New 9-Hydroxybriarane Diterpenoids from a Gorgonian Coral Briareum sp. (Briareidae).

Six new 9-hydroxybriarane diterpenoids, briarenolides ZI-ZVI (1-6), were isolated from a gorgonian coral Briareum sp. The structures of briaranes 1-6 were elucidated by spectroscopic methods and by comparison of their spectroscopic data with those of related analogues. Briarenolides ZII (2) and ZVI (6) were found to significantly inhibit the expression of the pro-inflammatory inducible nitric oxide synthase (iNOS) protein of lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells.

1. Introduction

The briarane-type diterpenoid (3,8-cyclized cembranoid), 2β-acetoxy-2-(debutyryloxy)-stecholide E, was first isolated from the gorgonian coral Briareum sp. in 1996 [1]. Since then, hundreds of compounds of this type have been obtained from various Taiwanese gorgonian corals, such as Briareum, Junceella and Ellisella spp. [2,3,4,5,6], that have been located off the coast of Taiwan. Recently, in a sample collected at the southern tip of Taiwan, as Briareum sp. (family Briareidae), we identified six new briaranes, briarenolides ZI–ZVI (1–6) (Figure 1). In this report, we isolate and determine the structures of these briaranes, in addition to studying their anti-inflammatory properties.

Figure 1

The structures of briarenolides ZI–ZVI (1–6), excavatolide F (7), 2β-acetoxy-2- (debutyryloxy)-stecholide E (8), excavatolide Z (9) and excavatolide E (10).

2. Results and Discussion

The molecular formula of a new briarane, briarenolide ZI (1), was determined as C24H33ClO11 (eight degrees of unsaturation) by high-resolution electrospray ionization mass spectrum (HRESIMS) at m/z 555.16025 (calcd. for C24H33ClO11 + Na, 555.16036). The IR of 1 showed absorptions at 1715, 1769 and 3382 cm−1, which were consistent with the presence of ester, γ-lactone and hydroxy groups. The 13C NMR spectrum (Table 1) suggested that 1 possessed an exocyclic carbon-carbon double bond based on signals at δC 138.6 (C-5) and 116.9 (CH2-16), which was confirmed by the 1H NMR spectrum of 1 (Table 1), which showed two olefin proton signals at δH 5.88 (1H, dd, J = 2.4, 1.2 Hz, H-16a) and 5.64 (1H, dd, J = 2.4, 1.2 Hz, H-16b). Three carbonyl resonances at δC 175.3 (C-19), 173.4 and 169.3 (2 × ester carbonyls) revealed the presence of one γ-lactone and two ester groups in 1; two acetyl methyls (δH 2.06, s, 2 × 3H) were also observed. According to the overall unsaturation data, it was concluded that 1 was a diterpenoid molecule possessing four rings.

Table 1

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data and 1H–1H COSY (correlation spectroscopy) and HMBC (heteronuclear multiple bond coherence) correlations for briarane 1.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	–	45.6, C	–	–
2	5.09 d (6.4)	73.4, CH	H2-3	C-1, -4, -15, acetate carbonyl
3	3.73 dd (16.0, 6.4); 1.46 d (16.0)	41.7, CH2	H-2	C-1, -2, -4
4	–	96.7, C	–	–
5	–	138.6, C	–	–
6	5.54 dt (2.8, 2.4)	56.2, CH	H-7, H2-16	n. o. a
7	4.73 d (2.8)	79.8, CH	H-6	n. o.
8	–	81.8, C	–	–
9	4.88 d (3.2)	76.9, CH	H-10, OH-9	n. o.
10	2.23 s	40.5, CH	H-9	C-1, -2, -8, -9, -15
11	–	78.5, C	–	–
12	3.50 br s	76.1, CH	H2-13, OH-12	n. o.
13	2.44 ddd (15.6, 4.0, 2.8); 1.98 ddd (15.6, 3.2, 2.8)	28.0, CH2	H-12, H-14	C-1
14	5.22 t (2.8)	76.3, CH	H2-13	n. o.
15	1.55 s	16.5, CH3	–	C-1, -2, -10, -14
16a/b	5.88 dd (2.4, 1.2); 5.64 dd (2.4, 1.2)	116.9, CH2	H-6	C-4, -5, -6
17	2.58 q (7.2)	50.4, CH	H3-18	C-9, -18, -19
18	1.33 d (7.2)	8.2, CH3	H-17	C-8, -17, -19
19	–	175.3, C	–	–
20	1.56 s	28.9, CH3	–	C-10, -11, -12
OAc-2	–	173.4, C	–	–
	2.06 s	21.3, CH3	–	Acetate carbonyl
OAc-14	–	169.3, C	–	–
	2.06 s	21.1, CH3	–	Acetate carbonyl
OH-4	6.50 s	–	–	C-3, -4, -5
OH-9	2.73 d (3.2)	–	H-9	C-8
OH-12	2.67 br s	–	H-12	n. o.

a n. o. = not observed.

