RU2665633C1 - New inhibitor of glutaminyl cyclase and its use - Google Patents

Abstract

FIELD: medicine.SUBSTANCE: invention relates to medicine, namely to pharmaceutical composition for modulating the activity of glutaminyl cyclase, and also for treatment of a disorder associated with glutaminyl cyclase activity containing an effective amount of a compound of N,N'-bis[2-(1H-imidazol-4-yl)ethyl]isophthalamide..EFFECT: implementation of invention will yield effective glutaminyl cyclase modulator having a high activity with respect to intracellular and/or secreted isoforms of glutaminyl cyclase and good pharmacokinetic characteristics.11 cl, 10 ex, 6 tbl

Description

Technical field

This invention relates to the chemistry of organic compounds, pharmacology and medicine, and relates to the treatment of diseases associated with the aberrant activity of cells of the immune system, preferably the treatment of diseases of the abdominal organs, in particular the treatment of peritonitis, nephropathy, Crohn’s disease and ulcerative colitis through the use of compounds with efficacy in modulation of the enzyme glutaminyl cyclase involved, in particular, in the processes of post-translational modification of chemokines and chemotaxis of immune cells Themes.

State of the art

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

CCL family chemokines (CCL2, CCL7, CCL8, CCL13), which are CCR2 receptor ligands, are the most potent chemotactic factors of monocytes and macrophages in mammals (Biochem J. 2012 Mar 1; 442 (2): 403-12). Chemokines of the CCL family constitute an important class of cytokines necessary for the activation of neutrophils and monocytes and the involvement of these cells in the focus of inflammation. However, high concentrations of chemokines typically cause an overflow of cells in the immune system. Aberrant activity of cells of the immune system 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, the involvement of 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 the aberrant activity of CCR2 +, CD14 + and CD16lo monocytes (rheumatoid arthritis, multiple sclerosis) is known. In addition, chemokines of the CCL family (in particular CCL2) are involved in the pathogenesis of metabolic syndrome, chronic pain, fibrosis and some forms of cancer.

Suppression of the aberrant activity of cells of the immune system, due to the inhibition of CCL-mediated chemotaxis, can be extremely useful for the treatment of a wide range of diseases, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy. For example, in the case of diabetic nephropathy, exposure to high glucose concentrations leads to increased secretion of CCL2 by the kidney tubular cells, which in turn causes monocyte migration (Kidney Int. 2006 Jan; 69 (1): 73-80). An increase in the concentration of monocytes in kidney tissues 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 surrounding kidney cells (Mediators Inflamm. 2012; 2012: 146154). Damage to kidney tissue leads to the development and maintenance of aberrant activity of cells of the immune system and further destruction of kidney tissue. In in vivo models of diabetic nephropathy, the blockade of CCL2 / CCR2 interaction by low molecular weight 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 the remainder of pyroglutamic acid (pE), whose role is to protect against degradation by aminopeptidases (Chem Immunol. 1999; 72: 42-56; Biochemistry. 1999 Oct 5; 38 (40): 13013-25). The 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) Biol. 2008 Jun 20; 379 (5): 966-80). Both isoforms of the enzyme are proteins with similar localization in structure and substrate specificity with different localization: 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 chemokines CCL2, CCL7, CCL8 and CCL13 (Biochem. J. (2012) 442, 403-412). Thus, the pyroglutamination of CCL family chemokines is a necessary step in CCL-mediated chemocytosis of monocytes, and the strategy aimed at inhibiting glutaminyl cyclase is a strategy for modulating the aberrant activity of immune 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, compounds of the invention, are given in Probiodrug Aktiengesellschaft (J Biol Chem. 2003 Dec 12; 278 (50): 49773-9). In this paper, glutaminyl cyclase inhibitors based on imidazole derivatives are described. However, in the structures of 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 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 cells of the immune system, so there remains a need to create and introduce into the clinic new effective drugs based on glutaminyl cyclase inhibitors.

