Chemistry and Bioactivity of Briaranes from the South China Sea Gorgonian Dichotella gemmacea

Abstract

Seven new briarane diterpenoids, gemmacolides AZ–BF (1–7), were isolated together with eight known analogues (8–15) from the South China gorgonian Dichotella gemmacea. Their structures were elucidated based on detailed spectroscopic analysis and a comparison with reported data. In an in vitro bioassay, these compounds exhibited different levels of growth inhibition activity against A549 and MG63 cells, giving continuous evidences about the biological contribution of functional groups at C-2, C-12, C-13, and C-16. These compounds were also evaluated for their antibacterial and antifungal activities. Compound 8 exhibited a potential antibacterial activity against both Gram-positive bacterium Bacillus megaterium and Gram-negative bacterium Escherichia coli.

1. Introduction

Briarane diterpenoids are a family of structurally intriguing metabolites that are mainly obtained from gorgonians [1]. These metabolites are reported to display a wide spectrum of bioactivities, including cytotoxic, anti-inflammatory, antiviral, antifouling, insecticidal, and immunomodulatory effects [1,2,3,4]. In the course of our searching for novel and bioactive secondary metabolites from the South China Sea invertebrates [5,6,7,8,9,10,11,12], the cluster of metabolites attracted our attention due to their significant tumor tell growth inhibitory activity [7,8,9,10,11,12]. Considering the structural complexity of the metabolites, specific research regarding the determination of absolute configuration was carried out using a solution TDDEFT/ECD approach [12]. Successive investigation on several collections of the gorgonian Dichotella gemmacea and Junceella gemmacea led to the isolation and structure elucidation of 48 new briaranes and 29 known analogues. On the basis of in vitro screening, a structure–activity relationship analysis of the metabolites was performed [7,8,9,10,11,12]. Three new briaranes, gemmacolides J, V, and Y, showed significant growth inhibitory activity towards A549 cells, being more active than the positive control adriamycin [10,11]. The initial research encourages continuous investigations of this class of metabolites, leading to the isolation and structure elucidation of seven new briaranes, namely gemmacolides AZ–BF (1–7), together with eight known analogues (8–15) [13,14,15,16,17,18,19] (Chart 1) from the gorgonian D. gemmacea. The structures of these compounds were elucidated on the basis of detailed spectroscopic analysis (Figures S1–S47) and a comparison with reported data. The isolates were tested in vitro for their antimicrobial and tumor cell growth inhibition activities. We herein report on the isolation, structure elucidation, and bioactivities of these compounds.

2. Results

Freshly collected specimens of D. gemmacea were immediately frozen to −20 °C and stored at this temperature before extraction. The usual workup for the extraction and isolation of briarane diterpenoids [8,9,10,11,12] yielded 15 pure compounds (1–15). The known compounds dichotelllides O and M (8, 9) were once reported from the gorgonian D. gemmacea [13], while gemmacolide C (12) [18] was previously isolated from the gorgonians J. gemmacea. Juncins P (10) [14] and ZI (11) [16] were firstly isolated from the gorgonian J. juncea, and then repeatedly obtained from the gorgonians D. gemmacea [13,20,21], D. fragilis [22], J. gemmacea [7], and J. fragilis [23]. Junceellolide D (13) [17], (−)-4-deacetoxy junceellolide D (14) [15], and junceellolide K (15) [19] were firstly isolated from the gorgonians J. fragilis and then re-isolated from many gorgonian corals, including Verrucella umbraculum [24], J. fragilis [23,25,26,27], D. gemmacea [8,13,20], J. gemmacea [7], Menella sp. [28], and J. juncea [14]. These metabolites displayed antifouling, anti-inflammatory, and cytotoxic activities in the in vitro bioscreening [13,16,21,22,29].

Gemmacolide AZ (1) was isolated as a white amorphous powder. The molecular formula C₃₁H₄₂O₁₃ was established by the HRESIMS. The IR spectrum showed strong absorption bands of hydroxyl (3468 cm⁻¹), γ-lactone (1775 cm⁻¹), and ester (1738 cm⁻¹) functionalities. This observation was in agreement with the signals in the ¹³C NMR and DEPT spectra (

Table 1. ¹³C NMR data for gemmacolides AZ–BF (1–7) ^a.

