1-(4'-bromphenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride

Abstract

FIELD: pharmaceuticals; medicine.SUBSTANCE: use of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride of the specified formula as a multifunctional agent possessing antioxidant (inhibition of lipid peroxidation, increase in the level of reduced glutathione), mitoprotective (slowing down calcium-induced depolarization and swelling of mitochondria) and antiaggregatory (reduction in the aggregation of pathological proteins TDP-43 and FUS) activities.EFFECT: preparation based on it could find use in the treatment of neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis.1 cl, 10 dwg, 1 tbl

Description

[0001] The present invention relates to the field of pharmaceuticals and medicine, in particular, to a chemical compound as a multifunctional neuroprotective agent and discloses the use of said compound for the creation of a medicinal product having a complex of activities: antioxidant, mitoprotective, antiaggregatory in relation to pathological proteins, which makes it a promising candidate for the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS).

[0002] The prior art discloses 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole derivatives for the treatment, alleviation or prevention of disorders associated with tau aggregates, such as Alzheimer's disease [EA 043389 B1, published 05 / 22 / 2022], characterized by the following chemical formula:

[0003]

[0004] or a pharmaceutically acceptable salt thereof;

[0005] where A is selected from the group consisting of

[0006] where and can be attached to Q in any available position, where , and also substituted by one or more substituents R j , and if A is , R j is not -CN, -halogen, or -CF3, and if A is , R j does not represent a -halogen; and where may be optionally substituted with one or more substituents R;

[0007] B is selected from the group consisting of O and NR a ;

[0008] E and V are independently selected from the group consisting of N, O and S;

[0009] G is selected from the group consisting of a benzene ring and a pyridine ring;

[0010] J is selected from the group consisting of O and NR 1 ;

[0011] Q represents N;

[0012] Y is selected from the group consisting of CZ and N, provided that when Y is N, B;

[0013] represents N-alkyl or O;

[0014] Y 1 represents CZ;

[0015] Y 2 represents CZ;

[0016] Y 3 represents CZ;

[0017] Z is independently selected from the group consisting of H, halogen, O-alkyl and alkyl;

[0018] R is independently selected from the group consisting of and -NR 3 R 4 ;

[0019] R a selected from the group consisting of H and alkyl;

[0020] R b , R c , R d , R e , R f and R g represent H;

[0021] R j independently selected from the group consisting of -halogen, -O-alkyl, -CF3, -CN, -NR 3 R 4 , and also , where the bridge containing the carbon atom C 1-2 , may be present between carbon atom a and carbon atom c or d, or a bridge containing carbon atom C 1-2, may be present between carbon atom b and carbon atom c or d;

[0022] R 1 selected from the group consisting of H and alkyl;

[0023] R 2 is an alkyl group, and if two R 2 are geminal, they can be combined to form a 3-6-membered ring;

[0024] R 3 and R 4 independently selected from the group consisting of H and alkyl, wherein alkyl may be optionally substituted with -O-alkyl;

[0025] n is 0, 1, 2, 3, or 4, and

[0026] where alkyl has from 1 to 6 carbon atoms.

[0027] The disadvantage of 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indole derivatives for the treatment, alleviation or prevention of tau aggregate-related disorders such as Alzheimer's disease is that they lack a comprehensive multifunctional effect, since they do not have a sufficient combination of antioxidant, mitoprotective and antiaggregation activities targeting the key pathological mechanisms of neurodegenerative diseases, which limits their therapeutic efficacy.

[0028] Also known from the prior art are copper chelators [EP2927215A1, published 07.10.2015], characterized by a chemical compound of the structure 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline, according to the formula:

[0029]

[0030] where n is a C1-C4 alkane and X is an aromatic ring or the corresponding heteronucleus of the formula

[0031]

[0032] or cyclopentadiene ortho-substituted formula

[0033]

[0034] and Y represents an aromatic ring or a corresponding heteronucleus of the formula

[0035]

[0036] The disadvantage of copper chelators is their narrowly targeted mechanism of action, associated primarily with the binding of copper ions, which does not provide a comprehensive effect on multiple pathological processes of neurodegenerative diseases, such as oxidative stress, mitochondrial dysfunction and pathological protein aggregation, as well as the potential risk of disruption of the metabolism of essential metals, which limits their therapeutic effectiveness.

