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Mar. Drugs 2017, 15(1), 23; doi:10.3390/md15010023

Article
Klyflaccicembranols A–I, New Cembranoids from the Soft Coral Klyxum flaccidum
Atallah F. Ahmed 1,2,3, Chia-Ruei Tsai 1, Chiung-Yao Huang 1, Sheng-Yang Wang 4 and Jyh-Horng Sheu 1,5,6,7,8,*
1
Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
2
Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
3
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
4
Department of Forestry, National Chung Hsing University, Taichung 40227, Taiwan
5
Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80756, Taiwan
6
Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan
7
Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
8
Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
*
Correspondence: Tel.: +886-7-5252-000 (ext. 5030)
Academic Editor: Kirsten Benkendorff
Received: 19 December 2016 / Accepted: 16 January 2017 / Published: 21 January 2017

Abstract

:
New cembranoids klyflaccicembranols A–I (19), 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.
Keywords:
soft coral; Klyxum flaccidum; cembranoid; NO inhibition; cytotoxicity

1. Introduction

The marine environment has long been recognized as an exceptional source of new natural products with a diverse range of chemical structures and bioactivities, including anti-cancer, antiviral, immunosuppressive and anti-inflammatory activities [1,2,3]. This structural diversity has supplied unique chemicals for the pharmaceutical, cosmetics, and even agrochemicals industry [4]. Soft corals belonging to the genus Klyxum (Alcyoniidae), including Klyxum flaccidum, are considered rich sources of eunicellin-based diterpenoids species [2,5,6,7,8,9,10], of which many compounds have been found to exert anti-inflammatory [7,8,9,10] and cytotoxic [8,11] effects. A series of cytotoxic and anti-inflammatory steroids was also discovered from K. flaccidum in our previous investigations [12,13]. Cembranoid-based diterpenoids have not hitherto been isolated from soft corals of the genus Klyxum. However, our continuing investigation into the chemical constituents of a Formosan soft coral K. flaccidum has led to the discovery of a series of new polyoxygenated cembranoids. Extensive spectroscopic analyses, including 2D NMR spectroscopy and spectral comparison, were applied to establish the structures of these new metabolites. The cytotoxicity of the isolated metabolites was further assessed against a limited panel of cancer cell lines via Alamar Blue assay [14,15], and anti-inflammatory activity was evaluated in terms of their potential to inhibit nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages.

