Synthesis and evaluation of cerebroprotective activity of novel 6,7-dimethoxyquinazolin-4(3H)-one derivatives containing residues of amino acids and dipeptides Chimica Techno Acta ARTICLE published by Ural Federal University 2022, vol. 9(2), No. 20229212 eISSN 2411-1414; chimicatechnoacta.ru DOI: 10.15826/chimtech.2022.9.2.12 1 of 12 Synthesis and evaluation of cerebroprotective activity of novel 6,7-dimethoxyquinazolin-4(3H)-one derivatives containing residues of amino acids and dipeptides Alexey S. Chiriapkin * , Ivan P. Kodonidi , Dmitry I. Pozdnyakov 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 Neurodegenerative processes of the central nervous system are an important so- cially significant problem of modern society. They cause many diseases, such as Alzheimer's disease and cerebral ischemia, which significantly reduce the quality of human life and can lead to disability or death. The aim of this study was to synthesize novel 6,7-dimethoxyquinazolin-4(3H)-one derivatives with the re- mains of neuroactive amino acids and dipeptides in order to investigate their cere- broprotective properties. As a result of the study, 13 novel 6,7-dimetho- xyquinazolin-4(3H)-one derivatives were synthesized. Cerebral ischemia in rats was reproduced by irreversible right-sided occlusion of the middle cerebral artery using the Tamura method, and the area of brain necrosis was evaluated. Cognitive functions were evaluated in the Y-maze test. Among the studied quinazolinone derivatives, compounds 3i, 3j and 3k have the most pronounced cerebrotropic ac- tivity, which is not inferior to ethylmethylhydroxypyridine succinate in terms of pharmacological activity, making them promising objects for further research. Keywords quinazolinones synthesis medicinal chemistry cerebroprotective activity ischemia amino acid dipeptide Received: 15.05.22 Revised: 10.06.22 Accepted: 13.06.22 Available online: 16.06.22 1. Introduction Disorders of the functioning of the central nervous system are an important socially significant problem of modern so- ciety. Every year there is an increase in patients with vari- ous cognitive impairments, which defines the urgency of developing new methods of their treatment. The main causes of cognitive dysfunctions are vascular pathologies (cerebral ischemia [1]), neurodegenerative processes (such as in the case of Alzheimer's disease) [2], traumatic brain injuries [3] and neuroinfections [4]. It is worth noting that disorders of cerebral circulation are the second most com- mon cause of cognitive dysfunction after Alzheimer's dis- ease [5]. Cerebral ischemia forms insufficient oxygen and glucose delivery for the metabolic processes of neurons, which leads to disruption of cellular homeostasis, oxidative stress, inflammation and death of brain cells [6]. Vascular pathologies of the brain can provoke an ischemic stroke, which often leads the patient to disability or death. The cause of a stroke can also be an acute traumatic brain injury or a brain dysfunction. Modern methods of treatment of is- chemic and other pathological conditions of the human cen- tral nervous system include intravenous thrombolysis and the use of neuroprotective and anti-inflammatory drugs. Despite this, in most patients with an unfavorable progno- sis of the course of the disease, there is a malfunction and rapid death of brain cells [7]. Based on this, an important task for pharmacy and medicine is the development of new highly effective and low-toxic drugs for the treatment of neurodegenerative processes. One of the most promising classes of organic compounds is quinazolinone derivatives, which have a diverse spec- trum of biological activity. So, series of kojyl thioether con- jugated to different quinazolinone derivatives were de- signed, synthesized, and evaluated for their inhibitory ac- tivity against mushroom tyrosinase. All derivatives dis- played better potency than kojic acid as the positive control [8]. Novel quinazolinone derivatives bear benzenesulfona- mide moiety with variable heterocyclic tail. One of them showed antioxidant and hepatoprotective activities in irra- diated mice [9]. Novel iodinated quinazolinones bearing sulfonamide were synthesized. Among them there is a com- pound that acts as a direct antioxidant by scavenging reac- tive oxygen species and inhibiting radiation-induced oxida- tive stress [10]. 3-Aryl-8-methylquinazolin-4(3H)-ones have promising properties for drug development against keratitis and brain infection causing free-living amoeba, A. http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2022.9.2.12 mailto:prk@pmedpharm.ru http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1016/j.bmc.2021.116044 https://doi.org/10.1016/j.ejmech.2020.112333 https://www.sciencedirect.com/topics/chemistry/antioxidant-agent https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/keratitis https://orcid.org/0000-0001-8207-2953 https://orcid.org/0000-0003-1333-3472 https://orcid.org/0000-0002-5595-8182 https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2022.9.2.12&domain=pdf&date_stamp=2022-6-16 Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 2 of 12 Castellanii [11]. Antimicrobial activity of substituted-6-me- thyl-1-thioxo-1,2-dihydro-3H-furo[3,2-g]pyrimido[1,6- a]quinazolin-3-ones was evaluated against Gram-positive, Gram-negative bacteria and fungi. Furothiazolo pyrimido quinazolines displayed results excellent for growth inhibi- tion of bacteria and fungi [12]. Most of the different substituted quinazolinones exhib- ited moderate to high anticonvulsant activity in all seizure models with no symptoms of neurotoxicity