[0037] The closest in technical essence are substituted pyrazolopyrimidines, the method for their production and their use as a medicine [RU2450004C2, published 10.05.2012], characterized by the following chemical formula (I):

[0038]

[0039] where Y 1 is N or C-,

[0040] Y 2 is N or C-,

[0041] Y 3 is N or C-,

[0042] Y 4 is N or C-,

[0043] where there are at least two groups of Y 1 -Y 4 represent a carbon atom,

[0044] R 1 is chlorine or bromine;

[0045] R 2 and R 3 each independently represents hydrogen, C 1‐6 alkyl,

[0046] C 3‐7 cycloalkyl or trifluoromethyl; or

[0047] R 2 and R 3, taken both together with the carbon atom of the ring, represent

[0048] carbonyl group;

[0049] R 4 and R 5 each independently represents hydrogen or C 1‐6 alkyl; or

[0050] R 4 and R 5 , taken both together with the carbon atom of the ring, represent

[0051] carbonyl group;

[0052] R 6 and R 7 independently represent hydrogen or C 1‐6 alkyl; or

[0053] R 6 and R 7 , taken both together with the ring carbon atom, represent a carbonyl group;

[0054] R 10 and R 11 independently represent hydrogen, halogen, amino, hydroxy, nitro, cyano, trifluoromethyl, trifluoromethoxy, C 1‐6 alkyl, C 1‐6alkoxy, a 5- or 6-membered heteroaryl ring containing one to four heteroatoms selected from oxygen and nitrogen, which may be optionally substituted with 1 or 2 substituents independently selected from halogen, C 1‐6 alkyl and C 1‐6 alkoxy, 5- or 6-membered saturated heterocyclyl containing 1 or 2 heteroatoms selected from oxygen and nitrogen, C 1‐6 alkylamino, di-C 1‐6 alkylamino, C 1‐6 alkylcarbonyl, morpholinocarbonyl, C 1‐6 alkylcarbonylamino, C 1‐6 alkoxycarbonyl, aminocarbonyl, C 1‐6 alkylaminocarbonyl, di-C 1‐6 alkylaminocarbonyl, C 1‐6 alkylsulfonyl;

[0055] or R 10 and R 11together with the two carbon atoms to which they are bound, represent a heteroaryl group containing 5 or 6 ring members and 1 or 2 heteroatoms selected from oxygen, sulfur and nitrogen, or a heterocyclyl group containing 5 or 6 ring members and 1 or 2 heteroatoms selected from oxygen and nitrogen, wherein said groups may be substituted by one or 2 substituents selected from C 1‐6 alkyl and oxo groups;

[0056] and its optical isomers and pharmaceutically acceptable salts, provided that the compound of formula (I) is not the compound (6-bromopyrazolo[1,5-a]pyrimidin-2-yl)-(3,4-dihydro-1H-isoquinolin-2-yl)-methanone.

[0057] The main technical problem of the prototype is their limited and narrowly targeted mechanism of action, which does not provide a comprehensive multifunctional neuroprotective effect, since they do not demonstrate significant antioxidant activity in biological systems, do not exhibit pronounced mitoprotective activity, and also do not have anti-aggregation activity against key pathological proteins of neurodegenerative diseases, and, as a result, have reduced therapeutic activity.

[0058] The objective of the invention is to eliminate the shortcomings of the prototype.

[0059] The technical result of the invention consists in obtaining the chemical compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride and in increasing the therapeutic effectiveness, in particular, increasing the antioxidant activity, mitoprotective activity and angioaggregation activity.

[0060] The technical result is achieved due to the fact that the chemical compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride is characterized by the following structural formula:

[0061]

[0062] Brief description of drawings

[0063] Fig. 1 shows the inhibition of spontaneous lipid peroxidation in the presence of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (10 μM) and trolox (10 μM).

