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Rumphellclovanes C–E, new clovane-type sesquiterpenoids from the gorgonian coral Rumphella antipathies
Tetrahedron 69 (2013) 2740e2744
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Rumphellclovanes CeE, new clovane-type sesquiterpenoids from the gorgonian coral Rumphella antipathies Hsu-Ming Chung a, b, Jui-Hsin Su a, b, c, Tsong-Long Hwang d, Jan-Jung Li b, Jih-Jung Chen e, Yung-Husan Chen b, Yu-Chia Chang b, f, Yin-Di Su a, b, Yu-Hsin Chen b, c, Lee-Shing Fang g, Jyh-Horng Sheu a, f, Wei-Hsien Wang a, b, *, Ping-Jyun Sung a, b, c, h, * a
Department of Marine Biotechnology and Resources and Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan Graduate Institute of Marine Biotechnology and Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan d Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan e Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 907, Taiwan f Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan g Department of Sport, Health, and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan h Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 January 2013 Received in revised form 25 January 2013 Accepted 29 January 2013 Available online 8 February 2013
Three new clovane-related sesquiterpenoids, rumphellclovanes CeE (1e3), and a new natural clovane, clovan-2,9-dione (4), were isolated from a gorgonian coral Rumphella antipathies. The structures of clovanes 1e4 were elucidated by spectroscopic methods and by comparison of the spectral data with those of related analogues. Rumphellclovane C (1) was proven to be the first clovane-type sesquiterpenoid containing an ε-lactone moiety. A plausible biosynthetic pathway between clovanes 1 and 2 was proposed. Clovane 4 displayed significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Clovane Rumphellclovane Rumphella Superoxide anion Elastase
1. Introduction The gorgonian corals of genus Rumphella (phylum Cnidaria, class Anthozoa, order Gorgonacea, suberorder Holaxonia, family Gorgoniidae),1,2 which are distributed in the tropical waters of the IndoPacific Ocean, have been investigated for ecological and medical uses.3,4 As part of our ongoing investigation into the isolation of new substances from the marine invertebrates collected in the waters of Taiwan, the chemical constituents of a gorgonian coral Rumphella antipathies was studied for its organic extract, which displayed meaningful signals in NMR studies. Previous chemical investigations on R. antipathies have yielded a series of caryophylleneand clovane-type sesquiterpenoid analogues, including kobusone,5
* Corresponding authors. Tel.: þ886 8 882 5001x5047; fax: þ886 8 882 4488 (W.-H.W.); tel.: þ886 8 882 5037; fax: þ886 8 882 5087 (P.-J.S.); e-mail addresses: [email protected] (W.-H. Wang), [email protected] (P.-J. Sung). 0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.01.087
isokobusone,6 rumphellolides AeI,7e10 rumphellatins AeD,11e13 rumphellaone A,14 2b-hydroxyclovan-9-one,15 9a-hydroxyclovan2-one16 and rumphellclovanes A and B.15,16 In the further studies on the chemical constituents of R. antipathies, four clovane-related derivatives, including three new compounds, rumphellclovanes CeE (1e3), and a new natural product, clovan-2,9-dione (4),17 were isolated. In this paper, we report the isolation, structure determination, and bioactivity of clovanes 1e4. 2. Results and discussion Rumphellclovane C (1) was isolated as a yellowish oil that gave a pseudomolecular ion (MþNa)þ at m/z 303.1934 in the HRESIMS, indicating the molecular formula C17H28O3 (calcd for C17H28O3þNa, 303.1936) and implying four degrees of unsaturation. An IR absorption at 1719 cm1 suggested the presence of an ester group in 1. The 13C NMR and DEPT spectra of 1 (Table 1) showed that clovane 1 was consisted of 17 carbons, including four methyls, seven sp3
H.-M. Chung et al. / Tetrahedron 69 (2013) 2740e2744 14 13 4 2
7
6
5
3
2'
15
H
12
11
CH3CH2O
H
H
8
O
1
1'
2741
O
9 10
O
O
R 2:R=OCH2CH3, 5:R=OH
1
OH
HO 3
H
H
O
OH HO
O 4
6
Table 1 1 H and 13C NMR data, 1He1H COSY and HMBC correlations for clovane 1
d Ha
C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
3.46 dd (7.2, 6.8)c 1.74 dd (12.0, 6.8); 1.51 dd (12.0, 7.2) 1.54 m 1.56 m 2.03 ddd (16.8, 4.0, 3.2); 1.83 dd (16.8, 3.2)
2.77 1.90 1.98 0.88 1.07 1.40 3.43 3.48 1.16
2-OCH2CH3 1' 2' a b c
dCb
m dd (14.8, 3.6) d (14.8); 1.61 d (14.8) s s s dq (8.8, 6.8) (H-1’a) dq (8.8, 6.8) (H-1’b) dd (6.8, 6.8) (H3-20 )
1
44.8 88.9 44.6 36.8 51.0 20.4 35.5 80.1 174.8 32.8 31.0 47.0 25.5 31.0 32.2 66.0
(C) (CH) (CH2) (C) (CH) (CH2) (CH2) (C) (C) (CH2) (CH2) (CH2) (CH3) (CH3) (CH3) (CH2)
15.6
(CH3)
He1H COSY
HMBC
H2-3 H-2
C-1, -3, -11, -12, -10 C-1, -2, -4, -5, -13, -14
H2-6 H-5, H2-7 H2-6
C-1, -3, -4, -6, -11, -13, -14 C-1, -4 C-5, -6, -8
H2-11 H2-10
C-1, C-1, C-1, C-3, C-3, C-7, C-2, C-2, C-10
H-1’b, H3-20 H-1’a, H3-20 H2-10
-9 -5, -5, -4, -4, -8, -20 -20
-9, -7, -5, -5, -9,
-10 -8, -11, -15 -14 -13 -12
Spectrum measured at 400 MHz in CDCl3 at 25 C. Spectrum measured at 100 MHz in CDCl3 at 25 C. J values (in hertz) in parentheses.
methylenes, two sp3 methines, three sp3 quaternary carbons and an sp2 quaternary carbon. From the 13C NMR data, a degree of unsaturation was accounted for (dC 174.8, C-9, an ester carbonyl) and 1 must be a tricyclic compound. From the 1He1H COSY spectrum of 1 (Table 1 and Fig. 1), it was possible to establish the spin systems that map out the proton sequences from H-2/H2-3, H-5/H2-6/H2-7, H210/H2-11 and H2-10 /H3-20 , which were assembled with assistance of an HMBC experiment. The key HMBC correlations between protons and quaternary carbons of 1 (Table 1 and Fig. 1), such as H-2, H2-3, H-5, H2-6, H2-10, H2-11, H2-12/C-1; H2-3, H-5, H2-6, H3-13, H3-14/C-
14
13
4 2
1'
O
15 8 5
12
O
1
: 1H-1H COSY : HMBC
9 O
2' Fig. 1. The 1He1H COSY and selective HMBC correlations (protons/quaternary carbons) for clovane 1.
