An amide compound containing a pyrimidine structure and application thereof in preparation of a TYK2 JH2 kinase inhibitor

Abstract

The application discloses an amide compound containing a pyrimidine structure and application thereof in preparation of a TYK2 JH2 kinase inhibitor, and relates to the technical field of compound preparation.The application provides an amide compound containing a pyrimidine structure, which can effectively block IL-12 / IL-23 and type I interferon signaling pathways by inhibiting TYK2 JH2 kinase activity with high selectivity, and significantly improve the pathological process of autoimmune diseases (such as psoriasis, colitis, systemic lupus erythematosus and the like); the compound has the advantages of low toxic side effect and high targeting, can precisely regulate abnormal immune response, and can provide various dosage forms (tablets, capsules, injection agents and the like), support oral or injection administration, and has both curative effect and medication convenience, and therefore provides a safe and efficient novel drug scheme for autoimmune disease treatment.

Description

Technical Field

[0001] This invention relates to the field of compound preparation technology, and in particular to an amide compound containing a pyrimidine structure and its application in the preparation of TYK2 JH2 kinase inhibitors. Background Technology

[0002] Autoimmune diseases are a group of illnesses caused by abnormalities in the body's immune system, leading to the mistaken attack of the body's own tissues or organs. They are increasingly becoming a significant threat to human health. Under normal circumstances, when the body is attacked by external pathogens, the immune system can recognize and eliminate these invaders, while simultaneously protecting its own tissues from attack through immune tolerance mechanisms. However, in patients with autoimmune diseases, this immune tolerance mechanism is disrupted, causing self-antigens, which should be considered "self," to be mistakenly identified as foreign substances and attacked, resulting in tissue damage.

[0003] With the increasing aging of the global population and changes in lifestyle, the incidence of autoimmune diseases is rising year by year, causing great suffering to patients and placing a heavy burden on medical resources. These diseases are diverse, including rheumatoid arthritis, Hashimoto's thyroiditis, systemic lupus erythematosus, inflammatory bowel disease, multiple sclerosis, and psoriasis. Their pathogenesis is complex, involving multiple factors such as genetics, environment, and immune dysregulation, and their clinical manifestations are diverse, thus posing many challenges in diagnosis and treatment.

[0004] The Janus kinase family comprises four members: JAK1, JAK2, JAK3, and TYK2. JAKs generally consist of four parts: the SH2 homology domain, the FERM domain, the kinase domain (JH1), and the pseudokinase domain (JH2). While structurally similar and all possess signal transduction functions, JAKs exhibit distinct characteristics in signal transduction and expression. JAK1 primarily mediates the signal transduction of cytokines such as IL-2, IL-6, and IFN-γ, and is widely expressed in various tissues, associated with autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. JAK2 primarily mediates the signal transduction of hematopoietic cytokines, mainly expressed in the hematopoietic system, and is associated with myeloproliferative disorders. JAK3 primarily mediates the signal transduction of cytokines such as IL-2, IL-4, and IL-7, mainly expressed in immune cells, and is associated with immunodeficiency and autoimmune diseases. TYK2 primarily mediates the signal transduction of IL-12, IL-23, and type I interferon. It is widely expressed in various tissues and is associated with autoimmune diseases such as psoriasis and inflammatory bowel disease. The STAT family has seven members: STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6. STAT proteins consist of six functional domains: ① N-terminal domain; ② coil-coil domain; ③ DNA-binding domain; ④ Scr homology 2 region (SH2); ⑤ tyrosine phosphorylation site; and ⑥ C-terminal transcription activation domain. In humans, the JAK-STAT signaling pathway is closely related to autoimmune diseases. The typical signal transduction process of the JAK-STAT pathway is as follows: ligands bind to transmembrane cytokine receptors and dimerize, thereby activating JAK kinase. Activated JAK kinase binds to STAT proteins, simultaneously phosphorylating STAT and activating the STAT protein. The phosphorylated STAT protein forms a dimer, enters the cell nucleus, and binds to the promoter of the corresponding target gene, enabling transcription of the target gene. The JAK-STAT pathway is involved in the pathogenesis of psoriasis, mainly through its influence on T cell differentiation, abnormal proliferation of keratinocytes, and abnormal angiogenesis.

[0005] There are two types of TYK2 inhibitors: one type acts on the JH1 domain and is an ATP-competitive inhibitor; the other type acts on the JH2 pseudokinase domain and is an allosteric inhibitor. JH1 has catalytic activity and is responsible for the binding of TYK2 to downstream STAT proteins. STAT is phosphorylated and dimerized before entering the cell nucleus. TYK2 inhibitors that act on the JH1 binding site include Brepocitinib, Ropsacitinib, and ICP-332. These ATP-competitive inhibitors can directly bind to the ATP-binding site of the JH1 domain, competitively blocking its binding to ATP, thereby inhibiting the kinase activity of TYK2 and preventing it from phosphorylating STAT. However, because the ATP-binding sites of Janus family members are very similar, the selectivity of ATP-competitive inhibitors is not high. While inhibiting TYK2, they may also inhibit JAK1, JAK2, and JAK3 simultaneously, thus causing some side effects during treatment, such as a significantly increased risk of infection.

[0006] The JH2 pseudokinase domain of TYK2 contains an allosteric binding pocket, unlike other members of the Janus family. This gives TYK2 allosteric inhibitors high selectivity, thus avoiding some off-target effects. Although JH2 has no catalytic activity, it plays a crucial role in regulating TYK2 activity. After the inhibitor binds to the allosteric binding pocket of JH2, it causes a conformational change in the JH2 pseudokinase domain, thereby affecting the activity of the JH1 domain and inhibiting TYK2 activation.

[0007] Therefore, it is urgent to provide a TYK2 inhibitor with high safety and low side effects. Summary of the Invention

[0008] The purpose of this invention is to provide an amide compound containing a pyrimidine structure and its application in the preparation of TYK2 JH2 kinase inhibitors, so as to solve the problems existing in the prior art.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] One of the technical solutions of this invention is to provide an amide compound containing a pyrimidine structure, which has the general formula shown in Formula I or Formula II:

[0011]

[0012] in:

[0013] R1 is

[0014] R2 is

[0015] One of them;

[0016] R3 is One of them;

[0017] R4 is One of them;

[0018] D stands for deuterium.

[0019] As a further preferred embodiment of the present invention, the amide compounds containing pyrimidine structures include the following compounds:

[0020] [1]N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)cyclopropaneformamide;

[0021] [2]N-(4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-2-(pyridin-2-ylamino)pyrimidin-5-yl)acetamide;

[0022] [3] 2-((3-Fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-ylacetamide;

[0023] [4]N-(2-((4-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide;

[0024] [5]N-(2-((5-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide;

[0025] [6]N-(2-((6-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide;

[0026] [7]N-(2-((5-trifluoromethyl-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide;

[0027] [8]N-(2-((5-cyanopyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide;

[0028] [9]N-(4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-2-((5-methoxypyridin-2-yl)amino)pyrimidin-5-yl)acetamide;

[0029]

[10] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)butyramide;

[0030]

[11] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)isobutyramide;

[0031]

[12] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)cyclobutaneformamide;

[0032]

[13] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)cyclohexylcarboxamide;

[0033]

[14] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)tetrahydro-2H-pyran-4-carboxamide;

[0034]

[15] 4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-N-(methyl-d3)-6-(tetrahydro-2H-pyran-4-carbamoyl)pyridazine-3-carboxamide;

[0035]

[16] 6-(cyclopropanecarbamate)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0036]

[17] 4-((6-(4-ethylpiperazin-1-yl)-5-methoxypyrimidin-4-yl)amino)-N-(methyl-d3)-6-(tetrahydro-2H-pyran-4-carbamoyl)pyridazin-3-carboxamide;

[0037]

[18] 6-(cyclopropanecarbamate)-4-((6-(4-ethylpiperazin-1-yl)-5-methoxypyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0038]

[19] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-(tetrahydro-2H-pyran-4-carbamoyl)pyridazine-3-carboxamide;

[0039]

[20] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-((5-methoxypyridin-2-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0040]

[21] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-(pyridin-2-ylamino)pyridazine-3-carboxamide;

[0041]

[22] 6-((3-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0042]

[23] 6-((4-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0043]

[24] 6-((5-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0044]

[25] 6-((6-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide;

[0045]

[26] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-((5-(trifluoromethyl)pyridin-2-yl)amino)pyridazine-3-carboxamide;

[0046]

[27] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-(pyridazin-3-ylamino)pyridazin-3-carboxamide.

