2 Zhuhai Key Laboratory of Fundamental and Applied Research in Traditional Chinese Medicine, Zunyi Medical University Zhuhai Campus, Zhuhai 519041, China
Aconitum brevicalcaratum Diels belongs to the family Ranunculaceae and is distributed mainly in the northwest of Yunnan Province in China at an altitude of 2800-3800 m . Its roots have been reputedly used as a folk medicine for the treatment of cough, cold, and various types of pain. Diterpenoid alkaloids (DAs), which possess complex structures and various bioactivities, are the main active ingredients of species from the genus Aconitum [2-4]. However, phytochemical research on A. brevicalcaratum is quite limited and only nine C19-diterpenoid alkaloids have been isolated from this plant [5-7]. In the course of our continued studies of diterpenoid alkaloids from Aconitum plants [8-9], three new C19-diterpenoid alkaloids brochyponines A-C (1-3) were isolated from the roots of A. brevicalcaratum. This paper reports the isolation and structural elucidation of these alkaloids (Fig. 1).
Compound 1 was obtained as amorphous solid and its molecular formula was deduced to be C25H41NO5 by HR-ESI-MS at m/z 436.3056 [M + H]+. The NMR spectra revealed the presence of an N-ethyl group (δH 1.44, t, J = 7.2 Hz; 2.94, 2H, br s; δC 13.8, q; 49.6, t) and four methoxy groups (δH 3.09, 3.18, 3.32, 3.43, each 3H, s; δC 48.9, q; 56.3, q; 56.4, q; 57.7, q) (Table 1). Compound 1 possessed nineteen carbons except for the above groups, including three characteristic quaternary carbons, in combination with biogenetic consideration, suggesting that 1 might be an aconitine-type C19-DA . The 13C NMR spectrum showed five oxygenated carbons (δC 66.6, t; 78.7, s; 81.0, d; 84.7, d; 83.5, d), corresponding to the molecular formula, which indicated the existence of one hydroxyl group in addition to four methoxyl groups. The oxygenated methylene was attributed to C-18 (δC 66.6, t), indicating a hydroxyl group substituted at C-18, which was confirmed by HMBC correlations from H-18 (δH 4.00, d, J = 11.3 Hz; 4.44, d, J = 11.3 Hz) to C-19 (δC 55.8, t), C-3 (δC 24.8, t) and C-5 (δC 39.0, d) . Four methoxyl groups were placed at C-1, C-8, C-14, and C-16 on the basis of the HMBC correlations from OCH3-1 (δH 3.18, s) to C-1 (δC 81.0, d), OCH3-8 (δH 3.09, s) to C-8 (δC 78.7, s), OCH3-14 (δH 3.43, s) to C-14 (δC 84.7, d), and OCH3-16 (δH 3.32, s) to C-16 (δC 83.5, d), respectively (Fig. 2). The ROESY correlations between H-14 and H-10, H-16 (δH 3.43, br s) and H-12α (δH 1.26, m) demonstrated the α-orientation of OCH3-14 and β-orientation of OCH3-16, respectively (Fig. 2) . The α-orientation of OCH3-1 was established according to the ROESY correlation between H-1 (δH 3.39, br s) and H-5 (δH 1.87, m) . Therefore, the structure of compound 1 was determined. Besides, the NMR spectroscopic data of salt form of compound 1 (Measured in CDCl3) are listed in Table 1. The difference in chemical shifts between compound 1 and its salt form was mainly at C-17, C-19, C-21, and C-22, which was in accord with the literatures [12-13]. Particularly, the uncommon upfield of C-22 (δC 10, q) could be utilized to distinguish the salt form.
