Structure and absolute stereochemistry of novel C15-halogenated acetogenins from the anaspidean mollusc Aplysia dactylomela

Tetrahedron 61 (2005) 7456–7460

Structure and absolute stereochemistry of novel C15-halogenated acetogenins from the anaspidean mollusc Aplysia dactylomela Emiliano Manzo,a,* M. Letizia Ciavatta,a Margherita Gavagnin,a Raffaella Puliti,a Ernesto Mollo,a Yue-Wei Guo,b,* Carlo Andrea Mattia,c Lelio Mazzarellad and Guido Ciminoa a

Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, I 80078 Pozzuoli (Na), Italy State Key Laboratory of Drug Research, Institute of Materia Medica, Shanghai, Institutes for Biological Sciences, Chinese Academy of Sciences, 201203, Shanghai, China c Dipartimento di Scienze Farmaceutiche, Universita’ di Salerno, via Ponte Don Melillo, I-84084 Fisciano, Salerno, Italy d Dipartimento di Chimica, Universita` “Federico II”, Complesso Universitario di Monte S. Angelo, via Cintia, I-80126 Napoli, Italy b

Received 8 March 2005; revised 5 May 2005; accepted 19 May 2005 Available online 15 June 2005

Abstract—Three novel halogenated C15-acetogenins, compounds 1–3, have been isolated, together with known metabolites, from a South China Sea collection of the anaspidean mollusc Aplysia dactylomela. The structures have been suggested by both NMR analysis and comparison with literature data. The structure of 1 was confirmed by a single crystal X-ray study, which also allowed the establishment of its absolute stereochemistry. q 2005 Elsevier Ltd. All rights reserved.

1. Introduction Opisthobranch molluscs, belonging to the order Anaspidea, are strictly herbivorous and widely distributed in both temperate and tropical waters. Generally these molluscs feed on red and brown algae from which they sequester selected bioactive secondary metabolites that are stored in the digestive gland and secreted in the mucus for defensive purpose.1 Among anaspideans, different species belonging to the family Aplysiidae, which consists of nine genera including Aplysia and Dolabella, have been the object of several chemical studies that resulted in the finding of a great number of dietary compounds, usually typical algal halogenated metabolites.2 We add here the isolation of three novel halogen-containing acetogenins, compounds 1–3, along with known related cyclic ethers 4 and 5, and brasilane-type sesquiterpenoid 6, from a South China Sea collection of Aplysia dactylomela Rang, 1828. Worldwide distributed, this large mollusc and its secondary metabolite pattern have been deeply investigated. Previous chemical reports on A. dactylomela from distinct geographical areas, including the finding of sesquiterpenes with eudesmane,3 chamigrane, 4 and cuparane 5 skeletons, dolabellane Keywords: Marine natural products; Molluscs; Acetogenins; Absolute stereochemistry. * Corresponding authors. Tel.: C39 0818675177; fax: C39 0818041770 (E.M.); e-mail: [email protected] 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.05.051

diterpenes6 as well as C15 ethereal lipids,7,8 suggest trophic relationship with red algae of the genus Laurencia. The function and the dynamics of the acquired algal metabolites have also been investigated in A. dactylomela.9

2. Results and discussion The mollusc (four specimens) was collected along the coast of Hainan Island, in the South China Sea, during January 2002. Frozen individuals were carefully dissected into mantle and internal organs that were separately extracted by acetone using ultrasound. The ethereal soluble fractions from the extracts of the two distinct anatomical parts were analysed by silica-gel TLC chromatography, showing similar secondary metabolite patterns. In particular, two series of apolar compounds [spots at Rf 0.9–0.8 (light petroleum ether/diethyl ether, 9:1) and at Rf 0.85–0.70 (light petroleum ether/diethyl ether, 1:1)] were detected, along with the usual lipids and sterols, in the extract of both mantle and internal glands by spraying TLC plates with CeSO4. A mixture of more polar terpenoid metabolites, the structure of which is under investigation, has been also found in both extracts. The mantle ether extract (606 mg) was subjected to a silica gel column eluted with light petroleum ether/diethyl ether gradient to give pure compounds 4 (4.0 mg), 2 (0.8 mg), 3

