Anti-Inflammatory Cembrane-Type Diterpenoids and Prostaglandins from Soft Coral Lobophytum sarcophytoides

Two new cembrane-type diterpenoids, lobophytins A (1) and B (3), and four new prostaglandins, (5E)-PGB2 (10), (5E)-13,14-dihydro-PGB2 (11), 13,14-dihydro-PGB2 (12) and 13,14-dihydro-PGB2-Me (13), together with ten known compounds were isolated from the soft coral Lobophytum sarcophytoides. The structures of these new secondary metabolites were identified by high resolution mass spectrometry (HR-ESIMS), nuclear magnetic resonance (NMR) and electron circular dichroism (ECD) analyses, as well as the modified Mosher’s method. Compounds 6, 7, 9, 10, 12, 13, 15 and 16 showed potential anti-inflammatory activity by inhibiting the production of nitric oxide (NO) in RAW264.7 cells that were activated by lipopolysaccharide, with IC50 values ranging from 7.1 to 32.1 μM and were better than the positive control indomethacin, IC50 = 39.8 μM.

Prostaglandins are biologically active lipid compounds, being 20-carbon fatty acids derived enzymically from the essential fatty acids by cyclization and oxidation [11,12]. They are widely distributed in animals and human-beings, which regulate a wide range of physiological activities.

Results and Discussion
Compound 1 was obtained as colorless oil and its molecular formula was established as C20H28O4 (seven degrees of unsaturation) according to the negative HR-ESIMS ion at m/z 331.1906 [M − H] − (calculated for C20 H27 O4, 331.1909) ( Figure S1). The IR spectrum of 1 revealed the presence of hydroxy (3450 cm −1 ) group and an α, β-unsaturated γ-lactone moiety (1737 and 1675 cm −1 ) ( Figure   S2). The 1 H NMR data (Table 1) (Table 1) revealed the presence of 20 carbons belonging to three methyls, seven methylenes, four methines and six quaternary carbons. Detailed analysis of the 1 H and 13 C NMR data ( Figure S4) suggested that 1 belongs to the cembrane-type diterpenoid class, which was quite similar to sarcomililatin A (2) [13].