[0064] Fig. 2 shows the inhibition of Fe 2+ -induced lipid peroxidation in the presence of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (10 μM) and trolox (10 μM).

[0065] Figure 3 shows the change in GSH level in SH-SY5Y cells. Medians with 95% confidence intervals are shown.

[0066] Figure 4 shows the inhibition of calcium-induced mitochondrial depolarization in the presence of 100 μM 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride.

[0067] Fig. 5 shows the inhibition of mitochondrial swelling by 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride.

[0068] Fig. 6 shows the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on the survival of SH-SY5Y neuroblastoma cells (MTT test).

[0069] Figure 7 shows the effect of the studied 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on the aggregation of TDP-43 (Δ1-192) as a representative photograph of SH-SY5Y cells after transfection with the EGFP-TDP-43 (Δ1–192) construct, with and without treatment with 10 μM of the substance.

[0070] Fig. 8 shows the effect of the studied 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on the aggregation of TDP-43 (Δ1–192) in the form of the results of the analysis of the number of cells with aggregates.

[0071] Fig. 9 shows the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on FUS-F15 aggregation as a representative photograph of SH-SY5Y cells after transfection with the EGFP-FUS-F15 construct, with and without treatment with 10 μM of the substance.

[0072] Fig. 10 shows the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on the aggregation of FUS-F15 as the results of the analysis of the number of cells with aggregates.

[0073] Implementation of the invention

[0074] The chemical compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride is characterized by the following structural formula

[0075]

[0076] is a multitarget compound with neuroprotective potential for the treatment of neurodegenerative diseases.

[0077] Molecular formula: C 17 H 19 BrClNO2

[0078] Molecular weight: 384.70 g / mol

[0079] Melting point: 134-135°C.

[0080] Elemental analysis:

[0081] C: 53.08

[0082] H: 4.98

[0083] Br: 20.77

[0084] Cl: 9.22

[0085] N: 3.64

[0086] O: 8.32

[0087] Spectra:

[0088] NMR 1 H (600 MHz, CDCl3, ppm, J / Hz): 2.71 (1H, dt, J= 4.9, 15.9, H a -4), 2.89 (1H, ddd, J= 5.3, 8.3, 14.8, H b -4), 3.01 (1H, ddd, J= 4.6, 8.3, 12.2, H a -3), 3.15 (1H, dt, J= 5.3, 12.2, H b -3), 3.62 (3H, s, 7-OSH3), 3.84 (3H, s, 6-OSH3), 4.98 (1H, s, H-1), 6.17 (1H, s, H-8), 6.61 (1H, s, H-5), 7.11 (2H, d, J=8.4, H-2',6'), 7.41 (2Н, d, J=8.4, H-3',5');

[0089] NMR 13C (150 MHz, CDCl3, ppm): 29.30 (C-4), 41.88 (C-3), 55.93 (6-OCH3), 55.98 (7-OCH3), 60.91 (C-1), 110.84 (C-8), 111.61 (C-5), 121.4 (C-4'), 127.76 (C-4a), 129.31 (C-8a), 130.76 (C-2',6'), 131.57 (C-3',5'), 144.04 (C-1'), 147.23 (C-7), 147.86 (C-6).

[0090] Method for producing 1-(4´-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride, C 17 N 18 O2NBr × HCl.

[0091] A mixture of 2.89 g (15.96 mmol) of 3,4-dimethoxyphenylethylamine, 2.94 g (15.96 mmol) of 4-bromobenzaldehyde in 10 ml of trifluoroacetic acid was refluxed for 4 hours.

[0092] The reaction progress was monitored by TLC (Thin Layer Chromatography).

[0093] The reaction mixture was then cooled and basified with a 5% aqueous sodium hydroxide solution to pH 9-10, then the product was exhaustively extracted with chloroform. After distilling off the chloroform, the crude product was dissolved in acetone, and the resulting precipitate was filtered. The yield was 5.22 g (94%), melting point 134-135°C (from CH3-CO-CH3)R f 0.82 (chloroform:methanol system, 12:1).

