Cembranoid-Related Metabolites and Biological Activities from the Soft Coral Sinularia flexibilis

Abstract

Five new cembranoid-related diterpenoids, namely, flexibilisins D and E (1 and 2), secoflexibilisolides A and B (3 and 4), and flexibilisolide H (5), along with nine known compounds (6–14), were isolated from the soft coral Sinularia flexibilis. Their structures were established by extensive spectral analysis. Compound 3 possesses an unusual skeleton that could be biogenetically derived from cembranoids. The cytotoxicity and anti-inflammatory activities of the isolates were investigated, and the results showed that dehydrosinulariolide (7) and 11-epi-sinulariolide acetate (8) exhibited cytotoxicity toward a limited panel of cancer cell lines and 14-deoxycrassin (9) displayed anti-inflammatory activity by inhibition of superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils.

1. Introduction

Soft corals have been known to be the organisms possessing secondary metabolites with high diversity in chemical structures [1]. Since 1975, many cembranoid-type natural products with diverse and important biological activities have been isolated from Sinularia flexibilis [2,3,4,5,6,7,8,9,10,11,12,13]. In previous studies of the chemical constituents of Taiwanese soft corals, numerous marine metabolites with cytotoxic [14,15], neuroprotective [16], and anti-inflammatory activities [17,18,19] were also found from Sinularia flexibilis. Some cembranolides, possessing an α-methylene lactone ring, have been discovered as potent cytotoxic agents to a limited panel of cancer cell lines, for example, 14-deoxycrassin [20], and as significant anti-inflammatory agents, for example, sinulariolone acetate [21]. Additionally, cembranoids and related compounds from other coral species were also found to have notable antiviral [22], anti-inflammatory [23,24], and antiproliferative [25,26,27,28] activities. A wide variety of chemical diversity and biological activity of cembranoids encouraged us to search for more natural products from soft coral S. flexibilis, collected off the waters of Taiwan. Herein, we report the isolation of five new cembrane-related metabolites, namely, flexbilisins D and E (1 and 2), secoflexibilisolides A and B (3 and 4), and a cembranolide flexibilisolide H (5) (Figure 1), and nine known compounds, including 6R-hydroxysinulariolide (6) [3], 11-dehydrosinulariolide (7) [18], 11-epi-sinulariolide acetate (8) [13], 14-deoxycrassin (9) [20], 3,4:8,11-bisepoxy-7-acetoxycembra-15(17)-en-1,12-olide (10) [6], sinulariolide (11) [2], sinulaflexiolide E (12) [10], querciformolide A (13) [21], and flexibilisquinone (14) [19] (Figure 2). Their structures were established by spectroscopic analysis including infrared (IR), mass spectrometry (MS), and nuclear magnetic resonance (NMR) data (Figures S1–S14), as well as chemical transformation. The biogenetic origins of 3 and 4 were postulated to demonstrate the relationship between stereochemistry and biogenetic implications. The cytotoxicity of the isolates toward a limited panel of cancer cell lines and their inhibition of superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils were also investigated.

Figure 1.

Structures of 1–5.

Figure 2.

Structures of metabolites 6−14.

