Synthesis and in vitro evaluation of new derivatives of 2-substituted- 6-fluorobenzo[d]thiazoles as cholinesterase inhibitors Aleš Imramovsky ´ a,⇑ , Vladimír Pejchal a , Šárka Šte ˇpánková b , Katarína Vorc ˇáková c , Josef Jampílek d , Ján Vanc ˇo d , Petr Šimu ˚ nek a , Karel Královec b , Lenka Bru ˚c ˇková b , Jana Mandíková e , František Trejtnar e a Institute of Organic Chemistry and Technology, Faculty of Chemical Technology, University of Pardubice, Studentská 573, CZ-532 10 Pardubice, Czech Republic b Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 53210, Czech Republic c Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, Studentská 573, Pardubice 53210, Czech Republic d Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic e Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic article info Article history: Received 3 December 2012 Revised 22 January 2013 Accepted 23 January 2013 Available online 1 February 2013 Keywords: 6-Fluorobenzo[d]thiazole Lipophilicity Acetylcholinesterase Butyrylcholinesterase Cholinesterase inhibition In vitro cytotoxicity Molecular docking Structure–activity relationships abstract A series of novel cholinesterase inhibitors based on 2-substituted 6-fluorobenzo[d]thiazole were synthes- ised and characterised by IR, 1 H, 13 C and 19 F NMR spectroscopy and HRMS. Purity was checked by ele- mental analyses. The novel carbamates were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The toxicity of the most active compounds was investigated using a standard in vitro test with HepG2 cells, and the ratio between biological activity and toxicity was determined. In addition, the toxicity of the most active compounds was evaluated against MCF7 cells using the xCELLigence system. Structure–activity relationships reflecting the dependence of cholinester- ase inhibitors on the lipophilicity of the compounds as well as on the Taft polar and steric substituent constants are discussed. The specific orientation of the inhibitors in the binding site of acetylcholinester- ase was determined using molecular docking of the most active compound. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Two important enzymes from the group of serine hydrolases, acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8; also known as pseudocholinesterase), are usually defined as cholinesterases (ChEs). Structurally, these serine hydro- lases belong to the class of proteins known as the esterase/lipase family within the a/b-hydrolase fold superfamily. 1 The major role of AChE is to catalyse the hydrolysis of acetylcholine (ACh) in cho- linergic synapses, whereas the function of BChE is less clearly de- fined because it can hydrolyse ACh as well as other esters. 2,3 The inhibition of these enzymes causes an increase in the concentra- tion of ACh in cholinergic synapses and can subsequently affect a number of pathogenic processes. 2 ChE inhibitors (ChEIs) are used in the treatment of various neuromuscular disorders and have pro- vided the first generation of drugs for the treatment of Alzheimer’s disease, 4–9 myasthenia gravis 10,11 and glaucoma. 12,13 An increase in the concentration of ACh can result in an alleviation of the symp- toms of these diseases. Thus, research on new ChEIs may be valu- able for further progress in the treatment of these diseases. (R)-1-(6-Fluorobenzo[d]thiazol-2-yl)ethanamine is a basic scaf- fold for antimicrobials, herbicides, plant desiccants and defoliant compounds. 14 Generally, benzothiazoles have been reported as a class of compounds with a wide range of biological activities, including antibacterial and antimicrobial, 15 anticancer, 16–18 antitu- mour, 19 antiviral, 20 anthelmintic, 21 antiparasitic, 22 anti-inflamma- tory, 23 fungicidal 24 and antioxidant 25 activity. The benzothiazole scaffold has provided several promising candidates, which were investigated as a clinical antitumor candidates. 26 Also other inter- esting bioactivities of these compounds were described in recent years, such as protein inhibitors 27,28 or biosensors. 29 The benzothi- azole ring is also present in various natural marine and terrestrial compounds which have interesting biological activities. 30–32 The moiety of these compounds can serve as a unique and versatile scaffold for experimental drug design. Benzothiazole has an impor- tant place in research, especially in synthetic and pharmaceutical chemistry, due to its potent pharmacological activities. Studies on structure–activity relationships have interestingly revealed that a change in the structure of the substituent group at the C (2) posi- tion commonly results in a change in bioactivity. Such derivatives 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.01.052 ⇑ Corresponding author. Tel.: +420 466037739; fax: +420 466038004. E-mail address: Ales.Imramovsky@upce.cz (A. Imramovsky ´ ). Bioorganic & Medicinal Chemistry 21 (2013) 1735–1748 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc