Design, Synthesis And Evaluation Of CC Chemokine Receptor4Antagonists

The CC chemokine receptor4(CCR4) belongs to the superfamily of Gprotein-coupled receptors, is broadly expressed on the cells of immunesystem,specificallyTreg cells and T helper2cells. It plays a key role inT cells migrateto the thymus andT cell maturation and education. Three natural chemokine ligandsfor CCR4have been identified as thymus and activation-regulated chemokine(TARC), macrophage-derived chemokine (MDC) and chemokine-like factor1(CKLF1). TARC and MDC are highly specific ligands with strong affinities forCCR4.Chemokine ligands binding to CCR4on the cell surface can activate coupledG-protein and a number of intracellular signaling pathways, including activation ofphospholipase C, protein kinase C,proteins Ras and Rho. CCR4plays a biologicaleffect to lead to the target cell migration to a specific location by the series ofinformation transmission. CCR4can regulate T cell migration to the sites ofinflammation in the body, including the skin and lungs.The studies have demonstratedthat CCR4and its ligands are closely related to the development of asthma, allergicrhinitis and atopic dermatitis, systemic lupus erythematosus and rheumatoid arthritisand other diseases. Meanwhile, CCR4is expressed in the tumor microenvironmentand play a role in regulating the tumor growth. CCR4can induce tumor immuneescape and promote metastasis of tumor cells.Therefore, CCR4has become an important potentialtherapeutic target forallergicinflammatory diseases and cancer immunotherapy.The selectively small molecularCCR4antagonists as drug will provide a new option for treatment of these diseases, inparticular treatment of asthma and CCR4-positive cancer so on.To date, only CCR4monoclonal antibody Mogamulizumab has been approved forlisting for the treatment of relapsed or refractory CCR4-positive adult T-cell leukemialymphoma in2012.The majority of small molecule CCR4antagonists are in bioassay or preclinical research stage.With in-depth research,there will be many smallmolecular CCR4antagonists used in clinical and provide new treatment options forpatients with asthma and CCR4-positivecancer in the future.To date, crystal structure of CCR4hasn’t been reported. CCR4does not have highhomology with other GPCRs. Binding sites of CCR4antagonists is not very clear.These disadvantage factors limit the use of computer-aided drug design in designingaimed compounds. Based on hits to leaddesign ideas and me-too strategy, wedesigned two types of novel trisubstituted pyrimidine amine derivatives as CCR4antagonists through summarizing structural features of many high activity pyrimidineamines CCR4antagonists and avoiding the existing compound patents. The publiclyreported CCR4antagonists can be subdivided into eight categories depending on thegeneral structure of the compounds. These categories are trisubstituted thiazolidinonederivatives, trisubstituted lactam derivatives,2-aminothiazole derivatives, aminopyrimidine derivatives, aryl sulfonamide derivatives,3-piperazinyl coumarinderivatives,2-phenyl morpholine derivatives and bipiperidinyl carboxylic acid amidederivatives so on. We take advantage of active fragment splicing and inverse syntheticanalysis to design novel naphthalene sulfonamide thiazolidinedione derivatives asCCR4antagonists on the basis of structural features of2-aminothiazole and arylsulfonamide derivatives. We have synthesized sixty five novel compounds, andoptimized methods of the synthesis of target compounds and key intermediates.Themolecular structures have been confirmed by1H-NMR, ESI-MS and HRMS.The activities of these compounds were evaluated by the chemotaxis inhibitionassay and label-free cellular assay. And the preferred compounds were studiedthrough CCR4receptor selective experiment. The cell cytotoxicity of compounds hasbeen evaluated by MTT experiment. Chemotaxis experiments indicated that most ofthe compounds at1μM showed ability to inhibit. Some of the compounds exhibithigher inhibitory activity than positive compounds.And inhibitory effects of TARCand MDC mediatedcells chemotaxis are the same. We tested IC50values of preferredcompounds. Compounds N-14, N-36and N-39(IC50values of64,77and69nM,respectively) showed similar activity with the positive control compound1(IC50value of78nM). Meanwhile the compounds show high selectivity for CCR4.Thelabel-freecellular assay experimental results show that the compound N-39(IC50value of1.913μM) has considerable inhibitory activity with the compound BMS-397(IC50valueof1.062μM).The MTT experiment results show that the compounds (excludeN-51)are non-cytotoxic at10μM.Compound N-39has shown higher activity, better safetyand receptor selectivity, andbeen worth further study.The preliminary structure-activity relationships of these trisubstituted pyrimidineamide derivatives were discussed based on the obtained experimental data.The resultsfrom the SAR study indicated that for maintaining the activity it was critical that thesubstituents which attached to piperazinyl group located in the2-position of thepyrimidine nucleus contain carbonyl groups. The substituents contain a saturatedcarbonyl group with the-position nitrogen atom, which attached to carbonyl group,and have high activity. The activity of the compounds which contain the substituentsthat don’t contain hetero atom or the aromatic substituents decreased significantly.The substituents which located in the4-position of the pyrimidine nucleus play animportant role in maintaining the activity. A benzyl amide group substituted withhalogen at the2-and4-positions is beneficial for maintaining the activity. The size ofthe spatial structure and the polar of the substituents which located in the6-position ofthe pyrimidine nucleus play an important role in maintaining the activity. Small andless polar substituents, Cl and methyl group, are more potent. Wherein the substituentis methyl, the compounds have stabilized activity of inhibiting TARC and MDCmediated cells chemotaxis.The discussion of SARs should be helpful for furtherdevelopment of the new CCR4antagonists which are more potent, lower toxicity andmore specificity.