Bioactive Isoprenoid-Derived Natural Products from a Dongsha Atoll

May 4, 2016 - During the course of our investigation into bioactive substances from this organism collected from Dongsha Atoll, which is located aroun...
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Bioactive Isoprenoid-Derived Natural Products from a Dongsha Atoll Soft Coral Sinularia erecta Chiung-Yao Huang,†,▽ Yen-Ju Tseng,†,▽ Uvarani Chokkalingam,† Tsong-Long Hwang,‡ Chi-Hsin Hsu,† Chang-Feng Dai,§ Ping-Jyun Sung,†,⊥ and Jyh-Horng Sheu*,†,∥,¶,# †

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Research Center for Industry of Human Ecology and Graduate Institute of Health Industry Technology, Chang Gung University of Science and Technology, Department of Anesthesiology, Chang Gung Memorial Hospital, Taoyuan 333, Taiwan § Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan ⊥ National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan ∥ Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan ¶ Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan # Frontier Center for Ocean Science and Technology, National Sun Yat-sen University, Kaohsiung 804, Taiwan ‡

S Supporting Information *

ABSTRACT: Four new isoprenoids, including two norcembranoids sinulerectols A and B (1 and 2), a cembranoid sinulerectol C (3), and a degraded cembranoid sinulerectadione (4), along with three known isoprenoids, an unnamed norcembrene (5), sinularectin (6), and ineleganolide (7), and a known nitrogen-containing compound (Z)-N-[2-(4hydroxyphenyl)ethyl]-3-methyldodec-2-enamide (8), were isolated from an extract of the marine soft coral Sinularia erecta. The structure of sinularectin (6) was revised, too. Compounds 3, 4, and 8 exhibited inhibitory activity against the proliferation of a limited panel of cancer cell lines, whereas 1, 2, and 8 displayed potent anti-inflammatory activity in fMLP/ CB-stimulated human neutrophils.

S

ectin (6), 2 1 ineleganolide (7), 2 and (Z)-N-[2-(4hydroxyphenyl)ethyl]-3-methyldodec-2-enamide (8),23 were discovered (Chart 1). Also, it is worthy to note that sinularectin (6) was previously isolated from the same specimen of S. erecta collected in Kenya.21 The in vitro cytotoxicities of 1−8 against four human cancer cell lines, promyelocytic leukemia (HL-60), chronic myelogenous leukemia (K-562), T cell lymphoblastlike (CCRF-CEM), and acute lymphoblastic leukemia (MOLT4), were assayed. The abilities of these compounds to inhibit superoxide anion generation and elastase release in N-formyl methionyl leucylphenylalanine/cytochalasin B (fMLP/CB)induced neutrophils were also evaluated.

oft corals, in particular, those belonging to the genus Sinularia, have proven to be rich sources of bioactive norcembranoids.1−14 Previous bioassay results of these macrocyclic metabolites demonstrated cytotoxic,1−5,11−14 antifungal,2 antiviral,9 and anti-inflammatory activities.9,10,14 Furthermore, some cembranoids15,16 and steroids17,18 with attractive bioactivities have also been discovered from corals of this genus. A literature search showed that previous chemical investigations of the soft coral Sinularia erecta (Klunzinger, 1877) afforded only a limited number of sesquiterpenes, cembranoids, and 18-norcembranoids,19−21 and until now, the biological activities of these compounds have not been investigated. Following the above investigations, with the aim of discovering bioactive substances from marine invertebrates, the chemical constituents of the soft coral S. erecta were examined by our group. During the course of our investigation into bioactive substances from this organism collected from Dongsha Atoll, which is located around the north of the South China Sea, two new 18-norcembranoids, sinulerectols A and B (1 and 2), a new cembranoid sinulerectol C (3), and a new degraded cembranoid sinulerectadione (4), along with four known compounds, an unnamed norcembrene (5),22 sinular© XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Bodies of S. erecta were sliced and extracted with EtOAc. Serial column chromatography including silica gel column chromatography along with normal and reverse-phase HPLC led to the Received: December 22, 2015

A

DOI: 10.1021/acs.jnatprod.5b01142 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

