Fitoterapia 104 (2015) 102–107
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Proaporphine and aporphine alkaloids with acetylcholinesterase inhibitory activity from Stephania epigaea Jian-Wei Dong a,1, Le Cai a,1, Yun-Shan Fang a, Huai Xiao a,b, Zhen-Jie Li a, Zhong-Tao Ding a,⁎ a b
Key Laboratory of Medicinal Chemistry for Nature Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China School of Pharmaceutical Sciences and Chemistry, Dali University, Dali 671000, PR China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 22 May 2015 Accepted 24 May 2015 Available online 29 May 2015 Chemical compounds studied in this article: Epiganine A Epiganine B Pronuciferine (PubChem CID: 200480) Dehydrodicentrine (PubChem CID: 3084326) Romerine (PubChem CID: 119204) Romeline N-methylcalycinine Phanostenine (PubChem CID: 12305138) Dicentrine (PubChem CID: 101300) N-methyllaurotetanine (PubChem CID: 16573)
a b s t r a c t An unusual proaporphine alkaloid bearing an isopropanenitrile group at isoquinoline nitrogen, named epiganine A (1) and a new aporphine alkaloid, epiganine B (2), together with eight known alkaloids, pronuciferine (3), dehydrodicentrine (4), romerine (5), romeline (6), N-methylcalycinine (7), phanostenine (8), dicentrine (9), and N-methyllaurotetanine (10), were isolated from the roots of Stephania epigaea. The absolute configuration of 1 was determined by calculating electronic circular dichroism (ECD) and comparing with experimental data. Compounds 2 and 4 showed strong acetylcholinesterase (AChE) inhibitory effects with the IC50 values of 4.36 and 2.98 μM, respectively. Compounds 5–9 also exhibited potent AChE inhibitory activities. © 2015 Elsevier B.V. All rights reserved.
Keywords: Stephania epigaea Proaporphine alkaloid Aporphine alkaloid Acetylcholinesterase inhibitory activity
1. Introduction The genus Stephania (Menispermaceae) is distributed mainly in Asia and Africa, with about thirty nine species of this genus growing in mainland China [1], which have been used as folk medicine for treating asthma, cancer, dysentery, fever, hyperglycemia, intestinal complaints, inflammation, sleep disturbances, tuberculosis, and so on [2]. Stephania epigaea Lo is an herbaceous liana mainly growing in Yunnan and Sichuan provinces, China [1]. Its tubers are frequently used by local people to treat fever and for sedation. Previous phytochemical researches showed that the major constituents of S. epigaea are aporphine [3], morphine [4], and bisbenzylisoquinoline alkaloids [2,5]. In order to explore the new and bioactive constituents from S. epigaea, a detailed phytochemical investigation on the roots of S. epigaea was carried out in the present study. An unusual proaporphine alkaloid bearing ⁎ Corresponding author. E-mail address: [email protected] (Z.-T. Ding). 1 The first two authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.fitote.2015.05.019 0367-326X/© 2015 Elsevier B.V. All rights reserved.
an isopropanenitrile group at isoquinoline nitrogen, named epigasine A (1) and a new aporphine alkaloid, epigasine B (2), together with eight known proaporphine/aporphine alkaloids, pronuciferine (3) [6], dehydrodicentrine (4) [7], romerine (5) [8], romeline (6) [9], Nmethylcalycinine (7) [10], phanostenine (8) [11,12], dicentrine (9) [13], and N-methyllaurotetanine (10) [14] (Fig. 1) were isolated from the roots of S. epigaea. Among the known compounds, compounds 3, 5, 8, and 10 were isolated from S. epigaea for the first time. The acetylcholinesterase (AChE) inhibitory activities of the isolated compounds have also been evaluated. 2. Results and discussions Compound 1 was obtained as a white amorphous powder and its molecular formula was deduced to be C21H22N2O3 by HRESIMS at m/z 351.1710 [M + H]+ (Calcd. for C21H23N2O3 351.1703). Its IR spectrum indicated the presence of CN_(2353 cm−1), C_O (1662 cm−1), and benzene ring (1450–1600 cm− 1). The NMR spectra of 1 (Table 1) showed the characteristic signals for a dienone moiety [δH 7.05 (1H,
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Fig. 1. Structures of compounds 1–10.
dd, J = 10.0, 2.8 Hz), 6.85 (1H, dd, J = 10.0, 2.8 Hz), 6.42 (1H, dd, J = 10.0, 2.8 Hz), 6.33 (1H, dd, J = 10.0, 2.8 Hz); δC 186.1 s, 152.8 d, 149.6 d, 128.7 d, 127.9 d], a penta-substituted phenyl unit [δH 6.65 (1H, s); δC 153.8 s, 144.7 s, 133.5 s, 127.5 s, 111.9 d], and two methoxy groups [δH 3.80 (3H, s), 3.59 (3H, s); δC 61.2 q, 56.4 q] [6]. A detailed comparison of the 1H and 13C NMR chemical shifts (Table 1) of 1 with pronuciferine (3) [6] which was also isolated from S. epigaea in this study, strongly suggested a proaporphine alkaloid for 1. The main difference between them was that compound 1 possesses no N-methyl group which exists in 3. Meanwhile, compound 1 exhibited the signals of an additional isopropanenitrile moiety [δH 3.86 (1H, q, J = 10.0, 2.4 Hz), 1.57 (3H, d, J = 6.4 Hz); δC 116.7 s, 48.3 d, 17.5 q] in its NMR spectra. HMBC correlations from H-2′ [δH 3.86 (1H, q, J = 10.0, 2.4 Hz)] in isopropanenitrile moiety to C-5 (δC 45.5 t) and C-6a (δC 62.1 d) confirmed the linkage of isoquinoline nitrogen with methine carbon in isopropanenitrile (Fig. 2). Herein, the planar structure of 1 was ascertained as N-(2′propanenitrile)-pronuciferine.
