Dactylomelane Diterpenes from the Sea Hare Aplysia depilans

Article pubs.acs.org/jnp

Dactylomelane Diterpenes from the Sea Hare Aplysia depilans Anastasia Petraki,† Efstathia Ioannou,† Panagiota Papazafiri,‡ and Vassilios Roussis*,† †

Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, School of Health Sciences, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece ‡ Department of Animal and Human Physiology, Faculty of Biology, School of Science, University of Athens, Panepistimiopolis Zografou, Athens 15784, Greece S Supporting Information *

ABSTRACT: A chemical investigation of the organic extract of the sea hare Aplysia depilans, collected off Skyros Island, Greece, yielded eight new brominated diterpenes (1−8), featuring the rare dactylomelane skeleton, together with the previously reported luzodiol (9). The structure elucidation and the assignment of the relative configurations of the new natural products were based on extensive NMR spectroscopic and MS spectrometric analyses. Compounds 1−9 were evaluated for their cytotoxic activities against five human tumor cell lines, but were proven inactive.

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hell-less mollusks of the genus Aplysia (Anaspidea, Aplysiidae) have been proven to be a rich source of secondary metabolites, mostly of dietary origin. These sea hares preferentially feed on red algae of the genus Laurencia (Ceramiales, Rhodomelaceae), and therefore their metabolites are often related to the chemistry of Laurencia species. The majority of the compounds isolated from sea hares of the genus Aplysia are halogenated sesquiterpenes and diterpenes, often exhibiting antibacterial, antiviral, cytotoxic, and/or antifeedant activity.1,2 Aplysia depilans Gmelin occurs in the eastern Atlantic Ocean and the Mediterranean Sea. It is usually found in shallow waters at a depth of 1.5 to 10 m. So far, there are only two previous studies focusing on the chemistry of A. depilans. From specimens collected at La Coruña (Spain) two 5,8-epidioxysterols have been isolated and characterized,3 while from specimens collected from Asturias and Cadiz, as well as from the Bay of Naples, aplyolides A−E, 16- and 18-membered fatty acid lactones, were obtained.4 In this report, we describe the isolation and structure elucidation of nine brominated diterpenes featuring the rare dactylomelane skeleton from the organic extract of A. depilans and the evaluation of their cytotoxic activities against five human tumor cell lines. Compounds 1−8 are new natural products, whereas metabolite 9 was identified as luzodiol by comparison of its spectroscopic and physical characteristics with those reported in the literature.5

extract was subjected to a series of chromatographic separations leading to the isolation of compounds 1−9. Compound 1, isolated as a colorless oil, had the molecular formula C20H34BrClO2, as deduced from the HRESIMS and NMR data. The mass spectrum exhibited adduct ion peaks [M + NH4]+ at m/z 438.1768, 440.1745, and 442.1715 with a relative intensity of 3:4:1, characteristic for the presence of one bromine and one chlorine atom. The fragment ions [M − H2O + H]+ at m/z 403.1396 and [M − 2H2O + H]+ at m/z 385.1291 indicated the presence of two hydroxy groups in the molecule, a hypothesis that was further confirmed by the

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RESULTS AND DISCUSSION Specimens of the opisthobranch mollusk A. depilans were collected off Skyros Island in the North Sporades complex of the Aegean Sea, Greece. The animals were exhaustively extracted with mixtures of CH2Cl2/MeOH, and the organic © XXXX American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of William Fenical Received: October 28, 2014

A

DOI: 10.1021/np500851w J. Nat. Prod. XXXX, XXX, XXX−XXX

B

1.32, m 3.42, dd (9.6, 1.7)

b

19 20 OMe

1.25, 4.92, 4.64, 0.82, 1.56,

17 18

s brs brs s s

1.58, s

a b a

1.99, m 2.05, m 2.24, m 4.25, dd (11.4, 4.3) 2.07, m 1.48, m 1.57, m

1.82, m 2.32, m

β α β

α

a b

a b a b

b

a b a

β α β

α

1.27, 4.93, 4.65, 0.81, 1.35,

s brs brs s s

1.37, s

1.52, m 3.93, d (10.6)

