Matrine-Type Alkaloids from the Roots of Sophora flavescens and

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Cite This: J. Nat. Prod. 2018, 81, 2259−2265

Matrine-Type Alkaloids from the Roots of Sophora flavescens and Their Antiviral Activities against the Hepatitis B Virus Yu-Bo Zhang,†,‡ Ding Luo,† Li Yang,† Wen Cheng,† Li-Jun He,† Guang-Kai Kuang,† Man-Mei Li,† Yao-Lan Li,*,† and Guo-Cai Wang*,†,‡

J. Nat. Prod. 2018.81:2259-2265. Downloaded from pubs.acs.org by UNIV OF SUNDERLAND on 10/26/18. For personal use only.

†

Institute of Traditional Chinese Medicine & Natural Products, Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, College of Pharmacy, Jinan University, Guangzhou 510632, People’s Republic of China ‡ Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou 510632, People’s Republic of China S Supporting Information *

ABSTRACT: Eight new matrine-type alkaloids, flavesines G−J (1−4), alopecurine B (5), 7,11-dehydro-oxymatrine (6), 10-oxy-5,6-dehydromatrine (7), and 10-oxysophoridine (8), along with nine known analogues (9−17) were isolated from the roots of Sophora f lavescens. Compounds 1−3 are the first natural matrine-type alkaloids with an open-loop ring D, while compound 4 represents an unprecedented dimerization pattern constructed from matrine and piperidine, and 5 is the first example of a matrine-type alkaloid with cleavage of the C-5−C-6 bond. The new structures were elucidated by means of spectroscopic data analysis (including NMR, MS, IR, and UV), and the absolute configurations were determined using single-crystal X-ray diffraction and ECD data. The isolated alkaloids were evaluated for their antiviral activity against hepatitis B virus, and compounds 1, 4, 5, 10, and 14 exhibited comparable antiviral potencies to matrine.

T

he perennial herbaceous plant Sophora flavescens Aiton (Leguminosae) is distributed widely in mainland China, Russia, Korea, Japan, and India.1 As a traditional Chinese medicine, the roots of S. f lavescens (“Kushen” in Chinese) have also been used for the treatment of dysentery, eczema, trichomonas vaginitis, and pyogenic infections of the skin.2 Previous phytochemical investigations of S. f lavescens have shown that it contains large amounts of quinolizidine alkaloids, especially matrine-type alkaloids.3−11 Matrine-type alkaloids were isolated initially from S. f lavescens in 1895 by a Japanese group11 and were found to have a variety of activities including antiviral,4,12,13 antitumor,14 anti-inflammatory,15 antibacterial,16 and insecticidal17,18 qualities. In particular, this kind of alkaloids exihibited potent antiviral activity against hepatitis B virus (HBV).4,12 Moreover, two representative matrine-type alkaloids, matrine and oxymatrine, have been used for the clinical treatment of hepatitis B in China.19,20 Recently, our group reported several novel matrine-based alkaloids from Sophora species, including five dimeric matrinetype alkaloids from S. f lavescens4 and four matrine-based alkaloids with new skeletons from Sophora alopecuroides.21 In an ongoing endeavor to search for antiviral and structurally novel compounds from Chinese medicinal plants, eight new matrine-type alkaloids (compounds 1−8) and nine known analogues (compounds 9−17) (Figure 1) were isolated from the roots of S. flavescens. Compounds 1−3 are the first © 2018 American Chemical Society and American Society of Pharmacognosy

Figure 1. Chemical structures of compounds 1−17.

naturally occurring matrine-type alkaloids with an open-loop ring D. Compound 4 represents an unprecedented dimerizaReceived: July 19, 2018 Published: October 9, 2018 2259