The absolute configuration of 1 was assigned by NOESY experiment and calculation of electronic circular dichroism (ECD) data. Because of the overlap of the signals of H-2′ and OCH3-2 in its 1H NMR spectrum tested in CDCl3, NOESY experiment with CD3OD as solvent (Fig. S10 in supplementary data) was also undertaken to observe the key NOE correlation from H-6a to H-2′. The presence of a key NOE correlation from H-6a [δH 3.98 (1H, q, J = 10.0, 2.0 Hz)] to H-2′ [δH 4.09 (1H, dd, J = 10.0, 6.0 Hz)] showed a syn-periplanar relationship between H-6a and the NCH which was consistent with pronuciferine (3) [6]. Possessing two additional chiral carbons, 1 has four possible absolute configurations [(6aS,2′S), (6aS,2′R), (6aR,2′R), and (6aR,2′S)]. To clarify the absolute configuration of C-6a and C-2′ in 1, the ECD data of four possible structures were calculated and compared with experimental spectrum. Four geometries were previously optimized by Density Functional Theory (DFT) methods at the B3LYP/6-31G(d) level [15]. Excitation energies and rotational strengths were calculated using time-dependent Density Functional Theory (TDDFT) at the B3LYP/6-31G(d,p) level in acetonitrile
Table 1 The NMR spectral data for 1 and 3. Position
Epigasine A (1) δCa
1 2 3 3a 3b 4
144.7 (s) 153.8 (s) 111.9 (d) 127.5 (s) 133.5 (s) 27.4 (t)
5
45.5 (t)
6a 7
62.1 (d) 46.7 (t)
8 9 10 11 12 13 13a OCH3-1 OCH3-2 1′ 2′ 3′ a 13
152.8 (d) 128.7 (d) 186.1 (s) 127.9 (d) 149.6 (d) 51.1 (s) 132.9 (s) 61.2 (q) 56.4 (q) 116.7 (s) 48.3 (d) 17.5 (q)
Pronuciferine (3) δHb
δH in MeOD
6.65 (1H, s)
6.79 (1H, s)
2.94 (2H, t, J = 3.4 Hz)
2.88 (2H, m)
3.28 (1H, ddd, J = 7.6, 4.8, 4.0 Hz) 2.70 (1H, ddd, J = 11.2, 8.8, 5.3 Hz) 3.98 (1H, dd, J = 10.0, 5.8 Hz) 2.39 (1H, dd, J = 11.6, 6.0 Hz) 2.19 (1H, t, J = 10.8 Hz) 6.85 (1H, dd, J = 10.0, 2.8 Hz) 6.42 (1H, dd, J = 10.0, 2.8 Hz)
3.36 (1H, m) 2.62 (1H, ddd, J = 10.8, 5.2, 4.0 Hz) 3.98 (1H, dd, J = 10.0, 2.0 Hz) 2.42 (1H, m) 2.21 (1H, t, J = 10.8 Hz) 7.01 (1H, dd, J = 10.0, 2.0 Hz) 6.38 (1H, dd, J = 10.0, 2.0 Hz)
6.33 (1H, dd, J = 10.0, 2.8 Hz) 7.05 (1H, dd, J = 10.0, 2.8 Hz)
6.28 (1H, dd, J = 10.0, 2.0 Hz) 7.22 (1H, dd, J = 10.0, 2.0 Hz)
3.59 (3H, s) 3.80 (3H, s)
3.59 (3H, s) 3.80 (3H, s)
3.86 (1H, q, J = 10.0, 2.4 Hz) 1.57 (3H, d, J = 6.4 Hz)
4.09 (1H, q, J = 10.0, 6.0 Hz) 1.52 (3H, d, J = 5.6 Hz)
C NMR spectra were performed in CDCl3 at 100 MHz. Multiplicity was determined by DEPT spectra. H NMR spectra were performed in CDCl3 at 400 MHz.
b 1
δCa 144.6 (s) 153.7 (s) 111.9 (d) 127.9 (s) 134.5 (s) 27.6 (t) 55.1 (t) 65.8 (d) 47.6 (t) 153.5 (d) 128.4 (d) 186.4 (d) 127.6 (d) 150.3 (d) 51.3 (s) 132.9 (s) 61.2 (q) 56.4 (q) 43.7 (q)
δHb
6.63 (1H, s)
3.02 (1H, ddd, J = 17.2, 11.6, 6.8 Hz) 2.84 (1H, dd, J = 16.8, 4.8 Hz) 3.14 (1H, dd, J = 11.6, 6.4 Hz) 2.53 (1H, td, J = 12.0, 6.6 Hz) 3.47 (1H, dd, J = 10.0, 6.0 Hz) 2.36 (1H, br d, J = 6.0 Hz, H-7a) 2.26 (1H, t, J = 11.8 Hz) 6.90 (1H, dd, J = 10.0, 2.2 Hz) 6.41 (1H, dd, J = 9.9, 2.2 Hz) 6.30 (1H, dd, J = 10.0, 2.2 Hz) 7.05 (1H, dd, J = 10.0, 2.2 Hz)
3.59 (3H, s) 3.84 (3H, s) 2.38 (3H, s)
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Fig. 2. Key 1H-1H COSY (
), HMBC (
with PCM model [16]. The ECD spectrum was simulated from electronic excitation energies and velocity rotational strengths (Fig. 3B). The results showed that the theoretical ECD data for 6aR,2′S-isomer was in good agreement with the experimental spectrum (Fig. 3A). Thus the structure of 1 was established as (6aR,2′S)-N-(2′-propanenitrile)pronuciferine and named epiganine A. It's worthy to note that epiganine A (1) is the first proaporphine alkaloid bearing an isopropanenitrile group at isoquinoline nitrogen. A plausible biogenetic pathway for unusual compound 1 is proposed as shown in Scheme 1. As shown, glutamic acid is converted to aldoxime through two successive N-hydroxylation of amino group of parent amino acid by an enzyme of cytochrome-P450 family. Aldoxime in turn is converted to cyanohydrin under the catalysis by another cytochrome-P450 enzyme [17–19]. Then, compound 3 and cyanohydrin undergo a substitution reaction [20] to produce 1a, which maintains βorientation of N-substitution as 3. Following oxidative decarboxylation [21,22] reactions of 1a affords 1. Compound 2 was isolated as a yellow amorphous powder and its molecular formula was deduced to be C21H19NO5 by HRESIMS at m/z 366.1338 [M + H]+ (Calcd. for C21H20NO5 366.1341), 388.1166 [M + Na]+ (Calcd. for C21H19NO5 Na 388.1161). The NMR spectra of compound 2 (Table 2) exhibited characteristic NMR features of an aporphine alkaloid [7,8,10] bearing an N-ethyl [δH 3.37 (3H, s); δC 49.5 q], two methoxyl groups [δH 4.06, 4.00 (each 3H, s); δC 56.0 (2C, q)], a methylenedioxy unit [δH 6.22 (2H, s); δC 101.4 t], and an aldehyde group [δH 10.26 (1H, s); δC 188.9 d]. Comparison of the NMR data of 2 with those of known 7-formyldehydrohernangerine [23,24] and 7formyldehydronornantenine [24] revealed that they shared the same skeleton and the aldehyde group was attached to its C-7 position, which was further supported by HMBC correlations from CHO [δH10.26 (1H, s)] to C-6a (δC 155.9 s), C-7 (δC 110 s), and C-7a (δC
) and NOESY (
) correlations of 1.
