Anti-Inflammatory Polyoxygenated Steroids from the Soft Coral Lobophytum michaelae

Three new polyoxygenated steroids, michosterols A–C (1–3), and four known compounds (4–7) were isolated from the ethyl acetate (EtOAc) extract of the soft coral Lobophytum michaelae, collected off the coast of Taitung. The structures of the new compounds were elucidated on the basis of spectroscopic analyses and comparison of the nuclear magnetic resonance (NMR) data with related steroids. The cytotoxicity of compounds 1–3 against the proliferation of a limited panel of cancer cell lines was assayed. Compound 1 was found to display moderate cytotoxicity against adenocarcinomic human alveolar basal epithelial (A549) cancer cells. It also exhibited potent anti-inflammatory activity by suppressing superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-stimulated human neutrophils. Furthermore, 3 could effectively inhibit elastase release, as well.


Results and Discussion
The frozen bodies of L. michaelae were sliced and exhaustively extracted with ethyl acetate (EtOAc).The EtOAc extract was separated by repeated gravity column chromatography and high-performance liquid chromatography (HPLC) to afford three new and four known triterpenoids steroids 1-7 (Figure 1).
Compound 1 possesses a double bond between C-16 and C-17 and an unusual side-chain with a hydroperoxyl group at C-20, whereas 2 has this group at C-16 and an uncommon olefinic structure at C-17 and C-20.The in vitro cytotoxicity of 1-3 against three cancer cell lines, adenocarcinomic human alveolar basal epithelial (A549), human colorectal adenocarcinoma (DLD-1) and human prostatic carcinoma (LNCap), was evaluated.The abilities of compounds 1-3 to inhibit superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLP/CB)-stimulated human neutrophils was also assayed.

.Results and Discussion
The frozen bodies of L. michaelae were sliced and exhaustively extracted with ethyl acetate (EtOAc).The EtOAc extract was separated by repeated gravity column chromatography and highperformance liquid chromatography (HPLC) to afford three new and four known triterpenoids steroids 1-7 (Figure 1).The molecular formula of 1, an amorphous solid, was determined as C31H52O7 based on the [M + Na] + ion peak obtained by high-resolution electron spray ionisation mass spectrometry (HRESIMS), implying six degrees of unsaturation.The 13 C NMR spectrum showed 31 carbon signals, including an ester carbonyl (δC 171.9, C), a double bond (δC 157.6, C and 127.0,CH), two oxymethines (δC 71.8 and 67.7, each CH) and three oxygenated sp 3 quaternary carbons (δC 87.2, 85.6, and 78.1) (Table 1).The 1 H NMR spectrum in conjunction with the heteronuclear single quantum coherence (HSQC) The molecular formula of 1, an amorphous solid, was determined as C 31 H 52 O 7 based on the [M + Na] + ion peak obtained by high-resolution electron spray ionisation mass spectrometry (HRESIMS), implying six degrees of unsaturation.The 13 C NMR spectrum showed 31 carbon signals, including an ester carbonyl (δ C 171.9, C), a double bond (δ C 157.6, C and 127.0,CH), two oxymethines (δ C 71.8 and 67.7, each CH) and three oxygenated sp 3 quaternary carbons (δ C 87.2, 85.6, and 78.1) (Table 1).The 1 H NMR spectrum in conjunction with the heteronuclear single quantum coherence (HSQC) spectrum revealed the presence of eight methyl groups (δ H 2.03 (3H, s), 1.50 (3H, s), 1.41 (3H, s), 1.31 (3H, s), 0.96 (6H, s), 0.93 (3H, d, J = 6.8 Hz) and 0.88 (3H, d, J = 7.2 Hz)), an olefinic methine proton (δ H 5.70 (1H, d, J = 2.0 Hz)) and a hydroperoxy group signal at δ H 8.06 (br s).Thus, the remaining four unsaturations of 1 corresponded to a tetracyclic skeleton.In the correlation spectroscopy (COSY) spectrum, it was possible to identify three different structural units extending from C-1 to C-4; C-6 to both C-12 and C-16 through C-8; and C-22 to both C-28 and C-29 through C-23 (Figure 2).From the heteronuclear multiple-bond correlation (HMBC) spectrum, the