2. Results and Discussion

Sliced bodies of the soft coral K. flaccidum were extracted exhaustively with ethyl acetate (EtOAc). The solvent-free extract was then fractionated over a silica gel 60 column to yield 26 fractions. Fractions that displayed terpenoidal methyl, olfeinic and oxymethine proton signals in the 1H NMR spectrum were selected and further purified using a successive series of silica gel 60 and RP-18 gel columns to yield compounds 110 (Figure 1).
Klyflaccicembranol A (1) was isolated as a colorless oil. Its molecular formula C20H32O4 was deduced from the sodium adduct ion peak (m/z 359.2196 [M + Na]+) in the high-resolution electrospray ionization mass spectrometry (HRESIMS) and NMR data measured in C6D6 (Table 1 and Table 2). Thus, five degrees of unsaturation were indicated. The infrared (IR) absorption band at νmax 3445 cm−1 revealed the presence of hydroxy functionality. The 13C NMR spectrum displayed 20 carbon signals (Table 1), which were characterized by distortionless enhancement by polarization transfer (DEPT) and heteronuclear single-quantum correlation (HSQC) spectra as five methyls, four methylenes, seven methines (including two olfeinic and four sp3 oxymethines), and four nonprotonated carbons (including two olfeinic and two sp3 oxycarbons). The NMR signals at δC 147.8, C; 132.1, C; 127.0, CH and 121.0, CH; δH 5.59, 1H, br s and 5.38, 1H, dd, J = 5.2, 5.2 Hz indicated the presence of two trisubstituted double bonds. Thus, the remaining three unsaturations suggested 1 to be a tricyclic diterpenoid. However, it was found that the NMR data of 1 differed from those of eunicellins discovered from Klyxum species [2,5,6,7,8,9,10] and resembled those of cembranoids isolated from Sinularia and Sarcophyton species [16,17,18], as no correlation spectroscopy (COSY) correlated ring-juncture sp3 methine protons were detected in 1. One trisubstituted epoxide (δC 61.9, C and 59.7, CH; δH 3.15, 1H, dd, J = 6.4, 2.0 Hz) and an ether bridge (δC 89.7, CH; δH 4.61, 1H, d, J = 4.8 Hz and δC 85.8, CH; δH 4.78, 1H, br d, J = 4.8 Hz) of a 2,3,5-trisubstituted dihydrofuran moiety [18] were also deduced. Thus, the remaining two oxygen atoms of the molecular formula were ascribed to two hydroxy groups linked to the carbon atoms resonating at 72.8 (CH) and 74.2 (C). Analysis of the COSY correlations of 1 indicated the presence of four proton-correlated partial structures, including those of an isopropyl group (Figure 2). By analysis of heteronuclear multiple bond correlation (HMBC) correlations observed from the methyl, olfeinic, and oxymethine protons, it was possible to connect the four partial structures and to assign the positions of the two hydroxy groups, one trisubstituted double bond, and a trisubstituted epoxide at C-4, C-13, C-7/C-8, and C-11/C-12, respectively (Figure 2). The ether linkage of the dihydrofuran was confirmed by the HMBC correlations found from H-2 (δH 5.59, br s) to C-1 (δC 147.8, C), C-3 (δC 89.7, CH) and C-14 (δC 85.8, CH) and from H-14 (δH 4.78, br d, J = 4.8 Hz) to C-3, respectively. The planar structure of compound 1 was thus established as 4,13-dihydroxy-3,14:11,12-bisepoxy-cembra-1,7-diene.
The relative configuration of 1 was determined by the analysis of nuclear Overhauser effect (NOE) correlations in a nuclear Overhauser enhancement spectroscopy (NOESY) experiment and with the assistance of 5JH,H coupling constants, in addition to molecular modeling using molecular mechanical parameters (MM2 force field) calculations (Figure 3). The high magnitude of the long-range coupling constant of H-3 and H-14 (J = 4.8 Hz) of the 2,5-dihydrofuran ring in 1 suggested the trans orientation of protons at C-3/C-14 [19]. Accordingly, no NOE correlation was detected between H-3 and H-14. From the NOESY spectrum of 1, it was found that H3-18 showed NOE interaction with H-14, and H-14 with H-13 and H3-20; therefore, due to the β-orientation of H3-18, H-13 and H-14 should also be positioned on the β-face. Furthermore, H-3 exhibited NOE correlation with the olefinic H-7, as did H-7 with H-11, while H-11 did not show NOE interaction with H3-20, revealing that H-3 and H-11 should be positioned on the α-face. Moreover, the NOE interactions exhibited for the olefinic H-2 with H3-17 and for H3-19 with H-9, but not with H-7, enabled the assignment of Z and E geometries of the double bonds at C-1/C-2 and C-7/C-8, respectively. As a result, the (3R*,4S*,11S*,12R*,13S*,14R*)-configuration of 1 was established.
After isolation and chemical identification of 1, related compound klyflaccicembranol H (8) was isolated from the less polar fraction of the same extract of the organism. Compound 8 possessed the molecular formula C22H34O5, as indicated by the HRESIMS (m/z 378.2404 [M]+) and NMR data (Table 1 and Table 3). IR absorptions at νmax 3310 and 1730 cm−1 and electron ionization mass spectroscopy EIMS ion peaks at m/z 318 [M − AcOH]+ and m/z 300 [M − AcOH − H2O]+ revealed the presence of hydroxy and acetoxy functionalities in the molecule of 8. On comparison of spectroscopic data, the NMR spectrum of 8 was found to be very similar to that of 1 (Table 1, Table 2 and Table 3), with the exception of the presence of signals of an additional methyl and an ester carbonyl carbon of an acetoxy group at δC 169.9, C and 20.6, CH3; and δH 2.06, 3H, s. These were associated with a downfield shift displayed by H-13 in 8H 5.16, d, J = 2.0 Hz) relative to that of 1H 3.58, br s). Therefore, 8 was suggested to be the 13-acetylated derivative of 1, as proven by complete 2D NMR correlations analyses (Figure 2). Moreover, compound 8, measured in CDCl3, showed the same NOE correlations that had been observed in the NOESY spectrum of 1, indicating the same relative configuration. Furthermore, hydrolysis of 8 yielded 1. To clarify the absolute configuration at C-13 in 1 (and hence also in 8), a modified Mosher’s method [20,21] was employed to prepare (S)- and (R)-α-methoxy-α-(trifluoromethyl)phenylacetic (MTPA) esters of 1 (1a and 1b, respectively) using the corresponding (−)- and (+)-MTPA-chloride, respectively. Calculation of ∆δ values (δH S − δH R) for the protons adjacent to C-13 led to the assignment of the S configuration at C-13 in 1, and consequently therefore in 8 (Figure 4). On the basis of the above findings and detailed NOESY correlations analyses (Figure 3), compound 1 was identified as (3R,4S,11S,12R,13S,14R,1Z,7E)-4,13-dihydroxy-3,14:11,12-bisepoxy-cembra-1,7-diene. Compound 8 (klyflaccicembranol H) was thus subsequently characterized as the C-13 acetyl derivative of 1.
Klyflaccicembranol B (2) was also isolated as a colorless oil. The sodium adduct ion peak [M + Na]+ appearing at m/z 417.2250 in the HRESIMS was appropriate for the molecular formula C22H34O6 with six unsaturations. The IR absorption bands at νmax 3443 and 1746 cm−1 indicated hydroxy and ester functionalities in the molecule. The NMR signals (δC 169.9, C and 21.0, CH3; δH 1.91, 3H, s) showed the ester functionality to be an acetoxy group. A trisubstituted-2,5-dihydrofuran moiety (δC 147.7, C; 121.2, CH; 91.1, CH and 85.0, CH; δH 5.65, br s; 5.05, d, J = 5.0 Hz and 4.78, d, J = 5.0 Hz) was also deduced, as in case of 1 and 8. However, the 13C NMR spectral comparison of compound 2 with 8 in the regions of δC 125–140 and 58–65 ppm indicated that 2 had an additional trisubstituted epoxide, instead of the 7,8-trisubstiututed double bond in 8. The gross structure of 2 was thus established by analysis of COSY and HMBC correlations (Figure 2). The relative configuration of 2 was determined by analysis of NOE correlations, as shown in Figure 3. The NOE interactions observed for H3-19 (δH 1.24, s) with H-6 (δH 1.59, m), H-7 (δH 3.07, dd, J = 6.0, 2.5 Hz) with H-3, and H-11 (δH 2.98, d, J = 7.5 Hz) with H3-20 (δH 1.42, s), indicated the trans and cis geometries of the trisubstituted 7,8- and 11,12-epoxides, respectively. As H-3 exhibited a NOE correlation with H-7, H-7 and H3-19 were thus α- and β-oriented, respectively. Moreover, the NOE correlations observed for H-14 with both H3-20 and H-13 disclosed the α-orientation of the acetyl group at C-13 and the β-orientation of the methyl group at C-12, and hence the β-oriented epoxide proton at C-11. On the basis of the above findings and the absolute configuration of biogenetically-related 1, compound 2 was thus determined as (3R,4S,7R,8R,11R,12R,13S,14R,1Z)-7,8:11,12:3,14-triepoxy-4-hydroxy-13- acetoxycembra-1-ene.
