Diterpenoid Alkaloids from Delphinium anthriscifolium var. majus

Extensive phytochemical investigation on the whole herbs of Delphinium anthriscifolium var. majus led to the identification of fourteen diterpenoid alkaloids, including three new C20–diterpenoid alkaloids (anthriscifolsines A–C, 1–3), six new C19–diterpenoid alkaloids (anthriscifolrines A–F, 4–9), and five know compounds (10–14). Among them, anthriscifolsine A represents a novel C20–diterpenoid alkaloid characterized by a seco C–ring. The structures of the isolated compounds were elucidated by extensive spectroscopic methods, including HR-ESI–MS, X–ray, and 1D and 2D NMR experiments. Bioactivity of compounds 3–6 was evaluated for their cytotoxicity against the MCF–7, HepG2 and H460 cancer cell lines.


Results
Anthriscifolsine A (1) was obtained as needles via crystallization from MeOH. Its molecular formula, C 29 H 31 NO 7 , was deduced from the HR-ESI-MS (m/z 506.2182 [M + H] + , calcd for C 29 H 32 NO 7 , 506.2179) and 13 C NMR spectroscopic data. The 1 H NMR data (Table 1)  were presented in the structure according to the NMR spectra. These characteristic spectroscopic data suggested that 1 was a typical skeleton of C 20 -diterpenoid alkaloid diester 9 . The proton and corresponding carbon resonances in the 2D NMR spectra of 1 were assigned by the HMQC experiment. The existence of three oxygenated carbons deduced from its 13 C NMR spectrum suggests that 1 has a hydroxyl group, in addition to two ester groups. The absence of a typical C-19 methylene signals in its NMR spectra suggested that a hydroxyl group might be located at C-19, which was confirmed by the HMBC correlations ( Fig. 2) from H-3, H 3 -18 and H-5 to C-19 9 . The acetoxy group could be assigned to C-2 and the benzoyl group at C-3 respectively, on the basis of the HMBC correlations from H-2 (δ H 5.59, m) to the carbonyl carbon of the acetyl group at δ C 169.8 and H-3 (δ H 5.18, d, J = 4.8 Hz) to the carbonyl carbon at δ C 165.5 of the benzoyl group. Compound 1 has the same macular formula and similar NMR spectraoscopic data with those of anthriscifolmine C (11) 9 , which also possesses an acetyl group at C-2 and a benzoyl group at C-3. However, compound 1 differs from anthriscifolmine C (11) mainly at C-11 where an aldehyde group and a trisubstituted double bond between C-12 and C-16 were deduced. Two methys group were shown to be attached at C-4 and C-16 according to the HMBC correlations from H 3 -18 to C-3, C-4, C-5 and C-19, and H 3 -17 to C-12, C-15 and C-16. The substitution pattern and the assigned planar structure of 1 were confirmed by complete 1 H− 1 H COSY and HMBC spectroscopic analysis.
The NOESY correlations of H-1β and H-3, H-3 and H-5, H-1α and H-11, H-1α and H-20, H-19 and H-20, proved that H-3 was β-oriented, H-11 and H-19 were in α-orientation (Fig. 3). The NOESY correlations indicated that H-2 was in an equatorial position, which indicated a β-orientation. Moreover, an X-ray diffraction experiment with a suitable crystal was conducted and the absolute configuration of 1 was established as H-2β, H-3β, H-11α, H-19α (19-s) (Fig. 4), consistent with the absolute configuration determined by NOESY correlations. Thus, the structure of 1 was assigned as shown in Fig. 1.
A possible biogenetic pathway of anthriscifolsine A (1) was proposed as shown in Fig. 5. Aldehyde A could be generated from the known alkaloid anthriscifolmine C (11) through a critical retro-aldol process involving the cleavage of C11-C12 bond. The latter has been also isolated from this plant, which was obtained as needles crystal (MeOH), and the structure of which was unambiguously confirmed by an X-ray crystallographic analysis (Fig. 6). The unstable intermediate A then underwent proton shift and epimerization of the C9 stereochemistry, thus leading to anthriscifolsine A (1). Finally, the artificial possibility of anthriscifolsine A (1) had been explicitly excluded used UPLC-HRESI-MS method and the detailed experiments were added the Supporting Information.
