Design,Synthesis,Antitumor Activity And Mechanism Of 2’-Deoxy-Rh₂

Ginseng is a traditional medicine in China,which can be used in the prevention and treatment of cancer in modern medicine.Ginsenoside is one of the main active components of ginseng,which has good antitumor activity.Protopanaxadiol can be divided into two configurations:20（S）-protopanaxadiol and 20（R）-protopanaxadiol,of which 20（S）-protopanaxadiol has stronger biological activity.In addition,20（S）-protopanaxadiol has relatively stable skeleton structure and C-3 hydroxyl and other reactive active sites,it is suitable for structural modification to further improve the anti-tumor activity.There are great differences in glucose metabolism between tumor cells and normal cells,so cancer can be treated by affecting cell metabolism.Cellular metabolism involves a variety of pathways,among which tumor cell metabolism is more dependent on the glycolytic pathway,the Warburg effect,even under the condition of sufficient oxygen,tumor cells will choose glycolysis pathway to convert glucose to lactic acid.Therefore,interfering with glycolysis pathway is a potential effective strategy to induce tumor cell death.2-deoxyglucose,a glycolytic inhibitor,is a synthetic glucose analogue that targets glucose metabolism and consumes energy of tumor cells.In addition,2-deoxyglucose can increase oxidative stress,induce autophagy,inhibit cell growth,and induce apoptosis of specific tumor cells.Although 2-deoxyglucose itself has limited therapeutic effect on a variety of tumor cells,it can be combined with other therapeutic drugs or radiotherapy to play a synergistic anti-cancer effect.20（S）-protopanaxadiol can inhibit the growth,proliferation,metastasis and apoptosis of tumor cells,and has inhibitory effects on a variety of tumor cells.2-deoxyglucose can play an anti-tumor role by affecting the metabolic pathway of tumor.Therefore,two compounds with antitumor activity,20（S）-protopanaxadiol and 2-deoxyglucose,were selected in this paper.The two compounds were combined into one molecule using the principle of combination to form a new hybrid molecule,in order to obtain compounds that can complement each other,synergize or reduce their toxic and side effects.The structure of ginsenoside Rh₂（Rh₂）contains 20（S）-protopanaxadiol and glucose,which is similar to the new compound 2’-Deoxy-Rh₂.Therefore,Rh₂ was used as a positive control drug to further explore the effect of 2’-Deoxy-Rh₂ on the apoptotic pathway and glycolytic pathway of tumor cells.The main innovative results of this paper are as follows:（1）Synthesis and process optimization of 2’-Deoxy-Rh₂2’-Deoxy-Rh₂ was synthesized by direct glycosidation.Two routes were designed.The first method is the direct glycosidation of glycodilution donor,in which 20（S）-propanaxadiol was used as starting material to react with pivcl,and the 12 hydroxyl group of propanaxadiol was protected to obtain single substituted propanaxadiol.3,4,6-Tri-O-acetyl-D-glucal was chosen as the glycosyl donor in a direct glycosidation reaction with the already synthesized monosubstituted protopanaxadiol to obtain the product.The second route is the direct glucosylation of trichloroacetylimidyl ester.After the catalytic hydration of triacetoglucoene,trichloroacetimide ester was introduced and used as a sugar donor to glycoside with monosubstituted protopanaxadiol to obtain the product.The structure of the product was identified by LC-MS,¹H NMR,¹³C NMR,HSQC,HMBC and H-H COSY spectra,and it was confirmed that the product was 2’-Deoxy-Rh₂.Because the synthesis conditions of the first route are more stringent,this paper focuses on the process investigation of the direct glycosidation of trichloroacetylimidyl ester.The optimization experiment was designed by single factor experiment and response surface analysis method,mainly for the reaction temperature,reaction feed ratio,catalyst dosage.After optimization,the optimal reaction process parameters were determined as follows:reaction temperature35℃,feed ratio 1:5,molar ratio of single substituted protoginseng diol to catalyst 1:0.2,and the total yield of 2’-Deoxy-Rh₂ was 84.28%.