Cystoseira usneoides: A Brown Alga Rich in Antioxidant and Anti

Article pubs.acs.org/jnp

Cystoseira usneoides: A Brown Alga Rich in Antioxidant and Antiinflammatory Meroditerpenoids Carolina de los Reyes,† María J. Ortega,† Hanaa Zbakh,‡,§ Virginia Motilva,‡ and Eva Zubía*,† †

Departamento de Química Orgánica, Facultad de Ciencias del Mar y Ambientales, Universidad de Cádiz, 11510 Puerto Real (Cádiz), Spain ‡ Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain § Department of Biology, Faculty of Sciences, University Abdelmalek Essâadi, 93030 Tetouan, Morocco S Supporting Information *

ABSTRACT: Twelve new meroditerpenoids, 1−12, along with eight known compounds, have been isolated from the brown alga Cystoseira usneoides collected off the coast of Tarifa (Spain). The structures of the new metabolites have been established by spectroscopic techniques. All of the new compounds consist of a toluhydroquinone-derived nucleus linked to a regular diterpenoid moiety, which can either be acyclic or contain an ether ring. Most structural diversity arises from the presence of different oxygenated functionalities and unsaturations along the two terminal isoprenoid units of the diterpene backbone. Twelve of the isolated meroditerpenes have been tested in antioxidant assays. All of them have shown radical-scavenging activity. The most active compounds were cystodiones G (1) and H (2), 11-hydroxyamentadione (15), and amentadione (16), which exhibited antioxidant activities in the range of 77−87% that of the Trolox standard. In anti-inflammatory assays, cystodiones G (1) and M (6), cystone C (9), 11-hydroxyamentadione (15), and amentadione (16) showed significant activity as inhibitors of the production of the proinflammatory cytokine TNF-α in LPS-stimulated THP-1 human macrophages.

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included other types of bioactivity such as antifouling, antioxidant, or anti-inflammatory properties, which have opened new perspectives for the research and potential applications of this class of algal natural products. In the field of natural antifoulants, several metabolites from C. baccata and C. tamariscifolia, including some phloroglucinol−meroterpenoid hybrids, together with related compounds from Halydris species (Sargassaceae), have shown antifouling activity against marine microorganisms and macrofoulers.12−14 The antioxidant properties of the polyprenyltoluquinols of Cystoseira, although expected from the presence of the hydroquinone-type ring, have only recently been demonstrated for the metabolites of C. crinita and C. usneoides by using various assays that have allowed the identification of compounds with antioxidative capacities comparable to those of α-tocopherol or its synthetic analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).15,16 In the field of the anti-inflammatory natural products, we have recently shown the in vitro activity of various meroditerpenes from C. usneoides as inhibitors of the production of tumor necrosis factor-α (TNF-α).16 This is a multifunctional cytokine whose dysregulation has been implicated in chronic inflammatory and autoimmune diseases such as rheumatoid arthritis, psoriatic arthritis, and inflamma-

atural products of mixed biogenesis consisting of a quinone or hydroquinone ring system attached to a terpenoid moiety have frequently been isolated from marine algae and invertebrates such as sponges and ascidians.1−4 Interestingly, structural features of the isolated meroterpenoids such as the positioning of the prenyl chain and other substituents on the quinone/hydroquinone ring have been noted to be associated with the phylum of the source organism.1 Thus, metabolites derived from a quinone or hydroquinone ring substituted by a prenyl chain at C-2 and a methyl group at C-6 (toluquinone or toluquinol) have almost exclusively been obtained from brown algae of the family Sargassaceae.1,5 Within this family, the species of the genus Cystoseira (formerly family Cystoseiraceae), which occur mostly in the Mediterranean Sea and along the Northeastern Atlantic coasts,6 have been the most prolific source of meroterpenoids. In particular, these algae produce an array of tetraprenyltoluquinol-derived metabolites that may exhibit regular isoprenoid frameworks, linear or cyclic, and up to three different rearranged skeletons.7−9 Aside from the knowledge of the structures and chemotaxonomic significance of the meroterpenoids produced by Cystoseira algae,7,8,10 the biological properties of these metabolites have been underexplored, and most of the described studies have been focused on the cytotoxic activity.9,11 However, recent reports of new metabolites have © 2016 American Chemical Society and American Society of Pharmacognosy

Received: December 2, 2015 Published: February 9, 2016 395

DOI: 10.1021/acs.jnatprod.5b01067 J. Nat. Prod. 2016, 79, 395−405

Journal of Natural Products

Article

at δH 2.15 (3H, s, 6′-Me), 1.84 (3H, d, J = 1.5 Hz, Me-19), 1.68 (3H, br s, Me-20), 1.32 (6H, s, Me-16 and Me-17), and 1.29 (3H, s, Me-18), while the 13C NMR spectrum (Table 1) contained two carbonyl resonances at δC 204.9 (C-12) and 201.6 (C-5), 12 signals between 165 and 110 ppm assigned to an aromatic ring and six olefinic carbons, and two signals at δC 79.5 (C-11) and 71.5 (C-15) due to oxygenated sp3 carbons. These data were consistent with a meroditerpenoid structure formed by a toluhydroquinone ring and an acyclic diterpenoid portion that bears two ketone carbonyls, three double bonds, and two tertiary alcohol groups. Further, the NMR data were closely related to those of usneoidone Z (13), a co-metabolite previously described,16 except for the absence in the spectra of 1 of the signals due to the methoxy group. Therefore, cystodione G (1) was the 1′-O-demethyl derivative of usneoidone Z (13). Cystodione H (2) possessed the molecular formula C27H38O5, determined by HRESIMS. The 1H NMR spectrum differed from that of 1 by the presence of a doublet due to a methyl group at δH 1.06 (J = 6.9 Hz) and a one-proton multiplet at δH 2.89, which were identified as Me-18 and H-11, respectively, from their mutual coupling in the COSY spectrum and the HMBC correlation of the methyl protons with the carbonyl at C-12 (δC 206.9) (Figure 1). Also, differing from 1, the 13C NMR spectrum of 2 contained only one signal due to an oxygenated sp3 carbon (δC 71.3). This was identified as C-15 from its HMBC correlations with the gem-dimethyl group at the end of the chain (δH 1.32, 6H, s) and with two olefinic protons at δH 6.95 (d, J = 15.9 Hz, H-14) and 6.35 (d, J = 15.9 Hz, H-13), which were in turn correlated with the carbonyl at C-12. All of these data led to the proposal of structure 2 for cystodione H. The NMR spectra of cystodiones I (3) and J (4) indicated that they were another two members of the cystodione series. A distinctive feature of the 1H NMR spectra of both compounds was the presence of the signal of a methoxy group at δH 3.64.

tory bowel disease.17,18 Nowadays, because of the serious side effects caused by the anti-TNF-α biological drugs in clinical use, great interest has been aroused in the development of small molecules that suppress TNF-α activity.19,20 In this context, our previous findings on the TNF-α inhibitory activity of the meroterpenoids of C. usneoides prompted us to investigate new compounds of this family and to obtain further data on their biomedical potential. For this purpose we have studied a new sample of C. usneoides that was collected in Tarifa, on the southern coast of Spain. Herein we describe the isolation and structure determination of 12 new meroditerpenoids: the cystodiones 1−6 and the cystones 7−12. In addition, eight known compounds were obtained: usneoidone Z (13),16 11hydroxy-1′-O-methylamentadione (14),16 11-hydroxyamentadione (15),21 amentadione (16),22 1′-O-methyl-6-cis-amentadione,23 1′-O-methylamentadione,23 cystomexicone A, and cystomexicone B.24 We also report the activities exhibited by compounds 1−6, 8−11, 15, and 16 in assays aimed to detect their radical-scavenging properties and their activity as inhibitors of the pro-inflammatory cytokine TNF-α.

