Antiviral Matrine-Type Alkaloids from the Rhizomes of Sophora

Article pubs.acs.org/jnp

Antiviral Matrine-Type Alkaloids from the Rhizomes of Sophora tonkinensis Qi-Ming Pan,† Yu-Huan Li,‡ Jing Hua,† Fu-Ping Huang,† Heng-Shan Wang,† and Dong Liang*,† †

State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, People’s Republic of China ‡ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Three new matrine-type alkaloids, (+)-5αhydroxyoxysophocarpine (1), (−)-12β-hydroxyoxysophocarpine (2), and (+)-5α-hydroxylemannine (3), along with 14 known analogues, (−)-sophocarpine (4), (−)-5αhydroxysophocarpine (5), (−)-9α-hydroxysophocarpine (6), (+)-12α-hydroxysophocarpine (7), (−)-12β-hydroxysophocarpine (8), (+)-oxysophocarpine (9), (+)-matrine (10), (+)-sophoranol (11), (+)-9α-hydroxymatrine (12), (−)-14β-hydroxymatrine (13), (+)-oxymatrine (14), (+)-5αhydroxyoxymatrine (15), (−)-14β-hydroxyoxymatrine (16), and (+)-sophoramine (17), were isolated from the rhizomes of Sophora tonkinensis. Their structures were elucidated via spectrometric data analyses, and the absolute configurations were established by single-crystal X-ray diffraction and ECD data. Alkaloids 2, 6, 11, and 13 exhibited antiviral activity against the Coxsackie virus B3 (CVB3), with IC50 values of 26.62−252.18 μM, and alkaloids 7, 8, and 17 inhibited influenza virus A/Hanfang/359/95 (H3N2) replication with IC50 values of 63.07− 242.46 μM.

T

he roots and rhizomes of Sophora tonkinensis Gepnep. (Leguminosae), commonly known as “Shan-Dou-Gen” in Chinese, have been used as a traditional Chinese medicine for the treatment of fever, throat inflammation, and tumors.1 The plant contains quinolizidine alkaloids, particularly matrine-type alkaloids, as principal constituents.2,3 Previous pharmacological studies showed that the crude alkaloids exhibited potent antiviral activity against Coxsackie virus B3 (CVB3) and B5 (CVB5), respiratory syncytial virus (RSV), and hepatitis B virus (HBV).3,4 Research toward the discovery of novel antiviral agents from the rhizomes of S. tonkinensis afforded three new (1−3) and 14 known matrine-type alkaloids (4−17). This paper deals with the isolation and structure elucidation of the new alkaloids, as well as the in vitro antiviral activities against CVB3 and influenza virus A/Hanfang/359/95 (H3N2) of the isolated alkaloids.

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RESULTS AND DISCUSSION Compound 1 was obtained as colorless needles from CH2Cl2− MeOH, [α]20 D +33 (c 0.8, MeOH). Its molecular formula was determined as C15H22N2O3 on the basis of 13C NMR data and the positive-ion HRESIMS (m/z 279.1695 [M + H]+, calcd 279.1703). The IR spectrum displayed absorption bands characteristic of hydroxy (3441 cm−1), α,β-unsaturated lactam (1661 and 1609 cm−1), and N→O (953 cm−1) functionalities.3 UV absorptions at 247 and 334 nm also suggested the presence of an α,β-unsaturated lactam group. The 1H NMR spectrum © XXXX American Chemical Society and American Society of Pharmacognosy

displayed signals of two olefinic protons (δH 6.69 and 5.85) and three deshielded aliphatic proton signals (δH 4.61, 4.04, and 3.87). Its 13C NMR and DEPT data (Table 1) revealed the Received: April 12, 2015

A

DOI: 10.1021/acs.jnatprod.5b00325 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Table 1. 1H and 13C NMR Data for Compounds 1−3a 1 (methanol-d4)

a1

position

δC

2a 2b 3a 3b 4a 4b 5 6 7 8a 8b 9a 9b 10a 10b 11 12a 12b 13 14 15 17a 17b

68.7 19.3 36.9 67.2 71.8 35.6 25.0 17.8 69.7 53.2 31.2 141.3 124.8 169.4 49.1b

δH (J in Hz) 3.34, 3.02, 2.44, 1.70, 1.95, 1.68,

m m m m m m

3.06, 2.43, 2.01, 1.67, 2.47, 1.55, 3.38, 3.14, 4.61, 2.72, 2.09, 6.69, 5.85,

d (3.6) m m m m m m m ddd (11.5, 5.5, 1.8) dt (17.8, 5.5) m dm (9.7) dd (9.7, 2.4)

