Cembranolides and Related Constituents from the Soft Coral Sarcophyton cinereum

In an attempt to explore the bioactive metabolites of the soft coral Sarcophyton cinereum, three new cembranolides, cinerenolides A–C (1–3), and 16 known compounds were isolated and identified from the EtOAc extract. The structures of the new cembranolides were elucidated on the basis of spectroscopic analysis, and the NOE analysis of cinerenolide A (1) was performed with the assistance of the calculated lowest-energy molecular model. The relative configuration of cinerenolide C (3) was determined by the quantum chemical NMR calculation, followed by applying DP4+ analysis. In addition, the cytotoxic assays disclosed that some compounds exhibited moderate to potent activities in the proliferation of P388, DLD-1, HuCCT-1, and CCD966SK cell lines.

In an extensive analysis of 1 H-1 H COSY, HSQC, and HMBC spectra (Figure 1), the planar structure of 2 was established and found to be quite similar to sartrolide D (Supplementary Materials, Table S1) [20]. A large coupling constant of 16.4 Hz indicated the E geometry for the Δ 1 double bond. The same 7S*,8R*-configuration as 1 was assigned for 2, as they showed similar NOEs neighboring the C-7 and C-8 stereogenic centers (Figure 2). Furthermore, the NOEs of H-7/H3-18 and H-11/H-15 indicated that H3-18 and the isopropyl group were cofacial (Supplementary Materials, Figures S10-S17). Accordingly, the structure of 2 was determined as shown (Chart 1).
Compound 3 was also obtained as a white powder with the same molecular formula, determined to be C20H32O5 from HRESIMS, as that of 2. Their NMR data were quite similar; however, differences were observed for the chemical shifts around C-1 and C-4. Its planar structure was confirmed by an analysis of the 1D and 2D NMR data (Figure 1). Compound 3 has the same 7S*,8R* configuration based on similar NOEs neighboring C-7 and C-8; however, the relative configurations of C-1 and C-4 remained unclear in an analysis of the NOEs (Figure 2) (Supplementary Materials, Figures S18-S25). Thus, the computational NMR data with DP4+ analysis [27,28] was applied for the establishment of the relative configuration of 3. The four possible isomers with two hydroxyl groups at C-1 and C-4, respectively, 1α4β, 1β4α, 1α4α, and 1β4β, were subjected for chemical shift calculations at the MPW1PW91/6-31+G(d,p)//B3LYP/6-31G(d) level with the polarizable continuum model (PCM). Then, the calculated NMR chemical shifts for the four possible isomers were compared with the experimental data of 3 and statistically analyzed using the  H-3)). The former was evidenced by the IR absorption band at 1653 cm -1 . Additionally, two hydroxy-containing quaternary carbons (δ C 77.0 (C, C-1); 74.9 (C, C-4)), one hydroxycontaining methine (δ C 71.7 (CH, C-7); δ H 3.92 (1H, d, J = 10.8 Hz, H-7)), and a down-field shifted quaternary carbon (δ C 86.1 (C, C-8)) were evidenced. Considering the molecular formula and the above functionality, the structure of 2 should be bicyclic.
In an extensive analysis of 1 H-1 H COSY, HSQC, and HMBC spectra (Figure 1), the planar structure of 2 was established and found to be quite similar to sartrolide D (Supplementary Materials, Table S1) [20]. A large coupling constant of 16.4 Hz indicated the E geometry for the ∆ 1 double bond. The same 7S*, 8R*-configuration as 1 was assigned for 2, as they showed similar NOEs neighboring the C-7 and C-8 stereogenic centers ( Figure 2). Furthermore, the NOEs of H-7/H 3 -18 and H-11/H-15 indicated that H 3 -18 and the isopropyl group were cofacial (Supplementary Materials, Figures S10-S17). Accordingly, the structure of 2 was determined as shown (Scheme 1).
Compound 3 was also obtained as a white powder with the same molecular formula, determined to be C 20 H 32 O 5 from HRESIMS, as that of 2. Their NMR data were quite similar; however, differences were observed for the chemical shifts around C-1 and C-4. Its planar structure was confirmed by an analysis of the 1D and 2D NMR data (Figure 1). Compound 3 has the same 7S*, 8R* configuration based on similar NOEs neighboring C-7 and C-8; however, the relative configurations of C-1 and C-4 remained unclear in an analysis of the NOEs (Figure 2) (Supplementary Materials, Figures S18-S25). Thus, the computational NMR data with DP4+ analysis [27,28] was applied for the establishment of the relative configuration of 3. The four possible isomers with two hydroxyl groups at C-1 and C-4, respectively, 1α4β, 1β4α, 1α4α, and 1β4β, were subjected for chemical shift calculations at the MPW1PW91/6-31+G(d,p)//B3LYP/6-31G(d) level with the polarizable continuum model (PCM). Then, the calculated NMR chemical shifts for the four possible isomers were compared with the experimental data of 3 and statistically analyzed using the DP4+ method, as shown in the Supplementary Materials. As a result, the conformer 1α4β was found to have a probability of 100% (Table 3) (Supplementary Materials, Tables  S2-S6), suggesting a 1S*, 4R* configuration for 3.
Molecules 2022, 27, x FOR PEER REVIEW 4 of 11 3 Å (Figure 4), implying that these protons are expected to have NOEs; however, these correlations were not found in compound 1, which further supports the 7S*,8R* configuration for 1. A comparison of the proton chemical shift of H3-19 (δH 1.41 s) in 1 to the literature data (1.38-1.41 ppm for 7S,8R analogues; 1.13-1.16 ppm for 7R,8R analogues) [21] also confirmed the relative configurations of C-7 and C-8 to be 7S* and 8R*, respectively. Accordingly, the structure of 1 was determined as shown (Chart 1).  As marine cembranoids have been proven to show a broad spectrum of biological activities, including anti-inflammatory [29], anti-oxidant [30], and cytotoxicity activities [30,31], compounds 2-19 were evaluated for their proliferation activities toward the P388, DLD-1, HuCCT-1, and CCD966SK cell lines (Table 4). Among the tested compounds, 18 exhibited the most potent activity to inhibit the proliferation of the HuCCT-1 cell with an IC 50 value of 2.0 µM, which is comparable to the positive control, doxorubicin (HuCCT-1, IC 50 = 1.9 µM), whereas compound 18 showed moderate anti-proliferation activity to P388 and DLD-1, with IC 50 s of 10.6 and 9.9 µM, respectively. In addition, compounds 5 and 6 were also found to show moderate activities toward P388 cells with IC 50 s of 15.2 and 11.8 µM, respectively. The other compounds, as shown in Table 4, were found to possess weak activities toward the above four cancer cell lines. In a comparison of the biological data between biscembranolids (16)(17)(18)(19), we found that the ∆ 22 double bond with a Z geometry in compound 18 dramatically and selectively increased the anti-proliferation activity toward HuCCT-1 cell line.

