Disulfides with Anti-inflammatory Activity from the Brown Alga

DOI: 10.1021/acs.jnatprod.5b01031. Publication Date (Web): March 4, 2016. Copyright © 2016 The American Chemical Society and American Society of Phar...
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Disulfides with Anti-inflammatory Activity from the Brown Alga Dictyopteris membranacea Maria Dimou,† Efstathia Ioannou,*,† Maria G. Daskalaki,‡ Leto A. Tziveleka,† Sotirios C. Kampranis,‡ and Vassilios Roussis*,† †

Department of Pharmacognosy and Chemistry of Natural Products, Faculty of Pharmacy, School of Health Sciences, University of Athens, Panepistimiopolis Zografou, Athens 15771, Greece ‡ Department of Biochemistry, School of Medicine, University of Crete, P.O. Box 2208, Heraklion 71003, Greece S Supporting Information *

ABSTRACT: Six new (1, 2, and 4−7) and two previously reported (3 and 8) disulfides, along with 4-butyl-2,6cycloheptadienone, γ-tocopherol, and δ-tocopherol, were isolated from an organic extract of the brown alga Dictyopteris membranacea, collected at Gerolimenas Bay, Greece. The structure elucidation of the isolated natural products was based on analysis of their spectroscopic data. Compounds 1, 3−6, and 8 were evaluated for their antibacterial and antiinflammatory activities. None of the compounds displayed antibacterial activity against two resistant strains of Staphylococcus aureus and one strain of Escherichia coli. In contrast, metabolite 5 was able to cause strong inhibition of NO production with an IC50 value of 3.8 μM using an LPS stimulation assay.

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RESULTS AND DISCUSSION A series of chromatographic separations of the organic extract of the brown alga D. membranacea, collected at Gerolimenas Bay, Greece, resulted in the isolation of the new disulfides 1, 2, and 4−7 and five previously reported natural products, which were identified as bis(3-oxo-undecyl) disulfide (3), 3-hexyl-4,5dithiocycloheptanone (8), 4-butyl-2,6-cycloheptadienone, γtocopherol, and δ-tocopherol by comparison of their spectroscopic and physical characteristics with those reported in the literature.7,9−13 To the best of our knowledge, only compound 8 has been found so far as a constituent of D. membranacea.7 Compound 1, isolated as a yellowish oil, displayed an ion peak at m/z 399.2389 (HRESIMS), corresponding to C22H39O2S2 and consistent with [M + H]+. However, the 13C NMR spectrum exhibited only 11 carbon signals, indicating that 1 is a symmetrical dimer. This hypothesis was supported further by the fragment ions at m/z 199 and 167 observed in the EIMS of 1, corresponding to [C11H19OS]+ and [C11H19O]+, respectively. According to the DEPT experiments, the carbon signals were attributed to one methyl, seven methylenes, two methines, and one quaternary carbon atom. Among them,

rown algae of the genus Dictyopteris (Dictyotaceae), some of which are edible, such as Dictyopteris plagiogramma (Montagne) Vickers with the common name “limu lipoa” in Hawaii, are distributed widely in the tropical and temperate regions of the world.1 Currently, the genus includes 35 taxonomically accepted species, which are characterized by their pleasant odor.2 Their aromatic note has been, at least partly, attributed to the presence of volatile C11 hydrocarbons, which usually constitute a large percentage of the algal essential oil.3 Besides C11 hydrocarbons, members of this genus have been reported to produce C11 sulfur-containing compounds and sesquiterpenes of either normal or mixed biosynthesis.4,5 A number of these C11 hydrocarbons act as sex pheromones, while several C11 metabolites, with or without sulfur, have been shown to deter grazing by some herbivores.6−8 Dictyopteris membranacea Batters is a commonly occurring species growing on sublittoral rocks of the Atlantic and Mediterranean coasts. In continuation of our research targeting the isolation of bioactive natural products from marine organisms found along the coastlines of Greece, we carried out a thorough investigation of the chemical composition of D. membranacea, collected at Gerolimenas Bay. Herein, we report the isolation and structure elucidation of six new (1, 2, and 4−7) and two previously reported (3 and 8) disulfides (Figure 1) and the evaluation of their antibacterial and anti-inflammatory activities, as well as the isolation of 4-butyl-2,6-cycloheptadienone, γtocopherol, and δ-tocopherol. © XXXX American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: November 14, 2015