4.04, d (13.5) 3.87, d (13.5)

2 (methanol-d4)

3 (CDCl3)

δC

δH (J in Hz)

δC

68.7c

3.72 (overlapped) 3.59 (overlapped) 2.38 (overlapped) 1.82 (overlapped) 1.95, m 1.84, m 2.14, m 3.85, br s 2.07, m 2.47, m 1.79, m 2.38 (overlapped) 1.82 (overlapped) 3.72 (overlapped) 3.59 (overlapped) 4.47, dd (11.7, 5.5) 4.34, dd (5.5, 4.0)

56.5

18.0d 25.6 34.9 70.7 41.4 24.3 17.9d 68.4c 60.9 65.9 142.1 124.2 165.4 43.9

6.55, dd (9.9, 4.0) 5.89, d (9.9) 4.28, dd (13.1, 5.5) 3.48, dd (13.1, 13.1)

H and 13C NMR were measured at 500 and 125 MHz, respectively. bSignal overlapped by solvent peaks.

presence of one carbonyl (δC 169.4), two sp2 methines (δC 141.3 and 124.8), and 12 sp3 carbons including eight methylenes, three methines, and one oxygenated tertiary carbon. The aforementioned data indicated that 1 was likely a matrine-type alkaloid. On the basis of literature data, the NMR data of 1 were similar to those of (+)-oxysophocarpine (9),5 except for the presence of a hydroxy group in 1. In the HMBC spectrum, the correlations from H2-4 (δH 1.95, 1.68)/ H-6 (δH 3.06)/H-17β (δH 3.87) to C-5 (δC 67.2) located the hydroxy group at the C-5 tertiary carbon. This was confirmed by the absence of 3JH,H couplings of H-5 with H-17α and H-17β of 1 (Table 1) compared to 9, while the 3JH‑5,H‑17α (6.5 Hz) and 3 JH‑5,H‑17β values (13.0 Hz) of 9 could be deduced from its 1H NMR spectrum (Supporting Information, Figure S26). The relative configuration of 1 was partially assigned by a NOESY spectrum, in which the correlation of H-6/H-7 showed that they were cofacial. Finally, the structure of 1 was confirmed by single-crystal X-ray diffraction (Cu Kα) analysis, which also determined its absolute configuration [a Flack parameter of 0.13(19)] (Figure 1). Thus, the structure of compound 1 was defined as (+)-5α-hydroxyoxysophocarpine. Compound 2, colorless oil ([α]20 D −90 (c 0.03, MeOH)), gave the same molecular formula of C15H22N2O3 as 1 from its 13 C NMR and HRESIMS data (m/z 279.1696 [M + H]+, calcd 279.1703). The 13C and 1H NMR data (Table 1) showed that alkaloid 2 was an isomer of 1, the only difference being the presence of a hydroxy group at C-12 (δC 65.9) in 2 rather than at C-5 in 1. This was confirmed by the COSY correlations of H11 (δH 4.47)/H-12 (δH 4.34)/H-13 (δH 6.55) and HMBC correlations from H-12 to C-7 (δC 41.4)/C-13 (δC 142.1)/C14 (δC 124.2). Collectively, the combination of these correlations and the shifts of the proton and carbon resonances confirmed that the hydroxy group was located at C-12 in 2. The relative configuration of 2 was defined from the NOESY

22.4 37.0 69.0 68.2 38.4 26.2 20.7 56.9 54.8 122.5 123.3 31.7 168.4 46.7 c,d

δH (J in Hz) 2.77, br d (11.4) 2.00, m 1.59, m 1.85, br d (11.5) 1.56, m 1.90, 2.14, 1.96, 1.35, 1.67, 1.46, 2.88, 2.03, 4.43, 5.86,

br s br d (10.3) m m m m br d (11.1) m dd (10.3, 3.8) dm (10.4)

5.77, dm (10.4) 2.97, br d (1.4) 4.38, d (13.4) 3.29, d (13.4)

Signals may be interchanged.