and hepatotoxi- city [13]. There are data indicating the ability of some quinazolinone inhibit activity of the urease [14]. Novel quinazolinones conjugated with indole acetamide, ibu- profen or thioacetohydrazide were designed to increase cy- clooxygenase-2 (COX-2) selectivity. The three synthesized series exhibited superior COX-2 selectivity compared with the previously reported quinazolinones and their nonsteroi- dal anti-inflammatory drug analogue and had equipotent COX-2 selectivity as celecoxib [15]. Some novel compounds of allyl/benzyl quinazolinone displayed remarkable anti-in- flammatory activity as compared to Diclofenac sodium [16]. Also, different 2,7-disubstituted[1,3,4]-tiadiazolov[2,3- b]quinazolin-5(4H)-ones were synthesized, and they showed good anti-inflammatory activity [17]. Various 2-(3- aryl-1H-pyrazol-1-yl)benzo[d]thiazole incorporated fused thiazolo[2,3-b]quinazolinones exhibited prominent anti- cancer and antibacterial activities. The potent compounds could serve as templates for further development of anti- cancer and antibacterial drugs [18]. A series of novel quinazoline derivatives were synthesized and evaluated for their anticonvulsant activity against electrically and chem- ically induced seizures, compared with that of the standard drugs methaqualone and sodium valproate [19]. A novel series of 4-anilinoquinazoline analogues, DW (1–10), were evaluated for anticancer efficacy in human breast cancer (BT-20) and human colorectal cancer cell lines (HCT116, HT29, and SW620). DW8 may represent a suitable lead for developing novel compounds to treat the human colorectal cancer [20]. The preparation and screen- ing of the focused libraries of 4-anilinoquinolines and 4-anilinoquinazolines for antiviral activity allowed identi- fying three potent compounds. N-(2,5-dimethoxyphenyl)-6- (trifluoromethyl)quinolin-4-amine effectively inhibited dengue virus infection, and N-(3,4-dichlorophenyl)-6-(tri- fluoromethyl)quinolin-4-amine and N-(3-ethynyl-4-fluoro- phenyl)-6,7-dimethoxyquinazolin-4-amine successfully in- hibited Venezuelan equine encephalitis virus. These results provide a prospect of developing a clinical compound against these emerging viral threats [21]. A series of novel histone deacetylase inhibitors using a substituted quinazo- line was designed and synthesized as the capping group, at- taching 3, 5-dimethyl pentyl as a potential metabolic site protector. One of representatives was proposed as an oral histone deacetylase inhibitor with a potential capacity of treating breast cancer [22]. There is evidence indicating that novel 1,2,3-triazole-quinazolines can be used as anti- proliferative agents displaying the extracellular signal-reg- ulated kinase inhibitory activity [23]. Approximately 150 different 6,7-dimethoxyquinazoline-2,4-diamines were synthesized, and via structure-activity relationship studies. Among them 6,7-dimethoxy-N4-(1-phenylethyl)-2-(pyrroli- din-1-yl)quinazo-lin-4-amine exhibits high antimalarial ac- tivity as a promising antimalarial drug lead [24]. Two new series of synthesized quinazolinone derivatives were inves- tigated as potential future antileishmanial agents, by as- sessing their activities against the Leishmania (L.) do- novani and L. major species. The two compounds that were found the most active are the mono quinazolinone and the bisquinazolinone with growth inhibitory efficacies of 35% and 29% for the L. major and L. donovani, respectively [25]. Based on quinazolin-4(3H)-one derivatives, a series of new dual-target inhibitors of poly (ADP-ribose) polymer- ase-1 (PARP 1) and bromodomain containing protein 4 (BRD4) were successfully synthesized. Among them are compounds that would be promising for the treatment of breast cancer [26]. As a result of the structural modification of the molecular-targeted agent sorafenib, a series of quinazolinyl-arylurea derivatives were synthesized and evaluated for their anti-proliferative activities against six human cancer cell lines. N-(3,4-dichlorophenyl)-N’-(3- ((quinazolin-4-ylamino)methyl)phenyl)urea could trigger three different cell death forms including apoptosis, ferrop- tosis and autophagy. This compound could be a promising lead for molecular-targeted anti-bladder cancer agents dis- covery [27]. Thus, quinazoline derivatives are promising compounds for the search for biologically active sub- stances, including those with neuroprotective properties. The most commonly used amino acid for the treatment of diseases of the central nervous system is glycine, acute ischemic stroke [28]. Also, in case of brain disorders, gamma-aminobutyric acid and its cyclic derivatives – race- tams are actively used [29]. The scientific literature pre- sents the results of studies that indicate high cerebropro- tective properties of a number of low-molecular-weight peptides. So, the novel 14-amino acid peptide, with stress- protein-like sequences exhibiting neuroprotection at un- precedented concentrations was revealed. This peptide pre- vented neuronal cell death associated with the envelope protein (GP 120) from Human immunodeficiency virus, with excitotoxicity (N-methyl d-aspartate), with the beta amyloid peptide (putative cytotoxin in Alzheimer's dis- ease), and with tetrodotoxin (electrical blockade) [30]. It was found that lipophilic peptide fragments offer bioavail- ability and stability, providing lead compounds for drug de- sign against neurodegenerative diseases [31]. Cationic argi- nine-rich peptides (CARPs) are an expanding and relatively novel class of compounds possessing intrinsic neuroprotec- tive properties [32]. The neuroprotective efficacy of differ- ent modifications to the poly-arginine-9 peptide (R9) was examined [33]. The new synthetic peptide, CMX-9236, can function as a neuroprotective agent and an activator of a https://doi.org/10.1016/j.ejmech.2019.111575 https://doi.org/10.1080/00397911.2018.1490433 https://doi.org/10.1080/14756366.2021.1956912 https://doi.org/10.1016/j.jics.2021.100033 https://doi.org/10.1039/C6RA01284A https://doi.org/10.1073/pnas.96.7.4143 https://doi.org/10.3389/fneur.2020.00108 Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 3 of 12 beneficial signal transduction pathway in in vitro and in vivo models of cerebral ischemia [34]. 