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DOI: 10.1021/acs.jnatprod.5b01031 J. Nat. Prod. XXXX, XXX, XXX−XXX

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assumed to be linear. The spectroscopic data of metabolite 1 (Table 1) were rather similar to those of the co-occurring bis(3oxo-undecyl) disulfide (3).7,9 The proposed structure was verified further by the analysis of its 2D NMR spectra (HSQC, HMBC, and COSY). Specifically, the correlations of H2-1, H22, H-4, and H-5 with C-3, of H2-7 with C-6 and C-8, and of H311 with C-9 and C-10 observed in the HMBC spectrum, in conjunction with the COSY cross-peaks of H2-1/H2-2, H-4/H5, H-5/H2-6, H2-6/H2-7, H2-7/H2-8, H2-8/H2-9, H2-9/H2-10, and H2-10/H3-11, were used to conclude the assignment of each monomer. The geometry of the Δ4 double bond was assigned as E on the basis of the value of the coupling constant between protons H-4 and H-5 (J = 15.8 Hz). Therefore, compound 1 was identified as bis(E)-(3-oxo-undec-4-enyl) disulfide. Compound 2, with the molecular formula C24H46O2S4, as deduced from HRESIMS measurements, was obtained as a yellowish oil. As in the case of 1, the 13C NMR spectrum displayed only 12 carbon signals, indicating that 2 is also a symmetrical dimer, a fact further verified by the diagnostic fragment ions at m/z 247 and 215 observed in the EIMS of 2, corresponding to [C12H23OS2]+ and [C12H23OS]+, respectively. Its 1H and 13C NMR spectroscopic data (Table 1) closely resembled those of 1 and 3, with the most prominent difference between 2 and 3 being the presence of a deshielded methine (δH/C 3.00/41.6) and a singlet methyl (δH/C 2.03/13.3), with the simultaneous absence of a methylene. The cross-peaks of H2-1/H2-2, H2-4/H-5, H-5/H2-6, H2-6/H2-7, H2-7/H2-8, H28/H2-9, H2-9/H2-10, and H2-10/H3-11 observed in the COSY spectrum, in combination with the HMBC correlations of H2-1, H2-2, H2-4, and H-5 with C-3, of the S-Me with C-5, of H2-8 with C-7, and of H3-11 with C-9 and C-10, supported the proposed structure of 2. Thus, compound 2 was identified as bis(5-methylthio-3-oxo-undecyl) disulfide. Compound 4, obtained as a yellowish oil, gave the molecular formula C22H40O2S2, as calculated from the HRESIMS measurement. Analysis of the spectroscopic data of 4 (Table 2) showed a high degree of similarity with metabolites 1−3. Nonetheless, the 13C NMR spectrum exhibited 22 carbon signals, thus indicating lack of a symmetry element in 4. In the EIMS of 4, the fragment ions originating from the cleavage of the disulfide bond were observed at m/z 201 and 199, consistent with [C11H21OS]+ and [C11H19OS]+, respectively. According to the DEPT experiments, the carbon signals

Figure 1. Structures of compounds 1−8.

resonances were evident for one carbonyl (δC 198.2) and two tertiary olefinic carbons (δC 130.1 and 148.5). The 1H NMR spectrum of 1 included signals for an aliphatic methyl on a secondary carbon (δH 0.87), three overlapping methylenes indicative of an aliphatic alkyl chain (δH 1.26−1.28), two additional methylenes (δH 1.44 and 2.20), two relatively deshielded methylenes (δH 2.90 and 2.96), and one trans 1,2disubstituted double bond (δH 6.09 and 6.86). Since the carbon−carbon double bond and the carbonyl functionality accounted for two of the four degrees of unsaturation and the molecule was considered a dimer, the structure of 1 was

