Additional Insights into the Obtusallene Family: Components of

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Additional Insights into the Obtusallene Family: Components of Laurencia marilzae Adrián Gutiérrez-Cepeda,†,‡ José J. Fernández,*,† Manuel Norte,† Matías López-Rodríguez,† Iván Brito,†,§ Christian D. Muller,⊥ and María L. Souto*,† †

Instituto Universitario de Bio-Orgánica “Antonio González” (IUBO), Centro de Investigaciones Biomédicas de Canarias (CIBICAN), Departamento de Química Orgánica, Universidad de La Laguna (ULL), Avenida Astrofísico Francisco Sánchez, 2, 38206 La Laguna, Tenerife, Spain ‡ Departamento de Química, Instituto de Química, Facultad de Ciencias, Universidad Autónoma de Santo Domingo, Ciudad Universitaria, 1355 Santo Domingo, Dominican Republic § Departamento de Química, Universidad de Antofagasta, Avenida Angamos 601, Antofagasta, Chile ⊥ Laboratoire d’Innovation Thérapeutique, UMR 7200 CRNS, Faculté de Pharmacie, Université de Strasbourg, 67401 Illkirch, France S Supporting Information *

ABSTRACT: The obtusallenes are a significant subset of C15-halogenated acetogenins that incorporate 12-membered cyclic ethers. We have recently reported the isolation from Laurencia marilzae of 12-epoxyobtusallene IV (1) and its related α,β-unsaturated carboxylate ester (2), both of special biogenetic relevance. Here we describe the final step of our study, the isolation of three new analogues (3−5), among these, the first bromopropargylic derivative (3) of this class of macrocyclic C15-acetogenins. The structures were elucidated by analysis of NMR and X-ray data. 12-Epoxyobtusallene IV (1), its new isomer 4, and known obtusallene IV (6) were evaluated for their apoptosis-inducing activities in a human hepatocarcinoma cell line.

R

bromopropargyl moiety may be produced biosynthetically by a bromoperoxidase-catalyzed reaction of the bromine atom on the double bond of a terminal enyne, followed by nucleophilic attack on the C-4 carbon.2

ed algae of the genus Laurencia (order Ceramiales, family Rhodomelaceae) are known to produce C15 acetogenins, a structurally diverse and complex group of halogenated metabolites, which arise from fatty acid metabolism.1 Structurally most of these compounds are characterized as cyclic ethers varying in ring size and usually containing a conjugated enyne or bromoallene terminus.2 An interesting group of these halogenated nonterpenoids incorporates 12-membered cyclic ethers in their structures. Twelve of them have been predominantly isolated from L. obtusa,3−7 and seven were recently identified by us from L. marilzae.8,9 Among these, the isolation of 12-epoxyobtusallene IV (1) and its related α,β-unsaturated carboxylate ester (2) led Braddock and co-workers to propose their biogenetic connection by epoxidation of bromoallenes.10 Previously, various studies had also focused on further understanding of the biogenesis of bromoallenes,11−13 based on the early pioneering work of Murai14 and leading to biosynthetically inspired syntheses.12,13,15 As the final stage of our study, we now report the isolation and structure characterization of three new compounds (3−5) ́ from extracts of this organism (Laurencia marilzae Gil-Rodriguez, ́ et M.T. Funji).16 One of these compounds contains a Senties bromopropargylic terminus, the first example for the macrocyclic family. The bromopropargylic unit is scarce even in marine organisms, and to date there have been just eight examples among more than 180 acetogenins.1,17−24 The terminal © XXXX American Chemical Society and American Society of Pharmacognosy

To evaluate their anticancer potential, compounds 1, 4, and obtusallene IV (6)8 were tested to determine their apoptosisinducing potential against the human hepatocellular carcinoma cell line HepG2. Received: December 5, 2015

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DOI: 10.1021/acs.jnatprod.5b01080 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

Table 1. 1H and 13C NMR Data (600 MHz, CDCl3) for Compounds 3−5 marilzanin (3) position