[0094] The resulting 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride was dissolved in acetone and acidified with concentrated HCl to pH 5-6. The resulting hydrochloride precipitate was filtered off and washed 3 times with acetone.

[0095] 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride is proposed as a potential multifunctional drug combining a number of primary activities – antioxidant, mitoprotective, and antiaggregatory – against a number of proteins characteristic of proteinopathies (β-amyloid protein, FUS, and TDP43 proteins). Therefore, it can be considered as a candidate for further development as a drug for the treatment of neurodegenerative diseases, in particular, possibly Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS).

[0096] The chemical compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride is understood under the conventional designation F46, the experimental activity of which, presented in the figures, includes inhibition of spontaneous and Fe²⁺-induced lipid peroxidation, an increase in the level of reduced glutathione (GSH), a slowdown in calcium-induced depolarization and swelling of mitochondria, low cytotoxicity, as well as a decrease in the aggregation of pathological proteins TDP-43 and FUS (Fig. 1-10).

[0097] Neurodegenerative diseases currently represent not only a medical problem but also a significant social and economic one. The main reason for the lack of significant progress in developing effective medications for the treatment of neurodegenerative diseases (NDs) is that they are diagnosed when the neurodegenerative process in the brain is already advanced, and their pathogenesis suggests different disease pathways depending on the precipitating factors and individual genetic characteristics.For virtually all neurodegenerative diseases, the progressive loss of neurons specific to each brain region is associated with uncontrolled pathological aggregation of specific proteins (proteinopathy), membrane lipid peroxidation (LPO), and impaired mitochondrial function, particularly decreased mitochondrial resistance to the key step in most cell death cascades—the opening of mitochondrial permeability pores (MPPs), as well as a number of other processes. Overall, neurodegenerative diseases have multiple pathological pathways, and these processes can be considered biotargets for potential neuroprotective drugs. We discovered precisely this activity profile for one of the isoquinoline derivatives, 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride.

[0098] 1. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride exhibits antioxidant activity.

[0099] 1.1. Suppresses both Fe 2+ -induced and spontaneous LPO in rat brain homogenate.

[0100] To evaluate the antioxidant properties of the compounds and their effect on mitochondrial characteristics, brain homogenate was obtained and liver mitochondria were isolated from nonlinear laboratory rats using standard differential centrifugation methods after euthanasia of the animals with carbon dioxide.

[0101] The method for measuring lipid peroxidation intensity is based on the formation of a complex between TBA and TBA-reactive lipid peroxidation products (mainly malondialdehyde) and measuring its light absorption. To measure the intensity of spontaneous lipid peroxidation, the brain homogenate was incubated with the test compounds or DMSO at 37°C for 2 and 4 hours. In the case of lipid peroxidation stimulation, 0.5 mM FeSO4 was added to the homogenate and incubated at 37°C for 1 hour or up to 4 hours without an oxidizing agent when measuring spontaneous lipid peroxidation. After incubation, 2.6 mM TBA and 0.92 M TCA (in a double volume of 250 mM HCl) were added to the homogenate and the mixture was incubated at 90°C for 1 hour. The samples were then centrifuged in an Avanti J-25 centrifuge (Beckman Coulter, USA) at 6000g for 15 minutes, after which the supernatant was transferred to a transparent 96-well plate to measure the light absorbance of the samples at a wavelength of 532 nm and 620 nm (background value) in an EnVision plate reader (PerkinElmer, Finland).The obtained values, with background subtraction, were used to assess the intensity of lipid peroxidation and the degree of its inhibition by the test compounds. In the case of spontaneous lipid peroxidation, the light absorbance of the samples was plotted against the incubation time; in the case of stimulated lipid peroxidation, the obtained values ​​were normalized to the control groups with and without the addition of ferrous iron.

