Cembrane-Based Diterpenoids Isolated from the Soft Coral Sarcophyton sp.

Abstract

This research explored the chemical composition of the soft coral Sarcophyton sp., leading to the discovery of six previously unreported cembranoids, sarcophynoids D–I (1–6), and three known analog compounds (7–9). Structural elucidation of the new metabolites was achieved by spectroscopic methods, including one- and two-dimensional (1D and 2D) NMR (COSY, HSQC and HMBC), high-resolution electrospray ionization mass spectrometry (HRESIMS), quantum mechanical NMR (QM-NMR) methods, electronic circular dichroism (ECD) calculations, and comparison with literature data. All isolated substances were screened for antibacterial activities, and most exhibited moderate inhibitory effects against six pathogenic bacterial strains, with MIC values between 8 and 64 μg/mL. In addition, the effects of these compounds on LPS and IFN-γ stimulated RAW264.7 cells, focused on the release of NO and TNF-α, were also evaluated, but were inactive at 20 μM.

1. Introduction

The genus Sarcophyton, a highly prevalent group of soft corals, is widely recognized as a prolific source of biologically active secondary metabolites [1,2]. Natural products derived from species within this genus exhibit substantial chemical diversity, particularly enriched in terpenoid classes such as cembrane-type diterpenes, tetraterpenes, triterpenes, and ceramides, alongside steroids, sesquiterpenes, and fatty acids [3].

Structurally, cembrane diterpenoids possess a 14-membered isoprenoid macrocyclic backbone characterized by an isopropyl group at C-1 and methyl groups located at C-4, C-8, and C-12 (Figure 1). The basic structure of this diterpene usually presents cyclic ether, lactone, or furan moieties around the macrocyclic ring. Due to extensive functional group variation, including lactone, epoxide, furan, ester, aldehyde, hydroxyl, and carboxyl moieties, as well as cyclization diversity, cembrane diterpenoids can be categorized into several subclasses, including simple cembranes, cembranolides, furanocembranoids, and biscembranoids [4]. The 14-membered carbocyclic nucleus of cembranolides is often bonded to a lactone ring with five, six, seven, or eight members [4]. Furanocembranoids feature a furan heterocycle and a 14-membered carbocyclic nucleus. Additionally, they include a butenolide moiety that includes C-10, C-12, and C-20. Biscembranoids have a tricyclic backbone of tetraterpenoids with 14–6–14 members. These compounds demonstrate a wide spectrum of biological activities, such as anti-cancer, neuroprotective, antimicrobial, antiviral, antidiabetic, antifouling, and anti-inflammatory effects [3,5,6].

In our continuous investigation of marine-derived bioactive compounds [7,8,9], we collected specimens of Sarcophyton sp. from the Xisha Islands. This study details the purification and structural characterization of six newly discovered cembranoids, designated as sarcophynoids D–I (1–6), along with three previously identified analogs (7–9) (Figure 1). Their antibacterial and anti-inflammatory properties were evaluated.

2. Results

The acetone extract of the title animals was partitioned between Et₂O and H₂O. The Et₂O-soluble portion of the acetone extracts yielded the purified compounds 1–9 (Figure 1). The known compounds were identified as crassumol A (7) [10], 5-episinuleptolide (8) [11], and arbolide C (9) [12].

