EA040699B1 - A NEW MODULATOR OF METABOTROPIC AND IONOTROPIC TRANSMEMBRANE RECEPTORS AND ITS APPLICATIONS - Google Patents

Abstract

The invention relates to the chemistry of organic compounds, pharmacology and medicine and concerns the treatment of inflammatory and autoimmune diseases such as psoriasis, atopic dermatitis, pruritus, Crohn's disease, colitis, gastrointestinal tract diseases such as diarrhea and irritable bowel syndrome, respiratory diseases such as asthma, COPD, bronchitis, rhinitis, as well as cough and a number of other diseases associated with the activity of opioid and tachykinin receptors and ion channels TRPV1 and TRPM8 through the use of the compound 2-phenylethylamide N-(p-hydroxyphenylacetyl)phenylalanine. This compound, as well as its pharmaceutically acceptable adducts, hydrates, solvates are agonists of opioid receptors, antagonists of tachykinin receptors and ion channels TRPV1 and TRPM8. The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of a compound of the invention.

Description

Technical field

This invention relates to chemistry of organic compounds, pharmacology and medicine and relates to the treatment of inflammatory and autoimmune diseases, diseases of the gastrointestinal tract, respiratory tract, cough, as well as a number of other diseases through the use of a compound that is a modulator of metabotropic and ionotropic transmembrane receptors involved in in particular, in the processes of nociception, vasodilation, development of neurogenic inflammation and chemotaxis of cells of the immune system.

State of the art

Metabotropic and ionotropic transmembrane receptors are the two largest groups of proteins that regulate nociception, vasodilation, development of inflammation, and other important processes in animals and humans. The biological effects of most transmembrane receptors are realized through interaction with endogenous modulators that activate or suppress the activity of the corresponding cellular receptors.

In particular, endogenous tachykinins and opioids are groups of neuropeptides involved in the development of neurogenic inflammation and pruritus, processes of nociception, vasodilation, contraction of muscle fibers, and chemotaxis of immune system cells. The biological effects of endogenous tachykinins and opioids are realized through interaction with tachykinin (neurokinin) and opioid metabotropic receptors. These G protein-coupled receptors are widely distributed in the central and peripheral nervous systems and are predominantly localized in primary afferent neurons located in the respiratory and urinary tracts (Life Sci., 2000, 66(23):2221-31), as well as in the gastrointestinal tract. -intestinal tract (Curr. Opin. Endocrinol. Diabetes Obes., 2016 Feb, 23(1):3-10; Cell Tissue Res., 2014 May, 356(2):319-32), papillary dermis and other skin layers (J. Comp. Neurol., 1999 Jun 14, 408(4):567-79; Physiol. Rev., 2014 Jan, 94(1):265-301). Modulation of the activity of tachykinin and opioid receptors in the peripheral nervous system is associated with a wide range of biological effects.

In the gastrointestinal (GI) tract, opioid and tachykinin receptors are predominantly expressed on mucosal muscularis cells, immune cells, and on submucosal and muscular plexus neurons (J. Comp. Neurol., 2007, 503, 381-91; Regul Pept 2009 Jun, 5, 155(1-3):11-7). Modulation of the activity of opioid and tachykinin receptors in the gastrointestinal tract affects intestinal motility, secretory and immune activities, visceral sensitivity and nociception (Holzer P. Tachykinins. In Handbook of Biologically Active Peptides (Second Edition); Kastin AJ, ed., Elsevier, 2013, pp. 1330-1337; Regul Pept., 2009 Jun, 5, 155(1-3):11-7). For example, activation of mu-opioid receptors (MOR) in the gastrointestinal tract leads to a decrease in abdominal pain and visceral hyperalgesia (Biochemical Pharmacology, 92(2014)448-456). At the same time, suppression of peripheral NK ₃ receptor activity has been shown in animals (Neurogastroenterol Motil, 2003, 15, 363-9; Neurogastroenterol Motil, 2004, 16, 223-31) to reduce colorectal distension-induced nociception, as well as hypersensitivity caused by stress. NK1 and NK2 tachykinin receptors, as well as mu-opioid receptors, have a pronounced effect on gastrointestinal motility (Pharmacol. Ther., 1997, 73, 173-217; Expert Opin. Investig. Drugs, 2007 Feb, 16(2):181-94 ), and therefore these receptors are the most promising targets for the treatment of functional bowel diseases and, in particular, diarrhea. Activation of opioid receptors and suppression of tachykinin receptor activity have been shown in a number of preclinical and clinical studies to decrease ion and fluid secretion, delay transit through the small and large intestine, and increase anal sphincter pressure (Expert Opin. Investig. Drugs, 2007 Feb, 16( 2):181-94; Pharmacol. Ther., 1997, 73, 173-217).

Importantly, dual and triple tachykinin receptor antagonists appear to be more effective than selective antagonists in the treatment of gastrointestinal disorders. For example, it has been shown that selective tachykinin receptor antagonists have a significant effect on animal intestinal motility only when blocking the cholinergic component (Holzer P., Role of tachykinins in the gastrointestinal tract. In: Holzer P., editor., Tachykinins. Handbook of experimental pharmacology, vol.164, Berlin: Springer, 2004, pp. 511-58). However, with simultaneous blocking of all three tachykinin receptors, peristalsis in the distal guinea pig colon was significantly reduced even without the participation of acetylcholine receptor antagonists (Gastroenterology, 2001, 120, 938-45).

Thus, modulators of opioid and tachykinin receptors can be used to treat a number of functional and inflammatory diseases of the gastrointestinal tract, such as diarrhea, irritable bowel syndrome, colitis, Crohn's disease, etc. chronic abdominal pain and functional disorders of the gastrointestinal tract (J. Med. Chem., 2011 Apr, 14, 54(7):2029-2038). Moreover, the simultaneous action on opioid and tachykinin receptor signaling potentially allows the use of low doses of the drug and reduces the likelihood of developing side effects characteristic of opioid therapy (Regul Pept., 2009 Jun, 5, 155(1-3):11-7).

