WO2022188796A1 - 一种含有三环杂芳基的化合物的用途 - Google Patents

一种含有三环杂芳基的化合物的用途 Download PDF

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WO2022188796A1
WO2022188796A1 PCT/CN2022/079887 CN2022079887W WO2022188796A1 WO 2022188796 A1 WO2022188796 A1 WO 2022188796A1 CN 2022079887 W CN2022079887 W CN 2022079887W WO 2022188796 A1 WO2022188796 A1 WO 2022188796A1
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Prior art keywords
lupus erythematosus
psoriasis
compound
skin
diseases
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PCT/CN2022/079887
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English (en)
French (fr)
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杨汉煜
张汉承
刘喜宝
蔡聪聪
秦宁
淡墨
吕璐
张丹丹
刘洁茹
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石药集团中奇制药技术（石家庄）有限公司
杭州英创医药科技有限公司
石药集团欧意药业有限公司
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Application filed by 石药集团中奇制药技术（石家庄）有限公司, 杭州英创医药科技有限公司, 石药集团欧意药业有限公司 filed Critical 石药集团中奇制药技术（石家庄）有限公司
Priority to CN202280000403.9A priority Critical patent/CN115697492A/zh
Priority to AU2022234495A priority patent/AU2022234495A1/en
Priority to US18/280,868 priority patent/US20240165123A1/en
Priority to EP22766319.2A priority patent/EP4306172A1/en
Priority to CA3224094A priority patent/CA3224094A1/en
Priority to JP2023555473A priority patent/JP2024509309A/ja
Publication of WO2022188796A1 publication Critical patent/WO2022188796A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the invention belongs to the field of medicine, in particular to the application of a compound containing a tricyclic heteroaryl group in the preparation of a medicine for treating diseases related to the high expression or abnormal activation of the kinases JAK and SYK.
  • JAK (Janus kinase), namely Janus kinase, is a class of non-transmembrane non-receptor tyrosine kinases, containing 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 Only present in the bone marrow and lymphatic system, mediates signaling of IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, TYK2 is involved in IFN- ⁇ , IL-6, IL-10 and IL-12 signaling. JAK inhibitors specifically inhibit the JAK-STAT (Signal transducers and activators of transcription) signaling pathway, block the cascade amplification of the above cytokines, and participate in immune regulation and other processes.
  • JAK-STAT Signal transducers and activators of transcription
  • SYK Stpleen tyrosine kinase
  • the spleen tyrosine kinase is a non-receptor tyrosine kinase that exists in the cell matrix.
  • SYK is widely expressed in hematopoietic cells, lymphocytes, fibroblasts, and vascular endothelial cells. It is 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.
  • SYK in the cytoplasm is the first recruited and activated target of ITAM, and activated The SYK then activates the transcription factor NF- ⁇ B through a CARD9-dependent pathway to produce a series of inflammatory factors.
  • this pathway can also activate Caspase-8, which cleaves IL-1 ⁇ precursor and promotes the maturation of immature IL-1 ⁇ .
  • the CARD9-independent pathway NLRP3 signaling also plays a role in immature IL-1 ⁇ maturation. Therefore, SYK is also an important target in autoimmune diseases.
  • Psoriasis is an immune-mediated chronic, relapsing, inflammatory skin disease.
  • the prevalence varies significantly around the world, ranging from 0.5% to 3.15% in the United States, 0.75% to 2.9% in Europe, 0.123% in China in 1984, and 0.123% in China in 2008.
  • the prevalence rate was 0.47%, and the prevalence rate in the four southwestern provinces in 2017 was 0.5%, and there were more than 6 million psoriasis patients in China.
  • Psoriasis can occur at any age, and there is no gender difference. About 2/3 of the patients develop the disease before the age of 40, and most patients are severe in winter and mild in summer.
  • psoriasis The etiology and pathogenesis of psoriasis have not been fully elucidated, and may involve a variety of factors such as genetics, immunity, and the environment. Through the immune response mainly mediated by T lymphocytes and the participation of various immune cells, excessive proliferation of keratinocytes is caused. , Inflammation of joint synovial cells and chondrocytes. The typical clinical presentation of psoriasis is scaly erythema or plaques, localized or widespread. Psoriasis can be combined with other system abnormalities, such as with visceral and joint damage. Patients with moderate to severe disease are at increased risk of developing metabolic syndrome and atherosclerotic cardiovascular disease.
  • Psoriasis vulgaris it is the most common type, with multiple acute onset.
  • the typical manifestation is a well-defined erythema of various shapes and sizes surrounded by an inflammatory flush. Slight infiltration thickening.
  • the surface is covered with multiple layers of silvery white scales. The scales are easy to scrape off, and the translucent film is reddish and shiny after scraping, and small hemorrhages can be seen when the film is scratched (Auspitz sign). Skin lesions occur on the head, sacrum, and extensor surfaces of the extremities. Some patients felt itching to varying degrees.
  • pustular psoriasis divided into generalized and palmoplantar type.
  • Generalized pustular psoriasis is a cluster of superficial sterile pustules on the erythema, some of which may coalesce into a lake of pus. The disease can occur all over the body. The flexors and folds of the limbs are more common, and the oral mucosa can be involved at the same time. Acute onset or sudden exacerbation is often accompanied by systemic symptoms such as chills, fever, joint pain, general malaise, and increased white blood cell count. Most of the episodes are cyclical, and psoriasis vulgaris skin lesions often appear during the remission period.
  • erythrodermic psoriasis also known as psoriatic exfoliative dermatitis
  • erythrodermic psoriasis also known as psoriatic exfoliative dermatitis
  • glucocorticoids rapid dose reduction or sudden drug withdrawal. It is manifested as diffuse flushing, swelling and desquamation of the whole body skin, accompanied by systemic symptoms such as fever, chills, malaise, swollen superficial lymph nodes, and increased white blood cell count.
  • joint disease psoriasis also known as psoriatic arthritis.
  • Rheumatoid arthritis-like joint damage occurs in patients with psoriasis at the same time, which can involve large and small joints of the whole body, but the most characteristic lesions of the intersegmental joints of the terminal fingers (toes).
  • the affected joint is red, swollen and painful, and the skin around the joint is often red and swollen. Joint symptoms often increase or decrease at the same time as skin symptoms. Blood rheumatoid factor negative.
  • Topical drugs are mainly: vitamin D3 analogs, glucocorticoids, anthralin, tretinoin gels and creams, tars, immunosuppressants, etc., as well as other external drugs such as tacrolimus, pimecrolimus, 0.03 % camptothecin ointment, 5% salicylic acid ointment, etc.
  • Systemic drugs include methotrexate, retinoids, cyclosporine, tacrolimus, mycophenolate mofetil, biologics such as etanercept, infliximab, and antibiotics.
  • the treatment dose of methotrexate is very close to the poisoning dose; the main side effect of tretinoin drugs is teratogenicity; Adverse reactions of cyclosporine A include nephrotoxicity, hypertension, nausea, vomiting, fatigue, muscle tremor and urinary tract irritation; adverse reactions of tacrolimus are similar to those of cyclosporine A; adverse reactions of mycophenolate mofetil include gastrointestinal tract Symptoms, anemia, leukopenia, increased risk of infection and tumor induction, etc.
  • Atopic dermatitis also known as atopic dermatitis and atopic dermatitis, is an allergic skin disease characterized by skin itching and polymorphic rash. Clinical manifestations, is a chronic, relapsing, inflammatory skin disease. Because patients often have other atopic diseases such as allergic rhinitis and asthma, it is considered to be a systemic disease.
  • AD Atopic dermatitis
  • the global prevalence of AD has gradually increased.
  • the prevalence of AD in children in developed countries has reached 10% to 20%.
  • the increase in the prevalence of AD in China is later than that in western developed countries, Japan and South Korea, but it has grown rapidly in the past 10 years. .
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • AD Alzheimer's disease
  • topical drug therapy topical glucocorticoids and topical calcineurin inhibitors, etc.
  • systemic therapy oral antihistamines, immunosuppressants, glucocorticoids, etc.
  • ultraviolet therapy antimicrobial therapy, among which external drugs, hormones and immunosuppressants are the main ones.
  • Mild-to-moderate patients are usually treated with a step-by-step approach.
  • the existing treatment methods can relieve symptoms, there are still some limitations and many adverse reactions. There are still many unmet needs for rapid onset of action, pruritus control, and prevention of recurrence.
  • Lupus erythematosus is a typical autoimmune connective tissue disease, which can be divided into discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), systemic lupus erythematosus (SLE), deep Subtypes of lupus erythematosus (LEP), neonatal lupus erythematosus (NLE), and drug-induced lupus erythematosus (DIL).
  • DLE discoid lupus erythematosus
  • SCLE subacute cutaneous lupus erythematosus
  • SLE systemic lupus erythematosus
  • LEP deep Subtypes of lupus erythematosus
  • NLE neonatal lupus erythematosus
  • DIL drug-induced lupus erythematosus
  • Discoid lupus erythematosus which mainly affects the skin, is the mildest type of lupus erythematosus. A few may have mild visceral damage, and a few cases can be transformed into systemic lupus erythematosus. Most patients have no symptoms, but it is difficult to completely subside. Subacute cutaneous lupus erythematosus, rare in clinical practice, is a special intermediate type. Skin lesions often recur repeatedly. Most patients have visceral damage, but there are very few severe cases. The main symptoms are joint pain and muscle pain. , repeated low fever, a few have nephritis, blood system changes.
  • Profound lupus erythematosus also known as lupus panniculitis, deep lupus erythematosus, is also an intermediate type of lupus erythematosus, unstable in nature, can exist alone, and later can be transformed into discoid lupus erythematosus or systemic erythematosus lupus, or both.
  • Neonatal lupus erythematosus manifested as annular erythematosus on the skin and congenital heart block, is self-limited and usually resolves spontaneously within 4 to 6 months after birth, and heart disease often persists.
  • Drug-induced lupus erythematosus is mainly manifested as fever, arthralgia, muscle pain, facial butterfly erythema, oral ulcers, and possibly serositis, which gradually improves after drug withdrawal.
  • SLE Systemic lupus erythematosus
  • SLE Systemic lupus erythematosus
  • a large number of autoantibodies are the main clinical feature. If not treated in time, it will cause irreversible damage to the involved organs and eventually lead to the death of the patient.
  • the prevalence of SLE varies greatly across regions. Currently, the global prevalence of SLE is 0-241/100,000, and the prevalence of SLE in mainland China is 30-70/100,000, with a male-female prevalence ratio of 1:10-12.
  • SLE SLE specific pathogenesis of SLE has not been fully clarified, and it is caused by the joint participation of multiple factors, mainly related to multiple factors such as heredity, infection, endocrine and environment.
  • some drugs such as methyldopa, phenytoin, penicillamine, quinidine, propranolol, etc.
  • the current drug treatment for SLE is based on glucocorticoids and hydroxychloroquine.
  • the treatment with the above-mentioned drugs will produce corresponding side effects, such as infection, impaired liver and kidney function, and abnormal metabolism, and sometimes have to reduce or stop the drug, and there is still no good drug to control the development of the disease, and it still needs to continue to develop new drugs.
  • SYK and JAK are upstream of two different signaling pathways that induce SLE, so SYK-JAK dual-channel inhibitors are expected to be an effective way to treat SLE.
  • the JAK-SYK dual-target inhibitor product R333 with DLE as the development indication has also been terminated on October 24, 2013 due to a phase II clinical failure.
  • autoimmune diseases especially immune-mediated skin diseases and autoimmune connective tissue diseases such as psoriasis, atopic dermatitis or lupus erythematosus.
  • compound (I) plays a regulatory role in the signal transduction, division and proliferation of cells by inhibiting JAK and SYK kinases, and has superior effects in the treatment of autoimmune diseases, especially in immune-mediated It shows satisfactory activity and low toxicity in the treatment of skin diseases and autoimmune connective tissue diseases such as psoriasis, atopic dermatitis or lupus erythematosus.
  • the present invention provides the use of compound (I), its optical isomer or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of diseases related to the high expression or abnormal activation of JAK and SYK kinases:
  • the JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinase, preferably JAK3 and/or TYK2 kinase.
  • the disease related to the high expression or abnormal activation of JAK and SYK kinases is an autoimmune disease.
  • the autoimmune disease is immune-mediated skin disease and autoimmune connective tissue disease.
  • the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is preferably psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis Psoriasis or arthropathic 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, profound lupus erythematosus, Neonatal lupus erythematosus, drug-induced lupus erythematosus, and more preferably systemic lupus erythematosus.
