Fragilides U–W: New 11,20-Epoxybriaranes from the Sea Whip Gorgonian Coral Junceella fragilis

Abstract

Three new 11,20-epoxybriaranes—fragilides U–W (1–3), as well as two known metabolites, junceellonoid D (4) and junceellin (5), were obtained from the octocoral Junceella fragilis. The structures of briaranes 1–3 were elucidated by spectroscopic methods and briaranes 3 and 5 displayed inhibition effects on inducible nitric oxide synthase (iNOS) release from RAW264.7.

1. Introduction

Gorgonian corals belonging to the genus Junceella (family Ellisellidae) [1,2,3], distributed abundantly in the coral reefs of tropical Indo-Pacific Ocean have been found to produce briarane diterpenoids, natural products of a marine origin, in abundance [4]. In our further research into the natural products of Junceella fragilis (Ridley 1884) (Figure 1), which was distributed extensively in the waters of Southern Taiwan, have resulted in the isolation of three new 11,20-epoxybriaranes– fragilides U–W (1–3) along with two know compounds junceellonoid D (4) [5] and junceellin (5) [6,7,8,9,10,11,12,13,14,15] (Figure 1). An anti-inflammatory assay was employed to evaluate the activity of these isolates against the release of inducible nitric oxide synthase (iNOS) from macrophage cells RAW264.7.

Figure 1.

Structures of fragilides U–W (1–3), junceellonoid D (4), junceellin (5), robustolide F (6), juncenolide M (= frajunolide S) (7), and a picture of Junceella fragilis.

2. Results and Discussion

Fragilide U (1) was isolated as an amorphous powder and displayed a sodiated pseudomolecular ion at m/z 589.22583 in the (+)-HRESIMS, which suggested that the molecular formula of 1 was C₂₈H₃₈O₁₂ (calcd. for C₂₈H₃₈O₁₂ + Na, 589.22555) (Ω = 10). The IR spectrum of 1 showed the presence of hydorxy (ν_max 3445 cm⁻¹), γ-lactone (ν_max 1780 cm⁻¹), and ester (ν_max 1733 cm⁻¹) groups. Analysis of the ¹H, ¹³C NMR, and distortionless enhancement by polarization transfer (DEPT) spectra together with the molecular formula, suggested that there must be an exchangeable proton. The ¹³C NMR spectrum (Table 1), in combination with DEPT and heteronuclear single quantum coherence (HSQC) spectra, revealed the presence of four acetoxy groups (δ_C 21.7, 21.2, 20.9, 20.8, 4 × CH₃; δ_C 170.1, 169.8, 169.4, 169.0, 4 × C), a γ-lactone moiety (δ_C 175.9), and a trisubstituted olefin (δ_C 142.0, C; 120.6, CH). Base on the ¹³C NMR data and numbers of unsaturation, 1 was established as a diterpenoid featuring with four rings. An exocyclic epoxy group was deduced from the signals of an oxygenated quaternary carbon and an oxymethylene at δ_C 62.5 and 59.1, respectively, and further supported by the chemical shifts of oxymethylene protons at δ_H 2.85 (1H, d, J = 4.4 Hz) and 2.98 (1H, dd, J = 4.4, 1.6 Hz). Moreover, a methyl singlet (δ_H 1.09, 3H, s), a methyl doublet (δ_H 1.15, 3H, d, J = 7.6 Hz), a vinyl methyl (δ_H 2.00, 3H, d, J = 1.6 Hz), three pairs of aliphatic methylene protons (δ_H 2.04, 1H, m; 2.62, 1H, ddd, J = 18.4, 4.0, 2.4 Hz; 1.13, 1H, m; 2.30, 1H, m; 2.11, 1H, m; 1.81, 1H, m), two aliphatic methine protons (δ_H 2.36, 1H, dd, J = 5.6, 1.6 Hz; 2.33, 1H, q, J = 7.6 Hz), five oxymethine protons (δ_H 5.91, 1H, br s; 5.65, 1H, d, J = 5.6 Hz; 5.04, 1H, d, J = 8.8 Hz; 5.03, 1H, br s; 4.71, 1H, d, J = 4.8 Hz), an olefin proton (δ_H 5.67, 1H, dq, J = 8.8, 1.6 Hz), four acetate methyls (δ_H 2.24, 2.01, 1.99, 1.98, each 3H × s), and a hydroxy proton (δ_H 4.85, 1H, br s) were observed in the ¹H NMR spectrum (Table 2).