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RESULTS AND DISCUSSION Frozen specimens of C. usneoides were extracted with MeOH, and after evaporation of the solvent under reduced pressure, the aqueous residue was extracted with Et2O. The resulting extract was subjected to column chromatography using as eluents nhexane/Et2O (1:1, v/v), then Et2O, CHCl3/MeOH mixtures, and finally MeOH. The fractions eluted with Et2O and CHCl3/ MeOH (9:1, v/v) were further separated by column chromatography, and the resulting subfractions subjected to repeated NP- and RP-HPLC to yield the new meroditerpenoids 1−12 together with the eight known compounds. Cystodione G (1) possessed the molecular formula C27H38O6, determined by HRESIMS, which indicated nine degrees of unsaturation in the molecule. The 1H NMR spectrum (Table 1) exhibited signals due to six methyl groups 396



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Table 1. 1H and 13C NMR Data (CD3OD) for Meroditerpenes 1−4a 1b position

δC, type

1 2 3 4 5 6 7 8

29.9, CH2 129.4, CH 131.2, C 56.2, CH2 201.6, C 124.3, CH 161.5, C 34.6, CH2

9

23.2, CH2

10

40.1, CH2

11 12 13

79.5, C 204.9, C 120.7, CH

14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 6′-CH3 −OCH3

156.2, CH 71.5, C 29.3,d CH3 29.2,d CH3 25.2, CH3 25.4, CH3 16.6, CH3 146.5, C 130.8, C 114.6, CH 151.5, C 115.9, CH 127.8, C 16.9, CH3

δH, m (J in Hz) 3.31, m 5.44, br t (7.3) 3.08, s 6.20, br s 2.57, 2.51, 1.52, 1.25, 1.76, 1.64,

ddd (11.7, 9.3, 6.4) ddd (11.7, 9.6, 6.1) m m m m

6.83, d (15.4) 7.01, d (15.4) 1.32, 1.32, 1.29, 1.84, 1.68,

s s s d (1.5) br s

6.39, br s 6.39, br s 2.15, s

2b

3b

4c

δC, type

δH, m (J in Hz)

δC, type

29.9, CH2 129.4, CH 131.3, C 56.2, CH2 201.6, C 124.2, CH 161.7, C 34.4, CH2

3.31, m 5.44, br t (7.3)

29.4, CH2 129.5, CH 131.4, C 56.2, CH2 201.3, C 124.1, CH 161.8, C 34.5, CH2

3.33, d (7.3) 5.42, br t (7.3)

2.57, m

29.4, CH2 129.6, CH 131.3, C 56.2, CH2 201.5, C 124.3, CH 161.6, C 34.6, CH2

26.7, CH2

1.41, m

23.3, CH2

33.6, CH2

1.71, m 1.32, m 2.67, m

38.5, CH2

26.8, CH2 34.2, CH2 44.9, CH 206.9, C 125.6, CH 155.2, CH 71.3, C 29.3, CH3 29.3, CH3 17.1, CH3 25.5, CH3 16.6, CH3 146.5, C 130.8, C 114.6, CH 151.5, C 115.9, CH 127.8, C 16.9, CH3

3.09, s 6.20, br s 2.58, m 2.55, m 1.39, m 1.64, m 1.30, m 2.89, m 6.35, d (15.9) 6.95, d (15.9) 1.32, 1.32, 1.06, 1.85, 1.68,

s s d (6.9) d (1.5) br s

6.39, s 6.39, s 2.15, s

47.6, CH 216.2, C 44.5, CH2 75.1, CH 73.2, C 26.4,e CH3 24.5,e CH3 16.4, CH3 25.6, CH3 16.7, CH3 150.7, C 135.6, C 115.0, CH 154.3, C 116.3, CH 132.7, C 16.4, CH3 60.9, CH3

δH, m (J in Hz)

3.09, s 6.20, br s

2.71, dd (16.6, 2.9) 2.61, m 3.85, dd (9.5, 2.7) 1.18, 1.13, 1.06, 1.86, 1.71,

s s d (6.9) d (1.5) s

6.43, br s 6.43, br s 2.19, s 3.64, s

δC, type

δH, m (J in Hz) 3.32, d (7.3) 5.42, br t (7.3) 3.09, s 6.20, br s 2.57, ddd (12.0, 9.4, 6.2) 2.50 ddd (12.0, 9.4, 5.9) 1.67, m 1.30, m 1.61, m 1.58, m

84.9, C 219.7, C 39.0, CH2 80.5, CH 71.8, C 25.8,f CH3 25.6,f CH3 20.8, CH3 25.4, CH3 16.6, CH3 150.8, C 135.6, C 115.0, CH 154.3, C 116.4, CH 132.7, C 16.4, CH3 61.0, CH3

2.45, dd (18.3, 9.4) 2.41, dd (18.3, 7.0) 4.04, dd (9.4, 7.0) 1.16, 1.24, 1.14, 1.85, 1.70,

s s s d (1.2) s

6.43, d (2.9) 6.44, d (2.9) 2.19, s 3.64, s

a

Assignments aided by COSY, HSQC, HMBC, and NOESY experiments. b1H at 500 MHz, 13C at 125 MHz. c1H at 600 MHz, 13C at 150 MHz. d−f Assignments marked with the same letter in the same column may be interchanged.

possessed the same structure as 1 and 2 from C-1 to C-10. Therefore, the structural differences had to be located on the terminal portion of the diterpenoid moiety. In particular, the remaining NMR data of cystodione I (3), together with the molecular formula C28H42O6 determined by HRESIMS, indicated that this compound must contain from C11 to C-18 of the diterpenoid framework a carbonyl group (δC 216.2), a secondary alcohol group [δC 75.1/δH 3.85 (C-14/H14)], and a tertiary alcohol group (δC 73.2, C-15). The HMBC correlations of both oxygenated sp3 carbons with the methyl groups Me-16 and Me-17 at the end of the chain [δH 1.18 (3H, s) and 1.13 (3H, s)] defined the location of the hydroxy groups at C-14 and C-15 (Figure 1). The COSY coupling of the oxymethine proton H-14 with two methylene protons at δH 2.71 (H-13a) and 2.61 (H-13b) that were correlated in the HMBC with the carbonyl group defined the location of the carbonyl at C-12. On the other hand, according to the NMR data of cystodione J (4), this compound accommodated from C-11 to C-18 a carbonyl group (δC 219.7) and three oxygenated sp3 carbons, one of them an oxymethine [δC 80.5/δH 4.04 (C-14/H-14)] and the other two fully substituted [δC 84.9 (C-11) and 71.8 (C-15)]. The location of a tertiary oxygenated sp3 carbon at C15 and the oxymethine at C-14 was supported by the HMBC

Figure 1. Key COSY (bold bond) and HMBC (→) correlations observed for the isoprenoid chain (C-9 to C-18) of cystodiones H (2), I (3), and J (4).