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

Disclosure of invention

An object of the present invention is to provide a new modulator of glutaminyl cyclase, more specifically an inhibitor of intracellular and / or secreted glutaminyl cyclase isoforms, and is effective for treating diseases associated with aberrant activity of immune 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 preparation of an effective glutaminyl cyclase modulator, in particular, a compound having high inhibitory activity against intracellular and / or secreted glutaminyl cyclase isoforms and good pharmacokinetic characteristics (in particular, a significant excess of the inhibitor exposure in target organs (kidney, spleen ) compared with 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 the cells of the immune system, in particular with aberrant cell chemotaxis 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 glutaminyl cyclase isoforms.

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

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 medicament for the prevention and / or treatment of a disorder associated with aberrant activity of cells of the immune system, in particular with aberrant chemotaxis of cells of the immune system.

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

The invention also includes a method of preventing and / or treating a disorder associated with glutaminyl cyclase activity in the body, comprising administering to the specified organism a medicament of the invention. Such a disorder associated with glutaminyl cyclase activity is a disease associated with aberrant activity of cells of the immune system, in particular with aberrant chemotaxis of cells of the immune system, in particular a disease of the abdominal organs. In some non-limiting embodiments of the invention, the disease of the abdominal organs is Crohn’s disease, ulcerative colitis, peritonitis, nephropathy (in particular diabetic nephropathy). In particular cases of the embodiment of the invention, the organism is a human or animal.

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

Detailed Disclosure of Invention

The preparation of Compound 1 , the medical use of which is the subject of the present invention, is described in the patent application RU 2013/116822. This patent application describes derivatives of dicarboxylic acid bisamides having the ability to complex or chelate metal ions, as well as their use as a means for the prevention and / or treatment of viral hepatitis, HIV infection, cancer, neurodegenerative, cardiovascular, inflammatory diseases, diabetes, gerontological diseases, diseases caused by toxins of microorganisms, as well as alcoholism, alcoholic cirrhosis of the liver, anemia, late porphyria, salt poisoning and transition metals.

In studies of the specific pharmacological activity of Compound 1 in models of various diseases, it was unexpectedly found that the use of Compound 1 significantly reduces the flow 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 cells of the immune system can be used in the treatment of a number of diseases associated with aberrant activity of cells of the immune system, in particular diseases of the abdominal cavity, such as Crohn's disease, ulcerative colitis, peritonitis and nephropathy (in particular, 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 methods of computational chemistry, molecular modeling and in vitro tests on the enzyme preparation revealed that the observed therapeutic effect of Compound 1 is associated with the ability of this compound to inhibit glutaminyl cyclase activity.

In 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 new 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 organs such as Crohn’s disease, ulcerative colitis, peritonitis, nephropathy, in particular, diabetic nephropathy, as well as other diseases of the abdominal organs associated with the aberrant activity of the cells of the immune system.

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 the test compound at which half-maximum inhibition of the enzyme is achieved.

The term " pharmaceutically acceptable salts " or "salts" includes salts of compound 1, which are obtained by interaction with relatively non-toxic acids. Examples of pharmaceutically acceptable salts are those formed with inorganic acids, such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or organic acids, such as acetic, oxalic, maleic, tartaric, succinic, citric or malonic acids, or obtained by other methods used in this field, for example, by ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, heptofluorosulfonate, glucose gluconate, formate glucose phosphate heptane, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate (mesylate), 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, pammit, palmit tension, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate hemifumarate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate (tosylate), undekanat, valerate, and the like.

The term “solvate” is used to describe a molecular complex comprising a compound of the invention and one or more pharmaceutically acceptable solvent molecules, for example ethanol. The term "hydrate" is used when the specified solvent is water.

