EA042698B1 - NEW GLUTAMINE CYCLASE INHIBITORS AND THEIR APPLICATION FOR THE TREATMENT OF VARIOUS DISEASES - Google Patents

Description

Technical field

This invention relates to chemistry of organic compounds, pharmacology and medicine and relates to 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. Also, the present invention relates to 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, which is 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 directed movement of cells of the immune system along the concentration gradient of some endogenous and exogenous substances (chemoattractants) is one of the most important components of the functioning of cells of the immune system. 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 process of chemotaxis at the molecular level can lead to new effective approaches in the treatment and prevention of a number of diseases associated with the aberrant activity of immune system cells.

CCL family chemokines (CCL2, CCL7, CCL8, CCL13), which are CCR2 receptor ligands, are the most potent monocyte and macrophage chemotaxis factors in mammals (Biochem J. 2012 Mar 1;442(2): 403-12). Chemokines of the CCL family constitute an important class of cytokines required 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 overabundance of immune system 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 damage to respiratory tissues: 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 CCL family chemokines in the pathophysiology of a number of autoimmune and allergic conditions mediated by aberrant activity of CCR2+, CD14+ and CD16lo monocytes (rheumatoid arthritis, multiple sclerosis) is known. In addition, CCL family chemokines (particularly CCL2) are implicated in the pathogenesis of obesity, metabolic syndrome, chronic pain, fibrosis, non-alcoholic fatty liver disease, and some forms of cancer.

Suppression of the aberrant activity of immune system cells by inhibiting CCL-mediated chemotaxis can be highly demanded 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, etc. .d.

For example, in the case of 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 tissues of the kidneys and their maturation into macrophages causes the development of an aberrant response associated with the release of more chemokines and reactive oxygen species that damage the surrounding kidney cells (Mediators Inflamm. 2012; 2012:146154). Damage to kidney tissue leads to the development and maintenance of aberrant activity of immune system cells and further destruction of kidney tissues, and blockade of the CCL2/CCR2 interaction by small molecule antagonists reduces the severity of the 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 that induces 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 that damage 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 residue (pE), whose role 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):966-80). Glutaminyl cyclase has a broad substrate specificity and is involved in the post-translational modification of a number of peptide molecules. In studies of the substrate specificity of glutamine 1 042698 nylcyclase, it was 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).

In experimental studies, it was 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 to treat 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 the monocytic 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 of bacterial lipopolysaccharides, lipoteichoic acid, or other irritants to the respiratory mucosa results in an increase in CCL2 secretion by bronchial smooth muscle cells (Am J Physiol Lung Cell Mol Physiol. 2012 Apr 15; 302(8):L785-92) and an increase in CCL2 concentration 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 that damage the 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 preservation of aberrant activity of cells of the immune system, and further destruction of respiratory tissues. 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;1 66(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 fever, pain is also an extremely common symptom of various diseases. Obviously, a decrease in the severity of the associated aberrant response associated with the release of a larger amount of reactive oxygen species that damage the surrounding tissues should in itself lead to a decrease in the severity of the pain syndrome. However, recent studies have shown the key role of 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.

Fractalkin 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. Fractalkin 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 dermal papillae and in antigen presenting cells (Br J Dermatol. 2001 Jun; 144(6):1105-13). The expression of fractalkine is enhanced by tumor necrosis factor-α and interferon-γ and in rheumatoid arthritis, promotes the migration of monocytes, T cells and osteoclast precursors into the synovial tissue (Mod Rheumatol. 2017 May; 27(3):392-397). Increased expression of fractalkin in 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). Fractalkin 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 made it possible to significantly alleviate 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 promising new therapeutic approach to 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). In animal models, it has been shown that the severity of radiation

- 2 042698 induced vascular dysfunction can be effectively reduced by inhibiting CCL-mediated signaling pathways. The use of CCL2 receptor knockout animals, as well as the use of antagonists of this receptor, blocks radiation-induced changes in the 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, diseases of the lungs, respiratory tract and abdominal cavity, radiation sickness and pain syndrome.

Glutaminyl cyclase inhibitors are currently 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 that is the subject of the present invention are given in the publications of the company Probiodrug Aktiengesellschaft (J Biol Chem. 2003 Dec 12; 278(50):49773-9). This paper describes glutaminylcyclase inhibitors based on imidazole derivatives. However, in the structures of the compounds published by Probiodrug Aktiengesellschaft, imidazole contains an aliphatic substituent at 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 immune system cells and can potentially lead to side effects.

To date, there is not a single drug that acts as an inhibitor of glutaminyl cyclase, which would be used in the treatment of diseases associated with the aberrant activity of immune system cells, therefore, there is still a need to create and introduce new effective drugs based on glutaminyl cyclase inhibitors for use in therapy.

This invention relates to the use of chemical compounds 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.

