Introduction

Diterpenoid alkaloids (DAs), which isolated mainly from the genera of Aconitum, Delphinium and Consolida (Ranunculaceae), are a group of structurally complex natural products displaying a wide range of biological activities. DAs could be divided into four subtypes: C₁₈-, C₁₉-, C₂₀- and bis-type, of which the C₁₉-type constitutes the major portion of DAs^{[1, 2]}. C₁₉-DAs possess complex cage-like skeletons composed of a 6/7/5/6/6-membered rings carbon framework, in addition to various oxygenated substituent groups such as hydroxyl, methoxyl, acetyl, aroyl groups and so on, which brings about difficulty in their structural identification. Nuclear magnetic resonance (NMR) spectroscopy plays a vital role in the structural identification of C₁₉-DAs. Hence, it is of great significance to provide more examples on the NMR spectral identification or assignments of C₁₉-DAs.

The spectral identification of a certain amount of DAs isolated from Aconitum plants have been reported in our previous studies^[3-5]. We have also summarized the NMR spectral features and identification method of C₁₉-DAs^[6], which is conducive to the further research of C₁₉-DAs. As parts of our continuous studies on the alkaloids from Aconitum plants, three known C₁₉-DAs, including two aconitine-type DAs taronenine E (1) and chasmaconitine (2), and a 7, 17-seco-type DAs vilmorisine (3), were isolated from the roots of Aconitum taronense Fletcher et Lauener (Fig. 1). This paper deals with the complete assignments and revision of NMR spectral data of those three C₁₉-DAs by extensive NMR spectroscopic analyses (i.e. ¹H NMR, ¹³C NMR, DEPT, ¹H-¹H COSY, HSQC, and HMBC).

1 Experimental section 1.1 Instruments and reagents

¹H NMR (400.13 MHz, CDCl₃) and ¹³C NMR (100.06 MHz, CDCl₃) spectra were acquired with a Bruker AM-400 spectrometer using tetramethylsilane (TMS) as the internal reference, and 2D NMR (CDCl₃) spectra were recorded at the standard conditions. High resolution mass spectra with electronic spray ionizer (HR-ESI-MS) were recorded with Agilent G3250AA (Agilent, Santa Clara, USA) and Auto Spec Premier P776 spectrometers (Waters, Milford, USA). Silica gel (300~400 mesh; Qingdao Haiyang, Qingdao, China) was used for column chromatography (CC). Fractions were monitored by thin layer chromatography (TLC) and visualized by spraying with modified Dragendorff's reagent.

1.2 Plant material

A. taronense roots were collected from Lushui County in Yunnan Province of China in December 2013, and identified by assistant professor Yang Shu-da from School of Pharmaceutical Science in Kunming Medical University. A voucher specimen (2013-nj-2) was deposited in the School of Chemical Science and Technology in Yunnan University of China.

1.3 Extraction and isolation of compounds 1~3

Air-dried and powdered A. taronense roots (6.0 kg) were percolated with 0.5% HCl (30 L). The aqueous acidic solution was basified with 10% ammonia to pH 9 and then extracted with ethyl acetate (50 L). Removal of the solvent under reduced pressure afforded the total crude alkaloids (41.5 g) as yellowish amorphous powder. The total alkaloids were subjected to silica gel CC eluted with a gradient system [V(CHCl₃):V(CH₃OH)=100:1~1:1] to give nine fractions (FrA~FrI). FrE (29.8 g) was further subjected to silica gel CC [V(petroleum ether):V(acetone):V(diethylamine)=100:7:1~100:50:1] to provide compound 1 (13 mg). Further silica gel CC purification of FrF (3.0 g) was accomplished by elution with a gradient system [V(petroleum ether):V(acetone):V(diethylamine)=100:7:1~100:50:1] to afford compound 2 (19 mg). FrH (7.1 g) was subjected to silica gel CC [V(petroleum ether):V(acetone):V(diethylamine)=100:7:1~ 100:50:1] to yield compound 3 (2 mg).

2 Results and discussion 2.1 NMR spectral assignment of taronenine E (1)

Compound 1 was isolated as a white powder, and its molecular formula was deduced to be C₂₆H₄₃NO₆ by HR-ESI-MS at m/z of 466.316 3 [M+H]⁺ (calculation for C₂₆H₄₄NO₆, 466.316 9).

Compound 1 first appeared as an artifact compound synthesized from forsticine in 2000^[7]. Li et al.^[7] provided the complete ¹³C NMR data but only assigned few protons in its ¹H NMR spectrum. Besides, during the structural identification of compound 1, we found there were several inconsistencies in the ¹³C NMR assignments.