The location of this group at C-1′ was supported by the correlations of the methoxy protons in the HMBC spectrum with the aromatic carbon C-1′ and in the NOESY spectrum with the methyl group at C-6′ (δH 2.19) and the benzylic methylene protons H-1. In addition, the analysis of the COSY and HMBC spectra indicated that compounds 3 and 4 397



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Table 2. 1H and 13C NMR Data (CD3OD) for Meroditerpenes 5, 6, and 15a 5b position

a

δC, type

1 2 3 4 5 6 7 8 9

29.4, CH2 129.5, CH 131.4, C 56.2, CH2 202.0, C 123.6, CH 160.8, C 41.9, CH2 22.6, CH2

10

38.0, CH2

11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 6′-CH3 −OCH3

84.9, C 219.7, C 39.0, CH2 80.5, CH 71.8, C 25.9,d CH3 25.6,d CH3 20.8, CH3 19.4, CH3 16.6, CH3 150.7, C 135.6, C 115.0, CH 154.3, C 116.4, CH 132.7, C 16.4, CH3 61.0, CH3

6c δH, m (J in Hz)

3.33, d (7.5) 5.43, br t (7.5) 3.11, s 6.18, br s 2.11, 1.73, 1.30, 1.57, 1.53,

dt (7.2, 3.1) m m ddd (13.7, 10.9, 5.5) ddd (13.7, 11.2, 5.3)

2.43, m 4.05, dd (8.2, 8.2) 1.17, 1.24, 1.13, 2.07, 1.71,

s s s br s s

6.43, d (3.1) 6.44, d (3.1) 2.19, s 3.64, s

15c

δC, type

δH, m (J in Hz)

δC, type

δH, m (J in Hz)

29.3, CH2 129.5, CH 131.5, C 56.4, CH2 202.3, C 122.3, CH 161.1, C 77.2, CH 29.9, CH2

3.34, d (6.9) 5.44, br t (7.3)


3.32, m 5.45, br t (7.3)

36.1, CH2 79.2, C 204.9, C 120.7, CH 156.3, CH 71.5, C 29.3, CH3 29.3, CH3 25.1, CH3 15.6, CH3 16.7, CH3 150.8, C 135.6, C 114.9, CH 154.3, C 116.4, CH 132.7, C 16.4, CH3 61.0, CH3

3.15, s 6.44, br s 3.97, 1.56, 1.48, 1.69,

m m m m

6.83, d (15.4) 7.02, d (15.4) 1.32, 1.32, 1.28, 2.00, 1.71,

s s s s s

6.44, br s 6.44, br s 2.19, s 3.64, s

39.6, CH2 79.4, C 204.9, C 120.6, CH 156.3, CH 71.5, C 29.3, CH3 29.3, CH3 25.2, CH3 19.3, CH3 16.6, CH3 146.5, C 130.8, C 114.6, CH 151.4, C 115.9, CH 127.7, C 16.9, CH3

3.11, s 6.19, br s 2.11, 1.56, 1.32, 1.72, 1.60,

m m m m m

6.83, d (15.7) 7.02, d (15.7) 1.32, 1.32, 1.29, 2.06, 1.69,

s s s s s

6.40, br s 6.40, br s 2.15, s

Assignments aided by COSY, HSQC, HMBC, and NOESY experiments. b1H at 600 MHz, 13C at 150 MHz. c1H at 500 MHz, 13C at 125 MHz. Assignments marked with the same letter in the same column may be interchanged.

d

correlations of the carbons at δC 71.8 (C-15) and 80.5 (C-14) with the methyl groups Me-16 and Me-17 at the end of the chain [δH 1.16 (3H, s) and 1.24 (3H, s)] (Figure 1). On the other hand, the HMBC correlations of Me-18 (δH 1.14, s) with the remaining oxygenated sp3 carbon (δC 84.9, C-11) and with the carbonyl group defined the location of a tertiary oxygenated function at C-11 and the carbonyl at C-12. Taking into account the molecular formula C28H40O6 determined by HRESIMS analysis of cystodione J (4), this compound should contain an additional ring. The presence in 4 of an ether linkage between C-11 and C-14, and not between C-11 and C-15, was proposed on the basis of the 13C chemical shift of C-12 (δC 219.7) and the IR absorption band at 1752 cm−1 that were characteristic of a carbonyl in a five-membered ring and significantly different from the values described for six-membered ketones and tetrahydropyran-3-ones (ca. δC 210−215 and ν 1715−1720 cm−1).16,25 The relative configuration 11R*,14R* was defined from the NOESY correlation between the oxymethine proton H-14 and Me-18. All of these data led to the proposal of structure 4 for cystodione J. The molecular formula of cystodione L (5), C28H40O6, indicated that it was an isomer of cystodione J (4). In particular, compound 5 was deduced to be the 6E isomer of 4. Key data were the shielding of the allylic methylene protons H-8 (δH 2.11) and the deshielding of the Me-19 (δH 2.07) in 5 (Table 2) with respect to 4. On the other hand, the 13C NMR

spectrum of 5 exhibited the resonance of C-8 at higher chemical shift (δC 41.9) and that of Me-19 at lower chemical shift (δC 19.4) than the corresponding carbons in 4. The 6E geometry was further supported by the NOESY correlation between the olefinic proton H-6 (δH 6.18) and the allylic methylene protons H-8 (δH 2.11). In a previous study of C. usneoides, Urones et al. reported the isolation of two compounds to which they assigned structures related to those of 4 and 5 but containing an ether linkage between C-11 and C-15, that is, a tetrahydropyran-3-one ring.26 However, the identities of the compounds isolated by Urones et al.26 may need revision, because the reported NMR data fit well with those of 4 and 5 recorded in CDCl3 (Supporting Information), while the specific rotation values are significantly different from those determined for 4 and 5. The last new member of the cystodione series was cystodione M (6), whose molecular formula C28H40O7 was determined by HRESIMS. The NMR spectra of 6 were closely similar to those of the co-metabolite 14,16 except for the higher chemical shift of the olefinic proton H-6 (δH 6.44) and the absence of the signals due to the allylic methylene at C-8, showing in turn those of an oxymethine at δC 77.2/δH 3.97. The presence of a carbonyl at C-5 and a double bond at C-6, C7 was confirmed by the HMBC correlations of the protons H-4 (δH 3.15) with the carbon signals at δC 202.3 (C-5) and 122.3 (C-6). The latter carbon was also correlated with Me-19 (δH 398



Article

Table 3. 1H and 13C NMR Data (CD3OD) for Meroditerpenes 7−9a,b 7 position

δC, type

1 2 3 4 5 6 7 8

29.9, CH2 129.4, CH 131.3, C 56.2, CH2 201.6, C 124.2, CH 161.6, C 34.5, CH2

9 10 11 12

27.4, CH2 126.2, CH 139.1, C 76.0, CH

13

35.7, CH2

14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 6′-CH3 −OCH3

63.4, CH 60.1, C 25.0, CH3 19.1, CH3 11.7, CH3 25.8, CH3 16.6, CH3 146.5, C 130.8, C 114.6, CH 151.5, C 115.9, CH 127.8, C 16.9, CH3

8 δH, m (J in Hz)

3.32, m 5.45, t (7.3) 3.10, s 6.22, br s 2.62, 2.60, 2.19, 5.45,

m m m t (7.3)

4.10, m 1.80, ddd (13.2, 8.3, 4.9) 1.62, m 2.85, dd (6.9, 4.9) 1.28, 1.26, 1.64, 1.89, 1.70,

s s s d (1.5) s

6.39, s 6.39, s 2.15, s

δC, type

9 δH, m (J in Hz)

29.4, CH2 129.6, CH 131.4, C 56.2, CH2 201.3, C 124.2, CH 161.6, C 34.5, CH2

3.33, d (6.9) 5.42, t (7.3) 3.10, s 6.21, br s 2.62, 2.59, 2.19, 5.45,

27.4, CH2 126.2, CH 139.1, C 76.0, CH

m m m t (7.3)

4.10, dd (8.3, 5.4)

