Use of a compound containing a tricyclic heteroaryl

Abstract

The present application provides the use of compound (I), its optical isomers or its pharmaceutically acceptable salts in the preparation of a medicament for treating diseases associated with high expression or abnormal activation of JAK and SYK kinases, including autoimmune diseases, such as immune-mediated skin diseases, especially psoriasis, atopic dermatitis and SLE. The compound (I), its optical isomers or its pharmaceutically acceptable salts of the present invention can improve skin lesions in psoriasis, atopic dermatitis and SLE mice, relieve kidney injury, inhibit the enlargement of immune organs, reduce the inflammation level, inhibit the increase of SLE-related antibodies and cytokines in serum, and have a certain safe treatment window, showing good clinical application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use of a compound containing a tricyclic heteroaryl in the preparation of a medicament for treating diseases related to high expression or abnormal activation of kinases JAK and SYK. Background Art

[0002] JAK (Janus kinase), namely Janus kinase, is a class of non-transmembrane non-receptor tyrosine kinases, including four subtypes: JAK1, JAK2, JAK3 and TYK2 (Tyrosine kinase 2). JAK1, JAK2 and TYK2 are widely present in various tissues and cells. JAK1 is involved in mediating inflammatory signaling pathways such as IL-6 and IFN. JAK2 can independently mediate cytokine signaling pathways such as IL-3, IL-5 and EPO. JAK3 is only present in the bone marrow and lymphatic system and mediates the signal transduction of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. TYK2 is involved in the signal transduction of IFN-α, IL-6, IL-10 and IL-12. JAK inhibitors participate in immune regulation and other processes by specifically inhibiting the JAK-STAT (Signal transducers and activators of transcription) signaling pathway and blocking the cascade amplification of the above cytokines.

[0003] SYK (Spleen tyrosine kinase), namely spleen tyrosine kinase, is a non-receptor tyrosine kinase present in the cell matrix. SYK is widely expressed in hematopoietic cells, lymphocytes, fibroblasts, vascular endothelial cells, highly expressed in B lymphocytes, and plays an important role in tumors and autoimmune diseases. Dectin-1 / ITAM is a classical pathway for antigen-stimulated immune cells to induce immune diseases. In normal B cells, when antigen-induced BCR cross-linking causes phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the cell, SYK in the cytoplasm is the first object recruited and activated by ITAM. The activated SYK then activates the transcription factor NF-κB through the CARD9-dependent pathway, generating a series of inflammatory factors. In addition, this pathway can also activate Caspase-8, and the activated Caspase-8 cleaves the precursor of IL-1β, promoting the maturation of immature IL-1β. The CARD9-independent NLRP3 signaling pathway also plays a role in the maturation of immature IL-1β. Therefore, SYK is also an important target for autoimmune diseases.

[0004] Psoriasis is an immune-mediated chronic, recurrent, inflammatory skin disease. The prevalence varies significantly around the world, with the prevalence in the United States being 0.5% - 3.15%, in Europe 0.75% - 2.9%, in China the reported prevalence of psoriasis in 1984 was 0.123%, the prevalence in 6 cities surveyed in 2008 was 0.47%, and the prevalence in 4 southwestern provinces in 2017 was 0.5%. The number of psoriasis patients in China is about 6 million or more. Psoriasis can occur at any age, without gender difference. Approximately 2 / 3 of the patients develop the disease before the age of 40, and most patients have more severe symptoms in winter and milder symptoms in summer.

[0005] The etiology and pathogenesis of psoriasis have not been fully elucidated and may involve multiple factors such as genetics, immunity, and environment. Through an immune response mainly mediated by T lymphocytes and participated by multiple immune cells, it causes excessive proliferation of keratinocytes and inflammation of synovial cells and chondrocytes in joints. The typical clinical manifestation of psoriasis is scaly erythema or plaques, which are distributed locally or widely. Psoriasis can be combined with other systemic abnormalities, such as visceral and joint damage. The risk of metabolic syndrome and atherosclerotic cardiovascular disease increases in moderate to severe patients.

[0006] According to the clinical manifestations and pathological features of psoriasis, it can be divided into the following types: 1. Plaque psoriasis: This is the most common type, often presenting an acute onset. The typical manifestation is erythema with well-defined boundaries and varying shapes and sizes, surrounded by an inflammatory red halo. It is slightly infiltrated and thickened. The surface is covered with multiple layers of silvery-white scales. The scales are easily scraped off, revealing a pale red, shiny, translucent film after scraping. Scratching the film can reveal small bleeding points (Auspitz sign). The skin lesions are prone to occur on the head, sacral region, and the extensor sides of the limbs. Some patients experience varying degrees of itching. 2. Pustular psoriasis: It is divided into generalized and palmo-plantar types. Generalized pustular psoriasis presents clusters of superficial sterile pustules on the erythema, and some can coalesce into pustular lakes. It can occur throughout the body, being more common on the flexor sides of the limbs and the flexural areas. The oral mucosa can be involved simultaneously. Acute onset or sudden exacerbation is often accompanied by systemic symptoms such as chills, fever, joint pain, general discomfort, and an increase in white blood cell count. It often shows periodic attacks, and in the remission period, plaque psoriasis skin lesions often appear. Palmoplantar pustulosis has skin lesions limited to the hands and feet, occurring symmetrically. The general condition is good, but the condition is stubborn and recurrent. 3. Erythrodermic psoriasis: Also known as psoriatic exfoliative dermatitis, it is a severe form of psoriasis. It is often caused by applying irritating drugs externally, long-term and large-dose use of glucocorticoids, too rapid reduction or sudden discontinuation of the drugs. It is manifested as diffuse flushing, swelling, and desquamation of the whole body skin, accompanied by systemic symptoms such as fever, chills, and discomfort, superficial lymph node enlargement, and an increase in white blood cell count. 4. Psoriatic arthritis: Also known as psoriatic arthropathy. Psoriasis patients simultaneously develop joint damage similar to rheumatoid arthritis, which can involve joints of all sizes throughout the body, but the interphalangeal joints of the distal fingers (toes) are the most characteristic. The affected joints are red, swollen, and painful, and the skin around the joints is often red and swollen. The joint symptoms often worsen or improve simultaneously with the skin symptoms. The blood rheumatoid factor is negative.

[0007] At present, there is no specific therapy for psoriasis. The main treatment methods include topical treatment, physical therapy, systemic treatment, traditional Chinese medicine treatment and other therapies. Among them, topical treatment includes topical drug treatment, physical therapy, etc. Topical drugs mainly include: vitamin D3 analogs, glucocorticoids, anthralin, retinoic acid gels and creams, tar preparations, immunosuppressants, etc., as well as other topical drugs such as tacrolimus, pimecrolimus, 0.03% camptothecin ointment, 5% salicylic acid ointment, etc. Systemic treatment drugs include methotrexate, retinoids, cyclosporine, tacrolimus, mycophenolate mofetil, biological agents such as etanercept, infliximab, and antibiotics. For moderate and severe patients, combined, alternating or sequential treatment is usually given when single treatment is not effective. Although the existing treatments have significant short-term effects, they will produce many side effects and have a high recurrence rate, and the long-term curative effect is poor. For example, the therapeutic dose of methotrexate is very close to the toxic dose; the main side effect of retinoids is teratogenesis; the adverse reactions of cyclosporine A include nephrotoxicity, hypertension, nausea, vomiting, fatigue, muscle tremors and urinary irritation symptoms, etc.; the adverse reactions of tacrolimus are similar to those of cyclosporine A; the adverse reactions of mycophenolate mofetil include gastrointestinal symptoms, anemia, leukopenia, and the risk of increasing infection and inducing tumors, etc.

[0008] Atopic dermatitis (AD), also known as atopic eczema and allergic dermatitis, is an allergic skin disease characterized by skin pruritus and polymorphic rashes. It has different clinical manifestations at different age stages and is a chronic, recurrent and inflammatory skin disease. Since patients often have other atopic diseases such as allergic rhinitis and asthma, it is considered a systemic disease. The prevalence of AD has gradually increased globally in the past 30 years. The prevalence of AD in children in developed countries reaches 10% - 20%. The increase in the prevalence of AD in China lags behind that in Western developed countries, Japan and South Korea, but has grown rapidly in the past 10 years. The overall prevalence of AD in school-age adolescents (6 - 20 years old) in 1998 was 0.70%. The prevalence of AD in preschool children (1 - 7 years old) in 10 cities in 2002 was 2.78%. The prevalence of AD in children aged 3 - 6 years in Shanghai reached 8.3% in 2012. The prevalence of AD in children aged 1 - 7 years in 12 cities in China reached 12.94% in 2014, and the prevalence of AD in infants aged 1 - 12 months reached 30.48%.