δC, type

1 2 3 4 5

77.0, 79.2, 37.7, 71.0, 31.9,

CH C CH CH CH2

6 7 8

78.9, CH 62.1, CH 38.9, CH2

9 10 11

77.9, CH 46.8, CH 35.5, CH2

12 13 14 15 OCH3

54.1, 61.1, 72.5, 10.1,

CH CH CH CH3

δH, mult. (J in Hz) 2.68, br s 4.42, br d (3.9) 4.06, ddd (1.8, 3.9, 11.2) β 2.31, ddd (1.8, 10.8, 14.0) α 1.92, ddd (1.8, 11.2, 14.0) 4.23, ddd (1.8, 1.8, 10.8) 4.44, br dd (1.8, 4.5) β 2.51, dd (6.8, 14.4) α 2.43, ddd (4.5, 9.7, 14.4) 4.80, ddd (5.8, 6.8, 9.7) 4.49, ddd (2.9, 5.8, 12.6) β 2.63, dd (2.9, 14.9) α 1.88, ddd (8.7, 12.6, 14.9) 3.14, dd (2.1, 8.7) 3.05, dd (2.1, 3.5) 4.59, dd (3.5, 7.1) 1.09, d (7.1)

12S,13S-epoxy-obtusallene IV (4) δC, type 73.9, 200.6, 103.8, 67.9, 37.3,

CH C CH CH CH2

77.5, CH 62.5, CH 41.9, CH2 79.1, CH 52.8, CH 35.7, CH2 57.3, 61.2, 77.3, 18.2,

CH CH CH CH3

δH, mult. (J in Hz) 6.04, dd (1.0, 5.6) 5.40, dd (5.7, 5.8) 4.52, m β 2.02, ddd (2.3, 11.2, 14.9) α 1.75, ddd (2.5, 11.5, 14.9) 4.47, m 4.56, m β 2.94, dt (6.5, 14.8) α 2.53, ddd (1.0, 8.0, 14.8) 4.57, m 4.29, m β 2.72, ddd (7.1, 8.9, 15.1) α 1.97, dd (6.1, 15.1) 3.28, m 3.29, m 3.29, m 1.45, d (6.1)

compound 5 δC, type 166.4, 121.8, 148.8, 70.7, 36.0,

C CH CH CH CH2

76.9, CH 60.8, CH 40.5, CH2 76.3, CH 47.5, CH 43.9, CH2 62.4, 76.7, 74.5, 11.6, 51.8,

CH CH CH CH3 CH3

δH, mult. (J in Hz) 6.05, dd (1.3, 15.8) 7.10, dd (6.2, 15.8) 4.55, dddd (1.3, 2.5, 6.2, 9.4) β 2.16, ddd (2.5, 11.1, 14.5) α 1.60, ddd (2.6, 9.4, 14.5) 4.22, ddd (2.6, 3.5, 11.1) 4.48, br dd (3.5, 6.1) β 2.56, ddd (6.1, 9.1, 14.4) α 2.39, dd (6.7, 14.4) 4.65, ddd (4.5, 6.7, 9.1) 4.51, ddd (2.4, 4.5, 11.7) α 2.67, ddd (1.1, 11.7, 15.5) β 2.57, ddd (2.4, 9.6, 15.5) 4.80, br dd (4.0, 9.6) 3.77, dd (3.4, 4.0) 4.19, dd (3.4, 6.9) 1.21, d (6.9) 3.76, s

Marilzanin (3) was isolated as colorless crystals. The molecular formula of 3 was deduced as C15H19Br2ClO3 from HRESIMS. The 1H and 13C NMR spectra of 3 (Table 1) compared to reported data for 1 revealed that both share the macrocycle core. However, compared to 1, compound 3 lacked the bromoallene unit. In its place was a bromopropargylic unit, supported by the 1H NMR resonances at δH 2.68 (H-1) and 4.42 (H-3) and the 13C NMR signals at δC 77.0 (C-1), 79.2 (C-2), and 37.7 (C-3). The connectivity of the bromopropargylic unit with the C-4 carbon was confirmed based on HMBC correlations from H-4 and H-1 to C-2/C-3 (Figure 1). The absolute

Figure 2. ORTEP diagram of marilzanin (3). The ellipsoids are shown at the 30% probability level. Figure 1. COSY () and selected HMBC (→) correlations of 3.

