Pinnisterols D–J, New 11-Acetoxy-9,11-secosterols with a 1,4-Quinone Moiety from Formosan Gorgonian Coral Pinnigorgia sp. (Gorgoniidae)

Seven new marine 11-acetoxy-9,11-secosterols, pinnisterols D–J (1–7), with a 1,4-quinone moiety, were discovered from the gorgonian coral Pinnigorgia sp. In this study, the structures of secosterols 1–7 were revealed by spectroscopic analysis. Bioactivity study showed that secosterol 1 treatment inhibited cell viability in a hepatic stellate cell line, HSC-T6, with an IC50 value of 3.93 μM; and secosterols 2, 5, and 7 reduced elastase enzyme release, and 3, 5, and 7 decreased the production of superoxide anions from human neutrophils.

Mar. Drugs 2017, 15, 11 4 of 11 There was a greater coupling constant between H-22 and H-23 (J = 14.8 Hz), which supported a trans relationship between H-22 and H-23. This implied that the configuration of C-24 should be R according to the 13 C NMR chemical shift of C-28 (δC 17.5). A previous study showed that, for a known sterol, (22E,24R)-24-methylcholesta-5,22-dien-3β-ol, with an identical chain, and the 24S epimer, (22E,24S)-24-methylcholesta-5,22-dien-3β-ol, the 13 C NMR value of C-28 resonates at δC 17.68 ppm in the 24R epimer, with a relative 0.4 ppm downfield chemical shift ( Figure 3) [10].  Pinnisterol E (2) was present as a colorless oil. From HRESIMS analysis, the signal at m/z 527.33410 (calcd. for C30H48O6 + Na, 527.33486) suggested the molecular formula of 2 to be C30H48O6 (7° of unsaturation), and the IR spectrum demonstrated the existence of hydroxy (νmax 3381 cm −1 ), ester (νmax 1740 cm −1 ), and α,β-unsaturated ketonic carbonyl (νmax 1686 cm −1 ) groups. The whole series of spectroscopic data demonstrated that secosterols 2 and 1 had an identical core structure, the difference being limited to the absence in 2 of the carbon-carbon double bond between C-22/23. The complete assignments of 1 H and 13 C NMR of 2 (Tables 2 and 3) were compared with the values of 1, and secosterol 2 was assigned as having structure 2, with the same configurations of the core rings A-C. In addition, both compounds had identical stereogenic centers at C-3, C-5, C-10, C-13, C-14, and C-17, and their 1 H and 13 C NMR chemical shifts and proton coupling constants were in concurrence also. Based on the 13 C NMR chemical shifts of C-25 (δC 31.5), C-26 (δC 17.6), and C-27 (δC 20.5), the configuration of the stereogenic center at C-24 was assigned as S. Previous study also showed that the 13 C NMR values of C-25, C-26, and C-27 resonates at δC 31.54, 17.68, and 20.56 ppm in a 24S epimer of a known sterol, (24S)-24-methylcholest-5-en-3β-ol, with an identical side chain, and the 13 C NMR values of C-25, C-26, and C-27 in a 24R epimer, (24R)-24-methylcholest-5-en-3β-ol, were observed at δC 32.49, 20.26, and 18.32 ppm, respectively ( Figure 4) [10].  Pinnisterol F (3) was present as a colorless oil. From HRESIMS analysis, the signal at m/z 583.32406 (calcd. for C32H48O8 + Na, 583.32469) suggested the molecular formula of 3 to be C32H48O8 (9° of unsaturation). The NMR signals of 3 (Tables 2 and 3) were similar to those of 1, except that the signals related to the C-21 methyl in 1 were substituted by signals for an acetoxymethylene group in 3. From the HMBC spectrum of 3, it was revealed that an ester carbonyl carbon at δC 171.1 correlated with a methyl signal at δH 2.04 and a pair of oxygenated methylene protons at δH 4.01 (1H, dd, J = 10.5, 7.0 Hz) and 3.96 (1H, dd, J = 10.5, 7.0 Hz), which revealed that an acetoxy group was at the position C-21 in the side chain of 3. Thus, pinnisterol F (3) was found to be the 21-acetoxy derivative of 1. Pinnisterol E (2) was present as a colorless oil. From HRESIMS analysis, the signal at m/z 527.33410 (calcd. for C 30 H 48 O 6 + Na, 527.33486) suggested the molecular formula of 2 to be C 30 H 48 O 6 (7 • of unsaturation), and the IR spectrum demonstrated the existence of hydroxy (ν max 3381 cm −1 ), ester (ν max 1740 cm −1 ), and α,β-unsaturated ketonic carbonyl (ν max 1686 cm −1 ) groups. The whole series of spectroscopic data demonstrated that secosterols 2 and 1 had an identical core structure, the difference being limited to the absence in 2 of the carbon-carbon double bond between C-22/23. The complete assignments of 