Cembrane- and Casbane-Type Diterpenes from the South China Sea Soft Coral Sinularia nanolobata

1. Introduction

Marine organisms, especially invertebrates, are shaped by their complex and competitive environments, leading them to biosynthesize an enormous diversity of secondary metabolites with unique chemical scaffolds and potent biological activities [1]. Soft corals of the genus Sinularia (phylum Cnidaria, class Anthozoa, subclass Octocorallia, order Alcyonacea, family Alcyoniidae) stand out as particularly rich source of such marine natural products, and serves as an amazing treasure trove of novel compounds ranging from sesquiterpenes, diterpenes, steroids, alkaloids to other miscellaneous compounds [2,3,4]. Numerous chemical investigations of the genus Sinularia revealed a vast array of structurally diverse secondary metabolites displayed highly promising biological activities including antibacterial, osteoclast inhibitory, anti-inflammatory, antithrombotic, antiviral, cytotoxic, and immunosuppressive activities [4,5].

Our team has been continuously dedicated to the search for bioactive secondary metabolites with novel structures from marine invertebrates, particularly soft corals from the South China Sea, and a large quantity of secondary metabolites with structural diversity and interesting bioactivities have been isolated and characterized [6,7,8]. Previously, an unpredented dieterpene with a tricyclo [10.3.0.0¹,²]pentadecane carbon skeleton, along with multiple polyoxygenated and unusual endoperoxide-bridged casbane-type diterpenes, were found from S. nanolobata collected off Ximao Island [9].

Inspired by our previous work, a new collection of the title species off the Ximao Island, South China Sea, were studied, which led to the discovery of 16 diterpenes. Among them, five previously unreported cembrane-type diterpenes and one previously described casbane-type diterpene were characterized. All isolated compounds were evaluated for their cytotoxicity against several human tumor cell lines and anti-inflammatory effects against murine macrophage cell line RAW264.7. Herein, the isolation, structural characterization, and bioactivity evaluation of these compounds were reported.

2. Results

Samples of S. nanolobata were frozen immediately to –20 ℃ after collection and stored at that temperature until exhaustive extraction with dichloromethane and methanol (1:1, v/v). The EtOAc-soluble portion of the organic extract was subjected to repeated column chromatography (CC) (silica gel, Sephadex LH-20, and reversed-phase HPLC), yielding five new cembrane-type diterpenes (1–5), one new casbane-type diterpene (7), along with ten known related compounds (6, 8–16). Those known compounds were rapidly characterized as 12α-methyl-1E,3E,7E-cembratrien-10-one (6) [10], nanolobatone B (8) [9], 10-oxo-11,12-dihydrodepressin (9) [11], 1-epi-10-oxo-11,12-dihydrodepressin (10) [11], 10-hydroxydepressin (11) [11], 1-epi-10-hydroxydepressin (12) [11], 2-epi-10-hydroxydepressin (13) [12], sinularcasbane A (14) [13], casbene (15) [14], and 5-oxo-casbene (16) [11], respectively, by comparison of their NMR data and optical rotation [α]_D values with those reported in the literature.