[0047] The second technical solution of the present invention provides a pharmaceutical composition comprising the above-mentioned amide compound containing a pyrimidine structure as an active component.

[0048] As a further preferred embodiment of the invention, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0049] As a further preferred embodiment of the present invention, the pharmaceutically acceptable excipients include one or more of the following: diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, suspending agents, flavoring agents, or preservatives.

[0050] As a further preferred embodiment of the present invention, the dosage form of the composition includes injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, or ointments.

[0051] The third technical solution of the present invention is to provide the application of the above-mentioned pyrimidine-containing amide compounds or the above-mentioned pharmaceutical compositions in the preparation of TYK2 JH2 kinase inhibitors.

[0052] The fourth technical solution of the present invention is to provide the use of the above-mentioned amide compounds containing pyrimidine structures or the above-mentioned pharmaceutical compositions in the preparation of medicaments for the treatment and / or prevention of inflammation.

[0053] Fifth technical solution of the present invention: providing the application of the above-mentioned pyrimidine-containing amide compounds or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating autoimmune diseases.

[0054] As a further preferred embodiment of the present invention, the autoimmune disease includes psoriasis, colitis, systemic lupus erythematosus, dermatomyositis, polymyositis, or scleroderma.

[0055] The compounds or compositions of the present invention inhibit T cell differentiation, abnormal proliferation of keratinocytes, and abnormal angiogenesis by inhibiting TYK2 JH2 kinase activity and regulating the IL-12 / IL-23 and type I interferon signaling pathways.

[0056] The compositions of the present invention are administered orally or via parenteral route, including intravenous, subcutaneous, intraperitoneal, or local administration.

[0057] This invention allows for the preparation of compositions by mixing the aforementioned compounds with pharmaceutically acceptable carriers or excipients, and to the formulation of clinically acceptable dosage forms. Pharmaceutically acceptable excipients refer to any diluent, adjuvant, and / or carrier that can be used in the pharmaceutical field. The compounds of this invention can be used in combination with other active ingredients, provided they do not produce other adverse effects, such as allergic reactions.

[0058] The clinical dosage of the compounds of this invention needs to be individually adjusted based on the in vivo therapeutic efficacy, bioavailability, metabolic and excretion characteristics of the active ingredients, combined with individual differences such as patient age, gender, and disease stage. The recommended daily dose for adults is 10–500 mg, preferably 50–300 mg. Specific medication should be administered under the guidance of a doctor or pharmacist, and divided into appropriate doses at appropriate intervals according to treatment needs, which can be taken 1 to 6 times daily.

[0059] The pharmaceutical compositions of this invention can be prepared into various pharmaceutical dosage forms, including a wide range of excipients commonly used in the pharmaceutical field. Specific dosage forms include injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, and ointments. The pharmaceutical carriers used in the compositions are conventional types used in the pharmaceutical field, including binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorants, flavoring agents, preservatives, solvents, and matrices. The pharmaceutical formulations can be administered orally or via parenteral routes (e.g., intravenously, subcutaneously, intraperitoneally, or locally). For drugs that are unstable in the acidic environment of the stomach, they can be further prepared as enteric-coated tablets to ensure drug release in the intestines, improving stability and bioavailability.

[0060] This invention also relates to the use of the above-mentioned compounds in the preparation of medicaments for the treatment and / or prevention of inflammation-related diseases. Based on their inhibitory activity against TYK2 JH2 kinases, the compounds can be used to prepare therapeutic drugs for inflammatory and autoimmune diseases, specifically including indications such as psoriasis, colitis, systemic lupus erythematosus, dermatomyositis, polymyositis, and scleroderma.

[0061] The present invention discloses the following technical effects:

[0062] This invention provides an amide compound containing a pyrimidine structure, which effectively blocks the IL-12 / IL-23 and type I interferon signaling pathways by highly selectively inhibiting TYK2 JH2 kinase activity, significantly improving the pathological process of autoimmune diseases (such as psoriasis, colitis, systemic lupus erythematosus, etc.). This compound has the advantages of low toxicity and high targeting, can precisely regulate abnormal immune responses, and provides multiple dosage forms (tablets, capsules, injections, etc.) to support oral or injectable administration, combining efficacy and convenience, and providing a safe and efficient new drug regimen for the treatment of autoimmune diseases. Attached Figure Description

[0063] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0064] Figure 1 The graph shows the trend of the inhibitory effect of the compound prepared in Example 21 of this invention on HaCaT cells over time and concentration.

[0065] Figure 2 The effect of compound 21 of this invention on apoptosis of HaCaT cells.

[0066] Figure 3The effect of compound 21 of this invention on the HaCaT cell cycle.

[0067] Figure 4 Flowchart for modeling and treating psoriasis in mice.

[0068] Figure 5 A diagram showing the treatment of skin wounds in mice with psoriasis.

[0069] Figure 6 This is a graph showing the weight of a mouse.

[0070] Figure 7 A represents the PASI score of mouse skin; B represents the mouse erythema score; C represents the mouse scaling score; and D represents the mouse skin thickness score.

[0071] Figure 8 The biochemical blood samples from psoriatic mice included: TP (total protein); BUN (blood urea nitrogen); GLU (blood glucose); CREA (creatinine); AST (aspartate aminotransferase); ALT (alanine aminotransferase); CK (creatine kinase); ALP (alkaline phosphatase); and LDH (lactate dehydrogenase).

[0072] Figure 9 The diagram shows mouse organs and organ indices; A represents the mouse heart; B represents the mouse liver; C represents the mouse spleen; D represents the mouse lungs; E represents the mouse kidneys; and F represents the mouse organ indices.

[0073] Figure 10 H&E staining of organs in psoriatic mice after treatment.

[0074] Figure 11 Image showing H&E staining of skin samples from psoriasis-affected mice.

[0075] Figure 12 Immunohistochemical staining for the treatment of psoriasis mice.

[0076] Figure 13 The values ​​represent the levels of inflammatory factors in mouse skin tissue; where a represents IL-17 and b represents IL-23. Detailed Implementation

[0077] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0078] H (400MHz) spectra were recorded on a Bruker Avance 400MHz NMR spectrometer using DMSO, CD3OD, CD3CN, and CDCl3 as solvents; mass spectrometry was performed using a Waters high-resolution quadrupole time-of-flight mass spectrometer; carbon spectrometry was performed using... 13C NMR was used for determination; purity was determined by high performance liquid chromatography. All reagents used were of analytical grade or chemically pure.

[0079] The following synthetic routes describe the preparation of compounds with the general formula structure shown in Formula I or Formula II of this invention. All starting materials are prepared by methods well known to those skilled in the art of organic chemistry, or are commercially available, as described in the synthetic routes. All final compounds of this invention are prepared by methods described in the synthetic routes, or by similar methods well known to those skilled in the art of organic chemistry. All variable factors used in the synthetic routes are defined below or as defined in the claims.

