Antifouling Eunicellin-Type Diterpenoids from the Gorgonian

Aug 20, 2012 - National Museum of Natural History Naturalis, 2300 RA Leiden, The ... astrogorgins B–M (1–12), were isolated from a Chinese gorgoni...
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Antifouling Eunicellin-Type Diterpenoids from the Gorgonian Astrogorgia sp. Daowan Lai,† Dong Liu,† Zhiwei Deng,‡ Leen van Ofwegen,§ Peter Proksch,⊥ and Wenhan Lin*,† †

State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, People's Republic of China Analytical and Testing Center, Beijing Normal University, Beijing, 100875, People's Republic of China § National Museum of Natural History Naturalis, 2300 RA Leiden, The Netherlands ⊥ Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, 40225 Duesseldorf, Germany ‡

S Supporting Information *

ABSTRACT: Twelve new eunicellin-based diterpenoids, astrogorgins B−M (1−12), were isolated from a Chinese gorgonian Astrogorgia sp., together with ophirin, muricellin, astrogorgin, calicophirins A and B, and 14-deacetoxycalicophirin B. The structures of the new compounds were elucidated by extensive spectroscopic analysis and by comparison with data reported in the literature. Significant antifouling activity was observed for 14-deacetoxycalicophirin B against the larval settlement of the barnacle Balanus amphitrite at nontoxic concentrations with an EC50 = 0.59 μg/mL, while the other analogues were effective within an EC50 range of 5.14−17.8 μg/mL.

E

unicellin-type diterpenoids are a group of structurally unique cembranoids widely distributed in the gorgonian corals, including the genera Briareum,1,2 Acalycigorgia,3 Eunicella,4−7 Muricella,8−10 Solenopodium,11 and Astrogorgia,12 and are also found from some soft corals.13−22 The structures are characterized by the presence of a cladiellane-based skeleton containing an ether bridge across C-3 and C-10. Although their exact ecological significance remains uncertain, some ecological- and agrochemical-related activities including molluscidal and mollusk repellant, hemolytic, inhibition of cell division in fertilized starfish eggs, and insect growth inhibitory activities have been reported. Pharmaceutically, these metabolites displayed a wide range of bioactivities, such as cytotoxicity toward cancer cell lines,9,10 anti-inflammatory,18,23 inhibitory activity against prostate cancer cell invasion and migration,17 and lethality toward brine shrimp.9,14 Astrogorgia is a genus of gorgonians whose secondary metabolites have been rarely examined, and only two eunicellin-type analogues, ophirin and astrogorgin, have been reported so far from Astrogorgia sp.12 During our chemical investigation of marine invertebrates inhabiting Beibuwan Bay of the South China Sea, an Astrogorgia sp. was collected. Chemical examination of this specimen resulted in the isolation of 18 minor eunicellin-based diterpenoids, including 12 new analogues, namely, astrogorgins B−M (1−12).24 Six known analogues were identified as ophirin (14),8,12 muricellin (15),10 astrogorgin (16),12 calicophirin A (17),25 calicophirin B (18),25 and 14-deacetoxycalicophirin B (19).9

Astrogorgin B (1) was isolated as a colorless oil, and its molecular formula was established as C24H36O6 on the basis of HRESIMS and NMR data, implying seven degrees of unsaturation. The IR absorptions (3449, 1735, and 1612 cm−1) suggested it contains hydroxy, carbonyl, and olefinic functionalities. The 13C NMR and APT spectra exhibited a total of 24 carbon resonances including two acetyl carbonyls and four olefinic carbons for two double bonds, while the others were attributed to aliphatic resonances. Thus, a tricyclic skeleton for 1 would account for the remaining degrees of molecular unsaturation. The COSY relationships established the spin systems for the subunits from C-1 to C-3, C-5 to C-7, C-9 to C-11, and C-14 to C-13 and C-1. Their connectivity was

■

RESULTS AND DISCUSSION The MeOH extract of the gorgonian Astrogorgia sp. was separated by column chromatography and semipreparativeHPLC purification to afford compounds 1−12 and 14−19. © 2012 American Chemical Society and American Society of Pharmacognosy

Received: June 11, 2012 Published: August 20, 2012 1595

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Figure 1. Key NOESY correlations of 1, 7, and 10.

