Krempfielins N–P, New Anti-Inflammatory Eunicellins from a Taiwanese Soft Coral Cladiella krempfi

Three new eunicellin-type diterpenoids, krempfielins N–P (1–3), were isolated from a Taiwanese soft coral Cladiella krempfi. The structures of the new metabolites were elucidated by extensive spectroscopic analysis and by comparison with spectroscopic data of related known compounds. Compound 3 exhibited activity to inhibit superoxide anion generation. Both 1 and3, in particular 1, were shown to display significant anti-inflammatory activity by inhibiting the elastase release in FMLP/CB-induced human neutrophils.


Introduction
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.

Results and Discussion
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 C 25 H 42 O 6 , 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 13 C NMR spectrum measured in CDCl 3 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 sp 3 methylenes, one sp 2 methylene, eight sp 3 methines (including four oxymethines), four quaternary carbons (including one ester carbonyl). The NMR spectroscopic data of 1 (Tables 1 and 2) showed the presence of one 1,1-disubstituted double bond (δ C 112.5 CH 2 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 CH 2 ; 18.4 CH 2 ; and 13.7 CH 3 ; 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 1 H-1 H 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 H 2 -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 C 28 H 44 O 9 (m/z 547.2880 [M + Na] + ). The NMR spectroscopic data of 2 (Tables 1 and 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, CH 3 and 21.6, CH 3 ), 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, CH 2 ; 18.5, CH 2 and 13.5, CH 3 ). 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).  (Tables 1 and 2). The 13 C 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 21.7CH 3 ) of an acetate group, respectively. The planar structure of 3 was determined by 1 H-1 H 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, CH 2 ; 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 same in 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,

General Experimental Procedures
Melting point was determined using a Fisher-Johns melting point apparatus. Optical rotations were measured on a JASCO P-1020 polarimeter. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. ESIMS were obtained with a Bruker APEX II mass spectrometer. The NMR spectra were recorded either on a Varian UNITY INOVA-500 FT-NMR and a Varian 400MR FT-NMR. Silica gel (Merck, Darmstadt, Germany, 230-400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Darmstadt, Germany, Kieselgel 60 F-254, 0.2 mm) were used for analytical thin layer chromatography (TLC). High performance liquid chromatography was performed on a Hitachi L-7100 HPLC apparatus with an octadecylsilane (ODS) column (250 × 21.2 mm, 5 µm).

Animal Material
C. krempfi was collected by hand using scuba off the coast of Penghu islands of Taiwan in June 2008, at a depth of 5-10 m, and stored in a freezer until extraction. A voucher sample (specimen No. 200806CK) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University.

Extraction and Separation
The octocoral (1.1 kg fresh wt) was collected and freeze-dried. The freeze-dried material was minced and extracted exhaustively with EtOH (3 × 10 L). The EtOH extract of the frozen organism was partitioned between CH 2 Cl 2 and H 2 O. The CH 2 Cl 2 -soluble portion (14.4 g) was subjected to column chromatography on silica gel and eluted with EtOAc in n-hexane (0%-100% of EtOAc, stepwise) and then further with MeOH in EtOAc with increasing polarity to yield 41 fractions. Fraction 31, eluted with n-hexane-EtOAc (1:10), was rechromatoraphed over a silica gel open column using n-hexane-acetone (3:1) as the mobile phase to afford eight subfractions (A1-A8). Subfraction