Anthriscifolsine B (2) was obtained as a white amorphous powder. Its molecular formula C 24 H 31 NO 7 was derived from a pseudomolecular ion at m/z 446.2196 [M + H] + in its HR-ESI-MS. It exhibited characteristic NMR features of a hetisine-type C 20 -diterpenoid alkaloid bearing groups including two acetyl groups, and an exocyclic double bond (Table 1) 12 . Two acetyl groups can be installed at C-2 and C-3, respectively, on the basis of the HMBC correlations from H-2 (δ H 5.35, m) to the carbonyl carbon of one acetyl group at δ C 170.2 and H-3 (δ H 4.93, d, J = 4.8 Hz) to the carbonyl carbon of another acetyl group at δ C 170.6. Along with the abovementioned signals, its 13 C NMR spectrum displayed five oxygenated carbon signals, suggesting that this compound possessed three additional hydroxyl groups in addition to two ester groups. Two hydroxyl groups were assigned at C-11 and C-13 based on the HMBC correlations from H-11 to C-10, C-13 and C -16, and H-13 to C -11, and COSY correlations of H-11/H-12/H-13/H-14. The observation of HMBC crosspeak between C-9 (δ C 80.2) and H-1    The relative configuration of 2 was deduced from the vicinal coupling constants and a NOESY experiment (Fig. 3). The coupling constant (J = 4.8 Hz) of H-2 with H-3, indicated that H-2 was in an equatorial position, which indicated a β-orientation 9 . The large coupling constant of H-13 (J = 8.4 Hz) with H-14α revealed that the dihedral angle between these two H-atoms was ca. 0 °C, which implied that H-13 was in an α-orientation 9 . In the NOESY spectrum of 2, the cross-peak between H-1β and H-3, H-3 and H-5, H-1α and H-11, H-1α and H-20, proved that H-3 was β-oriented and H-11was in α-orientation. Therefore, the structure of anthriscifolsine B was determined as shown in Fig. 1, and the full assignment of its spectroscopic data was achieved based on the 1Dand 2D NMR analysis (Table 1, Fig. 2).  The remaining 20 carbons were assigned based on 1D-and 2D-NMR data and exhibited characteristic NMR features of a hetisine-type C 20 -diterpenoid alkaloid 12 bearing five methylenes, nine methines (four oxygenated) and five quaternary carbons (one ester carbonyl), in addition to one methyl group that was attached to a quaternary carbon ( Table 1). The presence of an exocyclic double bond was evidenced by singals in the 1 H NMR spectrum (δ H 4.87, d, J = 1.8 Hz, 4.98, d, J = 1.8 Hz) and 13 C NMR spectrum (δ C 111.2 and 148.5). The locations of two 2-methylbutanoyloxy groups at C-2 and C-15 were determined by the correlations in the HMBC experiment (Fig. 2). The 13 C NMR spectrum showed a singlet at δ C 97.0, indicative of a carbinolamine carbon (C-6). Besides the two ester groups and the carbinolamine carbon, there were two OH groups in the molecule, which were placed at C-3 and C-11, respectively, according to the HMBC displayed in Fig. 2. The coupling constant (J = 5.4 Hz) of H-2 with H-3 indicated that H-2 was in an equatorial position, namely, a β-orientation.
The  6 . Its NMR data indicated seven methylene (one oxymethylene), seven (two oxymethines), and five quaternary carbons (a carbonyl and two oxygen-bearing), in addition to a methylenedioxy group, an N-ethyl, and three methoxy substituents, suggesting that 4 was a typical lycoctonine C 19 -diterpenoid alkaloid 13 . The 2D NMR and NOESY experiments confirmed the NMR data and configuration assignments of 4. In particular, HMBC correlations of C-14 with H-9, H-12, H-13, and H-16 confirmed the 14-keto group, while HMBC correlations of the protons of the methylenedioxy with C-7 and C-8, OCH 3 -1 with C-1, OCH 3 -16 with C-16, OCH 3 -18 with C-18, confirmed the locations of the methylenedioxy and three methoxy groups. The α-oriented 1-OCH 3 and β-oriented 16-OCH 3 in compound 4 were deduced from the vicinal coupling constants (Table 2)and a NOESY experiment (Fig. 7). The structure of anthriscifolrine A was thus established.