[0009] The onset of AD is closely related to factors such as genetics and environment. Although the exact pathogenesis is still unclear, currently, factors such as immune abnormalities, skin barrier dysfunction, and skin microbiota disorders are considered important links in the onset. AD usually begins in infancy, and about 50% of all patients develop the disease before the age of 1. It has a chronic course, with diverse clinical manifestations. The most basic features are dry skin, chronic eczematous lesions, and obvious pruritus. Some patients may also have other allergic diseases, such as allergic asthma and allergic rhinoconjunctivitis. In addition, due to long-term chronic inflammatory responses, the risk of chronic patients developing neurological diseases, inflammatory bowel disease, rheumatoid arthritis, cardiovascular diseases, and lymphoma is significantly increased.

[0010] Currently, the treatment of AD includes basic treatment (such as avoiding contact allergies), topical drug treatment (topical glucocorticoids and topical calcineurin inhibitors, etc.), systemic treatment (oral antihistamines, immunosuppressants, glucocorticoids, etc.), ultraviolet treatment, and antimicrobial treatment, etc. Among them, topical drugs, hormones, and immunosuppressants are the main ones. Usually, a stepped treatment approach is used to treat mild to moderate patients. Although the existing treatment methods can relieve symptoms, there are still certain limitations and many adverse reactions. There are still many unmet needs in terms of rapid onset, itching control, and recurrence prevention.

[0011] Lupus erythematosus (LE) is a typical autoimmune connective tissue disease, which can be divided into subtypes such as discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), systemic lupus erythematosus (SLE), lupus erythematosus profundus (LEP), neonatal lupus erythematosus (NLE), drug-induced lupus erythematosus (DIL), etc.

[0012] Discoid lupus erythematosus mainly affects the skin and is the mildest type of lupus erythematosus. A few may have mild visceral damage, and a few cases may transform into systemic lupus erythematosus. Most patients have no self-awareness symptoms of the skin lesions, but it is difficult to completely subside. Subacute cutaneous lupus erythematosus is clinically less common and is a special intermediate type. The skin lesions often recur, and the vast majority of patients have visceral damage, but severe cases are rare. The main symptoms are joint pain, muscle pain, and repeated low fever. A few have nephritis and blood system changes. Lupus erythematosus profundus, also known as lupus panniculitis and deep lupus erythematosus, is also an intermediate type of lupus erythematosus. Its nature is unstable and can exist alone. Later, it can either transform into discoid lupus erythematosus, systemic lupus erythematosus, or coexist with them. Neonatal lupus erythematosus presents as skin annular erythema and congenital heart block and is self-limiting. Generally, it subsides spontaneously within 4 to 6 months after birth, and the heart lesion often persists. Drug-induced lupus erythematosus mainly presents as fever, joint pain, muscle pain, facial butterfly erythema, oral ulcers, and may have serositis. It gradually improves after drug withdrawal. For patients with severe conditions, drug treatment can be given.

[0013] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease and the most severe type among various lupus erythematosus. Its main clinical features include multi-system and multi-organ involvement throughout the body, repeated relapses and remissions, and the presence of a large number of autoantibodies in the body. If not treated promptly, it will cause irreversible damage to the affected organs and ultimately lead to the death of the patient. The prevalence of SLE varies greatly by region. Currently, the global prevalence of SLE is 0 - 241 per 100,000, and the prevalence of SLE in China is 30 - 70 per 100,000, with the male-to-female ratio of 1:10 - 12. The specific pathogenesis of SLE has not been fully elucidated and is caused by the joint participation of multiple factors, mainly related to genetics, infection, endocrine, and environment. Defects in the immune regulation mechanism, such as the clearance of apoptotic cells and immune complexes, are also important factors in the occurrence of SLE. Loss of immune tolerance, increased antigen load, excessive T cell help, defects in B cell inhibition, and the transformation of Th1 cells to Th2 cells lead to over-activation of B cells and the production of pathogenic autoantibodies. In addition, some drugs such as methyldopa, phenytoin sodium, penicillamine, quinidine, and propranolol can directly cause drug-induced lupus and exacerbate lupus erythematosus. The vast majority of SLE patients show multi-system damage at the onset. A small number of patients develop from other types of lupus erythematosus, and some patients are also accompanied by other connective tissue diseases, such as scleroderma, dermatomyositis, and Sjogren's syndrome, forming various overlap syndromes.

[0014] Currently, the drug treatment of SLE is based on glucocorticoids and hydroxychloroquine. However, the use of the above drugs will produce corresponding side effects, such as infection, impaired liver and kidney function, and metabolic abnormalities. Sometimes, the dosage has to be reduced or the drug has to be discontinued. Moreover, there is still no good drug to control the development of the disease, and new drugs still need to be developed. SYK and JAK are respectively upstream of two different signaling pathways that induce SLE. Therefore, SYK-JAK dual-channel inhibitors are expected to be an effective way to treat SLE. Currently, there is no JAK-SYK dual-target inhibitor approved for marketing. The JAK-SYK dual-target inhibitor product R333, which is developed for DLE as an indication, also terminated its development on October 24, 2013, due to a Phase II clinical failure.

[0015] Therefore, there is a need to develop new drugs for the treatment of autoimmune diseases, especially immune-mediated skin diseases and autoimmune connective tissue diseases such as psoriasis, atopic dermatitis, or lupus erythematosus. Summary of the Invention

[0016] Compound (I) having the following structural formula (I):

[0017]

[0018] Its 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 the PCT international application with the publication number WO2018108084, and is known to be a highly selective dual-target inhibitor of JAK kinase and SYK kinase, and can be used for the treatment of cancer.

[0019] The inventors of the present invention further found through research that compound (I) regulates cell signal transduction, division and proliferation by inhibiting JAK and SYK kinases, and has excellent effects in the treatment of autoimmune diseases, especially in the treatment of immune-mediated skin diseases and autoimmune connective tissue diseases such as psoriasis, atopic dermatitis or lupus erythematosus, showing satisfactory activity and low toxicity and side effects.

[0020] Accordingly, the present invention provides the use of compound (I), its optical isomers or its pharmaceutically acceptable salts in the preparation of drugs for the treatment of diseases related to high expression or abnormal activation of JAK and SYK kinases:

[0021]

[0022] Preferably, in the above use, the JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinases, preferably JAK3 and / or TYK2 kinases.

[0023] Preferably, in the above use, the diseases related to high expression or abnormal activation of JAK and SYK kinases are autoimmune diseases.

[0024] Preferably, in the above use, the autoimmune diseases are immune-mediated skin diseases and autoimmune connective tissue diseases.

[0025] Preferably, in the above use, the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and 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, deep lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and more preferably systemic lupus erythematosus.

[0026] Furthermore, in the above use, the drug contains a therapeutically effective amount of compound (I), its optical isomers or its pharmaceutically acceptable salts, and optionally, a pharmaceutically acceptable excipient or carrier.

[0027] There is no particular limitation on the administration mode of the drug of the present invention. Representative administration modes include but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. Accordingly, the drug of the present invention can be made into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, topical dosage forms or external use dosage forms, etc.

[0028] Preferably, the drug of the present invention can be used alone clinically or in combination with other therapeutic components. For the convenience of clinical use, the compound (I) of the present invention, its optical isomers or its pharmaceutically acceptable salts can be combined with other therapeutic components to prepare compound drugs or combination products.

[0029] The present invention also provides a method for using the drug, that is, administering a therapeutically effective amount of the compound (I) of the present invention, its optical isomers or its pharmaceutically acceptable salts to a mammal (such as a human) in need of treatment.

[0030] The present invention provides a method for treating diseases related to high expression or abnormal activation of JAK and SYK kinases, which is characterized in that a therapeutically effective amount of the compound (I) of the present invention, its optical isomers or its pharmaceutically acceptable salts is administered to a mammal (such as a human) in need of treatment.

[0031] The JAK kinase described in the present invention is one or more of JAK1, JAK2, JAK3 or TYK2 kinases, preferably JAK3 and / or TYK2 kinases. The diseases related to high expression or abnormal activation of JAK and SYK kinases described in the present invention are autoimmune diseases, such as immune-mediated skin diseases and autoimmune connective tissue diseases. The immune-mediated skin diseases are selected from psoriasis or atopic dermatitis. The psoriasis is preferably vulgaris 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, deep lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and further preferably systemic lupus erythematosus.

