Novel salts of heterocyclic compound as protein kinase inhibitor and uses thereof

Abstract

To provide novel salts of a heterocyclic compound as a protein kinase inhibitor and uses thereof.SOLUTION: The present invention relates to novel salts of a heterocyclic compound as a protein kinase inhibitor and uses thereof. The novel salts are excellent in terms of water solubility and physical and chemical stability and thus can be usefully employed in formulating medicinal products. In addition, the heterocyclic compound or the salts thereof can effectively treat and prevent atopic dermatitis and inflammatory bowel disease.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to novel salts of heterocyclic compounds as protein kinase inhibitors and their use. [Background technology]

[0002] In general, it is an obvious fact that the same drug may have differences in pharmaceutically important properties such as solubility, dissolution characteristics, and bioavailability between its various forms, such as amorphous, one or more crystalline forms, salts, etc. Numerous types of pharmaceutically acceptable salts of drugs are known, and research is ongoing. However, even for the same salt, the properties vary from drug to drug, and it is difficult to obtain an ideal salt that satisfies all pharmaceutically important properties, such as solubility and stability.

[0003] Janus kinases (JAKs) are enzymes that phosphorylate other proteins, regulating their activity, location, and function, thereby controlling various intracellular processes. Janus kinases are located on the intracellular receptors of inflammatory cytokines. After inflammatory cytokines bind to and phosphorylate the receptors, they transduce the inflammatory cytokine signal into the cell through interaction with STAT molecules. Excessive signaling by various inflammatory cytokines can trigger the body's immune system to attack itself, resulting in autoimmune diseases. In recent years, phase II and III clinical trials of the selective JAK1 inhibitors upadacitinib and abrocitinib have shown that JAK1 inhibitors rapidly improve the severity and symptoms of Alzheimer's disease.

[0004] Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases. Elevated levels of Th (T helper) 2, Th22, and some Th1 and Th17 cytokines cause abnormal immune activation in atopic dermatitis skin lesions. Recently, the use of immune-targeted therapeutic agents for the treatment of atopic dermatitis has increased, and dupilumab, a monoclonal antibody targeting the IL-4 receptor, has been approved for the treatment of atopic dermatitis. As the number of patients suffering from atopic dermatitis continues to increase, there is a continuing demand for therapeutic agents that demonstrate excellent therapeutic effects. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korean Patent Publication No. 2019-0043437 Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide novel salts of heterocyclic compounds as protein kinase inhibitors.

[0007] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing atopic dermatitis, comprising a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor.

[0008] Another object of the present invention is to provide a pharmaceutical composition for treating or preventing inflammatory bowel disease, comprising a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor. That is the thing.

[0009] Another object of the present invention relates to the use of a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor for treating or preventing atopic dermatitis.

[0010] Another object of the present invention relates to the use of a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor for treating or preventing inflammatory bowel disease.

[0011] Another object of the present invention is to provide use of a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor in the manufacture of a medicament for the treatment or prevention of atopic dermatitis.

[0012] Another object of the present invention is to provide the use of a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor in the manufacture of a medicament for the treatment or prevention of inflammatory bowel disease.

[0013] Another object of the present invention is to provide a method for treating or preventing atopic dermatitis, which comprises administering to an individual a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor.

[0014] Another object of the present invention is to provide a method for treating or preventing inflammatory bowel disease, which comprises administering to an individual a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor. [Means for solving the problem]

[0015] The inventors of the present application have made extensive efforts to find compounds with improved physicochemical properties, which have pharmacological activity equivalent to or greater than that of conventional compounds, while improving stability against heat and moisture to minimize the generation of related substances. As a result, they have confirmed that the salts of the heterocyclic compounds according to the present invention not only have low hygroscopicity, excellent physicochemical properties, and stability against heat and moisture, but also have improved solubility, and have completed the present invention.

[0016] Furthermore, the present inventors have found that the heterocyclic compound according to the present invention and its pharmaceutically acceptable salts are useful in treating atopy and inflammatory bowel disease.

[0017] The heterocyclic compounds and pharmaceutically acceptable salts thereof according to the present invention were able to effectively inhibit skin severity similar to that of atopic dermatitis in a mouse model of atopic dermatitis by oral and topical administration compared to other selective JAK1 inhibitors. Furthermore, in vitro human whole blood analysis demonstrated that the heterocyclic compounds of the present invention are the best selective JAK1 inhibitors with safety advantages over other JAK inhibitors. Furthermore, the heterocyclic compounds and pharmaceutically acceptable salts thereof also demonstrated therapeutic effects in an inflammatory bowel disease model.

[0018] New salt and method for producing same In the novel salt of a heterocyclic compound as a protein kinase inhibitor according to the present invention, the heterocyclic compound is represented by the following chemical formula I:

[0019] [ka]

[0020] The name of the compound represented by chemical formula I is N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

[0021] The heterocyclic compound of formula I may be (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and (R)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, or a mixture thereof.

[0022] In one embodiment, the heterocyclic compound or a pharmaceutically acceptable salt thereof in the present invention may be a compound represented by the following chemical formula II or a pharmaceutically acceptable salt thereof:

[0023] [ka]

[0024] The heterocyclic compound represented by formula II is named (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

[0025] In the present invention, the pharmaceutically acceptable salt of the heterocyclic compound represented by Formula I may be an organic acid salt of the heterocyclic compound represented by Formula I. Here, the organic acid salt includes hydrochloride, hydrobromide, mesylate, phosphate, napadisylate, camsylate, or oxalate.

[0026] In the present invention, the pharmaceutically acceptable salt of the heterocyclic compound represented by Chemical Formula I is an organic acid salt comprising N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and an organic acid, where the organic acid salt can be hydrochloride, hydrobromide, mesylate, phosphate, napadisylate, camsylate, or oxalate.

[0027] The organic acid salt in the present invention may contain N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and the organic acid in an equivalent ratio of 1:1 to 1:1.3.

[0028] In the present invention, the pharmaceutically acceptable salt of the heterocyclic compound represented by the formula I can be obtained with high purity.

[0029] In addition, in the present invention, a pharmaceutically acceptable heterocyclic compound represented by the chemical formula I is The salt exhibits excellent stability even under high temperature and humidity conditions and also has excellent photostability, allowing it to be stably maintained against heat and moisture for a long period of time. Furthermore, the pharmaceutically acceptable salt of the heterocyclic compound represented by chemical formula I of the present invention exhibits excellent solubility and physicochemical properties, and also has excellent bioavailability. Furthermore, the pharmaceutically acceptable salt of the heterocyclic compound represented by chemical formula I of the present invention has low hygroscopicity and does not absorb surrounding moisture, allowing it to maintain its moisture content unchanged for a long period of time.

[0030] The salts of the heterocyclic compounds of formula I of the present invention may be administered orally or transdermally.

[0031] The salts of the heterocyclic compounds represented by formula I of the present invention can treat or prevent atopic dermatitis.

