Antifouling 26,27-Cyclosterols from the Vietnamese Marine Sponge

Article pubs.acs.org/jnp

Antifouling 26,27-Cyclosterols from the Vietnamese Marine Sponge Xestospongia testudinaria Xuan Cuong Nguyen,† Arlette Longeon,‡ Van Cuong Pham,† Félix Urvois,§ Christine Bressy,§ Thi Thanh Van Trinh,† Hoai Nam Nguyen,† Van Kiem Phan,† Van Minh Chau,† Jean-François Briand,§ and Marie-Lise Bourguet-Kondracki‡,* †

Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam ‡ Laboratoire des Molécules de Communication et Adaptation des Micro-organismes, UMR 7245 CNRS, Muséum National d’Histoire Naturelle, 57 rue Cuvier (C.P. 54), 75005 Paris, France § MAPIEM-EA 4223 - Biofouling & Marine Natural Substances, Université de Toulon, 83957 La Garde, France S Supporting Information *

ABSTRACT: Three new C29 sterols with a cyclopropane ring cyclized between C-26 and C-27 of the side chain, aragusterol I (1), 21-O-octadecanoyl-xestokerol A (4), and 7β-hydroxypetrosterol (5b), were isolated from the Vietnamese marine sponge Xestospongia testudinaria, along with the known compounds, aragusterol B (2), xestokerol A (3), 7αhydroxypetrosterol (5a), 7-oxopetrosterol (6), and petrosterol (7). The structures of the new compounds were established by analysis of spectroscopic data including 1D and 2D NMR, and high-resolution electrospray ionization mass spectrometry (HRESIMS). Their capacity to inhibit the adhesion of isolated bacteria from marine biofilms was evaluated against the bacterial strains Pseudoalteromonas sp. D41, Pseudoalteromonas sp. TC8, and Polaribacter sp. TC5. Aragusterol B (2) and 21-O-octadecanoyl-xestokerol A (4) exhibited the most potent antifouling activity with EC50 values close to these reported in the literature for tributyltin oxide, a marine anti-biofouling agent now considered to be a severe marine pollutant. Due to its comparable activity to tributyltin oxide and its absence of toxicity, the new 26,27cyclosterol, 21-O-octadecanoyl-xestokerol A (4) constitutes a promising scaffold for further investigations.

B

shown cardiovascular, cytotoxic, antitumor, HIV protease inhibitory, antimicrobial, and insecticidal activities.4 Previously, our group has focused its research activity on marine sponges of the genus Xestospongia, which led to the discovery of brominated polyacetylenic acids with Na+/K+ ATPase inhibitory activity from X. testudinaria collected at Mayotte;5 four alkaloids of the aaptamine class from an Indonesian sponge Xestospongia sp.;6 and a series of halenaquinone derivatives with anti-phospholipase A2, anti-farnesyltransferase, and antiplasmodial activities from South Pacific sponges Xestospongia sp. and X. testudinaria.7 As a part of our ongoing chemical investigations on marine sponges of the genus Xestospongia, we examined a Vietnamese sample. This paper deals with the isolation and structure elucidation of eight 26,27-cyclosterols from the Vietnamese marine sponge Xestospongia testudinaria; their in vitro antifouling activity was also evaluated. Successive chromatography of the MeOH extract of the sponge X. testudinaria on silica gel and RP-18 columns led to the isolation of three new C29 sterols with a cyclopropane ring cyclized between C-26 and C-27 of the side chain (1, 4, and 5b) and five known compounds, aragusterol B (2),8 xestokerol

iofouling refers to the natural colonization of submerged surfaces by a wide range of organisms from bacteria to invertebrates. Biofouling has a major economic impact, especially when it occurs on ship hulls or aquaculture facilities. The first stage of this process corresponds to the formation of a biofilm, initially constituted of pioneer bacteria, after which the adhesion of macrofoulers, such as algae or invertebrates, significantly increase the attached biomass.1 Biofilms are increasingly recognized to play an important role in biofouling processes, and inhibitors of biofilm formation are often potent antifoulants. Organotins, such as tributyltin (TBT) compounds and tributyltin oxide (TBTO), were the most effective antifouling agents ever developed, but also the most toxic biocides ever introduced to the marine environment.2 Recently, these biocides were banned by the International Maritime Organization. Therefore, the search for nontoxic (or environmentally benign) antifoulants is urgent. Although a wide range of marine natural products showing antifouling activity have been isolated from marine invertebrates such as sponges, soft corals, and ascidians,2,3 none of them is currently in development. Sponges of the genus Xestospongia have been proven to be a rich source of diverse secondary metabolites, including alkaloids, quinones, sterols, and brominated polyacetylenic acids. Some of them have © XXXX American Chemical Society and American Society of Pharmacognosy