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Opioid and tachykinin receptor modulators can also be used to treat respiratory diseases. In particular, tachykinins are potent airway smooth muscle constrictors. In addition, the action of tachykinins on vascular endothelial cells cause the development of vasodilation and increase the permeability of the vessels of the microvasculature of the respiratory tract (Drug News Perspect., 1998, 11(8):480; Navy Pulm. Med., 2011 Aug, 2, 11:41) . In addition, tachykinins increase the secretion of the mucous glands and epithelial cells of the respiratory tract (Pflugers. Arch., 2008 Nov, 457(2):529-37; Physiol. Rev., 2015 Oct, 95(4):1241-319) and are potent chemoattractants and activators of immune system cells in respiratory tissues (Drug News Perspect., 1998 Oct, 11(8):480-9; Trends Immunol., 2009 Jun, 30(6):271-6). Tachykinins and opioids modulate various pulmonary reflexes, including the cough reflex (Pharmacol. Ther., 2009 Dec, 124(3):354-75; Am. J. Physiol. Regul. Integr. Comp. Physiol., 2000 Oct, 279(4 ):R1215-23; Am. J. Respir. Crit. Care Med., 1998 Jul, 158(1):42-8), parasympathetic, cholinergic, bronchoconstrictor reflexes (Nat. Neurosci., 2012 Jul, 26, 15( 8):1063-7; Prog. Histochem. Cytochem., 2010 Feb, 44(4):173-202). The pathophysiological role of tachykinins in respiratory diseases seems to be mediated by activation of NK1 and NK2 receptors, while activation of NK ₂ and NK ₃ receptors is involved in the pathogenesis of cough (Am. J. Respir. Crit. Care Med. , 1998 Jul, 158(1):42-8; Eur J. Pharmacol., 2002 Aug, 23, 450(2):191-202). In addition, tachykinins seem to be able to effectively modulate the activity of ionotropic receptors (Neuropeptides., 2010 Feb, 44(1):57-61), in particular, TRPV1 and TRPM8 ion channels involved in determining and regulating sensory perception of temperature and expressed in primary afferent neurons and in surrounding airway tissues (Gut., 2008 Jul, 57(7):923-9; J. Neurosci., 2008 Jan, 16, 28(3):566-75). In addition to being involved in the pathogenesis of cough and rhinitis, TRPV1 plays an important role in the development of pain sensitivity (Expert Opin. Ther. Pat., 2012 Jun, 22(6):663-95; Recent. Pat. CNS Drug Discov., 2013 Dec, 8(3):180-204; Expert Opin Investig Drugs, 2012 Sep, 21(9):1351-69). In a number of clinical studies and animal models, administration of TRPV1 ion channel antagonists has been shown to increase the threshold for cough sensitivity (J. Allergy Clin. Immunol., 2014 Jul, 134(1):56-62) and also reduce the severity of symptoms of COPD (Am. J. Respir. Crit. Care Med., 2016 Jun, 15, 193(12):1364-72; Sci. Transl. Med., 2012 Nov, 7, 4(159):159ra147) and asthma (Br. J. Pharmacol., 2012 Jul, 166(6):1822-32). It is important to note that tachykinin and opioid receptors are associated with TRPV1 ion channels by a complex feedback system. For example, activation of TRPV1 leads to the development of tolerance to opioid analgesics (Channels (Austin), 2015, 9(5):235-43). For this reason, activation of opioid receptors with simultaneous suppression of the activity of TRPV1 ion channels is an effective strategy for the treatment of pain symptoms of various diseases. On the other hand, the suppression of TRPV1 ion channel activity leads to a decrease in the production of tachykinins and a decrease in the severity of neurogenic inflammation (Pulm. Pharmacol. Ther., 2018 Apr, 49:1-9). Unlike the high temperature activated TRPV1 ion channel, the TRPM8 ion channel is activated at surrounding tissue temperatures below 30°C. Activation of TRPM8 leads to increased expression of pro-inflammatory cytokines and mucus hypersecretion by human bronchial epithelial cells (Inflammation., 2018 Aug, 41(4):1266-1275) and nasal cavity (Medicine (Baltimore), 2017 Aug, 96(31):e7640) . Thus, activation of opioid receptors, together with suppression of the activity of tachykinin receptors and TRPV1 and TRPM8 ion channels, may have a synergistic effect in the treatment of cough, which is a characteristic symptom of a number of respiratory diseases, such as asthma, pulmonary fibrosis, COPD, and bronchitis. It is important to note that, according to the literature data, modulators of opioid and tachykinin receptors of TRPV1 and TRPM8 ion channels can have a direct pathogenetic effect on these respiratory diseases. Thus, in particular, in clinical studies, tachykinin receptor antagonists have been shown to suppress bronchospasm and reduce airway hyperreactivity in patients with asthma (Eur. Respir. J., 2004 Jan, 23(1): 76-81; Pulm. Pharmacol. Ther., 2006, 19(6):413-8; BMC Pulm. Med., 2011 Aug. 2, 11:41). In patients with COPD, inhaled morphine resulted in significant relief of dyspnoea and other symptoms of the disease (BMC Pulm. Med., 2017 Dec, 11, 17(1):186).

Modulators of opioid and tachykinin receptors and TRPV1 and TRPM8 ion channels may also be of use in the treatment of inflammatory and autoimmune diseases, in particular for the treatment of pruritus in psoriasis and atypical dermatitis (Br. J. Dermatol., 2019 Jan, 8; J. Am. Acad Dermatol., 2018 Mar, 78) and pain symptoms in Crohn's disease and ulcerative colitis (Pharmaceuticals (Basel). 2019 Mar, 30, 12(2); Inflamm. Bowel. Dis., 2015 Feb, 21(2):419 -27). Activation of TRPV1 ion channels located at the ends of sensory nerves and surrounding skin tissues leads to a significant increase in the production of substance P and other endogenous tachykinins and the development of neurogenic inflammation (Br. J. Dermatol., 2019 Jan, 8). Moreover, increased production of substance P leads to NK ₁ -mediated activation of mast cells, increased production of tumor necrosis factor and the development of pruritus (J. Am. Acad. Dermatol., 2018 Mar, 78(3 Suppl 1):S63-S66). In clinical studies, the involvement of substance P and NK ₁ -recipes in the development of prurigo (prurigo) was shown, while the antagonist of NK ₁ -recipes significantly reduced the severity of symptoms of the disease compared with baseline values (Acta Derm. Venereol., 2018 Jan, 12, 98 (1):26-31). Thus, suppression of activity

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Based on the literature data, it can be concluded that a strategy aimed at activating opioid receptors while simultaneously suppressing the activity of tachykinin receptors, TRPV1 and TRPM8 ion channels is a possible approach to the treatment of inflammatory and autoimmune diseases (such as psoriasis, atypical dermatitis, Crohn's disease and ulcerative colitis). ), diseases of the gastrointestinal tract (such as irritable bowel syndrome, colitis and postoperative ileus), respiratory tract (such as asthma, COPD, bronchitis, rhinitis) and cough, including cough in pulmonary fibrosis, bronchitis, asthma, COPD and other diseases.

To date, various opioid receptor agonists, tachykinin receptor antagonists, and TRPV1 and TRPM8 ion channel antagonists are known, including selective NK ₁ antagonists and mixed NK ₁ /NK2 receptor antagonists based on high-affinity derivatives of 3-cyano-1-naphthamide (WO 2001077089, WO 2002026724 ) or naphthoic acid amide (WO 2001077069, WO 2000059873). It is important to note that, to date, no compounds that are agonists of opioid receptors, antagonists of tachykinin receptors and TRPV1 and TRPM8 ion channels have been described in the scientific literature. Examples of related inventions include developments by Boehringer Ingelheim, mainly related to arylglycinamide derivatives (EP 1295599) for the treatment of inflammatory skin diseases. Menarini Group is developing a glycosylated bicyclic cyclohexapeptide NK ₂ receptor antagonist for the treatment of irritable bowel syndrome (Br. J. Pharmacol., 2001 Sep, 134(1):215-23, Eur. J. Pharmacol., 2006 Nov, 7, 549(1-3):140-8). The closest analogues of the compound that is the subject of the present invention are given in the publications of the company Ciba-Geigy (WO 1996026183). This paper describes non-selective tachykinin receptor antagonists based on phenylalanine derivatives for the treatment of diseases of the central nervous system. However, in the structures of the compounds published by Ciba-Geigy, the ligands contain two ester substituents, which significantly reduce the metabolic stability of the compound.

Thus, to date, there is no drug that acts as an opioid receptor agonist, tachykinin receptor antagonist and TRPV1 and TRPM8 ion channels, which would be used in the treatment of inflammatory and autoimmune diseases, diseases of the gastrointestinal tract, lungs and respiratory tract. Therefore, there remains a need to create and introduce into the clinic new effective drugs based on modulators of opioid and tachykinin receptors, as well as TRPV1 and TRPM8 ion channels.