4; H2-7, H2-12, H3-15/C-8; and H2-10, H2-11/C-9, permitted the elucidation of the main carbon skeleton. The tertiary methyl at C-8 was confirmed by the HMBC correlations between H3-15/C-7, -8, -9, -12 and H2-12/C-15. 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. The C-12 methylene bridge between C-1 and C-8 was linked by the HMBC correlations between H-2/C-12, H2-12/C-15 and H3-15/C-12. Based on the consideration of molecular formula, an additional oxygen atom had to be placed between C-8 and C-9 to form an ε-lactone moiety. A long-range J4-HMBC correlation between H3-15/C-9 further supporting this rationalization. The position of an ethoxy group at C-2 was confirmed by the HMBC correlations between the oxymethine proton at dH 3.46 (H-2)/dC 66.0 (CH2-10 ), and by the oxymethylene protons at dH 3.43, 3.48 (H2-10 )/dC 88.9 (CH-2). The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1 ascertained using molecular mechanics calculations (MM2),18 which suggested the most stable conformation, as shown in Fig. 2, in which the close contacts of atoms in space calculated were consistent the NOESY correlations. Because of the b-orientation of H-5, and this proton was found to show correlations with H3-14, but not with H-2, suggesting that Me-14 is located on the same face of H-5. H-2 was found to correlate with H3-13 and H2-12, indicating that H-2 and C-
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14
13
15 4
2 1'
8
5
: 1H-1H COSY : HMBC
12
1
9 O
O
2' Fig. 3. The 1He1H COSY and selective HMBC correlations (protons/quaternary carbons) for clovane 2.
Fig. 2. The stereoview of 1 (generated from computer modelling) and the calculated distances ( A) between selected protons having key NOESY correlations.
12 methylene bridge between C-1/8 was a-oriented. H3-15 showed correlations with H2-12, confirming the a-orientation for this tertiary methyl. Based on the above findings, the structure of 1 was established and the chiral carbons for 1 were assigned as 1S*, 2S*, 5S*, 8R*. The new clovane, rumphellclovane D (2), was isolated as a yellowish oil and its molecular formula C17H28O2 was established by HRESIMS (m/z 265.2158, calcd for C17H28O2þH, 265.2162). The IR spectrum of 2 showed a strong band at 1706 cm1, consistent with the presence of a keto carbonyl. It was found that the 1H and 13C NMR data of 2 were similar to those of a known clovane analogue, 2b-hydroxyclovan-9-one (5),15,19 that was also isolated from R. antipathies,15 except that the signals corresponding to a hydroxy group at C-2 in 5 (dH 3.87, 1H, dd, J¼7, 5.5 Hz; dC 80.2, CH-2)19 were replaced by an ethoxy group in 2 (dH 3.45, 1H, dd, J¼8.0, 5.6 Hz; dC 87.1, CH-2; dH 3.37, 1H, dq, J¼8.8, 6.8 Hz, H-10 a; 3.48, 1H, dq, J¼8.8, 6.8 Hz, H-10 b; 1.15, 3H, dd, J¼6.8, 6.8 Hz, H3-20 ; dC 65.4, CH2-10 ; 15.5, CH3-20 , ethoxy group) (Table 2). The 1He1H COSY and HMBC correlations observed fully supported the locations of functional groups (Table 2 and Fig. 3), and hence rumphellclovane D (2) was assigned as the structure 2 with the same relative stereochemistry as in rumphellclovane C (1) and 2b-hydroxyclovan-9-one (5),15,19 because for the chiral carbons that 2 has in common with
clovanes 1 and 5 and the relative configurations of 2 were assigned as 1S*, 2S*, 5S*, 8R*. The 1H and 13C NMR chemical shifts and proton coupling constants match well, and are further supported by a NOESY experiment (Fig. 4).
Fig. 4. The stereoview of 2 (generated from computer modelling) and the calculated distances ( A) between selected protons having key NOESY correlations.
A plausible biosynthetic pathway for 1 from 2 was proposed and clovane 2 was further lactonized to 1 by BaeyereVilliger oxidation. To the best of our knowledge, clovane-type analogues like 1 containing an ε-lactone moiety has not been found previously.
Table 2 1 H and 13C NMR data, 1He1H COSY and HMBC correlations for clovane 2 C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
2-OCH2CH3 1' 2' a b c d
d Ha 3.45 (8.0, 5.6)c 1.77 dd (12.8, 5.6); 1.60 dd (12.8, 8.0) 1.72 dd (7.6, 7.6) 1.56 md 1.50 md
2.64 1.87 1.55 0.96 1.07 1.01 3.37 3.48 1.15
He1H COSY
dCb
ddd (16.0, 12.4, 8.0); 2.34 ddd (16.0, 6.8, 2.4) ddd (12.4, 12.4, 6.8); 1.70 ddd (12.4, 8.0, 2.4) md s s s dq (8.8, 6.8) (H-1’a) dq (8.8, 6.8) (H-1’b) dd (6.8, 6.8) (H3-20 )
Spectrum measured at 400 MHz in CDCl3 at 25 C. Spectrum measured at 100 MHz in CDCl3 at 25 C. J values (in hertz) in parentheses. Signals overlapped.