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(0.2 mg), 5 (11.0 mg), 1 (6.4 mg), and 6 (4.0 mg), in order of increasing polarity. Compounds 1–3 were unprecedented enantiomers of known algal molecules, whereas the other metabolites were identified by their spectral data as (C)-3Epinnatifidenyne (4), previously isolated from Laurencia pinnatifida,7a (C)-laurenyne (5), already reported from Laurencia obtusa,8 and (C)-brasilenol (6), first isolated from both the mollusc Aplysia brasiliana and the red alga Laurencia obtusa.10 The absolute stereochemistry of compounds 4–6, previously established by either asymmetric synthesis7d,10b or X-ray analysis,8 has been assigned by comparison of their [a]D values with literature data. In order to get further amounts of minor metabolites 2 and 3, the diethyl ether extract (16 g) of the internal glands was submitted to silica-gel column purification in the same conditions as used for the mantle extract, obtaining in a similar manner compounds 4 (13 mg), 2 (0.9 mg), 3 (1.8 mg), 5 (19 mg), 1 (16 mg), and 6 (13 mg).

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methine at d 4.07 (dC 63.8, C-7) and the methylene at d 2.61–2.74 (dC 37.9, C-5) in turn coupled with the signal at d 6.18. The proton at d 4.07 was linked to another methylene at d 2.58–2.73 (dC 33.7, C-8) coupled with the double bond protons at d 5.87 (dC 129.9, C-9) and d 5.79 (dC 127.9, C-10) in turn correlated with the methylene signal at d 2.22 (dC 31.4, C-11). Another methine at d 4.07 (dC 78.7, C-12) was cross-peak coupled with the latter methylene and with the methine proton at d 3.96 (dC 62.1, C-13) which was linked to an ethyl group (dH 1.94–2.05, dC 29.3, H2-14; dH 1.09, dC 12.5, H3-15). Long range correlations between H-6 (d 4.34) and C-12 (d 78.7) suggested the presence of an ether linkage in agreement with the five degrees of unsaturation required by the molecular formula and by the absence of hydroxyl groups in the IR spectrum. The trans geometry of the C-3/ C-4 double bond was suggested by the value of the coupling constant (JZ16 Hz) between H-3 and H-4. The NMR data of compound 1 (all assigned as reported in Section 3) were identical with those of (C)-(3E)-13-epipinnatifidenyne, recently isolated from Laurencia obtusa,11 whereas the sign of [a]D value was opposite suggesting that 1 should be its enantiomer. With the aim of confirming the structure of 1, a suitable single crystal, obtained by careful crystallization from n-hexane, was used for an X-ray diffraction study. The results are shown in Figure 1 and in Section 3. X-ray analysis confirmed the proposed structure, and indicated the orientation of substituents at the oxocene ring as follows: the five-carbon chain present at C-6 and containing the terminal enyne group was equatorially oriented, while the chlorine atom at C-7 and the bromo-containing propyl residue at C-12 were axially oriented but in opposite directions. Furthermore, the diffraction analysis allowed the establishment of the absolute stereochemistry at the four chiral centres 6R,7R,12S,13S. Therefore, compound 1 is a diastereoisomer of known (C)-(3E)-pinnatifidenyne (4)7b differing in the absolute configurations at C-6 and C-7 carbons which in the algal metabolite were both determined to be S.

Compound 1 showed the molecular formula C15H20BrClO, deduced by the sodiated molecular peak in the HRESMS spectrum at m/z 353.0290 (MCNa)C. The 1H NMR spectrum exhibited signals for a terminal methyl group [d 1.09 (3H, t, JZ7.5 Hz, H3-15)], four olefinic protons [d 5.62 (1H, dd, JZ16, 5 Hz, H-3), d 5.79 (1H, m, H-10), d 5.87 (1H, m, H-9), and d 6.18 (1H, m, H-4)], four deshielded methine groups [d 3.96 (1H, m, H-13), d 4.07 (2H, m, H-7 and H-12), and d 4.34 (1H, m, H-6)], an acetylenic proton [d 2.84 (1H, d, JZ2 Hz, H-1)] and four methylenes [multiplets at d 2.8–1.9 integrating for 8H], strongly suggesting a non-terpenoid halogen-containing structure. According to the molecular formula, the 13C NMR spectrum contained 15 signals, attributed to two sp carbons, four sp2 carbons, and the remaining to sp3 carbons. The presence of a terminal conjugated enyne group was suggested by 1H–1H and long-range 1H–13C connectivities between the acetylenic proton at d 2.84 (dC 77.2, C-1) and the olefinic signals at d 5.62 (dC 112.0, C-3) and d 6.18 (dC 141.4, C-4) and confirmed by the IR absorption at 3280 and 2140 cmK1. The signal at d 4.34 (dC 73.8, C-6) was coupled with both a

Figure 1.