[0032] The salts of the heterocyclic compounds of formula I of the present invention can treat or prevent inflammatory bowel disease, which may include ulcerative colitis or Crohn's disease.

[0033] In the present invention, a method for preparing a pharmaceutically acceptable salt of the heterocyclic compound represented by the chemical formula I or II includes the steps of: (A) dissolving or suspending the heterocyclic compound of Formula I in an organic solvent; (B) adding any one organic acid selected from hydrochloric acid, bromic acid, methanesulfonic acid, phosphoric acid, 1,5-naphthalenedisulfonic acid, camphorsulfonic acid, and oxalic acid and stirring to form a salt of the heterocyclic compound; and (C) A step of stirring to solidify the salt formed in the previous step may be included.

[0034] The production method may further include a step (D) of adding an antisolvent to age the solid.

[0035] In step (A), the heterocyclic compound represented by formula I or II may be in an amorphous or crystalline form.

[0036] The organic solvent in step (A) includes ethyl acetate, acetone, methanol, ethanol, isopropanol, acetonitrile, 2-butanone, or a mixture thereof.

[0037] In the step (B), N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and the organic acid may be mixed in an equivalent ratio of 1:1 to 1:1.3.

[0038] Use in treating or preventing atopic dermatitis (1) The present invention provides a composition for treating or preventing atopic dermatitis, comprising, as an active ingredient, N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, a heterocyclic compound represented by chemical formula I, or a pharmaceutically acceptable salt thereof:

[0039] (2) The present invention provides a method for treating or preventing atopic dermatitis, which comprises administering a pharmaceutical composition containing a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof.

[0040] (3) The present invention provides use of a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof for treating or preventing atopic dermatitis.

[0041] (4) The present invention provides use of a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof for producing a medicament for treating or preventing atopic dermatitis.

[0042] (5) In the present invention according to (1), (2), (3), or (4), the term "atopic dermatitis" is used to mean all diseases classified as atopic dermatitis in the art, regardless of the direct or indirect cause of its onset. Atopic dermatitis is generally classified into infantile atopic dermatitis, childhood atopic dermatitis, adult atopic dermatitis, and atopic dermatitis in pregnant women, depending on the time of onset or the target of onset. In the present invention, atopic dermatitis is defined to include all of these atopic dermatitis.

[0043] (6) In the present invention according to (1), (2), (3), (4) or (5), the pharmaceutically acceptable salt may be a hydrochloride, a hydrobromide, a phosphate, a camsylate, an oxalate, a mesylate or a napadisylate.

[0044] Use in treating or preventing inflammatory bowel disease (7) The present invention provides a composition for treating or preventing inflammatory bowel disease, comprising, as an active ingredient, N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, which is a heterocyclic compound represented by chemical formula I, or a pharmaceutically acceptable salt thereof:

[0045] (8) The present invention provides a method for treating or preventing inflammatory bowel disease, comprising administering a pharmaceutical composition containing a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof.

[0046] (9) The present invention provides use of a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof for treating or preventing inflammatory bowel disease.

[0047] (10) The present invention provides use of a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof for producing a medicament for treating or preventing inflammatory bowel disease.

[0048] (11) In the present invention according to (7), (8), (9), or (10), "inflammatory bowel disease" refers to chronic inflammation of unknown cause that occurs in the intestine, and generally refers to idiopathic inflammatory bowel diseases such as ulcerative colitis and Crohn's disease, but may also include intestinal Behcet's disease, which is relatively common in Korea. Furthermore, inflammatory bowel disease is defined as a general term for all inflammatory diseases that occur in the intestinal tract, including infectious enteritis such as bacterial, viral, amoebic, and tuberculous enteritis, as well as ischemic enteritis and radiation enteritis.

[0049] (12) In the present invention according to (7), (8), (9), (10) or (11), the pharmaceutically acceptable salt may be a hydrochloride, a hydrobromide, a phosphate, a camsylate, an oxalate, a mesylate or a napadisylate.

[0050] The pharmaceutical composition for the prevention or treatment of atopic dermatitis or inflammatory bowel disease of the present invention according to (1) or (7) above may further contain a pharmaceutically acceptable additive, a commonly used suitable carrier, excipient, disintegrant, binder, lubricant or diluent.

[0051] The "pharmaceutically acceptable additive" may include a carrier, excipient, disintegrant, binder, lubricant, or diluent that does not stimulate the organism or inhibit the biological activity and properties of the compound to be injected. The type of additive used in the present invention is not particularly limited, and any pharmaceutically acceptable additive commonly used in the art may be used. Non-limiting examples of the additive include mannitol, microcrystalline cellulose, croscarmellose sodium, hydroxypropyl cellulose, colloidal silicon dioxide, microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, magnesium stearate, or a mixture thereof. Other conventional additives, such as antioxidants, buffers, and / or bacteriostatic agents, may also be added as needed.

[0052] The compound of the present invention that exhibits therapeutic or preventive effects against atopic dermatitis or inflammatory bowel disease is a heterocyclic compound represented by chemical formula I, N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, or a pharmaceutically acceptable salt thereof, wherein the salt may be hydrochloride, hydrobromide, phosphate, camsylate, oxalate, mesylate, or napadisylate.

[0053] In the present invention, the heterocyclic compound represented by Formula I or a pharmaceutically acceptable salt thereof may be administered orally or transdermally.

[0054] In the present invention, the dosage of the heterocyclic compound represented by Formula I or a pharmaceutically acceptable salt thereof must be a pharmaceutically effective amount. A "pharmaceutically effective amount" refers to an amount sufficient to prevent or treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and the effective amount can be selected in various ways by those skilled in the art depending on factors such as the formulation method, the patient's condition and weight, sex, age, severity, drug form, route and duration of administration, excretion rate, and sensitivity to the drug. As recognized by those skilled in the art, the effective amount will vary depending on the route of treatment, the use of excipients, and the possibility of coadministration with other drugs.

[0055] In one embodiment, the heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof may be administered as a transdermal formulation at a dose of 0.1 to 5% or as an oral formulation at a dose of 10 to 200 mg / kg, for example, as a transdermal formulation at a dose of 0.3 to 3% or as an oral formulation at a dose of 50 to 200 mg / kg.

[0056] As an example, the heterocyclic compound of formula I of the present invention or a pharmaceutically acceptable salt thereof may be administered orally at a dose of 10 to 180 mg / kg.

[0057] When formulated for oral administration, they may be formulated into tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs.

[0058] When formulated as a transdermal preparation, it may be manufactured into the dosage form of, for example, a solution, suspension, gel, cream, emulsion, or the like. [Effects of the Invention]

[0059] The novel salt of the heterocyclic compound represented by chemical formula I according to the present invention has excellent water solubility and excellent physical and chemical stability, and therefore can be usefully utilized in the formulation of pharmaceuticals.