[0032] The present invention also provides a medicament comprising a therapeutically effective amount of compound (I), its optical isomers or its pharmaceutically acceptable salts, characterized in that the medicament is used for treating diseases related to high expression or abnormal activation of JAK and SYK kinases in a subject, which are autoimmune diseases, such as immune-mediated skin diseases and autoimmune connective tissue diseases. The immune-mediated skin diseases are selected from psoriasis or 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, deep lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and more preferably systemic lupus erythematosus.

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

[0034] The present invention also provides a compound medicament or combination product, which comprises a therapeutically effective amount of compound (I), its optical isomers or its pharmaceutically acceptable salts, and other therapeutic components, characterized in that the compound medicament or combination product is used for treating diseases related to high expression or abnormal activation of JAK and SYK kinases in a subject, which are autoimmune diseases, such as immune-mediated skin diseases and autoimmune connective tissue diseases. The immune-mediated skin diseases are selected from psoriasis or 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, deep lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and more preferably systemic lupus erythematosus.

[0035] The JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinases, preferably JAK3 and / or TYK2 kinases.

[0036] The therapeutically effective amount described in the present invention refers to the pharmaceutically recognized effective dosage, that is, the amount of the active compound sufficient to significantly improve the condition without causing serious side effects. For a person weighing 60 kg, the daily dosage is usually 0.01 - 2000 mg, preferably 1 - 500 mg, more preferably 10 - 400 mg, and further preferably 15 - 360 mg or 15 - 250 mg, such as: 15 mg, 45 mg, 60 mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg, 360 mg. It can be administered as a single dose once a day, in multiple doses per day, or at intervals. The specific dosage and administration frequency should consider factors such as the administration route and the patient's health condition, which can be determined by a skilled physician based on routine skills. The administration method is not particularly limited, and representative administration methods include but are not limited to: oral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration. The amount of the active compound is calculated based on compound (I).

[0037] In vitro and in vivo studies have shown that (1) the compound (I) of the present invention can significantly inhibit the activities of JAK1, JAK2, JAK3, TYK2, and SYK kinases in vitro, and has a strong inhibitory effect on JAK3 and TYK2, with IC 50 being 1.43 nM and 0.82 nM respectively. The inhibitory effects on JAK2 and SYK are slightly weaker than those on JAK3 and TYK2, and the IC 50 is between 3 nM and 8 nM. The inhibitory effect on JAK1 is the weakest, with IC 50is 20.04 nM. (2) The compound (I) of the present invention can improve or significantly reduce the skin thickness, ear thickness, spleen weight, spleen index, PASI score (erythema, scale, thickness and total score), mouse skin epidermal thickness, skin pathology score, and the contents of IL-6 and TNF-α in the skin tissue of IMQ-induced psoriasis model mice. (3) The compound (I) of the present invention can improve or significantly reduce the skin thickness and skin clinical score of OXA-induced atopic dermatitis model mice; it can improve the aggregation of inflammatory cells, edema and capillary dilation in the skin of the modeled area, and significantly reduce the pathology score and epidermal thickness. (4) The compound I (compound (I)) of the present invention can dose-dependently improve the skin lesions of MRL / lpr lupus model mice, relieve kidney injury, reduce lymph node and spleen enlargement, and inhibit the increase of SLE-related antibodies and cytokines in the serum. Among them, the mice in the 20 mg / kg group could effectively inhibit the lymph node enlargement of SLE mice after 7 weeks of drug administration. The evaluation results of the endpoint indicators showed that 20 mg / kg administration could effectively inhibit spleen enlargement and lymph node enlargement, and there was a significant decrease in the total pathological score of both kidneys in the chronic index (CI). It could also inhibit the overexpression of serum IL-6 and TNF-α to a certain extent. The administration doses of 40 mg / kg and 60 mg / kg could comprehensively improve various symptoms of SLE mice in terms of improving skin lesions (skin injury score and skin pathology score), relieving kidney injury (reducing mouse urine protein and reducing kidney immune complex deposition), and inhibiting the enlargement of immune organs (improving lymph node pathology score and inhibiting spleen and lymph node enlargement).

[0038] The above research results indicate that the compound (I), its optical isomers or its pharmaceutically acceptable salts of the present invention can improve the skin lesions of psoriasis mice and atopic dermatitis mice, and inhibit the enlargement of immune organs; it can also dose-dependently improve the skin lesions of SLE mice, inhibit the enlargement of immune organs, relieve kidney function injury, reduce the inflammatory level, and has a certain safety treatment window, showing good clinical application prospects. Brief Description of the Drawings

[0039] Figure 1 : Spleen weight diagram of IMQ-induced psoriasis model mice at the research endpoint.

[0040] Figure 2 : Spleen index (spleen weight / body weight%) diagram of IMQ-induced psoriasis model mice at the research endpoint.

[0041] Figure 3 : IL-6 content diagram in the skin tissue of IMQ-induced psoriasis model mice at the research endpoint.

[0042] Figure 4:Content diagram of TNF-α in the skin tissue of mice at the research endpoint of the psoriasis model mice induced by IMQ.

[0043] Figure 5 :Skin pathology score diagram of the psoriasis model mice induced by IMQ at the research endpoint.

[0044] Figure 6 :Skin epidermal thickness diagram of the psoriasis model mice induced by IMQ at the research endpoint.

[0045] Figure 7 :Skin pathology score diagram of the atopic dermatitis model mice induced by OXA at the research endpoint.

[0046] Figure 8 :Skin epidermal thickness diagram of the atopic dermatitis model mice induced by OXA at the research endpoint.

[0047] Figure 9 :Skin pathology score diagram of SLE mice at the test endpoint.

[0048] Figure 10 :Lymph node weight diagram of SLE mice at the test endpoint, A: Total lymph node weight; B: Total lymph node weight / body weight %.

[0049] Figure 11 :Spleen weight diagram of SLE mice at the test endpoint, A: Spleen weight; B: Spleen weight / body weight %.

[0050] Figure 12 :Area under the curve diagram of urine protein of SLE mice treated for 16 weeks.

[0051] Figure 13 :Kidney weight diagram of SLE mice at the test endpoint, A: Total kidney weight; B: Kidney weight / body weight %.

[0052] Figure 14 :HE staining score diagram of the kidney tissue of SLE mice, A: Bilateral kidney HE score - activity index; B: Bilateral kidney HE score - chronic index; C: Bilateral kidney HE score - tubulointerstitial injury.

[0053] Figure 15 :IHC (IgG) staining score diagram of the kidney tissue of SLE mice.

[0054] Figure 16 :Serum anti-ds-DNA antibody concentration diagram of SLE mice.

[0055] Figure 17 :Serum cytokine level diagram of SLE mice, A: TNF-α concentration; B: IL-6 concentration. Specific implementation mode

[0056] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0057] Source or preparation of experimental materials:

[0058] 1. Compound (I): Self-prepared by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd.

[0059] 2. The positive control drug, reagents and raw materials used in the experiment are all commercially purchased or self-prepared.

[0060] 3. Preparation method of test substances (Compound (I) and positive control compound) for in vivo experiments:

[0061] After weighing, it is dissolved in an aqueous solution containing 0.4% Tween 80 and 0.5% methylcellulose. The concentrations of Compound (I) are configured to be 0.3 mg, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 6 mg / mL respectively; the positive control drug is configured with physiological saline at a concentration of 0.3 mg / mL or 0.6 mg / mL and diluted to the required concentration before use.

[0062] 4. Preparation of 1 L of solvent (0.4% Tween 80 / 0.5% methylcellulose):

[0063] Weigh 5.0 g of methylcellulose powder into a clean glass bottle, add 900 mL of sterile water, stir overnight until fully dissolved; suck 4.0 mL of Tween 80, stir well and make up to a final volume of 1000 mL. This solution is stored in a refrigerator at 4 °C.

[0064] 5. Preparation of OXA:

[0065] Preparation of acetone / olive oil (4 / 1) solvent: Mix 40 mL of acetone and 10 mL of olive oil and shake for 30 seconds until evenly mixed to obtain acetone / olive oil (4 / 1) solvent.

[0066] Preparation of 5% OXA: Weigh 100.00 mg of OXA and dissolve it in 2.00 ml of acetone / olive oil (4 / 1), shake for 30 seconds.

[0067] Preparation of 0.1% OXA: Weigh 15.69 mg of OXA and dissolve it in 15.69 mL of acetone / olive oil (4 / 1), shake for 30 seconds. Prepare it immediately before use and prepare it once every two days.

