Antiviral Chromones from the Stem of Cassia siamea - Journal of

Oct 18, 2012 - Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University o...
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Antiviral Chromones from the Stem of Cassia siamea Qiu-Fen Hu,†,§ Bin Zhou,†,‡ Xue-Mei Gao,† Li-Ying Yang,† Li-Dan Shu,† Yanqiong Shen,† Gan-Peng Li,*,† Chun-Tao Che,§ and Guang-Yu Yang*,†,‡ †

Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan University of Nationalities, Kunming 650031, Yunnan, People’s Republic of China ‡ Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, Kunming 650106, Yunnan, People’s Republic of China § Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, United States S Supporting Information *

ABSTRACT: Seven new chromones, siamchromones A−G (1−7), and 12 known chromones (8−19) were isolated from the stems of Cassia siamea. Compounds 1−19 were evaluated for their antitobacco mosaic virus (anti-TMV) and anti-HIV-1 activities. Compound 6 showed antitobacco mosaic virus (anti-TMV) activity with an inhibition rate of 35.3% and IC50 value of 31.2 μM, which is higher than that of the positive control, ningnamycin. Compounds 1, 10, 13, and 16 showed anti-TMV activities with inhibition rates above 10%. Compounds 4, 6, 13, and 19 showed anti-HIV-1 activities with therapeutic index values above 50.

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Cassia siamea Lam. (Fabaceae) is a tree growing in the southern regions of China. It has been widely used as a traditional Chinese medicine for the treatment of fever, malaria, arthritis, and swelling.1,2 Previous phytochemical studies of C. siamea have shown the presence of anthraquinones,3,4 alkaloids,5,6 triperpenoids,7,8 steroids,8,9 and chromones.10−12 Biological activities, including antiplasmodial5,6,10 and cytotoxicity,4 have been reported for the alkaloids, chromones, and anthraquinones. Motivated by a search for new bioactive metabolites from this plant, our group has investigated the chemical constituents of the stem of C. siamea, which led to the isolation and characterization of seven new chromones (1−7) and 12 known chromones (8−19). Their antitobacco mosaic virus (anti-TMV) and anti-HIV-1 activities were evaluated. Compound 6 showed an anti-TMV activity with an inhibition rate and IC 50 value higher than that of positive control, ningnamycin. Compounds 1, 10, 13, and 16 also showed anti-TMV activities, with inhibition rates above 10%. Compounds 4, 6, 13, and 19 showed anti-HIV-1 activities, with therapeutic index (TI) values above 50. © XXXX American Chemical Society and American Society of Pharmacognosy

RESULTS AND DISUSSION

The stems of C. siamea were extracted with 95% aqueous MeOH and chromatographed on silica gel, Sephadex LH-20, RP-18, and semipreparative RP-HPLC to afford seven new chromones, siamchromones A−G (1−7), and 12 known chromones (8−19). The structures of compounds 1−7 are as shown in Figure 1, and their 13C NMR data are listed in Table 1. The known compounds (Supporting Information, Figure S1), compared with literature data, were identified as 7hydroxy-2-methyl-5-(2-oxopropyl)-4H-chromen-4-one (8),11 O-methylalloptaeroxylin (9),12 perforatic acid (10),12 uncinoside A (11),13 8-methyleugenitol (12),13 11-hydroxy-sec-Oglucosylhamaudol (13),14 sec-O-glucosylhamaudol (14),15 barakol (15),16 urachromone A (16),17 peucenin-7-methyl ether (17),12 4-cis-acety1-3,6,8-trihydroxy-3-methyldihydronaphthalenone (18),18 and 2-methyl-5-(2′-hydroxypropy1)-7hydroxychromone-2′-O-D-glucopyranoside (19).19 Received: June 7, 2012

A

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Figure 2. Selected HMBC (↷) and 1H−1H COSY (−) correlations of 1 and 6.

