Flexibilins A–C, New Cembrane-Type Diterpenoids from the Formosan Soft Coral, Sinularia flexibilis

Three new cembrane-type diterpenoids, flexibilins A–C (1–3), along with a known cembrane, (−)-sandensolide (4), were isolated from the soft coral, Sinularia flexibilis. The structures of cembranes 1–4 were elucidated by spectroscopic methods. The structure of 4, including its absolute stereochemistry, was further confirmed by single-crystal X-ray diffraction analysis. Cembrane 2 displayed a moderate inhibitory effect on the release of elastase by human neutrophils.


Results and Discussion
Flexibilin A (1) was obtained as yellowish oil, and its molecular formula, C 20 H 32 O 5 , was determined according to a pseudomolecular ion [M + Na] + at m/z 375.2144 (calcd. for C 20 H 32 O 5 Na, 375.2147) identified by HRESIMS, as well as 13 C NMR coupled with DEPT spectra (Table 1), which indicated that the double bond equivalence (DBE) of 1 was five. The IR spectrum of 1 revealed the presence of carboxylic acid, hydroxy and ester functionalities from absorptions at 3750-2400, 3419 and 1711 cm −1 , respectively. The 13 C NMR data of 1 showed the presence of 20 carbon signals in total, which were assigned by the assistance of the DEPT spectrum to three methyls, seven sp 3 methylenes, three sp 3 methines, two sp 3 quaternary carbons, an sp 2 methylene, an sp 2 methine and three sp 2 quaternary carbons. 13  The tertiary methyls at C-4, C-8 and C-12 were confirmed by HMBC correlations between H 3 -18/C-3, -4, -5; H 3 -19/C-7, -8, -9; and H 3 -20/C-11, -12, -13. Thus, from the reported data, the skeleton of 1 was identified as a cembrane-type diterpenoid with two rings.  The relative configuration of 1 was elucidated from the interactions observed in a NOESY experiment, as shown in Figure 3. In the NOESY experiment of 1, H-1 was found to be correlated with H 2 -14, but not with H-13; this, plus, the lack of correlation between H-13 and H 3 -20 demonstrated that H-1, H-13 and Me-20 were α-, β-and α-oriented, respectively. Additionally, correlations between H-9/H-5 and H-9/H-13, and the absence of correlation between H-9/H 3 -19, reflected the E geometry of the double bond at C-8/9. From modeling analysis, H-9 was found to be close to H-5 and H-13, when H-5 and H-13 were β-oriented. H 3 -18 was found to be correlated with H-5, but not with H-1, indicating that the 4-hydroxy group was α-oriented. Based on the above findings, the structure of 1 was elucidated, and the chiral carbons of 1 were assigned as 1R*, 4R*, 5S*, 12S* and 13S*. The new cembrane diterpene, flexibilin B (2), had the molecular formula, C 20 H 30 O 5 , as deduced by HRESIMS (m/z calcd.: 373.1991; found 373.1989 [M + Na] + ) (6° of unsaturation). The IR spectrum of 2 revealed the presence of carboxylic acid (ν max 3700-2400 cm −1 ), hydroxy (ν max 3447 cm −1 ) and carbonyl (ν max 1713 cm −1 ) moieties. From the 13 C NMR and DEPT spectra of 2 (Table 2), a trisubstituted olefin (δ C 134.5, C-8; 126.5, CH-9), a α-exomethylene-carboxylic acid (δ C 171.3, C-16; 141.9, C-15; 126.2, CH 2 -17), a keto carbonyl (δ C 213.6, C-5) and a trisubstituted epoxide (δ C 60.8, C-12; 59.4, CH-13) were observed. The coupling information in the 1 H-1 H COSY spectrum of 2 enabled identification of the H-13/H 2 -14/H-1/H 2 -2/H 2 -3, H 2 -6/H 2 -7, H-9/H 2 -10/H 2 -11 and H-9/H 3 -19 (by allylic coupling) units. From these data, together with the results of an HMBC experiment for 2 ( Table 2 and Figure 4), the molecular framework of 2 could be established.  The relative stereochemistry of 2 was elucidated from the interactions observed in a NOESY experiment ( Figure 5). Due to the α-orientation of H-1, the epoxy proton, H-13, was identified as being β-oriented, as no correlation was observed between H-1 and H-13. H-9 displayed correlations with H-13 and one of the C-7 methylene protons (δ H 2.55), and there was a lack of correlation between H-9 and H 3 -19, which reflects the E geometry of the double bond between C-8/9. H 3 -20 showed correlations with H-9 and H-13, indicating that Me-20 is of a β-orientation at C-12. Furthermore, H-1 was found to be correlated with H 2 -3, and H 3 -18 exhibited a correlation with one of the C-3 methylene protons (δ H 1.78), which indicated the β-orientation of Me-18 at C-4 by molecular modeling analysis. Based on the above information, the chiral carbons of cembrane 2 were assigned as 1R*, 4R*, 12R* and 13S*.  (Table 3 and Figure 6). The key HMBC correlations between protons and quaternary carbons of 3, such as H 2 -2,     (4), was first isolated from a Chinese soft coral, Dendronephthya sp. [23], and its structure was elucidated by spectroscopic methods and by comparison of spectral (1D and 2D NMR) and physical (rotation value) data with those of its enantiomer, sandensolide [10,22]. In this study, the structure of 4, including its absolute stereochemistry, was further established by single-crystal X-ray diffraction analysis for the first time. The X-ray structure of 4 ( Figure 8) demonstrates the E geometry of the C-8/9 and C-11/12 double bonds, and the absolute configurations for all chiral carbons were assigned as 1R, 4R, 5S and 13S. As flexibilins A-C (1-3) were isolated, along with (−)-sandensolide (4), from the same organism, it is reasonable on biogenetic grounds to assume that cembranes 1-3 have the same absolute configurations as 4. The in vitro anti-inflammatory effects of cembranes 1-4 were examined, and cembrane 2 displayed a moderate inhibitory effect on the release of elastase by human neutrophils (Table 4) [24].