126.7 s) (Fig. 4). HMBC correlations from protons in methylenedioxy unit to C-1 (δC 147.7 s) and C-2 (δC 141.5 s) established the linkage of C(1)–O–CH2–O–C(2). Two methoxy groups were located at C-9 and C10 on the basis of the HMBC correlations from OCH3-9 [δH 4.06 (3H, s)] to C-9 (δC 56.0 q) and OCH3-10 [δH 4.00 (3H, s)] to C-10 (δC 56.0 q). NOE correlations (Fig. 4) from H-8 [δH 8.73 (1H, s)] to OCH3-9 and H-11 [δH 8.35 (1H, s)] to OCH3-10 also supported the substituted position of two methoxy groups. Unambiguous assignments of 1H and 13C NMR chemical shifts for 2 were accomplished by 2D NMR (1H-1H COSY, HSQC, and HMBC). Finally, the structure of 2 was identified as dehydrodicentrine-7-carbaldehyde and named epiganine B. Acetylcholinesterase (AChE) inhibitors could increase the efficiency of cholinergic transmissions by preventing from the hydrolysis of released acetylcholine, which is associated with the onset of Alzheimer's disease (AD) [25]. Thus, the enhancement of acetylcholine levels by using potent AChE inhibitors in the brain has been considered to be an effective approach for treating AD [26]. Previous researches revealed that most of aporphine alkaloids exhibit AChE inhibitory effects [27,28]. Herein, the AChE inhibitory activities of compounds 1–10 were measured using a modified Ellman method [29] and the results were showed in Table 3. The results revealed that compounds 2 and 4 showed strong AChE inhibitory effects with the IC50 values of 4.36 and 2.98 μM, respectively. Compounds 5–9 also exhibited potent AChE inhibitory activity with the IC50 values ranging 6.6–20.4 μM. And compounds 1, 3, and 10 showed weak AChE inhibitory activities (inhibition rates were 28.1%, 35.0%, and 29.3% at a concentration of 50 μM, respectively). The structure–activity relationship of these compounds for AChE inhibitory effects revealed that OCH2O and C6a_C7 might play important roles in AChE inhibitory activity of aporphine alkaloids. The present study is the first to report that proaporphine alkaloids were isolated from S. epigaea, which have much chemotaxonomic importance within the genus Stephania. Additionally, epiganine B (2) and dehydrodicentrine (4) were isolated as the candidates of AChE inhibitors for treating AD. 3. Experimental 3.1. Chemicals and instruments
Fig. 3. (A) Calculated and experimental ECD spectra of 1; (B) Calculated ECD spectra of four geometries of 1.
Melting points were determined on a XRC-1 Melting Point Apparatus and uncorrected. A Shimadzu UV–Vis 2550 spectrometer (Shimadzu, Tokyo, Japan) was used for UV–vis spectra determination; optical rotation was measured with a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Thermo Nicolet AVATAR 360 FTIR spectrometer (Thermo Nicolet Co., Madison, WI, USA) was used for scanning IR spectroscopy with KBr pellets. NMR spectra were recorded on Bruker Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. HRESIMS and ESIMS analyses were carried out with Agilent G3250AA (Agilent, Santa
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Scheme 1. Plausible biogenetic pathway of epiganine A (1).
Clara, CA, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, MA, USA). Electronic circular dichroism (ECD) spectrum was recorded by using a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., UK). Silica gel (200–300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China) was used for column chromatography (CC). Fractions were monitored by TLC and visualized by spraying with modified Dragendorff's reagent.
CC of silica gel, Sephadex LH-20 and yielded compounds 5 (150 mg) and 9 (850 mg). Fraction E (7.5 g) was further subjected to silica gel CC [PE–acetone (10:1–3:1)] to give 6 (4.8 mg), 7 (35.4 mg), and 8 (25.0 mg). Fraction F (15.0 g) was passed through a silica gel CC [CHCl3–MeOH (100:1–3:1)] and followed by Sephadex LH-20 chromatography to yield 2 (5.5 mg), 3 (10.0 mg), and 10 (32.7 mg).
3.2. Plant material
3.4. Inhibition of acetylcholinesterase activities
The roots of S. epigaea were collected from Kunming, Yunnan, China, in December 2014, and were identified by Prof. Shugang Lu from School of Life Sciences, Yunnan University. A voucher specimen (2014-dbr-01) has been deposited in the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Kunming, Yunnan, China.