Position	1	2	3	4	5	6	7
1	47.2, C	46.5, C	46.5, C	46.5, C	46.5, C	46.3, C	46.5, C
2	75.8, CH	75.6, CH	76.9, CH	74.2, CH	75.4, CH	74.4, CH	74.1, CH
3	131.5, CH	131.2, CH	130.5, CH	131.3, CH	130.5, CH	131.2, CH	132.2, CH
4	129.2, CH	129.6, CH	130.2, CH	128.6, CH	129.4, CH	127.8, CH	127.7, CH
5	144.7, C	144.5, C	144.6, C	141.7, C	141.5, C	138.9, C	139.0, C
6	123.4, CH	123.7, CH	124.4, CH	122.7, CH	122.6, CH	122.4, CH	122.3, CH
7	78.9, CH	78.7, CH	78.7, CH	79.0, CH	79.0, CH	78.7, CH	78.5, CH
8	81.2, C	81.0, C	81.1, C	81.1, C	81.0, C	81.0, C	81.1, C
9	64.1, CH	63.8, CH	63.8, CH	63.8, CH	63.9, CH	63.9, CH	64.0, CH
10	32.9, CH	32.8, CH	32.6, CH	32.7, CH	32.7, CH	32.7, CH	31.5, CH
11	59.2, C	58.4, C	58.3, C	58.4, C	58.2, C	58.7, C	60.4, C
12	73.0, CH	72.8, CH	73.1, CH	73.3, CH	72.8, CH	76.5, CH	75.3, CH
13	29.0, CH2	66.2, CH	66.2, CH	66.5, CH	66.4, CH	67.4, CH	67.3, CH
14	73.2, CH	73.8, CH	73.8, CH	73.4, CH	73.8, CH	76.6, CH	76.1, CH
15	14.2, CH3	14.5, CH3	14.4, CH3	14.5, CH3	14.4, CH3	14.8, CH3	14.6, CH3
16	63.9, CH2	63.9, CH2	63.9, CH2	72.1, CH2	71.9, CH2	62.8, CH2	62.8, CH2
17	44.2, CH	44.1, CH	44.1, CH	44.2, CH	44.2, CH	44.1, CH	44.0, CH
18	6.4, CH3	6.3, CH3	6.3, CH3	6.3, CH3	6.3, CH3	6.3, CH3	6.4, CH3
19	175.4, C	175.3, C	175.7, C	175.3, C	175.3, C	175.3, C	175.4, C
20	49.1, CH2	49.1, CH2	49.0, CH2	49.1, CH2	49.0, CH2	48.9, CH2	48.2, CH2
9-OAc	170.3, C	170.2, C	170.1, C	170.2, C	170.2, C	170.2, C	170.2, C
	21.6, CH3	21.6, CH3	21.5, CH3	21.5, CH3	21.5, CH3	21.5, CH3	21.6, CH3
R1	170.5, C	170.9, C	167.8, C	169.5, C	166.6, C	169.6, C	169.8, C
	21.3, CH3	21.6, CH3	61.0, CH2	20.6, CH3	60.8, CH2	21.3, CH3	21.3, CH3
			172.3, C		172.3, C
			42.7, CH2		42.7, CH2
			25.5, CH		25.6, CH
			22.3, 2 × CH3		22.5, 2 × CH3
R2	170.1, C	171.8, C	169.8, C	169.9, C	171.8, C	171.8, C
	21.3, CH3	43.5, CH2	20.8, CH3	21.1, CH3	43.5, CH2	21.0, CH3
		25.7, CH			25.7, CH
		22.4, 2 × CH3			22.5, 2 × CH3
R3		171.7, C	171.2, C	169.7, C	169.6, C		172.0, C
		42.6, CH2	42.6, CH2	21.3, CH3	20.5, CH3		42.8, CH2
		25.0, CH	25.0, CH				25.2, CH
		22.4, 2 × CH3	22.3, 2 × CH3				22.3, 2 × CH3
R4	172.0, C	170.1, C	170.1, C	172.3, C	170.2, C	173.7, C	169.8, C
	42.9, CH2	20.9, CH3	20.8 CH3	43.3, CH2	20.8, CH3	43.3, CH2	20.9, CH3
	24.8, CH			25.0, CH		25.6, CH
	22.6; 22.4, CH3			22.5, 2 × CH3		22.4, 2 × CH3
R5				58.5, CH3	58.5, CH3	172.1, C	172.0, C
						43.3, CH2	43.2, CH2
						25.7, CH	25.7, CH
						22.4, 2 × CH3	22.4, 2 × CH3

^a 100 MHz, in CDCl₃, assignments made by DEPT, ¹H–¹H COSY, HSQC, and HMBC.

