Tatsienensine,a Norditerpenoid Alkaloid From Delphinium tatsienense

Abstract

A novel C₂₀-diterpenoid alkaloid with an unprecedented 19 carbon atoms, designated as tatsienensine (1), was isolated from the whole plant of Delphinium tatsienense. The structure of the compound and its relative configuration were established by NMR techniques and single-crystal X-ray diffraction.

Delphinium, a large genus within the Ranunculaceae family, is estimated to contain about 350 species, mainly found in the northern hemisphere, of which 173 are found in mainland China. ¹ Diterpenoid alkaloids are believed to be the major bioactive components of the genus Delphinium. ^{2 -5} A large number of these compounds have been isolated from various species of Delphinium and Aconitum and are classified according to their structures as C₁₈-, C₁₉-, and C₂₀-diterpenoid alkaloids, ² with a wide range of fascinating bioactivities.

Delphinium tatsienense Franch grows mainly in China in southwest Sichuan and northwest Yunnan. ² Our previous investigation of the whole plants of this species resulted in the isolation of three new C₂₀-diterpenoid alkaloids. ⁶ Gratifyingly, further chemical investigation of this plant, collected in Miyi County, Sichuan Province, P. R. China, led to the discovery of a structurally unique diterpenoid alkaloid, tatsienensine (1), with an unprecedented skeleton, which is composed of a cage framework consisting of 6 rings (rings A to F). Most interestingly, the compound containing 19 carbon atoms has a similar structure to that of a C₂₀-diterpenoid alkaloid except for the absence of an exocyclic double bond.

Tatsienensine (1) was obtained as colorless monoclinic crystals. Its IR spectrum afforded evidence of hydroxyl (3720 cm⁻¹) and carbonyl (1738 cm⁻¹) groups. The HR-ESI-MS ion at m/z 300.1958 corresponded to the protonated molecular ion [M+H]⁺ (C₁₉H₂₅NO₂). The NMR and mass spectra indicated that the compound had a similar structure to that of a hetisine-subtype C₂₀-diterpenoid alkaloid with a N-C(6)-OH group, except for the absence of an exocyclic double bond. ² The NMR spectra (Table 1) of 1 showed the presence of a tertiary methyl group (δ _H 1.39, 3H, s; δ _C 30.4 q), a ketone carbonyl (δ _C 214.8), and a quaternary carbon (δ _C 99.2 s). Except for the above-mentioned signals, the NMR spectra showed that this compound lacked any other substituent group in addition to an acetyl group and a N-C(6)-OH group. The N-C(6)-OH group was also confirmed by HMBC experiment that showed correlations of H-19 to C-6, and H-20 to C-6 (Figure 1(b)). The ketone carbonyl (δ _C 214.8) was correlated with H-18 (δ _H 1.15, 3H, s) and H-19 (δ _H 2.20, 1H, δ _H 2.74, 1H), suggesting that the ketone carbonyl could be assigned to C-16 on the basis of the correlations of H-11 to C-16, and H-13 to C-16 (Figure 1(b)). It was highly desirable to obtain a single crystal for X-ray crystallography, which could unambiguously confirm the flat structure and relative configuration. Luckily, compound 1 was successfully recrystallized from acetone and subjected to X-ray crystallography (Figure 2). The structure of 1 was therefore characterized as shown in Figure 1.

Figure 1

Chemical structure of tatsienensine (1) and key ¹H-¹H COSY () and HMBC () correlations of tatsienensine (1).

Table 1

¹H and ¹³C NMR Spectroscopic Data and 2D NMR Correlations of 1 (400 MHz for ¹H NMR, 100 MHz for ¹³C NMR, CDCl₃, Δ in ppm, J in Hz).

Position	δ H	δ C	HMBC (H→C)
1	1.33, 1.47 m	35.7 t	C-20, C-3,C-5
2	1.48, 1.63 m	19.2 t	C-4,C-10
3	1.32, 1.78 m	27.9 t	C-5,C-18,C-19
4	-	37.7 s	-
5	1.53 s	61.7 d	C-7,C-9,C-18,C-19
6	-	99.2 s
7	2.15, 2.38 ABq (5.6)	42.1 t	C-5,C-9, C-15
8	-	44.9 s	-
9	1.74 m	47.9 d	C-20
10		50.9 s	-
11	1.74, 2.10 m	24.1 t	C-8,C-13,C-16
12	2.03 (hidden)	44.1 d
13	2.10 m	29.4 t	C-20, C-16
14	2.01 m	44.1 d	C-9, C-15
15	2.03, 2.07 ABq (5.6)	44.6 t	C-7,C-9, C-14,C-12
16	-	214.8 s	-
18	1.39 s	30.4 q	C-5,C-19
19	2.14, 3.16 ABq (12.0)	59.9 t	C-3,C-5,C-18,C-20
20	2.65 s	73.4 d	C-9,C-13,C-19

Figure 2

ORTEP drawing of tatsienensine (1).

Experimental

General

Column chromatography (CC): silica gel H (Qindao Marine Chemical, Inc., Qingdao, P. R. China). TLC: silica gel plates; detection by spraying with Dragendorff reagent. IR spectra: Nicolet FT-IR 200S spectrometer. Optical rotations: Perkin-Elmer 341 polarimeter. 1D and 2D NMR spectra were measured on a Varian Unity-INOVA-400 instrument with TMS as internal standard. ESI-MS: Finnigan LCQ; in m/z (rel.%). HR-ESI-MS: VG Auto Spec 3000 spectrometer.

Plant Material

Whole plants of D. tatsienense Franch were collected in Miyi County, Sichuan Province, P. R. China, in September 2007. The plant was authenticated by Professor Q. E. Yang of the Beijing Institute of Botany of the Chinese Academy of Sciences, where a voucher specimen (No. 200709-1) has been deposited.

Extraction and Isolation

Air-dried and powdered whole herbs of D. tatsienense (3.0 kg) were percolated with 0.1 M HCl (40 L). The obtained acid aqueous solution was basified with 10% aqueous NH₄OH to pH 9, then extracted with ethyl acetate (20 L × 3). Removal of the solvent under reduced pressure afforded the total crude alkaloids (12.1 g) as a yellowish amorphous powder, which was chromatographed over a silica gel column, eluting with a cyclohexane-acetone (8:1→1:2) gradient system, to give Frs. A (94 mg), B (399 mg), C (2.3 g), D (2.2 g), E (2.1 g), F (1.3 g) and G (2.8 g). Purification of Fr. G by silica gel CC using CHCl₃-CH₃OH (80:1 → 10:1) as eluent generated tatsienensine (1, 26 mg).

Tatsienensine

Colorless monoclinic crystal.

[α] ${}_{\text{D}}^{\text{20}}$ : +11.9 (c 1.0, CHCl₃).

IR (KBr) ν _max: 3720, 2919, 1738, 1233 cm⁻¹.

¹H and ¹³C NMR: Table 1.

ESI-MS m/z: 300 [M+H]⁺; HR-ESI-MS m/z: 300.1958 [M+H]⁺ (calcd for C₁₉H₂₅NO₂, 300.1963).