Received: April 5, 2013

A

dx.doi.org/10.1021/np400288j | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

A (3),9,10 7α-hydroxypetrosterol (5a),11 7-oxopetrosterol (6),11 and petrosterol (7).12 The known sterols were rapidly identified by comparison of their spectroscopic data with the literature values.

Compound 1 was isolated as a colorless solid. The HRESI mass spectrum of 1 exhibited a pseudomolecular ion peak at m/ z 469.3610 [M + Na]+ corresponding to a molecular formula of C29H50O3, which contains five degrees of unsaturation. Moreover, fragment ion peaks at m/z 429.3690 [M + H − H2O]+, 411.3603 [M + H − 2H2O]+, and 393.3501 [M + H − 3H2O]+ indicated the presence of three hydroxy groups in 1. This was confirmed by absorption band at 3298 cm−1 in its IR spectrum. The 1H and 13C NMR signals indicated that 1 had a sterol skeleton with a petrosterol-like side chain as previously isolated from marine sponges of the genus Xestospongia.8−10 In the 1H NMR spectrum, four high-field proton signals at δH 0.13 (1H, m, Ha-26), 0.23 (2H, m, H-25 and Hb-26), 0.47 (1H, m, H-27), and 0.62 (1H, m, H-24) suggested the presence of a cyclopropane ring. The presence of two oxymethine [δC 66.5 (C-3)/δH 4.03 (1H, t, J = 2.5 Hz, H-3) and δC 77.9 (C-12)/δH 3.36 (1H, dd, J = 4.5, 11.0 Hz, H-12)] and one oxygenated quaternary carbon [δC 75.7 (C-20)] signals were also observed in the 1H and 13C NMR spectra of 1. In addition, three singlet methyl proton signals were identified at δH 0.77 (CH3-19), 0.81 (CH3-18), and 1.13 (CH3-21), and two doublet (J = 6.0 Hz) methyl signals were at δH 0.90 (CH3-28) and 1.00 (CH3-29). Thus, the 1H and 13C NMR data of 1 were similar to those of aragusterol B (2).8 The difference between them is the appearance of an oxymethine group [δH 4.03 (1H, t, J = 2.5 Hz, H-3)/δC 66.5 (CH, C-3)] in 1 instead of a ketone group in 2. The HMBC cross-peaks of H-18 (δH 0.81) with C-12 (δC 77.9), C-13 (δC 48.6), C-14 (δC 54.9), and C-17 (δC 64.2) and those of H-21 (δH 1.13) with C-17 (δC 64.2), C-20 (δC 75.7), and C-22 (δC 33.9), confirmed the attachment of two hydroxy groups at C-12 and C-20. The 13C NMR chemical shifts for the rings A and B of 1 were essentially identical to those of aragusterol G.10 These data together with the HMBC correlations of the oxymethine proton at δH 4.03 with carbons C-1 (δC 32.3) and C-5 (δC 39.0) and those of methyl protons CH3-19 (δH 0.77) with carbons C-1 (δC 32.3), C-5 (δC 39.0), C-9 (δC 53.2), and C-10 (δC 36.0), confirmed the location of the remaining OH group at C-3. Detailed analysis of other HMBC and COSY correlations (Figure 1) clearly demonstrated that the planar structure of compound 1 is as shown. The relative configurations at C-12, C-20, C-25, and C-27 of 1 are suggested to be the same as those of aragusterol B (2)8 by complete agreement of the 13C NMR chemical shifts for the

Figure 1. Key HMBC and COSY correlations of compounds 1 and 4.

