Synthesis, in vitro and docking studies of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one derivatives as agents for the treatment of Alzheimer's disease Chimica Techno Acta ARTICLE published by Ural Federal University 2022, vol. 9(2), No. 20229204 eISSN 2411-1414; chimicatechnoacta.ru DOI: 10.15826/chimtech.2022.9.2.04 1 of 11 Synthesis, in vitro and docking studies of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one derivatives as agents for the treatment of Alzheimer's disease Alexey S. Chiriapkin * , Ivan P. Kodonidi , Dmitry I. Pozdnyakov , Alexander A. Glushko Pyatigorsk Medical and Pharmaceutical Institute, Branch of Volgograd State Medical University, Pyatigorsk 357532, Russia * Corresponding author: prk@pmedpharm.ru This paper belongs to a Regular Issue. © 2022, the Authors. This article is published open access under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Abstract Alzheimer's disease is a chronic neurodegenerative disease, which is characterized mainly by a progressive decrease in intellectual abili- ties, memory impairment and a change in a person's personality. Un- fortunately, there are practically no medicines that act on the patho- genesis of Alzheimer's disease. The development of new highly effec- tive medicines for the treatment of this pathology is one of the cru- cial areas of pharmaceutical research. The aim of this work is to search among 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one effective compounds with anticholinesterase and antiamyloid activities. As a result, it was found that compounds 4d, 4e and 4f have the highest anticholinesterase ability, containing in their structure the residues of hydroxy-methoxyphenyl fragments. Structures 4c, 4g, 4h, 4j, 4k, 4m, 4n and 4p showed slightly lower ac- tivity, the effect of which did not differ statistically from that of Donepezil. Compounds 4c, 4e, 4k and 4m have the greatest ability to inhibit the formation of the amyloid, comparable to GV-971. It should be noted that the molecular docking data are consistent with the re- sults of the determination of the anticholinesterase activity of the studied compounds obtained in vitro. Thus, the prospects for future studies of these compounds concerning the possibility of creating a pharmaceutical active substance for the treatment of neurodegenera- tive diseases have been revealed. Keywords Alzheimer's disease tetrahydrothienopyrimidine synthesis molecular docking AChE Acetylcholinesterase anticholinesterase action amyloid medicinal chemistry Received: 08.03.22 Revised: 21.04.22 Accepted: 21.04.22 Available online: 26.04.22 1. Introduction Alzheimer's disease (AD) is one of the most common neu- rodegenerative diseases in humans. Currently, there are practically no pathogenetic drugs that can cure the pa- tient. Drug therapy is aimed only at eliminating the symp- toms of the disease and slowing its progression. The most widely used anticholinesterase (AChE) drugs that can neu- tralize the symptoms of cholinergic insufficiency. Recent- ly, the development of antiamyloid drugs that can directly affect the pathogenesis of the disease and thereby signifi- cantly improve the patient's well-being has been intensi- fied. Thus, the search for new compounds with the above properties is a cutting-edge area of medicinal chemistry and neuropharmacology [1]. Research is actively underway to develop the new ace- tylcholinesterase inhibitors. Thus, new thiazolylhydrazone derivatives were designed and synthesized as acetylcholin- esterase and butyrylcholinesterase (BChE) inhibitors. All compounds showed a weak inhibitory effect on BChE; meanwhile, most of the compounds had a certain AChE in- hibitory activity [2]. Research was carried out to study the possibility of designing acetylcholinesterase inhibitors based on isoquinolone and azepanone derivatives. Overall, the compounds studied are weak AChE inhibitors, but, nonetheless, important insights were obtained on their mode of inhibition so that more potent analogues can be designed, prepared and tested [3]. There are literature data indicating that chalcone can be used as the scaffold for cho- linesterase inhibitor [4]. Pharmacophore based 3D QSAR http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2022.9.2.04 https://orcid.org/0000-0001-8207-2953 