AChE inhibition was performed spectrophotometrically by Ellman method [30] with slight modification [29]. Compounds were dissolved in 10% DMSO. The reaction mixture (totally 200 μL) containing 110 μL of phosphate buffer (PB, 0.1 M, pH 8.0), 10 μL of test compound (50 μM, final concentration), and 40 μL of AChE (0.02 U/mL, final concentration) was incubated for 20 min (30 °C). Then, the reaction was initiated by the addition of 40 μL of solution containing 5,5′dithiobis(2-nitrobenzoic acid) (DTNB, 0.625 mM) and acetylthiocholine iodide (ATCI, 0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 412 nm every 30 s for 30 min by using a Tecan Infinite® M1000 Pro microplate reader. Tacrine and huperzine A (Hup A) were used as the positive controls with final concentrations of 0.333 and 0.05 μM, respectively. All the reactions were performed in triplicate. The percentage inhibition (I%) was calculated as follows:
3.3. Extraction and isolation The dried and powdered roots (6.0 kg) of S. epigaea were percolated with 0.5% HCl. The aqueous acidic solution was basified with ammonia (10%) to pH 9.0 and then extracted with EtOAc. The removal of the solvent under reduced pressure afforded the total crude alkaloids (85.5 g) as a yellowish amorphous powder. The total alkaloids were chromatographed over silica gel CC eluting with petroleum ether (PE)–acetone (9:1–2:1) and chloroform–methanol (9:1–1:1) gradient systems to give Fractions A–G. Fraction A (0.7 g) was further subjected to silica gel CC [PE–acetone, (20:1–1:1)] to yield 4 (33.6 mg). Further CC purification of Fraction C (0.5 g) was accomplished by elution with PE–acetone–diethylamine (100:12.5:1) to afford 1 (13.5 mg). Fraction D (1.4 g) was separated by repeated Table 2 The NMR spectral data for 2. Position
δCa
1 1a 1b 2 3 3a 4 5 6a 7 7a
147.7 (s) 119.3 (s) 129.4 (s) 141.5 (s) 107.0 (d) 119.3 (s) 27.2 (t) 50.5 (t) 155.9 (s) 110 (s) 126.7 (s)
δHb
6.89 (1H, s) 3.11 (2H, br s) 3.52 (2H, br s)
Position
δCa
8 9 10 11 11a OCH3-9 OCH3-10 NCH3 OCH2O CHO-7
105.4 (d) 150.1 (s) 146.5 (s) 108.4 (d) 117.8 (s) 56.0 (q) 56.0 (q) 49.5 (q) 101.4 (t) 188.9 (d)
δHb
I% ¼ ðAE −AB Þ−ðAS –AB Þ=ðAE −AB Þ 100
where AE is the absorbance of the enzyme without test compound, and AS is the absorbance of enzyme with test compound, and AB is the absorbance of background. IC50 values of the potential AChE inhibitory compounds were calculated from inhibition curves obtained by plotting the percent inhibition at various inhibitor concentrations.
8.73 (1H, s)
3.5. Calculation 8.35 (1H, s) 4.06 (3H, s) 4.00 (3H, s) 3.37 (3H, s) 6.22 (2H, s) 10.26 (1H, s)
a 13 C NMR spectra were performed in CDCl3 at 100 MHz. Multiplicity was determined by DEPT spectra. b 1 H NMR spectra were performed in CDCl3 at 400 MHz.
The theoretical calculations of compound 1 were performed using Gaussian Program by Yunnan Electronic Computing Center. Four geometries of compound 1 [(6aS,2′S), (6aS,2′R), (6aR,2′R), and (6aR,2′S)] were previously optimized by Density Functional Theory (DFT) methods at the B3LYP/6-31G(d) level [15] and excitation energies and rotational strengths were calculated using time-dependent Density Functional Theory (TDDFT) at the B3LYP/6-31G(d,p) level in acetonitrile with PCM model [16]. The ECD spectrum is simulated from electronic excitation energies and velocity rotational strengths.
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Fig. 4. Key 1H-1H COSY (
), HMBC (
Epigasine A (1): white amorphous powder; m.p. 84–86 °C; [α]25 D − 8.89 (c 0.021, CHCl3); HRESIMS m/z 351.1710 [M + H]+ (Calcd. for C21H23N2O3 351.1703); UV λCH3CN max nm (ε) 210 (30030), 228 (19777), 280 (2184); FT-IR: νKBr max (cm−1) 3268 (= C–H), 2921 (− C–H), 2353 (C`N), 1662 (C_O), 1488 (− CH(CH3)CN), 1084 (C– O–C); 1H and 13C NMR spectral data see Table 1. Epigasine B (2): yellow amorphous powder; m.p. 146–147 °C; HRESIMS m/z 366.1338 [M + H]+ (Calcd. for C21H20NO5 366.1341), 388.1166 [M + Na]+ (Calcd. for C21H19NO5 Na 388.1161); UV λCH3OH max nm (ε) 201 (19253), 265 (23095), 429 (4480); FT-IR: νKBr max (cm−1) 2921 (− C–H), 2843 (O_C–H), 1620 (H–C_O), 1511, 1453 (benzene ring), 1392 (O_C–H), 1058 (C–O–C); 1H and 13C NMR spectral data see Table 2.
Conflict of interest The authors declared that there is no conflict of interest.
Acknowledgments This work was financially supported by a grant from the Natural Science Foundation of China (No. 81460648) and a grant from the Program for Changjiang Scholars and Innovative Research Team in University (IRT13095).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.05.019. Table 3 AChE inhibitory activity of compounds 1–10 and positive controls. Sample
IC50 (μM)
Sample
IC50 (μM)
1 2 3 4 5 6
N50 4.36 N50 2.98 8.32 13.9
7 8 9 10 Tacrinea HupAa
20.4 15.5 6.6 N50 0.25 0.04
Positive controls.
) correlations of 2.
