Article pubs.acs.org/jnp
Sequestered Fulvinol-Related Polyacetylenes in Peltodoris atromaculata M. Letizia Ciavatta,*,† Genoveffa Nuzzo,† Kentaro Takada,‡ Véronique 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 Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, 1050, Brussels, Belgium S Supporting Information *
ABSTRACT: The Mediterranean dorid nudibranch Peltodoris atromaculata that had been collected while feeding on Haliclona fulva was shown to sequester long-chain fulvinollike polyacetylene metabolites (compounds 2−5) from the prey. They were isolated along with previously reported bromorenierins from the diethyl ether extracts of both the mollusk and the sponge. Their structures were elucidated by NMR spectroscopy and tandem FABMS analysis. Compound 5 exhibited in vitro growth inhibitory effects against the SKMEL-28 melanoma cell line.
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extract of the sponge.6 In addition, a preliminary analysis of the diethyl ether extract of the sponge6 revealed the presence of less polar polyacetylene metabolites co-occurring with the renierines, brominated short chain polyacetylenes previously described from H. f ulva.7 Interestingly, the chromatographic comparison of the lipophilic extracts of the nudibranch with those of the sponge revealed the presence in the mollusk of ether-soluble polyacetylene sponge metabolites, whereas the butanol-soluble polyacetylene fraction was not detected.6 We report here the isolation and the structure determination of these polyacetylenes, compounds 2−5, which are structurally related to fulvinol (6), a C46 linear symmetric polyacetylene reported from a Spanish specimen of Reniera (=Haliclona) f ulva.8 The evaluation of their in vitro growth inhibitory properties in human cancer cells is also described.
he suborder Doridina is the largest nudibranch group containing the most varied and often brilliantly colored species.1 Many dorids are highly specialized sponge-feeders. Consequently, the chemistry of these mollusks is strictly related to that of the sponge diet from which they derive secondary metabolites. This is essential for understanding some basic biological phenomena, including chemical defense mechanisms.2 The spotted sea slug Peltodoris atromaculata is a dorid nudibranch distributed in the Mediterranean Sea and near Atlantic, exclusively occurring and very common in precoralligene and coralligene communities.3 Previous chemical studies conducted on P. atromaculata have reported the presence in the digestive gland of a complex mixture of dietary polyacetylene compounds, petroformynes, sequestered from the sponge Petrosia f iciformis.4 The same authors also demonstrated that the nudibranch is able to find its food by chemotaxis.5 Later, the preference of P. atromaculata for two selected haplosclerid sponges was shown on the basis of field observations and fecal analysis. In particular, it was reported that the nudibranch feeds on P. f iciformis and also on Haliclona f ulva. While P. f iciformis is more common and more frequently consumed, H. f ulva is preferred.3 We have recently investigated the secondary metabolites of a sample of H. f ulva collected off Procida Island (Gulf of Naples) on which three individuals of the nudibranch P. atromaculata were observed grazing. This study led us to characterize nine high molecular weight polyoxygenated acetylenes, fulvynes A−I (e.g., fulvyne A, 1), which were isolated from the butanolic © XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Frozen P. atromaculata (three individuals) were dissected into internal glands and mantles, which were separately extracted in acetone. A sample of the sponge H. f ulva on which the mollusk specimens were observed feeding was also extracted with acetone. After filtration and evaporation of the organic solvent under vacuum, the aqueous residues were partitioned with Et2O and subsequently with n-butanol. The ether extracts were analyzed by TLC and 1H NMR, revealing a similar metabolite Received: April 3, 2014
A
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Chart 1
spectra should be attributable to two magnetically equivalent nuclei. The 1H NMR spectrum contained a singlet at δH 3.21 due to the terminal acetylenic protons (H-1 and H-46) and a doublet at δH 6.18 (2H, d, J = 15.7 Hz, H-4 and H-43) that correlated 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 constant value (JH‑4/H‑5 = 15.7 Hz) indicated the E geometry for the double bond belonging to the terminal residue. The remaining signals at δH 1.98−2.06, 1.48−1.55, and 1.25−1.38 were assigned 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 triple bond, respectively. Moreover, the occurrence in the 13C NMR spectrum 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-1 and C-46) suggested that fulvindione (2) contained two (E)-1yn-3-oxo-4-ene moieties. These residues accounted for eight of the 11 degrees of unsaturation required by the molecular formula. The three remaining unsaturations were attributed to three double bonds, as indicated by the 1H NMR signal at δH 5.35 (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 double bonds was assigned by the value of the allylic methylene carbons9 at δC 27.1−27.2. The three double bonds in the chain
pattern characterized by the presence of polyacetylenes for both the sponge and the mollusk. In particular, in the nudibranch the metabolites of interest were observed to be mainly concentrated in the extract of the internal parts including the digestive gland. This latter extract was subjected to LH-20 Sephadex chromatography (CHCl3/MeOH, 1:1) to recover two selected fractions (A and B) containing polyacetylenes. In particular, fraction A consisted of fulvinol-like long-chain polyacetylenes, whereas fraction B contained short-chain brominated polyacetylenes. These fractions were further purified first by silica gel chromatography (light petroleum ether/Et2O gradient) and subsequently by RP-HPLC (MeOH). The new metabolites (2−5) were obtained from fraction A, whereas purification of fraction B led us to isolate known 18-hydroxyrenierin-1, renierin-2, and 18-hydroxyrenierin-2, which were identified by comparison of the spectroscopic data with values in the literature.7 Compounds 2−5 were also isolated by using the same purification procedures from the extract of the sponge H. f ulva. Compound 2, named fulvindione, had the molecular formula C46H72O2 deduced by ESIMS. Both the 1H and the 13C NMR spectra contained a smaller number of signals than those expected from the molecular formula, suggesting a highly symmetric structure. This implied that each resonance in the B
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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) position 1 3 4 5 6 7 8 9−13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37−39 40 41 42 43 44 46
δH, m (Hz)
fulvinone (3a/3b)b δH, m (Hz)
3.21, s
3.21, s
6.18, d (15.7) 7.25,dt, (15.7, 8.0) 2.31, m 1.55−1.48, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.55−1.48, m 2.31, m 7.25, dt (15.7, 8.0) 6.18, d (15.7)
6.17, d (15.7) 7.25, dt (15.7, 8.0) 2.31, m 1.55−1.48, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.40, m 2.07, m 5.92, dt (15.0, 7.3) 5.61, dd (15.0, 6.2) 4.83, br d (6.2) 2.56, d (2.2)
3.21, s
isofulvinol (4)
hydroxydehydroisofulvinol (5)
δH, m (Hz)
δH, m (Hz)
2.56, d (2.2) 4.83, br d (6.0) 5.60, dd (15.0,6.0) 5.92, dt (15.0,7.3) 2.07, m 1.40, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.40, m 2.07, m 5.92, dt (15.0, 7.3) 5.61, dd (15.0, 6.0) 4.83, br d (6.0) 2.56, d (2.2)
2.56, d (2.2) 4.83, br d (6.2) 5.60, dd (15.0, 6.2) 5.92, dt (15.0, 7.3) 2.07, m 1.40, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.79, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.54, m 4.17, m 5.96, dt (15.4, 7.3) 5.82, dd (15.4, 5.0) 4.90, br d (5.0) 2.58, d (2.2)
a
Assignments were based on COSY, HSQC, and HMBC (J = 10 Hz) experiments. bThe same shifts apply to structure 3b, although the numbering system would be reversed.
