New 8-Hydroxybriaranes from the Gorgonian Coral Junceella fragilis (Ellisellidae).

Three new 8-hydroxybriaranes-fragilides R-T (1-3) were obtained from a sea whip gorgonian coral Junceella fragilis. The structures of briaranes 1-3 were elucidated by using spectroscopic methods, including 1D (1H and 13C NMR), 2D (COSY, HSQC, HMBC, and NOESY experiments) NMR studies, and (+)-HRESIMS. Fragilides S and T (2 and 3) are the only briaranes known to possess 8α-hydroxy and 17β-methyl groups, respectively. Briarane 2 exerted an inhibition effect on iNOS release from RAW264.7; a macrophage cell line that originated from a mouse monocyte macrophage, stimulated with lipopolysaccharides.

1. Introduction

Gorgonian corals belonging to the genus Junceella (family Ellisellidae) [1,2,3] have been found to produce marine origin briarane-type diterpenoids in abundance [4]. Our recent research into the chemical constituents and properties of a gorgonian coral, Junceella fragilis (Ridley 1884) (Figure 1), which was distributed extensively in the waters of Orchid Island (= Lanyu Island), intersection of Kuroshio current and South China Sea surface current, has resulted in the isolation of three new 8- hydroxybriaranes–fragilides R–T (1–3) (Figure 1). A pro-inflammatory suppression assay was employed to assess the activity of these isolates against the release of inducible nitric oxide synthase (iNOS) from macrophage cells.

Figure 1

Structures of fragilides R–T (1–3), 9-O-deacetylumbraculolide A (4), and a picture of the gorgonian coral Junceella fragilis.

2. Results and Discussion

Fragilide R (1) was isolated as an amorphous powder and displayed a pseudomolecular ion at m/z 489.20971 in the (+)-HRESIMS, which indicated its molecular formula was C24H34O9 (calcd. for C24H34O9 + Na, 489.20950) (Ω = 8). Both the 1H and 13C NMR data (Table 1 and Table 2) indicated two acetates (δH 1.99, 1.94, each 3H × s; δC 21.3, 21.0, 2 × CH3; δC 170.8, 170.8, 2 × C). Besides the above ester carbonyls, the carbon signal at δC 176.7 (C) was assigned to a γ-lactone ring along with an oxymethine (δH 5.97, 1H, d, J = 10.2 Hz; δC 76.4, CH-7). The spectroscopic data, including 1D and 2D NMR experiments (Figure 2), were similar to those of a known metabolite, 9-O-deacetyl- umbraculolide A (4) [5] (Figure 1), except that the hydroxy group at C-4 in 1 was replaced by a proton in 4. It is interesting to note that an allylic coupling was observed between H-6 and H3-16 (J = 1.2 Hz) in the COSY spectrum (Figure 2). In the NOESY spectrum (Figure 2), one of the C-3 methylene protons (δH 2.96) exhibited a correlation to H-7 and not with H-2, suggesting the β- orientation of this proton. A correlation from H-4 to H-3α (δH 1.91) as well as the coupling constants between H-4 and H-3α/β (J = 5.4, 12.6 Hz), suggested that H-4 was α-oriented according to modeling study. Based on the above findings, the structure, including the relative configuration of stereogenic centers of 1 were assigned as 1R*,2S*,4R*,7S*,8R*,9S*,10S*,14S*, and 17R*, as those of 4 by correlations observed in a NOESY experiment.

Table 1

1H NMR data for briaranes 1–3.

Position	1 a	2 b	3 a
2	4.83 d (7.2) c	5.47 d (9.5)	6.33 d (10.8)
3α/β	1.91 ddd (15.6, 7.2, 5.4); 2.96 dd (15.6, 12.6)	5.67 dd (16.0, 9.5)	5.78 dd (12.0, 10.8)
4	4.18 dd (12.6, 5.4)	6.72 d (16.0)	5.96 d (12.0)
6	5.64 d (10.2, 1.2)	5.20 s	5.12 br s
7	5.97 d (10.2)	4.97 br s	4.88 d (4.2)
9	4.22 dd (5.4, 3.6)	5.53 s	5.59 s
10	3.20 d (3.6)	3.47 s	4.08 s
12α/β	2.22 m	2.24 m; 2.38 m	5.37 dd (5.4, 3.0)
13α/β	1.96 m; 1.81 m	1.84 m; 1.74 m	2.06 m
14	4.74 dd (4.2, 1.8)	4.84 dd (2.5, 2.0)	4.94 dd (4.2, 3.0)
15	1.25 s	1.18 s	1.16 s
16a/b	2.09 d (1.2)	5.49 s; 5.32 s	5.66 s; 5.48 s
17	3.11 q (7.2)	2.93 q (7.5)	2.90 q (7.2)
18	1.14 d (7.2)	1.18 d (7.5)	1.22 d (7.2)
20a/b	4.97 s; 4.88 s	4.93 s; 4.76 s	5.33 s; 4.72 s
OH-8	-	3.11 s	2.70 s
OH-9	2.09 d (5.4)	-	-
Acetoxy groups	1.94 s1.99 s	2.01 s2.04 s2.09 s	2.00 s2.16 s
Propionoxy group	-	-	1.08 t (7.2)2.26 m
Isovaleroxy group	-	-	0.96 d (6.6)0.98 d (6.6)2.10 m2.15 m

