RU2594154C2 - CRYSTALLINE FORM OF METHYL ESTER 3-(N-P-TOLYLACETAMIDO) METHYL)-2,3,4,5-TETRAHYDROBENZO[f][1,4]OXAZEPINE-4-CARBOXAMIDE)THIOPHENE-2-CARBOXYLIC ACID, ACTIVE COMPONENT, PHARMACEUTICAL COMPOSITION AND DRUG - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to a crystalline form of methyl ester 3-(7-((N-n-tolylacetamido)methyl)-2,3,4,5-tetrahydrobenzo[f][1,4]oxazepine-4-carboxamide)thiophene-2-carboxylic acid of formula 1, having receptor TGR5 agonist properties, as well as active component, pharmaceutical compositions and medicinal agents based thereon, which stimulate endocrine hormones secretion, for preventing and treating the diseases associated with glucose metabolism.

EFFECT: higher rate of dissolution and high storage stability as compared to the amorphous form.

4 cl, 2 tbl, 2 dwg, 7 ex

Description

The invention relates to a new physiologically active substance, a new non-steroidal agonist of TGR5 receptors - 2,3,4,5-tetrahydro-1,4-benzoxazepine carboxamide, its new crystalline form, an active component for pharmaceutical compositions, pharmaceutical compositions and medicines containing non-steroidal heterocyclic bile acid receptor agonist TGR5, which stimulates the secretion of incretin hormones, a method of treating metabolic diseases such as diabetes, obesity, metabolic syndrome, and kzhe concomitant cardiovascular and neurodegenerative diseases.

According to the World Health Organization, in 2008, 347 million people worldwide were ill with diabetes [WHO Newsletter No. 312. September 2012], while the number of patients more than doubled compared to 153 million in 1980 [Danaei G. et.al. National, regional, and global trends in fasting plasma glucose and diabetes prevalence since 1980: systematic analysis of health examination surveys and epidemiological studies with 370 country-years and 2 7 million participants. Lancet. 2011; 378 (9785): 31-40]. The spread of diabetes is accelerating. In 2010, diabetes was registered in 6.4% of the world population, in 2030 7.7% of patients in the world are projected. [Farag Y, Gaballa M. Diabesity: an overview of a rising epidemic. Nephrol Dial Transplant 2011; 26: 28-35]

Of particular danger is diabetes combined with overweight (obesity or obesity), which is the annual cause of death for more than 280 thousand people in the United States. The new scientific and medical term "Diabesity" (in the Russian version - "Diabetes") has become universally recognized, which means a combination of two interrelated pathologies: diabetes and obesity. At present, diabetes is considered as one of the most serious medical problems of mankind, which has become, in fact, the first non-infectious epidemic [Schmidt MI, Duncan BB. Diabesity: an inflammatory metabolic condition. Clin Chem Lab Med. 2003; 41: 1120-1130], [Dang MN, Hashem BE-S. The epidemiology of obesity. Gastroenterol Clin North Am. 2010; 39: 1-7]. Diabetes has become widespread and rapidly spread over the past century. The main causes of this epidemic are a sedentary lifestyle and an improper diet [Zimmet P, Alberti KG, Shaw J. Global and societal implications of the diabetes epidemic. Nature 2001; 414: 782-787], as well as a genetic predisposition [Ling C, Groop L. Epigenetics: a molecular link between environmental factors and type 2 diabetes. Diabetes 2009; 58: 2718-2725]. Diabetes is also an integral component of the metabolic syndrome.

Currently, such simple methods of treating diabetes, such as special diets, do not lead to a solution to the problem - 98% of those who lose weight gain it in excess for 5 years. Various surgical operations in the fight against diabetes (bariatric surgery) have a noticeable effect, but they are associated with a large number of side effects associated with impaired hormonal homeostasis [Tharakan G, Tan T, Bloom S. Emerging therapies in the treatment of ′ diabesity ′: beyond GLP-1. Trend Pharm Sci. 2011; 32: 8-15]. The many efforts of pharmaceutical companies to create highly effective diabetes and diabetes care products have not yet led to noticeable success. Despite the fact that at present more than 130 new drug candidates are undergoing various tests, no more than ten drugs are approved for use. Clinical trials of a large number of candidates have been suspended due to serious side effects. For example, Avandia, the most promising antidiabetic drug (Rosiglitazone, a PPARgamma receptor agonist), has been shown to increase the risk of heart attack and heart failure [Avorn J. Two centuries of assessing drug risks. New Engl J Med. 2012; 193-197]. The main reasons for the constant failure of researchers are a lack of understanding of the complex picture of the disease, in particular, the presence of its many signaling mechanisms. Exposure to only one effector target leads to the "escape" of the pathological process due to the activation of other mechanisms. Therefore, one of the main directions in the development of antidiabetic drugs is currently the search for pharmacological agents that can stop the development of pathology. In particular, a search is being made for stimulators of the secretion of incretin proteins, mainly GLP1, as well as YYP and GIP. These properties are possessed by bile acids (FAs), which specifically bind to TGR5 FA GPCR receptors.

TGR5 receptors (also known as GPBAR1, M-BAR, AXOR 109, GPR131 and GPCR19) are class A GPCR receptors linked to Gs proteins [Tiwari A, Maiti P. TGR5: an emerging bile acid GPCR target for the potential treatment of metabolic disorders. Drug Disc Today. 2009; 14: 523]. They are expressed mainly in the gall bladder, gastrointestinal tract, spleen, lungs and placenta [Maruyama T. et al. Identification of membrane-type receptor for bile acids (M-BAR). Biochem Biophys Res Commun. 2002; 298: 714]. The binding of FAs to TGR5 receptors activates adenylate cyclase, which leads to an increase in the intracellular concentration of cAMP followed by activation of the MAP kinase signaling cascade [Pols TWH, et al. The bile acid membrane receptor TGR5: a valuable metabolic target. Dig dis. 2011; 29: 37]. On the other hand, stimulation of TGR5 receptors regulates a number of metabolic processes [Chen X. et al. TGR5: a novel target for weight maintenance and glucose metabolism. Exp Diabetes Res. 2011; ID 853501]. In particular, it was shown that the action of agonists on these receptors of STC-1 enteroendocrine cells significantly increases the secretion of the GLP-1 peptide. [Knop FK. Bile-induced secretion of GLP-1: patophysiological implication in type 2 diabetes? Am J Physiol Endocrinol Metab. 2010; E10-E13]. In turn, the GLP-1 peptide improves glucose homeostasis in several ways, including stimulating insulin secretion and suppressing glucagon secretion, which is extremely important in the treatment of diseases such as diabetes, diabetes and obesity.

