RU2794090C1 - Membrane stabilizing effect of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-h-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the use of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride as membrane stabilizing agent for the prevention and treatment of diseases of the bronchopulmonary system.

EFFECT: invention provides an expansion of the arsenal of agents that simultaneously have a pronounced virus-specific effect and a membrane-stabilizing property.

2 cl, 5 dwg

Description

The invention relates to medicine, veterinary medicine, specifically to pharmacology, and concerns the use of hydrochloride 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4- benzylpiperazine as a membrane stabilizing agent for the prevention and treatment of diseases of the bronchopulmonary system, including viral and bacterial infections, including coronavirus infection caused by various strains of the SARS-COV-2 virus

It can also be used in the prevention and treatment of cancer patients under the influence of radiation exposure and chemotherapy, in other diseases associated with the stabilization of the cell membrane.

Numerous membrane structures of the cells of the body have been developed in the course of a long evolution of the forms of organization of the cell space, the fixation of enzyme systems and the regulation of metabolism. It has been proven that in many diseases, it is the cell membranes that suffer first of all, especially their lipid component, which normally ensures continuity, fluidity of the membranes, optimal conditions for the functioning of enzymes built into the membrane, transport proteins, and numerous receptors. The pathogenetic role of damage to the lipid component of the membranes of the epithelium of organs and tissues during the formation of various pathologies has been established. In this regard, the effectiveness of complex therapy largely depends on the degree of protection of the structure and function of cell membranes, therefore, for any pathology, the inclusion of membrane-stabilizing drugs in the treatment complex is justified. These drugs can also be used as monotherapy for the treatment and prevention of diseases based on primary damage to cell membranes, as a protection against various damaging effects.

The complex of membrane stabilizing agents can be quite wide, which is due to a wide variety of factors that damage the structure and function of cell membranes: peroxidation processes as a result of hypoxia, stress; ionizing radiation, heavy metal intoxication; processes that occur during osmotic, hemorrhagic shock; spontaneous and phospholipase hydrolysis of phospholipids; reduced energy supply for membrane-bound transport systems; autoimmune damage; excessive circulation of hormones (catecholamines, hypervitaminosis D); violation of cellular calcium homeostasis; viral and bacterial infections and others.

It is known that the cell membrane is a dynamic structure that continuously changes its shape. The most important cellular processes, such as exo- and endocytosis, intracellular vesicular transport, are associated with topological rearrangements of membranes, fusion and division, which require local disruption of the bilayer structure. These processes are associated with the formation of highly curved membrane surfaces, a certain configuration and lipid composition of which is maintained due to specific lipid-protein interactions. Similar processes of local changes in membrane morphology are realized under the action of radiation exposure and chemotherapy and at various stages of cell infection with bacteria and viruses, in particular, influenza viruses, whether it is the absorption of the virion during cellular endocytosis, the fusion of the viral and endosomal membranes, preceding the release of the genetic material of the virus into the cytoplasm of an infected cell, or the budding of daughter viral particles from the surface of its plasma membrane. It is clear that the energy costs required for the implementation of such topological rearrangements of the cell membrane structures will be determined by the mechanical properties of the membrane as a continuous medium. The necessary formalism for such a description was developed in the works of Helfrich [Helfrich W. Elastic properties of lipid bilayers: theory and possible experiments // Z. Naturforsch - 1973 - 28 - P. 693-703], and supplemented by Hamm and Kozlov [Hamm M. Elastic energy of tilt and bending of fluid membranes / Hamm M., Kozlov MM // Eur. Phys. J. - 2000 - P. 323-335]. In these works, it was proposed to consider the lipid bilayer of a cell as a two-dimensional liquid crystal and to use the theory of elasticity of liquid crystals to describe the mechanical properties of membranes. Within the framework of this approach, the average orientation of lipid molecules is described by a vector field of unit vectors, called the director, n. The field of directors is set on some surface passing inside the lipid monolayer. The shape of a surface is characterized by the vector field of unit normals to it, N. In membrane mechanics, as a rule, three main independent deformations are considered: transverse bending, inclination, and lateral tension/compression. The transverse bending deformation corresponds to the appearance of an angle between the directors at close points of the separating surface, and is quantified by the director divergence, div(n). The tilt deformation corresponds to the deviation of the director from the normal and is quantified by the tilt vector t = n - N. Lateral stretching/compression is characterized by the relative change in the area of the dividing surface of the monolayer, (a - a ₀ )/a ₀ , where a is the current area attributable to the lipid per dividing surface, a ₀ - the original area. The part of the free energy associated with deformations is represented by a Taylor series expansion in these deformation modes with respect to the spontaneous state, assuming that the deformations are small. The curvature of a monolayer in the spontaneous state can be nonzero. This curvature is called spontaneous. Spontaneous curvature is determined by the lipid composition of the membrane. It is believed that the spontaneous curvature of a multicomponent monolayer is equal to the concentration-weighted average spontaneous curvature of individual components. Thus, the deformation part of the free energy turns out to be related to the “chemical” contribution due to the dependence of spontaneous curvature on the concentrations of lipid components. Each of the deformations is characterized by its modulus, i.e. the energy that needs to be expended for its implementation. Experiments in various model lipid systems have shown that the greatest modulus is characteristic of the deformation of the lateral tension / compression of the membrane, and therefore such a deformation is practically not realized in the processes of cellular morphogenesis, and the deformation of the transverse bend has the greatest influence on the processes of topological rearrangements of the membrane [Galimzyanov T.R. Linear tension and structure of the raft boundary, calculated taking into account bending, tilt and tension/compression deformations /T.R. Galimzyanov, R.Yu. Molotkovsky, S.A. Akimov//Biol. Membranes - 2011-28(5) - P.415-422].

