Bioactive Capnosanes and Cembranes from the Soft Coral Klyxum flaccidum

Abstract

Two new capnosane-based diterpenoids, flaccidenol A (1) and 7-epi-pavidolide D (2), two new cembranoids, flaccidodioxide (3) and flaccidodiol (4), and three known compounds 5 to 7 were characterized from the marine soft coral Klyxum flaccidum, collected off the coast of the island of Pratas. The structures of the new compounds were determined by extensive spectroscopic analyses, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, and spectroscopic data comparison with related structures. The rare capnosane diterpenoids were isolated herein from the genus Klyxum for the first time. The cytotoxicity of compounds 1 to 7 against the proliferation of a limited panel of cancer cell lines was assayed. The isolated diterpenoids also exhibited anti-inflammatory activity through suppression of superoxide anion generation and elastase release in the N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-stimulated human neutrophils. Furthermore, 1 and 7 also exhibited cytotoxicity toward the tested cancer cells, and 7 could effectively inhibit elastase release. It is worth noting that the biological activities of 7 are reported for the first time in this paper.

1. Introduction

Chemical investigations on marine invertebrates have disclosed structural diversity in the terpenoid and steroid constituents of octocorals [1]. Cembranoid diterpenes, which contain a 14-membered macro cyclic skeleton, have been shown to exhibit a wide range of biological activities, including cytotoxic [2,3,4,5], antibacterial [6,7,8], anti-inflammatory [3,4,9,10,11], and antiviral [12,13,14] activities. The rarely found cembranoids with 3,7-fused carbobicyclic (capnosane) skeleton have also been found to possess inhibitory effects on protein tyrosine phosphatase 1B, a significant target in treating obesity and type II diabetes [15]; and against A2780 human ovarian tumor cells [16], human disease-related bacteria, and phytopathogens [17]. Additionally, cembranoid-derived diterpenoids are considered to be the main chemical defense of coral against natural predators [18]. Our previous investigation on the chemical constituents of soft corals belonging to the genus Klyxum [19,20,21,22,23,24,25,26,27,28,29,30,31,32,33] has shown a wealth of unique secondary metabolites, including cembrane- and eunicellin-based diterpenoids, and steroids. In this context, we isolated a series of cembrane-diterpenoids [32] and steroids [29,31,33] from the Formosan soft corals Klyxum flaccidum, some of which have been found to possess cytotoxic and anti-inflammatory activities. A further investigation on the secondary metabolites of the same coral has thus been conducted to discover new metabolites and their bioactivity. In this paper we report the isolation, structure determination, and bioactivity of four new cembrane-derived diterpenoids, flaccidenol A (1) and 7-epi-pavidolide D (2), flaccidodioxide (3), and flaccidodiol (4), and three known compounds, sarcophytol T (5) [34], flaccidoxide-13-acetate (6) [35], and 14-O-acetylsarcophytol B (7) [36]. The structures of compounds were established by extensive spectroscopic analyses (Supplementary Materials, Figures S1 to S28). The cytotoxicities of compounds 1 to 7 were assayed against the cancer cell lines; human lung adenocarcinoma (A549), human colorectal adenocarcinoma (DLD-1), and mouse lymphocytic leukemia (P388D1). Furthermore, the anti-inflammatory activities that occur due to the inhibition of superoxide anion (O₂^−•) generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-challenged human neutrophils, were evaluated.

2. Results and Discussion

The ethyl acetate (EtOAc) extract of K. flaccidum was initially fractionated over a silica gel column, and the eluted fractions displaying terpenoidal methyl, olefinic, and oxymethine proton signals in the ¹H NMR spectra were selected for further purification. One of these fractions was purified by repeated column chromatography to yield diterpenoids 1 to 7 (Figure 1), the structures were established on the basis of spectroscopic analyses (Supplementary Materials, Figures S1 to S28).