Disclosure of invention

The objective of the present invention is to develop new drugs that are inhibitors of glutaminyl cyclase and effective for the treatment of diseases associated with aberrant activity of cells of the immune system, in particular 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 inhibitors of glutaminyl cyclase, characterized by high inhibitory activity, allowing the use of these inhibitors for the treatment 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 pulmonary emphysema, bronchial obstruction); diseases of the abdominal cavity, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy (in particular diabetic nephropathy); radiation sickness and pain syndrome, as well as other diseases associated with aberrant activity of immune system cells, in particular with aberrant chemotaxis of immune system cells.

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

wherein

R ₁ is -C(O)-R ₂ -C(O)- or -R ₂ -C(O)-, where R ₂ is -(CH ₂ ) _p - optionally substituted with one or two C ₁ -C ₆ -alkyl, 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

- 3 042698

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

This technical result is also achieved by using compounds of formula (A) selected from any of the compounds IX 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 IX or their salts, hydrates, solvates to obtain a pharmaceutical composition for the prevention and/or treatment of a disorder associated with aberrant activity of immune system cells, in particular with aberrant chemotaxis of immune system cells.

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 with aberrant chemotaxis of cells of the immune system, and characterized in that they contain an effective amount of a compound according to 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 an organism, comprising administering a pharmaceutical composition of the invention to said organism. Such a disorder associated with glutaminyl cyclase activity 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 of the invention, the disease of the lungs and respiratory tract is bronchial asthma, acute and chronic bronchitis, pharyngitis, pulmonary emphysema, rhinitis, rhinosinusitis, or chronic obstructive pulmonary disease. In particular cases of embodiment of the invention, the organism is a human or an 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. This 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, oncological, neurodegenerative, cardiovascular, inflammatory diseases, diabetes, gerontological diseases, diseases caused by toxins of microorganisms, as well as alcoholism, alcoholic cirrhosis of the liver, anemia, late porphyria, poisoning with transition metal salts.

In the course of studies of the specific pharmacological activity of the Compound of formula (A), the authors of the present invention unexpectedly found that the compounds of formula (A) affect the chemotaxis of cells of the immune system. A decrease in 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, pulmonary emphysema, bronchial obstruction); as well as diseases of the abdominal cavity, 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 ability of a compound to complex or chelate metal ions, an attempt was made to find

- 4 042698 possible therapeutic targets. During the research of the author of the present invention found that the observed therapeutic effect of the compounds of formula (A) is associated with the ability of these compounds to inhibit the activity of glutaminylcyclase. In the course of 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 inhibitors of glutaminyl cyclase that affect the chemotaxis of cells of the immune system and can be used to treat 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 (particularly allergic rhinitis), rhinosinusitis, chronic obstructive pulmonary disease and its manifestations (particularly emphysema, bronchial obstruction); as well as diseases of the abdominal cavity, 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-(III-imidazol-4-yl)ethyl]oxalamide corresponding to the following structural formula:

^{0 ;} ...........= Y......V

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-(III-imidazol-4-yl)ethyl]malonamide, also represented by the structural formula

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

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

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

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

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

- 5 042698

The term Compound X refers to N,N'-bis[2-(1H-imidazol-4-yl)ethyl]terephthalamide corresponding to the following structural formula:

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

The term IC5o refers to the concentration of a test compound at which half-maximal enzyme inhibition is achieved.

The term pharmaceutically acceptable salts or salts includes salts of the active compounds which are prepared with relatively non-toxic acids. Examples of pharmaceutically acceptable non-toxic salts are salts formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, succinic, citric or malonic acids, or obtained by others. methods used in this area, for example, using ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanate, hexanate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate (mesylate), 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, hemi-fumarate, stearate, succinate, sulfate, tartrate, thiocyanate, ptoluenesulfonate (tosylate), undecanate, valeriate, and the like.

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

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

The term excipient means any pharmaceutically acceptable substance of inorganic or organic origin, which is part of the medicinal product or used in the manufacturing process, the manufacture of the medicinal product to give it the necessary physical and chemical properties.

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

The term glutaminyl cyclase refers to the enzyme aminoacyltransferase 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 to damaged tissue. The leading role in chemotaxis is played by chemotactic substances (chemoattractants). One of the most powerful chemoattractants for monocytes and macrophages is the CCL2 chemokine.

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. induction of regression of the disease, d) reversal of the disease or condition to which the term is applied, or one or more symptoms

- 6 042698 of this disease or condition.

The term prophylaxis encompasses the elimination of risk factors as well as the prophylactic treatment of subclinical stages of disease in a mammal, preferably in humans, to reduce the likelihood of occurrence of clinical stages of the disease. Patients for prophylactic therapy are selected on the basis of factors that, based on known data, entail an increased risk of clinical stages of the disease compared with the general population. Preventive therapy includes: a) primary prevention and b) secondary prevention. Primary prevention is defined as prophylactic treatment in patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of the recurrence of the same or a similar clinical disease state.

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); abdominal diseases such as Crohn's disease, ulcerative colitis, peritonitis and nephropathies (particularly diabetic nephropathy); radiation sickness and pain syndrome, both systemic and local in 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 may be used to treat other diseases associated with aberrant activity of cells of the immune system.

Method for Therapeutic Use of the Compounds

The subject matter of this 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 in 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 may 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 the treatment or prevention of the disease.