The NMR features of astrogorgin C (2) were closely related to those of muricellin, and it had the same molecular formula as the latter compound. The difference was found in ring A of 2, where the signals of an exomethylene group resonating at δH 4.92 (brs) and 4.89 (brs) were observed to replace the olefinic methyl group C-20. In addition, the oxymethine H-14 (δH 5.13) coupled to the methylene protons H2-13 (δH 2.37, 2.64) in the COSY spectrum and C-13 (δC 35.7) correlated with the exomethylene protons in the HMBC spectrum identified 2 as a rearranged olefin derivative of muricellin. Similar NOE correlations indicated 2 possesses the same configurations as muricellin, which was also isolated from this specimen. Astrogorgin D (3) has a molecular formula of C26H38O7 as determined by the HRESIMS data, one oxygen atom less than that of 2. Interpretation of 1D and 2D NMR data revealed that 3 shares the same partial structure with respect to rings A and B as that of 2, in which C-14 and C-15 are substituted by acetoxy groups. However, the NMR data of 3 exhibited three acetoxy groups. Comparison of the NMR data of ring C disclosed that C-4 (δC 90.4) of 3 shifted downfield dramatically in comparison with the hydroxy-substituted C-4 (δC 77.6) of 2, indicating the third acetoxy group of 3 is at C-4. This assignment was supported by the similar data of ophirin, which has an acetoxy group at C-4. Similar NOE relationships indicated 3 possesses the same relative configurations as 2. Astrogorgin E (4) has a molecular formula of C26H38O8, as determined by HRESIMS and NMR data, requiring eight degrees of unsaturation. The NMR spectroscopic data (Tables 1 and 2) were closely comparable to those of astrogorgin (16),12 except for the presence of three acetyl groups in place of four for the latter compound. The upfield shifted H-7 (δH 4.28) and the absence of an HMBC correlation between H-7 and an acetyl carbon suggested C-7 of 4 to be hydroxylated. The downfield-shifted C-6 (+1.8 ppm) and C-8 (+1.5 ppm) in comparison with the corresponding carbons of astrogorgin also supported 4 to be 7-deacetylastrogorgin. The NMR spectroscopic data of astrogorgin F (5) indicated it is closely related to 4 with the exception of the substituents at ring C. The COSY spectrum correlated H2-6 (δH 1.76, 3.10) to H-5 (δH 4.92, t, J = 2.8 Hz) and H-7 (δH 4.13, t, J = 3.0 Hz), indicating C-5 (δC 73.3) and C-7 (δC 73.1) to be oxygenated. The HMBC correlation between H-5 and an acetyl carbon at δC 171.1 positioned an acetoxy group at C-5. The remaining two acetoxy groups were deduced to be at C-14 and C-15 by the 2D NMR analysis and in comparison with the NMR data of 4. The upfield-shifted C-4 (δC 74.7) in contrast to the acetylated C-4 (δC 85.5) of 4 ascertained C-4 to be substituted with a hydroxy group. The NOE interaction between H-2 (δH 2.92, dd) and H-5 in a 1D GOESY experiment reflected H-5β. The HRESIMS data of astrogorgin G (6) was in accordance with a molecular formula of C26H38O10, showing one oxygen

determined through HMBC correlations. The HMBC relationships from H3-18 (δH 1.43, s) to C-3 (δC 88.2), C-4 (δC 77.1), and C-5 (δC 74.6), H3-19 (δH 1.91, s) to C-7 (δC 123.7), C-8 (δC 130.9), and C-9 (δC 44.4), and H3-20 (δH 1.79, s) to C-11 (δC 47.6), C-12 (δC 139.7), and C-13 (δC 139.7) allowed connection of the subunits to form a 14-membered carbocyclic ring, in which C-7/C-8 and C-12/C-13 were present as double bonds. An isopropyl group at C-1 was deduced by the HMBC interactions from H3-16 (δH 1.03, d, J = 6.8 Hz) and H3-17 (δH 0.99, d, J = 6.8 Hz) to C-1 (δC 41.2). An additional COSY cross-peak between H-2 (δH 2.64, brdd, J = 8.4, 10.0 Hz) and H-11 (δH 2.45, dd, J = 0.4, 8.4 Hz) connected C-2 to C-11 to form a cyclohexene ring, while the HMBC interactions between H-10 (δH 4.40, dd, J = 0.4, 5.6 Hz) and C-3 and in turn between H-3 (δH 4.63, d, J = 10.0 Hz) and C-10 (δC 80.7) revealed an ether bridge across C-3 and C-10. Thus, the basic skeleton of 1 is characteristic of a eunicellin-type diterpene, similar to ophirin and muricellin,10 from the same fraction. The positions of two acetoxy groups at C-5 and C-14 were determined on the basis of the HMBC interactions from H-5 and H-14 to the acetyl carbonyl carbons at δC 170.3 and 171.0, respectively. Finally, the presence of a hydroxy group at C-4 was evident from the chemical shift of the quaternary carbon C4 (δC 77.1) and the HMBC interaction between H3-18 and C4. The relative configurations of 1 were established by NOE interactions (Figure 1). The NOE relationship between H-2 and H-11 was in agreement with a cis-fusion between the cyclohexene and the tetrahydrofuran ring, with H-2 and H-11 arbitrarily assigned β-orientations. The α-orientations for H-1, H-3, and H-10 were evident from the NOE interactions from H-3 to H-1 and H-10, in addition to the interactions from H-2 to the protons of the isopropyl group and between H-11 and H-9a (δH 1.97). Additional NOE interactions observed from H14 to the protons of the isopropyl group and between H-2/H-5 and H-3/H3-18 clarified the β-orientations of H-5 and H-14 (Figure 1), whereas H3-18 was oriented to the α-face. The olefinic geometries were determined to be 7E and 12Z according to the NOE cross-peaks between H3-19 and H2-6 and between H3-20 and H-13 in association with the chemical shifts of C-19 (20 ppm). Because the absolute configuration of ophirin was determined by X-ray diffraction,8,12 the same sign and similar magnitudes of the specific rotations for 1 ([α]25D −136.7, CHCl3) and ophirin8 suggest that 1 shares the same absolute configuration as ophirin. This assignment is supported by the computed optical rotations of the isomers for (5S)-1 (−79.5), (5R)-1 (−88.3), and 5-unsubstituted 1 (−82.7) at the B3LYP/6-311++G(2d,p)//B3LYP/6-311+G(d) level in the gas phase,25,26 indicating that the configuration at C-5 could affect the magnitude of the specific rotation but not the sign. Thus, the absolute configuration of C-5 was assumed as 5S. 1596

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41.2, CH 37.6, CH 88.2, CH 77.1, C 74.6, CH 28.8, CH2 123.7, CH 130.9, C 44.4, CH2 80.7, CH 47.6, CH 139.7, C 120.4, CH 70.0, CH 30.7, CH 21.4,CH3 21.0, CH3 22.7, CH3 19.1, CH3 21.9, CH3 171.0, C, 170.3, C

21.6, CH3 21.3,,CH3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ac(CO)

Ac(CH3)