Table 1. NMR Data (1H 400 MHz, 13C 50 MHz, CDCl3) of Compounds 1−3 1 1, 1′ 2, 2′ 3, 3′ 4, 4′ 5, 5′ 6, 6′ 7, 7′ 8, 8′ 9, 9′ 10, 10′ 11, 11′ SMe a

2

δC

position 32.2, 39.3, 198.2, 130.1, 148.5, 32.5, 28.0, 28.9, 31.6, 22.5, 14.0,

δH (J in Hz) CH2 CH2 C CH CH CH2 CH2 CH2 CH2 CH2 CH3

2.90, m 2.96, m 6.09, 6.86, 2.20, 1.44, 1.27, 1.26, 1.28, 0.87,

d (15.8) dt (15.8, 7.1) q (7.1) m m m m t (6.7)

3

δ Ca 31.5, 42.8, 206.9, 48.5, 41.6, 34.5, 26.8, 29.1, 31.7, 22.6, 14.1, 13.3,

δH (J in Hz) CH2 CH2 C CH2 CH CH2 CH2 CH2 CH2 CH2 CH3 CH3

2.84, m 2.83, m 2.71, 3.00, 1.50, 1.39, 1.24, 1.26, 1.25, 0.86, 2.03,

dd (16.5, 7.5), 2.62, dd (16.5, 6.3) m m m m m m t (6.7) s

δC 31.7, 41.9, 209.0, 43.2, 23.8, 29.1, 29.2, 29.3, 31.8, 22.6, 14.1,

δH (J in Hz) CH2 CH2 C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

2.84, m 2.79, m 2.41, 1.55, 1.23, 1.23, 1.23, 1.22, 1.25, 0.85,

t (7.5) m m m m m m t (6.7)

Determined through HMBC correlations. B

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Table 2. NMR Data (1H 400 MHz, 13C 50 MHz, CDCl3) of Compounds 4−7 4 δC

position

5 δH (J in Hz)

1 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′

31.7, 41.9, 209.0, 43.2, 23.8, 29.1, 29.2, 29.3, 31.8, 22.6, 14.1, 32.2, 39.3, 198.2, 130.1,

CH2 CH2 C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH2 CH2 C CH

2.84, m 2.79, m

5′

148.5,

CH

32.5, 28.0, 28.9, 31.6, 22.5, 14.0,

CH2 CH2 CH2 CH2 CH2 CH3

6.86, dt (15.8, 7.1) 2.20, q (7.1) 1.44, m 1.27, m 1.26, m 1.28, m 0.87, t (6.7)

6′ 7′ 8′ 9′ 10′ 11′ SMe

2.41, 1.55, 1.23, 1.23, 1.23, 1.22, 1.25, 0.85, 2.90, 2.96,

t (7.5) m m m m m m t (6.7) m m

6.09, d (15.8)

δC

6 δH (J in Hz)

31.7, 41.9, 208.9, 43.2, 23.8, 29.1, 29.2, 29.3, 31.8, 22.6, 14.1, 31.5, 42.8, 206.9, 48.5,

CH2 CH2 C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH2 CH2 C CH2

41.6,

CH

2.71, dd (16.5, 7.5), 2.62, dd (16.5, 6.3) 3.00, m

34.5, 26.8, 29.1, 31.7, 22.6, 14.1, 13.3,

CH2 CH2 CH2 CH2 CH2 CH3 CH3

1.50, 1.39, 1.24, 1.26, 1.25, 0.86, 2.03,

δC

2.84, m 2.79, m 2.41, 1.55, 1.23, 1.23, 1.23, 1.22, 1.25, 0.85, 2.84, 2.83,

t (7.5) m m m m m m t (6.7) m m

m m m m m t (6.7) s

7 δH (J in Hz)