[0080] According to the structure of Formula I of this invention, R1 and R2, as defined in the summary section, can both be prepared by reacting according to the following method.

[0081]

[0082] According to the structure of Formula II of the present invention, R3 and R4, as defined in the inventive summary section, can both be prepared by the following steps of reaction.

[0083] Route 2:

[0084]

[0085] The structural formulas of the compounds prepared in Examples 1-27 of this invention are shown in Table 1.

[0086] Table 1

[0087]

[0088]

[0089]

[0090] Example 1

[0091] The present invention, general formula I, is based on N-(pyrimidin-5-yl)acetamide as a skeleton. Taking N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)cyclopropaneformamide as an example, the synthesis method is as follows.

[0092] Step 1: Preparation of intermediate A1

[0093] First, weigh 1 g of 2,4-dichloropyrimidine-5-amine and dissolve it in 10 mL of 1,4-dioxane solvent with stirring. Then, weigh acetyl chloride in a 1:1 ratio with 2,4-dichloropyrimidine-5-amine and acetamide, mix it in 10 mL of 1,4-dioxane solution, and add 1 mL of DIPEA. After mixing thoroughly, add the mixture dropwise to the 2,4-dichloropyrimidine-5-amine system using a constant pressure funnel. The reaction is carried out at room temperature. During the reaction, the progress is monitored by TLC until the reactants have completely reacted. After the reaction is complete, the solvent in the flask is evaporated to dryness using a rotary evaporator. Finally, the product is obtained as a white solid by rapid preparative liquid chromatography.

[0094] Step 2: Preparation of intermediate B1

[0095] First, 1 g of 6-chloro-5-methoxypyrimidine-4-amine was weighed into a 50 mL pear-shaped flask, followed by the addition of 10 mL of morpholine solution. The reaction mixture was heated to 100 °C in an oil bath and stirred continuously for 4 hours. Heating was stopped after the 6-chloro-5-methoxypyrimidine-4-amine had completely reacted. The reaction mixture was then extracted with dichloromethane and water to remove most of the morpholine solvent. The product was then separated and purified using preparative liquid chromatography (HPLC), yielding a light yellow powder.

[0096] Step 3: Preparation of intermediate C1

[0097] First, weigh 1 g of A1, and add 30 mL of ultra-dry tetrahydrofuran in a 1:1 ratio of N-(2,4-dichloropyrimidin-5-yl)acetamide to intermediate B1. Stir in an ice bath at 0°C, and then add 12.4 mL of NaHMDS dropwise to a flask. Protect the reaction system under nitrogen atmosphere and react at room temperature until the N-(2,4-dichloropyrimidin-5-yl)acetamide has completely reacted. After rotary evaporation to dryness, obtain a mixture. Separate and purify the mixture using rapid preparative liquid chromatography (HPLC) to obtain a fluorescent pale yellow solid.

[0098] Step 4: First, weigh 0.1 g of intermediate C1. Then, weigh cyclopropionamide, Pd2(dba)3, Xantphos, and Cs2CO3 in an equivalent ratio of 1:1:0.03:0.045:2. Place them in a flask, heat the flask in an oil bath to 120°C, and stir under nitrogen protection. After intermediate C1 has reacted completely, evaporate the solvent to dryness to obtain a mixture. Then, separate and purify the crude product using a rapid preparative liquid chromatography (HPLC) system to obtain a white solid.

[0099] 1H NMR(400MHz,DMSO-d6)δ10.38(s,1H),9.80(s,1H),8.98(s,1H),8.34(s,1H),8.09(s,1H),3.71- 3.68(t,4H),3.67-3.62(t,4H),3.55(s,3H),2.09(s,4H),0.78-0.74(m,2H),0.73-0.68(m,2H). 13 C NMR(151MHz,DMSO-d6)δ172.85,170.32,156.41,154.61,154.49,153.84,152.59,151.96, 131.02,117.12,66.67(2C),59.09,46.88(2C),23.55,14.20,8.56(2C).HRMS(M / Z):(M+H) + calcd for C 19 H 24 N8O4:429.1999, found,429.1999.

[0100] Example 2

[0101] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0102] N-(4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-2-(pyridin-2-ylamino)pyrimidin-5-yl)acetamide

[0103] 1 H NMR (400MHz, DMSO-d6) δ9.96(s,1H),9.58(s,1H),9.04(s,1H),8.53(d,J=8.6Hz,1H),8.30(s,1H),8.25(s,1H),8.24- 8.20(t,1H),7.63(t,J=7.9Hz,1H),6.94-6.88(t,1H),3.71-3.68(t,4H),3.67-3.64(t,4H),3.59(s,3H),2.11(s,3H). 13 C NMR(151MHz,Chloroform-d)δ169.03,156.32,155.63,155.28,153.03,152.65,152.13,151.12,147. 96,137.93,126.94,117.58,114.96,112.35,67.00(2C),58.91,46.68(2C),24.01.HRMS(M / Z):(M+H) +calcd for C 20 H 23 N9O3:438.2002,found,438.2003.

[0104] Example 3

[0105] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0106] 2-((3-Fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-ylacetamide

[0107] 1 H NMR(400MHz,DMSO-d6)δ9.64(s,1H),9.39(s,1H),8.76(s,1H),8.18-8.12(d,1H),8.10(s,1H),8.08(s,1H) ,7.61(t,J=9.2Hz,1H),7.25-7.17(d,1H),3.71-3.66(m,4H),3.61-3.56(m,4H),3.54(s,3H),2.05(s,3H).

[0108] Example 4

[0109] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0110] N-(2-((4-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide

[0111] 1 H NMR (400MHz, DMSO-d6) δ10.02(s,1H),9.89(s,1H),9.03(s,1H),8.72(d,J=13.9Hz,1H),8.23(t,J= 6.4Hz,3H),6.83(d,J=6.1Hz,1H),3.72-3.67(t,4H),3.67-3.62(t,4H),3.60(s,3H),2.11(s,3H). 13 C NMR(151MHz,DMSO-d6)δ170.69,156.12,156.03,155.99,154.82,153.76,152.58,15 0.55,150.49,129.64,114.02,105.41,100.07,66.64(2C),59.33,46.78(2C),23.35.

[0112] Example 5

[0113] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0114] N-(2-((5-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide

[0115] 1 H NMR(400MHz,DMSO-d6)δ9.82(s,1H),9.68(s,1H),8.95(s,1H),8.72(d,J=9.3Hz,1H),8.35(s,1H),8 .19(s,2H),7.65(d,J=11.2Hz,1H),3.73-3.69(t,4H),3.68-3.64(t,4H),3.60(s,3H),2.10(s,3H).

[0116] Example 6

[0117] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0118] N-(2-((6-fluoropyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide

[0119] 1 H NMR (400MHz, DMSO-d6) δ9.88(s,1H),9.84(s,1H),9.01(s,1H),8.52(d,J=8.3Hz,1H),8.32(s,1H),8.23(s,1H) ,7.81(t,J=8.4Hz,1H),6.60(d,J=7.7Hz,1H),3.72-3.68(t,4H),3.68-3.63(t,4H),3.59(s,3H),2.11(s,3H). 13 C NMR(151MHz,DMSO-d6)δ169.50,161.49,159.95,155.07,153.56,152.97,151.56,151.2 2,142.34,129.00,113.21,108.79,99.96,99.72,65.58(2C),58.20,45.77(2C),22.31.

