Note pubs.acs.org/jnp
Secondary Metabolites Isolated from the Amazonian Endophytic Fungus Diaporthe sp. SNB-GSS10 Hugues Mandavid,† Alice M. S. Rodrigues,†,⊥ Laila S. Espindola,‡ Véronique Eparvier,*,† and Didier Stien*,†,§ †
CNRS−Institut de Chimie des Substances Naturelles, Gif-sur-Yvette, France Laboratório de Farmacognosia, Universidade de Brasília, Brasília, DF, Brazil § UPMC Univ Paris 06, CNRS, Laboratoire de Biodiversité et Biotechnologies Microbiennes (LBBM), Sorbonne Universités, Observatoire Océanologique, 66650 Banyuls-sur-Mer, France ‡
S Supporting Information *
ABSTRACT: We describe a Sabicea cinerea endophytic fungus closely related to Diaporthe pseudomangiferae that produces two known metabolites, mycoepoxydiene (1) and altiloxin A (2), as well as enamidin (3) and eremofortin F (4), two compounds not previously described in the literature. The structure of these four metabolites was elucidated using spectroscopic analysis, and their cytotoxic activities were measured against the human cell lines KB, MRC-5, and MDA-MB-435.
M
and temperate woody plants. Their potential role in protecting plants from fungal diseases has been explored, and at least one endophytic Phomopsis from a woody tropical tree is known to produce toxins that affect the central nervous systems of vertebrates, suggesting an adaptive advantage to plants that harbor this fungus.10 In this paper, we investigate the compounds responsible for the cytotoxicity of the SBNGSS10 extract. The cytotoxicity-guided fractionation of an ethyl acetate extract of SNB-GSS10 led to the isolation of four compounds, among which mycoepoxydiene (1) and altiloxin A (2) had been described in previous articles, while compounds 3 and 4 were unknown (Figure 1). The structure of compounds 1 and 2 was ascertained based on spectroscopic data, and it was demonstrated that these data are in agreement with those reported previously.11−13 Compound 3 had a pseudomolecular ion peak at m/z 186.0773 in the negative ionization mode corresponding to the molecular formula C8H13NO4. The planar structure of 3 was deduced from interpretation of 1D and 2D NMR experiments (Table 1). The 1H NMR spectrum consisted of characteristic signals of a 2-hydroxyisovaleryl unit [3.85 (d, J = 3.5 Hz, 1H), 2.02 (septd, J = 6.8, 3.5 Hz,1H), 0.92 (d, J = 6.8 Hz, 3H), and 0.77 (d, J = 6.8 Hz, 3H)], one NH proton (11.74, d, J = 11.3 Hz), two olefinic protons [7.21 (dd, J = 11.3, 8.9 Hz, 1H) and 5.06 (d, J = 8.9 Hz, 1H)], and one hydroxy proton (5.93, br s).
icroorganisms constitute one of the most important forms of life that provide biotechnological tools to transform organic matter and produce useful chemicals and biochemicals.1 These microorganisms occupy virtually every living niche on earth, and it has been estimated that there may be as many as 5 million different fungal species on our planet.2,3 Thus, fungi have been an excellent source of bioactive molecules for pharmaceutical use. Antibacterial penicillin, cholesterol-lowering lovastatin, antifungal echinocandin B, and immunosuppressive cyclosporin A illustrate the importance of investigating fungal sources for new drugs.4,5 It has been demonstrated that endophytic fungi contribute to plant natural defenses by preventing herbivory and invasion from superficial pathogens.6−8 In general, a significant relative proportion of endophyte extracts are antimicrobial or cytotoxic.9 In a previous study, we reported 16 endophytic fungi obtained from