rings C, D and the side chain between these two compounds. A coupling constant analysis of H-3 (t, J = 2.5 Hz) indicated a βorientation of H-3 (versus tt, J = 4.7, 11.0 Hz for the αorientation as in case of aragusterol H10). This observation was further confirmed by comparison of the carbon chemical shift of C-3 of 1 with those of aragusterol G10 (with Hβ-3) and aragusterol H10 (with Hα-3): the chemical shift of C-3 of 1 (δC 66.5) is in agreement with that of aragusterol G (δC 66.4) and quite different from that of aragusterol H (δC 71.2). The relative configuration of 1 was also confirmed by a NOESY experiment, in which H-12 (δH 3.36) correlated to H-9 (δH 0.84), H-14 (δH 0.98), and H-17 (δH 1.63) indicating the αorientation for both H-12 and H-17. The geometry of the cyclopropane ring was deduced to be E by a NOE correlation of H-25 with H-29 (Figure 2). Thus, the structure of 1 was identified as 26,27-cyclo-24,27-dimethylcholestan-3α,12β,20βtriol, and compound 1 was named aragusterol I. The molecular formula of compound 4, isolated as a colorless solid, was defined as C47H82O6 by high-resolution electrospray ionization mass spectrometry (HRESIMS), which showed the protonated molecular ion at m/z 743.6232 [M + H]+. Its 1H NMR signals were also indicative of a sterol with a petrosterollike side chain showing four high-field protons characteristic of a cyclopropane ring at δH 0.13 (1H, m, Ha-26), 0.23 (2H, m, H25 and Hb-26), and 0.49 (1H, m, H-27). The 1D NMR signals of 4 were close to those of xestokerol A (3)9 with additional signals of a carbonyl [δC 174.6 (C-1′)], terminal methyl [δC 14.1 (C-18′)/δH 0.85 (3H, t, J = 7.0 Hz, H-18′)], and aliphatic methylene groups (δH 1.23, 28H, br s) indicating the presence of a saturated fatty acid moiety. A fragment ion at m/z 459.3486 [M + H − C18H36O2]+ was observed in the HRESI mass spectrum of 4 suggesting the presence of an octadecanoyl moiety. The COSY experiment allowed assignments of proton−proton correlations of H-1/H-2, H-4/H-5/H-6/H-7/ H-8/H-14/H-15/H-16/H-17, H-8/H-9/H-11/H-12, and H22/H-23/H-24/H-25/H-26/H-27. These data together with the HMBC cross-peaks of H-1 (δH 1.97 and 2.35), H-2 (δH 2.27 and 2.35), and H-4 (δH 2.02 and 2.25) with C-3 (δC 211.8); H-18 (δH 0.92) with C-12 (δC 77.3), C-13 (δC 49.0), C14 (δC 54.0), and C-17 (δC 55.1); H-19 (δH 1.00) with C-1 (δC B



Article

Figure 2. Key NOESY correlations of 1.

Table 1. The 13C and 1H NMR Data of 26,27-Cyclosterols 1, 4, and 5ba 1 position

a

δ C,

b,c

type

4

δ H,

b,d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

32.3, CH2 29.0, CH2 66.5, CH 35.6, CH2 39.0, CH 28.5, CH2 31.3, CH2 33.8, CH 53.2, CH 36.0, C 29.7, CH2 77.9, CH 48.6, C 54.9, CH 23.3, CH2 25.0, CH2 64.2, CH 9.6, CH3 11.1, CH3 75.7, C 28.6, CH3

1.33, 1.61, 4.03, 1.34, 1.53, 1.68, 0.87, 1.28, 0.84,

22 23 24 25 26 27 28 29 1′ 2′ 18′

33.9, 31.7, 39.0, 27.1, 11.5, 12.8, 20.2, 19.2,

1.53, 1.26, 0.62, 0.23, 0.13, 0.47, 0.90, 1.00,

CH2 CH2 CH CH CH2 CH CH3 CH3

mult. (J in Hz)

δ C,

m/1.45, m m t (2.5) m/1.46 m m m m/1.64, m m m

38.4, CH2 38.1, CH2 211.8, C 44.5, CH2 46.5, CH 28.8, CH2 31.0, CH2 33.8, CH 52.4, CH 35.6, C 29.1, CH2 77.3, CH 49.0, C 54.0, CH 23.4, CH2 22.3, CH2 55.1, CH 8.9, CH3 11.4, CH3 76.1, C 65.3, CH2

1.27, m/1.76, m 3.36, dd (4.5, 11.0) 0.98, 1.18, 1.47, 1.63, 0.81, 0.77,

m m/1.61, m m/1.61, m m s s

1.13, s m/1.74, m m/1.50, m m m m/0.23, m m d (6.0) d (6.0)

b,e

δ H,

type

5b b,f

mult. (J in Hz)

