Use of compound having tricyclic heteroaryl group

Abstract

To provide a compound in the manufacture of a medicament for treating diseases associated with overexpression or abnormal activation of JAK kinase and SYK kinase, including autoimmune diseases such as immune-mediated skin diseases (especially psoriasis, atopic dermatitis and SLE).SOLUTION: Provided is a compound of formula (I), an optical isomer thereof or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of medicine, and in particular, relates to the treatment of overexpression or abnormal activity of JAK kinase and SYK kinase. Use of a tricyclic heteroaryl group in the manufacture of a medicament for treating a disease associated with genitalization This relates to the use of compounds. [Background technology]

[0002] JAK kinase (Janus kinase), or Janus kinase, has four subtypes: JAK1 kinases, including JAK2 kinase, JAK3 kinase, and TYK2 kinase (Tyrosine kinase 2) JAK1 kinase, JAK2 kinase, and TYK2 are types of non-transmembrane non-receptor tyrosine kinases. Kinases are widely present in various tissues and cells, and JAK1 kinase is involved in the expression of inflammatory signals such as IL-6 and IFN. JAK2 kinase is involved in the signal transduction pathway of cytokines such as IL-3, IL-5, and EPO. JAK3 kinase is present only in the bone marrow and lymphoid system and can mediate the production of IL-2, IL-4, and TYK2 kinase can be involved in the signal transduction pathways of IL-7, IL-9, IL-15 and IL-21. Involved in the signal transduction pathways of IFN-α, IL-6, IL-10, and IL-12. JAK kinase inhibitors inhibit JAK -STAT (Signal transducers and activators of transcription) signaling pathway By specifically inhibiting this, the cascade amplification of the above cytokines is inhibited, resulting in immune regulation. It is involved in processes such as:

[0003] SYK kinase (Spleen tyrosine kinase), or spleen tyrosine kinase, is a SYK kinase is a non-receptor tyrosine kinase present in the substrate. It is widely expressed in leukocytes, fibroblasts, and vascular endothelial cells, is overexpressed in B lymphocytes, and is involved in tumor and autoimmune diseases. Dectin-1 / ITAM plays an important role in the regulation of immune disease by antigen-stimulated immune cells. In normal B cells, intracellular immune receptor tyrosinase activity is mediated by the classical pathway. When the ITAM is phosphorylated by antigen-induced BCR cross-linking, the cytoplasmic S YK kinase is the first target of ITAM recruitment and activation, and activated SYK kinase This enzyme then activates the transcription factor NF-κB through a CARD9-dependent pathway, leading to the release of a series of inflammatory factors. This pathway also activates caspase-8, which then converts IL-1β into IL-1β. CARD9-independent pathway NLRP3 signaling pathway SYK kinase also plays a role in the maturation of IL-1β. Therefore, SYK kinase is an important factor in the pathogenesis of autoimmune diseases. It is also a target.

[0004] Psoriasis is a chronic, relapsing, immune-mediated, inflammatory skin disease. varies significantly around the world, ranging from 0.5% to 3.15% in the United States, 0.75% to 2.9% in Europe, and 0.85% to 2.9% in China. reported a psoriasis prevalence of 0.123% in 1984 and a prevalence of 0.47% in 6 cities in 2008. In 2017, the prevalence of psoriasis in the four southwestern provinces was reported to be 0.5%, and the number of psoriasis patients in China was approximately 60. Psoriasis can occur at any age, and there is no gender predilection. Approximately two-thirds of patients have 4 The disease develops before the age of 0, and in most patients, symptoms are milder in the summer than in the winter.

[0005] The etiology and onset mechanism of psoriasis have not been fully elucidated, and various genetic, immunological, and environmental factors are thought to be involved. It may be involved in the development of HIV-1, and multiple immune cells, mainly T lymphocytes, are involved in this process. The immune response causes excessive proliferation of keratinocytes and inflammation of synovial cells and chondrocytes. The typical clinical manifestations of psoriasis are localized or widespread scaly erythema or plaque. Psoriasis can occur in conjunction with other system disorders, for example with damage to internal organs and joints. Severe cases may be associated with metabolic syndrome and atherosclerotic cardiovascular disease. There is an increased risk of contracting the disease.

[0006] Psoriasis is classified into three types based on clinical symptoms and pathological features: (1) plaque psoriasis; It is often acute in onset. Typically, a rash with a sharp border and irregular shape appears. There is inflammatory redness around the area. The area is slightly wet and thickened. The surface is covered with multiple layers of silvery white scales. The scales peel off easily and are covered with a light, translucent film with a slight reddish tint after removal. When the film is removed, small spots of bleeding (Auspitz) are seen. Skin breakdown is common on the extensor surfaces of the legs and feet. Some patients experience varying degrees of itching. (2) Pustular psoriasis, which is classified into generalized and palmoplantar types. Generalized pustular psoriasis is characterized by erythema on the skin. It is a group of shallow sterile pustules that may coalesce to form a lake of pus. It is often seen on the flexed sides of the limbs and in wrinkled areas, and the oral mucosa may also be affected at the same time. Acute onset or sudden exacerbation often presents with chills, fever, joint pain, general discomfort, white It is accompanied by systemic symptoms such as elevated blood counts. There are multiple cyclical episodes, with periods of remission and Palmoplantar pustulosis is limited to the hands and feet and occurs symmetrically. (3) Erythrodermic psoriasis, psoriasis Also known as dermabrasion, this is a severe form of psoriasis. Rather than applying large doses of glucocorticoids and tapering them too quickly or stopping the drugs suddenly, It is often characterized by diffuse redness, swelling, and scaling of the skin over the entire body. It is accompanied by systemic symptoms such as fever, chills, and discomfort, and the superficial lymph nodes become swollen and the white blood cell count increases. (4) Psoriatic arthritis, also known as psoriatic arthritis. Psoriasis patients have the same symptoms as rheumatoid arthritis. It simultaneously causes joint damage such as the above, affecting large and small joints throughout the body, but it is most common in the interphalangeal joints of the fingers and toes. Joint involvement is most characteristic. The joints become red and painful, and the skin around the joints also becomes red and swollen. Joint symptoms often worsen or improve at the same time as skin symptoms. It is negative.

[0007] To date, there is no specific treatment for psoriasis, and the main treatments are topical treatments and physical therapy. Local treatment includes many treatments such as external drug treatment, systemic therapy, and Chinese herbal medicine treatment. , physical therapy, etc. Topical medications are mainly vitamin D3 analogues, glucocorticoids, Anthralin, bitechoic acid gel and cream, cokes, immunosuppressants, and other topical Medications such as tacrolimus, pimecrolimus, 0.03% camptothecin ointment, 5% salicylic acid Ointments, etc. Systemic treatments include methotrexate, tretinoin, cyclosporin, etc. porin, tacrolimus, mycofenate, e.g. etanercept, infliximab These include chemotherapeutic agents, biologics, and antibiotics. For moderate to severe cases, they are used as a single treatment. When this is insufficient, combination, alternating or sequential treatments are usually used. The effect is remarkable, but the therapeutic dose and toxic dose of methotrexate are very close, and The main side effects of the drug are teratogenicity, and adverse reactions to cyclosporine A include nephrotoxicity, hypertension, Adverse reactions to tacrolimus include nausea, vomiting, weakness, muscle fibrillation, and urinary tract irritation. Similar to cyclosporine A, mycophenolate esters have adverse reactions to gastrointestinal It has many side effects, including rash, anemia, and leukopenia, and is at risk of inducing infection and tumors. The recurrence rate is high and the long-term treatment effect is insufficient.

[0008] Atopic dermatitis (AD) is also known as hereditary allergic dermatitis. It is an allergic skin disease characterized by itching and polymorphous skin rash, which occurs at different ages. It is a chronic, relapsing, inflammatory skin disease with different clinical manifestations. Since it often coexists with other atopic diseases such as rhinitis and asthma, it is considered a systemic disease. The prevalence of AD has gradually increased worldwide over the past 30 years, with the prevalence of AD increasing among children in developed countries. The prevalence of AD in China is 10% to 20%, and the increase in the prevalence of AD in China is comparable to that in Western developed countries, Japan, and South Korea. The number of school-age adolescents (6-20 years) in 1998 was 1.2%. The total prevalence of AD among preschool children (aged 1-7 years) in 10 cities in 2002 was 0.70%. The prevalence rate among children aged 3-6 years in the Shanghai area was 2.78% in 2012, and reached 8.3% in 2014. In 12 cities in our country, the prevalence of AD in children aged 1-7 years reached 12.94%, and the prevalence of AD in children from January to December was 30.48%. reached %.

