NPC
2013
Vol. 8
No. 8
1037 - 1040
Natural Product Communications
Natural Clovanes from the Gorgonian Coral Rumphella antipathies
a,b
a,b
c
Hsu-Ming Chung , Wei-Hsien Wang , Tsong-Long Hwang , Yang-Chang Wu
d,e,f,*
and Ping-Jyun Sung
a,b,g,h,*
a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan
National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan
c
Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan
d
School of Pharmacy, College of Pharmacy, China Medical University, Taichung 404, Taiwan
e
Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung 404, Taiwan
f
Center for Molecular Medicine, China Medical University Hospital, Taichung 404, Taiwan
g
Graduate Institute of Marine Biotechnology and Department of Life Science and Institute of Biotechnology,
National Dong Hwa University, Pingtung 944, Taiwan
h
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b
yachwu@mail.cmu.edu.tw; pjsung@nmmba.gov.tw
th
th
Received: March 29 2013; Accepted: May 13 , 2013
Three natural clovane-related sesquiterpenoids, 2-acetoxyclovan-9-ol (1), 9-acetoxyclovan-2-ol (2) and clovan-2,9-diol (3), were isolated from the
gorgonian coral Rumphella antipathies. The structures of clovanes 1-3 were elucidated by spectroscopic methods and by comparison of the spectral data with
those of known clovane analogues. This is the first time that clovanes 1-3 have been isolated from a natural source. Clovanes 1 and 2 displayed inhibitory
effects on the generation of superoxide anions and the release of elastase by human neutrophils.
Keywords: Clovane, Rumphella antipathies, Anti-inflammatory, Superoxide anion, Elastase.
The search for bioactive natural products from marine organisms
has been remarkably successful, and octocorals have been
demonstrated to be rich sources of interesting natural products
[1-3]. In continuation of our search for new natural substances from
marine invertebrates collected off the waters of Taiwan at the
intersection of the Kuroshio current and the South China Sea
surface current, the chemical constituents of the organic extract of
gorgonian coral Rumphella antipathies (phylum Cnidaria, class
Anthozoa, order Gorgonacea, suborder Holaxonia, family
Gorgoniidae) [4,5] were studied, which displayed meaningful
signals in NMR studies. Previous chemical investigations of
R. antipathies have yielded a series of caryophyllane- and
clovane-type sesquiterpenoid analogues [6-18]. In further studies of
the organic extract of R. antipathies, three natural clovane-related
sesquiterpenoids, 2-acetoxyclovan-9-ol (1) [19,20], 9-acetoxyclovan-2-ol (2) [21] and clovane-2,9-diol (3) [22], were
isolated. This is the first time that clovanes 1-3 have been isolated
from a natural source. In this paper, we report the isolation,
structure determination and bioactivity of clovanes 1-3 (Figure 1).
14
13
H
6
4
15
12
3
2
7
8
5
1
9
11
10
R2
1: R1 = OAc, R2 =-OH
2: R1 = OH, R2 =-OAc
3: R1 = OH, R2 = -OH
-1
spectrum of 1 showed bands at 3462 and 1738 cm , consistent with
1
the presence of hydroxy and ester carbonyl groups. From the H and
13
C NMR spectra, 1 was found to possess an acetoxy group
(H 2.04, 3H, s; C 171.0, acetate carbonyl; 21.3, acetate methyl).
1
1
From the H- H COSY spectrum of 1, it was possible to establish
the separate spin systems of H-2/H2-3, H-5/H2-6/H2-7 and H-9/H210/H2-11 (Figure 2). These data, together with the key HMBC
correlations between protons and quaternary carbons of 1, such as
H-3, H-5, H2-6, H2-10, H2-11, H2-12/C-1; H2-3, H-5, H2-6, H3-13,
H3-14/C-4; and H2-6, H2-7, H2-12, H3-15/C-8, permitted elucidation
of the main carbon skeleton (Figure 2). The tertiary methyl at C-8
was confirmed by the HMBC correlations between H3-15/C-7, -8,
-9, -12. Moreover, the two tertiary methyls at C-4 were elucidated
by the HMBC correlations between H3-13/C-3, -4, -5, -14 and
H3-14/C-3, -4, -5, -13. In addition, the carbon signal at C 171.0 (s)
was correlated with the signal of methyl protons at H 2.04 (3H, s)
in the HMBC spectrum and was assigned as the carbon atom of the
acetate carbonyl. The acetoxy group positioned at C-2, an oxygen1
1
bearing methine, as indicated by the key H- H COSY correlations
and characteristic NMR signal analysis. Thus, from the reported
data, the proposed skeleton of 1 was suggested to be a clovane
derivative with three rings.