2. Results and Discussion

Flexbilisin D (1) was obtained as a colorless oil and its high-resolution electrospray ionization mass spectrometry (HRESIMS) (m/z 371.2196 [M + Na]⁺) data established the molecular formula of C₂₁H₃₂O₄, indicating six degrees of unsaturation. The IR spectrum showed the absorption bands of carbonyl group (1716 cm⁻¹) and double bond (1647 cm⁻¹). The NMR data (Table 1), including ¹³C NMR and distortion less enhancement by polarization transfer (DEPT) spectra, displayed 21 carbin signals which can be classified as three methyls (δc 15.3, 17.0, 17.8), one methoxy (δc 52.0), seven sp³ methylene (δc 22.2, 24.6, 27.4, 31.5, 33.0, 36.0, 36.9), one sp² methylene (δc 124.4), three sp³ methine (δc 35.2, 60.1, 61.9), one sp² methine (δc 126.5), two sp³ quaternary (δc 60.2, 60.8), and three sp² quaternary (134.2, 142.7, 167.3) carbons. The ¹³C NMR signals at δc 142.7 (C), δc 124.4 (CH₂) and the ¹H NMR signals at δ_H 6.26 (1H, s) and δ_H 5.48 (1H, s) revealed the presence of a 1,1-disubstituted double bond, while those at δc 134.2 (CH), δc 126.5 (C), and δ_H 5.18 (1H, t, J = 5.6 Hz) are indicative of a trisubstituted double bond. Two trisubstituted epoxides were identified from the NMR signals at δc 61.9 CH, 60.8 C and 60.2 C, 60.1 CH; δ_H 2.65 dd, J = 10.0 and 3.2 Hz; 2.81 dd, J = 10.0 and 4.0 Hz. The molecular skeleton of 1 was established by the above results, as well as the correlations spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations as shown in Figure 3. By analysis of the COSY correlations, it was possible to identify three partial structures (a–c). The fact that the methyl ester group [CO (δc 167.3)/OMe (δc 52.0; δ_H 3.76)] was on C-15 (δc 142.7) was confirmed by the HMBC correlation from H₂-17 (δ_H 6.26, 5.48) to C-16 (δc 167.3). The two epoxides were assigned at 3,4- and 11,12-positions with methyl substituents at C-4 (δc 60.2) and C-12 (δc 60.8) according to HMBC correlations from H₃-18 (δ_H 1.29) to C-3 (δc 60.1), C-4, and C-5 (δc 36.9), as well as H₃-20 (δ_H 1.23) to C-11(δc 61.9), C-12, and C-13 (δc 33.0), respectively. Together with other key HMBC correlations from H₃-19 (δ_H 1.65) to C-7 (δc 126.5), C-8 (δc 134.2), and C-9 (δc 36.0), as well as H₂-17 to C-1 (δc 35.2), C-15, and C-16 permitted the establishment of the carbon skeleton.

Table 1.

¹H and ¹³C NMR spectroscopic data of 1 and 2.

Position	1		2
	δH a (J in Hz) c	δC b (mult.) d	δH (J in Hz)	δC (mult.)
1	2.85, m	35.2 (CH)	2.79, m	36.5 (CH)
2	2.05, m; 1.36, m	31.5 (CH2)	1.98, m; 1.40, m	32.5 (CH2)
3	2.81, dd (10.0, 4.0)	60.1 (CH)	2.77, dd; (9.6, 4.4)	59.4 (CH)
4		60.2 (C)		60.7 (C)
5	2.00, m; 1.59, m	36.9 (CH2)	1.99, m; 1.54, m	36.8 (CH2)
6	2.12, m; 2.08, m	22.2 (CH2)	2.10, m; 2.00, m	22.9 (CH2)
7	5.18, t (5.6)	126.5 (CH)	5.11, t (5.2)	126.4 (CH)
8		134.2 (C)		134.6 (C)
9	2.26, m; 2.09, m	36.0 (CH2)	2.55, m, 2.22, m	31.6 (CH2)
10	2.09, m; 1.40, m	24.6 (CH2	2.71, ddd (10.8, 8.0, 2.8)	34.3 (CH2)
			2.65, ddd (10.8, 8.0, 2.8)
11	2.65, dd (10.0, 3.2)	61.9 (CH)		213.6 (C)
12		60.8 (C)		78.8 (C)
13	1.70, m; 1.20, m	33.0 (CH2)	1.74, ddd (12.8, 5.6. 2.0) 1.48, m	36.1 (CH2)
14	1.70, m; 1.58, m	27.4 (CH2)	1.54, m, 1.37, m	25.2 (CH2)
15		142.7 (C)		142.2 (C)
16		167.3 (C)		167.4 (C)
17	6.26, s; 5.48, s	124.4 (CH2)	6.31, s, 5.49, s	124.4 (CH2)
18	1.29, s	17.8 (CH3)	1.27, s	18.2 (CH3)
19	1.65, s	15.3 (CH3)	1.66, s	17.1 (CH3)
20	1.23, s	17.0 (CH3)	1.34, s	25.7 (CH3)
16-OMe	3.76, s	52.0 (CH3)	3.76, s	52.0 (CH3)

^a Spectra recorded at 400 MHz in CDCl₃. ^b Spectra recorded at 100 MHz in CDCl₃. ^c J values (in Hz) in parentheses. ^d Attached protons were deduced by DEPT experiments.

Figure 3.

Key COSY and HMBC correlations for 1 and 2.