1.97, m/2.35, m 2.27, m/2.35, m 2.02, 1.49, 1.33, 0.87, 1.38, 0.83,

m/2.25, m m m m/1.70, m m m

1.34, m/1.76, m 3.38, dd (4.5, 11.0)

69.6, CH 36.3, CH2 34.9, CH 27.9, CH 12.5, CH2 12.3, CH 18.6, CH3 19.2, CH3 174.6, C 34.4, CH2 14.1, CH3

0.98, 1.20, 1.25, 1.75, 0.92, 1.00,

m m/1.67, m m/1.58, m m s s

4.16, 4.51, 3.51, 1.30, 0.90, 0.23, 0.13, 0.49, 0.90, 0.98,

d (12.0) d (12.0) br d (11.0) m/1.70, m m m m/0.23, m m d (6.0) d (6.0)

δ C,

b,c

type

37.0, CH2 31.6, CH2 71.4, CH 41.7, CH2 143.4, C 125.4, CH 73.3, CH 40.9, CH 48.2, CH 36.9, C 21.3, CH2 39.5, CH2 42.0, C 55.4, CH 26.4, CH2 28.5, CH2 55.9, CH 11.8, CH3 19.1, CH3 35.9, CH 18.7, CH3 33.5, 33.8, 38.7, 27.4, 11.6, 12.7, 19.8, 19.1,

CH2 CH2 CH CH CH2 CH CH3 CH3

δH,b,d mult. (J in Hz) 1.08, 1.50, 3.56, 2.22,

m/1.84, m m/1.83, m m m/2.27, m

5.27, 3.83, 1.47, 1.03,

br s (overlapped) m m

1.50, m 1.15, m/1.98, m 1.08, 1.65, 1.42, 1.16, 0.68, 1.03, 1.35, 0.99,

m m m/1.87, m m s s m d (6.0)

0.98, 1.24, 0.59, 0.11, 0.05, 0.42, 0.87, 0.91,

m/1.47, m m/1.30, m m m m/0.11, m m d (6.0) d (6.0)

2.33, m 0.85, t (7.0)

All the data were assigned by HSQC, HMBC, COSY, and NOESY experiments. bRecorded in CDCl3. c75 MHz. d400 MHz. e125 MHz. f500 MHz.

(Figure 1). The 1H and 13C NMR data at C-12 of 4 were identical to those of 1 (Table 1) indicating the β-orientation of the hydroxy group at C-12. The relative configurations at C-20, C-22, C-24, C-25, and C-27 of 4 were assumed to be identical with those of xestokerol A (3)9,10 on the basis of comparison of their chemical shifts as well as the coexistence of 3 and 4 in X.

38.4), C-5 (δC 46.5), C-9 (δC 52.4), and C-10 (δC 35.6); and H-21 (δH 4.16 and 4.51) with C-17 (δC 55.1), C-20 (δC 76.1), and C-22 (δC 69.6), located the ketone at C-3 and the hydroxy groups at C-12, C-20, and C-22. Furthermore, HMBC correlations of H-21 (δH 4.16 and 4.51) with C-1′ (δC 174.6) clearly indicated the linkage of the octadecanoyl moiety to C-21 C



Article

Table 2. Antiadhesion Activity of the 26,27-Cyclosterols 1−5a+5b and 7 against the Bacterial Strains Polaribacter sp. TC5, Pseudoalteromonas sp. TC8, and Pseudoalteromonas sp. D41a TC5 TC8 D41

EC50 R2 EC50 R2 EC50 R2

TBTO

1

2

3

4

5a+5b

7

10 0.90 12 0.95 14 0.72

114 0.77 70 0.91 171 0.92

23 0.78 20 0.90 60 0.74

65 0.98 111 0.79 138 0.71

25 0.89 10 0.84 36 0.94

>200

>200

>200

>200

>200

>200

EC50 expressed in μM as the concentration corresponding to 50% bacterial adhesion. R2 represents the goodness of fit of the sigmoid dose− response curve to experimental data. TBTO was used as standard biocide. a

