Bioorganic & Medicinal Chemistry Letters 25 (2015) 92–95
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
New anti-inflammatory tocopherol-derived metabolites from the Taiwanese soft coral Cladiella hirsuta Bo-Wei Chen a, Chokkalingam Uvarani a, Chiung-Yao Huang a, Tsong-Long Hwang b, Chang-Feng Dai c, Jyh-Horng Sheu a,d,e,f,⇑ a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan d Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan e Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan f Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan b c
a r t i c l e
i n f o
Article history: Received 4 August 2014 Revised 13 October 2014 Accepted 1 November 2014 Available online 11 November 2014 Keywords: Cladiella hirsuta Hirsutaquinone Anti-inflammatory activity
a b s t r a c t Two new tocopherol-derived metabolites, hirsutocospiro A (1) and hirsutocoquinone A (2), were isolated from the soft coral Cladiella hirsuta. The structures of 1 and 2 were determined by extensive spectroscopic analysis. The in vitro anti-inflammatory activity of compounds 1 and 2 was evaluated by measuring their ability in suppressing superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils. Compound 1 was shown to exhibit significant anti-inflammatory activity. Ó 2014 Elsevier Ltd. All rights reserved.
The chemical investigations on the soft coral of the genus Cladiella have led to the isolations of a series of biologically active natural products,1–18 including those of 18 eunicellin-based diterpenoids hirsutalins A–R15–17 and seven steroids hirsutosterols A–G18 discovered by our recent chemical study of a Taiwanese soft coral Cladiella hirsuta. As some of the metabolites from our investigation of C. hirsuta have been found to possess attracting cytotoxic and anti-inflammatory activities,15–17 we thus continued our chemical study of this marine animal with the aim of discovering new bioactive marine natural products. In the present study we further isolate two new tocopherol-derived metabolites, hirsutocospiro A (1)19 and hirsutocoquinone (2),19 from C. hirsuta (Fig. 1). The structures of new compounds were determined by extensive spectroscopic analysis. The anti-inflammatory activity of these two compounds to inhibit the generation of super oxide anion and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophiles was studied in order to discover bioactive compounds for future new drug development. Hirsutocospiro A (1) was isolated as a colorless oil. The HRESIMS (m/z 389.1938) of 1 established a molecular formula of C20H30O6, implying the presence of six degrees of unsaturation. ⇑ Corresponding author. Tel.: +886 7 5252000x5030. E-mail address: [email protected] (J.-H. Sheu). http://dx.doi.org/10.1016/j.bmcl.2014.11.002 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
The IR spectrum of 1 showed strong absorptions at 3436, 1736, and 1711 cm 1 for hydroxy and carbonyl groups, respectively. The 13C NMR spectroscopic data of 1 (Table 1) showed the presence of 20 carbon signals, which were assigned to six methyls, five sp3 methylenes, one sp3 methine, two quaternary sp2 carbons, three sp2 carbonyls and three sp3 oxygenated quaternary carbons. The NMR signals appearing at dC 205.0 (C), 163.2 (C), and 139.5 (C) were assigned to an a,b-unsaturated carbonyl system by the assistance of a DEPT spectrum. Furthermore, carbon signals of two carbonyls (dC 207.0 and 177.2), three oxygenated quaternary carbon (dC 92.6, 89.3, and 86.9), and one oxygen-bearing methyl (dC 51.5) were observed. The 1H NMR spectrum of 1 also showed signals of two olefinic methyl groups (d 1.84, s and 1.82, s), one acetyl group (dH 2.01, s), one tertiary group (dH 1.30, s) and one secondary methyl group (d 1.14, d, J = 6.8 Hz). The remaining two degrees of unsaturation identified 1 as a bicyclic compound. The planar structure of metabolite 1 was elucidated by analysis of COSY and HMBC correlations, as shown in Figure 2. The COSY spectrum of 1, revealed the presence of two structural units from H2-7 to H2-8 and H2-10 to H3-13a. Key HMBC correlations from H3-3a to C-3; H3-5a to C-4, C-5, and C-6; H3-6a to C-5, C-6 and C-1; H2-7 to C-1, C-2, C-4 and C-9; H3-9a to C-8, C-9, and C-10; H3-13a to C-12, C-13 and C-14; and CH3O to C-14, permitted the assembly of the carbon molecular of 1. Thus the planar structure of metabolite 1 (Fig. 2) was established by the analysis of COSY and HMBC
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O
3a 5a
OH
3 4
5
1
6
6a
3
7
10
12
9
O
6
O
14 COOMe
9a
2
1
13a
OH
7 9
13
COOMe
11
14
4
13
11
O
13a
9a
O
6a
3a
5a
O
1
2
Figure 1. The structures of hirsutocospiro A (1) and hirsutocoquinone A (2).