[0102] The ability of F46 to prevent the effects of oxidative stress in biological systems – lipid peroxidation of rat brain homogenate – was investigated as one of the mechanisms of neuroprotective action.

[0103] 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (10 μM) in the spontaneous lipid peroxidation test (Fig. 1) reduces the content of lipid oxidation products after 2-hour incubation, and this effect is somewhat less pronounced after 4 hours of incubation in the lipid peroxidation test induced by 500 μM Fe 2+ (Fig. 2).

[0104] 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride also has significant activity as a lipid peroxidation inhibitor and reduces the formation of lipid peroxidation products by more than 70%, although, as shown previously, this compound did not exhibit either radical-binding activity in the ABTS test or iron-reducing activity in the FRAP test.

[0105] Thus, the antioxidant activity of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride in a biological system (rat brain homogenate) is not associated with its own antioxidant activity and is determined by other mechanisms.

[0106] 1.2. Increases the content of reduced glutathione in SH-SY5Y neuroblastoma cells.

[0107] Reduced glutathione (GSH) is a tripeptide consisting of the amino acids L-glutamate, L-cysteine, and glycine. It plays a crucial role in maintaining intracellular redox potential, including in neuronal cells. GSH's importance in redox-dependent processes is determined by its role in regulating cellular redox signaling and transcription factor activity. It also serves as an intracellular antioxidant, acting as a free radical scavenger and a co-substrate in peroxide detoxification reactions catalyzed by glutathione peroxidase and glutathione transferase. GSH is known to play a key role in cellular oxidant defense and is essential for preventing lipid peroxidation.

[0108] The method for measuring GSH content in cells is based on the reaction of MCB binding to low molecular weight thiols and measuring the fluorescence of this reaction product. The cells were washed from the culture medium with HBSS buffer (1.26 mM CaCl2, 0.89 mM MgSO4, 5.3 mM KCl, 0.44 mM KH2PO4, 0.6 mM Na2HPO4, 137.9 NaCl, 4.2 mM NaHCO3, 10 mM HEPES, 5.55 mM glucose, pH = 7.4) and incubated in this buffer with the test compounds or at 37 ° C for 2 hours. Then MCB (50 μM) was added to the incubation medium and incubated at 37 ° C for 40 minutes. After the incubation, the cells were washed and, using a ZEISS LSM 900 confocal microscope with an Airyscan 2 system (Carl Zeiss AG, Germany), 5-7 z-stacks (a series of images along the z-axis) were taken on each cover glass (three glasses per connection) with excitation by 405 nm radiation and fixation in the region of 435-485 nm fluorescence filter.Using Fiji software, z-stacks were processed: cell regions were marked, images with the highest fluorescence intensity along the z-axis were selected, and fluorescence intensity values ​​for the selected regions were obtained. Fluorescence intensity was used to estimate GSH levels in SH-SY5Y neuroblastoma cells exposed to the test compounds.

[0109] The effect of the compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on the content of the reduced form of glutathione GSH in SH-SY5Y neuroblastoma cells was studied. It was shown that at low concentrations, 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride increases the GSH content and already at a concentration of 3 μM this increase reaches significance (Fig. 3).

[0110] 2. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride exhibits mitoprotective activity.

[0111] The prior art contains data on the toxic effects of isoquinoline derivatives on mitochondria. Several studies have concluded that isoquinoline analogs induce apoptosis through their effects on pMT. Isoquinoline derivatives have also been considered as structural analogs of MPTP, a known toxin that models Parkinson's disease.

[0112] Therefore, the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on mitochondrial potential and the process of opening of the mitochondrial membrane was evaluated to detect both possible mitoprotective and mitotoxic properties.

[0113] 2.1. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride does not cause signs of mitochondrial toxicity.

