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Tetrahedron 68 (2012) 3541e3544

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Tetrahedron

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Rossinone-related meroterpenes from the Antarctic ascidian Aplidium fuegiense

Marianna Carbone a, Laura N�u~nez-Pons b, Miriam Paone a, Francesco Castelluccio a, Conxita Avila b,Margherita Gavagnin a,*

aConsiglio Nazionale delle Ricerche, Istituto di Chimica Biomolecolare, Via Campi Flegrei 34, 80078-I Napoli, ItalybDepartament de Biologia Animal (Invertebrats), Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 6, 08028-E Barcelona, Catalunya, Spain

a r t i c l e i n f o

Article history:Received 4 January 2012Received in revised form 17 February 2012Accepted 5 March 2012Available online 13 March 2012

Keywords:Marine natural productsMeroterpenoidsQuinonesAscidianAntartic

* Corresponding author. Tel.: þ39 081 8675094; faaddresses: margherita.gavagnin@icb.cnr.it, mgavagnin@

0040-4020/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.tet.2012.03.013

a b s t r a c t

The chemical analysis of the ascidian Aplidium fuegiense resulted in the isolation of three novel mer-oterpenoids 2e4, structurally related to the main co-occurring known rossinone B (1). The structures ofthe new compounds were determined by interpretation of spectroscopic data. Compounds 1e4 werefound to be selectively localized in the viscera of the ascidian.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

The group of ascidians is one of the most compelling sources ofmetabolites both of chemical and biomedical interest in the marineenvironment.1 In particular, the species belonging to the genusAplidium (family Polyclinidae) are recognised as prolific producersof bioactive natural products exhibiting an extensive structuralvariability and including non-nitrogenous compounds, such asprenyl hydroquinones and prenyl quinones, and nitrogenous me-tabolites, like nucleosides, peptides andahighvariety of alkaloids.2,3

As a part of our continuing search for biologically active sec-ondarymetabolites of marine organisms from distinct geographicalareas,4e8 we have investigated the chemistry of the ascidian Apli-dium fuegiense, collected in Antarctica. Previous studies on Ant-arctic Aplidium species have resulted in the discovery ofmeridianins, which are a group of indole alkaloids showing a potentcdk inhibitor activity, and displaying feeding repellence towardssympatric sea stars.9e11 Moreover, bromoindole derivatives, theaplicyanins, with strong cytotoxic and antimitotic activities,12 aswell as meroterpenes, such as rossinone B (1), exhibiting anti-leukaemic, antiviral and anti-inflammatory properties have beenalso reported in southern ascidian representatives of this genus.13

We describe here the isolation of three novel meroterpenes(2e4) structurally related to the co-occurring known metabolite

x: þ39 081 8041770; e-mailicmib.na.cnr.it (M. Gavagnin).

All rights reserved.

rossinone B (1), from the ether extract of the visceral part of A.fuegiense. This extract exhibited significant feeding-deterrent ac-tivity against the generalist Antarctic asteroid predator Odontastervalidus.

Table 213C NMR dataaed of compounds 1e4

C 1d 2 3 4

1 190.3 n.d. 189.9 191.02 139.0 55.5 139.0 140.03 141.2 56.4 141.4 141.74 185.0 191.8 184.4 193.95 134.6 134.3 133.3 55.96 144.2 145.0 144.0 61.47 50.5 49.7 50.5 48.58 78.0 78.0 78.3 79.39 40.2 40.2 40.5 39.810 21.1 21.0 20.9 21.111 39.5 39.3 39.5 34.412 49.3 49.2 48.9 49.813 82.8 83.8 82.8 83.714 213.0 212.2 212.3 212.2