32.2, 39.3, 198.1, 130.1, 148.5, 32.5, 28.0, 28.9, 31.6, 22.5, 14.0, 31.5, 42.8, 206.9, 48.5,

CH2 CH2 C CH CH CH2 CH2 CH2 CH2 CH2 CH3 CH2 CH2 C CH2

2.90, m 2.96, m

41.6,

CH

2.71, dd (16.5, 7.5), 2.62, dd (16.5, 6.3) 3.00, m

34.5, 26.8, 29.1, 31.7, 22.6, 14.1, 13.3,

CH2 CH2 CH2 CH2 CH2 CH3 CH3

1.50, 1.39, 1.24, 1.26, 1.25, 0.86, 2.03,

6.09, d (15.8) 6.86, dt (15.8, 7.1) 2.20, q (7.1) 1.44, m 1.27, m 1.26, m 1.28, m 0.87, t (6.7) 2.84, m 2.83, m

m m m m m t (6.7) s

δC

δH (J in Hz)

31.5, 41.9, 208.9, 43.2, 23.8, 29.1, 29.2, 29.4, 31.8, 22.6, 14.0, 31.5, 42.7, 206.5, 48.0,

CH2 CH2 C CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3 CH2 CH2 C CH2

2.84, m 2.80, m

45.8,

CH

2.90, dd (17.2, 7.1), 2.63, dd (17.2, 6.2) 3.18, m

34.2, 26.9, 29.0, 31.7, 22.6, 14.1,

CH2 CH2 CH2 CH2 CH2 CH3

1.53, 1.34, 1.22, 1.26, 1.25, 0.85,

2.41, 1.54, 1.23, 1.23, 1.23, 1.22, 1.25, 0.85, 2.84, 2.83,

td (7.5, 2.1) m m m m m m t (6.7) m m

m m m m m t (6.7)

(δH/C 0.85/14.0 and 0.85/14.1), five relatively deshielded methylenes (δH/C 2.63, 2.90/48.0, 2.80/41.9, 2.83/42.7, 2.84/ 31.5, and 2.84/31.5), one deshielded methine (δH/C 3.18/45.8), and two carbonyls (δC 206.5 and 208.9). The spectroscopic data of 7 (Table 2) were similar to those of 1−6. In agreement with the molecular formula, it was apparent that 7 is a sulfenic acid derivative with a S-OH group at C-5 instead of a S-Me group as in 5. The 2D NMR correlations (see Supporting Information), in accordance with those observed for 5, supported the proposed structure of 7. Therefore, compound 7 was identified as 5-hydroxythio-1-(3-oxo-undecyl)disulfanylundecan-3-one. This substance was found to be unstable and gradually converted to 3. On the basis of the available spectroscopic data, the relative configuration at C-5/C-5′ for compounds 2 and 5−7 could not be determined. Compounds 1, 3−6, and 8 were evaluated for their antibacterial activities against the epidemic methicillin-resistant strain EMRSA-15 and the multi-drug-resistant effluxing strain SA1199B of Staphylococcus aureus, as well as the Escherichia coli strain NCTC-10418, but were proven inactive. The anti-inflammatory potential of compounds 1, 3−6, and 8 was evaluated by measuring the production of NO by RAW 264.7 mouse macrophages upon lipopolysaccharide (LPS) stimulation. Initial testing of the compounds at a fixed concentration of 10 μg/mL revealed that compounds 5 and 8 were able to cause strong inhibition of NO production (Figure 2). Further evaluation of these two compounds determined the IC50 value of 5 in the LPS stimulation assay to be 3.8 μM, compared to 2.2 μM for the strong anti-inflammatory and analgesic diterpene neorogioltriol (Figure 3).14,15 Compound 8 was 4 times less effective, with an IC50 value of 14.2 μM. Both compounds were evaluated for their ability to inhibit the

