Antioxidant and Anti-inflammatory Meroterpenoids from the Brown Alga

Article pubs.acs.org/jnp

Antioxidant and Anti-inflammatory Meroterpenoids from the Brown Alga Cystoseira usneoides Carolina de los Reyes,† 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: A chemical study of the alga Cystoseira usneoides has led to the isolation of six new meroterpenoids, cystodiones A−F (1−6), together with six known related compounds (7−12). The structures of the new metabolites have been established by spectroscopic techniques. In antioxidant assays all of the tested meroterpenes, and in particular cystodiones A (1) and B (2), 6-cis-amentadione-1′-methyl ether (7), and amentadione-1′-methyl ether (8), exhibited strong radical-scavenging activity. In antiinflammatory assays, usneoidone Z (11) and its corresponding 6E isomer (12) showed significant activity as inhibitors of the production of the proinflammatory cytokine TNF-α in LPS-stimulated THP-1 human macrophages.

T

he algal genus Cystoseira comprises about 50 species worldwide distributed through subtropical waters, with the biggest species diversity and abundance found in the Mediterranean Sea.1,2 Chemical studies of a variety of Cystoseira species from the eastern Atlantic and the Mediterranean coasts have shown that the characteristic secondary metabolites of this algal genus are compounds of mixed biogenesis, formed by a toluquinol nucleus linked to a diterpenoid moiety.1−3 Further, the metabolites so far isolated exhibit great structural diversity at the diterpenoid portion, ranging from compounds with a regular isoprenoid framework, linear or cyclic, to rearranged derivatives. Despite the number and structural variety of the meroditerpenoids obtained from Cystoseira algae, there are few data on their biological properties, in part because most of the compounds were isolated more than 20 years ago,1,2 when bioactivity assays were not as accessible. Nonetheless, the cytotoxic,4−6 antibacterial,7 antiviral,5,6 and antioxidant8,9 properties described for some Cystoseira meroterpenoids suggest that the biomedical potential of this class of algal metabolites deserves to be further explored. In particular, a main area of interest can be found in the potential of Cystoseira metabolites as antioxidant agents. It is well recognized that the oxidative modification of lipids, proteins, and DNA caused by free radicals such as superoxide, hydroxyl, or peroxyl radicals, and other reactive oxygen species, © 2013 American Chemical Society and American Society of Pharmacognosy

is involved in the aging process as well as in the pathogenesis of several human diseases, including atherosclerosis, rheumatoid arthritis, some neurological disorders, and some types of cancer.10−13 Natural antioxidant compounds exert radicalscavenging activity, reducing oxidative stress and protecting against oxidative damage, and may also provide an indirect protection by activating endogenous antioxidant defense systems.13,14 Considering that many meroterpenoids from Cystoseira are tetraprenyltoluquinol derivatives with chemical structures reminiscent of tocopherols, which are the main radical scavengers in biological lipid phases, it seems that Cystoseira metabolites could also feature significant antioxidative properties.8,9 On the other hand, a variety of phenolic and terpenoid natural products modulate or inhibit the activities of enzymes, cytokines, cytokine receptors, and other mediators involved in inflammatory cascades.15 In this context, the inhibition of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) represents an important therapeutic strategy, as enhanced TNF-α synthesis is associated with the development of a variety of chronic inflammatory diseases, such as rheumatoid arthritis, Received: November 29, 2012 Published: March 4, 2013 621

dx.doi.org/10.1021/np300833y | J. Nat. Prod. 2013, 76, 621−629

Journal of Natural Products

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Chart 1

psoriatic arthritis, and inflammatory bowel disease.16,17 Although various protein-based TNF-α inhibitors are in clinical use or in various stages of development, they have important shortcomings, including cost and potentially serious adverse side effects. Therefore, the development of small-molecule TNF-α inhibitors has emerged as a safer and more costeffective alternative to protein-based therapeutics.16 In this regard, natural products are considered as a broad resource for the discovery of new leads capable of modulating TNF-α production and activity.18,19 As part of our research on bioactive compounds from algae, we have studied specimens of the brown alga Cystoseira usneoides collected from the Gibraltar Strait. Herein we report the isolation and structure determination of the new meroterpenes cystodiones A−F (1−6), together with the compounds 6-cis-amentadione-1′-methyl ether (7),20 amentadione-1′-methyl ether (8),20 cystomexicone A (9),21 cystomexicone B (10),21 usneoidone Z (11),5,22 and its corresponding 6E isomer (12).2 The isolated compounds have been tested in radical-scavenging assays to assess their antioxidant activity and also in TNF-α inhibition assays to evaluate their antiinflammatory properties.

hexane/Et2O (3:7), Et2O, and CHCl3/MeOH (95:5) afforded the new meroterpenoids 1−6 together with the known compounds 7−12.5,20−22 Cystodione A (1) possessed the molecular formula C28H40O6, determined by HRMS, which indicated nine degrees of unsaturation in the molecule. The IR spectrum showed absorption bands for hydroxy (3407 cm−1) and conjugated carbonyl functions (1668 and 1606 cm−1). The 13C NMR spectrum, recorded in CD3OD, exhibited two carbonyl signals, at δC 206.9 and 201.7 (Table 1), assigned to two α,βunsaturated ketone functions, and 12 signals between 165 and 110 ppm that were attributable to the presence of three double bonds and an aromatic ring. In particular, the presence of an Omethyltoluquinol nucleus identical to that found in a large number of Cystoseira metabolites1,2 was inferred from the NMR resonances of two meta-coupled aromatic protons at δH 6.69 (d, J = 2.9 Hz, H-3′) and 6.50 (d, J = 2.9 Hz, H-5′), together with the signals at δH 3.60 (3H, s)/δC 61.4 and δH 2.18 (3H, s)/δC 16.2 attributable to a methoxy and a methyl group linked to C1′ and C-6′ of the aromatic ring, respectively. The location of the methoxy group at C-1′, and not at C-4′, was supported by the HMBC correlations of C-1′ (δC 150.7) with the methoxy group and with the aromatic methyl group as well as by the NOESY correlation between these two groups (Figure 1). The remaining substituent at C-2′ of the aromatic ring should be a C20H31O4 chain containing the two ketone groups and the three carbon−carbon double bonds mentioned above. On the basis of the 1H NMR data, two of these double bonds must be trans-disubstituted [δH 6.93 (d, J = 15.8 Hz)/6.33 (d, J = 15.8

■

RESULTS AND DISCUSSION Dried specimens of C. usneoides were extracted with acetone/ MeOH (1:1), and the resulting extract was subjected to column chromatography using as eluents n-hexane/Et2O mixtures of increasing polarity, then Et2O, CHCl3/MeOH mixtures, and finally MeOH. Repeated separation of fractions eluted with n622



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Table 1. 1H and 13C NMR Data (CD3OD) for Cystodiones A (1), C (3), and E (5)a,b 1

a1

3

5

δC

δH, m (J in Hz)

δC

δH, m (J in Hz)

δC

1 2 3 4

123.3, CH 137.7, CH 73.5, C 56.2, CH2

6.76, d (16.1) 6.29, d (16.1)

123.3, CH 137.7, CH 73.5, C 56.2, CH2

6.76, d (16.1) 6.29, d (16.1)

29.4, CH2 129.5, CH 131.4, C 56.2, CH2

5 6 7 8

201.7, C 126.4, CH 161.5, C 34.6, CH2

9

26.8, CH2

10

34.2, CH2

11 12 13

44.9, CH 206.9, C 125.6, CH

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

155.2, CH 71.3, C 29.3, CH3 29.3, CH3 17.0, CH3 25.6, CH3 28.6, CH3 150.7, C 131.9, C 111.0, CH 154.4, C 118.0, CH 133.1, C 16.2, CH3 61.4, CH3

position

2.79, d (14.2) 2.70, d (14.2)

2.80, d (14.4) 2.71, d (14.4)

201.7, C 126.8, CH 160.9, C 33.8, CH2

2.51, m

201.4, C 124.4, CH 161.5, C 34.6, CH2

23.1, CH2

1.67, m

23.0, CH2

43.7, CH2

2.44, t (7.2)

