Sequestered Fulvinol-Related Polyacetylenes in PeltodorisatromaculataM. Letizia Ciavatta,*,† Genoveffa Nuzzo,† Kentaro Takada,‡ Veronique Mathieu,§ Robert Kiss,§

Guido Villani,† and Margherita Gavagnin†

†Consiglio Nazionale delle Ricerche (CNR), Istituto di Chimica Biomolecolare (ICB), Via Campi Flegrei, 34, 80078 Pozzuoli, Naples,Italy‡Laboratory of Aquatic Natural Products Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo,Bunkya-ku, 113-8657 Tokyo, Japan§Laboratoire de Cancerologie et de Toxicologie Experimentale, Faculte de Pharmacie, Universite Libre de Bruxelles (ULB), Campusde la Plaine, Boulevard du Triomphe, 1050, Brussels, Belgium

*S Supporting Information

ABSTRACT: The Mediterranean dorid nudibranch Peltodorisatromaculata that had been collected while feeding onHaliclona fulva was shown to sequester long-chain fulvinol-like polyacetylene metabolites (compounds 2−5) from theprey. They were isolated along with previously reportedbromorenierins from the diethyl ether extracts of both themollusk and the sponge. Their structures were elucidated byNMR spectroscopy and tandem FABMS analysis. Compound5 exhibited in vitro growth inhibitory effects against theSKMEL-28 melanoma cell line.

The suborder Doridina is the largest nudibranch groupcontaining the most varied and often brilliantly colored

species.1 Many dorids are highly specialized sponge-feeders.Consequently, the chemistry of these mollusks is strictly relatedto that of the sponge diet from which they derive secondarymetabolites. This is essential for understanding some basicbiological phenomena, including chemical defense mecha-nisms.2

The spotted sea slug Peltodoris atromaculata is a doridnudibranch distributed in the Mediterranean Sea and nearAtlantic, exclusively occurring and very common in precor-alligene and coralligene communities.3 Previous chemicalstudies conducted on P. atromaculata have reported thepresence in the digestive gland of a complex mixture of dietarypolyacetylene compounds, petroformynes, sequestered fromthe sponge Petrosia f iciformis.4 The same authors alsodemonstrated that the nudibranch is able to find its food bychemotaxis.5 Later, the preference of P. atromaculata for twoselected haplosclerid sponges was shown on the basis of fieldobservations and fecal analysis. In particular, it was reportedthat the nudibranch feeds on P. f iciformis and also on Haliclonafulva. While P. f iciformis is more common and more frequentlyconsumed, H. fulva is preferred.3

We have recently investigated the secondary metabolites of asample of H. fulva collected off Procida Island (Gulf of Naples)on which three individuals of the nudibranch P. atromaculatawere observed grazing. This study led us to characterize ninehigh molecular weight polyoxygenated acetylenes, fulvynes A−I(e.g., fulvyne A, 1), which were isolated from the butanolic

extract of the sponge.6 In addition, a preliminary analysis of thediethyl ether extract of the sponge6 revealed the presence ofless polar polyacetylene metabolites co-occurring with therenierines, brominated short chain polyacetylenes previouslydescribed from H. fulva.7 Interestingly, the chromatographiccomparison of the lipophilic extracts of the nudibranch withthose of the sponge revealed the presence in the mollusk ofether-soluble polyacetylene sponge metabolites, whereas thebutanol-soluble polyacetylene fraction was not detected.6

We report here the isolation and the structure determinationof these polyacetylenes, compounds 2−5, which are structurallyrelated to fulvinol (6), a C46 linear symmetric polyacetylenereported from a Spanish specimen of Reniera (=Haliclona)fulva.8 The evaluation of their in vitro growth inhibitoryproperties in human cancer cells is also described.

