HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 6, 2011, pp. 1377-1383
Received, 16th February, 2011, Accepted, 23rd March, 2011, Published online, 11th April, 2011.
DOI: 10.3987/COM-11-12179

■ New Red Pigments from Lichen Lethariella sinensis

Ayako Tanaka, Kaoru Kinoshita, Koji Ichinose, Tadayasu Togawa, Kiyotaka Koyama, Takao Narui, Li-song Wang, and Kunio Takahashi*

Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan

Abstract

New red pigments were isolated from a lichen thallus, Lethariella sinensis by Wei & Jiang. The structures of the red pigments, rubrosinensiquinones A (1), B (2), and C (3), were determined by spectroscopic analyses.

The lichens Lethariella spp. are used for a health-promoting tea named Lu xing cha or Hong xue cha in Yunnan, China and Tibet.1 During our studies on secondary metabolites from Lethariella spp., we discovered three new red pigments, rubrocashmeriquinone (4), 7-chlororubrocashmeriquinone (5), and 7-chlorocanarione (6), along with one known yellow pigment, canarione (7) (Figure 1), and studied their antioxidant activity.2 In this paper, we report additional new red pigments, named rubrosinensiquinones A (1), B (2), and C (3) from Lethariella sinensis.

The MeOH and H2O extracts of L. sinensis were analyzed by TLC to reveal the presence of several red spots, which successfully guided the subsequent chromatographic separation of each extract. The two new red pigments (1 and 2) were isolated from the MeOH extract. Whereas, 3 was isolated from the H2O extract. The structures of 1, 2, and 3 (Figure 2) were determined to be 1,2-quinone derivatives based on the following spectroscopic data. Compound 1, dark red amorphous, was suggested to have the molecular formula C20H17NO8 by its positive HR-ESI (electronspray ionization) MS spectral data m/z 400.1014 [M+H]+, (calcd for C20H18NO8, [M+H]+ 400.1032). The IR spectrum exhibited absorptions for hydroxy (3440 cm-1) and carbonyl (1650, 1630 cm-1) groups.