[0009] The onset of AD is closely related to genetic and environmental factors. The mechanism of its onset is unclear. Although it is not certain, factors such as immune abnormalities, skin barrier dysfunction, and skin flora disorders are currently thought to be contributing to the onset of the condition. AD usually begins in infancy, and patients before the age of one year are at high risk for Alzheimer's disease. It accounts for approximately 50% of the body, shows a chronic course, and manifests clinically in a wide variety of ways. The most basic characteristics are The symptoms are dry skin, chronic eczema-like skin lesions, and pronounced pruritus. Patients may also have other allergic diseases such as asthma and allergic rhinoconjunctivitis. Furthermore, chronic inflammation can lead to neurological disorders, inflammatory bowel disease, and rheumatoid arthritis in chronic patients. The risk of developing cardiovascular disease and lymphoma is significantly increased.

[0010] Current treatments for AD include basic treatment (avoidance of contact hypersensitivity, etc.), topical medication (topical glucocorticoids), and and calcium-regulated neuronal phosphatase inhibitors), systemic treatment (oral antihistamines, immunosuppressants, etc.), These include topical medications, steroids, glucocorticoids, ultraviolet light therapy, and antibacterial therapy. Step therapy for mild and moderate cases is typically used. Existing treatments can alleviate symptoms, but they have certain limitations and many adverse reactions. The need for rapid action, relief of itching, and prevention of recurrence remains unmet.

[0011] Lupus erythematosus (LE) is a typical autoimmune connective tissue disease. Discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) , systemic lupus erythematosus (SLE), lupus erythematosus profundus (LEP), neonatal lupus It is classified into subtypes such as non-leukemia (NLE) and drug-induced lupus erythematosus (DIL).

[0012] Discoid lupus erythematosus primarily affects the skin and is the mildest form of lupus erythematosus. In a small number of cases, there is mild visceral damage, and in a small number, systemic lupus erythematosus Although the majority of patients are asymptomatic, it is difficult to completely eliminate the condition. Subacute cutaneous lupus erythematosus is a rare and unique intermediate form of the disease, characterized by the appearance of skin lesions. The disease occurs repeatedly, and most patients have visceral lesions, but few are severely affected. Joint pain, muscle pain, recurrent low fever, and in a small number of patients, nephritis and changes in the blood system may also occur. Lupus erythematosus is also known as lupus erythematosus, profundus lupus, and intermediate lupus erythematosus. It is unstable in nature and can exist alone or later develop into discoid lupus. It may transform into systemic lupus erythematosus, or may occur simultaneously with systemic lupus erythematosus. Neonatal lupus erythematosus is characterized by cutaneous loop erythema and congenital cardiac conduction blockade. It is self-limiting and generally resolves by 4-6 months of age, but is not always associated with cardiac disease. Drug-induced lupus erythematosus is characterized by fever, joint pain, muscle pain, facial rash, oral ulcers, and Tumors are the main symptom, and serositis is also possible. The condition gradually improves after drug discontinuation, and severe patients may need to undergo drug treatment. This may be done.

[0013] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease. It is an immune disease and the most severe of the various types of lupus erythematosus. It is characterized by damage to multiple organs in the body, repeated relapses and remissions, and the presence of large amounts of autoantibodies. The main clinical feature is that if treatment is not given in time, it can cause irreversible damage to the affected organs. The prevalence of SLE varies greatly depending on the region, but currently, Meanwhile, the prevalence of SLE worldwide is 0-241 people per 100,000 people, 30-70 people per 100,000 people in mainland China, and The male-to-female prevalence ratio is 1:10-12. The specific cause of SLE is not completely clear. However, multiple causes, including genetic, infectious, endocrine and environmental factors, are involved in combination. It is caused by the exposure of the immune system to apoptotic cells and the clearance of immune complexes. Defects in immune regulatory mechanisms are also important factors in the development of SLE. Increased T-cell helper activity, defective B-cell inhibition, and conversion of Th1 cells to Th2 cells B cell hyperactivation and the production of pathogenic autoantibodies are also contributing factors. Drug-induced steroids such as phenytoin sodium, penicillamine, quinidine, and Inderal May directly cause and progress lupus. Many SLE patients present with multisystem involvement. A small number of patients develop lupus erythematosus from other types of lupus, and some At the same time, other connective tissue diseases such as scleroderma, dermatomyositis, and Sjögren's syndrome may develop, and various It forms a comprehensive syndrome.

[0014] Current drug treatment for SLE is based on glucocorticoids and hydroxychloroquine. However, treatment with these drugs can lead to infections, liver and kidney dysfunction, and metabolic disorders. There may be side effects such as flu, which may require a reduction in dosage or discontinuation of the drug, and the progression of the disease may be delayed. Currently, there are no drugs that can inhibit SYK, and new drugs are being continuously developed. The kinases and JAK kinases are located upstream of two different signaling pathways that induce SLE, respectively. Therefore, SYK-JAK dual channel inhibitors are expected to be an effective approach for treating SLE. Currently, there are no approved JAK-SYK dual-target inhibitors on the market. The JAK-SYK dual-target inhibitor R333, which was approved for this indication, was discontinued due to a Phase II clinical trial failure. Development was announced to be terminated on October 24, 2013.

[0015] Therefore, autoimmune diseases, particularly psoriasis, atopic dermatitis, lupus erythematosus, etc. There is a need for the development of novel drugs that are useful for the treatment of immune-mediated skin diseases and autoimmune connective tissue diseases. There are. Summary of the Invention

[0016] A compound (I) having the following structural formula (I): [ka] The chemical name is (R)-4-(cyclopropylamino)-2-((3-(cyclopropylsulfonyl)-1,2,3 ,4,4a,5-Hexahydrobenzo[ b ]pyrazolo[1,2-d ][1,4]oxazin-8-yl) amino)pyrimidine-5-carboxamide, which was first disclosed in PCT International Publication WO2018108084 It is known as a highly selective dual-targeted inhibitor of JAK and SYK kinases, useful in the treatment of cancer. It is being done.

[0017] As a result of further research, the present inventors have found that compound (I) inhibits JAK kinase and SYK kinase. By suppressing the expression of β-glucan, it is possible to control cell signaling and proliferation, etc. Immune-mediated skin diseases such as psoriasis, atopic dermatitis, and lupus erythematosus, as well as autoimmune connective tissue disorders In the treatment of tissue diseases, it has excellent therapeutic effects on autoimmune diseases and also shows sufficient activity. It was also found to have few side effects.

[0018] The present invention relates to a method for treating JAK kinase of compound (I), its optical isomer, or a pharmaceutically acceptable salt thereof. and SYK kinase. Provides uses in manufacturing.

[0019] [ka]

[0020] Preferably, in the above-mentioned use, the JAK kinase is JAK1 kinase, JAK2 kinase, J AK3 kinase, or TYK2 kinase, preferably JAK3 kinase and / or TYK2 kinase.

[0021] Preferably, in the above applications, overexpression or abnormal activity of JAK kinase and SYK kinase Diseases associated with aging are autoimmune diseases.

[0022] Preferably, in the above use, the autoimmune disease is an immune-mediated skin disease and an autoimmune It is a sexually transmitted connective tissue disease.

[0023] Preferably, in the above use, the immune-mediated skin disease is psoriasis or atopic dermatitis. The psoriasis is preferably selected from plaque psoriasis, pustular psoriasis, erythrodermic psoriasis or psoriasis-related psoriasis. psoriatic arthritis, the autoimmune connective tissue disease is lupus erythematosus, the lupus erythematosus The preferred types of lupus erythematosus are discoid lupus erythematosus, subacute cutaneous lupus erythematosus, and systemic lupus erythematosus. Sexual lupus erythematosus, profundus lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus Preferably, the disease is systemic lupus erythematosus, more preferably systemic lupus erythematosus.

[0024] Furthermore, in the above-mentioned use, the medicament contains a therapeutically effective amount of compound (I), its optical isomer or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient or carrier. Includes.

[0025] The administration route of the pharmaceutical of the present invention is not particularly limited, and typical administration routes include oral, rectal, These include, but are not limited to, parenteral (intravenous, intramuscular, subcutaneous) and topical administration. The pharmaceutical of the present invention can be administered in various clinically acceptable forms, such as oral administration, injection, topical administration, and external application. It can be formulated into the following dosage forms.