■ RESULTS AND DISCUSSION

Frozen P. atromaculata (three individuals) were dissected intointernal glands and mantles, which were separately extracted inacetone. A sample of the sponge H. fulva on which the molluskspecimens were observed feeding was also extracted withacetone. After filtration and evaporation of the organic solventunder vacuum, the aqueous residues were partitioned withEt2O and subsequently with n-butanol. The ether extracts wereanalyzed by TLC and 1H NMR, revealing a similar metabolite

Received: April 3, 2014

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pattern characterized by the presence of polyacetylenes for boththe sponge and the mollusk. In particular, in the nudibranch themetabolites of interest were observed to be mainly concen-trated in the extract of the internal parts including the digestivegland. This latter extract was subjected to LH-20 Sephadexchromatography (CHCl3/MeOH, 1:1) to recover two selectedfractions (A and B) containing polyacetylenes. In particular,fraction A consisted of fulvinol-like long-chain polyacetylenes,whereas fraction B contained short-chain brominated poly-acetylenes. These fractions were further purified first by silicagel chromatography (light petroleum ether/Et2O gradient) andsubsequently by RP-HPLC (MeOH). The new metabolites(2−5) were obtained from fraction A, whereas purification offraction B led us to isolate known 18-hydroxyrenierin-1,renierin-2, and 18-hydroxyrenierin-2, which were identified bycomparison of the spectroscopic data with values in theliterature.7 Compounds 2−5 were also isolated by using thesame purification procedures from the extract of the sponge H.fulva.Compound 2, named fulvindione, had the molecular formula

C46H72O2 deduced by ESIMS. Both the 1H and the 13C NMRspectra contained a smaller number of signals than thoseexpected from the molecular formula, suggesting a highlysymmetric structure. This implied that each resonance in the

spectra should be attributable to two magnetically equivalentnuclei. The 1H NMR spectrum contained a singlet at δH 3.21due to the terminal acetylenic protons (H-1 and H-46) and adoublet at δH 6.18 (2H, d, J = 15.7 Hz, H-4 and H-43) thatcorrelated in the COSY spectrum with a signal at δH 7.25 (2H,dt, J = 15.7 and 8.0 Hz, H-5 and H-42). The coupling constantvalue (JH‑4/H‑5 = 15.7 Hz) indicated the E geometry for thedouble bond belonging to the terminal residue. The remainingsignals at δH 1.98−2.06, 1.48−1.55, and 1.25−1.38 wereassigned to an envelope of methylenes constituting the chain.The IR spectrum showed two bands at 1650 and 2098 cm−1

that were attributed to a conjugated carbonyl group and a triplebond, respectively. Moreover, the occurrence in the 13C NMRspectrum of signals at δC 177.8 (C, C-3 and C-44), 155.8 (CH,C-5 and C-42), 131.9 (CH, C-4 and C-43), and 78.8 (CH, C-1and C-46) suggested that fulvindione (2) contained two (E)-1-yn-3-oxo-4-ene moieties. These residues accounted for eight ofthe 11 degrees of unsaturation required by the molecularformula. The three remaining unsaturations were attributed tothree double bonds, as indicated by the 1H NMR signal at δH5.35 (6H, m) and by the 13C NMR sp2 signals at δC 130.0,129.8, and 129.7. The Z geometry of the three inner doublebonds was assigned by the value of the allylic methylenecarbons9 at δC 27.1−27.2. The three double bonds in the chain

Chart 1

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were located at C-15, C-21, and C-27 by tandem FABMS/MSfragmentation analysis (Figure 1), which was further supportedby the measurement of the negative FABMS ion afterozonolysis with oxidative workup (m/z 271 and 215,Supporting Information). 1H and 13C NMR values wereassigned as reported in Tables 1 and 2.Compound 3, named fulvinone, was an inseparable mixture

of two isomeric polyacetylenes that were indistinguishable byESIMS and NMR analysis. The molecular formula, C46H74O2,indicated an unsaturation degree less than fulvindione (2). The1H and 13C NMR spectra were very similar to those of

compound 2 (Tables 1 and 2). The only difference was due tothe presence of a carbinol function [δH 4.83 (1H, br d, J = 6.2Hz), δC 62.8 (CH)] rather than the carbonyl group in one ofthe two terminal moieties of the molecule. Analysis of theCOSY spectrum of 3 supported this structural hypothesis. Infact, the carbinol proton signal at δH 4.83 correlated with both adoublet of doublets at δH 5.61 (1H, dd, J = 15.0 and 6.2 Hz),which was in turn coupled with the signal at δ 5.92 (1H, dt, J =15.0 and 7.3 Hz), and a long-range coupled alkyne signal at δH2.56 (1H, d, J = 2.2 Hz). This was consistent with an (E)-1-yn-3-hydroxy-4-ene moiety.

