New alkaloids from Aconitum taipaicum and their cytotoxic activities

Three new aconitine-type C19-diterpenoid alkaloids, taipeinines A–C (1–3), were isolated from the roots of Aconitum taipaicum. The chemical structures of these three compounds were established as (1α,6α,8α,14α,16α)-20-ethyl-8,14-dihydroxy-1,6,16-trimethoxy-4-(methoxymethyl)-aconitane (1), (1α,6α,8α,14α,16β)-20-ethyl-8,14-dihydroxy-1,6,16-trimethoxy-4-(methoxymethyl)-aconitane (2) and (1α,6α,8α,14α,16α)-20-ethyl-1,8,14-trihydroxy-6,16-dimethoxy-4-(methoxymethyl)-aconitane (3), respectively, on the basis of spectroscopic analyses, mainly MS, 1D and 2D NMR. The cytotoxic activities of these compounds were also assayed, and the results were quite impressive.


Introduction
Herb Aconitum taipaicum Hand. -Mazz. (Ranunculaceae) is an endemic plant in the Taibai Mountains of Shaanxi Province in China and its roots have been used in Chinese folk medicine as anti-inflammatory, gout, rheumatic, cardiotonic, diuretic and analgesic drug for a long period of time (Pelletier & Mody 1980;Singhuber et al. 2009;Kawasaki et al. 2011). As an aconitum plant, alkaloids should be the main constituents of this herb, but in previous reports, only 10 alkaloids (Wang & Fang 1982;He et al. 2008), including yunaconitine, neoline, talatisamine, chasmanine, isodelelatine, atisine, delfissinol, liangshanine, hypaconitine and delelatine, have been reported. The potential medicinal importance of this herb and our interest in the chemistry of alkaloids prompted us to investigate more alkaloids from this plant. The result of our research is that three new aconitine-type C 19 -diterpenoid alkaloids (1-3, see Figure 1) and two other kinds of alkaloids which we reported (Xu et al. 2010) were isolated. The diterpenoid alkaloids from Aconitum plants are believed to be the main bioactive compounds (Gao et al. 2007). Therefore, in this study, the cytotoxicity of three new compounds against HL-60 and K-562 is assayed in order to find a suitable drug with low toxicity.
Compound 2 was obtained as a white amorphous powder. The MS ([M þ H] þ at m/z 452.2937) and 13 C NMR spectrum of the compound indicated a molecular formula of C 25 H 41 NO 6 . The 1 H NMR spectrum displayed signals for an N-ethyl group at d(H) 1.08 (t, J ¼ 7.2 Hz, 3H), 2.53 (m,1H) and 2.55 (m,1H), and four methoxyl groups at d(H) 3.25, 3.31, 3.34, 3.31 (each 3H, s). These characteristic data suggested that 2 was a C 19 -diterpenoid alkaloid (Chen et al. 2003;Gao et al. 2007;Jiang et al. 2012). The 1 H and 13 C NMR data of 2 were quite similar to those of compound 1 except that HZC(16) showed no correlation with any H b in the NOSEY experiment, which indicated that MeOZC(16) was b-configuration. Thus, the structure of compound 2 was established and it was named as taipeinines B.
Compound 3 was obtained as a white amorphous powder. The MS ([M þ H] þ at m/z 437.2774) and 13 C NMR spectrum of the compound indicated a molecular formula of C 24 H 39 NO 6 . The 1 H NMR spectrum displayed signals for an N-ethyl group at d(H) 1.12 (t, J ¼ 7.2 Hz, 3H), 2.48 (m,1H) and 2.57 (m,1H), and three methoxyl groups at d(H) 3.31, 3.31, 3.33 (each 3H, s). These characteristic data suggested that 3 was a C 19 -diterpenoid alkaloid (Chen et al. 2003;Gao et al. 2007;Jiang et al. 2012). The 1 H and 13 C NMR data were quite similar to those of compound 1 except that C(1) was substituted by hydroxyl group in compound 3 but by methoxyl group in compound 1. The deduction was further confirmed by another 2D NMR spectrum. Therefore, the structure of 3 was determined and named as taipeinines C.
Cytotoxicity of compounds 1-3 against cells HL-60 and K562 was examined and the IC 50 values are listed in Table 1 with adriamycin used as a positive control. Compound 1 potently suppresses the proliferation of cells HL-60 and K562. On the basis of these results, compounds from A. taipeicum could be potential anti-leukaemia agents.

Cell culture and cytotoxicity assay
In the cytotoxicity assays, adriamycin at concentration of 4 mg/mL was chosen as the reference drug. Cell line HL-60 and cell line K562 were maintained in RPMI-1640 medium (Gibco BRL) with 3.7 g/L sodium bicarbonate, supplemented with 10% heat-inactivated FBS, seeded in 96well tissue culture plates, and maintained in a humidified atmosphere of 5% CO 2 and 95% air at 378C for 3-6 days before experimentation. The cytotoxicity of the compounds was assayed by the methyl thiazolyl tetrazolium (MTT) method. The cells were diluted to 2 £ 10 3 cells/mL with fresh medium and mixed. the test compound was dissolved in dimethyl sulfoxide (DMSO). If the compound was active at 50 mg/mL, a series of solutions were prepared by twofold dilution, and exposed to cells as mentioned above, in order to obtain IC 50 values. Plates were incubated at 378C under 5% CO 2 atmosphere for 24 h. After incubation, 10% MTT was added and incubated at 378C for 4 h. The pure formazan product was solubilised with 150 mL DMSO for 10 min at room temperature. The plate was read at 590 nm in a plate reader, and all of the reported experiments were carried out at least three times. IC 50 value was calculated using non-linear regression analysis. The percent inhibitions were obtained from the equation: £ 100:

Conclusion
Three new aconitine-type C 19 -diterpenoid alkaloids, taipeinines A -C (1-3), were isolated and the structures of the three new compounds were elucidated. The cytotoxicity of the three compounds against cells HL-60 and K562 was assayed and compound 1 exhibited stronger cell growth inhibitory than adriamycin. On the basis of these results, compounds from A. taipaicum could be potential anti-leukaemia agents.

Supplementary material
The spectra of the three new compounds are available online, alongside Figures S1 and S2.