Table 1 NMR data for compound 1 Position 1 2 3 3a 4 5 5a 6 6a 7 8 9 9a 10 11 12 13 13a 14-COOMe 4-OH a b
O OH
dH (J in Hz)a
dC, Mult.b
2.01 s
1.82 s 1.84 s 2.37 dt (12.0, 6.0); 1.88 m 1.78 m 1.30 1.37 1.21 1.63
Ο
205.0, C 92.6, C 207.0, C 24.8, CH3 89.3, C 163.2, C 11.8, CH3 139.5, C 8.7, CH3 33.3, CH2 36.7, CH2 86.9, C 25.4, CH3 41.4, CH2 22.5, CH2 34.1, CH2
s m m dt (14.0, 7.6)
2.43 dq (13.6, 6.8) 1.14 d (6.8)
Ο
O
COOMe Ο
correlation, as shown in Figure 2. By comparison of 1H and 13C NMR data from C1–C10 in 1 with those of a-tocospiro B (3, Chart 1),20 it was found that both 1 and 3, possess nearly identical 1 H and 13C NMR spectral data. In the NOESY spectrum of 1 (Fig. 3), observation of the NOE correlations between 4-OH with both H-5a and one proton (dH 1.88) at C-7 suggested that both OH and CH2-7 were located on the same face, arbitrarily assigned as a face. The relative configuration of chiral center at C-9 was confirmed by the NOE correlations between H3-9a with H-7 (dH 2.37) and the chemical shift of H3-9a (d 1.30 for 1, 1.29 for 3) that suggested the a-orientations of 4-OH and CH3-9a. For the isomer of 3, a-tocospiro A, the proton signal of CH3-9a should appear at d
O OH
3
5a
6a
6
1
10
2
Ο
O
6a
7
4
5
13a
9 13
Ο
9a
1
14
COOMe
OH
O Ο
4
5
1.02.20 The relative configuration of the spiro rings of 1 was thus determined. However, the relative configuration at C-13 of the side chain remained unresolved. Hirsutocoquinone A (2) was also afforded as a colorless oil. Compound 2 has a molecular formula C20H30O6, appropriating for six degrees of unsaturation, as determined by HRESIMS. Thus, 1 and 2 have the same molecular formula. The NMR signals appearing at dC 194.6 (C), 194.3 (C), 144.9 (C), and 140.6 (C) indicated the presence of an a,b-unsaturated dicarbonyl system.21 Furthermore, carbon signal of a carbonyl (dC 177.2), three oxygenated quaternary carbons (dC 72.4, 63.1 and 63.1), and one oxygen-bearing methyl (dC 51.6) were observed (Table 2). The 1H NMR spectrum of 1 also showed signals of an olefinic methyl group (dH 1.99, s), three tertiary methyl groups (dH 1.59, 1.59, and 1.20, s) and one secondary methyl group (dH 1.16, d, J = 6.0 Hz). By comparison of the NMR data of 2 with those of a known compound flexibilisquinone (4, Chart 1),22 it was found that one double bond at C-5/C-6 in 4 was oxidized to one trisubstituted epoxide in 2. It was confirmed by the downfield shifted dc values of C-5/C-6 (dc 140.0 and 140.4) of 4, relative to that of 2 (dc 63.1), and the HMBC connectivity from H3-5a (dH 1.59) to the carbonyl carbon resonating at dC 194.3 (C) and the epoxide carbon at dc 63.1 (C). Moreover, comparison of the chemical shift values of 2 with those of 1,1-dimethyl-3-(2,3-epoxy-2,3,5-trimethylbenzoquinon-6-yl)propanol (5, Chart 1)21 revealed that 2 could be the
Recorded at 400 MHz in CDCl3. Spectra recorded at 100 MHz in CDCl3.