[0068] Example 1: Activity inhibition test of Compound (I) on kinases

[0069] 1. Kinases: SYK, JAK1, JAK2, JAK3, and TYK2

[0070] 2. Experimental methods

[0071] The protein kinase activity was determined by Mobility Shift Assay. Compound (I) was dissolved in DMSO and then prepared into a concentration gradient in 100% DMSO. Diluted with kinase buffer, 5 μL of 5-fold the final reaction concentration of Compound (I) (10% DMSO) was added to a 384-well plate. After adding 10 μL of 2.5-fold enzyme solution, it was incubated at room temperature for 10 minutes, and then 10 μL of 2.5-fold substrate solution was added. Incubated at 28 °C for 60 minutes, 30 μL of termination solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) was added to the reaction plate of the 384-well plate to terminate the reaction. The conversion rate data was replicated on a Caliper EZReader Ⅱ, and the conversion rate was converted into inhibition rate data: % Inhibition rate = (max - conversion rate) / (max - min) × 100%. Among them, "min" is the conversion rate of the control sample well without adding enzyme for the reaction; "max" is the conversion rate of the control well with DMSO added. Taking the compound concentration and inhibition rate as the horizontal and vertical coordinates, a curve was plotted, and the IC 50 value was calculated using the XLFit excel add-inversion 5.4.0.8. Fitting formula: Y = Bottom + (Top - Bottom) / (1 + (IC 50 / X)^HillSlope).

[0072]

[0073] 3. Experimental results

[0074] The test results of the inhibitory activity of Compound (I) on kinases are shown in the following table:

[0075] Table 1 Inhibitory activity of Compound (I) on kinases

[0076] Kinase JAK1 JAK2 JAK3 TYK2 SYK <![CDATA[IC 50 ,nM]]> 20.04 3.92 1.43 0.82 7.25 .

[0077] Example 2: Pharmacodynamic test of Compound (I) in a psoriasis model of mice induced by IMQ

[0078] 1. Test purpose

[0079] The purpose of this experiment is to evaluate the efficacy of compound (I) in an IMQ (5% imiquimod cream) - induced psoriasis mouse model. One of the clinical adverse reactions of imiquimod is the induction of psoriasis attacks. The imiquimod - induced psoriasis mouse model is simple to operate and easy to achieve, and its skin phenotype and pathological features have the advantages of complex interactions among tissues and are similar to clinical psoriasis.

[0080] 2. Test drugs

[0081] Test drug: Compound (I)

[0082] Positive control drug: Dexamethasone (Dex)

[0083] Solvent: 0.4% Tween80 / 0.5% methylcellulose.

[0084] 3. Test animals

[0085] 70 female Balb / c mice, 5 - 6 weeks old.

[0086] 4. Test grouping and dosing regimen

[0087] The animals were randomly divided into 7 groups according to their body weights one day before the start of the experiment. See Table 2 for details:

[0088] Table 2 Grouping of psoriasis efficacy experiment

[0089]

[0090]

[0091] Note: "PO" means oral administration; "bid" means twice a day; "qd" means once a day; mpk: mg / kg.

[0092] Model establishment:

[0093] One day before drug application, the hair on the backs of the animals was shaved to form a skin exposure area of about 2 cm × 3 cm. The mice were induced with IMQ, and 62.5 mg of 5% imiquimod cream was applied to the bare backs and right ears of the mice at a fixed time every day for 14 days. The mice in the normal control group were applied with petrolatum to the bare backs and right ears at a fixed time every day.

[0094] 5. Test results

[0095] 5.1 Effect of compound (I) on the skin thickness of IMQ - induced mice

[0096] The skin thickness was measured daily from the first day of the experiment until the end of the experiment.

[0097] The method for measuring skin thickness is as follows: Use the left thumb and index finger to pinch up the skin on the back of the mouse together with the subcutaneous tissue in the same direction as the body of the mouse; Hold the digital micrometer (model BK-3281, manufacturer: Shanghai Newhui) in the right hand and measure the skin fold thickness at a position 1 cm away from the pinched part of the left hand (the center near the modeling site). The thickness is in mm, and the actual thickness is half of the measured thickness (skin thickness = measured value / 2).

[0098] The experimental results showed that compared with the normal control group, the skin thickness of the mice in the model group increased significantly after being stimulated with 5% imiquimod cream for 14 consecutive days. At the end of the study, the treatments with compound (I) (3mpk, 10mpk, 30mpk, and 60mpk) and dexamethasone (3mpk) both significantly inhibited the increase in skin thickness (for all 5 groups: P < 0.001 vs the model group), as shown in Table 3 for details.

[0099] Table 3 Skin thickness of IMQ-induced mice after 14 days of treatment (mean ± standard error, unit: mm)

[0100] Group Day 1 Day 14 1 0.34±0.00 <![CDATA[0.26±0.00 *** > 2 0.33±0.00 0.60±0.02 3 0.32±0.00 <![CDATA[0.51±0.01 *** > 4 0.32±0.00 <![CDATA[0.53±0.02 *** > 5 0.32±0.00 <![CDATA[0.50±0.01 *** > 6 0.33±0.01 <![CDATA[0.43±0.02 *** > 7 0.32±0.00 <![CDATA[0.32±0.01 *** >

[0101] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs the model group.

[0102] 5.2 Effect of compound (I) on ear thickness of IMQ-induced mice

[0103] Measure the ear thickness daily (at the center of the auricle).

[0104] The experimental results showed that compared with the normal control group, the right ear thickness of the mice increased significantly after being stimulated with 5% imiquimod cream for 14 consecutive days. At the end of the study, the treatments with compound (I) at 3mpk, 10mpk, 30mpk, and 60mpk) and dexamethasone (3mpk) both significantly inhibited the increase in ear thickness (for all 5 groups: P < 0.001 vs the model group), and the inhibition of compound (I) on the increase in ear thickness showed a dose-dependent manner. See Table 4 for details.

[0105] Table 4 Ear thickness of IMQ-induced mice after 14 days of treatment (mean ± standard error, unit: mm)

[0106]

[0107] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs G2 (model group).

[0108] 5.3 Effect of compound (I) on skin PASI score of IMQ-induced mice

[0109] Skin photos were taken daily starting from the first day of the experiment until the end of the experiment.

[0110] Clinical symptoms were evaluated using the PASI scoring criteria, which included three indicators: erythema, scaling, and skin thickness. Scores ranged from 0 to 4, and the total score was obtained by adding the scores of the three indicators. The PASI scoring criteria were as follows: 0, no symptoms; 1, mild; 2, moderate; 3, severe; 4, extremely severe.

[0111] Erythema: 0 - no erythema visible; 1 - light red; 2 - red; 3 - dark red; 4 - extremely dark red.

[0112] Scaling: 0 - no visible scaling on the skin surface; 1 - some lesions covered with scales; 2 - most lesions covered with scales; 3 - almost all lesions covered with scales; 4 - all lesions covered with scales.

[0113] Skin thickness: 0 - smooth skin without wrinkles; 1 - slight wrinkles at the skin edge or rough skin; 2 - slight wrinkles or slight elevation throughout the lesion; 3 - further deepening of the wrinkle degree at the lesion or obvious thickening and elevation of the lesion; 4 - complete wrinkling of the lesion or highly thickened and significantly protruding lesion.

[0114] Dryness and itching were not included in the total score.

[0115] The experimental results showed that after continuous 14 - day stimulation with 5% imiquimod cream, the PASI scores of erythema, scaling, skin thickness, and the total score in the model group of mice were significantly increased. At the end of the study, the treatment with compound (Ⅰ) (3mpk) significantly improved the scaling score (P < 0.001 vs. model group) and the total score (P < 0.05 vs. model group); the treatment with compound (Ⅰ) (10mpk and 30mpk) significantly improved the scaling score (10mpk group: P < 0.01 vs. model group; 30mpk group: P < 0.001 vs. model group), the skin thickness score (both groups: P < 0.001 vs. model group), and the total score (10mpk group: P < 0.01 vs. model group; 30mpk group: P < 0.001 vs. model group); the treatment with compound (Ⅰ) (60mpk) and dexamethasone (3mpk) significantly improved the erythema score, scaling score, skin thickness score, and the total score (both groups: P < 0.001 vs. model group). See Table 5 for details.

[0116] Table 5 Skin PASI scores of IMQ - induced mice after 14 - day treatment (mean ± standard error)

[0117]

[0118] 5.4 Effects of Compound (I) on the Spleen Weight of IMQ-induced Mice

[0119] On the 14th day of the experiment, spleen weights were collected.

[0120] The experimental results showed that treatment with Compound (I) (10 mpk, 30 mpk, and 60 mpk) and dexamethasone (3 mpk) significantly inhibited the increase in spleen weight (for the 10 mpk group of Compound (I): P < 0.05 vs. the model group; for the other three groups: P < 0.001 vs. the model group); treatment with Compound (I) (30 mpk and 60 mpk) and dexamethasone (3 mpk) significantly inhibited the increase in spleen index (for all three groups: P < 0.001 vs. the model group), as shown in Table 6 for details.