This invention relates to the production and use of a new chemical compound that is effective in activating opioid receptors, as well as in suppressing the activity of tachykinin receptors and TRPV1 and TRPM8 ion channels, in the treatment of inflammatory, autoimmune diseases, diseases of the gastrointestinal tract, respiratory tract, and cough .

Disclosure of invention

The objective of the present invention is to develop a new drug that is an agonist of opioid receptors (mu, delta and kappa), an antagonist of tachykinin receptors (NK ₁ , NK2 and NK ₃ ) and ion channels TRPV1 and TRPM8, effective for the treatment of inflammatory and autoimmune diseases, diseases of the stomach. -intestinal tract, respiratory tract and cough.

The technical result of this invention is the development and production of an effective agonist of opioid receptors, an antagonist of tachykinin receptors and ion channels TRPV1 and TRPM8, allowing the use of this compound for oral and topical use for the treatment of cough, asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, Crohn's disease, colitis, psoriasis, atopic dermatitis, pruritus, as well as other diseases associated with the activity of opioid, tachykinin receptors and TRPV1 and TRPM8 ion channels.

The specified technical result is achieved by using the compound 2-phenylethylamide N-(p-hydroxyphenylacetyl)phenylalanine (compound I)

or its adduct hydrate, solvate as an opioid receptor agonist, tachykinin receptor antagonist and TRPV1 and TRPM8 ion channels.

The present invention also relates to a modulator of opioid and tachykinin receptors, as well as TRPV1 and TRPM8 ion channels, which is compound I.

The invention also relates to a process for the preparation of N-(p-hydroxyphenylacetyl)phenylalanine 2-phenylethylamide.

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The present invention also relates to the use of the compound 2-phenylethylamide N-(p-hydroxyphenylacetyl)phenylalanine or its adduct, hydrate, solvate for the preparation of a pharmaceutical composition for the prevention and/or treatment of inflammatory, autoimmune, diseases of the gastrointestinal tract, respiratory tract, cough , such as asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, Crohn's disease, colitis, as well as other diseases associated with the activity of opioid receptors, tachykinin receptors (NK1, NK ₂ and NK ₃ ), TRPV1 and TRPM8 ion channels.

In addition, the invention relates to a pharmaceutical composition for the prevention and/or treatment of diseases of the respiratory tract, urinary tract, gastrointestinal tract, cough, such as asthma, COPD, bronchitis, rhinitis, irritable bowel syndrome, Crohn's disease, colitis, psoriasis, atopic dermatitis, pruritus, as well as other diseases associated with the activity of opioid and tachykinin receptors, as well as TRPV1 and TRPM8 ion channels, containing an effective amount of compound I 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 the activity of opioid, tachykinin receptors and TRPV1 and TRPM8 ion channels in a subject in need of such treatment, comprising administering a pharmaceutical composition of the invention to said subject. In some non-limiting embodiments of the invention, the disease is cough, asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, colitis, psoriasis, atopic dermatitis, pruritus. In particular cases of embodiment of the invention, the organism is a human or animal organism.

The invention relates to a method for preventing and/or treating a disorder associated with the activity of opioid, tachykinin receptors and TRPV1 and TRPM8 ion channels in a subject in need of such treatment, comprising administering a therapeutically effective amount of Compound I to said subject.

The invention also relates to a method for preventing and/or treating cough, asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, Crohn's disease, colitis, psoriasis, atopic dermatitis, pruritus in a subject in need of such treatment, including the introduction of a therapeutically effective amount compound I to the specified subject.

The invention also relates to the use of compound I for the preparation of a medicament.

The present invention also relates to a combination containing compound I in combination with one or more other additional therapeutic agents.

Detailed disclosure of the invention

Compound I, which is the subject of the present invention, can be obtained using various well-known synthetic methods, including using the synthetic methods described below.

Compound I was surprisingly found to be a mu, delta and kappa opioid receptor agonist, a type 1, 2 and 3 tachykinin receptor antagonist and a blocker of the TRPV1 and TRPM8 ion channels during screening of the pharmacological targets of Compound I. In accordance with the spectrum of experimentally determined therapeutic targets of compound I, indications were identified in which the use of compound I seemed to be the most promising. It turned out that the use of compound I is promising for the treatment of inflammatory, autoimmune diseases (such as psoriasis, atopic dermatitis, Crohn's disease, ulcerative colitis), diseases of the gastrointestinal tract (such as diarrhea, irritable bowel syndrome), respiratory tract (such as cough, asthma, COPD, bronchitis, rhinitis), as well as pruritus.

Thus, compound I is a new opioid receptor agonist, antagonist of tachykinin receptors and TRPV1 and TRPM8 ion channels, which can be used for the treatment of cough, asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, Crohn's disease, colitis, psoriasis, atopic dermatitis, pruritus.

Terms and Definitions.

The term compound I refers to 2-phenylethylamide N-(p-hydrokCiphenylacetyl)phenylalanine, also represented by the structural formula

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

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

The term pharmaceutically acceptable adducts or adducts includes the product of direct addition of molecules to each other, which are obtained using relatively non-toxic compounds. Examples of pharmaceutically acceptable non-toxic adducts are those formed by non-toxic nitro derivatives or urea. Other pharmaceutically acceptable adducts include adducts of non-ionic surfactants, cyclodextrins and others, as well as charge transfer complexes (π-adducts). It should be noted that the term adducts also includes non-stoichiometric adducts.

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 stimulation of sensory nerve endings as used herein means stimulation significantly different from the baseline level in the body in the absence of pathology. Aberrant stimulation can be caused by an excessive influx of immune system cells to an organ or tissue, a disruption in the processes leading to stimulation of sensory nerve endings, 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 terms treatment, therapy encompass the treatment of pathological conditions in a mammal, preferably a human, and include

a) decrease

b) blocking (suspension) of the course of the disease,

c) relief of the severity of the disease, i.e. induction of disease regression,

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

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

Compound I, which is the subject of this invention, is promising for the treatment of diseases associated with aberrant stimulation of sensory nerve endings and the activity of mediators mediated by their action on opioid receptors, tachykinin receptors (NK _b NK2 and NK ₃ ), TRPV1 and TRPM8 ion channels, in particular for therapy of respiratory diseases (such as cough, asthma, chronic bronchitis, rhinitis), diseases of the gastrointestinal tract (such as irritable bowel syndrome, Crohn's disease, colitis and postoperative intestinal obstruction), urinary tract, both systemic and local in nature , including those caused by primary pathological changes 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 disorders associated with aberrant stimulation of sensory nerve endings.

Method for the therapeutic use of the compounds.

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

After mixing the drug with a particular suitable pharmaceutically acceptable carrier at the desired dosage, the compositions of the invention can be administered to humans or other animals orally, parenterally, topically (inhalation, intranasal, dermal) and the like.

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

When a compound of the invention 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 a compound of the invention (or a prodrug or other pharmaceutically acceptable derivative) and one or more pharmaceutically acceptable carriers, adjuvants, diluents and/or excipients, such as may be administered to a patient together with the compound , constituting the essence of this invention, and which do not affect the pharmacological activity of this compound and are non-toxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutical compositions of this invention contain a compound of this invention together with pharmaceutically acceptable carriers, which may include any solvents, diluents, dispersions or suspensions, surfactants, isotonic agents, thickeners and emulsifiers, preservatives, astringents, sliding materials, etc., suitable for the specific dosing 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 this invention are also dosage forms - a class of pharmaceutical compositions, the composition of which is optimized for a certain route of administration in the body in a therapeutically effective dose, for example, for administration to the body orally, topically, inhalation, for example in the form of an inhaled spray, or intravascularly, intranasally, subcutaneously , intramuscularly, as well as by infusion in the recommended dosages.

