Dibenzofurans and Pseudodepsidones from the Lichen

Note pubs.acs.org/jnp

Dibenzofurans and Pseudodepsidones from the Lichen Stereocaulon paschale Collected in Northern Quebec Claudia Carpentier,† Emerson Ferreira Queiroz,‡ Laurence Marcourt,‡ Jean-Luc Wolfender,‡ Jabrane Azelmat,§ Daniel Grenier,§ Stéphane Boudreau,⊥ and Normand Voyer*,† Département de Chimie and PROTEO and ⊥Centre d’Études Nordiques, Département de Biologie, Université Laval, 1045 Avenue de la Médecine, Québec G1V 0A6, Canada ‡ School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Rue Michel-Servet, CH-1211 Geneva 4, Switzerland § Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, 2420 Rue de la Terrasse, Québec G1V 0A6, Canada †

S Supporting Information *

ABSTRACT: Chemical investigation of the methanol extract of the lichen Stereocaulon paschale collected in Nunavik, Canada, led to the isolation and identification of two new dibenzofurans (1 and 3) and 11 known lichen metabolites. The structures of the new compounds were established by analysis of 1D and 2D NMR spectroscopic and high-resolution mass spectrometric data. Herein, the first isolation of ascomatic acid dibenzofuran derivatives (1−3) from a whole lichen organism is reported. In addition, some of the isolated metabolites showed antibacterial activity against the oral pathogens Porphyromonas gingivalis and Streptococcus mutans.

O

pressure liquid chromatography (MPLC-UV) fractionation of the methanol crude extract led to the isolation and identification of two new dibenzofurans, 1 and 3, and 11 known lichen metabolites. In this contribution, the antimicrobial properties of the isolated compounds against the major pathogens Streptococcus mutans, Porphyromonas gingivalis, and Candida albicans are also reported. The methanol crude extract was analyzed by HPLC-PDA and HPLC-TOF-HRMS to identify compounds that have already been isolated from the genus Stereocaulon.5,15−17 This dereplication procedure provided an unambiguous assignment of the molecular formula of compounds 4−13 (Figure S76 and Table S1, Supporting Information). Comparison of these data with those previously reported for compounds isolated from species of the genus Stereocaulon enabled the dereplication of methyl β-orsellinate (4), methyl hematommate (5), and lobaric acid (13) as metabolites frequently reported in Stereocaulon species. Similarly, compound 6 was identified as sakisacaulon A from Stereocaulon sasakii, and compounds 7−11 as diphenyl ethers described in S. azoreum and S. alpinum. Compounds 1− 3 exhibited molecular formulas that could not be associated with secondary metabolites previously identified in Stereocaulon

ver the last few decades, the expansion of shrub species in lichen-dominated ecosystems has had a negative impact on lichen abundance. In Nunavik (subarctic Québec), this expansion is mainly associated with the densification of dwarf birch (Betula glandulosa Michx.) stands.1,2 This species has seen its radial growth increase rapidly with the warming trend observed since the 1990s.3 As a result, lichens such as Stereocaulon paschale (L.) Hoffm. (Stereocaulaceae) appear to be under threat in a large portion of the subarctic region. Hence, it is important to carry out phytochemical investigations on lichens of northern Quebec prior to their decline. The genus Stereocaulon contains about 130 species; from the 40 species that have been studied phytochemically,4 only 75 metabolites have been identified.5 For example, only a few metabolites have been isolated from S. paschale previously, including ethyl hematommate, methyl-β-orsellinate, hematommate, lobaric acid, and heteroglycan.6−8 Moreover, several interesting biological activities were reported for metabolites isolated from S. alpinum (anti-inflammatory,9 antioxidant,10 antibacterial,10 tyrosinase protein phosphatase 1B inhibition,11,12 5-lipoxygenase inhibition,13 human tumor cell line cytotoxicity12), S. halei (antioxidant4), S. evolutum (antiviral, HCV5,14), and S. sasakii (antimitotic15). The methanol extract was chosen in this first thorough phytochemical study of the lichen S. paschale as more material was available than the other extracts produced. Medium© 2017 American Chemical Society and American Society of Pharmacognosy

