Withanolide-Based Steroids from the Cultured Soft ... - ACS Publications

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Withanolide-Based Steroids from the Cultured Soft Coral Sinularia brassica Chiung-Yao Huang,† Chih-Chuang Liaw,† Bo-Wei Chen,† Pei-Chin Chen,† Jui-Hsin Su,§ Ping-Jyun Sung,§ Chang-Feng Dai,⊥ Michael Y. Chiang,∥ and Jyh-Horng Sheu*,†,‡,▽ †

Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung 804, Taiwan Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 804, Taiwan § National Museum of Marine Biology & Aquarium, Pingtung 944, Taiwan ⊥ Institute of Oceanography, National Taiwan University, Taipei 106, Taiwan ∥ Department of Chemistry, National Sun Yat-sen University, Kaohsiung 804, Taiwan ▽ Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404, Taiwan ‡

S Supporting Information *

ABSTRACT: Seven novel withanolides, sinubrasolides A−G (1−7), have been isolated from the cultured soft coral Sinularia brassica. The structures of the new metabolites were determined by extensive spectroscopic analyses, and the absolute configuration of 1 was established by X-ray crystallographic analysis. The cytotoxicities of compounds 1−7 against a limited panel of cancer cell lines also were determined.

structure elucidation, and biological activities of marine withanolides 1−7 from the cultured soft coral S. brassica.

Previous chemical investigation of the wild-type soft coral Sinularia brassica (May 1898) identified only four steroids in this marine organism.1 Thus, it is likely that additional secondary metabolites can be discovered by further chemical investigation of this soft coral, as we have previously discovered that many new bioactive natural products can be obtained from cultured soft corals,2−5 as compared to wild-type organisms. We therefore carried out an investigation of the chemical constituents of cultured S. brassica. The study has led to the discovery of novel withanolides, sinubrasolides A−G (1−7), by conventional C18 reversed-phase HPLC and by tracing the characteristic fragmentation patterns of the marine withanolides by LC-MS/MS. The new structures were elucidated by extensive spectroscopic analysis, and that of 1 was confirmed by single-crystal X-ray diffraction analysis. Withanolides are steroidal lactones mostly found in terrestrial plants in which a C-22/C-26 δ-lactone is generally present in the side chain, and in some cases a C-23/C-26 γ-lactone also might occur.6−14 These steroidal lactones attracted the attention of many chemists and pharmacologists due to their interesting biological activities such as cytotoxic,6 antitumor,7,8 immunosuppresive,6,9,10 antimicrobial,11,12 anti-inflammatory,13 and chemopreventive14 activities. Withanolide-type compounds also have been discovered from the soft corals Paraminabea acronocephala13 and Minabea sp.,15 and some withanolides from P. acronocephala were found to exhibit interesting cytotoxic and anti-inflammatory activities.13 We report herein the isolation, © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSON Specimens of the cultured soft coral S. brassica were collected by hand in a cultivating pool located in the National Museum of Marine Biology and Aquarium, Taiwan, in January 2010, and stored in a freezer until extraction. The frozen bodies were minced and extracted with organic solvent and partitioned between EtOAc and H2O to yield an EtOAc-soluble product, which was further purified by column chromatography to afford compounds 1−7. Sinubrasolide A (1), isolated as a colorless prism, has a molecular formula of C28H38O4 as determined from its HRESIMS data, implying 10 degrees of unsaturation. The IR spectrum of 1 revealed the presence of carbonyl (νmax 1776 and 1661 cm−1) groups. Its 13C NMR data and DEPT (Table 1) spectroscopic data displayed 28 carbon signals, including five methyls, five sp3 methylenes, 10 sp3 methines (including three oxymethines), three sp2 methines, and two sp3 and three sp2 quaternary carbons (including one ester carbonyl and one ketone). The carbon NMR signals (Table 1) resonating at δC 186.3 (C), 155.6 (CH), 127.6 (CH), 123.9 (CH), and 168.8 (C) as well as the proton signals resonating at δH 7.05 (1H, d, J Received: June 13, 2013

A

dx.doi.org/10.1021/np400454q | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Chart 1

