Quinolizidine Alkaloids with Anti-HBV Activity from Sophora tonkinensis

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A new matrine-type alkaloid, (-)-14β-hydroxyoxymatrine (1), was isolated from the roots and rhizomes of Sophora tonkinensis Gapnep. (Leguminosae), together with five known matrine-type alkaloids, (-)-14β-hydroxymatrine (2), (+)-oxymatrine (3), (+)-matrine (4), (+)-sophoranol (5), and (-)-5α-hydroxysophocarpine (6), as well as with two known cytisine-type alkaloids, (-)-cytisine (7) and (-)-N-methylcytisine (8). Their structures were elucidated by spectroscopic methods. Compounds 1, 5 and 7 showed potent anti-HBV activity with an inhibitory potency against HBsAg secretion of 22.6 %, 31.1 % and 33.2 %, and against HBeAg secretion of 30.4 %, 26.3 % and 27.8 %, respectively.

As part of our continued screening for anti-HBV agents from Sophora medicinal plants [1], a phytochemical investigation on the alkaloid constituents of S. tonkinensis Gapnep. (S. subprostrata Chun et T. Chen) was carried out. The roots and rhizomes of the title plant are commonly used as the traditional Chinese medicine ‘Shan-dou-gen’ for the treatment of acute pharyngolaryngeal infections and sore throat [2]. Pharmacological studies showed that the crude alkaloids from this plant exhibited potent antiviral activity against CB₃V (Coxsackie virus), CB₅V, AdV₃ (adenovirus), AdV₇, and RSV (respiratory syncytial virus) [3]. Our present study on the roots and rhizomes of S. tonkinensis resulted in the isolation of a new matrine-type alkaloid (-)-14β-hydroxyoxymatrine (1), together with five known matrine-type alkaloids, (-)-14β-hydroxymatrine (2) [4], [5], (+)-oxymatrine (3) [6], (+)-matrine (4) [6], (+)-sophoranol (5) [7] and (-)-5α-hydroxysophocarpine (6) [8], as well as with two known cytisine-type alkaloids, (-)-cytisine (7) [6] and (-)-N-methylcytisine (8) [7], [9] (Fig. [1]). This paper reports the isolation and structure elucidation of the new alkaloid, as well as the in vitro anti-HBV activity of the isolated alkaloids.

(-)-14β-Hydroxyoxymatrine (1), pale yellow oil, [α]_D ²⁰: -5.3° (c 0.30, MeOH), was assigned the molecular formula C₁₅H₂₄N₂O₃ based on its HR-ESI-MS data. Its IR spectrum showed absorption bands characteristic of a hydroxy group (3416 cm^-1), a lactam C = O group (1626 cm^-1), and an N→O group (951 cm^-1) [6]. The EI-mass spectrum of 1 showed a base peak at m/z = 263, corresponding to [M - OH]⁺, and fragmentations very similar to those of 3 [6], indicating that it was a hydroxy derivative of 3. A fragment ion at m/z = 221 made up of rings A/C/D or B/C/D, which was 16 a. m. u. more than that of 3 (m/z = 205), as well as the fragment ions made up of rings A/B/C, A/B, and A or B, which were the same as those of 3, suggested that the hydroxy group was substituted on ring D. The ¹H-NMR spectrum of 1 agreed well with that of 3, except that there was an additional signal at δ _H = 3.91 (1H, dd, J = 12.0, 5.5 Hz) in 1 which could be assigned to a methine proton bearing a hydroxy group (Table [1]). In the ¹³C-NMR spectrum of 1, the signals corresponding to C-2 to C-11 and C-17 on rings A, B and C were consistent with those of 3 with Δδ _C ≤ 0.9 ppm (Table [2]). Based on the HMQC and HMBC data, the remaining signals at δ _C = 68.1, 27.0 and 26.3 were reasonably assigned to C-14, C-13 and C-12, respectively, which were shifted down-field by 35.2, 8.3 ppm and up-field by 2.3 ppm, respectively, in comparison with those of 3, by considering the substituent effects of a hydroxy group at C-14. The carbonyl carbon was also shifted down-field by 2.3 ppm (Table [2]). Thus, the hydroxy group was suggested to be at C-14, which was further confirmed by the HMBC correlations of the methine proton at δ _H = 3.91 (H-14) with C-15 (δ _C = 172.4), C-13 (δ _C = 27.0) and C-12 (δ _C = 26.3). An equatorial (β) hydroxy group at C-14 was determined by the coupling constants of J _H14a,H13a (12.0 Hz) and J _H14a,H13e (5.5 Hz) in the ¹H-NMR spectrum, which was further proven by the NOESY correlation between δ _H = 3.91 (H-14α) and δ _H = 1.28 (H-12α). Thus, 1 was presumed to be (7aS,11S,13aR,13bR,13cS)-dodecahydro-11-hydroxy-1H,5H,10H-dipyrido[2,1-f:3′,2′,1′-ij][1] [6] naphthyridin-10-one 4-oxide.

