RU2791703C2 - New glutamine cyclase inhibitor and its application - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: suggested is the use of N,N'-bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide or its pharmaceutically acceptable salt, hydrate or solvate for the treatment of a disorder, Crohn's disease, ulcerative colitis, peritonitis, nephropathy, or kidney disease, which is a side effect of the use of drugs. Also provided is a process for treating the disease, including administration of the said compound in a therapeutically effective amount.

EFFECT: technical result is the use of a compound that is effective in modulating glutaminyl cyclase in the treatment of diseases associated with aberrant activity of immune system cells.

4 cl, 6 tbl, 9 ex

Description

Technical field

The present invention relates to organic chemistry, pharmacology and medicine and relates to the treatment of diseases associated with aberrant activity of cells of the immune system, preferably the treatment of diseases of the abdominal organs, in particular the treatment of peritonitis, nephropathies, Crohn's disease and ulcerative colitis through the use of a compound effective in modulation of the glutaminyl cyclase enzyme, which is involved, in particular, in the processes of post-translational modification of chemokines and chemotaxis of immune system cells.

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. Excessive 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.

Chemokines of the CCL family (CCL2, CCL7, CCL8, CCL13), which are ligands of the CCR2 receptor, are the most potent factors of monocyte and macrophage chemotaxis 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). 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 metabolic syndrome, chronic pain, fibrosis, and some forms of cancer.

Suppression of the aberrant activity of immune system cells by inhibiting CCL-mediated chemotaxis may be highly demanded for the treatment of a range of diseases, such as Crohn's disease, ulcerative colitis, peritonitis, and nephropathies. 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 (ROS, TNFα, IL-1, IL-6) 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 tissue. In in vivo models of diabetic nephropathy, blockade of the CCL2/CCR2 interaction with small molecule antagonists has shown significant efficacy (Nephrol Dial Transplant. 2013 Jul;28(7):1700-10).

Members of the CCL family (CCL2, CCL7, CCL8, CCL13), as well as a number of other hormones and secreted proteins, contain a pyroglutamic acid (pE) residue 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 glutamine residue is a post-translational modification of the protein, and is catalyzed by the enzymes glutaminyl cyclase (QPCT or QC) (J Biol Chem. 2003 Dec 12;278(50):49773-9) and iso-glutaminyl cyclase (QPCTL or isoQC) ( J Mol Biol 2008 Jun 20;379(5):966-80). Both isoforms of the enzyme are proteins similar in structure and substrate specificity with different localizations: glutaminyl cyclase is a secreted protein, while isoglutaminyl cyclase is located inside the cell and is fixed in the Golgi apparatus (J Mol Biol. 2008 Jun 20;379(5):966- 80).

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). Thus, pyroglutamination of chemokines of the CCL family is a necessary step in CCL-mediated monocyte chemotaxis, and a strategy aimed at inhibiting glutaminyl cyclase is a strategy for modulating the aberrant activity of immune system cells.

Glutaminyl cyclase inhibitors are currently known, including sulfolipids (WO 2017/046256), flavanoid 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 to the compound of the present invention are given in 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 on one of the nitrogen atoms. 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 not required to suppress 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 aberrant activity of immune system cells, therefore, there is still a need to create and introduce new effective drugs based on glutaminyl cyclase inhibitors into the clinic.

This invention relates to the use of a compound having efficacy in modulating glutaminyl cyclase in the treatment of diseases associated with aberrant activity of cells of the immune system, preferably in the treatment of diseases of the abdominal organs, in particular the treatment of peritonitis, kidney disease (including those that are complications of other diseases (diabetic nephropathy) or a side effect of drugs), Crohn's disease, ulcerative colitis, as well as other diseases of the abdominal organs associated with aberrant activity of immune system cells.

Disclosure of invention

The object of the present invention is to develop a novel glutaminyl cyclase modulator, more specifically an inhibitor of intracellular and/or secreted isoforms of glutaminyl cyclase, and effective for the treatment of diseases associated with aberrant activity of immune system cells, in particular diseases of the abdominal organs, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy, as well as other diseases associated with aberrant activity of immune system cells.