Sarcophynoid D (1) was obtained as a colorless oil. Its molecular formula, C₂₀H₃₂O₃, was established through HRESIMS, which exhibited an ion peak at m/z 303.2325 [M − H₂O + H]⁺ (calculated 303.2324), indicating five degrees of unsaturation. Analysis of the ¹³C NMR and HSQC data suggested the presence of 20 carbon signals, including three double bonds, three nonprotonated sp³ carbons, two methine carbons, five methylene carbons, and four methyl groups. The planar framework of 1 was established by 2D NMR correlation analysis (Figure 2). The ¹H–¹H COSY correlations of H₂-13/H₂-14/H-1/H-2/H-3, H-1/H-2/H-3, H₂-5/H-6/H-7, and H₂-9/H₂-10/H-11 revealed three partial structures. These data, in combination with the crucial HMBC from H-2, H-3, H₂-5, and H₃-15 to C-4, from H-6, H-7, H₂-10, and H₃-19 to C-8, from H-11, H₂-13, H₂-14, and H₃-17 to C-12, and from H-1, H-2, H-3, H₂-16, and H₃-17 to C-15, established the skeleton of 1. A comparison of the 1D and 2D NMR spectral data for compound 1 (Figure 2) with those of (1S,2E,4R,6E,8R,11S,12S)-11,12-epoxy-2,6-cembrane-4,8-diol, a naturally occurring cembrane derived from Australian Sarcophyton sp., revealed that they possess the same planar structure [13]. The difference was the existence of the Δ^15,16 double bond of compound 1. The larger coupling constants (J_2,3 = 15.7 Hz, and J_6,7 = 15.6 Hz) supported the E configurations of the Δ^2,3 and Δ^6,7 double bonds. As reported by Quinn’s work, the change in configuration at C-8 is clearly reflected by the ¹³C chemical shift of the Me-19 group (27.8 ppm in 8S and 30.9 ppm in 8R), which suggested the 8R configuration of compound 1. The chemical shifts around the epoxide at C-11 and C-12 also suggested that this functional group should have the 11S,12S configuration. The 1D NOE correlation from H-13b (δ_H 1.01) to H-11, from H₃-20 to H-1, together with the absence NOEs of H-11/H₃-20, H-13b/H-1 and H-13b/H₃-20, indicated that H-13b and H-11 were oriented toward the same face, relative to H-1 and H₃-20 (Figure 3). Together with the quantum mechanical NMR (QM-NMR) approach [14,15], was applied to assign the relative configurations at C-1, C-4, C-8, C-11 and C-12. Four possible configurations were calculated: 1a (1R,4S,8R,11S,12S) and 1b (1R,4R,8R,11S,12S) (Figure S10). The computational and experimental data were correlated using the DP4+ procedure, and the relevant DP4+ probabilities were calculated [14,15]. According to Figure S10, configuration 1b showed a dominating probability, indicating that the relative configuration of 1 is (1R*,4R*,8R*,11S*,12S*). Ultimately, the absolute configuration of 1 was determined as 1R,4R,8R,11S,12S through time-dependent density functional theory (TDDFT) ECD calculations [16,17] (Figure 4).

Sarcophynoid E (2) was isolated as a colorless oil and shared the same molecular formula as 1. A comparison of the 1D NMR spectra of compounds 2 and 1 indicated that they possess similar structural groups, with the primary differences being the position of the double bonds, the presence of one more sp³ oxygenated methine carbon, and one less sp³ oxygenated quaternary carbon in 2. The structure and assignments were further validated through 2D-NMR experiments. The E geometry of the Δ^3,4 and Δ^6,7 double bonds in compound 2 was corroborated by 1D NOE data and a larger coupling constant (J_6,7 = 15.5 Hz). In the NOE experiment (Figure 3), it was found NOE correlations between H-13 and H₃-20, H-13 and H-1, and no NOE could be detected between H-11 and H₃-20, and H-11 and H-13, suggesting that H-13 and H₃-20, H-1 and H-13, were oriented toward the same face, as well as H-11 and H₃-20, and H-11 and H-13, were oriented toward the opposite face. Since there is no clear relationship between the stereocluster C11-C12-C13 and the other stereoclusters, four possible configurations were calculated: 2a (1S,8S,11R,12R,13S), 2b (1S,8R,11R,12R,13S), 2c (1S,8S,11S,12S,13R), and 2d (1S,8R,11S,12S,13R). The relative configuration of 2 was confirmed to be 1S*,8S*,11S*,12S*,13R* by DP4⁺ calculations (Figure S21). The absolute configuration of compound 2 was established as 1S,8S,11S,12S,13R through TDDFT/ECD calculations.