  • the medicament contains a therapeutically effective amount of Compound (I), its optical isomer or a pharmaceutically acceptable salt thereof, and optionally, a pharmaceutically acceptable excipient or carrier.
  • the mode of administration of the medicament of the present invention 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 medicament of the present invention can be prepared into various clinically acceptable dosage forms, including oral dosage forms, injection dosage forms, topical administration dosage forms or external dosage forms and the like.
  • the medicament of the present invention can be used clinically alone or in combination with other therapeutic components.
  • the compound (I) of the present invention, its optical isomer or its pharmaceutically acceptable salt can be combined with other therapeutic components to prepare a compound medicine or a combined product.
  • the present invention also provides a method of using the medicament by administering a therapeutically effective amount of the compound (I) of the present invention, its optical isomer or a pharmaceutically acceptable salt thereof to a mammal (eg, human) in need of treatment.
  • a mammal eg, human
  • the present invention provides a method for treating diseases related to high expression or abnormal activation of JAK and SYK kinases, characterized in that a therapeutically effective amount of Compound (I) of the present invention, an optical isomer or a pharmaceutically acceptable salt thereof is added.
  • Administration to a mammal eg, a human in need of treatment.
  • the JAK kinase described in the present invention is one or more of JAK1, JAK2, JAK3 or TYK2 kinase, preferably JAK3 and/or TYK2 kinase.
  • the diseases related to the high expression or abnormal activation of JAK and SYK kinases in the present invention are autoimmune diseases, such as immune-mediated skin diseases and autoimmune connective tissue diseases, and 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 erythema Lupus, the lupus erythematosus is preferably discoid lupus erythematosus, subacute cutaneous lupus erythematosus, systemic
  • the present invention also provides a medicament comprising a therapeutically effective amount of Compound (I), an optical isomer or a pharmaceutically acceptable salt thereof, characterized in that the medicament is used for the treatment of JAK and SYK kinases in a subject
  • autoimmune diseases such as immune-mediated skin diseases and autoimmune connective tissue diseases
  • said immune-mediated skin diseases are selected from psoriasis or atopic dermatitis, so
  • 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 erythemato
  • Said JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinase, preferably JAK3 and/or TYK2 kinase.
  • the present invention also provides a compound medicine or combination product comprising a therapeutically effective amount of compound (I), its optical isomer or a pharmaceutically acceptable salt thereof, and other therapeutic components, which are It is characterized in that the compound medicine or combination product is used to treat diseases related to high expression or abnormal activation of JAK and SYK kinases in subjects, which are autoimmune diseases, such as immune-mediated skin diseases and autoimmune connective tissue diseases , the immune-mediated skin disease is selected from psoriasis or atopic dermatitis, and the psoriasis is preferably psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis or arthropathy Psoriasis; the autoimmune connective tissue disease is lupus erythematosus, and the lupus erythematosus is preferably discoid lupus erythematosus, subacute cutaneous lupus erythemat
  • Said JAK kinase is one or more of JAK1, JAK2, JAK3 or TYK2 kinase, preferably JAK3 and/or TYK2 kinase.
  • the therapeutically effective dose in the present invention refers to the pharmaceutically effective dose, that is, the amount of the active compound is sufficient to significantly improve the condition without causing serious side effects.
  • the daily dosage is usually 0.01-2000mg, preferably 1-500mg, more preferably 10-400mg, even more preferably 15-360mg or 15-250mg, such as: 15mg, 45mg, 60mg, 90mg , 135mg, 180mg, 240mg, 300mg, 360mg. It may be administered in a single dose once daily, divided into multiple doses per day, or at intervals.
  • the specific dose and frequency of administration should take into account factors such as the route of administration, the patient's health status, etc., which can be determined by a skilled physician according to 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.
  • the amount of the active compound is based on compound (I).
  • the compound (I) of the present invention can significantly inhibit the kinase activities of JAK1, JAK2, JAK3, TYK2 and SYK in vitro, and has a strong inhibitory effect on JAK3 and TYK2, with IC 50 of 1.43 respectively nM and 0.82nM, the inhibitory effect on JAK2 and SYK is slightly weaker than that of JAK3 and TYK2, the IC50 is between 3nM and 8nM, and the inhibitory effect on JAK1 is the weakest, with an IC50 of 20.04nM.
  • 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) of IMQ-induced psoriasis model mice , mouse skin epidermal thickness, skin pathological score, and the content of IL-6 and TNF- ⁇ in skin tissue.
  • 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 accumulation of inflammatory cells, edema and telangiectasia in the modeled area.
  • Compound (I) of the present invention can dose-dependently improve skin lesions in MRL/lpr lupus erythematosus model mice, alleviate kidney damage, reduce lymph node and spleen enlargement, and inhibit serum Increased SLE-related antibodies and cytokines in Among them, the 20 mg/kg group of mice can effectively inhibit the lymph node enlargement in SLE mice after 7 weeks of administration.
  • the evaluation results of the end-point indicators of the test show that the 20 mg/kg administration can effectively inhibit the splenomegaly and lymph node enlargement, and in the chronic index ( The total pathological score of both kidneys in CI) was significantly decreased, and the overexpression of serum IL-6 and TNF- ⁇ could also be inhibited to a certain extent.
  • the doses of 40mg/kg and 60mg/kg can improve skin lesions (skin damage score and skin pathology score), alleviate kidney damage (reduce urine protein in mice, reduce renal immune complex deposition), inhibit immune organ enlargement ( The improvement of lymph node pathological score and inhibition of spleen and lymph node enlargement comprehensively improved various symptoms of SLE mice.
  • the compound (I) of the present invention can improve the skin lesions of psoriasis mice and atopic dermatitis mice, and inhibit the proliferation of immune organs. It can also improve the skin lesions of SLE mice in a dose-dependent manner, inhibit the enlargement of immune organs, relieve kidney function damage, and reduce the level of inflammation. It has a certain safety treatment window and has a good clinical application prospect.
  • Figure 1 Plot of spleen weights at the end of the study in IMQ-induced psoriasis model mice.
  • Figure 2 Plot of spleen index (spleen weight/% body weight) at the end of the study in IMQ-induced psoriasis model mice.
  • Figure 3 The content of IL-6 in mouse skin tissue at the end of the study in IMQ-induced psoriasis model mice.
  • Figure 4 The content of TNF- ⁇ in mouse skin tissue at the end of the study in IMQ-induced psoriasis model mice.
  • Figure 5 Graph of skin pathology scores at the end of the study in IMQ-induced psoriasis model mice.
  • Figure 6 Plot of skin epidermal thickness at the end of the study in IMQ-induced psoriasis model mice.
  • Figure 7 Graph of skin pathology score at the end of the study in OXA-induced atopic dermatitis model mice.
  • Figure 8 Plot of skin epidermal thickness at the end of the study in OXA-induced atopic dermatitis model mice.
  • Figure 9 Graph of the skin pathology score at the end of the SLE mouse test.
  • Figure 10 End-point lymph node weight graph in SLE mice, A: total lymph node weight; B: total lymph node weight/body weight %.
  • Figure 11 Graph of the spleen weight at the end point of the SLE mouse experiment, A: spleen weight; B: spleen weight/body weight %.
  • Figure 12 Urinary protein-area under the curve in SLE mice treated for 16 weeks.
  • Figure 13 Figure 13: Endpoint kidney weight chart in SLE mice, A: total kidney weight; B: kidney weight/body weight %.
  • Figure 14 SLE mouse kidney tissue HE staining score chart, A: bilateral kidney HE score - activity index; B: bilateral kidney HE score - chronic index; C: bilateral kidney HE score - tubulointerstitial injury.
  • FIG. 15 Score chart of IHC (IgG) staining in kidney tissue of SLE mice.
  • FIG. 16 Graph of serum anti-ds-DNA antibody concentrations in SLE mice.
  • Figure 17 Graph of serum cytokine levels in SLE mice, A: TNF- ⁇ concentration; B: IL-6 concentration.
  • Compound (I) self-made by CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd.
  • the positive control drug, reagents and raw materials used in the experiment were purchased commercially or prepared by themselves.
  • test substance compound (I) and positive control compound
  • Acetone/olive oil (4/1) solvent configuration 40 mL of acetone and 10 mL of olive oil are mixed and shaken for 30 seconds until the mixture is uniform to obtain an acetone/olive oil (4/1) solvent.
  • OXA preparation Weigh 100.00 mg of OXA, dissolve in 2.00 ml of acetone/olive oil (4/1), and shake for 30 seconds.
  • OXA preparation Weigh 15.69 mg of OXA, dissolve in 15.69 mL of acetone/olive oil (4/1), and shake for 30 seconds. Use now, with every two days.
  • Example 1 Activity inhibition test of compound (I) on kinases
  • Protein kinase activity was determined using the Mobility Shift Assay.
  • Compound (I) was dissolved in DMSO, and then prepared in a concentration gradient in 100% DMSO. Dilute with kinase buffer and add 5 ⁇ L of compound (I) (10% DMSO) at a 5-fold reaction final concentration in a 384-well plate. After adding 10 ⁇ L of 2.5 times enzyme solution, incubate at room temperature for 10 minutes, and then add 10 ⁇ L of 2.5 times substrate solution.
  • Example 2 Pharmacodynamic test of compound (I) on IMQ-induced mouse psoriasis model
  • the purpose of this test is to evaluate the efficacy of compound (I) in IMQ (5% imiquimod cream)-induced mouse psoriasis model.
  • IMQ 5% imiquimod cream
  • One of the clinical adverse reactions of imiquimod is to induce the onset of psoriasis.
  • the use of imiquimod-induced mouse psoriasis model is simple and easy to implement, and the skin phenotype and pathological characteristics have complex interactions between tissues and Similar advantages to clinical psoriasis.
  • Test drug Compound (I)
  • Vehicle 0.4% Tween 80/0.5% methylcellulose.
  • mice were randomly divided into 7 groups according to their body weight the day before the experiment, as shown in Table 2:
  • PO oral administration
  • bid means twice a day
  • qd means once a day
  • mpk mg/kg.
  • mice Animals were shaved on their backs one day before drug application to form an exposed skin area of about 2 cm x 3 cm in size. Mice were induced with IMQ, and 62.5 mg of 5% imiquimod cream was applied to the bare back and right ear of the mice every day for 14 days. For the mice in the normal control group, petrolatum was smeared on the bare back and right ear of the mice every day.
  • Ear thickness was measured daily (at the center of the pinna).
  • PASI scoring standard Clinical symptoms were evaluated by PASI scoring standard, which was evaluated from 3 indicators: erythema, scaling, and skin thickness. The scores were scored on a scale of 0 to 4, and the three scores were added to obtain the total score.
  • the PASI scoring criteria are as follows: 0, asymptomatic; 1, mild; 2, moderate; 3, severe; 4, very severe.
  • Erythema 0 - no erythema is visible; 1 - pale red; 2 - red; 3 - dark red; 4 - very deep red.
  • Scales 0 - no visible scales on the skin surface; 1 - some lesions covered with scales; 2 - most lesions covered with scales; 3 - almost all lesions covered by scales; 4 - all lesions covered by scales Scaly covered.
  • Skin thickness 0-smooth skin without wrinkles; 1-slightly wrinkled at the edge of the skin, or rough skin; 2-slightly wrinkled or slightly raised at all the skin lesions; The uplift is obvious; 4- The skin lesions are completely folded, or the skin lesions are highly hypertrophic and obviously raised.
  • mice were euthanized by excessive carbon dioxide inhalation, and the skin was collected and divided into four parts, one of which was placed in tissue fixative for 24 hours for pathological detection. After dehydration, they were embedded in paraffin and made into 4-micron sections. The skin sections were stained with hematoxylin and eosin to observe the infiltration of stratum corneum, epidermis, dermis and inflammatory cells. When measuring the thickness of the epidermis, the dyed skin sections were scanned with a Leica Aperio CS2 scanner at a magnification of 200, and then the histopathological changes were observed and scored.
  • pathological scoring The specific criteria for pathological scoring are as follows: Munro microabscess found in the epidermis 2.0 points; hyperkeratosis 0.5 points; parakeratosis 1.0 points; granular layer thinning or disappearance 1.0 points; acanthosis 1.0 points; Sudden extension and ups and downs were rated as 0.5, 1.0, and 1.5 points according to the severity of mild, moderate and severe. The infiltration of mononuclear or multinucleated cells in the dermis was scored as 0.5, 1.0, and 1.5 points according to the severity of mild, moderate and severe;
  • the dyed skin sections were scanned with a Leica Aperio CS2 scanner at a magnification of 200, and then the scanned images were opened with the HALO analysis software.
  • the epidermal thickness of each slice is represented by the average epidermal thickness.