a Spectra measured at 600 MHz in CDCl3. b Spectra measured at 500 MHz in CDCl3. c J values (in Hz) in parentheses.

Table 2

13C NMR data for briaranes 1–3.

Position	1 a	2 b	3 a
1	48.2, C c	47.5, C	47.7, C
2	73.3, CH	76.9, CH	70.4, CH
3	39.7, CH2	129.7, CH	130.4, CH
4	71.4, CH	135.4, CH	128.7, CH
5	146.9, C	142.6, C d	137.5, C
6	123.1, CH	66.0, CH d	64.5, CH
7	76.4, CH	79.5, CH	76.9, CH
8	83.4, C	81.8, C	80.3, C
9	74.1, CH	76.3, CH	74.8, CH
10	43.0, CH	41.8, CH	37.0, CH
11	152.3, C	149.3, C	146.3, C
12	29.0, CH2	29.1, CH2	74.3, CH
13	27.8, CH2	26.0, CH2	31.4, CH2
14	74.2, CH	74.4, CH	73.0, CH
15	15.6, CH3	16.4, CH3	14.7, CH3
16	26.2, CH3	118.8, CH2 d	116.4, CH2
17	43.8, CH	49.3, CH	50.7, CH
18	6.6, CH3	9.1, CH3	8.7, CH3
19	176.7, C	174.1, C d	173.9, C
20	111.3, CH2	111.4, CH2	115.2, CH2
Acetoxy groups	21.0, CH3170.8, C21.3, CH3170.8, C	21.2, CH3170.4, C21.2, CH3170.1, C21.2, CH3169.2, C	20.7, CH3169.7, C21.3, CH3169.6, C
Propionoxy group	-	-	8.8, CH327.8, CH2172.0, C
Isovaleroxy group	-	-	22.6, CH322.8, CH325.0, CH43.5, CH2172.6, C

a Spectra measured at 150 MHz in CDCl3. b Spectra measured at 125 MHz in CDCl3. c Multiplicity deduced by DEPT and HSQC spectra. d Chemical shfits were assigned by HSQC or HMBC experiments.

Figure 2

The COSY () correlations, selective HMBC (), and selective protons with key NOESY correlations () of 1.

One of the C-20 methylene protons (δH 4.88) showed an NOE correlation to H-9, demonstrating that this olefin proton was H-20b, and the other was assigned as H-20a (δH 4.97). The proton chemical shifts of the briarane derivatives containing an 11-methylidene group were summarized, and the difference between these two olefin protons (H-20a/b) was smaller than 0.2 ppm, whereas the methylidene-containing six-membered rings exhibited a twisted boat conformation [6]. Owing to the chemical shifts of C-20 methylene protons (δH 4.97 and 4.88), the conformation of six- membered ring in 1 was concluded to be twisted boat. In a previous study, the absolute configuration of a known chlorinated briarane, junceellin, was established by a single-crystal X-ray diffraction analysis [7]. As briaranes 1–3 were isolated along with junceellin from the same organism [7], it is reasonable on biogenetic grounds to assume that 1–3 have the same absolute configuration as that of junceellin. Therefore, the configuration of the stereogenic centers of 1 were elucidated as 1R,2S,4R,7S,8R,9S,10S,14S, and 17R (Figure 2), and this compound was found to be the 4β-hydroxy derivative of 4 (Supplementary Materials, Figures S1–S9).