Figure 1. X-ray ORTEP drawing of compound 1.

spectrum, in which the correlations of H-6/H-5 and H-6/H-7 revealed that they were cofacial and arbitrarily assigned to be αoriented. The NOESY cross-peaks of H-5/H-17α and H-11/H17β confirmed the β-orientation of H-11. Consequently, OH12 was fixed in a β-orientation by the key NOESY correlation of H-12/H-7. The absolute configuration of 2 was established on the basis of the ECD spectroscopic evidence. As shown in X-ray diffraction analyses using Cu Kα radiation (Figures 2 and 3), (+)-12α-hydroxysophocarpine (7) and (−)-12β-hydroxysophocarpine (8) were epimers at C-12. Comparison of the ECD curves of 7 and 8 (Figure 4) suggested that the C-12 absolute B



Article

Compound 3 was isolated as colorless crystals from CH2Cl2−MeOH, [α]20 D +25 (c 0.7, MeOH), and its molecular formula was established as C15H22N2O2 by the 13C NMR and HRESIMS data (m/z 263.1747 [M + H]+, calcd 263.1754). The IR spectrum showed absorption bands for a hydroxy (3388 cm−1), a lactam carbonyl (1614 cm−1), and a trans-quinolizidine moiety (2863, 2790, and 2741 cm−1).6 The 1H NMR spectrum showed signals of two olefinic protons (δH 5.86 and 5.77), and three deshielded aliphatic proton signals at δH 4.43, 4.38, and 3.29 were assigned to H-11, H-17α, and H-17β, respectively. Its 1 H and 13C NMR data were similar to those of (+)-lemannine,2 a known matrine-type alkaloid isolated from S. tonkinensis, except for a hydroxy group at a tertiary carbon in 3. The COSY cross-peaks of H-11 (δH 4.43)/H-12 (δH 5.86)/H-13 (δH 5.77)/H2-14 (δH 2.97), together with the HMBC correlations from H-13 to C-15 (δC 168.4) and from H2-14 to C-12 (δC 122.5)/C-13 (δC 123.3), located the double bond at C-12 in 3. Meanwhile, the absence of 3JH,H couplings of H-5 with H-17α and H-17β (Table 1) and the HMBC correlations from H2-4 (δH 1.85, 1.56)/H-6 (δH 1.90)/H-17α (δH 4.38) to C-5 (δC 69.0) suggested that the hydroxy group was located at C-5. The structure of 3 was subsequently confirmed by single-crystal Xray diffraction using Cu Kα radiation (Figure 5). Its absolute




configuration was then established through the refinement of the Flack parameter [χ = 0.07(17)]. Thus, the structure of compound 3 was defined as (+)-5α-hydroxylemannine. The known alkaloids were identified as (−)-sophocarpine (4),5 (−)-5α-hydroxysophocarpine (5),7 (−)-9α-hydroxysophocarpine (6), 8 (+)-12α-hydroxysophocarpine (7), 9 (−)-12β-hydroxysophocarpine (8),8 (+)-oxysophocarpine (9),5 (+)-matrine (10),2 (+)-sophoranol (11),10 (+)-9αhydroxymatrine (12), 11 (−)-14β-hydroxymatrine (13),2 (+)-oxymatrine (14),5 (+)-5α-hydroxyoxymatrine (15),10 (−)-14β-hydroxyoxymatrine (16),3 and (+)-sophoramine (17)12 by NMR analysis and comparison with literature data. Although the matrine-type alkaloids, the main active components of the genus Sophora, have been extensively investigated, three new and 14 known alkaloids of this type were isolated from S. tonkinensis. Compound 3 is the second lemannine derivative with a C-12 double bond. The single-

Figure 4. ECD spectra of compounds 2, 7, and 8.

configuration was the prime factor controlling the sign of the Cotton effects but not necessarily the magnitudes. As shown in Figure 4, the ECD curve of 2 matched that of 8, indicating that 2 shared the same absolute configuration at C-12 as 8. Together with the established relative configuration, the absolute configuration of (−)-12β-hydroxyoxysophocarpine (2) was assigned as depicted. C