CH3]. The HMBC correlations of H-12 (δH 3.33) with C-2 (δC 157.7) and of H-3 (δH 7.16) with C-11 (δC 198.6) indicated that the 3-hydroxypropan-1-one moiety was located at C-2. The HMBC correlations of H-14 (δH 4.15) with C-5 (δC 138.0), C6 (δC 118.9), and C-10 (δC 116.3) and of H-6 (δH 6.67) with C-14 (δC 50.2) indicated that the 2-oxopropyl group was attached to C-5. The attachment of a hydroxy group at C-7 was supported by the HMBC correlations of the hydroxy proton (δH 10.51) with C-6 (δC 118.9), C-7 (δC 163.9), and C-8 (δC 13.8). Thus, the structure of 1 was established as 2-(3-hydroxy1-oxopropyl)-7-hydroxy-5-(2-oxopropyl)-4H-chromen-4-one. Compound 2 was obtained as a yellow gum and showed a quasi-molecular ion at m/z 303.0862 [M − H]− in the HRESIMS (calcd m/z 303.0869), corresponding to the molecular formula C16H16O6. The 1H and 13C NMR spectra of 2 were similar to those of 1. The chemical shift differences resulted from the disappearance of a phenolic hydroxy proton signal and appearance of an O-methyl resonance (δC 55.8, δH 3.81) in 2. This indicated that the phenolic hydroxy group in 1 was converted into an O-methyl group in 2. The HMBC correlation of the O-methyl proton signal (δH 3.81) with C-7 (δC 166.8) indicated that the O-methyl group was located at C7. Thus, the structure of 2 was established as 2-(3-hydroxy-1oxopropyl)-7-methoxy-5-(2-oxopropyl)-4H-chromen-4-one. Compounds 3−5 were all obtained as yellow gums. Comparison of the NMR data of 2 with those of 3 and 4 disclosed that the main structural differences between these compounds were the substituents at C-2. Compound 3 possessed a molecular formula of C16H18O5, as deduced from

Figure 1. Structures of compounds 1−7 from C. siamea.

Compound 1 was obtained as a yellow gum and assigned the molecular formula C15H14O6 from its HRESIMS at m/z 289.0715 [M − H]− (calcd 289.0712). The IR absorption bands indicated the presence of hydroxy (3442, 3365 cm−1), carbonyl (1728, 1685, 1654 cm−1), and aromatic ring (1622, 1554, 1448 cm−1) groups, and UV absorptions at 219, 238, 268, and 352 nm suggested a conjugated aromatic ring system. Its 1 H, 13C, and DEPT NMR spectra displayed signals for 15 carbons and 14 hydrogen atoms, corresponding to one chromone ring system10,11 (δC 157.7, 117.9, 180.8, 138.0, 118.9, 163.9, 103.8, 160.1, 116.3) with three aromatic protons (δH 7.16, 6.67, and 6.79), two methylene (δC 42.1, 50.2), one oxygenated methylene (δC 58.2), two ketocarbonyls (δC 198.6, 209.6), one methyl (δC 30.7), and a phenolic hydroxy proton (δH 10.51). The 1H−1H COSY cross-peak of H-12/H-13, together with the HMBC correlations (Figure 2) of H-12 (δH 3.33) with C-11 (δC 198.6) and C-13 (δC 58.2) and of H-13 (δH 4.38) with C-11 (δC 198.6) and C-12 (δC 42.1), suggested the presence of a 3-hydroxypropan-1-one [−C(O)CH2CH2OH] moiety. The HMBC correlations of H-16 (δH 2.29) with C-14 (δC 50.2) and C-15 (δC 209.6) and of H-14 (δH 4.15) with C-15 (δC 209.6) and C-16 (δC 30.7) also revealed the existence of a 2-oxopropyl group [−CH2C(O)-

Table 1. 13C NMR Data of Compounds 1−7 (δ in ppm, in CDCl3) position 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17, 18 OMe-5 OMe-7 −OAc

1 157.7 117.9 180.8 138.0 118.9 163.9 103.8 160.1 116.3 198.6 42.1 58.2 50.2 209.6 30.7

2 s d s s d s d s s s t t t s q

157.6 117.8 180.0 138.3 117.1 166.8 102.6 160.9 116.3 198.1 42.3 58.5 50.5 208.9 30.5