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

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

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

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

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

Examples of pharmaceutical compositions.

The substance described in this invention can be used for the prevention and/or treatment of diseases in humans or animals in the form of the following compositions (substance means the active ingredient).

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Tablet I mg/tablet Substance 0.5 Microcrystalline cellulose 66.5 Sodium carboxymethyl starch 2.3 Magnesium stearate Tablet II Substance 0.7 mg/tablet 0.5 Microcrystalline cellulose 62.0 Sodium carboxymethyl starch 2.3 Magnesium stearate Tablet III 0.7 mg/tablet Substance 50 Microcrystalline cellulose Sodium carboxymethyl starch 620 23 Magnesium stearate Tablet IV 7 mg/tablet Substance 50 Lactose Ph. EUR 223.75 Croscarmellose sodium 6.0 Corn starch Polyvinylpyrollidone (5% w/v paste) 15 2.25 Magnesium Stearate Tablet V 3.0 mg/tablet Substance 200 Lactose Ph. EUR 182.75 Croscarmellose sodium Corn starch (5% w/v paste) magnesium stearate Capsule 12.0 2.25 3.0 mg/capsule Substance 10 Lactose Ph. EUR 488.5 Magnesia 1.5 Capsule mg/capsule Substance 10 Lactose Ph. EUR 488.5 Magnesia 1.5 Composition for intranasal administration I Substance mg/ml 1.0 Sodium citrate dihydrate 3.823 Citric acid monohydrate 0.609 Glycerol 25.0 Dextrose 5.5 benzyl alcohol 2.5 Water up to 100%

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Composition for intranasal administration II Substance mg/ml 1.0 Sodium citrate dihydrate Citric acid monohydrate Glycerol Dextrose Water Composition for intranasal administration II Substance 3.823 0.609 25.0 5.5 up to 100% mg/ml 1.0 Sodium dihydrogen phosphate dihydrate Disodium hydrogen phosphate dihydrate Glycerol Dextrose benzyl alcohol 3.38 2.08 25.0 5.5 2.5 Water Composition for intranasal administration II Substance up to 100% mg/ml 1.0 Sodium dihydrogen phosphate dihydrate Disodium hydrogen phosphate dihydrate 3.38 2.08 Glycerol Dextrose 25.0 5.5 Water Composition for inhalation up to 100% mg/ml Substance Glycerol Water for injections 2.0%w/v 20.0% w/v up to 100%

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

The use of compound I in combination therapy.

Although the compound I of this invention can be administered as a single active pharmaceutical agent, it can also be used in combination with one or more other agents, in particular, the other agent may be a cough reflex inhibitor (codeine, glaucine, butamirate , bithiodine), a mucolytic agent (bromhexine, ambroxol), a mucoregulatory agent (carbocysteine), an expectorant (thyme, potassium iodide, broncholithin), an antibiotic, an NSAID or other anti-inflammatory agent, etc. When co-administered orally, the therapeutic agents may be in different dosage forms administered simultaneously or sequentially at different times, or the therapeutic agents may be combined into a single dosage form.

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

Thus, the administration of the compound of this 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 and anti-inflammatory drugs, drugs to suppress symptoms, or side effects of one of the drugs.

If the dosage form is a fixed dose, such a combination uses the compound of this invention in an acceptable dose range. Compound I of the invention can also be administered sequentially to a patient with other agents when a combination of these drugs is not possible. The invention is not limited to the sequence of administration; the compound of this invention can be administered to the patient simultaneously, before or after administration of another drug.

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Obtaining compounds according to the invention.

Obtaining 2-phenylethylamide N-(p-hydroxyphenylacetyl)phenylalanine.

(S)-methyl 2-(2-(4-hydroxyphenyl)acetomido)-3-phenylpropanoate (04) (1.50 g, 5.29 mmol), phenylethylamine (0.86 g, 6.35 mmol, 1.2 eq .), TBTU (2-(III-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) (2.04 g, 6.35 mmol, 1.2 eq.) and triethylamine (0. 64 g, 6.35 mmol, 1.2 eq.) was dissolved in 20 ml of dry acetonitrile and stirred at room temperature for 5 hours. The reaction mixture was diluted with 3% potassium carbonate solution (500 ml) and extracted with dichloromethane (2x40 ml). The extract was washed with water, dried over sodium sulfate, and the solvent was removed in vacuo. The residue obtained after evaporation (1.65 g) was purified by preparative HPLC. The result was 1.1 g of product with a purity of at least 99% according to analytical HPLC.

APCI-MS (m/z (intensity)): 402.90 ([M+H] ⁺ , 100%).

1H NMR (400 MHz, DMSO-d ₆ ) δ 9.05 (s, 1H), 7.94-7.83 (m, 2H), 7.30-7.09 (m, 10H), 6.92 -6.84 (m, 2H), 6.64-6.56 (m, 2H), 4.43 (td, J=8.8, 5.2 Hz, 1H), 3.36-3.12 (m , 4H), 2.90 (dd, J=13.7, 5.2 Hz, 1H), 2.79-2.60 (m, 3H).

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

The biological activity of the compound I, which is the subject of the present invention, was studied in various in vitro and in vivo experiments. In particular, when studying the activity of compound I in various in vitro and in vivo models, the inhibitory effect of compound I was shown in a model of cough induced by the administration of capsaicin in guinea pigs.

Studies of the biological activity of compound I in vitro have shown that compound I is an agonist of opioid receptors, an antagonist of tachykinin receptors and ion channels TRPV1 and TRPM8. Apparently, the activity of compound I in models of cough, as well as in various models of disorders of the gastrointestinal tract, is associated with an action on the above proteins.

Example 1. Study of the effect of compound I on the activity of the first type tachykinin receptor.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was serially diluted with DMSO. The maximum starting concentration of the substance is -100 μM. The effect was determined at 8 concentrations of the test compound, each concentration was tested twice. The experiment used U373 cells expressing human NK1R, which, after preincubation with the [Sar9,Met(O2)11]-SP agonist (1 nM), were incubated with compound I. Receptor activity was determined by intracellular calcium concentration by fluorescence spectroscopy (Glia, 1992, 6(2):89-95).

As a result of the study, it was found that compound I is a type 1 tachykinin receptor antagonist with an IC ₅₀ =59 μM.

Example 2. Study of the effect of compound I on the activity of the second type tachykinin receptor.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was serially diluted with DMSO. The maximum starting concentration of the substance is -100 μM. The effect was determined at 8 concentrations of the test compound, each concentration was tested twice. The experiment used CHO cells expressing human NK2R, which, after preincubation with the [Nleu10]-NKA-(4-10) agonist (10 nM), were incubated with the test compound. Receptor activity was determined by intracellular calcium concentration by fluorescence spectroscopy (Biochem. Biophys. Res. Commun,. 1994 May, 16, 200(3):1512-20).

As a result of the study, it was found that compound I is a type 2 tachykinin receptor antagonist with an IC50=6.4 μM.