[0114] The effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on mitochondrial potential was carried out using the cationic dye safranin O, which penetrates into energized and "charged" mitochondria, which leads to quenching of its fluorescence. Upon depolarization of mitochondrial membranes, the dye leaves the organelles, which is recorded fluorometrically. The measurement medium contained 1 mM KH2PO4, substrates (10 mM glutamate, 2 mM malate and 5 mM succinate), 10 μM safranin O. Mitochondrial suspension was added to the medium (final protein concentration - 0.5 mg / ml). The resulting working suspension was pipetted into the wells of an opaque 96-well plate, and fluorescence was measured using an EnVision plate reader (PerkinElmer, Finland) with an excitation wavelength of 495 nm and an emission wavelength of 586 nm. After 2-3 minutes of measurement, the samples were supplemented with compounds or DMSO as a control, and the measurements were resumed.Once stable fluorescence values ​​were achieved, CaCl2 was sequentially added to the medium. During data processing, we determined whether and to what extent the studied compounds depolarized the mitochondrial membrane. The compound's ability to inhibit or stimulate Ca2 release was also assessed based on the change in fluorescence after the addition of CaCl2. 2+ -mediated depolarization.

[0115] When studying the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on mitochondrial potential, no mitochondrial depolarization was detected even at a concentration of 100 μM.

[0116] 2.2. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride inhibits calcium-induced opening of the AMP.

[0117] However, at this concentration, 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride inhibits calcium-induced mitochondrial depolarization (Fig. 4), which indicates the ability of this compound to suppress the opening of mitochondrial permeability pores, a key step in cell death, and may be the mechanism of the neuro(cyto)projector effect of this compound.

[0118] This conclusion is also confirmed by the discovered ability of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride in lower (3 and 10 μM) concentrations to slow down calcium-induced “swelling” of mitochondria (Fig. 5) – a change in the shape of mitochondria, and accordingly their light absorption at 540 or 620 nm, was carried out on an EnVision plate reader (PerkinElmer, Finland).

[0119] 3. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride has low cytotoxicity.

[0120] The potential cytotoxicity of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride was assessed using the standard MTT assay on SH-SY5Y neuroblastoma cells. The compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride significantly reduced cell viability to 55±5% at a concentration of 100 μM only, and was virtually indistinguishable from the control at a concentration of 10 μM (Fig. 6).

[0121] Thus, 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride exhibits low cytotoxicity in SH-SY5Y neuroblastoma cells and low mitochondrial toxicity—even at a concentration of 100 μM, it does not cause mitochondrial depolarization. At the same time, under conditions of calcium-induced mitochondrial permeability pore opening, it slows calcium-induced mitochondrial swelling and depolarization, and also exhibits mitochondrial permeability pore inhibitory properties, which may be the mechanism of its potential neuroprotective activity.

[0122] 4. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride exhibits antiaggregatory activity.

[0123] 4.1. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride F46 in vitro reduces the number of aggregates of beta-amyloid peptide (1-42), a protein characteristic of AD that is prone to aggregation.

[0124] Inhibitory effect of the studied compounds on the aggregation of beta-amyloid (1-42) (Aβ 42 ) was determined using a fluorescence method using Thioflavin T (ThT). The principle of the method is based on the specific interaction of the fluorescent dye Thioflavin T with the β-sheet structures of beta-amyloid fibrils formed as a result of its aggregation, which leads to a significant increase in the fluorescence intensity of the bound dye. Moreover, a decrease in the fluorescent signal in the presence of the studied compounds correlates with their inhibitory effect on the formation of Aβ aggregates. 42 .

[0125] 1 mg lyophilized Aβ preparation 42 , treated with hexafluoroisopropanol (HFIP), from GenicBio (China) was dissolved in DMSO to obtain a stable stock solution ([Aβ 42 ]=500 μM).