Table 1. 1H and 13C NMR Chemical Shifts for Compounds 1−4 1 position 1 2 3 4 5 6

δC, type 155.6, 127.6, 186.3, 123.9, 168.8, 32.7,

a

e

CH CH C CH C CH2

7

33.8, CH2

8 9 10 11 12

34.5, 52.4, 43.6, 22.5, 38.8,

13 14 15

41.5, C 54.2, CH 34.4, CH2

CH CH C CH2 CH2

16

83.0, CH

17 18 19 20 21 22 23

65.0, 14.3, 18.7, 35.2, 16.5, 80.0, 78.3,

24

36.8, CH

25 26 27 28

40.0, 179.3, 10.0, 8.5,

2 δH (J in Hz)

b

CH CH3 CH3 CH CH3 CH CH

CH C CH3 CH3

7.05, d (10.4) 6.23, d (10.4) 6.07, s 2.46, ddd (13.2, 13.2, 4.0) 2.36, ddd (13.2, 4.0, 2.4) 1.94, m 1.04, m 1.76, m 1.07, m 1.74, m 1.86, ddd (12.8, 3.2, 3.2) 1.14, m 0.99, m 2.08, ddd (13.2, 7.6, 6.0) 1.32, ddd (13.2, 13.2, 5.6) 4.61, ddd (7.6, 7.6, 5.6) 1.82, m 0.91, s 1.25, s 2.40, m 1.05, d (7.6) 4.03, dd (9.6, 5.6) 4.23, dd (9.6, 4.4) 2.61, qdd (7.2, 7.2, 4.4) 2.78, dq (7.2, 7.2) 1.17, d (7.2) 0.96, d (7.2)

δC, type d

155.6, 127.6, 186.3, 124.0, 168.8, 32.7,

CH CH C CH C CH2

33.8, CH2 34.5, 52.4, 43.6, 22.5, 38.9,

CH CH C CH2 CH2

3 δH (J in Hz) c

7.04, d (10.0) 6.23, d (10.0) 6.07, s 2.46, ddd (13.5, 13.5, 4.5) 2.36, m 1.92, 1.02, 1.72, 1.05,

m m m m

δC, type d

155.5, 127.6, 186.3, 124.0, 168.6, 32.7,

CH CH C CH C CH2

33.6, CH2 35.1, 52.2, 43.5, 22.6, 39.2,

1.70, m 1.83, m

CH CH C CH2 CH2

1.12, m 41.5, C 54.0, CH 34.4, CH2

64.6, 14.2, 18.7, 35.9, 16.4, 79.3, 78.6,

CH CH3 CH3 CH CH3 CH CH

42.5, CH 40.0, 179.8, 14.5, 14.5,

CH C CH3 CH3

δH (J in Hz) 7.04, d (10.0) 6.23, d (10.0) 6.07, s 2.47, ddd (14.0, 14.0, 4.5) 2.36, br d (14.0) 1.95, 1.06, 1.82, 1.08,

m m m m

1.68, m 1.81, m

δC, type d

155.4, 127.6, 186.2, 124.0, 168.6, 32.7,

CH CH C CH C CH2

33.6, CH2 35.2, 52.2, 43.5, 22.6, 39.2,

CH CH C CH2 CH2

1.20, m 41.1, C 55.2, CH 31.9, CH2

0.96, m 2.04, m 1.26, m

83.2, CH

4 c

1.14, m 2.02, m

2.27, m

81.8, CH 62.1, 16.3, 18.7, 38.4, 15.4, 108.8, 87.2,

CH CH3 CH3 CH CH3 C CH

33.6, CH

2.28, m

37.4, 179.8, 10.2, 15.7,

1.28, d (7.0) 1.13, d (6.5)

CH C CH3 CH3

61.1, 16.3, 18.8, 42.1, 14.5,

2.62, m

30.7, CH

1.14, d (7.5) 1.09, d (7.0)

6.07, s 2.48, ddd (13.5, 13.5, 4.5) 2.36, br d (13.5) 1.93, 1.06, 1.81, 1.08,

m m m m

1.68, m 1.76, m

1.12, m 2.00, m 1.30, m

4.62, ddd (8.0, 8.0, 7.0) 1.84, m 0.90, s 1.25, s 2.16, m 1.08, d (7.5) 115.6, C 4.00, d (2.0)

3.04, dq (7.5, 7.5)

7.02, d (10.0) 6.23, d (10.0)

1.19, m 40.8, C 55.1, CH 31.7, CH2

1.53, m

4.63, ddd (8.0, 8.0, 6.0) 1.80, m 0.89, s 1.25, s 2.37, m 1.06, d (6.5) 4.00, dd (10.0, 5.0) 4.38, dd (10.0, 6.0)

δHc (J in Hz)

82.8, CH CH CH3 CH3 CH CH3

38.4, CH2

43.2, 174.3, 14.2, 20.1,

CH C CH3 CH3

4.70, ddd (7.5, 7.5, 7.5) 1.90, m 0.85, s 1.25, s 2.04, m 1.06, d (6.5) 1.85, m 1.68, m 2.04, m 1.96, m 1.31, d (6.5) 1.05, d (6.5)

a Spectrum recorded at 400 MHz in CDCl3. b100 MHz in CDCl3. cSpectrum recorded at 500 MHz in CDCl3. d125 MHz in CDCl3. eDeduced from DEPT.