1.95, m 2.05, m 2.20, m 4.21, dd (11.2, 3.9) 1.89, m 1.67, m 1.93, m

5.90, dd (17.3, 10.7) 1.72, m 1.25, m 1.60, m 1.46, m 1.80, m 2.29, m

5.04, d (10.7)

5.04, d (17.2)

5.86, dd (17.2, 10.5) 2.07, m 1.48, m 1.56, m

5.20, d (17.3)

2 (J in Hz)

5.19, d (10.5)

1 (J in Hz)

16

15

14

13

12

10

9

6 8

5

4

2

1

position

b

a b a

β α β

α

a b a b

1.26, 4.92, 4.65, 0.81, 1.30,

s brs brs s s

1.32, s

1.39, m 3.75, brd (10.7)

1.95, m 2.05, m 2.20, m 4.21, dd (11.3, 4.0) 1.87, m 1.67, m 1.87, m

5.20, d (17.3, 1.2) 5.04, dd (10.7, 1.2) 5.89, dd (17.3, 10.7) 1.73, m 1.23, m 1.58, m 1.47, m 1.80, m 2.27, m

3 (J in Hz)

Table 1. 1H NMR Data (400 MHz, CDCl3) of Compounds 1−8

b

a b a

β α β

α

a b

a b

1.25, 4.92, 4.64, 0.82, 1.18,

s brs brs s s

1.21, s

1.26, m 3.29, brd (9.1)

2.02, m 1.47, m 1.41, m

1.84, m 2.31, ddd (11.8, 3.2, 3.2) 1.97, m 2.04, m 2.21, m 4.27, dd (11.2, 4.0)

5.86, dd (17.3, 10.7) 1.74, m 1.16, m 1.52, m

a b

β α β

α

1.25, 4.91, 4.63, 0.82, 1.11, 3.21,

s brs brs s s s

1.12, s

3.34, dd (8.9, 2.7)

1.98, m 2.05, m 2.21, m 4.26, dd (11.3, 4.2) 1.98, m 1.46, m 1.29, m

1.85, m 2.30, m

5.19, dd (17.3, 1.1) 5.03, dd (10.7, 1.1) 5.87, dd (17.3, 10.7) 1.74, m 1.18, m 1.52, m

5.19, d (17.3) 5.04, d (10.7)

5 (J in Hz)

4 (J in Hz)

b

a b a

β α β

α

a b a b

1.25, 4.92, 4.65, 0.82, 1.27,

s brs brs s s

1.30, s

1.25, m 2.72, dd (7.3, 5.6)

1.98, m 2.04, m 2.25, m 4.29, dd (9.4, 4.4) 1.85, m 1.48, m 1.66, m

5.85, dd (17.2, 10.5) 1.73, m 1.21, m 1.52, m 1.30, m 1.81, m 2.30, m

5.03, d (10.5)

5.18, d (17.2)

6 (J in Hz)

b

a b a

β α β

α

a b

m m m dd (11.1, 4.1)

1.25, 4.93, 4.65, 0.83, 1.09,

s brs brs s d (6.7)

2.63, dddd (13.7, 13.7, 6.7, 6.7) 1.09, d (6.7)

1.90, m 1.77, m 2.47, ddd (15.8, 12.3, 5.2) 2.24, m

1.99, 2.05, 2.22, 4.18,

1.79, m 2.34, m

1.74, m 1.21, m 1.53, m

5.87, dd (18.1, 10.8)

5.03, dd (10.8, 0.9)

5.18, dd (18.1, 0.9)

7 (J in Hz)

b

a b a

β α β

α

a b

4.95, 4.86, 1.25, 4.91, 4.63, 0.79, 1.72,

brs brs s brs brs s s

1.43, m 4.08, dd (5.7, 5.7)

