EA044574B1 - NEW GLUTAMINYL CYCLASE INHIBITORS AND THEIR APPLICATION FOR THE TREATMENT OF VARIOUS DISEASES - Google Patents

Description

Field of technology

This invention relates to the chemistry of organic compounds, pharmacology and medicine and concerns the treatment of diseases associated with aberrant activity of cells of the immune system, in particular for the treatment of diseases of the lungs, respiratory tract and abdominal cavity. The present invention also concerns the treatment of radiation sickness and pain syndrome, as well as other diseases through the use of compounds that are effective in inhibiting the enzyme glutaminyl cyclase, involved, in particular, in the processes of post-translational modification of chemokines and chemotaxis of cells of the immune system.

State of the art

Chemotaxis or the directed movement of immune system cells along the concentration gradient of certain endogenous and exogenous substances (chemoattractants) is one of the most important components of the functioning of immune system cells. An excess influx of immune system cells typically causes overactivity of immune system cells and damage to surrounding organs and tissues. Understanding the participants and processes associated with the chemotaxis process at the molecular level can lead to new effective approaches to the treatment and prevention of a number of diseases associated with aberrant activity of cells of the immune system.

Chemokines of the CCL family (CCL2, CCL7, CCL8, CCL13), which are ligands of the CCR2 receptor, are the most potent factors in the chemotaxis of monocytes and macrophages in mammals (Biochem J. 2012 Mar 1; 442(2):403-12). Chemokines of the CCL family constitute an important class of cytokines necessary for the activation of neutrophils and monocytes and the recruitment of these cells to the site of inflammation. However, high concentrations of chemokines tend to cause an excess influx of immune cells. Aberrant activity of immune system cells can lead to serious damage to surrounding organs and tissues. For example, in some cases, lipid oxidation products can activate vascular endothelial cells (intima), which leads to the release of CCL2, attracting macrophages which, in turn, release inflammatory markers that provoke damage to the arterial wall and the development of atherosclerosis (Mol Cell. 1998 Aug; 2( 2):275-81; Nature. 1998 Aug 27;394 (6696):894-7). A similar pathogenetic picture can be observed in the case of radiation-induced tissue damage of the respiratory tract: damage and activation of epithelial cells of the lungs and respiratory tract leads to the release of CCL2, which in turn causes extravasation of immune cells and circulating tumor cells into the lungs and respiratory tract (Antioxid Redox Signal. 2018 Apr 2. doi: 10.1089/ars.2017.7458).

The role of chemokines of the CCL family in the pathophysiology of a number of autoimmune and allergic conditions mediated by the aberrant activity of CCR2+, CD14+ and CD16lo monocytes (rheumatoid arthritis, multiple sclerosis) is known. In addition, CCL family chemokines (particularly CCL2) have been implicated in the pathogenesis of obesity, metabolic syndrome, chronic pain, fibrosis, nonalcoholic fatty liver disease, and some forms of cancer.

Suppression of aberrant activity of cells of the immune system, due to inhibition of CCL-mediated chemotaxis, may be of great demand for the treatment of a whole range of diseases, such as acute lung injury (ALI), bronchial asthma, bronchitis, chronic obstructive pulmonary disease, Crohn's disease, diabetic nephropathy, and etc.

For example, in diabetic nephropathy, exposure to high concentrations of glucose leads to increased secretion of CCL2 by renal tubular cells, which in turn causes monocyte migration (Kidney Int. 2006 Jan; 69(1):73-80). An increase in the concentration of monocytes in the kidney tissues and their maturation into macrophages causes the development of an aberrant response associated with the release of more chemokines and reactive oxygen species, damaging the surrounding kidney cells (Mediators Inflamm. 2012; 2012:146154). Damage to kidney tissue leads to the development and maintenance of aberrant activity of cells of the immune system and further destruction of kidney tissue, and blockade of the CCL2/CCR2 interaction with low molecular weight antagonists reduces the severity of pathology (Nephrol Dial Transplant. 2013 Jul; 28(7):1700-10). Similar processes are characteristic of the pathogenesis of non-alcoholic fatty liver disease: the accumulation of fatty acids in liver cells leads to the activation of the NF-κB signaling pathway and the development of an inflammatory response, inducing the release of pro-inflammatory cytokines, such as IL-6 and CCL2. The release of pro-inflammatory cytokines leads to the migration of monocytes to the site of inflammation, the development of an aberrant response associated with the release of more chemokines and reactive oxygen species, damaging surrounding cells (Int J Exp Pathol. 2013 Jun; 94(3):217-225).

Members of the CCL family (CCL2, CCL7, CCL8, CCL13), fractalkine, as well as a number of other hormones and secreted proteins, contain a pyroglutamic acid (pE) residue, the role of which is to protect against degradation by aminopeptidases (Chem Immunol. 1999; 72:42- 56; Biochemistry. 1999 Oct 5; 38(40):13013-25). Pyroglutamination of the N-terminal residue is catalyzed by the enzyme glutaminyl cyclase (QPCT or QC) (J Biol Chem. 2003 Dec 12;278(50):49773-9; J Mol Biol. 2008 Jun 20;379(5):96680). Glutaminyl cyclase has broad substrate specificity and is involved in the post-translational modification of a number of peptide molecules. Studies of the substrate specificity of glutaminyl cyclase have shown that the enzyme can catalyze the pyroglutamination of various substrates, regardless of the length of the polypeptide chain (FEBS Lett. 2004 Apr 9; 563 (1-3):191-6, J

Biol Chem. 2011 Apr 8; 286(14):12439-49).

Experimental studies have shown that inhibition of glutaminyl cyclases leads to a sharp decrease in the chemoattractor activity of non-pyroglutaminated forms of the chemokines CCL2, CCL7, CCL8 and CCL13 (Biochem. J. (2012) 442, 403-412) and fractalkine (Biosci Rep. 2017 Aug 23; 37(4)). Thus, glutaminyl cyclase inhibitors can obviously be used for the treatment of a wide range of diseases, and in particular, diseases of the lungs and respiratory tract, such as bronchial asthma, acute and chronic bronchitis, pharyngitis, emphysema, rhinitis, rhinosinusitis and chronic obstructive pulmonary disease. The pathogenesis of these diseases is associated with excessive production of cytokines and in particular monocyte chemoattractant proteins CCL2 and CCL7 (Am J Respir Cell Mol Biol. 2014 Jan; 50 (1): 144-57) and fractalkine (Expert Opin Ther Targets. 2010 Feb; 14( 2):207-19), which are substrates of glutaminyl cyclase (Biosci Rep. 2017 Aug 23; 37(4). pii: BSR20170712; EMBO Mol Med. 2011 Sep; 3(9):545-58). Neutralization of CCL2 and CCL7 using antibodies has been shown to significantly reduce the influx of leukocytes, monocytes and neutrophils into the airways of experimental animals (Am J Respir Cell Mol Biol. 2014 Jan; 50(1):144-57).

Exposure to bacterial lipopolysaccharides, lipoteichoic acid or other irritants on the mucous membrane of the respiratory system leads to an increase in the secretion of CCL2 by bronchial smooth muscle cells (Am J Physiol Lung Cell Mol Physiol. 2012 Apr 15; 302(8):L785-92) and an increase in the concentration of CCL2 in bronchoalveolar lavage (Mol Immunol. 2011 Jul; 48(12-13):1468-76). An increase in the concentration of CCL2 in turn causes the migration of eosinophils, monocytes and basophils and the development of an aberrant response associated with the release of more chemokines (TNFa, IL-1, IL-6, IL-4) and reactive oxygen species, damaging surrounding cells of the bronchi and organs respiration (Immunobiology. 2016 Feb; 221(2):182-7; Int J Biol Sci. 2012; 8(9):1281-90; Mol Immunol. 2013 Nov; 56(1-2):57-63). Damage to the bronchi leads to the development and persistence of aberrant activity of cells of the immune system, and further destruction of tissues of the respiratory organs. In in vivo models of allergic asthma, blockade of the CCL2/CCR2 interaction with small molecule antagonists has shown significant efficacy (Int Arch Allergy Immunol. 2015; 166(1):52-62).

It is important to note that the CCL2-mediated development of neutrophilic inflammation, the development of an aberrant response associated with the release of pyrogenic cytokines (TNFa, IL-1, IL-6) leads to an increase in temperature and the development of fever (J Infect Dis. 1999 Mar; 179 Suppl 2:S294 -304;Front Biosci.2004 May 1;9:1433-49).

In addition to fever and elevated temperature, pain is also an extremely common symptom of various diseases. Obviously, a decrease in the severity of the aberrant response associated with the release of more reactive oxygen species that damage surrounding tissues should in itself lead to a decrease in the severity of the pain syndrome. However, recent work has shown a key role for fractalkine in the pathogenesis of chronic pain (J Neurochem. 2017 May; 141(4):520-531).

Glutaminyl cyclase inhibitors can be used to treat various autoimmune diseases, in particular rheumatoid arthritis and psoriasis.

Fractalkine is one of the key pro-inflammatory mediators involved in the development of autoimmune diseases. The interaction between fractalkine and its unique receptor (CX3CR1) induces cell adhesion, chemotaxis and cell survival (Mol Interv. 2010 Oct; 10(5):263-70). Fractalkine levels are elevated in patients with rheumatoid arthritis (PA) (Mod Rheumatol. 2017 Mau; 27(3):392-397) and psoriasis (Ann Clin Lab Sci. 2015 Fall; 45(5):556-61) correlates with activity diseases. Fractalkine is expressed on fibroblast-like synoviocytes and endothelial cells in the synovial tissue of patients with rheumatoid arthritis. In psoriasis, high levels of fractalkine production are observed in the dermal papillae and antigen presenting cells (Br J Dermatol. 2001 Jun; 144(6):1105-13). Fractalkine expression is upregulated by tumor necrosis factor-α and interferon-γ and in rheumatoid arthritis, promotes the migration of monocytes, T cells and osteoclast precursors into synovial tissue (Mod Rheumatol. 2017 May; 27(3):392-397). Increased expression of fractalkine at dermal papillae provides a plausible explanation for the migration and accumulation of T cells at these sites in psoriasis (Br J Dermatol. 2001 Jun; 144(6):1105-13). Fractalkine also induces the production of inflammatory mediators by macrophages, T cells and fibroblast-like synoviocytes. Moreover, fractalkine promotes angiogenesis and osteoclastogenesis. In a model of collagen-induced arthritis in mice, the use of antibodies to fractalkine significantly alleviated the course of the pathology (Mod Rheumatol. 2017 May; 27(3):392-397).

Based on the results of recent scientific studies, it can be argued that inhibition of CCL-mediated chemotaxis is a new promising therapeutic approach for the treatment of radiation sickness (Antioxid Redox Signal. 2018 Apr 2. doi: 10.1089/ars.2017.7458, Int J Radiat Biol. 2015 Jun; 91 (6):510-8). Animal models have shown that radiation-induced vascular dysfunction can be effectively reduced by inhibiting CCL-mediated signaling pathways. The use of animals knockout for the CCL2 receptor gene, as well as the use of antagonists of this receptor, blocks radiation-induced changes in morphology and destruction of endothelial cells, and prevents the development of airway inflammation immediately after radiation exposure (Antioxid Redox Signal. 2018 Apr 2. doi: 10.1089/ ars.2017.7458). Moreover, suppression of CCL-mediated signaling pathways can significantly reduce the severity of radiation-induced pulmonary fibrosis, which is one of the main “delayed” side effects of radiation exposure.