The 1H and 13C NMR data of 1 are shown in Table 1. Both spectroscopic signal patterns of 1 are similar to those of 4–7. Therefore, we suggest that these are similar naphthoquinone derivatives. In addition, the UV spectrum (MeOH) of 1 showed a longer wavelength absorption (515 nm) than that of 1,4-naphthoquinone derivative 7. Generally, the UV absorptions of 1,2-quinones tend to be at longer wavelengths than 1,4-quinones. For example 1,4-benzoquinone absorbs at 242, 281, and 434 nm, whereas 1,2-benzoquinone absorbs at 390 and 610 nm.3 Thus, the structure of 1 was determined to be a 1,2-quinone derivative. HMBC (Hetero-nuclear Multiple-Bond Connectivity) and 1H-1H COSY data are presented in Figure 2. The methylene proton at δ 3.97 (H-7’), with an HMQC (Hetero-nuclear Multiple Quantum Coherence) correlation with the δ 47.5 (C-7’) methylene carbon, showed HMBC correlations with the aromatic quaternary carbons at δ 106.3 (C-7), 172.9 (C-6), 183.7 (C-8), the methylene carbon at δ 52.6 (C-5’), and the methine carbon at δ 67.7 (C-2’). HMQC and 1H-1H COSY data of 1 indicated the presence of the fragment, δ 67.7 (C-2’) - δ 28.3 (C-3’) - δ 23.3 (C-4’) - δ 52.6 (C-5’). Additionally, the structure of 1 contains nitrogen and carboxylic acid moieties according to the molecular formula of C20H17NO8. The methine carbon at δ 67.7 (C-2’) and methylene carbon at δ 52.6 (C-5’) shifted downfield because these carbons were near the nitrogen. As a result, the structure of the proline (amino acid) with a methylene group on the nitrogen atom was considered. Furthermore this proline moiety was connected via a methylene group to C-7 of the quinone moiety. Comparing to the optical rotation data of proline derivatives,4,5 it was indicated that compound 1 with negative rotation was a derivative of (-)-S-proline. The IR data of 1 (1620 cm-1) suggested the presence of a zwitter ion (COO- and NH+) on the proline moiety. Compound 1 was a new pigment, which we named rubrosinensiquinone A.
Compound 2, dark red amorphous, was suggested to have the molecular formula C18H15NO8 by its positive HR-ESIMS spectral data m/z 374.0856 [M+H]+, (calcd for C18H16NO8, [M+H]+ 374.0876). The IR spectrum exhibited presences of hydroxy (3450 cm-1) and carbonyl (1650, 1640, 1630, and 1610 cm-1) groups. The 1H and 13C NMR data of 2 are shown in Table 1. The UV spectrum (MeOH) of 2 was similar to that of 1. HMBC data is presented in Figure 2. The methylene proton at δ 4.05 (H-4’), with an HMQC correlation with the δ 49.6 (C-4’) methylene carbon, showed HMBC correlations with the aromatic quaternary carbons at δ 104.0 (C-7), 173.0 (C-6), 183.7 (C-8), the methylene carbon at δ 57.7 (C-2’), and the methyl carbon at δ 40.6 (1’-CH3). The methyl proton at δ 2.68 (1’-CH3) showed an HMBC correlation with the methylene carbons at δ 49.6 (C-4’) and δ 57.7 (C-2’). The methylene proton at δ 3.45 (H-2’) showed an HMBC correlation with the carbonyl carbon at δ 164.7 (C-3’). From the above, the 1-methylglycine with a methylene group on the nitrogen atom was considered as residual moiety. Furthermore 1-methylglycine moiety was connected via a methylene group to C-7 of the quinone moiety. Compound 2 was a new pigment, and named rubrosinensiquinone B.
Compound 3, dark red amorphous, was suggested to have the molecular formula C14H8O6 by its HR-EI (electron ionization) MS spectral data m/z 272.0319, (calcd for C14H8O6, [M]+ 272.0321). The IR spectrum exhibited presences of hydroxy (3440 cm-1) and carbonyl (1660 cm-1) groups. The 1H and 13C NMR data of 3 are shown in Table 1. Both spectroscopic signal patterns were similar to those of 4. The UV spectrum (MeOH) of 3 showed almost the same absorption at 503 nm compared to 1 and 2. HMBC and LSPD (long-range selective proton decoupling) data are presented in Figure 2. The aromatic methine proton of δ 6.06 (H-3) showed LSPD (long-range selective proton decoupling) correlations with the methyl carbon at δ 19.3 (2-CH3), the aromatic methine carbon at δ 111.8 (C-3), and the aromatic quaternary carbon at δ 164.4 (C-4). The hydroxyl proton at δ 17.34 (4-OH) showed HMBC correlations with aromatic quaternary carbons at δ 116.6 (C-4a), 164.4 (C-4), and the aromatic methine carbon at δ 111.8 (C-3). From the above, we deduced that the 5-OH of 4 switched positions with the carbonyl group at position 4 of 4 in 3 (Figure 2). Compound 3 was a new pigment, and named rubrosinensiquinone C.
Antioxidant activities of the isolated compounds were determined by the CUPRAC-BCS (copper (II) reduction assay with bathocuproine disulfonic acid disodium salt) method.5 The principle of this assay is based on Cu(II) reducing activity. As a result, 2 was found to have antioxidant activity (0.19), 3 showed equivalent antioxidant activity (0.10) to canarione (0.06), and 1 showed no effect (activity is expressed as mM trolox equivalents /mM).

EXPERIMENTAL
General
The IR spectra were measured with a JASCO IR Report-100 infrared spectrophotometer. The 1H and 13C NMR spectra and HMQC, HMBC, COSY, and LSPD were recorded using a JEOL JNM-AL-400 (1H 400 and 13C 100 MHz) and a JEOL JNM-LA 500 (1H 500 and 13C 125 MHz) spectrometer in DMSO-d6 using TMS as internal standard. The MS spectra were obtained using a JEOL JMS-700 and Thermo LTQ Orbitrap XL. Column chromatography was carried out on silica gel 60N (spherical, neutral, 63-210 µM, Kanto Chemical Co., Inc.) and PEGASIL PREP ODS-7515-12-A (Senshu Chemical Co., Inc.) and GE Healthcare Sephadex LH-20. HPLC was carried out using JASCO PU-2080 PLUS pump (flow late, 1.5 mL/min), equipped with a JASCO UV-2075 PLUS UV/VIS Detector.

Plant material
The lichen thalli (Useaceae), L. sinensis Wei and Jiang, was collected and identified by Dr. Wang Li-Song in Yunnan, China, in 2005. A voucher specimen is deposited at the Department of Pharmacognosy, Meiji Pharmaceutical University.