1.62, m 1.38, m 1.56, m

1.87, d (10.2) 2.29, ddd (12.0, 3.8, 3.8) 1.95, m 2.04, m 2.21, m 4.27, dd (11.4, 4.1)

5.88, dd (17.3, 10.6) 1.71, m 1.22, m 1.52, m

5.03, d (10.6)

5.20, d (17.3)

8 (J in Hz)

Journal of Natural Products Article


Journal of Natural Products

Article

Table 2. 13C NMR Data (50 MHz, CDCl3) of Compounds 1−8 position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe

1 111.9, 145.1, 73.2, 41.4, 19.9, 48.6, 145.3, 37.5, 35.5, 62.4, 43.9, 35.3, 24.5, 79.5, 75.4, 28.7, 27.8, 109.5, 17.3, 28.1,

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

112.0, 144.9, 73.3, 41.3, 20.0, 48.0, 145.0, 37.7, 35.3, 62.9, 43.9, 37.6, 27.0, 71.7, 72.6, 26.9, 27.8, 109.8, 17.1, 26.1,


112.0, 144.9, 73.3, 41.4, 19.9, 48.1, 145.1, 37.7, 35.3, 62.8, 43.9, 36.5, 26.9, 74.8, 72.9, 26.6, 27.8, 109.8, 17.1, 25.5,


111.8, 145.4, 73.3, 41.4, 19.8, 48.6, 145.0, 37.7, 35.3, 62.6, 44.0, 36.0, 24.4, 79.0, 72.7, 27.7, 27.8, 109.5, 17.1, 24.1,


absorption band at 3448 cm−1 observed in the IR spectrum. Moreover, the fragment ion [M − H2O − HBr + H]+ at m/z 323.2136 verified the presence of one bromine atom in the molecule, whereas the fragment ion [M − H2O − HCl + H]+ at m/z 367.1627 supported the presence of one chlorine atom. The 13C NMR spectrum revealed the presence of 20 carbon atoms, which according to the HSQC-DEPT experiment, corresponded to four nonprotonated, four methine, eight methylene, and four methyl carbons. The chemical shifts of four carbons resonating at δC 109.5, 111.9, 145.1, and 145.3 implied the presence of two double bonds, whereas four carbons resonating at δC 62.4, 73.2, 75.4, and 79.5 were attributed to carbon atoms directly bonded to halogen or oxygen atoms. The 1H NMR spectrum showed signals for four singlet methyls at δH 0.82, 1.25, 1.56, and 1.58, one terminal methylene group at δH 4.64 and 4.92, one monosubstituted double bond at δH 5.04, 5.19, and 5.86, and two deshielded methines at δH 3.42 and 4.25. Analysis of the 2D NMR spectra of compound 1 allowed for the assignment of the 1H and 13C chemical shifts (Tables 1 and 2) and led to the structure determination of the metabolite. In particular, the COSY crosspeaks of H2-8/H2-9 and H2-9/H-10, in combination with the correlations of H2-4, H2-18, and H3-19 with C-6, of H2-5, H-6, H2-8, and H2-18 with C-7, and of H-6, H-10, H2-12, and H3-19 with C-11 evident in the HMBC spectrum established the C6−C-11 ring closure of the monocyclic dactylomelane skeleton. The COSY correlations of H2-1/H-2, H2-4/H2-5, and H2-5/H6, as well as the HMBC correlations of H2-1, H-2, H2-4, and H3-17 with C-3, connected the first side chain at C-6 of the cyclohexane ring. Furthermore, the COSY cross-peaks of H212/H2-13 and H2-13/H-14, in conjunction with the HMBC correlations of H-14, H3-16, and H3-20 with C-15, completed the second side chain attached at C-11. The bromine atom was placed at C-10 on the basis of the chemical shift of the latter, resonating at δC 62.4, and the high degree of similarity to those of luzodiol and laurendecumtriol (δ C 63.3 and 63.9, respectively).5,6 The chlorine atom was placed at C-15 due to the chemical shifts of H3-16 and H3-20, resonating at δH 1.58