Results and Discussion
Compound 1 was obtained as colorless oil and its molecular formula was established as C 20 Figure S1). The IR spectrum of 1 revealed the presence of hydroxy (3450 cm −1 ) group and an α, β-unsaturated γ-lactone moiety (1737 and 1675 cm −1 ) ( Figure S2). The 1 H NMR data (Table 1) Figure S3). The 13 C NMR data (Table 1) revealed the presence of 20 carbons belonging to three methyls, seven methylenes, four methines and six quaternary carbons. Detailed analysis of the 1 H and 13 C NMR data ( Figure S4) suggested that 1 belongs to the cembrane-type diterpenoid class, which was quite similar to sarcomililatin A (2) [13]. The 1 H-1 H COSY spectrum ( Figure S5) revealed the appearance of four isolated proton spin systems as depicted in Figure 2. The key heteronuclear multiple bond correlation (HMBC) correlations from H-17 to C-1, C-15 and C-16; H-18 to C-3, C-4 and C-5; H-19 to C-7, C-8 and C-9; H-20 to C-11, C-12 and C-13; H-2 to C-1, C-15 and C-16; and H-14 to C-1 and C-15 established the cembrane-type diterpenoid skeleton as shown in Figure 2. The epoxy ring was located at C-11 and C-12 on the base of the HMBC correlations from H-20 to C-11 and C-12, as well as the chemical shift values of C-11 (δ C 62.1) and C-12 (δ C 61.1). The position of the remaining hydroxy group was assigned at C-7 (δ C 70.1) according to the HMBC correlations from H-19 to C-7 and the 1 H-1 H COSY correlation between H-6 and H-7. The relative configuration of 1 was deduced by analysis of the nuclear overhauser effect spectroscopy (NOESY) data ( Figure 3). The observed NOE correlations H-18 with H-2 and H-7 indicated they are α-orientation. NOE correlations H-11 with H-7, but not with H-20 suggested that H-11 and H-20 are β and α-orientation, respectively. The absolute configuration of 1 was determined by comparing experimental and calculated ECD spectra. The predicted ECD spectrum of (2S, 7R, 11R, 12R)-1 was in good agreement with that of the experimental one ( Figure 4). Finally, the gross structure of 1 was assigned as shown and was given the trivial name lobophytin A. spectrum of (2S, 7R, 11R, 12R)-1 was in good agreement with that of the experimental one ( Figure 4). Finally, the gross structure of 1 was assigned as shown and was given the trivial name lobophytin A.     Figure S9). The IR spectrum of 3 revealed the presence of hydroxy (3450 cm −1 ) group and an α, β-unsaturated γ-lactone moiety (1737 and 1675 cm −1 ) ( Figure S10). The 1 H and 13 C NMR data ( Table 1) of 3 revealed its structure possessed great similarity to the known cembrane-type diterpenoid, sarcomililatin B (4) [13]. The main difference between them was that the hydroperoxy group in 4 was replaced by the hydroxyl group in 3. This replacement caused the chemical shift of C- spectrum of (2S, 7R, 11R, 12R)-1 was in good agreement with that of the experimental one ( Figure 4). Finally, the gross structure of 1 was assigned as shown and was given the trivial name lobophytin A.    Figure S9). The IR spectrum of 3 revealed the presence of hydroxy (3450 cm −1 ) group and an α, β-unsaturated γ-lactone moiety (1737 and 1675 cm −1 ) ( Figure S10). The 1 H and 13 C NMR data (Table 1) of 3 revealed its structure possessed great similarity to the known cembrane-type diterpenoid, sarcomililatin B (4) [13]. The main difference between them was that the hydroperoxy group in 4 was replaced by the hydroxyl group in 3. This replacement caused the chemical shift of C- spectrum of (2S, 7R, 11R, 12R)-1 was in good agreement with that of the experimental one ( Figure 4). Finally, the gross structure of 1 was assigned as shown and was given the trivial name lobophytin A.    Figure S9). The IR spectrum of 3 revealed the presence of hydroxy (3450 cm −1 ) group and an α, β-unsaturated γ-lactone moiety (1737 and 1675 cm −1 ) ( Figure S10). The 1 H and 13 C NMR data (Table 1) of 3 revealed its structure possessed great similarity to the known cembrane-type diterpenoid, sarcomililatin B (4) [13]. The main difference between them was that the hydroperoxy group in 4 was replaced by the hydroxyl group in 3. This replacement caused the chemical shift of C-   Figure S9). The IR spectrum of 3 revealed the presence of hydroxy (3450 cm −1 ) group and an α, β-unsaturated γ-lactone moiety (1737 and 1675 cm −1 ) ( Figure S10). The 1 H and 13 C NMR data (Table 1) of 3 revealed its structure possessed great similarity to the known cembrane-type diterpenoid, sarcomililatin B (4) [13]. The main difference between them was that the hydroperoxy group in 4 was replaced by the hydroxyl group in 3. This replacement caused the chemical shift of C-8 to be shifted upfield ∆δ C 11.4 ppm (δ C 72.6 in 3; 84.0 in 4). The significant 1 H-1 H COSY ( Figure S13) and HMBC correlations ( Figure 2) allowed the complete assignment for the planar structure of 3.  