Animal Material
The animal material, S. cinereum, was collected from the coral reef of Xiaoliuqiu island of Taiwan in 2012. The specimen was identified by Prof. C.-F. Dai. A voucher specimen (specimen no. sheuCYJ-001) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan.

Extraction and Isolation
The animal tissues (107.4 g) were freeze-dried, minced, and extracted exhaustively with 700 mL of EtOAc for 2 h at room temperature, and the extraction was repeated 12 times. The concentrated EtOAc layer (14.6 g) was fractionated using silica gel column chromatography (CC) with a gradient system, comprising mixtures of hexane-EtOAc

Computational Method
The conformers found at the MMFF force field using Spartan'16 were selected within a 5 kcal/mol energy window. Twelve conformers were selected for 1 and subjected for geometry optimizations and frequency calculations at the CAM-B3LYP/6-31+G(d,p) level of theory with IEFPCM in CHCl 3 . The populations were calculated based on the Gibbs free energy obtained in the aforementioned frequency calculation. For DP4+ analysis, systematic conformational searches were performed for the possible isomers 1α4β, 1β4α, 1α4α, and 1β4β of 3, using the MMFF force field in gas phase. All conformers within a 5 kcal/mol energy window were subjected for geometry optimizations and frequency calculations at the B3LYP/6-31+G(d) level in gas phase. The conformers within 2 kcal/mol from the global minimum were subjected to chemical shift calculations using the gaugeindependent atomic orbital (GIAO) method at the mPW1PW91/6-31G+(d,p)//B3LYP/6-31G(d) level with PCM/MeOH. The Boltzmann-weighted NMR data of the four isomers and the experimental data of 3 were used for DP4+ probability analysis using the Excel sheet provided by Grimblat et al. [27,28].

Cytotoxicity Assay
The assay was implemented according to the published protocols [32,33]. In brief, the Alamar Blue assay was performed for compounds 2-19 by treating them with P388, DLD-1, HuCC-T1, and CCD966SK cancer cells, which were commercially available from the American Type Culture Collection (ATCC). The test was performed in triplicate, and doxorubicin was used as a positive control.

Conclusions
In total, 3 new and 16 known compounds were isolated from the soft coral S. cinereum. In the cytotoxicity assay, compound 18 was found to show potent and selective activity toward HuCCT-1 cell line, which is close to the control group, doxorubicin. The relative configuration of 1 was determined by an analysis of NOEs and by comparing the computational conformers with those of its possible epimer. The assignment of the relative configurations of 3, with the lack of crucial NOEs, was successfully attained by the assistance of quantum chemical NMR calculation and the DP4+ method. In this work, it was also found that some cembranolides were not so flexible, and they could be readily assigned the relative configurations by a careful analysis of NOEs based on a computational model. In contrast to the flexible molecules, the assignment of relative configuration was hindered by a lack of useful NOE data neighboring the stereogenic center. For this case, the computational NMR data coupled with the DP4+ approach could provide an alternative to elucidate the relative configurations of stereogenic centers.