Design, Synthesis And Biological Evaluation Of Pyrazole Derivatives As Spleen Tyrosine Kinase Inhibitors

The treatment of tumor has been a worldwide difficult issue. It is reported that more and more people are getting cancer. The conventional anticancer drugs have poor selectivity for tumor cells. When they kill or inhibit tumor cells they do some harm to normal cells at the same time. Currently available tyrosine kinase inhibitors,achieving a certain therapeutic effect and reducing adverse side effects, it is an effective way of screening anti-cancer drugs. Because the molecular targeted anti-tumor drugs have high selectivity, less adverse reaction, are becoming a hot research topic in this field.Spleen tyrosine kinase(Syk) is a non-receptor tyrosine kinase. Syk not only involves in many signal transduction pathways but also plays an important role in controlling differentiation, proliferation and spread of cells. Syk is effective in the treatment of autoimmune diseases and inflammation target. The main inhibitors are used in the treatment of allergic inflammation, autoimmune diseases and cancer etc.Although many Syk inhibitors are in the various stages of clinical research, but so far,we haven’t developed the activity well, less side effects, good Syk inhibitorWe chose compound 4 which is developed by Novartis as a lead compound and based on the study of molecular protein crystal structure of Syk, we designed two classes of pyrazole compounds which are pyrazolopyrazin-3-amine and pyrazolopyrimidin-3-amine. We started from 4-chloro-2–(methylthio)pyrimidine-5-carbonitrile and 3,5-dichloropyrazine-2-carbonitrile as starting materials,respectively. Through oxidation, cyclization, coupling and a series of reactions, we synthesized 20 target compounds. In vitro kinase inhibition experiments and in vitro cell proliferation inhibition experiments, we optimized these compounds and explored the antitumor effect of them. Originally designed pyrazolopyrimidine-3-amine compounds, have lower inhibition activity of Syk, thereby we abandoned this type of compounds. One of the most potent compounds 6m suppressed the enzymatic activity of Syk with an IC50 value of 75 nM. The compound also strongly inhibited the growth of Raji, Oci-ly3, PRMI-2286 and BC-1 cells with IC50 values of 2.38, 3.47, 2.48 and4.23 μM, respectively. Compound 6m also suppressed the activation of phosphorylation of Syk in Oci-ly3 cells.The computer molecular modeling shows that, compound 6m bound into the ATP binding site of Syk with a similar “U type” conformation to that of the lead compound 4. The aniline nitrogen formed a hydrogen bond with hinge Glu449 carbonyl oxygen, while the pyrazin nitrogen formed another hydrogen bond with hydroxyl of Ser511. The diaminocyclohexane could form two additional hydrogen bond networks with carboxyl of Asp512,which is similar with compound 4.Structure-activity relationship studies showed that the pyrazolopyrazin-3-amine compounds have better activitives than pyrazolopyrimidine-3-amine derivatives.Compounds 6a,6b,6c,6d,6e,6f,6g,6h,6l were also obviously less potent than compound 4. Encouragingly, 6’-indazole substituted compound 6j showed an improved Syk inhibitory activity. 6’-N-methyl indazole and 5’-indazole analogues also moderately suppressed the kinase activity of Syk.When the 1-positiuon was substituted by a 2-phenyltriazole group, the resulting compound 6m exhibited the best Syk inhibitory potency, which is comparable to the lead molecule 4.Compound 6m has a certain reference value for the development of novel Syk inhibitors.