correlations of H 3 -19 to C-1, C-5, C-9 and C-10, H 3 -18 to C-12, C-13, C-14 and C-17, H-6 to C-4 and C-5, H-16 to C-20, H 3 -21 to C-17, C-20 and C-22, both H 3 -26 and H 3 -27 to C-24 and H 3 -28 to C-25 permitted the establishment of the carbon skeleton of a 23,24-dimethycholestane (Figure 2).The hydroperoxy group positioned at C-20 was confirmed from the HMBC correlation of the hydroperoxy proton δ H 8.06 (br s) to the oxygenated carbon at δ C 85.6; hence, the acetoxy group was positioned at C-25 (δ C 87.2).The planar structure of 1 was thus established unambiguously.The relative configuration of 1 was deduced by interpretation of the nuclear Overhauser effect (NOE) correlations (Figure 3), analysis of 3 JH-H values and comparison of carbon chemical shifts.As depicted in Figure 3, it was found that the NOE interactions displayed by both H3-18 and H3-19 with H-8 and H3-19 with H-6, while one of the methylene protons at C-7 (δH 1.85, m), showed NOESY correlations with both of H-6 and H-8.Therefore, assuming the β-orientation of H3-19, H-6, H-7 at δH 1.85, H-8 and H3-18 should be positioned on the β-face, while the other H-7 (δH 1.09, q, J = 12.0 Hz) was assigned as H-7α.Moreover, H-14 showed NOESY correlations with H-7α, H-9 and one proton of H2-12 (δH 2.06, m); whereas the latter proton was NOE correlated with H3-21.This reflects theαorientations of H-9, H-14 and H3-21 and, consequently, the β-orientation of the hydroperoxy group at C-20 of the side chain.Further, H3-21 exhibited NOESY correlation with H-23; and H-23 expressed NOE interaction with H-24 as did H3-28 with H3-29, respectively, while no NOE interaction was found for H-23 with H3-28 and for H-24 with H3-29.Thus, the 23R*, 24S* relative configurations were revealed (Figure 3) and further supported by the comparison of the NOE interactions in 1 with those anticipated in its other three 23,24-rotamers (Figure 4).Finally, the configurations of C-3, C-5 andC-6 were elucidated by comparison of the 1 H NMR chemical shifts and coupling constants of H-3 and H-6 with those of related steroids (Table 2).The δ and J values of H-3 (δH 4.24, s) and H-6 (δH 3.74-3.81,dd, J = 12.0, 4.8 Hz) of known compound 5β-cholestane-3β,5,6α-triol [21,22] were found to be similar to the corresponding H-3 (δH 4.27, br s) and H-6 (δH 3.82, dd, J = 12.0, 4.8 Hz) of 1 (Table 2).Consequently, the relative configuration of 1 was determined unequivocally.Since it has been known for quite a long time that both H3-18 and H3-19 should be positioned on the β-face for natural steroids, thus the absolute configuration of 1 should be the same as shown in Structure 1.The relative configuration of 1 was deduced by interpretation of the nuclear Overhauser effect (NOE) correlations (Figure 3), analysis of 3 J H-H values and comparison of carbon chemical shifts.As depicted in Figure 3, it was found that the NOE interactions displayed by both H 3 -18 and H 3 -19 with H-8 and H 3 -19 with H-6, while one of the methylene protons at C-7 (δ H 1.85, m), showed NOESY correlations with both of H-6 and H-8.Therefore, assuming the β-orientation of H 3 -19, H-6, H-7 at δ H 1.85, H-8 and H 3 -18 should be positioned on the β-face, while the other H-7 (δ H 1.09, q, J = 12.0 Hz) was assigned as H-7α.Moreover, H-14 showed NOESY correlations with H-7α, H-9 and one proton of H 2 -12 (δ H 2.06, m); whereas the latter proton was NOE correlated with H 3 -21.This reflects theα-orientations of H-9, H-14 and H 3 -21 and, consequently, the β-orientation of the hydroperoxy group at C-20 of the side chain.Further, H 3 -21 exhibited NOESY correlation with H-23; and H-23 expressed NOE interaction with H-24 as did H 3 -28 with H 3 -29, respectively, while no NOE interaction was found for H-23 with H 3 -28 and for H-24 with H 3 -29.Thus, the 23R*, 24S* relative configurations were revealed (Figure 3) and further supported by the comparison of the NOE interactions in 1 with those anticipated in its other three 23,24-rotamers (Figure 4).Finally, the configurations of C-3, C-5 andC-6 were elucidated by comparison of the 1 H NMR chemical shifts and coupling constants of H-3 and H-6 with those of related steroids (Table 2).The δ and J values of H-3 (δ H 4.24, s) and H-6 (δ H 3.74-3.81,dd, J = 12.0, 4.8 Hz) of known compound 5β-cholestane-3β,5,6α-triol [21,22] were found to be similar to the corresponding H-3 (δ H 4.27, br s) and H-6 (δ H 3.82, dd, J = 12.0, 4.8 Hz) of 1 (Table 2).Consequently, the relative configuration of 1 was determined unequivocally.Since it has been known for quite a long time that both H 3 -18 and H 3 -19 should be positioned on the β-face for natural steroids, thus the absolute configuration of 1 should be the same as shown in Structure 1.The relative configuration of 1 was deduced by interpretation of the nuclear Overhauser effect (NOE) correlations (Figure 3), analysis of 3 JH-H values and comparison of carbon chemical shifts.As depicted in Figure 3, it was found that the NOE interactions displayed by both H3-18 and H3-19 with H-8 and H3-19 with H-6, while one of the methylene protons at C-7 (δH 1.85, m), showed NOESY correlations with both of H-6 and H-8.Therefore, assuming the β-orientation of H3-19, H-6, H-7 at δH 1.85, H-8 and H3-18 should be positioned on the β-face, while the other H-7 (δH 1.09, q, J = 12.0 Hz) was assigned as H-7α.Moreover, H-14 showed NOESY correlations with H-7α, H-9 and one proton of H2-12 (δH 2.06, m); whereas the latter proton was NOE correlated with H3-21.This reflects theαorientations of H-9, H-14 and H3-21 and, consequently, the β-orientation of the hydroperoxy group at C-20 of the side chain.Further, H3-21 exhibited NOESY correlation with H-23; and H-23 expressed NOE interaction with H-24 as did H3-28 with H3-29, respectively, while no NOE interaction was found for H-23 with H3-28 and for H-24 with H3-29.Thus, the 23R*, 24S* relative configurations were revealed (Figure 3) and further supported by the comparison of the NOE interactions in 1 with those anticipated in its other three 23,24-rotamers (Figure 4).Finally, the configurations of C-3, C-5 andC-6 were elucidated by comparison of the 1 H NMR chemical shifts and coupling constants of H-3 and H-6 with those of related steroids (Table 2).The δ and J values of H-3 (δH 4.24, s) and H-6 (δH 3.74-3.81,dd, J = 12.0, 4.8 Hz) of known compound 5β-cholestane-3β,5,6α-triol [21,22] were found to be similar to the corresponding H-3 (δH 4.27, br s) and H-6 (δH 3.82, dd, J = 12.0, 4.8 Hz) of 1 (Table 2).Consequently, the relative configuration of 1 was determined unequivocally.Since it has been known for quite a long time that both H3-18 and H3-19 should be positioned on the β-face for natural steroids, thus the absolute configuration of 1 should be the same as shown in Structure 1.      Analysis of the 13 C NMR and HRESIMS spectral data of 2 revealed that it has the same molecular formula as that of 1.The 1 H and 13 C NMR spectra of 2 are similar to those of 1, and the molecular skeleton of 2 was further established by HMBC correlations from H3-21 to C-17 (δC 143.6, C), C-20 (δC 132.8, C) and C-22 (δC 41.2, CH2); H-16 (δH 4.96, dd, J = 7.5, 7.5 Hz) to C-13 (δC 44.1, C) and C-20; and the COSY correlation between H2-15 and H-16 (Figure 2).The above results showed that an olefinic proton signal (δH 5.70, d, J = 2.0 Hz) in 1 was replaced by an oxymethine proton (δH 4.96, dd, J = 7.5, 7.5 Hz), which also showed correlation with the signal of C-16 (δC 86.1) in the HSQC spectrum of 2. Further, a proton signal appearing at δH 8.91 (br s) was found not to be correlated with any carbon signal in the HSQC spectrum.Thus, this should be the signal of a hydroperoxy group substituted at C-16, as confirmed by the downfield shift of this carbon in 2 in comparison with the chemical shifts of the corresponding carbons of two 16-hydroxy analogues, δC 72.3 for faccisteroid B [23] and 71.8 for hippuristerone L [24].The comparison of the 1 H NMR chemical shifts and the analysis