DOI: 10.1021/acs.jnatprod.8b00576 J. Nat. Prod. 2018, 81, 2259−2265

Journal of Natural Products

Article

olefinic carbons (δC 149.8, 115.4, and 114.3), two methines (δC 57.1 and 39.5), and two methylenes connected to heteroatoms (δC 53.1 and 52.4), indicating that its structure is similar to that of 1. The most notable differences were the olefinic carbons at C-7 (δC 115.4) and C-11 (δC 150.1) in 1 that were hydrogenated to two methines (δC 57.1 and 39.5) in 2, which was confirmed by the 1H−1H COSY correlations of H2-10 (δH 3.56)/H2-9 (δH 1.71)/H-8a (δH 2.04)/H-8b (δH 1.30)/H-7 (δH 2.71)/H-11 (δH 3.39)/H-12a (δH 2.77)/H2-13 (δH 1.84, 1.69)/H2-14 (δH 2.21), together with the HMBC correlations between H-8a and C-6 (δC 165.0), C-10 (δC 52.4), between H2-17 (δH 7.41) and C-4 (δC 23.8), C-5 (δC 97.8), C-6, C-11 (δC 57.1), and beween H2-14 (δH 2.21) and C-15 (δC 181.9). The relative configuration of 2 was deduced through the NOESY correlations between H-11 and H-8a, as well as between H-7 and H-12a (Figure 4). In addition, the absolute configuration of 2 was determined by comparing the experimental and calculated electronic circular dichroism (ECD) spectra using the time-dependent density functional theory (TDDFT) method. The obtained results indicated that the experimental ECD spectrum of 2 and calculated ECD spectrum of (7S,11S)-2 were in good agreement (Figure 5), allowing the assignment of the absolute configuration as 7S and 11S. Compound 3 (flavesine I) showed a [M + H]+ ion peak at m/z 263.1756, consistent with the molecular formula C15H22N2O2. The 1H and 13C NMR spectra of 3 (Table 1) were also similar to those of 1 except for the presence of an extra methine (δC 35.7), an extra methylene connected to a heteroatom (δC 45.5), and the absence of two olefinic carbons (δC 135.7 and 116.9), and the chemical shifts of C-4, C-6, C-7, and C-11 shifted from δC 25.2, 153.7, 115.4, 150.1 to δC 23.1, 163.7, 96.4, 169.9. These differences indicated that the double bond between C-5 and C-17 is hydrogenated. Subsquently, the 1 H−1H COSY correlations of H2-2 (δH 3.45)/H2-3 (δH 1.94)/ H2-4 (δH 2.01, 1.34)/H-5 (δH 2.79)/H2-17 (δH 3.51, 3.09), along with the HMBC correlations between H-17b and C-4 (δC 23.1), C-6 (δC 163.7), C-11 (δC 169.9) and between H12a and C-7 (δC 96.4), C-11, were used to confirm the structure of 3. Although there is a chiral center at C-5 in 3, the optical activity and Cotton effect for 3 were too weak to be detected, suggesting that 3 was a racemate. Compound 3 could not be separated by the chiral analytical conditions used. Thus, 3 is reported as being a racemic mixture. Compound 4 (flavesine J) was isolated as a yellowish oil. The molecular formula of 4 was determined as C20H29N3O by its HRESIMS (m/z 328.2396 [M + H]+, calcd for C20H30N3O, 328.2383). Comparison of the 1H and 13C NMR data of 4 (Table 1) with those of 1 showed many similarities, except for the presence of an additional piperidine ring (δC 48.0, 43.9, 27.6, 26.8, and 25.5), with the chemical shifts of C-14 and C15 shifting from δC 38.1 and 181.4 in 1 to δC 33.7 and 173.4 in 4. These differences indicated that a piperidine ring is connected to the matrine moiety through an amide bond in 4, which was further supported by the 1H−1H COSY correlations of H2-2′ (δH 3.45)/H2-3′ (δH 1.57)/H2-4′ (δH 1.65)/H2-5′ (δH 1.52)/H2-6′ (δH 3.52), together with the HMBC correlations between H2-2′/H2-4′ and C-15 (δC 173.4) (Figure 2). Compound 5 (alopecurine B) was obtained as colorless crystals from MeOH−H2O (2:1), mp 225−226 °C, [α]25 D +31.2 (c 1.0, MeOH). Its molecular formula was determined as C15H22N2O4 on the basis of the observed 13C NMR and

tion pattern constructed from matrine and piperidine via an amide bond, and compound 5 is the first example of a matrinetype alkaloid with cleavage of the C-5−C-6 bond. Herein are reported the isolation and structure elucidation of compounds 1−8. In addition, the antiviral activity of each compound against hepatitis B virus was determined using HepG2.2.15 cells.

