HETEROCYCLES

Paper | Regular issue | Vol. 85, No. 10, 2012, pp. 2485-2490
Received, 26th July, 2012, Accepted, 29th August, 2012, Published online, 11th September, 2012.
DOI: 10.3987/COM-12-12557

■ Two New Chromanone Derivatives from the Roots and Stems of Nicotiana tabacum and Their Cytotoxicity

Dingrong Mou, Wei Zhao, Tao Zhang, Lan Wan, Guangyu Yang, Yongkuan Chen, Qiufen Hu,* and Mingming Miao*

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

Two new chromanone derivatives, tabchromones A and B (1-2), together with four known compunds (3-6) were isolated from the roots and stems of Nicotiana tabacum. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D- NMR techniques. Compounds 1-6 were tested for their in vitro cytotoxicity against five human tumor (NB4, A549, SHSY5Y, PC3, and MCF7) cell lines. Compound 1 showed significant inhibitory effect against SHSY5Y cell line, with IC50 values of 2.8 μM, and compounds 2-4 showed moderate activity for some selected cell lines, with IC50 values in the range of 4.8-8.0 μM.

INTRODUCTION
Nicotiana tabacum L. is one of the most commercially valued agricultural crops in he world.1,2 Its leaves are the most important row material for cigarette industry. In addition to being used in cigarette industry, N. tabacum is also used as insecticides, anesthetics, diaphoretics, sedatives, and emetic agents in Chinese folklore medicines because of its containing many useful chemical compounds.1,3 The stems and roots of N. tabacum are rich in secondary metabolities and are normally used as organic fertilizer. The multipurpose utilization of the stems and roots of N. tabacum is an interesting focus, and receives more and more attentions.4,5
Our previous investigation of this species led to the discovery of a number of new compounds, and those compounds were found to show various bioactivaties.6-9 With the aim of continuing efforts to utilize N. tabacum and identify bioactive natural products the phytochemical investigation of the roots and stems of Honghua Dajinyuan (a variety of N. tabacum) was carried out, and led to two new chromanone derivatives (1-2), together with four known chromone derivatives (3-6). This paper reports the isolation, structural elucidation, and their cytotoxicity.

RESULTS AND DISCUSSION
The 90% aqueous ethanol extract prepared from the roots and stems of N. tabacum was subjected repeatedly to column chromatography on silica gel, sephadex LH-20, RP-18 and preparative HPLC to afford two new chromanone derivatives, tabchromones A and B (1-2), together with four known compunds (3-6), The structures of compounds 1-6 were as shown in Figure 1, and the 1H and 13C NMR data of the compounds 1 and 2 were listed in Table 1. The known compounds, compared with literature data, were identified as: 6-(3-hydroxy-4-methoxystyryl)-4-methoxy-2H-pyran-2-one (3),10 pestaloficiol G (4),11 takanechromone C (5),12 greveichromenol (6).13

Compound 1 was obtained as pale yellow oil. It gives a parent ion by HR-ESIMS at m/z 247.0976 [M-H]- (calcd for 247.0976) corresponding to a molecular formula C14H16O4, requiring seven degrees of unsaturation. The 1H and 13C NMR spectra of 1 along with analysis of the DEPT spectra (Table 1) displayed 14 carbon signals and 16 proton signals, respectively, corresponding to an chromanone nucleus10 (δC 79.2 s, 50.3 t, 192.0 s, 108.0 d, 151.0 s, 123.7 d, 131.6 s, 152.2 s, 120.9 s, 25.9 q (2C)), an acetonyl group (-CH2C(O)CH3) (δC 49.8 t, 206.8 s, 30.0 q; δH 4.08 s, 2.65 s), and a phenolic hydroxy group (δH 8.43 br. s). Strong absorption bands accounting for hydroxy (3436 cm-1), carbonyl group (1722, 1670 cm-1) and aromatic groups (1615, 1556, 1436 cm-1) could also be observed in its IR spectrum. The UV spectrum of 1 showed absorption maxima at 260 and 210 nm, which confirmed the existence of the aromatic functions. The HMBC correlations (Figure 2) of H-11 (δH 4.08) with C-7 (δC 123.7), C-8 (δC 131.6) and C-9 (δC 152.2), of H-7 (δH 6.89) with C-11 (δC 49.8) indicated that the acetonyl group should be located at C-8 on the chromone ring. The phenolic hydroxy group located at C-6 was supported by the HMBC correlations of the hydroxy proton (δH 8.43) with C-5 (δC 108.0), C-6 (δC 151.0), and C-7 (δC 123.7). Thus, the structure of 1 was established as shown, and given the name as tabchromone A.