Figure 1. Key tandem FABMS fragment ions derived from fulvindione (2) [M + Li]+.

Table 1. 1H NMR Dataa (400 and 600 MHz, CDCl3) for Compounds 2−5

fulvindione (2) fulvinone (3a/3b)b isofulvinol (4) hydroxydehydroisofulvinol (5)

position δH, m (Hz) δH, m (Hz) δH, m (Hz) δH, m (Hz)

1 3.21, s 3.21, s 2.56, d (2.2) 2.56, d (2.2)3 4.83, br d (6.0) 4.83, br d (6.2)4 6.18, d (15.7) 6.17, d (15.7) 5.60, dd (15.0,6.0) 5.60, dd (15.0, 6.2)5 7.25,dt, (15.7, 8.0) 7.25, dt (15.7, 8.0) 5.92, dt (15.0,7.3) 5.92, dt (15.0, 7.3)6 2.31, m 2.31, m 2.07, m 2.07, m7 1.55−1.48, m 1.55−1.48, m 1.40, m 1.40, m8 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m9−13 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m14 2.06−1.98, m 2.06−1.98, m 2.06−1.98, m 1.38−1.25, m15 5.35, m 5.35, m 5.35, m 1.38−1.25, m16 5.35, m 5.35, m 5.35, m 1.38−1.25, m17 2.06−1.98, m 2.06−1.98, m 2.06−1.98, m 1.38−1.25, m18 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m19 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 5.35, m20 2.06−1.98, m 2.06−1.98, m 2.06−1.98, m 5.35, m21 5.35, m 5.35, m 5.35, m 2.06−1.98, m22 5.35, m 5.35, m 5.35, m 1.38−1.25, m23 2.06−1.98, m 2.06−1.98, m 2.06−1.98, m 1.38−1.25, m24 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m25 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 5.35, m26 2.06−1.98, m 2.06−1.98, m 2.06−1.98, m 5.35, m27 5.35, m 5.35, m 5.35, m 2.06−1.98, m28 5.35, m 5.35, m 5.35, m 1.38−1.25, m29 2.06−1.98, m 2.06−1.98, m 2.06−1.98, m 1.38−1.25, m30 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m31 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 5.35, m32 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 5.35, m33 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.79, m34 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 5.35, m35 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 5.35, m36 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m37−39 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m40 1.55−1.48, m 1.40, m 1.40, m 1.54, m41 2.31, m 2.07, m 2.07, m 4.17, m42 7.25, dt (15.7, 8.0) 5.92, dt (15.0, 7.3) 5.92, dt (15.0, 7.3) 5.96, dt (15.4, 7.3)43 6.18, d (15.7) 5.61, dd (15.0, 6.2) 5.61, dd (15.0, 6.0) 5.82, dd (15.4, 5.0)44 4.83, br d (6.2) 4.83, br d (6.0) 4.90, br d (5.0)46 3.21, s 2.56, d (2.2) 2.56, d (2.2) 2.58, d (2.2)

aAssignments were based on COSY, HSQC, and HMBC (J = 10 Hz) experiments. bThe same shifts apply to structure 3b, although the numberingsystem would be reversed.