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RESULTS AND DISCUSSION Compound 1 was isolated as colorless block crystals from MeOH−H2O (2:1), mp 95−96 °C. Its molecular formula was deduced to be C15H20N2O2 by HRESIMS (m/z 261.1595 [M + H]+, calcd for C15H21N2O2, 261.1598). The IR absorption bands at 3435 and 1643 cm−1 suggested the presence of hydroxy and carbonyl groups. The 1H NMR spectrum of 1 displayed the presence of an olefinic proton at δH 7.63 (1H, s). The 13C NMR spectrum showed the presence of 15 carbons, including those for a carboxylic acid (δC 181.4) and a tetrasubstituted pyridine ring (δC 153.8, 150.1, 135.7, 116.9, and 115.4), and two methylenes connected to heteroatoms (δC 51.4 and 50.7). Besides the above signals, this alkaloid was found to possess seven methylene carbon atoms (δC 38.1, 31.4, 26.5, 25.2, 23.3, 21.0, and 20.9). With the aid of the 2D NMR experiments including 1H−1H COSY, HSQC, and HMBC (Figure 2), the 1H and 13C NMR data of 1 were assigned (Table 1).

Figure 2. Key 1H−1H COSY and HMBC correlations of 1, 4, 5, 6, and 8.

The 1H−1H COSY correlations of H2-2 (δH 3.45)/H2-3 (δH 1.98)/H2-4 (δH 2.71), H2-8 (δH 2.75)/H2-9 (δH 1.98)/H2-10 (δH 3.45), and H2-12 (δH 2.72)/H2-13 (δH 1.87)/H2-14 (δH 2.23) indicated the presence of three spin systems (C-2 to C-4, C-8 to C-10, and C-12 to C-14). When combined with the HMBC correlations between H2-8 and C-6 (δC 153.7), C-10 (δC 50.7), C-11 (δC 150.1), between H2-13 and C-11, C-15 (δC 181.4), between H2-14 and C-12 (δC 31.4), C-15, and between H2-17 (δH 7.63) and C-4 (δC 25.2), C-5 (δC 116.9), C-6, C-11, the planar structure of 1 was determined as shown in Figure 2. Finally, the structure of 1 was confirmed by a single-crystal X-ray diffraction (Cu Kα) analysis (Figure 3), and 1 was named flavesine G. Compound 2 (flavesine H) gave a molecular formula of C15H22N2O2 from its HRESIMS data. Its IR spectrum showed absorptions of carbonyl (1575 cm−1) and hydroxy (3426 cm−1) groups. The 13C NMR spectrum of 2 (Table 1) exhibited signals for a carboxylic acid group (δC 181.9), three 2260



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Table 1. . NMR Data of 1−4 (δ in ppm, J in Hz)a 1 δH

no. 2 3 4 5 6 7 8

2 δC

3.45 1.98 2.71

51.4 21.0 25.2

2.75

116.9 153.7 115.4 23.3

9 10 11 12

1.98 3.45 2.72

20.9 50.7 150.1 31.4

13

1.87 m

26.5

14 15 17

2.23 t (7.2)

38.1 181.4 135.7

7.63 s

3

δH 3.47 m 1.95 m a 2.38 b 1.61 m

2.71 a 2.04 m b 1.30 m 1.71 m 3.56 m 3.39 m a 2.77 b 2.38 a 1.84 m b 1.69 m 2.21 t (7.2) 7.41 s

δC 53.1 21.8 23.8 97.8 165.0 39.5 22.3 21.6 52.4 57.1 23.0 30.7 38.6 181.9 155.3

δH 3.45 1.94 a 2.01 m b 1.34 m 2.79 m

2.39 1.94 3.45 a 2.48 m b 2.39 m 1.82 m 2.21 t (7.1) a 3.51 m b 3.09 m

4 δC 52.3 21.8 23.1 35.7 163.7 96.4 23.5

δH

δC

3.24 1.92 2.64

50.6 22.2 25.5

2.68

115.4 149.8 114.3 24.2

22.1 52.6 169.9 33.5

1.94 3.24 2.61 m

22.2 49.8 156.4 34.8

25.5

1.83 m

26.1

38.2 181.3 45.4

2.41 t (7.3) 7.56 s

33.7 173.4 145.3

3.45 1.57 1.65 1.52 3.52

48.0 27.6 25.5 26.8 43.9

2′ 3′ 4′ 5′ 6′

m m m m m

a

Measured at 400 (1H) and 100 (13C) MHz in CD3OD. Overlapped signals are reported without designating multiplicity.