[0060] The present invention also provides a method for effectively inhibiting Janus kinase, a receptor for inflammatory cytokines, by using a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof. This allows for the effective treatment and prevention of atopic dermatitis and inflammatory bowel disease, respectively. [Brief explanation of the drawings]

[0061] [Figure 1] FIG. 1 is a graph showing the NMR analysis result of the hydrochloride salt of the compound represented by chemical formula II of the present invention. [Figure 2] FIG. 2 is a graph showing the results of NMR analysis of the hydrobromide salt of the present invention. [Figure 3] FIG. 3 is a graph showing the results of NMR analysis of the phosphate salt of the present invention. [Figure 4] FIG. 4 is a graph showing the results of NMR analysis of the camsylate salt of the present invention. [Figure 5] FIG. 5 is a graph showing the results of NMR analysis of the oxalate salt of the present invention. [Figure 6] FIG. 6 is a graph showing the NMR analysis results of the mesylate, oxalate and napadisylate salts of the present invention. [Figure 7] FIG. 7 is a graph showing the NMR analysis results of the mesylate, oxalate and napadisylate salts of the present invention. [Figure 8] FIG. 8 is a graph showing the experimental results of the ability of Compound 1 to inhibit IL-4 secretion in Basophil cells. [Figure 9] FIG. 9 is a graph showing the evaluation results of the ability of Compound 1 of the present invention to secrete IL-13, IL-10 and TNF-α. [Figure 10] FIG. 10 is a diagram illustrating the DNCB-induced animal model of Experimental Example 6. [Figure 11] FIG. 11 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 6. [Figure 12]FIG. 12 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 6. [Figure 13] FIG. 13 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 6. [Figure 14] FIG. 14 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 6. [Figure 15] FIG. 15 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 6. [Figure 16] FIG. 16 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 6. [Figure 17] FIG. 17 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 7. [Figure 18] FIG. 18 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 7. [Figure 19] FIG. 19 shows the results of confirming the therapeutic effect of atopic dermatitis in a DNCB-induced animal model in Experimental Example 7. [Figure 20] FIG. 20 is a diagram illustrating the DSS-induced animal model of Experimental Example 8. [Figure 21] FIG. 21 shows the results of examining the therapeutic effect of colitis in a DSS-induced animal model in Experimental Example 8. [Figure 22] FIG. 22 shows the results of examining the therapeutic effect of colitis in a DSS-induced animal model in Experimental Example 8. [Figure 23] FIG. 23 shows the results of examining the therapeutic effect on colitis in a DSS-induced animal model in Experimental Example 8. [Figure 24]FIG. 24 shows the results of examining the therapeutic effect of colitis in a DSS-induced animal model in Experimental Example 8. [Figure 25] FIG. 25 is a diagram illustrating the DNBS-induced animal model of Experimental Example 9. [Figure 26] FIG. 26 shows the results of examining the therapeutic effect of colitis in a DNBS-induced animal model in Experimental Example 9. [Figure 27] FIG. 27 is a diagram illustrating the HDM-induced animal model of Experimental Example 10. [Figure 28] FIG. 28 shows the results of confirming the therapeutic effect of atopic dermatitis in an HDM-induced animal model in Experimental Example 10. [Figure 29] FIG. 29 shows the results of examining the therapeutic effect of atopic dermatitis in an HDM-induced animal model in Experimental Example 10. [Figure 30] FIG. 30 shows the results of confirming the therapeutic effect of atopic dermatitis in an HDM-induced animal model in Experimental Example 10. DETAILED DESCRIPTION OF THE INVENTION

[0062] Hereinafter, the embodiments of the present invention will be described in detail. Unless otherwise defined, all terms used in this specification, including technical or scientific terms, have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the contextual meaning of the relevant art, and unless explicitly defined in this application, should not be interpreted as an ideal or overly formal meaning.

[0063] *p<0.05, **p<0.01, ***<0.001 in the respective figures.

[0064] Production Example 1: Synthesis of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide The title compound was prepared according to the method disclosed in US Pat. No. 5,629,999. 1 H NMR(400MHz,DMSO-d6)δ11.44(s,1H),10.57(s,1H),7.84(d,J=10.2Hz,1H),7.34(d,J=3.1Hz,1H),6.48(dd,J=1.8,3.7Hz,1H),6.17-6.0 3(m,1H),4.31-4.01(m,6H),3.96-3.62(m,2H),3.02(mJ=36.6Hz,1H),2.02(s,1H),0.88(s,3H),0.84-0.73(m,4H);MS(ESI+)m / z364(M+H) +

[0065] Example 1: Preparation of the hydrochloride salt 90 mL of methanol was added to 30 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, and the mixture was stirred and heated to 50°C. 1.1 eq. of hydrochloric acid (in MeOH) was then slowly added dropwise and stirred at 50°C for 2 hours to obtain a solution. The solution was cooled to room temperature to cause precipitation. The precipitate was then filtered, washed with methanol, and dried under vacuum to obtain the title compound (31.1 g, 94% yield) as a yellow powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 1.

[0066] Example 2: Preparation of the hydrobromide salt 300 mL of acetone was added to 30 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and stirred. 1.1 eq. of hydrobromic acid was then slowly added dropwise with stirring. The mixture was filtered, washed with methanol, and then dried under vacuum to give the title compound (34.4 g, 94% yield) as a yellow powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 2.

[0067] Example 3: Preparation of phosphate salts 90 mL of methanol was added to 30 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, and the mixture was stirred and heated to 50° C. 1.05 eq. of phosphoric acid was then added and stirred at 50° C. for 1 hour to obtain a solution. The solution was cooled to room temperature, filtered, washed with methanol, and then dried under vacuum to obtain the title compound (40 g, 85% yield) as an orange powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in FIG. 3.

[0068] Example 4: Preparation of camsylate salt 200 mL of ethanol was added to 20 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and stirred. 1.05 eq. of camphorsulfonic acid was then added and stirred at room temperature. The mixture was filtered, washed with ethanol, and vacuum dried to give the title compound (24.1 g, 92% yield) as a yellow powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 4.

[0069] Example 5: Preparation of oxalate salt 510 mL of acetonitrile was added to 30 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and stirred. 1.05 eq. of oxalic acid was then added and stirred at room temperature. The mixture was filtered, washed with acetonitrile, and then dried under vacuum to give the title compound (29.1 g, 66% yield) as a yellow powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 5.

[0070] Example 6: Preparation of mesylate salt 300 mL of ethyl acetate was added to 30 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, and the mixture was stirred and heated to 50° C. 1.1 eq. of methanesulfonic acid was then slowly added dropwise and the mixture was stirred at the same temperature. The mixture was filtered, washed with methanol, and dried under vacuum to give the title compound (35.3 g, 93% yield) as an orange powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in FIG. 6.