Since endogenous FAs, by virtue of their steroidal (hormonal) nature, are able to interact simultaneously with many biological targets [Makishima, M .; Okamoto, A.Y .; Repa, J.J .; Tu, H .; Learned, R.M .; Luk, A .; Hull, M.V .; Lustig, K.D .; Mangelsdorf, D.J .; Shanz, B. Science 1999, 284, 1362], including nuclear FXR receptors [Prawitt J. et al. Bile acid metabolism and the pathogenesis of type 2 diabetes. Curr Diab Rep. 2011; 11: 160], a search should be made for selective agonists of TGR5 receptors. The most reasonable is the search among small molecules of a non-steroidal nature. The prospects of this approach are confirmed by a series of drug candidates - non-steroidal TGR5 agonists, which are currently at different stages of clinical and laboratory development.

So it is known the use of sulfamide derivative SB-756050 in the treatment of metabolic disorders and the nervous system [WO 2007127505, SMITHKLINE BEECAM CORPORATION (US)].

Compound 780688 is used to treat or prevent diseases or disorders associated with TGR5 [WO 2012082947, IRM LLC (US)], and Example 99 is used to treat TGR5-mediated disease [WO 2008097976, KALYPSYS, INC. (US)].

New 1-hydroxyimino-3-phenyl-propanes are known, in particular Tauro-RO-5527239, which are GPBAR1 agonists and can be used as drugs for the treatment of diseases such as type II diabetes [WO 2012007365, F. HOFFMANN- LA ROCHE AG (CH)].

TGR5 agonists are known, in particular 735274, as well as their use for the treatment of obesity and type 2 diabetes [WO 2011071565, EXELIXIS, INC. (US)].

Derivatives of triazole and imidazole, in particular 709679, are known for use as TGR5 agonists in the treatment of diabetes and obesity [WO 2010093845, EXELIXIS, INC. (US)].

Agonists of the corresponding TGR5 receptors (GPBAR1, 109 AXOR, GPCR19) are known to be useful for the treatment of type 2 diabetes mellitus and capable of activating human GPBAR1 receptors expressed in CHO cells in the EU ₅₀ cAMP test ₅₀ = 0.001 μM, for example compound 742569 [US 8420647, F. HOFFMANN-LA ROCHE AG (CH)].

Known isoquinolines, GPR131 agonists for the treatment of diabetes, obesity and hyperlipidemia, for example, 712971 [WO 2010117084, MSD K.K. (JP)].

Derivatives of bile acids of the formula I are known as FXR ligands for the prevention or treatment of FXR-mediated diseases or conditions [US 7932244, INTERCEPT PHARMACEUTICALS, INC. (US)].

Heterocyclic compounds of the formula II are described [WO 2010014739, KALYPSYS, INC. (US)] or formula III [WO 2009026241, KALYPSYS, INC. (US)] used as TGR5 modulators and methods for treating or preventing metabolic, cardiovascular and inflammatory diseases.

Agonists of G-protein receptors for bile acids 1 (GPBAR1) of formula IV are known to be potentially useful for treating inflammatory and immune diseases such as rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, psoriasis, type II diabetes and obesity [WO 2008125627, NOVARTIS AG ( CH)].

Heterocyclic TGR5 modulators of Formula V are described for the treatment of insulin-resistant diabetes, rheumatoid arthritis, ulcerative colitis, inflammatory bowel disease, and obesity. The resulting compounds stimulate the production of cAMP in TGR5 transfected HEK-293 cells with an EC ₅₀ value below 10 μM [WO 2008067222, KALYPSYS, INC. (US)].

TGR5 receptor agonists are known containing condensed compounds represented by the general formula VI [WO 2004067008, Takeda Pharmaceutical Co., Ltd. (JP)].

Thus, the search for effective non-steroidal agonists of TGR5 receptors seems to be an extremely important and urgent task.

The listed, although by no means all, TGR5 receptor agonists represent various classes of compounds, but 2,3,4,5-tetrahydro-1,4-benzoxazepine carboxamides are not represented among them.

For the present invention, 1,4-benzoxazepin-3,5 (2H, 4H) dions were not considered as the closest analogues, since the latter have two different carbonyl groups in the oxazepine ring other than 2,3,4,5-tetrahydro- 1,4-benzoxazepines physical and chemical properties (due to changes in the donor-acceptor properties of the compounds) and spatial configurations, as well as excellent methods of preparation.

The research spectrum of 2,3,4,5-tetrahydro-1,4-benzoxazepines is quite wide. This includes treatment of Alzheimer's disease, stroke, neurodegenerative diseases, cognitive impairment due to the ability to regulate serotonin levels, treatment of osteoporosis, sleep disorders, arthritis, diseases mediated by the influence of serotonin 5-HT ₆ and 5-HT _2C receptors, which include obesity . In the vast majority of cases, 2,3,4,5-tetrahydro-1,4-benzoxazepines are studied as antitumor drugs for the treatment of diseases such as breast cancer, mantle cell lymphoma, renal cell cancer, acute myeloid leukemia, chronic myeloid leukemia, lymphoma , rhabdomyosarcoma, ovarian cancer, endometrial cancer, non-small cell lung cancer, small lung cancer, adenocarcinoma, colonel cancer, colon cancer, stomach cancer, hepatocellular carcinoma, melanoma, pancreatic cancer, prostate cancer, thyroid cancer gland, anaplastic large cell lymphoma, glioblastoma, etc.

Such compounds are mainly inhibitors of PI3 kinase and complex 1 mTOR. Perhaps this explains the rather rigid construction of the data structures of 2,3,4,5-tetrahydro-1,4-benzoxazepines, when on the side of the benzene ring all the substituents are aromatic aryl in nature and most of them on the side of the oxazepine ring (formula VII), which is characterized by a sufficiently low level of the number of rotatable bonds in the molecule [US 8648066, EXELIXIS, INC. (US)].