B



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= 10.4 Hz), 6.23 (1H, d, J = 10.4 Hz), and 6.07 (1H, s) were characteristic signals of steroids with a 1,4-dien-3-one moiety in ring A.13 In addition, proton resonances at δH 4.23 (1H, dd, J = 9.6, 4.4 Hz), 2.78 (1H, dq, J = 7.2, 7.2 Hz), 2.61 (1H, qdd, J = 7.2, 7.2, 4.4 Hz), 1.17 (3H, d, J = 7.2 Hz), and 0.96 (3H, d, J = 7.2 Hz) and carbon resonances at δC 179.3 (C), 78.3 (CH), 40.0 (CH), 36.8 (CH), 10.0 (CH3), and 8.5 (CH3) were characteristic signals of a γ-lactone, as compared to paraminabeolide D.13 All of the above data showed the presence of four double bonds and revealed the hexacyclic nature of 1. The gross structure of metabolite 1 was further established by the 2D NMR studies. The COSY correlations revealed five separated proton sequences (a−c), as depicted in Figure 1.

Figure 2. X-ray ORTEP drawing of 1.

then determined for 2. Compound 2 was thus found to be the C-25 epimer of 1. The ESIMS spectra of compounds 1 and 2 exhibited similar fragmentation patterns, in which the signals at m/z 269.1 (major in 1) and 267.0 (major in 2) were suspected to arise from cleavages between C-17/C-20 and C-16/OC-22 to lose the side chain with the lactone ring (Figure S7 in the Supporting Information). Tracking the characteristic fragment ion peak at m/z 267 in the Bruker LC-MS/MS extracted ion chromatogram, two related signals at tR 1.9 and 2.1 min with parent ions at m/z 477.2 were found in the fraction possessing compound 1 (subfraction 14B), and one signal at tR 7.6 min with a parent ion at m/z 519.1 was found in fraction 12. By C18 reversedphase HPLC, compounds 3, 5, and 6 were isolated and purified from these fractions, respectively (Figure S11 in the Supporting Information). Sinubrasolide C (3) was obtained as an amorphous solid. The HRESIMS (m/z 477.2620, [M + Na]+) and NMR data of 3 indicated a molecular formula of C28H38O5. A comparison of NMR spectroscopic data of 3 with 1 and 2 (Table 1) indicated that they differ only in the side chains. In particular, C-22 of 3 was found to be quaternary and more downfield-shifted (δC 108.8 for 3, 80.0 for 1, and 79.3 for 2), suggesting further oxidation of this carbon. The IR absorption at 1770 cm−1 and the δC of C-26 at 179.8 suggested that 3 also possesses a γlactone ring. Furthermore, the oxymethine proton H-16 exhibited an HMBC correlation with hemiketal C-22, and the oxymethine proton H-23 correlated with the ester carbonyl carbon C-26. These observations, together with the assistance of analysis of other 2D NMR (COSY and HMBC) correlations (Figure 1), established the planar structure of 3. The relative configuration of compound 3 was established by comparing the NOESY correlations to those of 1 (Figure 3). The NOE interactions of H3-18 with H-20, H-17 with H3-21, and H-23 with H-20, H3-21, and H-24 revealed the αorientations of H-23 and H-24, as shown in a molecular model in Figure 3. Finally, H-25 showed interactions with H-24, but not with H3-28, revealing the β-orientations of H3-27 and H328. Sinubrasolide D (4) possesses the same molecular formula as that of 1, based on the interpretation of the HRESIMS and 13C NMR spectroscopic data (Table 1). A comparison of NMR spectroscopic data of 1 and 4 indicated that they differ only in the nature of the side chains. The planar structure of the THF ring was elucidated mainly by the HMBC correlations from H321 to C-17, C-20, and C-22 and from H-16 to C-22. Also, in considering the molecular formula, the 10 degrees of unsaturation, the IR absorption at 1737 cm−1, and the signal

Figure 1. Selected COSY () and HMBC (→) correlations of 1−4.

