A novel of 40 pyrrolo[1,2-a]thiazolo[5,4-d]pyrimidinone derivatives were designed and synthesized as cholinesterase inhibitor agents. The in vitro enzyme assays proved that most of the compounds effectively inhibited cholinesterases in the micromolar range with little cytotoxicity. Compound G15 exhibited the best inhibitory activity against acetylcholinesterase with an IC50 of 2.69 +/- 0.17 mu M. Kinetic analysis and molecular modeling testified the competitive inhibition manner of G15 was more likely to bind to the active center of acetylcholinesterase. Thus, compound G15 can be considered as promising lead compounds or candidates for the development of novel drugs against Alzheimer's disease.