Compound 2 was isolated as a white powder and its molecular formula was deduced to be C32H47N2O6 by HR-ESI-MS at m/z 555.3428 [M + H]+. The NMR spectrum of 2 exhibited characteristic features of the aconitine-type C19-DA, bearing an N-ethyl group (δH 1.10, t, J = 7.2 Hz; 2.53, 2.53, each 1H, m; δC 13.4, q; 49.3, t), four methoxy groups (δH 3.28, 3.12, 3.35, 3.35, each 3H, s; δC 56.5, q; 48.3, q; 57.8, q; 56.5, q), and an anthranoyl group (δH 6.66, d, J = 8.6 Hz; 7.26, td, J = 9.1 Hz, 1.6 Hz; 6.65, d, J = 8.6 Hz; 7.80, dd, J = 1.4 Hz, 8.0Hz; δC 110.8, s; 150.7, s; 116.9, d; 134.3, d; 116.4, d; 131.1, d; 168.1, s; Table 1) . Four methoxy groups were placed at C-1, C-8, C-14 and C-16 on the basis of HMBC correlations from OCH3-1 (δH 3.28, s) to C-1 (δC 85.4, d), OCH3-8 (δH 3.12, s) to C-8 (δC 77.7, d), OCH3-14 (δH 3.35, s) to C-14 (δC 83.9, d), and OCH3-16 (δH 3.35, s) to C-16 (δC 83.8, d), respectively (Fig. 3). The orientation of OCH3-1 was determined as α according to ROESY correlation between H-1 (δH 3.15, m) and H-5 (δH 1.68, d, J = 7.3 Hz) along with its chemical shift (δC 85.4, d) . The anthranoyl group was located at C-18 on the basis of HMBC correlation from H-18 (δH 4.05, 3.94, ABq, J = 11.2 Hz) to OCO-18 (δC 168.1, s). Thus, the structure of compound 3 was determined as brochyponine B. Although compounds 2 and 1 possessed nearly the same substituent groups, the chemical shifts of 2 were very different from that of 1, which might be attributed to the solvent effect.
Compound 3 was isolated as an amorphous solid, whose molecular formula was deduced to be C29H38N2O6 by HR-ESI-MS at m/z 511.2803 [M + H]+. The IR spectrum revealed the presence of a hydroxyl group (3432 cm-1), an ester function (1720 cm-1) and an imino group (1677 cm-1) . The NMR spectra showed the presence of an anthranoyl group (δH 7.07, d, J = 8.3 Hz; 7.36, t, J = 8.2 Hz; 6.74, t, J = 7.0 Hz; 8.16, d, J = 8.1 Hz; δC 110.7, s; 153.1, s; 117.7, d; 135.1, d; 116.1, d; 131.9, d; 168.7, s) and three methoxy groups (δH 3.22, 3.43, 3.22, each 3H, s; δC 56.4, q; 57.9, q; 56.4, q; Table 1). Compound 3 contained nineteen carbons except for the above groups, including three characteristic quaternary carbons, in combination with biogenetic consideration, suggesting an aconitine-type C19-DA for 3 . The 13C NMR revealed five oxygenated carbons (δC 83.6, d; 73.7, s; 85.2, d; 83.4, d; 67.6, t), corresponding to the presence of a hydroxyl group except for the anthranoyl and methoxyl groups. Three methoxy groups were placed at C-1, C-14 and C-16 on the basis of HMBC correlations from OCH3-1 (δH 3.22, s) to C-1 (δC 83.6, d), OCH3-14 (δH 3.43, s) to C-14 (δC 85.2, d), and OCH3-16 (δH 3.22, s) to C-16 (δC 83.4, d), respectively (Fig. 3). The anthranoyl group was located at C-18 on the basis of HMBC from H-18 (δH 4.56, ABq, J = 11.4 Hz; 4.45, ABq, J = 11.4 Hz) to OCO-18 (δC 168.7, s), which was further supported by the fact that chemical shift of H-18 were upfield about 0.5- 1.0 ppm [17-18]. Besides, a rare imino group was identified in the NMR spectra of 3, which was verified by the HMBC correlations from olefinic H-19 (δH 7.59, s) to C-4 (δC 49.2, s), C-5 (δC 42.5, d) and C-17 (δC 63.0, d), respectively (Fig. 3). Hence, the structure of compound 3 was determined, with its assigned NMR spectroscopic data being listed in Table 1.