3a
O
Chart 1. Structures of known compounds 3–5.
39.3, CH 17.0, CH2 177.2, C 51.5, CH3
3.68 s 4.69 s
3
OH
O 5a
9a 7
6 1
9
11
14
3a
: COSY : HMBC Figure 2. COSY and HMBC correlations for 1 and 2.
13
COOMe
OH
4
Ο
13a
2
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3a 5a 4 7
10
9a
Figure 3. Key NOESY correlations of hirsutocospiro A (1).
Table 2 NMR data for compound 2 Position 1 2 3 3a 4 5 5a 6 6a 7 8 9 9a 10 11 12 13 13a 14-COOMe
O
dH (J in Hz)a
1.99 s
1.59 s 1.59 s 2.58 m; 2.34 m 1.52 m; 1.39 m 1.20 1.46 1.36 1.67 1.40 2.47 1.16
s m m m m m d (6.0)
3.68 s a b
dC, Mult.b 194.3, C 144.9, C 140.6, C 12.9, CH3 194.6, C 63.1, C 11.4, CH3 63.1, C 11.4, CH3 22.1, CH2 39.6, CH2 72.4, C 26.4, CH3 41.6, CH2 21.6, CH2 34.2, CH2
O
COOH
OH
4
[O] [Me] O
O O
O
O
COOMe
OH
6
O 2
COOMe
OH
H3 O+
O OH
COOMe
O OH H
39.3, CH 17.0, CH2 177.2, C 51.6, CH3
OH
H O
H
O
O
Recorded at 400 MHz in CDCl3. Spectra recorded at 100 MHz in CDCl3.
OH OH
HO
OH
OH
R O
R
H+ −H 2O
OH
1
Chart 2. Proposed biogenetic pathway for the formation of 1 and 2.
elongated derivative of 5. The C-5/C-6 epoxide was confirmed by the very similar chemical shifts of H3-5a/6a (DdH – 0.01 ppm) and C-5/6 (DdC 0 ppm) in 2 relative to those of 5. Thus, the structure of 2 was established. However, the relative configurations of the chiral centers C-9 and C-13 could not be elucidated. Epoxide 2 comes from the epoxidation of compound 4 at 5,6-double bond and the esterification of carboxy group. The biosynthesis of spiro compound 1 was assumed to be come from the acid-catalyzed ring opening of both the epoxide and sixmembered ring of 6 which is arisen from the 2,3-epoxidation and esterification of 4, and the subsequent five-membered ring formation via aldol condensation and the following ether ring formation, as proposed in a previous report20 (Chart 2). The neutrophil pro-inflammatory responses to compounds 1 and 2 were evaluated by suppressing N-formyl-methionyl-leucylphenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion (O2 ) generation and elastase release in human neutrophils, as shown in Table 3. From the results, hirsutocospiro A (1) showed strong inhibition (78.3 ± 2.2%) toward superoxide anion generation at a concentration of 10 lM. At the same concentration, 1 also exhibited potent inhibitory activity against elastase release with 89.5 ± 3.1% inhibition in the same fMLP/CB-stimulated cells. Compound 1 was found to show potent activity in inhibiting the superoxide generation (IC50 4.1 ± 1.1 lM) and elastase release
Table 3 Effect of 1 and 2 on superoxide anion generation and elastase release in fMLP/CBinduced human neutrophils Compounds
Superoxide anion Inhibition%
1 2
78.3 ± 2.2 8.5 ± 4.5
⁄⁄⁄
Elastase release
IC50 (lM)a
Inhibition% IC50 (lM)a
4.1 ± 1.1 >10
89.5 ± 3.1 21.2 ± 2.1
⁄⁄⁄ ⁄⁄⁄
3.7 ± 0.3 >10
Percentage of inhibition (Inh%) at 10 lM concentration. Results are presented as mean ± SEM (n = 3 or 4). ⁄⁄⁄p
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
New anti-inflammatory tocopherol-derived metabolites from the Taiwanese soft coral Cladiella hirsuta Bo-Wei Chen a, Chokkalingam Uvarani a, Chiung-Yao Huang a, Tsong-Long Hwang b, Chang-Feng Dai c, Jyh-Horng Sheu a,d,e,f,⇑ a
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Graduate Institute of Natural Products, Chang Gung University, Taoyuan 333, Taiwan Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan d Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan e Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan f Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan b c
a r t i c l e
i n f o
Article history: Received 4 August 2014 Revised 13 October 2014 Accepted 1 November 2014 Available online 11 November 2014 Keywords: Cladiella hirsuta Hirsutaquinone Anti-inflammatory activity
a b s t r a c t Two new tocopherol-derived metabolites, hirsutocospiro A (1) and hirsutocoquinone A (2), were isolated from the soft coral Cladiella hirsuta. The structures of 1 and 2 were determined by extensive spectroscopic analysis. The in vitro anti-inflammatory activity of compounds 1 and 2 was evaluated by measuring their ability in suppressing superoxide anion generation and elastase release in fMLP/CB-induced human neutrophils. Compound 1 was shown to exhibit significant anti-inflammatory activity. Ó 2014 Elsevier Ltd. All rights reserved.