Fragilide S (2) had a molecular formula C26H33ClO9 as deduced by (+)-ESIMS, which showed a pair of peaks at m/z 547/549 [M + Na]+:[M + 2 + Na]+ (3:1), suggesting a chlorine atom, and further confirmed by (+)-HRESIMS at m/z 547.17055 (calcd. for C26H3335ClO9 + Na, 547.17053). The IR spectrum indicated the presence of hydroxy (3447 cm−1), γ-lactone (1785 cm−1), and ester carbonyl (1733 cm−1) groups. The 13C NMR data (Table 2), showed the presence of a disubstituted olefin (δC 135.4, CH-4; 129.7, CH-3) and two methylidene groups (δC 142.6, C-5; 118.8, CH2-16; 149.3, C-11; 111.4, CH2-20). Moreover, four carbonyl resonances at δC 174.1, 170.4, 170.1, and 169.2 in the 13C spectrum confirmed the presence of a γ-lactone and three other ester groups. In the 1H NMR spectrum (Table 1), three acetate methyls (δH 2.09, 2.04, 2.01, each 3H × s) were observed. Moreover, a methyl singlet, a methyl doublet, two aliphatic methines, two pair of aliphatic methylenes, four oxymethines, a chlorinated methine, and a hydroxy proton were observed (Table 1).

Analyses of 2D NMR data established a tricyclic nucleus. This assignment was evident from the spin systems from H-2 to H-3, H-3 to H-4, H-6 to H-7, H-9 to H-10, H2-12 to H2-13, H2-13 to H-14, and H-17 to H3-18 (Figure 3), while the HMBC between protons and quaternary carbons such as H-2, H-9, H-10, H3-15/C-1; H-6, H-16a/C-5; H-6, H-9, H-10, H-17, H3-18, OH-8/C-8; H-9, H-10, H-20b/C-11; and H-17, H3-18/C-19, revealed the carbon skeleton (Figure 3). The methylidene groups at C-5 and C-11 were confirmed by the HMBC between H2-16 to C-4 and C-5, H2-20 to C-10, C-11, and C-12, respectively. The C-15 methyl group at C-1 was confirmed by the HMBC between H3-15 to C-1, C-2, C-10, and C-14. HMBC spectrum also revealed that the carbon signal at δC 170.4, 170.1, and 169.2 correlated with the signals of the methyl protons at δH 2.04, 2.01, and 2.09, respectively, and were assigned as the carbon atom of acetate carbonyl groups. The acetates at C-2, C-9, and C-14 were confirmed from the connectivity between H-2 (δH 5.47), H-9 (δH 5.53), and H-14 (δH 4.84) to the carbonyl carbons of the acetate groups at δC 170.1, 169.2, and 170.4, respectively. The hydroxy group at C-8 was deduced from the HMBC of a hydroxy proton (δC 3.11) to C-8 and C-9.

Figure 3

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

In the NOESY spectrum of 2 (Figure 3), one of the C-16 methylene protons (δH 5.49) showed a correlation to H-4, demonstrating that this olefinic proton was H-16a and the other was assigned as H-16b (δH 5.32). Moreover, one of the C-20 methylene protons (δH 4.76) correlated to H-10, indicating that this proton was H-20b and the other was assigned as H-20a. According to a summary for the chemical shifts of 11-methylidene groups, the configuration of six-membered ring was in a twisted boat conformation [6]. The E-geometry of C-3/4 double bond was determined by a large proton coupling constant (J = 16.0 Hz) between H-3 and H-4. Correlations between H-10 with H-2 and H-9, while no correlation was seen with Me-15, suggested that H-2, H-9, and H-10 were all in α-oriented. Meanwhile, a correlation of Me-15 with H-14 indicated that H-14 was β-oriented. Furthermore, OH-8 showed correlations with H-4 and H-17, indicating that the hydroxy group at C-8 and proton at C-17 were α-oriented. In addition, H-7 exhibited correlations with H-6 and H3-18 but not with OH-8, suggesting that H-7 was β-oriented. Based on above findings, the configuration of stereogenic carbons was determined as 1R,2S,6S,7R,8R,9S,10S,14S and 17S (see Figures S10–S18 in the Supplementary Materials).