Key HMBC correlations of H3-27 to C-24, C-25, and C-26, as well as from H3-28 to C-23, C-24, and C-25, corroborated the presence of a γ-lactone moiety, and H-16 to C-22 further confirmed the tetrahydrofuran (THF) ring fused to ring D (Figure 1). On the basis of the above analysis, the planar structure of 1 was established. The relative configuration of 1 was elucidated on the basis of the observed key NOE correlations. In the NOESY spectrum of 1, H-8 was found to show NOE interactions with both H3-18 and H3-19, suggesting the β-orientations of H-8, H3-18, and H319. Furthermore, NOE interactions could be observed between H3-18 and H-15β, H-20, and H-22; H-22 and H3-28; and H3-28 and H3-27, and H-9 showed NOE correlations with H-14 but did not correlate with either H-8 or H3-19. Also, NOE responses of H-14 with H-17, H-17 with H-16 and H3-21, and H-23 with H3-21, H-24, and H-25 reveal that H-9, H-14, H-16, H-17, H3-21, and H-23 should be placed on the α-face, while H-22, H3-27, and H3-28 should be β-oriented. The absolute configuration of 1 was assigned based on a Flack parameter of 0.12(14) in the X-ray analysis (Figure 2).16 Sinubrasolide B (2) was found to possess the same molecular formula, C28H38O4, as that of 1 from the HRESIMS and NMR spectroscopic data. The 13C NMR and 1H NMR spectroscopic data (Table 1) of 2 were found to be quite similar to those of 1, except for the C-27 and C-28 carbons resonating at δC 10.0 (CH3) and 8.5 (CH3) in 1 and at δC 14.5 (CH3) and 14.5 (CH3) in 2, revealing that 2 was the 24S or 25R isomer of 1. The presence of a γ-lactone ring also was evidenced by an IR absorption at 1775 cm−1. On the basis of above findings and by NOE correlations of H3-18 with H-22, H-22 with H-20 and H328, and H-23 with H3-21 and H3-27, the 25R configuration was C



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Figure 3. Selected NOE correlations for compounds 2−4.

of a ketal carbon (C-22, δC 115.6), the presence of a δ-lactone group was revealed. The above information, together with the HMBC information from H3-27 to C-24, C-25, and C-26 and from H3-28 to C-23, C-24, and C-25, established an unusual spiroketal unit between the THF and δ-lactone rings of 4. The relative configuration of 4 was established by the NOE correlations observed in a NOESY experiment and by examining a molecular model as shown in Figure 3. It was found that the NOE correlations of H-8, H-9, H-14, H-16, H17, H3-18, H3-19, H-20, and H3-21 are nearly the same as for 1−3, suggesting the same configurations at the corresponding carbons in 1−4. H-23β (δH 1.85, m) showed correlations with H-20 and H-24, and H-23α (δH 1.65, m) showed correlations with H3-21 and H3-28, which further interacted with H-25, revealing the β-orientations of the carbon side chain at C-22 and of H-24 and H3-27, and α-orientations of H-25 and H3-28. On the basis of the above findings and other detailed NOE correlations (Figure 3), the structure of 4 was determined. The ESIMS spectrum of compound 4 exhibited the same characteristic fragments as those in 1 and 2, suggesting the same cleavage between C-17/C-20 and C-16/OC-22 to lose the side chain with the lactone ring (Figure S15 in the Supporting Information). Sinubrasolide E (5) was isolated as an amorphous solid. It gave the same molecular formula, C28H38O5, as that of 3 from the HRESIMS spectrum. This compound was shown to possess a spiroketal functionality (δC 108.3) similar to compound 4. Compounds 4 and 5 have the same substituent patterns in rings A−D, as concluded by comparison of their 1D and 2D NMR spectroscopic data. Furthermore, it was found that the carbon chemical shift of 5 was shifted upfield at C-16 (δC 73.9 for 5; 82.8 for 4), suggesting the presence of a tetrahydropyran in 5, rather than a tetrahydrofuran. Moreover, a comparison of C-26 of 5 with 4 indicated that they differ from the lactone carbonyl carbons (δC 178.2 for 5; 174.3 for 4), again revealing the presence of a five-membered lactone ring in 5 as in compounds 1−3, rather than a six-membered lactone ring.13 Interpretation of the 2D NMR spectroscopic data of 5 confirmed the above elucidation and thus established its planar structure (Figure 4). In the NOESY spectrum of 5, the NOE correlations between H-20 and H3-18 and H-22 (δH 3.98, s) as well as between H-8 and both H3-18 and H3-19 indicated that these protons adopt β-orientations. Moreover, NOE responses between H-22 and

Figure 4. Selected 1H−1H COSY () and HMBC (→) correlations of 5−7.