1 H and 13 C NMR of 2 (Tables 2 and 3) were compared with the values of 1, and secosterol 2 was assigned as having structure 2, with the same configurations of the core rings A-C. In addition, both compounds had identical stereogenic centers at C-3, C-5, C-10, C-13, C-14, and C-17, and their 1 H and 13 C NMR chemical shifts and proton coupling constants were in concurrence also. Based on the 13 C NMR chemical shifts of C-25 (δ C 31.5), C-26 (δ C 17.6), and C-27 (δ C 20.5), the configuration of the stereogenic center at C-24 was assigned as S. Previous study also showed that the 13 C NMR values of C-25, C-26, and C-27 resonates at δ C 31.54, 17.68, and 20.56 ppm in a 24S epimer of a known sterol, (24S)-24-methylcholest-5-en-3β-ol, with an identical side chain, and the 13 C NMR values of C-25, C-26, and C-27 in a 24R epimer, (24R)-24-methylcholest-5-en-3β-ol, were observed at δ C 32.49, 20.26, and 18.32 ppm, respectively ( Figure 4) [10].
Mar. Drugs 2017, 15, 11 4 of 11 There was a greater coupling constant between H-22 and H-23 (J = 14.8 Hz), which supported a trans relationship between H-22 and H-23. This implied that the configuration of C-24 should be R according to the 13 C NMR chemical shift of C-28 (δC 17.5). A previous study showed that, for a known sterol, (22E,24R)-24-methylcholesta-5,22-dien-3β-ol, with an identical chain, and the 24S epimer, (22E,24S)-24-methylcholesta-5,22-dien-3β-ol, the 13 C NMR value of C-28 resonates at δC 17.68 ppm in the 24R epimer, with a relative 0.4 ppm downfield chemical shift ( Figure 3) [10].  Pinnisterol E (2) was present as a colorless oil. From HRESIMS analysis, the signal at m/z 527.33410 (calcd. for C30H48O6 + Na, 527.33486) suggested the molecular formula of 2 to be C30H48O6 (7° of unsaturation), and the IR spectrum demonstrated the existence of hydroxy (νmax 3381 cm −1 ), ester (νmax 1740 cm −1 ), and α,β-unsaturated ketonic carbonyl (νmax 1686 cm −1 ) groups. The whole series of spectroscopic data demonstrated that secosterols 2 and 1 had an identical core structure, the difference being limited to the absence in 2 of the carbon-carbon double bond between C-22/23. The complete assignments of 1 H and 13 C NMR of 2 (Tables 2 and 3) were compared with the values of 1, and secosterol 2 was assigned as having structure 2, with the same configurations of the core rings A-C. In addition, both compounds had identical stereogenic centers at C-3, C-5, C-10, C-13, C-14, and C-17, and their 1 H and 13 C NMR chemical shifts and proton coupling constants were in concurrence also. Based on the 13 C NMR chemical shifts of C-25 (δC 31.5), C-26 (δC 17.6), and C-27 (δC 20.5), the configuration of the stereogenic center at C-24 was assigned as S. Previous study also showed that the 13 C NMR values of C-25, C-26, and C-27 resonates at δC 31.54, 17.68, and 20.56 ppm in a 24S epimer of a known sterol, (24S)-24-methylcholest-5-en-3β-ol, with an identical side chain, and the 13 C NMR values of C-25, C-26, and C-27 in a 24R epimer, (24R)-24-methylcholest-5-en-3β-ol, were observed at δC 32.49, 20.26, and 18.32 ppm, respectively ( Figure 4)   Pinnisterol F (3) was present as a colorless oil. From HRESIMS analysis, the signal at m/z 583.32406 (calcd. for C32H48O8 + Na, 583.32469) suggested the molecular formula of 3 to be C32H48O8 (9° of unsaturation). The NMR signals of 3 (Tables 2 and 3) were similar to those of 1, except that the signals related to the C-21 methyl in 1 were substituted by signals for an acetoxymethylene group in 3. From the HMBC spectrum of 3, it was revealed that an ester carbonyl carbon at δC 171.1 correlated with a methyl signal at δH 2.04 and a pair of oxygenated methylene protons at δH 4.01 (1H, dd,   (Tables 2 and 3) were similar to those of 1, except that the signals related to the C-21 methyl in 1 were substituted by signals for an acetoxymethylene group in 3. From the HMBC spectrum of 3, it was revealed that an ester carbonyl carbon at δ C 171.1 correlated with a methyl signal at δ H 2.04 and a pair of oxygenated methylene protons at δ H 4.01 (1H, dd, J = 10.5, 7.0 Hz) and 3.96 (1H, dd, J = 10.5, 7.0 Hz), which revealed that an acetoxy group was at the position C-21 in the side chain of 3. Thus, pinnisterol F (3) was found to be the 21-acetoxy derivative of 1.