Compound 1 was isolated as a colorless oil with the molecular formula of C₂₀H₃₀O, which was established by the protonated molecular ion peak observed at m/z 287.2364 [M + H]⁺ (calcd. for C₂₀H₃₁O⁺, 287.2369) in the HRESIMS spectrum, implying six degrees of unsaturation. The ¹H NMR spectroscopic data of 1 (Table 1) revealed three vinyl methyls at δ_H 1.92 (3H, s), 1.78 (3H, s) and 1.60 (3H, s), one doublet methyl at δ_H 0.90 (3H, d, J = 6.7 Hz), and five olefinic protons at δ_H 6.31 (1H, d, J = 11.2 Hz), 6.02 (1H, d, J = 11.2 Hz), 5.25 (1H, t, J = 6.8 Hz), 5.03 (1H, s) and 4.95 (1H, s). The ¹³C NMR spectroscopic data of 1 (Table 2) showed 20 carbon signals, identified with the aid of HSQC spectrum, including four methyls (δ_C 16.6, 16.9, 21.1, 21.4), eight olefinic carbons (δ_C 112.4, 122.7, 123.0, 129.2, 130.3, 138.4, 139.2, 143.5), one carbonyl carbon (δ_C 209.8), and seven methylene carbons (δ_C 24.0, 25.6, 27.7, 35.9, 39.9, 47.1, 54.3). The above-mentioned four double bonds and one carbonyl accounted for five of the six degrees of unsaturation, indicating a monocyclic carbon framework for 1. The NMR data of 1 were almost identical to those of previously reported compound, 12α-methyl-1E,3E,7E-cembratrien-10-one (6) [10], except that the isopropyl with a methoxy group at C-15 in 6 was replaced by an isopropenyl group in 1. This replacement was corroborated by the HMBC correlations from H₂-16 to C-15 and C-17. The planar structure of 1 was finally determined as depicted in Figure 2 through analysis of ¹H–¹H COSY and HMBC spectra. The geometries of double bonds Δ¹, Δ³ and Δ⁷ were assigned as “E” based on the shielded carbon resonances of two vinyl methyls at δ_C 16.9 (C-18), 16.6 (C-19), as well as the observed NOESY correlations of H-2/Me-18, H-3/H₂-5, H-7/H₂-9 (Figure 3). The absolute configuration of compound 1 {$\left[\alpha \right]_{\mathrm{D}}^{20}$ -2.2 (c 0.18, CHCl₃)} was determined as same 12R as compound 6 {$\left[\alpha \right]_{\mathrm{D}}^{20}$ -13.5 (c 0.25, CHCl₃)} based on comparison of optical rotation values recorded under the same conditions. Therefore, the structure of 1 was established as shown in Figure 1 and named 12α-methyl-1E,3E,7E,15-cembratetraen-10-one.

Figure 1. Chemical structures of compounds 1–16.

Figure 1. Chemical structures of compounds 1–16.

Figure 2. ¹H–¹H COSY and selected key HMBC correlations of compounds 1–5 and 7.

Figure 2. ¹H–¹H COSY and selected key HMBC correlations of compounds 1–5 and 7.

Figure 3. The NOESY correlations of compounds 1–5 and 7.

Figure 3. The NOESY correlations of compounds 1–5 and 7.

Compound 2 was isolated as a colorless oil, and its molecular formula C₂₀H₃₂O₂ was established by the protonated molecular ion peak observed at m/z 305.2477 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475) in the HRESIMS spectrum, which was 18 mass units more than that of 1, corresponding to five degrees of unsaturation. A comparison of the 1D NMR data of 2 with those of 1 suggested that they were structural analogues, both being cembrane-type diterpenes. The ¹H NMR spectrum displayed three olefinic protons at δ_H 6.19 (1H, d, J = 10.0 Hz), 6.00 (1H, d, J = 10.0 Hz) and 5.06 (1H, t, J = 7.8 Hz), which were attributed to three trisubstituted double bonds. These moieties accounted for three of the five degrees of unsaturation, suggesting that 2 possessed a bicyclic system in its molecule structure. Moreover, the ¹³C NMR signals at δ_C 70.1 (CH), 74.7 (qC) and 76.0 (qC) confirmed the presence of a hydroxyl group and an additional oxygen-containing ring in 2, accounting for the remaining two oxygen atoms. The location of hydroxyl group at C-11 and the formation of the oxygen ring via the linkage of C-12 and C-15, respectively, as evidenced by the HMBC correlations from Me-16/17 to C-1 (δ_C 138.4), C-15 (δ_C 74.7) and C-17 (δ_C 29.9)/C-16 (δ_C 28.9), and from Me-20 to C-11 (δ_C 70.1), C-12 (δ_C 76.0), C-13 (δ_C 30.2). Thus, the planar structure of compound 2 was established on the basis of collective spectroscopic evidences (Figure 2).

