HETEROCYCLES

Short Paper | Regular issue | Vol. 92, No. 2, 2016, pp. 331-336
Received, 25th September, 2015, Accepted, 16th December, 2015, Published online, 25th December, 2015.

■ Two New Isoindolin-1-ones from the Leaves of Nicotiana tabacum and Their Anti-Tobacco Mosaic Virus Activities

Guang-Hui Kong, Yu-Ping Wu, Wei Li, Zhen-Yuan Xia, Qiang Liu, Kun-Miao Wang, Pei He, Rui-Zhi Zhu, Xiao-Xi Si, and Guang-Yu Yang*

Key Laboratory of Tobacco Chemistry of Yunnan Province, China Tobacco Yunnan Industry Company, Hongjin Road 181#, Kunming, 650231, China

Abstract

Two new isoindolin-1-ones, 2-(2-hydroxyethyl)-5-methyl-6-(3- methylbut-2-enyl)isoindolin-1-one (1) and 2,5-dimethyl-6-(3-methylbut-2-enyl) isoindolin-1-one (2), were isolated from the leaves of Nicotiana tabacum. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Compounds 1 and 2 were tested for their anti-tobacco mosaic virus (anti-TMV) activities. The results revealed that compounds 1 and 2 showed potential anti-TMV activities with inhibition rates of 48.2 and 45.6%, respectively.

Nicotiana tabacum, tobacco, is a stout herbaceous plant in the Solanaceae (nightshade family) and cultivated worldwide as the primary commercial source of tobacco, which is smoked or chewed as a drug for its mild stimulant effects.1,2 N. tabacum is also a kind of plant containing most complex secondary metabolites in nature, of which more than 2549 kinds of chemical compositions has been identified according to Dube and Green’s reports3 in 1982 while Perfetti and Rodgman reported4 that compounds found in tobacco, tobacco substitutes and cigarette smoke were up to 8700 kinds totally by 2008. In previous literatures, N. tabacum is used as insecticides, anesthetics, diaphoretics, sedatives, and emetic agents in Chinese folklore medicines because of its containing many useful chemical compounds.1,4-6 Previous phytochemical studies of tobacco have shown the presence of sesquiterpenes,6-8 alkaloids,9,10 lignans,11,12 flavonoids,13-15 phenylpropanoids,16,17 chromanones,18,19 biphenyls,20 phenolic amides,21 isocoumarins,22 and the homologous.
In continuing efforts to utilize N. tabacum and identify bioactive natural products, the phytochemistry investigation of the leaves of Yunyan 201 (a variety of N. tabacum) led to the isolation of two new isoindolin-1-ones (1 and 2). This paper deals with the isolation, structural elucidation, and their anti-TMV activities of these compounds.

A 70% aq. acetone extract prepared from the leaves of tobacco was subjected repeatedly to column chromatography on silica gel and preparative HPLC to afford two new isoindolin-1-ones, 2-(2- hydroxyethyl)-5-methyl-6-(3-methylbut-2-enyl)isoindolin-1-one (1) and 2,5-dimethyl-6-(3-methylbut-2- enyl)isoindolin-1-one (2). The structures of the compounds 1 and 2 were shown in Figure 1, and the 1H and 13C NMR data of 1 and 2 were listed in Table 1.