Received: September 9, 2016 Published: January 12, 2017 210

DOI: 10.1021/acs.jnatprod.6b00831 J. Nat. Prod. 2017, 80, 210−214

Journal of Natural Products

Note

Stereocaulon by the dereplication procedure used (Figure S76 and Table S1, Supporting Information). Compounds 4, 5, and 13 were isolated directly from the MPLC fractionation without any further purification. Methyl β-orsellinate (4), methyl hematommate (5), and lobaric acid (13) were confirmed by 1D and 2D NMR and HRMS.11,19 It has been reported that monoaryl compounds 4 and 5 can be artifacts from the methanolysis of the depside atranorin.19 However, no trace of the depside atranorin was detected by HPLC-PDA-MS in the hexanes, dichloromethane, and methanol extracts. In addition, no trace of atranorin was observed by HPLC-PDA-MS in a freshly prepared crude acetone extract. Therefore, compounds 4 and 5 are not artifacts from methanolysis. Further purification was achieved by semipreparative HPLC and led to the isolation at the milligram scale of 10 compounds (1−3 and 6−12). The additional spectroscopic data obtained by 1D and 2D NMR confirmed the conclusions made by dereplication of compounds 6−12: sakisacaulon A (6),15 methyllobarin (7),10 esterified lobarin (8),17 methylsakisacaulon (9),5,17 esterified sakisacaulon (10),17 anhydrosakisacaulon A (11),5,17 and norlobaric acid (12).5 In addition, the investigation showed that 2 corresponded to isostrepsilic acid, a dibenzofuran previously isolated from mycobiont culture of Usnea orientalis.20 Diphenyl ethers 6−11 are closely related to lobaric acid (13), the major compound of the crude extract, and norlobaric acid (12) is the nonmethylated derivative of 13. The co-occurrence in the lichen S. paschale of pseudodepsidones 6− 11 and the depsidone lobaric acid (13) suggests that they may be produced by the cleavage of the depsidone linkage of lobaric acid (Figure S81, Supporting Information).4 Compound 1 was isolated as a white-gray solid, and the HRESIMS showed a molecular ion at m/z 243.0662 [M − H]− (calcd for C14H11O4−, 243.0663), corresponding to C14H12O4. The 1H NMR and HSQC spectra revealed the presence of a methyl group (δH/δC 2.83/21.8, H-1′), a hydroxymethyl group (δH/δC 5.03/63.9, H-9′), and four aromatic protons (δH/δC 6.88/111.6, 6.81/96.7, 6.71/95.0, and 6.58/113.4 for H-8, H-6, H-4, and H-2, respectively). The coupling constant values between H-2 and H-4 (J = 2.0 Hz) and between H-6 and H-8 (J = 2.2 Hz) indicated that these four aromatic protons belong to two aromatic rings (A for H-2 and H-4 and B for H-6 and H8) and that they are in a meta position to each other. The

lichens and were isolated for de novo structure determination (Figure S76 and Table S1, Supporting Information). The methanol extract (2.5 g) was fractionated by MPLC to isolate the potentially novel compounds 1−3. To ensure similar selectivity for a precise separation prediction, an efficient scaleup from HPLC to MPLC was performed by geometric transfer of the analytical HPLC conditions to preparative MPLC using chromatographic calculations.18 This fractionation also led to the isolation of the other constituents (4−13), which were identified as compounds previously described in the genus

Table 1. 1H NMR and 13C NMR Data for Compounds 1−3 in CH3OH-d4 1 position

δC

1 2 3 4 4a 5a 6 7 8 9 9a 9b 1′ 2′ 9′

132.4, C 113.4, CH 155.7, C 95.0, CH 157.8, C 157.9, C 96.7, CH 155.6, C 111.6, CH 134.7, C 114.8, C 115.7, C 21.8, CH3 63.9, CH2

isostrepsilic acid (2)

3

δH (J in Hz)

HMBC

6.58 d (2.0)

3, 4, 9b, 1′

6.71 d (2.0)

2, 3, 4a, 9b

6.81 d (2.2)

5a, 7, 8, 9a

6.88 d (2.2)

6, 7, 9a, 9′

2.83 s

1, 2, 9b

5.03 s

8, 9, 9a

δC 136.2, C 116.7, C 160.2, C 95.9, CH 159.0, C 158.3, C 99.8, CH 156.4, C 110.8, CH 129.4, C 113.5, C 112.5, C 21,4, CH3 173.1, COOH 171.6, COOH 211

δH (J in Hz)

HMBC

6.89 s

2, 3, 4a, 9b

7.06 d (2.3)

5a, 7, 8, 9a

7.01 d (2.3)

6, 7, 9a, 9′

2.69 s

1, 2, 9b, 2′

δC

δH (J in Hz)

