Letter - spectral assignments Received: 15 March 2015 Revised: 14 July 2015 Accepted: 21 July 2015 Published online in Wiley Online Library: 25 August 2015 (wileyonlinelibrary.com) DOI 10.1002/mrc.4316 New phenolic compounds from the lichen Parmotrema praesorediosum (Nyl.) Hale (Parmeliaceae) Bui Linh Chi Huynh,a Duy Hoang Le,b Yukiko Takenaka,b Takao Tanahashib and Kim Phi Phung Nguyenc* Introduction Lichens, symbiotic combination of fungi and algae, comprise more than 20 000 species that are found in most of the environmental habitats from the tropics to polar regions Lichens produce a great variety of metabolites, most of them occur only in lichens and the others are also present in fungi and higher plants Characteristic secondary metabolites of lichens are depsides, depsidones, diphenyl ethers, benzofuran, usnic acid, and anthraquinone derivatives.[1–3] Parmotrema is one of the largest genera of the family Parmeliaceae This genus of foliose lichen is widely distributed in tropical regions and composed of c 350 species worldwide.[4] Previous phytochemical examination on Parmotrema praesorediosum (Nyl.) Hale, collected on a betel nut tree, in southern Thailand, revealed that this species contained some lactone fatty acids.[5] Sample collected in south Korea contained atranorin, chloroatranorin, and fatty acids, i.e protopraesorediosic acid and praesorediosic acid.[4] In the course of our systematic research on lichen substances from the Vietnamese flora, we have examined P praesorediosum (Nyl.) Hale that is distributed in the south of Vietnam In our previous study on this species, nine compounds, including methyl haematommate, butyl haematommate, methyl chlorohaematommate, methyl β-orsellinate, methyl divaricatinate, atranol, atranorin, (+)-(12R)isousnic acid, and (+)-(12R)-usnic acid were isolated.[6] Further chemical investigation on this species by using modern separation techniques has led to the isolation of seven novel phenolic compounds, four diphenyl ethers (1–4) and three phthalide derivatives (5–7) In this paper, we report the isolation and structure elucidation of these compounds carbonyl (1730 cmÀ1), and aromatic (1645, 1455 cmÀ1) functionalities Its 1H NMR spectrum showed one hydrogen bonded hydroxyl group at δ 12.06 (1H, s), a formyl group at δ 10.39 (1H, d, J = 1.0 Hz), an aromatic proton at δ 6.55 (1H, brs), two meta-coupled aromatic protons at δ 6.31 (1H, d, J = 2.5 Hz) and 6.17 (1H, brd, J = 2.5 Hz), a methoxy group at δ 3.50 (3H, s), and two methyl groups at δ 2.22 (3H, d, J = 0.5 Hz) and 2.00 (3H, brs) The combination of 13C and DEPT NMR spectra of (Table 1) revealed 17 carbons including a formyl group (δ 193.6), a methoxycarbonyl [δ 167.0 (―COO), 52.4 (―OCH3)], 12 aromatic carbons (δ 102–164), five of which were oxygenated, and two methyl groups (δ 20.8 and 17.0) Detailed analysis of HSQC, HMBC, and NOESY spectra identified two aryl units, A-ring and B-ring The HMBC spectrum (Fig 2) showed the correlations from the chelated hydroxyl group (δ 12.06, 4-OH) to carbon signals at δ 110.6 (C-3), 164.0 (C-4), and 113.9 (C-5) and from the aldehydic proton (δ 10.38) to C-3 and C4 The aromatic singlet proton (δ 6.55, H-5) showed cross-peaks with C-1 (δ 114.9), C-3, C-4, and C-9 (δ 20.8), and three protons of the methyl group (δ 2.22, H3-9), in turn, were correlated with C-1, C-5, and C-6 (δ 147.7) The methoxy signal showed HMBC crosspeak with the carboxyl carbon at δ 167.0 and NOESY correlation with H3-9, implying that the methoxycarbonyl group was situated at C-1 Furthermore, C-2 was supposed to be oxygenated from its