Synthesis, molecular docking and biological evaluation of N,N-disubstituted 2-aminothiazolines as a new class of butyrylcholinesterase and carboxylesterase inhibitors

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Abstract

A series of 31 N,N-disubstituted 2-amino-5-halomethyl-2-thiazolines was designed, synthesized, and evaluated for inhibitory potential against acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and carboxylesterase (CaE). The compounds did not inhibit AChE; the most active compounds inhibited BChE and CaE with IC50 values of 0.22–2.3 μM. Pyridine-containing compounds were more selective toward BChE; compounds with the para-OMe substituent in one of the two dibenzyl fragments were more selective toward CaE. Iodinated derivatives were more effective BChE inhibitors than brominated ones, while there was no influence of halogen type on CaE inhibition. Inhibition kinetics for the 9 most active compounds indicated non-competitive inhibition of CaE and varied mechanisms (competitive, non-competitive, or mixed-type) for inhibition of BChE. Docking simulations predicted key binding interactions of compounds with BChE and CaE and revealed that the best docked positions in BChE were at the bottom of the gorge in close proximity to the catalytic residues in the active site. In contrast, the best binding positions for CaE were clustered rather far from the active site at the top of the gorge. Thus, the docking results provided insight into differences in kinetic mechanisms and inhibitor activities of the tested compounds. A cytotoxicity test using the MTT assay showed that within solubility limits (<30 μM), none of the tested compounds significantly affected viability of human fetal mesenchymal stem cells. The results indicate that a new series of N,N-disubstituted 2-aminothiazolines could serve as BChE and CaE inhibitors for potential medicinal applications.

Introduction

Among functional types of enzymes, serine hydrolases constitute one of the largest and most varied groups. Of the approximately 240 serine hydrolases found in humans, about half are serine proteases whereas the remainder carry out metabolic functions. The metabolic serine hydrolases include diverse lipases, peptidases, esterases, thioesterases, and amidases that hydrolyze small molecules, peptides, or post-translational (thio)ester protein modifications.1 Because these enzymes serve important physiological and pathogenic functions and their mechanisms of action are well understood, they have been popular targets for drug development.2, 3

Acetylcholinesterase (AChE, EC 3.1.1.7) is a metabolic serine hydrolase that catalyzes the hydrolysis of acetylcholine, thus regulating cholinergic neurotransmission. Therefore, in disorders such as Alzheimer’s disease (AD), where there is diminished cholinergic activity, inhibition of AChE has been employed to treat some of the symptoms attributed to decreased acetylcholine levels.4, 5 Most of the currently available drugs on the market (tacrine, donepezil, rivastigmine and galanthamine) intended to treat AD are AChE inhibitors.4, 5, 6

However, during the progression of Alzheimer’s disease, brain AChE levels decline while butyrylcholinesterase (BChE, EC 3.1.1.8) activity increases, suggesting that acetylcholine hydrolysis may occur to a greater extent via BChE catalysis.7, 8 In this regard, it has been reported that highly selective inhibition of BChE is important in raising acetylcholine levels and improving cognition.8, 9, 10, 11, 12 Because selective BChE inhibitors do not exhibit the adverse cholinergic effects of AChE inhibitors, the search for selective BChE inhibitors is currently a promising direction in medicinal chemistry research.10, 13, 14, 15 Moreover, whereas some of the anticholinesterase compounds currently employed for AD treatment can inhibit both AChE and BChE12, 16, the highly selective AChE inhibitor (−) huperzine A was clinically ineffective.17 Likewise, the selective AChE inhibitor (−) phenserine was found to be potentially efficacious for mild to moderate AD, but because this drug also inhibits production of amyloid precursor protein, it is difficult to assess the degree to which its efficacy stems from AChE inhibition.18 Taken together, these findings suggest that the development of selective BChE inhibitors could be important for more effective treatment of AD.19

In recent years, interest in another group of metabolic hydrolases, the carboxylesterases (CaE, EC 3.1.1.1), has sharply increased. CaE play major roles in the activation, detoxification and biodistribution of xenobiotics and numerous classes of drugs including esters, thioesters, carbamates, and amides.20, 21 Representative types of pharmaceutical agents include angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, antiplatelet drugs, statins, antivirals, and central nervous system agents.22 Inhibitors of CaE decrease the rate of hydrolysis of such drugs and thus reduce the rate of conversion of pro-drug to active drug or, conversely, increase the half-life of the active drug. Consequently, CaE inhibitors have important therapeutic value23 and discovery of new CaE inhibitors with high selectivity is of considerable interest.24

