The Novel Method For Screening Inhibitors Of Protein Kinase And Palladium-catalyzed Suzuki Cross-coupling Of Arylhydrazines

The high-throughput screening of bioactive compounds is the key technique for the discovery of lead compound in medicinal chemistry. Protein kinases, G protein-couple receptors and Ion channels, are the three targets of drug design. Protein kinase inhibitors become focus of research owe to the potential in anti-cancer and anti-tumor. Recently, the methodologies for screening of protein kinase inhibitors including Stable-isotope Method, Enzyme-Linked ImmunoSorbent Assay, Capillary Electrophoresis and Fluorescence-peptide Method have its disadvantages and limitations. This paper intends to develop a novel method was applied for screening inhibitors of protein kinase based on γ-[18O4]-ATP and MALDI-TOF-MS, which offers many advantages, such as high sensitivity, inexpensive cost, high-throughput etc.Compared to the naturally existing γ-P-16O4-ATP (light-ATP), the foμar16O atoms attached to the γ-Phosphate are replaced by the stable isotope18O atoms. The biological equivalence of heavy-ATP to light-ATP was validated by using GST-ATF2, a substrate of protein kinase p38α, which can be randomly phosphorylated. Further experiments confirmed that heavy-ATP labeled GST-ATF2can also be recognized by the phosphor-antibody without bias. Two parallel protein kinase assays were carried out with heavy-ATP in H216O and H218O, respectively. No obivious difference was observed in comparison, which confirmed stability of heavy-ATP and the resulting phosphor-peptide and laid a robust foundation for the later quantitative measurements. Parallel kinase reactions were performed using light ATP or heavy ATP, in the absence or presence of an inhibitor, respectively. After the reactions, equal amounts of the reaction solution were mixed and measured directly by MS. Different resulting phosphor-products generated by corresponding kinases were separated based on their m/z. The effectiveness of a putative inhibitor could be readily determined by the suppression of the signal of the heavy phosphor-peptide peak and IC50determined by plotting inhibition curves based on the analysis of reactions using a series of concentrations of inhibitors.Biaryls is most important intermediates in organic synthesis, applied widely in medicine, dyestuff, organic materials. It can be synthesized by many methods involving electrochemical synthesis, intramolecular coupling and organometallic coupling. Among these methodologies, synthesis of biaryls via pallidium-catalyzed cross-coupling reaction occupied an extremely important position, such as Stille cross-coupling reaction, Hiyama cross-coupling reaction, Negishi cross-coupling reaction, Suzuki cross-coupling reaction etc. Herein, a novel and efficient protocol for the synthesis of biaryls has been developed, which is the first Pd-catalyzed Suzuki cross-coupling of readily available arylhydrazines with arylboronic acids. This method employed Pd(OAc)2as catalyst under mild condition and obtained good to excellent yield.23kinds of compounds was synthesized, and all of them has been confirmed by1H NMR,13C NMR, GC-MS and IR spectra.