chemical shift (δ 158.1) in the 13C NMR spectrum Therefore, the A-ring of was established as 3-formyl-4-hydroxy-1-methoxycarbonyl-6methyl-2-oxygenated benzene The second aryl unit, B-ring, was a tetrasubstituted benzene core The HMBC spectrum of showed correlations from a meta-coupled aromatic proton at δ 6.17 (H-1′) to carbon signals at δ 153.3 (C-2′), 102.1 (C-3′), 136.3 (C-5′), and 17.0 (C-7′), from another proton at δ 6.31 (H-3′) to C-1′ (δ 109.5), C-2′, C-4′ (δ 148.8), and C-5′, and from methyl protons (H3-7′) to C-1′, C-5′, and C-6′ (δ 131.1) Therefore, Results and discussion Magn Reson Chem 2016, 54, 81–87 * Correspondence to: Nguyen Kim Phi Phung, Department of Organic Chemistry, University of Science, National University—Ho Chi Minh City, 227 Nguyen Van Cu Str., Dist 5, Ho Chi Minh City, Vietnam E-mail: kimphiphung@yahoo.fr a Department of Science, Dong Nai University, Vietnam b Department of Organic Chemistry, Kobe Pharmaceutical University, Japan c Department of Organic Chemistry, University of Science, National University—Ho Chi Minh City, Vietnam Copyright © 2015 John Wiley & Sons, Ltd 81 The thalli of P praesorediosum were extracted with MeOH at room temperature A combination of chromatographic fractionation of the extract led to the isolation of seven phenolic compounds (1–7) (Fig 1) Their structures were elucidated as the following Compound was obtained as a yellow solid Its molecular formula was established as C17H16O7 through the protonated molecule at m/z 333.0970 [M + H]+ in the HR-ESI-MS spectrum IR absorptions implied the presence of hydroxyl (3383 cmÀ1), ester B L C Huynh et al Figure Structures of isolated compounds 1–7 82 the B-ring of was established as 2′,4′,5′-trioxygenated benzene bearing a methyl group at 6′ Comparison of the chemical shifts of C-1′–C-6′ of 2,4-dihydroxy6-methylphenoxy part with those of related compounds suggested that the A-ring was linked to the B-ring through an ether linkage between C-2 and C-5′.[7,8] This connection was well supported by the key NOESY correlation between H-8 of the A-ring and H3-7′ of the B-ring Thus, compound was characterized as methyl 2-(2,4dihydroxy-6-methylphenoxy)-3-formyl-4-hydroxy-6methylbenzoate and named praesorether A Compound was also obtained as a yellow solid Its molecular formula was determined as C27H26O11 from the HR-ESI-MS spectrum The 1H NMR spectral data of were similar to those of (Table 1) except for the absence of two meta-coupled aromatic protons of B-ring and the presence of two newly appeared singlets of aromatic protons and some additional signals, i.e one methoxy group (δ 3.88, 3H, s), one methylene group (δ 3.98 and 3.97, each 1H, br s), and one methyl group (δ 2.58, 3H, s) The 13C-NMR spectrum of exhibited, besides the signals because of the same A and B rings as 1, signals for one methoxycarbonyl group (δ 172.4, 52.2), six aromatic carbons including two oxygenated carbons [δC 161.5 (C-2″), 160.0 (C-4″)], three quaternary carbons [δC 142.5 (C-6″), 119.5 (C-5″), 109.4 (C-1″)], and one CH [δC 101.5 (C-3″)], one methylene carbon (δ 20.7) and one methyl carbon (δ 19.1) These data indicated the presence of a third aromatic C-ring linked to the B-ring in This C-ring was established as the 2″,4″-dihydroxy1″-methoxycarbonyl-6″-methylphenyl moiety with a methylene group at C-5″ by the analysis of its HMBC spectrum, which showed the correlations from the methyl protons (δ 2.58, H3-8″) to C-1″, C-5″, and C-6″, from the methylene (H2-8″) to C-4″, C-5″, C-6″, from an aromatic singlet proton (δ 6.33, H-3″) to C-1″, C-2″, C-4″, and C-5″, and