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

The introduction can be carried out as a single, and 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 period of days (eg 2-10 days) followed by a period without the substance (eg 1-30 days).

When the compounds of the invention are used as part of a combination therapy regimen, the dose of each of the components of the combination therapy is administered during the desired treatment period. The compounds constituting the combination therapy can be administered to the patient both at the same time, in the form of a dosage containing all the components, and 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 IX (or a prodrug or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, diluents and/or excipients, such that may be co-administered to a patient with a compound of the invention that does not interfere with the pharmacological activity of the compound and is non-toxic when administered at doses sufficient to deliver a therapeutic amount of the compound.

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, sliding materials, etc., suitable for a particular dosage form. Materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, mono- and oligosaccharides.

- 7 042698 reads, as well as their derivatives; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut, cottonseed, safrole, sesame, olive, corn and soybean oils; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffer substances such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solution, Ringer's solution; ethyl alcohol and phosphate buffer solutions. Other non-toxic compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, film formers, sweeteners, flavoring and flavoring agents, preservatives and antioxidants may also be included in the composition.

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

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

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

Tablets can be coated with sucrose, a cellulose derivative, or other materials suitable for coating. Tablets can be prepared in a variety of ways, such as direct compression, dry or wet granulation, or hot melting.

A pharmaceutical composition in the form of a gelatin capsule can be obtained 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 injectable solutions are used, which 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 diseases in humans or animals in the form of the following compositions (substance means the 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

Tablet IV mg/tablet

Substance 50

Lactose Ph. EUR 223.75

Croscarmellose sodium 6.0

Cornstarch 15

Polyvinylpyrolidon (5% mob paste) 2.25

Magnesium stearate 3.0

Tablet V mg/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

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Substance 10

Lactose Ph. EUR 488.5

Magnesia 1.5

Formulation 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-dodecylasecycloheptanone 50 cl

Propylene glycol up to 1 ml

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

Use of Compounds I-X in Combination Therapy

Although Compounds IX can be administered as the active pharmaceutical agent alone, they can also be used in combination with one or more other agents, in particular, the other agent may be an antibiotic, NSAID or other anti-inflammatory agent, antihypertensive agent, glucocorticosteroid, monoclonal antibody, etc. When co-administered orally, the therapeutic agents may be in different dosage forms 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, which in one way or another will provide a beneficial effect 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 IX can be carried out in combination with additional methods of treatment 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 dose range. Compounds IX of this invention can also be administered to the patient sequentially with other agents, when the 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 the patient together, before or after administration of another drug.

EXAMPLES Preparation of Compounds of 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.

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

The biological activity of compounds IX was 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 action of Compounds I and II cannot be predicted or explained on the basis of prior 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 shown that Compounds III-X are also inhibitors of the enzyme glutaminyl cyclase and thus the effect of Compounds III-X on chemotaxis of immune system cells may be mediated by inhibition of glutaminyl cyclase activity.

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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 IX, which are the subject of the present invention, on the enzymatic activity of glutaminyl cyclase in vitro, for the first time, a direct inhibitory effect of compounds IX on recombinant intracellular human glutaminyl cyclase was 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 -μ1 reaction mixture contained 50 µM 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 to the reaction mixture an aliquot of glutaminyl cyclase incubated with Compounds 1-X for 5 minutes.

Table 1. Effect of Compounds I-X on the enzymatic activity of human glutaminyl cyclase in vitro

The further course of the reaction was monitored spectrophotometrically (the excitation and emission wavelengths were 320 and 410 nm). The enzymatic activity was determined from the amount of 2-naphthylamide (bNA) released, calculated from the calibration curve. IC ₅₀ values were calculated using non-linear regression of the inhibitor concentration-enzymatic activity curve. The 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 Compounds I-X inhibit the activity of glutaminyl cyclase with an IC50 in the range from 0.8 to 20 μM (see table. 1)

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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)x10 ⁶ cells/ml on RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Biochem J. 2012 Mar 1 ; 442 (2): 403-12). Approximately 1x10 ⁷ U937 cells were incubated with various concentrations of Compounds I and II (total 7 concentrations for each of the compounds) at 37°C for 2 hours and then treated with E. coli lipopolysaccharide (O111:B4). The supernatant (conditioned medium) was used to study the migration of monocytes.

A fresh portion of U937 cells was stained with the fluorescent dye Calcein AM for 1 h at 37°C. An aliquot of stained cells was then placed in the top compartment of a BD FalconTM HTS FluoroBlok plate, the wells of which were separated by semi-permeable, optically opaque membranes. The conditioned medium obtained after incubation of the cells with the inhibitor and lipopolysaccharide was placed in the lower compartment of the wells of the tablet. The plates were incubated for 2 h at 37°C, the number (% relative to the experiment without inhibitor) of cells migrating into the lower compartment of the wells was determined fluorimetrically. The 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 the migration of monocytes in vitro. Compound I inhibits the migration of monocytes in a wide range of concentrations 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 in a model of bronchial asthma in guinea pigs

Induction of asthma in guinea pigs was carried out according to the standard method (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 intraperitoneally injected with saline. solution in a volume of 0.5 ml.