13
14
15
4
R1
1
Figure 1: The structures of clovanes 1-3.
The molecular formula of clovane 1 was established as C17H28O3
(four degrees of unsaturation) from a sodiated molecule at m/z 303
in the ESIMS spectrum and further supported by the HRESIMS ion
at m/z 303.1933 (calcd for C17H28O3 + Na, 303.1936). The IR
2
8
5
12
: 1H-1H COSY
: HMBC
9
OH
CH3CO
O
1
1
Figure 2: The H- H COSY and selective HMBC correlations (protons → quaternary carbons)
for clovane 1.
1038 Natural Product Communications Vol. 8 (8) 2013
The relative configuration of 1 was elucidated mainly from a
NOESY experiment as being compatible with that of 1 ascertained
using molecular mechanics calculations (MM2) [23], which
suggested the most stable conformation, as shown in
Figure 3, in which the close contacts of atoms in space calculated
were consistent with the NOESY correlations. Because of the
-orientation of H-5, and the fact that this proton was found to show
correlations with H3-14, but not with H-2, Me-14 was indicated to
be located on the same face as H-5. H-2 was found to be correlated
with H3-13 and H2-12, indicating that the H-2 and C-12 methylene
bridge between C-1/8 was -oriented. H3-15 showed correlations
with H2-12, confirming the -orientation of this tertiary methyl. It
was found that H-9 showed correlations with H2-10, H3-15 and one
of the C-7 methylene protons (H 1.13). Consideration of molecular
models revealed that H-9 is reasonably close to H2-10, H3-15 and
one of the C-7 methylene protons when H-9 is -oriented. Based on
the above findings, the structure of 1 was established and the chiral
carbons for 1 were assigned as 1S*, 2S*, 5S*, 8R*, 9R*. It was
found that the spectral data of 1 are identical to those of a known
synthetic compound 2-acetoxyclovan-9-ol, which was prepared
by Kikuchi’s group [19,20]. However, clovane 1 has not been
isolated previously from natural sources.
13
7
14
4
15
5S*
8R*
1S*
12
2S*
9R*
10
H/H
H-2/H-12
H-2/H-12'
H-2/H3-13
H-5/H3-14
H-7/H-9
H-9/H-10
H-9/H-10'
H-9/H3-15
H-12/H3-15
H-12'/H3-15
Distance (Å)
2.35
2.82
2.29
2.36
2.43
2.45
2.55
2.57
2.53
2.55
Figure 3: The stereoview of 1 (generated from computer modeling) and the calculated
distances (Å) between selected protons with key NOESY correlations.
The natural clovane 9-acetoxyclovan-2-ol (2) was found to have
the same molecular formula as 1, C17H28O3, as determined by the
HRESIMS ion at m/z 303.1934 (calcd for C17H28O3 + Na,
303.1936), with four degrees of unsaturation. The spectral data (IR,
1
13
MS, H and C NMR) of 2 were similar to those of 1. However, the
26
polarity and optical rotation value ([]D -103, c 0.03, CHCl3) of 2
24
were substantially different to those of 1 ([]D -7, c 0.09, CHCl3),
1
13
indicating that clovanes 1 and 2 are isomers. In the H and C
NMR spectrum of 2, an acetoxy group (H 2.05, 3H, s; C 171.0,
acetate carbonyl; 21.3, acetate methyl) was observed. From the
1
1
H- H COSY spectrum, it was possible to establish the proton
sequences from H-2/H2-3, H-5/H2-6/H2-7 and H-9/H2-10/H2-11
13
(Figure 4). The C NMR signal at C 171.0 (s) correlated with the
signal of the methyl protons at H 2.05 in the HMBC spectrum
(Figure 4) and was consequently assigned as the carbon atom of the
1
1
acetate carbonyl. The correlations observed in the H- H COSY
experiment of 2 further demonstrated that the hydroxy and acetoxy
groups should be positioned at the oxymethines C-2 (H 3.82, dd,
J = 10.0, 5.6 Hz; C 80.9) and C-9 (H 4.55, 1H, br s; C 76.9),
1
1
respectively, as indicated by the key H- H COSY correlations and
characteristic NMR signal analysis, although no HMBC correlation
1
1
was observed between H-9 and the acetate carbonyl. The H- H
COSY and HMBC correlations observed fully supported the
locations of the functional groups (Figure 4), and hence clovane 2
was assigned as structure 2 with same relative stereochemistry as 1.