2. Experimental 2.1. Chemistry All chemicals were acquired from Sigma-Aldrich (SigmaAl- drich, St. Louis, MO, USA), Carl Roth (Carl Roth, Karlsruhe, Germany) and Merck Chemicals (MerckKGaA, Darmstadt, Germany). Melting points (m.p.) were recorded using the PMP-M1 melting point apparatus (Himlaborpribor, Klin, Russia). All reactions were monitored by thin-layer chro- matography (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, Moscow, 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 tetramethylsilane as the internal standard. Coupling constant (J) values were measured in hertz (Hz) and spin multiplets are given as fol- lows: s (singlet), d (double), t (triplete), q (quartet), m (multiplet). 2.1.1. General procedure for synthesis of amides of 2- amino-4,5-dimethoxybenzoic acid (3, 4) 0.05 mol of the acetyl chloride or benzoyl chloride was slowly added to a solution of 9.85 g (0.05 mol) of 2-amino- 4,5-dimethoxybenzoic acid (2) in 35 ml of 10% sodium hy- droxide with stirring. As a result, a precipitate was formed, which was filtered and washed with water. Purification of the obtained substances is carried out by recrystallization from ethyl alcohol. 2.1.2. General procedure for synthesis of 2-alkyl-6,7-di- methoxy-3,1-benzoxazine-4-ones (2a–2e) 5.91 g (0.03 mol) of 2-amino-4,5-dimethoxybenzoic acid was dissolved at boiling in 25 ml of the corresponding an- hydride and the reaction was carried out for 5 hours. In the case of 2d-2e compounds, the reaction was carried out in a similar way, and 0.03 mol of amides of 2-amino-4,5-di- methoxybenzoic acid (3 and 4) were used as starting sub- stances. The reaction mixture was kept in the refrigerator for a day; the resulting precipitate was filtered and washed with water. The product was purified by recrystallization from ethyl alcohol. 2.1.3. General procedure for synthesis of 6,7-dimetho- xyquinazolin-4(3H)-one derivatives containing resi- dues of amino acids and dipeptides (3a–3m) A mixture of 0.01 mole of the corresponding 2-alkyl-6,7-di- methoxy-3,1-benzoxazine-4-one (2a–2e) and 0.01 mole of amino acid or dipeptide was dissolved at boiling in 10-15 ml of glacial acetic acid. Next, 0.5 ml of DMF was added and the reaction was carried out with stirring for 1.5 hours. The reaction mixture was kept in the refrigerator for a day; the resulting precipitate was filtered and washed with water. The product was purified by recrystallization from ethyl al- cohol. 2.2. Pharmacological study 2.2.1. Animals The study was performed on 96 mature male Wistar rats. The animals were obtained from the laboratory animal nursery "Rappolovo" (Leningrad region) and for the dura- tion of the experiment were kept in a vivarium, 6 individ- uals per cage at an air temperature of 20±2 0C, relative humidity of 60±5% and a twelve-hour daily cycle (12 hours day/12 hours night). Until the moment of inclu- sion in the work, the rats were kept in a quarantine room for 2 weeks. Animals received a complete dry food diet (granulated food) and water ad libitum. The design of the study and the manipulations performed with the animals were in accordance with the international ethical princi- ples of working with laboratory animals, as set out in the ARRIVE 2.0 guidelines [35]. 2.2.2. Cerebral ischemia model Cerebral ischemia in rats was reproduced by irreversible right-sided occlusion of the middle cerebral artery accord- ing to a Tamura, 1981. Animals were anesthetized by intra- peritoneal injection of chloral hydrate at a dose of 350 mg/kg, the area below and to the right of the eye was depilated, the skin and soft tissues were dissected. Next, a burr hole was made over the intersection of the artery with the olfactory tract, then the artery was coagulated by an electrocoagulator. The soft tissue topography was restored, the wound was sutured. The suture was treated with 10% povidone-iodine [36]. 2.2.3. Study design The cerebroprotective activity of the tested substances was evaluated during therapeutic administration. When setting up the study, the following experimental groups were dis- tinguished: SO – sham-operated animals, to which all se- quential procedures were applied with the exception of the coagulation of the middle cerebral artery; NC – a group of rats with cerebral ischemia, but without pharmacological correction (during the experiment, this group of animals received purified water); a group of rats that were treated by the reference drug and groups of rats that received the test substances. A total of 16 groups of 6 individuals each were formed. Ethylmethylhydroxypyridine succinate (EMHPS, «Mexidol», FARMASOFT, Russia) was used as a reference drug at a dose of 100 mg/kg, orally [37]. The test compounds were administered at a screening dose of 40 mg/kg orally. The reference and tested compounds were administered 30 min after ischemia modeling and then for 72 h (one administration per day). On the 4th day of the ex- periment, the changes of cognitive functions of rats in the Y-maze test were evaluated. After that, the animals were decapitated under anesthesia and the brain was removed, in which the change in the volume of necrotic tissue was assessed. Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 4 of 12 2.2.4. Y-maze test The setup consisted of three equal arms connected at an an- gle of 1200. The animal was placed in the center of the setup, and the number of animal movements between the arms was recorded for 8 minutes. At the same time, spon- taneous alternating entries into the arms (1–2–3, 3–1–2, 2–3–1) were recorded. Based on the data obtained, the per- centage of spontaneous alternation was determined (for- mula (1)), which reflects the change in the cognitive abili- ties of animals [38]: Percentage of spontaneous alternation = Number of alternating entries into the arms Total number of movements 100 (1) 2.2.5. Brane necrosis zone determination The size of the necrosis zone was determined by the change in the color intensity of triphenyltetrazolium chloride. The course of determination was as follows: after decapitation of the animals, the brain was removed, the cerebellum was cut off and the hemispheres were separated along the cen- tral sulcus. Both hemispheres were weighed, then individ- ually homogenized and placed in weighing bottles. To the homogenate was added 10 ml of a 1% solution of triphenyl- tetrazolium chloride in phosphate buffer (pH 7.4). The bot- tles were incubated in a water bath at 37 °C for 20 min. Af- ter that, the samples were centrifuged at 5000 RPM/10 min, and the supernatant was removed. 3 ml of phosphate buffer and 3 ml of cold chloroform were added to the precipitate. The mixture was shaken for 2 min and then incubated for 15 min at 4 °C, shaking every 5 min for 30 s, re-centrifuged in the same mode, and its optical density was measured against pure chloroform at 492 nm. The calculation of the necrosis zone was performed according to the formula (2) and expressed as a percentage of the total mass of the hem- ispheres: 𝑥 = 100 − 𝜀1𝑀1 + 𝜀2𝑀2 𝜀1(𝑀1 + 𝑀2) ∙ 100, (2) where x is the size of the necrosis zone as a percentage of the total brain mass; ε1 is the optical density of the sample with an undamaged hemisphere; ε2 is the optical density of the sample with the damaged hemisphere; M1 is the mass of the undamaged hemisphere; M2 is the mass of the dam- aged hemisphere. 2.2.6. Statistical analysis Statistical processing of the obtained results was carried out using the software package STATISTICA 6.0 (StatSoft, USA). The data were expressed as M (mean) ± SEM (stand- ard error of mean). The Gaussian distribution was tested by the Shapiro-Wilk test. The homogeneity of the variance was assessed in the Levene test. One-way analysis of variance (ANOVA) with post-processing by Tukey’s test (in the pres- ence of a Gaussian distribution) or Kruskal - Wallis (in the absence of Gaussian distribution), carried out by the com- parison of groups. The critical level of significance was taken as p<0.05. 3. Results and Discussion 3.1. Synthesis A convenient way to synthesize 6,7-dimethoxyquinazoline- 4(3H)-one is the replacement of the oxygen heteroatom with nitrogen in the intermediate 2-methyl-6,7-dimethoxy- 3,1-benzoxazine-4-one (Scheme 1), which was previously developed with the staff of The research-scientific Institute of Physical & Organic Chemistry of Rostov University of Southern Federal University. A new modification of the syn- thesis was proposed based on the interaction of ni- trohomoveratric acid (1) and polyphosphoric acid – PPA (2) with subsequent treatment of the reaction mixture with a small amount of acetic anhydride, which leads to the for- mation of 2-methyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2d) through the intermediate 2-acetamido-4,5-dimethoxy- benzoic acid (3) [39]. However, the yield of 2-methyl-6,7-dimethoxy-3,1-ben- zoxazine-4-one products is low, because a redox reaction of disproportionation occurs at the first stage of obtaining 2-acetamido-4,5-dimethoxybenzoic acid. Another negative factor in obtaining the intermediate 2-methyl-6,7-di- methoxy-3,1-benzoxazine-4-one is the use of acetic anhy- dride, which is a controlled substance (Narcotic drugs, psy- chotropic substances and their precursors, controlled in the Russian Federation), and this complicates the possibility of developing laboratory regulations for the synthesis of an active pharmaceutical substance. Ac 2 OAc2O 1 2 3 2d PPA Scheme 1 Synthesis of 2-methyl-6,7-dimethoxy-3,1-benzoxazine-4-one using acetic anhydride. Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 5 of 12 Based on this, for the synthesis of 2-substituted ethyl, propionyl and isopropionyl 6,7-dimethoxy-3,1-benzoxa- zine-4-one, an acylation process with further heterocycliza- tion should be used (Scheme 2). This process proceeds by boiling dimethoxyanthronylic acid in the corresponding an- hydride without the release of intermediate amides of di- methoxyanthranylic acid, which leads to the formation of intermediate 2-ethyl, 2-propyl and 2-isopropyl-6,7-di- methoxy-3,1-benzoxazine-4-one. At the first stage of syn- thesis, 2-amino-4,5-dimethoxybenzoic acid is heated in ex- cess of propionic, butyric and isobutyric anhydride. As a re- sult, 2-ethyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2a), 2-propyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2b) and 2-isopropyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2c) were obtained with yields of 90%, 84% and 75%, respectively. According to the yield of 2-substituted 6,7-dimethoxy- 3,1-benzoxazine-4-one, the reactivity decreases with the elongation of the