Phenolic Compounds from the Lichen Lobaria orientalis - Journal of

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Phenolic Compounds from the Lichen Lobaria orientalis Dung M. T. Nguyen,† Lien M. T. Do,§,∥ Vy T. Nguyen,‡ Warinthorn Chavasiri,§ Jacques Mortier,⊥ and Phung P. K. Nguyen*,† †

Department of Organic Chemistry and ‡Department of Genetics, University of Science, Vietnam National University−Ho Chi Minh City, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City 748355, Vietnam § Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan Bangkok 10330, Thailand ⊥ Institute for Molecules and Materials, University of Maine and CNRS, UMR 6283, Avenue Olivier Messiaen, 72085, Le Mans Cedex, France ∥ Department of Environmental Science, SaiGon University, Ho Chi Minh City 748355, Vietnam S Supporting Information *

ABSTRACT: The chemical investigation of the EtOAc extract of Lobaria orientalis collected in the southwestern part of Central Vietnam led to the isolation of new β-orcinol depsidones, lobarientalones A and B (1 and 2), and diphenyl ethers, lobariethers A−E (3−7). These types of structures are often reported in different lichen species, but the absolute configuration of the stereogenic acetal center has not been defined. This is the first assessment of the (1S) absolute configuration of the stereogenic acetal center using electronic circular dichroism (ECD) spectroscopic data and by comparison with the ECD data of analogous compounds. Based on the co-occurrence of the depsidones, 1,10-di-Omethylstictic acid (8) and (1−2), and diphenyl ethers (3−7), a plausible biosynthesis route toward 1−8 is proposed.

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the isolation and structural elucidation of seven new compounds are described. The absolute configurations of the stereogenic acetal center (Cγ) of the new compounds were assigned via electronic circular dichroism (ECD) spectroscopic data and by comparison with the ECD data of analogous compounds. A likely biosynthesis pathway toward the new compounds 1−7 from a readily accessible precursor, methyl βorcinolcarboxylate (10), is presented.

ichens are symbiotic products of a mycobiont (fungal partner) and photobiont (algal partner) and are known to produce a range of secondary metabolites, of which some are unique to lichen symbiosis, e.g., depsides, depsidones, and diphenyl ethers. These secondary metabolites have shown an impressive range of biological activities, including antibiotic, antifungal, antiviral, antitumor, and anticancer.1 Lobaria, a common foliose lichen genus belonging to the Lobariaceae, the second largest family of macrolichens in the Ascomycota,2 seems to be best developed in tropical and temperate regions.3,4 In folk medicine, seven Lobaria species including Lobaria orientalis are used as a remedy for the treatment of sore throats, indigestion, and lung ailments.5 Some 50 years ago, the phytochemical analysis of eight Lobaria sp. extracts, i.e., L. scrobiculata, L. amplissima,6 L. crassior, L. dentata, L. dissecta, L. erosa, L. laetevirens, and L. linita,7 resulted in the isolation of usnic acid, a mixture of two β-orcinoldepsidones, stictic acid and norstictic acid, depsides, and tridepsides. Subsequent studies of this genus, mostly L. pulmonaria samples collected in Spain,8 Turkey,9 Bosnia,10 Norway,11 and Canada,12,13 afforded other different types of lichen metabolites, such as monocyclic aromatic compounds, steroids, depsides, and depsidones. From the lichen L. kurokawae14 collected in China, five triterpenoidal acids were isolated. In the course of a systematic study on lichen substances from Vietnamese flora, L. orientalis, widely distributed in the southwestern part of Central Vietnam, was examined. Herein, © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The thalli of L. orientalis were collected in Lam Dong Province, Central Vietnam. Chromatographic fractionation of the EtOAc extract led to the isolation of 14 compounds. The known compounds included methyl orsellinate (9), methyl βorcinolcarboxylate (10), orcinol (11), orsellinic acid (12),15 lecanorin (13),16 isolecanoric acid (14), and 1,10-di-Omethylstictic acid (8).17 Compound 1 was isolated as a white, amorphous powder. The molecular formula of C20H18O9 of 1 was determined from the 13C NMR data and an HRESIMS sodium adduct ion at m/z 425.0842 [M + Na]+ (calcd for C20H18O9Na, 425.0849). The IR spectrum showed absorption bands for hydroxy (3537, 3439 cm−1) and lactone carbonyl (1740 cm−1) functionalities. The 1 H NMR data (Table 1) displayed characteristic resonances of Received: May 21, 2016 Published: February 9, 2017 261

DOI: 10.1021/acs.jnatprod.6b00465 J. Nat. Prod. 2017, 80, 261−268

Journal of Natural Products

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Table 1. 1H and 13C NMR Spectroscopic Data for 1, 2, and 8a 1b position 1 1a 3 3a 4 5 5a 7 7a 8 9 10 11 11a 12a 13 1-OCH3 10-OCH3 5-CH3 8-CH3 4-OH