1H NMR coupling information in the 1H–1H correlation spectroscopy (COSY) spectrum of 1 enabled identification of the H-2/H2-3, H-6/H-7, H-12/H2-13/H-14, H-6/H2-16 (by allylic coupling) and H-17/H3-18 units (Table 1). The heteronuclear multiple bond coherence (HMBC) correlations between protons and quaternary carbons of 1 (H-2, H2-3, H-10, H2-13, H3-15/C-1; H-2, H2-3, H2-16, OH-4/C-4; H-16b, OH-4/C-5; H-10, H3-18, OH-9/C-8; H3-20/C-11 and H-17, H3-18/C-19) permitted elucidation of the carbon skeleton (Table 1). HMBC correlations between H2-16/C-4, -5 and -6 indicated an exocyclic double bond at C-5, which was further confirmed by the allylic coupling between H2-16/H-6. HMBC correlations between H3-15/C-1, -2, -10 and -14 and H-2 and H-10/C-15, revealed that the ring junction C-15 methyl group was located at C-1. Furthermore, an HMBC correlation between H-2 (δH 5.09) and the acetate carbonyl (δC 173.4) revealed the presence of an acetate ester at C-2; and an HMBC correlation between a hydroxy proton (δH 6.50) and C-4 oxygenated quaternary carbon suggested the presence of a hydroxy group at C-4. The C-4 hydroxy group was determined to be part of a hemiketal constellation on the basis of a characteristic carbon signal at δC 96.7. 1H–1H COSY correlations between OH-9/H-9 and OH-12/H-12 suggested the presence of the hydroxy groups at C-9 and C-12. A carbon signal at δC 81.8 (C-8) indicated 3J-coupling with protons at δH 2.23 (H-10), 1.33 (H3-18) and 2.73 (OH-9). Therefore, the remaining hydroxy and acetoxy groups had to be positioned at C-11 and C-14, respectively, as indicated by analysis of 1H–1H COSY correlations and characteristic NMR signal analysis. The intensity of the sodiated molecules [M + 2 + Na]+ isotope peak observed in the ESIMS and HRESIMS spectra ([M + Na]+:[M + 2 + Na]+ = 3:1) was evidence of the presence of one chlorine atom in 1. The methine unit at δC 56.2 was more shielded than expected for an oxygenated carbon and was correlated to the methine proton at δH 5.54 (H-6) in the heteronuclear multiple quantum coherence (HMQC) spectrum, and this proton signal was 3J-correlated with H-7 (δH 4.73) in the 1H–1H COSY spectrum, which proved that a chlorine atom was attached at C-6. These data, together with the HMBC correlations between H-17/C-9, -18 and -19 and H3-18/C-8, -17 and -19, established the molecular framework of 1.

The relative configuration of 1 was elucidated on the basis of a nuclear Overhauser effect spectroscopy (NOESY) experiment and by vicinal 1H–1H proton coupling constant analysis. Most naturally-occurring briarane natural products have Me-15 in the β-orientation and H-10 in the α-orientation [2,3,4,5,6], which were verified by the absence of a correlation between these two groups. In the NOESY experiment of 1 (Figure 2), H-10 correlated with H-2, H-9 and H3-20, indicating that these protons were situated on the same face; they were assigned as α protons, as C-15 methyl was β-oriented at C-1. The oxymethine proton H-14 was found to exhibit a response with H3-15, but not with H-10, revealing that H-14 was β-oriented. H-12 correlated with each of the C-13 methylene protons and H3-20, but not with H-10, indicating that H-12 was β-oriented and was positioned on the equatorial direction in the cyclohexane ring by modeling analysis. H-17 exhibited correlations with H-9 and H-7 and was also found to be reasonably close to H-9 and H-7 by modeling analysis; thus, H-17 could therefore be placed on the β face in 1, and H-7 was β-oriented. One of the C-3 methylene protons (δH 3.73) displayed a correlation with H3-15; therefore, it was assigned as the H-3β proton, and the other was assigned as H-3α (δH 1.46). H-6 displayed correlations with H-3β and H-7, which confirmed that this proton was in the β-orientation, and the oxygen bridge between C-4 and C-8 was found to be α-oriented by modeling analysis. Based on the aforementioned results, the structure, including the relative configuration, of 1 was elucidated unambiguously.

Figure 2

Selected protons with key nuclear Overhauser effect spectroscopy (NOESY) correlations of 1.

