoll centrifugation. Superoxide generation
was carried out according to the procedures described previ-
ously.40,41 Briefly, superoxide anion production was assayed

2604
P.-J. Sung et al. / Tetrahedron 64 (2008) 2596e2604
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