https://orcid.org/0000-0003-1333-3472 https://orcid.org/0000-0002-5595-8182 https://orcid.org/0000-0001-7465-5657 mailto:prk@pmedpharm.ru http://creativecommons.org/licenses/by/4.0/ https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2022.9.2.04&domain=pdf&date_stamp=2022-4-26 Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 2 of 11 models for human acetylcholinesterase inhibitors with good significance, statistical values were generated. Virtual screening experiments and subsequent in vitro evaluation of promising hits revealed a novel and selective AChE inhib- itor [5]. A number of pyrimidine derivatives were synthe- sized, among which there are compounds that may be con- sidered as leaders for investigations in neurodegenerative diseases [6]. Some of diversely functionalized pyrimidine fused thiazolino-2-pyridones have an ability to inhibit the formation of amyloid-β fibrils associated with Alzheimer's disease, while others bind to mature amyloid-β and α- synuclein fibrils [7]. A new series of pyrimidine and pyri- dine diamines was designed as dual binding site inhibitors of cholinesterases, characterized by two small aromatic moieties separated by a diaminoalkyl flexible linker [8]. To obtain a multipotent framework that can target simultane- ously cyclooxygenase-2, arachidonate 5-lipoxygenase, ace- tylcholinesterase, and butyrylcholinesterase to treat neu- roinflammation, a series of derivatives containing pyrimi- dine and pyrrolidine cores were rationally synthesized and evaluated. Tacrine–pyrrolidine hybrids and tacrine– pyrimidine hybrid emerged as the most potent AChE inhibi- tors [9]. A series of 2,4-phenylsulfonyl-pyrimidine carbox- ylate derivatives was designed and synthesized. Two com- pounds among them exhibited promising AChE inhibition and significantly inhibited Aβ aggregation, that is important for anti-Alzheimer's action [10]. 2-Arylidene derivatives of thiazolopyrimidine with different linker size and target- anchoring functional groups for the treatment of AD were synthesized. Some of them showed excellent to good AChE and BChE inhibition potential at nanomolar to low mi- cromolar concentration [11]. A series of novel tetrahydropy- rimidin-4-yl)pyridine derivatives was designed and synthe- sized as inhibitors of AChE and BChE. The in vitro studies showed that all the synthesized derivatives showed signifi- cant BChE inhibitory activity and were more potent than donepezil as the standard. All the target compounds demon- strated good AChE inhibitory effects, comparable with donepezil as the reference drug [12]. 4-(Pent-4-yn-1- yloxy)phenyl)-2-phenylpyrimidine derivatives were synthe- sized and screened for monoamine oxidase and AChE inhibi- tory activities [13]. New triazolopyridopyrimidine was easi- ly prepared in good yields showing anticholinesterase inhi- bition and strong antioxidant power, which allows using new hit-triazolo pyridopyrimidine for AD therapy [14]. Previously, we studied the biological activity of azome- thine derivatives of 2-amino-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide, which are acyclic precur- sors of 2-substituted 5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one. The results show that some representatives of the studied azomethines have pronounced anticholinesterase and antiamyloid activities [15]. In this study, we decided to continue our research on finding candidates for the treat- ment of Alzheimer's disease. We decided to take 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one as the objects of the study, since their azomethine precursors demonstrated the ability to inhibit the acetylcholinesterase enzyme and the formation of the amyloid. The proposed class of organic compounds has various types of biological activity. It was found that 2-(4- Methoxyphenyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one may be an efficacious compound for the treatment of prostate cancer in advanced stages [16, 17]. Some thieno[2,3-d]pyrimidine-4(1H)-one-based analogs in- hibit the growth of human colon tumor cells [18]. Also, this class of organic compounds can suppress the production of inflammatory mediators [19]. Studies on thiophenpyrimidine derivatives with various conjugated cyclic systems showed that modification of 