It is known to use these compounds as gamma secretase inhibitors for the treatment of Alzheimer's disease [RU 2332408, F. HOFFMANN-LA ROCHE AG (CH)] or for the selective effect on the activation of serotonin 5-HT _2C receptors and used as a prophylactic and therapeutic agent for urinary tract obesity and / or pelvic organ prolapse [US 8247403, TAKEDA PHARMACEUTICAL COMPANY LIMITED (JP)], as well as the use of these compounds in the treatment of 5-HT ₆ mediated diseases such as Alzheimer's disease, cognitive impairment, cognitive impairment, associated with schizophrenia, obesity and Parkinson’s disease [WO 2007004959, ASTRAZENECA AB (SE)].

More traditional is the use of heterocyclodiazepines as CB2 modulators [WO 2009032754, KALYPSYS, INC. (US)]. Compounds close to them are used to treat autoimmune diseases [WO 2009032754, KALYPSYS, INC. (US)] or in the treatment of insulin resistance and related clinical conditions [US 8263587, PIRAMAL LIFE SCIENCES LIMITED (IN)], as well as ion channel modulators [WO 2013112932, GILEAD SCIENCES, INC. (US)] and its use in the treatment of various disease states, including cardiovascular disease and diabetes [US 8586732, GILEAD SCIENCES, INC. (US)].

It is of interest to use compounds of this class as selective inhibitors of the neuronal isoform of nitric oxide synthase [US 6489322, ASTRA AKTIEBOLAG (SE)], for the selective inhibition or counteraction of αVβ3 integrin [WO 2002018377, PHARMACIA CORPORATION (US)], orexin receptor antagonists [US 7192950 , ACTELION PHARMACEUTICALS LTD. (CH)], active against S1P receptors [EA 17691, GLAXO GROUP LIMITED (GB)], for the treatment of hyperlipidemia [WO 2006129859, TAKEDA PHARMACEUTICAL COMPANY LIMITED (JP)], for the relief of motor disorders associated with the treatment of Parkinson's disease with levodopa [ WO 2009069828, DAYITY SANKE COMPANY LIMITED (JP)], for modulating Btk activity and treating diseases associated with excessive Btk activity [JP 5497152, F. HOFFMANN-LA ROCHE AG (CH)] active to the MCH receptor [WO 2012036233, Shionogi & Co., Ltd. (JP)].

The listed, although by no means all, 2,3,4,5-tetrahydro-1,4-benzoxazepines exhibit various properties, but among them unexplored is stimulation of the secretion of incretin hormones, preferably agonism to TGR5 bile acid receptors.

Thus, the search for effective non-steroidal agonists of TGR5 receptors, namely 2,3,4,5-tetrahydro-1,4-benzoxazepine carboxamides, appears to be an extremely important, relevant and promising task.

In the present invention, the substituent on the side of the benzene ring has much higher mobility, but within the reasonable possibility of forming conformations for better interaction with the active site of the target under study. In the framework of this study, a bile acid receptor TGR5 belonging to a wide class of GPCR was chosen as a biotarget.

The authors of this invention have carried out the synthesis and extensive studies of the activity of various 2,3,4,5-tetrahydro-1,4-benzoxazepine carboxamides, among which 3- (7 - ((N- p- tolylacetamido) methyl ester is the most preferred and promising methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1.

The most relevant is the RF Patent [RU 2456287, ALLA KHEM, LLS (US)], which describes stimulators of the secretion of incretin hormones, non-steroid compounds derivatives of 2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepines, which are interest as medicinal substances for the creation of new drugs intended for the prevention and treatment of metabolic diseases.

The disadvantage of this series of compounds is the difficulty in obtaining a pure prodrug, since the preparation takes place in several stages — cyclization and reduction with lithium aluminum hydride to obtain an intermediate product, followed by subsequent acylation of the intermediate product with a biaryl acyl halide. This makes it difficult to obtain a medicinal candidate and conduct clinical trials.

It is also known that the 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophen-2- methyl ester compound carboxylic acid is registered in the CAS Registry under the number 951623-060-6 and is offered for sale by ChemDiv, Inc (US).

However, with respect to compound C AS 951623-060-6, so far there is only a message that this compound was obtained by the combinatorial method by purification using column chromatography, but neither its properties nor its crystalline form are known.

It is necessary to find out whether this compound is able to crystallize and have crystalline forms. Then, if crystalline forms exist, it is necessary to find reproducible methods for the preparation of chemically stable crystalline forms and to identify new crystalline forms of this compound.

Solid stability and shelf life of active components are very important factors. A drug compound and compositions comprising it must be able to be effectively stored for significant periods of time without showing a significant change in the physicochemical properties of the active component (for example, its chemical composition, density, hygroscopicity and solubility). In addition, it is also important to present the drug in a form that is as clean as possible. In this regard, amorphous compounds can present significant problems. For example, it is more difficult to handle such compounds and include them in dosage forms compared with the crystalline compound, due to dubious solubility, and it often turns out that they are unstable and chemically contaminated. One skilled in the art will understand that if a drug can be easily obtained in a stable crystalline form, then the above problems can be solved. It is usually impossible to predict only on the basis of molecular structure what the behavior of the compound will be during crystallization, and usually this can only be established empirically.

Thus, it is an object of the present invention to find new stable crystalline forms of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine- methyl ester 4-carboxamido) thiophene-2-carboxylic acid.

The technical result of the present invention is to improve the properties of this compound when it is used, in particular, a higher dissolution rate and increased storage stability.

The task and technical result are achieved by obtaining a new crystalline form of methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido ) thiophene-2-carboxylic acid of formula 1, or a pharmaceutically acceptable salt or hydrate, or racemic mixtures thereof or individual optical isomers, with diffraction peaks in a powder X-ray diffraction pattern, which characterize the following parameters: diffraction angles (2θ, °), intensity (I ,%).

The subject of the present invention is a new crystalline form of methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene 2-carboxylic acid of the formula 1, having characteristic peaks on the x-ray powder diffraction pattern at the following diffraction angles 2θ (± 0.10 °): 8.9; 12.5; 15.4; 17.8; 19.6; 21.8; 25.4; 27.1

The subject of the present invention is an active component having the property of an incretin hormone secretion stimulating agent, preferably the properties of a TGR5 bile acid receptor agonist, which is a new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) methyl ester -2,3,4 , 5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1. Nonsteroidal bile acid receptor agonists specifically bind to TGR5 receptors and stimulate the secretion of incretin hormones (GLP1, YYP, GIP), therefore nai olee is preferably TGR5 receptor agonists.

