Design,synthesis,and Structure-activity-relationship Of Small Molecule Chemokine Receptor CXCR4 Antagonist

The chemokine receptor CXCR4 belongs to the 7-transmembrane G-protein coupled receptors(GPCRs)superfamily.It is widely expressed in many types of cells,such as leukocytes,hematopoietic stem cells,tumor cells,endothelial and epithelial tissue cells.The activation of CXCR4 by its endogenous ligand CXCL12 can trigger multiple signaling pathways,including PLC,PI3K/AKT,mTOR,NF-?B,JAK/STAT,Ras/Raf,Erk1/2,that could result in a variety of physiological responses,such as cell proliferation,migration,invasion,adhesion,secretion,and survival.CXCR4 is also involved in many pathological processes such as HIV invasion,mobilization in stem cell transplantation,tumor growth and metastasis,inflammation and autoimmune diseases.Therefore,the development of CXCR4 antagonists is very significant.AMD3100,the only CXCR4 antagonist drug currently on the market,cannot be administered orally,and it is cardiotoxic and cannot be used for a long time.Many clinical or preclinical drugs such as AMD 11070,etc.,are striving to overcome the problems of oral administration and drug safety.However,the problems of strong CYP inhibition,high plasma protein binding,poor metabolic stability and pharmacokinetic properties are still common and have not been completely resolved.Therefore,more efforts are needed to develop new oral CXCR4 antagonists that are potent,safe,and orally bioavailable,in order to meet the urgent need for treatment of chemokine receptor CXCR4-related diseases.This thesis is based on the previous research results of our group(tetrahydroquinoline template CXCR4 antagonist).Through aromaticization and ring-opening transformation at the possible metabolic site,two novel templates(aminoquinoline-based and ethylpyridine-based CXCR4 small molecule antagonists)have been obtained.In the aminoquinoline series,we investigated the structure-activity relationship of multiple substitution sites and screened out compound 3,which has far better activity than the marketed drug AMD3100.It has excellent binding affinity(IC50=57 nM for 12G5 competitive binding assay)and functional activity(IC50=0.24 nM for calcium ion flow assay),and effective inhibition of chemotaxis induced by CXCL12,while shows no toxicity to multiple mouse and human cell lines at 1000 nM concentration.Compound 3 also has excellent receptor selectivity,good physicochemical properties(MW 362,clogP 2.13,PSA 48,pKa 7.0)and low CYP3A4 and 2D6 inhibition(13%and 15%at 10 ?M,respectively).However,it shows strong hERG inhibition(IC50=0.83 ?M,patch clamp).Compound 3 exhibits high permeability(45 × 10-6 cm/s,Caco-2)with low efflux ratio,and moderate plasma protein binding in mouse and rat,and high plasma protein binding in humans(99%).The t1/2 of compound 3 in rat and human liver microsomes were 3.8 minutes and 14 minutes,respectively.The ethylpyridine series was preliminarily derivatized to obtain many compounds with excellent biological activity(most of the IC50 data for calcium ion flow assay were below 1 nM),and all of them could significantly inhibit CXCL12 induced chemotaxis.Compound 58 with the best comprehensive activity had excellent physicochemical properties(MW 397,clogP 0.92,PSA 69,pKa 8.0)and good solubility(3364 ?g/mL).Its metabolic stability in liver microsomes is better than that of tetrahydroquinoline compound 1B and aminoquinoline compound 3.Compound 58 had minimal inhibition to the CYP isoenzymes 1A2,2C9,2C19,2D6,and 3A4,and solved the problem of hERG inhibition(IC50>30 ?M).At the same time,it has high permeability(37×10-6 cm/s,Caco-2),low efflux ratio,and greatly improved plasma protein binding rate(human PPB 80%).The PK study of compound 58 in rat showed that the key parameters such as AUC,Cmax,and t1/2 under intravenous injection were relatively good.Although the oral bioavailability is still low,the oral exposure of compound 58 is in a relatively acceptable level.Moreover,both AUC and Cmax for oral administration showed a good proportional correlation with dose.In summary,the safety issues in CYP and hERG inhibition commonly reported in the literatures and the CXCR4 antagonists developed earlier in our group have been successfully solved.Compound 58 has good physicochemical properties,the improved PPB property and pharmacokinetic properties.It is worthwhile to carry out more research and assessment on safety,drugability and in vivo pharmacodynamics in the future.