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Structural Elucidation and Structure−Anti-inflammatory Activity Relationships of Cembranoids from Cultured Soft Corals Sinularia sandensis and Sinularia flexibilis Tsung-Chang Tsai,†,‡ Hsueh-Yu Chen,§,∥ Jyh-Horng Sheu,† Michael Y. Chiang,⊥ Zhi-Hong Wen,† Chang-Feng Dai,# and Jui-Hsin Su*,§,∥ †

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Department of Beauty Science, Meiho University, Pingtung 912, Taiwan § National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan ∥ Graduate Institute of Marine Biology, National Dong Hwa University, Pingtung 944, Taiwan ⊥ Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan # Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan ‡

S Supporting Information *

ABSTRACT: New cembranoids 4-carbomethoxyl-10-epigyrosanoldie E (1), 7-acetylsinumaximol B (2), diepoxycembrene B (6), dihydromanaarenolide I (8), and isosinulaflexiolide K (9), along with 11 known related metabolites, were isolated from cultured soft corals Sinularia sandensis and Sinularia f lexibilis. The structures were elucidated by means of infrared, mass spectrometry, and nuclear magnetic resonance techniques, and the absolute configurations of 1, 4, 9, and 15 were further confirmed by single-crystal X-ray diffraction analysis. The absolute configurations of these coral metabolites and comparison with known analogues showed that one hypothesis (that cembrane diterpenes possessing an absolute configuration of an isopropyl group at C1 obtained from Alcyonacean soft corals belong to the α series, whereas analogues isolated from Gorgonacean corals belong to the β series) is not applicable for a small number of cembranoids. An in vitro anti-inflammatory study using LPSstimulated macrophage-like cell line RAW 264.7 revealed that compounds 9−14 significantly suppressed the accumulation of pro-inflammatory proteins, iNOS and COX-2. Structure−activity relationship analysis indicated that cembrane-type compounds with one seven-membered lactone moiety at C-1 are potential anti-inflammatory agents. This is the first culture system in the world that has successfully been used to farm S. sandensis. KEYWORDS: anti-inflammatory, Sinularia sandensis, Sinularia f lexibilis, soft coral, structure−activity relationship

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INTRODUCTION

Marine Biology & Aquarium) isolated several cembrane terpenes from cultured soft corals Sinularia f lexibilis,6 Sinularia gibberosa,7 Sinularia numerosa,8 Sarcophyton trocheliophorum,9 and Lobophytum crassum.10 Our recent study of the bioactive natural products derived from two cultured soft corals, S. sandensis and S. f lexibilis, resulted in the isolation of five new cembranoids, 4-carbomethoxyl-10-epigyrosanoldie E (1), 7acetylsinumaximol B (2), diepoxycembrene B (6), dihydromanaarenolide I (8), and isosinulaflexiolide K (9), as well as several known metabolites, 3−5, 7, and 10−16 (Figure 1). New metabolites 1 and 2 were isolated from S. sandensis, and compounds 6, 8, and 9 were obtained from S. f lexibilis. The structures of the new compounds were determined using one- and two-dimensional nuclear magnetic resonance (NMR) analyses, as well as data from mass spectroscopic study, and the absolute configurations of 1, 4, 9, and 15 were determined by single-crystal X-ray crystallography (Figure 2).11 Natural products 1−4 and 6−16 and derivatives 12a and 13a

Diterpenes of the cembrane type are some of the most frequently encountered metabolites in soft corals of the order Alcyonacean.1,2 Of particular interest, cembrane-type diterpenoids have been reported to display a variety of biological activities, including antitumor and antifouling activities.1,2 Moreover, cembrane diterpenes display potent anti-inflammatory activities; for example, 11-epi-sinulariolide acetate attenuates inflammation and bone damage in adjuvant-induced arthritis,3 sinularin inhibits nociceptive responses and spinal neuroinflammation,4 and flexibilide has anti-neuroinflammatory and analgesic effects.5 However, it is not practicable to extract large quantities of bioactive metabolites from wild-type species as the contents of anti-inflammatory secondary metabolites in soft corals are small. To overcome this problem, scientists are attempting to develop new ways in which to obtain new sources for larger amounts of materials. In recent years, the aquaculture technique of soft corals has been considerably improved, and researchers of marine natural products are able to prepare larger amounts of bioactive metabolites from cultured soft corals. This advanced technique has made in-depth pharmacological studies of metabolites of soft corals possible. In our previous research on bioactive metabolites, our group (National Museum of © XXXX American Chemical Society