Briarenolide ZII (2) was isolated as a white powder and had a molecular formula of C28H38O10 on the basis of HRESIMS at m/z 557.23552 (calcd. for C28H38O10 + Na, 557.23572). Carbonyl resonances in the 13C NMR spectrum of 2 (Table 2) at δC 173.0, 170.7, 170.4 and 169.9 demonstrated the presence of a γ-lactone and three other esters in 2. It was found that the NMR signals of 2 were similar to those of a known briarane analogue, excavatolide F (7) [7] (Figure 1), except that the signals corresponding to the 9-acetoxy group in 7 were replaced by signals for a hydroxy group in 2. The correlations from a NOESY experiment of 2 also revealed that the stereochemistry of this metabolite was identical to that of 7. Thus, briarenolide ZII (2) was found to be the 9-O-deacetyl derivative of 7.

Table 2

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for briarane 2.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	–	45.6, C	–	–
2	5.39 d (10.0)	75.9, CH	H-3	C-1, -3, -4, -14, -15, acetate carbonyl
3	5.76 dd (16.0, 10.0)	126.0, CH	H-2, H-4	C-5
4	6.82 d (16.0)	139.0, CH	H-3, H-6, H3-16	C-2, -3, -5, -6
5	–	140.4, C	–	–
6	5.44 dq (4.4, 1.6)	118.4, CH	H-4, H-7, H3-16	C-4, -8
7	5.10 d (4.4)	76.8, CH	H-6	C-5, -6
8	–	69.9, C	–	–
9	4.36 d (9.6)	74.6, CH	H-10	C-1, -8, -10, -11, -17
10	2.08 d (4.8)	38.5, CH	H-9, H-11	C-1, -2, -8, -9, -11, -14, -15, -20
11	2.23 m	39.2, CH	H-10, H-12, H3-20	C-1, -10, -12, -13, -20
12	4.98 m	70.3, CH	H-11, H2-13	C-20, -1′
13	2.03 m; 1.84 dt (14.4, 3.2)	26.3, CH2	H-12, H-14	C-12
14	4.95 t (3.2)	74.3, CH	H2-13	C-15, acetate carbonyl
15	1.43 s	16.1, CH3	–	C-1, -2, -10, -14
16	1.89 br s	23.5, CH3	H-4, H-6	C-4, -5, -6
17	–	63.4, C	H3-18	–
18	1.52 s	10.0, CH3	H-17	C-7, -8, -19
19	–	170.7, C	–	–
20	1.15 d (7.2)	10.5, CH3	H-11	C-10, -11, -12
OAc-2	–	169.9, C	–	–
	1.98 s	21.2, CH3	–	Acetate carbonyl
OAc-14	–	170.4, C	–	–
	2.09 s	21.3, CH3	–	Acetate carbonyl
OC(O)Pr-12 1′2′3′4′	–	–	–	–
1′	–	173.0, C	–	–
2′	2.26 t (7.2)	36.3, CH2	H2-3′	C-1′, -3′, -4′
3′	1.61 sext (7.2)	18.4, CH2	H2-2′, H3-4′	C-1′, -2′, -4′
4′	0.94 t (7.2)	13.7, CH3	H2-3′	C-2′, -3′

Briarenolide ZIII (3) had a molecular formula C24H32O10 as deduced from HRESIMS at m/z 503.18858 (calcd. for C24H32O10 + Na, 503.18877). The IR spectrum of 1 showed three bands at 3444, 1779 and 1732 cm−1, which were in agreement with the presence of hydroxy, γ-lactone and ester groups. Carbonyl resonances in the 13C NMR spectrum of 3 at δC 171.8, 170.7 and 170.6 revealed the presence of a γ-lactone and two esters (Table 3). Both esters were identified as acetates by the presence of two acetyl methyl resonances in the 1H (δH 2.01, 1.98, each 3H × s) and 13C (δC 21.1, 21.1) NMR spectra (Table 3).

Table 3

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for briarane 3.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	–	45.7, C	–	–
2	4.72 d (6.0)	73.8, CH	H2-3	C-1, -4, -10, -14, -15, acetate carbonyl
3	3.05 m; 1.92 m	40.8, CH2	H-2, H-4	C-1, -4, -5
4	4.23 dd (12.4, 5.2)	71.3, CH	H2-3	C-5, -6, -16
5	–	147.5, C	–	–
6	5.49 dt (9.6, 1.2)	122.0, CH	H-7, H3-16	C-4, -16
7	6.22 d (9.6)	73.4, CH	H-6	C-5, -6
8	–	71.0, C	–	–
9	4.45 dd (6.0, 3.6)	72.2, CH	H-10, OH-9	C-7, -8, -11
10	2.29 d (3.6)	42.5, CH	H-9	C-1, -8, -9, -11, -15
11	–	63.6, C	–	–
12	3.05 d (2.8)	61.4, CH	H2-13	n. o. a
13	2.08 m	25.2, CH2	H-12, H-14	n. o.
14	4.73 br s	73.8, CH	H2-13	C-1, -2, -10, -12, -15, acetate carbonyl
15	1.19 s	16.0, CH3	–	C-1, -10, -14
16	2.11 d (1.2)	25.5, CH3	H-6	C-4, -5, -6
17	–	62.5, C	–	–
18	1.67 s	9.4, CH3	–	C-8, -17, -19
19	–	171.8, C	–	–
20	1.35 s	24.5, CH3	–	C-10, -11, -12
OAc-2	–	170.7, C	–	–
	1.98 s	21.1, CH3	–	Acetate carbonyl
OAc-14	–	170.6, C	–	–
	2.01 s	21.1, CH3	–	Acetate carbonyl
OH-19	2.89 d (6.0)	–	H-9	C-8

a n. o. = not observed.