The technical result of this invention is the development and production of an effective glutaminyl cyclase modulator, in particular, a compound that has a high inhibitory activity against intracellular and/or secreted glutaminyl cyclase isoforms and good pharmacokinetic characteristics (in particular, a significant excess of inhibitor exposure in target organs (kidneys, spleen) compared to blood plasma), allowing the use of this compound for the treatment of diseases associated with aberrant activity of cells of the immune system, in particular diseases of the abdominal organs, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy (in particular diabetic nephropathy), as well as other diseases associated with aberrant activity of cells of the immune system, in particular with aberrant chemotaxis of cells of the immune system.

The specified technical result is achieved by using the compound N,N'- bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide of the formula

.

.

or a pharmaceutically acceptable salt, hydrate, solvate thereof as a modulator, in particular an inhibitor of intracellular and/or secreted isoforms of glutaminyl cyclase.

The specified technical result is also achieved by using the compound N,N'- bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide or its salt, hydrate, solvate for the manufacture of a drug for the prevention and/or treatment of a disorder mediated by activity glutaminyl cyclase.

The specified technical result is also achieved by using the compound N,N'- bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide or its salt, hydrate, solvate for the manufacture of a medicinal product for the prevention and/or treatment of a disorder associated with aberrant activity of cells of the immune system, in particular with aberrant chemotaxis of cells of the immune system.

Within the scope of the invention is a drug comprising a compound of the formula

.

.

or a pharmaceutically acceptable salt thereof.

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 drug according to the invention to said organism. Such a disorder associated with glutaminyl cyclase activity is a disease associated with aberrant activity of immune system cells, in particular, aberrant chemotaxis of immune system cells, especially a disease of the abdominal organs. In some non-limiting embodiments, the abdominal disease is Crohn's disease, ulcerative colitis, peritonitis, nephropathy (particularly diabetic nephropathy). In particular cases of embodiment of the invention, the organism is a human or an animal.

The compound N,N'-bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide is described in the patent application RU 2013/116822.

Detailed disclosure of the invention

The preparation of Compound 1 , the medical application of which is the subject of the present invention, 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 Compound 1 in models of various diseases, it was surprisingly found that the use of Compound 1 significantly reduces the influx of cells of the immune system. Thus, it was shown that Compound 1 affects the chemotaxis of cells of the immune system. Reducing the influx of immune system cells can be used in the treatment of a number of diseases associated with aberrant activity of immune system cells, in particular diseases of the abdominal cavity, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy (particularly diabetic nephropathy). Since the effect on chemotaxis cannot be predicted or explained by the ability of the compound to complex or chelate metal ions, an attempt was made to search for possible therapeutic targets for Compound 1 .

The search for possible therapeutic targets using computational chemistry, molecular modeling and in vitro tests on an enzyme preparation revealed that the observed therapeutic effect of Compound 1 is associated with the ability of this compound to inhibit the activity of glutaminyl cyclase.

In the course of further studies of the pharmacokinetics of Compound 1 , it turned out that as a result of oral administration of the substance, the highest concentrations are observed in the liver, kidneys and other organs of the abdominal cavity.

Thus, Compound 1 is a novel glutaminyl cyclase inhibitor that affects the chemotaxis of cells of the immune system and can be used to treat diseases associated with aberrant activity of cells of the immune system, preferably diseases of the abdominal cavity, such as Crohn's disease, ulcerative colitis, peritonitis, nephropathy, in particular, diabetic nephropathy, as well as other diseases of the abdominal organs associated with aberrant activity of immune system cells.

Terms and Definitions

The term "Compound 1" refers to N,N'- bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide represented by the structural formula:

.

.