The relative configuration of 1 was determined by the nuclear Overhauser effect spectroscopy (NOESY) experiment as shown in Figure 4. Assuming that H-1 is α-oriented, which showed NOESY correlations to one of H₂-2 (δ_H 1.36) and one of H₂-13 (δ_H 1.70); thus, another of H₂-2 (δ_H 2.05) and H₂-13 (δ_H 1.20) were β-oriented. Two trans epoxides were then assigned according to the NOESY correlations from H-11 to H-13β and from H-13α to H₃-20, as well as those from H₃-18 to H-2α. The proposed structure of 1 is in agreement with the most stable conformation (Figure 4) generated by an energy-minimized (MM2) force field calculation [29]. Consequently, the relative configurations of C-1, C-3, C-11, and C-12 were determined as 1R*, 3S*, 4S*, 11R*, 12R* (Figure 4).

Figure 4.

Selective NOESY correlations for 1 and 5.

Flexibilisin E (2) had a molecular formula C₂₁H₃₂O₅ as deduced from HRESIMS data. The ¹H and ¹³C NMR spectra showed that the structure of 2 closely resembled that of 1. By comparing their NMR data (Table 1), significant differences in chemical shifts were observed at C-11 (δc 61.9 for 1; 213.6 for 2), C-12 (δc 60.8 for 1; 78.8 for 2), and C-20 (δc 17.0 for 1; 25.7 for 2), suggesting that 2 is a 12-hydroxy-11-oxo derivative of 1. This was supported by the COSY and HMBC correlations (Figure 3). The structure and absolute configurations of the stereogenic centers of 2 were confirmed by an alkaline hydrolysis of 11-dehydrosinulariolide (7), whose absolute configurations at C-1, C-3, C-4, and C-12 were determined as 1R, 3S, 4S, and 12R, respectively, based on a single-crystal X-ray diffraction analysis [18]. Accordingly, the structure of 2 was determined as shown in Figure 1.

Secoflexibilisolide A (3) was isolated as a colorless oil and its molecular formula was established as C₂₀H₂₈O₅ by the observation of a sodiated molecular ion peak at m/z 371.1831 (calcd. for 371.1829 [M + Na]⁺) in the HRESIMS, indicating seven degrees of unsaturation. Its IR absorption bands suggested the presence of hydroxy (3450 cm⁻¹) and carbonyl (1765 and 1671 cm⁻¹) groups. Three spin systems (a–c), inferred by analysis of the COSY correlations, were constructed as shown in Figure 5. A bicyclo[4.3.0]nonane ring system was established by the HMBC correlations from H₃-20 (δ_H 1.39) to C-11 (δc 85.3), C-12 (δc 84.7), and C-13 (δc 30.4); from H₂-10 (δ_H 2.05, 1.68) to C-11 and C-12; and from H₂-17 (δ_H 2.26, 1.66) to C-1 (δc 34.5), C-15 (δc 60.9), and C-16 (δc 177.6). In addition, a γ–lactone ring between C-16 and C-11 was evidenced by the IR absorption band at 1765 cm⁻¹, which is consistent with perhydroindan analogues [30]. A methyl-substituted epoxy group was evidenced by the HMBC correlations from H₃-18 (δ_H 1.28) to C-3 (δc 61.9), C-4 (δc 58.8), and C-5 (δc 41.2), while an acetyl group was found to be located at C-7 by the HMBC correlations from H₃-19 (δ_H 2.26) to C-7 (δc 134.0) and C-8 (δc 198.3). Accordingly, the planar structure of 3 was deduced as shown in Figure 5.

Figure 5.

Key COSY and HMBC correlations and selective NOESY correlations for 3.

