Soft corals have been known to be rich sources of terpenoid metabolites [1]. For the purpose of discovering bioactive agents from marine organisms, we have previously investigated the chemical constituents and reported a series of bioactive natural products from Taiwanese soft corals [2,3,4,5]. In recent studies a series of bioactive eunicellin-based diterpenoids, have been isolated from the soft corals of the genera Cladiella, Klysum and Litophyton sp. [6,7,8,9,10,11,12,13,14]. The soft coral Cladiella krempfi has been found to produce several types of metabolites including eunicellin-type diterpenoids [15,16,17] and pregnane-type steroids [18,19]. Our previous chemical investigation of the Formosan soft coral Cladiella krempfi also resulted in the isolation of a series of new eunicellin-type diterpenoids, krempfielins A–M [20,21,22]. In this paper, we further report the discovery of three new eunicellin-based diterpenoids, krempfielins N–P (1–3) (Chart 1 and Supplementary Figures S1–S9). The ability of these compounds to inhibit the superoxide anion generation and elastase release in FMLP/CB-induced human neutrophils was also evaluated. The results showed that compound 3 could inhibit superoxide anion generation while 1 and 3, especially 1, effectively inhibited the generation of the elastase release in FMLP/CB-induced human neutrophils.

The new metabolite krempfielin N (1) showed the molecular ion peak [M + Na]⁺ at m/z 461.2882 in the HRESIMS and established a molecular formula of C₂₅H₄₂O₆, implying five degrees of unsaturation. The IR absorptions bands at ν_max 3445 and 1733 cm⁻¹ revealed the presence of hydroxy and ester carbonyl functionalities. The ¹³C NMR spectrum measured in CDCl₃ showed signals of 25 carbons (Table 1) which were assigned by the assistance of the DEPT spectrum to six methyls (including one oxgenate methyl δ_C 57.0), six sp³ methylenes, one sp² methylene, eight sp³ methines (including four oxymethines), four quaternary carbons (including one ester carbonyl). The NMR spectroscopic data of 1 (Table 1 and Table 2) showed the presence of one 1,1-disubstituted double bond (δ_C 112.5 CH₂ and 148.0 C; δ_H 5.03 s, and 4.86 s), one methoxy group (δ_H 3.34, 3H, s) and one n-butyryloxy group (δ_C 172.3 C; 37.4 CH₂; 18.4 CH₂; and 13.7 CH₃; δ_H 2.30 m, 2H; 1.67 m, 2H; and 0.98 t, 3H, J = 7.6 Hz). Therefore, taking account of the two degrees of unsaturation from double bonds, it was suggested that 1 should be a tricyclic compound from the remaining three degrees of unsaturation. The ¹H–¹H COSY and HMBC correlations (Figure 1) were further used for establishing the molecular skeleton of 1. The COSY experiment assigned three isolated consecutive proton spin systems. Above evidences and the analysis of HMBC spectrum (Figure 1) suggested that 1 is an eunicellin-based diterpenoid. Furthermore, the two hydroxy groups attaching at C-7 and C-12 were confirmed by the HMBC correlations from one methyl (δ_H 1.12 s, H-16) and one oxymethine (δ_H 4.12 m, H-6) to the oxygenated quaternary carbon appearing at δ 75.8 (C-7), and one methine (δ_H 2.91 t, H-10) and one proton of H₂-17 (δ_H 5.03 s) to the oxymethine carbon appearing at δ 71.0 (C-12). Thus, the remaining one n-butyryloxy group had to be positioned at C-3, an oxygen-bearing quaternary carbon resonating at δ 86.5 ppm. On the basis of above analysis, the planar structure of 1 was established. The stereochemistry of 1 was finally confirmed by the very similar NOE correlations of both 1 and krempfielin L [22].

Krempfielin O (2) was shown by HRESIMS to possess the molecular formula C₂₈H₄₄O₉ (m/z 547.2880 [M + Na]⁺). The NMR spectroscopic data of 2 (Table 1 and Table 2) showed the presence of two acetoxy groups (δ_H 2.07, s and 2.08, s, each 3H; and δ_C 170.7, C and 170.2, C; 21.4, CH₃ and 21.6, CH₃), and an n-butyryloxy group (δ_H 2.60 m and 2.50 m, each 1H; 1.67 m, 2H and 1.00 t, 3H, J = 7.5 Hz; and δ_C 173.0, C; 36.7, CH₂; 18.5, CH₂ and 13.5, CH₃). As demonstrated by the HMBC correlation from oxymethine proton H-8 (δ 5.19) to the ester carbonyl carbon appearing at δ_C 170.7 (Figure 1), one acetoxy group was positioned at C-8. The position of an n-butyryloxy group at C-3 was established by NOE interaction between the methylene protons (δ 1.67) of n-butyryloxy group with H-5 (δ 1.49). The remaining one acetoxy group was thus positioned at C-12. The relative configuration of 2 was further confirmed by NOE correlations (Figure 2).

The related metabolite, krempfielin P (3), had a molecular formula of C₂₆H₄₀O₇ as indicated by the HRESIMS (m/z 487.2675, [M + Na]⁺) and NMR data (Table 1 and Table 2). The ¹³C NMR spectrum of 3 revealed the appearance of two ester carbonyls (δ_C 172.5 and 170.1), which were correlated with one methylene (δ_H 2.12 m, 2H; and δ_C 37.4) of an n-butyrate and the methyl (δ_H 2.05 s, 3H; δ_C 21.7CH₃) of an acetate group, respectively. The planar structure of 3 was determined by ¹H–¹H COSY and HMBC correlations (Figure 1). Comparison of the NMR data of 3 with those of the compound krempfielin A [20] revealed that the only difference is the replacement of one methyl and one hydroxy group at C-7 in krempfielin A by the substitution of one olefinic methylene (δ_C 118.1, CH₂; δ_H 5.55, s and 5.23, s) in 3. The placement of one n-butyryloxy group and one acetoxy group at C-3 and C-12, respectively was established by comparison of the spectroscopic data with those of krempfielin A. The relative configuration of 3 was mostly determined to be the same as that of krempfielin A by comparison of the chemical shifts of both compounds and was further confirmed by NOE correlations (Figure 2).

Recently, we discovered several eunicellins showed anti-inflammatory activity by significantly inhibiting superoxide anion generation and elastase release in human neutrophiles induced by N-formyl-methionyl-leucyl-phenylalanine/cytochalasin B (FMLP/CB) [22,23]. The samein vitro anti-inflammatory effects of the diterpenoids 1–3 also were tested in this study (Table 3). At a concentration of 10 µM, 1 and 2 could not significantly reduce the generation of superoxide anion, however, 3 inhibited 23.32% ± 5.88% generation of superoxide anion, relative to the control cells stimulated with FMLP/CB only. At the same concentration, all of 1–3 were found to show anti-inflammatory activity by inhibiting the elastase release. Compound 1 displayed significant inhibition (73.86% ± 14.18%) at this concentration with IC₅₀ of 4.94 ± 1.68 µM in this assay.

New eunicellin-based diterpenoids were isolated from the soft coral Cladiella krempfi. Compounds 1 and 3, especially 1, could significantly inhibit the release of elastase in FMLP/CB-induced human neutrophils, and 3 inhibited 23% generation of superoxide anion. Thus, compounds 1 and 3 are promising anti-inflammatory agents and may warrant further biomedical investigation.