Example 3. Study of the effect of compound I on the activity of the third type tachykinin receptor.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was serially diluted with DMSO. The maximum starting concentration of the substance is -100 μM. The effect was determined at 5 concentrations of test compounds, each concentration was tested twice. The experiment used CHO-K1 cells expressing NK ₃ R, which, after preincubation with the [MePhe10]-NKB agonist (1 nM), were incubated with the test compound. Receptor activity was determined by intracellular calcium concentration by fluorescence spectroscopy (Br. J. Pharmacol., 1999 Oct, 128(3):627-36).

As a result of the study, it was found that compound I is a type 3 tachykinin receptor antagonist with an IC ₅₀ =15 μM.

Example 4 Investigation of the effect of compound I on the activity of the mu-opioid receptor.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was serially diluted with DMSO. The maximum starting concentration of the substance is 300 μM. The effect was determined at 10 concentrations of test compounds, each concentration was tested twice. The experiment used a recombinant human mu-opioid receptor, which, after preincubation with the [3H]DAMGO agonist (0.5 nM), was then incubated with the test compound for 120 min. Receptor activity was determined by the radioligand displacement method. As a result of the study, it was found that compound I is a mu-opioid receptor agonist with an IC ₅₀ =4.1 μM.

Example 5 Study of the effect of compound I on delta opioid receptor activity.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was serially diluted with DMSO. The maximum starting concentration of the substance is 300 μM. The effect was determined at 10 concentrations of test compounds, each concentration was tested twice. The experiment used a recombinant human delta-opioid receptor, which, after preincubation with the [3H]DADLE agonist (0.5 nM), was then incubated with the test compound for 120 min. Receptor activity was determined by the radioligand displacement method. As a result of the study, Compound I was found to be a delta opioid receptor agonist with an IC ₅₀ =64 μM.

Example 6 Study of the effect of compound I on kappa opioid receptor activity.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was serially diluted with DMSO. The maximum starting concentration of the substance is 300 μM. The effect was determined at 10 concentrations of test compounds, each concentration was tested twice. The experiment used a recombinant human delta-opioid receptor, which, after preincubation with the agonist [3H]U69593 (0.5 nM), was then incubated with the test compound for 120 min. Receptor activity was determined by the radioligand displacement method. As a result of the study, Compound I was found to be a kappa opioid receptor agonist with an IC ₅₀ =7.1 μM.

Example 7 Study of the effect of compound I on the activity of the TRPV1 ion channel.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was diluted with DMSO to prepare a test solution with a concentration of the substance - 50 μM. The experiment used CHO cells expressing TRPV1. On the day of the experiment, the cells were incubated with a 4 μM solution of the Fluo-4 AM fluorescent indicator. Cells after preincubation with Capsaicin (30 nM), a known TRPV1 ion channel agonist, were then incubated with the test compound. Receptor activity was determined by intracellular calcium concentration by fluorescence spectroscopy (Behrendt, H.J. et al. (2004), Br. J. Pharmacol., 141:737-745, FINAL).

As a result of the study, it was found that compound I is a TRPV1 ion channel blocker with an IC50=51 μM.

Example 8 Investigation of the effect of compound I on the activity of the TRPM8 ion channel.

Compound I was dissolved in DMSO to a concentration of 100 mm; then the stock solution was diluted with DMSO to prepare a test solution with a concentration of the substance - 50 μM. The experiment used HEK293 cells expressing TRPM8. On the day of the experiment, the cells were incubated with a 4 μM solution of the Fluo-4 AM fluorescent indicator. Cells after preincubation with Icilin (100 nM), a known TRPM8 ion channel agonist, were then incubated with the test compound.

Receptor activity was determined by intracellular calcium concentration by fluorescence spectroscopy (Behrendt, H.J. et al. (2004), Br. J. Pharmacol., 141:737-745, FINAL).

As a result of the study, it was found that compound I is a TRPM8 ion channel blocker with an IC ₅₀ =62 μM.

Example 9 Studies of the effect of compound I on gastrointestinal motility in vivo.

The effect of compound I on gastrointestinal motility was studied using a standard method (Li Y.Y., Li Y.N., Ni J.B., Chen C.J., Lv S., Chai S.Y., Wu R.H., Yuce B., Storr M., Involvement of cannabinoid-1 and cannabinoid -2 receptors in septic ileus//Neurogastroenterol Motil, 2010, vol.22, pp. 350-388). The study was conducted on male mice of the balb/c line, the individual weight of which deviated from the average value within the sex by no more than ±20%. Animals were intragastrically injected with a solution of activated charcoal (50 mg/ml, in a volume of 10 ml/kg) and the speed (in minutes) of movement of activated charcoal through the intestines of animals was evaluated. Compounds I were administered once intragastrically 1 hour before the administration of activated charcoal. Hyoscine butyl bromide (Buscopan) at a dose of 3 mg/kg, trimebutine (Trimedat) at a dose of 33 mg/kg, and mebeverine (Duspatalin) at a dose of 30 mg/kg were used as reference drugs. The data obtained were checked using the Grubbs test for the presence of the largest or smallest anomalous observation (outlier) in the sample. Values identified as outliers in this test were not used for further analysis. Descriptive statistics were used for all data: the mean (M) and the standard error of the mean (m) were calculated. Using the Kolmogorov-Smirnov criterion, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution to assess intergroup differences

- 10 040699 Student's t-test (t-test) was used. In the case of a non-normal distribution, the Kruskal-Wallis test (with Dunn's post-analysis) was used to compare multiple groups. Differences were determined at a 5% confidence level.

The results of the study are given in table. 1.

Table 1

Effect of Compound I on Gastrointestinal Motility

Groups The mode of administration of drugs η Activated carbon evacuation time, min Intact 10 73.8±3.7 Compound I (15 mg/kg) Once intragastrically 1 hour before administration 10 147.6±8.94* Compound I (7.5 mg/kg) 10 123.4±11.43* Compound I (3 mg/kg) 10 130.4±11.42* Compound I (1.5 mg/kg) activated carbon 10 108.7±9.28* hyoscine butyl bromide (3 mg/kg) 10 87.6±4.75* trimebutine (33 mg/kg) 10 110.8±6.52* mebeverine (30 mg/kg) 10 114.5±12.22*

Note.

* - Reliability of differences (P<0.05) with the intact group.

The introduction of compound I increased the time of activated charcoal evacuation in mice by 1.5-2 times. The data obtained allow us to conclude that compound I has a pronounced antispasmodic effect and is applicable for the treatment of diarrhea, irritable bowel syndrome, as well as other diseases associated with impaired gastrointestinal motility. In terms of the severity of the action of compound I, it surpasses the action of hyoscine butylbromide, trimebutine and mebeverine.

Example 10 Study of the activity of compound I in a stress-induced defecation model in rats.

The study of the activity of compound I on the model of stress-induced defecation was carried out using the standard method (Taguchi, R., Shikata, K., Furuya, Y., Hirakawa, T., Ino, M., Shin, K., Shibata, H ., Selective corticotropin-releasing factor 1 receptor antagonist E2508 reduces restraint stress-induced defecation and visceral pain in rat models//Psychoneuroendocrinology, 2017, pp. 110-115).

The rats were adapted to the room in which the experiment was conducted within 24 h. The study was conducted on fed rats. Compound I was administered once, intragastrically. After 1 hour, the rat was swaddled in cloth so that its front paws were pressed to the body. In this form, the rat was placed in an individual cage on a grid and left for 40 min. Then, in total, all the feces excreted during the observation period of 40 min were weighed.