35.7, CH2

1.80, ddd (13.7, 8.3, 5.4) 1.62, m 2.85, m

63.4, CH 60.1, C 25.0, CH3 19.1, CH3 11.7, CH3 25.8, CH3 16.7, CH3 150.7, C 135.6, C 115.0, CH 154.3, C 116.3, CH 132.7, C 16.4, CH3 60.9, CH3

1.28, s 1.26, s 1.64 s 1.89, d (1.5) 1.71, s

6.44, br s 6.43, br s 2.19, s 3.64, s

δC, type

δH, m (J in Hz)

29.4, CH2 129.5, CH 131.4, C 56.2, CH2 201.4, C 124.1, CH 162.0, C 34.9, CH2

3.33, d (7.3) 5.42, br t (7.3)

27.8, CH2 124.9, CH 136.9, C 37.9, CH2

2.15, td (7.8, 7.3) 5.21, br t (7.3)

30.7, CH2 79.0, CH 73.8, C 25.8,c CH3 24.9,c CH3 16.1, CH3 25.7, CH3 16.7, CH3 150.7, C 135.6, C 115.0, CH 154.3, C 116.3, CH 132.7, C 16.4, CH3 60.9, CH3

3.09, s 6.20, br s 2.59, t (7.8)

2.24, 2.00, 1.73, 1.33, 3.22,

ddd (14.2, 10.3, 4.4) m m m dd (10.5, 1.7)

1.15, 1.11, 1.63, 1.88, 1.71,

s s s d (1.5) s

6.43, s 6.43, s 2.19, s 3.64, s

a1 H at 500 MHz, 13C at 125 MHz. bAssignments aided by COSY, HSQC, HMBC, and NOESY experiments. cAssignments marked with the same letter in the same column may be interchanged.

(δH 2.62/2.60) (Figure 2). The HMBC correlation of the methyl group at δH 1.64 (Me-18) with the olefinic carbons C10 and C-11 and with the oxymethine carbon at δC 76.0 (C-12) supported the location of a secondary alcohol group at C-12. The NOESY correlation between the olefinic proton H-10 and the oxymethine proton H-12 supported the 10E configuration.

2.00). Following these assignments, the HMBC correlations of the oxymethine proton (δH 3.97, H-8) with the olefinic carbons C-6 (δC 122.3) and C-7 (δC 161.1), the Me-19 (δC 15.6), and a methylene carbon (δC 36.1, C-10), which in turn was correlated with Me-18 (δH 1.28), defined the presence of a secondary alcohol group at C-8. All of these data led to the proposal of structure 6 for cystodione M. The NMR data of cystone A (7) (Table 3) and its molecular formula C27H38O5, defined by HRESIMS, were consistent with a tetraprenyltoluhydroquinone structure. With regard to the functional groups on the diterpenoid framework, the 13C NMR spectrum exhibited a ketone carbonyl (δC 201.6), signals due to three trisubstituted double bonds [(δC 129.4 (CH, C-2), 131.3 (C, C-3), 124.2 (CH, C-6), 161.6 (C, C-7), 126.2 (CH, C-10), and 139.1(C, C-11)], and three oxygenated sp3 carbons, two of them oxymethines [δC 76.0/δH 4.10 (C-12/H-12) and δC 63.4/ δH 2.85 (C-14/H-14)] and the third one fully substituted (δC 60.1, C-15). The analysis of the COSY, HSQC, and HMBC spectra showed that compound 7 possessed the same structure as compound 1 from C-1 to C-8 of the isoprenoid framework, having a carbonyl at C-5 and two double bonds at Δ2E,6Z. The location of the third double bond of the molecule at Δ10 was defined from the COSY coupling of the olefinic proton at δH 5.45 (H-10) with the allylic methylene protons H-9 (δH 2.19), which were also coupled with the allylic protons H-8a/H-8b

Figure 2. Key COSY (bold bond), HMBC (→), and NOESY (dashed arrow) correlations observed in the isoprenoid chain (C-8 to C-18) for cystones A (7), C (9), and D (10). 399



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Table 4. 1H and 13C NMR Data (CD3OD) for Meroditerpenes 10, 11, and 12aa,b 10 position

δC, type

11

δH, m (J in Hz)

1 2 3 4 5 6 7 8 9


3.33, d (7.3) 5.42, br t (7.3)

10

35.1, CH2

1.62, m 1.55, m

11 12

86.0, C 79.0, CH

13

35.7, CH2

14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 6′-CH3 −OCH3 CH3COO−

84.0, CH 72.1, C 26.1,c CH3 25.6,c CH3 24.0, CH3 25.6, CH3 16.7, CH3 150.7, C 135.6, C 115.0, CH 154.3, C 116.7, CH 132.7, C 16.4, CH3 61.0, CH3

3.10, s 6.20, br s 2.60, m 1.55, m

3.87, m 2.12, m 1.77, ddd (13.0, 6.9, 3.9) 3.88, m 1.15, 1.09, 1.13, 1.89, 1.71,

s s s d (1.5) s

6.44, br s 6.44, br s 2.19, s 3.64, s

δC, type

12a

δH, m (J in Hz)

δC, type


3.33, d (7.3) 5.42, m

2.17, m 2.17, m


124.4, CH

5.14, m

35.5, CH2

137.2, C 37.9, CH2 30.8, CH2 79.0, CH 73.8, C 25.7,d CH3 24.9,d CH3 16.2, CH3 19.7, CH3 16.7, CH3 150.7, C 135.6, C 115.0, CH 154.3, C 116.4, CH 132.7, C 16.4, CH3 61.0, CH3

CH3COO−

3.11, s 6.19, br s

2.24, 2.00, 1.68, 1.34, 3.22,

m m m m dd (10.8, 1.5)

1.15, 1.11, 1.60, 2.10, 1.72,

s s br s d (1.5) s

6.44, br s 6.44, br s 2.19, s 3.64, s

85.3, C 81.2, CH 33.8, CH2 85.0, CH 71.7, C 26.1,e CH3 25.7,e CH3 25.3, CH3 19.4, CH3 16.7, CH3 155.5, C 136.1, C 121.4, CH 148.0, C 123.0, CH 133.3, C 16.3, CH3 60.9, CH3 172.1, C 171.4, C 21.0, CH3 21.0, CH3

δH, m (J in Hz) 3.41, d (7.3) 5.44, br t (7.3) 3.13, s 6.21, br s 2.16, 1.58, 1.48, 1.58, 1.48,

m m m m m

4.98, dd (6.1, 2.7) 2.25, m 1.80, ddd (13.7, 6.1, 2.7) 3.88 dd (9.8, 5.9) 1.17, 1.10, 1.18, 2.10, 1.71,

s s s d (1.5) br s

6.73, d (2.9) 6.76, (d, 2,4) 2.26, s 3.71, s

2.23, s 2.03, s

a1

H at 500 MHz, 13C at 125 MHz. bAssignments aided by COSY, HSQC, HMBC, and NOESY experiments. c−eAssignments marked with the same letter in the same column may be interchanged.