On the basis of these data the structure of (3E)-13-epipinnatifidenyne11 must be revised as the enantiomer of compound 1, this being the (C)-3E,12R,13R-pinnatifidenyne (ent-1). Compound 2 showed the molecular formula C15H20BrClO, the same as 1, deduced by the sodiated molecular peak in the HRESMS spectrum at m/z 353.0281 (MCNa)C. Analysis

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of 1H and 13C NMR spectra indicated a nine-membered cyclic ether skeleton also revealing some structural analogies with 1, in particular the same enyne-containing side-chain. Comparison of the spectral data of compound 2 with those reported by Norte et al. for (3E,12R,13R)obtusenyne,7b the absolute stereochemistry of which has been determined by total synthesis,12 indicated that 2 had the same structure, including the relative stereochemistry. However, the two molecules differed in the sign of the [a]D value, indicating that 2 was the enantiomer of (3E)-Norte’s obtusenyne, thus being the (C)-3E,6R,7R-obtusenyne. Compound 3 displayed a close structural relationship with 2, being the corresponding 3Z-isomer. Spectral data of 3 were identical with those reported by Norte et al. for (3Z,12R,13R)-obtusenyne,7b,12 with the exception of the [a]D value, that was opposite, thus indicating, analogously with 2, that 3 was the enantiomer of (3Z)-Norte’s obtusenyne. In conclusion, chemical analysis of the lipid content of a population of A. dactylomela from South China Sea has confirmed, according to the chemical studies so far reported, the ability of the mollusc to accumulate from its diet typical algal secondary metabolites, halogenated acetogenins and brasilane sesquiterpenoids. The observed transfer of the algal metabolites from the digestive glands to the external part, the mantle, is an indirect evidence of the potential defensive role of these compounds. The novel acetogenins 1–3 are enantiomers of known molecules previously isolated from red algae of genus Laurencia. The absolute stereochemistry of 1 has been secured by X-ray diffraction study whereas the absolute configuration of compounds 2 and 3 have been suggested by comparison of their optical rotation values with those reported in the literature for the corresponding enantiomers, the structure of which have been demonstrated by total synthesis.12 In order to test biological properties of these molecules and to establish the probable role as deterrents against predators, feeding experiments with Carassius auratus were carried out on all of the isolated compounds, as described in the literature.13 Compounds 1 and 5 were found to be active at a concentration of 50 mg/cm2. Even though no evidence of feeding preferences of Chinese A. dactylomela was detected in field, our results suggest, by chemical analogy, that this mollusc feeds on red algae belonging to the genus Laurencia, re-using algal metabolites in chemical defense. It could be a starting point for further field investigations aimed to assess the existence on the South China coast of a Laurencia species, which should contain enantiomers of C-15 halogenated acetogenins previously isolated from Laurencia obtusa. 3. Experimental 3.1. General experimental procedures TLC plates (Merk Silica Gel 60 F254) were used for analytical TLC and Merck Kieselgel 60 was used for preparative column chromatography. HRESIMS were