δH (J in Hz) 6.82, s

2c δC 103.0

δH (J in Hz)

δC

6.95, s

95.4

139.1

6.67, s

4.80, 4.99, 3.75, 3.94, 2.30, 2.53, 7.86,

d (12.0) d (12.0) s s s s s

169.8 108.1 152.4 121.0 150.0 161.8 113.9 145.7 111.1 161.6 118.1 159.2 131.9 54.0 56.4 57.0 9.3 21.8

δH (J in Hz)

δC

6.40, s

103.1

138.0

6.95, s

4.61, d (9.0) 4.80, d (9.0) 3.87, 2.18, 2.45, 10.04,

a c

8b

s s s s

166.6 109.0 151.6 120.4 148.3 161.7 112.7 144.3 111.6 161.6 118.5 159.0 135.9 51.4

56.3 9.6 20.9

138.8 169.7 107.9 152.6 121.3 149.6 160.9 114.5 151.5 112.1 163.7 115.1 163.0 132.2 187.0

6.74, s

10.50, s 3.70, 3.97, 2.30, 2.56, 7.90,

s s s s s

Chemical shifts (δ) are expressed in ppm, and J values are presented in Hz. bRecorded at 500 MHz for 1H and 125 MHz for Recorded at 500 MHz for 1H and 125 MHz for 13C in DMSO-d6.

56.8 58.0 9.3 22.4 13

C in CDCl3.

Figure 1. Chemical structures of 1−14.

169.8) carbons. These structural features were similar to those of 1,10-di-O-methylstictic acid17 (8), with the only difference being that the C-13 formyl group in 8 (δH 10.50; δC 187.0) was replaced by a hydroxymethylene (δH 4.80, 4.99; δC 54.0) group in 1. This deduction was supported by the HRESIMS data of 1 having two hydrogen atoms more than 8 and by the HMBC cross-peaks (Figure 2) from the sole aromatic proton signal at δH 6.67 (H-9) and from the hydroxymethylene protons to the same aromatic carbon signals at δC 118.1 (C-11) and 161.6 (C10). The NOESY correlations from H-13 to H-1 and from H-

two methyl [δH 2.30, 2.53 (each 3H, s)], two methoxy [δH 3.75, 3.94 (each 3H, s)], and two diastereotopic oxygenated methylene groups [δH 4.80, 4.99 (each 1H, d, J = 12.0 Hz)], two methine protons [δH 6.67, 6.82 (each 1H, s)], and one phenolic proton at δH 7.86 (1H, s). The combination of the 13C NMR and HSQC data of 1 (Table 1) exhibited 20 carbon resonances due to two methyl (δC 9.3, 21.8), an oxygenated methylene (δC 54.0), two methoxy (δC 56.4, 57.0), an aromatic methine (δC 111.1), six quaternary (δC 108.1, 113.9, 118.1, 121.0, 139.1, 145.7), five oxygenated sp2 (δC 131.9, 150.0, 152.4, 159.2, 161.6), and two ester-type carbonyl (δC 161.8, 262



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Figure 2. Key HMBC correlations observed for 1−7.

Figure 3. Experimental ECD spectra of 1−7.

13 to MeO-1 and the complete analysis of the HSQC and HMBC data for 1 support its structure as shown in Figure 1. Several β-orcinol depsidones and diphenyl ethers containing B-rings fused with a γ-butyrolactone moiety were isolated from different lichen species.6,8,17−19 However, the absolute configuration of the stereogenic acetal center was ignored, although the specific rotation of some were reported to be zero20,21 or near zero.22 The ECD spectroscopic data of compounds bearing an α,β-unsaturated-γ-lactone moiety have been described,23 but there are no reports that provide insight into the configuration of such a moiety fused through C-α and C-β to the B-ring of lichen metabolite depsidones or diphenyl ethers. The ECD spectrum in MeOH of 1 (Figure 3) contained negative Cotton effects (CEs) at (Δε) 320 (−1.2), 300 (−2.1), 283 (−2.5), and 250 (−1.2) nm, opposite of those of vermistatin [positive CEs at 327, 315, 302 nm],24 rubralide A [positive CEs at 302, 291 nm], rubralide C [positive CEs at 292, 276 nm], talaromycolide B [positive CEs at 287, 263 nm],25 and talaromycolide C [positive CEs at 296, 255 nm],26 possessing an R absolute configuration of the stereogenic acetal center of the α,β-unsaturated-γ-lactone moiety. Therefore, the (1S) absolute configuration of 1 was defined. The (1S) configuration was also assigned for the other compounds (2− 7) due to the similar CEs in their ECD spectra (Figure 3) as well as due to biosynthesis considerations. Accordingly, compound 1, lobarientalone A, was characterized as shown.