3 s d s s d s d s s s t t t s q

55.8 q

169.7 111.0 181.2 137.9 117.7 166.1 102.7 159.8 115.9 36.1 32.0 62.6 50.3 208.0 30.3

4 s d s s d s d s s t t t t s q

170.1 111.5 181.8 138.3 117.9 166.9 103.3 160.4 116.8 36.8 31.2 64.5 50.0 207.9 29.9

55.9 q

5 s d s s d s d s s t t t t s q

162.8 108.9 181.5 138.9 117.9 164.0 103.9 159.5 116.2 131.6 124.7 19.0 50.5 208.3 30.9

6 s d s s d s d s s d d q t s q

167.2 108.0 180.9 161.3 100.5 159.2 101.8 158.4 107.4 36.2 32.0 62.7 115.1 127.8 77.8 27.7

7 s d s s d s s s s t t t d d s q

167.9 107.8 181.3 164.1 99.9 159.7 102.1 159.1 106.2 36.5 32.2 62.9 115.0 127.6 78.1 27.6 55.9

s d s s d s s s s t t t d d s q q

55.7 q 21.0 q, B

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its HRESIMS m/z 289.1083 [M − H]− (calcd 289.1076) and NMR data. The 1D- and 2D-NMR spectra of 3 showed the existence of a 3-hydroxypropyl [−CH2CH2CH2OH] moiety (δC 36.2, 32.1, and 62.6; δH 2.53, 1.84, and 3.55). HMBC correlations of H-11 with C-2 and C-3 and of H-12 with C-2 established the location of the 3-hydroxypropyl group at C-2. Compound 3 was thus defined as 2-(3-hydroxypropyl)-7methoxy-5-(2-oxopropyl)-4H-chromen-4-one. Compound 4 had the molecular formula C18H20O6 as revealed by its HRESIMS at m/z 355.1159 [M + Na]+ (calcd 355.1158). The NMR spectra of 4, when compared to those of 3, displayed an O-acetyl group (δH 2.04; δC 21.0, 169.3), which was located at C-13, as indicated by the HMBC cross-peak between H-13 (δH 3.97) and the ester carbonyl carbon (δC 169.3). Thus, the structure of 4 was assigned as 3-(7-methoxy4-oxo-5-(2-oxopropyl)-4H-chromen-2-yl)propyl acetate. Accurate mass measurement of the pseudomolecular ion [M − H]− in the HRESIMS at m/z 257.0821 allowed a molecular formula of C15H14O4 to be assigned to compound 5. The NMR spectra of 5 were similar to those of 1 except for replacement of one 3-hydroxypropan-1-one moiety in 1 with an (E)-propenyl group (−CHCH−CH3; δC 131.6, 124.7, 19.0; δH 6.42, 1.71) in 5. In addition, HMBC correlations from the H-11 (δH 6.14, d) to C-2 implied that the (E)-propenyl group was attached at C-2. Therefore, the structure of compound 5 was assigned as 7hydroxy-5-(2-oxopropyl)-2-[(E)-prop-1-enyl]-4H-chromen-4one. Compound 6 was obtained as a yellow gum. It gave a parent ion by HRMS at m/z 301.1067 [M − H]− (calcd for 301.1076), corresponding to a molecular formula of C17H18O5. The NMR spectra, together with the UV and IR experiments, suggested that 6 was also a chromone derivative. The NMR spectra of 6 showed different substituents at C-5, C-7, and C-8 compared with compound 3. The presence of a gemdimethylchromene moiety in 6 was evidenced by a sharp sixproton singlet at δH 1.50 and an AB spin system at δH 5.54 (J = 10.0 Hz, H-15) and δH 6.63 (J = 10.0 Hz, H-14). Long-range correlations (Figure 2) of H-14 (δH 6.63) to C-7 (δC 159.2), C8 (δC 101.8), and C-9 (δC 158.4) and of H-15 (δH 5.64) to C-8 (δC 101.8) were observed. This led us to conclude that the gemdimethylchromene moiety was fused in an angular manner at C-7 and C-8. The signal at δH 10.73 (1H, s) was ascribed to a phenolic proton. The position of the phenolic group at C-5 in 6 was established by the HMBC correlation (Figure 2) of the hydroxy proton (δH 10.73) to C-5 (δC 161.3), C-6 (δC 100.5), and C-10 (δC 107.4). HMBC correlations of H-6 (δH 6.21) with the carbon signals of C-5 (δC 161.3), C-7 (δC 159.2), C-8 (δC 101.8), and C-10 (δC 107.4) further confirmed the above deduction. Three methylenes (δH 2.57, 2H, d, J = 8.1; 1.89, 2H, m; and 3.58 2H, d, J = 6.3) were assigned to a 3-hydroxypropyl group (−CH2CH2CH2OH) at C-2 by the HMBC correlations of H-11 (δH 2.57) with C-2 (δC 167.2) and C-3 (δC 108.0), of H-12 (δH 1.89) with C-2 (δC 167.2), and of H-3 (δH 6.34) with C-11 (δC 36.2). On the basis of the above evidence, the structure of 6 was established as 5-hydoxy-2-(3-hydroxypropyl)-8,8-dimethylpyrano[2,3-f ]chromen-4(8H)-one. Compound 7 was also obtained as a yellow gum. It was assigned the molecular formula C18H20O5 by its HRESIMS at m/z 315.1239 [M − H]−. The 1H and 13C NMR spectra of 7 were similar to those of compound 6. Analysis of the 1H and 13 C NMR data of 7 suggested that the difference was due to the disappearance of a phenolic hydrogen proton signal and appearance of an O-methyl resonance (δC 55.9, δH 3.82) in 7.