Figure 3. X-ray ORTEP drawings of 1, 5, and 7. The thermal ellipsoids are scaled to the 50% probability level.

bands for hydroxy (3384 cm−1) and carbonyl (1707 and 1645 cm−1) groups. The 1H and 13C NMR spectra of 5 (Table 2) displayed the presence of four characteristic downfield protons [δH 4.46 (1H, ddd, J = 14.2, 11.6, 3.0 Hz), 4.05 (1H, t, J = 6.0 Hz), 3.99 (2H, s)], three carbonyls (δC 205.3, 175.0, 174.6), an oxygenated quaternary carbon (δC 76.3), a methine connected to a heteroatom (δC 66.2), and three methylenes connected to heteroatoms (δC 57.8, 49.3, 49.1). According to the 2D NMR experiments including 1H−1H COSY, HSQC, and HMBC (Figure 2), the 1H and 13C NMR data of 5 could be assigned (Table 2). Detailed analysis of the 1D NMR data, molecular formula, and the presence of alkaloids in this plant suggested that the chemical structure of 5 possesses a great similarity to that of alopecurine A (9).22 Interestingly, while the 1D NMR spectra

Figure 4. Key NOESY correlations of 2, 6, and 7.

HRESIMS data (m/z 295.1675 [M + H]+, calcd for C15H23N2O4, 295.1693). The IR spectrum showed absorption 2261



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Figure 5. Experimental and calculated ECD spectra of 2 and 6.

Table 2. . NMR Data of 5−8 (δ in ppm, J in Hz)a 5

6

7

8

no.

δH

δC

δH

δC

δH

δC

δH

δC

2

a 4.46 ddd (14.2, 11.6, 3.0) b 2.64 dt (14.2, 3.0) a 2.31 m b 1.92 a 2.54 ddd (13.9, 11.6, 2.7) b 2.10 m

49.3

a 3.36 m b 3.25 m a 2.52 b 1.60 m a 1.94 b 1.82 m 2.06 m 3.68 d (4.0)

67.4

a 4.32 dt (12.0, 3.6) b 3.06 td (12.0, 2.8) a 1.96 m b 1.76 m a 2.05 m b 1.99 m

41.5

α 2.47 m β 4.69 m a 1.74 b 1.44 m α 1.30 m β 2.05 2.02 3.25 2.23 a 1.82 m b 1.65 a 2.40 b 2.33

44.1

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

a 1.80 b 1.77 a 1.81 b 1.78 a 3.10 m b 3.01 m 4.05 t (6.0) a 2.09 m b 1.98 m a 1.90 b 1.66 a 2.40 m b 2.37 m 3.99 s

26.4 38.5 205.3 174.6 76.3 28.7 19.5 49.1 66.2 23.8 20.0 33.4 175.0 57.8

17.6 26.0 32.0 68.7 108.9 28.3

a 2.67 m b 2.12 m a 1.90 b 1.77 m a 3.48 m b 3.10 m

24.0

2.38 m a 1.98 m b 1.45 m a 2.55 m b 2.36 m

70.1 136.8 25.9

a 2.74 m b 2.48 m a 1.88 b 1.74 m a 2.52 b 2.33 m

20.1 33.9

α 4.20 dd (12.3, 4.4) β 3.90 t (12.3)

171.8 42.6

22.4 25.8 111.4 130.8 40.4 24.3 32.5 169.9

3.38 m a 2.16 m b 1.77 m a 1.89 m b 1.72 m a 2.55 m b 2.36 m a 4.69 br d (17.7) b 3.42 br d (17.7)

25.6 32.1 32.8 59.5 39.9 23.0 31.7 172.1

58.9 26.8

3.46 a 2.04 b 1.72 a 1.85 m b 1.72 a 2.38 b 2.33

19.3 33.4 172.0 45.9

α 3.48 β 3.31

56.7 27.4 18.8 32.9 172.6 49.1

a

Measured at 400 (1H) and 100 (13C) MHz in CD3OD. Overlapped signals are reported without designating multiplicity.