H3-28, H3-28 with H3-27, and H-24 (δH 2.44, dq, J = 7.5, 7.5 Hz) with H-25 and H3-27, and that H-25 was not found to have a NOE correlation with H3-28, revealed the 20S, 22S, 23S, 24R, 25S configuration of 5, as shown in Figure 5. The molecular formula C30H40O6 of sinubrasolide F (6) was established from the HRESIMS spectrum. By comparison of the NMR data of 6 with those of 5, it was found that the 1H and 13C NMR data of 6 were similar to those of 5, with the difference that 6 contains one acetyl group. The chemical shift

Figure 5. Selected NOE correlations for compounds 5 and 7. D



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of H-22 in 5 (δH 3.98, s) was shifted downfield (δH 5.43, d, J = 1.5 Hz) in 6, suggesting that 6 is the 22-acetyl derivative of 5. Furthermore, acetylation of 5 gave a product that was found to be identical to 6 by comparison of their physical (specific rotation) and spectroscopic (IR, 1H and 13C NMR) data. Thus, 6 was determined to be the 22-acetyl derivative of 5. The molecular formula of sinubrasolide G (7) was found to be C30H42O3 as deduced from HRESIMS and 13C NMR data, appropriate for 10 degrees of unsaturation. The IR spectrum of 7 showed the presence of carbonyl (νmax 1733 and 1661 cm−1) groups, and the 13C NMR and DEPT spectra showed signals of five methyls, eight methylenes (including one oxymethylene), 11 methines, and six quaternary carbons (including one ester carbonyl and one keto-carbonyl). Compounds 1−7 were found to have the same A−D rings, by comparing their 1D and 2D NMR spectroscopic data. The gross structure of 7 was determined by a detailed analysis of COSY and HMBC correlations. The unusual spiro structure of the δ-lactone with a cyclopropane was elucidated mainly by the HMBC correlations from H3-27 (δH 1.18, d, J = 6.8) to C-24, C-25, and C-26, from H2-29 (δH 4.67, d, J = 10.8; 3.84, d, J = 10.8) to C-23, C-24, and C-26, and from H2-30 (δH 0.60, dd, J = 8.4, 5.2 Hz; 0.39, dd, J = 5.2, 5.2 Hz) to C-22, C-24, and C-29. The above observations together with the presence of a quaternary carbon signal at δC 27.3 showed the presence of a trisubstituted cyclopropane ring.17 The unusual spirocyclic linkage between the cyclopropane and an α,β-dimethyl-δ-lactone is proposed to be formed from the oxidation of the 26- and 29-methyls to a carboxylic acid and an alcohol, from a gorgosterol side chain, and thus subsequent lactone ring formation. The relative configuration of 7 was determined mainly by the assistance of a NOESY experiment. In the NOESY spectrum of 7, H-8 was found to show NOE correlations with H-7β (δH 1.98, m), H3-18, and H3-19, and H-9 showed NOE correlations with H-7α (δH 1.02, m) and H-14, while H-14 was correlated with H-17, suggesting the β-orientation of H-8 and the αorientations of H-9, H-14, and H-17. Furthermore, NOE correlations of H-17/H-22, H-17/H3-28, H3-28/H-29α (δH 4.67, d, J = 10.8 Hz), H-29α/H3-27, and H-29β (δH 3.84, d, J = 10.8 Hz)/H-20 and H-30α (δH 0.39, dd, J = 5.2, 5.2 Hz) suggested the β-orientation of CH2-29 in the cyclopropane and α-orientations of H3-27 and H3-28 in the α-lactonic ring and that the two methyl groups (H3-27 and H3-28) are cis to H-22 (Figure 5). On the basis of the above findings, the relative structure of 7 was determined. The cytotoxicities of compounds 1−7 against the proliferation of a limited panel of cancer cell lines, including murine leukemia (P388) cells, lymphoid T carcinoma (MOLT 4) cells, human erythroleukemia (K562) cells, and human colon carcinoma (HT-29) cells, were evaluated. The results showed compound 2 exhibited cytotoxicity toward P388, MOLT 4, and HT-29 cancer cell lines with ED50 values of 9.1 ± 1.4, 4.8 ± 0.9, and 4.8 ± 0.7 μM, respectively, while 5 was found to show cytoxicity toward MOLT 4 and HT-29 with ED50 values of 9.9 ± 1.8 and 7.5 ± 1.5 μM. Also, 1 showed cytotoxicity toward the K562 cell line (ED50 = 8.7 ± 1.4 μM). Withanolides with a 16,23-oxa-bridged tetrahydropyran ring, such as compounds 5 and 6 and a gorgosteroid with the δlactone ring in a spirocyclic linkage with a cyclopropane 7, are unusual in steroids. Our investigation demonstrated that the cultured soft coral S. brassica is a good source of novel bioactive withanolides. Several of the isolated compounds, in particular 1, 2, and 5, showed interesting cytotoxicities to cancer cell lines

