Briarane Diterpenoids from the Gorgonian Dichotella gemmacea

Abstract

Seven new briarane diterpenoids, gemmacolides AS-AY (1–7), were isolated together with ten known analogues (8–17) from the South China Sea gorgonian Dichotella gemmacea. The structures of the new compounds were elucidated by the detailed analysis of spectroscopic data and comparison with reported data. The absolute configuration of compounds was determined based on electronic circular dichroism (ECD) experiments and genetic correlations as well. Compounds 15 and 16 were reported for the first time for the gorgonian. In the preliminary in vitro bioassays, compound 5 showed potential growth inhibitory activity against MG63 cells.

1. Introduction

Gorgonian corals of the family Ellisellidae are proven to be rich source of briarane diterpenoids that are well known for displaying a wide spectrum of bioactivities, including cytotoxic, anti-inflammatory, antiviral, antifouling, insecticidal and immunomodulatory effects [1,2,3,4]. In the course of searching for novel and bioactive secondary metabolites from marine organisms of the South China Sea [5,6], the tumor cell growth inhibitory activity of briarane diterpenoids attracted our attention, leading to the isolation of a series of briarane-type diterpenes the gorgonians Dichotella gemmacea and Junceella gemmacea [7,8,9,10,11].

Chart 1. Structures for compounds 1–20.

Chart 1. Structures for compounds 1–20.

In particular, gemmacolides J, V and Y showed potential activity against A549 cells, being more active than the positive control adriamycin [9,10]. These results encourage subsequent studies of this class of metabolites, leading to the new collection of gorgonian D. gemmacea, a promising source of briarane diterpenoids. Chemical investigation on the acetone extract of these animals resulted in the isolation and structure elucidation of seven new briaranes, namely gemmacolides AS–AY (1–7), together with ten known analogues, gemmacolide L (12), gemmacolide X (13), gemmacolide AH (11), gemmacolide AQ (8), gemmacolide AJ (9), gemmacolide AO (10), Junceellolide C (17), junceellolide D (14), junceellin (15), and frajunolide K (16) [8,9,10,12,13,14] (Chart 1). The structures of these compounds were elucidated by detailed analysis of spectroscopic data and comparison with the reported analytical data for the known compounds. The isolates were tested in vitro for their tumor cell growth inhibitory activity. We report here the isolation, structure elucidation, and biological activity of these new compounds.

2. Results and Discussion

Freshly collected specimens of Dichotella 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 [7,8,9,10,11] yielded seventeen pure compounds (1–17).

Gemmacolide AS (1) was isolated as a white amorphous powder. Its molecular formula was established as C₃₆H₅₀O₁₄ by HRESI-MS. The IR spectrum showed absorption bands of hydroxy (3478 cm⁻¹), γ-lactone (1778 cm⁻¹), and ester (1739 cm⁻¹) functionalities. This observation was in agreement with the signals in the ¹³C NMR and DEPT spectra (Table 1) for ten sp² carbon atoms (6 × OC=O, CH=CH, CH=C) at lower field and twenty three sp³ carbon atoms at higher field (1 × C, 4 × CH, 3 × CH₂, 9 × CH₃, 2 × OC, 5 × OCH, 2 × OCH₂), accounting for eight double bond equivalents. The remaining double bond equivalents were due to the presence of four rings in the molecule.

Table 1. ¹³C NMR data of gemmacolides AS–AY (1–7) ^a.