carbon chain and even more so with an increase in the branching of anhydrides. Next, the heteroa- tom was replaced with nitrogen by the interaction of the corresponding benzoxazinone with amino acids or dipep- tides. Compounds 1c-10c were obtained by replacing the oxy- gen of the heterocycle with nitrogen by the interaction of the corresponding 6,7-dimethoxy-3,1-benzoxazine-4-one derivatives with glycine, glycylglycine, glycylleucine and glycyltryptophan in the medium of glacial acetic acid with the addition of 0.5 ml of dimethylformamide. At the same time, it is advisable to synthesize 2-methyl and 2-phenyl-6,7-dimethoxyquinazoline-4(3H)-ones (3k– 3m) through amides of 2-amino-4,5-dimethoxybenzoic acid (3 and 4), which in turn was synthesized by acylation of dimethoxyanthranilic acid (2) by acetyl chloride or benzoyl chloride (Scheme 3). 2a; R 1 = -C 2 H 5 2b; R 1 = -C 3 H 7 2c; R 1 = -CH(CH 3 ) 2 3e; R 1 = -C 3 H 7 , R 2 = -CH 2 -COOH 3f; R 1 = -C 3 H 7 , R 2 = -CH 2 -C(O)-NH-CH 2 -COOH 3i; R 1 = -CH(CH 3 ) 2 , R 2 = -CH 2 -C(O)-NH-CH 2 -COOH 3j; R1 = -CH(CH3)2, R2 = 3a-3j NH 2 -R 2 3g; R 1 = -C 3 H 7 , R 2 = AcOH, DMF 3h; R 1 = -C 3 H 7 , R 2 = (R 1 O) 2 O 3a; R 1 = -C 2 H 5 , R 2 = -CH 2 -COOH 3b; R 1 = -C 2 H 5 , R 2 = -CH 2 -C(O)-NH-CH 2 -COOH 3c; R 1 = -C 2 H 5 , R 2 = 3d; R 1 = -C 2 H 5 , R 2 = R 1 R 2 R 1 2 Scheme 2 Synthesis of 3a–3j. R 1 R 1R1 R 2 NH 2 -R 2 R 1 OCl NaOH 10% AcOH, DMF (EtO) 2 O 3k; R 1 = -CH 3 ; R 2 = -CH 2 COOH 3l; R 1 = Ph; R 2 = -CH 2 COOH 3m; R 1 = Ph; R 2 = -C 2 H 4 COOH 3; R 1 = -CH 3 4; R 1 = Ph 2d; R 1 = -CH 3 2e; R 1 = Ph 2 Scheme 3 Synthesis of 3k–3m. Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 6 of 12 The 2-methyl-6,7-dimethoxy-3,1-benzoxazine-4-one and 2-phenyl-6,7-dimethoxy-3,1-benzoxazine-4-one were obtained by two-stage synthesis. At the first stage, 2-acet- amido-4,5-dimethoxybenzoic acid was obtained by interac- tion in an alkaline medium by adding the acetyl chloride by drop while stirring. According to the method of the Schot- ten–Baumann acylation reaction, the pH of the medium should be controlled so that it remains alkaline. Further, the formation of the core of 3,1-benzoxazine-4-one was car- ried out by boiling 2-acetamido-4,5-dimethoxybenzoic acid in excess of propionic anhydride. The preparation of 2-phe- nyl-6,7-dimethoxy-3,1-benzoxazine-4-one was carried out in a similar way using benzoyl chloride. The substances 3k–3m were obtained by replacing oxygen with nitrogen in 3,1-benzoxazine-4-one by the interaction of the correspond- ing benzoxazinones with glycine or β-alanine in the me- dium of glacial acetic acid in the presence of catalytic amounts of DMF (Scheme 3). The mechanism of formation of 6,7-dimethoxyquinazo- line-4(3H)-one derivatives can be represented through the formation of an intermediate carbocation (Scheme 4). Possi- ble ways of protonation of the initial 2-phenylbenzoxazine- 4-one (I) were previously considered; the most reliable vari- ant of the reaction involves protonation of the oxygen het- eroatom (II), leading to a more stable carbocation (III). The attack by the carbocation amine reagent leads to the for- mation of another cation (IV), which further cleaves off the proton (V). Deprotonation preceding cyclization allows the formation of an intermediate adduct that cleaves off water molecules with the formation of a heterocycle (VI). 3.1.1. 2-acetamido-4,5-dimethoxy-benzoic acid (3) The grey crystals were obtained. Yield: 77%. M.p.: 136– 137 °C. UV spectrum (ethanol), λmax, nm: 231, 261, 324. IR spectrum (KBr), ν, cm–1: 3487 (OH), 3375 (NH), 16567 (C=O), 1624 (C=O), 999 (C–O). 3.1.2. 2-benzamido-4,5-dimethoxy-benzoic acid (4) The grey crystals were obtained. Yield: 91%. M.p.: 237– 238 °C. UV spectrum (ethanol), λmax, nm: 202, 242, 308. IR spectrum (KBr), ν, cm–1: 3450 (OH), 3157 (NH), 1676 (C=O), 1649 (C=O), 995 (C–O). 3.1.3. 2-ethyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2a) The white amorphous substance was obtained. Yield: 90%. M.p.: 142–143 °C. UV spectrum (ethanol), λmax, nm: 242, 275. IR spectrum (KBr), ν, cm–1: 1736 (C=O), 1026 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.23 (t, J = 7.5 Hz, 3H, CH3), 2.67 (q, J = 7.4 Hz, 2H, CH2), 3.87 (s, 3H, CH3), 3.92 (s, 3H, CH3), 7.07 (s, 1H, ArH), 7.37 (s, 1H, ArH). 3.1.4. 2-propyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2b) The white amorphous substance was obtained. Yield: 84%. M.p.: 184–185 °C. UV spectrum (ethanol), λmax, nm: 238, 271. IR spectrum (KBr), ν, cm–1: 1742 (C=O), 1018 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.93 (t, J = 7.4 Hz, 3H, CH3), 1.71–1.57 (m, 2H, CH2), 2.35 (t, J = 7.3 Hz, 2H, CH2), 3.76 (s, 3H, CH3), 3.81 (s, 3H. CH3), 7.42 (s, 1H, ArH), 8.33 (s, 1H, ArH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 14.00, 18.74, 55.96, 55.99, 103.43, 107.59, 113.13, 137.42, 143.74, 153.61, 169.83, 171.46. 3.1.5. 2-isopropyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2c) The white amorphous substance was obtained. Yield: 75%. M.p.: 214–215 °C. UV spectrum (ethanol), λmax, nm: 237, 271. IR spectrum (KBr), ν, cm–1: 1740 (C=O), 1012 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.17 (dd, J = 6.9, 1.0 Hz, 6H, CH3), 2.56 (qd, J = 6.9, 1.1 Hz, 1H, CH), 3.76 (s, 3H, CH3), 3.81 (s, 3H, CH3), 7.43 (d, J = 1.0 Hz, 1H, ArH), 8.35 (d, J = 1.1 Hz, 1H, ArH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 19.74, 36.95, 55.95, 55.99, 103.39, 107.61, 113.13, 137.57, 143.73. 153.64, 169.93, 175.33. 3.1.6. 