Design, Synthesis And Anti-tumor Activity Characterization Of Ivy Sapogenin-pyrazine Derivatives

Research Background:Hederagenin belongs to oleanane-type pentacyclic triterpenoid,which widely distributed in medicinal plants,such as Clematis,Honeysuckle,Pulsatilla,Dipsacus,Predictive,etc.Research revealed that hederagenin showed many biological activities,such as anti-tumor,anti-bacterial,anti-inflammatory,anti-depressant,anti-diabetes and liver protection,etc.But some problems limited the further development and application of hederagenin,include weak biological activities,expensive price,poor solubility and bioavailability,etc.In order to improve the solubility of triterpenoids and increase the antitumor activity,We introduced ligustrazine(2,3,5,6-tetramethylpyrazine)from chuanxiong into oleanolic acid and glycyrrhetinic acid based on "Compatibility and Combination"principle,and our previous work obtained some candidates with significant activity,low toxicity and remarkable selectivity.Therefore,in order to improve the antitumor activity of the hederagenin and find a novel hederagenin derivative with better antitumor activity,and poor side effects,we introduce ligustrazine and similar pyrazine fragments into hederagenin by chemical means.Research content:In this study,Ivy saponin was cleaved into hederagenin by acid,and the extraction method was determined by Response Surface Methodology.The pyrazine molecule is introduced into the carboxyl or hydroxyl group of the hederagenin by esterification reaction,oxidation reaction,free radical substitution reaction,etc.We evaluated the antitumor effect of derivatives by HepG2,A549 and MCF-7 cell lines,evaluated the cytotoxicity by H9c2 and MDCK.And we analyzed structure-activity relationship objectively by combining PLS-DA and IC50.Based on the above,H9was obtained.Furthermore,Giemsa and DAPI staining were used to observe the effect of H 9 on the A549 cells morphology.Flow cytometry and molecular docking techniques were used to investigate the antitumor mechanism of H9.Research result:We obtained hederagenin weighed more than 10 g and 26 novel hederagenin derivatives successfully,all of them were confirmed by 1H-NMR,13C-NMR and HRMS.H9was found to be the most likely drug candidate,showing ICs50value was3.45±0.59pM,while cisplatin was3.85±0.63 ?M.H9 exhibited excellent anti-tumor activity,but the kidney and myocardial toxicity was lower than cisplatin,showing the safe and low toxicity advantage of traditional Chinese medicine.As shown by Giemsa,DAPI,and Annexin V-FITC/PI staining,it was found that H9mainly acted by inducing early apoptosis.Moreover,the present research found that H9could induce cell-cycle arrest at the S phase.It was found that the proto-oncogene serine/threonine protein kinase PIM-1 may be a target of anti-tumor effect of hederagenin derivatives on the Pharm Mapper.We propose that the derivative may be a conjecture to promote the early cell apoptosis,and inhibit tumor mitosis by inhibiting the activity and expression of the PIM-1.Research conclusion:In this experiment,26 novel hederagenin derivatives were designed and synthesized,and the antitumor activity of the derivatives was evaluated in vitro.Studies have shown that the introduction of pyrazine structure can significantly enhance the anti-tumor activity of hederagenin.The structure-activity relationship showed that the derivatives with short carbon chain linked pyrazine structure at C-28 were better on A549 and MCF-7 cells lines,among them,derivative linked a chain with two carbon showed excellent performance.For HepG2 cells,derivatives linked a chain with multiple carbons performed well,which showed that four-carbons>two-carbons>one carbon.The difference in the modification sites of C28 carboxyl group and C23 hydroxyl group has different effect on the improvement of tumor activity.For MCF-7 and HepG2 cells,the introduction of pyrazine structure at C23 position may be more beneficial to enhance activity than C28 position.There is no obvious law between the number and site of methyl groups on the pyrazine ring and antitumor activity.We obtained the excellent compound H 9 by evaluating antitumor activity in vitro.Based on target data analysis,molecular docking analysis by the Pharm Mapper bioinformatics platform and molecular biological staining analysis,we propose that hederagenin derivatives may exert anti-tumor effects by inhibiting the activity and expression of PIM-1,enhancing the membrane permeability,promoting calcium influx and decreasing mitochondrial membrane potential to induce A549 tumors apoptosis,while blocking the mitosis of tumor cells in the S phase.