(Supporting Information, Figure S1). However, analysis of the 2D NMR spectra (COSY, HSQC, and HMBC) for 4 and 1 suggested the same planar structure. The relative configuration of 4 was established through the analysis of ROESY correlations, coupling constants, and comparison to those of 1. Owing to the overlapping 1H NMR signals observed in CDCl3, a different solvent (C6D6) was used to record the experimental data (Figure S1, Table S1). ROESY enhancements observed from H3-15 (δH 1.30) and H-4 (δH 4.22) to H-13 (δH 2.90) and from H-13 to H-10 (δH 3.48) and H-9 (δH 3.93) revealed an identical orientation for all of them, evoking a different relative configuration at C-13 in 4 vs 1. In addition, ROE enhancements between H-12 (δH 3.06) and H-6 (δH 4.05), H-7 (δH 3.86), and H-14 (δH 3.24) located these protons on the opposite face of the molecule (Figure 3A). The above data provide evidence of the trans-orientation of the epoxide protons. This was confirmed by the small coupling constant between H-12 and H-13 (3JH‑12,H‑13 = 1.9 Hz; Table S1) and thus determined the configuration of 4 to

configuration of 3 was established by means of a single-crystal X-ray diffraction study on crystals obtained from a CH2Cl2/ n-hexane solvent mixture. A computer-generated perspective drawing of the X-ray model of 3 is shown in Figure 2. On this basis, the absolute configuration was assigned as 3R, 4R, 6R, 7R, 9S, 10S, 12R, 13R, and 14S. 12S,13S-Epoxyobtusallene IV (4), an amorphous, white solid, displayed common bromoallene NMR data [δC 200.6 (C), 103.8 (CH), 73.9 (CH); δH 6.04 (dd, J = 1.0, 5.6 Hz), 5.40 (dd, J = 5.7, 5.8 Hz)] and physicochemical properties such as a 1958 cm−1 stretch in the IR spectrum. The molecular formula was established as C15H19Br2ClO3 based on HRESIMS data, identical to the molecular formula of 12-epoxyobtusallene IV (1).8 The most significant differences were observed at the deshielded 1H NMR chemical shifts of H-12, H-13, and H3-15 (δH 3.28, 3.29, and 1.45 in 4 vs δH 3.18, 3.03, and 1.08 in 1) and the shielded methine H-14 (δH 3.29 in 4 vs δH 4.54 in 1) B



Note

The apoptosis-inducing activities of the bromoallenes 1 and 4 were investigated against the human hepatocellular carcinoma cell line HepG2 (HB-8065). Interestingly, both of these acetogenins induced apoptosis by more than 65% and 70% of HepG2 cells at 50 μM, respectively. A sample of obtusallene IV (6) (reisolated in the previous study)8 was also evaluated but showed only weak activity (47% apoptotic HepG2 cells at 50 μM), which highlights epoxidation at C-12 and C-13 as a functionality that improves apoptosis induction activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Büchi 535 melting point apparatus and are uncorrected. Optical rotations were measured in CHCl3 on a PerkinElmer 241 polarimeter by using a Na lamp. Ultraviolet−visible spectra were run as MeOH solutions on a Jasco Inc. V-560 spectrophotometer. IR spectra were recorded on a Bruker IFS55 spectrometer. NMR spectra were recorded on either a Bruker Avance 600 instrument equipped with a 5 mm TCI inverse detection cryoprobe operating at 600/ 150 MHz (1H/13C nuclei). Chemical shifts were reported in ppm referenced to solvent signals (CDCl3: δH 7.26, δC 77.0; C6D6: δH 7.16, δC 128.4). Standard Bruker NMR pulse sequences were utilized. HRESIMS data were obtained on a Micromass Autospec spectrometer. Single-crystal X-ray diffraction analysis was measured on an Oxford Diffraction Supernova Dual diffractometer equipped with an Atlas CCD detector, using Cu Kα radiation. HPLC separations were carried out with an LKB 2248 system equipped with a photodiode array detector. Gel filtration flash chromatography was carried out using Sephadex LH-20 (Sigma-Aldrich). TLC were performed on AL Si gel Merck 60 F254 plates with visualization by spraying with phosphomolybdic acid reagent (10% in EtOH) and heating. Biological Material. Specimens of Laurencia marilzae were ́ Floral, Tenerife, collected by hand in the intertidal zone at Paraiso Canary Islands (28°07′12″ N, 16°46′45″ W). A voucher specimen was deposited at Departamento de Biologiá Vegetal, Botánica, Universidad de La Laguna, Tenerife (TFC Phyc 9860). Extraction and Isolation. Fresh alga (1.3 kg) was extracted with CH2Cl2/MeOH (1:1, v/v) at room temperature, and the solvent removed in vacuo to yield a dark green, viscous oil (35.1 g). The extract was first chromatographed using a Sephadex LH-20 column, 7 × 50 cm, with n-hexane/CH2Cl2/MeOH (2:1:1) as mobile phase. Selected fractions exhibiting similar TLC profiles were rechromatographed by medium-pressure normal-phase chromatography using a Lobar LiChroprep Si 60 column, 40−63 μm, 25 × 310 mm, with n-hexane/EtOAc/MeOH (14:5:1). Final purifications were achieved on a μ-Porasil HPLC column, 10 μm, 19 × 150 mm, using n-hexane/ EtOAc (9:1 and 7:3), yielding compounds 5 (1.0 mg), 4 (5.0 mg), and 3 (1.5 mg), in order of increasing polarity. Marilzanin (3): colorless needles; mp 120−122 °C; [α]25D −5 (c 0.1, CHCl3); IR (CHCl3) νmax 3289, 2854, 2117, 1732, 1570, 1450, 1384, 1261, 1092, 1029 cm−1; 1H and 13C NMR data (CDCl3), Table 1; HRESIMS m/z 462.9293, 464.9275, 466.9268, 468.9231 [M + Na]+ (46:100:71:15) (calcd for C15H1979Br235ClO3Na, 462.9287; C15H1979Br81Br35ClO3Na, 464.9267; C15H1981Br235ClO3Na, 466.9246; C15H1981Br237ClO3Na, 468.9217). (12S,13S)-Epoxyobtusallene IV (4): white, amorphous substance; [α]25D +127 (c 0.5, CHCl3); UV (MeOH) λmax (log ε) 203 (3.53) nm; IR (CHCl3) νmax 3442, 2973, 2930, 1958, 1729, 1377, 1265, 1129, 1087, 1044 cm−1; 1H and 13C NMR data (CDCl3), Table 1; HRESIMS m/z 462.9291, 464.9272, 466.9248, 468.9232 [M + Na]+ (45:100:71:15) (calcd for C 15 H 19 79 Br 2 35 ClO 3 Na, 462.9287; C15H1979Br81Br35ClO3Na, 464.9267; C15H1981Br235ClO3Na, 466.9246; C15H1981Br237ClO3Na, 468.9217). Compound 5: white, amorphous substance; [α]25D +9 (c 0.06, CHCl3); UV (MeOH) λmax (log ε) 203 (3.32) nm; IR (CHCl3) νmax 3443, 2919, 2850, 1721, 1656, 1438, 1349, 1278, 1168, 1087 cm−1; 1H and 13 C NMR data (CDCl3), Table 1; HRESIMS m/z 467.0000, 468.9984, 470.9964; [M + Na]+ (64:100:49) (calcd for C16H2379Br35Cl2O5Na,