[0121] Table 6 Spleen Weight and Spleen Index of IMQ-induced Mice after 14-day Treatment (Spleen Weight / Body Weight %)

[0122] (Mean ± Standard Error)

[0123] Group Spleen weight (mg) Spleen weight / body weight (mg / g) 1 73.11±3.20 4.24±0.19 2 216.20±13.22 13.12±0.77 3 231.44±12.72 14.46±0.71 4 <![CDATA[182.01±9.11 * > 11.71±0.56 5 <![CDATA[146.81±7.71 *** > <![CDATA[9.27±0.47 *** > 6 <![CDATA[125.73±4.63 *** > <![CDATA[8.01±0.28 *** > 7 <![CDATA[93.79±4.64 *** > <![CDATA[6.17±0.29 *** >

[0124] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs. G2 (model group).

[0125] 5.5 Determination of Skin Epidermal Thickness and Pathological Scoring

[0126] After the experiment, all mice were euthanized by excessive inhalation of carbon dioxide, and their skins were collected and divided into four parts. One part was placed in tissue fixative for 24 hours for pathological detection. After dehydration, it was embedded in paraffin to make 4-μm sections. The skin sections were stained with hematoxylin and eosin, and the stratum corneum, epidermis, dermis, and inflammatory cell infiltration could be observed. When measuring the epidermal thickness, the stained skin sections were first scanned with a Leica Aperio CS2 scanner at a magnification of 200, and then the tissue pathological changes were observed and scored.

[0127] The specific pathological scoring criteria are as follows: 2.0 points for the discovery of Munro microabscess in the epidermis; 0.5 points for hyperkeratosis; 1.0 point for parakeratosis; 1.0 point for thinning or disappearance of the granular layer; 1.0 point for acanthosis; for the elongation and undulation of rete ridges, 0.5 points, 1.0 point, and 1.5 points were counted according to mild, moderate, and severe degrees respectively. For the infiltration of mononuclear or multinuclear cells in the dermis, 0.5 points, 1.0 point, and 1.5 points were counted according to mild, moderate, and severe degrees respectively; 0.5 points for papillary elevation; 0.5 points for capillary dilation.

[0128] When measuring the thickness of the epidermal layer, first use a Leica Aperio CS2 scanner to scan the stained skin sections at a magnification of 200, and then open the scanned images with HALO analysis software. Using the "typing" template in the software, define the skin epidermis as the annotation layer. In the annotation layer, divide the epidermis into upper and lower line segments, and then use the "layer thickness" option to calculate the thickness at approximately 200 points in the line segment. The epidermal thickness of each section is represented by the average epidermal thickness.

[0129] The experimental results showed that in the normal control group of Group 1, a complete skin structure was visible under the microscope, the cell morphology was normal, and no obvious abnormal changes were seen. In the skin of the model group of Group 2, Munro microcysts were visible, with hyperkeratosis or parakeratosis, acanthosis, accompanied by obvious vasodilation, and moderate to severe inflammatory cell infiltration. Compared with the normal control group of Group 1, continuous stimulation with 5% imiquimod cream for 14 days significantly increased the pathological score of the skin tissue. Treatment with compound (Ⅰ) (60 mpk) and dexamethasone (3 mpk) significantly improved the pathological score of the 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 of Group 1, the epidermal thickness of the skin in the model group of Group 2 was significantly increased. Treatment with compound (Ⅰ) (30 mpk and 60 mpk) and dexamethasone (3 mpk) significantly reduced the epidermal thickness of the model mice (compound (Ⅰ) group 2: P < 0.05 vs model group; dexamethasone group: P < 0.001 vs model group). See Table 7 for details.

[0130] Table 7 Pathological scores and epidermal thickness of IMQ-induced mice after 14 days of treatment (mean ± standard error)

[0131]

[0132]

[0133] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs G2 (model group).

[0134] 5.6 Detection of inflammatory factors in skin samples

[0135] After the experiment, all mice were euthanized by excessive inhalation of carbon dioxide (CO2), the skin was collected and divided into four parts. Three of them were quickly frozen in liquid nitrogen and stored in a -80 °C refrigerator for the detection of inflammatory factors.

[0136] IL-6 and TNF-α were detected using the ELISA method.

[0137] Tissue specimen: Weigh a certain amount of skin tissue, add a certain amount of PBS (pH=7.4), and homogenize the specimen by hand or with a homogenizer. Centrifuge for 20 minutes (2000-3000 rpm). Carefully collect the supernatant. Aliquot one portion for testing and freeze the rest for later use.

[0138] Steps:

[0139] 1. Adding standard samples: Set up standard sample wells and sample wells, and add 50 μL of standard samples of different concentrations to each standard sample well.

[0140] 2. Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40 μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10 μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0141] 3. Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank well.

[0142] 4. Incubation: Seal the plate with a sealing film and incubate at 37 degrees Celsius for 60 minutes.

[0143] 5. Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0144] 6. Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0145] 7. Color development: First add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake to mix, and incubate at 37 degrees Celsius in the dark for 15 minutes.

[0146] 8. Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color immediately turns yellow).

[0147] 9. Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be carried out within 15 minutes after adding the stop solution.

[0148] 10. Calculation: Use the concentration and OD value of the standard to calculate the linear regression equation of the standard curve y=bx+a, where x is the concentration and y is the OD value. Substitute the OD value of the sample into the equation to calculate the sample concentration, and then multiply it by the dilution factor to get the actual concentration of the sample.

[0149] The experimental results showed that compound (I) (3mpk, 10mpk, 30mpk, and 60mpk) and dexamethasone (3mpk) significantly reduced the content of IL-6 in the skin (compound (I) 3mpk and 60mpk groups: P<0.001 vs model group; compound (I) 10mpk, 30mpk groups and dexamethasone group: P<0.01 vs model group) and the content of TNF-α in the skin (compound (I) 30mpk group: P<0.001 vs model group; compound (I) 10mpk group: P<0.05 vs model group; compound (I) 3mpk, 60mpk groups and dexamethasone group: P<0.01 vs model group). See Table 8 for details.

[0150] Table 8 Content of skin inflammatory factors in IMQ-induced mice after 14 days of treatment (mean ± standard error)

[0151] Group IL-6 (concentration pg / g) TNF-α (concentration pg / g) 1 <![CDATA[544.08±44.29 *** > <![CDATA[906.85±41.88 *** > 2 1000.87±72.20 1937.90±129.72 3 <![CDATA[554.74±88.14 *** > <![CDATA[1191.74±124.81 ** > 4 <![CDATA[641.83±96.28 ** > <![CDATA[1432.02±152.77 * > 5 <![CDATA[638.09±59.63 ** > <![CDATA[1236.41±100.8 *** > 6 <![CDATA[499.72±95.81 *** > <![CDATA[1188.81±159.63 ** > 7 <![CDATA[607.20±76.89 ** > <![CDATA[1172.53±115.72 *** >

[0152] Note: *P<0.05, **P<0.01, ***P<0.001 vs G2 (model group).

[0153] Example 3: Pharmacodynamic study of compound (I) on an OXA-induced atopic dermatitis mouse model

[0154] 1. Test objective

[0155] The objective of this study was to evaluate the pharmacodynamics of compound (I) in an OXA-induced atopic dermatitis model of Balb / c mice. Repeated OXA stimulation of the dorsal skin of mice can produce a long-term inflammatory response. This model is closer to clinical skin inflammation and is a commonly used model for screening and evaluating compounds with anti-inflammatory activity.

[0156] 2. Test drugs

[0157] Test drug: Compound (I)

[0158] Positive control drug: Dexamethasone (Dex)

[0159] Solvent (0.4% Tween80 / 0.5% methylcellulose).

[0160] 3. Test animals

[0161] 70 female Balb / c mice, 8-9 weeks old.

[0162] 4. Test grouping and dosing regimen

[0163] According to the body weight of each group of animals before dosing, they were randomly divided into 7 groups, with 10 animals in each group. See Table 9 for details.