OMe

1

position

47.9, 38.7, 91.6, 77.6, 75.0, 28.6, 123.9, 130.8, 44.1, 84.6, 49.8, 145.4, 35.7, 71.5, 84.2, 25.2, 24.8, 22.9, 18.8, 111.5, 170.3, 170.0, 169.9, 22.5, 21.5, 21.2,

2

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

47.3, CH 38.8, CH 90.5, CH 90.4, C 31.0, CH2 22.0, CH2 129.5, CH 126.2, C 44.7, CH2 85.6, CH 50.1, CH 145.9, C 35.6, CH2 71.3, CH 83.9, C 25.2, CH3 24.7, CH3 21.3, CH3 18.1, CH3 112.1,CH2 170.1, C 169.8, C 169.7, C 22.8, CH3 22.5, CH3 21.3, CH3

3 44.7, CH 36.0, CH 86.1, CH 85.5, C 23.5, CH2 26.6, CH2 74.8, CH 148.1, C 40.0, CH2 80.9, CH 46.1, CH 139.5, C 121.4, CH 66.5, CH 83.6, C 25.4, CH3 25.4, CH3 23.1, CH3 113.7,CH2 21.8, CH3 170.3, C 170.0, C 170.0, C 22.5, CH3 22.5, CH3 21.3, CH3

4 44.7, 35.6, 86.1, 74.7, 73.3, 37.1, 73.1, 148.1, 40.1, 81.0, 45.6, 139.4, 121.3, 66.7, 84.0, 25.4, 24.9, 22.2, 114.6, 21.7, 172.9, 171.1, 170.3, 22.6, 21.4, 21.3,

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

46.3, CH 36.3, CH 88.0, CH 75.4, C 71.5, CH 33.7, CH2 85.2, CH 143.7, C 40.5, CH2 82.6, CH 46.0, CH 139.7, C 121.4, CH 66.9, CH 83.9, C 25.2, CH3 24.7, CH3 22.3, CH3 116.6,CH2 22.0, CH3 171.7, C 170.2, C 170.2, C 22.5, CH3 21.3, CH3 21.3, CH3

6 45.2, 35.8, 85.3, 86.0, 28.8, 25.5, 84.7, 74.7, 45.8, 77.3, 47.9, 139.0, 122.1, 66.7, 83.5, 25.2, 25.1, 23.8, 23.6, 21.7, 170.2, 170.2, 170.2, 22.5, 22.5, 21.3, 57.5,

7

Table 1. 13C NMR Data for Astrogorgins B−K, M, and N (1−10, 12, and 13) (125 MHz, CDCl3) CH CH CH C CH2 CH2 CH C CH2 CH CH C CH CH C CH3 CH3 CH3 CH3 CH3 C C C CH3 CH3 CH3 CH3

CH CH CH C CH2 CH2 CH C CH2 CH CH C CH CH2 C CH3 CH3 CH3 CH3 CH3 C C 22.8, CH3 22.7, CH3

37.4, 35.7, 87.2, 90.0, 30.1, 22.0, 129.3, 126.0, 44.8, 79.6, 46.5, 58.3, 58.7, 20.5, 85.0, 25.7, 24.9, 21.2, 18.4, 22.5, 169.8, 169.6,

8 CH CH CH Cd CH CH2 CH C CH2 CH CH C CH CH C CH3 CH2 CH3 CH3 CH3 C C 21.4, CH3 21.3, CH3

41.9, 38.4, 88.7, 76.7, 74.5, 29.0, 123.9, 131.2, 44.0, 81.5, 46.7, 139.1, 121.0, 68.4, 58.5, 20.5, 53.3, 22.9, 19.1, 22.1, 170.7, 170.3,

9 46.1, CH 35.9, CH 83.5, CH 85.6, C 78.9, CH 47.0, CH2 105.9, C 154.6, C 41.5, CH2 80.9, CH 48.2, CH 139.5, C 122.0, CH 67.1, CH 83.7, C 25.0, CH3 24.7, CH3 22.1, CH3 114.5,CH2 21.7, CH3 170.4, C 170.2, C 169.9, C 22.6, CH3 21.2, CH3 21.0, CH3

10 CH CH CH C CH CH2 CH C CH2 CH CH C CH CH C CH3 CH3 CH3 CH3 CH3 C C 21.5, CH3 21.3, CH3

44.7, 38.5, 89.4, 76.7, 73.9, 28.8, 123.9, 131.1, 44.5, 80.5, 47.3, 135.9, 123.3, 70.2, 73.5, 30.0, 28.5, 22.5, 19.0, 21.2, 170.5, 170.0,

12

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

21.3, CH3

41.0, 38.9, 90.7, 76.6, 75.6, 29.1, 123.6, 131.2, 42.6, 85.4, 45.1, 145.2, 128.7, 129.7, 32.3, 20.9, 19.4, 23.1, 19.3, 112.7, 170.3,

13

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Table 2. 1H NMR Data for Astrogorgins B−H (1−7) (500 MHz, CDCl3) position

1

2

3

1 2

2.20, brd (7.3) 2.64, brdd (8.4, 10.0)

2.73, brd (2.4) 2.65, dd (8.8, 10.0)

2.83, brd (2.7) 2.59, dd (8.8, 9.7)

3 5

4.63, d (10.0) 5.19, dd (7.5, 10.0)

4.30, d (10.0) 5.15, dd (7.4, 10.2)

6

3.03, ddd (7.5, 8.9, 12.3)

3.02, ddd (8.8, 10.2, 12.0)

4.33, 2.39, 2.06, 2.40,

d (9.7) m m m

2.11, m

7

1.97, ddd (8.9, 10.0, 12.3) 5.46, dd (8.9, 8.9)