Article

HRESIMS data were obtained with a Thermo-Scientific Exactive spectrometer. X-ray data were collected on a Bruker APEX DUO diffractometer. Silica gel (200−300 mesh, Qingdao Marine Chemical Factory, China), Sephadex LH-20 gel (Pharmacia Biotech, Sweden), ODS (50 μm, YMC, Japan), and MCI gel (CHP20, 75−150 μm, Mitsubishi Chemical Corporation, Japan) were used for column chromatography (CC). TLC was carried out with GF254 plates (Qingdao Marine Chemical Factory). Spots were visualized by spraying with Dragendorff’s reagent and/or 10% H2SO4 acid in EtOH followed by heating. Plant Material. The rhizomes of S. tonkinensis were collected in December 2012 from Nanning, Guangxi Province, China, and identified by Professor Chun-Rui Lin of the Guangxi Institute of Botany, Chinese Academy of Sciences. A voucher specimen (No. ST2012018) was deposited in the State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin. Extraction and Isolation. The air-dried rhizomes of S. tonkinensis (8.3 kg) were extracted three times with 95% aqueous EtOH. The extract was partitioned between EtOAc and 3% tartaric acid−H2O. Water-soluble materials, which were adjusted to pH 9 with saturated Na2CO3, were extracted with CH2Cl2 and n-BuOH, successively. The CH2Cl2-soluble residue (42.0 g) was subjected to silica gel CC eluting with a gradient of petroleum ether−acetone−Et2NH (20:1:0.1 to 1:1:0.1), then CH2Cl2−MeOH−Et2NH (4:1:0.1 to 1:1:0.1) to afford fractions A−H. Fraction B (23.5 g) was further separated by RP-C18 silica gel CC (MeOH−H2O, 10:90 to 100:0) to afford four subfractions (B1−B4). Fraction B1 (50 mg) was purified by Sephadex LH-20 CC (MeOH) to yield 10 (32.4 mg). Fraction B2 (450 mg) was chromatographed over silica gel CC (petroleum ether−EtOAc− Et2NH, 11:1:0.1 to 7:1:0.1) to afford 4 (21.8 mg) and 13 (17.5 mg). Fraction C (3.4 g) was separated by silica gel CC (petroleum ether− EtOAc−Et2NH, 4:1:0.1 to 1:1:0.1) to give five subfractions (C1−C5). Fraction C3 (2.4 g) was subjected to RP-C18 CC (MeOH−H2O, 10:90 to 100:0) to afford 3 (7.0 mg), 5 (32.0 mg), and 17 (3.8 mg). Fraction C4 (808.1 mg) was also subjected to RP-C18 CC (MeOH− H2O, 10:90 to 100:0) to give 11 (20.6 mg). Fraction D (600 mg) was chromatographed on Sephadex LH-20 (MeOH) and then RP-C18 CC (MeOH−H2O, 20:80 to 100:0) to yield 7 (14.5 mg) and 8 (3.0 mg). Fraction E (1.0 g) was separated by RP-C18 CC (MeOH−H2O, 5:95 to 100:0) to afford seven subfractions (E1−E7). Fraction E5 (71.5 mg) was applied to Sephadex LH-20 CC (MeOH) to give 12 (10.3 mg), and fraction E6 (27.7 mg) was subjected to silica gel CC (CH2Cl2−MeOH−Et2NH, 26:1:0.1) to give 6 (20.0 mg). Fraction F (4.2 g) was chromatographed over repeated silica gel CC (petroleum ether−EtOAc−Et 2 NH, 5:4:0.1, and CH 2 Cl 2 −MeOH−Et 2 NH, 30:1:0.1 to 23:1:0.1) to afford 14 (17.0 mg). Compound 9 (18.6 mg) was purified from fraction G (4.0 g) by RP-C18 CC eluting with MeOH−H2O (10:90 to 100:0). The n-BuOH-soluble part (79.2 g) was applied to MCI gel CC eluting with a gradient of MeOH−H2O to give five fractions (N1−N5). Fraction N2 (2.8 g) was subjected to RPC18 CC (MeOH−H2O, 5:95 to 100:0) to afford subfractions N2a− N2g. Fraction N2b (136.7 mg) was purified by Sephadex LH-20 CC (MeOH) to afford 1 (45.0 mg). Fraction N2d (357.2 mg) was purified by Sephadex LH-20 (MeOH) and then silica gel CC (CH2Cl2− MeOH−Et2NH, 30:1:0.1 to 10:1:0.1) to give 2 (4.3 mg) and 15 (7.4 mg). Fraction N2f (48.2 mg) was separated by RP-C18 CC (MeOH− H2O, 5:95 to 15:85) and Sephadex LH-20 CC (MeOH) to afford 16 (3.8 mg). (+)-5α-Hydroxyoxysophocarpine (1): colorless needles (CH2Cl2− MeOH); mp 195−196 °C; [α]20 D +33 (c 0.8, MeOH); UV (MeOH) λmax (log ε) 247 (3.38), 334 (3.09) nm; IR (KBr) νmax 3441, 2938, 2863, 1661, 1609, 1416, 953, 822 cm−1; 1H NMR (methanol-d4, 500 MHz), 13C NMR (methanol-d4, 125 MHz) see Table 1; (+) HRESIMS m/z 279.1695 [M + H]+ (calcd for C15H23N2O3, 279.1703). (−)-12β-Hydroxyoxysophocarpine (2): colorless oil; [α]20 D −90 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 250 (3.34), 321 (2.98) nm; ECD (MeOH) λmax (Δε) 215 (− 17.70), 277 (− 2.16) nm; IR (KBr)