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

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

The term " pharmaceutically acceptable salts " or "salts" includes salts of Compound 1 which are formed by reaction with relatively non-toxic acids. Examples of pharmaceutically acceptable 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 other 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-hydroxy-ethanesulfonate, 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, p-toluenesulfonate (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 activity" of cells of the immune system, as used herein, means activity that is significantly different from the basic level of activity of immune system cells 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 "AUC" (area under the curve) means a pharmacokinetic parameter that characterizes the total concentration of the drug in the blood plasma during the entire observation period. It is mathematically defined as an integral from 0 to ∞ of the function of the concentration of the drug (pharmacokinetic curve) in blood plasma from time and is equal to the area of the figure limited by the pharmacokinetic curve and coordinate axes.

The term " RPMI medium " (eng. Roswell Park Memorial Institute medium) means a medium for cell and tissue cultures. 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 aminoacyltransferase enzyme involved in the conversion of the N-terminal group of glutamine to pyroglutamine in various peptide substrates. N-terminal pyroglutamate formation protects biologically active peptides, hormones, and chemokines (eg, thyrotropin-releasing hormone, β-chemokine ligand-2) from degradation by exopeptidases and, in some cases, may increase the affinity of ligands for their receptors.

The term " secreted glutaminyl cyclase" means one of the isoforms of the enzyme glutaminyl cyclase containing a secretory N-terminal domain and predominantly secreted by human cells. Secreted glutaminyl cyclase is predominantly expressed in cells of the nervous and immune systems.

The term " intracellular glutaminyl cyclase" means one of the isoforms of the enzyme glutaminyl cyclase containing an anchor N-terminal domain and predominantly associated with the Golgi apparatus. Intracellular glutaminyl cyclase is ubiquitous in mammalian and human cells.

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

The term " prophylaxis ", " prevention " encompasses the elimination of risk factors as well as the prophylactic treatment of subclinical stages of the disease in mammals, preferably in humans, aimed at reducing 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, cause 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.

Compound 1 is 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, preferably the treatment of diseases of the abdominal organs, such as Crohn's disease, ulcerative colitis, peritonitis, nephropathy, in particular diabetic nephropathy, having both systemic and local character, including those caused by primary pathological changes in these tissues, or associated with various diseases or long-term use of certain drugs. In some particular embodiments, the compounds of the invention may be used to treat other diseases of the abdominal organs associated with aberrant activity of cells of the immune system.

Method for Therapeutic Use of the Compounds

The subject matter of the present invention also includes a method of treatment comprising administering to a subject in need of appropriate treatment a therapeutically effective amount of Compound 1. By a therapeutically effective amount is meant that amount of a compound administered or delivered to a patient such that the patient is most likely to have the desired response to treatment ( prevention). 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 compound of the invention can be administered to the patient in any amount (preferably, the daily dose of the active substance is up to 0.5 g per patient per day, most preferably, the daily dose is 5-50 mg/day) and by any route of administration (preferably, oral route of administration), effective for the treatment or prevention of the disease.

After compound 1 has been mixed with a specific suitable pharmaceutically acceptable carrier at the desired dosage, the compositions of the invention may be administered to humans or other animals orally, parenterally, topically, and by other conventional means known to those skilled in the art.

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, the compound may be administered to the patient daily for a period of days (eg, 2-10 days) followed by a non-dose period (eg, 1-30 days).

When Compound 1 is 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 Compound 1 or a pharmaceutically acceptable salt, hydrate or solvate (or prodrug or other pharmaceutically acceptable derivative) thereof and one or more pharmaceutically acceptable carriers, adjuvants, diluents and/or excipients, such as may be co-administered to a patient with a compound for use in this 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 of this invention contain compound 1 or a pharmaceutically acceptable salt thereof, 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 the particular dosage form. Materials that can serve as pharmaceutically acceptable carriers include, but are not limited to, mono- and oligosaccharides, 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 the invention is also medicinal products, by which are meant 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 inhalation spray, or intravascular route. , intranasally, subcutaneously, intramuscularly, as well as by infusion, in recommended dosages.