Received: April 20, 2015 Revised: July 27, 2015 Accepted: August 4, 2015

A

DOI: 10.1021/acs.jafc.5b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of metabolites 1−16, 12a, and 13a. Supelco C18 column (φ 21.2 mm × 25 cm, 5 μm) were used for HPLC. Animal Material. The two light yellow cultured soft corals S. sandensis and S. f lexibilis were initially collected from the wild and subsequently grown for 8 years in an 80 ton cultivation tank located in the National Museum of Marine Biology & Aquarium. The tank was a flow-through seawater system, and deliberate feeding was not required. This was the first successful system that has been reported for the farming of S. sandensis. The specimens were collected by hand in January 2013, frozen immediately, and kept frozen until extraction. Two soft corals were identified by one of the authors (C.-F.D.). Voucher specimens were deposited in the National Museum of Marine Biology & Aquarium (Specimens 2013CSC-1 and 2013CSC-2). Extraction and Isolation. The frozen S. sandensis material (2.8 kg, wet weight) was freeze-dried, and the resulting material (320 g) was then minced and extracted exhaustively with EtOAc (6 × 1.5 L). The EtOAc extract was evaporated under reduced pressure to afford a residue (20.5 g), and the residue was subjected to column chromatography on silica gel, using n-hexane, an n-hexane/EtOAc mixture of increasing polarity, and finally pure acetone to yield 12 fractions: Fr-1 (eluted with n-hexane/EtOAc, 100:1), Fr-2 (eluted with n-hexane/EtOAc, 50:1), Fr-3 (eluted with n-hexane/EtOAc, 30:1), Fr4 (eluted with n-hexane/EtOAc, 20:1), Fr-5 (eluted with n-hexane/ EtOAc, 10:1), Fr-6 (eluted with n-hexane/EtOAc, 8:1), Fr-7 (eluted with n-hexane/EtOAc, 5:1), Fr-8 (eluted with n-hexane/EtOAc, 3:1), Fr-9 (eluted with n-hexane/EtOAc, 1:1), Fr-10 (eluted with n-hexane/

(Figure 1) were tested for anti-inflammatory activity to inhibit the protein expressions of inducible nitric oxide synthetase (iNOS) and cyclooxygenase-2 (COX-2). The structural determination and anti-inflammatory activities, in addition to the structure−activity relationships, of the isolates are reported herein.

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MATERIALS AND METHODS

General Method. Infrared (IR) spectra were obtained on a Fourier-transform IR spectrophotometer (Varian Digilab FTS 1000). 1 H and 13C NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR instrument at 500 and 125 MHz, respectively. Optical rotations were determined by a digital polarimeter (Jasco P-1010). Electrospray ionization mass spectrometry (ESIMS) analyses were performed on an APEX II Instrument (Bruker). Single-crystal X-ray analyses were performed on a Bruker APEX DUO diffractometer. Gravity column chromatography was performed with 230−400 mesh silica gel (Merck). TLC analyses were conducted on 0.2 mm thick precoated Kieselgel 60 F254 plates (Merck), and visualization of TLC spots was conducted by spraying the plate with a 10% aqueous H2SO4 solution followed by heating. High-performance liquid chromatography (HPLC) was performed using a system consisting of a Hitachi L-7100 pump and a Rheodyne 7725 injection port. A preparative normal phase column (φ 21.2 mm × 25 cm, silica gel 60, 5 μm) and a B

DOI: 10.1021/acs.jafc.5b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 2. Molecular plots of 1, 4, 9, and 15 with confirmed absolute configurations. mg). Fr-9 was separated by normal-phase HPLC (n-hexane/EtOAc, 2:3) to yield 12 (71.5 mg) and 14 (55.2 mg). Fr-10 was separated by normal-phase HPLC (n-hexane/acetone, 4:1) to afford 8 (3.2 mg), 13 (2150 mg), and 16 (105.3 mg). Fr-11 was separated by normal-phase HPLC with gradient elution (n-hexane/EtOAc, 1:2 to 1:3) to yield four subfractions (11A−11D). Subfraction 11A was separated by normal-phase HPLC (n-hexane/EtOAc, 2:3) to afford 9 (4.5 mg) and 10 (4.1 mg). Compound 11 (25.6 mg) was obtained from subfraction 11C using normal-phase HPLC (n-hexane/EtOAc, 1:1). Compound 15 (19.9 mg) was obtained from subfraction 11D using normal-phase HPLC (n-hexane/acetone, 2:1). 4-Carbomethoxyl-10-epigyrosanoldie E (1). Colorless needles: UV (MeOH) λmax (log ε) 213 (3.87) nm; [α]25D −28.0 (c 0.5, CHCl3); IR (neat) vmax 2935, 1755, 1715, 1642, 1443, 1383, 1280 cm−1; 13C and 1 H NMR data in Table 1; ESIMS m/z 413 [M + Na]+; HRESIMS m/z 413.1571 [M + Na]+ (calcd for C21H26O7Na, m/z 413.1570). 7-Acetylsinumaximol B (2). White powder: UV (MeOH) λmax (log ε) 235 (3.30), 260 (3.42), 290 (3.38) nm; [α]25D −56.0 (c 0.6, CHCl3); IR (neat) vmax 3480, 2948, 1749, 1715, 1645, 1610, 1569, 1441, 1228 cm−1; 13C and 1H NMR data in Table 1; ESIMS m/z 455 [M + Na]+; HRESIMS m/z 455.1675 [M + Na]+ (calcd for C23H28O8Na, m/z 455.1676). Diepoxycembrene B (6). Colorless oil: [α]25D +28.0 (c 0.5, CHCl3); IR (neat) vmax 2964, 2927, 2864, 1644, 1453, 1382 cm−1; 13 C and 1H NMR data in Table 2; ESIMS m/z 327 [M + Na]+; HRESIMS m/z 327.2295 [M + Na]+ (calcd for C20H32O2Na, m/z 327.2294).