Compound 2 has the molecular formula C19H14O8 according to HRESIMS analysis (m/z 371.0772 [M + H]+ (calcd 371.0767)). The IR spectrum revealed the presence of hydroxy (3430 cm−1) and carbonyl (1744 cm−1) functionalities. A comparison of the NMR spectroscopic data of 2 and 1 showed similarities, particularly in the A- and B-rings (Tables 1), but the former lacked a signal of one methoxy group. The chemical shift values of the oxygenated C-13 (δC 51.4) of the A-ring, the acetal C-1 (δC 95.4), and the C-3 carbonyl carbon (δC 166.6) of the γ-butyrolactone moiety in 2 were shielded compared to the corresponding carbons of 1 (δC 54.0, 103.0, and 169.8, respectively). In the HMBC experiments, the cross-peaks between H-1 and C-13 (Figure 2) confirmed that the oxymethylene group of the A-ring was connected to C-1 of the γ-butyrolactone moiety through an oxygen bridge. This type of linkage in depsidones was similar to that of verrucigeric acid, a β-orcinol depsidone isolated from the lichen Xanthoparmelia verrucigera.18 Therefore, compound 2, lobarientalone B, is a new β-orcinol depsidone possessing the structure shown in Figure 1. Lobariether A (3) was assigned the molecular formula C20H18O9 from its 13C NMR and HREIMS data. Its IR spectrum was similar to those of 1 and 2. The NMR data of 3 resembled those of 2, except for the presence of a methoxycarbonyl group, which was confirmed by the correlation between the methoxy protons at δH 3.76 and the ester carbonyl C-7 (δC 166.4). The remaining HMBC 263



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Table 2. 1H and 13C NMR Spectroscopic Data for 3−7 3b position 1 2 3 4 5 6 7 8 9 10 11 1′ 2′ 3′ 4′ 5′ 6′ 7′ 9′ 4-OCH3 7-OCH3 10-OCH3 9′-OCH3 2′-OH 4′-OH 6-CH3 3′-CH3

δH (J in Hz)

6.97, s

4.58, d (12.0) 5.04, d (12.0)

4a δC 118.3 152.5 114.0 159.2 110.8 140.7 167.3 56.4

δH (J in Hz)

6.64, s

4.71, d (12.0) 4.82, d (12.0)

5a δC 117.7 152.5 119.6 159.8 109.4 139.7 166.8 54.4

δH (J in Hz)

6.68, s

7.74, d (16.5) 7.09, d (16.5) 2.31, s

6.15, s 3.89, s 3.76, s

9.53, 10.27, 2.37, 2.06,

s s s s

101.9 151.4 115.9 152.4 134.3 129.2 166.4 96.5 56.7 52.4

20.2 8.9

5.30, s 3.93, s 3.41, s

102.6 151.9 115.3 153.2 127.1 133.7 170.2 102.2 56.3 52.1

5.22, s 3.97, s 3.43, s

6b δC 118.5 152.9 115.3 160.6 109.8 141.2 166.4 133.1 132.1 199.8 27.3 103.1 153.4 115.2 152.1 133.5 127.7 170.1 102.2 56.4 52.2

3.11, s 7.76, s

56.8

3.04, s 7.78, s

56.3

2.34, s 2.20, s

20.7 8.1

2.35, s 2.22, s

20.9 8.1

δH (J in Hz)

6.96, s

7.82, d (16.5) 6.74, d (16.5)

5.09, s 3.97, s 3.28, s 2.91, 9.45, 10.51, 2.27, 2.08,

s s s s s

7a δC 117.4 152.7 113.5 159.8 109.3 140.0 166.0 134.0 122.6 168.3 102.2 151.1 115.7 153.6 132.8 129.2 166.5 99.8 56.4 51.5

δH (J in Hz)

6.68, s

7.93, d (16.5) 6.84, d (16.5)