（2）Effects of 2’-Deoxy-Rh₂ on apoptosis and glycolysis of breast cancer cellsIn order to explore the anti-tumor mechanism of 2’-Deoxy-Rh₂,the effect of 2’-Deoxy-Rh₂ on the activity of various tumor cells was investigated by MTT assay.The effect of 2’-Deoxy-Rh₂ on cell membrane integrity was investigated by lactate dehydrogenase;.Apoptosis related proteins were detected by Western blot assay.Intracellular ROS levels and mitochondrial membrane potential changes were detected by DCFH-DA staining and JC-1 staining.Glucose uptake assay and XF metabolic flux analyzer were used to determine the changes of OCR and ECAR to investigate the effects of 2’-Deoxy-Rh₂ on cell glycolysis and mitochondrial function.MTT assay showed that 2’-Deoxy-Rh₂ had a more significant inhibitory effect on breast cancer MCF-7 cells in a concentration and time-dependent manner.Normal cells were less sensitive to 2’-Deoxy-Rh₂.Lactate dehydrogenase detection showed that the leakage rate of lactate dehydrogenase in 2’-Deoxy-Rh₂ group was high,indicating that the cell membrane was damaged.Western blot results showed that 2’-Deoxy-Rh₂promoted the expression of Pro-apoptotic proteins Bax and cleaved-caspase-9,and inhibited the expression of anti-apoptotic protein Bcl-2.The results of DCFH-DA staining and JC-1 staining showed that the intracellular ROS level increased and the mitochondrial membrane potential decreased in 2’-Deoxy-Rh₂ group,indicating that 2’-Deoxy-Rh₂ could induce intracellular ROS accumulation and change the mitochondrial membrane potential to induce cell apoptosis.The results of glucose uptake test showed that 2’-Deoxy-Rh₂ could inhibit the glucose uptake of MCF-7 cells.The results of OCR and ECAR measured by Seahorse XF metabolic flux analyzer showed that 2’-Deoxy-Rh₂ could inhibit the basic oxidative respiration,oxidative respiratory reserve and ATP production of MCF-7.In conclusion,2’-Deoxy-Rh₂ could inhibit the aerobic glycolysis of MCF-7 cells and then inhibit the growth of MCF-7 cells.（3）Anti breast cancer activity of 2’-Deoxy-Rh₂ in vivoIn order to explore the anti-tumor activity of 2’-Deoxy-Rh₂ in vivo,the changes of tumor volume and weight of mice were observed by constructing a mouse transplanted mouse breast cancer cell model.HE staining and TUNEL staining were used to investigate the effect of 2’-Deoxy-Rh₂ on tumor cell viability in vivo.The results showed that 2’-Deoxy-Rh₂ could inhibit the growth of mouse breast cancer 4T1 xenografts in vivo,and the inhibitory activity was stronger than Rh₂.2’-Deoxy-Rh₂ can induce apoptosis of 4T1 cells in mice.Compared with Rh₂,2’-Deoxy-Rh₂ has stronger antitumor effect.（4）Anti-osteosarcoma cell activity of 2’-Deoxy-Rh₂Breast cancer is prone to bone metastasis,and osteosarcoma is easily induced by radiotherapy.Therefore,we further investigated the effect of 2’-Deoxy-Rh₂ on osteosarcoma.In order to explore the mechanism of Rh₂,2’-Deoxy-Rh₂ on the activity of osteosarcoma cells and its mechanism.MTT assay and wound healing test were used to investigate the effect on the activity of osteosarcoma cells.TUNEL staining,JC-1staining,Annexin V/PI staining,and Western blot were used to explore the effect on osteosarcoma cell apoptosis.Western blot was used to further explore the relationship between apoptosis of Osteosarcoma cells and the expression of MAPK,PI3K/Akt/m TOR,NF‐κB signaling pathways.MTT assay showed that 2’-Deoxy-Rh₂ could inhibit the proliferation of U2OS cells in a concentration and time-dependent manner.Wound healing test showed that2’-Deoxy-Rh₂ could inhibit the migration of U2OS cells according to the results of relative wound density.Compared with the positive control group Rh₂,2’-Deoxy-Rh₂group had stronger ability to inhibit cell migration.TUNEL staining,JC-1 staining and Annexin V-FITC/PI staining showed that 2’-Deoxy-Rh₂ could induce early apoptosis of U2OS cells.Western blot analysis showed that the expression of pro-apoptotic protein Bax was significantly increased,while the expression of anti apoptotic protein Bcl-2was significantly decreased.The expression of caspase-3 and caspase-9 decreased.2’-Deoxy-Rh₂ could induce U2OS cell apoptosis by regulating apoptosis related proteins.2’-Deoxy-Rh₂ can promote the expression of MAPK signaling pathway and inhibit PI3K/Akt/m TOR and NF‐κB signaling pathway induces apoptosis in U2OS cells.In summary,2’-Deoxy-Rh₂ was designed and synthesized for the first time,and the synthesis process of the new compound 2’-Deoxy-Rh₂ was optimized by single factor and response surface methodology.The anti-breast cancer and osteosarcoma effects of 2’-Deoxy-Rh₂ and its mechanism were described for the first time.In this paper,a new compound 2’-Deoxy-Rh₂ with antitumor activity was synthesized.It can be used as a new drug candidate compound and has great development and application value.It provides experimental and theoretical basis for the research of innovative drugs.