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

When preparing a composition, for example in the form of a tablet, Compound 1 or a pharmaceutically acceptable salt thereof is mixed with one or more pharmaceutical excipients such as gelatin, starch, lactose, magnesium stearate, talc, silica, gum arabic, mannitol, microcrystalline cellulose, hypromellose or the like. connections.

Tablets may be coated with sucrose, cellulose or a derivative thereof, or other suitable coating agents. 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 prepared by mixing Compound 1 or a pharmaceutically acceptable salt thereof with the other ingredients 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

For the prevention and/or treatment of diseases in humans or animals, the following compositions are used, in particular (by "Substance" is meant Compound 1 or a pharmaceutically acceptable salt thereof):

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 223.75

Croscarmellose sodium 6.0

Cornstarch 15

Polyvinylpyrolidon (5% m/v paste) 2.25

Magnesium stearate 3.0

Tablet V mg/tablet

Substance 200

Lactose 182.75

Croscarmellose sodium 12.0

Corn starch (5% w/v paste) 2.25

Magnesium stearate 3.0

Capsule mg/capsule

Substance 10

Lactose 488.5

Magnesia 1.5

Formulation for injection I (50 mg/ml)

Substance 5.0% m/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 1 ml

Substance 40 mg

Ethanol 300 µl

Water 300 µl

1-dodecylasecycloheptanone 50 µl

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 Compound 1 in Combination Therapy

AlthoughCompound 1By of this invention can be administered as a single active agent, it can also be used in combination with one or more other agents, in particular, the other agent can be an antibiotic, non-steroidal anti-inflammatory drug (NSAID) or other anti-inflammatory agent, antihypertensive agent, α- blocker, cytostatic drug, 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 active 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 agents. Co-administration includes, in particular, co-delivery, for example, in a single tablet, capsule, injection or other form, having a fixed ratio of active agents, as well as simultaneous delivery in several, separate dosage forms for each compound, respectively.

Thus, the administration of Compound 1 or a pharmaceutically acceptable salt thereof of the present invention may be carried out in combination with additional treatments known to those skilled in the art of prevention and treatment of related diseases, including the use of antibacterial, cytostatic and cytotoxic drugs, drugs to suppress symptoms or side effects of one from medicines.

If the dosage form is a fixed dose, such a combination uses compound 1 or its pharmaceutically acceptable salt in an acceptable dosage range.Compound 1By of the present invention, or a pharmaceutically acceptable salt thereof, may also be administered to a patient sequentially with other agents, when co-administration of combinations of these agents is not possible. The invention is not limited to the sequence of administration; the compound of the present invention may be co-administered to the patient before or after administration of another drug.

Examples

1) Preparation of the compound of the invention

The preparation of Compound 1 is described in the application for invention RU 2013/116822. The same application describes the ability of Compound 1 to complex or chelate metal ions.

2) Characterization of the biological activity of the compound of the invention

The biological activity of Compound 1 has been studied in various in vitro and in vivo experiments. In particular, when studying the activity of Compound 1 in various in vitro and in vivo models, the inhibitory effect of Compound 1 on the chemotaxis of monocytes, macrophages and other cells of the immune system was shown. This biological action of Compound 1 cannot be predicted or explained on the basis of prior knowledge of the ability of Compound 1 to chelate metal ions.

Studies of the biological activity of Compound 1 in vitro showed that Compound 1 is an inhibitor of the enzyme glutaminyl cyclase and, thus, the effect of Compound 1 on the chemotaxis of cells of the immune system may be mediated by inhibition of glutaminyl cyclase activity.

2.1) Study of the effect of Compound 1 on the enzymatic activity of human intracellular glutaminyl cyclase in vitro .

In the course of studies of the effect of Compound 1 on the enzymatic activity of intracellular glutaminyl cyclase in vitro, a direct inhibitory effect of Compound 1 on recombinant human intracellular glutaminyl cyclase was discovered for the first time.

