Novel salts of heterocyclic compounds as protein kinase inhibitors and their applications

Abstract

The present invention relates to novel salts of heterocyclic compounds as protein kinase inhibitors and their uses, and the novel salts have excellent water solubility and excellent physical and chemical stability, and therefore can be usefully utilized in the formulation of pharmaceuticals. In addition, the heterocyclic compounds or their salts can effectively treat and prevent atopic dermatitis and inflammatory bowel disease, respectively.

Description

[Technical Field]

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

[0002] It is a well-known fact that, for the same drug, there can be differences in pharmaceutically important properties such as solubility, dissolution characteristics, and bioavailability depending on the form, such as amorphous, one or more crystalline forms, or salts. While numerous types of pharmaceutically acceptable salts of drugs are known and research is ongoing, even for the same salt, the properties differ from drug to drug, making it difficult to obtain an ideal salt that satisfies all the pharmaceutically important properties, including solubility and stability.

[0003] Janus kinases (JAKs) are enzymes that regulate the activity, location, and function of other proteins by phosphorylating them, thereby controlling various intracellular processes. Janus kinases are located at intracellular receptors for inflammatory cytokines. After inflammatory cytokines bind to these receptors and are phosphorylated, they transmit signals from inflammatory cytokines into the cell through interaction with STAT molecules. Excessive activation of signal transduction by various inflammatory cytokines can cause our body's immune system to attack itself, resulting in autoimmune diseases. In recent years, Phase II and Phase III clinical trials of selective JAK1 inhibitors, upadacitinib and abrocitinib, have reported that JAK1 inhibitors rapidly improve the severity and symptoms of Alzheimer's disease.

[0004] On the other hand, atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases. Elevated cytokine levels of Th(T helper)2, Th22, and some Th1 and Th17 cells trigger abnormal immune activation in the skin lesions of atopic dermatitis. Recently, the use of immunotargeted therapies for the treatment of atopic dermatitis has increased, and dupilumab, a monoclonal antibody that targets the IL-4 receptor, has been approved as a treatment for atopic dermatitis. As the number of patients suffering from atopic dermatitis continues to increase, the demand for therapeutic drugs that demonstrate superior efficacy continues. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Korean Published Patent Publication No. 2019-0043437 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] One objective 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 the treatment or prevention of atopic dermatitis, comprising a heterocyclic compound as a protein kinase inhibitor or a pharmaceutically acceptable salt thereof.

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

[0009] Another object of the present invention relates to the use of heterocyclic compounds or pharmaceutically acceptable salts thereof as protein kinase inhibitors for the treatment or prevention of atopic dermatitis.

[0010] Another object of the present invention relates to the use of heterocyclic compounds or pharmaceutically acceptable salts thereof as protein kinase inhibitors for the treatment or prevention of inflammatory bowel disease.

[0011] Another object of the present invention is to provide the use of heterocyclic compounds or pharmaceutically acceptable salts thereof as protein kinase inhibitors in the manufacture of agents for the treatment or prevention of atopic dermatitis.

[0012] Another object of the present invention is to provide the use of heterocyclic compounds or pharmaceutically acceptable salts thereof as protein kinase inhibitors in the manufacture of agents 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, comprising administering a heterocyclic compound or a pharmaceutically acceptable salt thereof as a protein kinase inhibitor to an individual.

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

[0015] The inventors of this application have made diligent efforts to find a compound with improved physicochemical properties that has pharmacological activity equal to or greater than that of conventional compounds, while also improving stability to heat and moisture and minimizing the generation of related substances. As a result, they have confirmed that the salt of the heterocyclic compound according to the present invention has low hygroscopicity, excellent physicochemical properties, stability to heat and moisture, and also improved solubility, thus completing the present invention.

[0016] Furthermore, we have identified therapeutic applications for the heterocyclic compounds and pharmaceutically acceptable salts thereof according to the present invention for the treatment of atopic dermatitis and inflammatory bowel disease.

[0017] The heterocyclic compound according to the present invention and its pharmaceutically acceptable salts were able to effectively inhibit skin severity similar to atopic dermatitis by oral and topical administration in an atopic dermatitis mouse model, more effectively than other selective JAK1 inhibitors. Also, in in vitro human whole blood analysis, the heterocyclic compound of the present invention was proven to be the most selective JAK1 inhibitor, advantageous in terms of safety compared to other JAK inhibitors. Furthermore, the heterocyclic compound according to the present invention and its pharmaceutically acceptable salts were able to confirm a therapeutic effect in an inflammatory bowel disease model.

[0018] Novel salt and method for producing the same In the novel salt of the heterocyclic compound as a protein kinase inhibitor according to the present invention, the heterocyclic compound is represented by the following Chemical Formula I.

[0019]

Chemical Formula

[0020] The name of the compound represented by the above 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 the above Chemical Formula I may be each 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 (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 may be 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]

Chemical formula

[0024] The name of the heterocyclic compound represented by the above Chemical Formula II is (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 the above Chemical Formula I may be an organic acid salt of the heterocyclic compound represented by the above Chemical 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-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide and an organic acid, where the organic acid salt may be a hydrochloride salt, hydrobromide salt, mesylate salt, phosphate salt, napadisylate salt, camusylate salt, or oxalate salt.