Sarcophynoid F (3) has a molecular formula of C₂₀H₃₀O₄ as determined by HRESIMS and NMR data, which indicates 6 degrees of unsaturation. The 1D NMR data (

Table 1. ¹H NMR data of compounds 1–6 at 600 MHz in CDCl₃.

No.	1	2	3	4	5	6
1	2.50, m	1.87, m	2.70, m	2.72, ov	2.68, m	2.78, m
2a	5.31, dd (15.7,9.3)	2.04, m	1.79, ov	1.94, m	1.77, m	1.78, m
2b		1.97, m	1.63, m	1.43, ddd (14.4, 9.5, 3.6)	1.37, ov	1.54, m
3	5.72, d (15.7)	5.05, tdd (5.8, 2.9, 1.4)	2.61, dd (7.4, 4.8)	2.72, ov	2.70, ov	2.79, dd (7.0, 5.0)
5a	2.54, dt (14.4, 2.6)	2.73, m	2.56, dd (4.5, 1.3)	2.61, m	2.03, ddd (13.9, 5.7, 3.6)	2.11, m
5b	2.14, dd (14.4, 10.7)	2.67, m	2.00, dd (14.5, 5.8)	1.84, m	1.41, ov	1.22, m
6a	5.94, ddd (15.6, 10.8, 2.9)	5.88, ddd (15.5, 9.4, 5.3)	5.44, ov	5.60, ov	2.14, m	2.19, m
6b					2.14, m	2.13, m
7	5.58, dd (15.6, 2.1)	5.54, d (15.5)	5.45, ov	5.60, ov	5.11, br.t (6.6)	5.14, br.t (9.2)
9a	1.79, m	1.88, m	1.79, ov	1.92, m	2.32, m	2.20, m
9b		1.73, m	1.61, m	1.84, ov	2.21, m	2.20, m
10a	1.96, ov	1.95, m	1.40, m	2.48, m	2.44, m	2.48, m
10b	1.22, m	1.41, m	1.35, m	2.33, m	2.44, m	2.40, m
11	2.99, dd (11.1, 1.9)	2.96, dd (9.4, 2.7)	2.95, dd (7.8,5.3)	6.72, ddd (8.0, 6.5, 1.5)	6.58, td (7.4, 1.4)	6.66, t (6.6)
13a	1.96, ov	3.59, dt (10.5, 4.0)
13b	1.01, m
14a	1.63, m	1.54, ddd (14.1, 11.1, 3.7)	2.80, dd (6.8, 0.8)	3.16, dd (14.7,8.8)	2.82, dd (12.7, 7.6)	3.47, dd (12.8, 8.8)
14b	1.25, m	1.36, m		2.42, dd (14.7, 6.0)	2.55, dd (12.7, 7.1)	2.33, dd (12.8, 6.5)
16a	4.70, d (0.9)	4.79, br.s	4.88, br.s	4.79, t (1.5)	4.76, br.s	5.14, br.s
16b	4.69, t (1.5)	4.76, br.s	4.77, br.s	4.69, br.s	4.71, br.s	4.88, br.s
17	1.69, s	1.77, s	1.76, s	1.77, s	1.73, s	4.10, s
18	1.38, s	1.64, s	1.28, s	1.17, s	1.16, s	1.77, s
19	1.30, s	1.35, s	1.28, s	1.34, s	1.64, s	1.62, s
20	1.20, s	1.33, s	1.42, s	1.74, s	1.71, s	1.23, s
13-OH		2.12, br.d (5.8)

and

Table 2. ¹³C NMR data of compounds 1–6 at 150 MHz in CDCl₃.