[0071] Example 7: Preparation of napadisylate 20 mL of acetonitrile was added to 1 g of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and stirred. The temperature was then raised to 50° C., and 1.05 eq. of 1,5-naphthalenedisulfonic acid was added and stirred. The mixture was filtered, washed, and then dried under vacuum to obtain the title compound. NMR analysis was performed to confirm the formation of the title compound. The results are shown in FIG. 7.

[0072] Analysis and Measurement Methods 1. Measurement of water solubility The aqueous solubility was measured by shaking a saturated solution (20 mg / 0.5 mL) at room temperature for 24 hours. The liquid in the solution was collected and filtered through a 0.22 μm PVDF filter. The filtrate was diluted 10 times for LC analysis.

[0073] LC analysis was carried out as follows.

[0074] [Table 1]

[0075] [Table 2]

[0076] 2. Physical stability evaluation experiment To assess physical stability, the samples were placed in a ((LPDE+N2)+Silica gel The samples were packaged in an Al-Bag (1g + LDPE) and stored under harsh conditions (60°C ± 2°C / 80% RH ± 5%) for 2 / 4 weeks, after which they were evaluated.

[0077] 3.Photostability evaluation experiment The specimen was wrapped in an LDPE bag and exposed to visible light (1.2M Lux-h) and UV light (200W-h / m 2 ) and UV+Visible conditions, and then evaluated.

[0078] 4. Moisture absorption evaluation experiment The specimens were placed in glass desiccators at relative humidity levels of 33%, 53%, 75% and 93% and then evaluated after storage for 2 and 4 weeks.

[0079] Analysis / measurement / evaluation results 1. Water solubility measurement results The results of measuring the water solubility of the salts obtained in Examples 1 to 6 are shown in Table 1 below.

[0080] [Table 3]

[0081] Referring to Table 1, it can be seen that the water solubility of the salts according to the present invention is at least 0.9 mg / mL or more. In particular, the hydrochloride, hydrobromide, camsylate, oxalate, and phosphate salts have excellent water solubilities of 1.9 mg / mL or more, and the hydrochloride, hydrobromide, and oxalate salts have extremely excellent water solubilities of more than 4 mg / mL.

[0082] 2. Physical stability evaluation results The results of evaluating the physical stability of the salts obtained in Examples 1 to 4 are shown in Table 2 below. In Table 2, "initial" refers to the result measured after the salt was produced, and "S2W" and "S4W" refer to the results measured after 2 weeks and 4 weeks, respectively, of exposure to harsh conditions.

[0083] [Table 4]

[0084] [Table 5]

[0085] Referring to Table 2, the purity and content before and after exposure to harsh conditions confirm that the salt of the present invention has excellent stability against moisture and heat. In particular, with regard to purity, it can be confirmed that even after exposure to harsh conditions, each of the individual related substances and the total related substances meets the quality standards of raw material drugs.

[0086] 3.Photostability evaluation results The results obtained for the salts according to Examples 1 to 5 of the present invention by the photostability evaluation method are as follows: This is as shown in Table 3 below.

[0087] [Table 6]

[0088] [Table 7]

[0089] Referring to Table 3, it can be seen that the salt of the present invention has excellent stability against ultraviolet and visible light. In particular, it can be seen that the purity of each of the individual related substances and the total related substances meets the quality standards of raw material drugs even after exposure to harsh conditions.

[0090] 4. Moisture absorption evaluation results The results obtained by the hygroscopicity evaluation method for the salts according to Examples 1 to 4 of the present invention are shown in Table 4 below.

[0091] [Table 8]

[0092] [Table 9]

[0093] Referring to Table 4, it can be seen that the salt according to the present invention has low hygroscopicity.

[0094] Experimental Example 1: ADP-Glo ​​Kinase Assay (1) JAK1 kinase assay 10 mM of (S)—N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide (hereinafter referred to as Compound 1), a heterocyclic compound represented by Chemical Formula II obtained in Production Example 1, was diluted in DMSO to prepare five concentrations of samples (1,000 nM, 200 nM, 40 nM, 8 nM, and 0.16 nM). ATP and 10mg / mL IRS (insulin receptor substrate) were diluted 1 / 40 and 1 / 50, respectively, with 1X kinase buffer. In addition, 222ng / μL of JAK1 enzyme was added to 1X kinase The mixture was diluted 1 / 8 with buffer. 1 μL of ATP, 1 μL of IRS, and 1 μL of compound 1 were mixed, and 2 μL of JAK1 enzyme was added. The mixture was incubated at 30°C for 40 minutes. A blank tube without JAK1 enzyme and a positive control tube without compound 1 were also prepared (final sample concentrations: 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM).

[0095] Next, 5 μL of ADP-glo was added to each tube and incubated at 30°C for 40 minutes, followed by 10 μL of kinase detection reagent and incubation at room temperature for 15 minutes. 18 μL of the reaction mixture was transferred to a 384-well plate, and luminescence was measured using a plate reader.

[0096] (2) JAK2 kinase assay 10 mM Compound 1 was diluted in DMSO to prepare four concentrations (5,000 nM, 500 nM, 50 nM, and 5 nM). 10 mM ATP was added to 1× kinase inhibitor. The ATP solution was diluted 1 / 40 with 1X kinase buffer. IGF1Rtide was used as a stock solution at 1 mg / mL. 100 ng / μL JAK2 enzyme was diluted 1 / 20 with 1X kinase buffer. 1 μL of the ATP solution, 1 μL of IGF1Rtide, and 1 μL of compound 1 were mixed, and 2 μL of JAK2 enzyme was added. The mixture was incubated at 30°C for 40 minutes. A blank tube without JAK2 enzyme and a positive control tube without compound 1 were also prepared (final sample concentrations: 1,000 nM, 100 nM, 10 nM, and 1 nM).

[0097] Next, 5 μL of ADP-glo was added to each tube and incubated at 30°C for 40 minutes, followed by 10 μL of kinase detection reagent and incubation at room temperature for 15 minutes. 18 μL of the reaction mixture was transferred to a 384-well plate, and luminescence was measured using a plate reader.

[0098] (3) JAK3 kinase assay 10 mM Compound 1 was diluted in DMSO to prepare four concentrations (50,000 nM, 5,000 nM, 500 nM, and 50 nM). 10 mM ATP was diluted 1 / 40 with 1X kinase buffer. Poly(Glu4, Tyr1) peptide was used at 1 mg / mL stock solution. 100 ng / μL JAK3 enzyme was diluted 1 / 20 with 1X kinase buffer. 1 μL of ATP, 1 μL of Poly(Glu4, Tyr1) peptide, and 1 μL of Compound 1 were mixed, and 2 μL of JAK3 enzyme was added. The mixture was incubated at 30°C for 40 minutes. A blank tube without JAK3 enzyme and a positive control tube without Compound 1 were also prepared (final sample concentrations: 10,000 nM, 1,000 nM, 100 nM, and 10 nM).