44.6 87.1 44.8 38.2 50.9 20.5 34.0 44.5 216.6 35.7 32.7 42.8 26.1 32.0 24.9 65.4
(C) (CH) (CH2) (C) (CH) (CH2) (CH2) (C) (C) (CH2) (CH2) (CH2) (CH3) (CH3) (CH3) (CH2)
15.5
(CH3)
1
HMBC
H2-3 H-2
C-4, -5, -11, -12, -10 C-1, -2, -4, -5, -13, -14
H2-6 H-5, H2-7 H2-6
C-1, -3, -4, -7, -13 C-1, -5, -7, -8 C-5, -6, -8
H2-11 H2-10
C-8, C-1, C-1, C-3, C-3, C-7, C-2, C-2, C-10
H-1’b, H3-20 H-1’a, H3-20 H2-10
-9, -2, -2, -4, -4, -8, -20 -20
-11 -5, -9, -10 -5, -7, -8, -9, -11 -5, -14 -5, -13 -9, -12
H.-M. Chung et al. / Tetrahedron 69 (2013) 2740e2744
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Rumphellclovane E (3) was isolated as a yellowish oil. The molecular formula of 3 was established as C15H24O3 (4 of unsaturation) from a sodium adduct at m/z 275 in the ESIMS spectrum and further supported by HRESIMS (m/z 275.1625, calcd for C15H24O3þNa, 275.1623). The presence of hydroxy and keto carbonyl groups were indicated by IR absorptions at 3433 and 1706 cm1. It was found that the spectral data of 3 were similar to those of a known clovane, clovan-2b,9a-diol (6), that was also isolated from Sindora summatrana,20 except that the signals corresponding to the C-12 methylene group in 6 (dH 0.91, 1H, br d, J¼12.5 Hz; 1.56, 1H, d, J¼12.5 Hz; dC 35.6)20 were replaced by a keto carbonyl in 3 (dC 219.4) (Table 3). Table 3 1 H and 13C NMR data, 1He1H COSY and HMBC correlations for clovane 3 C/H 1 2 3
2.18 dd (13.5, 5.0)
6 7
1.58 m 1.97 ddd (14.0, 14.0, 4.5); 1.66 m
8 9 10 11 12 13 14 15 b c d
He1H COSY
dCb
4.73 dd (12.0, 6.0)c 1.73 dd (14.0, 6.0); 1.53 dd (14.0, 12.0)
4 5
a
d Ha
3.93 br s 2.42 dddd (15.0, 15.0, 5.0, 3.0); 1.79 m 2.22 m; 1.62 m 0.78 s 1.07 s 1.12 s
1
HMBC
50.5 71.2 45.9
(C) (CH) (CH2)
H2-3 H-2
C-1, -11, -12 C-1, -2, -4, -5, -13, -14
36.9 57.4
(C) (CH)
H2-6
22.8 36.6
(CH2) (CH2)
H-5, H2-7 H2-6
C-4, -6, -11, -12, -13, -14 n.o.d C-6, -8, -9, -12
50.6 81.1 24.9
(C) (CH) (CH2)
H2-10 H-9, H2-11
C-12 n.o.
29.7 219.4 24.8 30.8 20.6
(CH2) (C) (CH3) (CH3) (CH3)
H2-10
C-5, -12 C-3, -4, -5, -14 C-3, -4, -5, -13 C-7, -8, -9, -12
2
8 : 1H-1H COSY : HMBC
12 O
9
Superoxide anion
Elastase release
IC50 (mg/mL)
IC50 (mg/mL)
Inh%a 14.071.76 16.886.13 2.731.78
2.720.93 0.800.21
Inh%a 22.514.47 12.885.40 21.323.81
6.730.85 31.955.92
Percentage of inhibition (Inh%) at a concentration of 10 mg/mL. DPI (diphenylene indonium) and elastatinal were used as positive control.
3. Experimental
15
1
1 2 3 4 DPIb Elastatinalb b
14
5
Table 4 Inhibitory effects of clovanes 1e4 on the generation of superoxide anion and the release of elastase by human neutrophils in response to FMLP/CB
a
The 2D NMR correlations observed fully supported the locations of functional groups (Table 3 and Fig. 5), and hence clovane 3 was assigned as the structure 3 with the same relative stereochemistry as in 6,20 because for the chiral carbons that 3 has in common with clovane 6 and were assigned as 1S*, 2S*, 5S*, 8S*. The 1H and 13C NMR chemical shifts and proton coupling constants match well, and are further supported by a NOESY experiment (Fig. 6).
4
The in vitro anti-inflammatory effects of clovanes 1e4 were tested (Table 4). Clovan-2,9-dione (4) was found to display significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils.
Compounds
Spectrum measured at 500 MHz in CDCl3 at 25 C. Spectrum measured at 125 MHz in CDCl3 at 25 C. J values (in hertz) in parentheses. n.o.¼not observed.
13
Fig. 6. The stereoview of 3 (generated from computer modelling) and the calculated distances ( A) between selected protons having key NOESY correlations.
OH
HO Fig. 5. The 1He1H COSY and selective HMBC correlations (protons/quaternary carbons) for clovane 3.
Our present study has also led to the isolation of a new natural clovane 4.17 Clovane 4 has the molecular formula C15H22O2 as determined by HRESIMS (m/z 235.1680, calcd for C15H22O2þH, 235.1693). The IR spectrum of 4 indicated the presence of keto carbonyl groups (1729 and 1708 cm1). It was found that the spectral data of 4 are identical with those of a known synthetic compound clovan-2,9-dione that was prepared by Collado’s group.17 However, clovane 4 has not been isolated previously from natural sources.
3.1. General experimental procedures Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer; peaks are reported in cm1. The NMR spectra were recorded on a Varian Mercury Plus 400 or on a Varian Inova 500 NMR spectrometers using the residual CHCl3 signal (dH 7.26 ppm) as internal standard for 1H NMR and CDCl3 (dC 77.1 ppm) for 13C NMR. Coupling constants (J) are given in hertz. ESIMS and HRESIMS were recorded on a Bruker APEX II or on a Finnigan/ Thermo Quest Mat 95XL mass spectrometers. Column chromatography was performed on silica gel (230e400 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 semi-preparative column (Hibar 25010 mm, LiChrospher Si 60, 5 mm) and a reverse phase semi-preparative column (Hibar 25010 mm, Purospher STAR RP-18e, 5 mm) were used for HPLC. 3.2. Animal material Specimens of the gorgonian coral R. antipathies (Nutting) were collected by hand using scuba equipment off the coast of the southern Taiwan and stored in a freezer until extraction. This organism was identified by comparison with previous descriptions.1,2 A voucher specimen (specimen no. NMMBA-TWGC-010) was
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H.-M. Chung et al. / Tetrahedron 69 (2013) 2740e2744
deposited in the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan.
release experiments were performed using MeO-Suc-Ala-Ala-ProValp-nitroanilide as the elastase substrate.
3.3. Extraction and isolation
Acknowledgements
Sliced bodies of the gorgonian coral R. antipathies (wet weight 402 g, dry weight 144 g) were extracted with a mixture of methanol (MeOH) and dichloromethane (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, 25:1epure EtOAc) to yield 29 fractions. Every fraction was checked by the 1H NMR spectra. Fraction 7 was purified by normal phase HPLC (NP-HPLC), using a mixture of n-hexane and EtOAc (9:1) as a mobile phase to afford 1 and 2. Fraction 18 was separated by NP-HPLC, using a mixture of n-hexane and EtOAc (5:1) to afford 4. Fraction 28 was separated by reverse phase HPLC using a mixture of MeOH and water (7:3) to afford 3.