All the geometries of the double bonds Δ¹, Δ³ and Δ⁷ were assigned to be “E” based on the NOESY correlations of H-2/Me-18, H-3/H₂-5, H-7/H₂-9 (Figure 3). The relative configuration of C-11 and C-12 was determined to be 11S*,12S* by the QM-NMR calculation and DP4+ probability analysis, where the experimentally observed NMR data of 2 gave the best match of 100% to those of the (11S*,12S*)-isomer (Figure S58). Subsequently, the absolute configuration of 2 was further elucidated to be 11S,12S via TDDFT-ECD calculation, showing that Boltzmann-averaged ECD curve of (11S,12S)-2 highly matched to the experimental ECD spectrum of 2, whereas the enantiomer exhibited a completely opposite curve (Figure 4). Accordingly, the structure of 2 was characterized as shown in Figure 1, and the compound was designated with the trivial name nanolobatol A.

Compound 3 was also isolated as a colorless oil, with the molecular formula C₂₁H₃₆O₃ established by HRESIMS from the molecular ion peak observed at m/z 337.2737 [M + H]⁺ (calcd. for C₂₁H₃₇O₃⁺, 337.2737), suggesting that 3 possessed four degrees of unsaturation. The ¹H, ¹³C NMR data and HSQC spectrum of 3 displayed 21 distinct carbon signals, including five singlet methyl groups (δ_H/δ_C 0.85/19.4, 1.07/19.2, 1.51/15.1, 1.60/15.0, 3.26/50.8), eight methylene groups (δ_C 22.4, 24.9, 25.3, 32.0, 36.8, 38.4, 39.5, 70.0), four methine groups (δ_C 30.9, 73.3, 127.7, 127.8), and four quaternary carbons (δ_C 79.0, 80.3, 133.4, 134.0). Based on the above data, the presence of two trisubstituted double bonds accounted for two of the four degrees of unsaturation, indicating that compound 3 was a bicyclic molecule. Furthermore, comprehensive analysis of the ¹H–¹H COSY spectrum revealed three distinct spin systems, as evidenced by the observed cross-peaks for H-3/H₂-2/H-1/H₂-14/H₂-13, H₂-5/H₂-6/H-7, H₂-9/H₂-10/H-11. Additionally, the key HMBC correlations from Me-18 to C-3, C-4, and C-5 revealed the substitution of the hydroxyl at C-3. The HMBC cross-peaks of H₂-16/C-4 were diagnostic for the ring linkage of C-4 and C-16. The methoxyl group at C-15 was indicated by the characteristic HMBC correlations from Me-21 to C-15 (Figure 2). Consequently, the planar structure of compound 3 was collectively established.

The geometries of the double bonds Δ⁷ and Δ¹¹ were both assigned as “E” based on the shielded carbon resonances of the two vinyl methyl groups at δ_C 15.1 (C-19) and 15.0 (C-20). This assignment was further supported by the NOESY correlations (Figure 3) of H-7/H-9 and of H-11/H-13. The relative configurations of C-1/C-3 and C-4/C-15 in compound 3 were determined to be 1R*,3S* and 4S*,15R*, respectively, based on the diagnostic NOESY correlations of H-1 (δ_H 1.96)/H-3 (δ_H 4.26) and Me-17 (δ_H 0.85)/H-16 (δ_H 3.64)/Me-18 (δ_H 1.07). However, the relationships of the relative configurations of two chiral domains could not be unambiguously determined by NOESY analysis. Assisted by the QM-NMR calculation and DP4+ probability analysis, the full relative configuration of 3 was established as 1R*,3S*,4S*,15R*, because the experimental NMR data of 3 showed the best match (100%) with those of the (1R*,3S*,4S*,15R*)-isomer (Figure S60). As shown in Figure 5, the experimental ECD spectrum of 3 was in good agreement with the calculated curve for (1R,3S,4S,15R)-3, while being completely opposite to that of its enantiomer. Consequently, the absolute configuration of 3 was determined to be 1R,3S,4S,15R by TDDFT-ECD calculation. Herein, the structure of 3 (the trivial name nanolobatol B) was proposed as depicted.