and should be further investigated. This study also demonstrates that cultured soft corals might be a useful source of bioactive natural products.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined using a Fisher-Johns melting point apparatus. Optical rotations were measured on a JASCO P1020 digital polarimeter. Ultraviolet spectra were recorded on a JASCO V-650 spectrophotometer. IR spectra were obtained on a JASCO FT/IR-4100 infrared spectrophotometer. The NMR spectra were 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 13 C in CDCl3. The values of 1H are in CDCl3 at 0.00 ppm from TMS, and the values of 13C are in CDCl3 at 77.0 ppm. LC-ESIMS/MS data were processed using a Dionex UltiMate 3000 UHPLC system (Thermo) and an AmaZon SL ion trap mass spectrometer (Bruker Daltonics) with ESI Compass DataAnalysis software, version 4.1 (Bruker Daltonik GmbH). ESIMS data were obtained with a Finnigan LCQ ion-trap mass spectrometer (or Bruker APEX II mass spectrometer). HRESIMS data were recorded on a LTQ Orbitrap XL mass spectrometer (or 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 TLC. High-performance liquid chromatography was performed on a Hitachi L-2455 HPLC apparatus with a Supelco C18 column (250 × 21.2 mm, 5 μm). Animal Material. The cultured soft coral Sinularia brassica used in this study was originally collected from the wild and cultured for five years in an 80-ton cultivation tank (height 1.6 m) located in the National Museum of Marine Biology and Aquarium, Taiwan. The tank was a semiclosed recirculating aquaculture system and did not require deliberate feeding. To the best of our knowledge, this is the first farming system for S. brassica in the world. The specimens were then collected by hand in January 2010 and were stored in a −20 °C freezer. The soft coral was identified by one of the authors (C.-F.D.). A voucher specimen (specimen no. 201001C1) was deposited in the Department of Marine Biotechnology and Resources, National Sun Yat-sen University. Extraction and Isolation. The frozen bodies of S. brassica (0.4 kg, wet wt) were minced and extracted exhaustively with CH2Cl2 and MeOH (1:1, 0.5 L × 6). The CH2Cl2 and MeOH extract of the soft coral S. brassica was partitioned between EtOAc and H2O to afford the EtOAc-soluble fraction. The EtOAc extract (3.7 g) was chromatographed over silica gel by column chromatography and eluted with EtOAc in n-hexane (0−100%, stepwise) and then with MeOH in EtOAc (5−50%, stepwise) to yield 24 fractions. Fraction 12, eluting with n-hexane−EtOAc (9:1), was further purified by reversed-phase HPLC using MeOH−H2O (3.5:1) to afford 6 (0.8 mg). Fraction 13, eluting with n-hexane−EtOAc (8:1), was further purified over silica gel using n-hexane−acetone (7:1) to afford five subfractions (13A−13E). Subfraction 13B was separated by reversed-phase HPLC using MeOH−H2O (3:1) to afford 2 (2.0 mg), 4 (1.2 mg), and 7 (2.4 mg). Fraction 14, eluted with n-hexane−EtOAc (7:1), was further purified over silica gel using n-hexane−acetone (6:1) to afford five subfractions (14A−14E). Subfraction 14B was separated by reversedphase HPLC using MeOH−H2O (2.2:1) to afford 1 (9.6 mg), 3 (2.1 mg), and 5 (2.4 mg). Sinubrasolide A (1): colorless prism; mp 179−181 °C; [α]25D −30 (c 0.53, CHCl3); UV (MeOH) λmax (log ε) 245 (4.5); IR (neat) νmax 2971, 2938, 1776, and 1661 cm−1; 13C and 1H NMR data, see Table 1; ESIMS m/z 439 [M + H]+; HRESIMS m/z 439.2849 [M + H]+ (calcd for C28H39O4, 439.2843). Sinubrasolide B (2): amorphous solid; [α]25D −167 (c 0.16, CHCl3); UV (MeOH) λmax (log ε) 245 (4.0); IR (neat) νmax 2935, 1775, and 1662 cm−1; 13C and 1H NMR data, see Table 1; ESIMS m/z 439 [M + H]+; HRESIMS m/z 439.2850 [M + H]+ (calcd for C28H39O4, 439.2843). E



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Table 2. 1H and 13C NMR Chemical Shifts for Compounds 5−7 5 position 1 2 3 4 5 6