[0120] Example 7

[0121] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0122] N-(2-((5-trifluoromethyl-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide

[0123] 1 H NMR (400MHz, DMSO-d6) δ10.30(s,1H),9.93(s,1H),9.06(s,1H),8.87(d,J=9.2Hz,1H),8.58(s,1H),8.41(s, 1H),8.29(s,1H),8.02(d,J=9.1Hz,1H),3.72-3.68(t,4H),3.66(t,J=5.3Hz,4H),3.60(s,3H),2.12(s,3H). 13 C NMR(151MHz,DMSO-d6)δ170.69,156.12,156.03,155.99,154.82,153.76,152.58,15 0.55,150.49,129.64,114.02,105.41,100.07,66.64(2C),59.33,46.78(2C),23.35.

[0124] Example 8

[0125] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0126] N-(2-((5-cyanopyridin-2-yl)amino)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-5-yl)acetamide

[0127] 1 H NMR (400MHz, DMSO-d6) δ10.41(s,1H),9.92(s,1H),9.07(s,1H),8.91(d,J=9.1Hz,1H),8.65(s,1H),8.40(s,1H),8.28(s, 1H),8.15(d,J=9.0Hz,1H),3.71(t,J=4.6Hz,4H),3.69-3.65(t,4H),3.61(s,3H),2.12(d,J=3.1Hz,3H).HRMS(M / Z):(M+H) + calcd for C 21 H 22 N 10 O3:463.1955,found,438.1963.

[0128] Example 9

[0129] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0130] N-(4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-2-((5-methoxypyridin-2-yl)amino)pyrimidin-5-yl)acetamide 1 H NMR (400MHz, DMSO-d6) δ9.78(s,1H),9.36(s,1H),8.92(s,1H),8.53(d,J=8.9Hz,1H),8.34(s,1H),8.16(s,1H), 7.95(s,1H),7.31(d,J=9.3Hz,1H),3.78(s,3H),3.73-3.68(t,4H),3.68-3.63(t,4H),3.59(s,3H),2.09(s,3H). 13 C NMR(151MHz,DMSO-d6)δ170.55,156.63,156.04,154.80,154.10,152.73,152.33,151.26,147.36,12 9.84,134.25,123.55,113.93,113.33,66.66(2C),59.22,56.26,46.86(2C),23.34.HRMS(M / Z):(M+H) + calcd for C 21 H 25 N9O4:468.2108,found,438.2104.

[0131] Example 10

[0132] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0133] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)butyramide

[0134] 1 H NMR(400MHz,DMSO-d6)δ10.09(s,1H),9.78(s,1H),9.00(s,1H),8.31(s,1H),8.14(s,1H),3.71-3.68(m,4H), 3.67-3.62(m,4H),3.53(s,3H),2.45(t,J=6.0Hz,2H),2.08(s,3H),1.53-1.43(m,2H),0.79(t,J=7.3Hz,3H). 13C NMR(151MHz,DMSO-d6)δ172.62,170.28,156.50,154.68,154.63,153.80,152.65,152.06,1 31.33,116.83,66.67(2C),59.09,46.90(2C),38.51,23.56,18.51,14.02.HRMS(M / Z):(M+H) + calcd for C 19 H 26 N8O4:431.2155,found,431.2155.

[0135] Example 11

[0136] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0137] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)isobutyramide

[0138] 1 H NMR(151MHz,DMSO-d6)δ10.09(s,1H),9.77(s,1H),8.98(s,1H),8.33(s,1H),8.13(s,1H),3.71-3 .69(t,4H),3.66-3.64(t,4H),3.53(s,3H),3.04-2.93(m,1H),2.08(s,3H),0.98(d,J=6.8Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ177.44,166.90,156.99,154.75,153.19,149.67,141.89,139.54,1 37.20,135.79,132.81,117.44,103.93,60.19,39.32,35.19,19.78(2C).HRMS(M / Z):(M+H) + calcd for C 19 H 26 N8O4:431.2155,found,431.2154.

[0139] Example 12

[0140] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0141] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)cyclobutaneformamide

[0142] 1 H NMR(400MHz,DMSO-d6)δ9.96(s,1H),9.75(s,1H),8.99(s,1H),8.29(s,1H),8.19(s,1H),3.81 (s,1H),3.69(d,J=8.4Hz,8H),3.54(s,3H),2.08(s,5H),1.95-1.87(m,2H),1.74-1.65(m,2H). 13 C NMR(151MHz,DMSO-d6)δ174.54,170.32,156.49,154.68,154.64,153.89,152.70,152.13, 131.38,116.60,66.68(2C),59.16,46.90(2C),24.72(2C),23.56,17.88.HRMS(M / Z):(M+H) + calcd for C 20 H 26 N8O4:443.2155,found,443.2155.

[0143] Example 13

[0144] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0145] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)cyclohexylcarboxamide

[0146] 1 H NMR(400MHz,DMSO-d6)δ9.99(s,1H),9.75(s,1H),8.96(s,1H),8.33(s,1H),8.14(s,1H),3.71-3.68(m,4H),3.66(s,4H),3 .53(s,3H),2.84-2.71(m,1H),2.08(s,3H),1.79-1.45(m,6H),1.26(q,J=10.8Hz,2H),1.11-1.01(m,2H).HRMS(M / Z):(M+H) + ,calcdfor C 22 H 30 N8O4:471.2468,found,471.2469.

[0147] Example 14

[0148] The reaction is carried out in the same manner as in Example 1 to obtain the product.

[0149] N-(5-acetamido-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)pyrimidin-2-yl)tetrahydro-2H-pyran-4-carboxamide

[0150] 13 C NMR(151MHz,DMSO-d6)δ174.11,170.28,156.53,154.61,154.56,153.77,152.61,152.09,131.4 1,117.02,66.71,(2C)66.68(2C),59.09,46.90(2C),40.88,29.04(2C),23.58.HRMS(M / Z):(M+H) + calcd for C 21 H 28 N8O5:473.2261,found,473.2267.

[0151] Example 15

[0152] The general formula II of this invention is based on the basic skeleton of N-(methyl-d3)pyridazine-3-carboxamide. Taking 6-(cyclopropanecarbamate)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide as an example, the synthesis method is as follows.

[0153] Step 1: Preparation of intermediate B1

[0154] Same as step two in Example 1

[0155] Step 2: Preparation of intermediate C2

[0156] First, weigh 1 g of 4,6-dichloro-N-(methyl-d3)pyridazine-3-carboxamide. Add 30 mL of ultra-dry tetrahydrofuran to the mixture of 4,6-dichloro-N-(methyl-d3)pyridazine-3-carboxamide and intermediate B1 in a 1:1 ratio. Stir the mixture in an ice bath at 0°C, and then add 12.4 mL of NaHMDS dropwise to a flask. Protect the reaction system under nitrogen atmosphere and react at room temperature until the 4,6-dichloro-N-(methyl-d3)pyridazine-3-carboxamide has completely reacted. After the reaction is complete, quench the reaction with dilute HCl solution. Remove the THF from the reaction system, then add 50 mL of ice water. A solid precipitates out; filter the solid and dry it in an oven to obtain a yellow solid.

[0157] Step 3: First, weigh 0.1 g of intermediate C2. Then, weigh tetrahydro-2H-pyran-4-carboxamide, Pd2(dba)3, Xantphos, and Cs2CO3 in an equivalent ratio of 1:1:0.03:0.045:2. Place them in a flask, heat the oil bath to 120°C, and then place the flask in the oil bath under reflux with stirring, all under nitrogen protection. After intermediate C2 has reacted completely, evaporate the solvent to dryness to obtain a mixture. Then, separate and purify the crude product using a rapid preparative liquid chromatography (HPLC) system to obtain a white solid.