Compounds 1 - 2 and 5 - 8 were investigated for in vitro antiviral activity against hepatitis B virus (HBV) (compounds 3 and 4 were reported previously [1]), and the results are summarized in Table [3]. Most of the alkaloids showed potent anti-HBV activity. Among them, compounds 1, 5 and 7 were the most potent, they significantly inhibited HBsAg secretion by 22.6 %, 31.1 % and 33.2 %, respectively, at the non-cytotoxic concentrations of 0.4 μmol/mL or 0.2 μmol/mL [29.6 % for lamivudine (3TC) used as positive control at 1.0 μmol/mL], and also inhibited HBeAg secretion by 30.4 %, 26.3 % and 27.8 % (35.4 % for 3TC control). Compounds 2 and 6 displayed slightly weaker HBsAg inhibitory effects of 28.9 % and 28.5 %, respectively, which were equivalent to 3TC control, but had very low or even no HBeAg inhibitory activity. Compound 8 was almost inactive. In addition, the inhibitory activities against HBsAg and HBeAg secretion, especially that against HBeAg secretion, of compounds 5 and 7 were found to be similar to or much stronger than those of 3 and 4 [1], which have been commonly used to treat hepatitis B in clinical practice [10], indicating that these two alkaloids might be valuable as potential anti-HBV agents.

Based on the analysis of the anti-HBV potency and the structural characteristics of the tested quinolizidine alkaloids from S. tonkinensis and those from S. flavescens [1], the structure-activity relationship (SAR) deduced in our previous paper [1] was further confirmed. Additionally, it was found that introduction of a β-substituted hydroxy group (2) into (+)-matrine reduced the anti-HBV activity. Compound 1 exhibited a stronger anti-HBeAg effect, but a weaker anti-HBsAg activity than (+)-matrine [1]. This might result from the complicated antagonistic effects of N→O and β-OH, since the former group mainly enhances the anti-HBeAg activity [1], while the latter dominantly reduces the anti-HBsAg effect. Furthermore, the structural characteristics and potency of 7 were compared with those of 8 and anagyrine [1]. Based on this limited SAR information, it could be noticed that alkylation on N-12 of cytisine leads to a decrease of anti-HBsAg efficacy, and even to a complete loss of anti-HBeAg activity.

Materials and Methods

NMR spectra: Bruker DRX-400 and 500 instruments; EI-MS: HP 5989A and 5973N mass spectrometers; HR-ESI-MS: AB QSTAR Pulsar mass spectrometer; UV spectra: Shimadzu UV-260 spectrophotometer; IR spectra: AVATAR 360 FT-IR spectrophotometer; Optical rotations: JASCO P-1020 digital polarimeter. Column chromatography was performed on silica gel (200 - 300 mesh; Yantai; Yantai, People’s Republic of China). Analytical (0.25 mm) and preparative (0.50 mm) TLC was conducted on GF₂₅₄ precoated silica gel plates (10 - 40 μm; Yantai).

