Use of piperazine derivative for treatment of dementia of alzheimer's type associated with calcium dysregulation

Abstract

FIELD: pharmaceuticals.SUBSTANCE: use of a piperazine derivative, namely N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide of formula (I) as a therapeutic agent intended for the restoration of cognitive and motor functions accompanying neurodegenerative diseases, such as Alzheimer's disease and dementias associated with impaired calcium regulation, is proposed.EFFECT: effective treatment Alzheimer's disease and / or dementia of the Alzheimer's type.1 cl, 16 dwg, 4 tbl, 5 ex

Description

[0001] The present invention relates to the fields of biology and medicine, more specifically to neurophysiology, and can be used to treat patients suffering from Alzheimer's disease (AD), as well as to prevent the worsening of symptoms and its progression. This invention concerns a new substance based on a piperazine derivative, which has shown positive neuroprotective results in AD mouse models and, therefore, can be further used to create a drug for the treatment of patients with AD and / or Alzheimer's-type dementia.

[0002] Currently, society needs to develop effective drug therapy for Alzheimer's disease. Therapy based on the "amyloid-β (Aβ) cascade hypothesis" is currently the leading edge of drug development for Alzheimer's disease. However, human clinical trials of anti-Aβ drugs have yielded conflicting results. Furthermore, Aβ oligomers and aggregates are present in the healthy aging brain [1].

[0003] The importance of finding new approaches to the treatment of Alzheimer's disease has been emphasized in many studies [2]. There is an active scientific debate about calcium dysregulation in neurons in the pathology of Alzheimer's disease [3-6]. The choice of neuronal store-operated calcium channels, such as the transient receptor cation channel, subfamily C, member 6 (TRPC6), as a target for action appears to be a promising direction for the development of Alzheimer's disease therapy [7, 8].

[0004] There is some genetic evidence for a role of TRPC6 in the pathogenesis of Alzheimer's disease. TRPC6 mRNA levels in blood cells are reduced in patients with Alzheimer's disease and mild cognitive impairment [9]. In addition, the level of TRPC6 expression in the blood is associated with stage 10 dementia. Knockdown of the TRPC6 gene has been found to interfere with store-operated calcium entry (nSOCE). Overexpression of TRPC6 or pharmacological activation of this channel restores nSOCE in hippocampal neurons [10-12]. Overexpression of TRPC6 has been shown to restore the loss of mushroom spines in a mouse model of Alzheimer's disease (mice with activated persenelin and amyloid precursor protein genes)

[10] , and also protects neurons from ischemic brain injury

[12] . Mice that overexpress TRPC6 in the brain have improved cognitive function and increased excitatory synapse formation.Pharmacological activation of TRPC6 protects hippocampal mushroom spines from amyloid toxicity in vitro and effectively restores synaptic plasticity and cognitive deficits in 5xFAD mice

[13] . Thus, positive modulators of TRPC6 could be proposed as a potential drug against Alzheimer's disease.

[0005] TRPC6 can be activated by many different compounds

[14] . Most of them exhibit either cross-specificity or toxicity. Hyperforin, a positive and specific TRPC6 agonist, has been tested in clinical trials for the treatment of mild to moderate depression [15, 16]. However, hyperforin is unstable, difficult to synthesize

[17] , and exhibits side effects and protonophore properties

[18] . Protonophore properties induce cytosolic oxidation, which in turn affects the plasma membrane Na-H+ exchanger. It also induces drug interactions through potent activation of the nuclear receptor PXR (NR1I2), a key transcriptional regulator of genes involved in drug metabolism and transport

[19] . These features limit its clinical application and require the search for other TRPC6 agonists.

[0006] Piperazine derivatives, [4-(5-chloro-2-methylphenyl)piperazin-1-yl](3-fluorophenyl)methanone (PPZ1) and 2-[4-(2,3-dimethylphenyl)-piperazin-1-yl]-N-(2-ethoxyphenyl)acetamide (PPZ2), have been described as TRPC6 activators and exhibit potent neurotrophic effects

[20] .

[0007] The closest analogue is N-(2-chlorophenyl)-2-(4-phenylpiperazin-1-yl)acetamide (51164 - found in the chemical library of InterBioScreen, Chernogolovka). Synaptoprotective properties of N-(2-chlorophenyl)-2-(4-phenylpiperazin-1-yl)acetamide have recently been demonstrated in mouse models of Alzheimer's disease. We have shown that 51164 activates TRPC6 through a store-operated mechanism, restores mushroom spines and induces long-term potentiation (LTP) in brain slices of 5×FAD mice

[11] . However, 51164 is unstable in blood plasma and does not penetrate the blood-brain barrier.

[0008] The technical challenge is to find piperazines that are structurally similar to 51164, bind to TRPC6, have synaptor-positive properties in culture and slices, penetrate the blood-brain barrier (BBB), and restore synaptic deficits in vivo.

[0009] The technical result of the claimed invention consists in expanding the arsenal of means for preventing neurodegenerative diseases, such as Alzheimer's disease and dementia associated with impaired calcium regulation.