This work was supported by grants from the National Dong Hwa University; the NMMBA (Grant No. 10120022); the Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, (Grant No. 00C-0302-05); and the National Science Council (Grant No. NSC 101-2325-B-291-001, 100-2325-B291-001 and 101-2320-B-291-001-MY3), Taiwan, awarded to P.-J.S.
3.3.1. Rumphellclovane C (1). Yellowish oil (0.4 mg); [a]25 D 45 (c 0.01, CHCl3); IR (neat) nmax 1718 cm1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data, see Table 1; ESIMS: m/z 303 (MþNa)þ; HRESIMS: m/z 303.1934 (calcd for C17H28O3þNa, 303.1936).
References and notes
3.3.2. Rumphellclovane D (2). Yellowish oil (10.9 mg); [a]25 D 63 (c 0.33, CHCl3); IR (neat) nmax 1706 cm1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data, see Table 2; ESIMS: m/z 265 (MþH)þ; HRESIMS: m/z 265.2158 (calcd for C17H28O2þH, 265.2162). 3.3.3. Rumphellclovane E (3). Yellowish oil (1.0 mg); [a]25 D 6 (c 0.05, CHCl3); IR (neat) nmax 3433, 1706 cm1; 1H (CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz) NMR data, see Table 3; ESIMS: m/z 275 (MþNa)þ; HRESIMS: m/z 275.1625 (calcd for C15H24O3þNa, 275.1623). 3.3.4. Clovan-2,9-dione (4). Yellowish oil (1.5 mg); [a]25 D 52 (c 0.08, CHCl3, Ref. 17, [a]25 D 97.0 (c 0.01325, CHCl3)); IR (neat) nmax 1729, 1708 cm1; 1H (CDCl3, 400 MHz) dH 2.52 (2H, m, H2-10), 2.40 (1H, d, J¼17.6 Hz, H-3), 2.23 (1H, d, J¼17.6 Hz, H-30 ), 1.97 (1H, dd, J¼6.8, 6.8 Hz, H-5), 1.75 (2H, m, H2-6), 1.67 (1H, m, H-11), 1.61 (2H, m, H2-12), 1.58 (1H, m, H-7), 1.49 (1H, m, H-110 ), 1.48 (1H, ddd, J¼14.0, 7.6, 7.6 Hz, H-70 ), 1.16 (3H, s, H3-14), 1.10 (3H, s, H3-13), 1.05 (3H, s, H3-15); 13C (CDCl3, 100 MHz) dC 220.6 (C-2), 214.5 (C-9), 52.4 (CH2-3), 49.9 (CH-5), 48.8 (C-1), 44.0 (C-8), 39.0 (CH2-12), 37.0 (C-4), 36.2 (CH2-10), 35.1 (CH2-11), 34.4 (CH2-7), 30.6 (CH3-14), 24.9 (CH3-13), 24.7 (CH3-15), 20.1 (CH2-6); ESIMS: m/z 235 (MþH)þ; HRESIMS: m/z 235.1680 (calcd for C15H22O2þH, 235.1699).17 3.4. Molecular mechanics calculations Implementation of the MM2 force field18 in CHEM3D PRO software from CambridgeSoft Corporation (Cambridge, MA, USA; ver. 9.0, 2005) was used to calculate molecular models. 3.5. Generation of superoxide anion 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.21e27 Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase
Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2013.01.087.
1. Bayer, F. M. Proc. Biol. Soc. Wash. 1981, 94, 902e947. 2. Fabricius, K.; Alderslade, P. Soft Corals and Sea Fansda Comprehensive Guide to the Tropical Shallow-water Genera of the Central-west Pacific, the Indian Ocean and the Red Sea, 1st ed.; Australian Institute of Marine Science: Queensland, Australia, 2001, pp 61, 214e215. 3. Puglisi, M. P.; Paul, V. J.; Biggs, J.; Slattery, M. Mar. Ecol.: Prog. Ser. 2002, 239, 105e114. 4. Nourry, M.; Urvois, P.-A.; Tomasoni, C.; Biard, J. F.; Verbist, J. F.; Roussakis, C. Anticancer Res. 1999, 19, 1881e1885. 5. Chuang, L.-F.; Fan, T.-Y.; Li, J.-J.; Sung, P.-J. Biochem. Syst. Ecol. 2007, 35, 470e471. 6. Chuang, L.-F.; Fan, T.-Y.; Li, J.-J.; Kuo, J.; Fang, L.-S.; Wang, W.-H.; Sung, P.-J. Platax 2007, 4, 61e67. 7. Sung, P.-J.; Chuang, L.-F.; Kuo, J.; Chen, J.-J.; Fan, T.-Y.; Li, J.-J.; Fang, L.-S.; Wang, W.-H. Chem. Pharm. Bull. 2007, 55, 1296e1301. 8. Sung, P.-J.; Chuang, L.-F.; Fan, T.-Y.; Chou, H.-N.; Kuo, J.; Fang, L.-S.; Wang, W.-H. Chem. Lett. 2007, 36, 1322e1323. 9. Hwang, T.-L.; Su, Y.-D.; Hu, W.-P.; Chuang, L.-F.; Sung, P.-J. Heterocycles 2009, 78, 1563e1567. 10. Sung, P.-J.; Su, Y.-D.; Hwang, T.-L.; Chuang, L.-F.; Chung, H.-M.; Chen, J.-J.; Li, J.-J.; Fang, L.-S.; Wang, W.-H. Chem. Lett. 2009, 38, 282e283. 11. Sung, P.-J.; Chuang, L.-F.; Kuo, J.; Fan, T.-Y.; Hu, W.-P. Tetrahedron Lett. 2007, 48, 3987e3989. 12. Sung, P.-J.; Chuang, L.-F.; Hu, W.-P. Bull. Chem. Soc. Jpn. 2007, 80, 2395e2399. 13. Sung, P.-J.; Su, Y.-D.; Hwang, T.-L.; Chuang, L.-F.; Chen, J.-J.; Li, J.-J.; Fang, L.-S.; Wang, W.-H. Chem. Lett. 2008, 37, 1244e1245. 14. Chung, H.-M.; Chen, Y.-H.; Lin, M.-R.; Su, J.-H.; Wang, W.-H.; Sung, P.-J. Tetrahedron Lett. 2010, 51, 6025e6027. 15. Chung, H.-M.; Chen, Y.-H.; Hwang, T.-L.; Chuang, L.-F.; Wang, W.-H.; Sung, P.-J. Tetrahedron Lett. 2010, 51, 2734e2736. 16. Chung, H.-M.; Hwang, T.-L.; Chen, Y.-H.; Su, J.-H.; Lu, M.-C.; Chen, J.-J.; Li, J.-J.; Fang, L.-S.; Wang, W.-H.; Sung, P.-J. Bull. Chem. Soc. Jpn. 2011, 84, 119e121. nchez, A. J.; Mobbs, D. J. Nat. Prod. 1998, 61, 17. Collado, I. G.; Hanson, J. R.; Macías-Sa 1348e1351 The HRESIMS data for clovane-2,9-dione (molecular formula C15H22O2) was reported as 234.1620 in this paper. However, the calculated data for this compound was reported as 252.2082 for C15H22O2. We suggested that the HRESIMS data for this compound should be examined. 18. Allinger, N. L. J. Am. Chem. Soc. 1977, 99, 8127e8134. 19. Heymann, H.; Tezuka, Y.; Kikuchi, T.; Supriyatna, S. Chem. Pharm. Bull. 1994, 42, 941e946. 20. Heymann, H.; Tezuka, Y.; Kikuchi, T.; Supriyatna, S. Chem. Pharm. Bull. 1994, 42, 138e146. 21. Yu, H.-P.; Hsieh, P.-W.; Chang, Y.-J.; Chung, P.-J.; Kuo, L.-M.; Hwang, T.