Compound 3 (fragilide T) has a molecular formula C32H43ClO11 according to its (+)-HRESIMS at m/z 661.23866 (calcd. for C32H4335ClO11 + Na, 661.23861). Both the 1H and 13C NMR (Table 1 and Table 2) indicated two acetates (δH 2.16, 2.00, each 3H × s; δC 21.3, 20.7, 2 × acetate methyls; 169.7, 169.6, 2 × acetate carbonyls), a propionate (δH 1.08, 3H, t, J = 7.2 Hz; 2.26, 2H, m; δC 8.8, CH3; 27.8, CH2; 172.0, propionate carbonyl), and an isovalerate (δH 0.96, 0.98, each 3H, d, J = 6.6 Hz; 2.10, 1H, m; 2.15, 2H, m; δC 22.6, 22.8, 2 × CH3; 25.0, CH; 43.5, CH2; 172.6, isovalerate carbonyl). Besides the above ester carbonyls, the carbon signal at δC 173.9 was assigned to a γ-lactone ring along with an oxymethine (δH 4.88, 1H, d, J = 4.2 Hz; δC 76.9, CH-7). Two pairs of proton signals at δH 5.66 and 5.48, and 5.33 and 4.72, correlating to the methylidene signals at δC 116.4 and 115.2 respectively, were ascribed to two methylidene groups. The tertiary methyl singlet at δH 1.16 (3H, s) was assigned to H3-15 while the secondary methyl doublet at δH 1.22 (3H, d, J = 7.2 Hz) was assigned to H3-18. In the HMBC spectrum (Figure 4), the propionoxy group at C-2 was confirmed by the connectivity between H-2 (δH 6.33) with the carbonyl carbon (δC 172.0) of propionoxy group. The HMBC also revealed that an acetoxy group at C-9 (Figure 4) and the remaining isovaleroxy and acetoxy groups should be positioned at C-12 or C-14, oxygen-bearing methines, by analysis of characteristic NMR signals (δH 5.37, 1H, dd, J = 5.4, 3.0 Hz; δC 74.3, CH-12; δH 4.94, 1H, dd, J = 4.2, 3.0 Hz; δC 73.0, CH-14). However, due to no HMBC detected between H-12 and H-14 and ester carbonyl, the positions of the isovalerate and remaining acetoxy group cannot be determined by HMBC.

Figure 4

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

Based on previous studies, while the difference between the two olefin protons (H-20a/b) was bigger than 0.3 ppm, the six-membered rings showed a chair conformation [6]. Owing to the chemical shifts of the C-20 methylene protons (δH 5.33 and 4.72 ppm), the configuration of the methylidene-containing six-membered ring was concluded to exist in a chair conformation. In the NOESY experiment (Figure 4), H-10 correlated to H-2, H-9, and OH-8, but not to H3-15, indicating that these protons are located on the same face and can be assigned as α-protons, as C-15 methyl group is a β-substituent at C-1. H-14 was found to exhibit a correlation with H3-15, showing that this proton is positioned on the equatorial direction and has a β-orientation at C-14. The cis geometry of the C-3/4 double bond was indicated by a 12.0 Hz coupling constant between H-3 (δH 5.78) and H-4 (δH 5.96). Moreover, a correlation between H-3 and H3-15, and there are correlations which were observed among H-4, H-6, and H-7, further supported the Z-form of C-3/4 double bond and indicated that H-6 and H-7 were on the β face. A correlation between OH-8 and H-17 showed that Me-18 at C-17 was β-oriented. The C-12 oxymethine proton (δH 5.37) was found to couple C-13 methylene protons with coupling constants J = 5.4, 3.0 Hz, showing that this proton should be positioned on the equatorial direction and has a β-orientation. Fortunately, a correlation between the methyl protons of acetoxy group at C-9 and methyl protons of isovaleroxy group, indicated that the isovaleroxy group should be located at C-12 by modeling analysis. Thus, based on the above findings, the stereogenic centers were assigned as 1R,2S,6S,7R,8R,9S,10R,12R,14S, and 17S (see Figures S19–S27 in the Supplementary Materials).

Using an in vitro pro-inflammatory suppression assay, the effects of briaranes 1–3 on the release of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein from lipopolysaccharides (LPS)-stimulated RAW264.7 macrophage cells were assessed. The results of pro-inflammatory suppression assay showed that briarane 2 at 10 μM suppressed the release of iNOS to 61.21 ± 9.61% as compared with results of the cells stimulated with LPS only (Table 3).

Table 3

Effects of briaranes 1–3 on LPS-induced pro-inflammatory iNOS and COX-2 protein expression in macrophages.

Compound	iNOS	COX-2	β-Actin	n
	Expression (% of LPS)
Negative Control	1.80 ± 0.21	1.04 ± 0.35	110.02 ± 5.23	2
LPS	100.01 ± 5.06	100.06 ± 0.43	100.07 ± 8.4	4
1	104.11 ± 16.63	100.51 ± 6.11	105.70 ± 7.05	4
2	61.21 ± 9.61	100.01 ± 5.11	99.29 ± 11.29	3
3	100.91 ± 24.08	96.36 ± 21.31	115.29 ± 3.4	4
Dexamethasone	5.54 ± 1.72	8.15 ± 5.13	105.21 ± 15.57	4

Data were normalized to those of cells treated with LPS alone, and cells treated with dexamethasone were used as a positive control. Data are expressed as the mean ± SEM (n = 2–4).