included two methyls, 16 methylenes, two methines, and two quaternary carbon atoms. Among them, two carbonyls resonating at δC 198.2 and 209.0 and two tertiary olefinic carbons (δC 130.1 and 148.5) were evident. Signals for two aliphatic methyls on secondary carbons (δH 0.85 and 0.87), one trans 1,2-disubstituted double bond (δH 6.09 and 6.86), and four relatively deshielded methylenes (δH 2.79, 2.84, 2.90, and 2.96) were observed in the 1H NMR spectrum of 4. The proposed structure of this compound was fully supported by the homonuclear and heteronuclear correlations observed in its 2D NMR spectra (see Supporting Information). Therefore, compound 4 was identified as (E)-1-(3-oxo-undecyl)disulfanylundec-4-en-3-one. Compounds 5 and 6, isolated as yellow oils, displayed ion peaks consistent with [M + Na]+ peaks at m/z 471.2397 and 469.2241 (HRESIMS), corresponding to C23H44O2S3 and C23H42O2S3, respectively. The structural elements displayed in the 1H and 13C NMR spectra of 5 and 6 (Table 2) exhibited close resemblances to those of metabolites 1−4. Analyses of their 2D NMR spectra (see Supporting Information) resulted in the establishment of two different monomeric units for each nonsymmetrical dimer. Specifically, monomers of 2 and 3 could be traced in 5, whereas monomers of 1 and 2 were identified in 6. Indeed, in the EIMS of 5 and 6 the fragment ions originating from the cleavage of the disulfide bond were observed at m/z 247 and 201 for 5 and at m/z 247 and 199 for 6. Thus, compounds 5 and 6 were identified as 5-methylthio-1-(3-oxoundecyl)disulfanylundecan-3-one and (E)-1-(5-methylthio-3oxo-undecyl)disulfanylundec-4-en-3-one, respectively. Compound 7, with the molecular formula C22H42O3S3, as calculated from the HRESIMS measurements, was obtained as a yellowish oil. The structural characteristics evident in the 1H and 13C NMR spectra included two overlapping triplet methyls C

