Tangutidines A–C, Three Amphoteric Diterpene Alkaloids from Aconitum tanguticum

Graphic Abstract Three new diterpene alkaloids, tangutidines A–C (1–3), and four known alkaloids (4–7) were isolated from the whole plant of Aconitum tanguticum, from which amphoteric diterpene alkaloids (1–3) were obtained for the first time. The structures of 1–3 were elucidated by detailed interpretation of spectroscopic data, including MS and NMR data. All of them were evaluated for their cytotoxic activities. Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00310-3.


Introduction
Plants in the genus Aconitum of the family Ranuculaceae are abundant in C 19 -and C 20 -diterpene alkaloids with diverse structural scaffolds and important biological activities, which have long attracted scientists' attention from chemistry and pharmacology communities [1][2][3]. Aconitum tanguticum (Maxim.) Stapf is mainly distributed in Tibet, Qinghai, Gansu, Sichuan and Yunnan Provinces in China [4]. Its whole plant has long been used as a traditional Tibetan medicine for treating fever caused by various infectious diseases, influenza and poisoning for thousands of years [5]. In a classic Tibetan Medical book Sman dpyad zla ba`i rgyal po, A. tanguticum is firstly recorded as one of the least toxic plants among other species in the genus Aconitum [5]. Previous 1 3 phytochemical investigation of A. tanguticum showed that it contained diterpene alkaloids, flavonoids, phenolic acids, glycosides, etc. [6][7][8][9][10][11][12][13][14][15][16]. However, most of the studies on diterpene alkaloids in A. tanguticum focused on its fat-soluble part and few focused on the water-soluble part. In our phytochemical investigation of the whole plant of A. tanguticum, it resulted in the discovery of three new C 20 -diterpene alkaloids with carboxyl groups, tangutidines A-C (1-3) from the n-BuOH extract, together with four known alkaloids (4-7) (Fig. 1). In this paper, we reported their isolation, structure determination, and cytotoxicity.  (Table 1). All the mentioned evidence suggested that 1 was a hetidine-type diterpene alkaloid containing three extra carbons. Compared with the structure of naviculine A [8] bearing a double bond between C-19 and N-atom, 1 had an extra 3-N-propanoic acid moiety and two hydroxyl groups located at C-5 and C-17, respectively. The fragment C-1'/C-2'/C-3' was identified by the 1 H-1 H COSY correlation of H 2 -1'/H 2 -2', along with the HMBC correlation from H 2 -1'a (δ H 3.31, m) to C-3' (δ C 178.7, s). The connection between the fragment C-1'/C-2'/C-3' and N-atom was confirmed by the key HMBC correlation from H 2 -1'a (δ H 3.31, m) to C-20 (δ C 79.4, d) (Fig. 2 (Fig. 3). The key HMBC correlations of H-18 (δ H 1.15, s) and H-6 (δ H 1.86 dd, J = 14.2, 7.0 Hz) with C-5 (δ C 73.7, s), and H-15 (δ H 5.83, br s) with C-17 (δ C 63.4, t) confirmed the attachment of the hydroxyl groups to C-5 and C-17. The linkage of the hydroxyl groups to C-5 and C-17 was confirmed by the HMBC correlations from a hydroxyl group (δ H 4.97, br s) to C-4, C-5, and C-6, from H-18 to C-5, from H-17 to C-12 and C-16, and from H-15 to C-17. The relative configuration of 1 was established based on the ROESY spectrum, which was the same as that of naviculine A (Fig. 3). Thus, the structure of 1 was elucidated with its assigned NMR spectroscopic data listed in Table 1.