red and green fluorescent SYTO dyes we used. Aragusterol I (1) exhibited moderate activity with EC50 values between 70 and 171 μM depending on the strain. In contrast, 7α- and 7βhydroxypetrosterol (5a+5b) and petrosterol (7) appeared inactive, suggesting the importance of the double bond at C5 or hydroxy groups at C-12 and on the side chain. Furthermore, while xestokerol A (3) revealed moderate activity (EC50’s between 70 and 138 μM), aragusterol B (2) and 21-Ooctadecanoyl-xestokerol A (4) exhibited the highest activity of the series with EC50 values between 10 and 60 μM depending on the strain, with these activities being close to those of TBTO. 21-O-octadecanoyl-xestokerol A (4) was the most potent molecule, with a similar activity to TBTO against the TC8 strain with an EC50 value of 10 μM (vs 12 μM for TBTO). Therefore, the presence of a secondary alcohol at C-3 rather than a ketone seemed to decrease the antiadhesion activity since compounds 1, 5a+5b, and 7 are inactive. Comparison of the activities of compounds 1 and 2 revealed that the C-3 oxidation is essential for the activity, but it was abrogated by two additional hydroxy groups at positions C-21 and C-22 (compound 3). The presence of a fatty acid chain at C-21 considerably increased the antiadhesion activity (compound 4), probably conferred by the additional hydrophobicity or lipid solubility of the molecule. Furthermore, growth inhibition and viability assays of aragusterol B (2) and 21-O-octadecanoylxestokerol A (4) on the most sensitive strain in this study, Polaribacter sp. TC5, did not show any toxicity up to 200 μM, indicating a specific antiadhesion mechanism. In conclusion, aragusterol B (2) and 21-O-octadecanoylxestokerol A (4) exhibited the most potent antifouling activity. Due to the significant activity of 21-O-octadecanoyl-xestokerol A (4), similar to that of TBTO, further complementary in situ macrofouling assays will be performed. In addition, the occurrence of petrosterol (7) in high quantity (3.5% of the MeOH extract) from this Xestospongia sponge could be used as starting material for the synthesis of antifouling analogues.

testudinaria and confirmed by NOESY experiment as done for compound 1. Consequently, the new compound 4 was named 21-O-octadecanoyl-xestokerol A as it appeared to be an ester derivative of xestokerol A. The mixture of 5a and 5b was isolated as a white solid. The molecular formula was identified to be C29H48O2 by analysis of its HRESI mass spectrum in which the pseudomolecular ion was observed at m/z 451.3612 [M + Na]+. The presence of two hydroxy groups was suggested by fragment ion peaks at m/z 411.3678 [M + H − H2O]+ and 393.3558 [M + H − 2H2O]+ and was also supported by a broad absorption band at 3325 cm−1 in the IR spectrum. The 1H and 13C NMR spectra showed two sets of signals with a ratio of 1:0.7, suggesting the presence of two isomers. Attempts to separate these isomers by normal and reversed-phase HPLC were unsuccessful. Due to their different proportions, the chemical shifts of these two isomers were separately assigned from 1D and 2D NMR spectroscopic analyses. The same skeleton as that described for the compounds above was attributed to both isomers, but with a double bond at C-5/C-6 and hydroxy groups at C-3 and C-7. Thus, these two isomers (5a and 5b) had identical planar structures. The carbon chemical shifts of C-3 of 5a (δC 71.3) and 5b (δC 71.4) indicated the β-orientation of the hydroxy at C-3 for both isomers, the same as for aragusterol H (δC 71.2).10 Moreover, the characteristic differences in the 1H and 13C NMR data, especially for the signals at the positions C-5, C-6, and C-7, between these two isomers indicated that they are epimeric at C-7. The data at δC 146.2 (C-5), δC 123.9 (C-6)/δH 5.58 (1H, dd, J = 5.0, 1.0 Hz, H-6), and δC 65.3 (C-7) of 5a were almost identical to those of 7α-hydroxypetrosterol11 at δC 146.2 (C-5), δC 123.9 (C-6)/δH 5.61 (1H, dd, J = 5.2, 1.6 Hz, H-6), and δC 65.4 (C-7) confirming the α-orientation of the hydroxy group at C-7. The data at δC 143.4 (C-5), δC 125.4 (C6)/δH 5.27 (1H, br s, H-6), and δC 73.3 (C-7) for 5b were essentially identical to those of (24S)-ergost-5-en-3β,7β-diol13 at δC 143.5 (C-5), δC 125.4 (C-6)/δH 5.29 (1H, br s, H-6), and δC 73.3 (C-7) indicating the β-orientation of the hydroxy group at C-7. From all the above evidence, compound 5a was characterized as the known compound 7α-hydroxypetrosterol,11 while its epimer at C-7 (5b,7β-hydroxypetrosterol) is a new compound. The antifouling activities of all compounds were evaluated using a fluorescent assay in comparison with that of TBTO, a marine biocide that has been banned since 2008 because of its toxicity against a wide range of marine organisms. Their antiadhesion capacity against bacterial strains Pseudoalteromonas sp. D41 and TC8 and against Polybacter sp. TC5 was determined after plotting of their sigmoid-dose−response curves (Table 2). No result is reported for 7-oxopetrosterol (6) because of nonspecific binding of the compound to both