New metabolite 3 was found to have the molecular formula C22H34O4, as deduced from the HRESIMS (m/z 385.2354 [M + Na]+) and NMR data (Table 1 and Table 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 (Table 1 and Table 2) of an acetoxy group (δC 170.7, C; 21.1, CH3; δH 2.10, 3H, s), a trisubstituted epoxide (δC 61.3, C and 59.7, CH) and three trisubstituted olefins (δC 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). The NOE interactions of H-14 with H3-20 (δH 1.22, 3H, s) and H-13 with H-11 (δH 2.47, dd, J = 7.2, 2.0 Hz) indicated the trans geometry of the 11,12-epoxide, and, consequently, the 11S and 12R configurations. Furthermore, other NOE interactions of H-2 with H3-18 (δH 1.80, 3H, s), H-3 with H-7 (δH 4.83, br d, J = 6.0 Hz), and H3-19 (δH 1.55, 3H, s) with H-6 (δH 2.27, m) established the E geometries of the C-3/C-4 and C-7/C-8 double bonds. The Z geometry of the double bond at C-14/C-1 in 3 was proven by the NOE correlation (Figure 5) of H-14 with the isopropyl methyls at C-15 (δH 1.05 and 1.09, each 3H, d, J = 6.8 Hz). The above observations thus established the structure of klyflaccicembranol C (3) as (2S,11S,12R,13S,3E,7E,14Z)-13-acetoxy-11,12-epoxy-2-hydroxycembra-3,7,14-triene.
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 C20H32O2 and five degrees of unsaturation. The IR absorption at νmax 3419 cm−1 and two sp3 oxycarbon signals appearing at δC 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 δC 146.3, C; 132.8, C; 126.7, CH; 122.0, CH; δH 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 J2,3 (16.4 Hz) and J6,7 (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) 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)-4,8-dihydroxycembra-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 345.2404 in the HRESIMS. The presence of hydroxy groups in 5 was demonstrated by an IR absorption band at νmax 3408 cm−1 and 13C NMR signals at δC 75.2 (C), 70.7 (CH), and 70.6 (CH). The NMR spectroscopic data comparison of 5 with semisynthetic product 11 (Figure 6), obtained by chromic acid oxidation of sarcophytol A [22], suggested that 5 (C20H34O3) was the hydrated derivative of 11 (C20H32O2). Therefore, the 3,14-dihydroxy compound 5, relative to the 3,14-epoxy compound 11, exhibited significant upfield (∆δC − 8.5, −6.5, −13.9 ppm) and downfield (∆δC + 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 (Figure 7) in combination with molecular modelling. The NOE correlations of the olefinic H-2 with protons of the isopropyl methyls (H3-16 and H3-17) revealed the Z geometry of the double bond at C-1/C-2. The chemical shift values of C-19 (15.1) and C-20 (17.1) reflected the E geometries of the trisubstituted double bonds (δC < 20 ppm) at C-7/C-8 and C-11/C-12 in the molecule of 5. Assuming an S-configuration at C-4 (as found in biogenetically-related metabolites 1, 2, and 8), the NOE interaction of H-2 with H3-18, but not with H-3, reflected the R-configuration at C-3. Consequently, the S-configuration was allocated for C-14, as H-14 exhibited strong NOE correlation with H-3. The above-mentioned findings, along with detailed analysis of other NOE correlations (Figure 7), identified klyflaccicembranol E (5) as (3R,4S,14S,1Z,7E,11E)-3,4,14-trihydroxycembra-1,7,11-triene.
Klyflaccicembranol F (6) displayed a pseudomolecular ion peak at m/z, 345.2405 ([M + Na]+) in the HRESIMS, consistent with a molecular formula of C20H34O3 and four degrees of unsaturation. Its IR spectrum also showed a broad absorption band at νmax 3392 cm−1, representing a hydroxy group. This was further supported by NMR signals resonating at δC 80.9, 75.1, and 71.9 (each C) of tertiary hydroxyl-bearing carbons and a hydroxy proton at δH 2.60 (1H, s). Moreover, carbon signals appearing at δC 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 H3-16 and H3-17 (δH 1.13 and 1.21, each 3H, s) to C-15 (δC 75.1, C) and C-1 (δC 80.9, C), 1-OH (δH 2.60, 1H, s) to C-1 and C-14, and H-2 (δH 5.61, 1H, d, J = 16.0 Hz) to C-1 and C-4 (δC 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 1H and 13C chemical shifts, except for the significant downfield shifts noticed at C-3, C-4, C-5 (∆δC + 1.1, +1.8, and +1.5, respectively) and H-2 (∆δH + 0.18), and upfield shifts at C-2 (∆δC − 0.9 ppm) and H-3 (∆δH − 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, H3-18 was found to exhibit correlation with H-2, and H-2 with both H3-16 and H3-17; therefore, assuming a β-orientation of H3-18, 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 (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)-1,4,15- trihydroxycembra-2,7,11-triene.
New metabolite 7 was also isolated as a colorless oil, with the molecular formula C20H32O3, 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 νmax 3419 cm−1 and 13C NMR signals at δC 73.0 (C) and 71.6 (CH). The two proton signals resonating at δH 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 δC 123.7 and 138.3 (each CH), respectively, attributable to a trans 1,2-disubstituted double bond. Moreover, carbon signals at δC 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 sp3 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) and NOESY correlations (Figure 7). In addition, the appearance of the 1H double doublet signal of H-13 (δH 4.59, 1H, dd, J = 8.0, 8.0 Hz) was due to vicinal coupling with the olefinic H-14 (δH 5.10, 1H, d, J = 8.0 Hz) and the free proton of the hydroxy group at C-13 (δH 1.74, 1H, d, J = 8.0 Hz). Comparison of the splitting patterns of H-13 and H-14 of 7 (d, J = 8.0 Hz) with those of known cembranoid 10 (d, J = 9.0 Hz) suggested the same stereochemistry at C-13. The structure of klyflaccicembranol G (7) was thus established as (4S,11S,12S,13S,2E,7E,14Z)-4,13-dihydroxy-11,12-epoxy-cembra-2,7,14-triene.
Klyflaccicembranol I (9) possessed the molecular formula C20H32O3, as revealed from the ESIMS (m/z 343 [M + Na]+) and NMR data (Table 1 and Table 3). The 1H and 13C NMR data demonstrated the characteristic features of non-lactonized cembranoids (C20 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 1H and 13C 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 ] D 25 +124.
Cytotoxicity of metabolites 16 and 810 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 (IC50 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 IC50 34.6–49.4 μM; however, it was found to be doubly-potent (IC50 49.4 μM) relative to the positive control fluorouracil (IC50 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.
The isolated compounds 16 and 810 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 IC50 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% (IC50 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%).

