Antiproliferative ent-abietane diterpenoids from Euphorbia fischeriana

Abstract Euphorfinoids M and N (1 and 2), two previously undescribed ent-abietane diterpenoids, together with seven known analogues (3−9), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, ECD, and comparison with structurally related known analogues. Bioassays against proliferative effects of HeLa cell line showed that compound 1 was the most active with IC50 3.62 ± 0.31 μM. Graphical Abstract


Introduction
Euphorbia fischeriana Steud., a perennial herb in the Euphorbiaceae family, is mainly distributed in Heilongjiang, Jilin, Liaoning, Qinghai provinces and Nei Monggol Autonomous Region of china.The roots of this plant are listed in Chinese pharmacopoeias officially and recommended for the treatment of edema, cough, asthma, and other inflammation-related diseases (Chinese Pharmacopoeia Commission 2020).The plants in the genus Euphorbia have been widely studied, leading to the isolation of a variety of diterpenoids with fascinating structural diversity and pharmacology activity, such as ent-abietane (Wei et al. 2021b), ent-atisane (Wei et al. 2021a), lathyrane (Yin et al. 2020), jatrophane (Mai et al. 2018), ent-kaurane (Liu et al. 2017;Wei et al. 2021c), tigliane (Wang et al. 2017), and ingol types (Li et al. 2009), et al.Some of these substances have displayed biological activities, including inhibitory activity against acetylcholinesterase (Wei et al. 2021a), anti-tumor (Wei et al. 2021b), antibacterial (Du et al. 2022) and anti-inflammatory (Sun et al. 2021).
As the study to seek interesting diterpenoids from E. fischeriana, two previously undescribed ent-abietane diterpenoids (euphorfinoids M and N, Figure 1) together with seven known analogues were obtained, and their anti-proliferative effects were also evaluated.

Structural elucidation
Compound 1 was isolated as a white amorphous powder.Its molecular formula was determined to be C 20 H 26 O 6 by (HRESIMS) at m/z 363.1797 respectively.These data, together with the vanish of a methoxy group signal, suggested that the ester group at C-3 in 3 was hydrolyzed to a carboxylic acid in 1, which was further elucidated by 2 D NMR data.In the NMR data, the chemical shift of H-1, d H 3.24 (1H, d, J ¼ 20.2 Hz, H-1a) and 2.69 (1H, d, J ¼ 20.2 Hz, H-1b), are remarkably different from those of known compound 3 (d H 2.67, overlap), in terms of this issue, we further checked the 2 D NMR data of this compound, and found the HMBC correlations from H-1 to C-2, from H-1 to C-9 and from H-1 to C-10, which demonstrated the correlations of C-1 protons with the structure and the correctness of hydrogen signal attribution.We speculate that it is related to the steric hindrance of the two compounds.The absence of methoxy group in the structure of compound 1 reduces the steric hindrance of H-1 to a certain extent, and then increases its chemical shift.Based on the high similarity between compounds 1 and 3 in NMR data, and the same biosynthesis pathway from E. fischeriana, it is reasonable to speculate that compound 1 is also an ent-abietane type diterpenoid.
In the 1 H-1 H COSY spectrum of 1, the H-5/H-6/H-7 and H-9/H-11/H-12 cross-peaks indicated the presence of fragments with vicinal couplings.The HMBC spectrum (Figure S2) showed two-and three-bond correlations between H-18, H-19/C-3; H-7, H-9, and H-12/C-14; H-11/C-13, which, in combination with the chemical shifts of these proton and carbon resonances, demonstrated the two carboxyl groups, an ester carbonyl, and two double bond groups are located at C-2, C-3, C-15, C8-C14, and C13-C15, respectively.In addition, the relative configuration of 1 was established by NOESY spectrum (Figure S2). the correlations of H-5/H-9 indicated that they were on the axial positions and were randomly designated as b-orientations.Consequently, the NOE correlation of H-12/Me-20 assigned Me-20 to be a-oriented.The absolute configuration was determined by experimental and calculated ECD and assigned as (5S,9R,10S,12R)absolute configuration (Figure S3).Therefore, the structure of 1 was determined to be new ent-abietane diterpenoid featuring a vicinal 2,3-seco-2,3-diacid moiety and named euphorfinoid M.
The HRESIMS of compound 2 showed a [M -H] -ion at m/z 345.1697 (calcd for C 20 H 25 O 5 -, 345.1707), suggesting a molecular formula of C 20 H 26 O 5 for 2. By comparing the 13 C NMR data of skeletal carbons with those of 3-oxo-8b,14b-epoxy-ent-abieta-11,13(15)-dien-16,12-olide (Yan et al. 2008), the main difference is that the NMR resonances of H-7a, H-7b, and H-14 and C-7, C-8, C-9, and C-14 in 2 were deshielded by Dd H þ0.19, þ0.05, and þ0.82 and Dd C þ5.6, þ9.7, þ7.9, and þ16.3 ppm, respectively, whereas the resonances of H-9 was shielded by Dd H À0.36 ppm, which suggested that a cycloreversion reaction was occurred in 8,14-epoxy group.All the above deductions on this structure were verified by the 2 D NMR experiments.In NOESY results (Figure S2), the correlation H-9/H-5/H-7b/H-14 indicated these protons were cofacial, conversely, two methyl groups at C-19 and C-20 are coplanar.It has been reported that a complex formed by Mo 2 (OAc) 4 with vic-diols has absorbance in the ultraviolet/visible region.The chirality of this Cotton effect relies completely on the configuration of the chiral molecules, and can be used to determine the absolute configuration of vic-diols.Thus, the absolute configuration of 2 was determined by the induced electronic circular dichroism (ECD) spectra using Mo 2 (OAc) 4 in DMSO solution (Snatzke's method) (G orecki et al. 2007).The negative Cotton effect at 310-340 nm in the experimental ECD spectrum of this complex indicated an 8 R configuration, on the contrary, which is 8S configuration.Therefore, based on this rule, the positive Cotton effect at 310 nmat different times supported the 8S,14S configuration (Figure S4).Therefore, the structure of compound 2 (euphorfinoid N) was therefore determined as shown.