[0026] Preferably, the medicament of the present invention is used clinically alone or in combination with other therapeutic ingredients. For convenience of clinical use, the compound (I) of the present invention, its optical isomers, The compound, or a pharmaceutically acceptable salt thereof, may be combined with other therapeutic ingredients to form a combination drug or composition. It can be prepared as a combination product.

[0027] The present invention also provides a therapeutically effective amount of the compound (I) of the present invention, its optical isomer or its pharmaceutical equivalent. administering a pharmaceutically acceptable salt thereof to a mammal (e.g., a human) in need of treatment; Provides instructions on how to use.

[0028] The present invention provides a method for treating diseases associated with overexpression or abnormal activation of JAK and SYK kinases. The method comprises administering to a patient a therapeutically effective amount of the compound (I) of the present invention, an optical isomer thereof, or a pharmaceutical composition thereof. and administering a physiologically acceptable salt thereof to a mammal (e.g., a human) in need of treatment. This provides a method for

[0029] In the present invention, the JAK kinase is JAK1 kinase, JAK2 kinase, JAK3 kinase, or TYK2 kinase, preferably JAK3 kinase and / or TYK2 In the present invention, the overexpression or abnormal activity of the JAK kinase and SYK kinase is Diseases associated with genitalization are autoimmune diseases, such as immune-mediated skin diseases and autoimmune complication. The immune-mediated skin disease is selected from psoriasis and atopic dermatitis, The psoriasis is preferably plaque psoriasis, pustular psoriasis, erythrodermic psoriasis, or psoriatic arthritis. The autoimmune connective tissue disease is lupus erythematosus, and the lupus erythematosus is preferably <Discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, lupus erythematosus profundus, neonatal lupus erythematosus, drug-induced lupus erythematosus, More preferably, it is systemic lupus erythematosus.

[0030] The present invention also provides a therapeutically effective amount of compound (I), its optical isomer, or a pharmaceutically acceptable salt thereof. and a pharmaceutical comprising an acceptable salt thereof for use in treating overexpression or is associated with abnormal activation of immune-mediated skin diseases and autoimmune connective tissue diseases. The present invention provides a pharmaceutical product for use in the treatment of immune diseases, and the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is preferably plaque psoriasis, pustular psoriasis, psoriasis, erythrodermic psoriasis, or psoriatic arthritis, and the autoimmune connective tissue disease is lupus erythema The lupus erythematosus is preferably discoid lupus erythematosus, subacute cutaneous lupus erythematosus, Cutaneous lupus erythematosus, systemic lupus erythematosus, profundus lupus erythematosus, neonatal lupus lupus erythematosus, drug-induced lupus erythematosus, and more preferably systemic lupus erythematosus It is.

[0031] The JAK kinase is JAK1 kinase, JAK2 kinase, JAK3 kinase, or TYK2 kinase. and preferably JAK3 kinase and / or TYK2 kinase.

[0032] The present invention also provides a therapeutically effective amount of compound (I), its optical isomer or a pharmaceutically acceptable salt thereof. and other therapeutic ingredients, Diseases associated with overexpression or abnormal activation of JAK and SYK kinases, e.g., immune-mediated A complex compound characterized by its application in the treatment of autoimmune diseases such as skin diseases and autoimmune connective tissue diseases. The present invention provides a method for treating an immune-mediated skin disease, such as psoriasis or atopic dermatitis, and a combination product. The psoriasis is preferably selected from plaque psoriasis, pustular psoriasis, erythrodermic psoriasis, or psoriasis-related skin diseases. psoriatic arthritis, the autoimmune connective tissue disease is lupus erythematosus, the lupus erythematosus The preferred types of lupus erythematosus are discoid lupus erythematosus, subacute cutaneous lupus erythematosus, and systemic lupus erythematosus. Sexual lupus erythematosus, profundus lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus Preferably, the disease is systemic lupus erythematosus, more preferably systemic lupus erythematosus.

[0033] The JAK kinase is JAK1 kinase, JAK2 kinase, JAK3 kinase, or TYK2 kinase. and preferably JAK3 kinase and / or TYK2 kinase.

[0034] In the present invention, the therapeutically effective amount is a dosage that is considered to be pharmaceutically effective, i.e., a dosage that is effective in preventing serious illness. By "effective dose" is meant an amount of active compound sufficient to significantly improve the condition without significant side effects. For a human weighing 60 kg, the daily dose is usually 0.01 to 2000 mg, preferably 1 to 500 mg. More preferably, it is 10 to 400 mg, and even more preferably, it is 15 to 360 mg or 15 to 250 mg. Available in 15 mg, 45 mg, 60 mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg, and 360 mg It can be administered as a single dose once a day, or in divided doses several times a day, or at intervals. The specific dosage and frequency of administration should be determined taking into consideration the route of administration, the patient's health condition, etc. The dosage can be determined by a skilled physician based on ordinary skill. Typical administration methods include oral, rectal, and parenteral (intravenous, intramuscular, subcutaneous). or topical administration. The amount of the active compound is expressed as Compound (I).

[0035] In vivo and in vitro tests have shown that (1) the compound (I) of the present invention inhibits JAK1 kinase, J Significant in vitro inhibition of AK2 kinase, JAK3 kinase, TYK2 kinase, and SYK kinase activity However, it showed more potent inhibition of JAK3 kinase and TYK2 kinase, and IC 50 are 1 each. 43 nM and 0.82 nM, respectively, and the inhibition of JAK2 kinase and SYK kinase was greater than that of JAK3 kinase and TYK kinase. Slightly weaker than 2 kinase, IC 50is 3-8 nM, the weakest inhibitor of JAK1 kinase There is an IC 50 (2) Compound (I) of the present invention showed IMQ Skin thickness, ear thickness, spleen weight, spleen index, and PASI score (red) of the induced psoriasis model mice The skin pathology scores and the epidermal thickness of the mice were also measured. The levels of IL-6 and TNF-α in skin tissues were improved or significantly reduced. Compound (I) from the study significantly increased the thickness and thickness of the skin in OXA-induced atopic dermatitis model mice. It can improve or significantly reduce clinical scores and reduce skin inflammatory symptoms in the modeling area. It can improve the phenomenon of cell aggregation, edema and capillary dilation, and improve the pathological score and epidermal thickness. (4) Compound (I) of the present invention was able to significantly reduce the severity of MRL / lpr lupus erythematosus. It improved skin lesions in a mouse death model in a dose-dependent manner and reduced kidney damage, It can reduce lymph node and spleen enlargement and reduce serum SLE-related antibodies and cytokines. In the 20 mg / kg group, the increase in It was subsequently found that the enlargement of lymph nodes in SLE mice was effectively inhibited, and the results of the endpoint evaluation were As a result, spleen enlargement and lymph node enlargement were effectively inhibited at 20 mg / kg, and chronic inflammatory bowel disease was observed. The total pathological score of both kidneys was significantly reduced in the number (CI), and serum IL-6 and TNF-α were overexpressed. At doses of 40 mg / kg and 60 mg / kg, skin lesions ( Improvement of skin lesion score and skin pathology score, reduction of kidney damage (reduction of urinary protein in mice, kidney damage) reduction of immune complex deposition), inhibition of immune organ proliferation (improvement of lymph node pathology scores, and spleen The results showed that the compound significantly improved the overall symptoms of SLE mice, including inhibition of liver and lymph node growth. Done.