3. Materials and Methods

3.1. General Experimental Procedures

NMR spectra were recorded on a 600 MHz Jeol NMR (model ECZ600R, Tokyo, Japan) or on 500 MHz Varian NMR (model Unity Inova-500, Palo Alto, CA, USA) spectrometers using the residual CHCl3 signal (δH 7.26 ppm) and CDCl3 (δC 77.1 ppm) as internal references 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, high-performance liquid chromatography (HPLC), IR spectra, and optical rotation were performed according to our earlier research [7].

3.2. Animal Material

Specimens of J. fragilis used for this study were collected in June 2017 by self-contained underwater breathing apparatus (SCUBA) divers off the coast of Orchid Island, 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-2017-08). 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 weight = 423 g) of the coral specimen were prepared and extracted with a 1:1 mixture of methanol (MeOH) and dichloromethane (CH2Cl2) to give 5.53 g of crude extract, which was partitioned between ethyl acetate (EtOAc) and H2O. The EtOAc extract (2.50 g) was then applied to a silica gel column 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 purified by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and EtOAc (3.5:1 of volume ratio) as solvent to obtain 14 subfractions (fractions: F1−F14). Fraction F10 was repurified by reverse-phase HPLC (RP-HPLC) using a mixture of MeOH and H2O (with volume:volume = 80:20; at a flow rate = 4.0 mL/min) to yield 2 (0.2 mg) and 3 (0.5 mg). Fraction G was separated by NP-HPLC using a mixture of n-hexane/EtOAc (1:1; volume ratio) to yield 9 fractions (fractions: G1−G9). Fraction G2 was purified by RP-HPLC using a mixture of MeOH and H2O (with volume:volume = 65:35; at a flow rate = 4.0 mL/min) to afford 1 (0.2 mg).

Fragilide R (1): Amorphous powder; [α] −288 (c 0.07, CHCl3); IR (ATR) νmax 3391, 1779, 1736 cm−1; 1H (600 MHz, CDCl3) and 13C (150 MHz, CDCl3) NMR data, see Table 1 and Table 2; ESIMS: m/z 489 [M + Na]+; HRESIMS: m/z 489.20971 (calcd. for C24H34O9 + Na, 489.20950).

Fragilide S (2): Amorphous powder; [α] +45 (c 0.16, CHCl3); IR (ATR) νmax 3447, 1785, 1733 cm−1; 1H (500 MHz, CDCl3) and 13C (125 MHz, CDCl3) NMR data, see Table 1 and Table 2; ESIMS: m/z 547 [M + Na]+, 549 [M + 2 + Na]+; HRESIMS: m/z 547.17055 (calcd. for C26H3335ClO9 + Na, 547.17053).

Fragilide T (3): Amorphous powder; [α] −168 (c 0.17, CHCl3); IR (ATR) νmax 1788, 1737 cm−1; 1H (600 MHz, CDCl3), and 13C (150 MHz, CDCl3) NMR data, see Table 1 and Table 2; ESIMS: m/z 661 [M + Na]+, 663 [M + 2 + Na]+; HRESIMS: m/z 661.23866 (calcd. for C32H4335ClO11 + Na, 661.23861).

3.4. In Vitro Anti-inflammatory Assay

The pro-inflammatory suppression assay was employed to assess the activities of the isolated compounds 1–3 against the release of iNOS and COX-2 from macrophage cells as the literature reported [8, 9, 10, 11].

4. Conclusions

J. fragilis has been demonstrated to have a wide structural diversity of briarane-type diterpenoids that possess various pharmacological properties, particularly in anti-inflammatory activity [12,13]. In our continued study of J. fragilis, three previously unreported fragilides R–T (1–3) were isolated. In the previous studies [14], all the Me-18 attached at C-17 was cis to the hydroxy group at C-8, and most of these two groups were α-oriented in briarane derivatives, respectively. Fragilides S and T (2 and 3) were proved to be the only two briaranes known to possess hydroxy group at C-8α and methyl group at C-17β, respectively. In the present study, the anti-inflammatory activity of 1–3 was assessed using inhibition of pro-inflammatory iNOS and COX-2 release from macrophages. The results indicated that fragilide S (2) showed the most potent suppressive effect on iNOS release.