Compound 4 was obtained as a colorless oil. The molecular formula of 4 was established as C₂₀H₃₂O₂ based on the protonated molecular ion peak observed at m/z 305.2471 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475) in its HRESIMS spectrum, implying five degrees of unsaturation. The ¹H NMR data displayed signals corresponding to one vinyl methyl at δ_H 1.61 (3H, s), two tertiary methyls at δ_H 1.26 (3H, s) and 1.33 (3H, s), two methyls appearing as doublets at δ_H 0.88 (6H, d, J = 6.8 Hz), and five olefinic protons at δ_H 5.24 (1H, t, J = 7.5 Hz), 5.51 (1H, d, J = 16 Hz), 5.52 (1H, dd, J = 16, 6 Hz), 5.64 (1H, d, J = 15.8 Hz) and 5.71 (1H, d, J = 15.8 Hz), which were attributed to two disubstituted and one trisubstituted double bonds. Based on the above, three double bonds accounted for three of the five degrees of unsaturation, indicating that a bicyclic ring system belonged to the molecular structure of 4. The HMBC correlations from Me-18 to C-2, C-3 and C-4 and from H-2 to C-1 corroborated the location of the hydroxyl group at C-4 and the settlement of double bond Δ². The characteristic HMBC cross-peaks of Me-20/C-12, H₂-13/C-1, along with the ¹H–¹H COSY cross-peaks for H₂-13/H₂-14, revealed the linkage of C-1 and C-12, forming the tetrahydrofuran ring. Finally, the planar structure of 4 was unequivocally established as shown in Figure 2.

The large coupling constants (J_2,3 = 15.8 Hz, J_10,11 = 16 Hz) established the “E” geometries of double bonds Δ² and Δ¹⁰. Additionally, the shielded carbon resonance of the vinyl methyl at δ_C 17.6 (C-19) assigned the “E” geometry to the double bond Δ⁷. These assignments were further confirmed by analysis of the NOESY correlations (Figure 3). The results of the QM-NMR/DP4+ calculations (Figure S62) confirmed the relative configuration of compound 4 to be 1S*,4R*,12S*. Moreover, the absolute configuration of 4 was also determined by comparing its experimental ECD spectrum with the TDDFT-ECD calculated curves of its two enantiomers, showing excellent agreement with the one of the (1S,4R,12S)-4 isomer (Figure 5). Thus, the structure of 4 (the trivial name nanolobatol C) was assigned as depicted.

Compound 5 was isolated as a colorless oil, possessing the same molecular formula of C₂₀H₃₂O₂ as 4 by HRESIMS ion peak at m/z 305.2472 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475). The ¹H NMR data revealed the presence of two vinyl methyls at δ_H 1.60 (3H, s) and 1.70 (3H, s), one tertiary methyl at δ_H 1.19 (3H, s), and five olefinic protons at δ_H 4.77 (2H, s), 4.86 (1H, s), 4.91 (1H, s) and 5.30 (1H, t, J = 7.6 Hz), assigned to one trisubstituted and two terminal double bonds. These structural fragments accounted for three of the five degrees of unsaturation, indicating that a bicyclic ring system was incorporated in the molecular structure of 5. Subsequently, comprehensive analysis of the key ¹H–¹H COSY cross-peaks and long-range HMBC correlations collectively established the connectivity of the spin systems and the assembly of all structural fragments. In detail, the ¹H–¹H COSY cross-peaks of H-3/H₂-2/H-1/H₂-14/H₂-13, H₂-5/H₂-6/H-7, and H₂-9/H₂-10/H-11 revealed three distinct spin systems. The HMBC correlations from Me-17 to C-1 (δ_C 41.4), C-15 (δ_C 150.2), and C-16 (δ_C 110.9); from Me-18 to C-3 (δ_C 73.6), C-4 (δ_C 84.9), and C-5 (δ_C 37.1); from H₂-19 to C-7 (δ_C 80.3), C-8 (δ_C 151.9), and C-9 (δ_C 34.3); and from Me-20 to C-11 (δ_C 126.3), C-12 (δ_C 134.5), and C-13 (δ_C 36.5) revealed the linkage between C-4 and C-7, forming the tetrahydrofuran ring. In addition, the hydroxy group was established at C-3, and the two terminal double bonds were assigned to C-8 and C-15, respectively, thereby unambiguously determining the planar structure of 5 (Figure 2).

