Design,Synthesis And Antitumor Activity Study Of Small Molecule Inhibitors Targeting JAK

JAK transduces cytokine and growth factor-mediated signaling pathways and plays important roles in cell growth,differentiation and proliferation.Herein,we used computer-aided drug design,molecular hybridization strategy and the drug Ruxolitinib as the lead compound to design and synthesize novel series of JAK inhibitors.Then,based on the molecular structure of JAK inhibitors,the dual-target inhibitors of JAK and HDAC with potent antitumor activity were designed and synthesized according to the structure-activity relationship of HDAC inhibitor SAHA.1.Research backgroundJAKs are involved in many important biological functions of the human body.Studies have confirmed that the different biological functions of JAK are determined by specific JAK and STAT combinations and their preferential binding to different cytokine receptors.JAK1 pairing with JAK2,JAK3 and TYK2 preferentially bind to the cytokine receptors such as gamma common(yc),type Ⅰ and type Ⅱ interferons,IL-6,and IL-10 family thereby are associated with inflammation,antiviral,myelofibrosis,immunodeficiency,leukemia and adaptive immunity.JAK2 pairing with JAK1,JAK2 and TYK2 preferentially bind to the cytokine receptors such as IL-3,IL-5,granulocyte macrophage colony-stimulating factor,erythropoietin and thrombopoietin thereby are associated with myelopoiesis,erythropoiesis and inflammation.JAK deficiency can cause many diseases.JAK1 mutations are often found in patients with rheumatoid arthritis and autoimmune diseases.Hematological disorders,myeloproliferative disorders are often associated with JAK2 mutations,JAK3 deficiency often leads to severe combined immunodeficiency disease.So far two JAK inhibitors Tofacitinib and Ruxolitinib have been approved by FDA for the treatment of arthritis and myelofibrosis in 2012 and 2011,respectively.In addition,many JAK inhibitors are in the clinical development stage.Therefore,research on JAK inhibitors has become a very promising strategy for anti-inflammatory,anti-cancer and blood diseases treatment.In addition,HDAC inhibitors are widely applied in the field of cancer treatment.HDACs have a wide impact on the development of tumor cells,including angiogenesis,metastasis,division and proliferation,differentiation,autophagy and apoptosis.HDAC inhibition can restore the expression of genes,which can achieve the purpose of cancer treatment.Studies have shown that,combination of JAK inhibitors with HDAC inhibitors can create synergies in the treatment of cancer.Therefore,JAK and HDAC dual target inhibitors have the potential to overcome the drug resistance of tumor resistant strains,and thus become a new treatment for malignant neoplasm such as myeloproliferative disorders and acute lymphoblastic leukemia.2.Design,synthesis and antitumor activity study of JAK inhibitorsA summary of the pharmacophore and structure-activity relationships of existing JAK inhibitors has revealed that almost all potent JAK inhibitors contain structures that match the hinge regions of JAK.Generally,the structure,which contains pyrimidine amine or pyrrolopyrimidine amine,can form strong hydrogen bonds with the hinge region of JAK.In addition to this extremely important binding site,JAK active sites also possess a methylation region,P-loop region and so on.According to the mode of action of JAK and its inhibitor,we designed three series of about 60 novel JAK inhibitors by means of computer aided drug design,combined with the design strategy of isosterism and privileged structures.All the target compounds are chemically synthesized using readily available starting materials,and the final target products are synthesized by a series of reactions such as Wittig-Homer reaction,Michael addition reaction,nucleophilic reaction,Suzuki reaction and reduction reaction.The preliminary biological assay of the target compounds was evaluated,including the determination of JAK inhibition(including JAK1,JAK2,JAK3 and TYK2),antitumor proliferation assay(MTT),and the western-blot experiment.The results showed that most of the compounds possessed significant JAK inhibitory activity,especially compounds Ⅱ-17m,Ⅲ-3f and Ⅲ-11b.In addition,we used western-blot to verify that compound Ⅱ-17m had a strong inhibitory effect on the phosphorylation of JAK2 in HeLa cells at the concentration of 5μM.MTT assay demonstrated that compound Ⅲ-3f exhibited a micromolar level of antiproliferative activity against different cell lines(PC-3,HEL,K562,MCF-7 and MOLT4),and compounds Ⅲ-11b exhibited a sub-micromolar level of antiproliferative activity against HEL and K562.3.Design,synthesis and antitumor activity study of JAK and HDAC dual target inhibitorsBased on the strong antitumor activity of compound Ⅲ-3f,we used this compound as a lead compound and then designed more than 20 JAK and HDAC dual target inhibitors in order to find compounds with better antitumor activity.The preliminary biological assay of the target compounds was evaluated,including the determination of enzyme activity of JAK(including JAK 1,JAK2,JAK3 and TYK2),the determination of HDAC inhibition in vitro,the determination of HDAC subtype selectivity(HDAC2,HDAC6 and HDAC8),antitumor proliferation assay(MTT),flow cytometry,in vivo nude mice anti-tumor proliferative assay.The results showed that the inhibitory activities of these compounds on JAK and HDAC were at nanomolar levels.MTT assay confirmed that the target compounds exhibited sub-micromolar levels of antiproliferative activity against hematopoietic HEL cell,K562 cell,MOLT4 cell and Jurkat cell,especially compounds Ⅳ-10e and Ⅳ-10s.In the flow cytometry experiment,we found that compounds Ⅳ-10e and Ⅳ-10s had a strong pro-apoptotic activity against HEL and K562 leukemia cell lines.In the nude mice animal model,we found that the compound Ⅳ-10e in vivo was effective to inhibit the growth of HEL cells.No significant liver and spleen toxicity was found.The above experiments confirmed that compound Ⅳ-10e was an orally antitumor lead compound.4.Conclusion and perspectiveIn conclusion,in this study,using the design strategy of dual targets,combined with the knowledge of medicinal chemistry,computer aided drug design and chemical biology,four series of more than 80 JAK inhibitors were designed and synthesized.Through the biological assay evaluation,we discovered a potent JAK and HDAC dual target inhibitor Ⅳ-10e,which showed a promising antitumor drug.The emergence of this compound opens up a new direction for the study of multi-target anti-tumor drug design.Next,we will have a further study such as pharmacokinetic properties,acute toxicity,anti-tumor mechanism at the molecular level of this compound in order to comprehensively examine its potential druglikeness.