36.2, CH2

6.33, d (15.8)

211.8, C 29.7, CH3 25.4, CH3

2.07, s 1.87, d (1.2)

84.5, C 219.5, C 37.7, CH2

6.93, d (15.8)

28.7, CH3

1.40, s

6.22, br s 2.55, m 2.51, m 1.37, m 1.65, m 1.33, m 2.82, m

1.31, 1.31, 1.02, 1.85, 1.40,

6.25, br s

s s d (6.8) d (1.0) s

6.69, d (2.9) 6.50, d (2.9) 2.18, s 3.60, s

150.7, C 131.9, C 111.0, CH 154.3, C 118.0, CH 133.1, C 16.1, CH3 61.4, CH3

6.69, d (2.9) 6.50, d (2.9) 2.18, s 3.60, s

81.1, CH 71.7, C 25.8,c CH3 25.6,c CH3 22.3, CH3 25.5, CH3 16.6, CH3 150.8, 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) 3.33, d (7.4) 5.42, br t (7.4) 3.09, s

6.21, br s 2.60, 2.54, 1.56, 1.48, 1.60, 1.46,

ddd (11.7, 9.1, 5.7) m m m m m

2.54, dd (18.2, 9.4) 2.43, dd (18.2, 6.6) 4.03, dd (9.4, 6.6) 1.12, 1.24, 1.18, 1.87, 1.70,

s s s d (1.1) br s

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

H at 600 MHz, 13C at 150 MHz. bAssignments aided by COSY, HSQC, HMBC, and NOESY experiments. cValues may be interchanged.

Following this, the additional correlation of H-1 with the oxygenated sp3 carbon at δC 73.5 (C-3) and the correlation of the olefinic carbon C-2 with the methyl group at δH 1.40 (s, Me-20) were consistent with the presence of a tertiary hydroxy group at C-3. The olefinic carbon C-2 was also correlated with an isolated methylene [δH 2.79 (d, J = 14.2, H-4a)/2.70 (d, J = 14.2, H-4b)], which in turn exhibited correlations with the carbonyl at δC 201.7 (C-5) and the olefinic methine at δC 126.4 (C-6). These data defined the location of a ketone group at C-5 and the trisubstituted double bond at C-6,C-7. The Z geometry of this double bond was indicated by the 13C NMR chemical shift of Me-19 at δC 25.6 and by the NOESY correlation of the olefinic proton H-6 (δH 6.22) with the Me-19.7a,9 The presence of a hydroxyisopropyl moiety at the end of the chain was deduced from the HMBC correlation of the six-proton singlet at δH 1.31 (Me-16 and Me-17) with the oxygenated carbon at δC 71.3 (C-15). The additional correlation of this carbon with the olefinic protons at δH 6.33 (H-14) and 6.93 (H-13), which in turn were correlated with the carbonyl at δC 206.9 (C-12), supported the location of the remaining trans-double bond at C-13,C-14, conjugated with the second ketone function of the molecule at C-12. This carbonyl group showed an additional HMBC correlation with Me-18, which displayed COSY coupling with the methine proton H-11 (δH 2.82). The

Figure 1. Key COSY (bold bond), HMBC (plain arrow), and NOESY (dashed arrow) correlations observed for cystodione A (1).

Hz) and δH 6.76 (d, J = 16.1 Hz)/6.29 (d, J = 16.1 Hz)], and the third was trisubstituted [δH 6.22 (br s)]. The chain should also accommodate two fully substituted sp3 carbons linked to oxygenated functions, whose 13C NMR resonances were observed at δC 73.5 and 71.3. The presence of 1H NMR signals accounting for five methyl groups [δH 1.85 (d, J = 1.0 Hz, Me-19), 1.40 (s, Me-20), 1.31 (s, Me-16 and Me-17), 1.02 (d, J = 6.8 Hz, Me-18)] was consistent with an isoprenoid backbone for the chain. The location of the various functions on the isoprenoid chain was defined with the aid of COSY and HMBC spectra (Figure 1). Thus, the HMBC correlation of the olefinic proton at δH 6.76 with the aromatic carbons C-1′ and C-3′ supported the location of a trans-double bond at C-1,C-2. 623



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carbons C-1′, C-2′, and C-3′ (Figure 2). On the other hand, the HMBC correlation of olefinic carbon C-2 with the protons of

connectivity between C-7 and the methine at C-11 through a sequence of three methylenes was consistent with the remaining correlations observed in the COSY and HMBC spectra. All these data defined the structure 1 for cystodione A. The molecular formula of cystodione B (2), C28H40O6, indicated that it was an isomer of cystodione A (1). Furthermore, the NMR spectra of 2 were similar to those of 1 except for the signals due to Me-19 and the methylene at C-8. In particular, the Me-19 proton resonance in 2 was downfield shifted [δH 2.05 (d, J = 1.1 Hz)] and the signal of protons H2-8 appeared upfield shifted [δH 2.11 (t, J = 7.5 Hz)] with respect to the corresponding signals in compound 1. These data, together with the chemical shift of Me-19 at δC 19.6, indicated that in compound 2 the double bond at C-6,C-7 possessed an E geometry. This assignment was further supported by the NOESY correlation between the olefinic proton H-6 (δH 6.20) and the methylene protons H2-8. Cystodione C (3) possessed the molecular formula C22H30O5, as determined by HRMS. The NMR spectra of compound 3 were related to those of cystodione A (1) and exhibited the signals corresponding to an O-methyltoluquinol moiety identical to that of 1. However, taking into account the molecular formula, the isoprenoid-derived side chain in compound 3 must contain only 14 carbon atoms. The NMR spectra showed the signals due to two ketone functions (δC 211.8 and 201.7), a trans-disubstituted double bond [δH 6.76 (d, J = 16.1 Hz, H-1)/6.29 (d, J = 16.1 Hz, H-2)], a trisubstituted double bond [δH 6.25 (br s, H-6)], and a quaternary sp3 carbon linked to an oxygenated function (δC 73.5, C-3). The positions of these functions on the chain were defined on the basis of the COSY and HMBC correlations, resulting in a structure identical to that of compound 1 from C1 to C-10. The only signal of the 1H NMR spectrum that remained to be assigned was a methyl singlet at δH 2.07, which showed HMBC correlations with the methylene at C-10 and with the carbonyl group at δC 211.8 (C-11). These data were consistent with the presence of a methyl ketone moiety at the end of the chain and defined the structure 3 for cystodione C. The HRMS pseudomolecular ion obtained for cystodione D (4) indicated that it was an isomer of cystodione C (3). Furthermore, the analysis of the NMR spectra showed that compound 4 differed from 3 only by the resonances of Me-13 (δH 2.06/δC 19.5) and the allylic methylene at C-8 (δH 2.10/δC 41.2). These chemical shifts, together with the NOESY correlation between the olefinic proton H-6 and the methylene protons H2-8, confirmed the E geometry of the double bond at C-6,C-7 in cystodione D (4). Cystodione E (5) possessed the molecular formula C28H40O6, obtained by HRMS analysis, which indicated nine degrees of unsaturation in the molecule. The 13C NMR spectrum exhibited the signals of two carbonyl groups at δC 219.5 and 201.4 and 10 signals between 165 and 110 ppm attributable to an aromatic ring and two double bonds. The presence in the 1H NMR spectrum of the signals of a methoxy group (δH 3.64) and a methyl linked to an aromatic ring (δH 2.19), together with another five methyl resonances [δH 1.87 (d J = 1.1 Hz), 1.70 (br s), 1.24 (s), 1.18 (s), 1.12 (s)] attributable to a C20 isoprenoid-derived moiety, indicated that compound 5 was another tetraprenyltoluquinol derivative of the cystodione family. The location of a double bond at C-2,C-3 was supported by the COSY coupling of the olefinic proton at δH 5.42 (br t, J = 7.4 Hz, H-2) with a methylene group at δH 3.33 (d, J = 7.4 Hz, H2-1) that exhibited HMBC correlations with the aromatic

