HETEROCYCLES

Short Paper | Regular issue | Vol. 92, No. 8, 2016, pp. 1462-1467
Received, 12th April, 2016, Accepted, 31st May, 2016, Published online, 20th June, 2016.
DOI: 10.3987/COM-16-13482

■ Two New 2-Arylbenzofurans from the Stems of Nicotiana tabacum and Their Bioactivities

Pei-Song Yang, Wei Zhang, Xiao-Feng Shen, Xin-Lin Wang, Chao Li, Xiao-Wei Gong, Xu-Dong Zheng, Dong-Lai Zhu, and Jia-Qiang Wang*

School of Chemical Science and Technology, Yunnan University, Kunming 650091, China

Abstract

Two new 2-arylbenzofurans, 4-(7-methoxy-3,5-dimethyl-benzofuran-2-yl)phenol (1), 4-(6-methoxy-3,5-dimethylbenzofuran-2-yl)-phenol (2), together with two known 2-arylbenzofurans (3 and 4) were isolated from the stems of Nicotiana tabacum. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Compounds 1-4 were tested for their anti-TMV and cytotoxicity activities. The results showed that compound 2 exhibited high anti-TMV activities with inhibition rates of 36.4% at the concentration of 20 μM. This rate is higher than that of positive control. Compounds 1-4 showed moderate-to-weak cytotoxicities against some tested human tumor cell lines with IC50 values in the range of 2.2–8.4 μM.

Nicotiana tabacum, tobacco is an important economic crop.1,2 Its leaves are used as a raw material for the tobacco industry, aerial plant as an insecticide, and also as anesthetic, diaphoretic, sedative, and emetic agents in Chinese folklore medicine.1-3 In previous literatures, many new bioactive compounds, such as, sesquiterpenes,4-6 alkaloids,7,8 lignans,9,10 flavonoids,11-14 phenylpropanoids,15,16 chromanones,17,18 biphenyls,19 phenolic amides,20 isocoumarins,21 were isolated from N. tabacum. The stems of N. tabacum are the main by-product in tobacco planting and are normally used as organic fertilizer. The multipurpose utilization of the roots and stems of N. tabacum is an interesting topic and has attracted more and more attentions.16,21 In continuing efforts to utilize N. tabacum and identify bioactive natural products, the phytochemistry investigation of the stems of Yunyan 201 (a variety of N. tabacum) led to the isolation of two new (1 and 2) and two known 2-arylbenzofurans (3 and 4). This paper deals with the isolation, structural elucidation, and their bioactivities of these compounds.

A 95% aq. MeOH extract prepared from the stems of tobacco was subjected repeatedly to column chromatography on silica gel and preparative HPLC to afford two new 2-arylbenzofurans, 4-(7-methoxy-3,5-dimethyl-benzofuran-2-yl)phenol (1) and 4-(6-methoxy-3,5-dimethylbenzofuran-2- yl)phenol (2), together with two known 2-arylbenzofurans (3 and 4). The structures of the compounds 1-4 were as shown in Figure 1, and the 1H and 13C NMR data of 1 and 2 were listed in Table 1. The known compounds, compared with literature, were identified as 2-[(2′-methoxy-4′-hydroxy)phenyl]-3- methy-6-hydroxybenzofuran (3) 22 and moracin M (4).23

Compound 1 was obtained as a yellow gum. Its molecular formula was determined as C17H16O3 by HRESIMS (m/z 291.0990 [M+Na]+; calcd C17H16NaO3 for 291.0997). It UV spectrum showed maximum absorption at 210, 270 and 309 nm. The IR absorptions at 3415, 1620, 1487, 1436 cm-1 indicated the presence of hydroxy and aromatic rings. The 1H, 13C NMR and DEPT spectra of 1 (Table 1) displayed 17 carbon and 16 proton signals, respectively, corresponding to a 1,2,3,5- tetrasubstituted phenyl ring (C-1~C-6, H-4 and H-6), a 1,4-substituted phenyl ring (C-1'~C-6', H2-2',6', and H2-3',5'), two methyl group (C-7 and C-9'; H3-7 and and H3-9'), two olefin carbons (C-7' and C-8'), one methoxy group (δC 56.1 q; δH 3.86 s), and one phenolic hydroxy group (δH 10.81). Its 1H and 13C NMR spectroscopic data [two phenyl rings, two olefin carbons (C-7' and C-8'), and methyl group (C-9' and H3-9')] were similar to those of known compound (3), which suggested that 1 should be a 3-methyl-2-arylbenzofuran.22 In addition, the 3-methyl-2-arylbenzofuran skeleton of 1 was also assigned by the HMBC correlations (Figure 2) of H3-9′ with C-1, C-7′, and C-8′; of H2-2′,6′ with C-7′; and of H-6 with C-8′. The position of the methyl group (C-7) at C-5 of the arylbenzofuran was elucidated by the HMBC correlations from H3-7 to C-4, C-5, and C-6; from H-4 to C-7; and from H-6 to C-7. The HMBC correlations from the methoxy proton (δH 3.86) to C-3 (δC 151.4) supported the methoxy group located at C-3. The phenolic hydroxy group located at C-4′ was supported by the HMBC correlation of phenolic hydroxy proton (δH 10.81) with C-4′ and C-3′,5′. Thus, the structure of 1 was established as 4-(7-methoxy-3,5-dimethylbenzofuran-2-yl)phenol.