Compound 1 was isolated as a yellow gum. The molecular formula of 1 was determined to be C16H21NO2 by the pseudomolecular ion peak at m/z 260.1658 [M+H]+ in its HRESIMS, suggesting 7 degrees of unsaturation. The UV spectrum showed absorption maxima at 210, 260 and 298 nm, and the IR spectrum showed absorption bands at 3312, 2930, 1665, 1610, 1547, 1460 cm-1, indicating the presence of hydroxy group, carbonyl group, and aromatic ring. The 1H, 13C NMR data (Table-1), and HSQC correlations of 1 showed resonances due to a isoindolin-1-one nucleus23 (C-1 to C-7a; H2-3, H-4, and H-7), a prenyl group22 (C-1' to C-5'; H2-1', H-2', H3-4', and H3-5'), a 2-hydroxyethyl group24 (C-7' and C-8'; H2-7' and H2-8'), and a methyl group (C-6' and H3-6'). The HMBC correlations (Figure 2) of H2-3 with C-1, C-3a, C-4, C-7a, and C-7', of H-4 with C-3, of H-7 with C-1, and of H2-7' with C-1 and C-3 also suggested that compound 1 should be an isoindolin-1-one. The HMBC correlations of H2-1′ (δH 3.36) with C-5 (δC 138.0), C-6 (δC 136.5), and C-7 (δC 126.5), and of H-2′ (δH 5.35) with C-6 (δC 136.5) indicated that the prenyl group was attached to C-6. The location of the methyl group was assigned to C-5 position on the basis of HMBC correlations of the methyl proton signal (δH 1.72) with C-4 (δC 128.2), C-5 (δC 138.0) and C-6 (δC 136.5). Finaly, the 2-hydroxyethyl group linked to nitrogen-atom (N-2) was confirmed by the HMBC correlation of H-7′ (δH 3.59) with C-1 (δC 166.5) and C-3 (δC 42.3). Thus, the structure of 1 was established as 2-(2-hydroxyethyl)-5-methyl-6-(3-methylbut-2-enyl)isoindolin-1-one.
Compound 2 was also obtained as yellow gum. A molecular formula C15H19NO was assigned from HRESIMS (m/z: 230.1540 [M+H]+, calcd 230.1545). The 1H and 13C NMR data of 2 (Table 1) displayed 15 carbon and 19 proton signals, corresponding to a isoindolin-1-one nucleus24 (C-1 to C-7a; H2-3, H-4, and H-7), a prenyl group (C-1' to C-5'; H2-1', H-2', H3-4', and H3-5'), a methyl group linked to aromatic ring (C-6 position), and a methyl group linked to nitrogen-atom (N-2 position). The 1H and 13C NMR spectral data of 2 were similar to those of 1. The obvious chemical shift differences resulted from the substituent group variation on the nitrogen-atom. The appearance of one methyl signals and the disappearance of a 2-hydroxyethyl signal were observed in compound 2. These changes indicated that the 2-hydroxyethyl group in 1 was replaced by a methyl group in 2. The detailed structures of 2 were also confirmed by further analysis of its HMBC correlations. Accordingly, the structure of 2,5-dimethyl-6-(3-methylbut- 2-enyl) isoindolin-1-one (2) was established.

Compounds 1 and 2 were tested for their anti-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 31.6%) as a positive control.25,26 The results revealed that compounds 1 and 2 showed high anti-TMV activities with inhibition rates of 48.2 and 45.6% at the concentration of 20 μM, respectively.

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 leaves of N. tabacum L (tobacco leaves) was collected from Yuxi 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 leaves (6.5 kg) were extracted three times with 70% aqueous acetone (3 × 8.0 L) at room temperature and filtered to yield a filtrate, which was successively evaporated under reduced pressure to obtain a crude extract (426 g). This crude extract was applied to Si gel (200-300 mesh) column chromatography eluting with a CHCl3-MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) to give six fractions A–F. The separation of fraction C (8:2, 20.5 g) was subjected to Si gel column chromatography eluting with CHCl3-(Me)2CO and then run on preparative HPLC (40% MeOH-H2O, flow rate 12 mL/min) to yield compounds 1 (15.2 mg) and 2 (10.8 mg).
2-(2-Hydroxyethyl)-5-methyl-6-(3-methylbut-2-enyl)isoindolin-1-one (1): Obtained as yellow gum; UV (MeOH) λmax nm (log ε) 210 (4.32), 260 (3.86), and 298 (3.05); IR (KBr) νmax 3312, 2930, 1665, 1610, 1547, 1460, 1354, 1213, 1152, 1068, 838, and 746 cm-1; positive ESIMS m/z 260 [M+H]+, positive HRESIMS m/z 260.1658 (calcd for C16H22NO2, 260.1651).
2,5-Dimethyl-6-(3-methylbut-2-enyl)isoindolin-1-one (2): Obtained as yellow gum; UV (MeOH) λmax nm (log ε) 210 (4.18), 258 (3.80), and 295 (3.11); IR (KBr) νmax 2935, 1672, 1612, 1536, 1465, 1357, 1215, 1168, 1060, 826, and 740 cm-1; positive ESIMS m/z 230 [M+H]+, positive HRESIMS m/z 230.1540 (calcd for C15H20NO, 230.1545).
ACKNOWLEDGEMENTS
This research was supported by the Foundation of Yunnan Tobacco Company (2014YN16), 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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