which resulted in a small Flack parameter of −0.03(8), allowing an explicit assignment of the absolute configuration of 5 as 7R and 11R. Compound 6 was obtained as a brown oil, and its molecular formula was established as C15H22N2O2 from its HRESIMS data (m/z 263.1766 [M + H]+, calcd for C15H23N2O2, 263.1754). The IR spectrum showed a band at 1637 cm−1, suggesting the presence of a carbonyl functionality. The 1H and 13C NMR data of 6 (Table 2) resembled those of oxymatrine (10)23 except for the absence of two methines at C-7 (δC 43.6) and C-11 (δC 55.0), the presence of an extra double bond (δC 136.8, 108.9), and the chemical shifts of C-5, C-6, C-8, and C-12 shifted from δC 35.8, 67.9, 25.2, 29.4 to δC 32.0, 68.7, 28.3, 25.9, indicating that the methines at C-7 and C-11 in 10 are replaced by a double bond in 6. This conclusion

of 5 and alopecurine A were very similar, their 2D NMR spectra showed a number of differences. In the 1H−1H COSY spectrum (Figure 2), the correlations of H-2a (δH 4.46)/H2-3a (δH 2.31, 1.92)/H-4a (δH 2.64), H-8a (δH 1.80)/H-9b (δH 1.78)/H-10a (δH 3.10), and H-11 (δH 4.05)/H2-12a (δH 2.09, 1.98)/H-13b (δH 1.66)/H-14a (δH 2.40) indicated the presence of the three fragments C-2 to C-4, C-8 to C-10, and C-11 to C-14. Moreover, the HMBC correlations between H2-3, H2-4, H2-17 and C-5 (δC 205.3), between H2-2, H2-8, H2-11 and C-6 (δC 174.6), between H2-11, H2-13, H2-14, H217 and C-15 (δC 175.0), and between H2-9, H-11, H2-12 and C-7 (δC 76.3) (Figure 2) supported the location of three carbonyls (δC 205.3, 174.6, 175.0) at C-5, C-6, and C-15 and an oxygenated quaternary carbon (δC 76.3) at C-7. Finally, the structure of 5 was verified by single-crystal X-ray diffraction, 2262



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was confirmed by 1H−1H COSY correlatons between H-17α (δH 4.20), H-17β (δH 3.90) and H-5 (δH 2.06), H-6 (δH 3.68) and HMBC correlations between H-6 and C-7 (δC 108.9), C11 (δC 136.8), C-17 (δC 42.6), between H-17α and C-6 (δC 68.7), C-11, and between H-8b (δH 2.12) and C-7, C-11 (Figure 2). The relative configuration of 6 was determined by analysis of its NOESY correlations (Figure 4) and the 1H NMR spectroscopic coupling constants. In the NOESY spectrum, the correlations between H-17α and H-5, H-4b (δH 1.82) and between H-6 and H-4a (δH 1.94), H-5, as well as the lack of any observed correlations between H-17β and H-5, H-6, together with the coupling constant between H-5 and H-6 (J5−6 = 4.0 Hz), implied that H-5 and H-6 have the same orientations. Good agreement between the calculated and the experimental CD spectra of 1S,5S,6S-6 (Figure 5) permitted the assignment of the absolute configuration of 6 as 1S, 5S, and 6S. Compound 6 was determined, therefore, as 7,11-dehydrooxymatrine. Compound 7, isolated as colorless crystals from MeOH−H2O (2:1), mp 157−158 °C, displayed a molecular formula of C15H20N2O2 by HRESIMS at m/z 261.1597 [M + H]+ (calcd for C15H21N2O2, 261.1598). Comparison of the 1H and 13C NMR data of 7 (Table 2) with those of matrine4 showed these compounds to be similar in structure. The most notable differences were the absence of a methylene (C-10, δC 57.3) and two methines (C-5, δC 35.4; C-6, δC 63.8) in matrine and the presence of signals for an extra carbonyl group (δC 169.9) and a double bond (δC 130.8, 111.4) in 7. In the HMBC spectrum, the observed correlations between H-2a (δH 4.32), H-8b (δH 1.45), H-11 (δH 3.38), H2-17 (δH 4.69, 3.42) and C6 (δC 130.8) and between H2-3 (δH 1.96, 1.76), H2-17 and C-5 (δC 111.4) (Figure 2) suggested that a double bond is located between C-5 and C-6. Moreover, the HMBC correlations between H-2a, H-8b and C-10 (δC 169.9) implied that the extra carbonyl was located at C-10. The complete structure of 7 was confirmed by a single-crystal X-ray diffraction with a indicative Flack parameter of −0.2(2) (Figure 3). Thus, compound 7 was established as 10-oxy-5,6-dehydromatrine. Compound 8 was assigned the molecular formula C15H22N2O2 from its HRESIMS peak at m/z 263.1752 [M + H]+ (calcd for C15H23N2O2, 263.1754). The 13C NMR data of 6 were similar to those of sophoridine (14).24 The main differences observed were the presence of an additional carbonyl group signal (δC 172.1) in 8 and the absence of a methylene connected to a heteroatom (δC 50.6) in 14, with the chemical shifts of C-2, C-6, and C-9 being shifted from δC 56.8, 63.6, 22.8 to δC 44.1, 59.5, 31.7. This indicated that the methylene (δC 50.6) at C-10 in 14 was replaced by a carbonyl (δC 172.1) in 8, which was verified by the 1H−1H COSY and HMBC correlations as shown in Figure 2. The relative configuration of 8 was deduced using the NOESY correlations between H-6 and H-2α, H-4α; H-17α and H-4α; and H-11 and H-8b (Figure 4). Accordingly, compound 8 was determined structurally as 10-oxysophoridine. The known alkaloids were identified as alopecurin A (9),22 oxymatrine (10),23 sophoranol (11),25 14β-hydroxymatrine (12),26 7,11-dehydromatrine (13),10 sophoridine (14),24 allomatrine (15),27 isosophoridine (16),28 and sophoramine (17),28 respectively, by comparison of their spectroscopic data with literature values. Since certain matrine-type alkaloids from S. f lavescens were reported to exhibit potent antiviral activity against HBV,4,9 compounds 1−17 were also evaluated for their anti-HBV