For all data, descriptive statistics were applied: the arithmetic mean (M) and the standard error of the mean (m) were calculated. Using the Shapiro-Wilk test, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution, 1-way ANOVA analysis (with Dunnett's post-analysis) was used to assess between-group differences. In the case of a non-normal distribution, 1-way ANOVA analysis (with Tukey's post-analysis) was used to compare multiple groups. Differences were determined at a 5% confidence level. The results of the study are given in table. 2.

The results of the study showed that intragastric administration of Compound I dose-dependently reduces stress-induced defecation in rats. The data obtained allow us to conclude that compound I has a pronounced antispasmodic effect and is applicable for the treatment of irritable bowel syndrome accompanied by diarrhea.

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table 2

Effect of compound I on fecal mass in a model of stress-induced defecation in rats (M±m, n=10)

Groups Dose, mg/kg η Mass of feces, g Intact - 10 0.24±0.05 Control - 10 2.65±0.35* Compound I oh, s 10 2.35±0.35* 1 10 1.43±0.12 3 10 1.00±0.06&

Note.

* - Reliability of differences (P<0.05) with the intact group;

& - significance of differences (P<0.05) with control.

Example 11 Study of the activity of compound I in an oxazolone-induced inflammatory bowel disease model.

The study of the activity of compound I on the model of oxazolone-induced inflammatory bowel disease (model of ulcerative colitis and Crohn's disease) was carried out using the standard method (Heller F., Fuss I.J., Nieuwenhuis E.E., Blumberg R.S., Strober W. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells//Immunity, 2002, pp. 629-638).

The study was conducted on female balb/c mice. Experimental animals were injected with a 3.5 F catheter to a depth of 3-4 cm into the colon. Then, 150 μl of a 1% solution of oxazolone in 50% ethanol was slowly injected into the lumen of the colon, the catheter was slowly removed and the mouse was kept in a vertical position (upside down) within 60 s to avoid leakage of the injected solution. The experimental animals were returned to their cages and kept warm. Compound I was administered intragastrically three times: 1, 25 and 49 hours after the rectal administration of oxazolone. 72 hours after rectal administration of oxazolone, a macroscopic assessment of damage to the intestinal walls was performed using a scoring scale:

points - no lesions, point - hyperemia, no ulcers, points - hyperemia and thickening of the intestinal wall, no ulcers, points - one ulcer without thickening of the intestinal wall, points - two or more sites of ulceration or inflammation, points - two or more serious sites of ulceration and inflammation, or one site of ulceration/inflammation involving >1 cm of bowel length,

6-10 points - the damage affects > 2 cm of the length of the intestine, the score increases by 1 point for each damaged 1 cm.

For all data, descriptive statistics were applied: the arithmetic mean (M) and the standard error of the mean (m) were calculated. Using the Shapiro-Wilk test, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution, 1-way ANOVA analysis (with Dunnett's post-analysis) was used to assess between-group differences. In the case of a non-normal distribution, 1-way ANOVA analysis (with Tukey's post-analysis) was used to compare multiple groups. Differences were determined at a 5% confidence level. The results of the study are given in table. 3.

The results of the study showed that in the model of inflammatory bowel diseases (ulcerative colitis and Crohn's disease), compound I, when administered intragastrically, has a pronounced therapeutic effect, in particular, it reduces damage to the walls of the large intestine to the level of intact animals. The data obtained allow us to conclude that compound I is applicable for the treatment of Crohn's disease and ulcerative colitis.

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Table 3

The effect of compound I on damage to the walls of the large intestine in a model of oxazolone-induced ulcerative colitis in mice (M±m, n=10)

Groups Dose, mg/kg η The degree of damage to the walls of the large intestine, points Intact - 10 0.00±0.00 Control - 10 2.00±0.21* Compound I 10 10 2.20±0.96 20 10 0.00±0.00& Prednisolone 10 10 1.40±0.37*

Note.

* - Reliability of differences (P<0.05) with the intact group;

& - significance of differences (P<0.05) with control.

Example 12 Study of the activity of compound I in a model of acute pain response to mustard oil administration in mice.

The study of the activity of the compound on the model of acute pain reaction was carried out using the standard technique (Laird M.A., Martinez-Cago L., Garcia-Nicas E., Cervero F., A new model of visceral pain and referred hyperalgesia in the mouse//J. Pain, 92 (2001), pp. 335-342).

The study was conducted on male balb/c mice, the individual weight of which deviated from the average value within the sex by no more than ±10%. Mice were first lightly anesthetized (after 24 hours of fasting). Animals were then injected with 1% mustard oil in saline rectally to a depth of 4 cm using a 3.5 F catheter. Healthy controls were injected with vehicle. Five minutes after the mustard oil injection, the presence of pain in the animal was assessed (number of abdominal licks, abdominal wall abductions, deformities of the lower abdomen to the floor, abdominal distention) during the first 20 minutes. Compound I was administered once intragastrically 1 hour before mustard oil administration. The data obtained were checked using the Grubbs test for the presence of the largest or smallest anomalous observation (outlier) in the sample. Values identified as outliers in this test were not used for further analysis. Descriptive statistics were used for all data: the mean (M) and the standard error of the mean (m) were calculated. Using the Kolmogorov-Smirnov criterion, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution, Student's t-test (t-test) was used to assess intergroup differences. In the case of a non-normal distribution, the Kruskal-Wallis test (with Dunn's post-analysis) was used to compare multiple groups. Differences were determined at a 5% confidence level.

The results of the study are given in table. 4.

Table 4

Effect of Compound I on the Amount of Pain in a Model of Acute Pain Response to Mustard Oil Administration in Mice

Groups pathology induction The introduction of the drug η 0-20 min Intact Phys. solution. 10 2.2±0.33 Control Mustard oil 1% in physical. solution Once intragastrically 1 hour before the introduction of mustard oil 10 10.4±1.59* Compound I (15 mg/kg) 10 2.56±0.78 & Compound I (7.5 mg/kg) 10 15.1±1.7* Compound I (3 mg/kg) 10 8.56±0.94* Compound I (1.5 mg/kg) 10 14.8±3.01*

Note.

* - significance of differences (P<0.05) with the intact group;

& -significance of difference (P<0.05) with control.

Administration of Compound I reduced to intact values the amount of pain caused by rectal administration of mustard oil to animals. The data obtained allow us to conclude that compound I has a pronounced analgesic effect in intestinal pain, and thus compound I is applicable for the treatment of pain symptoms in irritable bowel syndrome, ulcerative colitis, Crohn's disease and other gastrointestinal diseases.

Example 13 Activity studies of compound I by inhalation in a capsaicin cough model in guinea pigs.

The capsaicin cough model was implemented according to the standard method (Tanaka M., Mashuata K., Mechanisms of Capsaicin- and Citric-Acid-Induced Cough Reflexes in Guinea Pigs//J. Pharmacol. Sci., 2005, vol. 99, p. 77- 82). The study was conducted on Agouti guinea pigs, individual value

- 13 040699 masses of which deviated from the average value within the floor by no more than ±10%. The guinea pig was placed in a plastic chamber. To induce coughing, the animals were inhaled with a nebulizer a solution of capsaicin at a concentration of 30 μM for 5 min. The solution for inhalation was prepared as follows: 1.2 mg of capsaicin was diluted in 20 ml of a mixture of 10% ethanol and 10% tween-80. Compound I was administered once, by inhalation for 1 min 15 min before inhalation of the capsaicin solution. The number of coughing fits was counted within 15 min after capsaicin inhalation. The data obtained were checked using the Grubbs test for the presence of the largest or smallest anomalous observation (outlier) in the sample. Values identified as outliers in this test were not used for further analysis. Descriptive statistics were used for all data: the mean (M) and the standard error of the mean (m) were calculated. Using the Kolmogorov-Smirnov criterion, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution, Student's t-test (t-test) was used to assess intergroup differences. In the case of a non-normal distribution, the Kruskal-Wallis test with Dunn's post-analysis was used to compare several groups. Differences were determined at a 5% confidence level.