3. Materials and Methods

3.1. 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 1H and 125 MHz for 13C or on a Varian 400 FT-NMR at 400 MHz for 1H and 100 MHz for 13C or on a Bruker AMX-300 FT-NMR at 300 MHz for 1H and 75 MHz for 13C, 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).

3.2. 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.

3.3. Extraction and Separation

The frozen bodies of K. flaccidum (8.0 kg, wet weight) were sliced and exhaustively extracted with EtOAc and filtered off. The solvent-free residue (120 g) was fractionated by silica gel column chromatography, using EtOAc–n-hexane (0:100 to 100:0, gradient) and then MeOH–EtOAc (0:100 to 100:0, gradient) as eluting solvents, in order to yield 26 fractions (F1 to F26). F8 eluted with EtOAc–n-hexane (1:2) was further isolated on silica gel, using EtOAc–n-hexane (1:3 to 2:3, stepwise) to yield 3 (2.5 mg), 2 (1.5 mg), and 8 (40.5 mg), respectively. F10 eluted with EtOAc–n-hexane (1:1) was separated on silica gel, using EtOAc–n-hexane (1:2) to give four subfractions F101–F104. F101 was isolated on RP-HPLC using MeOH–H2O (3:1) to give 4 (10.2 mg), 9 (9.7 mg), and 10 (11.3 mg), respectively. F12 eluted with EtOAc–n-hexane (2:1) was re-chromatographed on silica gel column, using EtOAc–n-hexane (1:2–2:1), in order to give four subfractions F121–F124. F122 was isolated on RP-HPLC, using MeOH–H2O (15:1) as a mobile phase, to yield 1 (1.8 mg), 5 (3.4 mg), 6 (2.1 mg), and 7 (3.2 mg).

3.3.1. Klyflaccicembranol A (1)

Colorless oil; [ α ] D 25 +57.2 (c 0.5, CHCl3); IR (neat) νmax 3445, 2959, 2924, 2870, 1456, 1381, 1118, 1087, 1064, 850 and 738 cm−1; 13C (100 MHz, C6D6) and 1H NMR (400 MHz, C6D6) data, see Table 1 and Table 2, respectively; ESIMS m/z 359 [M + Na]+; HRESIMS m/z 359.2196 [M + Na]+ (calcd. for C20H32O4Na, 359.2198).

3.3.2. Klyflaccicembranol B (2)

Colorless oil; [ α ] D 25 +65.9 (c 0.4, CHCl3); IR (neat) νmax 3443, 2924, 2856, 1746, 1457, 1375, and 1234 cm−1; 13C (125 MHz, CDCl3) and 1H NMR (500 MHz, CDCl3) data, see Table 1 and Table 2, respectively; ESIMS m/z 417 [M + Na]+; HRESIMS m/z 417.2250 [M + Na]+ (calcd. for C22H34O6Na, 417.2253).

3.3.3. Klyflaccicembranol C (3)

Colorless oil; [ α ] D 25 +38.9 (c 0.7, CHCl3); IR (neat) νmax 3450, 2927, 2858, 1731, 1455, 1373, 1240 and 1016 cm−1; 13C (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1 and Table 2, respectively; ESIMS m/z 385 [M + Na]+; HRESIMS m/z 385.2354 [M + Na]+ (calcd. for C22H34O4Na, 358.2355).

3.3.4. Klyflaccicembranol D (4)

Pale yellow oil; [ α ] D 25 −1.51 (c 3.17, CHCl3); IR (neat) νmax 3419, 2953, 2925, 2867, 1456, 1375, 977, and 757 cm−1; 13C (100 MHz, C6D6) and 1H NMR (400 MHz, C6D6) data, see Table 1 and Table 2, respectively; ESIMS m/z 327 [M + Na]+; HRESIMS m/z 327.2200 [M + Na]+ (calcd. for C20H32O2Na, 327.2198).

3.3.5. Klyflaccicembranol E (5)

Colorless oil; [ α ] D 25 −70.5 (c 1.0, CHCl3); IR (neat) νmax 3408, 2956, 2924, 2869, 1455, 1381, 1002, and 757 cm−1; 13C (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1 and Table 2, respectively; ESIMS m/z 345 [M + Na]+; HRESIMS m/z 345.2404 [M + Na]+ (calcd. for C20H34O3Na, 345.2406).

3.3.6. Klyflaccicembranol F (6)

Colorless oil; [ α ] D 25 −32.0 (c 0.6, CHCl3); IR (neat) νmax 3392, 2924, 2855, 1455, and 1370 cm−1; 13C (125 MHz, CDCl3) and 1H NMR (500 MHz, CDCl3) data, see Table 1 and Table 3; ESIMS m/z 345 [M + Na]+; HRESIMS m/z 345.2405 [M + Na]+ (calcd. for C20H34O3Na, 345.2406).

3.3.7. Klyflaccicembranol G (7)

Colorless oil; [ α ] D 25 +18.0 (c 1.0, CHCl3); IR (neat) νmax 3419, 2953, 2925, 2867, 1460, 1375, 977 and 757 cm−1; 13C (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1 and Table 3, respectively; ESIMS m/z 343 [M + Na]+; HRESIMS m/z 343.2250 [M + Na]+ (calcd. for C20H32O3Na, 343.2249).