The remaining NMR data of cystone C (9) together with its molecular formula, C28H42O5, indicated that this compound had to contain from C-10 to C-18 a trisubstituted double bond [(δC 124.9/δH 5.21 (C-10/H-10) and 136.9 (C-11)], a secondary alcohol group [δC 79.0/δH 3.22 (C-14/H-14)], and a tertiary alcohol group (δC 73.8). The double bond was located at Δ10 from the HMBC correlations of both olefinic carbons with Me-18 (δH 1.63) and the correlation of the olefinic proton at δH 5.21 (H-10) with the allylic methylene carbon C-8 (δC 34.9) (Figure 2). The 10E geometry was supported by the NOESY correlation between Me-18 and the allylic methylene protons H-9 (δH 2.15). The HMBC correlations of the two oxygenated sp3 carbons abovementioned with the methyl groups Me-16 and Me-17 at the end of the chain [δH 1.15 (3H, s) and 1.11 (3H, s)] supported the presence of the tertiary alcohol function at C-15 and the secondary one at C-14. On the basis of the NMR data of cystone D (10) this compound was deduced to contain from C-10 to C-18 two oxymethines [δC 84.0/δH 3.88 (C-14/H-14), δC 79.0/3.87 (C-

The chemical shifts of the two remaining oxygenated carbons [δC 63.4 (C-14) and 60.1(C-15)], together with the HMBC correlations of both carbons with the methyl groups at the end of the chain [δH 1.28 (3H, s) and 1.26 (3H, s)] were consistent with the presence of an oxirane ring between C-14 and C-15, which also accounted for the remaining unsaturation degree indicated by the molecular formula of 7. All of these data led to the proposal of structure 7 for cystone A. Cystone B (8) exhibited NMR spectra almost identical to those of cystone A (7), except for the presence of the signals of a methoxy group at δC 60.9/δH 3.64. This group was located on C-1′ of the aromatic ring from the NOESY correlations with the methyl group on C-6′ (δH 2.19, s) and the benzylic methylene protons H-1 (δH 3.33). The NMR data of cystones C (9) (Table 3) and D (10) (Table 4) indicated that these compounds were another two members of the cystone series. Both compounds were deduced to contain a 1′-O-methyltoluhydroquinone and a diterpene portion identical to that of cystones A (7) and B (8) from C-1 up to C-9. 400



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12/H-12)] and two fully substituted oxygenated sp3 carbons [δC 86.0 (C-11), 72.1 (C-15)]. Further, taking into account the molecular formula C28H42O6 determined by HRESIMS, an additional ring must be accommodated. The COSY and HMBC correlations were consistent with the presence of a five-membered ring derived from an ether linkage between C11 and C-14, similar to that of compounds 4 and 5. Key data were the HMBC correlations of the oxygenated carbons at δC 84.0 (C-14) and 72.1 (C-15) with Me-16 and Me-17 [δH 1.15 (3H, s) and 1.09 (3H, s)] and those of the oxygenated carbons at δC 86.0 (C-11) and 79.0 (C-12) with Me-18 (δH 1.13) (Figure 2). The relative configuration 11R*,12R*,14S* was proposed on the basis of the NOESY correlations Me-18/H-12, H-12/H-13a, H-13b/H-14, and H-14/H-10. Cystodiones I (3) and M (6) as well as cystones A (7) and B (8) contained, at different positions of the chain, two asymmetric carbons whose relative configurations remain unassigned. Further, we tried to assign at least the configuration of carbons bearing a secondary hydroxy group through the corresponding MPA esters. However, all attempts at esterification led to decomposition of the substrates and/or to an intractable mixture of compounds. The molecular formula of cystone E (11), C28H42O5, was determined by HRESIMS and indicated that this compound was an isomer of cystone C (9). Further, the NMR data of 11 were closely related to those of 9 except for the chemical shifts of the allylic methylene C-8 (δC 42.1/δH 2.17) and Me-19 (δC 19.7/δH 2.10). These data, together with the NOESY correlation between the protons H-8 and the olefinic proton H-6 (δH 6.19 br s), were consistent with the configuration 6E for cystone E (11). The last member of this series of metabolites, cystone F (12), was isolated as the corresponding diacetyl derivative 12a. The treatment of a fraction containing an unseparable mixture of cystones with Ac2O/pyridine and subsequent purification led to the isolation of the derivatives 10a and 12a. The NMR spectra of compound 10a differed from those of 10 by the presence of two acetyl groups (δC 172.2, 171.1, 21.1, 21.0/δH 2.22 and 2.06), the higher chemical shifts of the oxymethine at C-12 (δC 81.1/δH 4.97), and the lower chemical shift of the aromatic carbon C-4′ (δC 148.0). Compound 10a was therefore identified as the 4′,12-di-O-acetyl derivative of cystone D (10). The molecular formula of compound 12a, C32H46O8, established by HRESIMS indicated that it was an isomer of diacetylcystone D (10a). The NMR data of both compounds differed by the chemical shifts of C-8 and Me-19, and, therefore, compound 12a was concluded to be the 6E analogue of 10a. In this study of C. usneoides we also obtained eight known compounds. Six of them had already been isolated by us from this species:16 usneoidone Z (13), 11-hydroxy-1′-O-methylamentadione (14), 1′-O-methyl-6-cis-amentadione, 1′-O-methylamentadione, and cystomexicones A and B. Compound 15 possessed the molecular formula C27H38O6 determined by HRESIMS analysis that indicated that it was an isomer of cystodione G (1). In particular, compound 15 was deduced to be the 6E isomer of 1 on the basis of the NMR chemical shifts of the allylic methylene at C-8 and the methyl group Me-19. Compound 15 has been previously described by Urones et al.21 and given the name 11-hydroxyamentadione. Herein we provide the full NMR data of 15 recorded in CDCl3 and revise some assignments of carbon signals and some proton chemical shifts and multiplicities described by Urones et al.

(Supporting Information). In addition, the NMR data of 15 in CD3OD have been included in Table 2 for comparison purposes. Compound 16 was identified as amentadione, a meroditerpenoid first isolated from C. stricta var. amentacea.22 This is the first report of amentadione (16) in C. usneoides. The polyprenyltoluquinols produced by brown algae of the family Sargassaceae are structurally related to the tocopherols and tocotrienols, a well-known family of lipid-soluble chainbreaking antioxidants, commonly known as vitamin E.27 We tested the radical-scavenging activity of the new isolated meroditerpenes 1−6 and 8−11 by using the ABTS (2,2′azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) assay.32 In addition, the known compounds 15 and 16, whose activity has not been reported, were also assayed. The results are expressed relative to the activity of Trolox, a synthetic analogue of α-tocopherol, that was measured in the same assay (Figure 3).

Figure 3. Antioxidant activities of meroditerpenes 1−6, 8−11, 15, and 16 in the ABTS assay. Trolox = 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. EC50(Trolox) = 12.0 ± 0.1 μM. Results are expressed as Trolox equivalents = EC50(Trolox)/EC50(compound).

All the tested compounds exhibited radical-scavenging activity with potencies that ranged from 37% to 87% of the activity of the Trolox standard. The most active compounds were cystodiones G (1) and H (2), 11-hydroxyamentadione (15), and amentadione (16), which exhibited antioxidant capacities 81%, 77%, 87%, and 77% that of Trolox, respectively. Cystodiones J (4) and M (6) and cystones C (9) and D (10) showed antioxidant activity in the range of 62−67% that of Trolox. The remaining compounds 3, 5, 8, and 11 displayed less activity, showing potencies between 53% and 37% that of Trolox. The radical-scavenging activity of algal toluquinols has been previously described only for a limited number of metabolites, including those of C. crinita,15 C. usneoides,16 and a few related compounds isolated from species of the genus Sargassum.28−31 The results herein described, taken together with those previously reported,16 indicate that C. usneoides contains a wide series of antioxidant metabolites and support the great potential of this species as a resource for the development of natural antioxidants. On the other hand, over the past decade a growing number of marine natural products, from a range of organisms and possessing very different chemical structures, have been investigated for their anti-inflammatory properties.33 Herein we report that 10 meroditerpenoids, namely, cystodiones G−I (1−3) and M (6), cystones B−E (8−11), 11-hydroxyamentadione (15), and amentadione (16), have been tested for their activity as inhibitors of the pro-inflammatory cytokine TNF-α. 401



Article

phils,35−38 the pro-inflammatory enzymes iNOS and COX-2 expression in LPS-stimulated RAW-264.7 macrophages,39 and the leukotriene B4 formation triggered by arachidonic acid in porcine leukocytes.40 In some instances, the anti-inflammatory effects have also been studied in animal models.41 On the other hand, despite the abundance of marine natural products reported to have pharmacological properties, only a few have been described to be TNF-α inhibitors, including a series of oxo fatty acids,34 a polyacetylene,42 the carotenoid astaxanthin,43 and various terpenoids,44,45 which in some instances have also been tested in animal models.45 We have examined the TNF-α inhibitory activity of a wide series of marine tetraprenyltoluquinols. In this study five compounds (1, 6, 9, 15, and 16) have shown to inhibit significantly the production of TNF-α in LPS-stimulated THP-1 human macrophages. These findings support the potential of this class of algal natural products in the anti-inflammatory area.