crystal X-ray diffraction analysis (Cu Kα) together with ECD data permitted the unambiguous definition of the absolute configurations of the new matrine-type alkaloids. Since certain matrine-type alkaloids and the crude alkaloids from S. tonkinensis were reported to exhibit potent antiviral activities,3,4 the isolated alkaloids were evaluated for their antiviral activities against Coxsackie virus B3 in Vero cells and influenza virus A/Hanfang/359/95 in MDCK cells. As shown in Table 2, compounds 2, 6, 11, and 13 exhibited strong antiviral activity against CVB3, with IC50 values of Table 2. Antiviral Activities of 2, 6, 11, and 13a against Coxsackie Virus B3 (CVB3) sample

TC50 (μM)

IC50 (μM)

SIb

2 6 11 13 RBVc

115.14 >762.34 >756.52 756.52 8189.67

26.62 197.22 252.18 184.14 1197.58

4.3 >3.9 >3.0 4.1 6.8

a

Compounds 1, 3−5, 7−10, 12, and 14−17 were inactive at their maximal nontoxic concentration. bSelectivity index value equaled TC50/IC50. cPositive control.

26.62−252.18 μM and selectivity index (SI) values all above 3.0. In particular, the most effective alkaloid, 2 (IC50 = 26.62 μM), showed ca. 45 times higher activity compared with the positive control ribavirin (RBV) (IC50 = 1197.58 μM), with therapeutic selectivity index similar to that of RBV. The data in Table 3 showed that compounds 7, 8, and 17 are the most potent inhibitors against H3N2, with IC50 values of Table 3. Antiviral Activities of 5, 7−9, and 17a against Influenza Virus A/Hanfang/359/95 (H3N2) sample

TC50 (μM)

IC50 (μM)

SIb

5 7 8 9 17 RBVc oseltamivirc

>762.34 478.83 >762.34 >762.34 272.86 4766.80 3070.18

440.14 84.70 242.46 402.82 63.07 5.86 1.07

>1.7 5.7 >3.1 >1.9 4.3 813.4 2869.3

a Compounds 1−4, 6, and 10−16 were inactive at their maximal nontoxic concentration. bSelectivity index value equaled TC50/IC50. c Positive control.

63.07−242.46 μM and therapeutic index values above 3.1. The most potent inhibitors, 7, 8, and 17, and moderately effective compounds 5 and 9 are sophocarpine or sophoramine derivatives, which suggested that the introduction of an α,β carbon−carbon double bond might play an important role in the anti-H3N2 activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an SGW X-4 micromelting point apparatus and are uncorrected. Optical rotations were recorded on a PerkinElmer 341 polarimeter, and UV spectra with a PerkinElmer 650 spectrophotometer. IR spectra were obtained on a PerkinElmer Spectrum Two FT-IR spectrometer with KBr disks. ECD spectra were recorded on a JASCO J-810 spectropolarimeter. NMR measurements were performed on Bruker AVANCE 500 MHz spectrometer. Chemical shifts are expressed in δ (ppm) and referenced to the residual solvent signals. D