EtOAc, 1:2), Fr-11 (eluted with EtOAc), and Fr-12 (eluted with acetone). Fr-9 was further purified with silica gel (n-hexane/acetone, 3:1 to 2:1) to afford 10 subfractions (9A−9J). Subfraction 9E was then separated by normal-phase HPLC (n-hexane/acetone, 3:1) to obtain 4 (560 mg). Subfraction 9G was separated by normal-phase HPLC (nhexane/EtOAc, 1:1) to afford 5 (1.2 mg) and 1 (10.2 mg). Fraction 10 was separated by normal-phase HPLC with gradient elution (nhexane/acetone, 3:1 to 2:1) to yield five subfractions (10A−10E). Subfraction 10C was separated by reverse-phase HPLC (CH3CN/ H2O, 7:3) to afford 2 (4.5 mg). Compound 3 (9.4 mg) was obtained from subfraction 10E using normal-phase HPLC (n-hexane/acetone, 1:1). The frozen S. f lexibilis (4.2 kg, wet weight) was freeze-dried, and the resulting material (450 g) was minced and extracted exhaustively with EtOAc (6 × 2 L). The EtOAc extract was evaporated under reduced pressure to afford a residue (30.5 g), and the residue was subjected to column chromatography on silica gel using n-hexane (H), an nhexane/EtOAc (E) mixture of increasing polarity, and finally pure acetone to yield 13 fractions: Fr-1 (eluted with H/E, 100:1), Fr-2 (eluted with H/E, 50:1), Fr-3 (eluted with H/E, 30:1), Fr-4 (eluted with H/E, 20:1), Fr-5 (eluted with H/E, 10:1), Fr-6 (eluted with H/E, 8:1), Fr-7 (eluted with H/E, 5:1), Fr-8 (eluted with H/E, 3:1), Fr-9 (eluted with H/E, 2:1), Fr-10 (eluted with H/E, 1:1), Fr-11 (eluted with H/E, 1:2), Fr-12 (eluted with EtOAc), and Fr-13 (eluted with acetone). By using acetone as the mobile phase, Fr-7 was purified by passage over a Sephadex LH-20 column to afford five subfractions (7A−7E). Subfraction 7C was separated by normal-phase HPLC (CH2Cl2/n-hexane/EtOAc, 2:2:0.5) to obtain 6 (3.0 mg) and 7 (10.2 C