The term "aberrant activity" of the cells of the immune system in this document means activity that is significantly different from the base level of activity of the cells of the immune system in the body in the absence of pathology. Aberrant activity can be caused by an excessive influx of cells of the immune system to an organ or tissue, disruption of processes leading to the activation of cells of the immune system, deregulation of processes associated with the death of cells of the immune system, as well as other factors.

The term "excipient" means any pharmaceutically acceptable substance of inorganic or organic origin that is part of the drug or used in the manufacturing process, manufacture of the drug to give it the necessary physico-chemical properties.

The term “AUC” (area under the curve) means a pharmacokinetic parameter characterizing the total concentration of a drug in a blood plasma during the entire observation period. It is mathematically defined as the integral from 0 to ∞ of the function of the concentration of the drug (pharmacokinetic curve) in blood plasma versus time and is equal to the area of the figure bounded by the pharmacokinetic curve and coordinate axes.

The term " RPMI medium " (eng. Roswell Park Memorial Institute medium) means an environment for cell and tissue cultures. RPMI has traditionally been used to grow human lymphoid cells. The medium contains a significant amount of phosphate and has a composition for growing in the atmosphere with a carbon dioxide content of 5%.

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

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

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

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

The term " prevention ", " prevention " covers 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 clinical stages of the disease. Patients for prophylactic therapy are selected on the basis of factors that, based on known data, increase the risk of the clinical stages of the disease compared with the general population. Preventive therapy includes a) primary prevention and b) secondary prevention. Primary prophylaxis is defined as prophylactic treatment in patients whose clinical stage of the disease has not yet occurred. Secondary prophylaxis is the prevention of the repeated onset of the same or similar clinical condition of the disease.

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 a systemic and local character, including those due to primary pathological changes in these tissues, or associated with various diseases or prolonged use of certain medications. In some particular embodiments, the compounds of the invention can be used to treat other diseases of the abdominal organs associated with aberrant activity of cells of the immune system.

Method for the therapeutic use of compounds

The subject of the present invention also includes a treatment method, which comprises administering to a subject in need of appropriate treatment a therapeutically effective amount of compound 1. By a therapeutically effective amount is meant an amount of a compound administered or delivered to a patient in which the patient is most likely to exhibit the desired response to the treatment ( prevention). The exact amount required can 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, combined treatment with other drugs, etc.

The compound of the invention can be administered to the patient in any quantity (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 treating or preventing a disease.

After mixing the compound 1 with a specific suitable pharmaceutically acceptable carrier in the desired dosage, the compositions of the invention can be administered orally, parenterally, topically, and other methods conventional to the person skilled in the art to humans or other animals.

The introduction can be carried out both once and several times a day, week (or any other time interval), or from time to time. In addition, the compound can be administered into the patient’s body daily for a certain period of days (for example, 2-10 days), and then a period without administration (for example, 1-30 days) follows.

In the case when compound 1 is used as part of a combination therapy regimen, a dose of each of the components of the combination therapy is administered during the required treatment period. The compounds that make up the combination therapy can be administered into the patient's body both at a 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 that contain compound 1 or a pharmaceutically acceptable salt, hydrate or solvate thereof (or a prodrug or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, solvents and / or excipients, such as may be introduced into the patient’s body together with the compound, the use of which is the subject of this invention, and which do not affect the pharmacological activity of this compound They are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

The pharmaceutical compositions of this invention comprise 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 a particular dosage form. Materials that may 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 oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic solution, Ringer's solution; ethyl alcohol and phosphate buffers. Also in the composition of the composition may be other non-toxic compatible moving substances, such as sodium lauryl sulfate and magnesium stearate, as well as dyes, film-forming agents, sweeteners, flavoring agents and flavorings, preservatives and antioxidants.

The subject of this invention is also pharmaceuticals, which are understood as pharmaceutical compositions, the composition of which is optimized for a specific route of administration into the body in a therapeutically effective dose, for example, for administration to the body orally, topically, by inhalation, for example, in the form of an inhalation spray, or by the intravascular method , intranasally, subcutaneously, intramuscularly, as well as by infusion, in the recommended dosages.