Figure 2. Key COSY (bold bonds), HMBC (plain arrows), and NOESY (dashed arrows) correlations observed for cystodione E (5).

an isolated methylene (δH 3.09, s, H2-4) that were additionally correlated with a carbonyl group (δC 201.4, C-5) and an olefinic methine carbon (δC 124.4, C-6) was consistent with the presence of a ketone function at C-5 and a double bond at C6,C-7. The E geometry of the double bond at C-2,C-3 was supported by the NOESY correlation between the olefinic proton H-2 and the allylic methylene protons H2-4, while the Z geometry of the double bond at C-6,C-7 was indicated by the NOESY correlation between the olefinic proton H-6 and Me19. The chemical shifts of Me-20 and Me-19 at δC 16.6 and 25.5, respectively, were in agreement with the proposed 2E,6Z configuration.7a,9 At this step of the structural determination, a carbonyl group (δC 219.5), an oxymethine (δC 81.1), and two fully substituted sp3 carbons linked to oxygenated functions (δC 84.5 and 71.7) remained to be accommodated. In the HMBC spectrum the fully substituted carbon at δC 71.7 (C-15) and the oxymethine at δC 81.1 (C-14) were correlated with the terminal methyl groups of the isoprenoid chain [δH 1.12 (Me-16) and 1.24 (Me-17)], thus defining the location of a tertiary oxygenated function at C-15 and the oxymethine at C-14. The oxymethine proton H-14 [δH 4.03 (dd, J = 9.4 and 6.6 Hz)] was coupled in the COSY spectrum with two protons at δH 2.54 (dd, J = 18.2 and 9.4 Hz, H-13a)/2.43 (dd, J = 18.2 and 6.6 Hz, H-13b) that were assigned to a methylene (C-13) adjacent to a carbonyl (C-12). The remaining oxygenated carbon at δC 84.5 was identified as C-11 from its HMBC correlation with Me-18 (δH 1.18, s) and the methylene protons at C-10 [δH 1.60 (m)/1.46 (m)]. Because one degree of unsaturation was unassigned, the molecule should contain an additional ring. Two possibilities were considered: an ether bridge either between C-11 and C-14 (five-membered ring) or between C-11 and C-15 (six-membered ring). The presence of a five-membered ring was proposed on the basis of the chemical shift of the carbonyl C-12 at δC 219.5 and the IR absorption at 1750 cm−1, which were characteristic of a cyclopentanone ring23 and significantly different from the values found for sixmembered ketones and tetrahydropyran-3-ones (ca. δC 210− 215, ν 1715−1720 cm−1).22,23 Furthermore, the NMR chemical shifts of carbons from C-11 to C-15 in compound 5 were closely related to those described for a similar tetrahydrofuran3-one ring present in various saponins.24 The relative configuration at C-11 and C-14 was proposed upon observation of the NOESY correlation between the oxymethine proton H14 and the methylene protons at C-10. The remaining COSY and HMBC correlations were consistent with a sequence of three methylenes between C-11 and C-7. All these data led to the proposal of structure 5 for cystodione E. Cystodione F (6) possessed the same molecular formula as for compound 5, as determined by HRMS. The NMR data of 624



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of usneoidone Z had been described as a two-proton doublet at δH 3.37 (J = 6.8 Hz), the protons H-1 in the spectrum of 11 were resolved into two signals at δH 3.41 (1H, dd, J = 16.4 and 7.5 Hz, H-1a) and 3.33 (1H, dd, J = 16.4 and 7.5 Hz, H-1b). Discrepancies were also detected for the signals of the methylene at C-4, described at δC 58.4/ δH 3.12 (2H, s) for usneoidone Z, while in compound 11 they appeared at δC 54.6/ δH 3.24 (d, J = 15.9 Hz, H-4a) and 3.10 (d, J = 15.9 Hz, H-4b). An inspection of the spectra of usneoidone Z, kindly provided by the authors that described this compound, revealed a mistake in the report of the chemical shift of C-4 and that protons at C-4 in usneoidone Z appeared, as in 11, as two doublets (ca. J = 16 Hz), but they had erroneously been reported as a singlet. The multiplicities observed for protons at C-1 and C-4 in 11 have been observed for other related meroterpenoids and can be due to the restricted rotation caused by intramolecular hydrogen bonding.3a,7a,25 The discrepancy between the molecular formula reported for usneoidone Z and that of compound 11 can be explained by the misidentification of an ion arising from the loss of water (m/z 454.2710) as the molecular ion of usneoidone Z.5 The use in our study of a softer ionization technique has allowed detecting the true molecular ion of the compound (m/z 473.2874, [M + H]+). All these data, together with the optical rotation measured for compound 11, led us to conclude that usneoidone Z was identical to compound 11, in agreement with the structural revision previously proposed by Desmaële et al.22 Compound 12 possessed the same molecular formula as 11, determined by HRMS. The NMR data of compound 12 were similar to those of 11, except for the chemical shifts of the methylene at C-8 (δC 42.1/δH 2.11) and the Me-19 (δC 19.3/ δH 2.06), which indicated that compound 12 differed from 11 by the geometry of the double bond at C-6,C-7. These data and the correlations observed in the COSY, HMBC, and NOESY spectra established that compound 12 was the 6E isomer of usneoidone Z (11). The 1H NMR data reported for usneoidone E5 matched those of compound 12 recorded in CDCl3, except for differences in the signals due to protons at C1 and C-4, which were attributable to the different resolution of the spectra. The comparison of the 13C NMR data revealed differences about 0.6−0.8 ppm for some carbon resonances (C1, C-6, C-7, C-10, C-13), while the 1H−13C long-range correlations reported for these carbons5 were similar to those observed in compound 12. These discrepancies in the NMR data could be attributable to an inaccurate measurement of some 13C NMR chemical shifts of usneoidone E. However, in the absence of further data, the possibility that the compound isolated by Urones et al. differs from usneoidone Z (11) not only by the geometry of the double bond at C-6,C-7 cannot be disregarded. Finally, it is worth noting that although a compound with the same planar structure of 12, namely, 11hydroxyamentadione-1′-methyl ether, has been cited as a metabolite of C. usneoides,2 we describe for the first time its spectroscopic characterization. The meroterpenoids 1−4 and 7−12 isolated from C. usneoides were tested for antioxidant activity by using the ABTS assay, which determines the radical-scavenging activity of compounds toward the ABTS•+ radical cation.26 In this method, the ABTS is chemically or enzymatically oxidized to its radical cation ABTS•+, which is intensely blue-green colored, and the antioxidant capacity of a compound is measured as its ability to reduce the color by reacting with the ABTS•+ radical. The results are expressed relative to the activity of Trolox, a