The roots and rhizomes of Sophora tonkinensis Gapnep. were purchased from Huayu Materia Medica Co., Ltd., Shanghai, in March of 2000, and verified by Dr. Dao-Feng Chen. A voucher specimen (SDG-SH-0003) is deposited in the Herbarium of Materia Medica, Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China.

A 20 g amount of the pulverized roots and rhizomes of S. tonkinensis Gapnep. was extracted with 500 mL of CHCl₃ and 20 mL of 25 % aqueous ammonia by ultrasonication at room temperature to give a residue of 1.9 g (yield 9.5 %). The residue was then subjected to silica gel (100 g) column chromatography eluting with CHCl₃-EtOAc-MeOH-NH₃·H₂O (8 : 2:1 : 0.1 and 8 : 2:2 : 0.2, each 1 L) to give 1 (5 mg). Another amount (9 kg) of the pulverized roots and rhizomes of the plant was extracted with aqueous 1 % H₂SO₄ (v/v) (15 L × 4) at room temperature. The filtered acid extract was basified to pH 10 - 11 with Na₂CO₃ and extracted with CHCl₃ (12 L × 6). The CHCl₃ extract was concentrated under vacuum to give the total alkaloids (120 g) in a yield of 1.33 %. The total alkaloids were subjected to silica gel (1500 g) column chromatography eluting with petroleum ether-EtOAc-EtOH-NH₃·H₂O (80 : 38 : 10 : 1.8, 40 : 30 : 40 : 3.4 and 30 : 30 : 60 : 8, each 20 L) gradiently to give fractions 1 - 6. Fraction 1 (20.5 g) was subjected to silica gel (500 g) chromatography using Et₂O-MeOH-NH₃·H₂O (10 : 0.2 : 0.05) as solvent system to give 4 (1700 mg). Fraction 3 (5.2 g) was eluted with Et₂O-MeOH-NH₃·H₂O (10 : 0.3 : 0.1) on silica gel (150 g) to give fractions 3-A, 3-B and 3-C. Fraction 3-C (2.2 g) was eluted with CHCl₃-EtOAc-MeOH-NH₃·H₂O (10 : 2:0.3 : 0.05) on silica gel (80 g) to yield 2 (50 mg). Fraction 4 (8.5 g) was applied to silica gel (250 g) column chromatography eluting with Et₂O-MeOH-NH₃·H₂O (13 : 0.4 : 0.1) to give 5 (80 mg) and 6 (10 mg). Fraction 5 (5.2 g) was chromatographed over silica gel (200 g) eluting with Et₂O-MeOH-NH₃·H₂O (10 : 1.5 : 0.3) to give fractions 5-A and 5-B. Fraction 5-A (300 mg) was purified by preparative TLC developed with Et₂O-MeOH-NH₃·H₂O (10 : 0.3 : 0.2) to give 8 (12 mg), and fraction 5-B (3.1 g) was chromatographed over silica gel (150 g) eluting with CHCl₃-EtOAc-MeOH-NH₃·H₂O (8 : 2:1 : 0.1) to give 7 (150 mg). Fraction 6 (20.8 g) was subjected to silica gel (300 g) column chromatography eluting with CHCl₃-EtOAc-MeOH-NH₃·H₂O (10 : 2:1 : 0.1) to yield 3 (2000 mg).

