Microtiter Plate-based Chemistry And In Situ Screening:SuFEx-enabled Lead Discovery Of Selective AchE Inhibitors

An approach derived from combinatorial chemistry has been attracting attention in the last two decades:microtiter plate-based synthesis coupled with in situ screening,and in situ click chemistry,which uses automation to rapidly detect biological or biochemical activity.This approach leverages high yield organic reactions with high selectivity,starting with a set of building blocks to generate a focused library in a microplate,in which a compound is ideally formed in each well.The reaction should ideally be carried out in water or a water miscible non-toxic solvent so that the final product can be tested directly against the target enzyme without any separation or purification.Su FEx click chemistry can achieve the high reactivity of sulfonyl fluorides with suitable amines.The formation of the intended sulfonamides would be efficient,with high purity and high yield.As a proof-of-concept,fragment splicing-based molecular design would be carried out using cholinesterases（Ch Es）,containing a peripheral anionic site（PAS）,a deep and narrow gorge and a catalytic active site（CAS）.Potent Ch E inhibitors can be designed through a proper linker between CAS and PAS to enable the hybrid to fall into both active site cavities.Here,we reported an efficient in situ screening method for building a library of sulfonamides on the picomolar scale using the Su FEx reaction between a sulfonyl fluoride（RSO₂F）core and primary or secondary amines.This biocompatible reaction condition would allow us to rapidly discover and synthesize Ch E inhibitors,and evaluate a focused drug library based on triazole-containing sulfonamide molecules.In an in situ screen against cholinesterases,the efficient ACh E inhibitor T14-A24 was identified.Compound T14-A24 in the drug-like evaluation satisfied Lipinski-Veber’s rule.Molecular docking results showed that compound T14-A24 could well insert into the active pocket CAS,and had aπ-S interaction with His447 in the catalytic triad,while the benzene ring structure of the compound had aπ-πinteraction with the core residue Trp286 of the peripheral anion site PAS.According to kinetic studies,compound T14-A24（K_i=22 n M）was a reversible and competitive ACh E inhibitor and showed remarkable neuroprotection,safe toxicological profile and BBB penetration.In vivo behavioral study showed that T14-A24 treatment improved the Aβ_1-42-induced cognitive impairment,significantly prevented the effects of Aβ_1-42toxicity.Therefore,selective ACh E inhibitor T14-A24 has potential to be further developed as AD therapeutics.