activity in HepG2.2.15 cells. As shown in Table 3, all isolated alkaloids showed the HBsAg inhibitory effects. In particular, Table 3. Inhibitory Activity of Compounds 1−17 against HBsAg and HBeAg Secretion compound

concentration (mM)

1a 2a 3a 4b 5a 6c 7b 8a 9a 10a 11b 12a 13a 14a 15a 16a 17c matrinea 3TCe

0.4 0.4 0.4 0.2 0.4 0.8 0.2 0.4 0.4 0.4 0.2 0.4 0.4 0.4 0.4 0.4 0.8 0.4 1.0

HBsAg (inhibition %) 37.2 30.8 29.8 44.3 46.0 25.4 33.0 32.5 14.1 38.3 33.8 33.2 22.4 40.2 26.3 13.8 29.5 34.7 32.6

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.1 1.8 1.1 3.5 2.8 1.5 2.2 0.5 1.2 1.8 2.5 3.6 1.7 3.8 1.6 0.5 0.7 0.9 1.7

HBeAg (inhibition %) ± ± ± ± ± ± ± ± -d 20.3 ± 28.4 ± 18.5 ± 29.9 ± 21.9 ± 17.7 ± -d 13.5 ± 16.8 ± 37.5 ± 33.2 16.8 15.1 38.0 40.4 22.6 38.8 18.3

4.3 0.7 1.6 2.4 2.0 0.6 1.8 1.3 2.9 1.0 2.8 1.5 0.4 0.6 1.3 1.2 2.4

a

This compound showed cytotoxicity against the HepG2.2.15 cell line at a concentration of 0.8 mM. Cell damage was assessed using an MTT assay, and a cell growth inhibition against the HepG2.2.15 cell line of ≥25% was considered as cytotoxic. bThese compounds showed cytotoxicity against the HepG2.2.15 cell line at a concentration of 0.4 mM. cThis compound showed cytotoxicity against the HepG2.2.15 cell line at a concentration of 1.0 mM. dShowed no inhibitory effect. e 3TC, or lamivudine, which was used as positive control.