5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one by replacing conjugated cyclohexane with 1-methylpiperidine can increase the ability of such compounds to be used in breast cancer therapy [20]. Derivatives of thieno[2,3- d]pyrimidine-4-one may have antioxidant properties [21]. Some new thieno[2,3-d]pyrimidine-4(3H)-one derivatives showed good analgesic activity by using Eddy´s hot plate method [22]. There are data indicating that tetrahydroben- zo[4,5]thieno[2,3-d]pyrimidine scaffolds may serve as mod- els for the development of antimalarial agents [23]. Some of the 5-alkoxytetrazolo[1,5-c]thieno[2,3-e]pyrimidine deriva- tives may exhibit anticonvulsant and antidepressant effects, which makes it possible to design compounds based on them with an effect on the central nervous system [24]. A method was proposed for the synthesis of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)- one by suspending 2-amino-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide in a small amount of butanol and the corresponding aldehyde with a catalytic amount of concentrated hydrochloric acid [17]. There is a technique for obtaining compounds of this series using ZnO-CeO2 nanocom- posite as a catalyst. ZnO-TiO2 nanocomposites were added to the mixture of aminoamide and aldehyde [25]. It is possible to carry out the chemical interaction of 2-amino-4,5,6,7- tetrahydro-1-benzothiophene-3-carboxamide with aldehydes in a DMF and piperidine medium when heated [26] and in the environment of hydrochloric acid and methanol [18]. A method was proposed for the preparation of 2-substituted 5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one by adding 2-amino-3-carbethoxythiophene in anhydrous dioxane saturated with hydrogen chloride gas nitrile to a solution of 2-amino-3-carbethoxythiophene in anhydrous dioxane [20]. 2. Experimental 2.1. Molecular modeling A virtual model of human acetylcholinesterase from the RCSB Protein Data Bank database was taken as an object for molecular docking with the identification number 4EY7 [27]. The three-dimensional structures of the studied compounds were constructed in the HyperChem 8.0.4 pro- Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 3 of 11 gram and then geometrically optimized by the MM+ meth- od. The final geometry optimization of the virtual struc- tures was calculated in the ORCA 4.1 program using the density functional theory (UB3LYP) method and the 6-311G** basis set. The docking study was performed us- ing the Autodock4 program. It was set to search for 200 energetically favorable conformations of the ligand- enzyme complex formation using the Lamarckian GA 4.2 scoring function for calculating the energy of the ligand- enzyme interaction. RMSD is 0.44 Å for donepezil. Molecu- lar docking is presented in more detail in the following work [15]. 2.2. Chemistry All chemicals were acquired from Sigma-Aldrich (Sig- maAldrich, St. Louis, MO, USA), Carl Roth (Carl Roth, Karlsruhe, Germany) and Merck Chemicals (MerckKGaA, Darmstadt, Germany). Melting points (m.p.) were record- ed using the PMP-M1 melting point apparatus (Him- laborpribor, Klin, Russia). All reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 F254 TLC plates (Merck, Darmstadt, Germany). Spectroscopic data were registered with the following instruments: IR, IR-Fourier FSM 1201 spectrophotometer (Spectrum, Mos- cow, Russia); UV, SF-2000 device (Spectrum, Moscow, Russia); 1H NMR and 13C NMR, Bruker Avance III 400 МHz spectrometer (Bruker, Germany) in DMSO-d6 using tetra- methylsilane as the internal standard. Coupling constant (J) values are measured in hertz (Hz) and spin multiplets are given as follows: s (singlet), d (double), t (triplete), q (quartet), m (multiplet). 2.2.1. General procedure for synthesis of azomethine deriv- atives of 2-amino-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide (3a–3s) 0.01 mol (1.92 g) of compound 1 and the equimolar amount of the corresponding aldehyde (2) were dissolved by heating in a minimum amount of ethanol. Then the so- lutions were combined. The reaction was carried out until a precipitate was formed. It took about 30 minutes. The precipitate was filtered and purified by recrystallization from ethanol [15, 28]. 