of NOESY correlations of 2 revealed the same configurations at C-3, C-5, C-6, C-8, C-9, C-10, C-13, C-14, C-23 and C-24 as those of 1. Further, NOESY correlations from H3-18 to H-8, H-15β (δH 1.69, m) and H-22β (δH 1.76 m) were observed, while H-15α (δH 2.08 m) was correlated with H-14 and H-16 and H-16 with H3-21, suggesting a β-orientation of hydroperoxy group at C-16 and the E geometry of Δ 17 (20) in 2. On the basis of the above findings and the careful analysis of NOESY correlations (Figure 5), the structure of 2 was determined as illustrated in Figure 1.Table 2.The 1 H NMR chemical shifts and coupling constants of the H-3 and H-6 of compound 1 and the related steroids [22].and the COSY correlation between H 2 -15 and H-16 (Figure 2).The above results showed that an olefinic proton signal (δ H 5.70, d, J = 2.0 Hz) in 1 was replaced by an oxymethine proton (δ H 4.96, dd, J = 7.5, 7.5 Hz), which also showed correlation with the signal of C-16 (δ C 86.1) in the HSQC spectrum of 2. Further, a proton signal appearing at δ H 8.91 (br s) was found not to be correlated with any carbon signal in the HSQC spectrum.Thus, this should be the signal of a hydroperoxy group substituted at C-16, as confirmed by the downfield shift of this carbon in 2 in comparison with the chemical shifts of the corresponding carbons of two 16-hydroxy analogues, δ C 72.3 for faccisteroid B [23] and 71.8 for hippuristerone L [24].The comparison of the 1 H NMR chemical shifts and the analysis of NOESY correlations of 2 revealed the same configurations at C-3, C-5, C-6, C-8, C-9, C-10, C-13, C-14, C-23 and C-24 as those of 1. Further, NOESY correlations from H 3 -18 to H-8, H-15β (δ H 1.69, m) and H-22β (δ H 1.76 m) were observed, while H-15α (δ H 2.08 m) was correlated with H-14 and H-16 and H-16 with H 3 -21, suggesting a β-orientation of hydroperoxy group at C-16 and the E geometry of ∆ 17 (20)  in 2. On the basis of the above findings and the careful analysis of NOESY correlations (Figure 5), the structure of 2 was determined as illustrated in Figure 1.Michosterol C (3) was isolated as a white powder and has a molecular formula of C30H52O5 by the analysis of HRESIMS.The IR spectrum of 3 also revealed the presence of hydroxy (3416 cm −1 ) and ester carbonyl (1718 cm −1 ) groups.Comparison of the 1 H and 13 C NMR data (Table 1) of compounds 2 and 3 pointed out that the A-C rings of 3 were similar to those of 2, with the exception of signals assigned to C-6, where the hydroxymethine (δH 3.80, dd, J = 12.0, 5.0 Hz; δC 71.7) in 2 was replaced by an acetoxymethine (δH 4.97, dd, J = 12.0, 4.8 Hz; δC 75.8) in 3. The acetoxy group substitution at C-6 was determined by the HMBC correlations of H-6 (δH 4.97) with C-4 (δC 31.1,CH2), C-5 (δC 77.4,C) and an acetoxy carbonyl carbon (δC 171.9, C), and further confirmed by the COSY spectrum (Figure 2).Furthermore, the signal of a hydroperoxy-bearing methine group at C-16 (δH 4.96, dd, J = 7.5, 7.5 Hz; δC 86.1) in 2 was replaced by signals of a methylene group (H2-16:δH 1.28 m and 1.84 m; δC 28.0).Comparison of the NMR data of 3, measured in CD3OD (see the Experimental Section) with those of (24S)-ergostane-6-acetate-3β,5α,6β,25-tetraol [21] indicated that the planar structure and the configurations of the side chain of both compounds are the same.Furthermore, the configurations of C-3, C-5 and C-6 were elucidated by comparison of the 1 H NMR coupling constants with the related steroids (Tables 1 and 2).The J values of H-3 (δH 4.11, br s) and H-6 (δH 4.97, dd, J = 12.0, 4.8 Hz) of compound 3 were identical to the corresponding H-3 (δH 4.27, br s) and H-6 (δH 3.82, dd, J = 12.0, 4.8 Hz) of 1.In addition, NOESY correlations of H3-19 with both H-6 and H-8, H-8 with H-7β (δH 1.81, m), H-7α (δH 1.07, m) with H-4α (δH 1.92, m) and H-9 and H-4α with H-3 confirmed the α-orientations of the H-3 and 6-OAc group and the β-orientation of the 5-OH group (Figure 5).Thus, the structure of 3 was established as shown in Figure 1.