Flaccidenol A (1) was isolated as a colorless oil and has a molecular formula of C₂₀H₃₄O₄ with four unsaturations, based on the sodium adduct ion peak [M + Na]⁺ obtained by positive high-resolution electrospray ionization mass spectroscopy (HRESIMS). The infrared (IR) spectrum revealed the presence of hydroxy and olefinic functionalities (ν_max 3391 and 1647 cm⁻¹). This was evidenced from the carbon signals of a trisubstituted and one 1,1-disubstituted carbon–carbon double bonds [δ_C 148.1 (C), 146.6 (C), 122.3 (CH), and 117.4 (CH₂)] and three sp³ oxygenated carbons [δ_C 90.7 (CH), 82.8 (C), and 74.5 (C)] (

Table 1. ¹³C nuclear magnetic resonance (NMR) data of compounds 1 to 4.

Position	1 a	2 b	3 b	4 c
1	146.6, C	144.4, C	149.6, C	78.6, C
2	122.3, CH d	121.8, CH	124.8, CH	131.5, CH
3	51.0, CH	51.1, CH	58.1, CH	136.5, CH
4	82.8, C	82.8, C	62.1, C	72.3, C
5	40.3, CH2	37.7, CH2	37.8, CH2	43.9, CH2
6	25.5, CH2	23.0, CH2	23.5, CH2	22.3, CH2
7	50.5, CH	54.3, CH	126.4, CH	128.4, CH
8	74.5, C	75.2, C	133.7, C	132.8, C
9	37.9, CH2	44.1, CH2	35.9, CH2	39.0, CH2
10	25.4, CH2	23.1, CH2	24.0, CH2	23.9, CH2
11	90.7, CH	127.1, CH	59.1, CH	126.5, CH
12	148.1, C	132.3, C	60.4, C	136.3, C
13	30.3, CH2	36.0, CH2	73.2, CH	36.2, CH2
14	28.6, CH2	28.3, CH2	67.8, CH	32.7, CH2
15	32.7, CH	32.9, CH	28.9, CH	38.7, CH
16	21.1, CH3	21.2, CH3	24.2, CH3	16.7, CH3
17	23.7, CH3	23.8, CH3	24.0, CH3	17.6, CH3
18	25.8, CH3	25.2, CH3	17.5, CH3	27.8, CH3
19	27.1, CH3	22.1, CH3	15.9, CH3	14.8, CH3
20	117.4, CH2	17.3, CH3	16.2, CH3	14.7, CH3
13-OAc			20.7, CH3
			170.4, C
14-OAc			20.7, CH3
			169.0, C

^a Spectrum recorded in C₆D₆ at 125 MHz at 25 °C, ^b Spectra recorded in CDCl₃ at 100 MHz at 25 °C, ^c Spectrum recorded in CDCl₃ at 125 MHz at 25 °C, ^d Attached protons were determined by distortionless enhancement by polarization transfer (DEPT) experiments.

), respectively, out of the 20 carbon signals in ¹³C NMR spectrum of 1. Moreover, the remaining two unsaturations indicated that compound 1 was a bicyclic diterpenoid. Similar to cembranoids [32], the ¹H NMR of 1, in conjunction with the heteronuclear single quantum coherence (HSQC) spectrum, revealed the presence of six exocyclic carbons of isopropyl [δ_H 0.97 (3H, d, J = 7.0 Hz), 0.90 (3H, d, J = 7.0 Hz), and 2.21 (1H, sept, J = 7.0 Hz)], two tertiary methyl [δ_H 1.05 and 0.99 (each 3H, s), and α-methylene [δ_H 4.95 and 4.93 (each 1H, s)] groups (

Table 2. ¹H NMR spectral data for compounds 1 to 4.