The relative configuration of 1 was determined by the analysis of NOE correlations observed in a NOESY experiment and with the assistance of 3JH,H coupling constants, in addition to molecular modeling using MM2 force field calculations. From the NOESY spectrum of 1, it was found that H-1 (δH 2.55, m) showed NOE interaction with H-13 (δH 4.73, dd, J = 12.4, 2.8 Hz); therefore, due to the β-orientation of H-1, H-13 should also be positioned on the β-face. Further, one of the methylene protons at C-14 (δH 1.60, ddd, J = 12.4, 2.8, 2.0 Hz) exhibited NOE correlations with H-1 and H-13 and was therefore characterized as H-14β, while the other (δH 0.66, ddd, J = 12.4, 12.4, 12.4 Hz) was assigned as H-14α, which is in the axial position. NOE correlations observed between H-14α and one proton of H2-2 (δH 1.89, dd, J = 12.4, 12.4 Hz) and H-11 (δH 4.31, s), and between H-11 and H-10 (δH 4.91, d, J = 8.4 Hz), together with the fact that no coupling between H-10 and H-11 was found, indicated that the dihedral angles of the C10−H and C-11−H bonds should be orthogonal (θ ≈ 90°) and trans-oriented on the lactone ring (Figure 2). Furthermore, the relatively deshielded H-11 also indicated that H-10 and the epoxide ring should be cis-oriented, as the signal of H-11 should be more shielded (e.g., δH 3.95, in scabrolide D) if the epoxide ring and H-10 are trans-oriented.3,10 Moreover, one H-4 proton (δH 2.28, dd, J = 15.2, 4.0 Hz) was found to interact with H-2β (δH 1.74, m) and H-5 (δH 5.21, dd, J = 4.4, 4.0 Hz) and H-7 with H-5 and H3-18, but H3-18 did not show an NOE correlation with H-10, revealing the α-orientations of H-10, the acetoxy group, and the epoxy oxygen. Also, both protons of H24 did not show any correlations with H-1 or H-13, revealing the β-orientation of the hydroxy group. The configuration of the trisubstituted double bond at C-7/C-8 is of the Z form, as it was found that H-7 (δH 6.20, br s) showed a significant NOE interaction with H3-18 (δH 2.01, s), and the chemical shift for C-18 was shifted to 29.9 ppm.25 The above results suggest a structure which was found to be in agreement with the most

isolation of four new compounds 1−4 and four known compounds 5−8. Sinulerectol A (1), obtained as an amorphous solid, was found to possess the molecular formula C21H26O8 as established by HRESIMS (m/z 429.1528 [M + Na]+), with nine degrees of unsaturation. The IR spectrum revealed the presence of hydroxy (3410 cm−1), γ-lactone (1782 cm−1), and α,β-unsaturated ketone carbonyl (1691 cm−1) groups. The 13C NMR and DEPT data (Table 1) indicated the presence of 21 carbons, including those of two vinylic methyl groups, one 1,1disubstituted double bond, one trisubstituted double bond, three carbonyls (including an acetoxy group), and one trisubstituted epoxide (δC 59.2, C and 58.9, CH). With the assistance of the HSQC spectrum, the proton signals in the 1H NMR spectrum (Table 1) showed the presence of three methyl groups, two sp2 methylene protons, four sp3 methylenes, one olefinic proton of a trisubstituted double bond, and one proton of a trisubstituted epoxide. Thus, the tetracyclic structure of 1 was revealed. In the COSY spectrum of 1, it was possible to identify three different structural units, from H2-2 to H-13 via H-1, H2-4 to H-5, and H2-9 to H-10, which were assembled with the assistance of an HMBC experiment (Figure 1). Key HMBC correlations of H2-2, H2-4, and H-5 to C-3; H-5 and H7 to C-6; H3-18 to C-7, C-8, and C-9; H2-9 and H-10 to C-11; H-10 and H-11 to C-19 (both correlations showing the presence of a γ-lactone ring); H-11 and H2-14 to C-12; H2-16 and H3-17 to C-1 and C-15, were used to establish the norcembranoid skeleton. Furthermore, the acetoxy group positioned at C-5 was confirmed by the HMBC correlations of H-5 (δH 5.21, dd, J = 4.4, 4.0 Hz) and protons of an acetate methyl (δH 2.17) to the ester carbonyl. In consideration of degrees of unsaturation and molecular formula, an ether linkage was placed between the hemiketal carbon C-3 (δC 96.7) and C13 (δC 64.2). On the basis of the molecular framework, the gross structure of 1 was established (Figure 1). B