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

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

[0029] Furthermore, in the present invention, the pharmaceutically acceptable heterocyclic compound represented by the chemical formula I is The salt exhibits excellent stability even under high temperature and high humidity conditions, and also has excellent photostability, allowing it to be maintained stably for long periods against heat and moisture. Furthermore, in this invention, the pharmaceutically acceptable salt of the heterocyclic compound represented by chemical formula I exhibits excellent solubility and physicochemical properties, and also has excellent bioavailability. Moreover, in this invention, the pharmaceutically acceptable salt of the heterocyclic compound represented by chemical formula I has low hygroscopicity and does not absorb surrounding moisture, allowing it to be maintained for long periods without changes in moisture content.

[0030] The salt of the heterocyclic compound represented by chemical formula I of the present invention may be administered orally or transdermally.

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

[0032] Salts of the heterocyclic compound represented by chemical formula I of the present invention can treat or prevent inflammatory bowel disease. Inflammatory bowel disease may include ulcerative colitis or Crohn's disease.

[0033] In the present invention, a method for producing a pharmaceutically acceptable salt of the heterocyclic compound represented by chemical formula I or chemical formula II is: (A) The step of dissolving or suspending the heterocyclic compound represented by chemical formula I in an organic solvent; (B) Adding one organic acid selected from hydrochloric acid, bromate, 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) The step may include stirring to solidify the salt formed in a previous step.

[0034] The above manufacturing method may further include the step of adding (D) an antisolvent to mature the solid.

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

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

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

[0038] Uses for the treatment or prevention of atopic dermatitis (1) The present invention provides a composition for the treatment or prevention of atopic dermatitis, comprising as an active ingredient N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-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, comprising administering a pharmaceutical composition comprising a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof.

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

[0041] (4) The present invention provides uses for a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof for producing a drug for the treatment or prevention of atopic dermatitis.

[0042] (5) In the present invention according to (1), (2), (3) or (4) above, "atopic dermatitis" is used to mean all diseases classified as atopic dermatitis in the industry, regardless of whether the cause of its occurrence is direct or indirect. Normally, atopic dermatitis is classified into infantile atopic dermatitis, childhood atopic dermatitis, adult atopic dermatitis, and pregnant atopic dermatitis depending on the time of onset or the person affected, but 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) above, the pharmaceutically acceptable salt may be a hydrochloride salt, a hydrobromide salt, a phosphate salt, a camusylate salt, an oxalate salt, a mesylate salt, or a napadisylate salt.

[0044] Uses for the treatment or prevention of inflammatory bowel disease (7) The present invention provides a composition for the treatment or prevention of inflammatory bowel disease, comprising as an active ingredient N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide, 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 comprising a heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof.

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

[0047] (10) The present invention provides uses for heterocyclic compounds represented by chemical formula I or pharmaceutically acceptable salts thereof for producing agents for the treatment or prevention of inflammatory bowel disease.

[0048] (11) In the present invention according to (7), (8), (9) or (10) above, "inflammatory bowel disease" means chronic inflammation of unknown cause occurring in the intestines, and usually 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 occurring in the intestinal tract, including infectious enteritis such as bacterial, viral, amoebic, and tuberculous enteritis, as well as ischemic bowel disease and radiation enteritis.

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

[0050] The pharmaceutical compositions of the present invention for the prevention or treatment of atopic dermatitis or inflammatory bowel disease according to (1) or (7) above may further comprise pharmaceutically acceptable additives, suitable carriers commonly used, excipients, disintegrants, binders, lubricants or diluents.

[0051] The aforementioned "pharmaceutically acceptable additives" may include carriers, excipients, disintegrants, binders, lubricants, or diluents that do not irritate living organisms or inhibit the biological activity and properties of the injected compound. The types of additives used in the present invention are not particularly limited, and any commonly used and pharmaceutically acceptable additives in the art may be used. Non-limiting examples of such additives include mannitol, microcrystalline cellulose, croscarmellose sodium, hydroxypropyl cellulose, colloidal silicon dioxide, microcrystalline cellulose, croscarmellose sodium, colloidal silicon dioxide, magnesium stearate, or mixtures thereof. In addition, other common additives such as antioxidants, buffers, and / or bacteriostatic agents may be added as needed.

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

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

[0054] In the present invention, the dose of the heterocyclic compound represented by chemical formula I or a pharmaceutically acceptable salt thereof must be a pharmaceutically effective dose. “Pharmaceutically effective dose” means an amount sufficient to prevent or treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective dose can be varied by those skilled in the art depending on factors such as the formulation method, the patient's condition and weight, sex, age, severity, form of the drug, route and duration of administration, excretion rate, and sensitivity to the drug. The effective dose varies, as is recognized by those skilled in the art, depending on the route of administration, the use of excipients, and the possibility of concomitant use with other drugs.