[0158] 4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-N-(methyl-d3)-6-(tetrahydro-2H-pyran-4-carbamate)pyridazine-3-carboxamide

[0159] 1 H NMR(400MHz, Acetonitrile-d3)δ12.26(s,1H),9.79(s,1H),9.03(s,1H),8.32(s,1H),8.09(s,1H),3.89-3.82(m,2 H),3.64(d,J=2.9Hz,7H),3.60-3.56(m,4H),3.32(t,J=11.0Hz,2H),2.69-2.58(m,1H),1.69(q,J=9.3,8.2Hz,4H). 13 C NMR (151MHz, Acetonitrile-d3) δ175.33,157.20,156.50,152.48,143.88,104.08,67.64(2C),67.60(2C),59.70,47.63(2C),43.57,29.90(2C).

[0160] Example 16

[0161] The reaction is carried out in the same manner as in Example 15 to obtain the product.

[0162] 6-(cyclopropaneformamido)-4-((5-methoxy-6-morpholinylpyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

[0163] 1 H NMR(400MHz,DMSO-d6)δ12.35(s,1H),11.38(s,1H),9.79(s,1H),9.26(s,1H),8.19(s,1H), 3.72(d,J=8.2Hz,7H),3.68-3.62(m,4H),2.12(p,J=6.4Hz,1H),0.88(q,J=8.0,6.4Hz,4H).

[0164] Example 17

[0165] The reaction is carried out in the same manner as in Example 15 to obtain the product.

[0166] 4-((6-(4-ethylpiperazin-1-yl)-5-methoxypyrimidin-4-yl)amino)-N-(methyl-d3)-6-(tetrahydro-2H-pyran-4-carbamate)pyridazin-3-carbamate

[0167] 1 H NMR(400MHz,Chloroform-d)δ12.29(s,1H),9.99(s,1H),8.53(s,1H),8.34(s,1H),8.24(s,1H),4.08(d,J=11.6Hz,2H),3.79(s,7 H),3.47(t,J=10.9Hz,2H),2.70-2.60(m,1H),2.59-2.50(m,4H),2.46(q,J=6.9Hz,2H),2.01-1.86(m,4H),1.13(t,J=7.2Hz,3H).

[0168] Example 18

[0169] The reaction is carried out in the same manner as in Example 15 to obtain the product.

[0170] 6-(cyclopropaneformamido)-4-((6-(4-ethylpiperazin-1-yl)-5-methoxypyrimidin-4-yl)amino)-N-(methyl-d3)pyridazin-3-carboxamide

[0171] 1 H NMR (400MHz, DMSO-d6) δ12.33(s,1H),11.37(s,1H),9.79(s,1H),9.26(s,1H),8.17(s,1H),3.69(s,3H),3.68-3. 63(m,4H),2.48-2.41(m,4H),2.36(q,J=6.9Hz,2H),2.16-2.08(m,1H),1.03(t,J=7.1Hz,3H),0.92-0.83(m,4H).

[0172] Example 19

[0173] Step 1: Preparation of intermediate B4

[0174] First, weigh 1g of 6-chloro-5-methoxypyrimidine-4-amine. Then, weigh other raw materials and catalyst according to the following ratio: 6-chloro-5-methoxypyrimidine-4-amine: 1-methylpyrazole-4-boronic acid pinacol ester: Pd(PPh3)2Cl2: K2CO3 = 1:2:0.03:2. Dissolve these in 20mL of a solution system of 1,4-dioxane: H2O = 3:1. When the oil bath temperature reaches 120℃, place the reaction flask in the oil bath, protect with nitrogen, and reflux until the reaction is complete. After the reaction is complete, extract the reaction solution with dichloromethane and water to obtain the crude product. Then, separate and purify the crude product using a rapid preparative liquid chromatography (HPLC) system to obtain a white solid.

[0175] Step Two: Same as Step Two in Example 15

[0176] Step 3: Same as Step 3 in Example 15

[0177] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-(tetrahydro-2H-pyran-4-carbamate)pyridazine-3-carboxamide

[0178] 13 C NMR(151MHz,Chloroform-d)δ173.39,166.52,155.90,154.58,153.27,149.86,142.72,140.14,137 .72,135.56,131.54,118.14,104.57,67.06(2C),59.92,43.50,39.30,28.91(2C).HRMS(M / Z):(M+H) + calcd for C 21 H 22 D3N9O4:471.2291,found,471.2293.

[0179] Example 20

[0180] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0181] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-6-((5-methoxypyridin-2-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

[0182] 1H NMR (400MHz, DMSO-d6) δ12.55(s,1H),10.25(s,1H),9.63(s,1H),9.28(s,1H),8.62(s,1H),8.44(s,1H),8. 11(s,1H),8.09(s,1H),7.66(d,J=9.0Hz,1H),7.45(d,J=9.1Hz,1H),3.96(s,3H),3.84(s,3H),3.83(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.27,157.47,153.81,152.16,150.31,148.34,146.71,140.04,13 8.42,136.01,132.69,132.64,131.63,123.71,116.46,113.28,99.68,59.03,55.26,38.23.

[0183] Example 21

[0184] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0185] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-(pyridin-2-ylamino)pyridazine-3-carboxamide

[0186] 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),10.43(s,1H),9.84(s,1H),9.31(s,1H),8.60(s,1H),8.45(s,1H),8.36(d,J= 4.4Hz,1H),8.11(s,1H),7.74(t,J=7.8Hz,1H),7.63(d,J=8.3Hz,1H),7.03-6.95(t,1H),3.95(s,3H),3.85(s,3H). 13 C NMR(151MHz,DMSO-d6)δ167.27,158.50,154.87,154.13,153.17,149.49,148.05,141.23 ,139.51,138.34,137.13,134.11,132.73,117.56,117.52,113.41,101.80,60.12,39.31.

[0187] Example 22

[0188] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0189] 6-((3-Fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

[0190] 1 H NMR(400MHz,DMSO-d6)δ12.59(s,1H),10.03(s,1H),9.71(s,1H),9.30(s,1H),8.56(s,1H),8.44(s,1H) ,8.24(d,J=4.8Hz,1H),8.11(s,1H),7.77-7.71(m,1H),7.17(d,J=4.4Hz,1H),3.95(s,3H),3.85(s,3H). 13 C NMR(151MHz,Chloroform-d)δ166.96,157.10,154.87,152.94,149.63,148.36,146.66,142.94,142. 16,140.11,137.74,134.14,131.49,122.17,118.17,117.42,102.33,59.82,39.31.HRMS(M / Z): (M+H) + ,calcd forC 20 H 16 D3FN 10 O2:454.1938,found,454.1942.

[0191] Example 23

[0192] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0193] 6-((4-Fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

[0194] 1H NMR (400MHz, DMSO-d6) δ12.58(s,1H),10.70(s,1H),9.80(s,1H),9.34(s,1H),8.54(s,1H),8.45(s,1H),8.12(s, 1H),7.94-7.88(m,1H),7.60(d,J=8.2Hz,1H),6.71(d,J=7.8Hz,1H),3.96(s,3H),3.85(s,3H).HRMS(M / Z):(M+H) + calcd for C 20 H 16 D3FN 10 O2:454.1938,foun d,454.1945.

[0195] Example 24

[0196] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0197] 6-((5-Fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

[0198] 1 H NMR(400MHz,DMSO-d6)δ12.57(s,1H),10.50(s,1H),9.72(s,1H),9.31(s,1H),8.63(s,1H ),8.45(s,1H),8.37(s,1H),8.11(s,1H),7.73(d,J=5.4Hz,2H),3.95(s,3H),3.85(s,3H).

