Synthesis and the activity assessment of adamantyl- containing thiazolium inhibitors of butyrylcholinesterase

Cholinesterase inhibitors can be used for treatment of neuropsychiatric symptoms and functional impairments in neurodegenerative pathologies such as Alzheimer’s and Parkinson’s diseases. Aim. To synthesize and assess the inhibitory activity of adamantyl-containing 5-substituted N-benzyl and N-phenacylthiazolium salts against butyrylcholinesterase and acetylcholinesterase. Results and discussion. The synthesis of 3-aroylmethyland 3-arylmethyl-5-(2-acyloxyethyl)-4-methylthiazolium salts included preparation of 5-acyloxyethyl thiazole derivatives by the reaction of 5-(2-hydroxyethyl)4-methyl-1,3-thiazole with the corresponding adamantoylor adamantylacetyl chlorides. The derivatives of 5-acyloxyethyl thiazole were quaternized in the reaction with benzyl or phenacyl halides. The studies in vitro have shown that the compounds synthesized inhibit butyrylcholinesterase with IC50 values in the micromolar range. Some of them exhibited selectivity over acetylcholinesterase. The molecular docking was performed for understanding the mechanisms of the enzyme-inhibitor complex formation. Experimental part. The synthesis of the intermediate and target compounds was carried out by the classical methods. The structures of compounds were proven by NMR 1H-spectroscopy and elemental analysis. The methods of enzymatic kinetics were used for determination of the inhibitory effects of the compounds synthesized. Calculations by molecular docking were carried out using Autodock 4.2 program. Conclusions. 3-Aroylmethyland 3-arylmethyl-5-(2-acyloxyethyl)-4-methylthiazolium salts with adamantylcontaining substituents in position 5 can selectively inhibit butyrylcholinesterase compared to their effect on acetylcholinesterase.

Ключевые слова: бутирилхолинестераза; ацетилхолинэстераза; ингибирование; соли тиазолия; адамантан; молекулярный докинг Alzheimer's disease (AD), the most common cause of dementia in the elderly, is associated with β-amyloid aggregation, neuron dysfunction and decrease of the acetylcholine level in cholinergic synapses of the brain [1,2]. This complex neurodegenerative disorder is characterized with progressive cognitive impairment, including the memory loss [3].
Cholinergic hypothesis is the most popular explanation of mechanisms of neurodegenerative disorders. According to it acetylcholinesterase (AChE; EC 3.1.1.7) as an enzyme, which is involved in the breakdown of acetylcholine in cholinergic synapses, can be a target for therapeutic intervention [2]. It should be also noted that AChE predominates in the healthy brain, while butyrylcholinesterase (BChE; EC 3.1.1.8) plays a minor role in regulation of the acetylcholine level [4]. However, the BChE activity in the brain of AD patients is increased, while the AChE activity remains constant or decreases. In case of the AChE knockout mice, BChE is able to hydrolyze acetylcholine and compensates the lack of the AChE activity [5]. The BChE exhibiting peptidase activity may be involved into AD progression due to production of β-amyloid plaques [6]. All these facts suggest that BChE can also be a therapeutic target in the treatment of neurodegenerative disorders. In addition, the inhibitors of BChE were found to increase the acetylcholine level in the cortex of the rat brain tissues, improve their learning [5], and they did not show significant adverse side effects.
A broad number of compounds were described as selective inhibitors of AChE. Some of them are used for the treatment of Alzheimer's disease [7] and show the activity against Parkinson disease, Lewy Body Dementia [8], and schizophrenia [9]. However, only a few compounds were described as selective inhibi-tors of BChE, for example, cymserine and its analogs [10], carbamate derivatives of isosorbide [11], and antiparkinson drug profenamine [12]. Rivastigmine, which is used in clinical practice, is a non-selective inhibitor of AChE and BChE [7]. Tacrine, which shows the side effect of hepatotoxicity, is a selective inhibitor of BChE [13].
In the present paper, we synthesized a series of 3-aroylmethyl-and 3-arylmethyl-5-(2-acyloxyethyl)-4-methylthiazolium salts with adamantoyloxyethyl or adamantylacetoxyethyl substituents in position 5 and assessed their inhibitory activity towards AChE and BChE. These compounds may be considered as structural analogs of the thiazolium part of vitamin B 1 which exhibits only a weak activity towards AChE [14] and BChE. The change of the substituent in position 3 of thiamine and introduction of a bulky adamantyl fragment in position 5 of the thiazolium ring was expected to improve the inhibitory properties of the compounds synthesized. Different adamantane derivatives are known to demonstrate the antiviral, antibacterial and anticancer activities [15][16][17]. Among them, amantadine, rimantadine and memantine show the anti-Parkinson's and anti-Alzheimer's effects [18][19][20], but their activities are not associated with inhibition of cholinesterases. At the same time, 6-O-demethyl-6-O-[(adamantan-1-yl)carbonyl]galanthamine and adamantine-substituted guanylhydrazones are described as inhibitors of AChE and BChE, respectively [21,22].