In connection with the development of an unfavorable situation associated with the spread of diseases caused by bacterial and viral infections, it is currently relevant to develop an effective drug to prevent the development of pneumonia, which is the main complication of influenza and other viral infections (ARVI, including coronavirus infection caused by various strains SARS-COV-2 virus).

It is known that for the chemical series of 5-hydroxyindoles, for example, umifenovir, in addition to virus-specific activity, a general membrane-stabilizing effect was revealed, which can ensure the preservation of the integrity and functionality of cells during viral and bacterial infections [Galiano V., Villalain J. The Location of the Protonated and Unprotonated Forms of Arbidol in the Membrane: A Molecular Dynamics Study// J. Membrane Biol. (2016). - 249:381-391].

Known compounds with antiviral activity, the method of their preparation /RF Patent No. 2387642, C07D401/06, publ. 10/31/2007/.

Known interferon-inducing agent for the treatment of acute respiratory viral infections (ARVI) /RF Patent No. 2445094, A61K 31/496, publ.20.03.2012/.

Known remedy against the influenza virus B /RF Patent No. 2435582, A61K 31/40, publ.10.12.2011/.

Known clathrate complexes of beta-cyclodextrin with 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-n-indol-3-yl]carbonyl}-4-benzylpiperazine with antiviral activity, their preparation and application / RF Patent No. 2448120, C08B 37/16, publ. 20.04.2012/.

These agents are 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride and have virus-specific activity.

The technical problem solved by the present invention is the expansion of the arsenal of agents that have a membrane-stabilizing effect, that is, the ability to change the flexibility and increase the resistance of the membranes of the epithelial cells of the body, including the bronchopulmonary system, to help create a specific immune defense in order to prevent the development of pneumonia, bacterial and viral etiology , including those caused by various strains of the SARS-COV-2 virus, as well as oncological diseases when exposed to radiation exposure and chemotherapy, and other diseases associated with the stabilization of the cell membrane.

The property of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride to change the flexibility and increase the resistance of the membranes of the epithelial cells of the body, including including the bronchopulmonary system, to promote the creation of specific immune protection is not obvious to a specialist and does not follow from the state of the art in this field, is not found in the patent and medical literature.

The above property of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride can be used in practical public health and veterinary medicine to improve the quality prevention and treatment. Thus, the proposed technical solution meets the criteria for patentability of the invention, namely "novelty", "inventive step" and "industrial applicability".