compound 2 (Tables 1 and 2). The only difference was due to the presence of a carbinol function [δH 4.83 (1H, br d, J = 6.2 Hz), δC 62.8 (CH)] rather than the carbonyl group in one of the two terminal moieties of the molecule. Analysis of the COSY spectrum of 3 supported this structural hypothesis. In fact, the carbinol proton signal at δH 4.83 correlated with both a doublet 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 δH 2.56 (1H, d, J = 2.2 Hz). This was consistent with an (E)-1-yn3-hydroxy-4-ene moiety.
were located at C-15, C-21, and C-27 by tandem FABMS/MS fragmentation analysis (Figure 1), which was further supported by the measurement of the negative FABMS ion after ozonolysis with oxidative workup (m/z 271 and 215, Supporting Information). 1H and 13C NMR values were assigned as reported in Tables 1 and 2. Compound 3, named fulvinone, was an inseparable mixture of two isomeric polyacetylenes that were indistinguishable by ESIMS and NMR analysis. The molecular formula, C46H74O2, indicated an unsaturation degree less than fulvindione (2). The 1 H and 13C NMR spectra were very similar to those of C
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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 2 3 4 5 6 7 8 9−13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37−39 40 41 42 43 44 45 46
78.8, CH 79.8, C 177.8, C 131.9, CH 155.8, CH 32.7, CH2 27.8, CH2 29.7,b CH2 29.7,b CH2 27.1,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.4,b CH2 29.2,b CH2 27.2,d CH2 130.0,c CH 129.8,c CH 27.1,d CH2 29.7,b CH2 29.8,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.4,b CH2 29.2,b CH2 29.7,b CH2 29.2,b CH2 29.7,b CH2 29.2,b CH2 29.4,b CH2 29.4,b CH2 27.8, CH2 32.7, CH2 155.8, CH 131.9, CH 177.8, C 79.8, C 78.8, CH
78.8, CH 79.8, C 177.8, C 131.9, CH 155.8, CH 32.7, CH2 27.8, CH2 29.8,b CH2 29.4,b CH2 27.1,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.3,b CH2 29.8,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.1,d CH2 29.7,b CH2 29.8,b CH2 27.2,d CH2 130.0,c CH 129.8,c CH 27.1,d CH2 29.4,b CH2 29.8,b CH2 29.6,b CH2 29.3,b CH2 29.6,b CH2 29.8,b CH2 29.6,b CH2 29.7,b CH2 28.8, CH2 31.9, CH2 134.6, CH 128.4, CH 62.8, CH 83.3, C 73.9, CH
74.0, CH 83.3, C 62.8, CH 128.4, CH 134.6, CH 31.9, CH2 28.8, CH2 29.4,b CH2 29.4,b CH2 27.1,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.8,b CH2 29.2,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.1,d CH2 29.8,b CH2 29.6,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.1,d CH2 29.2,b CH2 29.4,b CH2 29.7,b CH2 29.6,b CH2 29.7,b CH2 29.4,b CH2 29.6,b CH2 29.8,b CH2 28.8, CH2 31.9, CH2 134.6, CH 128.4, CH 62.8, CH 83.3, C 74.0, CH
73.9, CH 83.4, C 62.8, C 128.3, CH 134.6, CH 31.9, CH2 28.8, CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 27.1,c CH2 130.0,d CH 129.9,d CH 27.2,c CH2 29.8,b CH2 29.8,b CH2 27.1,c CH2 130.0,d CH 129.7,d CH 27.2,c CH2 29.8,b CH2 29.8,b CH2 27.1,c CH2 130.0,d CH 129.7,d CH 25.6, CH2 129.7,d CH 130.0,d CH 27.1,c CH2 29.4,b CH2 37.1, CH2 71.8, CH 135.9, CH 128.7, CH 62.1, CH 82.8, C 74.4, CH
a
Assignments were based on HSQC and HMBC experiments. b,c,dAssignments with the same superscript in the same column may be interchanged. The same shifts apply to structure 3b, although the numbering system would be reversed.
e
Figure 2. Key tandem FABMS fragment ions derived from isofulvinol (4) [M + Li]+.