-L. Free Radical Biol. Med. 2011, 50, 1737e1748. 22. Hwang, T.-L.; Wang, C.-C.; Kuo, Y.-H.; Huang, H.-C.; Wu, Y.-C.; Kuo, L.-M.; Wu, Y.-H. Biochem. Pharmacol. 2010, 80, 1190e1200. 23. Hwang, T.-L.; Su, Y.-C.; Chang, H.-L.; Leu, Y.-L.; Chung, P.-J.; Kuo, L.-M.; Chang, Y.-J. J. Lipid Res. 2009, 50, 1395e1408. 24. Hwang, T.-L.; Li, G.-L.; Lan, Y.-H.; Chia, Y.-C.; Hsieh, P.-W.; Wu, Y.-H.; Wu, Y.-C. Free Radical Biol. Med. 2009, 46, 520e528. 25. Hwang, T.-L.; Leu, Y.-L.; Kao, S.-H.; Tang, M.-C.; Chang, H.-L. Free Radical Biol. Med. 2006, 41, 1433e1441. 26. Hwang, T.-L.; Yeh, S.-H.; Leu, Y.-L.; Chern, C.-Y.; Hsu, H.-C. Br. J. Pharmacol. 2006, 148, 78e87. 27. Hwang, T.-L.; Hung, H.-W.; Kao, S.-H.; Teng, C.-M.; Wu, C.-C.; Cheng, S.-J. Mol. Pharmacol. 2003, 64, 1419e1427.
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Rumphellclovanes CeE, new clovane-type sesquiterpenoids from the gorgonian coral Rumphella antipathies Hsu-Ming Chung a, b, Jui-Hsin Su a, b, c, Tsong-Long Hwang d, Jan-Jung Li b, Jih-Jung Chen e, Yung-Husan Chen b, Yu-Chia Chang b, f, Yin-Di Su a, b, Yu-Hsin Chen b, c, Lee-Shing Fang g, Jyh-Horng Sheu a, f, Wei-Hsien Wang a, b, *, Ping-Jyun Sung a, b, c, h, * a
Department of Marine Biotechnology and Resources and Asia-Pacific Ocean Research Center, National Sun Yat-sen University, Kaohsiung 804, Taiwan National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan Graduate Institute of Marine Biotechnology and Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Pingtung 944, Taiwan d Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan e Department of Pharmacy and Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung 907, Taiwan f Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University and Academia Sinica, Kaohsiung 804, Taiwan g Department of Sport, Health, and Leisure, Cheng Shiu University, Kaohsiung 833, Taiwan h Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 January 2013 Received in revised form 25 January 2013 Accepted 29 January 2013 Available online 8 February 2013
Three new clovane-related sesquiterpenoids, rumphellclovanes CeE (1e3), and a new natural clovane, clovan-2,9-dione (4), were isolated from a gorgonian coral Rumphella antipathies. The structures of clovanes 1e4 were elucidated by spectroscopic methods and by comparison of the spectral data with those of related analogues. Rumphellclovane C (1) was proven to be the first clovane-type sesquiterpenoid containing an ε-lactone moiety. A plausible biosynthetic pathway between clovanes 1 and 2 was proposed. Clovane 4 displayed significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Clovane Rumphellclovane Rumphella Superoxide anion Elastase
1. Introduction The gorgonian corals of genus Rumphella (phylum Cnidaria, class Anthozoa, order Gorgonacea, suberorder Holaxonia, family Gorgoniidae),1,2 which are distributed in the tropical waters of the IndoPacific Ocean, have been investigated for ecological and medical uses.3,4 As part of our ongoing investigation into the isolation of new substances from the marine invertebrates collected in the waters of Taiwan, the chemical constituents of a gorgonian coral Rumphella antipathies was studied for its organic extract, which displayed meaningful signals in NMR studies. Previous chemical investigations on R. antipathies have yielded a series of caryophylleneand clovane-type sesquiterpenoid analogues, including kobusone,5
* Corresponding authors. Tel.: þ886 8 882 5001x5047; fax: þ886 8 882 4488 (W.-H.W.); tel.: þ886 8 882 5037; fax: þ886 8 882 5087 (P.-J.S.); e-mail addresses: [email protected] (W.-H. Wang), [email protected] (P.-J. Sung). 0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.01.087
isokobusone,6 rumphellolides AeI,7e10 rumphellatins AeD,11e13 rumphellaone A,14 2b-hydroxyclovan-9-one,15 9a-hydroxyclovan2-one16 and rumphellclovanes A and B.15,16 In the further studies on the chemical constituents of R. antipathies, four clovane-related derivatives, including three new compounds, rumphellclovanes CeE (1e3), and a new natural product, clovan-2,9-dione (4),17 were isolated. In this paper, we report the isolation, structure determination, and bioactivity of clovanes 1e4. 2. Results and discussion Rumphellclovane C (1) was isolated as a yellowish oil that gave a pseudomolecular ion (MþNa)þ at m/z 303.1934 in the HRESIMS, indicating the molecular formula C17H28O3 (calcd for C17H28O3þNa, 303.1936) and implying four degrees of unsaturation. An IR absorption at 1719 cm1 suggested the presence of an ester group in 1. The 13C NMR and DEPT spectra of 1 (Table 1) showed that clovane 1 was consisted of 17 carbons, including four methyls, seven sp3
H.-M. Chung et al. / Tetrahedron 69 (2013) 2740e2744 14 13 4 2
7
6
5
3
2'
15
H
12
11
CH3CH2O
H
H
8
O
1
1'
2741
O
9 10
O
O
R 2:R=OCH2CH3, 5:R=OH
1
OH
HO 3
H
H
O
OH HO
O 4
6
Table 1 1 H and 13C NMR data, 1He1H COSY and HMBC correlations for clovane 1
d Ha
C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
3.46 dd (7.2, 6.8)c 1.74 dd (12.0, 6.8); 1.51 dd (12.0, 7.2) 1.54 m 1.56 m 2.03 ddd (16.8, 4.0, 3.2); 1.83 dd (16.8, 3.2)