It's worth to note that compounds 1 and 3 bearing a methoxy group at C-8 were verified to be originated from plant by LC-MS-MS experiment (Fig. 4). Bioactivity assay revealed that compounds 1-3 exhibited no NO inhibitory, antimicrobial and cytotoxic activities.
Optical rotations were measured on a Jasco P-1020 digital polarimeter (Jasco, Tokyo, Japan). Melting points were determined on an XRC-1 Melting Point Apparatus (Sichuan University Science Instrument Co., Chengdu, China). A Nicolet Magna-IR 550 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 or Bruker Avance Ⅲ 600 MHz spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. ESI-MS analyses were recorded with Agilent G3250AA (Agilent, Santa Clara, CA, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, MA, USA). Silica gels (200-300 mesh and 300-400 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China) were used for column chromatography (CC). Fractions were monitored by TLC and visualized by spraying with modified Dragendorff's reagent.Plant materials
The roots of A. brevicalcaratum were collected from Yulong Snow Mountain in Yunnan Province of China in September 2016 and identified and authenticated by Assistant Prof. YANG Shu-Da from School of Pharmacy in Kunming Medical University. A voucher specimen (DJ-1) was deposited in the School of Chemical Science and Technology in Yunnan University of China.Extraction and isolation
Air-dried and powdered roots (1.8 kg) of A. brevicalcaratum were percolated with 0.5% HCl. The aqueous acidic solution was basified with ammonia to pH 9.0 and then extracted with ethyl acetate. Removal of the solvent under reduced pressure afforded the total crude alkaloids (20.0 g) as yellowish amorphous powder. The total alkaloids were subjected to silica gel CC eluted with CHCl3-CH3OH gradient system (100 : 1 to 1 : 1) to give five fractions (Frs.A-E). Fr. B (5.3 g) was further subjected to silica gel CC [petroleum ether-acetone-diethylamine, 100 : 2 : 1 to 100 : 20 : 1] to give four factions (Frs. B1-B4). Further silica gel CC purification of Fr. B1 (1.7 g) was subjected to silica gel CC (petroleum ether-acetone-diethylamine, 100 : 3 : 1 to 100 : 10 : 1) to yield compound2 (11 mg). Fr. B3 (1.2 g) was subjected to silica gel CC (petroleum ether-acetone-diethylamine, 100 : 5 : 1 to 100 : 20 : 1) to yield compounds 1 (5 mg). Fr. D (0.9 g) was accomplished by elution with CHCl3-CH3OH (30 : 1 to 3 : 1) to afford compound 3 (4 mg).Brochyponine A (1)
Amorphous solid; [α]D20 +37.9 (c 1.0, CH3OH); IR (KBr) νmax: 3425, 3329, 3193, 1657, 1614, 1456, 1400, 1074, 584 cm-1. For 1H-and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS m/z 436.3056 [M + H]+ (Calcd. for C25H42NO5, 436.3063).Brochyponine B (2)
White powder; mp 78-80 ℃, [α]D20 +12.5 (c 1.0, CH3OH); IR (KBr) νmax: 3715, 3672, 3432, 3424, 3330, 3241, 2500, 1720, 1614, 1457, 1401, 588, 519 cm-1. For 1H-and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS m/z 555.3428 [M + H]+ (Calcd. for C32H47N2O6, 555.3434).Brochyponine C (3)
Amorphous solid; [α]D20-14.8 (c 1.0, CH3OH); IR (KBr) νmax: 3432, 3331, 3253, 3189, 1720, 1677, 1611, 1456, 1401, 1180, 1091, 780, 592 cm-1. For 1H-and 13C NMR spectroscopic data, see Table 1. HR-ESI-MS m/z 511.2803 [M + H]+ (Calcd. for C29H39N2O6, 511.2808).
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