) for 9 sp² carbon atoms (5 × OC = O, CH = CH, CH = C) at lower field and 22 sp³ carbon atoms at higher field (1 × C, 3 × CH, 2 × CH₂, 7 × CH₃, 2 × OC, 5 × OCH, 2 × OCH₂), accounting for seven double bond equivalents. The remaining double bond equivalents were due to the presence of four rings in the molecule.

The ¹H and ¹³C NMR spectra data (Table 1 and

Table 2. ¹H NMR data for gemmacolides AZ–BC (1–4) ^a.

Position	1	2	3	4
2	5.68, d (9.5)	5.62, d (9.4)	5.71, d (9.3)	5.52, d (9.5)
3	5.60, dd (10.6, 9.5)	5.55, dd (9.5, 9.4)	5.63, dd (9.9, 9.3)	5.56, dd (9.8, 9.5)
4	6.33, d (10.6)	6.35, d (9.5)	6.39, d (9.9)	6.28, d (9.8)
6	5.82, d (8.5)	5.81, d (8.5)	5.84, d (8.6)	5.88, d (8.5)
7	4.98, ov	4.96, d (8.5)	4.95, d (8.6)	4.96, d (8.5)
9	4.80, br d (4.7)	4.74, br d (4.8)	4.74, br d (4.7)	4.74, br d (4.5)
10	3.66, br d (4.5)	3.60, br d (4.8)	3.62, ov	3.61, ov
12	4.54, br s	4.93, br d (2.3)	4.90, br d (2.4)	4.89, br s
13β	2.25, ov	5.11, t (3.4)	5.09, t (3.0)	5.10, br s
13α	1.96, ov
14	4.99, br s	5.23, br d (2.4)	5.19, br d (2.2)	5.25, br s
15	1.06, s	1.14, s	1.13, s	1.13, s
16a	4.49, br s	4.47, br s	4.49, d (16.0)	4.49, d (14.9)
16b	4.49, br s		4.42, d (16.0)	4.22, d (14.9)
17	2.31, ov	2.30, ov	2.30, ov	2.29, ov
18	1.16, d (7.0)	1.13, d (7.2)	1.14, d (7.1)	1.15, ov
20a	3.55, br d (2.0)	3.62, br d (2.3)	3.62, br s	3.61, br s
20b	2.79, br d (2.0)	2.95, br d (2.3)	2.92, br s	2.92, br s
9-OAc	2.19, s	2.20, s	2.19, s	2.19, s
R1	1.98, s	1.99, s	4.55, d (15.7)	1.95, s
			4.44, d (15.7)
			2.28, ov (×2)
			2.13, ov
			0.98, d (6.6) (×2)
R2	2.12, s	2.32, m	2.16, s	2.16, s
		2.26, m
		2.17, m
		1.01, d (6.2)
		0.99, d (6.2)
R3		2.07, m (×2)	2.08, ov (×2)	1.95, s
		2.00, m	1.98, m
		0.92, d (6.6) (×2)	0.92, d (6.5) (×2)
R4	2.25, ov,	2.09, s	2.09, s	2.28, ov,
	2.07, ov			2.15, ov
	2.15, ov			2.11, ov
	0.97, d (6.3)			1.00, d (6.5)
	0.94, d (6.4)			0.97, d (6.5)
R5				3.44, s

^a 400 MHz, in CDCl₃, assignments made by ¹H–¹H COSY, HSQC, HMBC, and NOESY.