The HMBC correlations of the O-methyl protons (δH 3.82) with C-5 (δC 164.1) indicated that the O-methyl group was located at C-5. Thus, the structure of compound 7 was determined as 2-(3-hydroxypropyl)-5-methoxy-8,8dimethylpyrano[2,3-f ]chromen-4(8H)-one. Since certain chromones exhibit potential antiphytoviral activities,20,21 and the extracts of some plants of the genus Cassia were reported to exhibit anti-TMV activity,22 compounds 1−19 were tested for their anti-TMV activity. The inhibitory activities of compounds 1−19 against TMV replication were tested using the half-leaf method.23,24 Ningnanmycin, a commercial biochemical pesticide against virus diseases on tomato, pepper, melons, tobacco, and many other crops with high efficiency, was used as a positive control. The antiviral inhibition rates of compounds 1−19 at the concentration of 20 μM are listed in Table 2. The results Table 2. TMV Infection Inhibition Activities of Compounds 1−19 on N. glutinosa in Vivoa compound 1 2 3 4 5 6 7 8 9 10 a

compound 15.2 9.8 8.6 7.5 9.2 35.3 5.7 6.7 9.2 13.6

± ± ± ± ± ± ± ± ± ±

3.5 2.8 3.2 2.4 2.6 3.8 2.7 2.2 3.0 2.1

10 12 13 14 15 16 17 18 19 ningnamycin

7.9 8.6 18.2 7.8 9.0 11.5 8.7 6.4 8.6 30.2

± ± ± ± ± ± ± ± ± ±

2.5 3.0 2.4 2.7 3.2 3.4 2.4 2.0 2.5 2.8

All results are expressed as mean ± SD; n = 3 for all groups.