Because of the chiral carbons that 2 has in common with 1, the
relative configurations of these were assigned as 1S*, 2S*, 5S*, 8R*,
Chung et al.
14
13
15
4
8
: 1H-1H COSY
: HMBC
5
12
1
9
2
OCCH3
O
HO
1
1
Figure 4: The H- H COSY and selective HMBC correlations (protons → quaternary carbons)
for clovane 2.
13
14
4
7
5S*
1S*
12
8R*
2S*
10
9R*
15
H/H
H-2/H-12
H-2/H-12'
H-2/H3-13
H-5/H3-14
H-7/H-9
H-9/H-10
H-9/H-10'
H-9/H3-15
H-12/H3-15
H-12'/H3-15
Distance (Å)
2.37
2.80
2.29
2.37
2.40
2.43
2.56
2.55
2.53
2.57
Figure 5: The stereoview of 2 (generated from computer modeling) and the calculated
distances (Å) between selected protons with key NOESY correlations.
9R*, and this rationalization was supported by a NOESY
experiment (Figure 5). It was found that the structure of 2 was
identical to that of an unnamed known synthetic clovane derivate
[21]. Clovane 2 was isolated for the first time from a natural source.
The present study also led to the isolation of a new natural clovane
3 [22]. Clovane 3 has the molecular formula C15H24O2 as
determined by the HRESIMS ion at m/z 237.1851 (calcd for
C15H26O2 - H, 237.1849). The IR spectrum of 3 indicated the
-1
presence of a hydroxy group at 3407 cm . It was found that the
spectral data of 3 are identical to those of a known synthetic
compound clovan-2,9-diol, which was prepared by Collado’s
group [22]. However, clovane 3 has not been isolated previously
from natural sources.
The in vitro anti-inflammatory effects of clovanes 1-3 were tested
and clovane 1 displayed a moderate inhibitory effect on the
generation of superoxide anions (inhibition rate 29.0%) by human
neutrophils. Moreover, clovanes 1 and 2 were found to exhibit
moderate inhibitory effects on the release of elastase (inhibition
rates 24.9 and 28.5%, respectively) by human neutrophils [24].
Experimental
General: Optical rotations were measured on a JASCO P-1010
digital polarimeter. Infrared spectra were recorded on a VARIAN
-1
DIGLAB FTS 1000 FT-IR spectrometer; peaks are reported in cm .
The NMR spectra were recorded on a VARIAN MERCURY Plus
400 spectrometer using the residual CHCl3 signal (H 7.26 ppm) as
1
13
the internal standard for H NMR and CDCl3 (C 77.1 ppm) for C
NMR. Coupling constants (J) are given in Hz. ESIMS and
HRESIMS were recorded on either a BRUKER APEX II or a
FINNIGAN/THERMO QUEST MAT 95XL mass spectrometer.
Column chromatography was performed on silica gel (230-400
mesh, Merck, Darmstadt, Germany). TLC was carried out on
precoated Kieselgel 60 F254 (0.25 mm, Merck); spots were
visualized by spraying with 10% H2SO4 solution followed by
heating. HPLC was performed using a system comprised of a
HITACHI L-7100 pump, a HITACHI L-7455 photodiode array
detector and a RHEODYNE injection port. A normal phase semipreparative column (Hibar 250×10 mm, LiChrospher Si 60, 5 m)
was used for HPLC.