Figure 3. Key ROESY correlations of compounds 4 (A) and 5 (B).

be 4R, 6R, 7R, 9S, 10S, 12S, 13S, and 14S, as well as displaying the bromoallene unit with an S configuration.8 Compound 5 has a molecular formula of C16H23BrCl2O5. The NMR data of 5 clearly showed a similarity to compound 2,8 indicating the presence of an α,β-unsaturated methyl ester [δC 166.4 (C), 148.8 (CH), 121.8 (CH), 51.8 (CH3); δH 7.10 (dd, J = 6.2, 15.8 Hz), 6.05 (dd, J = 1.3, 15.8 Hz); 3.76 (s)]. The main difference is the replacement of the epoxy group between C-12 and C-13 in 2 by a chlorohydrin in 5, C-12 (chloride) and C-13 (hydroxy) resonances, as shown by the shielded carbons at C-11 [δC 43.9 (CH2)], C-12 [δC 62.4 (CH)], and C-13 [δC 76.7 (CH)] in 5 vs δC 35.7, 54.0, and 61.6 in 2. 1H−1H J coupling and ROESY associations confirmed the same relative configurations at carbons C-4, C-6, C-7, C-9, C-10, and C-14 as in 2. The α-orientation (syn) of the C-12 chloro and C-13 hydroxy functions was inferred from the key ROE enhancements observed between H-12 and H-9/ H-13/H3-15 (Figure 3B). Thus, the relative configuration of 5 was determined to be 4R*, 6R*, 7R*, 9S*, 10S*, 12R*, 13S*, and 14S*. Biogenetically, it seems reasonable to suggest that 12S,13Sepoxyobtusallene IV (4) might be considered the biosynthetic precursor of compound 5 as well as that of marilzabicycloallene B9 recently isolated by us (Scheme 1). Thus, through the Scheme 1. Suggested Biogenesis of Compound 5 and Marilzabicycloallene B

formation of an oxonium ion, this intermediate could evolve to generate marilzabicycloallene B by nucleophilic attack by water or to give compound 5 in a sequence of chloride attack, epoxidation, attack by water, and HBr elimination.9,10 C