DOI: 10.1021/acs.jnatprod.5b01142 J. Nat. Prod. XXXX, XXX, XXX−XXX

C

a

76.0, CH 193.1, C 124.5, CH 150.6, C 33.7, CH2

77.6, 58.9, 59.2, 64.2, 29.4,

147.7, C 109.5, CH2 20.7, CH3 29.9, CH3 171.0, C

5 6 7 8 9

10 11 12 13 14

15 16 17 18 19 20 OAc

20.8, CH3 169.7, C

2.17 s

4.78, s; 4.70, s 1.73, s 2.01, s

4.73, dd (12.4, 2.8) α: 0.66, ddd (12.4, 12.4, 12.4) β: 1.60, ddd (12.4, 2.8, 2.0)

α: 4.05, d (13.2) β: 2.60, dd (13.2, 8.4) 4.91, d (8.4) 4.31, s

6.20, br s

2.48, dd (15.2, 4.4) 2.28, dd (15.2, 4.0) 5.21, dd (4.4, 4.0)

2.55, m α: 1.89, dd (12.4, 12.4) β: 1.74, m

δH, mult (J in Hz) b

CH CH C CH CH2

52.1, CH3

142.8, C 124.8, CH2 166.9, C 25.9, CH3 170.4, C

77.2, 65.1, 61.6, 66.6, 35.5,

76.7, CH 212.3, C 49.7, CH2 79.0, C 42.2, CH2

207.9, C 45.2, CH2

33.0, CH 47.6, CH2

δC, type a

b

3.78, s

1.48, s

6.30, s; 5.65, s

3.87, dd (5.2, 5.2) 2.20, m 2.12, m

β: 2.51, m α: 2.17, m 4.78, dd (6.8, 4.0) 4.22, s

2.48, m; 2.57 m

3.01, dd (14.8, 3.2) 2.62, dd (14.8, 8.4) 4.40, dd (8.4, 3.2)

3.54, m 2.60, m 2.52, m

δH, mult (J in Hz)

2

149.1, C 110.9, CH2 20.2, CH3 20.7, CH3 111.4, CH2 114.9, CH2

30.1, CH2 87.7, CH 145.7, C 30.2, CH2 28.8, CH2

37.8, CH 29.7, CH2 80.7, CH 148.7, C 31.1, CH2

73.3, CH 84.1, C

41.4, CH 38.0, CH2

δC, type c

m m ddd (14.5, 9.5, 5.5) br s

m m m; 1.78, m dd (8.0, 4.0)

s; 4.77, s s s s; 4.91, s s; 5.10, s

7.76, br s

4.84, 1.74, 1.11, 4.92, 5.14,

2.23, ddd (14.0, 8.0, 8.0), 2.06, m 1.75, m 1.54, m

2.40, 2.02, 2.08, 4.50,

2.00, m; 1.86, m 1.97, m; 1.84, m 4.22, dd (9.0, 4.0)

2.49, 1.84, 1.64, 3.56,

d

δH, mult (J in Hz)

3

CH2 C CH CH2 CH

145.7, C 113.3, CH2 18.0, CH3

26.8, CH2 41.2, CH2 208.6, C 30.1, CH3 17.2, CH3

41.5, 59.7, 61.8, 32.3, 44.7,

133.9, CH 142.4, CH

26.9, CH3 190.0, C

δC, type c

4.85, s; 4.78, s 1.62, s

2.13, s 1.26, s

1.75, m; 1.62 m 2.38, m

2.73, dd (6.0, 6.0) 1.54, m; 1.66 m 2.27, m

2.42, m

6.11, d (16.0) 6.73, dt (16.0, 7.0)

2.26, s

δH,d mult (J in Hz)

4

100 MHz in CDCl3. bSpectrum recorded at 400 MHz in CDCl3. c125 MHz in CDCl3. dSpectrum recorded at 500 MHz in CDCl3. eAttached protons were deduced by DEPT experiment.

OMe OOH

96.7, C 43.4, CH2

3 4

CH CH C CH CH2

35.3, CH 36.6, CH2

e

δC, type

1 2

position

a

1

Table 1. 1H and 13C NMR Spectroscopic Data for Compounds 1−4

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DOI: 10.1021/acs.jnatprod.5b01142 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 1. Selected COSY and HMBC correlations of 1 and 2.