It was found that the NMR data of 3 were similar to those of a known briarane analogue, 2β-acetoxy-2-(debutyryloxy)-stecholide E (8) [1] (Figure 1), except that the signals corresponding to the 4-hydroxy group in 3 were not present in 8. A correlation from the NOESY signals of 3 showed that H-4 correlated with H-2, but not with H3-15, indicating that the hydroxy group at C-4 was β-oriented. The results of 1H–1H COSY and HMBC correlations fully supported the positions of functional groups, and hence, briarenolide ZIII (3) was found to be the 4β-hydroxy derivative of 8.

Briarenolide ZIV (4) was obtained as a white powder, and the molecular formula of 4 was determined to be C28H40O11 (9° of unsaturation) by HRESIMS at m/z 575.24645 (calcd. for C28H40O11 + Na, 575.24628). The IR spectrum of 4 showed three bands at 3444, 1778 and 1732 cm−1, consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. Carbonyl resonances in the 13C NMR spectrum of 4 showed signals at δC 173.9, 173.2, 170.8 and 170.4, which revealed the presence of a γ-lactone and three esters in 4 (Table 4), of which, two of the esters were identified as acetates based on the presence of two acetyl methyl resonances in the 1H NMR spectrum of 4 at δH 1.97 (2 × 3H, s) (Table 4). The other ester was found to be an n-butyrate group based on 1H NMR studies, which revealed seven contiguous protons (δH 0.94, 3H, t, J = 7.2 Hz; 1.65, 2H, sextet, J = 7.2 Hz; 2.23, 2H, t, J = 7.2 Hz). According to the 1H and 13C NMR spectra, 4 was found to have a γ-lactone moiety (δC 173.9, C-19) and a trisubstituted olefin (δC 145.4, C-5; 121.6, CH-6; δH 5.32, 1H, d, J = 8.8 Hz, H-6). The presence of a tetrasubstituted epoxide that contained a methyl substituent was established based on the signals of two oxygenated quaternary carbons at δC 71.8 (C-8) and 63.7 (C-17) and confirmed by the proton signals of a methyl singlet at δH 1.51 (3H, s, H3-18). Thus, from the NMR data, five degrees of unsaturation were accounted for, and 4 was identified as a tetracyclic compound. From the 1H–1H COSY spectrum of 4 (Table 4), three different structural units, including C-2/-3/-4, C-6/-7 and C-9/-10/-11/-12/-13/-14, were identified. From these data and the HMBC correlation results (Table 4), the connectivity from C-1 to C-14 could be established. A methyl attached at C-5 was confirmed by an allylic coupling between H3-16/H-6 and by the HMBC correlations between H3-16/C-4, -5 and -6. The C-15 and C-20 methyl groups were identified as being positioned at C-1 and C-11 from the HMBC correlations between H3-15/C-1, -2, -10, -14 and H3-20/C-10, -11, -12, respectively. Furthermore, the acetate esters positioned at C-2 and C-14 were established by the HMBC correlations between δH 4.97 (H-2) and 4.70 (H-14) and the acetate carbonyls at δC 170.4 and 170.8, respectively. The location of an n-butyrate group in 4 was verified by an HMBC correlation between H-12 (δH 4.83) and the n-butyrate carbonyl carbon (δC 173.2) (Table 4). These data, together with the HMBC correlations between H3-18/C-8, -17 and -19, established the main molecular framework of 4. The NMR data of 4 were found to be similar to those of a known briarane, excavatolide Z (9) [8] (Figure 1), except that the signals corresponding to the 4-hydroxy group in 4 were not present in 9, and an 11β-hydroxy group was found in 9. The correlations from NOESY signals of 4 (Figure 3) also showed that the relative configurations of most chiral centers of 4 were similar to those of 9. H-10 exhibited interactions with H-2 and H-11, and H-2 correlated with H-4, indicating that the hydroxy group at C-4 and the methyl group at C-11 were β-oriented; additionally, briarenolide ZIV (4) was found to be the 4β-hydroxy-11-dehydroxy-11β-methyl derivative of 9.