(-)-14β-Hydroxyoxymatrine (1): pale yellow oil; [α]_D ²⁰: -5.3° (c 0.30, MeOH); UV (MeOH): λ _max (log ε) = 208 (3.66) nm; IR (CH₂Cl₂): ν _max = 3416, 2934, 2867, 1626, 1439, 1336, 1283, 1246, 1119, 951, 730 cm^-1; ¹H-NMR (CDCl₃, 500 MHz): δ = 5.03 (1H, dt, J = 10.5, 5.3 Hz, H-11β), 4.27 (1H, dd, J = 12.5, 5.5 Hz, H-17α), 4.19 (1H, t, J = 12.5 Hz, H-17β), 3.91 (1H, dd, J = 12.0, 5.5 Hz, H-14α), 3.28 (2H, br d, J = 11.0 Hz, H-2β, H-10β), 3.17 (2H, m, H-2α, H-10α), 3.11 (1H, br s, H-6α), 2.72 (1H, m, H-3β), 2.62 (1H, m, H-9β), 2.25 (2H, m, H-12β, H-13β), 2.05 (1H, br d, J = 13.7 Hz, H-8β), 1.88 (1H, m, H-5α), 1.81 (1H, m, H-4α), 1.72 (2H, m, H-4β, H-13α), 1.57 - 1.61 (4H, m, H-3α, H-7α, H-8α, H-9α), 1.28 (1H, m, H-12α); ¹³C-NMR (CDCl₃, 125 MHz): see Table [2]; EI-MS: m/z (rel. intensity) = 280 [M]⁺ (2), 264 (25), 263 (100), 262 (13), 245 (7), 221 (5), 217 (14), 150 (22), 149 (10), 148 (43), 138 (14), 136 (11), 117 (12), 96 (21), 59 (23), 55 (14); HR-ESI-MS: m/z = 281.1859 [M + H]⁺ (calcd for C₁₅H₂₅N₂O₃ : 281.1865).

Details of the in vitro anti-HBV assays were the same as those reported previously [1], [11]. Each test was performed three times, and the S.D. (standard deviation) of inhibition values varied by no more than 6 %.

Acknowledgements

This study was supported by the grant from the 9th Five-Year National Key Science and Technology Project from the Ministry of Science and Technology of P. R. China (99-929-01-31). Authors are grateful to Dr. Yu-Mei Wen at Fudan University for her kindly providing the HepG2 2.2.15 human hepatablastoma cell line for anti-HBV tests.

References

• 1 Ding P L, Liao Z X, Huang H, Zhou P, Chen D F. (+)-12α-Hydroxysophocarpine, a new quinolizidine alkaloid and related anti-HBV alkaloids from Sophora flavescens .  Bioorg Med Chem Lett. 2006;  16 1231-5
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  Search in Google Scholar
• 3 Fan H W, Lu J H, Zhang R. Studies on the antibacterial and antiviral activities of matrine-type alkaloids, and its effect on inducing interferon production.  Zhong Yi Yao Xin Xi. 2000;  17 75-6
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• 5 Xiao P, Kubo H, Komiya H, Higashiyama K, Yan Y N, Li J S. et al . (-)-14β-Acetoxymatrine and (+)-14α-acetoxymatrine, two new matrine-type lupin alkaloids from the leaves of Sophora tonkinensis .  Chem Pharm Bull. 1999;  47 448-50
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• 6 Zhang L Z, Li J S, Houghton P J, Jackson S, Twentyman P R. Alkaloids in Sophora alopecuroides seed and relevant tests for activity.  Zhongguo Zhong Yao Za Zhi. 1997;  22 740-3
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• 7 Zhao Y Y, Pang Q Y, Liu J B, Chen Y Y, Lou Z C. Studies on the alkaloids of Sophora flavescens .  Tian Ran Chan Wu Yan Jiu Yu Kai Fa. 1994;  6 10-3
  PubMedSearch in Google Scholar
• 8 Saito K, Arai N, Sekine T, Ohmiya S, Kubo H, Otomasu H. et al . (-)-5α-Hydroxysophocarpine, a new lupin alkaloid from the seeds of Sophora flavescens var. angustifolia .  Planta Med. 1990;  56 487-8
  PubMedSearch in Google Scholar
• 9 Takamatsu S, Saito K, Ohmiya S, Ruangrungsi N, Murakoshi I. Lupin alkaloids from Sophora exigua .  Phytochemistry. 1991;  30 3793-5
  CrossrefPubMedSearch in Google Scholar
• 10 Ding P L, Chen D F. The pharmacological activities, clinical applications and side effects of the Chinese drug ‘Shan-dou-gen’ and its preparations.  Zhongguo Lin Chuang Yao Xue Za Zhi. 2003;  12 315-8
  PubMedSearch in Google Scholar
• 11 Wu T, Huang H, Zhou P. The inhibitory effects of enduracidin on hepatitis B virus in vitro .  Zhongguo Bing Du Xue. 1998;  13 45-9
  PubMedSearch in Google Scholar