) of 1 showed great similarity to those of gemmacolide AQ (16) [9], except for the absence of an acetoxy group. The missing group was readily assigned at C-13 due to the proton sequence of H-12/H₂-13/H-14, established by ¹H–¹H COSY experiment (Figure 1). The assignment was fully supported by the ¹H–¹H COSY and HMBC spectra as shown in Figure 1. The relative configuration of 1 at chiral centers was proven the same as that of 16 by a NOESY experiment (Figure 2), showing a β configuration of H-7, H-12, H-14, Me-15, H-17, and CH₂-20, and an α configuration of H-2, H-9, H-10, and Me-18. The geometry of the Δ³ double bond was assigned as (Z) based on the proton coupling constant between H-3 and H-4 (J = 10.6 Hz), while Δ^5,6 was determined as (E) due to the NOESY correlation between H-6 and H₂-16. The NMR assignments of 1 were further supported by a comparison with the reported data of gemmacolide N (17), an analogue obtained from the same species of animals with its absolute stereochemistry being determined by a detailed TDDFT/ECD analysis [12]. Compound 17 differs from 1 in the functional groups at C-14 and C-16, having acetoxy and primary hydroxyl groups instead of isovaleryl and methoxy groups, respectively. As compound 1 contained the same lactone and diene chromophores as gemmacolide N [12], and they differed only in the nature of the ester groups and R₅, the ECD spectrum of gemmacolide N could be used as ECD reference for the configurational assignment. The absolute configuration of 1 is therefore determined as (1R,2S,7S,8S,9S,10S,11R,12R,14S,17R).

Gemmacolide BA (2), a white amorphous powder, had the molecular formula of C₃₆H₅₀O₁₅ on the basis of its HRESIMS. Its ¹H and ¹³C NMR spectra data (Table 1 and Table 2) were similar to those of gemmacolide AQ (16) [9]. However, an acetyl group in 16 was replaced by an isovaleryl group in 2. The three acetyl groups were assigned at C-2, C-9, and C-14 due to the obvious HMBC correlations from the secondary alcohol protons to the respective ester carbonyl groups. The location of two isovaleryl groups at C-12 and C-13 was supported by the obvious HMBC correlations from H-12 and H-13 to the respective carbonyl carbon of the isovaleryl groups. The relative configuration and the absolute configuration of 2 were proven to be the same as that of 16 on the basis of NOESY experiment and their similar ECD spectra.

Gemmacolide BB (3) was obtained as a white amorphous powder with the molecular formula of C₃₈H₅₂O₁₇ being established by HRESIMS. The ¹H and ¹³C NMR spectra of 3 showed similarity to those of compound 2 (Table 1 and Table 2). The structure of 3 differed from that of 2 with the presence of an isovaleric acetyl group instead of an acetyl group at C-2 and an acetyl group replace of the isovaleryl group at C-12 (Table 1 and Table 2). The conclusion was fully supported by the distinct HMBC correlations from H-2 to the carbonyl carbons of the isovaleric acetyl group at δ 167.8, and from H-12 to the acetyl ester carbon at δ 169.8, respectively. Its absolute configuration was proven to be the same as that of 2 on the basis of their similar ECD spectra.

Gemmacolide BC (4) was obtained as a white amorphous powder. Its HRESIMS demonstrated the same molecular formula as C₃₄H₄₆O₁₅ by HRESIMS. Comparison of overall ¹H and ¹³C NMR spectra data (Table 1 and Table 2) of 4 with those of gemmacolide AQ (16) [9] revealed great similarity with the only difference of an appearance of an oxygenated methyl group at C-16 (δ_H 3.44, s; δ_C 58.5, CH₃). The result was supported by the obvious HMBC correlations from the methoxyl protons to C-16. The absolute structure of 4 was proven to be the same as that of 16 by the analysis of NOESY and ECD spectra.

Gemmacolide BD (5) was obtained as a white amorphous powder and exhibited a molecular formula of C₃₉H₅₄O₁₇ as deduced from its HRESIMS. ¹H and ¹³C NMR spectra of 5 (Table 1 and

Table 3. ¹H NMR data for gemmacolides BD–BF (5–7) ^a.