compound 6 were very similar to those of 5, except for the chemical shifts of the Me-19 [δC 19.3/δH 2.08 (d, J = 1.5 Hz)] and the allylic methylene at C-8 [δC 42.0/δH 2.15 (t, J = 6.6 Hz)], which were consistent with an E geometry of the double bond at C-6,C-7. This assignment was also supported by the NOESY correlation between the olefinic proton H-6 and the allylic methylene protons H2-8. Compounds 7 and 8 were identified as 6-cis-amentadione-1′methyl ether and amentadione-1′-methyl ether, respectively, two meroditerpenoids that had been previously reported from Cystoseira tamariscifolia.20 The analysis of the 2D NMR data collected during our study has led us to revise some of the NMR assignments previously reported for 7 and 8. Compounds 9 and 10 were identified as cystomexicones A and B, respectively, previously obtained from Cystoseira abies marina.21 The NMR data of 9 and 10 matched those described in the literature,21 although the analysis of the HMBC spectra recorded in our study indicated that the NMR chemical shifts reported for C-1′and the methine C-3′ must be interchanged with those of C-4′ and the methine at C-5′, respectively. A previous chemical study of C. usnoides collected at the western coast of Portugal led to the isolation of usneoidones Z and E, two meroditerpenoids that were assigned the structures 13 and 14, respectively, by Urones et al.5 Later, Desmaële and co-workers synthesized compounds 13 and 14, but their spectroscopic data did not match those of the natural compounds, hence proposing the revision of the structures of usneoidones Z and E to the acyclic trans-enones 11 and 12, respectively.22 This proposal could not be fully confirmed, as the natural compounds had been described5 to possess the molecular formula C28H38O5, which indicated an unsaturation degree more than in 11 and 12. During our study of C. usneoides we isolated two compounds that, on the basis of their MS and NMR data, were concluded to possess the structures 11 and 12. The molecular formula C28H40O6 of compound 11 was defined by HRMS. The analysis of the NMR data, recorded in CD3OD, led to determine that compound 11 possessed the same O-methyltoluquinol ring as the compounds described above and an isoprenoid side chain identical to that of compound 5 from C-1 to C-10. Taking into account the molecular formula of 11 and the remaining NMR data, the chain fragment from C-11 to C-18 should contain a conjugated carbonyl group (δC 204.9), a disubstituted double bond (δC 156.2/δH 7.01 and δC 120.7/δH 6.83), and two hydroxy-bearing quaternary carbons (δC 79.5 and 71.5). In the HMBC spectrum, the carbon at δC 71.5 exhibited correlations with the methyl groups at the end of the chain (δH 1.32, Me-16 and Me-17) and with the olefinic protons at δH 7.01 (d, J = 15.5 Hz) and 6.83 (d, J = 15.5 Hz), which in turn were correlated with the carbonyl group at δC 204.9. These data supported the location of a hydroxy group at C-15, a double bond at C-13,C14, and a ketone function at C-12. The trans geometry of the double bond at C-13,C-14 was defined from the H-13,H-14 coupling constant of 15.5 Hz. The location of the remaining hydroxy group at C-11 was defined from the HMBC correlations of the oxygenated carbon at δC 79.5 with the methylene protons at C-10 and with Me-18 (δH 1.29), which were also correlated with the carbonyl at C-12. Although the article by Urones et al. does not indicate the solvent used for NMR, the data described for usneoidone Z5 were similar to the NMR data of 11 recorded in CDCl3. Differences observed for signals of the protons at C-1 were attributable to the use in our study of a higher field spectrometer. Thus, while protons at C-1 625



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α (Figure 3). During the incubation time of 24 h, control THP1 macrophages produced 16.77 ng/mL of TNF-α. After

synthetic vitamin E analogue, measured under the same conditions. All of the tested meroterpenoids exhibited strong radical-scavenging activity, as shown in Table 2. The most Table 2. Antioxidant Activities of Compounds 1−4 and 7− 12 in the ABTS Assay compound a

Trolox 1 2 3 4 7 8 9 10 11 12

EC50 (μM)b 25.9 22.5 24.4 55.9 44.7 26.3 24.5 51.6 33.3 43.1 33.1

TEc ± ± ± ± ± ± ± ± ± ± ±

0.5 2.1 0.9 9.9 1.1 2.3 1.6 4.8 2.3 3.1 5.1

1 1.15 1.06 0.46 0.58 0.98 1.06 0.50 0.78 0.60 0.78

Figure 3. Effects of compounds 1, 2, 4, 7, 11, and 12 isolated from C. usneoides on TNF-α production in THP-1 macrophages stimulated with LPS. The cells were treated with the compounds at concentrations of 10 μM for 4, 11, and 12, 8 μM for 1, 6 μM for 2, and 2 μM for 7 and then stimulated with LPS (1 μg/mL). TNF-α concentration in the supernatants was measured using the ELISA assay. Dexamethasone (Dex) was used as positive reference compound at 1 μM. Values are the mean ± SD of three determinations, *p < 0.5, **p < 0.01, ***p < 0.001.

a

Standard compound: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid. bConcentration that caused a 50% reduction of the absorbance. Values are mean ± SD of three determinations. cTrolox equivalents: EC50(Trolox)/EC50(compound).

active compounds were 1, 2, 7, and 8, which showed an antioxidant capacity slightly superior or equal to that of the Trolox standard. Compounds 10 and 12 were also strongly active, showing a potency of 78% that of Trolox, while compounds 3, 4, 9, and 11 displayed an antioxidant activity about 50−60% that of Trolox. From a structural point of view, the compounds possessing a 6E configuration were more active than the corresponding 6Z isomers, except for compounds 1 and 2. On the other hand, compounds with a C20 side chain were in general more active than the corresponding analogues with a C14 side chain, as evidenced by the comparison of the activity of the meroditerpenes 1, 2, 7, and 8 with that of the analogues 3, 4, 9, and 10, respectively. After an initial account on the properties of three Cystoseira-derived meroditerpenes as singlet oxygen and peroxyl radical scavengers,8 the antioxidant activities of a series of metabolites obtained from C. crinita have more recently been described.9 The meroditerpenes of C. crinita were found to be less active than Trolox in the ABTS assay, with the most active compound displaying an antioxidant activity about 40% that of the Trolox standard. The study herein described on the meroterpenes of C. usneoides has allowed the identification of new members of this family of algal metabolites, such as compounds 1, 2, 7, 8, 10, and 12, that possess more potent radical-scavenging properties, further supporting the potential of Cystoseira algae as a source of new natural antioxidants. Compounds 1, 2, 4, 7, 11, and 12 were also tested for their anti-inflammatory activity, in particular as inhibitors of TNF-α. This is a potent proinflammatory cytokine mainly produced by monocytes and macrophages in immunologic and inflammatory responses.19 Assays were performed on the human THP-1 macrophages, using lipopolysaccharide (LPS) as the triggering factor to stimulate the TNF-α production. In order to rule out cytotoxic effects, 1, 2, 4, 7, 11, and 12 were tested at maximum concentrations of 8, 6, 10, 2, 10, and 10 μM, respectively, which do not affect THP-1 cell viability in the sulforhodamine B assay. To test the effects of the compounds on TNF-α production, THP-1 macrophaghes were pretreated with the meroterpenes, then stimulated with LPS and finally analyzed to quantify TNF-

stimulation with LPS (1 μg/mL) TNF-α production increased about 10-fold, up to 153.16 ng/mL. Treatment of cells with compounds 11 and 12 significantly inhibited the production of TNF-α, causing 73% and 64% inhibition, respectively, upon comparison with LPS-stimulated THP-1 control cells. Cystodione A (1) was moderately active, causing a 33% decrease of the TNF-α level. Cystodiones B (2) and D (4) inhibited TNF-α production by only 11%, while compound 7 did not affect TNF-α production. These findings, and in particular the significant activities of compounds 11 and 12 as inhibitors of TNF-α production, are the first description of the anti-inflammatory properties of meroterpenes from Cystoseira algae and suggest that this class of marine natural products is worth exploring in the anti-inflammatory area.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. UV spectra were recorded on a GBC Cintra-101 spectrometer. IR spectra were recorded on a Perkin-Elmer FT-IR System Spectrum BX spectrophotometer. 1H and 13C NMR spectra were recorded on a Varian INOVA 600 or on an Agilent 500 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, using an RI-71 differential refractometer or L-7400 UV detector (Merck) working at 254 nm. All solvents were spectroscopic grade. Collection and Identification. Specimens of Cystoseira usneoides (class Phaeophyceae, order Fucales, family Sargassaceae) were collected by hand (1−4 m depth) along the Mediterranean coast of Morocco, at the Gibraltar Strait (35°50′52.58″ N, 5°33′39.04″ W). The algae samples were washed with sterile seawater, and epiphytes were removed. A voucher specimen (HTET.Phyc 545) is deposited at 626