The presence of a hydroxy group was further confirmed by the IR band at 3303 cm−1. In order to establish the position of the inner double bonds, fulvinone (3) was subjected to FABMS/MS. Analysis of the fragmentation ion pattern appeared to be inconsistent with the presence of a single compound, suggesting instead a mixture of two isomeric polyacetylenes, 3a and 3b, with the only difference being the location of the carbinol (C-3 or C-44). To confirm this
hypothesis, 3 was oxidized by 2,3-dichloro-5,6-dicyano-1,4benzoquinone (DDQ), and the single product obtained by reaction 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]+, corresponding to the molecular formula C46H76O2. The 1H and 13C NMR data D
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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 of Cell Culture with the Compound of Interest IC50 in vitro growth inhibitory concentration (μM) carcinoma a
compound
A549
MCF-7
fulvindione (2) fulvinone (3) isofulvinol (4) hydroxy-dehydro-isofulvinol (5) etoposide
37 >100 28 31 0.1
82 76 23 15 17
a
glioma a
PC-3
Hs683
86 >100 18 32 2
>100 >100 36 23 1
a
melanoma U373
a
>100 >100 19 15 7
SKMEL28a
mean ± SEM
>100 >100 11 3 1
>84 >96 22 ± 4 20 ± 5 5±3
a
The origin and histological type of each human cell line analyzed are as follows. Carcinoma models included the A549 NSCLC (DSMZ code ACC107), the MCF-7 breast (DSMZ code ACC115), and the PC-3 prostate (DSMZ code ACC465) cancer cell lines. Glioma models included the Hs683 oligodendroglioma (ATCC code HTB-138) and the U373 (ECACC code 08061901) cell lines. One melanoma model included the SKMEL28 (ATCC code HTB-72) cell line. DSMZ means Deutsche Sammlung von Mikroorganismen and Zellkulturen (Braunschweig, Germany), ATCC means American Type Culture Collection (Rockville, MD, USA), and ECACC means European Collection of Cell Cultures (Salisbury, UK).
the presence of a bis-allylic methylene group. Analysis of the C NMR spectrum and 2D NMR experiments confirmed the assignment of the two terminal moieties, whereas the fragmentation of tandem FABMS/MS suggested the position of 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 was applied on the main isofulvinol (4). An aliquot of 4 was treated with (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 at C-3 and C-44 indicated the S configuration of the two asymmetric centers (Figure 4), the same as determined for the related fulvinol (6).8 On the basis of biogenetic considerations, the same configuration was suggested for the corresponding carbons 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 the MTT colorimetric assay (Table 3). Compound 5 was active against SKMEL-28 melanoma cells (IC50 3 μM). The IC50 values for 5 ranged between 15 and 32 μM in the remaining five 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. f ulva. These compounds display a linear alkyl chain of 46 carbons with either a 1-yn-3-ol4-ene or a 1-yn-3-keto-4-ene moiety at each terminus, the same as for the petroformynes, previously described from the Mediterranean sponge Petrosia f iciformis4,11−16 and from P. atromaculata4,5 feeding on it. These metabolites have not been detected in any other Mediterranean sponges. Thus, the
of compound 4 seemed to be identical with those of known fulvinol (6),8 whereas the FABMS/MS data suggested a different position of the inner double bonds. In particular, the fragmentation ion pattern of compound 4 (Figure 2), the same as fulvindione (2), aided us in locating the inner double bonds as depicted. Accordingly, compound 4 was named isofulvinol. Compound 5, hydroxydehydroisofulvinol, had the molecular formula C46H74O3, as deduced from HRESIMS, consistent with the presence of an additional hydroxy group as well as a further unsaturation with respect to isofulvinol (4). The 1H NMR spectrum 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). The small difference observed in the chemical shifts of the two protons was ascribed to the presence of an additional hydroxy group in one of the two terminal moieties, as it was easily deduced 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 olefinic proton at δH 5.60 (1H, dd, J = 15.0 and 6.2 Hz, H-4). This latter proton was connected to the vicinal olefinic proton at δH 5.92 (1H, dt, J = 15.0 and 7.3 Hz, H-5), correlating with a methylene 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) was long-range coupled with the signal at δH 4.90 (1H, br d, J = 5.0 Hz, H-44). This carbinolic methine was connected to the olefinic 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 crosspeak with the methine proton at δH 4.17 (1H, m, H-41), which was 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
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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 and 2; ESIMS m/z 681 [M + Na]+ and HRESIMS m/z 681.5579 [M + Na]+ (calcd for C46H74O2Na, 681.5587); FABMS (NBA matrix) m/z 665 [M + Li]+. Isofulvinol (4): colorless oil; [α]25D −11 (c 0.04, CHCl3); IR (liquid film) 3309, 2924, 2854, 2103, 1713, 1462, 1381, 1018, 964 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS m/z 683 [M + Na]+ and HRESIMS 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 for C46H74O3Na, 697.5536); FABMS (NBA matrix) m/z 681 [M + Li]+. Ozonolysis of Fulvindione (2). An aliquot of compound 2 (0.5 mg) was dissolved in CH2Cl2 (1 mL) and treated with O3 at −78 °C for 15 min. After this time, the excess O3 was removed by a stream of N2, and the reaction mixture was treated with 90% HCOOH/35% H2O2 (2:1, 1 mL) at room temperature (rt) for 15 min. The reaction solution was concentrated and subjected to FABMS/MS analysis. Oxidation of Fulvinone (3a/3b). An aliquot of fulvinone (3) (0.5 mg) was dissolved in dioxane (200 μL) and treated with 2,3-dichloro5,6-dicyano-1,4-benzoquinone (2 mg) at 60 °C. After stirring for 2 h, the reaction mixture was cooled to rt. The reaction solution was filtered, and the filtrate was concentrated and then subjected to reversed-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. It was 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 separately added to two different aliquots of isofulvinol (4) (1.0 mg each) in dry CH2Cl2 (0.5 mL). The resulting mixtures were allowed to stand at rt for 12 h. After the evaporation of the solvent, the mixtures were purified 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 Concentrations in Vitro. The MTT colorimetric assay was used as detailed previously.17,18 Each experimental condition was assessed in six replicates. The origin of each cell line is detailed in the legend of Table 3.
