Seven New Lobane Diterpenoids from the Soft Coral Lobophytum catalai

Seven new lobane diterpenoids, namely, lobocatalens A–G (1–7), were isolated from the Xisha soft coral Lobophytum catalai. Their structures, including their absolute configurations, were elucidated via spectroscopic analysis, comparison with the literature data, QM-MNR, and TDDFT-ECD calculations. Among them, lobocatalen A (1) is a new lobane diterpenoid with an unusual ether linkage between C-14 and C-18. In addition, compound 7 showed moderate anti-inflammatory activity in the zebrafish models and cytotoxic activity against the K562 human cancer cell line.

The soft corals of the genus Lobophytum (family Alcyoniidae) are well known as a rich source of lobane diterpenoids [4][5][6][7][8][9], cembranolides [12][13][14][15], and prenylgermacranetype diterpenoids [16]. With the aim of seeking new bioactive lobane diterpenoids, our continuing investigation of the soft coral Lobophytum catalai collected from Yagong Island led to the isolation of seven new lobane diterpenoids. Considering that there have been no reports on lobane-based diterpenoids from the soft coral Lobophytum catalai, these new compounds, 1-7, were named lobocatalens A-G ( Figure 1). Among them, lobocatalen A (1) is a new lobane diterpenoid with an unusual ether linkage between C-14 and C-18. In addition, compound 7 showed moderate anti-inflammatory activity in the zebrafish models and moderate cytotoxic activity against the K562 human cancer cell line. Moreover, the isolation, structure elucidation, and biological activity of these isolated compounds are reported. Mar
In the NOESY spectrum of 1 (Figure 3), the clear correlations of H-3a (δ H 1.57)/H-2, H-3a (δ H 1.57)/H-4, H-3b (δ H 1.49)/H 3 -7, H 3 -7/H 3 -12, and H-2/H-8 indicated the βorientation of H-2 and H-4, and the α-orientation of H 3 -7. This deduction was further proven through a comparison of the chemical shifts with previously reported lobane-type diterpenoids [4][5][6][7]. The orientations of C-14 and C-17 were defined as 14R* and 17R* in the DP4 + calculations (Supplementary Materials, Figure S2) [17]. Finally, the absolute configurations of 1 were defined as 1R, 2R, 4S, 14R, and 17R in the TDDFT-ECD calculations ( Figure 4).  Lobocatalen B (2) was obtained as a colorless oil. The molecular formula of 2 was determined to be C 20 H 32 O 3 based on its HRESIMS ion peak at m/z 338.2685 [M + NH 4 ] + . The IR absorption at 3080 and 1635 cm −1 together with the UV spectrum at λmax = 193 (log ε 0.38) nm indicated the presence of a double-bond group. Additionally, the IR absorption at 3410 cm −1 indicated the presence of a hydroxy group. The 1D NMR data of 2 (Tables 1 and 2) were similar to those of lobatriene [18], a known lobane diterpenoid isolated from an Okinawan soft coral of the genus Sinularia flexibilis. The only difference between them is that one hydrogen atom of methylene at C-14 in lobatriene is replaced by a hydroxy group in 2. The key HMBC correlations ( Figure 2) from H-14 (δ H 5.48) to C-13 and C-17 and the significant downfield chemical shifts of C-14 (δ C 101.1) also supported the change in functional groups. Thus, the planar structure of 2 was constructed ( Figure 2). Through a comparison with the NMR data of previously reported lobane-type diterpenoids for which the cyclohexane systems all have the same stereochemistry of 1R*, 2R*, and 4S* [4][5][6][7], the relative configurations of C-1, C-2, and C-4 of 2 were the same as those reported for lobane-type diterpenoids. The NOESY correlations (Figure 3  Lobocatalen B (2) was obtained as a colorless oil. The molecular formula of 2 was determined to be C20H32O3 based on its HRESIMS ion peak at m/z 338.2685 [M + NH4] + . The IR absorption at 3080 and 1635 cm −1 together with the UV spectrum at λmax = 193 (log ε 0.38) nm indicated the presence of a double-bond group. Additionally, the IR absorption at 3410 cm −1 indicated the presence of a hydroxy group. The 1D NMR data of 2 (Tables 1  and 2) were similar to those of lobatriene [18], a known lobane diterpenoid isolated from an Okinawan soft coral of the genus Sinularia flexibilis. The only difference between them is that one hydrogen atom of methylene at C-14 in lobatriene is replaced by a hydroxy group in 2. The key HMBC correlations ( Figure 2) from H-14 (δH 5.48) to C-13 and C-17 and the significant downfield chemical shifts of C-14 (δC 101.1) also supported the change in functional groups. Thus, the planar structure of 2 was constructed (Figure 2). Through a comparison with the NMR data of previously reported lobane-type diterpenoids for which the cyclohexane systems all have the same stereochemistry of 1R*, 2R*, and 4S* [4][5][6][7], the relative configurations of C-1, C-2, and C-4 of 2 were the same as those reported for lobane-type diterpenoids. The NOESY correlations (Figure 3 Figure S4). Finally, the absolute configurations of 3 were defined as 1R, 2R, 4S, and 17R in the TDDFT-ECD calculations (Figure 4).
The relative configurations of 4 were determined through an analysis of its NOESY spectrum (Figure 3). The NOESY correlations of H-3a (δ H 1.52)/H-4 and H 3 -7/H-3b (δ H 1.64) indicated the β-orientation of H-4, and the α-orientation of H 3 -7. The E geometry of the ∆ 13 double bonds was established based on the NOESY correlations of H-15/H-4. Through a comparison of the NMR data with those of previously reported (-)-β-elemene-type diterpenoids and the co-isolates, the orientation of H-2 was found to be β. Then, the 1R*, 2R*, and 4S* configurations of the β-elemene ring system of 4 were further proven in the DP4 + calculations (Supplementary Materials, Figure S5). Finally, the absolute configurations of 4 were unambiguously determined in the TDDFT-ECD calculations as 1R, 2R, and 4S ( Figure 4).