133.0, C 117.4, C 157.3, C 95.7, CH 158.3, C 158.4, C 96.8, CH 156.5, C 112.6, CH 135.4, C 114.3, C 115.0, C 19.8, CH3

2.67 s

64.0, CH2

5.02 s

6.84 s

6.85 d (2.2) 6.94 d (2.2)



Note

HMBC correlations from the hydroxymethyl (H-9′) to C-8 and C-9 (δC 134.7) and C-9a (δC 114.8) allowed this methylene to be located on ring B in an ortho position to H-8, whereas the methyl group H-1′ was placed on ring A in an ortho position to H-2 due to its correlations with C-1 (δC 132.4) and C-2 and C9b (δC 115.7). The remaining carbons were assigned to oxygenated sp2 quaternary carbons, as indicated by their 13C NMR chemical shifts (δC 157.9, 157.8, 155.7, and 155.6 for C5a, C-4a, C-3, and C-7, respectively).21−23 On the basis of the molecular formula (C14H12O4), a dibenzofuran structure was proposed. The NOESY correlations from the hydroxymethyl to the methyl H-1′ and H-8 and from the methyl to H-2 confirmed the structure of 1 as a new dibenzofuran, 9(hydroxymethyl)-1-methyldibenzofuran-3,7-diol. The UV spectrum of 1 (218, 239, 256, and 309 nm) was consistent with a dibenzofuran structure. Similar UV spectra were obtained for norascomatic acid (or hypostrepsilic acid), hypostrepsilalic acid, and strepsilin, three dibenzofurans previously isolated from cultured mycobiont of Evernia esorediosa, S. japonium, and S. evolutum.5,21,22 The 1H NMR and 13C NMR spectra of compound 1 showed close similarities to analogous data for isostrepsilic acid (2) (Table 1).20 When compared with isostrepsilic acid (2), the 1H NMR spectrum of 1 displayed an additional aromatic proton (H-2), which replaced the carboxylic acid group. Compound 3 was isolated as a white solid. The HRESIMS showed a deprotonated molecular ion at m/z 301.0362 [M − H]− (calcd for C15H9O7−, 301.0364), consistent with a molecular formula of C15H10O7. The NMR data of 3 were very similar to those of isostrepsilic acid except that signals for a hydroxymethyl unit were missing.20 The HMBC correlations from H-8 (δH/δC 7.01/110.8) to C-7 (δC 156.4), C-6 (δC 99.8), C-9a (δC 113.5), and particularly C-9′ (δC 171.6) supported the carboxylic acid function being positioned at C-9. All these data indicated that 3 is a new dibenzofuran, 3,7-dihydroxy-1methyldibenzofuran-2,9-dicarboxylic acid. In this contribution, the phytochemical investigation of S. paschale has led to the first isolation of ascomatic acid dibenzofuran derivatives (1−3) from a whole lichen organism.20−24 Indeed, isostrepsilic acid (2), norsacomatic acid (or hypostrepsilic acid), and hypostrepsilalic acid have been isolated up to now only from mycobiont cultures of Usnea orientalis, Evernia esorediosa, and Stereocaulon japonicum.20−22,24 Miyagawa et al. have suggested that the dibenzofuran isostrepsilic acid is an intermediate compound in the oxidation pathway from norascomatic acid to hypostrepsilalic acid.21 On the basis of these results, compounds 1, 3, and isostrepsilic acid (2) appear to be derivatives of norsacomatic acid (or hypostrepsilic acid) and might be produced by final modification including decarboxylation and oxidation reactions. Isostrepsilic acid (2) could be produced by the oxidation of a methyl group of norsacomatic acid to form a hydroxymethyl. Sequential steps of oxidation of this hydroxymethyl would lead to an aldehyde (hypostrepsilalic acid) and finally to a carboxylic acid (3) (Scheme 1). Compound 1 could be formed from the decarboxylation of isostrepsilic acid (2). Structural similarities between compounds 1, 3, and isostrepsilic acid (2) were also indicative of this close biosynthetic relationship (Scheme 1). Miyagawa et al. also suggested that the osmotic stress due to the culture conditions could enhance the formation of ascomatic-type dibenzofurans.20−22 The fact that dibenzofurans 1−3 occur naturally in S. paschale suggests that the extreme growing conditions in the Nunavik region enhance the lichen’s