1.97, ddd (7.4, 8.8, 12.0) 5.42, dd (8.8, 8.8)

5.42, dd (8.2, 8.7)

9

2.57, dd (5.6, 13.2)

2.52, dd (6.4, 13.6)

2.46, dd (6.1, 13.8)

1.97, dd (0.4, 13.2)

2.02, brd (13.6)

2.04, brd (13.8)

10

4.40, dd (0.4, 5.6)

4.34, brd (6.4)

4.24, br.d (6.1)

11 13

2.45, brd (8.4) 5.61, d (5.2)

14

5.14, brd (5.2)

2.77, brd (8.8) 2.64, dd (5.6, 12.6) 2.37, dd (9.0, 12.6) 5.13, ddd (2.4, 5.6, 9.0)

2.72, brd (8.8) 2.66, dd (6.3, 12.9) 2.37, dd (10.0, 12.9) 5.04, ddd (2.7, 6.3, 10.0)

15 16 17 18 19

1.59, 1.03, 0.99, 1.43, 1.91,

1.54, 1.57, 1.48, 1.88,

s s s brs

1.42, 1.54, 1.84, 1.77,

s s s brs

20

1.79, brs

Ac

2.11, s 1.99, s

4.92, 4.89, 2.09, 2.02, 1.94,

brs brs s s s

4.92, 4.88, 2.02, 2.00, 1.93,

brs brs s s s

m d (6.8) d (6.8) s brs

4

5

6

2.93, brs 2.99, dd (8.0, 9.6) 4.41, d (9.6) 2.40, m 1.81, m 2.38, m

3.08, brs 2.92, brdd (7.8, 10.3) 4.38, d (10.3) 4.92, dd (2.8, 2.8)

2.93, brs 2.98, dd (8.6, 8.8)

3.10, dd (2.8, 14.0)

2.68, ddd (5.0, 6.0, 14.0)

1.89, m

1.76, ddd (2.8, 3.0, 14.0)

4.35, d (8.8) 5.34, brdd (4.0, 5.0)

7 2.96, brs 2.87, dd (6.7, 8.4) 4.40, d (8.4) 2.38, m 2.06, m 2.05, m 1.38, m

4.28, dd (2.4, 5.0) 2.34, dd (4.1, 14.3) 2.28, dd (3.3, 14.3) 4.42, brdd (3.3, 4.1) 2.68, brd (8.0) 5.68, d (5.6)

4.13, brs (3.0, 3.0)

2.13, ddd (2.0, 4.0, 14.0) 4.58, brdd (2.0, 6.0)

2.30, dd (3.0, 14.0)

2.43, dd (4.6, 14.0)

2.32, dd (2.8, 14.0)

2.35, dd (4.6, 14.0)

4.45, brdd (2.8, 3.0)

4.36, dd (4.6, 4.6)

2.67, brd (7.8) 5.68, d (5.3)

2.60, brd (8.6) 5.72, d (5.6)

1.75, dd (3.2, 12.0) 1.70, dd (11.5, 12.0) 4.67, dd (3.2, 11.5) 2.25, brd (6.7) 5.71, d (5.2)

5.42, d (5.6)

5.31, d (5.3)

5.38, d (5.6)

5.50, d (5.2)

1.59, 1.37, 1.72, 5.45, 5.15, 1.81,

1.47, 1.41, 1.39, 5.66, 5.19, 1.81,

1.49, 1.48, 1.35, 5.41, 5.21, 1.81,

s s s brs brs brs

1.63, 1.36, 1.74, 1.13,

2.11, 2.00, 1.99, 8.18,

s s s s

2.06, s 2.01, s 1.98, s

s s s brs brs brs

2.00, s 1.98, s 1.98, s

7-OOH 7-OMe

s s s brs brs br.s

2.15, s 2.01, s 2.01, s

4.04, brd (10.5)

s s s brs

1.82, brs

3.37, s

(δH 4.04, brd) resulted in the enhancement of H-10 (δH 4.67, 0.59%) and H3-18 (δH 1.74, 1.41%), while irradiation of H3-19 led to the enhancements of H-11 (δH 2.25, 0.23%) and OMe (0.62%). These findings led to the assignment of H-7α and H319β. The 2D NMR spectroscopic data of astrogorgin I (8) were mostly compatible with those of calicophirin A (16),28 characteristic of a 12,13-epoxy group. The distinction was recognized by the presence of two acetoxy groups in the NMR spectra instead of three. In the COSY spectrum, the epoxy proton H-13 (δH 3.04) correlated to the methylene protons H214 (δH 2.02, 2.12), indicating 8 to be a 14-deacetoxy analogue of calicophirin A. This assignment was also supported by the molecular weight of 8 showing 58 amu less than that of calicophirin A. The similar NOE interactions of 8 and calicophirin A led to the assignment of 8 as 14-deacetoxycalicophirin A. The NMR spectroscopic data (Tables 2 and 3) of astrogorgin J (9) were almost identical to those of muricellin,10 except for the substituent at C-1. The NMR resonances for the epoxide carbons C-15 (δC 58.5, s) and C-16 (δC 53.3, t) in addition to the HMBC correlations from H3-17 (δH 1.30, s) to C-1 (δC 41.9, d), C-15, and C-16 revealed an epoxyisopropyl group at C-1. Apart from C-15, the relative configurations of 9