leaves of the Rubiaceae species Sabicea cinerea, a vine found along forest edges in French Guiana.9 The ethyl acetate extract of one of the filamentous fungi isolated from this plant was strongly cytotoxic. The rDNA analysis for the ITS15.8S-ITS2 amplified region of this fungus and comparison with similar sequences in GenBank indicated that this isolate might be closely related to Diaporthe pseudomangiferae. The isolated endophytic strain was deposited in the Substances Naturelles et Biodiversité collection at the ICSN-CNRS, France, with the SNB-GSS10 access code. The Diaporthe genus is the sexual form of Phomopsis, the most prevalent endophytic fungus isolated from both tropical © XXXX American Chemical Society and American Society of Pharmacognosy
Received: December 19, 2014
A
DOI: 10.1021/np501029s J. Nat. Prod. XXXX, XXX, XXX−XXX
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Table 2. NMR Spectroscopic Data for Eremofortin F (4) in CDCl3 no. 1
13
C
1
31.6, CH2
eq: 2.42, m
H
31.4, CH2
C
1 2 3
170.4, C 102.3, CH 133.1, CH
1′ 2′
172.3, C 75.0, CH
3′
30.4, CH
4′ 5′ NH
18.9, CH3 16.1, CH3
1
H
COSY
HMBCa
NOESY
1, 3 1, 2
3 2
5.06, d (8.9) 7.21, dd (11.3, 8.9)
3 NH, 2
3.85, d (3.5)
3′
2.02, septd (6.8, 3.5) 0.92, d (6.8) 0.76, d (6.8) 11.74, br d (11.3) 5.93, br s
2′, 5′, 4′
1′, 3′, 4′, 5′ 2′, 4′, 5′
3
2′, 3′, 5′ 2′, 3′, 4′ 1′
47.2, CH
5 6
45.0, C 39.9, CH2
8 9 10 11 12
Table 1. 1H and 13C NMR Spectorscopic Data for Enamidin (3) in DMSO-d6 13
4
7
Figure 1. Structure of compounds 1−4 isolated from Diaporthe sp. SNB-GSS10, AA03390 (5), and eremofortin B.
no.
73.9, CH
3′, 4′, 5′ 2′, 4′, 5′ 2′, 3′ 2′, 3′
51.3, CH 198.9, 127.2, 163.1, 143.4, 114.6,
C CH C C CH2
13 14
19.7, CH3 10.7, CH3
15
65.7, CH2
1′ 2′
175.0, C 45.8, CH
3′
74.3, CH
4′
131.3, CH
HMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbons.
5′
132.4, CH
6′
132.3, CH
The determination of the 2-hydroxyisovaleryl unit was straightforward based on the proton−proton correlations, and this unit was linked to the 3-aminoacrylic acid moiety by an amide bond. Both H-2′ and NH protons correlate to the carbonyl at δ 172.3 (C-1′). The H-3 chemical shift and NH−H3 coupling suggest the presence of a vinylamide moiety. The double bond is disubstituted with two cis vinyl protons based on the H-2−H-3 coupling constant and their NOE correlation. In the HMBC experiment, both H-2 and H-3 correlate with a carboxylic acid at δ 170.4 (C-1). Interestingly, the very strong NH−H-3 coupling indicates that the NH bond is antiperiplanar to the C-3−H-3 bond and is most likely to be involved in a hydrogen bond with the CO2H group. Therefore, this compound is (Z)-3-(2-hydroxy-3-methylbutanamido)acrylic acid. It is an analogue of phomoenamide14 and is described here for the first time. It was given the trivial name enamidin. Compound 4 had a pseudomolecular ion peak at m/z 447.2747 in the positive ionization mode corresponding to the molecular formula C26H38O6. Initially, two partial planar structures were deduced from the analysis of 1H and 13C NMR, COSY, and HMBC spectral data (Table 2). Compound
7′
131.4, CH
2′-OH a
8′
42.5, CH2
9′
67.3, CH
10′ 11′
22.9, CH3 14.1, CH3
NOESY
10
ax: 1.94, br t (14.0) eq: 2.30, dd (13.5, 5.2) 3.48, dd (14.4, 5.2)
6eq, 7
4, 5, 7, 15 4, 6eq, 7, 14
6ax, 7 6eq, 6ax, 12a
5, 7, 10, 15 6, 8, 11, 12, 13
6ax, 7, 14, 15a 6eq, 6ax, 12a, 15
5.98, d (1.3)
1ax
1, 5, 7
1eq
a: 4.83, s b: 4.99, br s 1.73, s 1.04, d (6.9)
7, 13 13 12a, 12b 4
7, 7, 7, 3,