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C-10), 41.2 (CH2, C-8), 29.8 (CH3, Me-12), 28.7 (CH3, Me-14), 22.5 (CH2, C-9), 19.5 (CH3, Me-13), 16.1 (CH3, 6′-Me); HRESIMS (+) m/z 397.1983 [M + Na]+ (calcd for C22H30O5Na, 397.1991). Cystodione E (5): yellowish oil; [α]25D +6.4 (c 0.1, MeOH); UV (MeOH) λmax 224 (4.15), 245 (4.10), 286 (3.64) nm; IR (film) νmax 3398, 1750, 1681, 1610 cm−1; 1H NMR (CD3OD, 600 MHz) see Table 1; 13C NMR (CD3OD, 150 MHz) see Table 1; HRESIMS(−) m/z 471.2751 [M − H]− (calcd for C28H39O6, 471.2747). Cystodione F (6): yellowish oil; [α]25D +9.0 (c 0.1, MeOH); IR (film) νmax 3412, 1751, 1603 cm−1; 1H NMR (CD3OD, 500 MHz) δ 6.44 (1H, d, J = 2.6 Hz, H-5′), 6.43 (1H, d, J = 2.6 Hz, H-3′), 6.21 (1H, br s, H-6), 5.44 (1H, br t, J = 7.3 Hz, H-2), 4.00 (1H, dd, J = 9.5 and 6.2 Hz, H-14), 3.64 (3H, s, OCH3), 3.33 (2H, d, J = 7.0 Hz, H21), 3.11 (2H, s, H2-4), 2.54 (1H, dd, J = 18.3 and 9.5 Hz, H-13a), 2.41 (1H, dd, J = 18.3 and 6.6 Hz, H-13b), 2.19 (3H, s, 6′-Me), 2.15 (2H, t, J = 6.6 Hz, H2-8), 2.08 (3H, d, J = 1.5 Hz, Me-19), 1.71 (3H, br s, Me20), 1.59 (1H, m, H-9a), 1.55 (1H, m, H-10a), 1.49 (1H, m, H-9b), 1.40 (1H, m, H-10b), 1.24 (3H, s, Me-17), 1.18 (3H, s, Me-18), 1.12 (3H, s, Me-16); 13C NMR (CD3OD, 125 MHz) δ 219.5 (C, C-12), 202.0 (C, C-5), 160.6 (C, C-7), 154.3 (C, C-4′), 150.7, (C, C-1′), 135.6 (C, C-2′), 132.7 (C, C-6′), 131.5 (C, C-3), 129.5 (CH, C-2), 123.7 (CH, C-6), 116.3 (CH, C-5′), 114.9 (CH, C-3′), 84.4 (C, C11), 81.1 (CH, C-14), 71.7 (C, C-15), 61.0 (CH3, OMe), 56.2 (CH2, C-4), 42.0 (CH2, C-8), 37.7 (CH2, C-13), 35.8 (CH2, C-10), 29.4 (CH2, C-1), 25.7* (CH3, Me-16), 25.6* (CH3, Me-17), 22.4 (CH3, Me-18), 22.2 (CH2, C-9), 19.3 (CH3, Me-19), 16.6 (CH3, Me-20), 16.4 (CH3, 6′-Me), values marked with an asterisk may be interchanged; HRMS APCI m/z 473.2892 [M + H]+ (calcd for C28H41O6, 473.2903). 6-cis-Amentadione-1′-methyl ether (7): 1H NMR (CDCl3, 500 MHz) δ 6.97 (1H, d, J = 15.7 Hz, H-14), 6.88 (1H, br s, 4′-OH), 6.57 (1H, d, J = 2.9 Hz, H-3′), 6.55 (1H, d, J = 15.7 Hz, H-13), 6.53 (1H, d, J = 2.9 Hz, H-5′), 6.08 (1H, br s, H-6), 5.35 (1H, br t, J = 7.3 Hz, H2), 3.67 (3H, s, OMe), 3.51 (1H, br s, 15-OH), 3.36 (2H, d, J = 7.3 Hz, H2-1), 3.13 (2H, s, H2-4), 2.79 (1H, m, H-11), 2.59 (1H, ddd, J = 11.7, 9.8, and 6.4 Hz, H-8a), 2.47 (1H, ddd, J = 11.7, 9.8, and 6.4 Hz, H-8b), 2.24 (3H, s, 6′-Me), 1.85 (3H, d, J = 1.5 Hz, Me-19), 1.74 (1H, m, H-10a), 1.69 (3H, br s, Me-20), 1.54 (1H, m, H-10b), 1.38 (2H, m, H2-9), 1.38 (6H, s, Me-16, Me-17), 1.10 (3H, d, J = 6.8, Me-18); 13C NMR (CDCl3, 125 MHz) δ 205.0 (C, C-12), 199.8 (C, C-5), 160.2 (C, C-7), 152.8 (CH, C-14), 152.4 (C, C-4′), 149.7 (C, C-1′), 133.9 (C, C-2′), 131.9 (C, C-6′), 131.0 (C, C-3), 127.5 (CH, C-2), 124.1 (CH, C-13), 123.7 (CH, C-6), 115.4 (CH, C-5′), 113.6 (CH, C-3′), 71.3 (C, C-15), 60.4 (CH3, OMe), 54.9 (CH2, C-4), 44.3 (CH, C-11), 33.9 (CH2, C-8), 33.7 (CH2, C-10), 29.0* (CH3, Me-16), 28.9*(CH3, Me-17), 27.5 (CH2, C-1), 25.5 (CH2, C-9), 25.5 (CH3, Me-19), 16.9 (CH3, Me-18), 16.6 (CH3, Me-20), 16.2 (CH3, 6′-Me), values marked with an asterisk may be interchanged. Amentadione-1′-methyl ether (8): 1H NMR (CDCl3, 500 MHz) δ 6.94 (1H, d, J = 15.7 Hz, H-14), 6.54 (1H, d, J = 2.9 Hz, H-3′), 6.53 (1H, d, J = 2.9 Hz, H-5′), 6.39 (1H, d, J = 15.7 Hz, H-13), 6.39 (1H, br s, 4′-OH), 6.12 (1H, br s, H-6), 5.42 (1H, br t, J = 7.3 Hz, H-2), 3.67 (3H, s, OMe), 3.37 (2H, d, J = 7.3 Hz, H2-1), 3.12 (2H, s, H2-4), 2.72 (1H, m, H-11), 2.28 (1H, br s, 15-OH), 2.24 (3H, s, 6′-Me), 2.10 (2H, m, H2-8), 2.07 (3H, d, J = 1.5 Hz, Me-19), 1.70 (3H, br s, Me20), 1.67 (1H, m, H-10a), 1.40 (3H, m, H-10b, H2-9), 1.391* (3H, s, Me-16), 1.388* (3H, s, Me-17), 1.10 (3H, d, J = 6.9, Me-18), values marked with an asterisk may be interchanged; 13C NMR (CDCl3, 125 MHz) δ 204.3 (C, C-12), 200.1 (C, C-5), 158.6 (C, C-7), 152.9 (CH, C-14), 152.2 (C, C-4′), 149.9 (C, C-1′), 134.5 (C, C-2′), 132.0 (C, C6′), 131.0 (C, C-3), 127.8 (CH, C-2), 123.8 (CH, C-13), 122.8 (CH, C-6), 115.5 (CH, C-5′), 113.7 (CH, C-3′), 71.3 (C, C-15), 60.5 (CH3, OMe), 55.4 (CH2, C-4), 44.8 (CH, C-11), 40.9 (CH2, C-8), 32.1 (CH2, C-10), 29.27* (CH3, Me-16), 29.25* (CH3, Me-17), 27.9 (CH2, C-1), 24.5 (CH2, C-9), 19.3 (CH3, Me-19), 16.5 (CH3, Me-18), 16.4 (CH3, Me-20), 16.2 (CH3, 6′-Me), values marked with an asterisk may be interchanged. Usneoidone Z (11): yellowish oil; [α]25D −9.0 (c 0.2, CHCl3), lit.5 [α]25D −6.1 (c 0.3, CHCl3); IR (film) νmax 3407, 1684, 1611 cm−1; 1H NMR (CD3OD, 600 MHz) δ 7.01 (1H, d, J = 15.5 Hz, H-14), 6.83