Table 4

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for briarane 4.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	–	46.1, C	–	–
2	4.97 d (8.0)	74.9, CH	H2-3	C-1, -4, -10, -15, acetate carbonyl
3	3.22 dd (15.2, 12.0); 1.93 m	39.7, CH2	H-2, H-4	C-1, -4
4	4.16 dd (12.0, 5.2)	71.3, CH	H2-3	C-3, -5, -6, -16
5	–	145.4, C	–	–
6	5.32 d (8.8)	121.6, CH	H-7, H3-16	C-4, -16
7	6.14 d (8.8)	75.4, CH	H-6	C-5, -6, -19
8	–	71.8, C	–	–
9	3.79 br s	74.1, CH	H-10	C-1, -7, -8, -10, -11, -17
10	2.39 d (5.2)	37.2, CH	H-9, H-11	C-1, -2, -8, -9, -11, -12, -14, -15, -20
11	1.88 m	43.2, CH	H-10, H-12, H3-20	C-1, -10, -12, -20
12	4.83 br s	72.1, CH	H-11, H2-13	C-10, -14, -1′
13	2.11 m; 1.95 m	24.6, CH2	H-12, H-14	C-11, -12, -14
14	4.70 br s	74.2, CH	H2-13	C-1, -2, -10, -12, -15, acetate carbonyl
15	1.32 s	15.2, CH3	–	C-1, -2, -10, -14
16	2.05 d (1.2)	25.3, CH3	H-6	C-4, -5, -6
17	–	63.7, C	–	–
18	1.51 s	9.7, CH3	–	C-8, -17, -19
19	–	173.9, C	–	–
20	1.25 d (7.2)	15.2, CH3	H-11	C-10, -11, -12
OAc-12	–	170.4, C	–	–
	1.97 s	21.2, CH3	–	Acetate carbonyl
OAc-14	–	170.8, C	–	–
	1.97 s	21.5, CH3	–	Acetate carbonyl
OC(O)Pr-12 1′2′3′4′	–	–	–	–
1′	–	173.2, C		–
2′	2.23 t (7.2)	36.6, CH2	H2-3′	C-1′, -3′, -4′
3′	1.65 sext (7.2)	18.5, CH2	H2-2′, H3-4′	C-1′, -2′, -4′
4′	0.94 t (7.2)	13.6, CH3	H2-3′	C-2′, -3′

Figure 3

Selected protons with key NOESY correlations of 4.

Briarenolide ZV (5) was obtained as a white powder and had the molecular formula C24H30O10, as determined by HRESIMS at m/z 505.20460 (calcd. for C24H30O10 + Na, 505.20442) (10° of unsaturation). The IR spectrum of 5 showed bands at 3445, 1770 and 1732 cm−1, consistent with the presence of hydroxy, γ-lactone and ester carbonyl groups. Comparison of the 1H and distortioneless enhancement by polar transfer (DEPT) spectra with the molecular formula revealed that there must be three exchangeable protons, requiring the presence of three hydroxy groups. In addition, it was found that the spectral data (IR, 1H and 13C NMR) of 5 (Table 5) were similar to those of a known briarane, excavatolide Z (9) [8] (Figure 1), except that 9 exhibited signals representing an n-butyrate substitution, which were replaced by a hydroxy group in 5. The results of 1H–1H COSY and HMBC correlations fully supported the positions of functional groups, and hence, briarenolide ZV (5) was found to be the 12-O-debutyryl derivative of 9.

Table 5

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for briarane 5.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	–	48.6, C	–	–
2	5.02 d (7.2)	75.7, CH	H2-3	C-1, -3, -4, -10, -14, -15, acetate carbonyl
3	2.86 td (15.2, 5.2); 1.59 m	32.5, CH2	H-2, H2-4	n. o. a
4	2.50 br d (15.2); 1.91 m	28.7, CH2	H2-3	n. o.
5	–	146.0, C	–	–
6	5.28 d (9.6)	117.9, CH	H-7, H3-16	C-4
7	5.50 d (9.6)	75.1, CH	H-6	C-5
8	–	71.1, C	–	–
9	4.65 dd (5.6, 2.0)	69.7, CH	H-10, OH-9	C-7, -8, -10, -11, -17
10	2.13 br s	44.0, CH	H-9	C-9
11	–	78.6, C	–	–
12	3.43 br d (10.0)	76.6, CH	H2-13, OH-12	n. o.
13	2.32 m; 1.92 m	26.5, CH2	H-12, H-14	n. o.
14	4.99 t (2.8)	77.5, CH	H2-13	C-1, -10, -15, acetate carbonyl
15	1.42 s	15.9, CH3	–	C-1, -2, -10, -14
16	2.00 s	26.9, CH3	H-6	C-4, -5, -6
17	–	63.4, C	–	–
18	1.68 s	9.6, CH3	–	C-8, -17, -19
19	–	171.6, C	–
20	1.41 s	31.1, CH3	–	C-10, -11, -12
OAc-2	–	170.8, C	–	–
	1.99 s	21.4, CH3	–	Acetate carbonyl
OAc-14	–	169.8, C	–	–
	2.03 s	21.7, CH3	–	Acetate carbonyl
OH-9	2.45 br s	–	H-9	n. o.
OH-12	2.74 d (10.0)	–	H-12	n. o.

a n. o. = not observed.