a: 3.97, d (10.7) b: 4.03, d (10.7)
15b
2.58, quint (7.2) 4.24, br t (7.2) 5.59, dd (15.0, 7.0) 6.27, dd (15.0, 10.4) 6.12, dd (15.2, 10.4) 5.73, dt (15.2, 7.6) a: 2.23, br q (7.6) b: 2.29, m 3.87, br sxt (6.1) 1.21, d (6.1) 1.18, d (7.2)
3′, 11′
ax: 1.50, m eq: 2.21, m
3
HMBCb
1ax, 2eq, 2ax 1eq, 2eq, 2ax, 9 1eq, 1ax, 2eq, 3 1eq, 1ax, 2ax, 3 2ax, 2eq, 4 3, 14
ax: 2.63, m 2
COSYa
5.09, td (11.1, 4.7) 1.71, dq (11.1, 6.9)
15a
2, 9, 10
1ax, 2eq, 2ax, 9 1eq, 2eq
1
1eq, 2eq
14, 1′
1eq, 1ax, 2ax, 3 2eq, 14, 15a, 15b 6ax, 14
3, 5, 6, 14, 15
11, 13 13 11, 12 4, 5
7, 12b 12a, 13 12b 3, 4, 6ax, 6eq, 15a, 15b 4, 5, 6, 10 3, 6eq, 7, 14, 15b 4, 6, 10 3, 14, 15a
3′, 11′
3′, 5′
1′, 3′, 4′, 11′ 1′, 2′, 4′, 5′, 11′ 3′, 5′
4′, 6′
3′, 7′
3′, 4′, 6′
5′, 7′
4′
4′, 5′
2′, 4′, 5′
6′, 8′a, 6′, 8′ 8′b 7′, 8′b, 9′ 6′, 7′, 9′, 10′ 7′, 8′a, 9′ 6′, 7′, 9′ 8′a, 8′b, 10′ 9′ 8′, 9′ 2′ 1′, 2′, 3′
2′, 4′, 5′, 11′ 3′, 5′, 6′
10′ 11′ 2′, 3′
a Weak correlations are in italics. bHMBC correlations, optimized for 8 Hz, are from proton(s) stated to the indicated carbons.
4 contained an octahydronaphthalene ring system, indicated by the sequence of COSY and HMBC correlations. The double bond is placed between carbons 9 and 10, and the internal C5−C-10 bond is indicated by the HMBC correlation between both H-15 protons and C-10 and between H-6eq and C-10. An isopropenyl side chain is attached to C-7, and there is a carbonyl group at C-8. Carbon 3 bore a univalent acyloxy side chain indicated by the H-3−C-1′ HMBC correlation. This chain constitutes the other part of the molecule, which could easily be elucidated from the reconstruction of the sequence of B
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protons and carbons from the 1D and COSY NMR experiments. It contained two conjugated E double bonds, as indicated by the large coupling constants between H-4′ and H5′ (15.0 Hz) and between H-6′ and H-7′ (15.2 Hz). Overall, compound 4 had an eremofortin skeleton. Six eremofortins have been isolated in the past from Penicillium species.15 The closest analogues are eremofortin B and AA03390 (5). This last compound has been described only in a patent without any stereochemical assignment.16 It is the only eremofortin with the same unsaturated side chain on C-3. Compound 4 was given the trivial name eremofortin F. It was not possible to fully establish the stereochemistry of the flexible side chain of 4. However, on the basis of the examination of NMR data of 3-hydroxy-2-methylpropionic acid derivatives in the literature, it can be argued that the relative stereochemistry in 2′−3′ is anti (see Supporting Information). This statement is essentially based on the 13C chemical shift of 11′ (δ 14.1). The NOESY experiment provided conclusive evidence relative to the stereochemistry of the bicyclic unit. All of the correlations are listed in Table 2, and key NOESY correlations are represented in Figure 2. Sequential through-
Figure 3. Compared CD curves of eremofortins F (4) and B.
Table 3. Cytotoxic Activities of the Crude Extract (Percentage of Cell Growth Inhibition) and Compounds 1− 4 (IC50 μM) compound
KB
MRC5
MDA435
crude extract mycoepoxydiene (1) altiloxin A (2) enamidin (3) eremofortin F(4) docetaxel doxorubicin
100%a 7.5 >30 >30 13.9 0.0002
100%a 17.7 >30 >30 12.2 0.0005
97%a 15.8 >30 >30 >30 0.02
a
Percentage (%) cellular growth inhibition against target cells KB (cervical uterine cancer), MRC-5 (human lung fibroblasts), and MDAMB-435 (metastatic melanoma), at 1 μg/mL.