DOI: 10.1021/acs.jnatprod.5b01031 J. Nat. Prod. XXXX, XXX, XXX−XXX

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a PerkinElmer model 341 polarimeter with a 1 dm cell. UV spectra were obtained on a PerkinElmer Lambda 40 spectrophotometer. IR spectra were obtained on a Bruker Tensor 27 spectrometer. NMR spectra were recorded on Bruker AC 200 and Bruker DRX 400 spectrometers. Chemical shifts are given on a δ (ppm) scale using tetramethylsilane as internal standard. The 2D experiments (HSQC, HMBC, COSY, NOESY) were performed using standard Bruker pulse sequences. High-resolution ESI mass spectra were measured on a Thermo Scientific LTQ Orbitrap Velos mass spectrometer. Low-resolution EI mass spectra were measured on a Thermo Electron Corporation DSQ mass spectrometer using a directexposure probe. Column chromatography separations were performed with Kieselgel 60 (Merck). HPLC separations were conducted using a Pharmacia LKB 2252 liquid chromatography pump equipped with an RI-102 Shodex refractive index detector, using either a Supelcosil SPLC-Si 5 μm (250 × 10 mm i.d.; Supelco) or an Econosphere Silica 10 μm (250 × 10 mm i.d.; Grace) column. TLC was performed with Kieselgel 60 F254 (Merck aluminum-backed plates), and spots were detected after spraying with 15% H2SO4 in MeOH reagent and heating at 100 °C for 1 min. Plant Material. Specimens of Dictyopteris membranacea were collected by hand at Gerolimenas Bay (GPS coordinates 36°48′ N, 22°40′ E) in Peloponnese, Greece, at a depth of 0.5−2 m in July 2011. A voucher specimen of the alga has been deposited at the Herbarium of the Department of Pharmacognosy and Chemistry of Natural Products, University of Athens (ATPH/MP0193). Extraction and Isolation. Specimens of the fresh alga (1.1 kg) were exhaustively extracted with mixtures of CH2Cl2/MeOH at room temperature. Evaporation of the solvents in vacuo afforded a dark green, oily residue (23.0 g), which was subjected to vacuum column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc, followed by EtOAc with increasing amounts of MeOH as the mobile phase, to yield 11 fractions (1−11). Fraction 2 (15% EtOAc in cyclohexane, 2.0 g) was fractionated by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to yield eight fractions (2a− 2h). Fraction 2d (6% EtOAc in cyclohexane, 626 mg) was fractionated further by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford 14 fractions (2d1−2d14). Fraction 2d7 (6% EtOAc in cyclohexane, 105 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (95:5 and subsequently 97:3) as eluents, to yield 3 (4.6 mg), 4 (4.4 mg), 8 (11.9 mg), and γ-tocopherol (36.6 mg). Fraction 2d8 (6% EtOAc in cyclohexane, 63.0 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (93:7) and subsequently n-hexane/EtOAc (93:7) as eluents, to yield 5 (4.7 mg), 8 (3.3 mg), γ-tocopherol (11.4 mg), and 4-butyl-2,6-cycloheptadienone (3.0 mg). Fraction 2d9 (6% EtOAc in cyclohexane, 37.0 mg) was purified by normal-phase HPLC, using cyclohexane/EtOAc (94:6) and subsequently n-hexane/EtOAc (94:6) as eluents, to yield 1 (4.2 mg). Fraction 2e (10% EtOAc in cyclohexane, 273 mg) was fractionated further by gravity column chromatography on silica gel, using cyclohexane with increasing amounts of EtOAc as the mobile phase, to afford 11 fractions (2e1−2e11). Fractions 2e3 (7% EtOAc in cyclohexane, 56.4 mg) and 2e4 (7% EtOAc in cyclohexane, 50.9 mg) were purified separately by normal-phase HPLC, using cyclohexane/ EtOAc (91:9) and subsequently n-hexane/EtOAc (90:10) as eluents, to yield 1 (0.8 mg), 2 (0.8 mg), 6 (1.5 mg), 7 (1.2 mg), and δtocopherol (0.8 mg). Compound 1: yellowish oil; UV (CHCl3) λmax (log ε) 248 (3.64) nm; IR (thin film) νmax 2955, 2927, 2856, 1672 cm−1; 1H and 13C NMR data, Table 1; EIMS 70 eV m/z (rel int %) 398 (15), 199 (21), 167 (36), 151 (13), 139 (100), 115 (96), 83 (18), 69 (30), 55 (65); HRESIMS m/z 399.2389 [M + H]+ (calcd for C22H39O2S2, 399.2391). Compound 2: yellowish oil; [α]20 D +1.1 (c 0.05, CHCl3); UV (CHCl3) λmax (log ε) 250 (3.07) nm; IR (thin film) νmax 2956, 2924, 2856, 1712 cm−1; 1H and 13C NMR data, Table 1; EIMS 70 eV m/z

Figure 2. Screening for anti-inflammatory activity. The antiinflammatory potential of compounds 1, 3−6, and 8 was evaluated by measuring their effect on LPS-elicited macrophage activation. The concentration of compounds was fixed at 10 μg/mL (final molar concentration for each compound indicated in the figure), and their anti-inflammatory activity was compared to that of 2.5 μM neorogioltriol. Compounds were dissolved in Carbowax 400 (the final concentration of Carbowax was 0.1% v/v). Where added (bars indicated with “+”), the LPS concentration was 100 ng/mL. Control samples contained 0.1% v/v Carbowax 400 only. Graphical and statistical analyses were conducted using Prism (GraphPad Software, Inc.).