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The presence of a hydroxy group was further confirmed bythe IR band at 3303 cm−1. In order to establish the position ofthe inner double bonds, fulvinone (3) was subjected toFABMS/MS. Analysis of the fragmentation ion patternappeared to be inconsistent with the presence of a singlecompound, suggesting instead a mixture of two isomericpolyacetylenes, 3a and 3b, with the only difference being thelocation of the carbinol (C-3 or C-44). To confirm this

hypothesis, 3 was oxidized by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), and the single product obtained byreaction was analyzed by both 1H NMR and tandem FABMS/MS (Figure S28), revealing it to be identical with fulvindione(2).The HRESIMS spectrum of compound 4 contained a

sodium adduct ion at m/z 683.5736 [M + Na]+, correspondingto the molecular formula C46H76O2. The

1H and 13C NMR data

Table 2. 13C NMR Dataa (150 MHz, CDCl3) for Compounds 2−5

fulvindione (2) fulvinone (3a/3b)e isofulvinol (4) hydroxydehydroisofulvinol (5)

position δC, type δC, type δC, type δC, type

1 78.8, CH 78.8, CH 74.0, CH 73.9, CH2 79.8, C 79.8, C 83.3, C 83.4, C3 177.8, C 177.8, C 62.8, CH 62.8, C4 131.9, CH 131.9, CH 128.4, CH 128.3, CH5 155.8, CH 155.8, CH 134.6, CH 134.6, CH6 32.7, CH2 32.7, CH2 31.9, CH2 31.9, CH2

7 27.8, CH2 27.8, CH2 28.8, CH2 28.8, CH2

8 29.7,b CH2 29.8,b CH2 29.4,b CH2 29.7,b CH2

9−13 29.7,b CH2 29.4,b CH2 29.4,b CH2 29.7,b CH2

14 27.1,d CH2 27.1,d CH2 27.1,d CH2 29.7,b CH2

15 130.0,c CH 130.0,c CH 130.0,c CH 29.7,b CH2

16 129.7,c CH 129.7,c CH 129.7,c CH 29.7,b CH2

17 27.2,d CH2 27.2,d CH2 27.2,d CH2 29.7,b CH2

18 29.4,b CH2 29.3,b CH2 29.8,b CH2 27.1,c CH2

19 29.2,b CH2 29.8,b CH2 29.2,b CH2 130.0,d CH20 27.2,d CH2 27.2,d CH2 27.2,d CH2 129.9,d CH21 130.0,c CH 130.0,c CH 130.0,c CH 27.2,c CH2

22 129.8,c CH 129.7,c CH 129.7,c CH 29.8,b CH2

23 27.1,d CH2 27.1,d CH2 27.1,d CH2 29.8,b CH2

24 29.7,b CH2 29.7,b CH2 29.8,b CH2 27.1,c CH2

25 29.8,b CH2 29.8,b CH2 29.6,b CH2 130.0,d CH26 27.2,d CH2 27.2,d CH2 27.2,d CH2 129.7,d CH27 130.0,c CH 130.0,c CH 130.0,c CH 27.2,c CH2

28 129.7,c CH 129.8,c CH 129.7,c CH 29.8,b CH2

29 27.2,d CH2 27.1,d CH2 27.1,d CH2 29.8,b CH2

30 29.4,b CH2 29.4,b CH2 29.2,b CH2 27.1,c CH2

31 29.2,b CH2 29.8,b CH2 29.4,b CH2 130.0,d CH32 29.7,b CH2 29.6,b CH2 29.7,b CH2 129.7,d CH33 29.2,b CH2 29.3,b CH2 29.6,b CH2 25.6, CH2

34 29.7,b CH2 29.6,b CH2 29.7,b CH2 129.7,d CH35 29.2,b CH2 29.8,b CH2 29.4,b CH2 130.0,d CH36 29.4,b CH2 29.6,b CH2 29.6,b CH2 27.1,c CH2

37−39 29.4,b CH2 29.7,b CH2 29.8,b CH2 29.4,b CH2

40 27.8, CH2 28.8, CH2 28.8, CH2 37.1, CH2

41 32.7, CH2 31.9, CH2 31.9, CH2 71.8, CH42 155.8, CH 134.6, CH 134.6, CH 135.9, CH43 131.9, CH 128.4, CH 128.4, CH 128.7, CH44 177.8, C 62.8, CH 62.8, CH 62.1, CH45 79.8, C 83.3, C 83.3, C 82.8, C46 78.8, CH 73.9, CH 74.0, CH 74.4, CH

aAssignments were based on HSQC and HMBC experiments. b,c,dAssignments with the same superscript in the same column may be interchanged.eThe same shifts apply to structure 3b, although the numbering system would be reversed.