Glutaminyl cyclase activity at various concentrations of Compound 1 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 reaction mixture with a volume of 100 μl contained 50 μmol of the fluorogenic substrate; ~0.2 units of human pyroglutaminyl aminopeptidase (1 unit is defined as the amount that hydrolyzes 1 µmol 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 Compound 1 for 5 minutes. 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 a non-linear regression curve "inhibitor concentration"-"enzymatic activity". The known inhibitor of glutaminyl cyclases 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 1 inhibits the activity of intracellular glutaminyl cyclase with IC ₅₀ =4.7 μm.

2.2) Study of the effect of Compound 1 on the enzymatic activity of secreted human glutaminyl cyclase in vitro .

To the wells of a 96-well PCR plate (Bio-Rad) was added 25 µl of the solutionConnections 1. Each well containingCompound 1, 25 µl of a 320 µM solution of (2S)-2-amino-N-(4-methyl-2-oxo-chromen-7-yl)pentanediamide (Q-AMC) in 25 mM HEPES, pH 7.0 was placed and mixed. 50 μl of secreted glutaminyl cyclase solution was placed in each well (except for hydrolysis blank controls). (QPCT) at a concentration of 0.2 µg/ml in buffer (25 mM HEPES, pH 7.0) or similar buffer (hydrolysis blank control). The tablet was incubated at room temperature for 20 minutes, then placed in a thermostat, heated at 100°C for 5 minutes and then cooled in ice for 3 minutes. 100 μl of a 1 μg/ml solution of pyroglutaminyl peptylase (PGPEP) in buffer (0.1 mM trisaminomethane, 5 mM dithiothreitol, pH 9.0) was added to each well and incubated at room temperature for 10 minutes. From each well, 2 times 90 μl of the solution were taken, placed in 3 wells of a Greiner Bio fluorometry plate and fluorescence was determined (excitation - 355 nm, emission - 460 nm). The resulting inhibition curve (dependence of I on the concentration of the substance) was approximated by a dose-response curve (Dose-response curve) with lower asymptote = 0 and upper asymptote = 1 using Origin Pro 8.0 software, which made it possible to obtain the IC value₅₀.

As a result of the experiment, it was found that Compound 1 inhibits the activity of secreted glutaminyl cyclase with IC ₅₀ =2.9 μm.

2.3) Study of the effect of Compound 1 on monocyte migration in vitro

The effect of Compound 1 on monocyte migration in vitro was studied using U937 cells, concentration (2-3)×10 ⁶ cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Biochem J. 2012 Mar 1;442( 2):403-12). Approximately 1×10 ⁷ U937 cells were incubated with various concentrations of Compound 1 (total 7 concentrations) 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 hour at 37°C. Thereafter, an aliquot of stained cells was placed in the top well compartment of a BD FalconTM HTS FluoroBlok plate, the wells of which are 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 hours 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 comparison.

Data on the effect of Compound 1 on monocyte migration in vitro are shown in Table 1.

Table 1 - Influence of Compound 1 on the migration of a continuous culture of U937 monocytes in vitro

Concentration of Compound 1, μM 0 0.0003 0.003 0.03 0.3 3.0 30.0 Monocyte migration activity, in % of untreated control 100% 92% 65% 59% 56% 49% 32%

As a result of the experiment, it was found that Compound 1 inhibits the migration of monocytes with IC ₅₀ =2.9 μm.

2.4) Study of the effect of Compound 1 on macrophage chemotaxis in vivo on the carrageenan sac model

To assess the effectiveness of the action of a drug on the activity of immune system cells at the preclinical stage of studies, as a rule, various models of an acute inflammatory process are used. To date, the most commonly used models in which the pathological process develops in a limited cavity. These models make it possible to closely simulate a disease in which aberrant activity of immune system cells is localized in a certain cavity (peritonitis, cholangitis, arthritis) (J Pharmacol Toxicol Methods. 1994 Nov; 32(3): 139-47). 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 back of a mouse or rat. 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; TNFα, IL-8, prostaglandins and leukotrienes, NO) and also to migration of cells of the immune system into the sac.