3.3.8. Klyflaccicembranol H (8)

Colorless oil; [ α ] D 25 +83.0 (c 1.4, CHCl3); IR (neat) νmax 3310, 3070, 2964, 2958, 2931, 2867, 1730, 1644, 1460, 1441, 1371, 1251 and 1182 cm−1; 13C (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data, see Table 1 and Table 3, respectively; 1H NMR (CD3OD, 500 MHz) δH 5.65 (1H, d, J = 2.0 Hz, H-2), 5.37 (1H, dd, J = 5.5, 5.5 Hz, H-7), 5.15 (1H, d, J = 2.0 Hz, H-13), 5.07 (1H, ddd, J = 5.0, 2.0, 2.0 Hz, H-14), 4.57 (1H, d, J = 5.0 Hz, H-3), 2.95 (1H, dd, J = 7.5, 1.5 Hz, H-11), 2.21 (2H, m, H2-9), 2.19 (1H, m, H-6), 2.13 (1H, m, H-15), 2.10 (1H, m, H-6), 1.93 (3H, s, Ac), 1.87 (1H, dd, J = 14.0, 9.5 Hz, H-5), 1.81 (1H, m, H-10), 1.70 (1H, m, H-10), 1.64 (1H, dd, J = 14.0, 8.0 Hz, H-5), 1.60 (3H, s, H3-19), 1.39 (3H, s, H3-20), 1.10 (3H, dd, J = 7.0 Hz, H3-17), 1.06 (3H, dd, J = 7.0 Hz, H3-16), 0.96 (3H, s, H3-18); 13C NMR (CD3OD, 125 MHz) δC 171.7 (C, 13-OAc), 148.4 (C, C-1), 133.2 (C, C-8), 128.2 (CH, C-7), 122.9 (CH, C-2), 90.6 (CH, C-3), 86.2 (CH, C-14), 75.2 (C, C-4), 74.2 (CH, C-13), 62.3 (C, C-12), 61.0 (CH, C-11), 42.1 (CH2, C-5), 37.7 (CH2, C-9), 25.6 (CH2, C-10), 22.7 (CH2, C-6), 25.7 (CH, C-15), 22.7 (CH3, C-6), 22.7 (CH3, C-18), 22.6 (CH3, C-16), 21.3 (CH3, C-17), 20.6 (CH3, 13-OAc), 16.8 (CH3, C-19), 16.2 (CH3, C-20); ESIMS m/z 401 [M + Na]+, 385 [M − O + Na]+, 325 [M − AcOH − O + Na]+; EIMS m/z 379 [M + H]+, 361 [M − H2O]+, 318 [M − AcOH]+, 300 [M − AcOH − H2O]+; HRESIMS m/z 378.2404 [M]+ (calcd. for C22H34O5, 378.2406).

3.3.9. Klyflaccicembranol I (9)

Colorless oil; [ α ] D 25 +124.0 (c 2.7, CHCl3); IR (neat) νmax 3487, 3275, 2955, 2933, 2868, 1732, 1644, 1453, 1370, 1247 and 1183 cm−1; 13C (100 MHz, C6D6) and 1H NMR (400 MHz, C6D6) data, see Table 1 and Table 3, respectively; ESIMS m/z 343 [M + Na]+.

3.3.10. Hydrolysis of Klyflaccicembranol H (8)

A solution of 8 (3.2 mg, 8.12 μmol) in MeOH (2.0 mL) was stirred with K2CO3 (30 mg) overnight at room temperature. The reaction mixture was diluted with distilled water (3.0 mL) and followed by extraction with CH2Cl2. The organic extract was then purified on short silica gel CC, using 25% EtOAc in n-hexane as an eluting solvent, to afford 1 (2.4 mg, 6.82 μmol, 84% yield). 1: colorless oil; 1H NMR (CDCl3, 300 MHz). δH 5.64 (1H, br s, H-2), 5.32 (1H, dd, J = 5.5, 5.5 Hz, H-7), 3.81 (1H, d, J = 1.8 Hz, H-13), 4.92 (1H, br d, J = 5.5 Hz, H-14), 4.57 (1H, d, J = 5.5 Hz, H-3), 3.12 (1H, dd, J = 9.5, 3.6 Hz, H-11), 2.20 (2H, m, H2-9), 2.13 (2H, m, H2-6), 2.37 (1H, septet, J = 6.9 Hz, H-15), 1.89 (1H, m, H-5a), 1.83 (2H, m, H2-10), 1.65 (1H, m, H-5b), 1.59 (3H, s, H3-19), 1.36 (3H, s, H3-20), 1.10 (3H, dd, J = 6.9 Hz, H3-17), 1.08 (3H, dd, J = 6.9 Hz, H3-16), 1.01 (3H, s, H3-18); 13C NMR (CDCl3, 75 MHz) δC 148.4 (C, C-1), 132.8 (C, C-8), 126.8 (CH, C-7), 122.9 (CH, C-2), 89.6 (CH, C-3), 85.6 (CH, C-14), 74.9 (C, C-4), 72.6 (CH, C-13), 62.4 (C, C-12), 60.3 (CH, C-11), 41.6 (CH2, C-5), 36.9 (CH2, C-9), 24.8 (CH2, C-10), 22.1 (CH2, C-6), 26.6 (CH, C-15), 22.1 (CH3, C-6), 22.7 (CH3, C-18), 22.4 (CH3, C-16), 21.4 (CH3, C-17), 16.9 (CH3, C-19), 15.7 (CH3, C-20).