On days 29, 30, and 31 of the experiment, airway hyperreactivity was provoked by inhalation administration of ovalbumin at increasing concentrations of 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 the appearance of pronounced signs of asphyxia (falling on one side). On the 32nd day, the animals were injected with a resolving dose of ovalbumin - 1 mg/ml for 5 minutes with an 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.

Bronchoalveolar lavage (BAL) was taken from the animals 24 hours after the administration of a resolving dose of ovalbumin. BAL was taken under anesthesia by flushing the lungs with 5 ml of saline warmed up 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 bronchoalveolar lavage 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.

Cytological analysis of bronchoalveolar lavage (BAL) revealed a multiple increase in cellular elements in the BAL of sensitized guinea pigs (Fig. 1). Thus, this model is characterized by an inflammatory reaction of the respiratory tract of experimental animals. Analysis of individual cell types showed that the most pronounced influx of cells accounted for eosinophils. The results obtained confirm the literature data and allow us to conclude that the simulated inflammation is of an allergic 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 in the entire tested dose range (0.14-14 mg/kg), reduced both the total number of leukocytes and the number of individual cell types: eosinophils, neutrophils, macrophages (Fig. 1).

Meanwhile, in FIG. 1 sign * indicates differences that are statistically significant compared with the intact group (p<0.05); and sign - & - differences, statistically significant compared with 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 in a model of Sephadex-induced pulmonary bronchitis in rats

The model of Sephadex-induced lung bronchitis in rats was implemented according to the standard method (Int Arch Allergy Immunol. 2011; 154(4):286-94). Male Wistar rats were injected with Sephadex G-200 (Pharmacia, Sweden) at a dose of 5 mg/kg once by inhalation. The test compounds were administered intragastrically to animals four times: 24 and 1 h before and 24 and 45 h after the administration of Sephadex. The reference drug budesonide was administered according to the same scheme by inhalation at a dose of 0.5 mg/kg. Bronchoalveolar lavage was taken 48 hours after Sephadex inhalation. In 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 ones (Table 2).

Table 2. The number of cellular elements in bronchoalveolar lavage in the model of sephadex-induced bronchitis in rats (M±m, n=10)

Group The number of cellular elements in 1 µl of BIL 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) 38331525* 283+75 & 0+0 & 3127+351* 0+0 Compound I (1l 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 to the level of intact animals. The results obtained indicate that Compound I has a therapeutic effect in inflammation of the lower respiratory tract, in particular in bronchitis.

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

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

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

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.

Table 3. Results of a histological study on a model of non-infectious pneumonia __________ induced by cigarette smoke extract (M±w, n=12)_______

Group Compound dose, 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* 1.2 9+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 according to Student's t-test at p<0.05.

Study of the activity of Compound I in vivo in a model of pulmonary emphysema in mice

Pulmonary inflammation and emphysema were induced by a single intratracheal injection of porcine pancreatic elastase. Elastase was administered once intratracheally at a dose of 0 U/mouse in 30 µl NaCl 0.9%. Pentobarbital intraperitoneally at a dose of 30 mg/kg was used for anesthesia during the operation. The operating field was disinfected with 70% ethanol solution and freed from hair. The skin, subcutaneous tissue, and own fascia of the neck were dissected along the midline of the neck. Muscles on the ventral side of the trachea were separated by blunt tissue expansion. Porcine pancreatic elastase was injected with a Hamilton syringe along the course of air flow. 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 the lung tissue, histological preparations were made from the left lung. To do this, the lung was fixed in 10% neutral formalin solution 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 were obtained with a thickness of 5 μm and stained with hematoxylin and eosin according to the standard method. Next, photographs were taken of the apex of the lung, the middle lung field, and the lower lung field. The localization and area of emphysematous-dilated lung tissue (% of normal tissue) was studied using computer graphics processing tools, 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 the injection of elastase in all areas of the lung of mice revealed a moderately pronounced plethora of the vessels of the microcirculatory bed and capillaries of the interalveolar septa. In addition, elastase led to thickening of the walls of the alveoli due to lympho-macrophage infiltration, as well as inflammatory infiltration of the 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 passages and alveoli in the lung parenchyma, stretching of the alveoli and ruptures of the alveolar septa were noted, and diffuse emphysema developed. The analysis of 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 lung field (Table 4).

Table 4. Area of emphysematous enlarged lung tissue (% of normal) in mice under conditions of intratracheal injection of elastase on the 21st day of the experiment (M±m, n=5)

Groups Upper lung field Middle lung field Inferior lung 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 in comparison with the intact control (p<0.05);

- · - differences are significant in comparison with pathological control (p<0.05).

The introduction of Compound I reduced the intensity of inflammatory infiltration of the lung parenchyma and the severity of plethora of vessels of the microvasculature and capillaries of the interalveolar septa, reduced the relative area of emphysematous-dilated alveoli compared with the pathological control group (Table 4). The obtained data give grounds to conclude that Compound II has a therapeutic effect in pulmonary emphysema.

