Structurally Diverse Diterpenes from the South China Sea Soft Coral Sarcophyton trocheliophorum

The present investigation of the South China Sea soft coral Sarcophyton trocheliophorum resulted in the discovery of six new polyoxygenated diterpenes, namely sartrocheliols A–E (1, 3, 5–8) along with four known ones, 2, 4, 9, and 10. Based on extensive spectroscopic data analysis, sartrocheliol A (1) was identified as an uncommon capnosane diterpene, while sartrocheliols B–E (3, 5–8) were established as cembrane diterpenes. They displayed diverse structural features not only at the distinctly different carbon frameworks but also at the various types of heterocycles, including the epoxide, γ-lactone, furan, and pyran rings. Moreover, their absolute configurations were determined by a combination of quantum mechanical-nuclear magnetic resonance (QM-NMR) approach, modified Mosher’s method, and X-ray diffraction analysis. In the anti-tumor bioassay, compound 4 exhibited moderate cytotoxic activities against A549, H1975, MDA-MB-231, and H1299 cells with the IC50 values ranging from 26.3 to 47.9 μM.

In the last decades, we systematically carried out chemical and biological studies on South China Sea marine fauna and flora [36]. As a result, numerous terpenes with complex structures, some of which possessed unprecedented carbon frameworks, were found from In the last decades, we systematically carried out chemical and biological studies on South China Sea marine fauna and flora [36]. As a result, numerous terpenes with complex structures, some of which possessed unprecedented carbon frameworks, were found from the genus Sarcophyton [20,37,38]. During our continuous research, we frequently encountered the soft coral Sarcophyton trocheliophorum, one productive species belonging to the above-mentioned genus. Previously, our group disclosed a vast array of diterpenes with four skeletons, and these metabolites showed a broad spectrum of pharmacological activities such as protein tyrosine phosphatase 1B (PTP1B) inhibitory, antitumor, antibacterial and neuroprotective activities [39]. It was interesting to notice that the chemical profiles of diterpenes from the title soft corals varied upon temporal variations and geographical distributions. The samples collected in Yalong Bay, Hainan Island, South China Sea in February 2006 yielded diterpenes with an unprecedented carbon framework [37] together with sarsolenane, capnosane, and cembrane skeletons [39]. However, the specimen collected in the same water but in May 2006 only yielded cembrane diterpenes [40]. Of more interest, the soft corals collected from another different region, Ximao Island, South China Sea, merely afforded the cembranoids [41]. These findings probably reflected the existence of different metabolic processes in different seasons and inhabiting environments, which needs to be further investigated.
In order to obtain more evidence to disclose that the diterpenoid profile of the title animals was influenced by temporal variations, we made a new collection of S. trocheliophorum from Ximao Island. In the current study, six new polyoxygenated diterpenes, namely sartrocheliols A-E (1, 3, 5-8), and four known related analogs 2, 4, 9, and 10 were obtained ( Figure 1). These diterpenes displayed two distinctly different carbon frameworks: capnosane and cembrane, the former of which has rarely been found in soft corals. Meanwhile, various types of heterocycles, including the epoxide, γ-lactone, furan, and pyran rings were incorporated in their macrocyclic skeletons. Herein, we report the detailed structural elucidation of these isolates from the title soft corals, especially the challenging stereochemistry determination of the new compounds, which was dissolved by a combination of quantum mechanical-nuclear magnetic resonance (QM-NMR) approach, modified Mosher's method, and X-ray diffraction analysis. In addition, their biological evaluations including cytotoxicity against a panel of cancel cells and antibacterial against an array of bacteria are described.