[0010] The technical result is achieved by chemical synthesis (see Fig. 16) and the use of the compound (2S)-N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide of the general formula C 19 H 21 N3F2O as a therapeutic agent intended to restore cognitive and motor functions accompanying neurodegenerative diseases such as Alzheimer's disease and dementia associated with impaired calcium regulation.

[0011] No use of C has been found in the literature 19 H 21 N3F2O as a medicinal product for the treatment of Alzheimer's disease and / or Alzheimer's type dementia.

[0012]

[0013] Compound (2S)-N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide of general formula C 19 H 21 N3F2O.

[0014] This heterocyclic compound is a piperazine derivative, a positive regulator of TRPC6 channel activity. It binds to the central portion of the TRPC6 monomer similarly to hyperforin, based on binding parameters calculated in silico. This new piperazine derivative exhibits synaptoprotective properties in cell cultures and slices, penetrates the blood-brain barrier, alleviates synaptic plasticity deficits, and restores cognitive and motor functions in 5xFAD mice.

[0015] The essential features that form the basis of the invention: 1) the compound, a piperazine derivative, interacts with the TRPC6 channel at the same binding site as hyperforin, according to the results of molecular docking and molecular dynamics; 2) this compound is a selective activator of TRPC6 channels in hippocampal neurons; 3) stability in blood plasma and permeability of the compound through the blood-brain barrier; 4) absence of mutagenic and toxic properties; 5) improvement of synaptic plasticity indices in brain slices of laboratory animal models of AD after intraperitoneal injections at a dose of 10 mg / kg; 6) at a dose of 10 mg / kg, it restores cognitive and 7) motor functions in 5xFAD mice.

[0016] Brief description of illustrations:

[0017] The illustrations attached to the description show:

[0018] Fig. 1 - Molecular docking of the compound with the TRPC6 monomer. Designations: (1) The central binding site of TRPC6 (characteristic of C 19 H 21N3F2O) is highlighted in yellow, (2) the peripheral TRPC6 binding site is highlighted in blue.

[0019] Fig. 2 - Conformational map of complex formation of (a) hyperforin and (b) this piperazine derivative with TRPC6. Designations: (3) Van der Waals forces, (4) Hydrogen bond, (5) Carbon-hydrogen bond, (6) π-σ bond, (7) Bond between alkyl radicals, (8) π-alkyl bond.

[0020] Fig. 3 - RMSD for complexes of hyperforin with TRPC6 (c) and the considered piperazine derivative with TRPC6 (d).

[0021] Fig. 4 - Hydrogen bonds for complexes of hyperforin with TRPC6 (d) and the piperazine derivative under consideration with TRPC6 (e).

[0022] Fig. 5 - In vitro neuroprotective properties of C 19 H 21N3F2O. Calcium concentration fluctuations for HEK293 cells co-transfected with TRPC6 plasmids and GCamp5.3 calcium sensor plasmids. Colored lines represent the mean fluorescence value of the analyzed cells with error bars ±SEM. Legend: (9) DMSO (control), (10) C 19 H 21 N3F2O, (11) C20 positive control, (12) SKF negative control. The time of addition of chemical agents is indicated by bars above the graph: (I) 0.1 mM Ca 2+ (II) 2 mM Ca 2+ + 20 μM compound + 50 μM OAG.

[0023] Fig. 6 - In vitro neuroprotective properties of C 19 H 21 N3F2O. Average peak calcium entry, expressed as a change in GCamp5.3 fluorescence, for cells exposed to (13) DMSO (control), (14) C 19 H 21N3F2O, (15) C20 positive control, (16) SKF negative control. Results are presented as mean ± SD (cell number: 35 to 45) ***: p < 0.001, *: p < 0.05, ns: non-significant difference according to the Kruskal-Wallis test with Dunn's post hoc test. Normality of distribution was assessed by the Shapiro-Wilk test, homogeneity was assessed by the Bartlett test.

[0024] Fig. 7 - In vitro neuroprotective properties of C 19 H 21 N3F2O. Confocal microscopic images of dendritic fragments: for control neuronal cultures and cultures treated with amyloid β (Ab42). Scale bar is 4 μm.

[0025] Fig. 8 - The spine head area for each group is presented as median values ​​with interquartile ranges. The number of spines ≥ 210 from 3 hippocampal cultures. Designations: (17) control, (18) Ab42. The normality of distribution was assessed by the Shapiro-Wilk test, the homogeneity was assessed by the Bartlett test. Statistical analysis was performed using the Kruskal-Wallis test and Dunn's multiple comparison test; ns: insignificant difference, ***: p < 0.001.

[0026] Fig. 9. The mean slope of excitatory postsynaptic potential (epsp) at 30-40 min after high-frequency stimulation (values ​​as mean ± SD). Legend: (19) Wild type, (20) 5xFAD, (21) Wild type + C 19 H 21 N3F2O, (22) 5xFAD + C 19 H 21N3F2O. Normality of distribution was assessed using the Shapiro-Wilk test, homogeneity was assessed using the Bartlett test. Statistical analysis was performed using Welch-ANOVA with the Games-Howell multiple comparison post-hoc test, ns: insignificant difference, *: p < 0.05, ***: p < 0.001.