No.	1	2	3	4	5	6	7
1	47.2, C	46.5, C	46.4, C	46.6, C	47.0, C	47.1, C	48.8, C
2	74.5, CH	75.6, CH	74.3, CH	75.3, CH	71.9, CH	72.8, CH	70.4, CH
3	132.5, CH	131.2, CH	128.6, CH	131.2, CH	129.2, CH	65.3, CH	42.8, CH2
4	127.6, CH	129.6, CH	127.8, CH	129.1, CH	129.1, CH	78.9, CH	70.5, CH
5	140.0, C	144.5, C	139.3, C	139.8, C	136.8, C	135.0, C	140.0, C
6	122.4, CH	123.8, CH	122.8, CH	126.3, CH	64.3, CH	54.6, CH	52.0., CH
7	78.9, CH	78.7, CH	78.8, CH	78.4, CH	78.7, CH	80.2, CH	81.3, CH
8	81.2, C	81.1, C	81.2, C	81.0, C	80.0, C	83.8, C	81.3, C
9	64.1, CH	63.9, CH	64.1, CH	63.7, CH	75.7, CH	71.7, CH	70.8, CH
10	32.8, CH	32.7, CH	31.3, CH	32.7, CH	32.7, CH	41.2, CH	35.7, CH
11	59.3, C	58.4, C	60.1, C	58.2, C	57.0, C	56.9, C	57.2, C
12	73.0, CH	73.2, CH	76.0, CH	72.6, CH	73.3, CH	-	72.8, CH
13	29.0, CH2	66.3, CH	66.3, CH	66.3, CH2	66.6, CH	24.8, CH	29.5, CH
14	73.1, CH	73.9, CH	77.5, CH	73.7, CH	72.5, CH	74.0, CH	72.6, CH
15	14.2, CH3	14.4, CH3	14.6, CH3	14.3, CH3	14.7, CH3	15.8, CH3	14.0, CH3
16	62.8, CH2	63.8, CH2	63.1, CH2	44.4, CH2	117.1, CH2	118.8, CH2	118.4, CH2
17	44.2, CH	44.1, CH	44.2, CH	44.0, CH	48.5, CH	49.6, CH	51.3, CH
18	175.4, C	175.2, C	175.3, C	174.9, C	175.6, C	175.0, C	175.8, C
19	6.4, CH3	6.3, CH3	6.4, CH3	6.3, CH3	8.6, CH3	7.2, CH3	5.9, CH3
20	49.1, CH2	49.0, CH2	49.1, CH2	49.1, CH2	49.7, CH2	51.9, CH2	50.7, CH2
9-OAc	170.2, C	170.2, C	170.2, C	170.1, C	169.7, C	No.	169.3, C
	21.6, CH3	21.6, CH3	21.6, CH3	21.5, CH3	21.0, CH3		21.3, CH3
R1	169.1, C	170.9, C	169.3, C	171.8, C	see 1′–6′	170.3, C	172.9, C
	21.3, CH3	21.5, CH3	21.3, CH3	61.1, CH2		20.6, CH3	21.6, CH3
R2	170.1, C	169.7, C	No.	see 1′–5′	169.5, C	169.7, C	No.
	21.2, CH3	20.9, CH3			20.8, CH3	20.4, CH3
R3	No.	see 1′–5′	No.	169.7, C	170.5, C	No.	169.5, C
				20.5, CH3	20.6, CH3		21.3, CH3
R4	see 1′–5′	170.1, C	see 1′–5′	170.6, C	see 1ʺ–5ʺ	No.	see 1′–5′
		20.8, CH3		21.0, CH3
R5	see 1ʺ–5ʺ		170.2, C			170.1, C
			20.9, CH3			21.0, CH3
1′	172.0, C	171.8, C	173.7, C	171.4, C	166.1, C		172.9, C
2′	43.3, CH2	42.6, CH2	43.1, CH2	43.5, CH2	60.9, CH2		43.2, CH2
3′	25.7, CH	25.0, CH	25.2, CH	25.7, CH	172.4, C		24.7, CH
4′	22.4, CH3	22.4, CH3	22.6, CH3	22.4, CH3	42.8, CH2		22.7, CH3
5′	22.3, CH3	22.3, CH3	22.3, CH3	22.3, CH3	25.7, CH		22.5, CH3
6′					22.3, 2 × CH3
1ʺ	172.3, C				172.6, C
2ʺ	42.9, CH				43.3, CH2
3ʺ	24.8, CH3				25.1, CH
4ʺ and 5ʺ	22.5, 2 × CH3				22.4, 2 × CH3

^a 100 MHz, in CDCl₃, assignments made by CHEPT, HSQC, and HMBC.

Table 1. ¹³C NMR data of gemmacolides AS–AY (1–7) ^a.