compounds 1, 4, 5, 10, and 14 significantly inhibited HBsAg secretion by 37.2%, 44.3%, 46.0%, 38.3%, and 40.2%, respectively, at noncytotoxic concentrations of 0.2 or 0.4 mM, which suggested that these alkaloids had comparable potencies to matrine (34.7% at a concentration of 0.4 mM) and were more active than the positive control lamivudine (3TC, 31.5% at a concentration of 1.0 mM). Compounds 2, 3, 7, 8, 11, 12, and 17 exhibited less potent HBsAg inhibitory effects of 29.5% to 33.8%. Comparison of the structure and activity of 4 with those of 1 revealed that introduction of a pyridine ring to a carboxylic acid group at C-15 might enhance the anti-HBV activity and cytotoxicity. Additionally, the HBsAg inhibitory effects of 5 (46.0%) and 9 (14.1%) implied that the cleavage of the C-5−C-6 bond produces a positive influence on anti-HBV activity, whereas the cleavage of the C6−C-7 bond seems to be unfavorable for anti-HBV activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. The general experimental procedures are listed in the Supporting Information. Plant Material. The roots of Sophora f lavescens were collected from Xi’an, Shanxi Province, People’s Republic of China, in October 2011, and were authenticated by a senior herbalist at the Chinese Medicinal Material Company, Zhen-Qiu Mai. A voucher specimen (No. 20120712) was deposited at the Institute of Traditional Chinese Medicine and Natural Products, Jinan University, Guangzhou, People’s Republic of China. Extraction and Isolation. Dried roots of S. flavescens (25.0 kg) were extracted three times with 95% aqueous EtOH at room 2263



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Crystallographic data for 7: C 15H20N2O2, M = 260.33, monoclinic, a = 4.7951(2) Å, b = 10.4990(4) Å, c = 27.8473(12) Å, α = 90°, β = 90°, γ = 90°, V = 1401.93(10) Å3, T = 173(1) K, space group P212121, Z = 4, μ(Cu Kα) = 0.748 mm−1, 11 392 reflections collected, 2261 independent reflections (Rint = 0.0568). The final R1 values were 0.0521 [I > 2σ(I)]. The final wR(F2) values were 0.1362 [I > 2σ(I)]. The final R1 values were 0.0553 (all data). The final wR(F2) values were 0.1412 (all data). The goodness of fit on F2 was 1.070. Flack parameter: −0.2(2). CCDC number: 1844171. Anti-HBV Effect Assay. According to previous studies,4,19 matrine-type alkaloids were found to exhibit potent antiviral acitivity against HBV. Thus, the isolated alkaloids were also evaluated for their anti-HBV activity. The detailed procedure is described in the Supporting Information.