Pinnisterol G (4) had a molecular formula identical to that of 3, C 32 H 48 O 8 , with a HRESIMS signal located at m/z 583.32432 (calcd. for C 32 H 48 O 8 + Na, 583.32469) with nine degrees of unsaturation, indicating that secosterols 3 and 4 were isomers. Comparison of the NMR data of 4 with those of 3 (Tables 2 and 3) showed that both compounds possessed the same sterol nucleus and a similar side chain, but differed in terms of the location of one acetoxy group. From an HMBC experiment, it was revealed that one ester carbonyl carbon at δ C 171.3 correlated with one methyl signal at δ H 2.07 and a pair of oxymethylene protons signals at δ H 3.98 (dd, J = 10.5, 6.3 Hz) and 3.85 (dd, J = 10.5, 6.3 Hz), which indicated that an acetoxy group was located at C-27 in the side chain. The configurations at C-24 and C-25 were therefore designated as Sand R-forms, respectively, on the basis of the 13 C NMR chemical shifts of C-24 (δ C 38.2), C-25 (δ C 37.4), C-26 (δ C 13.1), C-27 (δ C 67.9), and C-28 (δ C 18.3). It was reported that the 13 C NMR values of C-24, C-25, C-26, C-27, and C-28 resonate at δ C 38.2, 37.6, 13.0, 68.1, and 18.6 ppm in a 24S and a 25R epimer of a known sterol, echrebsteroid C, with the same side chain, and the 13 C NMR values of C-24, C-25, C-26, C-27, and C-28 in a 24S and 25S epimer, echrebsteroid B, appeared at δ C 38. 8, 37.5, 14.1, 67.9, and 17.1 ppm ( Figure 5) [11].  80. 6, C 80.6, C 80.6, C 80.5, C 80.1, C 80.0, C 6 197.4, C 197.4, C 197.5, C 197.5, C 197.3, C 197.5, C 7 134 . Pinnisterol G (4) had a molecular formula identical to that of 3, C32H48O8, with a HRESIMS signal located at m/z 583.32432 (calcd. for C32H48O8 + Na, 583.32469) with nine degrees of unsaturation, indicating that secosterols 3 and 4 were isomers. Comparison of the NMR data of 4 with those of 3 (Tables 2 and 3) showed that both compounds possessed the same sterol nucleus and a similar side chain, but differed in terms of the location of one acetoxy group. From an HMBC experiment, it was revealed that one ester carbonyl carbon at δC 171.3 correlated with one methyl signal at δH 2.07 and a pair of oxymethylene protons signals at δH 3.98 (dd, J = 10.5, 6.3 Hz) and 3.85 (dd, J = 10.5, 6.3 Hz), which indicated that an acetoxy group was located at C-27 in the side chain. The configurations at C-24 and C-25 were therefore designated as S-and R-forms, respectively, on the basis of the 13 C NMR chemical shifts of C-24 (δC 38.2), C-25 (δC 37.4), C-26 (δC 13.1), C-27 (δC 67.9), and C-28 (δC 18.3). It was reported that the 13 C NMR values of C-24, C-25, C-26, C-27, and C-28 resonate at δC 38. 2, 37.6, 13.0, 68.1, and 18.6 ppm in a 24S and a 25R epimer of a known sterol, echrebsteroid C, with the same side chain, and the 13 C NMR values of C-24, C-25, C-26, C-27, and C-28 in a 24S and 25S epimer, echrebsteroid B, appeared at δC 38. 