Position	1 a	2 b	3 b	4 c
2	4.96 d (9.5) d	5.05 d (10.8)	5.25 m	5.59 d (16.0)
3	2.89 dd (10.0, 10.0)	2.37 dd (7.8, 9.6)	3.56 d (9.6)	6.05 d (16.0)
5	1.52 2H, m	1.72 m; 1.65 m	1.48 m; 2.11 m	1.52 m; 1.99 m
6	1.94 m; 1.64 m	1.88 m; 1.76 m	2.13 m; 2.23 m	2.22 m; 2.35 m
7	2.63 m	2.65 ddd (6.6, 7.8, 9.6)	5.22 m	5.34 dd (7.5, 7.5)
9	1.85 ddd (13.0, 13.0, 3.0); 1.20 ddd (13.0, 13.0, 4.5)	1.88 m; 2.00 m	2.14 m; 2.24 m	1.96 m; 2.18 m
10	1.94 m; 1.68 ddd (13.0, 10.5, 3.0)	2.14 m	1.09 m; 1.61 m	2.04 m; 2.23 m
11	4.27 dd (11.0, 4.5)	4.99 dd (7.6, 7.6)	3.12 dd (5.6, 5.6)	5.18 dd (9.0, 3.5)
13	2.66 m; 2.20 dd (11.5, 5)	2.03 m; 1.92 m	5.49 d (7.6)	2.10 m; 2.15 d (3.0)
14	2.79 ddd (11.5, 11.5, 4.5); 2.00 m	2.40 dd (14.4, 7.2); 1.94 ddd (14.4, 12.0, 3.0)	5.89 d (7.6)	1.86 td (12.5, 3.5); 1.60 m
15	2.22 sept (7.0)
16	0.97 3 H, d (7.0)	1.06 3H, d (7.2)	1.02 3H, d (6.4)	0.87 3H, d (7.0)
17	0.90 3 H, d (7.0)	1.02 3H, d (6.8)	1.02 3H, d (6.4)	0.87 3H, d (7.0)
18	0.99 3 H, s	1.14 3H, s	1.33 3H, s	1.40 3H, s
19	1.05 3 H, s	1.25 3H, s	1.64 3H, s	1.60 3H, s
20	4.93 s; 4.97 s	1.67 3H, s	1.26 3H, s	1.67 3H, s
13-OAc			2.14 3H, s
14-OAc			2.01 3H, s
1-OH				2.39 s
11-OOH	7.64 br s

^a Spectrum recorded in C₆D₆ at 500 MHz at 25 °C, ^b Spectra recorded in CDCl₃ at 400 MHz at 25 °C, ^c Spectrum recorded in CDCl₃ at 500 MHz at 25 °C, ^d J values (Hz) in parentheses.

). The analysis of correlation spectroscopy (COSY) of 1 indicated four partial structures bearing consecutive proton systems (Figure 2). One of the partial structures included two ring-juncture methines [δ_H/δ_C 2.89/51.0 (CH) and 2.63/50.5 (CH)] and were positioned at C-3 and C-7 from the analysis of heteronuclear multiple-bond correlation (HMBC) spectra (Figure 2), similar to those of 3,7-cyclized cembranoid (capnosane) diterpenoids [15,17,37,38]. Key HMBC correlations observed from H₃-18 to C-3 and C-5; H₃-19 to C-7 and C-9; H₂-20 to C-11 and C-13; H-15 to C-2 were used to connect the four COSY correlated partial structures and to further establish the 3,7-linkage in the capnosane molecular skeleton. Moreover, the NMR signals at δ_H 7.64 (1H, br s) and δ_C 90.7 (CH) pointed out the presence of an allylic hydroperoxy group [39,40,41]. Finally, the detailed HMBC correlation analysis confirmed the hydroperoxyl, the two hydroxyls, the exomethylene, and the trisubstituted double bond to be at C-11, C-4, C-8, C-12, and C-1/C-2, respectively, and consequently the planar structure of 1 as shown in Figure 2.