The study of the activity of Compound 1 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 1 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 solution containing 5.4 mmol of EDTA (ethylenediaminetetraacetic acid), 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, 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°C for 10 minutes. Smears were prepared from the cell sediment, which were further fixed in methanol (5 min) and stained according to the Romanovsky–Giemsa method (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.

Table 2 shows the number of cells of the immune system in the exudate from the carrageenan sac when studying the activity of Compound 1 in a model of macrophage chemotaxis.

Table 2 - Effect of Compound 1 on the migration of leukocytes and macrophages into the carrageenan sac (M±m, n=10)

Groups The number of cellular elements in 1 µl of exudate, ×10 ⁹ /l Leukocytes Macrophages Intact 0.9±0.2 0.6±0.1 Control 9.9±1.5* 7.7±1.5* Compound 1
(3 mg/kg) 1.0±0.1 & 0.5±0.1 & 14. * - differences are statistically significant compared to intact (p<0.05) & - differences are statistically significant compared to controls (p<0.05)

The conducted studies showed that the use of Compound 1 reduced the influx of leukocytes and macrophages by 10 times (to the level of intact control). Thus, Compound 1 is potentially useful in the treatment of a wide range of diseases such as Crohn's disease, ulcerative colitis, peritonitis and arthritis.

2.5) Study of the effect of Compound 1 on macrophage chemotaxis in vivo on the 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. Compound 1 was administered intragastrically 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 h, the animals were euthanized in a _CO2 chamber, the peritoneal area was moistened with 70% alcohol, the skin on the abdominal cavity was carefully cut off, and 5 ml of cold phosphate-buffered saline containing 0.1% EDTA was injected intraperitoneally with a syringe. After a light massage of the abdominal cavity, the exudate was collected with a syringe into test tubes, and the volume of the collected exudate was determined.

The number of cells of the immune system in the exudate from the abdominal cavity in the study of the activity of Compound 1 on the model of macrophage chemotaxis (thioglycolate peritonitis) is shown in table 3.

Table 3 - The effect of Compound 1 on the migration of leukocytes and monocytes into the peritoneal cavity of mice in the model of thioglycolate peritonitis M±m, n=10)

Groups The number of cellular elements in 1 µl of exudate, ×10 ⁹ /l Leukocytes Monocytes Intact 0.71±0.09 0.61±0.07 Control 3.99±0.42* 3.66±0.44* Compound 1 (1 mg/kg) 1.97±0.36*& 0.65±0.12& * - differences are statistically significant compared to intact (p<0.05) & - differences are statistically significant compared to controls (p<0.05)

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°C for 10 minutes. Smears were prepared from the cell sediment, which were subsequently fixed in methanol (5 min) and stained according to the Romanovsky–Giemsa method (40 min at t 20–22°C). On smears, the number of monocytes/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 useConnections 1 reduces thioglycolate-induced influx of macrophages into the peritoneal cavity of mice and has an anti-inflammatory effect. Thus,Compound 1 potentially useful in the treatment of peritonitis, Crohn's disease and ulcerative colitis.

2.6) Study of the activity of Compound 1 in the carrageenan edema model

To evaluate the effect of Compound 1 on the functional status of the immune system, a carrageenan-induced rat paw edema model 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 1 and the control substance (diclofenac) was evaluated by the degree of reduction in paw edema in comparison with the intact left paw. Paw volume was assessed before the administration of Compound 1 and 2 hours after the administration of carrageenan.

The effect of Compound 1 on the increase in rat paw edema (relative to control) in the carrageenan-induced edema model is shown in Table 4.

Table 4 - Effect of Compound 1 on the increase in rat paw edema (relative to control) in the carrageenan-induced rat paw edema model

Group pathology induction Introduction of drugs Timing of the evaluation Inhibition of edema growth, % Intact 2 hours after administration of carrageenan 0 Control 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 0 Compound 1 (1.8 mg/kg) 34 Compound 1 (0.18 mg/kg) 34 Diclofenac sodium (8 mg/kg) 42

From the above results, it can be seen that Compound 1 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.

2.7) Study of the activity of Compound 1 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 1 was started. The substance was administered daily intragastrically once a day at a dose of 50 mg/kg.

