Design, Synthesis And Biological Activity Evaluation Of DPP-Ⅳ Inhibitors Based On Piperazine Structur

Diabetes has become a health problem that seriously affects people’s quality of life.High blood glucose levels can lead to many complications and even death.Endogenously secreted enteroglucagon(e.g.Glucagon-Like Peptide-1,GLP-1)in the human body has a crucial role in maintaining glucose homeostasis.As a specific hydrolase of GLP-1,Dipeptidyl Peptidase-Ⅳ(DPP-Ⅳ)can degrade GLP-1.DPP-Ⅳ inhibitors are considered to be promising drugs for the treatment of Type 2 diabetes as a valuable oral hypoglycemic agent that can lower blood glucose by increasing the circulating concentration of GLP-1 in the body.In addition,the widely expressed DPP-Ⅳ can regulate a variety of biological peptides in vivo and has been shown to have many biological functions beyond glucose metabolism.Therefore,the design and synthesis of DPP-Ⅳ-based inhibitors have been a hot topic in the research of glucose-lowering drugs.In this thesis,we analyzed the structural features of DPP-Ⅳ active pockets and summarized the characteristics of the marketed DPP-Ⅳ inhibitors,and designed and synthesized a new parent nucleus structure compound containing a piperazine structure amide bond coupled to 3,3-difluoropyrrolidine by replacing the bioelectronic equivalent of 2-cyanopyrrolidine with 3,3-difluoropyrrolidine,using the molecular collocation principle(1-((±)-piperazine-2-carbonyl)-3,3-difluoropyrrolidine,5).The compound was synthesized in five steps by starting with ethylenediamine molecule,ring formation with ethyl 2,3-dibromopropionate under the protection of Tos,removal of Tos protecting group by strong acid at high temperature and pressure,then coupling with 3,3-difluoropyrrolidine under the protection of tert-butoxycarbonyl(Boc)to form peptide bond,and finally removal of Boc protecting group.The target compound 5 was obtained in 22.6% overall yield.The substituent modification and modification were carried out at the site(position 5of the piperazine ring)in the parent structure(5)that potentially enhances the inhibitory activity.The Boc-protected L-amino acids with L/D-serine methyl ester-hydrochloride were used as starting materials,coupled by EDCI condensation agent,catalyzed by trifluoroacetic acid to remove tert-butoxycarbonyl,then ring closure in the presence of base,then reduction,and then a total of 6 reactions of up-protection and oxidation to obtain 5-substituted-piperazine-2-carboxylic acid,and finally coupled with 3,3-difluoropyrrolidine to form peptide bonds and removal of Boc-protected groups to obtain8 new compounds(11a-h)in 3%-18% overall yield.All target compounds were characterized by NMR hydrogen and carbon spectra to determine the structures.The effect of reaction conditions on the synthesis and chiral conformation of the key intermediate piperazinedione(8a-h)was examined.The intermediate compound dipeptide-trifluoroacetate(7a-h)was found to react at room temperature with a water:methanol volume ratio of 4:1 and Na OH as the base p H = 10.0 to close the ring as the optimal reaction conditions,and the process required only an appropriate amount of acetonitrile for slurry treatment without column chromatography.The synthesis of compound 8a was carried out on a gram scale under the optimal reaction conditions,and the target product was obtained in 62% yield.This route is a better method for the synthesis of cyclic dipeptides and provides a synthetic route reference for the synthesis of this class of compounds.The in vitro biological activity of the target compounds was determined by using DPP-Ⅳ inhibitor fast screening kit,and the experimental results showed that the target compounds had no inhibitory activity.The analysis by molecular docking technique revealed that 3,3-difluoropyrrolidine was not tightly bound to the S1 pocket due to its inability to form covalent bonds with SER630,which affected the development of DPPⅣ inhibitors containing 3,3-difluoropyrrolidine structure.The piperazinecarboxylic acid fraction has a volume and charge rejection with the S2 pocket of DPP-Ⅳ,and also has low similarity to the S2 pocket filling fragment of the template compound,gostelatin,which requires special attention for future development.Later on,toxicity experiments were performed on compound 5 in GLP-1 highly expressed human embryonic kidney cells,and the cytotoxicity of compound(5)at a concentration of 10 μM was found to be almost indistinguishable from that of the 13 n M positive control drug selegiline by fluorescent staining for difference.A reverse virtual screening of nine new compounds in the pharmacophore database e Pharma Lib using a computer-aided drug design platform revealed that compounds 11 c and 11 h may have good inhibitory effects on CDK2,an important target of the cell cycle,and were subsequently validated by MTT colorimetric assay in hepatocellular carcinoma JHH-1 cells with high CDK2 expression,using 5-fluorouracil as positive control drug.The half-inhibitory concentration of compound 11 c on hepatocellular carcinoma JHH-1cells was calculated by the formula to be 78 n M,and the number of cancer cells in culture after administration at half-inhibitory concentration was observed significantly under the microscope,indicating good anti-cancer potential.In conclusion,compound 11 c has good potential for use in patients with hepatocellular carcinoma with concomitant hypoglycemia.