nudibranch P. atromaculata is highly specialized for feeding on the two sponges that contain these specific polyacetylenes.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO DIP 370 digital polarimeter. The UV spectra were recorded on an Agilent 8453 spectrophotometer. 1H and 13C NMR spectra were recorded on DRX 600, Avance 400, and DPX 300 MHz Bruker spectrometers in CDCl3, with chemical shifts reported in ppm 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 a Micromass Q-TOF Micro spectrometer coupled with a HPLC Waters Alliance 2695. The instrument was calibrated by using a PEG mixture from 200 to 1000 MW. Tandem FABMS/MS spectra were recorded on a JEOL JMS-700T at the University of Tokyo using m-nitrobenzyl alcohol (NBA) + LiCl as matrix. Sephadex LH-20 (GE Healthcare BioSciences) was carried out using an open prepacked column. Silica gel chromatography was performed using precoated Merck F254 plates and Merck Kieselgel 60 powder. HPLC purification was carried out on a Waters 510 pump equipped with a Waters differential refractometer R401 detector. Biological Material. Peltodoris atromaculata (three individuals, 6 cm length average) and Haliclona f ulva were collected by scuba diving at 40 m depth off Punta Pizzaco (Procida Island, Gulf of Naples) during May 2009. The animals were immediately transferred to ICB laboratories and frozen. The nudibranch and the sponge were identified by one of us (G.V.). A voucher specimen of P. atromaculata (code DA) and H. f ulva (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 internal glands (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 layers were evaporated under reduced pressure to give Et2O (50.0 mg from the mantle, 140.0 mg from internal glands) and n-BuOH (27.0 mg from the mantle, 100.0 mg from internal glands) residues. The frozen sample of H. f ulva (16 g, dry weight) was immersed in acetone (5 × 200 mL). After evaporation of the solvent under reduced pressure, the aqueous residue was partitioned with Et2O (4 × 400 mL) and subsequently with n-BuOH (3 × 100 mL). The organic layers were concentrated to give Et2O (800.0 mg) and n-BuOH (900.0 mg) residues. The extracts were analyzed by TLC in different solvent systems, revealing a similar secondary metabolite pattern in the two organisms. Compounds 2−5 were isolated from the ether extract of both 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 a column packed with Sephadex LH-20 and eluted with CHCl3/MeOH, 1:1, affording two fractions (A and B) of interest. Fraction A was submitted to SiO2-gel column chromatography (light petroleum ether/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 purified by reversed-phase HPLC (Supelco-Ascentis C18 column, 25 × 0.46 cm, 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 same purification 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 (1.9 mg). Fraction B was chromatographed by SiO2 column (light petroleum ether/Et2O gradient, then CHCl3, and finally MeOH) to afford 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 and 13 C NMR see Tables 1 and 2; ESIMS m/z 679 [M + Na]+ and HRESIMS m/z 679.5400 [M + Na]+ (calcd for C46H72O2Na, 679.5430); FABMS (NBA matrix) m/z 663 [M + Li]+.
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ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR spectra of compounds 2−5. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 0039 081 8675243. Fax: 0039 081 8041770. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The NMR spectra were recorded at the ICB NMR Facility, the staff of which is gratefully acknowledged. The authors thank F
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Mrs. D. Ricciardi for laboratory assistance, Mr. M. Zampa for ESIMS measurements, and Mr. C. Iodice for spectrophotometric measurements. Thanks are also due to T. Gras for the technical assistance in biological assays and H. Leclercqz for the calculation of IC50 concentrations. This research work was partially funded by PRIN 2009 KFMP7Z “Natural products and bioinspired molecules interfering with biological targets involved in control of tumor growth”.
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REFERENCES
(1) Rudman, W. B. In Mollusca: Southern Synthesis. Fauna of Australia; Beesley, P. I.; Ross, G. J. B.; Wells, A., Ed.; CSIRO Publishing: Melbourne, 1998; Vol. 5, Part B, pp 990−1001. (2) Cimino, G.; Ghiselin, M. T. Chemoecology 1999, 9, 187−207. (3) Gemballa, S.; Schermutzki, F. Mar. Biol. 2004, 144, 1213−1222. (4) Castiello, D.; Cimino, G.; De Rosa, S.; De Stefano, S.; Sodano, G. Tetrahedron Lett. 1980, 21, 5047−5050. (5) Castiello, D.; Cimino, G.; De Rosa, S.; De Stefano, S.; Izzo, G.; Sodano, G. In Biologie des Spongaires; Levi, C.; Boury-Esnault, N., Eds.; CNRS: Paris, 1979; pp 413−416. (6) Nuzzo, G.; Ciavatta, M. L.; Villani, G.; Manzo, E.; Zanfardino, A.; Varcamonti, M.; Gavagnin, M. Tetrahedron 2012, 68, 754−760. (7) Cimino, G.; De Stefano, S. Tetrahedron Lett. 1977, 15, 1325− 1328. (8) Ortega, M. G.; Zubia, E.; Carballo, J. L.; Salva, J. J. Nat. Prod. 1996, 59, 1069−1071. (9) Kalinowski, H.-O.; Berger, S.; Braun, S. In Carbon-13 NMR Spectroscopy; John Wiley & Sons: Great Britain, 1988; p 131. (10) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451− 2458. (11) Cimino, G.; Crispino, A.; De Rosa, S.; De Stefano, S.; Sodano, G. Experientia 1981, 37, 924−926. (12) Cimino, G.; De Giulio, A.; De Rosa, S.; De Stefano, S.; Sodano, G. J. Nat. Prod. 1985, 48, 22−27. (13) Cimino, G.; De Giulio, A.; De Rosa, S.; Di Marzo, V. Tetrahedron Lett. 1989, 30, 3563−3566. (14) Cimino, G.; De Giulio, A.; De Rosa, S.; Di Marzo, V. J. Nat. Prod. 1990, 53, 345−353. (15) Guo, Y.-W.; Gavagnin, M.; Trivellone, E.; Cimino, G. Tetrahedron 1994, 50, 13261−13268. (16) Guo, Y.-W.; Gavagnin, M.; Trivellone, E.; Cimino, G. J. Nat. Prod. 1995, 58, 712−722. (17) Van Goietsenoven, G.; Andolfi, A.; Lallemand, B.; Cimmino, A.; Lamoral-Theys, D.; Gras, T.; Abou-Donia, A.; Dubois, J.; Lefranc, F.; Mathieu, V.; Kornienko, A.; Kiss, R.; Evidente, A. J. Nat. Prod. 2010, 73, 1223−1227. (18) Lamoral-Theys, D.; Fattorusso, E.; Mangoni, A.; Perinu, C.; Kiss, R.; Costantino, V. J. Nat. Prod. 2011, 74, 2299−2303.