General Experimental Procedures
Optical rotations were measured on a Jasco P-1010 digital polarimeter. Infrared spectra were recorded on a Varian Diglab FTS 1000 FT-IR spectrometer; peaks are reported in cm −1 . The NMR spectra were recorded on a Varian Inova 500 or on a Varian Mercury Plus 400 NMR spectrometer using the residual CHCl 3 signal (δ H 7.26 ppm) as the internal standard for 1 H NMR and CDCl 3 (δ C 77.1 ppm) for 13 C NMR. Coupling constants (J) are given in Hz. ESIMS and HRESIMS were recorded on a Bruker APEX II mass spectrometer. Column chromatography was performed on silica gel (230-400 mesh, Merck, Darmstadt, Germany). TLC was carried out on precoated Kieselgel 60 F 254 (0.25 mm, Merck); spots were visualized by spraying with 10% H 2 SO 4 solution, followed by heating. HPLC was performed using a system comprised of a Hitachi L-7110 pump and a Rheodyne injection port. A normal phase semi-preparative column (Supelco Ascentis ® Si cat#:581515-U, 250 × 21.2 mm, 5 μm) was used for HPLC.

Animal Material
Specimens of the octocoral S. flexibilis were collected by hand using scuba equipment off the coast of southern Taiwan in July, 2011, and stored in a freezer until extraction. A voucher specimen (NMMBA-TWSC-11005) was deposited in the National Museum of Marine Biology and Aquarium, Taiwan.

Extraction and Isolation
Sliced bodies of the soft coral S. flexibilis (wet weight 3.0 kg, dry weight 950 g) were extracted with ethyl acetate (EtOAc). The EtOAc layer was separated by silica gel and eluted using n-hexane/EtOAc in a stepwise fashion from pure n-hexane-100:1-pure EtOAc to yield 11 fractions, A-K. Fraction J was chromatographed on silica gel and eluted using n-hexane/EtOAc (stepwise, 2:1-1:1) to afford subfractions 1-10. Fractions J3, J5 and J9 were separated by normal-phase HPLC (NP-HPLC) using a mixture of n-hexane and acetone as the mobile phase to afford 4 (3:1, 74.3 mg), 3 (2:1, 5.6 mg) and 2 (3:1, 16.6 mg), respectively. Fraction K was further purified by NP-HPLC using a mixture of n-hexane and acetone as the mobile phase to afford 1 (2:1, 22.4 mg). Intensity data were measured on a Bruker APEX-II CCD diffractometer equipped with a micro-focus Cu radiation source and Montel mirror up to θ max of 26° at 100 K. All 5454 reflections were collected. The structure was solved by direct methods and refined by a full-matrix least-squares procedure. The refined structural model converged to a final R1 = 0.0294, wR2 = 0.0759 for 5398 observed reflection [I > 2σ(I)] and 455 variable parameters. The absolute configuration was determined by Flack's method, with the Flack's parameter determined as 0.06(10) [26].

Generation of Superoxide Anions and Release of Elastase by Human Neutrophils
Human neutrophils were obtained by means of dextran sedimentation and Ficoll centrifugation. Measurements of superoxide anion generation and elastase release were carried out according to previously described procedures [27][28][29][30][31][32][33]. Briefly, superoxide anion production was assayed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. Elastase release experiments were performed using MeO-Suc-Ala-Ala-Pro-Valp-nitroanilide as the elastase substrate.

Conclusions
Cembrane-type diterpenoids are the major component of the organic extract of the soft coral, Sinularia flexibilis, collected from the waters of Taiwan, and compounds of this type have been shown to have the potential for use in medical applications. Our studies of S. flexibilis have led to the isolation of three new cembranes, flexibilins A-C (1-3), along with a known metabolite, (−)-sandensolide (4), and flexibilin B (2) displayed a moderate inhibitory effect on the release of elastase by human neutrophils. In addition to marine organisms, cembrane-type diterpenoids are also found in higher plants and bryophytes [34][35][36][37][38][39][40][41][42][43]. However, the carboxylic acid moieties exhibited in cembranes 1 and 2 are rarely found [35,44]. The soft coral, S. flexibilis, has begun to be transplanted to culturing tanks with a flow-through seawater system located in the National Museum of Marine Biology and Aquarium, Taiwan, for exhibition and the extraction of additional natural products in order to establish a stable supply of bioactive material.