Figure 2. Key NOESY correlations of 1 and 2.

correlation of the methoxy protons (δH 3.78, s) to the carbonyl carbon (δC 166.9, C-17). In addition, the oxymethine proton H-5 exhibited an HMBC correlation with another oxymethine carbon C-11, and the eight-membered ether ring was established. The above analyses revealed the presence of eight oxygen atoms in the molecular structure. One additional oxygen atom has to be attached to C-12 (δC 61.6, C) and C-13 (δC 66.6, CH) to form an epoxide. According to the above observations and by analysis of the HMBC spectrum, the molecular framework of 2 was established as shown in Figure 1. The configuration of 2 was deduced by analysis of the NOESY spectrum and the molecular model obtained from MM2 calculations (Figure 2). The NOE interaction between H-1 and H-5 indicated the β-orientation of H-5. NOE correlations of H3-18 with both H-5 and one proton of H2-9 (δH 2.51, m) were observed, indicating the β-orientations of H3-18 and this proton at C-9. Furthermore, H-10 exhibited significant correlations with H-11 and H-9α (δH 2.17, m) but did not interact with H3-18, and H-11 correlated with H-13 but not with H-5, revealing the α-orientations of H-10, H-11, and H-13 in the 12,13-epoxide ring. From the above findings and other correlations observed (Figure 2), the structure of sinulerectol B (2) was unambiguously established. The new metabolite sinulerectol C (3) exhibited a sodium adduct ion peak in the HRESIMS at m/z 359.2196 [M + Na]+, establishing the molecular formula C20H32O4 with five degrees of unsaturation. The IR spectrum suggested the presence of a hydroxy group (νmax 3395 cm−1) in 3. The 13C NMR spectrum of 3 measured in CDCl3 showed the presence of 20 carbon signals, which were assigned with the assistance of a DEPT

stable conformation of 1 (Figure 2) generated by MM2 force field calculations. Sinulerectol B (2) was obtained as an amorphous solid with the molecular formula C20H24O9, as indicated by HREIMS, with nine degrees of unsaturation. The IR spectrum showed absorptions at 3450 (hydroxy group), 1757 (lactone group), and 1709 cm−1 (ketone carbonyl group). The 13C NMR spectrum of 2 also revealed the presence of 20 carbon signals (including that of a methoxy carbon). The presence of seven carbon signals (Table 1) at δC 212.3 (C), 207.9 (C), 170.4 (C), 166.9 (C), 142.8 (C), 124.8 (CH2) and 52.1 (OCH3) and the presence of two vinylic protons and three carbomethoxy protons appearing in 1H NMR spectrum (Table 1) at δH 6.30 (1H, s), 5.65 (1H, s) and 3.78 (3H, s), were attributable to four carbonyl groups and one terminal methylene group of an αsubstituted α,β-unsaturated methyl ester. These data together with the presence of only one methyl (δH 1.48, 3H, s) indicated the possibility that 2 is a norditerpene. In consideration of the degrees of unsaturation and molecular formula, the tetracyclic structure of 2 was revealed. Furthermore, it was found that 2 displayed NMR signals of a trisubstituted epoxide (δC 61.6, C and 66.6, CH, and δH 3.87, dd, J = 5.2, 5.2 Hz). In the COSY spectrum, it was possible to identify three different structural units, which were further assembled with the assistance of an HMBC experiment (Figure 1). Key HMBC correlations of H22 and H-5 to C-3; H2-7 to C-5 and C-6; both H-10 and H-11 to C-19; H-13 to C-1, C-11, C-12, and C-14; H2-16 to C-1, C-15, and C-17; and H3-18 to C-7, C-8, and C-9 permitted the establishment of the 18-norcembranoid skeleton of 2. The methoxy group positioned at C-17 was confirmed from the D

DOI: 10.1021/acs.jnatprod.5b01142 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. Selected COSY and HMBC correlations of 3 and 4.

Figure 4. Key NOESY correlations of 3 and 6.