atom more than that of 5. The NMR data of 6 (Tables 1 and 2) were mostly identical to those of 5, with the exception that the resonances of C-7 shifted more downfield (δC 85.2, δH 4.58) than the hydroxyl-substituted C-7 (δC 74.8) of 4. This finding suggested C-7 of 6 was substituted by a hydroperoxide group. The fact that a downfield exchangeable proton (δH 8.18, s)27 showed an HMBC correlation to C-7, and its molecular formula presented 10 oxygen atoms, further supported this assignment. A comparison of NMR data revealed the A and B rings of astrogorgin H (7) to be the same as that of astrogorgin. Analysis of the NMR data of ring C disclosed the presence of a methyl singlet (δH 1.13, s, H3-19), a methoxy group (δH 3.37, δC 57.5), and the absence of an exomethylene group. The HMBC interactions from H3-19 to a methylene carbon (δC 45.8, C-9), a hydroxylated quaternary carbon (δC 74.7, C-8), and an oxymethine (δC 84.7, C-7), in association with the correlation between the methoxy protons and C-7, indicated a methoxy group was linked to C-7, while C-8 bears a methyl group and a hydroxy group. In addition, the chemical shift for C-4 (δC 86.0) is 10 ppm further downfield than a C-4 carbon with a hydroxy substituent such as in 5 and 6 and is closely similar to that of 4, indicating C-4 of 7 to be substituted by an acetoxy group. In 1D GOESY experiments, irradiation of H-7 1598

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Table 3. 1H NMR Data for Astrogorgins I−N (8−13) (500 MHz, CDCl3) position 1 2 3 5 6 7 9 10 11 13 14

8 2.49, 2.39, 3.99, 2.43, 1.89, 2.12, 2.40, 5.43, 2.54, 1.98, 4.39, 2.20, 3.04, 2.02, 2.12,

brd (7.4) dd (8.5, 10.0) d (10.0) m m m m dd (8.6, 9.2) dd (6.0, 13.2) brd (13.2) brd (6.0) brd (8.5) d (5.3) m m

9

11

dd (2.8, 2.8) ddd (2.8, 7.7, 8.4) d (8.4) dd (7.0, 10.0)

2.63, 3.09, 4.54, 5.10,

2.94, 2.01, 5.48, 2.54, 2.02, 4.34, 2.48, 5.60, 5.28,

ddd (7.6, 10.0, 12.3) ddd (7.0, 10.8, 12.3) dd (7.6, 10.8) dd (4.4, 14.0) dd (2.8, 14.0) dd (2.8, 4.4) brd (7.7) brd (4.5) dd (2.8, 4.5)

2.78, dd (10.0, 14.0) 2.80, dd (8.0, 14.0)

brd (4.6) brd (4.6) s s s

15 16

1.51, s

17 18 19

1.47, s 1.80, s 1.76, s

2.69, 2.55, 1.30, 1.43, 1.91,

20

1.33, s

1.79, s

Ac

2.00, s 1.91, s

2.11, s 2.02, s

7-OH a

10

2.32, 2.65, 4.42, 5.07,

2.83, 2.36, 4.36, 2.38, 5.74, 5.50,

brs dd (7.0, 7.6) d (7.6) dd (8.0, 10.0)

dd (9.6, 12.8) dd (6.8, 12.8) dd (6.8, 9.6) brd (7.0) d (5.6) d (5.6)

1.39, s 1.64, 1.40, 5.27, 5.09, 1.81,

s s brs brs s

2.07, s 2.00, s 2.00, s 6.27 sa

12

13

2.12, 2.61, 4.65, 5.22,

m dd (6.0, 10.0) d (10.0) dd (6.0, 8.0)

2.70, 2.94, 4.11, 5.27,

dd (1.8, 6.6) dd (7.7, 9.8) d (9.8) dd (7.3, 10.4)

2.41, 2.38, 3.83, 5.09,

m m d (8.0) dd (7.0, 9.5)

1.90, 3.02, 5.49, 1.96, 2.55, 4.39, 2.41, 5.65, 4.09,

m ddd (8.0, 8.0, 12.0) dd (6.4, 8.0) d (13.0) dd (6.0, 13.0) brd (6.0) brd (6.0) d (4.5) brd (4.5)

2.89, 2.03, 5.43, 2.53, 1.97, 4.22, 2.46, 5.41, 5.71,

m m brd (8.0) dd (5.9, 13.2) d (13.2) brd (5.9) brd (7.7) d (2.4) dd (2.4, 6.6)

2.97, 2.03, 5.52, 2.55, 2.10, 4.30, 2.82, 6.22, 5.72,

m m dd (5.0, 8.0) dd (6.0, 13.0) d (13.0) brd (6.0) m d (10.1) dd (2.0, 10.1)

1.60, m 1.01, d (7.0)

1.26, s

1.70, m 0.97, d (6.8)

0.97, d (7.0) 1.44, s 1.90, s

1.28, s 1.45, s 1.91, s

0.89, d (6.8) 1.42, s 1.92, s

1.76, s

1.72, s

2.12, s

2.10, s 2.08, s

5.01, brs 4.94, brs 2.12, s

Measured in DMSO-d6.