Natural clovanes from Rumphella antipathies
Animal material: Specimens of the gorgonian coral Rumphella
antipathies (Nutting) were collected by hand using scuba equipment
off the coast of southern Taiwan and stored in a freezer until
extraction. This organism was identified by comparison with previous
descriptions [4,5]. A voucher specimen (specimen no. NMMBATWGC-010) was deposited in the National Museum of Marine
Biology and Aquarium (NMMBA), Taiwan.
Extraction and isolation: Sliced bodies of R. antipathies (wet
weight 402 g, dry weight 144 g) were extracted with a mixture of
methanol (MeOH) and dichloromethane (CH2Cl2) (1:1). The extract
was partitioned with ethyl acetate (EtOAc) and H2O. The EtOAc
layer was separated by silica gel and eluted using n-hexane/EtOAc
(stepwise, 25:1-pure EtOAc) to yield 29 fractions. Every fraction
1
was checked by H NMR spectroscopy. Fraction 17 was purified by
normal phase HPLC (NP-HPLC), using a mixture of n-hexane
and EtOAc (4:1) as the mobile phase to afford 1 (2.6 mg) and 2
(0.5 mg). Fraction 22 was separated by NP-HPLC, using a mixture
of CH2Cl2 and EtOAc (10:1) to afford 3 (2.8 mg).
2-Acetoxyclovan-9-ol (1)
24
[α]D: -7 (c 0.09, CHCl3).
-1
IR (neat): νmax 3462, 1738 cm .
1
H NMR (400 MHz, CDCl3): H 4.84 (1H, dd, J = 8.8, 5.6 Hz, H-2),
3.32 (1H, br s, H-9), 2.04 (3H, s, acetate methyl), 1.98 (1H, m,
H-10), 1.78 (1H, dd, J = 12.4, 5.6 Hz, H-3), 1.63 (1H, m, H-10'),
1.58 (1H, m, H-11), 1.54 (1H, dd, J = 12.4, 8.8 Hz, H-3'), 1.53 (1H,
d, J = 13.2 Hz, H-12), 1.47 (1H, m, H-5), 1.47 (1H, m, H-6), 1.40
(1H, m, H-6'), 1.38 (1H, m, H-7), 1.21 (1H, m, H-11'), 1.13 (1H, m,
H-7'), 1.03 (1H, d, J = 13.2 Hz, H-12'), 1.05 (3H, s, H3-14), 0.94
(3H, s, H3-15), 0.91 (3H, s, H3-13).
13
C NMR (100 MHz, CDCl3): C 171.0 (acetate carbonyl), 82.1
(CH-2), 74.9 (CH-9), 50.2 (CH-5), 44.4 (C-1), 44.3 (CH2-3), 38.0
(C-4), 35.4 (CH2-12), 34.6 (C-8), 33.0 (CH2-7), 31.4 (CH3-14), 28.2
(CH3-15), 27.3 (CH2-11), 26.3 (CH2-10), 25.3 (CH3-13), 21.3
(acetate methyl), 20.7 (CH2-6).
+
ESIMS: m/z (%) = 303 [M + Na] .
+
HRESIMS: m/z [M + Na] calcd for C17H28O3 + Na: 303.1936;
found 303.1933.
9-Acetoxyclovan-2-ol (2)
Natural Product Communications Vol. 8 (8) 2013 1039
(C-4), 36.5 (CH2-12), 33.7 (C-8), 32.8 (CH2-7), 31.4 (CH3-14), 28.1
(CH3-15), 27.0 (CH2-11), 25.4 (CH3-13), 23.4 (CH2-10), 21.3
(acetate methyl), 20.5 (CH2-6).
+
ESIMS: m/z (%) = 303 [M + Na] .
+
HRESIMS: m/z [M + Na] calcd for C17H28O3 + Na: 303.1936;
found 303.1934.
Clovan-2,9-diol (3)
White powder.
MP: 154-155°C (ref. [22], MP: 172-174°C).
25 25
[α]D: +6 (c 0.007, CHCl3) (ref. [22], [α]D +15.3 (c 0.0028, CHCl3).
-1
IR (neat): νmax 3407 cm .