No.	1	2	3	4	5	6	7
1	47.2, C	46.5, C	46.4, C	46.6, C	47.0, C	47.1, C	48.8, C
2	74.5, CH	75.6, CH	74.3, CH	75.3, CH	71.9, CH	72.8, CH	70.4, CH
3	132.5, CH	131.2, CH	128.6, CH	131.2, CH	129.2, CH	65.3, CH	42.8, CH2
4	127.6, CH	129.6, CH	127.8, CH	129.1, CH	129.1, CH	78.9, CH	70.5, CH
5	140.0, C	144.5, C	139.3, C	139.8, C	136.8, C	135.0, C	140.0, C
6	122.4, CH	123.8, CH	122.8, CH	126.3, CH	64.3, CH	54.6, CH	52.0., CH
7	78.9, CH	78.7, CH	78.8, CH	78.4, CH	78.7, CH	80.2, CH	81.3, CH
8	81.2, C	81.1, C	81.2, C	81.0, C	80.0, C	83.8, C	81.3, C
9	64.1, CH	63.9, CH	64.1, CH	63.7, CH	75.7, CH	71.7, CH	70.8, CH
10	32.8, CH	32.7, CH	31.3, CH	32.7, CH	32.7, CH	41.2, CH	35.7, CH
11	59.3, C	58.4, C	60.1, C	58.2, C	57.0, C	56.9, C	57.2, C
12	73.0, CH	73.2, CH	76.0, CH	72.6, CH	73.3, CH	-	72.8, CH
13	29.0, CH2	66.3, CH	66.3, CH	66.3, CH2	66.6, CH	24.8, CH	29.5, CH
14	73.1, CH	73.9, CH	77.5, CH	73.7, CH	72.5, CH	74.0, CH	72.6, CH
15	14.2, CH3	14.4, CH3	14.6, CH3	14.3, CH3	14.7, CH3	15.8, CH3	14.0, CH3
16	62.8, CH2	63.8, CH2	63.1, CH2	44.4, CH2	117.1, CH2	118.8, CH2	118.4, CH2
17	44.2, CH	44.1, CH	44.2, CH	44.0, CH	48.5, CH	49.6, CH	51.3, CH
18	175.4, C	175.2, C	175.3, C	174.9, C	175.6, C	175.0, C	175.8, C
19	6.4, CH3	6.3, CH3	6.4, CH3	6.3, CH3	8.6, CH3	7.2, CH3	5.9, CH3
20	49.1, CH2	49.0, CH2	49.1, CH2	49.1, CH2	49.7, CH2	51.9, CH2	50.7, CH2
9-OAc	170.2, C	170.2, C	170.2, C	170.1, C	169.7, C	No.	169.3, C
	21.6, CH3	21.6, CH3	21.6, CH3	21.5, CH3	21.0, CH3		21.3, CH3
R1	169.1, C	170.9, C	169.3, C	171.8, C	see 1′–6′	170.3, C	172.9, C
	21.3, CH3	21.5, CH3	21.3, CH3	61.1, CH2		20.6, CH3	21.6, CH3
R2	170.1, C	169.7, C	No.	see 1′–5′	169.5, C	169.7, C	No.
	21.2, CH3	20.9, CH3			20.8, CH3	20.4, CH3
R3	No.	see 1′–5′	No.	169.7, C	170.5, C	No.	169.5, C
				20.5, CH3	20.6, CH3		21.3, CH3
R4	see 1′–5′	170.1, C	see 1′–5′	170.6, C	see 1ʺ–5ʺ	No.	see 1′–5′
		20.8, CH3		21.0, CH3
R5	see 1ʺ–5ʺ		170.2, C			170.1, C
			20.9, CH3			21.0, CH3
1′	172.0, C	171.8, C	173.7, C	171.4, C	166.1, C		172.9, C
2′	43.3, CH2	42.6, CH2	43.1, CH2	43.5, CH2	60.9, CH2		43.2, CH2
3′	25.7, CH	25.0, CH	25.2, CH	25.7, CH	172.4, C		24.7, CH
4′	22.4, CH3	22.4, CH3	22.6, CH3	22.4, CH3	42.8, CH2		22.7, CH3
5′	22.3, CH3	22.3, CH3	22.3, CH3	22.3, CH3	25.7, CH		22.5, CH3
6′					22.3, 2 × CH3
1ʺ	172.3, C				172.6, C
2ʺ	42.9, CH				43.3, CH2
3ʺ	24.8, CH3				25.1, CH
4ʺ and 5ʺ	22.5, 2 × CH3				22.4, 2 × CH3

^a 100 MHz, in CDCl₃, assignments made by CHEPT, HSQC, and HMBC.

¹H and ¹³C NMR spectra of 1 showed great similarity to those of gemmacolide N (18) [11], the difference was the acetyl group at C-14 and the methoxy group at C-16 in gemmacolide N (18) being replaced by two isovaleric acetyl in 1 due to the obvious HMBC correlations from the secondary alcohol protons to the respective ester carbonyl groups. The established planar structure of 1 was further supported by the COSY and HMBC spectra as shown in Figure 1. The relative configuration of 1 at the stereogenetic centers was proved the same as that of 18 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 = 10.1 Hz) while that of Δ⁵ was determined as E due to the NOESY correlation between H-6 and H₂-16. The relative configuration of 1 was thus determined as (1R*,2S*,7S*,8S*,9S*,10S*,11R*,12R*,14S*,17R*).

As gemmacolide AS (1) contained the same lactone and diene chromophores as gemmacolide N (18) and they differed only in the nature of substitutions for R₄ and R₅, the ECD spectrum of gemmacolide N could therefore be used as an ECD reference for the configurational assignment of gemmacolide AS (1) and its analogues. Because the absolute configuration of gemmacolide N had been unambiguously determined by a TDDFT calculation of its solution ECD spectrum [11], the absolute configuration of 1 was therefore determined as (1R,2S,7S,8S,9S,10S,11R,12R,14S,17R) due to the congruent ECD curves for 1 and that of gemmacolide N (18).