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Sequestered Fulvinol-Related Polyacetylenes in Peltodoris atromaculata M. Letizia Ciavatta,*,† Genoveffa Nuzzo,† Kentaro Takada,‡ Véronique 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 Cancérologie et de Toxicologie Expérimentale, Faculté de Pharmacie, Université Libre de Bruxelles (ULB), Campus de la Plaine, Boulevard du Triomphe, 1050, Brussels, Belgium S Supporting Information *
ABSTRACT: The Mediterranean dorid nudibranch Peltodoris atromaculata that had been collected while feeding on Haliclona fulva was shown to sequester long-chain fulvinollike polyacetylene metabolites (compounds 2−5) from the prey. They were isolated along with previously reported bromorenierins from the diethyl ether extracts of both the mollusk and the sponge. Their structures were elucidated by NMR spectroscopy and tandem FABMS analysis. Compound 5 exhibited in vitro growth inhibitory effects against the SKMEL-28 melanoma cell line.
T
extract of the sponge.6 In addition, a preliminary analysis of the diethyl ether extract of the sponge6 revealed the presence of less polar polyacetylene metabolites co-occurring with the renierines, brominated short chain polyacetylenes previously described from H. f ulva.7 Interestingly, the chromatographic comparison of the lipophilic extracts of the nudibranch with those of the sponge revealed the presence in the mollusk of ether-soluble polyacetylene sponge metabolites, whereas the butanol-soluble polyacetylene fraction was not detected.6 We report here the isolation and the structure determination of these polyacetylenes, compounds 2−5, which are structurally related to fulvinol (6), a C46 linear symmetric polyacetylene reported from a Spanish specimen of Reniera (=Haliclona) f ulva.8 The evaluation of their in vitro growth inhibitory properties in human cancer cells is also described.
he suborder Doridina is the largest nudibranch group containing the most varied and often brilliantly colored species.1 Many dorids are highly specialized sponge-feeders. Consequently, the chemistry of these mollusks is strictly related to that of the sponge diet from which they derive secondary metabolites. This is essential for understanding some basic biological phenomena, including chemical defense mechanisms.2 The spotted sea slug Peltodoris atromaculata is a dorid nudibranch distributed in the Mediterranean Sea and near Atlantic, exclusively occurring and very common in precoralligene and coralligene communities.3 Previous chemical studies conducted on P. atromaculata have reported the presence in the digestive gland of a complex mixture of dietary polyacetylene compounds, petroformynes, sequestered from the sponge Petrosia f iciformis.4 The same authors also demonstrated that the nudibranch is able to find its food by chemotaxis.5 Later, the preference of P. atromaculata for two selected haplosclerid sponges was shown on the basis of field observations and fecal analysis. In particular, it was reported that the nudibranch feeds on P. f iciformis and also on Haliclona f ulva. While P. f iciformis is more common and more frequently consumed, H. f ulva is preferred.3 We have recently investigated the secondary metabolites of a sample of H. f ulva collected off Procida Island (Gulf of Naples) on which three individuals of the nudibranch P. atromaculata were observed grazing. This study led us to characterize nine high molecular weight polyoxygenated acetylenes, fulvynes A−I (e.g., fulvyne A, 1), which were isolated from the butanolic © XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION Frozen P. atromaculata (three individuals) were dissected into internal glands and mantles, which were separately extracted in acetone. A sample of the sponge H. f ulva on which the mollusk specimens were observed feeding was also extracted with acetone. After filtration and evaporation of the organic solvent under vacuum, the aqueous residues were partitioned with Et2O and subsequently with n-butanol. The ether extracts were analyzed by TLC and 1H NMR, revealing a similar metabolite Received: April 3, 2014
A
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Chart 1
spectra should be attributable to two magnetically equivalent nuclei. The 1H NMR spectrum contained a singlet at δH 3.21 due to the terminal acetylenic protons (H-1 and H-46) and a doublet at δH 6.18 (2H, d, J = 15.7 Hz, H-4 and H-43) that correlated 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 constant value (JH‑4/H‑5 = 15.7 Hz) indicated the E geometry for the double bond belonging to the terminal residue. The remaining signals at δH 1.98−2.06, 1.48−1.55, and 1.25−1.38 were assigned 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 triple bond, respectively. Moreover, the occurrence in the 13C NMR spectrum 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-1 and C-46) suggested that fulvindione (2) contained two (E)-1yn-3-oxo-4-ene moieties. These residues accounted for eight of the 11 degrees of unsaturation required by the molecular formula. The three remaining unsaturations were attributed to three double bonds, as indicated by the 1H NMR signal at δH 5.35 (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 double bonds was assigned by the value of the allylic methylene carbons9 at δC 27.1−27.2. The three double bonds in the chain