No.	1	2	3	4	5	6
1	48.7, CH	44.7, CH	36.9, CH	41.1, CH	44.4, CH	40.4, CH
2	129.9, CH	33.0, CH2	31.9, CH2	30.6, CH2	31.6, CH2	31.7, CH2
3	137.9, CH	123.7, CH	58.7, CH	60.9, CH	63.0, CH	61.1, CH
4	72.3, C	136.8, C	60.6, C	60.7, C	60.5, C	60.3, C
5	46.9, CH2	41.5, CH2	41.6, CH2	42.1, CH2	37.3, CH2	38.7, CH2
6	122.2, CH	125.7, CH	123.8, CH	123.6, CH	23.6, CH2	24.0, CH2
7	139.4, CH	137.6, CH	139.9, CH	139.8, CH	126.5, CH	125.5, CH
8	73.4, C	73.2, C	72.8, C	72.8, C	134.1, C	134.3, C
9	38.0, CH2	38.1, CH2	38.6, CH2	41.2, CH2	38.7, CH2	38.2, CH2
10	22.4, CH2	22.1, CH2	25.2, CH2	24.8, CH2	25.8, CH2	25.6, CH2
11	65.6, CH	62.4, CH	64.2, CH	144.4, CH	143.5, CH	144.5, CH
12	61.1, C	63.6, C	66.7, C	136.5, C	136.4, C	138.3, C
13	36.1, CH2	70.6, CH	208.9, C	200.8, C	201.9, C	202.7, C
14	29.3, CH2	34.5, CH2	41.8, CH2	39.6, CH2	42.7, CH2	39.3, CH2
15	148.9, C	148.0, C	147.7, C	147.5, C	147.1, C	150.8, C
16	109.7, CH2	111.2, CH2	110.3, CH2	110.9, CH2	111.7, CH2	112.6, CH2
17	21.0, CH3	19.7, CH3	21.6, CH3	20.6, CH3	18.9, CH3	65.4, CH2
18	27.4, CH3	17.3, CH3	17.7, CH3	16.7, CH3	17.0, CH3	11.4, CH3
19	31.1, CH3	31.2, CH3	27.9, CH3	28.9, CH3	15.6, CH3	15.6, CH3
20	15.5, CH3	18.0, CH3	19.1, CH3	11.4, CH3	11.5, CH3	16.6, CH3

) and HSQC experiments revealed the presence of 20 carbon signals, including a ketone carbon, two disubstituted double bonds, three sp³ oxygenated quaternary carbons, three sp³ methine, five sp³ methylenes, and four methyl groups, indicating that compound 3 is also a cembranoid as compound 2, with differences in the functional group at the position of C-3 and C-4, and the oxidation of hydroxyl group in 2 to ketone group in 3. The proposed structure of 3 was supported by the comprehensive 1D and 2D NMR data. The Z geometry of the Δ^6,7 double bonds in compound 3 was corroborated by a smaller coupling constant (J_6,7 = 6.3 Hz). The critical NOE correlations (Figure 3) of H-3/H-1 and H-3/H₃-18 indicated H-1, H-3, and H₃-18 are on the same side, and NOE correlations of H-11/H₃-20 indicated H-11 and H₃-20 are on the same side. The relative configuration of 3 was further confirmed to be 1S*,3R*,4S*,8R*,11S*,12S* by DP4⁺ calculations. The absolute configuration of 3 was established as 1S,3R,4S,8R,11S,12S by TDDFT/ECD calculations.

Sarcophynoid G (4) was also a colorless oil. Its molecular formula was established as C₂₀H₃₀O₃ based on HRESIMS ion peak at m/z 319.2296 [M + H]⁺ (calculated for C₂₀H₃₁O₃, 319.2273), indicating six degrees of unsaturation. The NMR data for compound 4 closely resembled that of compound 3, suggesting that they are structural analogs. The main difference was that the functional group at C-11 and C-12 was a double bond (δ_C/δ_H 144.4/6.72 and δ_C 136.5) in 4 rather than the epoxy ring in 3. The proposed structure of 4 was supported by the comprehensive 1D and 2D NMR data. The Z and E geometry of the Δ^6,7 and Δ^11,12 double bonds in 4 was supported by a smaller coupling constant (J_6,7 = 7.9 Hz), as well as the 1D NOE enhancement of H-11/H₂-14, respectively. No NOE could be detected between H₃-18 and H-3, suggesting that H-3 and H₃-18 were oriented toward the opposite face. The relative configurations of compound 4 were indicated as 1R*,3R*,4R*,8S*, which were established by DP4⁺ calculations (Figure S40). Finally, the absolute configuration of 4 was established as 1S,3S,4S,8R by TDDFT/ECD calculations.