H-5α with H-3 and H-7 reflected the α-orientations of H-3 and H-7. According to the above NOE correlations, and the others shown in Figure 4, the relative configurations at C-1, C-3, C-4, C-7, and C-11 were suggested as 1R*, 3S*, 4R*, 7R*, and 11S*, respectively. Sinulerectadione (4) was isolated as a colorless oil. The HRESIMS spectrum of 4 exhibited a sodium adduct ion peak at m/z 301.1777 [M + Na]+ and established a molecular formula of C17H26O3, implying five degrees of unsaturation. Its 1H and 13 C NMR spectra clearly revealed the presence of two carbonyl carbons (δC 208.6, C and 190.0, C), two disubstituted double bonds (δC 145.7, C; 142.4, CH; 133.9, CH; and 113.3, CH2), and a trisubstituted epoxide substituted with a methyl group (δC 59.7, C; 61.8, CH; 17.2, CH3, and δH 2.73, dd, J = 6.0, 6.0 Hz and 1.26, s). The planar structure of 4 was established with the assistance of extensive 2D NMR analysis. The COSY spectrum was used to establish the proton sequences from H-3 to H2-5 and from H-7 to H2-11 (Figure 3). The methyl group (δH 1.26, s) positioned at C-6 was confirmed by the HMBC correlations from H3-14 to C-5, C-6, and C-7. Key HMBC correlations from H3-1 to C-2; H3-13 to C-12; H2-16 to C-9, C15, and C-17; and H3-17 to C-9, C-15, and C-16 permitted assembly of the carbon skeleton. Thus, the planar structure of 4 was established. The relative configuration of 4 was partially elucidated by the analysis of NOE correlations. The trans nature of the epoxide was established by NOE correlations between H3-14 and one proton of H2-8 (δH 1.54, m) and between H2-5 and H-7. Furthermore, the E configuration of the C-3/C-4 double bond was deduced from a 16.0 Hz coupling constant between H-3 and H-4. On the basis of the above findings, the structure of sinulerectol D was established, but the relative configuration of C-9 remains unknown.

spectrum to two methyls, seven sp3 methylenes, three sp2 methylenes, four sp3 methines (including three oxymethines), and one sp3 quaternary and three sp2 quaternary carbons (Table 1). The NMR signals at δC 87.7 (CH) and δH 7.76 (1H, br s) showed the presence of a hydroperoxy group at a methine carbon. Moreover, the HSQC spectrum revealed the presence of six olefinic methylene protons at δH 5.14 (s), 5.10 (s), 4.92 (s), 4.91 (s), 4.84 (s), and 4.77 (s). The planar structure and all of the assignments of 1H and 13C NMR data of 3 were determined with the assistance of 2D NMR studies, including COSY and HMBC experiments (Figure 3). The COSY spectrum revealed proton sequences from H-3 to H2-13 via H-1, H2-5 to H-7, and H2-9 to H-11, as shown by the bold lines in Figure 3. Key HMBC correlations of H2-16 to C-1, C-15, and C-17; H3-17 to C-1, C-15, and C-16; H3-18 to C-3, C-4, and C5; H2-19 to C-7, C-8, and C-9; and H2-20 to C-11, C-12, and C-13 permitted the elucidation of the cembrane carbon skeleton. In consideration of the degrees of unsaturation and 13 C NMR spectroscopic data, an ether linkage was placed between C-4 (δC 84.1) and C-7 (δC 80.7) and a hydroperoxy group was positioned at C-11 (δC 87.7). Thus, 3 was revealed to be a cembranoid possessing a 4,7-ether-linked tetrahydrofuran ring. On the basis of the above analyses, the gross structure of 3 was established. The relative configuration of 3 was examined by the analysis of NOE correlations, as shown in Figure 4. It was found that the β-oriented H-1 (δH 2.49, m) showed an NOE interaction with one proton of H2-14 (δH 1.75, m) and H3-18 (δH 1.11, s), and this proton of H2-14 showed an NOE correlation with H11; therefore, H-11 and H3-18 should also be positioned on the β-face. Furthermore, H3-18 exhibited an NOE correlation with one of the methylene protons at C-5 (δH 1.86, m), which was thus characterized as H-5β, while the other C-5 proton (δH 2.00, m) was assigned as H-5α. NOE correlations observed for E