Figure 2. Key NOESY correlations for the determination of relative stereochemistry in 4.
showed no effect (IC50 > 30 μM) on all tested cell lines, and compound 4 was cytotoxic on KB and MRC5 cells (IC50 = 13.9 and 12.2 μM, respectively). On KB and MDA-MB-435 cell lines, the positive control was docetaxel (0.2 and 0.5 nM, respectively). On the MRC5 cell line, the positive control was doxorubicin (20 nM). Mycoepoxydiene is a polyketide previously isolated from an undetermined fungal species (OS-F66617) cultured from dead plant material collected in Brazil11 and from endophytic Diaporthe species found on two different hosts, the first one in submerged rotten leaves of Kandelia candel in a mangrove forest and the second one in the Thai medicinal plant Hydnocarpus anthelminthicus.12,18 Little is known about the toxicity of eremofortins. PR toxin toxicity is linked to the presence of an aldehyde function at C12, and eremofortins A−E have never been reported to be cytotoxic. However, AA03390 (5) and many semisynthetic analogues with diverse acyloxy side chains have been patented for cancer treatment, although it is not clear what models have been tested.16 The side chain in eremofortin F may, therefore, impact its cytotoxic potential.
space correlations between H-7, H-15, H-3, and H-2eq indicate that these protons are on the same side of the molecule. On the other side, H-4 and H-6ax also correlated to one another, H6eq being unambiguously assigned by its correlation with H15a. Interestingly, in addition to not being hydroxylated on C15, the comparison of the chemical shifts and coupling constants between compounds 4 and 5 seemed to indicate that 5 was an epimer of 4 at C-3. In particular, the 3J coupling constant between H-3 and H-4 was 11.1 Hz in compound 4 and only 7.0 Hz in 5. In eremofortin B, the 3J coupling constant between H-3 and H-4 was also low (5.0 Hz), and Moreau et al. did not observe any NOE correlation between H-15 and H-3; they assigned eremofortin B as an epimer of compound 4 at C3.15 Altogether, these data allowed us to confirm that 4 and eremofortin B are epimers at C-3 and that AA03390 (5) is most likely in the same relative configuration as eremofortin B. The absolute configurations of the eremofortin series have been established for the PR toxin from the anomalous dispersion of the oxygen atom.17 PR toxin is an eremofortin analogue, and comparative circular dichroism analysis has demonstrated that PR toxin and all eremofortins possess the same absolute configuration.15 In the eremofortin series, the substitution at C-7 exerted a strong influence on the overall shape of the CD curve. Therefore, a comparison of the CD spectra of 4 and eremofortin B is relevant, and the superimposition of both curves (Figure 3) shows that the absolute configuration of 4 was the one shown in Figure 1, i.e., identical to that of eremofortin B. Mycoepoxydiene (1) showed cytotoxic activity with IC50 values of 7.5, 17.7, and 15.8 μM against KB, MDA-MB-435, and MRC5 cell lines, respectively (Table 3). Compounds 2 and 3
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured at 589 nm using an Anton Paar MCP 300 polarimeter in a 100 mm long, 350 μL cell. to NMR spectra were recorded on a Bruker 500 MHz spectrometer, or a Bruker 600 MHz spectrometer equipped with a 1 mm inverse detection probe. The chemical shifts (δ) are reported as ppm based on the TMS signal, and coupling constants are in hertz. The s stands for singlet, d for doublet, t for triplet, q for quartet, quint for quintet, sxt for sextet, sept for septet, m for multiplet, and br for broad. HRESIMS measurements were performed using a Waters Acquity UPLC system with a column bypass coupled to a Waters Micromass LCT Premier time-of-flight mass C