Table 1.

¹³C NMR data for briaranes 1–3.

Position	1 a	2 a	3 b
1	47.0, C c	46.1, C	49.1, C
2	72.2, CH	72.1, CH	75.4, CH
3	37.5, CH2	37.6, CH2	136.4, CH
4	70.6, CH	73.4, CH	128.2, CH
5	142.0, C	144.1, C	135.9, C
6	120.6, CH	58.0, CH	128.8, CH
7	76.5, CH	79.0, CH	80.0, CH
8	80.4, C	81.9, C	80.9, C
9	67.4, CH	68.9, CH	67.1, CH
10	39.9, CH	39.8, CH	39.5, CH
11	62.5, C	62.6, C	60.8, C
12	23.6, CH2	23.5, CH2	29.4, CH2
13	24.2, CH2	24.0, CH2	25.5, CH2
14	73.2, CH	73.5, CH	78.9, CH
15	14.6, CH3	15.4, CH3	16.0, CH3
16	21.4, CH3	121.0, CH2	48.0, CH2
17	42.4, CH	43.8, CH	45.2, CH
18	6.6, CH3	7.1, CH3	6.9, CH3
19	175.9, C	175.2, C	174.8, C
20	59.1, CH2	58.2, CH2	50.4, CH2
Acetate methyls	21.7, CH3	21.9, CH3	21.7, CH3
	21.2, CH3	21.0, CH3	21.4, CH3
	20.9, CH3	21.0, CH3	21.3, CH3
	20.8, CH3
Acetate carbonyls	170.1, C	171.0, C	170.4, C
	169.8, C	170.1, C	169.7, C
	169.4, C	169.7, C	169.3, C
	169.0, C

^a Spectra measured at 100 MHz in CDCl₃. ^b Spectra measured at 150 MHz in CDCl₃. ^c Multiplicity deduced by distortionless enhancement by polarization transfer (DEPT) and heteronuclear single quantum coherence (HSQC) spectra.

Table 2.

¹H NMR data (J in Hz) for briaranes 1–3.

Position	1 a	2 a	3 b
2	5.03 br s	4.96 dd (2.8, 2.8)	5.63 d (6.0)
3α/β	2.04 m; 2.62 ddd (18.4, 4.0, 2.4)	1.75 m; 2.38 m	6.04 dd (17.4, 6.0)
4	5.91 br s	4.85 m	6.42 d (17.4)
6	5.67 dq (8.8, 1.6)	5.49 br s	5.66 d (3.6)
7	5.04 d (8.8)	5.15 d (2.4)	5.62 d (3.6)
9	5.65 d (5.6)	5.52 d (6.4)	4.77 d (6.6)
10	2.36 dd (5.6, 1.6)	2.31 d (6.4)	3.32 d (6.6)
12α/β	1.13 m; 2.30 m	1.15 m; 2.30 m	1.29 m; 2.18 m
13α/β	2.11 m; 1.81 m	2.14 m; 1.78 m	2.08 m; 1.85 dddd (12.0, 12.0, 3.6, 1.8)
14	4.71 d (4.8)	4.86 d (4.4)	4.94 dd (3.6, 3.0)
15	1.09 s	1.13 s	0.87 s
16a/b	2.00 d (1.6)	5.74 s; 5.97 s	4.13 d (12.0); 4.19 d (12.0)
17	2.33 q (7.6)	2.29 q (7.6)	2.36 q (7.2)
18	1.15 d (7.6)	1.22 d (7.6)	1.18 d (7.2)
20a/b	2.85 d (4.4); 2.98 dd (4.4, 1.6)	2.83 d (4.0); 2.85 br d (4.0)	2.76 d (2.4); 3.40 dd (2.4, 2.4)
OH-8	4.85 br s	4.63 br s	3.13 d (1.2)
Acetate methyls	2.24 s	2.23 s	2.27 s
	2.01 s	2.03 s	2.25 s
	1.99 s	1.99 s	2.11 s
	1.98 s

^a Spectra measured at 400 MHz in CDCl₃. ^b Spectra measured at 600 MHz in CDCl₃.