111.8, 145.2, 73.2, 41.5, 19.9, 48.6, 145.5, 37.4, 35.4, 62.5, 43.9, 35.8, 23.9, 77.4, 77.6, 20.6, 27.6, 109.4, 17.4, 19.6, 49.1,

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

111.8, 145.0, 73.1, 41.1, 20.0, 48.1, 145.2, 37.5, 35.4, 62.2, 43.7, 34.4, 22.2, 64.1, 58.4, 24.8, 27.9, 109.6, 17.2, 18.7,


111.9, 145.1, 73.2, 41.2, 19.9, 48.5, 145.1, 37.7, 35.3, 62.3, 43.7, 32.5, 33.4, 214.5, 41.0, 18.4, 27.7, 109.9, 17.1, 18.2,

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

111.8, 145.2, 73.3, 41.2, 19.7, 48.3, 145.4, 37.7, 35.4, 62.7, 43.7, 33.2, 27.0, 75.7, 146.6, 111.2, 27.7, 109.4, 17.2, 17.8,

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

and 1.56, respectively, thus leaving the hydroxy group at C-14. The relative configurations of the stereogenic centers of 1 were proposed on the basis of the observed enhancements in the NOESY spectrum. The depicted relative configuration of 1 constitutes the only possible stereostructure that could justify the critical NOE enhancements of H-6/H-10, H-9α/H3-19, and H-9β/H-10. The relative configurations at C-3 and C-14 could not be established on the basis of NMR data. Compound 2, isolated as a colorless oil, displayed in the HRESIMS spectrum adduct ion peaks at m/z 487.0813, 489.0789, and 491.0768 with a relative intensity of 1:2:1, characteristic for the presence of two bromine atoms, consistent with [M + Na]+ and corresponding to the molecular formula C20H34Br2O2. Moreover, the absorption band at 3411 cm−1 observed in the IR spectrum was indicative of the presence of hydroxy groups. The signals of four singlet methyls at δH 0.81, 1.27, 1.35, and 1.37, one exomethylene group at δH 4.65 and 4.93, one monosubstituted double bond at δH 5.04, 5.20, and 5.90, and two deshielded methines at δH 3.93 and 4.21 observed in the 1H NMR spectrum suggested that metabolite 2 possessed the dactylomelane skeleton, as in the case of 1. This assumption was further supported by the correlations observed in the COSY and HMBC spectra. In comparison to compound 1, the main differences were the chemical shifts of H-14, H3-16, and H3-20, as well as of C-14 and C-15. In particular, the fact that H3-16 and H3-20 were shielded in 2 (δH 1.37 and 1.35 in contrast to 1.58 and 1.56 in 1) indicated that the hydroxy group was positioned at C-15 (δC 72.6), leaving the second bromine atom attached at C-14 (δC 71.7). The relative configurations of the stereogenic centers of 2 were retained as in 1, as deduced from the enhancements observed in the NOESY spectrum. Compound 3 was isolated as a colorless oil. A combination of its HRESIMS and 13C NMR data suggested the same molecular formula as in 2, and interpretation of its NMR spectroscopic data indicated that it was an isomer of the latter. Comparison of the 1H and 13C NMR data for the two metabolites revealed that the main difference between them was the chemical shifts of HC