[0027] Fig. 10 - Stability C 19 H 21 N3F2O in mouse plasma. Legend: (23) propantheline (control), (24) C 19 H 21 N3F2O. Data are presented as mean ± SD. n (plasma samples) = 2.

[0028] Fig. 11 - Pharmacokinetic profile of C 19 H 21 N3F2O in mouse plasma. Data are presented as mean ± SEM, n (mice) = 3.

[0029] Fig. 12 - Pharmacokinetic profile of C 19 H 21 N3F2O in mouse brain. Data are presented as mean ± SEM, n (mice) = 3.

[0030] Fig. 13 - Neuroprotective effect of intraperitoneal injection at a dose of 10 mg / kg C 19 H 21N3F2O for long-term potentiation. Legend: (25) Wild type, (26) 5xFAD, (27) 5xFAD + C 19 H 21 N3F2O. Mean EPSP slope values ​​at 30–40 minutes after high-frequency stimulation are presented as mean ± SD. Normality of distribution was assessed using the Shapiro-Wilk test, and homogeneity was assessed using the Bartlett test. Statistical analysis: one-way ANOVA with the Holm-Sidak multiple comparison test, *: p < 0.05.

[0031] Fig. 14 - C 19 H 21 N3F2O enhances recognition memory in 5xFAD mice. Key: (28) Subject A (old), (29) Subject B (new), (30) Wild type, (31) 5xFAD, (32) 5xFAD + C 19 H 21 N3F2O. Time of exploration of a new object B by mice from the 5xFAD + C group 19 H 21N3F2O was significantly higher than in the 5xFAD group. Results are presented as mean ± SD, with individual values ​​represented as dots. Number of mice tested per group: Wild type = 8, 5xFAD = 8, 5xFAD + C 19 H 21 N3F2O = 9. Normality of distribution was assessed using the Shapiro-Wilk test, homogeneity was assessed using the Bartlett test.** p < 0.01, ns: non-significant difference according to the Mann-Whitney test or t-test between two groups and one-way ANOVA test with Dunnett's post hoc test for comparison between 5xFAD + C 19 H 21 N3F2O and other groups.

[0032] Fig. 15 - C 19 H 21 N3F2O enhances contextual memory in 5xFAD mice. Key: (33) training, (34) context, (35) wild type, (36) 5xFAD, (37) 5xFAD + C 19 H 21N3F2O. Percentage of freezing in the conditioned freezing test on day 10 of testing. Results are presented as mean ± SD, with individual values ​​represented as dots. Number of mice tested per group: Wild type = 9, 5xFAD = 8 = 10, 5xFAD + C 19 H 21 N3F2O = 9. Normality of distribution was assessed using the Shapiro-Wilk test, homogeneity was assessed using the Bartlett test. ** p < 0.01, * p < 0.05, ns: insignificant difference according to the Mann-Whitney test (for comparing the level of freezing on the day of training and testing), according to the Kruskal-Wallis test and Dunn's post hoc multiple comparison test (for comparing differences between the 5xFAD and 5xFAD + C groups 19 H 21 N3F2O).

[0033] Fig. 16 - Scheme of synthesis of N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide.

[0034] The present invention presents in silico, in vitro, pharmacokinetic and in vivo studies of the newly discovered TRPC6-specific positive modulator N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide (C 19 H 21 N3F2O). An in silico comparison of the binding ability of hyperforin and C to TRPC6 was performed. 19 H 21 N3F2O, and a preliminary analysis of the suitability of this compound as a drug was also conducted. Then, using a HEK-293T cell model overexpressing the TRPC6 channel, it was shown that C 19 H 21 N3F2O selectively activates the TRPC6 channel, stimulating calcium influx across the cell membrane. Experiments on primary hippocampal cultures confirmed that C 19 H 21 N3F2O protects neuronal mushroom spines from amyloid toxicity in vitro. The effect was demonstrated in brain slices from 5xFAD:C mice. 19 H 21N3F2O effectively restored synaptic plasticity in brain slices. Its pharmacokinetic profile and ability to penetrate the blood-brain barrier were also determined.

[0035] C 19 H 21 N3F2O may be a potential TRPC6-selective compound for the treatment of synaptic failure in AD-affected hippocampal neurons.

[0036] During the drug development process, the properties of compounds are determined by testing them according to the criteria of "drug-likeness." Currently, pharmaceutical chemistry widely uses a method for primary testing of compound properties based on ADMET (absorption, distribution, metabolism, excretion, toxicity) parameters calculated using semi-empirical calculations

[21] . Compound C 19 H 21N3F2O exhibits high rates of gastrointestinal absorption and BBB permeability, and is a compound with high synthetic availability. According to toxicity prediction results, C 19 H 21 N3F2O does not exhibit toxicity at the levels of mutagenicity, carcinogenicity, hepatotoxicity and acute oral toxicity.

[0037] Molecular docking results show that C 19 H 21 N3F2O binds to TRPC6 at a central binding site, similar to the previously studied selective TRPC6 agonist hyperforin. The central binding site of TRPC6 is mediated by amino acid residues in the central part of the protein, forming a pocket between the α-helical S4, S5, and S6 (TMD domain,

[22] ). The peripheral binding site contains helices S2 and S3, in the peripheral zone of the TMD domain

[22] (Fig. 1). In this work, conformational maps of complex formation for hyperforin and C 19 H 21N3F2O. The biophysical and spatial-energetic parameters of the complex of this piperazine with TRPC6 were calculated.