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νmax 3417, 2932, 1667, 1600, 1385, 1029 cm−1; 1H NMR (methanold4, 500 MHz), 13C NMR (methanol-d4, 125 MHz) see Table 1; (+) HRESIMS m/z 279.1696 [M + H]+ (calcd for C15H23N2O3, 279.1703). (+)-5α-Hydroxylemannine (3): colorless crystals (CH 2Cl2− MeOH); mp 191−192 °C; [α]20 D +25 (c 0.7, MeOH); IR (KBr) νmax 3388, 2927, 2863, 2790, 2741, 1614, 1475, 1126 cm−1; 1H NMR (CDCl3, 500 MHz), 13C NMR (CDCl3, 125 MHz) see Table 1; (+) HRESIMS m/z 263.1747 [M + H]+ (calcd for C15H23N2O2, 263.1754). X-ray Diffraction Analysis. The X-ray crystallographic data of compounds 1, 3, 7, and 8 were collected on a Bruker APEX-II CCD diffractometer using Cu Kα radiation. The structures were solved by direct methods using SHELXS-9713 and refined with full-matrix leastsquares calculations on F2 using SHELXL-9713 via OLEX2.14 All nonhydrogen atoms were refined anisotropically. The hydrogen atom positions were geometrically idealized and allowed to ride on their parent atoms. Crystallographic data for 1, 3, 7, and 8 have been deposited in the Cambridge Crystallographic Data Centre (CCDC). Copies of these data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax: +44 (0) 1223-336033 or e-mail: [email protected]]. Crystallographic data for 1: C 15 H 22 N 2 O 3 , M = 278.35, orthorhombic, a = 9.052(3) Å, b = 10.577(3) Å, c = 14.128(4) Å, α = 90°, β = 90°, γ = 90°, V = 1352.6(7) Å3, T = 296.15 K, space group P212121, Z = 4, μ(Cu Kα) = 0.775 mm−1, 12 359 reflections measured, 2264 independent reflections (Rint = 0.0418). The final R1 values were 0.0301 [I > 2σ(I)]. The final wR(F2) values were 0.0763 [I > 2σ(I)]. The final R1 values were 0.0308 (all data). The final wR(F2) values were 0.0771 (all data). The goodness of fit on F2 was 1.125. Flack parameter: 0.13(19). CCDC number: 1054280. Crystallographic data for 3: C15H22N2O2, M = 262.35, monoclinic, a = 6.62720(10) Å, b = 14.2719(2) Å, c = 7.24420(10) Å, α = 90°, β = 103.27°, γ = 90°, V = 666.884(17) Å3, T = 296.15 K, space group P21, Z = 2, μ(Cu Kα) = 0.695 mm−1, 11 521 reflections measured, 2143 independent reflections (Rint = 0.0204). The final R1 values were 0.0261 [I > 2σ(I)]. The final wR(F2) values were 0.0689 [I > 2σ(I)]. The final R1 values were 0.0262 (all data). The final wR(F2) values were 0.0691 (all data). The goodness of fit on F2 was 1.074. Flack parameter: 0.07(17). CCDC number: 1054279. Crystallographic data for 7: C15H22N2O2·H2O, M = 280.36, orthorhombic, a = 7.9518(14) Å, b = 12.143(2) Å, c = 14.869(2) Å, α = 90°, β = 90°, γ = 90°, V = 1435.8(4) Å3, T = 296.15 K, space group P212121, Z = 4, μ(Cu Kα) = 0.731 mm−1, 36 759 reflections measured, 2430 independent reflections (Rint = 0.1291). The final R1 values were 0.0351 [I > 2σ(I)]. The final wR(F2) values were 0.0794 [I > 2σ(I)]. The final R1 values were 0.0773 (all data). The final wR(F2) values were 0.0859 (all data). The goodness of fit on F2 was 1.094. Flack parameter: −0.1(2). CCDC number: 1054281. Crystallographic data of 8: C 15 H 22 N 2 O 2 , M = 262.35, orthorhombic, a = 5.8201(6) Å, b = 14.8858(14) Å, c = 15.1911(15) Å, α = 90°, β = 90°, γ = 90°, V = 1316.1(2) Å3, T = 296.15 K, space group P212121, Z = 4, μ(Cu Kα) = 0.704 mm−1, 11 910 reflections measured, 2190 independent reflections (Rint = 0.0305). The final R1 values were 0.0270 [I > 2σ(I)]. The final wR(F2) values were 0.0658 [I > 2σ(I)]. The final R1 values were 0.0272 (all data). The final wR(F2) values were 0.0659 (all data). The goodness of fit on F2 was 1.103. Flack parameter: 0.1(2). CCDC number: 1054278. Cytotoxicity Assay. The cytotoxicity of compounds in Vero and MDCK cells was monitored by a cytopathogenic effect (CPE).15 Vero and MDCK cells were plated into a 96-well plate. After 24 h, the monolayer cells were incubated in the presence of various concentrations of test compounds. After 48 h of culture at 37 °C and 5% CO2 in a CO2 incubator, the cells were monitored by CPE. The median toxic concentration (TC50) was calculated by the methods of Reed and Muench.16 In Vitro Anti-CVB3 and H3N2 Activity Assays. Confluent Vero cells grown in 96-well microplates were infected with 100 median tissue culture infective doses (100TCID50) of CVB3 virus.15 MDCK cells were infected with H3N2.15 After 1 h adsorption at 37 °C, the