2.77 1.90 1.98 0.88 1.07 1.40 3.43 3.48 1.16
2-OCH2CH3 1' 2' a b c
dCb
m dd (14.8, 3.6) d (14.8); 1.61 d (14.8) s s s dq (8.8, 6.8) (H-1’a) dq (8.8, 6.8) (H-1’b) dd (6.8, 6.8) (H3-20 )
1
44.8 88.9 44.6 36.8 51.0 20.4 35.5 80.1 174.8 32.8 31.0 47.0 25.5 31.0 32.2 66.0
(C) (CH) (CH2) (C) (CH) (CH2) (CH2) (C) (C) (CH2) (CH2) (CH2) (CH3) (CH3) (CH3) (CH2)
15.6
(CH3)
He1H COSY
HMBC
H2-3 H-2
C-1, -3, -11, -12, -10 C-1, -2, -4, -5, -13, -14
H2-6 H-5, H2-7 H2-6
C-1, -3, -4, -6, -11, -13, -14 C-1, -4 C-5, -6, -8
H2-11 H2-10
C-1, C-1, C-1, C-3, C-3, C-7, C-2, C-2, C-10
H-1’b, H3-20 H-1’a, H3-20 H2-10
-9 -5, -5, -4, -4, -8, -20 -20
-9, -7, -5, -5, -9,
-10 -8, -11, -15 -14 -13 -12
Spectrum measured at 400 MHz in CDCl3 at 25 C. Spectrum measured at 100 MHz in CDCl3 at 25 C. J values (in hertz) in parentheses.
methylenes, two sp3 methines, three sp3 quaternary carbons and an sp2 quaternary carbon. From the 13C NMR data, a degree of unsaturation was accounted for (dC 174.8, C-9, an ester carbonyl) and 1 must be a tricyclic compound. From the 1He1H COSY spectrum of 1 (Table 1 and Fig. 1), it was possible to establish the spin systems that map out the proton sequences from H-2/H2-3, H-5/H2-6/H2-7, H210/H2-11 and H2-10 /H3-20 , which were assembled with assistance of an HMBC experiment. The key HMBC correlations between protons and quaternary carbons of 1 (Table 1 and Fig. 1), such as H-2, H2-3, H-5, H2-6, H2-10, H2-11, H2-12/C-1; H2-3, H-5, H2-6, H3-13, H3-14/C-
14
13
4 2
1'
O
15 8 5
12
O
1
: 1H-1H COSY : HMBC
9 O
2' Fig. 1. The 1He1H COSY and selective HMBC correlations (protons/quaternary carbons) for clovane 1.
4; H2-7, H2-12, H3-15/C-8; and H2-10, H2-11/C-9, permitted the elucidation of the main carbon skeleton. The tertiary methyl at C-8 was confirmed by the HMBC correlations between H3-15/C-7, -8, -9, -12 and H2-12/C-15. 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. The C-12 methylene bridge between C-1 and C-8 was linked by the HMBC correlations between H-2/C-12, H2-12/C-15 and H3-15/C-12. Based on the consideration of molecular formula, an additional oxygen atom had to be placed between C-8 and C-9 to form an ε-lactone moiety. A long-range J4-HMBC correlation between H3-15/C-9 further supporting this rationalization. The position of an ethoxy group at C-2 was confirmed by the HMBC correlations between the oxymethine proton at dH 3.46 (H-2)/dC 66.0 (CH2-10 ), and by the oxymethylene protons at dH 3.43, 3.48 (H2-10 )/dC 88.9 (CH-2). The relative configuration of 1 was elucidated mainly from a NOESY spectrum as being compatible with that of 1 ascertained using molecular mechanics calculations (MM2),18 which suggested the most stable conformation, as shown in Fig. 2, in which the close contacts of atoms in space calculated were consistent the NOESY correlations. Because of the b-orientation of H-5, and this proton was found to show correlations with H3-14, but not with H-2, suggesting that Me-14 is located on the same face of H-5. H-2 was found to correlate with H3-13 and H2-12, indicating that H-2 and C-
2742
H.-M. Chung et al. / Tetrahedron 69 (2013) 2740e2744
14
13
15 4
2 1'
8
5
: 1H-1H COSY : HMBC
12
1
9 O
O
2' Fig. 3. The 1He1H COSY and selective HMBC correlations (protons/quaternary carbons) for clovane 2.
Fig. 2. The stereoview of 1 (generated from computer modelling) and the calculated distances ( A) between selected protons having key NOESY correlations.
12 methylene bridge between C-1/8 was a-oriented. H3-15 showed correlations with H2-12, confirming the a-orientation for this tertiary methyl. Based on the above findings, the structure of 1 was established and the chiral carbons for 1 were assigned as 1S*, 2S*, 5S*, 8R*. The new clovane, rumphellclovane D (2), was isolated as a yellowish oil and its molecular formula C17H28O2 was established by HRESIMS (m/z 265.2158, calcd for C17H28O2þH, 265.2162). The IR spectrum of 2 showed a strong band at 1706 cm1, consistent with the presence of a keto carbonyl. It was found that the 1H and 13C NMR data of 2 were similar to those of a known clovane analogue, 2b-hydroxyclovan-9-one (5),15,19 that was also isolated from R. antipathies,15 except that the signals corresponding to a hydroxy group at C-2 in 5 (dH 3.87, 1H, dd, J¼7, 5.5 Hz; dC 80.2, CH-2)19 were replaced by an ethoxy group in 2 (dH 3.45, 1H, dd, J¼8.0, 5.6 Hz; dC 87.1, CH-2; dH 3.37, 1H, dq, J¼8.8, 6.8 Hz, H-10 a; 3.48, 1H, dq, J¼8.8, 6.8 Hz, H-10 b; 1.15, 3H, dd, J¼6.8, 6.8 Hz, H3-20 ; dC 65.4, CH2-10 ; 15.5, CH3-20 , ethoxy group) (Table 2). The 1He1H COSY and HMBC correlations observed fully supported the locations of functional groups (Table 2 and Fig. 3), and hence rumphellclovane D (2) was assigned as the structure 2 with the same relative stereochemistry as in rumphellclovane C (1) and 2b-hydroxyclovan-9-one (5),15,19 because for the chiral carbons that 2 has in common with
clovanes 1 and 5 and the relative configurations of 2 were assigned as 1S*, 2S*, 5S*, 8R*. The 1H and 13C NMR chemical shifts and proton coupling constants match well, and are further supported by a NOESY experiment (Fig. 4).
