HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 10, 2014, pp. 2369-2374
Received, 6th August, 2014, Accepted, 22nd September, 2014, Published online, 30th September, 2014.
DOI: 10.3987/COM-14-13068

■ Isocoumarins from the Bark of Lindera Caudata

Yan-qing Ye, Cong-Fang Xia, Juan-Xia Yang, Yu-Chun Yang, Xue-Mei Gao, Gang Du, Hai-Ying Yang, Yin-Ke Li, and Qiu-Fen Hu*

Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, School of Chemistry and Biotechnology, Yunnan University of Nationalities, Kunming, Jingming South Road, Chenggong New District, Kunming, Yunnan 650500, China

Abstract

Three new isocoumarins, caudacoumarins A - C (1 - 3), together with two known isocoumarins (4 - 5) were isolated from the bark of Lindera caudata. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D- NMR techniques. Compounds 1 - 5 showed modest anti-tobacco mosaic virus (anti-TMV) activity with inhibition rate of 14.8 - 24.5%, respectively.

The plants of Lindera family (Lauraceae) are mainly distributed in tropical, subtropical to temperate regions of Asia and the Midwest of United States.1 The plants from this family were traditionally used to treat stomach, urinary system diseases and rheumatic pain in Chinese folk.2,3 Lindera caudata (Nees) Hook. f., an evergreen plant belongs to Lindera family, Iteadaphne subgenus, had been widely used as herbal medicine with the effects of hemostatic, analgesic and antipyretic.4 However, the phytochemical studies on this plant had not been reported in literatures yet.
Isocoumarins are an important class of natural products widely occurring in plant kingdom and are known to exhibit a wide range pharmacological activities, such as antibacterial and antifungal,5-7 cytotoxic,8,9 antiviral,10,11 antioxidative,12 and anti-inflammatory13 properties. With the aim of multipurpose utilization of medicine plants and identification of bioactive natural products, the phytochemical investigation on the bark of L. caudata was carried out. As a result, three new (1 - 3), and two known (4 - 5) isocoumarins were isolated from this plant. The structures of new compounds were elucidated on the basis of a comprehensive analysis of the 1H, 13C and 2D NMR spectra. In addition, the anti-tobacco mosaic virus (anti-TMV) activities of 1 - 5 were evaluated. In this paper, we report the isolation, structure elucidation of the new isocoumarins, caudacoumarin A - C (1 - 3), as well as their anti-TMV activity.

A 70% aq. acetone extract prepared from the bark of L. caudata was subjected repeatedly to column chromatography on silica gel and preparative HPLC to afford compounds 1 - 5, including three new isocoumarins, caudacoumarins A - C (1 - 3), together with two known isocoumarins, exserolide D (4) 6 and exserolide F (5).6 The structures of the 1 - 5 were as shown in Figure 1, and the 1H and 13C NMR data of 1 - 3 were listed in Table 1. Compound 1 was obtained as pale yellow gum. Its molecular formula C16H18O5 was established on the basis of HRESIMS ([M+Na]+, 313.1057, calcd 313.1052 for C16H18NaO5), requiring 8 degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl group (3428 cm-1), unsaturated lactone (1738 and 1665 cm-1) and aromatic ring (1612, 1568, and 1476 cm-1). The 1H NMR signals revealed the presence of a 1,2,3,4,5-pentasubstituted benzene moiety (δH 7.40 s), a prenyl group [δH 3.40 d (7.2) 2H, 5.27 t (7.2) 1H, 1.54 s 3H, and 1.72 s 3H], an olefinic proton (δH 6.44 s), an oxygenated methylene proton (4.38 s 2H), a methoxy proton (δH 3.84 s), and a phenolic hydroxy proton (δH 9.10 s). Its 13C NMR showed the presence of a 1,2,3,4,5-pentasubstituted benzene moiety (δC 129.8 s, 132.2 s, 157.9 s, 146.4 s, 115.1 d, and 121.9 s), a prenyl group (δC 26.9 t, 122.9 d, 133.3 s, 17.3 q, 25.4 q), a ester carbonyl (δC 161.4 s), a pair of olefinic carbon signals (δC 155.9 s, 106.1 d), an oxygenated methylene carbon (δC 62.0 t), and a methoxy carbon (δC 61.0 q). Its 1H and 13C NMR spectroscopic data were similar to those of 6-hydroxy-3-hydroxymethyl-8-methoxyisocoumarin at the positions C-1 ∼ C-3, C-9,14

indicated that 1 should be a 3-hydroxymethyl-isocoumarin. This deduction was also supported by the HMBC correlations (Figure 2) of H-9 (δH 4.38) with C-3 (δC 155.9) and C-4 (δC 106.1), and of H-4 (δH 6.44) with C-3 (δC 155.9), C-9 (δC 62.0), C-4a (δC 129.8), C-5 (δC 132.2), and C-8a (δC 121.9). Moreover, the HMBC correlation of methoxy proton (δH 3.84) with C-6 (δC 157.9) suggested the methoxy group located at C-6. The prenyl group located at C-5 was supported by the HMBC correlations of H-1′ (δH 3.40) with C-4a (δC 129.8), C-5 (δC 132.2), and C-6 (δC 157.9), and of H-2′ (δH 5.27) with C-5 (δC 132.2). The phenolic hydroxy group was assigned to C-7 on the basis of the HMBC correlations between the hydroxy proton (δH 9.10) and C-6 (δC 157.9), C-7 (δC 146.4) and C-8 (δC 115.1). Thus, compound 1 was assigned as shown in Figure 1, and named caudacoumarin A.