showed that compound 6 showed anti-TMV activity with an inhibition rate of 35.3%, which is higher than that of ningnanmycin (30.2%). Compounds 1, 10, 13, and 16 showed anti-TMV activity with inhibition rates of 15.2%, 13.6%, 18.2%, and 11.5%, respectively. Since the inhibition rates of compounds 1, 6, 10, 13, and 16 were above 10%, the IC50 values of the five compounds were tested, and the results are listed in Table 3, with ningnanmycin as the positive control. Compound 1 exhibited the best activity, with an IC50 value of 31.2 μM; the efficiency was higher than that of ningnamycin. The protective effects of compounds 1, 6, 10, 13, and 16 on TMV were also evaluated by pretreating the tobacco leaf with solutions of compounds or a solution of DMSO for 6 h before inoculation with TMV.25 The results (Table 4) showed that at 20 μM all five tested compounds showed protective effects to the host plants, with the inhibition rates ranging from 10.2% to 42.7%. The results indicated that pretreatment with these chromones could increase the resistance of the host plant to TMV infection. Since certain chromones exhibit potential anti-HIV-1 activity,26,27 and the extracts from some plants of the genus Cassia were found to show inhibitory activities on HIV replication,28 compounds 1−19 were also tested for their anti-HIV-1 activity.29 The cytotoxicity assay against C8166 cells (CC50) and anti-HIV-1 activity were evaluated by the inhibition assay for the cytopathic effects of HIV-1 (EC50), using azidothymidine (AZT) as a positive control (EC50 = 0.034 μg/mL and CC50 > 200 μg/mL). The results are shown in Table 5. Compounds 4, 6, 13, and 19 showed anti-HIV-1 C

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Table 3. IC50 Values of Selected Compounds against TMV on N. glutinosa inhibition rate (%) compound

5 μm

10 μm

20 μm

40 μm

80 μm

160 μm

IC50 (μm)

1 6 10 13 16 ningnamycin

6.21 13.5 5.62 7.68 4.76 11.8

11.4 26.7 9.27 14.5 8.11 22.6

15.9 36.2 14.3 19.8 12.4 31.8

30.3 61.4 25.4 35.6 22.4 48.3

47.2 89.2 36.8 54.6 30.7 61.3

62.4 96.8 54.7 71.2 50.9 82.7

86.4 31.2 127.1 68.1 156.4 49.9

μm, Merck, Darmstadt, Germany), Sephadex LH-20 (Sigma-Aldrich, Inc., USA), or MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan). The fractions were monitored by TLC, and spots were visualized by heating the silica gel plates after spraying with 5% H2SO4 in EtOH. Plant Material. The stems of C. siamea were collected in the Xishuangbangna Prefecture, Yunnan Province, People’s Republic of China, in September 2010. The identity of the plant material was verified by Dr. N. Yuan of the Kunming Institute of Botany, Chinese Academy of Sciences. Voucher specimens (YNNU 10-9-22) have been deposited in the laboratory of the first author of this paper. Extraction and Isolation. The air-dried and powdered stems of C. siamea (4.5 kg) were extracted with 95% aqueous MeOH (5 L × 4 times) at room temperature and filtered. The filtrate was evaporated under reduced pressure, and the crude extract (426 g) was applied to silica gel (150−200 mesh) column chromatography, eluting with a CHCl3−MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to afford fractions A−F. Further separation of fraction B (42 g) on silica gel, eluted with petroleum ether−acetone (9:1−1:2), yielded fractions B1−B7. Fraction B2 (6.28 g) was subjected to silica gel column chromatography using petroleum ether−acetone followed by semipreparative HPLC (65% MeOH−H2O, flow rate 12 mL/min) to give 4 (15.8 mg), 9 (42.7 mg), and 15 (33.3 mg). Fraction B3 (5.72 g), upon separation on silica gel using petroleum ether−acetone and semipreparative HPLC (58% MeOH−H2O, flow rate 12 mL/min), afforded 2 (8.6 mg), 3 (11.8 mg), 5 (14.6 mg), 7 (15.9 mg), 8 (22.9 mg), and 17 (23.1 mg). Fraction B4 (11.6 g) was subjected to silica gel column chromatography using petroleum ether−acetone and semipreparative HPLC (52% MeOH−H2O, flow rate 12 mL/min) to yield 1 (16.5 mg), 6 (12.7 mg), 12 (22.4 mg), and 18 (20.6 mg). On the other hand, separation of fraction C (22.6 g) by silica gel column chromatography, eluted with CHCl3−(CH3)2CO, followed by semipreparative HPLC (40% MeOH−H2O, flow rate 12 mL/min) led to the purification of 10 (24.9 mg). Further separation of fraction D (46.3 g) by silica gel column chromatography, eluted with CHCl3− (CH3)2CO, followed by semipreparative HPLC (25−35% MeOH− H2O, flow rate 12 mL/min) gave 11 (18.4 mg), 13 (15.3 mg), 14 (26.8 mg), 16 (23.6 mg), and 19 (17.5 mg). 2-(3-Hydroxy-1-oxopropyl)-7-hydroxy-5-(2-oxopropyl)-4H-chromen-4-one (1): yellow gum; UV (MeOH) λmax (log ε) 219 (4.46), 238 (3.81), 268 (4.14), 352 (3.79) nm; IR (KBr) νmax 3442, 3365, 2945, 2834, 2753, 1728, 1685, 1654, 1622, 1554, 1448, 1327, 1137, 974, 768 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.16 (1H, s, H-3), 6.67 (1H, d, J = 2.2 Hz, H-6), 6.79 (1H, J = 2.2 Hz, H-8), 3.33 (2H, t, J = 6.1 Hz, H-12), 4.38 (2H, t, J = 6.1 Hz, H-13), 4.15 (2H, s, H-14), 2.29 (3H, s, H-16), 10.51 (1H s, Ar-OH-7); 13C NMR data (CDCl3, 125 MHz), see Table 1; negative ESIMS m/z 289 [M − H]−; HRESIMS m/z 289.0715 [M − H]− (calcd for C15H13O6, 289.0712). 2-(3-Hydroxy-1-oxopropyl)-7-methoxy-5-(2-oxopropyl)-4H-chromen-4-one (2): yellow gum; UV (MeOH) λmax (log ε) 216 (4.38), 240 (3.84), 270 (4.22), 356 (3.83) nm; IR (KBr) νmax 3439, 3367, 2948, 2832, 2759, 1725, 1687, 1658, 1618, 1552, 1449, 1329, 1122, 982, 769 cm−1; 1H NMR (CDCl3, 500 MHz) δ 7.17 (1H, s, H-3), 6.73 (1H, d, J = 2.4 Hz, H-6), 6.87 (1H, J = 2.4 Hz, H-8), 3.36 (2H, t, J = 6.1 Hz, H-12), 4.40 (2H, t, J = 6.1 Hz, H-13), 4.21 (2H, s, H-14), 2.27 (3H, s, H-16), 3.81 (3H s, −OMe-7); 13C NMR data (CDCl3, 125 MHz), see Table 1; negative ESIMS m/z 303 [M − H]−; HRESIMS m/z 303.0862 [M − H]− (calcd for C16H15O6, 303.0869).