δC 118.1 153.1 114.9 160.4 109.5 140.9 166.3 134.0 122.6 168.1

55.4

5.21, 3.98, 3.42, 3.76, 3.04,

s s s s s

102.9 151.8 115.2 152.6 127.4 133.3 170.1 102.1 56.2 52.1 51.7 56.2

19.8 8.7

2.35, s 2.22, s

20.7 8.0

protons of the former were upfield shifted (ΔδH = 0.08−0.80) due to the ring current effect. Compound 3, lobariether A, is thus a new diphenyl ether with the structure shown in Figure 1. Compound 4, lobariether B, was isolated as a yellowish oil. Its molecular formula was deduced as C21H22O10 from the HRESIMS data. The comparison of the HRESIMS and NMR data of compound 4 with those of 1 and 3 showed that the former was also composed of the A- and B-rings and a γbutyrolactone moiety (Tables 1 and 2), but it contained one more methoxy group and one more hydrogen atom. In the HMBC experiments, the interactions of the oxymethylene protons at δH 4.71 and 4.82 with C-2 (δC 152.5), C-3 (δC 119.6), and C-4 (δC 159.8), as well as the methoxy at δH 3.11 with C-9′ (δC 102.2), of the methoxy at δH 3.93 with C-4 (δC 159.8), and of the one at δH 3.41 with the C-7 carbonyl (δC 166.8), proved that the seven-membered lactone moiety in 1 was opened to afford a methoxycarbonyl group as in 3. Compared to 3, the diphenyl ether whose A- and B-rings were joined via two ether bridges, these two moieties of 4 were linked by a single ether bond. The molecule was therefore more flexible, and the ring current effect was more evident. Indeed, the chemical shift values in the same deuterated solvent of some protons of the three rings of 4, Me-6, Me-3′, and H-9′, were upfield shifted due to the ring current effect (ΔδH = 0.09− 1.51) compared to the corresponding chemical shifts of depsidone 1. The series of 2,3JH−C correlations present in the HMBC spectrum (Figure 2) confirmed the chemical structure of 4 as shown. Compound 5, lobariether C, was isolated as a yellowish oil. The HRESIMS analysis of 5 gave a sodium adduct ion at m/z

correlations were in accordance with the proposed structure (Figure 2). Although the relationship between the A- and Brings in 3 was not confirmed by the HMBC data, their connection through an oxygen bridge was well supported by the HRESIMS as well as by the two following observations. First, a literature search revealed that the chemical shift value of C-2 of 2-oxygenated depsides and 2-oxygenated depsidones recorded in CDCl3 or DMSO-d6 was in the low-field zone of 160−166 ppm in DMSO-d6 and 161−169 ppm in CDCl3 for depsides and at 163−167 ppm in DMSO-d6 and 160−164 ppm in CDCl3 for depsidones,15,19,27−30 compared to the corresponding carbon of 2-oxygenated diphenyl ethers at 156−158 ppm.21,31,32 In diphenyl ethers, C-2 of the A-ring is shielded by the B-ring, a bulky oxygenated arene moiety that could readily rotate around the axis between C-2 and oxygen atoms,33 while in depsides, C-2 is linked to a small hydroxy or methoxy group, and in depsidones, the molecules are relatively rigid.22,30 In 3, as well as in 4−7, C-2 resonated at a higher field of 152−154 ppm (Table 2); therefore these compounds should be diphenyl ethers. Second, in diphenyl ethers, the A- and B-rings are not coplanar,33 and with such a geometry of the system, the resonance of a nucleus located close to the face of an aromatic ring would be shifted to a higher field due to the ring current effect.31 In diphenyl ethers, each ring was affected by the other one through this effect, and these phenomena were observed for some protons of all three moieties of 3. A comparison of the chemical shift values of the protons of Me-6, Me-3′, and H-9′ of 3 with the corresponding ones of the depsidone 2, having a similar partial structure and being measured in the same deuterated solvent (Tables 1 and 2), clearly showed that the 264



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Scheme 1. Putative Biosynthetic Pathway for 1−8 from Methyl β-Orcinolcarboxylate (10)

495.1286 [M + Na]+, in accordance with the molecular formula of C24H24O10. The NMR spectroscopic data of 5 exhibited a similar pattern to those of 4, except for the lack of signals of the hydroxymethylene group and the appearance of signals corresponding to an (E)-3-oxobuta-1-ene group with a trans olefinic double bond (δH 7.09 and 7.74, each 1H, d, J = 16.5 Hz; δC 132.1 and 133.1), a conjugated carbonyl (δC 199.8, C10), and a terminal methyl group (δH 2.31, 3H, s; δC 27.3, C11). The connection of this side chain to C-3 was confirmed by the HMBC cross-peaks of the olefinic H-8 with C-2 (δC 152.9), C-3 (δC 115.3), C-9 (δC 132.1), and the C-10 carbonyl. The HMBC experiments also showed the correlations of the terminal methyl of this side chain at δH 2.31 (3H, s) with C9 and C-10. A complete analysis of the 2D NMR data of 5 (Figure 2) resulted in its identification as the diphenyl ether shown in Figure 1. Compound 6 was isolated as a yellowish oil and possessed a molecular formula of C23H22O11, as defined by the 13C NMR

and HRESIMS data at m/z 497.1033 [M + Na]+. A comparison of the NMR spectra of 6 and 5 showed that the carbonyl and the terminal methyl group in 5 were replaced by a hydroxycarbonyl carbon at δC 168.3 in 6. This was further confirmed by the HMBC cross-peaks of the two olefinic protons at δH 7.82 (1H, d, J = 16.5 Hz, H-8) and 6.74 (1H, d, J = 16.5 Hz, H-9) with the aromatic C-3 (δC 113.5) and with the C-10 hydroxycarbonyl (δC 168.3) (Figure 2). The other correlations were in agreement with the proposed structure of 6. Therefore, 6, lobariether D, is identified as the new diphenyl ether shown in Figure 1. Compound 7, lobariether E, isolated as a yellowish oil, exhibited a sodium adduct ion at m/z 511.1228 [M + Na]+ in its HRESIMS and was assigned a molecular formula of C24H24O11. A comparison of the NMR spectroscopic data of 7 and 6 showed similarities (Table 2) except for the appearance of an additional methoxy group (δH 3.76, 3H, s; δC 51.7) in 7. The HMBC cross-peaks of the methoxy protons as well as the 265