Compound 3 can be hypothesized to derive from a precursor with the cembranoid-type skeleton. As shown in Scheme 1, flexibilisolide D, which was also isolated from this coral [17], was suggested as a precursor. Flexibilisolide D was converted to intermediate I by oxidative cleavage and Michael addition. Reduction of the carboxylic acid of the cyclobutane intermediate (II), derived from I by the aldol condensation, would produce an alcohol functionality in III. Rearrangement of the hydroxymethyl cyclobutane through a reductive ring opening in III resulted in a formation of the cyclopentane ring in 3. This suggested that the configurations of C-1, C-3, C-4, and C-12 should be the same as those of flexibilisolide D and related analogues possessing a 3,4-epoxide group, isolated previously from this soft coral [17]. The relative configurations of C-11 and C-15 were determined by a combination of NOESY correlations (Figure 5) and pyridine-induced solvent shift experiment [31]. The NOESY correlations from H₃-20 to H₂-10 allowed the assignment of the lactone ring as α-oriented. However, the present NOESY data were unable to fully confirm the orientation of OH-11. As a result, the pyridine-induced solvent shift experiment was applied on 3. H-2β (δ_H 1.66 in pyridine), which is 1,3-diaxial to OH-11, was found to be downfield shifted by 0.28 ppm with respect to H-2β, measured in CDCl₃ (Table 2), suggesting the β-orientation of OH-11. Consequently, the relative configurations 1R*, 3S*, 4S*, 11S*, 12S*, 15R* were suggested for 3.

Scheme 1.

Proposed biosynthetic pathway for 3.

Table 2.

¹H, ¹³C NMR data, COSY, and HMBC correlations of 3.

Position	δH a (J in Hz) c	δC b (mult.) d	COSY	HMBC
1	2.13, m	34.5 (CH)	H-2, 14	C-3, 16
2	1.78, m; 1.38, m	30.7 (CH2)	H-1, 3	C-1, 3, 14, 15
3	2.74, dd (7.5, 6.5)	61.9 (CH)	H-2	C-2
4		58.8 (C)
5	2.44, d (7.5)	41.2 (CH2)	H-6	C-3, 4, 6, 7, 18
6	6.73, m	142.2 (CH)	H-5, 7	C-4, 5, 7, 8
7	6.12, d (16.0)	134.0 (CH)	H-6	C-8
8		198.3 (C)
9	1.82, m	20.8 (CH2)	H-10, 17	C-15
10	2.05, m; 1.68, m	34.1 (CH2)	H-9	C-9, 11, 12
11		85.3 (C)
12		84.7 (C)
13	2.10, m; 1.72, m	30.4 (CH2)		C-11, 12
14	1.96, m; 1.22, m	25.8 (CH2)		C-12, 15
15		60.9 (C)
16		177.6 (C)
17	2.26, m; 1.66, m	28.4 (CH2)	H-9	C-1, 15, 16
18	1.28, s	17.2 (CH3)		C-3, 4, 5
19	2.26, s	27.0 (CH3)		C-7, 8
20	1.39, s	18.7 (CH3)		C-11, 12, 13

^a Spectra recorded at 500 MHz in CDCl₃. ^b Spectra recorded at 125 MHz in CDCl₃. ^c J values (in Hz) in parentheses. ^d Attached protons were deduced by DEPT experiments.

The molecular formula of 4 was found to be C₂₀H₂₈O₆ as deduced from the HRESIMS and ¹³C NMR data, suggesting seven degrees of unsaturation. The ¹³C NMR data of 4 (Table 3) showed signals attributable to two double bonds (δc 139.5, 126.0, 136.9, 124.1), one ketone carbonyl (δc 205.6), and two ester carbonyls (δc 175.4, 165.3), which accounted for five degrees of unsaturation. Accordingly, the structure of 4 may contain two rings. From the COSY spectrum of 4, it was possible to suggest the presence of three proton sequences for H₂-13/H₂-14/H-1/H-2/H-3, H₂-5/H-6/H-7, and H₂-9/H₂-10 (a–c, respectively; Figure 6). Key HMBC correlations from H₃-20 (δ_H 1.64) to C-12 (δc 205.6) and C-13 (δc 39.4); from H₂-17 (δ_H 6.40, 5.12) to C-15 (δc 139.5), C-16 (δc 165.3), and C-1 (δc 35.9); from H₃-18 (δ_H 1.00) to C-3 (δc 83.0), C-4 (δc 72.5), and C-5 (δc 41.3); and from H₃-19 (δ_H 1.04) to C-7 (δc 136.9), C-8 (δc 83.9), and C-9 (δc 33.9); as well as H₂-10 (δ_H 2.05, 1.94) to C-11 (δc 175.4) were observed, allowing to establish the planar structure of 4 as shown in Figure 6.

Table 3.

¹H and ¹³C NMR data of compounds 4 and 5.