Figure 2. Key NOE interactions of 9.

were determined to be the same as that of muricellin on the basis of the NOE data and NMR resonances. The stereogenic center C-15 presents two possible configurations, representing “15S*” (9a) and “15R*” (9b). The NOE relationship was obseved from H3-17 to H-2 (δH 2.65, ddd, J = 2.8, 7.7, 8.4 Hz), H-14 (δH 5.28, dd, J = 2.8, 4.5 Hz), and H-16a (δH 2.55) and between H-16b (δH 2.69) and H-1, whereas the NOE interactions between H3-17/H-1 and H-16a/H-14 were absent (Figure 2). These findings suggest the 15S* configuration. Astrogorgin K (10) has a molecular formula of C26H36O9 as determined by HRESIMS and NMR data, requiring nine degrees of unsaturation. A comparison of NMR spectroscopic data disclosed the structure of 10 to be closely related to 6, except for different resonances found in ring C. The downfieldshifted C-7 (δC 105.9) was assigned to a hemiacetal quaternary carbon, which was confirmed by a D2O-exchanged proton at δH

6.26 (1H, s) showing HMBC correlations to C-6 (δC 47.0), C7, and C-8 (δC 154.6). The oxymethine H-5 (δH 5.10, dd, J = 8.0, 10.0 Hz) showed a COSY correlation with H2-6 and an HMBC interaction with an acetyl carbon, positioning an acetoxy group at C-5. The typical downfield-shifted C-4 (δC 85.6) in association with the molecular unsaturation led to the formation of an ether bridge across C-4 and C-7. The NOE correlations from H-2 to H-14 and H-11 and between H-1/H-3 and H-11/H-9a indicated rings A, B, and C were fused in the same configurations as that of 6. Additional NOE interactions from H3-18 to H-2 and H-5 and between H-5 and OH-7 (δH 6.27, s) assigned a β-orientation for H-5, while H3-18 and OH7 were also β-oriented (Figure 1). Astrogorgin L (11) has a molecular formula of C22H34O5 as determined by HRESIMS and NMR data, with 42 amu less than that of 1. The 1H NMR spectrum of 11 presented only 1599

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Figure 3. Conversion of 11 to 13 in CDCl3.

EC50) commonly used to assess the effectiveness of a compound in relation to its toxicity should be larger than 15 to select a nontoxic antifouling candidate.30 The large therapeutic ratio of 19 (>42) suggests that it might be useful as an environmentally benign antifouling agent. This is the second report for the antifouling activities of eunicellin analogues.

one acetyl group (δH 2.11, s), while the other resonances were comparable with those of 1, indicating 11 to be a deacetyl analogue. The upfield-shifted H-14/C-14 (δH 4.09, δC 67.1) in comparison with the corresponding signals of 1 indicated C-14 of 11 to be substituted with a hydroxy group. The similar NOE relationships between 11 and 1 confirmed its structure as the 14-deacetyl analogue of 1. It is noted that 11 is an unstable compound that converts readily to astrogorgin N (13) in CDCl3. Analysis of the NMR data of 13 revealed exomethylene protons at δH 4.94 (1H, brs, H-20a) and 5.01 (1H, brs, H-20b) and two vicinal olefinic protons at δH 6.22 (1H, d, J = 10.1 Hz, H-13) and 5.72 (1H, dd, J = 2.0, 10.1 Hz, H-14) in ring A. The olefinic positions were further supported by COSY and HMBC analyses. Because 11 is stable under the sodium-dried CDCl3, 13 is assumed as an artifact derived from 11 via acid-mediated dehydration of an allylic alcohol (Figure 3). Astrogorgin M (12) was isolated as a minor component. Analysis of 1D and 2D NMR spectroscopic data indicated that its structure contains two acetyl groups and is closely related to muricellin. Acetoxy groups at C-5 and C-14 were confirmed by HMBC correlations from H-5 and H-14 to the respective acetyl carbonyl carbon. Thus, the hydroxylated carbons were assigned to C-4 (δC 76.7) and C-15 (δC 73.5), and these assignments were supported by their 13C NMR shifts, which were upfield in comparison with the corresponding carbons linked to an acetoxy group. In contrast to the NOE data of muricellin, the observation of NOE relationships from H-14 to H-1 and H-3 led to the assignment of H-14α. In order to detect whether these compounds a play a chemoecological role in the host, some compounds were tested for antifouling activity. Compounds 1−3, 13, 15, 16, and 19 exhibited antifouling activity against the larval settlement of the barnacle Balanus amphitrite with EC50 values (Table 4) lower than the standard requirement of 25 μg/mL.29 Among them, 14-deacetoxycalicophirin B (19) was the most potent, with an EC50 = 0.59 μg/mL, whereas the other compounds exhibited moderate or weak inhibition. The therapeutic ratio (LC50/

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Table 4. Antifouling Activity of Compounds

a

compound

EC50 (μg/mL)