DOI: 10.1021/acs.jnatprod.5b01142 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. Inhibitory Effects of Compounds 1−8 on Superoxide Anion Generation and Elastase Release by Human Neutrophils superoxide anion

elastase release

compound

IC50 (μM)a

Inh %b

1 2 3 4 5 6 7 8 Idelalisib

2.3 ± 0.4 8.5 ± 0.3 >10 >10 >10 >10 >10 >10 0.07 ± 0.01

100 55 24 13 15 7 16 48 103

± ± ± ± ± ± ± ± ±

IC50 (μM)a *** *** * * * ** * *** ***

1 1 7 4 4 1 4 2 2

0.9 ± 0.1 3.8 ± 0.6 >10 >10 >10 >10 >10 1.0 ± 0.2 0.3 ± 0.1

Inh %b 113 93 33 17 19 14 18 102 100

± ± ± ± ± ± ± ± ±

3 4 3 3 7 5 3 2 4

*** *** *** **

** *** ***

Concentration necessary for 50% inhibition (IC50). bPercentage of inhibition (Inh %) at 10 μM concentration. Results are presented as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control value.

a

MOLT-4 with IC50 values of 6.3 ± 1.5 and 9.7 ± 3.6 μM, respectively. Also, 3 showed cytotoxicity toward the K-562 cell line with an IC50 value of 9.2 ± 3.3 μM (Table S6). The anti-inflammatory activities of compounds 1−8 on neutrophil pro-inflammatory responses were evaluated by measuring their ability to suppress fMLP/CB-induced superoxide anion (O2−•) generation and elastase release in human neutrophils, and the results are as shown in Table 2. From the results, 1, 2, and 8 exhibited strong inhibitory effects (100 ± 1, 55 ± 1, and 48 ± 2%, respectively) on superoxide anion generation at 10 μM. Compounds 1−3 and 8 exhibited potent inhibitory activity against elastase release, with 113 ± 3, 93 ± 4, 33 ± 3, and 102 ± 2% inhibition in the same fMLP/CBstimulated cells at the same concentration. Compounds 1 and 2 were found to show potent activities in inhibition of superoxide generation (IC50 = 2.3 ± 0.4 and 8.5 ± 0.3 μM, respectively) and elastase release (IC50 = 0.9 ± 0.1 and 3.8 ± 0.6 μM, respectively) in this assay. Although compound 8 did not exhibit strong activity in inhibiting superoxide anion generation, it displayed significant inhibitory activity in terms of elastase release (IC50 = 1.0 ± 0.2 μM). Chemical investigation of Sinularia erecta (Tixier-Durivault, 1945) led to the discovery of four new isoprenoids 1−4, along with three known isoprenoids 5−7, and a known nitrogencontaining phenolic compound 8. Cytotoxicity assays showed that compounds 3, 4, and 8 exhibited inhibitory activities against the proliferation of a limited panel of cancer cell lines. Compounds 1, 2, and 8 also exhibited potent anti-inflammatory activities in the inhibition of superoxide generation and elastase release in fMLP/CB-induced human neutrophils, whereas 3 only exhibited significant activity in inhibiting elastase release. Compounds 1, 2, and 8 are potently anti-inflammatory and might represent promising compounds in future marine antiinflammation drug development. Furthermore, as S. erecta produces bioactive natural products, and several studies have demonstrated that cultured soft corals can produce metabolites the same as or similar to those of wild-type marine organisms,26−29 we suggest that aquaculture of this soft coral should be undertaken for the preparation of large quantities of these useful compounds.

