Application of compound containing tricyclic heteroaryl

Abstract

The invention relates to application of a compound containing a tricyclic heteroaryl group. Specifically, the invention provides application of a compound (I), an optical isomer thereof or a pharmaceutically acceptable salt thereof in preparation of drugs for treating diseases related to 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 (systemic lupus erythematosus). The compound (I), the optical isomer thereof or the pharmaceutically acceptable salt thereof can improve psoriasis, atopic dermatitis and SLE mouse skin lesion, relieve renal injury, inhibit enlargement of immune organs, reduce the inflammation level and inhibit increase of SLE related antibodies and cell factors in serum, has a certain safe therapeutic window, and has a very good clinical application prospect.

Description

This application is a divisional application of the invention patent application with the application date of March 9, 2022, application number 202280000403.9, and name “Use of a compound containing a tricyclic heteroaryl group”. Technical Field

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

[0002] JAK (Janus kinase) is a non-transmembrane, non-receptor tyrosine kinase consisting of four subtypes: JAK1, JAK2, JAK3, and TYK2 (Tyrosine kinase 2). JAK1, JAK2, and TYK2 are widely present in various tissues and cells. JAK1 mediates inflammatory signaling pathways such as IL-6 and IFN, while JAK2 can independently mediate cytokine signaling pathways such as IL-3, IL-5, and EPO. JAK3 is exclusively present in the bone marrow and lymphatic system, mediating signaling of IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. TYK2 participates in signaling of IFN-α, IL-6, IL-10, and IL-12. JAK inhibitors specifically inhibit the JAK-STAT (Signal transducers and activators of transcription) signaling pathway, blocking the cascade of these cytokines and participating in immune regulation.

[0003] SYK (Spleen tyrosine kinase) is a non-receptor tyrosine kinase present in the cellular matrix. SYK is widely expressed in hematopoietic cells, lymphocytes, fibroblasts, and vascular endothelial cells, with high expression in B lymphocytes. It plays a crucial role in tumors and autoimmune diseases. The Dectin-1 / ITAM pathway is a classic pathway by which antigen-stimulated immune cells induce immune diseases. In normal B cells, when antigen-induced BCR crosslinking leads to phosphorylation of the intracellular immunoreceptor tyrosinase activation motif (ITAM), cytoplasmic SYK is the first target of ITAM recruitment and activation. Activated SYK then activates the transcription factor NF-κB through a CARD9-dependent pathway, leading to the production of a series of inflammatory factors. This pathway also activates Caspase-8, which cleaves the IL-1β proprotein, 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 a key target in autoimmune diseases.

[0004] Psoriasis is an immune-mediated, chronic, relapsing, inflammatory skin disease. Its prevalence varies significantly worldwide, ranging from 0.5% to 3.15% in the United States and 0.75% to 2.9% in Europe. In China, the prevalence was reported to be 0.123% in 1984, 0.47% in a 2008 survey of six cities, and 0.5% in four southwestern provinces in 2017. Approximately 6 million people suffer from psoriasis in China. Psoriasis can occur at any age, regardless of gender. Approximately two-thirds of patients develop the disease before the age of 40, and most experience more severe symptoms in winter and less severe symptoms in summer.

[0005] The etiology and pathogenesis of psoriasis are not yet fully elucidated. Multiple factors, including genetics, immunity, and the environment, may be involved. Through an immune response primarily mediated by T lymphocytes and involving multiple immune cells, it causes excessive proliferation of keratinocytes and inflammation of synovial cells and chondrocytes in joints. The typical clinical manifestations of psoriasis are scaly erythema or plaques, which can be localized or widespread. Psoriasis can be associated with other systemic abnormalities, such as visceral and joint damage. Patients with moderate to severe disease are at increased risk of metabolic syndrome and atherosclerotic cardiovascular disease.

[0006] Based on its clinical manifestations and pathological features, psoriasis can be divided into the following types: 1. Psoriasis vulgaris: This is the most common type and often presents with an acute onset. It typically presents with well-defined, erythematous plaques of varying shapes and sizes, surrounded by an inflammatory halo. These plaques may be slightly infiltrated and thickened. The surface is covered with multiple layers of silvery-white scales. These scales are easily scraped off, leaving a shiny, reddish, translucent film. Small hemorrhages may be observed when the film is broken (Auspitz sign). Lesions are most common on the head, sacrum, and extensor surfaces of the limbs. Some patients experience varying degrees of itching. 2. Pustular psoriasis: This type is classified as generalized or palmoplantar. Generalized pustular psoriasis is characterized by clusters of superficial, sterile pustules on an erythematous surface, some of which may coalesce into pus lakes. It can occur anywhere in the body, most commonly on the flexor surfaces and wrinkles of the limbs. The oral mucosa may also be affected. Acute onset or sudden exacerbation are often accompanied by systemic symptoms such as chills, fever, joint pain, malaise, and an increased white blood cell count. It usually recurs periodically, and psoriasis vulgaris lesions often appear during the remission period. Palmoplantar pustulosis lesions are limited to the hands and feet, occur symmetrically, and are generally in good condition. The condition is stubborn and recurrent. 3. Erythrodermic psoriasis: also known as psoriatic exfoliative dermatitis, is a severe form of psoriasis. It is often caused by the use of highly irritating topical medications, long-term and large-scale use of glucocorticoids, too-rapid dosage reduction, or sudden discontinuation of medication. It manifests as diffuse flushing, swelling, and desquamation of the skin all over the body, accompanied by systemic symptoms such as fever, chills, and malaise, superficial lymphadenopathy, and increased white blood cell count. 4. Arthritis psoriasis: also known as psoriatic arthritis. Psoriasis patients also develop rheumatoid arthritis-like joint damage, which can affect large and small joints throughout the body, but the most characteristic lesions are the interphalangeal joints of the distal digits. The affected joints are red, swollen, and painful, and the skin around the joints is often red and swollen. Joint symptoms often worsen or improve at the same time as skin symptoms. The blood rheumatoid factor is negative.

[0007] Currently, there is no specific treatment for psoriasis. The main treatment options include topical therapy, physical therapy, systemic therapy, and traditional Chinese medicine. Among them, topical treatment includes topical medications and physical therapy. Topical medications mainly include: vitamin D3 analogs, glucocorticoids, anthralin, tretinoin gel and cream, tars, immunosuppressants, etc., as well as other topical medications such as tacrolimus, pimecrolimus, 0.03% camptothecin ointment, 5% salicylic acid ointment, etc. Systemic treatment drugs include methotrexate, tretinoin, cyclosporine, tacrolimus, mycofenac, biological agents such as etanercept, infliximab, and antibiotics. For moderate to severe patients, combined, alternating, or sequential treatments are usually given when single treatment is inadequate. Although existing treatments have significant short-term effects, they can produce many side effects and have a high recurrence rate, and the long-term efficacy is poor. For example: the therapeutic dose of methotrexate is very close to the toxic dose; the main side effect of retinoic acid drugs is teratogenicity; the adverse reactions of cyclosporine A include nephrotoxicity, hypertension, nausea, vomiting, fatigue, muscle tremors and urinary tract irritation symptoms; the adverse reactions of tacrolimus are similar to those of cyclosporine A; the adverse reactions of mycophenolate mofetil include gastrointestinal symptoms, anemia, leukopenia, and an increased risk of infection and tumor induction.