The new briarane, briarenolide ZVI (6), had a molecular formula of C26H36O11 as determined by HRESIMS at m/z 547.21473 (calcd. for C26H36O11 + Na, 547.21498). Thus, nine degrees of unsaturation were therefore determined for the molecule of 6. In addition, the spectral data (IR, 1H and 13C NMR) (Table 6) of 6 were found to be similar to those of a known briarane, excavatolide E (10) [9] (Figure 1). However, the NMR spectra revealed that the signals representing the C-4 methylene group in 10 were replaced by those of an additional acetoxy group. In the NOESY experiment of 6, H-10 gives correlations to H-2, H-9, H-11 and H-12, but not to H3-15 and H3-20, and H-2 was found to show a correlation with H-4, indicating that these protons (H-2, H-4, H-9, H-10, H-11 and H-12) are located on the same face of the molecule and assigned as α-protons, since the C-15 and C-20 methyls are the β-substituents at C-1 and C-11, respectively. The signal of H3-20 showed a correlation with H3-18, indicating that H3-18 and 8,17-epoxide group were β- and α-oriented, respectively, in the γ-lactone ring in 6. H-4 correlated with H-2, but not with H-7 and H3-15, indicating that H-7 was β-oriented. H-14 was found to exhibit nuclear Overhauser effect (NOE) responses with H-2 and H3-15, but not with H-10, revealing the β-orientation of this proton. Thus, based on the above findings, Compound 6 was found to be the 4β-acetoxy derivative of 10, with a structure as described by Formula 6. Furthermore, the chemical shifts for H3-18 in briaranes 4, 5 and 6 were found to appear at δH 1.51, 1.68 and 1.57, respectively, indicating that the 11β-hydroxy group in 5 led to a downfield chemical shift for H3-18.

Table 6

1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data and 1H–1H COSY and HMBC correlations for briarane 6.

Position	δH (J in Hz)	δC, Multiple	1H–1H COSY	HMBC
1	–	46.1, C	–	–
2	4.87 d (8.0)	73.6, CH	H2-3	C-1, -3, -4, -10, -14, -15, acetate carbonyl
3	3.16 dd (15.6, 12.8); 1.91 m	37.6, CH2	H-2, H-4	C-1, -2, -4, -5
4	5.01 dd (12.8, 5.6)	72.7, CH	H2-3	C-3, -5, -6, -16, acetate carbonyl
5	–	144.1, C	–	–
6	5.39 d (9.2)	122.7, CH	H-7, H3-16	C-4, -16
7	5.92 d (9.2)	74.5, CH	H-6	C-5, -6, -19
8	–	71.7, C	–	–
9	3.91 br s	74.7, CH	H-10, OH-9	n. o. a
10	2.20 dd (4.8, 2.2)	41.6, CH	H-9, H-11	C-1, -2, -11, -15, -20
11	1.99 m	44.7, CH	H-10, H-12, H3-20	n. o.
12	4.04 dt (8.8, 3.6)	67.0, CH	H-11, H2-13	n. o.
13	1.84 m	29.0, CH2	H-12, H-14	C-1, -12
14	4.78 t (2.8)	76.2, CH	H2-13	C-10, -12, acetate carbonyl
15	1.31 s	15.4, CH3	–	C-1, -2, -10, -14
16	2.13 s	25.3, CH3	H-6	C-4, -5, -6
17	–	63.3, C	–	–
18	1.57 s	10.2, CH3	–	C-8, -17, -19
19	–	172.0, C	–	–
20	1.19 d (7.2)	9.5, CH3	H-11	C-10, -11, -12
OAc-2	–	170.2, C	–	–
	1.99 s	21.5, CH3	–	Acetate carbonyl
OAc-4	–	170.4, C	–	–
	2.01 s	21.0, CH3	–	Acetate carbonyl
OAc-14	–	170.5, C	–	–
	1.99 s	21.2, CH3	–	Acetate carbonyl
OH-9	2.95 br s	–	H-9	n. o.

a n. o. = not observed.

In an in vitro anti-inflammatory activity assay, Western blot analysis was used to evaluate the upregulation of the pro-inflammatory cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. At a concentration of 10 μM, briarenolides ZII (2) and ZVI (6) were found to significantly reduce the levels of iNOS to 47.2% and 55.7%, respectively, in comparison to the control cells stimulated with LPS only (Figure 4 and Table 7). By using trypan blue staining, it was observed that briarenolides ZI–ZVI (1–6) did not induce significant cytotoxicity in RAW264.7 macrophage cells.