Coupling information in the correlation spectroscopy (COSY) analysis enabled the proton sequences from H-2/H₂-3/H-4, H-6/H-7, H-9/H-10, H₂-12/H₂-13/H-14, and H-17/H₃-18 (Figure 2), which was assembled with a heteronuclear multiple bond correlation (HMBC) experiment (Figure 2). The HMBC between protons and quaternary carbons, such as H-2, H-10, H-13β, H-14, H₃-15/C-1; H-7, H₃-16/C-5; H-9, H-10, H₃-18/C-8; H-9, H-10, H₂-12, H-13α, H₂-20/C-11; and H-17, H₃-18/C-19, permitted elucidation of the carbon skeleton of 1. A vinyl methyl at C-5 was confirmed by the HMBC between H₃-16/C-4, C-5, C-6 and H-6/C-16; and further supporting by an allylic coupling between H-6 and H₃-16 (J = 1.6 Hz). The methyl group on C-1 (Me-15) was substantiated by the HMBC from H₃-15/C-1, C-2, C-10, C-14; and H-2, H-10, H-14/C-15. The epoxy group at C-11/20 was confirmed by the HMBC between H₂-20/C-10, C-11; and H-20a/C-12; and further supporting by a long range ⁴J-¹H–¹H correlation between H-10 (δ_H 2.36) and H-20b (δ_H 2.98) (J = 1.6 Hz). A hydroxy group attaching at C-8 was to infer that an HMBC of a hydroxy proton at δ_H 4.85 to C-7, C-8, and C-9. Moreover, HMBC from the oxymethine protons at δ_H 5.03 (H-2), 5.65 (H-9), and 4.71 (H-14) to the acetate carbonyls at δ_C 169.0, 169.4, and 170.1, placed the acetoxy groups on C-2, C-9, and C-14, respectively. Ten of the 12 oxygen atoms in the molecular formula of 1 could be accounted for the presence of a γ-lactone, three esters, an epoxide, and a hydroxy group. Thus, the remaining two oxygen atoms had to be positioned at C-4 as an acetoxy group, as indicated by its ¹H and ¹³C NMR chemical shifts (δ_H 5.91, 1H, br s; δ_C 70.6, CH), although no HMBC was observed from H-4 to any acetate carbonyl.

Figure 2.

The correlation spectroscopy (COSY) () correlations, selective heteronuclear multiple bond correlation (HMBC) experiment (), and protons with key nuclear Overhauser effect spectroscopy (NOESY) () correlations of 1.

Based on a summary of the ¹³C chemical shifts of 11,20-epoxy group in naturally occurring briarane analogues, with ¹³C NMR data for C-11 and C-20 at δ_C 62–63 and 58–60 ppm, the epoxide group was β-oriented and the cyclohexane ring existed in a twist boat conformation [16]; thus, the configuration of the 11,20-epoxy group in 1 should be β-oriented and the cyclohexane ring was found to be in a twist boat conformation for the ¹³C chemical shifts at δ_C 62.5 (C-11) and 59.1 (CH₂- 20). The relative stereochemistry of 1 was established by the analysis of correlations observed in a nuclear Overhauser effect spectroscopy (NOESY) experiment (Figure 2). In the NOESY spectrum of 1, NOE correlations between H-10/H-2, H-10/H-9, and H-10/OH-8, while no correlation was seen between H-10 and H₃-15, suggesting that these protons H-2, H-9, and H-10, and the hydroxy group at C-8 were α-oriented; meanwhile, an NOE correlation of H₃-15 with H-14 indicated that H-14 was β-oriented. The NOESY spectrum showed a correlation from H-6 to H₃-16, revealing the Z geometry of C-5/6 double bond. H₃-18 exhibited NOE correlations to OH-8 and H-9, suggesting the α-orientation of Me-18 at C-17. H-7 displayed NOE correlations with H-4 and H-17, which further confirmed that these three protons were in β-orientation at C-7, C-4, and C-17. Based on the above findings, the relative configurations of stereogenic carbons of 1 were elucidated as 1R*,2S*,4S*,7S*, 8R*,9S*,10S*,11S*,14S* and 17R*. However, as briaranes 1–4 were isolated along with the known chlorinated briarane, junceellin (5) [6], and the structure, including the absolute configuration of junceellin (5) was further confirmed by a single-crystal X-ray diffraction analysis [7,15]. It is reasonable, therefore, on biogenetic grounds to assume that briaranes 1–4 have the same absolute configuration as that of 5. Therefore, the configurations of the stereogenic carbons of 1 should be elucidated as 1R,2S,4S,7S,8R,9S,10S,11S,14S and 17R (Supplementary Materials, Figures S1–S8).