[0008] Atopic dermatitis (AD), also known as atopic dermatitis or genetic allergic dermatitis, is an allergic skin disease characterized by pruritus and a polymorphic rash. It presents differently at different ages and is a chronic, relapsing, inflammatory skin disease. Because patients often coexist with other atopic conditions such as allergic rhinitis and asthma, AD is considered a systemic disease. The prevalence of AD has gradually increased worldwide over the past 30 years, reaching 10% to 20% among children in developed countries. While the prevalence of AD in China has increased later than in Western developed countries, Japan, and South Korea, it has increased rapidly in the past decade. In 1998, the overall prevalence of AD among school-age adolescents (6 to 20 years) was 0.70%. In 2002, the prevalence among preschool children (1 to 7 years) in 10 cities was 2.78%. In 2012, the prevalence among children aged 3 to 6 years in Shanghai reached 8.3%. In 2014, the AD prevalence among children aged 1 to 7 years in 12 cities in my country reached 12.94%, and the AD prevalence among infants aged 1 to 12 months reached 30.48%.

[0009] The onset of AD is closely related to genetic and environmental factors. Although the exact pathogenesis is still unclear, it is currently believed that immune abnormalities, skin barrier dysfunction, skin flora disorders and other factors are important links in the onset of the disease. AD usually first occurs in infancy, and about 50% of patients develop the disease before the age of 1 year. It is a chronic course with a variety of clinical manifestations. The most basic characteristics are dry skin, chronic eczema-like lesions and obvious itching. Some patients may also have other allergic diseases, such as allergic asthma, allergic rhinoconjunctivitis, etc. In addition, due to long-term chronic inflammatory reactions, patients with chronic disease have a significantly increased risk of developing neurological diseases, inflammatory bowel disease, rheumatoid arthritis, cardiovascular disease and lymphoma.

[0010] At present, the treatment of AD includes basic treatment (avoiding contact allergies, etc.), topical drug treatment (topical glucocorticoids and topical calcineurin inhibitors, etc.), systemic treatment (oral antihistamines, immunosuppressants, glucocorticoids, etc.), ultraviolet treatment and antimicrobial treatment, among which topical medications, hormones and immunosuppressants are the main ones. Usually, a step-by-step treatment approach is used to treat mild to moderate patients. Although existing treatment methods can relieve symptoms, they still have certain limitations and many adverse reactions. There are still many unmet needs in terms of rapid onset of effect, itching control, and prevention of recurrence.

[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), profundus lupus erythematosus (LEP), neonatal lupus erythematosus (NLE), and drug-induced lupus erythematosus (DIL).

[0012] Discoid lupus erythematosus primarily affects the skin and is the mildest form of lupus erythematosus. A few may develop mild visceral damage, and a few cases may develop systemic lupus erythematosus. Most patients experience no symptoms, but complete resolution is difficult. Subacute cutaneous lupus erythematosus is a less common clinical condition and represents a special intermediate form. Skin lesions often recur, and the vast majority of patients experience visceral damage, though severe cases are rare. Symptoms include arthralgias, myalgias, and recurrent low-grade fever. A few may also experience nephritis and hematologic changes. Profundus lupus erythematosus, also known as lupus panniculitis or deep lupus erythematosus, is also an intermediate form. It is unstable and can exist in isolation, potentially transforming to either discoid lupus erythematosus or systemic lupus erythematosus, or both. Neonatal lupus erythematosus presents with annular erythema of the skin and congenital heart block. It is self-limited and generally resolves within 4 to 6 months of life, although cardiac lesions often persist. The main symptoms of drug-induced lupus erythematosus are fever, joint pain, muscle pain, butterfly rash on the face, oral ulcers, and may be serositis. The symptoms gradually improve after stopping the medication. Patients with severe conditions can be given drug treatment.

[0013] Systemic lupus erythematosus (SLE) is a systemic autoimmune disease, the most severe of the various forms of lupus erythematosus. Its main clinical features are multisystem and multiorgan involvement, repeated relapses and remissions, and the presence of numerous autoantibodies. Left untreated, it can cause irreversible damage to affected organs and ultimately lead to death. The prevalence of SLE varies significantly across regions. Currently, the global prevalence of SLE ranges from 0 to 241 per 100,000 individuals, while the prevalence in China ranges from 300 to 70 per 100,000 individuals, with a male-to-female prevalence ratio of 1:10 to 12. The specific etiology of SLE remains unclear, but it is multifactorial, primarily involving genetic, infectious, endocrine, and environmental factors. Defects in immune regulatory mechanisms, such as the clearance of apoptotic cells and immune complexes, are also important factors in the development of SLE. Loss of immune tolerance, increased antigen load, excessive T cell help, defective B cell suppression, and a shift from Th1 to Th2 cells lead to B cell hyperactivation and the production of pathogenic autoantibodies. In addition, some drugs, such as methyldopa, phenytoin, penicillamine, quinidine, and propranolol, can directly cause drug-induced lupus and exacerbate lupus erythematosus. The vast majority of SLE patients present with multisystem damage at onset, while a minority develop other forms of lupus erythematosus. Some also have other connective tissue diseases, such as scleroderma, dermatomyositis, and Sjögren's syndrome, resulting in various overlapping syndromes.

[0014] Current drug treatments for SLE are based on glucocorticoids and hydroxychloroquine. However, these drugs can cause side effects, such as infection, impaired liver and kidney function, and metabolic abnormalities, sometimes necessitating dose reduction or discontinuation. Furthermore, there are currently no effective treatments to control disease progression, necessitating the continued development of new drugs. SYK and JAK are upstream of distinct signaling pathways that trigger SLE, making SYK-JAK dual-pathway inhibitors a promising approach for the treatment of SLE. Currently, no JAK-SYK dual-target inhibitors have been approved for marketing. R333, a JAK-SYK dual-target inhibitor being developed for DLE, was terminated on October 24, 2013, following failure in a Phase II clinical trial.

[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] A compound (I) having the following structural formula (I): 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 publication number WO2018108084. It is known to be a highly selective JAK kinase and SYK kinase dual-target inhibitor that can be used to treat cancer.

[0017] After further research, the inventors found that compound (I) regulates cell signal transduction and division and proliferation by inhibiting JAK and SYK kinases, and has excellent effects in treating 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 toxic side effects.

[0018] The present invention therefore provides the use of compound (I), an optical isomer thereof, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating diseases associated with overexpression or abnormal activation of JAK and SYK kinases:

[0019] 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.

[0020] Preferably, in the above use, the disease associated with high expression or abnormal activation of JAK and SYK kinases is an autoimmune disease.

[0021] Preferably, in the above use, the autoimmune disease is an immune-mediated skin disease and an autoimmune connective tissue disease.

[0022] Preferably, in the above-mentioned use, the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is preferably psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis or arthritic psoriasis; 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.

[0023] Furthermore, in the above use, the medicine contains a therapeutically effective amount of compound (I), an optical isomer thereof or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient or carrier.

[0024] The administration method of the drug of the present invention is not particularly limited. Representative administration methods 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 prepared into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, topical dosage forms or external dosage forms.

[0025] Preferably, the drug of the present invention can be used alone or in combination with other therapeutic components in clinical practice. For the convenience of clinical use, the compound (I) of the present invention, its optical isomers or pharmaceutically acceptable salts thereof can be combined with other therapeutic components to prepare a compound drug or combination product.

[0026] The present invention also provides a method for using the drug, namely administering a therapeutically effective amount of the compound (I) of the present invention, its optical isomers or pharmaceutically acceptable salts thereof to a mammal (such as a human) in need of treatment.

[0027] The present invention provides a method for treating diseases associated with overexpression or abnormal activation of JAK and SYK kinases, characterized in that a therapeutically effective amount of the compound (I) of the present invention, an optical isomer thereof, or a pharmaceutically acceptable salt thereof is administered to a mammal (such as a human) in need of treatment.