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Perkin-Elmer 341 polarimeter. IR spectra were obtained on a Shimadzu 8400S FT-IR spectrometer. All NMR spectra were recorded on Bruker AC300, AC500, and AVANCE 400 FT-NMR spectrometers, TMS was used as an internal standard, and chemical shifts (δ) are reported in ppm calibrated to chloroform peaks at δH 7.25 and δC 77.0. Mass spectra were recorded on an ESI-Qq-TOF QSAR Pulsar spectrometer from Applied Biosystems. Column chromatography was performed on silica gel 230−400 mesh (0.040−0.063 mm, Merck) or RP-18 resins (Sepra C18-E 50 μm, 65A, Phenomenex). Thin layer chromatography (TLC) was performed on DC-Alufolien Si Gel 60 F254 (Merck) or Alugram RP18 F254s (Macherey-Nagel) plates. Compounds were visualized by D



Article

Mediterranean, France) by the MAPIEM laboratory,14 were used. Bacterial strains were grown on Vaatanen nine-salt solution (VNSS)16 at 20 °C under shaking conditions (120 rpm) and collected at the stationary phase. After centrifugation, cells were suspended in an adequate volume of sterile artificial seawater (ASW, Sigma-Aldrich) to reach an optical density of 0.2 at 600 nm. Molecules were tested at eight different concentrations in triplicate in microtiter plates (sterile black PS; Nunc, Fisher Scientific). The maximum percentage of solvent (DMSO) used for the dilution of tested compounds was also tested as a control. For the bacterial adhesion control, 100 μL of ASW was added in six wells. Then 100 μL of the bacterial suspension was inoculated in each of the wells except the border-row wells. The latter were filled out to 200 μL with ASW and constituted the nonspecific staining control (compounds with the fluorochrome but without bacteria). After 15 h for D41 and 24 h for TC8 and TC5, the nonadhered bacteria were removed by three successive washes (36 g L−1 sterile NaCl solution). Staining was performed by adding 200 μL of SYTO 61 (1 μM) for D41 and TC8 and SYTO 9 (1 μM) for TC5. After 20 min, the excess stain was removed by one wash (36 g L−1 NaCl solution). Fluorescence intensity (FI) was then directly measured (SYTO 61 or SYTO 9: λexc= 628 nm, λem= 645 nm) using an Infinit 200 microplate fluorescence reader (Tecan). The percentage of adhesion was calculated per well: (FIi − nsCi)/ (Mean FIc − Mean B) × 100, where FIi = fluorescence intensity in a treated well (tested compound + bacteria + fluorophore), FIc = fluorescence intensity in a control well (bacteria + fluorophore), nsCi = nonspecific control (tested compound without bacteria + SYTO 61 or SYTO 9), and B = blank, that is, stain control (only SYTO 61 or SYTO 9). After calculation of means and standard deviations of the triplicates at each concentration, a sigmoid dose−response curve was obtained by plotting % of adhesion against compound concentrations from which the EC50 values were calculated (GraphPad Prism 5, GraphPad Software). Bacterial growth inhibition and viability assays for compounds 2 and 4 were tested on Polaribacter sp. TC5 following the method described by Camps et al.14