The molecular formula of compound 5 (sarcophynoid H) was determined to be C₂₀H₃₀O₂ from its HRESIMS ion peak at m/z 303.2396 [M + H]⁺ (calculated for C₂₀H₃₁O₂, 303.2324). The 1D and 2D NMR analysis indicated that 5 should possess the same planar structure as eunicenone Ⅳ, a cembrane-type diterpene isolated from the lipid extract of the Caribbean gorgonian Eunicea mammosa [18]. The configurations of the Δ^7,8 and Δ^11,12 double bonds were assigned as E, which were supported by the 1D NOE enhancements of H-7/H₂-9 and H-11/H₂-14. The absence of NOEs between H₃-18 and H-3 suggested that H-3 and H₃-18 were oriented toward the opposite face. Additionally, the relative configuration of compound 5 was confirmed as 1R*,3S*,4S* by DP4⁺ calculations. The absolute configuration of compound 5 was established as 1S*,3R*,4R* by TDDFT/ECD calculations (Figure 4).

Sarcophynoid I (6) was also isolated as a colorless oil. Its molecular formula was determined to be C₂₀H₃₀O₃ via HRESIMS (m/z 319.2296 [M + H]⁺, calculated 319.2274), indicating six degrees of unsaturation. The NMR spectra of 6 (Table 1 and Table 2) showed great significance to those of 5, except that a primary alcohol (17-CH₂OH) in 6 rather than the methyl (17-CH₃) in 5. The configurations of the Δ^7,8 and Δ^11,12 double bonds were also assigned as E, based on the same method as for 5. The relative configuration of 6 was confirmed as 1S*,3S*,4R* by DP4⁺ calculations. The absolute configuration of 6 was established as 1S,3S,4R by TDDFT/ECD calculations.

Compounds 1–9 were evaluated for the inhibition of NO and TNF-α production in lipopolysaccharide (LPS) and interferon-gamma stimulated RAW 264.7 macrophages. However, all compounds were inactive at 20 μM. Furthermore, all compounds were evaluated for antibacterial activity against six pathogenic bacterial strains (Pseudomonas aeruginosa, Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, Staphylococcus saprophyticus, and Staphylococcus white). The results showed that most of the compounds displayed moderate inhibitory activity (MIC 8–64 μg/mL) (

Table 3. Inhibitory effects of 1–9 on six kinds of pathogenic bacteria.

No.	MIC (μg/mL)
	P. aeruginosa	B. subtilis	S. aureus	E. faecalis	S. saparophytics	S. white
1	>64	64	64	32	32	64
2	>64	>64	64	>64	8	64
3	>64	>64	64	>64	>64	8
4	>64	64	>64	>64	16	>64
5	32	>64	64	64	32	64
6	>64	64	64	>64	64	64
7	32	64	64	64	64	64
8	16	16	32	32	>64	>64
9	64	>64	>64	>64	64	64
Penicillin a	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5	≤0.5

^a Penicillin was used as a positive control.

).

3. Materials and Methods

3.1. General Chemical Experimental Procedures

NMR spectra were recorded on a Bruker AVANCE NEO 600 spectrometer (BrukerBiospin AG, Fällanden, Germany). ¹H chemical shifts were referenced to the residual CDCl₃ (7.26 ppm) and ¹³C chemical shifts were referenced to the CDCl₃ (77.2 ppm) solvent peaks. High-resolution electrospray ionization mass spectra (HRESIMS) were performed on an ultra-high-performance liquid chromatograph (UPLC) and TIMS-QTOF high-resolution mass spectrometry (Waters, Milford, MA, USA). The purification was performed by reversed-phase high-performance liquid chromatography using a Shimadzu LC-20AT system (Shimadzu Corporation, Tokyo, Japan). The solvents used for HPLC were all Fisher HPLC grade. A Cosmosil C₁₈-MS-II column (250 mm × 10.0 mm, id, 5 μm, Cosmosil, Nakalai Tesque Co. Ltd., Kyoto, Japan) was used for the preparative HPLC separation. Column chromatography was performed using silica gel (300–400 mesh, Qingdao Ocean Chemical Co. Ltd., Qingdao, China) and C₁₈ reversed-phase silica gel (75 µm, Nakalai Tesque Co. Ltd., Kyoto, Japan).