Scheme 1. Proposed Biosynthetic Relationship between Compounds 1−3

ability to produce new secondary metabolites. HPLC-PDA-MS analysis performed on a freshly prepared acetone extract demonstrated the presence of dibenzofurans 1−3, indicating that these compounds are indeed occurring naturally in the lichen S. paschale. Interestingly, Bhattari et al. have reported that lobastin, a pseudodepsidone isolated from S. alpinum, and lobaric acid were active against the Gram-positive bacteria B. subtilis and S. aureus.10 On the basis of these results, the antimicrobial activity of the isolated compounds from the methanol extract was investigated against the fungus Candida albicans and the two bacteria Porphyromonas gingivalis and Streptococcus mutans to identify new antimicrobial agents. Those pathogens were chosen, as they are involved in important oral infections, namely, candidiasis, periodontal disease, and dental caries, respectively.25−27 Active compounds endowed with a capacity to exert antimicrobial activity toward these oral pathogens have received considerable attention, as they may represent potential new therapeutic agents for the prevention/treatment of oral infections. The MIC and MBC values measured are presented in Table 2. None of the compounds studied were able to inhibit the growth of C. albicans at a concentration of 80 μM. However, all pseudodepsidone-type metabolites (6−11) and lobaric acid (13) showed moderate or weak antibacterial activities. The results suggest that the diphenyl ethers 8 and 11 and the depsidone 13 have potential as natural antibacterial agents and should be investigated further.

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EXPERIMENTAL SECTION

General Experimental Procedures. Analytical HPLC data were recorded on an Agilent 1260 Infinity equipped with a photodiode array detector. MPLC fractionation was performed using a Biotage Isolera Prime system equipped with a SiliCycle SiliaSep ISO120 (256 × 42 mm i.d.) flash cartridge loaded with C18 as the stationary phase (40− 63 μm). Semipreparative HPLC was carried out using an HP 1260 system with a photodiode array detector using an ACE C18 column (5 μm, 250 × 10.0 mm i.d). The NMR spectra were recorded on an Agilent DD2 500 MHz spectrometer. Complete assignment was performed using 2D experiments (COSY, HSQC, HMBC, and 212



Note

Rivières).31 The whole thallus was air-dried and stored at room temperature for approximately two months. Extraction and Isolation. The air-dried thallus (130.7 g) was ground in liquid nitrogen and extracted successively under maceration and agitation at room temperature with hexanes (3 × 600 mL), dichloromethane (4 × 600 mL), and methanol (4 × 600 mL). The extracts were concentrated under vacuum to yield respectively 0.39 g of a hexanes extract (0.29%), 3.22 g of a dichloromethane extract (2.5%), and 7.01 g of a methanol extract (5.4%). A portion of the methanol extract (2.5 g) was fractionated initially using a Biotage Isolera Prime system equipped with a SiliCycle SiliaSep ISO120 (256 × 42 mm i.d.) flash cartridge loaded with C18 silica gel as the stationary phase (40−63 μm). The crude extract was mixed with 7.50 g of C18 reversed-phase silica gel (40−63 μm, carbon 17%, SiliaBond C18 (17%), SiliCycle) and 2.25 g of Ottawa sand. To introduce the extract