the herbarium of the Laboratory of Applied Algology-Mycology, University of Abdelmalek Essâadi, Morocco. Extraction and Isolation. Shade-dried samples of C. usneoides (150.0 g) were ground and extracted with acetone/MeOH (1:1, 2L). The resulting extract (4.63 g) was subjected to column chromatography eluting with n-hexane/Et2O mixtures of increasing polarity (from 90:10 to 30:70), then Et2O, CHCl3/MeOH mixtures (95:5 and 80:20), and finally MeOH. The fractions of the general chromatography eluted with n-hexane/ Et2O (30:70) and Et2O were chromatographed over a silica gel column using n-hexane and n-hexane/EtOAc mixtures (80:20 to 30:70) as eluents. Repeated purifications of selected fractions by normal-phase HPLC using n-hexane/EtOAc (60:40) and n-hexane/iPrOH (90:10) yielded compounds 9 (29.6 mg, 2.0 × 10−2 % dry wt), 10 (73.2 mg, 4.9 × 10−2 % dry wt), 5 (5.3 mg, 3.5 × 10−3 dry wt), 7 (86.6 mg, 5.8 × 10−2 % dry wt), and 8 (85.1 mg, 5.7 × 10−2 % dry wt). The fraction from the general chromatography that eluted with CHCl3/MeOH (95:5) was further separated over a silica gel column using n-hexane/EtOAc mixtures (from 50:50 to 15:85) as eluents. Repeated separations of selected fractions by normal-phase HPLC nhexane/EtOAc (50:50) and n-hexane/i-PrOH (90:10) afforded compounds 11 (86.9 mg, 5.8 × 10−2 % dry wt), 6 (3.5 mg, 2.3 × 10−3 % dry wt), 12 (88.7 mg, 5.9 × 10−2 % dry wt), 3 (2.9 mg, 1.9 × 10−3 dry wt), 1 (29.5 mg, 2.0 × 10−2 % dry wt), 2 (16.7 mg, 1.1 × 10−2 % dry wt), 4 (4.9 mg, 3.3 × 10−3 % dry wt), and additional amounts of 8 (46.1 mg, total yield 8.7 × 10−2 % dry wt). Cystodione A (1): yellowish oil; [α]25D −1.0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 214 (4.39), 246 (4.22), 310 (3.55) nm; IR (film) νmax 3407, 1668, 1606 cm−1; 1H NMR (CD3OD, 600 MHz) see Table 1; 13C NMR (CD3OD, 150 MHz) see Table 1; HRESIMS(−) m/z 471.2758 [M − H]− (calcd for C28H39O6, 471.2747). Cystodione B (2): yellowish oil; [α]25D −2.6 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 213 (4.46), 245 (4.33), 305 (3.59) nm; IR (film) νmax 3398, 1660, 1605 cm−1; 1H NMR (CD3OD, 600 MHz) δ 6.93 (1H, dd, J = 15.9, 1.2 Hz, H-14), 6.76 (1H, d, J = 16.1 Hz, H-1), 6.70 (1H, d, J = 3.1 Hz, H-3′), 6.51 (1H, d, J = 3.1 Hz, H-5′), 6.34 (1H, d, J = 15.9 Hz, H-13), 6.30 (1H, d, J = 16.1 Hz, H-2), 6.20 (1H, br s, H-6), 3.60 (3H, s, OMe), 2.81 (1H, m, H-11), 2.80 (1H, d, J = 13.9 Hz, H-4a), 2.72 (1H, d, J = 13.9 Hz, H-4b), 2.18 (3H, s, 6′-Me), 2.11 (2H, t, J = 7.5 Hz, H2-8), 2.05 (3H, d, J = 1.1 Hz, Me-19), 1.61 (1H, m, H-10a), 1.40 (3H, s, Me-20), 1.38 (2H, m, H2-9), 1.32 (6H, s, Me-16 and Me-17), 1.30 (1H, m, H-10b), 1.04 (1H, d, J = 7.0 Hz, Me18); 13C NMR (CD3OD, 150 MHz) δ 206.7 (C, C-12), 202.1 (C, C5), 160.6 (C, C-7), 155.2 (CH, C-14), 154.4 (C, C-4′), 150.7 (C, C1′), 137.8 (CH, C-2), 133.1 (C, C-6′), 131.9 (C, C-2′), 125.9 (CH, C6), 125.6 (CH, C-13), 123.3 (CH, C-1), 118.0 (CH, C-5′), 111.0 (CH, C-3′), 73.5 (C, C-3), 71.3 (C, C-15), 61.4 (C, OMe), 56.4 (CH2, C-4), 45.0 (CH, C-11), 42.1 (CH2, C-8), 33.7 (CH2, C-10), 29.3 (CH3, Me-16), 29.3 (CH3, Me-17), 28.6 (CH3, Me-20), 26.2 (CH2, C-9), 19.6 (CH3, Me-19), 17.1 (CH3, Me-18), 16.2 (CH3, 6′Me); HRESIMS(−) m/z 471.2751 [M − H]− (calcd for C28H39O6, 471.2747). Cystodione C (3): yellowish oil; [α]25D −3.9 (c 0.1, MeOH); UV (MeOH) λmax 209 (4.35), 248 (4.16), 308 (3.54) nm; IR (film) νmax 3401, 1711, 1605 cm−1; 1H NMR (CD3OD, 600 MHz) see Table 1; 13 C NMR (CD3OD, 150 MHz) see Table 1; HRESIMS (+) m/z 397.1981 [M + Na]+ (calcd for C22H30O5Na, 397.1991). Cystodione D (4): yellowish oil; [α]25D +1.0 (c 0.1, MeOH); UV (MeOH) λmax 209 (4.31), 245 (4.15), 309 (3.47) nm; IR (film) νmax 3400, 1709, 1672, 1604 cm−1; 1H NMR (CD3OD, 600 MHz) δ 6.77 (1H, d, J = 16.2 Hz, H-1), 6.70 (1H, d, J = 2.9 Hz, H-3′) 6.50 (1H, d, J = 2.9 Hz, H-5′), 6.30 (1H, d, J = 16.2, H-2), 6.21 (1H, br s, H-6), 3.60 (3H, s, OMe), 2.81 (1H, d, J = 14.2 Hz, H-4a), 2.72 (1H, d, J = 14.2 Hz, H-4b), 2.43 (2H, t, J = 7.2 Hz, H2-10), 2.18 (3H, s, 6′-Me), 2.10 (2H, m, H2-8), 2.08 (3H, s, Me-12), 2.06 (3H, d, J = 1.2 Hz, Me-13), 1.68 (2H, m, H2-9), 1.41 (3H, s, Me-14); 13C NMR (CD3OD, 150 MHz) δ 211.3 (C, C-11), 202.2 (C, C-5), 160.2 (C, C-7), 154.3 (C, C4′), 150.7 (C, C-1′), 137.7 (CH, C-2), 133.1 (C, C-6′), 131.9 (C, C2′), 126.1 (CH, C-6), 123.3 (CH, C-1), 118.0 (CH, C-5′), 111.0 (CH, C-3′), 73.5 (C, C-3), 61.4 (CH3, OMe), 56.4 (CH2, C-4), 43.2 (CH2, 627