Assays were performed on human THP-1 macrophages using lipopolysaccharide (LPS) to stimulate the production of TNFα. As shown in Figure 4, the production of TNF-α in control

■

Figure 4. Effects of cystodiones G (1), H (2), I (3), and M (6), cystones B (8), C (9), D (10), and E (11), 11-hydroxyamentadione (15), and amentadione (16) on the TNF-α production in THP-1 macrophages stimulated with LPS. The cells were treated with the compounds (1, 2, 11 and 15 at 10 μM; 3, 6, 8, and 10 at 8 μM; 9 and 16 at 5 μM) and then stimulated with LPS (1 μg/mL). TNF-α concentration in the supernatants was measured using an ELISA assay. Dexamethasone (Dex) was used as a positive reference compound at 1 μM. Values are the mean ± SE of four determinations, +++p < 0.001 vs control; *p < 0.5, **p < 0.01, ***p < 0.001 vs LPS-stimulated cells.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer 341 polarimeter. UV−vis data were obtained on a VWR UV-1600PC. IR spectra were recorded on a PerkinElmer FT-IR System Spectrum BX spectrophotometer. 1H and 13 C NMR spectra were recorded on a Varian INOVA 600 spectrometer or on an Agilent 500 spectrometer using CD3OD or CDCl3 as solvent. Chemical shifts were referenced using the corresponding solvent signals [δH 3.30 and δC 49.0 for CD3OD, δH 7.26 and δC 77.0 for CDCl3]. COSY, HSQC, HMBC, and NOESY experiments were performed using standard Varian pulse sequences. High-resolution mass spectra (HRMS) were obtained on a Waters SYNAPT G2. Column chromatography was carried out on Merck silica gel 60 (70−230 mesh). HPLC separations were performed on a LaChrom-Hitachi apparatus equipped with LiChrospher Si-60 (Merck, 250 × 10 mm, 10 μm) and Luna Si (2) (Phenomenex, 250 × 4.6 mm, 5 μm) columns in normal phase and with Kromasil 100-5C18 columns (250 × 10 mm, 5 μm or 250 × 4.6 mm, 5 μm) in reversed phase and using a differential refractometer RI-71 or a UV detector L-7400 (Merck) working at 254 nm. All solvents were HPLC grade. ABTS diammonium salt and Trolox were purchased from Sigma. Collection and Identification. Specimens of Cystoseira usneoides (class Phaeophyceae, order Fucales, family Sargassaceae) were collected at the coast of Tarifa, Spain (36°00′41.2″ N, 5°35′48.3″ W). The alga samples were washed and immediately frozen. A voucher specimen (A-Cu-0611) is deposited at the Marine Natural Products Laboratory, Faculty of Marine and Environmental Sciences, University of Cadiz, Spain. Extraction and Isolation. Frozen samples of C. usneoides were extracted with MeOH (6 L) at room temperature (weight of alga after extraction and drying: 50.1 g). After evaporation of the solution under reduced pressure the aqueous residue was extracted three times with Et2O (0.8 L, 2 × 0.4 L). The organic layers were combined, dried over MgSO4, and evaporated under reduced pressure to yield 10 g of extract. This was subjected to column chromatography (25 × 7 cm) eluting with n-hexane/Et2O (1:1, v/v, 3 L), then Et2O (1 L), mixtures of CHCl3/MeOH (90:10 and 80:20, v/v, 2 L each), and finally MeOH (1.2 L). The fraction that eluted with Et2O was chromatographed over a silica gel column using n-hexane/EtOAc mixtures (80:20 to 30:70, v/ v) and EtOAc as eluents. The subfractions that in their respective 1H NMR spectra showed signals due to aromatic protons were selected for further purifications. Repeated purifications by normal-phase HPLC using n-hexane/EtOAc (70:30 and 60:40, v/v) and by reversedphase HPLC using MeOH/H2O (70:30, v/v) as eluents yielded compounds 2, 4, 5, 7, 8, 13, 14, 1′-O-methyl-6-cis-amentadione, 1′-Omethylamentadione, cystomexicone A, and cystomexicone B. The fraction that eluted with CHCl3/MeOH (90:10, v/v) was chromatographed over a silica gel column using as eluents mixtures of n-hexane/ EtOAc (50:50 to 30:70, v/v), then EtOAc, and finally MeOH. The

THP-1 macrophages was increased about 5-fold after stimulation with LPS (C vs C+LPS). Compounds were tested at concentrations in the range 5−10 μM, which do not affect THP-1 cell viability in the sulforhodamine B assay. The treatment of macrophages with cystodione G (1) and 11-hydroxyamentadione (15) at 10 μM and with cystodione M (6) at 8 μM caused great inhibition of the production of TNFα, which was decreased by 81%, 69%, and 79%, respectively, with respect to LPS-stimulated untreated cells. Cystone C (9) and amentadione (16) also exhibited significant activity and at 5 μM reduced the TNF-α level by 59% and 53%, respectively. The remaining compounds 2, 3, 8, 10, and 11 were moderately active, causing decreases of the TNF-α production between 21% and 35% at the concentration tested. When the activities of compounds that differ by the Δ6 geometry were compared, no general trend was observed. Thus, while the 6Z derivatives 1 and 9 were more active than their 6E analogues 15 and 11, respectively, the reverse is true for the pair 2/16, where compound 2 (6Z) was less active than 16 (6E). On the other hand, the inhibitory activities of compounds 1 and 15 were slightly higher than the activities previously found for their 1′O-methyl analogues 13 and 14, respectively.16 Taking into account that the structures of most of the tested compounds differ by only the functionalization on the two final isoprenoid units of the tetraprenyl chain, differences of activity among compounds could be ascribed to this portion of the molecule. Interestingly, four of the five more active compounds herein reported (1, 6, 15, 16) as well as the active compounds 13 and 14 previously described16 contain an α,β-unsaturated ketone at the final region of the terpenoid chain. This structural motif has been previously noted to be associated with higher levels of TNF-α and NO inhibitory activity.34 In recent years, other metabolites displaying a terpenoid−hydroquinone structure have been shown to inhibit in vitro several enzymes and mediators involved in inflammatory responses, including the superoxide production in PMA-stimulated human neutro402