The relative configurations of 1 at C-3, C-4, C-7, C-8, and C-11 were proposed from the analysis of nuclear Overhauser effect spectroscopy (NOESY) correlations in combination with molecular modeling using molecular mechanical parameters (MM2 force field) calculations (Figure 3). A strong NOE interaction of H-3 (δ_H 2.89, dd, J = 10.0, 10.0 Hz) with H-7 (δ_H 2.63, m) indicated the syn-orientation of both protons which were arbitrarily assigned as α-oriented. The olefinic proton H-2 (δ_H 4.96, d, J = 9.5 Hz) exhibited NOESY correlations with both of H₃-18 and H₃-19, but not correlated with H-3. Therefore, H₃-18 and H₃-19 should be placed on the β-face of the molecule. One of the methylene protons at C-10 (δ_H 1.68, ddd, J = 13.0, 10.5, 3.0 Hz, H-10α) displayed NOE interaction with H-3, while the other one (δ_H 1.94, m, H-10β) exhibited NOE correlation with H-11. H-11 is thus β-oriented, and the 11-hydroperoxy group is α-oriented accordingly. The NOE interaction shown by H-11 with H-9β (δ_H 1.20 ddd, J = 13.0, 13.0, 4.5 Hz) and by H-9α (δ_H 1.85 ddd, J = 13.0, 13.0, 3.0 Hz) with H-7 further confirmed the α-orientation of 11-OOH. The E-geometry of the 1,2-double bond was assigned from the NOE correlation of H-2 with H-15 of the isopropyl moiety. From the above findings and other detailed NOEs analysis (Figure 3), the relative configuration of 1 was determined as 1E, 3S *, 4S *, 7S *, 8S *, 11R *.

The molecular formula of compound 2 was found to be C₂₀H₃₄O₂ as deduced from HRESIMS and ¹³C NMR data, appropriate for four degrees of unsaturation. The IR spectrum showed the presence of the hydroxy group (ν_max 3391 cm⁻¹) and olefinic functionality (ν_max 1656 cm⁻¹). The NMR spectroscopic data were found to be similar to those of pavidolide D [37], while detailed analyses of 2D NMR correlations (COSY and HMBC) revealed that both two compounds possessed the same molecular skeleton (Figure 2). On careful comparison of the ¹H and ¹³C NMR spectroscopic data (Table 1 and Table 2, respectively) of 2 with those of pavidolide D, it was found that the ring-juncture protons H-3 (δ_H 2.37, dd, J = 9.6, 7.8 Hz) and H-7 (δ_H 2.65, ddd, J = 9.6, 7.8, 6.6 Hz) showed differentiable NMR data with the corresponding H-3 (δ_H 2.55, t, J =10.3 Hz) and H-7 (δ_H 1.98, m) of the known compound. Further NOE correlations analysis of 2, confirmed compound 2 to be a new capnosane diterpenoid that should be the 7-epimer of pavidolide D. The assignment of the cis ring juncture protons H-3 and H-7 in 2 was further supported by the similar upfield chemical shift C-7 in 2 (δ_C 54.3) with that of trocheliophol F (δ_C 54.2) [17] relative to those of the related capnosenes with trans fused rings (δ_C ≥ 55.5) [16,17,37]. Furthermore, the E-geometry of C-11/C-12 double bond was determined from the NOE correlations observed between H₃-20 and H-3 and between H-11 and H₃-19, and from the chemical shift of C-20 (δ_C < 20 ppm) [42].

Flaccidodioxide (3) was obtained as a colorless oil and had a molecular formula of C₂₄H₃₆O₆ as established by HRESIMS (m/z 443.2406 [M + Na]⁺, calcd 443.2404), corresponding to seven degrees of unsaturation. IR absorption at 1746 cm⁻¹ showed the presence of ester carbonyl group. Comparison of the NMR spectroscopic data of 3 (Table 1 and Table 2), with those of a known metabolite gibberosene C [43], revealed that both compounds possessed the same 3(4),11(12)-diepoxyl groups in the cembrane ring-structure. The hydroxyl group at C-13 of gibbrosene C was acetylated, and the methylene group of C-14 was converted to acetoxyl-bearing methine, as support by the downfield chemical shifts of H-13 (δ_H 5.49) and H-14 (δ_H 5.89) of 3, and the three-bond correlations of both protons to two acetate carbonyl carbons unveiled from HMBC spectrum of 3. The planar structure of 3 was further confirmed by the detailed analyses of ¹H-¹H COSY and HMBC spectra (Figure 2). These results, along with extensive analysis of NOE correlations of 3 (Figure 3), determined the structure of 3 unambiguously.