  • mice were euthanized by excessive carbon dioxide (CO2) inhalation, and the skin was collected and divided into four parts, three of which were quick-frozen in liquid nitrogen and stored in a -80 degree Celsius refrigerator for the detection of inflammatory factors.
  • CO2 carbon dioxide
  • IL-6 and TNF- ⁇ were detected using ELISA method.
  • sample set up blank wells respectively (no sample and enzyme labeling reagent are added to blank control wells, and the other steps are the same) and wells for samples to be tested.
  • sample diluent to the well of the sample to be tested on the enzyme-labeled coated plate, and then add 10 ⁇ L of the sample to be tested (the final dilution of the sample is 5 times).
  • Solution Dilute the 20-fold concentrated washing solution with distilled water 20-fold for later use.
  • washing Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let it stand for 30 seconds, and then discard, repeat 5 times, and pat dry.
  • Color development First add 50 ⁇ L of color developer A to each well, then add 50 ⁇ L of color developer B, gently shake and mix, and develop color at 37 degrees Celsius for 15 minutes in the dark.
  • Termination Add 50 ⁇ L of stop solution to each well to terminate the reaction (the blue turns to yellow at this time).
  • the purpose of this experiment was to evaluate the pharmacodynamics of compound (I) in the OXA-induced Balb/c mouse model of atopic dermatitis. Repeated OXA stimulation of the mouse's back skin can produce a long-term inflammatory response. This model is closer to clinical skin inflammation and is a common model for screening and evaluating compounds with anti-inflammatory activity.
  • Test drug Compound (I)
  • mice were randomly divided into 7 groups according to body weight, as shown in Table 9 above. OXA induction was performed on day 7. The induction method was to apply 5% OXA (dissolved in the solvent of acetone/olive oil (4/1)) on the back of the mouse near the neck, and apply 10 ⁇ L (1.5cm ⁇ 1.5cm). Mice in the normal control group were coated with 10 ⁇ L of acetone/olive oil (4/1) solvent in the same way. From day 1 to day 22, immunostimulation was performed, and 100 ⁇ L 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. Apply every two days.
  • mice in the normal control group were coated with 100 ⁇ L of acetone/olive oil (4/1) solvent in the same manner.
  • OXA smearing is performed after the above work is completed.
  • Compound (I) was administered by gavage on days 1 to 22, twice a day (PO, bid). Dosing was done in the morning, prior to skin thickness measurements, clinical scoring, photography, and OXA immunostimulation. The two doses were separated by 8 hours.
  • Body weight data were recorded twice a week.
  • Skin thickness (1.5 cm ⁇ 1.5 cm modeling area) was measured from day 1 to day 22 using a Mitutoyo digimatic indicator (Mitutoyo digital scale, model ID-C, USA), every two days. If skin thickness measurement and OXA immunostimulation are on the same day, skin thickness measurement will take precedence.
  • the skin of the modeling area was clinically scored from the 1st day to the 22nd day according to the skin scoring standard in Table 12, and the evaluation was performed every two days. If skin clinical scoring and OXA immunostimulation are on the same day, skin clinical scoring will take precedence.
  • tissue from the modeling area was collected for hematoxylin-eosin staining for skin pathology score and epidermal thickness measurement.
  • mice After the mice were sacrificed, the dorsal skin was removed and fixed in 10% neutral formalin for 24 hours. After dehydration, they were embedded in paraffin and made into 4-micron sections. The skin sections were stained with hematoxylin-eosin, and the inflammatory cell infiltration and tissue changes in the stratum corneum, epidermis and dermis were observed under a microscope, and pathological scoring was performed.
  • the scoring criteria are: inflammatory cell aggregation and edema, 1 point is mild, 2 points is moderate, 3 points is severe; telangiectasia is divided into 1 point is mild, 2 points is moderate, and 3 points is severe.
  • the specific scoring criteria for skin edema are as follows: (1) 1 point is mild: occasionally there are several edema cells at the junction of the epidermis and dermis, and the aggregated length of edema cells is less than 10% of the length of the dermis and epidermis junction; (2) 2 points is moderate: edema The cells occupy between 10% and 50% of the length of the dermis-epidermal junction; (3) 3 severe: edema cells occupy more than 50% of the dermis-epidermal junction.
  • telangiectasia The specific scoring criteria for telangiectasia are as follows: (1) 1 point is mild: 1-3 telangiectasias are occasionally seen; (2) 2 points are moderate: 3-6 points of telangiectasia; (3) 3 points are severe: more than 6 points telangiectasia.
  • the dyed skin sections were scanned with a Leica Aperio CS2 scanner at a magnification of 200, and then the scanned images were opened with the HALO pathological analysis software.
  • the epidermal thickness of each slice is represented by the average epidermal thickness.
  • the experimental results showed that there were inflammatory cell aggregation, edema and telangiectasia in the skin of the model group.
  • the pathological score of this group reached 6.8.
  • 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); after dexamethasone treatment, the pathological score was significantly reduced (P ⁇ 0.01 vs model group). 0.001 vs model group).
  • OXA stimulation significantly increased the thickness of the skin epidermis in the dorsal model area of mice.
  • compound (I) (10mpk, 30mpk and 60mpk) could significantly reduce the thickness of the epidermis (P ⁇ 0.05 vs the model group); the thickness of the epidermis in this area was significantly decreased after dexamethasone treatment (P ⁇ 0.001 vs the model group). ). See Table 14 for details.
  • Test drug Compound (I)
  • Positive control drug prednisone (national medicine approved word H33021207).
  • mice were randomly divided into 5 groups, namely model group: solvent (water solution containing 0.4% Tween80 and 0.5% methylcellulose) 10mL/kg BID, positive control group: Prednisone 6 mg/kg QD, compound (I) 20, 40 and 60 mg/kg BID groups, the administration volume was 10 mL/kg.
  • model group solvent (water solution containing 0.4% Tween80 and 0.5% methylcellulose) 10mL/kg BID
  • positive control group Prednisone 6 mg/kg QD
  • compound (I) 20, 40 and 60 mg/kg BID groups
  • the administration volume was 10 mL/kg.
  • female C57BL/6 mice served as normal controls.
  • Compound (I) and model group were intragastrically administered or solvent twice a day from the 5th week of age, with an interval of 8 hours between the two administrations; prednisone was administered by intragastric administration once a day from the 5th week of age; co-administration 17 weeks of medicine.
  • mice in the model group died at the 15th and 16th week of administration respectively, and 8 mice remained at the end of the test; 1 mouse in the compound (I) 20 mg/kg BID group died at the end of the 16th week of administration. The remaining 9 mice; the other groups did not appear to die.
  • mice 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.
  • Scoring system 1) skin redness, hemorrhage; 2) hair loss and skin dryness; 3) edema; 4) exfoliation/corrosion; 5) lichenoid plaques and other symptoms.
  • mice were dissected, the back skin tissues of the mice were taken for HE staining and pathological scoring.
  • lymph nodes of MRL/lpr mice were scored 17 times a week during the experiment.
  • the scoring standard is based on the diameter of the lymph node (cm), and the score is 0-6 points:
  • 3 points small (less than 1cm in diameter at the three side points);
  • both 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), low doses
  • Dosage group can significantly inhibit lymph node enlargement in SLE mice from 7 weeks to 11 weeks (7-9 weeks, ***P ⁇ 0.001vs model group; 11 weeks, *P ⁇ 0.05vs model group), while 12 weeks After the end of the experiment, the difference was no longer significant.
  • the positive control drug prednisone 6 mg/kg group also effectively inhibited the degree of lymph node enlargement in the systemic lupus erythematosus mice (***P ⁇ 0.001vs model group), see Table 17 for details.
  • mice were dissected and the lymph node tissue (submandibular, axillary and inguinal) was weighed. From the data of the lymph node tissue weighing, compared with the model group, both the high and middle doses of compound (I) could be effective. Effectively inhibited the degree of lymph node enlargement in systemic lupus erythematosus mice (***P ⁇ 0.001vs model group), see Table 18 and Figure 10 for details.
  • mice were dissected and the spleen tissue was weighed.
  • compound (I) high, medium and low doses could effectively inhibit the degree of spleen enlargement in mice with systemic lupus erythematosus (** *P ⁇ 0.001 vs model group), see Table 19 and Figure 11 for details.
  • the urine protein content of the mice was detected during the experiment, once a week, up to 16 weeks. According to the urine protein content at different times, the urine protein concentration-time curve was drawn, and the area under each curve was calculated.
  • mice were dissected and the kidney tissue was weighed.
  • the compound (I) high-dose group showed an extremely significant decrease in the total pathological score of both kidneys (***P ⁇ 0.001vs model group).
  • the total pathological score of both kidneys decreased significantly (**P ⁇ 0.01 vs model group), see Table 21 and Figure 13 for details.
  • TIL tubulointerstitial lesions
  • HE staining scoring results Compared with the model group, in the activity index (AI) score, the total pathological score of both kidneys in the middle and high doses of compound (I) group decreased significantly (**P ⁇ 0.01vs model group); In the chronic index (CI) score, the total pathological score of both kidneys in the compound (I) high-dose group decreased significantly (**P ⁇ 0.01 vs model group), and the compound (I) low-dose group showed a significant decrease in the total pathological score of both kidneys There was a significant decrease (*P ⁇ 0.05vs model group); in the score of tubulointerstitial damage (TIL), 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 for details.
  • the rate of IgG positive cells and the intensity of staining were scored by IHC staining of kidney tissue.
  • Kidney IHC (IgG) scoring criteria :
  • 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
  • the final score is the product of the two, 0 is negative (-); 1-3 is low expression (+); 4-8 is moderate expression (++); 9-12 is high expression (+++) .
  • each dose group of compound (I) improved IgG deposition in kidney 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 ⁇ 0.05 vs model group). See Table 23 and Figure 15 for details.
  • Mouse serum anti-ds-DNA antibody concentrations were measured during the experiment every 4 weeks to 16 weeks.
  • the results showed that the lupus erythematosus symptoms of mice in the model group continued to aggravate over time.
  • the compound (I) medium-dose group can effectively reduce the anti-ds-DNA antibody concentration level (16 weeks, *P ⁇ 0.05vs model group).
  • the positive control drug prednisone 6mg/kg can also effectively reduce the concentration of anti-ds-DNA antibody. See Table 24 and Figure 16 for details.
  • Compound (I) has no effect on the central nervous system, respiratory system and cardiovascular system of animals, and is expected to have no side effects on the central nervous system, respiratory system and cardiovascular system of humans;
  • the NOAEL of a single dose in rats is 2000mg/kg, Beagle
  • the MTD for a single dose in dogs is 500 mg/kg/time (1000 mg/kg/day)
  • the NOAEL for 28-day repeated dosing in rats is 10 mg/kg/time (20 mg/kg/day)
  • the NOAEL of the drug was 3 mg/kg/time (6 mg/kg/day)
  • Compound (I) was not genotoxic.
  • the safety window evaluation is shown in the following table.
  • the effective dose in rats is 3 mg/kg/time BID.
  • compound (I) has a safety window of 1 times in Beagle dogs and 8-13 times in SD rats.
  • the safety window of compound (I) was 3-fold in both Beagle dogs and SD rats according to the dose. It is proposed that the initial dose of compound (I) in the first human trial is 15 mg, and the NOAEL dose of compound (I) in the repeated dose toxicology study in Beagle dogs is 14 times safer than the starting dose in humans. Window, the human equivalent of the NOAEL dose in the repeated-dose toxicology study in SD rats has a safety window of 16 times the human starting dose.
  • the planned phase I clinical trial single-dose climbing doses are: 15mg, 45mg, 60mg, 90mg, 135mg, 180mg, 240mg, 300mg and 360mg.
  • compound (I) can improve the skin lesions of psoriasis and atopic dermatitis mice, inhibit the enlargement of immune organs, and reduce the level of inflammation; it can also improve the skin lesions of SLE mice in a dose-dependent manner, alleviate kidney damage, inhibit the The immune organs are enlarged, and the increase of SLE-related antibodies and cytokines in serum is inhibited; and it has a certain safety treatment window and has a good clinical application prospect.

