Klyflaccicembranols A–I, New Cembranoids from the Soft Coral Klyxum flaccidum

New cembranoids klyflaccicembranols A–I (1–9), along with gibberosene D (10), have been isolated from the organic extract of a Formosan soft coral Klyxum flaccidum. Their structures were established by extensive spectroscopic analyses, including 2D NMR spectroscopy, and spectral data comparison with related structures. The cytotoxicity of the isolated metabolites, as well as their nitric oxide (NO) inhibitory activity, were evaluated and reported. Metabolites 2, 4, 6, 8 and 9 were found to exhibit variable activities against a limited panel of cancer cell lines in a range of IC50 16.5–49.4 μM. Among the tested cembranoids, compounds 4, 5, 6, and 9 significantly inhibited NO production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages at a dose of 50 μg/mL.

New metabolite 3 was found to have the molecular formula C 22 H 34 O 4 , as deduced from the HRESIMS (m/z 385.2354 [M + Na] + ) and NMR data (Tables 1 and 2), implying six degrees of unsaturation. As in the cases of 2 and 8, the IR absorptions at 1731 and 3450 cm −1 further indicated the presence of an ester moiety and a hydroxy group. The NMR data (Tables 1 and 2) of an acetoxy group (δ C 170.7, C; 21.1, CH 3 ; δ H 2.10, 3H, s), a trisubstituted epoxide (δ C 61.3, C and 59.7, CH) and three trisubstituted olefins (δ C 155.9, 137.6, 133.2, each C, and 126.3, 125.2, 115.6, each CH) established five degrees of unsaturation in the molecule. By comparison of NMR spectroscopic data with those of gibberosene D (10) isolated from the soft coral S. gibberosa [16], compound 3 was suggested to be a regioisomer of 10. Interpretation of 2D NMR correlations ( Figure 2) indicated a secondary hydroxy group at C-2 and a trisubstituted double bond at C-3/C-4 in 3 instead of the disubstituted double bond and the tertiary hydroxy group at C-2/C-3 and C-4, respectively, in 10. Detailed COSY and HMBC correlations (Figure 2) further determined the C-7/C-8, C-1/C-14, C-11/C-12, and C-13 positions for the two other olefins, the epoxide, and the acetoxy group in the structure of 3, respectively, as were identified in 10 ( Figure 1). Thus, the gross structure of 3 was established as 13-acetoxy-11,12-epoxy-2-hydroxycembra-3,7,14-triene. The NOE interaction of H-13 with H-2 enabled assignment of their syn positions, and hence the α-orientation of the hydroxy group at C-2, based on the previously defined S configuration at C-13 (as in 1 and 8). Hz) reflected the E geometries of the two disubstituted double bonds at C-2/C-3 and C-6/C-7. Moreover, the NOE correlations ( Figure 5) found for H3-20 (1.60, 3H, s) with one proton of H2-10 (δH 2.37, m), but not with H-11, and for H-14 with the protons of the two methyls at C-15, indicated the E and Z geometries of the double bonds at C-11/C-12 and C-1/C-14, respectively. On the basis of the previously defined α-orientation of 4-OH in biogenetically-related compounds 1, 2, and 8, and the NOE interactions of the β-oriented H3-18 (δH 1.27, 3H, s) with the olefinic H-6 and the trans downward-oriented H-7 with H3-19 (δH 1.16, 3H, s), the β-orientation of the hydroxy group at C-8 was proven. These findings, together with detailed analysis of other NOE correlations (Figure 4), indicated the structure of klyflaccicembranol D (4) to be (4S,8R,2E,6E,11E,14Z)-4,8dihydroxycembra-2,6,11,14-tetraene.  Klyflaccicembranol E (5) was obtained as a colorless oil. It possessed the molecular formula C20H34O3 and four unsaturations, as concluded from the pseudomolecular ion peak [M + Na] + at m/z New metabolite klyflaccicembranol D (4) was isolated as a pale oil. A pseudomolecular ion peak [M + Na] + at m/z 327.2200 in the HRESIMS data was observed, corresponding to the molecular formula C 20 H 32 O 2 and five degrees of unsaturation. The IR absorption at ν max 3419 cm −1 and two sp 3 oxycarbon signals appearing at δ C 72.9 and 72.5 indicated the hydroxyl-bearing characteristic of the compound. Two trisubstituted double bonds were also identified