The chemical investigations on the soft coral of the genus Cladiella have led to the isolations of a series of biologically active natural products,1–18 including those of 18 eunicellin-based diterpenoids hirsutalins A–R15–17 and seven steroids hirsutosterols A–G18 discovered by our recent chemical study of a Taiwanese soft coral Cladiella hirsuta. As some of the metabolites from our investigation of C. hirsuta have been found to possess attracting cytotoxic and anti-inflammatory activities,15–17 we thus continued our chemical study of this marine animal with the aim of discovering new bioactive marine natural products. In the present study we further isolate two new tocopherol-derived metabolites, hirsutocospiro A (1)19 and hirsutocoquinone (2),19 from C. hirsuta (Fig. 1). The structures of new compounds were determined by extensive spectroscopic analysis. The anti-inflammatory activity of these two compounds to inhibit the generation of super oxide anion and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-induced human neutrophiles was studied in order to discover bioactive compounds for future new drug development. Hirsutocospiro A (1) was isolated as a colorless oil. The HRESIMS (m/z 389.1938) of 1 established a molecular formula of C20H30O6, implying the presence of six degrees of unsaturation. ⇑ Corresponding author. Tel.: +886 7 5252000x5030. E-mail address: [email protected] (J.-H. Sheu). http://dx.doi.org/10.1016/j.bmcl.2014.11.002 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
The IR spectrum of 1 showed strong absorptions at 3436, 1736, and 1711 cm 1 for hydroxy and carbonyl groups, respectively. The 13C NMR spectroscopic data of 1 (Table 1) showed the presence of 20 carbon signals, which were assigned to six methyls, five sp3 methylenes, one sp3 methine, two quaternary sp2 carbons, three sp2 carbonyls and three sp3 oxygenated quaternary carbons. The NMR signals appearing at dC 205.0 (C), 163.2 (C), and 139.5 (C) were assigned to an a,b-unsaturated carbonyl system by the assistance of a DEPT spectrum. Furthermore, carbon signals of two carbonyls (dC 207.0 and 177.2), three oxygenated quaternary carbon (dC 92.6, 89.3, and 86.9), and one oxygen-bearing methyl (dC 51.5) were observed. The 1H NMR spectrum of 1 also showed signals of two olefinic methyl groups (d 1.84, s and 1.82, s), one acetyl group (dH 2.01, s), one tertiary group (dH 1.30, s) and one secondary methyl group (d 1.14, d, J = 6.8 Hz). The remaining two degrees of unsaturation identified 1 as a bicyclic compound. The planar structure of metabolite 1 was elucidated by analysis of COSY and HMBC correlations, as shown in Figure 2. The COSY spectrum of 1, revealed the presence of two structural units from H2-7 to H2-8 and H2-10 to H3-13a. Key HMBC correlations from H3-3a to C-3; H3-5a to C-4, C-5, and C-6; H3-6a to C-5, C-6 and C-1; H2-7 to C-1, C-2, C-4 and C-9; H3-9a to C-8, C-9, and C-10; H3-13a to C-12, C-13 and C-14; and CH3O to C-14, permitted the assembly of the carbon molecular of 1. Thus the planar structure of metabolite 1 (Fig. 2) was established by the analysis of COSY and HMBC
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B.-W. Chen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 92–95
O
3a 5a
OH
3 4
5
1
6
6a
3
7
10
12
9
O
6
O
14 COOMe
9a
2
1
13a
OH
7 9
13
COOMe
11
14
4
13
11
O
13a
9a
O
6a
3a
5a
O
1
2
Figure 1. The structures of hirsutocospiro A (1) and hirsutocoquinone A (2).