Thus, based on the literature data, it can be concluded that a strategy aimed at inhibiting glutaminyl cyclase is a possible approach to the treatment of autoimmune diseases, obesity, lung, respiratory tract and abdominal diseases, radiation sickness and pain syndrome.

To date, glutaminyl cyclase inhibitors are known, including sulfolipids (WO 2017/046256), flavonoid derivatives (Bioorg Med Chem. 2016 May 15; 24(10):2280-6), pyridine derivatives (US 2015/0291632) and some small molecules, described in recent works (J Med Chem. 2017 Mar 23; 60(6):2573-2590; WO 2014/193974, US 2015/0291557). The closest analogues of the compound of the present invention are published by Probiodrug Aktiengesellschaft (J Biol Chem. 2003 Dec 12; 278(50):49773-9). This work describes glutaminyl cyclase inhibitors based on imidazole derivatives. However, in the structures of compounds published by Probiodrug Aktiengesellschaft, imidazole contains an aliphatic substituent on one of the nitrogen atoms of the imidazole ring. The introduction of an aliphatic substituent reduces the metabolic stability of the compounds. In addition, the introduction of an aliphatic substituent increases the hydrophobicity of the compounds and facilitates the penetration of the compound through the blood-brain barrier, which is clearly unnecessary for suppressing the aberrant activity of cells of the immune system and can potentially lead to side effects.

To date, there is not a single drug that acts as a glutaminyl cyclase inhibitor that would be used in the treatment of diseases associated with aberrant activity of cells of the immune system, so there remains a need for the creation and implementation of new effective drugs based on glutaminyl cyclase inhibitors for use in therapy.

This invention concerns the use of chemical compounds that are effective in inhibiting glutaminyl cyclase in the treatment of diseases of the respiratory tract and abdominal cavity, radiation sickness and various types of pain syndrome, as well as other diseases associated with aberrant activity of cells of the immune system.

Brief description of the figures

Figure - Influx of inflammatory cells into the bronchoalveolar space when studying the specific pharmacological activity of compound 1 in a model of acute asthma in guinea pigs (M±m, n=10).

Disclosure of the Invention

The objective of the present invention is to develop new drugs that are inhibitors of glutaminyl cyclase and are effective for the treatment of diseases associated with aberrant activity of cells of the immune system, especially diseases of the lungs, respiratory tract and abdominal cavity, radiation sickness and pain syndrome, as well as other diseases associated with aberrant activity of cells of the immune system.

The technical result of this invention is the development and production of effective glutaminyl cyclase inhibitors, characterized by high inhibitory activity, allowing the use of these inhibitors for the treatment of diseases associated with aberrant activity of cells of the immune system, in particular diseases of the lungs and respiratory tract, such as bronchial asthma, acute and chronic bronchitis , pharyngitis, rhinitis (in particular allergic rhinitis), rhinosinusitis, chronic obstructive pulmonary disease and its manifestations (in particular pulmonary emphysema, bronchial obstruction); diseases of the abdominal organs, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathies (in particular diabetic nephropathy); radiation sickness and pain syndrome, as well as other diseases associated with aberrant activity of cells of the immune system, in particular with aberrant chemotaxis of cells of the immune system.

The specified technical result is achieved by using compounds of formula (A)

- 3 044574 in which

R1 represents a group -C(O)-R2-C(O)- or -R2-C(O)-, where

R2 represents a -(CH2) _P- group, optionally substituted with one or two C ₁ -C ₆ alkyls, or phenyl, n is an integer from 0 to 4;

wherein said compounds of formula (A) are selected from the group consisting of any of compounds I-X, and combinations thereof

or pharmaceutically acceptable salts, hydrates, solvates thereof, as glutaminyl cyclase inhibitors.

The specified technical result is also achieved by using compounds of formula (A) selected from any of compounds I-X or their pharmaceutically acceptable salts, hydrates, solvates to obtain a pharmaceutical composition for the prevention and/or treatment of a disorder associated with glutaminyl cyclase activity.

The specified technical result is also achieved by using any of the compounds I-X or their salts, hydrates, solvates to obtain a pharmaceutical composition for the prevention and/or treatment of a disorder associated with aberrant activity of cells of the immune system, in particular with aberrant chemotaxis of cells of the immune system.

In addition, the invention provides pharmaceutical compositions for the prevention and/or treatment of a disorder associated with glutaminyl cyclase activity and/or aberrant activity of cells of the immune system, in particular aberrant chemotaxis of cells of the immune system, and characterized in that they contain an effective amount of a compound of the invention and at least one pharmaceutically acceptable excipient. In some embodiments, the excipient is a pharmaceutically acceptable carrier and/or excipient.

The invention also includes a method for preventing and/or treating a disorder associated with glutaminyl cyclase activity in the body, comprising administering to said body a pharmaceutical composition of the invention. This glutaminyl cyclase activity disorder is a disease associated with aberrant activity of cells of the immune system, in particular aberrant chemotaxis of cells of the immune system, especially a disease of the lungs and respiratory tract. In some non-limiting embodiments, the lung and airway disease is bronchial asthma, acute and chronic bronchitis, pharyngitis, emphysema, rhinitis, rhinosinusitis, or chronic obstructive pulmonary disease. In particular cases of embodiment of the invention, the organism is a human or animal.

Detailed Disclosure of the Invention

The preparation of compounds I-X, as well as a number of other chemical compounds, is described in the application for invention RU 2013/116822. The said patent application describes derivatives of dicarboxylic acid bisamides with the ability to complex or chelate metal ions, as well as their use as a means for the prevention and/or treatment of viral hepatitis, HIV infection, cancer, neurodegenerative, cardiovascular, inflammatory diseases, diabetes, gerontological diseases, diseases caused by microorganism toxins, as well as alcoholism, alcoholic cirrhosis of the liver, anemia, porphyria tarda, poisoning with transition metal salts.

In the course of studying the specific pharmacological activity of the compound of formula (A), the authors of the present invention unexpectedly discovered that the compounds of formula (A) affect the chemotaxis of cells of the immune system. Reducing the influx of immune system cells can be used in the treatment of a number of diseases associated with aberrant activity of immune system cells, in particular diseases of the lungs and respiratory tract, such as bronchial asthma, acute and chronic bronchitis, pharyngitis, rhinitis (in particular allergic rhinitis), rhinosinusitis , chronic obstructive pulmonary disease and its manifestations (in particular emphysema, bronchial obstruction); as well as diseases of the abdominal organs, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy (in particular diabetic nephropathy); and radiation sickness.

Since the effect on chemotaxis cannot be predicted or explained by the compound's ability to complex or chelate metal ions, an attempt was made to search for possible therapeutic targets. In the course of the research of the present inventor, it was discovered that the observed therapeutic effect of the compounds of formula (A) is associated with the ability of these compounds to inhibit the activity of glutaminyl cyclase. In further studies, the ability to inhibit the activity of glutaminyl cyclase was also shown for compounds III, IV, V, VI, VII, VIII, IX and X.

Thus, compounds I-X are new glutaminyl cyclase inhibitors that affect the chemotaxis of cells of the immune system and can be used for the treatment of diseases associated with aberrant activity of cells of the immune system, in particular diseases of the lungs and respiratory tract, such as bronchial asthma, acute and chronic bronchitis, pharyngitis, rhinitis (in particular allergic rhinitis), rhinosinusitis, chronic obstructive pulmonary disease and its manifestations (in particular pulmonary emphysema, bronchial obstruction); as well as diseases of the abdominal organs, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy (in particular diabetic nephropathy); as well as radiation sickness and pain syndrome.

Terms and Definitions

The term Compound I refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]oxalamide corresponding to the following structural formula:

The term Compound II refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]isophthalamide corresponding to the following structural formula:

The term Compound III refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]urea, also represented by the structural formula:

The term Compound IV refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]malonamide, also represented by the structural formula:

- 5 044574

The term Compound V refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]succinamide, also represented by the structural formula:

The term Compound VI refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]glutaramide corresponding to the following structural formula:

The term Compound VII refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl] adipamide corresponding to the following structural formula:

The term Compound VIII refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]-3methylpentanediamide corresponding to the structural formula:

The term Compound IX refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]-3,3dimethylpentanediamide, corresponding to the structural formula:

The term Compound X refers to K,K'-bis[2-(N-imidazol-4-yl)ethyl]terephthalamide corresponding to the following structural formula:

The term C, when used in reference to temperature, means the centigrade scale or the Celsius temperature scale.

The term IC ₅₀ means the concentration of the test compound at which half-maximal enzyme inhibition is achieved.

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The term pharmaceutically acceptable salts or salts includes salts of the active compounds that are prepared with relatively non-toxic acids. Examples of pharmaceutically acceptable non-toxic salts include those formed by inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or organic acids such as acetic, oxalic, maleic, tartaric, succinic, citric or malonic acids, or prepared by others. methods used in the art, for example, using ion exchange. Other pharmaceutically acceptable salts include adipinate, alginate, ascorbate, aspartate, benzophulus, benzoate, bisulfate, borage, butyrate, campforath, campf between citrate, citrate, cyclopentantanpropionate, excitement, dodecil sulfate, ethan sink, format, fumarat, glucoghephepo, glucoghephepo, glucoghepois, glucoghephepois, glucoghepois, glucoghephepois, glucoghephepois, glucoghephepois, glucoghephepois, glucoghephepois, glucoghepheth, glucoghepheth, glucoghephepas. on, glycerophosphate, gluconate, hemisufate, heptanate, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate (mesylate), 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, hemifumarate, stearate, succinate, sulfate, tartrate, thiocyanate, ptoluenesulfonate (tosylate), undecanate, valerate and the like.

The term solvate is used to describe a molecular complex containing a compound of the invention and one or more molecules of a pharmaceutically acceptable solvent, such as ethanol. The term hydrate is used when the specified solvent is water.

The term aberrant immune system cell activity as used herein means activity that deviates significantly from the baseline level of immune system cell activity in the body in the absence of pathology. Aberrant activity can be caused by excessive influx of immune system cells into an organ or tissue, disruption of processes leading to activation of immune system cells, deregulation of processes associated with the death of immune system cells, as well as other factors.

The term excipient means any pharmaceutically acceptable substance of inorganic or organic origin that is part of a medicinal product or used in the manufacturing process of a medicinal product to impart the necessary physicochemical properties to it.

The term RPMI medium (Roswell Park Memorial Institute medium) refers to cell and tissue culture medium. RPMI has traditionally been used to culture human lymphoid cells. The medium contains significant amounts of phosphate and is formulated for growth in an atmosphere containing 5% carbon dioxide.

The term glutaminyl cyclase refers to an aminoacyltransferase enzyme involved in the conversion of N-terminal glutamine to pyroglutamine in various peptide substrates. The formation of N-terminal pyroglutamate protects biologically active peptides, hormones and chemokines (eg, thyrotropin-releasing hormone, β-chemokine ligand-2) from degradation by exopeptidases and in some cases can increase the affinity of ligands for their receptors.

The term chemotaxis refers to the directed movement of cells in response to a chemical stimulus. Chemotaxis is based on the ability of a cell to respond to a concentration gradient of a chemotactic mediator. Chemotaxis is the process by which cells of the immune system leave the vascular bed and migrate into damaged tissue. Chemotactic substances (chemoattractants) play a leading role in chemotaxis. One of the most powerful chemoattractants for monocytes and macrophages is the chemokine CCL2.

The terms treatment, therapy cover the treatment of pathological conditions in mammals, preferably in humans, and include: a) reduction, b) blocking (suspension) of the course of the disease, c) alleviation of the severity of the disease, i.e. induction of disease regression, d) reversal of the disease or condition to which the term applies, or one or more symptoms of the disease or condition.