Figure 1. Key HMBC (arrow H→C) and COSY (bond) spin coupling systems for compound 1.

Figure 1. Key HMBC (arrow H→C) and COSY (bond) spin coupling systems for compound 1.

Figure 2. Key NOESY correlations for compound 1.

Figure 2. Key NOESY correlations for compound 1.

Gemmacolide AT (2), a white amorphous powder, had a molecular formula of C₃₃H₄₄O₁₅ as established by HRESI-MS. The ¹H and ¹³C NMR spectra data (Table 1 and Table 2) of 2 were almost identical to those of gemmacolide AQ (8) [8], showing the same functional groups for both compounds. The isovaleryl group, however, was found to be attached to C-13 of 2 instead of C-14 in gemmacolide AQ based on the analysis of HMBC spectra (see Supplementary Information). The structure of 2 had the same relative and absolute stereochemistry as that of 8 assessed by the NOESY and ECD measurements.

Table 2. ¹H NMR data for gemmacolides AS–AV (1–4) ^a.

No.	1	2	3	4
2	5.67, d (9.7)	5.62, ov	5.55, d (9.7)	5.64, ov
3	5.60, t (10.1, 9.7)	5.61, ov	5.60, t (9.7, 10.0)	5.65, ov
4	6.28, d (10.1)	6.35, d (9.1)	6.28, d (10.0)	6.41, d (7.7)
6	5.75, d (8.6)	5.83, d (8.5)	5.75, d (8.5)	6.07, d (8.7)
7	4.98, d (8.6)	4.96, d (8.5)	4.98, d (8.5)	4.94, d (8.7)
9	4.80, br d (4.7)	4.74, br d (4.8)	4.80, br d (4.5)	4.75, br d (3.8)
10	3.68, br d (4.7)	3.61, br d (4.8)	3.56, br d (4.5)	3.61, ov
12	4.53, br s	4.90, br d (2.8)	3.47, m	4.92, br d (2.9)
13β	1.96, ov	5.10, t (2.7, 2.8)	3.95, q (3.6)	5.07, t (2.8, 2.9)
13α	2.29, ov
14	4.96, br d (3.0)	5.22, br d (2.7)	5.30, m	5.18, br d (2.8)
15	1.05, s	1.14, s	1.08, s	1.14, s
16a	5.37, d (15.7)	4.47, br s	5.28, d (15.9)	4.67, d (13.5)
16b	4.68, d (15.7)	4.47, br s	4.69, d (15.9)	4.54, d (13.5)
17	2.30, ov	2.29, q (7.1)	2.31, ov	2.31, ov
19	1.16, d (7.1)	1.15, d (7.1)	1.17, d (7.1)	1.14, d (6.9)
20a	3.53, br d (2.4)	3.63, br d (2.5)	3.51, br d (2.8)	3.60, ov
20b	2.79, br d (2.4)	2.94, br d (2.5)	2.73, br d (2.8)	2.94, br d (2.0)
9-OAc	2.19, s	2.20, s	2.18, s	2.19, s
R1	1.95, s	1.99, s	1.97, s	4.16, d (16.8)
				4.02, d (16.8)
R2	2.11, s	2.16 s	No.	see 2′–5′
R3	No.	see 2′–5′	No.	1.93, s
R4	see 2′–5′	2.09 s	see 2′–5′	2.09, s
R5	see 2ʺ–5ʺ	No.	2.14, s	No.
2′a	2.28, ov	2.09, ov (×2)	2.30, ov (×2)	2.34, ov
2′b	2.01, ov			2.20, ov
3′	2.17, m	1.99, m	2.08, m	2.14, m
4′	0.99, d (6.6)	0.92, d (6.6) (×2 Me)	1.00, d (6.7)	0.99, d (6.8)
5′	0.99, d (6.6)		0.99, d (6.7)	1.01 (d, 6.8)
2ʺa	2.15, ov
2ʺb	2.06, ov
3ʺ	2.04, ov
4ʺ	0.96, d (6.6)
5ʺ	0.94, d (6.6)

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

Table 2. ¹H NMR data for gemmacolides AS–AV (1–4) ^a.