[0055] In one embodiment, the heterocyclic compound represented by chemical formula I of the present invention or a pharmaceutically acceptable salt thereof may be administered transdermally at a dose of 0.1-5% or orally at a dose of 10-200 mg / kg. For example, it may be administered transdermally at a dose of 0.3-3% or orally at a dose of 50-200 mg / kg.

[0056] As an example, the heterocyclic compound represented by chemical 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, it may be formulated as tablets, lozenges, water-soluble suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs.

[0058] When formulated as a transdermal agent, it may be manufactured in dosage forms such as solutions, suspensions, gels, creams, and lotions. [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 physical and chemical stability, and can therefore be usefully utilized in the formulation of pharmaceuticals.

[0060] Furthermore, the present invention effectively inhibits 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 drawing]

[0061] [Figure 1] Figure 1 is a graph of the NMR analysis results of the hydrochloride salt of the compound represented by chemical formula II of the present invention. [Figure 2] Figure 2 is a graph showing the NMR analysis results of the hydrobromide salt of the present invention. [Figure 3] Figure 3 is a graph showing the NMR analysis results of the phosphate of the present invention. [Figure 4] Figure 4 is a graph showing the NMR analysis results of the camusylate salt of the present invention. [Figure 5] Figure 5 is a graph showing the NMR analysis results of the oxalate salt of the present invention. [Figure 6] Figure 6 shows the NMR analysis results graph of the mesylate, oxalate, and napadisylate salts of the present invention. [Figure 7] Figure 7 shows the NMR analysis results graph of the mesylate, oxalate, and napadisylate salts of the present invention. [Figure 8] Figure 8 is a graph showing the experimental results of compound 1's ability to inhibit IL-4 secretion in basophil cells. [Figure 9] Figure 9 is a diagram showing the evaluation results of the IL-13, IL-10, and TNF-α secretion capacity of compound 1 of the present invention. [Figure 10] Figure 10 is a diagram illustrating the DNCB-induced animal model in Experimental Example 6. [Figure 11] Figure 11 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 6. [Figure 12]Figure 12 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 6. [Figure 13] Figure 13 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 6. [Figure 14] Figure 14 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 6. [Figure 15] Figure 15 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 6. [Figure 16] Figure 16 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 6. [Figure 17] Figure 17 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 7. [Figure 18] Figure 18 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 7. [Figure 19] Figure 19 shows the results of confirming the therapeutic effect of DNCB-induced atopic dermatitis in the animal model of Experimental Example 7. [Figure 20] Figure 20 is a diagram illustrating the DSS-induced animal model in Experimental Example 8. [Figure 21] Figure 21 shows the results of confirming the therapeutic effect of DSS-induced colitis in the animal model of Experimental Example 8. [Figure 22] Figure 22 shows the results of confirming the therapeutic effect of DSS-induced colitis in the animal model of Experimental Example 8. [Figure 23] Figure 23 shows the results of confirming the therapeutic effect of DSS-induced colitis in the animal model of Experimental Example 8. [Figure 24]Figure 24 shows the results of confirming the therapeutic effect of DSS-induced colitis in the animal model of Experimental Example 8. [Figure 25] Figure 25 is a diagram illustrating the DNBS-induced animal model in Experimental Example 9. [Figure 26] Figure 26 shows the results of confirming the therapeutic effect of DNBS-induced colitis in the animal model of Experimental Example 9. [Figure 27] Figure 27 is a diagram illustrating the HDM-induced animal model for Experimental Example 10. [Figure 28] Figure 28 shows the results of confirming the therapeutic effect of HDM-induced atopic dermatitis in the animal model of Experimental Example 10. [Figure 29] Figure 29 shows the results of confirming the therapeutic effect of HDM-induced atopic dermatitis in the animal model of Experimental Example 10. [Figure 30] Figure 30 shows the results of confirming the therapeutic effect of the HDM-induced animal model of atopic dermatitis in experimental example 10. [Modes for carrying out the invention]

[0062] The embodiments of the present invention will now be described in detail. Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as those generally understood by a person of ordinary skill in the art to which the present invention pertains. Terms as defined in commonly used dictionaries should be interpreted as having the meaning consistent with their meaning in the context of the relevant art, and not as ideal or overly formal unless explicitly defined herein.

[0063] In each drawing, *p<0.05, **p<0.01, and ***<0.001.

[0064] Manufacturing Example 1: Synthesis of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide The compound in the title was prepared according to the method disclosed in Patent Document 1. 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: Production of hydrochloride 30 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 90 mL of methanol and heated to 50°C. Then, 1.1 eq. of hydrochloric acid (in MeOH) was slowly added dropwise and stirred at 50°C for 2 hours to obtain a solution. The solution was cooled to room temperature and precipitated. The solution was then filtered, washed with methanol, and vacuum-dried to obtain the title compound (yield 31.1 g, yield 94%) 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: Production of hydrobromide 30 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 300 mL of acetone and stirred. Then, 1.1 eq. of hydrobromic acid was slowly added dropwise and stirred. After filtration and washing with methanol, the mixture was vacuum-dried to obtain the title compound (yield 34.4 g, 94%) 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: Production of phosphate 30 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 90 mL of methanol and heated to 50°C. Then, 1.05 eq. of phosphoric acid was added and the mixture was stirred at 50°C for 1 hour to obtain a solution. The solution was cooled to room temperature, filtered, washed with methanol, and then vacuum-dried to obtain the title compound (yield 40 g, yield 85%) as an orange powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 3.