2-methyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2d) The white amorphous substance was obtained. Yield: 86%. M.p.: 226–227 °C. UV spectrum (ethanol), λmax, nm: 236, 271. IR spectrum (KBr), ν, cm–1: 1734 (C=O), 1024 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.12 (t, J = 7.5 Hz, 3H, CH3), 3.78 (d, J = 20.3 Hz, 6H, CH3), 7.42 (s, 1H, ArH), 8.33 (s, 1H, ArH). R 1 R 2 R 1 + NH 2 -R 2 R 1 R 1 R 1 R 2R2R1 -H 2 O + H+ V III -H+ IVVI I II Scheme 4 Mechanism of formation of target 6,7-dimethoxykinazoline-4(3H)-one derivatives. Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 7 of 12 3.1.7. 2-phenyl-6,7-dimethoxy-3,1-benzoxazine-4-one (2e) The white amorphous substance was obtained. Yield: 96%. M.p.: 194–195 °C. UV spectrum (ethanol), λmax, nm: 202, 225, 263, 320. IR spectrum (KBr), ν, cm-1: 1740 (C=O), 1019 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 3.90 (s, 3H, CH3), 3.95 (s, 3H, CH3), 7.21 (s, 1H, ArH), 7.45 (s, 1H, ArH), 7.61 (dq, J = 14.4, 6.6 Hz, 3H, ArH), 8.15 (d, J = 8.1 Hz, 2H, ArH). 3.1.8. 2-(6,7-dimethoxy-2-ethyl-4-oxoquinazoline-3-yl)ace- tic acid (3a) The white amorphous substance was obtained. Yield: 75%. M.p.: 263–264 °C. UV spectrum (ethanol), λmax, nm: 242. IR spectrum (KBr), ν, cm–1: 3437 (OH), 1682 (C=O), 1610 (C=O), 1011 (C-O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.25 (t, J = 7.2 Hz, 3H, CH3), 2.75 (q, J = 7.3 Hz, 2H, CH2), 3.92 (s, 3H, CH3), 3.87 (s, 3H, CH3), 4.83 (s, 2H, CH2), 7.10 (s, 1H, ArH), 7.40 (s, 1H, ArH), 13.24 (s, 1H, COOH). 3.1.9. 2-[[2-(2-ethyl-6,7-dimethoxy-4-oxoquinazoline-3- yl)acetyl]amino]acetic acid (3b) The white amorphous substance was obtained. Yield: 70%. M.p.: 266–267 °C. UV spectrum (ethanol), λmax, nm: 242. IR spectrum (KBr), ν, cm–1: 3410 (OH), 3298 (C=O), 1736 (C=O), 1659 (C=O), 1612 (C=O), 1013 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.24 (t, J = 7.2 Hz, 3H, CH3), 2.73 (q, J = 7.0 Hz, 2H, CH2), 3.80 (d, J = 5.6 Hz, 2H, CH2), 3.86 (s, 3H, CH3), 3.91 (s, 3H, CH3), 4.82 (s, 2H, ArH), 7.08 (s, 1H, ArH), 7.39 (s, 1H, ArH), 8.63 (t, J = 5.7 Hz, 1H, NH), 12,72 (s, 1H, COOH). 3.1.10. 2-[[2-(2-ethyl-6,7-dimethoxy-4-oxoquinazoline-3- yl)acetyl]amino]-4-methyl-pentanoic acid (3c) The beige amorphous substance was obtained. Yield: 89%. M.p.: 225–226 °C. UV spectrum (ethanol), λmax, nm: 241. IR spectrum (KBr), ν, cm–1: 3419 (OH), 3309 (NH), 1732 (C=O), 1661 (C=O), 1612 (C=O), 1008,65 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.86 (d, J = 6.6 Hz, 3H, CH3), 0.92 (d, J = 6.5 Hz, 3H, CH3), 1.12 (t, J = 7.5 Hz, 1H, CH), 1.24 (t, J = 7.2 Hz, 3H, CH3), 1.62 – 1.49 (m, 2H, CH2), 2.70 (q, J = 8.1, 7.0 Hz, 2H, CH2), 3.86 (s, 3H, CH3), 3.92 (s, 3H, CH3), 4.24 (td, J = 8.9, 5.8 Hz, 1H.CH), 4.95 – 4.69 (m, 2H, CH3), 7.08 (s, 1H, ArH), 7.40 (s, 1H, ArH), 8.63 (d, J = 7.9 Hz, 1H, NH), 12,65 (s, 1H, COOH). 3.1.11. 2-[[2-(2-ethyl-6,7-dimethoxy-4-oxoquinazoline-3- yl)acetyl]amino]-3-(1H-indole-3-yl)propanoic acid (3d) The beige amorphous substance was obtained. Yield: 73%. M.p.: 265–266 °C. UV spectrum (ethanol), λmax, nm: 228, 239, 271. IR spectrum (KBr), ν, cm–1: 3383 (NH), 1730 (C=O), 1661 (C=O), 1641 (C=O), 1013 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.14 (t, J = 7.2 Hz, 3H, CH3), 3.05 (dd, J = 14.7, 8.7 Hz, 2H, CH2), 3.22 (dd, J = 14.6, 4.9 Hz, 2H, CH2), 3.85 (s, 3H, CH3), 3.91 (s, 3H, CH3), 4.53 (td, J = 8.3, 4.8 Hz, 1H, CH), 4.91–4.61 (m, 2H, CH2), 7.00 (t, J = 7.4 Hz, 1H, ArH), 7.07 (d, J = 7.6 Hz, 2H, ArH), 7.19 (d, J = 2.5 Hz, 1H, ArH), 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.39 (s, 1H, ArH), 7.56 (d, J = 7.7 Hz, 1H, ArH), 8.68 (d, J = 8.0 Hz, 1H, NH), 10.91 (d, J = 2.5 Hz, 1H, NH), 12,79 (s, 1H, COOH). 3.1.12. 2-(6,7-dimethoxy-4-oxo-2-propyl-quinazoline-3- yl)acetic acid (3e) The white amorphous substance was obtained. Yield: 58%. M.p.: 234–235 °C. UV spectrum (ethanol), λmax, nm: 241. IR spectrum (KBr), ν, cm–1: 3431 (OH), 1713 (C=O), 1676 (C=O), 1007 (C–O).1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.99 (t, J = 7.4 Hz, 3H, CH3), 1.83–1.69 (m, 2H, CH2), 2.74–2.66 (m, 2H, CH2), 3.87 (s, 3H, CH3), 3.92 (s, 3H, CH3), 4.82 (s, 2H, CH2), 7.09 (s, 1H, ArH), 7.40 (s, 1H, ArH), 13,25 (s, 1H, COOH). 13C NMR spectrum (100,6 MHz, DMSO- d6), δ, ppm: 14.12, 19,81, 36.17, 45.17, 56.15, 56.44, 105.57, 108.05, 112.96, 143.62, 148.84, 155.21, 155.74, 160.97, 170.16. 3.1.13. 2-[[2-(2-propyl-6,7-dimethoxy-4-oxo-quinazoline-3- yl)acetyl]amino]acetic acid (3f) The white amorphous substance was obtained. Yield: 78%. M.p.: 247–248 °C. UV spectrum (ethanol), λmax, nm: 242. IR spectrum (KBr), ν, cm–1: 3439 (OH), 3338 (NH), 1740 (C=O), 1662 (C=O), 1641 (C=O), 1011 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.99 (t, J = 6.7 Hz, 3H, CH3), 1.84–1.71 (m, 2H. CH2), 2.68 (t, J = 7.5 Hz, 2H, CH2), 3.83–3.78 (m, 2H, CH2), 3.88 (d, J = 20.3 Hz, 6H, CH3), 4.82 (s, 2H, CH2), 7.07 (s, 1H, ArH), 7.39 (s, 1H, ArH), 8.65 (t, J = 5.8 Hz, 1H, NH), 12,80 (s, 1H, COOH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 14.12, 19.65, 35.95, 41.26, 45.28, 56.13, 56.41, 105.72, 108.00, 113.10, 143.63, 148.70, 155.08, 156.09, 161.01, 167.86, 171.54. 3.1.14. 2-[[2-(2-propyl-6,7-dimethoxy-4-oxoquinazoline-3- yl)acetyl]amino]-4-methyl-pentanoic acid (3g) The beige amorphous substance was obtained. Yield: 69%. M.p.: 236–237 °C. UV spectrum (ethanol), λmax, nm: 242. IR spectrum (KBr), ν, cm–1: 3520 (OH), 3308 (NH), 1678 (C=O), 1655 (C=O), 1614 (C=O), 1007 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.86 (d, J = 6.5 Hz, 3H, CH3), 0.92 (d, J = 6.6 Hz, 3H, CH3), 0.98 (t, J = 7.4 Hz, 3H, CH3), 1.61–1.49 (m, 2H, CH2), 1.67 (dt, J = 12.9, 8.0 Hz, 1H, CH), 1.82–1.72 (m, 2H, CH2), 2.63 (t, J = 7.6 Hz, 2H, CH2), 3.86 (s, 3H, CH3), 3.91 (s, 3H, CH3), 4.30–4.20 (m, 1H, CH), 4.82 (q, J = 17.0 Hz, 2H, CH2), 7.07 (s, 1H, ArH), 7.40 (s, 1H, ArH), 8.64 (d, J = 8.0 Hz, 1H, NH), 12.72 (s, 1H, COOH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 14.12, 19.64, 21.66, 23.34, 24.80, 36.00, 45.04, 50.91, 56.14, 56.41, 105.74, 108.00, 113.08, 143.62, 148.71, 155.08, 156.04, 160.99, 167.44, 174.31. 