spraying with Lieberman reagent and heating for 3−5 min. Preparative TLC used glass plates coated with Si gel 60 F254 (Merck 1.05715), 0.25 mm thick. Biological Material. The sponge samples of Xestospongia testudinaria (phylum Porifera, class Desmospongia, order Haplosclerida, family Petrosiidae) were collected in the Truong Sa archipelago, Khanh Hoa, Vietnam, during February 2012 and identified by Prof. Do Cong Thung from the Institute of Marine Environment and Resources, VAST. Voucher specimens (No. XT-2012-01) were deposited at the Institute of Marine Biochemistry and the Institute of Marine Environment and Resources, VAST. Extraction and Isolation. The fresh frozen X. testudinaria samples (4 kg) were finely chopped and exhaustively extracted five times with MeOH under ultrasonic conditions (1 h each). The resulting mixtures were filtered, combined, and concentrated under vacuum to obtain 34 g of a MeOH extract. This extract was suspended in H2O (1 L) and partitioned in turn with CH2Cl2 (3 × 1 L) and BuOH (3 × 1 L) to obtain the corresponding extracts: CH2Cl2 (XC, 8 g), BuOH (XB, 1.5 g) and H2O layer. Extract XC (8 g) was crudely separated into nine fractions, XC1−XC9, subjected to silica gel column chromatography (CC), eluting with stepwise elution of CH2Cl2−acetone (98:2, 95:5, 90:10, 80:20, 50:50, v/v) and then CH2Cl2−MeOH (70:30, 50:50, 0:100, v/v). Fraction XC3 (1.4 g) was crystallized in MeOH to give compound 7 (1.2 g, white crystals). The remaining portion of fraction XC3 was combined with fraction XC4 (0.4 g) and separated on silica gel CC eluting with c-hexane−acetone 5:1 to obtain eight subfractions, XC4A−XC4H. Compound 6 (3 mg) was purified from subfraction XC4D (22 mg) by preparative TLC eluting with c-hexane−acetone, 2:1. Subfractions XC4F (13 mg) and XC4G (17 mg) were combined and purified on silica gel CC using CH2Cl2−acetone, 10:1, as eluent to obtain compounds 5a and 5b (2.8 mg). Fraction XC6 (573 mg) was separated into four subfractions, XC6A−XC6D, by silica gel CC eluting with CH2Cl2−MeOH (40:1, v/v). Compound 4 (5 mg) was purified from subfraction XC6B (260 mg) by RP-18 CC eluting with acetone−H2O (2.5:1, v/v), followed by preparative TLC with chexane−acetone (2:1, v/v). Fraction XC7 (90 mg) was subjected to silica gel CC eluting with CH2Cl2−MeOH, 40:1, to furnish compound 2 (2.7 mg). Fraction XC8 (0.96 g) was further separated into five subfractions, XC8A−XC8E, on silica gel CC eluting with c-hexane− acetone, 2:1. Fraction XC8B (35 mg) afforded compound 1 (4.5 mg), after chromatography on silica gel CC eluting with CH2Cl2−MeOH (30:1, v/v), followed by preparative TLC with CH2Cl2−MeOH (20:1, v/v). Finally, compound 3 (5 mg) was purified from fraction XC8C (150 mg) by RP-18 CC using acetone−H2O 1.5:1 as eluent followed by silica gel CC eluting with CH2Cl2−MeOH (20/1, v/v). Aragusterol I (1): colorless solid; [α]D20 + 6 (c 0.20, CH2Cl2); IR (film) νmax 3298, 2924 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3), see Table 1; HRESIMS m/z 469.3610 [M + Na]+ (calcd for C29H50O3Na, 469.3658), 429.3690 [M + H − H2O]+ (calcd for C29H49O2 429.3733), 411.3603 [M + H − 2H2O]+ (calcd for C29H47O 411.3627), 393.3501 [M + H − 3H2O]+ (calcd for C29H45 393.3521). 21-O-Octadecanoyl-xestokerol A (4): colorless solid; [α]D20 + 37 (c 0.20, CH2Cl2); IR (film) νmax 3298, 2924, 1732, 1716 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3), see Table 1; HRESIMS m/z 743.6232 [M + H]+ (calcd for C47H83O6, 743.6190), 459.3486 [M + H − C18H36O2]+ (calcd for C29H47O4, 459.3474). 7α- and 7β-hydroxypetrosterol (5a + 5b): white solid; [α]D20 − 74 (c 0.20, CH2Cl2); IR (film) νmax 3325, 2928 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) for 5b, see Table 1; HRESIMS m/z 451.3612 [M + Na]+ (calcd for C29H48O2Na, 451.3652), 411.3678 [M + H − H2O]+ (calcd for C29H47O, 411.3627), 393.3558 [M + H − 2H2O]+ (calcd for C29H45, 393.3521). Antiadhesion Assay. An antiadhesion assay was adapted for this study from Camps et al.14 Pseudoalteromonas sp. D41, isolated from a natural marine biofilm on a Teflon panel in Brest Bay (Brittany, France) by the IFREMER,15 and Pseudoalteromonas sp. TC8 and Polaribacter sp. TC5, both isolated from natural marine biofilms on a polydimethylsiloxane elastomer panel in Toulon Bay (North-Western