2.2.2. General procedure for synthesis of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one (4a–4s) Azomethine 3 (0.08 mol) was refluxed for 30–60 min in the glacial acetic acid. Then 1 ml DMSO was added and the reaction mixture was refluxed for 60 min. After cooling, the formed precipitate was filtered. In the filtrate the re- maining target product was precipitated with a 0.1 М cold water solution of sodium chloride. The precipitates were combined. Recrystallization of the obtained compounds was carried out from acetic acid. Compounds 4a–4j, 4p, 4q were obtained earlier [29]. 2.3. Pharmacological study 2.3.1. Evaluation of anti-amyloid activity in vitro The fragments Aß 1-42 were obtained from Sigma-Aldrich (Germany). GV-971 was provided by Hunan warrant pharm. (China). The aggregation process of amyloid parti- cles was evaluated in the reaction of the interaction of Aß with Congo red. 25 µl of a solution of the test compounds in dimethyl sulfoxide (the final concentration is 20 mg/ml, GV-971 in a similar concentration was used as a referent compound) was mixed with 225 µl of a 20 mM solution of congo red in phosphate buffer solution. The resulting mix- ture was incubated at room temperature. Then the ab- sorbance of the samples was recorded at wavelengths of 540 nm and 405 nm. after nine days of incubation. The number of aggregates Aß was calculated by the following equation on the 3rd, 6th and 9th day of the experiment: (1) where A405BL is the absorbance of the Congo red solution at a wavelength of 405 nm; A540 and A405 are the ab- sorbances of the solution containing the test substances at a wavelength of 540 nm and 405 nm, respectively. The difference between the compounds was evaluated by the ANOVA method with the Tukey post-test [30]. 2.3.2. Evaluation of anticholinesterase activity in vitro The activity of acetylcholinesterase was determined by the modified Ellman method. The analyzed medium contained 20 ml of acetylcholinesterase solution (3.2 U/l), 25 ml of a solution of the test compounds in various concentrations (30 mg/ml, 15 mg/ml, 7.5 mg/ml, 3.75 mg/ml and 1.875 mg/ml) and a potassium-phosphate buffer solution in a volume of up to 300 ml. Donepezil (KRKA, Slovenia) in similar concentrations was used as a reference sub- stance. The mixture was incubated for 5 minutes. The re- action was started by adding the acetylcholine chloride (25 µl, 0.02 M solution) and 5.5'-dithiobis-2-nitrobenzoic acid (25 µl, 0.02 M solution). The absorbance of the mix- ture was recorded after 5 minutes at 412 nm using the Infinite F50 microplate reader (Tecan, Austria). The tests were performed in a triplet version. IC50 (mg/ml) was cal- culated by probit analysis. The data is presented in the form of M±SEM (mean ± standard error of the mean). Statistical dif- ferences were evaluated at a significance level of p<0.05 by the ANOVA method with post-processing by Tukey [31]. 3. Results and Discussion 3.1. Synthesis As shown in Scheme 1, 2-amino-4,5,6,7-tetrahydro-1- benzothiophene-3-carboxamide 1 and aldehydes 2 were refluxed in ethanol to obtain azomethine derivatives 3. The reactions were performed in ethanol as a green sol- vent. Heterocyclization reaction was performed using gla- cial acetic acid and DMSO to afford the 2-substituted Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 4 of 11 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine- 4(3H)-one 4. The products 4a-4s were obtained with the high yields. The compounds were characterized by nuclear magnetic resonance and infrared spectroscopy. 3.1.1. 2-(3,5-di-tert-butyl-4-hydroxy-phenyl)-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4k) The beige crystals were obtained. Yield: 85%. M.p.: 293–294 °C. UV spectrum (ethanol), λmax, nm: 207, 337. IR spectrum (KBr), ν, cm–1: 3620 (NH), 3447 (OH, stretch- ing), 2951 (Csp3–H), 1649 (C=O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.44 (s, 18H, CH3), 1.87–1.71 (m, 4H, CH2), 2.72 (t, J = 6.0 Hz, 2H, CH2), 2.90 (t, J = 5.9 Hz, 2H, CH2), 7.60 (s, 1H, OH), 7.84 (s, 2H, ArH), 12.50 (s, 1H, NH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 22.26, 23.00, 24.96, 25.83, 35.21, 56.49, 120.46, 123.47, 131.14, 131.72, 139.13, 153.64, 157.60, 159.56, 164.00. 3.1.2. 