Position	4		5
	δH a (J in Hz) e	δC b (mult.) f	δH c (J in Hz)	δC d (mult.)
1	2.02, m	35.9 (CH)	2.92, m	34.6 (CH)
2	1.07, m; 1.02, m	28.2 (CH2)	2.14, m; 1.48, m	33.3 (CH2)
3	3.64, dd (11.6)	83.0 (CH)	3.07, dd (10.0, 4.0)	61.7 (CH)
4		72.5 (C)		59.6 (C)
5	2.14, m; 1.97, m	41.3 (CH2)	2.12, m; 1.48, m	34.3 (CH2)
6	5.66, m	124.1 (CH)	1.94, m; 1.91, m	25.5 (CH2)
7	5.20, dd (15.6, 6.8)	136.9 (CH)	4.53, d (10.0)	81.2 (CH)
8		83.9 (C)		145.8 (C)
9	1.44, m; 1.22, m	33.9 (CH2)	2.59, m; 2.23, m	29.7 (CH2)
10	2.05, m; 1.94, m	28.7 (CH2)	1.89, m; 1.78, m	26.5 (CH2)
11		175.4 (C)	5.60, dd (12.8, 2.4)	72.9 (CH)
12		205.6 (C)		87.1 (C)
13	1.76, m; 1.68, m	39.4 (CH2)	2.03, m; 1.92, m	32.8 (CH2)
14	1.62, m; 1.32, m	28.0 (CH2)	2.23, m; 1.38, m	30.1 (CH2)
15		139.5 (C)		143.4 (C)
16		165.3 (C)		168.1 (C)
17	6.40, s; 5.12, s	126.0 (CH2)	6.30, s; 5.50, s	125.0 (CH2)
18	1.00 s	22.4 (CH3)	1.40 s	24.0 (CH3)
19	1.04 s	26.3 (CH3)	5.09 s; 5.06 s	115.0 (CH2)
20	1.64 s	29.4 (CH3)	1.38 s	15.5 (CH3)
OAc			2.09 s	20.9 (CH3)
				170.6 (C)

^a Spectra recorded at 400 MHz in C₆D₆. ^b Spectra recorded at 100 MHz in C₆D₆. ^c Spectra recorded at 400 MHz in CDCl₃. ^d Spectra recorded at 100 MHz in CDCl₃. ^e J values (in Hz) in parentheses. ^f Attached protons were deduced by DEPT experiments.

Figure 6.

Key COSY and HMBC correlations for 4.

In the NOESY spectrum of 4, correlations from H-2α to both H-1 and H-3, and from H₃-18 to H-3 revealed that H-1 and H-3 are on the same face of the lactone ring. Biogenetically, 4 could be derived from sinuflexolide, which has been reported from the same coral [12], via an oxidative cleavage of the diol groups, epimerization of the vinyl alcohol, and subsequent esterification (Scheme 2). This suggested that the configuration of C-4 should be the same as that of sinuflexolide. Consequently, the relative configurations 1S*, 3S*, 4R* were suggested for 4. The configuration of C-8 remains undetermined.

Scheme 2.

Proposed biosynthetic pathway for 4.

The ¹³C NMR data (Table 3) and the HRESIMS of 5 showed that it has the same molecular formula as flexibilisolide G [17], that is, C₂₂H₃₂O₇. They also have similar functional groups, including an α-exomethylene-ε-lactone ring, an acetoxyl group, and a methyl-substituted epoxy group. However, the obvious difference between the two compounds is that the 6,7-double bond in flexibilisolide G is isomerized to an exocyclic double bond at C-8 in 5. In addition, the hydroperoxy group at C-7 in flexibilisolide G was found to migrate to C-8 in 5. The above findings were further confirmed by COSY and HMBC correlations. The 1R*, 12R* configuration of the α-exomethylene-ε-lactone ring was deduced based on the NOESY correlations from H-1 to H-14α, from H₃-20 to H-13β, and from H-14β to H-13β. In addition, NOESY correlations from H-11 to both H-7 and H-1 as well as H-1 to H-4 suggested the relative configurations of C-3, C-4, C-7, and C-11 as shown in Figure 4.