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subfractions that in their respective 1H NMR spectra showed signals due to aromatic protons were selected for further purifications. Repeated separations by normal-phase HPLC using n-hexane/EtOAc (60:40 and 50:50, v/v) or CHCl3/MeOH (99:1, v/v) mixtures as eluents and by reversed-phase HPLC using MeOH/H2O (70:30, v/v) as eluent yielded compounds 1, 3, 6, 9, 10, 11, 15, and 16 and additional amounts of compounds 2, 5, 13, 14, 1′-O-methyl-6-cisamentadione, and 1′-O-methylamentadione. The total amounts obtained of each compound were 1 (26.9 mg), 2 (95.2 mg), 3 (9.4 mg), 4 (110.8 mg), 5 (82.4 mg), 6 (5.0 mg), 7 (1.1 mg), 8 (2.3 mg), 9 (8.5 mg), 10 (4.6 mg), 11 (10.6 mg), 13 (267.4 mg), 14 (254.9 mg), 15 (21.6 mg), 16 (46.0 mg), 1′-O-methyl-6-cis-amentadione (187.6 mg), 1′-O-methylamentadione (231.0 mg), cystomexicone A (33.5 mg), and cystomexicone B (9.8 mg). Cystodione G (1): yellowish oil; [α]25D +2 (c 0.5, MeOH); IR (film) νmax 3394, 1680, 1610 cm−1; 1H NMR (CD3OD, 500 MHz) Table 1; 13 C NMR (CD3OD, 125 MHz) Table 1; HRESIMS m/z 457.2598 [M − H]− (calcd for C27H37O6, 457.2590). Cystodione H (2): yellowish oil; [α]25D −5 (c 0.1, MeOH); IR (film) νmax 3394, 1659, 1610 cm−1; 1H NMR (CD3OD, 500 MHz) Table 1; 13C NMR (CD3OD, 125 MHz) Table 1; HRESIMS m/z 441.2643 [M − H]− (calcd for C27H37O5, 441.2641). Cystodione I (3): colorless oil; [α]25D +8 (c 0.1, MeOH); IR (film) νmax 3416, 1680, 1603 cm−1; 1H NMR (CD3OD, 500 MHz) Table 1; 13 C NMR (CD3OD, 125 MHz) Table 1; HRESIMS m/z 473.2910 [M − H]− (calcd for C28H41O6 473.2903). Cystodione J (4): colorless oil; [α]25D −11 (c 0.1, MeOH), [α]25D −13 (c 0.2, CHCl3); IR (film) νmax 3399, 1752, 1685, 1602 cm−1; 1H NMR (CD3OD, 600 MHz) Table 1; 13C NMR (CD3OD, 150 MHz) Table 1; HRESIMS m/z 471.2755 [M − H]− (calcd for C28H39O6 471.2747). Cystodione L (5): colorless oil; [α]25D −25 (c 0.1, MeOH); [α]25D −28 (c 0.2, CHCl3); IR (film) νmax 3398, 1752, 1683, 1610 cm−1; 1H NMR (CD3OD, 600 MHz) Table 2; 13C NMR (CD3OD, 150 MHz) Table 2; HRESIMS m/z 471.2750 [M − H]− (calcd for C28H39O6, 471.2747). Cystodione M (6): colorless oil; [α]25D −2 (c 0.1, MeOH); IR (film) νmax 3390, 1681, 1622 cm−1; 1H NMR (CD3OD, 500 MHz) Table 2; 13C NMR (CD3OD, 125 MHz) Table 2; HRESIMS m/z 487.2709 [M − H]− (calcd for C28H39O7, 487.2696). Cystone A (7): yellowish oil; [α]25D +1 (c 0.1, MeOH); IR (film) νmax 3417, 1659, 1611 cm−1; 1H NMR (CD3OD, 500 MHz) Table 3; 13 C NMR (CD3OD, 125 MHz) Table 3; HRESIMS m/z 441.2649 [M − H]− (calcd for C27H37O5 441.2641). Cystone B (8): colorless oil; [α]25D +1 (c 0.1, MeOH); IR (film) νmax 3394, 1680, 1611 cm−1; 1H NMR (CD3OD, 500 MHz) Table 3; 13 C NMR (CD3OD, 125 MHz) Table 3; HRESIMS m/z 455.2813 [M − H]− (calcd for C28H39O5 455.2797). Cystone C (9): colorless oil; [α]25D −7 (c 0.1, MeOH); IR (film) νmax 3411, 1680, 1603 cm−1; 1H NMR (CD3OD, 500 MHz) Table 3; 13 C NMR (CD3OD, 125 MHz) Table 3; HRESIMS m/z 457.2956 [M − H]− (calcd for C28H41O5 457.2954). Cystone D (10): colorless oil; [α]25D −1 (c 0.1, MeOH); IR (film) νmax 3417, 1680, 1607 cm−1; 1H NMR (CD3OD, 500 MHz) Table 4; 13 C NMR (CD3OD, 125 MHz) Table 4; HRESIMS m/z 472.2903 [M − H]− (calcd for C28H41O6 473.2903). Cystone E (11): colorless oil; [α]25D −3 (c 0.2, MeOH); IR (film) νmax 3416, 1680, 1603 cm−1; 1H NMR (CD3OD, 500 MHz) Table 4; 13 C NMR (CD3OD, 125 MHz) Table 4; HRESIMS m/z 457.2969 [M − H]− (calcd for C28H41O5 457.2954). 11-Hydroxyamentadione (15): yellowish oil; [α]25D +3 (c 0.2, CHCl3), lit.21 [α]25D +1.66 (c 0.12, CHCl3); IR (film) νmax 3400, 1681, 1614 cm−1; 1H NMR (CD3OD, 500 MHz) Table 2; 13C NMR (CD3OD, 125 MHz) Table 2; 1H NMR (CDCl3, 500 MHz) δ 7.19 (1H, d, J = 15.1 Hz, H-14), 6.60 (H, d, J = 15.1 Hz, H-13), 6.54 (1H, d, J = 2.7 Hz, H-3′), 6.51 (1H, d, J = 2.7 Hz, H-5′), 6.09 (1H, br s, H6), 5.40 (1H, br t, J = 7.1 Hz, H-2), 3.36 (1H, d, J = 15.9 and 7.1 Hz, H-1a), 3.32 (1H, dd, J = 15.9 and 7.7 Hz, H-1b), 3.18 (1H, d, J = 15.4 Hz, H-4a), 3.14 (1H, d, J = 15.4 Hz, H-4b), 2.21 (3H, s, 6′-Me), 2.16