Extraction and isolation
The lichen thallus (1.0 kg) was extracted with Et2O, acetone, MeOH, and H2O three times each. The MeOH extract (75.4 g) was subjected to column chromatography (C. C.) on silica gel using a CHCl3-MeOH stepwise gradient system (CHCl3-MeOH 1:0 → 10:1 → 5:1 → 2:1 → 1:1 → 0:1 → CHCl3-MeOH-H2O 6:4:1) to yield Fraction 1 (CHCl3-MeOH 1:0), Fraction 2 (CHCl3-MeOH 10:1), Fraction 3 (CHCl3-MeOH 10:1), Fraction 4 (CHCl3-MeOH 5:1), Fraction 5 (CHCl3-MeOH 5:1 and 2:1), Fraction 6 (CHCl3-MeOH 2:1 and 1:1), Fraction 7 (CHCl3-MeOH 0:1), Fraction 8 (CHCl3-MeOH 0:1 and CHCl3-MeOH-H2O 6:4:1), and Fractions 9 (CHCl3-MeOH-H2O 6:4:1). Fr. 7 was repeatedly chromatographed on ODS using MeOH-H2O to obtain compound 1 (10.4 mg). Compound 2 (2.6 mg) was isolated from Fr. 8 by the combination of ODS column chromatography (eluant: MeOH-H2O 1:9 → 3:7 → 1:1 → 7:3 → 1:0) and ODS HPLC (eluant: 25% MeOH).
The H2O extract (64.3 g) was further extracted with MeOH. This MeOH extract (1.9 g) was partitioned into a MeOH-soluble part (1.4 g) and an insoluble part (0.5 g). The MeOH-insoluble part (0.5 g) was subjected to column chromatography on ODS using a MeOH-H2O stepwise gradient system (10% MeOH → 30% MeOH→ 50% MeOH →70% MeOH → 100% MeOH) to yield Fraction A (10% and 30% MeOH), Fraction B (30% MeOH), Fraction C (30% MeOH), Fraction D (30% and 50% MeOH), Fraction E (50% MeOH), and Fraction F (50 and 100% MeOH). Fr. E was subjected to Sephadex LH-20 column chromatography using MeOH to give compound 3 (0.5 mg).

Compound 1: rubrosinensiquinone A
Dark red amorphous. [α]D -941.2° (c 0.0034, DMSO); UV (DMSO) λmax (log ε) nm: 281 (3.86), 299 (sh, 3.78), 380 (3.32), 436 (sh, 2.97), 515 (3.03); IR υmax (KBr) cm-1: 3440, 1650, 1630, 1580, 1560, 1540, 1460, 1260, 1200, 1120, 1090, 960. Positive HR-ESIMS m/z 400.1014 [M+H]+ (calcd for C20H18NO8, [M+H]+ 400.1032). Positive ESIMS m/z 400 [M+H]+ 285, 257. The 1H and 13C NMR data are presented in Table 1. HMBC and 1H-1H COSY data are presented in Figure 2.

Compound 2: rubrosinensiquinone B
Dark red amorphous. UV (DMSO) λmax (log ε) nm: 277 (3.66), 378 (3.12), 524 (2.74); IR υmax (KBr) cm-1: 3450, 2350, 1650, 1640, 1630, 1610, 1580, 1560, 1540, 1500, 1450, 1090. Positive HR-ESIMS m/z 374.0856 [M+H]+ (calcd for C18H16NO8 [M+H]+ 374.0876). Positive ESIMS m/z 374 [M+H]+ 285, 257. The 1H and 13C NMR data are presented in Table 1. HMBC data is presented in Figure 2.

Compound 3: rubrosinensiquinone C
Dark red amorphous. UV (MeOH) λmax (log ε) nm: 227 (4.22), 279 (4.04), 378 (3.49), 503 (3.23); IR υmax (KBr) cm-1: 3440, 1660, 1580, 1480, 1270, 1185, 1110, 1090, 960. HR-EIMS m/z 272.0319 (calcd for C14H8O6 [M]+ 272.0321). EIMS m/z (rel. int. %): 272 ([M+] 100), 244 (86), 216 (21). The 1H and 13C NMR data are presented in Table 1. HMBC and LSPD data are presented in Figure 2.

ACKNOWLEDGEMENTS
This work was partially supported by a grant from the High-Tech Research Center Project, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (S0801043).

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