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Abstract

本申请提供化合物(I)、其光学异构体或其药学上可接受的盐在制备用于治疗JAK和SYK激酶高表达或异常激活相关疾病，包括自身免疫性疾病，如免疫介导的皮肤病，尤其是银屑病、特应性皮炎和SLE的药物中的用途。本发明所述化合物(I)、其光学异构体或其药学上可接受的盐可改善银屑病、特应性皮炎和SLE小鼠皮肤病变，缓解肾损伤，抑制免疫器官增大，降低炎症水平，抑制血清中SLE相关抗体和细胞因子的增高，且具有一定安全治疗窗，具有很好的临床应用前景。

Description

一种含有三环杂芳基的化合物的用途 技术领域
本发明属于医药领域，具体涉及一种含有三环杂芳基的化合物在制备治疗激酶JAK和SYK高表达或异常激活相关疾病的药物中的应用。
背景技术
JAK(Janus kinase)，即Janus激酶，是一类非跨膜型非受体型酪氨酸激酶，含四个亚型：JAK1、JAK2、JAK3和TYK2(Tyrosine kinase 2)。JAK1、JAK2和TYK2广泛存在于各种组织和细胞中，JAK1参与介导IL-6、IFN等炎症信号通路，JAK2可单独介导IL-3、IL-5和EPO等细胞因子信号通路，JAK3仅存在于骨髓和淋巴系统，介导IL-2、IL-4、IL-7、IL-9、IL-15和IL-21的信号传导，TYK2参与IFN-α、IL-6、IL-10和IL-12信号传导。JAK抑制剂通过特异性抑制JAK-STAT(Signal transducers and activators of transcription)信号通路，阻滞上述细胞因子的级联放大作用，参与免疫调节等过程。
SYK(Spleen tyrosine kinase)，即脾酪氨酸激酶，是一种非受体型酪氨酸激酶，存在于细胞基质中。SYK广泛表达于造血细胞、淋巴细胞、成纤维细胞、血管内皮细胞，在B淋巴细胞高表达，在肿瘤和自身免疫性疾病中发挥重要作用。Dectin-1/ITAM是抗原刺激免疫细胞诱导免疫性疾病的经典路径。在正常B细胞中，当抗原诱导的BCR交联，使胞内免疫受体酪氨酸酶活化基序(ITAM)发生磷酸化时，胞浆中的SYK是ITAM首先招募并活化的对象，活化的SYK再通过CARD9依赖性途径激活转录因子NF-κB，产生一系列炎性因子。另外，该途径还可激活Caspase-8，激活的Caspase-8剪切IL-1β前体，促进未成熟的IL-1β成熟。CARD9非依赖途径NLRP3信号通路也在未成熟IL-1β成熟中发挥作用。因此，SYK也是自身免疫性疾病的一个重要靶点。
银屑病(Psoriasis)是一种免疫介导的慢性、复发性、炎症性皮肤病。患病率在世界各地有显著差异，美国患病率为0.5％～3.15％，欧洲为0.75％～2.9％，中国1984年报告银屑病患病率为0.123％，2008年调查6个城市患病率为0.47％，2017年西南4省患病率为0.5％，中国银屑病患者约在600万以上。银屑病可发生于任何年龄，无性别差异， 约2/3的患者在40岁以前发病，大部分患者冬重夏轻。
银屑病的病因及发病机制尚未完全阐明，可能涉及遗传、免疫、环境等多种因素，通过以T淋巴细胞介导为主、多种免疫细胞共同参与的免疫反应，引起角质形成细胞过度增殖、关节滑膜细胞与软骨细胞炎症发生。银屑病的典型临床表现为鳞屑性红斑或斑块，局限或广泛分布。银屑病可以合并其他系统异常，如伴内脏及关节损害。中重度患者罹患代谢综合征和动脉粥样硬化性心血管疾病的风险增加。
根据银屑病的临床表现和病理特征，可分为以下几种类型：1、寻常型银屑病：为最常见的一型，多急性发病。典型表现为境界清楚、形状大小不一的红斑，周围有炎性红晕。稍有浸润增厚。表面覆盖多层银白色鳞屑。鳞屑易于刮脱，刮净后淡红发亮的半透明薄膜，刮破薄膜可见小出血点(Auspitz征)。皮损好发于头部、骶部和四肢伸侧面。部分患者自觉不同程度的瘙痒。2、脓疱型银屑病：分泛发型和掌跖型。泛发性脓疱型银屑病是在红斑上出现群集性浅表的无菌性脓疱，部分可融合成脓湖。全身均可发病。以四肢屈侧和皱褶部位多见，口腔黏膜可同时受累。急性发病或突然加重时常伴有寒战、发热、关节疼痛、全身不适和白细胞计数增多等全身症状。多呈周期性发作，在缓解期往往出现寻常型银屑病皮损。掌跖脓疱病皮损局限于手足，对称发生，一般状况良好，病情顽固，反复发作。3、红皮病型银屑病：又称银屑病性剥脱性皮炎，是一种严重的银屑病。常因外用刺激性较强药物，长期大量应用糖皮质激素，减量过快或突然停药所致。表现为全身皮肤弥漫性潮红、肿胀和脱屑，伴有发热、畏寒、不适等全身症状，浅表淋巴结肿大，白细胞计数增高。4、关节病性银屑病：又称银屑病性关节炎。银屑病患者同时发生类风湿性关节炎样的关节损害，可累及全身大小关节，但以末端指(趾)节间关节病变最具特征性。受累关节红肿疼痛，关节周围皮肤也常红肿。关节症状常与皮肤症状同时加重或减轻。血液类风湿因子阴性。
目前银屑病尚无特效疗法，主要治疗手段包括局部治疗、物理治疗、系统治疗、中医中药治疗等多种疗法。其中，局部治疗包括外用药物治疗、物理疗法等。外用药物主要为：维生素D3类似物、糖皮质激素、蒽林、维A酸凝胶和霜剂、焦油类、免疫抑制剂等，以及其他外用药如他克莫司，匹美莫司、0.03％的喜树碱软膏、5％的水杨酸软膏 等。系统治疗药物包括甲氨蝶呤、维A酸类、环孢素、他克莫司，霉芬酸酯，生物制剂如依那西普、英夫利昔单抗以及抗生素等。对于中、重度患者，单一治疗不佳时通常给予联合、交替或序贯治疗。现有治疗虽短期效果显著，但会产生诸多副作用且复发率高，远期疗效欠佳，如：甲氨蝶呤治疗量与中毒量很接近；维A酸类药主要副作用为致畸胎；环孢素A不良反应有肾毒性、高血压、恶心、呕吐、乏力、肌颤及尿路刺激症状等；他克莫司不良反应类似环孢素A；霉酚酸酯不良反应有胃肠道症状、贫血、白细胞减少，有增加感染和诱发肿瘤的风险等。
特应性皮炎(Atopic dermatitis，AD)，又称异位性皮炎、遗传过敏性皮炎，是一种以皮肤瘙痒、多形性皮疹为特征的变态反应性皮肤病，在不同年龄阶段有不同的临床表现，是一种慢性、复发性、炎症性皮肤病。由于患者常合并过敏性鼻炎、哮喘等其他特应性疾病，故被认为是一种系统性疾病。过去30年全球范围内AD患病率逐渐增加，发达国家儿童AD患病率达10％～20％，中国AD患病率的增加晚于西方发达国家和日本、韩国，但近10年来增长迅速。1998年学龄期青少年(6～20岁)AD的总患病率为0.70％，2002年10城市学龄前儿童(1～7岁)的患病率为2.78％，2012年上海地区3～6岁儿童患病率达8.3％。2014年我国12个城市1～7岁儿童AD患病率达到12.94％，1～12月婴儿AD患病率达30.48％。
AD的发病与遗传和环境等因素关系密切。虽然确切发病机制尚不清楚，但目前认为免疫异常、皮肤屏障功能障碍、皮肤菌群紊乱等因素是发病的重要环节。AD通常初发于婴儿期，1岁前发病者约占全部患者的50％，呈慢性经过，临床表现多种多样，最基本的特征是皮肤干燥、慢性湿疹样皮损和明显瘙痒。部分患者可同时有其他过敏性疾病，如过敏性哮喘、过敏性鼻结膜炎等。此外，由于长期慢性炎症反应，慢性病程患者合并发生神经系统疾病、炎性肠病、类风湿性关节炎、心血管疾病和淋巴瘤的风险明显增高。
目前AD的治疗包括基础治疗(避免接触过敏等)，外用药物治疗(外用糖皮质激素和外用钙调神经磷酸酶抑制剂等)，系统治疗(口服抗组胺药物、免疫抑制剂、糖皮质激素等)，紫外线治疗和抗微生物治疗等，其中以外用药、激素和免疫抑制剂为主。通常采用阶梯治 疗方式针对轻中度患者进行治疗。现有的治疗方法虽能缓解症状，但仍存在一定的局限性及诸多不良反应。在迅速起效、瘙痒控制、预防复发上仍有许多未被满足的需求。
红斑狼疮(lupus erythematosus，LE)是一种典型的自身免疫性结缔组织病，可分为盘状红斑狼疮(DLE)、亚急性皮肤型红斑狼疮(SCLE)、系统性红斑狼疮(SLE)、深在性红斑狼疮(LEP)、新生儿红斑狼疮(NLE)、药物性红斑狼疮(DIL)等亚型。
盘状红斑狼疮，主要侵犯皮肤，是红斑狼疮中最轻的类型。少数可有轻度内脏损害，少数病例可转变为系统性红斑狼疮，多数患者皮损无自觉症状，但很难完全消退。亚急性皮肤型红斑狼疮，临床上较少见，是一种特殊的中间类型，皮损常反复发作，绝大多数患者均有内脏损害，但严重者很少，主要症状为关节痛、肌肉痛、反复低热，少数有肾炎、血液系统改变。深在性红斑狼疮又称狼疮性脂膜炎、深部红斑狼疮，同样是中间类型的红斑狼疮，性质不稳定，可单独存在，以后既可转化为盘状红斑狼疮，也可转化为系统性红斑狼疮，或与它们同时存在。新生儿红斑狼疮，表现为皮肤环形红斑和先天性心脏传导阻滞，有自限性，一般在生后4～6个月内自行消退，心脏病变常持续存在。药物性红斑狼疮主要表现为发热、关节痛、肌肉痛、面部蝶形红斑、口腔溃疡，可有浆膜炎，停药后逐渐好转，病情较重者可给予药物治疗。
系统性红斑狼疮(Systemic lupus erythematosus，SLE)是一种系统性自身免疫性疾病,是红斑狼疮各类型中最为严重的一型，以全身多系统多脏器受累、反复的复发与缓解、体内存在大量自身抗体为主要临床特点，如不及时治疗，会造成受累脏器的不可逆损害，最终导致患者死亡。SLE患病率地域差异较大，目前全球SLE患病率为0～241/10万，中国大陆地区SLE患病率为30～70/10万，男女患病比为1:10～12。SLE具体发病因素尚未完全明确，是由多个因素共同参与引起的，主要与遗传、感染、内分泌及环境等多种因素有关。免疫调节机制的缺陷，如凋亡细胞和免疫复合物的清除也是SLE发生的重要因素。免疫耐受丧失、抗原负荷增加、过度的T细胞辅助、B细胞抑制缺陷以及Th1细胞向Th2细胞转变导致B细胞过活化和致病性自身抗体的产生。另外，有一些药物如甲基多巴、苯妥英钠、青霉胺、奎尼丁、心 得安等可以直接引起药物性狼疮和加重红斑狼疮。绝大多数SLE患者发病时即有多系统损害表现，少数病人由其他类型的红斑狼疮发展而来，部分病人还同时伴有其他的结缔组织病，如硬皮病、皮肌炎、干燥综合征等，形成各种重叠综合征。
目前SLE药物治疗以糖皮质激素、羟氯喹为基础用药。然而，应用上述药物的治疗会产生相应的副作用，如感染、肝肾功能受损和代谢异常等，有时不得不减量或停药，而且目前仍没有良好的药物控制病情发展，仍需继续开发新的药物。SYK和JAK分别处在诱发SLE两条不同信号通路的上游，因此SYK-JAK双通道抑制剂有望成为治疗SLE的有效途径。目前尚无被批准上市的JAK-SYK双靶点抑制剂。以DLE为开发适应症的JAK-SYK双靶点抑制剂产品R333，也已因一项Ⅱ期临床失败，于2013年10月24宣布终止开发。
因此，存在对开发新的用于治疗自身免疫性疾病，尤其是免疫介导的皮肤病和自身免疫性结缔组织病如银屑病、特应性皮炎或红斑狼疮等的药物的需要。
发明内容
具有如下结构式(I)的化合物(I)：
Figure PCTCN2022079887-appb-000001
其化学名称为：(R)-4-(环丙氨基)-2-((3-(环丙磺酰基)-1,2,3,4,4a,5-六氢苯并[b]吡唑[1,2-d][1,4]噁嗪-8-基)氨基)嘧啶-5-甲酰胺，首次公开于公开号为WO2018108084的PCT国际申请中，且已知是一种高选择性的JAK激酶和SYK激酶双靶点抑制剂，可用于治疗癌症。
本发明人经过进一步研究发现，化合物(I)通过抑制JAK和SYK激酶对细胞的信号转导和分裂增殖等起调控作用，在治疗自身免疫性疾病方面具有优越的效果，尤其是在免疫介导的皮肤病和自身免疫性 结缔组织病如银屑病、特应性皮炎或红斑狼疮等的治疗中表现出令人满意的活性和低的毒副作用。
本发明据此提供了化合物(I)、其光学异构体或其药学上可接受的盐在制备用于治疗JAK和SYK激酶高表达或异常激活相关疾病的药物中的用途：
