Two New Diterpenoids Formed by Transannular Diels-Alder Cycloaddition from the Soft Coral Sarcophyton tortuosum and Their Antibacterial and PPAR-β Agonist Activities

1. Introduction

Soft corals of the genus Sarcophyton (phylum, Cnidaria; class, Anthozoa; subclass, Octocorallia; order, Alcyonaceae; family, Alcyoniidae) dominated many coral reef areas, which are characterized by a smooth mushroom-shaped appearance. [1] Chemical investigation studies have shown that the soft corals of this genus furnished a wealth of secondary metabolites, which possess complex/intriguing structural characteristics and exhibit a range of biological activities.[2] Notable metabolites include sesquiterpenes, diterpenes, diterpene dimers, prostaglandins, and steroids. [3,4]

Sarcophyton tortuosum is widely distributed in the tropic oceans, especially the South China Sea, which have been continuously chemical investigated by our group for over a decade, leading to the isolation and characterization of a variety of structurally intriguing and biologically active diterpenes and diterpene dimers.[5,6,7,8,9] Among them, eunicellane-type diterpenoids, cembrane-type diterpenoids, and especially the biscembranoids (diterpene dimers) are characteristic secondary metabolites of this soft coral. Cembranoids, diterpenes with a 14-membered macrocycle, have been known to be common intermediates in the biogenesis of polycyclic diterpenes and biscembranoids by the intramolecular 2+2 cycloaddition and intermolecular 4+2 (Diels-Alder) cycloaddition,[10,11,12] respectively. However, no natural product example of polycyclic diterpenoid formed by intramolecular 4+2 (Diels-Alder) cycloaddition from cembranoids, and only a few biomimetic syntheses in natural products with Diels-Alder reaction,[13,14] which parked our great interest in searching for more evidences of natural products produced by intramolecular Diels-Alder cycloaddition from soft corals.

In this work, the chemical investigation on a new collection of soft coral S. tortuosum from Ximao Island, Hainan Province, China, has resulted in the isolation and full characterization of two previously undescribed compounds 1 and 2, along with two known compounds 3 and 4 (Figure 1). Herein, the isolation, structure elucidation, plausible biosynthetic pathway, and biological evaluation of these isolated compounds were described.

2. Results and Discussion

Freshly collected specimens of S. tortuosum were frozen to –20°C before exhaustively extracted with acetone. The Et₂O-soluble portion of the acetone extract of the title animal was subjected to repeated chromatography, including silica gel, Sephadex LH-20, and RP-HPLC to yield four compounds (1–4). The known compounds 3 and 4 were readily identified as 4-oxochatancin (3),[15] and (+)-chatancin (4),[16] respectively, by direct comparison of their NMR spectroscopic data and specific rotation value with those reported in the literatures.

Compound 1 was isolated as a colorless crystal. Its molecular formula of C₂₁H₃₂O₅ was established by the quasi-molecular ion peak at m/z 387.2129 ([M+Na]⁺, calc. 387.2147 for C₂₁H₃₂O₅Na) in the HR-ESIMS spectrum, indicating six degrees of unsaturation. The IR spectrum displayed strong absorption at 3460 cm⁻¹ and 1712 cm⁻¹, suggested the presence of hydroxyl and carbonyl groups. The ¹H NMR, ¹³C NMR, and DEPT spectra of 1 suggested the presence of one trisubstituted double bond [δ_H 7.27 (1H, s, H-9), δ_C 145.11 (CH, C-9) and δ_C 134.82 (qC, C-10)], one carbonyl carbon atom [δ_C 165.41 (qC, C-16)], and one oxymethine [δ_H 3.53 (1H, m, H-4), δ_C 77.0 (CH, C-4)]. Consequently, the remaining four degrees of unsaturation led to the fact that compound 1 was a tetracyclic structure.

Detailed comparison revealed that the NMR data (Table 1) of compound 1 were almost identical to those of co-occurred compounds 3 and 4, which had been previously isolated from the tropical marine slug Phyllodesmium longicirrum that prey on soft corals of the genus Sarcophyton. The only difference between 1 and 3 was that the carbonyl group at the C-4 position in 3 was replaced by a hydroxyl group in 1, which as evidenced by the 2 mass unit difference in their ESIMS data. Thus, the planar structure of 1 was determined as shown in Figure 1.