3.2. Animal Material

Soft coral Sarcophyton sp. was sampled off the coast of Xisha Islands, South China Sea, wet weight was 8.24 kg, and was frozen immediately after collection. The specimens (XSSC20190803) were deposited at the Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, Health Science Center, Ningbo University, China.

3.3. Extraction and Isolation

The soft coral samples were vacuum freeze-dried with a freeze-dryer, crushed in a pulverizer, and fully soaked in acetone at room temperature for 2 days each time, followed by ultrasonic extraction for an hour, repeated soaking and ultrasonic extraction for 4–5 times. The extract was filtered to remove the sample residue, and the extract was concentrated under reduced pressure. The extract was partitioned three times with Et₂O and water (1:1, v/v), and the Et₂O layer extract was concentrated under reduced pressure to obtain 67 g brown residue.

The 67 g of the extract was separated by gradient elution on a normal-phase silica gel column, yielding 11 fractions (FrA–FrK). Fr.E (4.7 g) was eluted with MeOH/H₂O (60:40 to 100:0, v/v) on reversed-phase column chromatography to obtain six subfractions (Fr.E.1–Fr.E.6). Purification of Fr.E.3 by semi-preparative HPLC (MeCN/H₂O, 55:45, 2 mL/min) gave compounds 1 (3.8 mg, t_R = 62 min) and 2 (5.8 mg, t_R = 67 min). Separation of Fr.F (2.3 g) on a reversed-phase column with MeOH/H₂O (55:45~100:0, v/v) afforded five subfractions (Fr.F.1~Fr.F.5). Fr.F.3 was purified by semipreparative reversed-phase HPLC (MeCN/H₂O, 53: 47, 2 mL/min) to afford compounds 3 (3.5 mg, t_R = 54 min), 4 (2.8 mg, t_R = 57 min) and 8 (3.8 mg, t_R = 63 min). Fr.F.4 was purified by semipreparative reversed-phase HPLC (MeCN/H₂O, 55: 45, 2 mL/min) to afford compound 5 (2.8 mg, t_R = 48 min). Separation of Fr.I (2.5 g) on a reversed-phase column with MeOH/H₂O (50:50~100:0, v/v) afforded seven subfractions (Fr.I.1~Fr.J.7). Fr.I.3 was purified by semipreparative reversed-phase HPLC (MeCN/H₂O, 42:58, 2 mL/min) to afford compounds 6 (6.7 mg, t_R = 63 min) and 7 (5.2 mg, t_R = 67 min). Fr.I.4 was purified by semipreparative reversed-phase HPLC (MeCN/H₂O, 44:56, 2 mL/min) to afford compound 8 (5.9 mg, t_R = 48 min) and 9 (8.3 mg, t_R = 52 min).

Sarcophynoid D (1): colorless oil; {[α${]}_{\mathrm{D}}^{25}$ −42.5 (c 0.5, MeOH)}; UV (MeOH): 205 (3.72); IR (KBr) ν_max 3362, 2973, 2928, 2858, 1635, 1445, 1394, 1094, 1043, 1016 cm⁻¹; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 303.2325 [M − H₂O + H]⁺ (calcd for C₂₀H₃₁O₂, 303.2324).

Sarcophynoid E (2): colorless oil; {[α${]}_{\mathrm{D}}^{25}$ −41.0 (c 0.5, MeOH)}; UV (MeOH): 204 (3.31); IR (KBr) ν_max 3395, 2957, 2832, 1673, 1463, 1375, 1094, 1028 cm⁻¹; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 303.2325 [M − H₂O + H]⁺ (calcd for C₂₀H₃₁O₂, 303.2324).