Figure 3. Determination of IC50 values for compounds 5 and 8. The compound concentrations resulting in 50% inhibition of NO production for neorogioltriol (open circles), 5 (solid circles), and 8 (open triangles) were determined. NO production levels were normalized to the concentration produced by cells treated with 0.1% v/v Carbowax 400 only (considered as 100%). Each data point is the average of four replicates, and the error bars represent one standard deviation.

growth of RAW 264.7 cells and were found to be cytostatic only at high concentrations. Growth inhibition was observed above 45 μM for 5 and above 85 μM for 8 (see Supporting Information). No significant inhibitory effect on cell viability or growth was observed at the concentration range where the antiinflammatory activity was studied. These results reveal the antiinflammatory potential of this group of compounds and also afford preliminary structure−activity relationships. The significantly higher activity of 5 compared to 3 suggests that the presence of the S-Me group at C-5′ is important for bioactivity. Moreover, the presence of a double bond at C-4 in 6 has a strong negative impact on the activity. D

DOI: 10.1021/acs.jnatprod.5b01031 J. Nat. Prod. XXXX, XXX, XXX−XXX

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(rel int %) 494 (9), 247 (100), 215 (42), 199 (46), 167 (58), 145 (25), 135 (32), 115 (12), 55 (30); HRESIMS m/z 517.2281 [M + Na]+ (calcd for C24H46NaO2S4, 517.2278). Compound 4: yellowish oil; UV (CHCl3) λmax (log ε) 251 (2.99) nm; IR (thin film) νmax 2954, 2925, 2854, 1714, 1679 cm−1; 1H and 13 C NMR data, Table 2; EIMS 70 eV m/z (rel int %) 400 (13), 201 (23), 199 (21), 167 (37), 139 (100), 115 (55), 70 (31), 55 (98); HRESIMS m/z 401.2544 [M + H]+ (calcd for C22H41O2S2, 401.2548). Compound 5: yellow oil; [α]20 D +2.6 (c 0.31, CHCl3); UV (CHCl3) λmax (log ε) 250 (3.04) nm; IR (thin film) νmax 2955, 2925, 2855, 1714 cm−1; 1H and 13C NMR data, Table 2; EIMS 70 eV m/z (rel int %) 448 (9), 247 (100), 215 (32), 201 (20), 199 (41), 167 (24), 135 (37), 103 (12), 55 (27); HRESIMS m/z 471.2397 [M + Na]+ (calcd for C23H44NaO2S3, 471.2401). Compound 6: yellow oil; [α]20 D +2.0 (c 0.10, CHCl3); UV (CHCl3) λmax (log ε) 248 (3.47) nm; IR (thin film) νmax 2955, 2927, 2857, 1710, 1681 cm−1; 1H and 13C NMR data, Table 2; EIMS 70 eV m/z (rel int %) 446 (9), 247 (84), 215 (37), 199 (62), 167 (47), 145 (35), 139 (45), 135 (47), 115 (59), 69 (41), 55 (100); HRESIMS m/z 469.2241 [M + Na]+ (calcd for C23H42NaO2S3, 469.2245). Compound 7: yellowish oil; 1H and 13C NMR data, Table 2; HRESIMS m/z 473.2191 [M + Na]+ (calcd for C22H42NaO3S3, 473.2194). Evaluation of Antibacterial Activity. The epidemic methicillinresistant S. aureus strain EMRSA-15, the S. aureus strain SA1199B that possesses the gene encoding the NorA quinolone efflux protein, and the E. coli strain NCTC-10418 were a generous gift of Prof. S. Gibbons (University of London, UK). All strains were cultured on nutrient agar and incubated for 24 h at 37 °C prior to the determination of minimum inhibitory concentration (MIC) values. Compounds 1, 3−6, and 8 were dissolved in DMSO and diluted subsequently in Mueller− Hinton broth (MHB) to give a starting concentration of 512 μg/mL. Bacterial inocula equivalent to the 0.5 McFarland turbidity standard were prepared in normal saline for each strain and diluted to a final inoculum density of 5 × 105 cfu/mL. MHB supplemented with 10 mg/L Mg2+ and 20 mg/L Ca2+ (125 μL/well) was dispensed into wells 1−11 of each row of 96-well microtiter plates. The compound solution (125 μL) was added to the first well of each row and was serially diluted across the row, leaving well 11 empty for growth control. The final volume was dispensed into well 12, which being free of MHB or inoculum served as the sterility control. The inoculum (125 μL/well) was added to wells 1−11 of each row, and the microtiter plates were incubated for 18 h at 37 °C. The lowest concentration at which no bacterial growth was observed was recorded as the MIC. The observation was confirmed by the addition of a 5 mg/mL methanolic solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 20 μL/well) and further incubation for 20 min at 37 °C. Bacterial growth was indicated by a color alteration from yellow to dark blue. Vancomycin and chloramphenicol were used as positive controls for S. aureus and E. coli strains, respectively. The highest concentration of DMSO remaining after dilution (3.125% v/v) caused no inhibition of bacterial growth. All samples were tested in triplicate. Evaluation of Anti-inflammatory Activity, Cell Growth, and Viability. RAW 264.7 mouse macrophage cells were cultured in DMEM medium (Gibco, 21885-025) supplemented with 10% heatinactivated fetal bovine serum (Gibco, 10270-106) and 1% penicillin− streptomycin (Gibco, 15070-063). Cells were grown at 37 °C and 5% CO2. Each compound used was diluted in Carbowax 400. The final concentration of Carbowax in culture was 0.1% (v/v). RAW 264.7 macrophages were seeded in 24-well plates and pretreated for 1 h with various concentrations of neorogioltriol (which was used as positive control), 5, and 8. Then, the cells were incubated with 100 ng/mL LPS for 48 h. The amount of nitrite, an oxidative product of NO, was measured in the supernatant of each culture using the Griess reaction. A 50 μL aliquot of the supernatant was mixed with 50 μL of sulfanilamide solution (1% sulfanilamide in 5% H3PO4) and incubated for 5 min at room temperature. Then, 50 μL of NED solution (0.1% N-1-naphthylethylenediamine dihydrochlorite in H2O) was added, and the absorbance was measured in an automated microplate reader (Infinite 200 PRO, Tecan) at 540 nm. Nitrite concentration was

