Identification of Diterpenoid Alkaloids from the Rootsof Aconitum kusnezoffii Reihcb

Three diterpenoid alkaloids, including an unreported compound, were isolated from the roots of Aconitum kusnezoffii Reichb. On the basis of spectral analysis, these three compounds were determined to be 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline, benzoylaconine and aconitine.


Introduction
Aconitum kusnezoffii Reichb., belonging to the genus Aconitum L., is distributed in the Xinjiang, Sichuan and Jilin provinces of China.Although recently there were some reports on flowers [1] and leaves [2] of this plant, roots of Aconitum kusnezoffii Reichb.had been long used in Traditional Chinese Medicine as an analgesic and cardiotonic herbal medicine.Diterpenoid alkaloids [3,4] and polysaccharide [5] have been isolated from its roots to date.To our knowledge, diterpenoid alkaloids extracted from the genus Aconitum L. have various pharmacological properties [6][7][8][9][10].In the search for biologically active alkaloids from the roots of Aconitum kusnezoffii Reichb., a detailed study was carried out.This led to the isolation of the new diterpenoid alkaloid 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline, along with two known diterpenoid alkaloids, benzoylaconine and aconitine.Herein, the structure of the new compound was determined based on MS, IR, and NMR spectral data, and the known ones were identified by comparing their NMR data with those in the literature [3].
The g 1 H-1 H COSY, gHSQC and gHMBC spectra allowed the complete assignment of the chemical shifts of compound 1 (Table 1) [12][13][14][15].The 1 H-NMR and 13 C-NMR resonances of compound 1 were similar to those of chasmanine [3], which was also isolated from roots of Aconitum kusnezoffii Reichb., except for the different configurations of C-3, C-15, C-16 and C-14.The main differences are that the chemical shifts of C-3, C-15, C-16 and H-14 are δ C 71.0, 85.8, 211.8 and δ H 5.44 (d, J = 5.0 Hz) respectively in compound 1, and δ C 35.0, 38.7, 81.9 and δ H 4.12 (t, J = 4.5 Hz) respectively in chasmanine, which suggested that C-3, C-15 and C-16 in compound 1 belong to an oxygen-containing group.In the gHMBC spectrum, the correlations between H-18 and C-3, and between H-15 and CH 3 O-15 were important to confirm the presence of oxygen groups at C-3 and C-15.Further analysis of the 13 C NMR data suggested that C-16 in compound 1 could be a carbonyl group, and not methoxyl group.The gHMBC correlations from C-1′′ to H-14 and H-3′′ confirmed the position of a benzoyl group.In addition, it is noteworthy that the chemical shift of H-14 in compound 1 was more deshielded than the same proton in chasmanine as the result of the steric effect of the carbonyl group at C-16.On the basis of the above spectral data, compound 1 was determined to be 1,15-dimethoxy-3-hydroxy-14-benzoyl-16-ketoneoline (Figure 1).

General
IR spectra were recorded using a Thermo Nicolet Nexus spectrophotometer.1D and 2D NMR spectra were obtained on Bruker AV400; δ H values are expressed in parts per million relative to the solvent (CDCl 3 ) signal.DEPT, g 1 H-1 H COSY, gHSQC, and gHMBC experiments were carried out with the pulse sequences given by Bruker.LC-MS were performed on a UPLC instrument (Waters, USA) coupled with a ZQ 2000 mass spectrometer (Waters, USA) using an XTerra MS C 18 2.1 × 150 mm column (Waters, USA) and a C 18 HCE 4.6 × 150 mm column (self-made).A Waters Auto-Purification Factory was used in this study and consisted of a sample inJector (Waters 2777 sample manager), a passive splitter, a compensation pump (515 HPLC pump), an eight-channel UV detector (MUX-UV 2488), a four-channel MS detector (Micromass ZQ2000), four Waters 2525 binary gradient modules, and four Waters 2757 sample managers using XTerra MS C 18 19 × 150 mm (Waters, USA) and C 18 HCE 10 × 150 mm (self-made) columns.Data were collected using a MassLynx workstation.

Figure 1 .
Figure 1.Structure and Key Correlations of Compound 1.
Aconitum kusnezoffii Reichb.were collected in YiLi, Xinjiang Province, China, in July 2008.The herb was authenticated by Xi Rong He, Institute of Medication, Xiyuan Hospital of China Academy of Traditional Chinese Medicine.A voucher specimen (CAOWU 200807) was deposited at the School of Chemical Engineering, Dalian University of Technology.