M. Carbone et al. / Tetrahedron 68 (2012) 3541e35443542

2. Results and discussion

The ascidian A. fuegiense was collected by dredging in the Wed-dell Sea (Antarctica), during the DecembereJanuary of 2003e2004.The biological material was frozen at �20 �C and transferred to thelaboratory in Spainwhere it was later dissected into external (tunic)and internal (viscera) parts. The two sections were separatelyextracted with acetone. In a further Antarctic campaign in Decem-bereJanuary 2008e2009, the Et2O soluble portion of the acetoneextracts of the two parts were tested in feeding-repellence testsagainst O. validus. Both extracts showed strong deterrent activity.Subsequently, these extracts were transferred to the laboratory inItaly for the chemical analysis. Comparative TLC of the extractsrevealed different secondary metabolite pattern for the two ana-tomical sections. Inparticular, amain spot at Rf 0.50 alongwith otherspots at Rf 0.10e0.60 (light petroleum ether/Et2O, 2/8) were selec-tively present in the viscera of the animal. With the aim to identifythese components, the visceral extract (117.0 mg) was submitted tofurther purification steps including silica-gel column and pre-parative TLC as well as reverse-phase HPLC chromatography (seeExperimental). Four structurally related pure molecules were re-covered: rossinone B (1, 9.0 mg), the main compound, and threeminor metabolites 2,3-epoxy-rossinone B (2, 1.0 mg), 8-epi-rossi-none B (3,1.1mg) and 5,6-epoxy-rossinone B (4, 0.5mg). Compound1hadbeen already reported fromanunidentifiedAntarcticAplidiumspecies,13 whereas compounds 2e4 were not previously described.

Preliminary NMR spectroscopic analysis of the minor co-occurring compounds 2e4 revealed the presence of a mer-oterpene carbon skeleton, the same as 1, characterized by an un-common molecular architecture with a 6-6-5 tricyclic core. Thestructure of rossinone B (1) has been recently confirmed by bio-mimetic total synthesis.14 Comparison of MS data indicated thatcompounds 2 and 4 contained an additional oxygen atom with re-spect to 1 whilst compound 3 was an isomer of 1 having the samemolecular formula.

The 1H NMR spectrum of 2,3-epoxy-rossinone B (2) closely re-sembled the spectrum of rossinone B (1) (Table 1). The relevant

Table 11H NMR dataaec of compounds 1e4

H 1c 2 3 4

dH (J in Hz) dH (J in Hz) dH (J in Hz) dH (J in Hz)

1 d d d d

2 6.79, d (10.5) 3.75, d (3.5) 6.79, d (10.3) 6.94, d (10.3)3 6.90, d (10.5) 3.80, d (3.5) 6.91, d (10.3) 7.05, d (10.3)4 d d d d

5 d d d d

6 7.44, d (1.9) 7.12, br s 7.48, d (1.5) 3.78, app. sd

7 2.06, dd(12.4, 1.9)

2.01, d (12.5) 2.48, dd(12.7, 1.5)

1.80, m

8 d d d d

9 1.92, m 1.95e2.08, m 1.80e2.18, m 1.90e1.8010 1.53, m 1.43e1.58, m 1.38e1.45, m 1.45e1.60 m

1.52e1.58, m11 2.67, m 2.45e2.62, m 2.05e2.15, m 2.48e2.58, m12 d d d d

13 d d d d

14 d d d d

15 4.55, d (8.8) 4.64, d (8.8) 4.53, d (8.4) 4.85, d (8.9)16 5.07, br d (8.9) 5.03, br d (8.8) 5.07, br d (8.4) 5.08, br d (8.9)17 d d d d

18 1.76, d (1.1) 1.75, br s 1.76, br s 1.77, br s19 1.54, s 1.50, s 1.40, s 1.51, s20 1.10, s 1.02, s 1.10, s 0.93, s21 1.73, d (1.1) 1.70, br s 1.73, br s 1.70, br s

a The spectra were recorded in CDCl3 at 400 MHz and 600 MHz.b Assignments made by 1He1H COSY, HSQC and HMBC (J¼10 Hz) experiments.c Values from Ref. 13.d H-6 exhibited a very small J (0.32 Hz), which was measured by acquiring the

high resolution spectrum of d 3.30e5.50 region (0.003426 Hz/pt).