In the NOESY spectrum, there was a clear correlation between H-4 (δ_H 3.53, m), CH₃-14 (δ_H 0.85, s), H-2 (δ_H 1.56) and H-10a (δ_H 2.79, d, J=1.73), indicating that H-2, H-4, CH₃-14 and H-10a are on the same side of the molecule, and randomly assigned as b-orientation (Figure 2). Subsequently, the interactions of H-4b (δ_H 1.60)/CH₃-15(δ_H 1.00, d, J=6.3), revealed that these protons and/or proton-bearing groups were positioned at the other side of the molecule, thus a-orientation. In the light of above observation, the relative configuration of C-2, C-4, C-4a, C-4b, C-7, and C-10a in 1 was assigned to be 2S*, 4S*, 4aR*, 4bR*, 7S, and 10aR*, respectively. In addition, the relative stereochemistry of C-1 and C-8a in 1 was tentatively determined to be the same 1R* and 8aR* as that of 3 on basis of their highly similar ¹³C NMR data.

The absolute configuration of 1 was determined by comparing its ECD spectra with that of 3. As shown in Figure 3, the cotton effects in the ECD spectra of the two compounds are consistent with each other. Therefore, the absolute configuration of 1 was ultimately determined as 1R, 2S, 4S, 4aR, 4bR, 7S, 8aR, 10aR.

In order to unambiguously confirm the structure and absolute configuration of 1, the suitable single crystals of 1 in MeOH were obtained. The X-ray crystallographic analysis using Cu Kα radiation (λ=1.54178 Å) firmly disclosed the planar structure of 1 and determined its absolute configuration as 1R, 2S, 4S, 4aR, 4bR, 7S, 8aR, 10aR with the Flack parameter of 0.08(7) (Figure 4, CCDC 2384793). Consequently, the structure of 1 was elucidated as depicted in Figure 1 and named as 4a-hydroxy-chatancin.

Sarcotoroid (2) was isolated as colorless oil, which possessed the molecular formula of C₂₁H₂₈O₅ as assigned by HR-ESIMS protonated molecular ion peak at m/z 361.2004 ([M+H]⁺, calc. for C₂₁H₂₉O₅, 361.2010), requiring eight degrees of unsaturation. The IR spectrum of 2 showed absorption bonds at 3300 cm^–1 and 1727 cm^–1 suggested the presence of hydroxy and easter carbonyl functionalities. The 1D and 2D NMR spectra of 2 revealed the presence of two trisubstituted double bonds [δ_H 5.86 (1H, s, H-6), δ_C 161.51 (qC, C-7), and δ_C 127.46 (CH, C-6); δ_H 5.89 (1H, s, H-9), δ_C 134.19 (qC, C-10), and δ_C 135.25 (CH, C-9)], one carbonyl group [δ_C 197.91 (qC, C-5)], one methyl ester group [δ_H 3.71 (3H, s, H-17) and δ_C 175.33 (qC, C-14)], and one hemiketal group [δ_C 106.36 (qC, C-3)]. The above functionalities accounted for four degrees of unsaturation, suggesting that compound 2 must possess a tetracyclic ring system accounting for the remaining four degrees of unsaturation.

Detailed analysis of ¹H‒¹H COSY spectrum of 2 revealed two sequential fragments (Figure 5) by the clear correlations of H-4b [δ_H 2.91 (d, J = 13.0)]/H-8a [δ_H 3.02, m]/H-8 [δ_H 2.25, m]/H-9 [δ_H 5.89, m], and CH₃-12 [δ_H 0.88 (d, J = 6.7)]/CH₃-13[δ_H 0.90 (d, J = 6.7)]/H-11 [δ_H 1.98, m]/H-2 [δ_H 2.18, m]/H₂-1 [δ_H 1.82 (dd, J = 13.1, 6.5), δ_H 1.46 (dd, J = 13.1, 8.3)]. The key HMBC correlations from H-4 to C-3, C-4a, and C-10a, from H-1 to C-2, C-3, C-10a, and C-4a, indicated the present of ring (a) in the structure. The second ring (b) was confirmed by HMBC correlations from H-4b to C-4a, C-8a, and C-9; as well as from CH₃-16 to C-9, C-10, and C-10a. The diagnostic HMBC correlation from H-4b to C-8a, C-8, and C-5 and from CH₃ to C-6, C-7, and C-8 agreed with the presence of the third ring (c). Considering the downfield shift of the two quaternary carbons C-3 (δ_C 106.36) and C-10a (δ_C 81.47), the remaining one degree of unsaturation must be given by the formation of an oxygen bridge between C-3 and C-10a. Finally, the planar structure of 2 was determined as shown in Figure 5.