[0028] 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 associated with 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, wherein the immune-mediated skin diseases are selected from psoriasis or atopic dermatitis, and the psoriasis is preferably psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis or arthritic psoriasis; 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.

[0029] The present invention also provides a drug comprising a therapeutically effective amount of compound (I), an optical isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the drug is used to treat a disease associated with high expression or abnormal activation of JAK and SYK kinases in a subject, which is an autoimmune disease, such as an immune-mediated skin disease and an autoimmune connective tissue disease, wherein the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is preferably psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis, or arthritic psoriasis; the autoimmune connective tissue disease is lupus erythematosus, and the lupus erythematosus is preferably discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, profundal lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and further preferably systemic lupus erythematosus.

[0030] The JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinases, preferably JAK3 and / or TYK2 kinases.

[0031] The present invention also provides a compound drug or combination product, comprising a therapeutically effective amount of compound (I), an optical isomer thereof or a pharmaceutically acceptable salt thereof, and other therapeutic components, characterized in that the compound drug or combination product is used to treat a subject's JAK and SYK kinase overexpression or abnormal activation-related disease, which is an autoimmune disease, 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 psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis or arthritic psoriasis; the autoimmune connective tissue disease is lupus erythematosus, the lupus erythematosus is preferably discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, profundus lupus erythematosus, neonatal lupus erythematosus, drug-induced lupus erythematosus, and further preferably systemic lupus erythematosus.

[0032] The JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinases, preferably JAK3 and / or TYK2 kinases.

[0033] The therapeutically effective amount of the present invention refers to the pharmaceutically effective dosage, i.e., the amount of active compound sufficient to significantly improve the condition without causing serious side effects. For a person weighing 60 kg, the daily dosage is generally 0.01 to 2000 mg, preferably 1 to 500 mg, more preferably 10 to 400 mg, and even more preferably 15 to 360 mg or 15 to 250 mg, such as 15 mg, 45 mg, 60 mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg, or 360 mg. It can be administered as a single dose once daily, divided into multiple doses per day, or administered at intervals. The specific dosage and frequency of administration should take into account factors such as the route of administration and the patient's health status, and can be determined by a skilled physician based on routine skills. The mode of administration is not particularly limited, and representative modes of administration include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular, or subcutaneous), and topical administration. The amount of the active compound is calculated as compound (I).

[0034] In vitro and in vivo studies have shown that (1) Compound (I) of the present invention can significantly inhibit the activity of JAK1, JAK2, JAK3, TYK2 and SYK kinases in vitro, with a stronger inhibitory effect on JAK3 and TYK2. 50 The inhibitory effects on JAK2 and SYK were slightly weaker than those on JAK3 and TYK2, with IC 50 The inhibitory effect on JAK1 is weakest between 3nM and 8nM, IC 50The results show that the compound (I) of the present invention can improve or significantly reduce the skin thickness, ear thickness, spleen weight, spleen index, PASI score (redness, scaling, thickness and total score), epidermal thickness, skin pathology score and IL-6 and TNF-α content in 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; improve the aggregation of inflammatory cells, edema and capillary dilation in the modeling area, and significantly reduce the pathological score and epidermal thickness. (4) The compound (I) of the present invention can dose-dependently improve the skin lesions of MRL / lpr lupus erythematosus model mice, alleviate kidney damage, reduce lymph node and spleen enlargement and inhibit the increase of SLE-related antibodies and cytokines in serum. Among them, the 20mg / kg group of mice was able to effectively inhibit lymph node enlargement in SLE mice after 7 weeks of administration. The results of the experimental endpoint evaluation showed that the 20mg / kg administration could effectively inhibit splenomegaly and lymph node enlargement, and the total kidney pathology score of the chronic index (CI) showed a relatively significant decrease. It could also inhibit the overexpression of serum IL-6 and TNF-α to a certain extent. The 40mg / kg and 60mg / kg dosages were able to comprehensively improve the various symptoms of SLE mice in terms of improving skin lesions (skin lesion score and skin pathology score), alleviating kidney damage (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).

[0035] The above research results show that the compound (I) described in the present invention, its optical isomers or pharmaceutically acceptable salts thereof can improve the skin lesions of psoriasis mice and atopic dermatitis mice and inhibit the enlargement of immune organs; it can also improve the skin lesions of SLE mice in a dose-dependent manner, inhibit the enlargement of immune organs, alleviate kidney function damage, reduce the level of inflammation, and has a certain safe treatment window, and has good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 : Spleen weight at the end point of the IMQ-induced psoriasis model mouse study.

[0037] Figure 2 : Graph showing spleen index (spleen weight / body weight %) at the end of the study in IMQ-induced psoriasis model mice.

[0038] Figure 3 : Graph showing the IL-6 content in mouse skin tissue at the end of the IMQ-induced psoriasis model mouse study.

[0039] Figure 4: TNF-α content in mouse skin tissue at the end point of the IMQ-induced psoriasis model mouse study.

[0040] Figure 5 : Skin pathology scores at the end of the study in IMQ-induced psoriasis model mice.

[0041] Figure 6 : Graph of skin epidermal thickness at the end point of the IMQ-induced psoriasis model mouse study.

[0042] Figure 7 : Skin pathology scores at the end of the study in OXA-induced atopic dermatitis model mice.

[0043] Figure 8 : Graph of skin epidermal thickness at the end point of the OXA-induced atopic dermatitis model mouse study.

[0044] Figure 9 : Diagram of skin pathology score at the end point of the SLE mouse experiment.

[0045] Figure 10 : Diagram of lymph node weight at the end point of the SLE mouse experiment, A: total lymph node weight; B: total lymph node weight / body weight%.

[0046] Figure 11 : Spleen weight of SLE mice at the end of the experiment, A: spleen weight; B: spleen weight / body weight%.

[0047] Figure 12 : Urinary protein-area under the curve of SLE mice after 16 weeks of treatment.

[0048] Figure 13 : Kidney weight at the end point of the SLE mouse experiment, A: total kidney weight; B: kidney weight / body weight %.

[0049] Figure 14 : HE staining scoring diagram of kidney tissue of SLE mice, A: HE score of bilateral kidneys-activity index; B: HE score of bilateral kidneys-chronic index; C: HE score of bilateral kidneys-tubular interstitial damage.

[0050] Figure 15 : IHC (IgG) staining score of kidney tissue in SLE mice.

[0051] Figure 16 : Graph of anti-ds-DNA antibody concentrations in serum of SLE mice.

[0052] Figure 17 : Serum cytokine levels in SLE mice, A: TNF-α concentration; B: IL-6 concentration. DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0054] Source or preparation of experimental materials: 1. Compound (I): produced by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd.

[0055] 2. The positive control drug, reagents, and raw materials used in the experiment were all purchased commercially or prepared independently.

[0056] 3. Preparation of in vivo test substances (Compound (I) and positive control compound): After weighing, the compound (I) was dissolved in an aqueous solution containing 0.4% Tween 80 and 0.5% methylcellulose. The concentrations of compound (I) were 0.3 mg, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL and 6 mg / mL, respectively. The positive control drug was prepared in normal saline at a concentration of 0.3 mg / mL or 0.6 mg / mL and diluted to the required concentration before use.

[0057] 4. Prepare 1 L of solvent (0.4% Tween 80 / 0.5% methylcellulose): Weigh 5.0 g of methylcellulose powder into a clean glass bottle, add 900 mL of sterile water, and stir overnight until fully dissolved. Pipette 4.0 mL of Tween 80, stir thoroughly, and dilute to a final volume of 1000 mL. Store the solution in a refrigerator at 4°C.

