Design And Synthesis Of Janus Kinase(JAK) Small Molecule Inhibitors Analogues

Janus kinase-signal transduction and activator of transcription(JAK-STAT)signaling pathway mainly composed of four parts:cytokines,transmembrane receptors,JAK and signal transduction activation factors.Many important cytokines including interleukin,interferon,granulocyte/macrophage colony-stimulating factor,erythropoietin,and thrombopoietin transmit signals via the JAK-STAT signaling pathway.JAK is a key component in this pathway.JAK are drug targets for many autoimmune diseases and some blood diseases.In present study,we focuseon discovery of new JAK inhibitors by analyzing of the molecular features of existing JAK inhibitors such as Tofatinib,Ruxolitinib,Barretinib,and Perficitinib,etc.as well their interactions with JAK.We designed and synthesized some new compounds and the JAK inhibition tests showed that some of them have low micromolar ragion inhibition potency.At first,a series of compounds were designed and synthesized by analyzing the crystal structure of the complex of tofacitinib,These compounds include:(R)-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine,(S)-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine,(1r,4r)-N1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclohexane-1 of 1,4-diamine,(1 r,4s)-N 1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)cyclohexane-1,4-diamine and their derivatives.During the synthesis of these compounds,4-chloropyrrole[2,3-d]pyrimidine was selected as the starting material for the reaction,it was protected with p-tolylsulfonyl chloride(TsCl),the results of single-factor optimization experiments show that when the molar ratio of reactants(4-chloropyrrole[2,3-d]pyrimidine:TsCl)is 1:1.4,the temperature is room temperature,the time is 2 h,and alkali sodium hydroxide is added to balance acid-base of the reaction system.The yield of the synthesized product was as high as 93%,after purification,the purity measured by high performance liquid chromatography was above 94%.Then reacted with R-(+)-1-Boc-3－aminopyrrolidine,S-(+)-1-Boc-3-aminopyrrolidine,tert-butyl((lr,4r)-4-aminocyclohexyl)carbamate or tert-butyl((1 s,4s)-4-aminocyclohexyl)carbamate.The single-factor optimization experiment results show that when the molar ratio of the reactant(A-Ts:nucleophile)is 1.1:1,the solvent is DMF,the temperature is 90℃,and the time is 5 h,the yield of(R)-N-(pyrrolidin-3-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine is 93%.After the reaction,the corresponding key skeleton compounds were obtained by removing Boc and Ts protecting groups.Kinase inhibition tests showed that the inhibition concentration of four skeleton compounds are 1404 nM,1454 nM,4182 nM,and 1011 nM,respectively,in the presence of 1 mM ATP in the reaction.Secondly,a new inhibitor N-((3s,5s,7s)-amantadine-l-group)-7H-pyrrole and[2,3-d]pyrimidine-4-amine was designed based on the molecular characteristics of Perficitinib and its cystal structure in complex with JAK.The designed molecule is predicted to bind to JAK by forming hydrogen bonds with the hinge region of the JAK and using the huge volume of the adamantyl group to occupy the space of the active site of the kinase.During synthesis,referring to the synthetic route of nucleophilic substitution reaction,1-adamantanamine and Ts-protected 4-chloropyrrole[2,3-d]pyrimidine compounds undergo a substitution reaction.The results of single-factor optimization experiments showed that the optimal solvent for this reaction was DMF,and the molar ratio of the reactant(A-Ts:1-adamantanamine)was 1.1:1,the designed small molecule structure was successfully synthesized.The purity after purification was 95%,the structure of the designed molecule was verified by 1H-NMR.The next step is to study its JAK inhibiton activity and how it is interacting with JAK.Finally,by summarizing the characteristics of existing JAK inhibitors,it is found that most of them have a common feature,which is that they form hydrogen bonds with the hinge region of JAK,followed by a hydrophobic group in teracts with the hydrophobic cavity of the active site of JAK.According to the above characteristics,a series of small molecular derivatives of pyrrole[2,3-d]pyrimidine were designed in this study.These molecules include N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine,N-benzyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine,N1-(7H-Pyrrolo[2,3-d]pyrimidin-4-yl)benzene-1,4-diamine,N1-(7H-pyrrolo[2,3-d]pyri-midin-4-yl)benzene-1,3-Diamine,N-methyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine,N-ethyl-N-phenyl-7H-pyrrolo[2,3-d]Pyrimidin-4-amine,N-benzyl-N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine and N-benzyl-N-ethyl-7H-pyrrolo[2,3-d]pyrimidin-4-4-amine.Inaddition to their potential activityfor JAK,these derivatives have features such as small molecular weight,easy to be synthesized and modified.These compunds will be used for the discovery of JAK inhibitors via fragments based on drug discovery.Durinthe synthesis,first,react with Ts-protected 4-chloro-pyrrole[2,3-d]pyrimidine by aniline,benzylamine,p-phenylene-diamine and m-phenylenediamine,respectively.Then using iodoethane and iodoethane to modify their structure,the final product yield is as high as 90%.