Note

467.0004; C16H2381Br35Cl2O5Na, 468.9983; C16H2379Br37Cl2O5Na, 470.9945). X-ray Crystal Structure Analysis. Colorless crystals of 3 were obtained in a solvent mixture of CH2Cl2 and n-hexane. Intensity data were collected at 293 K on an Oxford Diffraction Supernova Dual Atlas CCD diffractometer, using Cu Kα (λ = 1.5418 Å) radiation. Data collection, cell refinement, and data reduction were performed with the CrysAlisPRO25 set of programs. The structure was solved and refined using SHELX programs.26 The H atoms on C were placed at calculated positions with C−H distances 0.95−1.00 Å and refined using a riding model. The absolute structure is based on the refinement of the Flack parameter,27 x = 0.04(6), against 1395 Cu Kα Bijvoet pairs. Crystal data: C15H19Br2Cl1O3, Mw = 442.57, monoclinic, space group, P21, Z = 4, a = 15.775(5) Å, b = 7.137(3) Å, c = 16.382(5) Å; β = 107.97(3)°, V = 1754.4(11) Å3, μ(Cu Kα) = 7.32 mm−1, ρcalc = 1.68 g cm−3; S = 0.995, final R indices: R1 = 0.0647 and Rw = 0.1704 for 2329 observed from 3600 independent and 6132 measured reflections (θmax = 63.51, I > 2σ(I) criterion and 383 parameters); maximum and minimum residues are 0.36 and −0.59 e Å−3, respectively. Crystallographic data (excluding structure factor tables) have been deposited in the Cambridge Crystallographic Data Center as supplementary publication no. CCDC990739. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB1EZ, UK (fax: int. + (1223) 336 033); e-mail: deposit@ ccdc.cam.ac.uk). Apoptosis-Inducing Activity. The human hepatocellular carcinoma cell line HepG2 (ATCC HB-8065) was obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% FBS, penicillin/streptomycin (100 units/mL and 100 μg/mL), and glutamine (2 mM). HepG2 cells were grown in a humidified atmosphere with 5% CO2 at 37 °C in 75 cm2 flasks up to 70−80% confluence prior to treatment. Marine compounds were diluted in DMSO. For our purpose, cell lines were treated with the appropriate working concentrations mixed with the cell culture medium. The highest concentration of DMSO (for treated and untreated cells) never exceeded 0.25% (v/v) to avoid side effects such as cell toxicity or induction of differentiation. Apoptosis activities were assessed by evaluating the externalization of phosphatidylserine and toxicity by nucleus labeling by propidium iodide. For that purpose, cells were cultured and treated or not with our extracts in 96-well plates. Cell were washed with PBS, trypsinized, centrifuged, and resuspended in the preserved supernatant of the first wash so as to keep all nonadherent apoptotic cells present. A minimum of 2000 cells was acquired per sample and analyzed on the InCyte software (Guava/Millipore/Merck). Apoptosis rates were assessed by capillary cytometry (Guava EasyCyte Plus, Millipore Merck) using annexin V-FITC (ImmunoTools) and PI (MiltenyiBiotec Inc.) according to the manufacturer’s recommendations. Gates were drawn around the appropriate cell populations using a forward scatter vs side scatter acquisition dot plot to exclude debris. Cytometer performances were checked weekly using the Guava EasyCheck kit 4500-0025 (Merck/Millipore/Guava).

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants SAF2011-28883-C03-01 and CTQ2014-55888-C03-01-R (the Spanish MINECO), EU FP7-KBBE-3-245137 (MAREX), and EU FP7-REGPOT-2012CT2012-316137 (IMBRAIM). A.G.-C. acknowledges MAECAECID for a Doctoral Fellowship. The authors thank Dr. M. C. ́ Gil-Rodriguez for the taxonomic classification of the alga and Dr. J. Peluso (UMR7200 CNRS) for efficient technical support in cellular test.