[0199] Example 25

[0200] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0201] 6-((6-Fluoropyridin-2-yl)amino)-4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)pyridazine-3-carboxamide

[0202] 1H NMR (400MHz, DMSO-d6) δ12.58(s,1H),10.70(s,1H),9.80(s,1H),9.34(s,1H),8.54(s,1H),8.45(s,1H),8.12(s, 1H),7.94-7.88(m,1H),7.60(d,J=8.2Hz,1H),6.71(d,J=7.8Hz,1H),3.96(s,3H),3.85(s,3H).HRMS(M / Z):(M+H) + calcd for C 20 H 16 D3FN 10 O2:454.1938,foun d,454.1945.

[0203] Example 26

[0204] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0205] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-((5-(trifluoromethyl)pyridin-2-yl)amino)pyridazine-3-carboxamide

[0206] 1 H NMR (400MHz, DMSO-d6) δ12.61(s,1H),10.92(s,1H),9.82(s,1H),9.37(s,1H),8.73(s,1H),8. 66(s,1H),8.45(s,1H),8.12(d,J=6.2Hz,2H),7.87(d,J=8.7Hz,1H),3.95(s,3H),3.85(s,3H). 13 C NMR(151MHz,DMSO-d6)δ167.09,157.95,156.96,154.78,149.60,141.46,139 .52,137.15,135.61,134.79,132.74,117.50,112.91,102.78,60.16,39.31.

[0207] Example 27

[0208] The reaction is carried out in the same manner as in Example 19 to obtain the product.

[0209] 4-((5-methoxy-6-(1-methyl-1H-pyrazol-4-yl)pyrimidin-4-yl)amino)-N-(methyl-d3)-6-(pyridazin-3-ylamino)pyridazin-3-carboxamide

[0210] 1 H NMR (400MHz, DMSO-d6) δ12.61(s,1H),10.78(s,1H),9.52(s,1H),9.36(s,1H),8.88(d,J=4.6Hz,1 H),8.54(s,1H),8.45(s,1H),8.13(d,J=8.6Hz,2H),7.67-7.62(m,1H),3.96(s,3H),3.86(s,3H). 13 C NMR(151MHz,DMSO-d6)δ166.03,157.06,155.96,153.75,151.95,148.53,146.15,140.33,138 .45,136.09,133.69,131.70,127.48,117.29,116.39,101.53,59.08,38.24.HRMS(M / Z): (M+H) + calcd for C 19 H 16 D3N 11 O2:437.1984,found,437.1993.

[0211] Example of effect verification:

[0212] 1. TYK2 JH2 kinase activity (IC50) 50 (and inhibition rate measurement)

[0213] a. Kinase buffer preparation

[0214] Prepare 1x kinase buffer (25mM HEPES, pH 7.5, 150mM NaCl, 1mM DTT, 1mM MgCl2, 0.005% Tween-20).

[0215] b. Compound Preparation and Dilution: Dilute the compound to 100-fold the desired maximum inhibitor concentration using 100% DMSO to obtain a stock solution. In a 96-well storage plate, transfer the stock solution to the first well, then transfer 15-30 μl of the stock solution from the first well to the next well, diluting to the target concentration with 100% DMSO. Repeat this step to obtain a total of 5 concentration gradients. From the 96-well storage plate, transfer ≥40 μl of the compound dilution to each target well of a 384-well Echo plate. Transfer more than 40 μl of the compound to the 384-well Echo plate. Transfer 200 nmol of sample from each well of the 384-well Echo plate to a 384-well analytical plate, repeating once.

[0216] Protein-forming kinase solution was added to 1x kinase buffer, with 10 μL of kinase solution added to each well. For the control wells (without enzyme), 10 μL of kinase solution was added to each well. The plate was then mixed thoroughly. Next, JAK2 JH2 tracer and MAb Anti-6HIS Tb cryptate Gold were added to 1x kinase buffer to form a tracer solution, with 10 μL of tracer solution added to each well to initiate the reaction. After mixing the plate, it was incubated at room temperature for 60 min. Data were collected on an Envision microscope at an excitation wavelength of 340 nm and an emission wavelength of 495 nm. The percentage inhibition rate of the compounds was calculated.

[0217] Percentage inhibition rate = (max-sample Ratio) / (Max-Min)×100; "min" is the enzyme-free control ratio, and "max" is the DMSO control ratio.

[0218] The data is represented in MS Excel, and the curves are fitted using the XLFit Excel add-in version 5.4.0.8.

[0219] The formula for calculating IC50 is: Y = Bottom + (Top - Bottom) / (1 + (IC50 / X)^HillSlope)

[0220] Using Deucravacitinib as a positive control, Kinase-Glo and Kinase-Glo were used respectively. The inhibitory activity of the compounds against TYK2 JH2 kinases was tested using the Ultra enzyme activity evaluation method. The results are shown in Table 2, where NA indicates an inhibition rate of less than 1% and ND indicates no detection.

[0221] Table 2

[0222]

[0223]

[0224] The above experimental results show that the compounds of general formula I protected by this invention have good anti-TYK2JH2 kinase activity. Examples 20, 21, 25, and 27 exhibited excellent inhibition rates against TYK2JH2 kinase. Further kinase IC50 assays were conducted on the compounds with higher inhibition rates. 50 The measurements revealed that the IC in Example 21 50 IC with a value of 1.46 nM, Example 25 50 The value was 1.49 nM, comparable to that of a positive control drug. This suggests that the compound in Formula I of this invention holds promise as a potential inhibitor of the TYK2 JH2 kinase.

[0225] Further anti-inflammatory activity studies were conducted based on the compound from Example 21:

[0226] 2. Apoptosis and cell cycle experiments

[0227] HaCaT cells were resuscitated and cultured until they reached the logarithmic growth phase. After digestion and centrifugation, culture medium was added again to prepare a cell suspension, and cell counts were performed. Cells were added to 96-well plates at a density of 4000 cells per well and cultured for 24 hours. Then, solutions of the compound from Example 21 at concentrations of 100 μM, 33.3 μM, 11.1 μM, 3.7 μM, and 1.24 μM were added to the corresponding wells. After 72 hours, the culture medium was discarded, MTT solution was added, and the cells were cultured again. After 4 hours, the MTT solution was discarded, and DMSO solution was added. The optical density (OD) of each well was measured using a microplate reader at 570 nm. The anti-proliferation inhibition rate of the compound against HaCaT cells was determined by the OD value. The inhibition rate (%) was calculated using the following formula:

[0228] Inhibition rate (100%) = (OD) 对照 -OD 处理 ) / (OD 对照 -OD 空白 )×100%

[0229] The half-inhibitory concentration (IC50) of the compound on HaCaT cells was calculated using SPSS software. 50 ).

[0230] With the concentration of the compound from Example 21 as the x-axis, the IC50 of cells... 50 Plot the cell IC with the vertical axis as the ordinate. 50 The curve, the result is as follows Figure 1 As shown. Figure 1 The graph shows the trend of the inhibitory effect of the compound prepared in Example 21 on HaCaT cells over time and concentration. As can be seen from the line graph, when the concentration of the compound in Example 21 is 100 μM, the inhibition rate of compound 1 in Example 2 on HaCaT cells reaches 77.60% after 72 h of incubation. Furthermore, at each set concentration, the inhibition rate of the compound in Example 21 on HaCaT cells increases with increasing time. Within the same time period, the inhibition rate of the compound in Example 21 on HaCaT cells also increases with increasing dosage. In conclusion, the compound in Example 21 exerts an inhibitory effect on HaCaT cells in a time- and concentration-dependent manner.

