Six New Diterpene Glycosides from the Soft Coral Lemnalia bournei

A chemical study on the extracts of soft coral Lemnalia bournei resulted in the isolation and identification of six new bicyclic diterpene glycosides including three new lemnaboursides E–G (1–3), and three new lemnadiolboursides A–C (4–6), along with three known lemnaboursides (7–9). Their structures were elucidated by detailed spectroscopic analysis, ECD analysis, chemical methods, and comparison with the literature data. Lemnadiolboursides A–C (4–6) contained a lemnal-1(10)-ene-7,12-diol moiety compared with the lemnaboursides. All these compounds were evaluated for antibacterial activity; cell growth inhibition of A549, Hela, HepG2, and CCRF-CEM cancer cell lines; and inhibition of LPS-induced NO production in RAW264.7 macrophages. The results indicated that compounds 1, 2, and 4–6 exhibited antibacterial activity against Staphylococcus aureus and Bacillus subtilis (MIC 4–16 μg/mL); compounds 1–9 displayed low cytotoxicity on the CCRF-CEM cell lines (IC50 10.44–27.40 µM); and compounds 1, 2, and 5 showed weak inhibition against LPS-induced NO production (IC50 21.56–28.06 μM).

The diterpene glycosides from genus Lemnalia are all biflorane-type glycosides such as lemnaboursides, lemnaflavosides, lemnalosides, and their acetate derivatives [7][8][9][10]. Previous chemical investigations of Lemnalia bournei only provided four diterpene glycosides including lemnabourside and lemnaboursides B-D [9][10][11]. Lemnabourside was characterized with a D-glucose attached to a diterpene aldehyde through an acetal linkage, and lemnaboursides B and C were two analogs of lemnaboursides with monoacetylation of the sugar residue at different hydroxyl groups [1]. Biologically, these three metabolites only showed weak cytotoxicity.
To explore the bioactive secondary metabolites from marine organisms, soft coral L. bournei were collected from the coast of Xisha Island (7 m deep) by SCUBA diving.
age, and lemnaboursides B and C were two analogs of lemnaboursides with monoacetylation of the sugar residue at different hydroxyl groups [1]. Biologically, these three metabolites only showed weak cytotoxicity.