in the flash cartridge, the mixture was loaded in an empty solid-load cartridge (60 mL, SiliCycle), which was connected to the flash cartridge using a plunger for a solid load cartridge (SiliaSep plunger, 60 mL). The separation conditions for the MPLC fractionation were determined by performing a geometric transfer of the HPLC-PDA conditions.18 The solvent system used was (A) H2O containing 0.1% TFA and (B) MeOH. The UV absorbance was measured at 254 and 210 nm. A linear gradient from 40% to 100% in 350 min followed by 100% B for 18 min at a constant flow rate of 10 mL/min was performed and yielded 389 fractions. Fraction purity was monitored by HPLC-PDA. Those with a similar chromatogram were combined to afford nine fractions (A = Fr 104−134, B = Fr 140−168, C = Fr 199−220, D = Fr 264−273, E = Fr 273−287, F = Fr 288−292, G = Fr 303−313, H = Fr 314−328, and I = Fr 335−365). Fractions C, D, and I yielded 4 (100 mg), 5 (34 mg), and 13 (110 mg), respectively. Further purification was performed for fractions A, B, and E−H (Table S2, Supporting Information). Semipreparative HPLC was carried out using an HP 1260 system with a photodiode array detector. The analytical tubing (i.d. 0.17 mm) of the system was replaced by tubing with a larger internal diameter (0.25 mm) to reduce the internal pressure created by the flow rate. The final purification was performed with an Ace C18 column (5 μm, 250 × 10 mm i.d.) and the following solvent system: (A) H2O−0.l % TFA and (B) MeOH at a constant flow rate of 4.2 mL/min. 9-(Hydroxymethyl)-1-methyldibenzofuran-3,7-diol (1): amorphous, white-gray powder; UV (MeOH) λmax (log ε) 218 (3.99), 239 (3.91), 256 (3.73), 309 (3.75) nm; 1H and 13C NMR data (CH3OH-d4), Table 1; HRESIMS m/z 243.0662 [M − H]− (calcd for C14H11O4−, 243.0663). 3,7-Dihydroxy-1-methyldibenzofuran-2,9-dicarboxylic acid (3): amorphous, white powder; UV (MeOH) λmax (log ε) 240 (3.96), 261 (3.69), 309 (3.47) nm; 1H and 13C NMR data (CH3OH-d4), Table 1; HRESIMS m/z 301.0362 [M − H]− (calcd for C15H9O7−, 301.0364). Determination of Minimal Inhibitory Concentrations and Minimal Microbicidal Concentrations. Porphyromonas gingivalis ATCC 33277, Streptococcus mutans ATCC 25175, and Candida albicans ATCC 28366 were used. Bacteria were grown in Todd-Hewitt broth (BBL Microbiology Systems, Cockeysville, MD, USA) supplemented with 0.001% hemin and 0.0001% vitamin K (THBHK). Candida albicans was cultivated in yeast nitrogen base (YNB; BBL Microbiology Systems) medium supplemented with 0.5% glucose. P. gingivalis was incubated under anaerobic conditions (N2− H2−CO2/80:10:10), while S. mutans and C. albicans were grown aerobically, all at 37 °C. Overnight cultures of microorganisms were diluted in fresh broth medium to obtain an optical density at 660 nm (OD660) of 0.2. Samples (100 μL) were added to the wells of a 96-well tissue culture plate containing 100 μL of serial dilutions (80 to 5 μM) of compounds. Control wells with no substance but with substance vehicle were also inoculated. Penicillin G (Sigma-Aldrich Canada, Oakville, ON, Canada) and nystatin (EMD Biosciences Inc., San Diego, CA, USA) were used as reference controls for growth inhibition of bacteria and C. albicans, respectively. After incubation for 24 h, the concentration of compounds that caused complete growth inhibition