Anti-proliferative activities of above-mentioned compounds
Anti-proliferative effects of 1À9 was assessed against the MCF-7, HeLa, and Hcc1806 cell lines.As shown in Table S5, 1, 3, 5, 7, and 9 exhibited remarkable cytotoxic activities against MCF-7, HeLa, and Hcc1806 cell lines.In addition, compounds 1 and 3 showed displayed an approximately equal effect against Hela cell lines to cisplatin with IC 50 values of 3.62 ± 0.31 lM and 7.32 ± 0.98 lM, respectively.while compounds 2, 4, 6, and 8 showed no anti-proliferative effects.

Extraction and isolation
Organic compounds from powdered E. fischeriana roots (17 kg) were extracted with 85% ethanol (aq), refluxed, and then successively extracted with dichloromethane, ethyl acetate, and n-butanol.

ECD calculations
The ECD spectra were calculated according to the reported methods (Wei et al. 2021a).The computational data were fitted in the GraphPad Prism 7.0.The process is also described in detail in the Supporting Information (S3).

Mo 2 (OAc) 4 induced electronic circular dichroism
According to the published procedure (G orecki et al. 2007;Gao et al. 2020), about 1:1.2 mixture of diol-to-Mo 2 (OAc) 4 for compound 2 were prepared at the concentration of 1.0 mg/ml.Soon after mixing, the first ECD spectrum was recorded immediately, and its evolution was monitored until stationary (about 15 min after mixing).The inherent ECD of the diol was subtracted.The diagnostic band at around 310 nm in the induced ECD spectra were correlated to the absolute configuration of vicinal diol unit.

Anti-proliferative assay
The cytotoxicity assay for compounds 1-9 toward six human cancer cell lines MCF-7 (breast), HeLa (cervix), and Hcc1806 (breast) (National Infrastructure of Cell Line Resource, Beijing, China) was performed using the MTT method, as described formerly (Pang et al. 2019).Cisplatin was used as the positive control.

Conclusions
In summary, an intensive phytochemical study on the dichloromethane soluble partition of a traditional herb Euphorbia fischeriana afforded two previously undescribed ent-abietane diterpenoids, Euphorfinoids M and N, along with seven known diterpenoids.All isolates were evaluated for their cytotoxicity against MCF-7, HeLa, and HCC1806 cell lines.Majority of composition showed significant inhibitory activities, compound 1 displayed obviously anti-proliferative effects on HeLa cell line with IC 50 3.62 ± 0.31 lM.