The subject of the present invention is a pharmaceutical composition for the prophylaxis and treatment of metabolic diseases associated with glucose metabolism and related cardiovascular and neurodegenerative diseases, containing in an effective amount an active component having the property of a stimulator of the secretion of incretin hormones, preferably the properties of a TGR5 bile acid receptor agonist, the present invention.

The pharmaceutical composition may include pharmaceutically acceptable excipients. By pharmaceutically acceptable excipients are meant diluents, excipients and / or carriers used in the pharmaceutical field. The pharmaceutical composition along with the active component of the present invention may include other active ingredients, provided that they do not cause undesirable effects, such as allergic reactions. If it is necessary to use the pharmaceutical compositions of the present invention in clinical practice, they can be mixed for the manufacture of various forms, while they can include traditional pharmaceutical carriers; for example, oral forms (such as tablets, gelatine capsules, pills, solutions or suspensions); injection forms (such as injectable solutions or suspensions, or dry powder for injection, which only requires the addition of water for injection before use); local forms (such as ointments or solutions).

The carriers used in the pharmaceutical compositions of the present invention are carriers that are used in the pharmaceutical field to obtain common forms, including: in oral forms, binders, lubricants, disintegrants, solvents, diluents, stabilizers, suspending agents, colorless are used agents, flavoring agents of taste; antiseptic agents, solubilizers, stabilizers are used in injection forms; in local forms, bases, diluents, lubricants, antiseptic agents are used.

The pharmaceutical composition of the present invention can be obtained by mixing the active component, which is a new crystalline form of methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1, 4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1 with an inert excipient, solvent and / or other pharmaceutically acceptable excipients.

An object of the present invention is a medicament for the prophylaxis and treatment of metabolic diseases associated with glucose metabolism in the form of tablets, capsules, injections, rectal suspensions or gels in a pharmaceutically acceptable package containing in an effective amount an active ingredient or pharmaceutical composition of the present invention.

An object of the present invention is a method for the prevention and treatment of metabolic diseases associated with glucose metabolism, according to which a needy subject is administered an effective amount of an active component having the property of an incretin hormone secretion stimulating agent, preferably the properties of a TGR5 bile acid receptor agonist, or a pharmaceutical composition or drug of the present invention.

Medicines may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, topically or rectally). The clinical dosage of the active component (s), pharmaceutical composition or combination drug comprising a pharmaceutically effective amount of the active component in patients can be adjusted depending on the therapeutic efficacy and bioavailability of the active ingredients in the body, their metabolic rate and excretion from the body, as well as depending on the age, sex and stage of the patient’s disease, while the daily dose in adults is usually 10 ~ 500 mg, preferably 50 ~ 300 mg . Therefore, during the preparation of the pharmaceutical compositions of the present invention in the form of dosage units, the above effective dosage should be taken into account, with each dosage unit of the preparation containing 10 ~ 500 mg, preferably 50 ~ 300 mg. In accordance with the instructions of a doctor or pharmacist, these drugs can be taken several times during certain periods of time (preferably from one to six times).

The following are definitions of terms that are used in the description of the present invention.

“Agonists” means compounds that, by binding to receptors of a particular type, actively contribute to the transmission by these receptors of their specific signal and thereby elicit a biological response from the cell.

“Azaheterocycle” means an aromatic or non-aromatic monocyclic or polycyclic system containing at least one nitrogen atom in a ring.

"Active component" (drug substance, drug substance, drug-substance) means a physiologically active substance of synthetic or other (biotechnological, plant, animal, microbial and other) origin, having pharmacological activity and is the active principle of the pharmaceutical composition used for the manufacture and manufacture medicinal product (means).

“Antagonists” means ligands that bind to receptors of a particular type and do not elicit an active cellular response. Antagonists inhibit the binding of agonists to receptors and thereby block the transmission of a specific receptor signal.

“Annelated cycle” (condensed ring) means a bi- or polycyclic system in which the annelated cycle and the cycle or polycycle with which it is “annelated” have at least two atoms in common. Aromatic, non-aromatic systems and heterocyclic systems can be annelated.

“Aromatic cycle” (aromatic system) means a planar cyclic system in which all atoms of the cycle participate in the formation of a single conjugation system, which, according to the Hückel rule, includes (4n + π) π-electrons (n is a non-negative integer). Examples of aromatic cycles are benzene, naphthalene, anthracene, and the like. In the case of “heteroaromatic cycles”, π-electrons and p-electrons of heteroatoms participate in the conjugation system; their total number is also equal to (4n + 2). Examples of such cycles are pyridine, thiophene, pyrrole, furan, thiazole and the like. An aromatic ring may have one or more “ring system substituents” and may be anelated with a non-aromatic ring, heteroaromatic or heterocyclic ring.

“Heterocyclyl” means an aromatic or non-aromatic saturated monocyclic or polycyclic system comprising from 3 to 10 carbon atoms, preferably from 5 to 6 carbon atoms, in which one or more carbon atoms are replaced by a heteroatom such as nitrogen, oxygen, sulfur. The prefix "aza", "oxa" or "thia" before heterocyclyl means the presence in the cyclic system of a nitrogen atom, an oxygen atom or a sulfur atom, respectively. Heterocyclyl may have one or more “cyclic system substituents,” which may be the same or different. The nitrogen and sulfur atoms in the heterocyclyl can be oxidized to N-oxide, S-oxide or S-dioxide. Representative heterocyclyls are piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiophenyl, etc.

"Hydrate" means a stoichiometric or non-stoichiometric composition of a compound or its salt with water.

“Bile acids (endogenous bile acids, FAs)” - The main types of bile acids present in the human body are the so-called primary bile acids (primarily secreted by the liver): cholic acid (3α, 7α, 12α-trioxy-5β-cholanic acid) and chenodeoxycholic acid (3α, 7α-dioxi-5β-cholanic acid), as well as secondary (formed from primary bile acids in the colon under the influence of intestinal microflora): deoxycholic acid (3α, 12α-dioxi-5β-cholanoic acid), lithocholic (3α-manooxy-5β-cholanoic acid), allochole uric acid and ursodeoxycholic acid. Of the secondary ones in the intestinal-hepatic circulation, only deoxycholic acid, which is absorbed into the blood and then secreted by the liver as part of the bile, is involved in the amount that affects the physiology. Allocholic, ursodeoxycholic and lithocholic acids are stereoisomers of cholic and deoxycholic acids. All human bile acids have 24 carbon atoms in their molecules. Bile acids are designed to stimulate incretin hormones.