The cytotoxicitiy of 1, 2, and 4–14 against P-388 (murine leukemia), K-562 (human erythromyeloblastoid leukemia), and HT-29 (human colon carcinoma cells) cell lines was investigated. The results showed that 7–9 and 11 exhibited cytotoxic activity toward P-388 and K-562 cancer cell lines with half maximal inhibitory concentration (IC₅₀) values ranging from 6.9 μM to 26.7 μM (Table 4). Among them, 7 showed selective cytotoxicity toward P-388, while 8 was found to show potent activity and selectivity toward P-388 and HT-29 cancer cell lines. Compounds 1, 2, 4–6, 10, and 12–14 were nontoxic toward these cancer cell lines (IC₅₀ values > 40 μM). The anti-inflammatory effect of 1, 2, and 4–14 was also studied by measuring their ability to suppress N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF-CB)-induced superoxide anion (O₂^−•) generation and elastase release in human neutrophils. The results revealed that, at a concentration of 10 μM, 9 exhibited significant inhibition toward superoxide anion generation and elastase release with IC₅₀ values of 10.8 ± 0.38 and 11.0 ± 1.52 μM, respectively.

Table 4.

The cytotoxic activity of 7–9 and 11 ^a.

Compound	IC50 (μM)
	P-388 b	K-562 c	HT-29 d
7	9.3	23.4	15.9
8	6.9	12.2	9.6
9	16.0	26.7	(–)
11	(–) e	21.7	27.1
Doxorubicin hydrochloride f	0.3	1.0	0.9

^a Compounds 1, 2, 4–6, 10, and 12–14 were inactive toward the three cancer cell lines with IC₅₀ > 40 μM. ^b P-388: murine leukemia. ^c K-562: human erythromyeloblastoid leukemia. ^d HT-29: human colon adenocarcinoma. ^e (–): IC₅₀ > 40 μM. ^f Positive control.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations were measured on a JASCO P-1020 polarimeter (JASCO Corpotation, Tokyo, Japan). IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer (JASCO Corporation) (Varian Inc., Palo Alto, CA, USA). NMR spectra were recorded on 400 MHz (or 500 MHz) for ¹H and 100 MHz (or 125 MHz) for ¹³C in CDCl₃ or C₆D₆. LRMS or HRMS were obtained by electrospray ionization (ESI) on a Bucker APEX II mass spectrometer (Bruker, Bremen, Germany). Silica gel (230–400 mesh, Merck, Darmstadt, Germany) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical thin-layer chromatography (TLC). High-performance liquid chromatography was performed on a Hitachi L-7100 (HPLC) (Hitachi Ltd., Tokyo, Japan) apparatus with a Merck Hibar Si-60 column (250 mm × 21.2 mm, 8 μm) and on a Hitachi L-2455 (HPLC) (Hitachi Ltd., Tokyo, Japan) apparatus with a Sciences Inc. (GL Science, Tokyo, Japan) ODS-3 C18 column (250 mm × 20 mm, 5 μm).

3.2. Animal Material

The soft coral Sinularia flexibilis was collected by scuba diving off the coast of Liuqiu, Taiwan, in October 2011, at a depth of 10–15 m, and stored in the freezer until extraction. A voucher specimen was deposited in the Department of Marine Biotechnology and Resource, National Sun Yet-sen University.

3.3. Extraction and Separation

Sliced bodies of S. flexibilis were exhaustively extracted with EtOAc (2 L × 5). The EtOAc extract (40.0 g) was chromatographed over silica gel by column chromatography using hexane, EtOAc–hexane (1:100 and gradually increasing the proportion of EtOAc to 10:1), EtOAc, and then Me₂CO–EtOAc (1:100 and gradually increasing the proportion of Me₂CO to 10:1), and subsequently Me₂CO as eluents to yield 26 fractions. Fraction 16, eluting with hexane–EtOAc (3:1), was further applied on a silica gel column (240 g) using hexane–EtOAc (8:1) to yield five subfractions (A–E). Subfraction 16-C was purified by normal-phase HPLC using hexane–Me₂CO (8:1) to afford 1 (3.4 mg). Fraction 18, eluting with hexane-EtOAc (1:1), was further purified by RP-18 silica gel and using a mixture of MeOH-H₂O (1.5:1) to yield seven subfractions (A–G). Subfraction 18-B was purified by reversed-phase HPLC using MeCN-H₂O (1:2) to afford 3 (1.0 mg). Subfraction 18-D was purified over silica gel column (50 g) using hexane–EtOAc (2:1) to obtain 4 (3.2 mg). Fraction 19, eluting with hexane–EtOAc (1:2), was chromatographed on silica gel (240 g) using hexane–EtOAc (5:1) to yield six subfractions (A–F). Subfraction 19-B was purified on RP-18 silica gel using MeOH–H₂O (1:1) and subsequently by RP-HPLC with MeOH–H₂O (1.5:1) to afford 7 (280.4 mg) and 14 (2.9 mg). Subfraction 19-E was purified by RP-HPLC with MeCN–H₂O (2:1) to yield 2 (94.6 mg), 8 (84.5 mg), and 9 (11.2 mg). Fraction 20, eluting with hexane–EtOAc (2:1), was further purified over silica gel column (200 g) and eluted with hexane–EtOAc (2:1) to yield five subfractions (A–E). Compounds 5 (2.1 mg) and 10 (123.7 mg) were obtained from subfraction 20-C using NP-HPLC (hexane–Me₂CO, 4:1). Subfraction 20-E was isolated repeatedly over silica gel column (50 g) using hexane–Me₂CO (3:1) as eluent, followed by RP-HPLC (MeCN–H₂O, 1:1.5) to afford 6 (4.8 mg), 11 (9.5 mg), 12 (5.1 mg), and 13 (1.0 mg).