Compound 2 was also obtained as pale yellow oil, and showed quasi molecular ion at m/z 231.1027 [M-H]- in the HRESIMS (calcd m/z 231.1021), corresponding to the molecular formula of C14H16O3. The 1H and 13C NMR spectra of 2 were similar to those of 1 in C-2～C-10, C-14 and C-15. The obvious chemical shift differences resulted from the disappearance of an acetonyl group signals, and appearance of a 2-propenyl group signals (δC 39.0 t, 137.9 d, 117.8 t; δH 3.17 m, 5.96 m, 5.03 m) in 2. This indicated that the acetonyl group in 1 was substituted by a 2-propenyl group in 2. The HMBC correlations of H-11 (δH 3.17) with C-7 (δC 122.2), C-8 (δC 133.0) and C-9 (δC 152.4), of H-12 (δH 5.96) with C-8 (δC 133.0), of H-7 (δH 6.91) with C-11 (δC 39.0) indicated that the 2-propenyl group should be located at C-8 of the chromone ring. Thus, the structure of 2 was established and it was named tabchromone B.

Since certain of the chromone derivatives exhibit potential cytotoxicity,14-16 the compounds 1-6 were tested for their cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7) using the MTT method as reported previously.17 Taxol was used as the positive control.

The results were shown in Table 2. Compounds 5 and 6 showed low active (IC50 values >10 μM) for all tested tumor cell lines. Compound 1 showed high cytotoxicity against SHSY5Y cell with IC50 values of 2.8 μM. Compounds 2-4 also showed moderate cytotoxicity for some selected cell line with IC50 valve below 10 μM.

EXPERIMENTAL
General. 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 DRX-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 roots and stems of Honghua Dajinyuan (a variety of N. tabacum) were collected in Yuxi Prefecture, Yunnan Province, People’s Republic of China, in September 2010.
Extraction and Isolation. The air-dried and powdered roots and stems of N. tabacum (5.0 kg) were extracted four times with 90% aq. EtOH (4 ×5.0 L) at room temperature and filtered. The crude extract (298 g) was applied to silica gel (200 – 300 mesh) column chromatography, eluting with a CHCl3-(Me)2CO 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 C (8:2, 11.5 g) by silica gel column chromatography, eluted with CHCl3-MeOH (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures C1–C5. Fraction C1 (9:1, 1.57 g) was subjected to preparative HPLC (42% MeOH, flow rate 12 mL/min) to give 1 (10.6 mg), 2 (14.8 mg), 3 (15.2 mg), 4 (22.5 mg), and 6 (13.2 mg). The further separation of fraction E (6:4, 32.6 g) by silica gel column chromatography, and preparative HPLC (28% MeOH, flow rate 12 mL/min) gave 5 (18.2 mg).
Tabchromone A (1). Obtained as pale yellow oil; UV (MeOH) max (log ε) 210 (4.57), 260 (4.15), 358 (3.02) nm; IR (KBr) νmax 3436, 2918, 2872, 1722, 1670, 1615, 1556, 1436, 1358, 1137, 946, 853 cm-1; 1H NMR and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; negative ESIMS m/z 247 [M-H]-; negative HRESIMS m/z 247.0976 [M-H]- (calcd for C14H16O4, 247.0970).
Tabchromone B (2). Obtained as pale yellow oil; UV (MeOH), λmax (log ε) 210 (4.53), 254 (4.08), 355 (3.11) nm; IR (KBr) νmax 3418, 2975, 2926, 1669, 1614, 1538, 1469, 1247, 1154, 943, 862 cm-1; 1H NMR and 13C NMR data (CDCl3, 500 MHz and 150 MHz), see Table 1; negative ESIMS m/z 231 [M-H]-; negative HRESIMS m/z 231.1027 [M-H]- (calcd 231.1021 for C14H16O3).

ACKNOWLEDGMENT
This project was supported financially by the Excellent Scientific and Technological Team of Yunnan (2009CI014), and the Basic Research Foundation of Yunnan Tobacco Industry Co. Ltd (2011JC01).

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