No.	1	2	3	4
2	5.67, d (9.7)	5.62, ov	5.55, d (9.7)	5.64, ov
3	5.60, t (10.1, 9.7)	5.61, ov	5.60, t (9.7, 10.0)	5.65, ov
4	6.28, d (10.1)	6.35, d (9.1)	6.28, d (10.0)	6.41, d (7.7)
6	5.75, d (8.6)	5.83, d (8.5)	5.75, d (8.5)	6.07, d (8.7)
7	4.98, d (8.6)	4.96, d (8.5)	4.98, d (8.5)	4.94, d (8.7)
9	4.80, br d (4.7)	4.74, br d (4.8)	4.80, br d (4.5)	4.75, br d (3.8)
10	3.68, br d (4.7)	3.61, br d (4.8)	3.56, br d (4.5)	3.61, ov
12	4.53, br s	4.90, br d (2.8)	3.47, m	4.92, br d (2.9)
13β	1.96, ov	5.10, t (2.7, 2.8)	3.95, q (3.6)	5.07, t (2.8, 2.9)
13α	2.29, ov
14	4.96, br d (3.0)	5.22, br d (2.7)	5.30, m	5.18, br d (2.8)
15	1.05, s	1.14, s	1.08, s	1.14, s
16a	5.37, d (15.7)	4.47, br s	5.28, d (15.9)	4.67, d (13.5)
16b	4.68, d (15.7)	4.47, br s	4.69, d (15.9)	4.54, d (13.5)
17	2.30, ov	2.29, q (7.1)	2.31, ov	2.31, ov
19	1.16, d (7.1)	1.15, d (7.1)	1.17, d (7.1)	1.14, d (6.9)
20a	3.53, br d (2.4)	3.63, br d (2.5)	3.51, br d (2.8)	3.60, ov
20b	2.79, br d (2.4)	2.94, br d (2.5)	2.73, br d (2.8)	2.94, br d (2.0)
9-OAc	2.19, s	2.20, s	2.18, s	2.19, s
R1	1.95, s	1.99, s	1.97, s	4.16, d (16.8)
				4.02, d (16.8)
R2	2.11, s	2.16 s	No.	see 2′–5′
R3	No.	see 2′–5′	No.	1.93, s
R4	see 2′–5′	2.09 s	see 2′–5′	2.09, s
R5	see 2ʺ–5ʺ	No.	2.14, s	No.
2′a	2.28, ov	2.09, ov (×2)	2.30, ov (×2)	2.34, ov
2′b	2.01, ov			2.20, ov
3′	2.17, m	1.99, m	2.08, m	2.14, m
4′	0.99, d (6.6)	0.92, d (6.6) (×2 Me)	1.00, d (6.7)	0.99, d (6.8)
5′	0.99, d (6.6)		0.99, d (6.7)	1.01 (d, 6.8)
2ʺa	2.15, ov
2ʺb	2.06, ov
3ʺ	2.04, ov
4ʺ	0.96, d (6.6)
5ʺ	0.94, d (6.6)

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

Gemmacolide AU (3) was isolated as a white amorphous powder and found to have a molecular formula of C₃₁H₄₂O₁₄ established by HRESI-MS. ¹H and ¹³C NMR spectra of 3 were similar to those of compound gemmacolide AR (19) [8]. The acetyl group at C-12 and C-13 in 19 was replaced by two hydroxy groups in 3. This conclusion was supported by extensive 2D NMR (see Supplementary Information) and HRESIMS analysis. The structure of 3 was thus determined as (−)-(1S,2S,3Z,5E,7S,8S,9S,10S,11R,12R,13S,14R,17R) based on the ECD experiment.

Gemmacolide AV (4) was obtained as a white amorphous powder. Its HRESI-MS demonstrated the same molecular formula as C₃₃H₄₃O₁₅Cl by HRESI-MS. ¹H and ¹³C NMR spectroscopic data of 1 showed great similarity to those of gemmacolide AJ (9) [8], the only difference of the isovaleric acetyl at C-2 in 9 being replaced by a glycolyl group in 4. The location of the glycolyl group at C-2 was indicated by the distinct HMBC correlations (see Supplementary Information). The established structure of 4 was further supported by detailed analysis of its 2D NMR data. Its absolute configuration was proven the same as that of 9 based on their similar ECD spectrum.

Gemmacolide AW (5) was found to be a white amorphous powder, having the molecular formula C₃₈H₅₁ClO₁₆ based on the HRESIMS data. The ¹H and ¹³C NMR data of 5 greatly resembled those of gemmacolide L (12) [10]. However, the substitutions of isovaleric acetyl group at C-12 and acetyl group at C-14 in 12 had to be interchanged in 5 based on the HMBC experiment (see Supplementary Information). The relative configuration for all chiral centers remained intact due to the NOEY experiment. Its absolute configuration was proved the same as that of 12 due to their similarity in ECD spectrum.