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methyls at δH 0.83 and 1.25, two equivalent doublet methyls at δH 1.09, one 1,1-disubstituted double bond at δH 4.65 and 4.93, one monosubstituted double bond at δH 5.03, 5.18, and 5.87, and a brominated methine at δH 4.18. The homonuclear and heteronuclear correlations observed in the COSY and HMBC spectra of 7 and particularly the HMBC correlations of H3-16 and H3-20 with both C-14 and C-15 indicated the position of the carbonyl at C-14 and established its planar structure. The NOESY cross-peaks were in accordance with those observed for metabolites 1−6, thus indicating the same relative configurations at the stereogenic centers C-6, C-10, and C-11 for compound 7. Compound 8, obtained as a colorless oil, displayed the same molecular formula as 7, as calculated from the HRESIMS data. The most prominent differences in their spectroscopic data included the presence of a second 1,1-disubstituted double bond (δH 4.86 and 4.95), a vinylic methyl (δH 1.72), and an oxygenated methine (δH 4.08) with the simultaneous absence of the two doublet methyls and the carbonyl functionality. The HMBC correlations of H3-16 and H2-20 with C-14 and C-15 established the structure of the second side chain attached at C11 of the cyclohexane ring of metabolite 8, while the remaining parts of the dactylomelane skeleton were verified by the observed HMBC and COSY correlations, in accordance with compounds 1−7. The relative configurations at C-6, C-10, and C-11 were determined as 6R*, 10S*, and 11S*, respectively, as in the case of metabolites 1−7. Metabolites 1−9 were evaluated for their in vitro cytotoxic activity using the MTT colorimetric assay against five human tumor cell lines, namely, HeLa cervical adenocarcinoma, MCF7 breast adenocarcinoma, A431 epidermoid carcinoma, HepG2 hepatocellular carcinoma, and A549 alveolar adenocarcinoma cell lines, but they were proven inactive. The dactylomelane skeleton is an already known, but rarely encountered, diterpene skeleton featuring only one cyclization between carbons C-6 and C-11. Dactylomelol, isolated from Aplysia dactylomela, was the first metabolite bearing this carbon framework.7 Prior to the present report, 10 additional compounds bearing the dactylomelane skeleton have been isolated, one from the red alga Sphaerococcus coronopifolius, one from the mollusk Aplysia punctata, and eight from species of the genus Laurencia.5,6,8−10 While many Aplysia species predominantly feed on red algae of the genus Laurencia, the two previous investigations on the chemistry of A. depilans have reported that the collected specimens were feeding on green algae.3,4 In our case, the animals were collected from a coastal area dominated by Laurencia seaweeds, a fact that might insinuate the dietary origin of the isolated dactylomelanes, even though they could have undergone structural modifications by the sea hares.