The observed NOESY correlations between H-6 (δ_H 5.86)/CH₃-15 (δ_H 1.96) and H-9 (δ_H 5.89)/CH₃-16 (δ_H 1.86) demonstrated the Z geometries of the double bonds at Δ^6,7 and Δ^9,10. The NOE correlations of H-4b/H-8a suggested that H-4b and H-8a were in same orientation. The NOE correlations of H-8a/Ha-4 and Hb-4/H-2 indicated that H-2 and H-8a were in opposite orientation. Thus, the relative configuration of C-4b, C-8a, and C-2 was assigned as 2R*, 4bS*, and 8aS*. Due to the advantageous conformation of the five membered ring in the envelope form, the relative configuration of C-3, C-10a was determined to be 3R* and 10aS*. However, the relative configuration of C-4a remains a challenge task due to the absence of related NOESY correlations. Therefore, the quantum mechanical-nuclear magnetic resonance (QM-NMR) method, which has been widely used to define the relative configuration of multi stereogenic centers of natural products,[17] was applied. Four possible candidate structures, namely 2a (2R*, 3R*, 4aS*, 4bS*, 8aS*, 10aS*), 2b (2R*, 3R*, 4aR*, 4bS*, 8aS*, 10aS*), 2c (2R*, 3S*, 4aR*, 4bS*, 8aS*, 10aR*), and 2d (2R*, 3S*, 4aS*, 4bS*, 8aS*, 10aR*) were calculated for their theoretical NMR data. Following the DP4+ protocol, geometrical optimization at the DFT level was undertaken by using the B3LYP functional with the 6–311G(d,p) basis set.[18] Afterward, the NMR calculation was conducted at the mPW1PW91/6-31G* level, and the experimental NMR data of 2 gave the best match to that of 2a with over 99% probability. (Figure 6). Moreover, the TDDFT-ECD method was also performed to determine the absolute configuration of 2.[19,20] As shown in Figure 7, the calculated ECD curve of (2R, 3R, 4aS, 4bS, 8aS, 10aS)-2 fits good agreement with the experimental ECD curve of 2. Finally, the absolute configuration of 2 was assigned as 2R, 3R, 4aS, 4bS, 8aS, 10aS as shown in Figure 1.

It is worth pointing out that the structural similarities between compounds 1–4, and their co-occurrence in the same sample suggested that they may originate from the same biogenetic pathway. A plausible biosynthetic pathway for structural correlations among the related diterpenoids 1–4 was proposed. As shown in Scheme 1, compounds 1, 3 and 4 herein probably have the same precursor macrocyclic diketone a. Firstly, the C-6 of a undergoes enol interconversion, resulting in the formation of conjugated double bonds at Δ^3,4 and Δ^5,6 in b. Subsequently, these conjugated double bonds participate in a transannular Diels-Alder cycloaddition with the double bond at Δ^11,12, leading to the formation of the core structure featuring an uncommon 6/6/6-tricyclic ring system. Finally, the lone pair electrons of the oxygen atom in 6-OH attacked the C-2 carbon atom, resulting in the formation of 4 with an epoxy bridge between C-6 and C-2. Following by two steps of oxidation, compounds 1 and 3 are ultimately obtained. Similarity, the precursor methyl sarcoate (5)[21] undergoes enol interconversion yielded d, the conjugated double bonds at Δ^10,11 and Δ^12,13 of d undergo a transannular Diels-Alder cycloaddition with the double bonds at Δ^4,5 to generate e, followed by the nucleophilic attack of hydroxyl group 13-OH on C-2 formed 2.