carried out on a Micromass Q-TOF micro. HPLC purifications were carried out on a Waters liquid chromotograph equipped with Waters 501 pump and a Waters R401 RI detector. Normal-phase purifications were conducted by using Kromasil Silica, 5 m (250!4.60 mm, Phenomenex). NMR spectra, recorded at the NMR Service of Istituto di Chimica Biomolecolare of CNR (Pozzuoli, Italy), were acquired on a Bruker Avance-400 operating at 400 MHz, in CDCl3 (d values are reported referred to CHCl3 at 7.26 ppm) using an inverse probe fitted with a gradient along the Z-axis. 13C NMR were recorded on a Bruker DPX-300 operating at 300 MHz (d values are reported to CDCl3, 77.0 ppm) using a dual probe. Optical rotations were measured on a Jasco DIP 370 digital polarimeter. IR spectra were measured on a Biorad FTS 155 FTIR spectrophotometer. 3.2. Biological material A. dactylomela (four specimens) was collected by SCUBA diving at a depth of 20 m along the coast of Hainan Island, in South China Sea, during January 2002. Biological material was immediately frozen, then transferred to ICB Naples, and stored at K80 8C until extraction. A voucher specimen is stored for inspection at ICB (HN-59). 3.3. Extraction and isolation procedures A. dactylomela individuals were carefully dissected into mantle and internal organs that were separately extracted by acetone using ultrasound. Filtration of the two homogenates gave an aqueous Me2CO filtrate that was concentrated in vacuo to give a gummy residue. The residue was suspended in H2O and extracted sequentially with diethyl ether and n-BuOH. The mantle ether extract (606 mg) was subjected to a silica gel column eluting with light petroleum ether/ diethyl ether gradient to give the compounds 1 (6.4 mg), 4 (4.0 mg), 5 (11.0 mg), 6 (4.0 mg), and a less polar fraction that was purified on n-phase HPLC (eluent: n-hexane/ AcOEt, 99.5/0.5, flow rate 1 ml/min) yielded 2 (0.8 mg) and 3 (0.2 mg). The digestive gland ether extract (16 g) was purified by a silica gel column eluting with light petroleum ether/diethyl ether gradient to give, in the same way, the compounds 1 (16 mg), 2 (0.9 mg), 3 (1.8 mg), 4 (13 mg), 5 (19 mg), 6 (13 mg). The known compounds 4–6 have been identified by comparison of their spectral data (1H NMR, 13C NMR, MS) with those reported in the literature. Compound 4: [a]D C28.2 (CHCl3, c 1.7); [a]D lit.7aZC62.0 (CHCl3, c 8.9). Compound 5: [a]D C14.2 (CHCl3, c 3.0); [a]D lit.8Z C22.64 (CHCl3, c 2.3). Compound 6: [a]D C25.0 (CHCl3, c 1.7); [a]D lit.10aZC33.4 (CHCl3, c 1.6). 3.3.1. Compound 1. Crystal solid, Rf 0.8 (petroleum ether/ diethyl ether 1:1); [a]D K17.2 (CHCl3, c 2.2); IR (KBr) nmax 3280, 2140 cmK1; HRESIMS m/z 353.0290 (MCNa), 35 1 H NMR Calcd for C 15H 79 20 Br ClOCNa 353.0285. (CDCl3): d 6.18 (1H, m, H-4), d 5.87 (1H, m, H-9), d 5.79 (1H, m, H-10), d 5.62 (1H, dd, JZ5, 16 Hz, H-3), d 4.34 (1H, m, H-6), d 4.07 (1H, m, H-7), d 4.07 (1H, m, H-12), d 3.96 (1H, m, H-13), d 2.84 (1H, d, JZ2 Hz, H-1), d 2.74 (1H, m, H-5a), d 2.73 (1H, m, H-8a), d 2.61 (1H, m, H-5b), d

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2.58 (1H, m, H-8b), d 2.22 (2H, m, H2-11), d 2.05 (1H, m, H-14a), d 1.94 (1H, m, H-14b), d 1.09 (3H, t, 7.5, H3-15). 13 C NMR (CDCl3): d 141.4 (C-4, CH), d 129.9 (C-9, CH), d 127.9 (C-10, CH), d 112.0 (C-3, CH), d 81.7 (C-2, C), d 78.7 (C-12, CH), d 77.2 (C-1, CH), d 73.8 (C-6, CH), d 63.8 (C-7, CH), d 62.1 (C-13, CH), d 37.9 (C-5, CH2), d 33.7 (C-8, CH2), d 31.4 (C-11, CH2), d 29.3 (C-14, CH2), d 12.5 (C-15, CH3).

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˚ 3. The absolute configuration was determined using 1494 A Friedel pairs with a final Flack parameter of 0.041(14). Bond distances and angles agree well with generally accepted values and there were no abnormally short intermolecular contacts. All the crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (CCDC number: 266836). 3.5. Biological assays