14 (δH 3.75 in 3 and 3.93 in 2) and C-14 (δC 74.8 in 3 and 71.7 in 2). After analysis of the 2D NMR spectra, it was evident that metabolites 2 and 3 possessed the same planar structure. Because the relative configurations of 3 at C-6, C-10, and C-11, as established by analysis of the NOESY spectrum, were found in accordance with those of 2, in conjunction with the fact that the chemical shifts of C-3, C-17, and H3-17 were almost identical with those of 2, it was evident that compound 3 was the C-14 epimer of 2. This assumption was also supported by the noticeable difference in the chemical shifts of H2-13 observed for 2 and 3, which however was not accompanied by a change in the chemical shift of C-13. Compound 4, obtained as a colorless oil, had the molecular formula C20H35BrO3 as deduced from the HRESIMS and NMR data. The mass spectrum exhibited an adduct ion peak [M + Na]+ at m/z 425.1655 with an isotopic peak at m/z 427.1633 in a relative intensity of 1:1, characteristic for the presence of one bromine atom. The 1D and 2D NMR data of metabolite 4 displayed a close resemblance to those of compounds 2 and 3. In accordance with the molecular formula, it was obvious that the second bromine atom at C-14 was replaced by a third hydroxy group. This hypothesis was further supported by the deshielding of C-14 resonating at δC 79.0, while H-14 was shielded to δH 3.29. The NOE enhancements of H-6/H-10, H9α/H-10, and H-9β/H3 -19 observed for metabolite 4 confirmed that the relative configurations of the asymmetric centers on the cyclohexane ring remained unchanged. Compound 5, isolated as a colorless oil, had the molecular formula C21H37BrO3, as established from the HRESIMS and NMR data. The presence of a singlet methyl resonating at δH 3.21, in combination with the fact that the 1H and 13C NMR data of 5 were rather similar to those of metabolite 4, indicated that one of the three hydroxy groups was replaced by a methoxy group. The HMBC correlation of the protons of the methoxy group with C-14 placed the methoxy on C-14. The relative configurations at C-6, C-10, and C-11 were retained as in 1−4 on the basis of the correlations observed in the NOESY spectrum. Because MeOH was one of the solvents used for the extraction of the organism, the possibility of 5 being an artifact cannot be excluded. The structural elements displayed in the 1H and 13C NMR spectra of compound 6, isolated as a colorless oil, exhibited a high degree of similarity to those of the previously described metabolites. The molecular formula of 6 was determined as C20H33BrO2 due to fact that two equally intense isotopic adduct ion peaks [M + NH4]+ were detected in the HRESIMS spectrum at m/z 402.1999 and 404.1980. Because the two carbon−carbon double bonds accounted for two of the four degrees of unsaturation, the molecular structure of 6 was determined as bicyclic. The chemical shifts of an oxygenated methine at δH 2.72, as well as of two singlet methyls resonating at δH 1.27 and 1.30, in conjunction with the HMBC correlations of the latter two with two oxygenated carbons, one nonprotonated at δC 58.4 and one tertiary at δC 64.1, were indicative for the presence of an epoxide moiety between C-14 and C-15. The depicted relative configuration of 6 was suggested by the enhancements of H-6/H-10, H-9α/H3-19, and H-9β/H-10 observed in the NOESY spectrum. Compound 7, with the molecular formula C20H33BrO2, as deduced from the HRESIMS measurements, was obtained as a colorless oil. Among the 20 carbon atoms observed in the 13C NMR spectrum, a carbonyl carbon resonating at δC 214.5 was evident. The 1H NMR spectrum included signals for two singlet

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer model 341 polarimeter with a 1 dm cell. UV spectra were obtained on a PerkinElmer Lambda 40 spectrophotometer. IR spectra were obtained on a Bruker Tensor 27 spectrometer. NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers. Chemical shifts are given on a δ (ppm) scale using TMS as internal standard. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. High-resolution ESI mass spectra were measured on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer. Column chromatography separations were performed with Kieselgel 60 (Merck). HPLC separations were conducted using a

