HETEROCYCLES

Short Paper | Regular issue | Vol. 91, No. 10, 2015, pp. 1980-1985
Received, 6th August, 2015, Accepted, 2nd September, 2015, Published online, 17th September, 2015.

■ Chromones from the Twigs of Cassia fistula and Their Anti-Tobacco Mosaic Virus Activities

Qiu-Fen Hu, Li-Mei Li, Dong-Lai Zhu, Zhen-Hua Yu, Jian-Bo Zhan, Jie Lou, Ye-De Wang, Kun Zhou, Min Zhou, Yin-Ke Li, and Xue-Mei Gao*

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 chromones, 2-(3-hydroxypropanoyl)-8-methoxy-5-(2-oxopropyl)-4H-chromen-4-one (1), 8-hydroxy-2-(3-hydroxypropanoyl)-5-(2-oxopropyl)-4H-chromen-4-one (2), 2-(2-hydroxyethyl)-7-methoxy-5-(2-oxopropyl)-4H-chromen-4-one (3), together with four known chromones (3-7) were isolated from the twigs of Cassia fistula. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Compounds 1-3 were tested for their anti-tobacco mosaic virus (anti-TMV) activities. The results revealed that compound 1-3 showed high anti-TMV activities with inhibition rates of 26.6, 28.2 and 29.7%, respectively. These rates are close to that of positive control.

The plant of Cassia fistula L., (Leguminosae) belongs to the Cassia genus. It is widely grown as an ornamental plant in tropical and subtropical areas.1 In China, it also has been used as traditional Chinese medicine by people of Dai nationality, who lived in Xishuangbanna, Yunnan province for treatment of diarrhea, gastritis, ringworm, and fungal skin infections.2,3 Previous phytochemical studies of C. fistula have shown the presence of anthraquinones,4,5 steroids,6,7 chromones,8-10 flavonoids,11-13 naphtho[1,2-b]furan,14 and the like. In our continuing efforts to identify bioactive natural products from the medicinal plants, we now investigated the chemical constituents of the twigs of C. fistula. This leads to the isolation of three new (1-3), and four known chromones (4-7). The structures of 1-7 were elucidated by spectroscopic methods including extensive 1D and 2D NMR techniques. Compounds 1-3 were also evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. This article deals with the isolation, structural elucidation and biological activities of the new chromones.

A 70% aq. acetone extract prepared from the twigs of C. fistula was subjected repeatedly to column chromatography on silica gel and preparative HPLC to afford three new chromones, 2-(3-hydroxypropanoyl)-8-methoxy-5-(2-oxopropyl)-4H-chromen-4-one (1), 8-hydroxy-2-(3-hydroxy- propanoyl)-5-(2-oxopropyl)-4H-chromen-4-one (2), 2-(2-hydroxyethyl)-7-methoxy-5-(2-oxopropyl)- 4H-chromen-4-one (3), and four known chromones (4-7). The structures of the compounds 1-7 were as shown in Figure 1, and the 1H and 13C NMR data of compounds 1-3 were listed in Table 1. The known compounds, compared with literature, were identified as 7-hydroxy-2-methyl-5-(2-oxopropyl)-4H- chromen-4-one (4),15 8-methyleugenitol (5),16 barakol (6),17 and 5-hydroxy-9-methyl-1-(2-oxopropyl)- benzo[β]oxepin-7(2H)-one (7).18
Compound 1 was obtained as a yellow gum and assigned the molecular formula C16H16O6 from its HRESIMS at m/z 327.0852 [M+Na]+ (calcd 327.0845). The IR absorption bands indicated the presence of hydroxy (3418 cm-1), carbonyl (1730, 1682, 1650 cm-1), and aromatic ring (1610, 1558, 1436 cm-1) groups, and UV absorptions at 210, 238, 272, and 350 nm suggested a conjugated aromatic ring system. Its 1H, 13C, and DEPT NMR spectra displayed signals for 16 carbons and 16 hydrogen atoms, corresponding to one chromone ring system19 (C-2~C-10) with three aromatic protons (H-3, H-6, and H-7), one 2-oxopropyl moiety (CH3-CO-CH2-; C-14~C-16; H-14 and H-16),20 one 3-hydroxypropanoyl moiety 21 (-CO-CH2-CH2-OH; C-11~C-13; H-12 and H-13), and a methoxy group (δC 55.9, δH 3.83). The HMBC correlations of H-12 (δH 3.33) with C-2 (δC 156.8) and of H-3 (δH 7.12) with C-11 (δC 198.1) indicated that the 3-hydroxypropanoyl moiety was located at C-2. The HMBC correlations of H-14 (δH 4.18) with C-5 (δC 128.3), C-6 (δC 125.4), and C-10 (δC 120.1) and of H-6 (δH 6.81) with C-14 (δC 50.2) indicated that the 2-oxopropyl moiety was attached to C-5. The attachment of the methoxy group at C-8 was supported by the HMBC correlations of the methoxy proton (δH 3.83) with C-8 (δC 150.8). Thus, the structure of 1 was established as 2-(3-hydroxypropanoyl)-8-methoxy-5-(2-oxopropyl)-4H-chromen-4- one.