References
3.6. Spectral data
a
) and NOESY (
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Proaporphine and aporphine alkaloids with acetylcholinesterase inhibitory activity from Stephania epigaea Jian-Wei Dong a,1, Le Cai a,1, Yun-Shan Fang a, Huai Xiao a,b, Zhen-Jie Li a, Zhong-Tao Ding a,⁎ a b
Key Laboratory of Medicinal Chemistry for Nature Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China School of Pharmaceutical Sciences and Chemistry, Dali University, Dali 671000, PR China
a r t i c l e
i n f o
Article history: Received 22 April 2015 Received in revised form 22 May 2015 Accepted 24 May 2015 Available online 29 May 2015 Chemical compounds studied in this article: Epiganine A Epiganine B Pronuciferine (PubChem CID: 200480) Dehydrodicentrine (PubChem CID: 3084326) Romerine (PubChem CID: 119204) Romeline N-methylcalycinine Phanostenine (PubChem CID: 12305138) Dicentrine (PubChem CID: 101300) N-methyllaurotetanine (PubChem CID: 16573)
a b s t r a c t An unusual proaporphine alkaloid bearing an isopropanenitrile group at isoquinoline nitrogen, named epiganine A (1) and a new aporphine alkaloid, epiganine B (2), together with eight known alkaloids, pronuciferine (3), dehydrodicentrine (4), romerine (5), romeline (6), N-methylcalycinine (7), phanostenine (8), dicentrine (9), and N-methyllaurotetanine (10), were isolated from the roots of Stephania epigaea. The absolute configuration of 1 was determined by calculating electronic circular dichroism (ECD) and comparing with experimental data. Compounds 2 and 4 showed strong acetylcholinesterase (AChE) inhibitory effects with the IC50 values of 4.36 and 2.98 μM, respectively. Compounds 5–9 also exhibited potent AChE inhibitory activities. © 2015 Elsevier B.V. All rights reserved.
Keywords: Stephania epigaea Proaporphine alkaloid Aporphine alkaloid Acetylcholinesterase inhibitory activity
1. Introduction The genus Stephania (Menispermaceae) is distributed mainly in Asia and Africa, with about thirty nine species of this genus growing in mainland China [1], which have been used as folk medicine for treating asthma, cancer, dysentery, fever, hyperglycemia, intestinal complaints, inflammation, sleep disturbances, tuberculosis, and so on [2]. Stephania epigaea Lo is an herbaceous liana mainly growing in Yunnan and Sichuan provinces, China [1]. Its tubers are frequently used by local people to treat fever and for sedation. Previous phytochemical researches showed that the major constituents of S. epigaea are aporphine [3], morphine [4], and bisbenzylisoquinoline alkaloids [2,5]. In order to explore the new and bioactive constituents from S. epigaea, a detailed phytochemical investigation on the roots of S. epigaea was carried out in the present study. An unusual proaporphine alkaloid bearing ⁎ Corresponding author. E-mail address: [email protected] (Z.-T. Ding). 1 The first two authors contributed equally to this paper.
http://dx.doi.org/10.1016/j.fitote.2015.05.019 0367-326X/© 2015 Elsevier B.V. All rights reserved.
an isopropanenitrile group at isoquinoline nitrogen, named epigasine A (1) and a new aporphine alkaloid, epigasine B (2), together with eight known proaporphine/aporphine alkaloids, pronuciferine (3) [6], dehydrodicentrine (4) [7], romerine (5) [8], romeline (6) [9], Nmethylcalycinine (7) [10], phanostenine (8) [11,12], dicentrine (9) [13], and N-methyllaurotetanine (10) [14] (Fig. 1) were isolated from the roots of S. epigaea. Among the known compounds, compounds 3, 5, 8, and 10 were isolated from S. epigaea for the first time. The acetylcholinesterase (AChE) inhibitory activities of the isolated compounds have also been evaluated. 2. Results and discussions Compound 1 was obtained as a white amorphous powder and its molecular formula was deduced to be C21H22N2O3 by HRESIMS at m/z 351.1710 [M + H]+ (Calcd. for C21H23N2O3 351.1703). Its IR spectrum indicated the presence of CN_(2353 cm−1), C_O (1662 cm−1), and benzene ring (1450–1600 cm− 1). The NMR spectra of 1 (Table 1) showed the characteristic signals for a dienone moiety [δH 7.05 (1H,
J.-W. Dong et al. / Fitoterapia 104 (2015) 102–107
103
Fig. 1. Structures of compounds 1–10.
dd, J = 10.0, 2.8 Hz), 6.85 (1H, dd, J = 10.0, 2.8 Hz), 6.42 (1H, dd, J = 10.0, 2.8 Hz), 6.33 (1H, dd, J = 10.0, 2.8 Hz); δC 186.1 s, 152.8 d, 149.6 d, 128.7 d, 127.9 d], a penta-substituted phenyl unit [δH 6.65 (1H, s); δC 153.8 s, 144.7 s, 133.5 s, 127.5 s, 111.9 d], and two methoxy groups [δH 3.80 (3H, s), 3.59 (3H, s); δC 61.2 q, 56.4 q] [6]. A detailed comparison of the 1H and 13C NMR chemical shifts (Table 1) of 1 with pronuciferine (3) [6] which was also isolated from S. epigaea in this study, strongly suggested a proaporphine alkaloid for 1. The main difference between them was that compound 1 possesses no N-methyl group which exists in 3. Meanwhile, compound 1 exhibited the signals of an additional isopropanenitrile moiety [δH 3.86 (1H, q, J = 10.0, 2.4 Hz), 1.57 (3H, d, J = 6.4 Hz); δC 116.7 s, 48.3 d, 17.5 q] in its NMR spectra. HMBC correlations from H-2′ [δH 3.86 (1H, q, J = 10.0, 2.4 Hz)] in isopropanenitrile moiety to C-5 (δC 45.5 t) and C-6a (δC 62.1 d) confirmed the linkage of isoquinoline nitrogen with methine carbon in isopropanenitrile (Fig. 2). Herein, the planar structure of 1 was ascertained as N-(2′propanenitrile)-pronuciferine.