3.1.15. 2-[[2-(2-propyl-6,7-dimethoxy-4-oxoquinazoline-3- yl)acetyl]amino]-3-(1H-indole-3-yl)propanoic acid (3h) The beige amorphous substance was obtained. Yield: 57%. M.p.: 272–273 °C. UV spectrum (ethanol), λmax, nm: 229, 239, 271. IR spectrum (KBr), ν, cm–1: 3381 (NH), 1730 (C=O), 1662 (C=O), 1641 (C=O), 1009 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.91 (t, J = 7.4 Hz, 3H, CH3), 1.76–1.60 (m, 2H, CH2), 2.47 (dd, J = 7.6, 2.7 Hz, Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 8 of 12 2H, CH2), 3.25–3.01 (m, 2H, CH2), 3.86 (s, 3H, CH3), 3.91 (s, 3H, CH3), 4.52 (td, J = 8.2, 5.0 Hz, 1H, CH), 4.77 (q, J = 16.9 Hz, 2H, CH2), 7.00 (t, J = 6.9 Hz, 1H, ArH), 7.06 (s, 1H, ArH), 7.12–7.07 (m, 1H, ArH), 7.20 (d, J = 2.4 Hz, 1H, ArH), 7.41–7.30 (m, 2H, ArH), 7.56 (d, J = 7.8 Hz, 1H, ArH), 8.70 (d, J = 7.9 Hz, 1H, NH), 10.93 (s, 1H, NH), 12.83 (s, 1H, COOH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 14.07, 19.61, 27.58, 35.86, 45.16, 53.71, 56.14, 56.41, 105.72, 107.99, 110.12, 111.85, 113.06, 118.63, 118.86, 121.39, 124.12, 127.67, 136.54, 143.62, 148.69, 155.07, 156.06, 160.98, 167.26, 173.57. 3.1.16. 2-[[2-(2-isopropyl-6,7-dimethoxy-4-oxoquinazoline- 3-yl)acetyl]amino]acetic acid (3i) The white amorphous substance was obtained. Yield: 61%. M.p.: 219–220 °C. UV spectrum (ethanol), λmax, nm: 239. IR spectrum (KBr), ν, cm–1: 3425 (OH), 3298 (NH), 1732 (C=O), 1662 (C=O), 1614 (C=O), 1007 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.24 (d, J = 6.6 Hz, 6H, CH3), 3.06–2.92 (m, 1H, CH), 3.82 (d, J = 5.7 Hz, 2H, CH2), 3.86 (s, 3H, CH3), 3.92 (s, 3H, CH3), 4.87 (s, 2H, CH2), 7.07 (s, 1H, ArH), 7.40 (s, 1H, ArH), 8.66 (t, J = 5.9 Hz, 1H, NH), 12.68 (s, 1H, COOH). 13C NMR spectrum (100,6 MHz, DMSO-d6), δ, ppm: 21.69, 31.88, 41.26, 45.08, 56.15, 56.43, 105.72, 108.05, 113,10, 143.65, 148.76, 155.16, 160.82, 161.09, 167.93, 171.46. 3.1.17. 2-[[2-(2-isopropyl-6,7-dimethoxy-4-oxoquinazoline- 3-yl)acetyl]amino]-4-methyl-pentanoic acid (3j) The white amorphous substance was obtained. Yield: 50%. M.p.: 220–221 °C. UV spectrum (ethanol), λmax, nm: 241. IR spectrum (KBr), ν, cm–1: 3414 (OH), 3309 (NH), 1736 (C=O), 1678 (C=O), 1657 (C=O), 1007 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 0.86 (dd, J = 6.5, 1.9 Hz, 3H, CH3), 0.92 (dd, J = 6.6, 1.9 Hz, 3H, CH3), 1.17 (dd, J = 6.9, 2.0 Hz, 3H, CH3), 1.23 (d, J = 4.5 Hz, 3H, CH3), 1.63–1.49 (m, 2H, CH2), 1.73 – 1.65 (m, 1H. CH), 3.02–2.89 (m, 1H, CH), 3.86 (s, 3H, CH3), 3.92 (s, 3H, CH3), 4.31–4.20 (m, 1H, CH), 4.89 (q, J = 14.0 Hz, 2H, CH2), 7.07 (d, J = 1.9 Hz, 1H, ArH), 7.42 (dd, J = 13.1, 1.9 Hz, 1H, ArH), 8.64 (d, J = 8.0 Hz, 1H, NH), 12.72 (s, 1H, COOH). 3.1.18. 2-(6,7-dimethoxy-2-methyl-4-oxoquinazoline-3- yl)acetic acid (3k) The white amorphous substance was obtained. Yield: 72%. M.p.: 215–216 °C. UV spectrum (ethanol), λmax, nm: 242. IR spectrum (KBr), ν, cm–1: 3431 (OH), 16778 (C=O), 1610 (C=O), 1011 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 1.25 (td, J = 7.3, 1.6 Hz, 3H, CH3), 3.87 (s, 3H, CH3), 3.92 (s, 3H, CH3), 4.83 (s, 2H, CH2), 7.10 (t, J = 1.5 Hz, 1H, ArH), 7.40 (q, J = 6.3 Hz, 1H, ArH), 13.26 (s, 1H, COOH). 3.1.19. 2-(6,7-dimethoxy-4-oxo-2-phenyl-quinazoline-3- yl)acetic acid (3l) The white crystals were obtained. Yield: 66%. M.p.: 281– 282 °C. UV spectrum (ethanol), λmax, nm: 247, 295. IR spec- trum (KBr), ν, cm–1: 3437 (OH), 1716 (C=O), 1676 (C=O), 1005 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 3.91 (d, J = 2.1 Hz, 6H, CH3), 4.53 (s, 2H, CH2), 7.21 (s, 1H, ArH), 7.49 (s, 1H, ArH), 7.55 (s, 5H, ArH), 13.18 (s, 1H, COOH). 3.1.20. 2-(6,7-dimethoxy-4-oxo-2-phenyl-quinazoline-3- yl)propanoic acid (3m) The white crystals were obtained. Yield: 62%. M.p.: 202– 203 °C. UV spectrum (ethanol), λmax, nm: 246, 302. IR spec- trum (KBr), ν, cm–1: 3419 (OH), 1715 (C=O), 1670 (C=O), 1026 (C–O). 1H NMR spectrum (400 MHz, DMSO-d6), δ, ppm: 2.59 (t, J = 7.7 Hz, 2H, CH2), 3.90 (d, J = 3.7 Hz, 6H, CH3), 4.06 (t, J = 7.6 Hz, 2H, CH2), 7.16 (s, 1H, ArH), 7.49 (s, 1H, ArH), 7.57–7.50 (m, 3H, ArH), 7.63 (dd, J = 6.8, 2.9 Hz, 2H, ArH), 12.34 (s, 1H, COOH). 3.2. Pharmacological studies By assessing the change in cognitive deficits in animals, it was found that in the NC group of rats a pronounced cogni- tive impairment was observed, as evidenced by a decrease in the percentage of spontaneous alternations of the laby- rinth arms in this group of rats by 66.9% (p<0.05) in rela- tion to the SO animals (Figure 1). The use of EMHPS con- tributed to a significant decrease in cognitive deficit in rats by 80.1% (p<0.05) in relation to the NC group of animals. The administration of the studied compounds also led to a statistically significant decrease in the symptoms of depres- sion of the higher integrative functions of the brain; how- ever, the indicators of spontaneous movement vector change in the animals that were injected with the studied substances were significantly lower than those in the rats that were treated by EMHPS, with the exception of com- pounds 3i and 3j. The use of these substances contributed to the reduction (relative to the NC group of rats) of cogni- tive deficit in animals by 60.2% (p<0.05) and 57.9% (p<0.05), respectively, which did not statistically signifi- cantly differ from those in the rats receiving a reference drug. The size of the brain necrosis zone changed