What is new in the present invention is that as a membrane-stabilizing agent that is embedded in a bilayer lipid membrane, leading to a significant (3-fold) decrease in the flexural rigidity of the latter, its topological rearrangement, as a result of which it significantly complicates the penetration of a viral particle into epithelial cells and thereby prevents the development pneumonia caused by bacterial and viral infection, including those caused by various strains of the SARS-COV-2 virus, 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indole-3 hydrochloride can be used -yl]carbonyl}-4-benzylpiperazine.

The technical problem to be solved by the claimed invention is the expansion of the arsenal of agents that simultaneously have a pronounced virus-specific effect and a membrane-stabilizing property.

To achieve a technical result, the present invention proposes a membrane stabilizing agent that has the ability to change the flexibility and increase the stability of the membranes of the epithelial cells of the body, including the bronchopulmonary system, as a result of which it significantly hinders the penetration of the viral particle into the epithelial cells and thereby prevents the development of pneumonia caused by bacterial and viral infection, including those caused by various strains of the SARS-COV-2 virus.

The essence of the claimed invention is illustrated by the following figures:

Figure 1 - Change in the conductivity of the tube (curve 1) caused by the removal of the patch pipette from the BLM (curve 2, right scale).

Figure 2 - B - change in the conductivity of the NT, caused by a change in its length. Г - approximation of the experimental dependence of R on L by a linear function.

Figure 3 - A - current-voltage characteristic of NT at a fixed value of its length, B - approximation of the experimental dependence of r _NT ^-2 on U ² by a linear function.

Figure 4 - Image of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride adsorbed from an aqueous solution on the mica surface. All scales are given in nm.

Figure 5 - Image of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride adsorbed from a solution in chloroform on the mica surface. All scales are given in nm.

The essence of the invention is illustrated by an example of a specific implementation.

Model of a lipid nanotube and its application to the analysis of the mechanical properties of membranes

To carry out these studies, a lipid nanotube (NT) model was chosen, which was first proposed by the staff of the Laboratory of Bioelectrochemistry of the Institute of Physical Chemistry, Russian Academy of Sciences, and has proven itself in studies of the process of lipid membrane morphogenesis during cellular endocytosis [Bashkirov P.V. GTPase cycle of dynamin is coupled to membrane squeeze and release, leading to spontaneous fission / P.V. Bashkirov, S.A. Akimov, A.I. Evseev, S.L. Schmid, J.Zimmerberg, V.A. Frolov // Cell - 2008 - 135(7) - P. 1276-1286; Shnyrova A.V. Geometric catalysis of membrane fission driven by flexible dynamin rings. /P.V. Bashkirov, S.A. Akimov, T.J. Pucadyil, J. Zimmerberg, S.L. Schmid, V.A. Frolov // Science - 2013- 339 (6126) - P. 1433-1436].

To study the effect of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride on the mechanical properties of membranes, an experimental setup was used, consisting of universal oscillator PAR-175 (Princeton Applied Research, USA), patch-clamp amplifier EPC-8 (HEKA Elektronik, Germany), four-pole filter F-900 (Frequency Devices, USA) and oscilloscope OS-1420 (GOULD, England). The position of the pipette relative to the bilayer lipid membrane (BLM) was varied using Newport Motion Controller (Model 860-C2) motion controllers and a pre-calibrated Model ESA-CSA (Newport, USA) micromotion controller (a piezo controller that allows you to change the vertical position of the pipette with an accuracy of 0, 1 µm).