difference was in the chemical shift of the AB quartet due to thequinone protons resonating at higher fields [dH H-2: 3.75 (d,J¼3.5 Hz) in 2, 6.79 (d, J¼10.5 Hz) in 1; dHH-3: 3.80 (d, J¼3.5 Hz) in 2,6.90 (d, J¼10.5 Hz) in 1]. Bearing inmind that themolecular formulaof 2 (C21H28O5) contains an additional oxygen with respect to 1, itwas suggested the presence of an epoxy ring at C-2/C-3 in thestructure of 2. Accordingly, the 13C NMR spectrum of 2 displayedtwo additional sp3 carbons at dC 55.5 (C-2) and 56.4 (C-3) in theplace of the quinone resonances of 1 at dC 139.0 (C-2) and 141.2 (C-3)(Table 2). The remaining part of 2was suggested to be identical with1 including the relative stereochemistry of chiral centres on thebasis of the strong similarity of both proton and carbon NMR res-onances with those of 1 (Tables 1and 2). Analysis of a series of NOEexperiments confirmed this suggestion (see Supplementary data).Unfortunately, no steric effect was observed between either H-2 orH-3 with other protons of the structure preventing the definition ofthe orientation of the epoxide ring with respect to the plane of themolecule. Thus, this stereochemistry remained undetermined. Allproton and carbon resonances of 2,3-epoxy-rossinone B (2) wereassigned by 2D NMR experiments (see Supplementary data) andreported in Tables 1and 2.

15 77.2 77.4 77.4 77.016 118.6 118.7 117.8 118.517 142.5 142.3 143.0 142.718 25.9 25.9 25.6 25.919 27.1 27.1 26.7 27.420 8.8 8.7 8.6 11.021 18.7 18.9 18.6 18.7

a The spectra were recorded in CDCl3 at 300 MHz.b Assignments made by HSQC and HMBC (J¼10 Hz) experiments.c qC d’s measured by indirect detection.d Values from Ref. 13.

3-Epi-rossinone B (3), with themolecular formula C21H28O4, wasisomeric with themain co-occurring rossinone B (1). Analysis of the1He1H COSY spectrum of 3 revealed the presence of the same spinsystems as 1 (Table 1) indicating identical substitution patterns.Thus the two molecules had to differ in the configuration of one ormore chiral centres. The carbon values of 3 were quite similar tothose of 1 (see Table 2) suggesting that the stereochemistry of the7,11- and 12,13-junctions were the same as 1. Instead, significantdifferences were observed in the proton values of both H-7 and H-11 [dH H-7: 2.06 in 1, 2.48 in 3; dH H-11: 2.67 in 1; 2.10 in 3] thatcould be explained by the diverse steric influence of the hydroxylgroup in the two compounds. The analysis of a series of NOE ex-periments conducted on both compounds 3 and 1 was very in-dicative (see Supplementary data). In particular, in compound 3 theangular proton H-7 had NOE interactions only with H3-20 whereasin rossinone B (1) H-7 showed steric effects with both H3-19 andH3-20 according to the b-orientation of the twomethyl groups withrespect to the plane of the molecule. This suggested that in 3 the

M. Carbone et al. / Tetrahedron 68 (2012) 3541e3544 3543

methyl at C-8 should be a-oriented as it was further supported bythe diagnostic NOE effect observed between H-11 and H3-19. Thus 3was the C-8-epimer of rossinone B. Full proton and carbon as-signments are reported in Tables 1and 2.

5,6-Epoxy-rossinone B (4) was obtained in very small quantities.The 1H NMR spectrum immediately revealed the lack of the doublebond in ring B with respect to co-occurring compounds 1e3. In fact,the signal due to the olefinic proton H-6 observed for rossinones1e3 was replaced in 4 by an apparent singlet at dH 3.80 assigned toa proton, which was correlated in the HQSC spectrum to a CHcarbon resonating at dC 61.4. Analysis of the HMBC experimentrevealed a diagnostic correlation between H-3 (dH 7.05) and anadditional sp3 carbon resonance at dC 55.7, which was attributed tothe angular quaternary carbon C-5 directly connected to the qui-none moiety. Accordingly, due to the absence of the conjugateddouble bond, C-4 quinone carbonyl in 4 was observed down-fieldshifted (dC 193.9) with respect to 1 (dC 185.0) and 3 (dC 184.4).Thus, taking into account the additional oxygen atom required bythe molecular formula of 4 and the unsaturation degrees to besatisfied, an epoxide ring was located at C-5/C-6 being theremaining part of the molecule almost the same as 1(Tables 1and2). Significant differences were only observed in C-8 and C-20carbon values according to the presence of the additional sub-stituent in ring B. The connection with the ring C was supported bydiagnostic HMBC correlations between C-7 and both H-6 and H-11.As the vicinal coupling between H-6 and H-7 was not observed inthe 1He1H COSY spectrum due to the very small coupling constant,the 1H INADEQUATE experiment was performed on compound 4definitively evidencing this correlation. With the exception of theepoxide cycle, the overall relative stereochemistry of 4 was sug-gested to be the same as 1 by the similarity of NMR carbon andproton values (Tables 1and 2). Selected diagnostic NOE differenceexperiments confirmed this assignment (see Supplementary data).The relative configuration of C-5 and C-6 was suggested analyzingthe molecular models of the two possibleda- and b-epoxidedstereoisomers by Chem3D computer program. The JH6eH7 value(0.32 Hz, see Table 1) indicated that the dihedral angle between thetwo protons had to be about 90�. This geometry was observed inthe isomer exhibiting the b-oriented epoxide thus supporting theproposed structure 4.