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two olefinic protons at δH 7.93 (1H, d, J = 16.5 Hz, H-8) and 6.84 (1H, d, J = 16.5 Hz, H-9) with the ester carbonyl C-10 (δC 168.1) supported the connection of the methoxycarbonylethenyl side chain to the A-ring at C-3. A putative biosynthesis pathway for compounds 1−8 is shown in Scheme 1. Methyl β-orcinolcarboxylate (10) may serve as a precursor, which is modified by a sequence of phenolic oxidative coupling, oxidation, acetal formation, and Omethylation,28,30,34 to produce the diphenyl ether lobariether B (4) (i). This compound is susceptible to trans-esterification (intramolecular) involving the A-ring methoxycarbonyl and the B-ring phenolic group to form the corresponding depsidone lobarientalone A (1) (ii).25 The hydroxymethylene group of 4 may then be oxidized to afford a formyl group and finally to an (E)-3-oxobut-1-enyl group to form lobariether C (5) (iii).8,9,35,36 The (E)-3-oxobut-1-enyl group of lobariether C may have been the substrate for an enzymatically induced oxidation to afford lobariether D (6) (iv).37−45 Whether lobariether E (7) occurs naturally in the origin lichen or is merely an artifact formed during the isolation process remains to be determined. The aforementioned report suggested that within the β-orcinol chemosyndrome there was variation in the O-methylation of the hydroxycarbonyl group or in the degree of oxidation of the methyl group.18 A similar O-methylation of the 10-hydroxycarbonyl group of lobariether D (v) or the oxidation of the hydroxymethylene group of lobarientalone A (vi) may be assumed for the biosynthesis of lobariether E (7) or the known depsidone 1,10-di-O-methylstictic acid (8), respectively. In depsidones, the interplanar angle (θ) formed by the A- and B-rings is approximately 125°;22 thus the bulky substituents at C-3 (e.g., −CH2OH) of the A-ring and at C-6′ [e.g., −CH(O)OH] of the B-ring could be in close proximity to form an intramolecular lactonization under biosynthetic conditions. The acetal linkage from C-13 to C-1 in lobarientalone B (2) (vii) or from C-8 to C-9′ in lobariether A (3) (viii) could be formed in a similar manner. Compounds 2, 4, 5, 7, 8, 13, and 14 were evaluated for their in vitro cytotoxic potential against HeLa, HepG2, NCI-H460, and MCF-7 cancer cell lines using the sulforhodamine B method with camptothecin as the positive control. All compounds were inactive.