The absolute configuration of 1 was assigned by NOESY experiment and calculation of electronic circular dichroism (ECD) data. Because of the overlap of the signals of H-2′ and OCH3-2 in its 1H NMR spectrum tested in CDCl3, NOESY experiment with CD3OD as solvent (Fig. S10 in supplementary data) was also undertaken to observe the key NOE correlation from H-6a to H-2′. The presence of a key NOE correlation from H-6a [δH 3.98 (1H, q, J = 10.0, 2.0 Hz)] to H-2′ [δH 4.09 (1H, dd, J = 10.0, 6.0 Hz)] showed a syn-periplanar relationship between H-6a and the NCH which was consistent with pronuciferine (3) [6]. Possessing two additional chiral carbons, 1 has four possible absolute configurations [(6aS,2′S), (6aS,2′R), (6aR,2′R), and (6aR,2′S)]. To clarify the absolute configuration of C-6a and C-2′ in 1, the ECD data of four possible structures were calculated and compared with experimental spectrum. Four geometries were previously optimized by Density Functional Theory (DFT) methods at the B3LYP/6-31G(d) level [15]. Excitation energies and rotational strengths were calculated using time-dependent Density Functional Theory (TDDFT) at the B3LYP/6-31G(d,p) level in acetonitrile
Table 1 The NMR spectral data for 1 and 3. Position
Epigasine A (1) δCa
1 2 3 3a 3b 4
144.7 (s) 153.8 (s) 111.9 (d) 127.5 (s) 133.5 (s) 27.4 (t)
5
45.5 (t)
6a 7
62.1 (d) 46.7 (t)
8 9 10 11 12 13 13a OCH3-1 OCH3-2 1′ 2′ 3′ a 13
152.8 (d) 128.7 (d) 186.1 (s) 127.9 (d) 149.6 (d) 51.1 (s) 132.9 (s) 61.2 (q) 56.4 (q) 116.7 (s) 48.3 (d) 17.5 (q)
Pronuciferine (3) δHb
δH in MeOD
6.65 (1H, s)
6.79 (1H, s)
2.94 (2H, t, J = 3.4 Hz)
2.88 (2H, m)
3.28 (1H, ddd, J = 7.6, 4.8, 4.0 Hz) 2.70 (1H, ddd, J = 11.2, 8.8, 5.3 Hz) 3.98 (1H, dd, J = 10.0, 5.8 Hz) 2.39 (1H, dd, J = 11.6, 6.0 Hz) 2.19 (1H, t, J = 10.8 Hz) 6.85 (1H, dd, J = 10.0, 2.8 Hz) 6.42 (1H, dd, J = 10.0, 2.8 Hz)
3.36 (1H, m) 2.62 (1H, ddd, J = 10.8, 5.2, 4.0 Hz) 3.98 (1H, dd, J = 10.0, 2.0 Hz) 2.42 (1H, m) 2.21 (1H, t, J = 10.8 Hz) 7.01 (1H, dd, J = 10.0, 2.0 Hz) 6.38 (1H, dd, J = 10.0, 2.0 Hz)
6.33 (1H, dd, J = 10.0, 2.8 Hz) 7.05 (1H, dd, J = 10.0, 2.8 Hz)
6.28 (1H, dd, J = 10.0, 2.0 Hz) 7.22 (1H, dd, J = 10.0, 2.0 Hz)
3.59 (3H, s) 3.80 (3H, s)
3.59 (3H, s) 3.80 (3H, s)
3.86 (1H, q, J = 10.0, 2.4 Hz) 1.57 (3H, d, J = 6.4 Hz)
4.09 (1H, q, J = 10.0, 6.0 Hz) 1.52 (3H, d, J = 5.6 Hz)
C NMR spectra were performed in CDCl3 at 100 MHz. Multiplicity was determined by DEPT spectra. H NMR spectra were performed in CDCl3 at 400 MHz.
b 1
δCa 144.6 (s) 153.7 (s) 111.9 (d) 127.9 (s) 134.5 (s) 27.6 (t) 55.1 (t) 65.8 (d) 47.6 (t) 153.5 (d) 128.4 (d) 186.4 (d) 127.6 (d) 150.3 (d) 51.3 (s) 132.9 (s) 61.2 (q) 56.4 (q) 43.7 (q)
δHb
6.63 (1H, s)
3.02 (1H, ddd, J = 17.2, 11.6, 6.8 Hz) 2.84 (1H, dd, J = 16.8, 4.8 Hz) 3.14 (1H, dd, J = 11.6, 6.4 Hz) 2.53 (1H, td, J = 12.0, 6.6 Hz) 3.47 (1H, dd, J = 10.0, 6.0 Hz) 2.36 (1H, br d, J = 6.0 Hz, H-7a) 2.26 (1H, t, J = 11.8 Hz) 6.90 (1H, dd, J = 10.0, 2.2 Hz) 6.41 (1H, dd, J = 9.9, 2.2 Hz) 6.30 (1H, dd, J = 10.0, 2.2 Hz) 7.05 (1H, dd, J = 10.0, 2.2 Hz)
3.59 (3H, s) 3.84 (3H, s) 2.38 (3H, s)
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Fig. 2. Key 1H-1H COSY (
), HMBC (
with PCM model [16]. The ECD spectrum was simulated from electronic excitation energies and velocity rotational strengths (Fig. 3B). The results showed that the theoretical ECD data for 6aR,2′S-isomer was in good agreement with the experimental spectrum (Fig. 3A). Thus the structure of 1 was established as (6aR,2′S)-N-(2′-propanenitrile)pronuciferine and named epiganine A. It's worthy to note that epiganine A (1) is the first proaporphine alkaloid bearing an isopropanenitrile group at isoquinoline nitrogen. A plausible biogenetic pathway for unusual compound 1 is proposed as shown in Scheme 1. As shown, glutamic acid is converted to aldoxime through two successive N-hydroxylation of amino group of parent amino acid by an enzyme of cytochrome-P450 family. Aldoxime in turn is converted to cyanohydrin under the catalysis by another cytochrome-P450 enzyme [17–19]. Then, compound 3 and cyanohydrin undergo a substitution reaction [20] to produce 1a, which maintains βorientation of N-substitution as 3. Following oxidative decarboxylation [21,22] reactions of 1a affords 1. Compound 2 was isolated as a yellow amorphous powder and its molecular formula was deduced to be C21H19NO5 by HRESIMS at m/z 366.1338 [M + H]+ (Calcd. for C21H20NO5 366.1341), 388.1166 [M + Na]+ (Calcd. for C21H19NO5 Na 388.1161). The NMR spectra of compound 2 (Table 2) exhibited characteristic NMR features of an aporphine alkaloid [7,8,10] bearing an N-ethyl [δH 3.37 (3H, s); δC 49.5 q], two methoxyl groups [δH 4.06, 4.00 (each 3H, s); δC 56.0 (2C, q)], a methylenedioxy unit [δH 6.22 (2H, s); δC 101.4 t], and an aldehyde group [δH 10.26 (1H, s); δC 188.9 d]. Comparison of the NMR data of 2 with those of known 7-formyldehydrohernangerine [23,24] and 7formyldehydronornantenine [24] revealed that they shared the same skeleton and the aldehyde group was attached to its C-7 position, which was further supported by HMBC correlations from CHO [δH10.26 (1H, s)] to C-6a (δC 155.9 s), C-7 (δC 110 s), and C-7a (δC
) and NOESY (
) correlations of 1.
