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

Abstract

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 (δ_H 1.99, 1.94, each 3H × s; δ_C 21.3, 21.0, 2 × CH₃; δ_C 170.8, 170.8, 2 × C). Besides the above ester carbonyls, the carbon signal at δ_C 176.7 (C) was assigned to a γ-lactone ring along with an oxymethine (δ_H 5.97, 1H, d, J = 10.2 Hz; δ_C 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 (δ_H 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 (δ_H 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 s 1.99 s	2.01 s 2.04 s 2.09 s	2.00 s 2.16 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 m 2.15 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, CH3 170.8, C 21.3, CH3 170.8, C	21.2, CH3 170.4, C 21.2, CH3 170.1, C 21.2, CH3 169.2, C	20.7, CH3 169.7, C 21.3, CH3 169.6, C
Propionoxy group	-	-	8.8, CH3 27.8, CH2 172.0, C
Isovaleroxy group	-	-	22.6, CH3 22.8, CH3 25.0, CH 43.5, CH2 172.6, 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 (δ_H 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 (δ_H 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 (δ_H 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 (δ_C 135.4, CH-4; 129.7, CH-3) and two methylidene groups (δ_C 142.6, C-5; 118.8, CH₂-16; 149.3, C-11; 111.4, CH₂-20). Moreover, four carbonyl resonances at δ_C 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 (δ_H 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 δ_C 170.4, 170.1, and 169.2 correlated with the signals of the methyl protons at δ_H 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 (δ_H 5.47), H-9 (δ_H 5.53), and H-14 (δ_H 4.84) to the carbonyl carbons of the acetate groups at δ_C 170.1, 169.2, and 170.4, respectively. The hydroxy group at C-8 was deduced from the HMBC of a hydroxy proton (δ_C 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 (δ_H 5.49) showed a correlation to H-4, demonstrating that this olefinic proton was H-16a and the other was assigned as H-16b (δ_H 5.32). Moreover, one of the C-20 methylene protons (δ_H 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 (δ_H 2.16, 2.00, each 3H × s; δ_C 21.3, 20.7, 2 × acetate methyls; 169.7, 169.6, 2 × acetate carbonyls), a propionate (δ_H 1.08, 3H, t, J = 7.2 Hz; 2.26, 2H, m; δ_C 8.8, CH₃; 27.8, CH₂; 172.0, propionate carbonyl), and an isovalerate (δ_H 0.96, 0.98, each 3H, d, J = 6.6 Hz; 2.10, 1H, m; 2.15, 2H, m; δ_C 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 δ_C 173.9 was assigned to a γ-lactone ring along with an oxymethine (δ_H 4.88, 1H, d, J = 4.2 Hz; δ_C 76.9, CH-7). Two pairs of proton signals at δ_H 5.66 and 5.48, and 5.33 and 4.72, correlating to the methylidene signals at δ_C 116.4 and 115.2 respectively, were ascribed to two methylidene groups. The tertiary methyl singlet at δ_H 1.16 (3H, s) was assigned to H₃-15 while the secondary methyl doublet at δ_H 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 (δ_H 6.33) with the carbonyl carbon (δ_C 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 (δ_H 5.37, 1H, dd, J = 5.4, 3.0 Hz; δ_C 74.3, CH-12; δ_H 4.94, 1H, dd, J = 4.2, 3.0 Hz; δ_C 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 (δ_H 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 (δ_H 5.78) and H-4 (δ_H 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 (δ_H 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 (δ_H 7.26 ppm) and CDCl₃ (δ_C 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]$\underset{\mathrm{D}}{\overset{28}{}}$ −288 (c 0.07, CHCl₃); IR (ATR) ν_max 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]$\underset{\mathrm{D}}{\overset{28}{}}$ +45 (c 0.16, CHCl₃); IR (ATR) ν_max 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]$\underset{\mathrm{D}}{\overset{20}{}}$ −168 (c 0.17, CHCl₃); IR (ATR) ν_max 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.

Supplementary Materials

The Supplementary Materials are available online at https://www.mdpi.com/1660-3397/17/9/534/s1. Figure S1: ESIMS spectrum of compound 1, Figure S2: HRESIMS spectrum of compound 1, Figure S3: IR spectrum of compound 1, Figure S4: 1H NMR spectrum (600 MHz) of compound 1 in CDCl3, Figure S5: 13C NMR spectrum (150 MHz) of compound 1 in CDCl3, Figure S6: HSQC spectrum of compound 1 in CDCl3, Figure S7: 1H-1H COSY spectrum of compound 1 in CDCl3, Figure S8: HMBC spectrum of compound 1 in CDCl3, Figure S9: NOESY spectrum of compound 1 in CDCl3, Figure S10: ESIMS spectrum of compound 2, Figure S11: HRESIMS spectrum of compound 2, Figure S12: IR spectrum of compound 2, Figure S13: 1H NMR spectrum (600 MHz) of compound 2 in CDCl3, Figure S14: 13C NMR spectrum (150 MHz) of compound 2 in CDCl3, Figure S15: HSQC spectrum of compound 2 in CDCl3, Figure S16: 1H-1H COSY spectrum of compound 2 in CDCl3, Figure S17: HMBC spectrum of compound 2 in CDCl3, Figure S18: NOESY spectrum of compound 2 in CDCl3, Figure S19: ESIMS spectrum of compound 3, Figure S20: HRESIMS spectrum of compound 3, Figure S21: IR spectrum of compound 3, Figure S22: 1H NMR spectrum (600 MHz) of compound 3 in CDCl3, Figure S23: 13C NMR spectrum (150 MHz) of compound 3 in CDCl3, Figure S24: HSQC spectrum of compound 3 in CDCl3, Figure S25: 1H-1H COSY spectrum of compound 2 in CDCl3, Figure S26: MBC spectrum of compound 3 in CDCl3, Figure S27: NOESY spectrum of compound 3 in CDCl3.

Author Contributions

Conceptualization, L.-S.F. and Z.-H.W.; investigation, Y.-Y.C., Y.-H.C., B.-R.P., T.-P.S., T.-H.H.; N.-C.L., and J.-J.C.; data curation, F.-Y.L.; writing-original draft preparation, C.-Y.L.; writing-review and editing, J.-W.W. and P.-J.S.; project administration, C.-C.H.

Funding

This research was supported by grants from the National Museum of Marine Biology and Aquarium, the National Dong Hwa University, and the Ministry of Science and Technology, Taiwan (Grant Nos: MOST 104-2320-B-291-001-MY3 and 107-2320-B-291-001-MY3) awarded to Ping-Jyun Sung.

Conflicts of Interest

The authors declare no conflicts of interest.