3.3.2. Compound 2. Pale yellow oil, Rf 0.8 (petroleum ether/diethyl ether 9:1); [a]D C18 (CHCl3, c 0.17); IR (KBr) nmax 3320, 3000, 2920 cmK1; HRESIMS m/z 35 353.0281 (MCNa) Calcd for C 15H 79 20 Br ClOCNa 1 353.0285. H NMR (CDCl3): d 6.12 (1H, ddd, JZ7.6, 7.6, 15.5 Hz, H-4), d 5.64 (1H, m, H-3), d 5.61 (1H, m, H-10), d 5.59 (1H, m, H-9), d 4.21 (1H, m, H-12), d 4.08 (1H, m, H-7), d 3.51 (1H, m, H-6), d 3.20 (1H, m, H-13), d 2.84 (1H, d, JZ2.2 Hz, H-1), d 2.71 (1H, m, H-5a), d 2.57 (2H, m, H-11), d 2.51 (1H, m, H-5b), d 2.45 (2H, m, H-8), d 1.88 (2H, m, H-14), d 0.85 (3H, t, JZ7.5 Hz, H-15). 13C NMR (CDCl3): d 140.6 (C-4, CH), d 130.1 (C-10, CH), d 128.5 (C-9, CH), d 112.3 (C-3, CH), d 83.3 (C-13, CH), d 80.8 (C-6, CH), d 77.0 (C-1, CH), d 61.6 (C-7, CH), d 54.2 (C-12, CH), d 37.1 (C-5, CH2), d 34.2 (C-11, CH2), d 33.4 (C-8, CH2), d 27.7 (C-14, CH2), d 9.72 (C-15, CH3). 3.3.3. Compound 3. Pale yellow oil, Rf 0.8 (petroleum ether/diethyl ether 9:1); [a]D C10 (CHCl3, c 0.2); IR (KBr) nmax 3300, 3000, 2940 cmK1; HRESIMS m/z 353.0288 35 (MCNa) Calcd for C15H 79 20 Br ClOCNa 353.0285. 1 H NMR (CDCl3): d 6.03 (1H, dt, JZ7.5, 10.6 Hz, H-4), d 5.60 (1H, m, H-10), d 5.57 (1H, m, H-9), d 5.56 (1H, d, JZ10.6 Hz, H-3), d 4.26 (1H, m, H-12), d 4.06 (1H, m, H-7), d 3.58 (1H, m, H-6), d 3.22 (1H, m, H-13), d 3.15 (1H, d, JZ2.4 Hz, H-1), d 2.84 (2H, m, H-5), d 2.59 (2H, m, H-11), d 2.44 (2H, m, H-8), d 1.89 (2H, m, H-14), d 0.85 (3H, t, JZ7.4 Hz, H-15). 13C NMR (CDCl3): d 140.1 (C-4, CH), d 130.0 (C-10, CH), d 128.6 (C-9, CH), d 111.1 (C-3, CH), d 83.4 (C-13, CH), d 82.8 (C-1, CH), d 80.9 (C-6, CH), d 61.8 (C-7, CH), d 54.1 (C-12, CH), d 34.7 (C-5, CH2), d 34.1 (C-11, CH2), d 33.5 (C-8, CH2), d 27.6 (C-14, CH2), d 9.6 (C-15, CH3). 3.4. X-ray structure determination of 1 Crystal data: colourless prismatic crystal from n-hexane; C15H20BrClO, MW 331.67; crystal dimensions 0.32! 0.20!0.12 mm; crystal system orthorhombic; space group ˚ , bZ P2 121 21; unit cell dimensions aZ5.0780(3) A ˚ , cZ19.191(3) A ˚ ; VZ1574.9(3) A ˚ 3; ZZ4; 16.1610(6) A calculated density 1.399 g/cm3; F000Z680. A total of 10,798 reflections, 3614 of which were independent (RintZ0.0635), were measured at room temperature using ˚) graphite-monochromated Mo Ka radiation (lZ0.71073 A on a Bruker Kappa CCD diffractometer. Data were collected up to qZ27.678 (99.4% of completeness). The structure was solved by direct methods (SIR97) and refined with fullmatrix least-squares calculations on F2 using SHELXL. The R value was 0.0398 (RwZ0.1017) based on 2169 observed reflections (IO2s(I)) and 163 variable parameters. The nonhydrogen atoms were refined anisotropically and hydrogen atoms were included at the ideal positions but not refined. The residual electron density is in the range K0.31–0.36 e/

Feeding-deterrence tests against gold fish Carassius auratus were conducted according to literature procedures.13 Compounds 1–6 were assayed at 50 mg/cm2 and the activity was shown by compounds 1 and 5.