wileyonlinelibrary.com/journal/mrc from the hydroxyl (δ 10.56, 2″-OH) to C-1″, C-2″, and C-3″ The substitution pattern of the ring C was further supported by the ROESY correlation between H3-8″ and H2-8′ The HMBC experiments showed that these methylene protons (H2-8′) correlated to aromatic carbons of B-ring at δ 153.3 (C-2′), 113.1 (C-3′), and 149.2 (C-4′) From these findings, the methylene carbon of the C-ring was linked to the B-ring at C-3′ (Fig 2) Consequently, compound was established as a new diphenyl ether derivative and named praesorether B Compound was isolated as a yellow solid Its molecular formula was determined as C29H26O14 from the HR-ESI-MS spectrum that meant compound contained two carbon and three oxygen atoms more than that of The 1H NMR spectral data (Table 1) of closely resembled those of 2, suggesting that they had the same basic framework except for the lack of one aromatic methine proton and one methyl group and the appearance of an acetalic proton at δ 5.20 and a methoxy group at δ 3.13 The comparison of 13C NMR data of these two compounds showed that also possessed three aromatic rings connected thorough an ether linkage and a methylene bridge as in 2, but it lacked one methyl group and contained three more carbon atoms including one carboxyl carbon (δ 169.6), one acetalic carbon (δ 101.6), and one methoxy carbon (δ 56.8), implying the presence of a lactone ring in Although the HMBC spectrum could not afford further information relating to the position of this lactone ring, the ROESY correlations of the methoxy protons (7′-OCH3) with the acetalic proton (H-7′) and also with the formyl proton at δ 10.12 (H-8) of the Aring indicated the γ-lactone ring was fused to the B-ring at C-1′ and C-6′ (Fig 3) Complete analysis of the 2D NMR data for resulted in its formulation as shown, and it was named praesorether C Copyright © 2015 John Wiley & Sons, Ltd Magn Reson Chem 2016, 54, 81–87 New phenolic compounds from the Parmotrema praesorediosum Table NMR data for compounds 1À4 1a No 2b 3a 4b Moiety a δH 4-OH 7-OCH3 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 6.55 J (Hz) δC brs 114.9 158.1 110.6 164.0 113.9 147.7 167.0 193.6 20.8 10.39 2.22 12.06 3.50 6.17 d (1.0) d (0.5) s s brd (2.5) 6.31 d (2.5) 2.00 brs 9′ 7′-OCH3 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 2″-OH 7″-OCH3 52.4 109.5 153.3 102.1 148.8 136.3 131.1 17.0 δH J (Hz) 6.52 d (0.5) 10.44 2.15 12.11 3.18 6.24 brs s s s s 1.99 3.97 3.98 s brs brs 6.33 2.58 10.56 3.88 s s s s δC 115.5 158.9 111.0 164.3 112.9 148.2 166.6 195.6 20.5 52.0 108.9 153.3 113.1 149.2 135.8 129.9 16.7 20.7 109.4 161.5 101.5 160.0 119.5 142.5 172.4 19.1 52.2 δH J (Hz) δC 6.71 s 10.12 2.31 11.92 3.23 s s brs s 116.5 156.4 111.8 163.6 116.2 148.1 165.8 193.7 21.2 5.20 4.05 s brs 3.13 s 52.2 103.4 153.1 117.6 151.7 134.2 129.2 101.6 20.0 2.61 s 169.6 56.8 107.5 162.4 102.0 159.5 117.1 142.6 172.0 19.3 3.91 s 52.0 6.39 s δH Moiety b J (Hz) δC 6.48 s 10.45 2.11 12.09 3.00 6.22 s s brs s s 116.0 159.2 111.1 164.1 112.4 148.0 167.0 195.7 20.5 2.08 3.81 s s 51.9 108.9 152.7 114.9 149.1 136.3 130.5 16.6 19.3 brs brs 152.9 112.5 156.5 119.4 137.8 109.0 20.8 6.24 2.38 δH J (Hz) δC 6.47 s 10.43 2.13 12.09 3.00 6.16 s s brs s s 115.6 159.6 111.0 164.2 112.7 148.2 166.8 195.7 20.5 1.97 3.88 3.89 s brs brs 51.8 108.2 153.6 114.1 150.0 135.8 129.4 16.7 21.6 a CDCl3 acetone-d6 b Magn Reson Chem 2016, 54, 81–87 (C-3′a), 149.1 (C-4′a), and 152.7 (C-2′a) of the Ba-ring and to carbon signals at δ 112.5 (C-2″), and 152.9 (C-1″) of the C-ring, as well as correlations of the second methylene protons (δ 3.88 