Series of twenty-five benzyl (2S)-2-(arylcarbamoyl)pyrrolidine-1-carboxylates was prepared and completely characterized. All the compounds were tested for their in vitro ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and the selectivity of compounds to individual cholinesterases was determined. Screening of the cytotoxicity of all the compounds was performed using a human monocytic leukaemia THP-1 cell line, and the compounds demonstrated insignificant toxicity. All the compounds showed rather moderate inhibitory effect against AChE; benzyl (2S)-2-[(2-chlorophenyl)carbamoyl]pyrrolidine-1-carboxylate (IC50 = 46.35 μM) was the most potent agent. On the other hand, benzyl (2S)-2-[(4-bromophenyl)-] and benzyl (2S)-2-[(2-bromophenyl)carbamoyl]pyrrolidine-1-carboxylates expressed anti-BChE activity (IC50 = 28.21 and 27.38 μM, respectively) comparable with that of rivastigmine. The ortho-brominated compound as well as benzyl (2S)-2-[(2-hydroxyphenyl)carbamoyl]pyrrolidine-1-carboxylate demonstrated greater selectivity to BChE. The in silico characterization of the structure-inhibitory potency for the set of proline-based carbamates considering electronic, steric and lipophilic properties was provided using comparative molecular surface analysis (CoMSA) and principal component analysis (PCA). Moreover, the systematic space inspection with splitting data into the training/test subset was performed to monitor the statistical estimators performance in the effort to map the probability-guided pharmacophore pattern. The comprehensive screening of the AChE/BChE profile revealed potentially relevant structural and physicochemical features that might be essential for mapping of the carbamates inhibition efficiency indicating qualitative variations exerted on the reaction site by the substituent in the 3'-/4'-position of the phenyl ring. In addition, the investigation was completed by a molecular docking study of recombinant human AChE.

Department of Biological and Biochemical Sciences Faculty of Chemical Technology University of Pardubice Studentska 573 532 10 Pardubice Czech Republic

Department of Chemical Drugs Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Palackeho 1 612 42 Brno Czech Republic

Department of Human Pharmacology and Toxicology Faculty of Pharmacy University of Veterinary and Pharmaceutical Sciences Palackeho 1 612 42 Brno Czech Republic

Department of Pharmaceutical Chemistry Faculty of Pharmacy Comenius University Odbojarov 10 832 32 Bratislava Slovakia

Department of Synthesis Chemistry Faculty of Mathematics Physics and Chemistry University of Silesia Szkolna 9 40 007 Katowice Poland

Global Change Research Institute CAS Belidla 986 4a 603 00 Brno Czech Republic

Institute of Chemistry University of Silesia Szkolna 9 40 007 Katowice Poland

Institute of Organic Chemistry and Technology Faculty of Chemical Technology University of Pardubice Studentska 573 532 10 Pardubice Czech Republic

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