The formation of a model bilayer lipid membrane (BLM) was carried out by the Mueller-Rudin method [Mueller P. Methods for the formation of single bimolecular lipid membranes in aqueous solution / P. Mueller, D.O. Rudin, H.T. Tien, W.C. Wescott // J. Phys. Chem. - 1963 - 67 - P. 534-535] on holes in a copper grid (EMS, USA) with a diameter of ~100 μm, located in the volume of a Petri dish. The grid holes were treated with a solution of phospholipids (Avanti Polar Lipids Inc., USA) in a 1/1 octane/decane mixture (Sigma, USA) at a concentration of 10 mg/mL and dried in the atmosphere. After that, the Petri dish was filled with an electrolyte solution (10 mM KCl, 1 mM Hepes, 0.1 mM EDTA, pH = 7.0; 50 mM KCl, 5 mM Hepes, 0.5 mM EDTA, pH = 7.0 or 100 mM KCl, 10 mM HEPES, pH 7.0), and a drop of a solution of phospholipids in squalane (Sigma, USA) at a concentration of 10 mg/ml was applied to the holes in the lattice with a brush, which spontaneously formed BLM within several minutes. In this case, the solvent and excess lipid went into the bulk phase surrounding the BLM, that is, into the meniscus. The following lipid solutions were used in the experiments: 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), 10 mg/ml 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1.2 -dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS) and cholesterol (Cholesterol) (Avanti Polar Lipids Inc., USA) in chloroform, 10 mg concentration /ml, in the ratios indicated in the text.

After the formation of the BLM, a sawtooth signal from the generator was applied to the silver chloride electrodes using the patch clamps of the amplifier, and the capacitive current of the membrane was recorded in the potential clamping mode. Using a micromanipulator, a patch pipette was brought to the membrane, so that a tight contact was formed between the tip of the micropipette and the membrane (contact resistance 1–10 GΩ), which was seen from a sharp decrease in the capacitive current. To destroy the membrane under the pipette, the hydrostatic pressure changed abruptly, which led to the appearance of a conduction current. After that, removing the pipette from the flat membrane, the membrane tube was pulled out. The electrical conductivity of the tube was measured in the mode of fixing the potential difference between the measuring electrode inside the patch pipette and the ground electrode in the external electrolyte volume using a current amplifier. The potentials used are indicated in the text.

A smooth change in the length of the tube was carried out using a piezocontroller. The data obtained - the current through the tube, the patch-clamp amplifier converted into voltage (gain factors from 1mV / pA to 30mV / pA), the values of the voltage applied to the ends of the tube and the data from the indicator of the piezo controller - were recorded on the computer hard disk after preliminary digitization using ADC L-305/L-1210 (L-card, Russia). The sampling rate is 1 kHz. Before entering the computer, the signals were passed through a low-pass filter (F-900); cutoff frequency 0.5 kHz. The readings of the piezocontroller were recalculated into the values of the vertical displacement of the pipette using a calibration curve. During the transition of MT to NT, the conduction current dropped sharply to zero (Figure 1, curve 1), after which the movement of the pipette stopped. Further, by amplifying the signal from the conduction current by 10–30 times and bringing the pipette closer to the membrane with a piezocontroller, changes in the conduction current through the NT were recorded, and if an increase in current was observed, then one could say that there was an NT between the pipette and the membrane. After that, the dependence of the conduction current strength on the position of the micropipette was recorded.

The peculiarities of NT formation make it possible to monitor the change in the conductivity of the drawn tube depending on its length (Figure 3, C). The conductivity measured during the experiment includes the conductivity of the HT and the conductivity of the leak at the contact between the membrane and the patch pipette. Assuming that the shape of the NT differs little from the cylindrical one, and assuming that the leakage conductivity is constant (only the length L of the NT changes when the pipette moves), the electrical resistance R NT should depend linearly on the change in the length ΔL of the NT. Indeed, the linear function gives a good approximation of the dependence R(L) (Figure 3, D). Thus, since:

,

,

where ρ _sp is the specific resistance of the electrolyte, then both the length L of the NT and its radius r _NT can be calculated.

If the NT contains charged lipids, it is necessary to take into account the fact that the concentration of electrolyte ions inside the NT is higher than outside [10].

,

,

where ρ _NT /ρ _bulk is the specific conductivity of the electrolyte inside the NT/outside the NT, ϕ® is the potential distribution inside the NT.

The equilibrium radius of such an NT is defined as:

.

.