The results of the study are given in table. 5.

Table 5

The effect of compound I on the number of coughs within 15 min in a model of capsaicin cough in guinea pigs when inhaled 1 min before inhalation of capsaicin solution

Groups η Number of coughs within 15 minutes from the start of inhalation (experiment) Cough inhibition (% of control) Intact 16 0.0±0.0 - Control (placebo) 18 15.2±0.8* - Compound I (0.25 mg/kg) 18 8.1±0.2*& 46.7

Note.

* - Reliability of differences (P<0.05) with the intact group;

& - significance of differences (P<0.05) with control.

Compound I inhalation significantly reduced the number of coughs within 15 minutes of capsaicin inhalation in guinea pigs. The results obtained allow us to conclude that compound I has a pronounced antitussive effect.

Example 14 Inhalation activity studies of compound I in a citrate cough model in guinea pigs.

The citrate cough model was implemented according to the standard method (Tanaka M., Maruyama K., Mechanisms of Capsaicin- and Citric-Acid-Induced Cough Reflexes in Guinea Pigs//J. Pharmacol. Sci., 2005, vol. 99, p. 77- 82). The study was conducted on Agouti guinea pigs, whose individual weight value deviated from the average value within the sex by no more than ±10%. The guinea pig was placed in a plastic chamber. To induce coughing, the animals were inhaled with a nebulizer a solution of citric acid at a concentration of 0.4 M in physiological saline for 10 min. Compound I was administered once, by inhalation for 1 min 15 min before inhalation of citric acid solution. The number of coughing fits was counted during 10 min of citric acid inhalation. The data obtained were checked using the Grubbs test for the presence of the largest or smallest anomalous observation (outlier) in the sample. Values identified as outliers in this test were not used for further analysis. Descriptive statistics were used for all data: the mean (M) and the standard error of the mean (m) were calculated. Using the Kolmogorov-Smirnov criterion, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution, Student's t-test (t-test) was used to assess intergroup differences. In the case of a non-normal distribution, the Kruskal-Wallis test (with Dunn's post-analysis) was used to compare multiple groups. Differences were determined at a 5% confidence level.

Inhalation administration of compound I significantly reduced the number of coughs within 10 minutes after inhalation of citric acid solution in guinea pigs. The pharmacological effect was manifested as early as 15 minutes after the administration of the test compound to the animals.

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Table 6

The effect of compound I on the number of coughs within 10 min in a model of citrate cough in guinea pigs when administered by inhalation 15 min before inhalation of a citric acid solution

Groups η Number of coughs in the chamber during inhalation for 10 minutes Cough inhibition (% of pathology control) in chamber Intact 10 0.3±0.2 - Control (placebo) 10 48.8±4* - Compound I (0.25 mg/kg) 10 29.7±2.7*& 39.1

Note.

* - Reliability of differences (P<0.05) with the intact group;

& - significance of differences (P<0.05) with control.

The results obtained allow us to conclude that compound I has a pronounced antitussive effect upon inhalation administration, characterized by a high rate of onset of the effect. Thus, Compound I is useful in the treatment of cough as well as other respiratory diseases such as COPD, bronchitis and asthma.

Example 15 Pharmacokinetic and tissue bioavailability study.

Compound I after oral administration to rats.

Since compound I is an opioid receptor agonist, its high systemic bioavailability and penetration through the blood-brain barrier can potentially lead to the development of side effects.

To confirm the low systemic availability of Compound 1, a study was made of the pharmacokinetics and bioavailability of Compound 1 after oral administration to rats at a dose of 10 mg/kg. The study was carried out on 18 male Wistar rats. Blood samples were taken from animals at predetermined time points within 24 h after drug administration. The content of compound 1 in plasma samples was analyzed by HPLC-MS/MS, the limit of quantitation was 1 ng/ml. The results of the study are given in table. 7.

Table 7

Study of the pharmacokinetics and tissue bioavailability of compound I after oral administration to rats at a dose of 10 mg/kg

Textile η Maximum concentration Compounds I, ng/ml Average concentration within 24 hours, ng/ml blood plasma 6 32.6±30.5 5.5 Brain 6 0±0 0.0

Since in vitro studies have shown that Compound I has a modulating effect on opioid and tachykinin receptors at concentrations above 1 μM, it can be argued that Compound I will not have any toxic effects when administered systemically. Moreover, when administered at a therapeutic dose, Compound I does not enter the brain of rats and cannot have any effects on the CNS of animals.

Example 16 Study of the pharmacokinetics and tissue bioavailability of Compound I after oral administration to mice.

To confirm the low systemic availability of Compound 1 and assess the tissue bioavailability of Compound 1 upon oral administration, a study of the pharmacokinetics and bioavailability of Compound 1 after oral administration to mice at a dose of 10 mg/kg was performed. Blood samples were taken from animals at predetermined time points within 24 h after drug administration. The content of compound 1 in plasma samples was analyzed by HPLC-MS/MS, the limit of quantitation was 1 ng/ml. The results of the study are given in table. 8.

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Table 8

Study of the pharmacokinetics and tissue bioavailability of compound I after oral administration to mice at a dose of 10 mg/kg

Textile η Maximum concentration of Compound I, ng/ml Average concentration Compounds I within 24 hours, ng/ml blood plasma 6 14.2 0.8 Brain 6 8.62 1.5 Spinal cord 6 96 eleven The lymph nodes 6 81 9 Heart 6 32 3 muscles 6 86 9 Stomach 6 34053 1853 Small intestine 6 5949 1331 Colon 6 5299 989

Since in vitro studies have shown that compound I exhibits a modulating effect on opioid and tachykinin receptors at concentrations above 1 μM (i.e., more than 300 ng / ml), it can be argued that when administered orally, compound I will have a pharmacological effect. only receptors located in the tissues of the gastrointestinal tract. Moreover, the average concentration of a substance in the brain is about three orders of magnitude lower than the minimum effective concentration.

Example 17. Study of the effect of compound I on the functional state of the central nervous system of mice.

To finally confirm the safety of compound I when administered orally, a study was made of the effect of compound I on the functional state of the central nervous system of mice in an open field test. The Open Field test consists in assessing the motor and exploratory activity, orienting reaction and emotional reactivity when recording the spontaneous behavior of animals. The study was performed according to the standard method (Buresh Ya., Bureshova O., Joseph P. Houston, Methods and basic experiments for studying the brain and behavior, Moscow, 1991, pp. 119-122).

The study was conducted on male mice of the balb/c line, the individual weight of which deviated from the average value within the sex by no more than ±10%.

