New Secodaphnane-Type Alkaloids with Cytotoxic Activities from Daphniphyllum angustifolium Hutch

Graphic Abstract One new Daphniphyllum alkaloid, daphnioldhanol A (1), together with three known ones, were isolated from the stem part of Daphniphyllum angustifolium Hutch. Their structures were elucidated by spectroscopic methods and comparing with the literature data. Compound 2 is a new natural product, but known by synthesis as a racemate. Compound 1 exhibited week cytotoxic activity against Hela cell line with IC50 of 31.9 μM. Supplementary Information The online version contains supplementary material available at 10.1007/s13659-021-00309-w.

Qing-Yun Lu and Jia-Hui Zhang have contributed equally to this work.

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Herein, the isolation, structural elucidation, and bioactivities of these compounds are reported. , and an imine moiety (1631 cm −1 ). The 13 C NMR and DEPT data of 1 displayed 32 carbon signals (Table 1), due to three tetrasubstituted sp 2 carbon atoms at lower field and 29 sp 3 carbon atoms (5 × C, 6 × CH, 11 × CH 2 , 6 × CH 3 ) at higher field. According to the molecular formula and relative NMR data, one CH group (δ C 65.4, δ H 2.95) was ascribed to those bearing an N-atom, while one quaternary C-atom (δ C 84.4) were attributed to those bearing an O-atom. Additionally, three sp 2 quaternary carbons were attributable to one lactone carbonyl (δ C 180.2), one ester carbonyl (δ C 172.2), and one iminium group (δ C 168.4), while taking into account the three degrees of unsaturation. The remaining seven degrees of unsaturation were accounted for the presence of the heptacyclic system of 1.

Structure Elucidation of the Compounds
The 1 H and 13 C NMR spectra of 1 were closely related to those of the known compound Daphnioldhanine I [25], with the exception of the loss of signal for a CH in the latter and the addition of signal for quaternary carbon with a hydroxyl (δ C 84.4), which were supported by the HMBC correlations of H-11/C-9, H-13/C-9, H-15/C-9, H-16a/C-9, H-17/C-9.
To determine the orientation of the hydroxyl at C-9, we compared the 13 C NMR data of both 1 and daphnioldhanine I, which revealed that 9-OH substituent significantly shields the C-21 (5 ppm decrease) in the former. This indicated that the 9-OH in 1 should take a β-orientation. Moreover, the remaining relative configuration of 1 was elucidated from ROESY correlations as shown in computer-generated 3D drawing, which was the same as that of the daphnioldhanine I (Fig. 2).
The known compounds were identified as (−)-nitrone 17 (2) [26], daphnilactone A (3) [27], dapholdhamine B (4) [28], respectively, by comparison of their spectroscopic data with those reported in the literature (Fig. 1). Compound 2 was obtained as white amorphous powder. MS analysis of 2 revealed a [2 M + H] + peak at m/z 747. By comparison of its 1 H and 13 C NMR data with those of (±)-nitrone 17 in the literature, high similarity between them indicated that 2 shared the same structure as the latter. However, the compound 2 is a new chiral natural product with OR at − 31.75°, but known by synthesis is racemate.  A plausible biogenetic pathway for 1 and 2 was proposed as shown in Scheme 1. Biogenetically, both 1 and 2 should be the derivatives of secodaphnane-type alkaloid [13,29], which might be originated from sequalene, as proposed from Heathcock [26]. Then, 1 and 2 might be formed via different pathway.

Cytotoxic Activity
Both compounds 1 and 2 have been tested for their cytotoxicity against Hela, MCF-7, A549, MGC-803 and COLO-205 human cancer cell lines in vitro. The results indicated that 1 exhibited weak cytotoxic activity against Hela cell line with IC 50 of 31.9 μM (Table 2).

General Experimental Procedures
Optical rotations were measured with a Jasco P-1020 polarimeter. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for IR spectra as KBr pellets. 1D and 2D NMR spectra were recorded on Bruker spectrometer with TMS as internal standard. HRESIMS was performed on a triple quadrupole mass spectrometer. Semi-preparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Waters X-Bridge Prep Shield RP18 (10 × 150 mm) column. Column chromatography (CC) was performed using silica gel (100-200 mesh and 300-400 mesh, Qingdao Marine Chemical, Inc., Qingdao, P. R. China) and Sephadex LH-20 (40-70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden).

Extraction and Isolation
Air-dried stems of Daphniphyllum Angustifolium (20 kg) were powdered and extracted with MeOH (24 h × 3) at room temperature, and the solvent was evaporated in vacuo. The MeOH extract was then partitioned between EtOAc and TFA/H 2 O at pH 3.0. Water-soluble materials, after being adjusted at pH 10.0 with saturated Na 2 CO 3 , were partitioned with CHCl 3 . CHCl 3 -soluble materials (100.6 g) was subjected to silica gel column chromatography (CC) and eluted with gradient CHCl 3 /MeOH to yield five fractions F-1-F-5. F-4 was repeatedly submitted to silica gel CC and Sephadex LH-20, then purified by HPLC to afford compounds 1 (1.2 mg) and 2 (2.0 mg). Accordingly, 3 (18.0 mg) was obtained from F-1; 4 (2.5 mg) was obtained from F-5.

Concluding Remarks
In conclusion, one new Daphniphyllum alkaloid, daphnioldhanol A (1), together with three known ones, were isolated from the stem part of D. angustifolium Hutch. Compound 1 exhibited week cytotoxic activity against Hela cell line.