The cytotoxicity of the isolates 1-3 against A549, DLD-1 and LNCap cancer cells was assayed.The results showed that only compound 1 exhibited a moderate cytotoxicity effect against the A549 cell line with an IC50 value of 14.9 ± 5.7 μg/mL.The other compounds were found not to be cytotoxic against the above cancer cell lines (IC50's > 20 μg/mL).Michosterol C (3) was isolated as a white powder and has a molecular formula of C 30 H 52 O 5 by the analysis of HRESIMS.The IR spectrum of 3 also revealed the presence of hydroxy (3416 cm −1 ) and ester carbonyl (1718 cm −1 ) groups.Comparison of the 1 H and 13 C NMR data (Table 1) of compounds  1 and 2).The J values of H-3 (δ H 4.11, br s) and H-6 (δ H 4.97, dd, J = 12.0, 4.8 Hz) of compound 3 were identical to the corresponding H-3 (δ H 4.27, br s) and H-6 (δ H 3.82, dd, J = 12.0, 4.8 Hz) of 1.In addition, NOESY correlations of H 3 -19 with both H-6 and H-8, H-8 with H-7β (δ H 1.81, m), H-7α (δ H 1.07, m) with H-4α (δ H 1.92, m) and H-9 and H-4α with H-3 confirmed the α-orientations of the H-3 and 6-OAc group and the β-orientation of the 5-OH group (Figure 5).Thus, the structure of 3 was established as shown in Figure 1.
The cytotoxicity of the isolates 1-3 against A549, DLD-1 and LNCap cancer cells was assayed.The results showed that only compound 1 exhibited a moderate cytotoxicity effect against the A549 cell line with an IC 50 value of 14.9 ± 5.7 µg/mL.The other compounds were found not to be cytotoxic against the above cancer cell lines (IC 50' s > 20 µg/mL).
The anti-inflammatory activities of the new compounds 1-3 on pro-inflammatory responses were evaluated by measuring their ability to suppress fMLP/CB-induced superoxide anion (O 2 −• ) generation and elastase release in human neutrophils.From the results (Table 3), compound 1 showed significant inhibitory effects against superoxide anion generation and elastase release in fMLP/CB-stimulated cells at the 10 µM concentration tested, with the IC 50 values being 7.1 ± 0.3 µM and 4.5 ± 0.9 µM, respectively.Further, compound 3 exhibited stronger inhibitory activities against elastase release, with the IC 50 values of 0.9 ± 0.1 µM.

General Experimental Procedures
The specific optical rotation values and IR spectral absorptions were recorded on a JASCO P-1020 digital polarimeter (JASCO Corporation, Tokyo, Japan) and a JASCO J-815 spectrophotometer (JASCO Corporation), respectively.A Varian 400MR FT-NMR or Varian Unity INOVA-500 FT-NMR (Varian Inc., Palo Alto, CA, USA) was applied to record the 1 H and 13 C NMR spectra with the chemical shifts shown as ppm referenced to the solvent residue of CDCl 3 (δ H 7.26 ppm and δ C 77.0 ppm) and CD 3 OD (δ H 3.31 ppm and δ C 49.0 ppm), respectively.A Bruker APEX II mass spectrometer equipped with an ESI ionization source (Bruker, Bremen, Germany) was used for acquiring high-resolution mass data.The HPLC system used in this study was composed of a Hitachi L-2455 instrument (Hitachi Ltd., Tokyo, Japan) equipped with a reversed-phase (RP-18) column (ODS-3, 5 µm, 250 × 20 mm, Sciences Inc., Tokyo, Japan).