Table 2). This suggested that compound 10 was a geometric isomer of compound 15 with the replacement of 5E geometry of the double bond by 5Z ( Figure 5) on the base of the 13 C NMR chemical shifts of allylic methylene carbons (Z alkenes, δ C < 28 ppm; E alkenes, δ C > 30 ppm) of alkenes [14]. Detailed analysis of the 2D NMR spectroscopic data, the structure of 10 was established as (5E)-PGB 2 .
Mar. Drugs 2018, 16, x 5 of 11 8 to be shifted upfield ΔδC 11.4 ppm (δC 72.6 in 3; 84.0 in 4). The significant 1 H-1 H COSY ( Figure S13) and HMBC correlations ( Figure 2) allowed the complete assignment for the planar structure of 3. The relative configuration of 3 was deduced by analysis of the NOESY data ( Figure 3).  [13] showed quite similar ECD spectra (Figure 4), with a positive Cotton effect at 223 nm (Δε = +27.2) and a negative Cotton effect at 248 nm (Δε = −2.9), indicating that they have the same absolute configurations as 2S, 8R, 11R and 12R. Therefore, the structure of 3 was elucidated as depicted, named as lobophytin B.  Table 2). This suggested that compound 10 was a geometric isomer of compound 15 with the replacement of 5E geometry of the double bond by 5Z ( Figure 5) on the base of the 13 C NMR chemical shifts of allylic methylene carbons (Z alkenes, δC < 28 ppm; E alkenes, δC > 30 ppm) of alkenes [14]. Detailed analysis of the 2D NMR spectroscopic data, the structure of 10 was established as (5E)-PGB2.  Figure S25). The 1 H and 13 C NMR spectra ( Figure S27 and S28) of compound 11 were similar to those of prostaglandin, (5E)-PGB2 (10), except for the absence of two olefinic proton (δH 6.37 and 6.86; δC 124.3 and 143.7) and the presence of two additional methylenes (δH 2.53, 2.61; 1.58, 1.67). It means that compound 11 was an analogue of (5E)-PGB2 (10) with the replacement of two olefinic by two methylene group, which was supported by the 1 H-1 H COSY correlations of H-14 with H-13 and H-15 ( Figure 5). Detailed analysis of the 2D NMR spectroscopic data ( Figure S29-S31), the structure of 11 was established as shown in Figure 2 and named as (5E)-13,14-dihydro-PGB2.
Compound 12 was isolated as colorless oil. The molecular formula of 12 was established as C20H28O4 (five degrees of unsaturation) according to the negative HR-ESIMS ion at m/z 335.2231 [M − H] − (calculated for C20H31O4, 335.2227) ( Figure S32). The 1 H and 13 C NMR spectra ( Figure S34 and S35) of compound 12 were similar to those of (5E)-13,14-dihydro-PGB2 (11), except for chemical shift variation of two olefinic and two methylene carbons in 13 C NMR spectrum ( Table 2). This suggested that compound 12 was a geometric isomer of compound 11 with the replacement of 5Z geometry of the double bond by 5E according to the 13 C NMR chemical shifts of allylic methylene carbons (Z  (10), except for the absence of two olefinic proton (δ H 6.37 and 6.86; δ C 124.3 and 143.7) and the presence of two additional methylenes (δ H 2.53, 2.61; 1.58, 1.67). It means that compound 11 was an analogue of (5E)-PGB 2 (10) with the replacement of two olefinic by two methylene group, which was supported by the 1 H-1 H COSY correlations of H-14 with H-13 and H-15 ( Figure 5). Detailed analysis of the 2D NMR spectroscopic data ( Figures S29-S31), the structure of 11 was established as shown in Figure 2 and named as (5E)-13,14-dihydro-PGB 2 .
Compound 12 was isolated as colorless oil. The molecular formula of 12 was established as C 20 Figure S32). The 1 H and 13 C NMR spectra ( Figures S34 and S35) of compound 12 were similar to those of (5E)-13,14-dihydro-PGB 2 (11), except for chemical shift variation of two olefinic and two methylene carbons in 13 C NMR spectrum ( Table 2). This suggested that compound 12 was a geometric isomer of compound 11 with the replacement of 5Z geometry of the double bond by 5E according to the 13  carbons (Z alkenes, δ C < 28 ppm; E alkenes, δ C > 30 ppm) of alkenes. Therefore, the structure of 12 was established as 13,14-dihydro-PGB 2 .