[0058] 5. OXA preparation: Acetone / olive oil (4 / 1) solvent preparation: Mix 40 mL of acetone and 10 mL of olive oil and shake for 30 seconds until the mixture is uniform to obtain acetone / olive oil (4 / 1) solvent.

[0059] Preparation of 5% OXA: Weigh 100.00 mg of OXA, dissolve it in 2.00 ml of acetone / olive oil (4 / 1), and shake for 30 seconds.

[0060] 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 immediately before use, every two days.

[0061] Example 1: Activity inhibition test of compound (I) on kinase 1. Kinases: SYK, JAK1, JAK2, JAK3, and TYK2 2. Experimental Methods Protein kinase activity was measured using the Mobility Shift Assay. Compound (I) was dissolved in DMSO and prepared in a concentration gradient in 100% DMSO. After dilution with kinase buffer, 5 μL of compound (I) at 5x the final reaction concentration (10% DMSO) was added to a 384-well plate. After adding 10 μL of 2.5x enzyme solution and incubating at room temperature for 10 minutes, 10 μL of 2.5x substrate solution was added. After incubation at 28°C for 60 minutes, the reaction was terminated by adding 30 μL of stop solution (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA) to the 384-well plate. Conversion data were copied on a Caliper EZReader II and converted to inhibition data: % inhibition = (max - conversion) / (max - min) × 100%. Where "min" is the conversion rate of the control wells without enzyme; "max" is the conversion rate of the control wells with DMSO added. The curve was drawn with the compound concentration and inhibition rate as the horizontal and vertical coordinates, and the IC was calculated using XLFit Excel add-in version 5.4.0.8. 50 Fitting formula: Y=Bottom+(Top-Bottom) / (1+(IC 50 / X)^HillSlope).

[0062] 3. Experimental Results The results of the inhibitory activity test of compound (I) on kinases are shown in the table below: Table 1 Inhibitory activity of compound (I) against kinases Kinase JAK1 JAK2 JAK3 TYK2 SYK <![CDATA[IC 50 ,nM]]> 20.04 3.92 1.43 0.82 7.25

[0063] Example 2: Efficacy of Compound (I) in IMQ-induced psoriasis model in mice 1. Purpose of the study The purpose of this study was to evaluate the efficacy of compound (I) in an IMQ (5% imiquimod cream)-induced mouse psoriasis model. One of the clinical adverse effects of imiquimod is the induction of psoriasis flares. The imiquimod-induced mouse psoriasis model is simple and easy to implement, and its skin phenotype and pathological features demonstrate complex inter-organ interactions and are similar to those of clinical psoriasis.

[0064] 2. Experimental drugs Test drug: Compound (Ⅰ) Positive control drug: dexamethasone (Dex) Vehicle: 0.4% Tween 80 / 0.5% methylcellulose.

[0065] 3. Experimental Animals 70 female Balb / c mice, 5-6 weeks old.

[0066] 4. Trial Grouping and Dosage Regimen The animals were randomly divided into 7 groups according to their body weight the day before the experiment. See Table 2 for details: Table 2 Grouping of psoriasis efficacy experiments Note: “PO” means oral administration; “bid” means twice a day; “qd” means once a day; mpk: mg / kg.

[0067] Modeling: The animals' backs were shaved the day before application, leaving an exposed skin area approximately 2 cm x 3 cm. For IMQ-induced mice, 62.5 mg of 5% imiquimod cream was applied daily to the exposed backs and right ears for 14 days. For normal control mice, petroleum jelly was applied daily to the exposed backs and right ears.

[0068] 5. Test results 5.1 Effect of Compound (I) on IMQ-induced Skin Thickness in Mice Skin thickness was measured daily from the first day of the experiment until the end of the experiment.

[0069] The skin thickness measurement method is as follows: use the thumb and index finger of the left hand to pinch the mouse's back skin together with the subcutaneous tissue in the same direction as the mouse's body; hold a digital micrometer thickness gauge (model BK-3281, manufacturer: Shanghai Newhui) in the right hand and measure the skin fold thickness 1 cm away from the pinched part of the left hand (near the center of the modeling part). The thickness is in mm, and the actual thickness is half of the measured thickness (skin thickness = measured value / 2).

[0070] The experimental results showed that compared with the normal control group, the skin thickness of the model group mice increased significantly after 14 consecutive days of stimulation with 5% imiquimod cream. At the study endpoint, treatment with compound (I) (3 mpk, 10 mpk, 30 mpk, and 60 mpk) and dexamethasone (3 mpk) significantly inhibited the increase in skin thickness (all five groups: P < 0.001 vs. the model group). See Table 3 for details.

[0071] Table 3 Skin thickness of IMQ-induced mice after 14-day treatment (mean ± standard error, unit: mm) 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 *** ]]> Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0072] 5.2 Effect of Compound (I) on IMQ-induced ear thickness in mice Ear thickness was measured daily (center of the pinna).

[0073] The experimental results showed that compared with the normal control group, 14 consecutive days of 5% imiquimod cream stimulation resulted in a significant increase in right ear thickness in mice. At the study endpoint, treatment with Compound (I) (3, 10, 30, and 60 mpk) and dexamethasone (3 mpk) significantly inhibited the increase in ear thickness (all five groups: P < 0.001 vs. the model group). Compound (I) exhibited a dose-dependent inhibitory effect on ear thickness. See Table 4 for details.

[0074] Table 4 Ear thickness of IMQ-induced mice after 14-day treatment (mean ± standard error, unit: mm) Note: *P<0.05, **P<0.01, ***P<0.001 vs G2 (model group).

[0075] 5.3 Effect of compound (I) on IMQ-induced PASI score of mouse skin Skin photos were taken daily from the first day of the experiment until the end of the experiment.

[0076] Clinical symptoms are assessed using the PASI (Personal Indicator for Severity of Skin) scoring system, which uses three indicators: erythema, scaling, and thickness. The scores are calculated on a scale of 0 to 4, and the total score is calculated by adding the three indicators together. The PASI scoring system is as follows: 0, no symptoms; 1, mild; 2, moderate; 3, severe; and 4, very severe.

[0077] Erythema: 0 - no erythema visible; 1 - light red; 2 - red; 3 - deep red; 4 - very deep red.

[0078] Scaly: 0 - no visible scales on the skin surface; 1 - some lesions are covered with scales; 2 - most lesions are covered with scales; 3 - almost all lesions are covered with scales; 4 - all lesions are covered with scales.

[0079] Skin thickness: 0 - smooth skin without wrinkles; 1 - slightly wrinkled skin edges, or rough skin; 2 - slightly wrinkled skin edges, or slightly raised skin edges; 3 - the wrinkles at the skin edges are further deepened, or the skin edges are thickened and raised significantly; 4 - the skin edges are completely wrinkled, or the skin edges are highly thickened and raised significantly.

[0080] Dryness and itching are not included in the total score.

[0081] The experimental results showed that after 14 consecutive days of stimulation with 5% imiquimod cream, the PASI scores of erythema, scaling, skin thickness and total score of the model group mice were significantly increased. At the study endpoint, treatment with compound (I) (3 mpk) significantly improved the scaling score (P < 0.001 vs. model group) and total score (P < 0.05 vs. model group); treatment with compound (I) (10 mpk and 30 mpk) significantly improved the scaling 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 mpk group: P < 0.01 vs. model group; 30 mpk group: P < 0.001 vs. model group); and treatment with compound (I) (60 mpk) and dexamethasone (3 mpk) significantly improved the erythema score, scaling score, skin thickness score, and total score (both groups: P < 0.001 vs. model group). See Table 5 for details.