[0036] Based on the above-mentioned research results, the compound (I) according to the present invention, its optical isomer or its pharmaceutical use The naturally tolerable salt improved skin lesions in mice with psoriasis and atopic dermatitis, and enhanced immunity. It was shown to inhibit immune organ growth, improve skin lesions in SLE mice in a dose-dependent manner, and promote immune function. Inhibits organ growth, reduces kidney dysfunction, reduces inflammation levels, and provides a relatively safe treatment It has a window and has the potential for clinical application. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 shows a graph of spleen weight at the end of the study in IMQ-induced psoriasis model mice. [Figure 2] FIG. 2 shows a graph of the spleen index (spleen weight / body weight %) at the study endpoint in the IMQ-induced psoriasis model mice. [Figure 3] FIG. 3 shows a graph of IL-6 content in skin tissue of IMQ-induced psoriasis model mice at the end of the study. [Figure 4] FIG. 4 shows a graph of TNF-α content in mouse skin tissue at the end point of the IMQ-induced psoriasis model mouse study. [Figure 5] FIG. 5 shows a graph of the skin pathology scores at the end of the study in the IMQ-induced psoriasis model mice. [Figure 6] FIG. 6 shows a graph of the epidermal thickness of the skin at the end point of the study in the IMQ-induced psoriasis model mice. [Figure 7] FIG. 7 shows a graph of skin pathology scores at the end of the study in OXA-induced atopic dermatitis model mice. [Figure 8] FIG. 8 shows a graph of the epidermal thickness of the skin at the end point of the study in the OXA-induced atopic dermatitis model mouse. [Figure 9]FIG. 9 shows a graph of skin pathology scores at the study endpoint in SLE mice. [Figure 10] FIG. 10 shows graphs of lymph node weights at the study endpoint in SLE mice, A: total lymph node weight, B: total lymph node weight / body weight %. [Figure 11] FIG. 11 shows graphs of spleen weights at study endpoints in SLE mice: A: spleen weight, B: spleen weight / body weight %. [Figure 12] FIG. 12 shows a graph of urinary protein versus area under the curve in SLE mice treated for 16 weeks. [Figure 13] FIG. 13 shows graphs of kidney weights at study endpoints in SLE mice: A: total kidney weight, B: kidney weight / body weight %. [Figure 14] Figure 14 shows graphs of the scores of HE stained kidney tissues of SLE mice, A: HE score of both kidneys - activity index, B: HE score of both kidneys - chronicity index, C: HE score of both kidneys - tubulointerstitial damage. [Figure 15] FIG. 15 is a graph showing IHC (IgG) staining scores of kidney tissues from SLE mice. [Figure 16] FIG. 16 is a graph showing the concentration of anti-ds-DNA antibodies in the serum of SLE mice. [Figure 17] FIG. 17 shows graphs of serum cytokine levels in SLE mice: A: TNF-α concentration; B: IL-6 concentration. DETAILED DESCRIPTION OF THE INVENTION

[0038] The present invention will now be further described with reference to specific examples. It is understood that the above is for illustrative purposes only and is not intended to limit the scope of the present invention. In the following examples, experimental methods for which no specific conditions are given are generally conditions or those recommended by the manufacturer.

[0039] Source or preparation of experimental materials: 1. Compound (I): Self-manufactured by Shiyaku Group Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd. 2. The positive control drugs, reagents, and raw materials used in the experiments were either commercially purchased or self-prepared. Ta.

[0040] 3. Preparation of in vivo experimental subjects (compound (I) and positive control compound): After weighing, the compound ( I) at 0.3 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 6 mg / mL, respectively The positive control saline solution was prepared at a concentration of 0.3 mg / mL or 0.6 mg / mL. The solution was diluted to the required concentration before use.

[0041] 4. The solvent (0.4% Tween 80 / 0.5% methylcellulose) was prepared as 1 L: Weigh 5.0 g of methylcellulose powder into a clean glass bottle, add 900 mL of sterile water, and thoroughly The mixture was stirred overnight until dissolved in 1000 ml of Tween 80. 4.0 mL of Tween 80 was aspirated, mixed well, and the final volume was 1000 ml. The solution was stored in a refrigerator at 4°C.

[0042] 5, OXA preparation: Acetone / olive oil (4 / 1) solvent composition: 40 mL of acetone and 10 mL of olive oil The material was shaken for 30 seconds to mix the acetone / olive oil (4 / 1) solvent until it was homogeneous. To prepare 5% OXA: Weigh out 100.00 mg of OXA and dissolve it in 2.00 ml of acetone / olive oil (4 / 1). and shaken for 30 seconds. To prepare 0.1% OXA: Weigh out 15.69 mg of OXA and dissolve it in 15.69 mL of acetone / olive oil (4 / 1). The solution was dissolved in water and shaken for 30 seconds. The solution was prepared immediately before use and every two days.

[0043] Example 1: Kinase activity inhibition test by compound (I) 1. Kinases: SYK kinase, JAK1 kinase, JAK2 kinase, JAK3 kinase, TYK2 kinase Z

[0044] 2. Experimental Method Protein kinase activity was measured using a Mobility Shift Assay. After dissolving in DMSO, a concentration gradient was created in 100% DMSO. To each plate, 5 μL of compound (I) (10% DMSO) was added at 5 times the final concentration in the reaction. Add 10 μL of 2.5x kinase solution, then incubate at room temperature for 10 minutes, then add 10 μL of 2.5x substrate solution. After incubation at 28°C for 60 minutes, 30 μL of stop solution (100 (mM HEPES, pH7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50mM EDTA) The conversion data was replicated on a Caliper EZ Reader II and analyzed. The conversion rate was converted to inhibition rate: % inhibition rate = (max - conversion rate) / (max - min) x 100%. " is the conversion rate of the control wells reacted without adding kinase; "max" is the conversion rate of the control wells reacted without adding DMSO. The conversion rate was calculated using the control well as the compound concentration and inhibition rate. and IC fitting was performed using XLFit excel add-in version 5.4.0.8. 50 The values ​​were calculated using the fitting formula: Y = Bottom + (Top-Bottom) / (1 + (IC 50 / X )^ HillSlope ).

[0045] [Table 1]

[0046] 3. Experimental Results The results of the kinase inhibitory activity test of compound (I) are shown in the table below. [Table 2]

[0047] Example 2: Efficacy test of compound (I) in IMQ-induced mouse psoriasis model 1. Purpose of the test This study evaluated the efficacy of the compound in an IMQ (5% imiquimod cream)-induced mouse model of psoriasis. The purpose of this study was to evaluate the efficacy of imiquimod (I). One of the clinical adverse reactions of imiquimod is psoriasis. The imiquimod-induced mouse psoriasis model was simple and easy to perform. The skin phenotype and lesion profile are complex tissue interactions that affect clinical dry skin. It had the advantage of being similar to scabies.

[0048] 2. Test drug Acceptor: Compound (I) Positive control: dexamethasone (Dex) Solvent: 0.4% Tween 80 / 0.5% methylcellulose.

[0049] 3. Test Animals Seventy female Balb / c mice, 5–6 weeks old.

[0050] 4. Study Groups and Dosage Schedule The animals were randomly grouped into seven groups based on their body weight on the day before the start of the experiment, as detailed in Table 2. We divided it.

[0051] [Table 3]

[0052] modeling: The animals were shaved on the back one day before application to create an exposed skin area of ​​approximately 2 cm x 3 cm. Mice were treated with 62.5 mg of 5% imiquimod cream at fixed time points daily for 14 days. The normal control group was treated with IMQ on the bare back and right ear of the mice. Vaseline was applied to the right ear at fixed times daily.

[0053] 5. Test Results 5.1 Effect of Compound (I) on Skin Thickness in IMQ-Induced Mice Skin thickness was measured every day from the start of the experiment until the end of the experiment. The skin thickness was measured by pressing the skin on the back of the mouse with the thumb of the left hand in the same direction as the mouse's body. The subcutaneous tissue was pinched with the index finger and the digital thickness gauge (BK-3281, manufacturer) was held in the right hand. Using a car (Shanghai Niohui), the left hand was placed 1 cm from the pinched area (near the center of the model area). The thickness of the skin wrinkles was measured so that the actual thickness was halved (skin thickness = measured value / 2).

[0054] The experimental results showed that the 5% imiquimod clonidine was significantly increased in the model group compared with the normal control group. After 14 days of continuous stimulation with Ream, skin thickness increased significantly. Compound (I) (3 mpk, 10 mpk, 30 mpk and 60 mpk) and dexamethasone (3 mpk) Both treatments significantly inhibited the increase in skin thickness (all five groups, model group P < 0.001 v s), as detailed in Table 3.

[0055] [Table 4]

[0056] 5.2 Effect of Compound (I) on ear thickness in IMQ-induced mice Ear thickness was measured daily (at the center of the pinna).

[0057] The results of the study showed that 5% imiquimod cream for 14 consecutive days was associated with a significant improvement in blood cholesterol levels compared to the normal control group. After stimulation, the thickness of the right ear of the mice increased significantly. At the end of the study, compound (I ) 3 mpk, 10 mpk, 30 mpk and 60 mpk etc. and dexamethasone (3 mpk) treatment both in the ear Compound (I) significantly inhibited the increase in ear thickness (all five groups: P<0.001 vs. model group). The inhibition of the increase in vascular wall thickness was dose-dependent, as detailed in Table 4.