Figure PCTCN2022079887-appb-000002
优选地，上述用途中，所述的JAK激酶为JAK1、JAK2、JAK3或TYK2激酶中的一种或几种，优选为JAK3和/或TYK2激酶。
优选地，上述用途中，所述的JAK和SYK激酶高表达或异常激活相关疾病为自身免疫性疾病。
优选地，上述用途中，所述自身免疫性疾病为免疫介导的皮肤病和自身免疫性结缔组织病。
优选地，上述用途中，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
进一步地，上述用途中，所述药物含有治疗有效量的化合物(I)、其光学异构体或其药学上可接受的盐，以及任选的，药学上可接受的赋形剂或载体。
本发明药物的施用方式没有特别限制，代表性的施用方式包括但并不限于：口服、直肠、肠胃外(静脉内、肌肉内或皮下)和局部给药。相应地，本发明药物可制成临床上可接受的各种剂型，包括口服剂型、注射剂型、局部给药剂型或外用剂型等。
优选地，本发明药物在临床上可以单独使用，也可以与其他治疗 组分联合使用。为方便临床使用，本发明化合物(I)、其光学异构体或其药学上可接受的盐可以与其它治疗组分联合制备复方药物或组合产品。
本发明还提供所述药物的使用方法，即把治疗有效量的本发明化合物(I)、其光学异构体或其药学上可接受的盐施用于需要治疗的哺乳动物(如人)。
本发明提供了一种治疗JAK和SYK激酶高表达或异常激活相关疾病的方法，其特征在于，将治疗有效量的本发明化合物(I)、其光学异构体或其药学上可接受的盐施用于需要治疗的哺乳动物(如人)。
本发明所述的JAK激酶为JAK1、JAK2、JAK3或TYK2激酶中的一种或几种，优选为JAK3和/或TYK2激酶。本发明所述的JAK和SYK激酶高表达或异常激活相关疾病为自身免疫性疾病，如免疫介导的皮肤病和自身免疫性结缔组织病，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
本发明还提供一种包含治疗有效量的化合物(I)、其光学异构体或其药学上可接受的盐的药物，其特征在于，所述药物用于治疗受试者的JAK和SYK激酶高表达或异常激活相关疾病，其为自身免疫性疾病，如免疫介导的皮肤病和自身免疫性结缔组织病，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
所述的JAK激酶为JAK1、JAK2、JAK3或TYK2激酶中的一种或几种，优选为JAK3和/或TYK2激酶。
本发明还提供一种复方药物或组合产品，所述复方药物或组合产品包含治疗有效量的化合物(I)、其光学异构体或其药学上可接受的盐，和其它治疗组分，其特征在于，所述复方药物或组合产品用于治 疗受试者的JAK和SYK激酶高表达或异常激活相关疾病，其为自身免疫性疾病，如免疫介导的皮肤病和自身免疫性结缔组织病，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
所述的JAK激酶为JAK1、JAK2、JAK3或TYK2激酶中的一种或几种，优选为JAK3和/或TYK2激酶。
本发明所述的治疗有效量是指药学上认为的有效给药剂量，即活性化合物的量足以明显改善病情，而不至于产生严重的副作用。对于60kg体重的人而言，日给药剂量通常为0.01～2000mg，优选1～500mg，更优选为10～400mg，进一步优选为15～360mg或15～250mg，如：15mg、45mg、60mg、90mg、135mg、180mg、240mg、300mg、360mg。可以每日一次单剂量施用，可以每天分多次施用，也可以间隔使用。具体剂量和给药频率应考虑给药途径、病人健康状况等因素，这些都是熟练医师根据常规技能可以确定的。所述施用方式没有特别限制，代表性的施用方式包括但并不限于：口服、直肠、肠胃外(静脉内、肌肉内或皮下)和局部给药。所述活性化合物的量以化合物(I)计。
体内、体外研究显示，(1)本发明化合物(I)在体外能显著抑制JAK1、JAK2、JAK3、TYK2和SYK激酶活性，其中在JAK3和TYK2上有较强的抑制作用，IC 50分别为1.43nM和0.82nM，在JAK2和SYK上的抑制作用略弱于JAK3和TYK2，IC 50在3nM～8nM之间，针对JAK1的抑制作用最弱，IC 50为20.04nM。(2)本发明所述化合物(I)能够改善或显著降低IMQ诱导的银屑病模型小鼠的皮肤厚度、耳厚度、脾重量、脾指数，PASI评分(红肿，鳞屑，厚度与总分)、小鼠皮肤表皮厚度、皮肤病理评分，以及皮肤组织中IL-6和TNF-α的含量。(3)本发明所述化合物(I)能够改善或显著降低OXA诱导的特应性皮炎模型小鼠的皮肤厚度和皮肤临床评分；能够改善造模区域皮肤炎症细胞聚集，水肿及毛细血管扩张现象，显著降低病理评分和表皮厚度(4)本发明所述化合物I化合物(I)能够剂量依赖性改善MRL/lpr红斑狼 疮模型小鼠皮肤病变，缓解肾损伤，减少淋巴结和脾脏肿大以及抑制血清中SLE相关抗体和细胞因子的增高。其中20mg/kg组小鼠给药7周后能够有效抑制SLE小鼠的淋巴结肿大，在试验终点指标评价结果显示，20mg/kg给药可有效抑制脾肿大和淋巴结肿大，在慢性指数(CI)的双肾总病理评分出现比较明显的下降，也可一定程度抑制血清IL-6以及TNF-α的过度表达。40mg/kg和60mg/kg给药剂量能够在改善皮损病变(皮肤损伤评分及皮肤病理评分)、缓解肾损伤(降低小鼠尿蛋白，减少肾脏免疫复合物沉积)、抑制免疫器官增大(改善淋巴结病理评分以及抑制脾脏和淋巴结增大)等方面全面改善SLE小鼠的多种症状。
以上研究结果表明，本发明所述的化合物(I)、其光学异构体或其药学上可接受的盐可改善银屑病小鼠和特应性皮炎小鼠的皮肤病变，抑制免疫器官增大；还可以剂量依赖性改善SLE小鼠的皮肤病变，抑制免疫器官增大，缓解肾脏功能损伤，降低炎症水平，且具有一定安全治疗窗，具有很好的临床应用前景。
附图说明
图1：IMQ诱导的银屑病模型小鼠研究终点时的脾重量图。
图2：IMQ诱导的银屑病模型小鼠研究终点时的脾指数(脾重/体重％)图。
图3：IMQ诱导的银屑病模型小鼠研究终点时的小鼠皮肤组织中IL-6的含量图。
图4：IMQ诱导的银屑病模型小鼠研究终点时的小鼠皮肤组织中TNF-α的含量图。
图5：IMQ诱导的银屑病模型小鼠研究终点时的皮肤病理评分图。
图6：IMQ诱导的银屑病模型小鼠研究终点时的皮肤表皮厚度图。
图7：OXA诱导的特应性皮炎模型小鼠研究终点时的皮肤病理评分图。
图8：OXA诱导的特应性皮炎模型小鼠研究终点时的皮肤表皮厚度图。
图9：SLE小鼠试验终点皮肤病理评分图。
图10：SLE小鼠试验终点淋巴结重量图，A：总淋巴结重量；B： 总淋巴结重/体重％。
图11：SLE小鼠试验终点脾脏重量图，A：脾脏重量；B：脾重/体重％。
图12：SLE小鼠经治疗16周的尿蛋白-曲线下面积图。
图13：SLE小鼠试验终点肾脏重量图，A：总肾重；B：肾重/体重％。
图14：SLE小鼠肾脏组织HE染色评分图，A：双侧肾脏HE评分-活动指数；B:双侧肾脏HE评分-慢性指数；C：双侧肾脏HE评分-肾小管间质损伤。
图15：SLE小鼠肾脏组织IHC(IgG)染色评分图。
图16：SLE小鼠血清抗ds-DNA抗体浓度图。
图17：SLE小鼠血清细胞因子水平图，A：TNF-α浓度；B：IL-6浓度。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实验材料来源或配制：
1、化合物(I)：石药集团中奇制药技术(石家庄)有限公司自制。
2、阳性对照药、实验中所用试剂、原材料均为商业购买或自行配制。
3、体内实验受试物(化合物(I)和阳性对照化合物)配制方法：
称量后用含0.4％Tween80和0.5％甲基纤维素的水溶液溶解，化合物(I)配置浓度分别为0.3mg、1mg/mL、2mg/mL、3mg/mL、4mg/mL和6mg/mL；阳性对照药用生理盐水配置浓度为0.3mg/mL或0.6mg/mL，临用时稀释至所需浓度。
4、溶媒(0.4％Tween80/0.5％甲基纤维素)配制1L：
称取5.0g甲基纤维素粉末至干净玻璃瓶中，加入900mL的无菌水，过夜搅拌至充分溶解；吸取4.0mL Tween80，充分搅拌混匀，定 容至终体积1000mL。该溶液保存于4摄氏度冰箱。
5、OXA配制：
丙酮/橄榄油(4/1)溶媒配置：40mL丙酮和10mL橄榄油混合震荡30秒，至混合均匀得丙酮/橄榄油(4/1)溶媒。
5％OXA配制：称取100.00mg OXA，溶于2.00ml的丙酮/橄榄油(4/1)中，振荡30秒。
0.1％OXA配制：称取15.69mg OXA，溶于15.69mL的丙酮/橄榄油(4/1)中，振荡30秒。现用现配，每隔两天配一次。
实施例1：化合物(I)对激酶的活性抑制试验
1.激酶：SYK、JAK1、JAK2、JAK3和TYK2
2.实验方法
采用Mobility Shift Assay测定蛋白激酶活性。化合物(Ⅰ)用DMSO溶解后，在100％DMSO中配置成浓度梯度。以激酶缓冲液稀释，在384孔板中加入5μL的5倍反应终浓度的化合物(Ⅰ)(10％DMSO)。加入10μL的2.5倍酶溶液后室温孵育10分钟，再加入10μL的2.5倍底物溶液。28℃下孵育60分钟，向384孔板反应板中加30μL终止液终止反应(100mM HEPES，pH 7.5，0.015％Brij-35，0.2％Coating Reagent#3，50mM EDTA)。Caliper EZ ReaderⅡ上复制转化率数据，把转化率转化成抑制率数据：％抑制率＝(max-转化率)/(max-min)×100％。其中，“min”为不加酶进行反应的对照样孔转化率；“max”为加入DMSO作为对照孔转化率。以化合物浓度和抑制率为横纵坐标，绘制曲线，使用XLFit excel add-in version 5.4.0.8拟合计算IC 50值。拟合公式:Y＝Bottom+(Top-Bottom)/(1+(IC 50/X)^HillSlope)。
Figure PCTCN2022079887-appb-000003
3.实验结果
化合物(Ⅰ)对激酶的抑制活性测试结果见下表：
表1 化合物(Ⅰ)对激酶的抑制活性
激酶 JAK1 JAK2 JAK3 TYK2 SYK
IC 50,nM 20.04 3.92 1.43 0.82 7.25
实施例2：化合物(Ⅰ)对IMQ诱导的小鼠的银屑病模型中的药效试验
1.试验目的
本试验目的在于评估化合物(Ⅰ)在IMQ(5％咪喹莫特乳膏乳膏)诱导的小鼠银屑病模型中的药效。咪喹莫特的临床不良反应之一为诱导银屑病发作，利用咪喹莫特诱导的小鼠银屑病模型操作简单、易于实现，且皮肤表型、病理特征具有组织间复杂相互作用并与临床银屑病类似的优点。
2.试验药物
受试药：化合物(Ⅰ)
阳性对照药：地塞米松(Dex)
溶媒：0.4％Tween80/0.5％甲基纤维素。
3.试验动物
5-6周龄雌性Balb/c小鼠，70只。
4.试验分组及给药方案
根据实验开始前一天体重把动物随机分成7组，详见表2：
表2 银屑病药效实验分组
Figure PCTCN2022079887-appb-000004
备注：“PO”为口服；“bid”为每日2次；“qd”为每日1次；mpk：mg/kg。
造模：
动物于涂药前一天背部剃毛，形成约2cm×3cm大小的皮肤暴露区。IMQ诱导小鼠，每天定时在小鼠裸露的背部以及右耳涂抹5％咪喹莫特乳膏62.5mg，持续14天。正常对照组小鼠，每日定时在小鼠裸露的背部以及右耳涂抹凡士林。
5.试验结果
5.1化合物(Ⅰ)对IMQ诱导的小鼠的皮肤厚度的影响
从实验第一天开始每日进行皮肤厚度测量，直至实验结束。