DOI: 10.1021/acs.jafc.5b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Single-Crystal X-ray Analysis of 4.11 The ethyl acetate solution was allowed to slowly evaporate to generate a suitable colorless crystal. Diffraction intensity data were obtained on a Bruker APEX DUO Xray diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Crystal data for this compound: C21H24O6 (formula weight of 372.40), approximate crystal size of 0.30 mm × 0.20 mm × 0.15 mm, monoclinic, space group P21 (No. 4), T = 100(2) K, a = 8.862(3) Å, b = 12.539(4) Å, c = 16.776(5) Å, V = 1864.1(10) Å,3 Z = 4, Dcalcd = 1.327 mg/m3, F(000) = 792, and μ(MoKα) = 0.800 mm−1. A total of 6658 reflections were collected in the θ range of 5.646− 66.464°, with 3026 independent reflections [R(int) = 0.0225], and completeness to θmax was 94.7%; semiempirical from equivalents of absorption correction was applied. The refinement method was fullmatrix least squares on F2; the data, restraints, and parameters were 3026, 0, and 247, respectively. The goodness of fit on F2 = 1.175. Final R indices [I > 2σ(I)] R1 = 0.0330 and wR2 = 0.0960. R indices (all data) R1 = 0.0332 and wR2 = 0.0963. The largest difference peak and hole were 0.347 and −0.261 e/Å3, respectively; the absolute structure parameter was 0.12(5). Single-Crystal X-ray Analysis of 9.11 The ethyl acetate solution was allowed to slowly evaporate to generate a suitable colorless crystal. Diffraction intensity data were obtained on a Bruker APEX DUO Xray diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Crystal data for this compound: C22H32O5 (formula weight of 376.47), approximate crystal size of 0.60 mm × 0.35 mm × 0.30 mm, monoclinic, space group P21 (No. 4), T = 100(2) K, a = 8.0404(2) Å, b = 14.3426(4) Å, c = 17.9352(6) Å, V = 2068.29(10) Å,3 Z = 4, Dcalcd = 1.209 mg/m3, F(000) = 816, and μ(MoKα) = 0.681 mm−1. A total of 14442 reflections were collected in the θ range of 3.946− 66.656°, with 3651 independent reflections [R(int) = 0.0225], and completeness to θmax was 97.7%. Semiempirical from equivalents of absorption correction was applied. The refinement method was fullmatrix least squares on F2; the data, restraints, and parameters were 3651, 0, and 249, respectively. The goodness of fit on F2 = 1.046. Final R indices [I > 2σ(I)] R1 = 0.0263 and wR2 = 0.0677. R indices (all data) R1 = 0.0264 and wR2 = 0.0678. The largest difference peak and hole were 0.164 and −0.128 e/Å3, respectively; the absolute structure parameter was 0.09(3). Single-Crystal X-ray Analysis of 15.11 The ethyl acetate solution was allowed to slowly evaporate to generate a suitable colorless crystal. Diffraction intensity data were obtained on a Bruker APEX DUO Xray diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Crystal data for this compound: C44H68O14 (formula weight of 820.98), approximate crystal size of 0.40 mm × 0.30 mm × 0.30 mm, monoclinic, space group P21 (No. 4), T = 100(2) K, a = 9.7079(6) Å, b = 23.0869(14) Å, c = 9.72692(6) Å, V = 2172.1(2) Å,3 Z = 2, Dcalcd = 1.255 mg/m3, F(000) = 888, and μ(MoKα) = 0.760 mm−1. A total of 3825 reflections were collected in the θ range of 3.829− 66.665°, with 3825 independent reflections [R(int) = 0.0334], and completeness to θmax was 95.7%. Semiempirical from equivalents of absorption correction was applied. The refinement method was fullmatrix least squares on F2; the data, restraints, and parameters were 3825, 1, and 536, respectively. The goodness of fit on F2 = 1.032. Final R indices [I > 2σ(I)] R1 = 0.0289 and wR2 = 0.0751. R indices (all data) R1 = 0.0291 and wR2 = 0.0752. The largest difference peak and hole were 0.180 and −0.227 e/Å3, respectively; the absolute structure parameter was 0.14(8). Acetylation of 3. Ac2O (0.1 mL) was added to a solution of 3 (4.0 mg) in pyridine (0.2 mL), and the mixture was stirred at room temperature. After 24 h, pyridine and excess Ac2O were evaporated by adding EtOAc, and the residue was subjected to column chromatography over Si gel (n-hexane/EtOAc, 1:1) to yield acetyl derivative 2 (3.9 mg, 8%). The specific rotation [[α]25D −50.0 (c 0.05, CHCl3)] and 1H NMR spectrum were in full agreement with those of natural product 2. Hydrolysis of 12. A solution of 12 (16.0 mg) was dissolved in a 5% methanolic NaOH solution (1.0 mL). After being stirred at room temperature for 12 h, the mixture was neutralized with diluted HCl (0.1 M) and evaporated. The resulting residue was extracted with CHCl3. The CHCl3 soluble layers were dried over anhydrous NaSO4

Table 1. 1H and 13C NMR Data for 1 and 2 1 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Ac

δH (J in hertz)a 2.66 m 2.55 m; 2.45 m 3.97 d (3.5) 4.26 d (3.5) 2.66 m; 2.39 m 2.58 m; 2.38 m 5.19 s 7.16 s 2.38 m; 2.20 m 2.04 m; 1.78 m 4.79 s; 4.61 s 1.67 s 1.31 s 3.74 s

2

δC (mult.)b

δH (J in hertz)a

δC (mult.)b

42.0 (CH) 45.1 (CH2)

2.18 m 3.41 dd (14.5, 12.5); 2.73 dd (14.5, 2.0)

44.0 (CH) 31.9 (CH2)

202.5 (C) 61.5 (CH)

161.1 (C) 116.2 (C)

75.0 (CH)

6.66 s

109.5 (CH)

212.3 (C) 50.0 (CH2)

5.59 s

149.4 (C) 75.9 (CH)

79.9 (C) 42.3 (CH2) 79.0 151.9 130.4 20.8

(CH) (CH) (C) (CH2)

27.3 (CH2) 145.8 (C) 113.0 (CH2) 18.0 167.8 24.9 174.4 52.4

(CH3) (C) (CH3) (C) (CH3)

2.60 dd (15.0, 4.0); 1.95 dd (14.5, 11.5) 4.94 d (11.0) 5.81 s 2.33 m; 2.10 m 1.84 m; 1.52 m

4.84 s; 4.80 s 1.77 s 1.48 s 3.85 s 2.16 s

a

72.6 (C) 43.1 (CH2) 78.3 148.2 133.7 21.6

(CH) (CH) (C) (CH2)

28.1 (CH2) 146.0 (C) 112.6 (CH2) 19.0 163.7 21.0 173.2 51.7 169.4 21.0

(CH3) (C) (CH3) (C) (CH3) (C) (CH3)

At 500 MHz in CDCl3. bAt 125 MHz in CDCl3.