Results and Discussion
The frozen animals were cut into pieces and extracted exhaustively with acetone. Then an Et2O-soluble portion of the acetone extract was repeatedly chromatographed over silica gel, Sephadex LH-20, and RP-HPLC to yield six new compounds 1, 3, 5-8, and four
Sartrocheliol B (3) was isolated as optically active colorless oil. Its molecular formula C 22  , which was indicative of six degrees of unsaturation. The IR spectrum of 3 suggested the presence of lactone (ν max 1759 cm −1 ), ester (ν max 1736 cm −1 ) and hydroxyl (ν max 3442 cm −1 ) groups, while a strong UV absorption at λ max 231 nm (logε 3.83) suggested the presence of α,β-unsaturated γ-lactone group [43]. According to the 1 H and 13 C NMR data of 3 (Tables 1 and 2 (qC, C-18)), one trisubstituted double bond (δ H 5.05 (1H, t, J = 7.7 Hz, H-11), δ C 129.20 (CH, C-11), 130.80 (qC, C-12)) and one acetyl group (δ H 2.09 (3H, s), δ C 21.35 (CH 3 ), 171.13 (qC)). The remaining one degree of unsaturation implied the monocyclic nature of this molecule. Considering the co-isolated cembranoids 4, 9 and 10, metabolite 3 was likely a cembrane-type diterpene. Through detailed literature reviews on diterpenes from the genus Sarcophyton, the above-mentioned structural features were reminiscent of previously reported sarcophytonolide H (11) [43], a cembrane from South China Sea soft coral Sarcophyton latum. Our interpretation of the 2D NMR spectra (Figure 3) suggested they differed by the lack of the double bond ∆ 7 , which was supported by the significantly up-field shifted chemical shifts of C-7 and C-8 (∆δ C ca. 80 and 110 ppm, respectively). Similar patterns of NOE correlations in the NOESY spectra of  Figure 5) established the absolute configuration S for C-6 in 3. Considering the correlation between H-6 and H 3 -19, the absolute configuration of C-8 could be determined as S. It might be worth pointing out that the absolute configuration of sarcophytonolide H (4) had already been ambiguously confirmed as 1R,2R,6R,14S by the single crystal X-ray diffraction experiment in this study ( Figure 2). The CD spectrum of 3 displayed the Cotton effect resembling that of co-isolated 4 ( Figure 6), suggesting they shared the same absolute configuration R for the chrial carbon C-2 of the chromophore α,β-unsaturated γ-lactone. With the relationships of H-1, H-2, and H-14 in hand, the absolute configurations of C-1 and C-14 could be assigned as R and S, respectively. Consequently, the absolute configuration of 3 could be established as 1R,2R,6S,8S,14S.   Sartrocheliol B (3) was isolated as optically active colorless oil. Its molecular formula C22H34O5 was established by the HRESIMS protonated molecule peak at m/z 379.2482 ([M+H] + , calcd. for C22H35O5, 379.2479), which was indicative of six degrees of unsaturation. The IR spectrum of 3 suggested the presence of lactone (νmax 1759 cm −1 ), ester (νmax 1736 cm −1 ) and hydroxyl (νmax 3442 cm -1 ) groups, while a strong UV absorption at λmax 231 nm (logε 3.83) suggested the presence of α,β-unsaturated γ-lactone group [43]. According to the 1 H and 13 C NMR data of 3 (Tables 1 and 2       The protonated molecule peak at m/z 343.2237 ([M+Na] + , calcd. for C20H32O3Na, 343.2244) displayed in the HRESIMS spectrum of sartrocheliol C (5) revealed that compound 5 had the molecular formula C20H32O3, demonstrating the presence of an additional oxygen atom with respect to that of a dihydrofuran cembranoid 12 [46]. The IR spectrum of 5 showed the presence of olefinic (νmax 3726 cm -1 ) and hydroxyl (νmax 3441 cm -1 ) groups. A careful analysis of its NMR spectra revealed that the NMR spectroscopic features of 5 (Tables 1 and 2) highly resembled those of 12. In fact, the main difference between compounds 5 and 12 was that the CH2-13 in 12 was hydroxylated in 5. The presence of a hydroxyl group at C-13 was supported by the dramatically down-field shifted carbon chemical shift (ΔδC 56 ppm), and further confirmed by the diagnostic HMBC correlations of from H-11 (δH 5.32) to C-13 (δC 79.67) and from H3-20 (δH 1.56) to C-13 ( Figure 3). The crosspeak of H3-18 (δH 1.00)/H-2 (δH 5.51) observed in the NOESY spectrum of 5 (Figure 4) together with the small coupling constant (1.4 Hz) between H-2 and H-3 (δH 4.66) suggested the same orientation of H3-18 and H-3. Moreover, the coupling constant (4.6 Hz) between H-13 (δH 4.29) and H-14 (δH 4.84) indicated these two protons were cis-orientated. Whereas the lack of the NOE correlation between H-3 and H-14 revealed the trans-orientation of these two protons of the 2,5-dihydrofuran ring. Thus, the relative configuration of 5 could be assigned as 3R * ,4R * ,13S * ,14S * based on the extensive analysis of the NOESY spectrum. Meanwhile, the relative configuration of 5 could also be elucidated via QM-NMR protocol by using the DP4+ method, which has become one of the most popular and reliable methods to find the most likely structure from a set of putative candidates [34,47]. Four possible isomers (2R*,3R*,13R*,14S*)-5a, (2R*,3R*,13S*,14S*)-5b, (2R*,3S*,13R*,14S*)-5c and (2R*,3S*,13S*,14R*)-5d (Table S3) were subjected to QM-NMR calculations. As a result, the experimental NMR data of compound 5 gave the best match for 5b, with 100% probability (Figure 7, Table S4). Herein, the assigned relative configuration 3R * ,4R * ,13S * ,14S * was consistent with the observations deduced from the NOESY spectrum. As there was a secondary hydroxyl group at C-13, the modified Mosher's method was applied. The resulting distribution pattern of observed ΔδH(S-R) values ( Figure 5) established the absolute configuration R for C-13 in 5. Subsequently, the absolute configuration of 5 could be assigned as 3S,4S,13R,14R. The protonated molecule peak at m/z 343.2237 ([M + Na] + , calcd. for C 20 H 32 O 3 Na, 343.2244) displayed in the HRESIMS spectrum of sartrocheliol C (5) revealed that compound 5 had the molecular formula C 20 H 32 O 3 , demonstrating the presence of an additional oxygen atom with respect to that of a dihydrofuran cembranoid 12 [46]. The IR spectrum of 5 showed the presence of olefinic (ν max 3726 cm −1 ) and hydroxyl (ν max 3441 cm −1 ) groups. A careful analysis of its NMR spectra revealed that the NMR spectroscopic features of 5 (Tables 1 and 2) highly resembled those of 12. In fact, the main difference between compounds 5 and 12 was that the CH 2 -13 in 12 was hydroxylated in 5. The presence of a hydroxyl group at C-13 was supported by the dramatically down-field shifted carbon chemical shift (∆δ C 56 ppm), and further confirmed by the diagnostic HMBC correlations of from H-11 (δ H 5.32) to C-13 (δ C 79.67) and from H 3 -20 (δ H 1.56) to C-13 ( Figure 3). The cross-peak of H 3 -18 (δ H 1.00)/H-2 (δ H 5.51) observed in the NOESY spectrum of 5 ( Figure 4) together with the small coupling constant (1.4 Hz) between H-2 and H-3 (δ H 4.66) suggested the same orientation of H 3 -18 and H-3. Moreover, the coupling constant (4.6 Hz) between H-13 (δ H 4.29) and H-14 (δ H 4.84) indicated these two protons were cis-orientated. Whereas the lack of the NOE correlation between H-3 and H-14 revealed the trans-orientation of these two protons of the 2,5-dihydrofuran ring. Thus, the relative configuration of 5 could be assigned as 3R * ,4R * ,13S * ,14S * based on the extensive analysis of the NOESY spectrum. Meanwhile, the relative configuration of 5 could also be elucidated via QM-NMR protocol by using the DP4+ method, which has become one of the most popular and reliable methods to find the most likely structure from a set of putative candidates [34,47]. Four possible isomers (2R*,3R*,13R*,14S*)-5a, (2R*,3R*,13S*,14S*)-5b, (2R*,3S*,13R*,14S*)-5c and (2R*,3S*,13S*,14R*)-5d (Table S3) were subjected to QM-NMR calculations. As a result, the experimental NMR data of compound 5 gave the best match for 5b, with 100% probability (Figure 7, Table S4). Herein, the assigned relative configuration 3R * ,4R * ,13S * ,14S * was consistent with the observations deduced from the NOESY spectrum. As there was a secondary hydroxyl group at C-13, the modified Mosher's method was applied. ), which accounted for three degrees of unsaturation. The remaining two degrees of unsaturation strongly indicated one macrocyclic carbon