Results and Discussion
Lemnabourside E (1) was isolated as an amorphous solid. Its molecular formula was determined to be C 28 16), another four methyls, six methylenes, and six methines. The spectral data of 1 were similar to those of diterpene glycosides isolated from the soft coral Lemnalia bournei (Tables 1 and 2) with the differences reflecting the mono-acetylate position of the sugar residue [9]. Analysis of the COSY spectrum of 1 rapidly identified the sugar unit connection. In the HMBC experiment, the correlations from H-17 to C-3, and H-4 to C-17 suggested that C-3 is connected to C-17. The HMBC correlation between H-18 and C-10 indicated C-18 was located at C-9. In addition, the HMBC experiment showed correlations between H-10 and C-9, H-17 and C-2, H-20 and C-15, H-15 and C-1 , H-16 and C-6 , which indicated that C-9 was bonded to C-10, C-2 to C-3, C-20 to C-15, C-15 to C-16, and the presence of two acetal bonds. This evidence proved that 1 was a lemnabourside derivative, and the HMBC correlations from H-2 to acetyl carbon δ C 172.2 positioned the acetate group at C-2 ( Figure 2).  H-4 to C-17 suggested that C-3 is connected to C-17. The HMBC correlation between H-18 and C-10 indicated C-18 was located at C-9. In addition, the HMBC experiment showed correlations between H-10 and C-9, H-17 and C-2, H-20 and C-15, H-15 and C-1′, H-16 and C-6′, which indicated that C-9 was bonded to C-10, C-2 to C-3, C-20 to C-15, C-15 to C-16, and the presence of two acetal bonds. This evidence proved that 1 was a lemnabourside derivative, and the HMBC correlations from H-2′ to acetyl carbon δC 172.2 positioned the acetate group at C-2′ ( Figure 2).   The configuration of the sugar unit was assigned after the hydrolysis of 1 with H 2 SO 4 . The hydrolysate reaction mixture was partitioned between CH 2 Cl 2 and H 2 O. The CH 2 Cl 2 yielded the decalin-type bicyclic diterpene aldehyde (see 1 HNMR in Figure S56) according to the literature data [9]. The aqueous part was conducted with L-cysteine methyl ester and o-tolyl isothiocyanate and yielded methyl 2-(polyhydroxyalkyl)-3-(o-tolylthiocarbamoyl)thiazolidine-4(R)-carboxylates, according to the reported method [12]. The HPLC retention time (Rt = 7.8 min) of the sugar derivative was compared with the standard sample prepared in the same manner ( Figure S57). In this way, the sugar unit was determined to be D-glucose.
A cis-decalin configuration in the diterpene portion was deduced from the 13 C chemical shifts in C-5 (δ C 36.6) and C-10 (δ C 39.7) as well as the small coupling constant (∼2 Hz) between H-1 and H-6, which were very similar to those of the separated known lemnaboursides (7-9). Moreover, H 3 -19/H-5 showed NOE interactions, but there were no NOE correlations between H 3 -19 and H-6, which suggested H 3 -19 and H-5 were positioned on the same orientation. The absolute configurations of the decalin part could also be deduced from the DFT/ECD calculations. The experimental ECD spectrum exhibited a negative Cotton effect (CE) at 202 nm, which was in good agreement with the calculated ECD spectra of (5R,6S,10S,11S)-1 ( Figure S1). The β-configuration of the glucose was confirmed by the 1D-NOE method as the reported method, and it was in the boat form [10]. The torsion angle of H-1 and H-2 was close to 90 • , and the magnitude of the coupling was generally the smallest (close to 0). Moreover, H 3 -20/H-16 and H 3 -20/H-1 also showed NOE interactions, which positioned H 3 -20, H-16, and H-1 on the same face.
Lemnabourside F (2) was also a white solid with the chemical formula of C 30 H 48 O 8 as revealed by the HRESIMS ion peak, indicating seven degrees of unsaturation. Acid hydrol-ysis of 2 also yielded D-glucose. The 1 H NMR spectra of 2 exhibited most of the structural features found in 1, with the major difference of ring D opened, and two acetyl groups (δ C /δ H 172.3, 21.1/2.17 and δ C /δ H 171.6, 20.9/2.11) and oxygenated methylene (δ C /δ H 75.7/3.67/3.38) rather than oxygenated methine were present. The HMBC correlation of H-3 /3 -OAc and H-6 /6 -OAc suggested that the C-3 and C-6 hydroxyl groups of the glucose were acetylated (Figure 2). The 1 H-1 H COSY, HSQC, and HMBC experiments allowed for the complete assignment for structure 2. Acid hydrolysis of 2 was discriminated in the same manner as 1. The CH 2 Cl 2 part yielded the decalin-type bicyclic diterpene alcohol (see 1 HNMR in Figure S56). The absolute configurations of 2 were also proposed to be the same as that of 1 based on their identical 13 C chemical shifts of C-5, C-6, C-10, and C-11 as well as on biosynthetic considerations. The coupling constant of the anomeric proton was about 7 Hz, which suggested that ring C was in the chair form, and the torsion angle of H-1 and H-2 was close to 180 • .
Compound 3, a white solid, had the molecular formula C 32 H 50 O 9 as determined by HRESIMS. The 1 H NMR spectra of 3 showed a high similarity to those of 2 except for the presence of three acetyl groups instead of two. These three acetyl groups were located at 3 -OH, 4 -OH, and 6 -OH, as confirmed by the HMBC correlation from H-3 to 3 -OAc (δ C 170.7), from H-4 to 4 -OAc (δ C 169.7), and from H-6 to 6 -OAc (δ C 170.7). The acid hydrolysis of 3 also yields D-glucose. 1 H-1 H COSY, HSQC, HMBC, and 1D-NOE experiments allowed for the complete assignment of the structure of 3. Compound 3 was given the name lemnabourside G.
Compound 4, a white solid named lemnadiolbourside A, had the molecular formula C 41 H 64 O 8 , established by HRESIMS and NMR data. The 13 C and DEPT spectra exhibited a total of 41 carbon resonances (Table 2). Overall, the comparison of 1 H and 13 C NMR data of 4 and lemnabourside (7) revealed that 4 contained the structural unit of lemnabourside with 26 carbons [9]. The remaining 15 carbons belonged to a nardosinane-type sesquiterpenoid [13]. The analysis of the COSY spectrum of compound 4 revealed the presence of the lemnabourside unit and the nardosinane unit ( to C-2 allowed us to identify the nardosinane located at 2 -OH of the lemnabourside unit ( Figure 2). The acid hydrolysis of 4 was conducted following the above method, which yielded D-glucose and diterpene aldehyde, but failed to reveal the nardosinane moiety due to the inherent instability of lemnal-1(10)-ene-7,12-diol under strong acid and heating conditions. The relative configuration of 4 was deduced based onNOE correlations ( Figure 3).
All of the isolated compounds (1-9) were evaluated for antibacterial activity (against Bacillus subtilis, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Salmonella paratyphi), cell growth inhibition (against A549, Hela, HepG2, and CCRF-CEM cancer cell lines), and inhibition of LPS-induced NO production in RAW264.7 macrophages. As shown in Table 3, compounds 1, 2, and 7-9 exhibited antibacterial activity against S. aureus and B. subtilis (MIC 4−16 µg/mL), compounds 1-9 displayed low cytotoxicity on the CCRF-CEM cell lines (IC 50 10.44-27.40 µM); and compounds 1, 2, and 5 showed weak inhibition against LPS-induced NO production (IC 50 21.56-28.06 µM). The antibacterial activity of lemnaboursides disappeared if the glucose was condensed with the nardosinane moiety. Moreover, lemnabourside G (tri-acetylation of the sugar at different hydroxyl groups) was also inactive. It can be concluded that steric hindrance may decrease the antibacterial activity of lemnaboursides, however, it seems to not affect the weak cytotoxic activity (against CCRF-CEM cell lines).