[0231] HaCaT cells were seeded on 6-well plates and incubated overnight at 37°C. After complete cell adhesion, the culture medium was discarded, and culture media containing (2 μM, 4 μM, and 8 μM) of the drug were added for treatment. Medium containing Deucravacitinib (4 μM) served as a positive control, while the blank control group received the same volume of medium. Cells were cultured for another 48 hours. After incubation, the cells were centrifuged, the supernatant was discarded, and the cells were washed with PBS. Cells were resuspended in 1 mL of PBS, and 3 mL of pre-chilled anhydrous ethanol was added. The cells were incubated overnight at 4°C. The supernatant was discarded by centrifugation, and the cells were washed with PBS and collected. Staining buffer was added to the tubes, and the cells were incubated at 37°C in the dark for 15 min. The cells were then analyzed at 488 nm to detect red fluorescence and light scattering.

[0232] Figure 2 The effect of the compound in Example 21 on apoptosis in HaCaT cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns indicates no significant difference. Figure 3 The effect of compound 21 on the HaCaT cell cycle.

[0233] from Figure 2 The experimental results show that in the blank control group, the early apoptosis rate of HaCaT cells was 0.64%, the late apoptosis rate was 2.39%, and the total apoptosis rate was 3.03%. In contrast, the preferred compound Example 21, at 2 μM, induced early apoptosis in HaCaT cells at 2.32%, late apoptosis at 5.09%, and total apoptosis at 7.41%. At 4 μM, Example 21 induced early apoptosis in HaCaT cells at 1.25%, late apoptosis at 9.45%, and total apoptosis at 10.70%. At 8 μM, Example 21 induced early apoptosis in HaCaT cells at 3.65%, late apoptosis at 16.3%, and total apoptosis at 19.95%. In conclusion, Example 21 can induce apoptosis in HaCaT cells in a dose-dependent manner, with a more pronounced induction of late apoptosis.

[0234] exist Figure 3As can be seen, under the action of Deucravacitinib (4 μM), the proportion of HaCaT cells in the G0 / G1 phase increased (Deucravacitinib: 66.18%, Control: 49.69%), while the number of cells in the S phase decreased significantly. This indicates that Deucravacitinib can arrest HaCaT cells in the G0 / G1 phase. After treatment with compound Example 21 (2 μM), the proportion of HaCaT cells in the G0 / G1 phase increased compared to the control group (TS-41: 51.37%, Control: 49.69%). Furthermore, the proportion of cells in the G0 / G1 phase gradually increased with the increase of the concentration of compound Example 21 (2 μM: 51.37%, 4 μM: 56.36%, 8 μM: 57.74%). In summary, compound Example 21 preferably arrests HaCaT cells in the G0 / G1 phase in a dose-dependent manner, thereby inhibiting HaCaT cell proliferation.

[0235] 3. In vivo treatment of psoriasis in mice

[0236] Male BALB / c mice (NO. 110324251100417935) were randomly divided into four groups: a normal control group (blank group), a model group, a positive control group (20 mg / kg), a low-dose group (10 mg / kg) as described in Example 21, a medium-dose group (20 mg / kg) as described in Example 21, and a high-dose group (40 mg / kg) as described in Example 21, with six mice in each group. Two days before the start of the experiment, the hair on the backs of the mice was shaved and depilated with hair removal cream to create a 2 × 3.5 cm exposed area.

[0237] The positive control drug Deucravacitinib and the compound from Example 21 were dissolved in 5% Tween 80, 40% PEG, and 55% saline. The control group and the positive control group were given a homogeneous mixture of 5% Tween 80, 40% PEG, and 55% saline by gavage.

[0238] Control group: 62.5 mg of petroleum jelly was applied to the back skin of mice, followed by gavage with saline 4 hours later. Model group: 62.5 mg of 5% imiquimod cream (Sichuan Mingxin Pharmaceutical Co., Ltd., H20030129) was applied to the back skin of mice, followed by gavage with saline 4 hours later. Positive control group: 62.5 mg of 5% imiquimod cream was applied to the back skin of mice, followed by gavage at a dose of 20 mg / kg 4 hours later. Low-positive control group: 62.5 mg of 5% imiquimod cream was applied to the back skin of mice, followed by gavage at a dose of 10 mg / kg 4 hours later. Neutral control group: 62.5 mg of 5% imiquimod cream was applied to the back skin of mice, followed by gavage at a dose of 20 mg / kg 4 hours later. High-positive control group: 62.5 mg of 5% imiquimod cream was applied to the back skin of mice, followed by gavage at a dose of 40 mg / kg 4 hours later.

[0239] Skin tissues and organs were immersed in tissue fixative and sent for H&E staining. Immunohistochemical staining with Ki67 antibody and other methods was used to detect the effects of the selected compounds on cell proliferation, inflammatory factors, STAT3 phosphorylation and organs. Mouse skin tissues were subjected to ELISA to detect whether the compounds could regulate cytokine levels.

[0240] Figure 4 A flowchart for modeling and treating psoriasis in mice is provided. Before the experiment, mice were weighed, and their dorsal skin was photographed and scored according to the PASI scoring mechanism. After 7 days of modeling and treatment, the therapeutic effect of the preferred compound Example 21 on psoriasis mice was obtained. Figure 5 ). Figure 6 This is a graph showing the weight record of the experimental mice.

[0241] from Figure 6 The results show that during the experiment, the weight of the control group mice fluctuated less, while the weight of the model group mice decreased significantly. After drug treatment, the mice's weight slightly increased. The reason for this is that the model group mice experienced discomfort during the modeling process, which affected their normal growth and metabolism. In contrast, the treatment group mice experienced improved psoriasis symptoms through drug treatment, leading to an increase in their weight. Figure 5 and Figure 7 As can be seen, after induction with IMQ, the model group mice exhibited obvious psoriasis symptoms, including erythema, crusting, and thickened skin. Compared to the model group, the above symptoms were alleviated after administration of 10 mg / kg, 20 mg / kg, and 40 mg / kg in Example 21.

[0242] Mice were monitored during the experiment, and their erythema, scaling, and skin thickness were scored according to the PASI scoring criteria. The results are shown below. Figure 7 . Figure 7 In the table, A represents the mouse erythema score; B represents the mouse scaling score; C represents the mouse skin thickness score; and D represents the mouse PASI total score. ns p<1, p<*p<0.1, **p<0.01, ***p<0.001, ****p<0.0001.

[0243] After treatment, to evaluate the effect of the compound of Example 21 on psoriatic mice, ocular blood was collected from each group of mice. Serum samples were obtained from each mouse after centrifugation and biochemical analysis was performed using a preoperative ten-item test panel. Specific data are as follows: Figure 8 As shown:

[0244] from Figure 8As can be seen, the biochemical indicators of each experimental group were within the normal range, indicating that the mice's physical functions were not significantly affected during the psoriasis modeling and treatment process. Blood biochemical indicators in mice can comprehensively assess their physiological state and organ function. TP (total protein) effectively reflects the body's nutritional status, liver function, and fluid balance; CK (creatine kinase) is a sensitive indicator for detecting muscle or myocardial damage; BUN (blood urea nitrogen) and CREA (creatinine) jointly assess kidney function, with CREA being more specific for evaluating glomerular filtration function; GLU (glucose glucose) directly reflects the body's glucose metabolism status; combined analysis of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) can distinguish between liver damage and myocardial / muscle damage; abnormal ALP (alkaline phosphatase) suggests hepatobiliary disease or abnormal bone metabolism; and elevated LDH (lactate dehydrogenase), as a broad-spectrum marker of tissue damage, can be seen in various pathological conditions. These indicators provide important objective evidence for the establishment of mouse disease models, evaluation of experimental intervention effects, and toxicity studies. Overall, in the control group, after modeling with IMQ, the ALP level in mice showed a decreasing trend, while ALT, LDH, and BUN levels showed an increasing trend. This suggests that the renal function, liver function, and hepatocyte activity of the modeling mice were affected. These indicators improved after treatment with the positive control drug and a low dose of the compound from Example 21.