Table 4. Protective Effects of Selective Compounds on TMV Infectiona

a

compound

concentration (μm)

1 6 10 13 16 ningnamycin

20 20 20 20 20 20

inhibition rate (%) 18.4 42.7 10.2 22.8 13.7 27.4

± ± ± ± ± ±

2.2 2.8 2.4 2.0 2.5 2.0

All results are expressed as mean ± SD; n = 3 for all groups.

activity with TI values above 50, and compounds 1, 7, 14, 16, and 18 showed anti-HIV-1 activity with TI values above 20. Table 5. Anti-HIV Activities of the Chromones

a

compound

CC50 (μg/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 AZT

126.6 ± 5.8 85.24 ± 4.6 43.58 ± 4.2 >200 26.7 ± 3.9 196.5 ± 6.2 >200 88.68 ± 5.4 37.26 ± 4.0 26.5 ± 2.2 22.8 ± 2.5 57.45 ± 3.6 >200 86.82 ± XXXX 73.42 ± 4.0 69.5 ± 3.8 74.28 ± 4.0 88.34 ± 4.5 118.4 ± 4.7 >200

EC50 (μg/mL)

TIa

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

26.00 16.33 6.87 >106.9 9.37 67.99 >23.64 13.48 8.30 2.30 5.89 10.52 >108.7 49.61 17.15 31.03 15.19 27.96 63.32 >5881

4.87 5.22 6.34 1.87 2.85 2.89 8.46 6.58 4.49 11.54 3.87 5.46 1.84 1.75 4.28 2.24 4.89 3.16 1.87 0.034

0.08 0.12 0.38 0.14 0.22 0.18 0.56 0.42 0.38 0.87 0.36 0.54 0.36 0.22 0.35 0.27 0.53 0.42 0.29 0.02

TI = EC50/CC50, n = 3 for all groups.