(1H, m, H-8a), 2.03 (1H, m, H-8b), 2.01 (3H, d, J = 1.0 Hz, Me-19), 1.72 (3H, br s, Me-20), 1.70 (1H, m, H-10a), 1.66 (2H, m, H-9a and H-10b), 1.43 (3H, s, Me-16), 1.40 (3H, s, Me-17), 1.36 (3H, s, Me.18), 1.12 (1H, m, H-9b); 13C NMR (CDCl3, 125 MHz) δ 202.6 (C, C-12), 200.3 (C, C-5), 158.2 (C, C-7), 156.4 (CH, C-14), 149.5 (C, C-4′), 145.7 (C, C-1′), 132.0 (C, C-3), 127.4 (C, C-2′), 126.8 (CH, C-2), 125.4 (C, C-6′), 123.4 (CH, C-6), 117.8 (CH, C-13), 115.4 (CH, C-5′), 113.7 (CH, C-3′), 78.2 (C, C-11), 71.6 (C, C-15), 54.9 (CH2, C-4), 40.5 (CH2, C-8), 37.4 (CH2, C-10), 29.3 (CH3, Me16), 29.1 (CH3, Me-17), 29.0 (CH2, C-1), 25.3 (CH3, Me-18), 20.8 (CH2, C-9), 19.5 (CH3, Me-19), 16.8 (CH3, Me-20), 16.2 (CH3, 6′Me); HRESIMS m/z 457.2592 [M − H]− (calcd for C27H37O6 457.2590). Synthesis of Diacetyl Derivatives 10a and 12a. A fraction containing a mixture of compounds 10 and 12 and other metabolites (28 mg) was treated at room temperature with Ac2O in pyridine overnight. After evaporation under reduced pressure, the residue was separated by HPLC (n-hexane/EtOAc, 60:40, v/v) to yield the diacetyl derivatives 10a (2.4 mg) and 12a (3.6 mg). 4′,12-Di-O-Acetylcystone D (10a): yellowish oil; [α]25D −10 (c 0.1, MeOH); 1H NMR (CD3OD, 500 MHz) δ 6.75 (1H, d, J = 2.7 Hz, H5′), 6.73 (1H, d, J = 2.7 Hz, H-3′), 6.20 (1H, s, H-6), 5.43 (1H, br t, J = 7.3, H-2), 4.97 (1H, dd, J = 5.9 and 2.5 Hz, H-12), 3.89 (1H, dd, J = 10.0 and 5.9 Hz, H-14), 3.71 (3H, s, OCH3), 3.38 (2H, d, J = 7.3 Hz, H2-1), 3.10 (2H, s, H2-4), 2.60 (1H, m, H-8a), 2.55 (1H, m, H-8b), 2.26 (3H, s, 6′-CH3), 2.24 (1H, m, H-13a), 2.22 (3H, s, CH3COO−), 2.06 (3H, s, CH3COO−), 1.87 (3H, d, J = 1.5 Hz, Me-19), 1.80 (1H, ddd, J = 13.2, 5.9, and 2.5 Hz, H-13b), 1.70 (3H, br s, Me-20), 1.62 (1H, m, H-10a), 1.56 (1H, m, H-9a), 1.55 (1H, m, H-10b), 1.41 (1H, m, H-9b), 1.18 (3H, s, Me-18), 1.17 (3H, s, Me-16), 1.10 (3H, s, Me17); 13C NMR (CD3OD, 125 MHz) δ 201.0 (C-5), 172.2 (CH3COO−), 171.1 (CH3COO−), 161.8 (C-7), 155.5 (C-1′), 148.0 (C-4′), 136.2 (C-2′), 133.3 (C-6′), 132.3 (C-3), 128.7 (C-2), 124.2 (C-6), 122.9 (C-5′), 121.4 (C-3′), 85.5 (C-11), 85.0 (C-14), 81.1 (C-12), 71.7 (C-15), 60.8 (OCH3), 56.1 (C-4), 36.1 (C-10), 35.1 (C-8), 33.8 (C-13), 29.3 (C-1), 26.2* (C-16), 25.7* (C-17), 25.6 (C19), 25.3 (C-18), 24.1 (C-9), 21.1 (CH3COO−), 21.0 (CH3COO−), 16.7 (C-20), 16.3 (6′-CH3), values marked with an asterisk may be interchanged. 4′,12-Di-O-Acetylcystone F (12a): yellowish oil; [α]25D −6 (c 0.1, MeOH); IR (film) νmax 3451, 1736, 1683, 1614 cm−1; 1H NMR (CD3OD, 500 MHz) Table 4; 13C NMR (CD3OD, 125 MHz) Table 4; HRESIMS m/z 557.3116 [M − H]− (calcd for C32H45O8 557.3114). Antioxidant Assay. Antioxidant activity was determined by the ABTS free radical decolorization assay developed by Re et al.,32 with slight modifications. In brief, a solution of the radical cation ABTS•+ was prepared by mixing (1:1, v/v) a solution of ABTS diammonium salt (7 mM) and a solution of potassium persulfate (2.45 mM) in H2O. The mixture was kept in the dark at room temperature for 12−18 h before use. Then the solution was diluted with EtOH to an absorbance of 0.70 ± 0.02 at 734 nm. Trolox was used as standard. Solutions of Trolox and algal meroditerpenoids were prepared in EtOH. For the assay, 100 μL of tested compound were mixed with 2 mL of the ABTS•+ solution, and the absorbance at 734 nm was measured after 6 min. Controls were prepared by adding 100 μL of EtOH to 2 mL of ABTS•+ solution. All of the determinations were carried out in triplicate. The percentage of inhibition of the absorbance was calculated by the following equation: % Inhibition = [(A0 − A1)/A0] × 100, where A0 expresses the absorbance of control and A1 the absorbance of the tested compound. Cell Culture. The THP-1 human monocytic leukemia cell line was obtained from the American Type Culture Collection (TIB-202, ATCC, USA) and cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 μg/ mL streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Cell Proliferation Assay. Viability of THP-1 cells upon exposure to the meroterpenoids was determined by the sulforhodamine B assay.46 In brief, for differentiation into macrophages the cultured 403



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THP-1 cells in growth medium (1 × 104 cells/mL) were spiked with phorbol myristate acetate (PMA) dissolved in DMSO (0.8 mM) for a final concentration of 0.2 μM, transferred into 96-well plates (100 μL/ well), and incubated in a humidified atmosphere of 5% CO2 at 37 °C for 3 days. After that, the medium was removed, and cells were washed with phosphate saline buffer (PBS, 4 °C) and then incubated for 48 h with increasing concentrations of the test compounds (6.25, 12.5, 25, 50, and 100 μM) that were prepared by dilution of stock solutions in DMSO with fresh medium. Controls were incubated in fresh medium containing DMSO 0.1%, v/v, which did not affect cell viability. After 48 h the cells were fixed with 50 μL of trichloroacetic acid (50% v/v) and processed as described in the literature.46 Determination of TNF-α Production. Differentiation into macrophages was achieved by incubating for 3 days THP-1 cells (1 × 104 cells/mL) with PMA (final concentration of 0.2 μM) in 96-well plates (100 μL/well), as described above. After that, the medium was removed, and the cells were washed with PBS (4 °C) and then incubated for 1 h with solutions of the compounds (100 μL/well, final concentration of 5, 8, or 10 μM) that were prepared by dilution of stock solutions in DMSO with fresh medium. Positive controls were incubated with a dexamethasone solution (100 μL/well, final concentration 1 μM) that was prepared by dilution of a stock solution in DMSO with fresh medium. Control groups were incubated with growth medium (100 μL/well) containing DMSO 0.1%, v/v. The inflammatory response was induced by addition of lipopolysaccharide (LPS, final concentration of 1 μg/mL). The stock LPS solution (5 mg/mL) was prepared in DMSO and then diluted with fresh medium for a final volume of 5 μL/well. An unstimulated control containing DMSO 0.1%, v/v, without LPS, was also assayed. The viability of cells was greater than 95% throughout the experiments. After 24 h incubation, supernatant fluids were collected and stored at −80 °C until TNF-α measurement. A commercial enzyme-linked immunosorbent assay (ELISA) kit (Diaclone GEN-PROBE) was used to quantify TNF-α according to the manufacturer’s protocol.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01067. 1 H and 13C NMR spectra of compounds 1−10, 10a, 11, 12a, and 15 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +34 956016021. Fax: +34 956016193. E-mail: eva.zubia@ uca.es. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by grants from Junta de Andalucı ́a (FQM-169 and Proyecto de Excelencia P12-AGR-430). H.Z. acknowledges a fellowship from AECID-MAEC, Spain. We thank Dr. I. Hernández and Dr. R. Bermejo (University of Cadiz, Spain) for their collaboration in providing algal samples.

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Cystoseira usneoides: A Brown Alga Rich in Antioxidant and Anti

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