Figure 2. Key tandem FABMS fragment ions derived from isofulvinol (4) [M + Li]+.

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of compound 4 seemed to be identical with those of knownfulvinol (6),8 whereas the FABMS/MS data suggested adifferent position of the inner double bonds. In particular, thefragmentation ion pattern of compound 4 (Figure 2), the sameas fulvindione (2), aided us in locating the inner double bondsas depicted. Accordingly, compound 4 was named isofulvinol.Compound 5, hydroxydehydroisofulvinol, had the molecular

formula C46H74O3, as deduced from HRESIMS, consistent withthe presence of an additional hydroxy group as well as a furtherunsaturation with respect to isofulvinol (4). The 1H NMRspectrum showed two terminal alkyne protons at δH 2.56 (1H,d, J = 2.2 Hz, H-1) and 2.58 (1H, d, J = 2.2 Hz, H-46). Thesmall difference observed in the chemical shifts of the twoprotons was ascribed to the presence of an additional hydroxygroup in one of the two terminal moieties, as it was easilydeduced by the COSY experiment. In particular, H-1 (δH 2.56)was long-range coupled to the methine proton at δH 4.83 (1H,br d, J = 6.2 Hz, H-3), which in turn correlated with an olefinicproton at δH 5.60 (1H, dd, J = 15.0 and 6.2 Hz, H-4). Thislatter proton was connected to the vicinal olefinic proton at δH5.92 (1H, dt, J = 15.0 and 7.3 Hz, H-5), correlating with amethylene signal at δH 2.07 (2H, m, H2-6). On the other hand,the alkyne proton at δH 2.58 (1H, d, J = 2.2 Hz, H-46) waslong-range coupled with the signal at δH 4.90 (1H, br d, J = 5.0Hz, H-44). This carbinolic methine was connected to theolefinic proton at δH 5.82 (1H, dd, J = 15.4 and 5.0 Hz, H-43),which was coupled to the vicinal olefinic proton at δH 5.96 (1H,dd, J = 15.4 and 7.3 Hz, H-42). This latter signal had a cross-peak with the methine proton at δH 4.17 (1H, m, H-41), whichwas in turn correlated with the methylene signal at δH 1.54 (2H,m, H2-40). Finally, the presence of a signal at δH 2.79 (2H, m,H2-29) coupled only with vinyl protons at δH 5.35 suggested

the presence of a bis-allylic methylene group. Analysis of the13C NMR spectrum and 2D NMR experiments confirmed theassignment of the two terminal moieties, whereas thefragmentation of tandem FABMS/MS suggested the positionof the inner double bonds as depicted in structure 5 (Figure 3).With the aim of establishing the absolute configurations of

the stereogenic centers C-3 and C-44, Mosher’s method10 wasapplied on the main isofulvinol (4). An aliquot of 4 was treatedwith (R)- and (S)-MTPA chlorides to get the (S) and (R)-MTPA esters 4a and 4b, respectively. The Δδ (δSester − δRester)values observed for the protons close to the hydroxy groups atC-3 and C-44 indicated the S configuration of the twoasymmetric centers (Figure 4), the same as determined for therelated fulvinol (6).8 On the basis of biogenetic considerations,the same configuration was suggested for the correspondingcarbons in co-occurring compounds 3a, 3b, and 5.Compounds 2−5 were tested for in vitro growth inhibitory

activity against a panel of six human cancer cell lines using theMTT colorimetric assay (Table 3). Compound 5 was activeagainst SKMEL-28 melanoma cells (IC50 3 μM). The IC50

values for 5 ranged between 15 and 32 μM in the remainingfive cell lines analyzed (Table 3).In summary, new long-chain fulvinol-like polyacetylenes have

been isolated from the ether extract of both the mollusk P.atromaculata and the sponge H. fulva. These compoundsdisplay a linear alkyl chain of 46 carbons with either a 1-yn-3-ol-4-ene or a 1-yn-3-keto-4-ene moiety at each terminus, the sameas for the petroformynes, previously described from theMediterranean sponge Petrosia f iciformis4,11−16 and from P.atromaculata4,5 feeding on it. These metabolites have not beendetected in any other Mediterranean sponges. Thus, the

