A new C20-diterpenoid alkaloid from Aconitum soongaricum var. pubescens

Abstract A new denudatine-type C20-diterpenoid alkaloid, pubesine (1), along with seven known diterpenoid alkaloids, altaconitine (2), 14-benzoylaconine (3), spicatine A (4), 14-benzoylaconine-8-palmitate (5), 14-O-acetylsenbusine A (6), senbusine A (7) and 14-acetylneoline (8) were isolated from the whole plant of Aconitum soongaricum var. pubescens. Their structures were elucidated by means of extensive spectroscopic analyses (NMR and HR-ESI-MS) and comparison with data reported in the literature. All compounds were evaluated for their cytotoxicity against H460, MCF-7 and Hep G2 human cancer cell lines.


Introduction
Aconitum soongaricum var. pubescens (Ranunculaceae) is a perennial herb distributed mainly in the eastern part of Xinjiang of China, Kashmir and other places. The plant is used as a folk remedy for the treatment of arthritic and rheumatic pain (Wang & Warnock 1979).
The relative configuration was deduced from the ROESY spectrum ( Figure S10). The hydrogen group at C(1) was assigned an β-orientation based on the signal of H-1 at δ H 4.03 (d, J = 5.4 Hz) and the resonance of C-1 at δ C 68.3 in the NMR spectra (Pelletier et al. 1984), which was further supported by the cross-peaks between H-C(1)/H-C(20) and H-C(20)/H α -C(11) in the ROESY spectrum. Comparison of the NMR data of 1 with those of the known alkaloid Kirinine B (Feng et al. 1998) indicated that ring A possessed a boat conformation, which was confirmed by the cross-peaks between H-C(2)/H-C(5) and H-C(5)/H-C(9) in the ROESY experiment. The 16, 17-epoxy group was assigned α-orientation, since the epoxy moiety exhibited nearly identical NMR data to those of two known alkaloids bearing a 16, 17-epoxy segment, yesoxine (Bando et al. 1987) and gomandonine (Sakai et al. 1987) whose structures were unambiguously determined by X-ray crystallography. The cross-peaks between H-C(13) with H α -C(11) and H-C(15) with H α -C(14) in the ROESY experiment revealed that H-C(13) and H-C(15) were α-oriented. Thus, this compound was assigned to be pubesine.
As far as we know, there were only six denudatine-type C 20 -diterpenoid alkaloids that contain 16, 17-epoxy group reported before (Bando et al. 1987;Sakai et al. 1987;Liu et al. 2009;Zhang et al. 2010). Compound 1 was the first C 20 -diterpenoid with a denudatine skeleton bearing a 16, 17-epoxy group and a C-1-C-19 carbinolamine inner ether isolated from a natural source, providing a new candidate for further pharmacological investigation. The structures of compounds 2-8 were identified by comparison of their spectral data with those described in the literature.
All compounds were evaluated for their cytotoxicity against MCF-7, Hep G2 and H460 human cancer cell lines. Compound 5 displayed cytotoxicity against the above tumor cells with IC 50 values of 11.9, 27.6 and 31.8 μM, respectively, and other compounds were inactive (IC 50 > 100 μM, n = 3).

General experimental procedure
Optical rotation was measured on a Perkin-Elmer 341 polarimeter. 1D and 2D NMR spectra were recorded on a Bruker AV 600 NMR spectrometer, and IR spectrum on a ThermoFisher Nicolet 6700 spectrometer (KBr discs, cm −1 ). HR-ESI-MS was carried out on a Q-TOF micro mass spectrometer (Waters, uSA). Silica gel (Qingdao Haiyang Chemical Co., Ltd., China). TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co., Ltd, China) were visualised under a uV lamp at 254 nm or by spraying the Dragendorff's reagent or by iodine.

Plant material
Aconitum soongaricum var. pubescens were collected in Houxia, Xinjiang uygur Autonomous Region of China, in August 2014 and were identified by Prof Qing-Er Yang of the Institute of Botany, Chinese Academy of Sciences. A voucher specimen (C. Ren & L. Wang 705) was deposited in the School of Life Science and Engineering, Southwest Jiaotong university.

Extraction and isolation
The air-dried powder (6.5 kg) of A. soongaricum var. pubescens was extracted with 95% EtOH three times at room temperature, with each soaking process lasting three days.
After removal of the solvent by evaporation, the EtOH extract (280 g) was recovered. The extract was treated with 0.5 mol/L hydrochloric acid (2 L) and successively extracted with light petroleum (4 × 1 L) and ethyl acetate (4 × 1 L). Then, 28% aqueous ammonia soln. (2 L) was added to the aq. soln. to bring them to pH 10. The solutions were extracted with CH 2 Cl 2 (4 × 1 L). The CH 2 Cl 2 extract was concentrated to produce the crude alkaloid fraction (20 g).

Cell culture and cytotoxicity assay
The in vitro growth inhibitory activities of compounds 1-8 were assayed by the MTS method (Huang et al. 2013;Guo et al. 2014). H460 (human lung adenocarcinoma), MCF-7 (human breast carcinoma) and Hep G2 (hepatocellular carcinoma) cell lines were obtained from ATCC. The cells were cultured in a 96-well plate 24 h before treatment and then exposed to different concentrations of compounds or control for 72 h. Three replicates were prepared for each treatment. DMSO (0.1% v/v) was used as negative control.

Conclusion
A new diterpenoid alkaloid, pubesine (1), along with seven known diterpenoid alkaloids were isolated from the whole plant of Aconitum soongaricum var. pubescens. Compound 1 is the first C 20 -diterpenoid with a denudatine skeleton bearing a 16, 17-epoxy group and a C-1-C-19 carbinolamine inner ether isolated from a natural source. All compounds were evaluated for their cytotoxicity against MCF-7, Hep G2 and H460 cell lines. Compound 5 displayed cytotoxicity against the above tumor cells with IC 50 values of 11.9, 27.6 and 31.8 μM, respectively, and other compounds were inactive.