Synthesis,screening And Preliminary Establishment Of Quality Standards For Novel KCNQ Potassium Channels Openers

Cell membrane ion channel is one of the important drug targets.The distribution and activity of ion channels are important for the excitability and function of cells and tissues,and are important factors for physiological regulation.There are many ion channels in human tissue,and each channel contains many subtypes.Among them,the potassium channel has the most subtypes and complex functions.KCNQ potassium channel belongs to the voltage-gated Kv7 potassium channel subfamily.It has been proved that KCNQ2/3 potassium channel（M channel）is a new target for the treatment of epilepsy,and it has become one of the hot spots of drug development.The aim of this study was to discover novel KCNQ/M channel openers with high activity,high selectivity and to prepare them for further innovative drug development by conducting synthesis and quality control studies.Part one Synthesis and screening for new KCNQ potassium channels openersObjective:To design and synthesize a series of KCNQ potassium channel openers;To discover new drug candidate compounds through high-throughput screening;TO optimize the synthesis process of preferred compounds.Methods:1.We designed a series of benzoylamide compounds.The key intermediate II is obtained by a two-step reaction of substitution and reduction,and the intermediate II and the corresponding R different substituted carboxylic acid are charged in a ratio of 1:1 equivalent,and the target compound I is obtained under the conditions of EDC and DMAP hydrochloric acid.2.KCNQ channel open activity of target compounds was evaluated by high-throughput screening method based on ICR8000.3.For the obtained preferred compound 2,the synthesis scale is enlarged60 times to carry out repeated production of multiple batches,and the synthesis process parameters are optimized and determined.Result:A total of 4 compounds were designed,synthesized and identified.The activity screening results showed that the KCNQ potassium channel open activity EC₅₀ value of preferred compound 2 was 0.15±0.04μM.After optimization of the synthesis process,the compound purity was greater than99%and the yield was 82.3±1.8%（N=3）.Conclusion:In this part,a preliminary study on the structure-activity relationship of a series of KCNQ potassium channel openers was carried out,and one candidate compound with high activity new drug was obtained,and the amplification synthesis process was studied,which laid a foundation for the follow-up research.Part two Preliminary establishment of preferred compound quality standardsObjective:To calibrate the analytical reference of the compound 2 and to initially establish a quality control method for compounds 2.Method:The analytical reference of the compound 2 was calibrated by a dead-stop titration method with sodium nitrite.The compounds 2 were tested for traits,identification and content determination according to the Chinese Pharmacopoeia.Result:The titration of the sodium nitrite titration solution used was21.05 mg/mL,and for the titration of the compound 2,the linear correlation coefficient R² of the titration was 0.9998,and the purity of the finally calibrated compound 2 was 99.54%.The established HPLC method for determining the content of compound 2,the full method validation met the requirements.The linear relationship in the range of 0.05-0.4 mg/mL was good,R² was 0.9995,and the accuracy was between 99.0 and 100.7%.The measured contents of the three amplified batches were 99.3%,99.4%and99.3%,respectively.Conclusion:In this part,the calibration work of the analytical standard of compound 2 was completed,then the preliminary quality standard research was carried out,which provided guarantee for the quality reliability of the subsequent research reagent.