2-(2-hydroxy-5-nitro-phenyl)-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4l) The yellow crystals were obtained. Yield: 88%. M.p.: 285–286 °C. UV spectrum (ethanol), λmax, nm: 220, 370. IR spectrum (KBr), ν, cm–1: 3466 (OH, stretching), 2939 (Csp3-H), 1657 (C=O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.90–1.57 (m, 4H, CH2), 2.78 (t, J = 5.9 Hz, 2H, CH2), 2.91 (t, J = 6.0 Hz, 2H, CH2), 7.12 (d, J = 9.1 Hz, 1H, ArH), 8.25 (dd, J = 9.2, 2.9 Hz, 1H, ArH), 8.92 (d, J = 2.9 Hz, 1H, ArH), 12.84 (s, 1H, NH). 3.1.3. 2-(5-bromo-2-hydroxy-3-methoxy-phenyl)-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4m) The brown crystals were obtained. Yield: 95%. M.p.: T>300 °C. UV spectrum (ethanol), λmax, nm: 215, 235, 282. IR spectrum (KBr), ν, cm–1: 3455 (OH, stretching), 2928 (Csp3-H), 1657 (C=O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.86–1.72 (m, 4H, CH2), 2.77 (t, J = 6.1 Hz, 2H, CH2), 2.89 (t, J = 6.1 Hz, 2H, CH2), 3.88 (s, 3H, CH3), 7.30 (s, 1H, ArH), 7.84 (s, 1H, ArH), 11.84 (s, 1H, OH), 12.27 (s, 1H, NH). 3.1.4. 2-(3-bromo-2-hydroxy-5-methyl-phenyl)-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4n) The brown crystals were obtained. Yield: 93%. M.p.: 287–288 °C. UV spectrum (ethanol), λmax, nm: 210, 390. IR spectrum (KBr), ν, cm–1: 3458 (OH, stretching), 2928 (Csp3-H), 1658 (C=O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.86–1.70 (m, 4H, CH2), 2.28 (s, 3H, CH3), 2.78 (t, J = 5.8 Hz, 2H, CH2), 2.91 (t, J = 6.0 Hz, 2H, CH2), 7.60 (s, 1H, ArH), 8.07 (s, 1H, ArH), 12.93 (s, 1H, NH), 13.24 (s, 1H, OH). a (4d) b (4i) ( 3 ) (4n) (4l) ( 1 ) (4k) (4m) ( 2 ) (4g) ( 4 ) (4j) (4o) (4s) (4h) (4r) R1 4= R7 = R1 2 = (4a) R8 = R1 1 = R1 0 = R1 3 = R1 5= R9 = R1 9 = ; ; R1 8=; (4b) ; ; ; ; (4e) ; (4f) R4=R1 = R2 = R3 = R6 = R5 =; ; ; ; (4c) R1 6 = R1 7 = (4p) (4q) ; ; ; ; ; ; Scheme 1 Reagents and conditions: (a) ethanol, reflux; (b) glacial acetic acid, DMSO, reflux. Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 5 of 11 3.1.5. 2-(3,5-dibromo-2-hydroxy-phenyl)-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4o) The brown crystals were obtained. Yield: 92%. M.p.: 290–291 °C. UV spectrum (ethanol), λmax, nm: 211, 389. IR spectrum (KBr), ν, cm–1: 3429 (OH, stretching), 2928 (Csp3-H), 1653 (C=O). 1H NMR spectrum (400 MHz, DMSO- d6), δ, ppm: 1.88–1.72 (m, 4H, CH2), 2.78 (t, J = 5.9 Hz, 2H, CH2), 2.89 (t, J = 5.8 Hz, 2H, CH2), 7.94 (d, J = 2.4 Hz, 1H, ArH), 8.44 (d, J = 2.3 Hz, 1H, ArH), 13.04 (s, stretching, 2H, OH, NH). 3.1.6. 2-(5-iodo-2-furyl)-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4r) The brown crystals were obtained. Yield: 79%. M.p.: 297–298 °C. UV spectrum (ethanol), λmax, nm: 218, 283, 353. IR spectrum (KBr), ν, cm–1: 3436 (NH, stretching), 2928 (Csp3-H), 1649 (C=O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.84–1.74 (m, 4H, CH2), 2.75 (t, J = 6.1 Hz, 2H, CH2), 2.88 (t, J = 6.2 Hz, 2H, CH2), 6.97 (dd, J = 3.5, 1.6 Hz, 1H, ArH), 7.54–7.47 (m, 1H, ArH), 12.51 (s, 1H, NH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 22.20, 22.89, 25.02, 25.76, 98.87, 121.46, 131.53, 133.22, 143.14, 150.73, 158.55. 3.1.7. 2-[5-(4-nitrophenyl)-2-furyl]-5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3d]pyrimidine-4(3H)- one (4s) The brown crystals were obtained. Yield: 78%. M.p.: T>300 °C. UV spectrum (ethanol), λmax, nm: 204, 219, 399. IR spectrum (KBr), ν, cm–1: 3447 (NH, stretching), 2932 (Csp3-H), 1645 (C=O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.85–1.71 (m, 4H, CH2), 2.75 (q, J = 3.9, 2.3 Hz, 2H, CH2), 2.91 (t, J = 5.6 Hz, 2H, CH2), 7.53 (d, J = 3.7 Hz, 1H. ArH), 7.60 (d, J = 3.8 Hz, 1H, ArH), 8.35–8.25 (m, 4H, ArH), 12.84 (s, 1H, NH). 13C NMR spec- trum (100,6 MHz, DMSO-d6), δ, ppm: 22.19, 22.88, 25.05, 25.80, 113.03, 116.85, 121.65, 124.70, 125.76, 131.65, 133.27, 135.32, 143.75, 147.07, 147.33, 154.00, 158.65, 162.98. 3.2. Docking studies Based on the results of computational experiment, molecu- lar complexes were selected, in which the simulated com- pounds occupy the most energetically advantageous loca- tion in the active site of the acetylcholinesterase enzyme. 