[0068] Example 4: Production of camsilate 20 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 200 mL of ethanol. Then, 1.05 eq. of camphor sulfonic acid was added and the mixture was mixed at room temperature. After filtration and washing with ethanol, the mixture was vacuum-dried to obtain the title compound (yield 24.1 g, 92%) 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: Production of oxalates 30 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 510 mL of acetonitrile and stirred. Then, 1.05 eq. of oxalic acid was added and stirred at room temperature. After filtration and washing with acetonitrile, the mixture was vacuum-dried to obtain the title compound (yield 29.1 g, yield 66%) 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: Production of mesylate 30 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 300 mL of ethyl acetate and heated to 50°C. Then, 1.1 eq. of methanesulfonic acid was slowly added dropwise while continuing to stir at the same temperature. After filtration and washing with methanol, the mixture was vacuum-dried to obtain the title compound (yield 35.3 g, 93%) as an orange powder. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 6.

[0071] Example 7: Production of napadisylate 1 g of (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide was mixed with 20 mL of acetonitrile. The mixture was then heated to 50°C, 1.05 eq. of 1,5-naphthalenedisulfonic acid was added and mixed, filtered and washed, and then vacuum dried to obtain the title compound. NMR analysis was performed to confirm the formation of the title compound. The results are shown in Figure 7.

[0072] Analysis and measurement methods 1. Measurement of water solubility Water solubility was measured by shaking a saturated solution (20 mg / 0.5 mL) at room temperature for 24 hours. The liquid was collected from the solution and filtered using a 0.22 μm PVDF filter. The filtrate was diluted 10-fold for LC analysis.

[0073] LC analysis was performed using the following method.

[0074] [Table 1]

[0075] [Table 2]

[0076] 2. Physical Stability Evaluation Experiment To evaluate physical stability, the sample was prepared using ((LPDE+N2)+Silica gel The sample was packaged in 1g + LDPE + Al-Bag and stored under harsh conditions (60℃±2℃ / 80%RH±5%) for 2 / 4 weeks before being evaluated.

[0077] 3. Photostability evaluation experiment The sample was packaged in an LDPE bag and exposed to Visible (1.2M Lux-h) and UV (200W-h / m²) light. 2 The samples were evaluated after being stored under the respective conditions of ) and UV+Visible.

[0078] 4. Hygroscopicity evaluation experiment The samples were placed in glass desiccators adjusted to relative humidity levels of 33%, 53%, 75%, and 93%, stored for 2 and 4 weeks, and then evaluated.

[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 confirmed that the water solubility of the salts according to the present invention is at least 0.9 mg / mL. In particular, the water solubility of hydrochloride, hydrobromide, camusylate, oxalate, and phosphate is excellent at 1.9 mg / mL or higher, and it can be confirmed that hydrochloride, hydrobromide, and oxalate exhibit extremely excellent water solubility exceeding 4 mg / mL.

[0082] 2. Physical Stability Evaluation Results The results of the physical stability evaluation of the salts obtained in Examples 1 to 4 are shown in Table 2 below. In Table 2, "initial" represents the results measured after salt production, while "S2W" and "S4W" represent the results measured after 2 weeks and 4 weeks, respectively, under harsh conditions.

[0083] [Table 4]

[0084] [Table 5]

[0085] Referring to Table 2, the results regarding purity and content before and after exposure to harsh conditions confirm that the salt according to the present invention exhibits excellent stability against moisture and heat. In particular, in terms of purity, it can be confirmed that each individual related substance and the total related substance meet the quality standards of the raw material chemicals even after exposure to harsh conditions.

[0086] 3. Results of the photostability evaluation The results obtained by the above-mentioned photostability evaluation method for salts according to Examples 1-5 of the present invention are as follows: As shown in Table 3 below.

[0087] [Table 6]

[0088] [Table 7]

[0089] Referring to Table 3, it can be confirmed that the salt according to the present invention exhibits excellent stability against ultraviolet and visible light. In particular, in terms of purity, it can be confirmed that each individual related substance and the total related substance meet the quality standards of the raw material chemicals even after exposure to harsh conditions.

[0090] 4. Results of hygroscopicity evaluation The results for the salts obtained by the above-mentioned hygroscopicity evaluation method 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 confirmed that the salt according to the present invention has low hygroscopicity.