Figure 3. Key tandem FABMS fragment ions derived from 5 [M + Li]+.

Figure 4. Chemical shift differences Δδ (δSester − δRester) between (S)- and (R)-MTPA derivatives of compound 4.

Table 3. Determination of the IC50 in Vitro Growth Inhibitory Concentrations in Six Human Cancer Cell Lines after 72 h ofCell Culture with the Compound of Interest

IC50 in vitro growth inhibitory concentration (μM)

carcinoma glioma melanoma

compound A549a MCF-7a PC-3a Hs683a U373a SKMEL28a mean ± SEM

fulvindione (2) 37 82 86 >100 >100 >100 >84fulvinone (3) >100 76 >100 >100 >100 >100 >96isofulvinol (4) 28 23 18 36 19 11 22 ± 4hydroxy-dehydro-isofulvinol (5) 31 15 32 23 15 3 20 ± 5etoposide 0.1 17 2 1 7 1 5 ± 3

aThe origin and histological type of each human cell line analyzed are as follows. Carcinoma models included the A549 NSCLC (DSMZ codeACC107), the MCF-7 breast (DSMZ code ACC115), and the PC-3 prostate (DSMZ code ACC465) cancer cell lines. Glioma models included theHs683 oligodendroglioma (ATCC code HTB-138) and the U373 (ECACC code 08061901) cell lines. One melanoma model included the SKMEL-28 (ATCC code HTB-72) cell line. DSMZ means Deutsche Sammlung von Mikroorganismen and Zellkulturen (Braunschweig, Germany), ATCCmeans American Type Culture Collection (Rockville, MD, USA), and ECACC means European Collection of Cell Cultures (Salisbury, UK).

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nudibranch P. atromaculata is highly specialized for feeding onthe two sponges that contain these specific polyacetylenes.

■ EXPERIMENTAL SECTIONGeneral Experimental Procedures. Optical rotations were

measured on a JASCO DIP 370 digital polarimeter. The UV spectrawere recorded on an Agilent 8453 spectrophotometer. 1H and 13CNMR spectra were recorded on DRX 600, Avance 400, and DPX 300MHz Bruker spectrometers in CDCl3, with chemical shifts reported inppm referenced to CHCl3 (δH 7.26 for proton and δC 77.0 for carbon)as an internal standard. ESIMS and HRESIMS data were obtained on aMicromass Q-TOF Micro spectrometer coupled with a HPLC WatersAlliance 2695. The instrument was calibrated by using a PEG mixturefrom 200 to 1000 MW. Tandem FABMS/MS spectra were recordedon a JEOL JMS-700T at the University of Tokyo using m-nitrobenzylalcohol (NBA) + LiCl as matrix. Sephadex LH-20 (GE Healthcare Bio-Sciences) was carried out using an open prepacked column. Silica gelchromatography was performed using precoated Merck F254 platesand Merck Kieselgel 60 powder. HPLC purification was carried out ona Waters 510 pump equipped with a Waters differential refractometerR401 detector.Biological Material. Peltodoris atromaculata (three individuals, 6

cm length average) and Haliclona fulva were collected by scuba divingat 40 m depth off Punta Pizzaco (Procida Island, Gulf of Naples)during May 2009. The animals were immediately transferred to ICBlaboratories and frozen. The nudibranch and the sponge wereidentified by one of us (G.V.). A voucher specimen of P. atromaculata(code DA) and H. fulva (code RF) are available for inspection at ICB.Extraction and Isolation. Frozen specimens of P. atromaculata