3. Conclusion

The secondary metabolite pattern of the ascidian A. fuegiensewas characterized by the presence of meroterpenoids, according tothe literature data for the genus Aplidium. Three new members,rossinones 2e4, have been added to this interesting class of cyclicprenyl quinones. These compounds, along with the structurallyrelated main metabolite rossinone B (1), were found in the visceraextract of A. fuegiense that showed strong activity in the repellencyassay on O. validus. In situ ecological experiments on the pureisolated compounds 1e4 are still in progress to verify if the de-terrent activity of the extract could be in part ascribed to themeroterpenoid content. Biosynthetically, cyclic prenylated qui-nones, such as rossinone B (1) are suggested to derive from thecorresponding linear hydroquinone derivatives,14 which have beenreported to co-occur in the natural sources.13 Interestingly, neitheracyclic hydroquinones nor putative quinone-containing precursorsof rossinones 1e4 were detected in A. fuegiense extract.

4. Experimental section

4.1. General procedures

TLC plates (Merck Silica Gel 60 F254) were used for analytical TLCand Merck Kieselgel 60 was used for preparative column

chromatography. HPLC purification was carried out on a Shimadzuapparatus equipped with an LC-10ADVP pump and an UV SPD-10AVP detector by using reverse-phase semi-preparative column(250�10 mm, Phenomenex, Kromasil C18). 1D and 2D NMR spectrawere acquired in CDCl3 (shifts are referenced to the solvent signal)on a Bruker Avance-400 operating at 400 MHz, using an inverseprobe fitted with a gradient along the Z-axis and a Bruker DRX-600operating at 600 MHz, using an inverse TCI CryoProbe fitted witha gradient along the Z-axis. 13C NMR spectrawere recorded in CDCl3(d values are reported to the solvent signal) on a Bruker DPX-300operating at 300 MHz, using a dual probe. Optical rotations weremeasured on a Jasco DIP 370 digital polarimeter. IR spectra weremeasured on a Biorad FTS 155 FTIR spectrophotometer. BothESIMS and HRESIMS spectra were recorded on a Micromass Q-TOFmicroinstrument.

4.2. Collection and extraction of the animal material

Specimens of A. fuegiensewere collected in theWesternWeddellSea (Antarctica) at a depth of 597.6m, during the ANT XXI/2 cruise ofR/V Polarstern (AWI; Bremerhaven, Germany), fromNovember 2003to January 2004, using a bottom trawl. The biological material wasimmediately frozen at -20 �C transferred to the laboratory in Spain,and lateronsent to the ICB (Italy)where itwasdissected intoexternal(tunic) and internal (viscera) parts. The two sectionswere separatelyprocessed. Afterwards both sample parts were homogenized witha pestle and extractedwith acetone (150mL�3) by using ultrasoundvibration. The organic solvent was removed under reduced pressureand the residual water was partitioned with Et2O and subsequentlywith n-butanol. An aliquot of the Et2O extracts (10.1 mg visceral and2mg tunic) were used for the ecological tests. The remaining part (ofvisceral and 72.1 mg of tunic) was chemically analyzed. A voucherspecimen was fixed in 10% formalin for taxonomical determinationand it is stored at the Dept. of Animal Biology (Invertebrates), Uni-versity of Barcelona (Sample code #1093).