To measure the mechanical parameters, the method [Bashkirov P.V. Membrane nanotubes in an electric field as a system for measuring the mechanical parameters of a lipid bilayer // Biol. Membranes - 2007 - 24(2) - P. 183-192]. The essence of the method is to analyze the dependence of the measured NT radius on the potential difference applied to its ends. The point is that the equilibrium NT radius is determined by the root of the ratio of the NT membrane bending modulus to the BLM lateral tension, and according to the Lippmann electrocapillarity equation, the lateral tension decreases with the appearance of transmembrane stress. Thus, as a result of the electric potential drop along the NT, the transmembrane potential on the NT wall changes from the applied value at one end to 0 at the opposite end. This leads to the fact that the shape of the NT deviates from the cylindrical shape. However, as shown by our theoretical calculations, the electrical resistance of the NT retains a linear dependence on its length. Thus, the measured NT radius corresponds to the radius of some effective cylinder, which has the same conductivity and length as the NT. Moreover, the value of the effective radius of the NT is related to the mechanical parameters and the value of the potential difference applied to the ends of the NT by the following expression:

,

,

where K is the membrane bending modulus, σ ₀ is the lateral tension of the BLM, C _sp is the specific electrical capacitance of the BLM, U is the voltage applied to the ends of the NT.

Therefore, from the linearization of the dependence of the inverse square of the NT radius on the square of the applied stress, one can find both the membrane bending modulus and the lateral tension of the BLM (Figure 3).

Results of studies on the interaction of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride with lipid nanotubes.

To study the effect of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride on the mechanics of the lipid matrix of cell membranes, using the model lipid nanotubes, a lipid composition was chosen similar to that described for Maidin-Derby dog kidney cells (MDCK) (the main components are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and cholesterol), on which a detailed study of cellular and viral lipidoma, their common features and differences was carried out [Gerl M.J., Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane / M.J. Gerl, J.L. Sampaio, S. Urban, L. Kalvodova, J.-M. Verbavatz, B. Binnington, D. Lindemann, C.A. Lingwood, A. Shevchenko, C. Schroeder, K. Simons //J. Cell biol. - 2012 - 196(2) P. 213-221]. In addition, lipids with both saturated and unsaturated lipid tails were used, which is typical for cell membranes. In a number of experiments, when evaluating the effect of charged lipids on the interaction of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride with lipid membranes, the charged components of the membrane were removed and replaced with neutral dioleoylphosphatidylcholine (DOPC). Similarly, in the case of experiments with different amounts of cholesterol also departed from the cellular composition.

The addition of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride to the system was carried out in three different ways: from a solution in deionized water concentration 0.1 mg/ml; from a solution in 96% ethanol with a concentration of 1 mg / ml; or by adding a solution of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in chloroform at a concentration of 1 mg/ml directly to lipid mixture at the stage of BLM formation.

In the first case (aqueous solution with a concentration of 0.1 mg / ml), hydrochloride 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4- benzylpiperazine did not dissolve completely: a suspension remained in the water, which did not disappear for a long time (two weeks). Therefore, for further work, this solution was kept in an ultrasonic bath and filtered through pores with a diameter of 100 nm. The resulting solution was studied by atomic force microscopy (AFM) on a mica substrate. To do this, it was applied to mica (substrate diameter was 1.5 cm) in a volume of about 200 μl and incubated for 15 minutes at room temperature, then a drop of the solution was dried with an argon flow. The resulting sample was scanned in the resonant operating mode of the instrument (tapping mode).

From the image obtained (Figure 4) it can be seen that spherical particles of a substance are presented on the mica surface, the sizes of which vary greatly: the height of the particles is from 0.6 to 1.4 nm, the particle diameter lies in the range of 20-80 nm. The data obtained indicate that 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride is present in water mainly , in the form of agglomerates.

Both in chloroform and in 96% ethanol solution, hydrochloride 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4- benzylpiperazine at a concentration of 1 mg/ml dissolved completely. AFM images showed that in these cases, 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride is represented by particles whose heights range from 0.2 to 0.9 nm and whose diameter ranges from 12 to 20 nm (Figure 5).