The term prophylaxis includes the elimination of risk factors, as well as prophylactic treatment of subclinical stages of the disease in mammals, preferably in humans, aimed at reducing the likelihood of the occurrence of clinical stages of the disease. Patients for prophylactic therapy are selected based on factors known to increase the risk of clinical stage disease compared with the general population. Preventive therapy includes a) primary prevention and b) secondary prevention. Primary prevention is defined as preventive treatment in patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of reoccurrence of the same or similar clinical condition of the disease.

Compounds I-X are promising for the treatment of diseases associated with aberrant activity of cells of the immune system, in particular diseases associated with aberrant chemotaxis of cells of the immune system, in particular for the treatment of diseases of the lungs and respiratory tract, such as bronchial asthma, acute and chronic bronchitis, pharyngitis , rhinitis (in particular allergic rhinitis), rhinosinusitis, chronic obstructive pulmonary disease and its manifestations (in particular pulmonary emphysema, bronchial obstruction); diseases of the abdominal organs, such as Crohn's disease, ulcers

- 7 044574 colitis, peritonitis and nephropathy (in particular diabetic nephropathy); radiation sickness and pain syndrome of both systemic and local nature, including those caused by primary pathological changes, or associated with various diseases or long-term use of certain medications. In some particular embodiments, the compounds of the invention can be used to treat other diseases associated with aberrant activity of cells of the immune system.

Method for therapeutic use of compounds

The subject matter of the present invention also includes administering to a subject in need of appropriate treatment a therapeutically effective amount of one or more compounds of the invention. By a therapeutically effective amount is meant that amount of one or more compounds administered or delivered to a patient at which the patient is most likely to exhibit the desired response to treatment (prophylaxis). The exact amount required may vary from subject to subject depending on the age, body weight and general condition of the patient, the severity of the disease, the method of administration of the drug, combination treatment with other drugs, and the like.

The compounds of the invention or a pharmaceutical composition containing one or more compounds can be administered to the patient in any amount (preferably, the daily dose of the active substance is up to 0.5 g per patient per day, most preferably the daily dose is 5-50 mg /day) and by any route of administration (preferably the oral route) effective for treating or preventing the disease.

After mixing the drug with a particular suitable pharmaceutically acceptable carrier in the desired dosage, the compositions forming the essence of the invention can be administered to humans or other animals orally, parenterally, topically, or the like.

The administration can be carried out either once or several times a day, a week (or any other time interval), or from time to time. In addition, one or more compounds may be administered to the patient daily for a specified period of days (eg, 2-10 days), followed by a period without taking the substance (eg, 1-30 days).

When the compounds of the invention are used as part of a combination therapy regimen, a dose of each of the components of the combination therapy is administered for the required treatment period. The compounds that make up the combination therapy can be administered to the patient either at once, in the form of a dosage containing all the components, or in the form of individual dosages of the components.

Pharmaceutical compositions

The invention also relates to pharmaceutical compositions which contain formulas (A), in particular, compounds I-X (or a prodrug form or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, solvents and/or excipients, such that may be administered to a patient in conjunction with a compound of this invention and which do not interfere with the pharmacological activity of the compound and are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions claimed in this invention contain one or more compounds of formula (A) together with pharmaceutically acceptable carriers, which may include any solvents, diluents, dispersions or suspensions, surfactants, isotonic agents, thickeners and emulsifiers, preservatives , binders, glidants, etc., suitable for the particular dosage form. Materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, mono- and oligosaccharides, as well as derivatives thereof; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut, cottonseed, safrole, sesame, olive, corn and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solution, Ringer's solution; ethyl alcohol and phosphate buffer solutions. The composition may also contain other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, film formers, sweeteners, flavoring agents, preservatives and antioxidants.

The subject of this invention is also dosage forms - a class of pharmaceutical compositions, the composition of which is optimized for a specific route of administration into the body in a therapeutically effective dose, for example, for administration into the body orally, topically, inhalation, for example, in the form of an inhalation spray, or intravascularly, intranasally , subcutaneously, intramuscularly, and also by infusion, in recommended dosages.

The dosage forms of this invention may contain formulations prepared by liposome methods, microencapsulation methods, nanoformulation methods, or other methods known in the pharmaceutical art.

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When preparing the composition, for example in tablet form, the active principle is mixed with one or more pharmaceutical excipients such as gelatin, starch, lactose, magnesium stearate, talc, silica, acacia, mannitol, microcrystalline cellulose, hypromellose or similar compounds.

The tablets can be coated with sucrose, cellulose derivative or other substances suitable for coating. Tablets can be produced by various methods such as direct compression, dry or wet granulation or hot melt fusion.

A pharmaceutical composition in the form of a gelatin capsule can be prepared by mixing the active principle with other substances and filling the resulting mixture into soft or hard capsules.

For parenteral administration, aqueous suspensions, isotonic saline solutions, or sterile injection solutions are used that contain pharmacologically compatible agents, such as propylene glycol or butylene glycol.

Examples of Pharmaceutical Compositions

The compounds of formula (A) described in this invention can be used for the prevention and/or treatment of human or animal diseases in the form of the following compositions (by Substance is meant a compound of formula (A)):

Tablet I mg/tablet

Substance 0.5

Microcrystalline cellulose 66.5

Sodium carboxymethyl starch 2.3

Magnesium stearate 0.7

Tablet II mg/tablet

Substance 5.0

Microcrystalline cellulose 62.0

Sodium carboxymethyl starch 2.3

Magnesium stearate 0.7

Tablet III mg/tablet

Substance 50

Microcrystalline cellulose 620

Sodium carboxymethyl starch 23

Magnesium stearate 7

IV mg tablet/tablet

Substance 50

Lactose Ph. EUR 223.75

Croscarmellose sodium 6.0

Corn starch 15

Polyvinylpyrollidone (5% mobile paste) 2.25

Magnesium stearate 3.0

V mg tablet/tablet

Substance 200

Lactose Ph. EUR 182.75

Croscarmellose sodium 12.0

Corn starch (5% vol. paste) 2.25

Magnesium stearate 3.0

Capsule mg/capsule

Substance 10

Lactose Ph. EUR 488.5

Magnesia 1.5

Composition for injection I (50 mg/ml)

Substance 5.0% w/v

1M sodium hydroxide solution 15.0% w/v

1M hydrochloric acid solution to pH 7.6

Polyethylene glycol 400 4.5% w/v

Water for injection up to 100%

Ointment ml

Substance 40 mg

Ethanol 300 μl

Water 300 μl

1-dodecyl azacycloheptanone 50 μl

Propylene glycol up to 1 ml

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These compositions can be prepared in accordance with standard pharmaceutical techniques. Tablets (I)-(II) can be enteric-coated using, for example, cellulose acetate phthalate.

Use of compounds I-X in combination therapy

Although compounds I-X may be administered as a single active pharmaceutical agent, they may also be used in combination with one or more other agents, in particular the other agent may be an antibiotic, an NSAID or other anti-inflammatory agent, an antihypertensive agent, glucocorticosteroid, monoclonal antibody, etc. When administered together, the therapeutic agents may be different dosage forms that are administered simultaneously or sequentially at different times, or the therapeutic agents may be combined into a single dosage form.

The phrase combination therapy, in relation to the compounds of this invention in combination with other pharmaceutical agents, means the simultaneous or sequential administration of all agents that will somehow provide the beneficial effects of the combination of drugs. Co-administration means, in particular, co-delivery, for example, in a single tablet, capsule, injection or other form having a fixed ratio of active substances, as well as simultaneous delivery in several, separate dosage forms for each compound, respectively.

Thus, the administration of compounds I-X can be carried out in combination with additional treatment methods known to specialists in the field of prevention and treatment of relevant diseases, including the use of antibacterial, cytostatic and cytotoxic drugs, drugs to suppress symptoms or side effects of one of the drugs.

If the drug is a fixed dose, such a combination uses the compounds of this invention in an acceptable dosage range. Compounds I-X of this invention can also be administered to a patient sequentially with other agents in cases where a combination of these drugs is not possible. The invention is not limited to the sequence of administration; the compounds of this invention can be administered to a patient together, before or after the administration of another drug.

Examples

Preparation of compounds according to the invention

Methods for obtaining compounds I-X are disclosed in the application for invention RU 2013/116822. The same application describes the ability of such compounds to complex or chelate metal ions.

Characteristics of the biological activity of the compounds according to the invention

The biological activity of compounds I-X has been studied in various in vitro and in vivo experiments. In particular, when studying the activity of compounds I and II in various in vitro and in vivo models, the inhibitory effect of compounds I and II on the chemotaxis of monocytes, macrophages and other cells of the immune system was shown. This biological effect of compounds I and II cannot be predicted or explained on the basis of previous knowledge of the ability of compounds I and II to chelate metal ions.

Studies of the biological activity of compounds III-X in vitro have established that compounds III-X are also inhibitors of the enzyme glutaminyl cyclase and, thus, the effect of compounds III-X on the chemotaxis of cells of the immune system may be mediated by inhibition of glutaminyl cyclase activity.

Study of the effect of compounds I-X on the enzymatic activity of human glutaminyl cyclase in vitro.

In the course of studies of the effect of compounds I-X, which are the subject of the present invention, on the enzymatic activity of glutaminyl cyclase in vitro, a direct inhibitory effect of compounds I-X on recombinant intracellular human glutaminyl cyclase was first discovered.

Glutaminyl cyclase activity at various concentrations of compounds I-X was studied at 25°C using the fluorescent substrate L-glutaminyl 2-naphthylamide (Gln-bNA) (Anal Biochem. 2002 Apr 1; 303(1):49-56). The 100-μL reaction mixture contained 50 μΜ fluorogenic substrate; ~0.2 units of human pyroglutaminyl aminopeptidase (1 unit is defined as the amount that hydrolyzes 1 micromol pGlu-bNA per minute), and an aliquot of recombinant intracellular human glutaminyl cyclase (gQC) in 50 mM trisaminomethane-HCl and 5% glycerol, pH 8.0. The reaction was initiated by adding an aliquot of glutaminyl cyclase to the reaction mixture, incubated with compounds I-X for 5 min.

- 10 044574

Table 1

Effect of compounds I-X on the enzymatic activity of human glutaminyl cyclase in vitro

No. Compound IC50, μκΜ Κί, μκΜ I //^1 ^{0 9} iT^^f ) ⁰ 1.4 0.8 II —*NH ο ο < i A A O ]pj 2.9 1.61 III Η Η Η Η 3.5 2 IV η Η Η Η 6.3 3.50 V ν^, ο Ο 1 Η Η ^{n n} if I ) q U__'/ ^U --Ν 20 eleven VI Ο Ο ν'\II II/\ A/ Η Η 5.4 3 VII , ^Ν --. 0 ΟΙ Η Η ...AaX/V/'xZx/x ^{Η Η} I L> 4.4 2 VIII »Π ^Η ϊ 1 ϊ Π. Η Η 7.3 4 IX fi -ΝΗ Ο . . Ο ΗΝ~“Ά Η Η 5.7 3.17 X Ν·^\ ιΗ and ^N v_Z^V_y° ₀ \=/ ΗΝ^ Ο J Η 0.8 0.44

The further progress of the reaction was monitored spectrophotometrically (excitation and emission wavelengths were 320 and 410 nm). Enzyme activity was determined by the amount of 2-naphthylamide (bNA) released, calculated from the calibration curve. IC ₅₀ values were calculated using nonlinear regression of the inhibitor concentration-enzyme activity curve. The well-known glutaminyl cyclase inhibitor compound PBD150 (J Med Chem. 2006 Jan 26; 49(2):664-77) was used as a reference substance.