In bioassay, all the isolated compounds were tested for their antibacterial effects. Streptococcus parauberis is the primary pathogen responsible for fish-borne streptococcal disease, which is widely distributed and highly pathogenic, causing significant economic losses in fish aquaculture worldwide. S. parauberis can infect a diverse range of fish, including both freshwater and farmed or wild species, exhibiting clinical symptoms similar to septicemia. However, these strains have gradually developed drug resistance, including multidrug resistance. The S. parauberis SPOF3K strain is a drug-resistant isolate from aquaculture that exhibits significant resistance to tetracycline and oxytetracycline, with the MIC of over 24 μg/mL and 12.42 μg/mL, respectively. Notably, compound 1 exhibited good antibacterial activity against the S. parauberis KSP28 and oxytetracycline-resistant S. parauberis SPOF3K, with an MIC value of 9.10 μg/mL, which is superior to the antibacterial effect of oxytetracycline (MIC = 12.42 μg/mL).

Photobacterium damselae (formerly Vibrio damsela) is a halophilic bacterium associated with marine environments, known to cause skin ulcers in damselfish. It is a primary pathogen responsible for ulcers and hemorrhagic septicemia in various marine species, including dolphins, sharks, and shrimp, as well as both wild and cultivated fish. Additionally, this pathogen can lead to fatal infections in humans.[22] Compound 1 showed significant antibacterial activities against the P. damselae FP2244 with the MIC value of 18.21 μg/mL.

Besides, the isolated compounds were tested for antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, Enterobacter cloacae, Enterobacter hormaechei, Aeromonas sabnonicida, Photobacterium halotolerans, Lactococcus garvieae FP MP5245, and several strains of vancomycin-resistant Enterococcus faecium (G1, G4, G7, and G13), as well as Streptococcus agalactiae, Edwardsiella piscicida TH1, Vibrio parahaemolyticus, and Vibrio alginolyticus. However, these results were negative at the concentration of 100 μM.

Table 2. Antibacterial activities of compound 1 and antibiotics.^a.

Compounds	MIC (μg/mL)
	S. parauberis	S. parauberis SPOF3K	P. damselae FP2244
1	9.10	9.10	18.21
Tetracycline	3.01	＞24.00	0.02
Oxytetracycline	1.55	12.42	0.02
Levofloxacin	1.24	1.24	0.02
Ampicillin	4.64	0.58	0.02

^a Tetracycline, oxytetracycline, levofloxacin, and ampicillin were used as positive controls.

Table 2. Antibacterial activities of compound 1 and antibiotics.^a.

Compounds	MIC (μg/mL)
	S. parauberis	S. parauberis SPOF3K	P. damselae FP2244
1	9.10	9.10	18.21
Tetracycline	3.01	＞24.00	0.02
Oxytetracycline	1.55	12.42	0.02
Levofloxacin	1.24	1.24	0.02
Ampicillin	4.64	0.58	0.02

^a Tetracycline, oxytetracycline, levofloxacin, and ampicillin were used as positive controls.

Peroxisome proliferator-activated receptors (PPAR) include PPAR-α, PPAR-β, and PPAR-γ isotypes, which are transcription factors that regulate gene expression upon ligand activation. PPAR-α enhances cellular fatty acid uptake, esterification, and trafficking, while also regulating genes involved in lipoprotein metabolism. PPAR-β stimulates lipid and glucose utilization by increasing mitochondrial function and fatty acid desaturation pathways. While PPAR-γ promotes fatty acid uptake, triglyceride formation, and storage in lipid droplets, which enhances insulin sensitivity and glucose metabolism.[23] The compounds 1, 3 and 4 were evaluated for PPAR transcriptional activity using luciferase assay (Figure 8). As shown in Figure 8A, these compounds were first evaluated for their cytotoxicity against Ac2F cells. Compounds 1, 3 and 4 demonstrated no cytotoxic effects on Ac2F cells at a concentration of 10 μM after 24 h treatment. Notably, compound 3 exhibited significant and selective PPAR-β agonist activity at the concentration of 10 μM, comparable to that of the positive control GW501516 at 5 μM.