However, a solution of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in ethanol acquired a yellowish color after two weeks, the intensity of which depended on its concentration. This may indicate the transition of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride from the hydrochloride form to the base form.

A study was made of the interaction of an alcoholic solution of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride with a membrane of the following lipid composition: DOPC /DOPE/POPC/Cholesterol = 49/13/13/25 mol%. (DOPC - dioleoylphosphatidylcholine; DOPE - dioleoylphosphatidylethanolamine; POPC - palmitoyloleoylphosphatidyloline).

It has been shown that adding an alcoholic solution of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride to the system to a final concentration of 1 μg/ml (buffer 10 mM KCl, 1 mM Hepes, 0.1 mM EDTA, pH = 7.0) statistically significantly reduces the bending modulus of the membrane by half. A further increase in the concentration of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in solution to 2 μg/ml did not lead to a statistically significant change in the bending modulus. It was found that the lateral tension of the membrane did not depend on the presence of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in solution . In control experiments, it was shown that when ethyl alcohol was added to the membrane (the maximum value of the volume fraction of alcohol in the solution reached 0.2%, which corresponded to a similar addition of an alcoholic solution of 1-{[6-bromo-1-methyl-5-methoxy- 2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine) the membrane flexural modulus did not change. However, the fact that in an alcoholic solution of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride probably goes over from the hydrochloride form to the base form, does not allow us to draw a definite conclusion about the effect of the initial hydrochloride 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4 -benzylpiperazine on the membrane by adding it in an alcohol solution.

The membrane properties were studied in the presence of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride. For this, 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl] hydrochloride was added to a solution of DOPC/DOPE/POPC/Cholesterol lipids dissolved in chloroform. carbonyl}-4-benzylpiperazine, also dissolved in chloroform (concentration 1 mg/ml). The experiments were carried out for the following ratio of components: DOPC/DOPE/POPC/Cholesterol/ hydrochloride 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4 -benzylpiperazine = 49-X/13/13/25/X mol%, where X takes the values: 0.02; 0.1; 0.5 mol%. The obtained values of the membrane bending modulus did not depend on the initial concentration of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in the mixture, and amounted to (0.3±0.1)×10 ^-19 J. The value of the bending modulus in the absence of hydrochloride 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indole-3 -yl]carbonyl}-4-benzylpiperazine was (0.9±0.1)×10 ^-19 , i.e. 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H -indol-3-yl]carbonyl}-4-benzylpiperazine, initially added to the membrane, reduces its bending modulus by 3 times. Moreover, the fact that the bending modulus does not depend on the concentration of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in the membrane, rather In total, it indicates that already at a concentration of 0.02 mol%, its practically maximum possible concentration in the bilayer lipid membrane is reached.

An interaction study was conducted with an aqueous solution of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride, prepared as described above, with the membrane, depending on the concentration of cholesterol in it. Experiments were performed with a buffer solution: 50 mM KCl, 5 mM Hepes, 0.5 mM EDTA, pH = 7.0, lipid composition DOPC/DOPE/POPC/Cholesterol = 49/13/13/25 mol% and DOPC/DOPE/ POPC/Cholesterol = 34/13/13/40 mol%. At 25 mol% cholesterol in the absence of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride, the bending modulus is c, after adding 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride, the modulus decreased to (0.4±0, 1) × 10 ^-19 J, which, within the measurement error, corresponds to the results obtained when it was added directly to the membrane from a solution in chloroform (a drop in the bending modulus by 2-3 times). In the case of 40 mol% cholesterol, which corresponds to its level in the lipid envelope of the influenza virus [Gerl MJ, Quantitative analysis of the lipidomes of the influenza virus envelope and MDCK cell apical membrane / MJ Gerl, JL Sampaio, S. Urban, L. Kalvodova , J.-M. Verbavatz, B. Binnington, D. Lindemann, C.A. Lingwood, A. Shevchenko, C. Schroeder, K. Simons //J. Cell biol. - 2012 - 196(2) P. 213-221], in the absence of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}- hydrochloride 4-benzylpiperazine, the value of the bending modulus coincides with the value for the membrane at 25 mol% cholesterol (0.9±0.1)×10 ^-19 J. After adding an aqueous solution of hydrochloride 1-{[6-bromo-1-methyl-5- methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine, the value of this modulus was (0.6±0.1)×10 ^-19 J, i.e. its effect on the flexural stiffness of the membrane decreased proportional to the concentration of cholesterol in the membrane.