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Altiloxin A (2): [α]25D −20 (MeOH, c 0.106); 1H NMR (500 MHz, CDCl3) δ ppm 0.77 (s, 3H, H-13), 1.15 (s, 3H, H-14), 1.30 (m, 1H, H-1), 1.30 (s, 3H, H-12), 1.41 (s, 3H, H-15), 1.44 (m, 1H, H-3a), 1.49 (m, 1H, H-3b), 1.55 (m, 1H, H-2eq), 1.75 (qt, J = 13.7, 3.7 Hz, 1H, H2ax), 1.94 (br d, J = 13.1 Hz, 1H, H-1eq), 2.19 (d, J = 16.5 Hz, 1H, H7ax), 2.41 (dd, J = 16.5, 4.0 Hz, 1H, H-7eq), 3.15 (s, 1H, H-9), 3.17 (d, J = 4.0 Hz, 1H, H-6); 13C NMR (125.7 MHz, CDCl3) δ ppm 17.9 (C-15), 26.5 (C-14), 26.6 (C-13), 28.7 (C-12), 34.3 (C-4), 35.2 (C2), 36.2 (C-1), 36.7 (C-10), 38.2 (C-7), 38.3 (C-3), 53.3 (C-6), 60.9 (C-9), 66.4 (C-5), 69.7 (C-8), 174.8 (C-11); HRESIMS (negative) m/ z found 267.1596 [M − H]−; calcd for [C15H23O4]− 267.1602. Enamidin (3): [α]25D + 6.7 (MeOH, c 0.6); 1H NMR (500 MHz, DMSO-d6) see Table 1; 13C NMR (125.7 MHz, DMSO-d6) see Table 1; HRESIMS (negative) m/z found 186.0773 [M − H]−; calcd for [C8H12NO4]− 186.0772. Eremofortin F (4): [α]25D + 10 (CHCl3, c 0.6); 1H NMR (600 MHz, CDCl3) see Table 1; 13C NMR (125.7 MHz, CDCl3) see Table 1; HRESIMS (positive) m/z found 447.2747 [M + H]+; calcd for [C26H39O6]+ 447.2741. Cytotoxicity Evaluation. Cytotoxicity assays were conducted using human melanoma cells MDA-MB-435 (ATCC HTB-129), human uterine cervical carcinoma cells KB (ATCC CCL-17), and normal human lung fibroblast cells MRC5 (ATCC CCL-171), according to the procedure described in Tempête et al.19 Docetaxel (Sigma−Aldrich, France) and doxorubicin (Sigma−Aldrich, France) were used as positive controls. The extract obtained from the culture medium (blank) was tested in all of the assays and did not present any significant activity.
spectrometer equipped with an electrospray interface (ESI). Flash chromatography was performed on a Grace Reveleris system with dual UV and ELSD detection and equipped with a 40 g silica column. The flow rate was 50 mL/min, and the effluents were monitored at 254 and 280 nm. TLCs were conducted on 60 A F254 Merck plates and visualized using UV and phosphomolybdic acid. Analytical and preparative HPLCs were conducted using a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector, and a prepELSII electrospray nebulizer detector. The columns used for these experiments included a Phenomenex Luna C18 5 μm 4.6 × 250 mm analytical column and a Phenomenex Luna C18 5 μm 21.2 × 250 mm preparative one. The flow rate was set to 1 or 17 mL/min, respectively, using a linear gradient of H2O mixed with an increasing proportion of CH3CN. Both solvents were modified with 0.1% formic acid. All solvents were HPLC grade. Potato dextrose agar (PDA) was purchased from Fluka Analytical. Isolation and Identification of SNB-GSS10. The leaves of Sabicea cinerea J. B. Aublet (Rubiaceae) were collected in Roura, French Guiana, in January 2011. The plant was identified by one of the authors (V.E.), and a specimen voucher was deposited in the Cayenne Herbarium (CAY109390). The general procedures adopted for isolation and identification of the endophytic microorganisms followed the methodology described by Casella et al.9 Sixteen strains were isolated from Sabicea cinerea leaves. All strains were cultured and extracted with ethyl acetate, and the extracts were evaluated in a cytotoxicity assay.9 The most active strain was sequenced externally by BACTUP, France, for identification. The fungal rDNA sequences obtained were aligned with DNA sequences from GenBank, NCBI (http://www.ncbi.nlm.nih.gov/genbank/ consulted on November 19, 2014), and comparison with similar sequences indicated that SNBGSS10 is closely related to Diaporthe pseudomangiferae. The sequence was deposited in the GenBank with