Gemmacolide AX (6) was isolated as a white amorphous powder. The molecular formula C₂₆H₃₃ClO₁₁ was established by HRESI-MS. Comparison of overall ¹H and ¹³C NMR data (Table 1 and Table 3) of 6 with those of gemmacolide X (13) [9] revealed great similarity except for the absence of two acetyl group. The acetyl group at C-9 and C-12 in 13 was replaced by a hydroxy group and a hydrogen atom in 6. This conclusion was supported by extensive 2D NMR analysis. The relative configuration of all the asymmetric centers was determined as (1R*,2R*,3R*,4R*,6S*,7R*,8R*,9S*,10S*,11R*,14S*,17R*) based on a NOESY experiment (see Supplementary Information). The absolute configuration of 6 was tentatively suggested as (1R,2R,3R,4R,6S,7R,8R,9S,10S,11R,14S,17R) (Chart 1) due to its biogenetic correlation with compounds 1–5.

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

Proton	5	6	7
2	6.24, d (10.1)	5.38, d (6.8)	5.90, ov
3β	5.74, ov	6.13, dd (6.8, 10.6)	2.07, ov
3α			2.11, ov
4	6.00, d (11.5)	4.43, d (10.6)	4.43, ov
6	5.10, br s	5.48, br d (3.1)	4.43, ov
7	4.72, br d (3.6)	4.57, br d (3.1)	4.43, ov
9	4.95, br s	4. 37, br d (6.8)	5.79, br s
10	3.96, br s	2.44, br d (6.8)	3.62, br s
12	4.88, br d (3.4)	2.19, ov (×2)	4.61, br s
13β	5.23, t (3.4)	1.92, ov (×2)	2.19, ov
13α			2.09, ov
14	5.28, br s	4.97, br s	4.96, br s
15	1.31, s	1.33, s	1.21, s
16a	5.72, br s	5.54, br d (2.0)	6.04, br s
16b	5.70, br s	5.31, br d (2.0)	5.81, br s
17	2.93, ov	2.59, q (7.1)	2.99, ov
19	1.22, d (7.6)	1.30, d (7.1)	1.26, d (6.4)
20a	2.92, br d (2.9)	2.72, br d (3.0)	2.86, br d (2.1)
20b	2.64, br d (2.9)	2.82, br d (3.0)	2.35, br d (2.1)
9-OAc	2.17, s		2.23, s
R1	4.59, d (15.6)	2.04, s	2.03, s
	4.44, d (15.6)
	2.28, ov
	2.15, ov
	0.98, d (6.6) (×2 Me)
R2	2.11, s	1.98, s	3.08, ov
R3	1.95, s	No.	2.00, s
R4	2.25, m		2.18, ov
	2.15, m		2.06, ov
	2.15, m		2.04, ov
	0.98, d (6.6) (×2 Me)		0.98, d (6.3)
			0.94, d (6.3)
R5		2.07, s

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

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

Proton	5	6	7
2	6.24, d (10.1)	5.38, d (6.8)	5.90, ov
3β	5.74, ov	6.13, dd (6.8, 10.6)	2.07, ov
3α			2.11, ov
4	6.00, d (11.5)	4.43, d (10.6)	4.43, ov
6	5.10, br s	5.48, br d (3.1)	4.43, ov
7	4.72, br d (3.6)	4.57, br d (3.1)	4.43, ov
9	4.95, br s	4. 37, br d (6.8)	5.79, br s
10	3.96, br s	2.44, br d (6.8)	3.62, br s
12	4.88, br d (3.4)	2.19, ov (×2)	4.61, br s
13β	5.23, t (3.4)	1.92, ov (×2)	2.19, ov
13α			2.09, ov
14	5.28, br s	4.97, br s	4.96, br s
15	1.31, s	1.33, s	1.21, s
16a	5.72, br s	5.54, br d (2.0)	6.04, br s
16b	5.70, br s	5.31, br d (2.0)	5.81, br s
17	2.93, ov	2.59, q (7.1)	2.99, ov
19	1.22, d (7.6)	1.30, d (7.1)	1.26, d (6.4)
20a	2.92, br d (2.9)	2.72, br d (3.0)	2.86, br d (2.1)
20b	2.64, br d (2.9)	2.82, br d (3.0)	2.35, br d (2.1)
9-OAc	2.17, s		2.23, s
R1	4.59, d (15.6)	2.04, s	2.03, s
	4.44, d (15.6)
	2.28, ov
	2.15, ov
	0.98, d (6.6) (×2 Me)
R2	2.11, s	1.98, s	3.08, ov
R3	1.95, s	No.	2.00, s
R4	2.25, m		2.18, ov
	2.15, m		2.06, ov
	2.15, m		2.04, ov
	0.98, d (6.6) (×2 Me)		0.98, d (6.3)
			0.94, d (6.3)
R5		2.07, s

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

Gemmacolide AY (7) was obtained as a white amorphous powder. The molecular formula C₃₁H₄₃ClO₁₃ was established by HRE-SIMS. Comparison of overall ¹H and ¹³C NMR data (Table 1 and Table 3) of 7 with those of gemmacolide U (20) [9] revealed great similarity with the only difference of the acetyl group at C-4 in 20 being replaced by a hydroxy group. This conclusion was supported by extensive 2D NMR and HRESIMS analysis (see Supplementary Information). The absolute configuration of 20 was tentatively assigned as that drawn in Chart 1 due to its biogenetic correlation with the co-isolates.