3. Materials and Methods

3.1. General Experimental Procedures

Melting points were measured on an X-4 digital micro-melting point apparatus. The X-ray measurement was made on a Bruker D8 Venture X-ray diffractometer with Cu Kα radiation (Bruker Biospin AG, Fällanden, Germany). Optical rotations were measured on a Perkinelmer 241 MC polarimeter. IR spectrum was recorded on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Madison, WI, USA). ¹H and ¹³C NMR spectra were acquired on a Bruker DRX-600 spectrometer (Bruker Biospin AG, Fällanden, Germany). Chemical shifts are reported with the residual CDCl₃ (δ_H 7.26 ppm) as the internal standard for ¹H-NMR spectrometry and CDCl₃ (δ_C 77.26 ppm) for ¹³C-NMR spectrometry. The HR-ESI-MS spectra were recorded on a ZenoTOF7600 mass spectrometer (SCIEX). Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200–300, and 300–400 mesh) was used for column chromatography, and Sephadex LH-20 gel (Amersham Biosciences) were used for column chromatography (CC), and precoated-silica-gel-plates (G60 F-254, Yan Tai Zi Fu Chemical Group Co., Yantai, China) were used for analytical TLC. Spots were detected on TLC under UV light or by heating after spraying with anisaldehyde H₂SO₄ reagent. Reversed phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatograph equipped with a DAD G1315D detector at 210 nm (Agilent, Santa Clara, CA, USA). An Agilent semi-preparative XDB-C18 column (5 μm, 250×9.4 mm) was employed for the purification. 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

The soft coral S. tortuosum was collected from Ximao Island, Hainan Province, China, in 2023 at a depth of –20 meters, and identified by Professor Xiu-Bao Li from Hainan University. A voucher specimen (No. 23-YT-20) is available for inspection at the Shandong Laboratory of Yantai Drug Discovery.

3.3. Extraction and Isolation

The frozen animals (800.4 g, dry weight after extraction) were cut into pieces and extracted exhaustively with acetone at room temperature (4×2L). The organic extract was evaporated to give a brown residue, which was then partitioned between diethyl ether (Et₂O) and H₂O. The Et₂O solution was concentrated under reduced pressure to obtain a dark brown residue (58.0 g), which was classified by silica gel column chromatography (200-300 mesh) and eluted with a step gradient [0–100% Et₂O in petroleum ether (PE) solution to obtain six fractions (A–G). Fraction D (2.1 g) was subjected to chromatography on a Sephadex LH-20 column using a petroleum ether (PE)/CH₂Cl₂/MeOH (2:1:1) elution system, yielding subfractions DA to DC. Purification of the DB subfraction was performed using Sephadex LH-20 (CH₂Cl₂), followed by silica gel column chromatography with a PE/Et₂O (20:1) elution, yielding four subfractions (DBAA–DBAD). Further purification of DBAB by RP-HPLC [CH₃CN/H₂O (85:25), 3.0 mL/min] led to the isolation of compound 4 (4 mg, t_R = 29 min). Fraction F (2.3 g) was purified by chromatography on Sephadex LH-20, as described above, yielding subfractions FBA–FBE and FCA–FCH. Subfraction FBA was subjected to silica gel column chromatography, eluting with PE/Et₂O (15:1), to give five subfractions (FBAA–FBAE). Further purification of subfraction FBAB by semi-preparative RP-HPLC (CH₃CN/H₂O, 70:30) yielded compound 3 (2 mg, t_R = 23.5 min). Further purification of subfraction FCD by semi-preparative RP-HPLC (CH₃CN/H₂O, 52:48) yielded compound 1 (1.2 mg, t_R =11 min), and purification of subfraction FCF by semi-preparative RP-HPLC (CH₃CN/H₂O, 45:55) yielded compound 2 (1.7 mg, t_R = 16 min).

3.4. Spectroscopic Data of Compounds

4a-hydroxy-chatancin (1): Colorless crystal, (m. p. 122-124°C), ${\left[\alpha \right]}_D^{20}$ –2 (c 0.10, MeOH); IR (KBr): ν_max 3460, 2952, 2925, 2868, 1712, 1268 cm⁻¹; For ¹H and ¹³C NMR spectroscopic data, see Table 1; HR-ESI-MS m/z 387.2129 ([M+Na]⁺; calcd. for C₂₁H₃₂O₅Na, 387.2147).