Dihydromanaarenolide I (8). White powder: [α]25D −20.5 (c 0.6, CHCl3); IR (neat) vmax 3436, 2930, 2862, 1710, 1455, 1374 cm−1; 13C and 1H NMR data in Table 2; ESIMS m/z 359 [M + Na]+; HRESIMS m/z 359.2200 [M + Na]+ (calcd for C20H32O4Na, m/z 359.2198). Isosinulaflexiolide K (9). Colorless needles” [α]25D −73.6 (c 0.1, CHCl3); IR (neat) vmax 3462, 2976, 2943, 2858, 1742, 1698, 1645, 1451, 1373, 1235 cm−1; 13C and 1H NMR data in Table 2; ESIMS m/z 399 [M + Na]+; HRESIMS m/z 399.2139 [M + Na]+ (calcd for C22H32O5Na, m/z 399.2142). Single-Crystal X-ray Analysis of 1.11 The ethyl acetate solution was allowed to slowly evaporate to generate a suitable colorless crystal. Diffraction intensity data were obtained on a Bruker APEX DUO Xray diffractometer using graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Crystal data for this compound: C21H26O7 (formula weight of 390.42), approximate crystal size of 0.40 mm × 0.40 mm × 0.40 mm, monoclinic, space group P21 (No. 4), T = 100(2) K, a = 9.1141(2) Å, b = 8.9035(2) Å, c = 12.2954(3) Å, V = 962.98(4) Å,3 Z = 2, Dcalcd = 1.346 mg/m3, F(000) = 416, and μ(MoKα) = 0.838 mm−1. A total of 6772 reflections were collected in the θ range of 5.420− 66.673°, with 2603 independent reflections [R(int) = 0.0210], and completeness to θmax was 96.2%; semiempirical from equivalents of absorption correction was applied. The refinement method was fullmatrix least-squares on F2; the data, restraints, and parameters were 2603, 1, and 256, respectively. The goodness of fit on F2 = 1.080. Final R indices [I > 2σ(I)] R1 = 0.0266 and wR2 = 0.0707. R indices (all data) R1 = 0.0267 and wR2 = 0.0707. The largest difference peak and hole were 0.185 and −0.190 e/Å3, respectively; the absolute structure parameter was 0.04(5). D

DOI: 10.1021/acs.jafc.5b01931 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 2. 1H and 13C NMR Data for 6, 8, and 9 6 position

δH (J in hertz)a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OAc

2.07 m 1.96 m; 1.42 m 2.94 dd (8.5, 4.5) 2.10 m; 1.36 m 2.18 m 5.18 t (5.5) 2.28 m; 2.08 m 2.07 m; 1.33 m 2.61 dd (10.5, 3.5) 1.90 m; 0.85 m 1.55 m 4.78 1.67 1.26 1.66 1.27

s; 4.70 s s s s s

8 δc (mult.)b 44.9 33.0 61.2 60.3 38.1 23.1 126.1 134.5 36.6 24.5 62.3 61.7 37.6 28.1 146.8 111.4 19.1 16.9 15.0 16.2

(CH) (CH2) (CH) (C) (CH2) (CH2) (CH) (C) (CH2) (CH2) (CH) (C) (CH2) (CH2) (C) (CH2) (CH3) (CH3) (CH3) (CH3)

δH (J in hertz)a 1.61 m 1.94 m; 1.34 m 3.96 dd (11.5, 2.0) 1.78 m; 1.62 m 2.30 m; 1.92 m 5.06 t (8.0) 2.11 m; 1.96 m 1.98 m; 1.78 m 4.12 t (4.0) 2.20 m; 2.10 m 2.08 m; 1.28 m 2.10 m 1.39 1.41 1.63 5.25

d (7.0) s s s; 4.98 s

9 δc (mult.)b 35.3 27.6 84.6 74.3 38.4 22.6 124.0 135.8 32.3 30.2 73.0 148.1 28.2 30.8 42.5 174.7 15.9 25.1 16.1 108.7

(CH) (CH2) (CH) (C) (CH2) (CH2) (CH) (C) (CH2) (CH2) (CH) (C) (CH2) (CH2) (C) (C) (CH3) (CH3) (CH3) (CH2)

δH (J in hertz)a 2.43 m 1.86 m; 1.17 m 1.96 m; 1.80 m 5.46 d (10.0) 1.55 m; 1.38 m 2.04 m; 1.85 m 5.22 d (10.0) 3.18 m; 2.51 m 5.74 dd (11.5, 6.5) 4.69 dd (11.5, 3.0) 2.06 m; 1.70 m

6.20 1.32 1.66 1.69

s; 5.51 s s s s

2.11 s a

δc (mult.)b 31.8 29.3 33.0 86.1 71.4 27.5 34.6 129.6 127.9 26.8 129.5 134.6 66.2 39.1 145.0 169.0 124.4 23.7 16.0 16.7 170.7 21.1

(CH) (CH2) (CH2) (C) (CH) (CH2) (CH2) (C) (CH) (CH2) (CH) (C) (CH2) (CH2) (C) (C) (CH2) (CH3) (CH3) (CH3) (C) (CH3)

At 500 MHz in CDCl3. bAt 25 MHz in CDCl3.