[0099] Next, 5 μL of ADP-glo was added to each tube and incubated at 30°C for 40 minutes, followed by 10 μL of kinase detection reagent and incubation at room temperature for 15 minutes. 18 μL of the reaction mixture was transferred to a 384-well plate, and luminescence was measured using a plate reader.

[0100] (4) Tyk2 kinase assay 10 mM Compound 1 was diluted in DMSO to prepare five concentrations (5,000 nM, 500 nM, 50 nM, 5 nM, and 0.5 nM). 10 mM ATP was added to 1X kJ / mL. The compound 1 was diluted 1 / 40 with kinase buffer. Poly(Glu4, Tyr1) peptide was used at 1 mg / mL stock solution. 100 ng / μL Tyk2 enzyme was diluted 1 / 2 with 1X kinase buffer. 1 μL of the ATP, 1 μL of Poly(Glu4, Tyr1) peptide, and 1 μL of compound 1 were mixed, and 2 μL of Tyk2 enzyme was added. The mixture was incubated at 30°C for 40 minutes. A blank tube without JAK3 enzyme and a positive control tube without compound 1 were also prepared (final sample concentrations: 1,000 nM, 100 nM, 10 nM, 1 nM, and 0.1 nM).

[0101] Next, 5 μL of ADP-glo was added to each tube and incubated at 30°C for 40 minutes, followed by 10 μL of kinase detection reagent and incubation at room temperature for 15 minutes. 18 μL of the reaction mixture was transferred to a 384-well plate, and luminescence was measured using a plate reader.

[0102] Experimental Example 2: In vitro cell assay 1. Preparation of experimental cells (cell lines) UT-7 / EPO cells are an erythroleukemic cell line that expresses the EPO receptor and have been frequently used as a model to evaluate EPO-induced STAT5 phosphorylation mediated by JAK1 and JAK2 activity in many previous studies. Therefore, we prepared UT-7 / EPO cells to evaluate the activity of JAK1 and JAK2.

[0103] In addition, NK-92 cells are a natural killer cell line that grows in an IL-2-dependent manner and are derived from peripheral blood mononuclear cells, making them suitable for evaluating IL-2-induced JAK1 and JAK3 activation. Therefore, we prepared NK-92 cells to evaluate JAK1 and JAK3 activation.

[0104] Furthermore, there is literature that U2OS cells are associated with JAK1 / TYK2 signaling activity and STAT1 phosphorylation by IFN-alpha B2 and IFN-γ. Therefore, U2OS cells were prepared to evaluate JAK1 and TYK2, as they are a cell line that strongly expresses STAT1 signaling.

[0105] 2. Preparation of Test Substances and Stimuli (1) Preparation of test substances The test substance was dissolved in DMSO to prepare a 10 mM solution, and the solution was dispensed into 20 μL aliquots and stored at −20°C.

[0106] (2) Preparation of the stimulus source EPO: 2000 U / 0.5 mL stock was dispensed in 50 μL aliquots and stored in a refrigerator. IL-2: A 200 μg / mL stock was prepared and stored in 5 μL aliquots, and treated with 2 ng / mL (IL-2 1 μg / mL=13,000 U / mL). IFN-alpha B2: 1×10 U / mL stock was diluted and used to achieve 30,000 U / mL.

[0107] (3) Preparation of assay medium UT-7 / EPO cell assay: MEM-alpha (Gibco, cat#12561-056) was prepared to contain 5% FBS and 1% P / S. NK-92 cell assay: HBSS containing 1% FBS and 1% P / S It was prepared and used so that it could be used. U2OS cell assay: HBSS was prepared to contain 1% FBS and 1% P / S.

[0108] 3. Sample Preparation The cultured cells were centrifuged (1,000 rpm, 5 minutes) and then washed with PBS. The cell line size and appropriate reaction concentration were determined, and the cells were diluted to 1 × 10 5 ~106 96-well plates were seeded with 50 μL of test substance per well. Test substances were prepared at 4X the target concentration and serially diluted 1 / 5 to prepare five or six concentrations. Test substances were diluted 1:1000 in assay medium and applied at 20 μL per well. An untreated group was treated with 20 μL of assay medium per well and incubated in a CO2 incubator for 1 hour.

[0109] One to two hours after treatment with the test substance, the cells were treated with 10 μL / well of cytokines (final EPO concentration: 1 U / mL, final IL-2 concentration: 2 ng / mL, final IFN-alpha B2 concentration: 30,000 U / mL). The experiment was designed to include a cytokine-only treatment group and an untreated group. After seeding, the untreated group was treated with 10 μL of assay medium only.

[0110] After 20 minutes of stimulation with cytokines as described above, cells were lysed in 5X RIPA buffer for 30 minutes and centrifuged at 13,000 rpm for 5 minutes at 4°C. The supernatant was transferred to a new tube for ELISA or stored at -80°C until use.

[0111] 4. Performing ELISA The assay was carried out according to the protocol of Cell signaling ELISA KIT (Cat no. 7113, 7234C).

[0112] Experimental Example 3: Human whole blood assay 1. TEST1:CD4 + Evaluation of IL-6-induced STAT1 phosphorylation in cells The stock solution of Compound 1 obtained in Preparation Example 1 was diluted with distilled water and then serially diluted 1 / 5 with 4% DMSO. 100 μL of whole blood collected in a 1.7 mL Eppendorf tube was mixed with 5 μL of the test substance and then incubated at 37°C for 45 minutes.

[0113] IL-6 (10 μg / ml) was diluted to 1 μg / ml with 0.1% BSA / DW. Five μl of IL-6 (final concentration: 50 ng / ml) was added and cultured at 37°C for 15 minutes. Five μl of DPBS was added to the unstimulated control group. Lyse / fix buffer 5X was diluted to 1X with distilled water.

[0114] 900 μL of lyse / fix buffer pre-warmed at 37°C was added to each tube, and the tubes were incubated at 37°C for 20 minutes. After centrifugation at 500 x g for 8 minutes, the supernatant was removed, and the tubes were washed with 1 mL of FACS buffer. The washing process was repeated. Next, 400 μL of BD Phosflow® Perm buffer, which had been placed on ice, was added, and the tubes were incubated on ice for 30 minutes, followed by spinning down. After washing once with wash buffer, the tubes were resuspended in a buffer (BD Pharmingen® staining buffer).

[0115] For double staining of CD4 and pSTAT1, staining Anti-CD and anti-pSTAT1 were mixed in staining buffer and added to each sample. The tube containing the sample was gently tapped to thoroughly mix the antibody with the cells, and then left at 4°C overnight. The results were analyzed the next day using a FACSCanto II.

[0116] 2. TEST2~6 Tests 2 to 6 were each carried out in substantially the same manner as Test 1, except that the types and amounts of cytokines were as shown in Table 5 below.