4.3. Purification of compounds 1e4

A portion of the visceral Et2O extract (117.0 mg) was fraction-ated on silica-gel column using light petroleum ether with in-creasing amounts of Et2O as eluent, yielding four meroterpenoidscontaining fractions (AeD). Fraction A (4.2 mg) was subjected toreverse-phase HPLC purification using a Supelco Discovery C18column (25 cm�10mm, particle size¼5 mm) eluted with methanol/water 7:3 (flow rate¼2 mL/min) to give pure compounds 2(1.0 mg). Fraction B (14.8 mg) was submitted to preparative TLC(SiO2, light petroleum ether/Et2O, 2:8) to give compounds 1(9.0 mg). Finally, fractions C (1.1 mg) and D (0.5 mg) were directlyanalyzed by 1H NMR showing to contain pure compounds 3 and 4,respectively.

4.3.1. Rossinone B (1). Colourless oil; Rf (20% petroleum ether/diethyl ether) 0.50; �2.2 (c 0.82, CHCl3); UV (CH3OH) lmax (log ε)204 (4.20), 225 (4.21); 1H and 13C NMR data see Tables 1and 2.13

ESIMS m/z 379 (MþNa)þ; HRESIMS: (MþNa)þ, found 379.1518.C21H28 NaO4 requires 379.1521.

4.3.2. 2,3-Epoxy-rossinone B (2). Colourless oil; Rf (20% petroleumether/diethyl ether) 0.60; (c 0.1, CHCl3); UV (CH3OH) lmax (log ε)205 (4.08); 1H and 13C NMR data see Tables 1and 2. ESIMS m/z 395(MþNa)þ; HRESIMS: (MþNa)þ, found 395.1459. C21H28 NaO5 re-quires 395.1471.

4.3.3. 3-Epi-rossinone B (3). Colourless oil; Rf (20% petroleumether/diethyl ether) 0.35; 0 (c 0.1, CHCl3); UV (CH3OH) lmax (log ε)204 (4.03), 225 (4.00); 1H and 13C NMR data see Tables 1and 2.

M. Carbone et al. / Tetrahedron 68 (2012) 3541e35443544

ESIMS m/z 379 (MþNa)þ; HRESIMS: (MþNa)þ, found 379.1532.C21H28 NaO5 requires 379.1520.

4.3.4. 5,6-Epoxy-rossinone B (4). Colourless oil; Rf (20% petroleumether/diethyl ether) 0.20; (c 0.05, CHCl3); UV (CH3OH) lmax (log ε)205 (3.89); 1H and 13C NMR data see Tables 1and 2. ESIMS m/z 395(MþNa)þ; HRESIMS: (MþNa)þ, found 395.1433. C21H28 NaO5 re-quires 395.1471.

4.4. Biological assays

Individuals of the Antarctic omnivorous sea star O. validus werecollected in Deception Island (South Shetland Is.) on board of B/OHesp�erides during January 2006 for feeding-repellence assays.Experiments took place at the Spanish Base (BAE) ‘Gabriel de Cas-tilla’ in Deception Is., Antarctica, during the same period. Each assayconsisted of ten sea stars, each presented to one shrimp item. Forthe treatment tests, shrimp cubes containing Et2O extracts fromspecimens of A. fuegiense at the natural concentration (on a dryweigth basis), were presented to the sea stars. For the control teststhe shrimps were treated with solvent (Et2O) alone. After 24 h, thenumber of shrimp pieces eaten out of the 10 replicates was sta-tistically contrasted in treatment versus control assays (Fisher’sExact tests). The tests were carried out following the methodologyreported in previous studies.15,16

Acknowledgements

We wish to thank the R/V Polarstern, BIO Hesp�erides, BIO LasPalmas and the BAE ‘Gabriel de Castilla’ crews for their supportduring the Antarctic cruises. Funding was provided by the Min-istry of Science and Education of Spain through the ECOQUIMand ACTIQUIM Projects (REN2003-00545, REN2002-12006E ANT,CGL2004-03356/ANT and CTM2010-65453/ANT), and PRIN-MIUR2009 Project ‘Natural products and bioinspired molecules in-terfering with biological targets involved in control of tumour

growth’. Also thanks are due to J. V�azquez, D. Melck, S. Taboada,B. Figuerola, F. J. Cristobo and M. Varela for laboratory and fieldwork.

Supplementary data

Supplementary data associated with this article can be found inthe online version, at doi:10.1016/j.tet.2012.03.013. These data in-clude MOL files and InChiKeys of the most important compoundsdescribed in this article.

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