from the NMR signals at δ C 146.3, C; 132.8, C; 126.7, CH; 122.0, CH; δ H 5.48 (1H, dd, J = 8.0, 5.6 Hz), and 5.11 (1H, dd, J = 7.2, 7.2 Hz). Analysis of COSY correlations of 4 established five consecutive proton sequences, including those of the isopropyl group ( Figure 2). Careful study of the HMBC spectrum of 4 further established the connectivities of the five proton sequences with the diagnostic nonprotonated carbons (C-1, C-4, C-8, and C-12), as illustrated in Figure 2. Thus, the two 1,2-disubstuituted and the two trisubstituted double bonds were localized at C-2/C-3, C-6/C-7, C-11/C-12, and C-14/C-1, respectively, whereas the hydroxy groups were positioned at C-4 and C-8. Thus, the planar structure of 4 was established as 4,8-dihydroxycembra-2,6,11,14-tetraene. The large coupling constants of J 2,3 (16.4 Hz) and J 6,7 (15.6 Hz) reflected the E geometries of the two disubstituted double bonds at C-2/C-3 and C-6/C-7. Moreover, the NOE correlations ( Figure 5 . Therefore, the 3,14-dihydroxy compound 5, relative to the 3,14-epoxy compound 11, exhibited significant upfield (∆δ C − 8.5, −6.5, −13.9 ppm) and downfield (∆δ C + 3.6 ppm) shifts at C-2, C-3, C-14, and C-1, respectively. Furthermore, extensive interpretation of 2D NMR correlations further established the gross structure of compound 5 to be 3,4,14-trihydroxycembra-1(2),7,11-triene ( Figure 2). The geometries of the double bonds and stereochemistries at C-3, C-4, and C-14 were determined by careful investigation of NOE correlations exhibited in the NOESY spectrum (   β-face. Therefore, 1-OH and 4-OH should be α-oriented. The chemical shift values of C-19 (δC 14.7) and C-20 (δC 14.8) reflected the E geometries of the trisubstituted double bonds at C-7/C-8 and C-11/C-12 in the molecule of 6. Moreover, it should be noted that the large JHH values for H-2 and H-3 (δC 16.0 Hz) and the null NOE response reflected the trans positions of these two protons. On the basis of the above findings, metabolite 6 was determined to be (1R,4S,2E,7E,11E)-1,4,15trihydroxycembra-2,7,11-triene. The remaining ten carbons were assigned to five methyls, one sp 3 methine and four methylene groups. By NMR spectroscopic data comparison, it was found that this compound was the 13-hydroxy derivative of 10, isolated in this study and previously from S. gibberosa [16]. The hydroxy group at C-13 in 7 induced a significant upfield shift at H-13 (∆δH − 1.21 ppm) and a downfield shift at C-14 (∆δC + 5.0 ppm) relative to 10. The structure of 7 was unambiguously determined by the extensive analysis of COSY and HMBC ( Figure 2  Klyflaccicembranol F (6) displayed a pseudomolecular ion peak at m/z, 345.2405 ([M + Na] + ) in the HRESIMS, consistent with a molecular formula of C 20 H 34 O 3 and four degrees of unsaturation. Its IR spectrum also showed a broad absorption band at ν max 3392 cm −1 , representing a hydroxy group. This was further supported by NMR signals resonating at δ C 80.9, 75.1, and 71.9 (each C) of tertiary hydroxyl-bearing carbons and a hydroxy proton at δ H 2.60 (1H, s). Moreover, carbon signals appearing at δ C 129.2 (C-2, CH), 138.0 (C-3, CH), 128.6 (C-7, CH), 132.7 (C-8, C), 126.9 (C-11, CH), and 136.1 (C-12, C) indicated the presence of one disubstituted double bond and two trisubstituted olefins in 6, respectively. The analysis of COSY correlations (Figure 2) of 6 indicated four consecutive proton sequences. The connection of four partial structures was subsequently resolved by the HMBC. Furthermore, long-range correlations observed from both H 3 -16 and H 3 -17 (δ H 1.13 and 1.21, each 3H, s) to C-15 (δ C 75.1, C) and C-1 (δ C 80.9, C), 1-OH (δ H 2.60, 1H, s) to C-1 and C-14, and H-2 (δ H 5.61, 1H, d, J = 16.0 Hz) to C-1 and C-4 (δ C 71.9, C), enabled localization of the hydroxy groups at C-15, C-1, and C-4, respectively. The planar structure of compound 6 was thus described as 1,4,15-trihydroxycembra-2,7,11-triene ( Figure 2). Comparison of NMR data of 6 with the known cembranoid crassumol A (12) [23] revealed that 6 had similar 