Figure 4

Effects of briarenolides ZI–ZVI (1–6) on pro-inflammatory cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions in lipopolysaccharide (LPS)-stimulated murine macrophage cell line RAW264.7. (A) Relative density of the COX-2 Western blot; (B) relative density of the iNOS Western blot. The relative intensity of the LPS-stimulated group was taken to be 100%. Band intensities were quantified by densitometry and are indicated as the percentage change relative to that of the LPS-stimulated group. Briarenolides ZII (2) and ZVI (6) and DEX significantly inhibited LPS-induced iNOS protein expression (<60%) in macrophages. The experiments were repeated three times (* p < 0.05, significantly different from the LPS-stimulated group).

Table 7

The effect of briarenolides ZI–ZVI (1–6) on LPS-induced COX-2 and iNOS protein expression in macrophage.

Compounds	COX-2	iNOS
	Expression (% of LPS)	Expression (% of LPS)
Control	6.9 ± 2.1	8.7 ± 3.8
LPS	100 ± 0	100 ± 0
ZI (1)	85.1 ± 1.9	91.4 ± 16.6
ZII (2)	89.5 ± 4.0	47.2 ± 7.2
ZIII (3)	83.3 ± 3.3	63.7 ± 12.0
ZIV (4)	65.0 ± 6.4	76.4 ± 13.0
ZV (5)	77.5 ± 6.9	74.0 ± 9.4
ZVI (6)	72.2 ± 3.8	55.7 ± 6.1
DEX a	12.8 ± 0.6	14.2 ± 7.3

a Dexamethasone (DEX) was used as a positive control; COX-2: cyclooxygenase 2; iNOS: inducible nitric oxide synthase; LPS: liposaccharide.

3. Experimental Section

3.1. General Experimental Procedures

Melting points were determined using a Fargo apparatus (Panchum Scientific Corp. Kaohsiung, Taiwan), and the values were uncorrected. Optical rotation values were measured with a Jasco P-1010 digital polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan). IR spectra were obtained with an FT-IR spectrophotometer (Digilab FTS 1000; Varian Inc., Palo Alto, CA, USA); peaks are reported in cm−1. NMR spectra were recorded on a 400-MHz Varian Mercury Plus NMR spectrometer (Varian Inc.) using the residual CHCl3 signal (δH 7.26 ppm) as the internal standard for 1H NMR and CDCl3 (δC 77.1 ppm) for 13C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded using a Bruker 7 Tesla solariX FTMS system (Bruker, Bremen, Germany). Column chromatography was performed using 230–400 mesh silica gel (Merck, Darmstadt, Germany). TLC was carried out on precoated 0.25 mm-thick Kieselgel 60 F254 (Merck); spots were visualized by spraying with 10% H2SO4 solution followed by heating. Normal-phase HPLC (NP-HPLC) was performed using a system equipped with a Hitachi L-7110 pump (Hitachi Ltd., Tokyo, Japan), a Hitachi L-7455 photodiode array detector and an injection port (7725; Rheodyne LLC, Rohnert Park, CA, USA). A semi-preparative normal-phase LiChrospher column (Hibar 250 mm × 10 mm, Si 60, 5 μm, Merck) was used for HPLC. Reverse-phase HPLC (RP-HPLC) was performed with a system equipped with a Hitachi L-7100 pump, a Hitachi L-2455 photodiode array detector, a Rheodyne 7725 injection port and a 25 cm × 10 mm Polaris 5 C-18-A column (5 μm; Varian Inc., Palo Alto, CA, USA).

3.2. Animal Material

Specimens of Briareum sp. were collected by hand by scuba divers in an area off the coast of southern Taiwan in July 2011 and stored in a freezer. A voucher specimen was deposited in the National Museum of Marine Biology & Aquarium (NMMBA-TW-SC-2011-77) [10,11,12,13,14].