When charged lipids (phosphatidylserine) are introduced into the system to a level corresponding to the average content of these lipids in cell membranes (composition DOPC/DOPE/POPC/Cholesterol/DOPS = 34/13/13/25/15 mol%) (DOPS - dioleoylphosphatidylserine) effect 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride on the decrease in membrane flexural stiffness became most pronounced: a drop in the flexural modulus was observed from (0.88±0.14) × 10 ^-19 J to (0.22 ± 0.10) × 10 ^-19 J.

Thus, we studied the effect of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride on the mechanical properties of lipid membranes using the model lipid nanotubes. Three possible options for adding 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride to the system were considered: in the form of filtered aqueous solution, in the form of a solution in alcohol and by direct introduction into the membrane from a solution in chloroform. In all these cases, it was noted that when 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride was added to the lipid membrane, the bending the rigidity of the latter decreased. However, in the case of an alcoholic solution, 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride was converted from the hydrochloride form to the base form Therefore, these solutions were not used for further studies. The fact of the coincidence of the results for the case of adding 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in the form of an aqueous solution to the membrane washing buffer with results for its direct incorporation into BLM show that in all cases, incorporation of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl hydrochloride is observed }-4-benzylpiperazine into the membrane, resulting in a 3-fold decrease in the flexural rigidity of the latter. Moreover, the maximum concentration of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride in the composition of the lipid bilayer does not exceed 0.02 molar percent. Since the low bending stiffness of membranes, as mentioned above, simplifies local topological rearrangements of cell membranes [Campelo F. Helfrich model of membrane bending: from Gibbs theory of liquid interfaces to membranes as thick anisotropic elastic layers / F. Campelo, C. Arnarez, S.J. Marrink, M.M. Kozlov // Adv. colloid interface sci. - 2014 -208 P. 25-33], then 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride, can accelerate the processes of cell vital activity, synaptic transmission, phagocytosis, etc. It should be noted that the maximum effect of the decrease in flexural stiffness was observed in the presence of charged lipids in the membrane, i.e., precisely in the case when the lipid composition of nanotubes most closely reflected the composition of cell membranes.

Thus, 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride has not only a pronounced virus-specific effect, but also properties to change the flexibility and increase the stability of the membranes of the epithelial cells of the body, including the bronchopulmonary system, as a result of which it significantly complicates the penetration of the viral particle into the epithelial cells and thereby prevents the development of pneumonia caused by bacterial and viral infections, including those caused by various strains of the SARS-COV-2 virus . The results of the studies carried out allow us to consider 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride for use before and during viral epidemics, including to prevent the development of viral pneumonia, which is the main complication of influenza and other viral infections (SARS, including coronavirus infection caused by various strains of the SARS-COV-2 virus).

It should also be noted that 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride, having a membrane-stabilizing effect, can prevent damage to the lipid component of the membranes of the epithelium of organs and tissues by increasing their resistance to chemotherapy and radiotherapy of tumors in cancer patients and thereby increase the effectiveness of treatment and reduce the side effects of antitumor drugs and radiation.

Claims (3)

1. The use of 1-{[6-bromo-1-methyl-5-methoxy-2-phenylthiomethyl-1-H-indol-3-yl]carbonyl}-4-benzylpiperazine hydrochloride as a membrane stabilizing agent for the prevention and treatment of bronchopulmonary diseases systems. 2. Application according to claim 1, characterized in that diseases of the bronchopulmonary system in viral and bacterial infections. 3. The use according to claim 2, characterized in that the viral infections are a coronavirus infection caused by different strains of the SARS-COV-2 virus.

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