[0038] The main contribution to the energy ΔG is made by non-covalent (van der Waals (ΔG=-30.80±1.54 kcal / mol) and hydrogen bonds) and hydrophobic types of interactions. Based on the constructed conformational maps of complex formation, the amino acid residues Phe443; Ile613; Leu411; Ala616; Fe407; Phe440, which form a bond with this compound in the central site of TRPC6 (Fig. 2), were identified. C 19 H 21 N3F2O completely mimics the steric properties of hyperforin when interacting with the central region of TRPC6. Based on the calculated ΔG binding, it can be concluded that this compound is suitable for further study as a selective TRPC6 agonist.

[0039] The stability and dynamic behavior of TRPC6-piperazine complexes were evaluated using molecular dynamics simulations (Fig. 2, Fig. 3, Fig. 4). The results indicate that the interaction is stable during the first 5 ns, and the RMSD values ​​do not exceed 0.75 nm. Hyperforin remains stable up to 13.4 ns, after which a steric shift is observed within the central part of TRPC6, and the RMSD value varies within 1.85 nm. The calculated values ​​of the Coulomb forces for C 19 H 21 N3F2O (indicating the electrostatic type of interactions) do not exceed -3.43 kcal / mol. C 19 H 21 N3F2O, like hyperforin, can form a hydrogen bond with Asn460, the probability of which does not exceed 23.84% (Fig. 2). The combination of molecular dynamics studies is in good agreement with the molecular docking studies and confirms the choice of compound C. 19 H 21N3F2O as a lead for the development of TRPC6-specific positive modulators.

[0040] It was further demonstrated that the TRPC6 channel is indeed a target of this piperazine. Experiments were performed on HEK cell lines that were transfected with GCamp5.3 and TRPC6 plasmids. To assess the ability of the piperazine derivative to activate Ca 2+ entry, 2+ Calcium fluctuations were recorded via TRPC6 by assessing GCamp5.3 fluorescence. During the experiment, HEK-293T cells were incubated in a solution containing 0.1 mM extracellular Ca 2+ (depletion of calcium depots), then the solution was changed to a similar one with 2 mM Ca 2+, 50 μM OAG and 20 μM of the test compound (Fig. 5). Since hyperforin is very unstable in solution, we used another selective TRPC6 channel agonist, the compound TRPC6-PAM-C20 (C20, a previously described benzopyran derivative

[23] ), as a positive control. The non-selective TRPC channel antagonist SKF96365 (SKF) was used as a negative control.

[0041] Application C 19 H 21 N3F2O, like C20, contributed to an increase in the fluorescence intensity values ​​(indicating calcium influx) [1.10 ± 0.44 and 1.88 ± 0.3 for C structures 19 H 21 N3F2O and C20, respectively]. This is significantly higher than in the absence of drugs [0.91 ± 0.21 for the control group], or in the case of using the TRPC blocker SKF96365 [0.83 ± 0.25 for the control group] (Fig. 6); Statistical analysis - Kruskal-Wallis test, post hoc - Dunn's multiple comparison test: p < 0.001, *: p < 0.05, ns: insignificant difference). Thus, C 19H 21 N3F2O acts as a selective positive modulator of TRPC6.

[0042] To test the neuroprotective properties of the compounds under amyloid toxicity conditions, the model described previously

[24] was used. The mean dendritic spine head area (Fig. 7 and Fig. 8) is presented as median values ​​with interquartile ranges (n ≥ 210 spines from different neurons for each group of three culture batches). 24-hour incubation in the presence of Aβ42 [0.66 μM] negatively affects the morphology of dendritic spines. Specifically, Aβ42 caused a decrease in the spine head area (Fig. 8), indicating that the spines became thinner and weaker. In the case of incubation in the presence of Aβ42 with 100 nM C 19 H 21 N3F2O Aβ42-associated spine deformation was absent.

[0043] The influence of C was determined 19 H 21N3F2O on synaptic plasticity using an in vitro experiment measuring LTP. LTP was defined as the average slope of the excitatory postsynaptic potential (epsp) 30-40 minutes after high-frequency stimulation. Experiments were performed on hippocampal slices from 8-month-old mice. In wild-type mice, long-term potentiation (LTP) of 146.3 ± 12.4% of the baseline was recorded after high-frequency stimulation. LTP from slices of 5xFAD AD mouse model was significantly weaker (119.0 ± 5.4%). 19 H 21 N3F2O was added to the slice perfusion solution at a concentration of 100 nM 20 min before the start of baseline recording. Addition of C 19 H 21 N3F2O significantly increased LTP in 5xFAD slices compared to the untreated 5xFAD slice group (Fig. 9). No significant differences in potentiation were observed in the wild-type + C groups. 19 H 21N3F2O" and "wild type" were not observed.

[0044] Pharmacokinetic profile C 19 H 21 N3F2O.