Table 2. Antimicrobial Activity of the Isolated Compounds P. gingivalis 1 2 3 4 5 6 7 8 9 10 11 13 penicillin Ga

S. mutans

MICb (μm)

MBCc (μm)

MIC (μm)

MBC (μm)

− − − − − − 80 40 80 80 20 80 0.29

− − − − − − 80 40 80 80 20 80 2.3

− − − − − 80 80 80 80 80 10 20 0.15

− − − − − 80 80 − 80 − 20 80 4.7

a Positive control. bMIC, mininal inhibitory concentration. cMB(M)C, minimal bactericidal (microbial) concentration. −: no significant activity at 80 μM; MIC > 80 μM.

NOESY). The chemical shifts (δ) are given in ppm with respect to the residual solvent methanol-d4 signal (δH 3.31, δC 49.0 for 1H and 13C NMR, respectively), and coupling constants (J) are given in Hz. Highresolution mass spectra (HRMS) were obtained using an Agilent 6210 LC time of flight mass spectrometer with electrospray ionization (ESI) interface in direct injection mode. HPLC-PDA analyses were conducted on an Agilent 1260 Infinity equipped with an autosampler, a quaternary high-pressure mixing pump, and a photodiode array detector. Chromatographic analysis was performed with a Beckman ODS column (5 μm, 250 × 4.6 mm i.d.), and 20 μL (10 mg/mL) of the crude extract was injected. The gradient was performed at a flow rate of 1 mL/min with a mobile phase composed of H2O containing 0.1% TFA (A) and MeOH (B). A linear gradient of 40% to 100% B in 40 min followed by 100% B for 10 min was used to ensure a good separation. The column temperature was kept at 22.5 °C. The UV absorbance was recorded at 254, 210, 220, and 280 nm, and the UV spectra (PDA) were recorded between 190 and 400 nm (step, 2 nm). HPLC-TOF-HRMS metabolite profiling of the crude extract was obtained on an Agilent 6210 LC time-of-flight mass spectrometer with an ESI interface. The ESI conditions were as follows: capillary voltage 4000 V, fragmentor voltage 175 V, skimmer 65 V, octopole RF peak voltage of 250 V, gas temperature of 350 °C, gas flow of 8 L/min, and nebulizer gas pressure of 35 psi. Detection was performed in both the negative- and positive-ion mode with a m/z range of 120−1000 and a scan time of 1 s. The mass spectrometer was calibrated in the negative and positive modes using Agilent ESI-L low concentration tuning mix. The separation was performed with a Phenomenex Ultracarb ODS column (5 μm, 150 × 4.6 mm i.d.), and 15 μL (10 mg/mL) of the crude extract was injected. A splitter was used at the end of the column to let pass only 17% of the mobile phase to the MS. The following solvent system was used: H2O containing 0.1% formic acid (A) and MeOH (B). The constant flow rate was reduced to 0.5 mL/min, so a shorter column was used, and the separation conditions for the MS metabolite profiling were determined by doing a geometric transfer of the HPLC-PDA conditions. A linear gradient of 50% to 100% B in 48 min followed by 100% B for 12 min was used. The UV absorbance was recorded at 210 and 254 nm, and the UV spectra (PDA) were recorded between 190 and 400 nm (step, 2 nm). Plant Material. The lichen Stereocaulon paschale was collected in Umiujaq, Québec, Canada (56°32′55.77″ N, 76°32′26.19″ O), in August 2014. The Umiujaq region is located at the forest-tundra ecotone.28 In this region, lichens are progressively being replaced by shrub species,29,30 a likely consequence of recent temperature warming.3 The specimens were collected and identified by S.B. and Prof. Esther Lévesque (Centre d’Études Nordiques, Département des Sciences de l’Environnement, Université du Québec à Trois213



Note

(17) Gonzalez, A. G.; Rodriguez, E. M.; Bermejo, J. Ann. Quim. 1995, 91, 461−466. (18) Challal, S.; Queiroz, E.; Debrus, B.; Kloeti, W.; Guillarme, D.; Gupta, P.; Wolfender, J.-L. Planta Med. 2015, 81, 1636−1643. (19) Hylands, P. J.; Ingolfsdottir, K. Phytochemistry 1985, 24, 127− 129. (20) Kon, Y.; Iwashina, T.; Kashiwadani, H.; Wardlaw, J. H.; Elix, J. A. J. Jpn. Bot. 1997, 72, 67−71. (21) Miyagawa, H.; Yamashita, M.; Ueno, T.; Hamada, N. Phytochemistry 1997, 46, 1289−1291. (22) Miyagawa, H.; Hamada, N.; Sato, M.; Ueno, T. Phytochemistry 1993, 34, 589−591. (23) Kon, K.; Kashiwadani, H.; Wardlaw, J. H.; Elix, J. Symbiosis 1997, 23, 97−106. (24) Millot, M.; Dieu, A.; Tomasi, S. Nat. Prod. Rep. 2016, 33, 801− 811. (25) Takahashi, N.; Nyvad, B. J. Dent. Res. 2011, 90, 294−303. (26) Feng, Z.; Weinberg, A. Periodontol. 2000 2006, 40, 50−76. (27) Samaranayake, L. P.; Keung Leung, W.; Jin, L. Periodontol. 2000 2009, 49, 39−59. (28) Payette, S. Nordicana 1983, 47, 3−24. (29) Provencher-Nolet, L.; Bernier, M.; Lévesque, E. Écoscience 2014, 21, 419−433. (30) Paradis, M.; Lévesque, E.; Boudreau, S. Environ. Res. Lett. 2016, 11, 085005. (31) Brodo, I. M.; Sharnoff, S. D.; Sharnoff, S. Lichens of North America; Yale University Press: New Haven, CT, 2001.

was recorded as the minimal inhibitory concentration (MIC). Samples of 10 μL obtained from the wells showing no visible growth were spread on solid culture plates to determine the minimal microbicidal concentration (MBC) of the compounds. All the above assays were run in triplicate. Penicillin G was used for bacteria and nystatin for C. albicans as reference controls for growth inhibition (Table 2).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00831. Spectra and additional characterization of compounds (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +1 418 656 3613. Fax: +1 418 656 7916. E-mail: [email protected]. ORCID

Emerson Ferreira Queiroz: 0000-0001-9567-1664 Normand Voyer: 0000-0002-0429-9172 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSERC and the FRQNT. The authors thank PROTEO, FRQNT, and NSERC for graduate scholarships. The authors are also thankful to M. P. Audet for his help in NMR and MS analyses.

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REFERENCES

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Dibenzofurans and Pseudodepsidones from the Lichen

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