In ring C, Li et al.^[7] only reported the assignment of H-14 (δ_H 4.03, t, J=5.0 Hz, 1H). Through the analysis of consecutive ¹H-¹H COSY correlations between H-9 and H-16 (Fig. 2), and HMBC correlations between H-14 and C-10, C-16, and between H-16 and C-12, C-15 (Fig. 3), along with corresponding HSQC correlations, we were able to assign the exact chemical shifts of eight protons (H-9, H-10, H-12, H-13, H-16, and H-15) in rings C and D. Besides, the HMBC correlation between H-16′ and C-16, and HSQC correlation between δ_H 3.36 (s, 3H) and carbon resonating at δ_C 56.9 (q), supported the assignment of H-16′ and C-16′.

The N-ethyl group [δ_H 1.08 (t, J = 7.2 Hz, 3H), δ_C 13.6 (q); 49.4 (t)] and oxygenated methylene at C-18 [δ_H 3.09/3.19 (Abq, J = 9.2 Hz, 2H); δ_C 77.2 (t)] in ring A of DAs possessed characteristic NMR signals, which are often used as start points to assign the NMR data^[3]. The HMBC correlations between H-21 and C-17, C-19, and between H-18 and C-3, C-5, C-19, corresponding to HSQC correlations, supported the assignments of H-3, H-17, H-19 and reassignments of C-5 to δ_C 58.6 (d). The rest of protons in ring A could be assigned by the ¹H-¹H COSY correlations between H-3 and H-2, H-1. And the assignment of OCH₃-1′ was confirmed by HMBC correlation between H-1′ [δ_H 3.31 (s, 3H)] and C-1 [δ_C 84.5 (d)], and corresponding HSQC correlation.

The last proton signal was assigned to H-7 in ring B according to HMBC correlations between H-9, H-15, H-17 and C-7, along with HSQC correlation between H-7 and C-7. Chemical shift of C-7 was found to be inconsistent with that reported by Li et al.^[7], and was revised to δ_C 56.3 (d). Finally, the HMBC correlations between H-17 and the carbonyl carbon C-6 and oxygenated quaternary carbon C-8 confirmed the existence of ketone carbonyl substituted at C-6 and hydroxyl substituted in C-8.

Therefore, the ¹H and ¹³C NMR data of compound 1 were assigned and the wrongly assigned carbon signals were revised simultaneously (Table 1).

2.2 NMR spectral assignment of chasmaconitine (2)

Compound 2 was isolated as a white powder, whose molecular formula was deduced to be C₃₄H₄₇NO₉ by HR-ESI-MS at m/z of 614.333 7 [M+H]⁺ (calculation for C₃₄H₄₈NO₉, 614.332 9).

Compound 2 is an aconitine-type C₁₉-DAs and has been reported to be isolated from many Aconitum species^{[8, 9]}. During our phytochemical study on A. taronense, this compound was isolated and further investigated for its NMR spectral properties.

The characteristic ¹H NMR signals of H-18 [δ_H 3.59/3.16 (J=8.4 Hz, 2H)] and N-ethyl group [δ_H 1.09 (t, J=7.2 Hz, 3H)] were also used as start points for the assignments of compound 2. According to the HMBC correlations between H-18 and C-19, C-3, C-5, and between H-22 and C-17, C-19 (Fig. 4), along with corresponding HSQC correlations, the assignments of H-3, H-5, H-17 and H-19 were achieved. And H-1 and H-2 could be assigned by the ¹H-¹H COSY correlations between H-1, H-2 and H-3 (Fig. 2). Then, two methoxyl groups placed in C-1 and C-18 were assigned on the basis of HMBC correlations between H-18′ [δ_H 3.27 (s, 3H)] to C-18 [δ_C 80.6 (t)], and between H-1′ [δ_H 3.25 (s, 3H)] and C-1 [δ_C 85.2 (d)], along with the HSQC correlations.

In ring B, the ¹H NMR signal of H-6 [δ_H 3.96 (d, J=6.6 Hz, 1H)] was highly recognizable due to its downfield shift effected by methoxyl group, which was used to assign the rest protons. The ¹H-¹H COSY correlations between H-5, H-6 and H-7, in combination with HMBC correlations between H-5 and C-3, C-18 and C-19, between H-7 and C-8, C-9 and C-15, enabled us to assign the ¹H NMR signals of H-5 and H-6. And the OCH₃-6′ could be assigned on the basis of HMBC correlation between H-6′ [δ_H 3.13 (s, 3H)] and C-6 [δ_C 83.3 (d)].