- 11 044574

As a result of the experiment, it was found that compounds I-X inhibit the activity of glutaminyl cyclase with IC50 in the range from 0.8 to 20 μM (see Table 1)

Study of the effect of compounds I and II on monocyte migration in vitro

The effect of compounds I and II on monocyte migration in vitro was studied using U937 cells grown to a concentration of (2-3) x 10 ⁶ cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Biochem J. 2012 Mar 1 ;442(2):403-12). Approximately 1 x 10 ⁷ U937 cells were incubated with various concentrations of compounds I and II (a total of 7 concentrations for each compound) at 37°C for 2 hours and then treated with E. coli lipopolysaccharide (O111:B4). The supernatant (conditioned medium) was used to study monocyte migration.

A fresh portion of U937 cells was stained with the fluorescent dye Calcein AM for 1 hour at 37°C. After this, an aliquot of the stained cells was placed in the upper compartment of the wells of a BD FalconTM HTS FluoroBlok plate, the cells of which are separated by semi-permeable optically opaque membranes. The conditioned medium obtained after incubating the cells with the inhibitor and lipopolysaccharide was placed in the lower compartment of the wells of the plate. The plates were incubated for 2 hours at 37°C; the number (% relative to the experiment without inhibitor) of cells migrating to the lower compartment of the wells was determined fluorometrically. The well-known glutaminyl cyclase inhibitor compound PBD150 (J Med Chem. 2006 Jan 26;49 (2):664-77) was used as a reference substance.

As a result of the experiment, it was found that compound I and compound II in the micromolar concentration range have an inhibitory effect on monocyte migration in vitro. Compound I inhibits monocyte migration over a wide concentration range from 1 μM to 300 μM with an efficiency close to 60%. In the same concentration range, compound II inhibits monocyte migration with an efficiency of 50-70%.

Study of the effect of compound I on leukocyte chemotaxis in vivo using a model of bronchial asthma in guinea pigs

Induction of bronchial asthma in guinea pigs was carried out according to standard methods (Current Drug Targets. 2008 Jun; 9(6):452-65). Animals were immunized with a single intraperitoneal injection of 0.5 ml of a solution containing 100 μg/ml ovalbumin (Sigma) and 100 mg/ml aluminum hydroxide. Intact animals were injected intraperitoneally with saline. solution in a volume of 0.5 ml.

On days 29, 30 and 31 of the experiment, respiratory tract hyperresponsiveness was provoked by inhalation administration of ovalbumin in increasing concentrations - 0.1, 0.3 and 0.5 mg/ml (on days 29, 30 and 31, respectively). Inhalation was carried out for 5 minutes or until pronounced signs of asphyxia appeared (falling on the side). On the 32nd day, the animals were administered a permissive dose of ovalbumin - 1 mg/ml for 5 minutes with assessment of the bronchospastic reaction.

The test compound was administered to the animals intragastrically once a day for 10 days, ending 24 hours before the administration of the resolving dose of the antigen.

24 hours after administration of the permissive dose of ovalbumin, bronchoalveolar lavage (BAL) was collected from the animals. BAL fluid was taken under anesthesia by washing the lungs with 5 ml of saline heated to 37°C through the trachea using a syringe dispenser.

In the bronchoalveolar lavage fluid, the absolute number of cellular elements in 1 μl of lavage was counted using a Goryaev camera. Then the bronchoalveolar lavage was centrifuged at 200 g for 10 minutes. Smears were prepared from the cell sediment, which were subsequently fixed in methanol and stained according to Romanovsky-Giemsa to calculate the endopulmonary cytogram.

Cytological analysis of bronchoalveolar lavage (BAL) revealed a multiple increase in cellular elements in the BAL fluid of sensitized guinea pigs (Figure 1). Thus, this model is characterized by an inflammatory reaction in the respiratory tract of experimental animals. Analysis of individual cell types showed that the most pronounced cell influx was eosinophils. The results obtained confirm the literature data and allow us to conclude that the simulated inflammation is allergic in nature.

Daily intragastric administration of Compound I for 10 days reduced the influx of inflammatory cells into the bronchoalveolar space. Compound I had a therapeutic effect over the entire tested dose range (0.14-14 mg/kg), reducing both the total number of leukocytes and the number of individual cell types: eosinophils, neutrophils, macrophages (figure).

At the same time, in the figure, the sign * indicates differences that are statistically significant compared to the intact group (p<0.05); and the sign - & - differences that are statistically significant compared to the control group (p<0.05).

The results obtained indicate that compound I has a pronounced therapeutic effect in bronchial asthma.

Study of the effect of compound I on the chemotaxis of macrophages, neutrophils and eosinophils in vivo using a model of Sephadex-induced pulmonary bronchitis in rats

A model of Sephadex-induced pulmonary bronchitis in rats was implemented using standard methods

- 12 044574 (Int Arch Allergy Immunol. 2011; 154(4):286-94). Male Wistar rats were administered a single inhalation injection of Sephadex G-200 (Pharmacia, Sweden) at a dose of 5 mg/kg. The test compounds were administered intragastrically to animals four times: 24 and 1 hour before, and 24 and 45 hours after Sephadex administration. The reference drug budesonide was administered according to the same regimen by inhalation at a dose of 0.5 mg/kg. 48 hours after inhalation of Sephadex, bronchoalveolar lavage was collected. In the lavage, the total number of leukocytes was assessed and the leukocyte formula was determined.

Analysis of bronchoalveolar lavage showed that a single inhalation administration of Sephadex G-200 to rats causes a pronounced influx of leukocytes into the lung. The number of all cell types was increased in the control group compared to the intact group (Table 2).

table 2

Number of cellular elements in bronchoalveolar lavage in a model of Sephadex-induced bronchitis in rats (M±m, n=10)

Group Number of cellular elements in 1 μl of BAL fluid Leukocytes Neutrophils Eosinophils Macrophages Lymphocytes Intact 2209+348 270+54 0+0 1975+346 0+0 Control 4617+582* 989+135* 248+65* 3209+397* 0+0 Compound I (0.18 mg/kg) 3833+525* 283+75 & 0+0 & 3127+351* 0+0 Compound I (1.8 mg/kg) 4133+454* 846+101* 0+0 & 3287+381* 0+0

Notes:

* - difference from the intact group according to Student’s t-test at p<0.05 & - difference from the control group according to Student’s t-test at p<0.05

Intragastric administration of compound I to rats reduced the content of neutrophils and eosinophils in BAL fluid to the level of intact animals. The results obtained indicate that compound I has a therapeutic effect against inflammation of the lower respiratory tract, in particular bronchitis.

Study of the activity of compound I on a model of non-infectious lung inflammation induced by cigarette smoke extract

Induction of non-infectious inflammation of the lungs of mice was carried out according to standard methods [Exp Lung Res. Feb 2013; 39(1):18-31]. Male Balb/c mice were intraperitoneally injected with cigarette smoke extract (ESD, 0.45 ml/20 mg) on days 0, 11, 15, 17, 19 and 22. ESD was prepared as follows: 5 cigarettes were burned, the smoke was filtered using a vacuum pump to remove particles and collected in a vessel containing phosphate-buffered saline. Compound I was administered intragastrically, daily, once a day from days 7 to 27. Euthanasia was performed on the 28th day. The right lobe of the lung was fixed in a 10% solution of neutral formalin, passed through alcohols of increasing concentrations to xylene and embedded in paraffin according to the standard method. Dewaxed 5-μm-thick sections were stained with hematoxylin-eosin and histologically analyzed.

Each lesion was assessed on a 5-point scale: 1 point - inflammatory infiltrate occupies 0-20% of the area of the studied histological specimen, 2 points - inflammatory infiltrate occupies 21-40% of the area of the studied histological specimen, 3 points - inflammatory infiltrate occupies 41-60 % of the area of the histological specimen under study, 4 points - the inflammatory infiltrate occupies 61-80% of the area of the histological specimen under study, 5 points - the inflammatory infiltrate occupies 81-100% of the area of the histological specimen under study. The alveolar destruction index (DI), the percentage of damaged alveoli relative to the total number of alveoli, was also calculated.

The results of the study showed that repeated intraperitoneal administration of cigarette smoke extract to mice induces the formation of perivasculitis, peribronchitis, alveolitis and interstitial pneumonia (Table 3).

Intragastric administration of compound I significantly reduced the development of perivasculitis and alveolitis (Table 3). The results obtained allow us to conclude that compound I has a therapeutic effect in perivasculitis and alveolitis.

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Table 3

Results of a histological study on a model of non-infectious pneumonia induced by cigarette smoke extract (M±w, n=12)

Group Dose of compound, mg/kg Perivasculitis, points Peribronchitis, points Alveolitis (DI, %) Interstitial pneumonia, points Intact - 0.71+0.20 0.51+0.19 11.50+1.20 0.99+0.20 Control - 1.50+0.24* 1.29+0.18 30.90+2.30* 1.81+0.27* Compound I 0.3 0.38+0.13 & 1.09+0.24 17.70+2.00*& 1.08+0.26

Note:

* - difference from the intact group according to Student's t-test at p<0.05.

& - difference from the control group by Student's t-test at p<0.05.

Study of the activity of compound I in vivo in a mouse model of pulmonary emphysema

Inflammation and emphysema of the lungs were induced by a single intratracheal injection of porcine pancreatic elastase. Elastase was administered intratracheally once at a dose of 0.6 U/mouse in 30 μl of NaCl 0.9%. For anesthesia during surgery, pentobarbital was used intraperitoneally at a dose of 30 mg/kg. The surgical field was disinfected with a 70% ethanol solution and freed from hair. The skin, subcutaneous tissue, and cervical fascia were dissected along the midline of the neck. Using the method of blunt tissue spreading, the muscles on the ventral side of the trachea were pulled apart. Along the air flow during inspiration, an injection of porcine pancreatic elastase was made using a Hamilton syringe. After suturing the wound, the surgical field was treated with an antiseptic. The introduction of elastase was taken as day 0 of the experiment.

Compound I was administered at a dose of 3 mg/kg daily intragastrically once a day on days 8-21 of the experiment. On the 21st day of the study, the animals were euthanized in a CO ₂ chamber and the lungs were isolated. To assess the dynamics and severity of the development of emphysema in lung tissue, histological preparations were prepared from the left lung. To do this, the lung was fixed in a 10% solution of neutral formaldehyde and then embedded in paraffin according to the standard method. From deparaffinized sections, preparations of the apex of the lung, middle lung field and lower lung field with a thickness of 5 μm were prepared and stained with hematoxylin and eosin according to the standard technique. Next, photographs were taken of the apex of the lung, middle lung field, and lower lung field. Using computer processing of graphic data, the localization and area of emphysematous-dilated lung tissue (% of normal tissue) was studied; vessels and bronchi were excluded from the calculations (Int J Biomed Imaging. 2012; 2012:734734; Front Physiol. 2015 May 12;6: 14).