The molecular formula of anthriscifolrine B (5) 8 , 508.2910). The NMR spectroscopic data of 5 were similar to those of 4, suggesting 5 was also a lycoctonine C 19 -diterpenoid alkaloid with an acetyl group, an N-ethyl group, three methoxyl groups, and a methylenedioxy group 13 . In the HMBC spectrum of 5, critical correlations for the protons of the methylenedioxy/C-7 and C-8, OCH 3 -1/C-1, OCH 3 -16/C-16, OCH 3 -18/C-18, H-14/OAc, suggested  the location of three methoxyl groups at C-1, C-16 and C-18, the acetyl group at C-14, and the methylenedioxy group at C-7 and C-8. Its 13 C NMR spectrum displayed seven oxygenated carbon signals, suggesting that it possessed an additional hydroxyl group in addition to three methoxy groups, an ester group, and a methylenedioxy group. The additional hydroxyl group in 5 was assigned to C-10 on the basis of the correlations of C-10 (δc 79.8) with H-1 (δ H 3.57), H-9 (δ H 2.42), H-13 (δ H 2.76) and H-17 (δ H 2.98) in the HMBC spectrum. The configurations of 1α-OCH 3 , 16β-OCH 3 , 14α-OAc and constant18β-OCH 3 were deduced by the vicinal coupling constants (Table 2) and a NOESY experiment. Thus, the structure of anthriscifolrine B was confirmed by NMR experiments.
The molecular formula of anthriscifolrine C (6) was determined as C 27 H 41 NO 9 (HR-ESI-MS). The 1 H and 13 C NMR data ( Table 2) of 6 showed close structural similarity to compound 5, and the distinction between the two sets of spectra was demonstrated by the presence of an additional hydroxyl group signal in 6, which was validated by the additional 16 mass units in mass spectrometry. The proton signal of H-6 at [δ H 2.11 (1 H, m), 1.50 (1 H, m) and δ C 32.8 (t)] in compound 5 was shifted downfield to [δ H 5.52 (1 H, s), δ C 78.3(d)] in compound 6, suggesting that the hydroxyl group in 6 might be located at C-6, which was further confirmed by the HMBC correlations. The hydroxyl group at C-6 was determined to have a β-orientation based on the multiplicity of H-6 (singlet) in the 1 H NMR spectrum 2 . Thus, the structure of anthriscifolrine C was determined as shown in Fig. 1.
Anthriscifolrine D (7), a white amorphous powder, C 27 H 39 NO 9 (HR-ESI-MS), was also a lycoctonine-type C 19 -diterpenoid alkaloid. By comparison of the NMR data of 7 with those of 4, the main difference was the presence of a acetyl and a hydroxyl groups. In the HMBC experiment, long-range correlations were observed from H-6 (5.54) to the carbonyl carbon of the acetyl group at δ C 170.2, and H-1 (δ H 3.91), H-9 (δ H 3.49), H-13 (δ H 2.82) and H-17 (δ H 3.71) to C-10 (δc 79.8) supported the hydroxyl group at C-10. The structure of anthriscifolrine D (Fig. 1) was confirmed by the analysis of its 2D NMR data.
The molecular formula of anthriscifolrine E (8) was deduced to be C 26 H 39 NO 8 from its HR-ESI-MS at m/z 494.2752 [M + H] + . From its NMR data (Table 3), an N-ethyl group, two methoxy groups, an acetoxy group, and a methylenedioxy group could be easily recognized. Compound 8 shared highly similar 1 H-and 13 C-NMR spectral patterns with those of 5. The only difference is that the absence of a methoxy group and the presence of a hydroxy group at C-18 in 8, which was further supported by comparison of the NMR data: the C-18 signal in 8 appeared at δ C 68.3 instead of at δ C 79.1 in 5. The structure of anthriscifolrine E was unquestionably confirmed by extensive analyses of its 1D and 2D NMR spectra.