The drugs of this invention may contain formulations obtained by incorporation into liposomes, microencapsulation, preparation of nanoforms of the preparation, or other methods known in the pharmaceutical art.

In preparing the composition, for example in tablet form, 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.

The tablets may be coated with sucrose, cellulose or a derivative thereof, or other substances suitable for coating. Tablets can be prepared in various ways, such as direct compression, dry or wet granulation, or hot fusion.

A pharmaceutical composition in the form of a gelatin capsule can be prepared by mixing compound 1 or a pharmaceutically acceptable salt thereof with other components and filling the mixture with soft or hard capsules.

For parenteral administration, aqueous suspensions, isotonic saline solutions or sterile injectable solutions are used that contain pharmacologically compatible agents, for example propylene glycol or butylene glycol.

Examples of pharmaceutical compositions

For the prevention and / or treatment of diseases of 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

IV tablet / tablet

Substance 50

Lactose 223.75

Croscarmellose Sodium 6.0

Corn Starch 15

Polyvinylpyrrolidone (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% m / v paste) 2.25

Magnesium Stearate 3.0

Capsule mg / capsule

Substance 10

Lactose 488.5

Magnesia 1.5

Composition for injection I (50 mg / ml)

Substance 5.0% m / v

1M sodium hydroxide solution 15.0% m / v

1M hydrochloric acid solution to pH 7.6

Polyethylene glycol 400 4.5% m / v

Water for injection up to 100%

Ointment 1 ml

Substance 40 mg

Ethanol 300 μl

Water 300 μl

1-dodecylazacycloheptanone 50 μl

Propylene glycol up to 1 ml

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

The use of Compound 1 in combination therapy

Despite the fact thatCompound 1by this invention can be introduced as an individual 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 administered orally, the therapeutic agents can be different dosage forms that are administered simultaneously or sequentially at different times, or the therapeutic agents can 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 one tablet, capsule, injection, or in another 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 can be carried out in combination with additional treatment methods known to those skilled in the art of prophylaxis and treatment of relevant diseases, including the use of antibacterial, cytostatic and cytotoxic drugs, drugs to suppress symptoms or side effects of one from drugs.

If the dosage form is a fixed dose, such a combination uses compound 1 or a pharmaceutically acceptable salt thereof in an acceptable dosage range.Compound 1by this invention or its pharmaceutically acceptable salt can also be introduced into the patient’s body sequentially with other agents, in the case when the joint introduction of combinations of these agents is impossible. The invention is not limited to the sequence of administration; the compound of the present invention can be administered to the patient together, before or after the administration of another drug.

Examples

1) Preparation of a compound of the invention

The preparation of Compound 1 is described in patent application 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 compounds 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 effect of Compound 1 cannot be predicted or explained based on prior knowledge of the ability of Compound 1 to chelate metal ions.

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

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

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

Glutaminyl cyclase activity at various concentrations of Compound 1 was studied at 25 ° C using a fluorescent substrate of L-glutaminyl-2-naphthylamide (Gln-bNA) (Anal Biochem. 2002 Apr 1; 303 (1): 49-56). A 100 μl reaction mixture contained 50 μM fluorogenic substrate; ~ 0.2 units of human pyroglutaminyl aminopeptidase (1 unit is defined as the amount hydrolyzing 1 micromole pGlu-bNA per minute) and an aliquot of recombinant human intracellular 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 wavelength of excitation and emission was 320 and 410 nm). Enzymatic activity was determined by the amount of released 2-naphthylamide (bNA), calculated from the calibration curve. IC ₅₀ values were calculated using non-linear regression of the curve "inhibitor concentration" -enzymatic activity ". A known glutaminyl cyclase inhibitor, compound PBD150 (J Med Chem. 2006 Jan 26; 49 (2): 664-77) was used as a comparison substance.

As a result of the experiment, it was found that Compound 1 inhibits the activity of intracellular glutaminyl cyclase with an IC ₅₀ = 4.7 μM.

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

25 μl of solution was added to the wells of a 96-well PCR plate (Bio-Rad)Compounds 1. To each well containingCompound 125 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 a secreted glutaminyl cyclase solution was placed in each well (except for blank hydrolysis controls) (QPCT) with a concentration of 0.2 μg / ml in buffer (25 mm HEPES, pH 7.0), or a similar buffer solution (control of blank hydrolysis). The plate 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 solution of pyroglutaminyl peptylase (PGPEP) with a concentration of 1 μg / ml 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. 90 μl of solution was taken 2 times from each well, placed in 3 wells of a Greiner Bio fluorimetric plate, and fluorescence was determined (excitation 355 nm, emission 460 nm). The obtained inhibition curve (dependence of I on the concentration of the substance) was approximated by a dose-dependent curve (Dose-response curve) with the lower asymptote = 0 and the upper asymptote = 1 using the Origin Pro 8.0 software, which allowed us to obtain the IC value_fifty.

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

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

The effect of Compound 1 on in vitro monocyte migration was studied using cells of the U937 line, concentration (2-3) × 10 ⁶ cells / ml on RPMI 1640 medium supplemented with 10% thermally inactivated fetal bovine serum (Biochem J. 2012 Mar 1; 442 ( 2): 403-12). About 1 × 10 ⁷ U937 cells were incubated with different concentrations of Compound 1 (a total of 7 concentration values) 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 Calcein AM fluorescent dye for 1 hour at 37 ° C. An aliquot of stained cells was then placed in the upper compartment of the wells of a BD FalconTM HTS FluoroBlok plate, the cells of which were separated by semipermeable optically opaque membranes. In the lower compartment of the wells of the plate was placed conditioned medium obtained after incubation of cells with an inhibitor and lipopolysaccharide. The plates were incubated for 2 hours at 37 ° C; the number (% relative to the experiment without inhibitor) of the cells migrating to the lower compartment of the wells was determined fluorimetrically. As a comparison, a well-known glutaminyl cyclase inhibitor, compound PBD150, was used (J Med Chem. 2006 Jan 26; 49 (2): 664-77).

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

Table 1 - The effect of Compound 1 on the migration of transplantable culture of monocytes of the line U937 in vitro

The concentration of Compound 1, μm 0 0,0003 0.003 0,03 0.3 3.0 30,0 Monocyte migration activity,% of untreated control one hundred% 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) Investigation of the effect of Compound 1 on macrophage chemotaxis in vivo  on the model of a carrageenin bag

To assess the effectiveness of the drug on the activity of cells of the immune system at the preclinical stage of research, as a rule, various models of the 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 the aberrant activity of cells of the immune system is localized in a certain cavity (peritonitis, cholangitis, arthritis) (J Pharmacol Toxicol Methods. 1994 Nov; 32 (3): 139-47). The carrageenin 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 sac 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 back over several days leads to morphological changes in the cell lining of the sac. The pouch consists mainly of macrophages and fibroblasts and is well vascularized. On the sixth day from the moment of formation of the air sac, a λ-carrageenin solution is introduced into the cavity of the air sac. Carrageenin 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 migration of cells of the immune system into the sac.

A study of the activity of Compound 1 was performed on male Balb / c mice according to a standard procedure (Curr Protoc Pharmacol. 2012 Mar; Chapter 5: Unit5.6). Compound 1 was intragastrically administered to the experimental groups at a dose of 3 mg / kg immediately before the administration of carrageenin and then every 10-12 hours. The last administration was 12 hours before slaughter.

