Tigliane-and daphnane-type diterpenoids from the buds of Daphne genkwa with their cytotoxic activities

Abstract Two new tigliane- and daphnane-type diterpenoids, given the trivial names daphnegens A–B (1–2) were isolated from the buds of Daphne genkwa. Their structures were assigned on the basis of extensive spectroscopic. The absolute configurations of both compounds were determined by comparison of their calculated and experimental CD curves. In addition, compounds 1–2 were tested for their cytotoxic activities against MCF-7 and HepG-2 human cancer cell lines, and compound 2 showed remarkable cytotoxic activity against HepG-2 cell line with the IC50 value of 11.5 μM. Graphical Abstract


Introduction
Tigliane-and daphnane-type diterpenoids have attracted many scholars as their remarkable and extensive bioactive activities like anti-cancer, anti-HIV, anti-hyperglycemic, anti-leukemia, insecticidal effects and so on (Asada et al. 2011;Zhang et al. 2014;Wang et al. 2015;Minh et al. 2018;Hou et al. 2020). Due to their rich pharmacological activities, especially strong cytotoxicity activity, tigliane-and daphnane-type diterpenoids have been engaged in a range of clinical applications for a variety of clinical uses (Zhang et al. 2010;Wang et al. 2015;Yang et al. 2015). Until now, the extraction and isolation from medicinal plants are still the main method for discovering tigliane-and daphnane-type diterpenoids.
Daphne genkwa Sieb. et Zucc, a classical traditional Chinese Medicine, has long been used for the treatment of abortifacient, diuretic, antitussive, expectorant, and anticancer effects (Chen et al. 1997;Zhang et al. 2018), which distributed mainly in Shanxi, Hebei, Gansu, Jiangsu and Shandong provinces of China (Zhan et al. 2005;Jiang et al. 2015). Phytochemical studies indicated that the buds of D. genkwa wealth contained daphnane-type diterpenoids like neogenkwanines A-H, yuanhuacine, genkwadaphnin, and so on (Kasal et al. 1981;Zhang et al. 2006;Li et al. 2015;Han et al. 2016). With the purpose to find bioactive daphnane-type, we examined the ethanol extract of the buds of D. genkwa, and obtained two new diterpenoids named as daphnegens A-B ( Figure 1). Herein, we report the isolation and structural determination of the new constituents. Their cytotoxic activities are also described.  6, 129.7, 129.8, 133.6, and 167.9; four methyl signals at d H 0.94 (3H, d, J ¼ 6.6 Hz), 1.15 (3H, s), 1.31 (3H, s), and 1.78 (3H, s); seven oxygenated carbons at d C 61. 8, 64.5, 64.7, 65.8, 71.7, 75.5, and 72.5. All the proton signals were assigned to the corresponding carbons through direct 1 H and 13 C correlations in the HSQC spectrum. Partial structures of -(CH 2 ) 2 -and -(CH) 2 -were deduced from the 1 H-1 H COSY spectrum (Supporting Information, Figure  S17). Detailed NMR data suggested that compound 1 was a tigliane-type diterpenoid, which was similar with the reported one phorbol (Yan et al. 2022). The differences were the appearance of epoxy ether unit at C6/7 and benoyl group at C-13 in compound 1, which were further supported by 2 D NMR spectra. In the HMBC spectrum (Supporting Information, Figure S17), the correlations from d H 3.81 (1H, d, J ¼ 12.6 Hz, H-20a), 3.86 (1H, d, J ¼ 12.6 Hz, H-20b) and d H 2.91 (1H, d, J ¼ 7.2 Hz, H-8) to d C 61.8 (C-6) and d C 65.8 (C-7) indicated that the double bond at C-6/7 in compound phorbol was oxidized to epoxy ether group in compound 1. Considering the molecular formula and chemical shift value of C-13, we inferred that one benzoyl group was located at C-13 in compound 1. Furthermore, the hydroxyl group at C-5 in compound 1 was supported by the HMBC correlations between d H 3.81 (H-20a) and C-5 (d C 71.7). In the NOESY spectrum, the enhancements of H-5 and H-10, H-7 and H-8, H-8 and H-11, H-14 and H 3 -18 confirmed the b-orientation of hydroxyl group at C-5, and a-orientations of epoxy ether moiety at C-6, benzoxyl group at C-13. In fact, the conjunctions of A/B and B/C trans rings are essential (Bang et al. 2013;Zhang et al. 2014;Hou et al. 2020). In order to define the chiral carbons unambiguous, a comparison of the theoretically calculated and measured ECD curves was tested. The result showed that the calculated ECD spectrum of (4S, 5S, 6 R, 7S, 8 R, 9 R, 10S, 11 R, 13S, 14 R)-1 was identical with the measured CD spectrum (Supporting Information, Figure S15). The ECD spectrum was calculated using density functional theory (DFT) at the APFD/6-311 þ g(2d, p) level. As a result, compound 1 was established as shown and given the trivial name daphnegen A.