Position	5	6	7
2	5.64, d (9.6)	5.62, ov	5.66, ov
3	5.60, dd (10.3, 9.6)	5.61, ov	5.61, dd (10.4, 9.9)
4	6.33, d (10.3)	6.29, d (8.1)	6.28, d (10.4)
6	5.88, d (8.4)	5.71, d (8.3)	5.66, ov
7	4.98, d (8.4)	4.98, d (8.3)	4.98, ov
9	4.75, br d (4.5)	4.72, ov	4.78, br d (4.8)
10	3.62, br d (4.5)	3.59, br d (4.9)	3.56, br s
12	4.91, br d (2.2)	4.74, ov	3.48, br d (4.7)
13β	5.08, t (3.3)	4.07, br s	4.99, ov
13α
14	5.18, br d (2.6)	5.22, br s	5.33, br s
15	1.13, s	1.09, s	1.13, s
16a	4.52, d (15.3)	5.43, d (16.0)	5.46, d (16.6)
16b	4.11, d (15.3)	4.64, d (15.4)	4.55, d (15.6)
17	2.30, ov	2.29, ov	2.28, ov
18	1.14, ov	1.14, d (7.1)	1.16, d (7.2)
20a	3.59, br d (2.1)	3.60, br d (2.2)	3.56, br s
20b	2.92, br d (2.1)	2.87, br d (2.2)	2.78, br s
9-OAc	2.18, s	2.18, s	2.19, s
R1	4.52, d (15.7)	1.98, s	1.94, s
	4.43, d (15.7)
	2.29, ov (×2)
	2.11, ov
	0.98, ov (×2)
R2	2.19, ov,	2.10, s	3.31, br d (4.7)
	2.33, ov
	2.15, ov
	0.98, ov
	0.98, ov
R3	1.94, s	3.01, br s	2.16, ov (×2)
			2.06, ov
			0.96, d (6.5)
			0.94, d (6.5)
R4	2.05, s	2.32, ov (×2)	2.13, s
		2.14, ov
		0.99, ov (×2)
R5	3.44, s	2.32, ov (×2)	2.30, ov (×2)
		2.14, ov	2.13, ov
		0.99, ov (×2)	0.99, ov (×2)

^a 400 MHz, in CDCl₃, assignments made by ¹H–¹H COSY, HSQC, HMBC, and NOESY.

) were very similar to those of compound gemmacolide AB (18) [9]. The primary alcohol of the hydroxyacetyl group, however, was further isovalerianated. This conclusion was proved by the long-range correlation from secondary hydrogens of both the isovaleryl group and the hydroxyacetyl group to the carbonyl carbon of the isovaleryl group (δ 172.3), and from both H-2 and the secondary hydrogens of the hydroxyacetyl group to the carbonyl carbon of the hydroxyacetyl group (δ 166.6). The same absolute geometry was obtained on the basis of the ECD spectrum.

Gemmacolide BE (6) was found to be a white amorphous powder, having the molecular formula of C₃₆H₅₀O₁₅ based on the HRESIMS. ¹H and ¹³C NMR spectra (Table 1 and Table 3) of 6 resembled to those of gemmacolide AS (19) [8] except for a hydrogen at C-13 in 19 was replaced by a hydroxyl group in 6. The assignment was in agreement with its NMR shift values (δ_H-13 4.07, br s; δ_C-13 67.4, CH), compared to those of the ester group substitution in gemmacolide P, gemmacolide AC, gemmacolide AD, gemmacolide AF, gemmacolide AG, gemmacolide AR, and gemmacolide AT (δ_H-13 5.08–5.10, d, J = 2.7–3.5; δ_C-13 66.3–66.7, CH) [8,9,12], and further supported by the proton sequences from H-12 to H-14 established by the ¹H–¹H COSY experiment. The hydroxyl group was assigned as an α-orientation due to the NOESY correlation of H-13 with H-15. The two isovaleryl groups were deduced to be attached to C-14 and C-16 based on the 2D NMR (¹H–¹H COSY, HMBC) analysis and a comparison to those reported data of analogues [7,8,9,10,11,12]. The relative and absolute configuration of 6 was also proven to be the same as those of 19 by the NOESY and ECD experiments.

Gemmacolide BF (7) was a white amorphous powder and had the same molecular formula of C₃₆H₅₀O₁₅ as that of 6 as deduced from its HRESIMS. The structure of 7 differed from that of 6 only in the sequence of substituent groups. The hydroxyl, isovaleryl, and acetoxy groups at C-13, C-14, and C-12 in 6 were instead assigned at C-12, C-13, and C-14 in 7, respectively. The location of hydroxyl at C-12 was supported by ¹H and ¹³C NMR spectra data (δ_H-12 3.48, br d, J = 4.7; δ_C-12 75.3, CH), compared to those of ester group substitution (δ_H-12 4.88–4.93, br d, J = 2.8–3.5; δ_C-12 72.8–73.3, CH) [8,9,12]. A β-orientation of H-12 was deduced from its NOESY correlation with H-20b. Two isovaleryl groups were attached at C-13 and C-16 due to the HMBC correlations of H-13 and H-16 with the respective carbonyl carbon of the isovaleryl groups. The assignment was supported by the proton sequence of H-12/H-13/H-14, as deduced from the ¹H–¹H COSY experiment. The established structure of 7 was further supported by a detailed analysis of its 1D NMR and 2D NMR data. Its absolute configuration was determined as (−)-(1S,2S,3Z,5E,7S,8S,9S,10S,11R,12R,13S,14R,17R) based on NOESY and ECD experiments.