Flaccidodiol (4) was isolated as a gum. HRESIMS of 4 exhibited a sodiated molecular ion peak at m/z 329.2448, corresponding to the molecular formula C₂₀H₃₄O₂. IR absorptions at 3457 cm⁻¹ indicated the presence of a hydroxyl group in 4. The NMR data (Table 1 and Table 2) assigned the presence of an isopropyl group and three methyls, including two olefinic ones and one attaching to an oxygenated sp³ carbon atom. It was found that the proton and carbon chemical shifts of 4 were very similar to those of a known dihydroxycembrane, sarcophytol T [34]. Thus, both compounds should be diastereomers. Furthermore, the proton signal of 1-OH exhibited NOE correlation with H-3, while H₃-18 showed NOE correlation with H-2, revealing the position of both 1-OH and 4-OH to be in the same face of 4. Thus, the relative configuration for 4 was established.

Cytotoxicities of metabolites 1 to 7 against the growth of human lung adenocarcinoma (A549), human colorectal adenocarcinoma (DLD-1), and mouse lymphocytic leukemia (P388D1) cell lines were screened. Compounds 1, 2, and 7 exhibited inhibitory activity against the growth of the tested cancer cells (

Table 3. Cytotoxicity (IC₅₀ μg/mL) of compounds 1 to 7.

Compound	Cell Lines IC50 (μg/mL)
	A549 a	DLD-1 b	P388D1 c
1	9.7 ± 1.2	6.0 ± 0.4	7.2 ± 1.8
2	28.6 ± 3.8	31.6 ± 3.7	30.4 ± 4.8
3	− d	−	19.6 ± 8.3
4	−	−	−
5	−	−	−
6	−	−	−
7	10.8 ± 4.9	11.7 ± 4.8	8.9 ± 2.2
Doxorubicin e	0.3 ± 0.1	1.5 ± 0.2	0.9 ± 0.2

^a A549: Human lung adenocarcinoma. ^b DLD-1: Human colorectal adenocarcinoma. ^c P388D1: Mouse lymphocytic leukemia cell line. ^d IC₅₀ > 40 μg/mL. ^e Clinical anticancer drug used as a positive control.

). The hydroperoxyl (as in 1) seem to potentiate the cytotoxic effect of the diterpenoid molecules. However, the diepoxide compound 3 could selectively inhibit the growth of P388D1 cancer cells relative to the inactive monoepoxide derivative (6). These compounds also exhibited the potent anti-inflammatory activity by suppressing O₂^−• generation and elastase release in the fMLF/CB-stimulated human neutrophils (

Table 4. Inhibitory effects of compounds 2 to 7 on superoxide anion generation and elastase release in N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (fMLF/CB)-induced human neutrophils at 10 μM.

Compounds	Superoxide Anion			Elastase Release
	IC50 (μM) a	Inh % b		IC50 (μM) a	Inh % b
2	>10	24.46 ± 6.99	*	>10	29.96 ± 6.14	**
3	>10	8.88 ± 3.33		>10	27.18 ± 4.05	**
4	>10	3.29 ± 0.88	*	>10	14.43 ± 3.75	*
5	>10	8.89 ± 4.28		>10	14.89 ± 3.62	*
6	>10	4.73 ± 1.53	*	>10	3.09 ± 3.88
7	>10	11.95 ± 2.53	**	7.22 ± 0.85	59.66 ± 0.83	***
Idelalisib	0.07 ± 0.01	102.8 ± 2.2	***	0.3 ± 0.1	99.6 ± 4.2	***

^a Concentration necessary for 50% inhibition (IC₅₀). ^b Percentage of inhibition (Inh %) at 10 μM. Results are shown as mean ± S.E.M. (n = 3–4). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with the control value.