Our present study has also led to the isolation of a new briarane, fragilide V (2). The molecular formula of C₂₆H₃₅ClO₁₁ was deduced from (+)-HRESIMS at m/z 581.17589 (calcd. for C₂₆H₃₅³⁵ClO₁₁ + Na, 581.17601). Carbonyl resonances in the ¹³C NMR spectrum of 2 (Table 1) at δ_C 175.2, 171.0, 170.1, and 169.7 revealed the presence of a γ-lactone and three esters. In the ¹H NMR spectrum of 2 (Table 2), the signals for three acetate methyls were observed at δ_H 2.23, 2.03, and 1.99. It was found that the 1D (Table 1 and Table 2) and 2D NMR (Figure 3) data of 2 were similar to those of a known briarane, robustolide F (6) [17,18] (Figure 1), except that the signals corresponding to a hydroxy group in 2 were replaced by signals for a proton in 6. In the NOESY spectrum, one of the C-3 methylene protons (δ_H 2.38) showed a correlation to H-7 and not with H-2, suggesting the β-orientation of this proton by modeling study and the other was assigned as H-3α (δ_H 1.75). A correlation from H-4 to H-3α, suggested that H-4 was α-oriented according to modeling analysis. Therefore, the configuration of the stereogenic carbons of 2 were elucidated as 1R,2S,4R,6S,7R,8R,9S,10S,11S,14S, and 17R (Figure 3) (Supplementary Materials, Figures S9–S16).

Figure 3.

The COSY () correlations, selective HMBC (), and protons with key NOESY () correlations of 2.

Briarane 3 (fragilide W) was found to have a molecular formula of C₂₆H₃₃ClO₁₀ based on its (+)- HRESIMS at m/z 563.16554 (calcd. for C₂₆H₃₃³⁵ClO₁₀ + Na, 563.16545). Its absorption peaks in the IR spectrum showed ester, γ-lactone, and broad OH stretching at 1738, 1777, and 3459 cm⁻¹, respectively. The ¹³C NMR spectrum indicated that three esters and a γ-lactone were present, as carbonyl resonances were observed at δ_C 174.8, 170.4, 169.7, 169.3 (Table 1). The ¹H NMR spectrum indicated the presence of three acetate methyls (δ_H 2.27, 2.25, 2.11, each 3H × s) (Table 2). The ¹H and ¹³C NMR spectra of 3 was found to be similar with those of a known briarane, juncenolide M (= frajunolide S) (7) (Figure 1), isolated from J. juncea and J. fragilis [19,20], except that the signals corresponding to the 13-acetoxy and 3(Z)-ene moieties in 7 were disappeared and replaced by a proton and an (E)-ene moieties in 3, respectively. The locations of the functional groups were confirmed by 2D-NMR correlations (Figure 4), and hence the structure of fragilide W was assigned as 3, and the configurations of the stereogenic carbons were elucidated as 1R,2S,7S,8R,9S,10S,11R, 14S, and 17R (Figure 4) (Supplementary Materials, Figures S17–S24).

Figure 4.

The COSY () correlations, selective HMBC (), and protons with key NOESY () correlations of 3.

The known compound 4 was found to be identical with the known junceellonoid D, on the basis of the comparison of its physical and spectroscopic data with those of reported previously [5,21] (Supplementary Materials, Figures S25–S32).

Using an in vitro pro-inflammatory suppression assay, the activities of briaranes 1–5 on the release of iNOS and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW264.7 were assayed (Figure 5 and Table 3). The results showed that briaranes 3 and 5 reduced the release of iNOS to 28.55 and 33.72% at a concentration of 10 μM. Briarane 4 was found to be more weak in terms of reducing the expression of iNOS, indicating that the activity of briaranes 4 and 5 is largely dependent on the functional groups at C-2 and C-3. It is interesting to note that briarane 4 was found to enhance the expression of COX-2 to 130.88%, at a concentration of 10 μM.

Figure 5.

Activities of briaranes 1–5 on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins in LPS-treated murine RAW264.7 macrophage cells. Western blotting showed that briaranes 3 and 5 reduced the expression of iNOS. Data were normalized to the cells treated with LPS only, and cells treated with dexamethasone were used as a positive control. Data are expressed as the mean ± SEM (n = 3). * Significantly different from cells treated with LPS (p < 0.05).

Table 3.