Compounds 1–7 were evaluated for their tumor cell growth inhibitory activity against A549 and MG63 cells. Compounds 4 and 7 showed moderate growth inhibitory effect against A549 while compound 5 displayed a promising growth inhibitory activity toward MG63 (Table 4).

Table 4. Tumor cell growth inhibitory activity of compounds 1–7 (IC₅₀ in μM).

	1	2	3	4	5	6	7	Adriamycin a
A549	>30.0	>30.0	>30.0	25.3	>30.0	>30.0	24.6	3.6
MG63	>30.0	>30.0	>30.0	>30.0	7.2	>30.0	>30.0	2.6

^a Positive control.

Table 4. Tumor cell growth inhibitory activity of compounds 1–7 (IC₅₀ in μM).

	1	2	3	4	5	6	7	Adriamycin a
A549	>30.0	>30.0	>30.0	25.3	>30.0	>30.0	24.6	3.6
MG63	>30.0	>30.0	>30.0	>30.0	7.2	>30.0	>30.0	2.6

^a Positive control.

3. Experimental Section

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 anisaldehyde-sulphuric acid reagent. The NMR spectra were recorded at 300 K on a Bruker DRX 400 spectrometer (Bruker Biospin Inc., 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 (Rudolph, Flanders, NJ, USA) 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 (Varian, Palo Alto, CA, USA); peaks wavelengths are reported in nm. Circular dichroism spectra were recorded with a JASCO J-715 circular dichroism spectropolarimeter (JASCO, 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 (Agilent Technologies, Santa Clara, CA, USA) equipped with a refractive index detector using an YMC Pack ODS-A column (particle size 5 μm, 250 × 10 mm, YMC Co., Ltd., Kyoto, Japan).

3.2. Animal Material

The gorgonian coral Dichotella gemmacea. (2.0 kg, wet weight) was collected from the South China Sea (20°54′ N, 109°05′ E), in August 2007 and identified by Dr. Xiu-Bao Li, the South China Sea Institute of Oceanology, Academia Sinica. A voucher specimen was deposited in the Second Military Medical University, Shanghai, China.

3.3. Extraction and Isolation

The frozen specimen was extracted with acetone and methanol (3 × 2.0 L) by ultrasonication. The solvent was combined and removed in vacuo. The resultant residue was partitioned between H₂O and EtOAc. The layers were separated, and then EtOAc was removed to afford 14.0 g of residue. The crude extract was further partitioned between MeOH and hexane, affording 10.0 g from the MeOH fraction. This residue was subjected to silicagel CC and eluted with hexane/acetone (from 100:0 to 0:100) as eluent. Fraction 11 was further fractionated by RP-silical gel CC (MeOH/H₂O, 20:80 to 75:25, in 5% increments) to give three subfractions (A–C). Subfraction B was purified by HPLC (MeOH/H₂O, 55:45, 1.0 mL·min⁻¹) to yield 4 (2.9 mg, 40.8 min), 2 (2.0 mg, 36.5 min) and 7 (0.6 mg, 34.2 min). Fraction 10 was purified by HPLC (MeOH/H₂O, 58:42, 1.2 mL·min⁻¹), yielding 14 (10.3 mg, 36.4 min). Fraction 9 were repeatedly subjected to silica gel and Sephadex LH-20 CC, and then purified by HPLC (MeOH/H₂O, 60:40, 1.5 mL·min⁻¹) to yield 3 (0.7 mg, 25.3 min), 6 (0.5 mg, 28.5 min), 8 (1.1 mg, 32.6 min), and 17 (2.4 mg, 35.4 min). Fraction 8 was further fractionated by RP-silical gel CC (gradient elution from MeOH//H₂O, 3:7 to MeOH, in 5% increments) and purified by HPLC (MeOH/H₂O, 67:33, 1.2 mL·min⁻¹), yielding 1 (2.3 mg, 36.4 min), 9 (4.9 mg, 40.1 min), 10 (0.5 mg, 28.5 min), 15 (1.1 mg, 45.6 min), and 16 (2.4 mg, 49.4 min). Fraction 7 was purified by HPLC (MeOH/H₂O, 60:40, 1.5 mL·min⁻¹), yielding 5 (7.0 mg, 49.4 min), 11 (4.3 mg, 56.2 min), 12 (52.7 mg, 36.4 min).