and evaporated. The dried residue was purified by silica gel column chromatography (n-hexane/acetone, 6:1) to yield 12a (8.2 mg, 46.9%). Hydrolysis of 13. Using the same preparation method that was used for 12, the reaction of 13 (10.0 mg) with 1 mL of a 5% methanolic NaOH solution yielded a crude product, which was further separated by normal-phase HPLC (n-hexane/EtOAc, 1:2) to afford 13a (3.8 mg, 34.4%). In Vitro Anti-Inflammatory Assay. A macrophage-like mouse cell line, RAW 264.7, was purchased from ATCC. Using Western blot analysis,12,13 the in vitro anti-inflammatory activities of compounds 1− 4 and 6−16, and two derivatives 12a and 13a, were assessed by measuring their suppression effects on lipopolysaccharide (LPS)stimulated iNOS and COX-2 protein overexpression in RAW 264.7 cells.

single-crystal X-ray crystallographic analysis using Cu Kα irradiation at 100 K (Figure 2) confirmed the stereostructures of 1. The Flack parameter [0.04(5)] (CCDC 1028007) allowed the assignment of the absolute configuration of 1 as (1R,4S,5S,8S,10S)-5(8)-epoxy-4-carbomethoxyl-1-isopropenyl8-methyl-3,6-dioxocyclotetradec-11(12)-en-10,12-carbolactone. The HRESIMS spectrum of 7-acetylsinumaximol B (2) exhibited an ion peak at m/z 455.1675 [M + Na]+, which matched the formula C23H28O8. The 1H and 13C NMR spectral data (Table 1) of 2 were similar to those of 3,17 the difference being that the hydroxy group attached at C-7 in 3 was replaced with an acetoxy group in 2. Acetylation of 3 gave an acetyl derivative, whose 1H and 13C NMR spectral data were identical to those of the 7-acetyl derivative (2) derived from 3. Hence, 2 was the 7-acetyl derivative of 3. Therefore, 2 was established as (1R,7R,8R,10S,11Z)-3(6)-epoxy-7-acetoxy-4-carbomethoxyl-8hydroxy-1-isopropenyl-8-methyl-3(4),5(6),11(12)-trien-10,12carbolactone Diepoxycembrene B (6) had the same molecular formula (C20H32O2) as 7, as indicated by HRESIMS and NMR spectra (Table 2). The gross structure of 6 was established by interpretation of two-dimensional (2D) NMR data, especially by analysis of 1H−1H COSY and HMBC correlations (Figure 3). The 1H−1H COSY spectral data assigned three isolated consecutive proton spin systems. The aforementioned results indicated that 6 had the same molecular framework as known compounds diepoxycembrene A (7) [(1R*,3R*,4S*,11R*,12R*)-3,4,11,12-diepoxycembra-7,15diene]18 and diepoxycembrane A [(1R*,3S*,4S*,11S*,12S*3,4,11,12-diepoxycembra-7,15-diene],19 which were isolated previously from soft corals Sinularia sp. and S. flexibilis, respectively. NOE correlations observed in the NOESY spectrum were used to determine the relative configuration of 6 (Figure 4). The NOE correlations observed between H2-6 and H3-19 reflected the E configuration of the double bond

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RESULTS AND DISCUSSION 4-Carbomethoxyl-10-epigyrosanoldie E (1) was isolated as colorless needles. Its infrared (IR) absorptions showed the presence of both lactone (1755 cm−1) and ketone (1715 cm−1) functionalities. The molecular formula of 1 was determined to be C21H26O7 by HRESIMS (m/z 413.1571 [M + Na]+) and 13 C NMR data (Table 1), requiring nine degrees of unsaturation. Resonances due to two ketones (δ 212.3 and 202.5), two ester carbonyl carbons (δ 174.4 and 167.8), and two olefinic carbons (δ 151.9, CH; 145.8, C; 130.4, C; 113.0, CH2) in the 13C NMR and DEPT spectral data accounted for six double-bond equivalents, which indicated that 1 was a tricyclic compound. The 1H NMR spectrum of 1 (Table 1) revealed the presence of resonances of one olefinic methine proton (δ 7.16, s), two olefinic methylene protons (δ 4.79 and 4.61, each s), two oxygenated methines (δ 5.19, s, and δ 4.26, d, J = 3.5 Hz), and one oxymethyl group (δ 3.74, s). By comparison of the NMR data of 1 with those of 5,14−16 it was found that 1 differed from 5 only in the presence of a carbomethoxyl group instead of a proton at C-4. Moreover, E