δC,b type 155.6, 127.6, 186.3, 124.0, 168.8, 32.7,

6 δHa (J in Hz)

e

CH CH C CH C CH2

7

33.6, CH2

8 9 10 11 12 13 14 15α 15β 16 17 18 19 20 21 22 23 24 25 26 27 28 29

35.1, 52.3, 43.5, 22.6, 39.2, 41.4, 51.8, 33.4,

CH CH C CH2 CH2 C CH CH2

73.9, 53.0, 14.9, 18.8, 29.1, 17.6, 70.7, 108.3, 43.1, 39.2, 178.2, 9.8, 11.4,

CH CH CH3 CH3 CH CH3 CH C CH CH C CH3 CH3

7.04, d (10.0) 6.23, d (10.0) 6.08, s 168.7, C 2.48, ddd (13.5, 13.5, 5.0) 2.38, br d (13.5) 1.95, m 1.06, m 1.74, m 1.09, m 1.72, m 1.88, m; 1.22, m 0.94, 2.11, 1.18, 4.53, 1.37, 0.86, 1.25, 1.64, 1.18, 3.98,

m m m ddd (13.0, 7.5, 7.5) m s s m d (6.5) s

2.44, dq (7.5, 7.5) 3.29, dq (7.5, 7.5) 1.15, d (7.5) 0.93, d (7.5)

δC,b type 155.5, 127.6, 186.3, 124.0, 169.3, 32.7,

CH CH C CH C CH2

33.7, CH2 35.1, 52.4, 43.5, 22.6, 39.6, 41.5, 51.8, 33.2,


73.5, 53.4, 14.7, 18.7, 28.3, 17.1, 71.3, 107.0, 43.2, 38.8, 178.3, 10.0, 11.1,

CH CH CH3 CH3 CH CH3 CH C CH CH C CH3 CH3

7 δHa (J in Hz)

7.04, d (10.0) 6.24, d (10.0) 6.08, s 2.48, 2.38, 1.97, 1.07, 1.75, 1.08,

ddd (14.0, 14.0, 6.5) br d (14.0) m m m m

1.72, m 1.88, m; 1.22, m 0.96, 2.13, 1.22, 4.41, 1.31, 0.88, 1.25, 1.77, 1.04, 5.43,

m m m ddd (13.0, 7.5, 7.5) m s s m d (7.0) d (1.5)

2.47, m 3.15, dq (7.5, 7.5) 1.16, d (7.5) 1.05, d (7.5)

30 OAc

δC,d type 155.9, 127.5, 186.4, 123.8,

CH CH C CH

32.9, CH2 33.6, CH2 35.5, 52.3, 43.6, 22.8, 39.5, 43.1, 55.2, 24.5,


28.6, 57.6, 12.1, 18.7, 35.1, 20.9, 32.1, 27.3, 41.6, 40.3, 174.0, 13.7, 20.9, 71.7,

CH2 CH CH3 CH3 CH CH3 CH C CH CH C CH3 CH3 CH2

18.9, CH2 170.8, C 20.8, CH3

δHc (J in Hz) 7.05, d (10.0) 6.23, d (10.0) 6.07, s 2.47, 2.36, 1.98, 1.02, 1.60, 1.06,

ddd (13.6, 13.6, 5.2) br d (13.6) m m m m

1.65, m 2.02, ddd (12.4, 2.8, 2.8); 1.20, m 1.02, 1.62, 1.16, 1.98, 1.25, 0.70, 1.23, 0.90, 1.04, 0.79,

m m m m; 1.17, m m s s m d (6.8) m

1.16, m 2.90, dq (4.8, 6.8) 1.18, 1.04, 4.67, 3.84, 0.60, 0.39,

d (6.8) d (6.8) d (10.8), d (10.8) dd (8.4, 5.2) dd (5.2, 5.2)

2.12, s

a

Spectrum recorded at 500 MHz in CDCl3. b125 MHz in CDCl3. cSpectrum recorded at 400 MHz in CDCl3. d100 MHz in CDCl3. eDeduced from DEPT. Sinubrasolide C (3): amorphous solid; [α]25D −10 (c 1.00, CHCl3); UV (MeOH) λmax (log ε) 244 (3.9); IR (neat) νmax 3438, 2970, 2938, 2851, 1770, and 1660 cm−1; 13C and 1H NMR data, see Table 1; ESIMS m/z 477 [M + Na]+ ; HRESIMS m/z 477.2620 [M + Na]+ (calcd for C28H38O5Na, 477.2617). Sinubrasolide D (4): amorphous solid; [α]23D −52 (c 0.37, CHCl3); UV (MeOH) λmax (log ε) 244 (4.4); IR (neat) νmax 2936, 2852, 1737, and 1661 cm−1; 13C and 1H NMR data, see Table 1; ESIMS m/z 439 [M + H]+; HRESIMS m/z 439.2857 [M + H]+ (calcd for C28H39O4, 439.2843). Sinubrasolide E (5): amorphous solid; [α]23D −91 (c 0.24, CHCl3); UV (MeOH) λmax (log ε) 246 (3.9); IR (neat) νmax 2970, 2936, 1774, and 1661 cm−1; 13C and 1H NMR data, see Table 2; ESIMS m/z 477 [M + Na]+; HRESIMS m/z 477.2615 [M + Na]+ (calcd for C28H38O5Na, 477.2617). Sinubrasolide F (6): amorphous solid; [α]23D −95 (c 0.23, CHCl3); UV (MeOH) λmax (log ε) 245 (4.2); IR (neat) νmax 2970, 2934, 1781, 1747, 1716, 1662, and 1233 cm−1; 13C and 1H NMR data, see Table 2; ESIMS m/z 519 [M + Na]+; HRESIMS m/z 519.2725 [M + Na]+ (calcd for C30H40O6Na, 519.2722).