Study of Therapeutic Activity of Compound I in a Guinea Pig Model of COPD

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/cig, nicotine 1.4 mg/cig). 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 the smoke of a lit cigarette through PBS at a constant speed using a vacuum pump (0 ml/40 cigarettes). The burning time of one cigarette was 180 s. To remove particles, the obtained extract was filtered through a bacterial filter with a pore size of 45 nm. ESD was inhaled in guinea pigs (0.3 ml/min, 40 min) daily once a day on days 1-4, 6-9, 11-14, 16-19. LPS was inhaled in guinea pigs (25 μg/ml, 0.3 ml/min, 1 h) once a day on days 5, 10, and 15. Prior to 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 min). Immediately after the assessment of respiratory function, bronchoalveolar lavage (BAL) was taken. BAL was taken under anesthesia by flushing the lungs with 5 ml of saline warmed to 37°C through the trachea using a syringe dispenser.

In the bronchoalveolar lavage fluid, the absolute number of cellular elements per 1 μl of lavage (cytosis) was counted using a Goryaev camera. Then BAL 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 daily once a day on the 10-19th day of the study (the last injection was 1 hour before the last ESD inhalation). The false pathology group received physical inhalation instead of ETD and LPS. solution. Control animals received an equivalent volume of solvent instead of the test substance.

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

Table 5. Number of cellular elements in bronchoalveolar lavage in COPD model in guinea pigs (M±m, n=10)

Groups The number of cellular elements in 1 µl of bronchoalveolar lavage leukocytes Neutrofils Eosino phyla macro phages Lymphocytes 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 obtained results, we can conclude that Compound II has a pronounced therapeutic effect in COPD.

In Vivo Activity Study of Compound I in a Guinea Pig Model of Allergic Rhinitis

The allergic rhinitis model was implemented according to the standard method (Int Immunopharmacol. 2013 Sep;17(1):18-25). Guinea pigs (250-300 g) were immunized 4 times (on days 0, 7, 14 and 21) intraperitoneally with a mixture of ovalbumin (100 μg/gilt) and aluminum hydroxide (5 mg/gilt), diluted and suspended in saline. On the 28th day of the study, a solution of ovalbumin (60 mg/ml) was administered to the animals intranasally, 20 μl in each nostril. On the 35th day, the animals were injected with a solution of ovalbumin (200 µg/ml, 25 µl) intradermally, having previously shaved the area of the skin on the back. The presence of sensitization was confirmed by the formation of edema and redness at the injection site. On the 42nd day of the study, intranasal administration of an ovalbumin solution (60 mg/ml, 20 μl/nostril) was performed. In order to control the formation of precisely allergic inflammation, a group of falsely immunized animals was formed: on days 0, 7, 14 and 21, the pigs received a solution of aluminum hydroxide (5 mg/mumps), on the 28th and 35th days - physical. solution, on the 42nd ovalbumin (60 mg / ml, 20 μl / nostril).

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

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

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

Accounting for 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 scratching of the nose in experimental animals, which indicates the correctness of the implemented model of allergic rhinitis.

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Table 6. Clinical manifestations of allergic rhinitis in guinea pigs treated with ______ animals Compound I (M±m, n=10)_________

Group Quantity Quantity chikhov / 2 hours scratching nose/2 hours False immunization 5.9+1.3 12.5+3.1 Control 17+1.8* 69+7.4* Compound I (0.14 9.3+1.7 37.3+7.8*& mg/kg) Compound I (1.4 12.1+1.5 41.0+12.0 mg/kg) Notes: * - difference from the intact group according to Student's t-test with 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 treated with Compound III (M±m, 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 20.6+0.9* 75.4+6.5* 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*&

Table 8. Clinical manifestations of allergic rhinitis in guinea pigs treated with Compound IV (M±m, 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, IV to guinea pigs significantly 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 the model of formalin-induced acute rhinosinusitis in rats

Acute rhinosinusitis was induced in male Wistar rats by intranasal administration of 20 µl of 7.5% formalin into each nasal passage. Compound I was administered at 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, a nasal wash was taken. In the nasal wash, the total number of leukocytes was assessed and the leukocyte formula was determined.

Analysis of nasal wash showed that acute rhinosinusitis is accompanied by a pronounced influx of leukocytes into the nasal cavity. The maximum increase was noted in relation to 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. Compound I was as effective as dexamethasone (Table 9).

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Table 9. The number of cellular elements in the nasal wash of rats on the model of acute rhinosinusitis (M±m, n=10)

Groups The number of cellular elements in 1 µl of nasal flush Leukocytes Neutrofils 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 28+7& 0+0 Dexametazon (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 the model of non-infectious pharyngitis in rats The model of non-infectious pharyngitis was implemented on male Wistar rats. Rats were anesthetized with sodium thiopentone (50 mg/kg, ip) and the external jugular vein was cannulated with RenaSil® silicone rubber tubing (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 formalin solution (50 μl) was carefully applied 3 times with a sterile cotton swab for 5 seconds with each application. In the intact group, physiological saline was used.