“Substituent” means a chemical radical that binds to a scaffold (fragment), for example, “substituent alkyl”, “substituent of an amino group”, “substituent carbamoyl”, “substituent of the cyclic system”.

“Medicinal product (preparation)” - a combination of several medicinal substances for simultaneous use in the form of tablets, capsules, injections, ointments, rectal suspensions and gels and other finished forms, designed to restore, correct or alter physiological functions in humans and animals, and also for the treatment and prevention of diseases, diagnosis, anesthesia, contraception, cosmetology and other things. Medicinal substances in one set can be presented in the form of various ready-made forms intended for administration into an animal or human body in various ways, for example, orally and rectally.

"Ligands" (from Latin ligo - bind) are chemicals (small molecule, inorganic ion, peptide, protein, etc.) that can interact with receptors that transform this interaction into a specific signal.

"Metabolic diseases" - a group of diseases associated with metabolic disorders. These diseases can be caused by a hereditary predisposition, disruptions in the endocrine and nervous systems, as well as disturbances in the functioning of metabolically active organs. Currently, there are a large number of metabolic disorders, however, this invention mainly addresses diseases associated with the signaling cascade of TGR5 receptors, in particular, such as diabetes, obesity, diabetes, metabolic syndrome, hypercholesterolemia, dyslipidemia, etc.

“Neurodegenerative disease” means a specific condition and disease characterized by damage and primary death of populations of nerve cells in certain areas of the central nervous system. Neurodegenerative diseases include, but are not limited to, Alzheimer's and Parkinson's disease; Huntington's disease (chorea); multiple sclerosis; cerebellar degeneration; amyotrophic lateral sclerosis; dementia with Levy bodies; spinal muscular atrophy; peripheral neuropathy; spongiform encephalitis ("mad cow disease", Creutzfeld-Jakob Disease); AIDS-related dementia; multi-infarct dementia; frontotemporal dementia; leukoencephalopathy (a disease of endangered white matter); chronic neurodegenerative diseases; stroke; ischemic, reperfusion, and hypoxic brain damage; epilepsy; cerebral ischemia; glaucoma; traumatic brain injury; Down syndrome; encephalomyelitis; meningitis; encephalitis; neuroblastoma; schizophrenia; depression. In addition, neurodegenerative diseases include pathological conditions and disorders that develop with hypoxia, substance abuse, exposure to neurotoxins, infectious and oncological diseases of the brain, as well as neuronal damage associated with autoimmune and endocrine diseases; and other neurodegenerative processes.

"Receptors" (from the Latin recipere - to receive, to recognize) are biological macromolecules located on the cytoplasmic membrane of a cell or intracellular, capable of specifically interacting with a limited set of physiologically active substances (ligands) and transforming the signal about this interaction into a specific cellular response.

“TGR5 receptors” receptors (also known as GPBAR1, M-BAR, AXOR 109 and GPCR19) are GsR receptors associated with Gs proteins. They are expressed mainly in the gall bladder, gastrointestinal tract, spleen, lungs, placenta, as well as in immune cells (monocytes and dendritic cells).

“Signal cascade” (signal system, signal transmission cascade) means a set of interconnected sequential and parallel molecular processes of regulation of cellular metabolism by external (primary) signals that carry information into the cell, which fundamentally distinguishes them from other chemical compounds entering the cell that serve as its source matter and energy. The molecular mechanisms of transmission (transduction) of external signals into the cell imply not only the transmission of signals as such, but also the whole complex of events associated with it, including amplification, attenuation and suppression (or switching off) of signals.

"Steroids" are a group of natural, semi-synthetic and synthetic compounds - derivatives of partially or fully hydrogenated 1,2-cyclopentenophenanthrene, in the molecular skeleton of which 17 carbon atoms form 4 articulated rings. Steroids are widespread in nature, they are involved in the implementation of a wide variety of biological functions. The steroid nature is sex hormones, vitamin D, adrenal hormones, bile acids, metamorphosis hormones and arthropod repellents, plant phytosteroids, etc. Steroids with a similar structure exhibit very different physiological activity, they are widely used in medicine as anti-inflammatory, cardiac, contraceptive and other agents. When it is necessary to avoid the hormonal effects of steroids, so-called "non-steroidal" drug agents are developed.

"Pharmaceutical composition" means a composition comprising a compound of general formula 1 and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, excipients, distributing and perceiving agents, delivery vehicles such as preservatives, stabilizers, fillers, grinders, moisturizers, emulsifiers, suspending agents, thickeners, sweeteners, perfumes, flavors, antibacterials flax agents, fungicides, lubricants, and prolonged delivery controllers, the choice and suitable proportions of which depend on the nature and way of administration and dosage.

Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as mixtures of these substances. Protection against the action of microorganisms can be achieved using a variety of antibacterial and antifungal agents, for example, such as parabens, chlorobutanol, sorbic acid and the like. The composition may also include isotonic agents, for example, sugars, sodium chloride and the like. The prolonged action of the composition can be achieved using agents that slow down the absorption of the active principle, for example aluminum monostearate and gelatin.

Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, and also mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of excipients are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of grinders and distributors are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. The pharmaceutical composition for oral, sublingual, transdermal, intramuscular, intravenous, subcutaneous, local or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard administration form, in the form of a mixture with traditional pharmaceutical carriers.

Suitable unit dosage forms include oral forms such as tablets, gelatine capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, local, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms. Pharmaceutical compositions are generally prepared using standard procedures involving the mixing of the active compound with a liquid or finely divided solid carrier. For the manufacture of suppositories, in addition to the active components, cocoa butter, its alloys with paraffin and hydrogenated fats, vegetable and animal hydrogenated fats, solid fat, lanol, alloys of hydrogenated fats with wax, hard paraffin and other bases approved for medical use are also used.

“Pharmaceutically acceptable salt” means the relatively non-toxic organic and inorganic salts of the acids and bases of the present invention. These salts can be obtained in situ during the synthesis, isolation or purification of the compounds or prepared specially. In particular, base salts can be prepared on the basis of the purified free base of the claimed compound and a suitable organic or inorganic acid. Examples of salts thus obtained are hydrochlorides, hydrobromides, sulfates, bisulfates, phosphates, nitrates, acetates, oxalates, valeriates, oleates, palmitates, stearates, laurates, borates, benzoates, lactates, tosylates, citrates, maleates, fumarates, succinates, tartrates mesylates, malonates, salicylates, propionates, ethanesulfonates, benzenesulfonates, sulfamates and the like. (A detailed description of the properties of such salts is given in Berge S. M., et al., "Pharmaceutical Salts" J. Pharm. Sci. 1977, 66: 1-19).