[0094] Experimental Example 1: ADP-Glo ​​kinase assay (1) JAK1 kinase assay (S)-N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-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 at 10 mM to prepare five samples at different concentrations (1,000 nM, 200 nM, 40 nM, 8 nM, and 0.16 nM). 10 mM ATP and 10 mg / mL IRS (insulin receptor substrate) were diluted to 1 / 40 and 1 / 50, respectively, with 1X kinase buffer. The preparations were made as follows: Additionally, 222 ng / μL of JAK1 enzyme was administered to 1X kinase. The ATP was diluted to 1 / 8 with buffer to prepare the mixture. 1 μL of ATP, 1 μL of IRS, and 1 μL of compound 1 were mixed, and finally 2 μL of JAK1 enzyme was added and the mixture was reacted at 30°C for 40 minutes. At this point, a tube without JAK1 enzyme was prepared as a blank sample, and a tube without compound 1 was prepared as a positive control group (final sample concentrations: 200 nM, 40 nM, 8 nM, 1.6 nM, and 0.32 nM).

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

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

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

[0098] (3) JAK3 kinase assay Compound 1 at 10 mM was diluted in DMSO to prepare four samples at different 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 a concentration of 1 mg / mL. 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 finally 2 μL of JAK3 enzyme was added and the mixture was reacted at 30°C for 40 minutes. Here, a tube without JAK3 enzyme was prepared as a blank, and a tube without Compound 1 was prepared as a positive control (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 reacted at 30°C for 40 minutes, after which 10 μL of kinase detection reagent was added and reacted at room temperature for 15 minutes. 18 μL of the reaction mixture was transferred to a 384-well plate, and the luminescence was measured using a plate reader.

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

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

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

[0103] Furthermore, NK-92 cells are suitable for evaluating the activity of JAK1 and JAK3 mediated by IL-2 because they grow in an IL-2-dependent manner as a natural killer cell line and are derived from peripheral blood mononuclear cells. Therefore, NK-92 cells were prepared to evaluate the activity of JAK1 and JAK3.

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

[0105] 2. Preparation of test substances and irritants (1) Preparation of test material The test substance was dissolved in DMSO to prepare a 10 mM solution, which was then dispensed into 20 μL portions and stored at -20°C.

[0106] (2) Preparation of the stimulus EPO:2000U / 0.5mL stock was dispensed into 50μL portions and stored in the refrigerator. A stock of IL-2 at 200 μg / mL was prepared, aliquoted into 5 μL portions, stored, and treated with 2 ng / ml (IL-2 1 μg / ml = 13000 U / ml). A stock solution of IFN-alpha B2 at 1 × 10 U / mL was diluted and used to a concentration of 30,000 U / mL.

[0107] (3) Preparation of Assay medium For the 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: Contains HBSS, 1% FBS, and 1% P / S. It was prepared and used in such a way. U2OS cell assay: Prepared to contain HBSS, 1% FBS, and 1% P / S.

[0108] 3. Sample preparation After centrifuging the cultured cells (1,000 rpm, 5 minutes), they were washed with PBS. The cell line size and appropriate reaction concentration were set, and 1 × 10⁶ 5 ~106 Cells were seeded into a 96-well plate at a concentration of 50 μL / well. The test substance was prepared at 4X the concentration intended for treatment, and then serially diluted by 1 / 5 to prepare 5-6 different concentrations. The test substance was diluted 1:1000 in assay medium and treated at 20 μL / well. The untreated group (no drug treatment) was treated with 20 μL / well of assay medium and cultured in a CO2 incubator for 1 hour.

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

[0110] As described above, after stimulating with cytokines for 20 minutes, the 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 procedure followed the protocol of the 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 Production Example 1 was diluted with distilled water, and then serially diluted to 1 / 5 with 4% DMSO. 5 μL of the test substance was added to 100 μL of whole blood collected using a 1.7 mL Eppendorf tube and mixed, then incubated at 37°C for 45 minutes.

[0113] IL-6 (10 μg / ml) was diluted with 0.1% BSA / DW to 1 μg / mL each. 5 μL of IL-6 was added (final concentration 50 ng / mL) and incubated at 37°C for 15 minutes. For the non-stimulating control group, 5 μL of DPBS was added. Lyse / fix buffer 5X was diluted to 1X with distilled water and used.

[0114] 900 μL of lyse / fix buffer, pre-warmed at 37°C, was added to each tube and incubated at 37°C for 20 minutes. The tubes were then centrifuged at 500xg 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 pre-dissolved on ice, was added, and the tubes were incubated on ice for 30 minutes before spinning down. After washing once with wash buffer, the tubes were resuspended with a buffer (BD Pharmingen® staining buffer).

[0115] For double staining of CD4 and pSTAT1, staining Anti-CD and anti-pSTAT1 were mixed in a staining buffer and added to each sample. The tubes containing the samples were gently tapped by hand to ensure thorough mixing of antibodies and cells, and then left overnight at 4°C. The results were analyzed the following day using FACSCantoII.

[0116] 2. TEST2~6 TEST2 to TEST6 were each conducted in substantially the same manner as the experiment of TEST1, 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 numerical values analyzed by FlowJo were used with GraphPad Prism 5 software (product name) to calculate the IC 50 value.