[0117] [Table 10]

[0118] 3. Analysis method: Data collection and IC 50 calculation The values ​​analyzed by FlowJo were analyzed using GraphPad Prism 5 software (product name) to calculate IC 50 The value was calculated.

[0119] The no treatment response (NTR) was set as the baseline, and the degree of induction of STAT phosphorylation induced by cytokines was converted into a relative ratio (% control). Using GraphPad Prism (version 5.0), dose-response curves were obtained for the activity of each test substance at each concentration, and IC 50 The value was calculated.

[0120] Results of Experiments 1 to 3 The results obtained in the above-mentioned Experimental Examples 1 to 3 are as follows.

[0121] [Table 11]

[0122] From the above results, it can be seen that Compound 1 according to the present invention has excellent JAK1 inhibitory activity.

[0123] Experimental Example 4: Inhibitory effect of IL-4 secretion in Basophil cells RBL-2H3 cells were diluted in 10% FBS EMEM medium to 1x105 cells / well in a 24-well plate and dispensed. Incubate at 37°C. The cells were cultured in a hood for more than 18 hours.

[0124] The cells were treated with the test substance (Compound 1) at various concentrations (final concentrations of 0, 0.1, 0.5, and 1 μM) under stimulation with PMA (50 nmol / L) and A23187 (1 μmol / L). The cells were cultured in a 37°C incubator for 24 hours. The reaction was then terminated on ice, and the level of IL-4 secretion in the culture supernatant was measured using an IL-4 ELISA kit. The results are shown in Figure 8.

[0125] FIG. 8 is a graph showing the experimental results of the ability of Compound 1 to inhibit IL-4 secretion in Basophil cells.

[0126] 8, it can be seen that the IL-4 concentration was lower in the group treated with Compound 1 according to the present invention compared to the control group (vehicle). That is, Compound 1 according to the present invention inhibits IL-4 secretion, and in particular, when Compound 1 was treated at concentrations of 0.5 μM and 1 μM, the IL-4 secretion was significantly reduced to more than half compared to the negative control group (vehicle).

[0127] Experimental Example 5: IL-13, IL-10 and TNF-α secretion ability 50 μL of DNase I (2000 units / mL) was added to 50 mL of cell culture medium and mixed (the medium was warmed in a 37°C water bath before use to use the enzyme and cells). PMBC from an atopic patient was also quickly thawed in a 37°C water bath.

[0128] The PMBCs were slowly poured into the prepared medium and allowed to react for 5 minutes at 37°C. After centrifugation at 200 rpm for 15 minutes, the supernatant was removed and suspended in 1 mL of cell culture medium and counted.

[0129] 2×10 5 The cells were diluted in cell culture medium to give 100 μL / well of cells and seeded into a 96-well plate.

[0130] 50 μL of PBMC activation medium (0.5 μg / ml CD3 antibody, 5 μg / ml CD28 antibody) was added to each well except for the negative control wells (50 μL of cell culture medium was added to each negative control well).

[0131] 50 μL of the test substance was added to each well at each concentration (final concentrations: 1 nM, 10 nM, 100 nM, 1,000 nM; 50 μL of cell culture medium was added to each well of the negative control group).

[0132] After culturing for 24 hours in a 37°C, 5% CO2 incubator, the plate was centrifuged at 200 rpm for 15 minutes, and 150 μL of the suspension was placed in a round-bottom 96-well plate and stored in a deep freezer at -80°C.

[0133] After thawing the culture supernatant samples, ELISA was performed according to the protocol in the ELISA kit data sheet. The results are shown in Figure 9.

[0134] FIG. 9 is a graph showing the evaluation results of the ability of Compound 1 of the present invention to secrete IL-13, IL-10 and TNF-α.

[0135] 9, it can be seen that IL-13, IL-10, and TNF-α were each reduced in anti-CD3 and anti-CD28 activated PBMCs from atopic dermatitis patients. In particular, in the groups treated with Compound 1 at concentrations of 100 nM and 1,000 nM, IL-13, IL-10, and TNF-α were each significantly reduced. You can confirm that this is the case.

[0136] Experimental Example 6: DNCB-induced model (topical) 1. Induction of atopic model and treatment with test substances (Figure 10) NC / Nga mice were anesthetized with isoflurane, and the hair on their backs (from under the ears to the top of the tail) was removed. The mice were left for 24 hours to allow healing of the micro-injuries caused by shaving.

[0137] After healing, 200 μL of 1% 1-chloro-2,4-dinitrobenzene (DNCB) in acetone / olive oil (3:1) was applied topically to the dorsal skin (primary skin application). A secondary skin application was performed 3 days later for sensitization.

[0138] Starting 7 days after the primary dermal application, 150 μL of 0.4% DNCB in acetone / olive oil (3:1) was applied topically to the back skin three times a week for 5 weeks.

[0139] The phosphate salt of Compound 1 (hereinafter referred to as the phosphate compound) obtained in Example 3 was used as the test substance. The test substance was administered two weeks after the first skin application and before the application of DNCB. The dose of the test substance was calculated based on the body weight of the mice measured on the day of administration, and the test substance was topically applied to the atopic skin using a glass stick (twice daily for 28 days, a total of 56 times, with a 6-hour treatment interval). The control group (Vehicle) was administered 0.5% methylcellulose (0.5% MC).

[0140] 2. Autopsy Six weeks after the first skin application, the test animals were necropsied. Specifically, the test animals were anesthetized with isoflurane, and as much blood as possible was collected from the caudal vena cava using a syringe and stored in a 5 mL vacuum tube containing a clot activator. The blood was left at room temperature for 15-20 minutes to clot, and then centrifuged at 3000 rpm for 10 minutes.

[0141] 3. Evaluation Method (1) Clinical Atopic Dermatitis Score The atopic dermatitis score was assessed for each of five symptoms (erythema, dryness, skin edema and hematoma, erosion, and keratinization) on a scale of none (0), mild (1), moderate (2), or severe (3). The total clinical severity score was defined as the sum of all scores (maximum score: 15). Assessments were performed twice weekly by the same investigator at the same time.

[0142] (2) Itch behavior test To assess itch behavior at week 4, mice were placed in cages and their scratching frequency was assessed for 30 minutes.

[0143] (3) Serum IgE and histamine Quantitative analysis of serum IgE and histamine was performed using ELISA kits.

[0144] (4) Spleen / body weight The spleen and mouse body weights were measured, and the spleen-to-body weight ratio (relative organ weight) was calculated.

[0145] (5) qRT-PCR TARC, TSLP, IL-4, IL-1β, and TNF-α were quantified by qRT-PCR using skin tissue.

[0146] (6) Histopathological analysis On the day of necropsy, the skin in 10% neutral buffered formalin solution was analyzed by staining with H&E and toluidine blue.

[0147] 11 to 16, it can be seen that the phosphate compound of the present invention improves the symptoms of atopic dermatitis. In Figures 11 to 16, "compound 1" refers to the "phosphate compound" obtained in Example 3.