3.3.11. Preparation of (S)- and (R)-MTPA Esters of 1

To a solution of 1 (1.1 mg, 3.1 μmol) in pyridine (50 μL), R-(−)-MTPA chloride (5 μL) was added and allowed to react overnight at room temperature. The reaction was terminated by the addition of 1.0 mL of water, and then processed as previously described [27] to yield the (S)-MTPA ester 1a (1.2 mg, 2.1 μmol, 67.7%). Similarly, the correspondent (R) -MTPA ester 1b was also obtained from the reaction of S-(+)-MTPA chloride with 1. 1H NMR (CDCl3, 300 MHz) of 1a: δH 5.496 (1H, br s, H-2), 5.341 (1H, br s, H-13), 5.185 (1H, dd, J = 4.5, 4.5 Hz, H-7), 5.030 (1H, br d, J = 4.5 Hz, H-14), 4.324 (1H, br d, J = 4.5 Hz, H-3), 2.609 (1H, m, H-11), 2.177 (1H, m, H-15), 2.100 (4H, m, H2-6 and H2-9), 1.985 (1H, m, H-5α), 1.825 (1H, m, H-5β), 1.666 (2H, m, H2-10), 1.536 (3H, s, H3-19), 1.387 (3H, s, H3-20), 1.097 (3H, d, J = 6.9 Hz, H3-17), 1.040 (3H, d, J = 6.9 Hz, H3-16), 0.962 (3H, s, H3-18). 1H NMR (CDCl3, 300 MHz) of 1b: δH 5.639 (1H, br s, H-2), 5.347 (1H, br s, H-13), 5.223 (1H, dd, J = 4.5, 4.5 Hz, H-7), 5.075 (1H, br d, J = 4.5 Hz, H-14), 4.542 (1H, br d, J = 4.5 Hz, H-3), 2.623 (1H, m, H-11), 2.176 (1H, m, H-15), 2.111 (4H, m, H2-6 and H2-9), 1.994 (1H, m, H-5α), 1.858 (1H, m, H-5β), 1.637 (2H, m, H2-10), 1.543 (3H, s, H3-19), 1.382 (3H, s, H3-20), 1.122 (3H, d, J = 6.9 Hz, H3-17), 1.057 (3H, d, J = 6.9 Hz, H3-16), 1.003 (3H, s, H3-18).

3.4. Cytotoxicity Assay

Cancer cell (HT-29, A549, K562, and P388) lines were purchased from the American Type Culture Collection (ATCC). Evaluation of cytotoxicity for the isolated metabolites from K. flaccidum was performed according to Alamar Blue assay [14,15].

3.5. Nitric Oxide Inhibitory Assay

The inhibitory activity of isolated compounds on nitric oxide (NO) production by murine RAW 264.7 macrophage cells was assessed according to Griess reaction. Briefly, cells were cultured in 96-well plates for 1 h. The cells were challenged with LPS (5 μg/mL) and test samples for 48 h. The culture supernatant (100 μL) were reacted then with Griess reagent (1:1 mixture of 0.1% N-(1-naphthyl) ethylene-diamine dihydrochloride in water and 1% sulfanilamide in 5% phosphoric acid, 100 μL) in a 96-well plate, and absorbance was measured using the ELISA reader at 540 nm [28,29].

4. 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.

Supplementary Materials

HRESIMS, 1H, and 13C spectra of new compounds 19 are available online at www.mdpi.com/1660-3397/15/1/23/s1. Figure S1: HRESIMS spectrum of 1, Figure S2: 1H NMR spectrum of 1 in C6D6 at 400 MHz, Figure S3: 13C NMR spectrum of 1 in C6D6 at 100 MHz, Figure S4: HRESIMS spectrum of 2, Figure S5: 1H NMR spectrum of 2 in CDCl3 at 500 MHz, Figure S6: 13C NMR spectrum of 2 in CDCl3 at 125 MHz, Figure S7: HRESIMS spectrum of 3, Figure S8: 1H NMR spectrum of 3 in CDCl3 at 400 MHz, Figure S9: 13C NMR spectrum of 3 in CDCl3 at 100 MHz, Figure S10: HRESIMS spectrum of 4, Figure S11: 1H NMR spectrum of 4 in C6D6 at 400 MHz, Figure S12: 13C NMR spectrum of 4 in C6D6 at 100 MHz, Figure S13: HRESIMS spectrum of 5, Figure S14: 1H NMR spectrum of 5 in CDCl3 at 400 MHz, Figure S15: 13C NMR spectrum of 5 in CDCl3 at 100 MHz, Figure S16: HRESIMS spectrum of 6, Figure S17: 1H NMR spectrum of 6 in CDCl3 at 500 MHz, Figure S18: 13C NMR spectrum of 6 in CDCl3 at 125 MHz, Figure S19: HRESIMS spectrum of 7, Figure S20: 1H NMR spectrum of 7 in CDCl3 at 400 MHz, Figure S21: 13C NMR spectrum of 7 in CDCl3 at 100 MHz, Figure S22: HRESIMS spectrum of 8, Figure S23: 1H NMR spectrum of 8 in CDCl3 at 400 MHz, Figure S24: 13C NMR spectrum of 8 in CDCl3 at 100 MHz, Figure S25: ESIMS spectrum of 9, Figure S26: 1H NMR spectrum of 9 in C6D6 at 400 MHz, Figure S27: 13C NMR spectrum of 9 in C6D6 at 100 MHz.

Acknowledgments

This work was supported by grants from the Ministry of Science and Technology (MOST 102-2628-B-110-002-MY2 and 104-2320-B-110-001-MY2), Aim for the Top University Program (05C030205) from the Ministry of Education of Taiwan and the National Sun Yat-sen University-Kaohsiung Medical University (NSYSU-KMU) Joint Research Project (NSYSUKMU 105-I008) of Taiwan awarded to J.-H.S. Partial financial support from the Taiwan Protein Project (MOST 105-0210-01-12-01) to J.-H.S. is also acknowledged.