[0082] Table 5 PASI scores of skin of IMQ-induced mice after 14 days of treatment (mean ± standard error)

[0083] 5.4 Effect of Compound (I) on Spleen Weight of IMQ-Induced Mice On the 14th day of the experiment, spleen weight was collected.

[0084] The experimental results showed that treatment with compound (I) (10mpk, 30mpk and 60mpk) and dexamethasone (3mpk) significantly inhibited the increase in spleen weight (compound (I) 10mpk group: P < 0.05 vs. model group; the other three groups were all: P < 0.001 vs. model group); treatment with compound (I) (30mpk and 60mpk) and dexamethasone (3mpk) significantly inhibited the increase in spleen index (all three groups were: P < 0.001 vs. model group), see Table 6 for details.

[0085] Table 6 Spleen weight and spleen index (spleen weight / body weight %) of IMQ-induced mice after 14 days of treatment (mean ± standard error) 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 *** ]]> Note: *P<0.05, **P<0.01, ***P<0.001 vs G2 (model group).

[0086] 5.5 Skin epidermal thickness measurement and pathological scoring At the end of the experiment, all mice were euthanized by carbon dioxide overdose. Skin was collected and divided into four sections, one of which was fixed in tissue fixative for 24 hours for pathological examination. After dehydration, the sections were embedded in paraffin and prepared into 4-micron sections. The skin sections were stained with hematoxylin and eosin to observe the stratum corneum, epidermis, dermis, and inflammatory cell infiltration. To measure epidermal thickness, the stained skin sections were first scanned at 200x using a Leica Aperio CS2 scanner. Histopathological changes were then observed and scored.

[0087] Pathological scoring criteria are as follows: Munro microabscesses in the epidermis (2.0 points); hyperkeratosis (0.5 points); parakeratosis (1.0 points); thinning or loss of the stratum granulosum (1.0 points); thickening of the stratum spinosum (1.0 points); elongation and undulation of the cutaneous processes (0.5 points, 1.0 points, and 1.5 points, respectively, depending on severity); mononuclear or multinuclear cell infiltration in the dermis (0.5 points, 1.0 points, and 1.5 points, respectively, depending on severity); mastoid suprapubic region (0.5 points); and telangiectasia (0.5 points).

[0088] To measure epidermal thickness, stained skin sections were scanned at 200x magnification using a Leica Aperio CS2 scanner. The scanned images were then opened with HALO analysis software. Using the "Classification" template in the software, the epidermis was defined as an annotation layer. Within this annotation layer, the epidermis was divided into two segments, upper and lower. Using the "Layer Thickness" option, the thickness was calculated at approximately 200 locations within these segments. The average epidermal thickness for each section was used to represent the epidermal thickness.

[0089] The experimental results showed that the normal control group in group 1 had intact skin structure and normal cell morphology under the microscope, with no obvious abnormal changes. The skin of the model group in group 2 showed small Munro cysts, hyperkeratosis or incomplete keratinization, thickening of the stratum spinosum, accompanied by obvious vascular dilation, and moderate to severe inflammatory cell infiltration. Compared with the normal control group in group 1, continuous 14-day stimulation with 5% imiquimod cream significantly increased the pathological score of skin tissue. Treatment with compound (I) (60 mpk) and dexamethasone (3 mpk) significantly improved the skin tissue pathological score of 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 in group 1, the epidermal thickness of the skin in the model group in group 2 was significantly increased. Treatment with compound (I) (30 mpk and 60 mpk) and dexamethasone (3 mpk) significantly reduced the epidermal thickness of model mice (compound (I) 2 group: P < 0.05 vs. model group; dexamethasone group: P < 0.001 vs. model group). See Table 7 for details.

[0090] Table 7 Pathological scores and epidermal thickness of IMQ-induced mice after 14 days of treatment (mean ± standard error) Group Pathological scoring Epidermal thickness (μm) 1 0.00±0.00 25.32±0.93 2 8.25±0.56 138.78±9.69 3 7.75±0.39 136.10±5.96 4 7.45±0.80 131.63±7.15 5 5.55±0.90 <![CDATA[121.75±8.17 * ]]> 6 <![CDATA[5.05±0.88 ** ]]> <![CDATA[113.28±6.95 * ]]> 7 <![CDATA[3.95±0.50 *** ]]> <![CDATA[84.98±7.40 *** ]]> Note: *P<0.05, **P<0.01, ***P<0.001 vs G2 (model group).

[0091] 5.6 Detection of inflammatory factors in skin samples After the experiment, all mice were euthanized by inhaling excessive carbon dioxide (CO2), and their skin was collected and divided into four parts. Three of them were quick-frozen in liquid nitrogen and stored in a -80 degrees Celsius refrigerator for inflammatory factor detection.

[0092] IL-6 and TNF-α were detected by ELISA.

[0093] Tissue specimen: Weigh a certain amount of skin tissue and add a certain amount of PBS (pH 7.4). Homogenize the specimen thoroughly 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 remaining portion for future use.

[0094] Steps: 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.

[0095] 2. Sample Addition: Set up blank wells (blank control wells without sample or enzyme-labeled reagent; all other steps remain the same) and test sample wells. First, add 40 μL of sample diluent to the test sample wells on the enzyme-labeled plate, followed by 10 μL of the test sample (final sample dilution is 5x). Add the sample to the bottom of the plate well, avoiding contact with the well walls, and gently shake to mix.

[0096] 3. Add enzyme: Add 100 μL of enzyme-labeled reagent to each well, except for the blank well.

[0097] 4. Incubation: Seal the plate with sealing film and incubate at 37°C for 60 minutes.

[0098] 5. Liquid preparation: Dilute the 20-fold concentrated washing solution with 20-fold distilled water and set aside.

[0099] 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 it. Repeat this 5 times and pat dry.

[0100] 7. Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and incubate at 37°C in the dark for 15 minutes.

[0101] 8. Termination: Add 50 μL of stop solution to each well to terminate the reaction (the blue color will immediately turn yellow).

[0102] 9. Measurement: Use the blank well as the zero setting and measure the absorbance (OD value) of each well in sequence at a wavelength of 450 nm. The measurement should be performed within 15 minutes after adding the stop solution.

[0103] 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 obtain the actual concentration of the sample.

[0104] The experimental results showed that compound (I) (3 mpk, 10 mpk, 30 mpk, and 60 mpk) and dexamethasone (3 mpk) significantly reduced the content of IL-6 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 groups: P < 0.01 vs. model group) and the content of TNF-α in the skin (compound (I) 30 mpk group: P < 0.001 vs. model group; compound (I) 10 mpk group: P < 0.05 vs. model group; compound (I) 3 mpk, 60 mpk groups and dexamethasone groups: P < 0.01 vs. model group). See Table 8 for details.

[0105] Table 8 IMQ-induced skin inflammatory factor levels in mice treated for 14 days (mean ± standard error) 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 *** ]]> Note: *P<0.05, **P<0.01, ***P<0.001 vs G2 (model group).

[0106] Example 3: Efficacy of Compound (I) on OXA-induced atopic dermatitis model in mice 1. Purpose of the study This study aimed to evaluate the pharmacodynamics of compound (I) in the OXA-induced atopic dermatitis model in Balb / c mice. Repeated OXA stimulation of the dorsal skin of mice can produce a long-term inflammatory response. This model more closely resembles clinical skin inflammation and is commonly used to screen and evaluate compounds with anti-inflammatory activity.