Table 1 NMR data for compound 1 Position 1 2 3 3a 4 5 5a 6 6a 7 8 9 9a 10 11 12 13 13a 14-COOMe 4-OH a b
O OH
dH (J in Hz)a
dC, Mult.b
2.01 s
1.82 s 1.84 s 2.37 dt (12.0, 6.0); 1.88 m 1.78 m 1.30 1.37 1.21 1.63
Ο
205.0, C 92.6, C 207.0, C 24.8, CH3 89.3, C 163.2, C 11.8, CH3 139.5, C 8.7, CH3 33.3, CH2 36.7, CH2 86.9, C 25.4, CH3 41.4, CH2 22.5, CH2 34.1, CH2
s m m dt (14.0, 7.6)
2.43 dq (13.6, 6.8) 1.14 d (6.8)
Ο
O
COOMe Ο
correlation, as shown in Figure 2. By comparison of 1H and 13C NMR data from C1–C10 in 1 with those of a-tocospiro B (3, Chart 1),20 it was found that both 1 and 3, possess nearly identical 1 H and 13C NMR spectral data. In the NOESY spectrum of 1 (Fig. 3), observation of the NOE correlations between 4-OH with both H-5a and one proton (dH 1.88) at C-7 suggested that both OH and CH2-7 were located on the same face, arbitrarily assigned as a face. The relative configuration of chiral center at C-9 was confirmed by the NOE correlations between H3-9a with H-7 (dH 2.37) and the chemical shift of H3-9a (d 1.30 for 1, 1.29 for 3) that suggested the a-orientations of 4-OH and CH3-9a. For the isomer of 3, a-tocospiro A, the proton signal of CH3-9a should appear at d
O OH
3
5a
6a
6
1
10
2
Ο
O
6a
7
4
5
13a
9 13
Ο
9a
1
14
COOMe
OH
O Ο
4
5
1.02.20 The relative configuration of the spiro rings of 1 was thus determined. However, the relative configuration at C-13 of the side chain remained unresolved. Hirsutocoquinone A (2) was also afforded as a colorless oil. Compound 2 has a molecular formula C20H30O6, appropriating for six degrees of unsaturation, as determined by HRESIMS. Thus, 1 and 2 have the same molecular formula. The NMR signals appearing at dC 194.6 (C), 194.3 (C), 144.9 (C), and 140.6 (C) indicated the presence of an a,b-unsaturated dicarbonyl system.21 Furthermore, carbon signal of a carbonyl (dC 177.2), three oxygenated quaternary carbons (dC 72.4, 63.1 and 63.1), and one oxygen-bearing methyl (dC 51.6) were observed (Table 2). The 1H NMR spectrum of 1 also showed signals of an olefinic methyl group (dH 1.99, s), three tertiary methyl groups (dH 1.59, 1.59, and 1.20, s) and one secondary methyl group (dH 1.16, d, J = 6.0 Hz). By comparison of the NMR data of 2 with those of a known compound flexibilisquinone (4, Chart 1),22 it was found that one double bond at C-5/C-6 in 4 was oxidized to one trisubstituted epoxide in 2. It was confirmed by the downfield shifted dc values of C-5/C-6 (dc 140.0 and 140.4) of 4, relative to that of 2 (dc 63.1), and the HMBC connectivity from H3-5a (dH 1.59) to the carbonyl carbon resonating at dC 194.3 (C) and the epoxide carbon at dc 63.1 (C). Moreover, comparison of the chemical shift values of 2 with those of 1,1-dimethyl-3-(2,3-epoxy-2,3,5-trimethylbenzoquinon-6-yl)propanol (5, Chart 1)21 revealed that 2 could be the
Recorded at 400 MHz in CDCl3. Spectra recorded at 100 MHz in CDCl3.