[0119] Based on No treatment response (NTR), the degree induced by the phosphorylation of STAT induced by cytokines was converted to a relative ratio (% control). Using GraphPad Prism (version 5.0), a dose-response plot was obtained for the activity of each test substance by concentration, and the IC 50 value was calculated.

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

[0121]

Table 11

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

[0123] Experimental Example 4: Inhibitory ability of IL-4 secretion in basophil cells RBL-2H3 cells were diluted and dispensed in 10% FBS EMEM medium at 1X 105 cells / well in a 24-well plate. Incubated at 37 °C for 18 hours or more.

[0124] The test substance (compound 1) was treated with PMA (50 nmol / L) and A23187 (1 μmol / L) at different concentrations (final concentrations 0, 0.1, 0.5, and 1 μM). The samples were incubated at 37°C for 24 hours. After the reaction was terminated on ice, the culture supernatant was analyzed using an IL-4 ELISA kit to measure the level of IL-4 secretion. The results are shown in Figure 8.

[0125] Figure 8 is a graph showing the experimental results of compound 1's ability to inhibit IL-4 secretion in basophil cells.

[0126] Referring to Figure 8, it can be confirmed that the concentration of IL-4 in the group treated with compound 1 according to the present invention is lower than that of the control group (vehicle). In other words, it was found that 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, it was confirmed that the concentration was reduced to more than half compared to the negative control group (vehicle).

[0127] Experimental Example 5: IL-13, IL-10, and TNF-α secretion capacity 50 mL of cell culture medium was mixed with 50 μL of DNase I (2000 units / mL) (the medium was warmed in a 37°C water bath because it contained both enzymes and cells). In addition, PMBC from atopic patients was rapidly dissolved in a 37°C water bath.

[0128] PMBC was slowly added to the prepared culture medium and reacted at 37°C for 5 minutes. The mixture was centrifuged at 200 rpm for 15 minutes, the supernatant (supernatants) was removed, and the cells were resuspended in 1 mL of cell culture medium and counted.

[0129] 2 x 10 5 The cells were diluted in cell culture medium to a concentration of 100 μL / well 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 all wells except for the wells of the negative control group (50 μL of cell culture medium was added to the wells of the negative control group).

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

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

[0133] After dissolving the culture supernatant sample, ELISA was performed according to the ELISA kit datasheet protocol. The results are shown in Figure 9.

[0134] Figure 9 is a diagram showing the evaluation results of the IL-13, IL-10, and TNF-α secretion capacity of compound 1 of the present invention.

[0135] Referring to Figure 9, it can be confirmed that IL-13, IL-10, and TNF-α are reduced in anti-CD3 and anti-CD28 activated PBMCs of atopic dermatitis patients. In particular, in the groups experimented with compound 1 at concentrations of 100 nM and 1,000 nM, IL-13, IL-10, and TNF-α were all significantly reduced. You can confirm that it is happening.

[0136] Experimental Example 6: DNCB Induction Model (topical) 1. Induction of an atopic dermatitis model and treatment of test substances (Figure 10) NC / Nga mice were anesthetized with isoflurane, and the hair on their backs (from below the ears to the top of the tail) was removed. The NC / Nga mice were left for 24 hours to allow any micro-injuries to heal from the 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 skin of the back (primary skin application). A secondary skin application was performed 3 days later for sensitization.

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

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

[0140] 2. Autopsy Six weeks after the initial skin application, the test animals were necropsied. Specifically, after anesthetizing the test animals with isoflurane, as much blood as possible was collected from the posterior vena cava using a syringe and stored in a 5 mL vacuum tube containing a thrombolytic agent. The blood was left at room temperature for 15-20 minutes to allow it to coagulate, 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 the five symptoms (erythema, dryness, edema and hematoma, erosion, and keratinization) on a scale of none (0), mild (1), moderate (2), and severe (3). The total score indicating clinical severity was defined as the sum of all scores (maximum score: 15). Assessments were performed simultaneously by the same researcher twice a week.

[0142] (2) Itch behavior test In the fourth week, to assess itchy behavior, mice were placed in cages and the frequency of scratching was evaluated for 30 minutes.

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

[0144] (4) Spleen / Body weight After measuring the spleen and the body weight of the mice, the spleen's weight relative to body weight (relative organ weight) was calculated.

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

[0146] (6) Histopathological analysis On the day of the autopsy, skin samples in a 10% neutral buffered formalin solution were stained with H&E and toluidine blue and analyzed.

[0147] By referring to all the evaluation results of Experimental Example 6 in Figures 11-16, it can be confirmed that the phosphate compound according to the present invention improves the symptoms of atopic dermatitis. In Figures 11-16, "compound1" refers to the "phosphate compound" obtained in Example 3.

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

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

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

[0151] Figure 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 decreased in the group administered the phosphate compound.

[0152] Figure 16 shows the skin staining results of Experimental Example 6, confirming that treatment with phosphate compounds altered the histological characteristics of atopic dermatitis.