and 3.89, H2-8′b) to carbon signals at δ 114.1 (C-3′b), 150.0 (C-4′b), and 153.6 (C-2′b) of the Bb-ring and to signals at δ 119.4 (C-4″), 137.8 (C-5″) and 156.5 (C-3″) of the C-ring Thus, the first methylene carbon linked the C-ring to the Ba-ring at C-2″ and C-3′a and the second methylene carbon linked the C-ring to the Bb-ring at C-4″ and C-3′b This was further supported by the ROESY cross peak between H2-8′b (δ 3.88 and 3.89) and H3-7″ (δ 2.38) (Fig 3) These information fully established the chemical structure of compound as shown and it was named praesorether D Compound was obtained as a white amorphous solid Its molecular formula C12H14O6 was deduced from the protonated molecule [M + H]+ at m/z 255.0862 in the HR-ESI-MS spectrum The IR spectrum showed characteristic absorptions for a hydroxyl group (3243 cmÀ1), a lactone group (1785 cmÀ1) and substituted aromatic system (1630 and 1363 cmÀ1) The 1H NMR spectrum of displayed signals of three methoxy groups at δ 3.53, 3.62, and 3.86 (each 3H, Copyright © 2015 John Wiley & Sons, Ltd wileyonlinelibrary.com/journal/mrc 83 Compound was isolated as a yellow solid Its molecular formula was determined as C43H40O16 from the HR-ESI-MS The 1H NMR spectrum of (Table 1) was similar to that of 1; however, all signals ascribable to A and B-rings appeared in duplicate, and furthermore some additional signals of one aromatic methine proton (δ 6.24, 1H, s), two methylene protons (δ 3.89 and 3.88, each 1H, s; 3.81, 2H, s), and protons of a methyl group (δ 2.38, 3H, s) were observed The same observation was also recorded for the 13C and DEPT NMR spectra of with the appearance of six more aromatic carbons (δ 156.5, 152.9, 137.8, 119.4, 112.5, and 109.0), two methylenes (δ 21.6, 19.3), and a methyl (δ 20.8) These spectral features suggested that composed of two sets of praesorether A (1) basic skeleton (parts a and b) and a fifth aromatic ring C The position of functional groups in Aa, Ba, Ab, Bb, and C-rings was confirmed by analysis of HSQC, HMBC, and ROESY correlations The connection of the two units, Ba and Bb-rings of with the fifth aromatic ring C through two methylene groups, was elucidated by the HMBC spectrum with the correlations of the first methylene protons (δ 3.81, H2-8′a) to carbon signals at δ 114.9 B L C Huynh et al Figure HMBC and NOESY/ROESY correlations of and s), two methylene protons at δ 4.84 and 4.88 (each 1H, d, J = 14.0 Hz, H2-8), an aromatic proton at δ 6.88 (1H, s, H-7), an acetalic methine proton at δ 6.33 (1H, s, H-3), and a phenolic hydroxyl proton at δ 9.08 (1H, s, 4-OH) The combination of 13C and DEPT NMR spectra of revealed 12 carbons including one carboxyl carbon (δ 168.8), one acetalic methine carbon (δ 102.2), five aromatic quaternary carbons (δ 159.7, 153.3, 128.6, 124.5, and 116.1), one aromatic CH carbon (δ 97.6), one oxymethylene carbon (δ 70.0), and three methoxy groups (δ 59.3, 56.3, and 56.1) (Table 2) These spectral data suggested that could be a 3-oxyphthalide with three substituents on the benzene ring (Fig 4) The HMBC experiments showed the correlations from the methoxy protons at δ 3.53 to a methylene group (δ 70.0, C-8) and from the methylene protons (H2-8) to two oxygenated aromatic carbons (δ 159.7, C-6; 153.3, C-4), one aromatic quaternary carbon (δ 116.1, C-5), and also to this methoxy carbon (δ 59.3), indicating the methoxymethyl group at C-5 The sole aromatic proton at δ 6.88 was located at C7 by its HMBC correlations with the carboxyl carbon (δ 168.8, C-1) and other aromatic carbons (δ 159.7, C-6; 124.5, C-3a; and 116.1, C-5) The other substituents, methoxy