Results and discussion
Compound 2  . From the 13 C NMR APT spectrum, five methyls, two methylenes, eight methines, and nine quaternary carbons were observed. Among them, the carbon signals of d C 137.6 (C-2), 158.7 (C-1), and 209.4 (C-3) indicated the presence of a, b-unsaturated ketone moiety, and d C 18.6 (C-17), 113.2 (C-16), and 143.3 (C-15) suggested the existence of isopropenyl group. The NMR data were similar with the reported one daphneresiniferin A (Bang et al. 2013), except for the additional hydroxyl group at C-5 in compound 2. In the HMBC spectrum (Supporting Information, Figure S17), the correlations from H-5 (d H 4.35) to d C 73.1 (C-4) and 138.7 (C-6) together with the molecular formula of C 24 H 30 O 9 suggested that the C-5 was further oxygenated with an additional hydroxyl group in compound 2. In the NOESY spectrum, the enhancements between H-5 and H-10, H-8 and H-11, H-12 and CH 3 -18, suggested the b-orientations of hydroxyl group at C-5 and acetoxyl group at C-12. The R absolute configuration of C-5 in compound 2 was defined through the comparison between experimental CD and ECD data (Supporting Information, Figure S16). As a result, compound 2 was elucidated as shown and named as daphnegen B.
The two compounds were tested in vitro for their cytotoxic activities against MCF-7 and HepG-2 cell lines with adriamycin as positive control. The results displayed that compound 1 showed mild cytotoxic activities against both two cell lines with IC 50 values of 68.2 and 42.7 lM, respectively. Compound 2 showed moderate cytotoxic activity against MCF-7 cell lines with IC 50 value of 26.3 lM, while exhibited remarkable cytotoxic activity against HepG-2 cell lines with the IC 50 11.5 lM.
Adriamycin was given the IC 50 values of 6.72 and 14.7 lM against MCF-7 and HepG-2 cell lines, respectively.

General experimental procedures
Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter. IR spectrum was recorded on FTIR-8400S spectrometers, respectively. CD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained with a Bruker AV III 600 NMR spectrometer (chemical shift values are presented as d values with TMS as the internal standard). HRESIMS spectra were performed on a LTQ-Obitrap XL spectrometer. Purification was performed by semiprep-HPLC with a Waters apparatus equipped with UV detector under ODS column (250 Â 10 mm; i.d., 5 lM, YMC Co. Ltd., Japan). TLC was performed on silica gel GF254 (10 À 40 lm; Qingdao Marine Chemical, Inc.). Column chromatography was performed on silica gel (100-200 or 200-300 mesh; Qingdao Marine Chemical, Inc.)

Plant material
The buds of D. genkwa (1.5 kg) were collected in Korla, Xinjiang, China, and were identified by Prof. Wei Dong, (Pharmacy School, Qiqihar Medical University). A voucher specimen (No. 20210910) has been deposited in Qiqihar Medical University, Qiqihar, China.

ECD calculation methods
The ECD calculations were performed using the Gaussian 09 program. Conformers were generated by the MMFF94s force field, and each conformer was optimized with the HF/6-31G(d) method, and further optimized by the DFT method at the B3LYP/6-311 þ g(d, p) level. Frequency calculations were also performed at the same level to confirm that each optimized conformer was a true minimum and to estimate their relative thermal free energy (DG) at 298.15 K. Conformers with the Boltzmann distribution over 1% were chosen for ECD calculations in methanol at the APFD/6-311 þ g(2d, p) level. The ECD spectra were simulated by the SpecDis program. To obtain the final conformational averaged data, the simulated spectra of the predominant conformers were averaged according to the Boltzmann distribution theory.

Cytotoxicity assay
Compounds 1-2 were assessed for cytotoxic activities against human cancer cell lines MCF-7 and HepG-2. MTT method was used as described in previously published literature (Singh et al. 2017). Briefly, the cell lines were grown in DMEM supplemented with 10% fetal bovine serum and cultured at a density of 6 Â 10 4 cells/mL in a 96-well microtiter plate. Five different concentrations of each compound dissolved in dimethyl sulfoxide (DMSO) were subsequently added to the wells. Each concentration was tested in triplicate. After incubation under 5% CO 2 at 37 C for 48 h, 10 lL of MTT (4 mg/mL) was added to each well, and the cells were incubated for another four hours. Then, the liquid in each well was removed, and DMSO (200 lL) was added. The absorbance was recorded using a microplate reader at a wavelength of 570 nm.

Disclosure statement
No potential conflict of interest was reported by the authors.