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(1H, d, J = 15.5 Hz, H-13), 6.44 (1H, d, J = 2.9 Hz, H-5′), 6.43 (1H, d, J = 2.9 Hz, H-3′), 6.19 (1H, br s, H-6), 5.41 (1H, br t, J = 7.3 Hz, H2), 3.64 (3H, s, OMe), 3.32 (2H, d, J = 7.3 Hz, H2-1), 3.08 (2H, s, H24), 2.57 (1H, ddd, J = 12.0, 9.4, and 6.1 Hz, H-8a), 2.50 (1H, ddd, J = 12.0, 9.4, and 6.1 Hz, H-8b), 2.19 (3H, s, 6′-Me), 1.84 (3H, d, J = 1.3 Hz, Me-19), 1.75 (1H, ddd, J = 13.8, 12.3, and 4.6 Hz, H-10a), 1.70 (3H, br s, Me-20) 1.64 (1H, ddd, J = 13.8, 12.3, and 4.6 Hz, H-10b), 1.52 (1H, m, H-9a), 1.32 (6H, s, Me-16 and Me-17), 1.29 (3H, s, Me18), 1.26 (1H, m, H-9b); 13C NMR (CD3OD, 150 MHz) δ 204.9 (C, C-12), 201.3 (C, C-5), 161.5 (C, C-7), 156.2 (CH, C-14), 154.3 (C, C-4′), 150.8 (C, C-1′), 135.6 (C, C-2′), 132.7 (C, C-6′), 131.3 (C, C3), 129.6 (CH, C-2), 124.3 (CH, C-6), 120.7 (CH, C-13), 116.4 (CH, C-5′), 115.0 (CH, C-3′), 79.5 (C, C-11), 71.5 (C, C-15), 61.0 (CH3, OMe), 56.2 (CH2, C-4), 40.1 (CH2, C-10), 34.6 (CH2, C-8), 29.4 (CH2, C-1), 29.26* (CH3, Me-16), 29.25* (CH3, Me-17), 25.4 (CH3, Me-19), 25.2 (CH3, Me-18), 23.2 (CH2, C-9), 16.7 (CH3, Me-20), 16.4 (CH3, 6′-Me), values marked with an asterisk may be interchanged; 1H NMR (CDCl3, 500 MHz) δ 7.23(1H, br s, 4′OH), 7.15 (1H, d, J = 15.2 Hz, H-14), 6.88 (1H, d, J = 15.2 Hz, H-13), 6.65 (1H, d, J = 2.9 Hz, H-3′), 6.54 (1H, d, J = 2.9 Hz, H-5′), 6.09 (1H, br s, H-6), 5.34 (1H, br t, J = 7.8 Hz, H-2), 4.18 (1H, br s, 11OH), 4.08 (1H, br s, 15-OH), 3.68 (3H, s, OMe), 3.41 (1H, dd, J = 16.4 and 7.5 Hz, H-1a), 3.33 (1H, dd, J = 16.4 and 7.5 Hz, H-1b), 3.24 (1H, d, J = 15.9 Hz, H-4a), 3.10 (1H, d, J = 15.9 Hz, H-4b), 2.67 (1H, ddd, J = 11.2, 11.2, and 4.4 Hz, H-8a), 2.25 (3H, s, 6′-Me), 2.09 (1H, ddd, J = 11.2, 11.2, and 5.9 Hz, H-8b), 1.95 (1H, ddd, J = 14.3, 11.2, and 3.9 Hz, H-10a), 1.86 (1H, m, H-10b), 1.85 (3H, d, J = 1.5 Hz, Me19), 1.65 (3H, br s, Me-20) 1.59 (1H, m, H-9a), 1.40 (3H, s, Me-17), 1.361 (3H, s, Me-18), 1.356 (3H, Me-16), 1.09 (1H, m, H-9b) ; 13C NMR (CDCl3, 125 MHz) δ 203.2 (C, C-12), 200.3 (C, C-5), 160.7 (C, C-7), 155.8 (CH, C-14), 152.6 (C, C-4′), 149.6 (C, C-1′), 133.6 (C, C-2′), 131.9 (C, C-6′), 131.2 (C, C-3), 127.4 (CH, C-2), 124.0 (CH, C-6), 119.1 (CH, C-13), 115.4 (CH, C-5′), 113.4 (CH, C-3′), 78.6 (C, C-11), 71.6 (C, C-15), 60.3 (CH3, OMe), 54.6 (CH2, C-4), 38.5 (CH2, C-10), 34.5 (CH2, C-8), 29.5* (CH3, Me-16), 28.3* (CH3, Me-17), 27.3 (CH2, C-1), 26.3 (CH3, Me-18), 25.5 (CH3, Me-19), 22.9 (CH2, C-9), 16.5 (CH3, Me-20), 16.2 (CH3, 6′-Me), values marked with an asterisk may be interchanged; HRMS APCI m/z 473.2874 [M + H]+ (calcd for C28H41O6, 473.2903). 11-Hydroxyamentadione-1′-methyl ether (12): yellowish oil; [α]25D −6.4 (c 0.05, CHCl3); IR (film) νmax 3400, 1685, 1604 cm−1; 1 H NMR (CD3OD, 500 MHz) δ 7.02 (1H, d, J = 15.6 Hz, H-14), 6.83 (1H, d, J = 15.6 Hz, H-13), 6.44 (1H, d, J = 2.9 Hz, H-5′), 6.43 (1H, d, J = 2.9 Hz, H-3′), 6.18 (1H, br s, H-6), 5.42 (1H, br t, J = 7.3 Hz, H2), 3.64 (3H, s, OCH3), 3.33 (2H, d, J = 7.3 Hz, H2-1), 3.11 (2H, s, H2-4), 2.19 (3H, s, 6′-Me), 2.11 (2H, t, J = 7.3 Hz, H2-8), 2.06 (3H, d, J = 1.1 Hz, Me-19), 1.71 (3H, d, J = 1.1 Hz, Me-20), 1.70 (1H, m, H10a), 1.57 (1H, m, H-10b), 1.55 (1H, m, H-9a), 1.32 (6H, s, Me-16 and Me-17), 1.30 (m, H-9b), 1.28 (3H, s, Me-18); 13C NMR (CD3OD, 125 MHz) δ 204.9 (C, C-12), 201.9 (C, C-5), 160.7 (C, C7), 156.3 (CH, C-14), 154.3 (C, C-4′), 150.8, (C, C-1′), 135.6 (C, C2′), 132.7 (C, C-6′), 131.4 (C, C-3), 129.5 (CH, C-2), 123.8 (CH, C6), 120.6 (CH, C-13), 116.4 (CH, C-5′), 115.0 (CH, C-3′), 79.4 (C, C-11), 71.5 (C, C-15), 61.0 (CH3, OMe), 56.1 (CH2, C-4), 42.1 (CH2, C-8), 39.6 (CH2, C-10), 29.4 (CH2, C-1), 29.3 (2 x CH3, Me16 and Me-17), 25.2 (CH3, Me-18), 22.4 (CH2, C-9), 19.3 (CH3, Me19), 16.7 (CH3, Me-20), 16.4 (CH3, 6′-Me); 1H NMR (CDCl3, 500 MHz) δ 7.20 (1H, d, J = 15.2 Hz, H-14), 6.68 (1H, br s, 4′-OH), 6.58 (1H, d, J = 15.2 Hz, H-13), 6.56 (1H, d, J = 2.9 Hz, H-3′), 6.52 (1H, d, J = 2.9 Hz, H-5′), 6.11 (1H, br s, H-6), 5.42 (1H, br t, J = 7.3 Hz, H2), 4.13 (1H, br s, OH), 3.68 (3H, s, OCH3), 3.41 (1H, dd, J = 15.7 and 7.3 Hz, H-1a), 3.37 (1H, dd, J = 15.7 and 6.8 Hz, H-1), 3.19 (1H, d, J = 15.7 Hz, H-4a), 3.15 (1H, d, J = 15.7 Hz, H-4b), 2.91 (1H, br s, OH), 2.25 (3H, s, 6′-Me), 2.18 (1H, m, H-8a), 2.00 (1H, m, H-8b), 1.99 (3H, d, J = 1.2 Hz, Me-19), 1.71 (2H, m, H2-10), 1.70 (3H, br s, Me-20), 1.64 (1H, m, H-9a), 1.45 (3H, s, Me-16), 1.40 (3H, s, Me17), 1.36 (3H, s, Me-18), 1.09 (1H, m, H-9b); 13C NMR (CDCl3, 150 MHz) δ 202.6 (C, C-12), 200.8 (C, C-5), 158.0 (C, C-7), 156.4 (CH, C-14), 152.3 (C, C-4′), 149.8, (C, C-1′), 134.2 (C, C-2′), 131.9 (C, C6′), 131.1 (C, C-3), 127.6 (CH, C-2), 123.6 (CH, C-6), 117.8 (CH,