Salts of the claimed acids can also be specially prepared by reacting the purified acid with a suitable base, and metal and amine salts can be synthesized. Metal salts include sodium, potassium, calcium, barium, zinc, magnesium, lithium and aluminum salts, the most desirable of which are sodium and potassium salts. Suitable inorganic bases from which metal salts can be obtained are hydroxide, carbonate, sodium bicarbonate and hydride, potassium hydroxide and bicarbonate, potash, lithium hydroxide, calcium hydroxide, magnesium hydroxide, zinc hydroxide. As organic bases from which salts of the claimed acids can be obtained, amines and amino acids are selected that are sufficiently basic to form a stable salt and are suitable for medical use (in particular, they should have low toxicity). Such amines include ammonia, methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, benzylamine, dibenzylamine, dicyclohexylamine, piperazine, ethylpiperidine, tris (hydroxymethyl) aminomethane and the like. In addition, tetraalkylammonium hydroxides, for example, such as choline, tetramethylammonium, tetraethylammonium and the like, can be used for salt formation. As amino acids, the main amino acids can be used - lysine, ornithine and arginine.

"Pharmaceutically acceptable excipients" by pharmaceutically acceptable excipients refers to pharmaceutical diluents, excipients and / or carriers.

The invention is also illustrated by the drawings.

FIG. 1. X-ray powder diffraction pattern of a new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene methyl ester -2-carboxylic acid of the formula 1 obtained according to the present invention.

FIG. 2. Concentration dependence of the activity of the new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene methyl ester -2-carboxylic acid of formula 1 obtained according to the present invention, in relation to the hTGR5 receptor.

The following examples illustrate but do not limit the invention.

Example 1. The synthesis method of the compounds of formula 1. The synthesis is carried out in accordance with Scheme 1.

A solution of compound 2 (150 g, 0.75 mol) and ethanolamine (68.6 g, 1.5 equiv.) In toluene (1 l) is boiled with a Dean-Stark trap for 1 hour. Then the toluene is evaporated, another portion of toluene (0.5 L) is added and evaporated again. The resulting mass was dissolved in ethanol and NaBH ₄ (42.8 g, 1.5 equiv.) Was added portionwise with stirring. After 1 hour of stirring at room temperature, the solvent was evaporated, and the resulting viscous mass was dissolved in aqueous ammonia (0.6 L). The solution was separated from the insoluble precipitate and allowed to crystallize. To the resulting suspension of 3 (142.8 g, 0.58 mol) in dichloromethane (1 L), triethylamine (116.3 g, 2 equiv.) And boc2O (188.3 g, 1.5 equiv.) Were successively added and stirred overnight at room temperature. Then, the solvent and triethylamine were partially evaporated at 60 ° C, and hexane (0.5 L) was added to the resulting viscous mass. The precipitated solid precipitate was filtered off and washed with hexane (2 × 300 ml). 152.4 g of pure product 4 are obtained (yield 76%).

DIAD diisopropylazodicarboxylate (88.9 g, 0.44 mol) is added in small portions to a solution of 4 (152.4 g, 0.44 mol) and triphenylphosphine (114.7 g, 0.44 mol) in tetrahydrofuran (THF) (1 L) and stirred overnight at room temperature. Then the solvent is evaporated, and the resulting mass is chromatographed with ether: hexane (1: 3) on silica gel (6 liters). 136.7 g of product 5 are obtained (yield - 95%).

To a solution of compound 5 (6.3 g, 19 mmol) in 30 ml of THF at -70 ° C was added a 2.5 M solution of BuLi in hexane and after 1 h - DMF (2.2 ml, 28 mmol). The reaction mass is kept for 2 hours at room temperature, diluted with water and extracted with chloroform (2 × 100 ml). The organic layer is evaporated (78%) and applied to a column (height 70 mm and diameter 100 mm) with silica gel 230-400 M. Elute with a mixture (328 ml) of hexane: ether (70:30), isolate (after evaporation on a rotor and drying under vacuum) 3.5 g (70%) of compound 6. Next, a solution of compound 6 (1.2 g, 4.3 mmol), n-methylaniline (4.3 mmol) and TsOH (0.05 g) in toluene (20 ml) is boiled for 2 hours with a Dean nozzle -Star, then completely evaporated, the residue 7 is dissolved in ethanol (15 ml) and a twofold excess of sodium borohydride is added. After an hour, the reaction mass was diluted with 5% aqueous potash solution (100 ml) and extracted with chloroform (2 times 100 ml). The organic layer was dried with anhydrous sodium sulfate, evaporated completely and the resulting technical compound 8 was dissolved in 100 ml of CH ₂ Cl ₂ and triethylamine (0.62 ml, 4.3 mmol) and acetyl chloride (4.3 mmol) were added.

The reaction mass was incubated for 3 hours, completely evaporated, dissolved in 50 ml of methanol and 5 ml of concentrated hydrochloric acid was added. After 12 hours, the reaction mass was completely evaporated, diluted with water (100 ml), extracted with CH ₂ Cl ₂ (2 times 50 ml) and applied to a short column (height 35 mm and diameter 50 mm) with silica gel (230-400 M). Elute with a mixture (150 ml) of hexane: CH ₂ Cl ₂ : MeOH (5: 5: 1), 0.7 g (52%) of compound 9 is isolated.

To 0.063 g (0.000211 mol) of triphosgene dissolved in 1 ml of dichloromethane, 1 ml of a solution containing compound 9 (0.00057 mol) and diisopropylethylamine DIPEA (0.00126 mol, 0.163 g) are added. Stir the resulting mass at room temperature in an argon atmosphere for 30 minutes, and then add amine 10 (0.00057 mol) and DIPEA (0.081 g, 0.00063 mol) dissolved in 1 ml of dichloromethane. The mixture after stirring at room temperature (2 h) was diluted with 30 ml of dichloromethane, washed with 2 N. HCl solution (20 ml), dried over sodium sulfate and chromatographed with a mixture of CH ₂ O ₂ / EtOH (column height 4 cm, width 1 cm). Get 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid methyl ester of formula 1 , LC MS m / z 494 (M + 1), with a yield of 70%.

