Studies On Synthesis,Activity Evaluation And Molecular Dynamics Of Novel Tyrosine Phosphatase SHP2 Inhibitors

ObjectiveThe protein tyrosine phosphatase SHP2 has been recognized as a new target for anti-cancer drug.In order to search for more potent SHP2 inhibitor,two series of new compounds including 4-（2-substituted hydrazinyl）benzenesulfonic acid derivatives and 3,6-disubstitued pyrazin-2-amine derivatives were designed and synthesized;The inhibitory effects of benzenesulfonic acid derivatives on SHP2 protein were tested;The anti-proliferation activities of all synthesized compounds against MDA-MB-231 and NCI-H1975 cancer cell lines were evaluated,among which I_f and II_e were chosen to be analyzed by molecular dynamics simulations to explore their interaction mode with SHP2 and inhibitory selectivity over SHP2.Method1.Design and synthesis of target compounds:（1）12 analogues of GS493 were designed by keeping unchanged the phenylhydrazone pyrazolone and sulfonic acid groups while replacing nitro group on benzene ring at 1-position of GS493 with lactone or amide groups and the one at 3-position of GS493 with F or CH₃O groups.The preparing method:4-nitrobenzoic acid was transformed to 4-nitrobenzoyl chloride which reacted with various amines to afford amides.The amides was reduced,diazotized and reduced to produce the intermediate N-substitued-4-diazanyl benzamide;The 4-aminobenzenesulfonic acid was diazotized and then reacted with substituted ethyl benzoylacetate to yield another intermediate 4-{2-[1-ethoxy-3-（4-substituted）-1,3-dioxopropane-2-yl]hydrazinyl}benzenesulfonic acid which then reacted with N-substitued-4-diazanyl benzamide to get target compounds I_a-I_l;（2）12target compounds II_a-II_l were designed by keeping unchanged the pyrazin-2-amine moiety while introducing aromatic group or aromatic heterocyclic groups to 3-position and substituted piperazine groups with great steric effects to 6-position.The preparing method:3-bromo-6-chlorpyrazin-2-amine was coupled with substituted boric acids by the Suzuki reaction to afford the intermediate 3-substituted-6-chlorpyrazin-2-amine which then reacted with substituted piperazines to produce target compounds II_a-II_l.2.Biological activities evaluations of the target compounds:（1）The inhibitory activity of target compounds I_a-I_l against SHP2 protein was tested with SHP2 kit.（2）The proliferation inhibition activity of all target compounds on MDA-MB-231 and NCI-H1975 cell lines was evaluated by MTT assay.（3）After the optimized compound II_e acted on NCI-H1975 cancer cells for 48 h,flow cytometry instrument was used to detect cell apoptosis.3.Molecular docking and dynamic simulation:（1）Using molecular docking and dynamic simulation to explore the mode and strength of target compounds binding with proteins.（2）Molecular docking of all target compounds with SHP2 and its homogenous proteins PTP1B,TCPTP,SHP1 were performed by Autodock Vina software to evaluate their inhibitory selectivity against SHP2.Result1.In this study both 12 benzenesulfonic acid derivatives and 12 pyrazin-2-amino derivatives were designed and synthesized.The structures of all newly synthesized compounds were confirmed by ¹H-NMR,¹³C-NMR,IR and MS.2.The results of SHP2 inhibitory assay indicated:effect of I_f(IC₅₀=0.23±0.07μM)was more potent than PHPS1(IC₅₀=2.73±0.43μM),but weaken than GS493(IC₅₀=0.18±0.02μM).The results of antiprolifration activity evaluations against cancer cell lines by MTT assay indicated:compound I_f（IC50=3.38±1.85μM）was more potent than GS493(IC₅₀=20.92±1.23μM)in NCI-H1975 cells.Compounds II_e and II_i could obviously inhibit proliferation of cancer cells with IC50 values of 11.84±0.83μM,10.30±0.69μM respectively which were potent than GS493(IC₅₀=19.08±1.01μM).Compound II_e could induce apoptosis which was detected by flow cytometry instrument.In cell line MDA-MB-231,the prolifration inhibitory effect of compound I_f（28.94±6.23μM）was more potent than PHPS1（IC50=30.02±6.59μM）but weaken than GS493(IC₅₀=26.31±1.59μM).Compounds II_e、II_i、II_k all were potent than GS493（IC50=25.02±1.47μM）,among which II_e was the most potent one with IC₅₀ value of 5.66±2.39μM.3.The results of molecular docking and dynamic simulation indicated that I_f could form hydrogen bonds with amino acid in the active center of PTP of SHP2,while II_e acts on the inactive center of SHP2.It may be associated with allosteric sites.The naphthyl and dichlorophenyl group of II_e were able to form stable binding with SHP2and had higher affinity.II_e shown inhibitory selectivity to some degree over SHP2through their docking with the other homogenous proteins PTP1B,TCPTP,SHP1.Conclusions and innovationsIn this study,the reported SHP2 inhibitors GS493,SHP836 and SHP099 were used as lead compounds,and 24 novel compounds were designed and synthesized.The inhibitory activities against SHP2 protein of compounds I_a-I_l were tested and the antiprolifration effects of all synthesized compounds on human breast cancer cells MDA-MB-231 and non-small cell lung cancer NCI-H1975 were detected.The results indicated that 4-{（2Z）-2-[1-（（4-tert-butylcarbamoyl）phenyl）-3-（4-nitrophenyl）-5-oxo-1,5-dihydro-4H-pyrazol-4-ylidene]hydrazinyl}benzenesulfonic acid（If）and6-[4-（2,3-dichlorophenyl）piperazin-1-yl]-3-（naphthalen-1-yl）pyrazin-2-amine（II_e）exhibited good anticancer activity.Computer-aided molecular docking and dynamic simulation indicated that I_f could form hydrogen bonds with amino acid in the active center of PTP of SHP2,while II_e acts on the inactive center of SHP2,it may be associated with allosteric sites.II_e shown inhibitory selectivity to SHP2 through their docking with the other homogenous proteins PTP1B,TCPTP,SHP1.Compared to the inhibitor worked in the catalytic center of SHP2,the allosteric inhibitor shown more potent activity.