[0164] Table 9 Grouping of Pharmacodynamic Experiments for Atopic Dermatitis

[0165]

[0166] Induction of atopic dermatitis model:

[0167] The mice were randomly divided into 7 groups according to body weight as shown in Table 9 above. On the 7th day, OXA induction was performed. The induction method was to apply 5% OXA (dissolved in a solvent of acetone / olive oil (4 / 1)) to the back of the mice near the neck, applying 10 μL (1.5 cm × 1.5 cm). Mice in the normal control group were applied with 10 μL of the solvent of acetone / olive oil (4 / 1) in the same manner. From the 1st day to the 22nd day, immune stimulation was carried out, and 100 μL of 0.1% OXA (dissolved in a solvent of acetone / olive oil (4 / 1)) was evenly applied to the back of the mice near the neck. It was applied once every two days. Mice in the normal control group were applied with 100 μL of the solvent of acetone / olive oil (4 / 1) in the same manner. On the day of immune stimulation, skin thickness measurement, clinical scoring, and photography were required, and then OXA application was carried out after the above work was completed.

[0168] Drug administration plan:

[0169] The drug administration dose of the compound is shown in Table 9. Compound (Ⅰ) was administered by gavage from the 1st day to the 22nd day, twice a day (PO, bid). The morning dose was administered before skin thickness measurement, clinical scoring, photography, and OXA immune stimulation. The interval between the two doses was 8 hours.

[0170] 5. Test results

[0171] 5.1 Effect of Compound (Ⅰ) on the Body Weight of Mice with OXA-Induced Atopic Dermatitis

[0172] The body weight data was recorded twice a week.

[0173] The experimental results showed that OXA stimulation had no significant effect on the body weight of mice. At the end of the study, Compound (Ⅰ) (3 mpk, 10 mpk, 30 mpk, and 60 mpk) had no significant effect on the body weight of mice; the body weight of mice decreased significantly after dexamethasone treatment. See Table 10 for details.

[0174] Table 10 Body Weight of Mice Induced by OXA after 22 Days of Treatment (Mean ± Standard Error)

[0175]

[0176] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs the model group.

[0177] 5.2 Effect of Compound (I) on the skin thickness of mice with OXA-induced atopic dermatitis The skin thickness (in the modeling area of 1.5 cm × 1.5 cm) was measured using a Mitutoyo digimatic indicator (Mitutoyo digital display gauge, model ID-C, USA) from Day 1 to Day 22, once every two days. If the skin thickness measurement and OXA immune stimulation were on the same day, the skin thickness measurement would be prioritized.

[0178] The experimental results showed that after OXA immune stimulation, the thickness of the dorsal skin (in the modeling area of 1.5 cm × 1.5 cm) of the mice increased significantly. From Day 17, compared with the model group, Compound (I) (30 mpk and 60 mpk) significantly inhibited the increase in the skin thickness of the modeling area (Day 17 and 22 in the 30 mpk group: P < 0.05 vs. the model group; Day 17 and 22 in the 60 mpk group: P < 0.01 vs. the model group). From Day 7, compared with the model group, dexamethasone significantly inhibited the increase in skin thickness (Day 7: P < 0.01 vs. the model group; Days 17 and 22: P < 0.001 vs. the model group). See Table 11 for details.

[0179] Table 11 Skin thickness of OXA-induced mice after 1 - 22 days of treatment (mean ± standard error, unit: mm)

[0180]

[0181]

[0182] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs. the model group.

[0183] 5.3 Effect of Compound (I) on the skin clinical score of mice with OXA-induced atopic dermatitis The skin in the modeling area was clinically scored according to the skin scoring criteria in Table 12 from Day 1 to Day 22, once every two days. If the skin clinical score and OXA immune stimulation were on the same day, the skin clinical score would be prioritized.

[0184] Table 12 Skin scoring criteria

[0185] Standard Score Normal 0 Erythema 1 Edema 2 Desquamation 3 Exudation 4 。

[0186] The experimental results showed that after OXA immune stimulation, the clinical score of the skin in the modeled area of mice increased significantly. From the 19th day, compared with the model group, compound (I) (30 mpk and 60 mpk) significantly inhibited the increase in skin clinical score (both P < 0.05 vs model group). From the 9th day, compared with the model group, dexamethasone significantly inhibited the increase in skin clinical score (day 9: P < 0.05 vs model group; days 19 and 22: P < 0.001 vs model group). See Table 13 for details.

[0187] Table 13 Skin clinical scores of OXA-induced mice after 1 - 22 days of treatment (mean ± standard error)

[0188]

[0189] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

[0190] 5.4 Measurement of skin epidermal thickness and pathological score

[0191] At the end of the study, tissues from the modeled area were collected for hematoxylin - eosin staining for skin pathological scoring and measurement of epidermal thickness.

[0192] After the mice were sacrificed, the skin on the back was removed and placed in 10% neutral formalin for 24 hours. After dehydration, it was paraffin - embedded and made into 4 - micron sections. The skin sections were stained with hematoxylin - eosin, and the infiltration of inflammatory cells and tissue changes in the stratum corneum, epidermal layer, and dermal layer were observed under a microscope for pathological scoring. The scoring criteria were as follows: for inflammatory cell aggregation and edema, it was divided into 1 point for mild, 2 points for moderate, and 3 points for severe; for capillary dilation, it was divided into 1 point for mild, 2 points for moderate, and 3 points for severe.

[0193] The specific scoring criteria for inflammatory cell aggregation were as follows: (1) 1 point for mild: A small amount of <10% of the area occupied by inflammatory cells was visible in the dermal area; (2) 2 points for moderate: The area occupied by inflammatory cells was 10% - 50% of the dermal area of the skin; (3) 3 points for severe: The distribution area of inflammatory cells >= 50% of the dermal area of the skin.

[0194] The specific scoring criteria for skin edema were as follows: (1) 1 point for mild: Occasional cell edema was seen at the junction of the epidermis and dermis, and the length of the aggregated edematous cells was less than 10% of the length of the dermal - epidermal junction line; (2) 2 points for moderate: The length occupied by edematous cells was between 10% - 50% of the length of the dermal - epidermal junction line; (3) 3 points for severe: The length occupied by edematous cells was greater than 50% of the length of the dermal - epidermal junction line.

[0195] The specific scoring criteria for telangiectasia are as follows: (1) Score 1: Mild, with occasional 1 - 3 telangiectasias; (2) Score 2: Moderate, with 3 - 6 telangiectasias; (3) Score 3: Severe, with more than 6 telangiectasias.

[0196] When measuring the thickness of the epidermal layer, first scan the stained skin sections at 200 magnifications with a Leica Aperio CS2 scanner, and then open the scanned images with HALO pathology analysis software. Using the "typing" template in the software, define the skin epidermis as the annotation layer. In the annotation layer, divide the epidermis into upper and lower line segments, and then use the "layer thickness" option to calculate the thickness at approximately 100 points in the line segment. The epidermal thickness of each section is represented by the average epidermal thickness.

[0197] The experimental results showed that there were inflammatory cell aggregation, edema, and telangiectasia in the skin of the modeling area in the model group. According to the pathological scoring criteria mentioned in the experimental method, the pathological score of this group reached 6.8. Compared with the model group, after treatment with compound (I) (10 mpk, 30 mpk, and 60 mpk), the pathological score of the skin in this area could be significantly reduced (P < 0.01 vs. model group); after treatment with dexamethasone, the pathological score was significantly reduced (P < 0.001 vs. model group). OXA stimulation significantly increased the thickness of the epidermal layer in the skin of the modeling area on the back of mice. Compared with the model group, compound (I) (10 mpk, 30 mpk, and 60 mpk) could significantly reduce the epidermal thickness (P < 0.05 vs. model group); after treatment with dexamethasone, the thickness of the epidermal layer in this area was significantly decreased (P < 0.001 vs. model group). See Table 14 for details.

[0198] Table 14 Pathological scores and epidermal thickness (mean ± standard error) of OXA - induced mice after 22 days of treatment

[0199] Group Epidermal thickness (μm) Pathological score Normal control group <![CDATA[4.34 *** ±1.30]]> <![CDATA[0.0 *** ±0.00]]> Model group 86.76±3.55 6.8±0.33 Compound (Ⅰ) 3mpk group 75.79±3.36 6.4±0.34 Compound (Ⅰ) 10mpk group <![CDATA[68.84 * ±4.95]]> <![CDATA[5.1 ** ±0.43]]> Compound (Ⅰ) 30mpk group <![CDATA[75.08 * ±3.57]]> <![CDATA[5.0 ** ±0.42]]> Compound (Ⅰ) 60mpk group <![CDATA[72.90 * ±4.00]]> <![CDATA[5.0 ** ±0.52]]> Dexamethasone 3mpk group <![CDATA[54.53 *** ±2.98]]> <![CDATA[1.1 *** ±0.10]]>

[0200] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs. model group.

[0201] Example 4: Pharmacodynamic test of compound (I) on SLE model mice

[0202] 1. Test drugs

[0203] Tested drug: Compound (I)

[0204] Positive control drug: Prednisone (National Drug Approval No. H33021207).