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voucher specimen (No. US-B034) was deposited at the Herbarium of the Department of Organic Chemistry, University of Science, National University, Ho Chi Minh City, Vietnam. Extraction and Isolation. Before extraction, the lichen was carefully inspected for contaminants. Air-dried parts of L. orientalis (1.4 kg) were ground and extracted with MeOH (4 × 10 L) by the maceration method at ambient temperature, and the filtered solution was evaporated under reduced pressure to afford a MeOH residue (170.0 g). This crude extract was separated by quick column chromatography, first eluted with n-hexane to afford the n-hexane extract (15.5 g), then with a gradient of EtOAc and MeOH (stepwise, 10:0, 9:1, 8:2, and 5:5) to afford four EtOAc fractions, EA1 (13.5 g), EA2 (8.3 g), EA3 (7.7 g), and EA4 (86.5 g), and finally with MeOH to afford the MeOH residue (24.5 g). Fraction EA1 was subjected to silica gel column chromatography, eluted with n-hexane−CHCl3 (3:7) and then with CHCl3, to give 9 (1.30 g) and 10 (100 mg), respectively. Fraction EA2 was subjected to column chromatography, eluted with CHCl3, to afford 11 (800 mg), 8 (32 mg), 1 (30 mg), and 3 (5 mg); with CHCl3−acetone (98:2) to give 5 (20 mg); with CHCl3−acetone (95:5) to give 4 (10 mg); and with CHCl3−acetone (90:10) to give 6 (5 mg) and 7 (7 mg). Fraction EA3 was subjected to silica gel column chromatography, eluted with CHCl3−MeOH (stepwise, 98:2, 95:5, and 90:10), to give four compounds, 12 (20 mg), 13 (100 mg), 14 (200 mg), and 2 (30 mg). To verify that the new metabolites were not artifacts of the isolation process, the extraction of the lichen was repeated. Two samples of the lichen (100 mg each) were cut into small pieces and extracted with acetone or MeOH (20 mL) at ambient temperature for 48 h. The extraction with acetone was repeated once again to obtain the second crop. The filtered solutions were evaporated by a fan at room temperature to afford three concentrated solutions. The MeOH and acetone (first and the second crops) concentrated solutions and eight isolated metabolites (1−8) were spotted on an analytical TLC plate and developed using standard solvents46,47 A (toluene−dioxane− HOAc, 180:45:5), B (n-hexane−Et2O−formic acid, 130:80:20), and C (toluene−HOAc, 170:30). The spots were visualized by UV, and then the plate was sprayed with 5% vanillin in acidic aqueous solution followed by heating (Figures S1−S3, Supporting Information). These compounds were all evident in the acetone extract. Lobarientalone A (1): white, amorphous powder; [α]25 D = +267 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 220 (1.7), 270 (0.7), 310 (0.6) nm; ECD (Δε) (0.02 mg/mL, MeOH) 320 (−1.2), 300 (−2.1), 283 (−2.5), 250 (−1.2) nm; IR (KBr) νmax 3537, 3439, 1740, 1611, 1090 cm−1; 1H and 13C NMR (CDCl3), see Table 1; HRESIMS m/z 425.0842 [M + Na]+ (calcd for C20H18O9Na, 425.0849). Standard TLC Rf values: (A) 0.44; (B) 0.28; (C) 0.30. Vanillin/H2SO4 spray: orange (Figures S1−S3, Supporting Information). Lobarientalone B (2): white, amorphous powder; [α]25 D = +248 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 220 (4.2), 275 (2.2), 340 (1.1) nm; ECD (Δε) (0.1 mg/mL, MeOH) 310 (−13.0), 295 (−17.0), 270 (−7.0), 240 (−7.0) nm; IR (KBr) νmax 3430, 1744, 1613, 1136 cm−1; 1H and 13C NMR (DMSO-d6), see Table 1; HRESIMS m/ z 371.0772 [M + H]+ (calcd for C19H15O8, 371.0767). Standard TLC Rf values: (A) 0.26; (B) 0.14; (C) 0.11. Vanillin/H2SO4 spray: orange (Figures S1−S3, Supporting Information). Lobariether A (3): yellowish oil; [α]25 D = +211 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 220 (2.1), 270 (1.0), 310 (0.7) nm; ECD (Δε) (0.02 mg/mL, MeOH) 320 (−2.2), 300 (−3.1), 278 (−3.9), 250 (−2.9) nm; IR (KBr) νmax 3727, 3631, 1742, 1700, 1606, 1298 cm−1; 1 H and 13C NMR (DMSO-d6), see Table 2; HRESIMS m/z 425.0815 [M + Na]+ (calcd for C20H18O9Na, 425.0849). Standard TLC Rf values: (A) 0.51; (B) 0.40; (C) 0.44. Vanillin/H2SO4 spray: orange (Figures S1−S3, Supporting Information). Lobariether B (4): yellowish oil; [α]25 D = +282 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 238 (2.8), 270 (1.3), 315 (0.8) nm; ECD (Δε) (0.02 mg/mL, MeOH) 320 (−1.3), 300 (−1.8), 285 (−2.1), 250 (−1.5) nm; IR (KBr) νmax 3419, 3377, 1733, 1613, 1309 cm−1; 1H and 13 C NMR (CDCl3), see Table 2; HRESIMS m/z 433.1129 [M − H]− (calcd for C21H21O10, 433.1135). Standard TLC Rf values: (A) 0.37;

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a Kruss digital polarimeter. The UV spectra were recorded on a UV-2550 UV−vis spectrometer (Shimadzu, Kyoto, Japan). The ECD spectra were measured with a JASCO J-815 circular dichroism spectrometer (JASCO, Inc., Tokyo, Japan). The IR spectra were measured on a Bruker Tensor 37 infrared spectrometer. The NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 1H and 125 MHz for 13C). CDCl3 and DMSO-d6 were used both as a solvent and as an internal reference at δH 7.26, 2.50 and δC 77.2, 39.5, respectively. The HRESIMS data were obtained using a Bruker microOTOF Q-II. TLC was carried out on precoated silica gel 60 F254 or silica gel 60 RP-18 F254S (Merck KGaA, Germany). Gravity column chromatography was performed with silica gel 60 (0.040− 0.063 mm, Himedia Laboratories, Mumbai, India). The plates were analyzed under UV light or treated with a solution of 5% vanillin in acidic ethanolic solution followed by heating. Lichen Material. The thalli of the lichen were separated from the bark of various old trees at 2100−2200 m altitude in Bidoup Nui Ba National Park (12°26′ N, 108°30′ E), Dam Rong District, Lam Dong Province, Vietnam, in July−August 2012. The species was determined as Lobaria orientalis (Asahina) Yoshim by Dr. Robert Lücking (Department of Botany, the Field Museum, Chicago, IL, USA). A 266