similarly (Figure 2). In the NC group of rats, this parameter was 33.6±0.567%, while the use of the reference drug contrib- uted to a decrease in this indicator by 27.7% (p<0.05). Among the studied substances, the most pronounced effect on the change in necrotic processes in the brain tissue dur- ing ischemia was exerted by the administration of the com- pounds 3i, 3j and 3k, against the background of which the necrosis zone decreased relative to the NC group of rats by 23.1% (p<0.05), 27.9% (p<0.05) and 24.2% (p<0.05) and did not statistically significantly differ from the similar one in the group of animals that received the reference drug. The rest of the studied substances did not have a significant effect on the change in the area of brain necrosis. Currently, cerebrovascular accidents and, in particu- lar, ischemic stroke remain one of the main medical and social health problems. It was established that in 2019 stroke ranked second among the main non-communicable causes of death, second only to coronary heart disease and the terminal stage of this disease – myocardial infarction. Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 9 of 12 Figure 1 Influence of the studied compounds and the referent on the change in cognitive deficit in rats under the conditions of cerebral ischemia. Note: # – significant relative to SO (Tukey's test, p<0.05);* – significant relative to NC (Tukey's test, p<0.05);Δ – significant relative to EMHPS (Tukey's test, p<0.05). Figure 2 Influence of the studied compounds and the referent on the change of the brain tissue necrosis zone in rats under the condi- tions of cerebral ischemia. Note: in the SO group of rats, the brain necrosis zone was zero; * – significant relative to NC (Tukey's test, p<0.05); Δ – significant relative to EMHPS (Tukey's test, p<0.05). More than 6 million deaths from stroke occur annually, while there is a significant increase in cases of primary dis- ability and disability after an ischemic attack, which is more than 50% of the total number of stroke episodes. Ac- cording to WHO statistical reports, cerebral hemodynamic disorders are more often recorded in countries with middle and middle-high income levels, while with an increase in the well-being of the population and the possibility of ob- taining qualified and timely medical care, the incidence of stroke is significantly reduced, which is confirmed by data from high-income countries [40]. The high medical and socioeconomic role of ischemic stroke dictates the need to find new ways to treat this pathological condition. One of such relatively new strate- gies for adjuvant therapy of stroke may be “freezing” the Chimica Techno Acta 2022, vol. 9(2), No. 20229212 ARTICLE 10 of 12 activity of neurons in the ischemic penumbra, i.e. cerebro- protection. Cerebroprotectors include agents of various chemical structures: compounds of natural origin, which are most often represented by polyphenols: quercetin, ver- bascoside, aurapten, thymol, epigallocatechin gallate, cur- cumin [41], sex hormones, low molecular weight synthetic compounds such as lactic acid salts, acetyl-L-carnitine, an- giotensin II receptor antagonists. Several promising neuro- protectors have been discovered in the last decade: succinic acid derivatives, [42], chomones [43] uric acid, otaplimas- tat (an inhibitor of metalloproteinases) [44], drotrecogin-α (anticoagulant, PAR-1 agonist) [45], verapamil, nerinetide (anti-excitotoxic drug) [46]. The study showed that the investigated quinazolinone derivatives with fragments of amino acids and dipeptides also could be promising cerebroprotective agents. In this respect, especially noteworthy are the compounds 3i, 3j and 3k, the use of which not only led to the recovery of the cog- nitive functions of animals, but also reduced the size of the brain necrosis zone. Thus, the obtained results will allow expanding the range of potential cerebroprotective agents by three quinazolinone derivatives. 4. Conclusions In the course of the study, a number of novel 6,7-dimethox- yquinazolin-4(3H)-one derivatives with alkyl substituents and residues of neuroactive amino acids and dipeptides were synthesized. Among the studied quinazolinone deriv- atives, compounds 3i, 3j and 3k have the most pronounced cerebrotropic activity and are not inferior to ethylme- thylhydroxypyridine succinate in terms of pharmacological activity, which makes them promising objects for further research. Supplementary materials No supplementary materials are available. Funding The reported study was funded by RFBR, project No. 20-315-90060, https://www.rfbr.ru/rffi/eng. Acknowledgment None. Author contributions Conceptualization: I.P.K. Data curation: I.P.K. Formal Analysis: A.S.C., D.I.P., I.P.K. Funding acquisition: A.S.C., I.P.K. Investigation: A.S.C., I.P.K., D.I.P. Methodology: A.S.C., D.I.P., I.P.K. Project administration: I.P.K. Resources: A.S.C., I.P.K., D.I.P. Software: A.S.C., D.I.P. Supervision: I.P.K. Validation: D.I.P. Visualization: A.S.C., D.I.P. Writing – original draft: A.S.C., I.P.K., D.I.P. Writing – review & editing: A.S.C., D.I.P. Conflict of interest The authors declare no conflict of interest. Additional information Author IDs: A.S. Chiriapkin, Scopus ID 57218134815; I.P. Kodonidi, Scopus ID 10240218600; D.I. Pozdnyakov, Scopus ID 57190954589; Website: https://www.pmedpharm.ru/sveden_eng. References 1. Lin YF, Smith AV, Aspelund T, Betensky RA, Smoller, JW, Gudnason V, launer LJ, Blacker D. Genetic overlap between vascular pathologies and Alzheimer’s dementia and potential causal mechanisms. 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