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2; HRESIMS m/z 487.0813 [M + Na]+ (calcd for C20H34Br2O2Na, 487.0823). Compound 3: colorless oil; [α]20 D +43 (c 0.41, CHCl3); UV (CHCl3) λmax (log ε) 242 (2.40) nm; IR (thin film) νmax 3426, 2973, 1463, 1380, 735 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 487.0811 [M + Na]+ (calcd for C20H34Br2O2Na, 487.0823). Compound 4: colorless oil; [α]20 D +2.4 (c 0.41, CHCl3); UV (CHCl3) λmax (log ε) 241 (2.61) nm; IR (thin film) νmax 3406, 2966, 1463, 1379, 736 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 425.1655 [M + Na]+ (calcd for C20H35BrO3Na, 425.1667). Compound 5: colorless oil; [α]20 D +8.2 (c 0.33, CHCl3); UV (CHCl3) λmax (log ε) 241 (2.34) nm; IR (thin film) νmax 3460, 2970, 1464, 1381, 735 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 439.1815 [M + Na]+ (calcd for C21H37BrO3Na, 439.1824). Compound 6: colorless oil; [α]20 D +21.0 (c 3.72, CHCl3); UV (CHCl3) λmax (log ε) 241 (2.75) nm; IR (thin film) νmax 3446, 2966, 1461, 1380, 736 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 402.1999 [M + NH4]+ (calcd for C20H37BrNO2, 402.2008). Compound 7: colorless oil; [α]20 D +5.8 (c 0.36, CHCl3); UV (CHCl3) λmax (log ε) 242 (2.77) nm; IR (thin film) νmax 3467, 2969, 1709, 1464, 1376, 735 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 407.1554 [M + Na] + (calcd for C20H33BrO2Na, 407.1562). Compound 8: colorless oil; [α]20 D +16 (c 0.14, CHCl3); UV (CHCl3) λmax (log ε) 242 (2.99) nm; IR (thin film) νmax 3419, 2970, 1458, 1379, 737 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 407.1548 [M + Na]+ (calcd for C20H33BrO2Na, 407.1562). 5 20 Luzodiol (9): [α]20 D +11.6 (c 0.08, CHCl3); [α]D lit. +13.7 (c 0.12, CHCl3). Evaluation of Cytotoxic Activity. HeLa (human cervical adenocarcinoma), MCF7 (human breast adenocarcinoma), A431 (human epidermoid carcinoma), HepG2 (human hepatocellular carcinoma), and A549 (human alveolar adenocarcinoma) cells were routinely cultured in 60 mm tissue culture dishes as monolayers in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL), in a humidified 5% CO2 atmosphere at 37 °C. When 80−90% confluence was attained, the monolayers were subcultured using a 0.25% trypsin−1 mM EDTA solution. The cytotoxicity was determined with the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) dye reduction assay. Cells in their log phase of growth were harvested, counted, and plated into 96well tissue culture dishes at a density of 1 × 105 cells/mL DMEM (100 μL/well). After 24 h of incubation to allow cell attachment, cells were exposed to various concentrations of the compounds appropriately diluted in DMSO. After 48 h of incubation with the test compounds, MTT solution (at a final concentration of 0.5 mg/mL) was added in each well. Plates were further incubated for 4 h at 37 °C, and subsequently the MTT-containing culture medium was replaced by 0.1 N HCl solution in anhydrous 2-propanol (100 μL/well). Cell viability was determined by measuring the absorbance at 545 nm using a microplate reader. Three replicates were set up for each experimental condition, whereas untreated cells served as the control group. Percent growth in the presence of the test compounds was calculated considering the cell growth in the absence of any test compound as 100%. The IC50 values (μM; Supporting Information, Table S1) were calculated from three to four independent experiments, using Microsoft Office Excel software, as the concentration of the compound where 100(A0 − A)/A0 = 50, where A is the absorbance of the wells after 48 h of exposure to the test compound and A0 is the absorbance of the control wells. Miltefosine, an antiprotozoal drug, which was originally developed as an antineoplastic, was used as positive control, exhibiting IC50 values of 16.5, 9.2, 12.0, 26.3, and 8.5 against HeLa, MCF7, A431, HepG2, and A549 cells, respectively. All data were