Animal Material
Specimens of the soft coral Lemnalia bournei were collected from the coast of Xisha Island in the South China Sea by SCUBA diving at a depth of 7m, and frozen immediately after collection. The specimen was identified by Prof. Ping-Jyun Sung (National Museum of Marine Biology and Aquarium). The fresh sample was shown in Figure S58. The spicules from the cortex of the basal part of the stem, the cortex of the distal part of the stem, and the tentacles were extracted, then observed under a microscope ( Figure S59

Extraction and Isolation
The frozen soft coral (wet mass 2.5 Kg) was cut into pieces and freeze-dried, then extracted with acetone six times under ultrasound condition. The acetone extract was concentrated, and then partitioned between  Tables 1 and 2 .0 (c 0.10, MeOH)}; this structure was deduced by comparison with 1 H and 13 C NMR literature data of the known compound as well as the sign of its specific rotation [9].
Lemnabourside B (8): white solid; {[α] 25 D +27.0 (c 0.10, MeOH)}; this structure was deduced by comparison with 1 H and 13 C NMR literature data of the known compound as well as the sign of its specific rotation [9].
Lemnabourside C (9): white solid; {[α] 25 D +33.0 (c 0.10, MeOH)}; this structure was deduced by comparison with the 1 H and 13 C NMR literature data of the known compound as well as the sign of its specific rotation [9].

Acid Hydrolysis of Compounds 1-6
A solution of compound (3.0 mg) dissolved in dioxane (0.5 mL) and in 1 N H 2 SO 4 (0.5 mL) was refluxed (4 h), cooled, and extracted with CH 2 Cl 2 (3 × 2 mL). The CH 2 Cl 2 layer was condensed and washed with 5% NaHCO 3 and H 2 O five times respectively, and then purified on Si gel preparative thin layer chromatography [petroleum ether/EtOAc, 30:1, v/v] to give a diterpene aldehyde (for 1, 4-6) or diterpene alcohol (for 2 and 3). The aqueous layer was neutralized with 5% NaOH to pH 7 and then condensed as white solids to yield D-glucose. The D-glucose product and L-cysteine methyl ester hydrochloride (5 mg) was dissolved in pyridine (0.5 mL) and heated at 60 • C for 60 min, and then phenylisothiocyanate (5 µL) was added to the mixture and heated at 60 • C for 60 min. The reaction mixture was analyzed by UPLC and detected at 250 nm, 25 • C, using a Waters BEH C 18 (1.7 µm, 2.1 × 100 mm) column, eluting with MeCN/H 2 O (from 10:90 to 0:100, v/v, 0-10 min). Peaks of the glucose derivatives were detected by comparison with retention time. Standard D-glucose was treated in the same way as the sample.

Computational Methods
The theoretical electronic circular dichroism (ECD) spectra of the isolated compounds were calculated with the Gaussian09 program package based on the relative configurations determined by their 1D-NOE spectra (Gaussian Inc., Wallingford, CT, USA). Conformational analyses and density functional theory (DFT) calculations were used to generate and optimize the conformers at the B3LYP/6-31+G(d,p) level of theory as the method described in a reported article [14].

Antibacterial Assays
The antibacterial activities were evaluated with the broth dilution assay [15].

Cytotoxic Activity Assay
Cell lines were purchased from the American Type Cultural Collection (ATCC, Manassas, VA, USA). The cytotoxicity of the compounds was evaluated against the A549, HepG2, Hela, and CCRF-CEM with the Cell Counting Kit-8 (CCK-8) as the reported method [16]. Chidamide was used as the positive control.

Inhibition of Nitric Oxide Production Assay
The inhibition of the nitric oxide production assay was conducted as a previously reported protocol [17]. Dexamethasone in DMSO was used as the positive control.

Conclusions
In the course of the exploration of bioactive secondary metabolites from marine organisms, six new bicyclic diterpene glycosides (1-6) and three known lemnaboursides (7)(8)(9) were isolated and characterized from soft coral L. bournei. Based on the ECD spectroscopic data and the biogenetic consideration, the absolute configuration of the new compounds could be determined. In bioassay, compounds 1, 2, and 4-6 exhibited antibacterial activity against S. aureus and B. subtilis; compounds 1-9 displayed low cytotoxicity on CCRF-CEM cell lines; and compounds 1, 2, and 5 showed weak inhibition against LPS-induced NO production. The antibacterial activity of lemnabourside G (tri-acetylated glucose) and lemnadiolboursides disappeared, which indicated the steric hindrance may decrease the antibacterial activity of lemnaboursides. It is worth noting that the discovery of compounds 1-6, once again, enriched the chemical diversity and complexity of diterpene glycosides from marine organisms, and will stimulate further pharmacological studies due to their intriguing structural features and potent biological activities. As the main component of L. bournei, further pharmacological investigations of lemnabourside are still worth pursuing.