pattern characterized by the presence of polyacetylenes for both the sponge and the mollusk. In particular, in the nudibranch the metabolites of interest were observed to be mainly concentrated in the extract of the internal parts including the digestive gland. This latter extract was subjected to LH-20 Sephadex chromatography (CHCl3/MeOH, 1:1) to recover two selected fractions (A and B) containing polyacetylenes. In particular, fraction A consisted of fulvinol-like long-chain polyacetylenes, whereas fraction B contained short-chain brominated polyacetylenes. These fractions were further purified first by silica gel chromatography (light petroleum ether/Et2O gradient) and subsequently by RP-HPLC (MeOH). The new metabolites (2−5) were obtained from fraction A, whereas purification of fraction B led us to isolate known 18-hydroxyrenierin-1, renierin-2, and 18-hydroxyrenierin-2, which were identified by comparison of the spectroscopic data with values in the literature.7 Compounds 2−5 were also isolated by using the same purification procedures from the extract of the sponge H. f ulva. Compound 2, named fulvindione, had the molecular formula C46H72O2 deduced by ESIMS. Both the 1H and the 13C NMR spectra contained a smaller number of signals than those expected from the molecular formula, suggesting a highly symmetric structure. This implied that each resonance in the B
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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) position 1 3 4 5 6 7 8 9−13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37−39 40 41 42 43 44 46
δH, m (Hz)
fulvinone (3a/3b)b δH, m (Hz)
3.21, s
3.21, s
6.18, d (15.7) 7.25,dt, (15.7, 8.0) 2.31, m 1.55−1.48, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.55−1.48, m 2.31, m 7.25, dt (15.7, 8.0) 6.18, d (15.7)
6.17, d (15.7) 7.25, dt (15.7, 8.0) 2.31, m 1.55−1.48, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.40, m 2.07, m 5.92, dt (15.0, 7.3) 5.61, dd (15.0, 6.2) 4.83, br d (6.2) 2.56, d (2.2)
3.21, s
isofulvinol (4)
hydroxydehydroisofulvinol (5)
δH, m (Hz)
δH, m (Hz)
2.56, d (2.2) 4.83, br d (6.0) 5.60, dd (15.0,6.0) 5.92, dt (15.0,7.3) 2.07, m 1.40, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.40, m 2.07, m 5.92, dt (15.0, 7.3) 5.61, dd (15.0, 6.0) 4.83, br d (6.0) 2.56, d (2.2)
2.56, d (2.2) 4.83, br d (6.2) 5.60, dd (15.0, 6.2) 5.92, dt (15.0, 7.3) 2.07, m 1.40, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.38−1.25, m 2.06−1.98, m 5.35, m 5.35, m 2.79, m 5.35, m 5.35, m 2.06−1.98, m 1.38−1.25, m 1.54, m 4.17, m 5.96, dt (15.4, 7.3) 5.82, dd (15.4, 5.0) 4.90, br d (5.0) 2.58, d (2.2)
a
Assignments were based on COSY, HSQC, and HMBC (J = 10 Hz) experiments. bThe same shifts apply to structure 3b, although the numbering system would be reversed.
compound 2 (Tables 1 and 2). The only difference was due to the presence of a carbinol function [δH 4.83 (1H, br d, J = 6.2 Hz), δC 62.8 (CH)] rather than the carbonyl group in one of the two terminal moieties of the molecule. Analysis of the COSY spectrum of 3 supported this structural hypothesis. In fact, the carbinol proton signal at δH 4.83 correlated with both a doublet 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 δH 2.56 (1H, d, J = 2.2 Hz). This was consistent with an (E)-1-yn3-hydroxy-4-ene moiety.
were located at C-15, C-21, and C-27 by tandem FABMS/MS fragmentation analysis (Figure 1), which was further supported by the measurement of the negative FABMS ion after ozonolysis with oxidative workup (m/z 271 and 215, Supporting Information). 1H and 13C NMR values were assigned as reported in Tables 1 and 2. Compound 3, named fulvinone, was an inseparable mixture of two isomeric polyacetylenes that were indistinguishable by ESIMS and NMR analysis. The molecular formula, C46H74O2, indicated an unsaturation degree less than fulvindione (2). The 1 H and 13C NMR spectra were very similar to those of C
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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 2 3 4 5 6 7 8 9−13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37−39 40 41 42 43 44 45 46
78.8, CH 79.8, C 177.8, C 131.9, CH 155.8, CH 32.7, CH2 27.8, CH2 29.7,b CH2 29.7,b CH2 27.1,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.4,b CH2 29.2,b CH2 27.2,d CH2 130.0,c CH 129.8,c CH 27.1,d CH2 29.7,b CH2 29.8,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.4,b CH2 29.2,b CH2 29.7,b CH2 29.2,b CH2 29.7,b CH2 29.2,b CH2 29.4,b CH2 29.4,b CH2 27.8, CH2 32.7, CH2 155.8, CH 131.9, CH 177.8, C 79.8, C 78.8, CH
78.8, CH 79.8, C 177.8, C 131.9, CH 155.8, CH 32.7, CH2 27.8, CH2 29.8,b CH2 29.4,b CH2 27.1,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.3,b CH2 29.8,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.1,d CH2 29.7,b CH2 29.8,b CH2 27.2,d CH2 130.0,c CH 129.8,c CH 27.1,d CH2 29.4,b CH2 29.8,b CH2 29.6,b CH2 29.3,b CH2 29.6,b CH2 29.8,b CH2 29.6,b CH2 29.7,b CH2 28.8, CH2 31.9, CH2 134.6, CH 128.4, CH 62.8, CH 83.3, C 73.9, CH
74.0, CH 83.3, C 62.8, CH 128.4, CH 134.6, CH 31.9, CH2 28.8, CH2 29.4,b CH2 29.4,b CH2 27.1,d CH2 130.0,c CH 129.7,c CH 27.2,d CH2 29.8,b CH2 29.2,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.1,d CH2 29.8,b CH2 29.6,b CH2 27.2,d CH2 130.0,c CH 129.7,c CH 27.1,d CH2 29.2,b CH2 29.4,b CH2 29.7,b CH2 29.6,b CH2 29.7,b CH2 29.4,b CH2 29.6,b CH2 29.8,b CH2 28.8, CH2 31.9, CH2 134.6, CH 128.4, CH 62.8, CH 83.3, C 74.0, CH
73.9, CH 83.4, C 62.8, C 128.3, CH 134.6, CH 31.9, CH2 28.8, CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 29.7,b CH2 27.1,c CH2 130.0,d CH 129.9,d CH 27.2,c CH2 29.8,b CH2 29.8,b CH2 27.1,c CH2 130.0,d CH 129.7,d CH 27.2,c CH2 29.8,b CH2 29.8,b CH2 27.1,c CH2 130.0,d CH 129.7,d CH 25.6, CH2 129.7,d CH 130.0,d CH 27.1,c CH2 29.4,b CH2 37.1, CH2 71.8, CH 135.9, CH 128.7, CH 62.1, CH 82.8, C 74.4, CH
a
Assignments were based on HSQC and HMBC experiments. b,c,dAssignments with the same superscript in the same column may be interchanged. The same shifts apply to structure 3b, although the numbering system would be reversed.