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EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. 1D and 2D NMR spectra were recorded on a DRX-500 NMR spectrometer with TMS as internal standard. Chemical shifts (δ) are expressed in ppm with reference to the solvent signals. HRESIMS was performed on a VG Autospec-3000 spectrometer. Semipreparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph equipped with Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C18 (20 mm × 25 cm) columns. Column chromatography was performed using silica gel (200−300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (40−63 D

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For the half-leaf method,24 the virus was inhibited by mixing with the solution of compound. After 30 min, the mixture was inoculated on the left side of the leaves of N. glutinosa, whereas the right side of the leaves was inoculated with the mixture of DMSO solution and the virus as control. The local lesion numbers were recorded 3 or 4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula

2-(3-Hydroxypropyl)-7-methoxy-5-(2-oxopropyl)-4H-chromen-4one (3): yellow gum; UV (MeOH) λmax (log ε) 215 (4.45), 230 (3.86), 262 (4.17), 338 (3.82) nm; IR (KBr) νmax 3432, 2925, 2861, 1723, 1647, 1618, 1558, 1432, 1354, 1129, 1047, 962, 783 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.16 (1H, s, H-3), 6.70 (1H, d, J = 2.4 Hz, H-6), 6.84 (1H, J = 2.4 Hz, H-8), 2.53 (2H, t, J = 8.1 Hz, H-11), 1.84 (2H, m, H-12), 3.55 (2H, t, J = 6.3 Hz, H-13), 4.21 (2H, s, H-14), 2.28 (3H, s, H-16), 3.82 (3H s, −OMe-7); 13C NMR data (CDCl3, 125 MHz), see Table 1; negative ESIMS m/z 289 [M − H]−; HRESIMS m/z 289.1083 [M − H]− (calcd for C16H17O5, 289.1076). 3-(7-Methoxy-4-oxo-5-(2-oxopropyl)-4H-chromen-2-yl)propyl acetate (4): yellow gum; UV (MeOH) λmax (log ε) 216 (4.36), 232 (3.89), 260 (4.11), 335 (3.72) nm; IR (KBr) νmax 3430, 2927, 2864, 1726, 1685, 1652, 1620, 1554, 1436, 1358, 1125, 1055, 974, 785 cm−1; 1 H NMR (CDCl3, 500 MHz) δ 6.18 (1H, s, H-3), 6.72 (1H, d, J = 2.4 Hz, H-6), 6.83 (1H, J = 2.4 Hz, H-8), 2.52 (2H, t, J = 8.1 Hz, H-11), 1.97 (2H, m, H-12), 3.97 (2H, t, J = 6.3 Hz, H-13), 4.20 (2H, s, H-14), 2.28 (3H, s, H-16), 3.80 (3H s, −OMe-7), 2.04 (3H s, −OAc); 13C NMR data (CDCl3, 125 MHz), see Table 1; positive ESIMS m/z 355 [M + Na]+; HRESIMS m/z 355.1159 [M + Na]+ (calcd for C18H20O6Na, 355.1158). 7-Hydroxy-5-(2-oxopropyl)-2-((E)-prop-1-enyl)-4H-chromen-4one (5): yellow gum; UV (MeOH) λmax (log ε) 218 (4.42), 247 (3.92), 272 (4.38), 361 (3.98) nm; IR (KBr) νmax 3438, 2926, 2876, 1729, 1668, 1619, 1561, 1437, 1359, 1142, 957, 792 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.35 (1H, s, H-3), 6.64 (1H, d, J = 2.2 Hz, H-6), 6.80 (1H, d, J = 2.2 Hz, H-8), 6.42 (1H, d, J = 15.8 Hz, H-11), 6.14 (1H, m, H-12), 1.71 (3H, d, J = 6.5 Hz, H-13), 4.18 (2H, s, H-14), 2.28 (3H, s, H-16), 10.76 (1H s, Ar-OH-7); 13C NMR data (CDCl3, 125 MHz), see Table 1; negative ESIMS m/z 257 [M − H]−; HRESIMS m/z 257.0821 [M − H]− (calcd for C15H13O4, 257.0814). 