Preliminary Druggability Research Of A Highly Active C-Met Kinase Inhibitor

Objective:C-Met kinase is a protein encoded by the proto-oncogene C-Met.It is the only specific tyrosine receptor for hepatocyte growth factor.Moreover,it is overexpressed in tumor tissues,and thus C-Met kinase had become a research hotspot of antitumor drugs.BMS-2 is a small molecule C-Met kinase inhibitor developed by the Bristol-Myers Squibb Company and has preferable kinase inhibitory activity.However,BMS-2 was forced to terminate in clinical phase II trials due to its poor water solubility and clinically demonstrated nephrotoxicity.The research team designed a series of target compounds by modified the structure of the the lead compound BMS-2.The GTL-16human gastric cancer cell was used as a screening model to screen for the high activity target compound with an IC₅₀ of 29 nM,N-（3-Fluoro-4-（6,7-dimethoxyquinolin-4-yloxy）phenyl）-3-（4-fluorophenyl）-2-（3-morpholinoprop-ylamino）pyrimidinedine-4（3H）-one-5-carboxamide（A2）,which is superior to the lead compound BMS-2.It is intended to determine whether the highly active target compound A2 has the druggability potential to become a novel small molecule C-Met kinase inhibitor.The preliminary druggability research of the high activity target compound A2 was carried out from three aspects of physical and chemical properties,biochemical properties and pharmacokinetic properties,which provided theoretical basis for its follow-up research.Methods:The apparent solubility and lipid-water partition coefficient of compound A2 were investigated by the equilibrium solubility method and the shake flask method.The stability properties of compound A2 were investigated with high performance liquid chromatography from the aspects of light stability,thermal stability and pH stability.The binding rate of plasma protein of compound A2 and BMS-2 were measured by fluorescence spectroscopy.The gastrointestinal stability of compound A2 and BMS-2were researched by simulated determining the content of the compound at different time points in the artificial gastrointestinal fluid environment by high performance liquid chromatography.Rats were injected intraperitoneally with compound A2 and BMS-2and later taked blood from the eyelids and collected tissue samples at different time points.Determination of compound concentration by high performance liquid chromatography after pretreatment of biological samples.Using the Origin software to fit the blood concentration curve.The pharmacokinetic parameters were analyzed using DAS 2.0 software and the tissue distribution of the compounds were examined.Results:In the physicochemical properties,the apparent solubility of compound A2gradually increases with the range of pH=2 to pH=8.5.At pH=2,the apparent solubility was the minimum（9.63μg/mL）.At pH=8.5,the apparent solubility was the maximum（19.82μg/mL）.The lipid-water partition coefficient of compound A2 is with1.61-2.82 which is within the optimal interval.And less than the lead compound BMS-2（log P=4.82）,the water solubility is much improved compared to the lead compound BMS-2.In the biochemical study,the fluorescence quenching constant K_sv and the binding constant K_a of compound A2 and the lead compound BMS-2 at 296 K are(K_sv=0.950×10⁵ L/mol,K_a=3.09×10⁴ L/mol)and(K_sv=1.208×10⁵ L/mol,K_a=3.92×10⁴L/mol).At 310 K are(K_sv=0.667×10⁵ L/mol,K_a=2.15×10⁴ L/mol)and(K_sv=0.766×10⁵ L/mol,K_a=7.87×10⁴ L/mol).The number of binding sites for both the compound and HSA is close to 1.And the fluorescence quenching mechanism is a static quenching mechanism.The type of interaction between compound and HSA is mainly hydrogen bonding and van der Waals force.The plasma protein binding rate of compound A2 was 11.43%-13.20%,and the plasma protein binding rate of lead compound BMS-2 was 13.61%-16.12%.In the study of gastrointestinal stability,compound A2 and lead compound BMS-2 were extremely unstable in artificial gastric juice,and gradually degraded with the increase of contact time.The remaining percentage in 4 h was reduced to 30%and40%.However,both of them were stable in artificial intestinal fluid,and the remaining percentage at 80%and 60%respectively within 12 h.In the study of pharmacokinetic properties,the pharmacokinetic behavior of compound A2 and lead compound BMS-2 were consistent with the two-compartment model after intraperitoneal injection into rats.The pharmacokinetic parameters of the peak C_max of the drug peaks are（12.380 and 34.970μg/mL）;the peak times T_max are（1.5 and 2.0 h）;the absorption rate constants k_a are(4.177 and 1.061 h^-1);the absorption half-life t_1/2（a）are（0.166 and 0.653 h）;the distribution half-life t1/2（α）are（3.175 and3.314 h）;the elimination half-life t1/2（β）are（17.589 and 27.391 h）;the area under the curve of the drug of the AUC_（0-t）are（133.065 and 810.178μg/mL*h）.The results of tissue distribution showed that compound A2 and lead compound BMS-2 were distributed in various tissues of rats（heart,liver,spleen,lung,kidney,brain,stomach）and could pass through the blood-brain barrier.Compounds are distributed at higher levels in blood perfusion-rich tissues,such as liver,spleen,and lung tissue.Conclusion:The high-activity target compound A2 has a more reasonable lipid-water partition coefficient than the lead compound BMS-2,and the water solubility is increased.Moreover,there is no obvious accumulation of tissue compounds in rats.Compared with the lead compound BMS-2,it may have good renal safety and possess certain druggability potential,and it is expected to be a candidate compound of novel small molecule C-Met kinase inhibitors.