Flexibilisin D (1): colorless oil; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{19}$ −245 (c 0.85, CHCl₃); IR (KBr) v_max 2933, 1716, 1647, 1453, 1244, 1149, and 1074 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 371 [M + Na]⁺; HRESIMS m/z 371.2196 [M + Na]⁺ (calcd. for C₂₁H₃₂O₄Na, 371.2198).

Flexibilisin E (2): colorless oil; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ +26 (c 0.57, CHCl₃); IR (KBr) v_max 3481, 2927, 1713, 1627, 1460, 1438, 1387, 1252, 1159, 946, 816, and 755 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; ESIMS m/z 387 [M + Na]⁺; HRESIMS m/z 387.2145 [M + Na]⁺ (calcd. for C₂₁H₃₂O₅Na, 387.2147).

Secoflexibilisolide A (3): colorless oil; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{22}$ −580 (c 0.28, CHCl₃); IR (KBr) v_max 3450, 2924, 1766, 1672, 1627, 1380, 1232, 1175, 1101, 1078, 1030, and 1026 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; ESIMS m/z 371 [M + Na]⁺; HRESIMS m/z 371.1831 [M + Na]⁺ (calcd. for C₂₀H₂₈O₅Na, 371.1829).

Secoflexibilisolide B (4): colorless oil; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{19}$ −56 (c 0.91, CHCl₃); IR (KBr) v_max 3501, 2924, 1705, 1647, 1515, 1267, and 1153 cm⁻¹; ¹H and ¹³C NMR data, see Table 3; ESIMS m/z 387 [M + Na]⁺; HRESIMS m/z 387.1779 [M + Na]⁺ (calcd. for C₂₀H₂₈O₆Na, 387.1778).

Flexibilisolide H (5): white powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{19}$ −12 (c 0.60, CHCl₃); IR (KBr) v_max 3391, 2934, 1739, 1711, 1621, 1239, 1236, 1141, 1065, and 1048 cm⁻¹; ¹H and ¹³C NMR data, see Table 3; ESIMS m/z 431 [M + Na]⁺; HRESIMS m/z 431.2047 [M + Na]⁺ (calcd. for C₂₂H₃₂O₇Na, 431.2046).

3.4. Alkaline Hydrolysis of 7

Compound 7 (3.5 mg) was dissolved in 1 N methanolic NaOH solution (1 mL), and the mixture was stirred at 0 °C for 12 h. The reaction mixture was neutralized with 0.1 N HCl (aq). After evaporation of the solvent, the residue was extracted with CHCl₃, and subsequently purified by HPLC using MeOH–H₂O (3:1) as eluent to yield a methyl ester (1.1 mg, 31.4%; ${\left[\mathsf{\alpha }\right]}_D^{25}$ +48 (c 0.28, CHCl₃; ¹H and ¹³C NMR spectra, Supplementary materials, Figures S4 and S5), which was identified as 2.

3.5. Cytotoxicity Testing

Cell lines were purchased from the American Type Cultural Collection (ATCC). Cytotoxicity assay of 1, 2, and 4–14 were performed using Alamar Blue Assay [32,33].