Histological examination on the 21st day after elastase administration revealed a moderately pronounced congestion of the microcircular vessels and capillaries of the interalveolar septa in all areas of the lungs of mice. In addition, elastase led to thickening of the alveolar walls due to lymphomacrophage infiltration, as well as inflammatory infiltration of interstitial tissue. The lumen of individual alveoli is also filled with macrophages and lymphocytes. As a result of the action of the damaging agent on the elastic fibers of the bronchioles, alveolar ducts and alveoli in the lung parenchyma, stretching of the alveoli and ruptures of the alveolar septa were noted, and diffuse emphysema developed. Analysis of the preparations showed that on the 21st day of the experiment, a significant part of the tissue of the left lung was occupied by emphysematous-dilated alveoli with destruction of the interalveolar septa. Emphysema was localized in all lung fields. The most pronounced lesions were observed in the lower pulmonary field (Table 4).

Table 4

Area of emphysematously dilated lung tissue (% of normal) in mice under conditions of intratracheal administration of elastase on the 21st day of the experiment (M±w, n=5)__________

Groups Upper pulmonary field Middle pulmonary field Inferior pulmonary field Intact control 0 0 0 Pathological control 24.06 ± 6.12* 63.76±5.02* 82.90 ± 2.88 * Compound I (3 mg/kg) 11.76 ± 1.48 * 37.09 ± 5.86 * 35.28 ± 5.81 *

Notes:

- * - differences are significant compared to intact control (p <0.05);

- · - the differences are significant compared to the pathological control (p<0.05).

The administration of compound I reduced the intensity of inflammatory infiltration of the lung parenchyma and the severity of congestion of the microvasculature and capillaries of the interalveolar septa, reduced the relative area of emphysematous-dilated alveoli compared with

- 14044574 by the pathological control group (Table 4). The data obtained give grounds to conclude that compound II has a therapeutic effect in pulmonary emphysema.

Study of the therapeutic activity of compound I in a model of COPD in guinea pigs

The study was carried out on male guinea pigs. Chronic obstructive pulmonary disease was induced by a course of endotracheal administration of E. Coli cell wall lipopolysaccharide (LPS) and tobacco smoke extract (ETS) (Biol Pharm Bull. 2009 Sep; 32(9):1559-64). ETD was obtained from Hi-Lite brand cigarettes (Japan) (composition of 1 cigarette: tar 17 mg/cigar, nicotine 1.4 mg/cigar). Before obtaining the extract, the cigarette filter was removed, the length of the cigarette with the filter was 80 mm, while the filter was removed 55 mm. Extraction was performed by drawing smoke from a lit cigarette through PBS at a constant speed using a vacuum pump (40 ml/40 cigarettes). The burning time of one cigarette was 180 s. To remove particles, the resulting extract was filtered through a bacterial filter with a pore size of 45 nm. ESD was inhaled to guinea pigs (0.3 ml/min, 40 min) once a day on days 1-4, 6-9, 11-14, 16-19. LPS was inhaled to guinea pigs (25 μg/ml, 0.3 ml/min, 1 hour) once a day on the 5th, 10th and 15th days. Before the last ESD inhalation, immediately after the last ESD inhalation, and 1.5 hours after the last ESD inhalation, respiratory function was assessed (within 15 minutes). Immediately after assessing respiratory function, bronchoalveolar lavage (BAL) was collected. BAL fluid was taken under anesthesia by washing the lungs with 5 ml of saline heated to 37°C through the trachea using a syringe dispenser.

In the bronchoalveolar lavage fluid, the absolute number of cellular elements in 1 μl of lavage (cytosis) was counted using a Goryaev camera. Then the BAL fluid was centrifuged at 200 g for 10 min. Smears were prepared from the cell sediment, which were subsequently fixed in methanol and stained according to Romanovsky-Giemsa to calculate the endopulmonary cytogram.

Compound I was administered to animals intragastrically once a day on days 10-19 of the study (the last administration was 1 hour before the last ESD inhalation). Instead of ETD and LPS, the false pathology group was inhaled with saline. solution. Control animals received an equivalent volume of solvent instead of the test substance.

The study showed that in a COPD model, compound 1 reduces the influx of inflammatory cells into the bronchoalveolar space of guinea pigs. Compound I most significantly reduces the influx of neutrophils, macrophages and eosinophils (Table 5).

Table 5

Number of cellular elements in bronchoalveolar lavage in a COPD model in guinea pigs (M±m, n=10)

Groups Number of cellular elements in 1 μl of bronchoalveolar lavage Leukocytes Neutro Phils Eosino phyla Macro phages Lymph about quotes False pathology 2488+154 2876+27 184+65 2016+120 48+15 Control 7925+1458# 1632+319# 676+187# 4789+782# 168+60 Compound I (0.014 mg/kg) 2748+174 & 684+62#& 171+32 & 1805+123 # & 8 8+2 9 Compound I (0.14 mg/kg) 3422+434 & 937+141# 287+64 # 2064+313 & 134+60 Compound I (1.4 mg/kg) 4149+357#& 931+166# 245+55 # 2820+273& 71+2 9

Notes:

# - difference from the group of false pathology according to Student's t-test at p<0.05;

& - difference from the control group according to Student's t-test at p<0.05.

Summarizing the results obtained, we can conclude that compound II has a pronounced therapeutic effect in COPD.

Study of the activity of compound I in vivo in a model of allergic rhinitis in guinea pigs

The allergic rhinitis model was implemented according to standard methods (Int Immunopharmacol. 2013 Sep; 17(1):18-25). Guinea pigs (250-300 g) were immunized 4 times (on days 0, 7, 14 and 21) by intraperitoneal injection of a mixture of ovalbumin (100 μg/pig) and aluminum hydroxide (5 mg/pig), diluted and suspended in physiological solution. On the 28th day of the study, the solution

- 15 044574 ovalbumin (60 mg/ml) animals were injected intranasally, 20 μl into each nostril. On the 35th day, the animals were injected with an ovalbumin solution (200 μg/ml, 25 μl) intradermally, having previously shaved the skin on the back. Confirmation of the presence of sensitization was the formation of swelling and redness at the injection site. On the 42nd day of the study, intranasal administration of ovalbumin solution (60 mg/ml, 20 μl/nostril) was performed. In order to control the formation of allergic inflammation, a group of falsely immunized animals was formed: on days 0, 7, 14 and 21, pigs received an aluminum hydroxide solution (5 mg/pig), on days 28 and 35 - saline. solution, on the 42nd ovalbumin (60 mg/ml, 20 µl/nostril).

Compounds I, III, IV (0.14, 1.4 mg/kg) were administered intragastrically to animals once 3 hours before the last intranasal administration of ovalbumin.

Within 2 hours after the last administration of ovalbumin, the clinical manifestations of rhinitis were assessed: the number of sneezes and nose scratches was counted.

The research results are presented in table. 6-8.

Taking into account the clinical manifestations of allergic rhinitis within 2 hours after the last intranasal administration of ovalbumin to animals showed a pronounced increase in the number of sneezes and nose scratches in experimental animals, which indicates the correctness of the implemented model of allergic rhinitis.

Table 6 Clinical manifestations of allergic rhinitis in guinea pigs during animal therapy with compound I (M±w, n=10)

Group Number of sneezes/2 hours Number of nose scratches/2 hours False immunization 5.9+1.3 12.5+3.1 Control 17±1.8* 69±7.4* Compound I (0.14 mg/kg) 9.3+1.7 37.3+7.8*& Compound I (1.4 mg/kg) 12.1+1.5 41.0+12.0

Notes:

* - difference from the intact group according to Student's t-test at p<0.05 & - difference from the control group according to Student's t-test at p<0.05

Table 7 Clinical manifestations of allergic rhinitis in guinea pigs during animal therapy with compound III (M±t, n=10)

Group Number of sneezes/2 hours Number of nose scratches/2 hours False immunization 5, 9+0.3 10.3+2.2 Control 20, b±0.9* 75.4±b.5* Compound III (0.14 mg/kg) 17.8+0.7*& 32.5+8.0*& Compound III (1.4 mg/kg) 19.0+1.1* 39.9+9.5*&

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Table 8 Clinical manifestations of allergic rhinitis in guinea pigs during animal therapy with compound IV (M±t, n=10)

Group Number of sneezes/2 hours Number of nose scratches/2 hours False immunization 3.2+1.06 6.5+2.33 Control 15.2±2.1* 20.8±4.2* Compound IV (0.14 mg/kg) 1.4+0.67 & 5.3+1.14 Compound IV (1.4 mg/kg) 1.4±0.6 & 1.8±1&

Intragastric administration of compounds I, III, and IV expression to guinea pigs reduced the number of clinical manifestations of rhinitis. The results obtained give grounds to conclude that compounds I, III, IV have a pronounced therapeutic effect in allergic rhinitis.

Study of the activity of compound I in vivo on a model of formalin-induced acute rhinosinusitis in rats

Induction of acute rhinosinusitis was carried out in male Wistar rats by intranasal injection of 20 μl of 7.5% formaldehyde into each nasal passage. Compound I was administered in doses of 1.8 mg/kg and 18 mg/kg daily once a day, starting 24 hours after the administration of formalin, the last administration occurred on the 7th day. Dexamethasone (0.6 mg/kg) was administered in the same regimen. On the 8th day, nasal lavage was collected. In the nasal wash, the total number of leukocytes was assessed and the leukocyte formula was determined.

Analysis of nasal lavage showed that acute rhinosinusitis is accompanied by a pronounced influx of leukocytes into the nasal cavity. The maximum increase was noted in the number of macrophages (Table 9).

Intragastric administration of the test compound to rats reduced the content of macrophages and neutrophils in the nasal wash to the level of intact animals. In terms of the severity of action, compound I was not inferior to dexamethasone (Table 9).

Table 9

Number of cellular elements in the nasal wash of rats in a model of acute rhinosinusitis (M±w, n=10)

Groups The number of cellular elements in 1 μl of nasal lavage Leukocytes Neutro Phils Eosino phyla Macrophages Lymphocytes Intact 3420+641 3313+632 7+5 67+16 0+0 Control 5568+1015 5217+980 16+10 291+72* 0+0 Compound I (1.8 mg/kg) 5410+1143 5132+1096 11+11 231+49* 0+0 Compound I (18 mg/kg) 2490+339& 2461+339 & 0+0 2 8+7& 0+0 Dexamethase he (0.6 mg/kg) 1735+451* & 1664+433* & 0+0 68+23& 0+0

Notes:

* - difference from the intact group according to Student's t-test at p<0.05 & - difference from the control group according to Student's t-test at p<0.05

Thus, the results obtained showed that compound I has a pronounced therapeutic effect in rhinosinusitis.

Study of the activity of compound I in vivo on a model of non-infectious pharyngitis in rats

A model of non-infectious pharyngitis was implemented on male Wistar rats. The rats were anesthetized with sodium thiopentone (50 mg/kg, intraperitoneal) and inserted into the external jugular vein.

- 17044574 insert a RenaSil® silicone rubber tubing cannula (SIL 037, Braintree Scientific, Inc., Braintree, MA) with heparinized saline (40 U/mL). Evans Blue dye (EG) (30 mg/kg) was administered to all animals intravenously through a catheter; 10 minutes after the introduction of the EG dye, a 30% formalin solution was applied to the surface of the pharyngeal mucosa as follows: the tongue was slightly pulled out, the pharynx area was opened deep in the oral cavity using blunt forceps, and a formalin solution (50 μl) was carefully applied 3 times with a sterile cotton swab. for 5 s with each application. In the intact group, saline solution was used.

60 minutes after application of a 30% formaldehyde solution, the animals were euthanized by exsanguination. The head of each rat was perfused with heparinized saline (40 U/ml) to remove intravascular EG dye.

The degree of inflammation was assessed using the Evans blue dye exudation test (EG). The EG dye from the tissue was extracted into formamide at 55°C for 24 h, and the absorbance was determined spectrophotometrically at 620 nm. The amount of dye in the tissue was calculated using the standard curve for Evans blue dye and expressed as micrograms of dye per gram of tissue wet weight (μg/g).