Assessment of the anticholinesterase activity
Compounds 3-19 were tested as inhibitors of AChE and BChE using the Ellman's method [26]. The absence of the inhibitory activity of compound 3 suggests the importance of a benzyl or phenacyl substituent in position 3. The data represented in Table   Table  Compounds  show that compounds 4-18 exhibit inhibition of BChE with IC 50 values in the range from 0.42 to 10 µM. In case of AChE, this inhibition was less effective with IC 50 values higher than 2 µM. N-Benzylthiazolium derivatives 4-8, including compound 6 with the p-nitrobenzyl group, showed the micromolar inhibitory activity towards BChE and a modest selectivity over AChE. N-Phenacylthiazolium salts 9-13 were more effective inhibitors of BChE with greater selectivity over AChE. Among them, compounds 12 and 13 bearing the 3methyl-1-adamantyl and 3,5,7-trimethyl-1-adamantyl group had IC 50 values of 0.9 µM and 0.42 µM, respectively, and showed more than the 10-fold selectivity over AChE. p-Bromophenacyl derivatives 14-18 did not show better inhibitory effects and selectivity.
The dose-dependent curve of BChE inhibition by compound 13 is shown in Fig. 1. The calculated Hill slope is 0.90 ± 0.14. According to the Lineweaver-Burk plot (Fig. 2) compound 13 is a mixed inhibitor of BChE. This suggests that the inhibitor interacts with free enzyme and the enzyme-substrate complex. The apparent K i and K i ' values are 0.23 ± 0.05 µM and 0.76 ± 0.16 µM, respectively.

Molecular docking
The molecular docking was performed for understanding the mechanisms of the enzyme-inhibitor complex formation. Compound 13 was docked into the active site of human BChE (PDB code 4BDS [27]). The result obtained showed that compound 13 could be positioned at the active site of BChE (Fig. 3) with the estimated binding energy of -9.60 kcal/mol. According to the possible binding mode the 3,5,7-trimethyl-1-adamantyl fragment was located in the anionic binding site and surrounded by amino acid residues of Asp70, Gly78, Ser79, Trp82, Tyr332, Trp430, Met437, and Tyr440. The phenacyl fragment of the inhibitor occupied the ester binding site and provided electrostatic and van der Waals interactions with amino acid residues of Gly117, Trp231, Leu286, Val288, Phe329, Phe398, His438. The oxygen atom of the phenacyl substituent could form the hydrogen bond with Ser198 belonged to the catalytic triad (Ser198, His438 and Glu325). The binding mode suggests that hydrophobic, van der Waals and electrostatic interactions are responsible for stabilization of the enzyme-inhibitor complex.