The NMR spectroscopic data of three norcembranoids 5−7 and a known nitrogen-containing compound 8 were found to be identical to those of known compounds based on comparison of their physical and spectroscopic data with those reported in the literature.2,21−23 However, sinularectin (6) might have been assigned erroneously because H-11 and H10 were previously determined to be cis to each other on the lactone ring and the S configuration of C-13 of the given structure was not consistent with the proposed molecular model which showed an R configuration of this carbon.21 We completed a careful analysis of the NOE correlations for 6 in CDCl3, which revealed that H-1 (δH 2.85, dddd, J = 12.8, 12.0, 7.2, 5.2 Hz) showed NOE interactions with one proton of H2-2 (δH 2.35, dd, J = 12.0, 7.2 Hz) and H-5 (δH 4.32, d, J = 12.0 Hz), and H-5 showed NOE correlation with H3-18; therefore, due to the β-orientation of H-1, H-5 and H3-18 should also be positioned on the β-face. H-2β (δH 2.35, dd, J = 12.0, 7.2 Hz) showed an NOE correlation with H-11 and the other proton, H-2α (δH 1.82, dd, J = 12.0, 12.0 Hz), exhibited an NOE correlation with H-13 (δH 3.61, dd, J = 6.0, 5.2 Hz), but H-13 did not show an NOE correlation with H-1. Furthermore, H-11 showed only a weak NOE interaction with H-10, and H-10 and H-11 did not couple with each other; thus, H-10 and H-11 are trans-oriented on the lactone ring, and the 11S*,12S*,13R* configurations of 6 were re-established (Figure 4). These corrections were further confirmed by the NMR data and from the relative configuration of a known norcembranoid with a related 11,12-epoxide, for which the structure was confirmed by X-ray crystallography.22 The absolute configurations of the related norcembranoids 5epi-sinuleptolide, 6α-hydroxy-5-episinuleptolide,24 and a biogenetic-related cembranoid sinulochmodin B6 have been confirmed by X-ray crystallographic analyses. Because all Sinularia cembranoids and norcembranoids that have an absolute configuration experimentally established possess the C-1 R configuration,6,15,24,25 this configuration is also proposed for metabolites 1−3, 5, and 6, although experimental proof will be necessary to confirm this suggestion. The cytotoxicities of compounds 1−8 against the proliferation of a limited panel of human cancer cell lines, including promyelocytic leukemia (HL-60), chronic myelogenous leukemia (K-562), T cell lymphoblast-like (CCRF-CEM), and acute lymphoblastic leukemia (MOLT-4) cell lines, were evaluated. The results showed that compound 4 exhibited cytotoxicity toward K-562 and MOLT-4 cancer cell lines with IC50 values of 8.6 ± 1.1 and 9.7 ± 2.9 μM, respectively, while 8 was found to show cytotoxicity toward CCRF-CEM and

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations of isolates were measured on a JASCO P-2100 polarimeter and on a Horiba high sensitivity polarimeter SEPA-300. Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. IR spectra were recorded on a JASCO FT/IR-4100 infrared spectrophotometer. NMR spectra were F

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°C. After a 15 h culture, the test compounds in DMSO solutions were added. After 3 days in culture, attached cells were incubated with Alamar Blue (10 μL/well, 4 h). The absorption at 595 nm was then measured using a microplate reader. The IC50 values represent the concentration of tested compounds that reduced cell growth by 50% under the experimental conditions. Additional Bioassays. The preparation of human neutrophils, measurement of superoxide generation, measurement of elastase release, and statistical analysis protocols have been previously published.32

