Daphnane-Type Diterpenes from Stelleropsis tianschanica and Their Antitumor Activity

Four new daphnane-type diterpenes named tianchaterpenes C-F (1–4) and six known ones were isolated from Stelleropsis tianschanica. Their structures were elucidated based on chemical and spectral analyses. The comparisons of calculated and experimental electronic circular dichroism (ECD) methods were used to determine the absolute configurations of new compounds. Additionally, compounds 1–10 were evaluated for their cytotoxic activities against HGC-27 cell lines; the results demonstrate that compound 2 had strong cytotoxic activities with IC50 values of 8.8 µM, for which activity was better than that of cisplatin (13.2 ± 0.67 µM).


Introduction
The morbidity and mortality of malignant tumors are the highest in many countries and regions worldwide, seriously threatening human life and health [1]. However, many new anticancer drugs are expensive; long-term applications can easily cause drug resistance and side effects [2]. Therefore, searching for high-efficiency and low-toxicity anticancer drugs is the key to treating tumors. Daphnane-type diterpenes are a kind of diterpene with a 5/7/6-tricyclic ring system. They are mainly distributed in plants of the Thymelaeaceae and Euphorbiaceae families [3]. Modern pharmacological studies have shown that daphnanetype diterpenes have antitumor [4], antiHIV [5,6], antineuroinflammatory, insecticidal, and other effects [7,8]. In recent years, many daphnane-type diterpenes have attracted the interest of researchers on account of their remarkable anticarcinogenic and antiHIV activities [9][10][11][12]. As a result, finding and screening new daphnane-type diterpenes as active anticancer components or lead compounds from herbal medicine has become a research hotspot in the natural product field [13].
Stelleropsis tianschanica is a member of the genus Stelleropsis in the Thymelaeaceae family [14]. It grows on the hillside grassland at an altitude of 1700-2000 m. It is only distributed in Zhaosu County, Xinjiang, China [15]. S. tianschanica has a certain genetic relationship with Stellera chamaejasme Linn and has many common components, which are used as a counterfeit of traditional Chinese medicine Stellera chamaejasme in Xinjiang [16]. Due to the unique geographical environment and growth area, there is little research on the functional components of S. tianschanica. Our team found that S. tianschanica contained novel daphnane-type diterpenes ( Figure S37) [17]. To further -obtain potential daphnanetype diterpenes from S. tianschanica, its methanol, CH 2 Cl 2 , and EtOAc extractions were investigated, and ten daphnane-type diterpenes (Figure 1), including four new ones (1)(2)(3)(4) and six known compounds (5)(6)(7)(8)(9)(10), were obtained. Among them, compound 1 possessed a 5/7/6/3-tetracyclic skeleton, which is not common in daphnane-type diterpenes. In addition, the antitumor activity of the isolated compounds was tested. In this paper, we report the structure elucidation and biological screening in vitro of the obtained daphnane-type diterpenes from S. tianschanica.
Compound 2 was obtained as a white powder with its molecular formula assigned as C 32 H 42 O 10 according to the positive HR-ESI-MS peak at m/z [M + Na] + 609.2652 (calcd. for C 32 H 42 O 10 Na, 609.2670), exhibiting eleven degrees of unsaturation. Through the 1 H-and 13 C-NMR spectra, we inferred that the basic nucleus of compound 2 was a daphnane-type diterpene, which was further confirmed by the 1 H-1 H COSY and HMBC spectra ( Figure 2). Meanwhile, in the HMBC spectrum, the correlations from δ H 5.01 (1H, s, H-12) to δ C 169.4 (C-1') indicate that the acetoxyl group is attached to C-12, and the correlations of H-14 (δ H 4.83), H-2 (δ H 5.59, d, J = 15 Hz), and H-3 (δ H 6.63, dd, J = 10.8, 15 Hz) with C-1 (δ C 116.8) suggested that the adipose chain was linked to C-1 . Based on the olefinic proton signals with large coupling constants in the 1 H NMR data, the fatty group was assigned as trans-conjugated diene. In fact, the NMR data of compound 2 were similar to those reported for yuanhuadine [20][21][22], except for the different orientation of the epoxy group at δ C 75.2 (C-6) and 80.1 (C-7), which is located in the low field area compared with yuanhuadine. In the NOESY spectrum, the correlations of H-5/H-10, H-5/H-7, and H-7/H-20 indicated αorientation for these protons. Therefore, it is judged that the epoxy group has β-orientation, while the yuanhuadine group has α-orientation. In conclusion, the structure of compound 2 was determined as the isomer of yuanhuadine. The calculated and experimental ECD data also supported this deduction. Accordingly, the structure of compound 2 was established as shown and named tianchaterpene D.