Author Contributions

Jyh-Horng Sheu designed and guided the whole experiment. Atallah F. Ahmed contributed to structure elucidation and manuscript preparation. Chia-Ruei Tsai isolated the compounds and performed data acquisition and structure elucidation. Chiung-Yao Huang performed the structure elucidation and cytotoxicity assay. Sheng-Yang Wang performed the nitric oxide inhibitory assay.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Structures of cembranoids isolated from Klyxum flaccidum.
Figure 1. Structures of cembranoids isolated from Klyxum flaccidum.
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Figure 2. Correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations in 18.
Figure 2. Correlation spectroscopy (COSY) and heteronuclear multiple bond correlation (HMBC) correlations in 18.
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Figure 3. Key nuclear Overhauser effect ‎(NOE) correlations of 1, 8 and 2.
Figure 3. Key nuclear Overhauser effect ‎(NOE) correlations of 1, 8 and 2.
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Figure 4. 1H NMR chemical shift differences ∆δ (δS − δR) in ppm for the α-methoxy-α-(trifluoromethyl)phenylacetic ‎(MTPA) esters of 1.
Figure 4. 1H NMR chemical shift differences ∆δ (δS − δR) in ppm for the α-methoxy-α-(trifluoromethyl)phenylacetic ‎(MTPA) esters of 1.
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Figure 5. Key NOE correlations of 3 and 4.
Figure 5. Key NOE correlations of 3 and 4.
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Figure 6. Structures of semisynthetic cembranoid (11) and crassumol A (12).
Figure 6. Structures of semisynthetic cembranoid (11) and crassumol A (12).
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Figure 7. Key NOE correlations of 57.
Figure 7. Key NOE correlations of 57.
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Figure 8. Inhibitory effects of compounds 16 and 810 at 50 μg/mL on nitric oxide (NO) production in lipopolysaccharide‎ (LPS)-stimulated RAW264.7 cells.
Figure 8. Inhibitory effects of compounds 16 and 810 at 50 μg/mL on nitric oxide (NO) production in lipopolysaccharide‎ (LPS)-stimulated RAW264.7 cells.
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Table 1. 13C NMR spectral data of compounds 19.
Table 1. 13C NMR spectral data of compounds 19.
#1 a2 b3 c4 a5 c6 b7 c8 c9 a
1147.8 (C)147.7 (C)155.9 (C)146.3 (C)154.6 (C)80.9 (C)146.0 (C)147.4 (C)146.9 (C)
2121.0 (CH) d121.2 (CH)67.2 (CH)124.3 (CH)122.7 (CH)129.2 (CH)123.7 (CH)121.2 (CH)120.8 (CH)
389.7 (CH)91.1 (CH)126.3 (CH)136.2 (CH)70.7 (CH)138.0 (CH)138.3 (CH)89.1 (CH)121.0 (CH)
474.2 (C)74.6 (C)137.6 (C)72.9 (C)75.2 (C)71.9 (C)73.0 (C)74.6 (C)136.6 (C)
541.6 (CH2)40.5 (CH2)39.0 (CH2)45.5 (CH2)38.6 (CH2)43.8 (CH2)39.0 (CH2)41.3 (CH2)39.1 (CH2)
622.1 (CH2)25.2 (CH2)24.2 (CH2) 121.3 (CH)22.3 (CH2)22.3 (CH2)24.2 (CH2)21.8 (CH2)25.6 (CH2)
7127.0 (CH)64.6 (CH)125.2 (CH)141.4 (CH)127.0 (CH)128.6 (CH)127.6 (CH)126.3 (CH)126.5 (CH)
8132.1 (C)59.9 (C)133.2 (C)72.5 (C)133.9 (C)132.7 (C)131.9 (C)132.6 (C)133.8 (C)
936.8 (CH2)36.7 (CH2)36.9 (CH2)43.6 (CH2)39.0 (CH2)39.0 (CH2)36.7 (CH2)36.8 (CH2)36.6 (CH2)
1025.1 (CH2)23.6 (CH2)24.3 (CH2)23.5 (CH2)24.2 (CH2)23.8 (CH2)24.3 (CH2)24.5 (CH2)24.8 (CH2)
1159.7 (CH)59.0 (CH)59.7 (CH)126.7 (CH)125.6 (CH)126.9 (CH)61.3 (CH)59.2 (CH)59.9 (CH)
1261.9 (C)60.3 (C)61.3 (C)132.8 (C)131.9 (C)136.1 (C)64.7 (C)60.4 (C)62.9 (C)
1372.8 (CH)71.9 (CH)71.6 (CH)37.3 (CH2)44.1 (CH2)36.1 (CH2)71.6 (CH)72.7 (CH)75.2 (CH)
1485.8 (CH)85.0 (CH)115.6 (CH)122.0 (CH)70.6 (CH)29.9 (CH2)122.9 (CH)84.9 (CH)68.1 (CH)
1526.6 (CH)25.7 (CH)27.6 (CH)31.6 (CH)27.8 (CH)75.1 (C)32.1 (CH)25.7 (CH)28.2 (CH)