3.3. Extraction and Isolation

Sliced bodies of Briareum sp. (wet weight, 6.32 kg; dry weight, 2.78 kg) were extracted with a solvent mixture of methanol (MeOH) and dichloromethane (DCM) (1:1). The extract was partitioned between ethyl acetate (EtOAc) and H2O. The EtOAc layer was separated on silica gel followed by elution chromatography with a mixture of n-hexane/EtOAc (stepwise, 100:1, pure EtOAc) to yield 26 subfractions, A–Z. Fraction V was chromatographed on silica gel and eluted using a mixture of DCM/EtOAc (stepwise, 20:1, pure EtOAc) to afford 14 subfractions, V1–V14. Fraction V9 was separated by NP-HPLC using a mixture of DCM/EtOAc (1:1) to afford 25 subfractions, V9A–V9Y. Fraction V9J was further repurified by RP-HPLC, using a mixture of MeOH/H2O (40:60) as the mobile phase to afford 1 (3.7 mg). Fractions M, N, O and P were combined and further separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 4:1, pure EtOAc) to afford 30 subfractions, M1–M30. Fraction M4 was separated by NP-HPLC, using a mixture of DCM/acetone (40:1) to afford 17 subfractions, M4A–M4Q. Fraction M4B was purified by NP-HPLC, using a mixture of DCM/acetone (100:1) to afford 24 subfractions, M4B1–M4B24. Fraction M4B16 was further separated by RP-HPLC, using a mixture of MeOH/H2O (stepwise, 30/70–70/30) to afford 2 (60:40, 1.7 mg). Fraction M12 was chromatographed by silica gel and eluted using a mixture of DCM/MeOH (stepwise, 100:1, pure MeOH) to afford 34 subfractions, M12-1–M12-34. Fraction M12-31 was purified by RP-HPLC, using a mixture of MeOH/H2O (60:40) to afford 3 (2.7 mg) and 4 (5.0 mg), respectively. Fraction M18 was repurified by NP-HPLC, using a solvent mixture of DCM/acetone (15:1) to obtain 28 subfractions, M18-1–M18-28. Fraction M18-22 was separated by RP-HPLC, using a solvent mixture of MeOH/H2O (1:1) to afford 5 (1.0 mg). Fraction Q was separated on silica gel and eluted using n-hexane/EtOAc (stepwise, 4:1, pure EtOAc) to afford 25 subfractions, Q1–Q25. Fraction Q9 was further separated by reverse-phase C18 column, using a solvent mixture of H2O/MeOH (stepwise, 80:20, pure MeOH) to afford 18 subfractions, Q9A–Q9R. Fraction Q9G was separated on RP-HPLC and eluted with MeOH/H2O (1:1) as the mobile phase to afford 6 (2.0 mg).

Briarenolide ZI (1): white powder; mp 292–293 °C; −31 (c 0.2, CHCl3); IR (neat) νmax 3382, 1769, 1715 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 1); ESIMS: m/z 555 [M + Na]+, 557 [M + 2 + Na]+; HRESIMS: m/z 555.16025 (calcd. for C24H33ClO11 + Na, 555.16036).

Briarenolide ZII (2): white powder; mp 87–88 °C; −20 (c 0.1, CHCl3); IR (neat) νmax 3481, 1781, 1733 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 2); ESIMS: m/z 557 [M + Na]+; HRESIMS: m/z 557.23552 (calcd. for C28H38O10 + Na, 557.23572).

Briarenolide ZIII (3): white powder; mp 173–174 °C; +25 (c 0.1, CHCl3); IR (neat) νmax 3444, 1779, 1732 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 3); ESIMS: m/z 503 [M + Na]+; HRESIMS: m/z 503.18858 (calcd. for C24H32O10 + Na, 503.18877).

Briarenolide ZIV (4): white powder; mp 152–153 °C; +64 (c 0.3, CHCl3); IR (neat) νmax 3444, 1778, 1732 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 4); ESIMS: m/z 575 [M + Na]+; HRESIMS: m/z 575.24645 (calcd. for C28H40O11 + Na, 575.24628).

Briarenolide ZV (5): white powder; mp 192–193 °C; +15 (c 0.1, CHCl3); IR (neat) νmax 3445, 1770, 1732 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 5); ESIMS: m/z 505 [M + Na]+; HRESIMS: m/z 505.20460 (calcd. for C24H34O10 + Na, 505.20442).

Briarenolide ZVI (6): white powder; mp 173–174 °C; +70 (c 0.3, CHCl3); IR (neat) νmax 3446, 1772, 1734 cm−1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data (see Table 6); ESIMS: m/z 547 [M + Na]+; HRESIMS: m/z 547.21473 (calcd. for C26H36O11 + Na, 547.21498).

3.4. In Vitro Anti-Inflammatory Assay

The murine macrophage (RAW264.7) cell line was purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). The in vitro anti-inflammatory activities of Compounds 1−6 were measured by examining the inhibition of LPS-induced upregulation of pro-inflammatory iNOS and COX-2 protein expressions in the macrophage cell line using Western blotting analysis [15,16,17]. Briefly, an inflammation response in macrophages was induced by incubating cells in medium containing only LPS (10 ng/mL) without compounds for 16 h. For the anti-inflammatory activity assay, Compounds 1−6 and dexamethasone (10 μM) were added to the cells 10 min before LPS treatment. After incubation, the cells were lysed for Western blot analysis. The immunoreactivity data were calculated with respect to the average optical density of the corresponding (LPS)-stimulated group. Moreover, the effects of Compounds 1−6 on the viability of RAW 264.7 cells were also evaluated by trypan blue staining [16,17]. For statistical analysis, the data were analyzed by one-way analysis of variance (ANOVA), followed by the Student–Newman–Keuls post hoc test for multiple comparisons. A significant difference was defined as a p-value of <0.05.

4. Conclusions

Gorgonian corals belonging to the genus Briareum are proven to be rich sources of briarane-type compounds. Briarenolides ZII (2) and ZVI (6) are potentially anti-inflammatory compounds for future development [18,19]. This interesting species was transplanted to culture tanks located in the NMMBA, for extraction of natural material to establish a stable supply of bioactive substances.