accession number KF164383.1. Large-Scale Cultivation and Extraction of SNB-GSS10. Diaporthe sp. was cultivated on solid PDA medium at 26 °C for 15 days on 130 14 cm diameter Petri dishes (total 2 m2). The contents of the Petri dishes were cut into small pieces, transferred into a large container, and macerated with ethyl acetate for 24 h. The organic solvent was collected via filtration, washed with water in a separating funnel, and evaporated to dryness under reduced pressure, yielding 3.84 g of extract (1.92 g/m2). Isolation of Compounds 1−4. A portion of the ethyl acetate extract (720 mg) was fractionated using flash chromatography with a linear gradient of hexane mixed with an increasing proportion of ethyl acetate and methanol (Hex−AcOEt, 95/5 to 80/20 over 5.4 min; Hex−AcOEt, 80/20 to 0/100 over 5.4 min; pure AcOEt for 2.7 min; then AcOEt−MeOH, 100/0 to 70/30 over 2.7 min; and a plateau of AcOEt−MeOH, 70/30, for another 2.7 min). Seven fractions were collected based on their TLC profiles. Fractions 5 (33.3 mg) and 6 (89.0 mg) retained biological activity and were subjected to further fractionation by reversed-phase preparative HPLC using a linear gradient of H2O−CH3CN (80/20 to 60/40 in 10 min) followed by a 20 min isocratic plateau at 60/40, another linear gradient from 60/40 to 0/100 in 5 min, and another isocratic plateau at 0/100 for 8 min. This process allowed for the isolation of mycoepoxydiene (1) (2.4 mg, 6.4 mg/m2, tR = 12.55 min), altiloxin A (2) (1.2 mg, 3.2 mg/m2, tR = 13.46 min), and enamidin (3) (1.4 mg, 3.7 mg/m2, tR = 26.26 min) from fraction 5 and eremofortin F (4) from fraction 6 (0.6 mg, 1.6 mg/m2, tR = 15.80 min). Mycoepoxydiene (1): [α]25D +140 (MeOH, c 0.106); 1H NMR (500 MHz, DMSO-d6) δ ppm 1.00 (d, J = 6.4 Hz, 3H, H-14), 1.96 (s, 3H, H-16), 2.77 (m, 2H, H-6/13), 4.23 (d, J = 4.6 Hz, 1H, H-12), 4.43 (br t, J = 5.8 Hz, 1H, H-7), 4.64 (br d, J = 9.8 Hz, 1H, H-5), 5.20 (br d, J = 6.1 Hz, 1H, H-4), 5.86 (m, 2H, H-9/10), 5.96 (br dd, J = 9.9, 5.8 Hz, 1H, H-8), 6.10 (br dd, J = 11.4, 4.6 Hz, 1H, H-11), 6.22 (d, J = 9.5 Hz, 1H, H-2), 7.06 (dd, J = 9.5, 6.1 Hz, 1H, H-3); 13C NMR (125.7 MHz, DMSO-d6) δ ppm 13.3 (C-14), 20.0 (C-16), 48.9 (C-6), 51.9 (C-13), 62.6 (C-4), 74.0 (C-7), 76.4 (C-5), 84.9 (C-12), 123.2 (C-2), 123.4 (C-9), 124.9 (C-10), 137.3 (C-11), 137.4 (C-8), 140.9 (C-3), 161.6 (C-1), 169.1 (C-15); HRESIMS (positive) m/z found 291.1236 [M + H]+; calcd for [C16H19O5]+ 291.1227.
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ASSOCIATED CONTENT
S Supporting Information *
Supplementary Table S1: 13C and 1H NMR shifts of 3-hydroxy2-methylpropionic acid derivatives in CDCl3. Supplementary Figure S1: Significance of 13C and 1H chemical shift differences in 3-hydroxy-2-methylpropionic acid derivatives in CDCl3. Supplementary Figures S2−S13: 1H, 13C, COSY, HSQC, HMBC, and NOESY NMR spectra for compounds 3 (DMSO-d6) and 4 (CDCl3). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/np501029s.
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AUTHOR INFORMATION
Corresponding Authors
*Tel: +33 1 69 82 36 79. E-mail:
[email protected]. *Tel: +33 4 30 16 24 76. Fax: +33 1 69 82 37 84. E-mail: didier.
[email protected]. Present Address ⊥
Observatoire Océanologique, Banyuls-sur-mer, France.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by an “Investissement d’Avenir” grant managed by the Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-0025).
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REFERENCES
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D
DOI: 10.1021/np501029s J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
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DOI: 10.1021/np501029s J. Nat. Prod. XXXX, XXX, XXX−XXX
