Jatrophane Diterpenoids From Euphorbia Peplus With Inhibitory Effects On The ATR-Chk1 Pathway

DNA damage response(DDR)caused by anticancer drugs is one of the important mechanisms for multidrug resistance of tumor cells,which leads to failure of chemotherapy.The ATR-Chk1 pathway is a key component of the DDR,inhibiting the ATR-Chkl pathway is one of the important means to reverse the multidrug resistance of tumor cells.Euphorbia peplus L.,a temperate annual weed,belongs to the Euphorbia genus of the Euphorbiaceae family,which is rich in jatrophane diterpenoids.Previous studies reported that jatrophane diterpenoids can reverse the multidrug resistance of tumor cells.However,there are no reports about the inhibition of the ATR-Chkl pathway by jatrophane diterpenoids.Therefore,the study chose E.peplus as the research object,looking forward to discover jatrophane diterpenoids that can reverse the multidrug resistance in tumor cells by inhibiting the ATR-Chkl pathway.Macroporous resin was used to preliminary separate 95%ethanol extracts of the dried whole plant of Euphorbia peplus,and the jatrophane diterpenoids in the fractions were identified by TLC and HPLC-MS techniques.As a result,the macroporous resin 70%ethanol-water elution and 95%ethanol-water elution fractions were rich in jatrophane diterpenoids.Through various chromatographic techniques like silica gel column chromatography,reverse-phase column chromatography,recrystallization,semi-preparative HPLC chromatography and other separation methods for further separating and purifying,twenty-nine jatrophane diterpenoids were obtained.Various spectroscopic studies including high-resolution mass spectrometry,nuclear magnetic resonance spectroscopy,X-ray single crystal diffraction,CD spectroscopy,and ultraviolet absorption spectroscopy were used for the structural elucidation,of which compounds 1-17 are undescribed compounds.All the isolated jatrophane diterpenoids were tested for their ability to inhibit ATRChkl pathway by western blotting assay.The results showed that compounds 1*,2*,5*,8*,10*,13*,14*,15*,21 suppressed the camptothecin(CPT)-induced phosphorylation of Chk1.Furthermore,drug combination assay on active compounds was conducted,which showed that compound 10*increase the sensitivity of non-small cell lung cancer cell A549 to the cytotoxic reagent CPT.A preliminary structure-activity relationship(SAR)study of the isolates was conducted.It was speculated that the activity of the compounds is mainly related to the substituents at C-2,C-5,C-7,C-8 and C-9,which provided a basis for further structural optimization of jatrophane diterpenoids.This study discovered 17 new jatrophane diterpenoids that enriched the structural diversity of the jatrophane categories.Among them,compound 10*can inhibit the ATR-Chkl pathway and increase the sensitivity of A549 cells to cytotoxic agents,which is expected to develop into a new type multidrug resistance reversal agent against tumor cells.The above research provides a scientific basis for the further development and utilization of Euphorbia peplus.