Fig. 4. The stereoview of 2 (generated from computer modelling) and the calculated distances ( A) between selected protons having key NOESY correlations.
A plausible biosynthetic pathway for 1 from 2 was proposed and clovane 2 was further lactonized to 1 by BaeyereVilliger oxidation. To the best of our knowledge, clovane-type analogues like 1 containing an ε-lactone moiety has not been found previously.
Table 2 1 H and 13C NMR data, 1He1H COSY and HMBC correlations for clovane 2 C/H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
2-OCH2CH3 1' 2' a b c d
d Ha 3.45 (8.0, 5.6)c 1.77 dd (12.8, 5.6); 1.60 dd (12.8, 8.0) 1.72 dd (7.6, 7.6) 1.56 md 1.50 md
2.64 1.87 1.55 0.96 1.07 1.01 3.37 3.48 1.15
He1H COSY
dCb
ddd (16.0, 12.4, 8.0); 2.34 ddd (16.0, 6.8, 2.4) ddd (12.4, 12.4, 6.8); 1.70 ddd (12.4, 8.0, 2.4) md s s s dq (8.8, 6.8) (H-1’a) dq (8.8, 6.8) (H-1’b) dd (6.8, 6.8) (H3-20 )
Spectrum measured at 400 MHz in CDCl3 at 25 C. Spectrum measured at 100 MHz in CDCl3 at 25 C. J values (in hertz) in parentheses. Signals overlapped.
44.6 87.1 44.8 38.2 50.9 20.5 34.0 44.5 216.6 35.7 32.7 42.8 26.1 32.0 24.9 65.4
(C) (CH) (CH2) (C) (CH) (CH2) (CH2) (C) (C) (CH2) (CH2) (CH2) (CH3) (CH3) (CH3) (CH2)
15.5
(CH3)
1
HMBC
H2-3 H-2
C-4, -5, -11, -12, -10 C-1, -2, -4, -5, -13, -14
H2-6 H-5, H2-7 H2-6
C-1, -3, -4, -7, -13 C-1, -5, -7, -8 C-5, -6, -8
H2-11 H2-10
C-8, C-1, C-1, C-3, C-3, C-7, C-2, C-2, C-10
H-1’b, H3-20 H-1’a, H3-20 H2-10
-9, -2, -2, -4, -4, -8, -20 -20
-11 -5, -9, -10 -5, -7, -8, -9, -11 -5, -14 -5, -13 -9, -12
H.-M. Chung et al. / Tetrahedron 69 (2013) 2740e2744
2743
Rumphellclovane E (3) was isolated as a yellowish oil. The molecular formula of 3 was established as C15H24O3 (4 of unsaturation) from a sodium adduct at m/z 275 in the ESIMS spectrum and further supported by HRESIMS (m/z 275.1625, calcd for C15H24O3þNa, 275.1623). The presence of hydroxy and keto carbonyl groups were indicated by IR absorptions at 3433 and 1706 cm1. It was found that the spectral data of 3 were similar to those of a known clovane, clovan-2b,9a-diol (6), that was also isolated from Sindora summatrana,20 except that the signals corresponding to the C-12 methylene group in 6 (dH 0.91, 1H, br d, J¼12.5 Hz; 1.56, 1H, d, J¼12.5 Hz; dC 35.6)20 were replaced by a keto carbonyl in 3 (dC 219.4) (Table 3). Table 3 1 H and 13C NMR data, 1He1H COSY and HMBC correlations for clovane 3 C/H 1 2 3
2.18 dd (13.5, 5.0)
6 7
1.58 m 1.97 ddd (14.0, 14.0, 4.5); 1.66 m
8 9 10 11 12 13 14 15 b c d
He1H COSY
dCb
4.73 dd (12.0, 6.0)c 1.73 dd (14.0, 6.0); 1.53 dd (14.0, 12.0)
4 5
a
d Ha
3.93 br s 2.42 dddd (15.0, 15.0, 5.0, 3.0); 1.79 m 2.22 m; 1.62 m 0.78 s 1.07 s 1.12 s
1
HMBC
50.5 71.2 45.9
(C) (CH) (CH2)
H2-3 H-2
C-1, -11, -12 C-1, -2, -4, -5, -13, -14
36.9 57.4
(C) (CH)
H2-6
22.8 36.6
(CH2) (CH2)
H-5, H2-7 H2-6
C-4, -6, -11, -12, -13, -14 n.o.d C-6, -8, -9, -12
50.6 81.1 24.9
(C) (CH) (CH2)
H2-10 H-9, H2-11
C-12 n.o.
29.7 219.4 24.8 30.8 20.6
(CH2) (C) (CH3) (CH3) (CH3)
H2-10
C-5, -12 C-3, -4, -5, -14 C-3, -4, -5, -13 C-7, -8, -9, -12
2
8 : 1H-1H COSY : HMBC
12 O
9
Superoxide anion
Elastase release
IC50 (mg/mL)
IC50 (mg/mL)
Inh%a 14.071.76 16.886.13 2.731.78
2.720.93 0.800.21
Inh%a 22.514.47 12.885.40 21.323.81
6.730.85 31.955.92
Percentage of inhibition (Inh%) at a concentration of 10 mg/mL. DPI (diphenylene indonium) and elastatinal were used as positive control.
3. Experimental
15
1
1 2 3 4 DPIb Elastatinalb b
14
5
Table 4 Inhibitory effects of clovanes 1e4 on the generation of superoxide anion and the release of elastase by human neutrophils in response to FMLP/CB
a
The 2D NMR correlations observed fully supported the locations of functional groups (Table 3 and Fig. 5), and hence clovane 3 was assigned as the structure 3 with the same relative stereochemistry as in 6,20 because for the chiral carbons that 3 has in common with clovane 6 and were assigned as 1S*, 2S*, 5S*, 8S*. The 1H and 13C NMR chemical shifts and proton coupling constants match well, and are further supported by a NOESY experiment (Fig. 6).
4
The in vitro anti-inflammatory effects of clovanes 1e4 were tested (Table 4). Clovan-2,9-dione (4) was found to display significant inhibitory effects on the generation of superoxide anion and the release of elastase by human neutrophils.