Pharmacia LKB 2252 liquid chromatography pump equipped with an RI-102 Shodex refractive index detector, using either a Supelcosil LCSI Semiprep 5 μm (250 × 10 mm i.d.; Supelco) or a Chiralcel OD 10 μm (250 × 10 mm i.d.; Daicel Chemical Industries Ltd.) column. TLC was performed with Kieselgel 60 F254 (Merck aluminum-backed plates), and spots were detected after spraying with 15% H2SO4 in MeOH reagent and heating at 100 °C for 1 min. Animal Material. Specimens of Aplysia depilans were collected by hand off Skyros Island (GPS coordinates 38°87′ N, 24°57′ E), in the North Sporades complex of the Aegean Sea, Greece, at a depth of 2−4 m, in August of 2011. A voucher specimen of the mollusk has been deposited at the animal collection of the Department of Pharmacognosy and Chemistry of Natural Products, University of Athens (ATPH/MP0195). Extraction and Isolation. Three whole animals (750 g wet weight) were dissected and exhaustively extracted with mixtures of CH2Cl2/MeOH at room temperature. Evaporation of the solvents under vacuum afforded the organic residue (26.0 g), which was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as mobile phase, to yield 12 fractions (ADS1−ADS12). Fraction ADS6 (30% EtOAc in cyclohexane, 1.7 g) was further fractionated by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford 14 fractions (ADS6a−ADS6n). Fraction ADS6e (16% EtOAc in cyclohexane, 199.6 mg) was purified by normal-phase HPLC, using cyclohexane/ EtOAc (85:15) and subsequently cyclohexane/acetone (90:10) as eluent, to yield 7 (4.7 mg). Fraction ADS6f (16% EtOAc in cyclohexane, 258.7 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (85:15) as eluent, to yield 6 (48.7 mg). Fraction ADS6j (20% EtOAc in cyclohexane, 103.8 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (82:18) and subsequently cyclohexane/acetone (90:10) as eluent, to yield 3 (31.5 mg). Fraction ADS6m (30% EtOAc in cyclohexane, 81.0 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (82:18) as eluent, to yield 8 (2.5 mg). Fraction ADS7 (40% EtOAc in cyclohexane, 1.2 g) was further fractionated by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford 12 fractions (ADS7a−ADS7l). Fraction ADS7b (20% EtOAc in cyclohexane, 150.7 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (75:25) as eluent, to yield 1 (41.0 mg). Fraction ADS7e (20% EtOAc in cyclohexane, 173.6 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (75:25) as eluent, to yield 2 (44.4 mg). Fraction ADS8 (50% EtOAc in cyclohexane, 571 mg) was further fractionated by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford 11 fractions (ADS8a−ADS8k). Fraction ADS8g (25% EtOAc in cyclohexane, 66.2 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (75:25) and subsequently cyclohexane/acetone (90:10) as eluent, to yield 5 (4.3 mg). Fraction ADS8h (25% EtOAc in cyclohexane, 56.3 mg) was purified by normal-phase HPLC, using cyclohexane/acetone (90:10) and subsequently n-hexane/2-propanol (80:20) as eluent, to yield 9 (1.0 mg). Fraction ADS9 (60% EtOAc in cyclohexane, 176 mg) was further fractionated by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford 10 fractions (ADS9a−ADS9j). Fraction ADS9j (40% EtOAc in cyclohexane, 7.5 mg) was purified by normal-phase HPLC, using cyclohexane/acetone (80:20) as eluent, to yield 4 (5.3 mg). Compound 1: colorless oil; [α]20 D +3.8 (c 1.28, CHCl3); UV (CHCl3) λmax (log ε) 241 (2.84) nm; IR (thin film) νmax 3448, 2971, 1459, 1370, 738 cm−1; 1H NMR data, Table 1; 13C NMR data, Table 2; HRESIMS m/z 438.1768 [M + NH4]+ (calcd for C20H38BrClNO2, 438.1774). Compound 2: colorless oil; [α]20 D +36.0 (c 1.39, CHCl3); UV (CHCl3) λmax (log ε) 241 (2.57) nm; IR (thin film) νmax 3411, 2972, 1462, 1379, 736 cm−1; 1H NMR data, Table 1; 13C NMR data, Table E



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expressed as mean ± SD. Culture media and antibiotics were from Biochrom KG. All other chemicals were from Sigma-Aldrich.

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−8, 2D spectra of compound 1, table with the cytotoxic activity evaluation results for compounds 1−9. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +30-210-7274592. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. V. Smyrniotopoulos (University of Athens) for the collection of the mollusk specimens. This work was supported by the project GSRT-EPANII-09SYN-23-879 “SysTerp”, which is implemented under the “Cooperation 2009” Action of the Operational Programme “Competitiveness and Entrepreneurship” and is cofunded by the European Social Fund (ESF) and National Resources.

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DEDICATION Dedicated to Dr. William Fenical of Scripps Institution of Oceanography, University of California−San Diego, for his pioneering work on bioactive natural products.

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REFERENCES

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Dactylomelane Diterpenes from the Sea Hare Aplysia depilans

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