Results and Discussion
Compound 2 was obtained as colorless amorphous powder. The IR spectrum showed absorptions for hydroxyl (3428 cm −1 ) and carboxyl (1603 cm −1 ) groups. Its molecular formula was assigned as C 23 H 33 NO 4 by the analysis of its HRESIMS (m/z 388.2486 [M+H] + , calcd 388.2482), indicating eight degrees of unsaturation. Comparison of the 1 H and 13 C NMR spectra data of 2 with that of 1 (Table 1) revealed that 2 was an analogue of 1. The main difference between them was that an endo double bond at C-15/C-16 and a hydroxyl group at C-17 in 1 were replaced by a double bond at C-16/C-17 and a hydroxyl group at C-15 in 2. Compared with the 13 C NMR data of 1, some chemical shifts in that of 2 changed due to the shift of double bond: a high-field chemical shifts of C-7 (δ C 31.6, t, Δ − 1.5), C-9 (δ C 44.3, d, Δ − 6.2), C-11 (δ C 28.4, t, Δ − 0.7), C-13 (δ C 37.9, t, Δ − 4.9), C-14 (δ C 46.8, d, Δ − 2.5) and a low-field chemical shifts of C-8 (δ C 45.7, s, Δ + 1.3), C-10 (δ C 49.0, s, Δ + 1.6), and C-12 (δ C 35.5, d, Δ + 3.1). The main difference between 1 and 2 was further confirmed by the HMBC correlations from H-17 to C-12, C-16, and C-15, and from H-15 to C-7, C-12, and C-14. The presence of C-3' carboxyl group could be further confirmed by the HMBC correlation from both H 2 -1' and H 2 -2' to C-3'. The fragment C-1'/C-2'/C-3' connection with N-atom was confirmed by the HMBC correlation from H-20 to C-1'. The ROESY correlation of H-9β/Η-15 indicated that the hydroxyl group at C-15 was α-oriented ( Fig. 3). Combined with all the evidence, the structure of compound 2 was established.
Compound 3 was isolated as colorless amorphous powder. Its molecular formula was deduced as C 32   The carboxyl group was deduced to be linked with C-10', which accounted for the residual one degree of unsaturation and an IR absorption (1637 cm −1 ). Thus, the structure of 3 was established, and named as tangutidine C. The known alkaloids (4-7) were identified to be tanaconitine [10], heteratisine [14], trolline [17] and isocorydine [18], by comparison with published physical and spectroscopic data. Alkaloids 6 and 7 were first reported from A. tanguticum.

General
Optical rotations were measured with a JASCO P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401 PC spectrophotometer. A Tensor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. HRESIMS data was acquired on an Agilent 6540 QSTAR TOF time-of-flight mass spectrometer. 1D and 2D NMR spectra were recorded on Bruker DRX-600 spectrometers with TMS as internal standard. Chemical shifts (δ) are expressed in parts per million (ppm) with reference to the solvent signals. Semipreparative HPLC was performed on an Agilent 1260 liquid chromatograph with a COSMOSIL 5C18-MS-II (4.6ID × 250 mm) column. Column chromatography (CC) was performed with silica gel (80-100 and 100-200 mesh; Qingdao Marine Chemical, Inc., Qingdao, People's Republic of China), Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and SEPAFlash column (Spherical C-18, 20-45 μm, 100 Åm). Fractions were monitored by TLC (thin-layer chromatography) and spots were detected with the modified Dragendorff's reagent.

Plant Material
The

Extraction and Isolation
Dried whole plants of A. tanguticum. (8.8 kg) were powered and extracted with 70% EtOH (40 L each) for three times, each time for 3 days. The extract was filtered and concentrated under reduced pressure to give the crude extract.
The extract was suspended in water, solution was acidified with 5% aq. HCl to pH 2.0 and the acidic solution was successively extracted with petroleum ether (PE), CHCl 3 and n-BuOH. Then the acidic solution was was basified with saturated NaOH solution and extracted with CHCl 3 . The n-BuOH part was basified with saturated NaOH solution and extracted with CHCl 3 , EtOAc and n-BuOH. The EtOAc part was concentrated to yield the total crude alkaloids (28 g  need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.