Design,Synthesis And Immunosuppressive Activity Determination Of Dicloxacillinum Amide Derivatives

In recent years,with organ transplantation and self immune disease increased demand for immunosuppressant,a highly efficient,low toxicity and easy production of drug is needed.Immune inhibitors have been used in clinical for many years.Immunosuppressants of clinical use were all have different degrees of side effects,because effective therapeutic dose is very close to dose of side effects.Rapamycin was the most effective immune inhibitor.As the earliest discovered mTOR inhibitor,it has wide application range,small side effects.The mTOR signaling pathway has close relationship with immunosuppression.It works mainly by reducing cells factor receptor mRNA' synthesis of T cells to block the signal transduction,such as interleukin IL-1,IL-2,IL-3,IL-4,IL-6,IGF,PDGF and CSF.Then to block the T cells proliferation from G1 phase to S phase.But rapamycin has some disadvantages such as high cost of synthesis,low bioavailability(<15%)and poor water soluble.This paper studied small molecules belong to mTOR inhibitors which can overcome above disadvantages.In the early study,we used virtual screening of computer-aided drug design(CADD)to get the leading compound.According to the docking model,a series of compounds have been derived.Then inverse synthetic method was used to synthesis all dicloxacillinum amide derivatives.Through biological activity in vivo and in vitro experiments,it proved the rationality of the design of drugs.All of the above research laid a solid foundation for the development of new clinical immunosuppressant with high specificity,high efficiency,and low toxicity.The the research' main contents are as follows:1)Gain of leading compoundIn this study,nearly two million small compounds were downloaded from Zinc andPubMeddatabases.3-(2,6-dichlorophenyl-5-methyl)-N-6-methylsulfonyl-benzo[d]t hiazol-2-yl-isoxazole-4-carboxamide was selected as the lead compound used by Sybyl 2.0X.2)Designment of Dicloxacillinum Amide Derivatives We adopted surflex-docking method to simulate the binding mode between leading compound and mTOR acceptor.Through the analysis of potential relationship between chemical structure and biological activity,several novel dicloxacillinum amide derivatives were designed.3)Synthesis and purification of Dicloxacillinum Amide Derivatives Retrosynthesis Analysis was used to choose the best synthetic route.Then a series of derivatives of leading compound were synthesized.All the chemical structures were verified through LC-MS and NMR technology.4)Biological activity test of dicloxacillinum amide derivatives Vitro activity:The compounds were used by CCK8 technology to test the biological activity of Jurkat cell.A compound had highest immunosuppressive activity was selected and used in vivo.Vivo activity:We established mouse model which was immunized by Listeria monocytogenes.Then we conducted lavage and intraperitoneal injection of mice respectively with the above compounds.Seven days later,taking the mouse peripheral blood and separating lymphocytes to detect the ratio of CD8-IFN-?+ T cells by flow cytometry(FCM).5)Predict side effects of dicloxacillinum amide derivatives based on reverse dockingUsing the free online web server Pharm Mapper to let ligand compounds interact with more than 7000 kinds of protein receptors.Finally,we fished out the receptors that can predict side effects of target molecules.We got results through the research above as follows:1)Through virtual screening we receive 3-(2,6-dichlorophenyl)-5-methyl-N-(6-(methylsulfonyl)benzo[d]thiazol-2-yl)isoxazole-4-carboxamide as lead compounds.Reverse total synthesis method as the raw material via six steps reaction,nine compounds are obtained.Mass spectrometry data with high performance liquid chromatography(HPLC)data found that more than 99%,and nuclear magnetic resonance hydrogen spectrum/carbon data are consistent with the target compounds.2)From CCK-8 test we got a set of valid and reliable data which confirmed the inhibition of dicloxacillinum amide derivatives towards Jurkat T cells.It proved that this kind of compound had immunosuppressive activity in vitro.3)Compared with the control group,the mice which were immunized by Listeria monocytogenes generated a weaker primary CD8+ T cell response after taking drugs.4)Through reverse docking we got five grade higher receptor proteins.Thus inferred dicloxacillinum amide derivatives may cause neutral leukopenia,neurological disorders,skeletal dysplasia and decreased vision.