Caudacoumarin B (2) was assigned the molecular formula of C16H18O6 by its HRESIMS at m/z 329.1008 [M+Na]+. The 1H and 13C NMR spectra of 2 were similar to those of compound 1. The only difference was due to the disappearance of a methyl group (δC 25.4 q; δH 1.72 s) and appearance of an oxygenated methylene group (δC 67.2 t; 3.97 s) in 2. These changes suggested that one methyl in prenyl group was replaced by an oxygenated methylene.15 The HMBC correlations of H-5′ (δH 3.97) with C-2′ (δC 124.9), C-3′ (δC 133.1), and C-4′ (δC 13.8), provided further evidence for the structural assignment. Accordingly, the structure of 2 was determined as shown. The 1H and 13C NMR spectra of 3 were also similar to those of compound 2. The major difference resulted from the disappearance of a phenolic hydroxy group (δH 9.17 s). The remainder substituents, a 4-hydroxy-3-methylbutyl group at C-5 and a methoxy group at C-6 were deduced from the analysis of its HMBC collections (Figure 2). In addition, the HMBC correlations of H-8 with C-1, and the typical proton signals of C-7 [δH 6.79 (d) 8.2] and C-8 [δH 6.72 (d) 8.2] also supported the 5,6-substitution on the aromatic rings. Therefore, caudacoumarin C (3) was formulated as shown in Figure 1.

Since some isocoumarins are known to exhibit potential anti-virus activities,10,11 compounds 1 - 5 were tested for their anti-TMV activities. The anti-TMV activities were tested using the half-leaf method,16 using ningnanmycin (a commercial product for plant disease in China) as a positive control. Their antiviral inhibition rates at the concentration of 20 μM were listed in Table 2. The results revealed that compounds 1 - 5 showed modest anti-TMV activity with inhibition rate of 14.8 - 24.5%, respectively.

EXPERIMENTAL
General. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter. IR spectra were obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ESI-MS were measured on a VG Auto Spec-3000 MS spectrometer. 1H, 13C and 2D NMR spectra were recorded on Bruker 500 instrument with TMS as internal standard. Column chromatography was performed on silica gel (200-300 mesh), or on silica gel H (10 ∼ 40 µm, Qingdao Marine Chemical Inc., China). Second separate was used an Agilent 1100 HPLC equipped with ZORBAX-C18 (21.2 mm × 250 mm, 7.0 µm) column and DAD detector.
Plant Material. The bark of Lindera caudata (Nees) Hook. f. was collected in Xishuangbanna Prefecture, Yunnan Province, People’s Republic of China, in September 2012. The identification of the plant material was verified by Prof. Ren P. Y (Xishuangbanna Botanical Garden). A voucher specimen (YNNI-2012-97) has been deposited in our laboratory.
Extraction and Isolation. The bark of L. caudata (4.5 kg) was extracted four times with 70% acetone (4 ×6 L) at room temperature and filtered. The crude extract (158 g) was applied to silica gel (200 - 300 mesh) column chromatography, eluting with a CHCl3-Me2CO gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions A–F. The further separation of fraction B (9:1, 23.2 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures B1–B5. Fraction B2 (8:2, 5.84 g) was subjected to preparative HPLC (65% MeOH, flow rate 12 mL/min) to give 1 (11.4 mg), 3 (15.1 mg), and 5 (8.2 mg). Fraction B3 (7:3, 4.28 g) was subjected to preparative HPLC (60% MeOH, flow rate 12 mL/min) to give 2 (14.7 mg) and 4 (8.8 mg).
Anti-TMV Assays. The Anti TMV activities were tested using the half-leaf method,17 and ningnanmycin, a commercial product for plant disease in China, was used as a positive control.
Caudacoumarin A (1): C16H18O5, Obtained as a pale yellow gum; UV (MeOH) λmax (log ε) 210 (4.08), 270 (3.75), 295 (3.52), 335 (3.64) nm; IR (KBr) νmax 3428, 3062, 2935, 2864, 1735, 1668, 1615, 1562, 1498, 1382, 1210, 1132, 1080, 865, 752 cm–1; ESIMS m/z (positive ion mode) 313 [M+Na]+; HRESIMS (positive ion mode) m/z 313.1057 [M+Na]+ (calcd C16H18NaO5 for 313.1052).
Caudacoumarin B (1): C16H18O6, Obtained as a pale yellow gum; UV (MeOH) λmax (log ε) 210 (4.02), 272 (3.78), 295 (3.57), 336 (3.68) nm; IR (KBr) νmax 3432, 3068, 2939, 2862, 1738, 1665, 1612, 1568, 1476, 1389, 1248, 1135, 1068, 868, 759 cm–1; ESIMS m/z (positive ion mode) 329 [M+Na]+; HRESIMS (positive ion mode) m/z 329.1008 [M+Na]+ (calcd C16H18NaO6 for 329.1001).
Caudacoumarin C (3): C16H18O5, Obtained as a pale yellow gum; UV (MeOH) λmax (log ε) 210 (4.12), 268 (3.65), 292 (3.52), 330 (3.64) nm; IR (KBr) νmax 3438, 3065, 2937, 2867, 1740, 1667, 1610, 1560, 1479, 1385, 1246, 1138, 1067, 864, 756 cm–1; ESIMS m/z (positive ion mode) 313 [M+Na]+; HRESIMS (positive ion mode) m/z 313.1047 [M+Na]+ (calcd C16H18NaO5 for 313.1052).

ACKNOWLEDGMENT
This research was supported by the Applied Fundamental Foundation of Yunnan Province (No. 2012FD056), National Natural Science Foundation of China (No. 21302164), the Excellent Scientific and Technological Team of Yunnan High School (2010CI08).

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