Studies On The Design, Synthesis, Stability And Active Screening Of Several KCNQ2/3Openers As Photoactive Compounds

KCNQ2/3opener can be used in treating epilepsia and convulsion. Inour previous studies, many compounds that show positive activity as K+channel openers were designed and synthesized. However, the intensity ofthese compounds on opening K+channel are distinct. Unfortunately, themechanism of KCNQ2/3potassium channel opener remains unclear so far.The study is based on the principles of photoaffinity labeling bymodifying these active compounds without any changes on the essentialgroups of active compounds. We designed and synthesized severalderivatives by introducing photoactive group and radioisotipe to the activeopeners as parent compounds. The candidates were found in the study bydetermining chemical stability and activity as KCNQ2/3openers of thesynthetic derivatives for further studies.Part I: The pathway design and synthesis of m-trifluoromethylphenyl-acetic acidObjective: To investigate an appropriate synthetic route form-trifluoromethylphenylacetic acid in laboratory.Methods:4-Nitrophenylacetic acid was used as a starting materialwhich generated4-Aminophenylacetic acid via reduction by Pd/C, then byacetylization4-acetamidophenylacetic acid was obtained, this intermediatewas nitrified, then following acid hydrosis,4-amino-3-nitro-phenylaceticacid was provided, and then diazotization and reduction through phosphinicacid yielded3-amino-phenylacetic acid. After diazotization and Iodination,The iodine of3-iodine-phenylacetic acid can be substituted by sodiumtrifluoroacetate under the protection of N2in DMF. Finally,m-trifluorophenylacetic acid was obtained successfully.Result: By using4-nitrophenylacetic acid as an initial material, through eight steps reaction, the important intermediate, m-trifluorophenylacetic acid,for this study was obtained with a total yield of44.6%.Conclusion: This study begins with an economic material,4-nitrophenylacetic acid, involvs in eight-step reactions. All these reactionsand procedures are easy and simple to carry out. Therefore, this syntheticroute can be used in laboratory with its non-expensive and handy procedures.Part Ⅱ: Design and synthesis of several photoactive compounds ofKCNQ2/3openersObjective: To provide evidence for the optimization of KCNQ2/3openers or the development of new drugs, we designed and synthesizedseveral compounds of KCNQ2/3openers by introducing photoactive groupor radioisotope.Methods:(1)3,5-dinitrobenzoic acid was treated as a starting material,after four-step reactions including selective reduction of nitro, diazotizationof aryl-amino, iodine/azide-substitution and amidation with amantadine,subsequently3-azide-5-nitrobenzoyl amantadine(A-1) and3-iodine-5-nitrobenzoyl amantadine(A-2) were obtained.(2) Piperonylaldehyde in this study is an initial material after eight step reactions such asnitration, reduction of aldehyde, chlorination of benzyl hydroxyl, reductionof nitro, diazotization and azide-substitution, Gabriel reaction, andcondensation reaction with amantadineN-(6-azido-3,4-methylenedioxy-1-phenylmethyl)-N’-amantyl urea (B-1)wasobtained.(3)By using m-trifluorophenylacetic acid as an initial material,after five step reactions including nitration, reduction, diazotization,iodine/azide-substitution, and final amidation, four desired derivatives wereyielded, i.e.5-azide-3-trifluoromethylbenzoyl cyclopentylamine (C-1),2-azide-3-trifluoromethylbenzoyl cyclopentylamine (C-2),5-iodine-3-trifluoromethylbenzoyl cyclopentylamine (C-3), and2-iodine-3-trifluoromethylbenzoyl cyclopentylamine (C-4).Result: In this study, seven compounds are designed and synthesized,all of them are novel compouds. The chemical structures of six of them were confirmed by1H-NMR, while the rest was confirmed by the information ofMS and secondary MS basis on the analysis of the similar structures. All thechemical structures of these new compounds are shown below:Conclusion: In this study,7new photoactive/radioactive derivativeswere synthesized successfully as the potential KCNQ2/3potassium channelopeners.Part Ⅲ: The evaluation of chemical stability and avtivity screening ofthe synthetic photoactive compounds of KCNQ2/3potassiumchannel openersObjective: To investigate the chemical stability of3-azide-5-nitrobenzoyl amantadine; to evaluate the bioactivities of thesephotoactive derivatives by using activity screening of KCNQ2/3openers.Method: The solution of3-azide-5-nitrobenzoylamantadine (solvent：water) was prepared for the stability study. Ater determining the wavelengthof the maximum absorption of3-azide-5-nitrobenzoylamantadine andoptimazing the condition by HPLC, its purity was determined. A solution in aproper concentration which is used by high-throughput screening was thenprepared, and treated with variable factors, i.e. light, temperature and pH forseveral hours; By high-throughput screening, A-1, B-1and C-1are determined for the opening activity By using RTG as a positive control.Result： The chemical stability of the photoactive derivative(3-azide-5-nitrobenzoyl amantadine) was evaluated. The data showed thatthe chemical stability of synthetic derivative was well reserved whatever anyof the factors (water, light, pH and37℃); High-throughput screening datashowed different value of EC50on A-1, B-1and C-1, of which the valuesimplied a positive activity. Comparing with RTG with same concentration,the data of A-1showed a positive relevant relationship with its concentrationwithout a platform; the data of B-1implied it could be used as a candidatefor the ligand-receptor binding site study; the data of C-1indicated that theopening activity of C-1was irrelevant with its concentration.Conclusion：These compounds are tolerant with water, light, acid orbase and appropriate tempeture (37℃); A-1, B-1and C-1are activeKCNQ2/3openers, B-1could be applied in the further ligand-receptorbinding site study.