Euphorfinoids A and B, a pair of ent-atisane diterpenoid epimers from the roots of Euphorbia fischeriana, and their bioactivities

Abstract Euphorfinoids A and B (1 and 2), a pair of ent-atisane diterpenoid epimers with a vicinal 2,3-diol moiety, together with four known analogues (3–6), were isolated from the roots of wild Euphorbia fischeriana. Their structures were elucidated by spectroscopic analysis, including extensive NMR, HR-ESIMS, NMR calculations, X-ray diffraction, and comparison with structurally related known analogues. Our bioassays have established that compound 1 displayed moderate anti-proliferative effects on Hcc1806 cell line with IC50 15.53 ± 0.21 μM, and compound 5 showed remarkable inhibitory effects against AChE with IC50 32.56 ± 2.74 μM by an in vitro screened experiment. Graphical Abstract


Introduction
Euphorbia fischeriana Steud. (Euphorbiaceae) is a perennial herb mainly distributed in northeastern China (Uemura et al. 1975;Flora of China Editorial Committee 1997). Its roots are named 'Lang-du' and are used in traditional Chinese medicines for the treatments of tuberculosis and psoriasis (Liu 1988;Chinese pharmacopoeia commission 2015). Its chemical constituents have been widely studied, leading to the isolation of a variety of diterpenoids, such as ent-atisane (Wang et al. 2016), ent-abietane (Adelakun et al. 2019), ent-kaurane (Kuang et al. 2016a), rosane (Kuang et al. 2016a), lathyrane , pimarane (Wang et al. 2006), tigliane (Deng et al. 2019), ingenane (Wang et al. 2013), and ingol types (Zhao et al. 2018), as well as dimeric diterpenoids (He et al. 2021), and norditerpenoids (Wang et al. 2004). Among them, ent-atisanes are a big member of diterpenoids with diverse structural features. More than 180 ent-atisane diterpenoids have been isolated from different plants, especially from the species of family Euphorbiaceae. Recently, some novel ent-atisane diterpenoids such as crotobarin, crotogoudin (Rakotonandrasana et al. 2010), pharicusin C (Hu et al. 2018), aconicatisulfonines A and B (Wu et al. 2019) with unique skeletons, and variety of entatisnae diterpenoids with potent cytotoxicity toward human cancer cells were reported, which led to a new wave of research hot about ent-atisane diterpenoids.
It is known that one of the most promising approaches for the enhancement of the acetyl Cho line level in the brain is the use of inhibitors of acetylcholinesterase (AChE), an enzyme responsible for the metabolic hydrolysis of acetylcholinesterase. Thus, the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) has been proposed for the treatment of Alzheimer's disease (AD) and related neurodegenerative disorders based on the cholinergic hypothesis (Jiang et al. 2017;Rossor. 1983). In the previous study of diterpenoids, certain types of diterpenoids displayed a considerable AChE inhibitory effect (Murata et al. 2016;Liu et al. 2019;Yan et al. 2020).
As the study to seek interesting diterpenoids from E. fischeriana, a pair of undescribed ent-atisane diterpenoid epimers (euphorfinoids A and B, Figure 1) together with four known analogues were obtained, and their AChE inhibitory and anti-proliferative effects were also evaluated. . The 13 C NMR displayed 20 carbons, corresponding to 3 methyl groups, 7 methylene groups (one terminal double bond carbon at d C 107.0), 5 methine groups (two oxygenated carbon at d C 78.5 and 70.9), and 5 quaternary carbons (one olefinic carbon at d C 147.6 and one carbonyl carbons at d C 216.9). The above-mentioned spectroscopic data indicated that compound 1 was an ent-atisane type diterpenoid derivative similar with ent-(3a,5b,8a,9b,10a,12a)-3-hydroxyatis-16-en-14-one, previously isolated from E. fischeriana (Kuang et al. 2016b;Shi et al. 2017), except for an extra oxidized methylene group (d C 70.9; d H 4.08).
The absolute configuration of 1 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;Liu et al. 2010) and comparing the experimental and calculated 13 C NMR ( Figure S3). Based on the empirical helicity rule relating the sign of the Cotton effect of the diagnostic O-C-C-O moiety, the negative Cotton effect at 310 nm (De À 8.62) supported the 2 R,3S configuration ( Figure S4). Furthermore, the unusual structure of 1 was further proved by X-ray crystallography ( Figure S5) by application of the anomalous dispersion of Cu Ka radiation, using copper radiation (CCDC2074704), which indicated a 2 R,3S,5S,8S,9S,10R,12R. Therefore, the structure of 1 was determined to be new ent-atisane diterpenoid featuring a vicinal 2,3-diol moiety and named euphorfinoid A.