The compounds were evaluated for their antimicrobial and tumor cell growth inhibition activities. In the in vitro bioassays, compounds 1–9 exhibited a different level of growth inhibition against cell lines A549 and MG63 (

Table 4. Cytotoxic assay for compounds 1–9 (IC₅₀ μM).

Compound	A549	MG63	Compound	A549	MG63
1	28.3	72.0	6	25.8	30.6
2	24.7	15.8	7	13.7	34.8
3	34.1	43.2	8	25.5	36.8
4	26.8	11.4	9	>40.8	>40.8
5	>37.8	>37.8	Adriamycin	2.8	3.2

). The activity of 5 with 2-isovaleric acetyl substitution marked decreased compared with its 2-glycolyl analogue (IC₅₀ for A549 = 19.4 μM, for MG63 = 22.8 μM) [9]. The previous conclusion that 13-isovalerate may decrease the activity [9] was further supported by comparing the activity of 1 and 9 with those of 16 (IC₅₀ = 28.7 and > 100 μM for A549 and MG63, respectively) [9] and 8, respectively. Comparing the activity of 2 and 8 with respect to those of gemmacolides AT (IC₅₀ > 30.0 μM for both A549 and MG63) [8] and N (IC₅₀ > 50.5 μM for both A549 and MG63) [12] supported the conclusion that 12-O-isovalerate gave a positive contribution to the activity as mentioned previously [9,11,12]. The replacement of an isovaleryl group by an acetyl group at C-14 would increase the activity as observed in 4 and juncenolide D [12]. The 16-substituents displayed an activity order as 16-OH > 16-OMe when the activity of 2 and 3 was compared with those of 9 (IC₅₀ > 30.0 μM for both A549 and MG63) and gemmacolide AL (IC₅₀ > 37.8 μM for both A549 and MG63) [9], respectively.

In antimicrobial test in vitro, compounds 4 and 8 showed potential antibacterial activity against the Gram-negative bacterium Escherichia coli, while 8 exhibited significant antibacterial activity against the Gram-positive bacterium Bacillus megaterium. Compounds 1, 2, 4, 5, and 8 exhibited weak antifungal activity against both Septoria tritici and Microbotryum violaceum, while compound 9 only displayed weak activity against S. tritici (

Table 5. Agar diffusion assays for antibacterial and antifungal activities ^a,b.

	E. coli	B. megaterium	M. violaceum	S. tritici
1	16.0	8.0	10.0	19.0
2	14.0	11.0	11.0	11.0
3	/	/	/	/
4	18.0	11.0	11.0	17.0
5	11.0	11.0	13.0	13.0
6	/	/	/	/
7	/	/	/	/
8	21.0	19.0	11.0	14.0
9	14.0	0	0	14.0
ketoconazole	18.0	18.0	30.0	25.0
penicillin	26.0	18.0	14.0	12.0
streptomycin	18.0	11.0	16.0	11.0

^a 0.05 mg of the test or control substances dissolved in acetone were applied to a filter disc and sprayed with the respective test organism; ^b Radii of the zones of inhibition are given in mm. “/” not tested.

).