1622.2 (CH3)20.9 (CH3)23.7 (CH3)22.6 (CH3)22.8 (CH3)24.5 (CH3)22.1 (CH3)22.2 (CH3)24.0 (CH3)
1721.2 (CH3)20.6 (CH3)24.5 (CH3)22.6 (CH3)23.5 (CH3)24.7 (CH3)22.3 (CH3)20.9 (CH3)25.4 (CH3)
1823.1 (CH3)22.2 (CH3)15.7 (CH3)29.7 (CH3)25.1 (CH3)27.8 (CH3)30.0 (CH3)22.6 (CH3)16.7 (CH3)
1916.7 (CH3)16.8 (CH3)15.2 (CH3)29.6 (CH3)15.1 (CH3)14.7 (CH3)15.1 (CH3)16.7 (CH3)15.2 (CH3)
2015.5 (CH3)16.0 (CH3)15.2 (CH3)17.6 (CH3)17.1 (CH3)14.8 (CH3)15.5 (CH3)16.0 (CH3)16.1 (CH3)
OAc 169.9 (C)170.7 (C) 169.9 (C)
21.0 (CH3)21.1 (CH3) 20.6 (CH3)
Spectra recorded in a C6D6 at 100 MHz; b CDCl3 at 125 MHz; and c CDCl3 at 100 MHz at 25 °C; d Attached protons were determined by distortionless enhancement by polarization transfer (DEPT) experiments. Values are presented as ppm downfield from tetramethylsilane (TMS).
Table 2. 1H NMR spectral data for compounds 15.
Table 2. 1H NMR spectral data for compounds 15.
#1 a2 b3 c4 a5 c
25.59 br s5.65 br s5.70 d (10.0)6.19 d (16.4)5.41 d (7.6)
34.61 d (4.8) d4.78 d (5.0)5.25 d (10.0)5.83 d (16.4)4.34 d (7.6)
51.48 m; 1.85, m1.82 m; 1.92 m2.10 m; 2.24 m2.31 2H, d (6.8)1.55 m; 1.86 m
62.02 m; 2.15, m1.59 m; 1.86 m2.10 m; 2.27 m5.56 dd (15.6, 6.8)2.11 m; 2.34 m
75.38 dd (5.2, 5.2)3.07 dd (6.0, 2.5)4.83 br d (6.0)5.52 d (15.6)4.99 dd (6.0, 6.0)
92.04 m; 2.09 m2.10 m; 1.39 m2.16 m; 2.20 m1.58 m; 1.67 m1.98 m; 2.15 m
101.76 m; 1.83 m1.54 m; 1.95 m1.61 m; 1.86 m2.01 m; 2.37 m2.14 m; 2.18 m
113.15 dd (6.4, 2.0)2.98 d (7.5)2.47 dd (7.2, 2.0)5.11 dd (7.2, 7.2)4.93 dd (6.8, 6.0)
133.58 br s5.21 s5.52 d (10.4)2.71 2H, d (8.0) 2.27 m; 2.37 m
144.78 br d (4.8)5.05 d (5.0)5.03 d (10.4)5.48 dd (8.0, 5.6)4.78 dd (5.6, 5.6)
152.25 sept (6.8)2.17 m2.78 sept (6.8)2.53 sept (6.8)2.48 m
160.94 3H, d (6.8)1.05 3H, d (6.5)1.05 3H, d (6.8)1.09 3H, d (6.8)1.06 3H, d (6.8)
171.12 3H, d (6.8)1.10 3H, d (6.5)1.09 3H, d (6.8)1.10 3H, d (6.8)1.12 3H, d (6.8)
180.98 3H, s1.05 3H, s1.80 3H, s1.27 3H, s1.14 3H, s
191.48 3H, s1.24 3H, s1.55 3H, s1.16 3H, s1.55 3H, s
201.18 3H, s1.42 3H, s1.22 3H, s1.60 3H, s1.68 3H, s
OAc 1.91 3H, s2.10 3H, s
Spectra recorded in a C6D6, at 400 MHz; b CDCl3 at 500 MHz; and c CDCl3 at 400 MHz at 25 °C; d J values (Hz) in parentheses.
Table 3. 1H NMR spectral data for compounds 69.
Table 3. 1H NMR spectral data for compounds 69.
#6 a7 b8 b9 c
25.61 d (16.0) d6.24 d (16.0) 5.60 br s6.25 d (11.2)
36.10 d (16.0)5.75 d (16.0)4.57 d (5.2)5.85 d (11.2)
51.51 m; 2.01 m2.10 m; 2.24 m1.63 m; 1.89 m2.00 2H, m
62.22 m; 2.39 m2.10 m; 2.27 m2.16 m; 2.18 m1.97 m, 2.06 m
75.34 dd (7.5, 7.5)5.03 dd (6.0, 6.0)5.36 dd (5.5, 5.5)5.03 dd (6.0, 6.0)
91.95 m; 2.20 m2.16 m; 2.20 m2.04 m; 2.09 m1.95 m, 2.11 m
102.07 m; 2.24 m1.61 m; 1.86 m1.76 m; 1.83 m1.43 2H, m
115.19 br d (9.0)2.67 m2.89 dd (6.0, 3.6)3.08 dd (6.0, 6.0)
132.13 m; 2.19 m4.59 dd (8.0, 8.0)5.16 d (2.0)3.72 d (6.0)
141.63 m; 2.11 m5.10 d (8.0)5.00 dd (5.2, 2.0)4.60 d (6.0)
15 2.52 m2.13 m2.76 m
161.21 3H, s1.04 3H, d (6.8)1.03 3H, d (6.8)1.06 d (6.8)
171.13 3H, s1.07 3H, d (6.8)1.08 3H, d (6.8)1.28 d (6.8)
181.40 3H, s1.35 3H, s1.00 3H, s1.59 3H, s
191.62 3H, s1.60 3H, s1.59 3H, s1.32 3H, s
201.67 3H, s1.42 3H, s1.39 3H, s1.34 3H, s
15-OH2.60 s
13-OH 1.74 d (8.0)
OAc 2.06 3H,s
Spectra recorded in a CDCl3 at 500 MHz; b CDCl3 at 400 MHz; and c C6D6 at 400 MHz at 25 °C; d J values (Hz) in parentheses.
Table 4. Cytotoxicities (IC50 μM) of compounds 16 and 810.
Table 4. Cytotoxicities (IC50 μM) of compounds 16 and 810.
CompoundHT-29A549K562P388
1 a a a a
2 a16.534.6 a
3 a a a a
4 a a44.9 a
5 a a a a
6 a21.4 a a
8 a49.447.434.6
941.9 a a25.9
10 a a a a
Fluorouracil8.511031.55.5
a –: Compound was considered inactive when IC50 > 50 μM.
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