Sarcotoroid (2): Colorless oil, ${\left[\alpha \right]}_D^{20}$ –16 (c 0.10, MeOH); IR (KBr): ν_max 3300, 2952, 2918, 2850, 1727, 1627 cm⁻¹; For ¹H and ¹³C NMR spectroscopic data, see Table 1; HR-ESI-MS m/z 361.2004 ([M+H] ⁺; calc. for C₂₁H₂₉O₅, 361.2010).

3.5. Calculation Section

For the QM-NMR calculations of compounds, conformational search was performed by using the torsional sampling (MCMM) approach and the OPLS_2005 force field applying an energy window of 21 kJ/mol (5.02 kcal/mol). Reoptimize the conformers of populations exceeding 1% Boltzman populations at the B3LYP/6-311G (d, p) level. Subsequently, nuclear magnetic resonance calculations were performed at the PCM/mPW1PW91/6-31G (d) level. The shielding constant of NMR was calculated using the GIAO method. Finally, calculate the average shielding constant for each stereoisomer Boltzmann distribution and correlate it with experimental data.

Regarding the TDDFT-ECD calculations of compounds, conformational search was carried out according to the general protocols previously described for QM-NMR calculation. Reoptimized the conformers above 1% Boltzmann population and used the IEFPCM solvent model for TDDFT-ECD calculations, using Gaussian 09 at the theoretical B3LYP/6-311G (d, p) level. Finally, the SpecDis 171 software was used to obtain the calculated ECD spectrum and visualize the results.

3.6. X-ray crystallographic Analysis for Compound 1

The crystallographic data were collected on a Bruker D8 Venture diffractometer equipped with Cu Kα radiation (λ = 1.54178 Å). The structures were solved with the ShelXT structure solution program using Intrinsic Phasing and refined with the ShelXL refinement package using Least Squares minimisation.

Compound 1: colorless crystals (m.p. 122-124°C), monoclinic, C₂₁H₃₄O₆, Mr = 382.48, (including a water molecule) crystal size 0.2 × 0.15 × 0.04 mm³, space group C2, a = 19.2786(5) Å, b=11.3201(3)Å, c = 10.4695(3) Å, V = 2061.47(10) ų, Z = 4, ρcalc = 1.232 g/cm³, F (000) = 832.0, Independent reflections: 3908 [R_int = 0.0656, R_sigma = 0.0341]. R₁ = 0.0331, wR₂ = 0.0810 reflections with I ≥ 2σ (I), R₁ = 0.0331, wR₂ = 0.0810 for all unique data, Flack parameter: 0.08(7). The crystals of 1 were recrystallized from MeOH. These above crystal data were deposited in the Cambridge Crystallographic Data Centre (CCDC) and assigned the accession number (CCDC 2384793).

3.7. Antibacterial Activity Bioassays

The bacterial strains S. parauberis KSP28, S. parauberis SPOF3K, P. damselae FP2244, A. sabnonicida, P. halotolerans and L. garvieae FP MP5245, P. aeruginosa, E. coli, E. cloacae, E. hormaechei, A. salmonicida, S. agalactiae, V. parahaemolyticus, and V. alginolyticus were provided by National Fisheries Research & Development Institute, Korea. The vancomycin-resistant strains E. faecium bacteria G1, G4, G7 and G13, and S. aureus were provided by Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. The E. piscicida TH1 was provided by Chinese Academy of Tropical Agricultural Sciences. were also used in antibacterial experiments.

Minimal inhibitory concentration (MIC) for compounds were determined using the 0.5 McFarland standard method. Mueller–Hinton II broth (cation-adjusted, BD 212322) was used for bacterial culture. Compounds were generally dissolved in DMSO to a concentration of 2 mM as stock solutions. All samples were diluted in culture broth to an initial concentration of 100 µM. Serial 1:1 dilutions were then performed using culture broth to achieve concentrations ranging from 100 µM to 0.24 µM. A total of 5 µL of each dilution was distributed into 96-well plates, along with sterile controls, growth controls (containing culture broth and DMSO without compounds), and positive controls (containing culture broth with antibiotics). All wells except sterile control well were inoculated with 95 µL of an exponential-phase bacterial suspension (approximately 10⁵ CFU/well). The plates were incubated at 37°C for 12 h. MIC values were defined as the lowest concentration that completely inhibited bacterial growth. All MIC values were interpreted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. The MIC in molar concentration was ultimately converted to mass concentration based on the molecular weight of each compound.