[0058] [Table 5]

[0059] 5.3 Effect of Compound (I) on Skin PASI Score in IMQ-Induced Mice Photographs of the skin were taken daily from the start to the end of the experiment.

[0060] Clinical symptoms were scored using the PASI scoring criteria, with erythema, scaling, and The results were evaluated based on three indices: skin thickness, skin texture, and skin texture. The scores were 0 to 4 points. The PASI scoring criteria are as follows: 0, no symptoms; Symptoms: 1, mild; 2, moderate; 3, severe; 4, very severe. Erythema: 0 - no erythema, 1 - light red erythema, 2 - red, 3 - deep red, 4 - very Red. Scaling: No visible scales on the skin surface; 1- Part of the skin surface is covered with scales; 2- Most of the skin surface is covered with scales; 3 - Almost the entire skin surface is covered with scales; 4- The entire skin surface is covered with scales. Skin thickness: 0 - smooth, wrinkle-free skin; 1 - slight wrinkles or roughness at the skin edges; 2 - skin Slight wrinkles or slight elevations are present throughout the lesion; 3 - Deeper than the wrinkles in the skin lesion 4 - The entire skin lesion is wrinkled or the skin lesion is thickened and raised; It is highly thickened and prominently raised. Neither dryness nor itchiness is scored.

[0061] According to the experimental results, after 14 consecutive days of stimulation with 5% imiquimod cream, the model mice In the 12-month study, erythema, scaling, skin thickness PASI scores and total scores were significantly increased. In this respect, treatment with compound (I) (3 mpk) significantly reduced the scale score (P<0.001 vs. model group) and Compound I (10 mpk and 30 mpk) significantly improved the total score (P < 0.05 vs. model group). The treatment of 10 mpk group had a significant effect on the scale score (10 mpk group: P<0.01 vs. model group; 30 mpk group: P<0.001 vs. model group), skin thickness score (both groups, P<0.001 vs. model group), and total score (10 m pk group: P<0.01 vs. model group; 30 mpk group: P<0.001 vs. model group) Treatment with Compound I (60 mpk) and dexamethasone (3 mpk) resulted in an improvement in erythema score, scale score, Skin thickness score and total score (both groups, P<0.001 vs. model group) were significantly improved. The details are shown in Table 5.

[0062] [Table 6]

[0063] 5.4 Effect of Compound (I) on spleen weight in IMQ-induced mice On day 14 of the experiment, spleen weights were collected.

[0064] According to the results of the experiment, compound (I) (10 mpk, 30 mpk, 60 mpk) and dexamethasone (3 Treatment with Compound (I) 10 mpk significantly inhibited the increase in spleen weight (Compound (I) 10 mpk group: P<0.05 vs. model group, other 3 groups: P<0.001 vs. model group), Compound (I) (30 mpk, 60 mpk) Treatment with dexamethasone (3 mpk) significantly inhibited the increase in spleen index (all three groups: P <0.001 vs. model group), as detailed in Table 6.

[0065] [Table 7]

[0066] 5.5 Skin epidermal thickness measurement and pathology score After the experiment, all mice were euthanized by excessive carbon dioxide inhalation, and the skin was collected and analyzed for 4 min. The tissue was then left in a tissue fixative for 24 hours and used for pathological examination. The skin sections were embedded and cut into 4-micron sections. The skin sections were stained with hematoxylin and eosin. The thickness of the epidermis was measured using the dermal layer, epidermis layer, and dermis layer, and inflammatory cell infiltration was observed. At the time of the scan, the stained skin sections were scanned at 200x magnification with a Leica Aperio CS2 scanner to assess the histopathology of the tissue. Physical changes were observed and scored.

[0067] Specific pathological scoring criteria include the presence of Munro microabscesses in the epidermal layer. cess) found: 2.0 points; hyperkeratosis: 0.5 points; parakeratosis: 1.0 points; thinning or disappearance of the granular layer : 1.0 min; Increase in thickness of spinous layer: 1.0 min; Extension of skin processes and undulations vary depending on the severity. The time intervals were 0.5, 1.0, and 1.5 minutes, respectively. The degree of mononuclear or polynuclear cell infiltration in the dermis layer varied depending on the severity. The scores were 0.5, 1.0, and 1.5 points for each; suprapapilla: 0.5 points; and telangiectasia: 0.5 minutes.

[0068] To measure epidermal thickness, stained skin sections were first scanned with a Leica Aperio CS2 scanner. The scanned images were then analyzed using HALO analysis software. The skin epidermis was defined as an annotation layer in the software's "typing" template. The annotation layer is used to divide the epidermis into two lines, one above the other, and the thickness of the line is defined as the layer thickness. The skin thickness of each section was calculated at approximately 200 points, including the "thickness" option. expressed as a value.

[0069] The results of the experiment showed that in Group 1 (normal control group), the skin structure was completely visible under the microscope, and the cell morphology was The skin of the model group (Group 2) was normal and no obvious abnormal changes were observed. Abscesses, hyperkeratosis or hypokeratosis, thickening of the spinous layer, and moderate to severe inflammatory cell infiltration Compared with the normal control group 1, the 5% imiquimod cream was administered for 14 consecutive days. When stimulated with compound (I) (60 mpk), the pathological score of the skin tissue increased significantly. Treatment with samethasone (3 mpk) significantly improved the pathological scores of skin tissue in the model mice. (Compound (I) 60 mpk group: P < 0.01 vs. model group; dexamethasone group: P < 0.001 vs. model group) Compared with the normal control group (Group 1), the epidermal thickness of the skin in the model group (Group 2) was significantly increased. Treatment with compound (I) (30 mpk and 60 mpk) and dexamethasone (3 mpk) Compound (I) 2 significantly reduced the thickness of the epidermis of the model mice (P < 0.05 vs. The details are shown in Table 7.

[0070] [Table 8]

[0071] 5.6 Inflammatory Factor Assay of Skin Samples After the experiment, all mice were euthanized by excessive carbon dioxide (CO2) inhalation, and the skin was collected and stored for 4 days. The tissue was divided into 100 ml of tissue, 3 of which were rapidly frozen in liquid nitrogen and stored in a refrigerator at -80°C for the detection of inflammatory factors. Saved to. IL-6 and TNF-α were measured using ELISA.

[0072] Tissue preparation: A certain amount of skin tissue was measured, a certain amount of PBS (pH = 7.4) was added, and the tissue was then manually or The sample was thoroughly homogenized using a homogenizer and centrifuged for 20 minutes (2000-3000 rpm). The supernatant was carefully collected. After dispensing, a portion was detected and the rest was frozen as a backup. Ta.

[0073] Operation process: 1. Adding standard: Prepare a standard well and a sample well, and add the standard to the standard well. 50 μL of each solution was added at different concentrations. 2. Sample addition: Blank well (blank control well is the well to which the sample and labeled enzyme reagent are added) The other steps were the same (no enzyme labeling), and sample wells to be measured were prepared. First, add 40 μL of the diluent to the sample well of the plate to be measured, then add 10 μL of the sample to be measured. 1 μL was added (the final concentration of the sample to be measured was 5 times). The mixture was poured into the well and gently shaken to mix, avoiding contact with the well walls as much as possible. 3. Enzyme addition: Add 100 μL of enzyme-labeled reagent per well, excluding the blank well. Ta. 4. Incubation: After blocking the plate with blocking film, incubate at 37°C. and incubated for 60 minutes. 5. Liquid distribution: The 20-fold concentrated cleaning solution was diluted 20 times with distilled water to prepare a reserve.

[0074] 6. Washing: Carefully remove the blocking film, discard the liquid, shake off, and wash each well. Fill each container with cleaning solution, leave it for 30 seconds, discard, repeat the above procedure five times, tap, and dry. . 7. Color development: Add 50 μL of color developer A and 50 μL of color developer B to each well, shake gently to mix, and incubate at 37°C for 15 minutes. The color was developed while the plate was protected from light. 8. Termination: Add 50 μL of termination solution per well to terminate the reaction (when the blue color turns yellow). occurred). 9. Measurement: Zero the blank well and measure the absorbance (OD value) of each well at a wavelength of 450 nm. Measurement was performed within 15 minutes after the addition of the final solution. 10. Calculation: The linear regression equation for the calibration curve is y = bx + a (x is the concentration, y is the Calculate the OD value, then substitute the OD value of the sample into the formula to calculate the sample concentration, and multiply it by the dilution factor. The actual concentration of the sample was determined by the above.