were carefully dissected into mantles (3.5 g, dry weight) and internalglands (0.95 g, dry weight) and extracted separately with acetone (3 ×100 mL) using ultrasound. The extracts were concentrated in vacuo,and the aqueous residues were partitioned with Et2O (3 × 150 mL)and subsequently with n-BuOH (3 × 100 mL). The organic layerswere evaporated under reduced pressure to give Et2O (50.0 mg fromthe mantle, 140.0 mg from internal glands) and n-BuOH (27.0 mgfrom the mantle, 100.0 mg from internal glands) residues. The frozensample of H. fulva (16 g, dry weight) was immersed in acetone (5 ×200 mL). After evaporation of the solvent under reduced pressure, theaqueous residue was partitioned with Et2O (4 × 400 mL) andsubsequently with n-BuOH (3 × 100 mL). The organic layers wereconcentrated to give Et2O (800.0 mg) and n-BuOH (900.0 mg)residues. The extracts were analyzed by TLC in different solventsystems, revealing a similar secondary metabolite pattern in the twoorganisms. Compounds 2−5 were isolated from the ether extract ofboth the internal parts of the nudibranch and the sponge.Purification of Polyacetylenes 2−5. The ether extract (140 mg)

of the internal parts of P. atromaculata was chromatographed on acolumn packed with Sephadex LH-20 and eluted with CHCl3/MeOH,1:1, affording two fractions (A and B) of interest. Fraction A wassubmitted to SiO2-gel column chromatography (light petroleumether/Et2O gradient, then CHCl3, and finally MeOH, as eluent).Selected subfractions containing spots at Rf 0.80, 0.65, 0.50, and 0.10(light petroleum ether/Et2O, 6:4) were considered and further purifiedby reversed-phase HPLC (Supelco-Ascentis C18 column, 25 × 0.46cm, 100% MeOH; flow 1 mL/min) to afford 2 (1.0 mg), 3 (0.9 mg), 4(1.7 mg), and 5 (0.3 mg), in order of increasing polarity. The samepurification protocol was applied to the ether extract of the sponge(800.0 mg) to obtain 2 (4.5 mg), 3 (1.5 mg), 4 (10.0 mg), and 5 (1.9mg). Fraction B was chromatographed by SiO2 column (lightpetroleum ether/Et2O gradient, then CHCl3, and finally MeOH) toafford the known 18-hydroxyrenierin-1 (3.0 mg), renierin-2 (5.8 mg),and 18-hydroxyrenierin-2 (9.9 mg).Fulvindione (2): colorless oil; UV (MeOH) λmax (log ε) 244 (3.56);

IR (liquid film) 2925, 2854, 2098, 1650, 1455, 1232, 964 cm-1; 1H and13C NMR see Tables 1 and 2; ESIMS m/z 679 [M + Na]+ andHRESIMS m/z 679.5400 [M + Na]+ (calcd for C46H72O2Na,679.5430); FABMS (NBA matrix) m/z 663 [M + Li]+.

Fulvinone (3a/3b): colorless oil; [α]25D −23 (c 0.04, CHCl3); UV(MeOH) λmax (log ε) 244 (3.55); IR (liquid film) 3303, 2925, 2856,2087, 1644, 1443, 1370, 964 cm−1; 1H and 13C NMR see Tables 1 and2; ESIMS m/z 681 [M + Na]+ and HRESIMS m/z 681.5579 [M +Na]+ (calcd for C46H74O2Na, 681.5587); FABMS (NBA matrix) m/z665 [M + Li]+.

Isofulvinol (4): colorless oil; [α]25D −11 (c 0.04, CHCl3); IR (liquidfilm) 3309, 2924, 2854, 2103, 1713, 1462, 1381, 1018, 964 cm−1; 1Hand 13C NMR see Tables 1 and 2; ESIMS m/z 683 [M + Na]+ andHRESIMS m/z 683.5736 [M + Na]+ (calcd for C46H76O2Na,683.5743); FABMS (NBA matrix) m/z 667 [M + Li]+.