1 2 3 13 14 15 16 17 19

5.77 5.14 8.23 10.7 >25 8.73 17.8 >25 0.59

LC50 . >50 >50 >50 >50 UDa >50 >50 UD >25

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol III automatic polarimeter. IR spectra were determined on a Thermo Nicolet Nexus 470 FT-IR spectrometer. 1H, 13C, and 2D NMR spectra were recorded on Bruker Avance 400 NMR and Bruker Avance 500 NMR spectrometers (400/500 MHz for 1H and 100/125 MHz for 13C, respectively). Chemical shifts are expressed in δ (ppm) referenced to the solvent peaks at δH 7.26 and δC 77.0 for CDCl3 and at δH 7.22 and δC 123.9 for C5D5N, respectively, and coupling constants are in Hz. ESIMS and HRESIMS spectra were obtained from a Thermo Scientific LTQ Orbitrap XL instrument. Silica gel (160−200 and 200−300 mesh, Qingdao Marine Chemistry Co. Ltd.) and ODS (50 μm, YMC) were used for column chromatography. Precoated silica gel plates (Merck, Kieselgel 60 F254, 0.25 mm) were used for TLC analyses. HPLC chromatography was performed on an Alltech instrument (426-HPLC pump) equipped with an Alltech uvis-200 detector at 210 nm and semipreparative reversed-phased columns (YMC-packed C8, 5 μm, 250 mm × 10 mm). Animal Material. The gorgonian Astrogorgia sp. was collected from the inner coral reef at a depth of 10 m in Beibuwan Bay, GuangXi Province of China, in August 2010. The fresh samples were frozen immediately. The specimen was identified by L.v.O. The gorgonian specimen (GWB-32) was deposited at the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, China, and at the National Museum of Natural History Naturalis, The Netherlands (RMNH Coel. 40067). Taxonomic Description. Astrogorgia sp. (RMNH Coel. 40067) is a colony branched in one plane. Polyps were retracted in calyces, arranged all around the branches, closely set to each other. Calyces were about 1 mm high. Polyps with eight points were made up of spindles, up to 0.25 mm long, with simple tubercles. Tentacle sclerites are similar to those of the points but slightly shorter, more flattened, and less tuberculate. The surface of coenenchyme has spindles, up to 0.45 mm long, with simple or complex tubercles. The interior of the coenenchyme has capstans and rods, up to 0.10 mm long, with simple tubercles. The color of the colony is red. Tentacle sclerites are colorless, point sclerites yellow, interior coenenchymal sclerites colorless to pink, and surface coenenchymal sclerites red. Extraction and Isolation. The frozen gorgonian Astrogorgia sp. (wet weight 1.26 kg) was homogenized and then extracted with MeOH. The concentrated extract was desalted by redissolving in MeOH to yield a residue (49.7 g). This residue was partitioned between H2O and EtOAc, and the EtOAc layer was concentrated under vacuum to yield a residue of 12.8 g. The EtOAc fraction (11.5 g) was subjected to silica gel vacuum liquid chromatography, eluting with acetone−petroleum ether (from 0:1 to 1:0, gradient), to obtain 14 fractions (F1−F14). F4 (580 mg) was chromatographed over a Sephadex LH-20 column, using CHCl3−MeOH (1:1) as a mobile phase, to afford three portions (P1−P3). P2 (298 mg) was separated

>8.7 >9.7 >6.1 >4.7 >5.7 >2.8 >42

UD: undetected. 1600

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C 22 H 34 O 5 Na, 401.2304), 779.4703 [2M + Na] + (calcd for C44H68O10Na, 779.4710). Astrogorgin M (12): colorless oil; [α]25D −62 (c 0.08, CHCl3); IR (KBr) νmax 3448, 2970, 2933, 1733, 1627, 1445, 1372, 1246, 1140, 1076, 1023 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 459.2344 [M + Na]+ (calcd for C24H36O7Na, 459.2359), 895.4802 [2M + Na]+ (calcd for C48H72O14Na, 895.4820). Astrogorgin N (13): colorless oil; [α]24D −32.0 (c 0.019, MeOH); UV (MeOH) λmax 222, 235 nm; IR (KBr) νmax 3420, 2962, 2935, 2885, 1713, 1623, 1450, 1378, 1244, 1123, 1045, 1003 cm−1; 1H and 13 C NMR data, see Tables 1 and 3; HRESIMS m/z 361.2372 [M + H]+ (calcd for C22H33O4, 361.2379), 383.2190 [M + Na]+ (calcd for C22H32O4Na, 383.2198). Ophirin (14): needle crystal; mp 94−96 °C; [α]24D −127 (c 0.88, CHCl3) (lit.12 [α]25D −120 (c 1.0, CHCl3); lit.8b [α]25D −90.5 (c 1.2, CHCl3)). Antifouling Bioassay. The antilarval-attachment activity was determined using cyprids of the barnacle Balanus amphitrite Darwin. Adults of B. amphitrite exposed to air for more than 6 h were collected from the intertidal zone in Hong Kong (22°19′ N, 114°16′ E) and then were placed in a container filled with 0.22 μm filtered seawater (FSW) to release nauplii. The collected nauplii were reared to cyprid stage according to the method described by Thiyagarajan et al.31 When kept at 26−28 °C and fed with Chaetoceros gracilis, larvae developed to cyprids within four days. Fresh cyprids were used in the tests. Larval settlement assays were performed using 24-well polystyrene plates. Test compounds 1−3, 13, 15, 16, and 19 were dissolved in a small amount of dimethyl sulfoxide (DMSO) and then diluted with FSW to achieve final concentrations of 25 and 5 μg/mL for primary tests of their antifouling effects, and then the active ones were diluted to 25.0, 10.0, 5.0, and 1.0 μg/mL for further testing. About 15−20 competent larvae were added to each well with 1 mL of test solution in three replicates, and wells containing larvae in FSW with DMSO only served as a control. The plates were incubated for 48 h at 23 °C. The effects of the test samples against biofouling were determined by examining the plates under a dissecting microscope to check for (1) attached larvae, (2) unattached larvae, and (3) any possible toxic effects of the treatments, such as death or paralysis of larvae, which were also recorded. The percentage of larval settlement was determined by counting the number of attached individuals and expressed as a proportion of the total number of larvae added into each well. A concentration−response curve was then plotted, and a trend line was constructed for each compound. The EC50 was calculated as the concentration where 50% of the larval population had settling inhibited as compared to the control, while the LC50 was calculated as the concentration where 50% of the larval population was dead.