Chemistry
The 1 H NMR-spectra were recorded on a Varian M400 (400 MHz/100 MHz) spectrometer in DMSO-d 6 . 4-Methyl-5-(2-hydroxyethyl)thiazole (1), benzyl chloride, phenacyl bromide, and p-bromophenacyl bromide were commercially available. Compounds 2a-e were prepared by the reaction of 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole (1) with the appropriate acyl chlorides in benzene in the presence of triethylamine (Scheme). Adamantyl-containing acyl chlorides were obtained from the corresponding carboxylic acids.  The synthesis of compounds 3, 9-18. Add benzyl chloride, or phenacyl halogenide, or p-bromophenacyl bromide (5 mmol) to the solution of 4.5 mmol of 4-methyl-5-substituted thiazole 2a-e in acetone. Reflux the reaction mixture for 3-5 h and allow it to stand at the room temperature. Treat the product with acetone and diethyl ether, filter and recrystallize from the methanol-acetone-diethyl ether mixture to give compounds 9-18. Compound 3 was synthesized by the interaction of 2a with anhydrous hydrogen chloride in benzene at the room temperature.
The synthesis of compounds 4-8. Heat the mixture of benzyl chloride or p-nitrobenzyl chloride (5 mmol) and 4.5 mmol of 4-methyl-5-substituted thiazole (compounds 2a-d) at 100-115 o C for 1.5-4.5 h. After that add acetone and diethyl ether to wash the solidified product, and allow the mixture to stand at the room temperature. Recrystallize the product from the mixture of methanol, acetone, and diethyl ether to give compounds 4-8.

In vitro study of AChE and BChE inhibition
Acetylcholinesterase from electric еel, butyrylcholinesterase from the equine serum, and Ellman's reagent (DTNB) were purchased from Sigma-Aldrich. S-Acetylthiocholine iodide (Sigma-Aldrich) and S-butyrylthiocholine iodide (Fluka) were used as substrates for AChE and BChE, respectively.
Spectrophotometric detection of 5-thio-2-nitrobenzoate (TNB 2-) was performed at the wavelength of 412 nm using the molar extinction coefficient of TNB 2of 14150 M -1 cm -1 [28]. Before the in vitro study the inhibitor was dissolved in dimethyl sulfoxide. The assay solution (0.5 mL) contained of 25 mM phosphate buffer (pH 7.48), 0.1 mM S-acetylthiocholine iodide, 1 % DMSO, 1 mM DTNB, inhibitor, and water. AChE was added after incubation of the reaction mixture at 25 °C for 5 min. The inhibition of BChE was studied in the similar conditions with 0.5 mM S-butyrylthiocholine iodide.
The Hill coefficient for compound 13 was calculated from the dose-dependent inhibition curve (Fig. 1) using the equation with four parameters. The values of IC 50 presented in Table are concentrations of inhibitors, which reduce the enzyme activity by 50 %. The kinetic data of the enzymatic transformation of S-butyrylthiocholine iodide in the absence and the presence of compound 13 were calculated from Lineweaver-Burk plots (Fig. 2).

Molecular docking
The molecular docking calculations were performed by Autodock 4.2 using the Lamarckian Genetic Algorithm. Compound 13 was docked into the active site of human BChE (PDB code 4BDS [27]). Before the docking experiment the ligand and water molecules were removed from the protein structure. The three-dimensional structure of compound 13 was optimized by the AM1 semi-empirical quantum mechanical method in MOPAC program. MGLTools was used to prepare the docking files.

Conclusions
1. Adamantyl-containing N-benzyl-and N-phenacylthiazolium salts can exhibit the inhibitory properties against BChE with IC 50 values in the micromolar range and selectivity over AChE.
2. The most effective compound 13 is a mixed inhibitor of BChE.
3. The molecular docking calculations indicate that the compound occupies the anionic and esterase binding sites of the enzyme providing hydrophobic, van der Waals and electrostatic interactions.
Conflict of Interests: authors have no conflict of interests to declare.