Compound 2 was obtained as a yellow gum and showed a quasi-molecular ion at m/z 313.0680 [M+Na]+ in the HRESIMS (calcd m/z 313.0688), corresponding to the molecular formula C15H14O6. The 1H and 13C NMR spectra of 2 were similar to those of 1. The chemical shift differences resulted from the disappearance of a methoxy resonance (δC 55.9, δH 3.83) and appearance of a phenolic hydroxy proton signal (δH 10.60) in 2. This indicated that the methoxy group at C-8 in 1 was converted into a phenolic hydroxy group in 2. The HMBC correlations of the phenolic hydroxy proton signal (δH 10.60) with C-7 (δC 122.1), C-8 (δC148.1), and C-9 (δC 151.3) indicated that the phenolic hydroxy group was located at C-8. Thus, the structure of 2 was established as 8-hydroxy- 2-(3-hydroxypropanoyl)-5-(2-oxopropyl)-4H-chromen-4-one.

Compound 3 was also obtained as yellow gums. It had the molecular formula C15H16O5 as revealed by its HRESIMS at m/z 299.0897 [M+Na]+ (calcd 299.0895). Its 1H, 13C, and DEPT NMR spectra (Table 1) displayed signals for 15 carbons and 16 hydrogen atoms, corresponding to one chromone ring system (C-2~C-10) with three aromatic protons (H-3, H-6, and H-8), one 2-oxopropyl moiety (C-13~C-15; H-13 and H-15), one 2-hydroxyethyl [-CH2CH2OH; C-11 and C-12; H-11 and H-12] moiety,22 and a methoxy group (δC 56.0, δH 3.82). The HMBC correlations of H-11 with C-2 and C-3, of H-12 with C-2, of H-3 with C-11 indicated that the 2-hydroxyethyl moiety was located at C-2. The HMBC correlations of H-14 with C-5, C-6, and C-10, of H-6 with C-14 indicated that the 2-oxopropyl moiety was attached to C-5. The attachment of the methoxy group at C-7 was supported by the HMBC correlations of the methoxy proton with C-7. The typical proton signals of H-6 (H 6.70, d, J=1.8) and H-8 (H 6.83, d, J=1.8) also supported this substituents pattern. Compound 3 was thus defined as 2-(2-hydroxyethyl)-7-methoxy-5-(2- oxopropyl)-4H-chromen-4-one.
Since certain chromones exhibit potential anti-TMV activities,8,10,18,21 Compounds 1-3 were tested for their anti-TMV activity. The anti-TMV activity was tested using the half-leaf method. Ningnanmycin (a commercial product for plant disease in China) with inhibition rate of 30.8%, was used as a positive control.23,24 The results revealed that compounds 1-3 showed high anti-TMV activity with inhibition rates of 26.6, 28.2 and 29.7% at the concentration of 20 M, respectively. These rates are close to that of positive control.