and phenolic hydroxyl groups were located at C-6 and C-4, respectively, by the analysis of 2D NMR spectra (HMBC and NOESY) Finally, the HMBC correlations of the acetalic methine proton at δ 6.33 with the carboxyl carbon (C-1) and a methoxy carbon (δ 56.3) confirmed the methoxy group at C-3 on the five-member ring lactone The absolute configuration of C-3 was not determined Thus, compound was assigned as 4-hydroxy-3,6-dimethoxy-5-methoxymethylphthalide and named praesalide A Compounds and 7, designated praesalides B and C, were phthalide derivatives closely related to The HR-MS measurements of and established the molecular formulas of C13H16O6 and C14H18O6 The 1H and 13C NMR data of and (Table 2) were similar to those of except for the presence of an ethoxy group at C-8 in and two ethoxy groups at C-3 and C-8 in instead of methoxy groups in This was supported by the analysis of their 2D NMR (COSY, HSQC, HMBC, and NOESY) spectral features These results suggested the structures of and as 3-ethoxy-4hydroxy-6-methoxy-5-methoxymethylphthalide and 3-ethoxy-5ethoxymethyl-4-hydroxy-6-methoxyphthalide, respectively Experimental General experimental procedures The NMR spectra were measured on a Varian NMR System-500 or INOVA-500 spectrometer, at 500 MHz for 1H NMR and 125 MHz for 13 C NMR The HR-ESI-MS were recorded on an Exactive mass spectrometer (Thermo Fisher Scientific) The optical rotations were measured on a Jasco DIP-370 digital polarimeter The IR spectra were measured on Shimadzu FTIR-8200 infrared spectrophotometer TLC was carried out on silica gel 60F254 or silica gel 60 RP-18 F254S (Merck) and spots were visualized by spraying with a solution of 5% vanillin in ethanol, followed by heating at 100 °C Gravity column chromatography was performed with silica gel 60 (0.040– 0.063 mm, Merck) 84 Figure HMBC and ROESY correlations of and wileyonlinelibrary.com/journal/mrc Copyright © 2015 John Wiley & Sons, Ltd Magn Reson Chem 2016, 54, 81–87 New phenolic compounds from the Parmotrema praesorediosum Table NMR data for compounds 5–7 (CDCl3) No 3a 7a 1′ 2′ 1″ 2″ 4-OH δH J (Hz) 6.33 s 6.88 s 4.84 4.88 3.86 3.62 d (14.0) d (14.0) s s 3.53 s 9.08 s δC 168.8 102.2 124.5 153.3 116.1 159.7 97.6 128.6 70.0 56.1 56.3 59.3 δH J (Hz) 6.40 s 6.87 s 4.83 4.88 3.85 3.86 3.94 1.33 3.53 9.03 d (14.0) d (14.0) s dq (9.5, 7.0) dq (9.5, 7.0) t (7.0) s δC 169.0 101.5 124.9 153.3 116.0 159.6 97.5 128.6 70.0 56.1 65.3 15.1 59.3 brs δH J (Hz) 6.40 s 6.86 s 4.86 4.92 3.85 3.86 3.94 1.33 3.70 1.32 9.33 d (14.0) d (14.0) s dq (9.5, 7.0) dq (9.5, 7.0) t (7.0) q (7.0) t (7.0) s δC 169.0 101.5 124.8 153.3 116.2 159.5 97.4 128.5 68.1 56.1 65.3 15.2 67.6 15.0 Plant material The lichen thalli of P praesorediosum were collected on the bark of Dipterocarpus sp at Tan Phu forest, Dong Nai province, Vietnam in June 2009 The geographical location where the lichen was collected is at an altitude of 110 m, 11°20′–11°50′ N and 107°09′–107° 35′ E The botanical species of P praesorediosum (Nyl.) Hale (synonym of Parmelia praesorediosa Nyl.) was identified by MSc Vo Thi Phi Giao, Faculty of Biology, University of Science, National University—Ho Chi Minh City A voucher specimen (No US-B020) was deposited in the Herbarium of The Department of Organic Chemistry, Faculty of Chemistry, University of Science, National University—Ho Chi Minh City, Vietnam Figure HMBC and NOESY correlations of Magn Reson Chem 2016, 54, 81–87 Extraction and isolation The thallus material (5.0 kg) was washed under flow of tap water and then was air-dried at ambient temp to obviate thermally induced