3a
O
Chart 1. Structures of known compounds 3–5.
39.3, CH 17.0, CH2 177.2, C 51.5, CH3
3.68 s 4.69 s
3
OH
O 5a
9a 7
6 1
9
11
14
3a
: COSY : HMBC Figure 2. COSY and HMBC correlations for 1 and 2.
13
COOMe
OH
4
Ο
13a
2
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B.-W. Chen et al. / Bioorg. Med. Chem. Lett. 25 (2015) 92–95
3a 5a 4 7
10
9a
Figure 3. Key NOESY correlations of hirsutocospiro A (1).
Table 2 NMR data for compound 2 Position 1 2 3 3a 4 5 5a 6 6a 7 8 9 9a 10 11 12 13 13a 14-COOMe
O
dH (J in Hz)a
1.99 s
1.59 s 1.59 s 2.58 m; 2.34 m 1.52 m; 1.39 m 1.20 1.46 1.36 1.67 1.40 2.47 1.16
s m m m m m d (6.0)
3.68 s a b
dC, Mult.b 194.3, C 144.9, C 140.6, C 12.9, CH3 194.6, C 63.1, C 11.4, CH3 63.1, C 11.4, CH3 22.1, CH2 39.6, CH2 72.4, C 26.4, CH3 41.6, CH2 21.6, CH2 34.2, CH2
O
COOH
OH
4
[O] [Me] O
O O
O
O
COOMe
OH
6
O 2
COOMe
OH
H3 O+
O OH
COOMe
O OH H
39.3, CH 17.0, CH2 177.2, C 51.6, CH3
OH
H O
H
O
O
Recorded at 400 MHz in CDCl3. Spectra recorded at 100 MHz in CDCl3.
OH OH
HO
OH
OH
R O
R
H+ −H 2O
OH
1
Chart 2. Proposed biogenetic pathway for the formation of 1 and 2.
elongated derivative of 5. The C-5/C-6 epoxide was confirmed by the very similar chemical shifts of H3-5a/6a (DdH – 0.01 ppm) and C-5/6 (DdC 0 ppm) in 2 relative to those of 5. Thus, the structure of 2 was established. However, the relative configurations of the chiral centers C-9 and C-13 could not be elucidated. Epoxide 2 comes from the epoxidation of compound 4 at 5,6-double bond and the esterification of carboxy group. The biosynthesis of spiro compound 1 was assumed to be come from the acid-catalyzed ring opening of both the epoxide and sixmembered ring of 6 which is arisen from the 2,3-epoxidation and esterification of 4, and the subsequent five-membered ring formation via aldol condensation and the following ether ring formation, as proposed in a previous report20 (Chart 2). The neutrophil pro-inflammatory responses to compounds 1 and 2 were evaluated by suppressing N-formyl-methionyl-leucylphenylalanine/cytochalasin B (fMLP/CB)-induced superoxide anion (O2 ) generation and elastase release in human neutrophils, as shown in Table 3. From the results, hirsutocospiro A (1) showed strong inhibition (78.3 ± 2.2%) toward superoxide anion generation at a concentration of 10 lM. At the same concentration, 1 also exhibited potent inhibitory activity against elastase release with 89.5 ± 3.1% inhibition in the same fMLP/CB-stimulated cells. Compound 1 was found to show potent activity in inhibiting the superoxide generation (IC50 4.1 ± 1.1 lM) and elastase release
Table 3 Effect of 1 and 2 on superoxide anion generation and elastase release in fMLP/CBinduced human neutrophils Compounds
Superoxide anion Inhibition%
1 2
78.3 ± 2.2 8.5 ± 4.5
⁄⁄⁄
Elastase release
IC50 (lM)a
Inhibition% IC50 (lM)a
4.1 ± 1.1 >10
89.5 ± 3.1 21.2 ± 2.1
⁄⁄⁄ ⁄⁄⁄
3.7 ± 0.3 >10
Percentage of inhibition (Inh%) at 10 lM concentration. Results are presented as mean ± SEM (n = 3 or 4). ⁄⁄⁄p