48 hours after injection of λ-carrageenin, individual animal groups were euthanized by CO ₂ inhalation. Immediately after euthanizing, 1 ml of physiological saline containing 5.4 mM 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 pipette 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 chamber. Then, the exudate was centrifuged at 200 ° C for 10 minutes. Smears were prepared from the cell pellet, which were subsequently fixed in methanol (5 min) and stained according to the Romanovsky-Giemsa method (40 min at t 20-22 ° C). The number of macrophages was counted on smears in a routine manner under an Olympus bx51 microscope (at a magnification of 100). Cell counting was performed up to 100 pcs.

Table 2 shows the number of cells of the immune system in the exudate from the carrageenan sac in the study of the activity of Compound 1 on the model of chemotaxis of macrophages.

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 White blood cells Macrophages Intact 0.9 ± 0.2 0.6 ± 0.1 The 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 control (p <0.05)

Studies have shown that the use of Compound 1 10 times reduced the influx of leukocytes and macrophages (to the level of intact control). Thus, Compound 1 is potentially suitable for 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). The control group mice were intraperitoneally injected with 2 ml of 3% thioglycolic medium stored for 1 month. Intact animals were injected intraperitoneally with 2 ml of physiological saline. Compound 1 was intragastrically administered to experimental groups 1 hour before the injection 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 CO ₂ chamber, the peritoneal area was moistened with 70% alcohol, the skin on the abdominal cavity was carefully cut, 5 ml of cold phosphate-saline buffer containing 0.1% EDTA was intraperitoneally injected. 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 when studying the activity of Compound 1 on the model of macrophage chemotaxis (thioglycolate peritonitis) is shown in table 3.

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

Groups The number of cellular elements in 1 μl of exudate, × 10 ⁹ / l White blood cells Monocytes Intact 0.71 ± 0.09 0.61 ± 0.07 The 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 control (p <0.05)

In the exudate, the absolute number of cellular elements per 1 μl of washout (cytosis) was determined using a Goryaev chamber. Then the exudate was centrifuged at 200 ° C for 10 minutes. Smears were prepared from the cell pellet, which were subsequently fixed in methanol (5 min) and stained according to the Romanovsky-Giemsa method (40 min at 20-22 ° C). On smears in a routine manner under an Olympus bx51 microscope (at magnification 100), the number of monocytes / macrophages was counted. Cell counting was performed up to 100 pcs.

Studies have shown that the use ofCompounds 1 reduces thioglycolate-induced influx of macrophages into the peritoneal cavity of mice and has an anti-inflammatory effect. In this way,Compound 1 potentially useful in the treatment of peritonitis, Crohn's disease, and ulcerative colitis.

2.6) Study of the activity of Compound 1 on a model of carrageenin edema

A model of carrageenin-induced rat paw edema was used to evaluate the effect of Compound 1 on the functional status of the immune system. The experiments were performed on white nonlinear male rats weighing 250-270 g. Acute paw inflammation in rats was caused by intraplanar administration (subcutaneously) in a volume of 0.1 ml into the right paw of a 1.5% carrageenin solution. The test compound was administered intragastrically 1 hour before the administration of carrageenin. The effect of Compound 1 and the control substance (diclofenac) was evaluated by the degree of reduction of paw edema in comparison with the intact left paw. Paw volume was evaluated prior to administration of Compound 1 and 2 hours after administration of carrageenin.

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

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

Group Pathology induction Drug administration Assessment Dates Inhibition of growth of edema,% Intact 2 hours after administration of carrageenin 0 The control Intraplantation in a volume of 0.1 ml in the right paw of a 1.5% solution of carrageenin Intragastrically, once, 1 hour before the administration of carrageenin 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 rat diabetic nephropathy model

The rat diabetic nephropathy model was induced by prolonged 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, at the 9th week the rats were injected intraperitoneally with streptozotocin twice (2 consecutive days) 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 a biochemical analysis of urine after 6 weeks of intragastric administration of Compound 1 to rats in a model of diabetic nephropathy induced by a high-fat diet and the administration of streptozotocin (15 weeks of a high-fat diet)