decomposition prior to be ground into a fine powder The ground powder sample (3.0 kg) was macerated by methanol at room temperature to afford a crude methanol extract (450 g) This crude one (450 g) was applied to silica gel solid phase extraction, successively eluted with the following solvents: petroleum ether (60–90 °C) (PE), chloroform (C), ethyl acetate (EA), acetone (A), and methanol (M) to afford corresponding extracts: extract PE (40 g), extract C (105 g), extract EA (50 g), extract A (45 g), and extract M (37 g) The chloroform extract (105 g) was subjected to silica gel column chromatography, eluted by the solvent system of petroleum ether– ethyl acetate with increasing ethyl acetate to give 23 fractions from C1 to C23 Fraction C19 (6.1 g) was applied on silica gel column and eluted with a gradient solvent system of chloroform–acetone (95:5) to give three fractions (C19a, C19b, and C19c) Fraction C19a (1.0 g) was silica gel rechromatographed, eluted with chloroform–acetone (98:2) and subjected to pre TLC using chloroform–methanol (9:1 and 95:5) as eluent to afford (5.0 mg) Fraction C19b (3.2 g) was silica gel rechromatographed, eluted with chloroform–acetone (98:2) to give six fractions (C19ba to C19bf) Fraction C19bc (454.3 mg) Copyright © 2015 John Wiley & Sons, Ltd wileyonlinelibrary.com/journal/mrc 85 All NMR experiments were acquired at ambient temperature Chemical shifts are expressed in ppm with reference to the internal TMS (0.000) H and 13C NMR spectra were obtained using a Varian NMR System-500 or INOVA-500 spectrometer 1H spectra: On a Varian NMR System-500 spectrometer, spectral width (SW) 8012.8 Hz, acquisition time (AT) 2.045 s, number of data points (NP) 32 768 K, filter band width (FB) 4000 Hz, block size (BS) 32, steady-state transients (SS) 0, relaxation delay (D1) s, spectrometer frequency (SF) 499.73 MHz, pulse 90 width (PW) 7.9 μs, temperature (TE) 25 °C, line broadening (LB) not use; On a INOVA-500, SW 7996.8 Hz, AT 4.097 s, NP 65 530 K, FB 4000 Hz, BS 32, SS 1, D1 s, SF 499.83 MHz, PW 11.5 μs, TE 25 °C, LB not use 13C spectra: On a Varian NMR System-500 spectrometer, SW 31 250.0 Hz, AT 1.049 s, NP 65 536 K, FB 17 000 Hz, BS 32, D1 s, SF 125.671 MHz, PW 9.0 μs, TE 25 °C, LB 0.5 Hz; On a INOVA-500, SW 30 165.9 Hz, AT 1.3 s, NP 78 460 K, FB 17 000 Hz, BS 32, D1 1.7 s, SF 125.694 MHz, PW 17.5 μs, TE 25 °C, LB 0.5 Hz HSQC spectra were done using the INOVA-500: D1 1.301 s, AQ 0.199 s, width 7303.9 Hz, 2D width 30 165.9 Hz, TE 25 °C, FT size 4096 × 2048 HMBC was done using the INOVA-500: D1 1.000 s, AQ 0.128 s, width 7383.5 Hz, 2D width 30 165.9 Hz, TE 25 °C, FT size 2048 × 2048 ROESY: D1 1.000 s, AQ 0.140 s, width 7292.6 Hz, 2D width 7292.6 Hz, TE 25 °C, FT size 2048 × 2048 B L C Huynh et al was subjected to pre TLC (chloroform–methanol, 95:5) to afford (28.1 mg) Fraction C19ba (169.6 mg) was subjected to pre TLC (chloroform–methanol, 95:5, 9:1 and n-hexane–diethyl ether, 5:5) to afford (18.7 mg) and (7.0 mg) Fraction C20 (23.9 g) was repeatedly subjected to silica gel column chromatography, eluted with chloroform–methanol (10:0–9:1) to obtain eight fractions (from C20a to C20h) The fraction C20c (5.8 g) was subjected to silica gel chromatography, eluting with solvent of chloroform–methanol to get six fractions (from C20ca to C20cf) Fractions C20cb (979.3 mg) was silica gel rechromatographed, eluted with n-hexane–diethyl ether and continuously subjected to pre TLC (n-hexane–diethyl ether (2:8) and chloroform–methanol (98:2)) to afford three compounds (8.0 mg), (71.7 mg), and (6.2 mg) Praesorether A (1) [methyl 