Animal Material
The collection of the soft coral Lobophytum michaelae Tixier-Durivault (1956) was performed off the coast of Jihui Fishing Port, Taitung County, Taiwan, in March 2013.The organism was stored in a freezer at −20 • C until extraction.Prof. C.-F. Dai performed the species identification.A voucher specimen (JiH-201314) of this soft coral has been deposited at National Sun Yat-sen University.
Analysis of the13 C NMR and HRESIMS spectral data of 2 revealed that it has the same molecular formula as that of 1.The 1 H and13 C NMR spectra of 2 are similar to those of 1, and the molecular skeleton of 2 was further established by HMBC correlations from H 3 -21 to C-17 (δ C 143.6, C), C-20 (δ C 132.8, C) and C-22 (δ C 41.2, CH 2 ); H-16 (δ H 4.96, dd, J = 7.5, 7.5 Hz) to C-13 (δ C 44.1, C) and C-20;

2 and 3
pointed out that the A-C rings of 3 were similar to those of 2, with the exception of signals assigned to C-6, where the hydroxymethine (δ H 3.80, dd, J = 12.0, 5.0 Hz; δ C 71.7) in 2 was replaced by an acetoxymethine (δ H 4.97, dd, J = 12.0, 4.8 Hz; δ C 75.8) in 3. The acetoxy group substitution at C-6 was determined by the HMBC correlations of H-6 (δ H 4.97) with C-4 (δ C 31.1, CH 2 ), C-5 (δ C 77.4, C) and an acetoxy carbonyl carbon (δ C 171.9, C), and further confirmed by the COSY spectrum (Figure 2).Furthermore, the signal of a hydroperoxy-bearing methine group at C-16 (δ H 4.96, dd, J = 7.5, 7.5 Hz; δ C 86.1) in 2 was replaced by signals of a methylene group (H 2 -16:δ H 1.28 m and 1.84 m; δ C 28.0).Comparison of the NMR data of 3, measured in CD 3 OD (see the Experimental Section) with those of (24S)-ergostane-6-acetate-3β,5α,6β,25-tetraol [21] indicated that the planar structure and the configurations of the side chain of both compounds are the same.Furthermore, the configurations of C-3, C-5 and C-6 were elucidated by comparison of the 1 H NMR coupling constants with the related steroids (Tables

spectrum of 3 ,
Figure S16: 1 H NMR spectrum of 3 in CDCl 3 at 400 MHz, Figure S17: 13 C NMR spectrum of 3 in CDCl 3 at 100 MHz, Figure S18: HSQC NMR spectrum of 3 in CDCl 3 at 400 MHz, Figure S19: COSY NMR spectrum of 3 in CDCl 3 at 400 MHz, Figure S20: HMBC NMR spectrum of 3 in CDCl 3 at 400 MHz, Figure S21: NOESY NMR spectrum of 3 in CDCl 3 at 400 MHz, Figure S22: 1 H NMR spectrum of 3 in CD 3 OD at 400 MHz, Figure S23: 13 C NMR spectrum of 3 in CD 3 OD at 100 MHz, Figure S24: HSQC NMR spectrum of 3 in CD 3 OD at 400 MHz, Figure S25: COSY NMR spectrum of 3 in CD 3 OD at 400 MHz, Figure S26: HMBC NMR spectrum of 3 in CD 3 OD at 400 MHz.

Table 1 .
13C and 1 H NMR data of compounds 1

-3 in CDCl 3 . 1 2 3 Position δ C a , Mult. b δ H c , Mult. (J) d δ C e , Mult. δ H f , Mult. (J) δ C a , Mult. δ H c , Mult. (J)
a Spectrum recorded at 100 MHz; b attached protons were deduced by the DEPT experiment; c spectrum recorded at 400 MHz; d J values (in Hz) in parentheses; e spectrum recorded at 125 MHz; f spectrum recorded at 500 MHz.

Table 2 .
[22]1 H NMR chemical shifts and coupling constants of the H-3 and H-6 of compound 1 and the related steroids[22].