Effects of briaranes 1–5 on LPS-induced pro-inflammatory inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expression in macrophages at a concentration of 10 μM.

	iNOS	COX-2	β-Actin
Compound	Expression (% of LPS)
Lipopolysaccharides	100.01 ± 17.81	100.00 ± 3.04	100.00 ± 6.05
1	55.88 ± 11.42	64.73 ± 4.07	90.05 ± 7.11
2	77.58 ± 8.82	66.23 ± 6.59	99.29 ± 5.24
3	28.55 ± 5.35	86.46 ± 8.46	101.69 ± 6.46
4	67.25 ± 4.27	130.88 ± 13.94	103.20 ± 2.56
5	33.72 ± 2.57	64.15 ± 3.03	84.52 ± 7.78
Dexamethasone	12.11 ± 0.03	2.11 ± 0.44	123.86 ± 2.99

Data were normalized to those of cells treated with LPS alone, and cells treated with dexamethasone were used as a positive control (10 μM). Data are expressed as the mean ± SEM (n = 3).

3. Materials and Methods

3.1. General Experimental Procedures

Melting points were determined using a Fargo apparatus and the values were uncorrected. 1D and 2D NMR spectra were recorded on a 600 MHz Jeol NMR (model ECZ600R, Tokyo, Japan) or on a 400 MHz Jeol NMR (model ECZ400S) spectrometers using the residual CHCl₃ signal (δ_H 7.26 ppm) and CDCl₃ (δ_C 77.1 ppm) as internal standards for ¹H and ¹³C NMR, respectively. ESIMS and HRESIMS were obtained from the Bruker mass spectrometer with 7 Tesla magnets (model: SolariX FTMS system, Bremen, Germany). Column chromatography, HPLC, IR, and optical rotation were performed according to our earlier research [15].

3.2. Animal Material

Specimens of J. fragilis used for this study were collected in April 2017 by self-contained underwater breathing apparatus (SCUBA) at depths of 10−15 m off the coast of South Bay, Kenting, Taiwan. The samples were stored in a −20 °C freezer until extraction. A voucher specimen was deposited in the NMMBA (voucher no.: NMMBA-TW-GC-2017-022). Identification of the species of this organism was performed by comparison as described in previous studies [1,2,3].

3.3. Extraction and Isolation

Sliced bodies (wet/dry weight = 795/313 g) of the coral specimen were prepared and extracted with a 1:1 mixture of methanol (MeOH) and dichloromethane (CH₂Cl₂) (1:1) to give a crude extract (19.0 g) which was partitioned between ethyl acetate (EtOAc) and H₂O. The EtOAc extract (8.0 g) was then applied to a silica gel column chromatograph (C.C.) and eluted with gradients of n- hexane/acetone (stepwise from 50:1 to 1:2; volume ratio) to furnish 8 fractions (fractions: A−H). Fraction F was chromatographed on silica gel C.C. and eluted with gradients of n-hexane/EtOAc (2:1 to 1:2, stepwise) to furnish 4 fractions (fractions F1−F4). Fraction F3 was washed with a mixture of n-hexane/acetone (30:1) and the undissolved 5 (23.5 mg) was obtained. Fraction G was purified by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and EtOAc (4:1 to 1:1, stepwise) to afford 16 fractions (fractions G1−G16). Afterward, fraction G15 was separated by NP-HPLC using a mixture of CH₂Cl₂ and acetone (v:v = 10:1; at a flow rate = 2.0 mL/min) to yield 1 (1.7 mg). Fraction G14 was separated by NP-HPLC using a mixture of n-hexane/acetone (2:1; volume ratio) to yield 6 fractions (fractions: G14A−G14F). Fractions G14C and G14D were combined and purified by reverse-phase HPLC (RP-HPLC) using a mixture of MeOH and H₂O (v:v = 65:35; at a flow rate = 4.0 mL/min) to afford 2 (0.8 mg). Fraction G9 was purified by RP-HPLC using a mixture of MeOH and H₂O (v:v = 60:40; at a flow rate = 4.0 mL/min) to yield 16 fractions (fractions G9A−G9P), including compound 3 (0.8 mg, G9D). Fraction G9M was separated by RP-HPLC using a mixture of acetonitrile and H₂O (v:v = 55:45; at a flow rate = 4.0 mL/min) to obtain 4 (0.8 mg).

Fragilide U (1): Amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ −12 (c 0.09, CHCl₃); IR (KBr) ν_max 3445, 1780, 1733 cm⁻¹; ¹³C (100 MHz, CDCl₃) and ¹H (400 MHz, CDCl₃) NMR data, see Table 1; Table 2; ESIMS: m/z 589 [M + Na]⁺; HRESIMS: m/z 589.22583 (calcd. for C₂₈H₃₈O₁₂ + Na, 589.22555).