[0075] According to the results of the experiment, compound (I) (3 mpk, 10 mpk, 30 mpk, 60 mpk) and dexamethasone Tazone (3 mpk) significantly reduced the IL-6 content in the skin (compound (I) 3 mpk and 60 mpk groups: P< 0.001 vs. model group, compound (I) 10 mpk, 30 mpk groups and dexamethasone group: P<0.01 v s model group) and the content of TNF-α in the skin (compound (I) 30 mpk group: P<0.001 vs model group) Del group, Compound (I) 10 mpk group: P<0.05 vs. Model group, Compound (I) 3 mpk, 60 mpk The results are shown in Table 8. Ta.

[0076] [Table 9]

[0077] Example 3: Efficacy test of compound (I) on OXA-induced mouse atopic dermatitis model 1. Purpose of the test This study aims to evaluate the efficacy of compound (I) in an OXA-induced atopic dermatitis model in Balb / c mice. The purpose of this study was to evaluate the long-term effects of OXA on the dorsal skin of mice. This model more closely resembles clinical skin inflammation and has been shown to have anti-inflammatory activity. This has been a general model for screening and evaluating compounds with

[0078] 2. Test drug Acceptor: Compound (I) Positive control: dexamethasone (Dex) solvent (0.4% Tween 80 / 0.5% methylcellulose).

[0079] 3. Test Animals Seventy female Balb / c mice, 8–9 weeks old.

[0080] 4. Study Groups and Dosage Schedule The animals were randomly assigned to seven groups based on their pre-drug body weight, as detailed in Table 9. I did it.

[0081] [Table 10]

[0082] Induction model of atopic dermatitis: Mice were randomly divided into seven groups according to their body weight, as shown in Table 9 above. The induction method was to inject 5% OXA (dissolved in acetone / olive oil (4 / 1) solvent) into the 10 μL (1.5 cm × 1.5 cm) was applied to the back of the mice near the neck. Similarly, 10 μL of acetone / olive oil (4 / 1) solvent was applied to the mouse. 100 μL of 0.1% OXA (dissolved in acetone / olive oil (4 / 1) solvent) was evenly applied to the back of the mouse near the neck. The normal control group of mice was treated with acetone / oil solution. 100 μL of the solvent, Reeve's oil (4 / 1), was applied in the same manner. On the day of immune stimulation, the skin thickness was measured and clinical evaluation was performed. If spotting and photography were required, OXA was applied after the above procedures were completed.

[0083] Dosage regimen: The doses were as shown in Table 9. Compound (I) was administered twice a day ( The morning administration was performed for skin thickness measurement, clinical scoring, photography, and The two doses were administered 8 hours apart.

[0084] 5. Test Results 5.1 Effect of Compound (I) on Body Weight in Mice with OXA-Induced Atopic Dermatitis Body weight data were recorded twice weekly.

[0085] The results of the experiment showed that OXA stimulation did not significantly affect the body weight of the mice. At the end point, compound (I) (3 mpk, 10 mpk, 30 mpk and 60 mpk) There was no significant effect on body weight. After dexamethasone treatment, the mice lost a significant amount of weight. The details are shown in Table 10.

[0086] [Table 11]

[0087] 5.2 Effect of Compound (I) on Skin Thickness in Mice with OXA-Induced Atopic Dermatitis The skin thickness (1.5 cm × 1.5 cm modeling area) was measured using a Mitutoyo digimatic indicator (M Using the Itsutoyo Digimatic Indicator, Model ID-C, USA, from the 1st to the 22nd day When skin thickness measurement and OXA immunostimulation were performed on the same day, skin thickness was measured once every two days. Priority was given to measuring skin thickness.

[0088] According to the experimental results, after OXA immunostimulation, the dorsal skin of mice (modeling area 1.5 cm × 1.5 cm) ) significantly increased in thickness. From the 17th day, compound (I) (30 mpk and 60 mpk) The increase in skin thickness in the modeling area was significantly inhibited compared with the 30 mpk group (17 and 22 days). Day 17 and 22 of the 60 mpk group: P < 0.05 vs. model group; Day 17 and 22 of the 60 mpk group: P < 0.01 vs. model group). From day 7, zolanin significantly inhibited the increase in skin thickness compared to the model group (day 7: P <0.01 vs. model group; 17 and 22 days: P<0.001 vs. model group). The details are shown in Table 11.

[0089] [Table 12]

[0090] 5.3 Clinical Scoring of Mouse Skin with OXA-Induced Atopic Dermatitis Caused by Compound (I) G From day 1 to day 22, the skin of the modeling area was scored using the skin scoring criteria in Table 12. Clinical skin scores and OXA immunotherapy were assessed every two days. If irritation occurred on the same day, clinical skin scoring took precedence.

[0091] [Table 13]

[0092] The results of the experiment showed that after OXA immunostimulation, skin clinical symptoms were observed in the modeling area of ​​mice. From the 19th day onwards, the core was significantly increased compared with the model group. and 60 mpk) significantly inhibited the increase in skin clinical scores (both P < 0.05 vs. model group). Compared with the model group, dexamethasone significantly inhibited the increase in skin clinical scores from day 9 onwards. The results were statistically significant (day 9: P < 0.05 vs. model group, days 19 and 22: P < 0.001 vs. model group). The details are shown in Table 13.

[0093] [Table 14]

[0094] 5.4 Skin epidermal thickness measurement and pathology scoring At the end of the study, tissue from the modeling area was collected and skin pathology scores and epidermal thickness were evaluated. For the measurement, hematoxylin-eosin staining was performed.

[0095] After the mice were sacrificed, the dorsal skin was removed and fixed in 10% neutral formalin for 24 hours. After dehydration, the skin sections were embedded in paraffin and cut into 4 μm sections. Staining with osmoticum revealed inflammatory cell infiltration and tissue changes in the stratum corneum, epidermis, and dermis under a microscope. The pathological scoring criteria were based on the aggregation of inflammatory cells and edema. The degree of dilation of the capillaries is rated as 1 mild, 2 moderate, or 3 severe points. It was classified as 2 points, severe 3 points.

[0096] The specific scoring criteria for inflammatory cell aggregation are: (1) 1 point is mild: inflammatory cells appearing in the dermis area; (2) 2 points is moderate: the inflammatory cell area in the dermis is only <10%. (3) 3 points is severe: the area of ​​inflammatory cells in the dermis of the skin is ≥ 50%. And so it became.

[0097] The specific scoring criteria for skin edema are: (1) 1 point is mild: several areas at the junction of the epidermis and dermis are swollen; Edema is observed in some areas, but the length of the edema cell aggregates is less than 10% of the length of the dermis-epidermis boundary. (2) 2 points: moderate: edema cells occupy 10% to 50% of the borderline between the dermis and epidermis; (3) 3 points: severe: edematous cells occupy more than 50% of the length of the border between the dermis and epidermis.

[0098] The specific evaluation of capillary dilation is as follows: (1) 1 point is mild: Capillaries are dilated in 1-3 places; (2) 2 points for moderate: 3-6 capillary dilations; (3) 3 points for severe: more than 6 capillaries This resulted in the capillaries expanding.

[0099] To measure epidermal thickness, stained skin sections were first scanned with a Leica Aperio CS2 scanner. The scanned images were then analyzed using HALO analysis software. The skin epidermis was defined as an annotation layer in the software's "typing" template. The annotation layer is used to divide the epidermis into two lines, one above the other, and the thickness of the line is defined as the layer thickness. The skin thickness of each section was calculated at approximately 100 points, including the "thickness" option. expressed as a value.

[0100] The experimental results showed that the skin in the modeled area showed inflammatory cell aggregation, edema, and capillary dilation. According to the pathological scoring criteria mentioned in the experimental method, The chemical score reached 6.8. Compared with the model group, compound (I) (10 mpk, 30 mpk, and 60 m Treatment with pk significantly reduced the pathological score of the skin in that area (P<0.01 vs. model group). Dexamethasone treatment significantly reduced the pathological scores (P < 0.001 vs. model group). XA stimulation significantly increased the thickness of the epidermis in the modeled area on the back of the mice. In comparison with the control, compound (I) (10 mpk, 30 mpk, and 60 mpk) significantly reduced the epidermal thickness. Dexamethasone treatment increased the epidermal The thickness was significantly reduced (P < 0.001 vs. model group), as detailed in Table 14.