). Compound 7 could effectively inhibit elastase release (IC₅₀ = 7.22 ± 0.85 µM), reducing the level of elastase release to 59.66 ± 0.83% at a concentration of 10 µM relative to the control group.

3. Experimental Section

3.1. General Experimental Procedures

Optical rotations and IR spectra were measured on a JASCO P-1020 polarimeter and JASCO FT/IR-4100 spectrophotometer (JASCO Corporation, Tokyo, Japan), respectively. NMR spectra were recorded on a Varian 400 FT-NMR (Bruker, Bremen, Germany) at 400 and 100 MHz for ¹H and ¹³C, respectively; and on a Varian Unity INOVA 500 FT-NMR (Varian Inc., Palo Alto, CA, USA) at 500 and 125 MHz for ¹H and ¹³C, respectively, in C₆D₆ or CDCl₃ (δ in ppm, J in Hz). Silica and RP-18 silica gels (230 to 400 mesh), and precoated silica gel 60 F-254 (0.2 mm) plates (Merck, Darmstadt, Germany) were used for column and thin-layer chromatography, respectively. Preparative high-performance liquid chromatography (HPLC) was performed on a Hitachi L-2455 HPLC apparatus (Hitachi Ltd., Tokyo, Japan) with a Supelco C18 (250 × 21.2 mm, 5 μm) column (Supelco, Bellefonte, PA, USA).

3.2. Animal Material

The soft coral Klyxum flaccidum Tixier-Durivault was collected along the coast of Pratas island, Taiwan, and stored until extraction as described before [32]. The organism was identified by one of the co-authors (C.-F.D.). A voucher sample (No. LI6) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

3.3. Extraction and Isolation

The frozen bodies of K. flaccidum (8.0 kg, wet weight) were sliced and worked up as described previously to yield EtOAc extract (120 g) [32]. Twenty-six fractions were eluted by silica gel column chromatography, using n-hexane, EtOAc–n-hexane (0:100 to 100:0, gradient), and subsequently MeOH–EtOAc (1:100 to 100:1, gradient). Based on the analysis of ¹H NMR spectra, the terpenoids-containing fractions were selected for further chromatographic separation. Fraction 7 eluted with EtOAc–n-hexane (1:3) was purified by a Sephadex LH-20 column using acetone as eluent to obtain a mixture (7a to 7c), Subfraction 7a was further separated by reversed-phase RP-18 silica gel column using MeOH–H₂O (3:1) to give 3 (4.8 mg) and 5 (34.6 mg), and subfraction 7c using MeOH–H₂O (4:1) to give 4 (1.3 mg). Subfraction 7b was further separated by RP-18 silica gel column using MeOH–H₂O (4:1), and further semipreparative RP-18 HPLC (MeOH–H₂O, 3:1) to yield compounds 6 (3.4 mg) and 7 (2.1 mg). Fraction 13, eluted with EtOAc–n-hexane (3:1), was also rechromatographed by a Sephadex LH-20 column, using acetone as the mobile phase, and then was further purified by RP-18 HPLC using MeOH–H₂O (5:1) to afford 1 (1.5 mg) and 2 (9.7 mg).

Flaccidenol A (1): Colorless oil; ${\left[\alpha \right]}_D^{25}$ −10.3 (c 0.43, CHCl₃); IR (neat) ν_max 3391, 2957, 2870, 1647, 1455, 1376, 1109, 912, 754, and 614 cm^–1; ¹³C NMR data (125 MHz; C₆D₆), see Table 1; ¹H NMR data (500 MHz; C₆D₆), see Table 2; ESIMS m/z 361 [M + Na]⁺; HRESIMS m/z 361.2349 [M + Na]⁺ (calcd for C₂₀H₃₄O₄Na, 361.2349).