[0153] Experimental Example 7: DNCB Induction Model (PO) An animal model induced by DNCB was prepared substantially similarly to the DNCB model (topical) in Experimental Example 6, except that the phosphate compound was administered orally, and the phosphate compound obtained in Example 3 was administered. The phosphate compound was administered orally twice a day for 28 days, for a total of 56 doses, 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 administered into the stomach through the mouth of the mice using a 1 mL syringe and tube. The control group was administered 0.5% methylcellulose (0.5% MC).

[0154] Figure 17 is a graph showing the results of the clinical atopic dermatitis score for Experimental Example 7, Figure 18 is a graph showing the results of the itching behavior test for Experimental Example 7, and Figure 19 is a graph showing the calculation results of the spleen-to-body weight ratio for Experimental Example 7. In Figures 17 to 19, "compound1" refers to the "phosphate compound" obtained in Example 3.

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

[0156] Experimental Example 8: DSS-Induced Model (Figure 20) 1. Induction of colitis and handling of test material To induce colitis in C57BL / 6J mice, they were given drinking water treated with 3% Dextran Sodium Sulphate (DSS) every morning for 8 days, while the negative control group was given 0% DSS.

[0157] After grouping the mice into multiple groups (day 0), 0.5% methylcellulose and compound 1 obtained in Production Example 1 were orally administered to the mice at 9:00 AM and 5:00 PM daily from day 1 to day 7. The test animals were necropsied on day 8.

[0158] 2.Analysis method (1) Animal disease activity index The Disease Activity Index (DAI) was calculated using body weight, stool concentration, and fecal-blood score to represent the severity of the disease. Body weight and food intake were monitored and evaluated 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) Weight-to-length ratio of the colon After measuring the weight and length of the colon obtained through autopsy, the weight-to-length ratio was calculated.

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

[0162] mRNA was extracted from a portion of the colon tissue (at 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 The colon was fixed with 10% formalin, then cut using 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] In Figures 21 to 24, "compound1" refers to compound 1 (freebase) obtained in Production Example 1.

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

[0167] Referring to Figure 21, it can be seen that the disease activity index increased over time in all groups except the normal group. However, the increase in the disease activity index in the group administered compound 1 of the present invention was lower than that of the control group (Vehicle). In particular, in the group administered 90 mg / kg of compound 1, the disease activity index was reduced to about half compared to the control group (Vehicle).

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

[0169] Referring to Figure 22, it can be seen that in all groups other than the normal group, the weight-to-length ratio was higher than in the normal group. However, it can be confirmed that the group administered compound 1 had a lower value compared to the control group (Vehicle). This indicates that in the control group, the inflammation in the large intestine remained or worsened, with an increase in the weight of the large intestine and a decrease in its length, while in the group administered compound 1, the colitis improved and the symptoms became milder.

[0170] Figure 23 shows the detection results of plasma and colon biomarkers in Experimental Example 8.

[0171] Referring to Figure 23, it can be confirmed that the group administered compound 1 of the present invention showed a decrease in the concentrations of KC and MIP-2, which are biomarkers that increase in plasma in a colon inflammatory environment. Furthermore, it can be confirmed that the expression levels of MX2 and TNF-alpha in the colon were also decreased in the group administered compound 1 of the present invention. From these findings, it can be seen that the increase of inflammation-related biomarkers in the colon can also be prevented.

[0172] Figure 24 is a graph showing the results of the histopathology scores in Experimental Example 8, and it can be seen that the group administered compound 1 of the present invention had lower histopathology scores.

[0173] Experimental Example 9: DNBS-Induced Model (Figure 25) 1. Induction of colitis and handling of test material Before inducing colitis with DNBS each week, the experimental animals were fasted for 24 hours. The fasted animals were lightly anesthetized with isoflurane, and an 8 cm catheter tube was inserted into the rat's anus, after which DNBS [15 mg (1st week), 30 mg (2nd week), 45 mg (3rd week), and 60 mg (4th week) in 250 μL] was administered. Colitis was induced by slowly injecting 250 μL of 50% ethanol solution at a rate of 100 μL / min using a syringe pump. After injection, the animals were kept in a head-down position for 1 minute to ensure the solution spread evenly throughout the intestines and did not leak out of the body.

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

[0175] 2. Evaluation Method (1) Measurement of the ratio of weight and length of the large intestine The colon tissue extracted from the sacrificial experimental animals was photographed to measure the length of the colon. The photographs were then analyzed using image analysis software (Leica Application). The length of the large intestine was measured using Suite V4). After the tissue was photographed, the cecum was removed, the tissue was opened longitudinally, washed with saline solution, and its weight was measured. The measured weight and length were used to calculate the large intestine ratio (mg / cm) (= large intestine weight (mg) / large intestine length (cm)).

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

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

[0178] The results of the evaluation methods (1) to (3) described above are shown in Figure 26. In Figure 26, "compound1" refers to compound 1 (freebase) obtained in production example 1.

[0179] Referring to Figure 26, it can be confirmed that compound 1, the test substance, shows efficacy in the IBD rat model. It can be confirmed that the group administered compound 1 produced significantly lower levels of pro-inflammatory cytokines compared to the control group (vehicle).