[0107] 2. Experimental drugs Test drug: Compound (Ⅰ) Positive control drug: dexamethasone (Dex) Vehicle (0.4% Tween 80 / 0.5% methylcellulose).

[0108] 3. Experimental Animals 70 female Balb / c mice, 8-9 weeks old.

[0109] 4. Trial Grouping and Dosage Regimen According to the body weight of each group of animals before administration, they were randomly divided into 7 groups, with 10 animals in each group. See Table 9 for details.

[0110] Table 9 Grouping of atopic dermatitis efficacy trials

[0111] Induced atopic dermatitis model: Mice were randomly divided into 7 groups according to body weight, as shown in Table 9 above. OXA induction was performed on the 7th day. The induction method was to apply 5% OXA (dissolved in acetone / olive oil (4 / 1) solvent) to the back of the mouse near the neck, applying 10 μL (1.5 cm × 1.5 cm). The mice in the normal control group were applied with 10 μL of acetone / olive oil (4 / 1) solvent in the same way. From the 1st day to the 22nd day, immune stimulation was performed, and 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. Apply once every two days. The mice in the normal control group were applied with 100 μL of acetone / olive oil (4 / 1) solvent in the same way. On the day of immune stimulation, skin thickness measurement, clinical scoring and photography were required, and OXA application was performed after the above work was completed.

[0112] Dosage regimen: The compound dosages are shown in Table 9. Compound (I) was administered orally twice daily (PO, bid) from Days 1 to 22. Dosing was in the morning, prior to skin thickness measurement, clinical scoring, photography, and OXA immunostimulation. The two doses were separated by 8 hours.

[0113] 5. Test results 5.1 Effect of Compound (I) on Body Weight of Mice with OXA-Induced Atopic Dermatitis Body weight was recorded twice a week.

[0114] The experimental results showed that OXA stimulation had no significant effect on mouse body weight. At the study endpoint, compound (I) (3 mpk, 10 mpk, 30 mpk, and 60 mpk) had no significant effect on mouse body weight; however, dexamethasone treatment significantly decreased mouse body weight. See Table 10 for details.

[0115] Table 10 Body weight of OXA-induced mice after 22 days of treatment (mean ± standard error) Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0116] 5.2 Effect of Compound (I) on Skin Thickness in Mice with OXA-Induced Atopic Dermatitis Skin thickness (1.5 cm × 1.5 cm model area) was measured every two days from day 1 to day 22 using a Mitutoyo digimatic indicator (Model ID-C, USA). If skin thickness measurement and OXA immunostimulation were performed on the same day, skin thickness measurement was performed first.

[0117] The experimental results showed that after OXA immunostimulation, the thickness of the mouse back skin (1.5 cm × 1.5 cm modeling area) increased significantly. From day 17 onwards, compared with the model group, compound (I) (30 mpk and 60 mpk) significantly inhibited the increase in skin thickness in the modeling area (30 mpk group, days 17 and 22: P < 0.05 vs. model group; 60 mpk group, days 17 and 22: P < 0.01 vs. model group). From day 7 onwards, dexamethasone significantly inhibited the increase in skin thickness compared with the model group (day 7: P < 0.01 vs. model group; days 17 and 22: P < 0.001 vs. model group). See Table 11 for details.

[0118] Table 11 Skin thickness of OXA-induced mice treated for 1-22 days (mean ± standard error, unit: mm) Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0119] 5.3 Effect of Compound (I) on Skin Clinical Scoring of Mice with OXA-Induced Atopic Dermatitis The skin of the modeling area was clinically scored every two days from day 1 to day 22 according to the skin scoring criteria in Table 12. If skin clinical scoring and OXA immune stimulation were performed on the same day, skin clinical scoring was performed first.

[0120] Table 12 Skin scoring standards standard Score normal 0 redness 1 edema 2 molt 3 exudate 4

[0121] The experimental results showed that after OXA immunostimulation, the clinical scores of the skin in the modeling area of ​​the mice were significantly increased. From day 19 onwards, compared with the model group, compound (I) (30 mpk and 60 mpk) significantly inhibited the increase in skin clinical scores (both P < 0.05 vs. model group). From day 9 onwards, dexamethasone significantly inhibited the increase in skin clinical scores (day 9: P < 0.05 vs. model group; days 19 and 22: P < 0.001 vs. model group). See Table 13 for details.

[0122] Table 13 Skin clinical scores of OXA-induced mice treated for 1-22 days (mean ± standard error) Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0123] 5.4 Skin epidermal thickness measurement and pathological scoring At the end of the study, tissues from the modeling area were collected and stained with hematoxylin-eosin for skin pathological scoring and measurement of epidermal thickness.

[0124] After mice were sacrificed, dorsal skin was removed and fixed in 10% neutral formalin for 24 hours. After dehydration, the skin was embedded in paraffin and sliced ​​into 4-micron sections. Skin sections were stained with hematoxylin-eosin and microscopically observed for inflammatory cell infiltration and histological changes in the stratum corneum, epidermis, and dermis, and pathological scoring was performed. Scoring criteria included: inflammatory cell accumulation and edema (mild, 1 point; moderate, 2 points; severe, 3 points); and capillary dilation (mild, 2 points; moderate, 3 points).

[0125] The specific scoring criteria for inflammatory cell accumulation are as follows: (1) 1 point mild: a small number of inflammatory cells occupying <10% of the skin dermis area can be seen; (2) 2 points moderate: inflammatory cells occupy an area of ​​10%-50% of the skin dermis area; (3) 3 points severe: inflammatory cells distribute over ≥50% of the skin dermis area.

[0126] The specific scoring criteria for skin edema are as follows: (1) 1 point mild: edema is occasionally seen at the junction of the epidermis and dermis, and the length of the edema cells is less than 10% of the length of the dermis-epidermal junction; (2) 2 points moderate: edema cells occupy a length between 10% and 50% of the length of the dermis-epidermal junction; (3) 3 points severe: edema cells occupy a length greater than 50% of the length of the dermis-epidermal junction.

[0127] The specific scoring criteria for telangiectasia are as follows: (1) 1 point, mild: 1-3 telangiectasias are occasionally seen; (2) 2 points, moderate: 3-6 telangiectasias; (3) 3 points, severe: more than 6 telangiectasias.

[0128] To measure epidermal thickness, stained skin sections were scanned at 200x magnification using a Leica Aperio CS2 scanner. The scanned images were then opened with HALO pathology analysis software. Using the "Classification" template in the software, the epidermis was defined as an annotation layer. Within this annotation layer, the epidermis was divided into two segments, upper and lower. Using the "Layer Thickness" option, the thickness was calculated at approximately 100 locations within these segments. The average epidermal thickness for each section was used to represent the epidermal thickness.

[0129] The experimental results showed that the model group had inflammatory cell aggregation, edema and capillary dilation in the skin of the modeling area. 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, compound (I) (10mpk, 30mpk and 60mpk) treatment can significantly reduce the pathological score of the skin in this area (P < 0.01 vs. model group); dexamethasone treatment significantly reduced the pathological score (P < 0.001 vs. model group). OXA stimulation caused a significant increase in the thickness of the epidermis of the skin in the modeling area on the back of the mice. Compared with the model group, compound (I) (10mpk, 30mpk and 60mpk) can significantly reduce the epidermal thickness (P < 0.05 vs. model group); dexamethasone treatment significantly reduced the thickness of the epidermis in this area (P < 0.001 vs. model group). See Table 14 for details.