Compound I was administered intragastrically 24 and 1 hour before the application of formaldehyde at doses of 6 and 18 mg/kg. The control group was injected with a formaldehyde solution with a concentration of 30%. Dexamethasone and diclofenac were used as comparison drugs. Dexamethasone (0.6 mg/kg) and diclofenac (8 mg/kg) were administered intragastrically 24 and 1 hour before formaldehyde application. The results of the study are presented in table. 10.

From the table 10 shows that the introduction of formaldehyde (control) leads to a significant increase in the concentration of the dye in the tissue, which indicates inflammation of the pharynx in rats - pharyngitis. The test compound showed significant protection against formaldehyde-induced pharyngitis. Compound I had an effect at all tested doses (6 and 18 mg/kg) - the concentration of the dye in the tissue decreased by 2.8 and 6.4 times, respectively, compared to the control. The administration of dexamethasone (0.6 mg/kg) also led to a decrease in inflammation: the concentration of the dye decreased by 2.1 times. Diclofenac had no effect.

Thus, the results obtained showed that compound I has a pronounced anti-inflammatory effect in pharyngitis.

Table 10

Concentrations of Evans blue dye in rat pharyngeal tissue

Group Dye concentration in tissue, µg/mg Intact 0.029+0.004 Control (formaldehyde - 30%) 0.141+0.015* Compound I (6 mg/kg) 0.051+0.016*& Compound I (18 mg/kg) 0.022+0.003 & Dexamethasone 0.6 mg/kg 0.066+0.009*& Diclofenac 8 mg/kg 0.112+0.031*

Notes:

- * - differences are statistically significant compared to intact (p <0.05);

- & - differences are statistically significant compared to intact ones (p<0.05).

Study of the activity of compound II in a model of bleomycin-induced lung damage

The model of bleomycin-induced lung injury was carried out according to standard methods [Am J Respir Cell Mol Biol. 2009 V. 41(1). P. 50-58]. Male Balb/c mice were given a single endotracheal injection of bleomycin solution (4 units/kg in a volume of 50 μl). Compound II was administered to C57BL/6 mice intragastrically, twice - 1 before the administration of bleomycin and 12 hours after the administration of bleomycin. 24 hours after bleomycin administration, bronchoalveolar lavage was collected. In the lavage, the total number of leukocytes was assessed and the leukocyte formula was determined.

The results of the study showed that intragastric administration of compound II reduces the influx of inflammatory cells into the bronchoalveolar space (Table 11). This gives grounds to conclude that compound II has an anti-inflammatory effect in lung lesions.

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Table 11

The number of cellular elements in the bronchoalveolar lavage of mice in a model of bleomycin-induced acute lung injury (M±m, n=10)

Groups Dose of compound, mg/kg Number of cellular elements in 1 µl bronchoalveolar lavage Leukocytes Neutrophils Eosinophils Macrophages Lymphocytes Intact - 151.32+23.85 8.51±4.12 0.40+0.30 122.15+14.69 0.00±0.00 Control - 412.13+65.00* 212.39+37.00* 0.00+0.00 199.74+34.00 0.00+0.00 Compound II 3 122.34+15.96 & 82.77+16.87 *& 0.00+0.00 39.57+7.91*& 0.00+0.00 thirty 199, 12+46, 59 & 55.21+12.53* & 0.00+0.00 143.90+39.26 0.00+0.00

Notes:

* - difference from the intact group according to Student’s t-test at p<0.05 & - difference from the control group according to Student’s t-test at p<0.05

Study of the activity of compound IV in a model of lipopolysaccharide-induced acute lung injury in rats

The model of lipopolysaccharide-induced acute lung injury was implemented using standard methods [Lin Tong, Jing Bi, Xiaodan Zhu, Guifang Wang, Jie Liu, Linyi Rong, Qin Wang, Nuo Xu, Ming Zhong, Duming Zhu, Yuanlin Song, Chunxue Bai. Keratinocyte growth factor-2 is protective in lipopolysaccharide-induced acute lung injury in rats//Respiratory Physiology & Neurobiology. 2014. V. 201. P. 7-14]. Female Wistar rats were endotracheally injected with a solution of lipopolysaccharide (LPS) prepared in saline. Animals in the false pathology group were injected with physiological solution in the same volume. 48 hours after LPS administration, bronchoalveolar lavage (BAL) was collected from the animals. BAL fluid was taken under anesthesia by washing the lungs with 5 ml of physiological solution heated to 37C through the trachea using a syringe dispenser.

In the bronchoalveolar lavage fluid, the absolute number of cellular elements in 1 μl of lavage was counted using a Goryaev camera. Then the bronchoalveolar lavage was centrifuged at 200 g for 10 minutes. Smears were prepared from the cell sediment, which were subsequently fixed in methanol and stained according to Romanovsky-Giemsa to calculate the cytogram.

Compound IV was administered to rats twice intragastrically, 1 hour before LPS administration and 24 hours after LPS administration.

The results of the study showed that intragastric administration of compound IV reduces the influx of inflammatory cells into the bronchoalveolar space (Table 12). This gives grounds to conclude that compound IV has an anti-inflammatory effect in lung lesions.

Table 12

Number of cellular elements in bronchoalveolar lavage of rats in a model of LPS-induced acute lung injury (M±m, n=10)

Groups Dose connected II, mg/kg Number of cellular elements in 1 µl bronchoalveolar lavage Leukocytes Neutrophils Eosinophils Macrophages Lymphocytes Intact - 2263+311 199+59 0+0 2063+265 0+0 False pathology 3434+351 825+115 0+0 2609+302 0+0 Control - 12937+1837 , 5*# 3304+597 *# 0±0 9633+1502 0±0 Compound IV 18 4485+815* & 785±181& 0±0 3699±653& 0±0

Notes:

* - difference from the intact group according to the Kruskal-Wallis criterion at p<0.05 # - difference from the false pathology group according to the Kruskal-Wallis criterion at p<0.05

- 19 044574 & - difference from the control group according to the Kruskal-Wallis test at p<0.05

Study of the activity of compound II, compound IV, compound VI, compound I in a model of febrile reaction in rats

The fever response model was implemented according to standard methods [J Neurosci Methods. 2005. V. 147. P. 29-35]. Wistar rats were injected subcutaneously with a 20% baker's yeast suspension (12 ml/kg). Compounds I, II, IV, VI, VIII were administered once, intragastrically, 14 hours after the introduction of yeast. Rectal temperature was measured with an electric thermometer before the introduction of the pyrogen and at the highest point of development of the thermal reaction - 19 hours after it. The antipyrogenic effect of the compounds was studied in 2-7 experiments.

The results of the study showed that intragastric administration of the test compounds reduced the increase in rectal body temperature in rats (Tables 13-17). The data obtained allow us to conclude that compounds I, II, IV, VI, VIII have an antipyrogenic effect.

Table 13

Increase in rectal body temperature 19 hours after subcutaneous administration of yeast to rats, °C

Notes:

* - difference from the intact group according to the Kruskal-Wallis test at p<0.05 & - difference from the control group according to the Kruskal-Wallis test at p<0.05

Table 14

Increase in rectal body temperature 19 hours after subcutaneous administration of yeast to rats, °C (M±m, n=30-90)

Group Dose of compound, mg/kg Number of animals in Group Increase in rectal body temperature, °C Intact - 70 0.30+0.10 Control - 90 1.90+0.10* Compound 0.018 40 0.90+0.10*& VI 0.18 thirty 1.00±0.20*&

Notes:

* - difference from the intact group according to Student’s t-test at p<0.05 & - difference from the control group according to Student’s t-test at p<0.05

- 20 044574

Table 15

Increase in rectal body temperature 19 hours after subcutaneous administration of yeast to rats, °C ________________________(M±t, n=40-70)_______________________

Group Dose of compound, mg/kg Number of animals in the group Increase in rectal body temperature, °C Intact - 50 0.01+0.08 Control - 70 1.7 0+0.07* Compound I 0.018 40 1.01+0.09* & 0.06 40 0.88+0.11* & 0.18 50 1.01+0.08* & 0.6 40 0.88+0.11* & 1.8 50 1.14+0.09* &

Notes:

* - difference from the intact group according to the Kruskal-Wallis test at p<0.05 & - difference from the control group according to the Kruskal-Wallis test at p<0.05

Table 16

Increase in rectal body temperature 19 hours after subcutaneous administration of yeast to rats, °C (M±t, n=40-70)

Group Dose of compound, mg/kg Number of animals in the group Increase in rectal body temperature, °C Intact - 50 0.03±0.07 Control - 70 1.75±0.07* Connection tion IV 0.018 40 1.18+0.08*& 0.06 40 1.28+0.07*& 0.18 50 1.36±0.05* 0.6 40 1.27±0.09*&

Notes:

* - difference from the intact group according to the Kruskal-Wallis test at p<0.05 & - difference from the control group according to the Kruskal-Wallis test at p<0.05

-21 044574

Table 17

Increase in rectal body temperature 19 hours after subcutaneous administration of yeast to rats, °C

Notes:

* - difference from the intact group according to the Kruskal-Wallis test at p<0.05 & - difference from the control group according to the Kruskal-Wallis test at p<0.05

Study of the activity of compound I and compound IV on a model of a specific pain reaction using the method of chemical irritation of the peritoneum (vinegar writhing test)

A model of a specific pain reaction using chemical irritation of the peritoneum (vinegar writhing test) was implemented according to standard methods. To carry out the vinegar writhing test, Balb/c mice were intraperitoneally injected with 1% acetic acid in a volume of 10 ml per 1 kg of animal weight. Compounds I, IV, VII, IX, X were administered intragastrically, once, 1 hour before the introduction of acetic acid. The number of writhings (convulsive contractions of the abdominal muscles, accompanied by stretching of the hind limbs and arching of the back) was assessed 15 minutes after the administration of acetic acid.

The results of the study showed that intragastric administration of compounds I, IV, VII, IX, X significantly reduces the number of writhing in mice caused by intraperitoneal administration of acetic acid.

- 22 044574 acids (Table 18-19). The results obtained allow us to conclude that compounds I, IV, VII, IX, X have a pronounced therapeutic effect in pain.

Table 18

The number of vinegar cramps on a model of a specific pain reaction using the method of chemical irritation of the peritoneum (vinegar cramps test) (M±t, n=10-31)

Group Dose of compound, mg/kg Number of animals in the group Number of cramps in 15 minutes Control 31 34.68+2.40 Compound I 0.03 10 22.70+2.14* 0.3 20 13.90+2.23* 3 20 18.65+2.44* thirty 20 21.15+2.97* Compound IV 0.03 10 23.60+2.54* 0.3 10 15.50+2.63* 3 10 19.20+3.43* thirty 10 15.30+3.10* Ketorol 15 20 23.60+1.87*

Notes:

* - difference from the control group according to Student's t-test at p<0.05.

Table 19

The number of vinegar cramps in a model of a specific pain reaction using the method of chemical irritation of the peritoneum (vinegar cramps test) (M±t, n=10-31)_________

Group Dose of compound, mg/kg Number of animals in the group Number of cramps in 15 minutes Control 10 48+5.71 Compound VII 0.3 10 17.6+2.81* Compound IX 0.3 10 11.6+3.5* Connection X 0.3 10 17.2+3.4*

Notes:

* - difference from the control group according to Student's t-test at p<0.05.