3. Materials and Methods

3.1. General Experimental Procedures

Commercial silica gel (Yantai, China, 200–300; 400–500 mesh) and RP silica gel (Merck, Darmstadt, Germany, 43–60 μm) were used for column chromatography (CC). Precoated silica gel plates (Yantai, China, HSGF-254) and RP silica gel (Macherey-Nagel, Düren, Germany, RP-18 F254) were used for analytical thin-layer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with an anisaldehyde-sulphuric acid reagent. The NMR spectra were recorded at 300 K on a Bruker DRX 400 spectrometer (Ettlingen, Germany). Chemical shifts are reported in parts per million (δ), with use of the residual CHCl₃ signal (δ_H 7.26 ppm) as an internal standard for ¹H NMR and CDCl₃ (δ_C 77.0 ppm) for ¹³C NMR; Coupling constants (J) are reported in Hz. ¹H NMR and ¹³C NMR assignments were complemented ¹H–¹H COSY, HSQC, HMBC, and NOESY experiments. The following abbreviations are used to describe spin multiplicity: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet; br s = broad singlet; dd = doublet of doublets; ov = overlapped signals. Optical rotations were measured in CHCl₃ with an Autopol IV polarimeter at the sodium D line (590 nm). Infrared spectra were recorded in thin polymer films on a Nexus 470 FT-IR spectrophotometer (Nicolet, Madison, WI, USA); peaks are reported in cm⁻¹. UV absorption spectra were recorded on a Varian Cary 100 UV-Vis spectrophotometer (Palo Alto, CA, USA); peaks wavelengths are reported in nm. Circular dichroism spectra were recorded with a JASCO J-715 circular dichroism spectropolarimeter (Mary’s Court Easton, MD, USA). The MS and HRMS were performed on a Q-TOF Micro mass spectrometer (Bruker, Bremen, Germany), resolution 5000. An isopropyl alcohol solution of sodium iodide (2 mg/mL) was used as a reference compound. Semi-preparative RP-HPLC was performed on an Agilent 1100 system (Santa Clara, CA, USA) equipped with a refractive index detector using an YMC Pack ODS-A column (Kyoto, Japan, particle size 5 μm, 250 mm × 10 mm).

3.2. Animal Material

The South China Sea gorgonian coral Dichotella gemmacea (3.5 kg, wet weight) was collected from the South China Sea in August 2007 and identified by Xiu-Bao Li, South China Sea Institute of Oceanology, Chinese Academy of Sciences. A voucher specimen (ZS-3) was deposited in the Second Military Medical University.

3.3. Extraction and Isolation

The frozen specimen was extracted ultrasonically three times with acetone and MeOH, respectively. The combined residue was partitioned between H₂O and EtOAc to afford 16.1 g of an EtOAc extract. The EtOAc extract was further partitioned between MeOH and hexane, affording 11.2 g of MeOH soluble residue. The MeOH extract was subjected to column chromatography (CC) on silica gel to give 16 fractions, using hexane and acetone (from 100:0 to 0:100) as an eluent. Fraction 5 was repeatedly subjected to normal phase silica gel column chromatography, Sephadex LH-20, and RP-silical gel column chromatography, then purified via HPLC (eluent MeOH/H₂O, 75:25, 1.5 mL/min), yielding 5 (3.4 mg) at 29.6 min. Fraction 6 was chromatographed over Sephadex LH-20 to give four sub-fractions (A–D) eluted with CHCl₃ and MeOH (1:1). Sub-fractions A was purified via HPLC (eluent MeOH/H₂O, 70:30, 1.5 mL/min), yielding 2 (2.0 mg) at 34.0 min. Sub-fractions B was purified via HPLC (eluent MeOH/H₂O, 70:30, 1.5 mL/min), yielding 6 (1.4 mg) and 7 (1.6 mg) at 64.3 and 67.9 min. Sub-fractions C was purified via HPLC (eluent MeOH/H₂O, 70:30, 1.5 mL/min), yielding 3 (1.0 mg) at 62.3 min. Fraction 10 was repeatedly chromatographed over normal phase silica gel column and Sephadex LH-20 and purified via HPLC (eluent MeOH/H₂O, 70:30, 1.5 mL/min), yielding 4 (3.4 mg) at 51.7 min. Fraction 13 was further fractionated by RP-silical gel column chromatography (gradient elution from MeOH/H₂O, 2:7 to 2:1, in 5% increments) to afford 6 sub-fractions (A–F), and sub-fraction E was purified via HPLC (eluent MeOH/H₂O, 65:35, 1.5 mL/min), yielding 1 (2.3 mg) at 29.7 min.

Gemmacolide AZ (1): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −1.3 (c 0.07, CHCl₃); UV (MeOH) λ_max(log ε) 205 (1.71) nm; CD (CH₃CN, c 2.0 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 209 (−5.50) nm; IR (film) ν_max 3468, 1775, 1738 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 645 [M + Na]⁺; HRESIMS m/z 645.2527 [M + Na]⁺ (calcd. for C₃₁H₄₂O₁₃ Na, 645.2523).