skeleton fused with an oxacycle. Careful analysis of the 2D NMR spectrum (Figure 3) of 6 revealed this compound had almost the same gross bicyclic framework of sarcophytrol R (13) [44] except the hydroxyl group at C-11 in 13 was acetylated in 6. Due to the acetylation, the chemical shifts of H-11 and C-11 shifted down-field (ΔδH 1.5 ppm, ΔδC 3.2 ppm, respectively). It might be worth pointing out that the lists of 1 H and 13 C NMR data of sarcophytrols R and S in the reference [44] were exchanged inadvertently by the authors. The high similarity of the 1 H and 13 C NMR data as well as similar patterns of NOE correlations of compounds 6 and 13 suggested they shared the same relative configuration 3S*,4R*,11S*,12R*. With the relative configuration in hand, we tried to use the Mosher's method to establish the absolute configuration of this compound. However, to our disappointment, the left compound after bioassay was degraded although kept in a fridge.
Sartrocheliol E (7) was isolated as colorless oil, and its molecular formula was assigned as C20H34O2 by protonated molecule peak at m/z 329.2448 ([M+Na] + , calcd. for C20H34O2Na, 329.2451) in the HRESIMS spectrum. Its 13 C NMR data (  15.09). The presence of six olefinic carbons was attributed to three double bonds, which accounted for three degrees of unsaturation, indicating that 7 had a monocyclic carbon framework. In fact, the NMR spectroscopic characters of 7 were reminiscent of those of cembrendiol (14) [48]. Careful comparison of their NMR data disclosed their structures differed at the position of the secondary hydroxyl substituent. The secondary hydroxyl group substituted at C-10 in 7 was supported by the consecutive proton system extending ), which accounted for three degrees of unsaturation. The remaining two degrees of unsaturation strongly indicated one macrocyclic carbon skeleton fused with an oxacycle. Careful analysis of the 2D NMR spectrum (Figure 3) of 6 revealed this compound had almost the same gross bicyclic framework of sarcophytrol R (13) [44] except the hydroxyl group at C-11 in 13 was acetylated in 6. Due to the acetylation, the chemical shifts of H-11 and C-11 shifted down-field (∆δ H 1.5 ppm, ∆δ C 3.2 ppm, respectively). It might be worth pointing out that the lists of 1 H and 13 C NMR data of sarcophytrols R and S in the reference [44] were exchanged inadvertently by the authors. The high similarity of the 1 H and 13 C NMR data as well as similar patterns of NOE correlations of compounds 6 and 13 suggested they shared the same relative configuration 3S*,4R*,11S*,12R*. With the relative configuration in hand, we tried to use the Mosher's method to establish the absolute configuration of this compound. However, to our disappointment, the left compound after bioassay was degraded although kept in a fridge.
Sartrocheliol E (7) was isolated as colorless oil, and its molecular formula was assigned as C 20 15.09). The presence of six olefinic carbons was attributed to three double bonds, which accounted for three degrees of unsaturation, indicating that 7 had a monocyclic carbon framework. In fact, the NMR spectroscopic characters of 7 were reminiscent of those of cembrendiol (14) [48]. Careful comparison of their NMR data disclosed their structures differed at the position of the secondary hydroxyl substituent. The secondary hydroxyl group substituted at C-10 in 7 was supported by the consecutive proton system extending from H 2 -9 to H 2 -11 through H-10 in the 1 H− 1 H COSY spectrum (Figure 3). Due to the lack of the evidence regarding the orientations of H-10 and H 3 -18, it was hard to figure out the relative configurations of C-4 and C-10. In order to solve this problem, the QM-NMR method was applied (Table S5). By means of this approach, the experimentally observed NMR data for compound 7 gave the best match with the 1S*,4R*,10S* isomer (>90% probability) (Figure 8, Table S6). Thus, the relative configuration of compound 7 was determined as 1S*,4R*,10S*. Unfortunately, the application of Mosher's reaction failed, probably due to the insufficient amounts left after an array of bioassays.