C-13), 115.5 (CH, C-5′), 113.5 (CH, C-3′), 78.3 (C, C-11), 71.6 (C, C-15), 60.4 (CH3, OMe), 55.1 (CH2, C-4), 40.5 (CH2, C-8), 37.3 (CH2, C-10), 29.3* (CH3, Me-16), 28.9* (CH3, Me-17), 27.6 (CH2, C-1), 25.3 (CH3, Me-18), 20.8 (CH2, C-9), 19.4 (CH3, Me-19), 16.6 (CH3, Me-20), 16.2 (CH3, 6′-Me); HRESIMS(−) m/z 471.2758 [M − H] (calcd for C28H39O6, 471.2747); HRESIMS(+) m/z 495.2711 [M + Na]+ (calcd for C28H40O6Na, 495.2723). Antioxidant Assay. Antioxidant activity was determined by the ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)) free radical decolorization assay developed by Re et al.,26 with slight modifications. ABTS was dissolved in H2O to a 2.9 mM concentration. The ABTS•+ radical cation was produced by reacting the ABTS stock solution with 0.98 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12− 18 h before use. For the study of the algal compounds the ABTS•+ solution was diluted with EtOH to an absorbance of 0.7 ± 0.02 at 734 nm. Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as standard. After addition of 100 μL of ABTS•+ reagent to 90 μL of EtOH and 10 μL of the tested compound or Trolox standard (final concentration from 3.125 to 200 μM), the absorbance at 734 nm was taken 1 min after mixing and up to 6 min using a microtiter plate reader. Appropriate solvent blanks (controls) were run in each assay. All 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 the 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 (FBS), 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 was determined by the sulforhodamine B (SRB) assay.27 In brief, the cells were seeded into 96-well plates in the growth medium at 1 × 104 cells/well, and differentiation into macrophages was induced by 0.2 μM phorbol myristate acetate (PMA). Three days after differentiation into macrophages, cells were exposed to algal compounds in fresh medium and incubated for another 48 h. The cells were fixed with 50 μL of trichloroacetic acid (TCA, 50%) and processed as described in the literature.27 Determination of TNF-α Production. THP-1 cells (3 × 105 cells/mL) were incubated with PMA (0.2 μM) in 24-well plates for 3 days in a humidified atmosphere of 5% CO2 at 37 °C. The macrophages were incubated with the meroterpenes for 24 h (the viability of cells was greater than 95% throughout the experiment) and then exposed to lipopolysaccharide (LPS, 1 μg/mL) for another 24 h. Supernatant fluids were collected and stored at −80 °C until TNF-α measurements. Controls contained medium with equivalent amounts of solvent compared to treatments and were incubated with and without LPS. Dexamethasone was used as positive reference compound. Commercial enzyme-linked immunosorbent assay (ELISA) kits (Diaclone GEN-PROBE) were used to quantify TNFα according to the manufacturer’s protocol. The absorbance at 450 nm was read by a microplate reader. To calculate the concentration of TNF-α, a standard curve was constructed using serial dilutions of cytokine standards provided with the kit.

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR spectra of compounds 1−8, 11, and 12 and tables of 2D NMR data of compounds 1−6, 11, and 12. This information is available free of charge via the Internet at http:// pubs.acs.org. 628



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Article

(23) Pretsch, E.; Bü l hmann, P.; Badertscher, M. Structure Determination of Organic Compounds; Springer: Berlin, 2009; pp 130 and 312. (24) Hu, L.; Chen, Z.; Xie, Y. J. Nat. Prod. 1996, 59, 1186−1188. (25) Amico, V.; Cunsolo, F.; Piattelli, M.; Ruberto, G. Phytochemistry 1985, 24, 1047−1050. (26) Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Free Radical Biol. Med. 1999, 26, 1231−1237. (27) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107−1112.

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 Andaluciá (FQM-169 and Proyecto de Excelencia POLFANAT, P09AGR-5185). H.Z. acknowledges a fellowship from AECIDMAEC, Spain. We thank to Dr. H. Riadi (Abdelmalek Essâadi University) for his collaboration in providing algal samples. We are grateful to Prof. P. Basabe for kindly providing us with a copy of the NMR spectra of usneoidones.

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REFERENCES

(1) Amico, V. Phytochemistry 1995, 39, 1257−1279. (2) Valls, R.; Piovetti, L. Biochem. Syst. Ecol. 1995, 23, 723−745. (3) More recent examples of the isolation of meroterpenes from Cystoseira can be found in: (a) Mokrini, R.; Ben Mesaoud, M.; Daoudi, M.; Hellio, C.; Maréchal, J.-P.; El Hattab, M.; Ortalo-Magné, A.; Piovetti, L.; Culioli, G. J. Nat. Prod. 2008, 71, 1806−1811. (b) Navarro, G.; Fernández, J. J.; Norte, M. J. Nat. Prod. 2004, 67, 495−499. (4) (a) Francisco, C.; Banaigs, B.; Teste, J.; Cave, A. J. Org. Chem. 1986, 51, 1115−1120. (b) Fadli, M.; Aracil, J.-M.; Jeanty, G.; Banaigs, B.; Francisco, C. J. Nat. Prod. 1991, 54, 261−264. (c) Mesguiche, V.; Valls, R.; Piovetti, L.; Banaigs, B. Phytochemistry 1997, 45, 1489−1494. (5) Urones, J. G.; Basabe, P.; Marcos, I. S.; Pineda, J.; Lithgow, A. M.; Moro, R. F.; Brito Palma, F. M. S.; Araújo, M. E. M.; Grávalos, M. D. G. Phytochemistry 1992, 31, 179−182. A revision of the structures is proposed in ref 22. (6) Urones, J. G.; Araújo, M. E. M.; Brito Palma, F. M. S.; Basabe, P.; Marcos, I. S.; Moro, R. F.; Lithgow, A. M.; Pineda, J. Phytochemistry 1992, 31, 2105−2109. (7) (a) Amico, V.; Cunsolo, F.; Neri, P.; Piattelli, M.; Ruberto, G. Phytochemistry 1988, 27, 1327−1331. (b) Bennamara, A.; Abourriche, A.; Berrada, M.; Charrouf, M.; Chaib, N.; Boudouma, M.; Garneau, F. X. Phytochemistry 1999, 52, 37−40. (8) Foti, M.; Piattelli, M.; Amico, V.; Ruberto, G. J. Photochem. Photobiol. B 1994, 26, 159−164. (9) Fisch, K. M.; Böhm, V.; Wright, A. D.; König, G. M. J. Nat. Prod. 2003, 66, 968−975. (10) Finkel, T.; Holbrook, N. J. Nature 2000, 408, 239−247. (11) Valko, M.; Leibfritz, D.; Moncol, J.; Cronin, M. T. D.; Mazur, M.; Telser, J. Int. J. Biochem. Cell Biol. 2007, 39, 44−84. (12) Halliwell, B. Biochem. J. 2007, 401, 1−11. (13) Caputo, F.; Vegliante, R.; Ghibelli, L. Biochem. Pharmacol. 2012, 84, 1292−1306. (14) Masella, R.; Di Benedetto, R.; Varí, R.; Filesi, C.; Giovannini, C. J. Nutr. Biochem. 2005, 16, 577−586. (15) Gautam, R.; Jachak, S. M. Med. Res. Rev. 2009, 29, 767−820. (16) Palladino, M. A.; Bahjat, F. R.; Theodorakis, E. A.; Moldawer, L. L. Nat. Rev. Drug Discovery 2003, 2, 736−746. (17) Simmons, D. L. Drug Discovery Today 2006, 11, 210−219. (18) Paul, A. T.; Gohil, V. M.; Bhutani, K. K. Drug Discovery Today 2006, 11, 725−732. (19) Iqbal, M.; Verpoorte, R.; Korthout, H. A. A. J.; Mustafa, N. R. Phytochem. Rev. 2012, in press, DOI 10.1007/s11101-012-9251-7. (20) Amico, V.; Piattelli, M.; Neri, P.; Ruberto, G. Gazz. Chim. Ital. 1989, 119, 467−470. (21) Fernández, J. J.; Navarro, G.; Norte, M. Nat. Prod. Lett. 1998, 12, 285−291. (22) Danet, M.; Normand-Bayle, M.; Mahuteau, J.; D’Angelo, J.; Morgant, G.; Desmaële, D. Eur. J. Org. Chem. 2004, 1911−1922. 629


Antioxidant and Anti-inflammatory Meroterpenoids from the Brown Alga

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