Antimicrobial Terpenoids from South China Sea Soft Coral Lemnalia sp.

Chemical investigation of the South China Sea soft coral Lemnalia sp. afforded 13 structurally diverse terpenoids, including three new neolemnane sesquiterpene lineolemnenes E–G (1–3); a new aristolane-type sesquiterpenoid, 2-acetoxy-aristolane (4); four new decalin-type bicyclic diterpenes, named biofloranates A−D (5−8); a new serrulatane, named euplexaurene D (9); and a new aromadendrane-type diterpenoid cneorubin K (10), together with three known related compounds (11−13). The structures of the new compounds were elucidated by NMR spectroscopy, the Mosher’s method, and ECD analysis. Compounds 1–13 were tested in a wide panel of biological assays. Lineolemnene J (3) showed weak cytotoxicity against the CCRF-CEM cancer cell line. The isolated new diterpenes, except euplexaurene D (9), demonstrated moderate antimicrobial activity against Bacillus subtilis and Staphylococcus aureus with a MIC of 4−64 μg/mL. Compound 2 exhibited a mild inhibitory effect against influenza A H1N1 virus (IC50 = 5.9 µM).


Introduction
In the competition for food and living space for survival, marine organisms accumulate and secrete secondary metabolites that are deterrent, offensive, or even toxic to other organisms-that is, chemical defense substances [1]. Marine invertebrates, such as soft corals, grow slowly and lack physical defense capabilities, but they can survive in competitive marine environments. What they often depend on are chemical defense strategies, which represent a rich source of unexplored chemical structures and biological diversity with great potential for drug discovery [2].
The South China Sea is located in the tropics and subtropics, with a vast sea area and rich marine biological resources. It is a sea area with numerous coral distributions. Since the 1980s, researchers have studied the chemical composition of many kinds of corals collected from the South China Sea [3][4][5][6][7][8]. Many compounds with novel structures have been isolated, including steroids, terpenoids, alkaloids, etc. Among them, the marine soft coral genus Lemnalia has afforded structurally diverse novel terpenoids, including sesquiterpenes and diterpene glycosides, with promising biological activity [7].
Biofloranate A (5) was also a colorless oil, and the molecular formula was established as C 21 H 34 O 3 (HR-ESI-MS: m/z 357.2400 [M + Na] + ). The 1D NMR and HSQC spectra of 5 (Tables 2 and 3) disclosed two trisubstituted olefinic bonds (δ C /δ H 134.7 and 123.6/5.47; δ C /δ H 136.7 and 121.2/5.39), an acetate group (δ C /δ H 176.7 and 51.7/3.71), and an oxygenated methine (δ C /δ H 72.1/3.85), which also indicated a bicyclic structure. A comparison of the 1 H and 13 C NMR data of 5 with the aglycone of lemnabourside [12] showed that they had identical ring systems, including stereochemistry, and similar side chains. The planar structure was further established by the analysis of COSY and HMBC correlations. Compound 6 shared the same molecular formula as 5. The 1 H and 13 C NMR spectra of 6 (Tables 2 and 3) were also similar to those of 5, except that the oxygenated carbon was quaternary (δ C 74.8) instead of a methine. As a result, the hydroxy was connected to C-15 instead of C-14. The orientation of 15-OH present in 3 was hard to determine, leading to the unknown relative configuration of C-15. Compound 6 was given the name biofloranate B.   18 13.2,CH3 13.3,CH3 13.1,CH3 13.3,CH3 16.1,C 19 23.9,CH3 24.0,CH3 23.8,CH3 24.0,CH3 27.9,C 20 21.7,CH3 21.8,CH3 21.6,CH3 21.7,CH3 18.8,C OMe 51.7,CH3 52.7,CH3 51.7, CH3 Compound 6 shared the same molecular formula as 5. The 1 of 6 (Tables 2 and 3) were also similar to those of 5, except that the quaternary (δC 74.8) instead of a methine. As a result, the hydroxy instead of C-14. The orientation of 15-OH present in 3 was hard the unknown relative configuration of C-15. Compound 6 was ranate B.
Biofloranate C (7) had a molecular formula of C21H32O3 as de data, requiring six degrees of unsaturation. Comparing the molec and 6, two hydrogens were missing, corresponding to the presen tuted double bond (δC/δH 130. 4 and 146.1/6.88). An oxygenated 57.2/4.33) showed HMBC correlations with C-14, C-15, and C-16 COSY correlation and H-14/C-12/C-13/C-15/C-16/C-17 HMBC cor gested that the extra double bond was C-14−C-15. The H-14/16-O placed H-14 and the methoxy group on the same side ( Figure S59 7 was established unambiguously. Biofloranate D (8) was obtain the formula was assigned as C20H34O by HR-ESI-MS (m/z 313.23  (Tables 2 and 3) also corresponded closely to those of aglycone of lemnabourside [12]. Compared with aglycone, the 1D NMR data revealed the absence of an aldehyde group but the presence of an oxygenated methylene (δ C /δ Ha /δ Hb 68. 4/3.49/3.40). The structure of 8 was also confirmed by 1 H-1 H COSY and HMBC correlations, as shown in Figure 2. The AC of C-15 was also difficult to determine.
Euplexaurene D (9) was isolated as a white powder, and HR-EI-MS analysis (found 290.2604, M + ) showed the molecular formula C 20 H 34 O, suggesting four degrees of unsaturation. As the only sp 2 group, the trisubstituted olefinic bond (δ C /δ H 131. 3 and 125.0/5.12) was deduced from the 1D NMR data, indicating the tricyclic structure of 9. In the 13 C NMR spectrum (Table 3), 20 carbon signals were assignable to three quaternary carbons, six methines, six methylenes, and five methyls. Comparing the 13 C NMR spectrum of 9 with that of reported euplexaurene A [13] showed that they have identical ring systems, and the main difference was the position of the hydroxy group. Measuring the 2D NMR spectrum ( Figure 2) enabled the assignment of all resonances in the 1 H NMR and 13 C NMR spectra (Tables 2 and 3). The relative configurations of 9 were assigned by NOESY correlations between H .

Animal Material
Soft coral Lemnalia sp. (no. XSSC201907) was collected from the Xisha Islands of the South China Sea in April 2019 by scuba at a depth of 10 m. It was identified by Prof. Pingjyun Sung, National Museum of Marine Biology and Aquarium (NMMBA), Taiwan, China.