皮肤厚度测量方法如下：使用左手拇指和食指将鼠背部皮肤连同皮下组织捏起，方向与老鼠身体方向相同；右手握数显千分测厚规(型号BK-3281，生产厂家：上海纽辉)在距左手捏起部位1cm处(近造模部位的中心)测量皮褶厚度，厚度以mm为单位，实际厚度为测量厚度的一半(皮肤厚度＝测量值/2)。
实验结果显示，同正常对照组相比，模型组小鼠在连续14天给予5％咪喹莫特乳膏刺激之后，皮肤厚度显著升高。在研究终点时，化合物(Ⅰ)(3mpk、10mpk、30mpk和60mpk)和地塞米松(3mpk)治疗均显著抑制了皮肤厚度的升高(5组均为：P<0.001vs模型组)，详见表3。
表3 IMQ诱导小鼠经治疗14天的皮肤厚度(均值±标准误差，单位：mm)
组别 第1天 第14天
1 0.34±0.00 0.26±0.00 ***
2 0.33±0.00 0.60±0.02
3 0.32±0.00 0.51±0.01 ***
4 0.32±0.00 0.53±0.02 ***
5 0.32±0.00 0.50±0.01 ***
6 0.33±0.01 0.43±0.02 ***
7 0.32±0.00 0.32±0.01 ***
注：*P<0.05,**P<0.01，***P<0.001vs模型组。
5.2化合物(Ⅰ)对IMQ诱导的小鼠的耳厚度的影响
每日对耳厚度进行测量(耳廓中心位置)。
实验结果显示，同正常对照组相比，连续14天给予5％咪喹莫特乳膏刺激使得小鼠右耳厚度显著升高。在研究终点时，化合物(Ⅰ)3mpk、10mpk、30mpk和60mpk)和地塞米松(3mpk)治疗均显著抑制了耳厚度的增加(5组均为：P<0.001vs模型组)，化合物(Ⅰ)对耳厚度增加的抑制呈现剂量依赖性。详见表4。
表4 IMQ诱导小鼠经治疗14天的耳厚度(均值±标准误差，单位：mm)
Figure PCTCN2022079887-appb-000005
注：*P<0.05,**P<0.01，***P<0.001vs G2(模型组)。
5.3化合物(Ⅰ)对IMQ诱导的小鼠的皮肤PASI评分的影响
从实验第一天开始每日进行皮肤拍照，直至实验结束。
临床症状采用PASI评分标准，从红斑(Erythema)，鳞屑(Scaling)，皮肤厚度(Thickness)3项指标进行评价，以0～4分进行记分，将3者积分相加得到总积分。PASI评分标准如下：0，无症状；1，轻度；2，中度；3，重度；4，极重度。
红斑：0-无红斑可见；1-呈淡红色；2-红色；3-深红色；4-红色极深。
鳞屑：0-皮肤表面无可见鳞屑；1-部分皮损表面上覆有鳞屑；2-大多数皮损表面覆盖有鳞屑；3-皮损部位几乎全部被鳞屑覆盖；4-皮损部位全部被鳞屑覆盖。
皮肤厚度：0-皮肤光滑无褶皱；1-皮肤边缘轻微褶皱，或皮肤粗糙；2-皮损处全部出现轻微褶皱，或轻微隆起；3-皮损处褶皱程度进一步加深，或皮损肥厚、隆起明显；4-皮损处完全褶皱，或皮损高度肥厚，明显凸起。
干燥及瘙痒不计入总分。
实验结果显示，连续14天给予5％咪喹莫特乳膏刺激之后，模型组小鼠的红斑，鳞屑，皮肤厚度PASI评分以及总分均显著升高。在研究终点时，化合物(Ⅰ)(3mpk)的治疗显著改善了鳞屑评分(P<0.001vs模型组)与总分(P<0.05vs模型组)；化合物(Ⅰ)(10mpk和30mpk)的治疗显著改善了鳞屑评分(10mpk组：P<0.01vs模型组；30mpk组：P<0.001vs模型组)、皮肤厚度评分(2组均为：P<0.001vs模型组)与总分(10mpk组：P<0.01vs模型组；30mpk组：P<0.001vs模型组)；化合物(Ⅰ)(60mpk)以及地塞米松(3mpk)的治疗显著改善了红斑评分、鳞屑评分、皮肤厚度评分与总分(2组均为：P<0.001vs模型组)。详见表5。
表5 IMQ诱导小鼠经治疗14天的皮肤PASI评分(均值±标准误差)
Figure PCTCN2022079887-appb-000006
5.4化合物(Ⅰ)对IMQ诱导的小鼠的脾重量的影响
实验第14天，进行脾重量采集。
实验结果显示，化合物(Ⅰ)(10mpk、30mpk和60mpk)和地塞米松(3mpk)治疗显著抑制了脾重量的升高(化合物(Ⅰ)10mpk组为：P<0.05vs模型组；其它3组均为：P<0.001vs模型组)；化合物(Ⅰ)(30mpk和60mpk)和地塞米松(3mpk)治疗显著抑制了脾指数的升高(3组均为：P<0.001vs模型组)，详见表6。
表6 IMQ诱导小鼠经治疗14天的脾重量及脾指数(脾重/体重％)(均值±标准误差)
组别 脾重(mg) 脾重/体重(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
4 182.01±9.11 * 11.71±0.56
5 146.81±7.71 *** 9.27±0.47 ***
6 125.73±4.63 *** 8.01±0.28 ***
7 93.79±4.64 *** 6.17±0.29 ***
注：*P<0.05,**P<0.01，***P<0.001vs G2(模型组)
5.5皮肤表皮厚度测定及病理评分
实验结束后，所有小鼠过量吸入二氧化碳安乐死，采集皮肤，分为四份，其中一份放置于组织固定液固定24小时，用于病理检测。经脱水处理后石蜡包埋，制成4微米的切片。皮肤切片通过苏木伊红染色，可观察角质层、表皮层、真皮层和炎症细胞浸润情况。在测量表皮层厚度时，先用莱卡Aperio CS2扫描仪在200倍率下扫描染完色的皮肤切片，然后观察组织病理改变，进行评分。
病理评分具体标准如下:表皮层中发现芒罗小脓肿(Munro microabscess)2.0分；角化过度0.5分；角化不全1.0分；颗粒层变薄或消失1.0分；棘层增厚1.0分；皮突延长、起伏根据轻中重度分别计0.5分、1.0分、1.5分。真皮层中单一核或多核细胞浸润根据轻中重度分别计0.5分、1.0分、1.5分；乳突上顶0.5分；毛细血管扩张0.5分。
在测量表皮层厚度时，先用莱卡Aperio CS2扫描仪在200倍率下扫描染完色的皮肤切片，然后用HALO分析软件打开扫描后的图片。用软件中“分型”模板，将皮肤表皮定义为注释层，在注释层中将表皮分为上下两个线段，然后同“图层厚度”选项将线段中大约200处厚度计算出来。每个切片的表皮厚度是由表皮厚度平均值来表示。
实验结果表明，组1正常对照组镜下可见完整的皮肤结构，细胞形态正常，未见明显的异常改变。组2模型组的皮肤可见芒罗小囊肿，角化过度或不全，棘层增厚，并伴有明显的血管扩张，以及中度到重度的炎症细胞浸润。同组1正常对照组相比，连续14天给予5％咪喹莫特乳膏刺激显著升高了皮肤组织的病理评分。化合物(Ⅰ)(60mpk)和地塞米松(3mpk)的治疗显著改善了模型小鼠的皮肤组织病理评分(化合物(Ⅰ)60mpk组：P<0.01vs模型组；地塞米松组：P<0.001vs模型组)。同组1正常对照组相比，组2模型组的皮肤表皮厚度显著增加。化合物(Ⅰ)(30mpk和60mpk)和地塞米松(3mpk)的治疗显著降低了模型小鼠的表皮厚度(化合物(Ⅰ)2组：P<0.05vs模型组；地塞米松组：P<0.001vs模型组)。详见表7。
表7 IMQ诱导小鼠经治疗14天的病理评分与表皮厚度(平均值±标准误差)
组别 病理评分 表皮厚度(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±0.90 121.75±8.17 *
6 5.05±0.88 ** 113.28±6.95 *
7 3.95±0.50 *** 84.98±7.40 ***
注：*P<0.05,**P<0.01，***P<0.001vs G2(模型组)
5.6皮肤样本炎性因子检测
实验结束后，所有小鼠过量吸入二氧化碳(CO2)安乐死，采集皮肤，分成四份，其中三份于液氮中进行速冻，并保存于-80摄氏度冰箱，用于炎症因子检测。
使用ELISA方法对IL-6和TNF-α进行检测。
组织标本：称取一定量的皮肤组织，加入一定量的PBS(pH＝7.4)，用手工或匀浆器将标本匀浆充分。离心20分钟(2000-3000转/分)。仔细收集上清液。分装后一份待检测，其余冷冻备用。
操作步骤：
1.标准品的加样：设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL。
2.加样：分别设空白孔(空白对照孔不加样品及酶标试剂，其余各步操作相同)、待测样品孔。在酶标包被板上待测样品孔中先加样品稀释液40μL，然后再加待测样品10μL(样品最终稀释度为5倍)。将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混匀。
3.加酶：每孔加入酶标试剂100μL，空白孔除外。
4.温育：用封板膜封板后置37摄氏度温育60分钟。
5.配液：将20倍浓缩洗涤液用蒸馏水20倍稀释后备用。
6.洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30秒后弃去，如此重复5次，拍干。
7.显色：每孔先加入显色剂A 50μL，再加入显色剂B 50μL，轻轻震荡混匀，37摄氏度避光显色15分钟。
8.终止：每孔加终止液50μL，终止反应(此时蓝色立转黄色)。
9.测定：以空白孔调零，450nm波长依序测量各孔的吸光度(OD值)。测定应在加终止液后15分钟以内进行。
10.计算：用标准物的浓度与OD值计算出标准曲线的直线回归方程式y＝bx+a，其中x为浓度，y为OD值，将样品的OD值代入方程式，计算出样品浓度，再乘以稀释倍数，即为样品的实际浓度。
实验结果表明，化合物(Ⅰ)(3mpk、10mpk、30mpk和60mpk)和地塞米松(3mpk)显著降低了IL-6在皮肤中的含量(化合物(Ⅰ)3mpk和60mpk组：P<0.001vs模型组；化合物(Ⅰ)10mpk、30mpk组和地塞米松组：P<0.01vs模型组)和TNF-α在皮肤中的含量(化合物(Ⅰ)30mpk组：P<0.001vs模型组；化合物(Ⅰ)10mpk组：P<0.05vs模型组；化合物(Ⅰ)3mpk、60mpk组和地塞米松组：P<0.01vs模型组)。详见表8。
表8 IMQ诱导小鼠经治疗14天的皮肤炎性因子含量(平均值±标准误差)
组别 IL-6(浓度pg/g) TNF-α(浓度pg/g)
1 544.08±44.29 *** 906.85±41.88 ***
2 1000.87±72.20 1937.90±129.72
3 554.74±88.14 *** 1191.74±124.81 **
4 641.83±96.28 ** 1432.02±152.77 *
5 638.09±59.63 ** 1236.41±100.8 ***
6 499.72±95.81 *** 1188.81±159.63 **
7 607.20±76.89 ** 1172.53±115.72 ***
注：*P<0.05,**P<0.01，***P<0.001vs G2(模型组)
实施例3：化合物(Ⅰ)对OXA诱导的小鼠特应性皮炎模型的药效试验
1.试验目的
本试验目的在于评估化合物(Ⅰ)在OXA诱导的Balb/c小鼠特应性 皮炎模型中的药效学。反复多次的OXA刺激小鼠的背部皮肤，可以产生长期的炎症反应。该模型更贴近临床上的皮肤炎症，是筛选和评价具有抗炎症活性的化合物的常用模型。
2.试验药物
受试药：化合物(Ⅰ)
阳性对照药：地塞米松(Dex)
溶媒(0.4％Tween80/0.5％甲基纤维素)。
3.试验动物
8-9周龄雌性Balb/c小鼠，70只。
4.试验分组及给药方案
根据给药前，各组动物的体重，随机分为7组，每组10只动物，详见表9。
表9 特应性皮炎药效试验分组
Figure PCTCN2022079887-appb-000007
诱导特应性皮炎模型:
小鼠按体重随机分为7组，如上表9所示。第7天进行OXA诱导。诱导方式为将5％OXA(溶于丙酮/橄榄油(4/1)的溶剂中)涂抹于小鼠后背近脖颈处，涂抹10μL(1.5cm×1.5cm)。正常对照组的小鼠以相同的方式涂抹丙酮/橄榄油(4/1)的溶剂10μL。第1天到第22天，进行免疫刺激，将100μL 0.1％OXA(溶于丙酮/橄榄油(4/1)的溶剂中)均匀涂抹于小鼠后背近脖颈处。每两天涂抹一次。正常对照组的小鼠以相同的方式涂抹丙酮/橄榄油(4/1)的溶剂100μL。免疫刺激当天，需进行皮肤厚度测量、临床评分及拍照，则上述工作完成之后进行OXA涂抹。
给药方案：
化合物给药剂量如表9所示。化合物(Ⅰ)在第1到第22天以灌胃方式给药，每日两次(PO，bid)。早晨给药，在皮肤厚度测量、临床评分、拍照及OXA免疫刺激之前完成。两次给药间隔8小时。
5.试验结果
5.1化合物(Ⅰ)对OXA诱导的特应性皮炎的小鼠的体重的影响
每周记录两次体重数据。
实验结果显示，OXA刺激对小鼠体重无显著性影响。在研究终点时，化合物(Ⅰ)(3mpk、10mpk、30mpk和60mpk)对小鼠体重无显著性影响；地塞米松治疗后小鼠体重显著下降。详见表10。
表10 OXA诱导小鼠经治疗22天的体重(均值±标准误差)
Figure PCTCN2022079887-appb-000008
注：*P<0.05,**P<0.01，***P<0.001vs模型组
5.2化合物(Ⅰ)对OXA诱导的特应性皮炎的小鼠的皮肤厚度的影响
皮肤厚度(1.5cm×1.5cm造模区域)于第1天到第22天使用Mitutoyo digimatic indicator(三丰数显量表，型号ID-C，美国)进行测量，每两天测量一次。如果皮肤厚度测量和OXA免疫刺激在同一天，那么会优先对皮肤厚度进行测量。
实验结果显示，OXA免疫刺激之后，小鼠的背部皮肤(1.5cm×1.5cm造模区域)厚度显著升高。从第17天起，与模型组相比，化合物(Ⅰ)(30mpk和60mpk)显著抑制了造模区域皮肤厚度的升高(30mpk组第17天和22天：P<0.05vs模型组；60mpk组第17天和22天：P<0.01vs模型组)。从第7天起，与模型组相比，地塞米松显著抑制了皮肤厚度的增高(第7天：P<0.01vs模型组；第17和22天：P<0.001vs模型组)。详见表11。
表11 OXA诱导小鼠经治疗1-22天的皮肤厚度(均值±标准误差，单位：mm)
Figure PCTCN2022079887-appb-000009
注：*P<0.05,**P<0.01，***P<0.001vs模型组。
5.3化合物(Ⅰ)对OXA诱导的特应性皮炎的小鼠的皮肤临床评分
造模区域的皮肤于第1天到第22天参照表12皮肤评分标准进行 临床评分，每两天评价一次。如果皮肤临床评分和OXA免疫刺激在同一天，那么会优先进行皮肤临床评分。
表12 皮肤评分标准
标准 分值
正常 0
发红 1
水肿 2
蜕皮 3
渗液 4
实验结果表明，OXA免疫刺激之后，小鼠造模区域皮肤临床评分显著升高。从第19天起，与模型组相比，化合物(Ⅰ)(30mpk和60mpk)显著抑制了皮肤临床评分的升高(均为P<0.05vs模型组)。从第9天起，与模型组相比，地塞米松显著抑制了皮肤临床评分的升高(第9天：P<0.05vs模型组；第19天和22天：P<0.001vs模型组)。详见表13。
表13 OXA诱导小鼠经治疗1-22天的皮肤临床评分(均值±标准误差)
组别 第1天 第9天 第19天 第22天
正常对照组 0.0±0.00 0.0 ***±0.00 0.0 ***±0.00 0.0 ***±0.00
模型组 0.4±0.16 3.2±0.13 3.0±0.00 3.0±0.00
化合物(Ⅰ)3mpk组 0.4±0.16 3.1±0.10 2.9±0.10 3.0±0.00
化合物(Ⅰ)10mpk组 0.4±0.16 3.0±0.00 2.7±0.15 2.7±0.15
化合物(Ⅰ)30mpk组 0.5±0.17 3.0±0.00 2.5 *±0.17 2.5 *±0.17
化合物(Ⅰ)60mpk组 0.6±0.16 3.0±0.00 2.5 *±0.17 2.5 *±0.17
地塞米松3mpk组 0.4±0.16 2.7 *±0.15 1.2 ***±0.13 1.1 ***±0.10
注：*P<0.05,**P<0.01，***P<0.001vs模型组。
5.4皮肤表皮厚度测定及病理评分
研究终点时采集造模区域组织进行苏木素-伊红染色用于皮肤病理评分和表皮厚度的测量。
小鼠处死后，取下背部皮肤，将其放置于10％中性福尔马林固定24小时。经脱水处理后石蜡包埋，制成4微米的切片。皮肤切片通过苏木素-伊红染色，显微镜下观察角质层、表皮层和真皮层炎症细胞浸润和组织改变情况，进行病理评分。评分标准为：炎症细胞聚集和水肿，分为1分轻微，2分中度，3分严重；毛细血管扩张，分为1分轻微，2分中度，3分重度。
炎症细胞聚集具体评分标准如下：(1)1分轻微：可见少量<10％炎症细胞占据面积出现在真皮区域；(2)2分中度：炎症细胞占据面积在10％-50％的皮肤真皮区域；(3)3分严重：炎症细胞分布面积>＝50％的皮肤真皮区域。
皮肤水肿具体评分标准如下：(1)1分轻微：偶见几处表皮与真皮交界处细胞水肿，水肿细胞聚集长度小于10％的真皮与表皮交界线长度；(2)2分中度：水肿细胞占据长度介于10％-50％的真皮与表皮交界线长度；(3)3分严重：水肿细胞占据长度大于50％的真皮与表皮交界线长度。
毛细血管扩张具体评分标准如下：(1)1分轻微：偶见1-3处毛细血管扩张；(2)2分中度：3-6处毛细血管扩张；(3)3分重度：大于6处毛细血管扩张。
在测量表皮层厚度时，先用莱卡Aperio CS2扫描仪在200倍率下扫描染完色的皮肤切片，然后用HALO病理分析软件打开扫描后的图片。用软件中“分型”模板，将皮肤表皮定义为注释层，在注释层中将表皮分为上下两个线段，然后用“图层厚度”选项将线段中大约100处厚度计算出来。每个切片的表皮厚度是由表皮厚度平均值来表示的。
实验结果显示，模型组造模区域皮肤存在炎症细胞聚集，水肿及毛细血管扩张现象。根据实验方法中提及的病理评分标准，该组病理评分达到6.8。与模型组相比，化合物(Ⅰ)(10mpk、30mpk和60mpk)治疗后可以显著降低该区域皮肤的病理评分(P<0.01vs模型组)；地塞米松治疗后显著降低了病理评分(P<0.001vs模型组)。OXA刺激使得小鼠后背造模区域皮肤表皮层厚度显著增加。与模型组相比，化合物(Ⅰ)(10mpk、30mpk和60mpk)能够显著降低表皮厚度(P<0.05 vs模型组)；地塞米松治疗后该区域表皮层厚度显著下降(P<0.001vs模型组)。详见表14。
表14 OXA诱导小鼠经治疗22天的病理评分与表皮厚度(平均值±标准误差)
组别 表皮厚度(μm) 病理评分
正常对照组 4.34 ***±1.30 0.0 ***±0.00
模型组 86.76±3.55 6.8±0.33
化合物(Ⅰ)3mpk组 75.79±3.36 6.4±0.34
化合物(Ⅰ)10mpk组 68.84 *±4.95 5.1 **±0.43
化合物(Ⅰ)30mpk组 75.08 *±3.57 5.0 **±0.42
化合物(Ⅰ)60mpk组 72.90 *±4.00 5.0 **±0.52
地塞米松3mpk组 54.53 ***±2.98 1.1 ***±0.10
注：*P<0.05,**P<0.01，***P<0.001vs模型组
实施例4：化合物(Ⅰ)对SLE模型小鼠的药效试验
1.试验药物
受试药：化合物(Ⅰ)
阳性对照药：泼尼松(国药准字H33021207)。
2.试验动物
4周龄雌性MRL/lpr小鼠，50只；4周龄雌性C57BL/6小鼠，10只。
3.试验分组及给药方案
根据血清抗ds-DNA抗体浓度将雌性MRL/lpr小鼠随机分成5组，分别为模型组：溶剂(含0.4％Tween80和0.5％甲基纤维素的水溶液)10mL/kg BID，阳性对照组：泼尼松6mg/kg QD，化合物(Ⅰ)20、40和60mg/kg BID组，给药体积均为10mL/kg。另外，雌性C57BL/6小鼠作为正常对照组。化合物(Ⅰ)和模型组从第5周龄开始每天两次灌胃给药或溶剂，两次给药时间间隔8小时；泼尼松从第5周龄开始每天一次灌胃给药；共给药17周。
模型组有2只小鼠分别在给药15周和16周死亡，试验终点时剩余8只；化合物(Ⅰ)20mg/kg BID组有1只小鼠在给药16周后期死亡， 试验终点时剩余9只；其余各组未出现小鼠死亡现象。
4.试验结果
4.1化合物(Ⅰ)对MRL/lpr小鼠皮肤的影响
4.1.1皮肤损伤情况及评分
观察小鼠脸部、耳朵和背部皮肤损伤情况，对其进行评分，每周一次，共17次。
评分系统：1)皮肤红肿、出血；2)毛发缺失和皮肤干燥程度；3)水肿；4)表皮脱落/腐蚀；5)苔藓样硬化斑等症状。每项评分分值如下：正常＝0分；轻微＝1分；中度＝2；严重＝3分。皮肤损伤严重程度根据统计每项评估症状的总分决定。
结果显示，与模型组相比，从治疗14周开始，化合物(Ⅰ)高剂量和中剂量组能够有效抑制系统性红斑狼疮小鼠的皮肤损伤程度(14周-16周，*P<0.05vs模型组；17周，**P<0.01vs模型组)，阳性对照药泼尼松6mg/kg组也有效抑制系统性红斑狼疮小鼠的皮肤损伤程度(17周，**P<0.01vs模型组)，详见表15。
表15 小鼠经治疗0-17周皮肤评分
Figure PCTCN2022079887-appb-000010
注：*P<0.05,**P<0.01vs模型组
4.1.2皮肤HE病理评分
试验终点将小鼠解剖时，取小鼠背部皮肤组织HE染色，进行病理评分。
皮肤HE病理评分标准
评分 0 1 2 3 4
角化过度/角化不全 无 轻微 轻度 中度 重度
毛囊角栓 无 轻微 轻度 中度 重度
表皮、真皮萎缩 无 轻微 轻度 中度 重度
真皮浅层水肿 无 轻微 轻度 中度 重度
炎症细胞浸润 无 轻微 轻度 中度 重度
毛细血管扩张充血 无 轻微 轻度 中度 重度
表皮增生 无 轻微 轻度 中度 重度
结果显示，各给药组均可不同程度改善皮肤病理得分，化合物(Ⅰ)组高剂量组显示了统计学差异(**P<0.01vs模型组)，阳性对照药泼尼松6mg/kg组也显示了统计学差异(***P<0.001vs模型组)，实验结果详见表16及图9。
表16 小鼠经治疗17周后皮肤HE病理评分
Figure PCTCN2022079887-appb-000011
注：**P<0.01，***P<0.001vs模型组
4.2化合物(Ⅰ)对MRL/lpr小鼠淋巴结的影响
4.2.1淋巴结评分
在试验过程中对MRL/lpr小鼠淋巴结进行了评分，每周一次，共17次。
评分标准按淋巴结直径大小(cm)，评分分值为0-6分：
0分：正常；
1分：小(在一个两侧点位置直径小于1cm)；
2分：小(在两个两侧点位置直径小于1cm)；
3分：小(在三个两侧点位置直径小于1cm)；
4分：大(在一个两侧点位置直径大于1cm，另外两个两侧点位置直径小于1cm)；
5分：大(在两个两侧点位置直径大于1cm，另外一个两侧点位置直径小于1cm)；
6分：大(在三个两侧点位置直径大于1cm)。
从淋巴结评分数据来看，给药治疗7周后，化合物(Ⅰ)高、中剂量均能够有效抑制系统性红斑狼疮小鼠的淋巴结肿大程度(***P<0.001vs模型组)，低剂量组在给药7周至11周可显著抑制SLE小鼠淋巴结肿大(7-9周，***P<0.001vs模型组；；11周，*P<0.05vs模型组)，而12周后至实验终点则差异不再明显。阳性对照药泼尼松6mg/kg组也有效抑制系统性红斑狼疮小鼠的淋巴结肿大程度(***P<0.001vs模型组)，详见表17。
表17 小鼠经17周治疗淋巴结评分
Figure PCTCN2022079887-appb-000012
注：*P<0.05,***P<0.001vs模型组
4.2.2淋巴结重量