The “E” geometry of the Δ^11,12 double bond was assigned based on the shielded carbon resonances of the vinyl methyl at δ_C 17.2 (C-20). The four stereogenic centers (C-1, C-3, C-4, and C-7) in compound 5 were all located on a flexible macrocyclic ring and remote from each other, which prevented definitive configurational assignment by NOESY analysis and thus required QM-NMR/DP4+ calculations. Comparison of the calculated NMR data with the experimental values showed excellent agreement (99.99%) for the 1R*,3R*,4S*,7R* configuration (Figure S64). Following the same TDDFT-ECD calculation protocol as applied to the other compounds in this study, the absolute configuration of 5 was finally established as 1R,3R,4S,7R (Figure 5). Accordingly, the structure of 5 (the trivial name nanolobatol D) was characterized as depicted.

Compound 7 was isolated as a colorless oil. Its molecular formula was determined to be C₂₀H₃₂O₂, based on the HRESIMS ion peak observed at m/z 305.2475 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475). The ¹H and ¹³C NMR data of 7 were highly similar to those of the co-occurring known casbane-type diterpenoid, nanolobatone B (8) [9], with the main differences observed at C-1, C-2 and their adjacent carbons, suggesting that they were structural analogues and epimers at either the C-1 or C-2 position. The large Δδ_C value (more than 10 ppm) of the gem-dimethyls C-16 (δ_C 29.1) and C-17 (δ_C 15.9) indicated a 1,2-cis configuration for 7, which was further supported by the obvious NOESY correlations of H-1 (δ_H 0.61)/Me-16 (δ_H 1.06) and H-2 (δ_H 1.24)/H-1 (Figure 3). Careful comparison of the NMR data of 7 with those of the reported synthetic product S5 [15], particularly the chemical shifts of H-1/C-1 and H-2/C-2, indicated that 7 shared the same 1S*,2R* relative configuration as S5. Furthermore, the relative configuration of 5R*,12S* in 7 was confirmed to be identical to that of 8 based on the similarity of their NMR data. Therefore, the relative configuration of 7 was assigned as 1S*,2R*,5R*,12S*. Finally, the absolute configuration of 7 was determined to be 1S,2R,5R,12S by TDDFT-ECD calculations, as the experimental ECD spectrum matched well with the calculated curve for (1S,2R,5R,12S)-7 (Figure 5). Consequently, compound 7 was identified as the C-2 epimer of compound 8, named 2-epi-nanolobatone B.

In the in vitro bioassay, all isolated compounds were evaluated for their cytotoxic activities against MV-4-11, A-549, and 293T cell lines, as well as for their anti-inflammatory effects in RAW264.7 macrophages. Unfortunately, none of the compounds exhibited obvious bioactivity (IC₅₀ > 50 µM). Comparatively, previous studies have reported that certain cembrane- and casbane-type diterpenes from Sinularia species exhibit moderate cytotoxic and/or anti-inflammatory effects, with IC₅₀ values typically ranging from 10 to 50 µM. Despite the observed weak or negligible activities of the compounds in this study, evaluating these activities remains a routine and essential step for the full characterization of new natural products, whose potency may vary depending on structural substitution patterns. Notably, the lack of activity observed in the present study does not preclude the possibility of other biological effects, such as antimicrobial or neuroprotective activities, and additional bioassays to explore these possibilities are currently in progress.