2-(2,4-dihydroxy-6-methylphenoxy)-3-formyl-4-hydroxy-6-methylbenzoate] Yellow solid IR (KBr) νmax cmÀ1: 3383, 1730, 1645, 1455, 1265 HRESI-MS m/z 333.0970 [M + H]+, (Calcd for C17H16O7 + H, 333.0975) and m/z 355.0789 [M + Na]+, (Calcd for C17H16O7 + Na, 355.0794) H and13C NMR (CDCl3) data see Table HMBC and NOESY see Fig Praesorether B (2) Yellow solid IR (KBr) νmax cmÀ1: 3371, 1730, 1706, 1646, 1465, 1263 HR-ESI-MS m/z 527.1544 [M + H]+, (Calcd for C27H26O11 + H, 527.1554) and m/z 549.1363 [M + Na]+, (Calcd for C27H26O11 + Na, 549.1373) 1H and 13C NMR (acetone-d6) data see Table HMBC and ROESY see Fig Praesorether C (3) Yellow solid [α]D23 + 3.5 (c 0.68, CHCl3) IR (KBr) νmax cmÀ1: 3394, 1732, 1651, 1455, 1276 HR-ESI-MS m/z 599.1396 [M + H]+, (Calcd for C29H26O14 + H, 599.1402) and m/z 621.1213 [M + Na]+ (Calcd for C29H26O14 + Na, 621.1220) 1H and 13CNMR (CDCl3) data see Table HMBC and ROESY see Fig Conclusions From the chloroform soluble fraction of the methanol extract of the lichen P praesorediosum (Nyl.) Hale seven novel compounds are isolated, including four diphenyl ethers praesorether A (1), praesorether B (2), praesorether C (3), and praesorether D (4), as well as three stable phthalides praesalide A (5), praesalide B (6), and praesalide C (7) Their chemical structures were established primarily by NMR and MS spectroscopy Diphenyl ethers connected with benzyl moiety such as 2–4 are quite unique and have not been isolated from the lichens with exception of a depsidone furfuric acid of Pseudevernia furfuracea.[9] The one-pot synthesis of furfuric acid by the acid-catalysed alkylation led to the proposal that this compound is an artifact formed during isolation procedure.[10] Nevertheless, it is not excluded compounds 2–4 could be genuine lichen compounds Therefore, it is of great interest to study the mechanism of their formation A literature search revealed that just four natural phthalide derivatives have been reported from lichens: Buellolide and canesolide from Buellia canescens; 5,7-dihydroxy-6-methylphthalide from Anamylopsora pulcherrima; and 7-hydroxy-5-methoxy-6methylphthalide from Usnea aciculifera.[11–13] However, this type of compound was often found from fungi, such as rubralide C closely related to 5, which has been isolated from the marine sediment-derived fungus Penicillium pinophilum SD-272.[14] The majority of secondary metabolites found in lichens are produced by the fungal partner However, most of secondary metabolites known from lichens, so-called lichen substances such as depsides, depsidones, xanthones, diphenyl ethers, and pulvinic acid are unique to these organisms and related to the symbiosis as a small minority occur in other fungi or higher plants.[2,15] The isolation of the phthalides of unusual compound classes could offer some insights to the secondary metabolites of the lichens, and lichens could be a potent source for searching unusual compounds Acknowledgements Praesorether D (4) Yellow solid IR (KBr) νmax cmÀ1: 3366, 1706, 1645, 1458, 1268 HRESI-MS m/z 813.2394 [M + H]+, (Calcd for C43H40O16 + H, 813.2396) and m/z 835.2169 [M + Na]+, (Calcd for C43H40O16 + Na, 835.2214) H and 13C NMR (acetone-d6) data see Table HMBC and NOESY see Fig Praesalide A (5) (4-hydroxy-3,6-dimethoxy-5-methoxymethylphthalide) White amorphous solid [α]D26 + 24.7 (c 0.23, CHCl3) IR (KBr) νmax cmÀ1: 3243, 1785, 1630, 1363 HR-ESI-MS m/z 255.0862 [M + H]+, (Calcd for C12H14O6 + H, 255.0869) and m/z 277.0680 [M + Na]+, (Calcd for C12H14O6 + Na, 277.0688) 1H and 13C NMR (CDCl3) data see Table HMBC and NOESY see Fig We are grateful to