8, 37.5, 14.1, 67.9, and 17.1 ppm ( Figure 5) [11]. Pinnisterol H (5) was isolated as a colorless oil. Based on the HRESIMS signal located at m/z 585.33988 (calcd. for C32H50O8 + Na, 585.34034), it was concluded that the molecular formula of 5 was C32H50O8 (8° of unsaturation). The IR spectrum of 5 indicated the presence of hydroxy (νmax 3448 cm −1 ), ester (νmax 1736 cm −1 ) and α,β-unsaturated ketonic carbonyl (νmax 1686 cm −1 ) groups. The whole series of spectroscopic data showed that secosterol 5 and secosterol 1 shared the same core structure, with the exception of the addition of an acetoxy group to substitute the alkene at C-23 in 5. The complete assignments of the 13 C and 1 H NMR of pinnisterol H (5) (Tables 3 and 4) were compared with the values of 1, and the HMBC correlations fully supported the positions of the functional groups of 5, indicating that it had a structure of the same configuration as secosterols 14 in the core rings AC. The proton coupling constants and NMR chemical shift data also further supported this finding, though the configurations of C-23 and C-24 were not determined at this stage.
Pinnisterol I (6) was obtained as a colorless oil. The HRESIMS signal at m/z 585.33988 (calcd. for C32H50O8 + Na, 585.34034) suggested the molecular formula of 6 to be C32H50O8 (8° of unsaturation). The NMR signals of 6 (Tables 3 and 4) were very similar to those of 5, with the exception that 5 had signals corresponding to 3-hydroxy and 23-acetoxy groups, which were substituted by signals for acetoxy and hydroxy groups, respectively, in 6. From a NOESY experiment, the correlations of data Pinnisterol H (5) was isolated as a colorless oil. Based on the HRESIMS signal located at m/z 585.33988 (calcd. for C 32 H 50 O 8 + Na,585.34034), it was concluded that the molecular formula of 5 was C 32 H 50 O 8 (8 • of unsaturation). The IR spectrum of 5 indicated the presence of hydroxy (ν max 3448 cm −1 ), ester (ν max 1736 cm −1 ) and α,β-unsaturated ketonic carbonyl (ν max 1686 cm −1 ) groups. The whole series of spectroscopic data showed that secosterol 5 and secosterol 1 shared the same core structure, with the exception of the addition of an acetoxy group to substitute the alkene at C-23 in 5. The complete assignments of the 13 C and 1 H NMR of pinnisterol H (5) (Tables 3 and 4) were compared with the values of 1, and the HMBC correlations fully supported the positions of the functional groups of 5, indicating that it had a structure of the same configuration as secosterols 1-4 in the core rings A-C. The proton coupling constants and NMR chemical shift data also further supported this finding, though the configurations of C-23 and C-24 were not determined at this stage.