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were measured on a Perkin-Elmer 241MC polarimeter (PerkinElner, Fremont, CA, USA). NMR spectra were measured in CDCl₃ with a Bruker Advance 600 MHz spectrometer (Bruker Biospin AG, Fällanden, Germany). Chemical shifts are reported in parts per million (δ) in CDCl₃ (δ_H reported referred to CHCl₃ at 7.26 ppm; δ_C reported referred to CDCl₃ at 77.2 ppm), and coupling constants (J) are expressed in Hz. HRESIMS spectra were recorded on an Agilent 1290-6545 UHPLC-QTOF mass spectrometer (Agilent Technologies, CA, USA). Commercial silica gel (Qingdao Haiyang Chemical Group Co., Ltd., Qingdao, China, 200–300 and 300–400 mesh), Sephadex LH-20 gel (Amersham Biosciences, Uppsala, Sweden) were used for column chromatography, and precoated silica gel plates (Yan Tai Zi Fu Chemical Group Co., Yantai, China, G60 F-254) were used for analytical TLC. Reversed-phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatography equipped with a DAD G1315D detector at 210 and 254 nm. A semi-preparative ODS-HG-5 column [5 µm, 250 × 9.4 mm] was employed for the purifications. All solvents used for CC and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, China) and chromatographic grade (Dikma Technologies Inc., Beijing, China), respectively.

3.2. Animal Materials

Specimens of S. nanolobata were collected by scuba diving along the coast off Ximao Island (18°13.8′N, 109°22.1′E), Hainan Province, China, in May 2019, at a depth of 20 meters. A voucher specimen (19-XD-12) is deposited and available for inspection at the School of Medicine, Shanghai University.

3.3. Extraction and Isolation

The frozen animals (260.5 g, dry weight after extraction) were cut into pieces and extracted exhaustively with CH₂Cl₂/MeOH (1:1, v/v) at room temperature (2.0 L × 4). The organic extract was evaporated to give a brown residue, which was then partitioned between EtOAc and H₂O. The EtOAc solution was evaporated to give a dark brown residue (15.7 g). The obtained residue was subjected to gradient silica gel (200–300 mesh) column chromatography (CC) [EtOAc/petroleum ether (PE) 0→100%] to yield five fractions (Fr. A-E).

Fraction A (127.4 mg) was directly purified by semi-preparative RP-HPLC (CH₃CN/H₂O, 98:2, 3.0 mL/min) to afford compound 15 (6.0 mg, t_R = 27.5 min).

Fraction B (5.38 g) was separated by Sephadex LH-20 column chromatography eluted with PE/CH₂Cl₂/MeOH (2:1:1) to give four sub-fractions (B1–B4). Sub-fraction B4 (66.5 mg) was further purified by silica gel column chromatography (300–400 mesh) eluted with PE/EtOAc (40:1 and 15:1), followed by semi-preparative RP-HPLC (CH₃CN/H₂O, 87:13, 3.0 mL/min) to yield compound 16 (4.2 mg, t_R = 21.3 min).

Fraction C (1.26 g) was first separated by Sephadex LH-20 column chromatography eluted with PE/CH₂Cl₂/MeOH (2:1:1) to give three sub-fractions (C1–C3). Sub-fraction C2 was separated by silica gel column chromatography (300–400 mesh) eluted with PE/EtOAc (30:1, 15:1, 5:1, and 1:1) to afford five secondary fractions (C2a–C2e). Fraction C2a was further separated by RP-HPLC (CH₃CN/H2O, 80:20, 3.0 mL/min) to obtain compounds 4 (2.1 mg, t_R = 16.6 min) and 5 (1.6 mg, t_R = 19.2 min). Sub-fraction C2d was purified by semi-preparative RP-HPLC (CH₃CN/H₂O, 80:20, 3.0 mL/min) to yield compound 3 (2.6 mg, t_R = 18.7 min). Sub-fraction C2e was purified by semi-preparative RP-HPLC (CH₃CN/H₂O, 80:20, 3.0 mL/min) to give compounds 9 (12.3 mg, t_R = 10.8 min) and 10 (9.1 mg, t_R = 11.3 min).