Compound 13 was isolated as colorless oil. The molecular formula of 13 was assigned as C 20 Figure S40). The 1 H and 13 C NMR spectra (Figures S42 and S43) of compound 13 were quite similar to those of 13,14-dihydro-PGB 2 (12), except for the presence of an additional methyl group at δ H 3.62 (δ C 51.5) ( Table 2). The difference revealed that compound 13 was a methylated analogue of compound 12, which was supported by the HMBC correlations from −OCH 3 to C-1 ( Figure 5). Finally, 2D NMR spectroscopic data ( Figures S44-S46) further elucidated the structure of 13, as shown in Figure 5 and named as 13,14-dihydro-PGB 2 -Me.
All isolated prostaglandins 10-16 shared a secondary alcohol at C-15. Their absolute configurations at C-15 were assigned by the modified Mosher's method and comparison of optical activity sign, in combination with biosynthetic considerations. Initially, the known compound with most amount, PGB 2 (15) Figures S49 and S51). The chemical shift values (∆δ = δ S − δ R ) ( Figure 6) suggested that the absolute configuration of C-15 is S. Similarly, the absolute configuration of (5E)-PGB 2 (10) was clearly resolved by the modified Mosher's method and was characterized as S. Then, we also tried to use the modified Mosher's method to analyse the absolute configuration of (5E)-13,14-dihydro-PGB 2 (11), 13,14-dihydro-PGB 2 (12) and 13,14-dihydro-PGB 2 -Me (13), but the chemical shifts of H-14 and H-16 are overlapped and difficult to classify with the help of heteronuclear singular quantum correlation (HSQC) and 1 H-1 H COSY. Finally, optical rotations of all isolated prostaglandins 10-16 were measured and they have the same optical activity sign. Thus, the configuration at the hydroxy-bearing C-15 was deduced as S, as well as in combination with biosynthetic considerations.
All isolated compounds (except for 14 with limited amount) were tested for their inhibition activity against LPS-activated NO production in RAW264.7 cells using the Griess assay. Two cembrane-type diterpenoids 6 and 7 displayed promising inhibitory effects on the production of NO with IC 50 values 26.7 and 17.6 µM (the positive control indomethacin, IC 50 = 39.8 µM). apo-9 -Fucoxanthinone (9) had good inhibition activity against LPS-activated NO production in RAW264.7 cells with 32.1 µM. Most of the isolated prostaglandins, 10, 12, 13, 15 and 16 showed potential anti-inflammatory activity with IC 50 values 20.4, 24.8, 16.1, 15.9 and 7.1 µM, respectively. The other compounds were displayed weak or not anti-inflammatory. To evaluate the effects of all tested compounds on cell proliferation/viability, none of the compounds (up to 50 µM) showed any significant cytotoxicity with LPS treatment for 24 h using the thiazolyl blue tetrazolium bromide (MTT) method. The structure−activity relationships revealed that a methylene group at C-16 of five-member ring among cembrane-type diterpenoids was much more favorable than a carbonyl group for activity, as compound 5 was much more active than compounds 1−4. Among prostaglandins with 5E geometry, double bond at C-13 played an important role in their anti-inflammatory action because (5E)-PGB 2 (10) was much more active than (5E)-13,14-dihydro-PGB 2 (11). The side chain possessing a terminal carbonyl acid group made a more positive contribution to the anti-inflammatory activity than those with methoxycarbonyl group (12 vs. 13). The structure of PGA 2 made a more positive contribution to the anti-inflammatory activity than those of PGB 2 (16 vs . 15). In addition, the secondary metabolites (except for 14 with limited amount) were evaluated for their cytotoxicity using A549 (lung cancer), HepG2 (liver cancer) and MCF-7 (breast cancer) human cell lines and showed no cytotoxicity against all three cell lines at 50 µM.

Biological Material
Lobophytum sarcophytoides SYSU-MS001 were collected along the coast of Xisha Islands (17 • 06 14.50 N, 111 • 28 35.03 E), South China Sea, in July 2018, at a depth of −25 m and were frozen immediately after collection. The soft coral has been preserved at the school of marine sciences, Sun Yat-Sen University.

Extraction, Isolation and Characterization
The soft coral (0.9 kg) was cut into small pieces and extracted three times with CH 2 Cl 2 /MeOH (1:1, 1 L) to afford the organic extract (   The cytotoxic activities of the tested compounds were assayed according to the MTT method by using 96 well plates on the base of the previous reported procedures [24].

Anti-Inflammation Bioassays
The anti-inflammation activity of the isolated compounds was evaluated according to the reported procedures [25].