Compound 2 was also obtained as yellow gum and it gave an [M+Na]+ peak at m/z 291.0998 in HRESIMS, consistent with a molecular formula of C17H16O3. The 1H and 13C spectra data of 2 was very similar to these of 1 (see Table 1). The major differences were due to the substituent position variations on the tetrasubstituted phenyl ring (C-1~C-6). The methyl group (C-7) located at C-5 of was supported by the HMBC correlations from H3-7 to C-4, C-5, and C-6; from H-4 to C-7; and from H-6 to C-7. The HMBC correlations from the methoxy proton (δH 3.84) to C-4 (δC 155.6) supported the methoxy group located at C-4. The phenolic hydroxy group located at C-4′ was supported by the HMBC correlation of phenolic hydroxy proton (δH 10.90) with C-4′ and C-3′,5′. Thus, the structure of 4-(6-methoxy-3,5-dimethylbenzofuran-2-yl)phenol (2) was established.

Compounds 1-4 were tested for their anti-tobacoo mosaic virus (TMV) activities. The anti-TMV activities were tested by half-leaf method, using ningnanmycin (a commercial product for plant disease in China, with inhibition rate of 32.2%) as a positive control.24,25 The results showed that compounds 2 exhibited high anti-TMV activities with inhibition rates of 36.4% at the concentration of 20 μM. This rate is higher than that of positive control. The other compounds also showed potential activities with inhibition rates in the range of 20.6%~25.3 % at the concentration of 20 μM, respectively.
The cytotoxicities of compounds 1-4 were also tested using a previously reported procedure.26,27 The cytotoxic abilities against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) by MTT-assay were summarized in Table 3. The results revealed that compounds 1-4 showed moderate-to-weak inhibitory activities against some tested human tumor cell lines with IC50 values in the
range of 2.2–8.4 μM.

EXPERIMENTAL
General. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on DRX-500 spectrometers with TMS as internal standard, and the chemical shifts (δ) were expressed in ppm. HRESIMS was performed on an API QSTAR time-of-flight spectrometer and a VG Autospec-3000 spectrometer, respectively. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a ZORBAX PrepHT GF (21.2 mm × 25 cm, 7 m) column or a Venusil MP C18 (20 mm × 25 cm, 5 m) column. Column chromatography was performed with Si gel (200–300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China). The fractions were monitored by TLC, and spots were visualized by heating Si gel plates sprayed with 5% H2SO4 in EtOH.

Plant material. The stems of N. tabacum L (tobacco stems) was collected from Lijiang County, Yunnan Province, P. R. China, in September 2014. The tobacco variety is Yunyan-201, which had widely cultivated in China. The identification of the plant material was verified by Prof. H. W. Yang (School of Tobacco, Yunnan Agriculture University).
Extraction and Isolation. The air-dried and powdered tobacco stems (3.8 kg) were extracted with 95% MeOH, and the extract was partitioned between EtOAc. The EtOAc-soluble materials (63.8 g) were applied to silica gel (200–300 mesh) column chromatography, eluting with CHCl3/MeOH gradient system (9:1, 8:2, 7:3, 6:4, 5:5, 4:6) to give six fractions A-F. Further separation of fraction A (9:1, 13.6 g) by silica gel column chromatography, eluted with CHCl3/Me2CO (8:2-2:1) yielded mixtures A1–A7. Fraction A2 (7:3, 0.67 g) was subjected to silica gel column chromatography using petroleum ether/acetone, and then semi-preparative HPLC (48% MeOH/H2O, flow rate 12 mL/min) to give 1 (12.5 mg) and 2 (10.8 mg). Fraction A3 (6:4, 1.64 g) was subjected to silica gel column chromatography using petroleum ether/acetone, and then semi-preparative HPLC (44% MeOH/H2O, flow rate 12 mL/min) to give 3 (14.2 mg) and 4 (13.1 mg).
Anti-TMV Assays. The anti-TMV activity was tested using the half-leaf method,24,25 and Ningnanmycin (2% water solution), a commercial product for plant disease in China, was used as positive control.
Cytotoxicity Assay. The cytotoxicity tests for the isolates were performed by against NB4, A549, SHSY5Y, PC3, and MCF7 tumor cell lines by MTT-assay (with Taxol as the positive control).26,27
4-(7-Methoxy-3,5-dimethylbenzofuran-2-yl)phenol (1)：C17H16O3, obtained as a yellow gum; UV (MeOH) λmax (log ε) 210 (4.10), 270 (3.15), 309 (3.52) nm; IR (KBr) νmax 3415, 1620, 1487, 1436, 1379, 1158, 1069, 865, 793 cm–1; 1H and 13C NMR (500 and 125 MHz, in C5D5N, see Table-1; ESIMS m/z (positive ion mode) 291 [M+Na]+; HRESIMS (positive ion mode) m/z 291.0990 [M+Na]+ (calcd C17H16NaO3 for 291.0997).
4-(6-Methoxy-3,5-dimethylbenzofuran-2-yl)phenol (2): C17H16O3, obtained as a yellow gum; UV (MeOH) λmax (log ε) 210 (4.06), 268 (3.26), 306 (3.57) nm; IR (KBr) νmax 3420, 1618, 1482, 1440, 1382, 1153, 1062, 880, 764 cm–1; 1H and 13C NMR (500 and 125 MHz, in C5D5N, see Table-1; ESIMS m/z (positive ion mode) 291 [M+Na]+; HRESIMS (positive ion mode) m/z 291.0998 [M+Na]+ (calcd C17H16NaO3 for 291.0997).

ACKNOWLEDGEMENTS
This research was supported by the National Natural Science Foundation of China (No. 21562049, No. 31360081 and No. 31400303), and the Applied Fundamental Foundation of Yunnan Province (No. 2014FB163, No. 2015FB162).

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