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 twigs of C. fistula were collected in Dehong prefecture of Yunnan Province, People’s Republic of China, in September 2014. The identification of plant material was verified by Prof. Ning Yuan. A voucher specimen (Ynni-14-09-35) has been deposited in our Laboratory.
Extraction and Isolation. The air-dried and powdered twigs of C. fistula (4.8 kg) were extracted four times with 70% aqueous acetone (3×5 L) at room temperature and filtered. The solvent was evaporated in vacuo, and the crude extract was dissolved in H2O and partitioned with EtOAc. The EtOAc partition (122 g) was applied to silica gel (200–300 mesh) column chromatography, eluting with a CHCl3–MeOH gradient system (10:0, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions A–F. Further separation of fraction B (9:1, 18.5 g) by silica gel column chromatography, eluted with CHCl3 –Me2CO (9:1 - 2:1), yielded mixtures B1–B7. Fraction B3 (7:3, 2.85 g) was subjected to silica gel column chromatography using petroleum ether-acetone and semi-preparative HPLC (46% MeOH-H2O, flow rate 12 mL/min) to give 1 (12.2 mg), 3 (13.4 mg), and 6 (8.2 mg). Fraction B4 (6:4, 4.48 g) was subjected to silica gel column chromatography using petroleum ether-acetone and semi-preparative HPLC (38% MeOH-H2O, flow rate 12 mL/min) to give 2 (11.4 mg) , 4 (15.3 mg), 5 (13.7 mg), and 7 (10.3 mg).
Anti-TMV Assays. The anti-TMV activities were tested using the half-leaf method,23,24 and Ningnanmycin (2% water solution), a commercial product for plant disease in China, was used as a positive control.
2-(3-Hydroxypropanoyl)-8-methoxy-5-(2-oxopropyl)-4H-chromen-4-one (1): Obtained as yellow gum; UV (MeOH) max (log ) 210 (4.28), 238 (3.81), 272 (3.86), 350 (3.68) nm; IR (KBr) νmax 3418, 3087, 2936, 2854, 1730, 1682, 1650, 1610, 1558, 1436, 1318, 1142, 1057, 953, 876 cm–1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; positive ESIMS m/z 327 [M+Na]+; positive HRESIMS m/z 327.0852 [M+Na]+ (calcd for C16H16NaO6, 327.0845).
8-Hydroxy-2-(3-hydroxypropanoyl)-5-(2-oxopropyl)-4H-chromen-4-one (2): Obtained as yellow gum; UV (MeOH) max (log ) 210 (4.15), 240 (3.86), 275 (3.73), 352 (3.63) nm; IR (KBr) νmax 3452, 3092, 2941, 2850, 1732, 1680, 1653, 1608, 1562, 1435, 1357, 1162, 1049, 918, 852 cm–1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; positive ESIMS m/z 313 [M+Na]+; positive HRESIMS m/z 313.0680 [M+Na]+ (calcd for C15H14NaO6, 313.0688).
2-(2-Hydroxyethyl)-7-methoxy-5-(2-oxopropyl)-4H-chromen-4-one (3): Obtained as yellow gum; UV (MeOH) max (log ) 210 (4.22), 232 (3.65), 265 (3.96), 340 (3.70) nm; IR (KBr) νmax 3423, 2926, 2855, 1720, 1648, 1610, 1552, 1463, 1342, 1135, 1057, 946, 832 cm–1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), see Table 1; positive ESIMS m/z 299 [M+Na]+; positive HRESIMS m/z 299.0897 [M+Na]+ (calcd for C15H16NaO5, 299.0895).

ACKNOWLEDGEMENTS
This project was supported by the National Natural Science Foundation of China (No.21302164, 21562046, and 21562044) and the Applied Fundamental Foundation of Yunnan Province (No. 2013FB097).

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