[0045] To determine whether this compound is stable in blood plasma samples, C 19 H 21 N3F2O was incubated with mouse plasma samples for 4 hours at 37°C. To confirm plasma enzyme activity, the control substance propantheline, which is known to be unstable in plasma, was used. The study showed that the concentration of C 19 H 21 N3F2O remains stable in mouse plasma. After 4 hours, approximately 91.5% of the initial amount of the substance remains in mouse plasma (Fig. 10).

[0046] We investigated the pharmacokinetics of C 19 H 21 N3F2O in female C57BL / 6J mice. C 19 H 21N3F2O was administered intraperitoneally once at a dose of 10 mg / kg. Blood and brain samples were collected at 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours. Maximum C concentrations were observed 15 minutes after IP injection. 19 H 21 N3F2O: 7.69 ± 0.95 μg / mL (mean ± SEM) in plasma (Fig. 11) and 7.72 ± 2.37 μg / mL in brain (Fig. 12). C 19 H 21 N3F2O was not detected in plasma samples after one hour and in the brain after two hours. Thus, the results demonstrate rapid absorption of C. 19 H 21 N3F2O into the bloodstream and penetration through the BBB.

[0047] Since C 19 H 21 N3F2O is a positive modulator of TRPC6, stable in mouse plasma and able to penetrate the BBB, the effect of intraperitoneal injections of C was tested 19 H 21 N3F2O on the induction of LTP in synapses of Schaffer collaterals of the CA1 region of the hippocampus in vitro in living slices. For this purpose, 8-month-old mice were intraperitoneally injected with C19 H 21 N3F2O (10 mg / kg in saline with 10% kleptose) or control (equivalent amount of DMSO in saline with 10% kleptose) for 14 days. LTP was measured the day after the last injection. As expected, LTP was significantly reduced in slices from 5xFAD mice that received control injections compared to control slices from wild-type mice (Fig. 13). Injections of C 19 H 21 N3F2O rescued LTP deficiency in 5xFAD mice.

[0048] To study the effect of intraperitoneal injection of C 19 H 21To assess recognition memory in 5xFAD mice, a novel object recognition test was used. Mice behavioral characteristics were assessed after 17 days of intraperitoneal injections of a piperazine derivative at a dose of 10 mg / kg. The day before the test, the animals were placed in the test chamber for 10 minutes to familiarize themselves with the chamber to eliminate the possibility of exploratory behavior in unfamiliar conditions. On the first day of testing, two identical objects (Object A and Object B) were placed in opposite quadrants of the arena. On the second day of testing, Object B was replaced with a novel object.

[0049] All experimental groups showed the same preference for identical objects on the first day of testing. On the second day, WT and 5xFAD+C mice 19 H 21N3F2O mice showed significant interest in the novel object B compared to the familiar object A (Fig. 14). However, 5xFAD mice demonstrated reduced interest in the novel object (as measured by time spent in the object zone) compared to 5xFAD + C mice. 19 H 21 N3F2O.

[0050] To study the influence of C 19 H 21To study contextual and conditioned fear memory in 5xFAD mice, the conditioned freezing test was used. Mice behavior was assessed after 25 days of intraperitoneal injections of a piperazine derivative at a dose of 10 mg / kg. On day 1 (adaptation), mice were placed in the testing apparatus for 5 minutes without any auditory or tactile stimuli. On day 2 (training), each mouse was placed in the testing apparatus for 2 minutes without stimuli and then exposed to a sound (conditioned) stimulus, followed by an electric shock (unconditioned stimulus). This procedure was repeated three times for training. On days 3 and 10 of testing, mice were again placed in the familiar testing apparatus (without any stimuli), and their freezing time was assessed for 180 seconds to assess contextual fear memory.

[0051] All mice showed an increase in freezing time on day 3 of testing compared to the baseline freezing time on the day of training, indicating that all groups of mice learned the conditioned freezing test well. However, on day 10 of testing, the 5xFAD group did not show significant differences in freezing time compared to the day of training, indicating that contextual memory was impaired in 5xFAD mice. In contrast, the WT and 5xFAD+C groups 19 H 21 N3F2O such differences were observed (Fig. 15). Moreover, 5xFAD mice showed a significant deficit in fear memory compared to 5xFAD+C mice. 19 H 21 N3F2O (Fig. 15).

[0052] Example 1. Chemical synthesis of N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide

[0053] The synthesis of N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl)propanamide can be carried out through a series of sequential reactions shown in Fig. 16.

[0054] Stage 1

[0055] Synthesis methyl 2-(4-phenylpiperazin-1-yl)propanoate(2) implemented By method, specified V patent US2023279008A1 PTGR2 INHIBITORS AND THEIR USE.

[0056] 1-Phenylpiperazine (3 g 0.018 mol), methyl 2-bromopropionate (4.0 ml 0.036 mol), triethylamine (4.8 ml, 0.035 mol) and K2CO3 (9.7 g 0.07 mol) were dissolved in 10 ml DMF and heated at 60°C for 4 hours. After removing the solvent, the reaction mixture was diluted with ethyl acetate and washed successively with water and NaCl solution. The organic layer was dried over MgSO4, filtered and purified by column chromatography (gradient elution: 1-5% ethyl acetate in hexane), the final product was isolated as an oil (3.8 g 80%).