5'
7.60, t, (7.  Compound 2 was obtained as a white powder with its molecular formula assigned as C32H42O10 according to the positive HR-ESI-MS peak at m/z [M +Na] + 609.2652 (calcd. for C32H42O10Na, 609.2670), exhibiting eleven degrees of unsaturation. Through the 1 Hand 13 C-NMR spectra, we inferred that the basic nucleus of compound 2 was a daphnanetype diterpene, which was further confirmed by the 1 H-1 H COSY and HMBC spectra (Figure 2). Meanwhile, in the HMBC spectrum, the correlations from δH 5.01 (1H, s, H-12) to δC 169.4 (C-1') indicate that the acetoxyl group is attached to C-12, and the correlations of H-14 (δH 4.83), H-2" (δH 5.59, d, J = 15 Hz), and H-3" (δH 6.63, dd, J = 10.8, 15 Hz) with C-1" (δC 116.8) suggested that the adipose chain was linked to C-1". Based on the olefinic proton signals with large coupling constants in the 1 H NMR data, the fatty group was assigned as trans-conjugated diene. In fact, the NMR data of compound 2 were similar to those reported for yuanhuadine [20][21][22], except for the different orientation of the epoxy group at δC 75.2 (C-6) and 80.1 (C-7), which is located in the low field area compared with yuanhuadine. In the NOESY spectrum, the correlations of H-5/H-10, H-5/H-7, and H-7/H-20 indicated α-orientation for these protons. Therefore, it is judged that the epoxy group has β-orientation, while the yuanhuadine group has α-orientation. In conclusion, the structure of compound 2 was determined as the isomer of yuanhuadine. The calculated and experimental ECD data also supported this deduction. Accordingly, the structure of compound 2 was established as shown and named tianchaterpene D.    (Table 1) of 4 were similar to those of yuanhuacine. However, based on the coupling constant (J) in the 1 H NMR spectrum, the conjugated diene signals (H-2 to H-5 ) contain a trans, cis-conjugated diene, while yuanhuacine contains a trans, trans-conjugated diene unit, suggesting that the two compounds are geometric isomers. The correlations H-2 to H-10 in the 1 H-1 H COSY spectrum indicated that the side aliphatic group is a straight chain. In the HMBC spectrum (Figure 2), the correlations from δ H 5.21 (1H, s, H-12) to δ C 165.4 (C-1') suggested that the methyl benzoate group is attached to C-12. In the NOESY spectrum, correlations were observed among H-12/H-18, H-8/H-11, H-8/H-14, H-7/H-14, and H-5/H-10, which were analogous to those for compound 3, as supported by their identical ECD spectra ( Figure S36). As a result, compound 4 was elucidated as shown and named tianchaterpene F.

Antitumor Activity
Compounds 1-10 isolated from the Stelleropsis tianschanica were measured for their antitumor activities against HGC-27 human gastric cancer cells, with cisplatin as the positive control (Table 2). Among them, compound 2, which contain a 6,7-epoxide group of β-orientation, showed stronger inhibitory activity than the other compounds, indicating that the group may enhance the cytotoxic activity against HGC-27 gastric cancer cells, with an IC 50 of 8.8 µM. In addition, compounds 8-10 (IC 50 ≤ 17.5 µM), which each had an orthoester group and a decadiene group, showed stronger activity than all compounds except 6, suggesting that the orthoester and decadiene groups are both significant in the inhibitory activities against HGC-27 cells. This result is consistent with previous studies on daphnane-type diterpenoids [23]. At the same time, the experimental results also show that compound 2 had the best inhibitory effect; its activity was better than that of cisplatin, and the activity of tigliane skeleton diterpenes was lower than that of daphnane-type diterpenes.

General Experimental Procedures
Optical rotation data were measured using a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). UV and IR spectral data were recorded on Shimadzu UV2550 and FTIR-8400 spectrometers (Shimadzu, Kyoto, Japan). CD spectra were obtained using a JASCO J-815 spectropolarimeter. NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer with chemical shift values presented as δ values using TMS as the internal standard (Bruker, Billerica Germany). HR-ESI-MS was performed using an LTQ-Orbitrap XL spectrometer (Thermo Fisher Scientific, Boston, MA, USA); samples were dissolved in chromatographic methanol and treated through a membrane, single pump. Semipreparative HPLC was performed using an HPLC PUMP K-501, LC3000 highperformance liquid chromatograph (Beijing Tong Heng Innovation Technology Co., Ltd., Beijing, China), and Kromasil 100-5C18, 250 × 10 mm, E108850. Column chromatography (CC) was performed using silica gel

Plant Material
The Stelleropsis tianschanica Pobed. were collected in October 2020 from Zhaosu City, Xinjiang Autonomous Region, China and authenticated by Prof. Xiaoguang Jia. A voucher specimen (20201022) was deposited at the Xinjiang Institute of Chinese and Ethnic Medicine.

Antitumor Activity
The cytotoxic activities of compounds 1-10 against HGC-27 cells were tested using the MTT colorimetric method. HGC-27 cells were cultivated on DMEM medium at 37°C and 5% CO 2 . After diluting the DMEM medium, cells were seeded into 96-well sterile microplates (4 × 10 5 cells/well) and cultured with various concentrations of tested compounds or DDP (positive control) for 24 h at 37 • C. After incubation, all compounds were tested at five concentrations (10−100 µM) for 1 h. Following this, the supernatant was removed, and all components were dissolved in 100% DMSO at such an amount that there was a final DMSO concentration of 0.1% added to each well. The absorbance was measured using a microplate reader at a wavelength of 570 nm. Data are displayed as the means ± SD (n = 3). The cell growth assay was repeated three times, and the IC 50 values were calculated using Microsoft Excel software.

Conclusions
Ten diterpenoids, including four new ones, were obtained from Stelleropsis tianschanica, and the complete structures of all of these new compounds were eventually elucidated by extensive spectral analysis. The antitumor activities of all isolates were tested. The results show that compounds 2 and 8-10 displayed potential biological activities, with IC 50 values less than 20 µM, thus proving that daphnane-type diterpenes in Stelleropsis tianschanica can be used as potential antitumor drugs, which is worthy of attention. This manuscript also provides a theoretical basis for further clinical application, development, and utilization of the medicinal plant Stelleropsis tianschanica.