[0205] 2. Test animals

[0206] 50 female MRL / lpr mice at 4 weeks of age; 10 female C57BL / 6 mice at 4 weeks of age.

[0207] 3. Experimental grouping and dosing regimen

[0208] Female MRL / lpr mice were randomly divided into 5 groups according to the serum anti-ds-DNA antibody concentration. They were the model group: solvent (aqueous solution containing 0.4% Tween 80 and 0.5% methylcellulose) 10 mL / kg BID, positive control group: prednisone 6 mg / kg QD, and compound (Ⅰ) 20, 40, and 60 mg / kg BID groups. The dosing volume was 10 mL / kg for all groups. In addition, female C57BL / 6 mice were used as the normal control group. Compound (Ⅰ) and the model group were gavaged twice a day starting from the 5th week of age, with an 8-hour interval between the two doses; prednisone was gavaged once a day starting from the 5th week of age; the administration lasted for 17 weeks in total.

[0209] Two mice in the model group died at 15 weeks and 16 weeks of dosing respectively, and 8 mice remained at the end of the experiment; one mouse in the compound (Ⅰ) 20 mg / kg BID group died in the late stage of 16 weeks of dosing, and 9 mice remained at the end of the experiment; no mouse death occurred in the other groups.

[0210] 4. Experimental results

[0211] 4.1 Effects of compound (Ⅰ) on the skin of MRL / lpr mice

[0212] 4.1.1 Skin damage conditions and scoring

[0213] The skin damage conditions of the mice's faces, ears, and backs were observed and scored once a week for a total of 17 times.

[0214] Scoring system: 1) Skin redness, swelling, and bleeding; 2) Hair loss and skin dryness; 3) Edema; 4) Epidermal exfoliation / corrosion; 5) Symptoms such as lichenoid sclerosis patches. The score for each item was as follows: normal = 0 points; mild = 1 point; moderate = 2 points; severe = 3 points. The severity of skin damage was determined according to the total score of each evaluated symptom.

[0215] The results showed that compared with the model group, starting from the 14th week of treatment, the high-dose and medium-dose groups of compound (Ⅰ) could effectively inhibit the skin damage degree of systemic lupus erythematosus mice (14 weeks - 16 weeks, *P < 0.05 vs model group; 17 weeks, **P < 0.01 vs model group), and the positive control drug prednisone 6 mg / kg group also effectively inhibited the skin damage degree of systemic lupus erythematosus mice (17 weeks, **P < 0.01 vs model group). See Table 15 for details.

[0216] Table 15 Skin scores of mice after 0 - 17 weeks of treatment

[0217]

[0218] Note: *P<0.05, **P<0.01 vs model group.

[0219] 4.1.2 Skin HE pathological score

[0220] At the end of the experiment, when the mice were dissected, the skin tissues on the back of the mice were taken for HE staining and pathological scoring.

[0221] Skin HE pathological scoring criteria

[0222] Score 0 1 2 3 4 Hyperkeratosis / parakeratosis None Slight Mild Moderate Severe Follicular plug None Slight Mild Moderate Severe Atrophy of epidermis and dermis None Slight Mild Moderate Severe Superficial dermal edema None Slight Mild Moderate Severe Inflammatory cell infiltration None Slight Mild Moderate Severe Capillary dilation and congestion None Slight Mild Moderate Severe Epidermal hyperplasia None Slight Mild Moderate Severe 。

[0223] The results showed that the skin pathological scores in each drug administration group were improved to varying degrees. The high-dose group of compound (Ⅰ) showed statistical significance (**P<0.01 vs model group), and the positive control drug prednisone 6 mg / kg group also showed statistical significance (***P<0.001 vs model group). The experimental results are shown in Table 16 and Figure 9 。

[0224] Table 16 Skin HE pathological scores of MRL / lpr mice after 17 weeks of treatment

[0225]

[0226] Note: **P<0.01, ***P<0.001 vs model group.

[0227] 4.2 Effects of compound (Ⅰ) on lymph nodes of MRL / lpr mice

[0228] 4.2.1 Lymph node scoring

[0229] During the experiment, the lymph nodes of MRL / lpr mice were scored once a week for a total of 17 times.

[0230] The scoring criteria were based on the diameter of the lymph nodes (cm), and the score was 0-6 points:

[0231] 0 point: normal;

[0232] 1 point: small (diameter less than 1 cm at one bilateral point position);

[0233] 2 points: small (diameter less than 1 cm at two bilateral point positions);

[0234] 3 points: small (diameter less than 1 cm at three bilateral point positions);

[0235] 4 points: large (diameter greater than 1 cm at one bilateral point position and less than 1 cm at the other two bilateral point positions);

[0236] 5 points: large (diameter greater than 1 cm at two bilateral points, diameter less than 1 cm at the other bilateral point);

[0237] 6 points: large (diameter greater than 1 cm at three bilateral points).

[0238] From the lymph node scoring data, after 7 weeks of drug treatment, both high and medium doses of compound (Ⅰ) could effectively inhibit the degree of lymph node enlargement in systemic lupus erythematosus mice (***P < 0.001 vs model group). The low-dose group could significantly inhibit the lymph node enlargement in SLE mice from 7 to 11 weeks of drug administration (7 - 9 weeks, ***P < 0.001 vs model group; 11 weeks, *P < 0.05 vs model group), while there was no obvious difference after 12 weeks until the end of the experiment. The positive control drug prednisone 6 mg / kg group also effectively inhibited the degree of lymph node enlargement in systemic lupus erythematosus mice (***P < 0.001 vs model group), as shown in Table 17 for details.

[0239] Table 17 Lymph node scores of mice after 17 - week treatment

[0240]

[0241] Note: *P < 0.05, ***P < 0.001 vs model group.

[0242] 4.2.2 Lymph node weight

[0243] After dissecting the mice at the end of the experiment, the lymph node tissues (submandibular, axillary, and inguinal) were weighed. From the lymph node tissue weighing data, compared with the model group, both high and medium doses of compound (Ⅰ) could effectively inhibit the degree of lymph node enlargement in systemic lupus erythematosus mice (***P < 0.001 vs model group), as shown in Table 18 and Figure 10 .

[0244] Table 18 Lymph node weight (mean ± standard error)

[0245]

[0246] Note: ***P < 0.001 vs model group.

[0247] 4.3 Effects of compound (Ⅰ) on the spleen of MRL / lpr mice

[0248] After dissecting the mice at the end of the experiment, the spleen tissues were weighed. Compared with the model group, all high, medium, and low doses of compound (Ⅰ) could effectively inhibit the degree of spleen enlargement in systemic lupus erythematosus mice (***P < 0.001 vs model group), as shown in Table 19 and Figure 11 .

[0249] Table 19 Spleen weight and spleen weight / body weight (mean ± standard error)

[0250]

[0251] Note: ***P < 0.001 vs model group.

[0252] 4.4 Effects of compound (I) on the kidneys of MRL / lpr mice

[0253] 4.4.1 Urinary protein - area under the curve

[0254] During the experiment, the urinary protein content of mice was detected once a week until week 16. According to the urinary protein content at different times, a urinary protein concentration - time curve was plotted, and the area under each curve was calculated.

[0255] It can be found that the three dose groups of compound (I) had different degrees of improvement in urinary protein, and there were significant statistical differences in the area under the time curve at 16 weeks in the medium and high dose groups of compound (I) (*P < 0.05 vs model group). See Table 20 and Figure 12 .

[0256] Table 20 Urinary protein - area under the curve of mice after 16 weeks of treatment

[0257]

[0258] Note: *P < 0.05, ***P < 0.001 vs model group.

[0259] 4.4.2 Kidney weight

[0260] At the end of the experiment, the mice were dissected and the kidney tissues were weighed. Compared with the model group, the high dose group of compound (I) showed an extremely significant decrease in the total pathological score of both kidneys (***P < 0.001 vs model group), and the medium dose group of compound (I) showed a significant decrease in the total pathological score of both kidneys (**P < 0.01 vs model group). See Table 21 and Figure 13 .

[0261] Table 21 Total kidney weight and kidney weight / body weight of mice after 17 weeks of treatment

[0262]

[0263] Note: **P < 0.01, ***P < 0.001 vs model group.