temperature. The crude extract was suspended in H2O, acidified with 1% HCl to pH 4, and then partitioned with CHCl3 to remove the neutral components. Subsequently, the aqueous layer, which was adjusted to pH 9 with NH3·H2O, was re-extracted with CHCl3 to afford an alkaloidal extract (464 g). The detailed process of isolation is described in the Supporting Information. Flavesine G (1): colorless crystals in MeOH−H2O; mp 95−96 °C; UV (CH3OH) λmax (log ε) 206 (3.22), 230 (3.07), 296 (3.19) nm; IR (KBr) νmax 3435, 2925, 2859, 1643, 1565, 1409, 1303, 1216 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 261.1595 [M + H]+ (calcd for C15H21N2O2, 261.1598). Flavesine H (2): brown oil; [α]25 D +251.7 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 204 (3.13), 360 (3.33) nm; IR (KBr) νmax 3426, 2956, 1631, 1575, 1455, 1408, 1314, 1209 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 263.1753 [M + H]+ (calcd for C15H23N2O2, 263.1754). Flavesine I (3): brown oil; [α]25 D +0.4 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 204 (3.06), 356 (3.37) nm; IR (KBr) νmax 3416, 2945, 1647, 1561, 1408, 1315, 1209, 1127 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 263.1756 [M + H]+ (calcd for C15H23N2O2, 263.1754). Flavesine J (4): brown oil; UV (CH3OH) λmax (log ε) 208 (3.17), 295 (3.02) nm; IR (KBr) νmax 2931, 2866, 1640, 1598, 1565, 1450, 1312, 683 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 328.2396 [M + H]+ (calcd for C20H30N3O, 328.2383). Alopecurine B (5): colorless crystals in MeOH−H2O; mp 225−226 °C; [α]25 D +31.2 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 211 (3.38) nm; IR (KBr) νmax 3384, 2939, 2899, 1707, 1645, 1451, 1335, 1207, 1000, 638 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 295.1675 [M + H]+ (calcd for C15H23N2O4, 295.1693). 7,11-Dehydro-oxymatrine (6): brown oil; [α]25 D −28.2 (c 0.75, CH3OH); UV (CH3OH) λmax (log ε) 206 (3.41) nm; IR (KBr) νmax 2944, 2862, 1637, 1393, 1090 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 263.1766 [M + H]+ (calcd for C15H23N2O2, 263.1754). 10-Oxy-5,6-dehydromatrine (7): colorless crystals in MeOH− H2O; mp 157−158 °C; [α]D25 −119.5 (c 1.0, CH3OH); UV (CH3OH) λmax (log ε) 236 (2.16) nm; IR (KBr) νmax 2933, 2871, 2830, 1654, 1637, 1621, 943 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 261.1597 [M + H]+ (calcdfor C15H21N2O2, 261.1598). 10-Oxysophoridine (8): brown oil; [α]25 D −40.7 (c 0.7, CH3OH); UV (CH3OH) λmax (log ε) 206 (3.44) nm; IR (KBr) νmax 2931, 2856, 1631, 1457, 1417, 1336 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 263.1752 [M + H]+ (calcd for C15H23N2O2, 263.1754). X-ray Diffraction Analysis. X-ray crystallographic analysis of 1, 5, and 7 was performed on an Agilent Gemini S Ultra CCD diffractometer with Cu Kα radiation. The details of the protocols are described in the Supporting Information. Crystallographic data for 1: C 15H 20N2 O2, M = 260.33, monoclinic, a = 8.00779(9) Å, b = 11.80757(13) Å, c = 18.4571(2) Å, α = 90°, β = 101.2937(11)°, γ = 90°, V = 1711.38(3) Å3, T = 173(1) K, space group P21/n, Z = 4, μ(Cu Kα) = 0.499 mm−1, 26 656 reflections collected, 2731 independent reflections (Rint = 0.0250). The final R1 values were 0.0377 [I > 2σ(I)]. The final wR(F2) values were 0.1012 [I > 2σ(I)]. The final R1 values were 0.0389 (all data). The final wR(F2) values were 0.1017 (all data). The goodness of fit on F2 was 1.088. CCDC number: 1843522. Crystallographic data for 5: C 15H 22N2 O4, M = 294.34, orthorhombic, a = 6.89011(14) Å, b = 7.38722(16) Å, c = 26.7069(7) Å, α = 90°, β = 90°, γ = 90°, V = 1359.35(5) Å3, T = 173(1) K, space group P212121, Z = 4, μ(Cu Kα) = 0.861 mm−1, 9250 reflections collected, 2181 independent reflections (Rint = 0.0266). The final R1 values were 0.0276 [I > 2σ(I)]. The final wR(F2) values were 0.0683 [I > 2σ(I)]. The final R1 values were 0.0285 (all data). The final wR(F2) values were 0.0691 (all data). The goodness of fit on F2 was 1.073. Flack parameter: −0.03(8). CCDC number: 1844164.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00576.

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General experimental procedures, extraction and isolation, X-ray diffraction analysis, anti-HBV effect assay, detailed NMR, HRESIMS, UV, and IR spectra of 1−8; calculation details for 2 and 6 (PDF) X-ray crystallographic data (ZIP)

AUTHOR INFORMATION

Corresponding Authors

*Tel/fax: +86 20 85221559. E-mail: [email protected] (Y.-L. Li). *Tel/fax: +86 20 85221559. E-mail: [email protected] (G.-C. Wang). ORCID

Man-Mei Li: 0000-0003-2423-3119 Guo-Cai Wang: 0000-0002-4008-2640 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (Nos. 81803376, 81473116, 81673319, 81673670), the Science and Technology Planning Project of Guangdong Province (Nos. 2016A030303011, 2016B030301004), the Science and Technology Planning Project of Guangzhou City (201707010399), the Natural Science Foundation of Guangdong Province (No. 2017A030313732), and China Postdoctoral Science Foundation (No. 2017M620405).

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REFERENCES

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Matrine-Type Alkaloids from the Roots of Sophora flavescens and

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