calculated using a sodium nitrite standard curve. All incubations were performed in the dark. To identify potential cytotoxic or cytostatic effects of the compounds tested against RAW 264.7 mouse macrophages, cell viability was assessed using the MTT assay. The cells were seeded on 96-well plates and cultured overnight. The number of seeded cells was measured prior to treatment and used as control. Then, cells were treated and incubated with various concentrations of 5 and 8 for 48 h. The MTT solution was added to the cells at a final concentration of 0.5 mg/mL, and the cells were incubated for 48 h. The supernatant was removed, and the cells were lyzed in 2-propanol containing 0.4% HCl. The absorbance of each sample was measured in an automated microplate reader (Infinite 200 PRO, Tecan) at 610 nm. The average optical density (OD) of each treated sample was normalized to the OD of the control sample, and statistical analysis was performed using the one-way ANOVA test.

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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.5b01031. COSY and important HMBC correlations for compounds 1−8, 1D and 2D NMR spectra of compounds 1−8, and data on the evaluation of bioactivity (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel/Fax: +30-210-7274913. E-mail: [email protected]. *Tel/Fax: +30-210-7274592. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. S. Gibbons (University of London, UK) for the kind provision of the bacterial strains. DEDICATION Dedicated to Professors John Blunt and Murray Munro, of the University of Canterbury, for their pioneering work on bioactive marine natural products.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b01031 J. Nat. Prod. XXXX, XXX, XXX−XXX