Evaluation of cholinesterase inhibitory effects of ent-atisane diterpenoids
All the isolates were screened for inhibitory activities against acetylcholinesterase and butyrylcholinesterase, which are promising therapeutic targets in Alzheimer's disease (AD) (S aez-Valero and Small 2001; Montanari et al. 2016). As shown in Table S8, diterpenoids 4-6 displayed selective inhibition of cholinesterase, and exhibited moderate inhibitory activity against AChE with 50% inhibiting concentration (IC 50 ) values of 32.56-133.92 lM. Meanwhile, none of the assayed compounds inhibited BChE (IC 50 > 200 lM). Among them, 5 showed significant inhibitory effects with the IC 50 values of 32.56 ± 2.74 lM. In order to investigate the mechanism of the inhibitory effects of 5 toward AChE, docking analysis was also performed. As shown in Figure S6, binding site residues were observed for 5. The 3-C ¼ O of 5 interacted with ARG-289 and PHE-288 by H-bond, and the other two hydrogen bond was formed between ARG-289, TYR-70 and the 2-OH, 14-C ¼ O group of 5, respectively. For the active pocket, the docking analysis suggested that 5 could have been docked into the pocket with whole molecular.

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.

X-ray crystallographic analysis of 1
Colorless orthorhombic crystal of 1 were grown by slow evaporation in MeOH/H 2 O (19:1, v/v) solution. A suitable crystal was selected and measured on a Super Nova, Dual, Cu at zero, AtlasS2 diffractometer. The crystal was kept at 149.99(10) K during data collection.
The X-ray crystallographic data of compounds 1 have been deposited at the Cambridge Crystallographic Data Center with deposition numbers CCDC2074704, which can be obtained free of charge from the www.ccdc.cam.ac.uk.

Mo 2 (OAc) 4 induced electronic circular dichroism
According to the published procedure (Politi et al. 2002;G orecki et al. 2007;Liu et al. 2010), about 1:1.2 mixture of diol-to-Mo 2 (OAc) 4 for compound 1 and 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.

Bioassay of diterpenoids about their inhibitory effects on cholinesterase in vitro
The inhibitory effects of isolated compounds 1-6 on cholinesterase in vitro were determined according to the modified Ellman method using commercially available rivastigmine, a known inhibitor of cholinesterase, as the reference standard (Ellman et al. 1961;Çulhao glu et al. 2013;Posri et al. 2019). In individual plate of 96 microplate, 145 ll phosphate buffer saline (PBS) (0.1 M pH ¼ 8.0), 20 ll enzyme solution (AChE or BChE, 0.1 U/ml, pH ¼ 8.0 PBS), 5 ll test sample/reference standard of various concentrations were added and pre-incubated for 20 min at 37 C. Then, 10 ll acetylthiocholine (acetylthiocholine iodide or S-butyrylthiocholine iodide, 0.2 mmol/L) and 10 ll 5,5 0 -Dithiobis-(2-nitrobenzoic acid (DTNB 0.25 mmol/L) were added into the culture and incubated for 30 min at 37 C. After the addition of 10 ll 0.4% sodium dodecyl sulfate to stop the enzymatic reaction, the OD values were recorded at 405 nm using microplate reader. The inhibition rate of AChE was calculated by the formula [(A0 À A1/A0] Â 100, where A0 is the absorbance of the blank, and A1 is the absorbance of the compounds. All experiments were performed in triplicate, and IC 50 values were calculated graphically according to the logarithmic concentration inhibition curves (Graphpad Prism 7.0).

Molecular modeling
The Docking studies were performed for the interaction investigation between ent-2hydroxy-atis-1,16(17)-dien-3,14-dione (5) and AChE. The structure of 5 was drawn using ChemBioDraw. The conformers of 5 were generated using the OPLS2005 force field by Schrodinger. The crystallographic structure of AChE (PDB: 16EE) was downloaded from Protein Data Bank. The crystal structure of AChE was prepared by the Protein Preparation Wizard Workflow in the Schr€ odinger. The water and ligand were removed, and global energy was minimized using OPLS2005 force field. Shcrodinger2018 software was used to study the interactions. Discovery Studio 2016 Client was applied to analyze the docking results.

Conclusions
In summary, euphorfinoids A and B (1 and 2) was a pair of ent-atisane diterpenoid epimers from the roots of E. fischeriana. Notably, euphorfinoids A and B represent the first example of an ent-atisane epimers featuring a vicinal 2,3-diol moiety. All isolates were evaluated for their cytotoxicity against HeLa, Hcc1806, PC3, A549, H460, MCF-7, and LO2 cell lines, and some composition showed moderate anti-proliferative effects. Moreover, compound 5 showed significant inhibitory effects against AChE, and in silico docking analyses had been performed to reveal the interaction between 5 and AChE for developing the new inhibitors of AChE. All in all, AChE inhibitory activity and cytotoxicity of those compounds indicated their potential application in cancer and AChErelated neurodegenerative diseases, it was worthy of further investigation.

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

Funding
This work was supported by the National Natural Science Foundation of China (81903789).