Mar. Drugs 2023, 21, x 9 of 15 from H2-9 to H2-11 through H-10 in the 1 H− 1 H COSY spectrum (Figure 3). Due to the lack of the evidence regarding the orientations of H-10 and H3-18, it was hard to figure out the relative configurations of C-4 and C-10. In order to solve this problem, the QM-NMR method was applied (Table S5). By means of this approach, the experimentally observed NMR data for compound 7 gave the best match with the 1S*,4R*,10S* isomer (> 90% probability) (Figure 8, Table S6). Thus, the relative configuration of compound 7 was determined as 1S*,4R*,10S*. Unfortunately, the application of Mosher's reaction failed, probably due to the insufficient amounts left after an array of bioassays. The protonated molecule peak at m/z 403.2446 ([M + Na] + , calcd. for C22H36O5Na, 403.2455) in the HRESIMS spectrum of sartrocheliol F (8) suggested compound 8 and coisolate 9 [44] had the same molecular formula C22H36O5. Moreover, the NMR data of 8 (Tables 1 and 2) highly resembled those of 9, implying that they shared the same gross structure. The distinct difference was found as the chemical shift of C-4, which was δC72.47 ppm in 8 whereas δC 74.49 ppm in 9, disclosing the reverse configuration of the hydroxyl group at C-4. This reversion was further deduced from the NOE interactions between H3-18 and H-2 and between H-2 and H3-16 ( Figure 4). As the absolute configuration of the deacetylation derivative of 9 was determined as 1S,4S,11S,12R by the Mosher's method [44], the absolute configuration of 8 was consequently established as 1S,4R,11S,12R.
Although chemical investigations of the soft coral S. trocheliophorum have been well documented in the literature, the present study of this species collected from Ximao Island provided further intriguing results. In the current study, six new cembranoids diterpenoids, sartrocheliols A-E (1, 3, 5-8), along with four known related ones (2, 4, 9, and 10) were obtained. Among them, compound 1 was a capnosane diterpene, while others were cembrane diterpenes. They displayed diverse structural features not only at the distinctly different carbon frameworks but also at the various types of heterocycles, including the epoxide, γ-lactone, furan, and pyran rings. Compared with the previous research of the Ximao Island specimen [41], the major difference was the discovery of a capnosane diterpene in this study. This observation further indicated the possible impact of temporal variations on the different metabolic processes for the title soft corals.  (Tables 1 and 2) highly resembled those of 9, implying that they shared the same gross structure. The distinct difference was found as the chemical shift of C-4, which was δ C 72.47 ppm in 8 whereas δ C 74.49 ppm in 9, disclosing the reverse configuration of the hydroxyl group at C-4. This reversion was further deduced from the NOE interactions between H 3 -18 and H-2 and between H-2 and H 3 -16 ( Figure 4). As the absolute configuration of the deacetylation derivative of 9 was determined as 1S,4S,11S,12R by the Mosher's method [44], the absolute configuration of 8 was consequently established as 1S,4R,11S,12R.
Although chemical investigations of the soft coral S. trocheliophorum have been well documented in the literature, the present study of this species collected from Ximao Island provided further intriguing results. In the current study, six new cembranoids diterpenoids, sartrocheliols A-E (1, 3, 5-8), along with four known related ones (2, 4, 9, and 10) were obtained. Among them, compound 1 was a capnosane diterpene, while others were cembrane diterpenes. They displayed diverse structural features not only at the distinctly different carbon frameworks but also at the various types of heterocycles, including the epoxide, γ-lactone, furan, and pyran rings. Compared with the previous research of the Ximao Island specimen [41], the major difference was the discovery of a capnosane diterpene in this study. This observation further indicated the possible impact of temporal variations on the different metabolic processes for the title soft corals.