Study of the activity of compound I and compound II on the hot plate model of thermal pain stimulation

The hot plate model of thermal pain stimulation was implemented according to the standard method [Barrot M. Tests and models of nociception and pain in rodents. Neuroscience. 2012 Jun 1; 211:39-50]. Compound I and Compound II were administered intragastrically to Balb/c mice in a single dose. A hot plate test was performed 1 hour after drug administration. To perform the hot plate test, mice were placed on a hot plate whose temperature (+55±1°C) was constant. The time of the first manifestations of a pain reaction in mice (licking paws, jumping) was recorded and the average latent time of the pain sensitivity threshold (LPT, sec) was calculated in each group.

The results of the study showed that intragastric administration of the studied compounds increased the pain sensitivity threshold of mice by 1.5 times during the hot plate test (Table 20). The data obtained allow us to conclude that compound I and compound II have a pronounced analgesic effect in pain.

-23 044574

Table 20

Pain sensitivity threshold (PST) on the hot plate model of thermal pain stimulation, % of the values before drug administration (M±t, n=20)

Group Dose of compound, mg/kg After 1 hour, % to background Control 72.51±5.80 Compound I 0.3 101.13+9.64* 3 95.28+6.79* thirty 104.55±8.52* Compound II 0.3 97.43+6.43* 3 90.54±6.59* thirty 110.18±11.9*

Notes:

* - difference from the control group according to Student's t-test at p<0.05.

Study of the activity of compound II in a model of spontaneous obesity in db/db mice

To model spontaneous obesity, db/db mice were used, which carry the recessive leptin receptor-Leprdb-(db) gene (linkage group 8, chromosome 4) [Cardiovasc. Diabetol. 2012. V.ll. R. 139-147; Biochem. Biophys. Res. Commun. 2016. V. 472. P. 603-609].

Compound II was administered intragastrically starting from the 7th week of life of the animals. Animal body weight was measured weekly at 612 weeks of life.

The results of the study showed that in a model of obesity, intragastric administration of compound II at a dose of 7.5 mg/kg to mice reduces body weight gain in db/db mice (Table 21).

The results obtained indicate the therapeutic effect of compound II in obesity. The therapeutic effect begins already at 4 weeks of use of compound II.

Study of the activity of compound II in a model of metabolic syndrome

The metabolic syndrome model was implemented by keeping male Wistar rats for 16 weeks on a high-fat diet (cafeteria diet) in accordance with standard methods [Rothwell N.J., Stock M.J., Warwick V.R. Energy balance and brown fat activity in rats fed cafeteria diets or high-fat, semisynthetic diets at several levels of intake/ZMetabolism. 1985. Vol. 34(5). P. 474-480].

From the 10th to the 16th week of the diet, the animals of the experimental groups were administered intragastrically with compound II once a day. The activity of the test compound was assessed by weekly body weight measurements.

The results of the study showed that intragastric administration of compound II reduces body weight gain in animals.

The pharmacological effect begins to manifest itself starting from the 5th week of therapy (Table 22).

Thus, in a metabolic syndrome model, compound II reduces animal obesity.

Table 21

Increase in body weight of animals relative to the beginning of administration of compound II in a model of spontaneous obesity in db/db mice (M±m)

Groups Dose of compound, mg/kg Age of animals at the time of reading, weeks 6 7 8 9 10 eleven 12 Weeks of Compound 1 Administration 0 1 2 3 4 5 6 Intact (db/m) 100.0+4.4 100.0+0.9 103.3+0.9 106.0+0.9 109.8+1.5 113.3+1.1 116.5+1.5 Control (db/db) 100.0+2.9 109.6+3.1 118.3+3.3 126, 2+3,3 131.0+3.4* 134.2+4.2* 139.2+4, 8* Compound II 0.75 100.0+1.7 108.5+1.5 119.6+3.7 123.9+3.8 126.5+4.0* 127.3+4.2* 129.8+5, 2* 7.5 100.0+2.3 111.4+2.9 119.9+2.6 122.1+2.5 121.7+2.5* & 121.7+3.1* & 109.4+4 &

Notes:

* - difference from the intact group according to Student’s t-test at p<0.05 & - difference from the control group according to Student’s t-test at p<0.05

-24044574

Table 22

Body weight of rats in the metabolic syndrome model (M±m, n=12)

Weeks of Research Weeks of therapy Intact Control Compound II (1.5 mg/kg) 0 - 245.50+5.30 250.17+3.95 239.67+3.27 1 - 259.08+5.80 312.83+8.84*# 292.08+4.70* # 2 - 276.42+6.20# 325.33+7.60*# 307.33+5.53* # 3 - 305.00+7.30# 345.08+9.84*# 327.25+7.81* # 4 - 327.92+6.50# 374.75+11.28*# 355.50+9.05* # 5 - 348.00+5.70# 409.25+12.37*# 379.42+7.41* # 6 - 354.83+5.60# 471.33+12.88*# 443.00+7.70* # 7 - 321.00+5.60# 498.25+13.08*# 469.08+6.38* # 8 - 341.08+5.60# 520.83+13.77*# 491.83+6.04* # 9 - 359.17+6.00# 546.50+14.78*# 518.25+6.04* # 10 1 374.17+6.60# 559.08+16.87*# 528.08+6.01* # eleven 2 394.75+6.80# 571.92+18.62*# 547.92+8.27* # 12 3 393.33+7.90# 579.50+18.66*# 551.00+8.36* # 13 4 394.17+8.50# 592.58+18.47*# 553.58+8.52* # 14 5 396.83+10.20# 613.17+18.86*# 563, 67+9, 75*&# 15 6 398.42+9.50# 631.50+18.94*# 570.75+9.13*&# 16 7 397.42+8.30# 627.17+18.72*# 572.08+8.23*&#

Notes:

* - difference from the intact group according to Student's t-test at p<0.05 & - difference from the control group according to Student's t-test at p<0.05 # - difference from the values at week 0 of the study according to Student's t-test at p <0.05 Study of the activity of compound I and compound II in a model of psoriasis in mice Induction of psoriasis in mice was carried out according to standard methods [European Journal of Pharmacology. 2015. V. 756. P. 43-51]. Female Balb/c mice were treated with Aldara cream (5% imiquimod) on the inner side of the right ear once a day at a dose of 30 mg/mouse for 7 days (0-6 days). Vaseline was applied to intact animals. Compound I, compound II and a reference drug (cyclosporine) were administered to animals intragastrically, daily, once a day for 6 days (days 0-5). Euthanasia was performed 24 hours (day 6) after the last application of Aldara cream. Every day on days 0, 2, 3, 4, 5, in the morning before the next application of Aldara cream and before euthanasia, the thickness of the right ear was measured.

The severity of psoriasis was assessed by measuring the increase in the thickness of the affected ear over time.

The results of the study are presented in table. 23.

Table 23

Increase in the thickness of the affected ear on a certain day of the study to day 0 in a mouse model of psoriasis, % (M±m, n=10)

Group Dose of compound, mg/kg Increase in ear thickness on a certain day of the study to day 0, % 3 days 4 days 5 days 6 days Intact - 4.8 6+ 0.9 7.8+ 0.98 9.81+ 1.83 14, 8+ 1.3 Control - 40.3± 5, 3* 67.7± 7.9* 82.6± 9.5* 101± 9, 8* Compound I Compound II 0.3 38.3± 5, 8* 45.2± 4, 1*& 57.3± 3.9*& 62.8± 5, 4*& 0.3 35.3± 2.1* 48.1± 2.8*& 59, b± 4.5*& 7 0.6± 4.4*&

Notes * - difference from the intact group by Student's t-test at p<0.05 & - difference from the control group by Student's t-test at p<0.05

The results of the study showed that intragastric administration of compound I and compound II in a mouse model of psoriasis reduces the increase in the thickness of the affected ear of the animals. This indicates the therapeutic effect of compounds I and II in psoriasis.

Thus, in a mouse model of psoriasis, compounds I and II have a pronounced therapeutic effect.

Study of the activity of compound I and compound II in a mouse model of atopic dermatitis

The atopic dermatitis model was implemented according to standard methods [J Ginseng Res. 2011. Vol.4. P. 479-86]. Atopic dermatitis was modeled using male balb/c mice. 1-chloro-2,4-dinitrobenzene (DNCB) was used as an inducer of contact dermatitis. On days 0 and 12 of the experiment, 100 μl of a 2% DNCB solution was applied to the animals’ previously shaved back areas in order to sensitize the body. On the 17th day, 20 μl of a 2% alcohol solution of DNCB was applied to the animals’ right experimental ear twice with an interval of 1 hour. Ethanol was applied to intact animals instead of the DNCB solution. Animals in the sham immunization group were sensitized with ethanol. Compound I and Compound II were administered intragastrically once daily from days 8 to 17. On the 18th day, the animals were slaughtered. To assess the degree of edema after euthanasia, the weight of the experimental and control ears was determined. The reaction index (RI) was calculated, expressed as a percentage of the difference in the masses of the experimental and control ears.

The results of the study are presented in table. 24.

Table 24

Difference in the weight of the experimental and control ears of mice and the response index to a model of atopic dermatitis in mice (M±m, n= 12)

Group name Difference in weight of experimental and control ears (mg) Reaction Index (%) Intact -0.001+0.002 -0.82+5.3 False immunization 0.018+0.003* 57.8+11.3* Pathology control 0.029+0.002*# 83.3+6.1* Compound I (3 0.007+0.003 #& 25.4+10.2*#& mg/kg) Compound II (3 mg/kg) 0.013+0.002* & 38.1+7.7* &

Notes:

* - difference from the intact group according to Student's t-test at p<0.05 # - difference from the sham immunization group according to Student's t-test at p<0.05 & - difference from the control group according to Student's t-test at p<0 .05

The results of the study showed that intragastric administration of compound I and compound II in a mouse model of atopic dermatitis reduces the response index of the pathological process. This indicates the therapeutic effect of compounds I and II in atopic dermatitis.

Thus, in a mouse model of atopic dermatitis, compounds I and II have a pronounced therapeutic effect.

Study of the effect of compound II on macrophage chemotaxis in vivo using the carrageenan sac model

To assess the effectiveness of a drug on the activity of cells of the immune system at the preclinical stage of research, various models of acute inflammatory process are usually used. Today, the most commonly used models are those in which the pathological process develops in a limited cavity. These models make it possible to fairly closely simulate diseases in which the aberrant activity of cells of the immune system is localized in a certain cavity (peritonitis, cholangitis, arthritis) (J Pharmacol Toxicol Methods. 1994 Nov; 32(3):13947). The carrageenan pouch model is often used in preclinical research to study pathological processes associated with aberrant activity of immune system cells localized in an isolated cavity. In this model, an isolated sac is formed by subcutaneously injecting air into the intracapsular region of the dorsum of a mouse or rat. Subcutaneous injection of air into the back area leads to morphological changes in the cellular lining of the pouch over a few days. The sac consists mainly of macrophages and fibroblasts and is well vascularized. On the sixth day from the moment of formation of the air sac, a solution of λ-carrageenan is injected into the cavity of the air sac. Carrageenan interacts with TLR4 receptors on the surface of macrophages lining the intracapsular region of the pouch, which causes their activation and subsequent synthesis of chemokines and other mediators (IL-1; IL-6; TNFa, IL-8, prostaglandins and leukotrienes, NO) and also to migration of immune system cells into the pouch.

The study of the activity of compound II was carried out on male mice of the Balb/c line according to the standard

- 26 044574 method (Curr Protoc Pharmacol. 2012 Mar; Chapter 5:Unit5.6). In the experimental groups, animals were intragastrically administered compound II at a dose of 3 mg/kg immediately before the administration of carrageenan and then every 10-12

h. Last administration - 12 hours before slaughter.