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(1) Blunt, J. W.; Copp, B. R.; Keyzers, R. A.; Munro, M. H. G.; Prinsep, M. R. Nat. Prod. Rep. 2015, 32, 116−211 and earlier reviews in this series. (2) Erickson, K. L. In Marine Natural Products: Chemical and Biological Perspectives; Scheuer, P. J., Ed.; Academic Press: New York, 1979; Vol. 5, Chapter 4, pp 131−257. (3) Cox, P. J.; Imre, S.; Islimyeli, S.; Thomson, R. H. Tetrahedron Lett. 1982, 23, 579−580. (4) Ö ztunç, A.; Imre, S.; Wagner, H.; Norte, M.; Fernández, J. J.; González, R. Tetrahedron 1991, 47, 2273−2276. (5) Ö ztunç, A.; Imre, S.; Wagner, H.; Norte, M.; Fernández, J. J.; González, R. Tetrahedron Lett. 1991, 32, 4377−4380. (6) Braddock, D. C.; Rzepa, H. S. J. Nat. Prod. 2008, 71, 728−730. (7) Kokkotou, K.; Ioannou, E.; Nomikou, M.; Pitterl, F.; Vonaparti, A.; Siapi, E.; Zervou, M.; Roussis, V. Phytochemistry 2014, 108, 208− 219. (8) Gutiérrez-Cepeda, A.; Fernández, J. J.; Gil, L. V.; LópezRodríguez, M.; Norte, M.; Souto, M. L. J. Nat. Prod. 2011, 74, 441− 448. (9) Gutiérrez-Cepeda, A.; Fernández, J. J.; Norte, M.; Souto, M. L. Org. Lett. 2011, 13, 2690−2693. (10) Braddock, D. C.; Clarke, J.; Rzepa, H. S. Chem. Commun. 2013, 49, 11176−11178. (11) Braddock, D. C. Org. Lett. 2006, 8, 6055−6058. (12) Braddock, D. C.; Millan, D. S.; Pérez-Fuertes, Y.; Pouwer, R. H.; Sheppard, R. N.; Solanski, S.; White, A. J. P. J. Org. Chem. 2009, 74, 1835−1841. (13) Braddock, D. C.; Bhuva, R.; Pérez-Fuertes, Y.; Pouwer, R. H.; Roberts, C. A.; Ruggiero, A.; Sheppard, R. N.; Stokes, E. S. E.; White, A. J. P. Chem. Commun. 2008, 1419−1421. (14) Murai, A. In Comprehensive Natural Product Chemistry; Barton, D., Nakanishi, K., Meth-Cohn, O., Eds.; Pergamon: Elmsford, NY, 1999; Vol. 1, pp 303−324. (15) Braddock, D. C.; Bhuva, R.; Millan, D. S.; Pérez-Fuertes, Y.; Roberts, C. A.; Sheppard, R. N.; Solanski, S.; Stokes, E. S. E.; White, A. J. P. Org. Lett. 2007, 9, 445−448. (16) Gil-Rodríguez, M. C.; Sentíes, A.; Díaz-Larrea, J.; Cassano, V.; Fujii, M. T. J. Phycol. 2009, 45, 264−271. (17) Wang, B.-G.; Gloer, J. B.; Ji, N.-Y.; Zhao, J.-C. Chem. Rev. 2013, 113, 3632−3685. (18) González, A. G.; Martín, J. D.; Norte, M.; Pérez, R.; Rivera, P.; Rodríguez, M. L.; Fayos, J.; Perales, A. Tetrahedron Lett. 1983, 24, 4143−4146. (19) González, A. G.; Martín, J. D.; Norte, M.; Rivera, P.; Ruano, J. Z. Tetrahedron 1984, 40, 3443−3447. (20) Caccamese, S.; Toscano, R. M. Gazz. Chim. Ital. 1986, 116, 177−179. (21) Norte, M.; Fernández, J. J.; Ruano, J. Z.; Rodríguez, M. L.; Pérez, R. Phytochemistry 1988, 27, 3537−3539. (22) Norte, M.; Fernández, J. J.; Ruano, J. Z. Tetrahedron 1989, 45, 5987−5994. (23) Giordano, G.; Mayol, L.; Notaro, G.; Piccialli, V.; Sica, D. J. Chem. Soc., Chem. Commun. 1990, 22, 1559−1561.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01080. NMR spectra for the new compounds in CDCl3 and for compound 4 in C6D6 (PDF) Crystallographic data (CIF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail (J. J. Fernández): [email protected]. *E-mail (M. L. Souto): [email protected]. Tel: +34 922318586. Fax: +34 922318571. D



Note

(24) Imre, S.; Aydogmus, Z.; Guener, H.; Lotter, H.; Wagner, H. Z. Naturforsch., C, J. Biosci. 1995, 50, 743−747. (25) CrysAlisPro, Version 1.171.34.40, CrysAlis171.NET; Oxford Diffraction Ltd., 2010. (26) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122. (27) Flack, H. D. Acta Crystallogr. 1988, A41, 96−103.

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Additional Insights into the Obtusallene Family: Components of

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