[0057] Stage 2

[0058] 2-(4-Phenylpiperazin-1-yl)propanoic acid (3) received hydrolysis.

[0059] To a solution of methyl 2-(4-phenylpiperazin-1-yl)propanoate (3.5 g, 0.014 mol) in 10 ml of acetone, 10 ml of an aqueous solution of lithium hydroxide (0.67 g, 0.028 mol) was added. The reaction proceeded at room temperature for 24 h on an electromagnetic stirrer. 20 ml of distilled water were added to the reaction mixture, and the pH of the reaction medium was adjusted to 7. The resulting precipitate (3.15 g, 96%) was filtered off and dried in a vacuum dryer.

[0060] Stage 3

[0061] Synthesis N-(2,6-difluorophenyl)-2-(4-phenylpiperazin-1-yl) propanamide (5)

[0062] was carried out as follows: 2,6-difluoroaniline (1.1 g 0.0085 mol) was added to a solution of 2-(4-phenylpiperazin-1-yl)propanoic acid (2 g 0.0085 mol) in 40 ml of chloroform. N,N'-dicyclohexylcarbodiimide (1.75 g 0.0085 mol) was added to the reaction mixture with constant stirring and cooling. After 1 h, a precipitate of N,N'-dicyclohexylurea (4) begins to precipitate. The reaction mixture is stirred at room temperature for another 5-6 h. The precipitate of N,N'-dicyclohexylurea is filtered off and a solution of the reaction product in chloroform is washed successively with 1N hydrochloric acid, water and dried with anhydrous sodium sulfate. The solvent is distilled off in a vacuum at 35-40°C and the remaining substance is recrystallized from ethanol, the yield of which was 2.4 g 81.6%.

[0063] Example 2. Determination of mutagenic properties

[0064] For evaluation of mutagenic potential of C 19 H 21The Ames test was performed on N3F2O using histidine-dependent Salmonella typhimurium strains (TA98, TA1537 for frameshift mutations and TA100, TA1535 for point mutations) and tryptophan-dependent Escherichia coli strains (uvrA[pKM101] for point mutations). The test substance was assessed at six concentrations: 12, 40, 125, 400, 1265, and 4000 μM. The analysis was carried out both in the presence and absence of metabolic activation; known strain-specific mutagens were used as positive controls, and 2% DMSO as a negative control.

[0065] After 90 minutes of incubation of the piperazine derivative with bacteria of each strain in a histidine / tryptophan medium, the bacterial cultures were diluted in a pH indicator medium lacking histidine (for S. typhimurium) or tryptophan (for E. coli) and then incubated in 384-well microplates at 37°C for two days. Revertant cells (in which the mutation occurred) actively divided, changing the pH of the medium. The degree of mutagenicity of the compound at different concentrations was assessed by counting the number of wells with altered pH, which was compared with control values.

[0066] No increase in bacterial revertants was observed for strains TA98 and TA1537, either in the absence or presence of fraction S9, indicating that the test compound did not significantly affect the frequency of frameshift mutations. Moreover, this piperazine derivative was not mutagenic towards TA100, TA1535, and the combined E. coli strain with or without fraction S9 (point mutation assessment). For the positive control, a significant increase in the average number of revertant colonies was observed in all strains. These data indicate that C 19 H 21 N3F2O does not exhibit mutagenic potential under the conditions studied. Table 1 presents the results of the mutagenicity assessment of compound C. 19 H 21 N3F2O in the Ames test. Designations in Table 1: n - number of replicates; m - average number of revertant wells; m o / m к ±SD - the ratio of the average number of revertant wells in the experiment (mo ) to that in the negative control (m к ) - the number of times the zero line is exceeded (values ​​are rounded to the first decimal place). Statistical analysis was performed using the Kruskal-Wallis test with Dunn's post hoc multiple comparison test between the negative control and other groups, ***P < 0.001, ns - the difference is not significant.

[0067] Table 1.