Fraction D (1.36 g) was first separated by Sephadex LH-20 column chromatography eluted with PE/CH₂Cl₂/MeOH (2:1:1) to obtain four sub-fractions (D1–D4). Sub-fraction D2 (528.4 mg) was subjected to silica gel column chromatography (300–400 mesh) eluted with PE/EtOAc (30:1, 20:1, 10:1, 5:1, and 1:1) to afford compound 14 (2.6 mg) and five secondary fractions (D2a–D2e). Sub-fraction D2e was purified by semi-preparative RP-HPLC (MeOH/H₂O, 83:17, 3.0 mL/min) to yield compound 2 (1.8 mg, t_R = 16.4 min) and a tertiary fraction (10.2 mg, t_R = 24.8 min). Sub-fraction D3 (554.2 mg) was separated by silica gel column chromatography (300–400 mesh) eluted with PE/EtOAc (30:1, 20:1, 10:1, 5:1, 1:1) to yield compound 6 (3.9 mg) and three secondary fractions (D3a–D3c). Sub-fraction D3c was purified by semi-preparative RP-HPLC (CH₃CN/H₂O, 67:33, 3.0 mL/min) to afford compounds 7 (3.2 mg, t_R = 12.5 min) and 8 (2.8 mg, t_R = 13.3 min).

Fraction E (1.06 g) was initially separated by Sephadex LH-20 column chromatography eluted with PE/CH₂Cl₂/MeOH (2:1:1) to give three sub-fractions (E1–E3). Sub-fraction E3 was separated by silica gel column chromatography (300–400 mesh) eluted with PE/EtOAc (30:1, 20:1, 10:1, 5:1, 1:1) to afford compound 1 (2.4 mg) and three secondary fractions (E3a–E3c). Sub-fraction E3c was purified by semi-preparative RP-HPLC (CH₃CN/H₂O, 55:45, 3.0 mL/min) to afford compounds 11 (11.4 mg, t_R = 17.3 min), 12 (2.1 mg, t_R = 18.7 min), and 13 (4.1 mg, t_R = 19.3 min).

3.3.1. 12α-. Methyl-1E,3E,7E,15-Cembratetraen-10-One (1)

Colorless oil, $\left[\mathsf{\alpha }\right]_{\mathrm{D}}^{20}$ -2.2 (c 0.18, CHCl₃); For ¹H and ¹³C NMR spectroscopic data, see Table 1 and Table 2; HRESIMS m/z 287.2364 [M + H]⁺ (calcd. for C₂₀H₃₁O⁺, 287.2369).

3.3.2. Nanolobatol A (2)

Colorless oil, $\left[\mathsf{\alpha }\right]_{\mathrm{D}}^{20}$ -116.4 (c 0.14, CHCl₃); For ¹H and ¹³C NMR spectroscopic data, see Table 1 and Table 2; HRESIMS m/z 305.2477 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475).

3.3.3. Nanolobatol B (3)

Colorless oil, $\left[\mathsf{\alpha }\right]_{\mathrm{D}}^{20}$ +10.5 (c 0.43, CHCl₃); For ¹H and ¹³C NMR spectroscopic data, see Table 1 and Table 2; HRESIMS m/z 337.2737 [M + H]⁺ (calcd. for C₂₁H₃₇O₃⁺, 337.2737).

3.3.4. Nanolobatol C (4)

Colorless oil, $\left[\mathsf{\alpha }\right]_{\mathrm{D}}^{20}$ +20.5 (c 0.10, CHCl₃); For ¹H and ¹³C NMR spectroscopic data, see Table 1 and Table 2; HRESIMS m/z 305.2471 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475).