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

Groups Dose and regimen of streptozotocin administration Number of animals Diuresis, ml / 18 h. Daily protein, mcg / 18h ACR (protein (μg / L) / creatinine (mg / L) Intact - 8 4.35 ± 0.5 1041.2 ± 153.5 273.2 ± 68.0 The control 30 mg / kg, twice fourteen 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 control (p <0.05)

Thus, studies have shown that Compound 1 is effective in inhibiting the glutaminyl cyclase enzyme (IC ₅₀ = 4.7 μM for intracellular glutaminyl cyclase and IC ₅₀ = 2.9 μM for secreted glutaminyl cyclase) and inhibits in vitro macrophage chemotaxis ( IC ₅₀ = 2 9 μM ) . In the carrageenin sac and thioglycolate peritonitis model, Compound 1 inhibits macrophage chemotaxis in vivo. In the model of diabetic nephropathy, the effect of Compound 1 reduces the chemotaxis of monocytes and macrophages in the kidney tissue, stops the local pathological process in the glomeruli of the kidneys and normalizes the functional characteristics of the kidneys.

2.8) Study of the pharmacokinetics of Compound 1 with 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, it seems reasonable to achieve high concentrations of Compound 1 in the target organ to achieve the maximum therapeutic effect. At the same time, from the point of view of minimizing the possible negative effects associated with the systemic suppression of chemotaxis of the 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 the “accumulation” of Compound 1 , we selected the “tissue bioavailability” characteristic 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 AUC0-t value 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. The determination of the concentration of Compound 1 during 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 were observed in the abdominal organs - spleen, liver, and kidneys (see table 5). Thus, the use of Compound 1 is potentially suitable for the treatment of diseases of the abdominal organs.

Table 6 estimates the tissue bioavailability of Compound 1 with a single intragastric administration to rats of a dose of 18 mg / kg.

Table 6 - Tissue bioavailability of Compound 1 with a single intragastric administration to rats of a dose of 18 mg / kg.

Plasma Liver Spleen Heart Thymus Muscle Kidney Lungs Tissue bioavailability
nost one 188 23 9 12 2 38 13

Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the gist of the present invention.

Claims (15)

1. A pharmaceutical composition for modulating glutaminyl cyclase activity, comprising a pharmaceutically effective amount of a compound of the formula

or a pharmaceutically acceptable salt, hydrate or solvate thereof, and at least one pharmaceutically acceptable excipient. 2. The pharmaceutical composition according to claim 1, characterized in that the excipient is a carrier and / or excipient. 3. The pharmaceutical composition according to claim 1 or 2, where the modulation of glutaminyl cyclase activity is an inhibition of intracellular and / or secreted glutaminyl cyclase. 4. The pharmaceutical composition according to claim 1 or 2, further comprising another active agent. 5. The pharmaceutical composition according to claim 4, where the other active agent is selected from the group comprising antibacterial, cytostatic, cytotoxic agents, means of suppressing the symptoms or side effects of the active agents, and combinations thereof. 6. A pharmaceutical composition for treating a disorder associated with glutaminyl cyclase activity, comprising a pharmaceutically effective amount of a compound of the formula

or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. 7. The pharmaceutical composition according to claim 6, characterized in that the excipient is a carrier and / or excipient. 8. The pharmaceutical composition according to claim 6 or 7, characterized in that the disorder associated with the activity of glutaminyl cyclase is a disease of the abdominal organs. 9. The pharmaceutical composition of claim 8, wherein the disorder is Crohn’s disease, ulcerative colitis, peritonitis, nephropathy, in particular diabetic nephropathy, or kidney disease, which is a side effect of the use of drugs. 10. The pharmaceutical composition according to claim 6 or 7, further comprising another active agent. 11. The pharmaceutical composition of claim 10, where the other active agent is selected from the group comprising antibacterial, cytostatic, cytotoxic agents, means of suppressing the symptoms or side effects of the active agents, and combinations thereof.