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one mainly formed bonds with the following amino acid residues of the active site of AChE: Tyr 124, Trp 286, Val 294, Phe 295, Arg 296, Phe 297, Tyr 337, Phe 338, Tyr 341 and His 447. Table 1 shows the minimum energies for the formation of ligand complexes with the active site of AChE and the hydrogen bonds. Figure 1 and 2 shows locations of 4a–4s according to molecular docking. Table 1 Results of molecular docking experiments for compounds 4a–4s, donepezil and its hydrogen bonds. Compound AutoDock binding energy (kcal/mol) Residue Ligand atoms Distance (Å) 4a –9.05 – – – 4b –10.16 Arg 296 OH 1.979 Ser 293 OH 2.141 4c –9.23 Ser 293 OH 2.041 4d –9.55 Phe 295 OCH3 2.199 4e –10.29 Arg 296 OH 1.679 4f –10.29 Phe 295 OH 1.895 Arg 296 OCH3 2.146 4g –9.95 Ser 293 OH 1.804 Arg 296 OH 1.898 Arg 296 OH 1.895 4h –9.55 – – – 4i –9.48 – – – 4j –10.86 Phe 295 C=O 2.225 4k –10.30 – – – 4l –9.67 Phe 295 C=O 2.155 Arg 296 OH 2.064 Arg 296 OH 2.188 4m –11.64 Arg 296 OCH3 2.211 Phe 295 OH 2.153 4n –10.52 Phe 295 C=O 2.128 Ser 293 OH 1.822 4o –10.84 Phe 295 OH 2.104 4p –9.05 Arg 296 FurO 1.891 4q –8.93 Phe 295 C=O 1.700 4r –9.71 Phe 295 –S– 2.222 4s –10.67 Arg 296 NO2 1.903 donepezil –11.89 Phe 295 C=O 1.770 The compounds 4b, 4c, 4g and 4n form a hydrogen bond between their hydroxy groups and the amino acid residue Ser 293. The structures 4b, 4e, 4g and 4l by the same structural fragment can make a hydrogen bond with Arg 296. The compounds 4p and 4s form a hydrogen bond with Arg 296 by oxygen atoms of the furan heterocycle and the nitro group, respectively. It is often seen that the simulated compounds can form a hydrogen bond in a lig- and-enzyme complex with Phe 295. 4j, 4l, 4n and 4q inter- act with Phe 295 with their carbonyl groups, and 4r mole- cule forms a hydrogen bond between Phe 295 and the sul- fur atom of the thiophene heterocycle. Three compounds among the simulated structures 4f, 4m and 4o with the above amino acid residue interact with the oxygen atom of the hydroxy group of the aryl fragment of the molecule. It follows from the docking results that compounds 4f and 4m can form a hydrogen bond with the Arg 296 oxygen atom by the methoxy group, and in the structure of 4d similarly structural fragments interacts with Phe 295 by forming a hydrogen bond with a length of 2.199 Å. Accord- ing to the molecular docking data for 4a, 4h, 4i and 4k, the formation of hydrogen bonds is not observed. Donepezil makes a hydrogen bond between the oxygen atom of the carboxyl group of the five-membered cycle of the molecule and the amino acid Phe 295 of the active site of the en- Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 6 of 11 zyme. Many 2-substituted 5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one form a hydrogen bond with Phe 295, as does the donepezil. There is no non-bonding interactions such as van der Waals, π–π, π–alkyl according to this docking protocol. 3.3. Pharmacological studies The results of the antiamyloid activity evaluation of the test substances are presented in Table 3. As can be seen from the data in Table 3, the compounds 4c, 4e, 4k and 4m have the highest ability to inhibit the formation of β-amyloid in the model mixture. At the same time, the compounds 4c, 4e significantly suppressed the process of amyloidogenesis after 3 days of incubation. It is worth noting that on the 9th day of the experiment, all the leading compounds showed a comparable level of pharma- cological efficacy, which, however, was lower than that of GV-971. 4a 4b 4c 4d 4e 4f 4g 4h Figure 1 The location of 4a–4h according to molecular docking. Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 7 of 11 4i 4j 4k 4l 4m 4n 4o 4p 4q 4r 4s Figure 2 The location of 4i–4s according to molecular docking. As can be seen from the data obtained, the highest an- ticholinesterase activity was established for the com- pounds 4d, 4e and 4f, surpassing that of the referent. The substances 4c, 4g, 4h, 4j, 4k, 4m, 4n and 4p showed slight- ly lower activity, the effect of which did not differ statisti- cally from that of donepezil. 