[0130] Table 14 Pathological scores and epidermal thickness of OXA-induced mice after 22 days of treatment (mean ± standard error) Group Epidermal thickness (μm) Pathological scoring 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) 10 mpk group <![CDATA[68.84 * ±4.95]]> <![CDATA[5.1 ** ±0.43]]> Compound (I) 30 mpk group <![CDATA[75.08 * ±3.57]]> <![CDATA[5.0 ** ±0.42]]> Compound (I) 60 mpk group <![CDATA[72.90 * ±4.00]]> <![CDATA[5.0 ** ±0.52]]> Dexamethasone 3 mpk group <![CDATA[54.53 *** ±2.98]]> <![CDATA[1.1 *** ±0.10]]> Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0131] Example 4: Efficacy of Compound (I) on SLE Model Mice 1. Trial Drug Test drug: Compound (Ⅰ) Positive control drug: prednisone (National Medicine Approval No. H33021207).

[0132] 2. Experimental Animals 50 female MRL / lpr mice, 4 weeks old; 10 female C57BL / 6 mice, 4 weeks old.

[0133] 3. Trial Grouping and Dosage Regimen Female MRL / lpr mice were randomly divided into five groups based on serum anti-ds-DNA antibody concentrations: a model group (solvent (0.4% Tween 80 and 0.5% methylcellulose) 10 mL / kg daily), a positive control group (prednisone 6 mg / kg daily), and compound (I) 20, 40, and 60 mg / kg daily, all administered in a 10 mL / kg volume. Female C57BL / 6 mice served as a normal control group. Compound (I) and the model group were administered orally twice daily with an 8-hour interval starting at 5 weeks of age. Prednisone was administered once daily starting at 5 weeks of age for a total of 17 weeks.

[0134] Two mice in the model group died at 15 and 16 weeks of drug administration, respectively, leaving 8 mice at the end of the experiment. One mouse in the compound (I) 20 mg / kg BID group died late at 16 weeks of drug administration, leaving 9 mice at the end of the experiment. No mice died in the other groups.

[0135] 4. Test results 4.1 Effects of compound (I) on the skin of MRL / lpr mice 4.1.1 Skin damage condition and scoring The skin lesions on the face, ears and back of the mice were observed and scored once a week for a total of 17 times.

[0136] Scoring system: 1) skin redness, swelling, and bleeding; 2) hair loss and dryness; 3) edema; 4) excoriation / erosion; 5) lichenoid plaques. Each score is as follows: normal = 0; mild = 1; moderate = 2; severe = 3. The severity of skin damage is determined by summing the scores for each symptom assessed.

[0137] The results showed that compared with the model group, starting from 14 weeks of treatment, the high-dose and medium-dose groups of compound (I) were able to effectively inhibit the degree of skin damage in mice with systemic lupus erythematosus (14-16 weeks, *P<0.05 vs. model group; 17 weeks, **P<0.01 vs. model group). The positive control drug prednisone 6 mg / kg group also effectively inhibited the degree of skin damage in mice with systemic lupus erythematosus (17 weeks, **P<0.01 vs. model group). See Table 15 for details.

[0138] Table 15 Skin scores of mice treated for 0-17 weeks Note: *P<0.05, **P<0.01 vs. model group.

[0139] 4.1.2 Skin HE pathology scoring At the end of the experiment, the mice were dissected and the skin tissues on their backs were taken for HE staining and pathological scoring.

[0140] Skin HE pathology scoring standard score 0 1 2 3 4 Hyperkeratosis / parakeratosis none slight Mild Moderate severe Follicular keratin 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

[0141] The results showed that each drug-treated group could improve the skin pathology scores to varying degrees. The high-dose group of compound (I) showed statistical differences (**P<0.01 vs. model group), and the positive control drug prednisone 6 mg / kg group also showed statistical differences (***P<0.001 vs. model group). The experimental results are detailed in Table 16 and Figure 9 .

[0142] Table 16 HE pathological scores of the skin of mice after 17 weeks of treatment Note: **P<0.01, ***P<0.001 vs. model group.

[0143] 4.2 Effects of compound (I) on lymph nodes of MRL / lpr mice 4.2.1 Lymph node scoring Lymph nodes of MRL / lpr mice were scored once a week for a total of 17 times during the experiment.

[0144] The scoring criteria are based on the diameter of the lymph nodes (cm), with a score range of 0-6: 0 points: normal; 1 point: small (less than 1 cm in diameter at one bilateral point); 2 points: small (less than 1 cm in diameter at two bilateral points); 3 points: small (less than 1 cm in diameter at three bilateral points); 4 points: large (the diameter is greater than 1 cm at one bilateral point and less than 1 cm at the other two bilateral points); 5 points: large (the diameter at two side points is greater than 1 cm, and the diameter at another side point is less than 1 cm); 6 points: Large (diameter greater than 1 cm at three bilateral points).

[0145] Lymph node scoring data showed that after 7 weeks of treatment, both high and medium doses of Compound (I) effectively inhibited lymph node enlargement in SLE mice (***P<0.001 vs. model group). The low-dose group significantly inhibited lymph node enlargement in SLE mice from 7 to 11 weeks (7-9 weeks, ***P<0.001 vs. model group; 11 weeks, *P<0.05 vs. model group), but the difference was no longer significant after 12 weeks to the experimental endpoint. The positive control drug prednisone 6 mg / kg group also effectively inhibited lymph node enlargement in SLE mice (***P<0.001 vs. model group). See Table 17 for details.

[0146] Table 17 Lymph node scores of mice after 17 weeks of treatment Note: *P<0.05, ***P<0.001 vs. model group.

[0147] 4.2.2 Lymph node weight At the end of the experiment, the mice were dissected and the (submandibular, axillary and inguinal) lymph node tissues were weighed. The lymph node tissue weighing data showed that compared with the model group, both the high and medium doses of compound (I) could effectively inhibit the degree of lymph node enlargement in mice with systemic lupus erythematosus (***P<0.001vs model group), as shown in Table 18 and Figure 10 .

[0148] Table 18 Lymph node weight (mean ± standard error) Note: ***P<0.001 vs. model group.

[0149] 4.3 Effects of compound (I) on the spleen of MRL / lpr mice At the end of the experiment, the mice were dissected and the spleen tissue was weighed. Compared with the model group, the high, medium and low doses of compound (Ⅰ) were able to effectively inhibit the degree of spleen enlargement in mice with systemic lupus erythematosus (***P<0.001vs model group), as shown in Table 19 and Figure 11 .

[0150] Table 19 Spleen weight and spleen weight / body weight (mean ± standard error) Note: ***P<0.001 vs. model group.

[0151] 4.4 Effects of compound (I) on the kidneys of MRL / lpr mice 4.4.1 Urine protein - area under the curve During the experiment, the urine protein content of the mice was measured once a week until week 16. According to the urine protein content at different times, a urine protein concentration-time curve was drawn, and the area under each curve was calculated.

[0152] It can be found that the three dose groups of compound (I) have different degrees of improvement on urinary protein. The area under the curve of the 16-week time period showed a significant statistical difference in the medium and high dose groups of compound (I) (*P<0.05vs model group). See Table 20 and Figure 12 .

[0153] Table 20 Urine protein-area under the curve of mice treated for 16 weeks Note: *P<0.05, ***P<0.001 vs. model group.

[0154] 4.4.2 Kidney weight 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 .

[0155] Table 21 Total kidney weight and kidney weight / body weight of mice after 17 weeks of treatment Note: **P<0.01, ***P<0.001 vs. model group.