■

ASSOCIATED CONTENT

S Supporting Information *

The 1D (1H and JMOD) and 2D (COSY, HSQC, HMBC, and NOESY) NMR spectra compounds 1, 4, and 5a+5b and antiadhesion activity of the 26,27-cyclosterols 1−5a+5b and 7. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Tel: +33 1 40 79 56 06. Fax: +33 1 40 79 31 35. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank the Ministère des Affaires Etrangères et Européennes (MAEE) in France and the Museum National d’Histoire Naturelle (MNHN, Paris, France) for financial support in the framework of “MarSpongAsia” program. The authors are grateful to the Vietnam Ministry of Science and Technology and VAST for the sponge collection and to Prof. Do Cong Thung (Institute of Marine Environment and Resources, VAST) for the sponge identification, to A. Blond and A. Deville (MNHN, Paris, France) for NMR spectra, to A. Marie and L. Dubost (MNHN, Paris, France) for MS measurements, and to C. Compere (IFREMER Brest, France) for the supply of the bacterial strain D41. E



■

Article

REFERENCES

(1) Haras, D. Mater. Technol. 2005, 93, 27−41. (2) Fusetani, N. Nat. Prod. Rep. 2004, 21, 94−104. (3) Fusetani, N. Nat. Prod. Rep. 2011, 28, 400−410. (4) Zhou, X.; Xu, T.; Yang, X. W.; Huang, R.; Yang, B.; Tang, L.; Liu, Y. Chem. Biodiversity 2010, 7, 2201−2227. (5) Bourguet-Kondracki, M. L.; Rakotoarisoa, M. T.; Martin, M. T.; Guyot, M. Tetrahedron Lett. 1992, 33, 225−226. (6) Calcul, L.; Longeon, A.; Mourabit, A. A.; Guyot, M.; BourguetKondracki, M. L. Tetrahedron 2003, 59, 6539−6544. (7) Longeon, A.; Copp, B. R.; Roué, M.; Dubois, J.; Valentin, A.; Petek, S.; Debitus, C.; Bourguet-Kondracki, M. L. Bioorg. Med. Chem. 2010, 18, 6006−6011. (8) Iguchi, K.; Shimura, H.; Taira, S.; Yokoo, C.; Matsumoto, K.; Yamada, Y. J. Org. Chem. 1994, 59, 7499−7502. (9) Kobayashi, J.; Ishida, K.; Naitoh, K.; Shigemori, H.; Mikami, Y.; Sasaki, T. J. Nat. Prod. 1993, 56, 1350−1355. (10) Miyaoka, H.; Shinohara, M.; Shimomura, M.; Mitome, H.; Yano, A.; Iguchi, K.; Yamada, Y. Tetrahedron 1997, 53, 5403−5412. (11) Umeyama, A.; Ito, S.; Yoshigaki, A.; Arihara, S. J. Nat. Prod. 2000, 63, 1540−1542. (12) Mandeau, A.; Debitus, C.; Aries, M. F.; David, B. Steroids 2005, 70, 873−878. (13) Muralidhar, P.; Kumar, M. M.; Krishna, N.; Rao, C. B.; Rao, D. V. Chem. Pharm. Bull. 2005, 53, 168−171. (14) Camps, M.; Briand, J. F.; Guentas-Dombrowsky, L.; Culioli, G.; Bazire, A.; Blache, Y. Mar. Pollut. Bull. 2011, 62, 1032−1040. (15) Leroy, C.; Delbarre-Ladrat, C.; Ghillebaert, F.; Rochet, M. J.; Compère, C.; Combes, D. Lett. Appl. Microbiol. 2007, 44, 372−378. (16) Mårdén, P.; Tunlid, A.; Malmcroma-Friberg, K.; Odham, G.; Kjelleberg, S. Arch. Microbiol. 1985, 142, 326−332.

F


Antifouling 26,27-Cyclosterols from the Vietnamese Marine Sponge

Recommend Documents