Acknowledgements The authors are indebted to Professor V. Roussis for providing NMR spectra of (3E)-12R,13R-pinnatifidenyne. Thanks are due to the ICB NMR service and D. Melck of the staff service, C. Iodice for spectrophotometric measurements, R. Turco for the drawing support and G. Villani for the antifeedant bioassay. X-ray experiments were performed using the equipment of the ‘Centro Regionale di Competenza Nuove Tecnologie per le Attivita` Produttive’ of Campania Region. This research has been partially supported by an Italian-Chinese joint project (CNR/CAS 2001–2004) and by Regione Campania project: ‘Valorizzazione di risorse naturali non tradizionali della Campania: studio chimico di invertebrati marini’.

References and notes 1. (a) Cimino, G.; Ciavatta, M. L.; Fontana, A.; Gavagnin, M. In Tringali, C., Ed.; Bioactive compounds from natural sources; Taylor & Francis: London, 2001; Vol. 15, pp 577–637. (b) Carefoot, T. H. Oceanogr. Mar. Biol. Annu. Rev. 1987, 25, 167–284. (c) Yamada, K.; Kigoshi, H. Bull. Chem. Soc. Jpn. 1997, 70, 1479–1489. 2. Blunt, J. W.; Copp, B. R.; Munro, M. H. G.; Northcote, P. T.; Prinsep, M. R. Nat. Prod. Rep. 2004, 21, 1–49. 3. Yamamura, S.; Hirata, Y. Bull. Chem. Soc. Jpn. 1971, 44, 2560–2562. Baker, B.; Ratnapala, L.; Mahindaratane, M. P. D.; Dilip de Silva, E.; Tillekeratne, L. M. V.; Jeong, J. H.; Scheuer, P. J.; Seff, K. Tetrahedron 1988, 44, 4695–4701. Ojika, M.; Kigoshi, H.; Yoshikawa, K.; Nakayama, Y.; Yamada, K. Bull. Chem. Soc. Jpn. 1992, 65, 2300–2302. Dieter, R. K.; Kinnel, R.; Meinwald, J.; Eisner, T. Tetrahedron Lett. 1979, 19, 1645–1648. Ojika, M.; Yoshida, Y.; Okumura, M.; Ieda, S.; Yamada, K. J. Nat. Prod. 1990, 53, 1619–1622. 4. Fedorov, S. N.; Shubina, L. K.; Kalinovsky, A. I.; Lyakhova, E. G.; Stonik, V. A. Tetrahedron Lett. 2000, 41, 1979–1982. 5. Ichiba, T.; Higa, T. J. Org. Chem. 1986, 3364–3366. 6. Gonzalez, A. G.; Cataldo, F.; Fernande`z, J.; Norte, M. J. Nat. Prod. 1987, 50, 1158–1159. 7. (a) Gonzalez, A. G.; Martin, J. D.; Martin, V. S.; Norte, M.; Perez, R.; Ruano, J. Z. Tetrahedron 1982, 38, 1009–1014. (b) Norte, M.; Gonzalez, A. G.; Cataldo, F.; Rodriguez, M. L.;

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E. Manzo et al. / Tetrahedron 61 (2005) 7456–7460

Brito, I. Tetrahedron 1991, 47, 9411–9418. (c) King, T. J.; Imre, S.; Oztunc, A.; Thomson, R. H. Tetrahedron Lett. 1979, 16, 1453–1454. (d) Kim, H.; Choi, W. J.; Jung, J.; Kim, S.; Kim, D. J. Am. Chem. Soc. 2003, 125, 10238–10240. 8. Falshaw, C. P. Tetrahedron Lett. 1980, 21, 4951–4954. 9. Rogers, C. N.; De Nys, R.; Charlton, T. S.; Steinberg, P. D. J. Chem. Ecol. 2000, 26, 721–743. 10. (a) Stallard, M. O.; Fenical, W. Tetrahedron 1978, 34,

2077–2081. (b) Greene, A. E.; Serra, A. A.; Barreiro, E. J.; Costa, P. R. R. J. Org. Chem. 1987, 52, 1170–1172. 11. Iliopoulou, D.; Vagias, C.; Harvala, C.; Roussis, V. Phytochemistry 2002, 59, 111–116. 12. Awakura, D.; Fujiwara, K.; Murai, A. Chem. Lett. 1999, 461–462. 13. Cimino, G.; De Rosa, S.; De Stefano, S.; Sodano, G. Comp. Biochem. Physiol. 1982, 73B, 471–474.