3.4. Structure-activity relationship of the studied compounds In general, the results of molecular docking of the predict- ed structures are in a good agreement with the results of the primary pharmacological screening of anticholinester- Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 8 of 11 ase activity in vitro of 2-substituted 5,6,7,8- tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)-one. The most active compounds inhibiting AChE are thienopy- rimidines, containing in the second position of the hetero- cycle pyrimidine-4(3H)-one fragment with hydroxy and methoxyphenyl substituents (4d, 4e and 4f). Table 2 The effect of the studied compounds and GV-971 on the aggregation of amyloid particles. Compounds % of inhibition 3th day 6th day 9th day 4a 10.2±1.2* 34.5±1.6* 67.5±2.3* 4b 10.7±1.0* 42.5±2.5* 55.1±2.0* 4c 24.5±3.9* 32.2±3.8* 66.4±1.2* 4d 14.2±2.1* 38.6±2.4* 57.9±1.2* 4e 22.2±1.5* 39.1±2.2* 67.2±3.6* 4f 16.8±3.8* 38.1±1.5* 60.8±3.7* 4g 13.9±3.7* 33.7±2* 60.4±2.9* 4h 13.4±3.9* 42.9±3.6* 57.3±3.6* 4i 15.5±1.6* 36.5±3.7* 62.7±1.3* 4j 12.3±1.5* 45±2.9* 50±1.8* 4k 16.4±1.6* 55.3±2.3* 69.4±2.5* 4l 18.7±2.6* 38±2.9* 50±1.2* 4m 21.1±2.7* 49±3.5* 72.8±1.9* 4n 16.9±1.6* 31.2±3.3* 60.4±2.4* 4o 16.2±2.8* 34.9±3.1* 55.9±3.9* 4p 22.2±3.2* 43±1* 54.7±3.4* 4q 18.1±1.6* 32.2±2.8* 58±4* 4r 23.5±2.6* 41.3±3.4* 52.3±1.2* 4s 15.1±2.1* 41.2±3.4* 52.9±1.4* GV-971 33.5±2.4 65.2±3.9 86.3±2.5 * – statistically significant relative GV-971 (ANOVA with the Tuk- ey post-test, p<0,05) Table 3 The effect of the studied compounds and donepezil on the acetylcholinesterase activity. Compounds IC50, mg/ml 4a 6.31±0.091* 4b 5.36±0.087* 4c 3.10±0.031 4d 1.17±0.064* 4e 1.24±0.027* 4f 1.11±0.044* 4g 3.08±0.084 4h 3.75±0.058 4i 5.99±0.021* 4j 4.52±0.034 4k 3.19±0.044 4l 5.42±0.012* 4m 3.22±0.021 4n 3.68±0.092 4o 5.23±0.061* 4p 3.75±0.071 4q 5.82±0.025* 4r 4.92±0.074* 4s 4.57±0.096* donepezil 2.40±0.06 * – statistically significant relative donepezil (ANOVA with the Tukey post-test, p<0,05) These compounds are superior in the effectiveness to the drug Donezepil. It should be noted that for the acyclic pre- cursors of azomethine derivatives of 2-amino-4,5,6,7- tetrahydro-1-benzothiophene-3-carboxamide, substances with similar substituents showed better activity. This fact indicates the significance of these pharmacophores. To a lesser extent, the 4c and 4g substances containing only hydroxyphenyl groups as a pharmacophore fragments ex- hibit the anticholinesterase activity. Among the com- pounds having a furan heterocycle, the compound 4p has the greatest ability to inhibit AChE. The analysis of thecompounds 4j and 4k containing a tert-butyl radical in a hydroxyphenyl fragment allows us to judge its effect on the pharmacological properties of these structures. Partic- ularly interesting is the remainder of the sterically hin- dered phenol contained in the 4k compound. Among the halogen-derived target products, 4o containing two bro- mine atoms in the 3,5 positions of the phenyl substituent showed the least activity. Compounds that do not contain hydroxy, methoxy and bromophenyl substituents have weak inhibitory properties of AChE, which is in a good agreement with the results of molecular docking and con- firms the revealed tendency of the influence of electron- donating substituents in the 2-substituted phenyl fragment of the condensed thiophenpyrimidine system. Figure 3 shows the location of donepezil and 4d in the active site of AChE. Figure 3 The location of donepezil determined by X-ray diffraction analysis (blue color) and the location of 4d according to molecular docking (green color). Figure 4 shows the location of the donezepil molecule corresponding to the data of X-ray diffraction analysis in the 4EY7 molecular complex and the positions of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine-4(3H)- one with hydroxy-methoxyphenyl fragments. Figure 4 The location of donepezil determined by X-ray diffraction analysis (A – red color) and the location according to molecular dock- ing: 4d (B – blue color), 4e (C – green color), 4f (D – yellow color). Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 9 of 11 It can be seen that the aryl fragments 4d, 4e and 4f with methoxy and hydroxy groups occupy a similar posi- tion with the same structural element of Donezepil. Thus, it is possible to assume similar molecular mechanisms of inhibition of AChE in the predicted compounds and their prototypes, as well as the importance of the hydroxy- methoxyphenyl fragment for the process of inhibition of the enzyme. The study of the ability of synthesized compounds to aggregate amyloid particles allowed us to determine that the most active are tetrahydrothienopyrimidines with 5- bromo-2-hydroxy-3-methoxyphenyl (4m) and 3,5-di-tert- butyl-4-hydroxyphenyl (4k) substituents