recorded on a Varian 400MR FT-NMR (or Varian Unity INOVA500 FT-NMR) instrument at 400 MHz (or 500 MHz) for 1H and 100 MHz (or 125 MHz) for 13C in CDCl3 (or DMSO-d6). All NMR experiments were performed at room temperature using CDCl3 or DMSO-d6 as the solvent. Chemical shifts were referenced to residual solvent signals for TMS (δH 0.00 ppm) and CDCl3 (δC 77.0 ppm) or DMSO-d6 (δH 2.50, δC 39.5 ppm). ESIMS data were obtained with a Bruker APEX II mass spectrometer. HRESIMS data were recorded on a Bruker APEX II mass spectrometer. Silica gel (Merck, 230−400 mesh) was used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F-254, 0.2 mm) were used for analytical thin layer chromatography. High-performance liquid chromatography was performed on a Hitachi L-2455 HPLC (or Hitachi L-7100) apparatus with a Supelco C18 column (250 × 21.2 mm, 5 μm) [or Lichrosorb Si-60 (250 × 25 mm, 7 μm)]. Animal Material. The soft coral Sinularia erecta was collected by hand using SCUBA off the coast of Dongsha Atoll in March 2007, at a depth of 10−15 m, and stored in a freezer until extraction. A voucher sample (DS-2007) was deposited at the Department of Marine Biotechnology and Marine Resources, National Sun Yat-sen University. Extraction and Isolation. The frozen bodies of S. erecta (2.3 kg, wet wt) were sliced and exhaustively extracted with EtOAc (5 × 2 L). The EtOAc extract (14.8 g) was chromatographed over silica gel by column chromatography and eluting with EtOAc in n-hexane (0− 100%, stepwise) then with acetone in EtOAc (50−100%, stepwise) to yield 22 fractions. Fraction 15, eluting with n-hexane−EtOAc (1:1), was further purified over silica gel using n-hexane−EtOAc (5:1) to afford eight subfractions (A1−A8), including 3 (1.2 mg) and 8 (9.1 mg). Subfraction A2 was separated by reversed-phase HPLC using MeOH−H2O (3:1) to afford 7 (3.2 mg) and 4 (9.0 mg). Fraction 17, eluting with n-hexane−EtOAc (1:10), was further purified by normalphase HPLC using n-hexane−EtOAc (1:1) to afford 1 (3.5 mg) and 5 (4.5 mg). Fraction 20, eluting with pure EtOAc, was further purified by using n-hexane−EtOAc (1:5) to afford five subfractions (B1−B5), including 2 (3.5 mg). Subfraction B2 was separated by reversed-phase HPLC using MeOH−H2O (2:1) to afford 6 (16.5 mg). Sinulerectol A (1): amorphous solid; [α]25 D +58 (c 0.725, CHCl3); UV (MeOH) λmax (log ε) 213 (3.4); IR (neat) vmax 3410, 2927, 1782, 1746, 1691, 1628, and 1248 cm−1; for 13C and 1H NMR data, see Table 1; ESIMS m/z 429 [M + Na]+; HRESIMS m/z 429.1528 [M + Na]+ (calcd for C21H26O8Na, 429.1525). Sinulerectol B (2): amorphous solid; [α]24 D −51 (c 0.088, CHCl3); UV (MeOH) λmax (log ε) 204 (3.8); IR (neat) vmax 3450, 2925, 1757, and 1709 cm−1; for 13C and 1H NMR data, see Table 1; ESIMS m/z 431 [M + Na]+; HRESIMS m/z 431.1312 [M + Na]+ (calcd for C20H24O9Na, 431.1313). Sinulerectol C (3): amorphous solid; [α]24 D −142 (c 0.030, CHCl3); IR (neat) vmax 3395, 2924, 2853, and 1716 cm−1; for 13C and 1H NMR data, see Table 1; ESIMS m/z 359 [M + Na]+; HRESIMS m/z 359.2196 [M + Na]+ (calcd for C20H32O4Na, 359.2198). Sinulerectadione (4): colorless oil; [α]24 D −220 (c 0.008, CHCl3); UV (MeOH) λmax (log ε) 222 (3.6) and 203 (3.5); IR (neat) vmax 2923, 2852, 1714, and 1675 cm−1; for 13C and 1H NMR data, see Table 1; ESIMS m/z 301 [M + Na]+; HRESIMS m/z 301.1777 [M + Na]+ (calcd for C17H26O3Na, 301.1780). Unnamed Norcembrene (5): amorphous solid; [α]24 D −40 (c 1.0, CHCl3); lit. [α]D −47 (c 0.01, CHCl3).22 Sinularectin (6): amorphous solid; [α]27 D −18 (c 2.29, CHCl3); lit. [α]D −14 (c 0.001, CHCl3);21 for 13C and 1H NMR spectroscopic data, see Table S5-1. Ineleganolide (7): amorphous solid; [α]26 D +24 (c 0.71, CHCl3); lit. 2 [α]25 D +26.4 (c 0.05, CHCl3). Cytotoxicity Testing. Cytotoxicity assays were performed according to the published Alamar Blue assay protocol.30,31 Cell lines (K562, CCRF-CEM, and MOLT-4) were obtained from the American Type Culture Collection. Cancer cells were plated into a 96well microtiter plate with clear flat bottoms (Thermo Scientific Nunc MicroWell plate) at densities of 5 × 103 to 1× 104 cells per well and were incubated in a humidified, 5% CO2 atmosphere incubator at 37

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01142. 1 H, 13C, COSY, HSQC, HMBC, and NOESY NMR spectra for compounds 1−4 and 6, 1H and 13C NMR spectroscopic data for compound 6, and cytotoxicities of compounds 3, 4, and 8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +886-7-5252000, ext. 5030. Fax: +886-7-5255020. Email: [email protected]. Author Contributions ▽

C.-Y.H. and Y.-J.T. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the Ministry of Science and Technology (MOST102-2113-M-110-001-MY2, 104-2113-M-110-006, and 104-2811-M-110-026) awarded to J.H.S.

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REFERENCES

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