1 H and 13 C chemical shifts, except for the significant downfield shifts noticed at C-3, C-4, C-5 (∆δ C + 1.1, +1.8, and +1.5, respectively) and H-2 (∆δ H + 0.18), and upfield shifts at C-2 (∆δ C − 0.9 ppm) and H-3 (∆δ H − 0.18 ppm), which suggested klyflaccicembranol F (6) to be the 1-epimer of 12 ( Figure 6). Careful investigation of key NOE correlations (Figure 7) enabled us to prove the α-orientation of 1-OH. In the NOESY spectrum of 6, H 3 -18 was found to exhibit correlation with H-2, and H-2 with both H 3 -16 and H 3 -17; therefore, assuming a β-orientation of H 3 -18, H-2 and the isopropyl methyls should also be positioned on the β-face. Therefore, 1-OH and 4-OH should be α-oriented. The chemical shift values of C-19 (δ C 14.7) and C-20 (δ C 14.8) reflected the E geometries of the trisubstituted double bonds at C-7/C-8 and C-11/C-12 in the molecule of 6. Moreover, it should be noted that the large J HH values for H-2 and H-3 (δ C 16.0 Hz) and the null NOE response reflected the trans positions of these two protons. On the basis of the above findings, metabolite 6 was determined to be (1R,4S,2E,7E,11E)-1,4,15-trihydroxycembra-2,7,11-triene. New metabolite 7 was also isolated as a colorless oil, with the molecular formula C 20 H 32 O 3 , as indicated by HRESIMS (m/z 343.2250 [M + Na] + ). Substitution of the hydroxy group of this compound was revealed by the IR absorption band at ν max 3419 cm −1 and 13 C NMR signals at δ C 73.0 (C) and 71.6 (CH). The two proton signals resonating at δ H 6.24 and 5.75 (each 1H, d, J = 16.0 Hz) were found to represent two olefinic protons correlated in the HSQC spectrum with the carbon signals at δ C 123.7 and 138.3 (each CH), respectively, attributable to a trans 1,2-disubstituted double bond. Moreover, carbon signals at δ C 146.0 (C), 131.9 (C), 127.6 (CH), 122.9 (CH), 64.7 (C), and 61.3 (CH) indicated the presence of two trisubstituted double bonds and a trisubstituted epoxide in 7. The remaining ten carbons were assigned to five methyls, one sp 3 methine and four methylene groups. By NMR spectroscopic data comparison, it was found that this compound was the 13-hydroxy derivative of 10, isolated in this study and previously from S. gibberosa [16]. The hydroxy group at C-13 in 7 induced a significant upfield shift at H-13 (∆δ H − 1.21 ppm) and a downfield shift at C-14 (∆δ C + 5.0 ppm) relative to 10. The structure of 7 was unambiguously determined by the extensive analysis of COSY and HMBC ( Figure 2 (Tables 1 and 3). The 1 H and 13 C NMR data demonstrated the characteristic features of non-lactonized cembranoids (C 20 signals, including those of five methyls) isolated previously from soft corals of the genus Sinularia and Sarcophyton [16][17][18]24,25]. By careful spectral comparison, it was found that the 1 H and 13 C NMR data of 9 were identical to those of 11,12-epoxy-13,14-dihydroxycembrene obtained by hydrolysis of flaccidoxide [26], including the magnitude and sign of optical rotation [α] 25 D +124. Cytotoxicity of metabolites 1-6 and 8-10 against the growth of HT-29 (human colon adenocarcinoma), A549 (human lung adenocarcinoma), K562 (human erythromyeloblastoid leukemia), and P388 (mouse lymphatic leukemia) cell lines was evaluated. With the exception of inactive metabolites 1, 3, 5, and 10, all the other compounds exhibited variable potency against the tested cell lines (Table 4). Compounds 4 and 6 showed cytotoxicity against K562 and A549 (IC 50 44.9 and 21.4 µM, respectively). Compound 8 was capable of affecting the growth of three cancer cell lines (A549, K562, and P388) in the range of IC 50 34.6-49.4 µM; however, it was found to be doubly-potent (IC 50 49.4 µM) relative to the positive control fluorouracil (IC 50 110 µM) against A549 cancer cells. Compound 2 was cytotoxic against two cell lines (A549 and K562), being 6.5-fold more potent than the positive control against the growth of A549. In addition, compound 9 was cytotoxic against another pair of cancer cells (HT-29 and P338), being very potent against P388.

General Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). IR spectra were recorded on a JASCO FT/IR-4100 spectrophotometer (JASCO). Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. ESIMS and HRESIMS spectral data were recorded on a BRUKER APEX II mass spectrometer (Bruker, Bremen, Germany). The NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR at 500 MHz for 1 H and 125 MHz for 13 C or on a Varian 400 FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C or on a Bruker AMX-300 FT-NMR at 300 MHz for 1 H and 75 MHz for 13 C, in CDCl3 or C6D6 using TMS as internal standard (δ in ppm, J in Hz). Silica gel 60 (Merck, 230-400 mesh), precoated silica gel plates (Merck, Darmstadt, Germany, Kieselgel 60 F254, 0.2 mm) were used for open CC and analytical TLC analysis, respectively. Isolation by HPLC was performed by a Hitachi L-2455 instrument equipped with a reversed-phase (RP-18) column (GL Sciences Inc., Tokyo, Japan ODS-3, 5 μm, 250 × 20 mm).

Animal Material
The soft coral Klyxum flaccidum Tixier-Durivault (Alcyoniidea) was collected by hand via SCUBA off the coast of Hsiao Liuchiu Island (22°19′48″ N 120°21′55″ E; Pingtung County), in October 2011, at a depth of 10-15 m along the coast of the island of Pratas, Taiwan. The species identification is based on three levels of morphological characters, i.e., colony shape, polyps' morphology and the morphology of sclerites in different parts of the coral colony, and then stored at −20 °C until extraction. A voucher sample (specimen no. LI6) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University (Kaohsiung, Taiwan). The organism was identified by Professor Chang-Feng Dai, Institute of Oceanography, National Taiwan The isolated compounds 1-6 and 8-10 also were evaluated in terms of their ability to suppress NO in LPS-activated RAW264.7 macrophages (Figure 8). The results showed that cembranoids 5 and 9 strongly inhibited 88% and 87% of NO production at 50 µg/mL, respectively. However, compounds 4 and 6 at the same dose possessed moderate potency (65% and 64% NO inhibition), with IC 50 values of 46.7 and 47.0 µg/mL, respectively. The higher cell viability indexes attained by 4 and 6 (98% and 95%, respectively) relative to 5 and 9 represented an advantageous characteristic in addition to the NO inhibitory effect over 5 or 9. A positive control, curcumin, at 10 µg/mL succeeded under the same experimental conditions in reducing the NO level by 92.5% (IC 50 6.3 µg/mL), in association with 98% retention of cell viability. With the exception of the inactive metabolite 2, the rest of the tested compounds (1, 3, 8 and 10) showed weak NO inhibitory activity (12%-25%).

General Procedures
Optical rotations were measured on a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). IR spectra were recorded on a JASCO FT/IR-4100 spectrophotometer (JASCO). Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. ESIMS and HRESIMS spectral data were recorded on a BRUKER APEX II mass spectrometer (Bruker, Bremen, Germany). The NMR spectra were recorded on a Varian Unity INOVA 500 FT-NMR at 500 MHz for 1 H and 125 MHz for 13 C or on a Varian 400  FT-NMR at 400 MHz for 1 H and 100 MHz for 13 C or on a Bruker AMX-300 FT-NMR at 300 MHz  for 1 H and 75 MHz for 13 C, in CDCl 3 or C 6 D 6 using TMS as internal standard (δ in ppm, J in Hz). Silica gel 60 (Merck, 230-400 mesh), precoated silica gel plates (Merck, Darmstadt, Germany, Kieselgel 60 F254, 0.2 mm) were used for open CC and analytical TLC analysis, respectively. Isolation by HPLC was performed by a Hitachi L-2455 instrument equipped with a reversed-phase (RP-18) column (GL Sciences Inc., Tokyo, Japan ODS-3, 5 µm, 250 × 20 mm).

Animal Material
The soft coral Klyxum flaccidum Tixier-Durivault (Alcyoniidea) was collected by hand via SCUBA off the coast of Hsiao Liuchiu Island (22 • 19 48" N 120 • 21 55" E; Pingtung County), in October 2011, at a depth of 10-15 m along the coast of the island of Pratas, Taiwan. The species identification is based on three levels of morphological characters, i.e., colony shape, polyps' morphology and the morphology of sclerites in different parts of the coral colony, and then stored at −20 • C until extraction. A voucher sample (specimen no. LI6) was deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University (Kaohsiung, Taiwan). The organism was identified by Professor Chang-Feng Dai, Institute of Oceanography, National Taiwan University, Taipei 112, Taiwan.

Conclusions
For the first time, cembranoid-based compounds were isolated and identified from the soft corals of genus Klyxum by this study. Five metabolites (klyflaccicembranols B, D, F, H, and I) exhibited variable activities against a limited panel of cancer cell lines while klyflaccicembranols D-F and I showed strong anti-inflammatory effect through inhibition of NO production in LPS-stimulated RAW264.7 macrophages.