e
Figure 2. Key tandem FABMS fragment ions derived from isofulvinol (4) [M + Li]+.
The presence of a hydroxy group was further confirmed by the IR band at 3303 cm−1. In order to establish the position of the inner double bonds, fulvinone (3) was subjected to FABMS/MS. Analysis of the fragmentation ion pattern appeared to be inconsistent with the presence of a single compound, suggesting instead a mixture of two isomeric polyacetylenes, 3a and 3b, with the only difference being the location of the carbinol (C-3 or C-44). To confirm this
hypothesis, 3 was oxidized by 2,3-dichloro-5,6-dicyano-1,4benzoquinone (DDQ), and the single product obtained by reaction 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]+, corresponding to the molecular formula C46H76O2. The 1H and 13C NMR data D
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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 of Cell Culture with the Compound of Interest IC50 in vitro growth inhibitory concentration (μM) carcinoma a
compound
A549
MCF-7
fulvindione (2) fulvinone (3) isofulvinol (4) hydroxy-dehydro-isofulvinol (5) etoposide
37 >100 28 31 0.1
82 76 23 15 17
a
glioma a
PC-3
Hs683
86 >100 18 32 2
>100 >100 36 23 1
a
melanoma U373
a
>100 >100 19 15 7
SKMEL28a
mean ± SEM
>100 >100 11 3 1
>84 >96 22 ± 4 20 ± 5 5±3
a
The origin and histological type of each human cell line analyzed are as follows. Carcinoma models included the A549 NSCLC (DSMZ code ACC107), the MCF-7 breast (DSMZ code ACC115), and the PC-3 prostate (DSMZ code ACC465) cancer cell lines. Glioma models included the Hs683 oligodendroglioma (ATCC code HTB-138) and the U373 (ECACC code 08061901) cell lines. One melanoma model included the SKMEL28 (ATCC code HTB-72) cell line. DSMZ means Deutsche Sammlung von Mikroorganismen and Zellkulturen (Braunschweig, Germany), ATCC means American Type Culture Collection (Rockville, MD, USA), and ECACC means European Collection of Cell Cultures (Salisbury, UK).
the presence of a bis-allylic methylene group. Analysis of the C NMR spectrum and 2D NMR experiments confirmed the assignment of the two terminal moieties, whereas the fragmentation of tandem FABMS/MS suggested the position of 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 was applied on the main isofulvinol (4). An aliquot of 4 was treated with (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 at C-3 and C-44 indicated the S configuration of the two asymmetric centers (Figure 4), the same as determined for the related fulvinol (6).8 On the basis of biogenetic considerations, the same configuration was suggested for the corresponding carbons 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 the MTT colorimetric assay (Table 3). Compound 5 was active against SKMEL-28 melanoma cells (IC50 3 μM). The IC50 values for 5 ranged between 15 and 32 μM in the remaining five 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. f ulva. These compounds display a linear alkyl chain of 46 carbons with either a 1-yn-3-ol4-ene or a 1-yn-3-keto-4-ene moiety at each terminus, the same as for the petroformynes, previously described from the Mediterranean sponge Petrosia f iciformis4,11−16 and from P. atromaculata4,5 feeding on it. These metabolites have not been detected in any other Mediterranean sponges. Thus, the
of compound 4 seemed to be identical with those of known fulvinol (6),8 whereas the FABMS/MS data suggested a different position of the inner double bonds. In particular, the fragmentation ion pattern of compound 4 (Figure 2), the same as fulvindione (2), aided us in locating the inner double bonds as depicted. Accordingly, compound 4 was named isofulvinol. Compound 5, hydroxydehydroisofulvinol, had the molecular formula C46H74O3, as deduced from HRESIMS, consistent with the presence of an additional hydroxy group as well as a further unsaturation with respect to isofulvinol (4). The 1H NMR spectrum 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). The small difference observed in the chemical shifts of the two protons was ascribed to the presence of an additional hydroxy group in one of the two terminal moieties, as it was easily deduced 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 olefinic proton at δH 5.60 (1H, dd, J = 15.0 and 6.2 Hz, H-4). This latter proton was connected to the vicinal olefinic proton at δH 5.92 (1H, dt, J = 15.0 and 7.3 Hz, H-5), correlating with a methylene 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) was long-range coupled with the signal at δH 4.90 (1H, br d, J = 5.0 Hz, H-44). This carbinolic methine was connected to the olefinic 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 crosspeak with the methine proton at δH 4.17 (1H, m, H-41), which was 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