48 hours after injection of λ-carrageenan, separate groups of animals were euthanized by CO2 inhalation. Immediately after euthanasia, 1 ml of saline solution containing 5.4 mM EDTA, at room temperature, was injected into the bag with a sterile 25G syringe. After a light massage of the air sac area, a sagittal incision was made across the sac and the exudate was collected with a dispenser into sterile 15 ml tubes. In the exudate, the absolute number of cellular elements per 1 μl of washout (cytosis) was determined using a Goryaev camera. Then the exudate was centrifuged at 200 g for 10 minutes. Smears were prepared from the cell sediment, which were subsequently fixed in methanol (5 min) and stained according to Romanovsky-Giemsa (40 min at 20-22°C). The number of macrophages was routinely counted on the smears under an Olympus bx51 microscope (magnification 100). Cells were counted up to 100 cells.

Studies have shown that the use of compound II reduced the influx of leukocytes and macrophages by 10 times (to the level of intact control) (Table 25). Thus, Compound II may have potential for the treatment of a wide range of diseases, such as Crohn's disease, ulcerative colitis, peritonitis and arthritis.

Table 25

Number of immune system cells in the exudate from the carrageenan sac when studying the activity of compound II on the model of macrophage chemotaxis (carrageenan sac), x10 ⁹ /l (M±m, n=10)

Groups Number of cellular elements in 1 µl of exudate, x10 ⁹ /l Leukocytes Macrophages Intact 0.9+0.2 0.6+0.1 Control 9, 9+1.5* 7.7+1.5* Compound II (3 mg/kg) 1.0+0.1 & 0.5+0.1 &

* - differences are statistically significant compared to intact (p<0.05) & - differences are statistically significant compared to control (p<0.05)

Study of the effect of compound II on the chemotaxis of macrophages in vivo using a model of thioglycollate peritonitis

The study was performed on male balb/c mice using standard methods (J Leukoc Biol. 2009 Aug; 86(2):361-70). Mice in the control group were intraperitoneally injected with 2 ml of 3% thioglycollate medium, which was stored for 1 month. Intact animals were injected intraperitoneally with 2 ml of physiological solution. Animals in the experimental groups were intragastrically administered compounds II 1 hour before the administration of thioglycolate, 24 and 48 hours after the administration of thioglycolate at a dose of 1 mg/kg. After 72 hours, the animals were euthanized in a CO ₂ chamber, the peritoneal area was moistened with 70% alcohol, the skin on the abdominal cavity was carefully cut off, and 5 ml of cold phosphate-buffered saline containing 0.1% EDTA was injected intraperitoneally with a syringe. After a light massage of the abdominal cavity, the exudate was collected with a syringe into test tubes, and the volume of the collected exudate was determined. In the exudate, the absolute number of cellular elements per 1 μl of washout (cytosis) was determined using a Goryaev camera. Then the exudate was centrifuged at 200 g for 10 min. Smears were prepared from the cell sediment, which were subsequently fixed in methanol (5 min) and stained according to Romanovsky-Giemsa (40 min at 20-22°C). The number of monocytes/macrophages was routinely counted on the smears under an Olympus bx51 microscope (magnification 100). Cells were counted up to 100 cells.

Studies have shown that the use of compound II reduces the influx of macrophages into the peritoneal cavity of rats induced by 3% thioglycollate medium and reduces the severity of pathology induced by the administration of thioglycollate medium (Table 26). Thus, Compound II may have potential for the treatment of peritonitis, Crohn's disease and ulcerative colitis.

- 27 044574

Table 26 Number of immune system cells in exudate from the abdominal cavity when studying the activity of compound II on the model of macrophage chemotaxis (thioglycollate peritonitis), x10 ⁹ /l (M±m, nl 0) Groups Number of cellular elements in 1 µl of exudate, x10 ⁹ /l Leukocytes Monocytes Intact 0.71+0.09 0.61+0.07 Control 3.99±0.42* 3.66±0.44* Compound II (1 mg/kg) 1.97±0.Zb*& 0.65±0.12&

* - differences are statistically significant compared to intact (p<0.05) & - differences are statistically significant compared to control (p<0.05)

Study of the activity of compound II and compound I in a model of adjuvant arthritis induced by administration of Freund's complete adjuvant

The adjuvant arthritis model was implemented on outbred male rats according to the standard method [Chern Pharm Bull (Tokyo). 2018. V.66(4). R.410-415]. On days 0 and 5, Freund's Adjuvant, Complete (Pierce) was injected subplantarly into the right hind limb of the animals in a volume of 100 μl per animal. The volume of the right hind paw (affected) and left hind paw (contralateral) was measured on days 0, 14, 16, 18, 21, 25, 28 and 30 using a plethysmometer (Ugo Basel). The primary (inflammatory) reaction was judged by the change in the volume of the affected paw; the secondary (immune) reaction was judged by the change in the volume of the contralateral paw. Compound II was administered intragastrically, daily, once a day, from the 14th to the 30th day of the study.

The results of the study are presented in table. 27-28. The study showed that the use of compound II reduces the volume gain of both the affected paw and the contralateral paw. This allows us to conclude that compound II has both an anti-inflammatory effect and an immunotropic effect. Thus, compound II has high therapeutic potential for the treatment of rheumatoid arthritis and other types of arthritis, as well as arthrosis.

Table 27

Increase in the volume of the affected paw in a model of adjuvant arthritis induced by the introduction of Freund's complete adjuvant, % (M±t, n=10)

Group Dose of compound, mg/kg Increase in the volume of the affected paw,% to the initial level 14 days 16 days 18 days 21 days 25 days 28 days 30 days Intact 1.8+0.8 1.9+0.7 1.4+0.3 1.9+0.4 2.2+0.2 2.7+0.2 3+0.2 Control 122.3+6, 4* 118.7+5, 6* 114.3+2, 1* 98.9+3.2 100.6+2, 6* 102.5+1, 8* 101.4+2.8* Compound II 0.5 118.8+3, 6* 105+3.2* 104.4+2, 7*& 86.3+3.9 *& 88.5+2.4 *& 8 6, 9+2,3 *& 83.2+2.2*&

Note:

* - difference from the intact group according to Student's t-test at p<0.05.

& - difference from the control group by Student's t-test at p<0.05.

Table 28

Increase in the volume of the contralateral paw in the model of adjuvant arthritis induced by the introduction of complete Freund's adjuvant, % (M±w, n=10)

Group Dose of compound, mg/kg Increase in the volume of the contralateral paw, % to the initial level 14 days 16 days 18 days 21 days 25 days 28 days 30 days Intact 1.27+0.3 1.59+0.4 8 1.77+0.2 5 1.62+0.31 2.18+0.3 2.7 6+0.1 6 2.98+0.3 Control 5.48+0.5 8* 7.08+1.2 4* 21.69+1, 28* 25.29+1.6 9* 20.16+1, 66* 18.75+1, 13* 17.51+1.59* Compound II 0.5 5.39+1.6 7* 6, 75+1.5 8* 18.48+1* 19.77+1.6 7*& 15.45+1, 21*& 14.04+1.75*& 13.02+0.94* &

Note:

* - difference from the intact group according to Student's t-test at p<0.05.

& - difference from the control group by Student's t-test at p<0.05.

-28044574

Study of the activity of compound II on the model of carrageenan edema

To assess the effect of compound II on the functional immune status, a model of carrageenan-induced rat paw edema was used. The experiments were performed on male white nonlinear rats weighing 250-270 g. Acute inflammation of the paw in rats was caused by intraplantar (subcutaneous) injection of 0.1 ml into the right paw of a 1.5% solution of carrageenan. The test compound was administered intragastrically 1 hour before the administration of carrageenan. The effect of compound II and the control substance (diclofenac) was assessed by the degree of reduction in paw swelling in comparison with the intact left paw. Paw volume was assessed before administration of compound II and 2 hours after administration of carrageenan.

The results of the study are presented in table. 29.

Table 29

The effect of compound II on the inhibition of the increase in edema of the paw of rats (relative to the control) in the carcinoma model

ragenin-induced edema. Group Induction of pathology Administration of drugs Timing of the assessment Inhibition of edema growth, % Intact Intraplantar injection of 0.1 ml of 1.5% carrageenan solution into the right paw Intragastric, once, 1 hour before carrageenan administration 2 hours after administration of carrageenan 0 Control 0 Compound II (1.8 mg/kg) 34 Compound II (0.18 mg/kg) 34 Diclofenac sodium (8 mg/kg) 42

From the above results it is clear that compound II has a direct inhibitory effect on the growth of paw edema and can be used to treat a wide range of diseases associated with the activity of cells of the immune system.

Study of the activity of compound II in a model of diabetic nephropathy in rats

A model of diabetic nephropathy in rats was induced by long-term maintenance of animals on a high-fat diet and single or double intraperitoneal administration of streptozotocin at a dose of 30 mg/kg (Int J Clin Exp Med. 2015 Apr 15; 8(4):6388-96).

The animals were kept on a high-fat diet for 16 weeks; on the 9th week, the rats were intraperitoneally injected with streptozotocin twice (2 days in a row) at a dose of 30 mg/kg. 7 days after the administration of streptozotocin, the administration of compound II began. The substance was administered daily intragastrically once a day at a dose of 50 mg/kg.

Intragastric administration of compound II, starting from week 10 of the study, reduced the daily protein content in the urine of experimental animals to the level of the intact control (Table 30). The results obtained allow us to conclude that compound II has a therapeutic effect in diabetic nephropathy.

Table 30

Biochemical analysis of urine after 6 weeks of intragastric administration of compound II to rats in a model of diabetic nephropathy induced by a high-fat diet and administration of streptozotocin _____________________ (15 weeks of high-fat diet)_____________________

Groups Dose and regimen of streptozotocin administration Number of animals Diuresis, ml/18 hours. Daily protein, mcg/18h ACR (protein (µg/l)/creatinine (mg/l) Intact - 8 4.35+0.5 1041, 2+153, 5 273.2+68.0 Control 30 mg/kg, twice 14 8.32+1, 5 3718.2+572.7 952.8+186.4*

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Claims (2)

Compound II (5 30 mg/kg, mg/kg) twice 7.45±1.0 1846.3±337.6 *& 202.3+26.8 & * - differences are statistically significant compared to intact (p<0.05) & - differences are statistically significant compared to control (p<0.05) Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments detailed are provided for purposes of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications can be made without departing from the spirit of the present invention. CLAIM 1. Application of compounds of general formula (A): wherein R ₁ represents a group -C(O)-R ₂ -C(O)- or -R2-C(O)-, where R2 represents a -( _CH2 ) _n- group, optionally substituted with one or two C1-C6 alkyls, or phenyl, n is an integer from 0 to 4; wherein the compounds are selected from the group consisting of compounds of the formula I or pharmaceutically acceptable salts, hydrates or solvates thereof for the treatment of diseases of the lungs and respiratory tract, where the disease is selected from the group consisting of pain syndrome, bronchial asthma, acute or chronic bronchitis, pharyngitis, emphysema, rhinitis, rhinosinusitis, chronic obstructive pulmonary disease and acute lung damage, fever. 2. A method of treating a disease of the lungs and respiratory tract, comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of formula (A) wherein R1 represents a group -C(O)-R ₂ -C(O)- or -R2-C(O)-, where R2 represents a -(CH ₂ ) _p - group, optionally substituted with one or two C1-C6 alkyl-