Design,Synthesis And Druggability Evaluation Of Prodrug Of Anti-Ischemic Stroke Ligustrazine Derivative A11 And Discovery Of Novel Butylcholinesterase Selective Inhibitors

Part Ⅰ:Design,synthesis and preliminary druggability evaluation of All prodrugStroke is the first leading cause of death in China.Ischemic stroke is the most common type of stroke,accounting for 78%of the morbidity rate of stroke in China.Although the incidence,mortality and disability rate of ischemic stroke have decreased in recent years,there are no specific medicines for this disease.Therefore,the development of novel and effective anti-ischemic stroke drugs is still urgent.It is well known that natural products are important sources of new drug development.Based on natural product fragment hybrid strategy,ligustrazine chalcone derivative All was previously identified as a lead against ischemic stroke,which displayed far more potent antioxidant activity than ligustrazine.Besides,All could also significantly improve the behavior and reduce the area of cerebral infarction of rats with cerebral ischemia-reperfusion injury.However,All had a low oral bioavailability(F=6.35%)and short half-life(T1/2=2.24 h),which seriously limits its application.Prodrug strategies attracted much attention in drug development,which not only maintain the efficacy,but also improve the solubility,bioavailability,half-life and physical and chemical properties of the original drugs.To improve the pharmacokinetics of All,a novel carbonate prodrug(LL-1)of All was designed and synthesized,and its aqueous solubility and pharmacokinetics in rats were evaluated.The results of solubility test showed that,the water solubility of LL-1 was 32.9μg/mL and logD value was 3.46 in the phosphate buffer with the pH at 7.4,which is at least 70 times higher than that of All.The results of pharmacokinetic evaluation demonstrated that LL-1 could be rapidly converted to All in rats,and the plasma concentration of All was increased by 5 times(Cmax=982 ng/mL)after intravenous administration.However,LL-1 was quickly metabolized after converting to All,and it had had short half-life and low oral bioavailability(F=3.6%).Although the pharmacokinetic properties of prodrug LL-1 have not been significantly improved,it would provide new insights into further optimization of All in the future.Part Ⅱ:Discovery of novel butylcholinesterase selective inhibitorsThe cholinergic nervous system regulates the occurrence and development of Alzheimer’s disease,of which cholinesterase is regarded as an important target for the treatment of Alzheimer’s disease.The currently approved cholinesterase inhibitors are mostly acetylcholinesterase inhibitors,which can only delay the progression of the disease,rather than cure it,along with many adverse effects.It is worth noting that butyrylcholinesterase is a very promising target,which not only plays a critical role in the progression of AD,but also could maintain the cholinergic neurons function instead of AChE.Besides,BChE gene silencing dose not affect normal human survival.However,there are still no selective butyrylcholinesterase inhibitors applied in clinic,and more mechanism researches are lacking.Therefore,the development of butyrylcholinesterase inhibitors with highly selectivity and potent activities for the treatment and mechanism studies of AD is of great value.Based on the crystal structure of AChE and BChE,we chose tacrine as a lead compound and introduced structurally diverse substituents via oxime coupling reaction.Then,a 53-member oxime-containing compounds combinatorial library was built by microscale synthesis,followed by in situ screening for the discovery of selective and dual-site butyrylcholinesterase inhibitors.The preliminary screening results demonstrated that AlQ4,A2Q17,A2Q19,A2Q20 and A3Q19 exhibited effective inhibitory activity against cholinesterase.Then these compounds were synthesized in large quantities and their biological activities were further evaluated.The most potent butyrylcholinesterase inhibitor A3Q19(hBChE IC50=4 nM)exhibited the 8-fold greater potency than that of tacrine(hBChE IC50=31 nM),and also displayed high selectivity to hBChE(SI=62),as well as no significant cytotoxicity.Moreover,the enzyme binding kinetic studies indicated that A3Q19 appeared strong affinity for butyrylcholinesterase.But,A3Q19 could not inhibit Aβ1-42 peptide self-induced aggregation and was hard to penetrate into the CNS.In addition,A2Q17 could potently inhibit both of AChE and BChE,and Aβ1-42 peptide self-induced aggregation,and could penetrate into the CNS.Unfortunately,A2Q17 displayed high cytotoxicity.In a word,A2Q17 and A3Q19 deserve further development as potential anti-AD drug candidates.