Gemmacolide BA (2): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −43.0 (c 0.07, CHCl₃); UV (MeOH) λ_max(log ε) 208 (2.26) nm; CD (CH₃CN, c 4.2 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 209 (−4.77) nm; IR (film) ν_max 3469, 1775, 1744 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 745 [M + Na]⁺; HRESIMS m/z 745.3041 [M + Na]⁺ (calcd. for C₃₆H₅₀O₁₅Na, 745.3047).

Gemmacolide BB (3): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −28 (c 0.03, CHCl₃); UV (MeOH) λ_max(log ε) 204(1.47) nm; CD (CH₃CN, c 1.6 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 205 (−5.36) nm; IR (film) ν_max 3469, 1772, 1744 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 803 [M + Na]⁺; HRESIMS m/z 803.3107 [M + Na]⁺ (calcd. for C₃₈H₅₂O₁₇Na, 803.3102).

Gemmacolide BC (4): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −44.0 (c 0.24, CHCl₃); UV (MeOH) λ_max(log ε) 205 (1.55) nm; CD (CH₃CN, c 4.1 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 201.5 (−4.62) nm; IR (film) ν_max 3479, 1776, 1743 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 717 [M + Na]⁺; HRESIMS m/z 717.2730 [M + Na]⁺ (calcd. for C₃₄H₄₆O₁₅Na, 717.2734).

Gemmacolide BD (5): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −36.0 (c 0.12, CHCl₃); UV (MeOH) λ_max(log ε) 205 (1.61) nm; CD (CH₃CN, c 2.0 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 216 (−7.34) nm; IR (film) ν_max 3461, 1775, 1744 cm⁻¹; ¹H NMR spectroscopic data, see Table 3; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 817 [M + Na]⁺; HRESIMS m/z 817.3254 [M + Na]⁺ (calcd. for C₃₉H₅₄O₁₇Na, 817.3259).

Gemmacolide BE (6): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −22.0 (c 0.05, CHCl₃); UV (MeOH) λ_max(log ε) 204 (1.41) nm; CD (CH₃CN, c 3.5 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 198.0 (−5.43) nm; IR (film) ν_max 3467, 1776, 1741 cm⁻¹; ¹H NMR spectroscopic data, see Table 3; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 745 [M + Na]⁺; HRESIMS m/z 745.3043 [M + Na]⁺ (calcd. for C₃₆H₅₀O₁₅Na, 745.3047).

Gemmacolide BF (7): white amorphous powder; ${[\mathsf{\alpha }]}_{\mathrm{D}}^{24}$ −30.0 (c 0.05, CHCl₃); UV (MeOH) λ_max(log ε) 204 (1.53) nm; CD (CH₃CN, c 1.7 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 198.0 (−10.59) nm; IR (film) ν_max 3469, 1777, 1742 cm⁻¹; ¹H NMR spectroscopic data, see Table 3; ¹³C NMR spectroscopic data, see Table 1; ESIMS m/z 745 [M + Na]⁺; HRESIMS m/z 745.3051 [M + Na]⁺ (calcd. for C₃₆H₅₀O₁₅Na, 745.3051).

3.4. Cytotoxicity Assay

Cytotoxicity was tested against human lung adenocarcinoma (A549) and human osteosarcoma cell (MG63), using a modification of the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method [30]. DMSO was used as a vehicle control, and adriamycin was used as a positive control, IC₅₀ = 2.8 μM for A549 cells and 3.2 μM for MG63 cells.

3.5. Agar Diffusion Test for Biological Activity

Compounds 1–9 were dissolved in acetone at a concentration of 2 mg/mL; 25 μL of the solution (0.05 mg) were pipetted onto a sterile filter disc (Schleicher & Schuell, Dassel, Germany, 9 cm), which was placed onto an appropriate agar growth medium for the respective test organism and subsequently sprayed with a suspension of the test organize. The test organisms were the Gram-negative bacterium Escherichia coli (Coli), the Gram-positive bacterium Bacillus megaterium (Meg) (both grown on NB medium), the fungus Microbotryum violaceum (Vio), and Septoria tritici (Tri) (both grown on MPY medium). Commencing at the outer edge of the filter disc, the diameter of zone of inhibition was measured in mm. Reference substances were ketoconazole (0.05 mg), penicillin (0.05 mg), and streptomycin (0.05 mg), with the diameter (Ф) of zone of inhibition being 18.0, 18.0, 30.0, 25.0; 26.0, 18.0, 14.0, 12.0; and 18.0, 11.0, 16.0, 11.0 mm, respectively.