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(4) Ren, M. R.; Wang, X. Y.; Koh, Y. J.; Hur, J. S. Mycobiology 2012, 40, 1−7. (5) Rankovíc, B. Lichen Secondary Metabolites. Bioactive Properties, and Pharmaceutical Potential; Springer: Heidelberg, 2015. (6) Culberson, C. F. Phytochemistry 1967, 6, 719−725. (7) Culberson, C. F. Bryologist 1969, 72, 19−27. (8) Gonzáles, A. G.; Barrera, J. B.; Pérez, M. R.; Padrón, C. E. H. Biochem. Syst. Ecol. 1994, 22, 583−586. (9) Atalay, F.; Halici, M. B.; Mavi, A.; Ç akir, A.; Odabaşoğlu, F.; Kazaz, C.; Aslan, A.; Küfrevioğlu, Ö .I.̇ Turk. J. Chem. 2011, 35, 647− 661. (10) Pejin, B.; Tommonaro, G.; Iodice, C.; Tesevic, V.; Vajs, V.; De Rosa, S. J. Enzyme Inhib. Med. Chem. 2013, 28, 876−878. (11) Bidussi, M.; Goward, T.; Gauslaa, Y. Botany 2013, 91, 621−630. (12) Safe, S.; Safe, L. M.; Maass, W. S. G. Phytochemistry 1975, 14, 1821−1823. (13) Gauslaa, Y.; Bidussi, M.; Solhaug, K. A.; Asplund, J.; Larson, P. Phytochemistry 2013, 94, 91−98. (14) Wang, X. N.; Zhang, H. J.; Ren, D. M.; Ji, M.; Yu, W. T.; Lou, H. X. Chem. Biodiversity 2009, 6, 746−753. (15) Siegfried, H.; Yoshimura, I. Identification of Lichen Substances; Springer−Verlag: Berlin Heidelberg, 1977. (16) Rojas, I. S.; Hennsen, B. L.; Mata, R. J. Nat. Prod. 2000, 63, 1396−1399. (17) Papadopoulou, P.; Tzakou, O.; Vagias, C.; Kefalas, P.; Roussis, V. Molecules 2007, 12, 997−1005. (18) Elix, J. A.; Wardlaw, J. H. Aust. J. Chem. 2000, 53, 815−818. (19) Elix, J. A.; Kalb, K.; Wardlaw, J. H. Aust. J. Chem. 2003, 56, 315− 317. (20) Shimada, S.; Saitoh, T.; Sankawa, U.; Shibata, S. Phytochemistry 1980, 19, 328−330. (21) Ismed, F.; Dévéhat, F. L.; Delalande, O.; Sinbandhit, S.; Bakhtiar, A.; Boustie, J. Fitoterapia 2012, 83, 1693−1698. (22) Morita, H.; Tsuchiya, T.; Kishibe, K.; Noya, S.; Shiro, M.; Hirasawa, Y. Bioorg. Med. Chem. Lett. 2009, 19, 3679−3681. (23) Gawronski, J. K.; Chen, Q. H.; Geng, Z.; Huang, B.; Martin, M. R.; Mateo, A. I.; Brzostowska, M.; Rychlewska, U.; Feringa, B. L. Chirality 1997, 9, 537−544. (24) Fuska, J.; Uhrin, D.; Proska, B.; Voticky, Z.; Ruppeldt, J. J. Antibiot. 1986, 39, 1605−1608. (25) Kimura, Y.; Yoshinari, T.; Koshino, H.; Fujioka, S.; Okada, K.; Shimada, A. Biosci., Biotechnol., Biochem. 2007, 71, 1896−1901. (26) Zhai, M. M.; Niu, H. T.; Li, J.; Xiao, H.; Shi, Y. P.; Di, D. L.; Crews, P.; Wu, Q. X. J. Agric. Food Chem. 2015, 63, 9558−9564. (27) Berova, N.; Bari, L. D.; Pescitelli, G. Chem. Soc. Rev. 2007, 36, 914−931. (28) Ingolfsdottir, K.; Gissurarson, S. R.; Müller, J. B.; Breu, W.; Wagner, H. Phytomedicine 1996, 2, 243−246. (29) Elix, J. A.; Wardlaw, J. H.; Archer, A. W.; Obermayer, W. Aust. J. Chem. 1999, 52, 717−719. (30) Lang, G.; Cole, A. L. J.; Blunt, J. W.; Robinson, W. T.; Munro, M. H. G. J. Nat. Prod. 2007, 70, 310−311. (31) Duong, T. H.; Chavasiri, W.; Boustie, J.; Nguyen, K. P. P. Tetrahedron 2015, 71, 9684−9691. (32) Huynh, B. L. C.; Le, D. H.; Takenaka, Y.; Tanahashi, T.; Nguyen, K. P. P. Magn. Reson. Chem. 2016, 54, 81−87. (33) Feigel, M. J. Mol. Struct.: THEOCHEM 1996, 336, 83−88. (34) Okoye, F. B. C.; Lu, S.; Nworu, C. S.; Esimone, C. O.; Proksch, P.; Chadli, A.; Debbab, A. Tetrahedron Lett. 2013, 54, 4210−4214. (35) Piattelli, M.; Giudici de Nicola, M. Phytochemistry 1968, 7, 1183−1187. (36) Bo, L.; Zhongwen, L.; Handong, S. Act. Bot. Yunn. 1990, 12, 447−451. (37) Baker, C.; Elix, J. A.; Murphy, D. P. H.; Kurokawa, S.; Sargent, M. V. Aust. J. Bot. 1973, 21, 137−140. (38) Keogh, M. F. Phytochemistry 1977, 16, 1102. (39) Chester, D. O.; Elix, J. A. Aust. J. Chem. 1980, 33, 1153−1156. (40) Elix, J. A.; Evans, J. E.; Parker, J. L. Aust. J. Chem. 1987, 40, 2129−2131.