Hydroxydehydroisofulvinol (5): colorless oil; [α]25D +6.0 (c 0.6,CHCl3); IR (liquid film) 3303, 2925, 2856, 2087, 1644, 1443, 1370,964 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS m/z 697 [M+ Na]+ and HRESIMS m/z 697.5516 [M + Na]+ (calcd forC46H74O3Na, 697.5536); FABMS (NBA matrix) m/z 681 [M + Li]+.

Ozonolysis of Fulvindione (2). An aliquot of compound 2 (0.5mg) was dissolved in CH2Cl2 (1 mL) and treated with O3 at −78 °Cfor 15 min. After this time, the excess O3 was removed by a stream ofN2, and the reaction mixture was treated with 90% HCOOH/35%H2O2 (2:1, 1 mL) at room temperature (rt) for 15 min. The reactionsolution was concentrated and subjected to FABMS/MS analysis.

Oxidation of Fulvinone (3a/3b). An aliquot of fulvinone (3) (0.5mg) was dissolved in dioxane (200 μL) and treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (2 mg) at 60 °C. After stirring for 2 h,the reaction mixture was cooled to rt. The reaction solution wasfiltered, and the filtrate was concentrated and then subjected toreversed-phase HPLC (Supelco-Ascentis C18 column, 25 × 0.46 cm,100% MeOH, flow 1 mL/min). The single peak eluted was recovered,concentrated, and analyzed by 1H NMR and tandem FABMS/MS. Itwas identified as fulvindione (2).

Preparation of MTPA Esters of Compound 4. (R)- and (S)-MTPA-Cl (10 μL) and a catalytic amount of DMAP were separatelyadded to two different aliquots of isofulvinol (4) (1.0 mg each) in dryCH2Cl2 (0.5 mL). The resulting mixtures were allowed to stand at rtfor 12 h. After the evaporation of the solvent, the mixtures werepurified on a SiO2 Pasteur pipet (CH2Cl2), affording pure (S)- and(R)-MTPA esters of 4, respectively.

(S)-MTPA ester of 4: selected 1H NMR values (CDCl3, 600 MHz)δH 6.03 (2H, br d, J = 6.8 Hz, H-3 and H-44), 6.01 (2H, dt, J = 15.2,6.8 Hz, H-5 and H 42), 5.50 (2H, dd, J = 15.2, 6.8 Hz, H-4 and H 43),2.63 (2H, brs, H-1 and H-46); ESIMS m/z 1115 [M + Na]+.

(R)-MTPA ester of 4: selected 1H NMR values (CDCl3, 600 MHz)δH 6.01 (2H, br d, J = 6.6 Hz H-3 and H-44), 6.07 (2H, dt, J = 15.3,6.6 Hz, H-5 and H 42), 5.60 (2H, dd, J = 15.3, 6.6 Hz, H-4 and H 43),2.59 (2H, brs, H-1 and H-46); ESIMS m/z 1115 [M + Na]+.

Determination of the IC50 Growth Inhibitory Concentrationsin Vitro. The MTT colorimetric assay was used as detailedpreviously.17,18 Each experimental condition was assessed in sixreplicates. The origin of each cell line is detailed in the legend of Table3.

■ ASSOCIATED CONTENT*S Supporting Information1D and 2D NMR spectra of compounds 2−5. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*Tel: 0039 081 8675243. Fax: 0039 081 8041770. E-mail:lciavatta@icb.cnr.it.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe NMR spectra were recorded at the ICB NMR Facility, thestaff of which is gratefully acknowledged. The authors thank

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Mrs. D. Ricciardi for laboratory assistance, Mr. M. Zampa forESIMS measurements, and Mr. C. Iodice for spectrophoto-metric measurements. Thanks are also due to T. Gras for thetechnical assistance in biological assays and H. Leclercqz for thecalculation of IC50 concentrations. This research work waspartially funded by PRIN 2009 KFMP7Z “Natural products andbioinspired molecules interfering with biological targetsinvolved in control of tumor growth”.

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