Compounds
Spectrum measured at 500 MHz in CDCl3 at 25 C. Spectrum measured at 125 MHz in CDCl3 at 25 C. J values (in hertz) in parentheses. n.o.¼not observed.
13
Fig. 6. The stereoview of 3 (generated from computer modelling) and the calculated distances ( A) between selected protons having key NOESY correlations.
OH
HO Fig. 5. The 1He1H COSY and selective HMBC correlations (protons/quaternary carbons) for clovane 3.
Our present study has also led to the isolation of a new natural clovane 4.17 Clovane 4 has the molecular formula C15H22O2 as determined by HRESIMS (m/z 235.1680, calcd for C15H22O2þH, 235.1693). The IR spectrum of 4 indicated the presence of keto carbonyl groups (1729 and 1708 cm1). It was found that the spectral data of 4 are identical with those of a known synthetic compound clovan-2,9-dione that was prepared by Collado’s group.17 However, clovane 4 has not been isolated previously from natural sources.
3.1. General experimental procedures Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer; peaks are reported in cm1. The NMR spectra were recorded on a Varian Mercury Plus 400 or on a Varian Inova 500 NMR spectrometers using the residual CHCl3 signal (dH 7.26 ppm) as internal standard for 1H NMR and CDCl3 (dC 77.1 ppm) for 13C NMR. Coupling constants (J) are given in hertz. ESIMS and HRESIMS were recorded on a Bruker APEX II or on a Finnigan/ Thermo Quest Mat 95XL mass spectrometers. Column chromatography was performed on silica gel (230e400 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 semi-preparative column (Hibar 25010 mm, LiChrospher Si 60, 5 mm) and a reverse phase semi-preparative column (Hibar 25010 mm, Purospher STAR RP-18e, 5 mm) were used for HPLC. 3.2. Animal material Specimens of the gorgonian coral R. antipathies (Nutting) were collected by hand using scuba equipment off the coast of the southern Taiwan and stored in a freezer until extraction. This organism was identified by comparison with previous descriptions.1,2 A voucher specimen (specimen no. NMMBA-TWGC-010) was
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deposited in the National Museum of Marine Biology and Aquarium (NMMBA), Taiwan.
release experiments were performed using MeO-Suc-Ala-Ala-ProValp-nitroanilide as the elastase substrate.
3.3. Extraction and isolation
Acknowledgements
Sliced bodies of the gorgonian coral R. antipathies (wet weight 402 g, dry weight 144 g) were extracted with a mixture of methanol (MeOH) and dichloromethane (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, 25:1epure EtOAc) to yield 29 fractions. Every fraction was checked by the 1H NMR spectra. Fraction 7 was purified by normal phase HPLC (NP-HPLC), using a mixture of n-hexane and EtOAc (9:1) as a mobile phase to afford 1 and 2. Fraction 18 was separated by NP-HPLC, using a mixture of n-hexane and EtOAc (5:1) to afford 4. Fraction 28 was separated by reverse phase HPLC using a mixture of MeOH and water (7:3) to afford 3.
This work was supported by grants from the National Dong Hwa University; the NMMBA (Grant No. 10120022); the Division of Marine Biotechnology, Asia-Pacific Ocean Research Center, National Sun Yat-sen University, (Grant No. 00C-0302-05); and the National Science Council (Grant No. NSC 101-2325-B-291-001, 100-2325-B291-001 and 101-2320-B-291-001-MY3), Taiwan, awarded to P.-J.S.
3.3.1. Rumphellclovane C (1). Yellowish oil (0.4 mg); [a]25 D 45 (c 0.01, CHCl3); IR (neat) nmax 1718 cm1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data, see Table 1; ESIMS: m/z 303 (MþNa)þ; HRESIMS: m/z 303.1934 (calcd for C17H28O3þNa, 303.1936).
References and notes
3.3.2. Rumphellclovane D (2). Yellowish oil (10.9 mg); [a]25 D 63 (c 0.33, CHCl3); IR (neat) nmax 1706 cm1; 1H (CDCl3, 400 MHz) and 13C (CDCl3, 100 MHz) NMR data, see Table 2; ESIMS: m/z 265 (MþH)þ; HRESIMS: m/z 265.2158 (calcd for C17H28O2þH, 265.2162). 3.3.3. Rumphellclovane E (3). Yellowish oil (1.0 mg); [a]25 D 6 (c 0.05, CHCl3); IR (neat) nmax 3433, 1706 cm1; 1H (CDCl3, 500 MHz) and 13C (CDCl3, 125 MHz) NMR data, see Table 3; ESIMS: m/z 275 (MþNa)þ; HRESIMS: m/z 275.1625 (calcd for C15H24O3þNa, 275.1623). 3.3.4. Clovan-2,9-dione (4). Yellowish oil (1.5 mg); [a]25 D 52 (c 0.08, CHCl3, Ref. 17, [a]25 D 97.0 (c 0.01325, CHCl3)); IR (neat) nmax 1729, 1708 cm1; 1H (CDCl3, 400 MHz) dH 2.52 (2H, m, H2-10), 2.40 (1H, d, J¼17.6 Hz, H-3), 2.23 (1H, d, J¼17.6 Hz, H-30 ), 1.97 (1H, dd, J¼6.8, 6.8 Hz, H-5), 1.75 (2H, m, H2-6), 1.67 (1H, m, H-11), 1.61 (2H, m, H2-12), 1.58 (1H, m, H-7), 1.49 (1H, m, H-110 ), 1.48 (1H, ddd, J¼14.0, 7.6, 7.6 Hz, H-70 ), 1.16 (3H, s, H3-14), 1.10 (3H, s, H3-13), 1.05 (3H, s, H3-15); 13C (CDCl3, 100 MHz) dC 220.6 (C-2), 214.5 (C-9), 52.4 (CH2-3), 49.9 (CH-5), 48.8 (C-1), 44.0 (C-8), 39.0 (CH2-12), 37.0 (C-4), 36.2 (CH2-10), 35.1 (CH2-11), 34.4 (CH2-7), 30.6 (CH3-14), 24.9 (CH3-13), 24.7 (CH3-15), 20.1 (CH2-6); ESIMS: m/z 235 (MþH)þ; HRESIMS: m/z 235.1680 (calcd for C15H22O2þH, 235.1699).17 3.4. Molecular mechanics calculations Implementation of the MM2 force field18 in CHEM3D PRO software from CambridgeSoft Corporation (Cambridge, MA, USA; ver. 9.0, 2005) was used to calculate molecular models. 3.5. Generation of superoxide anion 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.21e27 Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase
Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2013.01.087.
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