Sinubrasolide G (7): amorphous solid; [α]23D +90 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 246 (4.4); IR (neat) νmax 2964, 2937, 1733, and 1661 cm−1; 13C and 1H NMR data, see Table 2; ESIMS m/z 451 [M + H]+; HRESIMS m/z 451.3216 [M + H]+ (calcd for C30H43O3, 451.3207). Acetylation of Sinubrasolide E (5). A solution of 5 (0.5 mg) in pyridine (0.5 mL) was mixed with Ac2O (0.1 mL), and the mixture was stirred at room temperature for 24 h. After evaporation of excess reagent, the residue was subjected to column chromatography over Si gel using n-hexane−acetone (5:1) to yield the acetyl derivative 6 (0.4 mg, 74%): [α]24D −100 (c 0.09, CHCl3); ESIMS m/z 519 [M + H]+. X-ray Crystallographic Analysis of 1. A suitable colorless crystal (0.50 × 0.45 × 0.40 mm3) of 1 was grown by slow evaporation from an acetone−n-hexane (2:1) solution. Diffraction intensity data were acquired with a CCD area detector with graphite-monochromated Cu Kα radiation (λ = 1.54178 Å). Crystal data for 1: C28H38O4 (formula weight 438.58), orthorhombic, space group, P212121 (#19), T = 100(2) K, a = 6.1962(2) Å, b = 12.5597(4) Å, c = 30.5348(11) Å, V = 2376.29(14) Å3, Dc = 1.226 Mg/m3, Z = 4, F(000) = 952, μ(Cu Kα) = 0.633 mm−1. A total of 17 041 reflections were collected in the range 2.89° < θ < 66.58°, with 4139 independent reflections [R(int) = F



Article

0.0239]; completeness to θmax was 99.1%; psi-scan absorption correction applied; full-matrix least-squares refinement on F2; the number of data/restraints/parameters were 4139/0/294; goodness-offit on F2 = 1.048; final R indices [I > 2σ(I)], R1 = 0.0270 wR2 = 0.0685; R indices (all data), R1 = 0.0272, wR2 = 0.0687, largest difference peak and hole, 0.166 and −0.130 e/Å3. Flack parameter = 0.12(14).18 Cytotoxicity Testing. Cell lines were purchased from the American Type Culture Collection (ATCC). Cytotoxicity assays of compounds 1−7 were performed using the Alamar Blue assay.19,20 Compounds 1−7 are considered inactive when ED50 > 10 μM, and the values are mean ± SEM in triplicate. 5-Fluorouracil exhibited cytotoxicity toward P388, MOLT 4, K-562, and HT-29 cancer cell lines with ED50 values of 6.2 ± 0.8, 7.7 ± 0.8, 21 ± 2.0, and 7.7 ± 0.8 μM, respectively.

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(15) Ksebati, M. B.; Schmitz, F. J. J. Org. Chem. 1988, 53, 3926− 3929. (16) Flack, H. D. Acta Crystallogr. 1983, A39, 876−881. (17) Tanaka, J.; Trianto, A.; Musman, M.; Yoshida, W. Y.; Ohtani, I. I.; Ichiba, T.; Higa, T.; Yoshida, W. Y.; Scheuer, P. J. Tetrahedron 2002, 58, 6259−6266. (18) Crystallographic data for compound 1 have been deposited with the Cambridge Crystallographic Data Centre (deposition number CCDC 865198). Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336033 or e-mail: [email protected]). (19) Nakayama, G. R.; Caton, M. C.; Nova, M. P.; Parondoosh, Z. J. Immunol. Methods 1997, 204, 205−208. (20) O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. I. Eur. J. Biochem. 2000, 267, 5421−5426.

ASSOCIATED CONTENT

* Supporting Information S

NMR spectra (including NOESY spectra) of compounds 1−7 and a cif file for 1 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Science Council of Taiwan (NSC100-2320-B-110-001-MY2) and Aim for the Top University Program (02C030205) from the Ministry of Education of Taiwan awarded to J.-H.S.

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REFERENCES

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