[0148] Specifically, Figure 11 is a graph showing the results of clinical atopic dermatitis scores in Experimental Example 6, and it can be seen from this that the skin severity score of the group administered with the phosphate compound was significantly reduced.

[0149] Figure 12 is a graph showing the results of the itching behavior test in Experimental Example 6. Referring to Figure 12, it can be seen that scratching behavior in the group administered with phosphate compounds was reduced by at least 30% compared to the control group (Vehicle). In particular, it can be seen that scratching behavior in the group administered with 3% phosphate compounds was reduced by more than 50% compared to the control group (Vehicle), significantly reducing to the level of normal mice (Normal).

[0150] Figures 13 and 14 show the cytokine analysis results (plasma, skin) of Experimental Example 6. The graph in Figure 13 (top) shows the plasma histamine concentration (ng / mL), and the graph in Figure 13 (bottom) shows the plasma IgE concentration (ng / mL). Figures 13 and 14 confirm that atopic dermatitis-related cytokines were reduced in the plasma and skin in the group administered with the phosphate compound.

[0151] FIG. 15 is a graph showing the calculation results of the spleen-to-body weight ratio in Experimental Example 6, and it can be seen that the spleen-to-body weight ratio value was reduced in the group administered with a phosphate compound.

[0152] FIG. 16 shows the results of skin staining in Experimental Example 6, and it can be seen that treatment with phosphate compounds changed the histological characteristics of atopic dermatitis.

[0153] Experimental Example 7: DNCB-induced model (PO) A DNCB-induced animal model was prepared in a manner substantially similar to the DNCB model (topical) in Experimental Example 6, except that the phosphate compound was administered orally. The phosphate compound was administered orally twice daily for 28 days, a total of 56 times, with an 8-hour interval between doses. The dose (10 mL / kg) was calculated based on the body weight measured on the day of administration, and the compound was administered orally into the stomach of the mouse using a 1 mL syringe and a probe. The control group was administered 0.5% methylcellulose (0.5% MC).

[0154] Fig. 17 is a graph showing the results of the clinical atopic dermatitis score in Experimental Example 7, Fig. 18 is a graph showing the results of the itch behavior test in Experimental Example 7, and Fig. 19 is a graph showing the calculation results of the spleen-to-body weight ratio in Experimental Example 7. In Figs. 17 to 19, "compound 1" refers to the "phosphate compound" obtained in Example 3.

[0155] Referring to all of the evaluation results of Experimental Example 7 in FIGS. 17 to 19, it can be seen that the phosphate compound according to the present invention improves the symptoms of atopic dermatitis.

[0156] Experimental Example 8: DSS induction model (Figure 20) 1. Induction of Colitis and Treatment of Test Substances To induce colitis in C57BL / 6J mice, 3% dextran sodium sulfate (DSS) was added to drinking water and provided every morning for 8 days, while the negative control group received 0% DSS.

[0157] After grouping the mice into several groups (day 0), 0.5% methylcellulose and Compound 1 obtained in Preparation Example 1 were orally administered to the mice as a test substance at 9:00 AM and 5:00 PM every day from day 1 to day 7. On day 8, the test animals were necropsied.

[0158] 2.Analysis method (1) Animal Disease Activity Index The Disease Activity Index (DAI) was calculated based on body weight, stool consistency, and stool blood score to represent the severity of the disease. Body weight and food intake were monitored and assessed at the start of the study and every 24 hours thereafter. Fecal samples were collected on days 0, 3, 5, and 7 and analyzed according to Table 6 below.

[0159] [Table 12]

[0160] (2) Colon weight-to-length ratio The weight and length of the colon obtained at autopsy were measured, and the weight-to-length ratio was calculated.

[0161] (3) Detection of biomarkers in plasma and the large intestine On day 8, blood was collected from the heart into a centrifuge tube containing EDTA-K2 anticoagulant. After centrifugation (7000 rpm, 10 min, 4°C), plasma was separated, and KC and MIP-2 were measured using the Meso Scale Discovery Kit.

[0162] mRNA was extracted from a portion of the colon tissue (the same location for each animal), and the expression levels of MX2 and TNF-alpha in the tissue were analyzed by qPCR.

[0163] (3) Histopathology score Colonic sections fixed in 10% formalin were cut into paraffin and stained with hematoxylin-eosin. The criteria for histological changes in the colon were as shown in Table 7 below.

[0164] [Table 13]

[0165] 21 to 24, "compound 1" means compound 1 (free base) obtained in Production Example 1.

[0166] FIG. 21 is a graph showing the analysis results of the disease activity index in Experimental Example 8.

[0167] 21, it can be seen that the disease activity index increased over time in all groups except the normal group, but the degree of increase in the disease activity index in the group administered with Compound 1 of the present invention was lower than that in the control group (vehicle). In particular, it can be seen that the disease activity index in the group administered with 90 mg / kg of Compound 1 decreased to about half that of the control group (vehicle).

[0168] FIG. 22 is a graph showing the results of colon weight to length ratio in Experimental Example 8.

[0169] 22, it can be seen that all groups except the normal group had a higher weight-to-length ratio compared to the normal group. However, it can be seen that the value was lower in the group administered with Compound 1 compared to the control group (vehicle). This indicates that the control group maintained or worsened the inflamed state of the colon, resulting in an increase in colon weight and a decrease in colon length, while the group administered with Compound 1 showed improvement in colitis and alleviated symptoms.

[0170] FIG. 23 shows the results of detecting biomarkers in plasma and the large intestine in Experimental Example 8.

[0171] 23, it can be seen that the group administered with Compound 1 of the present invention had reduced concentrations of KC and MIP-2, which are biomarkers that increase in plasma in a colonic inflammatory environment. It can also be seen that the expression levels of MX2 and TNF-alpha in the colon of the group administered with Compound 1 of the present invention were also reduced. These results indicate that the increase in biomarkers associated with inflammation in the colon can also be prevented.

[0172] FIG. 24 is a graph showing the results of the histopathological scores in Experimental Example 8, and it can be seen that the group administered with Compound 1 of the present invention had a lower histopathological score.

[0173] Experimental Example 9: DNBS-induced model (Figure 25) 1. Induction of Colitis and Treatment of Test Substances Before inducing colitis with DNBS every week, experimental animals were fasted for 24 hours. The fasted animals were lightly anesthetized with isoflurane, and an 8-cm-long catheter tube was inserted into the rat anus. DNBS was then administered in 250 μL at 15 mg (1st week), 30 mg (2nd week), 45 mg (3rd week), and 60 mg (4th week). Colitis was induced by slowly injecting 250 μL of a 50% ethanol solution into the intestines at a rate of 100 μL / min using a syringe pump. After the injection, the animals were kept in a head-down position for 1 minute to ensure that the solution was evenly distributed throughout the intestine and to prevent leakage.

