Biological Activity Determination And Quantitative Structure-Activity Relationship Studies Of Novel Quinazoline Compounds

CD⁴⁺Foxp³⁺Tregs induced by immunosuppressive agents, which play animportant role in induction and maintenance of immunological tolerance, willimprove the sensitivity of immune tolerance. It is conducive to long-term survival oforgan grafts and the establishment of transplantation tolerance. The currently usedimmunodepressant are CsA, FK506, MMF, and so on. As the representation of thethird generation immunosuppressive drug, rapamycin avoided the transplant rejectionand inducted the generation of Tregs. Therefore, the development of newimmunosuppressive agents which inducted regulatory T cells is an important directionin drugs discovery.In vivo, the functioning model of compounds is continuous and complicated. Soit is very important to explore immune suppression mode and molecular mechanism.In this paper, we had screened a kind of quinazoline compound which can induce Tlymphocytes differentiation to Tregs. In order to improve their effectiveness,molecular simulation and quantitative structure activity relationship study had beendone. Base on this information, a series of novel quinazoline compounds had beendesigned and synthesized. And this study also discussed the immune mechanism ofthese compounds in vivo and vitro. The above information provided theoreticalsupports for drugs discovery.The main contents of this project as follows，1) The design of novel quinazoline compoundsIn the present work, molecular docking method was used to simulate the bindingmode of drug molecules in JAK3active site. Using three-dimensional quantitativestructure-activity relationships（3D-QSAR） to analysis the interrelationship betweenstructural characteristics and bioactivity. Based on these results, novel quinazolinecompounds were designed. 2) Synthesis and structural identification of novel quinazoline compoundsThis study using2-amino-4,5-dime-thoxy benzonitrile and2-amino-5-R-benzonitrile （R: F, CI, Br, CF₃, CH3, NO₂） as raw materials to synthesistarget products. The analysis of the purity was performance by liquid chromatography（HPLC） and the molecular weight were tested by mass spectrometry （MS）.3) Determination of the biological activity of novel quinazoline compoundsActivity in vitro. Employing a combined detection method of CFSE labelingand Annexin V-FITC/PI staining to explore vitro biological activity for syntheticcompounds.Activity in vivo. To establish a mouse model which were immunized by Listeriamonocytogenes. Seven days later, taking the mouse peripheral blood and separatinglymphocytes to detect the ratio of CD8+IFN-γ+T cells by flow cytometry （FCM）.Through this research, we made the following results,1) Eight quinazoline compounds which purity is100%were design by moleculardocking method combined with3D-QSAR.2) The results of CFSE labeling indicate that the inhibitory activity of6,7-dimethoxy-4-Cyclopentyl aminoquinazoline and6-R-4-Cyclopentylaminoquinazoline （R: Br, CF₃） were stronger. Quinazoline compoundssynthesized in this study can inhibit proliferation of cells effectively with aconcentration-dependent manner.3) Annexin V-FITC/PI staining powerfully testify quinazoline compounds caninduce the apoptosis in a concentration-dependent way.4) Compared with the control group, the mice which were immunized by Listeriamonocytogenes generate a weaker primary CD8+T cell response after takingdrugs.In this study, the results show that a set of novel quinazoline compoundswhich have strong biological activity were designed. It can inhibit proliferation ofcells effectively and induce the apoptosis. After taking drugs, CD8T cell responsewill be significantly reduced. As a immunosuppressive, these compounds havevarious advantages such as high-effect, high specificity and safety. This studyconfirmed the possibility of clinical application for quinazoline compounds, andprovided theoretical support to a deeper level of scientific research.