[0245] After modeling and administering the drug to mice, the mice were dissected after blood was collected from their eyeballs to investigate the effects of the drug on their organs. Figure 9 The images show the organs of mice in each group, where A represents the mouse heart; B represents the mouse liver; C represents the mouse spleen; D represents the mouse lungs; E represents the mouse kidneys; and F represents the mouse organ index. Observation shows that all organs in each group of mice appear normal, with smooth surfaces, and are reddish-brown or dark red in color. Compared to the control group, the spleen volume in the model group was significantly increased, while in the treatment group, the spleen volume was noticeably reduced after drug treatment. The organ index graph shows that the indices of all organs in the model group increased relative to the model group, with the spleen index showing a particularly significant increase. In the compound treatment group of Example 21, the spleen also showed a decreasing trend compared to the model group, and this trend was dose-dependent. Changes in the spleen can indirectly reflect changes in the levels of inflammatory cells and related inflammatory factors in the treatment group after drug treatment.

[0246] Subsequently, H&E staining was performed on the dissected mouse organs to further determine the pathological morphology of the organ tissues in each experimental group. Specific staining results are as follows: Figure 10As shown in the figure, the H&E staining structures of the organs in each experimental group are clear, the tissue structures are intact, and the cell morphology is normal. Compared with the control group, there are no obvious abnormalities in the model group and the treatment group, indicating that neither the modeling drug nor the treatment drug has any toxic effect on the organs of mice during the mouse modeling and treatment process.

[0247] At the end of the experiment, skin samples from each group of mice were collected and stained with hematoxylin and eosin (H&E) for histological examination to verify the therapeutic effect of the compound in Example 21. Figure 11 Compared to the control group, skin samples from the IMQ-treated model group showed several significant histopathological changes. These changes included keratinocyte hyperplasia, characterized by excessive proliferation of keratinocytes leading to epidermal thickening. Furthermore, incomplete keratinization (parakeratosis) was observed, indicating abnormal keratinocyte maturation and the presence of preserved nuclei in the stratum corneum. Notably, the compound of Example 21 demonstrated significant therapeutic efficacy: at 10 mg / kg, a therapeutic effect was observed compared to the model group; at 20 mg / kg, skin condition was similar to the positive treatment group; and at 40 mg / kg, skin condition was superior to the positive treatment group.

[0248] Immunohistochemical staining of mouse skin sections was performed using Ki67 to assess cell proliferation levels in the epidermis and dermis of the skin tissue. Figure 12 The results showed that treatment with the compound of Example 21 resulted in a significant reduction in the number of Ki67 cells compared to the model group, particularly in the 40 mg / kg dose group. These findings highlight the strong therapeutic potential of the compound of Example 21 in alleviating histopathological changes and keratinocyte proliferation associated with IMQ-induced skin inflammation.

[0249] During the development of psoriasis, elevated levels of inflammatory factors lead to significant immune cell infiltration at the site of skin lesions. To further verify the effectiveness of the treatment, this invention analyzed the accumulation of immune cells in the skin of the back. The results showed that after administration, the number of Th17 cells was significantly reduced in the 20 mg / kg and 40 mg / kg dose groups, exhibiting a dose-dependent response. Figure 13 IL-17A is expressed in various pro-inflammatory cells. This invention measured the level of IL-17A in skin tissue and found that the cytokine level of IL-17A was also significantly reduced. Furthermore, this invention investigated whether the compound of Example 21 inhibited the phosphorylation of the downstream effector protein STAT3 in the skin after TYK2. Notably, the groups treated with the compound of Example 21 all showed a significant reduction in the number of phosphorylated signal transducer and activator of transcription 3 (STAT3) positive cells. Figure 13 This indicates that selective inhibition of TYK2 effectively inhibits the phosphorylation of STAT3 in the skin.

[0250] The crucial role of the IL-23 / Th17 axis in the development of psoriasis has been established. To investigate whether the treatment in Example 21 could normalize the levels of cytokines associated with the IL-23 / Th17 axis, this invention first used ELISA to detect the levels of IL-17A and IL-23 in mouse skin tissue. The results are as follows... Figure 13 As shown, a represents IL-17 and b represents IL-23. The results show that, compared to the control group, the levels of IL-17A and IL-23 in the model group were significantly increased. Compared to the model group, treatment with the compound of Example 21 downregulated the levels of IL-17A and IL-23 in mice, especially at a dose of 40 mg / kg, where the levels were significantly reduced. In conclusion, Example 21 effectively reduced the levels of key interleukins associated with the IL-23 / Th17 axis.

[0251] Application Example 1: Tablets

[0252] 10g of compound from Example 14 was mixed with 20g of excipients according to the general pharmaceutical tableting method and then compressed into 100 tablets, each weighing 300mg.

[0253] Application Example 2: Capsules

[0254] Using 5g of compound from Example 16, 10g of excipients were mixed according to the requirements for pharmaceutical capsules and then filled into empty capsules, each weighing 300mg.

[0255] Application Example 3: Ointment

[0256] The compound from Example 17 was prepared by grinding 5g of the compound into a fine powder and then mixing it with 500g of an oily matrix such as petrolatum.

[0257] Application Example 4: Aerosol

[0258] 10g of compound from Example 20 was dissolved in an appropriate amount of propylene glycol, and then distilled water and other additives were added to prepare a 500mL clear solution.

[0259] Application Example 5: Suppositories

[0260] 10g of compound from Example 25 was finely ground, and an appropriate amount of glycerin was added. After grinding evenly, melted glycerin gelatin was added and ground evenly. The mixture was then poured into a mold coated with lubricant to prepare 50 suppositories.

[0261] Application Example 6: Droplets

[0262] 5g of the compound from Example 26 was heated and melted with 25g of a matrix such as gelatin and mixed evenly. The mixture was then dripped into low-temperature liquid paraffin to prepare 1000 pellets.

[0263] Application Example 7: Injectable

[0264] Using 6g of compound from Example 27, the compound was adsorbed onto activated carbon according to conventional pharmaceutical methods, filtered through a 0.65μm microporous membrane, and then filled into a nitrogen tank to prepare an aqueous injection formulation. Each injection contained 2mL, and a total of 100 bottles were filled.

[0265] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention.

Claims

1. An amide compound containing a pyrimidine structure, characterized in that, Choose any one of the following structures: 、 、 、 ； D stands for deuterium.

2. A pharmaceutical composition, characterized in that, The active component includes the amide compound containing a pyrimidine structure as described in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that, This includes pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutically acceptable excipients include one or more of the following: diluents, binders, lubricants, disintegrants, solubilizers, stabilizers, suspending agents, flavoring agents, or preservatives.

5. The pharmaceutical composition according to claim 2, characterized in that, The dosage forms of the composition include injections, tablets, capsules, aerosols, suppositories, films, pellets, topical liniments, or ointments.

6. The use of the pyrimidine-containing amide compound of claim 1 or the pharmaceutical composition of any one of claims 2-5 in the preparation of TYK2 JH2 kinase inhibitors.

7. The use of the pyrimidine-containing amide compound of claim 1 or the pharmaceutical composition of any one of claims 2-5 in the preparation of a medicament for treating and / or preventing inflammation.

8. The use of the pyrimidine-containing amide compound as described in claim 1 or the pharmaceutical composition as described in any one of claims 2-5 in the preparation of a medicament for treating autoimmune diseases.

9. The application according to claim 8, characterized in that, The autoimmune diseases mentioned include psoriasis, colitis, systemic lupus erythematosus, dermatomyositis, polymyositis, or scleroderma.

Images