1 Introduction

Aconitum stapfianum Hand.-Mazz. belongs to the genus Aconitum of the Ranunculaceae, and is distributed mainly at an altitude of 2800–3400 m in the northwest of Yunnan Province in China [1]. Up to now, only four diterpenoid alkaloids have been isolated from A. stapfianum [2]. As part of our continuous work on the discovery of bioactive ingredients from the Aconitum plants [3, 4], an phytochemical investigation on the roots of A. stapfianum was carried out to afford nineteen alkaloids (Fig. 1), including a new C19-diterpenoid alkaloid stapfianine A (1), a new benzamide derivative stapfianine B (2), and a known amide 4-oxo-pentanoic acid dimethylamide (3) found in nature for the first time. Their structures were established on the basis of extensive spectroscopic analyses. In this paper, the isolation and structure determination of these alkaloids are described.

Fig. 1
figure 1

Structures of alkaloids isolated from Aconitum stapfianum

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2 Results and Discussion

Compound 1 was isolated as a white amorphous powder and its molecular formula was deduced to be C32H43NO6 by HRESIMS at m/z 538.3169 [M+H]+. The NMR spectra of 1 showed the presence of a ester carbonyl (δ C 166.6 s), a characteristic disubstituted double bond (δ H 6.41, d, J = 16.0 Hz, 7.65, d, J = 16.0 Hz; δ C 118.0 d, 145.4 d) and a mono-substituted benzene (δ H 7.36 m, 7.36 m, 7.50 m; δ C 128.3 d, 129.0 d, 130.5 d, 134.4 s), which were assigned to a cinnamoyl group [5]. Additionally, an N-ethyl group (δ H 1.10, t, J = 7.2 Hz; δ C 13.1 q, 48.6 t) and two methoxyl groups were identified in the NMR spectra as well. Compound 1 possesses 21 carbons except for the cinnamoyl and methoxyl groups, in combination with biogenetic consideration, suggest that 1 might be an aconitine-type C19-diterpenoid alkaloid [6]. The cinnamoyl group was placed at C-14 according to the HMBC correlation from H-14 (δ H 5.01, t, J = 4.8 Hz) to C-9′ (δ C 166.6 s) (Fig. 2), while the α-orientation of the cinnamoyl group was confirmed by the ROESY correlation between H-10 and H-14. Two methoxyl groups were placed at C-16 and C-18 on the basis of the HMBC correlations from OCH3-16 (δ H 3.26, s) to C-16 (δ C 82.3 d), from OCH3-18 (δ H 3.30, s) to C-18 (δ C 79.2 t), respectively. In addition, the ROESY correlations between H-13 and OCH3-16 demonstrated the β-orientation of OCH3-16. A hydroxyl group should be located at C-8 according to the HMBC correlations from H-15, H-6 and H-9 to C-8. Additionally, a signal at δ H 3.74 was attributed to H-1β, suggesting the presence of an OH-1α [7, 8], which was further supported by the ROESY correlation between H-1 and H-5. Therefore, the structure of compound 1 was determined as stapfianine A, with its assigned NMR data listed in Table 1.

Fig. 2
figure 2

Key 1H-1H COSY (), HMBC () and ROESY () correlations of compound 1

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Table 1 NMR spectroscopic data (400 MHz for 1H and 100 MHz for 13C, CDCl3) for compound 1
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Compound 2 was isolated as a white amorphous powder and its molecular formula was deduced to be C15H13NO5 with an unsaturation degree of ten by HRESIMS at m/z 310.0673 [M+Na]+. The 1H-NMR spectrum of 2 showed signals of a methoxyl group (δ H 3.95, s), a 1,2,4-trisubstituted aromatic ring (δ H 7.11, dd, J = 8.8 Hz, 2.4 Hz; 7.54, d, J = 2.4 Hz; 8.63, d, J = 8.8 Hz), and a 1,2-disubstituted aromatic ring (δ H 7.01, d, J = 8.4 Hz; 7.43, t, J = 7.6 Hz; 6.96, t, J = 7.6 Hz; 7.76, d, J = 8.0 Hz) (Table 2). The 13C-NMR spectrum revealed 15 carbons resonances, corresponding to the above protonated units and two carbonyl groups (δ C 168.8 s, 168.9 s). The data summarized above, in combination with biogenetic consideration, suggested that compound 2 might be a benzamide derivatives [9, 10]. A methyl ester group was placed at C-2 on the basis of the HMBC correlations from OCH3-7, H-3 to C-7 (δ C 168.9 s), and H-6 to C-2 (Fig. 3). Two hydroxyl groups could be located at C-4 and C-2′ on the basis of the HMBC correlations from H-6, H-3 to C-4, and OH-2′ to C-2′, respectively. A quaternary carbon (δ C 134.4 s) ortho to the methyl ester was assigned to connect with the N-atom of the amide group, which caused strong hydrogen bonding between –NH (δ H 11.94, s) and the carbonyl of the ester [11]. Similarly, the downfield shift of OH-2 (δ H 12.34, s) caused by intramolecular hydrogen bonding between the amide carbonyl and OH-2′ suggested that the amide group connected with C-1′, which was further supported by the HMBC correlation from H-6′ to C-7′ and the ROESY correlation between H-6 and –NH [12]. Therefore, the structure of compound 2 was determined as stapfianine B, with its assigned NMR spectroscopic data listed in Table 2.