[0101] [Table 15]

[0102] Example 4: Efficacy test of compound (I) on SLE model mice 1. Test drug Acceptor: Compound (I) Positive control drug: prednisone (China domestic approval number H33021207).

[0103] 2. Test Animals 50 4-week-old female MRL / lpr mice; 10 4-week-old female C57BL / 6 mice.

[0104] 3. Study Groups and Dosage Schedule Female MRL / lpr mice were divided into two groups according to the serum anti-ds-DNA antibody concentration: model group: vehicle (0.4% Tween Aqueous solution containing n80 and 0.5% methylcellulose) 10 mL / kg BID, positive control: prednisolone Compound (I) was administered QD at 6 mg / kg, Compound (I) was administered BID at 20 mg / kg, BID at 40 mg / kg, and BID at 60 mg / kg. The subjects were randomly divided into five groups, and the administration volume was 10 mL / kg in each group. Female C57BL / 6 mice were used. Compound (I) and model groups were administered 8-hour intervals from 5 weeks of age. Prednisone was administered intragastrically once a day from 5 weeks of age. The treatment was administered for a total of 17 weeks.

[0105] Two mice in the model group died at 15 and 16 weeks of treatment, respectively, and at the end of the study There were 8 mice remaining. One mouse was in the compound (I) BID 20 mg / kg group for 16 weeks. At the end of the study, nine mice remained. No mouse deaths occurred in the remaining groups. There wasn't.

[0106] 4. Test Results 4.1 Effects of Compound (I) on the Skin of MRL / lpr Mice 4.1.1 Skin damage status and score The condition of the skin damage on the face, ears, and back of the mice was observed once a week, for a total of 17 scores. I added. The scoring criteria were: 1) redness and bleeding of the skin, 2) absence of hair and dry skin, 3) edema, and 4) 4) Epidermal peeling / erosion, 5) lichen sclerosing plaques, etc. The score for each item was normal = 0 points, Mild = 1 point, moderate = 2 points, severe = 3 points. The degree of skin damage was assessed for each symptom. The evaluation was based on the total score.

[0107] As a result, after 14 weeks of treatment, the high and medium dose groups of compound (I) showed no significant improvement in the model. Compared with the control group, the degree of skin damage in systemic lupus erythematosus was effectively inhibited (14-16 weeks) , *P<0.05 vs. model group; 17 weeks, *P<0.01 vs. model group), positive control prednisone 6 mg / kcal g group also effectively inhibited the degree of skin damage in systemic lupus erythematosus (17 weeks, *P<0.01 vs model group), as detailed in Table 15.

[0108] [Table 16]

[0109] 4.1.2 Skin HE pathology score At the end of the study, the mice were dissected and their dorsal skin tissue was stained with HE to obtain a pathological score. went.

[0110] [Table 17]

[0111] As a result, the pathological scores of the skin were improved to some extent in each administration group, and the compound (I) group A statistically significant difference was observed in the high dose group of α-pred (*P <0.01 vs. model group), and in the positive control drug Pred A statistically significant difference was also observed in the 6 mg / kg nisone group (*P <0.001 vs. model group), and the experimental results were The details are shown in Table 16 and Figure 9.

[0112] [Table 18]

[0113] 4.2 Effect of Compound (I) on lymph nodes of MRL / lpr mice 4.2.1 Lymph node score Lymph nodes of MRL / lpr mice were scored weekly for a total of 17 times during the study.

[0114] The scoring criteria are determined by lymph node diameter (cm), with a score of 0 to 6: 0 points: normal; 1 point: small (one bilateral point less than 1 cm in diameter); 2 points: small (diameter less than 1 cm at two bilateral points); 3 points: small (diameter less than 1 cm at three bilateral points); 4 points: Large (diameter >1 cm at one bilateral point, diameter <1 cm at the other two bilateral points); 5 points: large (diameter >1 cm at two bilateral points, diameter <1 cm at the other bilateral point); 6 points: Large (over 1 cm in diameter at three bilateral points).

[0115] From the lymph node score data, it was found that compound (I) at high and medium doses Both drugs were found to effectively inhibit lymphadenopathy in patients with systemic lupus erythematosus (* * * P <0.001 vs. model group). In the low-dose group, the phospholipids in SLE mice were significantly reduced within 7 to 11 weeks of administration. It was found that the treatment significantly inhibited node swelling (7-9 weeks, *** P < 0.001 vs. model group; 11 weeks, *P<0.05 vs. model group), after 12 weeks, no difference was observed by the end of the experiment. The positive control group, prednisone 6 mg / kg, also effectively suppressed lymphadenopathy in patients with systemic lupus erythematosus. The results were shown in Table 17.

[0116] [Table 19]

[0117] 4.2.2 Lymph node weight At the end of the study, mice were dissected and lymph node tissue (submandibular, axillary, and inguinal) was weighed. However, from the data of lymph node tissue weight, it was found that the dose of compound (I) At both high and medium doses, it effectively treats lymphadenopathy in systemic lupus erythematosus. It was found to be inhibited (*P<0.001 vs. model group).

[0118] [Table 20]

[0119] 4.3 Effect of Compound (I) on the spleen of MRL / lpr mice At the end of the test, the mice were dissected and the spleen tissue was weighed. The dose of compound (I) was also determined to be high, medium and low, and the doses were also determined to be high, medium and low, and the doses were also determined to be low, and the doses were determined to be low, and the doses were also ... It was found that it effectively inhibited the degree of splenomegaly in mice with sexually transmitted lupus erythematosus (*P<0.0 01 vs. model group). Details are shown in Table 19 and Figure 11.

[0120] [Table 21]

[0121] 4.4 Effects of Compound (I) on the Kidneys of MRL / lpr Mice 4.4.1 Urine protein - area under the curve During the study, the urine protein content of the mice was measured once a week for 16 weeks. A graph of urinary protein concentration versus time is created from the urinary protein content at different times. For each graph, the area under the curve was calculated.

[0122] The three dose groups of Compound I showed varying degrees of improvement in urinary protein over a 16-week period. The area under the curve showed a statistically significant difference between the medium dose and high dose groups of Compound I ( *P<0.05 vs. model group), as detailed in Table 20 and Figure 12.

[0123] [Table 22]

[0124] 4.4.2 Kidney weight At the end of the test, the mice were dissected and the kidney tissues were weighed. In the high dose group of Compound (I), the total pathological score of both kidneys was significantly reduced ( *P<0.001 vs. model group), the total pathological scores of both kidneys were significantly higher in the medium dose group of compound (I). The results were shown in Table 21 and Figure 13.

[0125] [Table 23]

[0126] 4.4.3 Renal HE pathology score Scoring criteria for renal HE: [Table 24-1]

[0127] [Table 24-2]

[0128] [Table 25]

[0129] [Table 26]

[0130] According to the results of the HE staining score, the activity index (AI) score was higher than that of the model group. The high dose group of compound (I) showed a significant decrease in the total pathological score of both kidneys ( * * P<0.01 vs. model group), the chronicity index (CI) score was significantly higher in the high dose group of compound (I). The dose group showed a significant decrease in the total pathological score of both kidneys (**P<0.01 vs. The low dose group of compound (I) showed a significant decrease in the total pathological score of both kidneys. (**P<0.05 vs. model group), and the tubulointerstitial injury (TIL) score showed a significant improvement. The high dose group of compound (I) showed a significant decrease in the total pathological score of both kidneys ( * * P<0.01 vs. model group), as detailed in Table 22 and Figure 14.

[0131] [Table 27]

[0132] The kidney tissue IHC staining was performed and the rate of IgG-stained positive cells and the intensity of staining were scored. Renal IHC (IgG) scoring criteria: [Table 28]

[0133] [Table 29]

[0134] The final score is the product of the two, with 0 being negative (-) and 1 to 3 being low expression (+). 4 to 8 were classified as moderately expressed (++), and 9 to 12 were classified as highly expressed (+++).

[0135] As a result, compared with the model group, each dose group of compound (I) showed a significant difference in I in kidney tissue. The high dose group of compound (I) showed different degrees of improvement in IgG deposition, and the total IHC staining of both kidneys was significantly improved. The pathological score showed a relatively significant decrease (*P<0.05 vs. model group). Table 23 and Figure 1 5 is shown in detail.

[0136] [Table 30]

[0137] 4.5 Effect of Compound (I) on serum anti-ds-DNA antibody concentrations in MRL / lpr mice During the study period, serum anti-ds-DNA antibody concentrations in mice were measured every 4 weeks for up to 16 weeks.