60 min after applying a 30% formalin 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 by the Evans blue dye (EG) exudation test. The EG dye was extracted from the tissue into formamide at 55°C for 24 h, and the absorption was determined spectrophotometrically at 620 nm. The amount of dye in tissue was calculated using the standard Evans blue dye curve and expressed as micrograms of dye per gram wet weight of tissue (μg/g).

Compound I was administered intragastrically 24 and 1 h before 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 reference drugs. Dexamethasone (0.6 mg/kg) and diclofenac (8 mg/kg) were administered intragastrically 24 and 1 h before formaldehyde application. The results of the study are presented in table. 10.

From 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 with the control. The introduction 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.

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Table 10 Evans blue concentrations in rat pharyngeal tissue

Group Dye concentration in tissue, mcg/mg Intact 0.029+0.004 Control (formaldehyde - 30%) 0.141+0.015* Compound I (6 mg/kg) 0.051+0.01b*& Compound I (18 mg/kg) 0.022+0.003 & Dexamethasone 0.6 mg/kg 0,Obb+O,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 (p<0.05).

Study of the activity of Compound II in a model of bleomycin-induced lung injury

The model of bleomycin-induced lung injury was implemented according to the standard method [Am J Respir Cell Mol Biol. 2009 V. 41(1). P. 50-58]. Balb/c male mice received a single endotracheal injection of a bleomycin solution (4 units/kg in a volume of 50 µl). Compound II was administered to C57BL/6 mice intragastrically, twice - 1 hour before the administration of bleomycin and 12 hours after the administration of bleomycin. Bronchoalveolar lavage was taken 24 hours after the administration of bleomycin. In 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.

Table 11. The number of cellular elements in the bronchoalveolar lavage of mice in the model of bleomycin-induced acute lung injury (M±m, n=10)

Groups Dose of compound, mg/kg The number of cellular elements in 1 µl of 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 according to the standard method [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 inlipopolysaccharide-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 physiological saline. Animals of the false pathology group were injected with saline in the same volume. Bronchoalveolar lavage (BAL) was taken from the animals 48 hours after LPS administration. BAL was taken under anesthesia by flushing the lungs with 5 ml of saline warmed up 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 calculated using a Goryaev camera. Then bronchoalveolar lavage 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 cytogram.

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

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 reason to conclude that

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Compound IV has an anti-inflammatory effect in lung lesions.

Table 12. The number of cellular elements in the bronchoalveolar lavage of rats in the model

LPS-induced acute lung injury (M±m, n=10)

Groups Dose connected IA, mg/kg The 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 1.8 4485+815* & 785+181& 0+0 3699+653& 0+0

Notes * - difference from the intact group according to the Kruskal-Wallis test at p<0.05 # - difference from the group of false pathology according to the Kruskal-Wallis criterion at p<0.05 & - difference from the control group according to the Kruskal-Wallis criterion at p<0, 05

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

The febrile reaction model was implemented according to the standard method [J Neurosci Methods. 2005. V. 147. P. 29-35]. Wistar rats were injected subcutaneously with a 20% suspension of baker's yeast (12 ml/kg). Compounds I, II, IV, VI, VIII were administered once, intragastrically, 14 h after yeast injection. The rectal temperature was measured with an electrothermometer before pyrogen administration and at the highest point of thermal reaction development, 19 h 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 injection of _____________ yeast in rats, °С (M±w, n=30-90)___________

Group Compound dose, mg/kg Number of animals in the group Increase in rectal body temperature, °C Intact - 70 0.05+0.05 Control - 90 1.70+0.06* Compound II 0.18 40 1.18+0.08*& 0.6 thirty 0.78+0.07*& 1.8 70 1.09+0.06*& 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

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Table 14. Increase in rectal body temperature 19 hours after subcutaneous injection of yeast in ______ rats, °C (M±w, n=30-90)___________________

Group Compound dose, mg/kg Number of animals in Group Increase in rectal body temperature, °C Intact - 70 0.3 0+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__________________________________________________________________

Table 15. Increase in rectal body temperature 19 hours after subcutaneous injection of yeast in rats, °C (M±w, 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 1.70±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*& 18 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 injection of yeast in rats, °С (M±m, n=40-70)

Group Compound dose, 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* Connect- 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

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Table 17. Increase in rectal body temperature 19 hours after subcutaneous injection of yeast in rats, °С (M±m, n=40-70)

Group Compound dose, mg/kg Number of animals in the group Increase in rectal body temperature, °C Intact - 10 0.32+0.21 Control - 10 1.83+0.11* Compound VIII 1.8 10 1.16+0.12*&

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 Compounds I and Compounds IV on the model of a specific pain reaction by the method of chemical irritation of the peritoneum (acetic writhing test)

The model of a specific pain reaction by the method of chemical irritation of the peritoneum (acetic writhing test) was implemented according to the standard method. To perform the acetic 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 administration 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 reduced the number of writhing in mice caused by intraperitoneal administration of acetic acid (table. 18-19). The results obtained allow us to conclude that Compounds I, IV, VII, IX, X have a pronounced therapeutic effect in pain syndrome.