Table 2 NMR spectroscopic data (400 MHz for 1H and 100 MHz for 13C, CDCl3) for compound 2
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Fig. 3
figure 3

Key 1H-1H COSY (), HMBC () and ROESY () correlations of compound 2

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Based on spectroscopic analyses and comparison with the literature, the known alkaloids were identified as 4-oxo-pentanoic acid dimethylamide (3) [13], 6-epichasmanine (4) [14], talatisamine (5), sachaconitine (6), 14-acetylsachaconitine (7) [15], franchetine (8), vilmorrisine (9) [16], kongboendine (10) [17], vilmorrianine C (11), aconine (12) [18], crassicauline A (13) [19], yunaconitine (14), 8-deacetylyunaconnitine (15) [15], chasmaconitine (16), N-methylisosalsoline (17) [20], (−)-N-methylcoclaurine (18) [21] and tyramine (19) [22]. Compound 3 was isolated for the first time from a natural source in this study. Besides, compounds 4, 6–12, 15–19 were isolated from this species for the first time.

3 Experimental Section

3.1 General Experimental Procedures

Optical rotation was measured with a Jasco P-1020 digital polarimeter (JASCO, Tokyo, Japan). A Shimadzu UV–Vis 2550 spectrometer (Shimadzu, Kyoto, Japan) was used for collection of UV spectra. NMR spectra were acquired with a Bruker AM-400 spectrometer (Bruker, Karlsruhe, Germany) using TMS as the internal reference. A Nicolet Magna-IR 550 spectrometer (Thermo Nicolet, Madison, USA) was used for scanning IR spectroscopy with KBr pellets. Melting points were determined on a XRC-1 Melting Point Apparatus (Sichuan University Science Instrument, Chengdu, China) and were not corrected. ESI–MS analyses were recorded with an Agilent G3250AA (Agilent, Santa Clara, USA) and Auto Spec Premier P776 spectrometer (Waters, Milford, USA). Silica gel (200–300 mesh and 300–400 mesh; Qingdao Marine, Qingdao, China) and Sephadex LH-20 (GE Healthcare, Fairfield, USA) were used for column chromatography (CC). GF254 plates (Qingdao Marine, Qingdao, China) were used for thin layer chromatography, and spots were visualized by spraying with modified Dragendorff’s reagent or 10 % H2SO4 in ethanol followed by heating.

3.2 Plant Material

Roots of A. stapfianum were collected from Dali Bai Autonomous Prefecture of Yunnan Province in China in December 2012, and identified by professor Shu-Gang Lu from School of Life Sciences, Yunnan University. A voucher specimen (2012-yc-2) is deposited in the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, and Kunming, China.

3.3 Extraction and Isolation

Air-dried and powdered roots (8.0 kg) of A. stapfianum were percolated with 0.5 % HCl. The aqueous acidic solution was basified with ammonia (10 %) to pH 9.0 and then extracted with EtOAc. Removal of the solvent under reduced pressure afforded the total crude alkaloids (85 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 nine fractions (FrA–FrI). FrA (38.0 g) was further subjected to silica gel CC [petroleum ether (PE)–acetone–diethylamine, 100:5:1 to 100:20:1] to give five fractions (FrA1–FrA5). Further silica gel CC purification of FrA1 (0.7 g) was accomplished by elution with PE–acetone–diethylamine, (100:5:1 to 100:10:1) to afford compounds 6 (2.0 mg) and 7 (2.0 mg). FrA2 (4.7 g) was subjected to silica gel CC (PE–acetone–diethylamine, 100:10:1) to yield 1 (12.0 mg), 4 (37.0 mg) and 5 (3.5 g). FrA3 (0.9 g) was subjected to silica gel CC (CHCl3–CH3OH, 20:1) to yield compounds 11 (12.0 mg) and 13 (132.0 mg). FrA5 (27.0 g) was subjected to silica gel CC (PE–acetone–diethylamine, 100:5:1 to 100:20:1) to yield compounds 14 (23.0 g). Further silica gel CC purification of FrC (4.2 g) was accomplished by elution with PE–acetone–diethylamine, (100:10:1 to 100:20:1) to afford 15 (3.5 g), 16 (3.0 mg) and 17 (36.5 mg). FrE (1.9 g) was subjected to silica gel CC (CHCl3–CH3OH, 30:1 to 5:1) to yield compounds 2 (14.0 mg) and 3 (13.5 mg). FrF (2.7 g) was subjected to silica gel CC (PE–acetone–diethylamine, 100:5:1) to yield compounds 8 (2.0 mg), 9 (3.5 mg), and 10 (2.5 mg). FrG (0.7 g) was subjected to silica gel CC (PE–acetone–diethylamine, 100:5:1 to 100:10:1) to yield compounds 18 (8.5 mg), 19 (2.5 mg), and 12 (25.0 mg).

3.4 Stapfianine A (1)

White amorphous powder; m.p. 76–77 °C, [α]\( _{\text{D}}^{ 2 0} \) +4.19 (c 2.5, CH3OH), IR (KBr, cm−1): νmax 3437, 2927, 2354, 1714, 1633, 1452, 1175, 1101. For 1H- and 13C-NMR spectroscopic data, see Table 1. HRESIMS m/z: 538.3169 [M+H]+ (calcd for C32H44NO6, 538.3163).

3.5 Stapfianine B (2)

Yellow amorphous powder; m.p. 128–130 °C; IR (KBr, cm−1): νmax 3420, 1695, 1645, 1612, 1524, 1444, 1230, 1069, 979. For 1H- and 13C-NMR spectroscopic data, see Table 2. HRESIMS m/z: 310.0673 [M + Na]+ (calcd for C15H13NO5Na, 310.0691).