[0180] Experimental Example 10: HDM-Induction Model 1. Induction of an atopic dermatitis model and treatment of test substances (Figure 27) (1) Initial induction method After shaving the ears and the back of the neck of NC / Nga mice with a shaver, a suitable 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 uniformly applied to the ears and the back of the neck using a micropipette tip.

[0181] (2) Method of induction from the second time onward After shaving with a shaver as needed, 150 μL of 4% SDS aqueous solution was applied uniformly to the ears and back of the neck using a micropipette. After drying with a hairdryer on the cool setting, it was allowed to air dry for approximately 2-3 hours. Approximately 100 mg of AD induction reagent was applied uniformly 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 6 weeks.

[0182] (3) Preparation of the test substance As the test substance, an appropriate amount of the phosphate of compound 1 obtained in Example 3 was weighed and dissolved in a topical vehicle (acetone:DMSO = 7:1 v / v) to prepare the test substance. During preparation and administration, the test substance was kept protected from light and prepared every 3 days under light-shielded refrigeration conditions.

[0183] (4) Administration Administration of the test substance began when the atopic dermatitis score reached 2.1-2.3 points. The test substance was applied directly to the central part of the skin twice a day for three weeks, from the start of administration (Day 0) until the autopsy day.

[0184] 2. Autopsy The animals were sensitized by applying an AD-inducing substance 24 hours before necropsy, and the drug was administered and applied 1 hour before necropsy. After inducing atopic dermatitis, the animals were anesthetized 24 hours later, and after confirming anesthesia, the abdomen was opened and blood was drawn from the posterior vena cava using a syringe. After blood collection, the animals were euthanized, and the induced areas (skin and ears) were excised. The excised skin was divided into two halves; half was fixed in a 10% neutral buffered formalin solution, and the other half was stored in a cryogenic freezer (approximately -70°C) until analysis.

[0185] 3. Evaluation Method (1) Clinical Atopic Dermatitis Score To evaluate atopic dermatitis, a skin clinical index assessment (Matsuda et al., 1997) was performed from week 0 after initiation of atopic dermatitis induction. The skin clinical index assessment (Matsuda et al., 1997) evaluated erythema / hemorrhage, edema, excoriation / erosion, and scaling / dryness on a scale of none (0), mild (1), moderate (2), and severe (3), with the assessment stages determined based on Biostir visual assessment. Subsequently, the sum of the scores for each item was evaluated as the final score.

[0186] (2)ELISA analysis Serum obtained by centrifugation of blood collected on the day of autopsy was used to analyze blood IgE levels, and IL-1β, IL-4, IL-13, TNF-α, IL-6, and IL-31 were analyzed using excised skin. The analyses were performed 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, "compound1(phosphate)" refers to the "phosphate compound" obtained in Example 3.

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

[0189] Figure 29 is a graph showing the plasma IgE concentration in Experimental Example 10. Referring to Figure 29, it can be seen that the serum IgE levels were reduced in the group administered compound 1 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 group administered compound 1 had a decrease in atopic dermatitis-related cytokines compared to the control group.

[0191] Although preferred embodiments of the present invention have been described above, those skilled in the art may make various modifications without departing from the spirit and scope of the invention as described in the following claims. They will understand that it can be corrected and changed.

Claims

1. A pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide, The pharmaceutically acceptable salts are hydrochloride, hydrobromide, phosphate, camsilate, or oxalate. A pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide.

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

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

4. The pharmaceutically acceptable salt is the phosphate salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide according to claim 1. A pharmaceutically acceptable salt of carbboxamide.

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

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

7. A pharmaceutically acceptable salt of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide according to claim 1, wherein the pharmaceutically acceptable salt is contained in a ratio of 1:1 to 1:1.3 equivalents.

8. A composition for the treatment or prevention of atopic dermatitis, comprising N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof as an active ingredient.

9. The composition for the treatment or prevention of atopic dermatitis according to claim 8, wherein the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, phosphate, camsilate, or oxalate.

10. A composition for the treatment or prevention of inflammatory bowel disease, comprising N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof as an active ingredient.

11. The composition for the treatment or prevention of inflammatory bowel disease according to claim 10, wherein the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, phosphate, camsilate, or oxalate.

12. The composition for the treatment or prevention of inflammatory bowel disease according to claim 10, wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

13. Use of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof for the manufacture of agents for the treatment or prophylaxis of atopic dermatitis.

14. The use according to claim 13, wherein the pharmaceutically acceptable salt is a hydrochloride, hydrobromide, phosphate, camsilate, or oxalate.

15. Use of N-(4-(1-(2-cyanoacetyl)-3-methyl-1,2,3,6-tetrahydropyridine-4-yl)-1H-pyrrolo[2,3-b]pyridine-6-yl)cyclopropanecarboxamide or a pharmaceutically acceptable salt thereof for the manufacture of agents for the treatment or prophylaxis of inflammatory bowel disease.

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

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