3.6. In Vitro Anti-Inflammatory Assay

Human neutrophils were obtained from whole blood using dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were performed according to previously described procedures [34,35]. Idelalisib was used as a positive control, of which the IC₅₀ values for inhibition of superoxide anion generation and elastase release were 0.07 ± 0.01 and 0.28 ± 0.09 μM, respectively.

4. Conclusions

Five new diterpenoids and nine known compounds were isolated and characterized from the marine soft coral Sinularia flexibilis. The previously unreported 3, containing a bicyclo[4.3.0]nonane ring system, was proposed be derived from flexibilisolide D. Compounds 7 and 8 showed selective cytotoxicity toward P388 cancer cell line, while 8 also exhibited significant cytotoxicity toward HT-29 cancer cells. Compound 9 displayed weaker cytotoxicity than 7 and 8, but displayed potent inhibitory activities for superoxide anion generation and elastase release in (fMLF-CB)-induced human neutrophils.

Supplementary Materials

¹H and ¹³C spectra of compounds 1–14 and the hydrolyzed product of 7 are available online at http://www.mdpi.com/1660-3397/16/8/278/s1. Figure S1: ¹H NMR spectrum of compound 1 in CDCl₃, Figure S2: ¹³C NMR spectrum of compound 1 in CDCl₃, Figure S3: ¹H NMR spectrum of compound 2 in CDCl₃, Figure S4: ¹H NMR spectra of compound 2 and hydrolyzed product of 7 in CDCl₃, Figure S5: ¹³C NMR spectra of compound 2 and hydrolyzed product of 7 in CDCl₃, Figure S6: ¹³C NMR spectrum of compound 2 in CDCl₃, Figure S7: ¹H NMR spectrum of compound 3 in CDCl₃, Figure S8: ¹H NMR spectrum of compound 3 in pyridine-d₅, Figure S9: ¹³C NMR spectrum of compound 3 in CDCl₃, Figure S10: ¹H–¹H COSY spectrum of 3 in pyridine-d₅, Figure S11: ¹H NMR spectrum of compound 4 in C₆D₆, Figure S12: ¹³C NMR spectrum of compound 4 in C₆D₆, Figure S13: ¹H NMR spectrum of compound 5 in CDCl₃, Figure S14: ¹³C NMR spectrum of compound 5 in CDCl₃, Figure S15: ¹H NMR spectrum of compound 6 in CDCl₃, Figure S16: ¹³C NMR spectrum of compound 6 in CDCl₃, Figure S17: ¹H NMR spectrum of compound 7 in CDCl₃, Figure S18: ¹³C NMR spectrum of compound 7 in CDCl₃, Figure S19: ¹H NMR spectrum of compound 8 in CDCl₃, Figure S20: ¹³C NMR spectrum of compound 8 in CDCl₃, Figure S21: ¹H NMR spectrum of compound 9 in CDCl₃, Figure S22: ¹³C NMR spectrum of compound 9 in CDCl₃, Figure S23: ¹H NMR spectrum of compound 10 in CDCl₃, Figure S24: ¹³C NMR spectrum of compound 10 in CDCl₃, Figure S25: ¹H NMR spectrum of compound 11 in CDCl₃, Figure S26: ¹³C NMR spectrum of compound 11 in CDCl₃, Figure S27: ¹H NMR spectrum of compound 12 in CDCl₃, Figure S28: ¹³C NMR spectrum of compound 12 in CDCl₃, Figure S29: ¹H NMR spectrum of compound 13 in CDCl₃, Figure S30: ¹³C NMR spectrum of compound 13 in CDCl₃, Figure S31: ¹H NMR spectrum of compound 14 in CDCl₃, Figure S32: ¹³C NMR spectrum of compound 14 in CDCl₃.

Author Contributions

J.-H.S. designed and guided the whole experiment and contributed to manuscript preparation. T.-Z.H. and C.-H.C. make the structure elucidation and manuscript preparation. C.-H.W. and C.-Y.H. perform the experiment. C.-Y.H. and T.-L.H. performed bioassays. C.-F.D. identified the soft coral.

Funding

This work was supported by grants from Ministry of Science and Technology of Taiwan (MOST104-2113-M-110-006 and 105-2113-M-110-002), and the National Sun Yat-sen University-Kaohsiung Medical University (NSYSU-KMU) Joint Research Projects (NSYSUKMU 105-I008 and 106-I007) awarded to J.-H.S.

Conflicts of Interest

The authors declare no conflicts of interest.