5-Hydoxy-2-(3-hydroxypropyl)-8,8-dimethylpyrano[2,3-f ]chromen-4(8H)-one (6): yellow gum; UV (MeOH) λmax (log ε) 222 (4.51), 245 (3.97), 268 (4.22), 358 (4.02) nm; IR (KBr) νmax 3392, 2962, 2881, 1726, 1662, 1623, 1584, 1426, 1375, 1329, 1284, 1172, 1128, 853 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.34 (1H, s, H-3), 6.21 (1H, s, H-6), 2.57 (2H, d, J = 8.1 Hz, H-11), 1.89 (2H, m, H-12), 3.58 (2H, d, J = 6.3 Hz, H-13), 6.63 (1H, d, J = 10.0 Hz, H-14), 5.64 (1H, s, d, J = 10.0 Hz, H-15), 1.50 (6H, s, H-17 and H-18), 10.73 (1H, s, Ar-OH-5); 13C NMR data (CDCl3, 125 MHz), see Table 1; negative ESIMS m/z 301 [M − H]−; HRESIMS m/z 301.1067 [M − H]− (calcd for C17H17O5, 301.1076). 2-(3-Hydroxypropyl)-5-methoxy-8,8-dimethylpyrano[2,3-f ]chromen-4(8H)-one (7): yellow gum; UV (MeOH) λmax (log ε) UV (MeOH) λmax (log ε) 220 (4.56), 246 (3.95), 270 (4.28), 358 (4.05) nm; IR (KBr) νmax 3396, 2960, 2884, 1723, 1660, 1627, 1580, 1429, 1372, 1325, 1280, 1176, 1129, 858 cm−1; 1H NMR (CDCl3, 500 MHz) δ 6.38 (1H, s, H-3), 6.30 (1H, s, H-6), 2.56 (2H, d, J = 8.1 Hz, H-11), 1.85 (2H, m, H-12), 3.53 (2H, d, J = 6.3 Hz, H-13), 6.61 (1H, d, J = 10.0 Hz, H-14), 5.61 (1H, s, d, J = 10.0 Hz, H-15), 1.48 (6H, s, H-17 and H-18), 3.82 (3H s, −OMe-5); 13C NMR data (CDCl3, 125 MHz), see Table 1; negative ESIMS m/z 315 [M − H]−; HRESIMS m/z 315.1239 [M − H]− (calcd for C18H19O5, 315.1232). Anti-TMV Assays. TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, P. R. China. The virus was multiplied in Nicotiana tabacum cv. K326 and purified as described.30 The concentration of TMV was determined as 20 mg/mL with a UV spectrophotometer [virus concentration = (A260 × dilution ratio)/ ]. The purified virus was kept at −20 °C and was diluted to E0.1%,260nm 1cm 32 μg/mL with 0.01 M PBS before use. Nicotiana glutinosa plants were cultivated in an insect-free greenhouse. N. glutinosa was used as a local lesion host. The experiments were conducted when the plants grew to the 5−6-leaf stage. The tested compounds were dissolved in DMSO and diluted with distilled H2O to the required concentrations. A solution of equal concentration of DMSO was used as a negative control. The commercial antiviral agent ningnanmycin was used as a positive control.

inhibition rate (%) = [(C − T )/C ] × 100% where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment. Anti-HIV-1 Assay. The cytotoxicity assay against C8166 cells (CC50) was assessed using the MTT method, and anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV1 (EC50).29

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ASSOCIATED CONTENT

S Supporting Information *

The structure of known compounds, 1H and 13C NMR, HSQC, HMBC, and HRESIMS spectra of 1, 1H and 13C NMR spectra of 2−7. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-871-5913043, or 5910017. Fax: +86-871-5913043. E-mail: [email protected] (G.-Y.Y.); ganpeng_li@sina. com (G.-P.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the Excellent Scientific and Technological Team of Yunnan Higher Education (2010CI08), the Yunnan University of Nationalities Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and the Open Research Fund Program of Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan University of Nationalities) (2010XY08).

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