Synthesis And Antitumor Mechanism Of Biotin-substituted B-norcholesteryl Benzimidazole Derivatives

With the improvement of medical standards,more and more diseases can be effectively treated,but diseases such as cancer still have been plaguing us for a long time.Therefore,effective therapeutic drugs for cancer have long been a research hotspot in the field of pharmaceutical chemistry.Steroids,as one of the great potential antineoplastic drugs,have attracted much attention from pharmacists and organic chemists.The Cui team has been working on the synthesis of steroids for many years and has discovered several steroidal compounds that have good inhibitory activity against cancer cells in vitro.Among them,the B-norcholesteryl benzimidazole attracted our attention as their high inhibition of cancer cells while it showed low toxicity to HEK293T（human renal epithelial）cells.However,the target and mechanism of action of B-cholesterium benzimidazoles are still unclear.A series of B-cholesterium benzimidazole compounds with biotin probe structure were designed and synthesized in order to further explore the mechanism and target of this kind compounds.Based on the biological method,several suitable chemical structures were chosen and the anti-tumor mechanism of action and the pathway was validated by Western Blot were investigated as follow:（1）Three different 3′-oxyalcohol-5β′-hydroxyl-6′-B-norcholesteryl compounds were synthesized through changed structure of 3-hydroxyl cholesterol and broken the double bond of B ring and intramolecular condensation by Al₂O₃,three different 3′-side chain structure of 3′-oxyalcohol-5β′-hydroxyl-6′-B-norcholesteryl compounds were synthesis.The anti-tumor activity of all these compounds were tested in vitro,and the structure-activity relationship of the extended carbon chains was compared to screen out the appropriate carbon chain structure.（2）3′-oxyalcohol-5β′-hydroxyl-6′-B-norcholesteryl structure was chosen as raw materials,a series of benzimidazole structures were introduced into the 6-group,and the biotin probe was connected to the hydroxyl group of 3-oxy ethanol by esterification reaction.Four cholesteric heterocyclic compounds with biotin probes were prepared by the above reaction.（3）The antiproliferativity of 4 B-norcholesteryl benzimidazole biotin probe compounds were evaluated by MTT assays.the inhibitory to on the tumor cells including HeLa（human cervical cancer cells）,SK-OV-3（human ovarian cancer）cells,T47D（thymus gland cancer）,MCF-7（human breast cancer）cells and HEK293T（normal renal epithelial）cells,selective inhibition of SK-OV-3 is the best of these compounds,at the same time,compound 7d almost have no toxic effect on normal cells.（4）The apoptosis of compound 7d was detected by Annexin-FITC/PI and cell cycle methods on Flow Cytometry.after treatment on SK-OV-3 cells with compound 7d for 48 hours,those cells were used to explore Annexin-FITC/PI.It was found that SK-OV-3 cell proliferation inhibition was apoptotic.Then,cells were labeled by PI single staining method after the SK-OV-3 was treatment on compound 7d for 48 hours,and the proliferation of SK-OV-3 cells with compound7d was found to be blocked in the S phase.（5）According to western blot analysis,compound 7d down-regulated the relative level of Bcl-2 protein in mitochondria;Bax protein was up-regulated;caspase-9 apoptotic promoter was up-regulated,and its downstream caspase-3 apoptotic executor was up-regulated;inducing the intrinsic apoptotic pathway of SK-OV-3 cells.（6）Using UHPLC-QE-MS non-target metabolomics method,to analyze the effect of compound 7d on the metabolites of SK-OV-3 cells,it is most likely to affect the metabolism pathways:compound 7d in SK-OV-3 cells,inhibit the biosynthesis of acyl tRNA which was plays an important role in protein synthesis.In this paper,a total of B-norcholesteryl benzimidazole with 3-biotin were synthesized.All these compounds were characterized by IR,¹H NMR,¹³C NMR and HREIMS.compound 7d was the most suitable structure for further study by MTT assay.It was explored and proved that the mechanism of inhibiting cancer cells was apoptosis and blocked the proliferation of cancer cells in the S phase.By using the method of proteomics,the pathway of the compound 7d acting on the protein was verified,which provided a theoretical basis for the further exploration of the target.