[0126] For the assessment of Aβ aggregation 42and the degree of its inhibition by the test compounds, a portion of the prepared 500 μM beta-amyloid solution was dissolved in 215 mM Na-phosphate buffer pH 8.0 to a final concentration of Aβ 42 50 μM. The samples were then incubated for 24 h at 37°C in a thermostat without stirring in the absence of (baseline Aβ aggregation 42, control) or in the presence of test compounds. Myricetin and propidium iodide were used as reference compounds. The final concentration of all test compounds was 100 μM. After incubation, 5 μM Thioflavin T solution in 50 mM glycine-NaOH buffer pH 8.5 was added to the samples and the fluorescence spectrum was recorded (440 nm (exc.) and 485 nm (emis.)). The blank consisted of 215 mM Na-phosphate buffer pH 8.0 containing DMSO or test compounds, respectively. Measurements were performed on a FLUOstar OPTIMA multifunctional microplate reader (BMG Labtech, Germany). All studies were performed in triplicate, the results are presented as the mean ± SEM.

[0127] Degree of inhibition of Aβ aggregation 42 (%) was calculated using the following formula:

[0128] % inhibition = 100 – ((IF Aβ+ингибитор / IF Aβ ) x100),

[0129] where IF Aβ – fluorescence intensity in the absence of the test compound (taken as 100%), IF Aβ+ингибитор– the fluorescence intensity in the presence of the test compound after subtracting the fluorescence values ​​of the corresponding blanks.

[0130] The antiplatelet effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride was demonstrated. It does not exceed the effects of reference compounds and is only a quarter of their potency. However, when combined with other patented activities, it may provide a significant neuroprotective effect.

[0131] Table No. 1. Inhibitory effect of compounds on self-aggregation of β-amyloid (1-42)

[0132] Compound F-46 0.1 mM Myricetin 0.1 mM Propidium iodide, 0.1 mM % inhibition of Aβ42 aggregation (GenicBio), M±SEM, n=3 21.6±1.5 79.4±6.3 80.1±5.6

[0133] 4.2. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride reduces the amount of aggregates in neuronal cells (neuroblastoma SH-SY5Y) transfected with the EGFP-TDP-43 construct encoding a truncated form of TDP-43 (Δ1–192), a protein characteristic of ALS that is prone to aggregation.

[0134] An experiment was conducted to examine the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on cell aggregation. SH-SY5Y cells were grown on glass and then transfected with the EGFP-TDP-43 construct encoding a truncated form of TDP-43 (Δ1–192), an aggregation-prone protein fused with green fluorescent protein (GFP), using Lipofectamine 2000 reagent (ThermoScientific, USA) according to the manufacturer's instructions. Three hours later, the test compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride was added to the cells at a non-toxic concentration of 10 μM. Twenty-four hours after transfection, the cells were fixed and stained with DAPI (Sigma Aldrich, USA) to visualize the nuclei, after which the number of cells with aggregates was quantified: the number of TDP-43 (Δ1–192)-transfected cells with aggregates was counted in the control samples, as well as in the samples with F46-treated cells (Figs. 7–8).

[0135] Analysis of the number of cells with aggregates showed a decrease in such cells after their treatment with the compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride.

[0136] 4.3. 1-(4'-Bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride reduces the amount of aggregates in neuronal cells (neuroblastoma SH-SY5Y) transfected with the EGFP-FUS-F15 construct encoding a mutant form of FUS, a protein characteristic of ALS that is prone to aggregation.

[0137] An experiment was conducted to examine the effect of 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride on the aggregation of a mutant form of the FUS protein. SH-SY5Y cells were grown on glass and then transfected with the EGFP-FUS-F15 construct encoding a mutant form of FUS, an aggregation-prone protein fused with green fluorescent protein (GFP). Three hours later, the test compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride was added to the cells at a non-toxic concentration of 10 μM. After 24 hours, the cells were fixed, and the resulting preparations were examined microscopically. The number of cells with aggregates transfected with FUS-F15 without the addition of compound, in the presence of only an equal volume of solvent, as well as cells treated with 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride were counted (Fig. 9-10).

[0138] Analysis of the number of cells with aggregates showed a significant decrease in such cells when adding the compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride compared to control cells.

[0139] Thus, the compound 1-(4'-bromophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride increases therapeutic efficacy, in particular antioxidant activity, mitoprotective activity and angioaggregation activity.

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