[0156] 4.4.3 Renal HE pathology scoring Renal HE scoring criteria: Activity Index (AI) scoring criteria

[0157] Chronic Index (CI) scoring criteria

[0158] Tubulointerstitial lesions (TIL) scoring criteria

[0159] HE staining scoring results: Compared with the model group, in terms of activity index (AI) score, the compound (I) medium and high dose groups showed a significant decrease in the total pathological score of both kidneys (**P<0.01 vs. model group); in terms of chronic index (CI) score, the compound (I) high dose group showed a significant decrease in the total pathological score of both kidneys (**P<0.01 vs. model group), and the compound (I) low dose group showed a relatively obvious decrease in the total pathological score of both kidneys (*P<0.05 vs. model group); in terms of tubulointerstitial lesion (TIL) score, the compound (I) high dose group showed a significant decrease in the total pathological score of both kidneys (**P<0.01 vs. model group). See Table 22 and Figure 14 .

[0160] Table 22 HE pathological scores of kidneys in mice after 17 weeks of treatment Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0161] The IgG staining positive cell rate and staining intensity were scored by IHC staining of kidney tissue.

[0162] Kidney IHC (IgG) scoring criteria: IHC scoring standard 1 Staining intensity Rating value Negative 0 weak 1 middle 2 powerful 3

[0163] IHC scoring standard 2 Cell positive rate Rating value No positive cells 0 Cell positive rate ≤ 10% 1 10%<Cell positive rate≤50% 2 50%<Cell positive rate≤80% 3 Cell positive rate>80% 4

[0164] The final score is the product of the two, with 0 points indicating negative (-); 1-3 points indicating low expression (+); 4-8 points indicating moderate expression (++); and 9-12 points indicating high expression (+++).

[0165] The results showed that compared with the model group, all dose groups of compound (I) improved IgG deposition in renal tissue to varying degrees, and the high-dose group of compound (I) showed a significant decrease in the total IHC staining pathological score of both kidneys (*P<0.05vs model group). See Table 23 and Figure 15 .

[0166] Table 23 IHC pathological scores of kidneys in mice after 17 weeks of treatment Note: *P<0.05, ***P<0.001 vs. model group.

[0167] 4.5 Effect of compound (I) on serum anti-ds-DNA antibody concentration in MRL / lpr mice The serum anti-ds-DNA antibody concentration of mice was measured every 4 weeks until 16 weeks during the experiment.

[0168] The results showed that the lupus erythematosus symptoms in the model group mice worsened over time. In terms of anti-ds-DNA antibody concentration, after 3-4 weeks of treatment, the medium-dose group of compound (I) was able to effectively reduce the anti-ds-DNA antibody concentration level compared with the model group (16 weeks, *P<0.05 vs. model group). The positive control drug prednisone 6mg / kg was also able to effectively reduce the anti-ds-DNA antibody concentration level. See Table 24 and Figure 16 .

[0169] Table 24 Anti-Ds-DNA antibody concentrations in mice treated for 0-16 weeks Note: *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0170] 4.6 Effects of compound (I) on serum cytokines in MRL / lpr mice At the end of the experiment, the ELISA method was used to detect the level of cytokines in the serum of mice. The results showed that compared with the model group, the three low, medium and high dose groups of compound (I) all reduced the concentration level of TNF-α, and there was a significant difference between the medium and high dose groups and the model group (***P<0.001vs model group). The three 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 Table 25 and Figure 17 .

[0171] Table 25 Cytokine concentrations in serum at the end of the experiment Note: **P<0.01, ***P<0.001 vs. model group.

[0172] Example 5 Toxicology Experiment Toxicology studies were conducted on Compound (I) in compliance with the NMPA's "Good Nonclinical Research Practices" and FDA GLP regulations (21CFR Part 58), and in accordance with the requirements of the International Conference on Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and relevant NMPA guidelines. This provides safety data support for the use of Compound (I) in clinical patients. The administration method and experimental results are as follows:

[0173] Experimental results: Compound (I) has no effects on the central nervous system, respiratory system, or cardiovascular system in animals and is expected to have no side effects on the central nervous system, respiratory system, or cardiovascular system in humans. The NOAEL for a single dose in rats is 2000 mg / kg, the MTD for a single dose in Beagle dogs is 500 mg / kg / time (1000 mg / kg / day), the NOAEL for repeated doses over 28 days in rats is 10 mg / kg / time (20 mg / kg / day), and the NOAEL for repeated doses over 28 days in Beagle dogs is 3 mg / kg / time (6 mg / kg / day). Compound (I) is not genotoxic. The safety window assessment is shown in the table below. The effective dose in rats is 3 mg / kg / time BID. Based on exposure, Compound (I) has a 1-fold safety window in Beagle dogs and an 8-13-fold safety window in SD rats. Based on dose, Compound (I) has a 3-fold safety window in both Beagle dogs and SD rats. The proposed escalating dose for the first-in-human trial of Compound (I) is 15 mg. The NOAEL dose in repeated-dose toxicology studies of Compound (I) in Beagle dogs exhibited a 14-fold safety window compared to the human equivalent dose and the human starting dose, and the NOAEL dose in repeated-dose toxicology studies in SD rats exhibited a 16-fold safety window compared to the human equivalent dose and the human starting dose. Therefore, based on the preclinical toxicology data of Compound (I), the proposed single-dose escalating doses for the Phase I clinical trial are: 15 mg, 45 mg, 60 mg, 90 mg, 135 mg, 180 mg, 240 mg, 300 mg, and 360 mg.

[0174] Table 26 Safety window calculation of compound (I)

[0175] The results of the Phase I clinical trial showed that no safety issues above level 3 occurred when the single-dose escalation dose reached 240 mg.

[0176] In summary, compound (I) can improve skin lesions in mice with psoriasis and atopic dermatitis, inhibit the enlargement of immune organs, and reduce the level of inflammation; it can also dose-dependently improve skin lesions in SLE mice, alleviate renal damage, inhibit the enlargement of immune organs, and inhibit the increase of SLE-related antibodies and cytokines in serum; and it has a certain safe treatment window and has good clinical application prospects.

[0177] The full names and Chinese names of the English abbreviations used in this application are as follows:

Claims

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

2. The use according to claim 1, wherein the lupus erythematosus is selected from discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, profundal lupus erythematosus, neonatal lupus erythematosus and drug-induced lupus erythematosus; preferably, the lupus erythematosus is systemic lupus erythematosus.

3. The use according to any one of claims 1 to 2, characterized in that The medicament contains a therapeutically effective amount of compound (I), an optical isomer thereof or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient or carrier.

4. The use according to claim 3, characterized in that The drug is prepared into various clinically acceptable dosage forms.

5. The use according to claim 4, characterized in that The dosage form is selected from an oral dosage form, an injection dosage form, a local administration dosage form or an external use dosage form.

6. The use according to any one of claims 1 to 2, characterized in that The drug is used clinically alone or in combination with other therapeutic components.

7. Use of compound (I), an optical isomer thereof, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating lupus erythematosus in a subject in combination with other therapeutic components, wherein the structure of compound (I) is shown in the following formula:

8. The use according to claim 7, characterized in that The lupus erythematosus is selected from discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic lupus erythematosus, profundal lupus erythematosus, neonatal lupus erythematosus and drug-induced lupus erythematosus; preferably, the lupus erythematosus is systemic lupus erythematosus.

9. The use according to claim 3, characterized in that The therapeutically effective amount is 0.01-2000 mg, or 1-500 mg, or 10-400 mg, or 15-360 mg, or 15-250 mg.

10. The use according to claim 3, characterized in that 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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