The anti-tumor effects of all the ten secondary metabolites were evaluated against a list of tumor cells including A549 (human lung cancer cell), H1975 (human lung adenocarcinoma cell), MDA-MB-231 (human breast cancer cell) and H1299 (human non-small cell lung cancer cell). The results showed compound 4 displayed moderate cytotoxic activities against these four cells with the IC 50 values of 47.9, 26.3, 44.7, 33.1 µM, respectively, while 6 only showed moderate cytotoxicity against H1975 (IC 50 = 40.4 µM). Further, we also conducted molecular interaction experiments on all the compounds, looking for compounds that have the potential to bind to BRD4 and ROR1 anti-tumor targets, respectively. Unfortunately, we did not obtain satisfactory results. In addition, these compounds were tested for their antibacterial activities against a vast array of bacteria including the human pathogens Staphylococcus aureus ATCC27154, Enterococcus faecium, Escherichia coli ATCC25922, Enterobacter cloacae ZR042, Enterobacter hormaechei 2R043, Pseudomonas aeruginosa ATCC10145, methicillin-resistant Staphylococcus aureus (MRSA), and Candida albicans ATCC76485 and the marine strains Streptococcus parauberis KSP28, Streptococcus parauberis SPOF3K, Lactococcus garvieae MP5245, Aeromonas salmonicida AS42, Phoyobacterium damselae FP2244, Pseudomonas fulva ZXM181, Photobacterium halotolerans LMG22194T. To our disappointment, all of them were judged as inactive. Other bioassays such as neuroprotective and anti-inflammatory are currently on the way.

Subsection
Melting points were measured on an X-4 digital micromelting point apparatus. The X-ray measurements were made on a Bruker D8 Venture X-ray diffractometer with Cu Kα radiation (Bruker Biospin AG, Fällanden, Germany). IR spectra were recorded on a Nicolet iS50 spectrometer (Thermo Fisher Scientific, Madison, WI, USA). Optical rotations were measured on a PerkinElmer 241MC polarimeter (PerkinElmer, Fremont, CA, USA). CD & UV spectra were measured on a JASCO J-810 instrument (JASCO Corporation, Tokyo, Japan). 1 H and 13 C NMR spectra were acquired on a Bruker AVANCE III 400 and 600 MHz spectrometer (Bruker Biospin AG, Fällanden, Germany). Chemical shifts were reported with the residual CHCl 3 (δ H 7.26; δ C 77.16) as the internal standard for 1 H and 13 C NMR spectra. The LREIMS and HREIMS data were recorded on a Finnigan-MAT-95 mass spectrometer (Finnigan-MAT, San Jose, CA, USA). HRESIMS spectra were recorded on Agilent G6250 Q-TOF (Agilent, Santa Clara, CA, USA). Commercial silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200-300 mesh, 300-400 mesh) was used for column chromatography, and precoated silica gel GF254 plates (Sinopharm Chemical Reagent Co., Shanghai, China) were used for analytical TLC. Sephadex LH-20 (Pharmacia, Piscataway, NJ, USA) was also used for column chromatography. Reversed-phase (RP) HPLC was performed on an Agilent 1260 series liquid chromatography 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 column chromatography and HPLC were of analytical grade (Shanghai Chemical Reagents Co., Ltd., Shanghai, China) and chromatographic grade (Dikma Technologies Inc., Foothill Ranch, CA, USA), respectively.