Lobocatalen E (5) has a molecular formula of C 20 H 32 O 2 , as determined by its HRESIMS ion peak at m/z 305.2469 [M + H] + , suggesting five degrees of unsaturation. The IR absorption at 1711 cm −1 indicated the presence of a carbonyl group. Analysis of the 1 H and 13 C NMR spectra (Tables 1 and 2) indicated that 5 has a similar functional group to lobovarol G [4], a known lobane diterpenoid isolated from the soft coral Lobophytum varium. The only difference is that the 17-OH in lobovarol G is oxidized to a ketonic group in 5. Furthermore, the HMBC correlations from H-16 to C-17 (δ C 214.1) and from H 3 -20 to C-17, C-18 (δ C 76.4), and C-19 and the 1 H-1 H COSY correlations from H-15 to H-16 confirmed this variation in the functional groups ( Figure 2). The NOESY correlations (Figure 3) of H 3 -7/H-12 (δ H 1.71) and H-2/H-8, along with the similar NMR data compared with the co-isolates, indicated the 1R* and 2R* configurations of the β-elemene ring system. Then, the 13 C NMR chemical shift calculation in the DP4 + calculations clearly indicated that the relative configuration of C-4 is S*. Hence, the absolute configurations were confirmed as 1R, 2R, and 4S (Supplementary Materials, Figure S6) in the following TDDFT-ECD calculations.
Lobocatalen F (6) was isolated as a colorless oil. The HRESIMS ion peak at m/z 273.2213 [M + H] + of 6 indicated that its molecular formula is C 19 H 28 O, requiring six degrees of unsaturation. The IR absorption at 1706 cm −1 together with the UV spectrum at λmax = 287 (log ε 0.18) nm indicated the presence of an α, β-unsaturated carbonyl group. A survey of the literature revealed that the 1D NMR data (Tables 1 and 2) of compound 6 were similar to those of 3, 5-heptadien-2-one [21], a known lobane diterpenoid isolated from the soft coral Lobophytum microlobulatum from Havellock Island. The 2D NMR of 6 revealed that the planar structure of 6 is identical to that of 3, 5-heptadien-2-one, which suggested that 6 is a stereoisomer of 3, 5-heptadien-2-one. The same relative configurations of the β-elemene ring system were established in the NOESY experiment (Figure 3 [22], designated the Z-configuration of the ∆ 13 double bonds, revealing the only difference between 3, 5-heptadien-2-one and 6. Then, the 1R*, 2R*, and 4S* configurations of the β-elemene ring system of 6 were further proven in the DP4 + calculations (Supplementary Materials, Figure S7). Finally, the absolute configurations of 6 were unambiguously determined as 1R, 2R, and 4S in the TDDFT-ECD calculations (Figure 4).
Lobocatalen G (7), a colorless oil, possesses the molecular formula C 21 H 32 O 4 , which was established based on the HRESIMS ion peak at m/z 371.2192 [M + Na] + . The IR absorption at 1700 cm −1 together with the UV spectrum at λmax = 270 (log ε 0.03) nm indicated the presence of an α, β-unsaturated carbonyl group. The 1D NMR data (Tables 1 and 2) of 7 closely resembled those of (1R*,2R*,4S*,15E)-loba-8,10, 13 (14),15(16)-tetraen-17,18diol-17-acetate, a known lobane diterpenoid isolated from the Bowden Reef soft coral Sinularia sp. [8]. In fact, the structure of 7 is truly similar to that of the known compound, with the exception that the 1,1-disubstituted double bond at C-13 in the known compound is replaced by a carbonyl group in 7. This deduction was proven by the 1 H-1 H COSY correlations from H-14 to H-16 and the HMBC correlations from H-14 to C-13 (δ C 202.0) (Figure 3). Due to the absent HMBC correlations, the connection between C-4 and C-13 was established based on the molecular degrees of unsaturation.
In the NOESY experiment (Figure 3), the correlations of H-2/H-4, H-7/H-3a (δ H 1.60), and H-4/H-3b (δ H 1.72) established the 1R*, 2R*, and 4S* configurations of the β-elemene ring system. Moreover, the large coupling constants (J 14,15 = 18.0 Hz) established the E geometry of the ∆ 14 double bonds. In the relative configuration of C-16 in 7, the chiral center far away from the β-elemene ring system was difficult to assign in the NOESY experiment. Then, through the 13 C NMR chemical shift calculation in the DP4 + calculations (Supplementary Materials, Figure S8), the relative configuration of C-16 in 7 was deduced as 16S*. Finally, the absolute configurations of 7 were defined as 1R, 2R, 4S, and 16S in the TDDFT-ECD calculations (Figure 4).
On account of the fact that research on the anti-inflammatory activity of lobane diterpenoids in zebrafish models has not been reported up to now, we attempted to identify lobane diterpenoids with anti-inflammatory activity in zebrafish models. The anti-inflammatory effect of compounds 1-7 was assessed in CuSO 4 -induced transgenic fluorescent zebrafish. CuSO 4 can produce an intense acute inflammatory response in the neuromasts and mechanosensorial cells in the lateral line of zebrafish, stimulating the infiltration of macrophages [22,23]. Thus, the number of macrophages surrounding the neuromasts in zebrafish can be observed and counted under a fluorescence microscope. Therefore, the anti-inflammatory activity of these lobane diterpenoids can be evaluated. As shown in Figure 5 Moreover, the cytotoxic activity of these isolated compounds (1-7) was evaluated against the human leukemia K562, normal human hepatocyte L-02, human pancreatic cancer ASPC-1, and human breast cancer MDA-MB-231 cell lines. The results (Table 3) demonstrated that compound 7 exhibited modest cytotoxicity against the K562 cell line, with an IC 50 value of 27.96 µM.