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NMR data (1H−1H COSY and HMBC) further revealed that 8 possesses one methyl group at C-15 (Figure 3). The relative configurations of all stereocenters except C-15 of 8 were determined to be the same as those of manaarenolide I by comparisons of the coupling constants and proton shifts. Because of the NOE correlation between H3-17 and H-1, the methyl at C-15 was assigned a β-configuration primarily. Moreover, by comparison of the NMR data of 8 with those of similar known compounds, it was confirmed that all compounds have the same relative stereochemical configuration at C-15.20 According to the aforementioned results, the structure of 8 was established as (1R*,3R*,4S*,11S*,15S*,7E)-4,11-dihydroxy-cembr-7(8),12(20)-dien-16,3-olide. Isosinulaflexiolide K (9) was found to have the [M + Na]+ ion at m/z 399 in its ESIMS spectrum. The molecular formula was deduced to be C22H32O5 from HRESIMS and NMR data (Table 2). The structure and absolute stereochemistry of 9 were also confirmed by single-crystal X-ray diffraction (Figure 2) with Cu Kα irradiation [Flack parameter of 0.09(3)] (CCDC 1044093). On the basis of the results presented above, compound 9 was established as (1R,4R,5R,13S,8E,11Z)-5acetoxy-13-hydroxy-cembr-8(9),11(12),15(17)-trien-16,4-olide In addition, the 11 known compounds obtained were identified as sinumaximol B (3),17 pukalide (4),21 10epigyrosanoldie E (5),14,15 diepoxycembrene A (7),17 sinulaflexiolide K (10),22 (−)-sandensolide (11),23 11-dehydrosinulariolide (12),24 sinulariolide (13),25 3,4:8,11-bisepoxy-7acetoxycembra-15(17)-en-1,12-olide (14),26 dendronpholide F (15),27 and dendronpholide G (16)24 by comparison of spectroscopic data with published values. The absolute configurations of 4 and 15 were confirmed by single-crystal X-ray diffraction analysis with Cu Kα radiation [Flack parameters of 0.12(5) and 0.14(8), respectively] for the first time (Figure 2). In the previous report, compound 4 was obtained from Gorgonacean coral Leptogorgia rigida, and the absolute configuration of 4 was fully determined by CD spectroscopic data analysis.21 One hypothesis suggested that all cembranes of the known absolute configuration of an isopropyl group at C-1 obtained from Alcyonacean soft corals belong to the α series, whereas analogues isolated from Gorgonacean corals belong to the β series.28,29 However, compounds 9 and 15 isolated from an Alcyonacean soft coral have a β conformation, with the isopropyl group pointing upward at position C1, and 4 was obtained from Gorgonacean and Alcyonacean corals. Thus, this result showed that this hypothesis is not applicable for a small number of cembranoids. Using Western blotting, the effects of 1−4 and 6−16 on the inhibition of the accumulation of pro-inflammatory iNOS and COX-2 in LPS-stimulated RAW 264.7 cells were studied. The results presented in Figure 5 show that compounds 9−14 were found to significantly reduce the levels of iNOS to 30.9 ± 4.1, 37.4 ± 5.9, 61 ± 3.4, 31.9 ± 5.1, 47.7 ± 6.3, and 25.7 ± 5.2%, respectively, and the levels of COX-2 to 47.1 ± 3.8, 51.4 ± 5.6, 51.9 ± 7.2, 49 ± 5.6, 52.2 ± 5.1, and 55.3 ± 8.2%, respectively. Preliminary structure−activity relationships in diterpenes of the cembrane type may be deduced from anti-inflammatory bioassays. In the bioassays, the presence of one sevenmembered lactone functional group is critical for the antiinflammatory action of this class of compounds. Moreover, to confirm the active moiety, base-catalyzed hydrolysis of both 12 and 13 was performed, and the reactions were found to afford 12a and 13a. The two derivatives were inactive against the

Figure 3. Selected 1H−1H COSY () and HMBC (→) correlations of 6 and 8.

Figure 4. Selected NOE correlations of 6.

between C-7 and C-8. In addition, it was found that H-1 showed NOE interactions with one proton of the C-13 methylene (δ 0.85) and H3-18. Thus, assuming a β-orientation of H-1, H3-18 should be positioned on the β face. Moreover, H13β was found to exhibit correlations with H-11 (δ 2.61, dd, J = 10.3, 3.5 Hz), indicating that H-11 was situated on the β face; H-11 was assigned as a β proton, as the C-20 methyl was αoriented at C-12, which was verified by a correlation between H3-20 and H-13α (δ 1.90 m). This result shows that the epoxide at C-11/C-12 was of a trans geometry of the trisubstituted epoxide. Furthermore, the epoxide at C-3/C-4 was also of a trans geometry of the trisubstituted epoxide from the NOE correlations displayed by H-3 with the one proton of the C-5 methylene (δ 1.36), but not with H3-18. In light of these results and other detailed analyses of NOE correlations, 6 was unambiguously identified as (1R*,3S*,4S*,11R*,12R*)3,4,11,12-diepoxycembra-7,15-diene. Dihydromanaarenolide I (8) was isolated as a colorless amorphous powder. The HRESIMS (m/z 359.2200, [M + Na]+) and NMR data of 8 indicated the molecular formula C20H32O4. Both the 1H and 13C NMR signals of 8 (Table 2) were found to be very closely related to those of known compound manaarenolide I, which was isolated previously from the soft coral Sinularia manaarensis.20 NMR comparison of 8 with manaarenolide I demonstrated that the two exomethylene proton signals in manaarenolide I were replaced by a methyl proton signal (δH 1.39, d, J = 7.0 Hz) in 8. Additionally, 2D F

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Figure 5. Effects of compounds 1−4, 6−16, 12a, and 13a at 10 μM on pro-inflammatory iNOS and COX-2 protein expression in LPS-induced RAW 264.7 macrophage cells by Western blot analysis: (A) quantification of iNOS expression, (B) quantification of COX-2 expression, and (C) quantification of β-actin expression. Band intensities were quantified by densitometry and normalized to that of the group treated with LPS only (set to 100%). At a concentration of 10 μM, positive control, dexamethasone was found to significantly reduce the levels of iNOS to 30.8 ± 9.6 and 6.9 ± 3.4%, respectively, relative to that of the control cells stimulated with LPS only in this study. This experiment was repeated three times. *p < 0.05 as compared with the LPS-stimulated group.

expressions of both iNOS and COX-2 proteins (Figure 5). These results showed that the anti-inflammatory activity depended on the seven-membered lactone functional group at position C1.