Example 2. Obtaining a crystalline form of methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophen-2 -carboxylic acid of the formula 1.

10 mg of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid methyl ester of the formula 1 is dissolved in 10 ml of methanol, heating the mixture to 42 ° C. To the solution obtained as a result, 10 ml of water was added dropwise at the same temperature over 20 minutes, and the resulting mixture was cooled to 5 ° C during the day, after which it was filtered off and the crystalline material was collected. The crystals were dried at 50 ° C. under reduced pressure until the mass of crystals ceased to decrease upon further drying, resulting in the production of 9.52 mg of a white solid.

X-ray powder analysis of the obtained crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2 methyl ester β-carboxylic acid of formula 1 is carried out using a Rigaku diffractometer with a Saturn 944 HG detector and CuKα copper radiation. On the spectrum of the powder x-ray diffractogram of a crystalline substance (Fig. 1), characteristic peaks are observed at diffraction angles of 2θ (± 0.10 °): 8.9; 12.5; 15.4; 17.8; 19.6; 21.8; 25.4; 27.1.

Example 3. Determination of the agonistic activity of the compounds of formula 1 in relation to the hTGR5 receptor.

The agonistic activity of the library of compounds, including the compound of formula 1, is determined using the HEK-293 cell line in which the human TGR5 receptor (hTGR5) is stably expressed. The agonistic activity of the compounds is evaluated by increasing the intracellular concentration of cAMP, since it is known from the literature that activation of the hTGR5 receptor by subsequent activation of cellular adenylate cyclase leads to an increase in the intracellular content with AMP. As an experimental platform, the Lance Ultra cAMP test system (Perkin Elmer) is chosen. Known agonists of the hTGR5 receptor are used as a positive control and to determine the absolute value of the maximum signal of the experiment. The substances are tested at a final concentration of 10 μM, at one point. Substances exhibiting agonistic activity of more than 50% of the maximum signal, 3 × SD (minimum signal), are nominated for a repeat test at a concentration of 10 μM in replicates to confirm the activity shown.

For the most active compounds, the dependence of the agonistic activity of the substance on the concentration of the substance is analyzed. From the obtained dependency graphs, the EC ₅₀ values are determined (the concentration of the substance in micromoles, which causes 50% of the maximum effect).

Cell line: - an immortalized HEK 293 line stably expressing hTGR5, Millipore, catalog number HTS238L. The cell culture medium is a mixture of DMEM, 10% fetal bovine serum, 1% non-essential amino acids, 2 mM L-glutamine, and 1 μg / ml puromycin / 400 μg / ml G418 / 200 μg / ml hygromycin B as a selective mixture antibiotics.

Cells are thawed in a water bath at 37 ° C, resuspended in 5 ml of Hanks solution, centrifuged and again resuspended in 6 ml of Hanks solution. Thus, the cells are washed from serum and culture medium. The centrifugation step is repeated and the cells are resuspended in the stimulation buffer of the test system (SB). The final cell concentration was 2.00 × 10 ⁵ cells / ml, which yielded 1000 cells per well. cAMP antibodies are added to the cell suspension in a ratio of 1: 150.

The substances are dissolved in dimethyl sulfoxide (DMSO) to a concentration of 2.5 tM. Tablets with substances in DMSO are stored at -20 ° C. Experimental buffer (SB) was prepared: 1x HBSS, 5 mM HEPES, 0.1% BSA, pH 7.4. 45 ml of HBSS, 225 μl of 1M HEPES and 600 μl of 7.5% BSA were mixed.

A cell suspension including a 4-fold solution of anti-cAMP antibodies was added to 384-well experimental plates, 5 μl per well using a Biomek 2000 automated system (Beckman Coulter). Centrifuge at 300 rpm, 1 minute. A 2-fold mixture of [SB + substance] was added to the cell suspension using the Biomek FX 384 robotic system. To prepare a 2-fold mixture of [SB + substance], SB was transferred to a 384 well plate, 99 μl / well. 1 μl of a 200-fold working solution of substances in DMSO is transferred to the same 384-well plate. The resulting mixture was thoroughly mixed. 5 μl of a 2-fold mixture of [SB + substance] is added to the cell suspension at 5 μl per well. The experimental plate is centrifuged at 300 rpm, 1 minute. Next, the cell suspension is incubated with substances for 30 minutes at room temperature on a shaker, 300 rpm.

The original solution of the Eu-cAMP tracer is diluted 1: 100 in the buffer for signal detection, obtaining a 2-fold working solution of the tracer. Next, the solution was added to the experimental plate, 10 μl, using a Biomek 2000 robotic system. The cell suspension was incubated with a solution for signal detection for 60 minutes on a shaker, 300 rpm.

The essay signal is measured on a Wallac 1420 Victor ³ multifunction reader (Perkin Elmer) using a wavelength of 665 nm, LANCE protocol High Count_665_OptiPlate.

Agonistic activity of substances is calculated using a script for processing Grand CaLculate_0.1.1 data. As the minimum signal of the experiment, the averaged data of wells 23 of the column of the experimental plate containing unstimulated cells were used. As the maximum signal of the experiment, the averaged data of all wells of the 24th column of the experimental plate were used, containing cells stimulated by the known steroid agonist hTGR5.

Activation of the hTGR5 receptor by a new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2 methyl ester -carboxylic acid of the formula 1 at a given concentration of 10 μM is 94%. Data is analyzed using GraphPad Prizm software (GraphPad Software, Inc., San Diego, CA). To build the concentration dependence, choose the equation:

where Thor and Bottom are the maximum and minimum concentrations, HillScope is the Hill coefficient, and the EU ₅₀ is determined on the basis of the constructed graph as the concentration of a substance that causes 50% of the maximum effect. At a Hill coefficient of 4.117, the EC50 value of the new crystalline form of methyl ester is 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1 is 640 nM (Fig. 2).

Example 4. Metabolic model and study of the effect of the compounds of formula 1 on the secretion of the incretin peptide GLP-1.

The test compound is a new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2 methyl ester -carboxylic acid of formula 1 - (dose of 30 mg / kg) in an aqueous solution of carboxymethyl cellulose (0.5%) and Tween-20 (0.25%) are orally administered to C57B L / 6J DIO mice (6 mice per time point) 15 minutes before oral administration the introduction of dextrose (2 g / kg in saline). Blood for analysis is collected from the tail vein of animals 5, 10, 15 and 30 minutes after the introduction of sugar. As a control, an aqueous solution of carboxymethyl cellulose (0.5%) and Tween-20 (0.25%) are introduced.