[0174] Compound 1 obtained in Production Example 1 was used as the test substance, and the dose for each individual was calculated based on body weight, and the test substance was orally administered at 10 mL per kg of body weight once a day, 7 days a week, for a total of 4 weeks.

[0175] 2. Evaluation Method (1) Measurement of colon weight and length ratio Colon tissues were extracted from the sacrificed animals and photographed to measure the length of the colon. The photographs were analyzed using image analysis software (Leica Application Colon length was measured using a CT Suite V4. After photographing, the tissue was opened longitudinally, washed with saline, and weighed after removing the cecum. The measured weight and length were used to calculate the colon ratio (mg / cm) (= colon weight (mg) / colon length (cm)).

[0176] (2) Measurement of serum biomarkers Calprotectin & c-reactive protein (CRP) and IFN-γ in serum collected at necropsy were analyzed by ELISA.

[0177] (3) Immunohistochemical staining To observe immunohistochemical changes in the colonic tissue, each slide was reacted with antibody MX2, and the sites where MX2 was expressed in the tissue were calculated.

[0178] The results of the evaluation methods (1) to (3) are shown in Figure 26. In Figure 26, "compound 1" means compound 1 (free base) obtained in Production Example 1.

[0179] 26, it can be seen that the test substance, Compound 1, is effective in the IBD rat model. It can be seen that the Compound 1-treated group produced significantly lower levels of pro-inflammatory cytokines than the control group (vehicle).

[0180] Experimental Example 10: HDM induction model 1. Induction of atopic model and treatment with test substances (Figure 27) (1) First induction method The ears and back of the neck of NC / Nga mice were shaved with a shaver, and then an appropriate amount of depilatory agent was applied to remove the hair. After wiping off the depilatory agent, approximately 100 mg of atopic dermatitis induction reagent (Biostir, Japan) was applied evenly to the ears and back of the neck using a micropipette tip.

[0181] (2) Method of induction from the second time onwards After shaving with a shaver as needed, 150 μL of 4% SDS aqueous solution was applied evenly to the ears and back of the neck using a micropipette. The area was then dried with cool air using a hair dryer and allowed to air dry for approximately 2-3 hours. Approximately 100 mg of the AD induction reagent was applied evenly to the ears and back of the neck using a micropipette tip. All pretreatments were performed twice a week for a total of 11 treatments over a 6-week period.

[0182] (3) Preparation of test substance The test substance was prepared by weighing an appropriate amount of the phosphate salt of Compound 1 obtained in Example 3 and dissolving it in a topical vehicle (acetone:DMSO=7:1 v / v). During preparation and administration, the test substance was kept protected from light and refrigerated, and prepared every three days.

[0183] (4) Administration Administration of the test substance began when the atopy score reached 2.1 to 2.3 points, and the test substance was applied directly to the center of the skin twice a day for 3 weeks from the day administration began (Day 0) until the day of autopsy.

[0184] 2. Autopsy The animals were sensitized by application of an AD inducer 24 hours before autopsy, and the drug was administered and applied 1 hour before autopsy. 24 hours after induction of atopic dermatitis, the animals were anesthetized. After confirming anesthesia, the abdomen was opened and blood was collected from the caudal vena cava with a syringe. After blood collection, the animals were euthanized and the induced site (skin and ear) was excised. The excised skin was divided into two, one half was fixed in 10% neutral buffered formalin solution, and the other half was stored in an ultra-low temperature freezer (approximately -70°C) until analysis.

[0185] 3. Evaluation Method (1) Clinical atopic dermatitis score To evaluate atopic dermatitis, a skin clinical index evaluation (Matsuda et al., 1997) was performed starting from week 0 after the start of atopic dermatitis induction. The skin clinical index evaluation (Matsuda et al., 1997) was performed by rating erythema / hemorrhage, edema, excoriation / erosion, and scaling / dryness as none (0), mild (1), moderate (2), or severe (3). The evaluation stage was determined based on visual evaluation using a Biostir. The scores for each item were then summed to determine the final score.

[0186] (2)ELISA analysis On the day of autopsy, blood was collected and centrifuged to obtain serum, which was then analyzed for IgE levels. IL-1β, IL-4, IL-13, TNF-α, IL-6, and IL-31 were analyzed using a general-purpose kit.

[0187] The results are shown in Figures 28 to 30. In Figures 28 to 30, *p<0.05, **p<0.01, and ***p<0.001 (n=8). In Figures 28 to 30, "compound 1 (phosphate)" means the "phosphate compound" obtained in Example 3.

[0188] Figure 28 is a graph showing the results of clinical atopic dermatitis scores in Experimental Example 10, and it can be seen that the skin severity scores of all groups administered with phosphate compounds were significantly reduced.

[0189] Figure 29 is a graph showing plasma IgE concentrations in Experimental Example 10. Referring to Figure 29, it can be seen that the serum IgE level was reduced in the Compound 1 administration group compared to the control group.

[0190] Figure 30 shows the analysis results of atopic dermatitis-related cytokines in Experimental Example 10. Referring to Figure 30, it can be seen that the compound 1 administration group had a reduced level of atopic dermatitis-related cytokines compared to the control group.

[0191] While the present invention has been described with reference to preferred embodiments, those skilled in the art will recognize that various modifications may be made without departing from the spirit and scope of the present invention as set forth in the following claims. You will understand that this can be changed and improved.

Claims

1. A pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide, The pharmaceutically acceptable salt is a hydrochloride, hydrobromide, phosphate, camsylate, oxalate, mesylate or napadisylate. Pharmaceutically acceptable salts of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

2. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is hydrochloride of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

3. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is hydrobromide of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

4. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is a phosphate salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

5. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is a camsylate salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

6. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is the oxalate salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

7. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is a mesylate salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide. Pharmaceutically acceptable salts of (1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

8. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is napadisylate of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide.

9. The pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide according to claim 1, wherein N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide and the pharmaceutically acceptable salt are contained in an equivalent ratio of 1:1 to 1:1.

3.

10. A composition for treating or preventing atopic dermatitis, comprising N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof as an active ingredient.

11. The composition for treating or preventing atopic dermatitis according to claim 10, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, phosphate, camsylate, oxalate, mesylate, or napadisylate.

12. A composition for treating or preventing inflammatory bowel disease, comprising N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof as an active ingredient.

13. The composition for treating or preventing inflammatory bowel disease according to claim 12, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, phosphate, camsylate, oxalate, mesylate or napadisylate.

14. The composition for treating or preventing inflammatory bowel disease according to claim 12, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

15. Use of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of atopic dermatitis.

16. 16. The use according to claim 15, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, phosphate, camsylate, oxalate, mesylate or napadisylate.

17. Use of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating or preventing inflammatory bowel disease.

18. 18. The use according to claim 17, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, phosphate, camsylate, oxalate, mesylate or napadisylate.

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