Recently, N,S-containing heterocyclic compounds, especially derivatives of 2-amino-1,3-thiazole, have attracted increasing attention due to their chemical and biological properties. For example, it has become known that many compounds containing the thiazoline (4,5-dihydro-1,3-thiazole) fragment exhibit antibacterial, anticancer, antifungal, and other types of biological activities.25, 26 However, information about the inhibition of serine esterases by compounds containing the thiazoline fragment is practically absent in the literature. The ability to reversibly inhibit cholinesterases was shown for only some 2-substituted thiazolines and imidazo[2,1-b]thiazole derivatives.28, 29

Our preliminary study30 showed that N,N-disubstituted-2-aminothiazolines could inhibit BChE and CaE while exhibiting minimal inhibitory activity against AChE. A search of chemical reactivity of 5-Br-methyl substituted thiazolines for the past 40 years using Reaxys turned up very few reactions. The alkylation reactions for 5-Br-methyl thiazolines were not described in the literature. The same results were obtained with SciFinder. Thus, the ability of 2-amino-5-halomethylthiazoline derivatives to alkylate DNA is unlikely. Therefore, we extended our experimental search for inhibitors of serine esterases with potential biomedical application in the 2-aminothiazoline series.

Here, we report the synthesis of 31 derivatives of 2-aminothiazolines of general formula shown in Figure 1. The syntheses are outlined in Scheme 1 and the structures of the individual compounds are listed in Table 1. Our strategy for the design of the inhibitors is illustrated in Figure 2. We have also performed a biological evaluation of these compounds as inhibitors of three metabolic serine esterases (AChE, BChE and CaE) along with an optimization of their structures to yield selective inhibitors of BChE and/or CaE. To gain further insight into the molecular determinants responsible for the observed ability to inhibit BChE and CaE, molecular docking of the 9 most active compounds was carried out. In addition, the effect of the compounds on cell viability was evaluated in human fetal mesenchymal stem cells.

Section snippets

Chemistry

We have synthesized the hydrohalides of 2-amino-substituted 5-halomethyl-2-thiazolines 5am and 6at by traditional methods (Scheme 1): iodination or bromination of the corresponding N′,N′-derivatives of N-allylthiourea31 obtained from allylisothiocyanate and the corresponding amine.

Previous studies have shown that isomeric six-membered rings (thiazines) are formed in these reactions as well as thiazolines.32, 33 However, given that these reactions are usually performed in polar media with

Conclusions

In summary, a novel series of N,N-disubstituted 2-aminothiazolines has been designed, synthesized and evaluated as AChE, BChE, and CaE inhibitors. The results demonstrate that the tested compounds do not inhibit AChE; accordingly, they would be expected not to cause unwanted cholinergic side effects if they were employed, e.g., as co-drugs for ester-containing pharmacological agents. The relative selectivity of the most active compounds toward BChE and CaE is summarized in Figure 8. Whereas the

Chemistry

All solvents, chemicals, and reagents were obtained commercially and used without purification. 1H NMR (200 MHz) spectra were recorded on a Bruker CXP-200 NMR spectrometer using CDCl3, DMSO-d6, acetone-d6, CD3OD, CD3CN, or D2O as solvents with tetramethylsilane as an internal standard. Chemical shifts, d, are given in parts per million (ppm), and spin multiplicities are given as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet) or m (multiplet). Coupling constants, J, are

Acknowledgements

This work was partly supported by grant #14-03-01063 of the Russian Foundation for Basic Research, program ‘Medicinal Chemistry’ of the Russian Academy of Sciences, and a ‘Dynasty’ foundation grant to S.V.L. We thank Dr. Dmitry Suplatov for technical help, the Lomonosov Supercomputer center for computational time, and ChemAxon, Accelrys Software Inc., and ACD Labs, Inc. for free licensing of their products for academic users. We are also grateful to the Center of the Collective-Access Equipment

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