(B) 0.19; (C) 0.19. Vanillin/H2SO4 spray: orange-red (Figures S1−S3, Supporting Information). Lobariether C (5): yellowish oil; [α]25 D = +286 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 218 (4.2), 235 (4.0), 270 (2.5), 309 (2.4) nm; ECD (Δε) (0.02 mg/mL, MeOH) 320 (−1.2), 300 (−1.7), 285 (−1.9), 230 (−1.9) nm; IR (KBr) νmax 3424, 1733, 1607, 1299 cm−1; 1 H and 13C NMR (CDCl3), see Table 2; HRESIMS m/z 495.1286 [M + Na]+ (calcd for C24H24O10Na, 495.1267). Standard TLC Rf values: (A) 0.46; (B) 0.20; (C) 0.24. Vanillin/H2SO4 spray: orange-red (Figures S1−S3, Supporting Information). Lobariether D (6): yellowish oil; [α]25 D = +238 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 220 (4.2), 330 (1.2) nm; ECD (Δε) (0.1 mg/mL, MeOH) 305 (−6.0), 270 (−6.0), 240 (−6.0) nm; IR (KBr) νmax 3418, 1725, 1606, 1294 cm−1; 1H and 13C NMR (CDCl3), see Table 2; HRESIMS m/z 497.1033 [M + Na]+ (calcd for C23H22O11Na, 497.1059). Standard TLC Rf values: (A) 0.36; (B) 0.24; (C) 0.22. Vanillin/H2SO4 spray: orange (Figures S1−S3, Supporting Information). Lobariether E (7): yellowish oil; [α]25 D = +208 (c 0.002, MeOH); UV (MeOH) λmax (log ε) 233 (5.0), 265 (3.7), 300 (2.7) nm; ECD (Δε) (0.1 mg/mL, MeOH) 310 (−2.5), 270 (−2.5), 240 (−3.0) nm; IR (KBr) νmax 3415, 1730, 1605, 1287 cm−1; 1H and 13C NMR (CDCl3), see Table 2; HRESIMS m/z 511.1228 [M + Na]+ (calcd for C24H24O11 Na, 511.1216). Standard TLC Rf values: (A) 0.46; (B) 0.34; (C) 0.34. Vanillin/H2SO4 spray: orange (Figures S1−S3, Supporting Information). Cytotoxicity Assay. Compounds 2, 4, 5, 7, 8, 13, and 14 were subjected to cytotoxic evaluation against HepG2 (liver hepatocellular carcinoma), MCF-7 (human breast cancer), NCI-H460 (human lung cancer), and HeLa (human epithelial carcinoma) cell lines. The tested samples were performed at a concentration of 100 μg/mL using the sulforhodamine B assay with camptothecin as the positive control. The details were similar to those presented in our previous paper.31

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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.6b00465. IR, HRMS, and 1H, 13C, and 2D NMR spectra of compounds 1−8 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +84-8-01226966660. E-mail (P. P. K. Nguyen): [email protected]. ORCID

Phung P. K. Nguyen: 0000-0003-0868-2213 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Robert Lücking, Department of Botany, the Field Museum, 1400 South Lake Shore Drive, Chicago, Illinois 60605-2496, USA, for the determination of the scientific name for the lichen. The research was funded by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number #104.01−2013.17.

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REFERENCES

(1) Shrestha, G.; St. Clair, L. L. Phytochem. Rev. 2013, 12, 229−244. (2) Moncada, B.; Lücking, R.; Betancourt, M. L. Lichenologist 2013, 45, 203−263. (3) Yoshimura, I. Hattori Botanic. Lab. 1971, 34, 231−364. 267



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Phenolic Compounds from the Lichen Lobaria orientalis - Journal of

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