Dr. Dao-Feng Chen

Department of Pharmacognosy

School of Pharmacy

Fudan University

138 Yi Xue Yuan Road

Shanghai 200032

People’s Republic of China

Phone: +86-21-5423-7453

Fax: +86-21-6417-0921

Email: dfchen@shmu.edu.cn

References

• 1 Ding P L, Liao Z X, Huang H, Zhou P, Chen D F. (+)-12α-Hydroxysophocarpine, a new quinolizidine alkaloid and related anti-HBV alkaloids from Sophora flavescens .  Bioorg Med Chem Lett. 2006;  16 1231-5
  CrossrefPubMedSearch in Google Scholar
• 2 State Pharmacopoeia Commission of PRC. Pharmacopoeia of the People’s Republic of China. Vol. 1 Beijing; Chemical Industry Press 2005: p 19-20
  Search in Google Scholar
• 3 Fan H W, Lu J H, Zhang R. Studies on the antibacterial and antiviral activities of matrine-type alkaloids, and its effect on inducing interferon production.  Zhong Yi Yao Xin Xi. 2000;  17 75-6
  PubMedSearch in Google Scholar
• 4 Xiao P, Li J S, Kubo H, Saito K, Murakoshi I, Ohmiya S. (-)-14β-Hydroxymatrine, a new lupin alkaloid from the roots of Sophora tonkinensis .  Chem Pharm Bull. 1996;  44 1951-3
  PubMedSearch in Google Scholar
• 5 Xiao P, Kubo H, Komiya H, Higashiyama K, Yan Y N, Li J S. et al . (-)-14β-Acetoxymatrine and (+)-14α-acetoxymatrine, two new matrine-type lupin alkaloids from the leaves of Sophora tonkinensis .  Chem Pharm Bull. 1999;  47 448-50
  PubMedSearch in Google Scholar
• 6 Zhang L Z, Li J S, Houghton P J, Jackson S, Twentyman P R. Alkaloids in Sophora alopecuroides seed and relevant tests for activity.  Zhongguo Zhong Yao Za Zhi. 1997;  22 740-3
  PubMedSearch in Google Scholar
• 7 Zhao Y Y, Pang Q Y, Liu J B, Chen Y Y, Lou Z C. Studies on the alkaloids of Sophora flavescens .  Tian Ran Chan Wu Yan Jiu Yu Kai Fa. 1994;  6 10-3
  PubMedSearch in Google Scholar
• 8 Saito K, Arai N, Sekine T, Ohmiya S, Kubo H, Otomasu H. et al . (-)-5α-Hydroxysophocarpine, a new lupin alkaloid from the seeds of Sophora flavescens var. angustifolia .  Planta Med. 1990;  56 487-8
  PubMedSearch in Google Scholar
• 9 Takamatsu S, Saito K, Ohmiya S, Ruangrungsi N, Murakoshi I. Lupin alkaloids from Sophora exigua .  Phytochemistry. 1991;  30 3793-5
  CrossrefPubMedSearch in Google Scholar
• 10 Ding P L, Chen D F. The pharmacological activities, clinical applications and side effects of the Chinese drug ‘Shan-dou-gen’ and its preparations.  Zhongguo Lin Chuang Yao Xue Za Zhi. 2003;  12 315-8
  PubMedSearch in Google Scholar
• 11 Wu T, Huang H, Zhou P. The inhibitory effects of enduracidin on hepatitis B virus in vitro .  Zhongguo Bing Du Xue. 1998;  13 45-9
  PubMedSearch in Google Scholar

Dr. Dao-Feng Chen

Department of Pharmacognosy

School of Pharmacy

Fudan University

138 Yi Xue Yuan Road

Shanghai 200032

People’s Republic of China

Phone: +86-21-5423-7453

Fax: +86-21-6417-0921

Email: dfchen@shmu.edu.cn