Gemmacolide AS (1): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = −31 (c 0.13, CHCl₃); UV (MeOH) 204 nm; CD (CH₃CN, c 3.0 × 10⁻⁴) λ_max (Δε) positive below 195 nm, 197 (−15.41) nm; IR (film) ν_max 3478, 1778, 1739 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 729 [M + Na]⁺; HRESI-MS m/z 729.3094 [M + Na]⁺ (calcd for C₃₆H₅₀O₁₄Na, 729.3098).

Gemmacolide AT (2): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = −20 (c 0.075, CHCl₃); UV (MeOH) 207 nm; CD (CH₃CN, c 1.3 × 10⁻³) λ_max (Δε) positive below 190 nm, 201 (−14.26) nm, 217 (−13.70) nm; IR (film) ν_max 3468, 1774, 1743 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 703 [M + Na]⁺; HRESI-MS m/z 703.2576 [M + Na]⁺ (calcd for C₃₃H₄₄O₁₅Na, 703.2578).

Gemmacolide AU (3): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = −0 (c 0.04, CHCl₃); UV (MeOH) 204 nm; CD (CH₃CN, c 3.6 × 10⁻⁴) λ_max (Δε) positive below 190 nm, 217.5 (−10.22) nm; IR (film) ν_max 3467, 1777, 1740 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 661 [M + Na]⁺; HRESI-MS m/z 661.2470 [M + Na]⁺ (calcd for C₃₃H₄₄O₁₆Na, 661.2472).

Gemmacolide AV (4): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = −28 (c 0.15, CHCl₃); UV (MeOH) 205, 273 nm; CD (CH₃CN, c 1.3 × 10⁻³) λ_max (Δε) positive below 190 nm, 199.5 (−28.02) nm; IR (film) ν_max 3406, 1778, 1745 cm⁻¹; ¹H NMR spectroscopic data, see Table 2; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 737 [M + Na]⁺; HRESI-MS m/z 737.2195 [M + Na]⁺ (calcd for C₃₃H₄₃O₁₅ClNa, 737.2188).

Gemmacolide AW (5): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = −104 (c 0.5, CHCl₃); UV (MeOH) 203 nm; CD (CH₃CN, c 2.6 × 10⁻⁴) λ_max (Δε) positive below 197 nm, 194 (−31.04) nm, 219 (−21.22) nm; IR (film) ν_max 3550, 1782, 1744 cm⁻¹; ¹H NMR spectroscopic data, see Table 3; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 821 [M + Na]⁺; HRESI-MS m/z 821.2759 [M + Na]⁺ (calcd for C₃₈H₅₁O₁₆ClNa, 821.2763).

Gemmacolide AX (6): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = 0 (c 0.02, CHCl₃); UV (MeOH) 202 nm; CD (CH₃CN, c 1.4 × 10⁻⁴) λ_max (Δε) positive below 191 nm, 208.5 (−9.64) nm; IR (film) ν_max 3467, 1787, 1743 cm⁻¹; ¹H NMR spectroscopic data, see Table 3; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 579 [M + Na]⁺; HRESI-MS m/z 579.1613 [M + Na]⁺ (calcd for C₂₆H₃₃O₁₁ClNa, 579.1609).

Gemmacolide AY (7): white amorphous powder; ${[\alpha ]}_{\text{D}}^{\text{24}}$ = 0 (c 0.03, CHCl₃); UV (MeOH) 204 nm; CD (CH₃CN, c 1.4 × 10⁻⁴) λ_max (Δε) negative below 191 nm, 209 (15.06) nm; IR (film) ν_max 3488, 1788, 1742 cm⁻¹; ¹H NMR spectroscopic data, see Table 3; ¹³C NMR spectroscopic data, see Table 1; ESI-MS m/z 681 [M + Na]⁺; HRESI-MS m/z 681.2293 [M + Na]⁺ (calcd for C₃₁H₄₃O₁₃ClNa, 681.2290).

3.4. Cytotoxicity Assay

Compounds 1–7 were evaluated for cytotoxicity against human lung adenocarcinoma (A549) and human osteosarcoma cell (MG63), using a modification of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method [15]. Adriamycin was used as positive control.

4. Conclusions

This paper reported the isolation and structure elucidation of seven new briarane diterpenoids, gemmacolides AS–AY (1–7), from the gorgonian D. gemmacea. These compounds were tested for growth inhibition activity against A549 and MG63 cell lines. Compounds 4 and 7 showed a moderate growth inhibitory effect against A549 while compound 5 displayed a potential growth inhibitory activity toward MG63, being similar to that of positive control adriamycin. This research gives an additional example of briarane diterpenoids for their tumor growth inhibitory activity, and would continuously encourage further investigations on the chemistry and antitumor activity of this cluster of metabolites.