Table 18. The number of acetic writhing in the model of specific pain reaction by the method of chemical irritation of the peritoneum (acetic writhing test) (M±m, n=10-31)

Group Compound dose, 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 acetic writhing in the model of specific pain reaction by the method of chemical irritation of the peritoneum (acetic writhing test) (M±m, n=10-31)

Group Compound dose, 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* Compound X 0.3 10 17.2+3.4*

Notes:

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

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The study of the activity of Compounds I and Compounds II on the model of thermal pain stimulation hot plate

The model of thermal pain stimulation hot plate 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 to Balb/c mice intragastrically, once. A hot plate test was performed 1 hour after drug administration. For 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 the pain reaction in mice (licking paws, jumping) was recorded and the average latent time of the pain sensitivity threshold (TPB, sec) was calculated in each group.

The results of the study showed that intragastric administration of the studied compounds increased the threshold of pain sensitivity by 1.5 times in mice 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 syndrome.

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

Group Compound dose, 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.11. P. 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. The body weight of the animals was measured weekly at 612 weeks of life of the animals.

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

The results obtained indicate the therapeutic effect of Compound II in obesity. The therapeutic effect begins as early as 4 weeks of application of Compound II.

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

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

From the 10th to the 16th week of the diet, Compound II was administered intragastrically once a day to the animals of the experimental groups. 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 the weight gain of animals.

Pharmacological action begins to appear, starting from the 5th week of therapy (Table 22).

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

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Table 21. Increase in body weight of animals relative to the start of administration of Compound II in a model of spontaneous obesity in mice of the db/db line (M±m)

Groups Compound dose, mg/kg Age of animals at the time of reading, weeks ^{6 7 8} 1 ⁹ 10 ^eleven 12 Weeks of administration of Compound 1 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 10 9.6+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

Table 22. Body weight of rats in the metabolic syndrome model (M±w, n=12)

weeks Compound II (1.5 weeks of therapy Intact Control mg/kg) research 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 group of intact according to Student's t-test πρι 1 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 Compounds I and Compounds II in a mouse model of psoriasis

The induction of psoriasis in mice was carried out according to the standard method [European Journal of Pharmacology. 2015. V. 756. P. 43-51]. Balb/c female mice were treated with Aldara cream (5% imiquimod) on the inner side of the right ear once a day at 30 mg/mouse for 7 days (0-6 days). Vaseline was applied to intact animals. Compound I, Compound II and reference drug (cyclosporine) were administered to animals intragastrically, daily, 1 time per day for 6 days (0-5th day). Euthanasia was performed 24 hours (day 6) after the last application of Aldara cream. Daily 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 in dynamics.

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

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Table 23. Increase in the thickness of the affected ear on a certain day of the study to day 0 in a model of psoriasis in mice, % (M±w, n=10)

Group Compound dose, 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 - 4 0.3+ 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* 4 8, 1± 2.8*& 59.6± 4.5*& 7 0, 6± 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

The results of the study showed that intragastric administration of Compound I and Compound II in a mouse model of psoriasis reduced the increase in the thickness of the affected animal ear. 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 Compounds I and Compounds II in a model of atopic dermatitis in mice

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

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

Table 24. Difference in the mass of the experimental and control mouse ear and the response index to the model of atopic dermatitis in mice (M±w, n=12)

Group name Difference in mass of experimental and control ear (mg) Reaction index (%) Intact -0.001+0.002 -0.82+5.3 False immunization Pathology control Compound I (3 mg/kg) Compound II (3 mg/kg) 0.018+0.003* 0.029+0.002*# 0.007+0.003 #& 0.013+0.002* & 57.8+11.3* 83.3+6.1* 25.4+10.2*#& 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 by t-test Student 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 Compounds I and Compounds II in a model of atopic dermatitis in mice reduces the reaction 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.

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Study of the effect of Compound II on macrophage chemotaxis In vivo using a carrageenan sac model

To assess the effectiveness of the action of a drug on the activity of cells of the immune system at the preclinical stage of research, various models of an acute inflammatory process are usually used. To date, the most commonly used models in which the pathological process develops in a limited cavity. These models allow to closely simulate diseases in which the aberrant activity of immune system cells is localized in a certain cavity (peritonitis, cholangitis, arthritis) (J Pharmacol Toxicol Methods. 1994 Nov; 32(3):13947). The carrageenan sac model is often used at the preclinical stage of research to study pathological processes associated with aberrant activity of immune system cells localized in an isolated cavity. In this model, an isolated pouch is formed by subcutaneous injection of air into the intracapsular region of the mouse or rat back. Subcutaneous injection of air into the dorsal area leads to morphological changes in the cellular lining of the sac within a few days. The sac is composed 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 sac, 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 cells of the immune system inside the sac.

The study of the activity of Compound II was performed on Balb/c male mice according to the standard method (Curr Protoc Pharmacol. 2012 Mar;Chapter 5:Unit5.6). Experimental groups of animals were injected intragastrically with Compound II at a dose of 3 mg/kg immediately before the introduction of carrageenan and then every 10-12 hours. The last injection was 12 hours before slaughter.