the Government of Vietnam (Project 322, MOET) for the fellowship to B.L.C.H We are grateful to MSc Vo Thi Phi Giao for identification of the Parmotrema specimens Thanks are also due to Dr C Tode (Kobe Pharmaceutical University) for 1H and 13C NMR spectra, and to Dr A Takeuchi (Kobe Pharmaceutical University) for mass spectral measurements This research was financially supported by Vietnam’s National Foundation for Science and Technology Development (NAFOSTED) grant #104.01-2013.17 Conflict of interest The authors have declared that there is no conflict of interest Praesalide B (6) (3-ethoxy-4-hydroxy-6-methoxy-5-methoxymethylphthalide) White amorphous solid [α]D25 À2.0 (c 1.33, CHCl3) IR (KBr) νmax cmÀ1: 3240, 1769, 1625, 1341 HR-ESI-MS m/z 269.1018 [M + H]+, (Calcd for C13H16O6 + H, 269.1025) and m/z 291.0837 [M + Na]+ (Calcd for C13H16O6 + Na, 291.0845).1H and 13C NMR (CDCl3) data see Table Praesalide C (7) (3-ethoxy-5-ethoxymethyl-4-hydroxy-6-methoxyphthalide) 86 White amorphous solid [α]D26 + 9.5 (c 0.61, CHCl3) IR (KBr) νmax cmÀ1: 3235, 1766, 1624, 1339 HR-ESI-MS m/z 283.1174 [M + H]+ (Calcd for C14H18O6 + H, 283.1182) and m/z 305.0992 [M + Na]+ (Calcd for C14H18O6 + Na, 305.1001) 1H and 13C NMR (CDCl3) data see Table wileyonlinelibrary.com/journal/mrc References [1] V Ahmadjian, M E Hale, The Lichens, Academic Press, New York and London, 1973 [2] T H Nash III, Lichen Biology, 2nd edn, Cambridge Univ Press, Cambridge, New York, 2008 [3] S Huneck, I Yoshimura, Identification of Lichen Substances, SpringerVerlag Berlin Heidelberg, New York, 1996 [4] U Jayalal, P K Divakar, S Joshi, S.-O Oh, Y J Koh, J.-S Hur Mycobiology 2013, 41, 25–36 [5] F David, J A Elix, M W D Samsudin Aust J Chem 1990, 43, 1297–1300 Copyright © 2015 John Wiley & Sons, Ltd Magn Reson Chem 2016, 54, 81–87 New phenolic compounds from the Parmotrema praesorediosum [6] B L C Huynh, T H Duong, T Tanahashi, K P P Nguyen Vietnam J Chem 2010, 48, 332–337 [7] P Chomcheon, S Wiyakrutta, N Sriubolmass, N Ngamrojanavanich, S Kengtong, C Mahidol, S Ruchirawat, P Kittakoop Phytochemistry 2009, 70, 407–413 [8] F B C Okoye, S Lu, C S Nworu, C O Esimone, P Proksch, A Chadli, A Debbab Tetrahedron Lett 2013, 54, 4210–4214 [9] J Gunzinger, R Tabacchi, Helv Chim Acta 1985, 68, 1936–1939 [10] J A Elix, J E Evans, J L Parker Aust J Chem 1987, 40, 2129–2131 [11] T Sala, M V Sargent, J A Elix J Chem Soc Chem Commun 1978, 1041–1042 [12] S Huneck, J A Elix Herzogia 1993, 9, 647–651 [13] L T Tuong, T N Vo, T H Duong, B L C Huynh, K P P Nguyen Nat Prod Commun 2014, 9, 1179–1180 [14] Y Kimura, T Yoshinari, H Koshino, S Fujioka, K Okada, A Shimada Biosci Biotechnol Biochem 2007, 71, 1896–1901 [15] V Shukla, G P Joshi, M S M Rawat Phytochem Rev 2010, 9, 303–314 Supporting information Additional supporting information may be found in the online version of this article at the publisher’s website 87 Magn Reson Chem 2016, 54, 81–87 Copyright © 2015 John Wiley & Sons, Ltd wileyonlinelibrary.com/journal/mrc ... 59.3), indicating the methoxymethyl group at C-5 The sole aromatic proton at δ 6.88 was located at C7 by its HMBC correlations with the carboxyl carbon (δ 168.8, C-1) and other aromatic carbons... genuine lichen compounds Therefore, it is of great interest to study the mechanism of their formation A literature search revealed that just four natural phthalide derivatives have been reported from. .. (CDCl3) data see Table HMBC and ROESY see Fig Conclusions From the chloroform soluble fraction of the methanol extract of the lichen P praesorediosum (Nyl.) Hale seven novel compounds are isolated,