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Cif file for 15 (CIF)

AUTHOR INFORMATION

Corresponding Author

*Telephone: +886-8-8822370. Fax: +886-8-8825087. E-mail: [email protected].

ASSOCIATED CONTENT

S Supporting Information *

Funding

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01931. Cif file for 1 (CIF) Cif file for 4 (CIF) Cif file for 9 (CIF) NMR spectra of compounds 1, 2, 6, 8, and 9 (PDF)

This research was supported by grants from the National Museum of Marine Biology & Aquarium and the Ministry of Science and Technology (MOST 103-2320-B-291-001-MY3), Taiwan, awarded to J.-H. Su. Notes

The authors declare no competing financial interest. G

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production of pro-inflammatory cytokines in bone marrow-derived dendritic cells. Chem. Pharm. Bull. 2012, 60, 1581−1589. (18) Qin, S.; Huang, H.; Guo, Y.-W. A new cembranoid from the Hainan soft coral Sinularia sp. J. Asian Nat. Prod. Res. 2008, 10, 1075− 1079. (19) Hérin, M.; Colin, M.; Tursch, B. Chemical studies of marine invertebrates. XXV. Flexibilene, an unprecedented fifteen-membered ring diterpene hydrocarbon from the soft coral Sinularia f lexibilis (Coelenterata, Octocorallia, Alcyonacea). Bull. Soc. Chim. Belg. 1976, 85, 801−803. (20) Su, J.-H.; Ahmed, A. F.; Sung, P.-J.; Chao, C.-H.; Kuo, Y.-H.; Sheu, J.-H. Manaarenolides A−I, new diterpenoids from the soft coral Sinularia manaarensis. J. Nat. Prod. 2006, 69, 1134−1139. (21) Gutiérrez, M.; Capson, T. L.; Guzmán, H. M.; González, J.; Ortega-Barría, E.; Quiñ oá, E.; Riguera, R. Leptolide, a new furanocembranolide diterpene from Leptogorgia alba. J. Nat. Prod. 2005, 68, 614−616. (22) Wen, T.; Ding, Y.; Deng, Z.; van Ofwegen, L.; Proksch, P.; Lin, W. Sinulaflexiolides A-K, cembrane-type diterpenoids from the Chinese soft coral Sinularia f lexibilis. J. Nat. Prod. 2008, 71, 1133− 1140. (23) Hu, L.-C.; Su, J.-H.; Chiang, M.Y.-N.; Lu, M.-C.; Hwang, T.-L.; Chen, Y.-H.; Hu, W.-P.; Lin, N.-C.; Wang, W.-H.; Fang, L.-S.; Kuo, Y.H.; Sung, P.-J. Flexibilins A−C, new cembrane-type diterpenoids from the Formosan soft coral. Mar. Drugs 2013, 11, 1999−2012. (24) Hsieh, P.-W.; Chang, F.-R.; Mcphail, A. T.; Lee, K.-H.; Wu, Y.C. New cembranolide analogues from the Formosan soft coral Sinularia f lexibilis and their cytotoxicity. Nat. Prod. Res. 2003, 17, 409− 418. (25) Tursch, B.; Braekman, J. C.; Daloze, D.; Herin, M.; Karlsson, R.; Losman, D. Chemical studies of marine invertebrates−XI. Sinulariolide, a new cembranolide diterpene from the soft coral. Tetrahedron 1975, 31, 129−133. (26) Su, J.; Yang, J. S. R.; Kuang, Y.; Zeng, L. A new cembranoids from the soft coral Sinularia capillpsa. J. Nat. Prod. 2000, 63, 1543− 1545. (27) Ma, A.; Deng, Z.; van Ofwegen, L.; Bayer, M.; Proksch, P.; Lin, W. Dendronpholides A−R, cembranoid diterpenes from the chinese soft coral Dendronephthya sp. J. Nat. Prod. 2008, 71, 1152−1160. (28) Rodriguez, A. D.; Li, Y.; Dhasmana, H.; Barnes, C. New marine cembrane diterpenoids isolated from the Caribbean gorgonian Eunicea mammosa. J. Nat. Prod. 1993, 56, 1101−1113. (29) Pham, N. B.; Butler, M. S.; Quinn, R. J. Naturally occurring cembranes from an Australian Sarcophyton species. J. Nat. Prod. 2002, 65, 1147−1150.

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