Animal Material
The soft coral

X-ray Crystallographic Analysis for Compounds 1 and 4
The crystals of 1 and 4 were both recrystallized from methanol at 4 • C. X-ray analysis of 1 and 4 were carried out on a Bruker D8 Venture diffractometer with Cu Kα radiation (λ = 1.54178 Å) at 170 K, respectively. The acquisition parameters for 1 and 4 are provided in the Supplementary Materials, and crystallographic data for compounds 1 and 4 (deposition no. CCDC 2,196,207 and CCDC 2205721) have been deposited at the Cambridge Crystallographic Data Center. Copies of the data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (accessed on 11 August 2022). Theoretical calculations of all theoretical stereoisomers were carried out to determine the relative configuration of 5 and 7, based on the alignment of its 1 D NMR chemical shifts ( 13 C NMR chemical shifts herein) and calculation-generated chemical shifts. Confab was used to search the conformational space of 5a-5d and 7a-7d. Conformational searches were carried out using the torsional sampling (MCMM) method and OPLS_2005 force field in the Macromodel 9.9.223 software applying an energy window of 21 kJ/mol. Conformers above 1% population were re-optimized with Gaussian 09 at the B3LYP/6-311G(d,p) level with IEFPCM (Polarizable Continuum Model using the Integral Equation Formalism variant) solvent model for acetonitrile. The Boltzmann populations of the conformers were obtained based on the potential energy provided by the OPLS_2005 force field, leading to 11, 9, 13 and 24 conformers for 5a-5d; 11, 9, 13 and 24 conformers for 7a-7d above 1% population for further re-optimization, respectively. The obtained conformers were subjected to optimization and frequency calculations on B3LYP/6-311G(d,p) level of theory. GIAO DFT 13 C NMR calculations were calculated on mPW1PW91/6-31G* (CHCl 3 ) level of theory, and the calculated shielding tensors were Boltzmann averaged according to Gibbs free energy and then converted into chemical shifts following MSTD protocol. The experimental 13 C NMR data of 5 and 7 were compared with the calculated NMR chemical shifts of 5a-5d and 7a-7d using the mean absolute error (MAE) values, maximum deviation (MD) values, correlation coefficient (R 2 ), and DP4+ probability analysis. XYZ data for all conformations and detailed data for DP4+ analysis are provided in the Supplementary Materials.

Cytotoxic Bioassays
H1975, MDA-MB-231, A549, and H1299 cancer cell lines were purchased from the Procell Life Science & Technology Co., Ltd. Cells were cultured at 37 • C in a 5% CO 2 humidified incubator and maintained in high glucose Dulbecco's Modified Eagle Medium (DMEM, Nissui, Tokyo, Japan) containing 100 mg/mL streptomycin, 2.5 mg/L amphotericin B and 10% heat-inactivated fetal bovine serum (FBS). The cells were inoculated in 96-well culture plates for 12 h and then treated with different concentrations of compounds for 72 h. Water-soluble tezole (WST) reagent was added to each (10 µL) well and cultured at 37 • C for 2 h to assess cell viability. The absorbance was read with a microplate reader at 450 nm. Adriamycin (DOX) was the positive control.

Conclusions
In summary, the present investigation of the soft coral S. trocheliophorum from Ximao Island provided intriguing results including six new cembranoids diterpenoids, sartrocheliols A-E (1, 3, 5-8), along with four known related ones (2, 4, 9, and 10). This study not only extended the members of capnosane and cembrane diterpenes but also enriched the chemical diversity of the title species. In addition, a whole set of NMR computations, modified Mosher's method, and X-ray diffraction analysis were applied to assign the absolute configurations of compounds 1, 3, 4, and 5. However, the absolute configurations of other new metabolites remain undefined. To achieve it, a collection of the biological materials and accumulation of these new compounds should be conducted in future, which could supply sufficient amounts of metabolites for either the subsequent chemical transformations or recrystallization experiments. Anti-tumor and antibacterial bioassays were carried out. Among these secondary metabolites, compound 4 displayed moderate cytotoxicity against A549, H1975, MDA-MB-231, and H1299 cells (IC 50 = 47.9, 26.3, 44.7, 33.1 µM, respectively), while 6 only exhibited moderate cytotoxicity against H1975 (IC 50 = 40.4 µM). However, none of them were active in the antibacterial bioassay. Moreover, the bioactivities of these compounds such as anti-virus, will be evaluated in future.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.