Compound I was administered once, intragastrically. The evaluation of the effect on the central nervous system was carried out 2 hours after the administration of compound I in the study of the exploratory behavior of mice in an open field. The open field experimental setup was a 100x100x60 cm chamber with a square floor and white walls. The floor of the chamber is divided into 16 squares, in each square there is a round hole 6 cm in diameter. From above, the chamber is illuminated by a 100 V electric incandescent lamp located at a height of 1 m from the floor of the chamber. The animal was placed in one of the corners of the chamber, and for 15 min the number of horizontal squares it crossed (horizontal activity), rearing up on its hind legs (vertical activity), washing (grooming), defecation acts by the number of fecal pellets was recorded. Then the total motor activity was calculated, which was calculated as the sum of the crossed horizontal squares, standing up on the hind legs and washing.

The data obtained were checked using the Grubbs test for the presence of the largest or smallest anomalous observation (outlier) in the sample. Values identified as outliers in this test were not used for further analysis. Descriptive statistics were used for all data: the mean (M) and the standard error of the mean (m) were calculated. Using the Kolmogorov-Smirnov criterion, the normality of the distribution of the values obtained during the experiment was checked. In the case of a normal distribution, Student's t-test (t-test) was used to assess intergroup differences. In the case of a non-normal distribution, the Kruskal-Wallis test with Dunn's post-analysis was used to compare several groups. Differences were determined at a 5% confidence level. The results of the study are given in table. 9.

Compound I, when administered intragastrically, did not have a toxic effect on the indices of orienting-exploratory behavior in mice in the open field test, as evidenced by the absence of statistically significant differences between the indices of the experimental and control groups of animals.

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Claims (13)

Table 9 The effect of compound I on the functional state of the central nervous system of mice in the open field test after intragastric administration 2 hours before the test Group η Number of squares crossed within 15 minutes (horizontal activity) Number of standing up on hind legs within 15 minutes (vertical activity) Number of washes within 15 minutes (grooming) General physical activity for 15 minutes Number of bowel movements within 15 minutes Intact 10 446.4±19.4 72.5+2.9 7.0±2.2 525.9±20.0 0.5±0.3 Control 10 443.3±15.6 67.1±3.5 8.0±1.5 518.4±15.3 0.2±0.1 Compound I (0.75 mg/kg) 10 468.5±13.0 69.9±4.3 7.2±1.4 545.6±16.5 0.2±0.1 Compound I (1.5 mg/kg) 10 414.2±24.1 63.0±4.2 8.5±1.6 485.7±24.8 1.6±0.6 Compound I (2.5 mg/kg) 10 459.5±19.0 72.8±3.5 8.6±1.6 540.9±18.0 0.0±0.0 Compound I (5 mg/kg) 10 480.6±22.1 71.7±4.4 2.3±1.0 554.6±23.9 1.5±0.2 Compound I (10 mg/kg) 10 449.2±15.6 66.1±3.3 6.8±1.3 522.1±16.5 0.0±0.0 Compound I (20 mg/kg) 10 479.4±29.3 64.9±4.0 6.2±1.3 545.5±30.4 0.0±0.0 Compound I (30 mg/kg) 10 448.4±16.4 69.0±3.8 5.9±1.4 523.3±16.6 0.0±0.0 Example 18 Acute Toxicity Study of Compound I at a Single Intragastric Administration. An acute toxicity study was performed on 24 male rats and 24 female rats, as well as on 24 male mice and 24 female mice. Before the administration of compound I, the body weight of the animals was recorded; after the administration, the condition of the experimental animals was monitored for 1 hour. Further, within 14 days after the introduction of the substance, the body weight of rats and mice was recorded daily and examined in order to identify cases of death and deviations from the norm in the state of animals. Compound I was administered to rats and mice intragastrically at a dose of 5000 mg/kg, intraperitoneally at a dose of 2000 mg/kg. Control groups (females and males) received the solvent (0.1% solution of tween-80 in water). A dose of 2000 mg/kg was intraperitoneally administered 2 times with an interval of 15 minutes due to the poor solubility of the compound. The maximum possible doses of compound I administered intragastrically 5000 mg/kg and intraperitoneally 2000 mg/kg to male and female rats and mice did not cause the death of animals. It has been shown that with intragastric administration of compound I at a dose of 5000 mg/kg and with intraperitoneal administration at a dose of 2000 mg/kg, there is a lag in body weight gain in both male and female mice and rats. There were no differences in spontaneous behavior, responses to evoked reactions of experimental animals relative to the control group. Based on the obtained data, compound I is a moderately toxic compound and belongs to the III class of toxicity in accordance with GOST 12.1.007-76. Thus, in the course of the conducted studies, it was shown that compound 1 is a modulator of opioid and tachykinin receptors and TRPV1 and TRPM8 ion channels. The action on these therapeutic targets allows compound 1 to have a pronounced therapeutic effect in models of cough, abdominal pain, inflammatory and functional bowel diseases. Low systemic bioavailability and lack of penetration into the brain makes it possible to exclude the occurrence of side effects that could occur with the systemic use of such a multitarget agent. While the invention has been described with reference to the disclosed embodiments, it should be apparent to those skilled in the art that the specific experiments described in detail are for the purpose of illustrating the present invention only and should not be construed as limiting the scope of the invention in any way. It should be clear that various modifications are possible without departing from the essence of the present invention. CLAIM 1. Compound 2-phenylethylamide K-(p-hydroxyphenylacetyl)phenylalanine formula or its hydrate. - 17 040699 2. The use of a compound as defined in claim 1 for the prevention and/or treatment of a disorder associated with the activity of opioid and tachykinin receptors and TRPV1 and TRPM8 ion channels. 3. The method of obtaining the compound according to claim 1, including carrying out the reaction between (B)-methyl 2-(2-(4hydroxyphenyl)acetomido)-3-phenylpropanoate and phenylethylamine using a condensing agent in an acetonitrile medium. 4. An opioid receptor agonist, which is a compound according to claim 1. 5. Antagonist of tachykinin receptors and ion channels TRPV1 and TRPM8, which is a compound according to claim 1. 6. Pharmaceutical composition for the prevention and/or treatment of a disorder associated with the activity of opioid and tachykinin receptors and TRPV1 and TRPM8 ion channels, containing a therapeutically effective amount of a compound defined in claim 1 and at least one pharmaceutically acceptable carrier. 7. The pharmaceutical composition of claim 6 wherein the disorder associated with opioid and tachykinin receptor activity is cough, asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, Crohn's disease, colitis, psoriasis, pruritus, atopic dermatitis, and / or itching. 8. The use of a compound defined in claim 1 to obtain a pharmaceutical composition according to claim 6. 9. A method for preventing and/or treating a disorder associated with the activity of opioid and tachykinin receptors or TRPV1 and TRPM8 ion channels in a subject in need of such treatment, comprising administering to said subject a pharmaceutical composition according to claim 6. 10. A method for preventing and/or treating a disorder associated with the activity of opioid and tachykinin receptors or TRPV1 and TRPM8 ion channels in a subject in need of such treatment, comprising administering a therapeutically effective amount of a compound defined in π. 1 to the specified subject. 11. A method for preventing and/or treating cough, asthma, COPD, bronchitis, rhinitis, diarrhea, irritable bowel syndrome, Crohn's disease, colitis, psoriasis, atopic dermatitis, pruritus and/or itching in a subject in need of such treatment, comprising administering a therapeutically effective amount of a compound defined in claim 1 to said subject. 12. The use of a compound defined in claim 1 for the preparation of a medicament. 13. Use according to claim 12, wherein the drug is used to prevent and/or treat a disorder associated with the activity of opioid and tachykinin receptors or TRPV1 and TRPM8 ion channels. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2