试验终点将小鼠解剖后进行了(颌下、腋下和腹股沟)淋巴结组织称重，从淋巴结组织称重数据来看，与模型组相比，化合物(Ⅰ)高、中两个剂量均能够有效抑制系统性红斑狼疮小鼠的淋巴结肿大程度(***P<0.001vs模型组)，详见表18和图10。
表18 淋巴结重量(均值±标准误差)
Figure PCTCN2022079887-appb-000013
注：***P<0.001vs模型组
4.3化合物(Ⅰ)对MRL/lpr小鼠脾脏的影响
试验终点将小鼠解剖后进行了脾脏组织称重，与模型组相比，化合物(Ⅰ)高、中、低三个剂量均能够有效抑制系统性红斑狼疮小鼠的脾脏肿大程度(***P<0.001vs模型组)，详见表19和图11。
表19 脾脏重量及脾重/体重(均值±标准误差)
Figure PCTCN2022079887-appb-000014
注：***P<0.001vs模型组
4.4化合物(Ⅰ)对MRL/lpr小鼠肾脏的影响
4.4.1尿蛋白-曲线下面积
在试验过程中检测小鼠尿蛋白含量，每周一次，至16周。根据不同时间下尿蛋白含量，绘制尿蛋白浓度-时间曲线图，计算各曲线下面积。
可以发现化合物(Ⅰ)的3个剂量组对尿蛋白有不同程度的改善，16周的时间曲线下面积在化合物(Ⅰ)的中高剂量组出现明显统计学差异(*P<0.05vs模型组)。详见表20和图12。
表20 小鼠经治疗16周的尿蛋白-曲线下面积
Figure PCTCN2022079887-appb-000015
注：*P<0.05,***P<0.001vs模型组
4.4.2肾脏重量
试验终点将小鼠解剖后进行了肾脏组织称重，与模型组相比，化合物(Ⅰ)高剂量组在双肾总病理评分出现极其显著下降(***P<0.001vs模型组)，化合物(Ⅰ)中剂量组在双肾总病理评分出现显著性下降(**P<0.01vs模型组)，详见表21和图13。
表21 小鼠经治疗17周后总肾重及肾脏重/体重
Figure PCTCN2022079887-appb-000016
注：**P<0.01,***P<0.001vs模型组
4.4.3肾脏HE病理评分
肾脏HE评分标准：
Activity Index(AI，活动指数)评分标准
Figure PCTCN2022079887-appb-000017
Figure PCTCN2022079887-appb-000018
Chronic Index(CI，慢性指数)评分标准
Figure PCTCN2022079887-appb-000019
Tubulointerstitial lesions(TIL，肾小管间质损害)评分标准
Figure PCTCN2022079887-appb-000020
HE染色评分结果：与模型组相比，在活动指数(AI)评分上，化合物(Ⅰ)中高剂量组在双肾总病理评分均有出现显著性下降(**P<0.01vs模型组)；在慢性指数(CI)评分上，化合物(Ⅰ)高剂量组在双肾总病理评分出现显著性下降(**P<0.01vs模型组)，化合物(Ⅰ)低剂量组在双肾总病理评分出现比较明显的下降(*P<0.05vs模型组)；在肾小管间质损害(TIL)评分上，化合物(Ⅰ)高剂量组在双肾总病理评分中出现显著性的下降(**P<0.01vs模型组)。详见表22和图14。
表22 小鼠经治疗17周后肾脏HE病理评分
Figure PCTCN2022079887-appb-000021
注：*P<0.05,**P<0.01,***P<0.001vs模型组
通过肾脏组织IHC染色，对IgG染色阳性细胞率以及着色强弱进行了评分。
肾脏IHC(IgG)评分标准：
IHC评分标准一
染色强度 评分值
阴性 0
弱 1
中 2
强 3
IHC评分标准二
细胞阳性率 评分值
无阳性细胞 0
细胞阳性率≤10％ 1
10％＜细胞阳性率≤50％ 2
50％＜细胞阳性率≤80％ 3
细胞阳性率＞80％ 4
最终评分结果为二者乘积，0分为阴性(-)；1-3分为低表达(+)；4-8为中表达(++)；9-12分为高表达(+++)。
结果显示，与模型组相比，化合物(Ⅰ)各剂量组均不同程度的改善肾脏组织中IgG沉积，其中化合物(Ⅰ)高剂量组在双肾总IHC染色病理评分中出现比较明显的下降(*P<0.05vs模型组)。详见表23和图15。
表23 小鼠经治疗17周后肾脏IHC病理评分
Figure PCTCN2022079887-appb-000022
注：*P<0.05,***P<0.001vs模型组
4.5化合物(Ⅰ)对MRL/lpr小鼠血清抗ds-DNA抗体浓度的影响
在试验过程中检测小鼠血清抗ds-DNA抗体浓度，每4周一次，至16周。
结果显示，模型组小鼠红斑狼疮症状随着试验时间进行不断加重。从抗ds-DNA抗体浓度来看，经过3-4周治疗后，和模型组相比，化合物(Ⅰ)中剂量组可有效降低抗ds-DNA抗体浓度水平(16周，*P<0.05vs模型组)。阳性对照药泼尼松6mg/kg也可有效降低抗ds-DNA抗体浓度水平。详见表24和图16。
表24 小鼠经治疗0-16周抗Ds-DNA抗体浓度
Figure PCTCN2022079887-appb-000023
注：*P<0.05,**P<0.01,***P<0.001vs模型组
4.6化合物(Ⅰ)对MRL/lpr小鼠血清细胞因子的影响
试验终点，采用ELISA方法，检测小鼠血清细胞因子水平。结果显示，与模型组相比，化合物(Ⅰ)低中高三个剂量组均降低TNF-α浓度水平，并且中高剂量组与模型组相比有显著差异(***P<0.001vs模型组)。化合物(Ⅰ)低中高三个剂量组对IL-6因子也具有一定降低作用，高剂量组对IL-6降低效果显著。详见表25和图17。
表25 试验终点血清中细胞因子浓度
Figure PCTCN2022079887-appb-000024
注：**P<0.01,***P<0.001vs模型组
实施例5毒理学实验
遵从NMPA《药物非临床研究质量管理规范》和FDA GLP规范 (21CFR Part 58)，并符合国际人用药品注册技术要求协调会(ICH)和NMPA相关指导原则要求的一般毒理学研究，对化合物(I)进行了毒理学研究，为临床患者使用化合物(I)提供安全性数据支持。给药方式及实验结果如下：
Figure PCTCN2022079887-appb-000025
实验结果：
化合物(I)对动物中枢神经系统、呼吸系统，心血管系统没有影响，预计对人的中枢神经系统、呼吸系统和心血管系统没有副作用；大鼠单次给药的NOAEL为2000mg/kg，Beagle犬单次给药的MTD为500mg/kg/次(1000mg/kg/天)，大鼠28天重复给药的NOAEL为10mg/kg/次(20mg/kg/天)，Beagle犬28天重复给药的NOAEL为3mg/kg/次(6mg/kg/天)化合物(I)没有遗传毒性。安全窗评估见下表，大鼠起效剂量为3mg/kg/次BID，按照暴露量计算，化合物(I)在Beagle犬中有1倍的安全窗，在SD大鼠中有8-13倍的安全窗；按照剂量计算，化合物(I)在Beagle犬和SD大鼠中均有3倍的安全窗。拟待定化合物(I)首次人体试验爬坡起始剂量为15mg，化合物(I)在Beagle犬重复给药毒理研究中的NOAEL剂量的人体等效剂量与人体起始剂量比有14倍的安全窗，在SD大鼠重复给药毒理研究中的NOAEL剂量的人体等效剂量与人体起始剂量比有16倍的安全窗。因此，根据化合物(I)临床前毒理研究数据，拟开展的I期临床试验单次给药爬坡剂量为：15mg、45mg、60mg、90mg、135mg、180mg、240mg、 300mg和360mg。
表26 化合物(I)安全窗推算
Figure PCTCN2022079887-appb-000026
I期临床试验结果显示，单次给药爬坡剂量至240mg时，尚未出现3级以上安全性问题。
综上，化合物(Ⅰ)可改善银屑病和特应性皮炎小鼠的皮肤病变，抑制免疫器官增大，降低炎症水平；还可剂量依赖性改善SLE小鼠皮肤病变，缓解肾损伤，抑制免疫器官增大，以及抑制血清中SLE相关抗体和细胞因子的增高；且具有一定安全治疗窗，具有很好的临床应用前景。
本申请使用的英文缩略词的全称及中文名如下：
Figure PCTCN2022079887-appb-000027
Figure PCTCN2022079887-appb-000028

Claims (10)

  1. 化合物(I)、其光学异构体或其药学上可接受的盐在制备用于治疗JAK和SYK激酶高表达或异常激活相关疾病的药物中的用途，其中所述的JAK和SYK激酶高表达或异常激活相关疾病为自身免疫性疾病，所述化合物(I)的结构如下式所示：
    Figure PCTCN2022079887-appb-100001
  2. 如权利要求1所述的用途，所述自身免疫性疾病为免疫介导的皮肤病和自身免疫性结缔组织病。
  3. 如权利要求2所述的用途，其中所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；其中所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
  4. 如权利要求1-3任一所述用途，其特征在于，所述药物含有治疗有效量的化合物(I)、其光学异构体或其药学上可接受的盐，以及任选的，药学上可接受的赋形剂或载体。
  5. 如权利要求4所述用途，其特征在于，所述药物制成临床上可接受的各种剂型，包括口服剂型、注射剂型、局部给药剂型或外用剂型。
  6. 如权利要求1-3任一项所述用途，其特征在于，所述药物在临床上单独使用或与其他治疗组分联合使用。
  7. 一种包含治疗有效量的化合物(I)、其光学异构体或其药学上可接受的盐的药物，
    Figure PCTCN2022079887-appb-100002
    其中所述药物用于治疗受试者的JAK和SYK激酶高表达或异常激活相关疾病，其为自身免疫性疾病，如免疫介导的皮肤病和自身免疫性结缔组织病，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
  8. 一种治疗JAK和SYK激酶高表达或异常激活相关疾病的方法，包括将治疗有效量的化合物(I)、其光学异构体或其药学上可接受的盐施用于需要治疗的哺乳动物(如人)：
    Figure PCTCN2022079887-appb-100003
    所述的JAK和SYK激酶高表达或异常激活相关疾病为自身免疫性疾病，如免疫介导的皮肤病和自身免疫性结缔组织病，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
  9. 一种复方药物或组合产品，所述复方药物或组合产品包含治疗 有效量的化合物(I)、其光学异构体或其药学上可接受的盐，和其它治疗组分，
    Figure PCTCN2022079887-appb-100004
    其特征在于，所述复方药物或组合产品用于治疗受试者的JAK和SYK激酶高表达或异常激活相关疾病，其为自身免疫性疾病，如免疫介导的皮肤病和自身免疫性结缔组织病，所述免疫介导的皮肤病选自银屑病或特应性皮炎，所述银屑病优选寻常型银屑病、脓疱型银屑病、红皮病型银屑病或关节病性银屑病；所述自身免疫性结缔组织病为红斑狼疮，所述红斑狼疮优选盘状红斑狼疮、亚急性皮肤型红斑狼疮、系统性红斑狼疮、深在性红斑狼疮、新生儿红斑狼疮、药物性红斑狼疮，进一步优选为系统性红斑狼疮。
  10. 如权利要求4所述用途，其特征在于，所述治疗有效量为0.01～2000mg，优选1～500mg，更优选为10～400mg，进一步优选为15～360mg或15～250mg，如：15mg、45mg、90mg、135mg、180mg、240mg、300mg、360mg。
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