Animal Material
The soft coral Lobophytum catalai was collected from Xisha Island (YaGong Island) in the South China Sea in 2018 and frozen immediately after collection.

Extraction and Isolation
A frozen specimen of Lobophytum catalai (12.0 kg, wet weight) was homogenized and then exhaustively extracted with CH 3 OH six times (5 days each time) at room temperature. The combined solutions were concentrated in vacuo and subsequently desalted by redissolving with ethyl acetate to yield a residue (298.0 g). The crude extract was subjected to silica gel vacuum column chromatography eluted with a gradient of petroleum/ethyl acetate (200:1-1:1, v/v) and subsequently eluted with a gradient of CH 2 Cl 2 /MeOH (20:1-1:1, v/v) to obtain fourteen fractions (Frs.

Anti-Inflammatory Activity Assay
Healthy macrophage fluorescent transgenic zebrafish (Tg: zlyz-EGFP) were provided by the Biology Institute of Shandong Academy of Science (Jinan, China). The zebrafish maintenance and anti-inflammation assay were carried out as previously described [24]. Each zebrafish larva was photographed using a fluorescence microscope (AXIO, Zom.V16), and the number of macrophages around the nerve mound was calculated using Image-Pro Plus 6.0 software (Rockville, MD, USA). One-way analysis of variance was conducted using GraphPad Prism 7.00 software (San Diego, CA, USA). Lobocatalens A-G (1-7) were tested for anti-inflammatory activities in the zebrafish models. Three dpf (days postfertilization), healthy macrophage fluorescent transgenic zebrafish were used as animal models to evaluate the anti-inflammatory effects of compounds 1-7.

Cytotoxicity Activity Assay
The MTT method was used to evaluate cytotoxicity against the K562 (human leukemia) cell line, and the SRB method was used to evaluate cytotoxicity against the L-02 (normal human hepatocytes), ASPC-1 (human pancreatic cancer), and MDA-MB-231 (human breast cancer) cell lines. As a positive control, Adriamycin (doxorubicin) was used.

Conclusions
To the best of our knowledge, less than twenty new lobane diterpenoids have been discovered in the last two decades. In our continuing chemical investigation of the Xisha soft coral Lobophytum catalai, seven new lobane diterpenoids, named lobocatalens A-G (1-7), were isolated, enriching the chemical diversity of lobane diterpenoids. Among them, lobocatalen A (1) is a new lobane diterpenoid with an unusual ether linkage between C-14 and C-18. Moreover, extensive spectroscopic data analyses, spectral comparisons, quantum chemical calculations, and TDDFT-ECD calculations were combined to determine the structures and absolute configurations of 1-7. In the bioassay, only compound 7 showed moderate anti-inflammatory activity at 20 µM in the zebrafish models. This is the first report on the anti-inflammatory activity of lobane diterpenoids in zebrafish. In addition, compound 7 showed modest cytotoxic activity against the K562 human cancer cell line. This research suggests that this species has great potential for further evaluation.