1
H NMR (400 MHz, CDCl3): H 3.81 (1H, dd, J = 10.0, 5.6 Hz,
H-2), 3.21 (1H, dd, J = 10.8, 4.8 Hz, H-9), 1.77 (1H, m, H-10), 1.72
(1H, dd, J = 12.0, 5.6 Hz, H-3), 1.55 (1H, m, H-10'), 1.53 (1H, m,
H-11), 1.49 (1H, dd, J = 12.0, 10.0 Hz, H-3'), 1.40 (2H, m, H2-6),
1.34 (1H, dd, J = 10.4, 4.4 Hz, H-5), 1.28 (1H, m, H-11'), 1.24 (1H,
d, J = 12.8 Hz, H-12), 1.19 (2H, m, H2-7), 1.05 (3H, s, H3-14), 1.04
(1H, d, J = 12.8 Hz, H-12'), 1.00 (3H, s, H3-15), 0.86 (3H, s, H3-13).
13
C NMR (100 MHz, CDCl3): C 80.3 (CH-2), 78.0 (CH-9), 51.3
(CH-5), 47.9 (CH2-3), 43.9 (C-1), 42.0 (CH2-12), 37.2 (C-4), 35.2
(C-8), 31.5 (CH3-14), 31.1 (CH2-11), 28.7 (CH3-15), 27.8 (CH2-10),
27.2 (CH2-7), 25.5 (CH3-13), 20.3 (CH2-6).
+
ESIMS: m/z (%) = 237 [M – H] .
+
HRESIMS: m/z [M – H] calcd for C15H26O2 – H: 237.1849; found
237.1851.
Molecular mechanics calculations: Implementation of the MM2
force field [23] in CHEM3D PRO software from CambridgeSoft
Corporation (Cambridge, MA, USA; ver. 9.0, 2005) was used to
calculate the molecular models.
Generation of superoxide anions and release of elastase by
human neutrophils: Human neutrophils were obtained by means of
dextran sedimentation and Ficoll centrifugation. Measurements of
superoxide anion generation and elastase release were carried out
according to previously described procedures [25-31]. Briefly,
superoxide anion production was assayed by monitoring the
superoxide dismutase-inhibitable reduction of ferricytochrome c.
Elastase release experiments were performed using MeO-Suc-AlaAla-Pro-Valp-nitroanilide as the elastase substrate.
26
[α]D: -103 (c 0.03, CHCl3).
–1
IR (neat): νmax 3349, 1738 cm .
1
H NMR (400 MHz, CDCl3): H 4.55 (1H, br s, H-9), 3.82 (1H, dd,
J = 10.0, 5.6 Hz, H-2), 2.05 (3H, s, acetate methyl), 1.96 (1H, dddd,
J = 14.6, 14.6, 4.8, 3.2 Hz, H-10), 1.74 (1H, dd, J = 12.0, 5.6 Hz,
H-3), 1.70 (1H, m, H-10'), 1.56 (1H, d, J = 13.6 Hz, H-12), 1.54
(1H, m, H-11), 1.51 (1H, dd, J = 12.0, 10.0 Hz, H-3'), 1.44 (1H, m,
H-5), 1.44 (2H, m, H2-6), 1.43 (1H, m, H-7), 1.19 (1H, m, H-7'),
1.11 (1H, m, H-11'), 1.04 (3H, s, H3-14), 1.00 (1H, d, J = 13.6 Hz,
H-12'), 0.87 (3H, s, H3-13). 0.87 (3H, s, H3-15).
13
C NMR (100 MHz, CDCl3): C 171.0 (acetate carbonyl), 80.9
(CH-2), 76.9 (CH-9), 50.5 (CH-5), 44.7 (CH2-3), 44.0 (C-1), 37.2
Acknowledgments - This research was supported by grants from
the National Museum of Marine Biology and Aquarium; the
National Dong Hwa University; the Division of Marine
Biotechnology, Asia-Pacific Ocean Research Center, National Sun
Yat-sen University (Grant No. 00C-0302-05); the Department of
Health, Executive Yuan, Taiwan (Grant No. DOH101-TD-C-111002); and the National Science Council (Grant No. NSC 102-2325B-291-001, 101-2325-B-291-001, 100-2325-B-291-001 and 1012320-B-291-001-MY3), Taiwan, awarded to Y.-C.W. and P.-J.S.
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