Comparison of spectroscopic data of anthriscifolrine F (9) and E (8) indicated that an acetyl group in 8 was substituted by a methoxy group in 9. According to 2D NMR analysis, especially the HMBC correlation of OCH 3 /C-14, the OCH 3 group was attributed to C-14 in 9. The corresponding structure of 9 was confirmed by DEPT, HMQC, 1 H-1 H COSY, and HMBC experiments. Thus, anthriscifolrine F was assigned as shown in Fig. 1.
To evaluate the biological activities of these compounds isolated from the whole plant of D.anthriscifolium var. majus, compounds 3-6 were tested for their in vitro cytotoxicity against the MCF-7, HepG2 and H460 cancer cell lines. Unfortunately, all of the compounds were inactive (IC 50 > 50 μM, n = 3).

Discussion
Investigation on the whole plant of Delphinium anthriscifolium var. majus resulted in the isolation of nine new diterpenoid alkaloids named anthriscifolsines A-C (1-3) and anthriscifolrines A-F (4-9), together with five known alkaloids (10)(11)(12)(13)(14). Notably, anthriscifolsine A (1) is the first naturally occurring C 20 -diterpenoid alkaloid with a unique seco C-ring generated by an unprecedented C11-C12 bond cleavage, and its possible biogenetic pathway was proposed. Since the Sect. Anthriscifolium only comprises three species (D. anthriscifolium, D. anthriscifolium var. majus, and D. anthriscifolium var. savatieri), the present research would be particularly valuable in understanding their chemotaxonomical significance. The identification of various C 19 -and C 20 -diterpenoid alkaloids from D. anthriscifolium var. majus revealed its transitional position among the Delphinium plants.

Materials and Methods
General Experimental Procedures. Optical rotations were measured using a Perkin-Elmer 341 polarimeter. The IR spectra were obtained using a Thermo Fisher Nicolet 6700 spectrometer and KBr pellets in cm −1 . The HR-ESI-MS data were measured using a Q-TOF micro mass spectrometer (Waters). The 1D and 2D NMR spectra were recorded using a Bruker AV 600 with TMS. Silica gel (Qingdao Haiyang Chemical Co., Ltd., 200-300 mesh) was used for column chromatography (CC). The TLC plates were precoated with silica gel GF 254 (Qingdao Haiyang Chemical Co., Ltd., China), and it was visualized under a UV lamp at 254 nm or by spraying with Dragendorff 's reagent or iodine. Extraction and Isolation. Dried and powdered whole herbs of D. anthriscifolium (21.5 kg) were extracted with 95% EtOH four times at room temperature, with each soaking process lasting a week. After removal of the solvent by evaporation, the ethanol extract (2000 g) was recovered. The extract was suspended in H 2 O (3 L) and adjusted to pH 2 with HCl, and successively extracted with petroleum ether (4 × 1 L) and ethyl acetate (4 × 1 L). The pH of aqueous layer was adjusted to 10 with aqueous ammonia solution and extracted with CH 2 Cl 2 (4 × 1 L). The CH 2 Cl 2 extracts were concentrated to produce the crude alkaloid extract (28.5 g). Column chromatography of the crude alkaloid extract over silica gel, using a CH 2 Cl 2 :MeOH (80:1, v/v) mixture with increasing polarity afforded fractions A-D based on TLC analysis.

Spectroscopic data of 1-9. Anthriscifolsine
Cell Culture and Cytotoxicity Assay. The cytotoxicity of the compounds against cultured human tumor cell lines such as MCF-7, HepG2 and H460 cell lines was evaluated by the MTT method as described in our previous paper 14 . Cells treated with DMSO (0.1% v/v) were used as negative controls, whereas adriamycin (≥98%; Sigma Chemical Co., Ltd., Shanghai, China) was used as the positive control.