[0068] Strain; + / -S9 Experimental conditions n m±SD mo / mk±SD Statistical difference* TA98; -S9 Negative control 3 7,0±3,6 12 μM 3 6,3±2,3 0,60 ns 40 μM 3 12,7±6,0 1,19 ns 125 μM 3 8,3±2,9 0,79 ns 400 μM 3 12,0±1,7 1,13 ns 1265 μM 3 9,3±1,5 0,88 ns 4000 μM 3 13,3±3,5 1,26 ns 2-nitrofluorene (2 mcg / ml) 3 43,3±4,5 4,09 *** TA98; +S9 Negative control 3 2,0±1,7 12 μM 3 1,3±0,6 0,36 ns 40 μM 3 2,3±1,2 0,63 ns 125 μM 3 1,0±0,0 0,27 ns 400 μM 3 2,0±2,0 0,54 ns 1265 μM 3 1,7±2,1 0,45 ns 4000 μM 3 0,7±0,6 0,18 ns 2-aminoanthracene (5 μg / ml) 3 23,0±0.0 6,16 *** TA100; -S9 Negative control 3 13,7±3,1 12 μM 3 14,0±5,6 0,84 ns 40 μM 3 17,0±8,9 1,02 ns 125 μM 3 17,7±4,2 1,06 ns 400 μM 3 14,7±2,3 0,88 ns 1265 μM 3 15,3±2,5 0,92 ns 4000 μM 3 15,7±3,2 0,94 ns N-oxide-4-nitroquinoline (0.1 μg / ml) 3 48.0±0.0 2,87 *** TA100; +S9 Negative control 3 5,0±1,0 12 μM 3 3,0±1,0 0,50 ns 40 μM 3 3,3±4,9 0,56 ns 125 μM 3 4,3±2,1 0,72 ns 400 μM 3 3,3±1,5 0,56 ns 1265 μM 3 2,3±1,5 0,39 ns 4000 μM 3 2,3±2,3 0,39 ns 2-aminoanthracene (5 μg / ml) 3 46,0±1,0 7,67 *** TA1535; -S9 Negative control 3 2,4±2,5 12 μM 3 2,3±1,5 0,48 ns 40 μM 3 1,0±1,0 0,20 ns 125 μM 3 1,0±1,0 0,20 ns 400 μM 3 0,7±0,6 0,14 ns 1265 μM 3 1,0±1,0 0,20 ns 4000 μM 3 1,7±1,5 0,34 ns N4-aminocytidine (100 µg / ml) 3 48.0±0.0 9,80 *** TA1535; +S9 Negative control 3 2,4±3,8 12 μM 3 0,7±1,2 0,11 ns 40 μM 3 0,7±0,6 0,11 ns 125 μM 3 0,0±0,0 0,00 ns 400 μM 3 0,7±0,6 0,11 ns 1265 μM 3 0,7±0,6 0,11 ns 4000 μM 3 0,3±0,6 0,05 ns 2-aminoanthracene (5 μg / ml) 3 21,0±3,4 3,39 *** TA1537; -S9 Negative control 3 1,3±0,6 12 μM 3 3,7±1,2 1,92 ns 40 μM 3 2,3±1,2 1,22 ns 125 μM 3 1,7±1,2 0,87 ns 400 μM 3 3,7±1,2 1,92 ns 1265 μM 3 2,7±0,6 1,40 ns 4000 μM 3 2,0±1,7 1,05 ns 9-aminoacridine (15 μg / ml) 3 48.0±0.0 25,12 *** TA1537; +S9 Negative control 3 1,3±0,6 12 μM 3 0,7±0,6 0,35 ns 40 μM 3 0,3±0,6 0,17 ns 125 μM 3 0,3±0,6 0,17 ns 400 μM 3 0,7±1,2 0,35 ns 1265 μM 3 0,3±0,6 0,17 ns 4000 μM 3 0,7±0,6 0,35 ns 2-aminoanthracene (5 μg / ml) 3 25,7±7,6 13,43 *** E. coli combo; -S9 Negative control 3 4,3±1,5 12 μM 3 11,3±4,2 1,93 ns 40 μM 3 7,0±1,7 1,19 ns 125 μM 3 6,3±3,5 1,08 ns 400 μM 3 4,3±4,5 0,74 ns 1265 μM 3 5,3±2,9 0,91 ns 4000 μM 3 9,0±5,0 1,54 ns 4-nitroquinoline-N-oxide (2 μg / ml) 3 45,7±1,53 7,79 *** E. coli combo; +S9 Negative control 3 9,0±1,7 12 μM 3 6,3±1,5 0,59 ns 40 μM 3 3,3±1,5 0,31 ns 125 μM 3 5,3±2,3 0,50 ns 400 μM 3 6,3±2,1 0,59 ns 1265 μM 3 5,0±2,65 0,47 ns 4000 μM 3 4,3±1,5 0,40 ns 2-aminofluorene (400 µg / ml) 3 30,0±2,7 2,80 ***

[0069] Example 3. Determination of acute toxicity

[0070] For testing the acute toxicity properties of C 19 H 21N3F2O was administered as single intraperitoneal injections to 2-month-old C57BL / 6J mice at concentrations of 50 mg / kg and 100 mg / kg. The compound was dissolved in saline with 10% kleptose; the control group received injections of DMSO in a volume equivalent to the volume of compound in the experimental groups. No statistically significant differences in the weight of animals in the experimental and control groups were found during the 1-month observation period.

[0071] Acute toxicity assessment results C 19 H 21 N3F2O are presented in Table 2, where the change in the weight of mice after administration of C is estimated 19 H 21 N3F2O at doses of 50 and 100 mg / kg. Table 2 shows the relative weight of mice, normalized to pre-injection weight. Data are presented as mean ± SD (n=5 mice in each group). No statistically significant differences were found between groups. 19 H 21N3F2O does not show acute toxicity at a maximum dose of 100 mg / kg. No significant effect of C 19 H 21 No N3F2O was detected on the body weight of mice.

[0072]

[0073] Example 4. Use and effective dose of the claimed invention for the treatment of cognitive functions in Alzheimer's disease

[0074] The experiment involved 5xFAD mice carrying five mutations that cause the Alzheimer's disease phenotype. These mutations include changes in the APP (K670N / M671L + I716V + V717I) and PSEN1 (M146L + L286V) genes, which lead to the accumulation of β-amyloid 42 (Aβ42) and are responsible for cognitive impairment in these mice.