monolayers were washed by phosphate-buffered saline (PBS) and incubated at 37 °C in maintenance media (MEM plus 2% fetal bovine serum (FBS)) with or without different concentrations of test compounds. Viral CPE was observed when the viral control group reached 4+, and the antiviral activity of tested compounds was calculated by the methods of Reed and Muench.16

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR, 1H−1H COSY, HSQC, HMBC, NOESY, and HRESIMS spectra of compounds 1−3, DEPT-135 spectrum of compound 1, 1H NMR spectrum of compound 9, and ECD spectra of compounds 2, 7, and 8. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00325.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (21431001), the Ministry of Education of China (IRT1225), the Guangxi Scientific Research and Technology Development Program (1355004-3), the Talent’s Small Highland Project of Guangxi Medicinal Industry (1408), the State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources, Ministry of Science and Technology of China (CMEMR2012-A18 and CMEMR2013-C04), and the Fund of Guangxi Key Laboratory of Functional Phytochemicals Research and Utilization (FPRU2013-1).

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REFERENCES

(1) Chinese Pharmacopoeia Commission. Pharmacopoeia of the People’s Republic of China (Part I); China Medical Science Press: Beijing, 2010; pp 25−26. (2) Xiao, P.; Li, J. S.; Kubo, H.; Saito, K.; Murakoshi, I.; Ohmiya, S. Chem. Pharm. Bull. 1996, 44, 1951−1953. (3) Ding, P. L.; Huang, H.; Zhou, P.; Chen, D. F. Planta Med. 2006, 72, 854−856. (4) Fan, H. W.; Lu, J. H.; Zhang, R. Inf. Tradit. Chin. Med. 2000, 17, 75−76. (5) Qiao, L.; Huang, L. R.; Gao, C. Y.; Zhao, Y. Y.; Yang, X. B.; Zhang, L. H. J. Beijing Med. Univ. 1994, 26, 485−486. (6) Atta-ur-Rahman; Choudhary, M. I.; Parvez, K.; Ahmed, A.; Akhtar, F.; Nur-e-Alam, M.; Hassan, N. M. J. Nat. Prod. 2000, 63, 190−192. (7) Saito, K.; Arai, N.; Sekine, T.; Ohmiya, S.; Kubo, H.; Otomasu, H.; Murakoshi, I. Planta Med. 1990, 56, 487−488. (8) Xiao, P.; Kubo, H.; Komiya, H.; Higashiyama, K.; Yan, Y. N.; Li, J. S.; Ohmiya, S. Phytochemistry 1999, 50, 189−193. (9) Ding, P. L.; Liao, Z. X.; Huang, H.; Zhou, P.; Chen, D. F. Bioorg. Med. Chem. Lett. 2006, 16, 1231−1235. (10) Zhao, Y. Y.; Pang, Q. Y.; Liu, J. B.; Chen, Y. Y.; Lou, Z. C. Nat. Prod. Res. Dev. 1994, 6, 10−13. (11) Negrete, R.; Cassels, B. K.; Eckhardt, G. Phytochemistry 1983, 22, 2069−2072. (12) Liu, B.; Shi, R. B. Chin. J. Chin. Mater. Med. 2006, 31, 557−560. (13) Sheldrick, G. M. Acta Crystallogr., Sect. A: Found. Crystallogr. 2008, A64, 112−122. (14) Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339−341.

E



Article

(15) Schmidtke, M.; Schnittler, U.; Jahn, B.; Dahse, H. M.; Stelzner, A. J. Virol. Methods 2001, 95, 133−143. (16) Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497.

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