3.3.5. Nanolobatol D (5)

Colorless oil, $\left[\mathsf{\alpha }\right]_{\mathrm{D}}^{20}$ +6.5 (c 0.10, CHCl₃); For ¹H and ¹³C NMR spectroscopic data, see Table 1 and Table 2; HRESIMS m/z 305.2472 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475).

3.3.6. 2-Epi-Nanolobatone B (7)

Colorless oil, ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ +180.0 (c 0.16, CHCl₃); For ¹H and ¹³C NMR spectroscopic data, see Table 1 and Table 2; HRESIMS m/z 305.2475 [M + H]⁺ (calcd. for C₂₀H₃₃O₂⁺, 305.2475).

3.4. Calculation Section

Conformational searches were performed using the torsional sampling (MCMM) approach and OPLS_2005 force field within an energy window of 21 kJ/mol. Conformers with Boltzmann populations above 1% were re-optimized at the B3LYP/6-311G(d,p) level using the IEFPCM solvent model for chloroform. Frequency analysis was also carried out to confirm that the re-optimized geometries were at the energy minima. Subsequently, NMR calculations were performed at the PCM/mPW1PW91/6-31G(d) level, as recommended for DP4+ [16]. NMR shielding constants were calculated using the GIAO method. Finally, shielding constants were averaged over the Boltzmann distribution obtained for each stereoisomer and correlated with the experimental data. ECD spectra were obtained by TDDFT calculations with the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for CH₃CN. Finally, the Boltzmann-averaged ECD spectra of the compounds were obtained using SpecDis 1.62.

3.5. Cytotoxicity Assays

The in vitro cytotoxic activities of compounds 1–16 were evaluated against MV-4-11, A-549, and 293T cell lines using the CCK-8 assay, with doxorubicin (DOX) serving as the positive control. The human non-small cell lung carcinoma cell line (A-549) was obtained from National Collection of Authenticated Cell Cultures of China, while the human myeloid monocytic leukemia (MV-4-11) and human embryonic kidney (HEK) 293T cell lines were purchased from Zhejiang Meisen Cell Technology Co., Ltd. The growth inhibitory effects of compounds on cancer cells from different tissue sources were tested at five concentration gradients. The maximum tested concentration was 50 µM, diluted fivefold, and the cells were treated with the five concentration gradients for 72 h. Compounds showing an inhibition rate greater than 60% at 50 µM were selected for further screening, and their IC₅₀ values were calculated.

3.6. Anti-Inflammatory Assays

The murine macrophage cell line RAW264.7 was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The detailed experimental procedures were consistent with those described in our previous work [17]. The half maximal cytotoxic concentration (CC₅₀) and IC₅₀ values were estimated using the log (inhibitor) vs. normalized response nonlinear fit (GraphPad Prism 6.0). Dexamethasone was used as the positive control.

4. Conclusion

In this study, a total of 16 compounds were isolated and identified from the South China Sea soft coral S. nanolobata, which possessed the cembrane- and casbane-type carbon skeletons. Among them, compounds 1–5 were five new cembrane-type diterpenes and compound 7 was a new casbane-type diterpene. Interestingly, the new compounds 2–5 featured rare five- to seven-membered oxygen-containing heterocyclic rings as their structural highlights. In bioassays, all isolated compounds showed no significant cytotoxic or anti-inflammatory activities (IC₅₀ > 50 µM), suggesting that these novel structures may not be primarily responsible for such effects.

Given the structural novelty and the lack of cytotoxicity or anti-inflammatory activity observed, future investigations should explore alternative biological evaluations. For instance, antimicrobial, antiviral, neuroprotective, or enzyme inhibitory assays may reveal other potential bioactivities of these rare oxacyclic ring systems. Furthermore, chemical derivatization or semi-synthesis of these novel scaffolds might improve their bioactivity profiles by introducing pharmacophoric groups. Overall, the discovery of these structurally unique diterpenoids extended the chemical diversity of S. nanolobata and provided valuable leads for future marine natural product-based drug discovery efforts, particularly in the search for novel mechanisms of action beyond conventional cytotoxicity and anti-inflammatory pathways.