126.7 s) (Fig. 4). HMBC correlations from protons in methylenedioxy unit to C-1 (δC 147.7 s) and C-2 (δC 141.5 s) established the linkage of C(1)–O–CH2–O–C(2). Two methoxy groups were located at C-9 and C10 on the basis of the HMBC correlations from OCH3-9 [δH 4.06 (3H, s)] to C-9 (δC 56.0 q) and OCH3-10 [δH 4.00 (3H, s)] to C-10 (δC 56.0 q). NOE correlations (Fig. 4) from H-8 [δH 8.73 (1H, s)] to OCH3-9 and H-11 [δH 8.35 (1H, s)] to OCH3-10 also supported the substituted position of two methoxy groups. Unambiguous assignments of 1H and 13C NMR chemical shifts for 2 were accomplished by 2D NMR (1H-1H COSY, HSQC, and HMBC). Finally, the structure of 2 was identified as dehydrodicentrine-7-carbaldehyde and named epiganine B. Acetylcholinesterase (AChE) inhibitors could increase the efficiency of cholinergic transmissions by preventing from the hydrolysis of released acetylcholine, which is associated with the onset of Alzheimer's disease (AD) [25]. Thus, the enhancement of acetylcholine levels by using potent AChE inhibitors in the brain has been considered to be an effective approach for treating AD [26]. Previous researches revealed that most of aporphine alkaloids exhibit AChE inhibitory effects [27,28]. Herein, the AChE inhibitory activities of compounds 1–10 were measured using a modified Ellman method [29] and the results were showed in Table 3. The results revealed that compounds 2 and 4 showed strong AChE inhibitory effects with the IC50 values of 4.36 and 2.98 μM, respectively. Compounds 5–9 also exhibited potent AChE inhibitory activity with the IC50 values ranging 6.6–20.4 μM. And compounds 1, 3, and 10 showed weak AChE inhibitory activities (inhibition rates were 28.1%, 35.0%, and 29.3% at a concentration of 50 μM, respectively). The structure–activity relationship of these compounds for AChE inhibitory effects revealed that OCH2O and C6a_C7 might play important roles in AChE inhibitory activity of aporphine alkaloids. The present study is the first to report that proaporphine alkaloids were isolated from S. epigaea, which have much chemotaxonomic importance within the genus Stephania. Additionally, epiganine B (2) and dehydrodicentrine (4) were isolated as the candidates of AChE inhibitors for treating AD. 3. Experimental 3.1. Chemicals and instruments
Fig. 3. (A) Calculated and experimental ECD spectra of 1; (B) Calculated ECD spectra of four geometries of 1.
Melting points were determined on a XRC-1 Melting Point Apparatus and uncorrected. A Shimadzu UV–Vis 2550 spectrometer (Shimadzu, Tokyo, Japan) was used for UV–vis spectra determination; optical rotation was measured with a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). A Thermo Nicolet AVATAR 360 FTIR spectrometer (Thermo Nicolet Co., Madison, WI, USA) was used for scanning IR spectroscopy with KBr pellets. NMR spectra were recorded on Bruker Avance 400 MHz spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. HRESIMS and ESIMS analyses were carried out with Agilent G3250AA (Agilent, Santa
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105
Scheme 1. Plausible biogenetic pathway of epiganine A (1).
Clara, CA, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, MA, USA). Electronic circular dichroism (ECD) spectrum was recorded by using a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., UK). Silica gel (200–300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China) was used for column chromatography (CC). Fractions were monitored by TLC and visualized by spraying with modified Dragendorff's reagent.
CC of silica gel, Sephadex LH-20 and yielded compounds 5 (150 mg) and 9 (850 mg). Fraction E (7.5 g) was further subjected to silica gel CC [PE–acetone (10:1–3:1)] to give 6 (4.8 mg), 7 (35.4 mg), and 8 (25.0 mg). Fraction F (15.0 g) was passed through a silica gel CC [CHCl3–MeOH (100:1–3:1)] and followed by Sephadex LH-20 chromatography to yield 2 (5.5 mg), 3 (10.0 mg), and 10 (32.7 mg).
3.2. Plant material
3.4. Inhibition of acetylcholinesterase activities
The roots of S. epigaea were collected from Kunming, Yunnan, China, in December 2014, and were identified by Prof. Shugang Lu from School of Life Sciences, Yunnan University. A voucher specimen (2014-dbr-01) has been deposited in the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Kunming, Yunnan, China.