Sarcophynoid F (3): colorless oil; {[α${]}_{\mathrm{D}}^{25}$ +13.5 (c 0.5, MeOH)}; UV (MeOH): 203 (2.62); IR (KBr) ν_max 3478, 2926, 2775, 1729, 1655, 1417, 1124, 1095 cm⁻¹; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 335.2220 [M + H]⁺ (calcd for C₂₀H₃₁O₄, 335.2222).

Sarcophynoid G (4): colorless oil; {[α${]}_{\mathrm{D}}^{25}$ +36.0 (c 0.5, MeOH)}; UV (MeOH): 205 (3.25); IR (KBr) ν_max 3358, 2965, 1710, 1668, 1448, 1086, 1015 cm⁻¹; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 319.2296 [M + H]⁺ (calcd for C₂₀H₃₁O₃, 319.2273).

Sarcophynoid H (5): colorless oil; {[α${]}_{\mathrm{D}}^{25}$ −20.5 (c 0.5, MeOH)}; UV (MeOH): 207 (3.23); IR (KBr) ν_max 2957, 2843, 1728, 1659, 1443, 1396, 1228, 1132, 1081 cm⁻¹; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 303.2396 [M + H]⁺ (calcd for C₂₀H₃₁O₃, 303.2324).

Sarcophynoid I (6): colorless oil; {[α${]}_{\mathrm{D}}^{25}$ +21.4 (c 0.5, MeOH)}; UV (MeOH): 205 (3.80); IR (KBr) ν_max 3370, 2926, 1760, 1628, 1460, 1073 cm⁻¹; ¹H and ¹³C NMR data, Table 1 and Table 2; HRESIMS m/z 319.2296 [M + H]⁺ (calcd for C₂₀H₃₁O₃, 319.2274).

3.4. Computational Section

The NMR calculations method and the simulation of NMR spectra were referenced with the procedure described in the literature [14,15]. The ECD calculations method followed the protocol described in the literature [16,17].

3.5. Antibacterial Assays

Antibacterial activities of all isolated compounds were evaluated using standardized protocols [19]. The assay included six bacterial strains: Pseudomonas aeruginosa [CMCC (B) 10104], Bacillus subtilis [CMCC (B) 63501], Staphylococcus aureus [CMCC (B) 26003], Enterococcus faecalis [ATCC 29212], Staphylococcus saprophyticus [ATCC 49453], and Staphylococcus hominis [ATCC 8032], with penicillin G employed as the positive control. Compounds 1–9 were dissolved in DMSO and tested across serial concentrations of 64, 32, 16, 8, 4, 2, 1, and 0.5 μg/mL.

In brief, the bacterial strains were cultured in Mueller–Hinton (MH) medium for 24 h at 28 °C with shaking at 180 rpm, followed by dilution in sterile MH medium to a turbidity equivalent to the 0.5 McFarland standard. Subsequently, 100 μL of each bacterial suspension was combined with 100 μL of MH medium containing 0.002% 2,3,5-triphenyltetrazolium chloride and the corresponding test or control compound. After incubation, bacterial growth inhibition was quantified by optical measurements.

4. Conclusions

In conclusion, the phytochemical investigation of Sarcophyton sp., a soft coral collected from the South China Sea, resulted in the discovery of six previously undescribed cembranoids, named sarcophynoids D–I (1–6), together with three known analogues. Structural determination of these metabolites was achieved through high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR) spectroscopy, quantum mechanical NMR (QM-NMR) analyses, and electronic circular dichroism (ECD) calculations, in combination with comparison to reported data. Most of the isolated cembranoids demonstrated moderate antibacterial effects, displaying minimum inhibitory concentrations (MICs) between 8 and 64 μg/mL. The discovery of these new metabolites from marine soft corals highlights the remarkable chemical diversity of Sarcophyton species and emphasizes the importance of investigating their antimicrobial potential for future public health applications.