3.8. Cell Culture and Cell Viability

Rat liver Ac2F cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in Dulbecco's modified Eagle medium (DMEM) containing 2 mM L-glutamine, 100 mg/ml streptomycin, 2.5 mg/L amphotericin B, and 10% heat-inactivated fetal bovine serum (FBS) and were maintained in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were seeded in 96-well culture plates, cultured for 12 h, and then treated with various concentrations of samples for 24 h. Cell viabilities were evaluated using water-soluble tetrazolium (CCK-8) reagent, which was added to each well (10 mL) and incubated at 37°C for 1 h. Absorbances were read using an microplate absorbance reader at a wavelength of 450 nm.

3.9. Luciferase Transactivation Assays

For luciferase assays, the 3×AOX-TK-luciferase reporter plasmid, containing 3 copies of the PPRE in the acyl CoA oxidase promoter was a gift from Dr. Chistopher K. Glass (University of California at San Diego, La Jolla, CA, USA). The pcDNA3 expression vector and full-length human PPAR-α, PPAR-β, or PPAR-γ expression vector (pFlag-PPAR-α, pFlag-PPAR-β, or pFlag-PPAR-γ) were gifts from Dr. Chatterjee (the University of Cambridge, Addenbrooke's Hospital at Cambridge, UK). For the experiment, 1 μg of 3×AOX-TK-luciferase reporter plasmid were transfected into Ac2F cells in a 48-well plate (1×10⁵ cells/well) with 0.1 μg of effector plasmids, including pcDNA3, or pFlag-PPAR-α/β/γ by using Lipofectamine™ 2000 as the manufacturer's instructions. After transfection for 6 h, the conditioned media was removed and replaced with serum-free media, and the compounds were added. After an additional incubation for 7 h, cells were washed with PBS and assayed with the Luciferase Assay Systems (Promega, Madison, WI, USA). Luciferase activity was measured using a GloMax-Multi Microplate Multimode Reader (EnVision, PerkinElmer, Uk).

4. Conclusions

In summary, two previously unreported polycyclic diterpenoids (1 and 2) together with two structural analogues (3 and 4) were isolated and characterized from the soft coral S. tortuosum. Structurally, new compounds 1 and 2 represented the first example of cembrane-derived tricyclic diterpenes belonging to sarcophytin family that were isolated from S. tortuosum, which expanded the chemical diversity of diterpenes from soft coral of this species. To the best of our knowledge, only ten members have been reported from the soft corals of Sarcophyton genus and their predator sea slug Phyllodesmium longicirrum.[2,15] The stereochemistry of the new compounds was unambiguously determined using a combination of X-ray diffraction analysis, QM-NMR method, and TDDFT-ECD calculation. A plausible biosynthetic pathway for compounds 1–4 was proposed by undergoing an interesting transannular Diels-Alder cycloaddition. The intramolecular 4+2 cycloaddition reactions occurring in compounds 1 and 2 exhibit significant differences, manifested in different diene positions, which resulted in the carbomethoxy group being attached to the C-10 position of compound 1 and the C-4a position of compound 2, respectively. A study by Pierre and coworkers achieved an asymmertric total synthesis of (+)-chatancin via a transannular Diels-Alder reaction, demonstrated the rationality of above proposed biosynthetic pathway.[13] In bioassays, the new compound 1 exhibited antibacterial activities against fish pathogens S. parauberis KSP28, oxytetracycline-resistant S. parauberis, and P. damselae FP2244, while compound 3 showed selective PPAR-β agonist activity. These intriguing findings highlight the potential of sarcophytin-like tricyclic diterpenes as leader compounds for the treatment of microbial infections and metabolic diseases.

Statistical Analysis

Each experiment was repeated at least three times. The experimental data were analyzed using one-way analysis of variance (ANOVA) and are presented as the mean ± standard deviation after assessment.