AChE inhibition was performed spectrophotometrically by Ellman method [30] with slight modification [29]. Compounds were dissolved in 10% DMSO. The reaction mixture (totally 200 μL) containing 110 μL of phosphate buffer (PB, 0.1 M, pH 8.0), 10 μL of test compound (50 μM, final concentration), and 40 μL of AChE (0.02 U/mL, final concentration) was incubated for 20 min (30 °C). Then, the reaction was initiated by the addition of 40 μL of solution containing 5,5′dithiobis(2-nitrobenzoic acid) (DTNB, 0.625 mM) and acetylthiocholine iodide (ATCI, 0.625 mM) for AChE inhibitory activity assay, respectively. The hydrolysis of acetylthiocholine was monitored at 412 nm every 30 s for 30 min by using a Tecan Infinite® M1000 Pro microplate reader. Tacrine and huperzine A (Hup A) were used as the positive controls with final concentrations of 0.333 and 0.05 μM, respectively. All the reactions were performed in triplicate. The percentage inhibition (I%) was calculated as follows:
3.3. Extraction and isolation The dried and powdered roots (6.0 kg) of S. epigaea were percolated with 0.5% HCl. The aqueous acidic solution was basified with ammonia (10%) to pH 9.0 and then extracted with EtOAc. The removal of the solvent under reduced pressure afforded the total crude alkaloids (85.5 g) as a yellowish amorphous powder. The total alkaloids were chromatographed over silica gel CC eluting with petroleum ether (PE)–acetone (9:1–2:1) and chloroform–methanol (9:1–1:1) gradient systems to give Fractions A–G. Fraction A (0.7 g) was further subjected to silica gel CC [PE–acetone, (20:1–1:1)] to yield 4 (33.6 mg). Further CC purification of Fraction C (0.5 g) was accomplished by elution with PE–acetone–diethylamine (100:12.5:1) to afford 1 (13.5 mg). Fraction D (1.4 g) was separated by repeated Table 2 The NMR spectral data for 2. Position
δCa
1 1a 1b 2 3 3a 4 5 6a 7 7a
147.7 (s) 119.3 (s) 129.4 (s) 141.5 (s) 107.0 (d) 119.3 (s) 27.2 (t) 50.5 (t) 155.9 (s) 110 (s) 126.7 (s)
δHb
6.89 (1H, s) 3.11 (2H, br s) 3.52 (2H, br s)
Position
δCa
8 9 10 11 11a OCH3-9 OCH3-10 NCH3 OCH2O CHO-7
105.4 (d) 150.1 (s) 146.5 (s) 108.4 (d) 117.8 (s) 56.0 (q) 56.0 (q) 49.5 (q) 101.4 (t) 188.9 (d)
δHb
I% ¼ ðAE −AB Þ−ðAS –AB Þ=ðAE −AB Þ 100
where AE is the absorbance of the enzyme without test compound, and AS is the absorbance of enzyme with test compound, and AB is the absorbance of background. IC50 values of the potential AChE inhibitory compounds were calculated from inhibition curves obtained by plotting the percent inhibition at various inhibitor concentrations.
8.73 (1H, s)
3.5. Calculation 8.35 (1H, s) 4.06 (3H, s) 4.00 (3H, s) 3.37 (3H, s) 6.22 (2H, s) 10.26 (1H, s)
a 13 C NMR spectra were performed in CDCl3 at 100 MHz. Multiplicity was determined by DEPT spectra. b 1 H NMR spectra were performed in CDCl3 at 400 MHz.
The theoretical calculations of compound 1 were performed using Gaussian Program by Yunnan Electronic Computing Center. Four geometries of compound 1 [(6aS,2′S), (6aS,2′R), (6aR,2′R), and (6aR,2′S)] were previously optimized by Density Functional Theory (DFT) methods at the B3LYP/6-31G(d) level [15] and excitation energies and rotational strengths were calculated using time-dependent Density Functional Theory (TDDFT) at the B3LYP/6-31G(d,p) level in acetonitrile with PCM model [16]. The ECD spectrum is simulated from electronic excitation energies and velocity rotational strengths.
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J.-W. Dong et al. / Fitoterapia 104 (2015) 102–107
Fig. 4. Key 1H-1H COSY (
), HMBC (
Epigasine A (1): white amorphous powder; m.p. 84–86 °C; [α]25 D − 8.89 (c 0.021, CHCl3); HRESIMS m/z 351.1710 [M + H]+ (Calcd. for C21H23N2O3 351.1703); UV λCH3CN max nm (ε) 210 (30030), 228 (19777), 280 (2184); FT-IR: νKBr max (cm−1) 3268 (= C–H), 2921 (− C–H), 2353 (C`N), 1662 (C_O), 1488 (− CH(CH3)CN), 1084 (C– O–C); 1H and 13C NMR spectral data see Table 1. Epigasine B (2): yellow amorphous powder; m.p. 146–147 °C; HRESIMS m/z 366.1338 [M + H]+ (Calcd. for C21H20NO5 366.1341), 388.1166 [M + Na]+ (Calcd. for C21H19NO5 Na 388.1161); UV λCH3OH max nm (ε) 201 (19253), 265 (23095), 429 (4480); FT-IR: νKBr max (cm−1) 2921 (− C–H), 2843 (O_C–H), 1620 (H–C_O), 1511, 1453 (benzene ring), 1392 (O_C–H), 1058 (C–O–C); 1H and 13C NMR spectral data see Table 2.
Conflict of interest The authors declared that there is no conflict of interest.
Acknowledgments This work was financially supported by a grant from the Natural Science Foundation of China (No. 81460648) and a grant from the Program for Changjiang Scholars and Innovative Research Team in University (IRT13095).
Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.05.019. Table 3 AChE inhibitory activity of compounds 1–10 and positive controls. Sample
IC50 (μM)
Sample
IC50 (μM)
1 2 3 4 5 6
N50 4.36 N50 2.98 8.32 13.9
7 8 9 10 Tacrinea HupAa
20.4 15.5 6.6 N50 0.25 0.04
Positive controls.
) correlations of 2.
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3.6. Spectral data
a
) and NOESY (
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