DIO mice (Diet Induced Obesity mice) are widely used to model the metabolic syndrome in humans, which is characterized by abdominal obesity, high triglycerides, impaired glucose homeostasis and hyperinsulinemia [Hariri N, Thibault L. High-fat diet -induced obesity in animal models. Nutr Res Rev. 2010; 23 (2): 270-99]. To create a DIO model, C57B L / 6J mice are used, which are fed a high fat diet (HFD); food includes 58% lard. Diet D12492, Research Diet, New Brunswick, NJ; West D. et al. 1992). The HFD diet and water provide ad libitum animals for 28 days prior to the start of the experiment.

The plasma incretin peptide GLP-1 is determined by standard ELISA. As a result of stimulation of GLP-1 secretion, after 15 minutes of the experiment, the blood GLP-1 incretin peptide content in the blood exceeded 6 ng / mmol and 30 minutes after glucose administration, the blood GLP-1 incretin peptide content in the blood exceeded 5.5 ng / mmol (or 30 and 45 minutes after administration of the compound). Introduction methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1 (30 mg / kg) in all cases significantly and statistically significantly stimulates the secretion of the hormone GLP-1.

Example 5. The study of the effect of the compounds of formula 1 on glucose metabolism (glucose tolerance test GTT).

Test compound - 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid methyl ester formulas 1 - (doses from 5 to 100 mg / kg) in an aqueous solution of carboxymethyl cellulose (0.5%) and Tween-20 (0.25%) was administered orally to DIO mice C57B L / 6J (6 mice per time point) 15 minutes before oral administration the introduction of dextrose (2 g / kg in saline). Blood for analysis was collected from the tail vein of animals 0, 5, 10, 20, 30, 60, and 120 minutes after the introduction of sugar. An aqueous solution of carboxymethyl cellulose (0.5%) and Tween-20 (0.25%) was introduced as a control.

The data obtained from the experiment show a significant statistically significant decrease in the glucose content in the plasma of DIO mice under the action of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo methyl ester [f ] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1:

Plasma glucose 20 minutes after the start of the experiment - 1000 mg / l;

Plasma glucose after 60 minutes after the start of the experiment - 780 mg / l;

Plasma glucose after 120 minutes after the start of the experiment - 560 mg / l;

The results clearly indicate the promise of using 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2 methyl ester β-carboxylic acid of formula 1 in the approach to the treatment of metabolic diseases.

Example 6. Determination of the kinetics of dissolution of the crystalline form.

Dissolution kinetics of a new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophen-2- methyl ester carboxylic acid of the formula 1 obtained in example 2, is compared with the kinetics of dissolution of amorphous compounds With AS 951623-060-6. The device for determining the dissolution rate is a three-necked vessel with a capacity of 1 liter. A thermometer is introduced into one of the tubes, a glass tube for sampling and their integration in the other, and the main part of the device is a cylindrical basket 3.6 cm high and 2.5 cm in diameter, made of stainless steel in the form of a mesh with holes 40 mesh diameter (about 0.351 mm). The basket is mounted on the axis of the motor.

A dissolving medium (1000 ml) is poured into the vessel, in this experiment DMSO, since it is an important bipolar aprotic solvent and, due to its strong dissolving ability, DMSO is often used as a solvent in chemical reactions and a biological screening system. The test sample is placed in a cylindrical basket, which is installed at a distance of 2 cm from the bottom of the vessel.

For comparative sample 1, 200 mg of a new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido methyl ester is used ) thiophene-2-carboxylic acid of the formula 1, and for a comparative sample 2-200 mg of amorphous compound C AS 951623-060-6 to obtain, after complete dissolution, a solution containing 200 ppm Compounds C AS 951623-060-6.

The temperature of the solvent medium during the experiment is kept constant (37 ± 0.5 ° C). The rotation speed of the basket in the medium is regulated with an accuracy of ± 5%, it is 200 rpm. At set intervals, 1-2 ml samples are taken for analysis to determine the drug content. The taken volume of solvent is immediately replaced with a new one.

The results are shown in table 1 (where the values are the number (ppm) of samples 1 or 2 in solution:

The results demonstrate that the dissolution rate of the new crystalline form of 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido methyl ester) thiophene-2-carboxylic acid of formula 1 in DMSO is higher than the dissolution rate of the amorphous compound CAS 951623-060-6. In particular, the time during which 50% dissolution of the new crystalline form of methyl 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1 is two times less than for the comparative sample of the amorphous compound CAS 951623-060-6.

Example 7. Investigation of storage of crystalline form

All samples were stored in glass vials, sealed with rubber stoppers with aluminum caps, in a refrigerator at a temperature of ~ 4.7 ° C and in a freezer at a temperature of about -23 ° C. The amount of impurities was determined by the internal normalization method; detection was carried out by a UV detector at a wavelength of 220 nm.

Table 2 presents the test results of a new crystalline form of methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1, indicating the improvement of its stability at low temperature compared with the amorphous compound CAS 951623-060-6.

The results clearly indicate the promise of the proposed new crystalline form of methyl ester 3- (7 - ((N- p- tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1.

The present invention can be used in medicine, veterinary medicine, biochemistry.

Claims (4)

1. The crystalline form of the methyl ester of 3- (7 - ((Nn-tolylacetamido) methyl) -2,3,4,5-tetrahydrobenzo [f] [1,4] oxazepine-4-carboxamido) thiophene-2-carboxylic acid of the formula 1, having characteristic peaks on a powder X-ray diffractogram at the following angles 2θ (± 0.10 °): 8.9; 12.5; 15.4; 17.8; 19.6; 21.8; 25.4; 27.1

2. An active component having the property of a stimulator of the secretion of incretin hormones, preferably the properties of a TGR5 bile acid receptor agonist, which is a compound of formula 1 according to claim 1. 3. A pharmaceutical composition for the prevention and treatment of metabolic diseases associated with glucose metabolism, containing in an effective amount the active component according to claim 2. 4. A drug for the prevention and treatment of metabolic diseases associated with glucose metabolism in the form of tablets, capsules, injections, rectal suspensions or gels, placed in a pharmaceutically acceptable package containing in an effective amount an active ingredient according to claim 2 or a pharmaceutical composition according to claim 2 . 3.

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