[0264] 4.4.3 Kidney HE pathological score

[0265] Kidney HE scoring criteria:

[0266] Activity Index (AI) Scoring Criteria

[0267]

[0268] Chronic Index (CI) Scoring Criteria

[0269] Tubulointerstitial Lesions (TIL) Scoring Criteria

[0270] HE staining score results: Compared with the model group, in terms of the Activity Index (AI) score, the high and medium dose groups of compound (I) showed significant decreases in the total renal pathological scores of both kidneys (**P < 0.01 vs model group); in terms of the Chronic Index (CI) score, the high dose group of compound (I) showed a significant decrease in the total renal pathological scores of both kidneys (**P < 0.01 vs model group), and the low dose group of compound (I) showed an obvious decrease in the total renal pathological scores of both kidneys (*P < 0.05 vs model group); in terms of the Tubulointerstitial Lesions (TIL) score, the high dose group of compound (I) showed a significant decrease in the total renal pathological scores of both kidneys (**P < 0.01 vs model group). See Table 22 and Figure 14 。

[0271] Table 22 HE Pathological Scores of Kidneys in Mice after 17 Weeks of Treatment

[0272]

[0273] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

[0274] Through IHC staining of kidney tissues, the positive cell rate and staining intensity of IgG staining were scored.

[0275] Kidney IHC (IgG) Scoring Criteria:

[0276] IHC Scoring Criteria I

[0277] Staining intensity Score value Negative 0 Weak 1 Medium 2 Strong 3 。

[0278] IHC Scoring Criteria II

[0279] Positive cell rate Score value No positive cells 0 Positive cell rate ≤ 10% 1 10% < Positive cell rate ≤ 50% 2 50% < Positive cell rate ≤ 80% 3 Positive cell rate > 80% 4 。

[0280] The final score is the product of the two. A score of 0 is negative (-); a score of 1 - 3 is low expression (+); a score of 4 - 8 is medium expression (++); a score of 9 - 12 is high expression (+++).

[0281] The results showed that compared with the model group, each dose group of compound (Ⅰ) improved IgG deposition in the kidney tissue to varying degrees. Among them, the high-dose group of compound (Ⅰ) showed a significant decrease in the total IHC staining pathological score of both kidneys (*P < 0.05 vs model group). See Table 23 and Figure 15 .

[0282] Table 23 Pathological scores of kidney IHC in mice after 17 weeks of treatment

[0283]

[0284] Note: *P < 0.05, ***P < 0.001 vs model group.

[0285] 4.5 Effect of compound (Ⅰ) on the concentration of serum anti-ds-DNA antibody in MRL / lpr mice

[0286] During the experiment, the concentration of serum anti-ds-DNA antibody in mice was detected once every 4 weeks until 16 weeks.

[0287] The results showed that the lupus erythematosus symptoms of the mice in the model group gradually worsened with the progress of the experiment. From the perspective of the concentration of anti-ds-DNA antibody, after 3 - 4 weeks of treatment, compared with the model group, the medium-dose group of compound (Ⅰ) could effectively reduce the concentration level of anti-ds-DNA antibody (16 weeks, *P < 0.05 vs model group). The positive control drug prednisone 6 mg / kg could also effectively reduce the concentration level of anti-ds-DNA antibody. See Table 24 and Figure 16 .

[0288] Table 24 Concentrations of anti-Ds-DNA antibodies in mice treated for 0 - 16 weeks

[0289]

[0290] Note: *P < 0.05, **P < 0.01, ***P < 0.001 vs model group.

[0291] 4.6 Effect of compound (Ⅰ) on serum cytokines in MRL / lpr mice

[0292] At the end of the experiment, the ELISA method was used to detect the cytokine levels in the sera of mice. The results showed that compared with the model group, the low, medium, and high-dose groups of compound (I) all reduced the concentration of TNF-α, and there were significant differences between the medium and high-dose groups and the model group (***P < 0.001 vs the model group). The low, medium, and high-dose groups of compound (I) also had a certain effect on reducing the IL-6 factor, and the high-dose group had a significant effect on reducing IL-6. See Tables 25 and Figure 17 .

[0293] Table 25 Cytokine Concentrations in Sera at the End of the Experiment

[0294]

[0295] Note: **P < 0.01, ***P < 0.001 vs the model group.

[0296] Example 5 Toxicology Experiment

[0297] Compliant with the NMPA Good Laboratory Practice for Non-clinical Research on Drugs and the FDA GLP regulations (21 CFR Part 58), and in line with the general toxicology studies required by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and relevant NMPA guiding principles, a toxicology study was conducted on compound (I) to provide safety data support for the clinical use of compound (I) by patients. The administration methods and experimental results are as follows:

[0298]

[0299] Experimental Results:

[0300] Compound (I) has no effect on the central nervous system, respiratory system, and cardiovascular system of animals, and is not expected to have side effects on the central nervous system, respiratory system, and cardiovascular system of humans; the NOAEL for single-dose administration in rats is 2000 mg / kg, and the MTD for single-dose administration in Beagle dogs is 500 mg / kg / time (1000 mg / kg / day). The NOAEL for 28-day repeated-dose administration in rats is 10 mg / kg / time (20 mg / kg / day), and the NOAEL for 28-day repeated-dose administration in Beagle dogs is 3 mg / kg / time (6 mg / kg / day). Compound (I) has no genotoxicity. The safety window assessment is shown in the following table. The effective dose in rats is 3 mg / kg / time BID. Calculated based on the exposure amount, Compound (I) has a safety window of 1-fold in Beagle dogs and a safety window of 8 - 13-fold in SD rats; calculated based on the dose, Compound (I) has a safety window of 3-fold in both Beagle dogs and SD rats. The starting dose for the first-in-human trial ramp-up of the proposed Compound (I) is 15 mg. The ratio of the human equivalent dose of the NOAEL dose in the repeated-dose toxicology study of Compound (I) in Beagle dogs to the human starting dose has a safety window of 14-fold, and the ratio of the human equivalent dose of the NOAEL dose in the repeated-dose toxicology study of Compound (I) in SD rats to the human starting dose has a safety window of 16-fold. Therefore, based on the preclinical toxicology study data of Compound (I), the single-dose ramp-up doses for the planned Phase I clinical trial are: 15 mg, 45 mg, 60 mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg, and 360 mg.

[0301] Table 26 Calculation of the safety window of Compound (I)

[0302]

[0303] The results of the Phase I clinical trial showed that when the single-dose ramp-up dose reached 240 mg, no safety issues above Grade 3 occurred.

[0304] In summary, Compound (Ⅰ) can improve the skin lesions of psoriasis and atopic dermatitis mice, inhibit the enlargement of immune organs, and reduce the inflammation level; it can also dose-dependently improve the skin lesions of SLE mice, relieve kidney injury, inhibit the enlargement of immune organs, and inhibit the increase of SLE-related antibodies and cytokines in the serum; and it has a certain safety treatment window, showing good clinical application prospects.

[0305] The full names and Chinese names of the English abbreviations used in this application are as follows:

[0306]

[0307]

Claims

1. Use of a compound (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating psoriasis, wherein the structure of the compound (I) is shown by the following formula:

2. The use according to claim 1, wherein the psoriasis is selected from plaque psoriasis, pustular psoriasis, erythrodermic psoriasis and psoriatic arthritis.

3. The use according to claim 1 or 2, characterized in that, The medicament contains a therapeutically effective amount of the compound (I) or a pharmaceutically acceptable salt thereof.

4. The use according to claim 3, characterized in that, The medicament further contains a pharmaceutically acceptable excipient.

5. The use according to claim 1 or 2, characterized in that, The medicament is made into various clinically acceptable dosage forms.

6. The use according to claim 5, characterized in that, The dosage form is selected from oral dosage forms, injection dosage forms or topical dosage forms.

7. The use according to claim 5, characterized in that, The dosage form is selected from oral dosage forms, injection dosage forms or external use dosage forms.

8. The use according to claim 1 or 2, characterized in that, The medicament is used alone clinically or in combination with other therapeutic components.

9. Use of a compound (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating psoriasis of a subject in combination with other therapeutic components, wherein the structure of the compound (I) is shown by the following formula:

10. The use according to claim 9, characterized in that, The psoriasis is selected from plaque psoriasis, pustular psoriasis, erythrodermic psoriasis and psoriatic arthritis.

11. The use according to claim 3, characterized in that, The therapeutically effective amount is 0.01 - 2000 mg.

12. The use according to claim 3, wherein The therapeutically effective amount is 1 - 500 mg.

13. The use according to claim 3, characterized in that, The therapeutically effective amount is 10 - 400 mg.

14. The use according to claim 3, characterized in that, The therapeutically effective amount is 15 - 360 mg.

15. The use according to claim 3, characterized in that, The therapeutically effective amount is 15 - 250 mg.

16. The use according to claim 3, wherein, The therapeutically effective amount is 15 mg, 45 mg, 60 mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg or 360 mg.

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