[0138] As a result, the symptoms of lupus erythematosus in the model mice worsened over the test period. In terms of anti-ds-DNA antibody concentration, the compound (I) administration group showed a worsening of the anti-ds-DNA antibody concentration compared to the model group. effectively reduced anti-ds-DNA antibody levels after 3-4 weeks of treatment (16 weeks, *P<0.05 In addition, the positive control, prednisone 6 mg / kg, also significantly increased the concentration of anti-ds-DNA antibodies. The details are shown in Table 24 and Figure 16.

[0139] [Table 31]

[0140] 4.6 Effect of Compound (I) on serum cytokines in MRL / lpr mice At the end of the study, serum cytokine levels were measured using ELISA. As a result, compared with the model group, the three groups of low, medium and high doses of compound (I) TNF-α levels decreased, and significant differences were observed in the medium and high dose groups compared to the model group. (***P<0.001 vs. model group). Low, medium and high doses of compound (I) In the three dose groups, there was a certain reduction effect on IL-6 factor, and in the high dose group, there was no reduction effect on IL-6. The results were significant, as shown in Table 25 and Figure 17.

[0141] [Table 32]

[0142] Example 5 Toxicology Experiments Compound (I) was tested in accordance with the NMPA "Good Practice for Nonclinical Drug Research" and FDA GLP specifications (21 CFR Part 58) and in accordance with the International Council for Harmonisation (ICH) and NMPA related guidelines. Compound (I) was studied toxicologically in accordance with the principles of general toxicological studies. The dosage forms and experimental results were as follows:

[0143] [Table 33]

[0144] Test Results: Compound (I) does not affect the central nervous system, respiratory system, or cardiovascular system of animals, and does not affect the central nervous system, respiratory system, or cardiovascular system of humans. No adverse effects on the central nervous system, respiratory system, or cardiovascular system are expected. The NOAEL was 2000 mg / kg, and the MTD was 500 mg / kg / dose (1000 mg / kg) when administered once daily to Beagle dogs. mg / kg / day), and the NOAEL after repeated administration for 28 days to rats was 10 mg / kg / dose (20 mg / kg / day), and the NOAEL after repeated administration for 28 days to Beagle dogs was 3 mg / kg / dose (6 mg / kg / day). There was no genotoxicity. The effective dose in rats was 3 mg / kg / dose BID, and the exposure was calculated as Compound (I) has a safety window of 1 in Beagle dogs and According to the dosage calculation, Compound (I) had a safety window of 8 to 13 times. It has a three-fold safety window in both dogs and SD rats, and the safety window assessment The starting dose for the dose escalation phase of the initial clinical trial of Compound (I) was 15 mg. The human equivalent dose of the NOAEL dose in repeated dose toxicological studies of Compound (I) in Beagle dogs was Compared with the initial dose, the human starting dose had a 14-fold safety window, and the SD rat repeated dose toxicity Compared with the human equivalent dose of the NOAEL dose in the physical studies, the human starting dose was 16 times lower. Therefore, according to the preclinical toxicity test data of compound (I), The single-dose Phase 1 clinical development phase is available at dose escalation levels of 15 mg, 45 mg, and 60 mg. mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg and 360 mg.

[0145] [Table 34]

[0146] Based on the results of a Phase I clinical trial, a single dose of up to 240 mg in the dose escalation phase was administered in three stages. No safety issues were reported.

[0147] In summary, compound (I) improved skin lesions in mice with psoriasis and atopic dermatitis. , inhibiting the growth of immune organs, reducing inflammation levels, and also reducing skin lesions in SLE mice. Dose-dependent improvement, attenuated renal damage, inhibited the growth of immune organs, and reduced serum SLE-related It inhibited the increase of antibodies and cytokines and had a certain safety window, making it a promising candidate for clinical use. It has potential for floor applications.

[0148] The full names of the English contractions and Japanese names used in the present invention are as follows: [Table 35]

Claims

1. Compound (I) having the structural formula (I), its optical isomer or a pharmaceutically acceptable salt thereof for treating diseases associated with overexpression or abnormal activation of JAK kinase and SYK kinase in the manufacture of a medicament for the treatment of overexpression or abnormalities of the JAK kinase and SYK kinase. The use wherein the activation-related disease is an autoimmune disease. 【Chemistry 1】

2. 2. The method according to claim 1, wherein the autoimmune disease is an immune-mediated skin disease and an autoimmune connective tissue disease. Uses listed.

3. The immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is preferably Preferably, the psoriasis is plaque psoriasis, pustular psoriasis, erythrodermic psoriasis, or psoriatic arthritis, The autoimmune connective tissue disease is lupus erythematosus, and the lupus erythematosus is preferably Discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus lupus erythematosus, profundus lupus erythematosus, neonatal lupus erythematosus, and drug-induced lupus erythematosus. and more preferably systemic lupus erythematosus.

3. The use according to claim 2.

4. The medicament comprises a therapeutically effective amount of compound (I), an optical isomer thereof, or a pharmaceutically acceptable salt thereof.

4. The method of claim 1, further comprising administering to said patient a compound selected from the group consisting of hydroxybenzoates, ... The uses described in.

5. The medicament is a clinically acceptable formulation, including oral, injectable, topical, or external dosage forms. The use according to claim 4, characterized in that it is formulated into various dosage forms.

6. The medicament is clinically used alone or in combination with other therapeutic ingredients, 3. The use according to any one of the preceding items.

7. A therapeutically effective amount of compound (I), its optical isomer, or a pharmaceutically acceptable salt thereof. A pharmaceutical product, 【Chemistry 2】 A disease associated with overexpression or abnormal activation of JAK kinase and SYK kinase in a subject. and a pharmaceutical agent for treating an autoimmune disease caused by, for example, an immune-mediated skin disease and autoimmune connective tissue disease, wherein the immune-mediated skin disease is psoriasis or atopic dermatitis dermatitis, and the psoriasis is preferably selected from plaque psoriasis, pustular psoriasis, erythrodermic psoriasis or psoriatic arthritis, and the autoimmune connective tissue disease is lupus erythematosus, and the lupus Erythematosus is preferred, discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus Sexual lupus erythematosus, profundus lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus The most preferred is systemic lupus erythematosus, and more preferably systemic lupus erythematosus.

8. Methods for treating diseases associated with overexpression or aberrant activation of JAK and SYK kinases The present invention relates to a method for treating a mammal (e.g., a human) in need of such treatment, comprising administering to the mammal a therapeutically effective amount of Compound (I), or a pharmaceutically acceptable salt thereof, 【Transformation 3】 The diseases associated with the overexpression or abnormal activation of JAK kinase and SYK kinase include autoimmune diseases. The autoimmune diseases include, for example, immune-mediated skin diseases and autoimmune connective tissue diseases. wherein the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is Psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis, or psoriatic arthritis is preferred, and the autoimmune The connective tissue disease is lupus erythematosus, and the lupus erythematosus is preferably discoid lupus erythematosus. Lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, profundus lupus erythematosus lupus erythematosus, neonatal lupus erythematosus, and drug-induced lupus erythematosus are more preferred. It is systemic lupus erythematosus.

9. A therapeutically effective amount of compound (I), its optical isomer, or a pharmaceutically acceptable salt thereof, and other and a therapeutic ingredient of: 【Chemistry 4】 A disease associated with overexpression or abnormal activation of JAK kinase and SYK kinase in a subject. a combination drug or product for treating an autoimmune disease, said autoimmune disease being , such as immune-mediated skin diseases and autoimmune connective tissue diseases, wherein the immune-mediated skin diseases are The psoriasis is selected from psoriasis vulgaris and pustular psoriasis. psoriasis, erythrodermic psoriasis, or psoriatic arthritis, and the autoimmune connective tissue disease is lupus erythematosus The lupus erythematosus is preferably discoid lupus erythematosus or subacute cutaneous lupus erythematosus. Systemic lupus erythematosus, profundus lupus erythematosus, neonatal lupus erythematosus and drug-induced lupus erythematosus, and more preferably systemic lupus erythematosus.

10. The therapeutically effective amount is 0.01 to 2000 mg, preferably 1 to 500 mg, and more preferably 10 to 400 mg. mg, more preferably 15 to 360 mg or 15 to 250 mg, for example 15 mg, 45 mg, 90 mg , 135 mg, 180 mg, 240 mg, 300 mg, 360 mg. Uses listed.

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