Pinnisterol I (6) was obtained as a colorless oil. The HRESIMS signal at m/z 585.33988 (calcd. for C 32 H 50 O 8 + Na,585.34034) suggested the molecular formula of 6 to be C 32 H 50 O 8 (8 • of unsaturation). The NMR signals of 6 (Tables 3 and 4) were very similar to those of 5, with the exception that 5 had signals corresponding to 3-hydroxy and 23-acetoxy groups, which were substituted by signals for acetoxy and hydroxy groups, respectively, in 6. From a NOESY experiment, the correlations of data of 5 and 6 demonstrated that the configurations of the stereogenic centers in the core rings A-C were identical to those of 1. The configurations of stereogenic centers C-23 and C-24 of 6 were also not determined at this stage.  (Tables 3 and 4), compound 7 showed the same nuclear structure as that of compound 6. In the 13 C NMR data of 7, one additional disubstituted olefin was identified from signals of carbons at δ C 132.9 (CH-22) and 134.5 (CH-23). The presence of a 24-hydroxy group was evidenced by HMBC correlations between H-22, H-23, H-25, H 3 -26, H 3 -27, and H 3 -28/C-24 (δ C 75.0), a methyl-containing oxygenated tertiary carbon. There was a greater coupling constant between H-22 and H-23 (J = 15.6 Hz), suggesting that a trans relationship existed between H-22 and H-23. The configuration of the C-24 stereogenic center was assigned as S on the basis of the 13 C NMR chemical shifts of C-24 (δ C 75.0), C-25 (δ C 38.1), C-26 (δ C 17.5), C-27 (δ C 17.1), and C-28 (δ C 25.2). It was reported that the 13 C NMR values of C-24, C-25, C-26, C-27, and C-28 resonates at δ C 75. 1, 38.3, 17.7, 17.4, and 25.2 ppm in a 24S epimer of a known synthetic product, 24(S)-hydroxyvitamin D 2 , with the same side chain ( Figure 6) [12]. and H3-28/C-24 (δC 75.0), a methyl-containing oxygenated tertiary carbon. There was a greater coupling constant between H-22 and H-23 (J = 15.6 Hz), suggesting that a trans relationship existed between H-22 and H-23. The configuration of the C-24 stereogenic center was assigned as S on the basis of the 13 C NMR chemical shifts of C-24 (δC 75.0), C-25 (δC 38.1), C-26 (δC 17.5), C-27 (δC 17.1), and C-28 (δC 25.2). It was reported that the 13 C NMR values of C-24, C-25, C-26, C-27, and C-28 resonates at δC 75. 1, 38.3, 17.7, 17.4, and 25.2 ppm in a 24S epimer of a known synthetic product, 24(S)-hydroxyvitamin D2, with the same side chain ( Figure 6) [12].  Figure 6. Schematic diagrams of 13 C NMR chemical shift data of the side-chain of pinnisterol J (7) and 24(S)-hydroxyvitamin D2 (A) [12].
The hepatic stellate cell, a major cell type involved in liver fibrosis, is also responsible for liver damage by increasing proliferation and protein secretion associated with the formation of scar tissue. In cytotoxicity testing, secosterols 1-7 were examined in terms of their cytotoxic effects on HSC-T6, an immortalized rat hepatic stellate cell lines. At a concentration of 10 μM, secosterols 1, 3, and 5 significantly decreased the viability of HSC-T6 cells to 16.8 (IC50 = 3.93 μM), 56.9 and 37.1%, Figure 6. Schematic diagrams of 13 C NMR chemical shift data of the side-chain of pinnisterol J (7) and 24(S)-hydroxyvitamin D 2 (A) [12].
The hepatic stellate cell, a major cell type involved in liver fibrosis, is also responsible for liver damage by increasing proliferation and protein secretion associated with the formation of scar tissue. In cytotoxicity testing, secosterols 1-7 were examined in terms of their cytotoxic effects on HSC-T6, an immortalized rat hepatic stellate cell lines. At a concentration of 10 µM, secosterols 1, 3, and 5 significantly decreased the viability of HSC-T6 cells to 16.8 (IC 50 = 3.93 µM), 56.9 and 37.1%, respectively ( Figure 7). These results implied that the functional groups in the side chain of secosterols 1-7 play important roles in determining the activity of the compounds.

General Experimental Procedures
Optical rotations were measured with a digital polarimeter (P-1010; Japan Spectroscopic Corporation, Tokyo, Japan); and infrared spectra were recorded on a spectrometer (FT/IR-4100; Japan Spectroscopic Corporation); peaks are reported in cm −1 . NMR spectra were obtained on a 400 MHz NMR spectrometer (Mercury Plus; Varian, Palo Alto, CA, USA) and a 700 MHz NMR spectrometer (AVIIIHD700X; Bruker, Bremen, Germany), using the residual CHCl3 signal (δH 7.26 ppm) and CDCl3 (δC 77.1 ppm) as internal standards for 1 H NMR and 13 C NMR, respectively. Coupling constant values (J) are given in Hz. ESIMS and HRESIMS were performed using mass spectrometry (Tesla solariX Figure 7. Decreased cell viability in HSC-T6 cells treated with secosterols 1-7 for 24 h at a concentration of 10 µM. Cells were treated with secosterols, DMSO (control) or coral crude extract at 6 µg/mL. Cytotoxicity was monitored spectrophotometrically at OD 450 nm. Quantitative data are expressed as the mean ± S.E. M. (n = 3-4). ** p < 0.01, *** p < 0.001 compared to basal.