containing di- tert-butyl and bromine-substituted hydroxy- methoxyphenyl fragments in the second position of the pyrimidine-4(3H)-one heterocycle. Of the compounds with hydroxy-methoxyphenyl substituents, the substance 4e containing an isovaniline residue in its structure showed the greatest activity. The compound 4a, which has an unsubstituted phenyl substituent, also inhibits the aggregation of amyloid particles well. The resulting combination of pharmacological proper- ties of the studied objects, namely, the combination of the ability to suppress amyloidogenesis and anticholinestrease activity, opens up certain prospects in terms of the thera- peutic use of these compounds. So, it is known that amy- loidogenic processes underlie irreversible neurodegenera- tive diseases, in particular, Alzheimer's disease [32]. The development of drugs for the treatment of Alzheimer's disease is an extremely difficult task. Since 2003, exten- sive preclinical and clinical studies of promising molecules have been conducted, but none of them has been put into practice. As of 2021, not a single drug has been registered that directly affects the pathogenesis of the disease. But, at the same time, a purposeful search for substances that can prevent a neurodegeneration is ongoing [33]. Accord- ing to Cummings et al., the most promising direction for the development of new therapeutic agents for the treat- ment of Alzheimer's disease is the suppression of the for- mation of β-amyloid. The most promising in this regard are purposefully obtained monoclonal antibodies, which are at different stages of clinical trials: Solanezumab; Gan- tenerumab; Crenezumab; Aducanumab [34]. But it is im- possible to deny the possibility of using small molecules to suppress the formation of amyloid fragments. It should be emphasized that in addition to pathogenetic, symptomatic treatment is also important, which, as a rule, is aimed at eliminating cholinergic deficiency [35]. In this regard, the combination of pharmacological properties of 2- substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one may be a new vector of therapy for Alzheimer's disease, combining both the effect on the pathogenesis of the disease and the elimination of its lead- ing symptoms. 4. Conclusions In the course of the research, a method for the synthesis of 2-substituted 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3- d]pyrimidine-4(3H)-one was proposed, according to which new representatives of this class of organic compounds were obtained. Among the studied compounds there are substances with the high anticholinesterase activity. The most active are tetrahydrothienopyrimidine derivatives containing hydroxy-methoxyphenyl substituents in their structure. The compounds with fragments of 5-bromo-2- hydroxy-3-methoxyphenyl and 3,5-di-tert-butyl-4- hydroxyphenyl have the highest antiamyloid activity. As a result of the studies, the expediency of searching for new highly effective compounds for the treatment of neuro- degenerative diseases in the series of tetrahydro- benzthienopyrimidine-4(3H)-one was confirmed. Supplementary materials No supplementary materials are available. Funding The reported study was funded by RFBR, project No. 20-315-90060. Acknowledgment None. Author contributions Conceptualization: I.P.K Data curation: I.P.K. Formal Analysis: A.S.C., I.P.K., D.I.P., A.A.G. Funding acquisition: A.S.C., I.P.K. Investigation: A.S.C., I.P.K., D.I.P., A.A.G. Methodology: A.S.C., I.P.K., D.I.P. Project administration: I.P.K. Resources: A.S.C., I.P.K., D.I.P. Software: D.I.P., A.A.G. Supervision: I.P.K. Validation: A.S.C., D.I.P. Visualization: A.S.C., D.I.P., A.A.G. Writing – original draft: A.S.C., I.P.K., D.I.P., A.A.G. Writing – review & editing: A.S.C., A.A.G. Conflict of interest The authors declare no conflict of interest. Chimica Techno Acta 2022, vol. 9(2), No. 20229204 ARTICLE 10 of 11 Additional information Author ID’s: A.S. Chiriapkin, Scopus ID 57218134815; I.P. Kodonidi, Scopus ID 10240218600; D.I. Pozdnyakov, Scopus ID 57190954589; A.A. Glushko, Scopus ID 7003386007. Institute’s website: Pyatigorsk Medical and Pharmaceutical Institute, https://www.pmedpharm.ru/sveden_eng. References 1. Weber SA, Patel RK, Lutsep HL. 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