7-epi-Pavidolide D (2): Colorless oil; ${\left[\alpha \right]}_D^{25}$ −3.8 (c 2.77, CHCl₃); IR (neat) ν_max 3391, 2957, 2865, 1656, 1451, 1372, 1127, 908, and 754 cm^–1; ¹³C NMR data (100 MHz; CDCl₃), see Table 1; ¹H NMR data (400 MHz; CDCl₃), see Table 2; ESIMS m/z 329 [M + Na]⁺; HRESIMS m/z 329.2453 [M + Na]⁺ (calcd for C₂₀H₃₄O₂Na, 329.2452).

Flaccidodioxide (3): Colorless oil; ${\left[\alpha \right]}_D^{25}$ +13.0 (c 0.50, CHCl₃); IR (neat) ν_max 2962, 2930, 2872, 1746, 1455, 1373, 1223, 1029, 966, 877, and 757 cm^–1; ¹³C NMR data (100 MHz; CDCl₃), see Table 1; ¹H NMR data (400 MHz; CDCl₃), see Table 2; ESIMS m/z 443 [M + Na]⁺; HRESIMS m/z 443.2406 [M + Na]⁺ (calcd for C₂₄H₃₆O₆Na, 443.2404).

Flaccidodiol (4): Colorless oil; ${\left[\alpha \right]}_D^{25}$ −65.0 (c 0.25, CHCl₃); IR (neat) ν_max3457, 2925, 1450, 1368, 1095, and 986 cm^–1; ¹³C NMR data (125 MHz; CDCl₃), see Table 1; ¹H NMR data (500 MHz; CDCl₃), see Table 2; ESIMS m/z 329 [M + Na]⁺; HRESIMS m/z 329.2448 [M + Na]⁺ (calcd for C₂₀H₃₄O₂Na, 329.2451).

3.4. Cytotoxicity Assay

The cancer cell lines were purchased from American Type Culture Collection (ATCC). Cytotoxicity of the compounds were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method [44,45]. The positive control used was doxorubicin. The compound was considered inactive if IC₅₀ > 20 μg/mL.

3.5. In Vitro Anti-Inflammatory Assay

Human neutrophils were obtained from blood by dextran sedimentation, Ficoll-Hypaque centrifugation, and hypotonic lysis and then incubated as previously described [46]. Neutrophils (6 × 10⁵ cells mL⁻¹) incubated in Hank’s Balanced Salt Solution (HBSS) with MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (100 μM), and Ca²⁺ (1 mM) at 37 °C were treated with the tested compound or Dimethyl sulfoxide (DMSO) for 5 min. The activation of neutrophils was challenged for 10 min with fMLF (100 nM)/CB (0.6 and 0.5 μg/mL^‒1 for O₂^−• generation and elastase release, respectively). The anti-inflammatory activity of compounds 2 to 7 were assessed by measuring the inhibition of fMLF/CB-stimulated cells producing O₂^−• and elastase by using UV spectrometer detection at wavelength 550 nm and 405 nm, respectively [46,47].

4. Conclusions

Further chemical investigation on Formosan soft coral Klyxum flaccidum has led to the isolation and characterization of two new capnosanoids, flaccidenols A (1) and B (2); two new cembranoids, flaccidodioxide (3) and flaccidodiol (4); along with three known cembranoids. This is the first study to discover the rarely found capnosane diterpenoids from genus Klyxum. Diterpenoids 1, 2, and 7 exhibited significant cytotoxicity against a limited panel of cancer cell lines. The hydroperoxy group might be required to potentiate the cytotoxic effect, as shown by the capnosanoid 1. Moreover, 14-O-acetylsarcophytol B (7) could inhibit the elastase release in the fMLF/CB-stimulated human neutrophils effectively. This is the first time to disclose bioactivity for this known compound, which might be considered as an anti-inflammatory candidate.