Design, Synthesis And Evalution Of Long-acting Local Anesthetic Based On HCN Ion Channel

This dissertation focuses on design and synthesis of small molecules with long-acting local anesthetic activities, based on HCN ion channel. The structures of a clinical used non-selective HCN ion channel inhibitor ivabradine and its analogs were analyzed and a seven memberedheterocyclic skeleton was selected out as pharmacophore,, which was further connected with the main structure units of local anesthetic lidocaine and bupivacaine by butane or Z-2-butene, and finally 4 new compounds were designed and synthesized. The 4 new compounds were supposed to retain the local anesthetic activity and the ability to block the HCN ion channel.The anesthesia ability of these compounds was tested in the sciatic nerve of mice. Compound 1 and compound 2 were administrated at the concentration of 37 nM, and compound 3 at 15 mM. Acupuncture pain was used for preliminary judging the local anesthesia and the duration was used as the reference to determinate the local anesthesia ability. The results indicated that the durations of local anesthesia for compound 1, 2 and 3 were 39.4(34.6~44.1) min, 47.3(39.0~55.5) min and 23.5(3.6~43.4) min, respectively.The plasmid pcDNA3.0-HCN1, pcDNA3.0-HCN2 and pcDNA3.0-GFP were transformed into E. Coli DH5α by heat shock respectively, and then extracted by plasmid extraction kit. The plasmid was verified by double endonuclease restriction. pcDNA3.0-HCN1 was digested by HindⅢ and BglⅡ and pcDNA3.0-HCN2 by HindⅢ and Xho Ⅰ. The confirmed pcDNA3.0-HCN1 and pcDNA3.0-HCN2 were transfected into HEK293 cell lines respectively, together with pcDNA3.0-GFP, and the HCN ion channel subtype eukaryotic cell models were built. The HCN1 and HCN2 ion channel subtype eukaryotic cell models were checked by patch clamp technique, and hyperpolarizing current of transformed cell lines was recorded, respectively.In this thesis, based on the idea of single-molecule and dual targets, the active fragments of local anesthetic lidocaine and bupicacaine were linked with the active fragments of HCN ion channel inhibitor by carbon chain, and4 new compounds were designed and synthesized. The local anesthetic activity of the synthesized compounds was studied in mice. Compounds 1-3 showed local anesthesia activity very well. The HCN1 and HCN2 ion channel subtype eukaryotic cell models were built up successfully also, which can be used to evaluate the HCN ion channel inhibitory activity for the 4 synthesized compounds in the future.