48 hours after the injection of λ-carrageenan, separate groups of animals were euthanized by inhalation of CO ₂ . Immediately after euthanasia, 1 ml of saline containing 5.4 mM EDTA at room temperature was injected into the pouch with a sterile 25G syringe. After a light massage of the area of the air sac, 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 t 20–22°C). On smears, the number of macrophages was counted routinely under an Olympus bx51 microscope (at a magnification of 100). Cells were counted up to 100 pcs.

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 in the treatment of a wide range of diseases such as Crohn's disease, ulcerative colitis, peritonitis and arthritis.

Table 25. The number of cells of the immune system in the exudate from the carrageenan sac in the study of the activity of Compound II on the model of macrophage chemotaxis (carrageenan sac), x 109/l (M±m, n= 10)

Groups The 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 controls (p<0.05)

Study of the effect of Compound II on macrophage chemotaxis in vivo in a model of thioglycolate peritonitis

The study was performed on male mice of the balb/c line according to the standard method (J Leukoc Biol. 2009 Aug;86(2):361-70). Mice of the control group were intraperitoneally injected with 2 ml of 3% thioglycol medium, stored for 1 month. Intact animals were intraperitoneally injected with 2 ml of saline. Compounds II were intragastrically administered to experimental groups of animals 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 CO2 chamber, the peritoneal area was moistened with 70% alcohol, and the skin on the abdomen was carefully cut off.

- 24 042698 of the cavity, 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, using a Goryaev camera, the absolute number of cellular elements per 1 μl of washout (cytosis) was determined. 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 t 20–22°C).

On smears, the number of monocytes/macrophages was counted in a routine way under an Olympus bx51 microscope (at a magnification of 100). Cells were counted up to 100 pcs.

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

Table 26. The number of cells of the immune system in the exudate from the abdominal cavity in the study of the activity of Compound II on the model of macrophage chemotaxis (thioglycolate peritonitis), ____________________________x10 ⁹ /l (M±m, n=10) _______________________

Groups The 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.bb±0.44* Compound II (1 mg/kg) 1.97±0.36*& 0.65±0.12& * - differences are statistically significant compared to intact (p<0 _L 05) & - differences are statistically significant compared to controls (p<0.05)

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

The model of adjuvant arthritis was implemented on outbred male rats according to the standard method [Chem Pharm Bull (Tokyo). 2018. V.66(4). P.410-415]. On days 0 and 5, Freund's Adjuvant, Complete (Pierce) was subplantarly injected 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) were measured on days 0, 14, 16, 18, 21, 25, 28 and 30 using a plethysmometer (Ugo Basel). The change in the volume of the affected paw was judged on the primary (inflammatory) reaction, on the change in the volume of the contralateral paw was judged on the secondary (immune) reaction. 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 increase in volume 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 a 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 the model of adjuvant arthritis induced by the introduction of complete Freund's adjuvant, % (M±m, n=10)

Group Compound dose, mg/kg Increase in the volume of the affected paw, % to the initial level 14 day 16 day 18 day 21 days 25 day 28 day 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 *& 86.9±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 according to Student's t-test at p<0.05.

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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±m, n=10)

Group Compound dose, mg/kg Increase in the volume of the contralateral paw, % to the initial level 14 day 16 day 18 day 21 days 25 day 28 day 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 2.76+0.16 2.98±0.3 Control 5.48+0.58* 7.08+1.2 4* 21.69+1.28* 25.29+1.6 9* 2 0.16+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 according to Student's t-test at p<0.05.

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

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

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

Table 29. Effect of Compound II on the inhibition of the increase in rat paw edema (relative to control) in a model of carrageenan-induced edema

Group pathology induction Introduction of drugs Timing of the assessment Inhibition of edema growth, % Intact Intraplantar introduction in a volume of 0.1 ml into the right paw of 1.5% solution of carrageenan Intragastrically, once, 1 hour before the administration of carrageenan 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 these results, it can be seen 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 immune system cells.

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, streptozotocin was intraperitoneally injected 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 was started. The substance was administered daily intragastrically once a day at a dose of 50 mg/kg.

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

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Claims (2)

Table 30 Urinalysis after 6 weeks of intragastric administration of Compound II to rats in a model of diabetic nephropathy induced by high-fat diet and administration of streptozotocin (15 weeks of high-fat diet) Groups Dose and mode of administration of streptozotocin Number of animals Diuresis, ml/ 18 o'clock Daily protein, mcg/18h ACR (protein (mcg/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 -To 952.8±186.4* Compound II (5 mg/kg) 30 mg/kg, twice 12 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 controls (p<0.05) While the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for the purpose of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be clear that various modifications are possible without departing from the essence of the present invention. CLAIM 1. The use of a compound of formula IV or a pharmaceutically acceptable salt, hydrate or solvate thereof for the prevention and/or treatment of rhinitis. 2. A method of treating rhinitis comprising administering to a subject in need of treatment a therapeutically effective amount of a compound of formula IV or a pharmaceutically acceptable salt, hydrate or solvate thereof. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2