by semipreparative-HPLC eluting with MeOH−H2O (77:23) to afford ophirin (14, 201.7 mg), 3 (7.2 mg), and calicophirin B (18, 10.4 mg), while 14-deacetoxycalicophirin B (19, 52.1 mg) was prepared in the same manner using MeOH−H2O (83:17) from P3 (78.6 mg). F5 (89 mg) was subsequently subjected to an ODS column eluting with MeOH−H2O (from 80:20 to 100:0, gradient) to yield 8 (1.9 mg) and astrogorgin (16, 8.2 mg). Following the same protocol as for F5, F6 (78 mg) was separated on the same column with MeOH−H2O (71:29) as eluent to obtain 1 (5.6 mg), 12 (1.0 mg), 6 (2.1 mg), and 5 (2.1 mg). F7 (89 mg) was separated on a Sephadex LH-20 column eluting with MeOH−H2O (70−91% MeOH, gradient) to afford 7 (5.0 mg), 10 (0.5 mg), calicophirin A (17, 8.5 mg), 2 (5.3 mg), and muricellin (15, 46.9 mg). F8 (110 mg) was subsequently purified by semipreparative-HPLC with MeOH−H2O (78:22) as a mobile phase to yield 4 (1.8 mg), 9 (1.3 mg), and 11 (1.8 mg). Astrogorgin B (1): colorless oil; [α]25D −137 (c 0.28, CHCl3); IR (KBr) νmax 3449, 2959, 2926, 2859, 1735, 1612, 1458, 1373, 1241, 1076, 1018 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 443.2395 [M + Na]+ (calcd for C24H36O6Na, 443.2410), 863.4908 [2M + Na]+ (calcd for C48H72O12Na, 863.4921). Astrogorgin C (2): colorless oil; [α]25D −92.3 (c 0.27, CHCl3); IR (KBr) νmax 3449, 2929, 2869, 1737, 1621, 1454, 1371, 1246, 1137, 1077, 1050, 1020 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 501.2446 [M + Na]+ (calcd for C26H38O8Na, 501.2464), 979.5016 [2M + Na]+ (calcd for C52H76O16Na, 979.5031). Astrogorgin D (3): colorless oil; [α]25D −25.7 (c 0.36, CHCl3); IR (KBr) νmax 3442, 2929, 2864, 1736, 1650, 1625, 1541, 1457, 1371, 1252, 1135, 1078, 1021 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 485.2499 [M + Na]+ (calcd for C26H38O7Na, 485.2515), 947.5125 [2M + Na]+ (calcd for C52H76O14Na, 947.5133). Astrogorgin E (4): colorless oil; [α]24D −129 (c 0.11, CHCl3); IR (KBr) νmax 3425, 2929, 2854, 1733, 1629, 1441, 1371, 1254, 1131, 1078, 1019 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 501.2444 [M + Na]+ (calcd for C26H38O8Na, 501.2464), 979.5015 [2M + Na]+ (calcd for C52H76O16Na, 979.5031). Astrogorgin F (5): colorless oil; [α]24D −54.2 (c 0.13, CHCl3); IR (KBr) νmax 3434, 2925, 2856, 1736, 1602, 1459, 1376, 1259, 1130, 1072 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 517.2402 [M + Na]+ (calcd for C26H38O9Na, 517.2414), 1011.4927 [2M + Na]+ (calcd for C52H76O18Na, 1011.4929). Astrogorgin G (6): colorless oil; [α]24D −26.4 (c 0.125, MeOH); IR (KBr) νmax 3740, 3445, 2929, 2874, 1735, 1629, 1449, 1372, 1252, 1131, 1075, 1020 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 533.2338 [M + Na]+ (calcd for C26H38O10Na, 533.2363). Astrogorgin H (7): colorless oil; [α]29D −55.9 (c 0.23, CHCl3); IR (KBr) νmax 3431, 2929, 2866, 1734, 1651, 1621, 1459, 1370, 1254, 1134, 1079,1019 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 533.2709 [M + Na]+ (calcd for C27H42O9Na, 533.2727), 1043.5544 [2M + Na]+ (calcd for C54H84O18Na, 1043.5555). Astrogorgin I (8): colorless oil; [α]24D −12.3 (c 0.12, CHCl3); IR (KBr) νmax 3422, 2926, 2859, 1732, 1629, 1459, 1372, 1249, 1128, 1075, 1019 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 443.2387 [M + Na]+ (calcd for C24H36O6Na, 443.2410), 863.4893 [2M + Na]+ (calcd for C48H72O12Na, 863.4921). Astrogorgin J (9): colorless oil; [α]24D −96.0 (c 0.03, MeOH); IR (KBr) νmax 3419, 2933, 2864, 1732, 1622, 1443, 1373, 1244, 1124, 1050 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 457.2191 [M + Na]+ (calcd for C24H34O7Na, 457.2202), 891.4497 [2M + Na]+ (calcd for C48H68O14Na, 891.4507). Astrogorgin K (10): colorless oil; [α]24D −28.8 (c 0.06, MeOH); IR (KBr) νmax 3433, 2929, 2858, 1737, 1659, 1600, 1442, 1373, 1251, 1129, 1079, 1019 cm−1; 1H and 13C NMR data, see Tables 1 and 3; HRESIMS m/z 515.2230 [M + Na]+ (calcd for C26H36O9Na, 515.2257), 1007.4578 [2M + Na]+ (calcd for C52H72O18Na, 1007.4616). Astrogorgin L (11): colorless oil; 1H NMR (CDCl3,500 MHz) data, see Table 3; HRESIMS m/z 401.2298 [M + Na]+ (calcd for

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ASSOCIATED CONTENT

S Supporting Information *

MS, 1D NMR, and 2D NMR spectra for compounds 1−13 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-82806188. Fax: +86-10-82806188. E-mail: [email protected]. Notes

The authors declare no competing financial interest

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ACKNOWLEDGMENTS This work was supported by grants from the NSFC (No.30930109), National Key Innovation Project (2009ZX09501-014, 2011AA090701), and ICP (2010DFA31610). We would like to acknowledge Prof. P.-Y. Qian and his co-workers from Hong Kong University of Science and Technology for the antifouling bioassay. 1601

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REFERENCES

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NOTE ADDED AFTER ASAP PUBLICATION This paper was published on the Web on August 20, 2012. The Supporting Information has been updated and the corrected version was reposted on August 28, 2012. 1602

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