The ¹H NMR signals of H-9, H-10 and H-12 in ring C could be assigned according to the ¹H-¹H COSY correlation chain of H-14/H-9/H-10/H-12 by using the characteristic ¹H NMR signals of H-14 [δ_H 4.90 (d, J=5.2 Hz, 1H)]. These assignments could be further confirmed by corresponding HSQC and HMBC correlations. H-15 in ring D was assigned according to the ¹H-¹H COSY correlation between H-15 and H-16. The last methoxyl group (OCH₃-16′) was assigned by the HMBC correlation between H-16′ [δ_H 3.52 (s, 3H)] and C-16 [δ_C 83.9 (d)]. Finally, the location of OBz-14′ were confirmed by HMBC correlation between H-14 and the ester carbonyl carbon C-14′ [δ_C 166.6 (s)].

Consequently, all the ¹H and ¹³C NMR data of compound 2 were assigned and listed in Table 2.

2.3 NMR spectral assignment of vilmorisine (3)

Compound 3 was isolated as a white powder, whose molecular formula was deduced to be C₂₆H₃₉NO₆ by HR-ESI-MS at m/z of 462.269 9 [M+H]⁺ (calculation for C₂₆H₄₀NO₆, 462.265 6).

The 7, 17-seco-type DAs vilmorisine (3) was first isolated from A. vilmorinianum by Ding et al.^[10] in 1992, but its structure was wrongly identified. Later in 1997, Wang et al.^[11] revised its structure by comparison of the NMR data with its analogue franchetine. Vilmorisine has also been isolated from other species such as A. smirnovii^[12], A. kongboense ^[13] and A. handelianum^[14]. Its ¹³C NMR data had been assigned previously but the complete ¹H NMR data were not reported in literatures.

In ring A, only the ¹H NMR signal of H-22 [δ_H 0.94 (t, J=7.2 Hz, 3H)] in N-ethyl group has been assigned. The HMBC correlations between H-21 and C-17, C-19 (Fig. 5), in combination with HSQC correlations between H-17 and C-17, between H-19 and C-19, suggested the assignments of H-17 [δ_H 4.28 (s, 1H)] and H-19 [δ_H 2.35/1.96 (m, 2H)], respectively. Likewise, the HMBC correlations between H-19 and C-3, C-18, along with the corresponding HSQC correlations, supported the assignments of H-3 and H-18. And through the analysis of consecutive ¹H-¹H COSY correlations between H-3 and H-1 (Fig. 2), the ¹H NMR signals of H-1 and H-2 were assigned. Finally, two methoxyl groups were assigned by the HMBC correlations between H-1′ [δ_H 3.26 (s, 3H)] and C-1 [δ_C 86.9 (d)], between H-18′ [δ_H 3.22 (s, 3H)] and C-18 [δ_C 79.3 (t)], respectively.

When compared with aconitine-type or lycaconitine-type C₁₉-DAs, H-14 in franchetine-type C₁₉-DAs is distinctive for its peak shape. Normally, the triplet signal of H-14 in C₁₉-DAs could be observed in ¹H NMR spectrum if there is no substituent group in C-9 and C-13. However, it always appears as a broad singlet in franchetine-type C₁₉-DAs affected by the Δ^{7, 8 [15, 16]}. Using the characteristic H-14 as a start point, the rest ¹H NMR signals in ring C could be easily assigned on the basis of consecutive ¹H-¹H COSY correlations chain of H-9/H-10/H-12/H-13/H-15/H-16. And the remaining methoxyl group could be assigned according to HMBC correlation between H-16′ [δ_H 3.18 (s, 3H)] and C-16 [δ_C 85.6 (d)]^[17]. The location of OAc-14′ were also confirmed by HMBC correlation from H-14 to the eater carbonyl carbon in C-14′^[18].

The protons of H-6 and H-7 in ring B have been assigned before, which were verified by the HMBC correlation from H-6 to C-4, C-11 and C-8, and from H-7 to C-15, C-9 and C-5.

Thus, the ¹H and ¹³C NMR signals of compound 3 were assigned and reported (Table 3).

3 Conclusion

The full assignments and revision of NMR spectral data of three C₁₉-diterpenoid alkaloids from the roots of A. taronense Fletcher et Lauener were accomplished by extensive spectroscopic analyses (¹H NMR, ¹³C NMR, DEPT, ¹H-¹H COSY, HSQC and HMBC). The present study provides important references for the structural determination of diterpenoid alkaloids.