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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 and 2; ESIMS m/z 681 [M + Na]+ and HRESIMS m/z 681.5579 [M + Na]+ (calcd for C46H74O2Na, 681.5587); FABMS (NBA matrix) m/z 665 [M + Li]+. Isofulvinol (4): colorless oil; [α]25D −11 (c 0.04, CHCl3); IR (liquid film) 3309, 2924, 2854, 2103, 1713, 1462, 1381, 1018, 964 cm−1; 1H and 13C NMR see Tables 1 and 2; ESIMS m/z 683 [M + Na]+ and HRESIMS 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 for C46H74O3Na, 697.5536); FABMS (NBA matrix) m/z 681 [M + Li]+. Ozonolysis of Fulvindione (2). An aliquot of compound 2 (0.5 mg) was dissolved in CH2Cl2 (1 mL) and treated with O3 at −78 °C for 15 min. After this time, the excess O3 was removed by a stream of N2, and the reaction mixture was treated with 90% HCOOH/35% H2O2 (2:1, 1 mL) at room temperature (rt) for 15 min. The reaction solution was concentrated and subjected to FABMS/MS analysis. Oxidation of Fulvinone (3a/3b). An aliquot of fulvinone (3) (0.5 mg) was dissolved in dioxane (200 μL) and treated with 2,3-dichloro5,6-dicyano-1,4-benzoquinone (2 mg) at 60 °C. After stirring for 2 h, the reaction mixture was cooled to rt. The reaction solution was filtered, and the filtrate was concentrated and then subjected to reversed-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. It was 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 separately added to two different aliquots of isofulvinol (4) (1.0 mg each) in dry CH2Cl2 (0.5 mL). The resulting mixtures were allowed to stand at rt for 12 h. After the evaporation of the solvent, the mixtures were purified 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 Concentrations in Vitro. The MTT colorimetric assay was used as detailed previously.17,18 Each experimental condition was assessed in six replicates. The origin of each cell line is detailed in the legend of Table 3.
nudibranch P. atromaculata is highly specialized for feeding on the two sponges that contain these specific polyacetylenes.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on a JASCO DIP 370 digital polarimeter. The UV spectra were recorded on an Agilent 8453 spectrophotometer. 1H and 13C NMR spectra were recorded on DRX 600, Avance 400, and DPX 300 MHz Bruker spectrometers in CDCl3, with chemical shifts reported in ppm 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 a Micromass Q-TOF Micro spectrometer coupled with a HPLC Waters Alliance 2695. The instrument was calibrated by using a PEG mixture from 200 to 1000 MW. Tandem FABMS/MS spectra were recorded on a JEOL JMS-700T at the University of Tokyo using m-nitrobenzyl alcohol (NBA) + LiCl as matrix. Sephadex LH-20 (GE Healthcare BioSciences) was carried out using an open prepacked column. Silica gel chromatography was performed using precoated Merck F254 plates and Merck Kieselgel 60 powder. HPLC purification was carried out on a Waters 510 pump equipped with a Waters differential refractometer R401 detector. Biological Material. Peltodoris atromaculata (three individuals, 6 cm length average) and Haliclona f ulva were collected by scuba diving at 40 m depth off Punta Pizzaco (Procida Island, Gulf of Naples) during May 2009. The animals were immediately transferred to ICB laboratories and frozen. The nudibranch and the sponge were identified by one of us (G.V.). A voucher specimen of P. atromaculata (code DA) and H. f ulva (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 internal glands (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 layers were evaporated under reduced pressure to give Et2O (50.0 mg from the mantle, 140.0 mg from internal glands) and n-BuOH (27.0 mg from the mantle, 100.0 mg from internal glands) residues. The frozen sample of H. f ulva (16 g, dry weight) was immersed in acetone (5 × 200 mL). After evaporation of the solvent under reduced pressure, the aqueous residue was partitioned with Et2O (4 × 400 mL) and subsequently with n-BuOH (3 × 100 mL). The organic layers were concentrated to give Et2O (800.0 mg) and n-BuOH (900.0 mg) residues. The extracts were analyzed by TLC in different solvent systems, revealing a similar secondary metabolite pattern in the two organisms. Compounds 2−5 were isolated from the ether extract of both 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 a column packed with Sephadex LH-20 and eluted with CHCl3/MeOH, 1:1, affording two fractions (A and B) of interest. Fraction A was submitted to SiO2-gel column chromatography (light petroleum ether/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 purified by reversed-phase HPLC (Supelco-Ascentis C18 column, 25 × 0.46 cm, 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 same purification 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 (1.9 mg). Fraction B was chromatographed by SiO2 column (light petroleum ether/Et2O gradient, then CHCl3, and finally MeOH) to afford 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 and 13 C NMR see Tables 1 and 2; ESIMS m/z 679 [M + Na]+ and HRESIMS m/z 679.5400 [M + Na]+ (calcd for C46H72O2Na, 679.5430); FABMS (NBA matrix) m/z 663 [M + Li]+.
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ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR spectra of compounds 2−5. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 0039 081 8675243. Fax: 0039 081 8041770. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The NMR spectra were recorded at the ICB NMR Facility, the staff of which is gratefully acknowledged. The authors thank F
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Mrs. D. Ricciardi for laboratory assistance, Mr. M. Zampa for ESIMS measurements, and Mr. C. Iodice for spectrophotometric measurements. Thanks are also due to T. Gras for the technical assistance in biological assays and H. Leclercqz for the calculation of IC50 concentrations. This research work was partially funded by PRIN 2009 KFMP7Z “Natural products and bioinspired molecules interfering with biological targets involved in control of tumor growth”.
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REFERENCES
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