Table 5 shows biochemical analysis of urine after 6 weeks of intragastric administration of Compound 1 to rats in a model of diabetic nephropathy induced by high-fat diet and administration of streptozotocin (15 weeks of high-fat diet)

Table 5 - Effect of intragastric administration of Compound 1 for 6 weeks on the biochemical analysis of rat urine in a model of diabetic nephropathy induced by a high-fat diet and administration of streptozotocin

Groups Dose and mode of administration of streptozotocin Number of animals Diuresis, ml/18 hours 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* 952.8±186.4* Compound 1 (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)

Thus, the studies performed have shown that Compound 1 is effective in inhibiting the enzyme glutaminyl cyclase (IC ₅₀ =4.7 μM for intracellular glutaminyl cyclase and IC ₅₀ =2.9 μM for secreted glutaminyl cyclase) and inhibits macrophage chemotaxis in vitro ( IC ₅₀ =2 .9 µM ) . In the carrageenan sac and thioglycolate peritonitis model , Compound 1 inhibits macrophage chemotaxis in vivo. In the model of diabetic nephropathy, the action of Compound 1 leads to a decrease in the chemotaxis of monocytes and macrophages in the kidney tissue, stopping the local pathological process in the glomeruli of the kidneys and normalizing the functional characteristics of the kidneys.

2.8) Study of the pharmacokinetics of Compound 1 after a single oral administration

To analyze the applicability of Compound 1 as a drug, the pharmacokinetic parameters of this compound were investigated. Since the compound has a pronounced effect on the chemotaxis of cells of the immune system, in order to achieve the maximum therapeutic effect, it seems reasonable to achieve high concentrations of Compound 1 in the target organ. At the same time, from the point of view of minimizing possible negative effects associated with systemic suppression of chemotaxis of cells of the immune system, it seems reasonable to try to create the lowest possible concentrations of Compound 1 in the systemic circulation. Thus, the organs providing the "accumulation" of Compound 1 will be the most favorable target organs. As a measure of "accumulation" of Compound 1 , the characteristic "tissue bioavailability" was chosen, which is determined by the ratio of the area under the pharmacokinetic curve, which is calculated from the moment of administration to a specific time point (AUC0-t) in the tissue to the corresponding value of AUC0-t in blood plasma.

The pharmacokinetics of Compound 1 was studied after a single intragastric administration of Compound 1 to rats at a dose of 18 mg/kg. Determination of the concentration of Compound 1 in pharmacokinetic studies was carried out by high performance liquid chromatography with mass spectrometric detection (HPLC-MS/MS).

When studying the pharmacokinetics of Compound 1 , it turned out that the highest concentrations of Compound 1 are observed in the abdominal organs - spleen, liver, kidneys (see table 5). Thus, the use of Compound 1 is potentially suitable for the treatment of diseases of the abdominal cavity.

Table 6 assesses the tissue bioavailability of Compound 1 following a single intragastric dose of 18 mg/kg to rats.

Table 6 - Tissue bioavailability of Compound 1 at a single intragastric dose of 18 mg/kg in rats.

Plasma Liver Spleen Heart thymus muscles kidneys Lungs Tissue bioaccess
ness 1 188 23 9 12 2 38 13

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.

Claims (8)

1. Application of the compound of the formula

or a pharmaceutically acceptable salt, hydrate or solvate thereof for the treatment of a disorder of choice in Crohn's disease, ulcerative colitis, peritonitis, nephropathy or kidney disease which is a side effect of drugs. 2. Use according to claim 1, wherein the nephropathy is diabetic nephropathy. 3. A method of treating a disease, comprising administering a compound of formula

or a pharmaceutically acceptable salt, hydrate or solvate thereof in a therapeutically effective amount, wherein the disease is Crohn's disease, ulcerative colitis, peritonitis, nephropathy or kidney disease which is a side effect of drugs. 4. The method of claim 3 wherein the nephropathy is diabetic nephropathy.