Fragilide V (2): Amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −26 (c 0.04, CHCl₃); IR (KBr) ν_max 3444, 1779, 1738 cm⁻¹; ¹³C (100 MHz, CDCl₃) and ¹H (400 MHz, CDCl₃) NMR data, see Table 1; Table 2; ESIMS: m/z 581 [M + Na]⁺, 583 [M + 2 + Na]⁺; HRESIMS: m/z 581.17589 (calcd. for C₂₆H₃₅³⁵ClO₁₁ + Na, 581.17601).

Fragilide W (3): Amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −21 (c 0.04, CHCl₃); IR (KBr) ν_max 3459, 1777, 1738 cm⁻¹; ¹³C (150 MHz, CDCl₃) and ¹H (600 MHz, CDCl₃) NMR data, see Table 1; Table 2; ESIMS: m/z 563 [M + Na]⁺, 565 [M + 2 + Na]⁺; HRESIMS: m/z 563.16554 (calcd. for C₂₆H₃₃³⁵ClO₁₀ + Na, 563.16545).

Junceellonoid D (4): Amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ −18 (c 0.04, CHCl₃) (ref. [5] ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{ }$ −44.8 (c 0.10, CHCl₃, MeOH); ref. [21] ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −31 (c 0.05, CHCl₃)); IR (ATR) ν_max 3509, 1793, 1733 cm⁻¹; ¹³C and ¹H NMR data were found to be in full agreement with those reported previously [21]; ESIMS: m/z 521 [M + Na]⁺, 523 [M + 2 + Na]⁺; HRESIMS: m/z 521.15469 (calcd. for C₂₄H₃₁³⁵ClO₉ + Na, 521.15488).

Junceellin (5): Colorless crystals; mp 277–278 °C (ref. [15] mp 272–275 °C); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ −3 (c 1.18, CHCl₃) (ref. [15] ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ −2 (c 0.89, CHCl₃)); IR (KBr) ν_max 1794, 1743 cm⁻¹; ¹³C and ¹H NMR data were found to be in full agreement with those reported previously [6,8,9,12]; ESIMS: m/z 605 [M + Na]⁺, 607 [M + 2 + Na]⁺; HRESIMS: m/z 605.17612 (calcd. for C₂₈H₃₅³⁵ClO₁₁ + Na, 605.17601).

3.4. In Vitro Anti-Inflammatory Assay

The anti-inflammatory assay was employed to evaluate the activities of briaranes 1–5 reduce the release of iNOS and COX-2 from macrophage cells as the literature reported [22,23,24,25].

4. Conclusions

J. fragilis was proven to be a rich source to produce a wide structural diversity of briarane-type diterpenoids that possess various biomedical properties, particularly in anti-inflammatory activity [26,27]. In our continued study on J. fragilis, three previously unreported 11,20-epoxybriaranes, fragilides U–W (1–3), along with two known briaranes, junceellonoid D (4) and junceellin (5), were isolated. The exocyclic 11,20-epoxy group was proven to be a chemical marker for briarane-type natural products from the gorgonian corals belonging to the family Ellisellidae [28]. In the present study, the anti-inflammatory activity of 1–5 was assayed using inhibition of iNOS and COX-2 and the results indicated that fragilide W (3) and junceellin (5) showed the most potent suppressive effect on iNOS release.

Supplementary Materials

The Supplementary Materials are available online at https://www.mdpi.com/1660-3397/17/12/706/s1. HRESIMS, IR, 1D and 2D NMR spectra of compounds 1–4.

Author Contributions

Conceptualization, Z.-H.W.; investigation, Y.-M.J., B.-R.P. and Y.-J.W.; writing—original draft preparation, T.-P.S. and C.-H.Y.; writing—review and editing, T.-Y.W., H.-W.L. and P.-J.S.

Funding

This research was granted from the NMMBA; the NDHU; the Kaohsiung Armed Forces General Hospital (Grant No. 108-33); and the Ministry of Science and Technology, Taiwan (Grant Nos: MOST 108-2320- B-276-001, 104-2320-B-291-001-MY3, and 107-2320-B-291-001-MY3) awarded to Yu-Jen Wu and Ping-Jyun Sung.

Conflicts of Interest

The authors declare no conflicts of interest.