[0075] To assess the effect of the compound on spatial learning and memory, a Morris water maze test was performed. The behavioral characteristics of the mice were assessed after 20 days of intraperitoneal injections of a piperazine derivative at a dose of 10 mg / kg. Three groups of animals were formed for testing: 1) an experimental group of mice with an AD model, which received injections of the test compound (5xFAD + C 19 H 21 N3F2O), 2) a control group of AD mice that received injections of an equivalent amount of DMSO (5xFAD); 3) a control group of wild-type mice that received injections of an equivalent amount of DMSO (WT). The time spent searching for the platform was recorded for each trial during the 5-day training period.

[0076] The results of the Morris water maze test are shown in Table 3. C 19 H 21N3F2O improves spatial memory in 5xFAD mice in the Morris water maze test. The table shows the number of platform crossings by each mouse, as well as the mean ± SD. Number of mice tested per group: (1) 8 control mice (wild type), (2) 10 5xFAD mice, (3) 9 5xFAD + C mice 19 H 21 N3F2O. Normal distribution was tested using the Shapiro-Wilk test. Statistical analysis was performed using the Kruskal-Wallis test with post hoc or Dunn's one-way ANOVA after Dunnett's multiple comparisons test. *: P < 0.05, ns: not significant.

[0077] Table 3.

[0078] The number of platform crossings by animals from different experimental groups 1 2 3 4 5 6 7 8 9 10 mean±SD Statistical difference control 1 1 2 3 2 2 - 2 - 5 2,25± 1,28 - 5xFAD 0 3 1 0 0 0 4 0 2 0 1,00± 1,49 * 5xFAD+C19H21N3F2O 2 1 1 1 4 1 - 0 2 1 1,44± 0,38 ns

[0079] Search time tended to decrease in all groups. In the 5xFAD group, search time was significantly reduced compared to the WT group on the last day of training. However, there was no significant difference between the WT and 5xFAD + C groups. 19 H 21 There was no N3F2O.

[0080] On the fifth day of testing, the number of platform crossings, the time to the first platform crossing, and the time spent in the target quadrant were analyzed. The number of platform crossings was significantly reduced in the 5xFAD group compared to the WT group. There was no significant difference between the WT and 5xFAD + C groups. 19 H 21 N3F2O was not observed in this indicator. A similar pattern was observed for the indicators "time to first platform crossing" and "time spent in the target quadrant."

[0081] These results indicate that 20-day therapy with C 19 H 21 N3F2O at a dose of 10 mg / kg could potentially reduce spatial memory deficit in 8-month-old AD mice.

[0082] These experiments demonstrate that C 19 H 21N3F2O, being an activator of calcium entry through TRPC6, has neuroprotective properties, is able to limit synaptic loss and restore synaptic plasticity in 5xFAD mice when administered intraperitoneally at an effective dose of 10 mg / kg, this compound also lacks mutagenic and toxic properties, at a dose of 10 mg / kg it is able to reduce spatial memory deficit in 8-month-old 5xFAD mice. It is assumed that the use of C 19 H 21 N3F2O at a dose of 10 mg / kg will have a similar clinical effect (improving synaptic plasticity and reducing cognitive deficits) in patients with AD.

[0083] Example 5. Use and effective dose of the claimed invention for the treatment of motor functions in Alzheimer's disease.

[0084] The behavioral characteristics of mice were assessed after 34 days of intraperitoneal injections of a piperazine derivative at a dose of 10 mg / kg. Differences in motor functions were assessed in the "walking on a horizontal bar" test. The test apparatus is a round crossbar with a diameter of 8 mm, 1 m long, at the end of which there is a platform (the finish point). During the training stage, mice walked along the crossbar three times every day (for 3 days). On the 4th (test) day, the time spent walking along the crossbar and the number of paw slips (the average of 3 attempts) were measured. A score was also given for each attempt: "0" significant difficulties in moving along the crossbar, "crawling" gait; "100" normal gait, standing on all fours; "50" combined gait, partly normal and partly "crawling".

[0085] 5xFAD mice took significantly longer to walk on the 8 mm thick beam compared to wild-type mice (Table 4). Furthermore, 5xFAD mice had difficulty walking on the beam, as assessed by the "paw slip count" and "walking score" parameters (Table 4). C injections 19 H 21 N3F2O resulted in a significant reduction in the time required to walk the crossbar compared to the 5xFAD group not receiving C 19 H 21 N3F2O. Also, improved locomotor function in the 5xFAD + C group 19 H 21 N3F2O was noticeable on the 8 mm beam, as mice in this group walked with significantly fewer paw slips and a significantly better walking score (Table 4).

[0086] Table 4.

[0087] The transit time is average±SD The number of paw slips is average±SD Walking score mean±SEM control 17,87±5,02 0,10±0,31 46,67±8,62 5xFAD 22,49±3,72 1,17±1,58 3,33±2,32 5xFAD+C19H21N3F2O 19,89±4,33 0,43±0,86 25,00±7,09

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